Antigenic Analyses of Lactobacillus fermenti

Antigenic analyses of Lactobacillus fermenti were carried out by double immunodiffusion in agar using extracts prepared with cold trichloroacetic acid (TCA) or hot dilute hydrochloric acid (HCl). A common antigen of L. fermenti, designated as antigen f by the author, was extracted from whole cells with dilute HCl, but not with TCA. The antigen f was also observed in Lactobacillus casei. In addition, all strains isolated from human saliva contained antigen 6 in their cell walls, while the antigen was not observed in most of the isolates from human feces. Therefore, L. fermenti could be divided into two subgroups based upon the existence of antigen 6. Antigen 7 which was demonstrated in some strains of L. fermenti was shared by other species of lactobacilli belonging to the serological groups D and E. The common antigen 3 found in lactobacilli was extracted from all strains of L. fermenti. Sugar components of cell walls were mainly galactose, glucose and glucosamine (including N-acetylglueosamine), but a small amount of rhamnose was present in the cell wall of only one strain. Inhibition tests with various sugars showed that the serologically active sugars were galactose for antigen f and glucose for antigen 6.     

Grouping antigens of four Lactobacillus species and their characteristics.

Antigenic analyses of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus brevis and Lactobacillus buchneri were carried out by double immunodiffusion in agar. Antigens were extracted from whole cells and cell wall preparations with cold trichloroacetic acid. Most strains of the four species possessed antigen 9 in their cell walls. Another antigen, antigen 10, was found in the cell walls of all the strains of L. brevis and L. buchneri, and in some strains of L. lactis, but not in L. bulgaricus. Fractionation of the antigens was attempted using the cell wall extracts of L. lactis L-10 with only antigen 9 and of L. brevis X-1 with both antigens 9 and 10. The partially purified fractions of antigen 9 and of the complex of antigens 9 and 10 were obtained by zone electrophoresis. However, antigen 10 from the complex could not be separated by the same method or gel filtration on Sephadex G-100 since the two antigens 9 and 10 of the complex always behaved together. The fraction of antigen 9 consisted almost entirely of glycerol and glucose as sugar components, the molar ratio being 2: 1. The complex of antigens 9 and 10 also consisted of the same sugars, and the molar ratio of glycerol: glucose was 4: 1. Inhibition tests indicated that the immunodominant component of antigen 9 was a-methylglucoside (glucose), and most probably the determinant is a glycosylated glycerol teichoic acid. It was considered that the determinant of antigen 10 is a glycerol teichoic acid although glucosamine and galactosamine inhibited effectively the reaction between antigen 10 and its antibody.     

Grouping Antigens of Four Lactobacillus Species and Their Characteristics

Antigenic analyses of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus brevis and Lactobacillus buchneri were carried out by double immunodiffusion in agar. Antigens were extracted from whole cells and cell wall preparations with cold trichloroacetic acid. Most strains of the four species possessed antigen 9 in their cell walls. Another antigen, antigen 10, was found in the cell walls of all the strains of L. brevis and L. buchneri, and in some strains of L. lactis, but not in L. bulgaricus. Fractionation of the antigens was attempted using the cell wall extracts of L. lactis L-10 with only antigen 9 and of L. brevis X-1 with both antigens 9 and 10. The partially purified fractions of antigen 9 and of the complex of antigens 9 and 10 were obtained by zone electrophoresis. However, antigen 10 from the complex could not be separated by the same method or gel filtration on Sephadex G-100 since the two antigens 9 and 10 of the complex always behaved together. The fraction of antigen 9 consisted almost entirely of glycerol and glucose as sugar components, the molar ratio being 2:1. The complex of antigens 9 and 10 also consisted of the same sugars, and the molar ratio of glycerol: glucose was 4:1. Inhibition tests indicated that the immunodominant component of antigen 9 was α-methylglucoside (glucose), and most probably the determinant is a glucosylated glycerol teichoic acid. It was considered that the determinant of antigen 10 is a glycerol teichoic acid although glucosamine and galactosamine inhibited effectively the reaction between antigen 10 and its antibody.     

