CN114990186B - Coix seed multi-bacterium fermentation liquor and preparation method and application thereof - Google Patents
Coix seed multi-bacterium fermentation liquor and preparation method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9794—Liliopsida [monocotyledons]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Life Sciences & Earth Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
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- Genetics & Genomics (AREA)
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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
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Abstract
The invention discloses coix seed multi-bacterium fermentation liquor, a preparation method and application thereof, and relates to the technical field of fermentation. The coix seed is fermented by adopting mixed strains, wherein the mixed strains comprise lactococcus lactis, lactobacillus bulgaricus and streptococcus thermophilus. The method adopts the mixture of three strains of lactococcus lactis, lactobacillus bulgaricus and streptococcus thermophilus to ferment the coix seeds, wherein the lactococcus lactis is a fermentation dominant bacterium, the content of total phenols and total acids in fermentation liquor is obviously improved, the scavenging rate of hydroxy free radicals and the inhibition effect on tyrosinase activity are improved, and the oxidation resistance and the whitening performance of the fermentation liquor are excellent. Meanwhile, ethanol is not required to be used for washing in the fermentation process, the step of ethanol removal is not required to be processed, the risk of ethanol residue is avoided, and the final finished product contains fermentation liquor filtrate and multi-bacterial lysate and has more excellent performance.
Description
Technical Field
The invention relates to the technical field of fermentation, in particular to coix seed multi-bacterium fermentation liquor and a preparation method and application thereof.
Background
Lactic acid bacteria are a class of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid; the metabolites of lactobacillus fermentation mainly comprise organic acids, bacteriocins, aromatic substances such as acetaldehyde, and extracellular polysaccharide. Lactic acid is belonged to alpha-hydroxy acid (AHA), which can accelerate metabolism of cells, remove unhealthy cutin of skin, and improve skin color; the lactobacillus fermentation can convert carbohydrate and sugar into skin-friendly enzyme and amino acid, so that the effective components are decomposed and micronized. Lactococcus lactis is a gram positive, facultative anaerobe. When heme is present, it can alter the metabolic pattern of lactococcus lactis for aerobic respiration. Under the aerobic respiration mode, the lactococcus lactis has faster growth rate and higher survival rate, and the biomass of the lactococcus lactis can be obviously improved.
The coix seed is rich in nutrition, wherein the soluble sugar is 6.38-8.25%, the crude fat is 9.5-11.5%, the protein is 14.7-16.63%, the total amino acid amount is 12.57-12.75%, and the oil is 5%, and is known as 'the king of gramineous plants in the world', and researches show that the coix seed extract can inhibit melanin generation by inhibiting tyrosinase. The coix seed extract existing in the market has low extraction efficiency by adopting a water extraction method, alcohols with high irritation to human bodies can be introduced by adopting an alcohol extraction method, and other operations such as supercritical fluid extraction are complex.
Disclosure of Invention
The invention provides a coix seed multi-fungus fermentation broth, a preparation method and application thereof, which are used for improving the whitening and antioxidation effects of coix seed extracts, and have the advantages of small stimulation of components to human bodies and high safety performance.
In order to solve the technical problems, one of the purposes of the invention is to provide a preparation method of coix seed multi-bacterium fermentation broth, which adopts mixed bacteria to ferment coix seeds, wherein the mixed bacteria comprise lactococcus lactis, lactobacillus bulgaricus and streptococcus thermophilus.
In the fermentation process, the addition amount of the lactococcus lactis, the lactobacillus bulgaricus and the streptococcus thermophilus is 0.2-2 wt%, the concentration of the lactococcus lactis bacterial liquid is 8-10g/L, and the bacterial content of the lactobacillus bulgaricus and the streptococcus thermophilus is 100 hundred million CFU/g.
Preferably, the method comprises the following steps:
(1) Preparing strains;
(2) Saccharifying Coicis semen to obtain Coicis semen fermentation medium substrate;
(3) Inoculating the lactococcus lactis, the streptococcus thermophilus and the lactobacillus bulgaricus obtained in the step (1) into a coix seed fermentation medium, and fermenting to obtain a coix seed multi-bacterium fermentation liquid primary product;
(4) Adding lysozyme into the primary product of the coix seed multi-bacterium fermentation liquid, stirring and homogenizing, and centrifugally collecting the supernatant; filtering to obtain coix seed multi-fungus fermentation liquor.
