Graciela L. De Antoni

Chemical and microbiological characterisation of kefir grains

Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied. Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition. In contrast, protein and yeast content of AGK3 was higher than in the other grains. Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species. Lactococcus lactis subsp. lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain. While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains. All grains produced acid products with pH between 3.5 and 4.0. The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4. All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h. Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences. Despite their fermenting activity, AGK4 grains did not increase their weight.

Inhibition of Giardia intestinalis by Extracellular Factors from Lactobacilli: an In Vitro Study

ABSTRACT The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent.Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G1 phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation ofGiardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.

Lactobacillus johnsonii La1 Antagonizes Giardia intestinalis In Vivo

ABSTRACT This study describes the in vivo activity of Lactobacillus johnsonii La1 (NCC533) in Giardia intestinalis-infected gerbils (Meriones unguiculatus). Daily administration of lactobacilli in the drinking water from 7 days before inoculation with Giardia trophozoites efficiently prevented G. intestinalis strain WB clone C6 from infecting gerbils. More specifically, shedding of fecal Giardia antigens (GSA65 protein) was diminished in the La1-treated group, and resolution of infection was observed by 21 days postinoculation. Histology and analysis of enzymatic markers of microvillus membrane integrity revealed that probiotic administration also protected against parasite-induced mucosal damage. In addition, a cellular response to Giardia antigens was stimulated in spleen cells from La1-treated gerbils. Results show for the first time the antigiardial effect of probiotic lactobacilli in vivo and provide further insight into the antagonistic properties of lactic acid bacteria against protozoa involved in intestinal infections.

Isolation and characterization of Bifidobacterium strains for probiotic formulation

Twenty-five Bifidobacterium strains isolated from infant feces were identified by sugar fermentation patterns and whole-cell protein analysis. Using gradient SDS-PAGE, six characteristic protein bands of the genus were detected in 40 strains of bifidobacteria but not in lactobacilli. Computerized numerical analysis enabled strains to be grouped in two main clusters. Strains of Bifidobacterium bifidum belong to a well-differentiated cluster that joins the cluster of the remaining species at 0.582 similarity. The predominant species among isolated strains from infant feces were B. bifidum, B. longum, and B. breve. Probiotic and technological indicators such as surface properties, inhibitory capacity, resistance to bile and low pH, and ability to grow under aerobic conditions were studied. Not all desirable characteristics were present in a single strain. In general, adherent and inhibitory strains were not resistant to bile, low pH, and aerobic conditions. Only 10 of 40 strains were resistant to 0.5% bile.

Lactobacilli isolated from kefir grains: evidence of the presence of S-layer proteins

In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66-71 kDa as measured by SDS-PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.

Cellular injuries of spray-dried Lactobacillus spp. isolated from kefir and their impact on probiotic properties

The injuries caused by spray drying (SD) of three potential probiotic lactobacilli isolated from kefir grains and the impact on some probiotic properties, were evaluated. Results demonstrated that Lactobacillus plantarum 83114 and L. kefir 8321 showed a slight reduction of viability (0.11 and 0.29 log CFU/ml respectively) after SD process, and L. kefir 8348 was found to be more sensitive to the process with a reduction in viability of 0.70 log CFU/ml. Neither membrane damage, evaluated by increased sensitivity to NaCl, lysozyme, bile salt and penicillin G, nor changes in acidifying activity in MRS and milk by lactobacilli were detected after SD. L. plantarum 83114 and L. kefir 8321 after SD did not lose their capacity to adhere to intestinal cells. Nevertheless, L. kefir 8348 showed a significant loss of adhesion capacity after SD. In addition, rehydrated spray-dried L. kefir 8321 retained the ability to protect against Salmonella invasion of intestinal cells. This effect was observed when L. kefir is co-incubated with Salmonella before invasion assay. This work shows that the membrane integrity evaluated by indirect methods and some probiotic properties of lactobacilli isolated from kefir did not change significantly after SD, and these powders could be used in functional foods applications.

