Research on key technology of rice protein active peptide preparation

 Abstract: The experimental research is mainly based on the scraps of rice dregs left after the sugar production of broken rice saccharification as raw materials, in accordance with the advantages of high temperature denaturation of rice dregs protein, the full use of ultrasonic technology to modify the rice dregs protein, through the ultrasonic effect to make the tight protein molecules appear loose, and then the use of multi-enzymatic compound hydrolysis of rice dregs protein, control the hydrolysis conditions, to get the optimal hydrolysis degree. The hydrolyzed products were separated by membrane method, and the hydrolyzed products of rice residue protein were intercepted according to the molecular distribution level of active peptide, and then the physicochemical properties and nutritional value of the hydrolyzed products were evaluated, so as to design suitable product formulations and improve the market application value of active peptide of rice protein.

 

1 Significance of the preparation of rice protein active peptides

Protein is a nutrient with rich value, but protein is also a kind of biomolecule, so protein also has the function to change the protein composition of food. Therefore, in China's ecological botany protein development research, is trying to the protein structure of functional modification, in order to further broaden the market application of plant proteins, accompanied by the rapid development of enzyme industrial technology, with unique functions of peptide food is entering into a rapid development of a new era [1].

 

China is a rice grain production country, has a very rich rice protein resources, and through the rice protein research and technology development, can be conducive to the deep processing and comprehensive utilization of rice, thus improving the economic benefits of the rice market, but also for the development of China's food industry, for the nutritional health care of the people of our country to provide more protein production base material and protein supplements, therefore, the deep processing of rice protein has a very broad market development prospects [2]. The deep processing of rice protein has a very broad market development prospects [2].

 

The main content of this research is about the preparation of rice protein active peptide, belongs to the utilization of rice residue comprehensive research, can make rice protein resources to obtain a more scientific and reasonable development and utilization, but also solves the problem of high quality protein in China's food industry and food processing industry in the source problem and the demand for the problem, greatly enhance the level of cereal protein in the field of food production in the development and application of the level of food production, enhance the Rice protein bioeffectiveness and value, improve the added value of rice protein food, can make China's rice deep processing technology of science and technology content greatly improved, thus promoting the rapid development of rice protein food industry, promote the construction of the rural economy, and increase the income of farmers, which is also the study of this paper on the preparation of active peptides of rice protein is of great practical significance. Through the use of broken rice processing agricultural by-products of rice residue protein way, make full use of ultrasound - enzymatic digestion - membrane filtration technology, the rice residue protein to carry out complex processing, and then directional quantitative modification, you can get a special molecular distribution range of active peptide composition of rice protein [4].

 

2 Key technologies for the preparation of rice protein active peptides

2.1 Establishment of ultrasonic modification technology and process conditions for rice residue protein

The main content: through the processing of broken rice caramel production after the leftover scraps - rice residue for the preparation of rice protein active peptide raw materials, and then study the optimal process conditions of ultrasonic modification of rice residue protein.

Key technical problem: Screening and optimizing the process conditions of ultrasonic modification of rice residue protein.

In the experiment, the conditions of fixed enzyme digestion are ultrasonic power 500 W, ultrasonic time 10 min, the general mass fraction of substrate is 10%, the amount of enzyme is 2%, the enzyme temperature is 55 ℃, the enzyme pH value of 10, the enzyme time is 4h, and also need to optimize the ultrasonic auxiliary enzyme digestion of rice pomace protein active peptide preparation process, the use of response surface analysis, can be obtained Using response surface analysis, we can obtain the optimal conditions, i.e., ultrasonic power of 450W, ultrasonic time of 8min, substrate mass fraction of 12%, of which the amount of enzyme is 3.4%, the temperature of enzymatic digestion is 56℃, the pH value of the enzymatic digestion is 9.3, and we can carry out 5 parallel experiments in 4h, which can make the conversion rate of the protein maintain at about 75.78% [4].

 

2.2 Process conditions of ultrasonic compound enzyme digestion of rice residue protein to prepare rice active peptide

Main content: Select the rice residue protein hydrolysate after ultrasonic modification as the processing raw material, and then study the ultrasonic compound protease to enzyme digestion of silkworm pupa active peptide of the optimal process conditions.

Key technical problem: Screening and optimizing the best process conditions for the preparation of active peptide of silkworm pupa.