Preferably, in the step (2), the coix seed powder is mixed with water and then CaCl is added 2 Gelatinizing, cooling to 75-80deg.C, adding alpha-amylase, and liquefying at constant temperature; adjusting pH to 4-4.2, cooling to 60deg.C, adding saccharifying enzyme, maintaining at 75-80deg.C for saccharification, and sterilizing to obtain Coicis semen fermentation medium.
Preferably, in step (2), caCl 2 The addition amount of the catalyst is 1-5% of the mass of the substrate, and the gelatinization time is 0.5-2.5h; the enzyme dosage of alpha-amylase is 10-50U/g of Coicis semen powder, and the liquefying time is 1-5h; the enzyme dosage of saccharifying enzyme is 100-500U/g Coicis semen powder, and saccharifying time is 1-5 hr.
Preferably, in the step (4), the addition amount of lysozyme is 0.2-2g/L.
Preferably, in the step (4), the stirring time is 1-5h, and the homogenization times are 2-8.
Preferably, in step (4), the filtration is performed sequentially through 0.8 μm and 0.22 μm membranes.
Preferably, the lactococcus lactis is lactococcus lactis MG1363.
In order to solve the technical problems, the second purpose of the invention is to provide the coix seed multi-fungus fermentation liquor obtained by adopting the preparation method of the coix seed multi-fungus fermentation liquor.
In order to solve the technical problems, the invention provides an application of the coix seed multi-fungus fermentation liquor in cosmetics.
Preferably, the mass fraction of the coix seed multi-fungus fermentation liquor is more than 0.5 wt%.
Compared with the prior art, the embodiment of the invention has the following beneficial effects:
1. the method adopts the mixture of three strains of lactococcus lactis, lactobacillus bulgaricus and streptococcus thermophilus to ferment the coix seeds, wherein the lactococcus lactis is a fermentation dominant bacterium, the content of total phenols and total acids in fermentation liquor is obviously improved, the scavenging rate of hydroxy free radicals and the inhibition effect on tyrosinase activity are improved, and the oxidation resistance and the whitening performance of the fermentation liquor are excellent.
2. The fermentation process of the method does not need to use ethanol for washing, does not need to treat the step of ethanol removal, avoids the risk of ethanol residue, and has more excellent performance because the final finished product contains fermentation liquor filtrate and multi-bacterial lysate.
3. The fermentation process is carried out by adopting a high-density facultative anaerobic fermentation process, and meanwhile, the fermented lactococcus lactis MG1363 is a strain obtained by a genetic engineering culture technology, so that the propagation amount and activity of the strain are higher.
Drawings
Fig. 1: the preparation method is a flow diagram of a preparation method of coix seed multi-bacterium fermentation liquor in the first embodiment of the invention;
fig. 2: the electrophoresis band results obtained in the detection of the bacterial growth trend of coix seed multi-bacterial fermentation broth in the embodiment 1 of the invention (note: 1, 2 and 3 represent three biological repeats; the lactococcus lactis band is 991bp; the Lactobacillus bulgaricus band is 650bp; the Streptococcus thermophilus band is 797 bp);
fig. 3: the statistical result (note: P <0.05, P <0.01, P <0.001 compared to control) of the coix seed multi-fungus fermentation broth in the zebra fish test in example 1 of the present invention is shown.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The human body continuously generates free radicals due to continuous contact with the outside, including respiration (oxidation reaction), external pollution, radiation irradiation and other factors, and the free radicals are anti-oxidant, namely anti-free radicals. The oxidation is in the aspect of skin, and is easy to be dull after the skin is oxidized, so that free radicals on the skin are effectively removed, and the skin color can be lightened, and the purpose of whitening and brightening the skin can be achieved. Polyphenols are very strong antioxidants. The ortho-phenol in the phenol-base structure (catechol or pyrogallol) is easily oxidized into a wake structure, and meanwhile, the polyphenol has strong capturing capability on free radicals such as active oxygen and the like, so that the polyphenol has strong capability of scavenging the free radicals.
After the lactic acid bacteria are used for biotransformation of the coix seeds, macromolecules in the coix seeds can be decomposed into micromolecular substances, beneficial organic acids such as lactic acid, butyric acid and the like can be produced, the content of soluble dietary fibers is obviously increased, and the content of free fatty acids and free amino acids after fermentation is obviously increased. The lactic acid can mildly exfoliate waste cutin of skin, so as to achieve the purpose of whitening and brightening skin.
