CN114586845A - Method for preparing antimicrobial composite strain from kefir - Google Patents

Method for preparing antimicrobial composite strain from kefir Download PDF

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CN114586845A
CN114586845A CN202210159857.2A CN202210159857A CN114586845A CN 114586845 A CN114586845 A CN 114586845A CN 202210159857 A CN202210159857 A CN 202210159857A CN 114586845 A CN114586845 A CN 114586845A
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kefir
milk
whey
composition
fermentation
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刘树青
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
    • A23C11/106—Addition of, or treatment with, microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/60—Drinks from legumes, e.g. lupine drinks
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3571—Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor

Abstract

The invention relates to a method for preparing an antimicrobial composite strain by kefir, which comprises the following steps: step 1, providing and optionally filtering kefir or whey thereof; step 2, introducing fermentable sugars into kefir or whey thereof, wherein the fermentable sugars are used in an amount of at least a saturated amount by weight of kefir or whey thereof and allowing the curd to pool above, thereby forming a pre-fermented composition; step 3, fermenting the pre-fermented composition until a microbial film capable of growing is formed above and a whey liquid is formed below, and the whey liquid shows antimicrobial properties and has a pH of 3.0, during the fermentation kefir or kefir whey or fermentable sugar may be further added. The antimicrobial compositions prepared using natural materials according to the methods of the present invention can be used in a wide variety of industrial applications including personal care products, food applications, pharmaceuticals and health care.

Description

Method for preparing antimicrobial composite strain from kefir
Technical Field
The invention relates to a method for preparing an antimicrobial compound strain by kefir, in particular to a method for preparing an antibacterial, antifungal or antiviral compound strain by kefir.
Background
The term kefir, derived from turkish, indicates a nice taste, kefir is a fermented drink from the caucasian area that resembles a thin yogurt and is slightly frothy. Kefir is prepared by inoculating fermented animal milk, such as cow's or sheep's milk, with Kefir grains (or starter). In addition, kefir made by using soybean milk has been reported.
kefir grains are a group of symbiotic probiotic bacteria that promote human health, in which various microorganisms become naturally symbiotic organisms, specifically including various microorganisms such as lactobacilli and acetobacter xylinum, and lactose-fermenting yeasts such as kluyveromyces marxianus and non-lactose-fermenting yeasts such as monospora, saccharomyces cerevisiae, saccharomyces oligosporus, and in addition, tibetan mushroom is also known as kefir grains in tibetan and belongs to a strain of kefir grains.
Currently, there have been attempts to add food ingredients to kefir to obtain foods with better health maintenance and promotion effects, for example, WO2002/076240 discloses a food obtained by adding nattokinase (nattokinase) to kefir, and WO2010/021117 discloses an oral preparation for achieving beautiful skin, the oral preparation containing, for example, whey kefir obtained by culturing whey as an active ingredient, and may be a medicine, a food or a beverage.
In addition, WO2018/079728 discloses the administration of a preparation formed by comprising kefir as active ingredient for improving survival in cancer treatment, in particular survival resulting from chemotherapy and/or radiotherapy. Such as capsules, tablets, granules, powders, syrups, lozenges, sprays, emulsions, injectable preparations, suppositories, ointments, tapes and the like.
An antimicrobial substance is any substance of natural, semisynthetic or synthetic origin and which is capable of killing or inhibiting the growth of harmful microorganisms. The main types of antimicrobial substances include disinfectants, such as bleaches, antiseptics and antibiotics.
Generally, disinfectants, antiseptics and antibiotics are used somewhat differently. Disinfectants are non-selective and are typically applied to the surface of non-living objects to destroy microorganisms living on the non-living surface, antiseptics are used to destroy microorganisms on the surface of living tissue, such as during surgery, and antibiotics are used to destroy bacteria in the body. In addition, antifungal agents and antiviral agents are used to target fungi and viruses in vivo and in vitro, respectively.
