Ultralow-molecular-weight polysaccharide composition, preparation thereof and application of ultralow-molecular-weight polysaccharide composition in preparation of cosmetics

Abstract

The invention relates to an ultra-low molecular weight polysaccharide composition, which is prepared from the following components in percentage by mass: 45 to 75 percent of ultra-low molecular weight dendrobium officinale polysaccharide, 20 to 40 percent of ultra-low molecular weight tremella polysaccharide and 5 to 15 percent of ultra-low molecular weight hyaluronic acid, the molecular weight of the ultra-low molecular weight dendrobium officinale polysaccharide is less than or equal to 5000Da, the molecular weight of the ultra-low molecular weight tremella polysaccharide is less than or equal to 3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 400-1500Da. The ultra-low molecular weight polysaccharide composition is obtained by combining ultra-low molecular weight dendrobium officinale polysaccharide, ultra-low molecular weight tremella polysaccharide and ultra-low molecular weight hyaluronic acid according to a proper proportion. The molecular weight of each component in the composition is low, so that transdermal absorption is facilitated, and the skin care effect can be better exerted in the skin.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction and cosmetics, and in particular to an ultra-low molecular weight polysaccharide composition and its preparation and application in the preparation of cosmetics. Background Art

[0002] Polysaccharides (also called polysaccharides) are a class of natural high molecular biopolymers composed of multiple monosaccharides through glycosidic bonds. In recent years, plant, animal and microbial polysaccharides have received extensive attention from the medical and food industries as bioactive ingredients and food additives. It is generally believed that polysaccharides extracted from natural products have low toxicity and can be used as raw materials or supplements for functional food products. They have antioxidant, anti-fatigue, anti-tumor, anti-inflammatory, lipid-lowering and human immune regulation functions, as well as cell proliferation inhibition functions. Especially in terms of antioxidant, it can prevent free radicals from harming the human body. More and more studies have shown that the chemical composition and structural characteristics of polysaccharides, including molecular weight (Mw), monosaccharide composition, glycosidic bonds, chain conformation, conformational characteristics, etc., are closely related to their biological activity.

[0003] Dendrobium has a long history in the field of medicine and health care in my country. The stems of Dendrobium are rich in water-soluble Dendrobium polysaccharides. The main chain is composed of repeated β-1,3-D-mannopyranosyl and β-1,4-D-glucopyranosyl components, which is O-acetylglucomannan. Dendrobium polysaccharides have antioxidant, immunomodulatory, anti-tumor, and blood sugar-lowering effects.

[0004] Tremella is a traditional Chinese fungus edible medicine, known as the "king of fungi". Tremella is rich in Tremella polysaccharides, which account for up to 60% of Tremella. Tremella polysaccharides can induce the body to produce antibodies and have a variety of pharmacological effects such as anti-oxidation, anti-aging, anti-tumor, lowering blood pressure, lowering blood lipids, and enhancing immunity. Common Tremella polysaccharides are large molecular polysaccharides, mannans connected by α-(1→3) glycosidic bonds, and the side chains are composed of glucuronic acid and xylose.

[0005] Hyaluronic acid, also known as hyaluronic acid, is an acidic mucopolysaccharide with a unique molecular structure and multiple important physiological functions. It is widely distributed in various parts of the human body, especially in the skin, where it plays a key role in improving skin nutrition metabolism, making the skin soft, smooth, wrinkle-free, elastic, and anti-aging. It also has good transdermal absorption accelerator and moisturizing effects.

[0006] However, the molecular weight of common Dendrobium officinale polysaccharides, Tremella fuciformis polysaccharides, and hyaluronic acid is relatively large, generally between tens to millions of Daltons, and is difficult to be absorbed by the skin. Therefore, how to prepare ultra-low molecular weight Dendrobium officinale polysaccharides, Tremella fuciformis polysaccharides, and hyaluronic acid has become a technical problem that needs to be urgently solved in this field. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an ultra-low molecular weight polysaccharide composition, which has good transdermal absorption and excellent moisturizing, soothing and repairing effects.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides an ultra-low molecular weight polysaccharide composition, which comprises, by mass percentage, 45-75% of ultra-low molecular weight Dendrobium officinale polysaccharide, 20-40% of ultra-low molecular weight Tremella fuciformis polysaccharide and 5-15% of ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is ≤5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 400-1500Da.

[0010] Oligosaccharides are more easily absorbed by the skin due to their lower molecular weight, so they have stronger functionality when applied to the skin. The present invention uses ultra-low molecular weight Tremella polysaccharide as one of the components of the polysaccharide composition. The low molecular weight Tremella polysaccharide has a smaller molecular weight and is more conducive to transdermal absorption of the skin.

[0011] Hyaluronic acid (HA) is a type of viscous polysaccharide composed of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) linked by β-1,3 glycosidic bonds and β-1,4 glycosidic bonds to form unbranched disaccharide repeating units, commonly known as hyaluronic acid. Low molecular weight hyaluronic acid (Mr<1×10 4 ) has strong biological activity, can promote wound healing, promote angiogenesis and have immune regulation functions, and low molecular weight hyaluronic acid can easily penetrate into the dermis.

[0012] As a preferred embodiment of the first aspect, the ultra-low molecular weight polysaccharide composition comprises, by mass percentage, 60% of ultra-low molecular weight Dendrobium officinale polysaccharide, 30% of ultra-low molecular weight Tremella fuciformis polysaccharide and 10% of ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is 3000-5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is 2000-3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 700-900Da.

[0013] In a second aspect, the present invention provides a method for preparing the ultra-low molecular weight polysaccharide composition described in the first aspect, comprising the following steps:

[0014] S1: mixing the Dendrobium stem segment powder with water, and subjecting the mixture to ultrasonic cell disruption treatment;

[0015] S2: adding compound enzyme A to the mixture of step S1 for enzymolysis, filtering after the enzymolysis, and obtaining ultra-low molecular weight Dendrobium officinale polysaccharide; the compound enzyme A is a mixed enzyme preparation of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase;

[0016] S3: inoculating Lactobacillus johnsonii and / or two bifidobacterium seed liquids into Tremella fuciformis culture medium for fermentation to obtain a crude fermentation liquid;

[0017] S4: adding compound enzyme B to the crude fermentation liquid of step S3 for the first enzymolysis, after inactivating the enzyme, adding neutral protease for the second enzymolysis, after inactivating the enzyme, obtaining an enzymolysis solution, filtering, and obtaining ultra-low molecular weight Tremella polysaccharide; the compound enzyme B is a mixed enzyme preparation of cellulase and pectinase;

[0018] S5 dissolving hyaluronic acid in water, adding enzyme for enzymolysis, and obtaining enzymolysis solution;

[0019] S6 sequentially performs decolorization, protein removal, desalination, sterilization and drying on the enzymatic hydrolyzate of step S5 to obtain ultra-low molecular weight hyaluronic acid;

[0020] S7: the Dendrobium officinale polysaccharide liquid obtained in step S2, the ultra-low molecular weight Tremella fuciformis polysaccharide liquid obtained in step S4, and the ultra-low molecular weight hyaluronic acid obtained in step S6 are mixed according to mass percentage to prepare an ultra-low molecular weight polysaccharide composition.

[0021] As a preferred implementation of the second aspect, the Dendrobium stem segment powder in step S1 is dried Dendrobium huoshanense stem segment powder.

[0022] As a preferred implementation of the second aspect, in step S1, the mass ratio of the Dendrobium stem segment powder to water is 1:(15-25).

[0023] As a preferred embodiment of the second aspect, the frequency of the ultrasonic cell disruption treatment in step S1 is 20-24 kHz, the time is 40-90 s / time, the temperature is 40-60° C., and the number of times is 6-10 times.

[0024] In the present invention, the ultrasonic cell disruptor includes a generator and a transducer. The generator can generate high-frequency current, and the transducer converts electromagnetic oscillation into mechanical vibration. The high-energy mechanical vibration can disrupt the cells of the stem of Dendrobium officinale, which is beneficial to the extraction of intracellular active substances such as dendrobium polysaccharides. The purpose of multiple ultrasonic treatments is to maximize the disruption of the cells of the stem of Dendrobium officinale, so as to maximize the effective substances in the extract.

[0025] As a preferred implementation of the second aspect, the amount of the complex enzyme A added in step S2 is 0.4-1.2% of the mass fraction of the Dendrobium stem segment powder.

[0026] As a preferred embodiment of the second aspect, in step S2, the mass ratio of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase = (3-5): (1-3): (1-3).

[0027] As a preferred embodiment of the second aspect, the enzymatic hydrolysis temperature in step S2 is 40-60° C., and the enzymatic hydrolysis time is 3-5 hours. In the present invention, the purpose of enzymatic hydrolysis is to enzymatically hydrolyze the high molecular weight Dendrobium officinale polysaccharides.

[0028] As a preferred implementation of the second aspect, in step S2, enzymatic hydrolysis is terminated by inactivation, the inactivation temperature is 85-95° C., and the inactivation time is 15-30 min.

[0029] As a preferred implementation of the second aspect, the filtration in step S2 is performed using a diatom filter.

[0030] In the present invention, the above method can not only prepare ultra-low molecular weight Dendrobium officinale polysaccharide with a molecular weight of ≤5000Da, but also the extract is rich in other active substances in the stem of Dendrobium officinale.

