技术优化制备花生抗氧化肽的枯草芽孢杆菌固态发酵方法

Technology optimization on preparation of peanut antioxidant peptidesby bacillus subtilis solid state fermentation methodYu Lina 1, a*, Xu Tingting 1, b , Zhang Chushu 1, c , Sun Jie 1, d*, and Bi Jie 1, e1Shandong Peanut Research Institute, Qingdao 266100, China a lhtyln0626@ (Corresponding author), b 1940372672@, c zcs.2003@, d sj605@, e bj.baby@Keywords: Technology optimization, Peanut antioxidant peptides, Bacillus subtilis, Solid state fermentationAbstract. The purpose of this paper is to investigate the preparation of peanut antioxidant peptides by Bacillus subtilis solid state fermentation method and to provide a theoretical basis for further investigating deep processing products of fermentation peanut meal. The preparation technics for peanut antioxidant peptides optimized with the soluble nitrogen concentration of peptides, 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) and hydroxyl free radicals scavenging rates of fermentation broth as the indexes of investigation including the cell age was 20h, bacterial suspension volume was 3.0mL, fermentation temperature was 40°C, nutrient salt solution was 15mL, the fermentation time was 42h, water bath temperature was 45°C, and water bath time was 3h. The research shows that IC 50 values for peptides scavenging of DPPH, hydroxyl and superoxide anion free radicals, iron and copper ion chelating rates, lipid peroxidation inhibiting rate, iron and molybdenum reducing activity were 3.48mg/mL, 6.24mg/mL, 2.06mg/mL, 0.54mg/mL, 1.19mg/mL, 4.56mg/mL, 9.58mg/mL and2.17mg/mL, respectively.IntroductionPeanut is rich in protein of 25%-36% and it is a good dietary protein resource. However, protein denaturation will be found after thermal extracting oil. Therefore, high temperature peanut meal has low utilization rate and it is usually used as feed and fertilizer [1]. At present, enzyme hydrolysis protein to prepare peptides is a good method to use high temperature peanut meal. Peanut antioxidant peptides, ACE inhibitory peptides, α-glucosaccharase inhibitory peptides can be obtained from peanut protein hydrolysates [2-5]. In this paper, one more effective method of bacillus subtilis solid state fermentation method was introduced in order to prepare peanut antioxidant peptides. Many kinds enzyme system including protease, cellulose, β-amylase, lipase would participate in fermentation process. So, high quality, purity and activity peanut antioxidant peptides can be obtained by using solid state fermentation method, but not protease hydrolysis method [6, 7].Materials and MethodsMaterials and Chemicals. High temperature peanut meal was supplied by Shandong Luhua Group Co., Ltd. Bacillus subtilis (CICC10071) was purchased from China Center of Industrial Culture Collection. LB broth was purchased from Beijing Land Bridge Technology Co., Ltd. High temperature peanut meal was treated at 121°C sterilization for 20min.Preparation of Bacillus Subtilis Seed. The 2 ring vaccination activation of bacillus subtilis was added to 50ml LB broth and it was cultured under the condition of 30°C. When bacillus subtilis growth was at logarithmic phase, bacteria number was about 108/mL, it can be used to solid state fermentation experiment.Technology of Bacillus Subtilis Solid State Fermentation Peanut Meal. Bacillus subtilis seed, nutrient salt solution and high temperature peanut meal were mixed and placed in an incubator to solid state fermentation for a certain time. Distilled water would be added to the above mixture after Applied Mechanics and Materials Submitted:2015-11-04ISSN: 1662-7482, Vol. 835, pp 103-108Revised:2015-12-24doi:10.4028//AMM.835.103Accepted:2016-01-27© 2016 Trans Tech Publications, Switzerland Online:2016-05-11fermentation. Then the new mixture was treated in water bath for a certain time. The fermentation broth was obtained from the supernatant after the new mixture centrifugation. The fermentation broth would be ultrafiltered two times by using ultrafiltration membrane of 10kDa and 5kDa. In the end, the permeate solution of 5kDa ultrafiltration membrane was collected as antioxidant peptides and was freezed drying.Determination of Soluble Nitrogen Concentration (Lowery Method) [8].1.0mL sample solution was added to 5ml reagent A and stand reaction time 10 min at room temperature after mixing. The mixture was added to 0.5ml Folin- phenol reagent and reacted at 30°C water bath for 30min after mixing. In the end, soluble nitrogen concentration is determined by the absorption value at 750nm.Analysis Methods. All of these antioxidant activities DPPH, hydroxyl and superoxide anion free radicals scavenging rates, iron and copper ion chelating rates, lipid peroxidation inhibiting rate, iron and molybdenum reducing activity were determined in accordance with