Compound bacterial agent, Chinese wolfberry fruit fermentation liquor and preparation method and application of compound bacterial agent and Chinese wolfberry fruit fermentation liquor

Abstract

The invention provides a compound bacterial agent, Chinese wolfberry fermentation liquor and a preparation method and application thereof, and belongs to the technical field of biological fermentation, the bacterial agent comprises lactobacillus plantarum LZU-LJY-1Q1 and lactobacillus plantarum LZU-J-LZ1-1, the volume ratio of the lactobacillus plantarum LZU-LJY-1Q1 to the lactobacillus plantarum LZU-J-LZ1-1 is (1-3): (1-3), and the volume ratio of the lactobacillus plantarum LZU-LJY-1Q1 to the lactobacillus plantarum LZU-J-LZ1-1 is (1-3): (1-3). The concentration of total phenols contained in the lycium barbarum fermentation liquor obtained through fermentation of the complex microbial inoculant is high, the capacity of removing DPPH free radicals and OH-free radicals of the lycium barbarum fermentation liquor is improved, and the total ABTS antioxidant capacity is also improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a compound bacterial agent, wolfberry fermentation liquid, and a preparation method and application thereof. Background Art

[0002] Plant lactic acid bacteria fermentation is an environmentally friendly and sustainable technology used in food processing and healthcare. It not only diversifies product flavors but also provides health benefits. Therefore, research in this area is crucial. During the fermentation process, microorganisms use polysaccharides, proteins, fibers and other substances as nutrient sources. This process enhances the release of active ingredients and converts large molecules into small molecules, thereby increasing their biological activity. In addition, the growth of microorganisms during fermentation leads to the production of various metabolites, such as amino acids and organic acids, which further enhance the flavor.

[0003] Traditional Chinese medicine fermentation is the use of microbial fermentation methods to produce traditional Chinese medicine. In traditional Chinese medicine production, traditional Chinese medicine processing uses natural microorganisms to ferment traditional Chinese medicine to improve the efficacy of traditional Chinese medicine, eliminate the toxic and side effects of traditional Chinese medicine, and change the medicinal properties of traditional Chinese medicine. For example, traditional Chinese medicines such as Liushenqu, Dandouchi, and Banxiaqu are all fermented by natural microorganisms. However, in the prior art, there are few related studies on the use of fermentation to process traditional Chinese medicine.

[0004] Total phenolic acid is the main active ingredient of wolfberry, which has antioxidant and anti-aging effects. However, further research is still needed to increase the content of total phenolic acid and other active ingredients through fermentation technology. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a composite bacterial agent, wolfberry fermentation liquid and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a composite bacterial agent, which comprises Lactobacillus plantarum LZU-LJY-1Q1 and Lactobacillus plantarum LZU-J-LZ1-1;

[0008] The volume ratio of Lactobacillus plantarum LZU-LJY-1Q1 to Lactobacillus plantarum LZU-J-LZ1-1 is 1-3:1-3;

[0009] Lactobacillus plantarum LZU-J-LZ1-1, deposit number GDMCCNO:64796;

[0010] Lactobacillus plantarum LZU-LJY-1Q1, the deposit number is GDMCCNO:64797.

[0011] The present invention provides a method for preparing wolfberry fermentation liquid, comprising the following steps:

[0012] 1) mixing wolfberry powder, water and complex enzyme, performing enzymolysis and inactivating the enzyme to obtain enzymolysis solution;

[0013] 2) inoculating the composite bacterial agent into the enzymatic hydrolyzate for fermentation, and then inactivating the compound bacterial agent to obtain a wolfberry fermentation liquid;

[0014] Step 1) The mass volume ratio of wolfberry powder to water is 1g:10-20mL;

[0015] Step 1) The volume of the complex enzyme is 0.2% to 0.6% of the total volume of wolfberry powder and water;

[0016] Step 2) The inoculation volume of the composite bacterial agent is 1% to 5% of the total volume after the enzymatic hydrolysate and the composite bacterial agent are mixed, and the concentration of the bacterial cells of the composite bacterial agent is 10 6 ~10 10 CFU / mL;

[0017] Step 2) The fermentation temperature is 35-39° C. and the fermentation time is 12-18 hours.