One problem with antibiotics is that they indiscriminately sweep away all susceptible bacteria in the organism and/or microbiome, including both undesirable bacteria that they target and beneficial bacteria that strive to maintain the health of the human and animal body. In particular, beneficial bacteria can support the health of the human and animal body by helping to absorb nutrients, digest food, balance blood glucose, regulate the immune system, and even balance emotions. Another problem with antibiotics is resistance, which occurs when bacteria survive and continue to cause infection in the case of antibiotic use.
It has been desirable to obtain alternatives that have little or no effect on beneficial microorganisms in vivo while exhibiting inhibitory or biocidal activity against a wide range of harmful microorganisms. To this end, the art has attempted to use antimicrobial compositions prepared from natural materials. They are used in several industrial fields, such as personal care products, food applications, medicine and health care. For example, in personal care articles, antimicrobial agents are used to help prevent or eliminate the growth of surface pathogens as well as to protect the personal care article from damage or to substantially improve the shelf life of the article. US2016/0286820a1 discloses antimicrobial and antifungal compositions for personal care articles, which are prepared by: macerating the white pulp of the Cocos Nucifera coconut to produce a feedstock material; mixing the feedstock material with bacteria and fermenting the feedstock material using the bacteria to produce a fermented product; the fermented product is filtered to clarify the fermented product and to remove the remaining raw material, and the fermented product filtered and removed of the remaining raw material exhibits antibacterial and antifungal effects in use in personal care products. In addition, AU 2019100035 discloses a method for preparing antimicrobial complex strains from milk, raw eggs and plant parts.
However, there is a need for a simpler process for preparing antimicrobial complex strains that can be used for further strain propagation to provide compositions that have activity in inhibiting or destroying a wide range of harmful microorganisms, such as bacteria, fungi and/or viruses, but do not cause or cause little damage to beneficial microorganisms on the host.
In the prior published patent application, for example, chinese invention patent application No. CN201910091901.9 discloses the use of a sulfabisbenzimidazole compound and its pharmaceutically acceptable salts in the preparation of antimicrobial drugs. The sulfambisbenzimidazole compound shown as the general formula I or the general formula II and the medicinal salt thereof have stronger in-vitro antimicrobial activity, particularly show high inhibitory activity on gram-positive bacteria such as staphylococcus aureus, methicillin-resistant staphylococcus aureus, bacillus subtilis, micrococcus luteus and the like, gram-negative bacteria such as escherichia coli, proteus, pseudomonas aeruginosa, salmonella typhi and the like, and fungi such as candida utilis, aspergillus flavus, saccharomyces cerevisiae, candida albicans and the like, can be used for preparing antibacterial and/or antifungal medicaments, and are beneficial to solving the clinical treatment problems of increasingly severe drug resistance, stubborn pathogenic microorganisms, newly-appeared harmful microorganisms and the like.
As another example, chinese patent application No. cn201580042228.x1 discloses a method of preparing an antimicrobial particulate composition, the method comprising the steps of: i. mixing an aqueous dispersion of a fixing agent and an aqueous solution of a reducing agent, wherein the fixing agent is present in an amount of 1-5 wt% (based on the weight of the dispersion), and wherein the fixing agent is an inorganic porous material selected from zinc oxide, magnesium hydroxide or calcium carbonate, and wherein the fixing agent has a particle size in the range of 1-10 microns, and wherein the reducing agent is present in the aqueous solution in an amount of 10-30 wt% of the weight of the fixing agent in the aqueous dispersion; (ii) increasing the temperature of the mixture resulting from step (i) to a temperature in the range of 70-90 ℃; (iii) adding a water-soluble metal salt to the mixture resulting from step (ii) under mixing, the amount of said water-soluble metal salt corresponding to 0.05-3 wt% of metal by weight of said fixative.
None of the above-disclosed patent applications provides a composition, i.e., an antimicrobial complex strain, having activity to inhibit or destroy a wide range of harmful microorganisms without harming the beneficial microorganisms on the host.
