Complex microbial inoculant containing Lactobacillus brevis and application of complex microbial inoculant in fermentation of Lycium barbarum
By using the composite bacteria agent of Lactobacillus brevis and Trichoderma harziana to ferment wolfberry, the problem of insufficient improvement in the quality and efficacy of wolfberry fermentation products in the existing technology was solved, and the quality and efficacy of wolfberry fermentation products were significantly improved, and the quality and efficacy of wolfberry fermentation products were significantly improved. Zheimer's treatment provides new drug aids.
Patent Information
- Application Number
- CN202411263336.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2024-12-24
AI Technical Summary
The existing technology has conducted few research on the use of bacterial species for wolfberry fermentation, which has led to the failure to effectively improve the quality and efficacy of wolfberry fermentation products.
The wolfberry pulp was fermented by a composite bacterial agent containing Lactobacillus brevis and Trichoderma harzianum. By adjusting the effective live bacteria ratio and fermentation conditions of the bacterial agent, an efficient wolfberry fermentation product was prepared.
It significantly improves the quality and efficacy of wolfberry fermentation products, including carotenoids, vitamin C, and flavonoid content, and has significant acetylcholinesterase inhibitory activity, which is suitable for the treatment of Alzheimer's disease.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial fermentation, in particular to a composite bacterial agent containing Lactobacillus brevis and application thereof in wolfberry fermentation. Background Art
[0002] Lycium barbarum can enhance body skills, promote health recovery, improve the body's disease resistance, and has the effect of strengthening the body and delaying aging. Lycium barbarum can significantly increase the storage of muscle glycogen and liver glycogen, improve human vitality, and have an anti-fatigue effect. Lycium barbarum has the function of improving brain function and enhancing people's learning and memory ability. Eating Lycium barbarum can significantly reduce the content of serum cholesterol and triglycerides, reduce and prevent arteriosclerosis, and treat hypertension. It is a good health product for patients with coronary heart disease. Lycium barbarum has a significant effect of promoting the proliferation of hematopoietic cells, increasing the number of white blood cells, and enhancing the body's hematopoietic function. Lycium barbarum is a good medicine for strengthening the body, producing essence and marrow, nourishing yin and kidney, replenishing qi and calming the mind, strengthening the body, and delaying aging. It has been a top-grade tonic since ancient times. It effectively enhances the functions of various internal organs, can improve brain function and the function of fighting free radicals, and has a significant effect of delaying aging.
[0003] However, there are relatively few studies on the fermentation of wolfberries to further improve their quality, especially the lack of in-depth research and extensive exploration of the strains used in wolfberry fermentation. Therefore, how to prepare a composite bacterial agent for efficient fermentation of wolfberries and improve the quality and efficacy of wolfberry fermented products is one of the problems that need to be solved in this field. Summary of the invention
[0004] The purpose of the present invention is to provide a composite bacterial agent containing Lactobacillus brevis and its application in wolfberry fermentation to solve the problems existing in the above-mentioned prior art. The present invention uses the composite bacterial agent to ferment wolfberry puree, and the prepared wolfberry fermentation product achieves significant improvement in quality and efficacy.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] The present invention provides a composite bacterial agent, comprising Lactobacillus brevis and Trichoderma harzianum;
[0007] The Lactobacillus brevis has a deposit number of CGMCC NO.16125, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee on July 17, 2018, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;
[0008] The deposit number of the Trichoderma harzianum is CGMCC NO.14638, and it is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration. The deposit date is November 20, 2017, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.
[0009] Preferably, in the composite bacterial agent, the ratio of the effective viable counts of the Lactobacillus brevis to the Trichoderma harzianum is (4-8):1, and the total effective viable count is not less than 1.7×10 7 CFU / g.
[0010] The invention also provides application of the composite bacterial agent in wolfberry fermentation.
