CN114309008A - Method for promoting conversion of food waste into reducing sugar - Google Patents
Method for promoting conversion of food waste into reducing sugar Download PDFInfo
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- CN114309008A CN114309008A CN202111559989.6A CN202111559989A CN114309008A CN 114309008 A CN114309008 A CN 114309008A CN 202111559989 A CN202111559989 A CN 202111559989A CN 114309008 A CN114309008 A CN 114309008A
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- 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
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- 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
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Abstract
The invention provides a method for promoting food waste to convert into reducing sugar, which comprises the following steps: crushing the food waste, extracting oil and pulping to obtain high-concentration food waste thick slurry; directly mixing choline chloride and glycerol without adding water to prepare a deep eutectic solvent; uniformly mixing the thick slurry of the food waste with the deep eutectic solvent, and performing ultrasonic centrifugation to collect solid-phase sediment of the food waste; inoculating aspergillus into a container containing an enzyme production culture medium, extracting a crude enzyme solution after culture, and adding protease to obtain a mixed enzyme preparation; adding water into the solid phase precipitate to form a garbage homogeneous solution, and adding a mixed enzyme preparation to obtain a first mixture; placing the mixture in an enzymolysis tank, placing the mixture in a constant-temperature shaking table, oscillating to obtain a second mixture, and monitoring the concentration of fermentable sugar in the second mixture to regulate the enzyme amount. The invention can effectively shorten the reaction time and reduce the required temperature; the enzyme hydrolysis inhibition phenomenon caused by overhigh concentration of the product is reduced, the utilization rate of the enzyme is high, the cost is saved, and the reducing sugar can be quickly released from the biomass.
Description
Technical Field
The invention belongs to the field of solid waste treatment in environmental engineering, and particularly relates to a method for promoting conversion of food waste into reducing sugar.
Background
With the rapid development of the catering industry in China and the popularization of a garbage classification policy, the yield of wet garbage in China is improved year by year, wherein the kitchen garbage is one of main components of the wet garbage, and the national kitchen garbage yield in 2019 reaches 1.2 hundred million tons. The food waste is an organic raw material with strong biotransformation potential, contains a large amount of carbohydrates, proteins and oil nutrients, and can be used for producing biological energy sources such as methane, organic fertilizers, Volatile Fatty Acids (VFAs) and biomass fuel ethanol, so that the food waste has a great market prospect for recycling the food waste.
However, the kitchen waste particles are large, and organic matters are wrapped by solid phases, so that the kitchen waste particles are often difficult to be subsequently utilized by microorganisms, and the resource conversion efficiency of the kitchen waste is limited. The food waste contains a large amount of starch and cellulose which are not easy to dissolve and need to be decomposed for a long time, and the hydrolysis speed of the carbohydrate substances limits the fermentation starting time. In the biotransformation process, saccharides are preferentially utilized by microorganisms compared with proteins and lipids, and fermentable sugars are the most easily utilized organic matters in the saccharides and can be biotransformed in a short time, so that the increase of the concentration of reducing sugars in the food waste is one of important means for enhancing the biodegradability of the food waste.
In order to convert macromolecular saccharides (starch, cellulose and the like) into small-molecular fermentable sugars, the food waste is usually pretreated to solubilize and hydrolyze organic matters in the waste. Because of its stable structure, cellulose and hemicellulose rich in food waste have strong resistance to disintegration, and therefore, proper pretreatment is required to deconstruct their interwoven components and destroy the H-bond network of biomass, making polysaccharides more susceptible to hydrolysis. Although ionic liquids are considered to be ideal pre-treatment agents due to their specific solvating properties, their high synthesis cost, toxicity and biodegradability restrict their industrial use.
The deep eutectic solvent has very similar solvation characteristics to the ionic liquid, and has many advantages, such as good biocompatibility with saccharifying enzyme, easy preparation, biodegradability and the like, so that the deep eutectic solvent has a very good application prospect as a green and sustainable substitute of the ionic liquid. The deep eutectic solvent may be separated after pretreatment and reused in subsequent pretreatment cycles. However, the relatively high viscosity of the deep eutectic solvent may be a limiting factor that may hinder the speed of biomass pretreatment.
