Lactulose in combination with soybean lecithin has a cryoprotective effect on probiotic taxa of bifidobacteria and Lactobacillaceae.

Lactulose is commonly used in pharmacy for constipation and hepatic encephalopathy treatment. The prebiotic effect of lactulose is also often mentioned. However, its cryoprotective effect in combination with lecithin on the main representatives of probiotics has not been tested yet. The 12 taxa of bifidobacteria and Lactobacillaceae members were used for the purpose. These were mixed in a ratio of 1:1 with lactulose + lecithin (finally 5.0% and 1.25%, respectively; LL). The 25% glycerol (G+) solution and cultures themselves were applied as positive and negative controls, respectively. Bacterial suspensions were stored at a mild freezing temperature (-20°C) until the end of the experiment (210th day). The LL solution had a comparable (insignificant difference at the P-value = 0.05) cryoprotective effect as the positive control in five of six bifidobacteria and in three of six representatives of Lactobacillaceae. The better cryoprotective effect was revealed in other Lactobacillaceae. At the end of the experiment, the generally accepted therapeutic minimum (>107 Colony Forming Units/mL) was determined in LL solution in five bifidobacteria and four Lactobacillaceae strains. The presented results improve knowledge about long-term mild cryopreservation of the most commonly used probiotics and could contribute to developing new forms of (nutri)synbiotics.

Enteral Nutrition as a Growth Medium for Cultivable Commensal Bacteria and Its Effect on Their Quantity in the Stool of Children with Crohn's Disease.

Current studies indicate a link between the intake of exclusive enteral nutrition (EEN) and the induction of complex changes in the intestinal microbiota, as well as the clinical improvement of Crohn's disease (CD). The first aim of this study was to test the ability of various commensal bacterial strains (n = 19) such as bifidobacteria, lactobacilli, and Escherichia coli to grow on three different polymeric EN in vitro. Tested EN formulas were found to be suitable growth media for tested commensals. Furthermore, the counts of these bacteria and total counts of anaerobic bacteria in the fecal samples of children with CD (n = 15) before and after 6 weeks of EEN diet administration were determined using cultivation on selective media. The counts of cultivable commensal bacteria in the fecal samples of CD children were not significantly affected by EEN. However, tested bacteria showed some individual shifts in counts before and after EEN therapy. Moreover, cultured bifidobacteria were found to be in reduced counts in CD children. Therefore, the application of bifidogenic prebiotic compounds to EN for CD patients might be considered.

A new medium containing mupirocin, acetic acid, and norfloxacin for the selective cultivation of bifidobacteria.

Various culture media have been proposed for the isolation and selective enumeration of bifidobacteria. Mupirocin is widely used as a selective factor along with glacial acetic acid. TOS (transgalactosylated oligosaccharides) medium supplemented with mupirocin is recommended by the International Dairy Federation for the detection of bifidobacteria in fermented milk products. Mupirocin media with acetic acid are also reliable for intestinal samples in which bifidobacteria predominate. However, for complex samples containing more diverse microbiota, the selectivity of mupirocin media is limited. Resistance to mupirocin has been demonstrated by many anaerobic bacteria, especially clostridia. The objective was to identify an antibiotic that inhibits the growth of clostridia and allows the growth of bifidobacteria, and to use the identified substance to develop a selective cultivation medium for bifidobacteria. The susceptibility of bifidobacteria and clostridia to 12 antibiotics was tested on agar using the disk diffusion method. Only norfloxacin inhibited the growth of clostridia and did not affect the growth of bifidobacteria. Using both pure cultures and faecal samples from infants, adults, calves, lambs, and piglets, the optimal concentration of norfloxacin in solid cultivation media was determined to be 200 mg/L. Our results showed that solid medium containing norfloxacin (200 mg/L) in combination with mupirocin (100 mg/L) and glacial acetic acid (1 mL/L) is suitable for the enumeration and isolation of bifidobacteria from faecal samples of different origins.

Comparison of mupirocin-based media for selective enumeration of bifidobacteria in probiotic supplements.

