WO2022134293A1 - 一种猴头菇膳食纤维、制备方法及其应用 - Google Patents

一种猴头菇膳食纤维、制备方法及其应用 Download PDF

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WO2022134293A1
WO2022134293A1 PCT/CN2021/076781 CN2021076781W WO2022134293A1 WO 2022134293 A1 WO2022134293 A1 WO 2022134293A1 CN 2021076781 W CN2021076781 W CN 2021076781W WO 2022134293 A1 WO2022134293 A1 WO 2022134293A1
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hericium erinaceus
dietary fiber
preparation
residue
microwave
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PCT/CN2021/076781
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French (fr)
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刘婷婷
王大为
张艳荣
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吉林农业大学
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Priority to ZA2023/05100A priority Critical patent/ZA202305100B/en

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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
    • 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
    • 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
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
    • A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
    • 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
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
    • A23L5/34—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using microwaves
    • 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
    • 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
    • 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
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment

Definitions

  • the invention relates to the technical field of food processing, in particular to a Hericium erinaceus dietary fiber, a preparation method and an application thereof.
  • Hericium erinaceus also known as Hericium erinaceus and Hericium erinaceus, is a precious medicinal and edible fungus in my country. Chinese medicine believes that Hericium erinaceus is flat and sweet, which is beneficial to the five internal organs, aids digestion, and nourishes the body. In recent years, research on Hericium erinaceus has mainly focused on polysaccharides and proteins. Studies have shown that Hericium erinaceus polysaccharides have anti-constipation, antioxidant, hypoglycemic and other activities, and Hericium erinaceus protein has good anti-inflammatory and immune-enhancing properties. activity, and the relative molecular mass of the immunomodulatory protein is 10-14kDa.
  • Hericium erinaceus residues after the extraction of water-soluble polysaccharides and proteins, a large amount of Hericium erinaceus residues will be produced.
  • the main components include dietary fiber (DF), minerals, a small amount of protein and pigments. Therefore, the preparation of high value-added and high-quality Hericium erinaceus residues
  • the dietary fiber can not only avoid the large-scale waste of resources, but also open up a new way for the development of the Hericium erinaceus industry.
  • Dietary fiber can be divided into water-soluble dietary fiber (SDF) and water-insoluble dietary fiber (IDF) according to solubility.
  • SDF water-soluble dietary fiber
  • IDF water-insoluble dietary fiber
  • Many studies have found that high-quality dietary fiber can only be regarded as high-quality dietary fiber when the content of SDF in DF is above 10%. It is usually an important indicator to evaluate the quality of DF.
  • the functional properties of DF are often affected by its source, composition and treatment, so certain treatments are needed to improve the content of SDF and the function of DF, thereby improving the quality of natural DF.
  • the invention aims to provide a high-quality dietary fiber of Hericium erinaceus and a preparation method thereof, so as to solve the problems of low utilization rate of Hericium erinaceus resources and serious waste of raw materials in the current extraction process of Hericium erinaceus polysaccharide and protein, and improve the efficiency of Hericium erinaceus.
  • This cherished resource utilization efficiency creates greater economic and social benefits.
  • the present invention provides the following scheme:
  • the invention provides a preparation method of Hericium erinaceus dietary fiber, comprising the following steps:
  • step (2) adding distilled water to the Hericium erinaceus residue treated in step (1) according to a material-to-liquid ratio of 1:(30 ⁇ 40) (g/mL), and adding alkaline protease for enzymatic hydrolysis for 25 ⁇ 30min;
  • step (3) (4) adding ethanol solution to the material obtained in step (3), and obtaining dietary fiber of Hericium erinaceus through suction filtration, washing and drying.
  • step (1) the residue of Hericium erinaceus is pulverized to 40-120 mesh.
  • the residue of Hericium erinaceus is pulverized to 40 mesh, 60 mesh, 80 mesh, 100 mesh or 120 mesh. More preferably, in step (1), the residue of Hericium erinaceus is pulverized to 80 mesh.
  • the alkaline protease in step (2) comprises Alcalase FG 2.4L, with an enzyme activity of 20 ⁇ 10 4 U/g.
  • the amount of cellulase added in step (3) is 1-6 wt% of the residue of Hericium erinaceus, and the enzymatic hydrolysis time is 15-90 min.
  • the amount of cellulase added in step (3) is 1wt%, 2wt%, 3wt%, 4wt%, 5wt% or 6wt% of the residue of Hericium erinaceus, and the enzymatic hydrolysis time is 15min, 30min, 45min, 60min, 75min or 90min. More preferably, the amount of cellulase added is 3wt% of the residue of Hericium erinaceus, and the enzymatic hydrolysis time is 75min.
  • the microwave temperature is 35-60°C
  • the ultrasonic power is 250-500W.
  • the microwave temperature is 35°C, 40°C, 45°C, 50°C, 55°C or 60°C, and the ultrasonic power is 250W, 300W, 350W, 400W, 450W or 500W. More preferably, the microwave temperature is 55°C and the ultrasonic power is 300W.
  • the present invention also provides the Hericium erinaceus dietary fiber prepared by the preparation method of the Hericium erinaceus dietary fiber.
  • the present invention also provides the application of the Hericium erinaceus dietary fiber in the preparation of a drug for treating blood lipid lowering.
  • Ultrasonic-microwave synergistic extraction is helpful for the release of active ingredients in materials, and the enzymatic method has the advantages of mild action conditions, strong specificity and high purity.
  • Ultrasonic-microwave-assisted enzymatic modification as a combined technology, has the characteristics of short reaction time and good treatment effect. , random curl) changes favorably.
  • Ultrasonic-microwave-assisted enzymatic treatment of Hericium erinaceus residues to prepare high-quality dietary fiber of Hericium erinaceus is of great significance for preparing high-activity dietary fiber and improving the utilization efficiency of Hericium erinaceus.
  • Figure 1 shows the infrared spectra of HE-DF 1 and HE-DF 2 ;
  • Fig. 2 is the X-ray diffraction pattern of HE-DF 1 and HE-DF 2 ;
  • Figure 3 is the scanning electron microscope images of HE-DF 1 and HE-DF 2 , wherein a1, a2, and a3 are the magnifications of HE-DF 1 by 200, 1000, and 5000 times, and b1, b2, and b3 are the magnifications of HE-DF 2 , respectively. 200, 1000, 5000 times.
  • suction filtration, washing and drying of the present invention are conventional methods for preparing dietary fiber in the art, and are not the main points of the invention, and are not repeated here.
