Spina date seed fermented polypeptide beverage and preparation technology thereof

Abstract

The invention relates to the technical field of polypeptide preparation by a fermentation method, in particular to a spina date seed fermented polypeptide beverage and a preparation process thereof. The semen ziziphi spinosae fermented polypeptide beverage is prepared by taking semen ziziphi spinosae degreased powder as a raw material and adopting a microbial fermentation method, the molecular weight of polypeptide is generally distributed between 4 kDa and 10 kDa, and the molecular weight is smaller and more concentrated. The spina date seed fermented polypeptide beverage obtained by the invention can improve the compliance of consumers, is convenient for the consumers to use for a long time, achieves the effects of health preservation, health care and prevention and treatment of diseases, and has important practical significance for development of traditional Chinese medicine resources and improvement of economic value.

Description

Technical Field

[0001] The invention relates to the technical field of preparing polypeptides by fermentation, and in particular to a wild jujube seed fermented polypeptide beverage and a preparation process thereof. Background Art

[0002] Fatigue is a feeling of discomfort caused by excessive exercise, lack of sleep, etc. Long-term fatigue can cause low immunity and cause diseases related to the immune system, which seriously affects life and work. At present, fatigue is mainly relieved through physical therapy and nutritional supplements. Physical therapy includes acupuncture, massage and other methods. Although it can eliminate fatigue, it takes a long time and requires treatment in a specific place, which is extremely inconvenient. Nutritional supplements are the intake of some food-borne active ingredients, such as peptides that can reduce fatigue.

[0003] The seeds of the Chinese jujube seeds are the dried mature seeds of the plant Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou of the Rhamnaceae family. They mainly contain chemical components such as flavonoids, triterpenoid saponins, fatty oils, alkaloids, proteins, etc., and have sedative, anti-anxiety, and antioxidant activities. The seeds of the Chinese jujube seeds are important Chinese medicinal and edible materials for both medicine and food in my country. From the perspective of the development and registration of Chinese medicinal health products, the seeds of the Chinese jujube seeds are among the top 20, and the development prospects are broad. At present, the products related to the seeds of the Chinese jujube seeds have been developed, such as the seeds of the Chinese jujube seeds tea, the seeds of the Chinese jujube seeds beverage, and the seeds of the Chinese jujube seeds edible oil. The seeds of the Chinese jujube seeds are rich in protein, accounting for more than 20% of the quality of the seeds of the Chinese jujube seeds. Protein can provide energy for the body, supplement nutrition, and is beneficial to human health. It also has biological activities such as antioxidant, anti-fatigue, and immune regulation. However, the absorption and utilization rate of macromolecular proteins in the human body is low. If the macromolecular protein substances are converted into small molecular polypeptides, it will be more conducive to human absorption.

[0004] Fermentation is one of the methods to hydrolyze proteins in fermentation substrates into polypeptides with smaller molecular weights. Fermentation can utilize a variety of proteases secreted by bacteria to hydrolyze substrates. The hydrolyzate is rich in variety and can mask the bitterness of polypeptides, making them more palatable. In recent years, fermentation has become a research hotspot in the preparation of bioactive peptides. Compared with the enzymatic hydrolysis process of specific types of proteases, microbial fermentation has gradually been applied to the decomposition process of macromolecular substances due to its simple process, low cost, rich enzyme diversity, smaller and more concentrated molecular weight, etc. Summary of the invention

[0005] The present invention proposes a Chinese jujube seed fermented polypeptide beverage and a preparation process thereof. The fermented beverage obtained by the present invention can improve consumer compliance and has better sensory properties, is convenient for consumers to use for a long time, and achieves the effect of improving health care and preventing and treating diseases. It has important practical significance for the development of traditional Chinese medicine resources and the improvement of economic value.

[0006] The fermented polypeptide beverage of wild jujube seeds of the invention is prepared by using defatted wild jujube seeds powder as raw material and adopting a microbial fermentation method.

