CN113308419B

CN113308419B - Lactobacillus chaff for fermentation and application thereof

Info

Publication number: CN113308419B
Application number: CN202110828662.8A
Authority: CN
Inventors: 王凤忠; 李淑英; 高洁; 侯丽真; 范蓓; 佟立涛
Original assignee: Institute of Food Science and Technology of CAAS
Current assignee: Institute of Food Science and Technology of CAAS
Priority date: 2021-07-22
Filing date: 2021-07-22
Publication date: 2021-10-26
Anticipated expiration: 2041-07-22
Also published as: CN113308419A

Abstract

The invention provides lactobacillus chaff for fermentation and application thereof. The invention separates a lactobacillus from natural ferment, and the lactobacillus is identified as lactobacillus chaffLactobacillus farraginisIs named 84-M-Y-7 and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 22055. The strain is food-grade lactobacillus, and has short culture period and high growth and propagation speed. The invention also provides the application of the strain fermentation process, and beverage products are obtained through fermentation, after-ripening, centrifugation, filtration, seasoning and filling. The fermented beverage product prepared by the strain optimizes the original flavor of the traditional Chinese medicinal materials on the basis of having the special aroma and taste of natural plant beverages, so that the product has better taste, higher nutritional value, longer shelf life and good oxidation resistance.

Description

Lactobacillus chaff for fermentation and application thereof

Technical Field

The invention relates to the field of microbial technology and application thereof, in particular to a lactobacillus strain for food fermentation and application thereof in food and medicine homologous fermentation.

Background

Fermentation refers to the process by which a person produces the microbial cells themselves, or direct or secondary metabolites, by virtue of the life activities of the microorganism under aerobic or anaerobic conditions. At present, the microbial fermentation technology is widely applied to the food processing industry, compared with the production mode of traditional fermented products, the problem of the traditional fermented food in the industrial production can be effectively solved by utilizing the fermentation of specific strains, and the method has the advantages of strong fermentation activity, short fermentation time, capability of keeping the activity of microbial strains, effectively preventing mixed bacteria pollution, saving raw materials, reducing cost and the like, and simultaneously can ensure the stability of the quality of the fermented product, accurately control the fermentation engineering and reduce the production risk. Lactic acid bacteria are gram-positive bacteria which can metabolize carbohydrates into lactic acid and do not have a spore structure, are widely applied to various food processing industries, are used for producing and processing fermented products, improve the original flavor of materials, and have the effects of strengthening the gastrointestinal tract function of a human body, promoting the digestion and absorption of organisms on nutrient substances and the like.

"food and drug homologous" Chinese medicine refers to Chinese medicinal materials which have both medicinal effect and can be eaten as food, such as: ginseng, kudzuvine root, astragalus root and the like, which are mostly applied in the compatibility of traditional Chinese medicines and are less applied in the food processing industry. The fermented plant-based beverage is taken as a rising healthy beverage, the materials are fermented by microorganisms, macromolecular substances in the materials can be converted into small molecules, characteristic functional substances are enhanced, the digestion and absorption of a human body are facilitated, and the flavor of the product can be optimized. At present, few fermentation products related to 'food and drug homology' traditional Chinese medicines exist in the market. Therefore, the microbial fermentation technology is applied to the 'food and medicine homology' traditional Chinese medicinal materials, the nutrient substances in the traditional Chinese medicinal materials are retained to the maximum extent, the nutritional efficacy of the traditional Chinese medicinal materials is fully exerted, the flavor of the traditional Chinese medicinal materials can be effectively improved, and the quality guarantee period of the traditional Chinese medicinal materials is prolonged. Based on the method, the microbial fermentation technology is utilized to develop and research the 'food and drug homology' traditional Chinese medicine fermented beverage, so that the abundant traditional Chinese medicine resources in China are fully utilized, the types of processed products of the traditional Chinese medicine in China are increased and broadened, and the agricultural industrialization of the traditional Chinese medicine is promoted.

