01/04/57. Food Microbiology_Katekan Dajanta 1. Microbes used in food industry. Microflora in fermented foods

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1 1 Microbes used in food industry Nature uses microorganisms to carry out fermentation processes, and for thousands of years Fermentation in food processing is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeast, bacteria, or a combination under aerobic or anaerobic conditions Carbohydrates (sugar) microbes aerobic/ anaerobic Alcohols + Organic acids + CO 2 2 Microflora in fermented foods Bacteria o Lactic acid bacteria : Leuconostoc mesenteroides, Lactobacillus brevis, Pediococcus and Streptococcus sp. o Other bacteria : Bacillus, Acetobacter Mould o Aspergillus, Rhizopus, Mucor, Actinomucor, Amylomyces, Neurospora and Monascus Yeast o Saccharomyces 3 Food Microbiology_Katekan Dajanta 1

2 Benefits of fermentation Preservation of food: some microbes could be produced antimicrobial substances (organic acid, ethanol, bacteriocin) against spoilage and pathogenic microorganisms Fermentation to peel seed coat (eg. cocoa, coffee, pepper) with natural microorganisms (lactic acid bacteria, acetic acid bacteria and yeast) 4 Benefits of fermentation Biological enrichment: o Probiotic bacteria/lactic bacteria o Essential amino acids, vitamins, essential fatty acids Enrichment of the diet through development of a diversity of flavors, aromas, and textures in food substrates into new product 5 Fermentation 6 Food Microbiology_Katekan Dajanta 2

3 Type of fermentation Alcohol fermentation Acetic acid fermentation Lactic acid fermentation 7 Alcohol fermentation 8 Alcohol fermentation 9 Food Microbiology_Katekan Dajanta 3

4 Acetic fermentation acetification Alcohol + O 2 + Acetobacter spp. or Gluconobacter acetic acid + H 2 O 10 Lactic acid fermentation 11 ท มา : Lactic acid fermentation 12 Homofermenters o 85-90% of sugar were converted into lactic acid eg. Streptococcus faecalis, Pediococcus cerevisiae Heterofermenters o 50% of sugar were converted into lactic acid, 25% were converted into acetic acid and ethanol, and 25% were converted into CO 2 o Leuconostoc mesenteroides, Leu. fermenti, Lactobacilli o Foods : Yoghurt, fermented milk, cheese, fermented meat and vegetable Facultative heterofermenters o Some species of Lactobacillus could display both of homo- and heterofermenters o Lb. plantalum, Lb. casei and Lb. sake o Food: wine Food Microbiology_Katekan Dajanta 4

5 Ferment-inhibiting substances Production of organic acids and decreasing of ph Bacteriocins against other relative bacteria o nisin (Lactococcus lactis) Hydrogen peroxide (H 2 O 2 ) o Aerobic fermentation: LAB produce H 2 O 2 as electron acceptor o Anaerobic fermentation: small content of H 2 O 2 were produced by LAB o Raw milk: H 2 O 2 act as co-factor in Lactoperoxidase system (lactoperoxidase+ H 2 O 2 + thiocyanate) Ethanol in the heterofermenter at anaerobic condition Pickled fruits and vegetables LAB change sugar in fruit and vegetable to lactic acid Microbes involved in the fermentation process: Lactobacteriaceae (Streptococcus, Pediococcus, Diplococcus, Leuconostoc and Lactobacillus) Fermentation : o First stage of fermentation: LAB produce organic acids o Last stage of fermentation: aciduric yeasts and moulds 15 Food Microbiology_Katekan Dajanta 5

6 Pickled fruits and vegetables Change in appearance, texture, flavor, taste Factors affecting the quality of pickled fruits and vegetable o ph o Oxygen o Fermenting temperature o Salt concentration o Starter microorganisms 16 Kimchi Kimchi is a traditional fermented Korean side dish made of vegetables with a variety of seasoning Use different vegetable eg. cabbage, radish, cucumber, scallion Microbes involved in the fermentation process: o Leuconostoc sp. : Leu. mesenteroides, Leu. kimchi o Lactobacillus : Lb. plantarum, Lb. brevis 17 Change in LAB in fermented kimchi Leu. mesenteroides + vegetable + brine + ingredients (Facultative anaerobic condition) lactic acid + acetic acid + CO 2 + ethanol (ph = ) Growth of Lb. plantarum, Pediococcus cerevisiae, Lb. brevis, Lb. fermentum 18 Food Microbiology_Katekan Dajanta 6

