Fermentation culture medium for producing high-oxidation-resistance paecilomyces hypha micro powder and application of fermentation culture medium
By adding antioxidant precursor mixture of kiwi fruit peels, grape seeds and mushroom extracts to the fermentation medium, combined with deep liquid fermentation technology, the antioxidant activity of Penicillium mycelium powder of bat moth is improved, and the existing technology is solved The problem of insufficient antioxidant properties has achieved efficient enhancement of antioxidant properties and improved functionality, which is suitable for large-scale production.
Patent Information
- Application Number
- CN202411066594.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-05
- Publication Date
- 2024-12-06
AI Technical Summary
The prior art is difficult to effectively improve the antioxidant activity of Penicillium mycelium powder of bat moth, affecting its functionality and quality in health foods.
Antioxidant precursor mixtures containing kiwi fruit peel extract, grape seed extract and mushroom extract are prepared as part of the fermentation medium, and antioxidant precursor mixtures are prepared by water extraction and adsorption and desorption techniques, combined with glucose, peptone, Yeast powder, potassium dihydrogen phosphate and magnesium sulfate heptahydrate are cultured in deep liquid fermentation to improve the antioxidant activity of the mycelium.
It significantly improves the antioxidant activity of Penicillium mycelium powder, enhances its antioxidant ability, and at the same time improves the content of adenosine. It has the effects of enhancing immunity, anti-inflammatory, relieves physical fatigue, relieves cough, expectorant, and anti-asthma. , and suitable for large-scale industrial production.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of fungal fermentation and culture technology, and specifically discloses a fermentation medium for producing high-antioxidant Paecilomyces mycelium powder and an application thereof. Background Art
[0002] Paecilomyces hepiali is an entomogenous fungus isolated from wild Cordyceps sinensis. It was isolated from Cordyceps sinensis samples collected from Baima Snow Mountain in Diqing Prefecture, Yunnan Province by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences in 1989 (the insects were the larvae of the Cordyceps bat moth). It was identified and named "Paecilomyces hepiali" and belongs to the genus Penicillium of the family Combretaceae. The mycelium of Paecilomyces hepiali produced by deep liquid fermentation using the Paecilomyces hepiali strain contains functional ingredients consistent with those of Cordyceps sinensis. Its advantage is that it can artificially control the production of a large number of mycelium. At the same time, as the initial form of Paecilomyces hepiali, the content of various functional ingredients in the mycelium is higher than that in the fruiting body. The mycelium of Paecilomyces hepiali is yellow-brown, fragrant, and slightly bitter. It has been approved by the State Administration for Market Regulation as a fungal strain that can be used in health foods.
[0003] The mycelium of Paecilomyces batioides contains nutrients such as adenosine, polysaccharides, polypeptides, and unsaturated fatty acids. Among them, the adenosine content in the mycelium of Paecilomyces batioides is also one of the main indicators for evaluating the quality level of the mycelium powder of Paecilomyces batioides (the Chinese Pharmacopoeia stipulates that the adenosine content ≥ 0.2% is qualified Cordyceps sinensis powder); the polysaccharide of the mycelium of Paecilomyces batioides also has pharmacological activities such as antioxidant, anti-inflammatory, and immune improvement. Therefore, the large-scale, high-quality industrial fermentation production of Paecilomyces batioides is of great significance for promoting people's health and improving the quality of life. The Chinese patent "A method for increasing the content of adenosine in the fermented mycelium of Paecilomyces batioides" with publication number CN112322504A specifically discloses that inosinic acid is added to the fermentation medium to increase the adenosine content in the mycelium powder of Paecilomyces batioides, and can also increase the yield of the mycelium powder of Paecilomyces batioides. Summary of the invention
[0004] The technical problem to be solved by the present invention is to improve the antioxidant activity of Paecilomyces mycelium powder of Heterotrichum, and further provide a fermentation medium for producing Paecilomyces mycelium powder with high antioxidant activity and application thereof.