  • the particle size of the material, the amount of enzyme added, the microwave temperature, the ultrasonic power, and the enzymatic hydrolysis time were selected as single-factor factors.
  • the fixed test conditions were as follows: the particle size of the material was 80 meshes, the amount of enzyme added was 3%, the treatment temperature was 50°C, the ultrasonic power was 400W, and the treatment time was 45min.
  • weight coefficients are given according to the contribution of the indicators to the process impact.
  • the level of SDF content is usually an important indicator for evaluating the quality of DF, so the weight coefficient of SDF yield is set to 0.4. It is generally believed that the binding ability of dietary fiber to fat and cholesterol is an important factor in evaluating its ability to absorb lipophilic components. Therefore, in order to obtain a highly active dietary fiber with auxiliary hypolipidemic effect, the oil holding capacity and cholesterol adsorption of HE-DF 2 were used. The ability is used as the inspection index, and the weight coefficient is 0.3. The highest score of each index is 100 points, and then weighted and summed. The composite score is calculated according to the following formula.
  • the X-diffraction conditions were: Cu radiation, voltage 40KV, current 40mA, step size 0.02, scan rate 12deg/min, continuous scan 2 ⁇ from 3° to 90°.
  • V 1 is the measured volume of the sample after expansion (mL);
  • V 0 is the measured volume of the dry sample (mL);
  • m is the dry mass of the sample (g).
  • V 1 is the volume of NaOH solution used for titrating the sample (mL);
  • V 0 is the volume of NaOH solution used for titrating the blank sample (mL);
  • m is the mass of the dry sample (g);
  • 0.1 is the concentration of the titrated NaOH solution (mol /L).
  • the loss of cholate is generally slightly lower than the initial addition amount; the reagent blank group and the binding system do not add cholate, and the dietary fiber sample is replaced by an equal volume of 0.1mol/L PBS after simulated gastrointestinal digestion. Cholate, other operations were the same as the experimental group.
  • C3 sample blank group
  • C4 experimental group
  • C5 reagent blank group
  • TDF total dietary fiber
  • IDF insoluble dietary fiber
  • SDF soluble dietary fiber
  • the particle size of the material has a certain influence on the yield of SDF.
  • the surface area of the material in contact with the solvent increases, which is conducive to the dissolution of SDF and increases the yield of SDF.
  • the particle size of the material continues to increase, the particle size is too fine, which makes the material easy to agglomerate into agglomerates, which reduces the surface area in contact with the solvent, and the SDF yield tends to decline.
  • the oil holding capacity of HE-DF 2 showed a trend of rapid increase at first and then slow increase as the particle size became finer. The oil holding capacity was not only related to the particle size of the material, but also to the composition of dietary fiber.
  • the cholesterol adsorption capacity first increases and then decreases with the change of the particle size of the material.
  • the particle size is 80 meshes
  • the cholesterol adsorption capacity reaches the maximum, which may be because the structure becomes loose and the polar groups increase as the particle size of the material becomes finer. If the particle size is too fine, the structure will be damaged to a certain extent, and the adsorption capacity of cholesterol will be reduced.
  • Table 6 shows the effect of ultrasonic power on SDF yield, oil holding capacity and cholesterol adsorption capacity of HE-DF 2 . It can be seen from Table 6 that with the increase of ultrasonic power, the change trend of SDF yield, oil holding capacity and cholesterol adsorption capacity is similar, firstly increasing and then decreasing.
  • ultrasonic treatment When the ultrasonic power increases, ultrasonic treatment generates a large number of cavitation bubbles through intramolecular heating and cavitation effect, and the bubble burst generates energy to promote cell wall rupture and release of cellular substances, help solvent enter tissue cells, promote the dissolution of SDF and the change of material structure, so that the Its adsorption performance is enhanced.
  • Excessive ultrasonic power may cause more and more cellulose to be excessively hydrolyzed to form small molecules or fragments, which are easily removed in the subsequent alcohol precipitation process, resulting in a decrease in the yield of SDF and the adsorption capacity of dietary fiber.
  • the experimental results show that the enzymatic hydrolysis time has a certain influence on the yield and adsorption performance of SDF.
  • the yield of SDF reached the maximum
  • the oil holding capacity of HE-DF 2 also reached the highest
  • the cholesterol adsorption capacity was also better.
  • the degree of hydrolysis of cellulose is enhanced, the bonds between fibers are cut off, the three-dimensional network structure of dietary fiber may be destroyed and partially degraded, which affects the yield of SDF and makes HE-DF 2
  • the adsorption performance in vitro was affected accordingly.
  • the optimal solution is A 2 B 2 C 3 D 2 E 3 , which needs to be verified, compared with the sixth group of test combinations A 2 B 2 C 1 D 4 E 3 and treated according to A 2 B 2 C 3 D 2 E 3
  • the yield of SDF was (11.72 ⁇ 0.12)%
  • the oil holding capacity of HE-DF 2 was (2.05 ⁇ 0.01) g/g
  • the cholesterol adsorption capacity was (36.84 ⁇ 0.59) mg/g, which was generally higher than that of A 2 B 2
  • the effect of C 1 D 4 E 3 treatment was determined as the optimal solution was material particle size of 80 meshes, enzyme addition amount of 3%, microwave temperature of 55°C, ultrasonic power of 300W, and enzymatic hydrolysis time of 75min.
  • the infrared spectra of HE-DF 1 and HE-DF 2 are shown in Figure 1. Comparing the two spectra, it is found that the two have similar spectral distributions, and both have the characteristic absorption peaks of polysaccharides, with a broad peak at 3200-3600 cm -1 And the strong absorption peak, which is the characteristic peak of hydroxyl group, indicates that there are intermolecular hydrogen bonds in the associative state in the dietary fiber. After ultrasonic-microwave-assisted enzymatic treatment, the absorption peak of hydroxyl group in the fiber structure is lower.
  • the wave number shifts from 3417.05m -1 to 3383.73cm -1 , which indicates that the hydrogen bond formed by the hydroxyl group in HE-DF 2 is enhanced and the hydrophilicity is enhanced.
  • the more the wavenumber is shifted the more intense the absorption of the characteristic peaks and the more exposed the hydroxyl groups, possibly making the sample more hydrophilic.
  • the X-ray diffraction patterns of HE-DF 1 and HE-DF 2 are shown in FIG. 2 .
  • the crystalline regions of cellulose-based substances are generally composed of disordered amorphous regions (30%) and ordered crystalline regions (70%). It can be seen from Figure 2 that the X-ray diffraction patterns of HE-DF 1 and HE-DF 2 are similar in shape, and there are obvious crystalline diffraction peaks at 2 ⁇ of 19.15° and 20.05°, which are characterized by cellulose I. , which indicated that the crystal type of DF in Hericium erinaceus belonged to the cellulose I type.
