CN114958949A - Maca immunoregulation protein zymolyte or peptide and preparation method and application thereof - Google Patents

Maca immunoregulation protein zymolyte or peptide and preparation method and application thereof Download PDF

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CN114958949A
CN114958949A CN202210577461.XA CN202210577461A CN114958949A CN 114958949 A CN114958949 A CN 114958949A CN 202210577461 A CN202210577461 A CN 202210577461A CN 114958949 A CN114958949 A CN 114958949A
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吴晖
潘蕾蔓
张猛猛
贺萍
赖富饶
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South China University of Technology SCUT
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Abstract

The invention discloses a maca immunoregulation protein zymolyte or peptide and a preparation method and application thereof, belonging to the technical field of deep processing of agricultural products and comprehensive utilization of byproducts thereof. The maca immunomodulatory protein zymolyte with immunomodulatory activity is obtained by carrying out pre-enzymolysis and gastrointestinal digestion simulation on maca protein, and the maca immunomodulatory peptide is further obtained by ultrafiltration, sequencing, activity scoring, molecular docking and synthesis. The maca immunoregulatory protein zymolyte and peptide prepared by the method have high immunoregulatory activity and can be used as active ingredients for producing functional foods. The method has the characteristics of novelty, simplicity and high efficiency, is easy to realize industrial production, provides a new development direction for deep processing of maca, and provides new possibility for realizing high-value utilization of maca protein.

Description

Maca immunoregulation protein zymolyte or peptide and preparation method and application thereof
Technical Field
The invention belongs to the technical field of deep processing of agricultural products and comprehensive utilization of byproducts thereof, and particularly relates to a maca immunoregulatory protein zymolyte or peptide and a preparation method and application thereof.
Background
Maca is an annual herb of the genus Lepidium of the family Brassicaceae, native to Peru Andes plateau in south America. Maca grows in 3500-4500 m high-altitude areas, and can endure severe environments such as strong sunshine, strong wind and extreme weather which are not beneficial to survival of most species. To date, maca has been planted for over 2000 years; in the end of the world, maca is recommended to be planted and eaten by grain and agriculture organizations of the united nations, so that the danger of endangering extinction can be avoided. In China, maca is first approved to be introduced into China in 2002 and is planted in Yunnan, Xinjiang and Tibet regions, and at present, Yunnan is the main production place of the domestic maca. Among all parts, the maca rhizome is the main edible part of maca, the taste is slightly sweet and spicy, the smell is special fragrance similar to caramel, yellow maca and purple maca with medium taste number are the best, and maca with different skin colors has different main effects. Meanwhile, maca is eaten in various ways, including that fresh maca is directly eaten after being cooked or is eaten after being dried and processed, and the maca can be prepared into fruit juice and soup or can be compounded with other foods after being powdered. In recent years, maca products are regarded as fragrant cakes in the field of functional foods, and firstly, the maca has gradually increased public attention since 2011 is approved as a new resource food by the ministry of health and communications in China; secondly, the demand of people for immune enhancing food is increasing day by day. Currently, maca products are widely sold in the united states, canada, germany, china, japan and the netherlands, including various foods or medicines such as capsules, tablets and oral liquids. Researches show that maca has high nutritional value, contains main nutrients such as carbohydrates, proteins, vitamins and fats, and secondary metabolites such as alkaloids, glucosinolates, macaenes and macamides. Maca is also called "Peru ginseng" because of its nutritional components, like ginseng, in promoting vigor and endurance. Maca is reported in the literature to have a variety of biological activities, including fertility enhancement and libido promotion, nerve protection, memory enhancement, depression resistance, oxidation resistance, cancer resistance, anti-inflammation, and skin protection.
Protein is the main nutrient component of maca (9.56-21.90%), but the research related to maca protein at home and abroad is still in a lower level at present, and although the basic physicochemical characteristics, secondary structure and amino acid composition of maca protein and the biological activity of maca protein zymolyte or peptide are reported by respective research, the process optimization of maca protein extraction conditions is still the main concern. In addition, the current patents are only concerned with the extraction of maca peptides and do not describe the biological activity of maca peptides. Therefore, it is necessary to explore the biological activity of maca proteins to realize high-value utilization of maca proteins. In addition, maca protein has low digestibility and low bioavailability when taken directly.
The immune active peptide is a peptide capable of regulating cellular immunity and humoral immunity and further enhancing the immune function of an organism. Generally, the immune regulatory peptide is a hydrophobic small molecule peptide consisting of 2-10 amino acid residues. The characteristic amino acids of the immunoregulatory peptide include hydrophobic amino acids such as glycine and leucine, charged amino acids such as glutamic acid and arginine, and aromatic amino acids such as tyrosine and phenylalanine. Currently, methods for evaluating the activity of immunoregulatory peptides include in vitro methods as well as in vivo methods; in the in vitro activity evaluation, mouse macrophage RAW 264.7 is the most commonly used cell model, and the evaluation indexes comprise the expression levels of TNF-alpha, IL-6, NO and the like.
The method comprises the steps of digesting maca protein by pepsin and pancreatin in sequence (2 h at 37 ℃) to obtain zymolyte, performing ultrafiltration and Sephadex G-15 purification, and sequencing to obtain the peptide. The hydrolysate obtained by the method has low hydrolysis degree (22.91%), weak capability of promoting TNF-alpha expression of mouse macrophage RAW 264.7 by the hydrolysate, complex purification steps (He P, Pan L, Wu H, et al. isolation, identification, and immunology methods of peptides from Lepidium meyenii [ J ]. Journal of animal aggregation and food chemistry,2022,70(14): 4328-4341.).
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide maca immunoregulation protein zymolyte or peptide and a preparation method and application thereof.