[0005] The technical solution adopted by the present invention is as follows:
[0006] The invention provides a fermentation medium for producing high-antioxidant Paecilomyces mycelium powder, which is composed of the following raw materials in parts by weight: 20-60 parts of glucose, 10-40 parts of peptone, 10-40 parts of yeast powder, 1-4 parts of potassium dihydrogen phosphate, 1-3 parts of magnesium sulfate heptahydrate and 1-5 parts of an antioxidant precursor mixture; the pH of the fermentation medium is 5.0-6.5.
[0007] As a preferred embodiment, the fermentation medium for producing highly antioxidant Paecilomyces mycelium powder includes the following raw materials in parts by weight: 30 parts of glucose, 20 parts of peptone, 20 parts of yeast powder, 2 parts of potassium dihydrogen phosphate, 1 part of magnesium sulfate heptahydrate and 2 parts of antioxidant precursor mixture.
[0008] As a preferred embodiment, the antioxidant precursor mixture comprises kiwi fruit peel extract, grape seed extract and mushroom extract; the kiwi fruit peel extract, grape seed extract and mushroom extract are mixed in equal proportions.
[0009] As a preferred embodiment, the method for preparing the antioxidant precursor mixture comprises the following steps:
[0010] (1) drying kiwi fruit peels, grape seeds and mushrooms and crushing them into powder;
[0011] (2) extracting the Hericium erinaceus peel powder, grape seed powder and mushroom powder with water at least twice, with each extraction time of 1-1.5 hours, to obtain kiwi fruit peel extract, grape seed extract and mushroom extract;
[0012] (3) After combining the three extracts, adsorption and desorption were performed, and the desorbed liquid was concentrated and dried under the conditions of a temperature of 70° C. and a vacuum degree of ≥-0.06 MPa, and then sieved to obtain an antioxidant precursor mixture.
[0013] As a preferred embodiment, in the step (2), the solid-liquid ratio of the Hericium erinaceus peel powder, grape seed powder and mushroom powder for water extraction is 1:(4-5), and the water extraction temperature is ≥85°C.
[0014] As a preferred embodiment, in the step (3), silica gel is used as the adsorbent, and desorption is performed using an aqueous mobile phase as the eluent.
[0015] The present invention also provides a method for preparing high-antioxidant Paecilomyces mycelium powder, which uses the above-mentioned fermentation medium for producing high-antioxidant Paecilomyces mycelium powder to ferment and culture the Paecilomyces strain.
[0016] As a preferred embodiment, the method for preparing the mycelium powder of Paecilomyces oxysporum with high antioxidant capacity comprises the following steps:
[0017] S1. Add glucose, peptone, yeast powder, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and antioxidant precursor mixture into a fermentation tank according to weight, and steam sterilize at 120-125° C. for at least 30 minutes;
[0018] S2, after cooling the culture medium raw material in the fermentation tank to 25°C using a chilled water cycle, the seed liquid of the Paecilomyces strain in another fermentation tank is transferred to the fermentation tank containing the culture medium raw material through a pipeline, and the fermentation tank temperature is maintained at 20-28°C and the fermentation is cultured for 5-7 days;
[0019] S3. After the fermentation is completed, the obtained bacterial cake is subjected to low-temperature continuous vacuum drying and ultrafine grinding to obtain the highly antioxidant Paecilomyces mycelium and Paecilomyces mycelium powder.
[0020] As a preferred embodiment, the Paecilomyces seed solution in step S2 is Paecilomyces shepiali 3.14870 strain, with a deposit number of 11-137, which comes from the Institute of Microbiology, Chinese Academy of Sciences.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] 1. The present invention adds an antioxidant precursor mixture including kiwi fruit peel extract, grape seed extract and mushroom extract to the fermentation medium for producing high antioxidant Paecilomyces mycelium powder. The antioxidant precursor mixture is rich in flavonoids, polyphenols, selenium and the like, and can be directly incorporated into product molecules by microorganisms during the biosynthesis process. Under the premise of not changing the physical and chemical properties of the fermentation medium, fermentation culture using the fermentation medium can increase the content of antioxidant active substances in the Paecilomyces mycelium and enhance the antioxidant capacity of the Paecilomyces mycelium powder.
