Method for recycling bacterial residues for producing long-chain dibasic acid

Abstract

The invention provides a method for recycling mushroom dregs for producing long-chain dibasic acid. The method comprises the following steps: (1) pre-treating the mushroom dregs: separating the fermented mushroom dregs for producing the long-chain dibasic acid to obtain mushroom sludge; (2) releasing substances in the mushroom dregs: taking the mushroom sludge in the step (1), adding hydrolase and an activating agent, and respectively carrying out heat preservation twice at 35-50 DEG C and 52-70 DEG C; and (3) fermenting the treated mushroom dregs: using the mushroom dregs treated in the step (2) as a culture medium component to replace or partially replace a nitrogen source in the mushroom dregs for fermentation. A nitrogen source of the fermentation culture medium is replaced or partially replaced by the mushroom dregs, and after treatment of the method, protein is released in a high proportion, so that resource reutilization of the mushroom dregs is realized, and meanwhile, the problems of difficulty in discharging the mushroom dregs, high treatment cost and the like are solved.

Description

Technical Field

[0001] The invention relates to a method for recycling fungus residues producing long-chain dibasic acids, wherein intracellular substances, especially proteins, are released through treatment to increase their availability, and the method belongs to the technical field of biological waste recycling. Background Art

[0002] For many years, biological fermentation waste residues have been discarded as industrial waste residues at home and abroad. Since waste residues contain a large amount of protein, phosphorus, rich vitamins, amino acids and culture medium residues, discarding waste residues not only causes a large amount of resource waste, but also leads to serious environmental pollution.

[0003] As early as the 1950s, foreign countries began to study the physical and chemical properties and applications of antibiotic residues. In the late 1950s, domestic and foreign countries began to study the processing and utilization of antibiotic fermentation waste residues. Initially, a simple drying method was used to crush the dried waste residues and directly use them as ingredients or additives. Before the 20th century, the scale of projects for the high-value utilization of waste residues in my country's fermentation industry and pharmaceutical industry was relatively small, the equipment was relatively backward, the product conversion efficiency was low, and the technical methods for the high-value utilization of fermentation residues were immature, resulting in low efficiency and serious secondary pollution. In the past, my country's use of more waste resources was mainly concentrated on the research of anaerobic fermentation methods, resulting in a relatively slow development of other high-value technologies. After the mid-1960s, my country has increasingly attached importance to the protection of the ecological environment, and the technology for pollution control has developed rapidly, which is mostly used in the production of organic fertilizers, preparation of adsorbents, reuse of fermentation nitrogen sources, extraction of useful substances, biomass fuels, and anaerobic digestion.

[0004] At present, the research on the resource utilization of waste fungus residues is mainly focused on edible fungus residues, brewer's yeast residues and antibiotic fermentation residues, while there is still little research on waste fungus residues from bio-based chemical fermentation. CN106260605A invented a method for producing green Trichoderma residue fermented feed. The technology uses the fermented mixture to dry and crush; the crushed material is granulated to obtain the finished feed. This type of application is not suitable for bio-based chemical residue systems. CN106434770 invented a method for producing ethanol by fermenting chrysogenum residues. The technology uses acid and alkali for treatment. Long-chain dibasic acids are generally produced by biological fermentation. The fermentation process consumes nitrogen sources, and each ton of product produces about 0.8 tons of fungus residue with a moisture content greater than 90%. If the traditional incineration or landfill method is used, the cost is high and the environmental harm is great; as a fertilizer, as a non-edible fungus, there are problems of food safety and land pollution; if all are discharged into sewage, it will impact the sewage treatment plant, affect the stable operation of the device, and also affect water resources and the environment. Summary of the invention

[0005] In order to solve the problem that fermentation residues for producing long-chain dibasic acids are difficult to treat in the prior art, the present invention provides a method for recycling residues for producing long-chain dibasic acids. The treated residues are recycled to the long-chain dibasic acid fermentation section to reduce the input of nitrogen sources. This can not only solve the problem that the residues are difficult to treat and cause environmental pollution, and reduce the cost of residue discharge treatment, but also increase the utilization benefits of the residues and save production costs through the resource utilization of the residues.

