Many variables can affect the fermentation process of lactic acid bacteria (LAB) starters, including temperature, pH, strain capability, growth medium, inhibitors, bacteriophage, incubation period, heat treatment of milk, etc. To achieve the best activity of lactic acid bacteria during the manufacturing of fermented milks, caution is required. The following parameters significantly affect the growth and activity of lactic starter cultures:

1. Temperature

• Temperature is one of the key factors that directly affect the development of microorganisms.
• Although different kinds of LAB have varied ideal growth temperatures, the majority of Lactic starts, including L.lactis subsp.lactis and L.lactis subsp. cremoris«HWF, grow optimally between 27 and 32 degrees Celsius.
• In contrast, S.thermophilus and some lactobacilli thrive between 37 and 42 degrees Celsius. And Leuconostocs develop optimally between 20 and 30 degrees Celsius.
• Temperature change affects the dominance of strains in mixed and multiple starts.

2. pH

• Controlling the pH of the milk throughout the multiplication of the starting culture is essential, as excessive acidity might be damaging to the viability of LAB.
• Even though milk is believed to be the best diet for humans and bacteria, it must be enriched before it can be used as a mass starting medium.
• After their development in milk, lactic starters produce lactic acid at a rate more than 10 percent of their weight per minute; thus, the pH of milk is decreased.
• Extremely acidic pH may be harmful to the viability of LAB. Controlling the pH of milk during the propagation of starters is crucial.
• During commercial production of bulk starters, this feature is typically disregarded. Externally and internally pH-controlled media used for bulk starter preparation. pH can be changed via:
  1. Continuous neutralisation is used for the preparation of frozen concentrates.
  2. The elimination of S. cerevisiae’s hazardous metabolic byproducts using a diffusion culture approach.
  3. These organisms were genetically modified by inserting a pH-sensitive promoter that regulates structural genes involved in acid generation. 

3. Strain compatibility 

• For the creation of numerous fermented dairy products, a combination of starters was used.
• In any case, the maintenance of mixed strain starters in cheese processing plants is no longer a priority, in part due to the fact that repeated subculture of mixed strains of lactococci can lead to a decrease in number or loss of all but one strain, so that in the long run only one strain remains in the mixed starter preparation.
• Variations in development durations, acid sensitivity, the formation of antibiotic or bacteriocin by the component strains, and changes in ideal temperature all contribute to strain supremacy.
• In addition, antibiotic resistance of some strains in the mineral cultures, which is unaffected by the presence of inhibitory compounds in milk and milk fermentation cultures, plays a crucial role in bringing about the desired alterations in milk.
• In mixed cultures, if one strain is sick, the infection will not spread to the others. The associative activity between individual strains in mixed cultures plays a significant role in producing beneficial alterations in milk.

4. Growth medium

• Complex media are utilised for the development of LAB. Mediums MRS, M17, and Lactic or Eliker are used to cultivate LAB.
• Lactic medium and M-17 are excellent growth media and are often used for lactococci growth.
• L. lactis subsp. lactis, L. lactis subsp. cremoris, and L. lactis subsp. Diacetilactis have been separated using a few different plating media.
• Additionally, LAB thrive in milk, and during some seasons, more inoculum is required for milk fermentation.
• Late lactation milk and milk produced during the winter are typically low in substances that stimulate the growth of LAB; therefore, it is necessary to add stimulatory elements to these milks.

5. Inhibitors 

• The presence of residual antibiotics and sanitizers in milk and the generation of antibiotic-like compounds (bacteriocins) by certain wild strains of Lactococcus lactis subsp. lactis and other lactic cultures in raw milk inhibit the growth and activity of starter cultures in milk.
• Antibiotics, such as penicillin or streptomycin, may enter milk because of their unpredictability in treating mastitis or udder diseases.
• Before adding starter cultures, milk must be thoroughly examined for the presence of residual antibiotics.
• Immunological reactions and isotopic tracer dilution processes (Charm test) are highly effective techniques.

