Lactobacillus plantarum fermentation liquor and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum fermentation liquor and application thereof. The lactobacillus plantarum fermentation liquor provided by the invention is obtained by taking roses as a substrate and fermenting screened plant milk stalks under specific conditions. Experimental results show that the content of effective active ingredients such as total polyphenol, total polysaccharide and superoxide dismutase in rose fermentation liquor fermented by the lactobacillus plantarum is relatively high, and the fermentation effect of the rose fermentation liquor is far superior to that of commercially available common lactobacillus plantarum and other strains; besides, the rose fermentation liquor also shows obvious DPPH free radical and hydroxyl free radical scavenging ability, can effectively inhibit the activity of tyrosinase and improve the cell viability, has anti-aging and whitening effects, and can be widely applied to various foods and skin care products.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a lactobacillus plantarum fermentation liquid and application thereof. Background Art

[0002] With the development of society, people's requirements for beauty are gradually increasing, and the pursuit of green, natural, healthy food and cosmetics is increasing. In recent years, plant raw materials derived from natural plants have been favored by consumers. The consumption concept of "advocating green and returning to nature" has also become the mainstream of the cosmetics industry, which has also led to the functional raw materials derived from plants becoming a hot spot in the field of food and cosmetic raw materials.

[0003] However, being limited to extracting active ingredients from natural plants has, to a certain extent, restricted their application effects and scope; in addition, some active ingredients from plants may have certain side effects.

[0004] Therefore, obtaining efficient and safe plant-based products has become the focus of attention of scientific researchers.

[0005] Patent CN109247474A uses screened plant lactobacillus to ferment roses to produce a fermented rose beverage. However, the patent only uses a simple extraction operation to obtain the active ingredients in the roses. Due to the presence of the natural barrier of the plant cell wall, it is difficult to directly dissolve the active ingredients more fully during the extraction operation. Therefore, even if the extract is fermented, the types and contents of the rose active ingredients contained in the obtained fermentation liquid will be greatly limited.

[0006] In addition, although patent CN111588758A adopts the method of first enzymatic hydrolysis and then fermentation with Lactobacillus plantarum to prepare rose slurry, due to the uniqueness of different types of fermentation bacteria and the different methods of processing the fermentation substrate, the active ingredients in the fermentation liquid after the final fermentation are significantly different. Moreover, the patent aims to extract the active ingredients in roses and does not specifically describe the fermentation effect of Lactobacillus plantarum.

[0007] In the prior art, many methods for increasing the effective content of plant-derived active ingredients involve fermentation, but the effects of different fermentation strains and the effects of the same strain applied in different plant organs are different. Screening out a strain that can efficiently increase the content of effective active ingredients in flowers, and the fermentation liquid obtained after fermentation of the strain can effectively overcome the original toxic side effects of flowers has far-reaching significance for the wide application of flowers in the fields of food and cosmetics. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a Lactobacillus plantarum fermentation liquid and application thereof.

[0009] The present invention provides a Lactobacillus plantarum fermentation liquid, which is obtained by fermenting the screened Lactobacillus plantarum with rose as a substrate. The Lactobacillus plantarum was deposited in the China Center for Type Culture Collection on November 18, 2024, with a deposit address of Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M20242577, and a classification name of: Lactiplantibacillus sp. JHHY-1.

[0010] The raw materials for preparing the above-mentioned Lactobacillus plantarum fermentation liquid include, by weight, 2-6 parts of rose flowers, 3-9 parts of sugar, 0.1-1 parts of Lactobacillus plantarum and 70-85 parts of water.

[0011] Further, the method for preparing the Lactobacillus plantarum fermentation broth comprises the following steps: S1: drying rose flowers, crushing and sieving them to obtain rose dry pollen; S2: adding water to the dried rose pollen obtained in S1 and mixing them evenly, and homogenizing them to obtain a dried rose pollen wall-broken mixed solution; S3 first sterilizes the rose dry pollen wall-breaking mixed solution obtained in S2, and then sequentially performs enzymolysis using a mixed enzyme preparation of papain, cellulase and pectinase to obtain a rose enzymolysis solution; S4 adds sugar and water to the rose hydrolysate obtained in S3, sterilizes, then adds Lactobacillus plantarum to ferment and filter to obtain a clear liquid, sterilizes again and cools to obtain the Lactobacillus plantarum fermentation liquid.

