gottardi davide tesi

gottardi davide tesi
Alma Mater Studiorum – Università di Bologna
DOTTORATO DI RICERCA IN
SCIENZE E BIOTECNOLOGIE DEGLI ALIMENTI
Ciclo XV
Settore Concorsuale di afferenza: 07/F
Settore Scientifico disciplinare: AGR/16
PRODUCTION OF BIOACTIVE PEPTIDES THROUGH
SEQUENCIAL ACTION OF YARROWIA LIPOLYTICA
PROTEASES AND CHEMICAL GLYCATION
Presentata da:
Davide GOTTARDI
Coordinatore Dottorato
Relatore
Chiar.mo Prof. Claudio CAVANI
Dott.ssa Lucia VANNINI
Correlatore
Chiar.ma Prof.ssa
M. Elisabetta GUERZONI
Esame finale anno 2013
TABLE OF CONTENTS
CHAPTER 1. FOOD BY-PRODUCTS
1.1. Food by-products: current production and uses...................................................
1.2. Industrial examples of uses of food by-products...................................................
1.2.1. Vegetable by-products.........................................................................................
1.2.1.1. Starch production by-product......................................................................
1.2.1.2. By-products from vegetables canning.........................................................
1.2.1.3. By-products from legume...........................................................................
1.2.2. Animal by-products..............................................................................................
1.2.2.1. Alternative sourcing of gelatin....................................................................
1.2.2.2. Beef collagen fibre......................................................................................
1.2.2.3. Other successful examples on animal by-products.....................................
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CHAPTER 2. PEPTIDES AND FOOD
2.1. Properties of peptides..............................................................................................
2.1.1. Structure and functional properties......................................................................
2.2. Sensory properties....................................................................................................
2.3. Peptides bioactivities................................................................................................
2.3.1. Anti-hypertensive peptides..................................................................................
2.3.2. Antithrombotic peptides
2.3.3. Hypocholesterolemic and hypotriglyceridemic peptides.....................................
2.3.4. Antioxidant peptides............................................................................................
2.3.5. Opioid peptides....................................................................................................
2.3.6. Immunomodulatory peptides...............................................................................
2.3.7. Anticancer peptides..............................................................................................
2.3.8. Antimicrobial peptides.........................................................................................
2.3.9. Multifunctional peptides......................................................................................
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CHAPTER 3. GLYCOPEPTIDES
3.1. Structure...................................................................................................................
3.2. Antifreezing activity.................................................................................................
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3.3. Sensory properties....................................................................................................
3.4. Glycopetides bioactivities........................................................................................
3.4.1. Antioxidant activity..............................................................................................
3.4.2. Antitumor activity................................................................................................
3.4.3. Antimicrobial activity..........................................................................................
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CHAPTER 4. PROTEASES
4.1. Exopeptidase.............................................................................................................
4.2. Endopeptidase..........................................................................................................
4.2.1. Serine protease.....................................................................................................
4.2.2. Aspartic protease..................................................................................................
4.2.3. Cysteine protease.................................................................................................
4.2.4. Metalloprotease....................................................................................................
4.3. Functions and applications......................................................................................
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CHAPTER 5. Yarrowia lipolytica
5.1. Taxonomy and morphology....................................................................................
5.2. Metabolism...............................................................................................................
5.3. Industrial relevances for Y. lipolytica.....................................................................
5.4. Proteases of Y. lipolytica...........................................................................................
5.4.1. Extracelllular proteases........................................................................................
5.4.1.1. Alcaline protease.........................................................................................
5.4.1.2. Acidic protease............................................................................................
5.4.2. Intracellular protease............................................................................................
5.4.3. Proteases production............................................................................................
5.4.4. Industrial applications..........................................................................................
CHAPTER 6. OBJECTIVES
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CHAPTER 7. MATERIAL AND METHODS
7.1. Yarrowia lipolytica strains and culture conditions................................................
7.2. Protein matrices.......................................................................................................
7.2.1. Total meat protein extraction...............................................................................
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7.3. List of chemicals used..............................................................................................
7.4. Proteolytic activity of Y. lipolytica..........................................................................
7.4.1. Extracellular proteases recovery and characterization.........................................
7.4.2. Characterization of proteases by Zymography....................................................
7.4.3. Preliminary evaluation of the "cold attitude" of the proteolytic enzymes...........
7.4.4. Evaluation of the Proteolytic profiles generated by Y. lipolytica proteases.........
7.4.5. SDS-PAGE electrophoresis.................................................................................
7.5. Improvement of peptides bioactivity......................................................................
7.5.1. Model system ......................................................................................................
7.5.1.1. Model system preparation and UV spectra collection................................
7.5.1.2. LC/MS analysis...........................................................................................
7.5.1.3. Production of hydrolysates..........................................................................
7.5.1.4. Preparation of Glycated/Glycosylated peptides..........................................
7.6. Chemical characterization of the proteins and hydrolysates...............................
7.6.1. Degree of hydrolysis (DH)...................................................................................
7.6.2. Size exclusion chromatography...........................................................................
7.6.3. Determination of peptides and glycopeptides molecular weights by Matrixassisted laser desorption ionization-time of flight-mass spectrometry (MALDI/TOFMS) ...............................................................................................................................
7.7. Biological characterization of the peptides............................................................
7.7.1. Antioxidant properties..........................................................................................
7.7.1.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity...........
7.7.1.2 Inhibition of linoleic acid peroxidation .......................................................
7.7.2. Angiotensin I-converting enzyme (ACE) inhibitory activity...............................
7.7.3. Cytotoxicity of the hydrolyzed and conjugated samples on human HepG2 cells
7.7.4 Antimicrobial activity ..........................................................................................
7.8. Statistical Analysis.....................................................................................................
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CHAPTER 8. RESULTS
8.1. Evaluation of the proteolytic activity of Yarrowia lipolytica on different
proteins
8.1.1. Preliminary characterization of proteases by Zymogram....................................
8.1.2. "Cold attitude" of the proteolytic enzymes..........................................................
8.1.3. Proteolytic profiles generated by Y. lipolytica proteases.....................................
8.1.4. Proteolysis of gelatin............................................................................................
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8.1.5. Characterization of the products of gelatin hydrolysis obtained by Y. lipolytica
1IIYL4A protease..........................................................................................................
8.1.6. In vitro bioactivity evaluation of the peptides.....................................................
8.1.6.1. DPPH radical scavenging activity...............................................................
8.1.6.2. Inhibition of the linoleic acid peroxidation.................................................
8.1.6.3. Cytotoxicity activity in human cells...........................................................
8.1.6.4. Antimicrobial activity.................................................................................
8.2. Improvement of peptides bioactivity.......................................................................
8.2.1. Model system.......................................................................................................
8.2.2. Characterization of wheat gluten and its hydrolyzates........................................
8.2.3. Chemical evaluation of glycoconjugation of gluten peptides by MALDI/TOFMS..................................................................................................................................
8.2.4. In vitro bioactivity evaluation of glycopeptides mixtures ..................................
8.2.4.1. DPPH Radical Scavenging Activity...........................................................
8.2.4.2. Inhibition of Linoleic acid peroxidation.....................................................
8.2.4.3. Anti-ACE activity.......................................................................................
8.2.4.4. Antimicrobial activity.................................................................................
8.2.4.5. Cytotoxicity in human cells .......................................................................
8.3. Tables..........................................................................................................................
8.4. Figures........................................................................................................................
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CHAPTER 9. DISCUSSION AND CONCLUSIONS
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CHAPTER 10. REFERENCES
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ACKNOWLEDGMENTS
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CHAPTER 1.
FOOD BY-PRODUCTS
A by-product is a secondary product derived from food industries that does not represent the
primary service produced. In other words, a by-product is the "output of a process that has a minor
quantity and/or a net realizable value when compared to the main products". A by-product can be
useful and marketable or it can be considered waste.
1.1. Food by-products: current production and uses
The food processing industry, despite the great articulation of its productive sectors, is
characterized by a significant production of by-products (Federalimentare, 2006). Because of
complexity of the food system, estimating sizes and impacts of these streams is very difficult.
However, some estimates can be derived from literature. Wirsenius (2008) estimated the amounts of
by-products (used for feed) and wastes of food processing in terms of energetic values (i.e. J which
is expressed as energetic value per 100 energetic units food produced). In particular, in Western
Europe per 100J food, 56J ends in by-products, while 20J is lost from the food system. According
to a survey performed by Awarenet (2004), the European Food processing activities produce about
250 million tonnes per year of by-products and waste along with relevant amounts of high COD
effluents. Also in Italy the economic dimension of the management of by-products from food
processing industries is significant. The 2-3% of the dry volumes and the 7-10% of the moist
volumes produced are by-products. The estimated sale value is around 300 million euro per year
(Federalimentare, 2006).
Such waste streams are only partially valorised at different value-added levels (spread on
land, animal feed, composting), whereas the main volumes of them are managed as waste of
environmental concern, with relevant negative effects on the overall sustainability of the European
food processing industry. With increasing disposal costs, alternative uses of co-products are
increasingly being sought. The economic value of each product comes from its intrinsic nutritional
or applicability value. Moreover for a company the utilization of a by-product can give some
benefits from the economical point of view. The erroneous classification of such products as
"waste" is doubly negative: from one side, the producers are forced to manage the disposal of the
materials even if they are still susceptible to use, from the other side, the potential users are forced
to search expensive alternative sources of supply (Federalimentare, 2006).
More serious consequences are reflected on the ecosystem. In fact, disposed by-products
would finish in the landfill or through the drains, water courses, creating in this way serious
environmental problems (Smith, 1998).
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Due to the current promotion of sustainability in the production and consumption of food by
governments and international institutions, and, at the same time, considering that food systems
must satisfy increasing needs in quantitative and qualitative terms, because of the increment of the
world population (Mancini et al., 2011), it is clear that the exploitation and the use of food byproducts must be increasingly enhanced.
Although in the industrialized society various former by-products have been upgraded to the
food domain (for example whey protein isolates) and feed domain, still vast streams of by-product
exist that have potential for the food domain. With an eye on potential handling and application
purposes, primary and secondary by-products are distinguished.
Primary by-products (e.g. farming by-products such as straw and residues from land
management) are traditionally largely used for caring and feeding animals. With an eye on their
composition further enhancement of nutrient utilization efficiency for feed (FAO, 2012) and
utilization for renewable materials and energy are the most relevant development directions.
Also secondary by-products (processing residues) are largely used for feeding purposes.
With (historical) industrialisation and scaling up of food processing, the distance between the place
of generation and utilisation of the by-products has increased. Consequently, specialized byproducts trading companies have developed, who still mainly aim at feed markets. In general wet
by-products are transported directly from place of origin to the farmer, whereas parts of the dry byproducts are processed to feed concentrates.
Some examples food by-products are reported in table 1. Their reutilization can cover the
production of: animal and pet food (with sugar beet pulp, corn gluten, cereals used for the
production of beer, whey, by-products of the meat processing), pharmaceutical and cosmetic
compounds (collagen, gelatin), bio-fertilizer, food ingredients (gluten, germ and fiber), bio-fuels.
The chemo-physical properties, the shelf life, the availability and transportability of the by-products
are crucial to establish their possible use.
The sugar industry by-products are the easiest to be found and utilized (Smith, 1998). For
example the molasses has a sugar content close to the 50% and nowadays it is widely used for the
antibiotics, organic acids and bakers' yeast productions (Smith, 1998). Over 20 million tons of
animal by-products emerge annually from EU from slaughterhouses, plants producing food for
human
consumption,
dairies
and
as
fallen
stock
from
farms
(http://ec.europa.eu/food/food/biosafety/animalbyproducts/index_en.print.htm).
In large-scale food industry sectors, with voluminous homogeneous protein-rich by-product
streams (like dairy and meat processing), development of application in higher-value domains been
very successful in last decades. Some example are summarized in table 2. A number of new and
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more sophisticated possible exploitation of food processing by-products and waste have been
foreseen, tested and sometimes also scaled up (Kosseva, 2009; Galanakis, 2012).
1.2. Industrial examples of uses of food by-products
Notwithstanding the complexity of introducing new foods (or ingredients) based on byproducts, various examples of innovations in this field have been successfully introduced in
practice.
Current societal focus (and stimulating governmental arrangements) on bio-energy, bio-fuels
and other renewable bio-based solutions has resulted in a large number of practical in those areas,
which has overwhelmed applications in the food domain. Except for industrial uses of dietary fibres
from food by-products (Elleuch et al., 2011), the number of practical examples on innovative use of
the by-products presented in scientific literature lately is very limited. Yet, various appealing
examples exits. Below a brief summary of some of these successful practical examples in the sector
of animal and vegetable by-products and waste is reported; for each example key success factors
and obstacles are also briefly outlined.
1.2.1. Vegetable by-products
1.2.1.1. Starch production by-product
The processing of crops like potato, wheat, rice and corn results in considerable side stream
which contain notably potentially valuable proteins. Most prominent example is the food-grade
isolation potato protein.
Previously the protein was separated from the potato juice by thermal and acid denaturation,
which resulted in an insoluble aggregates with lost functionality, only suited for feed. Recently the
Solanic company (AVEBE, Netherlands) has developed a plant that successfully extracts native
potato proteins from potato juice (by-product from potato starch production), and the product is
marketed to food industries (e.g. bakery, meat, sport supplements). The key success factor is the
development of the isolation process combined with the high quality of the derived proteins
(solubility, emulsification, foaming and gelling quality, high nutritional value, low allergenicity)
compared to other commercial ones of different animal or vegetal origin.
Some other attempts to extract highly functional proteins from other crops are less
successful (e.g. extracting gluten from wheat and β-glucans from distillery grains).
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1.2.1.2. By-products from vegetables canning
Vegetable canning results in considerable amounts of by-products (peels, rejects, etc.).
These are traditionally traded as cattle feed or composted. Value for bio-energy production is
limited because of the high water content and limited energetic value. Therefore, interest in
application for human food is getting more attention. Recently the company Provalor has developed
a process for vegetable juice (natural colorant in food) and fibres extraction from the by-products.
The main key success factor for this process is represented by the development of an adequate
extraction process with highly appreciable yields. Moreover, also the increasing interest in ‘clean
labels’ (the natural colorant can replace synthetic colorants in food products) and Societal call for
sustainable valorisation of by-products have acted as external factors promoting the success of this
process.
1.2.1.3. By-products from legume
Grain legumes, also known as pulses, are plants belonging to the family Leguminacae,
which are grown primarily for edible grains or seeds. India is the fifth largest legume produced in
the world. Among the legumes, the soybean, also classed as an oilseed, is pre-eminent for its high
(38-45%) protein. By-products of legume include: hulls, husk, seeds etc. Microcore Research
Laboratories (India Pvt Ltd.) has developed a process and patented the technology for the
converting husk of Bengal gram to insoluble dietary fibers and micro-crystalline dietary cellulose
which can be used in daily diet to control obesity. The main success factors are represented by the
development of the adequate technology to obtain the insoluble fiber and the high demand for such
a functional component which has strongly increased worldwide.
1.2.2. Animal by-products
Traditionally the major parts of an animal are used as food, feed or materials. As such real
waste products from animal slaughter hardly exist. However, there is a continuous technical
innovation to maximize the value the refinement and splitting of streams. Stringent regulation
change with regard to BSE has been a game changer in these industries for the application of byproducts for food and feed.
1.2.2.1. Alternative sourcing of gelatin
Confidence in traditional sources of gelatin (amongst others bovine hides and bones) was
seriously damaged by BSE breakout. Increase of gelatin prices has been a trailblazer for alternative
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production processes. A successful example is the Dutch company Ten Kate Vetten that developed
a production process primarily aiming at extracting fats from pig slaughter by-products. Such a
process was innovated so that high-quality gelatin can be isolated from their processing water. The
(mild) fat extraction process furthermore enabled valorisation of other protein products in pet feed.
1.2.2.2. Beef collagen fibre
In leather production substantial amounts of animal material occur are cut off and wasted.
Hulsh of Protein Technologies in the Netherlands has innovated their process such that these cut
offs are kept in food-grade quality and processed to native collage fibres with superior water
binding and structuring properties compared to common thermally denatured collagen (due to mild
drying and grinding procedures that leave the collagen fibres in their native triple helix structure).
The collagen product is Halal certified.
1.2.2.3 Other successful examples on animal by-products
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Insuline from pancreas: pharmaceutical hormone for diabetic patients.
•
Mucine from pig bowels: ingredient for synthetic saliva.
•
Blood proteins processing and valorisation
•
Cholesterol from Lanoline (sheep wool fat).
•
Cholesterol as building block for the pharmaceutical industry, cosmetics
industry and crucial feed additive for shrimps.
Each of these examples was driven by internal factors: costs of wastage and/or value of the
product.
Table 1. By-products that could be exploited as substrates for biotechonological processes
(Smith, 1998)
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Table 2. Summary of common and potential uses of by-products in various agro-food sectors.
Sector (by-product)
Historical or
common use
Vegetable processing Dairy feed
(peels, reject, etc.)
Oil seeds (cake)
Protein-rich animal
feed
Fruits (seed, peel
pomace, kernel,
wastes)
Feed for livestock;
components are used
for cosmetics and
paints.
Cereals(bran, husks)
Feed for livestock
Dairy by-products
(whey, skim milk,
butter-milk, etc)
Protein-rich animal
feed
State-of-the-art use (and potentials) in food and
other high-value applications
Food-functional properties of various compounds
are broadly recognized.
Practical implementation is limited because of
technological and/or economic reasons. In a
limited number of practical situations, food
ingredients are produced out of the by-products
(e.g. food-grade trimmings for processed foods,
vegetable juices from food-grade peels and
rejects).
Vegetable wastes such as sugar beet leaves,
cauliflower leaves and gram plant with empty
pods can serve as a good source of essential
vitamins and antioxidants and serve as an organic
source of minerals.
Deriving attractive protein-rich ingredients for
food applications will require major changes in
oil extraction processes.
Fruit peels have relatively high contents of
functional food compounds. For example, apple
pomace is a rich source of polyphenols, minerals
and dietary fibre (Sudha et al., 2007), and banana
peels have high contents of pectins (including
glucose, galactose and xylose).
Beyond the food domain, a product like banana
peel can be used for biomethanation.
Furthermore, it can be used as a sorbent that
removes heavy metals from waste water.
Because of high contents of dietary fibres, bran is
traditionally used in amongst others bakery and
products and breakfast cereals.
Part of the rice bran is used for rice bran oil; 75%
of this oil is used in the food domain, whereas
25% is used for soap manufacturing. Rice bran
wax is an important by product of rice bran oil
industry. Rice bran wax can be used in the
preparation
candles,
polishes,
cosmetics,
emulsifiers and other industrial preparations.
Relatively new is the application in minced meat,
contributing to water binding capacity.
Wheat bran could be utilized in solid state, liquid
state fermentations and animal feeds.
Value of whey (powder) as high-value protein
product in food is very high.
Whey obtained as a by-product of cheese industry
has long been utilized in the production of
fermented beverages, both alcoholic and nonalcoholic (acidic).
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Sugar processing byproducts (sugarcane
bagasse)
Meat processing
(bone meal , poultry
by-products meal &
other animal byproducts)
Generation of steam
and power required to
operate the sugar
factory
Feed for livestock,
pets and aquaculture
(rich of essential
amino acids, fatty
acids, vitamins and
minerals)
Skim milk is a by-product obtained during the
manufacture of cream. It is rich in solids-not-fat
content and has high nutritional value. It is
regarded as a by-product only when it is either
not economically utilized or utilized for derived
by-products like casein and related products, coprecipitates, protein hydrolysates etc. E.g.: From
by-product of skim milk cultured butter milk and
Bulgarian butter milk has been prepared.
Amongst the alternative valorisation options are
use of fibres for paper and boards and bioethanol.
Because of danger of Bovine spongiform
encephalopathy (BSE), stringent limitations have
been formulated by governments on use of animal
by-products in feeds.
Next to feed applications, currently food,
pharmacy, pet food, compound feed, fertilisers
and technical applications are produced out of
meat by-products.
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CHAPTER 2.
PEPTIDES AND FOOD
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Peptides are short polymers of amino acids present in humans, animals, and plants, and
represent an important component of the innate immunity, as they may also participate in the
antioxidant, antimicrobial and signalling functions. They can be synthesized ex novo (like carnosine
or gluthatione), or they can be produced after proteolysis. In the first case, the new molecules are
synthesized to complete predetermined functions; in the second case, instead, they can follow
different pathways, such as the metabolic pathways of amino acids, or remain in a latent state within
the protein sequence. The peptides released from proteolytic processes during food processing are
related to the functional, nutritional and sensorial properties of the final product.
The desire for functional foods and the need to reduce chemical preservatives are connected
with the ever increasing health and nutrition concerns of the consumers (Mills et al., 2011) .
The concept that proteins can be tailored and their fragments modelled to achieve a
particular function, is now of great interest. In general, peptides generated from food proteins
present the great advantage to derive from harmless sources and therefore are considered safe.
Moreover, the production of peptides obtained from these sources could bring additional value to
food by-products, representing a breakthrough area for the industry of the future (Pellegrini, 2003) .