[0007] The specific steps are:

[0008] (1) Weighing defatted jujube seed powder, adding distilled water and glucose, sterilizing, cooling, and obtaining a fermentation medium;

[0009] (2) inoculating the bacterial strain into the fermentation matrix, fermenting, and obtaining a fermentation liquid; centrifuging the fermentation liquid to obtain a fermentation supernatant;

[0010] (3) Add xanthan gum, xylitol, anhydrous citric acid and potassium sorbate to the fermentation supernatant, stir evenly, let stand, check, filter, fill and sterilize to obtain the finished product.

[0011] In step (1), the weight ratio of defatted jujube kernel powder to distilled water is 1:14.

[0012] In step (2), the bacterial species is selected from Aspergillus niger.

[0013] In step (2), the fermentation conditions are: fermentation time 60h, material-liquid ratio 1:14, inoculation amount 1%.

[0014] In step (2), the fermentation liquid is centrifuged at a speed of 4000 r / min. -1 , time 20 minutes.

[0015] In step (3), the amount of xanthan gum added is 0.05% of the mass of the fermentation supernatant, calculated on a mass ratio basis.

[0016] In step (3), the amount of xylitol added is 5% of the mass of the fermentation supernatant, calculated on a mass ratio basis.

[0017] In step (3), the amount of anhydrous citric acid added is 0.05% of the mass of the fermentation supernatant, calculated on a mass ratio basis.

[0018] In step (3), 0.4 g of potassium sorbate is added per kilogram of fermentation supernatant on a mass basis.

[0019] The present invention mainly treats the protein with relatively rich content in the spinach kernel by microbial fermentation, optimizes the fermentation process with the polypeptide content as an indicator, optimizes the preparation process with the sensory score as an indicator, conducts quality standard research and stability research through small-scale tests, and finally prepares the spinach kernel fermented polypeptide beverage, which also provides a reference for the separation and purification of polypeptides. Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention uses microbial fermentation to convert the protein in the raw material into small molecular polypeptides that are easier to be absorbed and utilized by the human body. The operation is simple and the fermentation cycle is short. The molecular weight of the polypeptide is distributed in the range of 4 kDa to 10 kDa. The molecular weight is smaller and the distribution is more concentrated, and the fermentation efficiency is high.

[0021] (2) Liquid fermented Chinese medicine overcomes the shortcomings of Chinese herbal decoctions, such as large dosage, inconvenience in carrying, inability to store for a long time, and inability to produce commercially.

[0022] (3) Fermentation can significantly increase the content of active ingredients and antioxidant activity in defatted jujube kernel powder, which has the advantages of easier absorption, high bioavailability, and rapid effect, and can play a better role.

[0023] (4) Compared with the existing lactobacillus fermentation, the present invention adopts Aspergillus niger aerobic fermentation, and the polypeptide content in the product is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of SDS-PAGE electrophoresis, where M: protein marker; BSA: bovine serum albumin; 1: protein solution of Ziziphus jujuba seeds; 2: fermentation broth of Aspergillus niger;

[0025] Figure 2 The results of Tris-Tricine-SDS-PAGE electrophoresis, where 1: Ziziphus jujuba seed protein solution; 2-5: Aspergillus niger fermentation broth with different concentrations: 20, 30, 40, 50 mg / mL;

[0026] Figure 3 The effect of different stabilizer addition amounts on centrifugal sedimentation rate;

[0027] Figure 4 It is a process flow chart of the present invention. DETAILED DESCRIPTION

[0028] Example 1

[0029] The fermented polypeptide beverage of Chinese jujube kernels is prepared by aerobic fermentation of Aspergillus niger with the defatted powder of Chinese jujube kernels as raw material after the strains are selected.

