极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

Feather degradation by Kocuria rosea in submerged culture

A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A specific growth rate of 0.17 h⁻¹ was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in significant amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters

Aspergillus oryzae MTCC 5341, when grown on wheat bran as substrate, produces several extracellular acid proteases. Production of the major acid protease (constituting 34% of the total) by solid-state fermentation is optimized. Optimum operating conditions obtained are determined as pH 5, temperature of incubation of 30°C, defatted soy flour addition of 4%, and fermentation time of 120 h, resulting in acid protease production of 8.64 × 10⁵ U/g bran. Response-surface methodology is used to generate a predictive model of the combined effects of independent variables such as, pH, temperature, defatted soy flour addition, and fermentation time. The statistical design indicates that all four independent variables have significant effects on acid protease production. Optimum factor levels are pH 5.4, incubation temperature of 31°C, 4.4% defatted soy flour addition, and fermentation time of 123 h to yield a maximum activity of 8.93 × 10⁵ U/g bran. Evaluation experiments, carried out to verify the predictions, reveal that A. oryzae produces 8.47 × 10⁵ U/g bran, which corresponds to 94.8% of the predicted value. This is the highest acid protease activity reported so far, wherein the fungus produces four times higher activity than previously reported [J Bacteriol 130(1): 48–56, 1977].     

Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The production of cellulolytic enzymes by Aspergillus niger on lignocellulosic substrates groundnut fodder, wheat bran, rice bran and sawdust in solid state fermentation in a laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used to moisten lignocellulosic solid supports for cultivation of Aspergillus niger. The production of filter paperase, carboxymethyl cellulase and -glucosidase were monitored at daily intervals for 5 days. The peak production of the enzymes occurred within 3 days of incubation. Among solid supports used in the study, wheat bran was the best solid matrix followed by groundnut fodder in production of cellulolytic enzymes in solid state fermentation. Groundnut fodder supported significant production of FPase (2.09 FPU/g), CMCase (1.36 U/g) and -glucosidase activity (0.0117 U/g) in solid state fermentation. Considerable secretion of protein (5.10 mg/g) on groundnut fodder at peak time interval 1st day of incubation was recorded.     

Studies on the production of bacterial rennet in a pilot plant fermentor

An attempt was made to find out the optimum aeration and agitation rates on the production of bacterial rennet from Bacillus sublilis K-26 using 5% wheat bran medium in a 13 liter fermentor. The enzyme activity and the growth rate were shown to increase with an increase in the rate of agitation. The fermentation experiments carried out at an agitation rate of 400 rpm showed an approximate threefold increase in enzyme activity with a considerable decrease in the fermentation time over those agitated at 200 and 300 rpm. The beneficial effect of a higher oxygen rate was observed for enzyme production occurring at a lower agitation rate. The inoculum activity and the varying amounts of antifoam agent which were added showed no apparent effect either on the total incubation time or on the final enzyme activity. It has been suggested that an agitation rate of 400 rpm with an aeration level of 3000 cc/min are the optimum values for the efficient production of bacterial rennet from B. subtilis K-26 using 5% wheat bran medium in a 13 liter fermentor.     

Evaluation of Various Unstructured Kinetic Models for the Production of Protease by Bacillus sphaericus MTTC511

Bacillus sphaericus MTCC511 was used for the production of protease in submerged batch fermentation. Maximum protease activity of 1010 U/L was obtained during a fermentation period of 24 h under optimized conditions of 30 °C in a medium with an initial pH of 7 and at a shaking rate of 120 rpm. The maximum biomass obtained in the batch fermentation was 2.55 g/L after 16 h. Various unstructured models were analyzed to simulate the experimental values of microbial growth, protease activity and substrate concentration. The unstructured models, i.e. the Monod model for microbial growth, the Monod incorporated Luedeking‐Piret model for the production of protease and the Monod‐incorporated modified Luedeking‐Piret model for the utilization of substrate were capable of predicting the fermentation profile with high coefficient of determination (R²) values of 0.9967, 0.9402 and 0.9729, respectively. The results indicated that the unstructured models were able to describe the fermentation kinetics more effectively.     

