Probiyotik Ürünler Kime Ne Zaman Verilmeli, Probiyotik
Gıdaların Güvenirliliği
Prof. Dr. Şebnem Harsa, PhD
Gıda Müh. ve Biyoteknoloji Bölümü
İzmir Yüksek Teknoloji Enstitüsü
4. Ulusal Bağırsak Mikrobiyotası
ve Probiyotik Kongresi
Antalya, 2017
Related Projects by our Research Group in IZTECH
Effect of Trace Elements on the Production of Bakers’ Yeast, industrial project
(Pakmaya), 2000-2002 (project director). Molecular Characterization of Turkish Microflora, İYTE AF 2001 Fen 20, 2001-
2006 (researcher).
Isolation and Characterization of Lactic Acid Bacteria from Traditional
Dairy Products; Investigations on Starter Cultures and their Lactase
Activities, 2004 İYTE 20, 2004-2007 (project leader).
Investigations on the Starter Properties of Lactobacillus and
Formulation of Industrial Cheese Cultures, 2005İYTE36, 2005-2007
(project leader).
The Production of Cheese and Yogurt Starter Cultures and Lactase
Enzyme for the Dairy Industry: Traditional and Modern Solutions
Against Lactose Intolerance, 2005 K 120570, DPT-YUUP Project, 2005-
2008, Collaborated with 13 university and 4 industrial company,
(project leader).
Production, Purification and Immobilization of Lactase Enzymes from
Dairy Products, TOVAG-EU-COST 928 (104 O 270), 2005-2009 (project
leader).
Microencapsulation of Lactobacillus acidophilus in double emulsion system, BAP 2011 IYTE12, 2011- 2013 (project leader).
Development of Novel Yoghurt Starter Cultures for Özsüt, (Project No: BTSB 020217), İnoshe Co. Inc. Biotech Products, Food R&D Consultancy (project leader), 15/04/2013 – 15/09/2013.
European Network for Gastrointestinal Health Research, European Science Foundation Research Networking Programme (ENGIHR - 08-RNP-018), 10/2010 – 10/2014. Budget 403,401 Euro (project Leader from Turkey).
Production of Industrial Starter Cultures, (Project No: BTSB 020216),
İnoshe Co. Inc. Biotech Products, Food R&D Consultancy (project
leader), 01/12/2012 – 2016.
Development of Gluten-free bread, IZTECH – The University of Milan.
2015-2017.
Lactic Acid bacteria having ACE peptides, GABA, bile salt hydrolase,
lactase, protease activities..
Ongoing Projects
Development of Novel Yoghurt Starter Cultures, (Project No: BTSB 020214), İnoshe
Co. Inc. Biotech Products, Food R&D Consultancy (project leader), 01/01/2013 –
present.
Development of Functional Food Ingredients and Products for the Solution of
Malnutrition, (Project No: BTSB 03309), İnoshe Co. Inc. Biotech Products, Food R&D
Consultancy (project leader), 01/09/2015 – present.
Development of Oral Probiotic Lozenges, Industrial Project, İZTECH, 2017-
Development and Metagenomic Analysis of Physicobiotics, IZTECH – The Univ. Of
Reading, 2017 -
PATENTLER
"KATKISIZ, ENDÜSTRİYEL YOĞURT ÜRETİMİ İÇİN ÖZGÜN STARTER KÜLTÜRLERİN ELDE EDİLMESİ YÖNTEMİ" başlıklı patent başvurusu Enstitümüzce alınmış, Patent Siciline 30/12/2016 gün, 13.03:35 saat ve 2016/20493 sayı ile kayıt edilmiştir.
"PROBİYOTİK AYRAN KÜLTÜRÜ VE PROBİYOTİK AYRAN ÜRETİM METODU" başlıklı patent başvurusu Kurumumuzca alınmış, Patent Siciline 31/12/2016 gün, 14.52:11 saat ve 2016/20467 sayı ile kayıt edilmiştir.
Today foods are not intended to only satisfy hunger and to provide necessary nutrients for humans, but also to prevent nutrition-related diseases and improve consumers’ health (Siro et al., 2008; Gortzi et al., 2015).
ADA , the American Dietetic Association
12
• ADA, founded in 1917 defined a functional food as:
one that provides a ‘beneficial effect on health when consumed as part of a varied diet on a regular basis at effective levels’.
ADA classification:
13
• This organization has classified functional foods into four groups:
• conventional foods,
• modified foods,
• medical foods, and
• foods for special dietary use,
and has called for more research into their potential health benefits.
Different types of functional foods
14
Fortified products
Increasing the content of existing nutrients
Enriched products
Adding new nutrients or components not normally found in a particular food
Altered products
Replace existing components with beneficial components
Enhanced commodities
Changes in the raw commodities that have altered nutrient composition (Spence 2006)
Fortified foods
15
• The simplest types of functional foods are those products that are fortified with additional nutrients.
• grain products with folic acid
• fruit juices that are fortified with additional vitamin C
• This approach of fortification has proven to be an effective and economical way to improve nutrient quality and provide benefits to consumers.
Enriched foods
16
• To put additional components or components not normally found in great quantity in a particular food.
• orange juice that has added calcium
• the inclusion in margarines of plant sterol esters
Probiotics & Prebiotics
17
• Probiotics are live microbial food ingredients that have a beneficial effect on human health; they are traditionally found in fermented dairy products and fermented vegetables.
• Prebiotics are typically fermentable dietary fibres that provide a gastrointestinal environment in which beneficial bacteria can thrive.
Altered products
18
• Using different ingredients, food products can be developed whereby some potentially harmful or undesirable constituents could be replaced by more beneficial components, ideally without affecting product quality
• use of high fibre fat replacers, produced from grain products
Enhanced commodities
19
• Plant breeders can develop amazing varieties of products that have potentially important benefits to consumers.
• high lysine corn,
• fruits and vegetables with enhanced content of vitamins,
• Overproduction of phytonutrients in a variety of fruits and vegetables including the insertion of some of those components into food plants that do not normally produce those dietary components
• golden rice or carotenoid containing potatoes.
• Increasing consumer demand and interest in obtaining additional benefits from food has stimulated functional foods to emerge on the market, with USA, Europe, and Japan being
the dominant markets. Currently represent the largest segment of the functional foods market in Europe, Japan and Australia.
• Improving overall human health and well being & intestinal health etc.
• FFI are becoming one of the largest and fastest growing sectors because of
• Rapid advances in science and technology,
• Increasing healthcare costs,
• Changes in food laws affecting label and product claims,
• An aging population, and
• Rising interest in attaining wellness through diet
• Targeting gut health constitute
The main consumer motive for purchasing FFI:
• the growing desire to use foods either to help prevent chronic illnesses
such as cardiovascular disease, Alzheimer’s disease and osteoporosis,
• to optimize health, e.g. by increasing energy,
• boosting the immune system, generation of wellbeing.
Probiotics have been traditionally found in fermented foods (yogurt, cured meat, and vegetables)
And incorporated into new food vehicles such as cheese, ice cream, and chocolate in addition to yogurt drinks. Viable probiotic strains are supplied in the market either as fermented food commodities or in lyophilised form, both as supplements and as pharmaceutical preparations.
Comprising 65% of the functional food world market, probiotics represent the major and still growing segment of this huge market, estimated to exceed a total volume of US $ 75billion.
24
Value of Functional Food Market
• One of the fastest growing food sectors, with a compound annual growth rate of 8.6% in the 10 years to 2012 (Euromonitor, 2010).
• The emergence of a new market segment called ‘Health and Wellness’ reached a global value of US$625 billion in 2012.
• The value of U.S. functional food sales at $7.1 billion in 2009. Leatherhead Food International, UK, in its 2011 report, said global sales of functional foods in 2010 reached $24 billion.
• Predicted theU.S. Market could reach $9.1 billion by 2014.
BIOFUNCTIONAL FOODS
• Ideally, functional food refers to an existing traditional food product that is intended to be consumed as part of a normal diet and has a demonstrated added physiological benefit (Siro et al., 2008).
• Therefore, it could not be in the form of pill or capsule.
The concept of biofunctional foods is generally used when this desirable biological, medical, or physiological effect is exerted by microorganisms (Gobbetti et al., 2010).
The health benefits of these microorganisms can be exerted either directly through the interactions of ingested live microorganisms with the host (probiotic effect),
or indirectly by ingestion of the microbial metabolites synthesized during fermentation (bioactive effect)
(Stanton et al., 2005; Gobbetti et al., 2010; Joshi, 2015).
Probiotic Foods • Lactic acid bacteria (LAB) have been used to ferment foods for at least
4000 years..
• Although the probiotic concept has expanded more recently, we have been unconsciously ingesting beneficial microbes with traditional fermented foods since ancient times.
• Fermented foods are the main carriers to deliver probiotics Among them, dairy products (in particular fermented milks and yogurt) are by far the most efficient and widely used (Figure 1) (Giraffa, 2012).
FIGURE 1 | Beneficial effects resulting from the consumption of biofunctional fermented dairy foods. Lactic acid bacteria participating in milk fermentation in situ release and naturally enrich the fermented dairy product with a broad range of bioactive metabolites. Subsequent ingestion of this product can exert important health-promoting activities on the consumer, such as anti-hypertensive, and anti-diabetic, immune-modulatory, anti-cholesterolemic or microbiome modulation.
Cheese
• Cheese is a dairy product which has a good potential for the incorporation of probiotic cultures due to its specific chemical and physical characteristics compared to fermented milks (higher pH value and lower titrable acidity, higher buffering capacity, greater fat content, higher nutrient availability, lower oxygen content, and denser texture).