The enzymolysis method is also a method widely applied to increasing the concentration of fermentable sugar in various raw materials, has the advantages of no need of additional equipment, energy conservation, simple operation, no by-product and the like, can hydrolyze various macromolecular saccharides including cellulose, and can further increase the concentration of reducing sugar in the food waste slurry. The widely-researched fermentable sugar-producing substrates comprise commercial starch, crop straws, bamboos and the like at present, but have the problems of high raw material cost and low utilization of urban raw materials; in addition, the enzyme reagent mainly adopts saccharifying enzyme, cellulase and hemicellulase, has single composition and higher commercial enzyme price, and restricts the large-scale application of the enzymolysis method. The urban food waste is low in price and easy to obtain, but has the characteristic of complex components, and a compound enzyme reagent aiming at the characteristics of the food waste in China is lacked at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for promoting the conversion of food waste into reducing sugar. Aiming at the characteristics of high solid content and abundant starch and cellulose substances in food wastes in China, the invention utilizes mixed carbohydrase produced by Aspergillus niger to supplement extra protease, strengthens the hydrolysis of insoluble macromolecular sugar in the food wastes by a method of adding the enzyme in batches, greatly improves the concentration of fermentable sugar in the food wastes, controls the adding amount of enzyme liquid by monitoring the concentration of the fermentable sugar in real time by a control system, and provides a feasible method for solving the problems of low content of readily available carbohydrates in the food wastes, lack of configuration of targeted complex enzyme and high enzymatic treatment cost.
The technical scheme of the invention is as follows:
the invention provides a specific step for promoting the conversion of food waste into reducing sugar, which comprises the following steps:
(1) crushing the food waste, extracting oil and pulping to obtain high-concentration food waste thick slurry;
(2) directly mixing choline chloride and glycerol without adding water, heating and stirring the obtained mixture until uniform and transparent liquid is formed, and preparing a deep eutectic solvent with the components of choline chloride/glycerol;
(3) cooling the deep eutectic solvent obtained in the step (2) to room temperature before use, uniformly mixing the thick slurry of the food waste obtained in the step (1) with the deep eutectic solvent, carrying out ultrasonic pretreatment on the mixture, cooling the mixture to room temperature after the pretreatment, recycling the mixed solution containing the deep eutectic solvent after centrifugation for the next round of pretreatment, and collecting solid-phase sediment of the food waste for subsequent enzymatic treatment;
(4) inoculating aspergillus into a container containing an enzyme production culture medium, and extracting a crude enzyme solution after culture; adding protease to obtain mixed enzyme preparation;
(5) adding water into the solid phase precipitate obtained in the step (3) to form a garbage homogeneous liquid, adjusting the solid concentration TS, adjusting the pH value of the garbage homogeneous liquid, and adding the mixed enzyme preparation obtained in the step (4) to obtain a first mixture;
(6) placing the first mixture obtained in the step (5) in an enzymolysis tank, placing the enzymolysis tank in a constant-temperature shaking table, oscillating to obtain a second mixture, monitoring the concentration of fermentable sugar in the second mixture through a control system, supplementing a mixed enzyme preparation in an intermittent feeding mode, and regulating the enzyme amount through feedback.
Further, the grain diameter of the concentrated meal beverage garbage slurry in the step (1) is less than 1 mm; the oil content after oil extraction and pulping is lower than 4 percent; the concentration of TS is 200-300 g/L.
Further, the choline chloride and the glycerol in the step (2) are added into 3-12 mol of glycerol according to the proportion of 1mol of choline chloride, the obtained mixture is heated at 50-70 ℃ and stirred at 120-250 rpm for 1-3 h until uniform and transparent liquid is formed, and the deep eutectic solvent is prepared.
Further, in the step (3), the thick slurry of the food waste obtained in the step (1) and the deep eutectic solvent are uniformly mixed according to the volume ratio of 1: 1-5; the frequency of ultrasonic pretreatment is 15-30 kHz, the amplitude is 30-60%, the power is 30-50 w, the pulse period is 20-30 seconds on/10 seconds off, and the treatment time is 15-30 min.