[0018] Preferably, the complex enzyme is composed of the following raw materials in parts by weight:

[0019] 1 to 3 parts of cellulase and 1 to 3 parts of pectinase.

[0020] Preferably, the enzymatic hydrolysis temperature in step 1) is 50-70° C., and the enzymatic hydrolysis time is 40-80 min.

[0021] Preferably, the relative pressure of the enzyme inactivation in step 1) is 90-120 kPa, the enzyme inactivation temperature is 80-100° C., and the enzyme inactivation time is 40-80 min.

[0022] Preferably, the inactivation temperature in step 2) is 80-100° C. and the inactivation time is 15-45 min.

[0023] The invention provides wolfberry fermentation liquid prepared by the preparation method.

[0024] The invention provides application of the wolfberry fermentation liquid in preparing antioxidant drugs.

[0025] The present invention provides application of the wolfberry fermentation liquid in preparing anti-aging medicines.

[0026] The present invention provides application of the wolfberry fermentation liquid in preparing food or cosmetics, wherein the food includes health food.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a composite bacterial agent, including plant lactobacillus LZU-LJY-1Q1 and plant lactobacillus LZU-J-LZ1-1, which can obtain nutrients from components such as polysaccharides, fibers, and proteins in wolfberries. It is not only beneficial to promote the release of active ingredients in wolfberries, but also converts macromolecular active substances in wolfberries into small molecules, thereby enhancing its anti-aging and anti-oxidation effects. In addition, microorganisms produce various secondary metabolites during growth, including amino acids, organic acids, vitamins, phenols, and aroma components such as alcohols, aldehydes, and esters, which can improve the antioxidant activity of wolfberry fermentation liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram of the fermentation effects of different strains;

[0030] Figure 2 This is a diagram of the fermentation effect of different fermentation strain ratios;

[0031] Figure 3 This is the effect diagram of the fermentation time on the fermentation effect;

[0032] Figure 4 This is the effect diagram of the effect of inoculation amount on fermentation effect;

[0033] Figure 5 This is the effect diagram of the fermentation temperature on the fermentation effect;

[0034] Figure 6 This is a graph showing the changes in the number of live bacteria during the fermentation process;

[0035] Figure 7 This is a graph showing the changes in pH and titratable acid content during the fermentation process;

[0036] Figure 8 This is the content change diagram of total flavonoids and total phenolic acids during the fermentation process;

[0037] Fig. 9 This is the change diagram of total polysaccharide content during the fermentation process;

[0038] Fig.10 It is the regression equation graph of chromatographic peak area (y) versus concentration (x);

[0039] Fig.11 are chromatograms of standard and sample, where A is the chromatogram of standard and B is the chromatogram of sample;

[0040] Fig.12This is a graph showing changes in betaine content during fermentation;

[0041] Fig.13 This is a graph showing the changes in the total antioxidant capacity of ABTS and the DPPH and hydroxyl radical scavenging capacity during the fermentation process.

[0042] Biological Deposit Description

[0043] Lactobacillus plantarum LZU-LJY-1Q1 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, on July 1, 2024, and the deposit number is GDMCC NO: 64797;

[0044] Lactobacillus plantarum LZU-J-LZ1-1 was deposited in Guangdong Microbiological Culture Collection Center on July 1, 2024, with the deposit address at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC NO: 64796;

[0045] Lactobacillus plantarum LZU-J-Q21 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, on March 20, 2023, and the deposit number is GDMCC NO: 63277;

[0046] Lactobacillus plantarum LZU-J-TSL6 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The deposit date is October 23, 2020, and the deposit number is GDMCC NO: 61242;

[0047] Lactobacillus plantarum LZU-S-ZCJ is deposited in the Guangdong Provincial Microbiological Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The preservation time is December 31, 2020, and the preservation number is GDMCC NO:61402. DETAILED DESCRIPTION