[0012] In the above steps, preferably, the homogenization conditions in S2 are: homogenization pressure: 40-50Mpa, homogenization time: 5-10min.

[0013] Preferably, the specific operation of the step-by-step enzymatic hydrolysis described in S3 is: first, add papain at 55-60°C and pH 4.0-6.0, and perform enzymatic hydrolysis for 30-50 min; then cool to 45-50°C, add a mixed enzyme preparation of cellulase and pectinase at pH 4.5-5.5, and continue enzymatic hydrolysis for 20-40 min.

[0014] Preferably, the papain described in S3 accounts for 0.05-0.1% by weight of the rose dry pollen wall-breaking mixed solution, and the enzyme activity of the papain is ≥70,000 U / g.

[0015] Preferably, the cellulase described in S3 accounts for 0.05-0.1% by weight of the rose dry pollen wall-breaking mixed solution, and the enzymatic activity of the cellulase is ≥70,000 U / g.

[0016] Preferably, the pectinase described in S3 accounts for 0.5-0.1% by weight of the rose dry pollen wall-breaking mixed solution, and the enzyme activity of the pectinase is ≥80,000 U / g.

[0017] Preferably, the fermentation conditions described in S4 are: fermentation temperature of 25-35°C, pH of 4-6, and fermentation time of 28-32h.

[0018] Preferably, the sugar described in S4 is selected from at least one of glucose, oligofructose, honey, maple syrup, coconut sugar and maltose.

[0019] As a further preferred embodiment, the present invention provides the use of the Lactobacillus plantarum fermentation liquid prepared by the above steps in food and skin care products, which is also the key content protected by the present invention.

[0020] The beneficial effects of the present invention are: 1. The present invention ferments roses by using the screened Lactobacillus plantarum, thereby reducing the original toxic side effects of roses and obtaining safer rose food raw materials; 2. The content of effective active ingredients such as total polyphenols, total polysaccharides, and superoxide dismutase contained in the Lactobacillus plantarum rose fermentation liquid prepared by the present invention is relatively high, which is far superior to the fermentation effect of common Lactobacillus plantarum and other strains available on the market; 3. In addition, the plant lactobacillus fermentation broth provided by the present invention has obvious DPPH free radical and hydroxyl free radical scavenging ability, can effectively inhibit the activity of tyrosinase, improve cell viability, has anti-aging and whitening effects, and can be widely used in various foods and skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the content of active ingredients in the Lactobacillus plantarum fermentation broth obtained at different fermentation times in Example 1 of the present invention; Figure 2 The in vitro antioxidant properties of the Lactobacillus plantarum rose fermentation broth obtained after fermentation for 30 h in Example 4 of the present invention; Figure 3 The tyrosinase inhibition rate of the Lactobacillus plantarum rose fermentation broth obtained after fermentation for 30 h in Example 4 of the present invention. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0023] Example 1 A plant lactobacillus fermentation liquid, wherein the raw materials for preparation are, by weight, 2 parts of rose, 3 parts of glucose, 0.3 parts of plant lactobacillus, and 70-85 parts of water; Take 2 parts of rose dry powder, 3 parts of glucose and a certain amount of deionized water in a beaker and mix them evenly. Place them at 121.0℃ and sterilize them for 25min. Take them out and cool them to room temperature to obtain the culture medium. Under sterile conditions, take 30.0 mg of plant lactobacillus and inoculate them into the culture medium. Place them in a shaker at 40.0℃ and shake them to obtain rose fermentation products. After the products are sterilized at 85.0℃ for 30min, they are cooled and the inactivated rose fermentation liquid is centrifuged at 10000 r / min for 10 min to remove the bacteria and collect the supernatant. Then use a 0.45μm filter membrane to further remove the residue. The final supernatant is placed in an 80.0℃ water bath, sterilized for 20min, and cooled. Rose fermentation liquids with different fermentation times are prepared by the above method, which are 6 h, 12 h, 18 h, 24 h, 30 h, and 36 h, respectively.

