Fermentation broth and fermentation lysate of Lactobacillus plantarum, and preparation method and application thereof

Abstract

The present invention provides a Lactobacillus plantarum fermentation broth, a fermentation lysate and their preparation methods and applications. The present invention uses Lactobacillus plantarum to ferment pomegranate peel, and the obtained fermentation broth and fermentation lysate have superior antioxidant and whitening effects. There is a synergistic effect between Lactobacillus plantarum and pomegranate peel in the fermentation system. The active ingredients and functions of the two products have been greatly improved compared with traditional products, and it is expected to be further developed into a functional raw material for cosmetics. It can be used to prepare an antioxidant and whitening composition, and the preparation method is simple and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Lactobacillus plantarum fermentation broth, a fermentation lysate, and a preparation method and application thereof. Background Art

[0002] In the past 10 years, some domestic and foreign brand cosmetics have begun to add lactic acid bacteria lysates, and there have also been some patented products of lysates. However, due to the delicacy of lactic acid bacteria, which require various amino acids and growth factors, the culture media are mostly limited to MRS and dairy products.

[0003] China ranks first in the world in pomegranate production. However, the deep processing of the pomegranate industry lags seriously. In particular, pomegranate peels account for about 40% of the fresh weight of pomegranates, but they are often discarded or only sold and utilized as cheap traditional Chinese medicine, without fully realizing their value. Numerous studies have shown that pomegranate peels contain rich active substances, such as ellagic acid polyphenols, flavonoids, organic acids, polysaccharides, and various trace elements. In particular, ellagic acid has strong antioxidant properties and is considered by some experts to be the substance with the strongest anti-aging effect discovered so far. At present, domestic pomegranate deep processing-related skin care products mainly utilize pomegranate fruits and rarely utilize pomegranate peels. Summary of the Invention

[0004] The purpose of the present invention is to provide a Lactobacillus plantarum fermentation broth, a fermentation lysate, and a preparation method and application thereof in view of the above deficiencies of the prior art.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The first object of the present invention is to provide a preparation method of a Lactobacillus plantarum fermentation broth, comprising the following steps:

[0007] Step S1: Dry the washed pomegranate peels, cut them into blocks, add distilled water according to a preset solid-liquid ratio, adjust to a preset pH value, and sterilize to obtain a pomegranate peel fermentation medium;

[0008] Step S2: Quantitatively inoculate Lactobacillus plantarum into the pomegranate peel fermentation medium obtained in Step S1 for fermentation and proliferation;

[0009] Step S3: After Step S2 is completed, first filter out the pomegranate peel residues, then centrifuge and separate the proliferated Lactobacillus plantarum, and take the precipitate to obtain Lactobacillus plantarum cells; take the supernatant to obtain the Lactobacillus plantarum fermentation broth.

[0010] Further, in Step S1, the pomegranate peels are cut into small pieces of 1×1 cm, the pomegranate peel mass concentration of the pomegranate peel fermentation medium is 3 g / mL to 7 g / mL, the preset pH value is 2.5 to 4.5, the sterilization temperature is 115 to 120 °C, and the sterilization time is 10 to 15 min.

[0011] Further, in step S2, the process of Lactobacillus plantarum fermentation and proliferation is as follows: Take Lactobacillus plantarum and inoculate it into the pomegranate peel fermentation medium. Incubate at 30 - 37 °C for 48 - 72 hours, and the volume ratio of Lactobacillus plantarum to the pomegranate peel fermentation medium is 4% - 8%.

[0012] Further, in step S3, the centrifugation speed is 5000 - 6000 rpm, and the centrifugation time is 5 - 15 min.

[0013] The second object of the present invention is to provide a Lactobacillus plantarum fermentation broth, which is prepared by the above - mentioned preparation method.

[0014] The third object of the present invention is to provide a lysate of Lactobacillus plantarum fermentation, which is obtained by lysing Lactobacillus plantarum cells, and the Lactobacillus plantarum cells are prepared by the above - mentioned preparation method.

[0015] The fourth object of the present invention is to provide a preparation method of the above - mentioned lysate of Lactobacillus plantarum fermentation. The Lactobacillus plantarum cells are lysed by ice - bath ultrasonic disruption, and the lysed Lactobacillus plantarum cells are centrifuged and separated. The supernatant is taken to obtain the lysate of Lactobacillus plantarum fermentation; the ultrasonic power is 250 - 300 W, with an intermittent time of 1 s for every 2 s of working, and the disruption time is 25 - 30 min; the parameters of the centrifugal separation are: the centrifugation speed is 5000 - 6000 rpm, and the centrifugation time is 10 - 15 min.

[0016] The fifth object of the present invention is to provide an antioxidant and whitening composition, which comprises the above - mentioned Lactobacillus plantarum fermentation broth or comprises the above - mentioned lysate of Lactobacillus plantarum fermentation.