[0048] The present invention provides a composite bacterial agent, which comprises Lactobacillus plantarum LZU-LJY-1Q1 and Lactobacillus plantarum LZU-J-LZ1-1;

[0049] The volume ratio of Lactobacillus plantarum LZU-LJY-1Q1 to Lactobacillus plantarum LZU-J-LZ1-1 is 1-3:1-3;

[0050] Lactobacillus plantarum LZU-J-LZ1-1, deposit number GDMCCNO:64796;

[0051] Lactobacillus plantarum LZU-LJY-1Q1, the deposit number is GDMCCNO:64797.

[0052] In the present invention, the volume ratio of Lactobacillus plantarum LZU-LJY-1Q1 to Lactobacillus plantarum LZU-J-LZ1-1 is 1-3:1-3, preferably 1-2:1-2, and more preferably 1:1. The concentration of the bacterial cells of the composite bacterial agent is preferably 10 6 ~10 10 CFU / mL, more preferably 10 7 ~10 9 CFU / mL, more preferably 10 8 CFU / mL.

[0053] The present invention provides a method for preparing wolfberry fermentation liquid, comprising the following steps:

[0054] 1) mixing wolfberry powder, water and complex enzyme, performing enzymolysis and inactivating the enzyme to obtain enzymolysis solution;

[0055] 2) inoculating the composite bacterial agent into the enzymatic hydrolysate for fermentation, and then inactivating the compound bacterial agent to obtain a wolfberry fermentation liquid;

[0056] In the present invention, the mass volume ratio of the wolfberry powder to water in step 1) is preferably 1g:10-20mL, further preferably 1g:12-18mL, and further preferably 1g:15mL; the volume of the complex enzyme in step 1) is preferably 0.2%-0.6% of the total volume of wolfberry powder and water, further preferably 0.3%-0.5%, and further preferably 0.4%; the temperature of the enzymatic hydrolysis is preferably 50-70°C, further preferably 55-65°C, and further preferably 60°C; the enzymatic hydrolysis time is preferably 40-80min, further preferably 50-70min, and further preferably 60min; the enzyme inactivation pressure is preferably 90-120kPa, further preferably 95-115kPa, and further preferably 101kPa; the enzyme inactivation temperature is preferably 80-100°C, further preferably 85-95°C, and further preferably 90°C; the enzyme inactivation time is preferably 40-80min, further preferably 50-70min, and further preferably 60min.

[0057] In the present invention, the inoculation volume of the composite bacterial agent in step 2) is preferably 1% to 5% of the total volume after the enzymatic hydrolysate and the composite bacterial agent are mixed, more preferably 2% to 4%, and even more preferably 3%; the concentration of the bacterial cells of the composite bacterial agent is preferably 10 6 ~10 10 CFU / mL, more preferably 10 7 ~10 9 CFU / mL, more preferably 10 8 CFU / mL; the fermentation temperature is preferably 35-39°C, more preferably 36-38°C, and further preferably 39°C; the fermentation time is preferably 12-18h, more preferably 13-17h, and further preferably 15h; the inactivation temperature is preferably 80-100°C, more preferably 85-95°C, and further preferably 90°C; the inactivation time is preferably 15-45min, more preferably 20-40min, and further preferably 30min.

[0058] In the present invention, the complex enzyme is composed of the following raw materials in parts by weight:

[0059] The cellulase is preferably 1 to 3 parts, more preferably 1.5 to 2.5 parts, and even more preferably 2 parts;

[0060] The amount of pectinase is preferably 1 to 3 parts, more preferably 1.5 to 2.5 parts, and even more preferably 2 parts.

[0061] The invention provides wolfberry fermentation liquid prepared by the preparation method.

[0062] The present invention provides the use of the wolfberry fermentation liquid in the preparation of antioxidant drugs. The total phenolic acid in the wolfberry has an antioxidant effect, and after fermentation, the antioxidant capacity is improved.

