Method for preparing rapeseed meal polypeptide through mixed bacteria combined fermentation of rapeseed meal

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing rapeseed meal polypeptide through mixed bacteria combined fermentation of rapeseed meal. The mixed strain composition suitable for fermenting the rapeseed dregs is obtained through screening, and the rapeseed dregs are subjected to combined fermentation by adopting the mixed strain composition, so that the content of soluble peptides in a fermentation product can be increased, and the content of anti-nutritional factors such as glucosinolates in the fermentation product can be reduced; the antioxidant performance of the rapeseed meal fermentation peptide can be improved, and the rapeseed meal fermentation peptide feed has better nutritional efficacy and feed quality and is wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and particularly relates to a method for preparing rapeseed meal polypeptides by mixed bacteria combined fermentation of rapeseed meal. Background Art

[0002] Rapeseed meal is a byproduct of rapeseed as raw material, which is extracted by soaking or pre-pressing and then soaking to extract oil. Rapeseed meal is an important protein resource. It is rich in crude protein, methionine, lysine, choline, biotin, niacin, vitamin B and abundant trace elements. It has high potential nutritional value. Its quality is comparable to soybean meal. It is a high-quality and low-cost protein feed.

[0003] However, many factors limit the deep processing of rapeseed meal and its nutrition in the feed industry. For example, the crude protein in rapeseed meal has a large molecular weight and a low soluble peptide content, which is not easily digested and absorbed by animals. For another example, the presence of anti-nutritional ingredients in rapeseed meal, such as tannins, sinapines, phytic acid, crude fiber, etc., especially high levels of glucosinolates, can reduce palatability and lead to symptoms such as growth retardation, goiter, and organ tissue damage. The presence of anti-nutritional ingredients such as glucosinolates makes the utilization of rapeseed meal low, limiting the application of rapeseed meal as a plant protein resource in the feed industry. Therefore, the content of anti-nutritional ingredients is an important criterion for measuring the quality of rapeseed meal feed.

[0004] Therefore, how to further improve the nutritional value of rapeseed meal so that it is easier to digest and absorb while having lower anti-nutritional content and higher nutritional functionality has become a problem that needs to be solved. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to propose a method for preparing rapeseed meal polypeptides by mixed bacteria co-fermentation of rapeseed meal, wherein a mixed strain composition suitable for fermenting rapeseed meal is obtained by screening, and the mixed strain composition is used to co-ferment rapeseed meal, which can not only increase the soluble peptide content in the fermentation product, reduce the content of anti-nutritional factors such as glucosinolates in the fermentation product, but also improve the antioxidant properties of rapeseed meal fermentation peptides, has better nutritional efficacy and feed quality, and has broad application prospects.

[0006] To achieve the above-mentioned object, in a first aspect, the present invention provides a mixed strain composition suitable for fermenting rapeseed meal, wherein the mixed strain composition comprises Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus delbrueckii, and the ratio of the effective live counts of Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus delbrueckii is 39-48:14-24:13-19:13-21:13-20.

[0007] Furthermore, the ratio of the effective live bacteria count of Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus delbrueckii is 39:15:13:13:20.

[0008] In a second aspect, the present invention provides a bacterial agent, which includes the mixed strain composition or a fermentation product thereof described in the first aspect.

[0009] In a third aspect, the present invention provides a method for preparing rapeseed meal polypeptides by mixed bacterial co-fermentation of rapeseed meal, the method comprising the steps of inoculating the mixed bacterial strain composition of the first aspect for fermentation, wherein the inoculation density of the mixed bacterial strain composition is 1×10 6 CFU / mL

[0010] -1×10 8 CFU / mL, preferably 1×10 7 CFU / mL.

[0011] Furthermore, the method comprises the following steps:

[0012] S1. The rapeseed meal is crushed with a grinder and sieved to obtain rapeseed powder;

[0013] S2. The rapeseed meal obtained in step S1 is added with water to prepare a rapeseed meal suspension;

[0014] S3. Adding cellulase to the rapeseed meal suspension obtained in step S2 for enzymatic hydrolysis to obtain a rapeseed meal hydrolysis product;

[0015] S4. inoculating the mixed strain composition according to claim 1 or 2 into the rapeseed meal hydrolysate obtained in step S3 for fermentation to obtain a crude extract of rapeseed meal peptides;

[0016] S5. sterilize and extract the crude rapeseed meal peptide extract obtained in step S4 to obtain rapeseed meal polypeptide.

