Oyster mushroom bran polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses oyster mushroom bran polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention establishes an extraction method of six kinds of oyster mushroom bran polysaccharides, the extraction method comprises hot water extraction, acid extraction, ultrasonic-assisted extraction, alkali extraction, hot alkali extraction and acid-alkali continuous extraction, and the six kinds of polysaccharides, namely PORP-H, PORP-A, PORP-U, PORP-B, PORP-HB and PORP-AB, are extracted. Experiments prove that the six kinds of oyster mushroom bran polysaccharides have relatively high safety, oxidation resistance and prebiotic activity, and can be used as raw and auxiliary materials of foods, medicines and health-care products, so that oyster mushroom bran resources are fully developed and utilized, effective guarantee is provided for development of the mushroom industry, and the oyster mushroom bran polysaccharides have broad market prospects. And the method has relatively long-term scientific theory guiding significance and relatively high economic value significance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a polysaccharide from Pleurotus ostreatus spent mushroom substrate, a preparation method thereof, and an application thereof. Background Art

[0002] As a by-product generated during the cultivation of Pleurotus ostreatus, Pleurotus ostreatus spent mushroom substrate is usually used as a cheap by-product for feeding livestock and as a secondary cultivation substrate. Pleurotus ostreatus spent mushroom substrate is rich in active polysaccharides, proteins, fats and other nutrients. Polysaccharides are high-molecular compounds formed by polymerization of more than 10 monosaccharides of the same or different types through glycosidic bonds. Their structures are complex and diverse, and the molecular weights vary from several thousand to tens of thousands. Research shows that polysaccharides have various biological activities such as anti-fatigue, anti-hypoxia, antioxidant, and immune regulation. Currently, commercially available polysaccharide products such as lentinan, ginseng polysaccharide, and astragalus polysaccharide are used as adjuvant drugs for tumor radiotherapy and chemotherapy, and wolfberry polysaccharide functional food additives and polysaccharide masks have achieved good economic benefits, indicating that the research and development of polysaccharide products have broad application prospects. By extracting polysaccharides from Pleurotus ostreatus spent mushroom substrate, the economic value of Pleurotus ostreatus spent mushroom substrate can be greatly improved. However, there are few reports on polysaccharides from Pleurotus ostreatus spent mushroom substrate, and relatively little research has been done on the extraction methods of polysaccharides from Pleurotus ostreatus spent mushroom substrate. Therefore, how to use different methods to prepare polysaccharide products with different functional characteristics and biological activities and provide a new way for the in-depth utilization of Pleurotus ostreatus spent mushroom substrate resources is an urgent problem to be solved at present. Summary of the Invention

[0003] The purpose of the present invention is to provide a polysaccharide from Pleurotus ostreatus spent mushroom substrate, a preparation method thereof, and an application thereof to solve the problems existing in the above-mentioned prior art. The present invention has established six extraction methods for polysaccharides from Pleurotus ostreatus spent mushroom substrate, including hot water extraction, acid extraction, ultrasonic-assisted extraction, alkali extraction, hot alkali extraction, and acid-base continuous extraction, and six polysaccharides (PORP-H, PORP-A, PORP-U, PORP-B, PORP-HB, and PORP-AB) have been extracted. Through experiments, it is confirmed that these six polysaccharides from Pleurotus ostreatus spent mushroom substrate all have high safety, antioxidant activity, and prebiotic activity. The extraction method of the present invention provides technical support for the study of the physiological activity of polysaccharides from Pleurotus ostreatus spent mushroom substrate and provides a theoretical basis for the development of Pleurotus ostreatus spent mushroom substrate resources.

[0004] To achieve the above purpose, the present invention provides the following solutions:

[0005] The present invention provides an application of a polysaccharide from Pleurotus ostreatus spent mushroom substrate in the preparation of an antioxidant, a fermentation food additive, or a prebiotic food, and the polysaccharide from Pleurotus ostreatus spent mushroom substrate includes PORP-H, PORP-A, PORP-U, PORP-B, PORP-HB, and PORP-AB;

[0006] The molecular weights of PORP-H, PORP-A, PORP-U, PORP-B, PORP-HB, and PORP-AB are 581.00 kDa, 530.19 kDa, 432.05 kDa, 503.25 kDa, 590.10 kDa, and 449.16 kDa, respectively;

[0007] PORP-H is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 10.7:3.3:3.0:1.6:6.9:12.8:30.1:28.7:2.9; PORP-A is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 11.4:3.8:3.2:1.8:7.7:14.4:29.1:25.7:3.0; PORP-U is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 12.2:4.8:3.7:2.5:8.0:13.6:27.6:25.5:2.0; PORP-B is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 12.3:4.2:3.1:2.1:7.8:14.8:27.1:26.1:2.6; PORP-HB is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 13.7:4.3:3.7:2.1:7.6:13.1:25.6:27.9:1.9; PORP-AB is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 14.5:4.8:4.6:1.1:7.1:13.6:25.0:26.9:2.4.