2.1 Properties of peptides
2.1.1 Structure and functional properties
Peptides are polymers of at least two amino acids linked together by covalent bonds,
between the carboxyl group of one molecule and the amino group of the other molecule. All the
polymers containing more than 50 amino acids are considered proteins, whereas peptides are those
containing less than 50 units. Indeed, the complexity of the structure and the degree of activity are
supplementary methods to discriminate proteins from peptides (Van De Weert and Randolfh, 2012)
The length and the amino acid composition of these molecules will determine their
physicochemical properties. Each peptide presents a free amino group in the N-terminal region and
a free carboxylic group in the C-terminal with equalised magnetic charges. The elements
responsible for the final charge of the peptide, which can be ionized, belong to the inner amino acid
side chains. These side chains are very important in the food industry. Also the Maillard reaction,
generating colour and flavour compounds during the baking process, relies on the presence of these
amino acids side chains. At the same time, the inclusion of a peptide or their in situ production, can
improve the texture of the food product. For example the development of the texture in the Cheddar
cheese during ripening has been thought to depend upon the extent of proteolysis. Because more
peptide bonds are broken and more new charged groups (NH3 +/COO−) are available to compete
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for water, the “free” water content of maturing Cheddar cheese curd is reduced (O’Mahony et al.,
2005). The foaming capacity is another peptide-dependant property, which has been widely applied
in the alcoholic beverages industries (such as beer and sparkling wine) (Sharpe et al., 1981). The
liberation of hydrophobic sequences during hydrolysis can lead also to peptides with emulsifying
properties (Shimizu et al., 1984). Finally, peptides with antifreezing properties have been reported;
they have been obtained or isolated from Antarctic and Arctic fish and they were mainly alanineand cystine-rich (Wohrmann, 1996). Some others, such as alcalase hydrolizates of bovine gelatine,
ranging from 600 to 2700 Da, are also able to inhibit recrystallization of ice in frozen ice cream mix
as well as in frozen sucrose solutions (Wang and Damodaron, 2009)
2.2 Sensory properties
Peptides can contribute considerably to the final taste of food, in particular cheese and meat
products (Hansen-Møller et al., 1997) and they may cover the entire range of taste modalities:
sweet, bitter, umami, sour and salty (Temussi, 2012; Seki et al., 1990). Compounds with acidic-rich
residues have a sour taste, whereas those rich in hydrophobic residues have a bitter taste, and those
with a more balanced composition display little or no taste.
Sequence and conformation can also play an important role in flavour. Nowadays,
aspartame (L-aspartyl- phenylalanine methyl ester) is the most extensively used peptide to
substitute sugar in beverages. This peptide has the same calories of sucrose, but it is 200 times
sweeter; hence, aspartame can be used in a lower concentration, and it can be supplied to diabetics.
However, some disadvantages have been detected, such as: low stability at high temperature, low
solubility at neutral pH, and high sensitivity to proteolytic reactions. Moreover, phenylalanine, one
of its breakdown products, must be avoided by people suffering of phenylketonuria (PKU)
(Temussi, 2012). Mazur et al (1969) demonstrated that the molecule providing the sweetness to this
peptide is from one side the Asp residue, but also a very precise steric structure (H-L-Asp-L-PheOMe). All the other possible chiral isomers, i.e. D-L, L-D and D-D H-Asp-Phe-OMe, are bitter.
As mentioned above, hydrophobic peptides possess bitter taste. Bitter peptides are prevalent
in a wide variety of aged or fermented foodstuffs, because enzymatic hydrolysis frequently
generates bitterness; the development of bitter taste in cheese during maturation is a well-studied
example (Temussi, 2012). Otagiri et al. (1985) reported that a strong bitter taste is observed when
arginine is contiguous to proline). Finally, although the umami (taste enhancer) depends mainly to
the glutamate, novel "umami petides" have been studied and isolated. Yamasaki and Maekawa
(1987) isolated the "delicious peptide” from a beef soup (H-Lys-Gly-Asp-Glu- Glu-Ser-Leu-AlaOH), which produces a taste similar to that food product. However, there is no significant evidence
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to consider the small umami peptides as an independent class; it is possible that their taste is a
consequence of the presence of Asp or Glu. In this way, it is clear that the chemical nature of
peptides, particularly their incredible conformational versatility, plays a relevant role in determining
many structure–activity relationships, including those connected to food acceptance (Temussi,
2012).
2.3 Peptides bioactivities
The study of functional and bioactive peptides has been extensively promoted (Perez Espitia
et al., 2012). A peptide is considered bioactive if it can support health through a positive impact on
the functions or conditions of living beings (Korhonen and Pihlanto, 2006). The beneficial effects of
peptides depend on their antimicrobial (Reddy et al., 2004; Rajanbabu and Chen, 2011), antioxidant
(Sarmadi and Ismail, 2010), antithrombotic (Wang and Ng, 1999), anti-hypertensive (Erdmann et
al., 2008), opioid and immunomodulatory behaviour (St Georgiev, 1990; Gauthier et al., 2006). The
main peptides bioactivities are reported in figure 1.
Immunomodulatory
Anti-hypertensive
Anticancer
Antioxidant
Opioid
Food protein derived peptides
Antimicrobial
Antithrombotic
Multifunctional
Hypocholesterolemic
and hypotriglyceridemic
Figure 1. Bioactive properties of food protein-derived peptides.
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2.3.1. Anti-hypertensive peptides
Many of the physiological functions in an organism are mediated by peptides; for
instance, blood pressure can be regulated by peptides, like angiotensin-II or bradykinin. The
antihypertensive effect is defined by measuring the capability of a putative peptide to inhibit the
angiotensin-I-converting enzyme (ACE, EC 3.4.15.1). ACE is a constituent enzyme of the reninangiotensin system that plays a crucial role in blood pressure regulation and fluid and electrolyte
balance (Martínez-Maqueda et al., 2012) These processes are catalysed by two mechanisms:
either by the conversion of angiotensin I to angiotensin II, a potent vasoconstrictor, or by the
degradation of bradykinin, a potent vasodilator, and other vasoactive peptides. Some antihypertensive peptides are reported in table 3.
Most bioactive peptides generated from milk proteins have demonstrated ACE-I activity.
They can be released from two different pathways: milk protein hydrolysis or milk fermentation.
A number of peptides with antihypertensive properties have been identified from casein and
whey proteins with gastric and pancreatic enzymes.
One of the peptides with proven antihypertensive effect is a αs1-casein-derived peptide,
with sequence FFVAPFPGVFGK. The casein hydrolyzate containing this peptide has been
patented and commercialized as an antihypertensive product named Peptide C12®. Moreover αs1casein represents a source of bioactive peptides since two other sequences that reduce systolic
blood pressure (SBP) have also been identified (RYLGY and AYFYPEL) (Contreras et al.,
2009). The use of food-grade enzymes, derived from microorganisms, to release bioactive
peptides has also become a common strategy. In nature, the proteolytic activity of lactic acid
bacteria during fermentation of dairy products gives off peptides and amino acids, which are used
as nitrogen sources necessary for bacterial growth (Martínez-Maqueda et al., 2012). However,
some of these peptides, produced by Lactobacillus helveticus in combination with
Saccharomyces cerevisiae during milk fermentation, revealed blood pressure lowering properties
(i. e. VPP and IPP). Other strains responsible for the liberation of antihypertensive peptides are
Lactobacillus helveticus CPN4, Lactobacillus bulgaricus and Streptococcus thermophilus
(Nakamura et al., 1995)
Eggs are another important source of antihypertensive peptides. Ovokinin (FRADHPFL),
resulting from the pepsin hydrolysation, is one of them. These properties can be enhanced by
emulsification with egg yolk, because phospholipids promote its absorption and protect the
peptide from intestinal peptidases (Fujita et al., 1995).
An intriguing discovery about the
bioactive egg peptides is that even if they show ACE-I activity in vivo, many of them may not
work in vitro, demonstrating only partial correspondence between in vitro and in vivo effects.
13
Due to their optimal extracting conditions, collagen and gelatin are considered as good
sources of bioctive peptides. They are obtained mainly from porcine skin and bovine hide, as well
as bones, tendons and cartilages. Moreover, studies considering novel sources, such as meat,
poultry or fish and marine by-products and waste are already being developed (MartínezMaqueda et al., 2012). Some previously reported antihypertensive meat peptides are MNPPK
and ITTNP, which were released in the thermolysin hydrolysis of porcine muscle myosin. Other
peptides were found to be particularly active, such as AVF and VF, from an insect protein
digestion, YYRA from chicken bone hydrolysate and KRVIQY from porcine myosin
hydrolysate.
Antihypertensive peptides inhibitory to ACE can also be derived from vegetable proteins,
such as gluten, zein and hordein (Gobbetti et al., 1997). Indeed, hydrolysed or fermented soybean
proteins produced several sequences responsible for the in vitro antihypertensive activity (Tab.
3).
A daily consumption of a moderate amount of antihypertensive peptides from natural
sources could elicit a blood pressure reduction not far from that of synthetic drugs, because the
majority of patent drugs available in the market contain similar bioactive peptides to those found
in the above mentioned food products (Martínez-Maqueda et al., 2012).
2.3.2. Antithrombotic peptides
Cardiovascular diseases (CVDs) lead to the development of thrombosis, due to the
alterations in the coagulation mechanisms. Increased occurrence of thrombosis has been linked to
platelet hyperreactivity, high levels of haemostatic proteins (e.g., fibrinogen), defective
fibrinolysis and hyperviscosity of the blood (Erdmann et al., 2008). Therefore, antithrombotic
drugs are commonly used to reduce platelet aggregation and enhance fibrinolysis. Similarities
between the mechanisms of milk clotting, defined by the interaction of κ-casein with chymosin,
and blood clotting, defined by the interaction of fibrinogen with thrombin, have been reported. To
date, food derived peptides with antithrombotic properties are mainly the result of enzymatic
hydrolysis of κ-casein (Erdmann et al., 2008) (tab. 3). The dodecapeptide of fibrinogen and the
106-116 sequence of κ-casein show functional homologies. Another peptide, with
MAIPPKKNQDK sequence, functions mainly because of the presence of three amino acid
residues (Ile108, Lys112, Asp115). It inhibits both the aggregation of ADP activated platelets as
well as the binding of human fibrinogen γ-chain to its receptor region on the platelet surface
(Smacchi and Gobbetti, 2000).
14
Table 3. Examples of anti-hypertensive and antithrombotic peptides deriving from different food
sources.
sequence
Anti-hypertensive
Antithrombotic
source
reference
FFVAP
AVPYPQR
α-CN f23-27
β-CN f177-183
YGLF
α-LA f50-53
Mullally et al., 1996
ALPMHIR
β-LG f142.148
Mullally et al., 1997
KVLPVPQ
LVYPFPGPIPNSLPQNIPP
β-CN f169-174
β-CN f58-76
Maeno et al., 1996
Miguel et al., 2006
LHLPLP
β-CN f133138
Miguel et al., 2007
YP
α-CN f146-147
Maeno et al., 1996
FFVAPFPGVFGK
RYLGY
α-CN f23-34
α-CN f90-95
Keshmirian and Nogrady 1988
Contreras et al., 2009
AYFYPEL
α-CN f90-96
Contreras et al., 2009
VPP
IPP
β-CN f(84–86)
β-CN f(74–76)
Nakamura et al., 1995
Nakamura et al., 1995
Maruyama et al., 1985
Maruyama et al., 1986
YRGGLEPINF
Egg white
Miguel et al., 2007
ESIINF
YAEERYPIL
Egg white
Egg white
Miguel et al., 2008
Miguel et al., 2005
RADHPFL
Egg white
Miguel et al., 2006
IVF
Egg white
Miguel et al., 2007
Hydrolysate
FRADHPFL
Egg white
Ovalbumin
Miguel et al., 2008
Fujita et al., 1995
QVSLNSGYY
D-hordein
Gobbetti et al., 1997
SAYPGQITSN
FNQ
α-zein
α-zein
Gobbetti et al., 1998
Yano et al., 1996
LAY
α-zein
Yano et al., 1997
LLP
LNPA
α-zein
α-zein
Yano et al., 1998
Yano et al., 1999
LQQ
α-zein
Yano et al., 2000
AY
NWGPLV
α-zein
Soy (glycinin)
Yano et al., 2001
Kodera and Nio, 2006
IAP
Wheat (gliadin)
Motoi and Kodama, 2003
GA(Hyp)GL(Hyp)GP
Wheat protein hydrolysate
Chicken leg collagen hydrolysate
Kodera and Nio, 2006
Saiga et al., 2008
GF(Hyp)GP
Porcine skin collagen hydrolysate
Ichimura et al., 2009
LKP
IKW
Meat (chicken muscle)
Meat (chicken muscle)
Fujita et al., 2000
Fujita et al., 2001
LAP
Meat (chicken muscle)
Fujita et al., 2002
ITTNP
MNPPK
Meat (porcine muscle)
Meat (porcine muscle)
Nakashima et al., 2002
Nakashima et al., 2003
YYRA
Chicken bone extract hydrolysate
Nakade et al., 2008
KRVIQY
porcine myosin hydrolysate
Muguruma et al., 2009
DLTDY
Oyster protein hydrolysate
Shiozaki et al., 2010
DY
Oyster protein hydrolysate
Shiozaki et al., 2011
Hydrolysate
Oyster protein hydrolysate
Shiozaki et al., 2012
Hydrolysate
Salmon muscle protein hydrolysate
Ono et al., 2003
Hydrolysate
Squid skin collagen hydrolysate
Lin et al., 2012
VF
AVF
Insects hydrolysis
Insects hydrolysis
Vercruysse et al., 2010
Vercruysse et al., 2011
MAIPPKKNQDK
k-CN f106-116
Jolles et al., 1986
KDQDK
KRDS
k-CN f112-116
Lactotransferrin f39-42
Qian et al., 1994
Qian et al., 1995
k-CN f106-112
Battazzi, 1996
k-CN f106-116
k-CN f112-116
Battazzi, 1996
Battazzi, 1996
k-CN f113-116
Battazzi, 1996
15
2.3.3. Hypocholesterolemic and hypotriglyceridemic peptides
A destabilised blood lipids profile (hypercholesterolemia and/or hypertriglyceridemia) is
another risk factor for CVDs. It has been reported that dietary proteins with low ratios of
methionine/glycine
and
lysine/arginine,
such
as
soy
and
fish
protein,
favour
a
hypocholesterolemic effect, whereas bovine and casein, having higher amino acid ratios, tend to
elevate cholesterol levels (Erdmann et al., 2008). The most studied hypocholesterolemic peptides
derive from soy proteins (tab. 4). Peptides produced from this source revealed that a hydrophobic
region is required for biological activity. Moreover, a proline residue seems to be a key
component. Hypotriglyceridemic activities have been also detected in different animal species
when hydrolyzed globin has been provided. This effect may depend on the capability of the
peptides to decrease intestinal fat absorption and to enhance the lipolysis of triglycerides
(Erdmann et al., 2008).
Table 4. Examples of antioxidant, hypocholesterolemic and opioid peptides deriving from
different food sources.
sequence
Antioxidant
Hypocholesterolemic
Opioid
source
reference
MY
LLPHH
Fish (sardine muscle)
Soy (β-conglycinin)
Erdmann et al., 2006
Chen et a., 1995
YFYPEL
MHIRL
Milk (casein)
Milk (β-lactoglobulin)
Suetsuna et al., 2000
Hernandez-Ledesma et al., 2005
YVEEL
Milk (β-lactoglobulin)
Hernandez-Ledesma et al., 2006
WYSLAMAASDI
YAEERYPIL
Milk (β-lactoglobulin)
Egg (egg white)
Hernandez-Ledesma et al., 2007
Davalos et al., 2004
β-CN f98-105
β-CN f177-183
Rival et al., 2001
Rival et al., 2002
β-CN f169-176
Rival et al., 2003
LPYPR
β-CN f170-176
k-CN f96-106
Soy (glycinin)
Rival et al., 2004
Kudoh et al., 2001
Yoshikawa et al., 2000
IAVPGEVA
Soy (glycinin)
Pak et al., 2005
RYLGYLE
YIPIQYVLSR
α-CN f90-96
k-CN f25-34
Loukas et al., 1983
Chiba et al., 1989
β-CN f60-63
β-CN f60-70
Meisel, 1997
Fiat et al., 1993
gluten
Fukudome and Yoshikawa, 1992
Fukudome and Yoshikawa, 1993
GYYPT
GYYP
YGGTL
Fukudome et al., 1997
YGGT
YPISL
16
2.3.4. Antioxidant peptides
The use of antioxidants as preservatives has been systematically applied in the food
industry. Recently, the use as health products of food-derived peptides has also attracted interest,
because they can supplement endogenous antioxidants against oxidative stress (Fang et al.,
2002). Recent studies have shown that peptides with antioxidant properties can be released from
food sources such as milk casein, whey protein, egg and soy protein and food by-products
(Erdmann et al., 2008; Bougatef et al., 2010). Some examples of bioactive peptides derived from
different protein sources are listed in table 4.
Some suggested mechanisms influencing the antioxidant properties of peptides have been:
metal ion chelation, scavenging or quenching of reactive oxygen species (ROS), inhibition of
enzymatic (lipoxygenase- mediated) and nonenzymatic peroxidation of lipids and essential fatty
acids (Udenigwe and Aluko, 2012). The antioxidant activity has been attributed to certain amino
acid sequences, in particular histidine, proline, cysteine, methionine, and aromatic amino acids.
Chen and co-workers reported that histidine residues of peptides are able to chelate metal ion,
quench active oxygen, and scavenge OH (Chen et al., 1998) through its imidazole group, which can
participate in hydrogen atom transfer and single electron transfer reactions (Chan and Decker 1994).
This activity can be increased adding hydrophobic amino acids (such as proline and leucine) to the
N-terminus of a dipeptide His-His. This hydrophobic part is important because it can lead the
antioxidant peptides to interact with hydrophobic cellular targets (Chen et al., 1998). Although
selected information about the specific activities of single peptides has been reported, it is not clear
how protein hydrolysates contribute in these processes. Li et al. (2008) noticed that there is a dosedependent relationship between hydrolysate concentration and antioxidant activity. The highest
antioxidant activity was found in peptides between 500–1500 Da.
2.3.5. Opioid peptides
Opioid peptides are small molecules naturally produced in the central nervous system
(CNS) and in various glands throughout the body. They contribute to some behaviours, such as
motivation, emotion, and attachment, the control of food intake and the response to stress and
pain. They work in the same way of classic alkaloid opiates (such as morphine and heroin)
(Froehlich, 1997). Exorphins, a class of peptides with opioid activity, were found in pepsin
hydrolysates of wheat gluten and soy
-protein (Fanciulli et al., 2003; Zioudrou et al. 1979)
(tab. 4) and others, called cytochrophins and hemorphins, have been produced from
enzymatically treated bovine blood (Brantl et al., 1986).
17
2.3.6. Immunomodulatory peptides
Immunomodulatory peptides are able to enhance the functions of immune system,
including regulation of cytokine expression, antibody production, and ROS-induced immune
functions (Hartmann and Meisel 2007; Yang et al., 2009). It has been reported that a tryptic
digest of rice protein can promote phagocytosis and increase superoxide anion production in
human leukocytes (Takahashi et al., 1994). In addition, egg-derived peptides are used during
cancer immunotherapy to increase immune functions (Mine and Kovacs-Nolan, 2006).
(Udenigwe, 2012) Chicken meat proteins, especially myosin, tropomyosin and collagen, contain
bioactive peptide fragments with immune-stimulating properties which, theoretically, could be
released through the activity of proteinase K (Dziuba et al., 1996).
2.3.7. Anticancer peptides
A number of studies on anticancer peptides have been focused on soy peptides, in
particular in Lunasin (Hernandez-Ledesma et al., 2009). The anticancer property of lunasin is
predominantly against chemical and viral oncogene-induced cancers, and based on the
modulation of histone acetylation and deacetylation pathways specifically. The final effects are
repression of cell cycle and promotion of apoptosis in cancer cells (Hernandez-Ledesma et al.,
2009). Recently, 2 large peptides (Leu-Pro-His-Val-Leu-Thr-Pro-Glu-Ala-Gly-Ala-Thr and ProThr-Ala-Glu-Gly-Gly-Val-Tyr-Met-Val-Thr) from tuna dark muscle by-product hydrolyzed with
papain and protease XXII were isolated by Hsu and co-workers (2011). These peptides exhibited
dose-dependent antiproliferative activities against cultured breast cancer (MCF-7) (Hsu et al.,
2011). This result demonstrates the potential of meat products and meat by-products as valuable
sources of bioactive peptides for incorporation into functional foods (Ryan et al., 2011).
2.3.8. Antimicrobial peptides
One of the first lines of defence against infections used by animals, plants and insects is
the production of antimicrobial peptides. According to data already reported, antimicrobial
peptides should be relatively short (12 to 100 amino acids), positively charged (to interact with
lipids) and amphiphilic (to enter into the cell membrane). Despite their similar properties,
antimicrobial peptides have very limited sequence homologies with a wide range of secondary
structures. To date, hundreds of such peptides have been identified and their different
mechanisms of action have been elucidated (Jenssen et al., 2006). There are at least four major
groups (Mackintosh et al., 1998):
18
1) Linear helical peptides without cysteine, such as the cecropins from insects and pigs
(Boman, 1995);
2) Peptides with an antiparallel β-sheet structure stabilized by two or three intramolecular
disulfide bonds, such as defensins (Ganz, 2003);
3) Peptides with one intramolecular disulfide bond, such as bactenecin (Romeo et al.,
1988);
4) Linear peptides containing high proportions of one or more amino acids, in particular
tryptophan-, arginine- and proline-rich peptides, like drosocin (Bulet and Stöcklin, 2005).
Jennesen et al. (2006) reported that the antimicrobial action in peptides is given by
permeabilization through the microbial cells. In such case, a model has been proposed to describe
the mode of action (fig. 2): first, in the “aggregate” model (A), there is a reorientation of the
peptides spanning the membrane and forming an aggregate; then, on the “toroidal pore” model
(B), peptides form pores; in the “barrel-stave” model (C), peptides are inserted in a perpendicular
orientation to the plane of the bilayer, form the “staves” in a “barrel”-shaped cluster, and in the
“carpet” model (D), peptides aggregate in parallel to the lipid bilayer, showing a detergent-like
activity. Peptides that do not act by permeabilising the membrane can work at different levels:
inhibiting DNA and RNA synthesis (E), decreasing protein synthesis (F), reducing the enzymatic
activity (G), modifying aminoglycosides (H), or forming structural components (I) that can
disturb the normal bacterial growth. Bacteria antimicrobial peptides (bacteriocins) are among the
first ones to be isolated and characterized. They are produced as defence from other bacteria that
might compete in the same environment. One of the main studied bacteriocins is nisin, a
lanthionine containing a peptide, used for nearly 50 years as a food preservative (Jenssen et al.,
2006).
Even though it has not been as well-studied as the antioxidant capacity of food peptides,
the hydrolysis of food proteins can generate new and potent antimicrobial peptides (tab. 5). The
most investigated food-derived antimicrobial peptide is lactoferricin, the fragment 17-41 of the
iron-binding glycoprotein lactoferrin. This peptide has antimicrobial activity against Gramnegative bacteria and Candida albicans (Farnaud and Evans, 2003).
