WO2021174801A1 - 一种牛樟芝水不溶性膳食纤维的制备方法 - Google Patents
一种牛樟芝水不溶性膳食纤维的制备方法 Download PDFInfo
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- WO2021174801A1 WO2021174801A1 PCT/CN2020/114638 CN2020114638W WO2021174801A1 WO 2021174801 A1 WO2021174801 A1 WO 2021174801A1 CN 2020114638 W CN2020114638 W CN 2020114638W WO 2021174801 A1 WO2021174801 A1 WO 2021174801A1
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- Prior art keywords
- antrodia cinnamomea
- water
- insoluble dietary
- dietary fiber
- precipitate
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0288—Applications, solvents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2236/30—Extraction of the material
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to the technical field of industrial waste reuse, in particular to a method for preparing water-insoluble dietary fiber of Antrodia cinnamomea.
- Dietary fiber refers to the general term for plant food ingredients, carbohydrates and the like that are not digested and absorbed in the human small intestine but can be fully or partially fermented by microorganisms in the large intestine. It is known as the seventh nutrient. Dietary fiber has strong adsorption characteristics, including water-holding, oil-holding, water-absorbing, swelling, and gel-like characteristics, as well as the adsorption of heavy metals. According to its solubility, it can be divided into water-insoluble dietary fiber and water-soluble dietary fiber.
- water-insoluble dietary fiber can stimulate intestinal peristalsis, soften and accelerate bowel movement, reduce the re-collection of harmful substances in feces, and prevent obesity, constipation, hyperlipidemia, etc.
- the disease has a good preventive and auxiliary treatment effect, and it is an ideal functional food material.
- Water-insoluble dietary fibers are mainly plant cell wall components, including cellulose, hemicellulose, lignin, and chitosan. Studies have shown that insufficient dietary fiber intake can lead to a variety of health problems such as sugar and lipid metabolism disorders and cardiovascular diseases.
- Antrodia cinnamomea also known as Antrodia cinnamomea and Antrodia cinnamomea, has the reputation of "Forest Ruby" in Taiwan.
- Antrodia cinnamomea contains many active ingredients, including triterpenoids, polysaccharides, ubiquinones, superoxide dismutase, ergosterol, etc. It has physiological activities such as tumor, immunity enhancement, and antibacterial.
- Antrodia cinnamomea polysaccharides have good anti-tumor and immunity-enhancing activities. At present, most researches mainly focus on the fermentation, extraction and functional evaluation of water-soluble polysaccharides.
- the patent provides a method for preparing water-insoluble dietary fiber of Antrodia cinnamomea.
- the prepared dietary fiber has high extraction purity, is suitable for large-scale production, and can effectively reduce the lipid metabolism disorder caused by a high-fat diet.
- the present invention is made to solve the above-mentioned problems, and aims to provide a method for preparing water-insoluble dietary fiber of Antrodia cinnamomea.
- the present invention provides a method for preparing water-insoluble dietary fiber of Antrodia cinnamomea. It has such characteristics and includes the following steps: Step 1. Drying and superfinely pulverizing the Antrodia cinnamomea waste after extraction with ethanol and water to obtain Antrodia cinnamomea waste powder Step 2, using a NaOH solution containing NaBH 4 to extract the Antrodia cinnamomea waste powder twice to obtain two extracts with a material-to-liquid ratio of 1:5-1:25v/m; step 3, the two After the extracts are combined, an appropriate amount of glacial acetic acid is added for neutralization, and after standing for the first predetermined time, centrifugation is performed, and the precipitate is collected; step 4, the precipitate will be washed with ultrapure water 3 times, and then LiCl- The DMSO solution was dissolved, and the precipitate insoluble in the LiCl-DMSO solution was dialyzed for a second predetermined time, and freeze-dried to obtain the
- the Antrodia cinnamomea waste includes Antrodia cinnamomea fruiting bodies, Antrodia cinnamomea plate culture cells, Antrodia cinnamomea grain solid fermentation cells, and Antrodia cinnamomea Liquid fermentation mycelium of these Antrodia cinnamomea cultures.
- the method for preparing water-insoluble dietary fiber of Antrodia cinnamomea may also have the following characteristics: in step 2, the concentration of the NaOH solution is 0.1-1.0 mol/L, and the content of NaBH 4 is 0.1M.
- the method for preparing water-insoluble dietary fiber of Antrodia cinnamomea provided by the present invention may also have the following characteristics: wherein, in step 2, the extraction temperature is 2-15°C, and the extraction time is 4h-24h.
- the method for preparing water-insoluble dietary fiber of Antrodia cinnamomea provided by the present invention, it may also have the following characteristics: wherein, in step 3, the first predetermined time is 2 hours.
- the method for preparing water-insoluble dietary fiber of Antrodia cinnamomea may also have the following characteristics: wherein, in step 4, the second predetermined time is 3 days, and the concentration of LiCl in the LiCl-DMSO solution is 0.25 mol/L.
- the method for preparing water-insoluble dietary fiber of Antrodia cinnamomea provided by the present invention, it may also have the following characteristics: wherein, in step 5, the third predetermined time is 3 days.
