Different antimicrobial metabolites of lactic acid bacteria and its application in various system

1. Antimicrobial metabolites from Lactic Acid Bacteria
and its Application
Diwas Pradhan
Dairy Microbiology Division
Dec., 2019
National Dairy Research
Institute
Karnal-132001

2. Lactic Acid Bacteria
Gram +, non-sporeforming rods or cocci producing lactic acid as a major product
Old classification New classification
Lactobacillus Lactobacillus
Carnobacterium
Leuconostoc Leuconostoc
Oenococcus
Weisella
Streptococcus Streptococcus thermophilus
Lactococcus
Enterococcus
Vagococcus
Pediococcus Pediococcus
Aerococcus
Tetragenococcus

3. Lactic Acid Bacteria

4. Lactic acid
Propionic acid
Acetic acid
Antimicrobial
substances
LAB
produces
Organic acids
Low molecular
weight
compounds
Bacteriocins
Reuterin
Diacetyl & Acetaldehyde
H2O2
Fatty acids
Phenyl lactic acids
Nisin
Lacticin
Pediocin
Gassericin
Antimicrobial compounds produced by LAB

5. Antifungal compounds produced by LAB
Siedler et al., 2019

6. “Primary metabolic end products of carbohydrate metabolism”
Organic Acids
Most important: lactic acid and acetic acid
 General mechanism: Undissociated forms are lipid soluble and can penetrate
the cell membrane of target bacteria and release H+ ions inside. The internal
pH gets destabilized leading to alteration in the cellular functionality and
ultimately cell death.
 Specific mechanism:
 Formate and Acetate against E. coli: hampered bacterial stress response
 Lactate against B. cereus: Disruption of protein metabolism in addition to
inducing oxidative stress (Mols and Abee et al., 2011).

7. APPLICATIONS
Organic Acids
 Highly effective; inhibit wide range of microbial targets
 GRAS status
 As biopreservative (meat, animal feeds); Juices and bevarages: acidity
regulator
 Synergistic effect with other antimicrobial
 Forms complexes with metals and help permeabilize
 Salt form
 Organic acids when neutralized can form salts
 Stable, wide spectrum antimicrobial activity, Sodium lactate can
work as humectants and emulsifier.

8. “Bacteriocins are ribosomally-synthesized peptides or proteins with
antimicrobial activity, produced by different groups of bacteria”
BACTERIOCINS
 Bacteriocins of lactic acid bacteria play a defining role in the
preservation & microbial safety of foods
 Bacteriocins may have broad or narrow spectrum of activity
 Bacteriocins with broad spectrum of activity are of great importance
in food safety while Bacteriocins with narrow spectrum of activity
may be used for specified use

9. Bacteriocins of lactic acid bacteria
Safe and efficacious use of nisin for >40 years in several countries
(GRAS status)
Effective under wide pH & temperature range
Activity is not lost in the presence of food additives and effective in
dairy foods during storage
Effective in low concentrations
Consumer resistance to traditional chemical preservatives and
concern over the safety of existing food preservatives such as sulfites
and nitrites
Do not alter acceptance quality of food
What makes LAB bacteriocins as promising agent for their use in biopreservation

10. Alvarez-Seirio et al., 2016
Classification of bacteriocins

11. APPLICATIONS
Food Applications
•Alternative to chemical preservation
Medical applications
• Emergence of Antimicrobial resistance
•Veterinary applications

12. 1
Food Preservation and Safety
Using a purified/semi-purified bacteriocin
preparation as an additive in food
Incorporation of an ingredient previously
fermented with a bacteriocin-producing strain
Use of a bacteriocin-producing culture to
replace the starter culture in fermented foods
to produce the bacteriocin in situ
Traditional methods for incorporation of bacteriocin preparations in foods

13. 1. Use of purified/ semi purified bacteriocins
Commercially available purified / semipurified Bacteriocins
Nisaplin (Danisco) containing 1.82% Nisin
Lantibiotic produced by L. lactis
GRAS and approved as a food preservative by WHO
2) Use of an ingredient previously fermented with a bacteriocin-producing strain
MicroGARDTM ALTA™ 2341
DANISCO, Denmark
Skim milk fermentate by Propionibacterium
Approved by FDA (1990) and granted GRAS
status
Quest International, US
Fermentate of Pediococcus acidilactici
fermentation

