Isolation, growth on prebiotics and probiotic potential of novel bifidobacteria from pigs

Bifidobacteria were isolated from the faeces of pigs of various ages and examined for their potential use as probiotics in combination with di- and oligosaccharides. Ninty-six per cent of the isolates were found to have characteristics in common with Bifidobacterium boum, B. thermophilum and B. choerinum. B. thermophilum was most commonly isolated from sows, whereas most of the other strains were isolated from piglets. A few strains of each species were able to grow in the presence of air. A microplate assay was modified to allow comparison of growth on different substrates. Di- and oligosaccharides considered to promote bifidobacteria were screened for their ability to support growth of selected isolates in vitro. Growth on these substrates varied within and between species. Of the fructose oligosaccharides tested, Actilight P supported the best growth of the widest range of strains. The strains which grew best on the disaccharide lactulose were related to B. choerinum and some of these strains grew on xylo-oligosaccharides. It seems that prebiotic di- and oligosaccharides may have both a species and intra-species/strain selective effect. B. choerinum appeared to be well adapted to the gut of pre-weaned piglets.     

Response of the Microaerophilic Bifidobacterium Species, B. boum and B. thermophilum, to Oxygen

We investigated the effects of O₂ on Bifidobacterium species using liquid shaking cultures under various O₂ concentrations. Although most of the Bifidobacterium species we selected showed O₂ sensitivity, two species, B. boum and B. thermophilum, demonstrated microaerophilic profiles. The growth of B. bifidum and B. longum was inhibited under high-O₂ conditions accompanied by the accumulation of H₂O₂ in the medium, and growth was restored by adding catalase to the medium. B. boum and B. thermophilum grew well even under 20% O₂ conditions without H₂O₂ accumulation, and growth was stimulated compared to anoxic growth. H₂O-forming NADH oxidase activities were detected dominantly in cell extracts of B. boum and B. thermophilum under acidic reaction conditions (pH 5.0 to 6.0).     

Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain.

Symbiobacterium thermophilum is a strictly symbiotic thermophile, the growth of which is dependent on the coexistence of an associating thermophilic Bacillus sp., strain S. S. thermophilum grows only in mixed culture with the Bacillus strain in liquid media, and does not form visible colonies on solid media. To measure the growth of this symbiotic bacterium and to analyze its growth requirements, we developed a quantitative PCR method by using its specific sequences in a putative membrane translocator gene tnaT as primers. According to this method, independent growth of S. thermophilum was first confirmed in a dialyzing culture physically separated from Bacillus strain S with a cellulose membrane. Independent growth of S. thermophilum was also managed by adding conditioned medium prepared from the culture filtrate of the Bacillus strain, but the growth in the conditioned medium stopped at a very limited extent with appearance of filamentous cells, suggesting the uncoupling of cellular growth and cell division. Formation of micro-colonies of S. thermophilum was observed on the conditioned agar medium under both aerobic and anaerobic conditions, but the colony-forming efficiencies remained below 1%. Several other bacterial species, such as Bacillus stearothermophilus, Bacillus subtilis, Thermus thermophilus, and even Escherichia coli, were also found to support the growth of S. thermophilum. These results indicate that S. thermophilum essentially requires some ubiquitous metabolite(s) of low molecular weight produced by various bacterial species as growth factor(s) but coexistence of the living partner cells is still required, probably to maintain an effective level of the putative factor(s) in the medium.     

Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 micrograms/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na+, K+, Mg2+ or Ca2+ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

Role of 2-amino-3-carboxy-1,4-naphthoquinone, a strong growth stimulator for bifidobacteria, as an electron transfer mediator for NAD(P)(+) regeneration in Bifidobacterium longum.

