CN115747080A - Edible fungus mycelium extract powder, preparation method thereof and application of edible fungus mycelium extract powder as fermentation nitrogen source - Google Patents
Edible fungus mycelium extract powder, preparation method thereof and application of edible fungus mycelium extract powder as fermentation nitrogen source Download PDFInfo
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- CN115747080A CN115747080A CN202211463817.3A CN202211463817A CN115747080A CN 115747080 A CN115747080 A CN 115747080A CN 202211463817 A CN202211463817 A CN 202211463817A CN 115747080 A CN115747080 A CN 115747080A
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- fermentation
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- extract powder
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 118
- 239000000843 powder Substances 0.000 title claims abstract description 77
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 59
- 238000000855 fermentation Methods 0.000 title claims abstract description 57
- 230000004151 fermentation Effects 0.000 title claims abstract description 57
- 239000000284 extract Substances 0.000 title claims abstract description 47
- 241000233866 Fungi Species 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000001471 micro-filtration Methods 0.000 claims description 33
- 102000004190 Enzymes Human genes 0.000 claims description 29
- 108090000790 Enzymes Proteins 0.000 claims description 29
- 229940088598 enzyme Drugs 0.000 claims description 29
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 238000011282 treatment Methods 0.000 claims description 20
- 108010059892 Cellulase Proteins 0.000 claims description 19
- 239000004365 Protease Substances 0.000 claims description 19
- 108091005804 Peptidases Proteins 0.000 claims description 18
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- 229940106157 cellulase Drugs 0.000 claims description 14
- 240000008042 Zea mays Species 0.000 claims description 13
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- 238000012258 culturing Methods 0.000 claims description 11
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 10
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- 235000019419 proteases Nutrition 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 10
- 239000003513 alkali Substances 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- 241000307222 Clitocybe maxima Species 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
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- 108010059820 Polygalacturonase Proteins 0.000 claims description 8
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- 229940055729 papain Drugs 0.000 claims description 7
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- 108091005658 Basic proteases Proteins 0.000 claims description 6
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
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- 239000011702 manganese sulphate Substances 0.000 claims description 4
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- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 claims 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention belongs to the technical field of edible fungus fermentation, and particularly relates to edible fungus mycelium extract powder, a preparation method thereof and application of the edible fungus mycelium extract powder as a fermentation nitrogen source. Based on the edible fungus fermentation technology and method, the invention overcomes the influence of nutrition difference on microorganism preference, and prepares the edible fungus mycelium extract powder with richer nutrition and lower price. The edible fungus mycelium extract powder can be used as a fermentation nitrogen source to replace or combine with other nitrogen sources for fermentation of microorganisms, so that the production cost is greatly reduced, the industrial production is facilitated, and the application prospect is good.
Description
Technical Field
The invention belongs to the technical field of fermentation of edible fungi, and particularly relates to edible fungi mycelium extract powder, a preparation method thereof and application of the edible fungi mycelium extract powder as a fermentation nitrogen source.
Background
In the fermentation production and scientific research work of microbial products, because of different strains, different growth stages of the strains and different fermentation process conditions, the used culture media are different, and the raw materials of the culture media are summarized to have carbon sources, nitrogen sources, inorganic salts, trace elements, water, growth factors, precursors and the like.
The nitrogen source is one of the main raw materials used in the culture of microorganisms. Its main function is to constitute microbial cells and nitrogenous metabolites, which are divided into two major classes, organic nitrogen sources and inorganic nitrogen sources. Soybean cake powder, corn steep liquor, peptone, yeast powder, fish meal and the like belong to organic nitrogen sources. Urea, ammonia water, ammonium sulfate, ammonium chloride, and the like belong to inorganic nitrogen. The organic nitrogen source is a relatively ideal nutrient substance, which contains abundant proteins, peptides, free amino acids, and small amounts of sugars, fats, inorganic salts, growth factors, and the like.
The microorganisms are selective for the use of amino acids in organic nitrogen, such as valine, which is used both for the growth of Streptomyces erythromycin and for the biosynthesis of erythromycin as a nitrogen source. In addition, some amino acids contained in the organic nitrogen source are precursors for the synthesis of secondary metabolites by the bacterial cells. Such as a-aminoadipic acid and cysteine, are direct precursors for the synthesis of antibiotics such as penicillin.
In the laboratory, yeast extract related products are relatively common nitrogen sources, in the industry, for economic feasibility, plant source protein and inorganic nitrogen are mostly adopted, but the effect is not good as that of yeast extracts, and domestic yeast extracts and foreign related products have certain quality difference.
Disclosure of Invention
The invention aims to solve the technical problem of providing the mycelium extract powder which is prepared based on the edible fungus fermentation technology and has richer nutrition in order to overcome the influence of nutrition difference on microbial preference on the defects of the prior art.
