BACTERIA USED IN FOOD FERMENTATION INTORODUCTION ALL ARE CURRENTLY CLASSIFIED IN ONE OF THREE PHYLA ( 門, DIVISION): Proteobacteria: Gram negative bacteria involved in vinegar fermentation Firmicutes: Lactic acid bacteria, Bacillus spp. Brevibacterium spp. Actinobacteria: Bifidobacterium spp., Kocuria spp., Staphylococcus spp., Micrococcus spp. Actively growing microbial cells: milk yogurt Metabolic by-products: organic acids, bacteriocins, etc. Cellular components: SCP, dextran, cellulose, enzymes Should be safe (approved by regulatory agent, i.e. non-gmo), food grade (GRAS) MICROBIOLOGY OF FERMENTED FOODS (HISTORY ASPECT) Use food materials as substrate (metabolism) Long history Natural fermentation Back slopping technique Pure culture fermentation (starter culture) No official status in taxonomy Based on 16s rrna sequencing LACTIC ACID BACTERIA Phylum: Firmicutes Order: Lactobacillales Common characteristics of LAB Gram positive Fermentative (one exception) Catalase negative Facultative anaerobe Non-sporeforming Low mol% G+C Non-motile Produce large amount of lactic acid from carbohydrates Acid tolerant
Heterotrophic chemoorganotrophic: use organic C for growth and energy Fastidious with complex nutrients Recently, some LAB (Lactococcus lactis) do have respiration: Respiration as well as fermentation Heme (or heme precursor) must be added Grow better in respiring condition (ph remain high due to less lactic acid production) Light aeration in starter preparation 12 different genera : 1. Lactococcus 2. Leuconostoc 3. Pediococcus 4. Lactobacillus 5. Streptococcus 6. Enterococcus 7. Tetragenococcus 8. Carnobacterium 9. Weissella 10. Oenococcus 11. Aerococcus 12.Vagococcus
LACTIC AND FERMENTATIONS Lactic acid fermentation can occur by Homofermentative pathway Heterofermentative pathway Facultative homofermentative The fermentation of glucose in heterofermentative LAB is called
hexose monophosphate (HMP) shunt or Warburg-Dickens-Horecker pathway or phosphoketolase pathway Homofermentative LAB contain aldolase (2 lactates from glucose), while heterofermentative do not have it.
Lactococcus lactis Most widely used in dairy fermentation (cheese) 3 subspecies: Lc. lactis subsp. lactis Lc. lactis subsp. cremoris: found only in milk, diacetyl production Lc. lactis subsp. hordinae (not used as starter culture) 1 biovar: Lactococcus lactis sub. lactis biovar diacetylactis (CO2 + diacetyl from citrate) Pair, short chain Natural habitats: Originally: Green vegetation, silage New habitat: dairy environment, raw milk Grow rapidly in milk ph below 4.5 Obligate homofermentative L(+)-lactic acid Plasmid borne traits (acquired recently) Lactose transport and metabolism Casein hydrolysis and metabolism Selective pressure for the maintenance of plasmid in milk borne strains are needed Readily exchanged among other strains (via conjugal transfer) Plasmid can integrate within chromosome stabilized Closely related to Lc. lactis and Lc. cremoris Streptococcus Many diverse species with a wide array of habitats
Human and animal pathogens, oral commensals, intestinal commensals Only 1 species in dairy (yogurt) fermentation : Streptococcus thermophilus Pairs to long chain Obligate homofermentative L(+)-lactic acid Higher optimum temperature (40-42 C) Higher maximum growth temperature (52 C) Higher thermal tolerance (above 60 C) More fastidious than Lactococcus spp. for nutrients Weakly proteolytic (need pre-formed amino acids) Limited metabolic diversity Contain few plasmid; generally small and cryptic Leuconostoc Spherical or lenticular based on media (solid vs liquid) Heterofermentative D(-)-lactic acid, CO2, ethanol, acetic acid flavors CO2 reduce redox potential subsequent acid tolerant LAB growth Opt. temp: 18-25 C, some grow below 10 C Grow in milk w/o curding acidification is not major function Reduced or anaerobic environment enhance growth Plasmids are common: Lactose and citrate metabolism Bacteriocin production Leu. mesenteroides : Dextran formation from sucrose (dextran sucrase)
Plants, vegetables, silage, milk, raw meat 5 species: Leu. mesenteroides Leu. paramensenteroides Leu. lactis Leu. carnosum Leu. gelidum 3 subsp. in Leu. mesenteroides : Leu. mesenteroides subsp. mesenteroides Leu. mesenteroides subsp. dextranicum Leu. mesenteroides subsp. cremoris Dairy strains Ferment milk sugars (lactose, galactose, glucose) Leu. mesenteroides subsp. cremoris: produce diacetyl (buttery flavor) + CO2 from citrate (130-160 mg/100 ml) in milk
Leu. lactis citrate Vegetable strains Ferment plant sugars (fructose, sucrose, arabinose, trehalose) Leu. mesenteroides subsp. mesenteroides: initiate fermentation Leu. kimchii Leu. fallax (Identification and Characterization of Leuconostoc fallax Strains Isolated from an Industrial Sauerkraut Fermentation. 2002. Rodolphe Barrangou, Sung-Sik Yoon, Frederick Breidt, Jr., Henry P. Fleming and Todd R. Klaenhammer,* Appl. Environ. Microbiol. 78(19):2877-2884 Spoilage of refrigerated vacuum-packaged meat Leu. carnosum Leu. gelidum Leu. gasicomitatum Oenococcus oeni Previously Leuconostoc oeni More acid tolerant than Leuconostoc and most ethanol tolerant LAB (10% ethanol)
