A method for preparing lactobacillus fermentation products by two-step combined fermentation of prosopis

Abstract

The present invention discloses a method for preparing lactobacillus fermentation products by two-step combined fermentation of prosopis. The method of the present invention uses organic prosopis powder and arabinose as culture medium, and prepares lactobacillus fermentation products by two-step combined fermentation of prosopis by Lactobacillus buchneri and Lactobacillus plantarum. The present invention preferentially utilizes Lactobacillus buchneri to pre-ferment prosopis powder to promote the release of flavonoids, proteins, alkaloids, and short-chain fatty acids from plant fiber structures, and utilizes Lactobacillus plantarum to further express and modify small molecule active peptides by secondary fermentation. The two-step combination of homotypic fermentation and heterotypic fermentation is adopted to effectively improve the aerobic stability and bioavailability of the fermentation process, and the obtained lactobacillus fermentation product exhibits excellent free radical quenching ability and biological activity against peroxide damage.

Description

Technical Field

[0001] The invention relates to a method for preparing lactobacillus fermentation products by two-step combined fermentation of prosopis, and belongs to the technical field of preparing cosmetic raw materials by fermentation of probiotics. Background Art

[0002] Lactobacillus is a type of probiotic that can metabolize through the glycolysis pathway (EMP) to produce active factors such as lactic acid, short-chain fatty acids, short peptides, amino acids, etc. Lactobacillus fermentation products (LACTOBACILLUS FERMENT) contain bacterial lysis and all active ingredients synthesized during the fermentation process. It can provide rich prebiotics for the skin and has good antioxidant and anti-aging effects. It has been included in the "Catalogue of Used Cosmetic Raw Materials" (2021 edition), INCI number 02162.

[0003] Mesquite is a thorny leguminous shrub, which is called the "Tree of Life" by the American people because of its outstanding nutritional value. Mesquite pods are usually ground into powder, which is rich in amino acids, flavonoids, plant fiber and protein, which is very helpful for the body to build an active antioxidant immune system. However, the traditional extraction process has a poor effect on the degradation of lignin and cellulose structure of leguminous plants, and the content of probiotic flavonoid peptides in the product is generally low.

[0004] Studies have shown that fermentation of beans by lactic acid bacteria such as bifidobacterium, lactobacillus, and lactobacillus plantarum can promote the conversion of isoflavone monomers from glycoside types to aglycone types that are easier for the human body to absorb and have more antioxidant biological activity. In particular, lactobacillus plantarum also has a strong protease secretion ability, and can modify undesirable groups of small peptides directly or indirectly produced during the fermentation process through processing methods such as rearrangement and transfer, so that the small peptides have higher application value. However, lactobacillus plantarum usually has no ability to degrade fiber and has a weak ability to directly utilize plant matrix. To solve this problem, in existing patents, lactobacillus fermented plant fiber is basically pretreated by enzymatic hydrolysis, but the enzymatic hydrolysis process conditions are strictly controlled, the cost is high, and the fermentation substrate still needs to be loosened or pre-treated by adding water for homogenization.

[0005] At this stage, in the fermented skin care market segment, ingredients such as lactobacillus fermentation products, bifid yeast fermentation products, and yeast fermentation products are among the top ten microecological skin care ingredients that consumers are concerned about. The use of fermentation products has become the mainstream of fermented skin care products. The preparation of probiotic fermentation products based on modern industrial microbial fermentation regulation technology and their application in cosmetics has long-term value and significance. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a lactobacillus fermentation product by two-step combined fermentation of prosopis, in view of the deficiencies in the prior art. The method uses organic prosopis powder and arabinose as a culture medium, and prepares the lactobacillus fermentation product by two-step combined fermentation of Lactobacillus buchneri and Lactobacillus plantarum. The two-step combination of homotypic fermentation and heterotypic fermentation is adopted to effectively improve the aerobic stability and bioavailability of the fermentation process, and the obtained lactobacillus fermentation product exhibits excellent free radical quenching ability and biological activity against peroxide damage.