- the application of the Antrodia cinnamomea water-insoluble dietary fiber prepared according to the preparation method of the Antrodia cinnamomea water-insoluble dietary fiber provided in this article is a food additive with cholesterol and fat reduction and a meal replacement food.
- the process is simple and suitable for large-scale process production.
- the water-insoluble dietary fiber prepared has high extraction rate and high purity, suitable for large-scale production, and is a high-quality water-insoluble diet Fiber can also effectively absorb oil and cholesterol.
- water-insoluble dietary fiber can effectively alleviate the lipid metabolism disorder caused by high-fat diet, has the effect of lowering cholesterol and lipids, thereby reducing weight, and effectively using the waste extracted from Antrodia camphorata, with low cost and good economy Benefits and good development prospects.
- Figure 1 is a schematic diagram of the effect of Antrodia camphorata insoluble dietary fiber on the body weight of mice in an application example of the present invention.
- the preparation method of Antrodia cinnamomea water-insoluble dietary fiber of the present invention includes the following steps:
- step 1 the Antrodia cinnamomea waste after extraction with ethanol and water is dried and ultra-finely pulverized to obtain antrodia cinnamomea waste powder.
- Antrodia cinnamomea waste includes Antrodia cinnamomea fruiting bodies, Antrodia cinnamomea plate culture cells, Antrodia cinnamomea grain solid fermentation cells, and Antrodia cinnamomea liquid fermentation mycelium cultures.
- Step 2 Using NaOH solution containing NaBH 4 to extract the Antrodia cinnamomea waste powder twice to obtain two extracts with a material-to-liquid ratio of 1:5-1:25 v/m.
- the concentration of the NaOH solution is 0.1-1.0 mol/L, and the content of NaBH 4 is 0.1M.
- Step 3 Combine the two extracts and add an appropriate amount of glacial acetic acid for neutralization. After standing for a first predetermined time, centrifugation is performed, and the precipitate is collected.
- the extraction temperature is 2-15°C
- the extraction time is 4h-24h
- the first predetermined time is 2h.
- Step 4 Wash the precipitate with ultrapure water for 3 times, then dissolve it with LiCl-DMSO solution, dialyze the precipitate that is insoluble in LiCl-DMSO solution for a second predetermined time, and freeze-dry to obtain an Antrodia camphorata water-insoluble diet Fiber component ACA-IDK.
- the second predetermined time is 3 days, and the concentration of LiCl in the LiCl-DMSO solution is 0.25 mol/L.
- Step 5 the LiCl-DMSO solution is subjected to ethanol/DMSO graded precipitation, and the ethanol addition ratio is 30%%-90% v/v, and then the precipitate is collected, dialyzed for a third predetermined time, and freeze-dried to obtain an Antrodia camphorata water-insoluble diet Fiber component ACA-DK.
- the third predetermined time is 3d.
- the application of the Antrodia cinnamomea water-insoluble dietary fiber prepared according to the method for preparing the above-mentioned Antrodia cinnamomea water-insoluble dietary fiber is used in food additives and meal replacement foods for reducing cholesterol and lipids.
- food additives with cholesterol and fat reduction can be used in foods such as bread, biscuits, dairy products, meat products, beverages, etc.
- Meal replacement foods with cholesterol and fat reduction can be prepared into powders, granules, tablets, capsules, etc. .
- step 1 the Antrodia cinnamomea fruit body waste after extraction with ethanol and water is dried and ultrafinely pulverized to obtain an Antrodia cinnamomea waste powder.
- Step 2 Use 0.1 mol/L NaOH solution containing 0.01M NaBH 4 to extract the Antrodia cinnamomea waste powder twice, the extraction temperature is 2°C, the extraction time is 4h, and the material-to-liquid ratio is 1:5 (v/m) .
- Step 3 Combine the extracts and add an appropriate amount of glacial acetic acid for neutralization, and after standing for 2 hours, perform centrifugation, and collect the precipitate.
- Step 4 Wash the precipitate with ultrapure water three times and then dissolve it with a LiCl-DMSO solution with a LiCl concentration of 0.25 mol/L, and dialyze the precipitate insoluble in the LiCl-DMSO solution for 3 days, and freeze-dry to obtain Antrodia camphorata water Insoluble dietary fiber component ACA-IDK;
- Step 5 the LiCl-DMSO solution was subjected to ethanol/DMSO fractionation precipitation, the ethanol addition ratio was 30% (v/v), and then the precipitate was collected, dialyzed for 3 days, and freeze-dried to obtain the Antrodia camphorata water-insoluble dietary fiber component ACA-DK.
- the extraction rate of the insoluble dietary fiber component ACA-DK in this example is 3.6% and the purity is 68.5%; the extraction rate of ACA-IDK is 4.6% and the purity is 63.4%.
- Step 1 The Antrodia cinnamomea fruit body waste after extraction with ethanol and water is dried and ultra-finely pulverized to obtain Antrodia cinnamomea waste powder.
