Lactic acid bacteria and production of cultured dairy products, probiotics,
prebiotics
Hana Sýkorová
• Introduction - LAB characteristic and metabolism
• Role of LAB in industry
• Bio-preservatives - Bacteriocins
• Starter cultures (dairy, non-dairy products)
• Probiotic (definition, benefits)
• Prebiotics
• LAB as source of vitamins, enzymes…
LAB – characteristic, metabolism
• Gram positive
• Rods or cocci
• Non‐spore forming
• Catalase negative
• Acid tolerant
• Fastidious
• Non motile
• Low-GC
• Facultative anaerobes / Micro‐aerotolerant
• Sugar metabolism - fermentative with lactic acid as the major end product during sugar fermentation
Lactobacillus brevis
Streptococcus thermophilus
″milk souring organisms″
LAB – characteristic, metabolism
• Lactobacillus
• Leuconostoc
• Pediococcus
• Lactococcus
• Streptococcus
• Aerococcus
• Enterococcus
• Oenococcus
• Sporolactobacillus
• Weisella
• (Bifidobacterium – phylogenetically unrelated)
http://islab.tp.ugm.ac.id/files/2010/10/Biotechnology-of-Lactic-Acid-Bacteria-and-Their-Role-in-Food-Industry1.pdf
LAB – sugar metabolism
Homofermentative LAB • hexoses by Embden-Mayerhof pathway • end-product: lactate • key enzyme: aldolase • Lactococcus, Pediococcus, Streptococcus, some
of Lacobacillus
Heterofermentative LAB • hexoses by phosphoketolase pathway • end-products: lactate, ethanol, CO2
• key enzyme: phosphoketolase • Leuconostoc, Weisella, some of Lactobacillus
GLUCOSE
glucose-6-P
fructose6-P
fructose-1,6-diP
glyceraldehyde-3-P
2 pyruvate
2 lactate
dihydroxyacetone-P
glucose-6-P
6-phosphogluconate
ribulose-5-P
xylulose-5-P
glyceraldehyde-3-P
pyruvate
lactate
acetyl-P
acetaldehyde
ethanol
CO2
LAB – sugar metabolism
HOMOFERMENTATIVE HETEROFERMENTATIVE
↔
http://islab.tp.ugm.ac.id/files/2010/10/Biotechnology-of-Lactic-Acid-Bacteria-and-Their-Role-in-Food-Industry1.pdf
LAB – citrate metabolism
• aroma compounds
• diacetyl – butter aroma
• butter, cottage cheese, …
• Lactococcus var diacetylactis, Leuconostoc spp.
• characteristic flavor of yoghurt is associated with the production of acetaldehyde by Lactobacillus bulgaricus
http://islab.tp.ugm.ac.id/files/2010/10/Biotechnology-of-Lactic-Acid-Bacteria-and-Their-Role-in-Food-Industry1.pdf
LAB – bioactive proteins
• specific protein fragments that have a positive impact on body functions and conditions and may ultimately influence health
Bacteriocins - generally
• production by G+ and G-
• protein structure (ribosomal synthesis)
• antimicrobial activity
• usually low molecular weight
• frequent posttranslational modifications
• easily degraded by proteolytic enzymes
Bacteriocins x Antibiotics
• B – activity against related strains • A – wide activity spectrum
• B – produced during growth, primary metabolism • A – secondary metabolism
• B – no observation of acquired resistance • A – acquired resistance
LAB Bacteriocins - classification
Class I: Lantibiotics
Class II: non modified heat-stable bacteriocins
Class III: heat-labile bacteriocins
Class IV: cyclic (complex) bacteriocins
Class I - Lantibiotics
• small peptides (2-4 kDa)
• typical posttranslational modifications
• contain lanthionine or methylanthionine (modified AA)
lanthionin
Subgroups:
IA – relatively elongated, screw shaped, amphipathic, flexible molecules, positive charge
IB – globular, no charge or negative charge
thioether bonds lanthionin
Ala-S-Ala 3-methyl-lanthionin
Abu-S-Ala
Abu = 2-aminobutyric acid Dha = dehydrated Serine Dhb = dehydrated Threonine
IA:
IB:
Class I - Lantibiotics
Class I – Lantibiotics: Biosynthesis
• genes – in operon
• location – non specific (plasmid, chromosome, transposone)
• precursor (pre-bacteriocin)
• post-translational modifications
• transport
• final bacteriocin
• Precursor NisA
• NisB dehydratation
• NisC cyclization
• NisT transport
• NisP extracelular protease
nisin A
Class I – Lantibiotics: Biosynthesis
LAB Bacteriocins - classification
Class I: Lantibiotics
Class II: non modified heat-stable bacteriocins
Class III: heat-labile bacteriocins
Class IV: cyclic (complex) bacteriocins
• small peptides (max 10 kDa)
• heat stable
• non-lanthionin
• no posttranslational modifications
Class II
Subgroups:
IIA – pediocin-like
IIB – two-component
• strong activity against listeria
• N-terminal consensus sequence YGNGV (Tyr-Gly-Asn-Gly-Val)
• Cys-Cys bond on N-terminal
• Hydrofobic C-terminal domain
pediocin – Pediococcus spp.