Meanwhile, different species of bacteria have various interaction relations during co-fermentation, and a favorable symbiotic relation exists between the lactococcus lactis and the streptococcus thermophilus, so that the presence of the streptococcus thermophilus can better promote the growth and fermentation of the lactococcus lactis. The present application extends from lactococcus lactis and bifidobacteria to mixed systems of streptococcus thermophilus and lactobacillus bulgaricus and attempts to improve the performance of coix seed fermentation broths.
Example 1
A preparation method of coix seed multi-fungus fermentation liquor, as shown in figure 1, comprises the following steps:
(1) Strain preparation:
a. lactococcus lactis is obtained from NTCC collection for classical cultures MG1363, purchased from NIZO corporation, netherlands, and after activation by routine operation of M17 plates, monoclonal colonies were selected and inoculated into M17 medium for overnight culture at 30℃for 20h; inoculating the bacterial liquid obtained by overnight culture into a high-density culture medium for culturing for 48 hours at a ratio of 1%, wherein the formula of the high-density culture medium is shown in table 1, and obtaining the lactococcus lactis bacterial liquid with the concentration of 8-10 g/L;
b. lactobacillus bulgaricus and Streptococcus thermophilus are commercial Lactobacillus bulgaricus bacterial powder (Lactobacillus bulgaricus-100 hundred million CFU/g) and Streptococcus thermophilus bacterial powder (Streptococcus thermophilus-100 hundred million CFU/g) of Jia Yi bioengineering Co-Ltd in Shandong.
(2) Preparing a fermentation substrate:
crushing coix seeds into powder, sieving the powder with a 40-mesh sieve, and taking 3kg of the powder according to a feed liquid ratio of 1: mixing 10 proportion with 30L water, adding CaCl 2% of the substrate mass 2 Heating, boiling and gelatinizing for 1h to obtain gelatinized liquid; cooling the obtained gelatinized liquid to 75-80deg.C, adding alpha-amylase (enzyme dosage is 20U/g Coicis semen powder), and liquefying at 75-80deg.C for 2 hr to obtain liquefied liquid; regulating pH to 4-4.2 in liquefied solution, cooling to 60deg.C, adding saccharifying enzyme (enzyme usage amount is 300U/g Coicis semen powder), and saccharifying at 75-80deg.C for 2 hr to obtain saccharified solution; regulating pH of the obtained saccharified solution to 6.0, and sterilizing at 117 deg.C under high temperature and high pressure for 20min to obtain Coicis semen fermentation medium.
(3) And (3) industrialized fermentation:
cooling the coix seed fermentation medium to 37 ℃, adding the lactococcus lactis bacterial liquid, the lactobacillus bulgaricus bacterial powder and the streptococcus thermophilus bacterial powder obtained in the step (1) into the coix seed fermentation medium obtained in the step (2) according to the weight ratio of 1%, and carrying out anaerobic fermentation for 48 hours in a 50L fermentation tank to obtain a coix seed multi-bacterial fermentation liquid primary product.
(4) The final product is obtained:
adding 1g/L lysozyme into the primary product of the coix seed multi-fungus fermentation liquor, and stirring at 30 ℃ for 2 hours; collecting the product in the tank, homogenizing for 5 times (pressure 500-800 bar) by a homogenizer, centrifuging at 4deg.C and 18000rpm/min for 20min, and collecting supernatant; filtering with 0.8 μm and 0.22 μm filter membrane in turn, and obtaining filtrate which is the coix seed multi-fungus fermentation liquor.
TABLE 1 high Density Medium formulation
Comparative example one
The preparation method of the coix seed fermentation liquor, the steps, the reagents used in the steps and the technological parameters are the same as those in the first embodiment, and the differences are that: the lactobacillus added in the step (3) is bifidobacterium, the adding amount is 1% by weight, and the lactobacillus is derived from commercial fungus powder of Jiayi bioengineering Co-Ltd in Shandong, and the fungus content is 100 hundred million CFU/g.
Comparative example two
The preparation method of the coix seed fermentation liquor, the steps, the reagents used in the steps and the technological parameters are the same as those in the first embodiment, and the differences are that: the lactic acid bacteria added in the step (3) are lactic acid lactococcus bacteria liquid, the adding amount is 1% weight ratio, and the source of the lactic acid lactococcus is the same as that of the first embodiment.
