Lactose metabolism in Lactobacillus curvatus and Lactobacillus sake

Abstract The lactose metabolism was investigated in five strains of Lactobacillus curvatus and 14 strains of L. sake isolated from meat or meat-derived products. Strains with the ability to ferment lactose were found in both species. They exhibited either phospho-β-galactosidase (P-β-gal) or β-galactosidase (β-gal) activity, or both. P-β-gal activity of L. curvatus and L. sake was induced and detected only in the presence of lactose or galactose. Furthermore, catabolite repression by glucose was demonstrated. The immunological properties of the P-β-gal enzymes of these organisms resemble those of Lactococcus lactis . Several strains of L. sake but none of L. curvatus exhibited β-gal activity which was constitutive. In hybridisation experiments, the β-gal genes of L. sake and L. casei ATCC393 showed over 60% DNA-homology. The presence of β-gal genes in L. sake was demonstrated in both β-gal-producing and non-producing strains. This observations is consistent with a genetic potential of lactic acid bacteria exceeding their physiological capabilities.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

The occurrence of β-galactosidase and β-phosphogalactosidase in Lactobacillus casei strains

Abstract Several strains of Lactobacillus casei of different origins were compared and it was observed that lactose metabolism varied from one strain to the other. Certain strains contained a β-galactosidase, others a β-phosphogalactosidase and others contain both. It was shown that the activities present in these last strains are catalyzed by two proteins differing in their electrophoretic mobilities and M _r values. Genetic divergence of the studied strains is considered.     

Expression of Lactobacillus casei ATCC 393 β-galactosidase encoded by plasmid pLZ15 in Lactococcus lactis CNRZ 1123

Lactococcus lactis subsp. lactis CNRZ 1123, a Lac^- derivative of CNRZ 1122 was transformed by electroporation with the Lactobacillus casei ATCC 393 plasmid pLZ15, which bears a β-galactosidase gene. The transformants expressed a constitutive β-galactosidase activity at a higher level than in Lact. casei , and in the cell-free extract two additional protein bands were detected by SDS-PAGE which could correspond to lactose metabolism enzymes. Both plasmid and β-gal activity were stable in Lactococcus after 100 generations in glucose-containing medium.     

RAT BRAIN β-GALACTOSIDASES: OBSERVATIONS ON CEREBROSIDE-β-GALACTOSIDASE CATALYSED TRANSGALACTOSYLATION AND COMPARISON OF ASSAY METHODS FOR SPHINGOLIPID HYDROLYSIS

Abstract— A partially purified rat brain preparation, enriched in cerebroside β-galactosidase activity, was found to catalyze the synthesis of labelled galactosyl-ceramide from [¹⁴C]oleoyl-sphingosine as acceptor and several β -galactosides as donor. The following compounds in the order of their effectiveness served as galactose donors for this reaction: para-nitrophenyl-β -galactoside (PNP-β -gal), galactosyl-ceramide, lactosyl-sphingosine, lactosyl-ceramide, 4-methyl-umbelliferyl-β -galactoside (4-MU-β -gal). asialo-G_M1, galactosyl-sphingosine, G_M1 and monogalactosyl-diglyceride. It is believed that this transgalactosylation reaction is probably not a mere reversal of the hydrolytic reaction. Under optimal conditions the quantity of galactosyl-ceramide formed represented 10% of the amount of donor hydrolysed. These observations in conjunction with those on the hydrolyses provide further support for the possible existence of two β -galactosidase isoenzymes involved with the catabolism of G_M1 and/or lactosyl-ceramide, and a single form for galactosyl-ceramide. The activity of one isoenzyme can be 'selectively' measured simply by modifying the incubation mixture.     

β(1,3)-Glucanases from Geotrichum lactis: activity on its own nascent and preformed β(1,3)-glucan

Abstract Actively growing mycelium of Geotrichum lactis contains at least three β(1,3)-glucanase activities. Two of the activities have been characterized as exo- and the third as endo-hydrolytic. The action of the activities on β(1,3)-glucan synthesized in vitro by the β(1,3)-glucan synthase system from G. lactis has been studied. One of the exo-β-glucanases and the endo-β-glucanase were active on this β(1,3)-glucan and the degradation rates were higher on nascent than on preformed β(1,3)-glucan.     

