Biotransformation of corn bran derived ferulic acid to vanillic acid using engineered Pseudomonas putida KT2440

Abstract

Ferulic acid is a fraction of the phenolics present in cereals such as rice and corn as a component of the bran. Substantial amounts of waste bran are generated by the grain processing industry and this can be valorized via extraction, purification and conversion of phenolics to value added chemical products. Alkaline alcohol based extracted and purified ferulic acid from corn bran was converted to vanillic acid using engineered Pseudomonas putida KT2440. The strain was engineered by rendering the vanAB gene nonfunctional and obtaining the mutant defective in vanillic acid metabolism. Biotransformation of ferulic acid using resting Pseudomonas putida KT2440 mutant cells resulted in more than 95?±?1.4% molar yield from standard ferulic acid; while the corn bran derived ferulic acid gave 87?±?0.38% molar yield. With fermentation time of less than 24?h the mutant becomes a promising candidate for the stable biosynthesis of vanillic acid at industrial scale.     

Numerical simulation and deacidification of nanomagnetic enzyme conjugate in a liquid–solid magnetic fluidized bed

The conventional deacidification method is difficult to achieve a better refining effect due to the high acid value in the rice bran crude oil, and the enzymatic esterification deacidification method can effectively reduce the acid value without generating chemical waste. In this study, the free lipase was immobilized on a magnetic polymer carrier Fe₃O₄/SiOx-g-P (GMA: glycidyl methacrylate) to obtain a immobilized lipase with a particle size of 105.30 ± 1.1 nm and an enzyme activity of 6580 ± 9.6 PLU/g (PLU: enzyme activity unit). Based on the batch deacidification process parameters, a multi-stage magnetic fluidized bed continuous circulation deacidification system was designed, and then the motion law of nanomagnetic immobilized lipase particles in liquid–solid magnetic fluidized bed was simulated by computer. When the iterative step was 5 × 10⁻⁵ s, the open porosity of the porous plate was 35.0%, the rice bran oil flow rate was 3.0 mm/s, and the magnetic field strength was 25.0 mT, which was beneficial to the deacidification reaction of rice bran oil. Under the conditions of magnetic immobilized lipase dosage of 4.0%, the phytosterol dosage of 22.0%, the molecular sieve dosage of 10%, the esterification temperature of 78.0 °C and the FFA (free fatty acid) content in rice bran oil decreased to 1.5%, after 48 h of reaction. The conversion rate is 92.8%, which provides a theoretical basis for the subsequent guidance of magnetic fluidized bed enzymatic continuous deacidification.     

Production of vanillin from waste residue of rice bran oil by Aspergillus niger and Pycnoporus cinnabarinus

A new technology of transforming ferulic acid, which was from waste residue of rice bran oil, into vanillin was developed by a combination of fungal strains Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115. Various concentrations of ferulic acid were compared, and the highest yield reached 2.2 g l(-1) of vanillic acid by A. niger CGMCC0774 in a 25 l fermenter when concentration of ferulic acid was 4 g l(-1). The filtrate of A. niger CGMCC0774 culture was concentrated and vanillic acid in the filtrate was bio-converted into vanillin by P. cinnabarinus CGMCC1115. The yield of vanillin reached 2.8 g l(-1) when 5 g l(-1) of glucose and 25 g of HZ802 resin were supplemented in the bioconversion medium. The 13C isotope analysis indicated that delta13C(PDB) of vanillin prepared was much different from chemically synthesized vanillin.     

Covalently attached ferulic acid in a proteoglycan from rice bran

A water-soluble proteoglycan, precipitated with ammonium sulfate from the hot-water extract of rice bran, contained ferulic acid, which was liberated by alkaline treatment. Evidence for the linkage between the carboxyl group of ferulic acid and the proteoglycan was obtained by the characterization of ferulic acid hydroxamate after treatment of the proteoglycan with hydroxylamine.     