The purpose of the invention is realized by the following technical scheme:
the preparation method of the maca immunoregulation protein zymolyte comprises the following steps:
(1) preparing maca protein: taking maca rhizome powder as a raw material, and taking Tris-HCl aqueous solution (pH 7.0-pH 9.0) as an extracting solution for extraction, wherein the material-liquid ratio is 1: 20-1: 40, ultrasonic treatment (50-70 ℃, 200-250W, 20-30 min), wherein the extraction time is 30-60 min, and the extraction temperature is 50-70 ℃. Centrifuging for the first time (3000 rpm-4000 rpm, 5 min-15 min), filtering to obtain supernatant, adding 0.5 mol/L-1.0 mol/LHCl aqueous solution to ensure that the pH value of the supernatant reaches 3.5-4.0, standing for 30 min-40 min, centrifuging for the second time (3000 rpm-4000 rpm, 10 min-20 min), redissolving and precipitating with 0.5 mol/L-1.0 mol/LNaOH aqueous solution, dialyzing and desalting (1000 Da-5000 Da), and freeze-drying to obtain maca protein.
(2) Pre-enzymolysis: preparing the maca protein obtained in the step (1) into a 1% -2% (w/v, g/mL) maca protein solution, and heating in a boiling water bath for 10-20 min for the first time to inactivate a protease inhibitor in the maca protein. Adding protease, adjusting the temperature and pH to the optimum conditions of each protease, and preheating for 10-20 min. In the enzymolysis process, the pH is adjusted once every 5min to 15min for the first 10min to 30min, and then the pH is adjusted once every 10min to 30min, so that the pH is kept constant. The enzymolysis time is 3-4 h, and after the reaction is finished, the enzymolysis liquid is placed in a boiling water bath for the second time to be heated for 10-20 min so as to stop the reaction.
(3) Simulating gastrointestinal digestion: simulating the gastric digestion stage: dropwise adding 0.5-1.0 mol/L HCl aqueous solution to make the pH value reach 1.5-3.0, adding pepsin after the temperature reaches 36-37 ℃, and magnetically stirring for reaction for 2-3 h; simulating the intestinal digestion stage: dropwise adding 0.5-1 mol/L NaOH aqueous solution to make the pH value reach 6.5-7.5, stopping simulating gastric digestion, adding pancreatin, and magnetically stirring and reacting for 2-3 h at 36-37 ℃. And then adjusting the pH value of the solution to 7.0-7.5, boiling for 10-20 min to completely finish the reaction, cooling, centrifuging (3000-4000 rpm, 10-20 min), taking supernatant, dialyzing (the interception amount is 100-500 Da), and freeze-drying to obtain the maca immunomodulatory protein zymolyte.
Preferably, the concentration of the Tris-HCl aqueous solution in the step (1) is 0.05 mol/L.
Preferably, in step (1), the extraction conditions are as follows: the ratio of material to liquid is 1: 30, performing ultrasonic treatment (60 ℃, 250W and 20min), wherein the extraction time is 30min, and the extraction temperature is 60 ℃.
Preferably, in the step (1), the conditions of the first centrifugation are 4000rpm for 5 min; the conditions for the second centrifugation were 4000rpm, 10 min.
Preferably, the filtration in step (1) is filtration using 500 mesh filter cloth.
Preferably, in the step (1), the concentration of the HCl aqueous solution is 1.0mol/L, and the concentration of the NaOH aqueous solution is 0.6 mol/L.
Preferably, the pH of the system after the addition of aqueous HCl in step (1) is 3.75.
Preferably, the cut-off of the dialysis desalting in the step (1) is 2000 Da-5000 Da.
Preferably, the cut-off for the dialysis desalting in step (1) is 5000 Da.
Preferably, the protein content of the maca protein in step (1) is 87.02%.
Preferably, the concentration of the maca protein solution in step (2) is 1% (w/v, g/mL).
Preferably, in the step (2), in the enzymolysis process, the pH is adjusted for 20min to 30min, every 5min to 10min, and then every 10min to 20 min.
Preferably, in the step (2), the first boiling water bath heating time is 10min, the preheating time is 10min, the enzymolysis time is 3h, and the second boiling water bath heating time is 10 min.
Preferably, the enzyme activity of the protease in the step (2) needs to reach 2.0X 10 4 U。
Preferably, the category of the protease in the step (2) includes one of neutral protease, alkaline protease, papain, bromelain and complex protease.
Preferably, the optimal conditions for the protease in step (2) are respectively: the temperature of the neutral protease is 45 ℃, and the pH value is 7.5; the temperature of the alkaline protease is 40 ℃, and the pH value is 10.5; the temperature of the papain is 55 ℃, and the pH value is 7.0; the temperature of the bromelain is 55 ℃, and the pH value is 7.0; the temperature of the compound protease is 45 ℃ and the pH value is 7.0.
Preferably, in the step (3), the concentration of the HCl aqueous solution is 1.0mol/L, and the concentration of the NaOH aqueous solution is 1.0 mol/L.
Preferably, in the step (3), the enzyme activity of the pepsin is required to reach 3000U/mg, and the enzyme activity of the pancreatin is required to reach 8U/mg.
Preferably, in step (3), the conditions simulating the gastric digestion stage are: the pH value is 3.0, the temperature is 37 ℃, and the reaction time is 2 h; the conditions that mimic the intestinal digestion stage are: the pH was 7.0, the temperature was 37 ℃ and the reaction time was 2 h.
Preferably, in step (3), the dialysis cut-off is in the range of 100Da to 500 Da.
Preferably, in the step (3), the boiling time is 10 min; centrifuging at 4000rpm for 20 min; the dialysis cut-off was 100 Da.
Preferably, the yield of the maca immunomodulatory protein zymolyte in the step (3) is 39-56%, the hydrolysis degree is 56.38-57.06%, the yield of soluble peptides is 23.23-33.45%, and the content of free amino acids is 540.89-758.76 mg/100g maca protein.
Preferably, when the concentration of the maca immunomodulatory protein zymolyte in the step (3) is 500 mug/mL, the level of a mouse macrophage RAW 264.7 expression cytokine TNF-alpha is 17.08-20.43 ng/mL, and is increased by 280.40-355.01% compared with a blank group.
Preferably, the dialysis water in the steps (1) and (3) includes any one of distilled water, purified water, deionized water and tap water, and the dialysis temperature should be maintained at 4 ℃.
