Abstract Rice protein has anti-cancer and other health care functions, and its deep development and utilization have been receiving increasing attention. As one of the deep-processed products, antioxidant peptide has strong functionality and high safety, which can extend the rice processing industry chain. In this paper, we used β-cyclodextrin to de-bitter rice protein antioxidant peptides, and determined the optimal process conditions for de-bittering rice protein antioxidant peptides with β-cyclodextrin.
.jpeg)
China is the world's largest rice-growing country. In 2014, China produced 206 million tons of rice, accounting for 41.7% of the world's rice resources. Rice protein not only has nutritional values such as a reasonable amino acid ratio, hypoallergenicity, and a biomass-to-protein efficiency ratio comparable to that of animal proteins, but also has health functions such as anticancer. The deep development and utilization of rice protein has received increasing attention [1-5].
Bioactive peptides with hormone regulation, immune system regulation, antioxidant and other functions are important products in the deep processing of rice protein, and antioxidant peptides, as one of them, have strong functionality and high safety. As one of the peptides, antioxidant peptide has strong functionality and high safety. Enzymatic digestion is the first choice for the production of active peptides because of the mild production conditions, easy control of hydrolysis, production of specific products, and relatively low cost[6] . The preparation of rice protein antioxidant peptides by enzymatic hydrolysis can well extend the rice processing industry chain.
In the preliminary study of this paper, rice protein powder was used as the reaction substrate, and three kinds of proteases, papain, neutral protease and alkaline protease, were used as the tool enzymes. The scavenging rate of the enzyme solution on DPPH radicals and the degree of hydrolysis were used as the indexes, and the enzyme pH, enzyme temperature, enzyme dosage, and substrate concentration were selected for the one-way experiments, and the data were analyzed to determine the optimal enzymes, and then the optimal enzymatic process was optimized by the Box- Behnken Response Surface Design (RSD) method. The Box- Behnken response surface design method was used to optimize the enzymatic process.
Enzymatic digestion of proteins produces peptides containing hydrophobic amino acid residues, resulting in different bitter flavors. In order to obtain peptides with good nutrition and flavor, selective isolation, embedding and masking, enzymes, and microbial fermentation are often used to reduce the bitter taste of peptides. Compared with selective separation and enzymatic methods, the embedding and masking method will not affect the functionality of peptides, and the bitter amino acids can be embedded by the embedding agent to achieve the effect of de-bitterness, and the commonly used embedding agents include monosodium glutamate, gelatin, glycine, cyclic dextrin, etc. β-cyclic dextrin is a kind of oligosaccharides with the enzyme-like hydrophobic bonding portion, which is structurally stable, and its non-toxic and low-cost properties make it an excellent embedding agent. β-cyclodextrin is a kind of oligosaccharide with hydrophobic binding part like enzyme, and its structure stability, non-toxicity and low cost make it an excellent embedding agent.
In this paper, β-cyclodextrin was used to debitter rice protein antioxidant peptides. Based on the results of the one-way experiments on the addition of β-cyclodextrin, temperature, pH and time, and further optimized by orthogonal experiments, the optimal process conditions for the debittering of rice protein antioxidant peptides by β-cyclodextrin were determined by the debittering scores (in percent).
1 Experimental materials and equipment
1.1 Materials
Rice protein (food grade): Hunan Runtao Bio-technology Co., Ltd (protein ≥80%, fiber ≤6%, carbohydrate ≤10%, fat ≤2%, ash ≤2%);
Papain: Nanning Dongheng Huadao Biotechnology Co;
β-Cyclodextrin: Mengzhou Huaxing Biochemical Co.
1.2 Equipment
Water baths; spray dryers; centrifuges .