[0031] As a preferred embodiment of the second aspect, the tremella culture medium in step S3 includes the following components in mass concentrations: 1-3% tremella powder, 1-3% nitrogen source, 1-3% carbon source, 0.15-0.35% sulfate, 0.1-0.3% phosphate and the remainder water.

[0032] As a preferred embodiment of the second aspect, the peptone is soy peptone or trypsin; the carbohydrate compound is at least one of glucose, sucrose or fructose syrup; the phosphate is at least one of potassium dihydrogen phosphate or dipotassium hydrogen phosphate; the sulfate is at least one of magnesium sulfate, ammonium sulfate or manganese sulfate.

[0033] As a preferred embodiment of the second aspect, the fermentation temperature in step S3 is 33-40° C., the fermentation time is 36-60 h, and the fermentation method is closed fermentation.

[0034] As a preferred embodiment of the second aspect, the cell concentration of the Lactobacillus johnsonii or the two Bifidobacterium seed liquid in step S3 is 10 7 ~10 9 CFU / mL; the inoculation amount of Lactobacillus johnsonii and / or the two Bifidobacterium seed solutions in step S3 is 1-3% of the volume of the Tremella fermentation culture medium.

[0035] As a preferred implementation of the second aspect, the amount of the complex enzyme B added for the first enzymatic hydrolysis in step S4 is 0.05-0.13% of the mass fraction of the crude fermentation broth; the mass ratio of cellulase to pectinase in step S4 is (1-3):1.

[0036] As a preferred implementation of the second aspect, in step S4, the amount of cellulase added is 0.0375-0.065% of the mass fraction of the crude fermentation broth, and the amount of pectinase added is 0.0125-0.065% of the mass fraction of the crude fermentation broth.

[0037] As a preferred embodiment of the second aspect, the temperature of the first enzymatic hydrolysis in step S4 is 40-60° C., and the enzymatic hydrolysis time is 1.5-3 h.

[0038] As a preferred implementation of the second aspect, the amount of neutral protease added in the second enzymatic hydrolysis in step S4 is 0.08-0.15% of the mass of the crude fermentation broth.

[0039] As a preferred embodiment of the second aspect, the temperature of the second enzymatic hydrolysis in step S4 is 40-60° C., and the enzymatic hydrolysis time is 2.5-4 h.

[0040] As a preferred embodiment of the second aspect, in step S4, the enzymatic hydrolysis is terminated by inactivation, the inactivation temperature is 85-95° C., and the inactivation time is 15-30 min.

[0041] As a preferred embodiment of the second aspect of the present invention, the enzyme in step S5 is immobilized hyaluronidase, and the preparation method of the immobilized hyaluronidase is referred to CN118406676A.

[0042] As a preferred embodiment of the second aspect of the present invention, the mass ratio of hyaluronic acid to enzyme in step S5 is (1-3):1.

[0043] As a preferred embodiment of the second aspect of the present invention, in step S5, the mass ratio of hyaluronic acid to water is 1:(6-15), the temperature of the enzymatic hydrolysis is 40-48°C, and the time of the enzymatic hydrolysis is 4-8h.

[0044] As a preferred implementation of the second aspect, the filtration in step S4 is performed using a diatom filter.

[0045] The invention prepares ultra-low molecular weight Tremella polysaccharide with a molecular weight of ≤3000Da by a method of first fermentation and then enzymolysis. The purpose of fermentation is to extract the Tremella polysaccharide in Tremella fruiting bodies into a solvent to the greatest extent, thereby improving the extraction efficiency of Tremella polysaccharide. The purpose of enzymolysis is to hydrolyze the high molecular weight Tremella polysaccharide obtained by fermentation extraction, and finally prepare the ultra-low molecular weight Tremella polysaccharide with a molecular weight of ≤3000Da.

[0046] As a preferred implementation of the second aspect, the molecular weight of the hyaluronic acid in step S5 is between 1000 kDa and 1200 kDa.

[0047] In a third aspect, the present invention provides use of the ultra-low molecular weight polysaccharide composition described in the first aspect in the preparation of cosmetics.

[0048] As a preferred embodiment of the third aspect, the ultra-low molecular weight polysaccharide composition is added to the cosmetic in an amount of 0.1 to 30 wt %.

[0049] In a fourth aspect, the present invention provides the use of Lactobacillus johnsonii or Bifidobacterium bispinosa in the preparation of ultra-low molecular weight Tremella fuciformis polysaccharide, wherein the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da.

[0050] In a fifth aspect, the present invention provides an application of a composite bacterial strain in the preparation of ultra-low molecular weight Tremella polysaccharide, wherein the molecular weight of the ultra-low molecular weight Tremella polysaccharide is ≤3000Da, and the composite bacterial strain is a composite combination of Lactobacillus johnsonii and two Bifidobacteria;

[0051] As a preferred embodiment of the fifth aspect, the volume ratio of Lactobacillus johnsonii to the two bifidobacteria in the composite bacteria is: (1-2): (1-2).

[0052] In a sixth aspect, the present invention provides a culture medium for fermenting Lactobacillus johnsonii and / or two Bifidobacteria to prepare ultra-low molecular weight Tremella polysaccharides, wherein the culture medium comprises the following components in mass concentrations: 1-3% Tremella powder, 1-3% nitrogen source, 1-3% carbon source, 0.15-0.35% sulfate, 0.1-0.3% phosphate and the remainder water.

[0053] As a preferred embodiment of the sixth aspect, the peptone is soy peptone or trypsin; the carbohydrate compound is at least one of glucose, sucrose or fructose syrup; the phosphate is at least one of potassium dihydrogen phosphate or dipotassium hydrogen phosphate; the sulfate is at least one of magnesium sulfate, ammonium sulfate or manganese sulfate.

[0054] As a preferred embodiment of the sixth aspect, the culture medium comprises components in the following mass concentrations: 2% tremella powder, 2% soy peptone, 2% glucose, 0.25% magnesium sulfate, 0.2% potassium dihydrogen phosphate and balance water.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The present invention prepares ultra-low molecular weight dendrobium polysaccharide by a method combining ultrasound with complex enzyme enzymolysis; and prepares ultra-low molecular weight tremella polysaccharide by a method combining fermentation of Lactobacillus johnsonii and / or Bifidobacterium with secondary enzymolysis. The molecular weight of the prepared dendrobium polysaccharide is less than 5000Da, and the molecular weight of the tremella polysaccharide is less than 3000Da. A method for preparing ultra-low molecular weight dendrobium polysaccharide and tremella polysaccharide is constructed.

[0057] (2) The present invention combines ultra-low molecular weight Dendrobium officinale polysaccharide, ultra-low molecular weight Tremella polysaccharide and ultra-low molecular weight hyaluronic acid in a suitable ratio to obtain an ultra-low molecular weight polysaccharide composition. The molecular weight of each component in the composition is low, which is conducive to transdermal absorption and can better exert skin care effects inside the skin.

[0058] (3) In the present invention, there is a synergistic effect between the ultra-low molecular weight Dendrobium officinale polysaccharide, the ultra-low molecular weight Tremella fuciformis polysaccharide and the ultra-low molecular weight hyaluronic acid, and the composition has excellent moisturizing, soothing and repairing effects.

[0059] (4) The present invention uses a combination of multiple ultra-low molecular weight polysaccharides, thereby avoiding the disadvantage of limited efficacy of a single type of polysaccharide, and the final product has excellent skin care efficacy. DETAILED DESCRIPTION

[0060] The present invention is further described below in conjunction with the examples. It should be noted that the following description is only for the purpose of explaining the present invention and is not intended to limit its content. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained through commercial channels.

[0061] Unless otherwise specified, all percentages mentioned in the present invention are by mass percentages.

[0062] The hemicellulase, cellulase and pectinase used in the present invention are from Ningxia Xiasheng Industrial Group Co., Ltd., α-amylase and neutral protease are from Shanghai MacLean Biochemical Technology Co., Ltd., and Aspergillus niger acid β-mannanase is from Shanghai Huashang Xiangyang Biotechnology Co., Ltd.

[0063] The Lactobacillus johnsonii used in the present invention is numbered ATCC33200 and comes from Beijing Biobowei Biotechnology Co., Ltd., the Bifidobacterium bisporus is numbered BNCC336858 and comes from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., the Candida utilis is numbered GDMCC2.148 and comes from Guangdong Provincial Microbiological Culture Collection Center, and the Lactobacillus plantarum is numbered ATCC8014 and comes from Beijing Biobowei Biotechnology Co., Ltd.

[0064] The Lactobacillus bulgaricus (ACCC 10638) used in the comparative example of the present invention was sourced from Shanghai Fuxiang Biotechnology Co., Ltd.

[0065] The Lactobacillus acidophilus (ATCC 4356), Lactobacillus rhamnosus (ATCC 7469), Streptococcus thermophilus (ATCC 14485), and Lactobacillus paracasei (ATCC 334) used in the comparative example of the present invention are all from Beijing Biobo Biotechnology Co., Ltd.

[0066] It should be noted that the strains with numbers in the examples of the present invention are only used to verify the technical effects of the present invention and do not involve improvements to the strains, so there is no need to preserve the strains.