Osmoprotectant β-alanine betaine synthesis in the Plumbaginaceae: S-adenosyl- l-methionine dependent N-methylation of β-alanine to its betaine is via N-methyl and N,N-dimethyl β-alanines

β -Alanine betaine is an osmoprotective compound accumulated by most members of the plant family Plumbaginaceae. Leaf and root tissues of Limonium latifolium known to accumulate β -alanine betaine readily convert supplied β -alanine to β -alanine betaine. To identify the intermediates and the enzymes involved in β -alanine betaine synthesis, radiotracer experiments using [ 14 C] formate were employed. These studies demonstrate that β -alanine betaine is synthesized from β -alanine via N -methyl and N,N- dimethyl β -alanines. A rapid and sensitive radiometric assay was developed to measure N -methyltransferase (NMT) activities by using [methyl-¹⁴C] or [methyl-³H] S -adenosyl- l -methionine (AdoMet) as the methyl donor. Leaf extracts from β -alanine betaine accumulators – Armeria maritima , L. latifolium and L. ramosissimum – had detectable NMT activities while none were found in L. perezii , a species that does not accumulate β -alanine betaine. The NMT activities were further characterized from the leaves of L. latifolium . The activities had a pH optimum of 8.0, were soluble and inhibited by S -adenosyl- l -homocysteine. Extractable activities were similar from plants grown under control and salinity stress conditions. Radiolabeling with [ 14 C] l -aspartic acid indicated that, unlike in bacteria, decarboxylation of l -aspartic acid is not the source of β -alanine in the Plumbaginaceae.     

Presumptive involvement of methyl ricinoleate β-oxidation in the production of γ-decalactone by the yeast Pichia guilliermondii

Abstract Production of γ-decalactone by yeasts from fatty acids has been reported but little is known about the mechanisms involved in this process. This paper provides information about the mechanisms involved in the production of γ-decalactone by Pichia guilliermondii in the presence of a fatty acid methyl ester. Culturing of P. guilliermondii in media containing methyl ricinoleate (12(R)-hydroxy-9(Z)-octadecenoic acid) revealed a coordinated induction of β-oxidation activities and γ-decalactone production. However, no γ-decalactone synthesis was noted when methyl ricinoleate was changed into methyloleate or methyl linoleate, even though these fatty acid methyl esters are able to induce β-oxidation activities in P. guilliermondii . These observations led us to conclude that methyl ricinoleate is an inducer of β-oxidation and is probably the substrate for γ-decalactone production. The fatty acid ester β-oxidation should be involved, at least in part, in this production.     

Beta-galactosidase, phospho-beta-galactosidase and phospho-beta-glucosidase activities in lactobacilli strains isolated from human faeces

AIMS: Lactose intolerance, a serious health problem for Asians, can be solved using probiotic bacteria having high lactose hydrolysis activities. We determined the distribution of beta-galactosidase (beta-gal), phospho-beta-galactosidase (P-betagal) and phospho-beta-glucosidase (P-beta-glc) activities in species of lactic acid bacteria (LAB) isolated from human faeces to select strains for potential use in fermented dairy products, e.g. yogurt. METHODS AND RESULTS: The sugar substrates, o-nitrophenyl-beta-D- galactopyranoside 6-phosphate and o-nitrophenyl-beta-D-glucopyranoside 6-phosphate, were synthesized and used to measure respectively P-beta-gal and P-beta-glc activities. Sixty-five toluene-treated strains were examined for three lactase enzyme activities. Lactobacillus mucosae OLL2848 showed the highest beta-gal activity (107.09 U mg(-1) of protein) among the Lactobacillus strains from human faeces. Lactobacillus gasseri OLL2836 and OLL 2948 showed the highest P-beta-gal (46.58 U) and P-beta-glc (50.19 U)activity, respectively, with no beta-gal activity. CONCLUSIONS: The expression of P-beta-glc induced by lactose was characteristic of Lact. gasseri. Because this LAB is a major inhabitant of the human intestine. This enzyme is a key glycosidase involved in lactose utilization. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report describing the distribution of three glycosidase activities used in lactose metabolism in LAB isolated from human faeces for possible use in functional foods.     