[0007] In order to achieve the above object, the present invention provides a method for preparing a lactobacillus fermentation product by two-step combined fermentation of prosopis, comprising the following steps:

[0008] Step 1. Activating the strains: Lactobacillus buchneri and Lactobacillus plantarum are inoculated into the seed culture medium for activation culture to obtain Lactobacillus buchneri seed solution and Lactobacillus plantarum seed solution respectively; wherein the composition of the seed culture medium is: 2.00wt% glucose, 2.00wt% soy peptone, 0.20wt% potassium dihydrogen phosphate, 0.30wt% sodium chloride, 0.06wt% magnesium sulfate and the balance water;

[0009] Step 2. Preparation of fermentation medium: weigh arabinose and organic prosopis powder and add them into water, mix thoroughly and adjust the pH to 4.0-5.0, sterilize with steam and cool to room temperature;

[0010] Step 3. Two-step fermentation treatment: the fermentation medium prepared in step 2 is inoculated with the Lactobacillus buchneri seed solution obtained in step 1, and fermentation is carried out, the pH of the obtained fermentation solution is adjusted to 6.0-7.0, and the obtained fermentation solution is cooled to room temperature after steam sterilization, and then inoculated with the Lactobacillus plantarum seed solution obtained in step 1, and fermentation is continued;

[0011] Step 4. Preparation of fermentation product: The fermentation broth obtained through two-step fermentation treatment in step 4 is centrifuged and microfiltered in sequence, the supernatant is collected, and hexylene glycol, p-hydroxyacetophenone and ethylhexylglycerin are added to obtain the lactobacillus fermentation product.

[0012] Preferably, the Lactobacillus buchneri in step 1 is Lactobacillus buchneri MF060 with a preservation number of CGMCC No.19125 (i.e., the strain disclosed in the patent with publication number CN113288847A and invention name "A method for preparing a fermented extract of white aconite and its application").

[0013] Preferably, the amount of arabinose added to the fermentation medium in step 2 is 0.10wt%-2.00wt%, and the amount of organic prosopis powder added is 1.00wt%-3.00wt%.

[0014] Preferably, the Lactobacillus buchneri seed solution and the Lactobacillus plantarum seed solution in step 3 are diluted to a bacterial concentration of 0.5 to 2×10 9 cfu / mL.

[0015] Preferably, the inoculation amount of the Lactobacillus buchneri seed solution in step 3 is 3.00-5.00wt%, and the conditions for inoculating the Lactobacillus buchneri seed solution for fermentation and culture are: culturing in a shaking table at 37°C for 48-72h; the inoculation amount of the Lactobacillus plantarum seed solution is 1.00-2.00wt%, and the conditions for inoculating the Lactobacillus plantarum seed solution for fermentation and culture are: static culture at 30°C for 24-48h.

[0016] Preferably, the amounts of hexylene glycol, p-hydroxyacetophenone and ethylhexylglycerol added in step 4 are 1 wt%, 0.3 wt% and 0.2 wt% respectively.

[0017] The present invention also provides a lactobacillus fermentation product prepared by the above two-step combined fermentation method for preparing a lactobacillus fermentation product. The obtained lactobacillus fermentation product has a high content of flavonoids and polypeptides, and the obtained lactobacillus fermentation product has a high DPPH clearance rate and excellent ability to resist hydrogen peroxide damage, and therefore, can be used to prepare functional cosmetics.

[0018] The present invention also provides application of the lactobacillus fermentation product in preparing cosmetics.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention preferentially utilizes Lactobacillus buchneri to pre-ferment and treat prosopis powder to promote the release of flavonoids, proteins, alkaloids, and short-chain fatty acids from plant fiber structures, and utilizes Lactobacillus plantarum secondary fermentation to further express and modify small molecule active peptides. Compared with the prior art, the present invention has the following advantages: 1) the content of flavonoids and peptide active substances in lactobacillus fermentation products and their biological activities are specifically improved through microbial fermentation metabolism regulation; 2) the obtained lactobacillus fermentation products are rich in a variety of plant- and microbial-source prebiotics and alkaloid components, exhibit excellent free radical quenching and peroxide damage resistance capabilities, and can be widely used in moisturizing water, skin creams, and essences with antioxidant, radiation-resistant, sunscreen, and other properties; 3) the two-step combination of homotypic fermentation and heterotypic fermentation greatly improves the aerobic stability and bioavailability of the fermentation process, accelerates the dissolution of active factors, and reduces energy consumption at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the test results of the ability of lactobacillus fermentation products or prosopis przewalskii extracts obtained by different methods in Examples 1-3 and Comparative Examples 1-3 to resist damage from hydrogen peroxide. DETAILED DESCRIPTION

[0022] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0023] The Lactobacillus buchneri strain used in the following examples is Lactobacillus buchneri MF060 with a deposit number of CGMCC No.19125 (i.e., the strain disclosed in the patent with publication number CN113288847A and invention name "A method for preparing a fermented extract of white aconite and its application"); the Lactobacillus plantarum used is a conventional commercially available strain.

