Method for extracting active polysaccharides from Chinese wolfberry residues through compound microorganism fermentation

Abstract

The invention discloses a method for extracting active polysaccharides from Chinese wolfberry residues through compound microorganism fermentation. Relates to the technical field of microbial fermentation. Extraction methods and uses are provided. The method for extracting the lycium barbarum polysaccharide through the water extraction and alcohol precipitation method combined with the compound microorganism fermentation pretreatment has the advantages of being high in extraction efficiency, easy to operate and low in cost. The high-purity lycium barbarum polysaccharide can be obtained through subsequent deproteinization and refining purification steps. Meanwhile, the technological processes of multiple links are optimized and integrated, the use of solvents and the energy consumption are greatly reduced, and the production concept of green and environment-friendly is met. The biological activity of the polysaccharide subjected to biological fermentation is effectively improved, and the polysaccharide has relatively high medicinal value and market application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and more particularly to a method for extracting active polysaccharides from wolfberry residues by composite microbial fermentation. Background Art

[0002] Lycium barbarum L., as a traditional Chinese medicine and health food, has a history of thousands of years of consumption and medicinal use in China. Lycium barbarum contains rich bioactive ingredients, including Lycium barbarum polysaccharides (LBP), zeaxanthin, β-carotene, etc. Among them, Lycium barbarum polysaccharides have attracted much attention due to their antioxidant, immunomodulatory, anti-tumor and other biological activities. Modern medical research has also confirmed that Lycium barbarum polysaccharides have significant anti-fatigue effects, can improve physical strength and endurance, enhance the kidney's detoxification ability, and its antioxidant properties can also improve skin condition and delay the aging process.

[0003] China's wolfberry industry is huge in scale. However, the development of the wolfberry industry also faces problems such as low processing conversion rate and low by-product utilization rate. These problems limit the sustainable development of the industry.

[0004] As the main by-product of wolfberry processing, wolfberry residue is rich in polysaccharides, proteins and other biologically active substances and has high utilization value.

[0005] At present, there are limited ways to utilize wolfberry residue, mainly including the extraction of pigments, dietary fiber, and wolfberry seed oil. However, these utilization methods have not fully developed the potential of wolfberry residue, and traditional extraction methods are inefficient and may destroy the structure and function of active ingredients. Biofermentation is an emerging technology for extracting wolfberry polysaccharides from wolfberry residue. It uses specific microorganisms to decompose the complex components in wolfberry residue and improve the extraction efficiency and quality of polysaccharides. This method can not only improve the utilization efficiency of resources and reduce environmental pollution, but also increase the added value of products, which is of great significance to improving the economic benefits and environmental sustainability of the wolfberry industry.

[0006] Therefore, whether a method for extracting active polysaccharides from wolfberry residue by composite microbial fermentation can be provided is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a method for extracting active polysaccharides from wolfberry residue by composite microbial fermentation, aiming to explore a method for using biological fermentation technology to assist in extracting active polysaccharides from wolfberry residue, in order to solve the problems existing in the utilization of wolfberry by-products and promote the high-quality development of the wolfberry industry.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for extracting active polysaccharides from wolfberry residue by composite microbial fermentation, comprising the following steps:

[0010] (1) strain activation and mixed culture: Aspergillus niger, Trichoderma reesei, and Bacillus cereus were activated separately, and then mixed in a volume ratio of 1:1:1 to culture a composite microorganism;

[0011] (2) Biological fermentation pretreatment:

[0012] 21) Preparation of wolfberry residue: Collect the wolfberry residue after processing, dry it, crush it, add sterile water and mix it evenly to obtain a wolfberry residue aqueous solution;

[0013] 22) Inoculation and fermentation: inoculating the composite microorganism into the wolfberry residue aqueous solution for fermentation and culture;

[0014] 23) Fermentation end treatment: After fermentation, heating and ultrasonic treatment are performed; and the fermentation supernatant is collected;

[0015] (3) Deproteinization:

[0016] The pH value of the collected fermentation supernatant was adjusted, Sevag reagent was added, stirred and mixed, allowed to stand, centrifuged at 6000 rpm to remove the precipitate, and the fermentation supernatant was collected;

[0017] (4) Macroporous resin decolorization:

[0018] The pH value of the fermentation supernatant is adjusted, the supernatant is decolorized by passing through a resin column, deionized water is used for preliminary elution, and a 50% ethanol solution is used to elute the polysaccharide to obtain an eluate;

[0019] (5) Precipitation and drying of polysaccharides:

[0020] 95% anhydrous ethanol is added to the eluent, and the mixture is allowed to stand for precipitation, and then centrifuged. The precipitation is collected and the ethanol is recovered, and the precipitation is freeze-dried to obtain active polysaccharides.

