Some cultural conditions for the production of puromycin by Streptomyces alboniger

A simple synthetic medium was used in the fermentative production of puromycin to investigate the efficiencies of certain ingredients in stimulating the organism for higher yield. Addition of certain amino acids, especially L-histidine, L-glutamic acid, L-proline, L-aspartic acid, DL-methionine, L-leucine, β-alanine, L-isoleucine, DL-alanine, L-tyrosine and L-glycine, increased the antibiotic yield compared with the control. Lactic acid was more suitable for antibiotic production than the other organic acids used, i.e. maleic, acetic, malic, tartaric and fumaric acids. Addition of certain vitamins, namely cobalamin (vitamin B ₁₂), nicotinic acid, vitamin B ₁ and folic acid, did not markedly increase the antibiotic yield. Certain trace elements, such as zinc, copper, iron, manganese and cobalt, played an important role in the biosynthesis of puromycin by Streptomyces alboniger NRRL B-2403. The optimum concentrations of these trace elements were 0.2000, 0.0020, 0.0005, 0.3000, and 0.0050 g/litre respectively. The optimum concentration of K₂HPO₄ favouring bio-synthesis of puromycin was 1.5 g/litre. The amino acids formed in the microbial cells of Streptomyces alboniger NRRL B-2403 during the production of puromycin in the synthetic medium were determined quantitatively. Generally, all the amino acids recorded appeared in the microbial cells.     

Some cultural conditions for the production of puromycin by Streptomyces alboniger

A simple synthetic medium was used in the fermentative production of puromycin to investigate the efficiencies of certain ingredients in stimulating the organism for higher yield. Addition of certain amino acids, especially L-histidine, L-glutamic acid, L-proline, L-aspartic acid, DL-methionine, L-leucine, β-alanine, L-isoleucine, DL-alanine, L-tyrosine and L-glycine, increased the antibiotic yield compared with the control. Lactic acid was more suitable for antibiotic production than the other organic acids used, i.e. maleic, acetic, malic, tartaric and fumaric acids. Addition of certain vitamins, namely cobalamin (vitamin B₁₂), nicotinic acid, vitamin B₁ and folic acid, did not markedly increase the antibiotic yield. Certain trace elements, such as zinc, copper, iron, manganese and cobalt, played an important role in the biosynthesis of puromycin by Streptomyces alboniger NRRL B-2403. The optimum concentrations of these trace elements were 0.2000, 0.0020, 0.0005, 0.3000, and 0.0050 g/litre respectively. The optimum concentration of K₂HPO₄ favouring biosynthesis of puromycin was 1.5 g/litre. The amino acids formed in the microbial cells of Streptomyces alboniger NRRL B-2403 during the production of puromycin in the synthetic medium were determined quantitatively. Generally, all the amino acids recorded appeared in the microbial cells.     

The fermentative production of gentamicins by Micromonospora purpurea

For fermentative production of gentamicins by Micromonospora purpurea it was necessary to adjust the initial pH value of the medium to 7.0–7.5. Glucose was the preferred carbon source. The production of gentamicins was performed in two steps. The first step was to grow the microbial cells and the second step was to inoculate the fermentation medium with the growing cell culture (6.0% v/v). The organism produced more antibiotic with organic nitrogen sources than with inorganic nitrogen source. Fodder yeast (50 and 40% total nitrogen) was a good nitrogen source both for microbial growth and the antibiotic production. The suitable concentrations of fodder yeast (50 and 40%) were 2 and 6 g/1 respectively.     