14. 3)Use of a bacteriocin-producing culture in fermented foods to produce the bacteriocin in
situ
The use of cultures to produce bacteriocins in situ
A more natural method of shelf-life extension and improving the safety of foods
BS-10®
Nisin producing L. lactis spp. lactis, Chr. Hansen
BIOPROFIT™
L. rhamnosus LC705, BioGaia
BOVAMINE Meat CulturesTM
Texas Tech University
HOLDBAC™
L. plantarum, L. rhamnosus, L. sakei, L. paracasei and P.
freundenreichii ssp. shermanii, DANISCO

15. 1
Medical Applications
•Mostly on animal models with no clinical trails
•Nisin effective against H. pylori infection
•Commercialized for treatment H. pylori peptic ulcers (Dicks et al., 2011)
•Pediocin stopped translocation of listeria and reduced its levels in mice
•Oral health
•Nisin containing mouthwash against gingivitis in dogs
•Skin health
•Bacteriocin ESL5 containing topical lotion against acne causing P.
acnes (Kang et al., 2009)

16. 1
Veterinary Applications
•Use of bacteriocins to treat mastitis
•Clinical cure rate similar to that gentamycin (Cao et al., 2007)
•Development of nisin based teat sealer (comparative effect to
conventional treatment)
•Lacticin 3147 based teat dip (Klostermann et al., 2010)
•GI tract infection in animals
•Bacteriocin OR-7 reduced colonization of C. jejuni in chickens (Stern et
al., 2006)
•Clostridia infection in Swine by Pediocin PA-1 (Casadei et al., 2009)
•Bacteriocins as growth promoter in poultry (nisin and pediocin)

17. 1
Reuterin
•Produced by L. reuteri from glycerol
•L. brevis, L. coryniformis, L. collinoides and L. buchneri
•3-Hydroxy propionaldehyde, its hydrate and dimer
•Effective against a wide variety of target organisms
•Induction of oxidative stress in target cells.
• Toxicity issue
•Presence of acrolein in the reuterin mix

18. 1
Reutericyclin
•Antimicrobial N-acylated tetramic acid produced mainly by L.
reuteri
•proton-ionophore causing translocation of protons across cell
membrane by disrupting transmembrane proton gradient
•Mainly active against Gram positive
Reutericyclin

19. 1
Phenyllactate (PLA)
•First identified in L. plantarum 21B
•Potent antifungal as well as antibacterial agent
•No cytotoxicity; absence of any objectionable odour
•dual antibacterial targets
•Membrane and Genomic DNA

20. 1
Fatty Acids
•Some strains of LAB
•Antimicrobial Free FA or its hydroxyl derivative
•Both antibacterial and antifungal properties
•Longer chain length acids more active than short chain fatty acids
•Structure containing at least 1 hydroxyl group and 1 degree of
unsaturation in their carbon- very active
•Disrupts the membrane integrity by partitioning the lipid
bilayers of fungal species
•Yeast more responsive

21. 1
Volatile Compounds
•Diacetyl
•Mainly heterofermentative LAB from pyruvate (Citrate
metabolism)
•Important flavor compound in butter and Dahi
•Gram negative are more sensitive than Gram positive bacteria
•Carbon dioxide
•CO2 creates an anaerobic environment by replacing the
existent molecular oxygen
•inhibition of enzymatic decarboxylation.

22. 1
Volatile Compounds
•Ethanol
•Some Heterofermentative LAB ethanol from glucose via the
hexose monophosphate or pentose pathway.
•Ethanol denatures proteins and dissolves lipid membranes of
target bacteria.
•Hydrogen peroxide (H2O2)
•H2O2 is produced by many LAB strains in the presence of O2
•Strong oxidizing effect on the bacterial cell

23. 1
Application in Food Products
•DAIRY PRODUCTS
•Fermented milk products especially cheese and yoghurt
•BAKERY
•LAB as natural protectants as an alternative to chemical preservatives like benzoates and
sorbates
•FRUITS AND VEGETABLES
•Raw and processed fruits as well as vegetables
•ANIMAL FEED
•L. buchneri is used as silage additive
•Feedtech Silage F3000, a commercial bioprotectant as silage inoculant contains strain of L.
plantarum MiLAB 393.
•MISCELLANEOUS APPLICATIONS
•barley malt extract fermentation; other Beverages
•Rice cake and other processed foods
•Meat products (raw poultry meat and raw smoked sausages)
Kadyan & Pradhan, 2019