The invention also aims to solve the technical problem of providing a preparation method of the mycelium soaking powder.
The invention finally solves the technical problem of providing the application of the mycelium soaking powder as a fermentation nitrogen source.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a preparation method of edible fungus mycelium soaking powder comprises the following steps:
(1) Performing seed culture and fermentation culture on the pretreated edible fungus strain to obtain mycelium;
(2) Mechanically breaking the wall of the mycelium obtained in the step (1), performing alkali treatment, and adding complex enzyme for enzymolysis;
(3) Performing solid-liquid separation on the enzymatic hydrolysate obtained in the step (2) to obtain a first supernatant and bacterial residues, performing microfiltration treatment on the first supernatant to obtain microfiltration permeating liquid, uniformly mixing the microfiltration permeating liquid with the bacterial residues, performing solid-liquid separation to obtain a second supernatant, performing microfiltration treatment on the second supernatant, adding water, and performing percolation to obtain a microfiltration concentrated solution;
(4) Adding nuclease into the microfiltration concentrated solution obtained in the step (3) for enzymolysis, and then adding protease for enzymolysis to obtain enzymatic hydrolysate;
(5) And (4) carrying out vacuum concentration and spray drying on the enzymolysis liquid obtained in the step (4) to obtain the mycelium soaked powder of the edible fungi.
Wherein, in the step (1), the edible fungus strain comprises any one of pleurotus eryngii, pleurotus ostreatus and clitocybe maxima, and the preferred edible fungus strain is clitocybe maxima.
Wherein the steps(1) The pretreatment is to cut the fruiting body of the edible fungus into 2cm pieces 3 Wiping the surfaces of the left and right fungus blocks with alcohol in a super clean bench for disinfection, then cutting off the surfaces with a sterile blade, cutting a fungus block with the side length of 0.5cm on a slant plate, and culturing at 26 ℃ until the mycelium grows over the plate.
Wherein, in the step (1), the seed culture conditions are as follows: culturing at 25-28 deg.C for 4-7 days until the wet weight of corresponding thallus reaches 120-170g/L. Preferred culture conditions are: culturing at 25 deg.C for 5 days until the wet weight of corresponding thallus reaches 150g/L.
Specifically, the seed culture medium formula is as follows: 25g/L of glucose, 5g/L of yeast extract, 20g/L of corn steep liquor, 40.5g/L of MgSO4.7H2O, 0.08g/L of FeSO4.7H2O, 0.5g/L of KH2PO, 0.02g/L of VB, and pH =6.0.
Wherein, in step (1), said fermentation culture is carried out in an inoculum amount of 9-20%; the culture conditions are as follows: culturing at 25-28 deg.C for 4-7 days at 200-250 r/min. Preferably, the inoculation amount is 12% v/v, the culture conditions are: culturing at 25 deg.C for 6 days at 200 r/min.
Specifically, the formula of the fermentation medium is as follows: 10-30g/L of corn sugar, 10-25g/L of corn steep liquor, 1-6g/L of ammonium sulfate, 0.1-1g/L of magnesium sulfate, 0.1-0.4g/L of monopotassium phosphate, 0.0001-0.005g/L of manganese sulfate, 0.01-0.1g/L of VB and pH =6.0.
Preferably, the formula of the fermentation medium is as follows: 20g/L of corn sugar, 20g/L of corn steep liquor, 3g/L of ammonium sulfate, 0.4g/L of magnesium sulfate, 0.4g/L of monopotassium phosphate, 0.005g/L of manganese sulfate, 0.02g/L of VB and pH =6.0.
Wherein, in the step (2), the alkali treatment conditions are as follows: adjusting pH to 8-12 with NaOH, and bathing with hot water at 50-60 deg.C for 6-10 hr. Preferred process conditions are: adjusting the pH value to 11-12 with NaOH, and carrying out hot water bath at 60 ℃ for 8 hours.
Wherein, in the step (2), the complex enzyme is the combination of any several of cellulase, xylanase, glucanase, pectinase, helicase and glycosyl peptidase, the addition amount of each enzyme is 0.1-0.5 percent w/v (the mass of the added enzyme per unit volume, g/100 mL). Preferred complex enzymes are cellulase, glycosylpeptidase and pectinase, each added in an amount of 0.5% w/v (mass of enzyme added per unit volume, g/100 mL).
In the step (2), the enzymolysis conditions are as follows: adjusting the pH value to 5 by hydrochloric acid, hydrolyzing for 12 hours at 50 ℃, and stirring at the rotating speed of 450r/min.
Wherein, in the step (3), the microfiltration treatment is microfiltration by using a filter membrane with the pore diameter of 0.45 μm.