The invention provides a preparation method of maca immunoregulation peptide, which comprises the following steps:
screening and preparing maca immunoregulation peptide: selecting the maca immunomodulatory protein zymolyte subjected to protease pre-enzymolysis treatment in the step (3) to prepare a maca immunomodulatory protein zymolyte solution (0.25 mg/mL-1.0 mg/mL), sequentially grouping the maca immunomodulatory protein zymolyte solution by ultrafiltration tubes with the cut-off amount of 10kDa (centrifugation 4000 rpm-5000 rpm,30 min-60 min) and 3kDa (centrifugation 4000 rpm-5000 rpm,30 min-60 min), taking sample liquid at the lower part of the 3kDa ultrafiltration tube, and evaporating, concentrating, freezing and drying to obtain a graded maca immunomodulatory protein zymolyte with the molecular weight less than 3 kDa. Dissolving a graded maca immunomodulatory protein zymolyte with the molecular weight less than 3kDa by using a sample dissolving solution (0.1-0.2% formic acid, 2-3% acetonitrile, v/v), centrifuging (4-10 ℃, 10000-15000 rpm, 20-30 min), taking supernatant, carrying out liquid phase chromatographic separation, directly feeding the separated peptide segment into a mass spectrometer for on-line detection, and searching a library to obtain a sequence. Selecting peptides, performing activity scoring, performing molecular docking with a pattern recognition receptor to obtain a target peptide segment, and synthesizing the peptides according to the sequence.
Preferably, the maca immunomodulatory protein zymolyte is obtained by performing enzymolysis treatment on the maca immunomodulatory protein zymolyte by bromelain.
Preferably, the concentration of the maca immunomodulatory protein zymolyte is 0.25 mg/mL.
Preferably, the ultrafiltration conditions with a cut-off of 10kDa are centrifugation at 5000rpm for 30 min; the ultrafiltration conditions with a cut-off of 3kDa were centrifugation at 5000rpm for 60 min.
Preferably, the sample dissolving solution in the step (4) contains 0.1% (v/v) formic acid and 2% (v/v) acetonitrile.
Preferably, the centrifugation conditions after hydrolysis of the fractionated maca immunomodulatory protein hydrolysate having a molecular weight < 3kDa in step (4) with the sample lysate are 4 ℃, 13200rpm, 20 min.
Preferably, the search software is MASCOT and the search database is UniProt.
Preferably, the selected peptide is a peptide containing 2-10 amino acids.
Preferably, the means for activity scoring is the PeptideRanker website with the website http:// distilldeep.
Preferably, the tool for molecular docking is the HPEPDCOCK website with the website http:// huang glab. phys. host. edu. cn/hpeddock/.
Preferably, the proteins used for molecular docking are the pattern recognition receptors TLR2(PDB ID:1FYW) and TLR4(PDB ID:5 IJD).
Preferably, the peptide synthesis method is Fmoc solid phase synthesis.
Preferably, the molecular weight range of the maca immunomodulatory peptides is 900 Da-1300 Da.
Preferably, the activity score is immune activity evaluation, the immune activity evaluation method adopts mouse macrophage RAW 264.7 as an in vitro model, and the evaluation index is the expression level of the cytokine TNF-alpha.
The invention provides a maca immunomodulatory protein zymolyte with immunomodulatory activity, which is prepared by the preparation method.
The invention provides maca immunoregulation peptide which is prepared by the preparation method.
The invention also provides application of the maca immunoregulation protein zymolyte or the maca immunoregulation peptide with immunoregulation activity in functional food.
Further, the functional food is an immunoregulation functional food.
According to the invention, five different proteases are used for carrying out pre-enzymolysis and gastrointestinal digestion simulation to prepare the maca immunoregulation protein zymolyte with immunoregulation activity, and the maca immunoregulation peptide is obtained by adopting methods such as molecular docking and solid-phase synthesis, so that the process is simple and novel, the defect of rough maca processing is overcome, a new thought is provided for deep processing of maca, and a new direction is provided for utilization of maca resources.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the raw materials adopted by the maca nutritional food are maca roots and stems, and the maca nutritional food has the advantages of wide sources, rich nutrition and high protein content.
(2) The invention can obtain two products, namely maca immunoregulation protein zymolyte with an immunoregulation function and maca immunoregulation peptide with higher immunoregulation activity and easier absorption and utilization by organisms.
(3) According to the invention, maca roots and stems are used as raw materials, maca protein is extracted and then hydrolyzed by five different proteases respectively, and the protein zymolyte and peptide with immunological activity can be prepared simultaneously according to the same process flow.
(4) The invention creatively improves the digestibility of the maca protein in gastrointestinal digestion by a processing and treating mode of pre-enzymolysis on the premise of not weakening the immunoregulation activity of the maca protein.
(5) The preparation method disclosed by the invention is simple and efficient in preparation process, low in cost and suitable for industrial production, solves the problem of extensive maca processing, provides a new idea for deep processing of maca and provides a new direction for utilization of maca resources.
Drawings
FIG. 1 is a process flow chart for preparing maca immunomodulatory protein zymolyte by taking maca roots and stems as raw materials.
FIG. 2 is a flow chart of the preparation of maca immunomodulatory peptides provided by the invention.
FIG. 3 is a bar graph of the effect of the immunomodulatory protein hydrolysates of maca prepared in examples 1-5 on the viability of RAW 264.7 cells and the expression level of TNF- α.
FIG. 4 is a bar graph of the effect of maca immunomodulatory peptides prepared in example 6 on the viability of RAW 264.7 cells and the expression level of TNF- α.
Detailed Description
In order to better illustrate the present invention, the present invention is further described with reference to the following examples, which should be construed as limiting the scope of the present invention and referring to the conventional art for the process parameters not specifically mentioned. In the examples, the neutral protease is the neutral protease purchased from Shanghai-sourced leaf Biotechnology, Inc., the alkaline protease is the alkaline protease purchased from Shanghai-sourced leaf Biotechnology, Inc., the bromelain is the bromelain purchased from Shanghai-sourced leaf Biotechnology, Inc., and the papain is the papain purchased from Shanghai-sourced leaf Biotechnology, Inc.; the compound protease is a compound protease purchased from Shanghai-derived leaf Biotech Co., Ltd.
