A method for preparing polysaccharide from fermented Rosa roxburghii tratt and its application

Abstract

The present invention discloses a method for preparing polysaccharides from fermented Rosa roxburghii tratt and its application, belonging to the field of health foods, and comprising the following steps: Step 1: Sterilize Rosa roxburghii tratt by boiling slightly; Step 2: Inoculate Rosa roxburghii tratt with microbial strains for fermentation; Step 3: Add water to the mixture of Rosa roxburghii tratt and fermentation broth for pulping; Step 4: Filter the slurry to obtain Rosa roxburghii tratt residue and Rosa roxburghii tratt filtrate; Step 5: For the Rosa roxburghii tratt residue, first hydrolyze it with cellulase, secondly hydrolyze it with papain, then hydrolyze it with pectinase, perform enzyme inactivation operation, extract it by ultrasonic wave, and cool it to room temperature to obtain an extract; Step 6: Mix the Rosa roxburghii tratt filtrate and the extract to obtain a mixed solution, vacuum-concentrate the mixed solution, then add ethanol, and perform drying treatment to obtain Rosa roxburghii tratt polysaccharide. The present invention can effectively improve the fermentation efficiency by using a mixed strain, perform composite enzyme hydrolysis, enable the combined action of multiple enzymes, improve the molecular weight and group activity of the prepared Rosa roxburghii tratt polysaccharide, and improve the enzyme hydrolysis efficiency by ultrasonic extraction to assist enzyme hydrolysis.

Description

Technical Field

[0001] The invention relates to the field of health food, and in particular to a method for preparing polysaccharide after fermentation of roxburghii roxburghii and application thereof. Background Art

[0002] Rosarox bunghii is the fruit of the perennial deciduous shrub Rosaceae, also known as Mountain King Fruit, Thorny Berry, Frango, Ci Li, Wood Pear, and also known as Thorny Pineapple, Sending Spring Back, Thorny Sour Pear, Nine-headed Bird, Wenxian Fruit. It is a nutritious and rare fruit for nourishing and fitness. Rosarox has good edible and medicinal value. Pears are rich in B vitamins, which can protect the heart, relieve fatigue, enhance myocardial vitality, and lower blood pressure; the glycosides and tannic acid contained in them can remove phlegm and relieve cough, and have a nourishing effect on the throat; pears have more sugars and multiple vitamins, which are easily absorbed by the human body, increase appetite, and have a protective effect on the liver; pears are cool in nature and can clear away heat and calm the nerves. Regular consumption can restore blood pressure to normal and improve symptoms such as dizziness; eating pears can prevent atherosclerosis and inhibit the formation of carcinogenic nitrosamines, thereby preventing and fighting cancer; the pectin content in pears is very high, which helps digestion and promotes bowel movements. Seabuckthorn contains rich pharmacologically active polysaccharides, with a general content of 1.12% and 1.43% (dry basis). Yang Jiangtao, Yang Juan and others have conducted a relatively comprehensive study on seabuckthorn polysaccharides, including the separation and purification, physical and chemical properties and biological activity of seabuckthorn polysaccharides. The studies have shown that seabuckthorn polysaccharides have the effects of enhancing immunity, anti-complement activity, antioxidant effects in vitro, antioxidant effects in mice, anti-hypoxia, and neurotrophic activity. In addition, seabuckthorn polysaccharides also have obvious protective effects on sodium thiosulfate damage and glutamate damage to neural stem cells. Seabuckthorn polysaccharides have multiple biological activities and little toxicity to the body. They are a natural medicinal chemical component with great development potential.

[0003] A Chinese patent (patent number: CN109400734B) discloses a preparation method and application of roxburghii polysaccharide, which mainly includes raw material pretreatment, extraction, deproteinization, decolorization, alcohol precipitation and separation and purification; wherein, the extraction is to mix the pretreated roxburghii dry powder with water at a material-liquid mass volume ratio of 1:15 to 1:35, and extract at a temperature of 55 to 95°C; centrifugal separation is performed to obtain a roxburghii polysaccharide extract, which is concentrated under reduced pressure to 1 / 3 to 1 / 6 of the original volume to obtain a roxburghii polysaccharide concentrate. The roxburghii polysaccharide prepared by the invention is used as a prebiotic to inhibit the activity of α-glucosidase, and is used in health foods or medicines for the auxiliary treatment of intestinal diseases and diabetes; a Chinese patent (patent number: CN109400734B) discloses a method for ultrasonic extraction of roxburghii polysaccharide The preparation of the polysaccharide from pomace includes: (1) raw material processing: drying the pomace from the pomace of the pomaceous roxburghii fruit to constant weight and then crushing it to prepare pomace powder, which is then sealed and stored at low temperature; (2) extraction: weighing the pomace powder, adding distilled water to soak it, extracting the pomace polysaccharide from the pomaceous roxburghii by ultrasonic method, and filtering to obtain the extract; (3) removing lipids: concentrating the extract, adding anhydrous petroleum ether to the polysaccharide solution, stirring and shaking it to make it emulsified, standing it until the solution is separated into upper and lower layers, and removing the petroleum ether layer; (4) removing protein: adding Sevag reagent to fully dissolve it, shaking it, and then centrifuging it to remove the denatured protein layer and take the supernatant; (5) alcohol precipitation: adding anhydrous ethanol to the filtrate, stirring it, standing it, and collecting the precipitate; (6) drying: placing the precipitate in a culture dish on dry filter paper for drying, and obtaining the pomace polysaccharide from the pomaceous roxburghii fruit.

[0004] The above disclosed preparation method Chinese patent (patent number: CN109400734B) belongs to the acid-base extraction method with strict process requirements. If the pH range required by the process is exceeded, it may cause the glycosidic bonds in the polysaccharide to break. The Chinese patent (patent number: CN109400734B) belongs to the selective combination of ultrasonic extraction and acid-base extraction, which has a great influence on the composition and activity of the polysaccharide. As for the existing polysaccharide extraction methods, enzymatic extraction has significant advantages over other extraction methods, such as mild reaction conditions, products that are not easy to deteriorate, high extraction efficiency, low cost, environmental protection and energy saving, and optimization of effective components. In particular, the auxiliary extraction method combining enzyme method with other extraction methods can improve the extraction efficiency and reduce the extraction time. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of preparing roxburghii polysaccharide by composite enzyme enzymolysis with the assistance of ultrasound in order to solve the problems existing in the prior art.