Disclosure of Invention

Based on the analysis, the invention aims to provide a novel lactobacillus strain for food fermentation and a food and medicine homologous Chinese medicine fermented product processed by the novel lactobacillus, so as to overcome the defects of the prior art, furthest retain the nutrient components of the Chinese medicinal materials, optimize the flavor of the Chinese medicinal materials and strengthen the digestion and absorption of the nutrient substances of the Chinese medicinal materials by human bodies.

The invention provides a Lactobacillus chaff (Lactobacillus farraginis) 84-M-Y-7, which is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of Beijing western No. 1 institute of microbiology, institute of microbiology of China academy of sciences, zip code 100101) at 23 months 3 and 23 days 2021, and is classified and named as Lactobacillus chaff (CGMCC No. 22055).

Lactobacillus chaff of the present invention (L. farraginis) Bacteriological characteristics of the 84-M-Y-7 strain: lactobacillus chaff (L.) bacteriumL. farraginis) The 84-M-Y-7 single colony is milky white, round, wet, neat in edge, opaque, not connected, and positive in gram stain.

The invention relates to a method for preparing a protein using lactobacillus chaffL. farraginis) A method for producing a fermented product of Chinese medicines with homology of food and medicine by using 84-M-Y-7 strain is to research the fermentation temperature, pH value, time and the like of Chinese medicinal materials, and further to research a set of fermented beverage preparation process suitable for the strain: selecting raw materials → cleaning → blanching → juicing → boiling → fermenting → after-ripening → centrifuging → filtering → flavoring → filling → finished product.

The conditions for preparing the fermented beverage of the invention are as follows: said Lactobacillus chaff (L.), (L. farraginis) The inoculation amount of 84-M-Y-7 is 4-12%. The fermentation temperature is 20-40 deg.C, preferably the optimum temperature is 32-38 deg.C, more preferably 34-36 deg.C. The fermentation time is 30-80 h, preferably 40-55 h, more preferably 42-52 h. The after-ripening temperature is 4-10 deg.C, preferably the optimum temperature is 4 deg.C, and the after-ripening time is 20-80 h, preferably 40-55 h, more preferably 42-52 h. The fermentation mode is static culture.

An object of the present invention is to provide Lactobacillus chaffeensis (II)L.farraginis) 84-M-Y-7 in fermentation products. The fermented product is a fermented product of Chinese medicinal materials with food and medicine homology. The fermented product is one of food and medicine homologous traditional Chinese medicinal materials of kudzuvine root, dendrobium officinale, ginger, Chinese yam, ginseng and maca, and can also be other kinds of traditional Chinese medicinal materials fermented by using lactobacillus chaff strains. The fermented product can be fermented beverage, paste, powder, buccal tablet, etc.

The invention aims to provide a preparation process of a fermented beverage, which comprises the steps of raw material selection, cleaning, blanching, juicing, boiling, fermentation, after-ripening, centrifugation, filtration, seasoning, filling and finished product obtaining. The process comprises the steps of raw material selection, raw material treatment and the like, wherein the raw material treatment comprises the steps of blanching, juicing and boiling, and the after-ripening step is also included after the product is fermented. The process steps also comprise one or more steps of centrifugation, filtration, seasoning, filling, packaging and finished product.

The lactobacillus chaff provided by the inventionL. farraginisNumbered 84-M-Y-7, can be prepared into products or additives such as microbial inoculum, microbial inoculum composition, fermentation powder and the like.

The lactobacillus chaff 84-M-Y-7 in the product or the additive is cultured by using an MRS liquid culture medium, and the formula of the MRS liquid culture medium is as follows: 10g of peptone, 5g of beef powder, 4g of yeast powder and K₂HPO₄•7H₂O2 g, triammonium citrate 2g, CH₃COONa•3H₂O5 g, glucose 20g, Tween 801 mL, MgSO₄•7H₂O 0.2g、MnSO₄•4H₂0.05g of O, 15g of agar and 1000 mL of distilled water.

The strain 84-M-Y-7 is subjected to subculture for 3-4 times in MRS liquid medium. When the viable count reaches 1 × 10⁷When the concentration is more than CFU/mL, the fermented seed liquid is stored in a refrigerator at 4 ℃ for later use.