Disclosure of Invention
The invention provides a method for preparing an antimicrobial composite strain by kefir, which comprises the following steps:
step 1 providing the kefir or the kefir whey;
step 2 introducing fermentable sugars into the kefir or the kefir whey, wherein the fermentable sugars are used in an amount at least up to saturation by weight of the kefir or the kefir whey and such that curd pools above, thereby forming a pre-fermented composition;
step 3 the pre-fermented composition is fermented until a microbial film capable of growth is formed above and a whey liquid is formed below, and the whey liquid shows antimicrobial properties and has a pH of 3.0.
Further, in step 1, the kefir uses a screen with a pore size of 60-70 meshes to filter out particles with a particle size of more than 0.2 mm.
Further, in step 1, the Kefir is a slightly foamed yoghurt preparation obtained by fermenting milk with Kefir grains exhibiting small clusters of irregular shape, ivory or milky white and gummy.
Further, in step 1, the milk is selected from the group consisting of plant-based milk, animal-based milk, and combinations thereof.
Further, in step 1, the milk is selected from the group consisting of plant-based dairy products including soy milk, almond milk, or coconut milk, and combinations thereof.
Further, in step 1, the animal-based milk product includes cow's milk, goat's milk or sheep's milk and combinations thereof.
Further, in step 1, the kefir is obtained by a method comprising:
a) providing a dairy composition comprising said kefir grains, said milk and said fermentable sugars, wherein the weight of said kefir grains is from 0.5 wt% to 20 wt% based on the weight of said milk, and said fermentable sugars are used in an amount to at least saturation; b) fermenting the dairy composition into a yogurt texture.
Further, in the step 1, the kefir grains are Tibetan kefir.
Further, in step 1, sucrose is used as the fermentable sugar, and the amount thereof is 20% by weight relative to the weight of the milk at 20 ℃.
Further, in step 2, the fermentable sugar is a sugar fermented with the milk, including glucose, fructose, sucrose, maltose, galactose or raffinose, and a combination thereof.
Further, in step 2, the sucrose is cane sugar or beet sugar.
Further, in step 2), the fermentable sugars are used in an amount at least up to saturation based on the weight of the kefir or the kefir whey.
Further, in step 2, sucrose is used as the fermentable sugar in an amount of at least 20% by weight relative to the weight of the kefir or the kefir whey at 20 ℃.
Further, in step 2, after introducing the fermentable sugar into the kefir whey and standing for 20 minutes, the whey and the fermentable sugar bring the curd together above to form a pre-fermented composition; or by stirring the whey and the fermentable sugars followed by standing for 20 minutes such that curd pools above to form the pre-fermented composition.
Further, in the step 3, the fermentation of the pre-fermented composition is to place the material to be fermented in a closed container, and carry out the fermentation under the normal pressure and at the temperature of 16-35 ℃.
Further, in step 3, the temperature of the fermentation is preferably 28 ℃, and the time required for the fermentation is 2 to 3 days.
Further, in step 3, when the fermentation temperature is lower than 16 ℃, the time required for fermentation is 20 days.
Further, in step 3, the pre-fermented composition is fermented until a microbial film capable of growth is formed above and a whey liquid is formed below, which exhibits antimicrobial properties and has a pH of 3.0.
Further, in step 3, the kefir or the kefir whey and/or the fermentable sugar are/is added during the fermentation process of the pre-fermented composition, so that the microbial membrane can continue to grow and thicken, and gas can be observed to be rapidly generated during the fermentation process of the pre-fermented composition.
The method of the invention has the following excellent technical effects:
1. the kefir is fermented milk by kefir grains, the sources of the kefir grain fermented milk are wide, the kefir grain fermented milk comprises plant-based dairy products, animal-based dairy products and compositions thereof, the fermentable sugar comprises glucose, fructose, sucrose, maltose, galactose, raffinose and compositions thereof, the sources of the raw materials are wide, and the preparation of the high-efficiency antimicrobial compound strain is convenient.
2. The antimicrobial complex strains prepared by the method of the present invention can be used for further propagation, and have activity of inhibiting or destroying a wide range of harmful microorganisms such as bacteria, fungi and/or viruses, while not causing damage to beneficial microorganisms on the host.