[0067] The preparation method of the culture medium of the present invention comprises the following steps: uniformly mixing the raw materials in the culture medium, sterilizing at 121° C. for 20 minutes, and cooling the culture medium for standby use.

[0068] The cell concentration of the Lactobacillus johnsonii seed solution, the Bifidobacterium johnsonii seed solution and the seed solution of other strains in the comparative example is 10 7 ~10 9 CFU / mL can be prepared by the common seed solution preparation method in the prior art. The present invention does not limit the preparation method of Lactobacillus johnsonii seed solution, Bifidobacterium bispinosa seed solution, and seed solution of other bacterial species in the comparative example.

[0069] The high molecular weight hyaluronic acid (molecular weight between 1000 kDa and 1200 kDa) used in the present invention and the 3000-5000 Da hyaluronic acid used in the comparative example are derived from Dongguan Juwei New Material Technology Co., Ltd.

[0070] The present embodiment provides an ultra-low molecular weight polysaccharide composition, which comprises, by mass percentage, 45-75% of ultra-low molecular weight Dendrobium officinale polysaccharide, 20-40% of ultra-low molecular weight Tremella fuciformis polysaccharide and 5-15% of ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is ≤5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 400-1500Da.

[0071] As a preferred embodiment of this embodiment, the mass percentage of ultra-low molecular weight Dendrobium officinale polysaccharide can be 45%, 48%, 49%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 75% and any value between these values.

[0072] As a preferred embodiment of the embodiment, the mass percentage of ultra-low molecular weight Tremella polysaccharide can be 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40% and any value therebetween.

[0073] As a preferred embodiment of this embodiment, the mass percentage of ultra-low molecular weight hyaluronic acid can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% and any value in between these values.

[0074] As a preferred embodiment of this embodiment, the ultra-low molecular weight polysaccharide composition of the present invention comprises, by mass percentage, 60% of ultra-low molecular weight Dendrobium officinale polysaccharide, 30% of ultra-low molecular weight Tremella fuciformis polysaccharide and 10% of ultra-low molecular weight hyaluronic acid.

[0075] As a preferred embodiment of this embodiment, the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is 3000-5000 Da.

[0076] As a preferred embodiment of this embodiment, the molecular weight of the ultra-low molecular weight Tremella polysaccharide is 2000-3000 Da.

[0077] As a preferred embodiment of this embodiment, the molecular weight of the ultra-low molecular weight hyaluronic acid is 700-900 Da; more preferably, the molecular weight of the ultra-low molecular weight hyaluronic acid is 400-800 Da.

[0078] Example 1

[0079] The present embodiment provides an ultra-low molecular weight polysaccharide composition, which comprises, by mass percentage, 60% of ultra-low molecular weight Dendrobium officinale polysaccharide, 30% of ultra-low molecular weight Tremella fuciformis polysaccharide and 10% of ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is ≤5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 700-900Da.

[0080] The ultra-low molecular weight polysaccharide composition of this embodiment is prepared by the following method:

[0081] A. Preparation of ultra-low molecular weight Dendrobium officinale polysaccharide with molecular weight ≤ 5000Da:

[0082] A1. Mix the stem segment powder of Dendrobium huoshanense with 20 times the mass of water, and subject it to ultrasonic cell disruption treatment (frequency of 22 kHz, time of 60 s / time, temperature of 50°C, number of times of 8 times) to obtain a mixture;

[0083] A2, adding complex enzyme A (mass ratio of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase = 4:2:2) with a mass fraction of 0.6% of Dendrobium powder to the mixture, enzymolysis at 50°C for 4 hours, inactivation at 90°C for 20 minutes after the enzymolysis, filtering out the filter residue through a diatom filter, and obtaining an ultra-low molecular weight Dendrobium officinale polysaccharide;

[0084] B. Preparation of ultra-low molecular weight Tremella polysaccharide with molecular weight ≤ 3000Da:

[0085] B1, Lactobacillus johnsonii seed solution (bacterial concentration is about 10 8 CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 2%, and cultured in a closed manner at 37°C for 48h to obtain a crude fermentation liquid;

[0086] B2, in the crude fermentation liquid, add the complex enzyme B (cellulase and pectinase, cellulase 0.06%, pectinase 0.03%) of crude fermentation liquid mass fraction 0.09%, at 50 ℃, carry out the first enzymolysis 2h, after the temperature is raised to 90 ℃ of enzyme inactivations 20min, then add the neutral protease of crude fermentation liquid mass fraction 0.12%, at 50 ℃, carry out the second enzymolysis 3h, obtain enzymolysis solution, after the enzymolysis solution is deactivated at 90 ℃ for 20min and filtered through a diatom filter, obtain ultra-low molecular weight Tremella polysaccharide;

[0087] The Tremella culture medium comprises: 2% Tremella powder, 2% soy peptone, 2% glucose, 0.25% magnesium sulfate, 0.2% potassium dihydrogen phosphate and the balance water;

[0088] C. Preparation of ultra-low molecular weight hyaluronic acid with a molecular weight of 700-900 Da:

[0089] C1. Dissolve 1000 g of high molecular weight hyaluronic acid (molecular weight between 1000 kDa and 1200 kDa) in 10 L of water, mix well, add 500 g of immobilized hyaluronidase (the preparation method of immobilized hyaluronidase refers to Example 1 in the application publication number CN118406676A), perform enzymolysis at 45° C. for 6 h, remove the immobilized hyaluronidase, and obtain an enzymolysis solution;

[0090] C2. Add 1% activated carbon to the enzymolysis solution in step C1, stir at 100 rpm for 6 hours, and obtain a decolorized enzymolysis solution after filtering through a ceramic membrane; add an equal volume of deionized water to the decolorized enzymolysis solution, mix evenly, and obtain a dilution solution, filter the dilution solution through a 5 kDa ultrafiltration membrane to remove protein, and the obtained filtrate is the deproteinized enzymolysis solution; filter the deproteinized enzymolysis solution through a 300 Da nanofiltration membrane to obtain a desalted enzymolysis solution; filter the desalted enzymolysis solution through a 0.22 μm microfiltration membrane to obtain a sterilized enzymolysis solution, and spray dry the sterilized enzymolysis solution to obtain ultra-low molecular weight hyaluronic acid;

[0091] D. Preparation of ultra-low molecular weight polysaccharide composition:

[0092] The ultra-low molecular weight Dendrobium officinale polysaccharide prepared in step A, the ultra-low molecular weight Tremella fuciformis polysaccharide prepared in step B and the ultra-low molecular weight hyaluronic acid prepared in step C are uniformly mixed according to the above mass percentage to prepare the ultra-low molecular weight polysaccharide composition of the present invention.

[0093] Example 2

[0094] The present embodiment provides an ultra-low molecular weight polysaccharide composition, which comprises, by mass percentage, 45% of ultra-low molecular weight Dendrobium officinale polysaccharide, 40% of ultra-low molecular weight Tremella fuciformis polysaccharide and 15% of ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is ≤5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 700-900Da.

[0095] Example 3

[0096] The present embodiment provides an ultra-low molecular weight polysaccharide composition, which comprises, by mass percentage, 75% of ultra-low molecular weight Dendrobium officinale polysaccharide, 20% of ultra-low molecular weight Tremella fuciformis polysaccharide and 5% of ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is ≤5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 700-900Da.

[0097] The ultra-low molecular weight polysaccharide compositions of Examples 2 to 3 were prepared by the method of Example 1, except that the mass percentages of the components were different.

[0098] Example 4

[0099] The difference between Example 4 and Example 1 is that in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, the Lactobacillus johnsonii seed solution is replaced with two Bifidobacterium seed solutions (with a bacterial cell concentration of about 10 8CFU / mL), and the remaining steps and parameters were the same as in Example 1.

[0100] Example 5

[0101] The difference between Example 5 and Example 1 is that in step A1 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, 0.6% of complex enzyme A (mass ratio of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase = 4:2:2) is changed to 0.6% of complex enzyme A (mass ratio of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase = 5:1:1), and the remaining parameters and steps are the same as in Example 1.

[0102] Example 6

[0103] The present embodiment provides an ultra-low molecular weight polysaccharide composition, which comprises, by mass percentage, 55% ultra-low molecular weight Dendrobium officinale polysaccharide, 30% ultra-low molecular weight Tremella fuciformis polysaccharide and 15% ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is ≤5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 400-800Da.