The invention also provides a fermented beverage which uses the lactobacillus chaff (lactobacillus: (a)L. farraginis) 84-M-Y-7 is prepared by fermentation. The fermented beverage can be radix Puerariae, herba Dendrobii, rhizoma Zingiberis recens, rhizoma Dioscoreae, Ginseng radix or maca fermented beverage.

Lactobacillus chaff (L.) bacteriumL. farraginis) 84-M-Y-7, fermenting at 20-40 deg.C for 20-80 h, and standing for culturing.

In the fermentation step, the previously cultured cells are inoculated into the raw material solution in an inoculum size of 4-20%. The preparation process of the fermentation product also comprises an after-ripening step, wherein the after-ripening temperature is preferably 4-6 ℃, and the after-ripening time is preferably 36-60 h.

Through the technical scheme, the invention discloses and provides a lactobacillus strain for food fermentation and application thereof in fermentation of 'food and medicine homology' traditional Chinese medicines, and compared with the prior art, the invention has the following beneficial effects:

1. the strain can be used as a high-quality starter: the invention is used as a strain culture with high viable count to be inoculated into the traditional Chinese medicine stock solution with food and medicine homology, so that the feed liquid can be fully fermented, the fermentation time is short, the fermentation characteristic is good, the product quality is stable, and the flavor is excellent.

2. The leaven of the invention is convenient to use: the leaven of the invention can be directly added into materials without adding other additives, and has convenient use and small dosage.

3. The six fermented beverages prepared by the strain improve the taste of the traditional Chinese medicinal materials, furthest reserve the original nutrient components of the traditional Chinese medicinal materials while optimizing the flavor of the traditional Chinese medicinal materials, promote the release of combined active components by utilizing the action of lactic acid bacteria, effectively improve the functional characteristics of the products and enhance the digestive absorption of human bodies.

Drawings

FIG. 14 lactic acid bacteria colony morphology features and gram stain results.

FIG. 2 phylogenetic tree of 4 strains based on the 16S rDNA gene sequence.

FIG. 3 shows pH (A), total acid content (B) and OD of different strains of fermented dendrobe juice₆₀₀(C) And (4) changing.

FIG. 4 comparison of the ABTS free radical scavenging effect of fermented beverages and unfermented stock. A-F are the comparison of the fermented beverage of radix Puerariae, herba Dendrobii, rhizoma Zingiberis recens, rhizoma Dioscoreae, Ginseng radix and maca with the unfermented stock solution.

FIG. 5 comparison of hydroxyl radical scavenging effect of fermented beverages and unfermented stock solutions. A-F are the comparison of the fermented beverage of radix Puerariae, herba Dendrobii, rhizoma Zingiberis recens, rhizoma Dioscoreae, Ginseng radix and maca with the unfermented stock solution.

FIG. 6 comparison of reducing power of fermented beverages and unfermented stock solutions. A-F are the comparison of the fermented beverage of radix Puerariae, herba Dendrobii, rhizoma Zingiberis recens, rhizoma Dioscoreae, Ginseng radix and maca with the unfermented stock solution.

FIG. 7 pH value changes of six fermented beverages stored at 4 ℃ for 63 days.

FIG. 8 the total acid content of six fermented beverages stored at 4 ℃ for 63 days was varied.

FIG. 9 shows the change in viable count of six fermented beverages stored at 4 ℃ for 63 days.

FIG. 10 sensory evaluation results of six fermented beverages stored at 4 ℃ for 63 days.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 isolation and purification of lactic acid bacteria

And (3) carrying out gradient dilution on the collected folk ferment products by 10 times in an ultra-clean workbench by using sterile physiological saline, uniformly coating 1mL of bacterial liquid with a proper dilution ratio on a lactic acid bacteria identification culture medium by using a coating rod, and culturing for 48-72 h at constant temperature in an anaerobic incubator at 37 ℃. Then taking out the single colony with the diameter of 1-2 mm and smooth surface under the aseptic environment, inoculating the single colony to a new lactic acid bacteria identification culture medium by a streaking method, repeating for many times until pure colonies are separated and purified, and initially identifying the lactic acid bacteria as the strain and numbering the strain. The screened lactobacillus is subjected to gradient dilution by using sterile normal saline and is cultured for 72 hours at the constant temperature of 37 ℃ by using MRS solid culture medium to observe the colony morphological characteristics and the microscopic morphological characteristics (gram staining).