Wherein, in the step (4), the nuclease is nuclease P1, and the addition amount thereof is 0.01-0.1% w/v (mass of enzyme added per unit volume, g/100 mL); the protease is one or more of papain, flavourzyme, neutral protease, alkaline protease and yeast extraction enzyme, and the addition amount is 0.1-0.3% w/v. The preferred protease is a mixture of papain and alkaline protease, and the amount of each enzyme added is 0.3% w/v (mass of enzyme added per unit volume, g/100 mL).
Wherein, in the step (4), the microfiltration concentrated solution is heated to 68 ℃, nuclease is added for enzymolysis for 4-8 hours, the temperature is reduced to 50-60 ℃,1M sodium hydroxide is used for adjusting the pH value to 7-8, and protease is added for enzymolysis for 8-12 hours. Preferably, the microfiltration concentrated solution is heated to 68 ℃, nuclease is added for enzymolysis for 6 hours, the temperature is reduced to 50-60 ℃,1M sodium hydroxide is used for adjusting the pH value to 7.2, and protease is added for enzymolysis for 12 hours.
Wherein, in the step (5), the vacuum concentration and spray drying are carried out by concentrating the enzymatic hydrolysate to dry solids 40-55 w/v (mass of enzyme added per unit volume, g/100 mL), adding 0.1-1% w/v (mass of enzyme added per unit volume, g/100 mL) of modified starch, mixing well, and drying in a spray dryer, wherein the set parameters are: the feeding temperature is 25-35 ℃, the air inlet temperature is 135-160 ℃, the pressure is 20-25mmHg, and the feeding flow is 50-70ml/min.
The edible fungus mycelium extract powder prepared by the preparation method is also within the protection scope of the invention.
The application of the edible fungus mycelium extract powder as a fermentation nitrogen source is also within the protection scope of the invention.
Specifically, the edible fungus mycelium extract powder can be used as a fermentation nitrogen source instead of or in combination with other nitrogen sources for fermentation of microorganisms.
Specifically, the nutritional ingredients of the mycelium extract powder of the edible fungi (pleurotus eryngii, oyster mushroom and clitocybe maxima) are compared with the nutritional ingredients of the yeast extract powder and the soybean extract powder, and the results show that the contents of mycelium proteins, essential amino acids, VB6 and the like of the clitocybe maxima are higher than those of other samples, the levels of valine, methionine and lysine in the amino acids are generally higher, and the nutritional ingredients are rich.
Specifically, the pork tripe mushroom mycelium extract powder is compared with nitrogen sources such as corn steep liquor, yeast extract powder, yeast extract, peptone, fish meal peptone, beef extract, soybean peptone and the like, and the influence of different nitrogen sources on polysialic acid fermentation is researched. The mycelium extract powder is found to be beneficial to growth and metabolism of escherichia coli, the performance of the mycelium extract powder reaches the quality of imported yeast extract powder, and the mycelium extract powder can completely replace imported nitrogen sources.
Specifically, by taking the yield and the cost of cordycepin as fermentation evaluation indexes and nitrogen content as an addition amount index, the influence of nitrogen source peptone and yeast powder of different brands and the mycelium extract powder of the morchella esculenta on fermentation is investigated, and the fact that the mycelium extract powder is added into a nitrogen source has a promoting effect on cordycepin fermentation is found, the production cost is greatly reduced, and the industrial production is facilitated.
Has the advantages that:
1. the invention overcomes the influence of nutrition difference on microbial preference based on the edible fungus fermentation technology and method, and prepares the edible fungus mycelium extract powder with richer nutrition and lower price.
2. The edible fungus mycelium extract powder prepared by the invention can be used as a fermentation nitrogen source to replace or combine with other nitrogen sources, is used for fermentation of microorganisms, can greatly reduce the production cost, is beneficial to industrial production, and has good application prospect.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Example 1: liquid fermentation of Gaster Sus Domestica strain
Strain: procurement from the Taobao product world shop.
(1) Bacterial pre-treatmentAnd (3) treatment: cutting fruiting body into 2cm pieces 3 Wiping the surfaces of the left and right fungus blocks with alcohol in a super clean bench for disinfection, then cutting off the surfaces with a sterile blade, cutting a fungus block with the side length of 0.5cm on a slant plate, and culturing at 26 ℃ until the mycelium grows over the plate.
(2) Seed culture: 1 piece of colony with the diameter of 1 x 1cm is scraped from the slant strain of the clitocybe maxima to a 500mL triangular flask filled with 150mL seed culture medium, and the colony is cultured for about 5 days at the temperature of 25 ℃ until the corresponding wet weight of the strain reaches about 150g/L.
(3) Fermentation culture: the inoculum size was 12% v/v, inoculated into a 500mL Erlenmeyer flask containing 150mL of fermentation medium, and cultured at 25 ℃ for 6 days at 200r/min until the cell concentration became 14.7g/L.