[0104] The ultra-low molecular weight polysaccharide composition of this embodiment is prepared by the following method:

[0105] A. Preparation of ultra-low molecular weight Dendrobium officinale polysaccharide with molecular weight ≤ 5000Da:

[0106] A1. Mix the stem segment powder of Dendrobium huoshanense with 15 times the mass of water, and subject it to ultrasonic cell disruption treatment (frequency of 24 kHz, time of 90 s / time, temperature of 40° C., and number of times of 6 times) to obtain a mixture;

[0107] A2, adding complex enzyme A (mass ratio of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase = 3:3:1) with a mass fraction of 0.4% of Dendrobium powder to the mixture, enzymolysis at 60°C for 5h, inactivation at 85°C for 30min after the enzymolysis, filtering out the filter residue through a diatom filter, and obtaining ultra-low molecular weight Dendrobium officinale polysaccharide;

[0108] B. Preparation of ultra-low molecular weight Tremella polysaccharide with molecular weight ≤ 3000Da:

[0109] B1, Lactobacillus johnsonii seed solution (bacterial concentration is about 10 9 CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 1%, and cultured in a closed manner at 40°C for 36 hours to obtain a crude fermentation liquid;

[0110] B2, in the crude fermentation liquid, add the complex enzyme B (cellulase and pectinase, cellulase addition amount 0.065%, pectinase addition amount 0.065%) of crude fermentation liquid mass fraction 0.13%, at 40 ℃, carry out the first enzymolysis 3h, after the temperature is raised to 85 ℃ of enzyme inactivations 30min, then add the neutral protease of crude fermentation liquid mass fraction 0.08%, at 60 ℃, carry out the second enzymolysis 2.5h, obtain enzymolysis solution, after the enzymolysis solution is deactivated at 85 ℃ for 30min and filtered through a diatom filter, obtain ultra-low molecular weight Tremella polysaccharide;

[0111] The Tremella culture medium comprises: 1% Tremella powder, 3% soy peptone, 1% sucrose, 0.15% manganese sulfate, 0.3% dipotassium hydrogen phosphate and the balance water;

[0112] C. Preparation of ultra-low molecular weight hyaluronic acid with a molecular weight of 400-800 Da:

[0113] C1. Dissolve 1000 g of high molecular weight hyaluronic acid (molecular weight between 1000 kDa and 1200 kDa) in 10 L of water, mix well, add 500 g of immobilized hyaluronidase (the preparation method of immobilized hyaluronidase refers to Example 1 in CN118406676A), perform enzymolysis at 45° C. for 6 h, remove the immobilized hyaluronidase, and obtain an enzymolysis solution;

[0114] C2. Add 1% activated carbon to the enzymolysis solution in step C1, stir at 100 rpm for 7 hours, and obtain a decolorized enzymolysis solution after filtering through a ceramic membrane; add an equal volume of deionized water to the decolorized enzymolysis solution, mix evenly, and obtain a dilution solution, filter the dilution solution through a 5 kDa ultrafiltration membrane to remove protein, and the obtained filtrate is the deproteinized enzymolysis solution; filter the deproteinized enzymolysis solution through a 300 Da nanofiltration membrane to obtain a desalted enzymolysis solution; filter the desalted enzymolysis solution through a 0.22 μm microfiltration membrane to obtain a sterilized enzymolysis solution, and spray dry the sterilized enzymolysis solution to obtain ultra-low molecular weight hyaluronic acid;

[0115] D. Preparation of ultra-low molecular weight polysaccharide composition:

[0116] The ultra-low molecular weight Dendrobium officinale polysaccharide prepared in step A, the ultra-low molecular weight Tremella fuciformis polysaccharide prepared in step B and the ultra-low molecular weight hyaluronic acid prepared in step C are uniformly mixed according to the above mass percentage to prepare the ultra-low molecular weight polysaccharide composition of the present invention.

[0117] Example 7

[0118] The present embodiment provides an ultra-low molecular weight polysaccharide composition, which comprises, by mass percentage, 70% ultra-low molecular weight Dendrobium officinale polysaccharide, 20% ultra-low molecular weight Tremella fuciformis polysaccharide and 10% ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is ≤5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da; the molecular weight of the ultra-low molecular weight hyaluronic acid is between 900 and 1500Da.

[0119] The ultra-low molecular weight polysaccharide composition of this embodiment is prepared by the following method:

[0120] A. Preparation of ultra-low molecular weight Dendrobium officinale polysaccharide with molecular weight ≤ 5000Da:

[0121] A1. Mix the stem segment powder of Dendrobium huoshanense with 25 times the mass of water, and subject it to ultrasonic cell disruption treatment (frequency of 20 kHz, time of 40 s / time, temperature of 60°C, number of times of 10 times) to obtain a mixture;

[0122] A2, adding a composite enzyme (mass ratio of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase = 4:2:2) with a mass fraction of 1.2% of Dendrobium powder to the mixture, enzymolysis at 40°C for 3h, inactivation at 95°C for 15min after the enzymolysis, filtering out the filter residue through a diatom filter, and obtaining an ultra-low molecular weight Dendrobium officinale polysaccharide;

[0123] B. Preparation of ultra-low molecular weight Tremella polysaccharide with molecular weight ≤ 3000Da:

[0124] B1, two bifidobacterium seed liquid (bacterial concentration is about 10 7 CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 3%, and cultured in a closed manner at 33°C for 60h to obtain a crude fermentation liquid;

[0125] B2. Add 0.05% of the mass fraction of the crude fermentation broth of complex enzyme B (cellulase and pectinase, 0.0375% of cellulase, 0.0125% of pectinase) to the crude fermentation broth, carry out the first enzymolysis at 60°C for 1.5 hours, raise the temperature to 95°C to inactivate the enzymes for 15 minutes, then add 0.15% of the mass fraction of the crude fermentation broth of neutral protease, carry out the second enzymolysis at 40°C for 4 hours to obtain an enzymolysis solution, inactivate the enzymolysis solution at 95°C for 15 minutes, and filter it through a diatom filter to obtain an ultra-low molecular weight Tremella polysaccharide.

[0126] The Tremella culture medium comprises: 3% Tremella powder, 1% tryptone, 3% fructose syrup, 0.35% ammonium sulfate, 0.1% potassium dihydrogen phosphate and the balance water;

[0127] C. Preparation of ultra-low molecular weight hyaluronic acid with a molecular weight of 900 to 1500 Da:

[0128] C1. Dissolve 1000 g of high molecular weight hyaluronic acid (molecular weight between 1000 kDa and 1200 kDa) in 10 L of water, mix well, add 500 g of immobilized hyaluronidase (the preparation method of immobilized hyaluronidase refers to Example 1 in CN118406676A), perform enzymolysis at 45° C. for 5 h, remove the immobilized hyaluronidase, and obtain an enzymolysis solution;

[0129] C2. Add 1% activated carbon to the enzymolysis solution in step C1, stir at 100 rpm for 6 hours, and obtain a decolorized enzymolysis solution after filtering through a ceramic membrane; add an equal volume of deionized water to the decolorized enzymolysis solution, mix evenly, and obtain a dilution solution, filter the dilution solution through a 5 kDa ultrafiltration membrane to remove protein, and the obtained filtrate is the deproteinized enzymolysis solution; filter the deproteinized enzymolysis solution through a 300 Da nanofiltration membrane to obtain a desalted enzymolysis solution; filter the desalted enzymolysis solution through a 0.22 μm microfiltration membrane to obtain a sterilized enzymolysis solution, and spray dry the sterilized enzymolysis solution to obtain ultra-low molecular weight hyaluronic acid;

[0130] D. Preparation of ultra-low molecular weight polysaccharide composition:

[0131] The ultra-low molecular weight Dendrobium officinale polysaccharide prepared in step A, the ultra-low molecular weight Tremella fuciformis polysaccharide prepared in step B and the ultra-low molecular weight hyaluronic acid prepared in step C are uniformly mixed according to the above mass percentage to prepare the ultra-low molecular weight polysaccharide composition of the present invention.

[0132] Example 8

[0133] The difference between Example 8 and Example 1 is that in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, a composite seed solution of Lactobacillus johnsonii and two bifidobacteria (the cell concentrations of Lactobacillus johnsonii and two bifidobacteria are both 10 8 CFU / mL, the volume ratio of Lactobacillus johnsonii to the two bifidobacteria was 1:1, and the total inoculation volume ratio was 2%), and the other methods and parameters were the same as in Example 1.

[0134] Comparative Example 1

[0135] The difference between Comparative Example 1 and Example 1 is that ultra-low molecular weight hyaluronic acid is lacking, the mass percentage of ultra-low molecular weight Dendrobium officinale polysaccharide is 66.67%, the mass percentage of ultra-low molecular weight Tremella fuciformis polysaccharide is 33.33%, and the remaining methods and parameters are the same as Example 1.

[0136] Comparative Example 2

[0137] The difference between Comparative Example 2 and Example 1 is that the ultra-low molecular weight Tremella polysaccharide is missing, the mass percentage of ultra-low molecular weight Dendrobium officinale polysaccharide is 85.71%, the mass percentage of ultra-low molecular weight hyaluronic acid is 14.29%, and the remaining methods and parameters are the same as Example 1.

[0138] Comparative Example 3

[0139] The difference between Comparative Example 3 and Example 1 is that the ultra-low molecular weight Dendrobium officinale polysaccharide is missing, the mass percentage of ultra-low molecular weight Tremella fuciformis polysaccharide is 75%, the mass percentage of ultra-low molecular weight hyaluronic acid is 25%, and the remaining methods and parameters are the same as Example 1.

[0140] Comparative Example 4

[0141] The difference between Comparative Example 4 and Example 1 is that in step A1 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the ultrasonic cell disruption treatment method is changed to ordinary ultrasonic cleaning ultrasonic treatment with a frequency of 40 kHz, a power of 500 W, a temperature of 50° C., and a time of 1 h. The remaining methods and parameters are the same as in Example 1.

[0142] Comparative Example 5

[0143] The difference between Comparative Example 5 and Example 1 is that in step A2 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the complex enzyme A is replaced by a complex enzyme mixed with cellulase and pectinase (the mass ratio of cellulase to pectinase = 4:2, and the total addition amount of hemicellulase and pectinase is 6%), and the remaining methods and parameters are the same as those in Example 1.