• These conditions facilitate survivability of probiotic strains and tolerance to the low pH conditions encountered during gastric transit (Karimi et al., 2011).
• Utilization of probiotics has been optimized in several cheese varieties such as Cheddar, Gouda, Camembert, Cottage type, white-brined, and traditional cheeses, among others (Araujo et al., 2012; Giraffa, 2012; Martinovic et al., 2016; Oh et al., 2016).
• Kefir is another milk-fermented product that has health promoting bacteria (Prado et al., 2015).
• Other non-fermented dairy foods such as low-fat ice cream, chocolate mousse, coconut flan, or infant milk formula have also been supplemented with probiotic strains (Davidson et al., 2000; Aragon-Alegro et al., 2007; Correa et al., 2008; Baglatzi et al., 2016).
Health benefits promoted by probiotics
• supplied via dairy products are immunomodulatory effects (L. casei CRL431),
• reduction of serum cholesterol level (L. reuteri NCIMB 30242) and
• antihypertensive effects (L. plantarum TENSIATM) (EFSA, 2011; Jones et al., 2012; Aragon et al., 2014).
Probiotics are defined as ‘live micro-organisms, which when consumed in adequate amounts confer a health benefit on the host’ (FAO/WHO, 2001).
• However, regarding probiotic foods, some considerations are of paramount importance:
• Firstly, effective levels of the live probiotic in the corresponding food matrix at the time of ingestion are required. In this regard, the minimum effective dose which affects the intestinal environment and provides beneficial effects on human health is considered to be 106–109 live microbial cells per day, although this depends on the particular strain and foodstuff (Williams, 2010; Karimi et al., 2012; Watson and Preedy, 2015).
LIVE CELLS???
• Since probiotics show beneficial health effects on the host once consumed, another precondition for a bacterial strain to be called probiotic is the ability to survive and colonize (at least transiently) the gastrointestinal tract (GIT), which is in part helped by the buffering capacity of the food matrix. In some particular cases, bacterial viability may not be strictly required.
• As an example, inactivated and dead L. rhamnosus GG cells can maintain immunological and health-promoting effects (Ghadimi et al., 2008; Lopez et al.,2008).
Bioactive Compounds Derived from Microbes • Microorganisms involved in dairy fermentations can produce
biologically active molecules and enzymes, giving the final food product an additional health value.
• Unlike the probiotic concept (the bacteria must be ingested alive and produce the beneficial metabolite in the body),
• the biofunctional concept is generally used when the healthy metabolite emerge in the food product itself during the fermentation process as a consequence of the bacterial metabolic activity.
The bacteria can act as a microbial factory to enrich foodstuff • Consequently,, for which bacterial viability through the GIT or during
the product storage is not absolutely required (Farnworth and Champagne, 2015).
• The main bioactive compounds produced by LAB during dairy fermentation are vitamins, gamma-aminobutyric acid, bioactive peptides, bacteriocins, enzymes, conjugated linoleic acid, and exopolysaccharides (Table 1).
Vitamin K
• Vitamin K is an important promoter of bone and cardiovascular health. It has been associated with the inhibition of arterial calcification and stiffening, and the reduction of vascular risk.
• This vitamin is nearly non-existent in junk food, with little being consumed even in a healthy Western diet (Maresz, 2015). Its deficiency has been implicated in several clinical ailments such as intracranial hemorrhage in newborn infants and possible bone fracture resulting from osteoporosis (LeBlanc et al., 2011).
Vitamin K occurs in two forms:
• 1) Phylloquinone (vitamin K1), which is present in green plants,
• 2) Menaquinone (vitamin K2), which is produced by some intestinal bacteria (LeBlanc et al., 2011).
• Menaquinone can be biosynthesized by some LAB species (mainly Lactococcus lactis) commonly used in industrial fermentation of cheese, buttermilk, sour cream, cottage cheese, and kefir (Walther et al., 2013).
• Other LAB have been screened for the ability to produce menaquinone; these included strains from the genera Lactococcus, Bifidobacterium, Leuconostoc, and Streptococcus (Morishita et al., 1999).
Bacteriocin phenomena
• Bacteriocin-producer adjunct cultures are being used in Cheddar and semihard cheeses (Mills et al., 2011).
• Studies for the direct impact of dairy foods containing bacteriocins on human health and microbiome are still limited.
Enzymes
• Lactic acid bacteria associated to dairy fermentations possess enzymes which can be produced in situ during fermentation of dairy foods and have bioactive potential on the consumer. Examples are hydrolytic enzymes that may exert potential synergistic effects on digestion and alleviate symptoms of intestinal malabsorption (Patel et al., 2013).
• A well-known example is the Beta-galactosidase (LACTASE) activity, which can achieve lactose degradation and thereby improve health and reduce symptoms of lactose intolerant consumers.
• Yogurt and other conventional starter cultures and probiotic bacteria in fermented and unfermented milk products improve lactose digestion and alleviate symptoms of intolerance in lactose malabsorbers. These beneficial effects are due to microbial beta-galactosidase (de Vrese et al., 2001).
Conjugated Linoleic Acid
• Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid (PUFA) that can be biosynthesized by LAB and bifidobacteria through bioconversion of linoleic acid (LA; cis-9,cis-12 C18:2). The two isomers that have been shown to have bioactive potential are cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12).
• The health-promoting properties of CLA include anticarcinogenic, antiatherogenic, anti-inflammatory, and antidiabetic activity, as well as the ability to reduce body fat (Sosa-Castañeda et al., 2015). Although it is a native component of milk, the amount consumed in foods is far from that required in order to obtain desired beneficial effects.
• Thus, increasing the CLA content in dairy foods through milk fermentation with specific LAB offers a promising alternative. An effective way to increase CLA uptake in humans is to increase CLA levels in dairy products by using strains with high production potential (Lee et al., 2007). A number of food-grade LAB and bifidobacteria were reported to produce CLA in milk products (Sosa-Castañeda et al., 2015; Yang et al., 2015), as is the case of Lactococcus lactis LMG, L. rhamnosus C14, L. casei CRL431, L. acidophilus Lac1, L. plantarum-2, B. bifidum CRL1399 and B. animalis Bb12 (Van Nieuwenhove et al., 2007a; Florence et al., 2009).
• Some of these strains were also used as adjunct cultures for the manufacture of high CLA-content buffalo cheese (Van Nieuwenhove et al., 2007b). The CLA-producing starter culture of Lactococcus lactis CI4b enhanced levels of total CLA in Cheddar cheese (Mohan et al., 2013). Similarly, L. bulgaricus LB430 and S. thermophilus TA040 are suitable for production of CLA-enriched yogurt (Florence et al., 2009).
• In addition, it has been shown that specific microorganisms such as L. plantarum PL60 or B. breve NCIMB702258, are able to produce CLA following dietary administration in animal models (Wall et al., 2009, 2012) and following the administration as a freeze-dried product in humans (Lee and Lee, 2009). Thus, intestinal CLA production by bacteria may contribute to enhance CLA supply in addition to the CLA provided by the producing strain in fermented milks during the manufacture (Teran et al., 2015).
Exopolysaccharides • Exopolysaccharides (EPS) are complex extracellular carbohydrate
polymers that can be produced by some LAB in situ during dairy fermentations. Some of them promote selective growth of bifidobacteria, thus playing a role on the host microbiota and immune system (Fernandez et al., 2015; Salazar et al., 2016). In this regard, EPS derived from yogurt fermented with L. delbrueckii ssp. bulgaricus OLL1073R-1 exerted immunestimulatory effects in mice (Makino et al., 2016). Yogurt, Swiss-type, and Cheddar cheeses represent suitable food matrices for the delivery of the hypocholesterolemic EPS-producer strain L. mucosae DPC 6426 (Ryan et al., 2015).
REGULATORY ASPECTS • At present, the status of probiotic-based products is full of
ambiguities because various regulatory agencies in different countries are defining and categorizing probiotics differently.
• Despite the emerging interest of consumers toward probiotics and functional foods, in Europe the regulatory framework is still not harmonized and no health claim for probiotics alone (except yogurt starters) has been approved.
• Paradoxically, probiotics or bioactive bacteria have been introduced into the market as dietary supplements or natural health products (capsules, tablets, and powders) (Arora and Baldi, 2015).
• Japan was the very first global jurisdiction for implementing a regulatory system for functional foods and nutraceuticals in 1991, and is currently acting as global market leader where probiotics are available as both foods and drugs. The government has designated Foods for Specific Health Uses (FOSHU), which classifies health claims into different subcategories (gastrointestinal health, cholesterol moderation, hypertension moderation, lipid metabolism moderation, sugar absorption moderation, mineral absorption, and bone and tooth health).
• In China, State Food and Drug Administration (SFDA) has regulated all health foods including functional foods and nutraceuticals, and a well-developed market for functional foods is established (Arora and Baldi, 2015). Currently USA is regulating probiotics as a variety of products as per their intended usage and regulatory bodies are Dietary Supplement Health and Education Act (DSHEA) and Food and Drug administration (FDA). Dietary supplements are considered as ‘foods’ and are regulated by DSHEA and do not need FDA approval before being marketed.
• However, probiotics and dietary supplements containing a new dietary ingredient without a marketing history are regulated by FDA. In conclusion, a harmonized categorization of probiotics and functional foods may help to regulate these products whenever solid clinical documentation is available to support any health effects and health messages in human subjects. The appropriate level of evidence for determining a health benefit for probiotics should always be put ahead of commercial and labeling industrial interests.