[0017] The sixth object of the present invention is the application of the above - mentioned antioxidant and whitening composition in the preparation of daily cosmetics.

[0018] Compared with the prior art, the beneficial effects brought by the technical solution provided by the present invention are as follows:

[0019] The present invention uses Lactobacillus plantarum to ferment pomegranate peel, and the obtained fermentation broth and lysate of fermentation have excellent antioxidant and whitening effects. In the fermentation system, Lactobacillus plantarum and pomegranate peel have a synergistic effect. The active ingredients and functions of the two products are greatly improved compared with traditional products. It is expected to be further developed into a functional raw material for cosmetics, can be used to prepare an antioxidant and whitening composition, and the preparation method is simple and suitable for industrial production. Description of the Drawings

[0020] Figure 1 It is the colony map of Lactobacillus plantarum B;

[0021] Figure 2 It is the Gram staining microscopic examination diagram of Lactobacillus plantarum B. Specific implementation manners

[0022] To make the objectives, technical solutions and advantages of the present invention clearer, the specific implementation manners of the present invention will be further described in detail below in combination with specific embodiments and the accompanying drawings.

[0023] Example 1 is the information of two sources of Lactobacillus plantarum used in the present invention. Lactobacillus plantarum A: CICC 20038 Beijing Center for Biological Preservation; Lactobacillus plantarum B: CCTCC NO: AB2022276 China Center for Type Culture Collection.

[0024] Examples 2 - 7 are the fermentation broth of Lactobacillus plantarum prepared by fermenting pomegranate peel with Lactobacillus plantarum;

[0025] Examples 8 - 13 are the fermentation lysate products of Lactobacillus plantarum prepared by fermenting pomegranate peel with Lactobacillus plantarum;

[0026] Comparative Example 1 is the fermentation lysate product of Lactobacillus plantarum proliferated in MRS medium;

[0027] Formulation of MRS medium: peptone 10.0 g, beef powder 8.0 g, yeast powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 g, distilled water 1000 mL, pH value 5.7 ± 0.2, sterilized at 121 °C for 15 min.

[0028] Comparative Example 2 is the preparation of pomegranate peel decoction;

[0029] Comparative Example 3 is the preparation of pomegranate peel ultrasonic extract;

[0030] The detection methods used in the present invention are as follows:

[0031] (1) Determination of DPPH free radical (DPPH·) scavenging ability

[0032] It is carried out with reference to T / SHRH 006 - 2018 "Cosmetics - Experimental Method for Free Radical (DPPH) Scavenging".

[0033] Accurately weigh 12 mg of DPPH reagent and dissolve it in 100 mL of 95% ethanol to obtain a 0.12 mg / mL DPPH ethanol solution. Place it in a brown bottle and store it in the dark for later use.

[0034] Dilute the fermentation broth and cell lysate to multiple concentrations. Use 10 mL test tubes to set up sample tubes (T), sample blanks (T 0 ), DPPH tubes (C), and solvent blanks (C 0 ). Add 1 mL of the sample solution with the same concentration to each of the sample tubes (T) and sample blanks (T 0 ). Add water to all test tubes (T, T 0 , C, C 0 ) to make up to 3 mL, and mix well. Add 1 mL of DPPH ethanol solution to the sample tubes (T) and DPPH tubes (C). Replace the sample blank (T 0 ) and the solvent blank (C 0 ) with 95% ethanol, shake gently, and let stand at room temperature for 5 minutes. Transfer each reaction solution into a cuvette and measure the absorbance at 517 nm.

[0035] Measure each concentration 3 times and calculate the average value. Calculate according to the following formula:

[0036] DPPH scavenging rate (%) = [1 - (T - T 0 ) / (C - C 0 )] × 100%

[0037] (2) Determination of hydroxyl radical (·OH) scavenging ability

[0038] Take 1.0 mL of the diluted sample solution, add 1 mL of 6 mmol / L FeSO4·7H 2 O solution and 1 mL of 6 mmol / L H 2 O 2 solution, shake well, let stand for 10 min, then add 1 mL of 6 mmol / L salicylic acid solution, shake well and react in a 37 °C water bath for 30 min, and measure the absorbance of each tube at a wavelength of 520 nm as A 样品 . Replace salicylic acid with distilled water and measure the absorbance again according to the above method as A 对照 of the control group. Replace the sample solution with distilled water and measure the absorbance again according to the above method as A 空白 of the blank group. Repeat three times and calculate the average value. The scavenging rate calculation formula is as follows:

[0039] Hydroxyl radical scavenging ability (%) = [1 - (A 样品 - A 对照 ) / A 空白 × 100%

[0040] (3) Determination of tyrosinase inhibition rate

[0041] Performed according to T / GDCA 006-2021 "Test Method for Inhibitory Activity of Cosmetic Ingredients on Tyrosinase Activity (In Vitro Method)".