Wherein, the formula of the slant culture medium is as follows: 25g/L glucose, 5g/L yeast extract, 20g/L corn steep liquor, 0.5g/L MgSO4, feSO 4 .7H 2 O 0.08g/L，KH 2 PO 4 0.5g/L, VB 1.02 g/L, agar 15g/L, pH =6.0.
Wherein, the formula of the seed culture medium is as follows: 25g/L of glucose, 5g/L of yeast extract, 20g/L of corn steep liquor, 0.5g/L of MgSO4, 0.08g/L of FeSO4.7H2O, 0.5g/L of KH2PO4, 0.02g/L of VB1, and pH =6.0.
Wherein, the formula of the fermentation medium is as follows: 20g/L of corn sugar, 20g/L of corn steep liquor, 3g/L of ammonium sulfate, 0.4g/L of magnesium sulfate, 0.4g/L of monopotassium phosphate, 0.005g/L of manganese sulfate, 0.02g/L of VB and PH =6.0.
Example 2: influence of different alkali treatments and Complex enzymes on cell wall breaking hydrolysis
The mycelium obtained by fermentation in example 1 was washed twice with pure water, and then 5 times of water was added to the mycelium (water content 91.2%), and mechanical wall breaking was carried out for 2 minutes to slurry state with a high speed wall breaking machine (Jiuyang wall breaking machine). The mixture was dispensed into 1L beakers, the volume of the solution was 500mL (three parallel), the pH was adjusted to 8-12 with NaOH, and the mixture was then bathed in hot water at 60 ℃ for 8 hours. Adjusting pH to 5 with hydrochloric acid, adding cellulase, xylanase, glucanase, pectinase, helicase, etc., wherein each enzyme is added in an amount of 0.5% w/v (weight of enzyme added per unit volume, g/100mL, based on dry cell basis), hydrolyzing at 50 deg.C for 12 hr, and stirring at 450r/min. After enzymolysis, the mixture is centrifuged for 10 minutes at 5000r/min by a high-speed centrifuge to respectively obtain a centrifuged first supernatant and bacterial residue. And (3) performing microfiltration treatment (with a 0.45-micron filter membrane) on the first supernatant obtained by centrifugation, collecting microfiltration permeating liquid, discharging the microfiltration permeating liquid, mixing the microfiltration permeating liquid with the fungus residue obtained by centrifugation, uniformly stirring, and continuously centrifuging for 10 minutes at 5000 r/min. And performing microfiltration treatment again on the second supernatant obtained by the second centrifugation, adding pure water for percolation, and adding 3-4L of water each time for 3 times. And (4) finally, reserving the microfiltration concentrated solution, and drying and weighing filter residues.
TABLE 1 Effect of different alkali treatments and enzymatic hydrolysis on mycelium
| Numbering | Alkali treatment | Enzymolysis | Percentage of hydrolysis% |
| 0 | pH6.2 (not adjusted) | Without addition of enzyme | 27.23±1.25 |
| 1 | pH6.2 (not adjusted) | Cellulase (purchased from Novexin, same as the following) | 33.81±1.74 |
| 2 | pH8 | Cellulase enzymes | 36.93±1.32 |
| 3 | pH9 | Cellulase enzymes | 40.65±2.06 |
| 4 | pH10 | Cellulase enzymes | 45.74±2.27 |
| 5 | pH11 | Cellulase enzymes | 52.66±2.75 |
| 6 | pH12 | Cellulase enzymes | 53.72±2.44 |
| 7 | pH10 | Cellulase, xylanase (Youte, same below) | 46.23±2.50 |
| 8 | pH10 | Cellulase, dextranase (Richeng biology, same below) | 46.98±2.13 |
| 9 | pH10 | Cellulase, pectinase (Xiasang biology, the same below) | 50.76±2.34 |
| 10 | pH10 | Cellulase, snailase (Aopepti organisms, same as below) | 48.02±3.03 |
| 11 | pH10 | Cellulase, glycosyl peptidase (Ludger E-PNG01, same below) | 58.67±2.65 |
| 12 | pH10 | Cellulase, xylanase and glucanase | 46.87±1.76 |
| 13 | pH10 | Snail enzyme, xylanase and glucanase | 29.34±0.42 |
| 14 | pH10 | Glycosyl peptidase, pectase | 43.12±1.35 |
| 15 | pH11 | Cellulase, glycosylpeptidase, pectinase | 66.92±3.18 |
Note: hydrolysis rate = 100% of the mass of the cell residue after cell wall breaking/initial cell mass
Through different alkali treatments and the enzymolysis of the complex enzyme, the enzymolysis efficiency is the best under the action of pH11-12 and the complex enzyme (cellulase, glycosyl peptidase and pectinase). If the pH is adjusted up again, the hydrolysis effect will theoretically be better, but the amount of alkali used will increase significantly and the salt introduced will be higher. In addition, the supernatant before microfiltration, and the concentrate after microfiltration, which are numbers 0, 15, were dried and pulverized, and ash content was detected, and the results were (8.72 ± 0.33)%, (34.63 ± 1.45)% and (5.81 ± 1.45)%, respectively.