  • the peak shape and peak position of HE-DF 2 are basically unchanged, indicating that the crystalline structure properties of the fiber and the crystalline region of the fiber are basically unaffected, and the diffraction intensity is reduced, which may be due to ultrasonic-
  • the microwave-assisted enzymatic method partially destroys the crystalline area, for example, the hydrogen bonds between the cellulose molecules in the crystalline area are destroyed, the cellulose molecules are partially degraded, and the water-soluble components are dissolved.
  • the structure of dietary fiber is more loose and disordered, which leads to changes in physical properties such as water-holding and oil-holding properties.
  • HE-DF 1 and HE-DF 2 The scanning electron microscope images of HE-DF 1 and HE-DF 2 are shown in Figure 3.
  • a1, a2, and a3 are HE-DF 1 magnifications of 200, 1000, and 5000 times
  • b1, b2, and b3 are HE-DF 2 magnifications of 200, 200 1000, 5000 times.
  • the functional role of dietary fiber is often influenced by its structure.
  • the ultrastructures of HE-DF 1 and HE-DF 2 are shown in Figure 3.
  • HE-DF 1 is mostly irregular block-like structure with obvious surface
  • the folds are relatively dense, but the structural connection is relatively dense;
  • HE-DF 2 has a cluster-like structure, the surface structure is destroyed, and the internal capillary structure is exposed.
  • the specific surface area helps fibers absorb and retain moisture. It can be seen that ultrasonic-microwave-assisted enzymatic treatment can improve the microstructure of dietary fiber and make its surface area larger, thereby further improving the physical and chemical properties of dietary fiber and making it show stronger adsorption.
  • the ultrasonic-microwave-assisted enzymatic treatment has a certain improvement effect on the water holding capacity, oil holding capacity, swelling capacity and binding water capacity of the dietary fiber of Hericium erinaceus, and has a significant effect on the physical and chemical properties of the dietary fiber.
  • the water holding capacity, oil holding capacity, combined water holding capacity and swelling capacity of HE-DF 2 were increased by 51.21%, 72.70%, 59.77%, and 92.97%, respectively, which may be due to the ultrasonic-microwave-assisted enzymatic method.
  • the mechanical fragmentation and high-energy penetration effect of ultrasonic-microwave make the object break into more small fragments, and the cellulase can fully contact the substrate when it acts, and the enzymatic hydrolysis efficiency is improved.
  • This combined method makes the matrix of dietary fiber.
  • the structure is more loose and porous, thereby exposing more hydrophilic and lipophilic groups, and the small particle size allows easier entry of water and oil, and the water holding capacity, oil holding capacity and swelling force will increase.
  • Water holding capacity, oil holding capacity and swelling energy have been used as indicators to predict the cholesterol-lowering ability of dietary fiber, so it can be speculated that the ability of HE-DF 2 to bind cholesterol in the intestinal tract has also been improved, so it is better than HE-DF. 1 has better hypolipidemic function.
  • the side chain groups such as carboxyl group, hydroxyl group and amino group contained in the chemical structure of dietary fiber can produce the effect similar to weak acid cation exchange resin, and can exchange with cation reversibly.
  • Table 10 also shows that compared with HE-DF 1 , the cation exchange capacity of HE-DF 2 is significantly increased by 87.5%, indicating that the ultrasonic-microwave assisted enzymatic treatment can make some carboxyl and hydroxyl side chain groups in dietary fiber. The group is exposed, which is beneficial to improve the cation exchange capacity.
  • dietary fiber with high cation exchange capacity can bind and destroy fat emulsions, improve bile acid excretion in feces, improve bile acid metabolism, and delay the diffusion and absorption of lipids and cholesterol in the small intestine.
  • dietary fiber lowering blood lipids is its adsorption of cholesterol, which is excreted with the feces and reduces the cholesterol content in the body.
  • HE-DF 2 is better than HE-DF 1
  • the cholesterol adsorption capacity of the Hericium erinaceus was significantly enhanced, which may be due to the degradation of the insoluble dietary fiber in the Hericium erinaceus residue by the ultrasonic-microwave-assisted enzymatic treatment, which increased the content of soluble dietary fiber and loosened the structure of the dietary fiber.
  • the surface area is increased and thus its functional properties are enhanced.
  • the pH value also has a certain influence on the cholesterol adsorption capacity of HE-DF 1 and HE-DF 2.
  • the binding ability of dietary fiber to cholate can be used as one of the characterizations to measure the function of lowering blood lipids.
  • the binding of cholate in the small intestine and the promotion of cholate excretion are considered to be the main reasons for dietary fiber to lower cholesterol.
  • Table 12 shows the cholate binding capacity of Hericium erinaceus dietary fiber.
  • Hericium erinaceus dietary fiber has a certain adsorption capacity for cholate, and the binding ability of HE-DF 2 to sodium glycocholate and sodium taurocholate is significantly higher than that of HE-DF 2.
  • Hericium erinaceus residue was treated by ultrasonic-microwave-assisted enzymatic method to prepare high-quality dietary fiber of Hericium erinaceus.
  • the particle size of the material was 80 meshes
  • the amount of enzyme added was 3%
  • the microwave temperature was 55 °C
  • the highest SDF yield was (11.72 ⁇ 0.12)%
  • the oil holding capacity of HE-DF 2 was (2.05 ⁇ 0.01) g/g
  • the cholesterol adsorption capacity was (36.84 ⁇ 0.59) mg/g g.
  • the use of ultrasonic-microwave-assisted enzymatic treatment of Hericium erinaceus residue can improve the quality of Hericium erinaceus dietary fiber, and obtain high-quality dietary fiber of Hericium erinaceus with potential blood lipid-lowering ability.