α-Galactosidase Activity of Lactobacilli

alpha-Galactosidase (EC 3.2.1.22) activity was observed in cell-free extracts of Lactobacillus fermenti, L. brevis, L. buchneri, L. cellobiosis, and L. salivarius subsp. salivarius. The cultural conditions under which the enzyme activity was detected suggest that the enzyme is constitutive and present in the soluble fraction in the cell. The enzyme preparations readily hydrolyzed melibiose and other oligosaccharides containing alpha(1 --> 6) linked galactose. Although the cell-free extracts of L. fermenti and L. brevis are negative for beta-fructofuranosidase (EC 3.2.1.26), they hydrolyzed melibiose, stachyose, and raffinose in decreasing order of activity. The beta-fructofuranosidase-positive L. buchneri, L. cellobiosis, and L. salivarius preparations hydrolyzed melibiose, raffinose, and stachyose in decreasing rates of activity. The alpha-galactosidases from different lactobacilli showed optimum activity in pH range 5.2 to 5.9. L. fermenti and L. salivarius preparations exhibited maximum activity between 40 to 44 C and 48 to 51 C, respectively, whereas a 38 to 42 C range was observed for other lactobacilli. Cell-free extract of L. cellobiosis was studied for transgalactosylase activity. When incubated with melibiose, a new compound was detected and tentatively identified as manninotriose.     

THE NORMAL FLORA OF THE BOVINE RUMEN. IV. QUALITATIVE STUDIES OF LACTOBACILLI FROM COWS AND CALVES

SUMMARY: Two hundred and four samples of rumen contents were taken from eight fistulated cows receiving various diets, and seventy-one samples from twelve calves. From dilution counts of these samples, 91 isolates of Gram-positive catalase-negative rods were obtained, and by means of physiological and serological tests, 78 (95%) out of 82 lactobacilli were characterized as follows: Lactobacillus fermenti (33 strains), L. acidophilus (21), L. casei (17) and L. plantarum (7). Indications were obtained of progressive establishment of lactobacilli in the rumen of the growing calf, L. fermenti predominating. In the cow, L. acidophilus was generally associated with hay plus concentrates diets and L. casei with silage, whereas with grass small numbers of several species of lactobacilli were found.     

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.     

THE NORMAL INTESTINAL FLORA OF THE PIG. III. QUALITATIVE STUDIES OF LACTOBACILLI AND STREPTOCOCCI

SUMMARY: Streptococci and lactobacilli were isolated from the faeces and caecal contents of experimental pigs receiving various diets. Fifty-seven strains of streptococci and forty-one (representative of 220) lactobacilli were examined physiologically and serologically. Lactobacillus acidophilus and L. fermenti predominated among the lactobacilli, while the largest group of streptococci resembled the 'unclassified' strains of Barnes & Ingram (1955) and Barnes, Ingram & Ingram (1956) from bacon factory premises, pig faeces and canned hams.     

Distribution of indigenous Lactobacilli in the digestive tract of conventional and gnotobiotic rats.

Distribution of indigenous lactobacilli in the gastrointestinal tracts of rats was investigated at the species level. The indigenous lactobacilli isolated from conventional rats were divided into three groups, Lactobacillus acidophilus and its related strains, L. fermentum, and L. murini. Localization of the Lactobacillus groups in the gastrointestinal tracts could be distinguished clearly based on arabinose and glucose fermentation reaction of isolates from each part of the gastrointestinal tract. Group I (L. acidophilus and the related strains) and Group II (L. fermentum) were the major populations of lactobacilli on the walls of the non-glandular part and in the contents of the stomachs of both conventional and gnotobiotic rats. Group I predominated in all the parts of the digestive tract of conventional rats, whereas Group II was in the minority in the lower part of the gastrointestinal tracts of both groups of rats. Group III (L. murini) was the predominant population of Lactobacillus in the lower part of small intestine of conventional rats and in all parts of the gastrointestinal tracts of gnotobiotic rats except for the wall of the non-glandular part of the stomach.     