[0006] In order to achieve the above purpose, the technical solution adopted is:

[0007] A method for preparing polysaccharides after fermentation of roxburghii roxburghii and its application, comprising the following steps:

[0008] Step 1: Wash the sea buckthorn and soak it in water, sterilize it by slight boiling, and then drain it for later use;

[0009] Step 2: inoculating the roxburghii obtained in step (1) with microbial strains, and obtaining a mixture of roxburghii and fermentation liquid after fermentation;

[0010] Step 3: adding water to the mixture of the roxburghii and the fermentation liquid obtained in step (2) for grinding to obtain a uniform slurry;

[0011] Step 4: squeeze and filter the slurry to separate the roxburghii residue and the roxburghii filtrate;

[0012] Step 5: The roxburghii residue obtained in step (4) is first hydrolyzed with cellulase, then hydrolyzed with papain, and then hydrolyzed with pectinase, followed by enzyme inactivation, and finally ultrasonic extraction, and cooled to room temperature to obtain an extract;

[0013] Step 6: Mix the filtrate of the roxburghii obtained in step 2 with the extract obtained in step 3, and vacuum concentrate the mixture to a volume of 1 / 4 to 1 / 5 of the volume of the mixture, then add ethanol in an amount 3 to 4 times the volume of the concentrate, stir thoroughly and let stand for 12 hours; centrifuge, remove the lower precipitate, and spray dry to obtain roxburghii polysaccharide.

[0014] Preferably, in step 1, the volume ratio of the roxburghii to water is 1:1.5, the soaking is for 1 hour, and the mixture is slightly boiled for 20 minutes.

[0015] Preferably, the microbial strains in step 2 are a strain combination of 6-8 parts of Aspergillus niger and 5-10 parts of Kluyveromyces marxianus, and the amount of the strain combination added is 10-30% of the fermentation raw material.

[0016] Preferably, the microbial strains in step 2 are fermented and cultured at a temperature of 20-30° C. for 12 h.

[0017] Preferably, in the pulping operation of step 3, the amount of water added is 5 times the dry weight of the roxburghii.

[0018] Preferably, in step 5, the cellulase hydrolysis is performed with 1.5% to 2.5% of the mass of the roxburghii residue, and the hydrolysis time is 2-4 h. The papain hydrolysis is performed with 0.3% to 0.5% of the mass of the roxburghii residue, and the hydrolysis time is 2-4 h. The pectinase hydrolysis is performed with 0.5% to 1% of the mass of the roxburghii residue, and the hydrolysis time is 1-2 h.

[0019] Preferably, in the enzyme inactivation operation in step 5, the enzyme inactivation temperature is 90-95° C. and the enzyme inactivation time is 10-15 min.

[0020] Preferably, in the ultrasonic extraction in step 5, the ultrasonic frequency is 20kHz, the ultrasonic power is 20W, the pH value is 5-7, and the ultrasonic extraction time is 10 to 15 minutes.

[0021] Preferably, in the vacuum concentration in step 6, the vacuum degree is controlled to be 0.08-0.09 MPa and the temperature is controlled to be 65-85°C.

[0022] Preferably, the roxburghii polysaccharide is used in the preparation of liver health products.

[0023] The beneficial effects of adopting the above scheme are as follows: during the implementation of the present invention, microbial fermentation is preferred, and the use of mixed strains can effectively improve the fermentation efficiency, so that the roxburgh raw material can be pre-extracted, the fermentation efficiency is improved, and the effective ingredients of the roxburgh are further fully released.

[0024] During the implementation of the present invention, the mixture pre-fermented by microorganisms is hydrolyzed by composite enzymes, and cellulase, papain and pectinase are used for partial hydrolysis, so that multiple enzyme combinations interact with each other, the reaction conditions are mild, the product is not easy to deteriorate, and the molecular weight and group activity of the prepared roxburghii multiflora are improved.

[0025] During the implementation of the present invention, ultrasound is used to assist enzymolysis, thereby improving enzymolysis efficiency, improving extraction efficiency, and reducing extraction time. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention is clearly and completely described below in conjunction with the specific embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. Example 1

[0027] A method for preparing polysaccharides after fermentation of roxburghii roxburghii and its application, comprising the following steps:

[0028] Step 1: Take 50 portions of roxburghii, wash and soak the roxburghii in water, sterilize them by slight boiling, and then drain and set aside;

[0029] Step 2: inoculating the roxburghii obtained in step (1) with microbial strains, and obtaining a mixture of roxburghii and fermentation liquid after fermentation;

[0030] Step 3: adding water to the mixture of the roxburghii and the fermentation liquid obtained in step (2) for grinding to obtain a uniform slurry;

[0031] Step 4: squeeze and filter the slurry to separate the roxburghii residue and the roxburghii filtrate;

[0032] Step 5: The roxburghii residue obtained in step (4) is first hydrolyzed with cellulase, then hydrolyzed with papain, and then hydrolyzed with pectinase, followed by enzyme inactivation, and finally ultrasonic extraction, and cooled to room temperature to obtain an extract;

[0033] Step 6: Mix the filtrate of the roxburghii obtained in step 2 with the extract obtained in step 3, and vacuum concentrate the mixture to a volume of 1 / 4 of the volume of the mixture, then add ethanol in an amount 3 times the volume of the concentrate, stir well and let stand for 12 hours; centrifuge, remove the lower precipitate, and spray dry to obtain roxburghii polysaccharide.

[0034] Preferably, in step 1, the volume ratio of the roxburghii to water is 1:1.5, the soaking is for 1 hour, and the mixture is slightly boiled for 20 minutes.

