CN112011531A - Enzyme preparation produced by using immobilized cell technology and preparation method thereof - Google Patents
Enzyme preparation produced by using immobilized cell technology and preparation method thereof Download PDFInfo
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
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- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract
The invention relates to the technical field of production of enzyme preparations for kitchen waste, and discloses an enzyme preparation produced by using an immobilized cell technology and a preparation method thereof, wherein the enzyme preparation comprises a compound enzyme and a filler, and the filler adjusts the solid content of the compound enzyme to 5-10%; wherein the complex enzyme comprises amylase, protease and cellulase; the preparation method of the enzyme preparation comprises the following steps: treating wet thalli consisting of bacillus and actinomycetes by an immobilized cell technology to obtain immobilized thalli pellets, culturing the immobilized thalli pellets to obtain crude enzyme liquid, concentrating and refining the crude enzyme liquid to obtain refined enzyme liquid, drying the refined enzyme liquid, and adjusting the content of soluble solids to a certain extent by using a filler to prepare the enzyme preparation. The invention solves the technical problem that more than two kinds of complex enzymes cannot be produced by adopting the mixed fermentation of two kinds of strains in the existing production method of the enzyme preparation for kitchen garbage.
Description
Technical Field
The invention relates to the technical field of production of enzyme preparations for kitchen waste, in particular to an enzyme preparation produced by using an immobilized cell technology and a preparation method thereof.
Background
The kitchen waste is a mixture of various substances such as rice, flour, oil, fruits, vegetables, meat, bones and the like, has complex components, high water content and high organic matter content, is easy to decay and deteriorate, breeds bacteria, generates malodor, pathogenic bacteria, pathogenic microorganisms and the like, and has great influence on the healthy life of people. Because the kitchen waste contains a large amount of protein, cellulose, starch, fat and the like and is rich in various trace elements such as nitrogen, phosphorus, potassium, calcium and the like, products such as organic fertilizer and the like can be formed through reasonable treatment, so that the waste is changed into valuable, and the reutilization is realized.
The traditional kitchen waste treatment method, such as sanitary landfill and incineration, can not realize the reutilization of resources, and aggravates the problem of environmental pollution. With the updating and development of technical means, the novel kitchen waste treatment method can gradually realize harmless treatment and resource utilization of the kitchen waste. Such methods can be classified into three categories according to the presence or absence of microorganisms: the first type, microbial treatment, includes aerobic treatment and anaerobic treatment; a second complex enzyme treatment method, which directly utilizes the decomposition of microbial enzymes to treat the kitchen waste; and the third type is a comprehensive treatment method combining complex enzyme and microorganism, for example, firstly, organic garbage is pretreated by using a complex enzyme preparation, and then, pretreated products are decomposed by using biological enzyme.
The second and third harmless kitchen waste treatment methods both require the use of an enzyme preparation, which is a leavening agent using an enzyme as a main functional factor, wherein the enzyme mainly comprises amylase, protease, cellulase, pectinase and the like, and the enzyme is obtained by fermenting beneficial microorganisms such as bacillus, actinomycetes, filamentous fungi, lactic acid bacteria, saccharomycetes and the like, and is mainly used for fermenting organic waste gas in kitchen waste.
Microbial fermentation is the main production method of the current enzyme preparation, compared with the prior method of directly extracting from animal and plant tissues and organs, the method has high yield, low cost and simple process, and the produced enzyme has physical and chemical properties which are basically consistent with the enzyme naturally generated in animal bodies. The enzyme-producing microorganisms widely used in industrial production mainly include molds, bacilli, pseudomonads and some actinomycetes. Trichoderma and Aspergillus in the mold have strong enzyme production capacity and do not produce mycotoxin, so the method is widely applied to the fermentation industry. The bacillus has a strong enzyme production system, can utilize various nutrient substances, can secrete various extracellular enzymes such as neutral protease, alkaline protease, cellulase and the like, and compared with plant-source protease and animal-source protease, the bacillus protease has good biological properties such as high catalytic efficiency, mild action conditions, wide action range, safety and no toxic or side effect, and is widely applied to industrial production at present.
The mode of microbial fermentation mainly includes solid state fermentation and liquid state fermentation. The solid state fermentation refers to a process of fermenting substances to be fermented by utilizing the activity of microorganisms under the condition of no free water or extremely low water content, is the most common fermentation mode at present, and has the characteristics of low technical content, simple equipment requirement and low cost investment. Liquid fermentation refers to a process in which a substance to be fermented is fermented using the activity of microorganisms under the condition that a liquid is used as a medium, and is more suitable for industrial mass production than solid fermentation.
