A probiotic preparation and its preparation method and application

Abstract

The present invention discloses a probiotic preparation and a preparation method and application thereof, and relates to the field of biotechnology. The probiotic preparation is made of probiotic powder, Centella asiatica powder, malt extract and a probiotic protective agent; the probiotic powder is composed of Lactobacillus crispatus and Lactobacillus plantarum; the mass ratio of the Lactobacillus crispatus powder and the Lactobacillus plantarum powder is (3-4.5):1; the preservation number of the Lactobacillus crispatus is CGMCC No. 26680; the preservation number of the Lactobacillus plantarum is CGMCC No. 26679. The probiotic preparation of the present invention has high stability, can still maintain a high number of viable bacteria after being placed in low and high temperature environments for 3 months, and the probiotic preparation has good antibacterial performance, and has a good antibacterial effect on Staphylococcus aureus and Escherichia coli.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a probiotic preparation and a preparation method and application thereof. Background Art

[0002] Probiotics are living microorganisms that bring health benefits to the host when given in sufficient quantities. They have a variety of positive effects on the host, such as restoring the host's intestinal homeostasis and regulating the host's immune response. According to the morphological, physiological and biochemical characteristics of probiotics, probiotics can be divided into species such as Lactobacillus, Streptococcus, Bifidobacterium, and Saccharomyces. Lactobacillus includes Lactobacillus acidophilus ( Lactiplantibacillus acidophilus )、Lactobacillus casei( Lactobacillus cheese ), Lactobacillus bulgaricus ( Lactiplantibacillus bulgaricus )、Lactobacillus rhamnosus( Lactobacillus rhamnosus )、Lactobacillus crispatus( Lactobacillus crispatus ) and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) etc. Probiotics play a beneficial role in health in many aspects. Probiotics can play a lipid-lowering, blood sugar-lowering and anti-inflammatory role by decomposing and utilizing dietary fiber to produce short-chain fatty acids. They can express and secrete bile salt hydrolase, reduce the intestinal epithelial absorption of fat and cholesterol, promote the excretion of fat and cholesterol from feces, induce cell apoptosis, inhibit the proliferation of cancer cells to reduce inflammation, and probiotics can also delay the aging of the body.

[0003] Lactobacillus plantarum ( Lactiplantibacillus plantarum ) are mostly rod-shaped, anaerobic or facultative anaerobic, with an optimal growth temperature of 30-35°C and an optimal pH of about 6.5. Lactobacillus plantarum is widely present in fermented foods, has high safety, and has multiple probiotic functions, such as antibacterial, regulating intestinal flora, relieving colitis, regulating immune function, etc. At the same time, Lactobacillus plantarum has high tolerance and adaptability, and is widely used in the fermentation industry, food industry and other fields.

[0004] Lactobacillus crispatus ( Lactobacillus crispatus ) is a Gram-positive, non-spore-forming, sunny or microaerophilic bacterium belonging to the phylum Firmicutes. It can degrade a variety of carbohydrates and produce organic acids. Lactobacillus crispatus is a common flora in the human vagina. Lactobacillus crispatus can interfere with pathogenic bacteria through colonization, competitive exclusion, and the production of antimicrobial compounds.

[0005] During the application of probiotics, the activity of probiotics is easily affected by a variety of exogenous conditions, including temperature, humidity, processing methods, transportation routes, storage methods, etc., which may cause a significant decrease in the activity of probiotics and reduce the probiotic effect of its probiotic preparations.

[0006] At present, the storage stability of probiotics is relatively poor, and there is an urgent need to provide a probiotic preparation with stronger storage stability to improve its activity. Summary of the invention

[0007] The purpose of the present invention is to provide a probiotic preparation and a preparation method and application thereof.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a probiotic preparation, which is prepared from probiotic powder, Centella asiatica powder, malt extract and a probiotic protective agent;

[0010] The probiotic powder is composed of Lactobacillus crispatus and Lactobacillus plantarum;

[0011] The mass ratio of the Lactobacillus crispatus powder to the Lactobacillus plantarum powder is (3-4.5):1;

[0012] The preservation number of the Lactobacillus crispatus is CGMCC No. 26680; the preservation number of the Lactobacillus plantarum is CGMCC No. 26679.

[0013] Specifically, the probiotic preparation is prepared by weight from 8-18 parts of probiotic powder, 1.5-4 parts of Centella asiatica powder, 1-3 parts of malt extract and 0.5-2 parts of probiotic protective agent.