The experiment shows that the protein of silkworm pupa is composed of 43 ku molecules, and the molecular bands of the active peptide of silkworm pupa can not be seen in the electrophoresis graph of the enzyme digestion product, which proves that the enzymatic action of alkaline protease is the best, and the suitable time of hydrolysis for the optimization of alkaline protease for the preparation of the active peptide of silkworm pupa is 300min, the enzyme dosage is 0.3%, the concentration of the substrate is 8%, and the pH value of hydrolysis is 9.5, the temperature of hydrolysis is 50℃, and the DH value of the hydrolysis is 22.68%. The hydrolysis temperature is 50 ℃, the hydrolysis degree DH value is 22.68%, after the enzyme hydrolysis hydrolysis products are mainly composed of small peptide molecules, in general, after the defatting treatment of silkworm pupa raw material protein content is the highest, so the preparation of silkworm pupa active peptide is mainly based on the enzyme digestion and trypsin digestion of papaya protein [5].

 

2.3 Process conditions for separation of rice protein active peptides by membrane technology

Main content: Use the rice pomace protein hydrolysate after ultrasonic-assisted enzyme digestion as the processing raw material, and then use the membrane separation technology to carry out membrane separation of rice pomace protein hydrolysate, so as to get the active peptide product of rice protein with molecular mass distribution in a certain range and at the same time possessing activity.

Key technical problem: Screening the optimal process conditions for the separation of rice residue protein hydrolysate by membrane technology.

Through the ORAC method in membrane separation technology, the standard curve can be obtained, and after considering the factors of ACE inhibitory activity and in vitro antioxidant activity, the ultrafiltration membrane with relative molecular mass of 1KDa can be selected, therefore, when the ultrafiltration conditions are determined, the higher the ultrafiltration pressure, the larger the membrane permeability, so the optimal ultrafiltration pressure is 0.194MPa, and the optimal ultrafiltration temperature is 45℃, at this time the pH value of the ultrafiltration reaction is 6.8, the loss rate of the rice residue protein product after membrane separation and purification is 2% in the operation of nanofiltration. The optimal ultrafiltration temperature was 45 ℃, the pH value of the ultrafiltration reaction was 6.8, the loss rate of the rice pomace protein product after membrane separation and purification was 2% by nanofiltration, the ACE value was 0.78 mg/mL, and the ORAC value of the in vitro antioxidant activity was 2358.50±40.42 umol/g [6].

 

2.4 Evaluation of physicochemical properties of rice protein active peptides

Main content: To study a series of processing characteristics of rice protein active peptides, such as hydrolysis degree, solubility, foaming and emulsification, and then analyze the distribution of molecular weight, disulfide bond and sulfhydryl content, surface hydrophobicity and secondary structure.

Key technical issues: Detection and analysis of molecular weight distribution, disulfide bond and sulfhydryl content, surface hydrophobicity and secondary structure of rice protein active peptides.

Through the extrusion and expansion of rice protein active peptide, high temperature, high shear and high pressure equipment, can make the molecular structure of rice protein reorganization, stretching, and molecular surface charge distribution, so that the hydrogen bond between molecular proteins, disulfide bonds broken, so it will lead to the denaturation of proteins, so that the digestibility of proteins has been significantly improved, and can promote the enzyme on the protein decomposition [7]. The protein digestion rate is significantly improved, and the enzymatic decomposition of proteins is promoted [7].

 

2.5 Evaluation of nutritional and biological activities of rice protein active peptides

Main content: To investigate the amino acid composition and nutritional analysis of the active peptide of silkworm pupa, and to evaluate the in vitro digestibility, antioxidant property, ACE inhibitory value and immunomodulatory bioactivity of the active peptide of rice protein.

KEY TECHNICAL ISSUE: Understand the means of evaluating biological activities such as ACE inhibition and immunomodulation.

SepedexG-25 was used to separate the active peptides of silkworm pupa, and eight components from A to H could be obtained, and the ACE inhibitory activity of the F part was the strongest, which could reach 68.87%, and then the active peptides of the F group were separated by using the macroporous adsorbent resin DA201-C, and the F-60-6-1 protein molecules with high inhibitory rate could be obtained after two and a half times of the RP-HPLC separation [8]. -6-1 protein molecules, which have strong antioxidant and immunomodulatory functions [8].

 

2.6 Edible Dosage Forms and Formulations of Rice Protein-Activated Peptide Products

Main content: To explore the development of functional rice protein active peptide products, and to study the edible dosage forms and formulations of rice protein active peptide products. Key technical issues: Development of edible dosage forms and formulations of rice protein active peptide products.

Through the development of food dosage forms and formulations of rice protein active peptide products, it can be seen that after hydrolysis, rice protein can release a certain level of amino acids and biologically active peptides, which can increase the molecular polarity of proteins, and also increase the solubility, showing the properties of protein foaming and emulsification, so after hydrolysis of rice protein, protein is used as the substrate, so that the peptide bonds in protein molecules can be decomposed under the action of peptidases and peptidases to obtain relatively small peptide chain fragments [9]. Therefore, after hydrolysis of rice protein, the protein is used as a substrate so that the peptide bonds in the protein molecule can be decomposed by peptidases and peptidases to obtain smaller peptide fragments [9].