[0042] Add the sample solution / solvent and 20 μL of tyrosinase solution to each well in sequence, mix well, incubate in a constant temperature environment at 37 °C for 10 min, then add L-tyrosine solution and PBS buffer to each well in sequence. After mixing and reacting at 37 °C for 30 min ± 5 s, immediately place it in an enzyme-linked immunosorbent assay (ELISA) reader and measure at a wavelength of 475 nm. Note: The time from adding the tyrosinase solution to measuring the absorbance in each well should be consistent (5 min ± 5 s).

[0043] The inhibitory rate of the lysate tyrosinase activity is calculated according to the following formula:

[0044]

[0045] In the formula: Y——Inhibitory rate of tyrosinase activity;

[0046] A d ——Absorbance of the sample reaction well;

[0047] A c ——Absorbance of the sample background well;

[0048] A b ——Average value of the absorbance of the solvent reaction wells;

[0049] A a ——Average value of the absorbance of the solvent background wells.

[0050] If the standard deviation (SD) value between the inhibitory rates of each group of parallel wells is ≤ 15%, the test parallelism is considered effective.

[0051] (4) Determination of polysaccharide content

[0052] The phenol-sulfuric acid method was adopted. Prepare a standard glucose solution of 100 μg / mL, add 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL to the colorimetric tubes, and make up to 2 mL with distilled water. Then add 1.0 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid to each colorimetric tube and shake well. React at room temperature for 20 min and then cool in cold water. Use distilled water instead of the standard glucose solution as a blank control. Measure its absorbance value at a wavelength of 490 nm with a 722 spectrophotometer. Use the concentration of the standard glucose solution as the abscissa and the absorbance value as the ordinate to make a standard curve.

[0053] The sampling solution was added with 1 / 3 volume of Sevag reagent (chloroform:n-butanol = 4:1). After shaking for 30 min, it was centrifuged at 12000 r / min for 5 min. The upper aqueous solution was collected and the above operation was repeated once. Subsequently, 90% ethanol was added at a volume ratio of 1:3, precipitated at 4 °C for 15 h, centrifuged at 10000 r / min for 15 min. The precipitate was taken to volatilize the ethanol, dissolved in 2 mL of distilled water, 0.5 mL was taken and made up to 2 mL with distilled water, and the polysaccharide concentration was determined by the phenol-sulfuric acid method.

[0054] (5) Determination of polyphenol content

[0055] The Folin-Ciocalteu method was adopted. Exactly weigh 25 mg of gallic acid standard sample, dissolve it with water and make up to 250 mL to obtain a reference standard solution of 0.1 mg / mL. Pipette 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mL of the reference sample solution into stoppered test tubes, add distilled water to 10.2 mL, then add 0.3 mL of 2N Folin-Ciocalteu reagent, shake well and let stand for 5 min, add 1 mL of 15% Na 2 CO 3 solution, mix well and react in the dark at room temperature for 2 h, measure the absorbance at 746 nm, and draw the standard curve.

[0056] Take a stoppered test tube, first add 10 mL of distilled water, then add 0.2 mL of the fermentation broth diluted to an appropriate concentration and 0.3 mL of 2N Folin-Ciocalteu reagent, and proceed according to the method of the standard curve.

[0057] (6) Determination of protein content

[0058] First, protein was precipitated with saturated ammonium sulfate. 767 g of ammonium sulfate was slowly added to 1 L of distilled water while stirring, and the pH value was adjusted to 7.0 with ammonia water or sulfuric acid. This was the saturated ammonium sulfate solution. Take the fermentation broth, add an equal volume of saturated ammonium sulfate solution while adding and stirring. Place the solution on a magnetic stirrer and stir for 6 h to fully precipitate the protein, centrifuge, take the precipitate, and dissolve it with PBS.

[0059] Then, the protein was determined by the Coomassie brilliant blue method. Standard curve preparation: Prepare a 0.1 mg / mL bovine serum albumin standard protein solution. Respectively take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of the standard solution, make up to 1 mL, add 5 mL of Coomassie brilliant blue staining solution, mix well, let stand at room temperature for 5 minutes, and colorimetric at 595 nm. With the protein content (mg) as the abscissa and the absorbance as the ordinate, draw the standard curve. Sample determination: The sample was appropriately diluted, 1 mL was taken, 5 mL of Coomassie brilliant blue staining solution was added, mixed well, let stand at room temperature for 5 minutes, and colorimetric at 595 nm.