[0144] Comparative Example 6

[0145] The difference between this comparative example and Example 1 is that in step A of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, a method of first fermentation and then enzymolysis is adopted, and the specific method is as follows:

[0146] A1, Lactobacillus johnsonii seed solution (bacterial concentration is about 10 8 CFU / mL) was inoculated into the Dendrobium officinale culture medium at a volume ratio of 2%, and cultured in a closed manner at 37°C for 48h to obtain a crude fermentation liquid;

[0147] A2. Add 0.6% of the crude fermentation broth by mass of complex enzyme A (mass ratio of hemicellulase, α-amylase and pectinase = 4:2:2) to the crude fermentation broth, perform enzymolysis at 50°C for 4 hours, inactivate at 90°C for 20 minutes after the enzymolysis, filter out the residue through a diatom filter, and obtain ultra-low molecular weight Dendrobium officinale polysaccharide.

[0148] The Dendrobium officinale culture medium includes: 2% of Dendrobium huoshanense stem segment powder, 2% of soy peptone, 2% of glucose, 0.25% of magnesium sulfate, 0.2% of potassium dihydrogen phosphate and the balance of water.

[0149] The remaining methods and parameters are the same as in Example 1.

[0150] Comparative Example 7

[0151] The difference between Comparative Example 7 and Example 1 is that in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, Candida utilis is used instead of Lactobacillus johnsonii to prepare ultra-low molecular weight Tremella polysaccharide. The specific operation of step B1 is: Candida utilis seed liquid (cell concentration is about 10 8 CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 2%, and fermented for 48 hours at 30°C and a fermentation shake flask rotation speed of 150 rpm to obtain a crude fermentation liquid. The remaining methods and parameters were the same as in Example 1.

[0152] Comparative Example 8

[0153] The difference between Comparative Example 8 and Example 1 is that in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, Lactobacillus plantarum is used instead of Lactobacillus johnsonii to prepare ultra-low molecular weight Tremella polysaccharide, and the specific operation of step B1 is: Lactobacillus plantarum seed liquid (cell concentration is about 10 8 CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 2%, and the culture was sealed and fermented at 37°C for 48 hours to obtain a crude fermentation liquid; the remaining methods and parameters were the same as those in Example 1.

[0154] Comparative Example 9

[0155] The difference between Comparative Example 9 and Example 1 is that in step B2 of preparing ultra-low molecular weight Tremella polysaccharide, only one enzymatic hydrolysis using compound enzyme B is performed, and the specific operation is as follows:

[0156] B2, adding 0.09% of the crude fermentation broth mass fraction of complex enzyme B (cellulase and pectinase, cellulase 0.06%, pectinase 0.03%) to the crude fermentation broth, enzymolysis at 50°C for 5h to obtain an enzymolysis solution, deactivating the enzymolysis solution at 90°C for 20min, filtering through a diatom filter, and obtaining ultra-low molecular weight Tremella polysaccharide. The remaining methods and parameters are the same as in Example 1.

[0157] Comparative Example 10

[0158] The difference between Comparative Example 10 and Example 1 is that in step B2 of preparing ultra-low molecular weight Tremella polysaccharide, only one enzymatic hydrolysis is performed using neutral protease, and the specific operation is as follows:

[0159] B2. Add 0.12% neutral protease by mass to the crude fermentation broth, perform enzymolysis at 50° C. for 5 h to obtain an enzymolysis solution, inactivate the enzymolysis solution at 90° C. for 20 min, and filter through a diatom filter to obtain an ultra-low molecular weight Tremella polysaccharide; the remaining methods and parameters are the same as those in Example 1.

[0160] Comparative Example 11

[0161] The difference between Comparative Example 11 and Example 1 is that in step B of preparing ultra-low molecular weight Tremella polysaccharide, a method of enzymatic hydrolysis followed by fermentation is adopted, and the specific operation is as follows:

[0162] B1. Mix 2% Tremella powder and 98% water to obtain a mixture; add 0.09% cellulase and pectinase (0.06% cellulase and 0.03% pectinase) to the mixture, perform enzymolysis at 50°C for 2h, raise the temperature to 90°C and inactivate the enzymes for 20min, then add 0.12% neutral protease to the mixture, perform secondary enzymolysis at 50°C for 3h to obtain an enzymolysis solution;

[0163] B2, add 2% soy peptone, 2% glucose, 0.25% magnesium sulfate, 0.2% potassium dihydrogen phosphate in a mixture by mass fraction to the enzymatic hydrolyzate, mix well, sterilize at 121° C. for 20 min, and obtain Tremella fuciformis culture medium; after cooling, add Lactobacillus johnsonii seed solution (cell concentration is about 10 8 CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 2%, and the culture was sealed and fermented at 37°C for 48 hours to obtain a crude fermentation liquid, and the crude fermentation liquid was inactivated at 90°C for 20 minutes and filtered through a diatom filter to obtain ultra-low molecular weight Tremella polysaccharide. The remaining methods and parameters were the same as in Example 1.

[0164] Comparative Example 12

[0165] The difference between Comparative Example 12 and Example 1 is that the molecular weight of the ultra-low molecular weight hyaluronic acid (powder) is between 3000 and 5000 Da, and the other methods and parameters are the same as those in Example 1.

[0166] Comparative Example 13

[0167] The difference between Comparative Example 13 and Example 1 is that in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, Lactobacillus bulgaricus is used instead of Lactobacillus johnsonii to prepare ultra-low molecular weight Tremella polysaccharide. The specific operation of step B1 is: Lactobacillus bulgaricus seed liquid (cell concentration is about 10 8 CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 2%, and the culture was sealed and fermented at 37°C for 48 hours to obtain a crude fermentation liquid; the remaining methods and parameters were the same as those in Example 1.

[0168] Comparative Example 14

[0169] The difference between Comparative Example 14 and Example 1 is that in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, Lactobacillus acidophilus is used instead of Lactobacillus johnsonii to prepare ultra-low molecular weight Tremella polysaccharide. The specific operation of step B1 is: Lactobacillus acidophilus seed liquid (cell concentration is about 10 8CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 2%, and the culture was sealed and fermented at 37°C for 48 hours to obtain a crude fermentation liquid; the remaining methods and parameters were the same as those in Example 1.

[0170] Comparative Example 15

[0171] The difference between Comparative Example 15 and Example 1 is that in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, Lactobacillus rhamnosus is used instead of Lactobacillus johnsonii to prepare ultra-low molecular weight Tremella polysaccharide. The specific operation of step B1 is: Lactobacillus rhamnosus seed liquid (cell concentration is about 10 8 CFU / mL) was inoculated into Tremella culture medium at a volume ratio of 2%, and the culture was sealed and fermented at 37°C for 48 hours to obtain a crude fermentation liquid; the remaining methods and parameters were the same as those in Example 1.

[0172] Comparative Example 16

[0173] The difference between Comparative Example 16 and Example 1 is that, in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, a combination of thermophilic Streptococcus and Lactobacillus paracasei is used instead of Lactobacillus johnsonii to prepare ultra-low molecular weight Tremella polysaccharide. The specific operation of step B1 is: thermophilic Streptococcus and Lactobacillus paracasei seed liquid (cell concentration is about 10 8 CFU / mL, wherein the volume ratio of thermophilic Streptococcus to Lactobacillus paracasei is: 1:1) inoculated into Tremella culture medium at a volume ratio of 2%, and cultured in a closed fermentation at 37°C for 48h to obtain a crude fermentation liquid; the remaining methods and parameters are the same as those in Example 1.

[0174] Comparative Example 17

[0175] The difference between Comparative Example 17 and Example 1 is that, in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, a combination of Lactobacillus bulgaricus and Streptococcus thermophilus is used instead of Lactobacillus johnsonii to prepare ultra-low molecular weight Tremella polysaccharide. The specific operation of step B1 is: Lactobacillus bulgaricus and Streptococcus thermophilus seed liquid (the cell concentration of Lactobacillus bulgaricus and Streptococcus thermophilus is 10 8 CFU / mL, the volume ratio of Lactobacillus bulgaricus to Streptococcus thermophilus is 1:1) is inoculated into Tremella culture medium at a volume ratio of 2%, and the closed fermentation culture is carried out at 37°C for 48h to obtain a crude fermentation liquid; the remaining methods and parameters are the same as those in Example 1.

[0176] Comparative Example 18

[0177] The difference between Comparative Example 18 and Example 1 is that, in step B1 of preparing ultra-low molecular weight Tremella polysaccharide, a combination of Lactobacillus paracasei and Lactobacillus rhamnosus is used to replace Lactobacillus johnsonii to prepare ultra-low molecular weight Tremella polysaccharide. The specific operation is: Lactobacillus paracasei and Lactobacillus rhamnosus seed liquid (the cell concentration of Lactobacillus paracasei and Lactobacillus rhamnosus is 10 8CFU / mL, wherein the volume ratio of Lactobacillus paracasei to Lactobacillus rhamnosus is: 1:1) inoculated into Tremella culture medium at a volume ratio of 2%, and cultured in a closed fermentation at 37°C for 48h to obtain a crude fermentation liquid; the remaining methods and parameters are the same as those in Example 1.

