Preparation method of lotus stem fermentation product, lotus stem fermentation product and application of lotus stem fermentation product

Abstract

The invention discloses a preparation method of a lotus stem fermentation product, the lotus stem fermentation product and application of the lotus stem fermentation product, belongs to the technical field of plant fermentation, and provides hanseniaspora uvarium which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the hanseniaspora uvarium is CGMCC No.32471, the preservation date of the hanseniaspora uvarium is November 04, 2024, and the preservation number of the hanseniaspora uvarium is CGMCC No.32471. According to the present invention, the suggested classification name is hanseniaspora uvarium, and the lotus stem fermentation product is obtained by fermenting the lotus stem with the strain, and has a certain inhibition effect on the collagenase activity;

Description

Technical Field

[0001] The invention belongs to the technical field of plant fermentation, and in particular relates to a preparation method of lotus stem fermented product and the lotus stem fermented product and application thereof. Background Art

[0002] Lotus stalks are petioles or pedicels of the lotus plant (Nelumbo nucifera Gaertn.) of the Nymphaeaceae family. They are usually slender and cylindrical, with many vertical lines on the surface. They are generally green or yellow-green in color and have a relatively tough texture. Lotus stalks also have specific meanings and applications in some cultural traditions and medical fields. For example, lotus stalks are used in some traditional Chinese medicine prescriptions. They are believed to have certain medicinal value and can play a role in clearing away heat and relieving summer heat, ventilating and promoting water circulation. Their main components include plant fiber, starch, vitamins, flavonoids and other ingredients.

[0003] Collagenase can participate in the degradation of collagen, which is the main component of the skin and constitutes the elastic network of the skin, firmly locking in moisture. As we age, collagen will gradually be lost, causing aging phenomena such as dryness, wrinkles, and sagging of the skin. Collagenase may indirectly affect the moisturizing state of the skin by affecting the metabolism and renewal of collagen.

[0004] The survey results show that the research on lotus mainly focuses on lotus roots, lotus leaves and lotus core. In order to better utilize the whole lotus plant, it is of great significance to study the activity of its other parts.

[0005] At present, there are few research reports on the skin moisturizing function of petioles (lotus stalks) of Nelumbo plants. Therefore, in-depth exploration of the potential moisturizing function of lotus stalks is of research and development significance for the fields of skin science and cosmetics. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing lotus stem fermentation products.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for preparing lotus stem fermentation product, comprising:

[0010] enzymatically hydrolyzing lotus stem powder to obtain lotus stem enzymatic hydrolyzate;

[0011] The enzymatic hydrolysate is placed in a water bath at 90°C to 100°C for high temperature inactivation to obtain a lotus stem extract;

[0012] After activation, Hansenula viticola and Saccharomyces cerevisiae are inoculated into a fermentation medium containing lotus stem extract, and then placed in a shaking table for constant temperature culture to obtain lotus stem fermentation liquid;

[0013] The lotus stem fermentation liquid is centrifuged, the supernatant is taken, and after alcohol precipitation overnight, the precipitate is taken by centrifugation, and the lotus stem fermentation product is obtained after redissolution and membrane.

[0014] Among them, the grape spore Hansenia yeast is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, its deposit number is CGMCC No.32471, the deposit date is November 4, 2024, and the recommended classification name is: grape spore Hansenia yeast Hanseniasporauvarum; the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0015] The yeast Saccharomycopsis fibuligera was classified and named Saccharomycopsis fibuligera, purchased from China Industrial Microbiological Culture Collection Center (CICC), and the strain number was CICC33226.

[0016] As a preferred embodiment of the preparation method of the present invention, the enzymatic hydrolysis includes mixing papain and cellulase in a mass ratio of 2:1 to 4:1 to obtain a mixed enzyme, adding the mixed enzyme to a lotus stem powder solution, and performing enzymatic hydrolysis at 40 to 60° C. for 1 to 3 hours.

[0017] As a preferred embodiment of the preparation method of the present invention, the seed liquid OD obtained after the activation of Hansenula vitis spores is 600nm =1, Hansenula vitis spore seed solution, the inoculation amount is 1-10% based on the mass percentage of the fermentation system.