- Step 2 Use the 0.6mol/L NaOH solution containing 0.01MNaBH 4 to extract the Antrodia cinnamomea waste powder twice, the extraction temperature is 10°C, the extraction time is 12h, and the material-to-liquid ratio is 1:10 (v/m) .
- Step 3 Combine the extracts and add an appropriate amount of glacial acetic acid for neutralization, and after standing for 2 hours, perform centrifugation, and collect the precipitate.
- Step 4 Wash the precipitate with ultrapure water three times and then dissolve it with a LiCl-DMSO solution with a LiCl concentration of 0.25 mol/L, and dialyze the precipitate insoluble in the LiCl-DMSO solution for 3 days, and freeze-dry to obtain Antrodia camphorata water Insoluble dietary fiber component ACA-IDK;
- Step 5 the LiCl-DMSO solution was subjected to ethanol/DMSO fractionation precipitation, the ethanol addition ratio was 70% (v/v), and then the precipitate was collected, dialyzed for 3 days, and freeze-dried to obtain the Antrodia camphorata water-insoluble dietary fiber component ACA-DK.
- the extraction rate of the insoluble dietary fiber component ACA-DK in this example was 8.7% and the purity was 88.3%; the extraction rate of ACA-IDK was 9.2% and the purity was 82.7%.
- step 1 the Antrodia cinnamomea fruit body waste after extraction with ethanol and water is dried and ultrafinely pulverized to obtain an Antrodia cinnamomea waste powder.
- Step 2 Use 1.0 mol/L NaOH solution containing 0.01M NaBH 4 to extract the Antrodia cinnamomea waste powder twice, the extraction temperature is 20°C, the extraction time is 24h, and the material-to-liquid ratio is 1:25 (v/m) .
- Step 3 Combine the extracts and add an appropriate amount of glacial acetic acid for neutralization, and after standing for 2 hours, perform centrifugation, and collect the precipitate.
- Step 4 Wash the precipitate with ultrapure water three times and then dissolve it with a LiCl-DMSO solution with a LiCl concentration of 0.25 mol/L, and dialyze the precipitate insoluble in the LiCl-DMSO solution for 3 days, and freeze-dry to obtain Antrodia camphorata water Insoluble dietary fiber component ACA-IDK;
- Step 5 the LiCl-DMSO solution was subjected to ethanol/DMSO fractional precipitation, the ethanol addition ratio was 90% (v/v), and then the precipitate was collected, dialyzed for 3 days, and freeze-dried to obtain the Antrodia camphorata water-insoluble dietary fiber component ACA-DK.
- the extraction rate of the insoluble dietary fiber component ACA-DK in this example is 8.8%, and the purity is 77.3%; the extraction rate of ACA-IDK is 9.1%, and the purity is 70.8%.
- step 1 the Antrodia cinnamomea dish culture waste after extraction with ethanol and water is dried and ultra-finely pulverized to obtain antrodia cinnamomea waste powder.
- Step 2 Use the 0.6mol/L NaOH solution containing 0.01MNaBH 4 to extract the Antrodia cinnamomea waste powder twice, the extraction temperature is 10°C, the extraction time is 12h, and the material-to-liquid ratio is 1:10 (v/m) .
- Step 3 Combine the extracts and add an appropriate amount of glacial acetic acid for neutralization, and after standing for 2 hours, perform centrifugation, and collect the precipitate.
- Step 4 Wash the precipitate with ultrapure water three times and then dissolve it with a LiCl-DMSO solution with a LiCl concentration of 0.25 mol/L, and dialyze the precipitate insoluble in the LiCl-DMSO solution for 3 days, and freeze-dry to obtain Antrodia camphorata water Insoluble dietary fiber component ACA-IDK;
- Step 5 the LiCl-DMSO solution was subjected to ethanol/DMSO fractionation precipitation, the ethanol addition ratio was 70% (v/v), and then the precipitate was collected, dialyzed for 3 days, and freeze-dried to obtain the Antrodia camphorata water-insoluble dietary fiber component ACA-DK.
- the extraction rate of the insoluble dietary fiber component ACA-DK in this example is 8.3% and the purity is 89.1%; the extraction rate of ACA-IDK is 8.8% and the purity is 84.2%.
- step 1 the Antrodia cinnamomea liquid fermentation mycelium waste after extraction with ethanol and water is dried and ultrafinely pulverized to obtain Antrodia cinnamomea waste powder.
- Step 2 Use the 0.6mol/L NaOH solution containing 0.01MNaBH 4 to extract the Antrodia cinnamomea waste powder twice, the extraction temperature is 10°C, the extraction time is 12h, and the material-to-liquid ratio is 1:10 (v/m) .
- Step 3 Combine the extracts and add an appropriate amount of glacial acetic acid for neutralization, and after standing for 2 hours, perform centrifugation, and collect the precipitate.
- Step 4 Wash the precipitate with ultrapure water three times and then dissolve it with a LiCl-DMSO solution with a LiCl concentration of 0.25 mol/L, and dialyze the precipitate insoluble in the LiCl-DMSO solution for 3 days, and freeze-dry to obtain Antrodia camphorata water Insoluble dietary fiber component ACA-IDK;
- Step 5 the LiCl-DMSO solution was subjected to ethanol/DMSO fractionation precipitation, the ethanol addition ratio was 70% (v/v), and then the precipitate was collected, dialyzed for 3 days, and freeze-dried to obtain the Antrodia camphorata water-insoluble dietary fiber component ACA-DK.