Production of food yeast from whey ultrafiltrate by dialysis culture

Yeast (Kluyveromyces fragilis) concentrations up to 90 g dry weight/litre were obtained by dialysis culture in an 8 litre fermenter and the concentrations of lactose in the whey ultrafiltrate reservoirs were reduced to 5–8 g/litre during the cultivation period. A marked and progressive fall in cell yield was observed as the concentration of yeast increased. Modelling experiments indicated that this fall was consistent with the effects of maintenance energy requirements and that the effect could be minimised by a four-fold increase in the rate of dialysis. The feasibility of achieving the required increase with improved membranes was confirmed in experiments in which the rate of dialysis using existing membranes was shown to be doubled by either prior enzymic hydrolysis of the lactose or by doubling the membrane area.     

Generation and Characterization of a Soybean Line with a Vernonia galamensis Diacylglycerol Acyltransferase-1 Gene and a myo-Inositol 1-Phosphate Synthase Knockout Mutation

Soybean (Glycine max) meal is an important protein source. Soybean meal with lower phytate and oligosaccharides improves meal quality. A single recessive mutation in soybean myo-inositol 1-phosphate synthase (Gm-lpa-TW75-1) confers a seed phenotype with low phytate and increased inorganic phosphate. The mutant was crossed with high oil lines expressing a diacylglycerol acyltransferase1 (DGAT) gene from Vernonia galamensis (VgD). Gm-lpa-TW75-1 X VgD, designated GV, has 21%, and 22% oil and 41% and 43% protein from field and greenhouse seed production, respectively. No significant differences were found in mineral concentrations except for Fe which was 229 μg/g dry mass for GV followed by 174.3 for VgD and 162 for Gm-lpa-TW75-1. Phosphate (Pi) is higher in Gm-lpa-TW75-1 as expected at 5 mg/g, followed by GV at 1.6 mg/g whereas Jack, VgD, and Taiwan75 have about 0.3 mg/g. The Gm-lpa-TW75-1 line has the lowest phytate concentration at 1.4 mg/g followed by GV with 1.8 mg/g compared to Taiwan75, VgD, and Jack with 2.5 mg/g. This work describes a high oil and protein soybean line, GV, with increased Pi and lower phytate which will increase the nutritional value for human and animal feed.     

Biosynthesis of carbomycin,its extraction,purification and mode of action on Bacillus subtilis NRRL B-543

A fermentation medium containing (g dm^?3)—glucose, 10.0; soyabean meal, 10.0; NaCl, 5.0 and CaCO₃, 1.0 in distilled water—was used in the biosynthesis of the macrolide antibiotic carbomycin. Maximum yield of carbomycin was obtained after 120 h fermentation. Carbomycin was extracted by different organic solvents adjusted to different pH values. Extraction of the antibiotic was possible when the pH of the fermentation broth was neutral (6.5–7.5) with all organic acids used, but the most efficient organic solvent suitable for extraction of carbomycin was chloroform at a pH of 6.5. Further purification was carried out by preparative thin layer chromatography. The most efficient separation into carbomycin A and carbomycin B was obtained using a solvent system of ethanol-hexane-water [90:10:0.15 (v/v)]. Carbomycin inhibited the biosynthesis of the anabolising cellular constituents phosphoprotein, ribonucleic and deoxyribonucleic acids in Bacillus subtilis NRRL B-543.     

A study of antifungal antibiotic production by Thermomonospora sp MTCC 3340 using full factorial design

The three independent variables, viz concentration of carbon source (glucose), concentration of nitrogen source (soybean meal) and temperature of incubation were found to be the most important for production of antifungal antibiotic by the isolate Thermomonospora sp MTCC 3340 from one‐factor‐at‐a‐time study. These variables were varied at three levels in a total number of 27 experiments designed using full factorial design. The results on analysis using the statistical software SPSS (version 6.0) indicated that the optimum combination of the three factors for the maximum yield of the antibiotic was concentration of carbon source (glucose) 2%, concentration of nitrogen source (soybean meal) 1% and temperature of incubation 30 °C. A close fit between experimental and predicted values of the antifungal yield was obtained using one of the modes derived from the statistical analysis, indicating that this model was applicable to this production. Copyright © 2003 Society of Chemical Industry     

