Ribotyping of adherent Lactobacillus from weaning pigs: a basis for probiotic selection based on diet and gut compartment

Dietary inoculates for weaned pigs often include lacobacilli, but the effects of age and diet on the ecology of adherent lactobacilli are not well known. Four experimental treatments included 16 newborn piglets: a pre-weaning treatment (PW) included four pigs that were sacrificed at 28 days of age; a sow treatment (Sow) had four pigs that remained with the sow (Sow) from 28 to 38 days of age and were sacrificed at 38 days of age; and two treatments in which four pigs each, were placed on a corn-soy diet with (CSL), or without (CS) 40% lactose. Adherent lactobacilli were isolated from the pars esophagus, ileum, and cecum, and characterised phenotypically. A 23S rDNA probe hydrized toEcoRI orPstI digested DNA, and a 74.8% cut-off individualize isolates into 36 clusters. The most predominant strains found in the Sow, CSL, and CS treatments were the same, but different strains predominated in the PW animals. The most predominant strains found in the pars esophagus and ileum were the same, but different strains predominated in the cecum.     

Specific growth rate of bifidobacteria cultured on different sugars

The ability of six bifidobacterial strains (3 of human origin and 3 isolates from fermented milk products) to utilize glucose, lactose, melezitose, sucrose, raffinose, and stachyose was determined. Dairy-related bifidobacterial strains were identified asBifidobacterium animalis (2 strains) or asB. pseudolongum (1 strain). Human strains includedB. longum (2 strains) andB. breve (1 strain). All strains fermented lactose, sucrose, raffinose, and stachyose. Melezitose was utilized only byB. longum. B. pseudolongum did not ferment either glucose or melezitose. All isolates had a higher specific growth rate on raffinose and stachyose than on glucose. Dairy strains grew slowly on glucose compared to human strains.     

Effect of Dietary Supplementation With α-Galactosidase Preparation and Stachyose on Growth Performance,Nutrient Digestibility and Intestinal Bacterial Populations of Piglets

Two experiments were conducted to investigate the effect of dietary supplementation with f -galactosidase preparation and stachyose on growth performance, nutrient digestibility and intestinal bacterial populations of piglets. In Experiment I 72 crossbred piglets were allotted to three treatments, i.e. 1) control (basal) diet, 2) f -Gal1 and 3) f -Gal5, the basal diet supplemented with 0.01% and 0.05% f -galactosidase preparation ( f -Gal), respectively. Average daily gain, average daily feed intake and feed conversion ratio were not influenced by f -Gal supplementation. In Experiment II 72 crossbred weanling piglets were randomly assigned to four treatments with a complete factorial design, i.e. 1) basal diet, 2) basal diet with 0.01% f -Gal supplementation, 3) basal diet with 1% stachyose supplementation and 4) basal diet with 1% stachyose and 0.01% f -Gal supplementation. Average daily gain and feed conversion ratio were influenced by f -Gal and stachyose supplementation. The ileal digestibility of stachyose, raffinose, gross energy and crude protein was improved significantly by f -Gal supplementation. The microbial populations in the intestine were modified by both f -Gal and stachyose supplementation. The data suggested that stachyose supplementation had an adverse effect on the growth performance of piglets and f -Gal supplementation could ameliorate it, especially in younger pigs.     

A novel application of Cicer α-galactosidase in reduction of raffinose family oligosaccharides in soybean flour

Raffinose family oligosaccharide (RFOs) especially raffinose and stachyose are responsible for flatulence during intake of soybean derived products. This gives negative impression on the acceptance of soy-based foods. The effect of soaking, cooking and Cicer α-galactosidase treatment for reducing stachyose and raffinose content from the soybean flour was investigated. Cicer α-galactosidase treatment was found to be most effective in comparison to traditional methods, soaking and cooking. Enzymatic treatment for 6 h reduces raffinose and stachyose component of soybean flour by 80.0 % and 85.0 %, respectively. This can be utilized effectively for improving the nutritional quality of soy-based foods on large scale, which could be one of the best alternatives for lactose intolerant population for fulfilling their protein requirement.     