- the extraction rate of the insoluble dietary fiber component ACA-DK in this example was 7.7% and the purity was 86.4%; the extraction rate of ACA-IDK was 8.3% and the purity was 83.3%.
- Lipid-lowering functional experiment is animal experiment
- Antrodia cinnamomea water-insoluble dietary fiber component ACA-DK prepared as a suspension of a certain concentration, and placed at 4°C for use after preparation.
- mice 40 18-22g SPF C57BL/6 mice (approval number SCXK20180004) bred by Shanghai Jiesjie Experimental Animal Co., Ltd. were randomly divided into 4 groups, each with 10 mice.
- Main equipment; and reagents balance, centrifuge, Illumina MiSeq sequencer, microplate reader, RNA extraction kit.
- mice were randomly divided into 4 groups under the experimental environment, and the normal group NG (gavage of normal saline), the model group MG (high-fat diet), the positive control group PG (gavage of lovastatin), insoluble Dietary fiber group LG.
- the normal group NG was fed with basic feed, and the rest of the groups were fed with high-fat feed. Gavage mice with insoluble dietary fiber once a day for 8 weeks.
- mice were raised in an IVC system with a temperature of 18-22°C and a relative humidity of 40-70%.
- the experimental animal license number was SCXK20180004.
- mice were irradiated with sterile feed from Jiangsu Synergy Medical Bioengineering Co., Ltd.
- Basic feed flour 20%, bran 25%, corn 20%, rice flour 10%, soybean meal 20%, fish meal 2%, bone meal 2%, salt 0.9%, vitamin 0.1%;
- High-fat feed lard 10%, sucrose 5%, cholesterol 1%, bile salt 0.3%, basic feed 83.7%.
- SPSS 22.0 software was used to perform statistical analysis on the original data of each experiment, and the ANOVA program was used to test the significance of the data.
- Figure 1 is a schematic diagram of the effect of Antrodia camphorata insoluble dietary fiber on the body weight of mice in an application example of the present invention.
- mice after 8 weeks of high-fat diet increased their body weight significantly, and the weight gain rate reached 24.15%; while the mice were orally administered with Antrodia camphorata insoluble dietary fiber for 8 weeks. There was no significant difference from the normal group of mice, and the weight gain rate was only 16.54%. Antrodia cinnamomea insoluble dietary fiber can reduce weight.
- the effect of insoluble dietary fiber in Antrodia camphorata on serum lipids of mice is compared with that of mice in the normal group.
- the LDL-C of mice is significantly increased, up to 10.33 ⁇ 1.91mmol/L.
- HDL-C decreased slightly; after oral administration of Antrodia camphorata insoluble dietary fiber to mice for 8 weeks, LDL-C decreased significantly, only 2.43 ⁇ 0.28mmol/L.
- the risk factor of atherosclerosis (LDL-C/HDL-C) decreased from 4.83 in the MG group to 1.38 in the LG group.
- the insoluble dietary fiber of Antrodia cinnamomea can regulate the metabolism of serum lipoprotein.
- the total cholesterol (TC) content of the mice after 8 weeks of high-fat diet increased significantly, as high as 0.075 ⁇ 0.019mmol/g protein, while the total triglycerides (TG) There was no significant difference; after oral administration of Antrodia camphorata insoluble dietary fiber to mice for 8 weeks, the content of TC and TG decreased significantly, which were 0.023 ⁇ 0.007 and 0.042 ⁇ 0.015mmol/gprotein, respectively.
- Antrodia cinnamomea insoluble dietary fiber has the effect of reducing TC and TG.
- Example 4 and Example 5 when under the same conditions as in Example 2, the raw materials are Antrodia cinnamomea dish culture mycelium and Antrodia cinnamomea liquid fermentation mycelium, and the insoluble dietary fiber components ACA-DK and ACA are prepared.
- -IDK has high extraction rate and purity.
- the ACA-DK water-insoluble dietary fiber component of Antrodia cinnamomea has been further studied. It is found through animal experiments that the prepared water-insoluble dietary fiber of Antrodia cinnamomea has good water-holding, oil-holding and cholesterol-absorbing properties, which can effectively relieve The lipid metabolism disorder caused by a high-fat diet can also reduce the increase in blood lipids and cholesterol in mice caused by a high-fat diet, and can control or reduce body weight.
- the preparation method of Antrodia cinnamomea water-insoluble dietary fiber of the present invention has a simple process and is suitable for large-scale process production.
- the prepared water-insoluble dietary fiber has high extraction rate and high purity, and is suitable for large-scale production. It is a high-quality water-insoluble diet.
- Fiber can also effectively absorb oil and cholesterol.