Enhanced production of cellobiose dehydrogenase and β-glucosidase by Phanerochaete chrysosporium

The production of cellobiose dehydrogenase (CDH) and β-glucosidase by Phanerochaete chrysosporium ATCC 32629 was assessed during submerged fermentation. The maximum concentrations of CDH and β-glucosidase were obtained using rice straw as the carbon source. Organic nitrogen sources were more effective in enzyme production than inorganic nitrogen sources. Corn steep liquor (CSL) for CDH production and soy bean meal (SBM) for β-glucosidase production were the most appropriate organic nitrogen sources. Using optimum medium obtained by response surface methodology (RSM), the maximum concentrations of CDH and β-glucosidase achieved in the stirred-tank reactor (STR) were 204 U/L and 140 U/L, respectively. CDH productivity (22.7 U/L·day) was the highest at 9 days.     

抗生素AGPM摇瓶发酵条件的正交实验

采用正交实验设计考察了培养基组成对新型抗生素AGPM发酵的影响,改进后在培养基组成为(g/L)：葡萄糖5, 玉米淀粉40, 黄豆饼粉16, 玉米浆2 ml, K2HPO4 1.0, MgSO4×7H2O 0.5, NaCl 0.5, 淀粉酶0.05及pH 5.5的条件下,单位发酵液的活性提高了18.9倍;同时表明较高的碳氮比对抗生素AGPM的合成有利.     

Chemical and functional characterization of major protein fractions extracted from nontoxic Jatropha curcas byproduct meals

Jatropha curcas seeds are a suitable source of oil for biofuel, among other use. A protein-rich meal is obtained after oilseed extraction. The goals of this study were to determine the physicochemical and functional properties of a nontoxic genotype of J. curcas defatted meal (JCDM) and the seed storage protein fractions to identify future applications. Both glutelin and globulin were the predominant protein fractions obtained from JCDM (42.03 and 20.17 g/100 g of protein, respectively). Leucine, phenylalanine + tyrosine, and histidine content of JCDM and protein fractions met the Food and Agriculture Organization/World Health Organization recommendation for children. The protein solubility (PS) profiles showed minimum values (5.3%–59.7%) at pH 5–6 and maximum at pH 2 (79.7%–81.6%) and above pH 10 (84.6%–89.8%). These findings suggest that JCDM proteins could be used in the formulation of juice or protein-based beverages. All the proteins showed the highest values for foam expansion (231%–285%) at pH 9. JCDM and the albumin fraction formed highly stable foams at pH 9, while the globulin and glutelin foams were stable at pH 3 and 2, respectively. Protein with stable foams, like those from jatropha are suitable for application in ice cream, mousse, among others. The emulsion activity index had similar behavior as foam expansion, but did not follow a specific trend. Thus, the proteins are suitable for use in salad dressing, sausages, comminuted meats, and mayonnaise. Taken together, JCDM protein and its soluble protein fractions have strong promise as alternative proteins for food structuring.     

Influence of some compounds on gentamicin formation by Micromonospora purpurea

Production of gentamicin antibiotics by Micromonospora purpurea was carried out successfully on a synthetic medium which contained the following ingredients (g/litre): glucose 10.0, NaNO₃ 2.0, KH₂PO₄ 1.0, and CaCO₃ 1.0. The initial pH value of the fermentation medium was adjusted to 7.2. Effects of certain amino acids, organic acids, vitamins, purine and pyrimidine bases on the fermentative production of gentamicins were revealed. The ingredients which increased the antibiotic yields were phenylalanine, isoleucine, lysine, methionine, leucine, arginine, glycine, β-alanine, cystine, tryptophan, malic acid, maleic acid, cobalamin, folic acid, riboflavin, vitamin B₁, vitamin B₆, biotin, nicotinamide, uracil, adenine, guanine and adenosine. Trace elements (Co, Mo, Fe, Cu, Zn and Mn) exhibited their important role on the biosynthesis and formation of gentamicins by Micromonospora purpurea.     