sakacin – Lactobacillus sakei
Class IIA – ″pediocine-like″
• two different peptides (two structural genes)
• for activity – both must be present in approximately equal amounts
• mode of action – pore forming
• target – not identified
lactococcin G – Lactococcus lactis
plantaricin EF – Lactobacillus plantarum
Class IIB – ″two-component″
Bacteriocins – mode of action - summary
IA - elongated • pores • target lipid II • nisin
IB - globular • cell wall inhibition • target lipid II • mersacidin
IIA - pediocin like • pores • target: man-PTS • anti-listeria • sakacin
IIB – two component • pores • target: ?
Class I lantibiotics
Class II heat stable
LAB Bacteriocins - applications
• natural preservatives (food industry)
• generally considers as safe (GRAS status)
• application form – pure bacteriocin or producent culture
• packaging films with bacteriocins (poultry meat)
Bacteriocin Producing strain
Lactacin F L. johnsoni spp
Laktocin 705 L. casei spp.
Lactoccin G L. lactis spp.
Lactococcin MN Lactococcus lactis var. cremoris
Nisin Lacotoccus lactis spp.
Leucocin H Leuconostoc spp.
Plantacirin (EF, W, JK, S) L. plantarum spp.
LAB Bacteriocines - Nisin
• Class I – lantibiotics
• subgroup IA – flexible elongated molecule
• 34 aa (modifications)
• mode of action – pores formation (target - Lipid II)
• active against G+ (S. aureus, L. monocytogenes)
• heat stable at 121°C
• sensitive to chymotrypsin, resistant to trypsin and pepsin
• production: Lactococcus lactis
• application: food additive (E234) (cheeses, cheese spreads)
• commercial product – Nisaplin (2.5% nisin + NaCl)
Starter cultures
spontaneous fermentation (naturally present microflora)
addition of selected cultures
• improvement of nutritional, organoleptic, technological and shelf-life characteristics
• LAB initiate rapid acidification (lactic acid, acetic acid)
Starter cultures
dairy products
• yogurt
• butter milk
• kefir
• cheeses
non-dairy products
•meat and meat products
• fermented vegetables
• silages
Dairy Products
• starter cultures
• well defined or composed of different strains
• rotation of cultures at regular intervals - to avoid possible accumulation of LAB specific bacteriophages
• mesophiles : Lc. lactis subsp. lactis or subsp. cremoris
• thermophiles : Strep. thermophilus, Lb. helveticus, Lb. delbrueckii subsp. bulgaricus
Dairy Products
Product Type Micro-organism
Yogurt moderate acid St. thermophilus, Lb. bulgaricus
Cheddar cheese (UK) moderate acid St. cremoris, St. lactis, St. diacetylactis
Cultured buttermilk (USA) moderate acid Lc. cremoris, Lc. lactis citrovorum
Acidophlus milk (USA) high acid Lb. acidophilus
Bulgarican milk (Europe) high acid Lb. bulgaricus
Leben (Egypt) high acid streptococci, lactobacilli, yeasts
Kefir (Russia) high alcohol streptococci, Leuconostoc spp., yeasts
Koumis (Russia) high alcohol streptococci, Lb. bulgaricus, Saccharomyces lactis
Fermented milk products around the world:
Production of Yogurt
Raw milk
Pasteurisation
Cooling and homogenisation
Starter culture
Strep. thermophilus and Lb. bulgaricus (1:1)
Incubation 4-16 hrs, 30-45°C
Adding flavourings or fruit
Cooling
Packaging and storage
Raw milk
Pasteurisation
Starter culture
Addition of rennin
or protease from Mucor pusillis
Separate curds and whey
Salt
Ripening
Production of Cheese
http://www.nature.com/nature/journal/v444/n7122/fig_tab/4441009a_F1.html
Intestinal microflora
• 50 bacterial genera • 500 species • 1014 microbial cells
Functions
• synthesis of nutrients (vitamins)
• production of short chain fatty acids
• production of antimicrobial substances
• stimulation of immune system
• maintenance of gut barrier function
Probiotics
