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Table 5 Ingredients Used in Formulating Bulk Starter Media for Lactic Acid Bacteria

Carbohydrate

Nitrogen source

Vitamins and minerals

Phage-inhibitory agents

Antioxidants

Neutralizers

Lactose Maltose Sucrose Glucose

Milk protein Whey protein Hydrolyzed casein

Yeast extract Corn steep liquor

Phosphates Citrates

Ascorbic acid FeSO4

Carbonates Phosphates Hydroxides Oxides

Source: Whitehead et al., 1993.

Source: Whitehead et al., 1993.

<o media high enough to avoid acid injury is critical for producing cultures that consistently have sufficient activity for timely cheese manufacture. Acid injury in lactococci occurs when the pH declines below 5 (Harvey, 1965). Limited pH control can be achieved by addition of buffers to culture media. Buffering agents, such as phosphates and carbonate, allow development of higher cell concentrations, because the pH of the medium stays above 5 for a longer time. However, the neutralizing ability of the buffering agent is eventually overcome, exposing cells to excessive acidity. Also, high concentrations of buffers inhibit growth of some starter strains. Two approaches, internal and external pH control, are currently used to maintain growth media above pH 5 during culture preparation.

1. External pH Control

External pH control refers to a culture preparation system in which neutralizing agent is added to the medium during fermentation either manually or mechanically. There may be one or multiple additions of neutralizer. For one-step control, the pH of the medium is allowed to decrease to approximately 5, after which, sodium or potassium hydroxide is added to obtain a pH of 6.5-7 (Limsowtin et al., 1980). The culture is then allowed to incubate an additional 2 h before cooling. Multiple-step neutralization uses a mechanical system consisting of a pH electrode mounted in the bottom of the culture tank, a pump for adding ammonia to the tank, and a controller. When the pH declines below 5.8-6.2, the controller activates the pump to add ammonia until the pH is raised a certain amount (usually to 6-6.2). When acid production ceases because of lactose limitation, the culture is cooled (Thunell, 1988). External pH control has an additional advantage of requiring less phosphate for phage inhibition, because calcium is less soluble at higher pH. A disadvantage of external pH control is that the higher pH allows growth of nonstarter microflora even after lactose is depleted (Thunell, 1988). Therefore, a high degree of sanitation is required to implement this system. External pH control systems produce starter culture with 10 times greater cell concentration than phosphate-buffered media (Thunell, 1988). These cells are also healthier (i.e., they have no acid injury). The result is that a lower volume of starter can be used and milk ripening times are reduced. In addition, the culture produces acid more rapidly after salting (for Cheddar manufacture). More culture strains produce acceptable activity during cheese manufacture when external pH control is used for culture propagation as compared with conventional buffered media (Thunell, 1988).

2. Internal pH Control

Internal pH control describes a culture production system in which an insoluble neutralizing agent is added to the culture medium. The neutralizing agent is released in response to acid production. One means of achieving internal pH control is to use sodium carbonate encapsulated in magnesium stearate (Whitehead et al., 1993). Magnesium stearate dissolves at pH 5.2-5.3, releasing sodium carbonate. A similar effect is obtained by using buffer salts that are insoluble above a pH of 5.2 (Mermelstein, 1982). Sandine (1996) considered trimagnesium phosphate to be the most effective agent for this purpose. Internal pH control media have similar advantages to external pH control systems. In addition, a mechanism for adding neutralizing compound to the medium does not need to be installed. However, the fermentation tank must be stirred to keep the insoluble neutralizing agent suspended during fermentation. Agitation may lead to incorporation of sufficient oxygen into the medium to stimulate hydrogen peroxide production, resulting in autoinhibition of the culture (Mayra-Makinen and Bigret, 1993).

C. Incubation Conditions

Incubation temperature can affect activity and strain balance of the starter culture. Mesophilic cultures are grown at 21 °C if growth of leuconostocs is desired; otherwise, higher temperatures (up to 27°C) are used (McCoy and Leach, 1997). Incubation at 26°C helps maintain strain balance (Collins, 1976). Incubation is usually for 14-16 h or until a pH of 5 is reached. If pH control is not used, the final pH should be 4.8. Thermophilic cultures are incubated from 30 to 46°C for 8-10 h. A final pH value as low as 4.7 is acceptable, but this favors growth of lactobacilli (McCoy and Leach, 1997). Lower incubation temperatures favor growth of S. thermophilus and higher temperatures favor lactobacilli. Once the target pH is reached, the culture is cooled. Most cultures continue to produce acid during cooling. Mesophilic starters should be cooled to 5 to 7°C and thermophilic cultures to below 12°C (McCoy and Leach, 1997).

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