Carbohydrate-binding module 74 is a novel starch-binding domain associated with large and multidomain α-amylase enzymes.

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract.

Kinetic characterization of a novel endo-ß-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.

EndoBI-1 is a recently isolated endo-ß-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans.

Characterizing the release of bioactive N-glycans from dairy products by a novel endo-ß-N-acetylglucosaminidase.

Endo-ß-N-acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI-1) is a novel enzyme that cleaves N-N'-diacetyl chitobiose moieties found in the N-glycan core of high mannose, hybrid, and complex N-glycans. These conjugated N-glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45-8.45), temperature (27.5-77.5°C), reaction time (15-475 min), and enzyme/protein ratio (1:3,000-1:333) were evaluated on the release of N-glycans from bovine colostrum whey by EndoBI-1. A central composite design was used, including a two-level factorial design (2(4)) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N-glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan-free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI-1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies.

A novel endo-ß-N-acetylglucosaminidase releases specific N-glycans depending on different reaction conditions.

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N-glycans in protein functionality. Endo-ß-N-acetylglucosaminidase (EndoBI-1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat-stable enzyme that cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI-1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI-1 is able to cleave a high diversity of N-glycan structures. Nano-LC-Chip-Q-TOF MS data also revealed that different reaction conditions resulted in different N-glycan compositions released, thus modifying the relative abundance of N-glycan types. In general, more sialylated N-glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI-1 is able to release a wide variety of N-glycans, whose compositions can be selectively manipulated using different processing conditions.

Enhancement of ginsenoside Rg(1) in Panax ginseng hairy root by overexpressing the α-L-rhamnosidase gene from Bifidobacterium breve.

OBJECTIVES: To improve the production of ginsenoside Rg1 in Panax ginseng. RESULTS: The α-L-rhamnosidase gene from Bifidobacterium breve (BbRha) was overexpressed into hairy root culture system using Agrobacterium rhizogenes A4. Ginsenoside Rg1 in hairy roots was obtained following transformation via overexpressed gene representing 2.2-fold higher than those of control lines. Several overexpression transgenic hairy root lines were obtained exhibiting markedly increased levels of the corresponding α-L-rhamnosidase enzymatic activity relative to control. Ginsenoside Rg1 levels in the transgenic lines were higher (2.2-fold) than those of control after following 30 days culturing, while ginsenoside Re contents in tested transgenic lines were found to be lower. The transgenic hairy roots harboring α-L-rhamnosidase gene improved the accumulation of ginsenoside Rg1 up to 3.6 mg g(-1) dry weight. CONCLUSION: BbRha gene selectively enhances the production of ginsenoside Rg1 in P. ginseng hairy roots.

Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress.

Bifidobacteria are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). To improve oxidative-stress tolerance, the superoxide dismutase (SOD) gene from Streptococcus thermophilus (StSodA) and the heme-dependent catalase (KAT) gene from Lactobacillus plantarum (LpKatL) were heterologously expressed in Bifidobacterium longum strain NCC2705. Three types of strain NCC2705 transformants were obtained: with transgenic SOD expression, with transgenic KAT expression, and with coexpression of the two genes. Intracellular expression of the genes and their functional role in oxidative-stress resistance were evaluated. In response to oxidative stress, B. longum NCC2705/pDP401-LpKatL (expressing LpKatL) and NCC2705/pDP-Kat-Sod (coexpressing LpKatL and StSodA) rapidly degraded exogenous H2O2 and the peroxides generated as a byproduct of aerobic cultivation, preventing oxidative damage to DNA and RNA. Individual expression of StSodA or LpKatL both improved B. longum NCC2705 cell viability. Survival rate of strain NCC2705 was further improved by combining SOD and KAT expression. The two enzymes played complementary roles in ROS-scavenging pathways, and coexpression led to a synergistic beneficial effect under conditions of intensified oxidative stress. Our results illustrate that heterogeneous expression of heme-dependent KAT and Mn(2+)-dependent SOD is functional in the B. longum oxidative-stress response, and synergistic protection is achieved when their expressions are combined.

Synthetic disialyl hexasaccharides protect neonatal rats from necrotizing enterocolitis.

Two novel synthetic α2-6-linked disialyl hexasaccharides, disialyllacto-N-neotetraose (DSLNnT) and α2-6-linked disialyllacto-N-tetraose (DS'LNT), were readily obtained by highly efficient one-pot multienzyme (OPME) reactions. The sequential OPME systems described herein allowed the use of an inexpensive disaccharide and simple monosaccharides to synthesize the desired complex oligosaccharides with high efficiency and selectivity. DSLNnT and DS'LNT were shown to protect neonatal rats from necrotizing enterocolitis (NEC) and are good therapeutic candidates for preclinical experiments and clinical application in treating NEC in preterm infants.