[0030] The specific steps are:

[0031] The spiny jujube kernels were crushed and passed through a 60-mesh sieve. The spiny jujube kernel powder and petroleum ether were mixed at a solid-liquid ratio of 1:3 (g / mL), extracted in a 50°C water bath for 1 h (stirring with a glass rod from time to time during the extraction), and centrifuged (3000r / min, 20min) to remove the supernatant. This was repeated 4 to 5 times until the supernatant was clear and transparent. The powder was ventilated at room temperature for 12 h to obtain the spiny jujube kernel defatted powder, which was passed through a 40-mesh sieve and added to distilled water at a ratio of 1:14 and a concentration of 15 g / L. Water and anhydrous glucose are sterilized at 121°C for 30 min. After cooling to room temperature, Aspergillus niger is added at a ratio of 1%. After aerobic fermentation at 30°C for 60 h, samples are taken. The fermentation liquid is centrifuged at 4000 r / min for 20 min, the supernatant is taken out, 0.05% xanthan gum, 0.4 g / kg potassium sorbate, 5% xylitol and 0.05% anhydrous citric acid are added, mixed well, packaged, 100 mL per bottle, and sterilized.

[0032] Example 2 Screening of fermentation conditions

[0033] 1. Preparation of fermentation broth

[0034] Weigh 5g of defatted jujube kernel powder and place it in a 150mL conical flask, add a certain amount of glucose, add distilled water according to a certain material-liquid ratio, pH natural, autoclave at 121℃ for 20min, wait for the conical flask to cool to room temperature, inoculate according to a certain inoculation amount, ferment on a shaker at 200r / min at 30℃, take out samples fermented at different time points, and inactivate the enzyme at 100℃ for 20min. The single factor experimental conditions are shown in Table 1, the orthogonal experimental factor level design is shown in Table 2, and the orthogonal optimization conditions are shown in Table 3.

[0035] 2 Single factor experiment

[0036] (1) Under the conditions of fermentation time of 48 h, solid-liquid ratio of 1:10, and inoculation amount of 6%, the effect of glucose mass concentration (10 g / L, 20 g / L, 30 g / L, 40 g / L and 50 g / L) on polypeptide content was examined.

[0037] (2) Fermentation time: Under the conditions of glucose concentration of 10 g / L, solid-liquid ratio of 1:10, and inoculation amount of 6%, the effect of fermentation time (48 h, 72 h, 96 h, 120 h and 144 h) on the polypeptide content was examined.

[0038] (3) The effects of the solid-liquid ratio (1:8, 1:10, 1:12, 1:14 and 1:16) on the polypeptide content were investigated under the conditions of glucose concentration of 10 g / L, fermentation time of 48 h and inoculation size of 6%.

[0039] (4) Inoculation amount: Under the conditions of glucose concentration of 10 g / L, fermentation time of 48 h, and solid-liquid ratio of 1:10, the effect of inoculation amount (3%, 6%, 9%, 12% and 15%) on polypeptide content was examined.

[0040] 3 Orthogonal experiment

[0041] On the basis of single factor experiment, four factors and three levels were used to optimize orthogonal experiment, and verification experiment was carried out based on the best combination.

[0042] Table 1 Single factor experimental conditions

[0043]

[0044] Table 2 Orthogonal experimental factor level design

[0045]

[0046] Table 3 Orthogonal optimization conditions

[0047]

[0048]

[0049] The fermentation liquid was centrifuged at a speed of 4000 r / min for 15 min, and the supernatant was poured out to obtain the fermentation supernatant.

[0050] 4. Determination of polypeptide content

[0051] Take 2mL of fermentation supernatant and add an equal volume of 10% trichloroacetic acid, mix well and centrifuge at 16000r / min for 7min. Take 1mL of supernatant and add 4mL of biuret reagent, mix well, keep it away from light for 30min, and measure its absorbance at a wavelength of 540nm. Distilled water is used as blank. Using bovine serum albumin V as the standard, prepare 0, 2, 4, 6, 8, 10mg / mL standard sample solutions and make a standard curve. The polypeptide content is calculated according to the following formula:

[0052] Peptide content (mg / g) = C*V / m

[0053] Where: C is the mass concentration of the polypeptide in the supernatant, mg / mL; V is the total volume of the fermentation broth, mL; m is the mass of the sample weighed and measured after the raw material fermentation, g.

[0054] 5 Results and Analysis

[0055] Table 4 shows the results of single factor investigation, and Table 5 shows the results of orthogonal experiment.