3. The antimicrobial compositions prepared using natural materials according to the methods of the present invention can be used in a wide variety of industrial applications including personal care products, food applications, pharmaceuticals and health care.
4. The composition obtained according to the method of the invention can be used as a further basic strain for long-term propagation.
5. The antimicrobial composition obtained by the method is obtained through strict scientific experiments, is widely applied to the fields of medical treatment, health care, food preservation, fresh keeping and the like, and shows that all the components in the composition have synergistic effect through strict experimental tests, so that the antimicrobial composition is worthy of wide popularization and application and is beneficial to human beings.
Drawings
FIG. 1 shows the pre-fermented composition obtained from step 2 in the process according to the invention, wherein the curd converges on top.
FIG. 2 illustrates a microbial film or cake capable of growth formed by the method of the invention.
Figure 3 shows the composition obtained by fermenting grapes with antimicrobial complex species.
FIG. 4 shows an antimicrobial complex strain obtained by the method of the invention, wherein the microbial film or cake formation is located above.
Fig. 5 shows OD values of stock solutions and stock solutions diluted at each concentration after intervention in MAGI-CCR5 cells, where 1-18 correspond to 18 concentration samples of the stock solution up to 1/131072 fold concentration, respectively, and 19 is a blank control.
Fig. 6 shows the percentage inhibition of proliferation of MAGI-CCR5 cells by the stock solutions and the stock solutions diluted at each concentration, where 1-18 correspond to 18 concentration samples of the stock solutions up to 1/131072-fold concentration, respectively.
Detailed Description
The term "antimicrobial" as used herein refers to a microorganism that is capable of killing or inhibiting microorganisms harmful to the human and/or animal body, including, but not limited to, bacteria, fungi such as molds and viruses, in some embodiments of the present invention refers to infection-causing microorganisms, which refer to molds, particularly to green and black molds, which in the case of diabetes and its complications may include staphylococcus aureus, enterococcus, pseudomonas aeruginosa, escherichia coli, klebsiella and proteus vulgaris, and in the case of eczema may include herpesvirus and staphylococcus aureus; in addition, the term "antimicrobial" is intended to be associated with organic matter in the present invention and refers to an activity of killing or inhibiting harmful microorganisms such as mold like green mold and black mold, and it is important to detect whether the whey liquid becomes antimicrobial and can be confirmed simply by the description in the following specific examples. The organic matter includes, but is not limited to, various foods such as water, yogurt, fruit, and the like.
The term "complex bacterial species" as used herein refers to a preparation comprising a plurality of microbial species and wherein said bacterial species act synergistically. The "complex strain" in the present invention can be used for further strain propagation.
The term "microbial film capable of growth" is used herein to indicate that the microbial film can continue to grow thicker and form a soft mass, which is generally white, smooth and glossy, as shown in fig. 2.
The method comprises the following steps:
step 1, providing said kefir and/or said kefir whey;
step 2, introducing fermentable sugars into the kefir or the kefir whey, wherein the fermentable sugars are used in an amount at least up to saturation by weight of the kefir or the kefir whey and such that curd pools above, thereby forming a pre-fermented composition;
step 3, fermenting the pre-fermented composition until a microbial film capable of growth is formed above and a whey liquid is formed below and the whey liquid shows antimicrobial properties and has a pH of 3.0, wherein during the fermentation, if further needed, the kefir or kefir whey and/or the fermentable sugar are added, enabling the microbial film to continue to grow and thicken, and a sharp gas generation can be observed during the fermentation of the pre-fermented composition.
Further, in the kefir of the step 1, a sieve with a pore size of 60-70 meshes is used for filtering out particles with the particle size of more than 0.2 mm.
Further, in step 1, Kefir is a slightly foamed yogurt product obtained by fermenting milk with Kefir bacterial granules, wherein the Kefir bacterial granules are small clusters with irregular shapes, are ivory or milky white and are in a colloid shape.
Further, in step 1, the milk is selected from the group consisting of plant-based milk, animal-based milk, and combinations thereof.