[0063] The present invention provides the use of the wolfberry fermentation liquid in the preparation of anti-aging drugs. The total phenolic acid in the wolfberry has an anti-aging effect, and after fermentation, the anti-aging ability is improved.

[0064] The present invention provides application of the wolfberry fermentation liquid in preparing food or cosmetics, wherein the food includes health food.

[0065] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0066] Sources:

[0067] Wolfberry was purchased from Zhongning County Shengshi Trading Co., Ltd.;

[0068] Cellulase and pectinase were purchased from Nanning Pangbo Bioengineering Co., Ltd.;

[0069] Gallic acid and rutin were purchased from China Food and Drug Identification Institute.

[0070] Example 1: Preparation of Lycium barbarum enzymatic hydrolysate

[0071] Remove impurities from wolfberry, crush at 6000r / min for 1min and store in a desiccator for later use; weigh 6g of wolfberry powder, put it into a wall-breaking machine, add 90mL of purified water to make a homogenate according to the mass volume ratio of wolfberry powder and water of 1g:15mL, add 0.4% mixed enzyme (cellulase: pectinase = 1:1, v / v) by volume, and enzymolyze at 60°C for 1h. Subsequently, high pressure 101kPa, 90°C for 1h to inactivate the enzyme, and after cooling at room temperature, obtain an enzymolysis solution, which is stored at -4°C.

[0072] Example 2 Preparation of wolfberry fermentation broth

[0073] (1) Preparation of Lactobacillus plantarum liquid

[0074] Streak Lactobacillus plantarum on solid culture medium, culture and activate at 37℃ for 24h, inoculate the activated single colony of Lactobacillus plantarum into 8mL liquid culture medium containing 52.24g MRS broth / L, culture at 37℃ for 18h to obtain the first generation bacterial solution; take 200μL of the first generation bacterial solution and inoculate it into 9.8mL liquid culture medium containing 52.24g MRS broth / L, culture at 37℃ for 18h to obtain the second generation bacterial solution. Dilute the second generation bacterial solution to obtain 1×10 8 CFU / mL is the bacterial solution required for fermentation.

[0075] (2) Screening of fermentation strains

[0076] Take 5 sterile conical flasks, add 30mL of wolfberry enzymatic hydrolysate to each flask, and 8 CFU / mL, 2% volume inoculum was inoculated into Lactobacillus plantarum LZU-J-TSL6, LZU-LJY-1Q1, LZU-J-LZ1-1, LZU-J-Q21 and LZU-S-ZCJ, placed in a constant temperature incubator, cultured at 37°C, sampled every 6 hours, zeroed with wolfberry enzymatic hydrolysate, and the ΔOD600 value of the fermentation liquid was measured.

[0077] The results are as follows Figure 1 As shown, when the fermentation time reached 18h, the fermentation effects of Lactobacillus plantarum LZU-LJY-1Q1 and LZU-J-LZ1-1 were the best, and these two strains were selected to explore the fermentation strain ratio.

[0078] (3) Screening of fermentation strain ratio

[0079] The prepared Lactobacillus plantarum LZU-LJY-1Q1 and Lactobacillus plantarum LZU-J-LZ1-1 bacterial liquids were inoculated with wolfberry hydrolysate at volume ratios of 1:1, 1:3 and 3:1, and then inoculated into 30 mL of wolfberry hydrolysate at 2% by volume and cultured for 18 h, and the OD600 value of the fermentation liquid was determined.

[0080] The results are as follows Figure 2 As shown in the figure, when the fermentation time was 18h and the ratio of Lactobacillus plantarum LZU-LJY-1Q1 to Lactobacillus plantarum LZU-J-LZ1-1 was 1:1, the fermentation effect was the best. LZU-LJY-1Q1 and LZU-J-LZ1-1 (1:1) were selected for optimization of the subsequent fermentation process.