The immobilized cell technology is a method for obtaining enzymes and metabolites of cells, and is a technology in which biological cells having a certain physiological function, such as microbial cells, plant cells, or animal cells, are immobilized by a certain method and utilized as a solid biocatalyst. Because of their ability to perform normal growth, reproduction and metabolism, immobilized cells are also known as immobilized live cells or immobilized proliferating cells.
The enzyme preparation produced by the immobilized cell technology has the following advantages: the method has the advantages that the cell life activity is kept, the cell growth arrest time is short, a plurality of cells are generated, and the reaction is fast; secondly, the separation of cells and product enzyme is facilitated, the activity loss of the enzyme is reduced, and the stability of the enzyme is higher; the fermentation can be recycled, the strain expanding culture operation is omitted, the culture time is saved, the raw materials are saved, the production capacity is improved, and the production cost is reduced.
In the production process of enzyme preparations, most of the prior art adopts a single strain to produce one enzyme by fermentation, and the mode of producing more than two enzyme products by mixed fermentation of more than two strains is less, because different strains are easy to generate antagonism and have different enzyme production mechanisms and are difficult to realize synergistic symbiosis. However, in the actual use process, two or more kinds of complex enzyme preparations have better effects, so that a common production method is to prepare one kind of enzyme separately and then compound different enzymes to prepare the complex enzyme preparation, and the method has the defects of complex production process, low production efficiency, high production cost and the like, and needs technical improvement urgently.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides an enzyme preparation produced by using an immobilized cell technology and a preparation method thereof, aiming at solving the technical problem that more than two kinds of complex enzymes cannot be produced by adopting mixed fermentation of two kinds of strains in the existing production method of the enzyme preparation for kitchen garbage.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme:
an enzyme preparation produced by utilizing an immobilized cell technology comprises a compound enzyme and a filler, wherein the filler adjusts the solid content of the compound enzyme to 5-10%; the complex enzyme comprises amylase, protease and cellulase.
Preferably, the filler comprises starch, white carbon black and maltodextrin.
Preferably, the complex enzyme is produced by mixing and fermenting bacillus and actinomycetes by using an immobilized cell technology.
Preferably, the filler adjusts the solid content of the complex enzyme to 8%.
A preparation method of an enzyme preparation produced by using an immobilized cell technology comprises the following steps:
the method comprises the following steps: activating strains of bacillus and actinomycetes on an improved PDA inclined plane, inoculating the strains to an improved PDA liquid culture medium for culture, centrifugally washing the strains by using sterile normal saline, and taking a thallus precipitate for preservation at 4 ℃ for later use;
step two: treating wet thalli consisting of bacillus and actinomycetes by an immobilized cell technology to obtain immobilized thalli pellets;
step three: placing the immobilized bacteria pellets into an improved PDA liquid culture medium for pre-culture, and then placing the immobilized bacteria pellets into an enzyme production culture medium for culture to obtain a crude enzyme solution;
step four: concentrating the crude enzyme solution to obtain a refined enzyme solution;
step five: and (3) after the refined enzyme liquid is dried, adopting a filler to adjust the solid content of the complex enzyme to 5% -10%, and preparing the enzyme preparation.
Preferably, the preparation of the immobilized bacteria pellet in the second step is as follows: adding 2.5% sodium alginate solution into wet thallus, stirring, and dripping 2% CaCl from height of 10cm away from liquid surface with 8-gauge needle2Filtering out gel pellets from the solution, and replacing the gel pellets with 2% CaCl2In the solution, the solution is kept stand and hardened for 2 hours at 4 ℃.
Preferably, the preparation of the immobilized bacteria pellet in the second step is as follows: adding 1% of chitosan into 2.5% of sodium alginate solution to prepare sodium alginate chitosan colloidal solution; adding wet thallus into the solution, stirring, mixing, and dripping 2% CaCl from height of 10cm away from liquid surface with 8-gauge needle2Filtering out gel pellets from the solution, and adding 2% CaCl again2In the solution, the solution is kept stand and hardened for 2 hours at 4 ℃.
