Viable Intestinal Passage of a Canine Jejunal Commensal Strain Lactobacillus acidophilus LAB20 in Dogs

The strain Lactobacillus acidophilus LAB20 with immunomodulatory properties was previously found dominant in the jejunal chyme of four dogs, and the novel surface layer protein of LAB20 suggested its competitive colonization in canine gut. To evaluate the persistence and survival of LAB20 in healthy dogs, LAB20 was fed to five healthy pet dogs for 3 days, at a dosage of 10⁸ CFU daily as fermented milk supplement. The fecal samples, from 1 day prior to feeding, three continuous feeding days, and on day 5, 7, 14, and 21, were collected for strain-specific detection of LAB20 using real-time PCR. We found that LAB20 count was significantly increased in dog fecal samples at the second feeding day, but rapidly decreased after feeding ceased. The fecal samples from prior to feeding, during feeding, and post-cessation days were plated onto mLBS7 agar, from where LAB20 was recovered and distinguishable from other fecal lactobacilli based on its colony morphotype. Using strain-specific PCR detection, the colonies were further verified as LAB20 indicating that LAB20 can survive through the passage of the canine intestine. This study suggested that canine-derived strain LAB20 maintained at high numbers during feeding, viably transited through the dog gut, and could be identified based on its colony morphotype.     

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1_Lp and ram2_Lp) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramA_La). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1_Lp, Ram2_Lp, and RamA_La were able to efficiently hydrolyze rutin and other rutinosides, while RamA_La was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1_Lp and RamA_La enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1_Lp for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.Lactobacilli such as Lactobacillus plantarum have been used for centuries to ferment vegetables such as cabbage, cucumber, and soybean (34). Fruit pulps, for instance, those from tomato, have also been used as a substrate for lactobacilli for the production of probiotic juices (38). Recently, the full genomic sequences of several lactobacilli have become available (1, 22). A number of the plant-based substrates for lactobacilli are rich in rhamnose sugars, which are often conjugated to polyphenols, as in the case of cell wall components and certain flavonoid antioxidants. Utilization of these compounds by lactobacilli would involve α-l-rhamnosidases, which catalyze the hydrolytic release of rhamnose. Plant-pathogenic fungi such as Aspergillus species produce the rhamnosidases when cultured in the presence of naringin, a rhamnosilated flavonoid (24, 26). Bacteria such as Bacillus species have also been shown to use similar enzyme activities for metabolizing bacterial biofilms which contain rhamnose (17, 40).In food processing, rhamnosidases have been applied primarily for debittering of citrus juices. Part of the bitter taste of citrus is caused by naringin (Fig. ​(Fig.1),1), which loses its bitter taste upon removal of the rhamnose (32). More recently, application of rhamnosidases for improving the bioavailability of flavonoids has been described. Human intake of flavonoids has been associated with a reduced risk of coronary heart disease in epidemiological studies (19). Food flavonoids need to be absorbed efficiently from what we eat in order to execute any beneficial function. Absorption occurs primarily in the small intestine (12, 37). Unabsorbed flavonoids will arrive in the colon, where they will be catabolized by the microflora, which is then present in huge quantities. Therefore, it would be desirable for flavonoids to be consumed in a form that is already optimal for absorption in the small intestine prior to their potential degradation. For the flavonoid quercetin, it has been demonstrated that the presence of rhamnoside groups inhibits its absorption about fivefold (20). A number of flavonoids which are present in frequently consumed food commodities, such as tomato and citrus products, often carry rutinoside (6-β-l-rhamnosyl-d-glucose) or neohesperidoside (2-β-l-rhamnosyl-d-glucose) residues (Fig. ​(Fig.1).1). Therefore, removal of the rhamnose groups from such flavonoid rutinosides and neohesperidosides prior to consumption could enhance their intestinal absorption. With this aim, studies were recently carried out toward the application of fungal enzyme preparations as a potential means to selectively remove rhamnoside moieties (16, 30).Open in a separate windowFIG. 1.Chemical structures of rhamnose-containing flavonoids from plants. Relevant carbon atoms in glycoside moieties are numbered. (1) Rutin (quercetin-3-glucoside-1→6-rhamnoside); (2) narirutin (naringenin-7-glucoside-1→6-rhamnoside); (3) naringin (naringenin-7-glucoside-1→2-rhamnoside); (4) p-nitrophenol-rhamnose.In view of the frequent occurrence of lactobacilli on decaying plant material and fermented vegetable substrates, one could anticipate that their genomes carry one or more genes encoding enzymes capable of utilizing rhamnosilated compounds. In the work reported here, we describe the identification of three putative rhamnosidase genes in lactobacillus genomes. We expressed these genes in Escherichia coli and characterized their gene products. The activities of all three lactobacillus rhamnosidases on flavonoids naturally present in tomato pulp were then assessed. One of the L. plantarum genes, which encoded the enzyme with the highest activity and stability in E. coli, was then also expressed in Lactococcus lactis, with the aim of investigating the potential use of such a recombinant organism to improve the bioavailability of fruit flavonoids and thus their efficacy in common foodstuffs.     