7 Sauerkraut Sauerkraut = sour cabbage Finely cut cabbage fermented by various LAB: Leuconostoc, Lactobacillus, Pediococcus 19 Salt plays an important role in initiating the sauerkraut process and affects the quality of the final product o withdraw juice from the cabbage o Adjust anaerobic condition o Inhibit undesired microorganisms o making a more favourable environment for development of the desired bacteria o contribute to the firmness Microbes involved in the fermentation process of Sauerkraut Anaerobic LAB o Production of lactic acid, acetic acid, formic acid, succinic acid causing ph to decrease and inhibited spoilage aerobic bacteria Facultative anaerobic LAB Leu. Mesenteroides grow rapidly at low ph value, produce large contents of lactic and acetic acids 20 Microbes involved in the fermentation process of Sauerkraut Homofermentative lactobacilli Growing of Lactobacilli (Lb. plantarum, Lb. sakei, Lb. curvatus) and cocci (streptococci and pediococci) Converting of sugar (glucose, fructose, sucrose) into lactic acid Heterofermentative lactobacilli the last stage of sauerkraut fermentation ferment pentose sugar (arabinose, xylose) into lactic acid 21 Food Microbiology_Katekan Dajanta 7

8 Wine Wine is an alcoholic beverage made from fermented grapes or other fruits Production process: Grape (crushing) + S. cerevisiae alcohol fermentation (sugar) Bacterial fermentation Packaging aging/maturation malolactic fermentation (clarification) 22 Wine Source of fermenting microbials: o Starter: usually use S. cerevisiae o Grape : yeast, mould, LAB, AAB o Equipment 23 Malolactic fermentation L-malic acid Leuconostoc oenos fermentation Decaboxylation at o CO 2 L-lactic acid 24 Food Microbiology_Katekan Dajanta 8

9 Vinegar Vinegar is a liquid consisting mainly of acetic acid and water The acetic acid is produced by the fermentation of ethanol by acetic acid bacteria 25 Vinegar: 2 step of fermentation acetic acid bacteria (Aectobacter spp., Gluconobacter, A. pasteurianus) 26 Acetification Alcohol + Acetobacter spp. Alcohol dehydrogenase aldehydes Aldehyde dehydrogenase Acetic acid 27 Food Microbiology_Katekan Dajanta 9

10 Miso (Japanese) Also called Chiang (Chinese), Jang (Korean) Various types rice miso barley miso soybean miso 28 Miso fermentation 29 Koji making process K Brine fermentation process Polished rice Soaked Cooked Seed mold Incubated Koji Mixed Aspergillus oryzae Fermented Aged Mashed Miso Whole soybean Soaked Cooked Salt Saccharomyces rouxii, Yeast Zygosaccharomyces spp. Lactic acid bacteria Lb. acidophilus, Lb. delbrueckii, Pediococcus Koji 30 Food Microbiology_Katekan Dajanta 10

11 Tempeh 31 Tempeh is a fungal fermented soybean food originating from Indonesia The fungal enzyme activity causes significant decomposition of polymeric components, as well as a considerable modification of soybean flavonoids. Usually used Rhizopus oligosporus, other such as R. oryzae, R. stolonifer Tempeh offers a number of proven health benefits including excellent digestibility and protection against diarrhea and chronic degenerative diseases. 32 Tempeh 33 Food Microbiology_Katekan Dajanta 11

12 Tempeh 34 Potential Strains Selected for Improvement of Active Substances in Tempeh fermentation 35 Sufu 36 Su-fu (Chinese cheese) or tao-hu-yi in Thailand Sufu originated in China but is also prepared in other Asian countries where variations of sufu exist. Sufu is consumed as an appetizer and a side dish, e.g. with breakfast rice or steamedbread. Sufu is a highly flavoured, creamy cheese-like product, so it would be expected to be suitable for use in western countries as a healthy, non-cholesterol food from plant origin. Food Microbiology_Katekan Dajanta 12

13 Sufu Most sufu products are produced by a similar principle, which involves four main steps: (1) Preparation of tofu (2) Preparation of pehtze (3) Salting (4) Ripening 37 Sufu 38 According to the colour and flavour, sufu can be classified into four types Red sufu -mainly consists of salt, angkak (Monascus-fermented rice), alcoholic beverage, sugar, flour (or soybean) paste and some spices. White sufu - similar ingredients as red sufu in the dressing mixture but without angkak Grey sufu - contains the soy whey left over from making tofu, salt and some spices. Other types - made by adding various ingredients to the dressing, including vegetables, rice, bacon, and even higher concentrations of alcohol Sufu According to fermenting microbes, sufu can be classified into four types: 1) Mould-fermented sufu used Mucor racemosus, M. hiemalis and Actimomucor elegans in the pizi production 2) Naturally fermented sufu - used natural microbes in the pizi production 3) Bacteria-fermented sufu inoculated bacteria eg. Bacillus sp. or Micrococcus sp. into maturation process 4) Enzymatically ripened sufu 39 Food Microbiology_Katekan Dajanta 13