Preferably, the preparation of the immobilized bacteria pellet in the second step is as follows: preparing a thallus suspension from wet thallus; dripping 2.5% chitosan colloidal solution into coagulating liquid (20% methanol and 30% NaOH) with 8-gauge needle, reacting for 2h to obtain hollow chitosan ball, taking out, washing with deionized water, placing into thallus suspension, adsorbing for 2h, taking out, and washing with 0.75% sterile physiological saline.
(III) advantageous technical effects
Compared with the prior art, the invention has the following beneficial technical effects:
the invention processes wet thalli consisting of bacillus and actinomycetes by an immobilized cell technology to obtain immobilized thalli pellets, cultures the immobilized thalli pellets to obtain crude enzyme liquid, concentrates and refines the crude enzyme liquid to obtain refined enzyme liquid, adopts a filling material to adjust the refined enzyme liquid to a certain soluble solid content after drying, prepares an enzyme preparation, and tests show that the enzyme preparation comprises amylase, protease and cellulase, wherein the enzyme combination efficiency is 79.8-84.3%, and the enzyme activity recovery rate is 65.8-76.2%, thereby solving the technical problem that the existing production method of the enzyme preparation for kitchen garbage can not adopt two strains to mix and ferment to produce more than two kinds of complex enzymes.
Detailed Description
The strains of bacillus and actinomycetes involved in the invention are all from 'China Industrial microbiological culture Collection center Gansu Scoring center (GSICC)';
chitosan (degree of deacetylation is more than or equal to 80%), sodium alginate (viscosity range: 1.05-1.15), wherein NaOH, methanol, NaCl, CaCl2The reagents are all made in China and analytically pure;
improving a PDA culture medium: 200g of potato (peeled), 30g of glucose, 2g of peptone, 0.5gKH2PO4，0.5g(NH4)SO410mg of vitamin B11000mL of distilled water, and autoclaving at 115 ℃ for 30 min;
enzyme production culture medium: 5g KCl, 5g NaCl, 5g MgSO4·7H2O，0.75gK2HPO4，0.01gFeSO48.5g of peptone, 0.25g of beef extract, 10g of chitosan and 1000mL of distilled water, wherein the pH value is 7.5-8.0, and the mixture is sterilized at 121 ℃ for 30min under high pressure;
activating strains of bacillus and actinomycetes on an improved PDA inclined plane for 2 times, then inoculating the strains to an improved PDA liquid culture medium, culturing for 16h at 36 ℃ at 180r/min, then centrifugally washing for 2 times at 5000r/min by using 0.75% sterile physiological saline for 20min each time, and taking thalli precipitates to preserve at 4 ℃ for later use;
the first embodiment is as follows:
adopting a sodium alginate embedding method to carry out immobilization treatment on cells:
adding 2g of wet thallus into 50mL of 2.5% sodium alginate solution, stirring, mixing, and dripping 2% CaCl from height of 10cm away from liquid surface with 8-gauge needle2Filtering out gel pellets from the solution, and replacing the gel pellets with 2% CaCl2Standing and hardening the solution for 2 hours at 4 ℃;
putting the immobilized bacteria pellets into an improved PDA liquid culture medium, pre-culturing for 12h at 36 ℃ and 120r/min, then putting the immobilized bacteria pellets into an enzyme production culture medium, and culturing for 24h at 30 ℃ and 120r/min to obtain a crude enzyme solution;
dripping 3% chitosan colloidal solution into the condensate (20% methanol and 30% NaOH) with No. 8 needle, reacting for 2h to obtain hollow chitosan ball, taking out, washing with deionized water for 3 times, and drying with absorbent paper;
then, adding the crude enzyme solution until the crude enzyme solution just submerges the ball, adsorbing for 3 hours, taking out the crude enzyme solution, and washing with deionized water to obtain concentrated 5 times of refined enzyme solution;
spray drying the refined enzyme solution under the following spray drying conditions: the pressure of the atomizer is 0.4MPa, the air inlet temperature is set to be 140 ℃, the air outlet temperature is controlled to be 60 ℃, and then the solid content of the enzyme solution is adjusted to be 5% by adopting filler starch;
tests show that the enzyme preparation comprises amylase, protease and cellulase; wherein the enzyme combination efficiency is 84.3 percent, and the recovery rate of the enzyme activity is 65.8 percent;