Comparative example three
The preparation method of the coix seed multi-fungus fermentation liquor, each step and the reagent and the technological parameters used in each step are the same as those in the first embodiment, and the difference is that: the lactic acid bacteria added in the step (3) are bifidobacteria, streptococcus thermophilus and lactobacillus bulgaricus, the addition amount of each strain is 1% by weight, the sources of the streptococcus thermophilus and the lactobacillus bulgaricus are the same as those in the first embodiment, and the sources of the bifidobacteria are the same as those in the first comparative embodiment.
Comparative example four
The invention patent CN108078880B discloses a whitening composition containing coix seed fermentation broth, and the coix seed fermentation broth is obtained by adopting the preparation method of the coix seed fermentation broth disclosed in the invention.
Performance test
1. And (3) antioxidant function detection: the fermentation broths obtained in example 1 and comparative examples 1 to 3 were examined with reference to the total antioxidant capacity assay (FRAP method), the p-hydroxyl radical scavenging rate was examined with reference to the experiment for measuring the flavone p-hydroxyl radical scavenging capacity by salicylic acid, the super-oxygen anion scavenging rate was examined with reference to the experiment for super-oxygen anion scavenging capacity test by twenty-one chapter six experiment of the cosmetic efficacy evaluation experiment, and the results of the p-hydroxyl radical scavenging rate obtained by examining the coix seed fermentation medium samples before fermentation in step (2) are shown in table 2.
2. Whitening function detection: the results of the tyrosinase inhibition rate obtained by the fermentation broths obtained in example 1 and comparative examples 1 to 4, together with the coix seed fermentation medium sample before fermentation in step (2), were shown in Table 2, with reference to the standard test of the method for tyrosinase activity inhibition test by cosmetic raw materials (in vitro method) of T/GDCA 006-2021.
TABLE 2 antioxidant and whitening Property test results of Coix seed fermentation broths in example 1 and comparative examples 1-3
As shown in Table 2, the coix seed multi-fungus fermentation broth obtained in example 1 has the optimal effect of scavenging the hydroxyl radicals and superoxide anions and inhibiting the tyrosinase activity, which shows that the fermentation broth obtained in the application has the optimal antioxidation effect.
3. Physical and chemical property detection: the total acid content was detected by using the GB/T12456-2008 standard, the total phenol content was detected by using the GB/T8313-2018 standard, the total sugar content was detected by using the DNS method, the detection samples were the coix seed fermentation medium sample before fermentation and the coix seed multi-fungus fermentation broth obtained after fermentation in the step (2) of example 1, lactobacillus/coix seed fermentation broth produced by Gannuo chemical industry Co., ltd. In Shanshi was detected as a bid, and the coix seed fermentation broth obtained in comparative example 4 was detected as a reference, and the detection results are shown in Table 3.
4. Lactic acid content detection: referring to the high performance liquid chromatography (3.1 alpha-hydroxy acid first method) in the fourth chapter of physicochemical inspection method in cosmetic safety technical Specification-2015 edition, the lactic acid content of the sample was detected, the detected sample was the coix seed fermentation medium sample before fermentation and coix seed multi-fungus fermentation broth obtained after fermentation in step (2) of example 1, and the coix seed fermentation broth obtained in comparative example 4 was detected as a reference, and the detection results are shown in table 3.
TABLE 3 determination of the total sugar, total phenol and Total acid content of example 1 and the respective control groups
Note that: example 1 shows a significant difference from before fermentation (P<0.05) a The method comprises the steps of carrying out a first treatment on the surface of the Example 1 shows a significant difference from the reference (P<0.05) b The method comprises the steps of carrying out a first treatment on the surface of the Example 1 shows significant differences from the bid (P<0.05) c 。
As shown in table 3, the total sugar content of the coix seed multi-fungus fermentation broth obtained in example 1 is significantly different from that of the reference and the bid product; the content of total phenols and total acids is obviously different from the content of the total phenols and the total acids before fermentation, the content of the total acids after fermentation and the content of the total acids before fermentation; in addition, the content of lactic acid in the coix seed fermentation liquid after being fermented by lactococcus lactis, streptococcus thermophilus and lactobacillus bulgaricus is obviously increased compared with that before fermentation, and meanwhile, the content of lactic acid is higher than that of a reference content. The increase of the lactic acid and polyphenol content in the fermentation liquor can enhance the whitening effect.
