Composite wall-breaking enzymolysis fermentation product of ulva lactuca as well as preparation method and application of composite wall-breaking enzymolysis fermentation product

Abstract

The invention discloses an ulva lactuca composite wall-breaking enzymolysis fermentation product as well as a preparation method and application thereof, and relates to the field of cosmetics. The preparation method comprises the following steps: soaking ulva powder, and performing physical cell wall breaking treatment; mixed enzymes are added for an enzymolysis reaction, wherein the mixed enzymes comprise lipase, cell wall breaking enzyme, enzyme and protease; adding the enzymolysis product into a culture medium, and inoculating bacterial liquid for fermentation culture to obtain fermentation liquid. According to the application, active ingredients such as macromolecular polysaccharide and protein in the ulva lactuca are extracted and decomposed by adopting physical cell wall breaking, composite enzymolysis and bifidus yeast fermentation technologies, and the prepared fermentation product is rich in various composite micromolecular active ingredients such as oligosaccharide, polypeptide, amino acid, polyphenols and flavonoids, has high free radical scavenging capacity, and can be used for preparing the ulva lactuca beverage. The anti-wrinkle and anti-oxidation composition has good anti-wrinkle and anti-oxidation effects, is beneficial to skin absorption when being applied to cosmetics and has high absorption efficiency, and the prepared cosmetics have excellent anti-wrinkle, anti-oxidation, soothing and repairing effects.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular to a composite wall-breaking enzymatic hydrolysis and fermentation product of ulvae and a preparation method and application thereof. Background Art

[0002] Ulva lactuca, also known as sea lettuce and sea cabbage, is a large marine economic algae belonging to the genus Ulva of the family Ulva of the Chlorophyta. According to literature reports, Ulva algae are rich in carbohydrates, proteins and crude fiber, and contain a small amount of fat (mainly unsaturated fatty acids), polyphenols and flavonoids. Ulva polysaccharides, as the main active ingredient of Ulva, have a variety of biological activities, such as immunomodulation, anti-inflammatory, anticoagulant, anti-tumor, antioxidant, anti-high fat, and plant growth promotion. Therefore, Ulva polysaccharides have potential application prospects in biomedicine, cosmetics, food, health products and other industries.

[0003] However, the molecular weight of Ulva polysaccharides obtained by the current extraction methods is relatively large, and there are defects such as poor solubility and low bioavailability, which hinder the high-value development and utilization of Ulva polysaccharide resources. The current treatment of Ulva is mostly concentrated on simple extraction, enzymatic hydrolysis or fermentation. Due to the large molecular weight of Ulva polysaccharides and proteins in its extracted products and the difficulty in absorption, it is rarely used in the cosmetics industry. Its extract has little effect in cosmetics and is only used as a moisturizer and soothing agent. Summary of the invention

[0004] The present invention provides a composite cell wall-breaking enzymatic hydrolysis fermentation product of ulva and a preparation method and application thereof. Macromolecular proteins and active substances in ulva are effectively extracted and decomposed through cell wall breaking, composite enzymatic hydrolysis and bacterial fermentation. The molecular weight of the active ingredients of ulva in the obtained fermentation product is relatively small. The application of the product in cosmetics is beneficial to improving the absorption rate of the skin to the functional ingredients and the product has a strong antioxidant capacity.

[0005] In order to solve the above technical problems, one of the purposes of the present invention is to provide a method for preparing a product of Ulva composite wall-breaking enzymatic hydrolysis and fermentation, comprising the following steps:

[0006] (1) adding Ulva powder to water, and then performing physical cell wall breaking treatment after soaking;

[0007] (2) adding a mixed enzyme to the solution for enzymatic hydrolysis, followed by water extraction, centrifugation, and filtration to obtain an enzymatic hydrolysis product, wherein the mixed enzyme includes lipase, cell wall-breaking enzyme, carbohydrase, and protease;

[0008] (3) adding the enzymatic hydrolysis product to the culture medium, then inoculating the bacterial liquid into the culture medium, fermenting and culturing to obtain a fermentation liquid, and obtaining a fermentation product after centrifugation and filtration, wherein the bacterial species in the bacterial liquid is Bifidobacterium longum or Lactobacillus plantarum.