Solid‐state fermentation for the production of debittering enzyme naringinase using Aspergillus niger MTCC 1344

Aspergillus niger produced high levels of naringinase using easily available, inexpensive industrial waste residues such as rice bran, wheat bran, sugar cane bagasse, citrus peel, and press mud in solid‐state fermentation (SSF). Among these, rice bran was found to be the best substrate. Naringinase production was highest after 96 h of incubation at 27°C and at a substrate‐to‐moisture ratio of 1:1 w/v. Supplementation of the medium with 10% naringin caused maximum induction. An inoculum age of 72 h and an inoculum level of 15% resulted in maximum production of naringinase. Enzyme production was stimulated by the addition of nutrients such as naringin and peptone. Thus, A. niger produced a very high level of naringinase within a short time in solid‐state fermentation using inexpensive agro‐residues, a level that is much higher than reported for any other microbes.     

根际益生菌链霉菌S506固体发酵条件优化

链霉菌S506是一株具有广谱防病、促生和降解根系毒素功能的根际益生菌.采用液固两相发酵工艺,以活菌总数和分生孢子产生量为检测指标,研究了S506活菌制剂固体发酵物料的配方和工艺参数.获得固体发酵物料的最佳参数为:麸皮100 g、M-115g、磷酸氢二钾0.3 g、氯化钠0.25 g、硝酸钾0.1 g、碳酸钙0.8 g,自然pH值,液体菌种种龄84 h,接种量10%,发酵温度30℃,料水比10:6,发酵时间60 h.在最佳发酵条件下,S506固体菌剂的活菌总量达到8.27×109CFU/g,分生孢子数达到6.23×109CFU/g.     

Parametric optimization of feruloyl esterase production from Aspergillus terreus strain GA2 isolated from tropical agro-ecosystems cultivating sweet sorghum

A fungal strain, Aspergillus terreus strain GA2, isolated from an agricultural field cultivating sweet sorghum, produced feruloyl esterase using maize bran. In order to obtain maximum yields of feruloyl esterase, the solid state fermentation (SSF) conditions for enzyme production were standardized. Effective feruloyl esterase production was observed with maize bran as substrate followed by wheat bran, coconut husk, and rice husk among the tested agro-waste crop residues. Optimum particle size of 0.71- 0.3 mm and moisture content of 80% favored enzyme production. Moreover, optimum feruloyl esterase production was observed at pH 6.0 and a temperature of 30 degrees C. Supplementation of potato starch (0.6%) as the carbon source and casein (1%) as the nitrogen source favored enzyme production. Furthermore, the culture produced the enzyme after 7 days of incubation when the C:N ratio was 5. Optimization of the SSF conditions revealed that maximum enzyme activity (1,162 U/gds) was observed after 7 days in a production medium of 80% moisture content and pH 6.0 containing 16 g maize bran [25% (w/v)] of particle size of 0.71-0.3 mm, 0.6% potato starch, 3.0% casein, and 64 ml of formulated basal salt solution. Overall, the enzyme production was enhanced by 3.2-fold as compared with un-optimized conditions.     

Influence of cultivating conditions on the alpha-galactosidase biosynthesis from a novel strain of Penicillium sp. in solid-state fermentation

AIMS: The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Penicillium sp. in solid-state fermentation (SSF). METHODS AND RESULTS: Certain fermentation parameters involving incubation temperature, moisture content, initial pH value, inoculum and load size of medium, and incubation time were investigated separately. The optimal temperature and moisture level for alpha-galactosidase biosynthesis was found to be 30 degrees C and 50%, respectively. The range of pH 5.5-6.5 was favourable. About 40-50 g of medium in 250-ml flask and inoculum over 1.0 x 10(6) spores were suitable for enzyme production. Seventy-five hours of incubation was enough for maximum alpha-galactosidase production. Substrate as wheat bran supplemented with soyabean meal and beet pulp markedly improved the enzyme yield in trays. CONCLUSIONS: Under optimum culture conditions, the alpha-galactosidase activity from Penicillium sp. MAFIC-6 indicated 185.2 U g(-1) in tray of SSF. SIGNIFICANT AND IMPACT OF THE STUDY: The process on alpha-galactosidase production in laboratory scale may have a potentiality of scaling-up.     