Further, in step 1, the milk is selected from the group consisting of plant-based dairy products including soy milk, almond milk, coconut milk, and combinations thereof.
Further, in step 1, the animal-based milk product includes cow's milk, goat's milk or sheep's milk and combinations thereof.
Further, in step 1, the kefir is obtained by a method comprising the steps of:
a) providing a dairy composition comprising said kefir grains, said milk and said fermentable sugars, wherein the weight of said kefir grains is from 0.5 wt% to 20 wt% based on the weight of said milk, and said fermentable sugars are used in an amount to at least saturation;
b) fermenting the dairy composition until it becomes a yogurt texture.
Further, in step 1, said fermentable sugar of said step a) is sucrose, and the amount thereof is 20% relative to the weight of said milk at 20 ℃.
Further, in step 1, the kefir grains are Tibetan kefir.
Further, in step 2, the fermentable sugar is a sugar fermented with the milk, and the sugar comprises glucose, fructose, sucrose, maltose, galactose or raffinose and a combination thereof.
Further, in step 2, the sucrose is cane sugar or beet sugar.
Further, in step 2, the fermentable sugars are used in an amount at least up to saturation based on the weight of the kefir or the kefir whey.
Further, in step 2, sucrose is used as the fermentable sugar in an amount of at least 20% by weight relative to the weight of the kefir or the kefir whey at 20 ℃.
Further, in step 2, after introducing the fermentable sugar into the kefir whey and standing for 20 minutes, the whey and the fermentable sugar bring the curd together above to form a pre-fermented composition; or by stirring the whey and the fermentable sugars followed by standing for 20 minutes such that curd pools above to form the pre-fermented composition.
Further, in the step 3, the fermentation is to place the material to be fermented in a closed container, and the fermentation is carried out under normal pressure and at the temperature of 16-35 ℃.
Further, in step 3, the temperature of the fermentation is preferably 28 ℃, and the duration of the fermentation is 2-3 days.
Further, in step 3, when the fermentation temperature is lower than 16 ℃, the fermentation time is 20 days.
Further, in step 3, the pre-fermented composition is fermented until a microbial film capable of growth is formed above and a whey liquid is formed below, which exhibits antimicrobial properties and has a pH of 3.0.
Further, in step 3, during the fermentation of the pre-fermented composition, a sharp gas generation can be observed during the fermentation process by further adding the kefir or the kefir whey and/or the fermentable sugar to allow the microbial membrane to continue to grow and thicken.
It should be noted that: the method of detecting antimicrobial properties in the present invention comprises: covering a surface of a substrate with a predetermined amount of whey liquid of said complex bacterial species or a liquid portion of a derivative product thereof, wherein said derivative product is a product obtained by further propagating said antimicrobial complex bacterial species, and observing whether or not harmful microorganisms such as mold have grown after said whey liquid or said liquid portion of said derivative product and said substrate are placed under room temperature and open conditions for a period of time, and wherein an antimicrobial property test method for testing whether or not said antimicrobial complex bacterial species or said derivative product thereof have inhibitory or bactericidal activity against harmful microorganisms such as mold is used, and wherein a plurality of substrates such as yogurt can be tested for said antimicrobial property, specifically, a plurality of identical containers each containing a predetermined amount of yogurt are prepared, and then the surface of said yogurt is covered with a layer of a substance to be tested, wherein the covering thickness of the substance to be detected is 0.5-1 cm, and the substance to be detected comprises the whey liquid from the step 3 in the method. Alternatively, a control experiment may be performed using a control test substance comprising, for example, water, and then subjecting all samples to ambient and open conditions, wherein mold and other harmful microorganisms may grow as the liquid surface evaporates, and over a period of time, when no mold and other harmful microorganisms are observed in the container containing the test substance, and mold and other harmful microorganisms are observed in the container containing the control test substance, the test substance is preliminarily considered to have antimicrobial properties. In addition, the antimicrobial property of the substance to be tested can be judged according to the occurrence rate of mold or other harmful microorganisms, the size and number of microbial plaques, and the like.