Distribution and chemical characterization of regular arrays in the cell walls of strains of the genus Lactobacillus

Abstract The presence of regular arrays (RAs) in the cell walls of strains of the genus Lactobacillus was examined by electron microscopy. The RAs were found in 6 species including L. bulgaricus, L. helveticus, L. acidophilus, L. fermentum, L. brevis and L. buchneri . The RAs were composed of a protein with an apparent M _r ranging from about 41000 to 55000, depending on the species upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid composition of the RA proteins was shown to be acidic and hydrophobic. The antigenicity of the RA protein from L. buchneri appeared to be specific but not common among the RA proteins from the other lactobacilli.     

Immunochemical characteristics of Streptococcus mutans serotype h carbohydrate antigen

Serotype h carbohydrate antigen was prepared from cell walls of Streptococcus mutans strain MFe28 of monkey origin. The h antigen was extracted from the cell walls with 5% trichloracetic acid at 4 C, and purified by DEAE-Sephadex A-25 ion exchange chromatography followed by Sephacryl S-300 gel filtration. The purified antigen was composed of galactose (75%), glucose (16%), and rhamnose (3%). Although the antiserum against whole cells of S. mutans MFe28 gave a strong cross reaction with serotype d S. mutans, serotype h-specific antiserum could be obtained by adequate adsorption. The precipitin reactions and hapten inhibition test using serotype h-specific antiserum showed that galactose, glucose, and their derivative sugars were markedly potent inhibitors. It was concluded that the serotype h antigen is immunologically distinguishable from the known serotypes of S. mutans, although it is closely related to serotype d antigen of S. mutans.     

Amino acid profiles and presumptive nutritional assessment of single-cell protein from certain lactobacilli.

The amino acid profiles, modified essential amino acid (MEAA) indexes, and in vitro pepsin digestibilities were determined for single-cell protein (SCP) from certain industrially important lactobacilli. For the three parameters examined, substantial differences were seen between different Lactobacillus species and between strains with a given species. SCP from all of the lactobacilli examined appeared relatively high in MEAA indexes and pepsin digestibility. SCP from L. acidophilus 3205 and L. fermenti 3954 had the highest MEAA indexes, whereas L. bulgaricus 2217 and L. thermophilus 3863 had the highest percentage of digestible crude protein. SCP from L. plantarum strains had the lowest MEAA indexes. The essential amino acid compositions of SCP from different lactobacilli appear comparable to that of Food and Agriculture Organization reference protein and SCP from other sources.     

Transport and Metabolism of Lactose, Glucose, and Galactose in Homofermentative Lactobacilli

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained β-galactosidase activity, and no significant phospho-β-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.     

Amino acid profiles and presumptive nutritional assessment of single-cell protein from certain lactobacilli.

The amino acid profiles, modified essential amino acid (MEAA) indexes, and in vitro pepsin digestibilities were determined for single-cell protein (SCP) from certain industrially important lactobacilli. For the three parameters examined, substantial differences were seen between different Lactobacillus species and between strains with a given species. SCP from all of the lactobacilli examined appeared relatively high in MEAA indexes and pepsin digestibility. SCP from L. acidophilus 3205 and L. fermenti 3954 had the highest MEAA indexes, whereas L. bulgaricus 2217 and L. thermophilus 3863 had the highest percentage of digestible crude protein. SCP from L. plantarum strains had the lowest MEAA indexes. The essential amino acid compositions of SCP from different lactobacilli appear comparable to that of Food and Agriculture Organization reference protein and SCP from other sources.     

Structure of the cell wall of lactobacilli. Role of muramic acid phosphate in Lactobacillus fermenti

1. The polysaccharide and mucopeptide components of the cell wall of Lactobacillus fermenti, serological group F, were separated by mild conditions of acid hydrolysis; the polysaccharide was composed of glucose and galactose. 2. Soluble cell-wall products were isolated from cell wall lysed by lysozyme and a Streptomyces enzyme preparation. The lysozyme-dissolved fraction contained a greater proportion of mucopeptide. 3. The soluble preparations were heated in dilute acid to hydrolyse the linkage between the polysaccharide and mucopeptide components and then incubated with acid phosphatase. 4. Inorganic phosphate was released from products of Streptomyces enzyme action but not from products of lysozyme action. 5. The phosphate was shown to be present in the mucopeptide as muramic acid phosphate. It is concluded that in the intact wall polysaccharide is joined to muramic acid by a phosphodiester linkage.     