Potential of Aspergillus oryzae CFTRI 1480 for producing proteinase in high titres by solid state fermentation

Aspergillus oryzae CFTRI 1480, an isolate from a spoiled moist sample of casein, produced 59,105 units of an extracellular proteinase/g dry mouldy bran (DMB) at 72 h in an arbitrarily formulated wheat bran medium in a solid state fermentation system. The enzyme production was significantly affected by mineral salt content and pH of the liquid used for moistening the wheat bran. Enzyme titres were enhanced 1.34-fold with the addition of 0.4% corn starch. Optimization of key parameters, i.e., initial moisture content, age and size of inoculum, increased the enzyme production to 191,869 units/g DMB and reduced the fermentation time to 48 h. Such high titres in a simple medium, surpassing most of the literature reports, indicate the industrial importance of the culture. The properties of acetone-precipitated enzyme, viz, the optimum pH of 10.0, more than 95% activity between pH 7.0 and 10.0, temperature optimum at 55° C and more than 90% activity between 10 and 27°C, are similar to those of commercially available fungal proteinases employed in animal feed, leather and other industries. Correspondence to: B. K. Lonsane     

N+注入选育黑曲霉益生菌及其突变菌株产酶条件的研究

以益生菌株黑曲霉AN01为材料,经N 多次诱变得突变益生菌株AN03。结果表明,出发益生菌株AN01酸性蛋白酶、纤维素酶和果胶酶的酶活分别由原来的71.6Ug、141.7Ug和264.8Ug相继提高到996.5Ug、940.4Ug和906.5Ug。突变益生菌株AN03经传5代培养,产酶特性稳定。试验还研究了变突变益生菌株AN03最佳产酶条件,培养基为每升含麸皮105g,玉米芯105g,豆粕105g,氯化铵16g,pH5.0。30℃培养4d。     

Monacolin K production by citrinin‐free Monascus pilosus MS‐1 and fermentation process monitoring

Monacolin K (MK) is a naturally occurring hypocholesterolemic agent that specifically inhibits HMG‐CoA reductase. As a natural source of MK, Monascus‐fermented products are of special interest; however, some Monascus strains could produce citrinin, which is a nephrotoxin, as a contaminant in Monascus‐derived products. A Monascus pilosus strain (MS‐1) that produces high amounts of MK, but no citrinin, was screened in previous investigations. Herein, liquid‐state fermentation parameters of the MS‐1 strain were optimized using statistical methods to maximize the MK yield with potato juice as a basic medium. The maximum MK yield (326.74 μg/mL) was predicted with 50 mL of medium in a 250‐mL conical flask containing 30 g/L sucrose, 38.75 g/L soybean flour, 0.00105 mol/L Mg²⁺ at pH 5.48, and 8% v/v seed inoculum precultured for 42 h at 30°C, incubated at 30°C for 3 days, followed by further incubation for 11 days at 24.7°C. The verified MK yield was 390.68 μg/mL and the MK yield increased to 565.64 μg/mL after 21 days of fermentation. No citrinin was detected in MS‐1‐fermented products. The results suggest that citrinin‐free MK can be obtained from natural medium through liquid‐state fermentation in an economical way. This method will be of practical value to the industrial production of MK.     

Polyunsaturated fatty acids production by solid-state fermentation on polyurethane foam by Mortierella alpina

Polyunsaturated fatty acids (PUFAs) are essential in healthy diets and their production is extremely important. Natural sources of PUFAs includes animal and aquatic products such as marine fish oil, however there are several limitations such as the decrease of fish stocks throughout the world. Thus, microbial oils are a preferable source of PUFAs. Herein, it was studied the production of PUFAs by Mortierella alpina under solid-state fermentation (SSF) using polyurethane foam as inert substrate and synthetic medium or lignocellulosic hydrolysate as source of C, N, and other nutrients. Several parameters of fermentation conditions were evaluated as carbon source, inductors addition, ratio C/N and temperature. The highest amount of total PUFAs per mass of solid (535.41 ± 24.12 mg/g), linoleic acid (129.66 ± 5.84 mg/g), and α-linoleic acid (401.93 ± 18.10 mg/g) were produced when the culture medium contained 20 g/L glucose, 10% (w/v) linseed oil, the C/N ratio was adjusted to 25 and the incubation temperature was 25°C for 3 days decreasing to 16°C on the remaining 4 days of fermentation. In addition, a hemicellulosic hydrolysate can be used as low-cost substrate to produce PUFAs, although the production was lower than the achieved with synthetic medium. SSF showed an interesting technology for microbial PUFAs production.     