Survey of Some Actinomycetales for α-Galactosidase Activity

The enzyme alpha-galactosidase offers potential to (i) eliminate possibly the flatus-inducing factor(s) in edible beans, (ii) eliminate raffinose during beet-sugar processing, and (iii) determine raffinose analytically. Accordingly, 20 genera of the order Actinomycetales Buchanan 1917 were tested for evidence of alpha-galactosidase activity. Test filtrates were prepared with a medium containing D-galactose and soybean meal. Enzyme activity was demonstrated through cellulose thin-layer chromatography. Of 123 strains tested, 28 produced extracellular alpha-galactosidase. Almost all were streptomycetes. Members of the genera Actinoplanes Couch 1950, Micromonospora varphiOrskov 1923, and Promicromonospora Krasil'nikov et al. 1961 also exhibited alpha-galactosidase activity. Additional tests led to the selection of five strains whose filtrates degraded melibiose, raffinose, and stachyose but not lactose and sucrose. Tests also were made with several soybean preparations.     

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.     

Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus.

When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces alpha-galactosidase I which is different from alpha-galactosidase II which it produces when grown on galactose, melibiose, raffinose, or stachyose. We have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9 to 6.4 for alpha-galactosidase I, 6.3 to 6.5 for alpha-galactosidase II). However, alpha-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000, whereas alpha-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. alpha-Galactosidase I has a lower affinity for melibiose, raffinose, and stachyose (Km values of 20.8, 98.1, and 8.5 mM, respectively) than does alpha-galactosidase II (Km values of 2.3, 5.9, and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase. The increase in specific activity of alpha-galactosidase which was associated with growth on guar gum was due to an increase in the specific activity of enzyme I. Low, constitutive levels of enzyme II also were produced. By contrast, enzyme II was the only alpha-galactosidase that was detectable in bacteria which had been grown on galactose, melibiose, raffinose, or stachyose.     

大豆低聚糖对牙鲆营养效应的研究：Ⅰ摄食、生长和代谢酶活性

本试验比较研究了不同蛋白源(鱼粉,FM;大豆分离蛋白,SPI)基础饲料中添加大豆低聚糖(SBOS)对牙鲆(Paralichthys olivaceus)摄食率、生长性能和代谢酶活性的影响。分别以FM、SPI作为主要蛋白源,配制了4种等氮等能饲料。其中,饲料FM、SPI分别以FM、SPI作为主要蛋白源;饲料FMO、SPIO分别在饲料FM、SPI基础上添加10%SBOS(水苏糖：2.61%;棉籽糖：0.61%)。试验表明：①饲料中添加SBOS对牙鲆前两周摄食率无显著影响(p > 0.05);而显著降低了整个养殖周期FMO组牙鲆总摄食率,提高了SPIO组牙鲆总摄食率(p < 0.05);②FM基础饲料中添加SBOS对牙鲆特定生长率(SGR)、饲料效率(FER)和蛋白质效率(PER)均无显著影响(p > 0.05),但均呈明显下降趋势;SPI基础饲料中添加SBOS对FER和PER无显著影响,却显著提高了SGR(p < 0.05);③FM基础饲料中添加SBOS提高了牙鲆血浆谷草转氨酶(AST)、谷丙转氨酶(ALT)、碱性磷酸酶(AKP)、乳酸脱氢酶(LDH)和γ-L-谷氨酰转肽酶(γ-GT)的活性,但仅AST差异显著(p < 0.05),而对肝脏AST、ALT、AKP、LDH和γ-GT活性无显著影响(p > 0.05);SPI基础饲料中添加SBOS显著提高了血浆ALT和LDH活性,而对AST、AKP和γ-GT活性无显著影响;同时,SPI基础饲料中添加SBOS显著提高了肝脏AST和ALT活性,而对AKP、LDH和γ-GT活性无显著影响;④饲料中添加SBOS对牙鲆血浆中尿素氮和游离氨基酸浓度均无显著影响。试验结果表明,不同蛋白源基础饲料中添加SBOS表现出不同的生长效应;其中,FM基础饲料中添加SBOS表现出一定的抑制生长效应,而SPI基础饲料中添加SBOS却表现出促生长效应。     

Carbohydrate Changes during Maturation of Cucumber Fruit : Implications for Sugar Metabolism and Transport