[0024] Example 1

[0025] A method for preparing a lactobacillus fermentation product by two-step combined fermentation of prosopis, comprising the following steps:

[0026] (1) Bacteria activation:

[0027] Accurately weigh 2.00wt% glucose, 2.00wt% soy peptone, 0.20wt% potassium dihydrogen phosphate, 0.30wt% sodium chloride, 0.06wt% magnesium sulfate and the rest of water, fully dissolve and adjust the pH to 6.0±0.2, sterilize with high pressure steam at 121℃ for 15min, then cool to room temperature, inoculate Lactobacillus buchneri CGMCCNo.19125 and Lactobacillus plantarum for activation and culture for 48h, dilute the obtained seed solution to a concentration of 1×10 9 cfu / mL.

[0028] (2) Lactobacillus buchneri fermentation treatment:

[0029] Accurately weigh 2.00wt% arabinose and 3.00wt% organic prosopis powder, make up the rest with water, mix thoroughly and adjust the pH to 4.0-5.0, sterilize with high-pressure steam at 121°C for 15 min, then cool to room temperature, inoculate 5.00wt% of Lactobacillus buchneri seed liquid, and culture at 37°C, 200rpm shaking table for 72h.

[0030] (3) Lactobacillus plantarum fermentation treatment:

[0031] The pH value of the fermentation liquid was adjusted to 6.0-7.0, sterilized with high pressure steam at 121° C. for 15 min, cooled to room temperature, added with 2.00 wt% Lactobacillus plantarum seed liquid, and cultured at 30° C. for 48 h.

[0032] (4) Preparation of fermentation products:

[0033] The fermentation broth was centrifuged at 5000 rpm for 10 min to remove the fermentation residue, and the bacteria were removed by microfiltration with a 0.22 μm filter membrane. The supernatant was collected and added with 1 wt % hexylene glycol, 0.3 wt % p-hydroxyacetophenone, and 0.2 wt % ethylhexylglycerol to obtain a lactobacillus fermentation product.

[0034] Example 2

[0035] A method for preparing a lactobacillus fermentation product by two-step combined fermentation of prosopis, comprising the following steps:

[0036] (1) Bacteria activation:

[0037] Accurately weigh 2.00wt% glucose, 2.00wt% soy peptone, 0.20wt% potassium dihydrogen phosphate, 0.30wt% sodium chloride, 0.06wt% magnesium sulfate and the rest of water, fully dissolve and adjust the pH to 6.0±0.2, sterilize with high pressure steam at 121℃ for 15min, then cool to room temperature, inoculate Lactobacillus buchneri CGMCCNo.19125 and Lactobacillus plantarum for activation and culture for 48h, dilute the obtained seed solution to a concentration of 1×10 9 cfu / mL.

[0038] (2) Lactobacillus buchneri fermentation treatment:

[0039] Accurately weigh 1.00wt% arabinose and 2.00wt% organic prosopis powder, make up the rest with water, mix thoroughly and adjust the pH to 4.0-5.0, sterilize with high-pressure steam at 121°C for 15 min, then cool to room temperature, inoculate with 3.00wt%-5.00wt% Lactobacillus buchneri seed liquid, and culture at 37°C, 200rpm shaking incubator for 36h.

[0040] (3) Lactobacillus plantarum fermentation treatment:

[0041] The pH value of the fermentation liquid was adjusted to 6.0-7.0, sterilized with high pressure steam at 121° C. for 15 min, cooled to room temperature, added with 1.50 wt % Lactobacillus plantarum seed liquid, and cultured at 30° C. for 36 h.

[0042] (4) Preparation of fermentation products:

[0043] The fermentation broth was centrifuged at 5000 rpm for 10 min to remove the fermentation residue, and the bacteria were removed by microfiltration with a 0.22 μm filter membrane. The supernatant was collected and added with 1 wt % hexylene glycol, 0.3 wt % p-hydroxyacetophenone, and 0.2 wt % ethylhexylglycerol to obtain a lactobacillus fermentation product.

[0044] Example 3

[0045] A method for preparing a lactobacillus fermentation product by two-step combined fermentation of prosopis, comprising the following steps:

[0046] (1) Bacteria activation:

[0047] Accurately weigh 2.00wt% glucose, 2.00wt% soy peptone, 0.20wt% potassium dihydrogen phosphate, 0.30wt% sodium chloride, 0.06wt% magnesium sulfate and the rest of water, fully dissolve and adjust the pH to 6.0±0.2, sterilize with high pressure steam at 121℃ for 15min, then cool to room temperature, inoculate Lactobacillus buchneri CGMCCNo.19125 and Lactobacillus plantarum for activation and culture for 48h, dilute the obtained seed solution to a concentration of 1×10 9 cfu / mL.