Characterization of homofermentative lactobacilli isolated from kefir grains: potential use as probiotic

Considering that several health promoting properties are associated with kefir consumption and a reliable probiotic product requires a complete identification of the bacterial species, the present work evaluates several proved markers of probiotic potential of eleven isolates of homofermentative lactobacilli isolated from kefir grains and molecular identification and genotypic diversity. Using restriction analysis of amplified ribosomal DNA (ARDRA) and analysis of the 16S-23S rRNA internal spacer region we confirmed that all homofermentative lactobacilli belong to the species Lactobacillus plantarum. RAPD-PCR analysis allowed the discrimination of lactobacilli in five clusters. All isolates exhibited high resistance to bile salt. High survival after one hour of exposure to pH 2.5 was observed in Lb. plantarum CIDCA 8313, 83210, 8327 and 8338. All isolates were hydrophilic and non autoaggregative. Isolate CIDCA 8337 showed the highest percentage of adhesion among strains. All tested lactobacilli had strong inhibitory power against Salmonella typhimurium and Escherichia coli. Seven out of eleven isolates showed inhibition against Sal. enterica and five isolates were effective against Sal. gallinarum. Only CIDCA 8323 and CIDCA 8327 were able to inhibit Sal. sonnei. We did not find any correlation between the five clusters based on RAPD-PCR and the probiotic properties, suggesting that these isolates have unique characteristics.

Fatty acid composition and freeze–thaw resistance in lactobacilli

The fatty acid composition and freeze-thaw resistance of eight strains of thermophilic lactobacilli were studied. Seven of these contained the same polar and neutral lipids, the five major components making up 90% of the cellular fatty acid pool being 14:0, 16:0, 16:1, 18:1 and C19 cyclopropane (cyc19:0). Strain comparison by means of cluster analysis based on the fatty acid ratios using the overlap coefficient revealed two well defined clusters. One was formed by three strains of species Lactobacillus delbrueckii subsp. lactis and Lb. delbrueckii subsp. delbrueckii, the other included five strains of the species Lb. delbrueckii subsp. bulgaricus, Lb. acidophilus and Lb. helveticus. Resistance of strains with a high content of unsaturated fatty acids (66-70%) decreased with increasing cyc19:0 concentrations. In contrast, in strains with a low concentration of unsaturated fatty acids (42-49%), increasing cyc19:0 levels were associated with increased freeze-thaw resistance.

Characteristics of kefir prepared with different grain[ratio]milk ratios

Kefir is a traditional fermented milk originating many centuries ago in the Caucasian mountains. It is produced by fermentative activity of ‘kefir grains’ consisting mainly of lactococci, lactobacilli and yeasts in a protein–polysaccharide matrix. The grains contain a relatively stable and specific balance of microorganisms which exist in a complex symbiotic relationship. The grains grow in the process of kefir making only from pre-existing grains (Saloff-Coste, 1996). When kefir grains are allowed to grow in milk, microorganisms are shed from the grains into milk where they continue to multiply with the production of acid, flavour and physicochemical changes.The traditional method of kefir making is currently by adding kefir grains directly as starter to milk that has been pasteurized and cooled to 20–25°C. After a period of fermentation lasting ∼24 h, the grains are removed by filtration and the beverage is ready for consumption (Saloff-Coste, 1996). Kefir from which the grains have been removed may be used as starter. However, this fermented milk cannot be used for subsequent inoculations to make an acceptable product, because the original balance of microorganisms has been disrupted (Kroger, 1993).The complex microbiological composition of kefir grains explains why it is difficult to obtain starter with the optimal and constant composition necessary for a regular production of kefir of standard quality (Koroleva, 1988a). Studies have been undertaken to establish cultivation conditions[ratio ]grain[ratio ]milk ratio, cultivation temperature, period of time and conditions prior to separation of grains from the fermented milk, shaking conditions for agitation of milk with the grains in the course of fermentation, washing of kefir grains and so on. All these factors influence the microflora of the kefir starter and fermented milk. There are no rules about household manufacture of kefir. Different reports indicate a wide range of grain[ratio ]milk ratios for kefir making. Bottazzi & Bianchi (1980), Marshall & Cole (1985), Merin & Rosenthal (1986), Mann (1989), Hosono et al. (1990) and Kroger (1993) employed 20–50 g/l while Koroleva (1988a) employed 20–100 g kefir grain/l and Marshall et al. (1984) and Neve (1992) 50–100 g/l. Rea et al. (1996) used 1 g kefir grain/l as starter and 200 g starter in the form of kefir grains is recommended by Hansen for the fermentation of 1 l milk (Marshall & Cole, 1985). A critical control point in kefir manufacture to obtain a product with constant quality is the standardization of the kefir grain[ratio ]milk ratio. Koroleva (1988b) claimed that it is better to use kefir grains as starter for kefir production and, at the same time, to decrease the amount of inoculum.The purpose of this study was to evaluate the effects of changes in the kefir grain[ratio ]milk ratio (quantity of kefir grains inoculated into the milk) on microflora composition, acidity, apparent viscosity and carbon dioxide content of fermented milk.

Molecular identification and typing of lactobacilli isolated from kefir grains

Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.