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

Abstract

The application discloses a preparation method of lotus stem fermentation product, the lotus stem fermentation product and application thereof, and belongs to the technical field of plant fermentation. The application provides grape Hanseniaspora uvarum which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 32471 and a preservation date of November 4, 2024. The suggested classification name is grape Hanseniaspora uvarum. The lotus stem fermentation product is obtained by fermenting lotus stems by using the strain, and has a certain inhibitory effect on collagenase activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant fermentation, and particularly relates to a preparation method of lotus stem fermentation product, the lotus stem fermentation product and application thereof. BACKGROUND

[0002] Lotus stem is the petiole or peduncle of Nelumbo nucifera Gaertn. and usually has a slender cylindrical shape, with longitudinal lines on the surface and green or yellowish green color, and relatively tough texture. Lotus stem has specific significance and application in some cultural traditions and medical fields, such as being used in some Chinese medicine prescriptions, which is considered to have certain medicinal value and can play a role in clearing heat and resolving summer-heat, promoting qi and draining water, and the main components include plant fiber, starch, vitamins, flavonoids and the like.

[0003] Collagenase can participate in the degradation of collagen, which is the main component of the skin and forms the elastic network of the skin, firmly locking water. With the growth of age, collagen is gradually lost, resulting in dryness, wrinkles, relaxation and other signs of aging 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 is mainly focused on lotus rhizome, lotus leaf and lotus heart, and it is of great significance to study the activity of other parts of the whole plant for better utilization of lotus.

[0005] At present, there are few reports on the research of lotus stem (lotus stem) in the skin moisturizing function of lotus stem, therefore, it is of great significance to research and develop the potential moisturizing function of lotus stem for the field of skin science and cosmetics. SUMMARY

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

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

[0008] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art and provide a preparation method of lotus stem fermentation product.

[0009] To solve the above technical problems, the present application provides the following technical scheme: a preparation method of lotus stem fermentation product, comprising,

[0010] The lotus stem powder is subjected to enzymolysis to obtain lotus stem enzymolysis liquid;

[0011] The enzymolysis liquid is placed in a 90-100℃ water bath for high-temperature inactivation to obtain lotus stem extract;

[0012] After the Hanseniaspora uvarum and Saccharomycopsis fibuligera are activated, they are inoculated into a fermentation medium containing the lotus stem extract, and the fermentation medium is placed in a constant-temperature incubator to obtain lotus stem fermentation liquid;

[0013] The lotus stem fermentation liquid is centrifuged, and the supernatant is taken and subjected to overnight alcohol precipitation, and then the precipitate is obtained by centrifugation and redissolved and filtered to obtain lotus stem fermentation product;

[0014] The Hanseniaspora uvarum is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 32471, a preservation date of November 4, 2024, and a recommended classification name of Hanseniaspora uvarum.

[0015] The Saccharomycopsis fibuligera has a classification name of Saccharomycopsis fibuligera and is purchased from the China Industrial Microbial Culture Collection Center CICC, and has a strain number of CICC33226.

[0016] As a preferred solution of the preparation method, the enzymolysis includes mixing papain and cellulase at a mass ratio of 2:1-4:1 to obtain mixed enzymes, adding the mixed enzymes to a lotus stem powder solution, and subjecting the lotus stem powder solution to enzymolysis at 40-60℃ for 1-3h.

[0017] As a preferred solution of the preparation method, the seed liquid prepared after the activation of the Hanseniaspora uvarum has an OD 600nm =1, the Hanseniaspora uvarum seed liquid is inoculated at a quantity of 1-10% according to the mass percentage of the fermentation system.

[0018] As a preferred solution of the preparation method, the seed liquid prepared after the activation of the Saccharomycopsis fibuligera has an OD 600nm =1, the Saccharomycopsis fibuligera seed liquid is inoculated at a quantity of 1-10% according to the mass percentage of the fermentation system.

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

[0020] As a preferred scheme of the preparation method, the fermentation medium preparation method comprises,

[0021] The raw materials are mixed uniformly, sterilized at 121 DEG C and 0.1 MPa for 20 minutes to obtain the fermentation medium.

[0022] As a preferred scheme of the preparation method, the constant temperature culture comprises culturing in a constant temperature culture shaker at 28-32 DEG C and 180-220 rpm for 24-48 hours.

[0023] As a preferred scheme of the preparation method, the membrane filtration comprises 5-10 kD cellulose membrane filtration.

[0024] Still another object of the present application is to provide a lotus stem fermentation product having the effect of inhibiting collagenase activity, so as to overcome the defects in the prior art.