[0081] (4) Study on fermentation time

[0082] Add 30mL of wolfberry enzymatic hydrolysate to a sterile conical flask, inoculate 2% of the volume of the bacterial solution, and ferment at 37°C for 14h, 18h, 22h, 26h, and 30h. Detect the OD600 value at each time period, set 3 replicates for each ratio, and determine the optimal fermentation time.

[0083] The results are as follows Figure 3 As shown, when the fermentation time was 15 h, the OD600 value in the fermentation broth was significantly higher than that at other fermentation times, so the fermentation time was determined to be 15 h.

[0084] (5) Study on inoculation amount

[0085] Add 30 mL of wolfberry enzymatic hydrolysate into a sterile conical flask, and inoculate 1%, 2%, 3%, 4%, and 5% of the volume of the bacterial solution respectively. Ferment at 37°C for 18 h, and detect the OD600 value of the fermentation liquid at each inoculation amount. Set 3 replicates for each ratio to determine the optimal inoculation amount.

[0086] The results are as follows Figure 4 As shown, when the inoculum dosage was 3%, the number of live bacteria in the fermentation broth was significantly higher than that at other inoculum dosages, so the inoculum dosage was determined to be 3%.

[0087] (6) Research on fermentation temperature

[0088] Add 30 mL of wolfberry enzymatic hydrolysate into a sterile conical flask, access 3% volume of bacterial solution, and ferment at 33°C, 35°C, 37°C, 39°C, and 41°C for the optimal time. Detect the OD value of the fermentation liquid, set 3 replicates for each ratio, and determine the optimal fermentation temperature.

[0089] The results are as follows Figure 5 As shown, when the fermentation temperature was 37°C, the OD600 value in the fermentation broth was significantly higher than that at other temperatures, so the fermentation temperature was determined to be 37°C.

[0090] In summary, the fermentation conditions were determined as follows: fermentation time 15h, fermentation temperature 37℃, inoculation amount 3%. After fermentation, the mixture was inactivated at 90℃ for 30min, cooled at room temperature, and stored at -4℃ for later use.

[0091] Example 3 Changes in physicochemical properties during the fermentation of wolfberry hydrolysate

[0092] (1) Changes in viable bacterial count, pH, and total acid during fermentation

[0093] The number of live bacteria of Lactobacillus plantarum increased significantly during the fermentation process (see Figure 6 During the fermentation process, the number of live bacteria increased significantly. When the fermentation reached 15 hours, the number of live bacteria reached 10.28 CFU / mL. Bacterial growth will produce certain organic acids, such as lactic acid, which will reduce the pH of the fermentation liquid and increase the total acid content of the fermentation. These are two important indicators for evaluating the degree of fermentation. The pH and total acid content of the wolfberry fermentation liquid during the fermentation process were determined using an acidometer and according to the national standard for the determination of total acid in food (GB12456-2021).

[0094] The results are as follows Figure 7 As shown in the figure, the horizontal axis represents the fermentation time (h), and the vertical axis represents the pH value; the titratable acid content was measured, and different letters indicate significant differences (P<0.05). During the fermentation process, the pH gradually decreased, while the titratable acid content gradually increased. Compared with the fermentation 0h, the total acid content increased by 223.07% at 18h of fermentation. The results show that Lactobacillus plantarum LZU-LZ1-1 and LZU-LJY-1Q1 can grow in wolfberry enzymatic hydrolysate and produce metabolites.

[0095] (2) Changes of total flavonoids and total phenolic acids during fermentation

[0096] (2.1) Preparation of reference solution

[0097] Accurately weigh 8.52 g of gallic acid reference substance (mass fraction 91.5%) and place it in a 10 mL volumetric flask, add purified water to dissolve it, dilute to the mark, shake well, and obtain a reference substance standard solution with a gallic acid concentration of 0.780 mg / mL. Store at 4°C for later use.

[0098] Accurately weigh 10.27 mg of rutin reference substance (mass fraction 92.4%) and place it in a 50 mL volumetric flask, add methanol to dissolve it, dilute to the scale, shake well, and obtain a reference substance standard solution with a rutin concentration of 0.190 mg / mL. Store at 4°C for later use.