Further, in the step (4), the Aspergillus is Aspergillus niger;
further, the culture mode is solid state fermentation; the culture method comprises the steps of adding 10 g-20 g of enzyme production culture medium into a triangular flask, sterilizing in a sterilization pot at 121 ℃ for 20min, cooling to 30 ℃, and inoculating 1 mL-3 mL of Aspergillus niger spore suspension; wherein the concentration of the Aspergillus niger spore suspension is 0.8-1.2 multiplied by 10-8CFU/mL；
Further, the method for extracting the crude enzyme solution comprises the steps of taking 10g of the cultured bacterial solution after 3-6d, adding 40-60 mL of sodium acetate buffer solution with the pH value of 4.5-4.7, putting the mixture into a shaking table with the temperature of 38-45 ℃ and the rotating speed of 150-180 rpm for leaching for 1-2 h, and filtering and centrifuging to obtain the crude enzyme solution;
further, the enzyme production culture medium formula is 10g/L, MgSO g of glucose4·7H2O 1.0g/L、(NH4)2SO43.0g/L、KH2PO4 3.0g/L、NaH2PO4·2H2O 1.69g/L、MnSO4·H2O 0.04g/L、ZnCl2 0.02g/L、CaCl2 0.076g/L、CuSO4·5H2O 0.015g/L、CoCl2·6H2O 0.015g/L、FeSO4·7H20.3g/L of O and 0.67g/L of EDTA-2 Na;
further, the activity of the crude enzyme liquid enzyme is 14000-22000U/mL, the hydrolysis capacity of the crude enzyme liquid on macromolecular sugar substances is strong, wherein 1 enzyme activity unit is defined as the enzyme amount required for forming 1mg of reducing sugar in 1 hour;
further, the protease activity is 350-700 LAPU/g, wherein 1LAPU namely leucine aminopeptidase unit is the enzyme amount required for hydrolyzing 1 millimole of L-leucine-p-nitroaniline per minute;
further, the proportion of the mixed enzyme preparation is as follows: the crude enzyme solution is 1g of protease, and the weight ratio of the protease is 0.2-0.5 g.
Further, in the step (5), the concentration of TS is 50-200 g/L, and the pH of the garbage homogenized liquid is adjusted to 4.5-5.5.
Further, in the step (5), the adding ratio of the mixed enzyme preparation is as follows: the addition amount of the mixed enzyme preparation is 1g, 100 g-250 g of the wet weight of the garbage homogenized solution.
Further, in the step (6), the temperature of the constant-temperature shaking table is 45-55 ℃; the rotating speed is 120-200 rpm, and the time is 6-24 h.
Further, in the step (6), the control system adopts an automatic sampling mode, and a full-automatic reducing sugar tester connected with the enzymolysis tank is used for periodically testing the fermentable sugar concentration of the second mixture every 15-60 min;
further, the mode of intermittently adding the enzyme preparation is as follows: detecting the total sugar concentration of the garbage homogenized liquid, setting the total sugar concentration as an upper limit value T of the reducible sugar, and automatically supplementing the mixed enzyme preparation when detecting that the fermentable sugar concentration of the second mixture is less than 50% of the value T, wherein the supplementing amount is 1/4 of the adding amount of the initial mixed enzyme preparation; when the concentration of the fermentable sugar is detected to be 50-70% of the T value, 1/8 of the addition amount of the initial mixed enzyme preparation is supplemented; when the concentration of the fermentable sugar is detected to be 70-90% of the T value, enzyme is not supplemented, and the concentration is measured next time; and (3) when the concentration of the fermentable sugar is detected to exceed 90 percent T, ending the enzymatic process, discharging the food waste slurry with high concentration of the fermentable sugar, adding a fresh first mixture obtained in the step (5) into the enzymolysis tank, repeating the step (6), and carrying out a plurality of enzymatic processes.
The beneficial technical effects of the invention are as follows:
aiming at the characteristics of high solid content and abundant starch and cellulose substances in the food waste in China, the invention degrades the cellulose in the food waste by utilizing a combined pretreatment mode of deep eutectic solvent and ultrasound, can effectively shorten the reaction time and reduce the required temperature, and realizes the effective pretreatment of more rapid release of sugar from biomass. The method has the advantages of simple and convenient operation and remarkable effect of promoting the conversion to form reducing sugar.
The deep eutectic solvent prepared by the invention is simple in preparation method, environment-friendly and biodegradable, does not cause inhibition to subsequent enzymatic treatment, has recoverability, and can be separated after pretreatment and reused in subsequent pretreatment circulation. Realizes the reutilization of the raw materials and reduces the cost.
In order to reduce the cost of enzyme reagents, Aspergillus niger is utilized to produce crude enzyme with polysaccharide hydrolysis capacity, and in addition, a part of carbohydrate substances cannot be released due to the coating of protein, so that the concentration of fermentable sugar after the enzymolysis of the food waste can be further improved by additionally adding protease.