[0178] Comparative Example 19

[0179] The difference between Comparative Example 19 and Example 1 is that in step A2 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the complex enzyme A is a complex enzyme mixed with cellulase, pectinase and Aspergillus niger acid β-mannanase, wherein the mass ratio of hemicellulase, pectinase and Aspergillus niger acid β-mannanase is 4:2:2, the total amount of complex enzyme A added remains unchanged, and the other methods and parameters are the same as Example 1.

[0180] Comparative Example 20

[0181] The difference between Comparative Example 20 and Example 1 is that in step A2 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the complex enzyme A is a complex enzyme mixed with pectinase, α-amylase and Aspergillus niger acid β-mannanase, wherein the mass ratio of hemicellulase and α-amylase is 4:2:2, the total amount of complex enzyme A added remains unchanged, and the other methods and parameters are the same as Example 1.

[0182] Comparative Example 21

[0183] The difference between Comparative Example 21 and Example 1 is that in step A2 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the complex enzyme A is a mixed complex enzyme of neutral protease, α-amylase, and Aspergillus niger acid β-mannanase, wherein the mass ratio of α-amylase to Aspergillus niger acid β-mannanase is 4:2:2, the total amount of complex enzyme A added remains unchanged, and the other methods and parameters are the same as Example 1.

[0184] Comparative Example 22

[0185] The difference between Comparative Example 22 and Example 1 is that in step A2 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the complex enzyme A is a complex enzyme mixed with hemicellulase, neutral protease and α-amylase, wherein the mass ratio of hemicellulase and Aspergillus niger acid β-mannanase is 4:2:2, the total amount of complex enzyme A added remains unchanged, and the other methods and parameters are the same as Example 1.

[0186] Comparative Example 23

[0187] The difference between Comparative Example 23 and Example 1 is that in step A2 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the compound enzyme A is a compound enzyme mixed with hemicellulase and α-amylase, wherein the mass ratio of hemicellulase to α-amylase is 4:2, the total amount of compound enzyme A added remains unchanged, and the other methods and parameters are the same as Example 1.

[0188] Comparative Example 24

[0189] The difference between Comparative Example 24 and Example 1 is that in step A2 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the complex enzyme A is a complex enzyme mixed with α-amylase and Aspergillus niger acid β-mannanase, wherein the mass ratio of α-amylase to Aspergillus niger acid β-mannanase is 2:2, the total amount of complex enzyme A added remains unchanged, and the other methods and parameters are the same as Example 1.

[0190] Comparative Example 25

[0191] The difference between Comparative Example 25 and Example 1 is that in step A2 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the complex enzyme A is a complex enzyme mixed with hemicellulase and Aspergillus niger acid β-mannanase, wherein the mass ratio of hemicellulase and Aspergillus niger acid β-mannanase is 4:2, the total amount of complex enzyme A added remains unchanged, and the other methods and parameters are the same as Example 1.

[0192] Test Example 1 Polysaccharide Molecular Weight Determination

[0193] Test samples: molecular weights of Dendrobium officinale polysaccharides and Tremella fuciformis polysaccharides of Examples 1 to 8 and Comparative Examples 1 to 25.

[0194] Test method: A calibration curve was established using dextran as a reference substance, and the molecular weight and molecular weight distribution of the test sample were calculated by gel chromatography (GPC). The molecular weight described in this test is the number average molecular weight, and the molecular weight test results are shown in the following table:

[0195] Table 1: Molecular weight of polysaccharide molecules in Examples 1 to 8 and Comparative Examples 1 to 25

[0196]

[0197]

[0198] As can be seen from Table 1, the molecular weights of Dendrobium officinale polysaccharide and Tremella fuciformis polysaccharide are different in Examples 4 to 8 and Comparative Examples 4 to 11 and 13 to 25 due to different preparation processes. Dendrobium officinale polysaccharide with a molecular weight of ≤5000Da and Tremella fuciformis polysaccharide with a molecular weight of ≤3000Da can be obtained by the preparation methods of Examples 4 to 8. The molecules of Dendrobium officinale polysaccharide obtained by the preparation methods of Comparative Examples 4 to 6 and 19 to 25 are greater than 5000Da, and the molecules of Tremella fuciformis polysaccharide obtained by Comparative Examples 7 to 11 and 13 to 18 are greater than 3000Da. It can be seen that ultra-low molecular weight polysaccharides can only be prepared through a specific preparation process and using specific enzyme preparations and fermentation strains.

[0199] In comparative example 4, the process parameters of ultrasonic treatment of Dendrobium officinale were changed in step A1 of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, resulting in a weight of 14600Da for Dendrobium officinale polysaccharide. It can be seen that the ultrasonic cell disruption treatment parameters are crucial to the molecular weight of Dendrobium officinale polysaccharide. In the present invention, the ultrasonic cell disruptor includes a generator and a transducer, the generator can generate high-frequency current, the transducer converts electromagnetic oscillation into mechanical vibration, and the high-energy mechanical vibration can disrupt the cells of the stem of Dendrobium officinale, which is beneficial to the extraction of intracellular active substances such as Dendrobium polysaccharide. In the present invention, the purpose of multiple ultrasonic treatments is to maximize the disruption of the cells of the stem of Dendrobium officinale, so as to maximize the efficacy of the extract.

[0200] In comparative examples 5 and 19 to 25, the compatibility of the composite enzyme A was changed in step A2 of ultra-low molecular weight Dendrobium officinale polysaccharide production, wherein comparative examples 19 to 22 combined hemicellulase, α-amylase, pectinase, Aspergillus niger acid β-mannanase and other enzymes to form composite enzyme A, and comparative examples 5 and 23 to 25 changed the type or compatibility of the composite enzyme A of the present invention; it can be seen from the results in the above table that all of them resulted in the weight of Dendrobium officinale polysaccharide being greater than 5000Da, which shows that not any enzyme combined with other enzymes can prepare low molecular weight Dendrobium officinale polysaccharide, and the compatibility of the composite enzyme A screened out by the present invention has a synergistic effect on the enzymatic hydrolysis of Dendrobium officinale, can improve the enzymatic hydrolysis efficiency, and generate more small molecule Dendrobium officinale.

[0201] In comparative example 6, in step A of preparing ultra-low molecular weight Dendrobium officinale polysaccharide, the Dendrobium officinale polysaccharide was treated by fermentation followed by enzymatic hydrolysis, but the molecular weight of the obtained Dendrobium officinale polysaccharide was greater than that in the embodiment. It can be seen that the combination of fermentation and enzymatic hydrolysis did not achieve the desired effect on Dendrobium officinale.

[0202] In the step B1 of preparing ultra-low molecular weight Tremella polysaccharide, comparative examples 7-8, 13-18 changed the strain and strain combination of fermented Tremella, and the molecules of Tremella polysaccharide of comparative examples 8, 13-18 were measured to be much larger than 3000Da, which failed to meet the requirements of the present invention. The molecular weight of Tremella polysaccharide prepared in comparative example 7 is 827Da. Since the fermentation broth contains many other substances, the molecular weight is too low and is diluted by other substances in the fermentation broth. At the same time, the molecular weight is too low and will be filtered out in the subsequent filtration step, thereby affecting the efficacy of the combination. It can be seen that selecting suitable strains for fermentation is a key factor in preparing ultra-low molecular weight Tremella polysaccharide.

[0203] Comparative Examples 9 and 10 changed the enzymolysis step in step B2 of preparing ultra-low molecular weight Tremella polysaccharide, and only performed enzymolysis once, and the molecular weight of the obtained Tremella polysaccharide was greater than 3000Da, which did not meet the needs of the present invention. Therefore, the enzymolysis step also affects the molecular weight preparation of Tremella polysaccharide.

[0204] In comparative example 11, the preparation order was changed in step B2 of preparing ultra-low molecular weight Tremella polysaccharide, and the fermentation step and the enzymatic hydrolysis step were swapped. The molecular weight of the obtained Tremella polysaccharide was 368000Da, which was much larger than that of the embodiment and did not meet the requirements of the present invention. Therefore, the preparation step order of Tremella polysaccharide is a key factor in obtaining Tremella polysaccharide with a molecular weight of less than 3000Da.

[0205] In summary, in the method for preparing ultra-low molecular weight polysaccharides, the parameters set for each process in the preparation process, the type and combination of enzymes, the selection of fermentation strains and the sequence of steps in each process all affect the molecular weight of the polysaccharide.

[0206] Test Example 2 Anti-allergic efficacy test (Relative expression of COX-2 mRNA)

[0207] Test principle: Skin allergies are mainly manifested as tingling, burning, and itching. The physiological mechanisms of these symptoms are related to damaged skin barriers, abnormal skin nerve perception, and skin inflammation. Inflammatory pain caused by skin allergies is a reaction produced under the stimulation of various inflammatory and pain-causing factors. Mitochondrial function is impaired, lysosomes and lipid membranes are damaged, and tissue and organ damage is aggravated. Cyclooxygenase (COX) is a key enzyme in the synthesis of PGs from arachidonic acid metabolism. COX-2 is an inducible enzyme. The activity of COX-2 in normal tissue cells is extremely low. When cells are stimulated by inflammation, its expression level in inflammatory cells can increase to 10-80 times the normal level, causing an increase in the content of PGE2, PGI2, and PGE1 in the inflammatory site, leading to inflammatory reactions and tissue damage. This test used sodium dodecyl sulfate (SDS)-induced mouse fibroblasts L929 as an in vitro sensitive cell model, added the test substance as an anti-allergic agent into the cells, and measured the relative expression level of COX-2 mRNA to evaluate whether the test substance had in vitro anti-allergic efficacy.