[0009] The present application pre-treats the Ulva cells to break the cell wall, so that the proteins, active substances and other effective ingredients in the cells are fully dissolved in water, the cell wall is broken by enzymatic hydrolysis of the cell wall enzyme so that the polysaccharides and proteins are fully extracted, and are decomposed into small molecular active substances by lipase, carbohydrase and protease, and the enzymatic hydrolysate is used as a substrate, and the unique bifida yeast fermentation technology is utilized to enrich more small molecular active oligosaccharides and polypeptides through microbial metabolism, while also retaining the active ingredients of phenolic and flavonoid substances. The fermentation product finally prepared contains a variety of complex small molecular active ingredients such as oligosaccharides, polypeptides, amino acids, polyphenols and flavonoids, has high free radical scavenging ability, strong antioxidant ability, and when used in cosmetics, the small molecular weight is conducive to skin absorption, thereby improving the absorption efficiency of the effective ingredients, and the resulting product is highly safe, and the prepared cosmetics have high anti-wrinkle, antioxidant, soothing and repair effects.

[0010] As a preferred embodiment, the culture medium includes 0.5wt%-3wt% soy peptone, 0.02wt%-0.05wt% histidine, 0.1wt%-0.3wt% dipotassium hydrogen phosphate, 0.05wt%-0.1wt% magnesium chloride, 0.01wt%-0.02wt% calcium chloride, 0.002wt%-0.008wt% manganese sulfate, 0.2wt%-0.8wt% sodium acetate, 0.02wt%-0.08wt% L-cysteine ​​and the balance water.

[0011] The enzymatic hydrolysate of Ulva ulvae in the present application is added to the culture medium as a carbon source. By controlling the addition amount of the carbon source and the nitrogen source soy peptone, the growth and reproduction of the fermentation bacteria are effectively promoted, the fermentation effect is improved, and more small-molecule active oligosaccharides and polypeptides are enriched, so that the antioxidant activity content of the fermentation product is increased, the free radical scavenging efficiency is improved, and the antioxidant capacity is strong.

[0012] As a preferred embodiment, in step (1), ulva powder is added into water at a ratio of 1 wt% to 5 wt%.

[0013] As a preferred embodiment, in step (1), the cell wall is broken by using a cell wall breaking machine for 10 min-30 min, with a stirring power of 800-1200 W and a stirring speed of 30000-40000 r / min; followed by ultrasonic treatment for 10 min-30 min, with an ultrasonic power of 120-160 W and an ultrasonic frequency of 30-50 kHz.

[0014] As a preferred embodiment, in step (2), a mixed enzyme is added to the solution in a ratio of 3.5-15wt% for enzymatic hydrolysis reaction, and the mixed enzyme includes 14wt%-45wt% of protease, 2wt%-28wt% of cell wall-disrupting enzyme, 10wt%-55wt% of carbohydrase and 3wt%-36wt% of lipase.

[0015] As a preferred embodiment, the cell wall-disrupting enzyme is cellulase, the carbohydrase is β-glucanase, and the protease is neutral protease.

[0016] As a preferred embodiment, the enzyme mixture comprises 22wt%-44wt% neutral protease, 2wt%-13wt% cellulase, 43wt%-44wt% β-glucanase and 11-22wt% lipase.

[0017] The present application controls the ratio of adding different enzymes during the enzymatic hydrolysis reaction stage, thereby increasing the content of antioxidant active molecules in the enzymatic hydrolysis product, which is beneficial for the cell wall-breaking enzyme and lipase to fully dissolve the cells, so that the active substances are dissolved in the solution. At the same time, the addition amount of neutral protease and carbohydrase is controlled to be high, which helps to improve the enzymatic hydrolysis efficiency of large molecules, increase the concentration of active ingredients, and improve antioxidant capacity.

[0018] As a preferred embodiment, in step (3), the enzymatic hydrolysis product is added to the blank culture medium at a ratio of 20wt%-80wt%.

[0019] As a preferred solution, in step (3), the concentration of the bacterial solution is 2.8*10 8 CFU / mL and above.

[0020] As a preferred embodiment, in step (3), the fermentation culture time is 12-36 hours.

[0021] As a preferred embodiment, in step (2), the water extraction temperature is 40°C-60°C, the extraction time is 2h-5h, and the filtration is performed using 0.3μm-0.8μm filter paper.

[0022] In order to solve the above technical problems, the second purpose of the present invention is to provide a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva.

[0023] In order to solve the above technical problems, the third purpose of the present invention provides an application of a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva in the field of preparing cosmetics.

[0024] As a preferred solution, the mass fraction of the Ulva composite wall-breaking enzymatic hydrolysis and fermentation product in the cosmetics is 0.1wt%-2.5wt%.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present application adopts physical cell wall breaking, composite enzymatic hydrolysis and bifid yeast fermentation technology to extract and decompose active ingredients such as macromolecular polysaccharides and proteins in Ulva. Cell wall breaking enzymes, proteases, lipases and carbohydrases fully dissolve the cells in the broken wall extracts, and the macromolecular substances are dissolved in the solution, which are converted into small molecular substances using composite enzymes. Subsequently, Bifidobacterium longum is used to ferment the culture medium containing the enzymatic hydrolysate. The prepared fermentation product contains rich oligosaccharides, polypeptides, amino acids, polyphenols, flavonoids and other composite small molecule active ingredients, has high free radical scavenging ability, and strong antioxidant ability.