[0048] (2) Lactobacillus buchneri fermentation treatment:

[0049] Accurately weigh 0.10wt% arabinose and 1.00wt% organic prosopis powder, make up the rest with water, mix thoroughly and adjust the pH to 4.0-5.0, sterilize with high-pressure steam at 121°C for 15 min, then cool to room temperature, inoculate 3.00wt% of Lactobacillus buchneri seed liquid, and culture at 37°C, 200rpm shaking for 48h.

[0050] (3) Lactobacillus plantarum fermentation treatment:

[0051] The pH value of the fermentation liquid was adjusted to 6.0-7.0, sterilized with high pressure steam at 121° C. for 15 min, cooled to room temperature, added with 1.00 wt % Lactobacillus plantarum seed liquid, and cultured at 30° C. for 24 h.

[0052] (4) Preparation of fermentation products:

[0053] The fermentation broth was centrifuged at 5000 rpm for 10 min to remove the fermentation residue, and the bacteria were removed by microfiltration with a 0.22 μm filter membrane. The supernatant was collected and added with 1 wt % hexylene glycol, 0.3 wt % p-hydroxyacetophenone, and 0.2 wt % ethylhexylglycerol to obtain a lactobacillus fermentation product.

[0054] Comparative Example 1

[0055] Accurately weigh 2.00wt% arabinose and 3.00wt% organic prosopis powder, make up the rest with water, mix thoroughly and adjust the pH to 4.0-5.0, sterilize with high pressure steam at 121°C for 15 min, cool to room temperature, centrifuge at 5000rpm for 10 min to remove plant residues, collect the supernatant through a 0.22μm microporous filter membrane, add 1wt% hexylene glycol, 0.3wt% p-hydroxyacetophenone and 0.2wt% ethylhexylglycerin to obtain a prosopis extract.

[0056] Comparative Example 2

[0057] A method for preparing a lactobacillus fermentation product by fermenting prosopis, the specific steps are the same as those of implementation 1, except that the fermentation treatment of Lactobacillus buchneri in step 2) is omitted, that is, 2.00wt% of arabinose and 3.00wt% of organic prosopis powder are accurately weighed, the rest is supplemented with water, the pH is adjusted to 4.0-5.0 after fully mixing, the product is sterilized with high-pressure steam at 121°C for 15 minutes, cooled to room temperature, and then directly inoculated with 2.00wt% of Lactobacillus plantarum seed liquid, and cultured at 30°C for 48 hours.

[0058] Comparative Example 3

[0059] A method for preparing a lactobacillus fermentation product by fermenting prosopis przewalskii, wherein the specific steps are the same as those in implementation 1, except that step 3) the Lactobacillus plantarum fermentation treatment is omitted.

[0060] The samples obtained from the above Examples 1 to 3 and Comparative Examples 1 to 3 were tested as follows:

[0061] 1. Determination of flavonoid content:

[0062] Prepare 0.6g / L rutin standard solution with 60% methanol aqueous solution, take 2.5mL of standard solution with different dilution multiples, add 400μL 5% NaNO2 and let stand for 5min, add 400μL 10% Al(NO3)3 and let stand for 6min, add 600μL 10% NaOH and let stand for 6min, add 30% ethanol to 10mL and let stand for 10min, and measure the absorbance at 510nm. Draw a standard curve with the concentration of standard solution as the horizontal axis and the absorbance value as the vertical axis. Substitute the absorbance value of the test solution into the standard curve according to the same steps to obtain the flavonoid content.

[0063] 2. DPPH clearance determination:

[0064] Refer to GB / T 39100-2020 "Determination of Antioxidant Activity of Peptides"

[0065] 3. Determination of the ability to resist hydrogen peroxide damage:

[0066] HaCat cells in the logarithmic growth phase were collected and seeded in 96-well plates. The density of HaCat cells was adjusted to 1×10 5 Each well was cultured for 24 hours, and the culture medium was discarded after adherence. 100 μL of DMEM culture medium containing different concentrations of lactobacillus fermentation products was added, with 6 replicates per group. After 24 hours of drug action, 0.2 mM hydrogen peroxide was added for artificial damage treatment. The DMEM culture medium without sample and hydrogen peroxide was used as the blank control group, and the DMEM culture medium without sample and containing 0.2 mM hydrogen peroxide was used as the positive control group. After continuing to culture for 24 hours, 10 μL of CCK-8 solution was added to each well, incubated for 2 hours, and the absorbance of each well was measured at 450 nm to calculate the cell proliferation rate.