[0021] Preferably: Step (1) Cultivation: Cultivation is carried out at 30° C., 200 rpm in a mixed culture medium containing 1% pectin, 1% cellulose, 1% soy peptone, 0.5% glucose, 0.5% yeast extract, 0.1% K2HPO4, and 0.05% MgSO4.

[0022] Preferably, step 21) is specifically as follows: collecting the wolfberry residue after processing, drying at 50° C. to a moisture content of less than 10%, crushing to a particle size of less than 1 mm, adding 20 times the mass of sterile water to the crushed wolfberries and mixing evenly to obtain a wolfberry residue aqueous solution.

[0023] Preferably, step 22) is specifically as follows: inoculating 1% of the composite microorganism into the wolfberry residue aqueous solution, and controlling the fermentation conditions to be 37° C. and 200 rpm.

[0024] Preferably, step 23) is specifically as follows: after step 22) fermentation reaches 48 hours, heating to 100° C. and ultrasonically treating at 200W / 40Hz for 20 minutes; collecting the fermentation supernatant.

[0025] Preferably, step (3) is specifically as follows: adding glacial acetic acid to the collected fermentation supernatant to adjust the pH value to 4-5, adding Sevag reagent according to 1 / 20-1 / 10 of the volume of the fermentation liquid, stirring and mixing, standing for 4-8 hours, centrifuging at 6000 rpm for 30 minutes to remove the precipitate, and collecting the fermentation liquid supernatant.

[0026] Preferably, step (4) is specifically as follows: adjusting the pH value of the fermentation supernatant to 4.5, and decolorizing it through a D-101 resin column at 15 to 40° C., controlling the loading flow rate at 1 to 2 times the column volume / h, using 2 times the column volume of deionized water for preliminary elution, and then using 5 to 10 times the column volume of 50% ethanol solution to elute the polysaccharide to obtain an eluate.

[0027] Preferably, step (5) is specifically as follows: an equal volume of 95% anhydrous ethanol is added to the eluent, and the mixture is allowed to settle at 4°C for 12 hours, and then centrifuged at 6000 rpm for 30 minutes, the precipitate is collected and the ethanol is recovered, and the precipitate is freeze-dried at -20°C to -50°C and 10 mBar to 1 mBar vacuum for 24 hours to obtain active polysaccharides.

[0028] The present invention also provides application of any of the above methods in fermentation production.

[0029] The present invention also provides the use of active polysaccharides prepared by any of the above methods in the preparation of anti-oxidation, immunomodulatory and anti-tumor drugs.

[0030] It can be known from the above technical scheme that compared with the prior art, the present invention discloses a method for extracting active polysaccharides from wolfberry residues by composite microbial fermentation. The technical effect achieved is the method for extracting wolfberry polysaccharides by water extraction and alcohol precipitation combined with composite microbial fermentation pretreatment provided by the present invention, which has the advantages of high extraction efficiency, simple operation and low cost. High-purity wolfberry polysaccharides can be obtained through subsequent deproteinization and refining and purification steps. At the same time, the process flow of multiple links is optimized and integrated. The present invention greatly reduces the use of solvents and energy consumption, which is in line with the green and environmentally friendly production concept. The polysaccharides after biological fermentation have effectively improved biological activity and have high medicinal value and market application prospects. DETAILED DESCRIPTION

[0031] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The embodiment of the present invention discloses a method for extracting active polysaccharides from wolfberry residue by composite microbial fermentation

[0033] The raw materials and strains in the examples are all commercially available, and their sources and specific strains are not limited, as long as they meet the process requirements. The methods not mentioned are conventional treatments and will not be repeated here, for example:

[0034] Source and activation of composite microorganisms in the embodiments:

[0035] Aspergillus niger AS3.3289: Cultured in a culture medium containing 2% pectin, 0.5% yeast extract, 0.1% KH2PO4, and 0.05% MgSO4 at 30°C and 200 rpm for 48 h.