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Abstract

本发明公开了一种猴头菇膳食纤维、制备方法及其应用，属于食品加工技术领域，所述制备方法包括以下步骤：将提取多糖和蛋白后的猴头菇残渣水洗，粉碎，干燥，备用；按照1g：(30～40)mL的料液比加入蒸馏水，加入碱性蛋白酶酶解25～30min；调节pH至5～6，加入纤维素酶进行酶解，酶解的同时进行微波-超声处理，沸水浴灭酶10～15min；加入乙醇溶液，经抽滤、洗涤、干燥得到猴头菇膳食纤维。所述猴头菇膳食纤维呈簇状结构，表面结构被破坏，可以束缚、破坏脂肪乳状液，提高粪便中胆汁酸排泄能力，改善胆汁酸的代谢，在小肠内延迟对脂类和胆固醇的扩散和吸收。

Description

一种猴头菇膳食纤维、制备方法及其应用 技术领域
本发明涉及食品加工技术领域，特别是涉及一种猴头菇膳食纤维、制备方法及其应用。
背景技术
猴头菇又称猴头菌、猴头蘑，是我国珍贵的药食兼用真菌。中医认为，猴头菇性平味甘，有利五脏、助消化、滋补身体等功效。近年来，关于猴头菇的研究主要集中在多糖、蛋白质等方面，研究表明，猴头菇多糖具有抗便秘、抗氧化、降血糖等活性，猴头菇蛋白具有良好的抗炎和提升免疫的活性，且免疫调节蛋白的相对分子质量为10-14kDa。但是提取水溶性多糖、蛋白质后会产生大量的猴头菇残渣，其主要成分包括膳食纤维(DF)、矿物质、少量蛋白质和色素，因此，从猴头菇残渣中制备高附加值和高品质的膳食纤维，不仅能够避免资源的大规模浪费，还可以开辟猴头菇产业发展的新途径。
膳食纤维根据溶解性可以分为水溶性膳食纤维(SDF)和水不溶性膳食纤维(IDF)，许多研究发现，DF中SDF含量在10％以上时才能视为高品质膳食纤维，因此SDF含量的高低通常是评价DF品质优劣的重要指标。此外，DF的功能特性往往受到其来源、组成和处理的影响，因此需要通过一定的处理来提升SDF的含量和DF的功能，进而提升天然DF的品质。
发明内容
本发明旨在提供一种猴头菇高品质膳食纤维及其制备方法，以解决目前猴头菇多糖及蛋白质提取过程中造成猴头菇资源利用率低、原料浪费严重等问题，提高猴头菇这一珍惜资源的利用效率，创造更大的经济与社会效益。
为实现上述目的，本发明提供了如下方案：
本发明提供一种猴头菇膳食纤维的制备方法，包括以下步骤：
(1)将提取多糖和蛋白后的猴头菇残渣水洗，粉碎，干燥，备用；
(2)在步骤(1)处理后的猴头菇残渣按照1：(30～40)(g/mL)的料液比加入蒸馏水，加入碱性蛋白酶酶解25～30min；
(3)调节pH至5～6，加入纤维素酶进行酶解，酶解的同时进行微波-超声处理，沸水浴灭酶10～15min；
(4)在步骤(3)得到的物质中加入乙醇溶液，经抽滤、洗涤、干燥得到猴头菇膳食纤维。
优选地，步骤(1)中将猴头菇残渣粉碎至40～120目。
优选地，步骤(1)中将猴头菇残渣粉碎至40目、60目、80目、100目或120目。更优选步骤(1)中将猴头菇残渣粉碎至80目。
优选地，步骤(2)所述碱性蛋白酶包括Alcalase FG 2.4L，酶活20×10 4U/g。
优选地，步骤(3)纤维素酶的加入量为猴头菇残渣的1～6wt％，酶解时间为15～90min。
优选地，步骤(3)纤维素酶的加入量为猴头菇残渣的1wt％、2wt％、3wt％、4wt％、5wt％或6wt％，酶解时间为15min、30min、45min、60min、75min或90min。更优选纤维素酶的加入量为猴头菇残渣的3wt％，酶解时间为75min。
优选地，步骤(3)微波温度为35～60℃，超声功率为250～500W。
优选地，步骤(3)微波温度为35℃、40℃、45℃、50℃、55℃或60℃，超声功率为250W、300W、350W、400W、450W或500W。更优选微波温度为55℃，超声功率为300W。
本发明还提供由所述猴头菇膳食纤维的制备方法制备得到的猴头菇膳食纤维。
本发明还提供所述的猴头菇膳食纤维在制备治疗降血脂药物中的应用。
本发明公开了以下技术效果：
超声-微波协同萃取有助于物料中活性成分的释放，酶法具有作用条件温和、专一性强、纯度高等优点。超声-微波辅助酶法改性作为一种联合技术，具有反应时间短、处理效果好等特点，超声和酶同时使用时，还可以改变纤维素酶结构，使酶的二级结构(α-螺旋、无规则卷曲)发生有利地变化。利用超声-微波辅助酶法对猴头菇残渣进行处理制备猴头菇高品质膳食纤维，对于制备高活性膳食纤维和提高猴头菇的利用效率具有重要意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发 明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为HE-DF 1与HE-DF 2的红外光谱图；
图2为HE-DF 1与HE-DF 2的X-射线衍射图；
图3为HE-DF 1与HE-DF 2的扫描电子显微镜图，其中，a1、a2、a3为HE-DF 1放大200、1000、5000倍，b1、b2、b3分别为HE-DF 2放大200、1000、5000倍。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
本发明抽滤、洗涤、干燥为本领域常规制备膳食纤维的方法，且并非发明要点，在此不做赘述。
准确称取提取多糖和蛋白后的猴头菇残渣5.0g，粉碎，按料液比1：40(g/mL)加入蒸馏水，60℃经碱性蛋白酶酶解30min，调节pH至5.5，加入纤维素酶，在 一定的温度、时间和功率下对猴头菇残渣进行微波-超声处理，然后沸水浴灭酶15min，加入总反应液体积4倍的95％(体积分数)乙醇溶液醇沉，经抽滤、洗涤、干燥得到猴头菇膳食纤维。
单因素试验：
选取物料粒度、酶添加量、微波温度、超声功率、酶解时间为单因素考察因素。物料粒度40、60、80、100、120目，纤维素酶添加量1％、2％、3％、4％、5％、6％，微波温度35、40、45、50、55、60℃，超声功率250、300、350、400、450、500W，酶解时间15、30、45、60、75、90min，以SDF得率、HE-DF 2的持油力和胆固醇吸附能力为指标进行试验，平行做3组试验。单因素试验中固定试验条件为物料粒度80目，酶添加量3％，处理温度50℃，超声功率400W，处理时间45min。