[0014] Furthermore, the probiotic preparation is prepared by weight from 8-16.5 parts of probiotic powder, 1.5-4 parts of Centella asiatica powder, 1-3 parts of malt extract and 0.5-2 parts of probiotic protective agent.

[0015] Furthermore, the probiotic preparation is prepared by weight from 10 parts of probiotic powder, 2.5 parts of Centella asiatica powder, 2 parts of malt extract and 1 part of probiotic protective agent.

[0016] Specifically, the preparation method of the malt extract comprises the following steps:

[0017] (1) Grinding malt and adding water to obtain malt mash;

[0018] (2) adding α-amylase to the mash for enzymatic hydrolysis and filtering;

[0019] (3) The filtrate obtained in step (2) is subjected to enzyme inactivation, concentration and drying to obtain a malt extract.

[0020] Furthermore, the malt in step (1) is wheat malt.

[0021] Furthermore, in step (1), the mass volume ratio of wheat malt to water is 1:(30-50).

[0022] Furthermore, in step (1), the mass volume ratio of wheat malt to water is 1:40.

[0023] Furthermore, in step (2), the mass volume ratio of α-amylase to malt slurry is (0.01-0.08):1.

[0024] Furthermore, in step (2), the mass volume ratio of α-amylase to malt slurry is (0.05-0.08):1.

[0025] Furthermore, in step (2), the mass volume ratio of α-amylase to malt slurry is 0.05:1.

[0026] Furthermore, the enzymatic hydrolysis conditions in step (2) are 60-90°C and the enzymatic hydrolysis time is 10-40 minutes;

[0027] Furthermore, the enzymatic hydrolysis conditions in step (2) are 80° C. and 20 min.

[0028] Furthermore, the enzyme inactivation condition in step (3) is 80-100° C. for 10-20 min.

[0029] Furthermore, the concentration conditions in step (3) are 60-80° C. and 0.08-0.09 MPa.

[0030] Furthermore, the concentration conditions in step (3) are 65° C. and 0.09 MPa.

[0031] Furthermore, in step (3), the concentration is stopped when the solid content reaches 60%-80%.

[0032] Furthermore, in step (3), the concentration is stopped when the solid content reaches 80%.

[0033] Specifically, the probiotic protective agent consists of dextran, casein and marigold pollen.

[0034] Furthermore, the mass ratio of glucan, casein and marigold powder in the probiotic protective agent is (10-20): (1-6): (1-5).

[0035] Furthermore, the mass ratio of glucan, casein and marigold powder in the probiotic protective agent is (10.5-15): (3.5-4): 1.

[0036] According to some embodiments of the present invention, the mass ratio of glucan, casein and marigold powder in the probiotic protective agent is 15:4:1 or 10.5:3.5:1.

[0037] Specifically, the total viable count of probiotics in the probiotic preparation is 1×105 cfu / g-1×10 12 cfu / g.

[0038] Specifically, the probiotic preparation is in the form of tablets, powders, capsules, granules, ointments, suspensions, gels, suppositories or aerosols.

[0039] In another aspect, the present invention provides a method for preparing the above-mentioned probiotic preparation, comprising the following steps:

[0040] (1) mixing Centella asiatica powder, malt extract, Lactobacillus crispatus powder and Lactobacillus plantarum powder;

[0041] (2) Dissolve the probiotic protective agent in sodium chloride solution and mix well;

[0042] (3) Add the solution obtained in step (2) to the powder obtained in step (1), mix well, and freeze-dry to obtain a probiotic preparation.

[0043] Specifically, in step (2), the mass volume ratio of the probiotic protective agent to the sodium chloride solution is 2:(3-5).

[0044] According to some embodiments of the present invention, in step (2), the mass volume ratio of the probiotic protective agent to the sodium chloride solution is 2:3, 2:4 or 2:5.

[0045] In yet another aspect, the present invention provides use of the above-mentioned probiotic preparation in preparing antibacterial products.

[0046] Specifically, the antibacterial activity is the inhibition of Staphylococcus aureus and Escherichia coli.