FIG. 1 is a process flow chart for preparing maca immunomodulatory protein zymolyte by taking maca roots and stems as raw materials.
FIG. 2 is a flow chart of the preparation of maca immunomodulatory peptides provided by the invention.
Example 1
The embodiment is a preparation method of maca immunomodulatory protein zymolyte with immunomodulatory activity (neutral protease pre-enzymolysis combined with simulated gastrointestinal digestion), and the preparation method comprises the following specific steps:
(1) preparing maca protein: taking 50g of maca rhizome powder as a raw material, and taking 1LTris-HCl solution (0.05mol/L, pH 7.0) as an extracting solution for extraction, wherein the material-liquid ratio is 1 g: 20mL, ultrasonic treatment (50 ℃, 200W, 20min), extraction time of 30min and extraction temperature of 60 ℃. Centrifuging for the first time (3000rpm, 5min), filtering (500 mesh filter cloth), collecting supernatant, adding 0.5mol/LHCl aqueous solution until the pH of the supernatant reaches 3.5, standing for 30min, centrifuging for the second time (3000rpm, 10min), redissolving with 0.5mol/LNaOH aqueous solution for precipitation, dialyzing for desalination (2000Da), and lyophilizing to obtain maca protein.
(2) Pre-enzymolysis: dissolving 0.5g of maca protein in 50mL of distilled water to prepare a 1% (w/v, g/mL) maca protein solution, and heating in a boiling water bath for 10min for the first time to inactivate a protease inhibitor in the maca protein. Adding 438.60mg neutral protease (enzyme activity is 114U/mg), adjusting temperature to 45 deg.C, adjusting pH to 7.5, and preheating for 10 min. During the enzymolysis, the pH is adjusted every 5min for the first 10min, and then every 10min, so that the reaction system keeps constant pH at 7.5. The enzymolysis time is 3h, and after the reaction is finished, the enzymolysis liquid is placed in a boiling water bath for heating for 10min for the second time to stop the reaction.
(3) Simulating gastric digestion: dropwise adding 0.5mol/L HCl aqueous solution to make the pH value reach 1.5, adding 143.90mg pepsin (enzyme activity is 250U/mg) after the temperature reaches 36 ℃, and magnetically stirring to react for 2 hours;
(4) simulating intestinal digestion: 0.5mol/L NaOH aqueous solution is added dropwise to make the pH value reach 6.5, the simulated gastric digestion is stopped, 625.00mg of pancreatin (enzyme activity is 8U/mg) is added, and the mixture is magnetically stirred and reacted for 2 hours at 36 ℃.
(5) Adjusting the pH value of the enzymolysis system to 7.0, boiling for 10min to complete the reaction, cooling, centrifuging (3000rpm for 10min), collecting the supernatant, dialyzing (interception amount of 100Da), and freeze-drying to obtain the maca immunomodulatory protein zymolyte.
Example 1 the final yield of the maca immunomodulatory protein hydrolysate was 40.00%, the degree of hydrolysis was 56.84%, the yield of soluble peptides was 23.23%, and the free amino acid content was 564.51mg/100g maca protein; the maca immunomodulatory protein hydrolysate prepared in example 1 at a concentration of 500 μ g/mL promoted the expression of the cytokine TNF- α by the mouse macrophage RAW 264.7 at 17.08ng/mL, which was increased by 280.40% over the blank, which was a sample-free medium (89% (v/v) DMEM + 10% (v/v) FBS + 1% (v/v) diabody, which was penicillin and streptomycin), as shown in B1 in fig. 3. The maca immunomodulatory protein hydrolysate prepared in example 1 can be used to prepare capsules, tablets, pills, granules or oral liquids.
Example 2
The embodiment is a preparation method of maca immunomodulatory protein zymolyte with immunomodulatory activity (alkaline protease pre-enzymolysis combined with simulated gastrointestinal digestion), and the preparation method comprises the following specific steps:
(1) preparing maca protein: taking 50g of maca rhizome powder as a raw material, and extracting by taking 1.5L Tris-HCl solution (0.05mol/L, pH 8.0) as an extracting solution, wherein the material-to-liquid ratio is 1 g: 30mL, ultrasonic treatment (60 ℃, 225W, 25min), extraction time of 45min and extraction temperature of 60 ℃. Centrifuging for the first time (3500rpm, 10min), filtering (500 mesh filter cloth), collecting supernatant, adding 0.75mol/LHCl aqueous solution until the pH of the supernatant reaches 3.75, standing for 35min, centrifuging for the second time (3500rpm, 15min), redissolving with 0.75mol/LNaOH aqueous solution for precipitation, dialyzing for desalination (3500Da), and lyophilizing to obtain maca protein.
(2) Pre-enzymolysis: dissolving 0.75g of maca protein in 50mL of distilled water to prepare 1.5% (w/v, g/mL) of maca protein solution, and heating in a first boiling water bath for 15min to inactivate a protease inhibitor in the maca protein. Adding 193.80mg alkaline protease (enzyme activity is 258U/mg), adjusting temperature to 40 deg.C, adjusting pH to 10.5, and preheating for 15 min. In the enzymolysis process, the pH is adjusted every 10min for the first 20min, and then the pH is adjusted every 20min, so that the reaction system keeps constant pH at 10.5. The enzymolysis time is 3.5h, and after the reaction is finished, the enzymolysis liquid is placed in a boiling water bath for heating for 15min for the second time so as to stop the reaction.
(3) Simulating gastric digestion: dropwise adding 0.75mol/L HCl aqueous solution to make pH reach 2.25, adding 143.90mg pepsin (enzyme activity is 250U/mg) after the temperature reaches 36.5 ℃, and magnetically stirring to react for 2.5 h;
(4) simulating intestinal digestion: 0.75mol/L NaOH aqueous solution is added dropwise to make the pH value reach 7.0, the simulated gastric digestion is stopped, 625.00mg of pancreatin (enzyme activity is 8U/mg) is added, and the mixture is magnetically stirred and reacted for 2.5 hours at the temperature of 36.5 ℃.
