植物乳杆菌ZJ316生产细菌素

[目的]研究植物乳杆菌ZJ316生长和产细菌素的最佳培养基成分和发酵条件,以提高该菌产plantaricin ZJ316的能力.[方法]改变培养基成份和发酵条件,考察不同氮源、碳源等培养基成分和不同的发酵温度等条件对ZJ316生长和产细菌素的影响.[结果]最佳培养基为MRS培养基;优化后的培养基配方为葡萄糖10 g/L,麦芽糖10 g/L,酵母提取物10 g/L,蛋白胨10 g/L,柠檬酸三铵2 g/L,吐温80为1 Ml/L,K2HPO4·3H2O 6 g/L,乙酸钠5 g/L,硫酸镁0.2 g/L,硫酸锰0.05 g/L.培养基初始Ph6.5,30℃静置培养24 h.[结论]通过培养基成分和发酵条件的优化,细菌素产量提高了2.3倍,为进一步研究和规模化生产奠定基础.     

Effect of medium components on bacteriocin production by Lactobacillus plantarum strains ST23LD and ST341LD, isolated from spoiled olive brine

Bacteriocin ST23LD levels of 2930AU/OD were recorded in MRS broth (pH of 6.5) and in the presence of tryptone and yeast extract as sole nitrogen sources. Growth in MRS broth at an initial pH of 6.0 yielded only 1460AU/OD bacteriocin ST23LD. Activities of 5861AU/OD were recorded with maltose (20, 30 and 40 g/l) as sole carbon source and 9036AU/OD with the addition of 2.0-10.0 g/l KH2PO4. Bacteriocin ST341LD levels of 2850 and 2841AU/OD were recorded in MRS broth at an initial pH of 6.0 or 5.5, respectively. Only 709AU/OD was recorded in the same medium with an initial pH of 6.5. Bacteriocin ST341LD production was stimulated by the presence of tryptone. However, glucose at 10 and 40 g/l, or the presence of 5.0 or 10.0 g/l K2HPO4, resulted in a 50% reduction of bacteriocin activity. Glycerol in the growth medium repressed bacteriocin production. No increased bacteriocin production was recorded in medium supplemented with vitamins.     

Statistical screening of medium components by Plackett-Burman design for lactic acid production by Lactobacillus sp. KCP01 using date juice

Statistical screening of media components for production of lactic acid by Lactobacillus sp. KCP01 using date juice as a sugar source was carried out by Plackett-Burman design. Date juice at 5% sugar concentration when used alone showed 2.6 g/l of lactic acid production. Increase in lactic acid production (15.1 g/l) was observed with supplementation of salts and organic nitrogen sources of MRS medium and after optimization of pH and temperature using date juice as a C-source. Plackett-Burman design showed peptone, K2HPO4, sodium acetate and date juice as significant components influencing the lactic acid production.     

Optimization of nutritional requirements for gentamicin production by Micromonospora echinospora

Effect of various fermentation media, carbon sources, nitrogen sources, phosphate concentration and culture requirements includes inoculum levels and age were determined on gentamicin production and biomass dry weight production for Micromonospora echinospora, a gentamicin producing strain. Of the substrates tested, starch as a sole carbon source promoted maximal gentamicin production, while maltose promoted maximal growth. Yeast extract as a sole nitrogen source promoted maximal growth, while soyabean meal for gentamicin production. Increasing phosphate concentration enhanced gentamicin production and observed optimum production at 1.2 g/1 (6% v/v) of phosphate having 72 h old inoculum in the medium. Highest gentamicin production was obtained after cultivation with shaking for 120 h in a medium containing starch 0.75% (w/v), soyabean meal 0.5%, K2HPO4 0.12%, CaCO3 0.4%, FeSO4 0.003% and CoCl2 0.0001%. The gentamicin production was 1.2-fold in this medium as compared to basal medium.     