正交试验：
根据单因素实验结果，以SDF得率、HE-DF 2的持油力、胆固醇吸附能力及综合评分为评价指标，用L 16(4 5)设计正交试验优化处理条件。每组实验重复3次，因素与水平如表1所示。
运用综合评价的方法，根据指标对工艺影响的贡献，给予不同的权重系数。SDF含量的高低通常是评价DF品质优劣的重要指标，因此将SDF得率的权重系数设为0.4。通常认为，膳食纤维与油脂和胆固醇的结合能力是评估其吸收亲脂性成分能力的重要因素，因此为获得具有辅助降血脂效果的高活性膳食纤维,将HE-DF 2的持油力和胆固醇吸附能力作为考察指标，权重系数均为0.3，各指标中以最高者为100分，再进行加权求和。按照下列公式计算综合评分。
综合评分＝0.4×SDF得率/SDF得率最大值×100+0.3×持油力/持油力最大值×100+0.3×胆固醇吸附能力/胆固醇吸附能力最大值×100。
表1正交试验设计因素和水平
Figure PCTCN2021076781-appb-000001
对比例1
将提取多糖和蛋白后的猴头菇残渣用大量水洗，在55℃条件下干燥，备用。参照GB 5009.88-2014《食品中膳食纤维的测定》制备猴头菇膳食纤维，称取5.0g猴头菇残渣按1:40(g/mL)料液比加入蒸馏水，60℃经碱性蛋白酶酶解30min，沸水浴灭酶15min，用总反应液体积4倍的95％(体积分数)乙醇溶液醇沉，经抽滤、洗涤、干燥后得到HE-DF 1，作为对照组。
实施例1
准确称取提取多糖和蛋白后的猴头菇残渣5.0g，粉碎至80目，按料液比1：40(g/mL)加入蒸馏水，60℃经碱性蛋白酶酶解30min，调节pH至5.5，加入3wt％纤维素酶，在微波温度55℃、超声功率300W的条件下对猴头菇残渣进行微波-超声处理75min，然后沸水浴灭酶15min，加入总反应液体积4倍的95％(体积分数)乙醇溶液醇沉，经抽滤、洗涤、干燥得到猴头菇膳食纤维，记为HE-DF 2。
SDF得率的测定：
Figure PCTCN2021076781-appb-000002
持油力的测定：
将50mL的离心管干燥至恒重，进行称量，然后准确称取0.5g(精确到0.001g)膳食纤维样品置于离心管中，再加入玉米油10g，在37℃水浴锅中静置3h，在3800r/min的转速下离心20min，弃其上层油，并且用滤纸小心擦拭吸干游离的油，进行称重。持油力的计算公式为：
Figure PCTCN2021076781-appb-000003
式中：m 0为样品干质量(g)；m 1为离心管质量(g)；m 2为吸油后样品和离心管质量和(g)。
胆固醇吸附能力的测定：
(1)标准曲线的绘制：采用邻苯二甲醛法，以胆固醇为标准品，绘制标准曲线，标准曲线方程为：y＝0.012x+0.0499。
(2)胆固醇吸附能力的测定：取市售新鲜蛋黄混合搅拌均匀后，加入9倍体积的水，用超声波破壁机搅打成蛋黄乳液，分别取30mL乳液用1mol/L NaOH和1mol/L HCL溶液调pH＝7.0(模拟肠的环境)，37℃水浴震荡2h，4000r/min离心20min，收集上清液，定容至100mL，取1mL用冰醋酸稀释10倍后，取1mL 稀释液测定胆固醇含量。根据反应前后的浓度差计算吸附量。
Figure PCTCN2021076781-appb-000004
傅里叶红外光谱分析：
将膳食纤维样品置于烘箱中烘干至恒重后，称取干燥样品2mg与KBr粉末200mg于研钵中，充分混匀，研磨均匀后进行压片处理，采用傅立叶红外光谱仪进行分析，扫描波长为4000cm -1～400cm -1。
X-射线衍射扫描：
取适量膳食纤维过200目筛，在65℃下烘干，在室温条件下置于干燥器中备用，取1～2g样品，在特定玻璃样品板(MiniFlx 600台式X射线衍射仪自带的玻璃样品板，尺寸为50mm×35mm)凹槽内将其装填好，用光滑干净的玻璃板将样品压紧，刮去玻璃板表面多出的粉末，重复2次左右后将制好的样品放入衍射仪扫描。
X衍射条件为：Cu福射，电压为40KV，电流为40mA，步长为0.02，扫描速率为12deg/min，连续扫描的2θ从3°到90°。
扫描电子显微镜观察：
将膳食纤维样品过筛后置于供箱中干燥至恒重，取适量进行黏台，采用溅射镀膜法对膳食纤维样品进行表面镀金，表面镀金的样品置于扫描电镜下观察，进行200、1000、5000倍微观结构观察拍照。
理化性质测定：
持水力的测定：
将50mL的离心管干燥至恒重，进行称量，然后准确称取0.5g(精确到0.001g)膳食纤维样品干粉置于离心管中，加入20mL蒸馏水，震荡摇匀，室温下浸泡24h后，在3800r/min的转速下离心20min，小心弃其上清液后进行称重。持水力按下式计算：
Figure PCTCN2021076781-appb-000005
式中：m 0为样品干质量(g)；m 1为离心管质量(g)；m 2为吸水后样品和离心管质量和(g)。
持油力的测定：
同上述持油力的测定。
膨胀力的测定：
准确称取膳食纤维样品0.2g(精确到0.001g)，置于10mL的量筒中，测得样品干样体积，再加入5mL的蒸馏水，混匀，在室温条件下静置12h，观察记录样品吸水后的体积的变化情况。膨胀力的计算公式为：
Figure PCTCN2021076781-appb-000006
式中：V 1为样品膨胀后的测定体积(mL)；V 0为样品干样的测定体积(mL)；m为样品干质量(g)。
结合水力的测定：
称取膳食纤维干样品0.2g(精确到0.001g)，置于50mL的烧杯中，加入25mL蒸馏水，充分搅拌并密封，放在4℃冰箱中静置24h，在3800r/min条件下离心30min，弃去上清液，将其抽滤后进行称重，105℃条件下干燥至恒重，两者差值即为样品结合水的质量，结合水力计算公式如下：
Figure PCTCN2021076781-appb-000007
式中：m 0为样品干重(g)；m 1为离心静置后湿样品质量(g)；m 2为干燥后样品质量(g)。