[0027] 2. During the physical cell wall breaking, compound enzymatic hydrolysis and bifid yeast fermentation process of the present application, the macromolecular polysaccharides and proteins in the Ulva cells are decomposed into small molecular active functional ingredients. When applied to cosmetics, the small molecular weight is conducive to skin absorption, effectively improving the absorption efficiency of the functional ingredients, and the resulting products are highly safe. The prepared cosmetics have excellent anti-wrinkle, antioxidant, soothing and repairing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The Ulva wall-breaking products prepared in Test Examples 1-7 of the present invention (Note: from left to right are Test Examples 1, 3, 2, 4, 5, 7, 6);

[0029] Figure 2 : the Ulva wall-breaking enzymatic fermentation products prepared in Test Examples 30, 41-44 of the present invention (Note: the first one is Test Example 32, the third one is Test Example 44, the fifth one is Test Example 42, the sixth one is Test Example 43, and the seventh one is Test Example 41);

[0030] Figure 3 : The enzymatic fermentation products of Ulva wall-breaking prepared in Test Examples 36, 38-40 of the present invention (Note: Test Examples 36, 38, 39, 40, and 41 are from top right to bottom). DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Test Example 1

[0033] A method for preparing a ulva product comprises the following steps:

[0034] (1) adding ulva powder to deionized water, wherein the proportion of ulva powder in the solution is 2 wt%, and soaking for 20 minutes;

[0035] (2) The mixture was then stirred and extracted at a constant temperature of 55°C for 4 h, and then centrifuged at a speed of 10,000 rpm for 15 min. The supernatant was filtered using a 5 μm filter paper to obtain the Ulva product.

[0036] Test Example 2

[0037] A method for preparing a Ulva wall-breaking product comprises the following steps:

[0038] (1) adding ulva powder to deionized water, wherein the proportion of ulva powder in the solution is 2 wt%, and soaking for 20 minutes;

[0039] (2) Use a wall-breaking machine at high setting to break the wall for 20 min, with a stirring power of 1000 W and a stirring speed of 35000 r / min, then stir and extract with water at a constant temperature of 55°C for 4 h, centrifuge at a speed of 10000 rpm for 15 min, and filter the supernatant with 5 μm filter paper to obtain the Ulva wall-breaking product.

[0040] Test Example 3

[0041] A method for preparing a Ulva wall-breaking product comprises the following steps:

[0042] (1) adding ulva powder to deionized water, wherein the proportion of ulva powder in the solution is 2 wt%, and soaking for 20 minutes;

[0043] (2) Then, ultrasonic treatment was performed for 20 min at an ultrasonic power of 150 W and an ultrasonic frequency of 40 kHz. The mixture was stirred and extracted with water at a constant temperature of 55° C. for 4 h. The mixture was centrifuged at a speed of 10,000 rpm for 15 min. The supernatant was filtered using a 5 μm filter paper to obtain the Ulva wall-breaking product.

[0044] Test Example 4

[0045] A method for preparing a Ulva wall-breaking product comprises the following steps:

[0046] (1) adding ulva powder to deionized water, wherein the proportion of ulva powder in the solution is 2 wt%, and soaking for 20 minutes;

[0047] (2) The mixture was then frozen at -80°C and then thawed, and the freeze-thaw process was repeated three times for 24 hours. The mixture was then stirred and extracted with water at a constant temperature of 55°C for 4 hours. The mixture was centrifuged at a speed of 10,000 rpm for 15 minutes, and the supernatant was filtered using a 5 μm filter paper to obtain the Ulva wall-breaking product.

[0048] Test Example 5

[0049] A method for preparing a Ulva wall-breaking product comprises the following steps:

[0050] (1) adding ulva powder to deionized water, wherein the proportion of ulva powder in the solution is 2 wt%, and soaking for 20 minutes;

[0051] (2) Use a wall-breaking machine at High setting to break the wall for 20 min, with a stirring power of 1000 W and a stirring speed of 35000 r / min, followed by ultrasonication for 20 min, with an ultrasonic power of 150 W and an ultrasonic frequency of 40 kHz, followed by stirring and water extraction at a constant temperature of 55°C for 4 h, centrifuging at a speed of 10000 rpm for 15 min, and filtering the supernatant with 5 μm filter paper to obtain the Ulva wall-breaking product.