Malo-lactic fermentation: Deacidification (Increase ph by 0.1 to 0.3 unit, decrease titratable acidity by 0.01 to 0.03g/L) and decarboxylation full, smooth flavor and texture Important for high acid wine (May be undesirable in low acid situation)
Slow growing Ferment limited number of sugars Weissella Heterofermentative Kimchi isolates Weissella cibaria (Weissella kimchii) Weissella koreensis
Tetrads, in pairs Pediococcus Obligate homofermentative L(+) or DL-lactic acid Tolerate high acid (ph 4.2) and salt (6.5% NaCl) Lactose is not fermented do not grow in milk Ripening of cheese: secondary flora Plants, vegetables, silage, beer, kimchi, sauerkraut, fermented meat, fish Two major species in vegetable (sauerkraut, kimchi) and meat fermentations: P. acidilactici P. pentosaceus Beer spoilage P. damnosus Diacetyl serious flavor defect for beer Plasmids are frequently found Sugar (raffinose, sucrose) metabolism Bacteriocin (pediocin) production Importance of pediocin producing P. acidilactici inhibit meat-associated pathogens (Listeria monocytogenes, Staphylococcus aureus, Clostridium botulinum) Starter culture Tetragenococcus New species (previously Pediococcus) Extremely halophilic Tolerate 25% NaCl Require 3-10% NaCl for growth
No growth when salt is absent Contribute to flavors 4 species T. halophilus: soysauce fermentation, homofermentative on glucose. Heterofermentative on pentose (xylose) T. koreensis: kimchi T. muriaticus: fish sauce T. solitaries Grow well in low aw Compatible sugars Betaine carnitine Lactobacillus > 80 species: ubiquitous in nature Fastidious to grow Various morphologies Very short (coccobacillus) to very long rod (often bent) Single to long chain Large round colonies, small or irregular colonies Wide range of habitats Dairy Meat Vegetable Cereal Some are probiotics L. acidophilus L. reuteri L. casei subsp. rhamnosus L. johnsonii Facultative heterofermentative Dairy strains L. helveticus L. delbrueckii subsp. bulgaricus L. casei L. acidophilus L. kefir (kefir fermentation) Sausage strains L. plantarum L. sakei subsp. sakei Vegetable strains (Kimchi, sauerkraut, pickle) L. plantarum
L. brevis L. sakei: rice wine fermentation L. sanfranciscensis : San Francisco sourdough bread Plasmids Lactose metabolism Bacteriocin production Antibiotic resistance http://www.youtube.com/watch?v=bch1_ep5m1s
Bifidobacterium Not classical LAB Different morphologies (pleomorphism) Bifid: V, Y, or X- shaped Sugar metabolism fructose-6-phosphate phosphoketolase Lactic acid: acetic acid = 2:3 w/o CO2 production Human origins: B. bifidum B. longum B. brevis B. infantis B. adolescentis Vitamin synthesis of human origin Thiamine (B1) Riboflavin (B2)
Pyridoxine (B6) Folic acid (B9) Large intestine Added to dairy products (do not grow well in milk) Beneficial role (probiotic) High number in breast fed infant (B. infantis) Host specificity: B. animalis is unsuitable for human Age specificity: B. infantis for the infant, B. adolescentis for the young. Need bifidigenic factors( 숙제 ) Strict anaerobe Need reducing agent (L-cysteine and thioglycolate that have thiol (-SH) on it) Cystein Thioglycolate High cost for cultivation
Propionibacterium Phylum Actinobacteria Not LAB High G+C: 53-68 mol% Non-sporeforming Gram positive Non-motile Pleomorphic rod (coccoid, bifid or branched) Mesophilic Anaerobic to aerotolerant Catalase positive Neutraphilic and grow slowly at ph 5-5.2 (ph of Swiss type cheese) Two types Dairy Cutaneous : causing acne Dairy (Swiss type cheese, Emmental cheese): P. freudenreichii subsp. freudenreichii P. freudenreichii subsp. shermanii P. thoenii P. acidipropionici P. jensenii Strong lipolytic activity to form free fatty acids flavor Lactate fermentation of dairy strain (Propionic acid pathway) Lactobacillus helveticus grows first provides peptides and amino acids and also produce lactic acid Dairy strains use lactate as carbon and
energy source to form acetate, propionate and CO2 Impart nutty and sweet flavor and eye formation (CO2) Small amount of vitamin B12 Probiotic effect
ACETIC ACID BACTERIA, AAB Only Gram negative bacteria used in food fermentation Not taxonomical name Peritrichous flagella Family Acetobacteraceae Mesophilic (opt. temp. of 25-30 C) Three genus Acetobacter Gluconobacter Gluconoacetobacter Obligate aerobe Only respiratory metabolism Surface filn Produce acetic acid from ethanol in the presence of oxygen Some species overoxidizes acetic acid to water and CO2 Vinegar production Acetobacter aceti: most commonly used A. orleanensis A. pasteurianus subsp. pasteurianus Gluconobacter europaeus G. xylinus Fruit flies or vinegar eels are considered as common vector in propagating acetic acid bacteria in nature (mother of vinegar) Some are spoilage microorganisms in wine, beer, and sake fermentation Acetobacter xylinum synthesize microbial cellulose Audio speaker Wound dressing Paper and paper products Dessert food Filter
P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107 124 (2009)