Changes in the carbohydrate profiles in the mesocarp, endocarp, and seeds of maturing cucumber (Cucumis sativus, L.) fruit were analyzed. Fruit maturity was measured by a decrease in endocarp pH, which was found to correlate with a loss in peel chlorophyll and an increase in citric acid content. Concentrations of glucose and fructose (8.6-10.3 milligrams per gram fresh weight, respectively) were found to be higher than the concentration of sucrose (0.3 milligrams per gram fresh weight) in both mesocarp and endocarp tissue. Neither raffinose nor stachyose were found in these tissues. The levels of glucose and fructose in seeds decreased during development, but sucrose, raffinose, and stachyose accumulated during the late stages of maturation. Both raffinose and stachyose were found in the seeds of six lines of Cucumis sativus L. This accumulation of raffinose saccharides coincided with an increase in galactinol synthase activity in the seeds. Funiculi from maturing fruit were found to be high in sucrose concentration (4.8 milligrams per gram fresh weight) but devoid of both raffinose and stachyose. The results indicated that sucrose is the transport sugar from the peduncle to seed, and that raffinose saccharide accumulation in the seed is the result of in situ biosynthesis and not from direct vascular transport of these oligosaccharides into the seeds.     

Influence of Diet and Age on Bacterial Counts of Ileal Digesta and Feces Obtained from Young Calves

The numbers of total aerobes, total anaerobes, and facultatively aerobic lactobacilli, streptococci, and coliform bacteria were determined in samples of ileal digesta and feces from calves 0, 3, 6, 9, and 12 hr after feeding milk, or milk with sucrose or starch added. Differences in the flora could be attributed to diurnal variation (time from feeding to sampling), source of the sample (ileal versus fecal), nature of the dietary carbohydrate (lactose versus lactose plus sucrose versus lactose plus starch) and age of the animal (on a diet of milk plus sucrose).     

In-feed administered sub-therapeutic chlortetracycline alters community composition and structure but not the abundance of community resistance determinants in the fecal flora of the rat

The impact of continuous sub-therapeutic chlortetracycline on community structure, composition and abundance of tetracycline resistance genes in the rat fecal community was investigated. Rats were fed a standard diet containing chlortetracycline at 15 μg g⁻¹ diet for 28 days, followed by 30 μg g⁻¹ diet to completion of the study on day-56. These levels are similar to those administered to swine during the grow-out phase. Sub-therapeutic chlortetracycline affected the fecal community as determined through change in the cultivable anaerobic community and through molecular-based analyses including denaturing gradient gel electrophoresis profiles of the variable 2–3 region community 16S rRNA genes over time and through comparative sequence analysis of 16S rRNA gene community libraries. Significant decreases in fecal phylotype diversity occurred in response to sub-therapeutic chlortetracycline, although total bacterial output remained constant over the entire feeding trial. Chlortetracycline at 15 μg g⁻¹ diet resulted in significant change in community composition, but only modest change to the fecal community structure in terms of the distribution of individual phylotypes among the major fecal lineages. Chlortetracycline at 30 μg g⁻¹ diet significantly altered the distribution of phylotypes among the major fecal lineages shifting the overall community such that Gram-negative phylotypes aligning within the phylum Bacteroidetes became the dominant lineage (>60% of total community). While chlortetracycline impacted both fecal community structure and composition, there was no significant effect on the abundance of community tetracycline resistance genes [tet(Q), tet(W), tet(O)] or on the emergence of a new putative tetracycline resistance gene identified within the fecal community. While sub-therapeutic chlortetracycline provides sufficient selective pressure to significantly alter the fecal community, the primary outcome appears to be the development of a community which may have a higher inherent tolerance to sub-therapeutic levels of chlortetracycline rather than an overgrowth of the tetracycline resistant bacteria already present within the community.     

Naturally occurring prebiotic oligosaccharides in poultry feed mixtures

The presence of raffinose series oligosaccharides (RSO) was determined by an enzymic method in three commercially available chicken feed mixtures. All feed mixtures contained RSO at a concentration of 2.1–2.2 %. Soya meal was identified as the exclusive source of RSO. Subsequently, the bifidogenic effect of stachyose (main soya bean RSO) was also assigned on the growth of poultry intestinal bifidobacteria. Bifidobacteria were counted in chicken intestinal tract using cultivation and FISH methods. Four out of 6 bifidobacterial strains tested grew significantly better on stachyose than on glucose. It can be thus concluded that chicken feed mixtures naturally contain prebiotic oligosaccharides in the form of RSO in higher levels (>2 %) compared with the concentration (usually up to 1 %) recommended for artificially added prebiotics. Our results therefore indicate that there is no reason for the supplementation of chicken feed mixtures with prebiotics with bifidogenic properties.     