[0024] The added papain accounts for 0.08% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥70,000 U / g; the added cellulase accounts for 0.08% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥70,000 U / g; the added pectinase accounts for 0.08% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥80,000 U / g; The contents of total polysaccharides, total phenols and total acid contained in the Lactobacillus plantarum rose fermentation liquid prepared in this embodiment were measured, and the results are shown in the attached Figure 1 shown.

[0025] As can be seen from the figure, when the fermentation time is 30 h, the content of active ingredients in the obtained Lactobacillus plantarum rose fermentation liquid is the highest, so the fermentation time in the following experiments is 30 h.

[0026] Example 2 A plant lactobacillus fermentation liquid, wherein the raw materials for preparation are, by weight, 2 parts of rose, 3 parts of oligofructose, 0.1 parts of plant lactobacillus, and 70-85 parts of water; The specific preparation method is as follows: S1: Take 2 parts of roses, air-dry them, crush the air-dried roses, and pass them through a 100-mesh sieve to obtain dried rose pollen; S2 adds 5 times the amount of water to the dried rose pollen obtained in S1 and mixes the mixture, and homogenizes the mixture using a high-pressure homogenizer at a homogenization pressure of 50 MPa for 8 min to obtain a dried rose pollen wall-broken mixed solution; S3 first puts the rose dry pollen wall-breaking mixed solution obtained in S2 into a sterilization tank, sterilizes at 95°C for 20 min, then cools to 60°C, adds papain at pH 5.5, and keeps the temperature for enzymolysis for 30 min; then cools to 50°C, adds cellulase and pectinase at pH 5.0, and keeps the temperature for enzymolysis for 30 min to obtain rose enzymolysis solution; The added papain accounts for 0.05% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥70,000 U / g; the added cellulase accounts for 0.1% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥70,000 U / g; the added pectinase accounts for 0.08% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥80,000 U / g; S4 adds 3 parts of oligofructose and 10 times of water to the rose hydrolysate obtained in S3, sterilizes at 95°C for 20 minutes, then cools to 30°C, adds Lactobacillus plantarum for fermentation at a pH of 5.0 for 30 hours, obtains a clear liquid after filtration, sterilizes the clear liquid at a second high temperature, and cools it to obtain Lactobacillus plantarum rose fermentation liquid.

[0027] Example 3 A plant lactobacillus fermentation liquid, wherein the raw materials for preparing the liquid include, by weight, 2 parts of roses, 4.5 parts of honey, and 0.1 parts of plant lactobacillus; The specific preparation method is as follows: S1: Take 2 parts of rose flowers, air-dry them, and crush them through a 100-mesh sieve to obtain rose dry pollen; S2: adding 10 times the amount of water to the dried rose pollen obtained in S1, mixing well, and passing through a high-pressure homogenizer to obtain a dried rose pollen wall-broken mixed solution; S3: Put the rose dry pollen wall-breaking mixed solution obtained in S2 into a sterilization tank, sterilize it at 95°C for 25 min, then cool it down to 58°C, add papain at pH 6.0, and keep it warm for 30 min; continue to cool it down to 45°C, add cellulase and pectinase at pH 4.8, and keep it warm for 30 min to obtain rose enzymolysis solution; The added papain accounts for 0.08% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥70,000 U / g; the added cellulase accounts for 0.08% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥70,000 U / g; the added pectinase accounts for 0.08% of the weight of the rose dry pollen wall-breaking mixture, and its enzyme activity is ≥80,000 U / g; S4 adds 5 times of water and 4.5 parts of honey to the rose hydrolysate obtained in S3, sterilizes at 95°C for 20 min, cools to 28°C, adds Lactobacillus plantarum for fermentation at pH 5.0 for 30 h, obtains a clear liquid after filtration, sterilizes the clear liquid at high temperature for a second time, and cools it to obtain Lactobacillus plantarum rose fermentation liquid.