 

3 Technical route and experimental program for the preparation of rice protein active peptide

This experimental research is mainly based on the residue of rice dregs as raw material after the sugar production of broken rice saccharification, in accordance with the high temperature denaturation of rice dregs of protein advantages, make full use of ultrasonic technology to rice dregs of protein modification, trying to make the tight protein molecules loosened through the ultrasonic effect, and then use the multi-enzyme compound hydrolysis of rice dregs of protein, control the hydrolysis conditions, to get the optimal hydrolysis degree, the product of hydrolysis to carry out membrane separation, and then based on the molecular distribution level of active peptides to intercept the rice dregs of protein hydrolysis product rice dregs of protein hydrolysate, and then the physical and chemical properties and nutritional value of the liquid The product of hydrolysis to carry out membrane separation, and then based on the molecular distribution level of active peptides to intercept the rice residue protein hydrolysis product rice residue protein hydrolysate, and then for rice residue protein hydrolysate to carry out the physicochemical properties and nutritional value assessment, design of rice residue protein hydrolysis products, experiments after the hydrolysis products for the nutritional and digestive characteristics of the development of the evaluation, in the mathematical modeling simulation of the modification process, to be able to carry out the design and positioning of the quality of the final product. In the process of mathematical modeling simulation of modification, the quality of the final product can be designed and positioned, proving that the experimental preparation program and technical implementation route of rice protein active peptide is feasible [10].

 

3.2 Experimental program

3.2.1 Establishment of ultrasonic modification technology and process conditions for rice residue protein

The rice pomace protein, which is the waste material of caramel production from broken rice, is mainly used as the raw material for the preparation of rice protein active peptide, and the hydrolysis degree of rice protein is used as the evaluation index, and then one-way experiments and response surface analyses are carried out on the factors of ultrasonic cell crushing, processing time, material-liquid ratio, pH, and initial temperature, in order to obtain the optimal process conditions for the ultrasonic treatment of the rice protein active peptide properties of the rice pomace protein. On the basis of one-factor analysis, ultrasonic power, ultrasonic time, substrate mass fraction, enzyme addition, enzyme digestion temperature, enzyme digestion pH value as independent variables, and the conversion rate of rice pomace protein as the response value to carry out the response surface process optimization, to obtain the linear relationship between the independent variables of the functional equation and the dependent variable, and in the regression model, R2 = 0.9877.

 

3.2.2 Process conditions for the preparation of rice active peptides from rice pomace protein by ultrasonic complex enzyme digestion

After the ultrasonic modification of rice pomace protein as the raw material, then through the protease screening and enzymatic treatment, so as to obtain the substrate protein concentration, enzyme dose, enzymatic temperature, the experimental factors to carry out a one-way analysis, multifactorial experiments and response surface analysis, in order to obtain the optimal conditions for the optimization of the enzymatic method and the preparation of active peptides from rice. In the rice pomace protein hydrolysis experiment, we need to fully consider the interactions among the influencing factors, and optimize the experiment according to the -1, 0, 1 three-level factor model designed by Response Surface Design Expert 8.0 software, which shows that the DH value and the DPPH clearance rate of rice pomace protein peptide decrease with the increase of the enzyme hydrolysis temperature, and the DH value decreases from -1 to -1 at the concentration of enzyme and substrate 0. When the enzyme-substrate concentration is 0, the DH value will increase from -1 to 1. When the pH value is 0, the DH value will also increase and then decrease, and when the temperature level is 0, the DH value will increase with the enzyme-substrate concentration level, therefore, the interaction between enzymatic digestion temperature and enzyme-substrate concentration is the most obvious, and the role of the substrate concentration and the pH value is the most obvious.

 

3.2.3 Process conditions for separation of rice protein active peptides by membrane technology

The membrane separation technology was applied to the hydrolysate of rice pomace protein, and then appropriate membrane pore size, separation time, separation pressure, separation temperature and membrane flux were analyzed by one-way analysis, so as to find the optimal process conditions for the separation of active peptides from rice pomace protein by the membrane method. When the molecular weight of the short peptide in the permeate is 1KDa, the ACE IC50 value of the permeate is 0.82mg/ml, and when the number of times of adding water is increasing, the removal rate of Na+ ions will increase, and when it comes to the 10th time, the removal rate of Na+ ions is the largest, so the amount of adding water can be 3 times of the amount of water is the most appropriate, to ensure that the permeability of the short peptide is more than 65%, and then to reduce the loss of short peptide rate.