Peptides released from milk proteins are the richest source of antimicrobial peptides
(AMPs). Peptides obtained from bovine αs1-casein and produced by Lactobacillus acidophilus
DPC6026, are active against both Gram-positive and Gram-negative bacteria; these have been
proposed as bioprotective compounds if supplied in milk food products (Hayes et al., 2006)
19
Figure 2. Mechanisms of action of antibacterial peptides proposed by Jenssen et al., 2006.
Muscle foods as source of antimicrobial peptides are less documented. A peptide obtained
from bovine meat (GLSDGEWQ) inhibited the growth of both Gram-positive and Gram-negative
pathogens. It was also reported that GFHI and FHG inhibited the growth of the pathogen P.
aeruginosa (Ryan et al., 2011). Moreover, a cysteine rich antimicrobial peptide was produced
from the digestion of oyster muscle, using a combination of alcalase and bromelin (Liu et al.,
2008). This peptide also resulted active inhibiting the growth of the fungi Botrytis cinerea and
Penicillium expansum (Ryan et al., 2011)
20
Table 5. Examples of antimicrobial peptides deriving from different food sources.
sequence
Antimicrobial
YQEPVLGPVRGPFPIIV
YQEPVLGPVRGPFPI
source
reference
colostrum
colostrum
Birkemo et al., 2009
Birkemo et al., 2010
RPKHPIKHQGLPQEVLNENLLRF colostrum
IKHQGLPQE
casein
Birkemo et al., 2011
Hayes et al., 2004
VLNENLLR
casein
Hayes et al., 2005
SDIPNPIGSENSEK
GLSDGEWQ
casein
beef sarcoplasmic protein hydrolysate
Hayes et al., 2006
Ryan et al., 2011
GFHI
FHG
beef sarcoplasmic protein hydrolysate
beef sarcoplasmic protein hydrolysate
Ryan et al., 2012
Ryan et al., 2013
2.3.9. Multifunctional peptides
The functions described above can be detected in different peptides, but sometimes one
peptide show multiple activities at the same time. In this case it is defined as a multifunctional
peptide (Udenigwe and Aluko, 2012). A hexapeptide (TTMPLW) derived from αS1-casien (f194–
199), through trypsin-catalyzed digestion, exhibited both ACE-inhibitory and immunomodulatory
activities (Meisel, 2004); in addition, a β-lactoglobulin-derived β-lactorphin (YLLF) inhibited
ACE activity and also possessed opioid-like activity (Mullally et al., 1997). Moreover, crude
chemotryptic α-casein hydrolysates displayed several in vitro bioactivities such as ACE
inhibition, antioxidant, Zn2+-binding, and antibacterial activities (Srinivas and Prakash, 2010).
Four peptides (GFHI, DFHING, FHG, and GLSDGEWQ) present in beef sarcoplasmic protein
hydrolysates have been reported to possess anticancer, antimicrobial, and ACE-inhibitory
properties (Jang et al., 2008).
21
CHAPTER 3.
GLYCOPEPTIDES
22
Glycopeptides are molecules present and produced in nature. The study of sugars bounded
to amino acids has become increasingly interesting, since these structures have been showed to play
important roles in fertilization, the immune system, brain development, the endocrine system and
inflammation (http://en.wikipedia.org/wiki/Glycopeptide). Some glycopeptides, isolated from
microorganisms, are already used as antibiotics (Kahne et al., 2005), while some others instead are
produced by the insects as the first defence against bacterial infections (Bulet and Stöcklin, 2005).
The synthesis of novel glycopeptides can be useful to elucidate glycan function in nature, but the
current major challenge is to uncover their therapeutic and biotechnological applications in food
(http://en.wikipedia.org/wiki/Glycopeptide).
3.1. Structure
Glycopeptides are short sequences of amino acids (minimum 7-8) bound to one or more
mono-, di- and oligosaccharides. The function of the sugar group has not been fully elucidated;
even though the absence of that sugar inside the chain can reduce around 100 times the
glycopeptide bioactivity (Otvos et al., 2002). In general it could be hypothesized that the presence
of the sugar determines a chemical and physical rearrangement of the peptide conformation,
similarly to what occurs in proteins, as described above.
Different studies that tried to design non-glycosylated analogues of glycopeptides in order to
explore the role of glycosylation have been published. Kaur et al. (2006), for example, reported that
it is possible to obtain a functionally equivalent non-glycosylated analogue from a native
glycosylated peptide just by performing strategic modifications of the sequence. This means that the
presence of the sugar provides the peptide with a new spatial distribution. At the same time, the
peptide, and in particular the type of amino acid, can influence the special sugar conformation once
it is linked (Hindley et al., 2005).
Some of the most studied glycopeptides belong to insects and they are produced as a
mechanism of defence against bacterial invasion. These peptides, most of them rich in proline, can
undergo glycosylation with one, two, or three glycan residues, forming a O-glycosylated
substitution with a conserved threonine residue (Gobbo et al., 2002). The first characterized
glycopeptide was Drosocin, a O-glycosylated peptide, consisting of 19 amino acid residues (of
which nearly one third are proline), and three characteristic Pro-Arg-Pro motifs. Drosocin is
glycosylated on Thr 11 by either a monosaccharide (such as N-acetyl-galactosamine) or a
disaccharide. The presence of the disaccharide in the middle of the molecule may open the turn
comprising residues 10-13 to a more extended conformation, thus helping drosocin to assume the
23
most suitable orientation to bind to its putative intracellular target. In many cases the integrity of the
carbohydrate side chain is also necessary for the maximum activity of the glycopeptides (such as
formaecin and diptericin); however, it has also been reported that unglycosylated peptides, like
pyrrhocoricin, appear to be more potent than the native glycosylated form. To date, it has not been
possible to define a specific structure for "the" glycopeptide, and in particular many studies are still
in progress (Gobbo et al., 2002).
A well-studied glycopeptide, already patented as a potent drug is Vancomycin. It was first
isolated in 1953 from a soil sample and it is produced by the bacteria Amycolatopsis orientalis. It is
a 1449.3 Da molecule and from its structure several novel compounds were isolated, produced and
designed. The common structure in all, called "vancomycin-related", contains a homologous
heptapeptide scaffold. Five out of the seven residues in vancomycin are aromatic, and the remaining
two residues are modified tyrosines, with chlorine at the meta position of the aromatic ring, and an
OH substitute at the benzylic carbon of the side chain. The electron-rich side chains of these
aromatic amino acid residues facilitate the oxidative crosslinking, leading to a rigid architecture of
the heptapeptide scaffolds. Finally, the peptide framework is glycosylated, by a mono- or
disaccharide on residue 4 (Kahne et al., 2005). Another glycopeptide with bioactive properties was
isolated by Yang et al. (2009) from a fermentation broth of Penicillium sp. M03. This molecule is a
1017 Da and contains five aminoacids (Ala, Glu, Gly, Asp and Ile) plus two monosaccharides
(glucose and xylose). PGY is also a low molecular-weight glycopeptide; it was isolated by Wu and
Wang (2009) from the fruit Ganoderma lucidumwas. This molecule possess a peptide part with a
Ser-Arg-[(Ala)2(Gly)2] sequence, and a carbohydrate part, coupled by O-linkage via Ser,
constituted of
a backbone of (1 → 3)-β-glucan with (1 → 6)-linked Araf branches. The
carbohydrate moiety, especially the side chains of terminal α-L-Araf residues, is essential for the
preservation of the activity of this class of glycopeptides.
3.2. Antifreezing activity
It is already well known that many O-glycan-rich glycoproteins, especially isolated from
artic fish, act as in vivo “antifreezer”, preventing nucleation of ice and allowing them to survive at
temperatures of -2 °C (Gamblin et al., 2009). In fact, antifreeze glycopeptides (AFGP) and peptides
have been isolated from 37 species of Antarctic fish. The glycopeptides are made up of a tripeptide
repeat (alanine-alanine-threonine), with a disaccharide moiety attached to the threonyl residues
(Wohrmann, 1996). Their molecular weights range from 2600 Da to 34000 Da (Wohrmann, 1996).
These compounds kinetically depress the temperature at which ice grows, in a non-colligative
24
manner, and hence exhibit thermal hysteresis, i.e. a positive difference between the equilibrium
melting point and the ice growth temperature (the temperature at which seed ice crystals will grow
in the solution) (Harding et al., 2003)
3.3. Sensory properties
Certain glycopeptides and peptides provide an improved taste function, in particular for the
kokumi taste function. These molecules could be used in a wide range of industrial applications and
enhance the basic tastes, as well as impart thickness, spread, continuity, and unity of the final
product (https://usgene.sequencebase.com/patents/US20060083847).
3.4. Glycopetides bioactivities
Just like peptides, the glycopeptides have revealed different properties and bioactivities. The
main studied property is the antimicrobial one. Nevertheless, their additional properties have been
reported to a lesser extent.
3.4.1. Antioxidant activity
Wu and Wang (2009) isolated from the fruiting bodies of G. lucidum a new water-soluble
glycopeptide (PGY) with higher levels of antioxidant properties against superoxide radicals,
compared with a selected antioxidant (BHT). This activity strictly depends on the carbohydrate
moiety, while the peptide moiety appears to be nonessential for their antioxidant activity.
Furthermore, they demonstrated that PGY provides higher levels of antioxidant characteristics
against superoxide radicals, in comparison with that of a selected antioxidant (BHT).
3.4.2. Antitumor activity
Natural ecosystems play an important role as a source of new antitumor compounds. The
antitumor bleomycin (BLM) was first isolated from Streptomyces verticillus; it provided the name
to a class of natural antitumor glycoconjugates, made of complex polypeptide aglycones with a
variety of mono-, di-, and tetra-saccharides attached. The BLM, clinically known as Blenoxane
(Bristol-Myers Squibb), treats several types of tumours, such as testicular cancer (90% efficiency),
Hodgkin’s lymphoma, and carcinomas of the skin, head and neck. However, the cytotoxic
therapeutic efficacy of BLM is limited, due to induction of lung fibrosis. The mechanism of action
of BLM depends on a metal-dependent oxidative cleavage of DNA and RNA, which leads to cell
25
death. Three functional domains can be recognized inside the structure of BLM: the N-terminal
domain, represented by pyrimidoblamic acid (PBA) along with β-hydroxy-histidine, is the metalbinding domain and enables the activation of molecular oxygen; the C-terminus is the DNA-binding
domain, and it is formed from a bisthiazole moiety with a cationic tail; and the hydroxyl group of bOH-His, which that participates in cell recognition and uptake and in metal ion coordination.
NC0604 is a BLS analogue isolated from Streptomyces verticillus var. pingyangensis, with
enhanced antitumor activity and lower pulmonary toxicity (La Ferla et al., 2011).
Other natural antitumor glycopeptides have been found in macromycetes (macro-fungi). PSP
and PSK, from the macro-fungus Coriolus versicolor, for example, are well known for their
immunomodulatory and antitumoral activities. Both glycopeptides stimulate the T-cell activation,
and induce cytokine production in vitro and in vivo, enhancing ‘‘killer’’ cytotoxic activity against
tumours; they are considered biological response modifiers. The glycopeptides 220-GP and 120-GP
obtained from pronase- treated ovomucin (an egg-white glycoprotein) were found to have high
direct antitumor activity at low concentrations; both glycopeptides have shown to promote complete
rejection of direct tumours and slight growth inhibition of distant tumours. A number of muramyl
glycopeptides (fragments of murein, a bacterial peptidoglycan from the cell wall) are known to
cause growth inhibition and necrosis of experimental tumours. For example, glucosaminylmuramyl
dipeptide (GMDP) was reported to enhance tumoricidal activity of macrophages, by inducing the
secretion of TNF-α, one of the key players of macrophage cytotoxicity against tumoural cells.
GMDP also augmented the cytotoxic effect on tumour cell lines, when combined with TNF-a and
cisplatin; in this case, 100% tumour cells were killed (La Ferla et al., 2011).
3.4.3. Antimicrobial activity
Vancomycin was the first glycopeptide antibiotic to be discovered. Despite its toxicity
profile, it gained prominent use to face the problem of the emergence of a large-scale resistant S.
aureus. The mechanism by which vancomycin exerts its action depends on the inhibition of cell
wall synthesis in Gram-positive bacteria, as opposed to the Gram-negative, because the outer
membrane in the Gram-negative bacterial membrane keeps the glycopeptides from reaching their
targets at the periplasmic face of the cytoplasmic membrane (Kahne et al., 2005). The large
hydrophilic molecule is able to form hydrogen bond interactions with the terminal D-alanyl-Dalanine moieties of the NAM/NAG-peptides (fig. 3). In this way, it prevents the transglycosylation
and subsequently affects the transpeptidation, which is essential for bacterial cell wall cross-linking
(Sujatha and Praharaj, 2012). Several studies attempting to develop new molecules mimicking
vancomycin have been reported (Chen et al., 2002). Hence, glycopeptides having effect on Gram26
negative microorganism have been isolated as well. In fact, Drosocin, a glycopeptide isolated from
insects, works in a low micromolar range of concentration, and it is mainly active towards Gramnegative bacteria (Bullet and Stocklin, 2005).
Figure 3. Mechanisms of action of Vancomycin towords sensitive and not sensitive bacteria
(http://en.wikipedia.org/wiki/Vancomycin).
The relatively slow-killing kinetics of drosocin and the observation that an all-D analogue is
50 to 150-fold less active than the native isomer suggested that the peptide do not seem to have a
membrane permeabilization mechanism, which is thus markedly dissimilar to that of lytic peptides
such as cecropins.
As already indicated, deglycosylation significantly reduces the antimicrobial activity of
drosocin. The syntheses of differently glycosylated peptide analogues showed that the antimicrobial
activity against several Gram-negative bacteria is affected by the type of sugar and the type of
glycosidic linkage, particularly in the case of E. coli D21 and the K. pneumoniae strains (Gobbo et
al., 2002).
The mechanism of action of these proline-rich glycopeptides could depend on the interaction
with the lipopolysaccharides (LPS) of Gram-negative bacteria and/or with the bacterial
chaperone/heat shock proteins GroEL and DnaK; such an interaction inhibits protein folding
(Kragol et al., 2001). The fact that some of these glycopeptides (i.e. pyrrhocoricin analogues) do not
show selectivity towards Gram-negative or Gram-positive strains, has confirmed that their toxicity
for bacteria is not strongly related to membrane binding. At the same time, the specificity to certain
bacterial strains may derive from altered binding to DnaK (Otvos et al., 2000). An additional effect
of glycopeptides is the protection of host cells from pathogen adhesion. The anti-adhesive capacity
of the glycopeptides was reported by Yang and co-workers when β-conglycinin hydrolysates were
produced. These glycopeptides have both a D-mannosyl residue and a hydrophobic region; it was
27
suggested that the presence of the mannose substructure of glycopeptides prevents E. coli and
Salmonella adhesion to the intestinal epithelium (Yang et al., 2008). Glycopeptides with antibiofilm
properties have been also reported. In particular, peptides with an amino acid sequence bound to a
casein or a fragment of casein, comprising at least one glycosylated amino acid, can prevent dental
caries, gingivitis and periodontitis (http://www.freepatentsonline.com/y2012/0283174.html).
28
CHAPTER 4.
PROTEASES
29
According to the Enzyme Commission (EC) classification, proteases belong to the hydrolase
family (group 3), which hydrolyse peptide bonds (sub-group 4). The processing of many food
products depends on proteolytic activities, because they can enrich the characteristics of the final
product or generate innovative food and ingredients (Sumantha et al., 2006). Until fairly recently,
proteases were considered primarily to be protein-degrading enzymes. However, this view
dramatically changed when their important roles in physiological processes such as generating
signalling and/or functional molecules was discovered. Consequently, they immediately attracted
the interest for commercial applications, becoming one of the three largest groups of industrial
enzymes, and accounting for approximately the 60% of the total worldwide sales of enzymes.
(http://acd.ufrj.br/proteases/ProteaseApres.htm). In food processing, proteases can be derived from
the food matrix, generated by microorganisms or supplied as additives. Because of their wide
applicability, the microbial proteases represent the 40% of the commercial enzymes currently
present in the market (Rao et al., 1998).
Proteases can be separated into two major groups based on their ability to cleave peptide
bonds: when they act at the terminus of polypeptide chains (amino- or carboxy- peptidases), they
are classified as exopeptidases (EC 3.4.11-19); if they act internally, they are considered
endopeptidases or proteinases (EC 3.4 21-99). The exopeptidases are used for their debittering
action and they are becoming increasingly available in the industrial enzyme market (Raksakulthai
and Haard, 2003). Additionally, proteases can also be classified according to: their optimal pH (as
acidic, neutral or alkaline); substrate specificity (collagenase, keratinase, elastase, etc.); or their
homology with well-studied proteins (trypsin-like, pepsin-like, etc.) (Sumantha et al., 2006).
A more detailed classification is presented in Figure 4.
4.1. Exopeptidases
The exopeptidases act only near the ends of the polypeptide chains at the N or C terminus.
The aminopeptidases act at on the free N terminus of the polypeptide chain and liberate a single
amino acid residue, a dipeptide, or a tripeptide; in general, the aminopeptidases are intracellular
enzymes. The commercial Aminopeptidse I and II have been generated from Escherichia coli and
Bacillus licheniformis, respectively. In contrast, the exopeptidases act on the free C terminus, to
liberate a single amino acid or a dipeptide; they have been isolated mainly from Penicillium spp.,
Saccharomyces spp., and Aspergillus spp. Additionally, the omega peptides are enzymes working
on isopeptide bonds, i.e. an amide bond not present on the main chain of the protein (Rao et al.,
1998).
30
Protease
Aminopeptidase
Dipeptidyl peptidase
Tripeptidyl peptidase
Carboxypeptidase
Endopeptidase
Serine protease
Cysteine protease
Aspartic protease
Metalloprotease
Figure 4. Classification of proteases. Arrows show the sites of action of the enzyme.
4.2. Endopeptidases
The endopeptidases work preferentially on the peptide bonds away from the N and C
termini. The presence of free α-amino or α- carboxyl groups has a negative effect on the activity of
the enzyme. According to the reactive groups at the active site involved in catalysis, they can also
be divided into serine- (EC 3.4.21), cysteine- (EC 3.4.22), aspartic-peptidases (EC.3.4.23) and
metallo-peptidases (EC 3.4 24). Among these, the alkaline proteases are the most industrially
significant (Sumantha et al., 2006).
4.2.1. Serine proteases
They are characterized by the presence of a serine group in their active site, and they are the
most conserved proteases among eukaryotic and prokaryotic organisms. Even if the primary
structure is different among the members of this class, their general catalytic reaction depends on
three common amino acids, i.e. serine (nucleophile), aspartate (electrophile), and histidine (base).
They are active at neutral and alkaline pH (between pH 7 and 11), and they have molecular masses
between 18 and 35 kDa. Subtilisin is one of the most well-studied serine proteases isolated from
Bacillus subtilis, although other enzymes have also been discovered in S. cerevisiae (Mizuno and
Matsuo, 1984) Y. lipolytica (Li et al., 2009), Conidiobolus spp. (Phadatare et al.,1993) and
Aspergillus spp. (Hajji et al., 2008). Some of these enzymes have already been produced for
31
industrial purposes, such as Alcalase® (from Bacillus licheniformis), Savinase® (from Bacillus
lentus), Esperase® (Bacillus spp.), etc. (Novozymes, Denmark) (Rao et al., 1998).
4.2.2. Aspartic proteases
The aspartic proteases owe their name to the presence of aspartic acid residues necessary for
their catalytic activity. The active-site aspartic acid residue is situated within the motif Asp-X-Gly,
where X can be Ser or Thr. Because most of the aspartic proteases show a maximal activity at low
pH (pH 3 to 4), they are also known as acidic proteases; their molecular masses are in the range of
30 to 45 kDa. Most of the acidic proteases have been studied in fungi and yeasts (Rao et al., 1998).
A significant property of aspartic proteases is the ability to coagulate milk, as it is evidenced by
their widespread application in the dairy industry to coagulate casein during the manufacturing of
cheese (Yegin et al., 2010).
4.2.3. Cysteine proteases
The activity of cystein proteases depends on the presence of a dyad of Cys and His in the
catalytic site. These kinds of enzymes occur in both prokaryotes and eukaryotes and have an
optimal neutral pH. Papain (from Carica papaya), bromelain (from Ananas comorus), ficin (from
Ficus sp.) are the most studied proteases from food origin; Clostripain and Streptopain are produced
by Clostridium histolyticum and Streptococcus spp., respectively (Sumantha et al., 2006).
4.2.4. Metalloproteases
Enzymes belonging to this class are the most heterogeneous proteins according to the
catalytic site structure. The only common characteristic among them is the requirement for a
divalent metal ion to perform their activities; thus, they can be endo- or exopeptidases. Collagenase,
a member of this family, has been discovered in bacteria (Clostridium hystolyticum and
Achromobacter iophagus), yeasts (Candida albicans, Lima et al., 2009) and fungi, and it is very
specific for the proteolysis of collagen and gelatine (Rao et al., 1998).
4.3. Functions and Applications
The estimated value of the worldwide sales of industrial enzymes is $1 million a year, with
proteases accounting for about 60 % the total (Godfrey and West, 1996). Proteases have been
widely applied, mainly in the detergent and food industries. For example, acidic proteases are used
in the dairy industry for their ability to coagulate milk protein (casein) to form curds, from which
32
cheeses are prepared (Neelakantan et al., 1999). Alcaline proteases, instead, are useful in baking
processes (for biscuits, crackers and cookies, for instance), because they can hydrolyse flour
proteins and thus enhance the texture, flavour, and colour of the final product. Commercial enzymes
commonly used in the food industry include Alcalase®, Neutrase®, and Novozym®. In addition,
mixtures of enzymes are available, such as Flavourzyme™ and Kojizyme™. These are fungal
complexes of exopeptidases and endoproteases derived from the fermentation of soy sauce by
Aspergillus oryzae. Proteases from Bacillus subtilis have been used to deproteinize crustacean
waste and obtain chitin (Yang et al., 2000)
Other fields where proteases have been successfully applied are in pharmaceutical, cosmetic
and bioremediation processes. Even though applied biotechnological methods have significantly
improved the production of these enzymes in the past few years, the search for innovative sources
of enzymes, revolutionary production techniques, and novel applications of such enzymes in
unexplored fields has continued uninterrupted (Sumantha et al., 2006).