[0006] To achieve the above technical objectives, the technical solution of the present invention is as follows:

[0007] A method for recycling fungus residues for producing long-chain dibasic acids, comprising:

[0008] (1) Pretreatment of bacterial residue: The fermentation residue producing long-chain dibasic acids is separated to obtain bacterial sludge;

[0009] (2) Release of substances in bacterial residue: Take the bacterial sludge in (1), add hydrolase and activator, and keep it warm twice at 35℃-50℃ and 52℃-70℃ respectively;

[0010] (3) Using the treated bacterial residue for fermentation: Using the treated bacterial residue in (2) as a component of the culture medium to replace or partially replace the nitrogen source therein for fermentation.

[0011] Furthermore, in step (2), a hydrolase and an activator are added and kept warm to increase the permeability of the yeast cell wall and promote the release of intracellular substances, wherein the hydrolase is selected from at least one of lysozyme, snail enzyme and protease, and the addition amount is 5-50 mg / g of bacterial mud, wherein snail enzyme is preferred; the activator is selected from at least one of glucose, sucrose, acetic acid, sodium acetate, sodium chloride, potassium chloride, microorganism B2, cysteine ​​and sodium sulfite, and the addition amount is 0.1-1 mg / g of bacterial mud, wherein acetic acid is preferred. The activator is added in the form of an aqueous solution, and the concentration of the activator solution is 1-10wt%.

[0012] Furthermore, the process of adding the hydrolase and the activator in step (2) and keeping the temperature at 35°C-50°C and 52°C-70°C twice respectively does not have a strict order control, as long as it is avoided that the hydrolase is immediately kept at high temperature after adding, so that it is inactivated and cannot function. According to the above principles, one of the specific implementation methods is: first add the hydrolase, keep the temperature at 35°C-50°C for the first time, then add the activator, and keep the temperature at 52°C-70°C for the second time; the second specific implementation method is: add the hydrolase and the activator at the same time, keep the temperature at 35°C-50°C for the first time, and then heat it to 52°C-70°C for the second time; the third specific implementation method is: first add the activator, keep the temperature at 52°C-70°C for the first time, then cool it to 35°C-50°C, add the hydrolase, and keep the temperature at 35°C-50°C for the second time. Among them, the first method is more preferred.

[0013] Furthermore, the temperature and time of the two insulations in step (2) are preferably 35°C-42°C for 2-10 h, and 55°C-65°C for 6 h-14 h, respectively.

[0014] Furthermore, the fermentation residue for producing long-chain dibasic acids in step (1) is the residue produced by aerobic fermentation of Candida tropicalis to produce long-chain dibasic acids.

[0015] Furthermore, the separation in step (1) is centrifugal separation or flocculation separation.

[0016] Furthermore, the centrifugal separation is performed at a centrifugal rate of 2000rpm-8000rpm for 3-10min, and the supernatant is separated and discarded to obtain bacterial sludge. The flocculation separation is performed by allowing the fermentation liquid to stand for 2h-4h, removing residual alkanes, and then using 1%-5% high-viscosity chitosan and 1%-3% sodium alginate for flocculation, and discarding the supernatant to obtain bacterial sludge. Among them, centrifugal separation is preferred.

[0017] Furthermore, the main elements in the bacterial sludge obtained by centrifugal separation and flocculation separation in step (1) include C, H, N, etc., wherein the C content is generally 30%-50%, the H content is generally 6%-9%, the N content is generally 40000-50000 mg / kg, the S content is 0.1%-2%, the Ca content is 100-2000ppm, the Mg content is 900-1500ppm, the Ni content is 0.01-5ppm, the Pb content is 1-8ppm, the Zn content is 10-50ppm, and the protein content in the dry matter is 30%-70%.