Effect of alteration of aeration and temperature on production of α-amylase by Bacillus subtilis

α-Amylase production by Bacillus subtilis in fermentation was markedly increased by reduction of aeration at the end of the exponential growth phase. The resultant decrease in pH seems to be a limiting factor and without pH control the aeration rate could be reduced from 1 litre/litre/min to 0.2 litre/litre/min for optimum α-amylase production. Alteration of temperature at the same stage did not result in improved yields.     

Influence of some compounds on gentamicin formation by Micromonospora purpurea

Production of gentamicin antibiotics by Micromonospora purpurea was carried out successfully on a synthetic medium which contained the following ingredients (g/litre): glucose 10.0, NaNO₃ 2.0, KH₂PO₄ 1.0, and CaCO₃ 1.0. The initial pH value of the fermentation medium was adjusted to 7.2. Effects of certain amino acids, organic acids, vitamins, purine and pyrimidine bases on the fermentative production of gentamicins were revealed. The ingredients which increased the antibiotic yields were phenylalanine, isoleucine, lysine, methionine, leucine, arginine, glycine, β′-alanine, cystine, tryptophan, malic acid, maleic acid, cobalamin, folic acid, riboflavin, vitamin B₁, vitamin B₆, biotin, nicotinamide, uracil, adenine, guanine and adenosine. Trace elements (Co, Mo, Fe, Cu, Zn and Mn) exhibited their important role on the biosynthesis and formation of gentamicins by Micromonospora purpurea.     

Use of Cottonseed Meal for Producing Eicosapentaenoic Acid by Pythium irregulare

Cottonseed meal (CSM), a common agricultural by‐product, was used as a nutrient source for the production of eicosapentaenoic acid (EPA) by Pythium irregulare. CSM can support good cell growth performance, as can yeast extract (YE). In terms of the maximum EPA content and EPA yield, CSM is superior to YE. Low concentrations of CSM are beneficial to lipid synthesis, and high concentrations favor the EPA content. Utilizing response surface methodology (RSM) analysis, the optimum contents of glucose and CSM in the fermentation medium were determined to be 40.2 and 16.1 g/l, respectively. After 6 days of fermentation at 25 °C and optimal conditions, the EPA yield and productivity were 245.3 and 40.9 mg/l day, respectively. Particle size of CSM was found to affect the EPA production, and a finely ground CSM (100 mesh) was determined to be best for EPA production. The variation in the fatty acid content of total fatty acid (TFA) indicates that EPA was synthesized through the n‐6 route in P. irregulare and Δ12 desaturase was the key enzyme for EPA biosynthesis. Sodium carbonate was determined to be notably good at removing free gossypol attached to biomass. After fungal biomass from each flask had been harvested from Na₂CO₃‐supplemented medium, 1 % (w/v) Na₂CO₃ solution was used to wash the mycelia three times; free gossypol (FG) was not detected (detection limit 0.0018 %). This work provides a new approach using cottonseed meal to produce EPA through fungal fermentation.     

Barley grain as carbon substrate for fungal protein production in a farm process

A simple, non-aseptic, submerged aerobic fermentation was sought in which microbial protein could be synthesised from non-protein nitrogen, at the expense of barley carbohydrate, to supplement the protein content of barley grain for non-ruminant feeding. Screening of 23 isolates of fungi in shake flask cultures showed that microbial protein could be successfully produced by a number of different species in a medium containing barley as a source of carbohydrate. Protein synthesis was markedly affected by the addition of different sources of non-protein N to the fermentation; and (NH₄)₂SO₄ or urea proved to be good N sources in this respect. Aspergillus oryzae was selected as the most suitable species for protein synthesis, based on its performance when supplemented with the various N sources. The growth rate of A. oryzae in stirred culture was such that high yields of mycelial protein could be achieved within 24 h from a culture inoculated with a spore suspension. Under the conditions of growth used in these experiments there appeared to be little advantage in adding more than 2% barley to the medium. Higher yields of protein could be achieved at barley concentrations in excess of 2%, but only if the incubation period was extended. The tolerance of A. oryzae to low pH values was exploited to enable fungal cultures to be grown under non-aseptic conditions. Good yields of protein were obtained in 40 litre cultures grown at pH 3.5 in a modified domestic washing machine. Although protein yields were poor, non-aseptic growth was also sucesssful at pH 3.5 in 1000 litre cultures grown in an agricultural feed-mixing vessel.     

