Regulatory and Safety Requirements for Food Cultures.

The increased use of food cultures to ferment perishable raw materials has potentiated the need for regulations to assess and assure the safety of food cultures and their uses. These regulations differ from country to country, all aimed at assuring the safe use of food cultures which has to be guaranteed by the food culture supplier. Here we highlight national differences in regulations and review a list of methods and methodologies to assess the safety of food cultures at strain level, at production, and in the final product.

The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis BB-12(®).

This review presents selected data on the probiotic strain Bifidobacterium animalis subsp. lactis BB-12(®) (BB-12(®)), which is the world's most documented probiotic Bifidobacterium. It is described in more than 300 scientific publications out of which more than 130 are publications of human clinical studies. The complete genome sequence of BB-12(®) has been determined and published. BB-12(®) originates from Chr. Hansen's collection of dairy cultures and has high stability in foods and as freeze dried powders. Strain characteristics and mechanisms of BB-12(®) have been established through extensive in vitro testing. BB-12(®) exhibits excellent gastric acid and bile tolerance; it contains bile salt hydrolase, and has strong mucus adherence properties, all valuable probiotic characteristics. Pathogen inhibition, barrier function enhancement, and immune interactions are mechanisms that all have been demonstrated for BB-12(®). BB-12(®) has proven its beneficial health effect in numerous clinical studies within gastrointestinal health and immune function. Clinical studies have demonstrated survival of BB-12(®) through the gastrointestinal tract and BB-12(®) has been shown to support a healthy gastrointestinal microbiota. Furthermore, BB-12(®) has been shown to improve bowel function, to have a protective effect against diarrhea, and to reduce side effects of antibiotic treatment, such as antibiotic-associated diarrhea. In terms of immune function, clinical studies have shown that BB-12(®) increases the body's resistance to common respiratory infections as well as reduces the incidence of acute respiratory tract infections.

Evaluation of reuterin production in urogenital probiotic Lactobacillus reuteri RC-14.

Classified as a distinct species in 1980, Lactobacillus reuteri strains have been used in probiotic formulations for intestinal and urogenital applications. In the former, the primary mechanism of action of L. reuteri SD2112 (ATCC 55730) has been purported to be its ability to produce the antibiotic 3-hydroxypropionaldehyde (3-HPA), also known as reuterin. In the vagina, it has been postulated that probiotic Lactobacillus reuteri RC-14 does not require reuterin production but mediates a restoration of the normal microbiota via hydrogen peroxide, biosurfactant, lactic acid production, and immune modulation. The aim of the present study was to determine whether strain RC-14 produced reuterin. Using PCR and DNA dot blot analyses, numerous Lactobacillus species, including RC-14, were screened for the presence of the gene encoding the large subunit of glycerol dehydratase (gldC), the enzyme responsible for reuterin production. In addition, lactobacilli were grown in glycerol-based media and both high-performance liquid chromatography and a colorimetric assay were used to test for the presence of reuterin. L. reuteri RC-14 was determined to be negative for gldC sequences, as well as for the production of reuterin when cultured in the presence of glycerol. These findings support that the probiotic effects of L. reuteri RC-14, repeatedly demonstrated during numerous studies of the intestine and vagina, are independent of reuterin production.

Susceptibility of Streptococcus thermophilus to antibiotics.

A total of 74 Streptococcus thermophilus isolates collected between 1948 and 2005 from different environments were investigated to assess erythromycin, clindamycin, streptomycin, gentamicin, tetracycline and ampicillin susceptibility by means of microdilution, Etest and disk diffusion methods. For this purpose a new S. thermophilus Susceptibility test Medium (SSM) was developed. This medium allowed a better identification of strains with atypical tetracycline resistance. The recipe is a mixed formulation of Iso-Sensitest medium (90% v/v) and M17 medium (10% v/v) supplemented with lactose (0.5% w/v). The overall agreement of the techniques was good with exception of tetracycline, for which Etest provided lower MICs than the microdilution method. Most strains were susceptible to all the antibiotics tested while a few erythromycin, tetracycline and streptomycin resistant strains were detected.

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis strains isolated between 1936 and 1995.

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strains.

Susceptibility of Lactobacillus spp. to antimicrobial agents.

Bacteria used as probiotics or in starter cultures may serve as hosts of antibiotic resistance genes, which can be transferred to pathogenic bacteria. Before launching a starter culture or a probiotic product into the market, it is therefore important to verify that the single bacterial isolates (strains) do not contain transferable resistance genes. A study has been undertaken to establish the levels of susceptibility of Lactobacillus spp. to various antimicrobial agents. This is a prerequisite for differentiating putative transferable resistance from natural resistance. A selection of 62 strains has been screened with the use of the Etest (ABBiodisk, Stockholm, Sweden) for their susceptibility to 25 antimicrobial agents. The strains belonged to the following species: Lactobacillus plantarum/pentosus, L. rhamnosus, L. paracasei, L. sakei, L. curvatus and species of the L. acidophilus group: L. johnsonii, L. crispatus, L. gasseri, and L. acidophilus. The results from the Etests have shown that the level of susceptibility to the antimicrobial agents is species-dependent. For the following antimicrobial agents, susceptibility varied several folds between species: vancomycin, teicoplanin, tetracycline, norfloxacin, ciprofloxacin, fusidic acid, and clindamycin. The differences between the species were more subtle for the rest of the tested antimicrobial agents. On the basis of the result, it was possible to suggest minimal inhibition concentrations (MICs) for the individual Lactobacillus species to be used as a microbiological breakpoint when screening strains for transferable resistance genes.