example two:
adopting a sodium alginate chitosan embedding method to carry out immobilization treatment on cells:
adding 1% of chitosan into 2.5% of sodium alginate solution to prepare sodium alginate chitosan colloidal solution; adding 2g wet thallus into 50mL of the solution, stirring, mixing, and dripping 2% CaCl from a height of 10cm away from the liquid surface with a No. 8 needle2Filtering out gel pellets from the solution, and adding 2% CaCl again2Standing and hardening the solution for 2 hours at 4 ℃;
putting the immobilized bacteria pellets into an improved PDA liquid culture medium, pre-culturing for 12h at 36 ℃ and 120r/min, then putting the immobilized bacteria pellets into an enzyme production culture medium, and culturing for 24h at 30 ℃ and 120r/min to obtain a crude enzyme solution;
mixing 5mL of sodium alginate-chitosan colloidal solution containing 5% sodium alginate and 2% chitosan with the same amount of the above crude enzyme solution, and dripping into 2% CaCl from 10cm away from the liquid surface with 8-gauge needle2In solution; the gel beads were filtered off and replaced with 2% CaCl2Standing and hardening the solution for 2h at 4 ℃, filtering out gel pellets, and washing the gel pellets with deionized water to obtain 5 times concentrated fine enzyme solution;
spray drying the refined enzyme solution under the following spray drying conditions: the pressure of an atomizer is 0.4MPa, the air inlet temperature is set to be 180 ℃, the air outlet temperature is controlled to be 80 ℃, and then the solid content of the enzyme solution is adjusted to be 8% by using filler white carbon black to obtain an enzyme preparation;
tests show that the enzyme preparation comprises amylase, protease and cellulase; wherein the enzyme combination efficiency is 79.8 percent, and the recovery rate of the enzyme activity is 70.7 percent;
example three:
immobilizing cells by adopting a chitosan adsorption method:
2g of wet thalli is taken to prepare a thallus suspension;
dripping 2.5% chitosan colloidal solution into coagulating liquid (20% methanol and 30% NaOH) with No. 8 needle, reacting for 2h to obtain hollow chitosan ball, taking out, washing with deionized water, placing into thallus suspension, adsorbing for 2h, taking out the chitosan ball, and washing with 0.75% sterile physiological saline;
putting the immobilized bacteria pellets into an improved PDA liquid culture medium, pre-culturing for 12h at 36 ℃ and 120r/min, then putting the immobilized bacteria pellets into an enzyme production culture medium, and culturing for 24h at 30 ℃ and 120r/min to obtain a crude enzyme solution;
adding 1% (w/v) of activated carbon into the crude enzyme solution for decolorization, sequentially filtering the filtrate by qualitative filter paper through water-phase membranes with the pore diameters of 0.8um, 0.45um and 0.22um, and performing sterilization filtration to obtain a refined enzyme solution concentrated by 5 times;
spray drying the refined enzyme solution under the following spray drying conditions: the pressure of the atomizer is 0.4MPa, the air inlet temperature is set to be 160 ℃, the air outlet temperature is controlled to be 70 ℃, and then the solid content of the enzyme solution is adjusted to 10 percent by adopting maltodextrin as a filling material;
tests show that the enzyme preparation comprises amylase, protease and cellulase; wherein the enzyme combination efficiency is 72.4 percent, and the recovery rate of the enzyme activity is 76.2 percent.
Claims (8)
1. An enzyme preparation produced by utilizing an immobilized cell technology is characterized by comprising a compound enzyme and a filler, wherein the filler adjusts the solid content of the compound enzyme to 5% -10%; the complex enzyme comprises amylase, protease and cellulase.
2. The enzyme preparation produced by using the immobilized cell technology as claimed in claim 1, wherein the filler comprises starch, white carbon black and maltodextrin.
3. The enzyme preparation produced by using the immobilized cell technology as claimed in claim 2, wherein the complex enzyme is produced by mixed fermentation of two strains of bacillus and actinomycetes by using the immobilized cell technology.
4. The enzyme preparation produced by using the immobilized cell technology as claimed in claim 3, wherein the filler adjusts the solid content of the complex enzyme to 8%.