[0060] (7) Determination of Amino Acid Nitrogen Content

[0061] The formaldehyde titration method was adopted. Pipette 5.0 mL of the test sample into a 50 mL beaker, wash it into a 100 mL volumetric flask several times with water, add water to the scale, mix well, then pipette 20.0 mL into a 200 mL beaker, add 60 mL of water, start the magnetic stirrer, and titrate with a standard sodium hydroxide solution [c(NaOH) = 0.050 mol / L] until the pH meter indicates a pH of 8.2, record the milliliters of the standard sodium hydroxide titrant consumed, and the total acid content can be calculated. Add 10.0 mL of formaldehyde solution, mix well. Then continue to titrate with the standard sodium hydroxide titrant until the pH is 9.2, and record the milliliters of the standard sodium hydroxide titrant consumed. At the same time, take 80 mL of water, first adjust it to a pH of 8.2 with the standard sodium hydroxide solution [c(NaOH) = 0.050 mol / L], then add 10.0 mL of formaldehyde solution, and titrate with the standard sodium hydroxide titrant until the pH is 9.2 to conduct a reagent blank test. The calculation formula for the amino acid nitrogen content is as follows:

[0062]

[0063] In the formula:

[0064] m—the mass of the test sample for determination, in grams (g)

[0065] V1—the volume of the standard sodium hydroxide solution consumed when titrating the sample dilution to the end point (pH 9.2) after adding formaldehyde, in milliliters (mL)

[0066] V2—the volume of the standard sodium hydroxide solution consumed when titrating the blank test to the end point (pH 9.2) after adding formaldehyde, in milliliters (mL)

[0067] X1—the content of amino acid nitrogen in the test sample, in grams per hundred grams (g / 100g)

[0068] c—the concentration of the standard sodium hydroxide solution, in moles per liter (mol / L)

[0069] 0.014—the millimolar mass of nitrogen, in grams per millimole (g / mmol).

[0070] (8) Determination of Glutathione Peroxidase Content

[0071] A kit (Nanjing Jiancheng, GSH-PX Assay Kit, colorimetric method A005) was used and carried out according to the kit instructions. The GSH-PX activity in the cell lysate was calculated as follows:

[0072]

[0073] Wherein: GSH-PX activity — unit is U / mL;

[0074] Standard product concentration — 20 μmol / L;

[0075] Dilution factor — 5.

[0076] Example 1

[0077] Lactobacillus plantarum A: CICC 20038, obtained by purchase from Beijing Center for Biological Preservation.

[0078] Lactobacillus plantarum B: CCTCC NO: AB2022276, China Center for Type Culture Collection (Lactobacillus plantarum), was isolated by the present inventor from self-made pickles, named LP8, and the isolation and identification process is as follows:

[0079] Take the self-made pickle broth, after gradient dilution, take an appropriate amount and coat it on MRS medium, anaerobically culture at 37 °C for 72 h, pick colonies with conforming morphology for Gram staining microscopy and preliminary determination of antioxidant capacity, and send them to Sangon Biotech (Shanghai) Co., Ltd. for strain identification. As Figure 1 shown is the colony map of Lactobacillus plantarum B, and as Figure 2 shown is the Gram staining microscopy map of Lactobacillus plantarum B.

[0080] On June 16, 2022, it was sent to and preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: AB2022276.

[0081] Taking the scavenging rate of DPPH free radicals as the primary screening index, select the strain with the highest antioxidant property, and through strain identification (16S rRNA identification), it is Lactobacillus plantarum, and its 16S rRNA sequence is as shown in SEQ ID NO: 1.

[0082] Measure the antioxidant and antibacterial abilities of LP8, and conduct a safety evaluation.

[0083] The results are shown in Tables 1 - 3. As can be seen from Table 1, the fermentation broth of Lactobacillus plantarum B has good antioxidant property, and its scavenging rates for hydroxyl free radicals and DPPH free radicals can both reach about 90% of the effect of 1 mg / mL VC. As can be seen from Table 2, Lactobacillus plantarum B has certain antibacterial effects on three pathogenic bacteria, and has better effects on Escherichia coli and Staphylococcus aureus, and these two bacteria are common harmful bacteria on the skin. As can be seen from Table 3, Lactobacillus plantarum B shows high sensitivity to 5 common antibiotics, and the hemolysis test shows that it does not have hemolytic property, so its safety is preliminarily evaluated as good and it can be used.