2-Amino-3-carboxy-1,4-naphthoquinone (ACNQ) is a novel growth stimulator for bifidobacteria. The role of ACNQ as a mediator of the electron transfer from NAD(P)H to dioxygen (O(2)) and hydrogen peroxide (H(2)O(2)), proposed in our previous paper, was examined using the cell-free extract and whole cells of Bifidobacterium longum. Continuous monitoring of ACNQ, O(2) and H(2)O(2) by several amperometric techniques has revealed that ACNQ works as a good electron acceptor of NAD(P)H diaphorase and that the reduced form of ACNQ is easily autoxidized and also acts as a better electron donor of NAD(P)H peroxidase than NAD(P)H. The generation of H(2)O(2) by B. longum under aerobic conditions is effectively suppressed in the presence of ACNQ. These ACNQ-mediated reactions would play roles as NAD(P)(+)-regeneration processes. The accumulation of ACNQ in the cytosol has been also suggested. These characteristics of ACNQ seem to be responsible for the growth stimulation of bifidobacteria. Vitamin K(3), which has an extremely low growth-stimulating activity and was used as a reference compound, exhibits much lower activity as an electron transfer mediator. The difference in the activity is discussed in terms of the redox potential and partition property of the quinones.     

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.     

The effect of probiotics and organic acids on Shiga-toxin 2 gene expression in enterohemorrhagic Escherichia coli O157:H7

Probiotics are known to have an inhibitory effect against the growth of various foodborne pathogens, however, the specific role of probiotics in Shiga-toxin-producing Escherichia coli (STEC) virulence gene expression has not been well defined. Shiga toxins are members of a family of highly potent bacterial toxins and are the main virulence marker for STEC. Shiga toxins inhibit protein synthesis in eukaryotic cells and play a role in hemorrhagic colitis and hemolytic uremic syndrome. STEC possesses Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), both of which have A and B subunits. Although STEC containing both Stx1 and Stx2 has been isolated from patients with hemorrhagic colitis, Stx2 is more frequently associated with human disease complications. Thus, the effect of Lactobacillus, Pediococcus, and Bifidobacterium strains on stx2A expression levels in STEC was investigated. Lactic acid bacteria and bifidobacteria were isolated from farm animals, dairy, and human sources and included L. rhamnosus GG, L. curvatus, L. plantarum, L. jensenii, L. acidophilus, L. casei, L. reuteri, P. acidilactici, P. cerevisiae, P. pentosaceus, B. thermophilum, B. boum, B. suis and B. animalis. E. coli O157:H7 (EDL 933) was coincubated with sub-lethal concentrations of each probiotic strain. Following RNA extraction and cDNA synthesis, relative stx2A mRNA levels were determined according to a comparative critical threshold (Ct) real-time PCR. Data were normalized to the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the level of stx2A expression between treated and untreated STEC was compared. Observed for all probiotic strains tested, stx2A was down-regulated, when compared to the control culture. Probiotic production of organic acids, as demonstrated by a decrease in pH, influenced stx2A gene expression.     

Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 μg/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na⁺, K⁺, Mg²⁺ or Ca²⁺ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

Wolinella recta, Wolinella curva, Bacteroides ureolyticus, and Bacteroides gracilis are microaerophiles, not anaerobes.

Although the nonfermentative, asaccharolytic, putative anaerobes Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis are phylogenetically related to the true campylobacters, the type strains of these species exhibited O2-dependent microaerophilic growth in brucella broth and on brucella agar. The optimum O2 levels for growth of these strains ranged from 4 to 14% in brucella broth and from 2 to 8% on brucella agar, when H2 was provided as the electron donor. No growth occurred under 21% O2, and scant or no growth occurred under anaerobic conditions unless fumarate or nitrate was provided as a terminal electron acceptor. Aspartate, asparagine, and malate also served as apparent electron acceptors. The organisms were catalase negative and, except for B. gracilis, oxidase positive. Catalase added to brucella broth enhanced growth. O2 uptake by all species was inhibited by cyanide and 2-heptyl-4-hydroxyquinoline N-oxide. We concluded that these organisms are not anaerobes but instead are microaerophiles, like their campylobacter relatives.     