[0018] As a preferred embodiment of the preparation method of the present invention, the seed liquid OD obtained after the activation of the yeast Saccharomyces cerevisiae is 600nm =1, the seed solution of Saccharomyces cerevisiae flavus, calculated by the mass percentage of the fermentation system, the inoculation amount is 1-10%.

[0019] As a preferred embodiment of the preparation method of the present invention, the fermentation medium, in g / L, comprises 20-40 lotus stems, 15-25 peptone, 5-15 yeast extract, and 15-25 glucose; the fermentation medium has a pH of 6.5-7.5 and contains 1.0 L of pure water.

[0020] As a preferred embodiment of the preparation method of the present invention, the fermentation medium preparation method comprises:

[0021] The raw materials are mixed evenly and sterilized at 121°C and 0.1Mpa for 20 minutes.

[0022] As a preferred embodiment of the preparation method of the present invention, the constant temperature culture includes culturing in a constant temperature culture shaker at 28-32°C and 180-220 rpm for 24-48 hours.

[0023] As a preferred embodiment of the preparation method of the present invention, the membrane passing includes passing through a 5-10 kD cellulose membrane.

[0024] Another object of the present invention is to overcome the deficiencies in the prior art and provide a lotus stem fermentation product having the effect of inhibiting the activity of collagenase.

[0025] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for using lotus stem fermentation as a moisturizing ingredient in cosmetics.

[0026] Beneficial effects of the present invention:

[0027] (1) The present invention provides Hansenia sporauvarum, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No.32471 and a deposit date of November 4, 2024. The proposed classification name is: Hansenia sporauvarum. Lotus stem fermentation is obtained by fermenting lotus stems with the strain, which has a certain inhibitory effect on collagenase activity.

[0028] (2) The present invention provides a method for preparing lotus stem fermentation products. The preparation method uses Hansenula vitis spores and Saccharomyces cerevisiae to mix and ferment lotus stems. The fermentation process is mild. The prepared lotus stem fermentation products have a good collagenase inhibition rate. The lotus stem fermentation products can be prepared into a facial cream to effectively improve the transepidermal water loss of the skin, and have broad application prospects in the moisturizing of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0030] Figure 1This is a graph showing the collagenase inhibition activity results of lotus stem fermentation broths made from five yeasts in Example 1 of the present invention.

[0031] Figure 2 This is a graph showing the collagenase inhibition results of lotus stem fermentation products of 19 H.uvarum mutants in Example 2 of the present invention.

[0032] Figure 3 This is a graph showing the collagenase inhibition results of the lotus stem fermentation product produced by mixed fermentation in Example 3 of the present invention.

[0033] Figure 4 This is a graph showing the inhibition of collagenase by lotus stem fermentation products of different molecular weights in Example 4 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0037] Example 1

[0038] Screening process of single yeast fermentation of lotus stems:

[0039] The strains of lotus stem fermentation products were screened by yeast from five different species, including Rhodosporidium toruloides (CICC31643), Hanseniasporauvarum (BNCC193162), Saccharomycopsis fibuligera (CICC33226), Candida bombicola (BNCC340735), and Saccharoymces cerevisiae (CICC1210).

[0040] Lotus stem pre-treatment:

[0041] The lotus stems were soaked and washed in an appropriate amount of clean water, dried in a 60°C oven to a constant weight, crushed with a grinder, and passed through a 200-mesh sieve to obtain lotus stem powder;

[0042] 1.00 g lotus stem powder was added to 50 mL phosphate buffer (pH 5-6), 10% papain (enzyme activity 200 U / mg) and 5% cellulase (enzyme activity 400 U / mg) were added, and the lotus stem was enzymolyzed in a 50°C water bath for 2 h to obtain lotus stem enzymolysis solution;

[0043] The enzymatic hydrolysate was placed in a 95°C water bath for high temperature inactivation to obtain the lotus stem extract.