The antimicrobial compound strain prepared by the method of the invention comprises the following components: 1) the microbial film capable of growing can be formed when the further strain is propagated and/or the microbial film in the composite strain can grow; 2) can inhibit or kill harmful microorganisms; 3) capable of fermenting organic matter; 4) can be fermented to a pH of 3.0, and especially both the whey liquid and the microbial film (or microbial cake formed by continued growth of the microbial film) can inhibit or kill harmful microorganisms.
The antimicrobial complex strain prepared by the method of the present invention ferments grape to obtain a composition, wherein the fermentation is performed on the basis of a composition comprising the antimicrobial complex strain or whey liquid of the complex strain, fermentable sugar, grape and optionally water until a growing microbial film is formed above and/or the microbial film in the complex strain continues to grow and a liquid part is formed below, and the liquid part shows antimicrobial properties and has a pH of 3.0. In some embodiments, when grapes are fermented with the antimicrobial complex strain, new microbial films will form during fermentation if agitated, and the microbial films in the complex strain will continue to grow if not agitated.
The antimicrobial complex strain produced by the method of the present invention can be used to ferment grapes, such as, but not limited to, grapes from the cultivars such as, but not limited to, cilazar, cabernet sauvignon, reynia, chardonnay, melanopino, to provide a composition having inhibitory or biocidal activity against a wide range of harmful microorganisms, such as bacteria, fungi, and/or viruses, but that does not cause or causes little damage to beneficial microorganisms on the host. The fermentation process can be carried out under closed conditions, and the fermentation temperature is not lower than 16 ℃ and not higher than 35 ℃ under normal pressure. Preferably, the fermentation is carried out at 18 ℃ to 28 ℃, for example 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃ or greater than 28 ℃. More preferably, the fermentation is carried out at 22 ℃. When the fermentation temperature is lower than 16 ℃, a longer time is required to achieve the desired fermentation effect. The fermentation time may vary depending on environmental factors, for example 20 days. In certain embodiments, the fermentation is performed on the basis of a composition consisting of whey liquid, fermentable sugar, water and grapes of the antimicrobial complex strain. In some embodiments, the fermentation may employ a weight ratio of 1: 1 and water containing fermentable sugars, and an appropriate amount of grapes. The fermentable sugar is used in an amount at least up to saturation with respect to the liquid component. The amount of the grapes can be selected according to the container used. When the pulp of the grapes in the container is consumed during the fermentation, new grapes should be added in time to replace the grapes present in the container. The composition thus obtained can be used as a basic strain for long-term propagation.
The composition prepared by the method is used for health care and food preservation and fresh keeping. The ingredients in the composition have synergistic effect through experimental test, can obviously improve metabolism in vivo and maintain beneficial microbial community in intestinal tract, and therefore can be called as "vitality element", also called as "LEV", and represents life, energy and vitality. In certain embodiments, the compositions can be used to help alleviate or ameliorate conditions such as ovarian cancer, type II diabetes and its complications, as well as eczema and skin wounds.
Examples
Example 1, preparation of antimicrobial complex strains:
as shown in fig. 1-3, a 3-liter fermentor was charged with 2 liters of whole fresh milk, 400g of sucrose and 100g of kefir grains to provide a dairy composition, the dairy composition was fermented at room temperature under closed conditions for 12 hours until it substantially became a yogurt texture to obtain kefir, the kefir was filtered with a 70-mesh screen, after which 500g of sucrose was added thereto, stirred well, left to stand with the curd therein on top to form a pre-fermented composition, the pre-fermented composition was closed fermented at room temperature for about 12 days while forming a biofilm capable of growth on top and a whey liquid on the bottom, and the whey liquid showed antimicrobial properties and had a pH of about 3.0. Fig. 4 shows the antimicrobial complex strain obtained from this example, with the microbial film or cake on top.
Example 2, composition obtained by further fermentation/propagation of the complex species:
the composition was prepared by fermenting grapes in whey liquid of the antimicrobial complex strain obtained from example 1. The fermentation was carried out in the following manner: 500g of whey liquid obtained from example 1, 500g of water, 200g of sucrose and 100g of grapes are charged into a fermenter to form a mixture, the mixture is allowed to ferment at room temperature under closed conditions until a microbial film capable of growth is formed above the composition and a liquid fraction is formed below, and the liquid fraction exhibits antimicrobial properties and has a pH of 3.0.