Effect of electroporation on viability and bioconversion of isoflavones in mannitol-soymilk fermented by lactobacilli and bifidobacteria.

BACKGROUND: The aim of this study was to evaluate the effect of electroporation (2.5-7.5 kV cm⁻¹ for 3.0-4.0 ms) on the growth of lactobacilli and bifidobacteria, membrane properties and bioconversion of isoflavones in mannitol-soymilk. RESULTS: The viability of lactobacilli and bifidobacteria decreased immediately after electroporation. This was attributed to lipid peroxidation, which led to alterations in the membrane phospholipid bilayer, specifically at the polar head, interface and apolar tail regions. Such alterations also resulted in decreased membrane fluidity and increased membrane permeability upon electroporation (P < 0.05). However, the effect was reversible and treated cells showed better growth than the control upon fermentation for 24 h at 37 °C (P < 0.05). Additionally, electroporation increased the bioconversion of glucosides to bioactive aglycones in mannitol-soymilk, which was attributed to increased intracellular and extracellular ß-glucosidase activities of cells upon treatment (P < 0.05). CONCLUSION: Application of electroporation on lactobacilli and bifidobacteria could be beneficial for the development of fermented soymilk with enhanced bioactivity. Considering the enhanced bioactive aglycones, this soymilk could be useful for the prevention of hormone-dependent disorders.

Metabolism of ginsenoside Rb1 by human intestinal microflora and cloning of its metabolizing ß-D-glucosidase from Bifidobacterium longum H-1.

To understand the role of intestinal microflora in expressing the pharmacological effect of ginsenoside Rb1, the metabolic activity of ginsenoside Rb1 by 148 fecal specimens was measured and its metabolizing ß-glucosidase was cloned. The average activities for p-nitrophenyl-ß-D-glucopyranoside and ginsenoside Rb1 were 0.097±0.059 µmol/min/mg and 0.311±0.118 pmol/min/mg, respectively. These enzyme activities were not different between male and female, or between ages. A gene encoding ß-D-glucosidase (BglX) was cloned from Bifidobacterium longum H-1, which transformed ginsenoside Rb1 to compound K. The probe for cloning was synthesized from the genes encoding a ß-D-glucosidase of previously reported B. longum DJO10A. The sequences of the cloned gene revealed 2364 bp open reading frame (ORF) encoding a protein containing 787 amino acids (molecular weight of 95 kDa). The gene exhibited 99% homology (identities) to that of B. longum. The cloned gene was expressed under T7 promoter of the expression vector, pET-39b(+), in Escherichia coli BL21(DE3), and the expressed enzyme was purified by using HiTrap immobilized metal affinity chromatography (IMAC) HP. The enzyme potently biotransformed ginsenoside Rb1, loganin, arctiin and arbutin to ginsenoside Rd, loganetin, arctigenin and hydroquinone, respectively, but was not active in the case of hesperidin, and kakkalide. This is the first report on cloning and expression of ß-D-glucosidase from B. longum. Based on these findings, ginsenoside Rb1 may be metabolized to bioactive compound(s) by exo-ß-D-glucosidase(s) produced from the intestinal bacteria and its pharmacological effects may be dependent on intestinal bacterial exo-ß-D-glucosidase(s) activity.

Metabolism of a plant derived galactose-containing polysaccharide by Bifidobacterium breve UCC2003.

In this study, we describe the functional characterization of the Bifidobacterium breve UCC2003 gal locus, which is dedicated to the utilization of galactan, a plant-derived polysaccharide. Using a combination of molecular approaches we conclude that the galA gene of B. breve UCC2003 encodes a ß-1,4-endogalactanase producing galacto-oligosaccharides, which are specifically internalized by an ABC transport system, encoded by galBCDE, and which are then hydrolysed to galactose moieties by a dedicated intracellular ß-galactosidase, specified by galG. The generated galactose molecules are presumed to be fed into the fructose-6-phosphate phosphoketolase pathway via the Leloir pathway, thereby allowing B. breve UCC2003 to use galactan as its sole carbon and energy source. In addition to these findings we demonstrate that GalR is a LacI-type DNA-binding protein, which not only appears to control transcription of the galCDEGR operon, but also that of the galA gene.

The ability of green tea to positively modulate key markers of gastrointestinal function in rats.

