CN110607287A - Method for producing laccase and co-producing animal feed by industrially utilizing pleurotus eryngii mushroom bran solid fermentation - Google Patents
Method for producing laccase and co-producing animal feed by industrially utilizing pleurotus eryngii mushroom bran solid fermentation Download PDFInfo
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Abstract
The invention discloses a method for producing laccase and co-producing animal feed by industrially utilizing pleurotus eryngii mushroom bran solid fermentation. The invention relates to a method for producing laccase and co-producing animal feed by solid fermentation under certain conditions by using pleurotus eryngii fungus chaff as a strain and a base material, bagasse, oil-tea-cake meal and ginger slag as carbon sources, ammonium nitrate as a nitrogen source and mixed inorganic salt as an inducer. The method for industrially co-producing the laccase and the animal feed by using the pleurotus eryngii dregs as the main raw material has the advantages of high yield, mild process conditions, short preparation period, no use of expensive raw materials such as peptone and the like, low cost, simple operation and industrial production. The invention also provides an effective way for high-value utilization of waste biomass.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing laccase and co-producing animal feed by industrially utilizing pleurotus eryngii mushroom bran solid fermentation.
Background
The edible fungus chaff, called fungus chaff, fungus dreg, leftover and the like for short, refers to the culture medium waste after the edible fungus goes out of the mushroom, contains a large amount of components such as edible fungus hypha residues, extracellular enzyme, polysaccharide, crude fiber degraded by biological enzyme, lignin which is not completely utilized and the like, and is a high-quality biomass resource. According to statistics of edible fungus society in China, the total yield of edible fungi in China in 2017 is 3712 ten thousand tons, and the total yield of fungus residues is 5568 ten thousand tons according to the average biotransformation rate of the edible fungi of 40 percent. Due to the lack of corresponding treatment technology, poor supervision and the like of edible fungus cultivators, a large amount of fungus residues are not reasonably applied but are randomly discarded, so that biomass resource waste, cultivation environment and water body pollution are caused, and meanwhile, harm is caused to human health.
During the growth and fruiting process of mycelium, the edible fungi can secrete extracellular enzymes for degrading lignocellulose, including laccase, lignin peroxidase, cellulase, xylanase and the like, wherein the laccase is the most widely researched. Laccases are a class of copper ion-containing polyphenol oxidases. It was first discovered by Japanese scientists in Lacca Lacquertree and was formally named laccase in 1894. The laccase has wide acting substrates, can utilize various compounds such as phenols, amines and the like as the substrates, finally reduces molecular oxygen into water, does not generate other byproducts, is also called as a green catalyst, and has very good application in the degradation aspect of toxicants, the construction of biosensors, the paper industry and other fields. And the laccase has good thermal stability, so that the laccase can be reused in various special fields, such as the fields of functional polymer synthesis, sewage treatment and the like.
The fungi can synthesize laccase in the growth process and secrete the laccase to the outside during the hypha growth process. Such as Pleurotus ostreatus, Pleurotus eryngii, Pleurotus citrinopileatus and the like, but laccase enzyme activity is not high generally. The invention patent application CN201310712411.9 'a method for preparing laccase', the production strain is tiger skin mushroom, the laccase fermentation enzyme activity is 462.59U/mL; the invention patent application CN200910083514.7 'A laccase and its preparation method and special production strain', the production strain is dense red hole fungus, the laccase fermentation enzyme activity is 300U/mL; the invention patent application CN 101942421B' a method for producing laccase by fermenting Shiraia bambusicola, wherein the laccase fermentation enzyme activity is 500U/mL; the invention patent application CN200810198678.X discloses a lucid ganoderma strain for producing laccase, wherein the laccase fermentation enzyme activity is 8620U/mL, and the lucid ganoderma strain is a strain with higher enzyme activity at present. The laccase is obtained by fermenting fungi, a large amount of fresh culture medium is required to be added, and the production cost is high.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a method for producing laccase and co-producing animal feed by solid fermentation of pleurotus eryngii bran.