[0052] Test Example 6

[0053] A method for preparing a Ulva wall-breaking product comprises the following steps:

[0054] (1) adding ulva powder to deionized water, wherein the proportion of ulva powder in the solution is 2 wt%, and soaking for 20 minutes;

[0055] (2) The cells were then broken with a wall-breaking machine at high setting for 20 min, with a stirring power of 1000 W and a stirring speed of 35,000 r / min. The cells were then frozen at -80°C and thawed three times for 24 h. The cells were then stirred and extracted with water at a constant temperature of 55°C for 4 h. The cells were centrifuged at a speed of 10,000 rpm for 15 min. The supernatant was filtered with a 5 μm filter paper to obtain the Ulva wall-breaking product.

[0056] Test Example 7

[0057] A method for preparing a Ulva wall-breaking product comprises the following steps:

[0058] (1) adding ulva powder to deionized water, wherein the proportion of ulva powder in the solution is 2 wt%, and soaking for 20 minutes;

[0059] (2) Then, ultrasonic treatment was performed for 20 min at an ultrasonic power of 150 W and an ultrasonic frequency of 40 kHz. The mixture was then frozen at -80°C and then thawed. The freeze-thaw process was repeated three times for 24 h. The mixture was stirred and extracted with water at a constant temperature of 55°C for 4 h. The mixture was centrifuged at a speed of 10,000 rpm for 15 min. The supernatant was filtered using a 5 μm filter paper to obtain the Ulva wall-breaking product.

[0060] The solid content of the Ulva wall-breaking products prepared in Test Examples 1-7 was measured, and the dry matter yield was used as a characterization index of the wall-breaking technology. The measurement results are shown in Table 1 below. It can be found that the solid content of the Ulva water extract product without cell wall breaking treatment was only 32.58%, while the cell wall breaking efficiency was optimal after the cell wall was broken by combining homogenization with a wall-breaking machine and ultrasonic treatment, and the content of active substances, protein, fat and other extracted substances in the extract was the highest, and the solid content could reach 49.58%.

[0061] Among them, the solid content test method is as follows: accurately weigh 2.0g of the sample liquid, accurate to 0.001g, place it in a glass culture dish that has been weighed and kept constant at 105℃, put it in a 105℃ forced air constant temperature oven and dry it for 3 hours until constant weight, take out the sample, put it in a dryer to cool to room temperature, and weigh its mass.

[0062] The solid content is calculated according to the formula: solid content % = sample mass after heating / sample mass before heating × 100. The test result is the average of two parallel tests, and the experimental result is rounded to two decimal places.

[0063] Table 1 - Solid content test results of Ulva wall-breaking products in test examples 1-7 of this application

[0064]

[0065]

[0066] Test Example 8-26

[0067] A method for preparing a composite wall-breaking enzymatic hydrolyzate of Ulva ulvae comprises the following steps:

[0068] (1) adding ulva powder to deionized water, the proportion of ulva powder in the solution is 2wt%, soaking for 20min, breaking the wall with a wall breaking machine at High gear for 20min, stirring power of 1000W, stirring speed of 35000r / min, and then ultrasonicating for 20min, ultrasonic power of 150W, and ultrasonic frequency of 40kHz;

[0069] (2) Enzyme was added to the solution (water was used as the blank group in Test Example 8) to carry out enzymatic hydrolysis. The enzymatic hydrolysis reaction was carried out at 55° C. for 4 h. The mixed liquid after the reaction was completed was centrifuged at a rate of 10,000 rpm for 15 min. The supernatant was filtered using a 5 μm filter paper to obtain the enzymatic hydrolysis product.

[0070] Among them, the enzyme components and contents added in step (2) of Test Example 8-26 are shown in Table 2 below. The obtained enzymatic hydrolysate was added with water to a concentration of 25wt%, and a 50ppm ABTS antioxidant scavenging rate test was performed. The ABTS antioxidant scavenging rate was tested with reference to the national standard "GB / T 39100---2020 Determination of Antioxidant Activity of Polypeptides by DPPH and ABTS Method". The test results are shown in Table 2 below, and the specific commodity source information of the added enzyme is shown in Table 3.