[0056] Table 4 Peptide content results of single factor experiment ( n=3)

[0057]

[0058]

[0059] Table 5 Orthogonal experiment table

[0060]

[0061]

[0062] The results show that the optimal solution for the polypeptide content of defatted jujube kernel powder fermented by Aspergillus niger is A3B3C2D1.

[0063] 6 Validation Experiment

[0064] The optimal combination of Aspergillus niger was repeated three times, and the average polypeptide content was 45.9302 mg / g original medicinal material, which was higher than that of other groups of experiments in each orthogonal optimization table. Therefore, the optimal fermentation conditions for Aspergillus niger were glucose concentration of 15 g / L, fermentation time of 60 h, solid-liquid ratio of 1:14, and inoculation amount of 1%.

[0065] Example 3 Changes in polypeptides before and after fermentation and determination of antioxidant capacity

[0066] 1. Preparation of fermentation samples

[0067] Weigh 5g of defatted jujube kernel powder and place it in a 250mL conical flask. Add distilled water and glucose according to the solid-liquid ratio of 1:14 and the glucose mass concentration of 15g / L. The pH is natural and sterilized at 121℃ for 20min. After the conical flask is cooled to room temperature, inoculate it at 1% inoculation amount and ferment it on a shaker at 200r / min at 30℃ for 60h. Take out the fermentation product after a certain period of culture and inactivate the enzyme at 100℃ for 20min. Centrifuge at 4000r / min for 20min, measure the volume of the supernatant, and store the supernatant at 4℃ for later use.

[0068] 2 Preparation of unfermented samples

[0069] Weigh 5 g of defatted jujube kernel powder and place it in a 150 mL conical flask, add water at a solid-liquid ratio of 1:10, do not inoculate, autoclave at 121°C for 20 min, centrifuge at 4000 r / min for 20 min, and store the supernatant at 4°C for later use.

[0070] 3SDS-PAGE electrophoresis

[0071] Place the prepared gel block in the electrophoresis tank, fill the inner tank with electrophoresis buffer, and place it at room temperature for 10 minutes. If there is no liquid seeping out from the bottom, it means there is no leakage. Then fill the outer tank with electrophoresis buffer, add the treated sample to the sample well, and load 5μL of the color pre-stained protein molecular weight standard (10-170kD) for electrophoresis. The electrophoresis conditions are 70V for concentrated gel voltage, 20mA for current, 140V for separation gel voltage, and 30mA for current. The end point of electrophoresis is when all the bands of the color pre-stained protein molecular weight standard (10-170kD) run out. After the electrophoresis is completed, turn off the electrophoresis device, cut off all the concentrated gel, and take the separation gel for Coomassie Brilliant Blue staining. Soak the separation gel in Coomassie brilliant blue staining solution, stain at a constant speed for 40 minutes on a horizontal shaker, take out the separation gel, soak the separation gel in Coomassie brilliant blue destaining solution, destain at a constant speed on a horizontal shaker, change the destaining solution every 1 hour, and when the separation gel changes from blue to colorless and transparent, pour out the destaining solution and take out the separation gel. Place the destaining separation gel in an automatic gel imager for imaging, take pictures for analysis and save.

[0072] 4Tris-Tricine-SDS-PAGE electrophoresis

[0073] Put the prepared gel block into the electrophoresis tank, fill the inner tank with cathode buffer, and place it at room temperature for 10 minutes. If there is no liquid seeping out from the bottom, it means that there is no leakage. Then add anode buffer to the outer tank, put the electrophoresis tank into an ice water bath, add the treated sample to the sample loading hole, pre-stain ultra-low molecular weight protein Marker (3.3kD-31kD) with a sample amount of 10μL for electrophoresis, and the electrophoresis conditions are voltage 30V, current 20mA electrophoresis for 1h, voltage 100V, current 20mA electrophoresis until the indicator reaches the bottom of the gel as the electrophoresis end point. After the electrophoresis is completed, turn off the electrophoresis device, cut off all the concentrated gel and interlayer gel, and take the separation gel for Coomassie brilliant blue staining. Soak the separation gel in Coomassie brilliant blue staining solution, stain at a constant speed for 30 minutes on a horizontal shaker, take out the separation gel, soak the separation gel in Coomassie brilliant blue destaining solution, destain at a constant speed on a horizontal shaker, replace the destaining solution every 30 minutes, and when the separation gel changes from blue to colorless and transparent, pour out the destaining solution and take out the separation gel. Place the destaining separation gel in an automatic gel imager for imaging, take pictures for analysis and save.