the Yu et al [2] method. Results and AnalysisEffect of Cell Age on the Fermentation Result. Effect of cell age on the fermentation result was shown in Fig. 1. In the range of 12-32h cell age, soluble nitrogen concentration increased at first and then decreased, the maximum value was observed at 20h; DPPH free radical scavenging rate overall showed a trend of decrease after the first increase, the maximum value presented at 24h; hydroxyl free radical scavenging rate presented decreased first, increased then and constant trend, the minimum value was at 20h. Bacillus subtilis logarithmic phase was at 12-32h. In the range of 12-20h, as the cell age time extended, bacillus subtilis grew faster so that the enzyme quantity increased and hydrolysis efficiency sped up and soluble nitrogen concentration increased the maximum value. When cell age was more than 20h, the growth speed of bacillus subtilis further speed up. The amount of protein to growth increased and protein to hydrolysis decreased result in decreasing the soluble nitrogen concentration. DPPH and hydroxyl free radical scavenging rates changed with the change of soluble nitrogen. So,the optimum cell age was 20h.Fig. 1 Effect of cell age on the fermentation result.Effect of Bacterial Suspension Volume on the Fermentation Result. Fig. 2 showed effect of bacterial suspension volume on the fermentation result. In the range of 1-6mL bacterial suspension volume, soluble nitrogen concentration increased at first and then decreased, the maximum value was observed at 2, 3mL; DPPH free radical scavenging rate showed a trend of increase after the first decrease, the minimum value presented at 2mL; hydroxyl free radical scavenging rate presented overall upward trend, the minimum value was at 6mL. When bacterial suspension volume was in the range of 1-3mL, quantity of bacillus subtilis increased and bacteria growth sped up to cause enzyme and soluble nitrogen concentration increased. When bacterial suspension volume was more than 3mL, quantity of bacillus subtilis in fermentation system was too much to cause bacteria growth restrained.So,the optimum bacterial suspension volume was 3mL.Fig. 2 Effect of bacterial suspension volume on the fermentation result.Effect of Fermentation Temperature on the Fermentation Result. Fig. 3 showed effect of fermentation temperature on the fermentation result. In the range of 25-50°C fermentation temperature, soluble nitrogen concentration showed the trend of decreased, increased and decreased, the maximum value was observed at 40°C; DPPH free radical scavenging rate showed a trend of gradual increase, the maximum value presented at 50°C; hydroxyl free radical scavenging rate presented overall reduce trend, the maximum value was at 25°C. Temperature is one of the important factors that affect the growth of bacillus subtilis. With the increase of fermentation temperature, the growth sped up and quantity of enzyme increased result in obtaining high hydrolysis efficiency and the maximum soluble nitrogen concentration. When the fermentation temperature continued to rise, high temperature inhibited the growth of bacillus subtilis. Growth rate declined to cause quantity of enzyme and soluble nitrogen concentration decreased. So,the optimum fermentation temperature was 40°C.Fig. 3 Effect of fermentation temperature on the fermentation result.Effect of Nutrient Salt Solution Volume on the Fermentation Result. Fig. 4 showed effect of nutrient salt solution volume on the fermentation result. In the range of 10-35mL nutrient salt solution volume, soluble nitrogen concentration showed the trend of increased and decreased, the maximum value was observed at 15mL and 20mL; DPPH free radical scavenging rate showed a trend of gradual decreased, the maximum value presented at 10mL; hydroxyl free radical scavenging rate presented first increased and then decreased trend, the maximum value was at 25mL. In the range of 10-20mL, the gradually increased nutrient salt solution volume was beneficial to the growth of bacillus subtilis to cause the quantity of enzyme and soluble nitrogen concentration increased. When nutrient salt solution volume was more than 20mL, too much solution exceeded the demand of bacillus subtilis and the growth of bacillus subtilis flattened out. In addition, increasing nutrient salt diluted the enzyme in the fermentation system result in enzyme and substrate reducing chance to contact and making enzymatic hydrolysis efficiency lower. Then soluble nitrogen concentration decreased. So,the optimum nutrient salt solution volume was 15mL.Fig. 4 Effect of nutrient salt solution volume on the fermentation result.Effect of Fermentation Time on the Fermentation Result. Fig. 5 showed effect of fermentation time on the fermentation result. In the range of 