[0008] The present invention also provides a method for preparing the Pleurotus ostreatus spent mushroom substrate polysaccharide described in the above application, which includes the following steps:

[0009] (1) Degrease the Pleurotus ostreatus spent mushroom substrate powder;

[0010] (2) Extract the degreased Pleurotus ostreatus spent mushroom substrate powder with hot water, and after purification, obtain the Pleurotus ostreatus spent mushroom substrate polysaccharide PORP-H;

[0011] Extract the degreased Pleurotus ostreatus spent mushroom substrate powder with hydrochloric acid solution, and after purification, obtain the Pleurotus ostreatus spent mushroom substrate polysaccharide PORP-A;

[0012] Mix the defatted Pleurotus ostreatus residue powder with water and then perform ultrasonic treatment. After purification, Pleurotus ostreatus residue polysaccharide PORP-U is obtained.

[0013] Extract the defatted Pleurotus ostreatus residue powder with an alkali solution. After purification, Pleurotus ostreatus residue polysaccharide PORP-B is obtained.

[0014] Extract the defatted Pleurotus ostreatus residue powder with a hot alkali solution. After purification, Pleurotus ostreatus residue polysaccharide PORP-HB is obtained.

[0015] Extract the defatted Pleurotus ostreatus residue powder successively with a hydrochloric acid solution and an alkali solution. After purification, Pleurotus ostreatus residue polysaccharide PORP-AB is obtained.

[0016] Further, the step of extraction with hot water is as follows: Mix the defatted Pleurotus ostreatus residue powder and water at a ratio of 1 g: 40 - 60 mL, then soak and extract at 85 - 90 °C for 2 - 3 h to obtain an extract.

[0017] Further, the step of extraction with a hydrochloric acid solution is as follows: Mix the defatted Pleurotus ostreatus residue powder and a hydrochloric acid solution with a pH of 3.0 at a ratio of 1 g: 40 - 60 mL, then stir and extract at 45 - 55 °C for 2 - 3 h to obtain an extract.

[0018] Further, the step of ultrasonic treatment is as follows: Ultrasonically extract the mixture of the defatted Pleurotus ostreatus residue powder and water at 150 - 250 W and 45 - 55 °C for 2 - 3 h to obtain an extract.

[0019] The mixing ratio of the defatted Pleurotus ostreatus residue powder and water is 1 g: 40 - 60 mL.

[0020] Further, the step of extraction with an alkali solution is as follows: Mix the defatted Pleurotus ostreatus residue powder and an alkali solution with a pH of 10.0 at a ratio of 1 g: 40 - 60 mL, then stir and extract at 45 - 55 °C for 2 - 3 h to obtain an extract.

[0021] Optionally, the alkali solution is a sodium hydroxide solution.

[0022] Further, the step of extraction with a hot alkali solution is as follows: Mix the defatted Pleurotus ostreatus residue powder and water at a ratio of 1 g: 40 - 60 mL, adjust the pH to 8.0, then stir and extract at 85 - 90 °C for 2 - 3 h to obtain an extract.

[0023] Further, the steps of sequentially extracting with hydrochloric acid solution and alkali solution are as follows: Mix the defatted Pleurotus ostreatus residue powder with hydrochloric acid solution at pH 3.0 in a ratio of 1 g: 20 - 35 mL, soak and extract at 25 - 30 °C for 30 - 45 min; after protein removal by Sevag method, dialysis treatment and freeze-drying, obtain freeze-dried powder; mix the freeze-dried powder with alkali solution at pH 8.0 in a ratio of 1 g: 20 - 35 mL, stir and extract at 30 - 35 °C for 8 - 12 min to obtain an extract.

[0024] Optionally, the alkali solution is sodium hydroxide solution.

[0025] Further, the purification steps are as follows: subject the extract to ethanol precipitation, protein removal by Sevag method and dialysis treatment in sequence;

[0026] The ethanol precipitation is carried out with 75% ethanol aqueous solution at 4 °C for 12 - 16 h;

[0027] When carrying out the dialysis treatment, the cut-off molecular weight of the dialysis bag is 8 - 14 KDa.

[0028] The present invention also provides Pleurotus ostreatus residue polysaccharide prepared by the above preparation method.

[0029] The present invention discloses the following technical effects:

[0030] The present invention has established an extraction method for six Pleurotus ostreatus residue polysaccharides, including hot water extraction, acid extraction, ultrasonic-assisted extraction, alkali extraction, hot alkali extraction and acid-base continuous extraction, and six polysaccharides (PORP-H, PORP-A, PORP-U, PORP-B, PORP-HB and PORP-AB) have been extracted. It is confirmed by experiments that these six Pleurotus ostreatus residue polysaccharides all have high safety, antioxidant activity and prebiotic activity, and can be used as raw and auxiliary materials for food, medicine and health products, so that the Pleurotus ostreatus residue resources have been fully developed and utilized, providing effective guarantee for the development of the mushroom industry, and having relatively long-term scientific theoretical guiding significance and high economic value significance. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a flow chart of the extraction method for six Pleurotus ostreatus residue polysaccharides;

[0033] Figure 2The monosaccharide composition of polysaccharides from six Pleurotus ostreatus spent substrates;

[0034] Figure 3 The molecular weights of polysaccharides from six Pleurotus ostreatus spent substrates;

[0035] Figure 4 The infrared spectral analysis of polysaccharides from six Pleurotus ostreatus spent substrates;

[0036] Figure 5 The microscopic morphological characteristics of polysaccharides from six Pleurotus ostreatus spent substrates magnified 1000 times (50μm), 5000 times (10μm) and 10000 times (5μm), respectively; among them, A is the microscopic morphological characteristic of PORP-H; B is the microscopic morphological characteristic of PORP-U; C is the microscopic morphological characteristic of PORP-B; D is the microscopic morphological characteristic of PORP-HB; E is the microscopic morphological characteristic of PORP-A; F is the microscopic morphological characteristic of PORP-AB; the magnification multiples of 1-3 are 1000 times, 5000 times and 10000 times in sequence;