Then selecting single bacterial colony which is milky white and has smooth surface according to the size, color, luster and transparency of the microbial colony, streaking and purifying for 2-3 times on an MRS plate by using a streaking method, and storing the strain in a refrigerator at 4 ℃.

Example 2 identification of Lactobacillus species

The phenotypic characteristics of the purified strain and 16S rDNA were systematically identified.

Firstly, phenotypic characteristics and phenotypic observation:

the colony morphology characteristics of the 4 separated lactic acid bacteria are shown in figure 1, and as can be seen from figure 1A, the colony morphologies of the strains are all milky white, the edges are regularly circular, the surface is smooth, the diameter of the colony is in the range of 1.0-1.5 mm, and the colony morphologies are respectively named as 84-M-Y-7, 83-M-Y-6, 57-M-H-7 and 102-M-H-8. The 4 lactic acid bacteria were identified by gram staining, and the characteristics of the cells were observed under an oil microscope of 100 times. Microscopic examination results show that all strains are gram-positive strains, and the thalli of the strains are rod-shaped and have various arrangement modes.

Two, 16S rDNA sequence analysis

Inoculating the separated lactobacillus into an MRS liquid culture medium to activate for 24 h, taking 1mL of bacterial liquid into a 1.5 mL sterile centrifuge tube, extracting genome by adopting Plant Zol (full-scale gold kit), amplifying the extracted genome DNA by using bacterial universal primers 27F (SEQ ID NO: 15 '-AGAGT TTGAT CCTGG CTCAG-3') and 1492R (SEQ ID NO: 25 '-CTACG GCTAC CTTGT TACGA-3') to obtain bacterial 16SrDNA, and amplifying the amplified template by adopting a 30 mu L system. The PCR conditions were as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 57 ℃ for 30 s, extension at 72 ℃ for 1.5 min, and 25 cycles; extending for 7 min at 72 ℃; 3% agarose electrophoresis followed by sequencing the amplified samples by Biotech, Inc., Boxing Ke Borui Beijing.

Logging in NCBI website, performing BLAST homology analysis and comparison on sequencing result of 4 separated lactobacillus 16S rDNA and related information in the website database, identifying strains and constructing phylogenetic tree, wherein the result is shown in figure 2. As can be seen from FIG. 2, the strain numbered 84-M-Y-7 is Lactobacillus chaff, the strain numbered 83-M-Y-6 is Lactobacillus harabini, the strain numbered 57-M-H-7 is Lactobacillus paracasei, and the strain numbered 102-M-H-8 is Lactobacillus buchneri.

Example 3 Lactobacillus chaffeensis (II)L. farraginis) Screening of 84-M-Y-7

Selecting fresh herba Dendrobii, removing impurities and rotten leaves, cleaning herba Dendrobii with flowing clear water, cutting into segments (about 2-3 cm), blanching (about 10-20 s), juicing, boiling (about 10-20 s), and filling into 250 mL conical bottles. A250 mL Erlenmeyer flask was sterilized at 121 ℃ for 15 min.

Inoculating activated 4 strains of lactobacillus in ultra-clean bench to MRS liquid culture medium sterilized in advance, and subculturing for 3-4 times until viable count reaches 1 × 10⁷When the concentration is more than CFU/mL, preparing seed liquid for later use.

Respectively inoculating 4 strains of lactobacillus seed liquid into the dendrobium juice according to the inoculation amount of 10% for fermentation, wherein the fermentation conditions are as follows: standing at 37 deg.C for 48 h, and then transferring into a low-temperature refrigerator at 4 deg.C for post-maturation for 48 h. During the maturation period after fermentation, the pH value, total acid content and OD were measured every 12 h₆₀₀And selecting the suitable fermentation strain by comparing the measurement results of multiple indexes.