Phytochemical characterization of defatted rice bran and optimization of a process for their extraction and enrichment

The aim of this study was to characterize the defatted rice bran (DRB) employing HPLC for identifying the major phytochemicals in DRB and to examine its commercial potential as a source of bioactive phytochemicals leading to value addition of DRB otherwise used as cattle feed. Various solvent extracts showed the presence of oryzanols, tocols, and ferulic acid. Methanol was the most effective extractant under the optimized conditions of a material-solvent ratio of 1:15 (wt./vol.) and a time of extraction of 10h. The yields of total phenols, oryzanols and ferulic acid from DRB with methanol were 2204, 316, and 233 ppm, respectively. Enrichment of antioxidants in the crude methanolic extract (CME) was achieved by sequential extraction and fractionation, resulting in three enriched fractions, viz., acetone extract (AE), acetone extract-lipophilic fraction (AE-LP) and acetone extract-polar fraction (AE-PP). While AE-LP was enriched in oryzanols and tocols by about 65 times, AE-PP was enriched in ferulic acid by 70 times as compared to their contents in DRB.     

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.     

Rice bran stabilization and rice bran oil extraction using ohmic heating

Ohmic heating has been shown to increase the extraction yields of sucrose from sugar beets, apple juice from apples, beet dye from beet root, and soymilk from soybeans. Rice bran is a byproduct of the rice milling process that has economic potential by virtue of highly nutritious rice bran oil contained within the bran. In this study, ohmic heating was used to stabilize rice bran and to improve rice bran oil extraction yield as compared to microwave heating and a control (no heating). Results showed that ohmic heating is an effective method for rice bran stabilization with moisture addition. Free fatty acid concentration increased more slowly than the control for raw bran samples subjected to ohmic heating with no corresponding temperature rise, indicating that electricity has a non-thermal effect on lipase activity. Ohmic heating increased the total percent of lipids extracted from rice bran to a maximum of 92%, while 53% of total lipids were extracted from the control samples. Lowering the frequency of alternating current significantly increased the amount of oil extracted, probably due to electroporation. Ohmic heating was successfully applied to rice bran despite its high oil content. This could have important implications for the enhanced extraction of non-polar constituents.     

Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472

Aspergillus niger I-1472 was grown on sugar beet pulp to produce cell wall polysaccharide-degrading enzymes, including feruloyl esterases. Compared to enzymatic activities measured in commercially available mixtures previously used for the release of ferulic acid, the A. niger enzymes were more various. These enzymes were tested to release ferulic acid from sugar beet pulp, maize bran, or autoclaved maize bran. They were as efficient as the commercial mixture to release ferulic acid from sugar beet pulp. On the other hand, they were much more efficient to release ferulic acid from maize bran after autoclaving pretreatment, as 95% of ferulic acid ester were solubilized. Thus, A. niger enzymes exhibited a high interest in the release of ferulic acid from various agro-industrial by-products.     

Mechanism of rice bran lipase inhibition through fermentation activity of probiotic bacteria

Rice bran oil is known as wonder oil and it is the most important vegetable oil in Asia. Rice bran oil is extracted from bran that is the outer hard layer of rice. It is an emerging category in edible oil with a lot of nutritional properties and health benefits. Rice bran oil is heart-friendly, boosts up immunity, and prevents from other diseases occurring commonly in Pakistan. The current study aimed to stabilize rice bran oil through different probiotic isolates and to assess the nutritional content of rice bran oil after stabilization. The study was aimed to inactivate naturally occurring lipases that can hydrolyze oil into glycerol and free fatty acid which is a serious problem that gives it a rancid taste and smell. Antilipase activity was used to inactivate naturally occurring lipases that are a huge threat to the stabilization process. The fermentation process utilizes antilipase activity without affecting the nutritional value of oil. Lactobacillus strains were used for the stabilization of rice bran oil. Rice bran oil was extracted in the Soxhlet apparatus. The probiotic lab isolates Lactobacillus delbrueckii S2, Lactobacillus casei S5 and Lactobacillus plantarum S13 were applied to it to increase its shelf life and prevent oxidative rancidity. The extraction temperature of rice bran oil was maintained above 40 °C to inhibit lipase activity. Rice bran oil samples were stored at refrigeration temperature to arrest lipase activity. Probiotics maintained acidic pH to keep oil stabilization. Qualitative analysis was done to confirm rice bran oil stabilization. Determination of Free Fatty Acid (FFA) and saponification value confirmed that oxidative rancidity of rice bran oil was controlled by probiotics. FFA count was less than 10% and Saponification Value (SV) was 180. GC analysis was performed to analyze the FFA profile. Gas Chromatography results have shown 3 fatty acids. Statistical analysis has shown non-significant effect on different incubation temperatures of Lactobacillus isolates. Among the biological methods of stabilization, the use of probiotics is a novel concept and recommended for commercial application.     