[0037] Figure 6 The Congo red staining of polysaccharides from six Pleurotus ostreatus spent substrates;

[0038] Figure 7 The I 2 -KI test of polysaccharides from six Pleurotus ostreatus spent substrates;

[0039] Figure 8 The particle size of polysaccharides from six Pleurotus ostreatus spent substrates;

[0040] Figure 9 The Zeta potential of polysaccharides from six Pleurotus ostreatus spent substrates;

[0041] Figure 10 The thermogravimetric analysis of polysaccharides from six Pleurotus ostreatus spent substrates;

[0042] Figure 11 The differential scanning calorimetry analysis of polysaccharides from six Pleurotus ostreatus spent substrates;

[0043] Figure 12 The oil-holding and water-holding capacities of polysaccharides from six Pleurotus ostreatus spent substrates;

[0044] Figure 13 The foaming and foam stability capacities of polysaccharides from six Pleurotus ostreatus spent substrates;

[0045] Figure 14 The emulsifying and emulsion stability capacities of polysaccharides from six Pleurotus ostreatus spent substrates;

[0046] Figure 15 The DPPH radical scavenging capacity of polysaccharides from six Pleurotus ostreatus spent substrates;

[0047] Figure 16 The ABTS radical scavenging capacity of polysaccharides from six Pleurotus ostreatus spent substrates;

[0048] Figure 17 is the hydroxyl radical scavenging ability of polysaccharides from six Pleurotus ostreatus spent mushroom substrates;

[0049] Figure 18 is the determination result of the prebiotic activity of polysaccharides from six Pleurotus ostreatus spent mushroom substrates; among them, A - D are the growth rates of probiotic concentrations in the cultures at 6, 12, 24, and 36 h in sequence. Specific Embodiments

[0050] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0051] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0052] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0053] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0054] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open - ended terms, meaning including but not limited to.

[0055] Example 1 Extraction of Polysaccharides from Pleurotus ostreatus Spent Mushroom Substrates

[0056] In this example, six methods are used to extract polysaccharides from Pleurotus ostreatus spent mushroom substrates. The extraction process is shown in Figure 1 . The specific process is as follows:

[0057] 1. Collect Pleurotus ostreatus spent mushroom substrate (the spent mushroom substrate culture medium consists of wood chips, wheat bran, corn straw, calcium carbonate and Pleurotus ostreatus mycelium), dry it and then crush it. Pass it through an 80-mesh sieve, take the material under the sieve, and perform defatting treatment with 95% ethanol to obtain Pleurotus ostreatus spent mushroom substrate powder.

[0058] 2. Polysaccharide extraction

[0059] 2.1 Hot water extraction: Add the defatted Pleurotus ostreatus spent mushroom substrate powder to deionized water at a ratio of 1:50 (g / mL), and soak it in a water bath at 90 °C for 2 hours. Concentrate the extract with a rotary evaporator, precipitate it with 75% ethanol at 4 °C overnight, and centrifuge it at 8000 r / min for 10 min. After resuspending the precipitate with deionized water, mix it with Sevag reagent (a mixture of chloroform and n-butanol in a volume ratio of 4:1) for 1 h and then centrifuge it. Take the supernatant to remove proteins. Dialyze the supernatant after protein removal with a dialysis bag (8 - 14KDa), and after freeze-drying, obtain Pleurotus ostreatus spent mushroom substrate polysaccharide PORP-H.

[0060] 2.2 Acid extraction: Mix the defatted Pleurotus ostreatus spent mushroom substrate powder with hydrochloric acid solution at pH 3.0 at a ratio of 1:50 (g / mL), and continuously stir it at 50 °C for 2 hours. Concentrate the extract with a rotary evaporator, precipitate it with 75% ethanol at 4 °C overnight, and centrifuge it at 8000 r / min for 10 min. After resuspending the precipitate with deionized water, remove proteins by the Sevag method (the same as above). Dialyze the supernatant after protein removal with a dialysis bag (8 - 14KDa), and after freeze-drying, obtain Pleurotus ostreatus spent mushroom substrate polysaccharide PORP-A.

[0061] 2.3 Ultrasonic-assisted extraction method: Add the defatted Pleurotus ostreatus spent mushroom substrate powder to deionized water at a ratio of 1:50 (g / mL), and perform ultrasonic-assisted extraction with 200 W. Extract it at 50 °C for 2 hours. Concentrate the extract with a rotary evaporator, precipitate it with 75% ethanol at 4 °C overnight, and centrifuge it at 8000 r / min for 10 min. After resuspending the precipitate with deionized water, remove proteins by the Sevag method (the same as above). Dialyze the supernatant after protein removal with a dialysis bag (8 - 14KDa), and after freeze-drying, obtain Pleurotus ostreatus spent mushroom substrate polysaccharide PORP-U.

[0062] 2.4 Alkali extraction: Mix the defatted Pleurotus ostreatus spent mushroom substrate powder with sodium hydroxide solution at pH 10.0 at a ratio of 1:50 (g / mL), and stir and extract it at 50 °C for 2 hours. Concentrate the extract with a rotary evaporator, precipitate it with 75% ethanol at 4 °C overnight, and centrifuge it at 8000 r / min for 10 min. After resuspending the precipitate with deionized water, remove proteins by the Sevag method (the same as above). Dialyze the supernatant after protein removal with a dialysis bag (8 - 14KDa), and after freeze-drying, obtain Pleurotus ostreatus spent mushroom substrate polysaccharide PORP-B.