[0035] Preferably, the microbial strains in step 2 are a strain combination of 6 parts of Aspergillus niger and 5 parts of Kluyveromyces marxianus, and the amount of the strain combination added is 10% of the fermentation raw material.

[0036] Preferably, the microbial strains in step 2 are fermented at a temperature of 20° C. and for a time of 12 h.

[0037] Preferably, in the pulping operation of step 3, the amount of water added is 5 times the dry weight of the roxburghii.

[0038] Preferably, in step 5, the cellulase hydrolysis is performed with 1.5% of the mass of the roxburghii residue and the hydrolysis time is 2 h, the papain hydrolysis is performed with 0.3% of the mass of the roxburghii residue and the hydrolysis time is 2 h, and the pectinase hydrolysis is performed with 0.5% of the mass of the roxburghii residue and the hydrolysis time is 1 h.

[0039] Preferably, in the enzyme inactivation operation in step 5, the enzyme inactivation temperature is 90° C. and the enzyme inactivation time is 10 min.

[0040] Preferably, in the ultrasonic extraction in step 5, the ultrasonic frequency is 20 kHz, the ultrasonic power is 20 W, the pH value is 5, and the ultrasonic extraction time is 10 min.

[0041] Preferably, in the vacuum concentration in step 6, the vacuum degree is controlled to 0.08 MPa and the temperature is controlled to 65°C. Example 2

[0042] A method for preparing polysaccharides after fermentation of roxburghii roxburghii and its application, comprising the following steps:

[0043] Step 1: Take 50 portions of roxburghii, wash and soak the roxburghii in water, sterilize them by slight boiling, and then drain and set aside;

[0044] Step 2: inoculating the roxburghii obtained in step (1) with microbial strains, and obtaining a mixture of roxburghii and fermentation liquid after fermentation;

[0045] Step 3: adding water to the mixture of the roxburghii and the fermentation liquid obtained in step (2) for grinding to obtain a uniform slurry;

[0046] Step 4: squeeze and filter the slurry to separate the roxburghii residue and the roxburghii filtrate;

[0047] Step 5: The roxburghii residue obtained in step (4) is first hydrolyzed with cellulase, then hydrolyzed with papain, and then hydrolyzed with pectinase, followed by enzyme inactivation, and finally ultrasonic extraction, and cooled to room temperature to obtain an extract;

[0048] Step 6: Mix the filtrate of the roxburghii obtained in step 2 with the extract obtained in step 3, vacuum concentrate the mixture to a volume of 1 / 4 of the volume of the mixture, add ethanol in an amount 3 times the volume of the concentrate, stir well and let stand for 12 hours; centrifuge, remove the lower precipitate, and spray dry to obtain roxburghii polysaccharide.

[0049] Preferably, in step 1, the volume ratio of the roxburghii to water is 1:1.5, the soaking is for 1 hour, and the mixture is slightly boiled for 20 minutes.

[0050] Preferably, the microbial strains in step 2 are a strain combination of 7 parts of Aspergillus niger and 8 parts of Kluyveromyces marxianus, and the amount of the strain combination added is 20% of the fermentation raw material.

[0051] Preferably, the microbial strains in step 2 are fermented at a temperature of 20° C. and for a time of 12 h.

[0052] Preferably, in the pulping operation of step 3, the amount of water added is 5 times the dry weight of the roxburghii.

[0053] Preferably, in step 5, the cellulase hydrolysis is performed with 1.5% of the mass of the roxburghii residue and the hydrolysis time is 2 h, the papain hydrolysis is performed with 0.3% of the mass of the roxburghii residue and the hydrolysis time is 2 h, and the pectinase hydrolysis is performed with 0.5% of the mass of the roxburghii residue and the hydrolysis time is 1 h.

[0054] Preferably, in the enzyme inactivation operation in step 5, the enzyme inactivation temperature is 90° C. and the enzyme inactivation time is 10 min.

[0055] Preferably, in the ultrasonic extraction in step 5, the ultrasonic frequency is 20 kHz, the ultrasonic power is 20 W, the pH value is 5, and the ultrasonic extraction time is 10 min.

[0056] Preferably, in the vacuum concentration in step 6, the vacuum degree is controlled to 0.08 MPa and the temperature is controlled to 65°C. Example 3

[0057] A method for preparing polysaccharides after fermentation of roxburghii roxburghii and its application, comprising the following steps:

[0058] Step 1: Take 50 portions of roxburghii, wash and soak the roxburghii in water, sterilize them by slight boiling, and then drain and set aside;

[0059] Step 2: inoculating the roxburghii obtained in step (1) with microbial strains, and obtaining a mixture of roxburghii and fermentation liquid after fermentation;

[0060] Step 3: adding water to the mixture of the roxburghii and the fermentation liquid obtained in step (2) for grinding to obtain a uniform slurry;

[0061] Step 4: squeeze and filter the slurry to separate the roxburghii residue and the roxburghii filtrate;

[0062] Step 5: The roxburghii residue obtained in step (4) is first hydrolyzed with cellulase, then hydrolyzed with papain, and then hydrolyzed with pectinase, followed by enzyme inactivation, and finally ultrasonic extraction, and cooled to room temperature to obtain an extract;

[0063] Step 6: Mix the filtrate of the roxburghii obtained in step 2 with the extract obtained in step 3, and vacuum concentrate the mixture to a volume of 1 / 4 of the volume of the mixture, then add ethanol in an amount 3 times the volume of the concentrate, stir well and let stand for 12 hours; centrifuge, remove the lower precipitate, and spray dry to obtain roxburghii polysaccharide.

[0064] Preferably, in step 1, the volume ratio of the roxburghii to water is 1:1.5, the soaking is for 1 hour, and the mixture is slightly boiled for 20 minutes.

[0065] Preferably, the microbial strains in step 2 are a strain combination of 8 parts of Aspergillus niger and 10 parts of Kluyveromyces marxianus, and the amount of the strain combination added is 30% of the fermentation raw material.