Example 3 effect of the composition obtained from example 2 on the proliferation activity of MAGI-CCR5 cells:
the effect of the composition obtained in example 2 on the proliferation activity of MAGI-CCR5 cells was examined by the MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) method, the composition was centrifuged, the precipitate was removed, the supernatant was collected and sterilized by filtration through a 0.22 μm filter to obtain a stock solution.
The stock was diluted to 1/131072 with a 2-fold gradient with complete medium and the following samples were obtained: the stock solution comprises 1/2 stock solution, 1/4 stock solution, 1/8 stock solution, 1/16 stock solution, 1/32 stock solution, 1/64 stock solution, 1/128 stock solution, 1/256 stock solution, 1/512 stock solution, 1/1024 stock solution, 1/2048 stock solution, 1/4096 stock solution, 1/8192 stock solution, 1/16384 stock solution, 1/32768 stock solution, 1/65536 stock solution and 1/131072 stock solution. Inhibition (blank control OD value-sample OD value/blank control OD value) × 100%, where OD value is optical density value. After intervention for 24 hours, the results show that the stock solutions, 1/2 stock solutions, 1/4 stock solutions and 1/8 stock solutions have extremely strong inhibition effects on the proliferation activity of MAGI-CCR5 cells, and the inhibition rates are all over 90%; 1/16 stock solution and 1/32 stock solution have obvious inhibition effect on the proliferation activity of MAGI-CCR5 cells; 1/64 stock solution and stock solution with subsequent concentration have no obvious inhibition effect on the proliferation activity of MAGI-CCR5 cells. The results are shown in Table 1 (percent (%) cytotoxic effect of the stock solutions and diluted stock solutions at each concentration on MAGI-CCR 5) and in FIGS. 5-6.
TABLE 1
Sample (I) OD value Proliferation inhibition ratio (%)
Stock solution 0.18±0.01 91.54
1/2 stock solution 0.12±0.01 94.43
1/4 stock solution 0.14±0.00 93.46
1/8 stock solution 0.19±0.03 91.22
1/16 stock solution 1.18±0.08 45.53
1/32 stock solution 1.42±0.05 34.60
1/64 stock solution 2.03±0.50 6.19
1/128 stock solution 2.10±0.43 3.03
1/256 stock solution 2.09±0.20 3.36
1/512 stock solution 2.10±0.19 2.88
1/1024 stock solution 2.39±0.16 -10.46
1/2048 stock solution 2.01±0.03 7.26
1/4096 stock solution 2.37±0.28 -9.61
1/8192 stock solution 2.21±0.12 -2.07
1/16384 stock solution 2.14±0.20 1.04
1/32768 stock solution 2.24±0.32 -3.68
1/65536 stock solution 2.13±0.06 1.63
1/131072 stock solution 1.98±0.02 8.33
Blank control sample 2.16±0.13
Although embodiments of the present invention have been described in the foregoing detailed description, it will be understood that the invention is not limited to the examples disclosed herein, which are capable of numerous rearrangements, modifications, and substitutions without departing from the scope of the invention.

Claims (9)

1. A method of preparing an antimicrobial complex strain from kefir, the method comprising the steps of:
step 1, providing kefir or kefir whey;
step 2. introducing fermentable sugars into the kefir or the kefir whey, wherein the fermentable sugars are used in an amount of at least a saturation amount by weight of the kefir or the kefir whey and the curd is allowed to pool above, thereby forming a pre-fermented composition;
step 3. fermenting the pre-fermented composition until a microbial film capable of growing is formed above and a whey liquid is formed below and showing antimicrobial properties and having a pH of 3.0, wherein during the fermentation the kefir or the kefir whey and/or the fermentable sugar is further added to increase the thickness of the microbial film.
