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 ie 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)

LACTIC ACID BACTERIA

No official status in taxonomy Based on 16s rRNA sequencing

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

12Vagococcus

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

12Vagococcus

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 45 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 - 42degC)

Higher maximum growth temperature (52degC)

Higher thermal tolerance (above 60degC)

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 18-25degC some grow below 10degC Grow in milk wo 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 mg100 ml) in milk

citrate

Leu lactis 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 Fermentationdagger 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)

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 45 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 - 42degC)

Higher maximum growth temperature (52degC)

Higher thermal tolerance (above 60degC)

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 18-25degC some grow below 10degC Grow in milk wo 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 mg100 ml) in milk

citrate

Leu lactis 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 Fermentationdagger 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)

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 45 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 - 42degC)

Higher maximum growth temperature (52degC)

Higher thermal tolerance (above 60degC)

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 18-25degC some grow below 10degC Grow in milk wo 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 mg100 ml) in milk

citrate

Leu lactis 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 Fermentationdagger 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)

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 - 42degC)

Higher maximum growth temperature (52degC)

Higher thermal tolerance (above 60degC)

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 18-25degC some grow below 10degC Grow in milk wo 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 mg100 ml) in milk

citrate

Leu lactis 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 Fermentationdagger 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)

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 mg100 ml) in milk

citrate

Leu lactis 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 Fermentationdagger 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)

citrate

Leu lactis 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 Fermentationdagger 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 01 to 03 unit decrease titratable acidity

by 001 to 003gL) 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

Pediococcus Tetrads in pairs

Obligate homofermentative L(+) or DL-lactic acid Tolerate high acid (pH 42) and salt (65 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

gt 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

httpwwwyoutubecomwatchv=bch1_Ep5M1s

Plasmids

Lactose metabolism Bacteriocin production Antibiotic resistance

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

gt 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

httpwwwyoutubecomwatchv=bch1_Ep5M1s

Plasmids

Lactose metabolism Bacteriocin production Antibiotic resistance

L brevis L sakei rice wine fermentation L sanfranciscensis San Francisco sourdough bread

httpwwwyoutubecomwatchv=bch1_Ep5M1s

Plasmids

Lactose metabolism Bacteriocin production Antibiotic resistance

Bifidobacterium Not classical LAB Different morphologies (pleomorphism) Bifid V Y or X- shaped Sugar metabolism

fructose-6-phosphate phosphoketolase Lactic acid acetic acid = 23

wo 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

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 5-52 (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 ldquoeyerdquo 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 25-30degC)

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

PR CHAWLA et al Fermentative Production of Microbial Cellulose Food

Technol Biotechnol 47 (2) 107ndash124 (2009)