[0044] YPD seed culture medium: peptone 20.0 g / L, yeast extract 10.0 g / L, glucose 20.0 g / L, pure water 1.0 L, pH = 7.0; sterilize at 121°C, 0.1 MPa for 20 min;

[0045] Seed liquid culture: Take out the yeast glycerol cryopreservation tube from the -80℃ refrigerator, inoculate it into YPD liquid culture medium, place it in a 30℃ constant temperature shaker, and culture it at 200r / min for 24h to obtain yeast seed liquid.

[0046] Fermentation medium: lotus stem extract 20.0 g / L, peptone 20.0 g / L, yeast extract 10.0 g / L, glucose 20.0 g / L, pure water 1.0 L, adjust pH = 7.0; 121°C, 0.1 MPa sterilization for 20 min.

[0047] Preparation process of lotus stem fermentation: dilute the cultured yeast seed liquid to OD 600nm =1, inoculated into a 250mL triangular shake flask containing 100mL fermentation medium at a ratio of 10%, and cultured in a constant temperature culture shaker at 30°C and 200rpm for 48h. The fermentation broth was inactivated and centrifuged at the end of fermentation, and the supernatant was precipitated with tetraploid 95% ethanol to obtain lotus stem fermentation product.

[0048] Test method for collagenase inhibitory activity: N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA; Sigma) was used as collagenase substrate, and 10mM PBS phosphate buffer (pH=7.2) was used to construct the buffer system. 50μL collagenase (final concentration 500μg / mL; Product No. SCR103; Sigma) was taken and mixed thoroughly with 50μL sample solution, and placed in a 37℃ environment for reaction for 10min. Subsequently, 100μL substrate solution (final concentration 0.5mM) was added and reacted at 37℃ for 30min. After the reaction, the absorbance value at 335nm was measured with an enzyme reader, and the blank group used buffer instead of sample solution.

[0049] The inhibitory activity of the sample on collagenase was calculated by the following formula: Inhibition rate (%) = [1-(test-blank) / (control-blank)].

[0050] Collagenase inhibition effect of lotus stem fermentation products with five yeast species Figure 1 It can be seen that according to the test method of collagenase inhibitory activity, the collagenase inhibitory activity of the samples was tested. According to the test results, H.uvarum had the best inhibitory activity on collagenase, followed by S.fibuligera.

[0051] Example 2

[0052] NTG mutagenesis of H. uvarum:

[0053] In order to further improve its collagenase inhibitory activity, H.uvarum was mutagenized using NTG.

[0054] Nitrosoguanidine (NTG) mutagenesis method:

[0055] (1) NTG solution: Prepare a 1 mg / mL solution using sterile pH 7.0 phosphate buffer.

[0056] (2) Preparation of strains: Take the bacterial suspension in the logarithmic growth phase and centrifuge it at 3000 rpm for 5 min, wash it three times with saline, and resuspend it with saline to a bacterial suspension with an OD600 of 1.

[0057] (3) Mutagenesis treatment: Take 1 mL of bacterial suspension and add 100 μL of NTG solution. After reacting at 37°C for 1 h, apply 50 μL on MRS medium.

[0058] (4) After mutagenesis, H. uvarum was obtained on YPD medium for subculture and seed preservation. Lotus stems were fermented and ethanol precipitated according to the method of Example 1 to obtain lotus stem fermentation products, and the collagen inhibitory activity of the supernatant was detected.

[0059] The collagenase inhibition effect of lotus stem fermentation products of 19 H. uvarum mutants can be found in Figure 2 The results showed that HU9 was a positive mutagenesis with the highest inhibitory activity on collagen. It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.32471 and a deposit date of November 4, 2024. The recommended classification name is: Hanseniasporauvarum.

[0060] Example 3

[0061] Screening process of single yeast fermentation of lotus stems:

[0062] Compared with single-bacteria fermentation, mixed-bacteria fermentation has many advantages, such as improving fermentation efficiency, increasing product variety and yield, and reducing costs. Therefore, HU9 and S. fibuligera were used for mixed-bacteria fermentation of lotus stems.