Example 3: the influence of protease on the content of amino acid nitrogen is not regulated
The microfiltration concentrate obtained under the treatment condition of number 15 in example 2 was heated to 68 ℃ and subjected to enzymatic hydrolysis with nuclease P1 (purchased from Kyoto Kagaku Biopsis, nanjing) for 6 hours. Cooling to 50-60 deg.C, adjusting pH to 7.2, and adding different proteases for enzymolysis at the same temperature for 12 hr. Concentrating the enzymolysis solution under rotary vacuum (50 deg.C, rotation speed of 65r/min, vacuum degree of about-0.1 Mpa), oven drying, and pulverizing. The content of free amino acid nitrogen is measured (the standard of the measurement method set adopts national standard GB/T20886.2-2021 appendix C) to characterize the proteolysis degree.
TABLE 2 Effect of different proteases on proteolysis
| Number of | Enzymolysis | Amino acid nitrogen% | Reduced protein content% | Nucleic acid hydrolysis Rate% |
| 0 | Without addition of enzyme | 3.22±0.21 | 20.12 | 27.87 |
| 1 | Papain | 6.01±0.54 | 37.56 | 45.12 |
| 2 | Flavourzyme protease | 4.13±0.32 | 25.81 | 39.43 |
| 3 | Neutral protease | 4.45±0.26 | 27.81 | 41.23 |
| 4 | Alkaline protease | 5.44±0.29 | 34.00 | 46.12 |
| 5 | Yeast extraction enzyme | 4.68±0.31 | 29.25 | 42.09 |
| 6 | Papain, a,Alkaline protease | 11.02±0.44 | 68.88 | 52.91 |
Note: the above enzymes were purchased from Pompe, guangxi, and the amounts of each enzyme added were 0.3% w/v (mass of enzyme added per unit volume, g/100mL, on a dry cell basis); amino acid nitrogen%: in percentage of oven dry mass, i.e. g/100g.
From the results in table 2, the results of the protease enzymolysis using papain and alkaline protease are better, the content of amino acid nitrogen can reach (11.02 ± 0.44)%, the reduced protein content is about 68.88%, and the nucleic acid hydrolysis rate is 52.91%.
Example 4: preparation of pork tripe mushroom soaked powder
Crushing the fresh pork tripe mushroom fruiting body by a crusher. Then weighing 1200g of wet morchella mycelium and pulped fruit body (calculated by absolute dryness), adding pure water to the volume of 30L, and breaking the wall for 2 minutes by a high-speed beater respectively. The pH was adjusted to 11 with NaOH and the mixture was then bathed in hot water at 60 ℃ for 8 hours. Adjusting pH to 5 with hydrochloric acid, adding cellulase, glycosyl peptidase, and pectinase in an amount of 0.5% by weight, based on the total weight of enzyme added per unit volume, g/100mL, dry weight of cells, hydrolyzing at 50 deg.C for 12 hr, and stirring at 450r/min. After enzymolysis, the mixture is centrifuged for 10 minutes at 5000r/min by a high-speed centrifuge to respectively obtain a first centrifuged supernatant and bacterial residues. Centrifuging to obtain the first supernatant, performing microfiltration treatment (with a filter membrane having a pore diameter of 0.45 μm), collecting microfiltration permeate, discharging the microfiltration permeate, mixing with the residue obtained by centrifuging, stirring, and centrifuging at 5000r/min for 10 min. And performing microfiltration treatment on the second supernatant obtained by the second centrifugation, adding pure water for percolation, adding 3-4L of water each time, and percolating for 3 times. Heating the microfiltration concentrated solution to 68 ℃, adding nuclease P1, and carrying out enzymolysis for 6 hours. Cooling to 50-60 deg.C, adjusting pH to 7.2, adding papain and trypsin at the same temperature (50-60 deg.C) for enzymolysis for 12 hr. Concentrating the enzymatic hydrolysate by rotary vacuum and spray drying (concentrating the enzymatic hydrolysate to dry content of 40-55 g/100mL, adding 0.1-1% of modified starch, mixing, and drying in spray dryer with the parameters of feeding temperature of 25-35 deg.C, air inlet temperature of 135-160 deg.C, pressure of 20-25mmHg, and feeding flow rate of 50-70 mL/min) to obtain mycelium extract powder and fruiting body extract powder of Coryngium Sus Domestica respectively.
TABLE 3 comparison of nutrients in mycelium extract and fruiting body extract of Morchella
| Gaster Sus Domestica | Crude protein% | Amino acid nitrogen% | Nucleic acid% | Ash content% | Others% |
| Mycelium | 67.32 | 7.54 | 10.23 | 7.82 | 14.63 |
| Fruit body | 58.23 | 6.01 | 8.28 | 9.25 | 24.24 |
Note: in percentage of oven dry mass, i.e. g/100g.
