Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis

Abstract-Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5-15 nm) or the transformation in the presence of randomly methylated beta-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The technical product ADD was obtained in 75% yield, based on phytosterol. It contained as impurity 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment-the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD-no by products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5 AD was combined. The yield of 9-OH-AD (m.p. 218-220 degrees C) based on transformed phytosterol was 56%.     

Production of arabitol from enzymatic hydrolysate of soybean flour by Debaryomyces hansenii fermentation

Arabitol is a low-calorie sugar alcohol with anti-cariogenic properties. Enzymatic hydrolysate of soybean flour is a new renewable biorefinery feedstock containing hexose, pentose, and organic nitrogen sources. Arabitol production by Debaryomyces hansenii using soybean flour hydrolysate was investigated. Effects of medium composition, operating conditions, and culture stage (growing or stationary phase) were studied. Production was also compared at different culture volumes to understand the effect of dissolved oxygen concentration (DO). Main factors examined for medium composition effects were the carbon to nitrogen concentration ratio (C/N), inorganic (ammonium) to organic nitrogen ratio (I/O-N), and sugar composition. Arabitol yield increased with increasing C/N ratio and a high I/O-N (0.8–1.0), suggesting higher yield at stationary phase of low pH (3.5–4.5). Catabolite repression was observed, with the following order of consumption: glucose > fructose > galactose > xylose > arabinose. Arabitol production also favored hexoses and, among hexoses, glucose. DO condition was of critical importance to arabitol production and cell metabolism. The yeast consumed pentoses (xylose and arabinose) only at more favorable DO conditions. Finally, arabitol was produced in fermentors using mixed hydrolysates of soy flour and hulls. The process gave an arabitol yield of 54%, volumetric productivity of 0.90 g/L-h, and specific productivity of 0.031 g/g-h.

     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria <Emphasis Type="Italic">Mycobacterium neoaurum,Pimelobacter simplex</Emphasis>, and <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.     

Use of soybean flour (dehulled, solvent-extracted soybean) as a fish meal substitute in practical diets for African catfish, Clarias gariepinus (Burchell 1822): growth, feed utilization and digestibility

Growth and digestibility trials were conducted using African catfish, Clarias gariepinus (Burchell 1822): (1) to obtain apparent digestibility coefficient (ADC) values for capelin fish meal, soybean flour and corn meal; (2) to formulate diets based on ADC values of the protein feedstuffs; and (3) to evaluate the effects of replacing 25%, 50% and 75% of fish meal in control diets with soybean flour on growth, feed utilization efficiency and carcass composition. Supplemental methionine was added to the diet formulation in which soybean flour replaced 75% of the diet. Diets were formulated (400 g digestible protein kg⁻¹ and 15 kJ digestible energy g⁻¹ dry diet) and fed to catfish fingerlings (13.1 ± 0.5 g) to apparent satiation twice daily for 70 days. The protein and energy digestibilities of fish meal and soybean flour were high (>90% and >80%, respectively; P < 0.05). At 75% fish meal replacement with soybean flour (without methionine supplementation), growth and feed utilization efficiency indicators were depressed compared with other diet treatments which had a similar (P > 0.05) growth and feed utilization efficiency to those fed the control diet. The carcass compositions of catfish in all diets were similar (P > 0.05) and the liver histology of catfish fed any of the diets showed no alterations. The results obtained indicate that 50% of fish meal protein in practical catfish diets can be replaced with soybean flour and that catfish can effectively utilize supplemental methionine, thereby allowing up to 75% of the dietary fish meal protein to be replaced by soybean flour.     

Proteins and saponins in the lipid preparation obtained by extraction of soybean flour

A complex lipid preparation was obtained by extraction of soybean flour with organic solvents. This preparation was shown to include not only phospholipids (major components), but also up to 30% saponins. These compounds were identified by TLC, HPLC, and 1H-NMR spectroscopy. Minor components of the lipid extract were represented by polypeptides associated with phospholipids via electrostatic or hydrophobic forces.     