[0063] 2.5 Hot alkali extraction: The defatted Pleurotus ostreatus spent mushroom substrate powder was mixed with distilled water at a ratio of 1:50 (g / mL). The pH of the mixture was adjusted to 8.0 with sodium hydroxide solution, and then continuously stirred and extracted at 90 °C for 2 h. The extract was concentrated by a rotary evaporator, precipitated with 75% ethanol at 4 °C overnight, and centrifuged at 8000 r / min for 10 min. After the precipitate was resuspended with deionized water, protein was removed by the Sevag method (the same as above). The supernatant after protein removal was dialyzed with a dialysis bag (8 - 14KDa), and after freeze-drying, Pleurotus ostreatus spent mushroom substrate polysaccharide PORP-HB was obtained.

[0064] 2.6 Acid-base sequential extraction: The defatted Pleurotus ostreatus spent mushroom substrate powder was soaked in hydrochloric acid solution with pH 3.0 (ratio 1:30 g / mL) at 28 °C for 40 min. After protein removal by the Sevag method (the same as above), dialysis (8 - 14KDa), and freeze-drying, it was soaked in sodium hydroxide solution with pH 8.0 (ratio 1:30 g / mL) and stirred at 32 °C for 10 min. The extract was concentrated by a rotary evaporator, precipitated with 75% ethanol at 4 °C overnight, and centrifuged at 8000 r / min for 10 min. After the precipitate was resuspended with deionized water, protein was removed by the Sevag method (the same as above). The supernatant after protein removal was dialyzed with a dialysis bag (8 - 14KDa), and after freeze-drying, Pleurotus ostreatus spent mushroom substrate polysaccharide PORP-AB was obtained.

[0065] Example 2 Determination of the Yield, Physicochemical Properties and Monosaccharide Composition of Pleurotus ostreatus Spent Mushroom Substrate Polysaccharides (PORPs)

[0066] The contents of polysaccharide, protein, uronic acid and total phenol in the six PORPs extracted in Example 1 were determined by the phenol-sulfuric acid method, Coomassie brilliant blue method, m-hydroxydiphenyl sulfate method and Folin-Ciocalteu method, respectively. Monosaccharide composition analysis was performed using a Thermo ICS5000+ ion chromatography system with a Dionex TM CarboPac TM PA20 column (150×3.0 mm, 10 μm). The homogeneity and molecular weight of the polysaccharide were determined by high performance gel permeation chromatography (HPGPC-ELSD) and a TSK-gel G-3000PWXL column (7.8×300 mm). The measurement results are shown in Table 1, Figure 2 and Figure 3 as shown.

[0067] Table 1 Yield, Chemical Composition and Monosaccharide Composition of Pleurotus ostreatus Spent Mushroom Substrate Polysaccharides

[0068]

[0069] Note: Man: Mannose, GlcA: Glucuronic acid, Rha: Rhamnose, GalA: Galacturonic acid, Glc: Glucose, Gal: Galactose, Xyl: Xylose, Ara: Arabinose, Fuc: Fucose. Different letters indicate significant differences between different treatments (P<0.05).

[0070] As can be seen from Table 1 Figure 2 and Figure 3 , the polysaccharide yields of the hot water extraction method and the hot alkali extraction method are relatively high. The molecular weights of PORP-H, PORP-A, PORP-U, PORP-B, PORP-HB, and PORP-AB are 581.00 kDa, 530.19 kDa, 432.05 kDa, 503.25 kDa, 590.10 kDa, and 449.16 kDa, respectively. Among them, PORP-H is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 10.7:3.3:3.0:1.6:6.9:12.8:30.1:28.7:2.9; PORP-A is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 11.4:3.8:3.2:1.8:7.7:14.4:29.1:25.7:3.0; PORP-U is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 12.2:4.8:3.7:2.5:8.0:13.6:27.6:25.5:2.0; PORP-B is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 12.3:4.2:3.1:2.1:7.8:14.8:27.1:26.1:2.6; PORP-HB is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 13.7:4.3:3.7:2.1:7.6:13.1:25.6:27.9:1.9; PORP-AB is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 14.5:4.8:4.6:1.1:7.1:13.6:25.0:26.9:2.4.

[0071] Example 3 Determination of the Microscopic Morphology and Structural Characteristics of Pleurotus ostreatus Mushroom Residue Polysaccharides (PORPs)

[0072] In this example, the microscopic morphology and structural characteristics of six PORPs were determined.