[0063] The lotus stems were fermented by the method in Example 1, and the preparation process of the lotus stem fermentation product in Example 1 was modified as follows:

[0064] Dilute the cultured yeast seed solution to OD 600nm =1, HU9 and S.fibuligera were inoculated into a 250mL triangular shake flask containing 100mL fermentation medium at a ratio of 5%, and cultured in a constant temperature culture shaker at 30°C and 200rpm for 48h. The fermentation broth was inactivated and centrifuged at the end of fermentation. The supernatant was taken and precipitated with tetraploid 95% ethanol to obtain the lotus stem fermentation product, which was recorded as S1.

[0065] Comparative Example 1

[0066] Referring to the preparation process of lotus stem fermentation product in Example 3, the fermentation process was modified as follows:

[0067] The HU9 strain was first inoculated into the fermentation medium at a ratio of 5% and fermented for 24 hours, then sterilized, and then inoculated with 5% S. fibuligera and fermented for 24 hours. The lotus stem fermentation product obtained was recorded as S2.

[0068] Comparative Example 2

[0069] Referring to the above lotus stem fermentation preparation process, the fermentation process is modified as follows:

[0070] The S. fibuligera strain was first inoculated into the fermentation medium at a ratio of 5% and fermented for 24 hours, then sterilized, and then inoculated with 5% HU9 and fermented for 24 hours to obtain the lotus stem fermentation product, which was recorded as S3.

[0071] The lotus stem fermented product was diluted to 5%, and the collagenase inhibition activity of the sample was detected according to the collagenase inhibition rate test method in Example 1. The collagenase inhibition effect of the lotus stem fermented product by mixed bacteria fermentation is shown in Figure 3 The collagenase inhibitory activity of the samples was tested according to the test method for collagenase inhibitory activity. According to the test results, S1 has the best inhibitory activity on collagenase.

[0072] Example 4

[0073] Detection of the inhibitory activity of lotus stem fermentation products with different molecular weights on collagenase:

[0074] The lotus stem fermentation product was diluted to 5%, and the collagenase inhibition activity of the sample was detected according to the collagenase inhibition rate test method in Example 1.

[0075] The lotus stem fermentation product obtained in Example 3 was filtered using a cellulose membrane-10KDa to collect a filtrate with a molecular weight of <10kD and a filtrate with a molecular weight of >10kD;

[0076] The filtrate 1 is then filtered using a cellulose membrane-5KDa membrane to collect a filtrate 2 having a molecular weight <5kD and a filtrate 2 having a molecular weight of 5kD to 10kD;

[0077] The filtrate 2 was collected to obtain lotus stem fermentation product with a molecular weight less than 5 kD.

[0078] Comparative Example 3

[0079] The filtrate 2 obtained in Example 4 was used.

[0080] Comparative Example 4

[0081] The filtrate 1 obtained in Example 4 was used.

[0082] The inhibitory effect of lotus stem fermentation products with different molecular weights on collagenase can be found in Figure 4 It can be seen that the lotus stem fermentation product with a molecular weight less than 5kD has the best inhibitory effect on collagenase.

[0083] Example 5

[0084] Evaluation of the efficacy of lotus stem fermentation products on human body:

[0085] In order to better evaluate the activity of the lotus stem fermented product, the lotus stem fermented product was prepared into a facial cream, and the effect of the lotus stem fermented product was evaluated on human body. The components of the facial cream refer to Table 1.

[0086] The preparation method is as follows: weigh the components of water phase A and oil phase B respectively as shown in Table 1, and heat them in a water bath at 80°C to dissolve them;

[0087] Then add phase B into phase A and stir to make it emulsified evenly;

[0088] When the temperature drops to about 40°C, add the lotus root fermentation product of phase C and stir evenly;

[0089] The facial cream prepared by adding the lotus stem fermentation product described in Example 4 was the sample group;

[0090] The cream prepared by adding unfermented product served as the control group.