According to the invention, the crude enzyme liquid is produced by the aspergillus, so that the enzyme cost can be greatly reduced, the produced crude enzyme liquid contains a large amount of abundant carbohydrases which can be used for hydrolyzing cellulose and starch in food and beverage garbage, and the protease is added in an auxiliary manner to release the wrapped saccharides, so that the purpose of producing a large amount of reducing sugar is achieved. The method has the advantage of remarkable effect of promoting the conversion to form reducing sugar.
According to the invention, through fed-batch feeding and a mode of automatically detecting the concentration of reducing sugar and automatically supplementing a mixed enzyme preparation, the phenomenon of enzyme hydrolysis inhibition caused by overhigh product concentration can be effectively reduced, the utilization rate of enzyme is high, the addition amount of enzyme can be reduced, and the cost is saved. The method has the advantages of simple and convenient operation and remarkable effect of promoting the conversion to form reducing sugar.
Drawings
FIG. 1 shows the specific steps of a process for promoting the conversion of food waste to form reducing sugars.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
(1) as shown in figure 1, the food waste is crushed by a food processor to the particle size of less than 0.5mm, oil is extracted by a cooking method until the oil content is 1.8%, and tap water is added to adjust the TS to 260 g/L.
(2) The deep eutectic solvent with the components of choline chloride/glycerol is prepared by adding 1mol of choline chloride into 6mol of glycerol without adding water, heating the obtained mixture at 58 ℃ and stirring at 200rpm for 2h until a uniform and transparent liquid is formed.
(3) And (2) cooling the prepared deep eutectic solvent to room temperature, uniformly mixing the thick slurry of the food waste obtained in the step (1) with the deep eutectic solvent according to the volume ratio of 1:3, and then carrying out ultrasonic treatment on the mixture, wherein the frequency of the ultrasonic pretreatment is 20kHz, the amplitude is 50%, the power is 38w, the pulse period is 25 seconds on/10 seconds off, and the treatment time is 22 min. Cooling the mixed solution to room temperature, centrifuging the mixed solution, recovering the mixed solution containing the deep eutectic solvent for the next round of pretreatment, and collecting the food waste solid-phase precipitate for subsequent enzymatic treatment.
(4) Adding 15g of enzyme production culture medium into a 250mL triangular flask, sterilizing in a sterilizing pot at 121 ℃ for 20min, cooling to 30 ℃, and inoculating 2mL of spore suspension; inoculating Aspergillus niger (Aspergillus niger) into a container containing enzyme-producing culture medium, and performing solid-state fermentation, wherein the concentration of Aspergillus niger spore suspension is 1.0 × 10-8CFU/mL; taking 10g of the bacterial liquid after 5d of culture, adding 50mL of sodium acetate buffer (pH4.6), leaching in a shaking table at the temperature of 40 ℃ and the rotating speed of 160rpm for 1.5h, filtering and centrifuging to obtain a crude enzyme liquid, wherein the activity of the obtained crude enzyme liquid is 20000U/mL, wherein 1 enzyme activity unit is defined as the enzyme amount required for forming 1mg of reducing sugar in 1 h. Separately prepared was a protease with an activity of 500LAPU/g, wherein 1LAPU (leucine aminopeptidase unit) hydrolyzes 1 mmol L-leucine-p-nitro group per minuteThe amount of enzyme required for aniline. Mixing a crude enzyme solution prepared by aspergillus niger and protease according to a certain proportion to prepare a mixed enzyme preparation, wherein the proportion is that the crude enzyme solution: the protease is 1g: 0.40 g.
(5) Adding water into the solid phase precipitate obtained in the step 3 to form a homogeneous solution, adjusting the solid concentration (TS) to be 100g/L, adjusting the pH of the garbage homogeneous solution to be 4.8, and then adding enzyme preparations according to the following ratio: adding the mixed enzyme preparation prepared in the step 3 into the food waste with the wet weight of 1g to 180g to obtain a first mixture.
(6) And (3) putting the first mixture obtained in the step (5) into a constant-temperature shaking table at the temperature of 50 ℃ and the rotating speed of 150rpm to obtain a second mixture. Sampling is carried out periodically by a control system, and the fermentable sugar concentration of the second mixture is periodically determined every 30min by using a full-automatic reducing sugar determinator connected with the enzymolysis tank. Measuring the total sugar concentration in the garbage homogenizing solution to be 44.8g/L, setting the target concentration of reducing sugar to be 40g/L, and supplementing the mixed enzyme preparation in an intermittent feeding mode, wherein the specific operation is that when the concentration of fermentable sugar is detected to be less than 20g/L, the mixed enzyme preparation is automatically supplemented, and the supplementing amount is 1/4 of the adding amount of the initial mixed enzyme preparation; detecting 1/8 that the supplement amount is the addition amount of the initial mixed enzyme preparation when the concentration of the fermentable sugar is 20 g/L-28 g/L; when the concentration of the fermentable sugar is detected to be 28 g/L-36 g/L, enzyme is not supplemented, and the concentration is measured next time.