The beneficial effects of selenium-containing green tea (Se-GTE, 1.44 mg selenium/kg dry leaves) and China green tea (CH-GTE, 0.13 mg selenium/kg leaves) on the population size of lactobacilli and bifidobacteria and the activity of two microbial enzymes in the caeca of rats have been investigated. Oral gavage of rats with Se-GTE extract for 6 days resulted in a significant increase in caecal counts of lactobacilli and bifidobacteria (p < 0.05) while significantly reducing the caecal counts of bacteroides and clostridial bacteria. In contrast, gavaging the rats with CH-GTE extract for 6 days resulted in a slight but not significant increase in the numbers of caecal lactobacilli and bifidobacteria but decreased significantly the numbers of bacteroides (p < 0.05) and clostridia (p < 0.05). In addition, rats gavaged with CH-GTE and Se-GTE showed a 17.2% and 21.3% reduction in the activity of the bacterial enzyme ß-glucuronidase, respectively, when compared with the rats gavaged with water only. ß-glucuronidase is considered to be one of the enzymes that increases the risk for colorectal cancer. Moreover, gavaging rats with these teas resulted in 19% and 25.5% increments in the activity of ß-glucosidase, respectively. In conclusion, Se-GTE showed both bifidogenic and lactogenic effects and the high level of selenium may be behind the superiority of this tea over CH-GTE.

Production of beta-glucosidase and hydrolysis of isoflavone phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei in soymilk.

The study determined beta-glucosidase activity of commercial probiotic organisms for hydrolysis of isoflavone to aglycones in fermenting soymilk. Soymilk made with soy protein isolate (SPI) was fermented with Lactobacillus acidophilus LAFTI L10, Bifidobacterium lactis LAFTI B94, and Lactobacillus casei LAFTI L26 at 37 degrees C for 48 h and the fermented soymilk was stored for 28 d at 4 degrees C. beta-Glucosidase activity of organisms was determined using rho-nitrophenyl beta-D-glucopyranoside as a substrate and the hydrolysis of isoflavone glycosides to aglycones by these organisms was carried out. The highest level of growth occurred at 12 h for L. casei L26, 24 h for B. lactis B94, and 36 h for L. acidophilus L10 during fermentation in soymilk. Survival after storage at 4 degrees C for 28 d was 20%, 15%, and 11% greater (P < 0.05) than initial cell counts, respectively. All the bacteria produced beta-glucosidase, which hydrolyzed isoflavone beta-glycosides to isoflavone aglycones. The decrease in the concentration of beta-glycosides and the increase in the concentration of aglycones were significant (P < 0.05) in the fermented soymilk. Increased isoflavone aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk.

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N-acetylglucosamine metabolism in Bifidobacterium.

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial D: -fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the D: -fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.

Estrogenic effect of main components kakkalide and tectoridin of Puerariae Flos and their metabolites.

To understand the relationship between the metabolism and estrogenic activity of kakkalide and tectoridin, main isoflavones in the flower of Pueraria thunbergiana (family Leguminosae), these isoflavones and their metabolites by human intestinal microflora as well as their estrogenic effects were investigated. All human fecal specimens metabolized kakkalide and tectoridin. All isolated kakkalide-hydrolyzing intestinal bacteria also hydrolyzed kakkalide and tectoridin to irisolidone and tectorigenin, respectively. When the estrogenic effects of kakkalide and tectoridin were compared with those of their metabolites irisolidone and tectorigenin, the metabolites more potently increased proliferation of MCF-7 cells than kakkalide and tectoridin. These metabolites also potently induced estrogen-response c-fos and pS2 mRNA expression. These results suggest that kakkalide and tectoridin may be metabolized mainly to irisolidone and tectorigenin, respectively, by intestinal microflora in the intestines, and which may be subsequently absorbed into the blood where they can express their estrogenic effect.

Effect of dietary honey on intestinal microflora and toxicity of mycotoxins in mice.