[0023] 2. The mycelium of Paecilomyces prepared by the culture medium and preparation method provided by the present invention and its flattened form have high antioxidant activity and high uridine content, and also have the effects of enhancing immunity, anti-inflammatory and analgesic, relieving physical fatigue, relieving cough, expectorant, relieving asthma, and preventing respiratory infections.
[0024] 3. The present invention utilizes the provided fermentation medium to carry out fermentation culture of Paecilomyces mycelium and preparation of related micro-powder products, which has low production cost, short cycle, is not restricted by environmental conditions, and is suitable for large-scale factory production and promotion.
[0025] 4. When the present invention utilizes the provided fermentation medium to ferment and culture mycelium, a continuous vacuum dryer is used to dry the filtered mycelium to protect the important active substances of the fungus from being destroyed by high temperature, thereby effectively retaining the antioxidant activity of the active substances in the mycelium. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 A schematic diagram showing the comparison of the scavenging activities of the mycelium powder of Paecilomyces prepared in the present invention and other Cordyceps water extracts on DPPH (30 μg / ml);
[0027] Figure 2 The figure is a schematic diagram comparing the hydroxyl radical scavenging activities of the mycelium powder of Paecilomyces prepared in the present invention and other Cordyceps water extracts. DETAILED DESCRIPTION
[0028] The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0029] The present invention will be further described below in conjunction with the accompanying drawings and preferred embodiments. The given embodiments are only for illustrating the present invention, rather than for limiting the scope of the present invention.
[0030] The water used in the water extraction in the following examples is any water that can be used to prepare water extracts, including medicinal water, such as distilled water and deionized water.
[0031] The preparation of the water extract in the following examples can be performed at least three times as needed.
[0032] The following examples use several Cordyceps products for comparative studies on antioxidant activity: BMB-ZST (Hirsutella sinensis), BMB-JF (Hirsutella sinensis), artificial Cordyceps fruiting bodies (YCC, Cordyceps militaris), BLJN (Hirsutella sinensis), JSBJN (Paecilomyces Hepiali), and natural Cordyceps sinensis (DCXC, Cordyceps sinensis).
[0033] The reagents and instruments used in the following examples are commonly used in the art, and the determination methods used are commonly used in the art, unless otherwise specified.
[0034] Example 1
[0035] This embodiment provides a fermentation medium composed of 30 parts of glucose, 20 parts of peptone, 20 parts of yeast powder, 2 parts of potassium dihydrogen phosphate, 1 part of magnesium sulfate heptahydrate and 2 parts of an antioxidant precursor mixture; the pH of the fermentation medium is 6.0.
[0036] The antioxidant precursor mixture comprises kiwi fruit peel extract, grape seed extract and mushroom extract mixed in equal proportions; the preparation method of the antioxidant precursor mixture comprises the following steps:
[0037] (1) drying kiwi fruit peels, grape seeds and mushrooms and crushing them into powder;
[0038] (2) The solid-liquid ratio of Hericium erinaceus peel powder, grape seed powder and mushroom powder for water extraction is 1:(4-5), and the water extraction is performed at least twice, the water extraction temperature is ≥85°C, and the extraction time for each time is 1-1.5h to obtain kiwi fruit peel extract, grape seed extract and mushroom extract;
[0039] (3) After combining the three extracts, adsorption and desorption were performed. Silica gel was used as the adsorbent and water (water-ethanol) mobile phase was used as the eluent for desorption. The desorbed liquid was concentrated and dried at a temperature of 70°C and a vacuum degree of ≥-0.06MPa. After sieving, an antioxidant precursor mixture was obtained.