And (4) detecting that when the concentration of the fermentable sugar exceeds 40g/L, finishing the enzymatic process, and discharging the food waste slurry with high concentration of the fermentable sugar. The first round of enzymatic time is 8 hours, the concentration of the reducing sugar in the food waste after the first round of enzymatic time is measured to be 41.9g/L, the concentration of the reducing sugar in the original food waste slurry under the same condition is 8.1g/L, and the concentration of the reducing sugar is 517% of the original food waste slurry, so that the aim of converting the food waste into the reducing sugar is fulfilled. After this round, fresh first mixture is re-dosed, and step 4 is repeated for a number of enzymatic processes.
Example 2:
(1) as shown in figure 1, the food waste is crushed by a food processor to the particle size of less than 0.5mm, the oil content is extracted by a cooking method to 4%, and tap water is added to adjust the TS to 300 g/L.
(2) The deep eutectic solvent with the components of choline chloride/glycerol is prepared by adding 1mol of choline chloride into 12mol of glycerol without adding water, heating the obtained mixture at 70 ℃ and stirring at 120rpm for 1h until a uniform and transparent liquid is formed.
(3) And (2) cooling the prepared deep eutectic solvent to room temperature, uniformly mixing the thick slurry of the food waste obtained in the step (1) with the deep eutectic solvent according to the volume ratio of 1:5, and then carrying out ultrasonic treatment on the mixture, wherein the frequency of the ultrasonic pretreatment is 30kHz, the amplitude is 30%, the power is 30w, the pulse period is 20 seconds on/10 seconds off, and the treatment time is 15 min. Cooling the mixed solution to room temperature, centrifuging the mixed solution, recovering the mixed solution containing the deep eutectic solvent for the next round of pretreatment, and collecting the food waste solid-phase precipitate for subsequent enzymatic treatment.
(4) Adding 20g of enzyme production culture medium into a 250mL triangular flask, sterilizing in a sterilizing pot at 121 ℃ for 20min, cooling to 30 ℃, and inoculating 3mL of spore suspension; inoculating Aspergillus niger (Aspergillus niger) into a container containing enzyme-producing culture medium, and performing solid-state fermentation, wherein the concentration of Aspergillus niger spore suspension is 1.0 × 10-8CFU/mL; taking 10g of the cultured bacterial liquid after 6d, adding 50mL of sodium acetate buffer (pH4.6), leaching in a shaking table at the temperature of 40 ℃ and the rotating speed of 160rpm for 1.5h, filtering and centrifuging to obtain a crude enzyme liquid, wherein the activity of the obtained crude enzyme liquid is 20000U/mL, wherein 1 enzyme activity unit is defined as the enzyme amount required for forming 1mg of reducing sugar in 1 h. A protease having an activity of 500LAPU/g, wherein 1LAPU (leucine aminopeptidase unit) is an amount of enzyme required for hydrolyzing 1 mmol L-leucine-p-nitroaniline per minute, was prepared. Mixing a crude enzyme solution prepared by aspergillus niger and protease according to a certain proportion to prepare a mixed enzyme preparation, wherein the proportion is that the crude enzyme solution: the protease is 1g: 0.50 g.
(5) Adding water into the solid phase precipitate obtained in the step 3 to form a homogeneous solution, adjusting the solid concentration (TS) to 200g/L, adjusting the pH of the garbage homogeneous solution to 5.5, and then adding enzyme preparations according to the following ratio: adding the mixed enzyme preparation prepared in the step 3 into the food waste with the wet weight of 1g to 250g to obtain a first mixture.
