Page 11 of 15
Enumeration of probiotic bacteria
Consumers buy probiotic products on the understanding that the genus and species designations are correct and that high concentrations of viable bacteria are present.
Traditional dairy products including yoghurts and cheese are used to deliver probiotic bacteria. This means that enumeration protocols must ensure that the selective agar media inhibit the growth of lactococci, Str. thermophilus and Lb. delbrueckii subsp. bulgaricus. However, they must also allow the selective enumeration of probiotic bacteria. In addition these bacteria have demanding nutritional requirements with the consequence that complex and relatively undefined basal media must be used.
The selective agents used include antibiotics, carbohydrates, 'salts' and bile. These can be used to develop selective agar media that when used with the correct gaseous environment and incubation temperature can give reliable quality assurance information.
Gentamicin, nalidixic acid, and neomycin are amongst the antibiotics that can be used to inhibit the growth of particular probiotic bacteria or LAB. In general Bifidobacterium species are relatively resistant to gentamicin, nalidixic acid and sensitive to neomycin (although less sensitive than starter bacteria and Lb. casei) while Str. thermophilus is sensitive to nalidixic acid and neomycin. Lb. acidophilus and Lb. bulgaricus are sensitive to all three antibiotics. In general lactococci are sensitive to low concentrations of these agents. Lb. casei, Lb. acidophilus and Lb. bulgaricus, lactococci and Str. thermophilus are sensitive to mupirocin while bifidobacteria are resistant.
Media containing maltose, raffinose, salicin or melibiose and other carbohydrates have been studied for their potential to select for bifidobacteria or Lb. acidophilus. Media containing maltose will permit the growth of many probiotic bacteria but will not enable the growth of most strains of Str. thermophilus and Lb. delbrueckii subsp. bulgaricus.
Relatively low concentrations of sodium chloride and other salts can be used to prevent the growth of Str. thermophilus, Lb. delbrueckii subsp. bulgaricus and Lc. lactis subsp. cremoris. Lithium chloride has also been found to be a useful selective agent.
Lactococci, Str. thermophilus and Lb. delbrueckii subsp. bulgaricus are inhibited by bile salts.
Since most strains of Lb. casei, unlike other probiotics, will grow at 15ºC temperature of incubation has generally been used as a selective factor to differentiate this organism from other probiotic bacteria. During studies of NSLAB growth in cheese, the author and colleagues noted the very high salt (sodium chloride) tolerance of Lb. casei and developed modified media that facilitated the isolation of this bacterium.