Update on the Regulatory Landscape of Probiotics – Preview of Dr Elinor McCartney’s Presentation at IPC2016, Posted 1 June, 2016 • Regulation of probiotics and prebiotics in the EU – what’s new?
• The big news this year is undoubtedly the increased pressure to reduce antibiotic use in both humans and animals. This is driving food, feed and pharmaceutical players to seek creative opportunities with probiotics. Creativity and regulation are not easy partners, so it is important to recognise and address legal obstacles and practical considerations at an early stage of project planning for new probiotic development or renewal of old registration dossiers.
• Most live microbial strains used in EU foods and food supplements do not require a premarket safety assessment, due to traditional and safe use in fermented foods. However, EU regulators look to EFSA (European Food Safety Authority) for guidance on strain safety, particularly for food supplements, since these must be notified prior to marketing in most Member States. All live microorganisms used in animal nutrition must pass EFSA on strain safety prior to marketing and face a stringent safety assessment:
• Strain identity – the use of modern molecular techniques to identify probiotic strains frequently results in taxonomy updates. A change in the taxonomy of a strain can result in regulatory challenges, especially if the new strain name is not listed as QPS (Qualified Presumption of Safety), updated annually by EFSA, or is no longer recognised as having a traditional use in fermented foods.
• QPS – EFSA lists absence of toxin and virulence factors and antimicrobial resistance (AMR) as key qualifiers for strain safety. Current EFSA guidance on Bacillus safety illustrates the difficulty of deciding when a toxin is a toxin. The latest EFSA update on AMR was published in 2012, and sets cut-off values based on published data. There are also technical challenges in this area, for example the difficulty of setting appropriate cut-off values for “new” taxons with relatively few members.
• Full genome – Exotic probiotic strains may be classed as “novel”, and could face evaluation by EFSA. For such strains, EFSA will require the full strain genome.
• Strain identity – the use of modern molecular techniques to identify probiotic strains frequently results in taxonomy updates. A change in the taxonomy of a strain can result in regulatory challenges, especially if the new strain name is not listed as QPS (Qualified Presumption of Safety), updated annually by EFSA, or is no longer recognised as having a traditional use in fermented foods.
• What’s new on strain efficacy? • Probiotics have still not succeeded in achieving a claim under the nutrition
and health regulation, other than the generic claim for yoghurt bacteria, production of lactase and aid in digesting lactose in subjects with intolerance. It is interesting that the only approved probiotic claim in the EU is generic, since EFSA rejected most other generic probiotic claims on the grounds of lack of definitive strain identity.
• EFSA also insisted that probiotic efficacy is strain-specific. On the other hand there are around 40 probiotic strains approved for use in animal feeds, with several strains under EFSA evaluation. Demonstrating probiotic efficacy in animals to EFSA’s satisfaction is challenging, but possible. Perhaps there is room for more coherence across EFSA food and feed panels to allow best practices from both areas – human and animal?
Health claims by the European Food Safety Authority (EFSA) and recent recommendations regarding designs for probiotic trials with the goal of substantiating health claims.
• EC Regulation No. 1924/2006 was established to generate approval of health claims made for food, including an evaluation of dossiers by EFSA. Potential probiotic claims have not met with a favorable reaction from EFSA. Therefore, a panel of independent academic scientists with proven track records in probiotic research made the following recommendations for the design of :
Probiotic studies intended to substantiate health claims in EU • Discriminate between a trial to test a hypothesis compared with a trial to
substantiate a health benefit claim.
• Use a dose available in commercial products.
• Ensure that trials are appropriately powered with an adequate sample size, based on the expected magnitude of effect. Otherwise, statistically significant conclusions cannot be obtained and meaningful conclusions drawn. Therefore, >1 recruitment site may be needed.
• Ensure that trials are of appropriate duration to determine endpoints that are tested
• Avoid evaluation of multiple parameters unless they are hypothesis driven.
• Volunteers should reflect the general population (e.g., age, sex, BMI) • Characterize the probiotic product, including demonstration of viability at
beginning, middle, and end of study, as well as detailed biological and genetic description of the probiotic strain(s).
• Ensure that strain(s) used in studies is (are) the same as those in the intended product.
• Use multiple sample times. • Take and store additional physiological samples for future tests. • Preferably use validated endpoints with clinical support, e.g., eczema,
immune aspects, lipids, microbiota, transit, gut assessments, metabolites, geno/cytotoxicity, other biomarkers.
• Include biomarker(s) to help mechanisms/causality.
CHALLENGES IN INDUSTRY
• A goal of the dairy industry is to develop novel dairy products with increased nutritional and/or health promoting properties. Food-grade bacteria have the potential to fortify fermented dairy food products with bioactive metabolites by a natural process, thereby reducing the need for fortification with costly chemically synthesized supplements.
• Nowadays, a number of commercial sources have available synthetic formulations of bioactive substances for their use as a dietary supplement. The use of health-supporting bacteria for naturally enriching dairy foods with bioactives could be a suitable alternative to food fortification with chemical formulations.
The starter cultures
• The starter cultures must be carefully selected, since the ability of microbial cultures to produce bioactive metabolites is generally a strain dependent trait and varies considerably among strains within the same species. The yield of bioactive synthesis and the concentration of such compound in dairy products is another critical strain-dependent factor. In this regard, the dose of bioactives ingested with the corresponding food product should remain over the minimum required to meet the human requirements and/or have the claimed therapeutic level on the consumer, according to existing clinical recommendations and studies.
• An open question when using co-cultures or strain combinations is their interaction in terms of
• nutrient availability,
• bacterial growth,
• as well as the bioactive production yield.
In some cases, metabolites (i.e., vitamins etc.) produced by one of the strains could be consumed by the other strains, thus decreasing the final content in food.
• Generally, the biosynthetic pathways are genetically encoded. In this regard, the increasing availability of bacterial genome sequences over the last decade has provided a major contribution to the knowledge about microbial production of bioactive molecules.
• However, the presence of the genes required for the biosynthesis of a particular biomolecule should not be assumed as synonym of metabolite production.
• Typical exceptions to the correlation genotype-phenotype occur when the genes are not active or when the metabolite is intracellularly biosynthesized and a release system is lacking. This is indeed one of the major bottlenecks during biosynthesis of some vitamins that needs to be overcome through the use of alternative strategies such as autolytic mutants and metabolic engineering (Basavanna and Prapulla, 2013).
• Consideration should also be given by manufacturers to the optimum conditions for bioactive compound biosynthesis by LAB during technological processes.
The content and activity of a bioactive compound in the dairy fermented foodstuffs is the result of the
type of food matrix,
the individual bacterial strain properties as well as
the processing conditions and
storage time.
• In this regard, it should be noted that the high bioactive biosynthetic rates observed in culture media might not always be extrapolated to dairy products.
• Therefore, factors such as optimal temperature for microbial growth and viability, food composition or bioactive stability and shelf-life in the final foodstuff are of paramount importance to reach the maximum concentration and activity in the final product.
• Overall, the current review updates knowledge about LAB, bifidobacteria and propionibacteria with potential to enrich dairy food products with health-promoting bio-metabolites.
• Promising applications at commercial level emerge; however, adequate selection of strains is vital to increase the concentration and bioavailability of such biomolecules in fermented foods. The use of LAB and bifidobacteria able to synthesize bioactive components in fermented foods could help to provide these compounds in foods, this being in compliance with current regulatory rules.
Scientific challenges for future developments of probiotics and prebiotics
FCT 2015 - S.HARSA
FCT 2015 - S.HARSA
1) Very common mechanisms (shared by most probiotics)
• Resistance to colonization.
• Production of short chain fatty acids and acidification ofthe medium.
• Regulation of gastrointestinal transit.Normalization of microbiota.Increased enterocyte regeneration.
• Competitive exclusion of pathogens.
2) Frequent mechanisms common to some species
• Vitamin synthesis.
• Direct antagonism of other bacteria.
• Reinforcement of intestinal barrier.
• Bile salt metabolism.Enzymatic activities.
• Neutralization of carcinogens.
3) Rare mechanisms specific to different strains
• Neurological effects.
• Immunological effects.
• Endocrine effects.
• Production of bioactive substances.
LWT - Food Science and Technology
Volume 85, Part A, November 2017, Pages 151-157
Microbial diversity of traditional kefir grains and their role on kefir aroma
Author links open overlay panelEnesDertliaAhmet HilmiÇonb a
Department of Food Engineering, Faculty of Engineering, Bayburt University, Bayburt, Turkey b
Department of Food Engineering, Faculty of Engineering, Ondokuz Mayıs University, Samsun, Turkey
Received 1 June 2017, Revised 7 July 2017, Accepted 11 July 2017, Available online 14 July 2017.
HIGHLIGHTS
•A pyrosequencing approach was applied for the identification of bacterial and
fungal microflora of four Turkish kefir grains.
•
Lactobacillus kefiranofaciens was the dominant kefir associated bacterial strain in
kefir grains.
•
Yeast strains dominated the fungal microflora in which Dipodascaceae family was
dominant.
•
Richness in microflora resulted in a diverse volatile compounds in kefir samples
produced with these grains.
Areas of knowledge involved in the development of probiotics and prebiotics.