As can be seen from Table 3, the extract powder prepared from the mycelia of morel showed higher crude protein content in the final product, indicating that the mycelia are easier to break the wall than the fruit bodies. After proteolysis, the mycelium extract powder has higher content of amino acid nitrogen. In addition, the mycelium extract powder obtained by fermentation has higher nucleic acid content. Therefore, the mycelium is adopted to prepare the extract powder, and the quality is relatively better.
Example 5: comparison of different fungal mycelia with Yeast and Soybean Nutrition
According to the preparation method of the example 4, different fungal mycelium (pleurotus eryngii, pleurotus ostreatus and clitocybe maxima) extract powders, yeast extract powders and soybean extract powders are prepared, and the nutritional ingredients of the fungal mycelium extract powders, the yeast extract powders and the soybean extract powders are compared, so that the results are shown in table 4, and the contents of the mycelium proteins, the essential amino acids, the VB6 and the like of the clitocybe maxima are higher than those of other samples, and the levels of valine, methionine and lysine in the amino acids are generally higher.
TABLE 4 comparison of the nutrient content of the mycelium fermented by different fungi with that of yeast and soybean (unit: g/100 g)
Example 6: effect of different extract powders on fermentation of polysialic acid
The prepared morchella esculenta mycelium extract powder is used as a fermentation nitrogen source, and compared with nitrogen sources such as corn steep liquor, yeast extract powder, yeast extract, peptone, fish meal peptone, beef extract, soybean peptone and the like, the influence of different nitrogen sources on polysialic acid fermentation is researched.
Selecting a single colony of Escherichia coli SA-1 (mutagenized and screened and preserved by a microorganism breeding platform of Jiangsu Collection Industrial biotechnology research institute, ltd., preservation number of CGMCC 20415), inoculating the single colony to a primary seed solution (liquid containing amount of 20 mL), and culturing at 37 ℃ at 200r/min overnight. Transferring the first-order seed solution into a 500mL shake flask containing 100mL shake flask medium according to the inoculation amount of 2% v/v, and culturing at 37 ℃ and 200r/min for 72h. The content of polysialic acid (PSA) was measured by the resorcinol method, and the results are shown in Table 5.
Wherein, the formula of the seed liquid is as follows: 10g/L of peptone, 10g/L of sodium chloride and 5g/L of yeast powder; the pH is natural before sterilization.
Wherein, the formula of the shake flask culture medium is as follows: sorbitol 40g/L, ammonium sulfate 4g/L, potassium dihydrogen phosphate 5g/L, organic nitrogen source several g/L (according to the table concentration added), magnesium sulfate 1.2g/L, before sterilization pH7.2.
TABLE 5 Effect of different Nitrogen sources on polysialic acid fermentation
| Number of | Nitrogen source | Brand | Specification of | The addition amount is g/L | PSA yield g/L |
| 1 | Corn steep liquor | Ningxia Yipin | The total nitrogen is more than or equal to 3 percent | 30 | 0.31±0.01 |
| 2 | Yeast extract powder | Olympic star | The total nitrogen is more than or equal to 9 percent | 10 | 0.78±0.03 |
| 3 | Yeast extract | Group of traditional Chinese medicines | The total nitrogen is more than or equal to 7 percent | 13 | 1.02±0.06 |
| 4 | Yeast extract powder | Angel yeast | The total nitrogen is more than or equal to 9 percent | 10 | 1.13±0.05 |
| 5 | Yeast extract powder | OXOID | The total nitrogen is more than or equal to 10 percent | 9 | 1.39±0.10 |
| 6 | Peptone | OXOID | The total nitrogen is more than or equal to 12.7 percent | 7 | 1.15±0.08 |
| 7 | Fish meal peptone | Group of traditional Chinese medicines | The total nitrogen is more than or equal to 12 percent | 7.5 | 0.68±0.02 |
| 8 | Beef extract | Group of traditional Chinese medicines | The total nitrogen is more than or equal to 13 percent | 7 | 0.54±0.01 |
| 9 | Soybean peptone | Good moistening effect | The total nitrogen is more than or equal to 8 percent | 11 | 0.43±0.02 |
| 10 | Mycelium extract powder | The scheme is | The total nitrogen is more than or equal to 10 percent | 9 | 1.42±0.09 |
Comparison of 10 nitrogen sources shows that the effect of corn steep liquor and soybean peptone is poor, and the effect of plant-derived protein hydrolysate on promoting fermentation of escherichia coli SA-1 is poor. The yeast source protein extract has a remarkable improvement effect on polysialic acid fermentation, particularly the yield of polysialic acid in an imported (OXOID) yeast extract powder experimental group is 4.5 times of that of a corn steep liquor group, and the mycelium extract powder is equivalent to the level of the mycelium extract powder, so that the mycelium extract powder is beneficial to growth and metabolism of escherichia coli, the performance of the mycelium extract powder reaches the quality of the imported yeast extract powder, and an imported nitrogen source can be completely replaced.