Existence of phosphoenolpyruvate: carbohydrate phosphotransferase systems in Lactobacillus fermentum, an obligate heterofermenter.

The presence or absence of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) in obligately heterofermentative group III lactobacilli including Lactobacillus brevis (3 strains), L. buchneri (2 strains) and L. fermentum (3 strains) was surveyed systematically for a series of sugars utilizable by these organisms. Contrary to common expectation, PTSs were found in two strains of L. fermentum: sucrose-PTS in one strain; sucrose- and mannose-PTSs in the other. All these activities were found to be constitutive.     

A Synthetic Medium for Comparative Nutritional Studies of Lactobacilli

The composition of a synthetic medium supporting the growth of lactobacilli is given (Table 1). The medium, containing glucose, amino acids, vitamins, mineral salts, purines and pyrimidines, allows the study of nutritional requirements of different strains of lactobacilli under identical environmental conditions. It was found that all the strains tested needed L-glutamic acid, L-valine and L-leucine, and a group of them also required L-arginine, L-tyrosine and L-tryptophan. Some strains required vitamins, e.g. L. bulgaricus (pantothenic acid), L. fermenti (pantothenic acid and niacin). These results are compared with those found by others employing different media.     

Utilisation of maltose and glucose by lactobacilli isolated from sourdough

Abstract The utilisation of glucose and maltose was investigated with Lactobacillus strains isolated from sourdough starters. These preparations have been in continuous use for a long period to produce sourdough from rye, wheat and sorghum. The major metabolic products formed by resting cells from glucose or maltose were lactate, ethanol and acetate. Upon fermentation of maltose, resting cells of Lactobacillus sanfrancisco, L. reuteri, L. fermentum and Lactobacillus ep. released up to 13.8 mM glucose after 8 h. The ratio of released glucose per mol of utilised maltose was up to 1:1. Glucose formation was high when starved cells of L. sanfrancisco and Lactobacillus sp. were used. This is consistent with maltose utilisation via maltose phosphorylase which phosphorylates maltose without the expenditure of ATP and thus allows the cell to waste glucose in the presence of abundant maltose. The glucose formed may be utilised by the lactobacilli or other microorganisms, e.g. yeasts. However, the release of glucose into the medium by sourdough lactobacilli prevents competitors from utilising the abundant maltose by glucose repression. In strains of L. sanfrancisco , maltose utilisation was very effective and not subject to glucose repression. Therefore, they overgrow other microorganisms sharing this habitat. Wild isolates of L. sanfrancisco were initially unable to grow on glucose. Upon growth on maltose such strains required adaptation times of up to 150 h to grow on glucose. After subsequent transfer of glucose-grown cells to fresh medium the strains resumed growth both on glucose or maltose. They readily lost their ability to grow on glucose upon exposure to maltose. L. sanfrancisco exhibited biphasic growth characteristics on media containing glucose, maltose or both carbon sources. Evidence is provided that biphasic growth and metabolite formation are dependent on the redox potential.     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

阴道乳酸杆菌的分子生物学鉴定方法研究

目的鉴定女性生殖道乳杆菌种类及各种乳杆菌在阴道中的分布差异。方法通过扩增和测序乳酸杆菌16S rRNA序列中特异性区段（8f926r）,并利用Vector NTI 8.0序列分析软件对各菌株的基因序列与标准菌株比对进行。结果 26株乳酸菌,L1株、L25株和L46株为乳酸杆菌属中的格氏乳酸杆菌（Lactobacillus gasseri）;L2株、L5株、L6株、L7株、L8株、L11株、L13株、L15株、L19株、L20株、L24株、L26株、L28株、L29株、L31株、L32株、L33株、L34株、L35株、L36株、L40株与L45株均为乳酸杆菌属中的卷曲乳酸杆菌（Lactobacillus crispatus）,而L21株未鉴定为乳酸球菌。结论女性阴道乳酸杆菌的优势菌种是卷曲乳杆菌、格氏乳杆菌。     

Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.