BACKGROUND: Bee honey is a functional food which has a unique composition, antimicrobial properties and bifidogenic effect. In order to assess whether honey can inhibit the toxic effect of mycotoxins, the present study was undertaken. METHODS: Production of biomass and toxins by Aspergillus parasiticus and Aspergillus ochraceus were followed in media without and with honey. Although aflatoxins and ochratoxin A. were administrated to male Swiss albino mice up to 1 mug and 10 ng/kg body weight/day respectively. The experimental animals were fed diets without our with 10% honey for two months. The changes in colonic probiotic bacteria, determintal colon enzyme glucuronidases, and genotoxicity were followed. RESULTS: Addition of 32% in its media increased the biomass of A parasiticus, while the biomass of A. ochraceus decreased and Ochratoxin A. was not produced. When the honey was added at the ratio of 32 and 48% in the medium. No relationship was found between mycelium weight and production of mycotoxins. Oral administration of aflatoxins (mixture of B1, B2, G1 and G2) and Ochratoxin A. induced structural and numerical chromosomal aberrations in bone marrow and germ cells of male mice, whereas, honey treatment reduced the genotoxicity of mycotoxins. Also both toxins induced histopathological changes in liver and kidney. Feeding on diet supplemented with honey improved the histopathological changes in case of aflatoxin group, but not in the case of ochratoxin A. group (except of kidney in two cases). No significant differences were found in the activity of colon beta-glucuronidase between group fed diet with or without honey. On the other hand, the colon bifido bacteria and lactobacilli counts were increased markedly in group receiving diet supplemented with honey. CONCLUSION: Substituting sugars with honey in processed food can inhibit the harmful and genotoxic effects of mycotoxins, and improve the gut microflora.

[Inhibition of carbohydrate metabilsm enzymes by pentachlorophenol in cells of pectinolytic bacteria from gastrointestinal tract of animals and effect of clinoptilolite adsorbents on this process].

Changes in functional activity of specific enzyme reactions in the cells of pectinolytic bacteria from the gastrointestinal tract of animals in vitro cultivated in the medium containing pectin or glucose were studied against a background of the low dose effect of the wide spread biocide pentachlorophenol alone as well as in combination with the natural sorbents clinoptilolites. Regardless of the absence of transketolase reaction in the cells of all studied strains, they metabolized highly the above substrates that are dissimilar in chemical structure and produced different products of their degradation. It has been shown that the high metabolic level in the cells is provided by the function of the unique enzymatic reaction catalyzed by 2-keto-3-desoxy-6-phosphogluconate aldolase (EC 4.1.2.14) that permits to use effectively the metabolic pathway of Entner-Doudoroff. Cells could also utilize the same substrates via the Embden-Meyerhof-Parnas pathway, therefore they possess the other key reaction that is catalyzed by fructosobiphosphate aldolase (EC 4.1.2.13). Even a low dose of PCP (20 microM) decreased sharply activity of the mentioned key enzymes and intermediates' production in the cells of the studied strains with the use of both substrates. However, presence of clinoptilolites in the medium reduced significantly the biocide inhibition effect. Furthermore, in the medium with glucose, protection of intracellular metabolism with the help of sorbents was registered more clearly than with pectin. This can evidence for more mobile and simpler possibilities of accelerated production of necessary intermediates from glucose that are capable to induce activation of the key enzymatic reactions in cells utilizing selectively the substrates (which are different in accessibility and other characteristics) under the toxic agent effect.

Transformation of ginsenosides Rb1 and Re from Panax ginseng by food microorganisms.

Ginsenosides Rb1 and Re, respectively belonging to the major protopanaxadiol and protopanaxatriol ginsenosides, were transformed using cell-free extracts from food microorganisms. Rb1 was transformed into compound K via Rd and F2 by Bifidobacterium sp. Int57, Bif. sp. SJ32, Aspergillus niger and A. usamii. Lactobacillus delbrueckii, and Leuconostoc paramesenteroides transformed Rb1 into Rh2 via Rd and F2. Bifidobacterium sp. SH5 transformed Rb1 into F2 via Rd. Re was transformed into Rh1 via Rg2 by Bif. sp. Int57 and Bif. sp. SJ32. A. niger transformed Re into Rh1 via Rg1. A. usamii transformed Re into Rg2. Transformation of Rb1 proceeded at a higher rate and needed less amount of enzymes than that of Re. Taken together, these processes would allow a specific bioconversion process possible to obtain specific ginsenosides using an appropriate combination of ginsenoside substrates and specific microbial enzymes.

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bifidobacterium pseudolongum.

