STARTER CULTURES AND FERMENTED MILK PRODUCTS

Module 5. Quality and activity of starters

Lesson 7
ANTIMICROBIAL COMPOUNDS PRODUCED BY STARTERS AND INTERACTIONS AMONG STARTER CULTURES

7.1 Introduction

Starter cultures are known to produce a number of antimicrobial compounds during the preparation of fermented milk products. These antimicrobial compounds are very effective against many Gram positive bacteria and exhibit therapeutic properties. The starter cultures grow in association with other bacterial cultures and such association will vary depending upon the characteristics of the organisms grown in a particular environment.

7.2 Antimicrobial Compounds

Fermented milks are well known for their therapeutic properties. This is primarily due to their ability to fight against intestinal pathogens. The effect is exerted by LAB through production of several antimicrobial compounds and /or creation of unfavourable conditions for other bacteria. The primary compounds responsible for such effects are,

1. Organic acids: Lactic acid, acetic acid, propionic acid etc
2. Hydrogen ion concentration (pH)
3. Low oxidation reduction potential
4. H₂O₂ and CO₂
5. Aroma compounds- diacetyl, acetaldehyde
6. Fatty acids
7. Bacteriocins

7.2.1 Organic acids

Lactic acid is the major metabolite of LAB fermentation. At low pH it is found in its undissociate form and is toxic to many microorganisms.

Acetic acid and propionic acid are more antimicrobial than lactic acid due to higher pKa values.

The antimicrobial effect of organic acid lies in the reduction of pH as well as the undissociate forms of molecules. The low external pH causes acidification of cell cytoplasm, while the undissociate form of molecule may cause alteration in the cell membrane permeability, collapsing the electrochemical proton gradient that results in disruption of substrate transfer system.

7.2.2 H₂O₂ and CO₂

Some lactobacilli are known to produce H₂O₂ in the presence of flavoprotein oxidase or NADH peroxidase from glycerol, under aerobic conditions. The antimicrobial activity of H₂O₂ results from the oxidation of –SH groups causing denaturation of number of enzymes and from the peroxidation of membrane lipids thus the increased membrane permeability. H₂O₂ also acts as a precursor for the production of bactericidal free radicals such as superoxide O₂^- , hydroxides –OH that can damage DNA.

Flavoprotein Oxidase

Glycerol ------------------------------------ H₂O₂

Xanthine oxidase

Acetaldehyde ------------------------------- H₂O₂

Glucose oxidase

Lactobacillus species and Lactococcus species can produce H₂O₂ while CO₂ is mainly produced by heterofermentative LAB. The precise mechanism of its action is still unknown, but it is believed that it may play a role in creating an anaerobic environment which inhibits enzymatic decarboxylation reactions and the accumulation of CO₂ in the membrane lipid bilayer may cause defunctioning in permeability.

7.2.3 Aroma compounds

Diacetyl produced by LAB inhibits the growth of Gram negative bacteria by reacting with the arginine (Arg) binding protein, thus affecting Arg utilization & finally interfere with protein synthesis.

Acetaldehyde can also be toxic to many bacteria and it is converted to H₂O₂ in the presence of xanthine oxidase or glucose oxidase activities.

Xanthine oxidase

Acetaldehyde -------------------------------- H₂O₂

glucose oxidase

7.2.4 Fatty acids

Under certain conditions some lactobacillus and lactococci possessing lipolytic activity may produce significant amount of fatty acids. The unsaturated fatty acids are active against many Gram +ve bacteria and antimicrobial action may be due to the undissociated molecule.

7.2.5 Bacteriocins

These are proteinaceous compounds produced by bacteria which are inhibitory to many bacterial species.

As shown in Table 7.1 many LAB produces bacteriocins & other antibiotic like proteineous substances. These substances are found to be active against many spoilage and pathogenic microorganisms. Their nature and structure is not well known, except for nisin. However, many reports claim them to be proteinous in nature, which are stable at low pH, high temperature and many other adverse conditions. The pathways of their metabolism are still to be studied. Further it is known that production of such compounds is encoded by plasmid DNA.