Multidisciplinary research projects cover a range of topics:
The development of efficacious probiotic foods and understanding probiotic
mechanisms at a molecular level;
Developing technologies to ensure delivery of stable products; and
demonstrating safety and efficacy of specific probiotics in defined treatment
targets
An enhanced understanding of the human intestinal microbiota’s role in
health and disease,
New approaches and products to tackle a variety of intestinal problems.
Novel research is in high demand for the development of novel functional food & ingredients and their influences on gastrointestinal health. Research topics in IZTECH include,
• Developing starter cultures, novel probiotics, prebiotics, synbiotics for gut health;
• Lactase enzyme production by LAB for Lactose Intolerance
• Sourdough LAB bacteria for Celiac Disease and Gluten Intolerance
• New probiotic strains, in-vitro studies for the efficacy of strains
• The stability - viability by designing new food manufacturing biotechnologies such as micro-encapsulation for probiotics and enzymes
• Prebiotics from agricultural and dairy wastes
• Application such as yoghurt, ayran, cheese and novel functional foods
KEFİR
• 3 farklı parti numarasından, farklı marka kefirlerden 3’ şer adet kefir örneği alınarak Türk Gıda Kodeksi’ne göre;
• Toplam canlı mikroorganizma ; Kok, Basil, Maya, Küf ve Fekal Koliform olarak analiz yapıldı.
• Ekteki tabloda ortalama sonuçlar ayrıntılı olarak verilmiştir, aşağıda analiz sonuçlarının kısa bilgilendirmesi yer almaktadır. Yapılan çalışmalar sonucu ve TÜRK GIDA KODEKSİ FERMENTE SÜT ÜRÜNLERİ TEBLİĞİ (TEBLİĞ NO: 2009/25)’da belirtildiği üzere, adı geçen kefirin yeterli miktarda yararlı spesifik mikroorganizma içerdiği sonucu bulgulanmıştır. Ancak, küf ile fekal koliform da bulgulanmıştır.
• LAB, KOK: • Ornek1: 73 x 107
• Ornek 2: 58 x 107
• Ornek 3: 36x 108
• LAB, BASİL: • Ornek1: 1x 107
• Ornek 2: 10 x 107
• Ornek 3: 18x 107
• Toplam MAYA:
• Ornek1: 2 x 107
• Ornek 2: 2 x 107
• Ornek 3: 4x 107
•
• KÜF:
• Ornek1: 5 x 105
• Ornek 2: 3 x 105
• Ornek 3: 0
•
• Fekal Koliform
• Ornek1: 1
• Ornek 2: 2
• Ornek 3: 0
•
Psychobiotics
Department of Food & Nutritional Sciences
University of Reading
Date of Registration: 16st of March 2017
Date report completed: 16st of September 2017
Investigations of the Impacts of the Gut Microbiota Composition and the Diet Intervention on
Attention-Deficit-Hyperactivity-Disorder (ADHD)
Six-Month Assessment Report
Buket Sagbasan
Project Supervisors: Dr Gemma Walton, Professor Claire Williams and Professor Dr Sebnem Harsa
PROJECT OBJECTIVES:
The aim of this study is to investigate the mechanisms impact probiotics and prebiotics on Attention-
Deficit-Hyperactivity-Disorder (ADHD) through their interaction with the intestinal microbiota and the
brain axis. In this regard, ADHD will be researched in the manner of a cognitive disorder in psychological
aspects and the potential treatments of ADHD with specific probiotic species and prebiotics.
*(Dinan et al., 2013) Figure 1. A symbolic image of the impacts of chronic stress and depression on brain-gut axis activity. The bi-directional connection allows signals from the brain corticolimbic structures to adjust gastrointestinal function. The key regulators of this function is the HPA axis and immune system.
such, during this PhD
The impact of pre and probiotics on the microbiota of children with ADHD will be studied in
vitro
Tests for cognition and ADHD screening will be used to assess symptoms of a pilot group of
volunteers before and after a treatment regime.
Metagenomic and metabonomic screening tools will be used to monitor the microbiota and
its end products and how these change after intervention. These changes will be mapped to
changes in cognitive function
Geleneksel Fermente Ürünlerimizden İzole Edilen Laktik Asit Bakterileri ile; Angiotensin-I enzimi (ACE)-inhibitörü peptidleri ve gamma-aminobütirik asit (GABA) içeren; Biyofonksiyonel bir Fermente Peyniraltı Suyu İçeceği Geliştirilmesi
Proje Özeti
Hipertansiyon, kardiyovasküler hastalıkların (damar sertliği, inme ve miyokard infarktüsü) ve daha ileriki aşamada böbrek hastalıklarının gelişmesi yönündeki başlıca risk faktörlerinden biri olarak görülmektedir. Ülkemizde toplam nüfusta görülme sıklığı %30,3 (THBHD, 2012) olan hipertansiyon önlenebilir ve tedavi edilebilir bir hastalıktır. Doğru yaşam biçimi ve diyetle birlikte, bazı fonksiyonel bileşiklerin ilavesi ile zenginleştirilmiş fermente ürünlerin tüketilmesi de hipertansiyon sıklığının azalmasında ve önlenmesinde önemli bir yardımcı olabilir. Laktik asit bakterileriyle fermentasyon sırasında oluşan biyojenik bileşikler başta antihipertansif etki olmak üzere sağlık üzerine birçok olumlu etkisi olduğu çalışmalarla belirlenmiştir. Sunulan proje ile peynir üretimi yan çıktısı olan peyniraltı suyunun (PAS) fermentasyonu ile biyojenik bileşiklerden olan Anjiyotensin-I Dönüştürücü Enzimi (ACE) inhibe eden peptitler ve nörotransmitter olarak bilinen, hayvanlarda ve insanlarda antihipertansif etki de dahil olmak üzere çeşitli fizyolojik fonksiyonlara sahip olduğu belirlenmiş Gamma-aminobütirik asit (GABA) içeriğince zengin yeni bir fermente içecek geliştirilmesi planlanmaktadır. Böylelikle ülkemizde her yıl ciddi tonajlarda üretilen, atık niteliği taşıyan ve doğaya ciddi zararları bulunan peyniraltı suyuna katma değer kazandırılacaktır. Fermantasyon sırasında kullanılacak kültürler İzmir Yüksek Teknoloji Enstitüsü Gıda Mühendisliği Bölümü Moleküler Gıda Mikrobiyolojisi Laboratuvarı’nın kültür koleksiyonunda bulunan yöresel fermente süt ürünlerinden izole edilmiş laktik asit bakterilerinden GABA üretim kabiliyetine ve ACE inhibitörü aktivitesine sahip olduğu belirlenen kültürlerden seçilerek kullanılacaktır. Peyniraltı suyu geliştirilecek süt bazlı aşı kültürü ile fermente edilecektir. GABA içeriği ve ACE inhibitorü aktivitesi aşı kültüründe ve peynirlaltı suyu bazlı fermente içecekde analiz edilecektir. Bunun yanı
FCT 2015 - S.HARSA
Lactic Acid Bacteria (LAB)
The lactic acid bacteria are a functionally related group of organisms known primarily for their bioprocessing roles in food and beverages.
Commercial probiotics are mostly members of the Lactobacillus genus, which have been used for centuries to create fermented food products.
Selected members of the LAB have been implicated in a number of probiotic roles that impact general health and well-being.
LAB as Starter Cultures
Starter cultures are of great industrial significance in that they play a vital role in the manufacturing, flavor, and texture development of fermented dairy foods.
Additional interest in starter bacteria has been generated because of the data accumulating on the potential health benefits of these organisms.
Much research has been published on the health benefits associated with ingesting cultured dairy foods and probiotics, particularly their role in modulating immune function.
Lactic Fermentations
The use of lactic cultures is a strong tradition for yoghurt and cheese manufacturing.
They can be isolated from raw milk and artisanal dairy products, complicated characterizations of purified bacteria and technological properties should be determined before using as starter cultures in dairy industry.
Lactic Culture Products
Increased interest in the production of L(+) lactic acid is which a potential feedstock for poly-L(+) lactic acid, has long been used in the food, chemical, textile, pharmaceutical and other industries.
The worldwide production is 80,000 tons/year and 90% of it is produced by lactic acid bacterial fermentation.
C
COOH
CH3
H HO
L(+)
LAB were isolated from Turkish artisanal yogurt and cheese. They were characterized using biochemical and molecular techniques.
LAB as Starter Culture
Source: O. Erkus, 2007,MsC thesis
Scanning electron microscobic image of S. thermophilius cTY25
Scanning electron microscobic image of Lb. delb. ssp. bulgaricus bTY30
Gram staining
Catalase activity
Gas production from glucose
Growth at 15-45 °C
Growth at 4-6,5% NaCl
Carbohydrate fermentation
Biochemical Identification
Genomic DNA Isolation
Characterization by 16S-ITS rDNA region (Bulut et al, 2005)
16S rRNA Sequencing
Molecular Characterization
113 cocci 21 bacilli were isolated from Turkish artisanal cheese (from Cappadocia region).
54 Lactococcus lactis ssp lactis 59 Enterococcus (30 E. faecium 8 E. faecalis, 3 E. avium, 2 E.
durans, 16 E. Sp.) 3 Lactobacillus paracasei paracasei 3 Lactobacillus casei 15 Lactobacillus sp.
Cheese Starter Cultures
66 Streptecoccus thermophilus & 71 Lactobacillus delbrueckii ssp bulgaricus were isolated from Turkish artisanal yogurt (from Toros region).
The strains were evaluated for their technological properties, appropriate strains were used in the industry for yogurt production.
