Different techniques were tested for studying the synergism between the micro‐organisms of sugary kefir grains. Agar cultures in Petri dishes did not give reproducible results. In sequential cultures, i.e. growing one organism, sterile filtering and then inoculating the other, 10 of 18 selected lactic acid bacteria/yeast pairs revealed stimulation of bacterial growth in a poor glucose medium. In mixed culture, Saccharomyces florentinus supported better survival of Lactobacillus hilgardii and a significant increase in lactic acid production; at the same time, the growth and alcoholic fermentation of S. florentinus were drastically reduced. The inter‐relationships between these two strains were the same when immobilized in calcium alginate beads, even though total metabolite production was always lower than with free cells. The stimulation of Lact. hilgardii by Candida lambica in sequential culture was not confirmed in mixed culture, where the two organisms grew as in pure culture, and bacterial growth and lactic acid production were inhibited in the immobilized system.

Stimulation of the growth of Lactobacillus hilgardii and production of lactic acid in the presence of Saccharomyces florentinus have already been observed in a poor glucose medium. In this study we showed that CO2, pyruvate, propionate, acetate and succinate excreted by the yeast were responsible for this phenomenon, whereas ethanol, fumarate and a cocktail of vitamins had no effect. Fermentation by the yeast did not enrich the total nitrogen or free amino acid content of the medium. The use of proteases confirmed that it was improbable that any nitrogenous compound could be responsible for the bacterial activation. Between values of 0.1 and 5 %, the initial yeast : bacteria ratio had no effect on the stimulation of Lact. hilgardii, even though yeast fermentation was drastically affected by a low initial value. The interactions differed greatly according to the sugar source. With monosaccharides, stimulation began after 48 h of culture, and was higher on glucose than on fructose. With sucrose, stimulation appeared at the beginning of fermentation. Different mechanisms seemed to be involved.

A variant ofLactobacillus hilgardii was immobilized by its own production of dextran gel, forming grains. The best rate of weight increase of the gel in continuous fermentation was 16.3±3.3%/h, at pH 4.8±0.1 and with a dilution rate of 0.22 to 0.26/h. Observation by scanning electron microscopy located most of the bacteria as microcolonies on the surface. A similar arrangement appeared in calcium alginate beads. The best population density (10(10) cells/g) was obtained in grains at pH 5.8, after 30h. At a similar pH value, 4.8, the growth rate was higher in alginate beads than in dextran gel but the final population density was approximately the same. Acidification rate increased faster with mixed gel at pH 5.2 than with dextran at pH 5.8.

Homo- and heterofermentative species of Lactobacillus have been isolated from sugary kefir grains. Most of the homofermentative strains fermented tagatose and aldonitol and presented 48-54% of homology with Lactobacillus paracasei ssp. paracasei NCDO 151 (ex Lactobacillus casei). The two variants of a hetero-fermentative species, although fermenting arabinose, were related to Lactobacillus hilgardii NCDO 264 (type strain) with 88% of homology. One of them produced polysaccharide from sucrose at pH 4.8 and 30 degrees C; the best glucose conversion into polysaccharide was obtained from 3% of sucrose (81.8%), and the maximum production occurred about 35 hours after the end of the log phase of growth, in MRS sucrose broth. Polysaccharide formation did not occur above 40 degrees C, a temperature at which no growth was observed. The two variants were forming minicells by abnormal divisions.

The bacterium Lactobacillus hilgardii (previously named L. brevis), isolated from sugary kefir grains, has been found to produce a gelling extracellular polysaccharide. Methylation analysis and 1H- and 13C-NMR analysis revealed a dextran-like structure for the polysaccharide. A high proportion of α-(1,6)-Glc and small proportions of α-(1,3,6)-Glc, α-(1,4,6)-Glc and terminal α-Glc were detected. The L. hilgardii polysaccharide differed from normal dextrans in containing a small proportion of α-(1,3)-Glc. The structure of the polysaccharide was found to be independent of harvesting time and unaffected by changes in the growth medium. The development of the gel structure has been followed by transmission electron microscopy.

The microflora of sugary kefir grains was principally mesophilic and consisted chiefly of lactic acid bacteria [Lactobacillus casei, Lactobacillus hilgardii (=brevis),Leuconostoc mesenteroides ssp.dextranicum, Streptococcus lactis] and a small proportion of yeasts (Zygosaccharomyces florentinus, Torulospora pretoriensis, Kloeckera apiculata, Candida lambica andC. valida). Few coliforms and faecal streptococci were observed. Observation by scanning electron microscopy revealed that the filamentous yeasts adhered to the bacteria on the periphery of the grain.Lactobacillus hilgardii, the single microorganism isolated which was able to produce a gelling polysaccharide, was important in the biosynthesis of the grain. Pieces of gel produced by this strain, and transferred in a yeast extract-sucrose solution, grew and resembled the household kefir grains. This represents a new, cheap way of producing immobilized cells by self-embedding in a neutral polysaccharide.

The gel-forming polysaccharide of the sugary kefir grains (11.5% of dry matter) or one taken from aLactobacillus brevis culture were identified as dextrans with some 1)-Gp-(3 links in the main chain, with a ratio (branched/total units) of 0.19 and 0.14 respectively, instead of 0.07 for the non-gelling polysaccharide.