[0084] Table 1. Experimental results of antioxidant capacity

[0085]

[0086] Table 2. Experimental results of antibacterial ability

[0087]

[0088] Table 3. Experimental results of antibiotic sensitivity

[0089]

[0090] Example 2

[0091] Preparation of Lactobacillus plantarum fermentation broth using Lactobacillus plantarum A and Lactobacillus plantarum B from two sources

[0092] Step S1: Dry the washed pomegranate peel, cut it into small pieces about 1×1 cm, add distilled water at a mass concentration of 3 g / mL of pomegranate peel, adjust the preset pH value to 3.5, and sterilize at 115 °C for 15 min to obtain the pomegranate peel fermentation medium;

[0093] Step S2: Quantitatively inoculate Lactobacillus plantarum A or Lactobacillus plantarum B into the pomegranate peel fermentation medium obtained in Step S1, culture at 37 °C for 48 h, and the inoculation amount is 5% (OD600 = 2.0) for fermentation and proliferation;

[0094] Step S3: After Step S2 is completed, first filter out the pomegranate peel residue, then centrifuge the proliferated Lactobacillus plantarum at a centrifugation speed of 6000 rpm for 10 min, and take the precipitate to obtain the Lactobacillus plantarum cells; take the supernatant to obtain the Lactobacillus plantarum fermentation broth.

[0095] Example 3

[0096] Preparation of Lactobacillus plantarum fermentation broth using Lactobacillus plantarum A and Lactobacillus plantarum B from two sources

[0097] Step S1: Dry the washed pomegranate peel, cut it into small pieces about 1×1 cm, add distilled water at a mass concentration of 3 g / mL of pomegranate peel, adjust the preset pH value to 2.5, and sterilize at 115 °C for 15 min to obtain the pomegranate peel fermentation medium;

[0098] Step S2: Quantitatively inoculate Lactobacillus plantarum A or Lactobacillus plantarum B into the pomegranate peel fermentation medium obtained in Step S1, culture at 30 °C for 48 h, and the inoculation amount is 4% for fermentation and proliferation;

[0099] Step S3, after step S2 is completed, first filter out the pomegranate peel residue, then centrifuge the proliferated Lactobacillus plantarum at a centrifugation speed of 6000 rpm for 10 min. Take the precipitate to obtain Lactobacillus plantarum cells; take the supernatant to obtain the Lactobacillus plantarum fermentation broth.

[0100] Example 4

[0101] Preparation of Lactobacillus plantarum fermentation broth using Lactobacillus plantarum A and Lactobacillus plantarum B from two sources

[0102] Step S1, dry the washed pomegranate peel, cut it into small pieces about 1×1 cm, add distilled water at a mass concentration of 5 g / mL of pomegranate peel, adjust the preset pH value to 3.5, and sterilize at 115 °C for 15 min to obtain the pomegranate peel fermentation medium;

[0103] Step S2, quantitatively inoculate Lactobacillus plantarum A or Lactobacillus plantarum B into the pomegranate peel fermentation medium obtained in step S1, and culture at 37 °C for 48 h with an inoculation amount of 6% for fermentation and proliferation;

[0104] Step S3, after step S2 is completed, first filter out the pomegranate peel residue, then centrifuge the proliferated Lactobacillus plantarum LP8 at a centrifugation speed of 6000 rpm for 10 min. Take the precipitate to obtain Lactobacillus plantarum cells; take the supernatant to obtain the Lactobacillus plantarum LP8 fermentation broth.

[0105] Example 5

[0106] Preparation of Lactobacillus plantarum fermentation broth using Lactobacillus plantarum A and Lactobacillus plantarum B from two sources

[0107] Step S1, dry the washed pomegranate peel, cut it into small pieces about 1×1 cm, add distilled water at a mass concentration of 7 g / mL of pomegranate peel, adjust the preset pH value to 3.5, and sterilize at 115 °C for 15 min to obtain the pomegranate peel fermentation medium;

[0108] Step S2, quantitatively inoculate Lactobacillus plantarum A or Lactobacillus plantarum B into the pomegranate peel fermentation medium obtained in step S1, and culture at 30 °C for 72 h with an inoculation amount of 8% for fermentation and proliferation;

[0109] Step S3, after step S2 is completed, first filter out the pomegranate peel residue, then centrifuge the proliferated Lactobacillus plantarum at a centrifugation speed of 6000 rpm for 10 min. Take the precipitate to obtain Lactobacillus plantarum cells; take the supernatant to obtain the Lactobacillus plantarum fermentation broth.

[0110] Example 6

[0111] Preparation of Lactobacillus plantarum fermentation broth using Lactobacillus plantarum A and Lactobacillus plantarum B from two sources Preparation of Lactobacillus plantarum fermentation broth

[0112] Step S1: Dry the washed pomegranate peel, cut it into small pieces about 1×1 cm, add distilled water according to the mass concentration of pomegranate peel of 3 g / mL, adjust the preset pH value to 4.5, and sterilize at 115 °C for 15 min to obtain the pomegranate peel fermentation medium;

[0113] Step S2: Quantitatively inoculate Lactobacillus plantarum A or Lactobacillus plantarum B into the pomegranate peel fermentation medium obtained in Step S1, and culture at 37 °C for 72 h with an inoculation amount of 5% for fermentation and proliferation;

[0114] Step S3: After Step S2 is completed, first filter out the pomegranate peel residue, then centrifuge the proliferated Lactobacillus plantarum at a centrifugation speed of 6000 rpm for 10 min, take the precipitate to obtain Lactobacillus plantarum cells; take the supernatant to obtain Lactobacillus plantarum fermentation broth.