Yogurt Starter Cultures
Technological Properties
LAB were screened for the production ability of lactic acid,
Exopolysaccharides (EPS) and
Aroma compounds mainly acetaldehyde to contribute on appearance, texture, and aroma.
S. thermophilus Isolates
4
4,5
5
5,5
6
6,5
7
1 2 3 4 5 6 7
Time(h)
pH
in
Milk M
ed
ium
97-1 95-2
95-1 94a
90b 79
50 52
66a 66b
74 77a
77b 78
TY8 TY15
TY21 TY24
TY47 TY55
TY31 TY53
TY72 TY75
TY79 TY82
TY27 TY45
TY70 71
TY17 TY25
TY26 TY29
TY32 TY38
TY41 TY44
TY63-2 TY65
TY71 60
62 65
85 94
97-2 TY9
TY10
Acetaldehyde production of L.delbrueckii spp.bulgaricus
isolates
0
5
10
15
20
25
30
35
1
L.delbrueckii spp. bulgaricus isolates
Aceta
ldehyde form
atio
n
(mg/l)
16 22 22b
24 25 26
30 34 33
44 48 53
54 57 77
79 30b 33b
TY5b TY9b TY14a
TY14b TY16 TY24
TY30 TY77a TY77b
TY80 TY90 TY43
For L. delbrueckii ssp. bulgaricus isolates the concentration of acetaldehyde synthesis in yoghurt samples ranged from 10.4-31.5 mg/ L.
For S. thermophilus isolates, acetaldehyde synthesis are below 10mg/L except one isolate (47→35,7 mg/L).
Out of 48 L. delbrueckii ssp. bulgaricus isolates, 22
isolates have EPS synthesis in the range of 30 mg/L – 200mg/L.
Out of 38 S. thermophilus isolates, 14 isolates have
EPS synthesis in the range of 30 mg/L – 200mg/L.
Bulk Production of Yogurt Starter Cultures
Biotechnology and Bioengineering Central
Research Lab. (IZTECH)
oTemperature: 42ºC
oAgitation speed: 110 rpm oMinerals: Fe+2, Mn+2, Mg+2
oSalts: Phosphate salts oNitrogen source: Yeast extract
oCarbohydrate source: Lactose
Constants µmax (1/h) Ks (g/L ) Substrate saturation
(g/L)
Pure culture fermentations
S.therm. 0,85 31,3 90
L.bulg. 0,66 30,9 70
Mixed culture fermentations
S.therm. 0,92 24,5 120
L.bulg. 0,73 20,7 80
Comparison of pure and mixed lactic culture fermentations
Lactic yogurt cultures were screened for their mineral requirements to prevent phage adsorption in whey based media. Na2HPO4 and KH2PO4salt mixture with 2% of the media has been determined to stimulate the growth of bacteria.
Symbiotic growth of yoghurt bacteria was
observed according to the fermentation constants with higher values of µmax (1/h) when comparing with pure culture constants.
Maximum lactose requirements up to 120 g/L
were determined.
LAB as Probiotic Culture from Human Milk
200 isolates were obtained from 15 different human milk samples; 2 bacilli of 200 isolates showed probiotic properties.
These isolates showed resistance to stomach pH (pH 3.0),
tolerance against 0.3% bile salt and antimicrobial activity against
Salmonella thyphimurium CCM 5445, Escherichia coli O157:H7 NCTC 129000 Escherichia coli NRRL B-3008.
These isolates were biochemically identified and then characterized by using restriction fragment length polymorphism (RFLP) of 16S rDNA and 16S sequencing.
Two lactobacilli, probiotic strains, were identified as Lactobacillus oris and Lactobacillus fermentum from human milk origin.
Gram staining
Catalase activity
Gas production from glucose
Growth at 15-45 °C
Growth at 4-6,5% NaCl
Carbohydrate fermentation
Biochemical Identification
Genomic DNA Isolation
Characterization by 16S-ITS rDNA region (Bulut et al, 2005)
16S rRNA Sequencing
Molecular Characterization
Isolation, Characterization and Screening Probiotic Properties of Artisanal Yoghurt Starters from Urla
Region PhD Thesis by Burcu OKUKLU
Advisor: Prof. Dr. Şebnem HARSA
Thirteen artisanal samples were screened
In total 253 LAB strains isolated
Five S. thermophilus, 26 Lb. bulgaricus were identified as probiotic using different probiotic screening tests Tolerance the low pH
Tolerance the simulated gastic juice
Tolerance the simulated intestinal juice
Bile salt tolerance
Autoaggregation
Antibiotic resistance
Cholesterol assimilation
Cell surface hydrophobicity
Growth with different prebiotics
Adhesion capabilities
Autoaggretion
Growth in lactulose
These starters were differentiated by using biochemical methods
Gram staining
Catalase reaction
Growth in different carbohydrates
Growth in different NaCl concentrations
Growth in different temperatures
Proteolytic activity
Gelatinase activity
Urease activity
B-Galactosidase activity
Indole production
B-Galactosidase activity; yellow (+), white (-)
Proteolytic activity; yellow (+), purple(-)
Gelatinase activity
Urease activity, yellow (-), pink (+)
Probiotic candidates were also characterized by genetic methods
Differentiated by 16S-ITS region
Pulsed Field Gel Electrophoresis (PFGE)
Genomic DNA of strains Amplification of 16S-ITS region EcoRI RFLP of 16S-ITS region
PFGE RFLP using SmaI
Probiotic yoghurt production Assessment of coagulation properties of probiotics
Milk acidifying activity
Yoghurt production and Selection of yoghurt combinations
Investigation technological properties
Titratable acidity Syneresis Apparent viscosity Aroma profiles by GC-FID Textural analysis by Texture Analyzer
Milk acidifying activity
• All isolates were differtiated as Gr(+), Catalase (-) and homofermantative
• According to the genetic methods cocci isolates were classified as S. thermophilus and bacilli isolates Lb. bulgaricus
• Two cocci, eight bacilli were paired as probiotic-starter strain combinations
• They are now recently found usage for the production of functional yoghurt in dairy industry
Lactobacillus delbrueckii subsp. bulgaricus & Streptococcus salivarus subsp.
thermophilus species
previously isolated from local Turkish Dairy products in Iztech
carried from Iztech laboratories to Rowett Institute of Nutrition and Health in aseptic conditions.
Fecal inoculum provided by the host Institute
Study funded by ESF –ENGIHR Committee.
Fermenter systems to assess the impact of dietary changes on the microbial
community and all the techniques required has been routinely used in
Microbial Ecology Laboratory of Rowett Institute, University of Aberdeen, U.K.
Studies on isolation of lactobacillus species from Turkish dairy products have
been continuing for many years in IzTech.
Cultivation of probiotics
Providing and processing of fecal inoculum
Selection of Optimum Prebiotics
Fermentation
Sampling and processing of Samples
DNA Extraction DGGE SCFA Enumeration of Probiotics on Selective Media
To develop a suitable delivery system such as emulsification in water-in-oil-in-water double emulsion for efficient survival of lactic acid bacteria under simulated gastrointestinal (GI) conditions and producing a functional food product (yoghurt) supplemented with developed emulsion system in order to stimulate the proliferation or the activity of the probiotic bacterial population in the colon.
Microencapsulation of Functional Food Ingredients
The double emulsion may serve as a suitable wrapper to encapsulate and protect probiotic bacteria in human gastrointestinal tract, it may be used as a potential biocapsule to encapsulate bacteria for commercial utilization in dairy products.
In this research, whey protein/pullulan (WP/pullulan) microcapsules were developed in order to assess its protective effect on the viability of Lactobacillus acidophilus NRRL-B 4495 under in vitro gastrointestinal conditions. Results demonstrated that WP/pullulan microencapsulated cells exhibited significantly (p £ 0.05) higher resistance to simulated gastric acid and bile salt. Pullulan incorporation into protein wall matrix resulted in improved survival as compared to free cells after 3 h incubation in simulated gastric solution. Moreover WP/pullulan microcapsules were found to release over 70% of encapsulated L. acidophilus NRRL-B 4495 cells within 1 h. The effect of encapsulation during refrigerated storage was also studied. Free bacteria exhibited 3.96 log reduction while, WP/pullulan encapsulated bacteria showed 1.64 log reduction after 4 weeks of storage.
Survived cell counts of free (circles) and encapsulated Lactobacillus acidophilus NRRL B-4495 in WP (squares) and WP/pullulan (triangles) after exposure to simulated bile salt solution at 37˚C. Values shown are means standard deviations (n = 3).
Optical microscopy images of WP/pullulan microcapsules after (A) SGJ, (B) bile salt and (C) SIJ exposure at 100x magnifications. Scale bars represent 20 mm.
Abstract
In this research, pullulan was incorporated in protein-based encapsulation matrix in order to assess its cryoprotective effect on the viability of freeze-dried (FD) probiotic Lactobacillus acidophilus NRRL-B 4495. This study demonstrated that pullulan in encapsulation matrix resulted in a 90.4% survival rate as compared to 88.1% for whey protein (WPI) encapsulated cells. The protective effects of pullulan on the survival of FD-encapsulated cells in gastrointestinal conditions were compared. FD WPI-pullulan capsules retained higher survived cell numbers (7.10 log CFU/g) than those of FD WPI capsules (6.03 log CFU/g) after simulated gastric juice exposure. Additionally, use of pullulan resulted in an increased viability after bile exposure. FD-free bacteria exhibited 2.18 log CFU/g reduction, while FD WPI and FD WPI-pullulan encapsulated bacteria showed 0.95 and 0.49 log CFU/g reduction after 24 h exposure to bile solution, respectively. Morphology of the FD microcapsules was visualized by scanning electron microscopy
Viable counts of free and microencapsulated L. acidophilus NRRL-B 4495 in wet and FD forms under in vitro (A) acid conditions at 37 C at pH 2.0 for 3 h, (B) bile salt at 37 C for 24 h. Note: Values shown are mean ± standard deviations (n = 3).