- water-insoluble dietary fiber can effectively alleviate the lipid metabolism disorder caused by high-fat diet, has the effect of lowering cholesterol and lipids, thereby reducing weight, and effectively using the waste extracted from Antrodia camphorata, with low cost and good economy Benefits and good development prospects.
Abstract
一种牛樟芝水不溶性膳食纤维的制备方法，包括如下步骤：步骤1，将经过乙醇、水提取后的牛樟芝废弃物进行干燥、超微粉碎，得到牛樟芝废弃物粉末；步骤2，采用含有NaBH 4的NaOH溶液对牛樟芝废弃物粉末浸提两次，得到两份料液比为1:5-1:25v/m的浸提液；步骤3，将两份浸提液合并后加入适量冰醋酸进行中和，静置第一预定时间后，进行离心，并收集沉淀；步骤4，将沉淀将用超纯水洗涤3次后，采用LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析第二预定时间，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK；步骤5，将LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，收集沉淀，透析第三预定时间，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。制备得到的牛樟芝水不溶性膳食纤维可用于降胆固醇降脂的食品添加剂以及代餐食品中。
Description
本发明涉及工业废物再利用技术领域，具体涉及一种牛樟芝水不溶性膳食纤维的制备方法。
膳食纤维是指在人体小肠中不被消化吸收而在大肠中能完全或部分被微生物发酵利用的植物性食品成分、碳水化合物及其类似物的总称，被誉为第七营养素。膳食纤维具有较强的吸附特性，包括持水、持油、吸水膨胀呈凝胶状以及吸附重金属等特征。按其溶解性可分为水不溶性膳食纤维和水溶性膳食纤维，其中水不溶性膳食纤维能够刺激肠道蠕动，软化并加速排便，减少粪便中有害物质的重新收，对肥胖、便秘、高血脂等疾病有很好的预防和辅助治疗作用，是一种比较理想的功能性食品原料。水不溶性膳食纤维主要为植物细胞壁成分，包括纤维素、半纤维素、木质素以及壳聚糖等。研究表明膳食纤维摄入不足，会导致糖、脂质代谢紊乱、心血管疾病等多种健康问题。
牛樟芝，又名牛樟菇、樟菇，有着台湾“森林红宝石”的美誉。牛樟芝含有很多活性成分，包括三萜类化合物、多糖、泛醌类化合物、超氧歧化酶、麦角固醇等，具有肿瘤、增加免疫能力、抗细菌等生理活性。牛樟芝多糖具有良好的抗肿瘤以及提高免疫力活性，目前大多数研究主要集中在水溶性多糖发酵、提取以及功能评价方面。工业生 产过程中，牛樟芝子实体或是菌丝体经过乙醇、水提取之后，醇溶性化合物以及水溶性多糖基本都被提取，但是残渣中任然含有大量水不溶性膳食纤维。本专利提供一种牛樟芝水不溶性膳食纤维的制备方法，制备得到的膳食纤维提取纯度高，适合规模化生产，并且能够有效降低高脂饮食导致的脂质代谢紊乱。
发明内容
本发明是为了解决上述问题而进行的，目的在于提供一种牛樟芝水不溶性膳食纤维的制备方法。
本发明提供了一种牛樟芝水不溶性膳食纤维的制备方法，具有这样的特征，包括如下步骤：步骤1，将经过乙醇、水提取后的牛樟芝废弃物进行干燥、超微粉碎，得到牛樟芝废弃物粉末；步骤2，采用含有NaBH
4的NaOH溶液对所述牛樟芝废弃物粉末浸提两次，得到两份料液比为1:5-1:25v/m的浸提液；步骤3，将两份所述浸提液合并后加入适量冰醋酸进行中和，静置第一预定时间后，进行离心，并收集沉淀；步骤4，将所述沉淀将用超纯水洗涤3次后，采用LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析第二预定时间，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK；步骤5，将所述LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，且乙醇添加比例为30％％-90％v/v，而后收集沉淀，透析第三预定时间，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。
在本发明提供的牛樟芝水不溶性膳食纤维的制备方法中，还可以 具有这样的特征：其中，步骤1中，牛樟芝废弃物包括牛樟芝子实体、牛樟芝皿培菌体、牛樟芝谷物固态发酵菌体以及牛樟芝液态发酵菌丝体这些牛樟芝培养物。
在本发明提供的牛樟芝水不溶性膳食纤维的制备方法中，还可以具有这样的特征：其中，步骤2中，NaOH溶液的浓度为0.1-1.0mol/L，NaBH
4的含量为0.1M。
在本发明提供的牛樟芝水不溶性膳食纤维的制备方法中，还可以具有这样的特征：其中，步骤2中，浸提温度为2-15℃，浸提时间为4h-24h。