The basic index determination comprises the following steps:

and (3) pH value measurement: measuring by using a pH meter;

and (3) total acid content determination: measuring by a potentiometric titration method;

OD₆₀₀and (3) determination: and measuring by using an ultraviolet-visible spectrophotometer.

Data were processed and analytically plotted using Graph Pad Prism 7.0 plotting software, and analyzed for variance using SPSS 11.

And (4) analyzing results: pH value, total acid content and OD of dendrobium juice fermented by different strains₆₀₀The change of (A) is shown in FIG. 3, wherein the change trend of three indexes of 37 ℃ fermentation is from 0 h to 48 h, and the change trend of three indexes of 4 ℃ after-ripening is from 48 h to 96 h. As can be seen from FIG. 3, the pH change is inversely related to the total acid content change throughout the fermentation after-ripening process, while OD₆₀₀The trend is that the temperature rises first and then falls. The pH value of the dendrobium natural juice is 5.46, the total acid content is 0.45 mL/L, the pH value is sequentially reduced to 3.12, 3.54, 3.43 and 3.39 after the dendrobium natural juice is fermented by lactobacillus chaff 84-M-Y-7, lactobacillus harbin 83-M-Y-6, lactobacillus paracasei 57-M-H-7 and lactobacillus buchneri 102-M-H-8, and the lactic acid content is sequentially increased to 4.95 mL/L, 1.35 mL/L, 3.45 mL/L and 4.35 mL/L. After the post-maturation stage is started after 48 h of fermentation, the growth and metabolism speed of the lactic acid bacteria is reduced, so that the pH value and the total acid content are slightly changed, after 96 h of post-maturation, the pH values of the dendrobium juice fermented by 4 strains are 3.03, 3.5, 3.3 and 3.27 in sequence, and the total acid content is 3.03, 3.5, 3.3 and 3.27 in sequence6.45 mL/L, 2.3 mL/L, 4.35 mL/L, and 5.25 mL/L. Combining the change of the pH value and the total acid content of the dendrobium juice in the fermentation stage, the change of the pH value and the total acid content of the dendrobium juice fermented by lactobacillus chaff 84-M-Y-7 is the largest, the change of lactobacillus buchneri and lactobacillus paracasei are the second, and the change of the minimum is that the dendrobium juice is fermented by lactobacillus harabii.

Experiment with OD₆₀₀The method is used as an index for measuring the change of viable count in the process of fermenting the dendrobium juice by different strains. OD₆₀₀The larger the value is, the higher the thallus concentration is relatively, and the stronger the adaptability of the strain in the dendrobium juice is. As can be seen from FIG. 3C, in the whole fermentation after-ripening process, the OD of the dendrobium nobile juice is fermented by Lactobacillus chaff 84-M-Y-7₆₀₀The value is obviously higher than that of other strains, the lactobacillus buchneri and lactobacillus paracasei are used as the second strains, the lactobacillus harbini is used as the last strain, and the OD of the four strains in the dendrobium juice₆₀₀The change in (c) corresponds to a change in pH and total acid content. Therefore, according to the experimental results, the lactobacillus chaff 84-M-Y-7 has stronger adaptability in the dendrobium juice, and the effect of fermenting the dendrobium juice is better than that of other strains. The strain is selected and preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of West Lu No. 1 of Beijing university Hokko Yang district, No. 3 of China academy of sciences, postal code 100101) at 23 months and 3 days of 2021, and the strain is classified and named as lactobacillus chaff (the strain is a strain of Lactobacillus chaff)L.farraginis) The preservation number is CGMCC No.22055, and subsequent research is carried out.

Example 4 Lactobacillus chaffeensis ((II))L. farraginis) Application of 84-M-Y-7 in fermentation with different Chinese medicinal materials as base materials

(1) Pretreatment of raw materials: selecting fresh and mildew-free radix Puerariae, herba Dendrobii, rhizoma Zingiberis recens, rhizoma Dioscoreae, Ginseng radix and maca, respectively, peeling, repeatedly cleaning with flowing clear water for several times, removing surface dirt, peeling, cutting into small pieces, and blanching with boiling water for about 20-30 s.