The technical scheme of the invention is summarized as follows:
a method for producing laccase and co-producing animal feed by industrially utilizing pleurotus eryngii fungus chaff solid fermentation comprises the following steps:
crushing fresh pleurotus eryngii fungus chaff according to the proportion of 1 Kg: adding 1-2L of fresh culture medium in a mass-volume ratio, fermenting for 3-7 days at 25-30 ℃ in a dark place, and then fermenting according to the weight-volume ratio of 1 Kg: adding water in a mass-volume ratio of 10L, stirring for 6h, and filtering to obtain a filtrate, namely laccase crude enzyme solution; 1Kg of filter residue: 1X 106Inoculating lactobacillus into the CFU, uniformly mixing, putting into an anaerobic fermentation tank, compacting and fermenting for 7-14 days to obtain the feed suitable for the ruminant.
The formula of the fresh culture medium is as follows: 7-16 wt% of carbon source, 8-15 g/L of nitrogen source and KH2PO4 0.2～0.6g/L，ZnSO4·7H2O0.05g/L，MgSO4·7H2O 0.03g/L，CuSO4·5H2O 0.2～0.6g/L，CaCl2·2H2O 0.008g/L，MnSO4·H2O0.03～0.08g/L。
Preferably, the pleurotus eryngii fungus chaff is fresh fungus chaff within 3 days of picking the fungus sticks.
Preferably, the carbon source in the culture medium is a combination of 4-7 wt% of bagasse, 3-6 wt% of oil-tea camellia cake and 0-3 wt% of ginger residue.
Preferably, the camellia oleifera cake in the culture medium is camellia oleifera cake after the tea saponin is extracted.
Preferably, the ginger residue in the culture medium is ginger residue after extraction of ginger essential oil and gingerol.
Preferably, the nitrogen source in the culture medium is ammonium nitrate with concentration of 10-12 g/L.
Preferably, the culture medium formula is as follows: the combination of 6wt% of bagasse as a carbon source, 4 wt% of oil-tea-cake meal and 2 wt% of ginger residue as a carbon source, 10g/L of ammonium nitrate as a nitrogen source, KH2PO4 0.5g/L，ZnSO4·7H2O 0.05g/L，MgSO4·7H2O 0.03g/L，CuSO4·5H2O 0.4g/L，CaCl2·2H2O 0.008g/L，MnSO4·H2O 0.05g/L。
Preferably, the anaerobic fermentation time is 8-10 days.
Has the advantages that:
the invention relates to a method for producing laccase and co-producing animal feed by solid fermentation at a certain temperature by using pleurotus eryngii fungus chaff as a strain and a base material, bagasse, oil-tea-cake meal and ginger slag as carbon sources, ammonium nitrate as a nitrogen source and mixed inorganic salt as an inducer as auxiliary materials. Compared with the prior art, the invention has the following remarkable progress:
1. the method for producing laccase and co-producing animal feed by fermenting pleurotus eryngii dregs has low production cost, can prolong the pleurotus eryngii industrial chain and increase the income of enterprises.
2. The base materials used in the method are all industrial waste biomass, so that waste biomass resources are effectively utilized, and the method is green and environment-friendly.
3. The enzyme activity of the laccase obtained by the invention is as high as 812U/g, which is nearly 40 times of the initial enzyme activity of 21U/g of pleurotus eryngii dregs.
4. The method prepares the animal feed by fermenting the pleurotus eryngii dregs after producing the laccase, can effectively shorten the fermentation time and simultaneously improve the nutritional value of the feed.
5. The method provided by the invention utilizes pleurotus eryngii dregs as a main raw material to industrially co-produce laccase and animal feed, has the advantages of high yield, mild process conditions, short preparation period, no use of expensive raw materials such as peptone and the like, low cost and simple operation, and is suitable for industrial popularization and application.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.