Contribution of NADH oxidase to aerobic metabolism of Streptococcus pyogenes

An understanding of how the heme-deficient gram-positive bacterium Streptococcus pyogenes establishes infections in O(2)-rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of S. pyogenes and other lactic acid bacteria which directly catalyzes the four-electron reduction of O(2) to H(2)O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O(2) conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O(2) conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O(2) tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H(2)O(2) in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H(2)O(2) and rescued the growth defect of the mutants under high-O(2) conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O(2) with carbohydrate limitation.     

Development of a membrane dialysis bioreactor and its application to a large-scale culture of a symbiotic bacterium,Symbiobacterium thermophilum

A simple membrane dialysis bioreactor was developed for a large-scale axenic culture of Symbiobacterium thermophilum, a symbiotic thermophile that requires co-cultivation with an associating thermophilic Bacillus strain S for normal growth. The bioreactor consisted of an outer- and an inner-coaxial cylindrical compartment bordered across a dialyzing membrane, which enabled a 1 l-scale dialysis culture with exchange of low molecular metabolites between the two compartments to be performed. Using the bioreactor, growth characteristics of S. thermophilum and Bacillus strain S were assessed under two medium conditions. The growth of S. thermophilum was measured by quantitative PCR because the bacterium formed no visible colonies and gave abnormally low turbidity. In medium containing 2% tryptone peptone, S. thermophilum proliferated up to 4x10(7) cells/ml, and strict dependence on the co-culture with Bacillus strain S was observed. On the other hand, medium containing 0.5% yeast extract not only facilitated the growth of S. thermophilum in the co-culture (6x10(7) cells/ml), but also allowed limited pure growth independent of Bacillus strain S (1x10(7) cells/ml), implying that some component of yeast extract can partially replace the growth requirement of S. thermophilum supplied by Bacillus strain S. Both the oxidative redox potential values and the cell morphology in the independently growing culture suggested the occurrence of marked unbalanced growth possibly caused by significant metabolic changes. The bioreactor is applicable to the analyses of culturing characteristics in symbiotic systems between free-living microorganisms.     

Expression of uptake hydrogenase and hydrogen oxidation during heterotrophic growth of Bradyrhizobium japonicum.

Strains I-110 ARS, SR, USDA 136, USDA 137, and AK13 1c of Bradyrhizobium japonicum induced Hup activity when growing heterotrophically in medium with carbon substrate and NH4Cl in the presence of 2% H2 and 2% O2. Hup activity was induced during heterotrophic growth in the presence of carbon substrates, which were assimilated during the time of H2 oxidation. Strains I-110 ARS and SR grown heterotrophically or chemoautotrophically for 3 days had similar rates of H2 oxidation. Similar rates of Hup activity were also observed when cell suspensions were induced for 24 h in heterotrophic or chemoautotrophic growth medium with 1% O2, 10% H2, and 5% CO2 in N2. These results are contrary to the reported repression of Hup activity by carbon substrates in B. japonicum. Bradyrhizobial Hup activity during heterotrophic growth was limited by H2 and O2 and repressed by aerobic conditions, and CO2 addition had no effect. Nitrogenase and ribulosebisphosphate carboxylase activities were not detected in H2-oxidizing cultures of B. japonicum during heterotrophic growth. Immunoblot analysis of cell extracts with antibodies prepared against the 65-kilodalton subunit of uptake hydrogenase indicated that Hup protein synthesis was induced by H2 and repressed under aerobic conditions.     