[0017] Furthermore, step S2 also includes the steps of sterilizing and cooling the rapeseed powder suspension; preferably, the sterilization step is heating at 85° C. for 30 minutes.

[0018] Furthermore, in step S3, the mass ratio of the addition amount of cellulase to the rapeseed powder suspension is 0.2%;

[0019] Furthermore, the enzymatic hydrolysis condition in step S3 is 45-55° C. for 4-8 hours, and high temperature enzyme inactivation is performed at the end of the enzymatic hydrolysis. For example, the enzymatic hydrolysis temperature is 50° C., and the enzymatic hydrolysis time is 4 hours, 5 hours, 6 hours, 7 hours or 8 hours.

[0020] In a fourth aspect, the present invention provides a rapeseed meal polypeptide prepared by the method described in the third aspect.

[0021] In a fifth aspect, the present invention provides the use of the mixed strain composition described in the first aspect, the bacterial agent described in the second aspect, the method described in the third aspect, or the rapeseed meal polypeptide described in the fourth aspect in improving the nutritional value of rapeseed meal or preparing feed.

[0022] In a sixth aspect, the present invention provides use of the mixed strain composition of the first aspect, the bacterial agent of the second aspect, the method of the third aspect, or the rapeseed meal polypeptide of the fourth aspect in increasing the peptide content in a rapeseed meal fermentation product;

[0023] In a seventh aspect, the present invention provides use of the mixed strain composition of the first aspect, the bacterial agent of the second aspect, the method of the third aspect, or the rapeseed meal polypeptide of the fourth aspect in reducing the content of anti-nutritional factors in a rapeseed meal fermentation product;

[0024] In an eighth aspect, the present invention provides use of the mixed strain composition described in the first aspect, the bacterial agent described in the second aspect, the method described in the third aspect, or the rapeseed meal polypeptide described in the fourth aspect in improving the antioxidant properties of rapeseed meal fermentation products.

[0025] Furthermore, the anti-nutritional factor is glucosinolate.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) The present invention screens out a mixed strain composition suitable for fermenting rapeseed meal. The mixed strain composition is used to co-ferment rapeseed meal, which can not only increase the content of soluble peptides in the fermentation product, but also reduce the content of anti-nutritional factors such as glucosinolates in the fermentation product, but also has better nutritional efficacy and feed quality, and has broad application prospects.

[0028] (2) The four strains in the strain composition of the present invention are mixed in a specific ratio, which can exert a better joint fermentation effect, further increase the soluble peptide content in the fermented rapeseed meal, and reduce the anti-nutritional factor content of the fermented rapeseed meal.

[0029] (3) The present invention optimizes the culture medium components and fermentation conditions of the mixed strain composition, further increasing the soluble peptide content in the fermented rapeseed meal and reducing the anti-nutritional factor content in the fermented rapeseed meal.

[0030] (4) DPPH free radical scavenging capacity IC of the rapeseed meal fermented peptide prepared by the present invention 50 It can reach 0.31mg / mL, and the reducing capacity of trivalent iron ions can reach 0.57mM / mL FeSO4·7H2O, which is better than the reducing capacity of vitamin E (analogues) and has excellent antioxidant properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shown is the peptide yield of rapeseed meal fermented with different strains of bacteria.

[0032] Figure 2 The figure shows the degradation rate of glucosinolates in rapeseed meal fermented by different strains.

[0033] Figure 3 Shown is the effect of different carbon sources on the colony count of Lactobacillus casei.

[0034] Figure 4 Shown is the effect of different maltose addition amounts on the number of Lactobacillus casei colonies.

[0035] Figure 5 The figure shows the effect of different trace element additions on the colony count of Lactobacillus casei.

[0036] Figure 6 Shown are the peptide yields in the Examples and Comparative Examples.

[0037] Figure 7 Shown are the degradation rates of glucosinolates in the examples and comparative examples.

[0038] Figure 8 Shown is the DPPH radical scavenging rate of fermented rapeseed meal at different concentrations.