[0091] Table 1 Composition of lotus stem fermented cream

[0092]

[0093]

[0094] Ten volunteers aged between 25 and 40 were organized to evaluate the efficacy of the product. The inner sides of the left and right forearms were selected as the test areas for immediate testing. The changes in stratum corneum water content and transepidermal water loss before and after use were compared.

[0095] The test conditions were: constant temperature of 20°C and humidity of 60%. After the volunteers cleaned their arms, they applied the samples to the different areas and waited for 60 minutes in a constant temperature and humidity room before starting the test. The results are shown in Table 2.

[0096] The lotus stem fermentation product obtained in Example 4 was used as the experimental group, water was used to replace the lotus stem fermentation product as the blank group, and the unfermented lotus stem extract was used as the comparative example 5.

[0097] Table 2 Water content of stratum corneum and transepidermal water loss (TEWL) of volunteers

[0098]

[0099] It can be seen that compared with the blank group without adding lotus stem active ingredients, after using this product, the water content of the volunteers' stratum corneum increased, the transepidermal water loss decreased, and the activity of lotus stems after fermentation was better than before fermentation.

[0100] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing lotus stem fermentation product, characterized in that: include, enzymatically hydrolyzing lotus stem powder to obtain lotus stem enzymatic hydrolyzate; The enzymatic hydrolysate is placed in a water bath at 90°C to 100°C for high temperature inactivation to obtain a lotus stem extract; After activation, Hansenula viticola and Saccharomyces cerevisiae are inoculated into a fermentation medium containing lotus stem extract, and then placed in a shaking table for constant temperature culture to obtain lotus stem fermentation liquid; The lotus stem fermentation liquid is centrifuged, the supernatant is taken, and after alcohol precipitation overnight, the precipitate is taken by centrifugation, and the lotus stem fermentation product is obtained after redissolution and membrane. The grape spore Hansenia yeast is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No.32471 and a deposit date of November 4, 2024. The recommended classification name is: grape spore Hansenia yeast Hanseniasporauvarum; The yeast Saccharomycopsis fibuligera was classified and named Saccharomycopsis fibuligera, purchased from China Industrial Microbiological Culture Collection Center (CICC), and the strain number was CICC33226.

2. The preparation method according to claim 1, characterized in that: The enzymolysis comprises: mixing papain and cellulase in a mass ratio of 2:1 to 4:1 to obtain a mixed enzyme, adding the mixed enzyme to a lotus stem powder solution, and performing enzymolysis at 40 to 60° C. for 1 to 3 hours.

3. The preparation method according to claim 1 or 2, characterized in that: The seed liquid OD obtained after the activation of Hansenula viticola 600nm =1, Hansenula vitis spore seed solution, the inoculation amount is 1-10% based on the mass percentage of the fermentation system.

4. The preparation method according to claim 3, characterized in that: The seed liquid OD of the activated Saccharomyces cerevisiae was 600nm =1, the seed solution of Saccharomyces cerevisiae flavus, calculated by the mass percentage of the fermentation system, the inoculation amount is 1-10%.

5. The preparation method according to claim 1 or 4, characterized in that: The fermentation medium, in g / L, comprises 20-40 lotus stems, 15-25 peptone, 5-15 yeast extract, and 15-25 glucose; the pH value of the fermentation medium is 6.5-7.5, and the pure water is 1.0 L.

6. The preparation method according to claim 5, characterized in that: The method for preparing the fermentation medium comprises: The raw materials are mixed evenly and sterilized at 121°C and 0.1Mpa for 20 minutes.

7. The preparation method according to claim 1 or 6, characterized in that: The constant temperature culture includes culturing in a constant temperature culture shaker at 28-32° C. and 180-220 rpm for 24-48 hours.

8. The preparation method according to claim 7, characterized in that: The membrane passing includes passing through a 5-10 kD cellulose membrane.

9. The lotus stem fermentation product obtained by the preparation method according to any one of claims 1 to 8, characterized in that: The lotus stem fermentation product has the effect of inhibiting the activity of collagenase.

10. Use of the lotus stem fermentation product according to claim 9 as a moisturizing ingredient in cosmetics.

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