[0074] 5. Determination of polypeptide content

[0075] Take 2mL of fermentation supernatant and add an equal volume of 10% trichloroacetic acid, mix well and centrifuge at 16000r / min for 7min. Take 1mL of supernatant and add 4mL of biuret reagent, mix well, keep it away from light for 30min, and measure its absorbance at a wavelength of 540nm. Distilled water is used as blank. Using bovine serum albumin V as the standard, prepare 0, 2, 4, 6, 8, 10mg / mL standard sample solutions and make a standard curve. The polypeptide content is calculated according to the following formula:

[0076] Peptide content (mg / g) = C*V / m

[0077] Where: C is the mass concentration of the polypeptide in the supernatant, mg / mL; V is the total volume of the fermentation broth, mL; m is the mass of the sample weighed and measured after the raw material fermentation, g.

[0078] 6 Antioxidant activity assay

[0079] 6.1DPPH free radical scavenging activity

[0080] Take 0.5mL VC sample solution and 0.5mL DPPH solution (0.1mmol / L, prepared on the spot, diluted with anhydrous ethanol) and mix well. Place it at room temperature and avoid light for 30min, and measure its absorbance at 517nm. Repeat the test for each sample 3 times, and calculate the average value of the 3 groups of data as the result. Use VC as a control, draw a standard curve based on the relationship between different VC concentrations and clearance rates, and calculate the DPPH free radical clearance rate according to the following formula.

[0081] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100%

[0082] Wherein, A1 is the absorbance after 0.5mL sample solution and 0.5mL DPPH solution are mixed; A2 is the absorbance after 0.5mL sample solution and 0.5mL anhydrous ethanol are mixed; A0 is the absorbance after 0.5mL DPPH solution and 0.5mL distilled water are mixed.

[0083] The regression equation is y=10029x+16.141, R 2 =0.997. The absorbance of the test solution was determined by the same method, and the DPPH radical scavenging rate of the test solution was calculated using the regression equation.

[0084] 6.2 ABTS free radical scavenging activity

[0085] Prepare 7mmol / L ABTS solution and 2.6mmol / L potassium persulfate solution with distilled water, mix equal volumes of ABTS solution and potassium persulfate solution, place at room temperature in the dark for 12-16h, dilute with distilled water until the OD value at 734nm is 0.70±0.02, and obtain ABTS reaction solution. Mix 0.5mL VC sample solution with 0.5mL ABTS reaction solution, place at 37℃ in the dark for 10min, and measure the absorbance at 734nm. Repeat the test for each sample 3 times, and average the 3 sets of data as the result. Calculate the ABTS free radical scavenging rate according to the following formula.

[0086] ABTS free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100%

[0087] Wherein, A1 is the absorbance after 0.5 mL sample solution and 0.5 mL ABTS reaction solution are mixed; A2 is the absorbance after 0.5 mL sample solution and 0.5 mL distilled water are mixed; A0 is the absorbance after 0.5 mL ABTS reaction solution and 0.5 mL distilled water are mixed.

[0088] According to the relationship between different VC concentrations and clearance rates, a standard curve was established, and the linear regression equation was y = 10029x + 16.141, R 2 =0.997. This indicates that VC has a good linear relationship in the concentration range of 0.001059 to 0.007413 mg / mL. Dilute the fermentation broth 60 times as the sample solution to be tested, and take 0.5 mL of the sample solution. The rest of the steps are the same as above. Calculate the VC equivalent antioxidant capacity of the sample solution according to the standard curve equation, and the result is expressed in VC equivalent (mg VC / g original medicinal material).