33
CHAPTER 5.
Yarrowia lipolytica
34
A "non-conventional" yeast is defined as “a microorganism easily distinguished from the
well-studied and widely used Saccharmyces cerevisae and Schizosaccharomyces pombe”.
Among the "non-conventional" ones, Yarrowia lipolytica is one of the most studied as a model
system in physiology, genetics, dimorphism, gene manipulation, protein expression, and lipid
accumulation research works (Bankar et al., 2009). Since a few amount of sugar is needed for its
growth, it has been isolated from diverse environments rich in lipids and proteins; its natural
habitats include oil-polluted environments, rivers and foods such as cheeses, yogurt, kefir, shoyu,
meat, and poultry products (Bankar et al., 2009). It is considered a non-pathogenic organism and
it is generally recognized as safe (GRAS) by the Food and Drug Administration (FDA, USA)
(Cohelo et al., 2010). Isolated for the first time in 1928 by Nannizzi, it just reached industrial
interest by the late '40s, especially for the dairy industry and later as a producer of citric acid.
5.1. Taxonomy and morphology
Y. lipolytica is a yeast belonging to the Fungi kingdom, division Ascomycota, class
Saccharomycetes, order Saccharomycetales. The wild form of Y. lipolytica presents different
morphology ranging from smooth and shiny to wrinkled colonies. These characteristics depend
on the growth conditions (oxygen, carbon, ammonia availability), but also on the genotype and
strain (Rodriguez and Domínguez, 1984). It is a dimorphic fungus that can come in the form of
single cells, pseudo-hyphae, or septate hyphae (Barth and Gaillardin, 1997). The yeast-tomycelium transition is associated with unipolar growth, asymmetric division, large polar-located
vacuoles, and repression of cell separation after division. It is believed that yeast dimorphism is
related to a defence mechanism against adverse conditions, such as temperature and nutritional
changes (Cohelo et al., 2010). The name Y. lipolytica was given in 1980 by van der Walt and von
Arx, but it is still possible to find it with the previous nomenclature, as follows: Mycotorula
lipolytica (1928), Candida lipolytica (1942), Candida olea (1949), Azymoprocandida lipolytica
(1961), Candida paralipolytica (1963) Candida pseudolipolytica (1973) (Kurtzman and Fell
1998).
5.2. Methabolism
Y. lipolytica is a unique, strictly aerobic yeast, with the ability of efficiently degrade
hydrophobic substrates such as n-alkanes, fatty acids, fats and oils, for which it has specific
metabolic pathways (Fickers et al., 2005). Even if, from the genome point of view, it is related to
35
Saccharomyces cerevisiae, it has significantly different genetic mechanisms. Particularly, the
genome displays an expansion of protein families and genes involved in hydrophobic substrate
utilization (Cohelo, 2010).
5.3. Industrial relevance of Y. lipolytica
There are two reasons for Y. lipolytica to be extensively studied: its secreted metabolites
and enzymes, and its activities during the cell growth. Without a doubt, the main interesting
feature about this microorganism is the expression of its extracellular and cell-bonded lipases
(Ota et al., 1982). Lipases are lipolytic enzymes able to hydrolyze the triglycerides in glycerol
and fatty acids, and they are also exploitable for several applications in the detergent (substituting
chemical surfactants), food, and environmental industries. Some of these lipases possess also the
capability to work under cold conditions (Parfene et al., 2011) and this aspect is progressively
getting interesting for industrial applications (Joseph et al., 2007). Being capable to consume nalkanes, isoprenoids and aromatic hydrocarbons as the group of naphthalenes and the group of
phenanthrene, Y. lipolytica could be also applied in bioremediation of contaminated environments
(Bankar et al., 2009) or for the treatment of olive mill wastewaters (Lanciotti et al., 2005).
Yarrowia lipolytica is recognized as one of the most frequent species associated with milk
(Lanciotti et al., 2004; Gardini et al., 2006) and meat products (Patrignani et al., 2011 a; 2011b) ,
due to its enzymatic activities, it has been regarded as a good candidate for accelerating ripening
(Guerzoni et al., 1998; van den Tempetl and Jakobsen, 2000; Lanciotti et al., 2005; Patrignani et
al., 2007). In fact, thanks to its high proteolytic and lipolytic activities, some yeast species may
play an important role in the production of aroma precursors from amino acids, fatty acids and
esters (Suzzi et al., 2001). γ-Decalactone is a peach-like aroma compound, reported in several
food and beverages, that can be produced biotechnologically, e.g. by Y. lipolytica which is able to
biotransform ricinoleic acid (12-hydroxy-octadec-9-enoic acid) into the lactone (Cohelo, 2010).
Another important application of this yeast is the production of citric acid. Currently, the
conventional procedure depends on the bioconversion of molasses into citric acid by Aspergillus
niger. This production is estimated to be approximately 1.6 million tons per year (Sauer et al.,
2007) However, the use of Y. lipolytica may bring some more benefits: a larger substrate variety
(n-paraffin, fatty acids, glucose), a smaller sensitivity to low dissolved oxygen concentrations and
heavy metals, and higher product yields. One potential disadvantage of this process is the
secretion of isocitric acid (ICA) as by-product; when it is above 5% of the citric acid
36
concentration can generate problems in the crystallization of CA during the purification process
(Cohelo, 2010).
5.4. Proteases of Yarrowia lipolytica
The production of proteases by Y. lipolityca and their possible applicability are two
aspects that need to be studied in depth. Y. lipolytica is a natural secretor of proteins, and it was
used especially for this property as eukaryotic host for secretion of heterologous proteins. In
common with many strains of yeast, Y. lipolytica expresses also proteolytic enzymes
(extracellular and intracellular proteases). In particular, an acidic extracellular protease (AXP)
and an alkaline extracellular protease (AEP) have been mainly studied (Young et al., 1996). From
1 to 2% of the total cell proteins belong to AEP; hence, over 1 g of AEP per litre has been
estimated (Matoba et al., 1988) at high cell densities.
5.4.1. Extracellular proteases
5.4.1.1. Alkaline protease
The gene xpr2, coding for the AEP, has been cloned and sequenced (Davidow et al., 1987).
It has a functional promoter region greater than 700 bp, maybe as a result of the complex regulation
of the gene. The regulatory region, consisting in a TATA box and other two major activation
sequences, is deeply influenced by the environment (Young et al., 1996). In fact, the gene is
transcripted during nitrogen or sulphur starvation, lack of carbon sources, and the presence of
extracellular proteins (Davidow et al., 1987). According to the mRNA sequence, AEP is originally
synthesized in an immature form, having some pro-regions added (Hernández-Montañez et al.,
2007). Most probably a 55 kDa precursor is synthesized first, and then after different cleavages and
maturating steps, 52, 44, and 36 kDa polypeptides are generated. In the final step, in which an
intracellular protease participate, the 32-kDa mature AEP is formed (Matoba et al., 1988). It has
been also reported that the pro-region is glycosilated (Young et al., 1996). Studies regarding the
AEP sequence showed a strong homology with the subtilisin family serine proteases. The highest
homologies belong to the region referring to the active-site of the enzyme (Davidow et al., 1987); in
particular, a 32% homology with the Bacillus subtilis DY subtilisin (Nedkov et al., 1983), 32%
homology to Thermoactinomyces vulgaris thermitase (Meloun et al., 1985), and 42.6% homology
with the Tritirachium album proteinase K (Jany et al., 1986). Once the enzyme is in its mature
form, it has been reported that the AEP secretion process is rapid.
37
5.4.1.2. Acidic protease
Initially, three acidic proteases were reported (Yamada and Ogrydziak, 1983), but upon
additional studies and the development of more sophisticated techniques, it was demonstrated
that Y. lipolytica produces just one acidic protease under specific growth conditions. The AXP1
gene encoding the acid protease has been characterized and genetic control of the synthesis has
been reported (Young et al., 1996; González-López et al., 2002). As the AEP, the AXP is also
synthesised as a pro-enzyme, having 397 residues and a molecular weight of 42 kDa. The mature
37 kDa enzyme is produced by cleavage of the precursor between Phe44 and Ala45. In this case no
glycosylation of the pro region has been reported (Young et al., 1996). According to the final
primary sequence, AXP shows a 44.94% identity with a 36 amino acid overlap with
Endothiapepsin, an aspartyl protease from the chestnut blight fungus, Cryphonectria parasitica
(Razanamparany et al., 1992), and Candidapepsin, the aspartic endopeptidase of Candida
albicans (Young et al., 1996).
5.4.2. Intracellular proteases
Although there is scarce evidence, the presence of some intracellular proteases in Y.
lipolytica has been reported. An aminopeptidase (yylAPE), belonging to the metalloprotease
group, has been intracellularly detected in a soluble form, whereas a dipeptidyl aminopeptidase
(yylDAP) activity has been reported both in the soluble and in the membrane fractions. The
membrane form of yylDAP is a serine protease maybe involved in the maturation process of
AEP; in contrast, the soluble form yylDAP is a metalloprotease probably involved in the
dimorphic transition of the yeast. Finally, no specific functions have been described for the
discovered soluble carboxypeptidase (yylCP) belonging to the serine protease family (HernándezMontañez et al., 2007).
5.4.3. Proteases production
The main element that discriminates the production of AEP or AXP is the environmental
pH. In general, neutral and high pH values (6-9) lead to AEP production, while low pH (2-6)
favours AXP (Ogrydziak, 1993).
Also carbon and sulphur compounds, as well as nitrogen sources (ammonium ions, amino
acids) can play a role in repressing the protease production. On the other hand, their production
can be induced if a protein source is present in the growth medium (Nelson, 1986). Moreover, it
has been reported that an extra carbon source (glucose) reduces AEP production (Akpınar et al.,
38
2011). Thus, if the type of synthesized protease is strictly dictated by the environmental pH, both
proteases are similarly induced at the end of the exponential phase on complex media (GonzálezLópez et al., 2002).
5.4.4. Industrial applications
Due to its lipolytic and proteolytic activities, Y. lipolytica has a high industrial potential.
In fact, its use as a co-starter for the production of some cheese varieties has already been
proposed by several Authors (Bintsis and Robinson, 2004; Lanciotti et al., 2005). The
extracellular proteases have important commercial value and multiple applications can be found
in various industrial settings. Although there are many microbial sources available for protease
production, only few are recognized as commercial producers. Since Yarrowia lipolytica is a
GRAS microorganism it could be used in different industrial processes; however there is missing
evidence referring to the applicability of Y. lipolytica proteases. One of the first studies by Nelson
and Young (1986) considered the use of these enzymes in the brewing industry as a chillproofing agent for beer. Additional work reported the use of Y .lipolytica enzymes for meat
tenderisation; hence Krasnowska et al. (2006) showed that yeast preparations had better
proteolytic (and collagenolytic) activities than pepsin. All enzyme preparations showed different
activity against beef meat proteins according to the pH. Instead, Patrignani et al. (2007)
demonstrated that the surface-inoculated Y. lipolytica did not have a deep effect on the immediate
proteolysis of dried fermented sausage when compared to Debaryomyces hansenii.
39
CHAPTER 6.
OBJECTIVES
40
Microorganisms elaborate a large array of proteases, which are intracellular and/or
extracellular. Intracellular proteases are important for various cellular and metabolic processes, such
as sporulation and differentiation, protein turnover, maturation of enzymes and hormones and
maintenance of the cellular protein pool. Extracellular proteases are important for the hydrolysis of
proteins in cell-free environments and enable the cell to absorb and utilize hydrolysis products
(Kalisz, 1988). At the same time, the extracellular proteases have also been widely commercially
exploited to assist protein degradation in various industrial processes (Kumar and Takagi, 1999;
Outtrup and Boyce, 1990; Gupta et al., 2002).
Commercial protease preparation usually consist of a mixture of various enzymes. They are
largely utilised in food processing to
•
improve the workability of dough (as backing enzymes particularly for crackers and
biscuits);
•
optimize and control the aroma formation of cheese and milk products;
•
improve the texture of fish products;
•
tenderize meat;
•
stabilize beer
Due to their large consumption and applications commercial preparations do not have a
specific action apart from the protease used for the production of hypoallergenic foods. In Europe
there are about forty commercial protease preparations on the market. Fifteen of these preparation
are produced with genetically modified Aspergillus and Bacillus spp. Their optimal activity is
between 35 and 50 °C.
The resource that I have explored to identify proteases with different promising attitudes has
been the collection of about 112 strains of Yarrowia lipolytica, isolated from different ecosystems
(cured meat, Po river alkanes contaminated waters, commercial chilled foods, cheeses, irradiated
poultry meats or light butter) and characterized during the last 20 years. All the strains belong to the
DISTAL Alma Mater Studiorum, University of Bologna. Preliminary research on 112 strains
showed great strain-related differences in the protein breakdown profiles (Badiali, 2004). In
particular, the cluster analysis of electrophoretic profiles obtained when α- and β-caseins had been
separately exposed to cell free supernatants of the various strains showed that the profiles of strains
isolated from the same ecosystem clustered together. On the basis of these results I selected for each
cluster one strain having the typical proteolytic profile representative of the group. In particular, I
selected the following strains of Y. lipolytica: 1IIYL4A and 1IIYL8A, both isolated from speck, but
41
characterized by different RAPD-PCR profiles (Badiali, 2004); 16B from irradiated poultry meat;
PO19 from Po river water; CLCD from salami; Y16 from commercial chilled food.
The main objectives of my research on these strains have been to:
1) evaluate the suitability of proteases released by Y. lipolytica to hydrolyse proteins of
different origins available as industrial food by-products. In particular I have taken into
consideration gluten, gelatin, milk and meat proteins;
2) identify proteases with "cold attitude", i.e. proteolytic activity at temperature ≤ 10 °C, in
order to save energy consumption during industrial processes. In fact, the main technological
criterium to assess the suitability of a biological process to exploit by-products regards the energy
consumption during all the phases. Therefore the enzyme to be selected for the biotransformation
should be active within a temperature range between 4 and 20°C;
3) obtain and characterize peptides having specific biological activity (namely antioxidant,
anti-hypertensive, antimicrobial, cytotoxic)
4) improve the bioactivity of the peptides through a novel process based on their
glycation/glycosylation.
The sequence of the activities performed on the selected strains can be summarized in the
following scheme:
42
Selection of 7 different
strains and hydrolysis of
different matrix
Protease
characterization
Gelatin
Gluten
Meat extract
proteins
Caseins
Selection of one strain and
hydrolysis of one matrix
Gelatin
Bioactivities
Improvement of
bioactivities of peptides
through
glycation/glycosilation
Bioactivities
43
CHAPTER 7.
MATERIAL AND METHODS
44
7.1. Yarrowia lipolytica strains and culture conditions
The strains of Yarrowia lipolytica used in this Ph.D. thesis belong to the Department of
Agri-Food Science and Technologies (DISTAL) - Alma Mater Studiourum, University of Bologna.
The strains selected have different isolation habitats: salami (CLCD), speck (1IIYL4A,
1IIYL8A), irradiated poultry meat (16B), commercial chilled foods (Y10, Y16) and superficial
waters of Po river (PO19). The strains were cultivated in Sabouraud broth (Oxoid, Basingstoke,
England) at 27°C for 72-96 h.
7.2. Protein matrices
The proteolytic activity of Y. lipolytica was evaluated on proteins of different origin also
available as industrial food by-products, and namely gelatin, meat protein, dairy proteins (skimmilk, α- and β-caseins), and gluten from wheat.
In particular, α- and β-caseins were purchased from Sigma-Aldrich, skim-milk from a local
dairy processing plant, gelatin and meat proteins were extracted from meat by-products used for the
production of salami.
7.2.1. Total meat protein extraction
Total proteins were extracted from 2 g of sample (i.e. meat by-products) with 40 ml of 1.1
M potassium iodide, 0.1 M sodium phosphate, pH 7.4 buffer (Cordoba, 1994). The sample was
homogenized for 3 minutes and then centrifuged (Beckman Coulter Avanti J-10, Fullertn, CA,
USA) at 8000 x g for 15 min at 4°C. The supernatant was filtered with 0.22 µm filter and then used
for the enzymatic activity assay (paragraph 7.4.4.).
7.3. List of chemicals used
Alcalase (Alc, EC 3.4.21.14, from Bacillus licheniformis) and Flavourzyme (Flv, from
Aspergillus oryzae), Glycine-Glutamine (Gly-Gln), Glucosamine hydrochloride (GlcN), 2,2diphenyl-1-picrylhydrazyl (DPPH), Transglutaminase (TGase) from guinea pig liver, Hanks’
Balanced Salt Solution (HBSS) were purchased from Sigma-Aldrich (St. Louise, MO). All
chemicals used in SDS-PAGE electrophoresis, Size Exclusion Chromatography (SEC), LC-MS/MS
and MALDI-TOF were of HPLC grade supplied by Sigma-Aldrich (St. Louise, MO), whereas other
45
chemicals were of analytical grade. Fetal bovine serum (FBS) was purchased from Atlanta
Biologicals (Lawrenceville, GA).
7.4. Proteolytic activity of Y. lipolytica
7.4.1. Extracellular proteases recovery and characterization
For the production and the recovery of extracellular proteases, the protocol used by Vannini
et al. (2001) was followed with some modifications. The strains of Y. lipolytica were pre-cultured
for 72 h at 27°C in Sabouraud Broth under agitation (200 rpm). After centrifugation at 8000 x g
(Beckman Coulter Avanti J-10, Fullerton, CA, USA) at 4°C for 15 min the supernatants were
collected and were used as the source of the protease enzymes and thus referred to the
“supernatants”.
A preliminary characterization of the extracellular proteases released by the various strains
was made by analyzing the cell-free supernatants with the technique of substrate-incorporated
polyacrylamide gel electrophoresis (zymography). Such a technique was used to separate and
characterize the enzymes produced by cultures of Y. lipolytica and to evaluate their activity on
substrates (i.e. gelatine and casein) incorporated into the gel.
7.4.2. Characterization of proteases by Zymography
The characterization of the extracellular proteases was performed by the technique of
substrate-incorporated polyacrylamide gel electrophoresis (Zymography). In particular two different
precast Zymogram gels, containing gelatin or caseins as substrate incorporated into the gels (Bio
Rad), were used to see if proteases showed a different specificity for the substrates. 50 µL of the
cell-free supernatants of the various strains of Y. lipolytica were mixed with an equal volume of
Zymogram sample buffer 2X (Bio Rad) and let to rest for 10 min at room temperature. 35 µL of
each sample was loaded on the gels. A 10 µL aliquot of Precision Plus Protein Standard All Blue
(Bio Rad) was used as standard. Gels were run in a Mini Protean Cell System with a Tris-Glycine
SDS Running Buffer at 100 V for the first 10 min and at 120 V for 1 h. After running, the gel was
soaked into the Zymogram Renaturing Buffer (Bio Rad) and incubated with a gentle agitation for
40 minutes at room temperature. Subsequently, the Zymogram Renaturing Buffer was washed
before (30 min) and then replaced with the Zymogram Developing Buffer (Bio Rad). The gel was
incubated in this buffer overnight at 37°C to allow enzymatic digestion of the protein.
46
Gel was stained for 1 hour with the Staining solution (0.5 % Bromophenol blue, 50 %
methanol and 7% glacial acetic acid) and de-stained for 2 hours with the Destaining solution (40%
Glacial acetic acid, 10% methanol).
7.4.3. Preliminary evaluation of the "cold attitude" of the proteolytic enzymes
The ability of the extracellular proteases to hydrolyse the different proteins was evaluated
both at 27 and 6°C. In particular, the supernatants of the various strains were tested on gluten agar,
skim-milk agar and gelatin.
Gluten agar was prepared according to Wiese (1995) with some modifications (1.5 % gluten,
1 % glucose, 0.5 % universal peptone, 1.7 % agar from Oxoid). The media was boiled in stirring
conditions and then poured into Petri plates. Similarly, skim-milk agar (1 % skim-milk and 1.7 %
agar from Oxoid) was boiled in stirring conditions and then poured into the Petri plates. Both the
media contained suspended solid particles which made the agar plates cloudy.
Gelatin liquefaction was estimated preparing a solution of 12 % gelatin, 1 % glucose, 0.5 %
universal peptone. After autoclaving it was dispensed into glass tubes.
Aliquots of 10 µL of the cell-free supernatants were spotted on the centre of the agarized
media. The plates were incubated at 6 or 27°C in order to assess enzyme activity at cold and control
temperatures, respectively. After 3 days (when incubation was at 27°C) or 10 days (for the cold
conditions) plates were checked in respect to growth, contaminants and enzymatic digestion of the
proteins. In particular proteolytic activity resulted in the formation of clear zones onto the agarized
media (for skim milk and gluten) or liquefaction (for gelatin). In general, the proteolytic activity
was carried out at least in triplicate.
7.4.4. Evaluation of the proteolytic profiles generated by Y. lipolytica proteases
Enzymatic activities of the extracellular proteases of the various strains were determined by
using the following substrates: α-casein and β-casein from bovine milk (Sigma), commercial
gelatine and meat protein extract.
The assay mixture contained Y. lipolytica supernatants, sterile distilled water (1:2) and 1%
(w/v) of gelatin or meat protein extract, or 0.1% (w/v) of caseins. The hydrolysis was performed in
stirring conditions. After 48, 72 and 96 hours of incubation at 27°C, the reaction was stopped by
heating at 100 °C for 5 min and the collected samples were stored at -20°C before their use for
protein analysis by SDS-PAGE electrophoresys (paragraph 7.4.5), chemical (paragraph 7.6) and
biological (paragraph 7.7) characterization.
