Optimization of Ultrasonic-Assisted Extraction of Flavonoid Compounds and Antioxidants from Alfalfa Using Response Surface Method.

Ultrasonic-assisted extraction (UAE) was used to extract flavonoid-enriched antioxidants from alfalfa aerial part. Response surface methodology (RSM), based on a four-factor, five-level central composite design (CCD), was employed to obtain the optimal extraction parameters, in which the flavonoid content was maximum and the antioxidant activity of the extracts was strongest. Radical scavenging capacity of the extracts, which represents the amounts of antioxidants in alfalfa, was determined by using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) methods. The results showed good fit with the proposed models for the total flavonoid extraction (R² = 0.9849), for the antioxidant extraction assayed by ABTS method (R² = 0.9764), and by DPPH method (R² = 0.9806). Optimized extraction conditions for total flavonoids was a ratio of liquid to solid of 57.16 mL/g, 62.33 °C, 57.08 min, and 52.14% ethanol. The optimal extraction parameters of extracts for the highest antioxidant activity by DPPH method was a ratio of liquid to solid 60.3 mL/g, 54.56 °C, 45.59 min, and 46.67% ethanol, and by ABTS assay was a ratio of liquid to solid 47.29 mL/g, 63.73 °C, 51.62 min, and 60% ethanol concentration. Our work offers optimal extraction conditions for total flavonoids and antioxidants from alfalfa.

Waste vinegar residue as substrate for phytase production.

Waste vinegar residue, the by-product of vinegar processing, was used as substrate for phytase production from Aspergillus ficuum NTG-23 in solid-state fermentation to investigate the potential for the efficient re-utilization or recycling of waste vinegar residue. Statistical designs were applied in the processing of phytase production. First, a Plackett-Burman (PB) design was used to evaluate eleven parameters: glucose, starch, wheat bran, (NH(4))(2)SO(4), NH(4)NO(3), tryptone, soybean meal, MgSO(4)·7H(2)O, CaCl(2)·7H(2)O, FeSO(4)·7H(2)O, incubation time. The PB experiments showed that there were three significant factors: glucose, soybean meal and incubation time. The closest values to the optimum point were then derived by steepest ascent path. Finally, a mathematical model was created and validated to explain the behavioural process after these three significant factors were optimized using response surface methodology (RSM). The best phytase activity was attained using the following conditions: glucose (7.2%), soybean meal (5.1%), and incubation time (271 h). The phytase activity was 7.34-fold higher due to optimization by PB design, steepest ascent path design and RSM. The phytase activity was enhanced 0.26-fold in comparison with the results by the second step of steepest ascent path design. The results indicate that with waste vinegar residue as a substrate higher production of phytase from Aspergillus ficuum NTG-23 could be obtained through an optimization process and that this method might be applied to an integrated system for recycling of the waste vinegar residue.

Effects of dietary taurine on egg production, egg quality and cholesterol levels in Japanese quail.

BACKGROUND: Taurine is a semi-essential amino acid and has many biological properties. The objective of this study was to determine the effect of dietary supplementation with taurine on egg production, egg quality, and cholesterol level in serum and egg yolk of quails. A total of 108 quails aged 6 weeks were randomly allocated to three dietary treatments. Each treatment consisted of four replicates of nine quails. The diets were supplemented with 0, 100, and 500 mg kg(-1) of taurine for 8 weeks. RESULTS: Dietary 500 mg kg(-1) taurine significantly affected egg production rate and feed conversion ratio, but had no significant effects on body weight gain, feed consumption, or egg weight. Dietary taurine had no significant effect on egg quality parameters studied. Serum triglyceride concentration was reduced significantly with supplementation of taurine at 100 and 500 mg kg(-1). Egg yolk cholesterol content was reduced significantly, and the contents of serum taurine and egg yolk taurine were increased significantly with taurine supplementation at 500 mg kg(-1). CONCLUSION: Results of the present study indicated that adding 500 mg kg(-1) taurine reduced yolk cholesterol concentration and increased yolk taurine content without adverse effects on performance and egg quality of laying quails.

Vinegar production residue as substrates for phytase production by Aspergillus ficuum.

Two kinds of vinegar production residues, sorghum vinegar residue (SVR) and corn vinegar residue (CVR), were used as a substrate for phytase production in solid-state fermentation (SSF) by Aspergillus ficuum. Various process parameters influencing phytase production were evaluated by single factor design experiments; further study involved CVR and its goodness-of-fit levels. An incubation time of 48 hours, initial moisture of 55% and an inoculum of 1.2 x 10(7) spores per millilitre were the optimum cultural conditions for the production of phytase in waste corn vinegar residue, without adding in vitro nutrient elements by orthogonal design experiment. Results showed that vinegar production residue as substrates for phytase production by Aspergillus ficuum through microbiology fermentation could be applied to treatment of an integrated system for waste from vinegar processing.

Purification, characterisation and protective effects of polysaccharides from alfalfa on hepatocytes.

The objective of this study was to determine the preliminary characteristics and protective effects of alfalfa polysaccharides (APS) on hepatocytes in vitro. The crude APS was purified by DEAE-cellulose and Sephadex G-100 chromatography, resulting in the four purified fractions: APS-1, APS-2, APS-3 and APS-4. The results indicated that APS-3 had higher carbohydrate and uronic acid contents and that APS-4 had a more complicated monosaccharide composition compared to the other purified fractions. The average molecular weights of APS-1, APS-2, APS-3 and APS-4 were 48,536, 6,221, 66,559 and 13,076 Da, respectively. Furthermore, APS (crude and its purified fractions) restored the activities of antioxidant enzymes and increased the total antioxidant capacity of hepatocytes subjected to H2O2-induced oxidative stress. Furthermore, APS treatment counteracted the increases in lactic dehydrogenase and malonaldehyde in the culture supernatant. These results clearly demonstrate that APS possesses a protective effect against oxidative injury in hepatocytes.