[0028] Example 4 The Lactobacillus plantarum rose fermentation broth obtained after fermentation for 30 h in Example 1 was taken, and its performance was verified.

[0029] (1) In vitro antioxidant properties Take the plant lactobacillus rose fermentation liquid obtained after fermentation for 30h in Example 1, set the concentration to 1%, 5%, and 10% respectively, and use 0.5% vitamin C (VC) as a control at the same time, and measure the scavenging rate for DPPH free radical and the scavenging rate of hydroxyl free radical respectively. The experimental results are shown in Table 2 and Attached Figure 2 shown.

[0030] Table 2 In vitro antioxidant properties of Lactobacillus plantarum rose fermentation broth concentration(%) 1% 5% 10% VC (0.5%) DPPH free radical scavenging rate (%) 80.769 85.452 85.618 89.298 Hydroxyl radical scavenging rate (%) 82.818 87.972 91.924 81.443

[0031] When the concentration of Lactobacillus plantarum rose fermentation liquid was 1%, 5%, and 10%, the scavenging rates of DPPH free radicals were 80.769%, 85.452%, and 85.618%, respectively; the scavenging rates of hydroxyl free radicals were 82.818%, 87.972%, and 91.924%, respectively. By comparison, it was found that the antioxidant capacity of Lactobacillus plantarum rose fermentation liquid was consistent with that of 0.5% VC, and the results proved that Lactobacillus plantarum rose fermentation liquid had good antioxidant effect.

[0032] (2) Tyrosinase inhibitory activity Take the Lactobacillus plantarum rose fermentation liquid obtained after fermentation for 30h in Example 1, set the concentration to 1%, 5%, and 10%, respectively, and use 0.5% arbutin as a control, and measure the inhibition of tyrosinase activity, respectively. The results are shown in Table 3 and Attached Figure 3 .

[0033] Table 3 Tyrosinase activity inhibition of Lactobacillus plantarum rose fermentation broth concentration(%) 1% 5% 10% Arbutin (0.5%) Tyrosinase activity inhibition rate (%) 78.8 82.8 107.2 71.2

[0034] The synthesis of melanin is affected by tyrosinase, which can catalyze the hydroxylation of L-tyrosine into DOPA and oxidize DOPA into DOPAquinone to form the final reaction product, melanin.

[0035] From the experimental results, it can be seen that when the concentration of Lactobacillus plantarum rose fermentation liquid is 1%, 5%, and 10%, the activity inhibition rate of tyrosinase is 78.800%, 82.800%, and 107.200% respectively. After comparative analysis, the activity inhibition rate of Lactobacillus plantarum rose fermentation liquid on tyrosinase is higher than that of 0.5% arbutin, proving that Lactobacillus plantarum rose fermentation liquid has a certain whitening effect.

[0036] Comparative Example 1 Commercially available common Lactobacillus plantarum was used to ferment in the manner of Example 1 for 30 h to prepare Lactobacillus plantarum rose fermentation liquid.

[0037] Comparative Example 2 Rose was fermented with lactobacillus in the manner of Example 1 to prepare Lactobacillus plantarum rose fermentation liquid.

[0038] Comparative Example 3 A Lactobacillus plantarum fermentation liquid was prepared using the fermentation conditions of Example 1. The difference from Example 1 was that the types and proportions of the raw materials were slightly adjusted, as shown in Table 4 below.

[0039] Table 4 Types and proportions of raw materials (mass fractions) Rose glucose Fructo-oligosaccharides Honey Maltose Group 1 2 2 0 1 0 Group 2 2 0 0 0 3 Group 3 2 2 0 1 1 Group 4 2 2 1 0 1

[0040] The in vitro antioxidant properties of the Lactobacillus plantarum rose fermentation broth obtained in Comparative Examples 1-3 and the inhibition of tyrosinase activity are shown in Table 5 below.