Galactomyces geotrichum Y25产脂肪酶条件的优化

应用响应面法对Galactomyces geotrichumY25液体发酵产脂肪酶的条件进行了优化。首先采用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出黄豆粉、玉米浆和发酵时间3个对产酶影响显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面设计对显著因素进行优化,得出黄豆粉、玉米浆最佳质量分数分别为2.51%、2.12%,最佳发酵时间101.95 h。优化后液体发酵液中脂肪酶活力提高到34.65 U/mL,比初始酶活力9.6 U/mL提高了3.61倍。表明响应面法可显著优化Galactomyces geotrichumY25液体发酵产脂肪酶条件。     

Greater enhancement of Bacillus subtilis spore yields in submerged cultures by optimization of medium composition through statistical experimental designs

Bacillus subtilis spore preparations are promising probiotics and biocontrol agents, which can be used in plants, animals, and humans. The aim of this work was to optimize the nutritional conditions using a statistical approach for the production of B. subtilis (WHK-Z12) spores. Our preliminary experiments show that corn starch, corn flour, and wheat bran were the best carbon sources. Using Plackett–Burman design, corn steep liquor, soybean flour, and yeast extract were found to be the best nitrogen source ingredients for enhancing spore production and were studied for further optimization using central composite design. The key medium components in our optimization medium were 16.18 g/l of corn steep liquor, 17.53 g/l of soybean flour, and 8.14 g/l of yeast extract. The improved medium produced spores as high as $ 1.52 \pm 0.06 \times {10^{10}}{\text{spores}}/{\text{ml}} $ under flask cultivation conditions, and $ 1.56 \pm 0.07 \times {10^{10}}{\text{spores}}/{\text{ml}} $ could be achieved in a 30-l fermenter after 40 h of cultivation. To the best of our knowledge, these results compared favorably to the documented spore yields produced by B. subtilis strains.     

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

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

Induction of cytochrome P-450 in Streptomyces griseus by soybean flour

Soybean flour and the isoflavonoid genistein, were found to induce cytochrome P-450 in Streptomyces griseus. The chromophore was found in the 105,000xg supernatant and gave a reduced CO-difference spectrum with an absorption maximum of 448 nm. Almost 70% of the P-450 could be precipitated at 35-45% ammonium sulfate saturation. SDS-gel electrophoresis revealed the presence of a 45,000 dalton polypeptide in extracts induced by either soybean or genistein. S. griseus generated the free isoflavonoids genistein and daidzein when grown on soybean flour medium.     

Influence of medium design on lovastatin and mevastatin production byAspergillus terreus strains

In order to investigate the influence of medium design on lovastatin and mevastatin production byAspergillus terreus strains, several nitrogen complex sources, such as vegetal flour and peptones of different origin (animal and vegetal) were tested, together with the addition of methionine, an aminoacid that is directly involved in lovastatin biosynthetic pathway. Soybean peptone generally allowed the best lovastatin yields to be achieved (250–280 mg l^?1), particularly in the presence of soybean and peanut flours. For mevastatin, the best results (300–320 mg l^?1) were obtained at 7 days fermentation with modified base medium (CLD), and at 14 days with standard medium (STD), not being possible in this case to associate the best yield with a defined flour and/or peptone. The results show that lovastatin production is influenced by the presence of soybean peptone and by the addition of methionine; instead, the production of mevastatin appears more strictly strain-associated and not directly dependent on the complex ingredients employed.     

Relationship between population growth of the red flour beetle Tribolium castaneum and protein and carbohydrate content in flour and starch