14 40 Thua Nao/Natto Thua Nao Natto Thua Nao (Thai)/Natto (Japanese) are Bacillus-fermented soybean Bacillus subtilis and related bacilli (i.e., B. licheniformis and B. pumilus) are usually found to be dominant microflora of the fermented soybeans A major activity in the fermentation is the hydrolysis of proteins to peptides and amino acids which are responsible for the typical umami taste in fermented soybeans Traditional Natto Process 41 Traditional Thua Nao Fermentation 42 Food Microbiology_Katekan Dajanta 14

15 Thua Nao VS Natto Production 43 Thua Nao Thua Nao Kab Roasted Thua Nao Mer Steamed Thua Nao Mer Nam Ngearw Curry Chilli paste 44 Natto 45 Food Microbiology_Katekan Dajanta 15

16 Natto Foods 46 Thua Nao/Natto The utilisation of soybean protein by bacteria leads to free amino acids formation Typical characteristics of traditional fermented soybeans are distinct ammoniacal smell, brownish colour, palatable taste and seed covered with mucous substance. Isoflavones are phytoestrogens that are abundant in soy-fermented foods and have been related to several health benefits. Pharmacological and antioxidant properties of soy isoflavones are involved in prevention of breast cancer, antihypertensive effect, reduced risk of cardiovascular diseases, improvement of bone health, reduced menopause symptoms, antimutagenic effects and antidiabetic effects. 47 Improvement of Thua Nao Production 48 Food Microbiology_Katekan Dajanta 16

17 Starter culture/thua Nao powder Fermented ground pork (Nham) Nham is an uncooked, fermented semi-dry Thai sausage It is made from fresh lean pork, pork skins, cooked rice, fresh garlic and eye bird chillies The sausage is wrapped in banana leaves or synthetic casings and fermented for 3-5 days Rice act as a source of carbohydrates for lactic acid production during fermentation LAB fermentation : o early stage of fermentation usually found Pediococcus cerevisiae, hetrofermentative LAB o last stage - Lb. plantarum and Lb. brevis fermentation 51 Food Microbiology_Katekan Dajanta 17

18 Improved Nham Fermentation Pure starter culture for fermenting Nham : o Lb. plantarum o Lb. curvatus o Pediococcus pentosaceus o Ped. acidilactici Mixed 3 microbes: o Lb. plantarum o Pediococcus serevisiae o Micrococcus varians 52 Salami Salami is cured sausage, fermented and air-dried meat Ingredients included pork or meat, minced fat, red wine, garlic, spices LAB fermentation 53 Fish sauce Fish sauce is a clear, amber to reddish brown liquid with a predominantly salty taste and characteristic aroma. It is obtained from the hydrolysis of fish with salt through natural fermentation for 6 12 months Fish sauce is well recognized as a food flavoring by the people in Southeast Asia for many centuries. Hydrolysis of fish protein results in small-molecule peptides, nitrogen compounds, and amino acids. Fish sauce contains about 20 g/l of total nitrogen, of which 80% is in the form of amino acids. 54 Food Microbiology_Katekan Dajanta 18

19 Local Names of Fish Sauce in Different Countries Food Microbiology_Katekan Dajanta 19

20 Food Microbiology_Katekan Dajanta 20

21 Fermented milk Dairy starter cultures are microorganisms that are intentionally added to milk in order to create a desired outcome in the final product, most often through their growth and fermentation processes. 61 Fermented milk Fermented milk products such as yogurt, kefir, buttermilk, and acidophilus milk are gaining increasing attention. Fermented milks containing probiotics could be used to prevent and/or cure some important diseases (such as intestinal- and immune-associated diseases). 62 Fermented milk The most common use of starter cultures is for the production of lactic acid from lactose (milk sugar), which in most cases causes or assists in the coagulation of milk protein by lowering its ph value. Cultures that produce lactic acid are generally referred to as lactic acid bacteria (LAB) such as Lactobacillus, Lactococcus and Leuconostoc. 63 Milk + LAB Lactic acid + Acetic acid + CO 2 + (lactose) Flavor compounds (diacetyl, acetaldehyde) Food Microbiology_Katekan Dajanta 21