Effect of long term feeding of rice bran oil upon lipids and lipoproteins in rats

The effects of feeding two levels of rice bran oil (RBO) on the growth, lipid parameters, and fatty acid composition of the plasma and liver of rats (Wistar strain) were compared with those produced on animals which had been fed the same levels of peanut oil (PNO). The control animals were fed synthetic diets containing 5 and 20% peanut oil (PNO) and the experimental groups were fed similar diets, containing the same level of rice bran oil (RBO). There was no significant difference with respect to the organ weights between the control and the experimental groups. In general, groups fed 20% oil gained more weight than groups fed 5% oil. The animals which received rice bran oil in their diet had, in general, comparatively lower levels of cholesterol, triglycerides and phospholipids. On the other hand, animals receiving 20% rice bran oil in their diet, showed an increase of 20% in high density lipoproteins (HDL-C), within 18 weeks (p<0.05), when compared to the animals fed with peanut oil. Similarly, low density lipoprotein cholesterol (LDL-C) and very low density lipoprotein cholesterol (VLDL-C) were lower in RBO-fed groups, than in the PNO-fed groups. There was, however, no significant differences in the cholesterol/phospholipid (C/P) ratio of the two groups. Analysis of plasma and of liver fatty acids indicated, in a general way, the type of fat consumed. There were no significant difference in the P/S ratio, nor any in the oleic/linoleic, oleic/stearic, palmitoleic/palmitic, oleic/palmitic, and oleic/palmitoleic ratios. Furthermore, levels of saturated (SAFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids were identical in both the groups. Thus, our results suggest that feeding a high level of rice bran oil (RBO) has no deleterious effect on the growth and blood lipid profile of rats.Abbreviations PNO peanut oil - RBO rice bran oil - HDL-C high density lipoprotein cholesterol - LDL-C low density lipoprotein cholesterol - VLDL-C very low density lipoprotein cholesterol - SAFA saturated fatty acids - MUFA mono-unsaturated fatty acids     

Rice bran oil and oryzanol reduce plasma lipid and lipoprotein cholesterol concentrations and aortic cholesterol ester accumulation to a greater extent than ferulic acid in hypercholesterolemic hamsters

Our laboratory has reported that the hypolipidemic effect of rice bran oil (RBO) is not entirely explained by its fatty acid composition. Because RBO has a greater content of the unsaponifiables, which also lower cholesterol compared to most vegetable oils, we wanted to know whether oryzanol or ferulic acid, two major unsaponifiables in RBO, has a greater cholesterol-lowering activity. Forty-eight F(1)B Golden Syrian hamsters (Mesocricetus auratus) (BioBreeders, Watertown, MA) were group housed (three per cage) in cages with bedding in an air-conditioned facility maintained on a 12-h light/dark cycle. The hamsters were fed a chow-based hypercholesterolemic diet (HCD) containing 10% coconut oil and 0.1% cholesterol for 2 weeks, at which time they were bled after an overnight fast (16 h) and segregated into 4 groups of 12 with similar plasma cholesterol concentrations. Group 1 (control) continued on the HCD, group 2 was fed the HCD containing 10% RBO in place of coconut oil, group 3 was fed the HCD plus 0.5% ferulic acid and group 4 was fed the HCD plus 0.5% oryzanol for an additional 10 weeks. After 10 weeks on the diets, plasma total cholesterol (TC) and non-high-density lipoprotein cholesterol (HDL-C) (very low- and low-density lipoprotein) concentrations were significantly lower in the RBO (-64% and -70%, respectively), the ferulic acid (-22% and -24%, respectively) and the oryzanol (-70% and -77%, respectively) diets compared to control. Plasma TC and non-HDL-C concentrations were also significantly lower in the RBO (-53% and -61%, respectively) and oryzanol (-61% and -70%, respectively) diets compared to the ferulic acid. Compared to control and ferulic acid, plasma HDL-C concentrations were significantly higher in the RBO (10% and 20%, respectively) and oryzanol (13% and 24%, respectively) diets. The ferulic acid diet had significantly lower plasma HDL-C concentrations compared to the control (-9%). The RBO and oryzanol diets were significantly lower for plasma triglyceride concentrations compared to the control (-53% and -65%, respectively) and ferulic acid (-47% and -60%, respectively) diets. Hamsters fed the control and ferulic acid diets had significantly higher plasma vitamin E concentrations compared to the RBO (201% and 161%, respectively) and oryzanol (548% and 462%, respectively) diets; the ferulic acid and oryzanol diets had significantly lower plasma lipid hydroperoxide levels than the control (-57% and -46%, respectively) diet. The oryzanol-fed hamsters excreted significantly more coprostenol and cholesterol in their feces than the ferulic acid (127% and 120%, respectively) diet. The control diet had significantly greater aortic TC and FC accumulation compared to the RBO (115% and 89%, respectively), ferulic acid (48% and 58%, respectively) and the oryzanol (74% and 70%, respectively) diets. However, only the RBO and oryzanol diets had significantly lower aortic cholesterol ester accumulation compared to the control (-73% and -46%, respectively) diet. The present study suggests that at equal dietary levels, oryzanol has a greater effect on lowering plasma non-HDL-C levels and raising plasma HDL-C than ferulic acid, possibly through a greater extent to increase fecal excretion of cholesterol and its metabolites. However, ferulic acid may have a greater antioxidant capacity via its ability to maintain serum vitamin E levels compared to RBO and oryzanol. Thus, both oryzanol and ferulic acid may exert similar antiatherogenic properties, but through different mechanisms.     