The effects of eight diets (atta flour, wheat flour, self-rising flour, rice flour, custard powder, corn flour, tapioca starch, and potato starch) on the development of the red flour beetle, Tribolium castaneum (Herbst), reared at 29-31 degrees C and 66-70% RH were assessed. Five pairs of male and female T. castaneum were reared on the respective diets for 28 d before the experimental setup was dismantled and adult counts were recorded. In another experiment, the insects were allowed to mate and oviposit in each flour or starch type over a period of 7 d before being removed. The counting of pupae and adult emergence began on the day of emergence and was continued on a daily basis until day 140. Proximate analysis was performed for chemical composition of each diet, and the numbers of new adults that developed were found to be positively correlated (r2 = 0.97; P < 0.05) with the protein content and negatively correlated (r2 = 0.93; P < 0.05) with the carbohydrate content. For T. castaneum, the suitable diets were ranked as follows: atta flour > wheat flour > self-rising flour > rice flour > custard powder > corn flour > tapioca starch > potato starch. T. castaneum larval development to the pupal and adult stages developed significantly faster in atta flour (P < 0.05) than in the other diets, and the greatest number of progeny was produced from beetles reared on atta flour. Fewer adults emerged from wheat flour, self-rising flour, and rice flour, and no new emergences were recorded for the remaining diets. Developmental rate was much slower in beetles reared on diets in which a low number in progeny was produced. These data illustrate that different diets can influence the sustainability of these insects and affect their development and growth.     

Improvement of ethanol yield from raw corn flour by <Emphasis Type="Italic">Rhizopus</Emphasis> sp.

A novel raw-starch-digesting glucoamylase producer, Rhizopus sp. W-08, and Saccharomyces cerevisiae Z-06 were used in a fed batch process for simultaneous saccharification and fermentation of raw corn flour. Ethanol concentration of 21% (v/v) was obtained after 48 h. The conversion efficiency of raw corn flour to ethanol was 94.5% of the theoretical ethanol yield.     

Overexpression of cytochrome p450 125 in <Emphasis Type="Italic">Mycobacterium</Emphasis>: a rational strategy in the promotion of phytosterol biotransformation

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD⁺/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD⁺ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD⁺ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD⁺/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.     

Rotylenchulus reniformis Management in Cotton with Crop Rotation

One-year crop rotations with corn or highly resistant soybean were evaluated at four locations for their effect on Rotylenchulus reniformis population levels and yield of a subsequent cotton crop. Four nematicide (aldicarb) regimes were included at two of the locations, and rotation with reniform-susceptible soybean was included at the other two locations. One-year rotations to corn or resistant soybean resulted in lower R. reniformis population levels (P ≤ 0.05) than those found in cotton at three test sites. However, the effect of rotation on nematode populations was undetectable by mid-season when cotton was grown the following year. Cotton yield following a one-year rotation to resistant soybean increased at all test locations compared to continuous cotton, and yield following corn increased at three locations. The optimum application rate for aldicarb in this study was 0.84 kg a.i./ha in furrow. Side-dress applications of aldicarb resulted in yield increases that were insufficient to cover the cost of application in 3 of the 4 years.     

Control of the Soybean Cyst Nematode by Crop Rotation in Combination with a Nematicide

An experiment to evaluate the control of soybean cyst nematodes compared 1-year, 2-year, and 3-year nonhost rotations with continuous soybeans (Glycine max) in 0.2-ha plots. In a second 1-year rotation, the plots were planted to soybean or corn (Zea mays) after fumigation in the spring with a split application of 1,3-dichloropropene (748.2 liters/ha). The effects of the nematicide were apparent the first year. Soybean yield was 1,482 kg/ha compared to 233 kg/ha in the untreated plots. In the second year, the highest yielding plants (2,035 kg/ha) were those following 1 year of corn that had been treated the previous year; plants in untreated plots yielded 288 kg/ha. Average yield of soybean following 1 year of corn was 957 kg/ha compared to 288 kg/ha for continuous soybean. In the third year, the effects of the nematicide were still evident. Soybean plants in plots treated the first year, followed by corn, then soybean, yielded 1,044 kg/ha compared to 761 kg/ ha for soybean following 1 year of corn and 991 kg/ha for soybean following 2 years of corn. Plots planted to soybean for 3 consecutive years yielded 337 kg/ha. Nematicidal effects were no longer evident during the fourth year. Yields were most improved by the greatest number of years in the nonhost crop; highest yields in descending order were from plants following 3 years of corn, 2 years of corn, and 1 year of corn. Plots planted to soybean for 4 consecutive years yielded 130 kg/ha. Highly significant negative correlations occurred each year between initial nematode population densities and seed yield.     