Élie Metchnikoff Russian scientist, Nobel laureate in 1907 suggested that it would be possible to modify the gut flora and to replace harmful
microbes with useful microbes
The World Health Organization's 2001 definition of probiotics
"live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host"
pro - "for" + bios - "life"
alive health benefit taxonomically defined safe
• mainly - Lactobacillus and Bifidobacterium genus
Probiotics - safety
Probiotics must be safe for their intended use.
The 2002 FAO/WHO guidelines recommend that, though bacteria may be Generally Recognized as Safe (GRAS), the safety of the potential probiotic should be assessed by the minimum required tests:
• Determination of antibiotic resistance patterns
• Assessment of certain metabolic activities (e.g., D-lactate production)
• Assessment of side-effects during human studies
• Epidemiological surveillance of adverse incidents in consumers (post-market)
• If the strain under evaluation belongs to a species that is a known mammalian toxin producer, it must be tested for toxin production.
• If the strain under evaluation belongs to a species with known hemolytic potential, determination of hemolytic activity is required
Probiotics - safety
Probiotics must be safe for their intended use.
The 2002 FAO/WHO guidelines recommend that, though bacteria may be Generally Recognized as Safe (GRAS), the safety of the potential probiotic should be assessed by the minimum required tests:
• Determination of antibiotic resistance patterns
• Assessment of certain metabolic activities (e.g., D-lactate production)
• Assessment of side-effects during human studies
• Epidemiological surveillance of adverse incidents in consumers (post-market)
• If the strain under evaluation belongs to a species that is a known mammalian toxin producer, it must be tested for toxin production.
• If the strain under evaluation belongs to a species with known hemolytic potential, determination of hemolytic activity is required
Probiotics – mechanism of action
antimicrobial activity
• decreasing pH
• antimicrobial compounds – bacteriocins
• hydrogen peroxide
• organic acids
• competition for epithelial binding sites
immunomodulation (increase of IgA)
Schematic diagram illustrating potential or known mechanisms whereby probiotic bacteria might impact on the microbiota. These mechanisms include (1) competition for dietary ingredients as growth substrates, (2) bioconversion of, for example, sugars into fermentation products with inhibitory properties, (3) production of growth substrates, for example, EPS or vitamins, for other bacteria, (4) direct antagonism by bacteriocins, (5) competitive exclusion for binding sites, (6) improved barrier function, (7) reduction of inflammation, thus altering intestinal properties for colonization and persistence within, and (8) stimulation of innate immune response (by unknown mechanisms). IEC: epithelial cells, DC: dendritic cells, T:T-cells
Probiotics - advantages
Produce lactic acid - lowers the pH of intestines and inhibiting bacterial pthogenes (Clostridium, Salmonella, Shigella, E. coli, etc.)
Decreases the production of a variety of toxic or carcinogenic metabolites.
Aid absorption of minerals, especially calcium, due to increased intestinal acidity.
Production of β- D- galactosidase enzymes that break down lactose.
Produce a wide range of antimicrobial substances -acidophilin and bacteriocin etc. help to control pathogenic bacteria .
Produce vitamins (especially vitamin B and vitamin K).
Act as barriers to prevent harmful bacteria from colonizing the intestines.
Probiotics – health benefits
• diarrheal diseases (infective d., antibiotic d., travelers d.)