12-42h fermentation time, soluble nitrogen concentration showed the trend of increased, the maximum value was observed at 42h; DPPH free radical scavenging rate showed a trend of first increased then decreased, the maximum value presented at 30h; hydroxyl free radical scavenging rates presented small changes in different fermentation periods. In the range of 12-30h, both the growth of bacillus subtilis and the amount of enzyme increased slowly in the fermentation system to cause soluble nitrogen concentration increased slowly. When fermentation time was more than 30h, bacillus subtilis growth rate increased rapidly. Then the amount of enzyme and soluble nitrogen concentration increased rapidly. So,the optimum fermentation time was 42h.Fig. 5 Effect of fermentation time on the fermentation result.Effect of Water Bath Temperature on the Fermentation Result. Fig. 6 showed effect of water bath temperature on the fermentation result. In the range of 25-50°C water bath temperature, soluble nitrogen concentration showed the trend of first increased and then decreased, the maximum value was observed at 45°C; DPPH free radical scavenging rate showed a trend of decreased, the maximum value presented at 25°C; hydroxyl free radical scavenging rates presented the tendency of increase with the decrease of the first, the minimum value was observed at 35°C. In the range of 25-40°C, enzyme system in the fermentation system continued to hydrolysate protein. Both the digestion rate and soluble nitrogen concentration increased slowly. When the temperature of the water bath was more than 40°C, digestion rate was accelerated and soluble nitrogen concentration increased. So,the optimum water bath temperature was 45°C.Fig. 6 Effect of water bath temperature on the fermentation result.Effect of Water bath Time on the Fermentation Result. Fig. 7 showed effect of water bath time on the fermentation result. In the range of 1-6h water bath time, soluble nitrogen concentration showed the trend of first increased and then decreased, the maximum value was observed at 3h; DPPH free radical scavenging rate showed a trend of decreased, the maximum value presented at 1h; hydroxyl free radical scavenging rates presented the tendency of decrease with the increase of the first, the maximum value was observed at 3h. In the range of 1-3h, enzymolysis reaction increased and soluble nitrogen concentration increased. When water bath time was more than 3h, the amount of enzyme in the fermentation system gradually reduced result in soluble nitrogen concentration decreased. So, the optimum water bath time was 3h.Fig.7 Effect of water bath time on the fermentation result.Results of Antioxidant Activities of Peanut Antioxidant Peptides. Antioxidant activities of peanut antioxidant peptides were determined and results was seen in Table 1. Peanut antioxidant peptides had better antioxidant activities.Table 1. Results of antioxidant activities.Regression equation R 2 IC 50 [mg/mL] DPPH free radical scavenging ratey = -0.8228x 2 + 17.116x + 0.4392 0.9961 3.48 Hydroxyl free radical scavenging ratey = -0.3053x 2 + 7.3445x + 16.058 0.9945 6.24 Superoxide anion free radical scavenging ratey = -0.549x 2 + 9.7286x + 32.278 0.9971 2.06 Lipid peroxidation inhibiting ratey = -1.092x 2 + 17.219x - 5.7877 0.9923 4.56 Iron reducing activityy = -0.0031x 2 + 0.0816x + 0.0027 0.9990 9.58 Molybdenum reducing activityy = -0.0114x 2 + 0.2594x - 0.0088 0.9961 2.17 Iron ion chelating ratey = -0.7159x 2 + 11.541x + 43.926 0.9933 0.54 Copper ion chelating rate y = -2.7671x 2 + 38.633x + 7.9102 0.99071.19SummaryIn this paper, the technology optimization on preparation of peanut antioxidant peptides by bacillus subtilis solid state fermentation method was investigated. Peanut antioxidant peptides obtained from ultrafiltration separation had better antioxidant activities. Therefore, these results indicate that peanut antioxidant peptides is valuable for exploiting new hygienical food.AcknowledgmentsThis work was supported by the Shandong Key Research and Development Program (2015GGX108006), the Qingdao People's Livelihood Science and Technology Project (14-2-3-65-nsh), and the Shandong Independent Innovation and Achievement Transformation special plan (2014CGZH0709), Youth Scientific Research Fund Project of Shandong Academy of Agricultural Sciences, Young Talents Training Program of Shandong Academy of Agricultural Sciences.References[1] Q.Q. Ming, L.N. Yu, Q.L. Yang, W.Q. Yang, J. Sun, F. Zhu, J. Bi and C.S. Zhang, Preparation and antioxidant activities of peanut (Arachin conarachin L.) protein peptides by Aspergillus niger solid state fermentation method, Food Sci. Technol. 2 (2014) 17-22.[2] L.N. Yu, J. Sun, S.F. Liu, J. Bi, C.S. Zhang and Q.L. 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