[0025] Another object of the present application is to provide the application of the lotus stem fermentation product as a moisturizing ingredient in cosmetics, so as to overcome the defects in the prior art.

[0026] The present application has the following beneficial effects:

[0027] (1) The present application provides Hanseniaspora uvarum, which is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 32471 and the preservation date November 04, 2024, and the recommended classification name is Hanseniaspora uvarum. The lotus stem fermentation product obtained by fermenting lotus stems with the strain has a certain inhibitory effect on collagenase activity.

[0028] (2) The present application provides a lotus stem fermentation product preparation method, which uses Hanseniaspora uvarum and Kloeckera apiculata to mix ferment lotus stems. The fermentation process is mild, and the prepared lotus stem fermentation product has good collagenase inhibition rate. The preparation of the cream can effectively improve the transdermal water loss of the skin, and has broad application prospects in cosmetic moisturizing. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor. Among them:

[0030] Figure 1The collagenase inhibitory activity result chart of five kinds of yeast fermentation broth of lotus stem in the embodiment 1 of the present application.

[0031] Figure 2 The collagenase inhibition result chart of 19 kinds of H. uvarum mutant lotus stem fermentation in the embodiment 2 of the present application.

[0032] Figure 3 The collagenase inhibition result chart of mixed bacteria fermentation lotus stem fermentation in the embodiment 3 of the present application.

[0033] Figure 4 The collagenase inhibition result chart of different molecular weight lotus stem fermentation in the embodiment 4 of the present application. DETAILED DESCRIPTION

[0034] In order to make the above objectives, features and advantages of the present application more apparent, the specific embodiments of the present application will be described in detail below with reference to the description and embodiments.

[0035] In the following description, a lot of specific details are set forth in order to give a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the concept of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0036] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an independent or alternative embodiment that excludes other embodiments.

[0037] Embodiment 1

[0038] Screening process of single bacteria fermentation of lotus stem by yeast:

[0039] The strains of lotus stem fermentation product are screened by using yeast from five different species, which are Rhodosporidium toruloides (CICC31643), Hanseniaspora uvarum (BNCC193162), Saccharomycopsis fibuligera (CICC33226), Candida bombicola (BNCC340735) and Saccharoymces cerevisiae (CICC1210).

[0040] Pre-treatment of lotus stem:

[0041] Lotus stem is soaked in appropriate amount of water, dried in a 60℃ oven to constant weight, and then ground into powder through a 200 mesh sieve;

[0042] 1.00g of lotus stem powder is added to 50mL of phosphate buffer (pH 5-6), 10% papain (enzyme activity 200U / mg) and 5% cellulase (enzyme activity 400U / mg) are added, and the lotus stem is enzymatically hydrolyzed in a 50℃ water bath for 2h to obtain a lotus stem enzymatic hydrolysate;

[0043] The enzymatic hydrolysate is placed in a 95℃ water bath for high temperature inactivation to obtain a lotus stem extract.

[0044] YPD seed culture medium: 20.0g / L of peptone, 10.0g / L of yeast extract, 20.0g / L of glucose, 1.0L of pure water, pH adjusted to 7.0; sterilized at 121℃, 0.1Mpa for 20min;

[0045] Seed liquid culture: the yeast glycerol stock tube is taken out from the-80℃ freezer, inoculated into YPD liquid medium, and placed in a 30℃ constant temperature incubator at 200r / min for 24h to obtain a yeast seed liquid.

[0046] Fermentation medium: 20.0g / L of lotus stem extract, 20.0g / L of peptone, 10.0g / L of yeast extract, 20.0g / L of glucose, 1.0L of pure water, pH adjusted to 7.0; sterilized at 121℃, 0.1Mpa for 20min.

[0047] Lotus stem fermentation product preparation process: the prepared yeast seed liquid is diluted to OD 600nm =1, inoculated into a 250mL triangular flask containing 100mL of fermentation medium at a ratio of 10%, and cultured in a 30℃, 200rpm constant temperature incubator for 48h. The fermentation broth is inactivated and centrifuged after fermentation is completed, and the supernatant is precipitated with four times 95% ethanol to obtain a lotus stem fermentation product.