(2) Preparing a traditional Chinese medicine stock solution: adding purified water and a certain amount of sodium carboxymethylcellulose into the processed Chinese medicinal materials, squeezing, boiling the stock solution of the squeezed Chinese medicinal materials for 15-20 s, and cooling.

(3) Preparing a fermentation strain: the strain lactobacillus chaff for fermentation (b)L. farraginis) 84-M-Y-7 is subcultured in MRS liquid medium for 3-4 times. When the viable count reaches 1 × 10⁷When the concentration is more than CFU/mL, the fermented seed liquid is stored in a refrigerator at 4 ℃ for later use.

(4) After-ripening in fermentation: inoculating the seed solution into the stock solution of the Chinese medicinal materials according to the inoculation amount of 6-12% in an aseptic environment, culturing in a constant temperature incubator at 34-37 deg.C for 36-60 h, and after-ripening in a low temperature refrigerator at 4-6 deg.C for 36-60 h.

(5) Centrifugal filtration: centrifuging the fermentation liquor, wherein the centrifugation conditions are as follows: 4000 r/min, 10 min, taking supernatant, and filtering through 300-mesh filter cloth to obtain fermentation stock solution.

(6) Seasoning and filling: adding small amount of honey water (honey: water volume is 1: 1) and purified water into the fermentation stock solution for flavoring, and aseptically packaging in a sterilized container to obtain the Chinese medicinal material fermented beverage.

Example 5 fermented beverage Performance testing

Six fermented beverages were prepared according to the above method and their basic indexes, oxidation resistance and storage stability were measured, and the specific experimental methods were as follows:

the experimental method for measuring basic indexes of fermented beverage comprises the following steps:

(1) measurement indexes are as follows: pH value of beverage

The determination method comprises the following steps: direct measurement with pH meter

(2) Measurement indexes are as follows: total acid content

The determination method comprises the following steps: the determination was carried out by potentiometric titration according to GB12456-2018 "determination of total acids in food".

(3) Measurement indexes are as follows: number of viable bacteria

The determination method comprises the following steps: gradient dilution plate counting method

(4) Measurement indexes are as follows: water content of beverage

The determination method comprises the following steps: the measurement was carried out by the direct drying method in GB5009.3-2010 "measurement of moisture in food".

(6) Measurement indexes are as follows: fat content of beverage

The determination method comprises the following steps: the measurement was carried out by Soxhlet extraction method in GB5009.6-2016 (determination of fat in food).

(7) Measurement indexes are as follows: ash content of beverage

The determination method comprises the following steps: the measurement is carried out according to the direct ashing method in GB5009.4-2010 determination of ash content in food.

(8) Measurement indexes are as follows: sodium and calcium content of beverage

The determination method comprises the following steps: the measurement is carried out according to flame atomic absorption spectrometry in GB5009.92-2016 (determination of calcium in food) and GB5009.91-2017 (determination of potassium and sodium in food).

(9) Measurement indexes are as follows: beverage coliform determination

The determination method comprises the following steps: the determination is carried out according to the MPN method in GB4789.3-2016 (food microbiology test for coliform bacteria count)

(10) Measurement indexes are as follows: beverage pathogenic flora determination

Because the pathogenic bacteria most frequently contained in the food are salmonella, the salmonella is selected as the pathogenic bacteria for detection in the experiment.

The determination method comprises the following steps: measured according to GB4789.3-2016 Salmonella test for food microbiology.

Secondly, measuring the oxidation resistance of the six fermented beverages, wherein the specific measuring method comprises the following steps:

(1) determination of ABTS free radical scavenging ability

The sample groups were prepared by diluting the fermented beverages with purified water to 0.1 mL of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5625% by volume, and adding 3.5 mL of ABTS diluent. The control group was 0.1 mL of fermented beverage of the same volume fraction, 3.5 mL of 10 mM PBS solution pH7.4 was added, the blank group was 0.1 mL of purified water, and 3.5 mL of ABTS dilution was added. Shaking the three groups of samples, placing in dark place for reaction for 6 min, and measuring the absorbance at 714 nm. The unfermented stock was tested in the same manner. All experiments were repeated three times, averaged and calculated according to the formula.