Abstract In this study, whey protein isolate-pullulan (WP/pullulan) microspheres were developed to entrap the probiotic Lactobacillus acidophilus NRRL-B 4495 by spray-drying technique. Microcapsules were analyzed for physicochemical characteristics including morphology, particle size, moisture content, water activity, dissolution time, and color properties. Results revealed that microcapsules were spherical in shape and obtained particle sizes between 5 and 160 lm, with an average size of around 50 lm. Blending pullulan with WP provided enhanced survival of probiotic bacteria during spray drying with a final viable cell number of 8.81 log CFU/g of microcapsule. Encapsulated probiotics were also found to have significantly (p 0.05) higher survived cell numbers compared to free probiotics under detrimental gastrointestinal conditions. Moreover, dissolution analysis suggested that protein-polysaccharide powdered microcapsules showed pH-sensitive dissolution properties in simulated gastric juice and simulated intestinal juice.
Nanoencapsulation
Characteristics
particle sizes starting from 10nm
chemically functional surfaces
hydrophobic or hydrophilic payloads
high surface area particles Applications
protein stabilization
small molecule delivery
clear liquid formulations
stable colloid dispersions
controlled release
targeted delivery
triggered release
Encapsulation techniques
Micelles a self-assembly process due to hydrophobic and hydrophilic
interactions
5-100 nm in diameter
able to encapsulate nonpolar molecules ( eg. lipids,
vitamins, antioxidants…)
Liposomes
are spherical, polymolecular aggregates with a bilayer
vary in size btw 20 nm and few hundred micrometer
able to encapsulate both water and lipid-soluble molecules (
e.g. proteins)
Layer-by-layer deposition
charged surfaces are coated with
interfacial films consisting of multiple
nanolayers of different materials
enables creation of thin films (1-100 nm per layer)
Nanolaminates
consist of two or more layers of material
with nanometer dimensions (1-100 nm) that
are physically and chemically bonded
used as edible coatings and films in foods
(eg. fruits, vegetables, meats, candies,…)
a nanolaminate material formed from protein and polysaccharide
Abstract • Functionality of the whey-based a-lactalbumin (a-La) may be increased when assembled
in the form of nanotubes, promising novel potential applications subject to investigation. The purpose of this study was to extract highly pure a-La from whey protein isolate (WPI) and whey powder (WP) and to construct protein nanotubes from them for industrial applications.
• For protein fractionation, WPI was directly fed to chromatography, however, WP was first subjected to membrane filtration and the retentate fraction, whey protein concentrate (WPC), was obtained and then used for chromatographic separation. a-La and, additionally b-Lg, were purified at the same batches with the purities in the range of 95%–99%. After enzymatic hydrolysis, WPI-based a-La produced chain-like and long nanotubules with 20 nm width while WPC-based a-La produced thinner, miscellaneous, and fibril-like nanostructures by self-assembly.
• Raman and FT-IR spectroscopies revealed that a-La fractions, obtained from both sources and the nanostructures, developed using both fractions have some structural differences due to conformation of secondary structure elements. Nanotube formation induced gelation and nanotubular gel network entrapped a colorant uniformly with a transparent appearance. Dairy-based a-La protein nanotubules could be served as alternative gelling agents and the carriers of natural colorants in various food processes.
HPLC chromatograms of whey proteins. Insets: (a) a-La and b-Lg extracted from WPI (I-a-La, I-b-Lg) and (b) a-La and b-Lg extracted from WPC (C-a-La, C-b-Lg).
• AFM images of nanotubes constructed by purified a-La proteins (a, b) WPI-based a-La nanotubes (I-a-LaNTs); (c, d) WPCbased
• a-La nanotubes (C-a-LaNTs).
• (a, c) Height images; (b, d) Phase images.
Prebiotics and Microfibrillated cellulose from agricultural
wastes Merve Şamlı (PhD Student, IZTECH)
ARTICHOKE
Cynara cardunculus var. scolymus
10/30/12 166
Artichoke By-products
• Leaves
• Bracts
• Stems
• Root
80% of plant mass
10/30/12 167
Polymer Extraction
Ground dry artichoke residue
Extraction with ethanol by Soxhlet method
Dewaxed sample
Treatment with alkali and 2% H2O2 (pH=11.5, T=50oC, t=16 h)
Cellulose Lignin - Hemicellulose
Neutralization to pH 5.5
Concentration Precipitation with
ethanol
Lignin Pellet
Washing with ethanol Hemicellulose
10/30/12 168
Prebiotic oligosaccharides from artichoke waste • The artichoke canning industry generates a solid waste consisting
mainly of the stems and external parts of the flowers (bracts),which are about 70% of the total artichoke flower
• These wastes are generally used in the production of animal feed or as manure.
• However, the bracts can contain bioactive compounds, as fructooligosaccharides (FOS)
Prebiotic oligosaccharides from artichoke waste • FOS are natural fructose oligomers with a degree of polymer-ization
(DP) varying from 3 to 10
• These sugars play an important role in human nutrition, as they affect the host by selectively stimulating the growth and/or activity of some microorganism in the colon, showing prebiotic effect
Hemicellulose as prebiotic
• Hemicellulose is an untapped natural resource, which can be produced economically in large quantities, therefore, the study of probiotics and hemicellulose together could also have potential for symbiotic formulations
• purified non-digestible oligosaccharides with prebiotic properties suitable to be used as ingredients for functional foods or feed applications
Hemicellulose as prebiotic
• Among the prebiotic compounds, xylooligosaccharides appears to be a promising one, since these can be sourced from agricultural crop residues that are inexpensive, abundant and renewable in nature.
• Advantages of consumption : • Low calorie sugar alcohol having prebiotic effects
• No elevation of blood glucose; suitable for diabetic patients
• Antioxidant,cyto-protective
• Offers scope for dietary supplements,beneficial drug or drug adjuvant
Seperation of Microfibrillated cellulose
• Artichoke leaves ( which is classified as waste in food industry ) were used as raw material
• Cellulose was obtained from that wasted with % efficiency
• All of the cellulose was converted into microfibrillated cellulose with no loss
• Microfibrillated cellulose pulp was freeze dried for long term storage
• Freeze dried for of microfibrillated cellulose could be considered as partially water soluble micro/nano fiber
• That’s why its prebiotic function should be investigated in advance!!
Overall procedure of methodology
Health Impact: Lactose Intolerance
Among lactic cultures there have been some thermophilic β-galactosidase (lactase) producers.
This enzyme catalyses the hydrolysis of lactose to glucose and galactose; it has potential importance due to various applications of lactose-reduced ingredients (galactooligosaccharides) in the food and dairy industry.
FCT 2015 - S.HARSA
Map shows the lactose
intolerance percentages
in Europe (Vesa et. al., 2000)
FCT 2015 - S.HARSA
β-galactosidase can be obtained from a wide variety of sources such as microorganisms,
plants and animals; however, according to their source, their properties differ markedly.
Yeast
• Kluyveromyces lactis
• Kluyveromyces fragilis
• Kluyveromyces marxianus
Bacteria
• Escherichia coli
• Lactobacillus bulgaricus
• Streptococcus thermophilus
Fungi
• Aspergillus niger
• Aspergillus oryzae
• Alternaria palmi
Sources of β-galactosidase
FCT 2015 - S.HARSA
Among lactic acid bacteria, yoghurt bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus) are the highest β-galactosidase producers.
β-galactosidase of these cultures show high stability and activity at high temperatures.
Lactic Cultures for Lactic Acid, Galactosidase Production
FCT 2015 - S.HARSA
Purification and Immobilization of LAB Lactases Thermostable bacterial lactase preparations (free and immobilized) for dairy industry especially to be used for lactose free milk.
Purification of thermostable lactase was produced from lactic acid bacteria isolated from traditional yoghurt.
Preparation of stable immobilized β-galactosidase through covalent attachment on chitosan-hydroxyapatite composite matrix.
FCT 2015 - S.HARSA
Ab
sorb
an
ce, 280 n
m
Act
ivit
y,
U/m
l
Purification of β-galactosidase
Purification step Total activity (U) Total protein (mg) Specific activity (U/mg) Recovery (%) Purification fold
Enzyme extract 45,36 7,76 5,85 100 1
(NH4)2SO4 precipitation 42,114 1,416 29,74 92,84 5,08
DEAE-A-50 Sephadex 38,22 0,72 53,1 84,26 9,08
Ion-Exchange Purification
FCT 2015 - S.HARSA
Figure shows the Native-PAGE analysis of various enzyme preparations with Native-PAGE. It was noted that most protein impurity was removed after precipitation. The molecular weight of the enzyme, was approximately 200 kDa as determined by Native-PAGE.
Among β-galactosidases from thermophilic microorganisms, molecular weights of 150, 240, 440 and 700 kDa were reported. In other studies, the molecular weight of β-galactosidase from Streptococcus thermophilus was estimated to be 530 kDa by gel-permeation chromatography. β-galactosidases from Streptococcus lactis and Lactobacillus thermophilus were reported with the a molecular weight of 540 kDa.