[0071] Table 2 - Test results of 50ppm ABTS antioxidant scavenging rate of enzymatic hydrolyzates in Experimental Examples 8-26 of this application

[0072]

[0073]

[0074]

[0075] Table 3 - Product information of different enzymes added in the examples and comparative examples of this application

[0076]

[0077]

[0078] The inventors conducted multiple tests on the enzymatic hydrolysis of Ulva and found that the enzymatic hydrolysis product of Ulva wall-breaking solution was subjected to enzymatic hydrolysis by neutral protease, cellulase, β-glucanase and lipase, and had a high ABTS antioxidant scavenging rate, and the enzyme addition amount was controlled within the range of 3.6wt%-12wt% to have a high ABTS antioxidant scavenging rate; in addition, the enzyme was controlled to contain 14wt%-45wt% of neutral protease, 2wt%-28wt% of cellulase, 10wt%-55wt% of β-glucanase and 3wt%-36wt% of lipase, which could ensure that the enzymatic hydrolysis product had a high ABTS antioxidant scavenging rate and improve the antioxidant capacity. When the enzyme was added in the proportion of 43.5wt% of neutral protease, 2wt% of cellulase, 43.5wt% of β-glucanase and 11wt% of lipase, the enzymatic hydrolysis product had the highest ABTS antioxidant scavenging rate.

[0079] Test Examples 27-32

[0080] A method for preparing a composite wall-breaking enzymatic fermentation product of Ulva ulvae comprises the following steps:

[0081] (1) The concentration of Bifidobacterium longum obtained by activating the culture of strain ATCC15707 was 2.0×10 8 Bacterial solution with CFU / mL or above;

[0082] (2) Adding the test example 20 to the blank medium at a ratio of 0wt% to 80wt% to prepare an enzymatic hydrolysate, the specific amount of the enzymatic hydrolysate added is shown in Table 3 below, and then inoculating 3wt% of the bacterial liquid into the medium, fermenting and culturing at 37°C for 26h to obtain a fermentation liquid;

[0083] (3) The fermentation broth was centrifuged at 10,000 rpm for 15 min, and the supernatant was collected and filtered using a 5 μm filter paper to obtain the fermentation product.

[0084] Wherein, the blank culture medium of step (2) comprises 1wt% soy peptone, 0.02wt% histidine, 0.2wt% dipotassium hydrogen phosphate, 0.05wt% magnesium chloride, 0.015wt% calcium chloride, 0.005wt% manganese sulfate, 0.5wt% sodium acetate, 0.05wt% L-cysteine ​​and the balance water.

[0085] The fermentation product prepared in step (3) of Test Example 27-32 was measured for OD value by absorbance method. 200ul of the fermentation product was taken into a 96-well plate by a pipette, and the absorbance was measured at 600nm on a microplate reader. At the same time, the fermentation product was diluted 8 times (i.e., the concentration was 12.5%) and then the 50ppm DPPH scavenging rate was measured. The DPPH free radical scavenging ability was tested with reference to the national standard "GB / T 39100-2020 Determination of the Antioxidant Activity of Polypeptides by DPPH and ABTS Method". The measurement results are shown in Table 3 below.

[0086] Table 3 - Test results of Ulva composite wall-breaking enzymatic fermentation products in test examples 27-32 of this application

[0087]

[0088] Test Examples 33-38

[0089] A method for preparing a composite wall-breaking enzymatic fermentation product of Ulva ulvae comprises the following steps:

[0090] (1) The concentration of Bifidobacterium longum obtained by activating the culture of strain ATCC15707 was 2.0×10 8 Bacterial solution with CFU / mL or above;

[0091] (2) adding 60 wt % of the enzymatic hydrolyzate prepared in Test Example 20 to a blank medium, then inoculating 3 wt % of the bacterial solution into the medium, and fermenting and culturing at 37 ° C for 26 h to obtain a fermentation broth;

[0092] (3) The fermentation liquid was centrifuged at 1000 rpm for 15 min, and the supernatant was collected and filtered through a 5 μm filter paper to obtain the fermentation product.

[0093] Wherein, the blank culture medium of step (2) comprises 0-3wt% soy peptone, 0.02wt% histidine, 0.2wt% dipotassium hydrogen phosphate, 0.05wt% magnesium chloride, 0.015wt% calcium chloride, 0.005wt% manganese sulfate, 0.5wt% sodium acetate, 0.05wt% L-cysteine ​​and the balance water, and the specific content of soy peptone is shown in Table 4 below.

[0094] The fermentation product prepared in step (3) of Test Examples 33-38 was measured for OD value by absorbance method. The fermentation product was diluted 8 times and then the 50 ppm DPPH scavenging rate was measured. The DPPH free radical scavenging ability was tested with reference to the national standard "GB / T 39100-2020 Determination of Antioxidant Activity of Polypeptides by DPPH and ABTS Method". The measurement results are shown in Table 4 below.