[0115] Example 7

[0116] Preparation of Lactobacillus plantarum fermentation broth using Lactobacillus plantarum A and Lactobacillus plantarum B from two sources Preparation of Lactobacillus plantarum fermentation broth

[0117] Step S1: Dry the washed pomegranate peel, cut it into small pieces about 1×1 cm, add distilled water according to the mass concentration of pomegranate peel of 3 g / mL, adjust the preset pH value to 3.5, and sterilize at 115 °C for 15 min to obtain the pomegranate peel fermentation medium;

[0118] Step S2: Quantitatively inoculate Lactobacillus plantarum A or Lactobacillus plantarum B into the pomegranate peel fermentation medium obtained in Step S1, and culture at 34 °C for 60 h with an inoculation amount of 5% for fermentation and proliferation;

[0119] Step S3: After Step S2 is completed, first filter out the pomegranate peel residue, then centrifuge the proliferated Lactobacillus plantarum at a centrifugation speed of 6000 rpm for 10 min, take the precipitate to obtain Lactobacillus plantarum cells; take the supernatant to obtain Lactobacillus plantarum fermentation broth.

[0120] Example 8

[0121] Preparation of Lactobacillus plantarum fermentation lysate

[0122] The Lactobacillus plantarum cells prepared in Example 2 were lysed by ice bath ultrasonic disruption with an ultrasonic power of 300 W, intermittent for 1 s every 2 s of work, and the disruption time was 30 min; the parameters of the centrifugal separation were: centrifugation speed of 6000 rpm and centrifugation time of 10 min; take the supernatant to obtain the Lactobacillus plantarum fermentation lysate.

[0123] Example 9

[0124] Preparation of cell lysate of Lactobacillus plantarum

[0125] The obtained Lactobacillus plantarum cells prepared in Example 3 were lysed by ice bath ultrasonic disruption with an ultrasonic power of 300 W, intermittent for 1 s every 2 s of operation, and the disruption time was 30 min; the parameters of the centrifugal separation were: the centrifugal speed was 6000 rpm and the centrifugal time was 10 min; the supernatant was taken to obtain the cell lysate of Lactobacillus plantarum.

[0126] Example 10

[0127] Preparation of cell lysate of Lactobacillus plantarum

[0128] The obtained Lactobacillus plantarum cells prepared in Example 4 were lysed by ice bath ultrasonic disruption with an ultrasonic power of 300 W, intermittent for 1 s every 2 s of operation, and the disruption time was 30 min; the parameters of the centrifugal separation were: the centrifugal speed was 6000 rpm and the centrifugal time was 10 min; the supernatant was taken to obtain the cell lysate of Lactobacillus plantarum LP8.

[0129] Example 11

[0130] Preparation of cell lysate of Lactobacillus plantarum

[0131] The obtained Lactobacillus plantarum cells prepared in Example 5 were lysed by ice bath ultrasonic disruption with an ultrasonic power of 300 W, intermittent for 1 s every 2 s of operation, and the disruption time was 30 min; the parameters of the centrifugal separation were: the centrifugal speed was 6000 rpm and the centrifugal time was 10 min; the supernatant was taken to obtain the cell lysate of Lactobacillus plantarum.

[0132] Example 12

[0133] Preparation of cell lysate of Lactobacillus plantarum

[0134] The obtained Lactobacillus plantarum cells prepared in Example 6 were lysed by ice bath ultrasonic disruption with an ultrasonic power of 300 W, intermittent for 1 s every 2 s of operation, and the disruption time was 30 min; the parameters of the centrifugal separation were: the centrifugal speed was 6000 rpm and the centrifugal time was 10 min; the supernatant was taken to obtain the cell lysate of Lactobacillus plantarum.

[0135] Example 13

[0136] Preparation of cell lysate of Lactobacillus plantarum

[0137] The obtained Lactobacillus plantarum cells prepared in Example 7 were lysed by ultrasonic disruption in an ice bath. The ultrasonic power was 300 W, with a 1 s interval for every 2 s of operation, and the disruption time was 30 min. The parameters for centrifugal separation were: a centrifugal speed of 6000 rpm and a centrifugal time of 10 min. The supernatant was taken to obtain the fermentation lysate of Lactobacillus plantarum LP8.

[0138] The products prepared in Examples 2 - 13 were studied for their DPPH radical scavenging rate. Among them, for Examples 2 - 7, the fermentation broth was diluted 30 - fold before measurement, and for Examples 8 - 13, the lysate was diluted 5 - fold before measurement. The results are shown in Table 4.