A NEWLY ANTI-Streptococcus suis BACTERIOCIN PRODUCING STRAIN FROM UNWEANED PIGLETS FECAL MATTER: ISOLATION, PRELIMINARY IDENTIFICATION, AND OPTIMIZATION OF MEDIUM COMPOSITION FOR ENHANCED BACTERIOCIN PRODUCTION

A newly isolated anti-Streptococcus suis bacteriocin-producing strain LPL1-5 was obtained from healthy unweaned piglets' fecal matter, and was designated as Lactobacillus pentosus LPL1-5 based on morphology, biochemical properties, and 16S rDNA sequencing analysis. The medium composition for enhanced bacteriocin production by L. pentosus LPL1-5 was optimized by statistical methodology. Yeast extract, K(2)HPO(4)?·?3H(2)O, and MnSO(4)?·?H(2)O were identified as significant components influencing pentocin LPL1-5 production using the Plackett-Burman method. Response surface methodology was applied for further optimization. The concentrations of medium components for enhanced pentocin LPL1-5 production were as follows (g/L): lactose 20.00, tryptone 10.00, beef extract 10.00, yeast extract 14.00, MnSO(4)?·?H(2)O 0.84, K(2)HPO(4)?·?3H(2)O 4.92, triammonium citrate 2.00, Na-acetate 5.00, MgSO(4)?·?7H(2)O 0.58, Tween 80 1.00. Under the optimized condition, a value of 3154.65?±?27.93 IU/mL bacteriocin activity was achieved, which was 4.2-fold that of the original medium.     

Medium optimization for the production of cyclic adenosine 3',5'-monophosphate by Microbacterium sp. no. 205 using response surface methodology

Response surface methodology was employed to optimize medium composition for the production of cyclic adenosine 3',5'-monophosphate (cAMP) with Microbacterium sp. no. 205. A fractional factorial design (2(11-7)) was applied to evaluate the effects of different components in the medium. K(2)HPO(4), MgSO(4) and NaF were found to significantly influence on the cAMP production. The steepest ascent method was used to access the optimal region of the medium composition. The concentrations of the three factors were optimized subsequently using central composite design and response surface methodology. The optimal medium composition to achieve the optimal cAMP production was determined (g/L): K(2)HPO(4), 12.78; MgSO(4), 3.53 and NaF, 0.18. The corresponding cAMP concentration was 8.50 g/L, which was about 1.8-fold increase compared with that using the original medium. Validation experiments were also carried out to prove the adequacy and the accuracy of the model obtained. The cAMP fermentation in 5L fermenter reached 9.87 g/L.     

浙贝母内生真菌Nectria sp. JZ6分泌抑菌活性代谢产物的初步研究

Nectria sp.JZ6是从浙贝母新鲜鳞茎中首次分离到的一株内生真菌,其发酵液具有抑菌活性。为建立该真菌稳定分泌抑菌活性成分的发酵体系,本研究利用单因素实验和正交实验确定了发酵培养基的配方并初步优化了发酵条件。结果表明,将活化后的菌种接种于改良查氏培养基(8%葡萄糖,0.5%蛋白胨,0.05%KCl,0.1%K2HPO4,0.15%MgSO4,pH6.5)中,28℃、150r/min振荡培养6d后获得的发酵液抑菌活性比优化前提高了26%。该发酵液经100℃水浴30min不失活,在pH1-5时抑菌活性相对稳定,其后逐渐减弱,并在pH9.0及以上时丧失。Nectriasp.JZ6发酵液的乙酸乙酯浸膏抑菌活性最强,对金黄色葡萄球菌、藤黄八叠球菌、枯草芽孢杆菌、绿脓杆菌和大肠杆菌的最低抑菌浓度(MIC)分别为0.625、0.625、1.25、1.25和1.25mg/mL。     

Optimization of bacteriocin production by Lactobacillus plantarum ST13BR, a strain isolated from barley beer