[0066] Preferably, the microbial strains in step 2 are fermented at a temperature of 20° C. and for a time of 12 h.

[0067] Preferably, in the pulping operation of step 3, the amount of water added is 5 times the dry weight of the roxburghii.

[0068] Preferably, in step 5, the cellulase hydrolysis is performed with 1.5% of the mass of the roxburghii residue and the hydrolysis time is 2 h, the papain hydrolysis is performed with 0.3% of the mass of the roxburghii residue and the hydrolysis time is 2 h, and the pectinase hydrolysis is performed with 0.5% of the mass of the roxburghii residue and the hydrolysis time is 1 h.

[0069] Preferably, in the enzyme inactivation operation in step 5, the enzyme inactivation temperature is 90° C. and the enzyme inactivation time is 10 min.

[0070] Preferably, in the ultrasonic extraction in step 5, the ultrasonic frequency is 20 kHz, the ultrasonic power is 20 W, the pH value is 5, and the ultrasonic extraction time is 10 min.

[0071] Preferably, in the vacuum concentration in step 6, the vacuum degree is controlled to 0.08 MPa and the temperature is controlled to 65°C. Example 4

[0072] A method for preparing polysaccharides after fermentation of roxburghii roxburghii and its application, comprising the following steps:

[0073] Step 1: Take 50 portions of roxburghii, wash and soak the roxburghii in water, sterilize them by slight boiling, and then drain and set aside;

[0074] Step 2: inoculating the roxburghii obtained in step (1) with microbial strains, and obtaining a mixture of roxburghii and fermentation liquid after fermentation;

[0075] Step 3: adding water to the mixture of the roxburghii and the fermentation liquid obtained in step (2) for grinding to obtain a uniform slurry;

[0076] Step 4: squeeze and filter the slurry to separate the roxburghii residue and the roxburghii filtrate;

[0077] Step 5: The roxburghii residue obtained in step (4) is first hydrolyzed with cellulase, then hydrolyzed with papain, and then hydrolyzed with pectinase, followed by enzyme inactivation, and finally ultrasonic extraction, and cooled to room temperature to obtain an extract;

[0078] Step 6: Mix the filtrate of the roxburghii obtained in step 2 with the extract obtained in step 3, vacuum concentrate the mixture to a volume of 1 / 4 of the volume of the mixture, add ethanol in an amount 3 times the volume of the concentrate, stir well and let stand for 12 hours; centrifuge, remove the lower precipitate, and spray dry to obtain roxburghii polysaccharide.

[0079] Preferably, in step 1, the volume ratio of the roxburghii to water is 1:1.5, the soaking is for 1 hour, and the mixture is slightly boiled for 20 minutes.

[0080] Preferably, the microbial strains in step 2 are a strain combination of 8 parts of Aspergillus niger and 10 parts of Kluyveromyces marxianus, and the amount of the strain combination added is 30% of the fermentation raw material.

[0081] Preferably, the microbial strains in step 2 are fermented at a temperature of 30° C. and for a time of 12 h.

[0082] Preferably, in the pulping operation of step 3, the amount of water added is 5 times the dry weight of the roxburghii.

[0083] Preferably, in step 5, the cellulase hydrolysis is performed with 1.5% of the mass of the roxburghii residue and the hydrolysis time is 2 h, the papain hydrolysis is performed with 0.3% of the mass of the roxburghii residue and the hydrolysis time is 2 h, and the pectinase hydrolysis is performed with 0.5% of the mass of the roxburghii residue and the hydrolysis time is 1 h.

[0084] Preferably, in the enzyme inactivation operation in step 5, the enzyme inactivation temperature is 90° C. and the enzyme inactivation time is 10 min.

[0085] Preferably, in the ultrasonic extraction in step 5, the ultrasonic frequency is 20 kHz, the ultrasonic power is 20 W, the pH value is 5, and the ultrasonic extraction time is 10 min.

[0086] Preferably, in the vacuum concentration in step 6, the vacuum degree is controlled to 0.08 MPa and the temperature is controlled to 65°C. Example 5

[0087] A method for preparing polysaccharides after fermentation of roxburghii roxburghii and its application, comprising the following steps:

[0088] Step 1: Wash the sea buckthorn and soak it in water, sterilize it by slight boiling, and then drain it for later use;

[0089] Step 2: inoculating the roxburghii obtained in step (1) with microbial strains, and obtaining a mixture of roxburghii and fermentation liquid after fermentation;

[0090] Step 3: adding water to the mixture of the roxburghii and the fermentation liquid obtained in step (2) for grinding to obtain a uniform slurry;

[0091] Step 4: squeeze and filter the slurry to separate the roxburghii residue and the roxburghii filtrate;

[0092] Step 5: The roxburghii residue obtained in step (4) is first hydrolyzed with cellulase, then hydrolyzed with papain, and then hydrolyzed with pectinase, followed by enzyme inactivation, and finally ultrasonic extraction, and cooled to room temperature to obtain an extract;

[0093] Step 6: Mix the filtrate of the roxburghii obtained in step 2 with the extract obtained in step 3, and vacuum concentrate the mixture to a volume of 1 / 4 of the volume of the mixture, then add ethanol in an amount 3 times the volume of the concentrate, stir well and let stand for 12 hours; centrifuge, remove the lower precipitate, and spray dry to obtain roxburghii polysaccharide.

[0094] Preferably, in step 1, the volume ratio of the roxburghii to water is 1:1.5, the soaking is for 1 hour, and the mixture is slightly boiled for 20 minutes.

[0095] Preferably, the microbial strains in step 2 are a strain combination of 8 parts of Aspergillus niger and 10 parts of Kluyveromyces marxianus, and the amount of the strain combination added is 30% of the fermentation raw material.

[0096] Preferably, the microbial strains in step 2 are fermented at a temperature of 30° C. and for a time of 12 h.

[0097] Preferably, in the pulping operation of step 3, the amount of water added is 5 times the dry weight of the roxburghii.