[0039] Note: Different lowercase letters represent significant differences between values ​​(p < 0.05). DETAILED DESCRIPTION

[0040] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0041] In the following examples and figures, the abbreviations and sources of the various strains are as follows:

[0042]

[0043] Bacteria activation and counting method:

[0044] The frozen stock of the strain was activated, and after 2-3 generations of culture, the culture was inoculated and fermented. The colony density was counted using a hemocytometer. The calculation formula is as follows:

[0045] Colony density (CFU / mL) = total number of bacteria in 80 cells × 5 × 10 4 × Dilution factor

[0046] Method for detecting peptide content and calculating peptide yield:

[0047] The content of polypeptide in the supernatant was determined by the biuret method: 1.5g CuSO4·5H2O and 6g KNaC4H4O6·4H2O were weighed, dissolved in 500mL distilled water, 300mL 10% NaOH was added under stirring, and the volume was adjusted to 1000mL with water to obtain the biuret reagent. Take 1mL of sample solution and add 1mL 10% trichloroacetic acid (TCA) aqueous solution. After mixing, let it stand for 8min and centrifuge at 7000r / min for 10min. Then take 1mL of supernatant and add 4mL of biuret reagent. Similarly, mix the solution and let it stand for 30min. At the same time, mix 1mL of water and 4mL of biuret reagent as a blank, and measure the absorbance value at 540nm. The polypeptide concentration C (mg / mL) in the hydrolyzate was calculated using the standard regression equation. The polypeptide yield is the percentage of soluble peptides obtained by fermentation hydrolysis in the total protein.

[0048] Peptide yield calculation formula:

[0049]

[0050] Example 1 Preparation of rapeseed meal polypeptide

[0051] Process flow:

[0052] Rapeseed meal → 80 mesh sieving → enzyme inactivation, sterilization (85℃, 30min) → inoculation → mixed fermentation → sterilization → extraction → rapeseed meal fermentation peptide

[0053] The specific steps are as follows:

[0054] (1) The rapeseed meal is crushed by a crusher and passed through an 80-mesh sieve for later use.

[0055] (2) Rapeseed meal pretreatment: Rapeseed meal was suspended in water and mixed, heated at 85°C for 30 min to reduce endogenous microbial flora, and then allowed to cool to room temperature.

[0056] (3) Enzymatic hydrolysis: Cellulase (0.2% w / w) was added to the rapeseed meal aqueous solution at room temperature to initiate the enzymatic hydrolysis reaction, which was carried out at 50°C for 6 h and then heated at 85°C for 30 min to terminate the enzymatic hydrolysis reaction.

[0057] (4) Fermentation: After the seed meal enzymatic hydrolysate was placed at room temperature, mixed bacteria were inoculated at an inoculation density of 1×10 7CFU / mL, after inoculation with the bacteria, fermentation was carried out in a shaker at 37°C, and the peptide content was tested every 8 hours until the peptide content tended to stabilize or decrease (about 48h), and the crude extract of rapeseed meal peptides was obtained.

[0058] (5) The fermented sample is sterilized at 85° C. for 30 min and then extracted. The extracted clear liquid is the rapeseed meal polypeptide.

[0059] Example 2 Bacterial strain screening

[0060] The rapeseed meal polypeptide was prepared by the method of Example 1. In step (4), 15 strains of lactic acid bacteria were inoculated at a density of 1×10 7 CFU / mL. 15 strains were initially screened using peptide yield as an indicator. The peptide content was determined every 8 hours and the peptide yield was calculated. The end point of the fermentation was when the peptide content stabilized or decreased. The peptide yield at 32h was selected for differential analysis based on the fermentation results. Figure 1 It can be seen that Lactobacillus casei has the highest peptide yield of 42.11%, while Lactobacillus pentosus has the lowest yield of 38.56%. According to the difference comparison results, 8 dominant strains with higher peptide yields were selected, namely Lactobacillus casei, Pediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus reuteri, and Lactobacillus paracasei.

[0061] After lactic acid bacteria fermentation, the glucosinolate content in rapeseed meal has decreased significantly. Figure 2 As shown in the figure, the degradation rates of glucosinolates after fermentation of different strain groups are as follows: Lactobacillus casei>Lactobacillus rhamnosus>Lactobacillus delbrueckii>Lactobacillus brevis>Lactobacillus paracasei>Pediococcus acidilactici>Lactobacillus fermentum>Lactobacillus reuteri, and the differences among the treatment groups are significant. It can be seen that Lactobacillus casei has a greater degradation effect on the toxic substance glucosinolates in rapeseed meal, and the degradation rate of glucosinolates is 97.01%. Combined with the peptide yield results, 5 lactic acid bacteria with a glucosinolate degradation rate of more than 80% were selected as the dominant strains for fermenting rapeseed meal, and finally the suitable mixed strains for preparing rapeseed meal fermentation peptides were determined to be Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus delbrueckii, Lactobacillus brevis and Lactobacillus paracasei.