Example 7: influence of different soaking powders on cordycepin biological fermentation
Slant strain culture: transferring the cordyceps militaris strain C.militaris CGMCC 18581 preserved in the China general microbiological culture collection center to a solid culture medium, and culturing at the constant temperature of 25 ℃ for 5-7 days to obtain a slant strain.
Seed culture: scraping the cultured slant mycelia, inoculating into a 1L triangular flask containing 200mL seed culture medium, and performing constant temperature shaking culture at 25 deg.C and 180rpm for 3-4 days.
Fermentation culture: inoculating into a 1L triangular flask containing 200mL of fermentation medium according to 10% v/v inoculation amount, and standing and fermenting at 26 deg.C for 22 days.
The method takes the yield and the cost of the cordycepin as fermentation evaluation indexes and nitrogen content as an addition index, and inspects the influence of the combination of nitrogen source peptone and yeast powder of different brands and the immersed powder of the mycelium of the morchella esculenta on fermentation.
Wherein, glucose, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and sodium chloride are purchased from chemical reagents of national medicine group, inc.; yeast peptone and Angel yeast powder are purchased from the market; both peptone and yeast powder are OXOIDs; adenine and adenosine are both purchased from Shanghai Biotechnology engineering (Shanghai) GmbH.
Solid medium: 200g of potato is boiled and filtered to obtain juice, 20g of glucose and KH of potato 2 PO 4 2g，MgSO 4 ·7H 2 O1.5 g, vitamin B 1 0.1g of agar and 15g of agar, the volume is determined to be 1L, and the pH is natural.
Seed culture medium: 30g/L glucose, 10g/L peptone, 15g/L yeast powder, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate and pH5.8.
Fermentation medium: glucose 40g/L, combined nitrogen source several g/L (see table 6 specifically), potassium dihydrogen phosphate 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, magnesium sulfate 0.2g/L, tween-80.5 g/L, adenine 4g/L, pH5.8.
TABLE 6 influence of different nitrogen source combinations on cordycepin fermentation
Note: the cost is calculated by the cost of the nitrogen source in each liter of fermentation liquor.
Cost accounting formula: the addition amount of nitrogen source per liter system is multiplied by the price of unit mass
As can be seen from Table 6, the cordycepin yields of groups 1, 6, 10, 11 and 12 are high (all exceeding 5.7 g/L), i.e., the imported yeast powder, the imported peptone, the Angel yeast powder FM139 and the mycelium extract powder of the scheme have a significant improvement effect on cordycepin fermentation, wherein the fermentation effect of the scheme is better. In addition, in consideration of cost factors, the mycelium extract powder is used as a fermentation nitrogen source, so that the cost is greatly reduced, and the industrial production is facilitated. In this example, we refer to the previously published paper "Zhouyan et al cordycepin production medium optimized by shallow fermentation of Cordyceps militaris. Jiangxi agricultural journal, 2021, 33:97-102", in which it was analyzed that the amino acid (glutamic acid and aspartic acid) components may be beneficial for cordycepin synthesis. When the nutrient components in the embodiment 5 are compared, the concentration of the two amino acids is higher, so that the addition of the mycelium soaking powder in the nitrogen source has a promoting effect on cordycepin fermentation.
The invention provides a method for soaking edible fungus mycelium powder, a preparation method thereof and an application concept and method thereof as a fermentation nitrogen source, and a plurality of methods and ways for realizing the technical scheme are provided. All the components not specified in the present embodiment can be realized by the prior art.
Claims (14)
1. The preparation method of the edible fungus mycelium soaking powder is characterized by comprising the following steps:
(1) Performing seed culture and fermentation culture on the pretreated edible fungus strain to obtain mycelium;
(2) Mechanically breaking the wall of the mycelium obtained in the step (1), performing alkali treatment, and adding complex enzyme for enzymolysis;
(3) Carrying out solid-liquid separation on the enzymatic hydrolysate obtained in the step (2) to obtain a first supernatant and bacterium residues, carrying out microfiltration treatment on the first supernatant to obtain a microfiltration permeating liquid, uniformly mixing the microfiltration permeating liquid with the bacterium residues, carrying out solid-liquid separation to obtain a second supernatant, carrying out microfiltration treatment on the second supernatant, adding water, and carrying out percolation to obtain a microfiltration concentrated solution;
(4) Adding nuclease into the microfiltration concentrated solution obtained in the step (3) for enzymolysis, and then adding protease for enzymolysis to obtain enzymolysis solution;
(5) And (4) carrying out vacuum concentration and spray drying on the enzymolysis liquid obtained in the step (4) to obtain the mycelium soaked powder of the edible fungi.