The cell-free supernatant containing bacteriocin ST13BR, produced by Lactobacillus plantarum ST13BR, inhibits the growth of L. casei, Pseudomonas aeruginosa, Enterococcus faecalis, Klebsiella pneumoniae and Escherichia coli. Based on tricine-SDS-PAGE, bacteriocin ST13BR is 10 kDa in size. Complete inactivation or significant reduction in bacteriocin activity was observed after treatment with Proteinase K, trypsin and pronase, but not with catalase or alpha-amylase. Low bacteriocin activity (200 AU/ml) was recorded in BHI medium, M17 broth, 10% (w/v) soy milk, and 2% and 10% (w/v) molasses, despite good growth. Maximal bacteriocin activity (6,400 AU/ml) was recorded after 23 h in MRS broth, but only at 30 degrees C. Tween 80 in MRS broth increased bacteriocin production by more than 50%. Meat extract or yeast extract as sole nitrogen source, or a combination of the two (1 : 1) in MRS broth, stimulated bacteriocin production (6,400 AU/ml). Only 50% activity (3,200 AU/ml) was recorded with tryptone as sole nitrogen source, whereas a combination of tryptone, meat extract and yeast extract yielded 6,400 AU/ml. Bacteriocin production was not stimulated by the addition of glucose at 2.0% w/v (3,200 AU/ml), nor 2% (w/v) fructose, sucrose, lactose or mannose, respectively (800 AU/ml). Activity levels less than 200 AU/ml were recorded in the presence of 0.05% to 0.5% (w/v) maltose. Maximal bacteriocin production (6,400 AU/ml) was recorded in the presence of 2% (w/v) maltose. Maltose at 4.0% (w/v) led to a 50% reduction of bacteriocin activity. The presence of 1.0% (w/v) and higher KH(2)PO(4), or glycerol at 0.2% (w/v) suppressed bacteriocin production.     

假单胞菌属No.2120生产D-甘露糖异构酶发酵培养基的优化

通过单因子实验、Plackett-Burman实验设计、响应面分析法对假单胞菌属No.2120产D-甘露糖异构酶的培养基进行优化,确定发酵优化条件:果糖15.26 g/L,牛肉膏20 g/L,酵母膏2 g/L,K2HPO42 g/L,MgSO4.7H2O0.5 g/L,NaCl 0.5 g/L,Tween-80 1.54 g/L。采用优化配方异构酶比酶活可以达到68.28 U/mL。     

The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5

Aims:  To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5.
Methods and Results:  Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l⁻¹ of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l⁻¹, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation.
Conclusions:  Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem.
Significance and Impact of the Study:  The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.     

产γ-谷氨基甲酰胺合成酶菌株的筛选及产酶条件研究

以假单胞菌(Pseudomonas sp.)为出发菌株,通过紫外诱变筛选得到一株γ-谷氨基甲酰胺合成酶高产菌株UV-19,其酶活提高32.54%。以突变株UV-19为供试菌株,对γ-谷氨基甲酰胺合成酶的发酵条件进行优化。首先利用Plackett-Burman设计筛选出影响较大的4个因素:葡萄糖、蛋白胨、起始pH值、装液量。在此基础上再利用CCD响应面分析法进行优化,得到最佳产酶培养条件为(g/L):葡萄糖15、蛋白胨12、NaCl 5.0、MgSO4.7H2O 0.2、K2HPO4.3H2O 0.5、甲胺盐酸盐1.0g/L、起始pH值6.5、装液量72mL/250mL。该优化条件下进行产酶培养,假单胞菌发酵产γ-谷氨基甲酰胺合成酶酶活力可达32.68U/mL。     

Thermostable alpha-amylase production by an extreme thermophile Bacillus thermooleovorans