在本发明提供的牛樟芝水不溶性膳食纤维的制备方法中，还可以具有这样的特征：其中，步骤3中，第一预定时间为2h。
在本发明提供的牛樟芝水不溶性膳食纤维的制备方法中，还可以具有这样的特征：其中，步骤4中，第二预定时间为3d，LiCl-DMSO溶液中的LiCl浓度为0.25mol/L。
在本发明提供的牛樟芝水不溶性膳食纤维的制备方法中，还可以具有这样的特征：其中，步骤5中，第三预定时间为3d。
根据本分提供的牛樟芝水不溶性膳食纤维的制备方法制备得到的牛樟芝水不溶性膳食纤维在具有降胆固醇降脂的食品添加剂以及代餐食品中的应用。
发明的作用与效果
根据本发明所涉及的牛樟芝水不溶性膳食纤维制备方法，工艺简 洁，适合大规模工艺生产，制备得到的水不溶性膳食纤维提取率高、纯度高，适合规模化生产，是一种高品质水不溶性膳食纤维，还能够有效吸附油脂、胆固醇。此外，水不溶性膳食纤维能够有效缓解高脂饮食引起的脂质代谢紊乱，具有降胆固醇降脂的功效，从而能够降低体重，并且有效利用了牛樟芝提取后的废弃物，成本低，具有良好的经济效益和良好的开发前景。
图1是本发明的应用实施例中牛樟芝不溶性膳食纤维对小鼠体重的影响示意图。
为了使本发明实现的技术手段与功效易于明白了解，以下结合实施例以及附图对本发明作具体阐述。
本发明的一种牛樟芝水不溶性膳食纤维的制备方法，包括如下步骤：
步骤1，将经过乙醇、水提取后的牛樟芝废弃物进行干燥、超微粉碎，得到牛樟芝废弃物粉末。
本发明中，牛樟芝废弃物包括牛樟芝子实体、牛樟芝皿培菌体、牛樟芝谷物固态发酵菌体以及牛樟芝液态发酵菌丝体这些牛樟芝培养物。
步骤2，采用含有NaBH
4的NaOH溶液对所述牛樟芝废弃物粉末 浸提两次，得到两份料液比为1:5-1:25v/m的浸提液。
本发明中，NaOH溶液的浓度为0.1-1.0mol/L，NaBH
4的含量为0.1M。
步骤3，将两份所述浸提液合并后加入适量冰醋酸进行中和，静置第一预定时间后，进行离心，并收集沉淀。
本发明中，浸提温度为2-15℃，浸提时间为4h-24h，第一预定时间为2h。
步骤4，将所述沉淀将用超纯水洗涤3次后，采用LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析第二预定时间，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK。
本发明中，第二预定时间为3d，LiCl-DMSO溶液中的LiCl浓度为0.25mol/L。
步骤5，将所述LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，且乙醇添加比例为30％％-90％v/v，而后收集沉淀，透析第三预定时间，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。
本发明中，第三预定时间为3d。
根据上述牛樟芝水不溶性膳食纤维的制备方法制备得到的牛樟芝水不溶性膳食纤维在具有降胆固醇降脂的食品添加剂以及代餐食品中的应用。
本发明中，具有降胆固醇降脂的食品添加剂可用于面包、饼干、乳制品肉制品、饮料等食品中，具有降胆固醇降脂的代餐食品可以制备成粉剂、颗粒剂、片剂、胶囊等。
下述实施实例中所使用的实验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
<实施例1>
步骤1，将经过乙醇、水提取后的牛樟芝子实体废弃物干燥、超微粉碎后，得到牛樟芝废弃物粉末。
步骤2，采用含有0.01MNaBH
4的0.1mol/L的NaOH溶液对牛樟芝废弃物粉末浸提两次，浸提温度2℃，浸提时间4h，且料液比为1:5(v/m)。
步骤3，合并浸提液加入适量冰醋酸进行中和，静置2h后，进行离心，并收集沉淀。
步骤4，将沉淀用超纯水洗涤3次后用LiCl浓度为0.25mol/L的LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK；
步骤5，将LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，乙醇添加比例为30％(v/v)，而后收集沉淀，透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。
本实施例的不溶性膳食纤维组分ACA-DK提取率为3.6％，纯度为68.5％；ACA-IDK提取率为4.6％，纯度为63.4％。
<实施例2>
步骤1，将经过乙醇、水提取后的牛樟芝子实体废弃物干燥、超 微粉碎后，得到牛樟芝废弃物粉末。
步骤2，采用含有0.01MNaBH
4的0.6mol/L的NaOH溶液对牛樟芝废弃物粉末浸提两次，浸提温度10℃，浸提时间12h，且料液比为1:10(v/m)。
步骤3，合并浸提液加入适量冰醋酸进行中和，静置2h后，进行离心，并收集沉淀。
步骤4，将沉淀用超纯水洗涤3次后用LiCl浓度为0.25mol/L的LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK；