[0018] Furthermore, in step (3), the treated fungus residue is used for fermentation, preferably for fermentation of tropical Candida to produce long-chain dibasic acids with alkane. The alkane is a C10-C16 alkane. The fermentation temperature is 30°C-40°C, the pH is 6.5-8.0, the fermentation time is 96h-192h, and the ventilation volume is 0.1-1.5VVM.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The fungus residue is used to replace or partially replace the nitrogen source of the fermentation medium. After the treatment of the present invention, a high proportion of protein is released, thereby realizing the resource recycling of the fungus residue and solving the problems of difficulty in discharging the fungus residue and high processing cost.

[0021] (2) According to the yeast cell wall structure, the present invention introduces exogenous hydrolases under certain conditions to help enhance the destruction of the cell wall structure, promote the release of intracellular substances, and activate the cell's own enzyme system by using an activator, so that the cell wall's glucanase system is degraded to increase cell permeability. The technology has mild operating conditions, does not produce other wastewater compared to acid-base treatment, and has low equipment requirements.

[0022] (3) The fungus residue of the present invention can completely replace the nitrogen source, and the fermentation result is equivalent to that of the commercially available yeast extract.

[0023] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .Concentration of dibasic acid in the fermentation broth during the fermentation process of Example 1 and Comparative Example 4. DETAILED DESCRIPTION

[0025] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0026] Example 1

[0027] (1) Pretreatment of fungus residue:

[0028] Tropical Candida was used for dodecanedioic acid fermentation. The fermentation broth was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded to obtain yeast mud, in which the C content was 38.37%, the H content was 7.06%, the N content was 44619 mg / kg, the sulfur content was 0.50%, the Ca content was 111.10 ppm, the Mg content was 988.90 ppm, the Ni content was 0.23 ppm, the Pb content was 8.37 ppm, the Zn content was 11.91 ppm, and the protein content was 45%.

[0029] (2) Release of substances from fungal residue:

[0030] Take the yeast paste obtained in step (1), add snail enzyme at a rate of 10 mg snail enzyme / g yeast paste, keep warm at 35°C for 4 hours, then add acetic acid aqueous solution at a rate of 0.1 mg acetic acid / g yeast paste, the concentration of the acetic acid aqueous solution is 1%, and keep warm at 60°C for 12 hours to activate the yeast cell's own enzyme system.

[0031] The protein content in the product after the treatment in the above step (2) was determined, and the protein release rate was calculated: the supernatant was taken out by centrifugation, and the protein content therein was detected. The protein release rate was 85.1%.

[0032] The protein determination method is as follows: the BCA protein concentration determination kit of Biyuntian is used. A series of standard protein solutions are prepared, and the absorbance is measured at 562nm using an enzyme reader and a standard curve is drawn. For the determination of protein in fresh cells, 0.1g-2.0g of frozen cells are weighed and added to 200mL of 0.1M NaOH in a boiling water bath for 5min to extract protein. After cooling, centrifuge at 5000rpm for 5min, take the supernatant and measure the absorbance at 562nm to calculate the protein content in fresh cells. After the sample yeast mud is pretreated, an appropriate amount of sample is centrifuged to take the supernatant and measure the absorbance at 562nm to calculate the soluble protein content in the supernatant of the pretreated yeast mud. Protein release rate = soluble protein content in the supernatant of the pretreated yeast mud / protein content in fresh cells.

[0033] (3) Fermentation of fungus residue

[0034] The culture medium is prepared with dodecane as the raw material, and the nitrogen source content in the yeast mud obtained in step (1) is used as the basis, and the bacterial mud treated in step (2) is added as the fermentation nitrogen source, sterilized at 121° C. for 20 minutes, and tropical Candida is inoculated in the fermentation medium at a 10% inoculation amount. The tropical Candida used is preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the preservation number is CGMCC No. 20459. The dibasic acid is fermented in a 3L fermenter at 35° C. and 300 rpm to produce the dibasic acid. The fermentation is stopped after 144 hours, and the dibasic acid concentration in the fermentation liquid is 143 g / L (acid-base titration method). By adopting this technology, the fungus residue is reused to replace the nitrogen source and the fungus residue is used as waste solid treatment costs, which can reduce the fermentation cost by about 1,000 yuan / ton of dibasic acid.

[0035] Example 2

[0036] (1) Same as Example 1.