[0095] Test Example 39

[0096] A method for preparing a composite wall-breaking enzymatic fermentation product of Ulva ulvae comprises the following steps:

[0097] (1) adding ulva powder to deionized water, wherein the proportion of ulva powder in the solution is 2 wt%, and soaking for 20 min, adding enzymes in a ratio of 4.6 wt% to the solution for enzymatic hydrolysis, wherein the enzymes include 43.5 wt% neutral protease, 2 wt% cellulase, 43.5 wt% β-glucanase and 11 wt% lipase, and performing enzymatic hydrolysis at 55° C. for 4 h, centrifuging the mixed liquid after the reaction at a rate of 10,000 rpm for 15 min, and filtering the supernatant with a 5 μm filter paper to obtain an enzymatic hydrolysis product;

[0098] (2) The concentration of Bifidobacterium longum obtained by activation culture of strain ATCC15707 was 2.0×10 8 Bacterial solution with CFU / mL or above;

[0099] (3) Adding the enzymatic hydrolyzate prepared in Test Example 20 at a ratio of 60 wt % to the blank culture medium, then inoculating 3 wt % of the bacterial solution into the culture medium, and fermenting and culturing at 37 ° C for 26 h to obtain a fermentation broth;

[0100] (4) The fermentation liquid was centrifuged at 1000 rpm for 15 min, and the supernatant was collected and filtered through a 5 μm filter paper to obtain the fermentation product.

[0101] Wherein, the blank culture medium of step (3) comprises 1.5wt% soy peptone, 0.02wt% histidine, 0.2wt% dipotassium hydrogen phosphate, 0.05wt% magnesium chloride, 0.015wt% calcium chloride, 0.005wt% manganese sulfate, 0.5wt% sodium acetate, 0.05wt% L-cysteine ​​and the balance water.

[0102] Test Example 40

[0103] A method for preparing a composite wall-breaking enzymatic fermentation product of Ulva ulvae comprises the following steps:

[0104] (1) adding ulva powder to deionized water, the proportion of ulva powder in the solution is 2wt%, soaking for 20min, breaking the wall with a wall breaking machine at High gear for 20min, stirring power of 1000W, stirring speed of 35000r / min, and then ultrasonicating for 20min, ultrasonic power of 150W, and ultrasonic frequency of 40kHz;

[0105] (2) adding water in a ratio of 4.6 wt % to the solution for enzymatic hydrolysis reaction, and performing the enzymatic hydrolysis reaction at 55° C. for 4 h. After the reaction, the mixed liquid was centrifuged at a rate of 10,000 rpm for 15 min, and the supernatant was filtered using a 5 μm filter paper to obtain the enzymatic hydrolysis product;

[0106] (3) Adding the enzymatic hydrolyzate prepared in Test Example 20 at a ratio of 60 wt % to the blank medium, then adding 3 wt % of water to the medium, and fermenting and culturing at 37 ° C for 26 h to obtain a fermentation broth;

[0107] (4) The fermentation liquid was centrifuged at 1000 rpm for 15 min, and the supernatant was collected and filtered through a 5 μm filter paper to obtain the fermentation product.

[0108] Wherein, the blank culture medium of step (3) comprises 1.5wt% soy peptone, 0.02wt% histidine, 0.2wt% dipotassium hydrogen phosphate, 0.05wt% magnesium chloride, 0.015wt% calcium chloride, 0.005wt% manganese sulfate, 0.5wt% sodium acetate, 0.05wt% L-cysteine ​​and the balance water.

[0109] Table 4 - Test results of the Ulva composite wall-breaking enzymatic fermentation products in Test Examples 33-40 of this application

[0110]

[0111]

[0112] As shown in Table 4, compared with Test Example 36, the Ulva powder in Test Example 39 was not subjected to cell wall disruption treatment. During the subsequent enzymatic hydrolysis and fermentation process, the intracellular polysaccharides and proteins could not be fully extracted, and the content of active ingredients in the enzymatic hydrolysis and fermentation products was low, resulting in low efficiency in scavenging free radicals. After the enzymatic hydrolysis products in Test Example 40 were mixed with the culture medium, Bifidobacterium longum was not introduced for fermentation. The content of various complex small molecule active ingredients such as oligosaccharides, polypeptides, amino acids, polyphenols and flavonoids in the products was low, and the antioxidant capacity was insufficient.

[0113] Test Examples 41-44

[0114] A method for preparing a composite wall-breaking enzymatic fermentation product of Ulva ulvae comprises the following steps:

[0115] (1) using bacterial strains for activation culture to obtain bacterial liquid, the bacterial strains are Bifidobacterium longum, Saccharomyces cerevisiae, and Bacillus subtilis of ATCC15707;

[0116] (2) adding 60 wt % of the enzymatic hydrolyzate prepared in Test Example 20 to a blank medium, then inoculating 3 wt % of the bacterial solution into the medium, and fermenting and culturing at 37 ° C for 26 h to obtain a fermentation broth;

[0117] (3) The fermentation liquid was centrifuged at 1000 rpm for 15 min, and the supernatant was collected and filtered through a 5 μm filter paper to obtain the fermentation product.