SDS–PAGE profiles of crude β-galactosidase extracts: Lane M; marker proteins, Lane S; commercial β-galactosidase from Saccharomyces fragilis, Lane 1; crude enzyme, Lane 2; β- galactosidase from (NH4)2SO4 precipitation precipitation, Lane 3; purified β-galactosidase from Sephadex DEAE A25 chromatography
M S 1 2 3
FCT 2015 - S.HARSA
Health Impact: Gluten Intolerance
Lactic cultures in sourdough fermentation may eliminate the toxicity of gluten fractions of bread.
These bacteria shows promises also for gluten intolerance and celiac disease since there can be a noticeable decrease in toxic gliadin fractions.
Celiac disease (CD), gluten-sensitive enteropathy, is an autoimmune disease of the small intestine caused by the ingestion of gluten proteins from food sources such as wheat, rye, and barley.
Recently, the capacity of probiotic lactic acid bacteria (LAB) in hydrolysation of wheat flour gliadins, including polypeptides responsible for CD, has been explored as a tool to increase tolerability in CD patients.
Sourdough contains metabolically active yeast and LAB strains. Gliadin and glutenin fractions are hydrolyzed by their complex protease and peptidase system, also by the wheat proteolytic enzymes.
To evaluate the changes in the structure of gliadins involved in gluten intolerance during sourdough fermentation with selected Lactobacillus acidophilus NRRL-B 1910, Lb. casei D4, Lb. delbrueckii ssp. bulgaricus TY30 and to obtain an intermediate product safe for CD patients.
Also a dough with no LAB inoculation and a chemically acidified dough employed.
Sourdough Fermentation
Gliadin Protein Bands on SDS-PAGE
55
43
34
26
M MW (kDa)
Lactic Cultures for Health
39-38
kDa
29-21
kDa
According to SDS-PAGE gel, some changes were observed in gliadin patterns of all sourdough samples. Some bands around 38-22 kDa disappeared and new bands around 21-27 kDa observed after 24 h of fermentations carried out by using individual Lb. acidophilus NRRL-B 1910, Lb. casei D4 strains and mixed cultures; the same changes happened after 48 h for sample fermented with Lb. delbrueckii ssp. bulgaricus TY30 and control dough.
2-D Electrophoresis gel of Lb. casei D4 and 3D view of the gel
FCT 2015 - S.HARSA
Some changes were observed in all protein patterns of electrophoresis data.
These changes obtained throughout the sourdough fermentation period with or without selected LAB and may not be specific to any bacterial species used and can be attributed to the enzymes present in wheat flour. They are aspartic proteinases and serine carboxypeptidases which work at pH range of 3.0-4.5 and 4.0-6.0, respectively (Loponen et al., 2006; Bleukx et al., 1997).
Lactic Cultures for Health
Gluten-Free Bread (GFB) Quality
Caprilles et al., 2014
189
the studies on the action mechanisms of LAB in gastrointestinal system,
investigation of improved techniques for analysis of the gut microbiota,
developing new food manufacturing biotechnologies such as micro-encapsulation, effects on diseases, infections and allergies, the stability - viability and safety of functional food
ingredients.
Future trends of functional food/food ingredients incl. Lactic cultures
KAHRAMAN, G., CAPPA, C., LUCISANO, M., & HARSA, Ş., 2016. “Optimization of Gluten-Free Bread Formulation Containing Leblebi Flour and Evaluation of Dough and Bread Properties”, 15th International Cereal and Bread Congress (15th ICBC), 18-21 April, Istanbul, Turkey, Abstract Book, 54 (Oral Presentation).
KAHRAMAN, G., CAPPA, C., CASIRAGHI, M.C., HARSA, S., & LUCISANO, M. (2016). Use of response surface methodology for optimization of gluten-free bread formulation containing leblebi flour and evaluation of quality and digestibility parameters. 30th EFFoST International Conference, 28-30 November, Vienna, Austria (Oral Presentation).
HARSA, T.S. “Functional Food Products&Ingredients for Gut Health” Editorial, Journal of Nutritional Therapeutics, 2016, Vol. 5, No. 2. 1-2.
CABUK, B. and HARSA, T. S. “Protection of Lactobacillus acidophilus NRRL-B 4495 under in vitro Gastrointestinal Conditions with Whey Protein/Pullulan Microcapsules”, Journal of Bioscience and Bioengineering, accepted for publication, (2015).
CABUK, B. and HARSA, S. "Whey protein-pullulan (WP/pullulan) polymer blend for preservation of viability of Lactobacillus acidophilus", Drying Technology, DOI:10.1080/07373937.2015.1021008, accepted for publication online 05 May, 33 (10): 1223-1233, (2015).
CABUK, B. and HARSA, T. Ş. “Improved viability of Lactobacillus acidophilus NRRL-B 4495 during freeze-drying in whey protein-pullulan microcapsules”, Journal of Microencapsulation, Early Online: 1–8, DOI: 10.3109/02652048.2015.1017618, published online 16 March, (2015). 32 (3) : 300-307, (2015).
TARHAN, O. and HARSA, Ş. “Nanotubular Structures Developed from Whey-Based a-Lactalbumin Fractions for Food Applications” (DOI 10.1002/btpr.1956) Biotechnology Progress, Published online August 6, 2014 in Wiley Online Library (wileyonlinelibrary.com) 1301-1310.
CABUK, B., OKUKLU, B., STANCIUC, N. and TELLIOGLU HARSA, S. “Nanoencapsulation of Biologically Active Peptides from Whey Proteins”, Journal of Nutritional Health & Food Science, 2 (3): 1-4, (2014).
CABUK, B., TARİ, C. and TELLIOGLU HARSA, S. “β-Galactosidase Immobilization on Chitosan-Hydroxyapatite Complex: Effects of Immobilization Conditions”, Journal of Nutritional Health & Food Engineering, 1(1): 00004, (2014).
SAMLI, M. and TELLIOGLU HARSA, S. “Traditional Fermented Foods from Turkey” ENGIHR Conference: The Gut Microbiota Throughout Life, Max Rubner-Insitut, Karlsruhe, Germany. September 24th-26th 2014, http://www.engihr.eu/wp-content/uploads/2014/03/Samli-M.-and-Harsa-S..pdf (Short Communications).
ERKUS, O., OKUKLU, B. YENIDUNYA, F. and HARSA, S. “High Genetic and Phenotypic Variability of Streptococcus thermophilus Strains Isolated from Artisanal Yuruk Yoghurts”, LWT-Food Science and Technology, 58, 348-354, (2014).
TARHAN, O., TARHAN, E. and HARSA, Ş., “Investigation of the structure of alpha-lactalbumin protein nanotubes using optical spectroscopy”, Journal of Dairy Research, Volume 81 (01): 98-106 (2013).
KOMEN, G., BAYSAL, A.H. and HARSA, S., “Gliadin degradation ability of
artisanal lactic acid bacteria, The potential probiotics from dairy products”, Journal of Nutritional Therapeutics, 2, 163-172, (2013).
KOMEN, G. and HARSA, S. “Development of functional food products with “free from” food concept” VI th International Bioengineering Congress, 12-15 November, İzmir, Turkey, Abstract Book, 92, 2013.
OKUKLU, B., YENIDUNYA, A.F. and HARSA, S., "Probiotic screening of yoghurt starters of traditional samples from Urla region", 2 nd Traditional Foods from Adriatic to Caucasus, Struga Macedonia, 23-26 October 2013.
OKUKLU, B., YENIDUNYA, A.F. and HARSA, S., "Investigations on probiotic properties of Streptecoccus thermophilus isolated from yogurts from Urla region" TGDF Gıda Kongresi 2013, Antalya, Turkey, 13-14 October, 2013 (in Turkish).
TARHAN, Ö., and HARSA, Ş. “Analysis of the Growth of Alpha-Lactalbumin Protein Nanotubes Functional for Food Applications”, PPM, International Porous and Powdered Materials Symposium and Exhibition, October 3–6 2013, İzmir, TURKEY, Congress Proceedings p.616-619 (full article, poster presentation).
ERKUS, O., OKUKLU, B., YENIDUNYA, A:F: and HARSA, S., "Genotypic identification of Lactobacillus delbrueckii ssp. bulgaricus strains isolated from artisanal yoghurts in Turkey" The Intestinal Microbiota and Gut Health: Contribution of the Diet, Bacterial Metabolites, Host Interactions and Impact on Health and Disease : 2013 ENGIHR Conference, Valencia, Spain, Proceedings pp 70-73, 18th-20th September 2013.
OKUKLU, B., YENIDUNYA, A.F. and HARSA, S., "Probiotic properties screening of Streptecoccus thermophilus from Urla region" The Intestinal Microbiota and Gut Health: Contribution of the Diet, Bacterial Metabolites, Host Interactions and Impact on Health and Disease: 2013 ENGIHR Conference, Valencia, Spain, Proceedings pp 122-125, 18th-20th September 2013.
OKUKLU, B., GURSOZ, S., DAGISTAN F., A. PEYNIRCI, A. and HARSA, S. “The Yoghurt Cherishes At Our Tender Hands” 2013 ENGIHR Conference, Valencia 18th-20th September, 2013.
SAMLI, M., SUREK, E., BUYUKKILECI, A.O., HARSA, S. and NIRANJAN, K., "Extraction of lignocellulosic compounds from artichoke: Their potential in packaging applications" International Conference on Food and Biosystems Engineering- Conference Proceedings, Greece, 30 May-2 June, 2013 (Full article and Poster Presentation).