[0036] Trichoderma reesei ATCC24449: Cultured in a medium containing 1% cellulose, 0.5% yeast extract, 0.1% K2HPO4, and 0.05% MgSO4 at 30°C and 200 rpm for 48 h.

[0037] Bacillus cereus ACCC11077: Cultured in a medium containing 1% soy peptone, 0.5% glucose, 0.1% K2HPO4, and 0.05% MgSO4 at 30°C and 200 rpm for 24 h.

[0038] The content determination method of polysaccharide and protein refers to the determination method of wolfberry polysaccharide and protein in the national standard wolfberry (GB / T 18672-2014). The calculation method of polysaccharide purity is the ratio of the measured polysaccharide mass m1 to the crude polysaccharide mass m0, that is, polysaccharide purity (%) = m1 / m0×100%.

[0039] The yield of wolfberry polysaccharides was calculated as the ratio of the measured polysaccharide mass m1 obtained by extraction to the measured polysaccharide mass m in the wolfberry residue: yield (%) = m1 / m×100%.

[0040] The colorimetric detection of polysaccharides was measured by spectrophotometry, and the absorbance value A450 at 450 nm was used as the measurement.

[0041] The DPPH free radical scavenging rate and ABTS free radical scavenging rate were used to evaluate the antioxidant activity of Lycium barbarum polysaccharides. The specific detection method referred to the national standard polypeptide antioxidant activity determination DPPH and ABTS method (GB / T39100-2020).

[0042] The activities of cellulase, pectinase and protease were determined according to NY / T 912-2020 Spectrophotometric determination of cellulase activity in feed additives, GB / T 44738-2024 Determination of pectinase activity in feed, and SB / T 10317-1999 Protease activity determination method.

[0043] Example 1

[0044] A method for extracting active polysaccharides from wolfberry residue by composite microbial fermentation, comprising the following steps:

[0045] (1) Strain activation and mixed culture: The three activated strains (Aspergillus niger, Trichoderma reesei, and Bacillus cereus) were mixed in a volume ratio of 1:1:1 and cultured in a mixed culture medium containing 1% pectin, 1% cellulose, 1% soy peptone, 0.5% glucose, 0.5% yeast extract, 0.1% K2HPO4, and 0.05% MgSO4 at 30°C and 200 rpm;

[0046] (2) Biological fermentation pretreatment:

[0047] 21) Preparation of wolfberry residue: Collect the wolfberry residue after processing, dry it at 50°C to a moisture content of less than 10%, crush it to a particle size of less than 1 mm (to increase the contact area of ​​microorganisms), add 20 times the mass of sterile water to the crushed wolfberries and mix them evenly to obtain a wolfberry residue aqueous solution;

[0048] 22) Inoculation and fermentation: inoculate 1% of the mixed cultured composite microorganisms into the above wolfberry residue aqueous solution, and control the fermentation conditions to be 37° C. and 200 rpm;

[0049] 23) Treatment at the end of fermentation: After fermentation for 48 hours, the temperature was raised to 100° C. and ultrasonically treated at 200W / 40Hz for 20 minutes (on the one hand, it can kill the microorganisms and terminate the fermentation, and on the other hand, it can more fully extract the undissolved polysaccharides in the wolfberry residue); the fermentation supernatant was collected;

[0050] (3) Deproteinization:

[0051] The collected fermentation supernatant was added with glacial acetic acid to adjust the pH value to 4-5 (4 in this embodiment), Sevag reagent (a mixture of chloroform and n-butanol, in a ratio of 1:4) was added according to 1 / 20-1 / 10 (1 / 15 in this embodiment) of the volume of the fermentation broth, stirred and mixed, and allowed to stand for 4-8 hours (6 hours in this embodiment) to fully precipitate the protein, centrifuged at 6000 rpm for 30 minutes to remove the precipitate, and the fermentation broth supernatant was collected;

[0052] (4) Macroporous resin decolorization:

[0053] The pH value of the fermentation supernatant is adjusted to 4.5, and the supernatant is decolorized by passing through a D-101 resin column (pretreated and activated, specifically, rinsed with deionized water to a pH value of 6.5 to 8 (7 in this embodiment), and then rinsed and activated with 2 column volumes of 70% ethanol) at 15 to 40° C. (20° C. in this embodiment), and the sample flow rate is controlled at 1 to 2 column volumes / h to ensure sufficient adsorption, and then, 2 column volumes of deionized water are used for preliminary elution to remove small molecular impurities, and then 5 to 10 (8 in this embodiment) column volumes of 50% ethanol solution are used to elute the polysaccharide to effectively recover and obtain an eluate;

[0054] (5) Precipitation and drying of polysaccharides

[0055] An equal volume of 95% anhydrous ethanol was added to the eluate, and the mixture was allowed to stand at 4°C for 12 hours to precipitate. The mixture was then centrifuged at 6000 rpm for 30 minutes. The precipitate was collected and the ethanol was recovered. The precipitate was freeze-dried (-20°C to -50°C, 10mBar to 1mBar vacuum freeze-dried for 24 hours, and in this embodiment, -25°C, 5mBar vacuum freeze-dried for 24 hours) to obtain (wolfberry) active polysaccharides.

[0056] Comparative test

[0057] Compare the effects of microbial fermentation through comparative examples

[0058] Embodiment: The strains are matched with those in embodiment 1;

[0059] Comparative Example 1: No microbial fermentation step;

[0060] Comparative Example 2: Aspergillus niger AS3.3289 only;

[0061] Comparative Example 3: Aspergillus niger AS3.3289 + Trichoderma reesei ATCC 24449;

[0062] Comparative Example 4: Trichoderma reesei ATCC24449+Bacillus cereus ACCC11077;

[0063] Comparative Example 5: Aspergillus niger AS3.3289 + Bacillus cereus ACCC11077;

[0064] Detection of enzyme activity and its effect on the extraction of Lycium barbarum polysaccharides

[0065] The results are shown in Tables 1 and 2

[0066] Table 1

[0067]

[0068] Table 2

[0069]

[0070] The results showed that Aspergillus niger produced a large amount of pectinase and a small amount of protease, Trichoderma reesei produced a large amount of cellulase and a small amount of pectinase and protease, and Bacillus cereus produced a large amount of protease. The combination of the three bacteria can effectively decompose the pectin, cellulose and protein components in the wolfberry residue, effectively improving the extraction rate of polysaccharides in the wolfberry residue. At the same time, under the biotransformation of microorganisms, the DPPH free radical scavenging ability and ABTS free radical scavenging ability of wolfberry polysaccharides have been significantly improved.

[0071] Exploration 23) The effect of process changes in the final fermentation step on polysaccharide extraction

[0072] Embodiment is the same as embodiment 1;

[0073] Comparative Example 6: No heating, no ultrasonic treatment;

[0074] Comparative Example 7 was heated only without ultrasonic treatment;

[0075] The results are shown in Table 3

[0076] Table 3

[0077]

[0078] Comparative Example 6 shows that heating and ultrasonic treatment after the microbial fermentation can significantly improve the extraction rate of polysaccharides. On the one hand, the heating and ultrasonic treatment can further promote the dissolution of polysaccharides in wolfberry residues. On the other hand, timely killing of microorganisms can terminate the fermentation process, thereby avoiding the further decomposition of polysaccharides by microorganisms due to excessive action for a long time. Comparative Example 7 shows that ultrasonic treatment can effectively improve the extraction rate of polysaccharides.

[0079] The examples for investigating the effects of (3) deproteinization treatment and (4) macroporous resin decolorization steps on polysaccharide purity are the same as those in Example 1;

[0080] Comparative Example 8: only without decolorization treatment;

[0081] Comparative Example 9: only without deproteinization treatment;

[0082] The results are shown in Table 4

[0083] Table 4

[0084]

[0085]

[0086] It can be seen from Comparative Example 8 that the deproteinization treatment can effectively remove the protein in the polysaccharide and improve the purity of the polysaccharide. Comparative Example 9 shows that the decolorization treatment effectively reduces the chromaticity of the polysaccharide and removes impurities in the polysaccharide, thereby improving the purity of the polysaccharide.