[0098] Preferably, in step 5, the cellulase hydrolysis is performed with an enzyme mass of 2% of the roxburghii residue and a hydrolysis time of 3 h, the papain hydrolysis is performed with an enzyme mass of 0.4% of the roxburghii residue and a hydrolysis time of 3 h, and the pectinase hydrolysis is performed with an enzyme mass of 0.75% of the roxburghii residue and a hydrolysis time of 1.5 h.

[0099] Preferably, in the enzyme inactivation operation in step 5, the enzyme inactivation temperature is 93° C. and the enzyme inactivation time is 12.5 min.

[0100] Preferably, in the ultrasonic extraction in step 5, the ultrasonic frequency is 20 kHz, the ultrasonic power is 20 W, the pH value is 5, and the ultrasonic extraction time is 10 min.

[0101] Preferably, in the vacuum concentration in step 6, the vacuum degree is controlled to 0.08 MPa and the temperature is controlled to 65°C. Example 6

[0102] A method for preparing polysaccharides after fermentation of roxburghii roxburghii and its application, comprising the following steps:

[0103] Step 1: Wash the sea buckthorn and soak it in water, sterilize it by slight boiling, and then drain it for later use;

[0104] Step 2: inoculating the roxburghii obtained in step (1) with microbial strains, and obtaining a mixture of roxburghii and fermentation liquid after fermentation;

[0105] Step 3: adding water to the mixture of the roxburghii and the fermentation liquid obtained in step (2) for grinding to obtain a uniform slurry;

[0106] Step 4: squeeze and filter the slurry to separate the roxburghii residue and the roxburghii filtrate;

[0107] Step 5: The roxburghii residue obtained in step (4) is first hydrolyzed with cellulase, then hydrolyzed with papain, and then hydrolyzed with pectinase, followed by enzyme inactivation, and finally ultrasonic extraction, and cooled to room temperature to obtain an extract;

[0108] Step 6: Mix the filtrate of the roxburghii obtained in step 2 with the extract obtained in step 3, and vacuum concentrate the mixture to a volume of 1 / 4 of the volume of the mixture, then add ethanol in an amount 3 times the volume of the concentrate, stir well and let stand for 12 hours; centrifuge, remove the lower precipitate, and spray dry to obtain roxburghii polysaccharide.

[0109] Preferably, in step 1, the volume ratio of the roxburghii to water is 1:1.5, the soaking is for 1 hour, and the mixture is slightly boiled for 20 minutes.

[0110] Preferably, the microbial strains in step 2 are a strain combination of 8 parts of Aspergillus niger and 10 parts of Kluyveromyces marxianus, and the amount of the strain combination added is 30% of the fermentation raw material.

[0111] Preferably, the microbial strains in step 2 are fermented at a temperature of 30° C. and for a time of 12 h.

[0112] Preferably, in the pulping operation of step 3, the amount of water added is 5 times the dry weight of the roxburghii.

[0113] Preferably, in step 5, the cellulase hydrolysis is performed with 2.5% of the mass of the roxburghii residue and the hydrolysis time is 4 h, the papain hydrolysis is performed with 0.5% of the mass of the roxburghii residue and the hydrolysis time is 4 h, and the pectinase hydrolysis is performed with 1% of the mass of the roxburghii residue and the hydrolysis time is 2 h.

[0114] Preferably, in the enzyme inactivation operation in step 5, the enzyme inactivation temperature is 93° C. and the enzyme inactivation time is 12.5 min.

[0115] Preferably, in the ultrasonic extraction in step 5, the ultrasonic frequency is 20 kHz, the ultrasonic power is 20 W, the pH value is 5, and the ultrasonic extraction time is 10 min.

[0116] Preferably, in the vacuum concentration in step 6, the vacuum degree is controlled to 0.08 MPa and the temperature is controlled to 65°C.

[0117] Comparative Example 1

[0118] The difference from Example 1 is that step (2) of inoculating microbial strains is omitted, and the other steps are the same as those in Example 1.

[0119] Comparative Example 2

[0120] The difference from Example 3 is that the microbial strains in step 2 are 10 parts of Aspergillus niger and 12 parts of Kluyveromyces marxianus, and the amount of the strain combination added is 40% of the fermentation raw material. The other steps are the same as in Example 3.

[0121] Comparative Example 3

[0122] The difference from Example 4 is that, in step 5, the cellulase hydrolysis is performed with a cellulase mass of 0.5% of the mass of the roxburghii residue, and the hydrolysis time is 1 h; the papain hydrolysis is performed with a papain mass of 0.2% of the mass of the roxburghii residue, and the hydrolysis time is 1 h; the pectinase hydrolysis is performed with a pectinase mass of 0.3% of the mass of the roxburghii residue, and the hydrolysis time is 0.5 h; and the other steps are the same.

[0123] Comparative Example 4

[0124] The difference from Example 6 is that, in step 5, the cellulase hydrolysis is 3% of the mass of the roxburghii residue, and the hydrolysis time is 4.5 h; the papain hydrolysis is 1% of the mass of the roxburghii residue, and the hydrolysis time is 4.5 h; the pectinase hydrolysis is 1.5% of the mass of the roxburghii residue, and the hydrolysis time is 2.5 h; and the other steps are the same.

[0125] Comparative Example 5

[0126] The difference from Example 6 is that, in step 5, cellulase hydrolysis, papain hydrolysis, pectinase hydrolysis, and mixed enzymolysis are carried out, the mass of the cellulase is 2.5% of the mass of the roxburghii residue, the mass of the papain is 0.5% of the mass of the roxburghii residue, the mass of the pectinase is 1% of the mass of the roxburghii residue, and the mixed enzymolysis is carried out for 2 hours, and the other steps are the same.

[0127] Experimental Example 1 Determination of total sugar content of roxburghii polysaccharides

[0128] The total sugar content of Rosa roxburghii polysaccharides was determined by phenol-sulfuric acid method.