[0118] Wherein, the blank culture medium of step (2) comprises 1wt% soy peptone, 0.02wt% histidine, 0.2wt% dipotassium hydrogen phosphate, 0.05wt% magnesium chloride, 0.015wt% calcium chloride, 0.005wt% manganese sulfate, 0.5wt% sodium acetate, 0.05wt% L-cysteine ​​and the remainder water, and the specific content of soy peptone is shown in Table 5 below.

[0119] The fermentation product prepared in step (3) of Test Examples 41-44 was diluted 16 times and then the 50 ppm DPPH clearance rate was measured. The ABTS clearance rate was tested with reference to the national standard "GB / T 39100-2020 Determination of Antioxidant Activity of Polypeptides by DPPH and ABTS Method". The results are shown in Table 5 below.

[0120] Table 5 - Test results of Ulva composite wall-breaking enzymatic fermentation products in Test Examples 41-44

[0121]

[0122]

[0123] Table 6 - Information on the source of bacteria used in the fermentation process of this application

[0124]

[0125] The fermentation products prepared in Test Examples 36, 38, 39, and 40 were used for cytotoxicity tests and cosmetic efficacy tests:

[0126] 1. The cell irritation test method is based on the chicken embryo chorioallantoic membrane test of SN / T 2329-2009 Cosmetic Eye Irritation / Corrosion. The test results are shown in Table 7 below.

[0127] 2. Cytotoxicity test method: Take cell culture and inoculate it in a 96-well plate at an appropriate density. The cell culture is human skin fibroblast HFF-1 and mouse mononuclear macrophage leukemia cell line RAW264.7, respectively. The inoculation volume per well is 100 μL, and it is cultured in a carbon dioxide incubator for 24 hours. After culture, discard the culture medium in the 96-well plate, add 0.625%, 1.25%, 2.5%, 5%, 10%, 20% of the test solution of different concentrations, 100 μL / well, and set up a blank control group and a positive control group at the same time. The positive substances are 100 ng / mL TGF-β (Okai Biotechnology) and 2 mg / ml dexamethasone sodium phosphate, respectively. Each concentration is added to 6 replicate wells and placed in a carbon dioxide incubator for 24 hours. After the culture is completed, observe and record the cell state, discard the culture medium, wash 1-2 times with PBS, add MTT working solution, 50 μL / well, and place in a carbon dioxide incubator for 4 hours. The culture medium was discarded, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 min. The absorbance at 570 nm was measured using an ELISA reader, and 630 nm was used as the reference wavelength. The cell survival rate was calculated based on the measurement results. When the cell survival rate of the sample at the corresponding concentration in the 96-well plate was ≥90%, and there was no significant difference in the morphology between the cells in the sample group and the cells in the blank group, it was indicated that the sample had no significant cytotoxicity at this concentration. The test results are shown in Table 7 below.

[0128] Cell survival rate formula: Cell survival rate (%) = OD sample / OD blank × 100%,

[0129] In the formula: OD sample-the mean OD value of the sample group; OD blank-the mean OD value of the blank control group.

[0130] 3. Cell anti-wrinkle and firming test method:

[0131] 1) Cell inoculation: Human skin fibroblasts HFF-1 were inoculated into a 96-well plate at an appropriate density (the degree of confluence reached 60% 24 hours after inoculation), 100 μL per well, and cultured in a CO2 incubator for 24 hours;

[0132] 2) Administration: discard the culture medium in the 96-well plate, dilute the test substance to 2.5% with cell culture medium and add it to the test substance wells, add culture medium containing 100 ng / mL TGFβ1 to the positive control wells, and add cell culture medium to the blank control wells, 100 μL per well. After administration, place the 96-well plate in a CO2 incubator for 24 hours;

[0133] 3) ELISA detection: After the culture is completed, the cell culture supernatant in each well is collected and detected according to the instruction manual of the human type I collagen enzyme-linked immunosorbent assay kit.

[0134] Calculation of type I collagen content: Use professional curve making software to calculate the regression equation of the standard curve using the concentration of the standard in the kit and the OD450 value, substitute the OD450 value of the sample into the equation, and calculate the type I collagen content of the sample. The average value of the three replicate wells in each group is used as the final type I collagen result. The test results are shown in Table 7 below.

[0135] 4. The cell soothing test method is:

[0136] 1) Cell inoculation: Mouse mononuclear macrophage leukemia cell line RAW264.7 was inoculated into a 96-well plate at an appropriate density (the degree of confluence reached 90% 24 hours after inoculation), 100 μL per well, and cultured in a CO2 incubator for 24 hours.