22 Fermented milk 64 The fermentation process increases the shelf-life of the product, while enhancing the taste and improving the digestibility of milk. Starter organisms can also influence flavor and texture of cultured and/or aged products through the breakdown of proteins, fats and other milk constituents in addition to the ph effect. The lower ph of cultured products can be inhibitory to certain spoilage organisms, although inhibition is also associated with other by-products of growth with some starters. Fermented milk Probiotic cultures are organisms that have some claimed health benefit for those that consume them, e.g., better digestion, anti-cancer compounds, and prevention of heart disease. Probiotic cultures may be added as adjuncts or they may be directly involved in the fermentation process. 65 Lactic acid bacteria Lactic acid production: Streptococcus lactis, S. cremoris and S. thermophilus Flavour production : S. diacetilactis and Leuconostoc citrovorum Fermented milk usually use mixed starter culture o Starter D: S. cremoris+ S. lactis +S. diacetilactis o Starter DL: S. cremoris +S. lactis+ S. diacetilactis +Leuconostoc citrovorum o Starter L: S. cremoris +S. lactis+ Lb. citrovorum 66 Food Microbiology_Katekan Dajanta 22

23 Dairy Starter Cultures Mesophilic starters Major Function Product Use 67 Dairy Starter Cultures Thermophilic starters Major Function Product Use 68 Type of fermented milk Mesophilic starter fermented milk 69 Butter milk o Fermented pasteurized skim milk with mesophilic LAB eg. Lactococcus and Leuconostoc at C for h Sour cream o Fermented milk butter with Streptococcus and Leuconostoc at C until ph decreased at 4.5 Fermented milk o Langfil o Filmjolk o Ymer Food Microbiology_Katekan Dajanta 23

24 Type of fermented milk Mesophilic and thermophilic starter fermented milk Yoghurt o Fermented milk with Strep. thermophilus (mesophilic) and Lb. bulgaricus (thermophilic) (ratio 1:1) Yakult o Fermented skim milk with Lb. casei at 37 C for 4 days Bulgarian butter milk o High acid fermented butter milk, fermented by 2% Lb. bulgaricus at C for h 70 Type of fermented milk Mesophilic and thermophilic starter fermented milk Acidophilus milk o Fermented sterilized milk with 2-5% Lb. acidophilus at 38 C for h Probiotic fermented milk o Fermented milk with LAB (Lb. acidophilus, Lb. casei) and probiotic (Bifidobacterium) acidophilus at 38 C for h 71 Type of fermented milk Alcohol-lactic fermentation Kefir o Fermented milk with LAB (Lactococci, Leuconostoc, Lactobacilli) and yeast (Candida, Kluyveromyces, Saccharomyces) at C and 8-10 C Kumiss o Russian fermented milk, fermented with Lb. bulgaricus and Candida kefir at C and 4-6 C 72 Food Microbiology_Katekan Dajanta 24

25 73 Health benefit microorganisms Usually according probiotic (meaning for life) Probiotics are live microorganisms, which when administered in adequate amounts, confer a health benefit on the host Prebiotic a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health Prebiotic = oligofructose, fructooligosaccharides, inulin and polyfructose 74 Probiotics Lactobacillus species Bifidobacterium species Other L. acidophilus B.bifidum Bacillus cereus L. casei B.longum Eschericia coli L. reuteri B.breve Saccharomyces cerevisiae L. bulgaricus B.infantis Saccharomyces boulardii L. plantarum B.lactis Enterococcus faecalis L. johnsonii B.adolescentis Strep. thermophilus L. lactis Trichuris suis 75 Food Microbiology_Katekan Dajanta 25

26 Mechanisms of Action of Probiotics Produce antimicrobial substances (organic acids or bacteriocins) Upregulate immune response to possible pathogens or to vaccines Downregulate inflammatory response 76 Assist in early programming g of the immune system to result in a better balanced immune response and reducing risk of development of allergy Improve gut mucosal barrier function Enhance stability or promote recovery of commensal microbiota Deliver functional proteins (eg, lactase) or enzymes Decrease pathogen adhesion Health benefits Prevention and treatment of diarrhea Treatment of chronic inflammatory bowel disease Treatment of ischemic bowel disease and bacterial infections in children born prematurely The immune system of the body Treatment of allergies Prevention of colon cancer Treatment of fungal vaginitis 77 Reduces blood cholesterol levels Food Microbiology_Katekan Dajanta 26