[0048] Test method of collagenase inhibitory activity: N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA; Sigma) was used as the collagenase substrate, and 10 mM PBS (pH = 7.2) was used to construct the buffer system. 50 μL of collagenase (final concentration 500 μg / mL; item number SCR103; Sigma) and 50 μL of sample solution were mixed well and placed in a 37°C environment for 10 min. Then, 100 μL of substrate solution (final concentration 0.5 mM) was added and reacted at 37°C for 30 min. After the reaction, the absorbance value at 335 nm was measured by a microplate 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] The collagenase inhibitory effect of the five yeast rhizome fermentations is shown in Table 1. Figure 1 As can be seen, the collagenase inhibitory activity of the sample was detected according to the test method of collagenase inhibitory activity, and according to the detection results, H. uvarum had the best collagenase inhibitory activity, followed by S. fibuligera.

[0051] Example 2

[0052] NTG mutagenesis of H. uvarum:

[0053] In order to further improve its collagenase inhibitory activity, NTG was used for mutagenesis of H. uvarum.

[0054] Nitrosoguanidine (NTG) mutagenesis method:

[0055] (1) NTG solution: a sterile pH 7.0 phosphate buffer solution was prepared with a concentration of 1 mg / mL solution.

[0056] (2) Preparation of the strain: the logarithmic growth phase bacterial solution was centrifuged at 3000 rpm for 5 min, washed with physiological saline for 3 times, and resuspended with physiological saline to an OD600 of 1.

[0057] (3) Mutagenesis treatment: 1 mL of bacterial suspension was added with 100 μL of NTG solution, and after 1 h of reaction at 37°C, 50 μL was plated on MRS medium.

[0058] (4) After mutagenesis, H. uvarum was obtained on YPD medium, and after subculture and preservation, the rhizome was fermented and alcohol precipitated according to the method of Example 1 to obtain rhizome fermentation, and the collagenase inhibitory activity of the supernatant was detected.

[0059] 19 kinds of H. uvarum mutant lotus stem fermentations collagenase inhibition effect see Figure 2 , the results show that HU9 is positive mutagenesis, and its collagen inhibition activity is significantly improved. It is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 32471, the preservation date is November 04, 2024, and the recommended classification name is: Hanseniaspora uvarum.

[0060] Example 3

[0061] Screening process of single strain fermentation of lotus stem by yeast:

[0062] Compared with single strain fermentation, mixed strain fermentation has many advantages such as improving fermentation efficiency, increasing product types and yield, and reducing cost, etc., so HU9 and S. fibuligera are used for mixed strain fermentation of lotus stem.

[0063] The method in Example 1 is used to ferment lotus stem, and the preparation process of lotus stem fermentation in Example 1 is modified as follows:

[0064] The cultured yeast seed liquid is diluted to OD 600nm =1, HU9 and S. fibuligera are inoculated at a ratio of 5% at the same time into a 250 mL triangular flask containing 100 mL fermentation medium, and cultured in a constant temperature incubator at 30°C, 200 rpm for 48 h. The fermentation broth is inactivated and centrifuged, and the supernatant is precipitated with four times 95% ethanol to obtain lotus stem fermentation, marked as S1.

[0065] Comparative Example 1

[0066] Reference Example 3 lotus stem fermentation preparation process, the fermentation process is modified as follows:

[0067] First, inoculate HU9 strain into fermentation medium at a ratio of 5% for 24 h, sterilize, then inoculate 5% S. fibuligera for 24 h, and obtain the lotus stem fermentation, marked as S2.

[0068] Comparative Example 2

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

[0070] First, inoculate S. fibuligera strain into fermentation medium at a ratio of 5% for 24 h, sterilize, then inoculate 5% HU9 for 24 h to obtain the lotus stem fermentation, marked as S3.

[0071] The lotus stem ferment was diluted to 5%, and the collagenase inhibitory activity of the sample was detected according to the collagenase inhibitory activity detection method in Example 1. The collagenase inhibitory effect of the mixed bacteria fermented lotus stem ferment is shown in Table 1. Figure 3 The collagenase inhibitory activity of the sample was detected according to the collagenase inhibitory activity detection method. According to the detection results, S1 has the best collagenase inhibitory activity.

[0072] Example 4

[0073] Detection of collagenase inhibitory activity of lotus stem ferment with different molecular weights:

[0074] The lotus stem ferment was diluted to 5%, and the collagenase inhibitory activity of the sample was detected according to the collagenase inhibitory activity detection method in Example 1.

[0075] The lotus stem ferment obtained in Example 3 was filtered by cellulose membrane-10KDa, and the filtrate 1 with molecular weight <10kD and the filtrate 2 with molecular weight >10kD were collected.

[0076] The filtrate 1 was further filtered by cellulose membrane-5KDa, and the filtrate 2 with molecular weight <5kD and the filtrate 2 with molecular weight 5kD-10kD were collected.