[0011] The present invention also provides a method for preparing a wolfberry fermented product, comprising the following steps:
[0012] Inoculating the composite bacterial agent into wolfberry pulp, fermenting it, and obtaining a fermentation liquid;
[0013] The fermentation liquid is centrifuged and the supernatant is collected to obtain the wolfberry fermentation product.
[0014] Preferably, the inoculation amount of the composite bacterial agent is 3-5%.
[0015] Preferably, the fermentation temperature is 29-34°C, the time is 24-36h, and the rotation speed is 150r / min.
[0016] Preferably, the centrifugal speed is 5000 rpm and the time is 20 min.
[0017] The invention also provides a wolfberry fermented product prepared by the preparation method.
[0018] The present invention also provides the use of the wolfberry fermentation product in preparing a medicine for inhibiting the activity of acetylcholinesterase.
[0019] The present invention also provides the use of the wolfberry fermentation product in preparing a medicine for auxiliary treatment of Alzheimer's disease.
[0020] The present invention discloses the following technical effects:
[0021] The present invention prepares a wolfberry fermented product by fermenting a composite bacterial agent prepared by inoculating Lactobacillus brevis and Trichoderma harzianum. Experiments have shown that the wolfberry fermented product of the present invention is superior to wolfberry puree and wolfberry products prepared using other fermented bacteria in terms of quality and efficacy, indicating that the composite bacterial agent of the present invention plays a synergistic role in improving the quality of wolfberry fermentation. The wolfberry fermented product of the present invention can inhibit the activity of acetylcholinesterase and is used to prepare a drug for the treatment of Alzheimer's disease. The present invention provides a new fermentation bacteria combination for wolfberry fermentation, expands the method of wolfberry processing, and has broad application prospects. DETAILED DESCRIPTION
[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0027] In the present invention, Lactobacillus brevis with a preservation number of CGMCC NO.16125, Trichoderma harzianum with a preservation number of CGMCC NO.14638, and Torulaspora delbrueckii with a preservation number of CGMCC NO.29537 were all purchased from the General Microbiological Center of China National Committee for the Preservation of Microorganisms. The public can purchase the above strains from the above preservation units.
[0028] MRS liquid culture medium: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 10.0 g / L, diammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, diammonium phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1 mL / L, solvent is deionized water, pH 5.0, sterilized by high pressure steam at 121 ° C for 30 min.
[0029] MRS solid culture medium is based on the MRS liquid culture medium formula with 18g / L agar added, and the remaining ingredients and processing are the same as MRS liquid culture medium.
[0030] PDA culture medium: add 300 g potato powder, 20 g glucose and 14 g agar into 1 L distilled water, heat and boil to dissolve, divide into portions and sterilize by high pressure steam at 121°C for 30 min.
[0031] PDB culture medium: add 300 g potato powder and 20 g glucose into 1 L distilled water, heat and boil to dissolve, divide into portions, and sterilize with high pressure steam at 121°C for 30 min.
[0032] Example 1
[0033] 1. Preparation of wolfberry puree
[0034] Take 300 g of freshly picked Ningxia wolfberry, wash, dry, add 1000 mL of sterile water, grind thoroughly with a grinder, add 0.3 g of carrageenan as a stabilizer, concentrate to 1000 mL, sterilize with high-pressure steam at 121° C. for 20 min, and cool to obtain.
[0035] 2. Preparation of Bacterial Liquid
[0036] Lactobacillus brevis with the accession number of CGMCC NO.16125 was activated on MRS solid medium, inoculated into MRS liquid medium and cultured to the logarithmic growth phase, the cells were collected by centrifugation, washed, and diluted to 2×10 7 CFU / mL, for bacterial solution A;
[0037] The Trichoderma harzianum with the accession number of CGMCC NO.14638 was activated on PDA medium, inoculated on PDB medium and expanded to the logarithmic growth phase, the cells were collected by centrifugation, washed, and diluted to 1×10 7 CFU / mL, for bacterial solution B.