5. The microbial biomass growth trend of the three lactic acid bacteria in example 1 at different fermentation stages was examined, comprising the following steps:
1) Sample treatment: in the process of preparing coix seed multi-fungus fermentation liquor by adopting the method of the embodiment 1, respectively extracting fermentation liquor in fermentation time of 0h, 4h, 8h, 24h, 28h, 32h and 48h, filtering a fermentation liquor sample by two layers of sterile gauze, filtering filtrate by a 100 mu m filter, filtering filtrate by a 40 mu m filter, and centrifuging the obtained filtrate at 15000rpm/min for 5min to obtain a bacterial precipitate sample.
2) Genomic DNA extraction: the procedure was as per OMEGA bacterial DNA extraction kit Bacteria DNA KitD 3550-1.
3) Specific primer design: specific primers were designed based on the variable region sequences of the 16S rRNA genes of the three species using Premier 5, as shown in Table 4.
4) PCR reaction procedure: pre-denatured at 94℃for 4min, denatured at 94℃for 30sec, annealed at 55℃for 30sec, extended at 72℃for 1min (30 cycles), extended at 72℃for 5min.
5) Electrophoresis: the 1% agarose gel was run with 10. Mu.L loading per lane, and the detection results of the run bands are shown in FIG. 2.
TABLE 4 PCR primer sequences for three lactic acid bacteria
As can be seen from the brightness of the electrophoresis bands shown in FIG. 2, the bands of the strains were higher when fermentation was carried out for 4 hours, wherein the amount of the lactococcus lactis among the three strains was the largest, and the fermentation time was continuously increased for the first 24 hours, so that the lactococcus lactis was the dominant strain in the multi-strain fermentation.
6. Zebra fish test: according to the test of the method for measuring the whitening effect of cosmetics by using zebra fish embryo of the group label T/HPCIA 005-2021, the mass concentration of 0.5%, 1% and 2% of the coix seed multi-bacterium fermentation broth obtained in the example 1 is used as a test sample, and blank (Control) and solvent (Arbutin 1.0 mg/mL) are used as a Control group, and the statistical data of the test results of the melanin generation inhibition rate of the zebra fish embryo are shown in figure 3.
As shown in fig. 3, the melanogenesis inhibition rate of the zebra fish embryo treated by the sample of example 1 increases with the increase of the concentration, which indicates that the zebra fish embryo can be obviously whitened, the distribution of melanin particles can be reduced, and the statistically significant difference indicates that the coix seed multi-fungus fermentation broth obtained in example 1 has whitening effect when the mass concentration is 0.5% -2%.
7. The evaluation of the human body freckle removing efficacy comprises the following steps:
1) Sample preparation: the human body efficacy evaluation was performed after preparing the coix seed fermentation broth sample with the mass concentration of 5% in example 1, and the specific formulation is shown in the following table 5.
Table 5-5% mass concentration Coix seed fermentation broth formulation
2) Sample application method: before use, cleaning the face, firstly smearing basic moisturizing water, then taking a proper amount of samples to be uniformly smeared on the product side until the samples are completely absorbed, and using blank matrix samples on the control side; 2 times a day, each time in the morning and evening, and continuously used for 56 days; during the trial, the subject was prohibited from using the formulation that affected the whitening and freckle-removing test; subjects were prohibited from dropping, injecting, orally taking or otherwise ingesting formulations that affected the whitening and freckle-removing test; the subject is mainly subjected to indoor activities, so that long-term exposure to outdoor illumination is avoided; no insolation is possible during the test and the test site should be well-protected from sunlight.
3) Objective quantitative evaluation: after face cleaning for 30min, face photographing was performed using a VISIA-CR face image analyzer, spot analysis was performed in an equally large Area (AOI, area of Interest, r=1 cm) of the spot position using IPP software, and skin ITA ° values and Melanin Index (MI) were detected at the test site using CL400 and MX18, both of which were taken as baseline values (D0), and then product dispensing was performed and the method of use was instructed; subjects were asked to return for and to perform the same index test 14 days (D14), 28 days (D28) and 56 days (D56) after the product; the test of the same subject is completed by the same instrument and the same tester, the front and back of the tested part is consistent between the two testing time points, and the test results are shown in table 6.
Table 6-5% mass concentration of coix seed multi-bacterium fermentation broth freckle-removing test results in example 1 (n=33)
Note that: significant differences (P < 0.01) compared to D0.