(5) Adjusting the pH value of the enzymolysis system to 7.25, boiling for 15min to complete the reaction, cooling, centrifuging (3500rpm, 15min), collecting the supernatant, dialyzing (interception amount of 200Da), and freeze-drying to obtain maca immunomodulatory protein zymolyte.
The yield of the final maca immunomodulatory protein zymolyte obtained in the embodiment is 47.00%, the hydrolysis degree is 56.61%, the yield of soluble peptides is 28.39%, and the content of free amino acids is 670.49mg/100g of maca protein; the level of promoting mouse macrophage RAW 264.7 to express cytokine TNF-alpha is 17.41ng/mL, which is increased by 287.75% compared with blank group, as shown in B2 in figure 3. The zymolyte can be used for preparing capsule, tablet, pill, granule or oral liquid.
Example 3
The embodiment is a preparation method of maca immunomodulatory protein zymolyte with immunomodulatory activity (papain pre-enzymolysis combined with simulated gastrointestinal digestion), and the specific steps are as follows:
(1) preparing maca protein: taking 50g of maca rhizome powder as a raw material, and extracting with 2L of Tris-HCl solution (0.05mol/L, pH 9.0) as an extracting solution, wherein the material-liquid ratio is 1 g: 40mL, ultrasonic treatment (70 ℃, 250W, 30min), extraction time of 60min and extraction temperature of 70 ℃. Centrifuging for the first time (4000rpm, 15min), filtering (500-mesh filter cloth), collecting supernatant, adding 1.0mol/LHCl aqueous solution until the pH of the supernatant reaches 4.0, standing for 40min, centrifuging for the second time (4000rpm, 20min), redissolving with 1.0mol/LNaOH aqueous solution for precipitation, dialyzing for desalination (5000Da), and lyophilizing to obtain maca protein.
(2) Pre-enzymolysis: dissolving 1.0g of maca protein in 50mL of distilled water to prepare a 1% (w/v, g/mL) maca protein solution, and heating in a boiling water bath for 20min for the first time to inactivate a protease inhibitor in the maca protein. 118.20mg of papain (enzyme activity 423U/mg) was added, the temperature was adjusted to 55 ℃, the pH was adjusted to 7.5, and the mixture was preheated for 20 min. During the enzymolysis, the pH is adjusted every 15min for the first 30min, and then every 30min, so that the reaction system keeps constant pH at 7.5. The enzymolysis time is 4h, and after the reaction is finished, the enzymolysis liquid is placed in a boiling water bath for heating for 20min for the second time so as to stop the reaction.
(3) Simulating gastric digestion: dripping 1mol/L HCl aqueous solution to make the pH value reach 3.0, adding 143.90mg pepsin (enzyme activity is 250U/mg) after the temperature reaches 37 ℃, and magnetically stirring for reaction for 3 hours;
(4) simulating intestinal digestion: adding 1mol/L NaOH aqueous solution dropwise to make pH reach 7.5, stopping simulating gastric digestion, adding 625.00mg pancreatin (enzyme activity is 8U/mg), and reacting for 3h at 37 deg.C under magnetic stirring.
(5) Adjusting the pH value of the enzymolysis system to 7.5, boiling for 20min to complete the reaction, cooling, centrifuging (4000rpm, 20min), collecting supernatant, dialyzing (interception amount of 500Da), and freeze-drying to obtain maca immunomodulatory protein zymolyte.
The yield of the maca protein immunoregulation white zymolyte finally obtained in the embodiment is 42.00%, the hydrolysis degree is 56.38%, the yield of soluble peptides is 27.19%, and the content of free amino acid is 584.53mg/100g maca protein; the level of promoting mouse macrophage RAW 264.7 to express cytokine TNF-alpha is 18.74ng/mL, which is increased by 317.37% compared with blank group, as shown in B3 in figure 3. The zymolyte can be used for preparing capsule, tablet, pill, granule or oral liquid.
Example 4
The embodiment is a preparation method of maca immunomodulatory protein zymolyte with immunomodulatory activity (bromelain pre-enzymolysis combined with simulated gastrointestinal digestion), and the specific steps are as follows:
(1) preparing maca protein: taking 50g of maca rhizome powder as a raw material, and extracting with 1.5L of Tris-HCl solution (0.05mol/L, pH 9.0) as an extracting solution, wherein the material-to-liquid ratio is 1 g: 30mL, ultrasonic treatment (50 ℃, 200W, 20min), extraction time of 30min and extraction temperature of 60 ℃. Centrifuging for the first time (3000rpm, 5min), filtering (500 mesh filter cloth), collecting supernatant, adding 0.5mol/LHCl aqueous solution until the pH of the supernatant reaches 3.75, standing for 30min, centrifuging for the second time (3000rpm, 10min), redissolving with 0.5mol/LNaOH aqueous solution for precipitation, dialyzing for desalination (5000Da), and lyophilizing to obtain maca protein.
(2) Pre-enzymolysis: 0.5g of maca protein is dissolved in 50mL of distilled water to prepare 1% (w/v, g/mL) of maca protein solution, and the maca protein solution is heated in a boiling water bath for 10min for the first time to inactivate a protease inhibitor in the maca protein. Adding 135.50mg bromelain (enzyme activity 369U/mg), adjusting temperature to 55 deg.C, adjusting pH to 7.0, and preheating for 10 min. In the enzymolysis process, the pH is adjusted every 5min for the first 10min, and then the pH is adjusted every 10min, so that the reaction system keeps constant pH at 7. The enzymolysis time is 3h, and after the reaction is finished, the enzymolysis liquid is placed in a boiling water bath for heating for 10min for the second time so as to stop the reaction.
(3) Simulating gastric digestion: dripping 1mol/L HCl aqueous solution to make the pH value reach 3.0, adding 143.90mg pepsin (enzyme activity is 250U/mg) after the temperature reaches 37 ℃, and magnetically stirring for reaction for 2 hours;
(4) simulating intestinal digestion: adding 1mol/L NaOH aqueous solution dropwise to make pH reach 7.0, stopping simulating gastric digestion, adding 625.00mg pancreatin (enzyme activity is 8U/mg), and reacting for 2h at 37 deg.C under magnetic stirring.