[0073] 1. The dry PORPs powder was uniformly mixed with spectroscopic grade potassium bromide powder and then pressed into tablets. FT-IR detection was carried out on a Nexus 470 FT-infrared spectrometer (Thermo Nicolet, USA). The results are as Figure 4 shown. It can be seen that there are no significant differences in the FT-IR of PORPs, indicating that the structures of PORPs extracted by the 6 extraction methods are the same. The strong absorption band of this polysaccharide at 3410.25 cm -1 is O-H stretching, and the weak absorption band at about 2924.57 cm -1 is C-H stretching. The characteristic absorption peak at 1628.21 cm -1 is related to the carboxyl-related products of the polysaccharide, which is related to the vibration of the presence of GalA. This result is consistent with the results of monosaccharide composition analysis, proving that PORPs involve GalA. The characteristic absorption peak at 1415.51 cm -1 is due to the symmetric stretching of C-O, and the weaker absorption peak at 1242.05 cm -1 belongs to the bending of the oxygen bridge (O-O), indicating the presence of oxalic acid. PORPs all show multiple inconsistent small absorption peaks, and the characteristic peak is mainly the peak at 1041.34 cm -1 , which is due to the presence of polysaccharide pyranose ring-related compounds, usually showing asymmetric stretching vibration of C-O-C. In addition, there are weak absorption bands at 892.66 cm -1 and 867.88 cm -1 , indicating the simultaneous presence of α and β configurations in the polysaccharide. Therefore, PORPs are all pyran polysaccharides with α and β configurations.

[0074] 2. The PORPs powder was evenly sprayed with gold powder and analyzed using a high-resolution field emission scanning electron microscope system (Hitachi, Regulus 8230, Japan) to obtain the surface morphology at a voltage of 5.0 kV, and the image magnification was 1000, 5000, and 10000 times respectively. The results are as Figure 5As shown, it can be seen that PORP-H exhibits a network-like porous structure in the low-power lens, while in the high-power lens, there are a small number of spherical protrusions and network connections. This is mainly because the polysaccharide by the HWE method can largely maintain the original structure of the polysaccharide, thus enabling better polymerization performance of the polysaccharide chains and maintaining the integrity of the long-chain polysaccharide molecules. PORP-U shows a dense sponge-like structure under the low-power lens, while obvious spherical particles and some irregular cavities can be seen under the high-power lens, which may also be caused by the cavitation of polysaccharides induced by ultrasonic waves. PORP-B has a loose sponge-like honeycomb network structure with irregular surface pores and a diameter of nearly 20 μm. According to previous results, this is caused by the corrosion of sodium hydroxide. PORP-HB has a dense network structure, which may be caused by high temperature and alkaline conditions. Under the high-power microscope, the irregular spherical protrusions are connected together, indicating that PORP-HB contains rich side chains. PORP-A consists of continuous and dense spherical structures, similar to the microstructure of polysaccharides extracted by the acid method. PORP-AB has an overall sheet-like porous structure, which may be due to the polysaccharide being first degraded by hydrochloric acid and then corroded by sodium hydroxide during the extraction process. Different extraction methods will damage the original connections of polysaccharides to varying degrees, resulting in changes in their surface morphology. These results confirm that different extraction methods will affect the hydrolysis degree of polysaccharide chains, thereby affecting the microstructure of porp.

[0075] 3. Mix 200 μL of Congo red solution (200 μg / mL) and 200 μL of PORPs solution (2 mg / mL) with sodium hydroxide solution until the concentration of sodium hydroxide in the mixture is 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L, respectively. Then, use a microplate reader (TECAN-Spark, Switzerland) to scan the above mixtures in the range of 400 - 700 nm to obtain the maximum absorption wavelength (λmax) of Congo red and the Congo red-polysaccharide complex. The results are as Figure 6 shown. It can be seen that the λmax values of the 6 PORPs show a downward trend with the increase of sodium hydroxide concentration, without obvious red shift or spin phenomenon, indicating that PORPs do not have a triple helix structure.

[0076] 4. Mix 2 mL of PORPs (1 mg / mL) with 1.2 mL of potassium iodide reagent (0.2% KI and 0.02% I 2 , w / v) at room temperature for 10 min. The spectrum is detected by a microplate reader (TECAN-Spark, Switzerland) in the range of 300 - 700 nm. The results are as Figure 7As shown, it can be seen that all PORPs have no absorption peak at 565 nm, indicating that PORPs have a tight molecular chain structure and are abundant in the long side chains. On the other hand, PORP-U, PORP-H, PORP-AB, and PORP-A have large absorption peaks at 350 nm. PORP-B and PORP-HB have no absorption peak at 350 nm, indicating that the PORPs obtained by ultrasonic-assisted extraction, hot water extraction, sequential acid-base extraction, and acid method have more complete long branched chains than those obtained by alkali method and hot alkali method. In addition, the reaction of PORPs with I2-KI did not show the characteristic color reaction of starch, indicating that PORPs are non-starch polysaccharides.

[0077] 5. Prepare a PORPs solution (1.0 mg / mL) and detect the particle size and zeta potential value on a nanoparticle size and zeta potential analyzer (NANO ZS90, Malvern, UK). The results are as Figure 8 and Figure 9 shown. It can be seen that PORP-HB has the smallest average particle size (93.205 ± 3.44 nm), while PORP-AB has the largest average particle size (476.45 ± 2.90 nm). The zeta potential of PORPs is distributed in the range of -15.73 to -5.22 mV, which may be due to the negative charge characteristics of polysaccharides mainly due to their abundant uronic acid. The absolute value of the zeta potential of PORP-HB is the highest, reaching 15.73 ± 0.67 mV. The absolute values of PORP-A and PORP-U are the closest (14.1 ± 0.70 mV and 13.73 ± 0.64 mV, respectively), while the absolute value of PORP-B is the lowest, at 5.22 ± 0.92 mV. Therefore, PORP-HB has weak colloidal flocculation ability and strong stability, showing good application potential for the development and utilization of gelling agents and stabilizer materials.