47
7.4.5. SDS-PAGE electrophoresis
The hydrolysis of the proteins was detected by sodium dodeceyl sulphate polyacrylamide
(SDS-PAGE) gel electrophoresis. For the denaturation, 70 µL of each sample were mixed with an
equal volume of Laemmli Sample Buffer 2X (Bio-Rad Laboratories, Milan, Italy) containing βmercaptoethanol. The mixture was incubated at 100 °C for 10 min and then charged on a precast
gel. Different gels (Bio Rad) were used according to the protein analysed: a gradient 4-20 % gel
was used for the casein samples and a 4-15% gel for the meat protein and the gelatin samples. 10
µL of Precision Plus Protein Standard All Blue (Bio Rad) was used as standard, and 35 µL of each
sample were loaded on the gels. Gels were run in a Mini Protean Cell System with a Tris-Glycine
SDS Running Buffer at 100 V for the first 10 min and at 200 V for 1 h.
Gels were stained for 1 hour with the Staining solution (0.1 % Bromophenol blue, 50 %
methanol and 7% glacial acetic acid). Subsequently, 2 hours of Destaining solution (40% Glacial
acetic acid, 10% methanol) were performed.
7.5. Improvement of peptides bioactivity
7.5.1. Model System
7.5.1.1.Model system preparation and UV spectra collection
A model system consisting of a dipeptide Gly-Gln (0.03 M) and GlcN with different molar
ratios of 1:1, 1:3 and 1:10 was prepared. Samples were dissolved in 0.05 M (NH4)HCO3/NH4OH
buffer at pH 8.8 with or without TGase (2 unit/g). TGase was activated with 5 mM calcium chloride
solution. In addition, 0.03 M and 0.12 M solutions of GlcN in the absence of the peptide were
prepared separately as control. These solutions were treated in the same way as the mixtures of
peptide and GlcN. About 5 mL of the mixture was poured into screw-cap tubes (15 mL) and
incubated at 25 and 37°C with shaking (200 rpm) for 0, 3.5 and 7 h. All aliquots were kept at -20ºC
upon incubation for further use. The UV-Vis absorbance spectra (250 - 500 nm) of the samples
were measured using a spectrophotometer SpectroMax M3 plate reader (Molecular devices,
Sunnyvale, CA) in order to create the UV profiles.
7.5.1.2. LC/MS analysis
Samples prepared as above described were diluted in aqueous 25 % (v/v) acetonitrile and 0.2
% (v/v) formic acid, and ionized by using nanoflow HPLC (Easy-nLC II, Thermo Scientific,
Mississauga, ON) coupled to the LTQ XL-Orbitrap hybrid mass spectrometer (Thermo Scientific,
Mississauga, ON). Nanoflow chromatography and electrospray ionization were accomplished by
48
using a PicoFrit fused silica capillary column (ProteoPepII, C18) with 100 µm inner diameter (300
Å, 5 µm, New Objective). Samples were injected onto the column at a flow rate of 3000 nL/min and
resolved at 500 nL/min using 30 min linear acetonitrile gradients from 5 to 50 % (v/v) aqueous
acetonitrile in 0.2 % (v/v) formic acid. Mass spectrometer was operated in data-dependent
acquisition mode, recording high-accuracy and high-resolution survey Orbitrap spectra using
external mass calibration, with a resolution of 60 000 and m/z range of 100–2000.
7.5.1.3. Production of hydrolysates
Exactly 5 % (w/v) of gluten was suspended in 0.05 M (NH4)HCO3/NH4OH buffer, the final
pH of the mixture was pH 7.5 – 8. It was homogenised with an Ultra-Turrax homogenizer, model T
25 (IKA Works, Inc. Staufen, Germany) at 10,000 rpm for 1 min. Then it was heated at 80 ºC for 10
min. The gluten mixture was cooled to 50 ºC before adding Alc or Flv, respectively at 1:10 volume
ratio of enzyme to buffer, followed by incubation in a shaker (50 ºC, 3.5 h, 200 rpm). Incubation
was terminated at 80 ºC for 10 min, the hydrolysates were centrifuged at 10,000 × g (10 ºC) for 15
min and filtered by using Whatman No. 1 filter paper. The filtrate was collected, lyophilized and
stored at -18 ºC before their chemical and biological characterization.
7.5.1.4. Preparation of Glycated/Glycosylated peptides
Samples of the lyophilized hydrolysate powders (from Alc or Flv respectively) were
weighed at 1.5 g each and added to GlcN at the weight ratio of 1:1. Each of the weighed powders
was dissolved in 30 mL of 0.05 M (NH4)HCO3/NH4OH buffer (pH 7.0 ± 0.5) and each incubated at
25 ºC and 37 ºC for 3.5 h. Samples with GlcN were subjected to incubation with or without TGase
(2 unit/g of lyophilized hydrolysate) at pH 7.5. 5 mM calcium chloride were added prior to use in
order to activate TGase. Controls consisiting of the lyophilized hydrolysate were incubated at the
same temperature without GlcN. At the end of incubation, all the mixtures were passed through a
0.2 µm nylon syringe filter (13 mm, Mandel, Ontario) followed by ultra-filtration with a molecular
weight cut-off membrane of 10 kDa (3,900 × g, 20 min, 10 ºC, Amicon Ultra Centrifugal filters
(Millipore, Cork, Ireland)). Excess of GlcN was removed by dialysis membrane with a molecular
weight cut-off membrane of 100 – 500 Da (Spectrum Laboratories, TX). The retentates were
collected and lyophilized, samples were then stored at -18 ºC before their chemical and bioactive
characterization.
49
7.6. Chemical characterization of the proteins and hydrolysates
7.6.1. Degree of hydrolysis (DH)
The measurement of DH was carried out according to the OPA method as stated by Nielsen
et al. (2001) by using serine as a standard for hydrolysis determination. Protein contents of gluten
samples were assessed. The percent of DH was calculated according to Alder-Nissen (1986).
7.6.2. Size exclusion chromatography
After filtration with 0.2 nm filters, the samples were subjected to size exclusion
chromatography using a 120 mL HiLoad 16/60 Superdex 200 pg column (GE Healthcare
Amersham Biosciences) connected to a fast protein liquid chromatography (GE Healthcare
Amersham Biosciences). A sample volume of 100 µL (1 mg/mL of the freeze-dried peptide
mixture) was injected and eluted isocratically at a flow rate of 0.5 mL/min with 50 mM phosphate
buffer containing 0.15 M NaCl. Eluted molecules were detected at 215 nm (for gelatin) or 280 nm
(for gluten). The mass calibration was performed using a protein mixture (200 to 12.4 kDa) of βAmilase, Alcohol Deidrogenase, Albumin, Carbonic Anhydrase and Cytocrome C (Sigma Aldrich).
7.6.3. Determination of peptides and glycopeptides molecular weights by Matrixassisted laser desorption ionization-time of flight-mass spectrometry (MALDI/TOF-MS)
For the MALDI-TOF-MS analysis, the protein hydrolysate samples were diluted tenfold in
50 % (v/v) acetonitrile/water + 0.1 % (v/v) trifluoroacetic acid/water. One microliter of each sample
was mixed with 1 µL of a-cyano-4-hydroxycinnamic acid (matrix) (4-HCCA, 10 mg/ml in 50 %
acetonitrile/water + 0.1 % trifluoroacetic acid/water). One microliter of the sample/matrix solution
was then spotted onto a stainless steel target plate and allowed to air dry. All mass spectra were
obtained using a Bruker Ultraflex MALDI/TOF-MS (Bruker Daltonic, GmbH). Ions were analyzed
in positive mode after acceleration from the ion source 174 by 25 kV. External calibration was
performed by use of the following standard peptide mixture ((M+H)+ monoisotopic masses are
reported in Da): Bradykinin fragment 1-7 (757.3997), Angiotensin II (human) (1,046.5423),
synthetic peptide (1,533.8582), ACTH fragment 18-39 (human) (2,465.1989), Insulin oxidized B
chain (bovine) (3,494.6513), Insulin (bovine) (5,730.6087) (Sigma Aldrich).
50
7.7. Biological characterization of the peptides
7.7.1 Antioxidant properties
7.7.1.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
DPPH radical scavenging activity was assessed according to the method of Yen and Wu
(1999) and modified by Hsu (2010). Exactly 200 µL of each sample (10 mg/mL for gelatin and
meat extract or 1 mg/ml for caseins, referring to the starting protein utilized) were mixed into a test
tube containing 400 µL of 0.5 mM DPPH and 1.4 mL of 99.5 % methanol. L-ascorbic acid (0.1
mg/mL) was used to replace sample and referred to as positive control. The whole mixture was
mixed thoroughly and incubated for 30 min in the dark at room temperature. Then the absorbance at
517 nm was measured using a spectrophotometer. The percentage of inhibition of radical
scavenging activity was calculated as follows:
% DPPH scavenging activity = ((Abs control - Abs sample) / Abs control)) x 100
where: Abs control is the absorbance of reference solution containing only DPPH and water, and Abs
sample
is the absorption of the DPPH solution with sample after 30 min. Methanol was used as a
blank. A lower absorbance represents a higher DPPH scavenging activity.
When reported, the EC50 value (concentration required for 50 % reduction of activity) was
calculated through software Prism 5.
7.7.1.2. Inhibition of linoleic acid peroxidation
Linoleic acid was oxidised in a model system according to Mendis et al. (2005) with
modifications from Li et al. (2007) Reagent mixtures consisting of 1.5 mL of 0.1 M sodium
phosphate buffer (pH 7.0), 1.5 mL of ethanol containing 50 mM linoleic acid and 2 mL of 10
mg/mL of gelatin or gluten hydrolyxed samples were prepared. For the positive control, peptide
mixture samples were replaced with 2 mM alpha-tocopherol and 2 mM butylated hydroxytoluene
(BHT); for the negative control, samples were replaced by water. All the mixtures were kept sealed
and incubated in the dark at 40 ºC. The oxidation level was measured every 24 h over 7 days by
using a ferric thiocyanate method as described by Osawa and Namiki (1981). Exactly 50 µL of the
mixture were mixed with 100 µL of 1 M HCl, 50 µL of 30 % (w/v) aqueous ammonium
thiocyanate, 50 µL of 20 mM ferrous chloride in 3.5 % HCl and 2.25 mL of 75 % ethanol. The
mixture was gently agitated and incubated at room temperature for 5 min before reading the optical
51
density at 500 nm. The linoleic acid peroxidation was monitored for 7 days following the increase
of the absorbance at 500 nm.
7.7.2. Angiotensin I-converting enzyme (ACE) inhibitory activity
The hydrolysis of N-[3-(2-furyl) acryloyl]-L-phenylalanylglycylglycine (FA-PGG), which
represents a substrate for angiotensin converting enzyme, was used to assess the angiotensin
converting enzyme inhibitory activity (Bunning et al., 1983). The assay was performed according to
Vermeirssen et al. (2002) and Shalaby et al. (2006). Buffers were made according to Hou et al.
(2003) and the volumes were reduced to fit into 96-well plates. FA-PGG was dissolved at a
concentration of 1.75 mM in 50 mM Tris-HCl buffer, pH 7.5, containing 0.3 M NaCl. The ACE
solution (0.25 units/mL) was freshly prepared by adding purified water to a vial containing 0.25
units of enzyme (Sigma). The assay was performed in a 96-well, clear, flat-bottomed polystyrene
plate. Exactly 10 µL of ACE solution and 10 µL of protein hydrolysate samples were placed
separately in the well at room temperature. 150 µL of pre-heated (37 °C) substrate solution (FAPGG) was added quickly to each well with an eight channel pipette to start the reaction. The plate
was immediately transferred into a SpectroMax M3 plate reader. Enzyme activity at 37 °C was
based on the initial linear rate of change in absorbance at 340 nm, recorded every 3 min for 30 min.
The control contained all reaction components, and water instead of the protein hydrolysate sample.
Blanks with no enzyme (substituted by water) or with no substrate (substituted by 50 mM Tris-HCl
buffer, pH 7.5, containing 0.3 M NaCl) were used. The ACE activity was expressed as the slope (m)
of the decrease in absorbance at 340 nm and the ACE inhibition (%) was calculated from the ratio
of the slope in the presence of sample to the slope obtained without added sample, according to the
formula:
% ACE inhibition = (1- (msample / mcontrol ) x 100
7.7.3. Cytotoxicity of the hydrolyzed and conjugated samples on human HepG2 cells
HepG2 cells were grown in growth medium (EMEM supplemented with 10 % Fetal Bovine
Serum - FBS), 50 units/mL penicillin and 50 µg/mL streptomycin) and were maintained at 37°C
and 5 % CO2 as described by Wolfe and Liu (2007). Cells used in this study were between passages
5 and 10. Cytotoxicity was determined using the protocols of the Water Soluble Tetrazolium Salts
(WST1) assay by using protein hydrolyzed samples at 3 different concentration, i.e. 0.1, 0.5 and 1
mg/ml. The inhibition of cell growth by the tested protein hydrolyseate samples was expressed as
52
the percentage of cell viability with respect to control. Concentrations of samples that decreased the
absorbance by >10 % when compared to the control were considered to be cytotoxic.
7.7.4. Antimicrobial activity
The antimicrobial activity of the protein hydrolysate samples was determined by the
modified micro-dilution technique against some bacteria, i.e. Escherichia coli 555, Listeria
monocytogenes 56Ly, Bacillus subtilis FAD 110 and Salmonella enteritidis 155, and yeasts, i.e.
Pichia membranaefaciens OC70, P. membranaefaciens OC71, Pichia anomala CBS 5759 and P.
anomala DBVPG 3003. The bacterial strains were grown at 37°C in Brain Heart Infusion broth
(BHI, Oxoid), while those of yeasts at 27°C in Sabouraud (Oxoid). The microbial cultures were
prepared at 104 cfu/mL in the 96-well microplates. Diluted sample solutions of the protein
hydrolysates were dispensed into the wells providing final concentrations in the range of 16 to 0.06
mg/mlfor gelatin and 40 to 0.097 mg/ml for gluten. The same tests were performed simultaneously
to check for the growth in control conditions (media + microorgaism) using water instead of the
samples, and sterility control (media + the protein hydrolysate samples tested). The final volume in
each well was 200 µL; plates were incubated at the optimal growth temperatures typical for each
microorganism for 24 or 48 hours. The MIC values were defined as the lowest concentrations
preventing any discernible growth. From the same MIC well, 10 µL of the suspension was plated in
Luria Broth LB agar plate and incubated at 37 ºC for 24 h (for bacteria) or 27°C for 48 h (for
yeasts). The absence of growth in the plate represented the MBC value, thus the capacity to
completely kill all the bacteria or yeasts. All the MIC and MBC values were evaluated in triplicate.
In order to obtain information on the antimicrobial activity of protein fractions with specific
molecular weights, samples of gelatine, hydrolysed with the strain 1IIYL4A and collected after 96
hours, were also ultra-filtered with a molecular weight cut-off membrane of 3 kDa (3,900 × g, 20
min, 10 ºC) (Amicon Ultra Centrifugal filters (Millipore, Cork, Ireland)). The retentates
corresponded to the fraction > 3 kDa, whereas the filtered part corresponded to the < 3kDa. Both
the fractions were used for MIC analysis.
7.8. Statistical Analysis
Data were reported as means ± standard deviation of at least triplicates. Means obtained
were analysed by one way analysis of variance (ANOVA), separated by Duncan test using
STATISTICA statistics software (ver. 6.0). Means were considered significant when p < 0.05. As
53
for model study, the results from UV–Vis spectra were analysed by using bidimensional
hierarchical clustering analyses (heat map) as reported by Hrynets et al. (2013).
54
CHAPTER 8.
RESULTS
55
8.1. Evaluation of the proteolytic activity of Yarrowia lipolytica on different
proteins
Due to its physiological attributes and biotechnological potentialities, Yarrowia lipolytica is
one of the most studied "non-conventional" yeasts and it is classified as “generally recognized as
safe.” It is able to growth in different environments and many strains have been isolated from oilpolluted environments, rivers and foods such as refrigerated foods, cheese, irradiated meats,
sausages, salami and other products (Bankar et al., 2009).
In addition to its potential use as adjunct as ripening agent of dairy and meat products
(Suzzi et al., 2001; Lanciotti et al., 2005; Patrignani et al., 2007; 2011a; 2011b), Y. lipolytica has
been exploited also for the production of single cells proteins for feeds and the treatment of olive
mill wastewaters (Lanciotti et al., 2005). Without any doubt, the most studied features of this
microorganism are the production of lipases (Ota et al., 1982), citric acid and γ-decalactone
(Cohelo, 2010). In particular some strains have been described as producers of "cold" lipases
(Parfene et al., 2011) and this aspect is progressively getting interesting for industrial applications
(Joseph et al., 2007). Proteases are another class of enzymes that are produced and secreted by Y.
lipolytica. In particular, an acidic extracellular protease (AXP) and an alkaline extracellular
protease (AEP) have been mainly studied (Young et al., 1996). The main element that
discriminates the production of AEP or AXP is the environmental pH. In general, neutral and
high pH (6-9) lead to AEP production, while low pH (2-6) favours AXP (Ogrydziak, 1993).
However, both proteases are similarly induced at the end of the exponential phase (GonzálezLópez et al., 2002). The screening of novel enzymes that are capable of generating compounds
with specific and improved functional bioactivities is constantly necessary. In particular this kind
of research can have a relevance for the food industry as a possible strategy to transform cheap
food by-products into active compounds with an increased market value.
In my research, seven strains of Y. lipolytica have been used to evaluate the capability of
the extracellular proteases to produce bioactive peptides from different matrices. In particular
gelatin, gluten, milk and meat by-product proteins have been considered. The possibility to obtain
biologically active peptides, endowed with technological properties from different matrices
through the use of lactic acid bacteria has been reported. But on the other hand the biotechonolgal
properties of yeast concerning this aspect have been not explored. The wide diversity and
specificity of proteases are used to great advantage in developing effective therapeutic agents.
Proteases from Aspergillus oryzae have been used as a digestive aid to correct certain lytic
enzyme deficiency syndromes. An asparaginase isolated from E. coli is used to eliminate
56
asparagine from the blood stream in various forms of leukaemia. In addition microbial proteases
have been used for the synthesis of dipeptides (Barros et al., 1999) and tripeptides (So et al.,
2000) Nutritionally enhanced meat products have been obtained by using microbial proteases
able to restructure and improve appearance, texture and nutritional quality of animal parts of
poorer technological quality (Marques et al., 2010)
8.1.1. Preliminary characterization of proteases by Zymogram
The Zymograms of the 72 h old supernatants of the 7 strains obtained both on casein and
gelatin are shown in figures 5A and 5B, respectively. In figure 5A, two main bands were detected,
one at low molecular waight (MW) nearby 37 kDa (1IIYL4, 16B, Y10) and one at high MW higher
than 200 kDa (Y16, 1IIYL8 and Y10). While the first one corresponds to the acid protease typical
of Y. lipolytica reported by many Authors (Young et al., 1996), the other one presents a band with
an unusual MW never reported in this species. The strains 1IIYL4 and 16B were characterized by
an intense protein clearness zone nearby 37 kDa, while the strain PO19 showed a weak band
suggesting either a lower activity or a lower release of the protease. The strain CLCD, isolated from
salami, did not show any apparent activity. A similar behaviour was observed in the presence of
gelatin although the bands were less intense (fig. 5B).
8.1.2. "Cold attitude" of the proteolytic enzymes
In order to evaluate the cold attitude of the proteases, the supernatants were spotted onto
agar plates containing gluten or skim milk as substrates for the enzymatic reactions. The activity
was evaluated on the basis of the clearance of the media after 10 days of incubation at 6 °C.
A 72 h sample incubated at 27 °C was used as a control. In addition, the ability to hydrolyse gelatin
in the same conditions was assessed in tubes measuring the depth of liquefaction.
At the optimal temperature (27 °C), 1IILY4A, 16B, Y16 and 1IIYL8A were the strains with
the highest ability to hydrolyse the skim milk and gluten, whereas CLCD was not active under these
conditions. The incubation at 6 °C did not affect the final capability of all the strains to work at this
low temperature, except for 1IIYL8A on gluten agar (tab. 6a and 6b)
Regarding gelatin, similar behaviours were observed at 27 °C for all the strains, except for
Y16 and 1IIYL8A which showed a reduction of their activity. On the other hand, the strain Y10,
which was unable to hydrolyze both skim milk and gluten, showed a significant activity. The
reduction of the temperature moderately affected the final degree of the proteolysis (tab. 6c). In fact
the strains 1IIYL4 and 16B presented a band at 37 kDa while the 1IIYL8A and Y16 showed an
57
intense band at 200 kDa. By the comparison of these results with the two Zymogram it is evident
that the two proteins visualized display their activity both on gelatin and casein.
8.1.3. Proteolytic profiles generated by Y. lipolytica proteases
Four different protein matrices (meat proteins, α-casein, β-casein and gelatin) were
resuspended in water and incubated with the supernatants of the 7 strains of Y. lipolytica for 72 h at
27 °C. The proteolytic profiles were evaluated by SDS-PAGE electrophoresis.
Figure 6 reports the proteolysis of α-casein. α-casein includes two main isoforms αs1 and
αs2 that migrate in the SDS PGAE electrophoretogram with a final MW of around 34 kDa. After
incubation with Y. lipolytica supernatants, a significant proteolytic activity was detected. In
particular, 1IIYL4A, 16B and Y10 significantly reduced the intensity of the casein bands. A partial
hydrolysis, with the formation of several detectable bands having different low MWs, was observed
with CLCD, PO19 and Y16. Comparing these strains, CLCD and PO19 were the least active since
both the bands of αs1- and αs2- caseins were still present. Taking into consideration the bands
present in the control, the bands at 30 and 34 kDa were still visible in the samples of the strains
1IIYL4A CLCD, PO19 Y16 and 1IIYL8A while they disappeared in 16B and Y10. The band of 24
kDa disappeared in all the samples except for strains PO19, whereas the band at 22 kDa was
detected in CLCD, PO19 and Y16. New bands at 20 and 21 kDa were formed with PO19 and Y16,
and a band at 18 kDa was detected with CLCD, Y16, 1IIYL8A and PO19. It can be noticed that the
strains 1IILY4A and 16B, which showed in the zymograms a 37 kDa protease, were characterized
by similar breakdown profiles and a strong proteolysis. On the other hand the strains Y16 and
1IIYL8A, which displayed in SDS-PAGE electrophoretogram a lower proteolytic activity, were
characterized in the zymograms by a protease having a MW higher than 200 kDa.