[0087] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0088] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting active polysaccharides from wolfberry residue by composite microbial fermentation, characterized in that: The following steps are involved: (1) strain activation and mixed culture: Aspergillus niger, Trichoderma reesei, and Bacillus cereus were activated separately, and then mixed in a volume ratio of 1:1:1 to culture a composite microorganism; (2) Biological fermentation pretreatment: 21) Preparation of wolfberry residue: Collect the wolfberry residue after processing, dry it, crush it, add sterile water and mix it evenly to obtain a wolfberry residue aqueous solution; 22) Inoculation and fermentation: inoculating the composite microorganism into the wolfberry residue aqueous solution for fermentation and culture; 23) Fermentation end treatment: After fermentation, heating and ultrasonic treatment are performed; and the fermentation supernatant is collected; (3) Deproteinization: The pH value of the collected fermentation supernatant was adjusted, Sevag reagent was added, stirred and mixed, allowed to stand, centrifuged at 6000 rpm to remove the precipitate, and the fermentation supernatant was collected; (4) Macroporous resin decolorization: The pH value of the fermentation supernatant is adjusted, the supernatant is decolorized by passing through a resin column, deionized water is used for preliminary elution, and a 50% ethanol solution is used to elute the polysaccharide to obtain an eluate; (5) Precipitation and drying of polysaccharides: 95% anhydrous ethanol is added to the eluent, and the mixture is allowed to stand for precipitation, and then centrifuged. The precipitation is collected and the ethanol is recovered, and the precipitation is freeze-dried to obtain active polysaccharides.

2. The method according to claim 1, characterized in that The culture in step (1) is carried out at 30° C. and 200 rpm in a mixed culture medium containing 1% pectin, 1% cellulose, 1% soy peptone, 0.5% glucose, 0.5% yeast extract, 0.1% K2HPO4, and 0.05% MgSO4.

3. The method according to claim 2, characterized in that Step 21) is specifically as follows: collecting the wolfberry residue after processing, drying at 50° C. to a moisture content of less than 10%, crushing to a particle size of less than 1 mm, adding 20 times the mass of sterile water to the crushed wolfberries and mixing evenly to obtain a wolfberry residue aqueous solution.

4. The method according to claim 3, characterized in that Step 22) is specifically as follows: inoculating 1% of the composite microorganism into the wolfberry residue aqueous solution, and controlling the fermentation conditions to be 37° C. and 200 rpm.

5. The method according to claim 4, characterized in that Step 23) is specifically as follows: after step 22) fermentation reaches 48 hours, heating to 100° C. and ultrasonically treating at 200W / 40Hz for 20 minutes; collecting the fermentation supernatant.

6. The method according to claim 5, characterized in that Step (3) is specifically as follows: adding glacial acetic acid to the collected fermentation supernatant to adjust the pH value to 4-5, adding Sevag reagent according to 1 / 20-1 / 10 of the volume of the fermentation liquid, stirring and mixing, standing for 4-8 hours, centrifuging at 6000 rpm for 30 minutes to remove the precipitate, and collecting the fermentation liquid supernatant.

7. The method according to claim 6, characterized in that Step (4) is specifically as follows: adjusting the pH value of the fermentation supernatant to 4.5, and decolorizing it through a D-101 resin column at 15 to 40° C., controlling the loading flow rate at 1 to 2 column volumes / h, using 2 column volumes of deionized water for preliminary elution, and then using 5 to 10 column volumes of a 50% ethanol solution to elute the polysaccharide to obtain an eluate.

8. The method according to claim 7, characterized in that Step (5) is specifically as follows: add an equal volume of 95% anhydrous ethanol to the eluent, place it in a 4°C environment to precipitate for 12 hours, then centrifuge at 6000 rpm for 30 minutes, collect the precipitate and recover the ethanol, freeze-dry the precipitate at -20°C to -50°C and 10mBar to 1mBar vacuum for 24 hours to obtain active polysaccharides.

9. Use of the method according to any one of claims 1 to 8 in fermentation production.

10. Use of the active polysaccharide prepared by the method according to any one of claims 1 to 8 in the preparation of antioxidant, immunomodulatory and anti-tumor drugs.

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