[0089] 6.3 Total antioxidant capacity (FRAP)

[0090] Take 0.3mL of FeSO4·7H2O reference solution of different concentrations, add 3mL of FRAP solution, mix thoroughly, react for 10min, measure its absorbance at 593nm wavelength, and draw a standard curve based on the relationship between different concentrations of FeSO4 and absorbance. According to the relationship between different concentrations of FeSO4 and absorbance, establish the regression equation y=4.1111x+0.186, R 2 =0.9979. The absorbance of the test solution was determined by the same method, and the FRAP value of the test solution was calculated using the regression equation. The FRAP value of the sample is equal to the number of mmol of FeSO4·7H2O extracted per gram of dry sample.

[0091] 7 Results and Analysis

[0092] 7.1 SDS-PAGE electrophoresis

[0093] Test results see Figure 1 .

[0094] The results showed that the protein in the jujube kernel was mainly distributed above 10KDa. After fermentation by Aspergillus niger, the protein was mainly decomposed into polypeptides below 10KDa. The reason may be that the acidic protease and neutral protease secreted by Aspergillus niger during its growth acted on the protein in the jujube kernel, making the proteolysis of the jujube kernel more complete.

[0095] 7.2 Tris-Tricine-SDS-PAGE electrophoresis

[0096] Test results see Figure 2 .

[0097] The results showed that the 20mg / mL to 30mg / mL fermentation broth did not show electrophoresis bands. In the concentration range of 40mg / mL to 50mg / mL, there were bands, but they were not very clear. It is speculated that this is because the small molecular polypeptides after fermentation are not much different, mainly concentrated in the range of 4kDa to 10kDa. This shows that after fermentation by Aspergillus niger, the large molecular weight jujube kernel protein was enzymatically hydrolyzed into smaller molecular weight polypeptide fragments.

[0098] 7.3 Analysis of peptide content determination results

[0099] The polypeptide content after fermentation was 45.93 mg / g of original medicinal material, while the polypeptide content of unfermented medicinal material was 23.98 mg / g of original medicinal material. The polypeptide content after fermentation increased by 91.53%.

[0100] 7.4 Results of DPPH free radical scavenging rate, ABTS free radical scavenging rate and FRAP total iron ion reducing capacity determination

[0101] The measurement results are shown in Table 6.

[0102] Table 6 DPPH free radical scavenging rate, ABTS free radical scavenging rate and FRAP iron ion total reducing ability determination results ( n=3)

[0103]

[0104] The results showed that the antioxidant activity of defatted jujube kernel powder after fermentation by Aspergillus niger was improved to a certain extent. Compared with the unfermented defatted jujube kernel powder, the DPPH free radical scavenging rate after fermentation increased by 49.41%; the ABTS free radical scavenging rate increased by 25.46%; and the total FRAP iron ion reducing capacity increased by 61.18%.

[0105] Example 4 Study on the Preparation Technology of Fermented Polypeptide Beverage from Ziziphus jujuba Seed

[0106] 1. Screening of stabilizer types and addition amounts

[0107] Different amounts (mass fractions) of CMC-Na, carrageenan and xanthan gum (0.05%, 0.10%, 0.15%, 0.20% and 0.25%) were added to the fermented polypeptide beverage of Chinese jujube seeds, heated and stirred at 55°C to dissolve, and the centrifugal sedimentation rate was measured after the solution was cooled.

[0108] 2. Choice of sweetener type

[0109] Taking sensory scores as indicators, 0.05% anhydrous citric acid and 0.4g / kg potassium sorbate were added, stabilizers were added according to the above experimental results, and 5% and 10% white sugar and xylitol were added to compare the effects of white sugar and xylitol on the sensory scores.

[0110] 3. Determination of the amount of sweetener added

[0111] After determining the type of sweetener, 0.05% anhydrous citric acid and 0.4 g / kg potassium sorbate were added based on the sensory score. Stabilizers were added based on the above experimental results, and the effects of the added amounts of sweetener (1%, 3%, 5%, 7%, 9%) on the sensory score were compared.

[0112] 4 Determination of the amount of anhydrous citric acid added

[0113] Taking the sensory score as an indicator, 0.4 g / kg of potassium sorbate and 5% of sweetener were added, and stabilizers were added according to the above experimental results. The effects of the addition amount of anhydrous citric acid (0%, 0.05%, 0.10%, 0.15%, 0.20%) on the sensory score were compared.