In figure 7 the β-casein profile of hydrolysis is reported. Also against this protein, the strains
1IIYL4A and 16B, characterzed by the 37 kDa protease, displayed a very strong proteolytic
activity. In fact, not only the bands of β-casein including the principal one at 31 kDa but also all the
bands having MWs ranging from 25 to 15 kDa disappeared. Also the strain PO19, having a weaker
37 kDa protease in the zymogram, displayed a good proteolytitc activity also if at lower extent than
the former. The comparison of all the proteolytic profiles of α- and β-caseins demonstrated that a
strong activity was displayed by all the strains of Y. lipolytica, except for Y10, against β-casein. In
fact, also the strains Y16 and 1IIYL8A, characterized by the protease > 200 kDa, showed a strong
proteolytic activity against β-casein.
In figure 8, the meat protein breakdown profiles obtained with the supernatants of the
various strains are showed. With this protein matrix, two different behaviours can be identified: the
58
strains 1IIYL4A, 16B and PO19, characterized by the 37 kDa protease, displayed a strong
proteolysis with the formation of peptides having MW lower than 10 kDa and the fading of higher
MW peptides. In particular, the 75, 70, 47, 26, 17 kDa MW bands faded, whereas those at 65, 37
and 24 kDa disappeared. On the countrary CLCD, Y10, Y16, 1IIYL8A showed only a weak
hydrolytic activity as most of th bands were as intense ad the control samples.
8.1.4. Proteolysis of gelatin
The strains having the highest activity in gelatin agar plate (tab. 6), were incubated with
gelatin for 72 h and the corrisponding hydrlysed samples were analysed through SDS-PAGE gel
electrophoresis (fig. 9).
Figure 9 showed that not all the strains were able to hydrolyze gelatin at the same extent.
Looking at the higher MW, above 100 kDa, it can be observed that the intensities of the two typical
bands of gelatine were reduced in the samples 16B and 1IIYL8A, while they were completely
absent in 1IIYL4A. As far as the bands with the intermediate MWs (75-20 kDa), none of the
samples had the 37 kDa band. Moreover some differences can be detected in the sample 16B, where
the band at 23 kDa disappeared, while the 25 kDa one became more intense. The same behaviour
was partially shown by the sample Y16. Comparing all the samples together only the sample
1IIYL4A showed a general increment in the intensities of the bands in the range 37-20 kDa.
Instead, molecules below 10 kDa were generated by all the samples, also if with different extents.
In order to have a wider overview on these profiles, size exclusion chromatography was
applied to the gelatin hydrolyzed samples. The chromatograms reported in figure 10 showed how in
many samples, the peak corresponding to gelatin (having an elution volume of 40 mL) reduced
comparedo figure 10 A, which reprents the gelatin before its hydrolysis. As the extent of the
hydrolysis increased, more peacks having lower MWs appeared on the left side of the
chromatogram with higher elution volumes. In some samples, such as CLCD, Y10 and PO19 (fig.
10 D, F, G) there was mainly an increment of the peaks eluting between 50 and 100 mL, whereas
peaks eluting between 90 and 140 mL represented the main one for the samples 1IIYL4A, 16B and
1IIYL8A (fig. 10 B, C, E). Among all these conditions, the sample that showed the best hydrolysis
with the formation of smaller MWs was the 1IIYL4A. An overlay of the two chromatograms
(before and after hydrolysis) is reported in figure 11.
On the basis of the comparative results of the size exclusion chromatograms the strain
1IIYL4A was chosen for a more detailed characterization of the peptides obtained.
59
8.1.5. Characterization of the products of gelatin hydrolysis obtained by Y. lipolytica
1IIYL4A protease
The gelatin hydrolysis by proteases from Y.lipolytica 1IIYL4A was followed over time (time
0, 48, 72, 96 hours) in stirring conditions (useful for the enzyme activity). In figures 12 the SDSPAGE electrophoretograms (A) and size exclusion chromatograms (B) of the hydrolysates collected
over time are reported, respectively. In particular, figure 12 B represents a closeup of the final
elution volume of the size exclusion analysis in order to show better the progressive increment of
the lowest MWs (lower than 10 kDa). It is possible to see that three main regions are
distinguishable: the first one, ranging between 90-115 mL, the second one between 115-125 mL and
the third one between 125-135 mL. These three areas were integrated and the relative abundances
are reported in the figure 13. Among the three areas, the first one, ranging between 90-115 mL,
reached its maximum abundance already at 48 h and it seamed constant at 72 and 96 h. However,
looking at figure 12B it is possible to see that even if the final area of 72 h and 96 h samples is the
same, the peak at 48 h is smaller and broader. This latter aspect has to be taken into consideration
because it suggests that different MWs are present in that area. On the contrary, in the peaks of 72
and 96 h samples ranging between 90-115 mL, the MWs should be more similar among them. A
sensible decrease in the abbundance of the second peak (ranging between 115-125 mL) and a
sensible increase of the third peak (ranging between 125-135 mL) was observed over time.
The following analysis were performed only on the peptides with low MWs because it has
been reported that many peptides having antioxidant, anti-hypertensive, antimicrobial, and cell
modulating activities are characterized by MWs below 10 kDa (Li et al. 2008; Ryan et al., 2011).
However, the size exclusion technique is not able to separate and identify the exact MW of the
peptides, and particularly those lower than 10 kDa. For this reason the samples were analysed also
with MALDI-TOF after ionization (fig. 14 A and B). The reduction of MWs is well evidenced by a
shift of the signals towards the left side of the spectrum (fig. 14 B). In particular all the peaks
ranging between 7 and 3.3 kDa disappeared, while new signals manly ranging from 4 to 1 kDa
appeared.
Taking into consideration all the signals detected, table 7 reports the flow over time of the
peptide MWs. In this table the evolution of some peptides, present in at least two sampling times, is
reported. All the red numbers derive from the time 0, in particular from the native gelatin added
with the supernatant of Y. lipolytica 1IIYL4A; in green the peptides produced after 48 h, and in blue
that ones released at 72 h.
The MALDI-TOF results (fig. 14, tab. 7) are comparable with the SDS-PAGE image (fig.
12A) where it was highlighted that strong differences in the hydrolytic profiles occurred only after
60
how 72 h. Moreover, at 96 h the main peptides are either newly originated or deriving from those
detected at 72 h, although the number of peptides identified at 72 and 96 h was similar.
8.1.6. In vitro bioactivity evaluation of the peptides
Preliminary investigations on the biological properties of the peptides obtained were
performed on the gelatin hydrolysates produced by extracellular protease of Y. lipolytica 1IIYL4A.
8.1.6.1. DPPH radical scavenging activity
A DPPH assay was performed to establish if the degree of proteolysis, and in particular the
mixture of peptides whose MWs had been determined with MALDI-TOF analysis, affected the
antioxidant properties of the hydrolysates collected after 0, 48 72 and 96 h of incubation.
According to figure 15 a progressive increase of the DPPH activity with the hydrolysis time
was observed. In particular the antioxidant activity, which was 19.7 ± 6% at time 0 with respect to
the ascorbic acid chosen as a reference sample, attained values of 39.4 ± 1.4 and 48.3 ± 0.5% after
72 and 96 h, respectively. The latter differences were significant (p < 0.05).
8.1.6.2. Inhibition of the linoleic acid peroxidation
The capability to inhibit the peroxidation of linoleic acid by the peptide mixture collected
over time was also evaluated. As previously shown by the DPPH assay, the progressive incubation
time gave rise to a significant increase (p < 0.05) of the antioxidant activity in particular for the
samples at 72 and 96 h (fig. 16). On the other hand the sample collected at time 48 h showed a
behaviour much more similar to that at time 0. The α-tocopherol, chosen as a reference for the
inhibition of the peroxidation, has been used at 0.89 mg/ml. Its activity was higher than that of the
peptide mixture, but it is necessary to outline that I compared the antioxidant properties of a pure
compound with a mixture of peptides of 20 mg/ml (freeze-dried powder/water) having a peptide
content of about 1.3 mg/ml. Moreover, it can be assumed that only few peptides included in the
mixture were endowed with antioxidant properties.
8.1.6.3. Cytotoxicity activity in human cells
The assessment of the cytotoxicity is a prerequisite for the use of a new ingredients, such as
peptide mixtures having technological properties, in food formulations. On the basis of the figure
17 potential cytotoxic molecules are generated only when the proteolysis was extended over 72 h
and the cells HepG2 were exposed to a concentration of 1 mg/ml.
61
8.1.6.4. Antimicrobial activity
The bacteria species used for evalutation of the antimicrobial activity were Escherichia coli
555, Salmonella enteritidis 155, Listeria monocytogenes 56Ly, Bacillus subtilis FAD 110, Pichia
membranaefaciens OC71, P. membranaefaciens OC70, Pichia anomala CBS 5759, and P. anomala
DBVPG 3003. None of the bacteria resulted sensitive to the compounds at any concentration tested.
However, it has been observed a progressive inhibition of the growth of some strains of Pichia
membranefaciens in relation to the degree of hydrolysis. In particular MIC values of 8 mg/ml were
observed for gelatin hydrolysates collected at 48 and 72 h. The extension of the hydrolysis up to 96
h resulted in a two fold reduction of the MIC value. Moreover, when the sample at 96 h was
separated into fractions with MWs higher than 3 kDa, a MIC value of 2 mg/ml was detected. On the
contrary, no activity was observed for the fraction below 3 kDa.
8.2. Improvement of peptides bioactivity
The technological attitudes of the peptide mixtures can be regarded as promising, but need
to be improved. A new frontier to be explored, to enhance the properties of peptides, is their
transformation into glycopeptides. The enhancement of the functional and biological properties in
the glycoproteins with respect to the native form has been explored (Bielikowicz et al., 2010; Liu et
al., 2012) and the importance of this peptide transformation on some physiological processes (such
as immune-system, inflammation, brain development, endocrine system and fertilization) has been
emphasised (Spiro, 2002).
The reaction involving sugars and amino acids can be triggered either through enzymes or
spontaneously occur under specific conditions. Glycosylation, as an enzyme driven process, is one
of the main processes occurring in eukaryotic and prokaryotic cells. In fact, it is known that the
majority of proteins are subjected to post-translational modifications, such as the attachment of
glycans. This reaction is crucial in many biological pathways (Spiro, 2002). On the contrary,
"glycation" is the term universally used to define the chemical bounding of sugars with proteins or
peptides. This second reaction is spontaneous, and can occur both in human body and in food
systems (Liu et al., 2012). As reviewed by Oliver et al. (2006), glycation via the Maillard reaction
can improve several important functional properties of food proteins (Liu et al., 2012). One of the
main requirements for the glycation through the Maillard reaction is the use of high temperatures or
a prolonged heat treatment, in order to favour the reaction between sugars and aminoacids. A
controlled Maillard-induced glycosylation is fundamental to limit the progress of the reaction into
undesired advanced stages; advanced Maillard reactions may result in reduced food digestibility
62
(Erbersdobler et al., 1981), formation of mutagenic compounds (Brands et al., 2000), development
of off-flavors (Moor and Ha, 1991), and excessive browning (Guerra- Hernandez et al., 2002).
(Wang and Ismail, 2012). Due to the low reactivity of sugars and amino acids, high temperatures
are necessary to promote the reaction (Wang and Ismail, 2012). On the other hand, amino-sugars
demonstrated to have a higher reactivity comparing to normal sugars (Kraehenbuehl et al.,2008).
The amino-sugar glucosamine (GlcN) can be obtained through the hydrolysis of chitosan, the main
by-product from shrimp and other crustacean shells processing. The acetylated form of GlcN is
fundamental for the building the bacterial cell wall and the particular cartilage in the human body
(Wang SX et al., 2007).
Aminosugars binding, exploited through the transglutaminase (TGase) enzymatic process,
has been already proposed, in particular between GlcN and peptides. However, results have been
inconclusive (Jiang and Zhao, 2010). The above mentioned enzyme (TGase), discovered in
eukaryotic and prokaryotic cells, and extensively used in food processing, is responsible for the
acyl-transfer reaction between the γ-carboxyamide group of glutamine residue and the primary
amino group. Moreover, this reaction can be driven towards the formation of inter- and intramolecular cross-linkages (if the primary amino group derives from other amino acids, such as
lysine) or deamidation (in absence of primary amino groups, a molecule of H2O is used). The first
two types of bonds are stable and resist to proteolysis (Greenberg et al., 1991)
Wheat gluten, a by-product of the wheat starch industry, is massively produced worldwide.
Due to its modest price, it can industrially compete with milk and soy proteins, as an economic
protein source (Kong et al., 2007). Because of their functional properties, such as solubility,
foaming and emulsifying capacity, the wheat gluten peptides obtained by hydrolysis attracted the
interest of food industries (Kong et al., 2007, Wang et al., 2007) focused on the preparation of
hypoallergenic nutritional mixtures (Daya et al., 2006). Additional studies have reported antioxidant
properties of gluten hydrolyzates, including the capability to inhibit the linoleic acid peroxidation or
to quench DPPH radical. Moreover, Koo et al., (2011) demonstrated that gluten hydrolysates
exhibited taste-enhancing properties. Wang and Ismail (2012), instead, reported the production of
some partially glycosylated wheat protein. My strategy to improve the boactivity of peptides
obtained by enzymatic proteolysis of gluten and other proteic by-products was based on
glycosylation/glycation. The experiment has been developed in two steps:
1) a single peptide was employed to prove the occurrence of the glycation (through a mild
Maillard reaction) and glycosylation (TGase-mediated), and to optimize the main reaction
parameters to be pursued (such as concentration, temperature). This process is totally new and never
ever exploited before,
63
2) a more complex system, based on gluten hydrolysates, was taken into consideration and
the bioactivities of the glycopeptides mixture obtained was assessed.
8.2.1 Model system
To establish the novel conjugation method, and in order to determine the most appropriate
conditions for the reaction, a simplified model system involving glucosamine (GlcN) and the
dipeptide glycine-glutamine (Gly-Gln) was considered. It has been reported that GlcN at high
temperature is able to undergo rearrangements, auto-condensation processes and Maillard reaction
(in presence of amino-acids), with a yield higher than with the simple sugars. Hence, in the first part
of this model, different concentrations of GlcN were tested to detect the threshold conditions for the
formation of its side-products and hence to avoid their production.
The reaction involving the peptide Gly-Gln (chosen because of its simple structure and the
presence of glutamine) and GlcN (molar ratio 1:1, 1:3 and 1:10, dipeptide/glucosamine) was
monitored over time (0, 3.5 and 7 hours) at 25 or 37°C. As control, GlcN alone was incubated at the
same conditions and monitored over time. The UV-spectra (280 - 420nm) of the samples during
incubation are shown in figure 18. The temperature and the sugar concentration played a
fundamental role in both the increased and progressive shift of absorbance to high wavelength
values. The highest absorbance was revealed with the 1:10 ratio at 37°C (fig. 18 A). As the sugar
ratio increased, its augmented by-products created interferences when monitoring the sugar-peptide
interaction and evolution. On the other hand, the ratios 1:3 and 1:1 at 25 or 37 °C promoted a
suitable arrangement (fig. 18 B and 18 C, respectively). At 1:3 ratio, an increase in the absorbance,
due to the sugar auto-condensation and rearrangement, was reported. However, these absorbance
increases were < 1 after 3.5 hours (compared to 1:10, where Abs ranged from 1 to values higher
than 2.5). To monitor the reaction behaviour, the wavelengths corresponding to the typical Maillard
markers were selected, as follows: 280 nm for furosine, 320 nm for the soluble pre-melanoidin
compounds, and 420 nm for the melanoidins (Rada-Mendoza et al., 2002; Wijewickreme et al.,
1997). The results are summarized in a heat map (fig. 19). The conditions able to generate
significant increases of the optical densities at 280, 320 and 420 nm clustered together and were
characterised by a high amount of sugar (1:10 molar ratio peptide/sugar), long incubation time (>
3.5 h) and high temperature (37°C). The sugar concentration (1:10 peptide/sugar ratio) promoted
the highest increase in the selected wavelengths, independently from the peptide presence. For
instance, following the sample N37C or C37C over time, a progressive increase was initially
observed in the absorbance at 280 nm, and then at 320 and 420 nm, from the 3.5 h time point. In
contrast, N37B_7 and C37B_7 reached a comparable absorbance at 280 nm after 7 h. This results
64
confirms that glucosamine generates either Maillard reaction intermediate products (MRP) or
condensation products; thus, less native sugar would be available for the conjugation. Temperature
reduction to 25°C required a longer incubation time (7 h) to show an increase of the absorbance
(which was detectable in N25C_7 and C25C_7). Samples with a low peptide/sugar ratio (1:3 and
particularly 1:1) had lower or no detectable glucosamine degradation/rearrangement products.
Therefore, the lowest peptide/sugar ratio (1:1) and the lowest incubation time (3.5 h) were selected
as the most suitable conditions to carry on the glycosylation reaction.
To confirm the conjugation (both glycation and glycosylation), the peptide was incubated
with and without TGase in the presence of glucosamine, and then analysed using Orbitrap-LC/MS.
The spectrum of peptide plus glucosamine and TGase after incubation is reported in Figures 20.
The MW of reactants are indicated as follows: m/z 180.08 for GlcN and m/z 204.09 for Gly-Gln. To
calculate the glycated form of the peptide, the typical release of H2O in the Maillard reaction was
considered. Hence, a mass shift of +161 Da (i.e. 179-18 Da) from the original MW of the peptide
was predicted. One mole of ammonia was released from TGase-catalysed reactions (Ramos et al.,
2001). Thus, the MW of glycopeptides resulting from glycosylation of peptides with GlcN, in the
presence of TGase, can be predicted based on a mass shift of +162 Da (179 - 17 Da).
In figure 20, products of both glycation and glycosylation are reported: m/z 366.15 is
related to the MW of the glycosylated glycopeptide, whereas m/z 365.16 corresponds to the
glycated glycopeptide. The Orbitrap-mass spectrum shows also a protonated ion at 162.07 m/z,
which is a typical adduct of glucosamine. As showed in figures 20, the intensity of glycoconjugates
produced are low compared to the intensity of the reagents. However, this result does not reflect
the actual concentration of glycopeptides produced. In fact, according to Itoh et al. (2009),
glycopeptides are poorly ionized in mass spectrometry. Moreover, their MS signal can be reduced
or suppressed if a mixture of peptides and glycopeptides is analysed. The final hypothesised
mechanism of the glycosylation and glycation involving GlcN and Gly-Gln is summarised in figure
21.
8.2.2. Characterization of wheat gluten and its hydrolyzates
The first step of the process was the gluten hydrolysis. In this phase, focused on the
optimisation of the process, I preferred to use commercial proteases (Alcalase and Flavourzyme)
instead of Yarrowia lipolytica enzymes which have not been purified yet. The chromatograms
obtained by size exclusion analysis of the hydrolysates confirmed the enzyme activities. In figure
22, it is possible to observe the shift of the peaks from high to low molecular weights after
65
hydrolysis. The estimated degree of hydrolysis (DH) by Alcalase was 4.70 ± 0.2%, whereas the
Flavourzyme DH was 11.85±1.2%.
8.2.3. Chemical evaluation of glycoconjugation of gluten peptides by MALDITOF/TOF-MS
After the glycation/glycosylation process, the MW profiles of the samples were determined
using MALDI-TOF-MS. The analysis of the controls (GAH and GFH) revealed different profiles
when Alcalase or Flavourzyme were used. The Alcalase peptides ranged from 500 to 3000 Da,
whereas those in Flavourzyme were from 300 to 4100 Da. This distribution was expected, because
Alcalase is an endopeptidase that generates a homogeneous distribution of MW, while Flavourzyme
produces a more scattered profile including low and higher MW, due to its mixture of endo- and
exo-peptidases.
To estimate the glycation of the peptides, all the signals present in the spectra have been
checked for their mass before and after the reaction. In particular the MWs of glycopeptides were
calculated adding a mass shift of 161 Da (or any multiple of it, in case of additional sugars attached)
to the original peptide MWs, due to the production of 1 mole of water. Hrynets et al., (2013)
proposed that a potential glycation reaction, with an initial release of water, and a subsequent
ammonia molecule, may occur with an additional mass shift of 144 Da to be add to the peptide
MW. Hence, a mass shift of 162 Da in the MW of the peptides was considered to identify the
glycosylated peptide in the presence of TGase.
The total amount of glycopeptides estimated when the ratio peptide:GlcN was 1:1 (both at
25 and 37°C) was 5 fold higher than the amount of glycopeptides observed at 1:3 ratio (5 vs. 25); in
general, GAT25 (1:3) and GFT25 (1:3) presented a minor number of glycopeptides. This would be
because GlcN tends to auto-condensate resulting in a lower free GlcN amount available for the
glycation/glycosylation process when present at higher concentrations (Zhu et al., 2007). Among
the samples with the lowest (1:1) ratio, at least three different glycopeptides were produced through
glycation, independently from the temperature used (fig. 22 and 23). Only one glycosylated peptide
was estimated in GFT37 (fig. 24). The MW profiles of the glycopeptides were different if their
origin was considered: gluten Alcalase glycopeptides were mainly constituted of 1322 and 1676 Da
glycopeptides, whereas gluten Flavourzyme glycopeptides presented MWs ranging from 1775 to
3112 Da.
66
8.2.4. In vitro bioactivity evaluation of glycopeptides mixtures
8.2.4.1. DPPH Radical Scavenging Activity
The quenching activity of the samples against DPPH was assessed and the EC50 values
estimated are reported in table 8. As shown in table 8, all the samples that underwent glycation
improved their antioxidant activity, because neither GAH nor GFH (peptides not glycated) had a
concentration-dependent activity. Among GA samples, GAC37 was the most active, followed by
GAT25, GAT37 and GAC25. Nevertheless the final EC50 values were not significantly different
amongst them. GFT37 was the most active among GF samples. Anyway all the samples incubated
with TGase (GFT25 and GFT37) had a EC50 lower than the corresponding control with no enzyme
added (GFC37 and GFC25). The reaction mixture with 1:3 peptide/sugar ratio, where glycopetides
had not been generated, was used as control, in order to assess if other Maillard reaction products
could prevent oxidation. None of the tested samples showed any activity similarly to GAC37 and
GFC37, as reported in table 8.