(5) Adjusting the pH value of the enzymolysis system to 7.0, boiling for 10min to complete the reaction, cooling, centrifuging (3000rpm for 10min), collecting the supernatant, dialyzing (interception amount of 100Da), and freeze-drying to obtain the maca immunomodulatory protein zymolyte.
The yield of the final maca immunomodulatory protein zymolyte obtained in the embodiment is 39.00%, the hydrolysis degree is 56.50%, the yield of soluble peptides is 26.55%, and the content of free amino acids is 540.89mg/100g maca protein; the level of promoting mouse macrophage RAW 264.7 to express the cytokine TNF-alpha is 20.43ng/mL, which is increased by 355.01% compared with the blank group, as shown in B4 in figure 3. The zymolyte can be used for preparing capsule, tablet, pill, granule or oral liquid.
Example 5
The embodiment is a preparation method of maca immunomodulatory protein zymolyte with immunomodulatory activity (the combination of compound protease pre-enzymolysis and simulated gastrointestinal digestion), and the specific steps are as follows:
(1) preparing maca protein: taking 50g of maca rhizome powder as a raw material, and extracting by taking 1.5L Tris-HCl solution (0.05mol/L, pH 9.0) as an extracting solution, wherein the material-to-liquid ratio is 1 g: 30mL, ultrasonic treatment (55 ℃, 225W, 25min), extraction time of 45min and extraction temperature of 65 ℃. Centrifuging for the first time (3500rpm, 10min), filtering (500 mesh filter cloth), collecting supernatant, adding 0.75mol/LHCl aqueous solution until the pH of the supernatant reaches 3.75, standing for 35min, centrifuging for the second time (3500rpm, 15min), redissolving with 0.75mol/LNaOH aqueous solution for precipitation, dialyzing for desalination (5000Da), and lyophilizing to obtain maca protein.
(2) Pre-enzymolysis: dissolving 0.75g of maca protein in 50mL of distilled water to prepare 1.5% (w/v, g/mL) of maca protein solution, and heating in a first boiling water bath for 15min to inactivate a protease inhibitor in the maca protein. Adding 806.50mg of compound protease (enzyme activity is 62U/mg), adjusting temperature to 45 deg.C, adjusting pH to 7.0, and preheating for 15 min. In the enzymolysis process, the pH is adjusted every 10min for the first 20min, and then the pH is adjusted every 20min, so that the reaction system keeps constant pH at 7. The enzymolysis time is 3.5h, and after the reaction is finished, the enzymolysis liquid is placed in a boiling water bath and heated for 15min in a second boiling water bath to stop the reaction.
(3) Simulating gastric digestion: dropwise adding 0.75mol/L HCl aqueous solution to make pH reach 2.25, adding 143.90mg pepsin (enzyme activity is 250U/mg) after the temperature reaches 36.5 ℃, and magnetically stirring to react for 2.5 h;
(4) simulating intestinal digestion: 0.75mol/L NaOH aqueous solution is added dropwise to make the pH value reach 7.0, the simulated gastric digestion is stopped, 625.00mg of pancreatin (enzyme activity is 8U/mg) is added, and the mixture is magnetically stirred and reacted for 2.5 hours at the temperature of 36.5 ℃.
(5) Adjusting the pH value of the enzymolysis system to 7.25, boiling for 15min to complete the reaction, cooling, centrifuging (3500rpm, 15min), collecting the supernatant, dialyzing (interception amount of 200Da), and freeze-drying to obtain maca immunomodulatory protein zymolyte.
The yield of the final maca immunomodulatory protein zymolyte obtained in the embodiment is 56.00%, the hydrolysis degree is 57.06%, the yield of soluble peptides is 33.45%, and the content of free amino acids is 758.76mg/100g maca protein; the level of promoting mouse macrophage RAW 264.7 to express cytokine TNF-alpha is 17.43ng/mL, which is increased by 288.20% compared with blank group, as shown in B5 in figure 3. The zymolyte can be used for preparing capsule, tablet, pill, granule or oral liquid.
Example 6
The embodiment is a preparation method of maca immunoregulation peptide, which comprises the following specific steps:
(1) preparing maca protein: taking 50g of maca rhizome powder as a raw material, and extracting with 1.5L of Tris-HCl solution (0.05mol/L, pH 9.0) as an extracting solution, wherein the material-to-liquid ratio is 1 g: 30mL, ultrasonic treatment (50 ℃, 200W, 20min), extraction time of 30min and extraction temperature of 60 ℃. Centrifuging for the first time (3000rpm, 5min), filtering (500 mesh filter cloth), collecting supernatant, adding 0.5mol/LHCl aqueous solution until the pH of the supernatant reaches 3.5, standing for 30min, centrifuging for the second time (3000rpm, 10min), redissolving with 0.5mol/LNaOH aqueous solution for precipitation, dialyzing for desalination (5000Da), and lyophilizing to obtain maca protein.
(2) Pre-enzymolysis: dissolving 0.5g of maca protein in 50mL of distilled water to prepare a 1% (w/v, g/mL) maca protein solution, and heating in a boiling water bath for 10min for the first time to inactivate a protease inhibitor in the maca protein. 135.50mg bromelain (enzyme activity 369U/mg), adjusting the temperature to 55 deg.C, adjusting pH to 7.0, and preheating for 10 min. During the enzymolysis, the pH is adjusted every 5min for the first 10min, and then every 10min to keep the reaction system at a constant pH of 7. The enzymolysis time is 3h, and after the reaction is finished, the enzymolysis liquid is placed in a boiling water bath for heating for 10min for the second time to stop the reaction.