[0047] The beneficial effects of the present invention are:

[0048] The probiotic preparation of the present invention is prepared from Lactobacillus crispatus powder, Lactobacillus plantarum powder, Centella asiatica powder, malt extract and a probiotic protective agent. The prepared probiotic preparation can still maintain a high number of viable bacteria after being placed at 4° C. and 50° C. for 3 months, has high stability, and has good antibacterial performance, and has a good antibacterial effect on Staphylococcus aureus and Escherichia coli.

[0049] Collection Instructions

[0050] Deposit number: CGMCC No. 26680;

[0051] Classification name: Lactobacillus crispatus Lactobacillus crispatus ;

[0052] Storage time: February 27, 2023;

[0053] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0054] Abbreviation of depository unit: CGMCC;

[0055] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0056] Collection Instructions

[0057] Deposit number: CGMCC No. 26679;

[0058] Classification name: Lactobacillus plantarum Lactiplantibacillus plantarum ;

[0059] Storage time: February 27, 2023;

[0060] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0061] Abbreviation of depository unit: CGMCC;

[0062] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0063] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0064] Example 1 Preparation of probiotic preparation

[0065] The probiotic preparation is prepared from 10 parts of probiotic powder, 2.5 parts of Centella asiatica powder, 2 parts of malt extract and 1 part of probiotic protective agent in terms of weight.

[0066] The mass ratio of Lactobacillus crispatus powder to Lactobacillus plantarum powder is 4:1.

[0067] The preparation method of the malt extract comprises the following steps:

[0068] (1) Grind 20g of wheat malt and add 800ml of water to obtain malt slurry;

[0069] (2) Add α-amylase to the malt mash at a mass volume ratio of α-amylase to malt mash of 0.05:1, perform enzymolysis at 80°C for 20 min, and filter;

[0070] (3) The filtrate obtained in step (2) is inactivated at 100° C. for 15 min, concentrated at 65° C. and a pressure of 0.09 MPa until the solid content reaches 80%, and then dried to obtain a malt extract.

[0071] The mass ratio of glucan, casein and marigold pollen in the probiotic protective agent is 15:4:1.

[0072] The preparation method of the probiotic preparation comprises the following steps:

[0073] (1) mixing corresponding parts by weight of Centella asiatica powder, malt extract, Lactobacillus crispatus powder and Lactobacillus plantarum powder;

[0074] (2) Dissolve the probiotic protective agent in 0.9% sodium chloride solution and mix well;

[0075] (3) Add the solution obtained in step (2) to the powder obtained in step (1), mix well, and freeze-dry to obtain a probiotic preparation.

[0076] In step (2), the mass volume ratio of the probiotic protective agent to the sodium chloride solution is 2:3.

[0077] Example 2

[0078] The probiotic preparation is prepared from 8 parts of probiotic powder, 1.5 parts of Centella asiatica powder, 1 part of malt extract and 0.5 part of probiotic protective agent in terms of weight.

[0079] The mass ratio of Lactobacillus crispatus powder to Lactobacillus plantarum powder is 3:1.

[0080] The preparation method of the malt extract comprises the following steps:

[0081] (1) Grind 20g of wheat malt and add 1000ml of water to obtain malt slurry;

[0082] (2) Add α-amylase to the malt mash at a mass volume ratio of α-amylase to malt mash of 0.06:1, perform enzymolysis at 80°C for 20 min, and filter;

[0083] (3) The filtrate obtained in step (2) is inactivated at 100° C. for 15 min, concentrated at 65° C. and a pressure of 0.09 MPa until the solid content reaches 80%, and then dried to obtain a malt extract.

[0084] The mass ratio of glucan, casein and marigold powder in the probiotic protective agent is 10.5:3.5:1.

[0085] The preparation method of the probiotic preparation comprises the following steps:

[0086] (1) mixing corresponding parts by weight of Centella asiatica powder, malt extract, Lactobacillus crispatus powder and Lactobacillus plantarum powder;

[0087] (2) Dissolve the probiotic protective agent in 0.9% sodium chloride solution and mix well;

[0088] (3) Add the solution obtained in step (2) to the powder obtained in step (1), mix well, and freeze-dry to obtain a probiotic preparation.

[0089] In step (2), the mass volume ratio of the probiotic protective agent to the sodium chloride solution is 2:4.

[0090] Example 3

[0091] The probiotic preparation is prepared from 16.5 parts of probiotic powder, 4 parts of Centella asiatica powder, 3 parts of malt extract and 2 parts of probiotic protective agent in terms of weight.

