Factors Affecting Fermentation Process of Starters

The fermentation process of LAB starters can be influenced by several factors such as temperature, pH, strain capability, growth medium, inhibitors, bacteriophage, incubation period, heat treatment of milk, etc. There is a need to take adequate precautions to achieve the optimal activity of lactic acid bacteria during the manufacture of fermented milks. Some of the important factors affecting the growth and activity of lactic starter cultures as discussed below:

i. Temperature

The performance of lactic acid bacteria during milk fermentation is influenced by a number of factors. Temperature is one of the important factors, which directly effect the growth of microorganisms. Although different type of lactic acid bacteria have different optimal temperatures for their growth, the majority of lactic starters such asL. lactis subsp lactis, L. lactis subsp cremoris, etc. grow optimally at 27-32oC.On the other hand, S. thermophilus and some lactobacilli grow best at 37-42oC temperature range. However, Leuconostocs have their optimal temperature at 20- 30oC. This variation in optimal temperature requirement may be a very important factor in strain dominance in mixed and multiple starters.

ii. pH

The pH is one of the most important factors which can influence the growth and metabolic activity of lactic starter cultures during milk fermentations. Although milk is considered to be nearly the perfect food for man as well as bacteria, it still needs some enrichment for use as a bulk starter medium. Lactic acid bacteria produce lactic acid at the level of more than 10 per cent of their weight per minute after their growth in milk and thus, pH of milk is lowered. The extreme acidic pH could be detrimental for the viability of LAB. Hence, control of pH in milk during propagation of starters is very important. This fact is generally ignored during the commercial production of bulk starters. Both externally and internally pH controlled media used for the preparation of bulk starters. Another method for controlling pH is consisting of diffusion culture technique for the removal of toxic end products of metabolism of starter cultures by dialysis. In this context, the preparation of starter culture concentrates in low lactose milk is also promising as this can eliminate the possibility that acid from lactose could be detrimental to their own viability and survival. When starter cultures are allowed to grow till the pH falls below 5.0, considerable injury to the cells results, which could adversely affect the performance of these organisms.

In externally pH controlled whey medium, the pH drop is controlled by an insoluble buffer so that the lactic acid produced is immediately neutralized. Another promising and practical approach to tackle this problem could be through genetic manipulation of these organism by introducing a pH sensitive promoter for regulation of structural genes involved in acid production.

iii. Strain Compatibility

Mixed starters have been used for the preparation of several fermented dairy products. However, maintenance of mixed strain starters in cheese factories is not much practiced any longer, partially because repeated subculture of mixed strains of lactococci may result in decrease in number or loss of all but one of the strains that eventually a single strain remains in the mixed starter preparation. Some of the factors responsible for strain dominance or overgrowth in a mixed culture by one strain include differences in generation times, acid sensitivities, the production of antibiotics or bacteriocins by the component strains, differences in optimum temperature and rates of plasmid loss etc. The main objective of using a mixture of strains in mixed culture is that even if one strain become infected by a specific bacteriophage, the other strains would be unaffected and enable the culture to produce adequate acid during bulk culture preparation. Same thing may hold good for antibiotic resistance of some strains in the mineral cultures which will not be affected by the presence of inhibitory substances in milk and the milk fermentation cultures also plays a very important role in bringing about the desirable changes in milk.

iv. Growth Medium

The media used for the cultivation of LAB are quite complex. The most widely used growth media are MRS, M-17 and Lactic or Elliker’s medium. Although Elliker’s medium is of choice, M-17 is also an excellent growth medium and is extensively used for the growth of lactococci. Several plating media have been described for differentiation of different species of lactococci, namely, L. lactis subsp. lactis, L. lactis subsp. cremoris and L. lactis subsp. diacetilactis. Another medium on which Lactococci can be differentiated is one to distinguish between fast and slow growing cultures. The medium is designated as Fast Slow Differential Agar (FSDA). Fast colonies (lac+, prt+) on this medium appear 1-3 mm in diameter, shiny white, convex and surrounded by a red zone against the blue background of the medium indicating lactose fermentation. Slow colonies (lac+, prt+) are 0.2-0.5 mm in diameter, translucent and flat. Apart from these media, lactic acid bacteria also grow very well in milk. However,it is pointed out that during specific seasons of the year, more inoculum may be needed during milk fermentation in order to achieve the same type of acid production obtained with lower inoculum rates at other times of the year.