Sites of Cellular Autolysis in Lactobacillus acidophilus

Ultrastructural changes which occur during cellular autolysis of Lactobacillus acidophilus strain 63AM Gasser in 0.05 M citrate buffer, pH 5.0, were examined. Early in the process, randomly distributed electron-dense patches were seen on the wall surface, along with an accompanying eversion of mesosomes. Later, after a loss of about 20% of the initial cellular turbidity, dissolution from the outside of nascent cross walls was seen. This observation was related to the normal process of cell separation. After this stage, short lengths of the cylindrical portion of the wall appeared to be completely removed in a random manner over the entire surface. This dissolution produced gaps in the wall which allowed the extrusion of membrane and cytoplasm. Although membrane was usually extruded through one major, polar, subpolar, or septal site, other secondary points of membrane extrusion were also frequently seen in the same cell section.     

Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri.

Sixteen strains of Lactobacillus reuteri and 20 strains of Lactobacillus acidophilus were tested for resistance to 22 antibiotics by using commercially available sensitivity disks. Evidence suggesting linkage of these resistances to plasmids was obtained by "curing" experiments with acridine dyes and high growth temperatures. Examination of plasmid patterns of agarose gel electrophoresis provided further evidence of loss in plasmid DNA under curing conditions in some of the strains examined.     

Degradation of Thymidine by Lactobacillus acidophilus

Whole cells of Lactobacillus acidophilus are capable of degrading thymidine to thymine, suggesting the presence of thymidine phosphorylase (or thymidine hydrolase). This activity was also demonstrated in cell-free extracts.     

降胆固醇嗜酸乳杆菌的诱变选育

通过诱变获得具有降胆固醇功能的优良嗜酸乳杆菌新菌株。利用亚硝基胍(作用浓度为1 g/L)对嗜酸乳杆菌进行诱变。突变菌株测定其耐渗透压和抗胆盐能力后,在含有0.3 g/L胆固醇的培养基中培养48 h,测定降胆固醇率。挑选优良突变菌株制成酸奶并喂养高脂大鼠模型,28 d后测定血清及粪便胆固醇指标。嗜酸乳杆菌突变后获得的60个突变菌株中有8株具有良好的耐渗透压和抗胆盐能力,其中突变株Y48的清除胆固醇能力最高,清除率达到(61.44±1.8)%。Y48发酵酸奶喂养高脂大鼠模型28 d后与对照大鼠模型相比,血清中TC、TG明显减少(P<0.05),粪便TC明显增加。通过诱变获得了优良的嗜酸乳杆菌突变菌株,为今后获得优质降胆固醇乳酸制品提供良好的候选菌种。     

Assimilation of cholesterol by Lactobacillus acidophilus

Considerable variation was found among strains of Lactobacillus acidophilus isolated from the fecal flora of pigs with regard to the ability to grow well in the presence of bile and to assimilate cholesterol from a laboratory growth medium. The uptake of cholesterol occurred only when the culture(s) was growing in the presence of bile under anaerobic conditions. Consumption of L. acidophilus RP32, which was selected for its ability to grow well in the presence of bile and to assimilate cholesterol from the laboratory medium, significantly inhibited increases in serum cholesterol levels of pigs (P less than 0.05) fed a high-cholesterol diet. Consumption of L. acidophilus P47, which was selected for its ability to grow in the presence of bile and lack of ability to remove cholesterol from the growth medium, failed to have a similar effect. This indicates that certain strains of L. acidophilus act directly on cholesterol in the gastrointestinal tract, and thus may be beneficial in reducing serum cholesterol levels.     

Specific antigens of Lactobacillus acidophilus.

Antigens specific for Lactobacillus acidophilus were investigated by double immunodiffusion in agar-gel. Antigenic materials were extracted from whole bacteria and some walls with cold trichloroacetic acid. Antisera were prepared by intravenous injection into rabbits of suspensions of whole organisms in solutions of bovine serum albumin, which had been heated and then washed. Four specific antigens were found as precipitinogens and denoted as antigens 11, 12, 13 and 14. Of 43 strains of L. acidophilus studied, 33 strains possessed antigen 11, six strains antigen 12, two strains antigen 13 and two strains antigen 14. Sugar compositions of wall preparations were analysed in an attempt to characterize the determinants of antigens 11 and 12. The walls contained glucose, galactose, hexosamine and sometimes glycerol, but no rhamnose was found. It was considered that alpha-glucopyranose was the major component of the determinant of antigen 11 since trehalose and maltose significantly inhibited the reaction between antibody 11 and its antigen; the determinant of antigen 12 was not clarified.     