ABTS free radical scavenging rate/% = [1- (Ai-Aj)/AO ]. times.100

In the formula: ai is the absorbance of the mixed solution of each volume fraction sample and the ABTS diluent, Aj is the absorbance of the mixed solution of each volume fraction sample and 10 mM PBS with pH7.4, and AO is the absorbance of the mixed solution of purified water and the ABTS solution.

(2) Determination of the hydroxyl radical scavenging Capacity

The fermented beverage is diluted to 1mL with purified water until the volume fractions are 100%, 50%, 25%, 12.5%, 6.25%, 3.125% and 1.5625%, and then 1mL of 0.75 mmol/L ortho-diazaphenanthrene solution, 2 mL of 0.1 mol/L PBS solution with pH of 7.4 and 1mL of 0.75 mmol/L ferrous sulfate are added. After mixing well, 1mL of 0.01% hydrogen peroxide solution was added as a sample group. The control group uses 1mL of purified water to replace 1mL of hydrogen peroxide, the blank group uses 1mL of purified water to replace 1mL of samples, the three groups of samples are placed in a water bath kettle at 37 ℃, water bath is carried out for 60 min, and the light absorption value is measured at 536 nm. The unfermented stock was tested in the same manner. All experiments were repeated three times, averaged and calculated according to the formula.

Hydroxyl radical clearance/% = (AS-AP)/(AB-AP)

In the formula: AS is the absorbance of the mixed solution of the samples with each volume fraction and the reagents such AS phenanthroline, AB is the absorbance of the control group, and AP is the absorbance of the blank group.

Thirdly, determination of reducing power

The fermented beverage was diluted with purified water to 1mL of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5625% by volume, and 2.5 mL of 0.2 mol/LpH 6.6 PBS solution and 2.5 mL of 1% potassium ferricyanide solution were added, and after 20 min of 50 ℃ water bath reaction, 2.5 mL of 10% TCA solution and 0.5 mL of 0.1% ferric chloride were added. After reacting for half an hour in the dark, centrifuging at 2000 r/min for 5 min, and taking the supernatant to measure the light absorption value at 700 nm. The unfermented stock was tested in the same manner. All experiments were repeated three times and averaged.

Fourth, storage stability of fermented beverage

The specific determination method is as follows:

the fermented beverage prepared by the method is placed at 4 ℃ for 60 days, the pH value, the total acid content and the viable count of the beverage are measured every 7 days, and the storage stability of the fermented beverage is measured by sensory evaluation. The measuring method of the pH value, the total acid content and the viable count is the same as the measuring method of the basic indexes of the fermented beverage, and sensory evaluation is carried out by a plurality of professional teachers according to a sensory evaluation table which is as follows:

EXAMPLE 6 basic index measurement results of fermented beverages

The results of the measurement of the basic indexes of the six fermented beverages according to the measurement method are shown in the following table:

example 7 measurement of antioxidant Properties of fermented beverages

The reducing power of the fermented beverage and the unfermented stock solution is compared with the reducing power of the fermented beverage and unfermented stock solution for scavenging ABTS free radicals and hydroxyl free radicals, and the results are shown in FIGS. 4 and 5.

ABTS radicals are green radicals formed by the reaction of 2, 2-diaza-bis (3-ethyl-benzothiazole-6-sulphonic acid) diammonium salt with potassium peroxodisulfate, which have an absorption maximum at 714 nm. After the ABTS free radical solution is added into the sample, if the absorbance at 714nm is reduced, the substance has free radical scavenging activity, and the antioxidant capacity of the substance can be evaluated by comparing the scavenging effect of the sample on the ABTS free radicals. As can be seen from FIG. 4, with increasing volume fraction, the clearance of ABTS free radicals from the fermented beverage and the unfermented stock solution is gradually increased, and the clearance of ABTS free radicals from the fermented beverage and the unfermented stock solution with different volume fractions is higher than that of the unfermented stock solution.