[0078] 6. Determine the thermal properties of PORPs by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) on a synchronous thermal analyzer TGA / DSC1 / 1600LF (Switzerland). The results are as Figure 10 and Figure 11As shown, it can be seen that the TGA curves of the six polysaccharides are similar and can be divided into three stages. In the first stage, the weights of the six polysaccharides decreased to varying degrees at low temperatures (<100 °C), mainly due to the evaporation of free water and bound water. In the second stage (200 - 450 °C), the polysaccharides underwent thermal decomposition, with the highest weight loss rate. The PORPs were 46.186% (PORP-H), 48.189% (PORP-A), 46.485% (PORP-U), 49.054% (PORP-B), 48.373% (PORP-HB), and 48.306% (PORP-AB), respectively. When the pyrolysis temperature rose to 500 - 800 °C, the weight of the polysaccharides gradually decreased due to the carbonization of the polysaccharides. In the third stage, the weight of the polysaccharides hardly changed. Among them, the maximum mass loss rate of PORP-U was the lowest (46.485%), indicating that the mass damage of PORP-U was relatively stable. The final weight order was PORP-U (23.949%) > PORP-AB (20.553%) > PORP-H (18.345%) > PORP-B (17.203%) > PORP-A (17.077%) > PORP-HB (13.496%), indicating that PORP-U had the highest thermal stability and PORP-HB had the lowest thermal stability.

[0079] The DSC curves of the PORPs were similar, with two absorption peaks. The first absorption peak shifted between 30 - 100 °C, which might be related to the loss of bound water. The second absorption peak transitioned from 500 °C to 800 °C, indicating that the PORPs were degraded, which was consistent with the experimental results of TGA. Therefore, PORP-U had the strongest thermophysical stability and could be applied to certain materials.

[0080] Example 4 Determination of the Functional Properties of Pleurotus ostreatus Mushroom Substrate Polysaccharides

[0081] In this example, the functional properties of the six PORPs were determined, and the functional properties included oil and water holding activities, foaming properties, and emulsifying properties:

[0082] 1. Oil and water holding activities of polysaccharides: 20 mg of PORPs were mixed with deionized water (1 mL) or soybean oil (1 mL). After centrifugation at 8500×g for 15 min, the supernatant was removed and equilibrated for 30 min, and the mass of the centrifuge tube and the remaining PORP precipitate was measured. The water holding capacity (WHC) and oil holding capacity (OHC) of the PORPs were calculated according to the following formulas.

[0083]

[0084] In the formula, W 0 / O 0(g) is the mass of PORPs; W 1 / O 1 (g) is the mass of the plastic centrifuge tube; W 2 / O 2 (g) is the mass of the plastic centrifuge tube and the PORPs precipitate.

[0085] The results are as Figure 12 shown. It can be seen that PORP-A has the best water-holding capacity, PORP-U has the best oil-holding capacity, and PORP-HB has poor water-holding and oil-holding capacities.

[0086] 2. Determination of foaming properties

[0087] The PORPs solution (10 mg / mL) was homogenized with an F6 / 10 handheld homogenizer (Jingxin Co., China) at 35000 rpm for 60 s, then left to stand for 30 min, and the initial volume of the PORPs solution, and the foam volumes at 0 min and 30 min after homogenization were measured. The foaming capacity (FC) and foam stability (FS) of PORPs were calculated according to the following formula.

[0088]

[0089] Wherein, V (mL), V 0 (mL) and V 30 (mL) are the initial volume of the PORPs solution, and the foam volumes at 0 min and 30 min after homogenization, respectively.

[0090] The results are as Figure 13 shown. It can be seen that both the foaming capacity and foam stability of PORP-H are good; the foaming capacity of PORP-AB is poor, and the foam stability of PORP-A is poor.

[0091] 3. Emulsification properties

[0092] 0.75 mL of the PORPs solution (10 mg / mL) was mixed with 0.25 mL of soybean oil and homogenized with an F6 / 10 handheld homogenizer at 35000 rpm for 60 s. 2 μL of the emulsion was taken from the bottom at 0 min and 10 min after homogenization, diluted 100-fold with 0.1% SDS, and the absorbance at 500 nm was measured using a microplate reader. The emulsification activity index (EAI) and emulsifying stability index (ESI) of the emulsion were calculated using the following formula.

[0093]

[0094] Wherein, A 0 and A 10 are the absorbances of the emulsion at 0 and 10 min, DF is the dilution factor (100), C is the polysaccharide concentration (g / mL), is the oil volume fraction (0.25).

[0095] The results are as Figure 14 shown. It can be seen that the emulsifying activity index of PORP-U is the highest, but the emulsion stability index is the lowest; the emulsifying activity index of PORP-H is the lowest, but the emulsion stability index is the highest.

[0096] Example 5 Determination of the antioxidant properties of Pleurotus ostreatus spent mushroom substrate polysaccharides (PORPs)

[0097] In this example, the antioxidant activities of six PORPs were evaluated by measuring their ABTS radical scavenging ability, DPPH radical scavenging ability and hydroxyl radical scavenging ability.