[0092] The mass ratio of Lactobacillus crispatus powder to Lactobacillus plantarum powder is 4.5:1.

[0093] The preparation method of the malt extract comprises the following steps:

[0094] (1) Grind 20g of wheat malt and add 600ml of water to obtain malt slurry;

[0095] (2) Add α-amylase to the malt mash, with the mass volume ratio of α-amylase to malt mash being 0.08:1, perform enzymolysis at 80°C for 20 min, and filter;

[0096] (3) The filtrate obtained in step (2) is inactivated at 100° C. for 15 min, concentrated at 65° C. and a pressure of 0.09 MPa until the solid content reaches 80%, and then dried to obtain a malt extract.

[0097] The mass ratio of glucan, casein and marigold pollen in the probiotic protective agent is 15:4:1.

[0098] The preparation method of the probiotic preparation comprises the following steps:

[0099] (1) mixing corresponding parts by weight of Centella asiatica powder, malt extract, Lactobacillus crispatus powder and Lactobacillus plantarum powder;

[0100] (2) Dissolve the probiotic protective agent in 0.9% sodium chloride solution and mix well;

[0101] (3) Add the solution obtained in step (2) to the powder obtained in step (1), mix well, and freeze-dry to obtain a probiotic preparation.

[0102] In step (2), the mass volume ratio of the probiotic protective agent to the sodium chloride solution is 2:5.

[0103] Comparative Example 1

[0104] The only difference from Example 1 is that the mass ratio of Lactobacillus crispatus powder to Lactobacillus plantarum powder is different. In Comparative Example 1, the mass ratio of Lactobacillus crispatus powder to Lactobacillus plantarum powder is 6:1, and the rest are the same.

[0105] Comparative Example 2

[0106] The only difference from Example 1 is that an equal amount of Lactobacillus crispatus powder (CGMCC No. 26680) is used to replace Lactobacillus plantarum powder, and the rest is the same.

[0107] The probiotic preparation is prepared from 10 parts of Lactobacillus crispatus powder, 2.5 parts of Centella asiatica powder, 2 parts of malt extract and 1 part of probiotic protective agent in parts by weight.

[0108] The mass ratio of glucan, casein and marigold pollen in the probiotic protective agent is 15:4:1.

[0109] The preparation method of the probiotic preparation comprises the following steps:

[0110] (1) mixing corresponding parts by weight of Centella asiatica powder, malt extract, and Lactobacillus crispatus powder;

[0111] (2) Dissolve the probiotic protective agent in 0.9% sodium chloride solution, with the mass volume ratio of the probiotic protective agent to the sodium chloride solution being 2:3, and mix well;

[0112] (3) Add the solution obtained in step (2) to the powder obtained in step (1), mix well, and freeze-dry to obtain a probiotic preparation.

[0113] Comparative Example 3

[0114] Compared with Example 1, the only difference is that the content of each component in the probiotic preparation is different. The probiotic preparation in Comparative Example 3 is prepared by 20 parts of probiotic powder, 5 parts of Centella asiatica powder, 5 parts of malt extract and 4 parts of probiotic protective agent in terms of weight, and the rest are the same.

[0115] Comparative Example 4

[0116] Compared with Example 1, the only difference is that the components of the probiotic preparation are different, the Centella asiatica powder in the probiotic preparation is replaced by Calendula officinalis powder, and the rest are the same.

[0117] The probiotic preparation is prepared from 10 parts of probiotic powder, 2.5 parts of marigold powder, 2 parts of malt extract and 1 part of probiotic protective agent according to weight.

[0118] The mass ratio of Lactobacillus crispatus powder to Lactobacillus plantarum powder is 4:1.

[0119] The preparation method of malt extract is the same as that in Example 1.

[0120] The mass ratio of glucan, casein and marigold pollen in the probiotic protective agent is 15:4:1.

[0121] The preparation method of the probiotic preparation comprises the following steps:

[0122] (1) mixing corresponding parts by weight of marigold powder, malt extract, Lactobacillus crispatus powder and Lactobacillus plantarum powder;

[0123] (2) Dissolve the probiotic protective agent in 0.9% sodium chloride solution, with the mass volume ratio of the probiotic protective agent to the sodium chloride solution being 2:3, and mix well;

[0124] (3) Add the solution obtained in step (2) to the powder obtained in step (1), mix well, and freeze-dry to obtain a probiotic preparation.