[0037] (2) Take the yeast slurry obtained in step (1), add snail enzyme at a rate of 5 mg snail enzyme / g yeast slurry, incubate at 35° C. for 4 hours, then add acetic acid aqueous solution at a rate of 0.5 mg acetic acid / g yeast slurry, the concentration of the acetic acid aqueous solution is 5%, and continue incubating at 60° C. for 12 hours. The protein release rate was measured to be 70.5%.

[0038] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 128 g / L.

[0039] Example 3

[0040] (1) Same as Example 1.

[0041] (2) The yeast slurry obtained in step (1) was added with snail enzyme at a rate of 10 mg snail enzyme / g yeast slurry, and incubated at 35° C. for 4 hours, and then a glucose aqueous solution was added at a rate of 10% glucose / g yeast slurry, and the incubation was continued at 60° C. for 12 hours. The protein release rate was measured to be 65.6%.

[0042] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 115 g / L.

[0043] Example 4

[0044] (1) Same as Example 1.

[0045] (2) Take the yeast slurry obtained in step (1), add snail enzyme at a rate of 10 mg snail enzyme / g yeast slurry, incubate at 35° C. for 4 hours, then add sodium acetate solution at a rate of 0.1 mg sodium acetate / g yeast slurry, the concentration of the sodium acetate aqueous solution is 1%, and continue incubating at 60° C. for 12 hours. The protein release rate was measured to be 76.5%.

[0046] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 125 g / L.

[0047] Example 5

[0048] (1) Same as Example 1.

[0049] (2) Take the yeast paste obtained in step (1), add snail enzyme at a rate of 50 mg snail enzyme / g yeast paste, incubate at 35° C. for 4 hours, then add sodium chloride aqueous solution at a rate of 0.1 mg sodium chloride / g yeast paste, the concentration of the sodium chloride aqueous solution is 1%, and continue incubating at 60° C. for 12 hours. The protein release rate was measured to be 80.5%.

[0050] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 131 g / L.

[0051] Example 6

[0052] (1) Same as Example 1.

[0053] (2) Take the yeast slurry obtained in step (1), add snail enzyme at a rate of 20 mg snail enzyme / g yeast slurry, incubate at 35° C. for 4 hours, then add acetic acid aqueous solution at a rate of 0.1 mg acetic acid / g yeast slurry, the concentration of the acetic acid aqueous solution is 1%, and continue incubating at 60° C. for 12 hours. The protein release rate was measured to be 80.1%.

[0054] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 135 g / L.

[0055] Example 7

[0056] (1) Same as Example 1.

[0057] (2) Take the yeast slurry obtained in step (1), add snail enzyme at a rate of 50 mg snail enzyme / g yeast slurry, incubate at 35° C. for 4 hours, then add acetic acid aqueous solution at a rate of 0.1 mg acetic acid / g yeast slurry, the concentration of the acetic acid aqueous solution is 1%, and continue incubating at 60° C. for 12 hours. The protein release rate was measured to be 83.2%.

[0058] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 139 g / L.

[0059] Example 8

[0060] (1) Same as Example 1.

[0061] (2) Take the yeast paste obtained in step (1), add snail enzyme at a rate of 10 mg snail enzyme / g yeast paste, and then add acetic acid aqueous solution at a rate of 0.1 mg acetic acid / g yeast paste, the concentration of the acetic acid aqueous solution is 1%, first keep it warm at 35°C for 4 hours, and then keep it warm at 60°C for 12 hours. The protein release rate is measured to be 75.1%.

[0062] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 130 g / L.

[0063] Example 9

[0064] (1) Same as Example 1.

[0065] (2) Take the yeast paste obtained in step (1), add acetic acid aqueous solution at a rate of 0.1 mg acetic acid / g yeast paste, the concentration of acetic acid aqueous solution is 1%, keep it at 60°C for 12 hours, cool it to 35°C, add snail enzyme at a rate of 10 mg snail enzyme / g yeast paste, keep it at that temperature for 4 hours. Centrifuge at 5000 rpm for 5 minutes, take the supernatant and test the protein content, and calculate the protein release rate to be 69.8%.