5. A method for preparing an enzyme preparation produced by using an immobilized cell technology is characterized by comprising the following steps:
the method comprises the following steps: activating strains of bacillus and actinomycetes on an improved PDA inclined plane, inoculating the strains to an improved PDA liquid culture medium for culture, centrifugally washing the strains by using sterile normal saline, and taking a thallus precipitate for preservation at 4 ℃ for later use;
step two: treating wet thalli consisting of bacillus and actinomycetes by an immobilized cell technology to obtain immobilized thalli pellets;
step three: placing the immobilized bacteria pellets into an improved PDA liquid culture medium for pre-culture, and then placing the immobilized bacteria pellets into an enzyme production culture medium for culture to obtain a crude enzyme solution;
step four: concentrating the crude enzyme solution to obtain a refined enzyme solution;
step five: and (3) after the refined enzyme liquid is dried, adopting a filler to adjust the solid content of the complex enzyme to 5% -10%, and preparing the enzyme preparation.
6. The method for preparing enzyme preparation using immobilized cell technology as claimed in claim 5, wherein the immobilized bacteria pellet in step two is prepared by: adding 2.5% sodium alginate solution into wet thallus, stirring, and dripping 2% CaCl from height of 10cm away from liquid surface with 8-gauge needle2Filtering out gel pellets from the solution, and replacing the gel pellets with 2% CaCl2In the solution, the mixture is kept stand at 4 ℃ to harden.
7. The method for preparing enzyme preparation using immobilized cell technology as claimed in claim 5, wherein the immobilized bacteria pellet in step two is prepared by: adding 1% of chitosan into 2.5% of sodium alginate solution to prepare sodium alginate chitosan colloidal solution; adding wet thallus into the solution, stirring, mixing, and dripping 2% CaCl from height of 10cm away from liquid surface with 8-gauge needle2Filtering out gel pellets from the solution, and adding 2% CaCl again2In the solution, the mixture is kept stand at 4 ℃ to harden.
8. The method for preparing enzyme preparation using immobilized cell technology as claimed in claim 5, wherein the immobilized bacteria pellet in step two is prepared by: preparing a thallus suspension from wet thallus; dripping 2.5% chitosan colloidal solution into coagulating liquid (20% methanol and 30% NaOH) with 8-gauge needle to obtain hollow chitosan ball, taking out, washing with deionized water, placing into thallus suspension, adsorbing, taking out, and washing with sterile normal saline.
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Cited By (3)
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| CN112746090A (en) * | 2020-12-29 | 2021-05-04 | 南宁东恒华道生物科技有限责任公司 | Kitchen waste enzymolysis treatment process |
| CN112808257A (en) * | 2021-01-14 | 2021-05-18 | 宁波林叶生物科技有限公司 | Preparation method of sodium alginate affinity chromatography medium and application of sodium alginate affinity chromatography medium in extraction of hyaluronidase |
| CN113862249A (en) * | 2021-10-20 | 2021-12-31 | 徐州工程学院 | Preparation and application of recombinant beta-1, 4-endoglucanase immobilized cell |
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| US9605247B2 (en) * | 2013-12-23 | 2017-03-28 | Hunan Hong Ying Biotech Co., Ltd. | Strain and a method to produce cellulase and its use |
| CN109554302A (en) * | 2018-12-26 | 2019-04-02 | 江苏牧之歌生态农业科技有限公司 | A method of utilizing immobilized cell technology fermenting and producing fodder enzyme preparation |
| CN111440785A (en) * | 2020-02-28 | 2020-07-24 | 浙江工业大学 | Method for immobilizing glucose isomerase-containing cells by using modified diatomite |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103602656A (en) * | 2013-11-12 | 2014-02-26 | 柳州市净元生物科技有限公司 | Method for preparing immobilized enzymes and immobilized strains |
| US9605247B2 (en) * | 2013-12-23 | 2017-03-28 | Hunan Hong Ying Biotech Co., Ltd. | Strain and a method to produce cellulase and its use |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112746090A (en) * | 2020-12-29 | 2021-05-04 | 南宁东恒华道生物科技有限责任公司 | Kitchen waste enzymolysis treatment process |
| CN112808257A (en) * | 2021-01-14 | 2021-05-18 | 宁波林叶生物科技有限公司 | Preparation method of sodium alginate affinity chromatography medium and application of sodium alginate affinity chromatography medium in extraction of hyaluronidase |
| CN112808257B (en) * | 2021-01-14 | 2022-05-20 | 宁波林叶生物科技有限公司 | Preparation method of sodium alginate affinity chromatography medium and application of sodium alginate affinity chromatography medium in hyaluronidase extraction |
| CN113862249A (en) * | 2021-10-20 | 2021-12-31 | 徐州工程学院 | Preparation and application of recombinant beta-1, 4-endoglucanase immobilized cell |
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Application publication date: 20201201 |