(6) And (3) putting the first mixture obtained in the step (5) into a constant-temperature shaking table at the temperature of 55 ℃ and the rotating speed of 120rpm to obtain a second mixture. Sampling is carried out periodically by a control system, and the fermentable sugar concentration of the second mixture is periodically determined every 30min by using a full-automatic reducing sugar determinator connected with the enzymolysis tank. Measuring the total sugar concentration in the garbage homogenizing solution to be 84.7g/L, setting the target concentration of reducing sugar to be 80g/L, and supplementing the mixed enzyme preparation in an intermittent feeding mode, wherein the specific operation is that when the concentration of fermentable sugar is detected to be less than 40g/L, the mixed enzyme preparation is automatically supplemented, and the supplementing amount is 1/4 of the adding amount of the initial mixed enzyme preparation; detecting 1/8 that the supplement amount is the addition amount of the initial mixed enzyme preparation when the concentration of the fermentable sugar is 40 g/L-56 g/L; when the concentration of the fermentable sugar is 56 g/L-72 g/L, enzyme is not supplemented, and the concentration is measured next time.
And (4) detecting that when the concentration of the fermentable sugar exceeds 80g/L, finishing the enzymatic process, and discharging the food waste slurry with high concentration of the fermentable sugar. The first round of enzymatic time is 12 hours, the concentration of the reducing sugar in the food waste after the first round of enzymatic time is determined to be 80.6g/L, the concentration of the reducing sugar in the original food waste slurry under the same condition is 19.4g/L, and the concentration of the reducing sugar is 415 percent of the original food waste slurry, so that the aim of converting the food waste into the reducing sugar is fulfilled. After this round, fresh first mixture is re-dosed, and step 4 is repeated for a number of enzymatic processes.
Example 3:
(1) as shown in figure 1, the food waste is crushed by a food processor to the particle size of less than 0.8mm, the oil content is extracted by a cooking method to 2.5%, and tap water is added to adjust the TS to 200 g/L.
(2) The deep eutectic solvent with the components of choline chloride/glycerol is prepared by adding 1mol of choline chloride into 3mol of glycerol without adding water, heating the obtained mixture at 50 ℃ and stirring at 250rpm for 3h until a uniform and transparent liquid is formed.
(3) And (2) cooling the prepared deep eutectic solvent to room temperature, uniformly mixing the thick slurry of the food waste obtained in the step (1) with the deep eutectic solvent according to the volume ratio of 1:1, and then carrying out ultrasonic treatment on the mixture, wherein the frequency of the ultrasonic pretreatment is 15kHz, the amplitude is 60%, the power is 50w, the pulse period is 30 seconds on/10 seconds off, and the treatment time is 30 min. Cooling the mixed solution to room temperature, centrifuging the mixed solution, recovering the mixed solution containing the deep eutectic solvent for the next round of pretreatment, and collecting the food waste solid-phase precipitate for subsequent enzymatic treatment.
(4) Adding 15g of enzyme production culture medium into a 250mL triangular flask, sterilizing in a sterilizing pot at 121 ℃ for 20min, cooling to 30 ℃, and inoculating 2mL of spore suspension; inoculating Aspergillus niger (Aspergillus niger) into a container containing enzyme-producing culture medium, and performing solid-state fermentation, wherein the concentration of Aspergillus niger spore suspension is 1.0 × 10-8CFU/mL; taking 10g of the cultured bacterial liquid after 3d, adding 50mL of sodium acetate buffer (pH4.6), leaching in a shaking table at the temperature of 40 ℃ and the rotating speed of 160rpm for 1h, filtering and centrifuging to obtain a crude enzyme liquid, wherein the activity of the obtained crude enzyme liquid is 20000U/mL, and 1 enzyme activity unit is defined as the enzyme amount required for forming 1mg of reducing sugar in 1 h. A protease having an activity of 500LAPU/g, wherein 1LAPU (leucine aminopeptidase unit) is an amount of enzyme required for hydrolyzing 1 mmol L-leucine-p-nitroaniline per minute, was prepared. Mixing a crude enzyme solution prepared by aspergillus niger and protease according to a certain proportion to prepare a mixed enzyme preparation, wherein the proportion is that the crude enzyme solution: the protease is 1g: 0.20 g.
(5) Adding water into the solid phase precipitate obtained in the step 3 to form a homogeneous solution, adjusting the solid concentration (TS) to 50g/L, adjusting the pH of the garbage homogeneous solution to 4.5, and then adding enzyme preparations according to the following ratio: adding the mixed enzyme preparation prepared in the step 3 into the food waste with the wet weight of 1g to 100g to obtain a first mixture.