阳离子交换能力的测定：
将样品干燥至恒重，向50mL烧杯中加入0.500g样品，倒入20mL的0.1mol/LHCl溶液，放置在室温下，密封静置12小时，抽滤并用蒸馏水多次洗涤滤渣，再将滤渣放入250mL锥形瓶中，分别加入100mL质量分数5％Nacl溶液，充分搅拌30min，滴入少量0.5％的酚酞-乙醇溶液作为反应指示剂，用0.1mol/L的NaOH溶液进行滴定，边振荡锥形瓶边滴定，以溶液变色后3min不褪色为滴定终点，用蒸馏水代替0.1mol/L HCl溶液做空白试验。计算公式如下：
Figure PCTCN2021076781-appb-000008
公式中：V 1为滴定样品用的NaOH溶液体积(mL)；V 0为滴定空白样用的NaOH溶液体积(mL)；m为干样质量(g)；0.1为滴定NaOH溶液的浓度(mol/L)。
胆固醇吸附能力的测定
同上，并同时测定HE-DF 1和HE-DF 2在pH＝2(模拟胃的环境)时的胆固 醇吸附能力。
胆酸盐结合能力测定
(1)标准曲线的绘制：以pH 6.3的0.1mol/L PBS配制浓度为0.3mmol/L的甘氨胆酸钠、0.3mmol/L的牛磺胆酸钠溶液，分别取其0、0.1、0.5、1.0、1.5、2.0、2.5mL于25mL的比色管中，用pH 6.3的0.1mol/L PBS补加至2.5mL，分别加入7.5mL体积分数为60％的硫酸溶液，70℃下水浴20min，取出后冰浴5min，在387nm处测定吸光值。以胆酸盐含量为横坐标，吸光度为纵坐标，绘制胆酸盐含量的标准曲线，由标准曲线求得样液中胆酸盐的浓度。甘氨胆酸钠标准曲线：y＝0.624x+0.045；牛磺胆酸钠标准曲线：y＝0.043x+0.055。
(2)胆酸盐结合试验:分别称取150mg HE-DF 1和HE-DF 2于50mL锥形瓶中，加入1mL 0.01mol/L的HCL溶液，3mL 10mg/mL胃蛋白酶溶液(以pH6.3 0.1mol/L PBS配制)，在37℃条件下恒温水浴振荡1h，模拟胃环境；用0.1mol/L NaOH溶液调节pH6.3，随后加入4mL 10mg/mL胰蛋白酶溶液(以pH 6.3 0.1mol/L PBS配制)，在37℃条件下恒温振荡1h，模拟肠道环境。像每个样品中加入4mL 0.3mmol/L甘氨胆酸钠或牛磺胆酸钠，37℃恒温水浴振荡1h。将其倒入离心管中，3800r/min离心20min，吸取上清液2.5mL，加入7.5mL体积分数60％的硫酸，在387nm处测定吸光度值，此为实验组。此外，还需做样品空白组和试验空白组，样品空白组即不加膳食纤维样品，其他操作同实验组，样品空白组测得的胆酸盐加入量，通过设置样品空白组可以扣除样品转移带来的胆酸盐损失，一般会比初始加入量略低；试剂空白组即及结合体系不加胆酸盐，膳食纤维样品经模拟胃肠消化后，以等体积的0.1mol/L PBS代替胆酸盐，其他操作同实验组。
Figure PCTCN2021076781-appb-000009
式中：C 0—样品空白组；C 1—实验组；C 2—试剂空白组。
Figure PCTCN2021076781-appb-000010
式中：C 3—样品空白组；C 4—实验组；C 5—试剂空白组。
猴头菇渣基础成分分析见表2。
表2猴头菇渣基础成分
Figure PCTCN2021076781-appb-000011
Figure PCTCN2021076781-appb-000012
注：TDF为总膳食纤维；IDF为不溶性膳食纤维；SDF为水溶性膳食纤维。
猴头菇渣经干燥、粉碎后，其基础成分含量结果见表2。由表2可知，猴头菇渣中TDF含量高达87.35％，其中主要为IDF，SDF只达到3.63％。
超声微波辅助酶法处理条件的确定：
单因素试验结果：
物料粒度的影响：
表3物料粒度对SDF得率、HE-DF 2的持油力和胆固醇吸附能力的影响
Figure PCTCN2021076781-appb-000013
注：同列小写字母不同表示差异显著(P＜0.05)，下同。
由表3可知，物料粒度对SDF得率有一定影响，随着粒度的变细，物料与溶剂接触的表面积增大，有助于SDF溶出，使得SDF得率增加。但当物料粒度继续增加，粒度过细使得物料容易粘聚成团，减小了与溶剂接触的表面积，SDF得率呈下降趋势。HE-DF 2的持油力随粒度的变细呈先快速增大后缓慢增大趋势，持油力不仅与物料粒度有关，还与膳食纤维的组成有关。胆固醇吸附能力随着物料粒度的变化先上升又下降，当粒度为80目时胆固醇吸附能力达到最大，可能是由于随着物料粒度的变细，其结构变得疏松，极性基团增多，但粒度过细又会对其结构造成一定程度的破坏，使胆固醇吸附能力下降。
酶添加量的影响
表4酶添加量对SDF得率、HE-DF 2的持油力和胆固醇吸附能力的影响
Figure PCTCN2021076781-appb-000014
由表4可知，随着纤维素酶添加量的增加，样品与酶的接触机会增大，可溶性部分因细胞纤维壁的破裂而大量溶出，并且得到的产物更加疏松，SDF得率、HE-DF 2的持油力和胆固醇吸附能力都有升高。当纤维素酶添加量到一定程度时， 其于底物作用完全，不会再有大量相应的细胞壁随纤维素酶用量的增加而被降解，因此HE-DF 2的体外吸附性能基本保持稳定。酶添加量过度时，大分子聚合物被降解成不能被乙醇沉淀的小分子物质，导致SDF得率下降。
微波温度的影响
表5微波温度对SDF得率、HE-DF 2的持油力和胆固醇吸附能力的影响
Figure PCTCN2021076781-appb-000015
由表5可知，随微波温度的升高，SDF得率和HE-DF 2的胆固醇吸附能力先升高后降低。较高的温度可以加快酶的催化速率，从而促进IDF水解为SDF，增加胆固醇吸附能力。而较高的温度会破坏酶的结构并使酶的活性降低或丧失，这对纤维素的水解不利，因而导致胆固醇吸附能力有所降低。整个过程中，HE-DF 2的持油力随处理温度的变化没有发生特别显著的升降趋势。
超声功率的影响：