[0208] Test sample: The ultra-low molecular weight polysaccharide compositions of Examples 1 to 8 and Comparative Examples 1 to 25 were prepared into a solution with a mass fraction of 1% using H-DMEM culture medium containing 10% FBS.

[0209] Test steps: Take L929 cells in the logarithmic phase and add 2×10 5cells, add H-DMEM culture medium containing 10% FBS (hereinafter referred to as culture medium), and culture for 24 hours at 37°C, 5% CO2, and saturated humidity; aspirate and discard the culture medium, add 1mL culture medium to the negative control group, and add 1mL 1% SDS solution to the model control group and the sample group to be tested for 1 hour to induce modeling, aspirate and discard the culture medium, add 1mL culture medium to the negative control group and the model control group, and add 1mL corresponding sample solution to the sample group to be tested, and continue to culture for 24 hours. After the culture is completed, extract the total RNA from each well of the culture plate, perform RNA reverse transcription and RT-qPCR, and use the formula to calculate the gene expression multiple (n).

[0210] n=2^(-z)

[0211] z=xy

[0212] x = ct (sample group target gene) - ct (sample group housekeeping gene)

[0213] y = ct (control group target gene) - ct (control group housekeeping gene)

[0214] Each test sample was tested in parallel for 4 times, and the test results were calculated as the arithmetic mean. The results were retained to two significant figures. The test results are shown in the following table:

[0215] Table 2: Anti-allergic efficacy test results of the compositions of Examples 1 to 8 and Comparative Examples 1 to 25

[0216]

[0217]

[0218] As shown in the results of the above table, after SDS was added to the model control group, the COX-2 gene expression in L929 cells increased significantly, indicating that an allergic reaction was occurring in the cells. After SDS stimulation in the test sample group, the samples of the embodiments or comparative examples were added for culture, and the COX-2 gene expression in L929 cells decreased, indicating that the ultra-low molecular weight polysaccharide compositions of the embodiments and comparative examples have anti-allergic effects. Compared with the samples of the comparative examples, after using the samples of the embodiments, the COX-2 gene expression of the cells decreased to a greater extent, which means that the samples of the embodiments have better anti-allergic effects.

[0219] Test Example 3 Soothing Efficacy Test (TRPV1 Protein Content)

[0220] TRPV1 is an ion channel receptor, also known as capsaicin receptor. As a multiphasic receptor widely distributed in the peripheral nervous system, TRPV1 can be directly or indirectly activated by various ligands such as capsaicin, causing intracellular Ca 2+Increased, triggering action potentials. Therefore, TRPV1 is closely related to burning and pain in the skin. The higher the expression of TRPV1, the higher the skin's nerve sensitivity. Inhibiting the expression of TRPV1 can have a certain nerve-relieving effect. When the test substance acts on TRPV1, it will cause intracellular Ca 2+ The decrease in concentration indicates that the test substance achieves soothing effect by inhibiting TRPV1. This test is based on capsaicin stimulating keratinocytes and detecting the TRPV1 protein content to evaluate whether the test substance achieves soothing effect by inhibiting TRPV1.

[0221] Test samples: Examples 1 to 8, Comparative Examples 1 to 25, prepared with H-DMEM culture fluid containing 10% FBS into a solution with a mass fraction of 2%.

[0222] Test steps: 2×10 capsaicin-stimulated keratinocytes in the logarithmic phase were added to each well of a 24-well culture plate. 5 cells, add H-DMEM culture medium containing 10% FBS (hereinafter referred to as culture medium), and culture for 24 hours at 37°C, 5% CO2, and saturated humidity. When the cell plating rate in the 24-well plate reaches 40% to 60%, group administration is carried out, 1 mL of culture medium is added to the negative control group, 1 mL of culture medium containing 10 μM capsaicin is added to the model control group, and 1 mL of the corresponding sample solution is added to the sample group to be tested. Three replicate wells are set up for each group, and culture is continued for 24 hours. After the culture is completed, the cells are collected, the supernatant is discarded, and the cells are rinsed with PBS solution for 3 times. The immunofluorescence staining process is performed and photographed using a fluorescence microscope. The relative IOD value of the negative control group is 1.00, and the IOD values ​​of the model group and the sample group to be tested are calculated. The test results are shown in the following table.

[0223] Table 3: Soothing efficacy test results of the compositions of Examples 1 to 8 and Comparative Examples 1 to 25

[0224] Group Relative IOD average Group Relative IOD average Negative control group 1.00 Model control group 2.35 Example 1 1.13 Comparative Example 9 1.60 Example 2 1.38 Comparative Example 10 1.65 Example 3 1.36 Comparative Example 11 1.57 Example 4 1.28 Comparative Example 12 1.55 Example 5 1.24 Comparative Example 13 1.88 Example 6 1.37 Comparative Example 14 1.98 Example 7 1.30 Comparative Example 15 1.79 Example 8 1.16 Comparative Example 16 1.81 / / Comparative Example 17 1.91 Comparative Example 1 1.78 Comparative Example 18 1.78 Comparative Example 2 2.05 Comparative Example 19 1.98 Comparative Example 3 1.94 Comparative Example 20 1.63 Comparative Example 4 1.72 Comparative Example 21 1.77 Comparative Example 5 1.88 Comparative Example 22 1.54 Comparative Example 6 1.70 Comparative Example 23 1.63 Comparative Example 7 1.74 Comparative Example 24 1.74 Comparative Example 8 1.62 Comparative Example 25 1.53

[0225] As shown in the results in the table above, after the culture medium containing 10 μM capsaicin was added to the model control group, the average value of the relative IOD of keratinocytes was 2.35 times that of the negative control group. After the test sample was added, the average value of the relative IOD of keratinocytes decreased, indicating that TRPV1 was inhibited and the test sample showed a soothing effect. The average value of the relative IOD of the group treated with the example sample was lower than that of the control group, indicating that the soothing effect of the test example sample was better.

[0226] Test Example 4 Anti-wrinkle effect test

[0227] Test principle: Elastase is an enzyme in the matrix metalloproteinase family, which is mainly synthesized and secreted by fibroblasts. It can degrade elastin in the skin, leading to skin aging and loss of elasticity. If the test sample can inhibit the activity of elastase, it means that the test sample can alleviate the degradation of elastin in the skin and has an anti-wrinkle effect.

[0228] Test samples: The ultra-low molecular weight polysaccharide compositions of Examples 1 to 8 and Comparative Examples 1 to 25 were prepared into solutions with a mass concentration of 2% using deionized water.

[0229] Test steps: 85 μL of 50 mmol / L Tris-HCl buffer (pH 8.0) was mixed with 15 μL of sample solutions of different mass concentrations, and then 25 μL of elastase solution (60 mU / mL) was added, and incubated at 25°C for 15 min, followed by the addition of 25 μL of 1.015 mmol / L (N-succinyl-alanine-alanine-alanine-p-nitroaniline) AAAPAN solution. After 15 min, the absorbance value was measured at 410 nm. Both the elastase solution and the AAAPAN solution were prepared with 50 mmol / L Tris-HCl buffer (pH 8.0), and the determination was repeated 3 times. The inhibition rate of the sample on elastase was calculated according to formula (2).

[0230] Reaction solution without sample: Take 85 μL 50 mmol / L Tris-HCl buffer (pH 8.0) and mix with 15 μL purified water, then add 2.5 μL elastase solution (6.0 mU / mL), incubate at 2.5°C for 1.5 min, then add 2.5 μL 1.015 mmol / L AAAPAN solution, measure the absorbance at 410 nm after 15 min, and measure three times in parallel.

[0231] Reaction solution containing sample and enzyme: 85 μL 50mmol / L Tris-HCl buffer (pH 8.0) was mixed with 15 μL sample solution of different mass concentrations, and then 25 μL elastase solution (60 mU / mL) was added. The mixture was incubated at 25°C for 15 min, followed by adding 25 μL 1.015mmol / L AAAPAN solution. After 15 min, the absorbance value was measured at 410 nm, and the measurement was repeated 3 times. The elastase solution and N-succinylalanine-alanine-alanine-p-nitroaniline (AAAPAN) solution were both prepared with 50mmol / LTris-HCl buffer (pH 8.0). The inhibition rate of the sample on elastase was calculated according to the following formula.

[0232]

[0233] Among them, A is the absorbance of the reaction solution without sample; B is the absorbance of the reaction solution without sample and enzyme; C is the absorbance of the reaction solution with sample and enzyme; D is the absorbance of the reaction solution without enzyme. The test results are shown in the following table.