(6) And (3) putting the first mixture obtained in the step (5) into a constant-temperature shaking table at the temperature of 45 ℃ and the rotating speed of 200rpm to obtain a second mixture. Sampling is carried out periodically by a control system, and the fermentable sugar concentration of the second mixture is determined periodically every 15min by using a full-automatic reducing sugar tester connected with the enzymolysis tank. Measuring the total sugar concentration in the garbage homogenizing solution to be 23.5g/L, setting the target concentration of reducing sugar to be 20g/L, and supplementing the mixed enzyme preparation in an intermittent feeding mode, wherein the specific operation is that when the concentration of fermentable sugar is detected to be less than 10g/L, the mixed enzyme preparation is automatically supplemented, and the supplementing amount is 1/4 of the adding amount of the initial mixed enzyme preparation; detecting 1/8 that the supplement amount is the addition amount of the initial mixed enzyme preparation when the concentration of the fermentable sugar is 10 g/L-14 g/L; when the concentration of the fermentable sugar is detected to be 14 g/L-18 g/L, enzyme is not supplemented, and the concentration is measured next time.
And (3) detecting that when the concentration of the fermentable sugar exceeds 20g/L, finishing the enzymatic process, and discharging the food waste slurry with high concentration of the fermentable sugar. The first round of enzymatic time is 6 hours, the concentration of the reducing sugar in the food waste after the first round of enzymatic time is determined to be 20.2g/L, the concentration of the reducing sugar in the original food waste slurry under the same condition is 4.31g/L, and the concentration of the reducing sugar is 469% of that of the original food waste slurry, so that the aim of converting the food waste into the reducing sugar is fulfilled. After this round, fresh first mixture is re-dosed, and step 4 is repeated for a number of enzymatic processes.
Wherein, the enzyme production culture medium adopted in the examples 1-3 has the following formula:
glucose 10g/L, MgSO4·7H2O 1.0g/L、(NH4)2SO4 3.0g/L、KH2PO4 3.0g/L、NaH2PO4·2H2O 1.69g/L、MnSO4·H2O 0.04g/L、ZnCl2 0.02g/L、CaCl2 0.076g/L、CuSO4·5H2O 0.015g/L、CoCl2·6H2O 0.015g/L、FeSO4·7H2O0.3 g/L and EDTA-2Na 0.67 g/L.
While the embodiments of the present invention have been disclosed above, it is not limited to the applications listed in the description and embodiments, but is fully applicable to various fields suitable for the present invention, and it will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the principle and spirit of the present invention, and therefore the present invention is not limited to the specific details without departing from the general concept defined in the claims and the scope of equivalents thereof.
Claims (10)
1. A method for promoting food waste to convert into reducing sugar is characterized by comprising the following steps:
(1) crushing the food waste, extracting oil and pulping to obtain high-concentration food waste thick slurry;
(2) directly mixing choline chloride and glycerol without adding water, heating and stirring the obtained mixture until uniform and transparent liquid is formed, and preparing a deep eutectic solvent with the components of choline chloride/glycerol;
(3) cooling the deep eutectic solvent obtained in the step (2) to room temperature before use, uniformly mixing the thick slurry of the food waste obtained in the step (1) with the deep eutectic solvent, carrying out ultrasonic pretreatment on the mixture, cooling the mixture to room temperature after the pretreatment, recycling the mixed solution containing the deep eutectic solvent after centrifugation for the next round of pretreatment, and collecting solid-phase sediment of the food waste for subsequent enzymatic treatment;
(4) inoculating aspergillus into a container containing an enzyme production culture medium, and extracting a crude enzyme solution after culture; adding protease to obtain mixed enzyme preparation;
(5) adding water into the solid phase precipitate obtained in the step (3) to form a garbage homogeneous liquid, adjusting the solid concentration TS, adjusting the pH value of the garbage homogeneous liquid, and adding the mixed enzyme preparation obtained in the step (4) to obtain a first mixture;
(6) placing the first mixture obtained in the step (5) in an enzymolysis tank, placing the enzymolysis tank in a constant-temperature shaking table, oscillating to obtain a second mixture, monitoring the concentration of fermentable sugar in the second mixture through a control system, supplementing a mixed enzyme preparation in an intermittent feeding mode, and regulating the enzyme amount through feedback.
2. The method of claim 1, wherein: the grain diameter of the Chinese food and beverage garbage concentrated slurry in the step (1) is less than 1 mm; the oil content after oil extraction and pulping is lower than 4 percent; the concentration of TS is 200-300 g/L.
3. The method of claim 1, wherein: and (3) adding 3-12 mol of glycerol into 1mol of choline chloride in the step (2), heating the obtained mixture at 50-70 ℃, and stirring at 120-250 rpm for 1-3 h until a uniform and transparent liquid is formed, thereby preparing the deep eutectic solvent.