[0038] Example 2
[0039] 20 mL of the bacterial solution A and 10 mL of the bacterial solution B prepared in Example 1 were taken and mixed to obtain 30 mL of a composite bacterial agent, in which the effective viable bacterial count ratio of Lactobacillus brevis to Trichoderma harzianum was 4:1.
[0040] The above composite bacterial agent was inoculated into the wolfberry puree prepared in Example 1 (inoculation amount 3%, v / v), fermented at 150 r / min at 31° C. for 24 h, sterilized at 100° C. for 10 min, cooled, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the wolfberry fermented product.
[0041] Example 3
[0042] 30 mL of the bacterial solution A and 10 mL of the bacterial solution B prepared in Example 1 were taken and mixed to obtain 40 mL of a composite bacterial agent, in which the effective viable bacterial count ratio of Lactobacillus brevis to Trichoderma harzianum was 6:1.
[0043] The above composite bacterial agent was inoculated into the wolfberry puree prepared in Example 1 (inoculation amount 4%, v / v), fermented at 29° C. and 150 r / min for 36 h, sterilized at 100° C. for 10 min, cooled, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the wolfberry fermented product.
[0044] Example 4
[0045] 40 mL of the bacterial solution A and 10 mL of the bacterial solution B prepared in Example 1 were taken and mixed to obtain 50 mL of a composite bacterial agent, in which the effective viable bacterial count ratio of Lactobacillus brevis to Trichoderma harzianum was 8:1.
[0046] The above composite bacterial agent was inoculated into the wolfberry puree prepared in Example 1 (inoculation amount 5%, v / v), fermented at 34°C and 150r / min for 30h, sterilized at 100°C for 10min, cooled, centrifuged at 5000rpm for 20min, and the supernatant was collected to obtain the wolfberry fermented product.
[0047] Comparative Example 1
[0048] Take 40 mL of the bacterial solution A prepared in Example 1, inoculate it into the wolfberry puree prepared in Example 1 (inoculum amount 4%, v / v), ferment it at 150 r / min at 34° C. for 30 h, sterilize it at 100° C. for 10 min, cool it, centrifuge it at 5000 rpm for 20 min, collect the supernatant, and obtain the wolfberry fermented product.
[0049] Comparative Example 2
[0050] Take 30 mL of the bacterial solution B prepared in Example 1, inoculate it into the wolfberry puree prepared in Example 1 (inoculum amount 3%, v / v), ferment it at 150 r / min at 29° C. for 36 h, sterilize it at 100° C. for 10 min, cool it, centrifuge it at 5000 rpm for 20 min, collect the supernatant, and obtain the wolfberry fermented product.
[0051] Comparative Example 3
[0052] 50 mL of sterile water was added to the wolfberry puree prepared in Example 1, treated at 31° C. and 150 r / min for 24 h, heated at 100° C. for 10 min, cooled, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain a wolfberry product not inoculated with microorganisms.
[0053] Comparative Example 4
[0054] Delb's spore yeast with the accession number of CGMCC NO.29537 was activated on PDA medium, inoculated on PDB medium and expanded to the logarithmic growth phase, the cells were collected by centrifugation, washed, and diluted to 2×10 7 CFU / mL is the C of bacterial solution.
[0055] 50 mL of the bacterial solution C was inoculated into the wolfberry puree prepared in Example 1 (inoculation amount 5%, v / v), fermented at 30° C. and 150 r / min for 36 h, sterilized at 100° C. for 10 min, cooled, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the wolfberry fermented product.
[0056] Effect example
[0057] The products prepared in the above Examples 2-4 and Comparative Examples 1-4 were tested for quality and efficacy according to the following methods.