[0129] (1) Preparation of solution:

[0130] 5% phenol solution: Accurately weigh 50 mg of phenol, place it in a 1 mL brown volumetric flask, add water to dissolve and dilute to the mark.

[0131] Glucose series standard solutions: Accurately pipette 0, 40, 80, 120, 160, 200 μL of standard solution (1 mg / mL) into a 2 mL volumetric flask, add water to dilute to the scale to obtain glucose series standard solutions of 0, 20, 40, 60, 80, 100 μg / mL.

[0132] Rosa roxburghii polysaccharide test solution: Take the Rosa roxburghii polysaccharides prepared in Examples 1-3 and Comparative Examples 1-2, prepare 10 mL of 10 mg / mL test solution respectively, take 0.01 mL of the prepared test solution and dilute it to 2 mL, to obtain a reaction solution of 50 μg / mL.

[0133] (2) Phenol-sulfuric acid method to measure polysaccharide content:

[0134] Take 200 μL of the prepared roxburghii polysaccharide test solution and glucose series standard solution into a stoppered test tube, add 100 μL of phenol in an ice water bath, shake well, add 500 μL of sulfuric acid, shake well, heat in a boiling water bath for 15 minutes, take out, and ice water bath for 10 minutes. Measure the absorbance at 490 nm with an enzyme marker.

[0135] The results and analysis of Experimental Example 1 are shown in Table 1 below.

[0136] Table 1 Determination results of total sugar content of roxburghii polysaccharides,

[0137] Group Total sugar content (%) Example 1 96.61% Example 2 97.21% Example 3 98.81% Comparative Example 1 67.52% Comparative Example 2 80.55% .

[0138] According to the test data in Table 1 above, it can be seen that the total sugar content in Examples 1-3 of the present invention is relatively high. By controlling the ratio of added microbial strains inoculated between the components, the content of polysaccharides can be effectively increased, and the extraction effect is improved. Comparative Example 1 does not adopt the method of microbial strain fermentation, and directly performs enzyme extraction. The mass ratio of added microbial strains inoculated in Comparative Example 2 is not within the protection scope disclosed in the present invention. It can be seen from the test data that Comparative Examples 1-2 affect the extraction effect, thereby verifying that the use of mixed strains for fermentation and then extraction of polysaccharides can fully release the effective ingredients of Rosa roxburghii, effectively improve the fermentation efficiency, and achieve beneficial effects.

[0139] Experimental Example 2 Determination of protein content in roxburghii polysaccharides

[0140] The protein content in the total sugar of Rosa roxburghii polysaccharide was determined by Coomassie Brilliant Blue method.

[0141] (1) Preparation of solution:

[0142] Coomassie Brilliant Blue G250 solution: Accurately weigh 10.00 mg of Coomassie Brilliant Blue G250, add the weighed Coomassie Brilliant Blue into a 100-mL volumetric flask, then add 5 mL of 95% ethanol, and once again add 10 mL of 85% phosphoric acid. Finally, make up to volume with distilled water and shake well. The final reagent contains 0.01% Coomassie Brilliant Blue G250, 4.7% ethanol, and 8.5% phosphoric acid. Place it in a brown bottle for later use.

[0143] Protein series standard solution: accurately weigh 10.00 mg of bovine serum albumin, add the weighed bovine serum albumin into a 10 mL volumetric flask, first add a small amount of distilled water to dissolve and mix, then add distilled water to make up to volume to obtain a 1 mg / mL protein standard mother solution. Accurately pipette 0, 40, 80, 120, 160, 200, 240 μL of the mother solution into a 2 mL volumetric flask, make up to volume, mix well, and obtain a bovine serum albumin series standard solution of 0, 20, 40, 60, 80, 100 μg / mL.

[0144] Rosa roxburghii polysaccharide test solution: Take the Rosa roxburghii polysaccharides prepared in Examples 4-6 and Comparative Examples 3-5, weigh 10 mg of the test sample into a 1 mL volumetric flask, dilute to the scale line, and obtain a 10 mg / mL test solution for later use.

[0145] (2) Coomassie Brilliant Blue Method for Determination of Protein Content in Polysaccharides

[0146] Take 100 μL of bovine serum albumin series standard solution and 100 μL of roxburghii polysaccharide test solution into stoppered test tubes, add 500 μL of Coomassie Brilliant Blue G-250 solution, mix well, let stand for 10 minutes, and measure the absorbance at 595 nm using an enzyme reader.

[0147] Experimental Example 3 Determination of molecular weight distribution of roxburghii polysaccharide

[0148] Gel chromatography column and high performance liquid chromatography were used to determine the molecular weight distribution and purity of the polysaccharide.

[0149] (1) Drawing of standard curve:

[0150] Molecular weight standard curves were prepared using dextran with molecular weights of 9750, 13050, 36800, 64650, 135350, 300600, and 20000000.

[0151] (2) Sample determination: Take appropriate amounts of the roxburghii polysaccharides prepared in Examples 4-6 and Comparative Examples 3-5, respectively, and then dry the polysaccharide samples, dissolve them in ultrapure water, and prepare a sugar solution with a concentration of 2 mg / mL. After filtering with an aqueous microporous filter membrane, the solution is measured on an instrument.

[0152] (3) High pressure liquid chromatography conditions: injection volume of 20 μL, mobile phase of 20 mM ammonium acetate, flow rate of 0.5 mL / min, chromatographic column of gel chromatography, column temperature of 40°C, and detector of evaporative light scattering detector.

[0153] (4) Evaporative light scattering detector conditions: nebulizer temperature was 30°C, drift tube temperature was 115°C, and nitrogen pressure was 1.6 SLM.

[0154] The results and analysis of Experimental Examples 2 and 3 are shown in Table 2 below.

[0155] Table 2 Protein content determination and molecular weight distribution determination results of roxburghii polysaccharides.

[0156] Group Protein content (%) Molecular weight (Da) Example 4 0.11% 6025Da Example 5 0.08% 6532Da Example 6 0.06% 7011Da Comparative Example 3 2.5% 3220Da Comparative Example 4 2.3% 2958Da Comparative Example 5 1.2% 4122Da .