步骤5，将LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，乙醇添加比例为70％(v/v)，而后收集沉淀，透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。
本实施例的不溶性膳食纤维组分ACA-DK提取率为8.7％，纯度为88.3％；ACA-IDK提取率为9.2％，纯度为82.7％。
<实施例3>
步骤1，将经过乙醇、水提取后的牛樟芝子实体废弃物干燥、超微粉碎后，得到牛樟芝废弃物粉末。
步骤2，采用含有0.01MNaBH
4的1.0mol/L的NaOH溶液对牛樟芝废弃物粉末浸提两次，浸提温度20℃，浸提时间24h，且料液比为1:25(v/m)。
步骤3，合并浸提液加入适量冰醋酸进行中和，静置2h后，进行离心，并收集沉淀。
步骤4，将沉淀用超纯水洗涤3次后用LiCl浓度为0.25mol/L的LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK；
步骤5，将LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，乙醇添加比例为90％(v/v)，而后收集沉淀，透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。
本实施例的不溶性膳食纤维组分ACA-DK提取率为8.8％，纯度为77.3％；ACA-IDK提取率为9.1％，纯度为70.8％。
<实施例4>
步骤1，将经过乙醇、水提取后的牛樟芝皿培菌体废弃物干燥、超微粉碎后，得到牛樟芝废弃物粉末。
步骤2，采用含有0.01MNaBH
4的0.6mol/L的NaOH溶液对牛樟芝废弃物粉末浸提两次，浸提温度10℃，浸提时间12h，且料液比为1:10(v/m)。
步骤3，合并浸提液加入适量冰醋酸进行中和，静置2h后，进行离心，并收集沉淀。
步骤4，将沉淀用超纯水洗涤3次后用LiCl浓度为0.25mol/L的LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK；
步骤5，将LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，乙醇添加比例为70％(v/v)，而后收集沉淀，透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。
本实施例的不溶性膳食纤维组分ACA-DK提取率为8.3％，纯度为89.1％；ACA-IDK提取率为8.8％，纯度为84.2％。
<实施例5>
步骤1，将经过乙醇、水提取后的牛樟芝液态发酵菌丝体废弃物干燥、超微粉碎后，得到牛樟芝废弃物粉末。
步骤2，采用含有0.01MNaBH
4的0.6mol/L的NaOH溶液对牛樟芝废弃物粉末浸提两次，浸提温度10℃，浸提时间12h，且料液比为1:10(v/m)。
步骤3，合并浸提液加入适量冰醋酸进行中和，静置2h后，进行离心，并收集沉淀。
步骤4，将沉淀用超纯水洗涤3次后用LiCl浓度为0.25mol/L的LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK；
步骤5，将LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，乙醇添加比例为70％(v/v)，而后收集沉淀，透析3天，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。
本实施例的不溶性膳食纤维组分ACA-DK提取率为7.7％，纯度为86.4％；ACA-IDK提取率为8.3％，纯度为83.3％。
应用实施例：降脂功能性实验即动物实验
1、实验条件
样品：牛樟芝水不溶性膳食纤维组分ACA-DK，配置成一定浓度悬浊液，制备好后放置4℃备用。
实验动物：选用上海杰思捷实验动物有限公司繁殖的18-22g SPF级C57BL/6小鼠(批准号为SCXK20180004)40只，随机分为4组，每组10只。
主要仪器；与试剂：天平，离心机，Illumina MiSeq测序仪，酶标仪，RNA提取试剂盒。
实验方法：在实验环境下小鼠随机分成4组，分别设定能够正常组NG(灌胃生理盐水)、模型组MG(高脂饮食)、阳性对照组PG(灌胃洛伐他汀)、不溶性膳食纤维组LG。正常组NG用基础饲料喂养，其余分组均用高脂饲料喂养。不溶性膳食纤维灌胃小鼠每日一次，持续8周。
饲养条件：小鼠在温度为18-22℃、相对湿度为40-70％的IVC系统中饲养，实验动物使用许可证号SCXK20180004，小鼠辐照无菌饲料自江苏协同医药生物工程有限公司。
基础饲料：面粉20％，麸皮25％，玉米20％，米粉10％，豆料20％，鱼粉2％，骨粉2％，食盐0.9％，维生素0.1％；
高脂饲料：猪油10％，蔗糖5％，胆固醇1％，胆盐0.3％，基础 饲料83.7％。
数据分析：采用SPSS 22.0软件对各实验原始数据进行统计学分析，利用ANOVA程序对数据显著性进行检验。
2、结果分析
(1)牛樟芝不溶性膳食纤维对小鼠体重的影响
图1是本发明的应用实施例中牛樟芝不溶性膳食纤维对小鼠体重的影响示意图。