8.2.4.2. Inhibition of Linoleic acid peroxidation
The capability of the samples to inhibit linoleic acid peroxidation was evaluated in
comparison to α-tocopherol, generally used as positive control. The results obtained are reported in
Figures 25 and 26. On the basis of the monitoring of the absorbance over time, GAC25 (1:1) was
the only sample with a low lipid oxidation level (fig. 25). It was observed that during the first three
days of the trial, no differences were detectable between GAH and GAC25. However, the oxidation
curves significantly diverged over time (p < 0.05), reflecting the improved capability of the
conjugated sample to inhibit the linoleic acid peroxidation. All the other GA samples (both 1:1 and
1:3 ratio) did not show any significant antioxidant activity (p < 0.05).
The GF samples (1:1) (fig. 26) not only did not show any antioxidant activity (p < 0.05), but
also one of them, GFC25, resulted as the most significantly pro-oxidative sample. In fact, it reached
the highest absorbance after three days, followed by a rapid decline associated with the
decomposition of (hydro) peroxide.
8.2.4.3. Anti-ACE activity
Samples were tested for their anti-hypertensive activity, measuring the capacity to inhibit the
ACE enzyme (fig. 27). In the samples GAH and GFH (with a concentration of 10 mg/ml) the
enzyme inhibition was 64.1 ± 1.95 % and 50.3 ± 1.00%, respectively. The glycation had negative
effects in almost all the samples, in particular in those corresponding to the GF. In fact, a half
67
reduction in the final activity was measured in all the GF samples except for the GFC37, whereas in
the GAC37, GAT25 and the GAT37 a significantly lower reduction was detected (p < 0.05).
8.2.4.4. Antimicrobial activity
The antimicrobial activity of the samples, before and after glycation/glycosilation, was
tested against two bacterial strains: Escherichia coli and Bacillus subtilis (Gram negative and Gram
positive, respectively). The peptide mixtures before the treatment (GAH and GFH) did not show
any antimicrobial activity. However, despite the absence of activity in the 1:3 samples, the majority
of the 1:1 ratio samples resulted active against E. coli (tab. 9). Specifically, all the conjugated GF
samples inhibited the cell growth at 40 mg/ml, and GFC25 and GFT37 were also bactericidal.
Among the GA samples, GAT37 was both inhibitor and bactericidal (40 mg/ml) for E. coli. None of
the samples (1:1) was active against B. subtilis.
It can be outlined that the peptide mixture includes peptides not glycated and about three
glycopeptides. Therefore the MICs values are overextimated and presumably the MIC really
effective against E. coli are lower than that obtained.
8.2.4.5. Cytotoxicity in human cells
The cytotoxicity of the peptides and glycopeptides (1:1 peptide/sugar ratio) was tested
against human hepatocyte HepG2, using three different concentrations (1 mg/ml, 0.5 mg/ml and
0.1 mg/ml). When comparing GAH vs. GFH, no effects on cell viability were shown on human
carcinoma cells (fig. 28), even at 1 mg/ml (p < 0.05). However, enhanced cytotoxicity was detected
in all the conjugated GF samples, resulting in a significant reduction of the cell viability of
approximately 50% at 1mg/ml (p < 0.05). High cytotoxicity was detected in GFT25 and GFT37 at
0.5 mg/ml. Similarly GAT37 showed a strong activity at 1 mg/ml (p < 0.05), while the citotoxicity
of the other GA samples was not significant.
68
8.3 Tables
Table 6. Proteolytic activity of different strains of Y. lipolytica towards three different
matrices (a, milk proteins; b, gluten; c, gelatin) at two different temperature (27 and 6 °C). The
proteolytic activities on skim milk and gluten agar were evaluated measuring the clear zone formed.
The proteolytic activity on gelatin was estimated measuring the volume of liquefied gelatin. The
values were obtained after three (for 27 °C) or ten (for 6 °C) days of incubation.
(a) skim milk agar
(b) gluten agar
strain
27 °C
27 °C
CLCD
1IILY4A
16B
PO19
Y10
Y16
1IIYL8A
0.5*
3
2.5
0.5
0.5
3.2
3
6 °C
0.5*
2.6
2.3
0.5
0.5
2.3
2.3
0.5*
2.3
2
1.3
0.5
2.3
2
6 °C
0.5*
2.3
2
0.5
0.5
2.5
0.5
(c) gelatin
27 °C
0.5**
1.5
1.8
0.5
1.9
0.8
0.5
6 °C
0.5**
0.8
0.7
0.5
0.5
0.5
0.5
* refers to the clear zone diameter expresses in cm; ** refers to liquefied volume in a glass
tube expresses in cm
69
Table 7. The evolution of some peptides, present in at least two sampling time, are reported.
All the red cells correspond to the time 0, in particular to the native gelatine added with the
supernatant of Y. lipolytica 1IIYL4A; the green ones correspond to the peptides produced after 48 h,
the blue ones correspond to those released at 72 h.
MW+ (Da)
Y0
Y 48
Y 72
Y 96
1143
1449
1606
1748
2078
2224
2766
3298
3371
3535
3766
3839
3921
4678
4842
5059
70
Table 8. DPPH scavenging activity of Alcalase (GAH) and Flavourzyme (GFH)
hydrolysates (control samples not conjugated) and the corresponding conjugated peptides obtained
without enzyme (GAC25, GAC37, GFC25, GFC37) or with enzyme (GT25, GAT37, GFT25,
GFT37). The EC50 values (mg/ml) are reported. All these samples refer to the mixture obtained
incubating peptide/glucosamine in a 1:1 ratio. Since all the 1:3 ratio did not show any activity,
GAC37 1:3 is just reported as an example.
Alcalase
sample
EC50 (mg/ml)
Flavourzyme
R2
sample
EC50 (mg/ml) R
2
GAH
nd *
GFH
nd
GAC37
nd
G FC37
nd
After glycation
GAC25
GAC37
27.45
20.25
0.98
0.99
GFC25
GFC37
32
35.3
0.99
0.97
After +Tgase
GAT25
GAT37
21.82
25.15
0.99
0.99
GFT25
GFT37
22.1
10
0.99
0.64
Before glycation
After glyaction with higer
ammount of sugar ( 1:3)
* not detectable.
71
Table 9. Antimicrobial activity of Alcalase (GAH) and Flavourzyme (GFH) hydrolysates
(control samples not conjugated) and the corresponding conjugated peptides obtained without
enzyme (GAC25, GAC37, GFC25, GFC37) or with enzyme (GT25, GAT37, GFT25, GFT37)
against E. coli. MIC and MBC (both expressed in mg/ml) refer to effect exerted by the mixture
obtained incubating peptide/glucosamine in a 1:1 ratio.
E. coli
sample
MIC
MBC
sample
MIC
MBC
GAH
-
-
GFH
-
-
After glycation
GAC25
GAC37
-
-
GFC25
GFC37
40
40
40
-
After +Tgase
GAT25
GAT37
40
40
GFT25
GFT37
40
40
40
Before glycation
"-" no activity was observed for concentration ≤ 40 mg/ml
72
Y10
1IIYL8A
16B
PO19
Y16
M
CLCD
1IIYL4A
8.4 Figures
250
150
100
75
50
37
Figure 5A. Casein zymography of different Y. lipolytica culture supernatants. Culture
supernatants were recovered by centrifugation of a 4 days culture. The lable of the strains is
Y10
1IIYL8A
16B
PO19
Y16
1IIYL4A
CLCD
M
reported above each lane. M: Molecular weight markers are reported in kDa.
250
150
100
75
50
37
Figure 5B. Gelatin zymography of different Y. lipolytica culture supernatants. Culture
supernatants were recovered by centrifugation of a 4 days culture. The lable of the strains is
reported above each lane. M: Molecular weight markers are reported in kDa.
73
Figure 6. SDS-PAGE electrophoresis of α-casein hydrolyzed by different strains of Y.
lipolytica at 72 h of incubation. C: α-casein alone, M: Molecular weight markers are reported in
kDa.
CLCD 1IIYL4 16B PO19 Y10 Y16 1IIYL8A
C
M
250
150
100
75
50
37
25
20
15
10
Figure 7. SDS-PAGE electrophoresis of β-casein hydrolyzed by different strains of Y.
lipolytica at 72 h of incubation. C: β-casein alone, M: molecular weight markers were marked in
kDa.
74
M
C
1
2
3
4
5
6
7 C
250
150
100
75
50
37
25
20
15
10
Figure 8. SDS-PAGE electrophoresis of meat protein extracts hydrolyzed by different
strains of Y. lipolytica at 72 h of incubation. C: meat protein extracts alone, M: molecular weight
markers were marked in kDa. 1, CLCD; 2, 1IIYL4A; 3 16B; 4, PO19; 5, Y10; 6, Y16; 7, 1IIYL8A.
Figure 9.
SDS-PAGE electrophoresis of gelatin hydrolyzed by different strains of Y.
lipolytica at 72 h of incubation. C: gelatin alone, M: molecular weight markers were marked in kDa.
75
Absorbance at 215
A
B
C
Elution volume (mL)
76
Absorbance at 215
D
E
F
Elution volume (mL)
77
Absorbance at 215
G
H
Elution volume (mL)
Figure 10. Size exclusion chromatograms of gelatin incubated for 72 h with the supernatant
of different strains of Y.lipolytica: Gelatin alone (A), 1IIYL4A (B), 16B (C), CLCD (D), 1IIYL8A
(E), Y10 (F), PO19 (G), Y16 (H).
78
Absorbance at 215
Elution volume (mL)
Figure 11. Overlay of two size exclusion chromatograms representing the gelatin at the
starting point (time 0) and after hydrolysis with the supernatant of 1IIYL4A (time 72 h).
79
t0
t48
t72
t96
M
200
116.25
97.4
Absorbance at 215
45
Elution volume (mL)
Figure 12. Gelatin hydrolysed by Y. lipolytica 1IIYL4A collected over time (time 0, 48, 72
and 96 h). (A) SDS-PAGE electrophoretogram of the samples after different incubation times. M:
molecular weight markers are marked in kDa. (B) Overlay of size exclusion chromatograms of the
gelatin hydrolysed by Y. lipolytica 1IIYL4A collected over time time 0 (blue and brown), 48
(green), 72 (red), 96 h (light blue). In particular a closeup of the MW < 25 kDa (estimated on the
basis of a standard mixture previously injected) is shown.
80
Figure 13. Relative abundance of the three main area referred to the figure 8B. In particular,
range between 90-115 mL (1), 115-125 mL (2) and 125-135 mL (3) for each chromatogram were
considered.
50 0 0
A
Intensity (arbitrary units)
30 0 0
10 0 0
5 0 00
B
3 0 00
1 0 00
10 00
2 00 0
3 0 00
40 0 0
50 0 0
6 0 00
7 00 0
80 0 0
9 00 0
m /z
Figure 14. MALDI-TOF spectra of ultrafiltered (cut-off < 10 kDa) gelatin hydrolysed by Y.
lipolytica 1IIYL4A. The spectrum A refers to time 0, the spectrum B refers to time 96 h.
81
DPPH activity
120
% DPPH
100
80
60
DPPH activity
40
20
0
ascorbi
acid
0
48
72
96
sample
Figure 15. DPPH radical scavenging activity of gelatin incubated with the supernatant of Y.
lipolyica 1IIYL4A and collected over time (0, 48, 72 and 96 h). Ascorbic acid (0.1 mg/ml) was used
as reference standard. Values are expressed as means ± SD in triplicate experiments.
Linoleic acid peroxidation
2
Absorbamce (500 nm)
1.8
1.6
Y0
1.4
Y48
1.2
Y72
1
Y96
0.8
BHT
0.6
α-Tocopherol
0.4
Control
0.2
0
0
1
2
3
4
5
6
7
tim e (day)
Figure 16. Antioxidant activity, in a linoleic acid oxidation model system, played by gelatin
hydrolysates by Y. lipolyica 1IIYL4A protease collected over time (Y0, 48, 72, 96 h). BHT and αtocopherol were used as reference standards. Control was prepared with linoleic acid and water
instead of the sample. Values are expressed as means ± SD in triplicate experiments.
82
120
cell survival (%)
100
80
1
60
0.5
0.1
40
20
0
control
0
48
72
96
gelatin
samples
Figure 17. The cytotoxic effect of the gelatin hydrolysates, obtained at different times,
against Hepatocellular carcinoma (HepG2). Cells were treated with different concentrations of the
gelatin hydrolyzed (1, 0.5 ad 0.1 mg/ml). Values are expressed as means ± SD in quadruplicate
experiments. Control refers to untreated cells; gelatin refers to cells treated just with gelatin.
83
wavelength (nm)
0.5
0
0
wavelenght (nm)
4
4
3.5
3.5
3
3
2.5
2
N25 0
1.5
N25 3.5
1
N25 7
0.5
0.5
0
0
490
0.5
490
PN25 7
470
1
470
PN25 3.5
450
1.5
450
PN25 0
430
2
430
3
410
2.5
390
4
3.5
410
4
3.5
390
wavel enght (nm)
490
470
450
430
410
390
370
1
N25 7
370
1.5
N25 3.5
370
N25 0
350
3
350
2.5
330
wavelenght (nm)
350
4
330
3.5
330
490
470
450
430
410
390
370
350
330
310
0
290
0
310
0.5
310
0.5
310
2
270
PN25 7
290
PN25 3.5
Absorbace (Abs)
PN25 0
Absorbance (Abs)
490
470
450
430
410
390
370
350
330
1
270
490
470
450
430
410
390
370
350
310
3
290
0
330
290
2
270
0.5
0
310
270
4
3.5
290
0.5
290
Absorbance (Abs)
1.5
Absorbance (Abs)
270
Absorbace (Abs)
2.5
Absorbace (Abs)
490
470
450
430
410
390
370
350
330
310
290
270
Absorbance (Abs)
4
3.5
270
490
470
450
430
410
390
370
350
330
310
290
270
Absorbance (Abs)
A
2.5
3
1.5
2
PN37 0
1
PN37 3.5
PN37 7
wavel eght (nm)
3.5
4
2.5
3
2
N37 0
1.5
N37 3.5
1
N37 7
wavel enght (nm)
B
2.5
3
2
PN37 0
1.5
PN37 3.5
1
PN37 7
wavelenght (nm)
2.5
1.5
2
N37 0
1
N37 3.5
N37 7
wavelength (nm)
84
4
3.5
wavelength (nm)
wavelength (nm)
4
4
3.5
1
490
470
450
430
410
390
350
370
330
0
310
0
290
0.5
270
N37 7
1
0.5
wavelength (nm)
N37 3.5
1.5
470
490
N25 7
410
430
450
1.5
N37 0
2
350
370
390
N25 3.5
290
310
330
N25 0
2
3
2.5
270
3
2.5
Absorbance (Abs)
3.5
Absorbance (Abs)
490
270
290
490
430
450
470
0
390
410
0.5
0
330
350
370
0.5
310
PN37 7
1
450
470
1
PN37 3.5
1.5
430
PN25 7
410
1.5
PN37 0
2
370
390
PN25 3.5
330
350
PN25 0
2
3
2.5
310
2.5
Abs orbance (Abs)
4
3.5
3
270
290
Absorbance (Abs)
C
wavelenght (nm)
Figure 18. UV-spectra of samples absorbance (Abs) of the Glucosamine (N) and peptide
plus glucosamine (PN) were monitored during incubation at two different temperatures (25 or
37°C), for different incubation times (0, 3.5 and 7h). A, B and C represents the 1:10, 1:3 and 1:1
molar ratio of peptide/GlcN or GlcN alone, respectively. Values are expressed as
triplicate
experiments.
85
280 nm
320 nm
420 nm
Figure 19. Hierarchal clustering of peptide–sugar (or just sugar) UV–Vis absorbance data
for Gly-Gln and glucosamine (GlcN). The vertical axis represent individual wavelengths (280, 320
and 420 nm). The horizontal axis represents individual glycation treatments. The colours in each
cell indicate the absorbance of a particular sample relative to the mean level from all samples for
the specific wavelength. The colour scale extends from bright green (maximum absorbance) to
bright red (minimum absorbance). The samples GlcN (N), GlcN plus peptide (C) were followed
during the incubation, under different temperatures (25 or 37°C), different peptide:GlcN ratios (1:1
(A) , 1:3 (B) or 1:10 (C)) and incubation times (0, 3.5 and 7h).
86
Figure 20. Orbitrap LC-MS spectrum of products resulting from TGase mediated
Glycosylation of peptides with GlcN.
Figure 21. Proposed mechanism of reaction at 37 ºC for 3.5 h: glycosylation and glycation
87
Absorbance at 215
Elution volume (mL)
Figure 22. Size exclusion chromatogram of gluten (solid line) and the hydrolysates
subjected to Alcalase (GA, dots) and Flavourzyme (GF, dotted line) hydrolysis, respectively.
88
2
GAH
1
Intensity (arbitrary units)
0
2
GAC25
1
0
2
GAT25
1
1
*
3
*
4
*
0
0
1000
2000
3000
m/z
89
2
GAH
1
2
4
3
Intensity (arbitrary units)
0
2
GAC37
1
1
2
*
*
3
*
0
2
GAT37
1
1
2
*
*
4
*
0
0
1000
2000
3000
m/z
Figure 23. MALDI-TOF-MS spectra of gluten hydrolysated by Alcalase (GAH), gluten
hydrolysate glycated at 25 or 37 °C (GAC25 and GAC37), gluten hydrolysate enzymatically
glycosylated at 25 and 37 °C (GAT25 and GAT 37). Glycated peptides are marked with an asterisk
(*), while glycosylated peptides are marked with a filled circle (•).
90
GFH
1
12
34
5
Intensity (arbitrary units)
0
2
GFC25
1
1
*
2
* 4
*
5
*
0
2
GFT25
1
3
*
24
* *
0
0
1000
2000
3000
4000
m/z
91
2
GFH
2
1
3
1
4
5
Intensity (arbitrary units)
0
2
GFC37
1
3
*
4
*
5
*
0
2
GFT37
1
2
1
*
0
0
1000
°
5
*
2000
3000
4000
m/z
Figure 24. MALDI-TOF-MS spectra of gluten hydrolysated by Flavourzyme (GFH), gluten
hydrolysate glycated at 25 or 37 °C (GFC25 and GFC37), gluten hydrolysate enzymatically
glycosylated at 25 and 37 °C (GFT25 and GFT 37). Glycated peptides are marked with an asterisk
(*), while glycosylated peptides are marked with a filled circle (•).
92
Absorbance (500 nm)
4
3.5
H20+LA
3
NO LA
2.5
Alpha-Toco
GAH 1:1
2
1.5
GAC25 1:1
1
GAC37 1:1
0.5
GAT25 1:1
0
GAT37 1:1
0
1
2
3
4
5
6
7
time (day)
Figure 25. Antioxidant activities of Alcalase gluten hydrolysate and its
glycacted/glycosylated samples in a linoleic acid oxidation system. The antioxidant activity was
measured on the basis of the ability to keep constant the absorbance at 500 nm over time. αtocopherol was used as reference positive control. Linoleic acid plus water was used as negative
control. Values are expressed as means ± SD of at least triplicate experiments.
Absorbance (500 nm)
4
H20+LA
3.5
NO LA
3
2mM Alpha-Toco
2.5
GFH 1:1
2
GFC25 1:1
1.5
GFC37 1:1
1
GFT25 1:1
0.5
GFT37 1:1
0
0
1
2
3
4
5
6
7
time (day)
Figure 26. Antioxidant activities of Flavourzyme gluten hydrolysate and its
glycacted/glycosylated samples in a linoleic acid oxidation system. The antioxidant activity was
measured on the basis of the ability to keep constant the absorbance at 500 nm over time. αtocopherol was used as reference positive control. Linoleic acid plus water was used as negative
control. Values are expressed as means ± SD of at least triplicate experiments.
93
Figure 27. Anti-ACE activity of gluten samples hydrolysed with Alcalase (GA) and
Flavourzyme (GF). The samples were incubated with GlcN (C) and glucosamine + TGase (T) at 25
and 37°C. Values are expressed as means ± SD of at least triplicate experiments.
Gluten peptides cytotoxicity
140
130
120
110
100
cell survival (%)
90
80
70
cell survival
60
50
40
30
20
10
GFT37 0.1
GFT37 1
GFT37 0.5
GFT25 0.1
GFT25 1
GFT25 0.5
GFC37 0.1
GFC37 1
GFC37 0.5
GFC25 0.1
GFC25 0.5
GFH 0.1
GFC25 1
GFH 1
GFH 0.5
GAT37 0.1
GAT37 1
GAT37 0.5
GAT25 0.1
GAT25 1
GAT25 0.5
GAC37 0.1
GAC37 1
GAC37 0.5
GAC25 0.1
GAC25 0.5
GAH 0.1
GAC25 1
GAH 1
GAH 0.5
control
0
sample
Figure 28. The cytotoxic effect of of Alcalase (GAH) and Flavourzyme (GFH) hydrolysates
(control samples not conjugated) and the corresponding conjugated peptides obtained without
enzyme (GAC25, GAC37, GFC25, GFC37) or with enzyme (GT25, GAT37, GFT25, GFT37)
against Hepatocellular carcinoma (HepG2). Cells were treated with different concentrations (1, 0.5,
0.1 mg/ml) of the mixture obtained incubating peptide/glucosamine in a 1:1 ratio. Values are
expressed as means ± SD in quadruplicate experiments. Control refers to cells not treated.
94
CHAPTER 9.