Note : Cyclohexenoesculetin-β-D-glucoside: a new substrate for the detection of bacterial β-D-glucosidase

A new substrate for the detection of bacterial β- D -glucosidase was evaluated as an alternative to aesculin. This substrate, 3,4-cyclohexenoesculetin-7-β- D -glucoside, was compared with aesculin for the detection of β- D -glucosidase in 150 enterococci, 40 streptococci, 12 Listeria sp. and 250 strains of Enterobacteriaceae. In the Gram-positive strains tested, aesculin hydrolysis correlated with hydrolysis of 3,4-cyclohexenoesculetin-7-β- D -glucoside. In the Gram-negative strains the new substrate was hydrolysed by all aesculin-positive strains and also by four strains (10%) of Escherichia coli which gave a negative aesculin reaction. 3,4-Cyclohexenoesculetin-7-β- D -glucoside was shown to be a reliable alternative to aesculin and was shown to have significant advantages over aesculin when incorporated into solid media. This was due to the non-diffusible end product produced by hydrolysis of 3,4-cyclohexenoesculetin-7-β- D -glucoside in the presence of iron.     

The influence of the host on expression of intestinal microbial enzyme activities involved in metabolism of foreign compounds

The activities of four enzymes (β-glucosidase, β-glucuronidase, nitrate reductase and nitroreductase) in selected intestinal bacteria ( Escherichia coli, Clostridium sp., Streptococcus sp., Bacteroides sp. and Lactobacillus salivarius ) were measured after growth in vitro and in vivo . The five strains differed in their activites with Clostridium sp. being the most active for β-gjucosidase, β-glucuronidase and nitroreductase, and E. coli the most active producer of nitrate reductase. Enzyme activity in vivo tended to be higher than in vitro but there were instances where the comparative activities were reversed.     

Expression of β-galactosidase in Rhizobium japonicum

Abstract The plasmid pGC91.14 was used to introduce via conjugation the Escherichia coli lac operon into fast-growing and slow-growing strains of Rhizobium japonicum . Exconjugants now expressed higher levels of β-galactosidase activity which was still inducible by isopropyl-β- d -thiogalactoside (IPTG). The presence of the lac operon allowed the slow-growing strain 61A76 to grow on lactose as the sole carbon source; the fast-growing strains grew poorly on lactose but growth was not inhibited by lactose as had been reported for Rhizobium meliloti . β-galactosidase could be detected in nodule extracts and bacteroid preparations from soybean plants ( Glycine max L. Merrill) infected with the strain 61A76 (pGC91.14).     

Formation and regeneration of protoplasts and spheroplasts of gastrointestinal strains of lactobacilli.

Methods were developed for the formation of protoplasts and spheroplasts of gastrointestinal strains of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus salivarius. Attempts to regenerate vegetative cells from protoplasts were not successful, but spheroplasts could be regenerated consistently for five of six strains.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Characterization of β-glucosidase activity in yeasts of oenological origin

I. ROSI, M. VINELLA AND P. DOMIZIO. 1994. Three hundred and seventeen strains representing 20 species of yeasts were screened for the presence of β-glucosidase activity. All of the strains of the species Debaryomyces castellii, Deb. hansenii, Deb. polymorphus, Kloeckera apiculata and Hansenula anomala showed β-glucosidase activity, but only one of 153 strains of Saccharomyces cerevisiae. The other species behaved differently, depending upon the strain. The strains that hydrolysed arbutin were checked to localize the β-glucosidase activity. A strain of Deb. hansenii exhibited the highest exocellular activity and some wall-bound and intracellular activity. The β-glucosidase synthesis from this yeast was enhanced by aerobic conditions of growth, was repressed by high glucose concentration (9%) and occurred during exponential growth. The optimum conditions for enzymatic preparations of Deb. hansenii were between pH 4.0 and 5.0 and 40C. A high concentration of ethanol and glucose did not reduce the ezymatic activity. The enzymatic preparations of Deb. hansenii released monoterpenols and other alcohols from a grape glycoside extract.     