如图1所示，与正常组小鼠相比，小鼠经过8周高脂饮食饲养，体重显著提高，体重增加率达到24.15％；而经口服给与小鼠牛樟芝不溶性膳食纤维8周，体重与正常组小鼠无显著差异，体重增加率仅为16.54％。牛樟芝不溶性膳食纤维能够减轻体重。
(2)牛樟芝不溶性膳食纤维对小鼠血清血脂的影响
如表1所示，牛樟芝不溶性膳食纤维对小鼠血清血脂的影与正常组小鼠相比，小鼠经过8周高脂饮食饲养，LDL-C显著提高，高达10.33±1.91mmol/L，而HDL-C稍有下降；经口服给与小鼠牛樟芝不溶性膳食纤维8周，LDL-C显著下降，仅为2.43±0.28mmol/L。动脉粥样硬化危险因子(LDL-C/HDL-C)由MG组的4.83降低到LG组的1.38。牛樟芝不溶性膳食纤维具有调节血清脂蛋白代谢的作用。
表1牛樟芝不溶性膳食纤维对小鼠血清血脂的影响
(3)牛樟芝不溶性膳食纤维对小鼠TG以及TC的影响
如表2所示，与正常组小鼠相比，小鼠经过8周高脂饮食饲养，总胆固醇(TC)含量显著提高，高达0.075±0.019mmol/g protein，而总甘油三酯(TG)无显著差异；经口服给与小鼠牛樟芝不溶性膳食纤维8周，TC与TG含量均显著下降，分别为0.023±0.007和0.042±0.015mmol/gprotein。牛樟芝不溶性膳食纤维具有降低TC和TG的功效。
表2牛樟芝不溶性膳食纤维对小鼠TG以及TC的影响
实施例的作用与效果
由实施例1至实施例3可知，当以牛樟芝子实体为原料，且NaOH溶液的浓度为0.6mol/L，浸提温度为10℃，浸提时间为12h时，不溶性膳食纤维组分ACA-DK的提取率和纯度均较高，且当乙醇添加 比例为70％(v/v)时的不溶性膳食纤维组分ACA-IDK的提取率和纯度均较高。
由实施例四和实施例五可知，当在与实施例2相同的条件下，原料采用牛樟芝皿培菌体以及牛樟芝液态发酵菌丝体是，制备得到的不溶性膳食纤维组分ACA-DK和ACA-IDK的提取率和纯度均较高。
由应用实施例可知，对牛樟芝水不溶性膳食纤维组分ACA-DK做了进一步研究，通过动物实验发现，制备得到牛樟芝水不溶性膳食纤维具有良好的持水、持油以及吸附胆固醇特性，能够有效缓解高脂饮食导致的脂质代谢紊乱，同时能够降低高脂饮食导致的小鼠血脂以及胆固醇升高，并且能够控制或减轻体重。
综上，本发明的牛樟芝水不溶性膳食纤维制备方法，工艺简洁，适合大规模工艺生产，制备得到的水不溶性膳食纤维提取率高、纯度高，适合规模化生产，是一种高品质水不溶性膳食纤维，还能够有效吸附油脂、胆固醇。此外，水不溶性膳食纤维能够有效缓解高脂饮食引起的脂质代谢紊乱，具有降胆固醇降脂的功效，从而能够降低体重，并且有效利用了牛樟芝提取后的废弃物，成本低，具有良好的经济效益和良好的开发前景。
上述实施方式为本发明的优选案例，并不用来限制本发明的保护范围。
Claims (8)
- 一种牛樟芝水不溶性膳食纤维的制备方法，其特征在于，包括如下步骤：步骤1，将经过乙醇、水提取后的牛樟芝废弃物进行干燥、超微粉碎，得到牛樟芝废弃物粉末；步骤2，采用含有NaBH 4的NaOH溶液对所述牛樟芝废弃物粉末浸提两次，得到两份料液比为1:5-1:25v/m的浸提液；步骤3，将两份所述浸提液合并后加入适量冰醋酸进行中和，静置第一预定时间后，进行离心，并收集沉淀；步骤4，将所述沉淀将用超纯水洗涤3次后，采用LiCl-DMSO溶液进行溶解，并将不溶于LiCl-DMSO溶液的沉淀物透析第二预定时间，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-IDK；步骤5，将所述LiCl-DMSO溶液进行乙醇/DMSO分级沉淀，且乙醇添加比例为30％％-90％v/v，而后收集沉淀，透析第三预定时间，冷冻干燥后得到牛樟芝水不溶性膳食纤维组分ACA-DK。
- 根据权利要求1所述的牛樟芝水不溶性膳食纤维的制备方法，其特征在于：其中，所述步骤1中，牛樟芝废弃物包括牛樟芝子实体、牛樟芝皿培菌体、牛樟芝谷物固态发酵菌体以及牛樟芝液态发酵菌丝体这些牛樟芝培养物。
- 根据权利要求1所述的牛樟芝水不溶性膳食纤维的制备方法， 其特征在于：其中，所述步骤2中，NaOH溶液的浓度为0.1-1.0mol/L，NaBH 4的含量为0.1M。
- 根据权利要求1所述的牛樟芝水不溶性膳食纤维的制备方法，其特征在于：其中，所述步骤2中，浸提温度为2-15℃，浸提时间为4h-24h。
- 根据权利要求1所述的牛樟芝水不溶性膳食纤维的制备方法，其特征在于：其中，所述步骤3中，第一预定时间为2h。
- 根据权利要求1所述的牛樟芝水不溶性膳食纤维的制备方法，其特征在于：其中，所述步骤4中，第二预定时间为3d，LiCl-DMSO溶液中的LiCl浓度为0.25mol/L。
- 根据权利要求1所述的牛樟芝水不溶性膳食纤维的制备方法，其特征在于：其中，所述步骤5中，第三预定时间为3d。
- 根据权利要求1所述的牛樟芝水不溶性膳食纤维的制备方法制 备得到的牛樟芝水不溶性膳食纤维在具有降胆固醇降脂的食品添加剂以及代餐食品中的应用。
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