• inflammatory bowel disease (Crohn´s disease)
• prevention of colon cancer
• Helicobacter pylori (inhibition of growth)
• lactose intolerance
• blood cholesterol
• atopic dermatitis
• bacterial vaginosis and vaginal candidosis
properties strain-specific, no strain with all proposed benefits
Able to survive the passage through the digestive system.
Able to adhere to the intestinal epithelium and colonize.
Able to maintain good viability.
Able to utilize the nutrients and substrates in a normal diet.
Non pathogenic and non toxic.
Stabile the intestinal microflora and be associated with health benefits.
Stability of desired characteristics during processing, storage and transportation.
Characteristic of effective probiotics
Minimum Consumption:
100g of a probiotic food with 107 CFU/g (108-1011 CFU per day)
most probiotics do not permanently adhere in the intestine, but exert their effects as they metabolize and grow during their passage through the intestine (colonization). Thus, daily consumption of these bacteria is probably the best way to maintain their effectiveness
Probiotics - consumption
X
Prebiotics
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 (Marcel Roberfroid, 2007)
non-digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial to health (Marcel Roberfroid, 1995)
• trans-galacto-oligosascharides (TOS)
• inulin
• fructo-oligosaccharides (FOS)
inulin
Prebiotics
• non-digestible carbohydrates (mainly oligosaccharides and non-starch polysaccharides)
• act by promoting the growth and/or activity of probiotic bacteria in the gut
• they are found in various vegetables and fruit (tomatoes, onions, garlic, leeks, asparagus, bananas…)
• Prebiotics are relatively stable and, unlike probiotics, can be relied on to arrive relatively unchanged in the gut despite the presence of digestive enzymes.
Synbiotics
prebiotics and probiotics in the same product
Prebiotics – natural sources
Food Prebiotic Fiber
Arabic Gum (stabilizer E 414) 85.6%
Raw Chicory Root 64.6%
Raw Jerusalem Artichoke (topinambour) 31.5%
Raw Dandelion Greens 24.3%
Raw Garlic 17.5%
Raw Leek 11.7%
Raw Onion 8.6%
LAB – source of vitamins
• Folate (B-group, participate in DNA and RNA biosynthesis)
• Vitamin B12 (cobalamin, required for metabolism of fatty acids, AA, NA…)
• Vitamin K (involved in blood clotting, tissue calcification, bones function…)
• Riboflavin (B2, necessary in cellular metabolism)
cobalamin
folate
LAB – source of enzymes
• enzymes with a potential to influence the composition and the processing, organoleptic properties and quality of foods and feeds
• LAB release various enzymes into GIT – synergistic effect on digestion (alleviation of intestinal malabsorption)
• Lactococcus, Pediococcus
• natural improvement of bread texture (amylases), sensory quality of cheese improvement (peptidases)
• wine-making (flavor and aroma)
LAB – source of exopolysaccharides (EPS)
• long chain sugar polymers
• protection from toxic or limiting environmental conditions
• application – GRAS status
• improvement of rheology of fermented food (viscosity and elasticity), natural emulsifiers, gelling agents, stabilizers
• negative effect – food spoilage (fermentation of wine or cider) – final product with undesirable properties
http://www.scielo.br/scielo.php?pid=S0101-20612012000400011&script=sci_arttext dextran
LAB – source of low-calorie sweeteners
• mannitol • sorbitol • xylitol • tagatose
polyols (sugar alcohols)
GRAS substances
diabetic foods, sugar-free candies
Leuconostoc, Lactobacillus
sorbitol
xylitol
tagatose
mannitol
http://islab.tp.ugm.ac.id/files/2010/10/Biotechnology-of-Lactic-Acid-Bacteria-and-Their-Role-in-Food-Industry1.pdf
Homemade Yogurt
http://janasjournal.com
1 L of whole milk ¼ cup of plain milk yogurt
(live and active) (2 TS of dry milk)
• Heat the milk 82°C
• When the temperature drops to 45°C, stir in the ¼ cup of yogurt (and dry milk)
• pour the mixture into jars
• put in a slightly warm place for 8-12 hours (longer fermentation will yield a more tart yogurt)
• chill the yogurt (at least 3 hrs)
Bon Appetite !!!