| Cottonseed meal (CSM) is the world’s leading plant protein resource. However, the application of CSM in feed industry is limited due to the gossypol and other anti-nutrients, as well as a relatively low protein digestibility. Recently, several detoxification procedures have been developed including the solid-state fermentation. However, studies for improving the protein quality of CSM are still scarce, particularly those involving the evaluation of functional properties of the cottonseed peptides (CPs). In the present study, a Bacillus subtilis strain was screened out for the solid-state fermentation of CSM. Then, one protease was selected as fermentation adjuvant to further improve the quality of CSM. The CPs were extracted from treated-CSM and its antioxidant activity was evaluated in vitro. The stability of CPs was accessed after a simulated gastrointestinal digestion. Then, two antioxidant peptides from CPs were successfully isolated, purified and their amino acid sequences were identified. Furthermore, the protective effects of CPs against hydrogen peroxide (H2O2)-induced oxidative damage in murine macrophages (RAW264.7), as well as the effects on oxidative status of cholesterol-fed rats were accessed. The findings are as follows:1. Improvement of the nutritional quality of CSM by Bacillus subtilis and the addition of papainBased on the results of free gossypol detoxification and degree of protein hydrolysis, seven Bacillus stains from our laboratory were screened. The fermentation conditions including the inoculum size, fermentation time and temperature were optimized using the response surface method. Five different kinds of proteases including the acid protease, neutral protease, alkaline protease, flavor protease and papain were also screened for CSM fermentation. The nutrient composition, protein degradation, in vitro digestibility and antioxidant activity of CSM treated with Bacillus and papain were determined. The results showed that:(1) B. subtilis BJ-1 could reduce (p<0.05) the free gossypol content and improve (p<0.05) the degree of protein hydrolysis (DH) among the seven candidate strains.(2) The optimal fermentation conditions by B. subtilis BJ-1were as follows:inoculum size2%, fermentation time48h, and fermentation temperature of37℃. Under these conditions, the detoxification rate of free gossypol was up to66.74%.(3) The addition of0.1%(w/v) papain further increased (p<0.05) the DH, but had no significant effect on the content of free gossypol.(4) Compared with the untreated meal, the contents of crude protein and amino acids increased (p<0.05) in CSM treated with B. subtilis BJ-1and papain, whereas the amount of crude fiber, crude fat and free gossypol were decreased (p<0.05).(5) Co-treatment by bacteria and enzyme also effectively degraded the9S and5S subunits of cottonseed protein, increased (p<0.05) the in vitro protein digestibility and1,1-diphenyl-2-picryl hydrazine (DPPH) free radical scavenging activity. The results suggest that the B. subtilis BJ-1fermentation with papain supplementation may offer an effective strategy to improve the quality of CSM.2. Preparation and antioxidant properties of CPsTo further access the influence of fermentation with papain addition on the antioxidant activity of CSM, the CPs with molecular weight below3kD were extracted from CSM samples after B, subtilis BJ-1fermentation with papain supplementation and its chemical composition and molecular weight distribution were determined. CPs were further detected for their in vitro antioxidant activity including DPPH radical scavenging activity, hydroxyl radical scavenging activity, metal chelating ability and reducing power. The protein degradation, molecular weight distribution and antioxidant function of CPs were also measured after a simulated gastrointestinal digestion. The results showed that:(1) the in vitro antioxidant activity of CPs was closely related to its dosage and the fermentation time of B. subtilis BJ-1. The lowest half effect concentrations of DPPH radical, hydroxyl radical scavenging activity, metal chelating ability and reducing power were2.15,2.93,2.06, and2.18mg/mL, respectively, after a48-h treatment.(2) Amino acid analysis showed that CPs contained a large amount of glutamic acid, aspartic acid and arginine. The total hydrophobic amino acids were up to28.31%. The components of molecular weight less than1kDa accounted for56.52%of the total protein content.(3) The simulated gastrointestinal digestion increased (p<0.05) the DH as well as further reduced molecular weight distribution of CPs.(4) In vitro digestion decreased (p<0.05) DPPH radical scavenging activity and total reducing power by40.3%and7.69%, respectively. However, the digestion increased (p<0.05) hydroxyl radicals scavenging activity and metal chelating ability by36.64%and17.62%, respectively. The results suggest that the CPs may be used as a natural antioxidant and its antioxidant activity could be maintained after the digestion with pepsin and pancreatin.3. Purification and identification of antioxidant peptides in CPsThe antioxidant peptides in CPs were isolated, purified by gel filtration chromatography and preparative liquid chromatography. Their amino acid sequences were further identified by using the matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS). The results showed that (1) the CPs were fractionated into four fractions (A-D) by gel filtration on Sephadex G-15column.(2) After investigated by in vitro methods, the fraction A demonstrated the highest DPPH scavenging activity and it can be further divided into main components (A-1-A-9) by the preparative high performance liquid chromatography. The fractions with the strongest antioxidant activity were named A-8and A-9.(3) The amino acid sequence of peptide A-8is STFAPSGSA, with a molecular weight of823.37Da. The amino acid sequence of peptide A-9is GAAANDRDNPVRDDGPGG, with a relative molecular mass of1752.78Da.4. Effects of CPs on the antioxidant function of RAW264.7cells and Sprague-Dawley (SD) ratsTo determine the protective effect of CPs on oxidative damage RAW264.7cells and its possible mechanism, RAW264.7cell culture was added with H2O2to a final concentration of600μM to induce oxidative stress. The effects of CPs and its A-8fraction (STFAPSGSA) on the cell viability, genomic DNA protection, intracellular antioxidant enzyme activity, and p53gene expression were measured. On the other hand, in order to study the impact of CPs on oxidative status of cholesterol-fed rats,18male SD rats (108.47±5.42g) were divided into three groups. The control rats were fed a basal diet and orally administrated physiological saline (1mL/d). Rats in the high-fat group and CPs-treated group were fed high-fat diets and oral administrated saline or2%CPs (1mL/d). The experiment lasted for14d. The results showed that:(1) CPs and purified peptide A-8could protect cell genomic DNA from in vitro oxidant damadge.(2) Pre-cultured cells with0.5and2.0mg/mL of CPs or50μg/mL of A-8reduced (p<0.05) the inhibition of RAW264.7cell viability.(3)2.0mg/mL of CPs and50μg/mL A-8components improved (p<0.05) catalase activity and superoxide dismutase activity.0.5,2.0mg/mL of CPs and50μg/mL of A-8fraction increased (p<0.05) intracellular activity of glutathione peroxidase (GSH-Px).(4) CPs (0.5mg/mL) and50μg/mL of A-8component reduced (p<0.05) the level of malondialdehyde (MDA).(5) Additionally, adding0.5or2.0mg/mL of CPs and50μg/mL of A-8purified fraction decreased (p<0.05) the p53gene expression.(6) In vivo tests showed that rats treated with2%CPs (1mL/d) had an increased (p<0.05) GSH-Px activity in serum and liver, as well as a lower (p<0.05) MDA and NO content in serum. These results suggest that the CPs could improve the antioxidant functions in both RAW264.7cells and rats under oxidant stress, and the protection may be related to the increase of antioxidant enzymes. |
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