表6为超声功率对SDF得率、HE-DF 2的持油力和胆固醇吸附能力的影响。由表6可知，随着超声波功率的增加，SDF得率、持油力和胆固醇吸附能力的变化趋势相似，先升高后降低。当超声波功率增加，超声处理通过分子内加热和空化效应产生大量空化气泡，气泡破裂产生能量促进细胞壁破裂和细胞物质释放，帮助溶剂进入组织细胞，促进SDF的溶出和物料结构的变化，使其吸附性能增强。超声波功率过大可能会使越来越多的纤维素被过度水解形成小分子或片段，后续醇沉过程中容易被除去，导致SDF得率和膳食纤维的吸附能力降低。
酶解时间的影响：
表7酶解时间对SDF得率、HE-DF 2的持油力和胆固醇吸附能力的影响
Figure PCTCN2021076781-appb-000016
实验结果表明，酶解时间对SDF得率和吸附性能有一定影响，SDF得率、 持油力和胆固醇吸附能力都随酶解时间的延长先升高后降低。酶解时间为60min时，SDF得率达到最大，HE-DF 2的持油力也达到最高，胆固醇吸附能力也较好。随着酶解时间的继续延长，纤维素的水解度增强，纤维之间连接的键被切断，膳食纤维的三维网络结构可能被破坏部分发生降解，影响SDF得率，并使HE-DF 2的体外吸附性能随之受到一定影响。
正交试验结果：
表8 L 16(4 5)正交试验结果
Figure PCTCN2021076781-appb-000017
表9正交试验方差分析结果
Figure PCTCN2021076781-appb-000018
注：*.差异显著，P＜0.05；**.差异极显著，P＜0.01。
根据表8中的R值可知，各因素对综合评分的影响主次为A>C>E>B>D。由表9的方差分析结果可知，物料粒度、酶添加量、微波温度、酶解时间为极显著因素(P＜0.01)，超声功率为显著因素(P＜0.05)，这一结果与极差分析结果一致。最优方案为A 2B 2C 3D 2E 3，需进行验证试验，与第6组试验组合A 2B 2C 1D 4E 3对比，按照A 2B 2C 3D 2E 3处理得到SDF得率为(11.72±0.12)％，HE-DF 2的持油力为(2.05±0.01)g/g，胆固醇吸附能力为(36.84±0.59)mg/g，总体高于A 2B 2C 1D 4E 3处理的效果，所以确定最优方案为物料粒度80目、酶添加量3％、微波温度55℃、超声功率300W、酶解时间75min。此外，对16组正交试验得到的SDF得率、HE-DF 2的持油力和胆固醇吸附能力进行相关性分析，结果显示，SDF得率与HE-DF 2的胆固醇吸附能力存在正相关关系(r＝0.46，P<0.01)，即随着SDF得率增加，HE-DF 2的胆固醇吸附能力有增加的趋势，与持油力未呈现明显的相关关系(P>0.05)，有研究发现木聚糖酶处理可以改变膳食纤维的组成和微观结构，并形成更可溶多糖与膳食纤维的胆固醇吸收能力呈正相关，这与本结果一致。综上，猴头菇高品质膳食纤维具有良好的吸附油脂和胆固醇的能力。
HE-DF 1与HE-DF 2的红外光谱图见图1，对比两个图谱发现，二者有相似的光谱分布，均具有多糖的特征吸收峰，在3200～3600cm -1处有一个较宽且较强的吸收峰，这是羟基的特征峰，说明其膳食纤维中存在着处于缔合状态的分子间氢键，经过超声-微波辅助酶法处理后，纤维结构中的羟基吸收峰向低波数移动，由3417.05m -1变为了3383.73cm -1，这说明HE-DF 2中羟基所形成的氢键增强，亲水性增强。一般而言，波数移动越多，特征峰的吸收强度就越大，羟基就暴露的越多，可能会使样品的亲水性增强。HE-DF 2没有明显新的化学基团出现，不同的只是峰强度都明显增加，说明超声-微波辅助酶法主要是破坏纤维素和半纤维素的链间和链内氢键，使膳食纤维的结构疏松，大分子降解为小的分子片断，表面积增加，从而表现出更强的吸附力。
HE-DF 1与HE-DF 2的X-射线衍射图见图2。纤维素类物质的晶区一般是由无序的无定形区(30％)和有序的结晶区(70％)组成。由图2可看出，HE-DF 1与HE-DF 2的X-射线衍射图形在形状上相似，分别在2θ为19.15°和20.05°处有明显的结晶衍射峰，表现为纤维素I特征，这表明猴头菇中DF的晶体类型属于纤维素I型。与HE-DF 1相比，HE-DF 2的峰形和出峰位置基本没有变化，说明该 纤维的结晶结构属性和纤维晶区基本未受影响，衍射的强度有所降低，可能是超声-微波辅助酶法对结晶区造成部分破坏，如使结晶区纤维素分子间的氢键破坏，纤维素分子发生部分降解，水溶性成分得以溶出，而非结晶区的无序性、无定型特性导致了膳食纤维结构更加松散、无序，从而导致持水性、持油性等物理性质的变化。
HE-DF 1与HE-DF 2的扫描电子显微镜图见图3，a1、a2、a3为HE-DF 1放大200、1000、5000倍，b1、b2、b3分别为HE-DF 2放大200、1000、5000倍。膳食纤维的功能作用往往受其结构的影响。HE-DF 1和HE-DF 2的超微结构如图3所示，两种膳食纤维表面都出现了褶皱，但是相比而言，HE-DF 1多为不规则块状结构，表面有明显的褶皱，但结构联系相对较密实；HE-DF 2则呈簇状结构，表面结构被破坏，暴露内部的毛细管结构，整体更加疏松，凹凸不平，形成了较多大的空腔，从而具有更大的比表面积，帮助纤维吸收和保留水分。可见，超声-微波辅助酶法处理可以改善膳食纤维的微观结构，使其表面积更大，从而可以进一步改善膳食纤维的理化性质，使其表现出更强的吸附作用。
HE-DF 1与HE-DF 2的纤维样品的理化特性见表10。
表10纤维样品的理化特性
Figure PCTCN2021076781-appb-000019
由表10可知，超声-微波辅助酶法处理对猴头菇膳食纤维持水力、持油力、膨胀力、结合水力均有一定的改善作用，且对膳食纤维的理化性质均有显著性影响。与HE-DF 1相比，HE-DF 2的持水力、持油力、结合水力、膨胀力分别提高了51.21％、72.70％、59.77％、92.97％，原因可能是因为超声-微波辅助酶法处理时，超声-微波的机械破碎作用和高能穿透效应使得物体断裂成更多小碎片，纤维素酶作用时能充分与底物接触，酶解效率提高，这种联合方法使膳食纤维的基质结构更加松散、多孔，从而暴露出更多的亲水和亲油基团，并且粒度小可以使水分和油更易进入，持水力、持油力和膨胀力会升高。持水力、持油力和膨胀作用能促已被用来作为预示膳食纤维降胆固醇能力的指标，由此可以推测HE-DF 2在肠道内结合胆固醇的能力也得到了提高，因而比HE-DF 1具有更佳的降血脂功能。