AIMS: In a rabbit caecal bacterium Bifidobacterium pseudolongum, metabolites of pectin and glucose, and activities of enzymes involved in the degradation of pectin were assayed. Simultaneously, activities of these enzymes were assayed in a rumen pectinolytic strain of Streptococcus bovis. METHODS AND RESULTS: A strain B. pseudolongum P6 which grew best on pectin was selected among bifidobacteria isolated from the rabbit caecum. Cultures of B. pseudolongum P6 grown on pectin produced significantly less formate, lactate and ethanol, and more acetate and succinate than cultures grown on glucose. No CO2 production on pectin was observed. Pectin macromolecule was degraded by extracellular pectinase (EC 3.2.1.15). Cell extracts possessed the activity of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). Streptococcus bovis X4, possessed activity of exopectate lyase and pectinase, but not that of KDPG aldolase. CONCLUSIONS: Our results are consistent with the assumption that in B. pseudolongum P6 acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway, as shown previously in rumen pectin-utilizing bacteria. The missing KDPG aldolase activity in Strep. bovis X4 seems to be the reason for the absence of growth of this bacterium on pectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on polysaccharide metabolism in bifidobacteria is fragmentary. This study extends the knowledge on pectin metabolism in intestinal bacteria.

Microbiological and biochemical properties of canestrato pugliese hard cheese supplemented with bifidobacteria.

Canestrato Pugliese cheeses from ewe milk were produced according to a traditional protocol and by adding 7.0 log10 cfu of fresh cells per gram of Bifidobacterium bifidum Bb02, Bifidobacterium longum Bb46, or both species. The traditional technology was modified slightly to favor the survival of probiotic microorganisms. After 56 d of ripening, the survival of B. bifidum Bb02 and B. longum Bb46 was 6.0 and 5.0 log10 cfu/g, respectively. After 19 d cheeses contained ca. 7.0 log10 cfu/g of bifidobacteria. Compared to traditional cheese, the addition of bifidobacteria seemed to support the growth and survival of mesophilic lactobacilli and Streptococcus thermophilus, used as starter, during ripening. No significant differences were observed in the main chemical composition, and only a slightly higher concentration of acetic acid was found in cheeses with bifidobacteria added. On the contrary, alpha- and beta-galactosidase activities were markedly more pronounced in the presence of bifidobacteria, especially with B. bifidum Bb02. In contrast with traditional cheese, the lactose was completely hydrolyzed in cheeses made with bifidobacteria. Urea-PAGE electrophoresis of the pH 4.6-soluble and pH 4.6-insoluble N fractions did not show appreciable variations. Only the reversed-phase-HPLC analysis of the pH 4.6-soluble N showed a slightly more complex profile in the presence of bifidobacteria. This finding was in agreement with the higher value of the pH 4.6-soluble N/total N ratio and with the more pronounced amino-, imino-, and dipeptidase activities found in all the cheeses with the bifidobacteria added, especially B. bifidum Bb02. No differences were found in the free amino acid and free fatty acid contents. The amino acids glutamic acid, valine, proline, leucine, and lysine and the fatty acids butyric, caproic, capric, and oleic acids were found at the highest concentrations. The sensory evaluation did not show significant differences, and Canestrato Pugliese cheeses were characterized by small and uniformly distributed eyes, were pale yellow, had an elastic consistency and a Pecorino-like smell, were very salty, and tended to be moderately piquant.

Purification and characterization of a novel sennoside-hydrolyzing beta-glucosidase from Bifidobacterium sp. strain SEN, a human intestinal anaerobe.

A novel beta-glucosidase, which is inducible and capable of catalyzing the hydrolysis of sennosides, was purified from Bifidobacterium sp. strain SEN with Triton X-100 solubilization and DEAE-cellulose column chromatography, by which hydrolytic activities toward sennoside B, 4-methylumbelliferyl beta-glucoside (MUG), and p-nitrophenyl beta-glucoside (pNPG) were obtained together in the same eluted fractions. The activity was stable against detergents such as sodium dodecyl sulfate (SDS) and Triton X-100, but was denatured by SDS and beta-mercaptoethanal when heated. The final preparation was shown to be nearly homogeneous on SDS-polyacrylamide gel electrophoresis (PAGE) either after the enzyme was denatured or when it was not denatured. In the non-denaturing SDS-PAGE, a single protein band hydrolyzed MUG on the gel. In the denaturing SDS-PAGE, the subunit mass of the enzyme was estimated to be 110 kDa. The enzyme was optimally active at pH 6.0 for hydrolysis of sennoside B and MUG. Km values for sennoside B and MUG are 0.94 and 0.53 mM, respectively. The enzyme also catalyzed the hydrolysis of pNPG, amygdalin, geniposide and salicin. It was less active against methyl beta-glucoside and incapable of hydrolyzing cellobiose. The beta-glucosidase activity was inhibited by deoxynojirimycin and p-chloromercuribenzenesulfonic acid, but was less susceptible to several metals (FeSO4, ZnCl2, and CuSO4), and 5,5'-dithio-bis(2-nitrobenzoic acid).