[0062] Example 3 Optimization of culture medium

[0063] Taking colony density as an indicator, 1×10 activated 7CFU / mL Lactobacillus casei, based on MRS medium, 2% glucose, maltose, sucrose, lactose, starch were added as the carbon source of the medium for single factor experiments, and after the carbon source was determined, 0.5%, 0.8%, 1.1%, 1.4%, 1.7%, 2%, 2.3% carbon source were added for single factor experiments. Based on the previous step, different concentrations of FeSO4·7H2O, ZnSO4·7H2O, CuSO4·5H2O, MgSO4·7H2O, and NaCl trace elements were added for single factor experiments.

[0064] Rapeseed meal has a high protein content, and microorganisms grow and metabolize slowly, so some nutrients need to be added to promote the growth of microorganisms. Carbon source is one of the essential nutritional factors for the growth and reproduction of microorganisms. It can regulate the growth metabolism and metabolic characteristics of microorganisms and has a direct impact on the fermentation process. Figure 3 It can be seen that among these five carbon sources, maltose has the most significant effect on the growth and reproduction of Lactobacillus casei. Figure 4 It can be seen that after adding different concentrations of maltose, the addition amount reached the maximum at 0.8%, and then the colony density showed a downward trend, but there was no significant difference when the addition amount was 0.5% and 0.8%. Therefore, 0.5% was selected as the addition amount of the carbon source.

[0065] The presence of trace elements can promote the growth of microorganisms, enhance the activity of probiotics, change their physical and chemical properties, and are components of certain active substances. Figure 5 It can be seen that adding Na + , Cu 2+ 、Zn 2+ , Fe 2+ The effect of adding MgSO4·7H2O on promoting the growth and reproduction of Lactobacillus casei is not as obvious as that of adding MgSO4·7H2O. When the added amount is 0.12%, the colony density reaches the highest, but when the added amount is 0.09%, there is no significant difference from 0.12%. Therefore, 0.09% is preferred as the added amount of trace elements.

[0066] The final optimized conditions of the culture medium were determined as follows: 0.5% maltose as the carbon source and 0.09% MgSO4·7H2O as the trace element.

[0067] Example 4 Optimization of fermentation process

[0068] Taking peptide yield as the main evaluation index, the key parameters of the optimal fermentation process for preparing rapeseed meal peptides were determined by orthogonal experiments as follows: strain activation time of 24 h, fermentation temperature controlled at 37 °C, fermentation time of 48 h, material-liquid ratio of 1:15, and initial bacterial density controlled at 1×10 7 CFU / mL.

[0069] Example 5 Optimization of strain ratio

[0070] According to the experimental results of polypeptide content and glucosinolate degradation rate, the five selected dominant strains were mixed and fermented. The optimization experiment adopted a unified design table, with 10 factors of five dominant strains as levels (10 different mixed bacteria ratios), and polypeptide yield as the response value. The design experiment is shown in Table 1.

[0071] Table 1. Soluble peptide yields after fermentation of various strains

[0072]

[0073] As can be seen from Table 1, the soluble polypeptide yields of Group 1, Group 3, Group 4, Group 6, Group 9 and Group 10 exceeded 44%, with the highest reaching 46.64%, and the soluble polypeptide yields of other groups were lower. Therefore, when the ratio of the effective viable counts of Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus delbrueckii was 39-48:16-24:13-19:13-21:13-20, the polypeptide yield was high.

[0074] Taking the peptide yield as the response value, Minitab software combined with Excel planning solution was used for analysis, and the regression equation was established as follows:

[0075] Y=105.54-83.6X1-469.9X3-193.7X1 2 -600.9X3 2 +206.3X5 2 +1586.4X1X3-24.08X2X4-457.8X3X5

[0076] R in the regression equation 2 =0.99, and the optimal mixed bacteria ratio finally determined is 39% of Lactobacillus brevis, 15% of Lactobacillus casei, 13% of Lactobacillus paracasei, 13% of Lactobacillus rhamnosus, and 20% of Lactobacillus delbrueckii. The predicted polypeptide yield is now 48.54%. According to the predicted optimal conditions, a verification experiment was conducted, and the polypeptide yield after the fermentation of rapeseed meal was 47.07%. Compared with the predicted value, the error value of the polypeptide yield was 3.03%, indicating that the equation can well predict the optimal ratio, verifying the validity of the model.