An international standard already exists for the selective enumeration of bifidobacteria in milk products. This standard uses Transgalactosylated oligosaccharides (TOS) propionate agar supplemented with mupirocin. However, no such standard method has been described for the selective enumeration of bifidobacteria in probiotic supplements, where the presence of bifidobacteria is much more variable than in milk products. Therefore, we enumerated bifidobacteria by colony count technique in 13 probiotic supplements using three media supplemented with mupirocin (Mup; 100mg/l): TOS, Bifidobacteria selective medium (BSM) and modified Wilkins-Chalgren anaerobe agar with soya peptone (WSP). Moreover, the potential growth of bifidobacterial strains often used in probiotic products was performed in these media. All 13 products contained members of the genus Bifidobacterium, and tested mupirocin media were found to be fully selective for bifidobacteria. However, the type strain Bifidobacterium bifidum DSM 20456 and collection strain B. bifidum DSM 20239 showed statistically significant lower counts on TOS Mup media, compared to BSM Mup and WSP Mup media. Therefore, the TOS Mup medium recommended by the ISO standard cannot be regarded as a fully selective and suitable medium for the genus Bifidobacterium. In contrast, the BSM Mup and WSP Mup media supported the growth of all bifidobacterial species.

Prebiotic effects of a novel combination of galactooligosaccharides and maltodextrins.

Prebiotics are used for stimulating the growth of beneficial microorganisms in the gut. However, it is very difficult to find a suitable prebiotic mixture that exclusively supports the growth of beneficial microbes such as bifidobacteria and lactobacilli. We tested the effects of a prebiotic mixture in vitro by incubating it with fecal samples and in vivo by administration of the prebiotic supplement to healthy adult volunteers, followed by analysis of their fecal microbiota. The effect of the oligosaccharides on bacterial metabolism was studied by analyzing short-chain fatty acid (SCFA) production in vitro and the SCFA pattern for the stool samples of volunteers. In the in vitro test, a higher proportion of bifidobacteria (25.77%) was seen in the total bacterial population after cultivation on a prebiotic mixture than on the control medium (7.94%). The gram-negative anaerobe count significantly decreased from 8.70 to 6.40 log CFU/g (from 35.21% to 0.60%) and the Escherichia coli count decreased from 7.41 to 6.27 log CFU/g (from 1.78% to 0.44%). Administration of a prebiotic mixture in vivo (9 g of galactooligosaccharides [GOS]+1 g of maltodextrins; daily for 5 days) significantly increased the fecal bifidobacterial count from 9.45 to 9.83 log CFU/g (from 40.80% to 53.85% of total bacteria) and reduced the E. coli count from 7.23 to 6.28 log CFU/g (from 55.35% to 45.06% of total bacteria). The mixture comprising GOS and maltodextrins thus exhibited bifidogenic properties, promoting the performance of bifidobacteria by boosting their growth and inhibiting the growth of undesirable bacteria.

Mupirocin-mucin agar for selective enumeration of Bifidobacterium bifidum.

Bifidobacterium bifidum is a bacterial species exclusively found in the human intestinal tract. This species is becoming increasingly popular as a probiotic organism added to lyophilized products. In this study, porcine mucin was used as the sole carbon source for the selective enumeration of B. bifidum in probiotic food additives. Thirty-six bifidobacterial strains were cultivated in broth with mucin. Only 13 strains of B. bifidum utilized the mucin to produce acids. B. bifidum was selectively enumerated in eight probiotic food supplements using agar (MM agar) containing mupirocin (100 mg/L) and mucin (20 g/L) as the sole carbon source. MM agar was fully selective if the B. bifidum species was presented together with Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium longum subsp. longum species and with lactic acid bacteria (lactobacilli, streptococci). Isolated strains of B. bifidum were identified using biochemical, PCR, MALDI-TOF procedures and 16S rRNA gene sequencing. The novel selective medium was also suitable for the isolation of B. bifidum strains from human fecal samples.