Characteristics of the Jejunal Microbiota in 35-Day-Old Saba and Landrace Piglets

The balanced microbiological system is a significant hallmark of piglet health. One of the crucial factors affecting intestinal microbiota is the host’s genetics. This study explored the difference in the diversity of jejunal microbiota between Saba (SB) and Landrace (LA) piglets. Nine Saba and nine Landrace piglets were fed with sow’s milk until day 35. Jejunal contents were harvested for 16S rRNA sequencing. The birth weight, body weight, and average daily gain of Saba piglets were lower than those of Landrace piglets (p < 0.01). Firmicutes were the main phylum in Saba and Landrace piglets, and the Saba piglets had a higher (p < 0.05) abundance of Bacteroidetes compared with Landrace piglets. The two most abundant genera were Lactobacilli and Clostridium XI in the jejunum of Landrace and Saba piglets. Compared with Landrace piglets, the Saba piglets had significantly lower (p < 0.05) abundance of Veillonella, Streptococcus, and Saccharibacteria genera incertae sedis. The functional prediction showed that “d-glutamine and d-glutamate metabolism” and “one carbon pool by folate” pathways were enriched in Saba piglets, while “limonene and pinene degradation”, “tryptophan metabolism”, and “sulfur relay system” pathways were enriched in Landrace piglets. In summary, the growth performance was higher for Landrace piglets compared with Saba piglets due to their genetic characteristics. The rich diversity and fewer infection-associated taxa were observed in Saba piglets, partially accounting for their higher adaptability to environmental perturbations than Landrace piglets. Furthermore, different pig breeds may regulate their health through different metabolic pathways.Key words: Saba piglets, Landrace piglets, jejunal content, the 16S rRNA gene, diversity     

Adaptive acid tolerance response in Lactobacillus acidophilus

Stationary phase cultures of Lactobacillus acidophilus CRL 639 are naturally acid resistant to pH 3.0 while exponential phase cells induce an acid tolerance response upon exposure to sublethal pH (3.8–6.0). Maximal response was achieved after adaptation at pH 4.2 and pH 5.0. Protein synthesis was required in the latter case. © Rapid Science Ltd. 1998     

Temperature adaptation and cryotolerance in Lactobacillus acidophilus

The response of Lactobacillus acidophilus CRL 639 to cold-shock (freezing) depends on the initial growth temperature. Cells cultured at 25°C (long filaments with a value of 0.02 h–1) show a higher -galactosidase activity and develop cryotolerance, compared with cells grown at 37°C. One-dimensional proteins gel electrophoresis shows the over-expression of six proteins (22.5–105.0 kDa) and the repression of eight proteins (4.3–48.0 kDa) at 25°C. © Rapid Science Ltd. 1998     

Genetic transformation of rifampicin resistance in Lactobacillus acidophilus

Lactobacillus acidophilus strain 100-33, originally isolated from swine faeces, was transformed to rifampicin resistance with DNA from spontaneous rifampicin-resistant mutants derived from it. Cells of the recipient strain were treated with lysozyme and mutanolysin, mixed with donor DNA and polyethylene glycol and grown on a regeneration medium overnight. After 48 h incubation, the numbers of rifampicin-resistant cells in the populations of regenerated cells were estimated from numbers of colonies. Efficiency of the lysozyme/mutanolysin treatment (the ratio of the number of osmotically fragile cells after the enzyme treatment to the initial cell number) was about 99%. The regeneration frequency of the enzyme-treated cells varied from 5 to 67%. The transformation frequency varied from about 0.2 X 10(-8) to 8.0 X 10(-8) transformants per regenerated cell per microgram DNA. To our knowledge, this method for genetic transformation is the first to be reported for a Lactobacillus strain.     

Growth of Lactobacillus acidophilus in the absence of folic acid

Soska, Jirí (Kansas State University, Manhattan). Growth of Lactobacillus acidophilus in the absence of folic acid. J. Bacteriol. 91:1840-1847. 1966.-A chemically defined medium, containing no folic acid, was used for the cultivation of Lactobacillus acidophilus R-26. In such a medium, the organism required thymine in addition to a deoxyriboside, purines, pyrimidines, and most amino acids. If thymine was present in this medium, an unlimited exponential growth was possible. The influence of the components of this medium on the growth is described. The concentration and type of adenine compounds in this medium were most important. Adenine and adenosine inhibited utilization of thymine, but not of thymidine, whereas adenylic acid inhibited recovery from amino acid starvation. In the absence of thymine or deoxyribosides, cells continued to grow in length, and after 3 hr a slow decline in viable count ensued.     

On the biogenesis of cytokinins in Lactobacillus acidophilus ATCC 4963

The biosynthesis of free cytokinins in the mevalonic acid auxotrophic Lactobacillus acidophilus , ATCC 4963 has been investigated. After a short pulse labelling with [¹⁴C]-mevalonic acid the labelled free cytokinins of bacteria and media and the labelled cytokinin-nucleotide moiety of tRNA and oligonucleotides were determined and compared. tRNA is the main precursor for cytokinin production. Bacteria previously starved for mevalonic acid showed the presence of at least one additional cytokinin precursor. A fraction of oligonudeotides shows rapid incorporation of ¹⁴C and contains labelled cytokinin nucleotides. There are no indications for a direct isopentenylation of adenine, adenosine or its phosphate derivatives.