AIMS: alpha-Amylase production by a newly isolated thermophile, Bacillus thermooleovorans, was studied under different cultivation conditions. METHODS AND RESULTS: The influence of various carbon and nitrogen sources on alpha-amylase production was quantified in batch fermentation in shake flasks. Starch and tryptone were observed to be the ideal carbon and nitrogen sources, respectively. Cultivation of the organism in a chemically defined medium consisting of glucose, riboflavin, cysteine, MgSO4, K2HPO4 and NaCl led to a near twofold increase in the production of alpha-amylase in comparison with that in the complex medium. The increase in enzyme production was achieved using vitamins and amino acids. When the organism was grown in a laboratory fermenter in the optimized complex medium, the noticeable effects were the near abolition of the lag phase, a 2.2-fold increase in enzyme production and a reduction in optimal production time from 12 to 4-5 h. CONCLUSION: Enhancement of amylase production was achieved under various cultivation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thermooleovorans produces a calcium-independent and thermostable amylase which can find use in starch saccharification.     

Design and optimization of fermentation medium for enhanced bacteriocin production by probiotic bacterium Enterococcus faecium MC13

Statistics-based experimental designs were used to develop a cost-effective medium for enhanced production of viable cells and bacteriocin by probiotic Enterococcus faecium MC13. Carbon, nitrogen, and mineral sources were first screened by one-variable-at-a-time (OVAT) methods. In order to increase yield production, the selected variables were further statistically optimized using response-surface methodology (RSM) with central composite design (CCD). The maximum and minimum levels of the selected variables were determined and a set of 34 experimental runs was performed. The optimum concentrations of the tested variables for production of viable cells (12.24 log CFU mL(-1)) and bacteriocin activity (25,600 AU mL(-1)) were tryptone (10.0 g/L), peptone (6.0 g/L), maltose (3.0 g/L), glucose (9.0 g/L), NaCl (15.0 g/L), sodium citrate (2.5 g/L), sodium acetate (1.0 g/L), and dipotassium PO(4) (0.1 g/L). Threefold increased yield of bacteriocin was achieved in optimized medium compared to the unoptimized counterpart, and this was two times less cost than commercial MRS medium.     

Optimization of a fermentation medium for the production of Penicillin G acylase from Bacillus sp

AIMS: Optimization of Penicillin G acylase (PAC) production from a novel isolate of Bacillus sp. METHODS: Fermentation medium for PAC production was optimized using a two-level fractional factorial design with seven components. RESULTS: A maximum production of 9.5 U ml(-1) of PAC was obtained in an optimized medium containing (g l(-1): K2HPO4, 1.0; MgSO4.7H2O, 0.1; CaCl2.2H2O, 0.1; PAA, 2.0; tryptone, 5.0; yeast extract, 3.0; and sucrose, 50.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The two-step medium optimization resulted in a twofold increase in PAC production. Since the strain Bacillus sp. PGS10 produces a high level of PAC, it could be a potential candidate for industrial production of PAC.     

Improvement of xylanase production in solid-state fermentation by alkali tolerant Aspergillus versicolor MKU3

AIMS: To optimize the media components for xylanase production by Aspergillus versicolor MKU3 in solid-state fermentation (SSF). METHODS AND RESULTS: Medium optimization was carried out using De Moe's fractional factorial design with seven components. Maximum production of xylanase 3249.9 U g(-1) was obtained in SSF with an optimized medium containing (g l(-1)): NaNO(3), 20; K(2)HPO(4), 20; MgSO(4), 10; FeSO(4), 0.001; KCl, 1; peptone, 10 and yeast extract, 10. Four components namely NaNO(3), MgSO(4), peptone and K(2)HPO(4) significantly increased the xylanase production by A. versicolor MKU3. CONCLUSIONS: Fractional factorial design was used to optimize the seven components in the fermentation medium for SSF. The optimized media increased xylanase production by 3.4-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus versicolor MKU3 produced maximum xylanase after two steps of media optimization under alkaline condition. This medium will be significant value for xylanase production in SSF.     