(3) Simulating gastric digestion: dropwise adding 0.5mol/L HCl aqueous solution to make the pH value reach 1.5, adding 143.90mg of pepsin (enzyme activity is 250U/mg) after the temperature reaches 36 ℃, and carrying out magnetic stirring reaction for 2 hours;
(4) simulating intestinal digestion: 0.5mol/L NaOH aqueous solution is added dropwise to enable the pH value to reach 7.0, digestion simulation is stopped, 625.00mg of pancreatin (enzyme activity is 8U/mg) is added, and the mixture is magnetically stirred and reacted for 2 hours at 36 ℃.
(5) Adjusting the pH value of the enzymolysis system to 7.0, boiling for 10min to complete the reaction, cooling, centrifuging (3000rpm for 10min), collecting the supernatant, dialyzing (interception amount of 100Da), and freeze-drying to obtain the maca immunomodulatory protein zymolyte.
(6) Dissolving the 20mg maca immunomodulatory protein zymolyte obtained in the step (4) in 80mL of ultrapure water to prepare a solution (0.25mg/mL), sequentially grouping by using ultrafiltration tubes with cut-off amounts of 10kDa (centrifugation 5000rpm,30min) and 3kDa (centrifugation 5000rpm,60 min), taking sample liquid at the lower part of the 3kDa ultrafiltration tube, and evaporating, concentrating, freezing and drying to obtain the graded maca immunomodulatory protein zymolyte with the molecular weight less than 3 kDa. Dissolving the hierarchical maca immunomodulatory protein zymolyte with the molecular weight less than 3kDa by using a dissolving solution (0.1% (v/v) formic acid and 2% (v/v) acetonitrile), centrifuging (4 ℃, 13200rpm, 20min), taking the supernatant, separating by liquid chromatography, directly feeding the separated peptide segment into a mass spectrometer for on-line detection, and searching the library to obtain a sequence. Selecting peptides containing 2-10 amino acids, carrying out PeptideRanker website activity scoring and HPEPDCCK website molecule butt joint (respectively butt joint with pattern recognition receptors TLR2 and TLR 4) to obtain 10 peptide segments with the highest expected immune regulation activity, and synthesizing the peptides by an Fmoc solid phase synthesis method according to sequences, wherein the specific sequences are NPYPFFGFSI, VSMWRYGIW, LFNPIFSIF, SVPWVARF, AIIFIFIRR, LSVDFVFVMC, IGFWNSNGSF, KFGDLFHLRM, CGRTPFLVWG and SMHLPRPF.
(7) And (3) selecting a mouse macrophage RAW 264.7 to evaluate the immunoregulatory activity of the 10 peptide fragments obtained in the step (5). The peptide segments No.1 NPYPFFGFSI and No.4 SVPWVARF with the highest activity promote the TNF-alpha secretion of RAW 264.7 cells at 250 μ g/mL with 4.93 plus or minus 0.03pg/mL and 4.21 plus or minus 0.04pg/mL respectively, as shown in FIG. 4 (1-10 in FIG. 4 respectively represent NPYPFFGFSI, VSMWRYGIW, LFNPIFSIF, SVPWVARF, AIIFIFIRR, LSVDFVFVMC, IGFWNSNGSF, KFGDLFHLRM, CGRTPFLVWG and SMHLPRPF, and the blank in FIG. 4 is DMEM medium containing 10% (v/v) fetal calf serum and 1% (v/v) double antibody).
Comparative example 1
(1) Preparing maca protein: taking 50g of maca rhizome powder as a raw material, and extracting with 1.5L of Tris-HCl solution (0.05mol/L, pH 9.0) as an extracting solution to obtain an extracting solution, wherein the material-to-liquid ratio is 1 g: 30mL, ultrasonic treatment (50 ℃, 200W, 20min), extraction time of 30min and extraction temperature of 60 ℃. Centrifuging for the first time (3000rpm, 5min), filtering (500-mesh filter cloth), collecting supernatant, adding 0.5mol/LHCl aqueous solution until the pH of the supernatant reaches 3.75, standing for 30min, centrifuging for the second time (3000rpm, 10min), redissolving with 0.5mol/LNaOH aqueous solution for precipitation, dialyzing for desalination (5000Da), and lyophilizing to obtain maca protein.
(2) Dissolving 0.5g of maca protein in 50mL of distilled water to prepare a 1% (w/v, g/mL) maca protein solution, and heating in a boiling water bath for 10min to inactivate a protease inhibitor in the maca protein. Simulating gastric digestion: dropwise adding 1mol/L HCl aqueous solution to make the pH value reach 3.0, adding 143.90mg of pepsin (enzyme activity is 250U/mg) after the temperature reaches 37 ℃, and carrying out magnetic stirring reaction for 2 hours;
(3) simulating intestinal digestion: adding 1mol/L NaOH aqueous solution dropwise to make pH reach 7.0, stopping simulating gastric digestion, adding 625.00mg pancreatin (enzyme activity is 8U/mg), and reacting for 2h at 37 deg.C under magnetic stirring.
(4) Adjusting pH of the enzymolysis system to 7.0, boiling for 10min to complete reaction, cooling, centrifuging (3000rpm, 10min), collecting supernatant, dialyzing (interception amount of 100Da), and freeze drying to obtain maca protein zymolyte.
The yield of the maca protein hydrolysate obtained finally in comparative example 1 is 28.00%, the hydrolysis degree is 22.91%, the yield of soluble peptides is 17.20%, and the content of free amino acids is 395.56mg/100g maca protein; the level of promoting mouse macrophage RAW 264.7 to express the cytokine TNF-alpha is 19.14ng/mL, which is increased by 326.28% compared with the blank group, as shown in MCPH in figure 3.
A comparison of the yields, degree of hydrolysis, soluble peptide yield, free amino acid content, and enhanced expression of the cytokine TNF-. alpha.by the mouse macrophage RAW 264.7 of example 4 and comparative example 1 is shown in Table 1. Under the same concentration, the yield of the maca immunomodulatory protein zymolyte obtained in example 4 of the invention is 11% higher than that of the maca proteolytic zymolyte obtained in comparative example 1, and the level of promoting mouse macrophage RAW 264.7 to express cytokine TNF-alpha is 28.73% higher than that of comparative example 1, so that the immunomodulatory activity of the maca immunomodulatory protein zymolyte obtained in example 4 is higher.