[0098] 1. DPPH radical scavenging activity

[0099] Take 200 μL of PORPs solutions with different concentrations (0.5, 1, 2, 3, 4 mg / mL), add 100 μL of DPPH solution (prepared with absolute ethanol, 0.04 mg / mL), place in the dark for 10 min, measure the OD value 3 times at 517 nm, and take the average value, denoted as A1; in addition, repeat the above operation with absolute ethanol instead of the DPPH solution, measure the absorbance value, denoted as A2; use distilled water instead of the PORPs solution, measure the absorbance value, denoted as A3; use distilled water instead of the PORPs solution and absolute ethanol instead of the DPPH solution, measure the absorbance value, denoted as A4. Finally, calculate the DPPH radical scavenging ability according to the following formula:

[0100] DPPH radical scavenging rate (%) = [1 - (A1 - A2) / (A3 - A4)] × 100% (7)

[0101] Using VC as the positive control group, the results are as Figure 15 shown.

[0102] 2. ABTS radical scavenging ability

[0103] Dissolve 0.3841 g of ABTS and 0.0662 g of potassium persulfate in water and make up to 100 mL to prepare the ABTS stock solution, and store it in the dark at room temperature overnight; dilute the stock solution to OD 734nmThe value is approximately 0.75 ± 0.02, which is used as the working solution. Add 200 μL of ABTS working solution to 50 μL of PORPs samples with different concentrations (0.5, 1, 2, 3, 4 mg / mL), react in the dark for 10 min, measure the OD value 3 times at 734 nm, and take the average value, denoted as A1; in addition, repeat the above operation with deionized water instead of ABTS to measure the absorbance value of PORPs itself, denoted as A2; use water instead of PORPs to measure the absorbance value of ABTS, denoted as A3, and then measure the absorbance value of water, denoted as A4. Finally, calculate the ABTS radical scavenging ability according to the following formula:

[0104] ABTS radical scavenging rate (%) = [1 - (A1 - A2) / (A3 - A4)] × 100% (8)

[0105] Use VC as the positive control group, and the results are as Figure 16 shown.

[0106] 3. Hydroxyl radical scavenging activity: Prepare PORPs solutions with concentrations of 0.5, 1, 2, 3, and 4 mg / mL with distilled water, and add 50 μL of 9 mmol / L FeSO 4 and 50 μL of 9 mmol / L salicylic acid-ethanol solution respectively. Finally, add 50 μL of 1.2 mmol / L H 2 O 2 to initiate the reaction, react at 37 °C (oven) for 30 min, zero with deionized water, measure the OD value 3 times at a wavelength of 510 nm, and take the average value, denoted as A1; in addition, repeat the above operation with deionized water instead of H 2 O 2 to measure the absorbance value of PORPs itself, denoted as A2; at the same time, use water instead of the PORPs solution to measure the absorbance value, denoted as A0. Finally, calculate the hydroxyl radical scavenging rate according to the following formula:

[0107] Hydroxyl radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100% (9)

[0108] Use VC as the positive control group, and the results are as Figure 17 shown.

[0109] 4. Statistically analyze the IC 50 values of the six PORPs for ABTS radical, DPPH radical, and hydroxyl radical scavenging abilities, as shown in Table 2.

[0110] Table 2 IC 50 values of the in vitro antioxidant ability of polysaccharides from Pleurotus ostreatus spent mushroom substrate

[0111]

[0112] Note: Different letters indicate significant differences between different treatments (P<0.05).

[0113] From Table 2, Figures 15 - 17 it can be seen that among the six Pleurotus ostreatus spent mushroom substrate polysaccharides, PORP-B has the strongest scavenging ability for DPPH free radicals; PORP-HB has the strongest scavenging ability for ABTS free radicals; and PORP-A has the strongest scavenging ability for hydroxyl free radicals.

[0114] Example 6 Determination of the prebiotic activity of Pleurotus ostreatus spent mushroom substrate polysaccharides (PORPs)

[0115] In this example, the prebiotic activities of six PORPs were determined, and the prebiotic activities of PORPs were evaluated by measuring the promoting effect of PORPs on the growth of probiotics. The probiotics selected were Lactobacillus rhamnosus LR1 (L. rhamnosus) and Lactobacillus plantarum LP1 (L. plantarum), and these two strains of bacteria have been disclosed in the literature (Li H, Liu S, Liu Y, et al. Effects of invitro digestion and fermentation of Nostoc commune Vauch. polysaccharides onproperties and gut microbiota[J]. Carbohydrate Polymers, 2022, 281:119055-. DOI:10.1016 / j.carbpol.2021.119055.).

[0116] The probiotics were cultured in the laboratory until OD 600 = 0.8 to obtain the probiotic solution. MRS containing 0.75% (w / v) PORPs was prepared as the experimental group (PORPs), and MRS containing 0.5% glucose was used as the blank control (CK), and MRS containing 0.75% (w / v) fructooligosaccharide (FOS) was used as the positive control (FOS). The probiotic solution was inoculated into different MRSs at a ratio of 2% (v / v) and cultured, and the OD 600 values of the cultures were measured at 0, 6, 12, 24, and 36 h, respectively, and the growth rate of the probiotic concentration was calculated compared with the initial probiotic concentration (0 h).

[0117] The results are as Figure 18As shown in the figure, where A - D are the growth rates of probiotic concentrations in the cultures at 6, 12, 24, and 36 hours respectively. It can be seen that as the culture time extends, the growth rate of the probiotic concentration in the blank control group stabilizes at about 10%, while the growth rates of the probiotic concentrations in the positive control group and the PORPs groups continue to increase. After 6 hours, the growth rates basically stabilize, indicating that the six PORPs have the effect of promoting the growth of probiotics. Among the six PORPs, the order of the promoting effect on the growth of probiotics from strong to weak is PORP - A, PORP - H, PORP - B, PORP - HB, PORP - AB, and PORP - U.

