Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.

We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.     

Osmotically Regulated Transport of Proline by Lactobacillus acidophilus IFO 3532

[This corrects the article on p. 2831 in vol. 57.].     

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.     

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.     

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.     

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.     

Inhibition of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei and Lact. acidophilus against Shigella sonnei was studied. There was a 100% survival rate in mice fed for 8 d with fermented milk and then dosed orally with Sh. sonnei. The survival rate in control mice was approximately 60% after 21 d. Colonization of the liver and spleen with Sh. sonnei was markedly inhibited by pretreatment with fermented milk. Differences in cell counts of 2-3 log units between treated and control mice were always obtained, shigellas were not detected in these organs by the 10th day in treated mice, while high levels were maintained in the controls. Higher levels of anti-shigella antibodies were found both in sera and in small intestinal fluid of mice treated with fermented milk, suggesting that the protective immunity could be mediated by the mucosal tissue. These results suggest that milk fermented with Lact. casei and Lact. acidophilus could be used as a prophylactic against gastrointestinal infections by shigellas.     

Inhibition of Shigella sonnei by Lactobacillus casei and Lact. acidophilus

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei and Lact. acidophilus against Shigella sonnei was studied. There was a 100% survival rate in mice fed for 8 d with fermented milk and then dosed orally with Sh. sonnei. The survival rate in control mice was approximately 60% after 21 d. Colonization of the liver and spleen with Sh. sonnei was markedly inhibited by pretreatment with fermented milk. Differences in cell counts of 2–3 log units between treated and control mice were always obtained, shigellas were not detected in these organs by the 10th day in treated mice, while high levels were maintained in the controls. Higher levels of anti-shigella antibodies were found both in sera and in small intestinal fluid of mice treated with fermented milk, suggesting that the protective immunity could be mediated by the mucosal tissue. These results suggest that milk fermented with Lact. casei and Lact. acidophilus could be used as a prophylactic against gastrointestinal infections by shigellas.     

嗜酸乳杆菌产细菌素生物学特性的研究

嗜酸乳杆菌在MRS培养基中培养得到的细菌素经30、60、90、121℃处理20min后,活性几乎不变,对以金黄色葡萄球菌为代表的革兰阳性菌、大肠埃希菌为代表的革兰阴性菌有明显的抑制作用,Tricine—SDS—PAGE和不同pH值抑菌实验测定结果证明,嗜酸乳杆菌细菌素是一组低分子量,并在pH2—4时有抑菌作用的活性物质。     

Plasmid-linked Galactose Utilization by Lactobacillus acidophilus TK8912

A gramicidin S analog ([Orn^1,1′]GS·4HCl) containing L-oroithine in place of L-valine at the 1,1′ positions was synthesized by the conventional solution method in order to examine whether this analog had antibacterial activity toward Gram-negative bacteria. In the synthesis of [Orn^1,1′]GS·4HCl, two intermediate analogs ([Orn^1,1′, Orn(For)^2,2′]GS·2HCl and [Orn(Z)^1,1′]GS·2HCl) were obtained. [Orn^1,1′]GS·4HCl and [Orn,^1,1′, Orn(For)^2,2′]GS·2HCl showed no activity toward either Gram-negative or Gram-positive bacteria, whereas [Orn(Z)^1,1′]GS 2HCl showed appreciable activity toward only Gram-positive bacteria.     

Heterogeneity of S-layer proteins of Lactobacillus acidophilus strains.

An S-layer (surface regular array) was found in the cell wall from six out of ten strains of Lactobacillus acidophilus examined by electron microscopic observations. All of the six strains which were shown to carry the S-layers belonged to the deoxyribonucleic acid (DNA) homology group A, but not to B, which had been classified by Johnson et al (Int. J. Syst. Bacteriol. 30: 53-68, 1980). On the other hand, the other four strains which possessed no S-layers were in the homology group B. The apparent molecular weights of the S-layer proteins ranged from 41 to 49 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the S-layer proteins from the six strains, three were susceptible to chemical cleavage with N-chlorosuccinimide, giving different peptide maps. All of the six S-layer proteins were fragmented by limited proteolysis with Staphylococcus aureus V8 protease, and gave markedly different peptide patterns by the subsequent peptide mapping analysis, except that the peptide maps of the S-layer proteins from the two strains which were in the same subgroup were identical.     

Production and properties of alpha-glucosidase from Lactobacillus acidophilus.

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.     

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.     

Interferon induction in murine peritoneal macrophage by stimulation with Lactobacillus acidophilus.

Induction of interferon for a kind of dairy lactic acid bacteria, Lactobacillus acidophilus (L. acidophilus), was investigated in murine peritoneal macrophage (M phi) cultures. Lactobacillus acidophilus JCM 1034, 1132T, 1229 and 2125 induced IFN (12-34 I.U./ml) in M phi cultures in vitro. Strain 1132T- and 2125-induced IFNs were characterized as IFN alpha/beta by treatment with anti-IFNs serum. The results indicate that the inducing activity of IFNs may be one of the available biological parameters for designating the dairy products containing L. acidophilus as "physiologically functional foods."