TABLE 1 yield, degree of hydrolysis, soluble peptide yield, free amino acid content and TNF-. alpha.expression in RAW 264.7 cells
Figure BDA0003662733800000131
Sequence listing
<110> university of southern China's science
<120> maca immunoregulation protein zymolyte and peptide, and preparation method and application thereof
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> maca (Lepidium meyenii Walp.)
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Asn Pro Tyr Pro Phe Phe Gly Phe Ser Ile
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<213> maca (Lepidium meyenii Walp.)
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Val Ser Met Trp Arg Tyr Gly Ile Trp
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Leu Phe Asn Pro Ile Phe Ser Ile Phe
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<210> 4
<211> 8
<212> PRT
<213> maca (Lepidium meyenii Walp.)
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Ser Val Pro Trp Val Ala Arg Phe
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<213> maca (Lepidium meyenii Walp.)
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Ala Ile Ile Phe Ile Phe Ile Arg Arg
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Leu Ser Val Asp Phe Val Phe Val Met Cys
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<210> 8
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<213> maca (Lepidium meyenii Walp.)
<400> 8
Lys Phe Gly Asp Leu Phe His Leu Arg Met
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<210> 9
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<213> maca (Lepidium meyenii Walp.)
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Cys Gly Arg Thr Pro Phe Leu Val Trp Gly
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Ser Met His Leu Pro Arg Pro Phe
1 5

Claims (10)

1. The preparation method of the maca immunoregulation protein zymolyte is characterized by comprising the following steps:
(1) preparing maca protein: taking maca rhizome powder as a raw material, taking a Tris-HCl solution with the pH of 7.0-9.0 as an extracting solution for extraction, performing ultrasonic post-extraction, performing first centrifugation, filtering to obtain a supernatant, adding a 0.5 mol/L-1.0 mol/LHCl solution to adjust the pH to 3.5-4.0, standing for 30-40 min, performing second centrifugation, re-dissolving and precipitating with a 0.5 mol/L-1.0 mol/LNaOH solution, performing dialysis desalination with the cut-off of 2000 Da-5000 Da, and performing freeze-drying to obtain maca protein;
(2) pre-enzymolysis: preparing a maca protein solution by using the maca protein obtained in the step (1), heating in a first boiling water bath for 10-20 min, adding protease, adjusting the temperature and the pH, preheating for 10-20 min, adjusting the pH every 5-15 min in the first 10-30 min during enzymolysis, adjusting the pH every 10-30 min later to keep the pH constant, wherein the enzymolysis time is 3-4 h, and after the reaction is finished, placing an enzymolysis liquid in the boiling water bath, and heating in a second boiling water bath for 10-20 min to stop the reaction;
(3) simulating gastrointestinal digestion: simulating the gastric digestion stage: adding pepsin, magnetically stirring for reaction, adjusting pH, and stopping gastric digestion simulation; simulating the intestinal digestion stage: and adding pancreatin, carrying out magnetic stirring reaction, then adjusting the pH value of an enzymolysis system to 7.0-7.5, boiling for 10-20 min to completely finish the reaction, cooling, centrifuging, taking supernatant, dialyzing to obtain the interception amount of 100-500 Da, and freeze-drying to obtain the maca immunomodulatory protein zymolyte.
2. The preparation method of the maca immunomodulatory protein zymolyte of claim 1, wherein the ultrasound conditions in step (1) are 200W-250W, 20 min-30 min, 50-70 ℃; the extraction conditions in the step (1) are 30-60 min and 50-70 ℃.
3. The method for preparing maca immunomodulatory protein zymolyte according to claim 1, wherein the conditions of the first centrifugation in step (1) are 3000rpm to 4000rpm, 5min to 15 min; the conditions of the second centrifugation are 3000 rpm-4000 rpm and 10 min-20 min.
4. The method for preparing maca immunomodulatory protein zymolyte of claim 1, wherein in step (2) the protease is one of neutral protease, alkaline protease, papain, bromelain and complex protease.
5. The method for preparing maca immunomodulatory protein zymolyte according to claim 1, wherein in step (2), the temperature is adjusted to 40-55 ℃ and the pH is adjusted to 7.0-10.5.
6. The preparation method of the maca immunomodulatory protein zymolyte according to claim 1, wherein in the step (3), the reaction pH of the simulated gastric digestion stage is 1.5-3.0, the temperature is 36-37 ℃, and the reaction time is 2-3 h; the reaction pH value of the simulated intestinal digestion stage is 6.5-7.5, the temperature is 36-37 ℃, and the time is 2-3 h; the centrifugation conditions in the step (3) are 3000 rpm-4000 rpm and 10 min-20 min.
7. The preparation method of the maca immunoregulation peptide is characterized by comprising the following steps:
screening and preparing maca immunoregulation peptide: selecting the maca immunomodulatory protein zymolyte which is subjected to protease pre-enzymolysis treatment in the step (3) of the step (1) to prepare a maca immunomodulatory protein zymolyte solution, performing ultrafiltration grouping, performing evaporation concentration and freeze drying to obtain graded maca proteolytic zymolyte with the molecular weight less than 3kDa, dissolving the graded maca proteolytic zymolyte with the molecular weight less than 3kDa, centrifuging to obtain supernatant, performing liquid chromatography separation, directly feeding the separated peptide segment into a mass spectrometer for online detection and searching a library to obtain a sequence, selecting peptide, performing activity scoring, performing molecular docking with a mode recognition receptor to obtain a target peptide segment, and synthesizing the peptide according to the sequence.
8. The preparation method of the maca immunomodulatory peptide of claim 7, wherein the selected peptide is a peptide containing 2-10 amino acids; the peptide synthesis method is Fmoc solid phase synthesis.
9. The maca immunomodulatory protein hydrolysate or the maca immunomodulatory peptide prepared by the preparation method of claim 1 or claim 7.
10. Use of a maca immunomodulatory protein hydrolysate or a maca immunomodulatory peptide of claim 9 in an immunomodulatory functional food.
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