Purification of mitomycins produced by Streptomyces caespitosus

Mitomycins were fermentatively produced using certain agricultural raw by-products by Streptomyces caespitosus NRRL 2564. The excreted antibiotics were thoroughly extracted with ethyl acetate at pH 6.0–8.0. The ethyl acetate extract was extensively purified by paper and thin-layer chromatographic techniques. The most efficient solvent systems in the complete separation of mitomycins were water-benzene-methanol (2:1:1 by vol.) in the case of paper chromatography, while acetone-ligroin-n-octanol (5:5:2 by vol.) was suitable for silical gel and alumina thin-layer plates. Pure samples of mitomycins A, B and C were obtained by preparative thin-layer chromatography using the solvent system composed of acetone-ligroin-n-octanol (5:5:2 by vol.). The active principles of mitomycins A, B and C, when biologically assayed on Bacillus subtilis ATCC 6622, were 980, 955 and 1000 μg/mg respectively. Mitomycin A can be converted into mitomycin C by exposing thin-layer chromatograms to concentrated ammonia vapour for 20 min.     

Antioxidant capacity of rapeseed meal and rapeseed oils enriched with meal extract

Response surface methodology (RSM) was used to evaluate the quantitative effects of two independent variables: solvent polarity and temperature of the extraction process on the antioxidant capacity (AC) and total phenolics content (TPC) in meal rapeseed extracts. The mean AC and TPC results for meal ranged between 1181–9974 µmol TE/100 g and 73.8–814 mg sinapic acid/100 g of meal. The experimental results of AC and TPC were close to the predicted values calculated from the polynomial response surface models equations (R² = 0.9758 and 0.9603, respectively). The effect of solvent polarity on AC and TPC in the examined extracts was about 3.6 and 2.6 times greater, respectively, than the effect of processing temperature. The predicted optimum solvent polarity of ε = 78.3 and 63.8, and temperature of 89.4 and 74.2°C resulted in an AC of 10 014 µmol TE/100 g and TPC of 863 mg SAE/100 g meal, respectively. The phenolic profile of rapeseed meal was determined by an HPLC method. The main phenolics in rapeseed meal were sinapine and sinapic acid. Refined rapeseed oils were fortified with an extract – rich in polyphenols – obtained from rapeseed meal. The supplemented rapeseed oil had higher AC and TPC than the refined oil without addition of meal extracts. However, AC and TPC in the enriched oils decreased during storage. The TPC in the studied meal extracts and rapeseed oils correlated significantly (p<0.0000001) positively with their AC (R² = 0.9387). Practical applications: Many bioactive compounds extracted from rapeseed meal provide health benefits and have antioxidative properties. Therefore, it seems worth to consider the application of antioxidants extracted from the rapeseed meal for the production of rapeseed oils with potent AC. Moreover, antioxidants extracted from the rapeseed meal were added to refined rapeseed oil in order to enhance its AC. AC was then tested by FRAP assay. FRAP method is based on the reduction of the ferric tripyridyltriazine (Fe³⁺‐TPTZ) complex to the ferrous tripyridyltriazine (Fe²⁺‐TPTZ), and it is simple, fast, low cost, and robust method. FRAP method does not require specialized equipment and can be performed using automated, semi‐automatic, or manual methods. Therefore the proposed FRAP method can be employed by the fat industry laboratories to asses the AC of rapeseed oils and meal.     