[0058] 1. Product quality measurement
[0059] 1.1 Determination of carotenoid content
[0060] Weigh 2g of the product, add it to a 100mL brown reagent bottle, add 30mL of tetrahydrofuran (containing 0.1% BHT), extract for 45min under ultrasonic conditions of W=7, F=4, repeat twice, and combine the extracts; pass the extract through 3g of anhydrous sodium sulfate (with filter paper) into a new round-bottom flask, and rotary evaporate to 6mL at 35°C; add 30mL of petroleum ether and 6mL of 10% sodium chloride, pass through 5g of anhydrous sodium sulfate (with filter paper) into the original round-bottom flask, and rotary evaporate to 6mL at 35°C; add 6mL 10% KOH-CH3OH, saponify in the dark; after saponification, transfer the liquid into a 60mL separatory funnel, add 10mL petroleum ether and 10mL sodium chloride, wash the organic phase with water until neutral (PH=7), evaporate to dryness at 35°C, dilute the residue to 10mL with dichloromethane, and measure the absorbance value at 448nm with an ultraviolet spectrophotometer. Calculate the carotenoid content in the sample using the formula.
[0061] 1.2 Determination of vitamin C content
[0062] Weigh 2g of the product, add 8mL of 2.5% metaphosphoric acid, centrifuge at 10000rpm for 10min at 4℃, repeat twice, combine the supernatant, and dilute to 20mL. Filter with a 0.45μm water filter membrane before testing. On a high performance liquid chromatograph, photodiode array detector, mobile phase: 0.1mol / L dipotassium hydrogen phosphate, 0.08mol / L potassium dihydrogen phosphate, methanol volume ratio of 55:25:20, isocratic elution; chromatographic column is Agilent (4.6×250mm), 0.5μm C18 column, column temperature is 25℃, flow rate is 1.0mL / min, detection wavelength is 245nm, injection volume is 25μL. Calculate the vitamin C content in the sample based on the sample peak area and the standard curve of vitamin C content.
[0063] 1.3 Determination of flavonoid content
[0064] Weigh 2g of the product, add 10mL of 70% ethanol, filter and dilute to 25mL, mix well, take 5mL of the extract, blow dry with nitrogen at 60℃, re-dissolve with 2mL of chromatographic methanol, filter through a 0.22μm organic fiber filter membrane and test. On the high performance liquid chromatograph, the chromatographic column is a C18 reverse phase column (4.6×250mm, 5μm); the mobile phase A is 50mM phosphoric acid solution (pH2.5), the mobile phase B is 100% acetonitrile for elution, the elution program is: 50mM phosphoric acid: acetonitrile (80 / 20); the column temperature is 35℃, the flow rate is 1.0mL / min, the injection volume is 10μL, and the detection wavelength is 365nm. The flavonoid content in the sample is calculated based on the standard curve of the sample peak area and rutin content.
[0065] 1.4 Determination of betaine content
[0066] Weigh 1g of product, add 25mL of ultrapure water, let stand for 2min, centrifuge at 10000rpm for 3min, filter and absorb 5mL of filtrate, add to the activated MCX column, maintain natural flow rate, after the sample enters the adsorption layer of the extraction column, elute with 5mL of methanol and 5mL of methanol in turn; then elute with 5mL of ammoniated methanol, and repeat the elution once. Dry the collected eluate with nitrogen, dissolve it with 2mL of acetonitrile solution to make up the volume, filter it through a 0.45μm filter membrane and then test it. On the high performance liquid chromatograph, the chromatogram is a PC HILIC column (4.6×250 mm, 5 μm); mobile phase A: water, mobile phase B: acetonitrile, gradient elution program: 0-2 min, 95%-90% B; 2 min-3 min, 90%-70% B; 3 min-4 min, 70%-50% B; 4-9 min, 50% B; 9-10 min, 50%-95% B; 10-15 min, 95% B. The column temperature is 25°C, the flow rate is 1.0 mL / min, the detection wavelength is 195 nm, and the injection volume is 10 μL. The betaine content in the sample is calculated based on the sample peak area and the standard curve of the betaine content.
[0067] The data results were normalized, that is, the measurement result of Comparative Example 3 (wolfberry product not inoculated with microorganisms) was taken as 100%, and the test results of the remaining groups were expressed as a ratio to Comparative Example 3.