[0137] 2) Administration: Discard the culture medium in the 96-well plate, set up a positive control group, a negative control group, and a blank control group, with 6 replicate wells in each group, and add 50 μL of the test solution and LPS working solution to each well. Add 100 μL of 2 mg / ml dexamethasone sodium phosphate working solution to each well of the positive control group, add 50 μL of test culture medium and 50 μL of LPS working solution to each well of the negative control group, and add 100 μL of test culture medium to each well of the blank control group. After adding the sample, place the 96-well plate in a CO2 incubator and culture for 24 hours.

[0138] 3) Griess test: After the culture was completed, 50 μL of cell culture supernatant was collected from each well and placed in a new 96-well plate. 50 μL of Griess solution was added to each well, mixed and reacted in the dark for 10 minutes. The OD value was detected at 540 nm using an ELISA reader. The relative content of NO (%) = OD sample / OD negative control group × 100%. The test results are shown in Table 7 below.

[0139] Table 7 - Cytotoxicity and cosmetic efficacy test results of fermentation products of Experimental Examples 36, 38-40

[0140]

[0141] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. It is particularly pointed out that for those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a product of Ulva composite wall-breaking enzymatic hydrolysis and fermentation, characterized in that: The following steps are involved: (1) adding Ulva powder to water, and then performing physical cell wall breaking treatment after soaking; (2) adding a mixed enzyme to the solution for enzymatic hydrolysis, followed by water extraction, centrifugation, and filtration to obtain an enzymatic hydrolysis product, wherein the mixed enzyme includes lipase, cell wall-breaking enzyme, carbohydrase, and protease; (3) adding the enzymatic hydrolysis product to the culture medium, then inoculating the bacterial liquid into the culture medium, fermenting and culturing to obtain a fermentation liquid, and obtaining a fermentation product after centrifugation and filtration, wherein the bacterial species in the bacterial liquid is Bifidobacterium longum or Lactobacillus plantarum.

2. The method for preparing a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva as claimed in claim 1, characterized in that: The culture medium includes 0.5wt%-3wt% soy peptone, 0.02wt%-0.05wt% histidine, 0.1wt%-0.3wt% dipotassium hydrogen phosphate, 0.05wt%-0.1wt% magnesium chloride, 0.01wt%-0.02wt% calcium chloride, 0.002wt%-0.008wt% manganese sulfate, 0.2wt%-0.8wt% sodium acetate, 0.02wt%-0.08wt% L-cysteine ​​and the balance water.

3. The method for preparing a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva according to claim 1, characterized in that: In step (1), ulva powder is added into water at a ratio of 1 wt% to 5 wt%.

4. The method for preparing a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva as claimed in claim 1, characterized in that: In step (1), the cell wall is broken by using a cell wall breaking machine for 10 min-30 min, with a stirring power of 800-1200 W and a stirring speed of 30000-40000 r / min; followed by an ultrasonic treatment for 10 min-30 min, with an ultrasonic power of 120-160 W and an ultrasonic frequency of 30-50 kHz.

5. The method for preparing a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva as claimed in claim 1, characterized in that: In step (2), a mixed enzyme is added to the solution at a ratio of 3.5-15wt% for enzymatic hydrolysis reaction, wherein the mixed enzyme includes 14wt%-45wt% of protease, 2wt%-28wt% of cell wall-breaking enzyme, 10wt%-55wt% of carbohydrase and 3wt%-36wt% of lipase.

6. The method for preparing a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva according to claim 1, characterized in that: The mixed enzyme comprises 22wt%-44wt% of neutral protease, 2wt%-13wt% of cellulase, 43wt%-44wt% of beta-glucanase and 11-22wt% of lipase.

7. The method for preparing a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva as claimed in claim 1, characterized in that: In step (3), the enzymatic hydrolysate is added to the blank culture medium at a ratio of 20 wt% to 80 wt%, and the concentration of the bacterial solution is 2.8*10 8 CFU / mL and above, the fermentation culture time is 12-36h.

8. The method for preparing a composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva as claimed in claim 1, characterized in that: In step (2), the water extraction temperature is 40°C-60°C, the time is 2h-5h, and the filtration is performed using 0.3μm-0.8μm filter paper.

9. A Ulva composite wall-breaking enzymatic hydrolysis and fermentation product prepared by the method for preparing the Ulva composite wall-breaking enzymatic hydrolysis and fermentation product as described in any one of claims 1 to 8.

10. Use of the composite wall-breaking enzymatic hydrolysis and fermentation product of Ulva as claimed in claim 9 in the preparation of cosmetics.

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