[0114] 5. Determination of the amount of potassium sorbate added

[0115] GB 2760-2014 "Food Additives Edible Standard" stipulates that the amount of potassium sorbate added shall not exceed 0.5g / kg (calculated as sorbic acid). Taking the sensory score as an indicator, 0.05% anhydrous citric acid and 5% sweetener were added, and stabilizers were added according to the above experimental results to compare the effects of the addition of potassium sorbate (0.2g / kg, 0.3g / kg, 0.4g / kg, 0.5g / kg, 0.6g / kg) on ​​the sensory score.

[0116] 6 Determination of centrifugal sedimentation rate

[0117] Take 10mL of fermented beverage in a 15mL centrifuge tube, centrifuge at 4000r / min for 20min, pour out the supernatant, turn the centrifuge tube upside down for 5min, weigh the mass of the sediment after centrifugation, and calculate the centrifugal sedimentation rate of the fermented beverage according to the following formula.

[0118] Centrifugal sedimentation rate (%) = m1 / m2*100

[0119] Where m1 is the mass of the sediment, and m2 is the mass of 10 mL of the fermented beverage.

[0120] 7 Sensory Rating

[0121] Invite 10 students to form an evaluation team, rinse their mouths with water, and conduct sensory evaluation on the taste, color, smell and tissue distribution of the fermented polypeptide beverage from Chinese date seeds, with a full score of 100. The sensory evaluation standards are shown in Table 7.

[0122] Table 7 Sensory evaluation scoring standards

[0123]

[0124] 8 Verification Experiment

[0125] According to the optimal formulation process conditions obtained from the previous experimental results, three repeated experiments were carried out to verify the reliability of the optimal formulation.

[0126] 9 Results and Analysis

[0127] 9.1 Effect of stabilizer type and amount on centrifugal sedimentation rate

[0128] Effects of different stabilizer addition amounts on centrifugal sedimentation rate Figure 3 shown.

[0129] With the increase of stabilizer additives, the centrifugal sedimentation rate of the beverage increased significantly. When the addition amount of xanthan gum, carrageenan and CMC-Na was 0.05%, the sedimentation rate was the lowest, the stability was the best, and the xanthan gum had the best effect. Therefore, xanthan gum was selected as the stabilizer to be added, and the addition amount was 0.05%.

[0130] 9.2 Effect of sweeteners on sensory scores

[0131] The sensory evaluation results of white sugar and xylitol are shown in Table 8, and the effect of xylitol addition on the sensory score is shown in Table 9.

[0132] Table 8 Effect of sweetener types on sensory scores of fermented peptide beverages from Chinese date seeds

[0133]

[0134] Table 9 Effect of xylitol addition on sensory scores of fermented polypeptide beverages from Chinese date seeds

[0135]

[0136] 9.3 Effect of anhydrous citric acid addition on sensory scores

[0137] The effect of the amount of anhydrous citric acid added on the sensory score of the fermented polypeptide beverage from Chinese jujube seeds is shown in Table 10.

[0138] Table 10 Effect of anhydrous citric acid addition on sensory scores of fermented polypeptide beverages from Chinese date seeds

[0139]

[0140] 9.4 Effect of potassium sorbate addition on sensory scores

[0141] The effect of potassium sorbate addition on the sensory score of the fermented polypeptide beverage from Chinese jujube seeds is shown in Table 11.

[0142] Table 11 Effect of potassium sorbate addition on sensory scores of fermented peptide beverages from Chinese date seeds

[0143]

[0144] 9.5 Verification Experimental Results

[0145] The optimal formula is 0.05% xanthan gum, 0.05% anhydrous citric acid, 5% xylitol, and 0.4 g / kg potassium sorbate. The experiment was repeated three times, and the drink was brown-yellow in color, with a fermented smell, and a suitable sweet and sour taste. The average sensory score was 79. The experimental results were not much different from the previous results, indicating that the process is stable and feasible.