[0041] Table 5 Activity of Lactobacillus plantarum rose fermentation broth (5%) obtained by different methods concentration(%) Comparative Example 1 Comparative Example 2 Group 1 Group 2 Group 3 Group 4 DPPH free radical scavenging rate (%) 60.960 70.142 80.490 83.425 82.231 79.019 Hydroxyl radical scavenging rate (%) 68.126 76.301 82.703 86.039 83.066 82.528 Tyrosinase inhibition rate (%) 53.3 64.2 80.6 81.3 80.2 79.6

[0042] The above results show that the Lactobacillus plantarum rose fermentation broth obtained by the method of the present invention has good anti-aging and oxidation effects and tyrosinase inhibitory activity.

Claims

1. A Lactobacillus plantarum fermentation broth, characterized in that The plant lactobacillus was deposited in the China Center for Type Culture Collection on November 18, 2024, with a deposit number of CCTCC NO: M20242577 and a classification name of: Lactiplantibacillus sp. JHHY-1.

2. A plant lactobacillus fermentation liquid as claimed in claim 1, characterized in that, The plant lactobacillus fermentation liquid is obtained by fermenting with rose as substrate.

3. A plant lactobacillus fermented liquid as claimed in claim 2, characterized in that, The raw materials for preparing the plant lactobacillus fermentation liquid include, by weight, 2-6 parts of rose flowers, 3-9 parts of sugar, 0.1-1 parts of plant lactobacillus and 70-85 parts of water.

4. A method for preparing a plant lactobacillus fermented liquid as claimed in claim 1, characterized in that: The steps include: S1: air-drying, crushing and sieving rose flowers to obtain rose dry pollen; S2: adding water to the dried rose pollen obtained in S1 and mixing them evenly, and homogenizing them to obtain a dried rose pollen wall-broken mixed solution; S3 sterilizes the rose dry pollen wall-breaking mixed solution obtained in S2, and then sequentially hydrolyzes the mixed enzyme preparation of papain, cellulase and pectinase step by step to obtain rose enzymatic hydrolysate; S4 adds sugar and water to the rose hydrolysate obtained in S3, sterilizes, then adds Lactobacillus plantarum to ferment and filter to obtain a clear liquid, sterilizes again and cools to obtain the Lactobacillus plantarum fermentation liquid.

5. The preparation method according to claim 4, characterized in that: In S2, the homogenization conditions are: homogenization pressure: 40-50 MPa, homogenization time: 5-10 min.

6. The preparation method according to claim 4, characterized in that: In S3, the specific operation of the step-by-step enzymatic hydrolysis is: first, add papain at 55-60°C and pH 4.0-6.0, and perform enzymatic hydrolysis for 30-50 min; then cool to 45-50°C, add a mixed enzyme preparation of cellulase and pectinase at pH 4.5-5.5, and continue enzymatic hydrolysis for 20-40 min.

7. The preparation method according to claim 4, characterized in that: The papain described in S3 accounts for 0.05-0.1% of the weight of the rose dry pollen wall-breaking mixed solution, and the enzyme activity of papain is ≥70,000 U / g. The cellulase described in S3 accounts for 0.05-0.1% of the weight of the rose dry pollen wall-breaking mixed solution, and the enzyme activity of cellulase is ≥70,000 U / g. The pectinase described in S3 accounts for 0.5-0.1% of the weight of the rose dry pollen wall-breaking mixed solution, and the enzyme activity of pectinase is ≥80,000 U / g.

8. The preparation method according to claim 4, characterized in that: In S4, the fermentation conditions are: fermentation temperature is 25-35°C, pH is 4.0-6.0, and fermentation time is 28-32 h.

9. The preparation method according to claim 4, characterized in that: In S4, the sugar is selected from at least one of glucose, oligofructose, honey, maple syrup, coconut sugar, and maltose.

10. Use of the Lactobacillus plantarum fermentation liquid according to claim 1 in food and skin care products.

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