Ferulic acid,a bioactive component of rice bran,improves oxidative stress and mitochondrial biogenesis and dynamics in mice and in human mononuclear cells

The aim of the study was to characterize the vascular effects of rice bran enzymatic extract (RBEE). ApoE−/− mice were fed a high-fat/cholesterol diet (HFD) or HFD supplemented with 5% RBEE for 21 weeks. RBEE prevented development of atherosclerotic plaques and oxidative stress in mouse aorta as well as the down-regulation of markers of mitochondrial biogenesis. Analysis of the bioactive components identified ferulic acid (FA) as responsible component. In healthy human volunteers, FA intake reduced NADPH oxidase activity, superoxide release, apoptosis and necrosis in peripheral blood mononuclear cells. Differentiation and proliferation of endothelial progenitor cells were improved. In summary, the study identifies FA as a major active component of rice bran, which improves expression of mitochondrial biogenesis and dynamics markers and reduces oxidative stress in a mouse model of vascular damage as well as in endothelial cells and human mononuclear cells.     

A novel process for extraction of edible oils: Enzyme assisted three phase partitioning (EATPP)

Three phase partitioning (TPP), a technique used in protein purification has been evaluated, for extraction of oil from three different plant sources viz: mango kernel, soybean and rice bran. The process consists of simultaneous addition of t-butanol (1:1,v/v) and ammonium sulphate (w/v) to a crude preparation/slurry. Under optimized condition, the protein appears as an interfacial precipitate between upper t-butanol containing oil and lower aqueous phase. Pretreatment of the slurries with a commercial enzyme preparation of proteases, Protizyme, followed by three phase partitioning resulted in 98%, 86% and 79% (w/w) oil yields in case of soybean, rice bran and mango kernel, respectively. The efficiency of the present technique is comparable to solvent extraction with an added advantage of being less time consuming and using t-butanol which is a safer solvent as compared to n-hexane used in conventional oil extraction process.     

A two-step acid-catalyzed process for the production of biodiesel from rice bran oil

A study was undertaken to examine the effect of temperature, moisture and storage time on the accumulation of free fatty acid in the rice bran. Rice bran stored at room temperature showed that most triacylglyceride was hydrolyzed and free fatty acid (FFA) content was raised up to 76% in six months. A two-step acid-catalyzed methanolysis process was employed for the efficient conversion of rice bran oil into fatty acid methyl ester (FAME). The first step was carried out at 60 degrees C. Depending on the initial FFA content of oil, 55-90% FAME content in the reaction product was obtained. More than 98% FFA and less than 35% of TG were reacted in 2 h. The organic phase of the first step reaction product was used as the substrate for a second acid-catalyzed methanolysis at 100 degrees C. By this two-step methanolysis reaction, more than 98% FAME in the product can be obtained in less than 8 h. Distillation of reaction product gave 99.8% FAME (biodiesel) with recovery of more than 96%. The residue contains enriched nutraceuticals such as gamma-oryzanol (16-18%), mixture of phytosterol, tocol and steryl ester (19-21%).     