[0077] The filtrate 2 with molecular weight <5kD was collected.

[0078] Comparative Example 3

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

[0080] Comparative Example 4

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

[0082] The collagenase inhibitory effect of lotus stem ferment with different molecular weights is shown in Table 1. Figure 4 It can be seen that the lotus stem ferment with molecular weight <5kD has the best collagenase inhibitory effect.

[0083] Example 5

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

[0085] In order to better evaluate the activity of the lotus stem ferment, the lotus stem ferment was prepared into a cream, and the effect of the lotus stem ferment on human body was evaluated. The components of the cream are shown in Table 1.

[0086] The preparation method is as follows: the components of the water phase A and the oil phase B are weighed according to Table 1, and heated in a water bath at 80°C to dissolve;

[0087] Then, B phase is added to A phase and stirred to emulsify uniformly;

[0088] When the temperature drops to about 40℃, add the C-phase lotus stamen ferment and stir until uniform;

[0089] The cream prepared by adding the lotus stem ferment of Example 4 is the sample group;

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

[0091] Table 1 Composition of the lotus stem ferment cream group

[0092]

[0093]

[0094] Ten volunteers aged 25-40 years old were selected for efficacy evaluation. The inner sides of the left and right forearms were selected as the test area, and immediate testing was performed. The changes in the water content of the stratum corneum and the trans-epidermal water loss before and after use were compared.

[0095] The test conditions were: constant temperature of 20℃ and humidity of 60%. After the volunteers cleaned their arms, the samples were applied to the designated areas. After waiting for 60 minutes in a constant temperature and humidity room, the test was started. The results are shown in Table 2.

[0096] Among them, the lotus stem ferment obtained in Example 4 was used as the experimental group, water was used to replace the lotus stem ferment as the blank group, and the unfermented lotus stem extract was used as Comparative Example 5.

[0097] Table 2 Water content of the stratum corneum and trans-epidermal water loss (TEWL) of the volunteers

[0098]

[0099] It can be seen that compared with the blank group without adding lotus stem active substances, after using the product, the water content of the stratum corneum of the volunteers increased, and the trans-epidermal water loss decreased. The activity of the fermented lotus stem was better than that before fermentation.

[0100] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the present application.

Claims

1. A method for preparing lotus stem ferment, characterized in that: include, Lotus stem powder was enzymatically hydrolyzed to obtain lotus stem hydrolysate, wherein the enzymes used in the hydrolysis were papain and cellulase. The enzymatic hydrolysate was inactivated at high temperature in a water bath at 90℃~100℃ to obtain lotus stem extract; After activating Hansenula polymorpha and Bacillus subtilis, they were simultaneously inoculated into a fermentation medium containing lotus stem extract and cultured in a shaker at a constant temperature to obtain lotus stem fermentation broth. Centrifuge the lotus stem fermentation broth, collect the supernatant, precipitate it overnight with alcohol, centrifuge again to collect the precipitate, redissolve it, and pass it through a membrane to obtain the lotus stem fermentation product. The *Hansenula polymorpha* species described herein is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32471, and the deposit date is November 4, 2024. The proposed classification name is *Hansenula polymorpha*. Hanseniaspora uvarum ; The classification of *Cytosporum tobira* is named as follows: Saccharomycopsis fibuligera The strain was purchased from the China Industrial Microbial Culture Collection Center (CICC), with strain number CICC33226.

2. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis includes mixing papain and cellulase in a mass ratio of 2:1 to 4:1 to obtain a mixed enzyme, adding it to a lotus stem powder solution, and hydrolyzing 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 prepared by activating *Hansenula polymorpha* of grapes 600nm =1, grape Hansenula polysaccharide seed culture, the inoculum amount is 1~10% by weight percentage of fermentation system.

4. The preparation method according to claim 3, characterized in that: The seed liquid OD prepared after activation of the *Saccharomyces cerevisiae* 600nm =1, the inoculum of *Saccharomyces cerevisiae* seed culture, based on the mass percentage of the fermentation system, 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 g of lotus stem extract, 15-25 g of peptone, 5-15 g of yeast extract, and 15-25 g of glucose; the pH 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 includes, The raw materials are mixed evenly and then sterilized at 121℃ and 0.1 MPa for 20 minutes to obtain the final product.

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

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

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

10. The application of lotus stem ferment as described in claim 9 in the preparation of cosmetics, wherein the lotus stem ferment is used as a moisturizing ingredient.

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