[0040] Example 2
[0041] This embodiment provides a fermentation medium composed of 20 parts of glucose, 10 parts of peptone, 10 parts of yeast powder, 1 part of potassium dihydrogen phosphate, 2 parts of magnesium sulfate heptahydrate and 1 part of antioxidant precursor mixture; the pH of the fermentation medium is 5.0.
[0042] The antioxidant precursor mixture comprises kiwi fruit peel extract, grape seed extract and mushroom extract mixed in equal proportions; the preparation method of the antioxidant precursor mixture comprises the following steps:
[0043] (1) drying kiwi fruit peels, grape seeds and mushrooms and crushing them into powder;
[0044] (2) The solid-liquid ratio of Hericium erinaceus peel powder, grape seed powder and mushroom powder for water extraction is 1:(4-5), and the water extraction is performed at least twice, the water extraction temperature is ≥85°C, and the extraction time for each time is 1-1.5h to obtain kiwi fruit peel extract, grape seed extract and mushroom extract;
[0045] (3) After combining the three extracts, adsorption and desorption were performed. Silica gel was used as the adsorbent and water (water-ethanol) mobile phase was used as the eluent for desorption. The desorbed liquid was concentrated and dried at a temperature of 70°C and a vacuum degree of ≥-0.06MPa. After sieving, an antioxidant precursor mixture was obtained.
[0046] Example 3
[0047] This embodiment provides a fermentation medium composed of 60 parts of glucose, 40 parts of peptone, 40 parts of yeast powder, 4 parts of potassium dihydrogen phosphate, 3 parts of magnesium sulfate heptahydrate and 5 parts of antioxidant precursor mixture; the pH of the fermentation medium is 6.5.
[0048] The antioxidant precursor mixture comprises kiwi fruit peel extract, grape seed extract and mushroom extract mixed in equal proportions; the preparation method of the antioxidant precursor mixture comprises the following steps:
[0049] (1) drying kiwi fruit peels, grape seeds and mushrooms and crushing them into powder;
[0050] (2) The solid-liquid ratio of Hericium erinaceus peel powder, grape seed powder and mushroom powder for water extraction is 1:(4-5), and the water extraction is performed at least twice, the water extraction temperature is ≥85°C, and the extraction time for each time is 1-1.5h to obtain kiwi fruit peel extract, grape seed extract and mushroom extract;
[0051] (3) After combining the three extracts, adsorption and desorption were performed. Silica gel was used as the adsorbent and water (water-ethanol) mobile phase was used as the eluent for desorption. The desorbed liquid was concentrated and dried at a temperature of 70°C and a vacuum degree of ≥-0.06MPa. After sieving, an antioxidant precursor mixture was obtained.
[0052] Example 4
[0053] This embodiment provides a method for preparing a high antioxidant Paecilomyces mycelium powder, which uses the fermentation medium prepared in the above embodiment 1 to ferment and culture the Paecilomyces strain, comprising the following steps:
[0054] S1. Add glucose, peptone, yeast powder, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and antioxidant precursor mixture according to the weight parts of Example 1 into a 2T fermentation tank, and steam sterilize at 121° C. for 30 min;
[0055] S2, after cooling the culture medium raw material in the fermentation tank to 25°C using a chilled water cycle, the seed liquid of the Paecilomyces strain in another 50L fermentation tank is transferred to the fermentation tank containing the culture medium raw material through a pipeline, and the fermentation tank temperature is maintained at 20-28°C and the fermentation is cultured for 5-7 days;
[0056] S3. After the fermentation is completed, the obtained bacterial cake is subjected to low-temperature continuous vacuum drying and ultrafine grinding to obtain highly antioxidant Paecilomyces Hepialis mycelium powder (NQM-JF, Paecilomyces Hepiali).