[0077] Therefore, when the ratio of the effective live counts of Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus delbrueckii is 39-48:14-24:13-19:13-21:13-20, the yield of soluble peptides in the polypeptides obtained by fermenting rapeseed meal is higher.

[0078] Example 6 Optimization of the number of bacterial strains

[0079] In order to verify that the selected strains mixed and fermented with the above-mentioned optimal mixed ratio are the best strain combination for fermenting rapeseed meal polypeptides, other mixed forms were designed for fermentation and verified by polypeptide yield. Four strains (uniform design experiment as shown in Table 2), five strains (uniform design experiment as shown in Table 3) and six strains (uniform design experiment as shown in Table 4) were selected respectively, and the optimal ratios were obtained through uniform experiments.

[0080] (1) 4 kinds of mixed bacteria fermentation mode

[0081] The rapeseed meal polypeptide was prepared by the method of Example 1, wherein the composition of the mixed strains inoculated in step (4) is as shown in Table 2.

[0082] Table 2 Soluble peptide yields after 4 mixed bacteria fermentation

[0083]

[0084]

[0085] Taking the peptide yield as the response value, Minitab software combined with Excel planning solution was used for analysis, and the regression equation was established as follows:

[0086] Y=164.7-816X2+364.2X1 2 +661X2 2 -2343X1X3

[0087] +3076X2X3

[0088] R in the regression equation 2 =0.99, and the optimal mixed bacteria ratio finally determined is 45% of Lactobacillus brevis, 34% of Lactobacillus paracasei, 10% of Pediococcus acidilactici, and 11.50% of Lactobacillus delbrueckii. At this time, the predicted polypeptide yield is 36.57%. According to the predicted optimal conditions, the verification experiment was carried out, and the polypeptide yield after rapeseed meal fermentation was 37.38%. Compared with the predicted value, the error value of the polypeptide yield was 2.21%, indicating that the equation can well predict the optimal ratio and verify the effectiveness of the model.

[0089] (2) 5 kinds of mixed bacteria fermentation mode

[0090] The rapeseed meal polypeptide was prepared by the method of Example 1, wherein the composition of the mixed strains inoculated in step (4) is as shown in Table 3.

[0091] Table 3 Soluble peptide yields after mixed fermentation of five strains of bacteria

[0092]

[0093]

[0094] Taking the peptide yield as the response value, Minitab software combined with Excel planning solution was used for analysis, and the regression equation was established as follows:

[0095] Y=106.5-272.9X2-566.8X3-13X2 2 +713X3 3 +61.6X1X2+1665X2X3-50.8X2X4

[0096] R in the regression equation 2 =0.99, and the optimal mixed bacteria ratio finally determined is 48% of Lactobacillus brevis, 24% of Lactobacillus paracasei, 22% of Pediococcus acidilactici, 13% of Lactobacillus delbrueckii, and 17.70% of Lactobacillus rhamnosus. At this time, the predicted polypeptide yield is 43.49%. According to the predicted optimal conditions, the verification experiment was carried out, and the polypeptide yield after rapeseed meal fermentation was 42.19%. Compared with the predicted value, the error value of the polypeptide yield was 2.99%, indicating that the equation can well predict the optimal ratio and verify the effectiveness of the model.

[0097] (3) 6-strain mixed fermentation mode

[0098] The rapeseed meal polypeptide was prepared by the method of Example 1, wherein the composition of the mixed strains inoculated in step (4) is as shown in Table 4.

[0099] Table 4 6 Soluble peptide yield after mixed culture fermentation

[0100]

[0101] Taking the peptide yield as the response value, Minitab software combined with Excel planning solution was used for analysis, and the regression equation was established as follows:

[0102] Y=42.8886+954.54X4 2 +1274.93X5 2 +2528.26X6 2 -327.155X1X5-28.424X2X3-11.478X3X4-279.88X3X5-3468.49X4X6

[0103] R in the regression equation 2=0.99, and the optimal mixed bacteria ratio finally determined is 36% of Lactobacillus brevis, 16% of Lactobacillus paracasei, 8% of Pediococcus acidilactici, 17% of Lactobacillus delbrueckii, 12% of Lactobacillus rhamnosus, and 12% of Lactobacillus casei. The predicted polypeptide yield is 37.14%. According to the predicted optimal conditions, the verification experiment was carried out, and the polypeptide yield after rapeseed meal fermentation was 36.36%. Compared with the predicted value, the error value of the polypeptide yield was 2.10%, indicating that the equation can well predict the optimal ratio and verify the validity of the model.