Antimicrobial potential of four Lactobacillus strains isolated from breast milk

AIMS: The antimicrobial potential of four lactobacilli (Lactobacillus salivarius CECT5713, Lactobacillus gasseri CECT5714, L. gasseri CECT5715 and Lactobacillus fermentum CECT5716), isolated from fresh human breast milk, was evaluated in this study and compared with Lactobacillus coryniformis CECT5711, a reuterin-producing strain isolated from an artisan goat's cheese. METHODS AND RESULTS: Agar diffusion tests, competitive adhesion assays and mucin expression assays were carried out in order to value the antibacterial properties of the lactobacilli strains. The antibacterial capability of the strains was tested in vivo by using a murine infection model with Salmonella choleraesuis. The results revealed that all the strains studied, displayed antibacterial properties against pathogenic bacteria. However, the antibacterial potential varied among the lactobacilli tested and, in fact, L. salivarius CECT5713 showed not only the best in vitro antibacterial activity, but also the highest protective effect against a Salmonella strain in the murine infection model. CONCLUSION: The four breast-milk lactobacilli, and particularly L. salivarius CECT5713, possess potent antibacterial activities that result in a higher protection against S. choleraesuis CECT4155 in a mouse infection model. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that lactobacilli from breast milk could contribute to an anti-infective protection in neonates and would be excellent candidates for the development of infant probiotic products.     

Formation and regeneration of protoplasts and spheroplasts of gastrointestinal strains of lactobacilli

Methods were developed for the formation of protoplasts and spheroplasts of gastrointestinal strains of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus salivarius. Attempts to regenerate vegetative cells from protoplasts were not successful, but spheroplasts could be regenerated consistently for five of six strains.     

Expression of mRNA encoding IFNα in macrophages stimulated with Lactobacillus gasseri

Abstract The ability of Lactobacillus gasseri , a dairy lactic acid bacterium, to induce interferon (IFN) was investigated in murine macrophage cultures. IFN α was substantially induced by some strais of L. gasseri and the titers were the highest at a concentration of 100 μg ml⁻¹ of L. gasseri DSM20243^T. The expression of mRNA encoding IFN α was detected in spleen-macrophages (SP-M θ ) and Peyer's patch-adherent cells stimulated with L. gasseri DSM20243^T. Actinomycin D and cycloheximide added to SP-M θ cultures showed that the mRNA was synthesized by 0.5 h, and that IFN α was produced within 3 to 6 h after the stimulation with L. gasseri DSM20243^T. The results support the notion that dairy products containing L. gasseri can be 'physiologically functional foods'.     

Characterization of β-mannanase and β-mannosidase secreted from the yeast Trichosporon cutaneum JCM 2947

Y. ODA AND K. TONOMURA. 1996. β-Mannanase and β-mannosidase were purified from the culture fluid of the yeast Trichosporon cutaneum JCM 2947 (= T. beigelii CBS 5790). The molecular weights of the two enzymes were estimated to be 49 900 and 114000 by SDS-PAGE and 4500 and 193 000 by gel filtration, respectively. β-Mannanase contained 43% molecular weight as carbohydrate. The K _m and V _max values of β-mannanase for konjac glucomannan were 2.7 (mg ml^-1) and 10.6 (U mg protein^-1), and those of β-mannosidase for p -nitrophenyl β-D-mannopyranoside were 0.25 (mmol l^-1) and 91.7 (U mg protein^-1). Maximal activities were observed between pH 4.0 and 6.5 at 50°C for β-mannanase and around 6.5 at 40°C for β-mannosidase.     

Secretion of β-galactosidases by Aureobasidium pullulans and Penicillium brevicompactum on polygalacturonate medium

An assay has been developed to detect production of extracellular β -galactosidases by fungi during growth on a defined medium containing polygalacturonate. Using this method strains of the yeast-like fungus Aureobasidium pullulans and the filamentous fungus Penicillium brevicompactum which secrete β -galactosidase have been isolated. Seventy strains of yeast were tested but none secrete detectable extracellular β -galactosidase during growth on polygalacturonate agar medium.