[0057] Example 5
[0058] Sample preparation: Take 0.1 g of each artificial Cordyceps fruiting body powder BMB-ZST, BMB-JF, YCC, BLJN, JSBJN, natural Cordyceps powder DCXC and the pseudo-Paecilomyces mycelium powder NQM-JF prepared according to Example 4, add 10 mL of deionized water, ultrasonically extract for 50 min, filter, take 2 mL of the filtrate, put it in a 10 mL volumetric flask, add water to the scale, shake well, and obtain 10 mg crude drug / mL test solution, and then dilute according to the multiple to obtain 2 mg crude drug / mL, 0.4 mg crude drug / mL, 0.08 mg crude drug / mL, 0.016 mg crude drug / mL test solution.
[0059] Prepare DPPH working solution with reference to the prior art, accurately weigh 6.0 mg of DPPH, place in a 50 mL volumetric flask, add anhydrous ethanol to dissolve, set to volume, and then dilute with anhydrous ethanol to obtain 30 μg / mL DPPH working solution. Weigh 6.0 mg of VC and place in 50 mL to dissolve in water. The concentration of VC is 124 μg / mL, and then dilute to 20.8 μg / mL, 4.16 μg / mL, 0.832 μg / mL, and 0.166 μg / mL. Pipette 150 μL of each concentration gradient of the above test solution, add 150 μL of DPPH working solution to prepare the reaction solution, mix for 30 minutes, and determine the absorbance value A1; replace DPPH with ethanol, and determine the absorbance value A2 in the same way. Pipette 150 μL of DPPH working solution and ethanol in the same way to determine the absorbance value of blank A0. The absorbance of each reaction solution was measured at a wavelength of 520 nm using an enzyme marker, and the DPPH free radical scavenging rate was calculated according to the following formula:
[0060] Free radical scavenging rate % = (1-(A1-A2) / A0) × 100%;
[0061] The DPPH clearance rate was plotted with the logarithm of the sample concentration as the horizontal axis and the absorbance value as the vertical axis. The amount of sample required to clear 50% DPPH, i.e., the IC50 value, was calculated using the software prism8.0 (Nonlinear regression). The IC50 values of each sample group were compared.
[0062] The statistical results of the median effective dose of each sample for DPPH removal are shown in Table 1 and Figure 1 The IC50 value of the pseudo-Paecilomyces mycelium powder (NQM-JF) prepared by the present invention is the lowest, and the DPPH free radical scavenging effect is the best.
[0063] Table 1 Comparison of in vitro antioxidant activity of each sample
[0064]
[0065] Example 6
[0066] The hydroxyl radical scavenging ability was determined by referring to the prior art test method; 50 μL of FeSO4 (0.911 mg / mL), 50 μL of salicylic acid solution (0.828 mg / mL, 50% ethanol), 50 μL of each sample solution (10 mg crude drug / mL) (including the sample prepared in Example 4) and 50 μL of H2O2 (0.03%) were added to a 96-well plate, reacted in a 37°C water bath for 1 h, cooled to room temperature, and the absorbance (A1) was measured at a wavelength of 510 nm; 1 mL of 50% ethanol solution was used instead of salicylic acid solution to determine the absorbance (A2), and 1 mL of deionized water was used instead of Cordyceps sample solution to determine the absorbance (A0), 1 mg / mL L-ascorbic acid was used as a control, and the experiment was repeated 3 times in parallel, and the hydroxyl radical scavenging rate (%) was calculated according to the following formula.
[0067] Hydroxyl radical scavenging rate (%) = (1-(A1-A2) / A0) × 100%;
[0068] The results of scavenging hydroxyl radicals by each Cordyceps sample are shown in Table 2 and Figure 2 The results showed that among several Cordyceps products at a concentration of 10 mg / mL, the hydroxyl scavenging rate of the mycelium powder of Paecilomyces prepared by the present invention was the highest.