[0104] Comparative analysis of different mixed bacteria modes:

[0105] Example 6-1

[0106] The method of Example 1 was used to prepare rapeseed meal polypeptides, wherein the mixed strains inoculated in step (4) were composed of the following:

[0107] Lactobacillus brevis 39%, Lactobacillus casei 15%, Lactobacillus paracasei 13%, Lactobacillus rhamnosus 13%, Lactobacillus delbrueckii 20%.

[0108] Comparative Example 6-1

[0109] The method of Example 1 was used to prepare rapeseed meal polypeptides, wherein the mixed strains inoculated in step (4) were composed of the following:

[0110] Lactobacillus brevis 45%, Lactobacillus paracasei 34%, Pediococcus acidilactici 10%, Lactobacillus delbrueckii 11.50%.

[0111] Comparative Example 6-2

[0112] The method of Example 1 was used to prepare rapeseed meal polypeptides, wherein the mixed strains inoculated in step (4) were composed of the following:

[0113] Lactobacillus brevis 48%, Lactobacillus paracasei 24%, Pediococcus acidilactici 22%, Lactobacillus delbrueckii 13%, and Lactobacillus rhamnosus 17.70%.

[0114] Comparative Example 6-3

[0115] The method of Example 1 was used to prepare rapeseed meal polypeptides, wherein the mixed strains inoculated in step (4) were composed of the following:

[0116] Lactobacillus brevis 36%, Lactobacillus paracasei 16%, Pediococcus acidilactici 8%, Lactobacillus delbrueckii 17%, Lactobacillus rhamnosus 12%, Lactobacillus casei 12%.

[0117] According to Example 6-1 and Comparative Examples 6-1, 6-2 and 6-3, the peptide yield ( Figure 6 ), glucosinolate degradation rate ( Figure 7), and finally determined that Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus delbrueckii of Example 6-1 were selected as the dominant strains for fermenting rapeseed meal peptides, that is, these five mixed bacteria modes were fermented as the optimal mixed bacteria mode for mixed bacteria fermentation of rapeseed meal polypeptides. The peptide yield and glucosinolate degradation rate of the polypeptides prepared by fermentation with one less bacteria, one more bacteria, or one more bacteria could not reach the above-mentioned optimal mixed bacteria mode.

[0118] Performance Testing Evaluation of Antioxidant Activity of Rapeseed Meal

[0119] Performance test 1DPPH free radical scavenging ability evaluation

[0120] Prepare 38ug / mL DPPH solution. Take the rapeseed meal peptide fermentation broth prepared in Example 6-1 and dilute it into sample solutions of different concentrations (0.13, 0.19, 0.26, 0.32, 0.39, 0.52mg / mL). Take 1mL of the sample solution and add 4mL of DPPH solution to a brown stoppered test tube and mix well. React at room temperature in the dark for 30 minutes. Measure the absorbance value of each concentration at a wavelength of 517nm as sample A. Replace the sample solution with an equal amount of distilled water and mix with DPPH. Measure its absorbance value as control A. Draw a regression equation based on the concentration (C) and absorbance (A) to calculate the sample concentration required when the clearance rate is 50%, and use IC 50 The scavenging rate (R) of DPPH is calculated by the following formula:

[0121]

[0122] Different concentrations (10, 12, 14, 16, 18, 20, 22, 24 ug / mL) of Vc were used as positive control to evaluate the antioxidant activity of rapeseed meal peptide fermentation broth.

[0123] from Figure 8 It can be seen that the DPPH free radical scavenging ability of rapeseed meal peptide is basically positively correlated with the concentration. The free radical scavenging rate increased from 27.04% to 72.95% between 0.13-0.52 mg / mL, indicating that its DPPH free radical scavenging ability increased with the increase of concentration. 50 It is 0.31 mg / mL, that is, when the mass concentration of rapeseed meal peptide reaches 0.31 mg / mL, the free radical scavenging rate reaches 50%.