[0125] Comparative Example 5

[0126] The difference between Comparative Example 5 and Example 1 is that the components of the probiotic protective agent are different, and the marigold pollen in the probiotic protective agent is replaced with skimmed milk powder, and the rest are the same.

[0127] Comparative Example 6

[0128] The only difference between Comparative Example 6 and Example 1 is that the mass ratios of the components in the probiotic protective agent are different. In Comparative Example 6, the mass ratio of glucan, casein and marigold powder is 25:8:1.

[0129] Experimental Example 1 Stability of probiotic preparations

[0130] The content of probiotics in the examples and comparative examples was detected according to the method of GB4789.35. The probiotic preparations prepared in the examples and comparative examples were placed under 4°C and high temperature 50°C for experimental investigation. The number of live bacteria in the probiotic preparations was detected by sampling on the 0th day and the third month. The test results are shown in Tables 1 and 2 below:

[0131] Table 1 Number of viable cells of probiotic preparations at 4°C

[0132]

[0133] Table 2 Number of viable bacteria of probiotic preparations at 50°C

[0134]

[0135] It can be seen from Tables 1 and 2 above that the probiotic preparations prepared in Examples 1-3 can still maintain a high number of viable bacteria after being placed at 4°C and 50°C for 3 months, and the viable bacteria rate is significantly higher than that of Comparative Examples 1-6, indicating that the probiotic preparations of the present invention have high stability under refrigeration and high temperature conditions.

[0136] Experimental Example 2 Antibacterial properties of probiotic preparations

[0137] 1×10 9 CFU / mL 30 μL of Staphylococcus aureus and Escherichia coli O157 were coated in LB solid culture medium and cultured at 37°C for 24 hours. 200 μL of probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-6 were added (the probiotic preparations added here were probiotic preparations prepared without freeze drying, and the total bacterial counts of the examples and comparative examples were the same), each was repeated 3 times, and the total culture was 4 hours. Then, the diameter of the inhibition zone was measured at a constant temperature of 37°C for 24 hours.

[0138] Table 3 Antibacterial properties of probiotic preparations

[0139]

[0140] It can be seen from the above table that compared with the comparative examples 1-6, the probiotic preparation prepared in Example 1 has significantly better antibacterial effect on Staphylococcus aureus and Escherichia coli.

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A probiotic preparation, characterized in that The probiotic preparation is made of probiotic powder, Centella asiatica powder, malt extract and probiotic protective agent; The probiotic powder is composed of Lactobacillus crispatus and Lactobacillus plantarum; The mass ratio of the Lactobacillus crispatus powder to the Lactobacillus plantarum powder is (3-4.5):1; The preservation number of the Lactobacillus crispatus is CGMCC No. 26680; the preservation number of the Lactobacillus plantarum is CGMCC No. 26679; The probiotic preparation is prepared by weight from 8-18 parts of probiotic powder, 1.5-4 parts of Centella asiatica powder, 1-3 parts of malt extract and 0.5-2 parts of probiotic protective agent; The preparation method of the malt extract comprises the following steps: (1) Grinding malt and adding water to obtain malt mash; (2) adding α-amylase to the mash for enzymatic hydrolysis and filtering; (3) inactivating the enzyme in the filtrate obtained in step (2), concentrating and drying the filtrate to obtain a malt extract; The probiotic protective agent is composed of glucan, casein and marigold powder; The mass ratio of glucan, casein and marigold powder in the probiotic protective agent is (10.5-15): (3.5-4):

1.

2. The probiotic preparation according to claim 1, characterized in that The probiotic preparation is prepared by weight from 10 parts of probiotic powder, 2.5 parts of Centella asiatica powder, 2 parts of malt extract and 1 part of probiotic protective agent.

3. The probiotic preparation according to claim 1, characterized in that In step (2), the mass volume ratio of α-amylase to malt slurry is (0.01-0.08):

1.

4. The method for preparing the probiotic preparation according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) mixing Centella asiatica powder, malt extract, Lactobacillus crispatus powder and Lactobacillus plantarum powder; (2) Dissolve the probiotic protective agent in sodium chloride solution and mix well; (3) Add the solution obtained in step (2) to the powder obtained in step (1), mix well, and freeze-dry to obtain a probiotic preparation.

5. Use of the probiotic preparation according to any one of claims 1 to 3 in the preparation of antibacterial products.

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