Isolation of oryzanol from crude rice bran oil

The isolation of oryzanol from crude rice bran oil (RBO) was achieved by a two-step crystallization process. In the first crystallization, oryzanol was concentrated in the liquid phase along with free fatty acid (FFA), monoacylglycerol (MG), squalene, tocols, and phytosterols, whereas the solid phase contained mainly triacylglycerol (TG) and steryl esters. Oryzanol-rich product obtained from the first crystallization was subjected to the second crystallization where the oryzanol-rich product was kept at room temperature (20.5+/-1.5 degrees C) for 24h. Hexane was added as anti-solvent to the oryzanol-rich product and kept at 5+/-1 degrees C for another 48h. Parameters that affect the isolation of oryzanol from crude RBO were systematically investigated. Under optimal operation conditions, oryzanol with purity and recovery of 93-95% and 59%, respectively, was obtained from RBO with an initial FFA content of ca. 5%.     

高效石油降解菌群的构建及对芳香烃化合物萘的协同降解性能

【背景】石油作为一类混杂有机化合物,一旦产生污染就会对人类和环境造成严重的危害。【目的】从新疆石油污染土壤中分离筛选石油降解菌,为石油污染土壤的生物修复提供数据支持及技术参考。【方法】以石油为唯一碳源,通过富集培养、筛选分离得到123株单菌,根据菌落形态挑选出30个不同形态菌株,通过16S rRNA基因序列确定其种属,构建系统发育树;通过原油降解实验筛选出高效石油降解菌,以芳香烃的标志化合物萘为唯一碳源筛选出高效降解菌株,并分别筛选可降解水杨酸、邻苯二酚的菌株。【结果】分离筛选出5株高效石油降解菌,降解率高于85%;萘、水杨酸和邻苯二酚降解菌株各获得一株,将3种菌株按照1:1:1的接种比例对萘进行降解,萘的降解率从单菌60.74%提升到89.40%,菌株间的分工协作可以提高有机物的降解效率。【结论】筛选得到的菌株丰富了石油降解微生物菌种库,不同微生物菌株之间的分工协作为石油污染物的降解提供了新思路,为进一步研究石油污染治理提供参考。     

Effects of fibre on digestibility and passage time in callithricidae

The effects of fibre in a pelleted diet on food intake, digestibility of crude fibre, dry matter and energy, on passage time and consistency of faeces were studied in 2 species of Callithricidae, Callithrix jacchus and Saguinus fuscicollis. Coarse cellulose, microcellulose, wheat bran and shrimp meal (chitin = crude fibre) were tested in diets containing 2,4 and 6% total crude fibre, respectively. Digestibility and passage time were determined by inclusion of 0.5% Cr2O3 in the diet. Both celluloses had little influence on the digestibility of energy and dry matter. Digestibility of crude fibre was very low. Wheat bran led to evident depression of energy and dry matter digestibility. High digestibility of crude fibre occurred at the higher levels of inclusion in the diet. Shrimp meal was highly digested with little influence on digestibility of energy and dry matter, indicating considerable degradation of chitin. Wheat bran showed a marked effect, while microcellulose had no effect on passage time, consistency and volume of faeces.     

Production of xylanases from a newly isolated alkalophilic thermophilic Bacillus sp.

A new thermophilic strain of Bacillus SPS-0 which produces thermostable xylanases was isolated from a hot spring in Portugal. Xylanase production was 50 nkat/ml in the presence of wheat bran arabinoxylan. The temperature and pH for optimum activity were 75°C and 6–9, respectively. The hydrolysis patterns demonstrated that crude xylanases yield mainly xylose and xylobiose from xylan, whereas xylose and arabinose were produced from destarched wheat bran. An increase in xylose release was observed when SPS-0 xylanase was supplemented by a ferulic acid esterase. © Rapid Science Ltd. 1998     

Ferulic Acid Esterase Activity from Schizophyllum commune

Schizophyllum commune produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase, p-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate.     

Fractionation and Identification of Volatile Acids and Phenols in the Steam Distillate of Rice Bran

Fractionation of the volatile concentrate obtained from steam-distillate of rice bran and organoleptic tests of each fraction were carried out. The neutral fraction gave a much higher yield than others and was assumed to be indispensable for the reproduction of rice bran odor. The acidic fraction methylated with diazomethane and the phenolic fraction were analyzed by glass capillary gas chromatography to identify 17 carboxylic acids and 7 phenols. 4-Vinylguaiacol and 4-vinylphenol identified in the steam volatile concentrate were considered to be produced from ferulic and p-coumaric acids during steam-distillation. 4-Vinylphenol was the main component in the steam volatile concentrate of rice bran, having a characteristic unpleasant odor.