Optimization of culture media for L-asparaginase production by newly isolated bacteria,Bacillus sp. GH5

The enzyme L-asparaginase has been extensively studied by many researchers mainly because of its considerable therapeutic properties. Producing a convenient quantity of L-asparaginase can be conducted either by discovering new microbial sources with higher enzyme production or by manipulating the medium components for known microbial sources. The present paper discusses the studies carried out in order to enhance the production of L-asparaginase by newly isolated bacteria, Bacillus sp. GH5. Based on the results obtained from media optimization studies, a modified media was developed for optimal L-asparaginase production. Concisely, screening of the nutrients using a proper statistical design showed that tapioca starch, gelatin, ammonium oxalate, CaCO₃, and L-asparagine were respectively the most important sources for carbon, organic nitrogen, inorganic nitrogen, mineral salt, and amino acids. The composition of the optimized medium was the following (per 1 L): 5.0 g L-asparagine; 0.5 g MgSO₄ · 7H₂O; 6.0 g NaHPO₄ · 2H₂O; 3.0 g (NH₄)₂C₂O₄; 0.5 g CaCO₃; 0.014 g CaCl₂ · 2H₂O; 2.0% w/v tapioca starch; 5.0 g gelatin; and 15.0 g agar.     

Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1

AIMS: Optimization of the growth of Pediococcus damnosus NCFB 1832 and the production of pediocin PD-1 by traditional fermentation methods. METHODS AND RESULTS: Fermentation studies were conducted in De Man Rogosa and Sharpe (MRS) broth (Oxoid), preadjusted to specific pH values, and in MRS broth supplemented with various nitrogen sources, MnSO4, MgSO4 and Tween 80. The production of pediocin PD-1 closely followed the growth curve of Ped. damnosus NCFB 1832. Maximum levels of bacteriocin activity (3249 AU ml(-1)/O.D.max) were recorded in MRS broth with an initial pH of 6.7. In media with an initial pH of 4.5 bacteriocin activity as low as 222 AU ml(-1)/O.D.max was recorded. The highest bacteriocin activity was recorded in growth conditions allowing the greatest pH variation (highest DeltapH). The addition of bacteriological peptone (1.7%, w/v), MnSO4 (0.014%, w/v) and Tween 80 (3%, v/v) to MRS and adjustment of the medium pH to 6.7 resulted in a further increase in activity (from 3249 to 5078 AU ml(-1)/O.D.max). The same medium, but with an initial pH of 6.2, resulted in an 82.5% decrease in bacteriocin activity. CONCLUSIONS: Pediocin PD-1 production is not only stimulated by the presence of specific growth factors (e.g., bacteriological peptone, MnSO4 or Tween 80), but may also be stimulated by the lowering in pH during growth (highest DeltapH), and thus also the amount of organic acids produced. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of pediocin PD-1 by the wild-type producer strain was significantly improved by using a defined medium and traditional fermentation methods.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

Partial characterisation of two bacteriocins produced byLactobacillus paracasei subsp.paracasei ST242BZ and ST284BZ and the effect of medium components on their production

The influence of medium components on production of bacteriocins ST242BZ (10.0 kDa) and ST284BZ (3.5 kDa) byLactobacillus paracasei subsp.paracasei ST242BZ and ST284BZ have been studied. Growth in MRS broth (pH of 6.5) yielded bacteriocin levels of 12800 AU/ml. Modified MRS with tryptone as the only nitrogen source, MRS supplemented with KH₂PO₄ (10–100 g/l), or MRS supplemented with thiamine increased bacteriocin ST242BZ production to 25600 AU/ml. Tryptone, combinations of tryptone, meat extract and yeast extract, or thiamine did not increase bacteriocin ST284BZ production. However, MRS supplemented with K₂HPO₄ (50–100 g/l) increased bacteriocin ST284BZ production up to 25600 AU/ml. Our results suggest that production of bacteriocins ST242BZ and ST284BZ are stimulated by potassium ions.     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.