Production,partial characterization,and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus <Emphasis Type="Italic">Myceliophthora</Emphasis> sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.     

Production of raw-starch digesting amyloglucosidase by Aspergillussp GP-21 in solid state fermentation

Aspergillus sp GP-21 produced a raw-starch digesting amyloglucosidase which showed optimum activity at 65°C and pH 5.0–5.5. At 50°C the enzyme converted about 40% of raw corn starch to glucose within 48 h. Enzyme production was studied in solid state fermentation using wheat bran. Productivity was affected by the level of moisture, incubation temperature and the presence or absence of supplements. Maximum enzyme production was observed at a moisture level of 75% and at 30°C. Enzyme production was stimulated by supplementing wheat bran with 0.25% proteose peptone, 1% trace mineral solution, 0.01% CaCl₂ and 0.01% MgSO₄. Received 27 October 1998/ Accepted in revised form 15 March 1999     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

土壤中高产蛋白酶菌株产酶条件及酶学性质

【背景】微生物蛋白酶已经成为工业用蛋白酶的主要来源,筛选具有特殊环境适应性的微生物成为生物酶资源的开发热点。【目的】通过对青藏高原土壤微生物产蛋白酶菌株的筛选、优化及相关特性研究,寻找新的蛋白酶资源,为高原菌种资源利用提供科学依据。【方法】采用形态学和分子生物学对筛选菌株进行菌种鉴定,利用单因素试验和正交试验对菌株进行发酵条件优化及酶学性质的探究。【结果】筛选出一株高产蛋白酶菌株XC2,经鉴定菌株XC2为枯草芽孢杆菌(Bacillus subtilis)。XC2最优产酶条件:可溶性淀粉4.0%,牛肉膏1.0%,K~+0.6%,培养温度34°C、初始pH 7.0、接种量2.0%的条件下200 r/min振荡培养13 h,所产蛋白酶活力最高为638.5 U/mL。XC2所产蛋白酶最适反应温度60°C,最适pH9.0;40-50°C、pH8.0-10.0条件下酶活稳定性较高;Mn~(2+)对酶活力有明显激活作用,而Zn~(2+)、Cu~(2+)、Fe~(2+)、Fe3+对酶活力有明显抑制作用。【结论】枯草芽孢杆菌XC2有较强的产碱性蛋白酶的能力,具有较好的应用前景。     

Endocellulase Production by <Emphasis Type="Italic">Cotylidia pannosa</Emphasis> and its Application in Saccharification of Wheat Bran to Bioethanol

For efficient bioconversion of lignocellulosic materials to bioethanol, the study screened 19 white-rot fungal strains for their endocellulolytic activity and saccharification potential. Preliminary qualitative and quantitative screening revealed Cotylidia pannosa to be the most efficient endocellulase producing fungal strain when compared to the standard strain of Trichoderma reesei MTCC 164. Ensuing initial screening, the production of endocellulase was further optimized using submerged fermentation to recognize process parameters such as temperature, time, agitation pH, and supplementation of salts in media required for achieving maximum production of endocellulase. The strain C. pannosa produced the maximum amount of endocellulase (8.48 U/mL) under submerged fermentation with wheat bran (2%) supplemented yeast extract peptone dextrose (YEPD) medium after an incubation time of 56 h at 30 °C and pH 5.0 at an agitation rate of 120 rpm with a saccharification value of 50.5%. The fermentation of wheat bran hydrolysate with Saccharomyces cerevisiae MTCC 174 produced 4.12 g/L of bioethanol after 56 h of incubation at 30 °C. The results obtained from the present investigation establish the potential of white-rot fungus C. pannosa for hydrolysis and saccharification of wheat bran to yield fermentable sugars for their subsequent conversion to bioethanol, suggesting its application in efficient bioprocessing of lignocellulosic wastes.