Example 1
Crushing 10Kg of fresh pleurotus eryngii fungus chaff (collected from Yuxiu biological science and technology limited company in Hunan province, with initial enzyme activity of 21U/g, the same below) within 3 days after picking the fungus sticks in a clean workshop section, and then sieving with a 70-mesh sieve according to the weight ratio of 1 Kg: adding 1L of culture medium according to the mass-volume ratio, fermenting for 7 days at 25 ℃ in a dark place, and then adding 1Kg of culture medium: adding 10L of water according to the mass-volume ratio, stirring for 6h, and filtering to obtain a filtrate, namely laccase crude enzyme solution, wherein the enzyme activity of the solid fermentation culture is 812U/g; 1Kg of filter residue: 1X 106Inoculating commercial lactobacillus into the CFU, uniformly mixing, putting into an anaerobic fermentation tank, compacting, and fermenting for 8 days to obtain the feed suitable for the ruminant.
The culture medium is as follows: 6wt% of bagasse, 4 wt% of oil-tea-cake meal and 2 wt% of ginger residue, 10g/L of ammonium nitrate and KH2PO4 0.5g/L，ZnSO4·7H2O 0.05g/L，MgSO4·7H2O 0.03g/L，CuSO4·5H2O 0.4g/L，CaCl2·2H2O 0.008g/L，MnSO4·H2O 0.05g/L。
TABLE 1 test values of indexes before and after fermentation of Pleurotus eryngii residue,% n-3
Example 2
10kg of fresh pleurotus eryngii fungus chaff within 3 days of picking the fungus sticks is put on a vehicleCrushing in a clean interval, and then sieving by a 90-mesh sieve according to the weight ratio of 1 Kg: adding 2L of culture medium according to the mass-volume ratio, fermenting for 5 days at 28 ℃ in the dark, and then adding 1Kg of culture medium: adding 10L of water according to the mass-volume ratio, stirring for 6h, and filtering to obtain a filtrate, namely crude laccase enzyme solution, wherein the enzyme activity of the solid fermentation culture is 675U/g. 1Kg of filter residue: 1X 106Inoculating commercial lactobacillus into the CFU, uniformly mixing, putting into an anaerobic fermentation tank, compacting, and fermenting for 7 days to obtain the feed suitable for the ruminant.
The culture medium is as follows: a combination of carbon source 4 wt% bagasse and 3 wt% ginger residue, nitrogen source 12(g/L) ammonium nitrate, KH2PO40.6(g/L)，ZnSO4·7H2O 0.05(g/L)，MgSO4·7H2O 0.03(g/L)，CuSO4·5H2O 0.4(g/L)，CaCl2·2H2O 0.008g/L)，MnSO4·H2O 0.05(g/L)。
TABLE 2 Pleurotus eryngii residue before and after fermentation, each index test value,% n ═ 3
Example 3
Crushing 10Kg of fresh pleurotus eryngii fungus chaff within 3 days after picking the fungus sticks in a clean workshop interval, and sieving the crushed material through a 80-mesh sieve according to the weight ratio of 1 Kg: adding 1L of culture medium according to the mass-volume ratio, fermenting for 7 days at 25 ℃ in a dark place, and then adding 1Kg of culture medium: adding 10L of water according to the mass-volume ratio, stirring for 6h, and filtering to obtain a filtrate, namely laccase crude enzyme solution, wherein the enzyme activity of the solid fermentation culture is 783U/g. 1Kg of filter residue: 1X 106Inoculating commercial lactobacillus into the CFU, uniformly mixing, putting into an anaerobic fermentation tank, compacting, and fermenting for 9 days to obtain the feed suitable for the ruminant.