[0099] (2.2) Establishment of standard curve

[0100] (2.2.1) Standard curve of total flavonoids

[0101] Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of rutin reference solution and dilute to 2 mL, shake well, add 2 mL of 0.14 moL / L AlCl3 solution and 1 mL of buffer solution, the buffer solution is a mixture of 0.2 moL / L anhydrous sodium acetate solution and 0.2 moL / L glacial acetic acid in a 1:1 ratio, add methanol to make the volume to 10 mL, mix well, and color develop at 40°C in a water bath for 10 min. Take 200 μL and measure the OD value of each well in a 96-well plate at 421 nm using an enzyme reader. Use the absorbance value as the ordinate (Y) and the rutin reference solution as the abscissa (X) to establish a standard curve.

[0102] The standard curve is Y = 11.461X + 0.0015, and R 2 =0.9965, and the linear range is 0.044-0.259 mg / mL, which can be used for further experimental analysis.

[0103] (2.2.2) Total phenolic acid standard curve

[0104] Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of gallic acid reference solution and dilute to 10 mL, shake well, pipette 500 μL of different concentrations of standard, add 1.5 mL of folin solution to each, mix well, place in the dark for 15 min, add 2 mL of 8% anhydrous sodium carbonate solution, mix well, water bath at 50°C for 15 min, color development, stand at room temperature for 5 min, take 200 μL in a 96-well plate, measure the OD value of each well at 760 nm with an enzyme reader, and establish a standard curve with absorbance as the ordinate (Y) and gallic acid reference solution as the abscissa (X).

[0105] The standard curve is Y = 79.923X + 0.0312, and R 2 =0.9984, and the linear range is 0.191-0.955 mg / mL, which can be used for further experimental analysis.

[0106] (2.3) Result analysis

[0107] Total phenolic acids and total flavonoids are the main active substances in wolfberry. They will undergo biotransformation during the fermentation process, and some total flavonoids will be converted into total phenolic acids. Figure 8 As shown in the figure, the horizontal axis represents the fermentation time (h), the vertical axis represents the total flavonoids and total phenolic acid content (mg / mL), and different letters indicate significant differences (P<0.05). The total polyphenol content increased significantly during the fermentation process, and the total polyphenols increased by 1.31% from 0h to 15h of fermentation. The total flavonoids were fermented to 15h, and the mass concentration was 0.19mg / mL. However, both the total flavonoids and total phenolic acid contents were significantly improved compared to before fermentation.

[0108] (3) Changes of total polysaccharides during fermentation

[0109] Polysaccharides are the main active substances in wolfberry. Macromolecular substances have low oral availability. Microbial fermentation can convert macromolecular substances into small molecules, improve bioavailability and thus improve effectiveness. Fig. 9 It can be seen that the content of total polysaccharides decreased significantly during the fermentation process. After 15 hours of fermentation, the total polysaccharide content was 1.04 mg / mL. It may be that the sugar substances in the fermentation process provide energy for the fermentation of microorganisms. In addition, the total sugar content in the wolfberry pulp was significantly reduced by Lactobacillus plantarum fermentation. The total sugar content of the wolfberry fermentation liquid obtained after fermentation was reduced, which is suitable for use by diabetics and increases the scope of application. At the same time, the sugar-acid ratio of the wolfberry fermentation liquid is reduced, which helps to improve the flavor of the wolfberry fermentation liquid.

[0110] (4) Changes of betaine during fermentation

[0111] (4.1) Chromatographic conditions

[0112] CAPCELL PAKNH2 chromatographic column (250mm×4.6mm, 5μm); mobile phase: acetonitrile (A)-water (B), isocratic elution (0-30min, 90% A, 10% B); volume flow rate: 0.7mL / min; column temperature: 30°C; detection wavelength: 195nm, injection volume: 10μL.