2 Experimental Methods
2.1 Preparation of rice protein antioxidant peptides
Weigh a certain amount of rice protein, with a certain amount of distilled water to form a substrate mass concentration of 5 g /100 mL of protein solution, adjust the pH value of 5.25, according to the amount of enzyme 5.6 U/mL to add papain, placed in a constant temperature water bath 61.4 ℃ enzyme digestion for 1 h, after the end of the boiling water bath to destroy the enzyme for 10 min, and then centrifuged at 3,500 r/min for 10 minutes, the supernatant was extracted. The supernatant was spray dried to obtain peptide powder.
2.2 Evaluation methods for bitter taste
Sensory evaluation and instrumental evaluation are the two main methods to evaluate the bitterness of protein hydrolysis products. Although sensory evaluation is simple, it is easy to produce large differences due to subjective factors. Instrumental evaluation is the future development trend, but it is limited by technology, and new methods are needed to evaluate the bitterness of protein hydrolysates. At present, sensory analysis is still used as the main bitter taste evaluation method, and instrumental analysis is used as an auxiliary evaluation method. In this study, the sensory analysis method was chosen to evaluate the bitterness of the protein hydrolysis products.
2.3 Determination of Bitterness Evaluation Criteria
The concentration of rice protein antioxidant peptide was artificially divided into 10 grades, corresponding to 10 scores, and the bitterness value of rice protein antioxidant peptide with a concentration of 10.0% was set at 10, and so on, with a total of 10 standard bitterness values, as shown in Table 1. Take 2-3 mL of the solution to be evaluated and put it into the mouth for full tasting, and then compare it with the standard bitter taste value solution, and repeat the tasting until the bitter taste value is determined. The final bitterness value was taken from the average of more than 5 people.
Table 1 Bitterness evaluation criteria
2.4 One-factor experiments on β-cyclodextrin desiccated rice protein antioxidant peptides
2.4.1 Effect of β-cyclodextrin addition on the debittering effect of rice protein antioxidant peptides
Five 50 mL beakers were taken and 1.0 g of accurately weighed rice protein antioxidant peptide was put into each beaker. 9.0 mL of distilled water was added and the pH was adjusted to 6.0, and 0.1 g, 0.2 g, 0.3 g, 0.4 g, and 0.5 g of β-cyclodextrin was added respectively, and the reaction was put into a 40℃ water bath for 60 min. cyclic dextrin on the de-bittering effect of rice protein antioxidant peptides. 2.4.2 Effect of temperature on the de-bittering effect of rice protein antioxidant peptides
Five 50 mL beakers were taken, and 1.0 g of rice protein antioxidant peptide and 0.3 g of β-cyclodextrin were put into each beaker, and 9.0 mL of distilled water was added to each beaker respectively, and the pH was adjusted to 6.0, and the reaction was carried out for 60 min in a water bath with the temperatures of 20 ℃, 30 ℃, 40 ℃, 50 ℃, and 60 ℃ respectively, and the effect of the temperatures on the bitterness of rice protein antioxidant peptide was investigated according to the bitter taste evaluation criteria in Table 1. The effect of temperature on the de-bittering effect of rice protein antioxidant peptide was investigated according to the bitter taste evaluation criteria in Table 1.
2.4.3 Effect of pH on the de-bittering effect of rice protein antioxidant peptides
Five 50 mL beakers were taken and 1.0 g of rice protein antioxidant peptide and 0.3 g of β-cyclodextrin were put into each beaker, and 9.0 mL of distilled water was added into each beaker, and the pH was adjusted to 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, and then the reaction was carried out at a constant temperature for 60 min in a water bath at 40°C. The effect of pH on the de-bittering effect of the rice protein antioxidant peptide was investigated in accordance with the evaluation criteria for the bitterness in Table 1. The effect of pH on the de-bittering effect of rice protein antioxidant peptides was investigated according to the bitter taste evaluation criteria in Table 1.