The culture medium is as follows: a combination of 5 wt% (w/w) bagasse as a carbon source, 6wt% oil-tea-cake and 3 wt% ginger slag as a nitrogen source, 14(g/L) ammonium nitrate, KH2PO4 0.5(g/L)，ZnSO4·7H2O 0.05(g/L)，MgSO4·7H2O 0.03(g/L)，CuSO4·5H2O 0.6(g/L)，CaCl2·2H2O 0.008g/L)，MnSO4·H2O 0.05(g/L)。
TABLE 3 test values of indexes before and after fermentation of Pleurotus eryngii residue,% n is 3
Example 4
Crushing 10Kg of fresh pleurotus eryngii fungus chaff within 3 days after picking the fungus sticks in a clean workshop interval, and sieving the crushed material through a 70-mesh sieve according to the weight ratio of 1 Kg: adding 1L of culture medium according to the mass-volume ratio, fermenting for 7 days at 25 ℃ in a dark place, and then adding 1Kg of culture medium: adding water in a mass-volume ratio of 10L, stirring for 6h, and filtering to obtain a filtrate, namely laccase crude enzyme solution; the enzyme activity of the solid fermentation culture is 766U/g. 1Kg of filter residue: 1X 106Inoculating commercial lactobacillus into the CFU, uniformly mixing, putting into an anaerobic fermentation tank, compacting, and fermenting for 10 days to obtain the feed suitable for the ruminant.
The culture medium is as follows: a combination of 2 wt% of bagasse as a carbon source, 6wt% of oil-tea-cake meal and 3 wt% of ginger residue as a carbon source, 11(g/L) of ammonium nitrate as a nitrogen source, KH2PO4 0.3(g/L)，ZnSO4·7H2O 0.05(g/L)，MgSO4·7H2O 0.03(g/L)，CuSO4·5H2O 0.5(g/L)，CaCl2·2H2O 0.008g/L)，MnSO4·H2O 0.08(g/L)。
TABLE 4 Pleurotus eryngii residue before and after fermentation, each index test value% n is 3
Example 5
Crushing 10Kg of fresh pleurotus eryngii fungus chaff within 3 days after picking the fungus sticks in a clean workshop interval, and sieving the crushed material through a 70-mesh sieve according to the weight ratio of 1 Kg: adding 1L of culture medium according to the mass-volume ratio, fermenting for 7 days at 25 ℃ in a dark place, and then adding 1Kg of culture medium: adding 10L of water according to the mass-volume ratio, stirring for 6h, and filtering to obtain a filtrate, namely crude laccase solution, wherein the enzyme activity of the solid fermentation culture is 778U/g. 1Kg of filter residue: 1X 106The ratio of CFU was inoculated with commercial lactobacillus and mixedAfter being mixed evenly, the mixture is put into an anaerobic fermentation tank, compacted and fermented for 8 days, and the feed suitable for being eaten by the ruminants is obtained.
The culture medium is as follows: a combination of 6wt% of bagasse as a carbon source, 6wt% of oil-tea-cake meal and 1 wt% of ginger residue as a carbon source, 11(g/L) ammonium nitrate as a nitrogen source, KH2PO4 0.3(g/L)，ZnSO4·7H2O 0.05(g/L)，MgSO4·7H2O 0.03(g/L)，CuSO4·5H2O 0.5(g/L)，CaCl2·2H2O 0.008g/L)，MnSO4·H2O 0.08(g/L)。
TABLE 5 test values of indexes before and after fermentation of Pleurotus eryngii residue,% n is 3
The enzyme activity determination method in the above examples is as follows:
(1) enzyme activity determination of crude enzyme liquid laccase
DMP is taken as a substrate, and the reaction system of 3mL contains 10 mmol.L-1DMP 0.5mL，20mmol·L-12.4mL of a phosphate-sodium citrate buffer solution having a pH of 3.0 and 0.1mL of the crude enzyme solution were measured for the change in absorbance of the reaction solution within 1min at 469 nm. 1 enzyme activity unit is defined as the amount of enzyme that oxidizes 1. mu. mol DMP per minute under the current reaction conditions. According to the definition of enzyme activity and the beer-Lambert law, the calculation formula of the enzyme activity of the crude enzyme liquid is as follows:
crude enzyme liquid enzyme activity
Wherein Δ OD represents the difference in absorbance over the reaction time; vGeneral assemblyRepresents the total volume of the reaction system, in units of: mL; n represents the dilution factor; Δ t represents reaction time in units: min, VEnzymeThe volume of the crude enzyme solution added to the reaction system is expressed in units of: mL; ε is the molar extinction coefficient of the oxidation product 3,5,3 ', 5' -tetramethoxydiphenylquinone of DMP469＝4.96×104L·mol-1·cm-1(ii) a b represents the distance light travels in the medium, i.e. the cuvette width.