[0157] According to the test data in Table 2 above, it can be seen that the protein content in Examples 4-6 of the present invention is low, and the molecular weight of polysaccharides is high. By controlling the ratio of adding composite enzymes between the components, the activity of polysaccharides can be effectively improved, and the extraction effect is improved. The mass ratio of adding composite enzymes in Comparative Example 3 and Comparative Example 4 is not within the protection scope disclosed in the present invention. Comparative Example 5 mixes the composite enzymes for enzymolysis. From the test data, it can be seen that Comparative Examples 3-5 affect the extraction effect, thereby verifying that enzymolysis is carried out in parts in a sequential order, so that a variety of enzyme combinations interact with each other, the reaction conditions are mild, and the product is not easy to deteriorate, thereby improving the molecular weight and group activity of the prepared Rosa roxburghii.

[0158] Experimental Example 4 Study on the effect of roxburghii polysaccharide in protecting the liver from alcoholic damage

[0159] Test method: Male SD rats weighing 100±20g were selected as test subjects and kept in an SPF animal room with a room temperature of 25±3°C, a relative humidity of 60%-70%, and a 12h light and 12h dark environment. After one week of adaptive feeding, the rats were randomly divided into 4 groups, with 6 rats in each group.

[0160] Normal control group: rats were given free diet and intragastric administration of distilled water for 7 consecutive days.

[0161] Model control group: rats were fed with free diet and gavaged with distilled water for 7 consecutive days, and finally gavaged with 12 mL / kg (based on body weight) of 50% ethanol solution to establish the mouse alcoholic liver injury model.

[0162] Rosa roxburghii polysaccharide low-dose group: Rats were fed ad libitum and administered with distilled water (100 mg / (kg·d, based on body weight) of Rosa roxburghii polysaccharide, which was selected from Example 6, for 7 consecutive days, and finally administered with 12 mL / kg (based on body weight) of 50% ethanol solution.

[0163] Rosa roxburghii polysaccharide high-dose group: Rats were fed ad libitum and administered with distilled water (500 mg / (kg·d, based on body weight) of Rosa roxburghii polysaccharide, which was selected from Example 6, for 7 consecutive days, and the last administration was with 12 mL / kg (based on body weight) of 50% ethanol solution.

[0164] After the last oral gavage, all groups were fasted but not watered for 12 h to collect serum samples and the animals were killed to collect liver samples for structural examination.

[0165] Histopathological examination: The same part of the left lobe of the liver was fixed with 10% neutral formaldehyde, embedded in paraffin, and stained with hematoxylin-eosin (HE) to observe the pathological changes of liver tissue under an optical microscope.

[0166] Serum related index detection: blood was collected from the eyeball in a 1.5 mL centrifuge tube, and the blood was allowed to stand for 2 hours, centrifuged at 4°C, 3000 r / min for 10 minutes, and the supernatant was divided into 3 parts and stored at -20°C. When measuring ALT, AST and TG levels, a serum sample was taken out for testing according to the method of the kit.

[0167] Detection of liver homogenate-related indicators: Accurately weigh about 0.1 g of liver, wash the blood on the surface with ice saline and wipe it dry with filter paper, put the liver in a 1.5 mL centrifuge tube, add about 0.9 mL of pre-cooled saline to make the ratio of liver to saline 1:9, use a homogenizer to prepare 10% liver homogenate, centrifuge at 4°C, 3000r / min for 10 min, and divide it into 3 portions for subsequent detection of MDA, SOD and GSH-Px activity.

[0168] Experimental Example 4 Results and Analysis

[0169] 1. Effect of roxburghii polysaccharide on pathological changes of liver tissue in mice with alcoholic liver damage

[0170] The results of pathological sections of mouse liver tissue are as follows: the liver tissue structure of the normal control group is intact, the size is relatively uniform, the nucleus is round and clear, and the cytoplasm is abundant. The liver lobules are normal, and neatly radially arranged liver cell cords can be seen.

[0171] Compared with the normal control group, the model control group showed disordered liver tissue structure, hepatocyte swelling, vacuolar degeneration, a large number of round lipid droplets of varying sizes in the cytoplasm, unclear boundaries of liver lobules, hepatocyte necrosis, unclear cell boundary morphology, red blood cells between liver cords, and a large number of inflammatory cells infiltration. Therefore, it was concluded that the model was successful.

[0172] Compared with the model control group, the two roxburghii polysaccharide groups showed improved hepatocyte swelling and vacuolar degeneration, and fat vacuoles were visible. The central vein of the liver was dilated, and red blood cells were occasionally seen between the hepatic cords. Inflammatory cell infiltration was significantly reduced. Therefore, it can be concluded from the pathological sections that roxburghii polysaccharides have a certain protective effect on acute alcoholic liver injury in mice, and the high-dose effect is slightly better than the low-dose group.

[0173] 2. Effects of roxburghii polysaccharides on liver index and liver function indices in mice with acute alcoholic liver injury

[0174] As shown in Table 3, compared with the normal control group, the liver index, ALT (alanine aminotransferase), and AST (aspartate aminotransferase) of the model control group were significantly increased, indicating that the liver cells were significantly damaged; while the liver index of the low-dose and high-dose roxburghii polysaccharide groups was significantly decreased, and ALT and AST were also significantly decreased, which was statistically significant compared with the model control group (P<0.05, P<0.01), but there was no statistically significant difference between the two groups. This shows that roxburghii polysaccharide has a protective effect on acute alcoholic liver injury in mice in terms of liver index and liver function indicators, but there is no dose dependence.