[0146] Example 5 Quality Standard and Stability Study of Fermented Polypeptide Beverage from Ziziphus jujuba Seed

[0147] The Chinese jujube seed fermented polypeptide beverage was prepared according to the optimal fermentation process and the optimal preparation process, and was subjected to sensory evaluation, property inspection, pH value inspection, relative density inspection, filling quantity difference inspection, microbial limit inspection, thin layer identification, main active ingredient content determination (polypeptide content), nutrient content determination (fat, protein, moisture, ash, dietary fiber, carbohydrate, energy, sodium ion content), light effect test, temperature effect test and accelerated test.

[0148] It was finally determined that the fermented polypeptide beverage of Chinese jujube kernel is a brown-yellow liquid with a glossy texture, a slightly sweet and sour taste, a fermented aroma and a soft mouthfeel; the liquid preparation is a clear liquid and must not become moldy, rancid or other deteriorate during storage, and a small amount of precipitation that can be dispersed by shaking is allowed; the pH value is between 4.0 and 4.5; the relative density should not be lower than the minimum value of 1.0230 (35°C); spots corresponding to Chinese jujube kernel should appear during thin layer identification; the polypeptide content per 1mL of this product must not be less than 4.6085mg; the nutritional components are all within a reasonable range; it can be stored in sunlight at room temperature, and the shelf life is initially set at 2 years.

Claims

1. A preparation process of a fermented polypeptide beverage of wild jujube seeds, characterized in that it is prepared by microbial fermentation using wild jujube seeds defatted powder as raw material, and the specific steps are as follows: (1) Weighing defatted jujube seed powder, adding distilled water and glucose, sterilizing, cooling, and obtaining a fermentation medium; (2) inoculating the bacterial strain into the fermentation matrix, fermenting, and obtaining a fermentation liquid; centrifuging the fermentation liquid to obtain a fermentation supernatant; (3) Add xanthan gum, xylitol, anhydrous citric acid and potassium sorbate to the fermentation supernatant, stir evenly, let stand, check, filter, fill and sterilize to obtain the finished product.

2. The preparation process of the fermented polypeptide beverage from Chinese date seeds according to claim 1, characterized in that: In step (1), the mass ratio of defatted jujube kernel powder to distilled water is 1:14; and the mass concentration of glucose in the fermentation matrix is ​​15 g / L.

3. The preparation process of the fermented polypeptide beverage from Chinese date seeds according to claim 1, characterized in that: In step (2), the bacterial species is Aspergillus niger.

4. The preparation process of the fermented polypeptide beverage from Chinese date seeds according to claim 1, characterized in that: In step (2), the fermentation conditions are: fermentation time 60h, material-liquid ratio 1:14, inoculation amount 1%.

5. The process for preparing the fermented polypeptide beverage from Chinese date seeds according to claim 1, characterized in that: In step (2), the fermentation liquid is centrifuged at a speed of 4000 r / min. -1 , time 20 minutes.

6. The process for preparing the fermented polypeptide beverage from Chinese date seeds according to claim 1, characterized in that: In step (3), the amount of xanthan gum added is 0.05% of the mass of the fermentation supernatant, calculated on a mass ratio basis.

7. The process for preparing the fermented polypeptide beverage from Chinese date seeds according to claim 1, characterized in that: In step (3), the amount of xylitol added is 5% of the mass of the fermentation supernatant, calculated on a mass ratio basis.

8. The process for preparing the fermented polypeptide beverage from Chinese date seeds according to claim 1, characterized in that: In step (3), the amount of anhydrous citric acid added is 0.05% of the mass of the fermentation supernatant, calculated on a mass ratio basis.

9. The process for preparing the fermented polypeptide beverage from Chinese date seeds according to claim 1, characterized in that: In step (3), 0.4 g of potassium sorbate is added per kilogram of fermentation supernatant on a mass basis.

10. A fermented polypeptide beverage made from Chinese date seeds, characterized in that: The preparation process of the fermented polypeptide beverage of Chinese jujube seeds according to any one of claims 1 to 9 is adopted, and the molecular weight of the Chinese jujube seeds polypeptide is distributed in the range of 4kDa to 10kDa.

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