(2) Measurement of Water content of fermentation culture
And (3) putting 1g of fermented mushroom dreg culture in a 60 ℃ oven, drying to constant weight, and calculating to obtain the water content beta.
(3) Calculating the enzyme activity of the solid fermentation culture: 1g of fermented mushroom dreg culture is taken and placed in a 60 ℃ oven, and dried to constant weight to obtain dry yeast; the enzyme activity of the solid fermentation culture is expressed by the enzyme activity U/g contained in each gram of the dry yeast. The enzyme activity calculation formula is as follows:
u/g wet yeast is crude enzyme liquid enzyme activity x 10
U/g dry yeast ═ U/g wet yeast ÷ (1-beta)
(4) In the examples, the crude protein assay was performed according to GB/T15673; coarse ash determination reference GB/T12532; crude fiber determination
Reference GB/T5515; lignin assay is referred to GB/T20805.
Claims (8)
1. A method for producing laccase and co-producing animal feed by industrially utilizing pleurotus eryngii fungus chaff solid fermentation is characterized by comprising the following steps:
crushing fresh pleurotus eryngii mushroom bran, adding a fresh culture medium according to the mass volume ratio of 1Kg to 1 ~ 2L, fermenting for 3 ~ 7 days at 25 ~ 30 ℃ in the dark, then adding water according to the mass volume ratio of 1Kg to 10L, stirring for 6h, filtering to obtain a filtrate, namely a laccase crude enzyme solution, and filtering residues according to the mass volume ratio of 1Kg to 1 × 106Inoculating lactobacillus into the CFU, mixing, placing into an anaerobic fermentation tank, compacting, and fermenting for 7 ~ 14 days to obtain feed suitable for ruminant;
the fresh culture medium comprises 7wt% of carbon source 7 ~ 16, 8 g/L of nitrogen source 8 ~ 15g/L and KH2PO4 0.2~0.6g/L， ZnSO4·7H2O 0.05g/L，MgSO4·7H2O 0.03g/L， CuSO4·5H2O 0.2~0.6g/L，CaCl2·2H2O 0.008g/L，MnSO4·H2O 0.03~0.08g/L。
2. The method according to claim 1, wherein the Pleurotus eryngii mushroom bran is fresh mushroom bran within 3 days of picking mushroom sticks.
3. The method according to claim 1, wherein the carbon source in the culture medium is a combination of 4 ~ 7wt% bagasse, 3 ~ 6wt% oil tea cake, and 0 ~ 3 wt% ginger residue.
4. The method according to claim 3, characterized in that the oil tea cake in the culture medium is oil tea cake after extraction of tea saponin.
5. The method according to claim 3, wherein the ginger residue in the culture medium is ginger residue after extraction of ginger essential oil and gingerol.
6. The method according to claim 1, wherein the nitrogen source in the medium is ammonia nitrate at 10 ~ 12 g/L.
7. The method according to any one of claims 1 to 6, wherein the culture medium is: the combination of 6wt% of bagasse as a carbon source, 4 wt% of oil-tea-cake meal and 2 wt% of ginger residue as a carbon source, 10g/L of ammonium nitrate as a nitrogen source, KH2PO4 0.5g/L，ZnSO4·7H2O 0.05g/L，MgSO4·7H2O 0.03g/L，CuSO4·5H2O 0.4g/L，CaCl2·2H2O 0.008g/L，MnSO4·H2O 0.05g/L。
8. The method of claim 1, wherein the anaerobic fermentation time is 8 ~ 10 days.
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| CN112680365A (en) * | 2021-02-02 | 2021-04-20 | 吉林农业大学 | Liquid culture medium for beauveria bassiana and preparation method of beauveria bassiana microbial inoculum |
| CN115125152A (en) * | 2022-03-28 | 2022-09-30 | 湖南科技学院 | Mixed bacteria for degrading lignocellulose, mixed enzyme and degradation method |
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