H2O2参与水杨酸诱导丹参培养细胞中丹酚酸B合成的信号转导

过氧化氢(Hydrogen peroxide,H2O2)为活性氧(Reactive oxygen species,ROS)的一种,存在于许多生物体系中并介导植物中多种生理和生化过程。为了探讨H2O2作为信号分子在水杨酸(Salicylic acid,SA)诱导丹参培养细胞合成丹酚酸B(Salvianolic acid B,Sal B)过程中的作用,分别考察了SA和H2O2、过氧化氢酶(Catalase,CAT)、二甲基硫脲(2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,DMTU)及咪唑(Imidazole,IMD)对苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)和酪氨酸氨基转移酶(Tyrosine aminotransferase,TAT)的活性及Sal B含量的影响。结果表明,SA处理可有效地诱导丹参培养细胞中H2O2产生、PAL和TAT活性升高以及Sal B合成积累量的增加;外源施加10～30 mmol/L H2O2也可以有效促进PAL、TAT活性升高和Sal B合成积累量的增加;用H2O2的清除剂CAT处理发现,CAT对SA或外源H2O2诱导的Sal B合成积累具有消除作用,说明H2O2可能作为SA诱导Sal B积累过程中的上游信号分子起作用;用H2O2淬灭剂DMTU处理,可以有效抑制SA对Sal B合成的促进作用;用质膜烟酰胺腺嘌呤二核苷酸(Nicotinamide vadenine dinucleotide phosphate,NADPH)氧化酶(H2O2来源的主要酶)抑制剂IMD处理,可以抑制Sal B的合成,但这种抑制效果可以部分被外源施加的SA削弱,说明通过HADPH氧化酶产生的H2O2受阻时,SA诱导的Sal B合成积累也会受到抑制。表明H2O2是介导SA诱导丹参培养细胞中Sal B合成积累的信号分子。     

Quantification of human fecal bifidobacterium species by use of quantitative real-time PCR analysis targeting the groEL gene

Quantitative real-time PCR assays targeting the groEL gene for the specific enumeration of 12 human fecal Bifidobacterium species were developed. The housekeeping gene groEL (HSP60 in eukaryotes) was used as a discriminative marker for the differentiation of Bifidobacterium adolescentis, B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. dentium, B. gallicum, B. longum, B. pseudocatenulatum, B. pseudolongum, and B. thermophilum. The bifidobacterial chromosome contains a single copy of the groEL gene, allowing the determination of the cell number by quantification of the groEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given Bifidobacterium species. Independent of the Bifidobacterium species tested, the proportion of groEL copies recovered from fecal samples spiked with 5 to 9 log(10) cells/g feces was approximately 50%. The quantification limit was 5 to 6 log(10) groEL copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3 Bifidobacterium species and B. longum frequently detected. The predominant species in infant and adult fecal samples were B. breve and B. adolescentis, respectively. It was possible to distinguish B. catenulatum and B. pseudocatenulatum. We conclude that the groEL gene is a suitable molecular marker for the specific and accurate quantification of human fecal Bifidobacterium species by real-time PCR.     

Evaluation of the PCR method for identification of Bifidobacterium species

AIMS: Bifidobacterium species are known for their beneficial effects on health and their wide use as probiotics. Although various polymerase chain reaction (PCR) methods for the identification of Bifidobacterium species have been published, the reliability of these methods remains open to question. METHODS AND RESULTS: In this study, we evaluated 37 previously reported PCR primer sets designed to amplify 16S rDNA, 23S rDNA, intergenic spacer regions, or repetitive DNA sequences of various Bifidobacterium species. CONCLUSIONS: Ten of 37 experimental primer sets showed specificity for B. adolescentis, B. angulatum, B. pseudocatenulatum, B. breve, B. bifidum, B. longum, B. longum biovar infantis and B. dentium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that published Bifidobacterium primer sets should be re-evaluated for both reproducibility and specificity for the identification of Bifidobacterium species using PCR. Improvement of existing PCR methods will be needed to facilitate identification of other Bifidobacterium strains, such as B. animalis, B. catenulatum, B. thermophilum and B. subtile.     