[0113] (4.2) Preparation of sample solution

[0114] According to the fermentation conditions of Example 2, "fermentation time 15h, fermentation temperature 37°C, inoculation amount 3%. After the fermentation, inactivate at 90°C for 30min and cool at room temperature", prepare wolfberry fermentation liquid, centrifuge at 6000r / min for 5min after cooling, take the supernatant and filter out impurities through 0.22μm microporous filter membrane to obtain the sample solution.

[0115] (4.3) Preparation of reference solution

[0116] Accurately weigh 24.08 mg of betaine reference substance (mass fraction 98%), place it in a 100 mL volumetric flask, add purified water to dissolve it, dilute to the scale, shake well, and obtain a reference substance standard solution with a betaine mass concentration of 0.13 mg / mL, and store it at 4°C for later use.

[0117] (4.4) Methodological considerations

[0118] (4.4.1) Linear relationship results

[0119] The reference solution prepared under 4.3 was diluted into solutions of different concentrations, injected into a high performance liquid chromatograph, and the peak area was measured. A linear regression was performed between the chromatographic peak area (y) and the concentration (x), and the regression equation Y = 321027X + 51952 was obtained. (See Fig.10 ).

[0120] (4.4.2) Results of precision test, stability test and repeatability test

[0121] Accurately aspirate the same test solution, inject it according to the chromatographic conditions described in step (4.1), repeat the injection 6 times, and calculate the RSD of the retention time (tR) and peak area (S) of each target compound. Accurately aspirate the same test solution, inject it according to the chromatographic conditions under item "3", measure it at 0, 3, 6, 9, 12, and 18h, respectively, and calculate the RSD of the retention time (tR) and peak area (S) of each target compound. Accurately weigh 6 portions of the same batch of samples, prepare the test solution, inject 10μL of each portion, and calculate the RSD of the retention time (tR) and peak area (S) of each target compound. The results are shown in Table 1. The RSD values ​​of the peak area (S) and retention time (tR) of betaine were less than 3% in the precision, repeatability, and stability experiments. The HPLC analysis charts of the reference and samples at 195nm are shown in Fig.11 .

[0122] Table 1 Methodological results review

[0123]

[0124] (4.5) Analysis of betaine results during fermentation

[0125] Depend on Fig.12 The results show that the content of betaine decreased significantly during the fermentation process, and the mass concentration was 3.72 mg / mL at 18 hours of fermentation. It may be that the microorganisms use betaine as their source of carbon and nitrogen. During the fermentation process, betaine can serve as a methyl donor to ensure the metabolic function of the strain. At the same time, betaine can also regulate the osmotic pressure to ensure the stability of the cells, and can also improve the ability to tolerate osmotic pressure.

[0126] (5) Changes in the total antioxidant capacity and DPPH and OH- free radical scavenging capacity of ABTS during fermentation

[0127] (5.1) Total antioxidant capacity of ABTS

[0128] The ABTS free radical stock solution is a mixture of 50 mL of 7 mM ABTS solution and 880 μL of 140 mM potassium persulfate solution, which is stored at room temperature in the dark for 18 h, and diluted with distilled water until the absorbance at 734 nm reaches 0.70 ± 0.02 to obtain the ABTS free radical working solution. Sample: 100 μL of sample (prepared in step (4.2)) is mixed with 100 μL of ABTS working solution, control: 100 μL of sample is mixed with 100 μL of distilled water, blank: 100 μL of distilled water is mixed with 100 μL of ABTS free radical working solution (3 portions each), react at room temperature in the dark for 30 min, and measure the absorbance values ​​at 734 nm using an ELISA reader, which are A1, A2, and A0, respectively.

[0129] The results were expressed as the percentage inhibition of antioxidant capacity (% ABTS clearance), calculated as follows:

[0130]

[0131] (5.2) DPPH free radical scavenging rate

[0132] The DPPH free radical scavenging ability detection method is as follows: sample: 1mL sample (prepared in step (4.2)) mixed with 1mL 0.2mMDPPH (dissolved in anhydrous ethanol); control: 1mL sample mixed with 1mL distilled water; blank: 1mL distilled water mixed with 1mL DPPH solution, shaken thoroughly, centrifuged at 4000r / min for 5min, and kept in dark for 30min. Reacted in dark place at room temperature for 30min, and the absorbance values ​​measured at 517nm by an enzyme marker were A1, A2, and A0 respectively.