DISCUSSION AND CONCLUSIONS
95
The extracellular proteases are widely studied in Y. lipolytica, in particular genes encoding
for an alkaline protease (AEP) and an acidic protease (AXP) have been cloned and sequenced
(Hernández-Montañez et al., 2007). However, since Y. lipolytica is an attractive host for the
production of foreigner proteins, its proteolytic system must be further studied further because
unspecific proteases may degrade the heterologous proteins or in many case protolytic processing is
necessary for the expression of activities such as aminopeptidase, or dipeptidil-aminopeptidase by
the host cells. Young et al. (1996) cloned and sequenced the gene and potential regulatory region of
the extrcellular acid protease of a strain of Y. lipolytica and showed that the transcription of both the
genes for acid and alkaline is regulated by the pH of the medium. Investigation relative to the strain
diversity, the effect of the temperature or the occurence of dimeric or complex forms of the
enzymes are lacking.
A novel finding resulting from my experimental results is the detection of a new high MW
protease in strains of Y. lipolytica. On the basis of the results some of the tested strains released
prevalently or only a protease having MW higher than 200 kDa active both on casein and gelatin,
while others released only protease, active on gelatin and casein, having a MW of 37 kDa. Both the
proteins are released at the end of the exponential phase. Proteases having MW higher than 150 kDa
have never been reported in Y. lipolytica and other yeasts, except for an extracellular protease
having a MW of approximately 200 kDa has been described in M. pulcherrima (Reid et al., 2012).
Reid et al., (2012) reported a protein, or a protein complex, having a protease activity characterized
by 180-200 kDa MW. Moreover Chen et al., (1997) characterized, in Cryiptococcus spp., an
extracellular proteolytic activity in vitro as a serine proteinase and found it associated with proteins
of approximately 200 kDa. Goodley and Hamilton (1993) isolated a 200 kDa proteinase from the
filtrate of C. neoformans.
It has been reported that the initial size of intracellular and extracellular enzymes can change
over time. In fact the MW of a protease can change because of the removal of a molecular fragment
that obstruct the catalytic size. This has been documented as a common activation step of some
mammalian proteases (Nagase et al., 1990). In order to avoid dangerous degradation of intracellular
proteins, proteases are often synthesised with an additional segment that obstructs the catalytic site.
This latent form (called either pro-enzyme or zymogen) displays up to 60% of the activity of the
fully active molecule (Woessner, 1995). Outside the cells the fragment is cleaved off and full
activity is achieved. This hypothesis does not fit the experimental results. In fact, generally the MW
of the removed fragment did not significantly change the MW of the active protease (Hoffman and
Decho, 2000). Moreover an activity of 37 kDa was not observed in the supernatant of strains
showing proteolytic activity at 200 kDa, and vice versa.
96
Microorganisms evoluted in stressful environments, like marine ones, produce an array of
enzymes having different MWs. In particular the marine bacterium Pseudomonas atlantica releases
high MW (103 kDa) proteases during the exponential phase. These enzymes are degraded over time
and new low MW enzymes (34, 31, 75, 69 kDa) are formed (Hoffman and Decho, 2000).
Also this temporal evolution of released enzymes does not fit my data because both the
enzymes at 200 and 37 kDa are alternatively produced in samples processed the same time, stored
and run under the same conditions and using the same reagents. The possibility of artefacts can be
considered negligible.
In Y. lipolytica the aminoacid sequence, deduced by Young et al., (1996) from the
nucleotide sequence encoding for AXP, consists of 353 aminoacids and a MW of 37 kDa.
According to Young et al. (1996), the mature extracellular enzyme is produce from a propeptide by
cleavage between Phenylalanine and Alanine. Likewise Y. lipolytica releases an alkaline
extracellular protease from a 55 kDa precursor produced after cleavage of a 15 aminocid signal
peptides. This precursor is then processed by a diamonopeptidase to generate a 52 kDa proprotein
which is subsequently cleaved to give the mature 32 kDa secreted protein. Generally a
propolypeptide is inactive and can be converted to mature active polypeptide by catalytic or
autocatalytic cleavage of the propeptide.
If, as suggested by Young et al. (1996), Y. lipolytica is characterized by just one specific
gene for an acidic protease of a MW of 37 kDa, it can be hypothesised that the band having
proteolytic activity and having a MW higher than 200 kDa corresponds to a complex of the acid
proteases. However, further studies would be necessary to demonstrate why a such a complex of
proteins is active. Moreover all the proprotein or the obstructed proteases generally present a
significantly lower MW. In any case a proteolyticic activity of a protein or protein aggregate having
MW higher than 200 kDa has not been reported in Y. lipolytica and in genetically related yeast yet
(Rao et al., 1998). Moreover our preliminary results showed that the two hypothetical acid
proteases (with a MW of 37 and higher than 200 kDa), associated to different strains, generated
different profiles from caseins, meat extract and gelatin. It could be wondered why a protease coded
by the same gene from the same organism produces different profiles on the same protein. Y.
lipolytica strains used in my thesis presented different RAPD profiles and different physiological
characteristics as well as different ecological origins. Therefore it can be postulated that, due to
subsequent environment-induced mutations, the protease acquires a different substrate specificities
compared to the starting one (Craik et al., 1985). It must be considered that the genome of Y.
lipolytica has been sequenced only in the strain Y. lipolytica CLIB122 provided by the Genolevures
97
Consortium. Therefore it can hypothesised that other strains of the same species harbour different
genes and protein/enzymes not described yet.
Concerning the biotechnological properties, most of the strains used in my work were
endowed with proteolytic activity on gelatin, skim milk and gluten at T 6°C. The intracellular and
extracellular proteolytic systems of Y. lipolytica have been object of several investigation and
exploited for different technological uses (González-López et al., 2002). Different strains of Y.
lipolytica used as cheese and dried sausage ripening adjuncts (Guerzoni et al., 1998, Vannini et al.,
2001, Lanciotti et al., 2005; De Wit et al., 2005; Patrignani et al., 2007) gave rise to different
proteolysis patterns as well as strain dependent sensory and texture properties.
In any case the physiological attribute that mosly distinguishes Y. lipolytica or its ecological
biotypes from other yeasts species having biotechnological potential such as Debaryomyches
hensenii, Geotrichum spp., Candida spp., (Vakhlu and Kour 2006; Bankar et al., 2009) is the ability
to produce cold active enzymes. The possible application of cold active enzymes in the food
industries are numerous (Gerday et al., 2000). Cold proteases offer potential economic benefits
particularly through substantial energy saving in large scale processes that would not require the
expensive heating of reactors. The typical example is the industrial peeling of leather by proteases
which can be done with cold enzymes at temperature under 10°C instead of 37°C. In general the
cold active enzyme have a more flexible structure and undergo the conformational changes
necessary for catalysis with a lower energy demand. More recently enzymes from psychrotropic
species have become interesting for industrial applications partially because of the ongoing efforts
to decrease energy consumption during industrial processes. When enzymes from psychrotropic
species are used it becomes feasible to develop for instant laundry applications that can be
performed at lower temperatures.
Concerning the valorisation of meat by-products, the enzymes currently used are
characterized by mesophylic or thermophylic characteristics. The quest for valuable proteases with
distinct specificity for industrial applications is always a continuous challenge. Proteolytic enzymes
from plant and fungal sources have received special attention for being active over a wide range of
temperatures and pH. In general the commercial enzymes currently used present an optimal activity
near 50-60°C.
The strains of Y. lipolytica taken into consideration are characterized by cold attitude,
versatile proteases and diversity as far as the peptides profiles obtained on all the protein sources
considered.
One of the strains taken into consideration has been selected, due to its intense proteolytic
activity in the range from 4 to 28°C, to evaluate its performance in the production of peptides
98
having antioxidant, antimicrobial and cytotoxic, anti-ACE properties. The selected protein was
gelatin due to its large availability as industrial by-product. The whole peptide mixture obtained
during proteolysis over time were characterized by an increasing antioxidant activity. The peptides
playing this effect are formed during the last 72 and 96 h. The latter samples were characterized by
the presence of peptides having prevalently a MW lower than 3 kDa.
The selection of strains having good biotechnological potential is a fundamental tool to
identify natural active compound. However, wild strains and reactions performed with their
enzymes generally give rise to a low level of active molecules. In order to increase the bioactivities
of the peptides produced by the enzymes released by Y. lipolytica, I developed a chemico/enzymatic
approach that has only been suggested in literature, but not explored yet. I would like to highlight
that this process can be exploited not only for the peptides produced by proteases from Y. lipolytica,
but also for peptides obtained from other protein hydrolysates of food by-products, thus increasing
their added-value.
The formation of a bound between a sugar and an amino acid occurs through enzymatic
(glycosylation) and non-enzymatic (glycation) reactions. Although various techniques are available
to prepare synthetic glycoproteins or glycopeptides (Caer et al., 1990; Christopher et al., 1980;
Colas et al., 1993; Hattori et al., 1996; Kitabatake et al., 1985), the glyco-conjugates of proteins and
polysaccharides obtained by the Maillard reaction have received much attention only in recent years
(Liu et al., 2012). In fact the role of Maillard reaction on the improvement of functional properties
of food proteins has been only recently proposed (Oliver et al., 2006). The main disadvantage of the
Maillard reaction is the use of high temperature or prolonged heat treatments, to obtain the
glycation (Liu et al., 2012). In my work, I proposed a novel procedure to conjugate sugars and
peptides, exploiting the particular behaviour of the amino-sugar GlcN, and the use of temperature
not exceeding 37 °C. In the model system development the conditions and the validation of the
glycation between GlcN and Gly-Gln at 25-37°C were shown. Further, the same model system was
used to demonstrate the feasibility of using GlcN as a substrate for TGase, and for the production of
glycosylated peptides. TGase is a universal enzyme capable of modifying proteins through the
incorporation of amines; it is used in the food industry to improve the texture of vegetable and
animal proteins and to create innovative food products with different properties (Seguro et al.,
1996).
Based on the results of the UV spectra, the experimental conditions optimal to reduce, to a
feasible extent the formation of GlcN side-products generated by its high reactivity, were
determined. Lanciotti et al. (1999) reported that Maillard reactions (> 90°C) between sugars and
aminoacids produced antimicrobial and antioxidant compounds. For this reason, even though lower
99
absorbance curves were detected in 1:1 ratio samples, 1:3 ratio was subsequently considered to
assess the possible interference with the final activities. In general, conjugation leads to the
formation of compounds with different properties, compared with the native structure. For example,
it has been reported that the presence of sugars bounded to the peptides enhances the antimicrobial
effect of the native peptide by approximately 100 times (Otvos et al., 2002).
Once proved the conjugation in a model system, I tried to transfer the process into a more
complex mixture of peptides. Wheat gluten, a by-product of the wheat industry, is a heterogeneous
mixture of peptides with more than 60 different molecular weights ranging from 30 to 90 kDa. In
particular, the main abundant forms are represented by the polymeric glutenin and the monomeric
gliadin (Wang J. et al., 2007). Glutenin can also rearrange forming disulfide bonds and create
structures with MW of 50 to 2000 kDa (Bietz and Wall, 1972). In my work, gluten was employed
after proteolysis with Alcalase and Flavourzyme.
The estimated degree of hydrolysis (DH) was not elevated for Alcalase. In fact higher DH
for wheat gluten has been previously reported (Kong et al., 2007). On the contrary, the
Flavourzyme generated a more common DH already described for protein hydrolysis. Even if
differences in DH may depend on the selected method for its assessment, the low solubility of
gluten at neutral pH could have also played an important role (Takeda et al., 2001).
The hydrosylated samples obtained were then employed for the conjugation. All the samples
were mainly glycated rather than glycosylated, and at least three glycopeptides were present in all
samples. The excess of GlcN in the 1:3 ratio samples did not interfere with the tested activities. In
these samples very low glycopeptides were detected and no interferences, due to possible GlcN
side-products, were induced. Hence, it can be suggested that the reactivity of glucosamine at higher
concentrations can form auto-condensed and rearrangement products that were not suitable for the
glycation/glycosylation process. On the other hand, low amounts of sugar (1:1) generated more
glycopeptides, and showed enhanced modulation of the final bio-activities.
The assessed bioactivities were: DPPH scavenging activity, inhibition of linoleic acid
peroxidation, anti-ACE, antimicrobial and cytotoxicity. As already mentioned, DPPH measures the
free radical scavenging capacity of a sample, based on a combination of hydrogen atom and
electron transfer reactions (Huang et al., 2005). With this assay all the conjugated samples showed
an improvement of the final activity compared to the control ones (GAH and GFH). In particular
GFT37, the sample having a glycopeptide obtained through glycosylation, was the most active. I
would like to outline that in all the samples the contribution of the classical Maillard products can
be regarded as negligible. These activities may depend on the sugar moiety of glycopeptides, which
could be both electron donors and electron acceptors (van Boekel, 2001). The 1:3 peptide/sugar
100
ratio was also tested to determine whether the increased sugar content in the solution could
influence the reducing power. However, no activity was detected in any sample when comparing
GAH and GFH (data not shown) because of the lower content or absence of glycation of the
peptides. It was also confirmed that under these reaction conditions, the possibile formation of some
Maillard Reaction Products (MRPs) did not influence the antioxidant activity.
The linoleic acid assay measures the capability of a specific sample to inhibit or reduce the lipid
oxidation. However, the antioxidant reactions that occur in this case are different from those in the
DPPH analysis. Zhang et al. (2008) reported that hydrophobic amino acids represent a key element
to protect against lipid derived-radicals, due to their ability to interact with lipids (Ajibola et al.,
2011). In contrast, the presence of the sugar moiety within a glycopeptide (glycoprotein) could lead
to an increase on the overall hydrophilicity (Wang and Ismail, 2012). This could explain the prooxidative activity found in GFC25, the highest glycated sample as compared to the others. On the
other hand, the antioxidant activity of GAC25 may depend on the relatively lower MW (1095 Da),
with respect to all the other glycated peptides. Li et al. (2008) reported that protein hydrolysates
prepared from corn gluten meal had increased effective antioxidant capacity when the MWs of
peptides were between 500 - 1500 Da. Hence, glycopeptides with low MW may also be more
effective in the linoleic acid assay.
The ability of a peptide to inhibit ACE was also tested. This activity is strongly linked to the
peptide amino acid composition and their primary sequence. Anti-ACE gluten peptides have been
already reported (Kim et al., 2004); in particular, the main activity of the hydrolyzates can be
attributed to the low molecular weight peptides. In fact, after fractionation of the samples, the
inhibitory activity increased by fourf-old. Kinoshita et al. (1993) reported that high molecular
weight peptides fraction reduced the blood pressure in hypertensive rats. The conjugation of gluten
peptides had shown negative effects in almost all the samples. Je et al. (2004) described that the
presence of hydrophobic amino acids in the sequence enhances the ACE-inhibitory ability of the
peptides. As a result, the perturbation of the final hydrophobicity of the peptide (once glycation has
occurred) can have a negative effect in all activities depending on the proportion of
hydrophobic/hydrophilic region inside the molecule.
The antimicrobial activity of these samples demonstrated that the glycation was essential for
the improvement of the antimicrobial activity. In particular, this effect is more relevant in GF than
GA. Although the antimicrobial activity of glycopeptides has been reported, (Kahne et al., 2005;
Bullet and Stocklin, 2005) the mechanisms of action have not been completely described.
Glycopeptides active specifically against Gram-negative bacteria have been reported; for example,
Drosocin, a well-studied glycopeptide isolated from Drosophila melanogaster, is a small proline-
101
rich peptide (around 2-4 kDa) with a disaccharide (galactose and N-acetylgalactosamine) (Bulet and
Stocklin, 2005). Gluten, on the other hand, is mainly constituted by glutamine/glutamic acid
(around 40%), and proline (around 17-20%) (Wieser, 2007). The sugar moiety enhances the
antimicrobial effect of the native peptide by approximately 100 times in Drosocin (Otvos et al.,
2002). Thus, the peptide sequence and the presence of a hydrophilic part in a specific position of the
peptide are suggested to be important for the antimicrobial action. Kragol et al. (2001) reported that
these glycopeptides may inhibit the protein folding through a mechanism involving the bacterial
chaperonine/heat shock proteins GroEL and DnaK, once entered into the cells.
The bioactivity of the samples was also assessed in relation to human tumor cells (HepG2).
The enhanced cytotoxicity detected in the conjugated peptides implies that the formation of
glycopeptides through a glycation process can be responsible for the main production of molecules
with higher cytotoxicity. The low TGase-dependent effect could depend on the low/absent
glycosylation reported or on the low affinity of the peptides for TGase. The cytotoxicity of
glycopeptides has been previously described; although the role of sugar remains unknown, the
carbohydrate moiety is likely to be involved in cell recognition, cellular uptake and DNA binding
(La Ferla et al., 2011). Indeed, Brahim et al. (2008) demonstrated that the deglycosylated
Bleomycin-A2 (a cytotoxic glycopeptide) was less toxic than its native form, due to the inability to
induce ROS formation. Therefore, the two main elements influencing the studied properties were:
1) the type of starting peptides (which are affected by the enzyme used for the hydrolysis and the
MWs of the resulting peptides); 2) the reduction of the final hydrophobicity of the peptides.
Although it was demonstrated the formation of glycopeptides through TGase both in the
model system and in the gluten system, these results pointed out that the enzymatic process was not
able to create sufficient glycosylated gluten peptides with improved activities. Indeed, studies
regarding TGase and gluten have been reported. Dekking et al. (2008) demonstrated that
transglutaminase can deamidate gluten peptides by introducing negative charges. This process was
responsible for the enhanced immunogenicity of gluten. Similarly, Elli et al. (2012) reported the use
of TGase as a tool to incorporate lysine, the first limiting amino acid in wheat products, into gluten.
Cross-linking with glutamine using TGase promoted lysine availability. However, the incorporation
of molecules to "protect" glutamines as a way to reduce the toxicity of gluten has not been exploited
yet. The attachment of GluN can represent a potent tool in this direction. In this way, the
conjugation method could be highly effective to modify peptides. The use of GluN and TGase can
be employed to create innovative functional peptides or mixtures of peptides, using a mild
temperature process. Additional studies have to be performed to improve the final yield of
glycation/glycosylation process and to test the stability of these new compounds over time.
102
I can summarize the results of my thesis taking into consideration the steps included in the
original project.
Selection of 7 different
strains and hydrolysis of
different matrix
Protease
characterization
Gelatin
Gluten
Different proteolytic profiles
Meat extract
proteins
Casein
Detection of proteases with 2
MWs
Enzymes able to work
at 6 °C
Selection of one strain and
hydrolysis of one matrix
Gelatin
Bioactivities
Production of peptide
mixtures having antioxidant
and antimicrobial activities
Improvement of
bioactivities of peptides
through
glycation/glycosilation
Development of a new
method to
glycate/glycosylate peptides
Bioactivities
Improved activities of the
peptide mixtures after
glycation
103
My research can not be regarded as exhaustive. In fact several points need to be further
elucidated and developed: i.e. the nature of the high MW protease release by Y. lipolytica, the
identification of the most active peptides in relation to their specific bioactivities, and the
optimisation of the glycation reaction in relation to the food by-products and their hydrolysis when
proteases released by Y. lipolytica are employed. However, I tried to integrate the selection and
development
of
strains
endowed
with
valuable
biotechnological
potential
with
a
chemico/technological approach aimed at enabling the enhancement of the bioactivities of the
resulting compounds.
104
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ACKNOWLEDGMENTS
Another step of my life has arrived at the end. Such a long way since I arrived at the Food
Microbiology Lab but it is quite satisfactory to see the outcome of my adventure. I would like to
acknowledge myself because I reached this result but I have to admit that everything was possible
just because of the assistance of the following people.
I am particularly grateful to my supervisor, Prof. Maria Elisabetta Guerzoni, who dared to
trust in me and whose incredible creativity and support gave me feedback and inspirations during
my entire PhD career. Her time and continuous guidance are hugely appreciated.
I am also thankful to my other supervisor, Dott.ssa Lucia Vannini, who always believed in
me and gave me complete freedom as well as her support to conduct my research.
My deep appreciation is for my Canadian supervisor Prof. Mirko Betti, who gave me the
chance to live the most professional experience of my life. Without his support and his trust in me I
could not have been able to further my investigation. I will always keep with me all his suggestions.
A sincere thanks to Dr. Maurice Ndagijimana for all the scientific discussions we had and
his constant and respectful presence, constantly next to me with precious suggestions.
Prof. Fausto Gardini and Prof.ssa Rosalba Lanciotti, my gratitude is also extended to you.
I would like to thank all my friends and colleagues of the Food Microbiology Lab at the
University of Bologna: Diana Serrazanetti, Chiara Montanari, Giulia Tabanelli, Pamela Vernocchi,
Francesca Patrignani, Giorgia Gozzi, Lucia Camprini, Lorenzo Siroli, Rodrigo Troncoso, Lucilla
Dei Più and Danielle Taneyo. We lived, worked and suffered together and we always supported
each other.
A personal thanks to "my" technician Luciana Perillo, that followed my "first steps" in the
microbiology lab and she has always supported me during these years.
I would also like to show my appreciation to Danka Bukvicki and Amit Kumar Tyagi
because they became some of my best friends rather than colleagues, and they have continuously
provided me with their consideration as well as with moral support through the distance.
Since I spent almost one year in Canada, I have to offer my gratitude for the support and
friendship received from all my friends and colleagues at the University of Alberta, in Prof. Betti's
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Lab: Pui Khoon (Kimi) Hong, Zied Khiari, Yulija Hrynets, Lihui (Mavis) Do, Henan Wang; and
those in the Microbiology lab: Alma Fernanda Sanchez Maldonado, Petya Koleva and Xiaoji
(Christine) Liu. A thanks goes also to all the other people that I met in Edmonton and that made my
permanence there as familiar as possible in some way: Lynda and Alan Jones, Sam Cheng, Soledad
Urrutia, Maricruz Ormeño Urrutia, Sahar Navidghasemizad and also all the "Chinese basketball
players".
My old friends Andrea Bonora, Luca Vanini, Luca Brochetto, Caterina Villani who have
always encouraged and granted me with their friendship.
A special and affectionate thanks to Emma Hernandez Sanabria, because from the beginning
she has been patient and supportive with me, giving me strength and love.
I would like to express my sincere admiration to my grandparents, but most importantly to
my parents, who have been incredibly encouraging along my Ph.D. They all have undoubtedly
made sacrifices and it is due to them that I have found the motivation and resolution to continue
straight and committed.
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