Degradation of stachyose,raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi

Forty-six strains of tempe-forming Rhizopus species were screened for their ability to grow on raffinose as the sole carbon source. Six of the strains showed good growth and sporulation. These isolates were one Rhizopus oligosporus, one Rhizopus microsporus var. chinensis, three Rhizopus oryzae and one Rhizopus stolonifer. These six moulds and R. oligosporus strain NRRL 2710 were investigated for their metabolism of the raffinose family of -galactoside carbohydrates. Degradation experiments were performed in submerged culture in a medium containing soybean -protein, sodium phytate and either stachyose, raffinose or melibiose. R. oryzae and R. stolonifer completely consumed the tested carbohydrates as carbon source. R. microsporus var. chinensis failed to hydrolyse the -galactosidic bonds of raffinose, stachyose or melibiose, whereas it was able to use sucrose and the fructose moiety of raffinose or stachyose for growth. R. oligosporus NRRL 2710 was unable to hydrolyse any of the tested carbohydrates. The results of the oligosaccharide degradation experiments could be verified during tempe production from soybeans with the selected fungal species.     

Nonstructural carbohydrates in dormant and afterripened wild oat caryopses

Nonstructural carbohydrates were determined in both embryo and endosperm of dormant (nongerminating) and afterripened (germinating) intact caryopses of wild oat ( Avena fatua L.). No changes in endosperm starch or soluble sugar were observed at the onset of germination (18 h). No changes in glucose, fructose, sucrose or starch within dormant or afterripened embryos correlated with onset of visual germination. In afterripened embryos, depletion of raffinose (18 h), stachyose (18 h) and galactose (24 h) was correlated with germination. In contrast, raffinose-family oligosaccharide levels in dormant embryos remained constant for 7 days following imbibition. Germination of isolated dormant embryos on 88 m M galactose-containing media was accompanied by decreased endogenous levels of raffinose and stachyose. Isolated embryos from dormant caryopses incorporated ¹⁴C from ¹⁴C-fructose into both raffinose and stachyose during 24 h of imbibition. In contrast, no ¹⁴C incorporation into stachyose was observed in embryos from afterripened caryopses. No ¹⁴C incorporation into raffinose was observed at 18 and 24 h. When in vitro activities of α galactosidase were measured, no temporal differences between dormant or afterripened caryopses were detected in either embryo or endosperm tissue. Although the mechanism associated with differences in utilization of raffinose and stachyose is yet unidentified, alterations in raffinose-family oligosaccharide metabolism in the embryo appear to be a unique prerequisite for afterripening-induced germination.     

Galactinol synthase activity and soluble sugars in developing seeds of four soybean genotypes

Galactinol synthase (UDP-galactose:inositol galactosyltransferase) is the first unique enzyme in the biosynthetic pathway of raffinose saccharides. Its role as a regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean (Glycine max L. Merrill) seeds was examined. Galactinol synthase activity and concentrations of sucrose, stachyose, and raffinose were compared during seed development between two genotypes that were high and two genotypes that were low in mature seed raffinose saccharide concentration. In all genotypes, sucrose concentration increased as seed development progressed, but in both low raffinose saccharide genotypes, greater increases in sucrose concentration were observed late in seed development. Sucrose to stachyose ratios in mature seeds were 2.3-fold greater in low raffinose saccharide genotypes than in the high raffinose saccharide genotypes. During seed development, higher levels of galactinol synthase activity were observed in the high raffinose saccharide genotypes than in the low raffinose saccharide genotypes. A common linear relationship for all four soybean genotypes was shown to exist between galactinol formed estimated from galactinol synthase activity data and the concentration of galactose present in raffinose saccharides. Results of this study implied that galactinol synthase is an important regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean seeds.     

Conditions of formation, purification, and characterization of an alpha-galactosidase of Trichoderma reesei RUT C-30.

Trichoderma reesei RUT C-30 formed an extracellular alpha-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain alpha-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced alpha-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of alpha-galactosidase formation by galactose. alpha-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 +/- 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several alpha-galactosides (p-nitrophenyl-alpha-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei beta-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50 degrees C resulted in recovery of 70% of activity), and its highest level of activity occurred at 60 degrees C. Its action on galactomannan was increased by the presence of beta-mannanase.     

Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.