Myostatin knockout using zinc-finger nucleases promotes proliferation of ovine primary satellite cells in vitro.

Myostatin (MSTN) has previously been shown to negatively regulate the proliferation and differentiation of skeletal muscle cells. Satellite cells are quiescent muscle stem cells that promote muscle growth and repair. Because the mechanism of MSTN in the biology of satellite cells is not well understood, this study was conducted to generate MSTN mono-allelic knockout satellite cells using the zinc-finger nuclease mRNA (MSTN-KO ZFN mRNA) and also to investigate the effect of this disruption on the proliferation and differentiation of sheep primary satellite cells (PSCs). Nineteen biallelic and four mono-allelic knockout cell clones were obtained after sequence analysis. The homologous mono-allelic knockout cells with 5-bp deletion were used to further evaluations. The results demonstrated that mono-allelic knockout of MSTN gene leads to translation inhibition. Real-time quantitative PCR results indicated that knockout of MSTN contributed to an increase in CDK2 and follistatin and a decrease in p21 at the transcript level in proliferation conditions. Moreover, MSTN knockout significantly increased the proliferation of mutant clones (P < 0.01). Consistent with the observed increase in CDK2 and decrease in p21 in cells lacking MSTN, cell cycle analysis showed that MSTN negatively regulated the G1 to S progression. In addition, knockout of myostatin resulted in a remarkable increase in MyoD and MyoG expression under differentiating conditions but had no effect on Myf5 expression. These results expanded our understanding of the regulation mechanism of MSTN. Furthermore, the MSTN-KO ZFN mRNA system in PSCs could be used to generate transgenic sheep in the future.

Optimization of enzyme-assisted extraction of polysaccharides from alfalfa and its antioxidant activity.

In this present study, an efficient complex enzyme-assisted extraction technology was developed and optimized to extract polysaccharides from alfalfa using four factors at five levels central composite rotatable response surface design (CCRD). The experimental data was fitted to a second order polynomial equation with high coefficient of determination values (R(2)>0.95). The results of statistical analysis showed that the linear and quadratic terms of these four variables had significant effects (P<0.05) on the yield of polysaccharides from alfalfa. The optimum conditions were as follows: enzyme concentration of 2.5%, 2.0%, 3.0% (weight of alfalfa) of cellulase, papain and pectase, extraction temperature 52.7 °C, extraction pH 3.87, ratio of water to raw material 78.92 mL/g and extraction time 2.73 h. Under the optimal conditions, the experimental extraction yield of alfalfa polysaccharides was 5.05 ± 0.02%, which was well matched with the value (5.09%) predicted by the CCRD model. Moreover, evaluation of the antioxidant activity of polysaccharides from alfalfa in vitro suggested that the polysaccharides had good antioxidant effect, especially scavenging activity for hydroxyl radical and DPPH radical, which indicated that the polysaccharides from alfalfa may be explored as a novel natural antioxidant.

Influence of chestnut tannins on welfare, carcass characteristics, meat quality, and lipid oxidation in rabbits under high ambient temperature.

A study was conducted to evaluate the influence of chestnut tannins (CT) on welfare, carcass characteristics, meat quality, and lipid oxidation in rabbits under high ambient temperature. Rabbits in one group were raised at 20°C and fed with basal diet (N) and other three groups (33°C) were fed basal diet with 0 (C), 5 (CT5), and 10 g (CT10) of CT/kg of diet. Compared with the C group, rabbits in CT10 had higher pH(24) and lower cooking loss and thiobarbituric acid reacting substance values at 0, 30, and 60 min of forced oxidation. Rabbits in C group had higher cortisol levels, creatine kinase activities, white blood cell counts, neutrophil percentage, neutrophil:lymphocyte ratio and lower T(3), T(4) levels, lymphocyte percentage than N and CT10 groups. Supplementation of CT seemed to have a positive effect on growth performance, welfare, and meat quality of rabbits under high ambient temperature.

Assessment of melamine contamination in crop, soil and water in China and risks of melamine accumulation in animal tissues and products.

The scandal of melamine-adulterated infant formula in China in September 2008 demanded the need to assess the extent of melamine contamination in the environment and food products and possible risks of consuming melamine-contaminated diets. In this work, our extensive work tested water, soil and crop samples from 21 provinces in China. Soils nearby and waste waters from melamine-manufacturing factories were examined, and the highest melamine concentrations in waste water and soil samples were 226.766 and 41.136 mg/kg, respectively. Six of 94 irrigation water samples had melamine at a concentration of 21-198 microg/L. Only 1 sample collected from 124 farmlands farther than 150 km from melamine factories was detected for melamine at a content of 176 microg/L. Only 3 out of 557 crop samples contaminated more than 1mg/kg melamine, with the highest level of 2.05 mg/kg in a wheat sample. When basal diets contained 2mg/kg melamine were fed to various animals, deposition of melamine in animal tissues and products was all lower than 122 microg/kg. The melamine deposition was much higher (e.g., 4483 microg/kg in the kidney of chicken) when diets contained 100 mg/kg melamine but was found to be completely depleted after 96 h for all animals after switching to the basal diets. Our work may be valuable to regulate melamine production and monitor the safety of food and animal products.