Table 7.1 Bacteriocins produced by lactic acid bacteria

Sr. No.	Species	Compound
1.	Lactococcus lactis subsp lactis	Nisin
2.	Lactococcus lactis subsp cremoris	Diplococcin
3.	Lb. reuteri	Reuterin (non-protein)
4.	Lb. acidophilus	Acidolin Acidophilin Lactocidin
5.	Lb. brevis	Lactobacillin Lactobrevin
6.	Lb .bulgaricus	Bulgaricin
7.	Lb .fermenti	Bacteriocin
8.	Lb. helveticus	Lactacin 27 Helveticin J
9.	Lb. plantarum	Lactolin
10.	Pediococcus acidolactici	Pediocin Bacteriocin
11.	S. thermophilus	Unnamed

Nisin: It has a narrow spectrum anti-bacterial activity and does not inhibit Gram –ve bacteria, yeast, or fungi. Closely relative organisms like Lactococcus lactis subsp cremoris are more sensitive. It is effective against spore formers. Its synthesis occurs as pronisin in the cell and it converts form pronisin to nisin in the outer layer of the cell.

7.3 Types of Interactions among Starter Cultures

Microbial association in the same environment can be Neutralism, Antagonistic (negative) and Symbiosis (Positive)

a) Neutralism: This type of association is most unlikely as the two organisms living in a close proximity are not affected by each other. This may exist between two organisms whose growth requirements are quite different and hence affect neither kind as there will be no competition between these two for nutrients.

b) Antagonistic association: When an organism adversely affects the environment of another organism it is said to be antagonistic. Antibiosis is antagonistic association between two organisms in which one is adversely affected

Eg. Production of antibiotic or inhibitory substances by one organism that affect the growth or survival of another organism. Lactic acid bacteria produce lactic acid that is inhibitory to spoilage organisms

c) Symbiosis: Symbiosis is defined in the dictionary as the relationship between two (or more) organisms that live in a close association that may but is not necessarily of benefit to each. This dictionary definition is a bit misleading. In the vast majority of symbioses one or both partners gain something positive from the association. A pair of symbionts may be able to live separately, but they almost always do better in the long run by living together.

d) Mutualism – each organism benefits from the association but the manner in which benefit is derived varies

i) Exchange of nutrients between two species (Syntrophism)

ii) Association results in metabolic end products which are different from association as compared with sum of the products of separate species.

eg. Proteus vulgaris ferment lactose and produce acid, Staphylococcus aureus

ferment lactose and produce acid but together they produce gas and acid

e) Commensalisms: It refers to a relationship between microorganisms in which one organism benefits from the association but the other organism is not affected. Host organism by its growth affects the physical or physiological environment in such a way that the commensal species is favoured.

f) Facultative organism grows and produces anaerobic conditions that favour growth of anaerobes

g) Growth of yeasts in sugar solutions reduce the concentration of sugar thus permitting growth of bacteria

h) Synergism: The ability of two or more organisms to bring about an effect greater than the sum of their individual effects, (or the changes usually chemical in nature that neither can accomplish alone)

The growth association between the two organisms (S. thermophilus and Lactobacillus delbrueckii subsp bulgaricus) of the yoghurt starter culture is termed a symbiosis. The two lactic acid bacteria grown in association in milk and their growth is considered symbiotic because the rate of acid development was greater when mixed yoghurt cultures of S. thermophilus and Lactobacillus delbrueckii subsp bulgaricus bacteria were used as compared with the single strains.

When mixture is inoculated into the milk, the cocci grow much more rapidly than rods often out numbering the rods by three or four to one at the end of the first one hour of the incubation at 45°C. Thus the initial acid production is mainly due to activity of S. thermophilus. Gradually rods increase in number and at the end of incubation period they again approximate the cocci numbers. Acid production during that latter part of the incubation period is accomplished by Lactobacillus delbrueckii subsp bulgaricus.

Lactobacillus delbrueckii subsp bulgaricus stimulates S. thermophilus by releasing several amino acids (example histidine, leucine, lysine, cystine, valine etc.,) and the most important being Valine. S. thermophilus produces formic acid which promote the growth of Lactobacillus delbrueckii subsp bulgaricus. The other stimulatory compounds produced by S. thermophilus are pyruvic acid and carbon dioxide.