[0067] Table 1 shows the test results of flavonoids, polypeptide contents and DPPH free radical scavenging rate in samples obtained by different methods in Examples 1-3 and Comparative Examples 1-3. Figure 1 These are the test results of the ability of samples obtained by different methods in Examples 1-3 and Comparative Examples 1-3 to resist damage from hydrogen peroxide.

[0068] Table 1 Detection results of flavonoids and polypeptide contents in samples

[0069] Experimental Group Flavonoids (g / L) Peptide (g / L) DPPH clearance rate (%) Example 1 1.24 3.15 92.14％ Example 2 1.03 2.74 85.32％ Example 3 0.98 2.31 73.98％ Comparative Example 1 0.06 0.58 12.15％ Comparative Example 2 0.14 1.97 32.19％ Comparative Example 3 0.46 1.06 45.76％

[0070] The above results show that the flavonoids and polypeptide contents in the lactobacillus fermentation product prepared by the method of preparing lactobacillus fermentation product by two-step combined fermentation of prosopis are higher than those in the prosopis extract prepared by the traditional water extraction method or the lactobacillus fermentation product prepared by one-step fermentation, and the DPPH clearance rate and the ability to resist hydrogen peroxide damage are both optimal.

[0071] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing lactobacillus fermentation products by two-step combined fermentation of prosopis, characterized in that: The following steps are involved: Step 1. Activation of strains: Lactobacillus buchneri and Lactobacillus plantarum are inoculated into seed culture medium for activation culture to obtain Lactobacillus buchneri seed solution and Lactobacillus plantarum seed solution respectively; wherein the composition of the seed culture medium is: 2.00 wt% glucose, 2.00 wt% soy peptone, 0.20 wt% potassium dihydrogen phosphate, 0.30 wt% sodium chloride, 0.06 wt% magnesium sulfate and the balance water; Step 2. Preparation of fermentation medium: weigh arabinose and organic prosopis powder and add them into water, mix thoroughly and adjust the pH to 4.0-5.0, sterilize with steam and cool to room temperature; Step 3. Two-step fermentation treatment: the fermentation medium prepared in step 2 is inoculated with the Lactobacillus buchneri seed solution obtained in step 1, and fermentation is carried out, the pH of the obtained fermentation solution is adjusted to 6.0-7.0, and the obtained fermentation solution is cooled to room temperature after steam sterilization, and then inoculated with the Lactobacillus plantarum seed solution obtained in step 1, and fermentation is continued; Step 4. Preparation of fermentation product: centrifuging and microfiltering the fermentation broth obtained by two-step fermentation treatment in step 4 in sequence, collecting the supernatant, adding hexylene glycol, p-hydroxyacetophenone and ethylhexylglycerol, and obtaining a lactobacillus fermentation product; The Lactobacillus buchneri in step 1 is Lactobacillus buchneri MF060 with a preservation number of CGMCC No.19125.

2. The method for preparing lactobacillus fermentation products by two-step combined fermentation of prosopis as claimed in claim 1, characterized in that: The amount of arabinose added to the fermentation medium in step 2 is 0.10 wt%-2.00 wt%, and the amount of organic prosopis powder added is 1.00 wt%-3.00 wt%.

3. The method for preparing lactobacillus fermentation products by two-step combined fermentation of prosopis przewalskii according to claim 1, characterized in that: In step 3, the Lactobacillus buchneri seed solution and the Lactobacillus plantarum seed solution are diluted to a bacterial concentration of 0.5 to 2×10 9 cfu / mL.

4. The method for preparing lactobacillus fermentation products by two-step combined fermentation of prosopis as claimed in claim 1, characterized in that: In the step 3, the inoculation amount of the Lactobacillus buchneri seed solution is 3.00-5.00 wt%, and the conditions for inoculating the Lactobacillus buchneri seed solution for fermentation and culture are: culturing in a shaking table at 37° C. for 48-72 h; the inoculation amount of the Lactobacillus plantarum seed solution is 1.00-2.00 wt%, and the conditions for inoculating the Lactobacillus plantarum seed solution for fermentation and culture are: static culture at 30° C. for 24-48 h.

5. The method for preparing lactobacillus fermentation products by two-step combined fermentation of prosopis as claimed in claim 1, characterized in that: The amounts of hexylene glycol, p-hydroxyacetophenone and ethylhexylglycerin added in step 4 are 1 wt %, 0.3 wt % and 0.2 wt % respectively.

6. The lactobacillus fermentation product obtained by the method for preparing lactobacillus fermentation product by two-step combined fermentation of prosopis przewalskii according to any one of claims 1 to 5.

7. Use of the lactobacillus fermentation product according to claim 6 in the preparation of cosmetics.

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