24. 1
Application in Food Products
Product Name Application Scope Protective Cultures Company
Holdbac Culture
Series
Fermented dairy products including cheese and
yoghurt
L. rhamnosus, L. plantarum and P.
freudenreichii subsp. shermanii
DuPont Danisco
FreshQ Culture
Series
Cottage cheese, Yoghurt, sour cream L. rhamnosus and L. paracasei CHR Hansen
MicroGard Fermented Sausages, Bakery/ Dairy products
and Cured meat
Skim milk-Fermentate of P.
freudenreichii subsp. shermani
DuPont Danisco
Hi Shield P Bakery and Salad dressings Corn-Fermentate of LAB and
yeasts
HI-FOOD S.p.A.
Befresh Fresh fermented milk products L. paracasei and P. freudenreichii
subsp. shermanii
Handary
Inhibit Culture
Series
Cheese dips and spreads, Bakery products, Meats Fermentate (wheat, whey, brown
rice) of P. freudenreichii
Mezzoni Foods
DelvoGuard Yogurt, sour cream and fresh cheese L. rhamnosus and L. sakei DSM
BIOPROX® RP 80 Stirred/SetYoghurt, fermented milk and cheeses L. rhamnosus and L. plantarum
Bioprox
BIOPROX® RP 83 Stirred/SetYoghurt, fermented milk and cheeses
L. plantarum
BIOPROX® RP 94 Fresh/continental cheeses and cheddar cheese
M-CULTURE Safe
3100 SSL
Raw Sausages L. plantarum and L. curvatus Meat Cracks
Technologie
GmbH
Dairy Safe™ Cheeses Defined Mesophilic D-type lactic
acid bacteria
CSK Food
Enrichment B.V.
Salas et al., 2017

25. Development of resistance against
antimicrobial
Natural degradation over time
Complex interaction with food matrix
Consumption of whole of the
antimicrobial in killing of target
microbes
(Balasubramanian et al., 2011)
Drawbacks of instant addition of antimicrobial
Antimicrobial becomes ineffective due to:-

26. Antimicrobial
packaging
(AMP)
Microencapsulation
Nanoencapsulation
Hurdle
technology
1 2
3 4
Contd…….
Newly emerged techniques for incorporation of bacteriocin preparations in
foods

27. 1) ANTIMICROBIAL PACKAGING (AMP)
“Anti Microbial Packaging (AMP) is the packaging system that is able to kill or inhibit
spoilage and pathogenic organism that are contaminating foods”
AMP
Bacteriocins of
LAB
Pre-existing
Packaging
concept
Antimicrobial packaging film prevents microbial
growth on food surface by direct contact of the
package with the surface of foods
The classical protective function of packaging is
supported by the antimicrobial action of relevant
bacteriocins

28. 2) Microencapsulation
“It is a technology whereby the target molecule is packaged in miniature, sealed capsules
to protect it from external factors and deliver in the targeted site under specific
conditions. ”
Minimizes bacteriocin resistance development and helps
to achieve controlled release of bacteriocins

29. 3) Nano-encapsulation
Small metabolites may not be efficiently retained in porous microcapsules which may
lead to leakage or inefficient delivery at the target site.
Hence many times primarily encapsulation of bacteriocins in nanosized liposomes is
popularly followed.
Nanoliposomes provide more
surface area
Improve the distribution in the
food system along with the
bioavailability of the
encapsulated bacteriocins
Added directly to the food
items
Malheiros et al. 2012

30. 4) Bacteriocins as part of Hurdle Technology
“ Use of hurdles of differing levels of intensity to bring
microbiological growth under control”

31. WAY FORWARD
Identify new antimicrobial compounds for application in foods
Altering the specificity of existing antimicrobial compounds
Increasing the level of antimicrobial compounds production
Development of antimicrobial compounds producing lactic starters
through gene transfer system
Continued study of physical and chemical properties

32. Winter School, NDRI, 2015
THANK Y
U