[0139] Table 4: DPPH radical scavenging rate % of fermentation broths of Lactobacillus plantarum from two sources

[0140] Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Lactobacillus plantarum A 46.70％ 39.72％ 39.70％ 38.34％ 41.08％ 40.19％ Lactobacillus plantarum B 45.58％ 34.21％ 40.57％ 43.95％ 41.72％ 39.08％ Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Lactobacillus plantarum A 58.90％ 51.81％ 52.81％ 45.60％ 52.00％ 56.73％ Lactobacillus plantarum B 61.97％ 46.59％ 53.70％ 47.29％ 53.44％ 54.32％

[0141] As can be seen from Table 4, the fermentation broth of Lactobacillus plantarum prepared in Example 2 had the highest DPPH radical scavenging rate, and the fermentation lysate of Lactobacillus plantarum prepared in Example 8 had the highest DPPH radical scavenging rate.

[0142] Comparative Example 1

[0143] Prepared using two sources of Lactobacillus plantarum A and Lactobacillus plantarum B

[0144] The lysate was prepared by fermenting Lactobacillus plantarum with MRS as the medium

[0145] Step S1: Quantitatively inoculate Lactobacillus plantarum A or Lactobacillus plantarum B into the MRS medium obtained in Step S1, and culture at 37 °C for 48 h with an inoculation amount of 5% for fermentation and proliferation.

[0146] Step S2: After Step S1 is completed, first filter out the pomegranate peel residue, and then centrifuge the proliferated Lactobacillus plantarum LP8 at a centrifugal speed of 6000 rpm for 10 min. Take the precipitate to obtain Lactobacillus plantarum cells.

[0147] S3: After Step S2 is completed, lyse the obtained Lactobacillus plantarum cells by ultrasonic disruption in an ice bath. The ultrasonic power is 300 W, with a 1 s interval for every 2 s of operation, and the disruption time is 30 min. The parameters for centrifugal separation are: a centrifugal speed of 6000 rpm and a centrifugal time of 10 min. Take the supernatant to obtain the fermentation lysate of Lactobacillus plantarum.

[0148] Comparative Example 2

[0149] Preparation of pomegranate peel decoction

[0150] Take 3 g of dried pomegranate peel, soak it in distilled water for 2 h, then boil it on an induction cooker for 30 min, filter it with a filter cloth, add the filter residue to distilled water again and boil for 30 min, combine the two filtrates, and make up to 100 mL with distilled water to obtain the pomegranate peel decoction.

[0151] Comparative Example 3

[0152] Preparation of ultrasonic extract of pomegranate peel

[0153] Take 3 g of dried pomegranate peel, add 40 mL of distilled water, extract ultrasonically at 50 °C for 30 min and then filter with an 800-mesh filter cloth; add another 40 mL of distilled water to the filter residue, ultrasonically vibrate for 30 min, combine the two filtrates, and make up to 100 mL with distilled water to obtain the ultrasonic extract of pomegranate peel.

[0154] In order to better illustrate the functional effects of the fermentation lysate and fermentation broth obtained by Lactobacillus plantarum fermenting pomegranate peel, the applicant also conducted the following performance studies:

[0155] 1. Comparative study of the fermentation broth of Lactobacillus plantarum prepared in Example 2 with the pomegranate peel decoction in Comparative Example 2 and the ultrasonic extract of pomegranate peel in Comparative Example 3

[0156] 1.1 Comparison of active ingredient contents

[0157] Compare the contents of polyphenols, polysaccharides, proteins and amino acid nitrogen in the different fermentation broths of Lactobacillus plantarum obtained in Example 2 with the pomegranate peel decoction in Comparative Example 2 and the ultrasonic extract of pomegranate peel in Comparative Example 3. The results are shown in Table 5.

[0158] As can be seen from Table 5, the contents of polyphenols and proteins in the fermentation broth of Lactobacillus plantarum are not much different from those of the traditional extracts, but the contents of its polysaccharides and amino acids have been greatly improved. This is related to the role of Lactobacillus plantarum. During the fermentation process, Lactobacillus plantarum releases many extracellular enzymes, which promote the rupture of cell walls and the release of active substances, and at the same time can convert macromolecular substances into small-molecular substances with better activity (such as decomposing proteins into amino acids).

[0159] Table 5:

[0160]

[0161] 1.2 Comparison of main efficacy

[0162] Compare the DPPH free radical scavenging activity, tyrosinase inhibitory activity and antibacterial ability against Staphylococcus aureus of the different fermentation broths of Lactobacillus plantarum obtained in Example 2 with the pomegranate peel decoction in Comparative Example 2 and the ultrasonic extract of pomegranate peel in Comparative Example 3. The concentration for measuring the antioxidant index is 1 mg / mL (the stock solution is diluted 30 times), and the rest are measured with the stock solution. The results are shown in Figure 6.