Reduction of vitamin K concentration by salivary Bifidobacterium strains and their possible nutritional competition with Porphyromonas gingivalis

AIMS: To assess the possibility that bifidobacteria compete with Porphyromonas gingivalis for their mutual growth factor vitamin K. This study also examined whether salivary Bifidobacterium species decrease vitamin K concentration in the growth medium. METHODS AND RESULTS: Sixty-five strains of Bifidobacterium were obtained from 20 of 24 periodontally healthy subjects. Bifidobacterium dentium was most frequently detected in the saliva of subjects, followed by Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium urinalis. The growth of most Bifidobacterium isolates, except that of B. urinalis, was stimulated by vitamin K. Moreover, the isolates were capable of decreasing vitamin K after incubation, which suggests that bifidobacteria compete with P. gingivalis for vitamin K. In a co-culture, a representative strain -B. adolescentis S2-1 - inhibited the growth of P. gingivalis if it was inoculated in the medium before P. gingivalis. CONCLUSIONS: B. adolescentis S2-1 decreased vitamin K concentration and inhibited the growth of P. gingivalis by possibly competing for the growth factor. SIGNIFICANCE AND IMPACT OF THE STUDY: Salivary bifidobacteria may possess the potential to suppress the growth of P. gingivalis by reducing the growth factor(s) in the environment.     

Identification of the bacteriochlorophylls, carotenoids, quinones, lipids, and hopanoids of "Candidatus Chloracidobacterium thermophilum"

"Candidatus Chloracidobacterium thermophilum" is a recently discovered chlorophototroph from the bacterial phylum Acidobacteria, which synthesizes bacteriochlorophyll (BChl) c and chlorosomes like members of the green sulfur bacteria (GSB) and the green filamentous anoxygenic phototrophs (FAPs). The pigments (BChl c homologs and carotenoids), quinones, lipids, and hopanoids of cells and chlorosomes of this new chlorophototroph were characterized in this study. "Ca. Chloracidobacterium thermophilum" methylates its antenna BChls at the C-8(2) and C-12(1) positions like GSB, but these BChls were esterified with a variety of isoprenoid and straight-chain alkyl alcohols as in FAPs. Unlike the chlorosomes of other green bacteria, "Ca. Chloracidobacterium thermophilum" chlorosomes contained two major xanthophyll carotenoids, echinenone and canthaxanthin. These carotenoids may confer enhanced protection against reactive oxygen species and could represent a specific adaptation to the highly oxic natural environment in which "Ca. Chloracidobacterium thermophilum" occurs. Dihydrogenated menaquinone-8 [menaquinone-8(H(2))], which probably acts as a quencher of energy transfer under oxic conditions, was an abundant component of both cells and chlorosomes of "Ca. Chloracidobacterium thermophilum." The betaine lipid diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine, esterified with 13-methyl-tetradecanoic (isopentadecanoic) acid, was a prominent polar lipid in the membranes of both "Ca. Chloracidobacterium thermophilum" cells and chlorosomes. This lipid may represent a specific adaptive response to chronic phosphorus limitation in the mats. Finally, three hopanoids, diploptene, bacteriohopanetetrol, and bacteriohopanetetrol cyclitol ether, which may help to stabilize membranes during diel shifts in pH and other physicochemical conditions in the mats, were detected in the membranes of "Ca. Chloracidobacterium thermophilum."     

枯草芽孢杆菌B47菌株高产抗菌物质的培养基及发酵条件优化

枯草芽孢杆菌Bacillus subtilis B47菌株为番茄内生细菌, 也是玉米小斑病拮抗菌, 能产生对玉米小斑病菌有强烈抑制作用的抗菌物质。以B47菌株发酵液的无菌滤液对玉米小斑病菌的抗菌活性为检测指标, 测定B47菌株产抗菌物质培养所需的最佳碳、氮源和无机盐, 并通过正交试验法对该菌株产抗菌物质的培养基配方和摇瓶发酵条件进行优化。研究结果表明, B47菌株产抗菌物质最佳碳、氮源和无机盐分别为蔗糖、酵母浸膏和MgSO4·7H2O, 最优培养基是YSB (Yeast extract-sucrose-beef extract)培养基, 其配方为: 蔗糖2%, 酵母浸膏2%, 牛肉浸膏1.5%, MgSO4?7H2O 0.06%, FeSO4·7H2O 0.000 9%, 最优发酵条件组合为: 30 °C, pH 7.0, 170 r/min摇床培养6 d, 接种量为1%, 装液量为40 mL/200 mL。