2.4.4 Effect of time on the effect of rice protein antioxidant peptides on the desiccation of bitterness
Five 50 mL beakers were taken, and 1.0 g of rice protein antioxidant peptide and 0.3 g of β-cyclodextrin were put into each beaker, and 9.0 mL of distilled water was added into each beaker respectively to adjust the pH to 6.0, and then the reaction was carried out at a constant temperature of 40 ℃ in a water bath for 10 min, 30 min, 60 min, 90 min, and 120 min, respectively, and then the effects of time on the bitterness removal effect of the rice protein antioxidant peptide were investigated according to the bitter taste evaluation criteria in Table 1. The effect of time on the de-bittering effect of rice protein antioxidant peptide was investigated according to the bitter taste evaluation criteria in Table 1.
2.4.5 Determination of optimal conditions for β-cyclodextrin debittering of antioxidant peptides from rice proteins
Based on the one-factor experiments on the de-bittering of rice protein antioxidant peptides, an orthogonal experimental table was designed to determine the optimal process for de-bittering rice protein antioxidant peptides with β-cyclodextrin using four factors: β-cyclodextrin additive amount, temperature, pH, and time, and the de-bittering scores were determined by the percentage of the de-bittering effect.
3 Experimental results and analysis
3.1 One-way experimental results of β-cyclodextrin desiccated rice protein antioxidant peptides
3.1.1 Effect of β-cyclodextrin addition on rice protein antioxidant peptide degradation and degradation of rice protein antioxidants
Impact of bitter effects
As shown in Figure 1, the bitter value of rice protein antioxidant peptides decreased with the addition of β-cyclodextrin in the range of 0.1-0.3 g. The bitter value was lowest when the amount of β-cyclodextrin was 0.3 g, and increased when the amount of β-cyclodextrin was 0.3 g. The bitter value of the rice protein antioxidant peptides decreased with the addition of β-cyclodextrin.
After adding a certain amount of β-cyclodextrin, it will produce a burnt taste, and the burnt taste will be aggravated with the increase of the added amount. Therefore, 0.25~0.35 g was selected as a more suitable range of β-cyclodextrin addition to carry out subsequent experiments.
Fig. 1 Effect of β-cyclodextrin addition on antioxidant peptide removal from rice proteins
Impact of bitter effects
3.1.2 Effect of temperature on the de-bittering effect of rice protein antioxidant peptides
From Fig. 2, it can be seen that the bitterness value of rice protein antioxidant peptide decreases with the increase of temperature at the beginning, and the bitterness value decreases to the lowest when the temperature is 40℃, and then the bitterness value rises with the increase of temperature after 40℃. This may be related to the energy absorbed by β-cyclodextrin, and when too much β-cyclodextrin is added, the energy absorbed will be too much, which will lead to changes in the spatial structure of the molecules, and reduce the embedding capacity. Therefore, 30~50℃ was chosen as the more suitable reaction temperature range for β-cyclodextrin to carry out the subsequent experiments.
Fig. 2 Effect of temperature on the desiccation effect of rice protein antioxidant peptides
3.1.3 Effect of pH on the de-bittering effect of rice protein antioxidant peptides
From Figure 3, it can be seen that the bitterness value of rice protein antioxidant peptides decreased with the increase of pH from pH 4 to 6, and the bitterness showed the lowest value at pH 6.0, but the bitterness value rebounded when pH>6.0 and had a tendency to increase with the increase of pH. This may be due to the fact that the microstructure of β-cyclodextrin is affected by pH, the spatial structure of the molecule is changed, which reduces the efficiency and selectivity of bitterness removal, thus affecting its embedding ability. Therefore, the pH value of 5.5-6.5 was chosen as the more suitable pH range for β-cyclodextrin to carry out the subsequent experiments.
Fig. 3 Effect of pH on the de-bittering effect of rice protein antioxidant peptides
3.1.4 Effect of time on the effect of rice protein antioxidant peptides on the desiccation of bitterness
From Fig. 4, it can be seen that the bitterness value of rice protein antioxidant peptide did not change significantly with the time of debittering, especially in the range of 60-100 min, which was almost unchanged, and then the bitterness value increased with the increase of debittering time, which may be attributed to the change of the microstructure of β-cyclodextrin molecule due to the prolonged debittering time, which may affect the debittering ability of the β-cyclodextrin. Therefore, 50-70 min was chosen as a suitable reaction time range for β-cyclodextrin to carry out the subsequent experiments.