v. Inhibitory Substances

The growth and activity of the starter cultures in milk is adversely affected due to the presence of residual antibiotics and sanitizers in milk as well as the production of antibiotic-like substances (bacteriocins) by certain wild strains of Lactococcus lactis subsp. lactis and other lactic cultures in raw milk. Antibiotics such as penicillin or streptomycin may enter milk as a result of their indiscriminate use in the treatment of mastitis or udder diseases. Hence, milk must be thoroughly monitored for the presence of residual antibiotics before addition of starter cultures. The methods based on immunological reactions as well as isotopic tracer dilution procedures (Charm test)

are very effective.

vi. Bacteriophages

Bacteriophages are considered as one of the single most important factors causing slow acid production by lactic acid bacteria in the commercial environment. The most promising solution to this problem could be by replacing the phage sensitive strains with phage resistant one. The use of defined single strains and their phage resistant mutants is becoming increasingly popular throughout the world for the manufacture of various fermented foods.

vii. Incubation Period

The period of incubation is another important factor, which can affect the growth of lactic acid bacteria. The higher the temperature upto a certain limit, the faster does the culture pass through its growth phases. Normally, 16-24 hr incubation is adequate for the maximal growth of these organisms at their optimal temperatures. However, storage of the ripened starters for about 18 hrs at low temperature does not appear to affect their activity, although over-ripened cultures are adversely affected on prolonged storage.

viii. Heat Treatment of Milk

Heat treatment of milk usually improves its value as a medium for starter organisms and other lactic acid bacteria. Adequate heat treatment of milk drives out the dissolved oxygen, brings about formation of sulphydryl compounds (acting as growth factors), destruction of inhibitory substances naturally present in milk and killing of antagonistic bacteria. However, with more severe heating, slight protein breakdown may occur with the formation of peptides and amino acids, which act as nutrients. In addition to these, different species of lactic acid bacteria appears to behave differently in heattreated milks. For example, the growth of S. thermophilus appears to be favored, while L. lactis subsp. cremoris disfavored by drastically heat treatment of milk.

ix. Degree of Aeration

The lactic acid bacteria appear to be indifferent to aeration of the medium or slightly preferring a reduced oxygen tension. It can be stated that acid production is faster at the bottom of the container or under controlled oxygen tension condition (i.e. reduced oxygen tension than that of atmosphere). Possibly, a reduced level of oxygen tension is favorable for the initiation of growth as it affords energy for growth by a mechanism somewhat more efficient than the lactic fermentation, which releases only a small fraction of the energy available in the lactose. Aeration, agitation and the surface culture usually depress the activity of lactic acid bacteria. However, agitation is obviously a vague condition and may sometimes accelerate souring. It clearly includes two quite different factors, namely, oxygenation and movement of the medium, which appear to have opposing effects on the starter cultures. Although excessive aeration may be the cause of slow starters, its effects may be neutralized by heating milk or by adding sulphydryl compounds.

x. Effect of Carbon Dioxide

A minimum concentration of carbon dioxide is essential for the initiation of bacterial growth. Complete removal of carbon dioxide from a medium results in extended lag phase until the bacteria have slowly produced sufficient carbon dioxide to sustain normal growth. For most of the lactic acid bacteria, the optimum initial concentration of CO2 varies from 0.2 – 2.3% by volume. Sterilized skim milk may contain only 0.3-0.5% of CO2 and this may account for the prolonged lag phase of the given starter culture. However, incorporation of yeast extract in milk at a concentration of 0.5% can get rid of this problem.

xi. Storage conditions

Storage of lactic acid bacteria is yet another important factor affecting their performance during the manufacture of fermented milk products. It is desirable not to store the mature cultures in presence of acid that they have produced during growth. Storage under these conditions will result into cellular injury and promote the loss of plasmids, which may increase the proportion of slow cells in the population. Such cultures will become slow and can no longer be useful for the preparation of fermented products. Therefore, it is important that during the maintenance of such cultures, they should be transferred to fresh milk and in refrigerator without incubation. When a new culture is needed, it is removed from the refrigerator, incubated and transferred to fresh medium and placed in the refrigerator again. However, mature cultures may also be stored at 2-5oC either in milk with added calcium carbonate or in stabs of selective media. Cultures can also be frozen and stored at -40oC or below in the freeze-dried state. Frozen storage in liquid nitrogen (-196oC) in the form of starter concentrates also affords stability to cultures.