高效降解复合菌系的廉价降解培养基筛选

考察了麸皮、鱼粉、玉米粉、棉籽粉、花生粉、豆粕及尿素对偶氮染料降解复合菌系的生长、产酶及底物降解等方面的影响,从而筛选出最适宜的廉价氮源。研究表明,采用麸皮和鱼粉作为廉价氮源并控制其含氮量的质量比为4∶2时,复合菌系对偶氮染料的降解效果最佳。同时还发现,最佳的廉价培养基(BFS)组合为:麸皮18.76g/L、葡萄糖5.00 g/L、鱼粉3.08 g/L、KH_2PO_4 1.80 g/L、NaH_2PO_4 3.50 g/L、FeCl_3 0.01 g/L、MnSO_4 0.02 g/L、MgSO_4 0.20 g/L、pH 7.2。筛选出的廉价培养基(BFS)成本仅为原蛋白胨染料降解培养基(PDS)成本的8.77%。     

Exploring seed and meal composition and protein solubility in soybean genotypes from different domestication periods

Traditionally, commercial soybean breeding has focused on increasing seed yield by crossing elite cultivars and limiting the genetic diversity within commercial germplasm. Wild and ancestral soybean genotypes have higher seed protein concentrations than commercial ones. Different seed protein concentrations and compositions result in diverse functional properties of soybean meal, in particular solubility is important for beverages and protein isolates production. The objectives of our study were (i) to characterize seed protein concentration and composition in genotypes from different soybean domestication periods (types) and (ii) to evaluate the protein concentration and solubility profiles of the defatted meals obtained from these genotypes. Variation within seed and meal protein concentration, composition, and solubility was evident along the domestication process. Wild relative (G. soja) and Elite genotypes had the maximum and minimum seed protein concentrations, respectively (42.9 and 36.3 g 100 g⁻¹). Soybean meal protein concentrations were 55.1, 47.7, 48.4 and 44.1 g 100 g⁻¹ for Wild relative (G. soja), Asian landraces, North American (Nam) ancestors and Elite, respectively. Ample genotypic variation was observed for β-conglycinin components, such as for β, α, and α′ subunits and for total glycinin and its components. Asian landraces had the highest protein solubility. Wild and ancestral germplasm are a reservoir of useful traits to improve soybean seed quality. This study opens the gates to the introduction of ancestral germplasm to breeding programs focused on protein quality and functionality.     

Conversion of Canola Meal into a High-Protein Feed Additive via Solid-State Fungal Incubation Process

The study goal was to determine the optimal fungal culture to reduce glucosinolates (GLS), fiber, and residual sugars while increasing the protein content and nutritional value of canola meal. Solid‐state incubation conditions were used to enhance filamentous growth of the fungi. Flask trials were performed using 50 % moisture content hexane‐extracted (HE) or cold‐pressed (CP) canola meal with incubation for 168 h at 30 °C. Incubation on HE canola meal Trichoderma reesei (NRRL‐3653) achieved the greatest increase in protein content (23 %), while having the lowest residual levels of sugar (8 % w/w) and GLS (0.4 μM/g). Incubation on CP canola meal Trichoderma reesei (NRRL‐3653), A. pullulans (NRRL‐58522), and A. pullulans (NRRL‐Y‐2311‐1) resulted in the greatest improvement in protein content (22.9, 16.9 and 15.4 %, respectively), while reducing total GLS content from 60.6 to 1.0, 3.2 and 10.7 μM/g, respectively. HE and CP canola meal GLS levels were reduced to 65.5 and 50.7 % by thermal treatments while solid‐state microbial conversion further reduced GLS up to 99 and 98 %, respectively. Fiber levels increased due to the concentration effect of removing oligosaccharides and GLS.