3.2 Determination of optimal conditions for β-cyclodextrin debitterization of rice protein antioxidant peptides
3.2.1 Orthogonal experimental factor level design
Based on the results of one-way experiments on the de-bittering of rice protein antioxidant peptides by β-cyclodextrin, and the effects of β-cyclodextrin addition, temperature, pH and time on the de-bittering of rice protein antioxidant peptides, we designed a four-factor, three-level experiment according to the orthogonal experimental table shown in Table 2 (L9(34)), to determine the optimal process for the de-bittering of rice protein antioxidant peptides by β-cyclodextrin.
Table 2 Antioxidant peptides of β-cyclodextrin desiccated rice protein L9 (34)
3.2.2 Experimental results and analysis
Through orthogonal experiments, 10 food students (5 males and 5 females) conducted sensory evaluation of the products to obtain the comprehensive average sensory scores (in order to facilitate the calculation, on the basis of the design of Table 1, it was expanded into a percentage system, i.e. the bitter value of rice protein antioxidant peptide concentration of 10.0% was set as 100, and so on, with a total of 10 standardized bitter values), and the results were as shown in Table 3. Through the polar analysis, it was concluded that the influence of each factor on the experimental results in the order of C→D→B→A, i.e., the factor that has the greatest influence on the effect of rice protein antioxidant peptide de-bitterness is pH, followed by time, temperature, and β-cyclodextrin addition has the smallest influence; In addition, it can be seen from the analysis that the optimal combination of formula factors for the de-bitternization of rice protein antioxidant peptide by β-cyclodextrin was A2B1C2D3, i.e., when the amount of β-cyclodextrin addition was 100, there were 10 standard bitter values, and the results were as shown in Table 3. The optimal combination of formulation factors was A2B1C2D3, i.e., when the addition of β-cyclodextrin was 0.30 g of rice protein antioxidant peptide per gram, and the temperature was controlled at 30 ℃, pH 6.0, and the constant temperature was 70 min, the experimental effect was optimal.
4 Conclusion
(1) A one-way experiment was carried out by selecting the amount of β-cyclodextrin added, temperature, pH and time, and after analyzing the experimental data, a range of β-cyclodextrin addition of 0.25-0.35 g, a range of reaction temperature of 30-50°C, a range of reaction pH of 5.5-6.5, and a range of reaction time of 50-70 min were selected to carry out the subsequent experiments.
(2) On the basis of the one-way experiment, a four-factor, three-level L9(34) orthogonal experiment was designed to optimize the process conditions of rice protein antioxidant peptide debittering: 0.30 g of β-cyclodextrin was added to each gram of rice protein antioxidant peptide, the reaction temperature was 30 ℃, the pH was 6.0, and the reaction time was 70 min.
References:
[1] ZHOU Hui, ZENG Xiaohong .Review of domestic and international rice markets in 2014 and outlook for 2015[J]. Agricultural Outlook, 2015, (2): 4-8.
[2] CHEN Datu, LI Rui, HU Guanbo, et al. Nutritional value and utilization of rice protein[J]. China Feed, 2013, (10): 37-40.
[3] Pan Minyao . Development of rice protein concentrate and protein modification [D]. Hangzhou: Zhejiang University, 2006.
[4] GUO Yan, ZENG Li . Research Progress on the Nutritional Value and Extraction and Separation Methods of Rice Protein[J]. Food and Fermentation Science and Technology, 2010, 46(1):31-34.
[5] WANG Zhangcun , SHEN Ruiling , YAO Huiyuan . Progress of rice protein research [J]. China Journal of Cereals and Oils , 2004, 19(2):11-15.
[6] Li Ping. Research on Soybean Peptide Debitterization and Its Physiological Functions [D]. Jinan: Jinan University, 2014.
没有评论:
发表评论