[0124] Performance Test 2Fe 3+ Determination of reducing power

[0125] The rapeseed meal peptide fermentation broth prepared in Example 6-1 was diluted to 1.2 mM, and 180 μL of FRAP working solution was added to the detection wells of a 96-well plate. 5 μL of sample and the same concentration of Trolox were added to the sample detection wells as positive controls. 5 μL of distilled water was added to the blank control wells. The mixture was gently mixed and incubated at 37°C for 5 minutes before measuring the absorbance at a wavelength of 593 nm. The absorbance was measured under the same conditions using 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM FeSO4·7H2O solutions as controls and a standard curve was drawn, y=0.2666x+0.0025(R 2 =0.9976), and the iron ion reducing capacity of the sample was calculated and expressed as mM / mL FeSO4·7H20. Meanwhile, vitamin E analogs were used as the positive control group.

[0126] The results showed that the rapeseed meal peptide prepared by fermentation in Example 6-1 had a strong affinity for iron ions (Fe 3+ ) has a reducing power equivalent to 0.57 mM / mL FeSO4·7H2O, which is comparable to the positive control (vitamin E analogue) of iron ions (Fe 3+ ) reducing capacity 0.29mM / mLFeSO4·7H2O. In comparison, the iron ion (Fe 3+ ) has better restoration ability.

[0127] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A mixed strain composition suitable for fermenting rapeseed meal, characterized in that: The mixed strain composition comprises Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus delbrueckii, and the ratio of the effective live bacteria counts of Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus delbrueckii is 39-48:14-24:13-19:13-21:13-20.

2. The mixed strain composition according to claim 1, characterized in that: The effective live bacterial count ratio of Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus delbrueckii is 39:15:13:13:

20.

3. A bacterial agent, characterized in that: The bacterial agent comprises the mixed strain composition or a fermentation product thereof according to claim 1 or 2.

4. A method for preparing rapeseed meal polypeptides by mixed bacteria combined fermentation of rapeseed meal, characterized in that: The method comprises the steps of inoculating the mixed strain composition according to claim 1 or 2 for fermentation, wherein the inoculation density of the mixed strain composition is 1×10 6 CFU / mL -1×10 8 CFU / mL, preferably 1×10 7 CFU / mL.

5. The method according to claim 4, characterized in that The method comprises the following steps: S1. The rapeseed meal is crushed with a grinder and sieved to obtain rapeseed powder; S2. The rapeseed meal obtained in step S1 is added with water to prepare a rapeseed meal suspension; S3. Adding cellulase to the rapeseed meal suspension obtained in step S2 for enzymatic hydrolysis to obtain a rapeseed meal hydrolysis product; S4. inoculating the mixed strain composition according to claim 1 or 2 into the rapeseed meal enzymatic hydrolysate obtained in step S3 for fermentation to obtain a crude extract of rapeseed meal peptides; S5. sterilizing and extracting the crude rapeseed meal peptide extract obtained in step S4 to obtain rapeseed meal polypeptide.

6. The method according to claim 5, characterized in that Step S2 also includes the steps of sterilizing and cooling the rapeseed powder suspension; preferably, the sterilization step is heating at 85° C. for 30 minutes.

7. The method according to claim 5, characterized in that In step S3, the mass ratio of the added amount of cellulase to the rapeseed powder suspension is 0.2%; preferably, the enzymatic hydrolysis conditions in step S3 are enzymatic hydrolysis at 45-55°C for 4-8h, and high temperature enzyme inactivation at the end of the enzymatic hydrolysis; preferably, enzymatic hydrolysis at 50°C for 6h.

8. The rapeseed meal polypeptide prepared by the method according to any one of claims 4 to 7.

9. Use of the mixed strain composition according to claim 1 or 2, the bacterial agent according to claim 3, the method according to any one of claims 4 to 7, or the rapeseed meal polypeptide according to claim 8 in improving the nutritional value of rapeseed meal or preparing feed.

10. Use of the mixed strain composition according to claim 1 or 2, or the bacterial agent according to claim 3, or the method according to any one of claims 4 to 7, or the rapeseed meal polypeptide according to claim 8, wherein the use comprises at least one of the following: (1) Increasing the soluble peptide content in rapeseed meal fermentation products; (2) reducing the content of anti-nutritional factors in rapeseed meal fermentation products; (3) Improving the antioxidant properties of rapeseed meal fermentation products, preferably, the anti-nutritional factor is glucosinolate.

Images