Lactic acid bacteria and production of cultured dairy …old-biomikro.vscht.cz/vyuka/ifm/Lactic_probiotics_prebio...LAB – sugar metabolism Homofermentative LAB •hexoses by Embden-Mayerhof

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

Type IA – pore formation

wedge type barrel-stave type

Class I – Lantibiotics: Mode of action

Type IB – inhibition of cell wall synthesis

Class I – Lantibiotics: Mode of action

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″

Class IIA – structure x action

receptor = man-PTS (mannose phosphotransferase system)

Class IIA – mode of action

pore forming

• 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″

pore formation transmembrane „helix-helix“ structure

Class IIB – mode of action

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

Starter cultures

P Florou-Paneri, E Christaki, E Bonos - 2013 - cdn.intechopen.com

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

ATB resistance

Probiotics - safety

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

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

Parvéz et al: J.App.Microb. 100 (2006) 1171–1185

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

Characteristic of effective probiotics

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 !!!

Hana Sýkorová [email protected]

Thank you for your attention!