膳食纤维化学结构中所含有的羧基、羟基和氨基等侧链基团，可产生类似弱酸性阳离子交换树脂的作用，可与阳离子进行可逆的交换。表10还显示了，与HE-DF 1相比，HE-DF 2的阳离子交换能力显著增加了87.5％，说明超声-微波辅助酶法处理可以使膳食纤维中的一些羧基、羟基的侧链基团显露出来，有利于提高阳离子交换能力。有研究发现阳离子交换能力高的膳食纤维可以束缚、破坏脂肪乳状液，提高粪便中胆汁酸排泄能力，改善胆汁酸的代谢，在小肠内延迟对脂类和胆固醇的扩散和吸收。
膳食纤维降血脂的机理之一就是其对胆固醇的吸附，使其随粪便排出，降低体内胆固醇含量。表11是猴头菇膳食纤维的胆固醇吸附能力，胆固醇吸附能力主要模拟了胃部环境pH＝2和肠道环境pH＝7，由表中可以看出，HE-DF 2相比HE-DF 1的胆固醇吸附能力明显增强，这可能是由于超声-微波辅助酶法处理对猴头菇渣中的不溶性膳食纤维起到降解作用，增加了可溶性膳食纤维含量，并且使膳食纤维的结构变得疏松，表面积增大，因而使其功能特性有所增强。此外，pH值对HE-DF 1和HE-DF 2的胆固醇吸附能力也有一定影响，pH＝7.0时胆固醇吸附能力均大于pH＝2.0时的胆固醇吸附能力，由此可推测，膳食纤维对胆固醇的吸附能力主要在肠道中起作用。
表11纤维样品的胆固醇吸附能力
Figure PCTCN2021076781-appb-000020
膳食纤维对胆酸盐的结合能力可以作为衡量降血脂功能特性的表征之一，在小肠中束缚胆酸盐和促进胆酸盐的排出被认为是膳食纤维降低胆固醇的主要原因。表12是猴头菇膳食纤维的胆酸盐结合能力，猴头菇膳食纤维对胆酸盐有一定的吸附能力，并且HE-DF 2结合甘氨胆酸钠和牛磺胆酸钠能力明显高于HE-DF 1结合能力。这可能是由于通过超声-微波辅助酶法处理，猴头菇膳食纤维的内部结构被破坏，变得疏松多孔，增加了胆酸盐的结合位点，因而易于HE-DF 2的吸附性。同时与HE-DF 1相比，HE-DF 2中SDF含量增加，使得黏度增加，导致胆酸盐的扩散速率降低。
表12纤维样品的胆酸盐结合能力
Figure PCTCN2021076781-appb-000021
Figure PCTCN2021076781-appb-000022
通过超声-微波辅助酶法处理猴头菇渣，以制备猴头菇高品质膳食纤维，通过单因素和正交试验，确定了在物料粒度80目、酶添加量3％、微波温度55℃、超声功率300W、酶解时间75min时，SDF得率最高为(11.72±0.12)％，HE-DF 2持油力为(2.05±0.01)g/g，胆固醇吸附能力为(36.84±0.59)mg/g。红外光谱、X-射线衍射扫描显示超声-微波辅助酶法处理可以改变膳食纤维的组成和微观结构，这些组成及微观结构的变化与膳食纤维体外吸附性能的提高之间可能有密切的关系。通过理化性质分析发现，HE-DF 2的持水力、结合水力、膨胀力分别为(10.76±0.10)g/g、(5.47±0.15)g/g、(13.82±0.29)mL/g，均符合高品质膳食纤维的要求，且具有良好的阳离子交换能力、胆固醇吸附能力和胆酸盐结合能力。综上，利用超声-微波辅助酶法处理猴头菇渣，可以改善猴头菇膳食纤维的品质，得到具有潜在降血脂能力的猴头菇高品质膳食纤维。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (10)

  1. 一种猴头菇膳食纤维的制备方法，其特征在于，包括以下步骤：
    (1)将提取多糖和蛋白后的猴头菇残渣水洗，粉碎，干燥，备用；
    (2)在步骤(1)处理后的猴头菇残渣按照1g：(30～40)mL的料液比加入蒸馏水，加入碱性蛋白酶酶解25～30min；
    (3)调节pH至5～6，加入纤维素酶进行酶解，酶解的同时进行微波-超声处理，沸水浴灭酶10～15min；
    (4)在步骤(3)得到的物质中加入乙醇溶液，经抽滤、洗涤、干燥得到猴头菇膳食纤维。
  2. 根据权利要求1所述的一种猴头菇膳食纤维的制备方法，其特征在于，步骤(1)中将猴头菇残渣粉碎至40～120目。
  3. 根据权利要求2所述的一种猴头菇膳食纤维的制备方法，其特征在于，步骤(1)中将猴头菇残渣粉碎至40目、60目、80目、100目或120目。
  4. 根据权利要求1所述的一种猴头菇膳食纤维的制备方法，其特征在于，步骤(2)所述碱性蛋白酶为Alcalase FG 2.4L，酶活20×10 4U/g。
  5. 根据权利要求1所述的一种猴头菇膳食纤维的制备方法，其特征在于，步骤(3)纤维素酶的加入量为猴头菇残渣的1～6wt％，酶解时间为15～90min。
  6. 根据权利要求5所述的一种猴头菇膳食纤维的制备方法，其特征在于，步骤(3)纤维素酶的加入量为猴头菇残渣的1wt％、2wt％、3wt％、4wt％、5wt％或6wt％，酶解时间为15min、30min、45min、60min、75min或90min。
  7. 根据权利要求1所述的一种猴头菇膳食纤维的制备方法，其特征在于，步骤(3)微波温度为35～60℃，超声功率为250～500W。
  8. 根据权利要求7所述的一种猴头菇膳食纤维的制备方法，其特征在于，步骤(3)微波温度为35℃、40℃、45℃、50℃、55℃或60℃，超声功率为250W、300W、350W、400W、450W或500W。
  9. 一种根据权利要求1～8任一项所述的猴头菇膳食纤维的制备方法制备得到的猴头菇膳食纤维。
  10. 如权利要求9所述的猴头菇膳食纤维在制备治疗降血脂药物中的应用。
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CN116369504A (zh) * 2023-05-22 2023-07-04 湖北省农业科学院农产品加工与核农技术研究所 一种富含香菇嘌呤的香菇提取液、香菇残渣蛋白水解肽液及其制备方法和应用

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