FIG. 5 is a comparison of hydroxyl radical scavenging effect of fermented beverages and unfermented stock solutions. Hydroxyl radical (-OH) is an important active oxygen, and has a very strong electron-gaining ability, i.e., an oxidizing ability. The experiment adopts o-diazaphenanthrene-Fe²⁺The oxidation method is used for measuring the scavenging capacity of the sample to the hydroxyl radical, and the oxidation resistance of the sample is reflected by the change of the color. As can be seen from fig. 5, the removal rates of hydroxyl radicals of the fermented beverage and the unfermented stock solution are concentration-dependent, the higher the sample concentration is, the better the removal effect of hydroxyl radicals is, and the removal rates of hydroxyl radicals of the fermented beverage are higher than those of the unfermented stock solution.

The reduction force measurement means that the antioxidant gives electrons to scavenge free radicals through the reduction action of the antioxidant, and the stronger the reduction force is, the stronger the oxidation resistance is. The absorbance values of the fermented beverage and the unfermented stock solution measured at 700 nm are shown in FIG. 6, and the larger the absorbance value, the stronger the reducing power of the sample. According to the experimental result, the reducing power is gradually improved along with the increase of the volume fraction of the sample, and the reducing power of the fermented beverage is obviously superior to that of the unfermented stock solution. In summary, Lactobacillus chaffeensis (II) is usedL. farraginis) The antioxidant property of the fermented beverage prepared by fermenting kudzuvine root, dendrobium officinale, ginger, Chinese yam, ginseng and maca is obviously enhanced.

Example 8 measurement results of storage stability of fermented beverage

The pH value, total acid content, viable count, and sensory evaluation results of the six fermented beverages stored at 4 deg.C for 63 days are shown in FIGS. 7-10. As can be seen from FIGS. 7 to 10, the pH, total acid content and viable cell count of the fermented beverage did not change significantly with the increase in storage time. According to sensory evaluation results, the fermented beverage has good taste after being refrigerated and stored for about two months, and has longer shelf life compared with the same type of fermented products in the market.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art can change or modify the technical content disclosed above into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

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<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctacggctac cttgttacga 20

<210> 3

<211> 434

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tggacgaagt ctgatggagc aacgccgcgt gagtgatgaa gggtttcggc tcgtaaaact 60

ctgttgttgg agaagaacag gtgatagagt aactgttatc atcttgacgg tatccaacca 120

gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc 180

cggatttatt gggcgtaaag cgagcgcagg cggtttttta ggtctgatgt gaaagccttc 240

ggcttaaccg gagaagggca tcggaaaccg ggagacttga gtgcagaaga ggacagtgga 300

actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaaggcggc 360

tgtctggtct gtaactgacg ctgaggctcg aaagcatggg tagcgaacag gattagaaac 420

cccgggtagt ccaa 434

Claims

1. A Lactobacillus chaff is prepared fromLactobacillus farraginis84-M-Y-7, wherein the lactobacillus chaff is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 22055.

2. A microbial preparation comprising the Lactobacillus chaffeensis according to claim 1.

3. A composition comprising the lactobacillus chaffeensis of claim 1.

4. Use of lactobacillus chaffeensis according to claim 1 or of a bacterial preparation according to claim 2 or of a composition according to claim 3 in a fermented product.

5. The use of claim 4, wherein the fermented product is a fermented product of a Chinese medicinal material that is food and drug homologous.

6. The use of claim 5, wherein the fermented product is one of kudzu root, Dendrobium officinale, ginger, yam, ginseng, maca.

7. A fermented beverage prepared by fermentation using the Lactobacillus chaffeensis according to claim 1.

8. The fermented beverage according to claim 7, wherein the fermented beverage is a kudzu root, dendrobium officinale, ginger, yam, ginseng or maca fermented beverage.

9. The process for preparing a fermented beverage according to claim 7 or 8, comprising the steps of raw material selection and cleaning, raw material treatment, fermentation, filtration and flavoring.

10. The process for preparing a fermented drink according to claim 9, wherein the lactobacillus chaff according to claim 1 is fermented at a temperature of 20 to 40 ℃ for 30 to 80 hours, and then is subjected to static culture.