 

3.2.4 Evaluation of physicochemical properties of rice protein active peptides

The hydrolysis degree, solubility, foaming and emulsifying properties of rice protein active peptides were analyzed, the disulfide bond and sulfhydryl content of rice protein active peptides were detected by chemical methods, a kinetic model of controlled enzymatic hydrolysis was established, the molecular weight distribution of proteins was detected by the SDS-PAGE technique and protein spectroscopy, and the ultrastructure of protein molecules was observed by transmission electron microscopy, and the surface hydrophobicity and secondary structure of peptide bonds were analyzed by far-infrared spectroscopy and ultraviolet spectroscopy. The molecular weight distribution of the proteins was examined by SDS-PAGE and protein spectroscopy, and the ultrastructure of the protein molecules was observed by transmission electron microscopy.

 

3.2.5 Evaluation of nutritional and biological activities of rice protein active peptides

RP-HPLC chromatography and amino acid autoanalyzer were used to study the composition of the active peptides of silkworm pupa, and then nutritional evaluation was carried out, and then the digestibility of rice protein was used as an evaluation index to analyze the in vitro digestibility of the active peptides, and the antioxidant capacity of the active peptides was investigated by using the level of elimination of free radicals of DPPH, the total reducing power, and the chelating power of ferrous ions as an index. Research shows that if the ACE activity value of the active peptide is too high, it will damage the blood pressure system of human body. Therefore, from the rice residue hydrolysate, we can separate the rice residue protein and soy protein according to a certain ratio, and produce protein powder with reasonable amino acid composition, and we can also use plant protease to hydrolyze the rice residue, and then we can produce a new type of amino acid nutritious oral liquid.

 

3.2.6 Edible Dosage Forms and Formulations of Rice Protein-Activated Peptide Products

Through the use of rice protein active peptide powder as a carrier, further compounding of milk powder and soy protein powder, so as to obtain the edible dosage forms and formulations of rice protein active peptide products. After hydrolyzing the rice hydrolysis product by protease, special processing can be carried out for the preparation of protein nutritional supplements, and the protein nutritional supplements can also be made into powder and oral liquid, which is very convenient to consume, and is also conducive to the digestion and absorption of the human body.

 

4 Conclusion

Through this experimental research, we can solve the problem of deep processing and utilization of by-products of rice dregs in the process of deep processing of broken rice, and combined with ultrasonic "pretreatment - enzymatic compound treatment - membrane separation" and other technological means, greatly improving the enzymatic hydrolysis of rice dregs protein efficiency, improve the hydrolysis efficiency. And the combination of ultrasonic "pretreatment - enzyme complex treatment - membrane separation" and other technical means, greatly improving the enzyme digestion efficiency of rice residue protein, improve the efficiency of hydrolysis, the establishment of rice protein bioactive peptide preparation process technology, the development of functional rice protein active peptide food, and then for the deep processing of rice residue resources to open up a new path of utilization.

 

References:

[1] Cai J. Complex enzymatic preparation and physicochemical properties of antioxidant peptides from rice [D]. Wuhan: Wuhan University of Light Industry, 2016

[2] WANG Lu, CHEN Yuehua, XU Zhou, et al. Screening of enzymes for hydrolysis of rice immunoreactive peptides[J]. Food and Machinery, 2015,31(2):38-42

[3] LU Le, LIU Dong, WAN Hongxia, et al. Kinetic study on the preparation of small molecule peptides by enzymatic hydrolysis of rice protein by alkaline protease Alcalase[J]. Modern Food Science and Technology, 2014, 30(7):149-153,123

[4] Bu H-P. Preparation and isolation and purification of immunoactive peptides from rice [D]. Changsha: Changsha University of Science and Technology, 2014

[5] Guo Liwei. Enzymatic digestion of rice dregs for the preparation of ACE inhibitory peptide[D]. Changsha: Hunan Agricultural University, 2012

[6] Jiang Yan. Preparation of antioxidant peptides and ACE inhibitory peptides by enzymatic method of broken rice[D]. Hefei: Hefei University of Technology, 2012.

[7] SHIN Yanhao, LIU Fang. Enzymatic preparation of active peptides and antioxidant properties of rice[J]. Modern Agricultural Science and Technology, 2010 (23): 319-321

[8] MA Hailer, LIU Bin, LI Shujun, et al. Protease screening for enzymatic preparation of antioxidant peptides from rice[J]. Journal of Agricultural Machinery, 2010,41(11):119-123

[9] LucaAmagliani, JonathanO'Regan, AlanL.Kelly, JamesA.O'Mahony.Composition and proteinprofile ana lysis ofrice proteining redients[J].Journal of Food Composition and Analysis, 2016, 74 (5): 662-668

[10]Luca Amagliani, Jonathan O'Regan, AlanL.Kelly, JamesA. O'Mahony.Physicalandflowpropertiesofriceproteinpowders[J]. Journal of Food Engineering, 2016(98): 45-48