2. The preparation method according to claim 1, wherein the edible fungi is any one of pleurotus eryngii, pleurotus ostreatus and clitocybe maxima.
3. The method according to claim 1, wherein the pretreatment in step (1) is carried out by purifying the edible fungi to obtain the edible fungi species.
4. The method according to claim 1, wherein in the step (1), the seed is cultured under the following conditions: culturing at 25-28 deg.C for 4-7 days until the wet weight of corresponding thallus reaches 120-170g/L.
5. The process of claim 1, wherein in step (1), the fermentation culture is carried out in an inoculum size of 9-20%; the culture conditions are as follows: 200-250r/min, culturing for 4-7 days at 25-28 ℃, and the formula of the fermentation medium is as follows: 10-30g/L of corn sugar, 10-25g/L of corn steep liquor, 1-6g/L of ammonium sulfate, 0.1-1g/L of magnesium sulfate, 0.1-0.4g/L of monopotassium phosphate, 0.0001-0.005g/L of manganese sulfate, 0.01-0.1g/L of VB and pH =6.0.
6. The method according to claim 1, wherein in the step (2), the alkali treatment is carried out under the following conditions: adjusting pH to 8-12 with NaOH, and bathing with hot water at 50-60 deg.C for 6-10 hr.
7. The preparation method of claim 1, wherein in the step (2), the complex enzyme is a combination of any several of cellulase, xylanase, glucanase, pectinase, helicase and glycosyl peptidase, and the addition amount of each enzyme is 0.1-0.5% g/100mL.
8. The preparation method according to claim 1, wherein in the step (2), the enzymolysis is carried out under the conditions: adjusting the pH value to 5 by hydrochloric acid, hydrolyzing for 12 hours at 50 ℃, and stirring at the rotating speed of 450r/min.
9. The method according to claim 1, wherein in the step (3), the microfiltration treatment is microfiltration with a filter membrane having a pore size of 0.45 μm.
10. The method according to claim 1, wherein in the step (4), the nuclease is nuclease P1 in an amount of 0.01 to 0.1% w/v; the protease is one or more of papain, flavourzyme, neutrase, alkaline protease and yeast extraction enzyme, and the addition amount is 0.1-0.3% by weight g/100mL.
11. The preparation method according to claim 1, wherein in the step (4), the microfiltration concentrated solution is heated to 68 ℃, nuclease is added for enzymolysis for 4-8 hours, the temperature is reduced to 50-60 ℃,1M sodium hydroxide is used for adjusting the pH value to 7-8, and protease is added for enzymolysis for 8-12 hours.
12. The method according to claim 1, wherein in the step (5), the vacuum concentration and spray drying are performed by concentrating the enzymatic hydrolysate to a dry content of 40-55% by weight, adding 0.1-1% by weight of modified starch per 100mL, mixing uniformly, and drying in a spray dryer, wherein the parameters are as follows: the feeding temperature is 25-35 ℃, the inlet air temperature is 135-160 ℃, the pressure is 20-25mmHg, and the feeding flow is 50-70ml/min.
13. An immersion powder of mycelia of an edible fungus prepared by the method of any one of claims 1 to 11.
14. Use of the mycelium extract powder of edible fungi of claim 12 as a source of nitrogen for fermentation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111607528A (en) * | 2020-06-28 | 2020-09-01 | 广西壮族自治区农业科学院微生物研究所 | Pleurotus californicus strain Pg-w01 and application thereof |
| PH22021051100U1 (en) * | 2021-09-23 | 2022-02-16 | Central Luzon State Univ | SUBSTRATE USED FOR PROPAGATION OF Pleurotus giganteus FRUITING BODIES |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111607528A (en) * | 2020-06-28 | 2020-09-01 | 广西壮族自治区农业科学院微生物研究所 | Pleurotus californicus strain Pg-w01 and application thereof |
| PH22021051100U1 (en) * | 2021-09-23 | 2022-02-16 | Central Luzon State Univ | SUBSTRATE USED FOR PROPAGATION OF Pleurotus giganteus FRUITING BODIES |
Non-Patent Citations (2)
| Title |
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| ALEX GRACA CONTATO 等: ""Comparison between the aqueous extracts of mycelium and basidioma of the edible mushroom Pleurotus pulmonarius:chemical composition and antioxidant analysis"", 《JOURNAL OF FOOD MEASUREMENT AND CHARACTERIZATION》, vol. 14, pages 830 - 837 * |
| 黄忠 等: ""超声波提取灰树花菌丝体蛋白工艺优化"", 《中国酿造》, vol. 35, no. 7, pages 3 - 4 * |
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