[0118] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An application of Pleurotus ostreatus husk polysaccharide in the preparation of antioxidants, fermented food additives or prebiotic foods, characterized in that: The oyster mushroom waste polysaccharide includes PORP-H, PORP-A, PORP-U, PORP-B, PORP-HB and PORP-AB; The molecular weights of PORP-H, PORP-A, PORP-U, PORP-B, PORP-HB, and PORP-AB are 581.00 kDa, 530.19 kDa, 432.05 kDa, 503.25 kDa, 590.10 kDa, and 449.16 kDa, respectively; The PORP-H is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 10.7:3.3:3.0:1.6:6.9:12.8:30.1:28.7:2.9; the PORP-A is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose. The molar ratio is 11.4:3.8:3.2:1.8:7.7:14.4:29.1:25.7:3.0; the PORP-U is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 12.2:4.8:3.7:2.5:8.0:13.6:27.6:25.5:2.0; The PORP-B is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 12.3:4.2:3.1:2.1:7.8:14.8:27.1:26.1:2.6; the PORP-HB is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose. The composition is in a molar ratio of 13.7:4.3:3.7:2.1:7.6:13.1:25.6:27.9:1.9; the PORP-AB is composed of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a molar ratio of 14.5:4.8:4.6:1.1:7.1:13.6:25.0:26.9:2.

4.

2. The method for preparing the oyster mushroom husk polysaccharide according to claim 1, characterized in that: The steps include: (1) defatting the oyster mushroom chaff powder; (2) extracting the defatted Pleurotus ostreatus bran powder with hot water, and obtaining Pleurotus ostreatus bran polysaccharide PORP-H after purification; Extracting the defatted Pleurotus ostreatus bran powder with a hydrochloric acid solution, and obtaining Pleurotus ostreatus bran polysaccharide PORP-A after purification; The defatted Pleurotus ostreatus bran powder is mixed with water and subjected to ultrasonic treatment to obtain Pleurotus ostreatus bran polysaccharide PORP-U after purification; Extracting the defatted Pleurotus ostreatus husk powder with an alkali solution, and obtaining Pleurotus ostreatus husk polysaccharide PORP-B after purification; Extracting the defatted Pleurotus ostreatus husk powder with hot alkali solution, and obtaining Pleurotus ostreatus husk polysaccharide PORP-HB after purification; The defatted Pleurotus ostreatus bran powder is extracted with a hydrochloric acid solution and an alkaline solution in sequence, and after purification, the Pleurotus ostreatus bran polysaccharide PORP-AB is obtained.

3. The preparation method according to claim 2, characterized in that: The step of extracting with hot water is as follows: mixing the defatted Pleurotus ostreatus bran powder with water in a ratio of 1 g:40-60 mL, and soaking and extracting at 85-90° C. for 2-3 hours to obtain an extract.

4. The preparation method according to claim 2, characterized in that: The step of extracting with hydrochloric acid solution is: mixing the defatted Pleurotus ostreatus bran powder with a hydrochloric acid solution with a pH of 3.0 in a ratio of 1g:40-60mL, and stirring and extracting at 45-55°C for 2-3h to obtain an extract.

5. The preparation method according to claim 2, characterized in that: The ultrasonic treatment step comprises: subjecting the defatted oyster mushroom bran powder and water to ultrasonic extraction for 2-3 hours at 150-250W and 45-55°C to obtain an extract; The mixing ratio of the defatted Pleurotus ostreatus bran powder and water is 1g:40-60mL.

6. The preparation method according to claim 2, characterized in that: The step of extracting with alkali solution is: mixing the defatted Pleurotus ostreatus bran powder with alkali solution with a pH value of 10.0 at a ratio of 1 g:40-60 mL, and stirring and extracting at 45-55° C. for 2-3 hours to obtain an extract.

7. The preparation method according to claim 2, characterized in that: The step of extracting with hot alkali solution is: mixing the defatted Pleurotus ostreatus bran powder with water in a ratio of 1g:40-60mL, adjusting the pH to 8.0, and stirring and extracting at 85-90°C for 2-3h to obtain an extract.

8. The preparation method according to claim 2, characterized in that: The steps of sequentially extracting with hydrochloric acid solution and alkali solution are as follows: mixing the defatted oyster mushroom bran powder with a hydrochloric acid solution with a pH value of 3.0 at a ratio of 1 g: 20-35 mL, and then soaking and extracting for 30-45 min at 25-30° C.; removing protein with Sevag, dialysis treatment and freeze-drying to obtain freeze-dried powder; mixing the freeze-dried powder with an alkali solution with a pH value of 8.0 at a ratio of 1 g: 20-35 mL, and then stirring and extracting for 8-12 min at 30-35° C. to obtain an extract.

9. The preparation method according to any one of claims 2 to 8, characterized in that: The purification steps are: subjecting the extract to ethanol precipitation, Sevag method protein removal and dialysis treatment in sequence; The ethanol precipitation is carried out by using 75% ethanol aqueous solution at 4°C for 12-16 hours; The molecular weight cut-off of the dialysis bag during the dialysis treatment is 8-14KDa.

10. Pleurotus ostreatus bran polysaccharide prepared by the preparation method according to any one of claims 2 to 9.

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