4. The method of claim 1, wherein: in the step (3), the thick slurry of the food waste obtained in the step (1) and the deep eutectic solvent are uniformly mixed according to the volume ratio of 1: 1-5;
the frequency of ultrasonic pretreatment is 15-30 kHz, the amplitude is 30-60%, the power is 30-50 w, the pulse period is 20-30 seconds on/10 seconds off, and the treatment time is 15-30 min.
5. The method of claim 1, wherein: in the step (4), the Aspergillus is Aspergillus niger;
the culture mode is solid state fermentation; the culture method comprises the steps of adding 10 g-20 g of enzyme production culture medium into a triangular flask, sterilizing in a sterilization pot at 121 ℃ for 20min, cooling to 30 ℃, and inoculating 1 mL-3 mL of Aspergillus niger spore suspension; wherein the concentration of the Aspergillus niger spore suspension is 0.8-1.2 multiplied by 10-8CFU/mL；
Taking 10g of the cultured bacterial liquid after 3-6d, adding 40-60 mL of sodium acetate buffer solution with the pH value of 4.5-4.7, putting the mixture into a shaking table with the temperature of 38-45 ℃ and the rotating speed of 150-180 rpm, leaching for 1-2 h, and filtering and centrifuging to obtain a crude enzyme liquid;
the formula of the enzyme production culture medium is 10g/L, MgSO g of glucose4·7H2O 1.0g/L、(NH4)2SO4 3.0g/L、KH2PO43.0g/L、NaH2PO4·2H2O 1.69g/L、MnSO4·H2O 0.04g/L、ZnCl2 0.02g/L、CaCl2 0.076g/L、CuSO4·5H2O 0.015g/L、CoCl2·6H2O 0.015g/L、FeSO4·7H20.3g/L of O and 0.67g/L of EDTA-2 Na;
the activity of the crude enzyme liquid enzyme is 14000-22000U/mL, the hydrolysis capacity of the crude enzyme liquid on macromolecular saccharide substances is strong, wherein 1 enzyme activity unit is defined as the enzyme amount required for forming 1mg of reducing sugar in 1 hour;
the protease activity is 350-700 LAPU/g, wherein 1LAPU (leucine aminopeptidase) unit is the enzyme amount required for hydrolyzing 1 millimole of L-leucine-p-nitroaniline per minute;
the proportion of the mixed enzyme preparation is as follows: the crude enzyme solution is 1g of protease, and the weight ratio of the protease is 0.2-0.5 g.
6. The method of claim 1, wherein: in the step (5), the concentration range of TS is 50-200 g/L, and the pH value of the garbage homogeneous liquid is adjusted to 4.5-5.5.
7. The method of claim 1, wherein: in the step (5), the adding proportion of the mixed enzyme preparation is as follows: the addition amount of the mixed enzyme preparation is 1g, 100 g-250 g of the wet weight of the garbage homogenized solution.
8. The method of claim 1, wherein: in the step (6), the temperature of the constant temperature shaking table is 45-55 ℃; the rotating speed is 120-200 rpm, and the total reaction time is 6-24 h.
9. The method of claim 1, wherein: in the step (6), the control system adopts an automatic sampling mode, and a full-automatic reducing sugar tester connected with the enzymolysis tank is used for periodically testing the fermentable sugar concentration of the second mixture every 15-60 min;
the mode of the intermittent feeding of the enzyme preparation is as follows: detecting the total sugar concentration of the garbage homogenized liquid, setting the total sugar concentration as an upper limit value T of the reducible sugar, and automatically supplementing the mixed enzyme preparation when detecting that the fermentable sugar concentration of the second mixture is less than 50% of the value T, wherein the supplementing amount is 1/4 of the adding amount of the initial mixed enzyme preparation; when the concentration of the fermentable sugar is detected to be 50-70% of the T value, 1/8 of the addition amount of the initial mixed enzyme preparation is supplemented; when the concentration of the fermentable sugar is detected to be 70-90% of the T value, enzyme is not supplemented, and the concentration is measured next time; and (3) when the concentration of the fermentable sugar is detected to exceed 90 percent T, ending the enzymatic process, discharging the food waste slurry with high concentration of the fermentable sugar, adding a fresh first mixture obtained in the step (5) into the enzymolysis tank, repeating the step (6), and carrying out a plurality of enzymatic processes.
10. A high fermentable sugar concentration food waste slurry produced by the process of any one of claims 1 to 9.
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