4) Determination criteria: human body efficacy evaluation is carried out according to the second method of human body open use freckle removing and whitening efficacy test method issued by the national drug administration 2021. The test group (side) uses any visit point instrument before and after the product to test or analyze the change result of any parameter in the relevant parameters obviously (P < 0.05), or the test value result after using the sample is obviously better than the control group (side) result (P < 0.05), then the test product is identified to have the freckle removing and whitening effects, otherwise, the test product is identified to have no damp-heat freckle removing and whitening effects.
As shown in the results of table 6, after the subject uses 5% concentration of coix seed multi-bacterium fermentation broth, the skin ITA ° value has an ascending trend at each return visit time point (D14, D28 and D56), D28 has a significant difference from the baseline value, and D56 and D28 have no significant difference, which indicates that the whitening effect can be maintained after 56 days of use without blackening; the mottle MI values were progressively lower at each return visit time point (D14, D28, and D56), with significant differences between D28 and D56 and baseline values, and no significant differences between D14 and baseline and control sides; according to the method for testing the freckle-removing and whitening effects of cosmetics in the second method, 3.8 of the method for testing the freckle-removing and whitening effects of human body open use can judge that 5% of coix seed multi-fungus fermentation liquor has the freckle-removing and whitening effects.
The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present invention, and are not to be construed as limiting the scope of the invention. It should be noted that any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art without departing from the spirit and principles of the present invention are intended to be included in the scope of the present invention.
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Claims (9)
1. A preparation method of coix seed multi-bacterium fermentation broth is characterized by adopting mixed strains to ferment coix seeds, wherein the mixed strains are lactococcus lactis, lactobacillus bulgaricus and streptococcus thermophilus; the method comprises the following steps:
(1) Preparing strains;
(2) Saccharifying Coicis semen to obtain Coicis semen fermentation medium substrate;
(3) Inoculating the lactococcus lactis, the streptococcus thermophilus and the lactobacillus bulgaricus obtained in the step (1) into a coix seed fermentation medium, and fermenting to obtain a coix seed multi-bacterium fermentation liquid primary product;
(4) Adding lysozyme into the primary product of the coix seed multi-bacterium fermentation liquid, stirring and homogenizing, and centrifugally collecting the supernatant; filtering to obtain coix seed multi-fungus fermentation liquor.
2. The method for preparing coix seed multi-bacterium fermentation broth according to claim 1, wherein the adding amount of lactococcus lactis, lactobacillus bulgaricus and streptococcus thermophilus is 0.2-2 wt% in the fermentation process, the concentration of the lactococcus lactis bacteria solution is 8-10g/L, and the bacterial contents of the lactobacillus bulgaricus and the streptococcus thermophilus are 100 hundred million CFU/g.
3. The method for preparing a coix seed multi-fungus fermentation broth according to claim 2, wherein in the step (2), coix seed powder is mixed with water and then CaCl is added 2 Gelatinizing, cooling to 75-80deg.C, adding alpha-amylase, and liquefying at constant temperature; adjusting pH to 4-4.2, cooling to 60deg.C, adding saccharifying enzyme, maintaining at 75-80deg.C for saccharification, and sterilizing to obtain Coicis semen fermentation medium.
4. A method for preparing a coix seed multi-fungus fermentation broth according to claim 3, wherein in the step (2), caCl is added 2 The addition amount of the catalyst is 1-5% of the mass of the substrate, and the gelatinization time is 0.5-2.5h; the enzyme dosage of alpha-amylase is 10-50U/g of Coicis semen powder, and the liquefying time is 1-5h; the enzyme dosage of saccharifying enzyme is 100-500U/g Coicis semen powder, and saccharifying time is 1-5 hr.
5. The method for preparing a coix seed multi-fungus fermentation broth according to claim 2, wherein in the step (4), the lysozyme is added in an amount of 0.2-2g/L.
6. The method of claim 2, wherein in the step (4), the stirring time is 1-5 hours, the homogenization time is 2-8 times, and the filtration is performed by passing through 0.8 μm and 0.22 μm membranes in sequence.
7. The method for preparing coix seed multi-bacterium fermentation broth according to claim 1, wherein the lactococcus lactis is lactococcus lactis MG1363.
8. A coix seed multi-fungus fermentation broth obtained by the preparation method of the coix seed multi-fungus fermentation broth according to any one of claims 1 to 7.
9. Use of the coix seed multi-fungus fermentation broth according to claim 8 in cosmetics.
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