[0234] Table 4: Anti-wrinkle efficacy test results of the compositions of Examples 1 to 8 and Comparative Examples 1 to 25

[0235] Group Inhibition rate (%) Group Inhibition rate (%) Example 1 31.5 Comparative Example 9 24.2 Example 2 26.3 Comparative Example 10 23.9 Example 3 28.4 Comparative Example 11 21.5 Example 4 29.2 Comparative Example 12 20.1 Example 5 30.6 Comparative Example 13 23.8 Example 6 26.1 Comparative Example 14 21.2 Example 7 28.2 Comparative Example 15 21.6 Example 8 30.5 Comparative Example 16 24.1 / / Comparative Example 17 22.6 Comparative Example 1 18.4 Comparative Example 18 22.3 Comparative Example 2 20.3 Comparative Example 19 23.3 Comparative Example 3 12.8 Comparative Example 20 21.5 Comparative Example 4 22.3 Comparative Example 21 22.3 Comparative Example 5 20.5 Comparative Example 22 21.5 Comparative Example 6 19.6 Comparative Example 23 20.7 Comparative Example 7 20.8 Comparative Example 24 24.7 Comparative Example 8 23.0 Comparative Example 25 22.2

[0236] As shown in the results in the above table, the ultra-low molecular weight polysaccharide composition of the embodiment has a higher inhibition rate on hyaluronidase, indicating that the ultra-low molecular weight polysaccharide composition of the embodiment has better anti-wrinkle effect.

[0237] It can be seen from Tables 2 to 4 that the anti-allergic, soothing and anti-wrinkle effects of the ultra-low molecular weight polysaccharide compositions of Examples 1 to 8 are better than those of Comparative Examples 1 to 3, indicating that in the present invention, there is a synergistic effect between the ultra-low molecular weight Dendrobium officinale polysaccharide, the ultra-low molecular weight Tremella polysaccharide and the ultra-low molecular weight hyaluronic acid, and the three are used together to improve the anti-allergic, soothing and anti-wrinkle effects of the composition.

[0238] The ultra-low molecular weight polysaccharide compositions of Examples 1 to 8 have better anti-allergic, soothing and anti-wrinkle effects than Comparative Examples 4 to 6 and 19 to 25, indicating that the preparation process of ultra-low molecular weight Dendrobium officinale polysaccharide affects the content of active substances in the final product of ultra-low molecular weight Dendrobium officinale polysaccharide, and further affects the comprehensive anti-allergic, soothing and anti-wrinkle effects of the final composition. In the present invention, the ultra-low molecular weight Dendrobium officinale polysaccharide is preferentially crushed by ultrasonic cell disruption technology to the stem segments of Dendrobium officinale, and then enzymolyzed by hemicellulase, α-amylase and Aspergillus niger acid β-mannanase complex enzyme.

[0239] The anti-allergic, soothing and anti-wrinkle effects of the ultra-low molecular weight polysaccharide compositions of Examples 1 to 8 are better than those of Comparative Examples 7 to 11 and 13 to 18, indicating that in the process of preparing ultra-low molecular weight Tremella polysaccharide, the strain used for fermentation, the step of enzymolysis and the type of enzymolysis have a great influence on the comprehensive anti-allergic, soothing and anti-wrinkle effects of the ultra-low molecular weight Tremella polysaccharide finally prepared. In the process for preparing ultra-low molecular weight Tremella polysaccharide of the present invention, it is preferred to ferment first and then enzymolyze, the strain of fermentation is preferably Lactobacillus johnsonii and / or Bifidobacterium bifidum, the enzymolysis step is preferably twice, the enzyme types of the first enzymolysis are cellulase and pectinase, and the enzyme type of the second enzymolysis is neutral protease.

[0240] The anti-allergic, soothing and anti-wrinkle effects of the ultra-low molecular weight polysaccharide compositions of Examples 1 to 8 are better than those of Comparative Example 12. The molecular weight of the ultra-low molecular weight hyaluronic acid in the ultra-low molecular weight polysaccharide composition affects the anti-wrinkle effect of the final product. The present invention preferably uses an ultra-low molecular weight hyaluronic acid with a molecular weight of 400 to 1500 Da.

[0241] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. An ultra-low molecular weight polysaccharide composition, characterized in that: The composition comprises, by mass percentage, 45-75% of ultra-low molecular weight Dendrobium officinale polysaccharide, 20-40% of ultra-low molecular weight Tremella fuciformis polysaccharide and 5-15% of ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is ≤5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is ≤3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 400-1500Da.

2. The ultra-low molecular weight polysaccharide composition according to claim 1, characterized in that The composition comprises, by mass percentage, 60% of ultra-low molecular weight Dendrobium officinale polysaccharide, 30% of ultra-low molecular weight Tremella fuciformis polysaccharide and 10% of ultra-low molecular weight hyaluronic acid; the molecular weight of the ultra-low molecular weight Dendrobium officinale polysaccharide is 3000-5000Da, the molecular weight of the ultra-low molecular weight Tremella fuciformis polysaccharide is 2000-3000Da, and the molecular weight of the ultra-low molecular weight hyaluronic acid is 700-900Da.

3. A method for preparing the ultra-low molecular weight polysaccharide composition according to claim 1 or 2, characterized in that: The steps include: S1: mixing the Dendrobium stem segment powder with water, and subjecting the mixture to ultrasonic cell disruption treatment; S2: adding compound enzyme A to the mixture of step S1 for enzymolysis, filtering after the enzymolysis, and obtaining ultra-low molecular weight Dendrobium officinale polysaccharide; the compound enzyme A is a mixed enzyme preparation of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase; S3: inoculating Lactobacillus johnsonii and / or two bifidobacterium seed liquids into Tremella fuciformis culture medium for fermentation to obtain a crude fermentation liquid; S4: adding compound enzyme B to the crude fermentation liquid of step S3 for the first enzymolysis, after inactivating the enzyme, adding neutral protease for the second enzymolysis, after inactivating the enzyme, obtaining an enzymolysis solution, filtering, and obtaining ultra-low molecular weight Tremella polysaccharide; the compound enzyme B is a mixed enzyme preparation of cellulase and pectinase; S5 dissolving hyaluronic acid in water, adding enzyme for enzymolysis, and obtaining enzymolysis solution; S6 sequentially performs decolorization, protein removal, desalination, sterilization and drying on the enzymatic hydrolyzate of step S5 to obtain ultra-low molecular weight hyaluronic acid; S7: mixing the Dendrobium officinale polysaccharide liquid obtained in step S2, the ultra-low molecular weight Tremella fuciformis polysaccharide liquid obtained in step S4, and the ultra-low molecular weight hyaluronic acid obtained in step S6 according to mass percentage to prepare an ultra-low molecular weight polysaccharide composition; Preferably, the enzymatic hydrolysis temperature in step S2 is 40-60°C, and the enzymatic hydrolysis time is 3-5h; Preferably, in step S3, the inoculation volume ratio of Lactobacillus johnsonii or / and the two bifidobacteria seed liquid is 1-3%, and the cell concentration of Lactobacillus johnsonii or the two bifidobacteria seed liquid is 10 7 ~10 9 CFU / mL, fermentation temperature is 33-40°C, and fermentation time is 36-60h; Preferably, the Tremella culture medium in step S3 comprises the following components in mass concentrations: 1-3% Tremella powder, 1-3% nitrogen source, 1-3% carbon source, 0.15-0.35% sulfate, 0.1-0.3% phosphate and the balance water; In step S4, the temperature of the first enzymolysis is 40-60° C., and the enzymolysis time is 1.5-3 h. The temperature of the second enzymolysis is 40-60° C., and the enzymolysis time is 2.5-4 h.

4. The method for preparing the ultra-low molecular weight polysaccharide composition according to claim 3, characterized in that: The frequency of the ultrasonic cell disruption treatment in step S1 is 20-24 kHz, the time is 40-90 s / time, the temperature is 40-60° C., and the number of times is 6-10 times.

5. The method for preparing the ultra-low molecular weight polysaccharide composition according to claim 3, characterized in that: The amount of compound enzyme A added in step S2 is 0.4-1.2% of the mass fraction of the dendrobium powder; the amount of compound enzyme B added in step S4 is 0.05-0.13% of the mass fraction of the crude fermentation liquid.

6. The method for preparing the ultra-low molecular weight polysaccharide composition according to claim 3, characterized in that: The mass ratio of hemicellulase, α-amylase and Aspergillus niger acid β-mannanase in step S2 is (3-5):(1-3):(1-3); the mass ratio of cellulase and pectinase in step S4 is (1-3):

1.

7. Use of the ultra-low molecular weight polysaccharide composition according to claim 1 or 2 in the preparation of cosmetics.

8. Use of Lactobacillus johnsonii or Bifidobacterium bifidum in the preparation of ultra-low molecular weight Tremella polysaccharide, wherein the molecular weight of the ultra-low molecular weight Tremella polysaccharide is ≤3000Da.

9. Use of a composite bacterial strain in the preparation of ultra-low molecular weight Tremella polysaccharide, wherein the molecular weight of the ultra-low molecular weight Tremella polysaccharide is ≤3000Da, and the composite bacterial strain is a composite combination of Lactobacillus johnsonii and two Bifidobacteria; Preferably, the volume ratio of Lactobacillus johnsonii to the two bifidobacteria in the composite bacteria is: (1-2): (1-2).

10. A culture medium for preparing ultra-low molecular weight Tremella polysaccharide by fermentation of Lactobacillus johnsonii and / or two Bifidobacteria, characterized in that: The culture medium comprises the following components in mass concentrations: 1-3% tremella powder, 1-3% nitrogen source, 1-3% carbon source, 0.15-0.35% sulfate, 0.1-0.3% phosphate and the balance of water.

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