[0163] As can be seen from Table 6, after Lactobacillus plantarum ferments pomegranate peel, the DPPH free radical scavenging activity, tyrosinase inhibitory activity of pomegranate peel and the antibacterial ability against Staphylococcus aureus are greatly improved.

[0164] Table 6:

[0165]

[0166] 2. Comparative study on the active ingredients and efficacy of the lysate of Lactobacillus plantarum prepared in Example 8 and the lysate of Lactobacillus plantarum prepared in Comparative Example 1

[0167] 2.1 Content and comparison of active ingredients

[0168] The contents of polysaccharides, proteins, amino acid nitrogen and glutathione peroxidase in the lysate of Lactobacillus plantarum obtained in Example 8 and the lysate of Lactobacillus plantarum prepared in Comparative Example 1 were compared, and the results are shown in Tables 7(a) and 7(b):

[0169] Table 7(a):

[0170]

[0171] Table 7(b):

[0172]

[0173] As can be seen from Table 7, it shows that pomegranate peel greatly increases the content of intracellular active substances of Lactobacillus plantarum, making its skin care effect stronger. Pomegranate peel and Lactobacillus plantarum have a synergistic effect. The various nutrients such as polysaccharides, trace elements and vitamins contained in pomegranate peel can act as prebiotics to promote the growth of Lactobacillus plantarum.

[0174] 2.2 Comparison of main efficacy

[0175] The antioxidant properties, whitening efficacy and antibacterial efficacy of the different lysates of Lactobacillus plantarum obtained in Example 8 and the lysate of Lactobacillus plantarum prepared in Comparative Example 1 were compared. Among them, the antioxidant index was measured by diluting the stock solution 5 times, and the rest were measured with the stock solution. The results are shown in Tables 8(a) and 8(b):

[0176] Table 8(a):

[0177]

[0178] Table 8(b):

[0179]

[0180] As can be seen from Table 8, pomegranate peel greatly enhances the ability of the lysate of Lactobacillus plantarum to scavenge DPPH free radicals and the tyrosinase inhibition rate.

[0181] In summary, Lactobacillus plantarum and pomegranate peel have a synergistic effect in the fermentation system. The active ingredients and functions of the two products have been greatly improved compared with traditional products, and are expected to be further developed into cosmetic functional raw materials.

[0182] Without conflict, the above embodiments and the features in the embodiments in this article can be combined with each other.

[0183] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0184]

[0185]

Claims

1. A preparation method of a fermented lysate of Lactobacillus plantarum, characterized in that, it includes lysing Lactobacillus plantarum cells by ultrasonic disruption in an ice bath, centrifugally separating the lysed Lactobacillus plantarum cells, and taking the supernatant to obtain the fermented lysate of Lactobacillus plantarum; the ultrasonic power is 300 W, with a 1 s interval for every 2 s of operation, and the disruption time is 30 min; the parameters of the centrifugal separation are: the centrifugal speed is 6000 rpm and the centrifugal time is 10 min; the Lactobacillus plantarum cells are prepared by the following method, and the specific steps are: S1. Dry the washed pomegranate peel, cut it into 1 cm×1 cm pieces, add distilled water according to a preset solid-liquid ratio, adjust the pH value to a preset 3.5, and sterilize to obtain a pomegranate peel fermentation medium; S2. Quantitatively inoculate Lactobacillus plantarum CICC 20038 into the pomegranate peel fermentation medium obtained in step S1 for fermentation and proliferation; the process of fermentation and proliferation of Lactobacillus plantarum is: take Lactobacillus plantarum, inoculate it into the pomegranate peel fermentation medium, and culture it at 37°C for 48 h, and the inoculation amount of Lactobacillus plantarum CICC 20038 is 5%; S3. After step S2 is completed, first filter out the pomegranate peel residue, then centrifugally separate the proliferated Lactobacillus plantarum, take the precipitate as Lactobacillus plantarum cells, and take the supernatant to obtain the fermented liquid of Lactobacillus plantarum.

2. The preparation method according to claim 1, characterized in that, in step S3, the centrifugal speed is 5000 - 6000 rpm and the centrifugal time is 5 - 15 min.

3. A fermented lysate of Lactobacillus plantarum, characterized in that, it is obtained by lysing the Lactobacillus plantarum cells prepared by using the preparation method according to any one of claims 1 - 2.

4. An antioxidant and whitening composition, characterized in that, it includes the fermented lysate of Lactobacillus plantarum according to claim 3.

5. Use of an antioxidant and whitening composition according to claim 4 in the preparation of daily-use cosmetics.

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