6. Bacteriophage

• Bacteriophages or phages are bacteria-infecting viruses. They are supposed to represent the most numerous organisms.
• These bacterial viruses are found in habitats containing bacteria, including ecological niches created by humans, such as food fermentation vats.
• These bacteriophages inhibit LAB acid synthesis. Phage infection of starter LAB cultures is the most prevalent cause of slow or incomplete fermentation in the dairy sector, despite intensive efforts.
• Infection by phages is the most influential biological influence on enterprises reliant on bacterial growth and metabolic activity.
• Depending on the stage of the process at which the infection spreads, the implications may range from slowed acid production to batch loss.
• Phage assaults that occur during the early stages of fermentation result in high pH values, excessive residual lactose concentration, and insufficient lactic acid content. Replacement of phage-sensitive bacteria with phage-resistant variants is a promising option.
• In various regions of the world, the usage of identified single strains and their phage-resistant mutations is widespread.

7. Incubation period 

• The length of the incubation period is another crucial aspect that can affect the proliferation of lactic acid bacteria.
• Up to a point, the greater the incubation temperature, the quicker the development of S. coli.
• Typically, 16 to 24 hours at optimal temperature is sufficient for maximum growth of these organisms. Storage of ripened starters at a low temperature for around 18 hours has little effect on their activity, however prolonged storage has an effect on overripe cultures.

8. Heat treatment of milk 

• Generally, milk that has been heated is a better medium for starting organisms and other lactic acid bacteria.
• Different LAB species respond differently to heat-treated milk. Numerous benefits emerge from the proper heating of milk, including:
  • It eliminates dissolved oxygen.
  • It results in the production of sulphydryl chemicals (acting as growth factors).
  • Destruction of naturally occurring inhibitory compounds in milk and elimination of antagonistic bacteria. The synthesis of peptides and amino acids, which function as nutrition, may occur when proteins are subjected to more intense heating. In addition, several kinds of lactic acid bacteria appear to function differently in milks that have been heated. By significantly heating milk, the growth of S. thermophilus appears to be encouraged, but L. lactis subsp. cremoris is inhibited.

9. Degree of aeration 

• The lactic acid bacteria (LAB) prefer a medium with a lower oxygen tension than the atmosphere, therefore acid generation is more rapid toward the bottom of the container or under regulated oxygen conditions (reduced oxygen tension).
• Possibly, a low oxygen tension is optimal for the commencement of growth because it controls energy for growth through a mechanism that is somewhat more efficient than lactic fermentation, which releases just a small fraction of the energy available in lactose.
• However, agitation is undeniably a precarious state and can occasionally accelerate deterioration. It comprises two distinctive variables, namely oxygenation and medium mobility, which appear to have opposing impacts on the beginning cultures.
• Although high aeration may be the cause of a sluggish starter, its effects can be countered by heating the milk or adding sulphydryl chemicals.

10. Effect of carbon dioxide 

• A minimum concentration of carbon dioxide is required for bacterial growth to begin.
• Complete removal of carbon dioxide from the medium prolongs the lag phase until the bacteria create slowly enough carbon dioxide to support normal development.
• The optimal concentration of carbon dioxide for LAB ranges between 0.2% to 2.3%. minuscule amount of CO2 Sterilized skim milk may prolong the lag phase of a particular starting culture.
• However, incorporating yeast extract into milk at a 0.5% dosage can eliminate this issue.

11. Storage conditions

• During the production of fermented milk products, the storage of lactic acid bacteria is a crucial aspect that affects their function.
• It is advised against storing mature cultures in the presence of the acid they created during growth.
• Under these conditions, cellular damage will occur, and the cultures will become sluggish and unusable for the preparation of fermented products.
• When a new culture is required, it is removed from the refrigerator, incubated, and then transferred to fresh medium before being placed back in the refrigerator.
• Mature cultures may also be kept between 2 and 5 degrees Celsius in milk containing calcium carbonate.
• Freeze-dried cultures can also be frozen and stored at temperatures below -40 °C. Starter concentrations frozen in liquid nitrogen (-196 °C) at a temperature of -196 °C.