[0175] Table 3 Effects of roxburghii polysaccharide on liver index and liver function indexes in mice with acute alcoholic liver injury (x±s, n=6),

[0176] Group dose Liver index (%) ALT (U / L) AST (U / L) Normal control group - 0.038±2.16 18.652±1.85 38.522±6.75 Model control group - 0.047±4.99** 35.357±3.11** 50.119±5.29** Low-dose group of roxburghii polysaccharide 100 0.39±5.84# 20.254±3.15## 39.951±2.65# High-dose group of roxburghii polysaccharide 500 0.048±2.11# 23.595±2.54## 39.552±2.22#

[0177] Compared with normal control, *P<0.05, **P<0.01; compared with normal control, #P<0.05, ##P<0.01.

[0178] 3. Effect of Rosa roxburghii polysaccharide on serum TG in mice with acute alcoholic liver injury

[0179] As shown in Table 4, compared with the normal control group, the TG content in the serum of the mice in the model control group was significantly increased, indicating that the model group was successfully established in terms of this index. The TG content of mice in the low-dose and high-dose roxburghii polysaccharide groups was significantly reduced, and the difference was statistically significant compared with the model group (P<0.01), but there was no statistically significant difference between the two groups. This shows that roxburghii polysaccharide has a protective effect on acute alcoholic liver injury in mice in terms of serum TG indicators, but there is no dose dependence.

[0180] Table 4 Effects of roxburghii polysaccharide on serum TG in mice with acute alcoholic liver injury (x±s, n=6),

[0181] Group dose TG (mmol / L) Normal control group - 0.876±8.26 Model control group - 2.927±8.85** Low-dose group of roxburghii polysaccharide 100 1.389±2.84## High-dose group of roxburghii polysaccharide 500 1.718±1.91##

[0182] Compared with normal control, *P<0.05, **P<0.01; compared with normal control, #P<0.05, ##P<0.01;.

[0183] 4. Effects of roxburghii polysaccharides on oxidative stress-related indicators in liver tissue of mice with acute alcoholic liver injury

[0184] As can be seen from Table 5, the activities of SOD and GSH-Px in the model control group were significantly reduced, and the content of MDA was significantly increased, which was statistically significant compared with the normal control group (P<0.01); while the activities of SOD and GSH-Px in the low-dose group and the high-dose group of roxburghii polysaccharide were significantly enhanced, and the content of MDA was significantly decreased, which was statistically significant compared with the model control group (P<0.05, P<0.01), but the difference between the two groups was not statistically significant. This shows that roxburghii polysaccharide has a protective effect on acute alcoholic liver injury in mice in terms of oxidative stress indicators, but it is not dose-dependent.

[0185] Table 5 Effects of roxburghii polysaccharides on oxidative stress-related indicators in liver tissue of mice with acute alcoholic liver injury (x±s, n=6),

[0186] Group dose MDA (nmol / L) SOD (U / mg) GSH-Px (U) Normal control group - 83.885±2.85 18.652±1.85 38.522±6.75 Model control group - 146.525±33.11** 152.634±14.85** 365.985±74.29** Low-dose group of roxburghii polysaccharide 100 108.524±8.24# 176.552±13.25## 435.584±25.65# High-dose group of roxburghii polysaccharide 500 108.298±10.12# 178.533±12.14## 436.502±23.28#

[0187] Compared with normal control, *P<0.05, **P<0.01; compared with normal control, #P<0.05, ##P<0.01.

[0188] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0189] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing polysaccharides after fermentation of roxburghii ..., It is characterized in that The following steps are involved: Step 1: Wash the sea buckthorn and soak it in water, sterilize it by slight boiling, and then drain it for later use; Step 2: inoculating the roxburghii obtained in step (1) with microbial strains, and obtaining a mixture of roxburghii and fermentation liquid after fermentation; Step 3: adding water to the mixture of the roxburghii and the fermentation liquid obtained in step (2) for grinding to obtain a uniform slurry; Step 4: squeeze and filter the slurry to separate the roxburghii residue and the roxburghii filtrate; Step 5: The roxburghii residue obtained in step (4) is first hydrolyzed with cellulase, then hydrolyzed with papain, and then hydrolyzed with pectinase, followed by enzyme inactivation, and finally ultrasonic extraction, and cooled to room temperature to obtain an extract; Step 6: Mix the filtrate of the roxburghii obtained in step (4) with the extract obtained in step (5) to obtain a mixed solution, vacuum concentrate the mixed solution to a volume of 1 / 4 of the volume of the mixed solution, add ethanol in an amount 3 times the volume of the concentrated solution, stir well and let stand for 12 hours; centrifuge, remove the precipitate, and spray dry to obtain roxburghii polysaccharide; The step 1 is soaked in water with a volume ratio of 1:1.5 of roxburghii to water, soaked for 1 hour, and slightly boiled for 20 minutes; The microbial strains in step 2 are composed of 6-8 parts of Aspergillus niger and 5-10 parts of Kluyveromyces marxianus, and the amount of the combined strains added is 10-30% of the fermentation raw material; The microbial strain in step 2 is fermented at a temperature of 20-30°C and a culture time of 12 hours; In the grinding operation of step 3, the amount of water added is 5 times the dry weight of the roxburghii; In step 5, the cellulase hydrolysis is performed, the mass of the cellulase is 1.5% to 2.5% of the mass of the roxburghii residue, and the hydrolysis time is 2-4 hours; the papain hydrolysis is performed, the mass of the papain is 0.3% to 0.5% of the mass of the roxburghii residue, and the hydrolysis time is 2-4 hours; the pectinase hydrolysis is performed, the mass of the pectinase is 0.5% to 1% of the mass of the roxburghii residue, and the hydrolysis time is 1-2 hours; In step 5, the enzyme inactivation temperature is 90-95°C and the enzyme inactivation time is 10-15 min; In step 5, the ultrasonic extraction is carried out at a frequency of 20 kHz, a power of 20 W, a pH value of 5-7, and a time of 10-15 min. In step 6, the vacuum concentration is carried out with the vacuum degree controlled at 0.08-0.09 MPa and the temperature controlled at 65-85°C.

2. The method for preparing polysaccharides after fermentation of roxburghii as claimed in claim 1 is used for preparing health products with auxiliary protective effects on alcoholic liver damage.