[0069] Table 2 Comparison of in vitro antioxidant activity of each sample
[0070]
[0071] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A fermentation medium for producing highly antioxidant Paecilomyces mycelium powder, characterized in that: The fermentation medium is composed of the following raw materials in parts by weight: 20-60 parts of glucose, 10-40 parts of peptone, 10-40 parts of yeast powder, 1-4 parts of potassium dihydrogen phosphate, 1-3 parts of magnesium sulfate heptahydrate and 1-5 parts of an antioxidant precursor mixture; the pH of the fermentation medium is 5.0-6.
5.
2. The fermentation medium for producing high antioxidant Paecilomyces mycelium powder according to claim 1, characterized in that: The invention comprises the following raw materials in parts by weight: 30 parts of glucose, 20 parts of peptone, 20 parts of yeast powder, 2 parts of potassium dihydrogen phosphate, 1 part of magnesium sulfate heptahydrate and 2 parts of antioxidant precursor mixture.
3. The fermentation medium for producing high antioxidant Paecilomyces mycelium powder according to claim 1, characterized in that: The antioxidant precursor mixture comprises kiwi fruit peel extract, grape seed extract and mushroom extract; the kiwi fruit peel extract, grape seed extract and mushroom extract are mixed in equal proportions.
4. The fermentation medium for producing high antioxidant Paecilomyces mycelium powder according to claim 1, characterized in that: The method for preparing the antioxidant precursor mixture comprises the following steps: (1) drying kiwi fruit peels, grape seeds and mushrooms and crushing them into powder; (2) extracting the Hericium erinaceus peel powder, grape seed powder and mushroom powder with water at least twice, with each extraction time of 1-1.5 hours, to obtain kiwi fruit peel extract, grape seed extract and mushroom extract; (3) After combining the three extracts, adsorption and desorption were performed, and the desorbed liquid was concentrated and dried under the conditions of a temperature of 70° C. and a vacuum degree of ≥-0.06 MPa, and then sieved to obtain an antioxidant precursor mixture.
5. The fermentation medium for producing high antioxidant Paecilomyces mycelium powder according to claim 1, characterized in that: In the step (2), the material-liquid ratio of the Hericium erinaceus peel powder, the grape seed powder and the mushroom powder for water extraction is 1:(4-5), and the water extraction temperature is ≥85°C.
6. The fermentation medium for producing highly antioxidant Paecilomyces mycelium powder according to claim 1, characterized in that: In the step (3), silica gel is used as the adsorbent, and desorption is performed using an aqueous mobile phase as the eluent.
7. A method for preparing a micropowder of mycelium of Paecilomyces with high antioxidant activity, characterized in that: The Paecilomyces strain is fermented and cultured using the fermentation medium for producing Paecilomyces mycelium powder with high antioxidant capacity as claimed in any one of claims 1 to 6.
8. The method for preparing the mycelium powder of Paecilomyces spp. producing high antioxidant activity according to claim 7, characterized in that: The following steps are included S1. Add glucose, peptone, yeast powder, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and antioxidant precursor mixture into a fermentation tank according to weight, and steam sterilize at 120-125° C. for at least 30 minutes; S2, after cooling the culture medium raw material in the fermentation tank to 25°C using a chilled water cycle, the seed liquid of the Paecilomyces strain in another fermentation tank is transferred to the fermentation tank containing the culture medium raw material through a pipeline, and the fermentation tank temperature is maintained at 20-28°C and the fermentation is cultured for 5-7 days; S3. After the fermentation is completed, the obtained bacterial cake is subjected to low-temperature continuous vacuum drying and ultrafine grinding to obtain the highly antioxidant Paecilomyces mycelium and Paecilomyces mycelium powder.
9. The method for preparing the mycelium powder of Paecilomyces spp. producing high antioxidant activity according to claim 8, characterized in that: The Paecilomyces hepialis seed solution in step S2 is Paecilomyces hepiali 3.14870, with a preservation number of 11-137, and is sourced from the Institute of Microbiology, Chinese Academy of Sciences.