[0068] Table 1 Statistics of wolfberry quality test results in each group
[0069]
[0070] The results are shown in Table 1. After the wolfberry pulp is inoculated with microorganisms for fermentation, the contents of carotenoids, vitamin C, flavonoids and amino acids can be increased. In particular, the active substance content of the wolfberry fermented product prepared by fermentation with the composite bacterial agent prepared by the present invention is significantly higher than that of the fermentation using Lactobacillus brevis (Comparative Example 1) or Trichoderma harzianum (Comparative Example 2) alone, and is significantly higher than that of the fermentation using Delbur's spore yeast (Comparative Example 4).
[0071] 2. Product efficacy determination
[0072] The inhibition of acetylcholinesterase activity in vitro was determined using a modified Ellman method.
[0073] The total reaction volume was 260 μL, containing 1 U mL -1 AChE 10μL, wolfberry fermentation product 20μL, 15mmol·L -1 DTNB 100 μL, 15 mmol·L -1100 μL of ATch was added and reacted at 37℃ for 6 min. 30 μL of 4% SDS was quickly added to terminate the reaction. The OD value was measured at 412 nm. Each sample was repeated 4 times. Considering the color of the wolfberry fermentation product, 10 μL PBS was used instead of AChE for background subtraction in each experiment. The control group was replaced with 20 μL PBS instead of fermentation liquid, which was recorded as A control.
[0074] The calculation formula is as follows:
[0075]
[0076] As shown in Table 2, the data are presented in the form of mean ± standard deviation. The results show that the use of microbial fermentation can improve the acetylcholinesterase inhibitory activity of wolfberry products, especially the acetylcholinesterase inhibitory activity of the wolfberry fermented product prepared by the composite bacterial agent prepared by the present invention is significantly better than that of the fermentation using Lactobacillus brevis or Trichoderma harzianum alone, and significantly better than that of the fermentation using Psoralea delbrueckii.
[0077] Acetylcholinesterase is usually an important target for treating Alzheimer's disease. Therefore, the wolfberry fermentation product prepared by the present invention can be used to prepare a drug for treating Alzheimer's disease.
[0078] Table 2 Statistical table of wolfberry efficacy test results in each group
[0079]
[0080] Different letters indicate significant differences (P<0.05).
[0081] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A composite bacterial agent, characterized in that: These include Lactobacillus brevis and Trichoderma harzianum; Wherein, the preservation number of the Lactobacillus brevis is CGMCC NO.16125; the preservation number of the Trichoderma harzianum is CGMCC NO.14638.
2. The composite bacterial agent according to claim 1, characterized in that In the composite bacterial agent, the ratio of the effective viable count of the Lactobacillus brevis to the Trichoderma harzianum is (4-8):1, and the total effective viable count is not less than 1.7×10 7 CFU / g.
3. Use of the composite bacterial agent as claimed in claim 1 or 2 in wolfberry fermentation.
4. A method for preparing a wolfberry fermented product, characterized in that: The following steps are involved: Inoculating the composite bacterial agent according to claim 1 or 2 into wolfberry pulp, fermenting it, and obtaining a fermentation liquid; The fermentation liquid is centrifuged and the supernatant is collected to obtain the wolfberry fermentation product.
5. The preparation method according to claim 4, characterized in that: The inoculation amount of the composite bacterial agent is 3-5%.
6. The preparation method according to claim 4, characterized in that: The fermentation temperature is 29-34°C, the time is 24-36h, and the rotation speed is 150r / min.
7. The preparation method according to claim 4, characterized in that: The centrifugal speed is 5000 rpm and the time is 20 min.
8. The wolfberry fermented product prepared by the preparation method according to any one of claims 4 to 7.
9. Use of the wolfberry fermented product according to claim 8 in the preparation of a drug for inhibiting acetylcholinesterase activity.
10. Use of the wolfberry fermented product according to claim 8 in preparing a drug for auxiliary treatment of Alzheimer's disease.