The effect of seaweed composite flour on the textural properties of dough and bread

Seaweeds as food and seaweed-derived food flavors, colors, and nutrients are attracting considerable commercial attention. In the baking industries, hydrocolloids are of increasing importance as bread making improvers, where their use aims to improve dough handling properties, increase the quality of fresh bread, and extend the shelf life of stored bread. Seaweeds contain a significant amount of soluble polysaccharides and have the potential function as a source of dietary fiber. In this study, red seaweed (Kappaphycus alvarezii) powder was incorporated (2–8 %) with wheat flour and used to produce bread. The effect of seaweed composite flour on dough rheological properties and the quality of bread was investigated using various techniques. Farinograph tests were applied to determine the effect of seaweed powder on the rheological properties of wheat flour dough, while texture profile analysis (TPA) was used to measure the textural properties of dough as well as the final product. The results showed that the additions of seaweed powder (2–8 %) increased the water absorption of the dough. TPA results showed that the addition of seaweed powder decreased stickiness properties. Bread produced with seaweed composite flour showed higher values of firmness.     

Medium optimization for the production of thermal stable beta-glucanase by Bacillus subtilis ZJF-1A5 using response surface methodology

Polysaccharides, such as barley flour, dextrin and soluble starch, were better carbon sources than monosaccharides and disaccharides, such as glucose and maltose, for cell growth of Bacillus subtilis ZJF-1A5 and beta-glucanase production. beta-Glucanase produced by B. subtilis ZJF-1A5 was associated partially with cell growth and increased significantly when cells entered stationary phase; yeast extract was the best nitrogen source, followed by soybean flour. All inorganic nitrogen sources chosen in the experiments were not favorable for cell growth and enzyme production. A fractional factorial design (2(6-2)) was applied to elucidate medium components that significantly affect beta-glucanase production. The concentration of barley flour, corn flour and soybean flour in medium were significant factors. The steepest ascent method was used to locate the optimal domain and a central composite design was used to estimate the quadratic response surface from which the factor levels for maximum production of beta-glucanase were determined. The composition of fermentation medium optimized with response surface methodology was (g/l): barley flour, 63.5; corn flour, 44.8; KH2PO4, 1.0; MgSO4 x 7H2O, 0.1; CaCl2, 0.1. beta-Glucanase activity was 251 U/ml at 48 h using optimized medium, 1.4 times higher than that in original medium.     

Effect of soybean lipoxygenae on baking properties of wheat flour

Changes in bread-baking properties of wheat flour caused by soybean lipoxygenase and polyunsaturated fatty acids were studied. A positive effect of soybean flour added during dough kneading in an amount of 2% was demonstrated. A method for dough fermentation increasing the loaf volume and improving organoleptic characteristics and total bread-baking estimate is recommended.     

Effect of soybean lipoxygenase on baking properties of wheat flour

Changes in bread-baking properties of wheat flour caused by soybean lipoxygenase and polyunsaturated fatty acids were studied. A positive effect of soybean flour added during dough kneading in an amount of 2% was demonstrated. A method for dough fermentation increasing the loaf volume and improving organoleptic characteristics and total bread-baking estimate is recommended.     

Efficient production of protopectinases by Bacillus subtilis using medium based on soybean flour

We have developed a culture system for efficient production of protopectinases (PPases) by Bacillus subtilis. PPase shows the pectin-releasing activity and is expected to be utilized in the enzymatic cotton scouring. B. subtilis IFO3134 was cultivated using defatted soybean flour as a main component of culture media. This strain produced three different types of PPases, namely, PPase-C, -N and -R performing endo-arabinase activity, pectate-lyase activity and pectin-lyase activity, respectively. The effects of alkaline solubilization and autoclave treatments to extract nutrients from soybean flour and initial soybean flour concentration (20-80g/l) on production of PPases in batch fermentation were investigated. Alkaline solubilization of soybean flour with NaOH remarkably reduced enzyme productivity. In addition, a higher initial concentration of soybean flour reduced the enzyme productivity of cells. The pectin-releasing activity was the largest and reached up to 2200-2400U/ml, when the culture medium containing an initial soybean flour concentration of 40g/l was autoclaved for 45-60min without alkaline solubilization treatment.