[0133] (5.3) OH- free radical scavenging rate

[0134] OH- free radical scavenging ability The OH- scavenging ability was analyzed using a hydroxyl radical assay kit (Nanjing Jiancheng Bioengineering Institute). The hydroxyl radical scavenging activity of all samples was determined according to the assay kit. The percentage of OH- scavenging activity was calculated using the equation. The results are expressed as the percentage of antioxidant capacity inhibition (% OH- scavenging).

[0135] The results were expressed as the percentage of antioxidant capacity inhibition (% DPPH scavenging), and the calculation formula was as follows:

[0136]

[0137] The results are as follows Fig.13As shown, the Lactobacillus plantarum LZU-J-LZ1-1: LZU-LJY-1Q1 (1:1) fermented wolfberry hydrolysate groups each have a certain antioxidant capacity. As time changes, the antioxidant activity of the fermented wolfberry hydrolysate increases with time, the ability to inhibit OH- increases by 1.93%, the ability to inhibit DPPH free radicals increases by 1.37%, and the total antioxidant capacity increases by 3.37%, indicating that metabolites may be transformed during the fermentation process and the content of substances related to antioxidant activity increases.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent includes Lactobacillus plantarum LZU-LJY-1Q1 and Lactobacillus plantarum LZU-J-LZ1-1; The volume ratio of Lactobacillus plantarum LZU-LJY-1Q1 to Lactobacillus plantarum LZU-J-LZ1-1 is 1-3:1-3; Lactobacillus plantarum LZU-J-LZ1-1, deposit number is GDMCC NO: 64796; Lactobacillus plantarum LZU-LJY-1Q1, the deposit number is GDMCC NO:64797.

2. A method for preparing wolfberry fermentation broth, characterized in that: The following steps are involved: 1) mixing wolfberry powder, water and complex enzyme, performing enzymolysis and inactivating the enzyme to obtain enzymolysis solution; 2) inoculating the composite bacterial agent according to claim 1 into the enzymatic hydrolyzate, fermenting, and then inactivating the mixture to obtain wolfberry fermentation liquid; Step 1) The mass volume ratio of wolfberry powder to water is 1g:10-20mL; Step 1) The volume of the complex enzyme is 0.2% to 0.6% of the total volume of wolfberry powder and water; Step 2) The inoculation volume of the composite bacterial agent is 1% to 5% of the total volume after the enzymatic hydrolysate and the composite bacterial agent are mixed, and the concentration of the bacterial cells of the composite bacterial agent is 10 6 ~10 10 CFU / mL; Step 2) The fermentation temperature is 35-39° C. and the fermentation time is 12-18 hours.

3. The preparation method according to claim 2, characterized in that: The complex enzyme is composed of the following raw materials in parts by weight: 1 to 3 parts of cellulase and 1 to 3 parts of pectinase.

4. The preparation method according to claim 3, characterized in that: Step 1) The enzymolysis temperature is 50-70° C. and the enzymolysis time is 40-80 min.

5. The preparation method according to claim 4, characterized in that: In step 1), the relative pressure of the enzyme inactivation is 90-120 kPa, the temperature of the enzyme inactivation is 80-100° C., and the enzyme inactivation time is 40-80 min.

6. The preparation method according to claim 4, characterized in that: The inactivation temperature of step 2) is 80-100° C. and the inactivation time is 15-45 min.

7. The wolfberry fermentation liquid prepared by the preparation method according to any one of claims 2 to 6.

8. Use of the wolfberry fermentation broth according to claim 7 in the preparation of antioxidant drugs.

9. Use of the wolfberry fermentation liquid according to claim 7 in the preparation of anti-aging drugs.

10. Use of the wolfberry fermented liquid according to claim 7 in preparing food or cosmetics, characterized in that: The food includes health food.

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