2. The method of claim 1, wherein said kefir in step 1 is obtained by:
step 1.1 providing a dairy composition comprising kefir grains, milk and fermentable sugars, wherein the kefir grains are present in an amount of 0.5 to 20 wt% based on the weight of the milk, the fermentable sugars being present in an amount of at least a saturating amount;
step 1.2 fermenting the dairy composition such that the dairy composition substantially becomes a yoghurt texture.
3. The method according to any one of claims 1 to 2, wherein the antimicrobial complex strain has: microbial films capable of growing are formed when the dairy composition is propagated through further compound strains, the microbial films in the compound strains can further grow, harmful microorganisms can be inhibited or killed, organic substances are fermented, and the pH value can be raised to 3.0.
4. The method according to claim 1 or 2, characterized in that the antimicrobial complex bacterial species prepared is a composition obtained by fermenting grape, said fermentation being performed on the basis of a composition comprising said antimicrobial complex bacterial species or whey liquid of said antimicrobial complex bacterial species, fermentable sugar, grape and optionally water, until a biofilm capable of growing is formed above and/or the biofilm in said complex bacterial species continues to grow and a liquid fraction is formed below said biofilm, and said liquid fraction exhibits antimicrobial properties and has a pH of 3.0.
5. The method of claim 1, wherein said kefir in step 1 filters particles having a size greater than 0.2mm using a screen having a pore size of 60-70 mesh.
6. The method according to claim 1, wherein said Kefir in step 1 is a slightly foamed yoghurt preparation obtained by fermenting milk with Kefir grains, said Kefir grains being small clusters of irregular shape, ivory or milky white and gelatinous; said milk is selected from plant-based milk or animal-based milk and combinations thereof; the milk is selected from plant-based dairy products including soy milk, almond milk or coconut milk, and combinations thereof; the animal-based milk product comprises cow's milk, goat's milk or sheep's milk and combinations thereof; the kefir is obtained by a method comprising the steps of:
a) providing a dairy composition comprising said kefir grains, said milk and said fermentable sugars, wherein the weight of said kefir grains is from 0.5 wt% to 20 wt% based on the weight of said milk, and said fermentable sugars are used in an amount to at least saturation;
b) fermenting the dairy composition until it becomes a yogurt texture; the kefir grains are Tibetan mushroom; said fermentable sugar in step a) is sucrose, and is used in an amount of 20% by weight relative to the weight of said milk at 20 ℃;
in step 2, the fermentable sugar is a sugar fermented with the milk, and comprises glucose, fructose, sucrose, maltose, galactose or raffinose and a composition thereof; the sucrose is cane sugar or beet sugar;
the fermentable sugars are used in step 2 in an amount at least up to saturation by weight of the kefir or the kefir whey;
step 2 after introducing the fermentable sugar into the kefir whey, standing for 20 minutes, the whey and the fermentable sugar bringing the curd together above to form a pre-fermented composition;
in the step 3, the fermentation is to place the material to be fermented in a closed container, and carry out fermentation under normal pressure and at the temperature of 16-35 ℃; the fermentation temperature in the step 3 is preferably 28 ℃, and the time required by the fermentation is 2-3 days;
when the fermentation temperature in the step 3 is lower than 16 ℃, the fermentation time is 20 days;
during the fermentation process of the pre-fermentation composition in the step 3, the kefir or the kefir whey and/or the fermentable sugar are added, so that the microbial membrane can grow and thicken continuously, and gas is generated during the fermentation process;
the pre-fermented composition in step 3 is fermented until a microbial film capable of growth is formed above and a whey liquid is formed below, which exhibits antimicrobial properties and has a pH of 3.0.
7. A method according to claim 6, characterized in that said sucrose is used as said fermentable sugar in an amount of at least 20% by weight relative to the weight of said kefir or said kefir whey at 20 ℃.
8. A process according to claim 1 wherein curd is pooled above to form the pre-fermented composition, either by stirring the kefir whey and the fermentable sugars and allowing to stand for 20 minutes.
9. The method of claim 4, wherein the composition is used in health care and/or preservation of food.
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