OKUKLU, B. and HARSA, Ş. “Determination of probiotic properties of yoghurt starter bacteria isolated from Urla region” Süt Endüstrisinde Yenilikçi Yaklaşımlar Sempozyumu, Denizli, Turkey, 15-16 October 2012.
KOMEN, G. and HARSA, S., “Artisanal lactic acid bacteria utilization for the development of gluten-free sourdough” V Symposium on Sourdough, Cereal Fermentation for Future Foods, 10-12 October, Helsinki, Finland, Abstract Book, 124 (2012).
BARAN, E., BUYUKKILECI, A.O. and HARSA, S., “Screening of aroma profiles for artisanal yogurt starter cultures” XV. European Congress on Biotechnology, 23-26 September, İstanbul, Turkey, 29, S: 233, (2012).
CABUK, B. and HARSA, S., “Entrapment of Lactobacillus acidophilus NRRL B-1910 in soy milk based water-in-oil-in-water (W1/O/W2) emulsion” XV. European Congress on Biotechnology, 23-26 September, İstanbul, Turkey, 29, P: S66 (2012).
SAMLI, M., ERKUS, O., HARSA, S., SCOTT, K., BUYUKKILECI, O. and OKUKLU, B., “Simulation of human colon system using potential probiotic yoghurt cultures from Toros region of Turkey”, 8th INRA-Rowett Symposium: Gut microbioata: Friend or Foe? , 17-20 July, Clermont-Ferrand, France, 2012.
SAMLI, M., SCOTT, K., BUYUKKILECI, A.O., and HARSA, S., “Simulation of human colon system using potential Probiotic yoghurt cultures from Toros region of Turkey” ENGIHR, Diet and Gut Microbiota: New directions, 2nd ENGIHR Workshop, 2-4 May, Helsinki, Finland (2012).
HARSA, S. “Impact of Lactic Probiotic Cultures for Food and Bio Processing; Simulation of Human Colon System Using Potential Probiotic Yoghurt Cultures from Toros Region of Turkey” The First North Eastern European Food Congress, April, St. Petersburg, Russia, Proceedings Book, 2012 (oral presentation).
KOMEN, G.; BAYSAL, A. H. and HARSA, S., “Recent developments in formulating gluten-free food products” International Symposium on Health Benefits of Foods- From Emerging Science to Innovative Products, 5-7 October, Prague, Czech Republic, Abstract Book, 87 (2011).
KOMEN, G.; BAYSAL, A. H. and HARSA, S., “Sourdough fermentation and
evaluation of gliadin degradation” Novel Approaches in Food Industry (NAFI 11), International Food Congress, May 26-29, Cesme-İzmir, Turkey, Abstract Book, 215 (2011).
TARHAN, Ö., GÖKMEN V. and HARSA, S., “Alpha-Lactalbumin protein nanotubes”
Novel Approaches in Food Industry (NAFI 11), International Food Congress, May 26-29, Cesme-İzmir, Turkey, Congress Proceedings, Vol. 1, 106-109 (2011).
TARHAN, Ö., GÖKMEN V. and HARSA, S., “Purification of major proteins in whey” Novel Approaches in Food Industry (NAFI 11), International Food Congress, May 26-29, Cesme-İzmir, Turkey, Congress Proceedings, Vol. 2, 562 (2011).
KOMEN, G.; BAYSAL, A. H. and HARSA, S., “Structural changes of gliadins during sourdough fermentation” 11th International Congress on Engineering and Food (ICEF11), May 22-26, Athens, Greece, Congress Proceedings, Vol. I, 219-220 (2011).
TARHAN, Ö.; GÖKMEN, V. and HARSA, S., “Protein nanotubes constructed from whey based α-lactalbumin” 11th International Congress on Engineering and Food (ICEF11), May 22-26, Athens, Greece, Congress Proceedings, Vol. 2, 1039-1040 (2011).
HARSA, S. and SAMLI, M., “Impact of lactic cultures for food and bio processing” 1st Workshop of the European Network for Gastrointestinal Health Research (ENGIHR), 15-16 February, Braga, Portugal (2011).
OKUKLU B.; ERKUS, O.; YENIDUNYA, A. F. and HARSA, S., “Isolation, phenotypic and genotypic characterization of starter bacteria from Toros yoghurts” 1st Workshop of the European Network for Gastrointestinal Health Research (ENGIHR), 15-16 February, Braga, Portugal (2011).
Komen, G., Baysal, H. and S. Harsa, “Degradation of toxic gliadin peptides during sourdough fermentation by using lactic acid probiotic bacteria” COST 928 Final Workshop (31), Naples, Italy, 2-4 March 2010.
Ustok, F.I., Tarı C. and S. Harsa, “Biochemical and Thermal Properties of Beta-galactosidase Enzymes Produced by Novel Yoghurt Cultures”, Food Chemistry, 119, 1114-1120 (2010).
Tarı, C., Ustok, F.I. and S. Harsa, “Production of Food Grade β-galactosidase from Artisanal Yogurt Strains”, Food Biotechnology, 24:78–94 (2010).
Tarı, C., Ustok, F.I. and S. Harsa, “Optimization of the Associative Growth of Novel Yoghurt Cultures in the Production of Biomass, Beta-galactosidase and Lactic Acid Using Response Surface Methodology”, International Dairy Journal, 19, 236-243 (2009).
Harsa, Ş. “Functional Food Ingredients for Gut Health” International Symposium on Biotechnology Developments and Trends”, Biotech2009, METU Ankara, Abstract Book p. 46, Turkey, 27-30 September 2009, (invited speaker).
Çabuk, B., Dağbağlı, S., Kadiroğlu, P., Tarı, C. Göksungur, Y. Hamamcı, H. and Ş. Harsa, “Purification and Covalent Immobilization of Lactobacillus bulgaricus β-Galactosidase” COST 928 Action 3rd Annual Workshop (35), Krakow, Poland, 23-25 September 2009.
Komen, G., Baysal, H. and S. Harsa, “Sourdough Fermentation as a Promising Approach to Gluten-free Diet”, Xth International Gluten Workshop, Book of Abstracts p.257, Clermont-Ferrand, France, 7-9 September 2009.
Yavuzdurmaz, H. and S. Harsa, “Characterization and Determination of Some Probiotic Properties of Lactic Acid Bacteria Isolated from Human Milk”, Food Micro2008, Programme and Abstract Book, PFF49, 503, Aberdeen, Scotland, 1-4 September 2008.
Ustok, F. I., Tari, C. and S. Harsa, “Effect of Symbiotic Relationship of Lactobacillus bulgaricus 77 and Streptococcus thermophilus 95/2 on β-galactosidase and Lactic Acid Production”, Journal of Biotechnology, Volume 131, Issue 2S, S224 (2007).
Ustok, F.I., Tari, C. and S. Harsa, “Effect of Symbiotic Relationship of Lactobacillus bulgaricus 77 and Streptococcus thermophilus 95/2 on β-galactosidase and Lactic Acid Production”, 13th European Congress on Biotechnology, Barcelona, Spain. 16-19 September 2007.
Altıok, D., Tokatlı, F. and S. Harsa, “Kinetic Modelling of Lactic Acid Production from Whey by Lactobacillus casei (NRRL B-441)”, Journal of Chemical Technology and Biotechnology, 81, 1190-1197 (2006).
Erkuş, O., Celik, E.S., Yavuzdurmaz, H., Okuklu, B. and S. Harsa, “Isolation and Molecular Characterization of Artisanal Yoghurt Starter Bacteria”, The 20th International ICFMH Symposium on Food Safety and Food Biotechnology: “Diversity and Global Impact”, Bologna, Italy. Preprints, 29 August- 2 October 2006.
Celik, E.S., Erkuş, O., Yavuzdurmaz, H., Harsa, S. and F. Korel, “Scanning and Screening of Aroma and Exopolysaccarides Formation Ability of Traditional Turkish Yoghurt Starter Cultures”, The 20th International ICFMH Symposium on Food Safety and Food Biotechnology: “Diversity and Global Impact”, Bologna, Italy. Preprints, 29 August-2 October 2006.
Goksungur, Y., Gunduz, M., and S. Harsa, “Production and Optimization of Lactic Acid from Whey by Lactobacillus casei NRRL B-441, Immobilized in Chitosan Stabilized Ca-Alginate,” Journal of Chemical Technology and Biotechnology, 80, 1282-1290 (2005).
Bulut, C., Gunes, H., Okuklu, B., Harsa, S., Kilic, S., Coban, H.S. and Yenidunya, A.F. “ Homofermentative Lactic Acid Bacteria of Traditional Cheese, Çömlek Peyniri, from Cappadocia Region” Journal of Dairy Reseach, 72:1 (2005): 19-24.
Okuklu, B., Yenidunya, A.F., Bulut, C., Harsa, S. and H. Gunes, “Chromosome and Plasmid
Profiling Studies on Lactococcus lactis ssp. lactis Strains Isolated from an Artisanal Cheese “Comlek Peyniri” Sample”, 8th Symposium on Lactic Acid Bacteria, Egmond aan Zee, The Netherlands, 28 August-1 September 2005.
Yavuz, E., Gunes, H., Bulut, C., Harsa, S. and A. F. Yenidunya, “RFLP of 16S rDNA-ITS Region to Differentiate Lactobacilli at Species Level,” World Journal of Microbiology and Biotechnology, 20, 535-537 (2004).
•TEŞEKKÜRLER !!!!!