[0066] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 116 g / L.

[0067] Comparative Example 1

[0068] (1) Same as Example 1.

[0069] (2) Take the yeast paste obtained in step (1), add acetic acid aqueous solution at a concentration of 1% at a rate of 0.1 mg acetic acid / g yeast paste, and incubate at 60° C. for 12 hours to activate the yeast cell's own enzyme system. Centrifuge at 5000 rpm for 5 minutes, take the supernatant and test the protein content therein, and calculate the protein release rate to be 45.7%.

[0070] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 100 g / L.

[0071] Comparative Example 2

[0072] (1) Same as Example 1.

[0073] (2) The yeast paste obtained in step (1) was kept at 35°C for 4 hours, and then an acetic acid aqueous solution was added thereto at a concentration of 1% at a rate of 0.1 mg acetic acid / g yeast paste, and kept at 60°C for 12 hours to activate the yeast cell enzyme system. The mixture was centrifuged at 5000 rpm for 5 minutes, and the protein content of the supernatant was measured. The protein release rate was calculated to be 46.3%.

[0074] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 102 g / L.

[0075] Comparative Example 3

[0076] (1) Same as Example 1.

[0077] (2) Take the yeast slurry obtained in step (1), add snail enzyme at a rate of 10 mg snail enzyme / g yeast slurry, keep the mixture at 35°C for 4 hours, then raise the temperature to 60°C and keep the mixture for 12 hours. Centrifuge at 5000 rpm for 5 minutes, take the supernatant and test the protein content, and calculate the protein release rate to be 60.3%.

[0078] (3) The operation was the same as in Example 1. After 144 h of fermentation, the concentration of dibasic acid in the fermentation broth was 108 g / L.

[0079] Comparative Example 4

[0080] The culture medium was prepared with dodecane as the raw material, yeast extract was added as the fermentation nitrogen source so that the nitrogen content in the culture medium was the same as that in Example 1, and the culture medium was sterilized at 121° C. for 20 min. Candida tropicalis was inoculated into the fermentation medium at a 10% inoculation rate, and the dibasic acid was fermented in a 3L fermentor at 35° C. and 300 rpm to produce the dibasic acid. The fermentation was stopped after 144 h, and the dibasic acid concentration in the fermentation broth was 140 g / L.

[0081] The concentration of dibasic acid in the fermentation broth during the fermentation process of Example 1 and Comparative Example 4 is shown in FIG. Figure 1 .

[0082] Comparative Example 5

[0083] The culture medium was prepared with dodecane as the raw material, and the yeast paste obtained in step (1) of Example 1 was used as the standard. The yeast paste obtained in step (1) of Example 1 was added to make the nitrogen content in the culture medium the same as that in Example 1. The culture medium was sterilized at 121° C. for 20 min, and tropical Candida was inoculated into the fermentation medium at a 10% inoculation rate. The dibasic acid was fermented in a 3L fermentor at 35° C. and 300 rpm to produce the dibasic acid. The fermentation was stopped after 144 h to obtain a fermentation broth with a dibasic acid concentration of 135 g / L.

Claims

1. A method for recycling fungus residues for producing long-chain dibasic acids, comprising: (1) Pretreatment of bacterial residue: The fermentation residue that produces long-chain dibasic acids is separated to obtain bacterial sludge; (2) Release of substances in bacterial residue: Take the bacterial sludge in (1), add hydrolase and activator, and keep it warm twice at 35℃-50℃ and 52℃-70℃ respectively; (3) Using the treated bacterial residue for fermentation: Using the treated bacterial residue in (2) as a component of the culture medium to replace or partially replace the nitrogen source therein for fermentation.

2. The method according to claim 1, characterized in that The hydrolase is selected from at least one of lysozyme, helicase and protease.

3. The method according to claim 1, characterized in that: The amount of the hydrolase added is 5-50 mg / g of bacterial mud.

4. The method according to claim 1, characterized in that The activator is selected from at least one of glucose, sucrose, acetic acid, sodium acetate, sodium chloride, potassium chloride, microorganism B2, cysteine ​​and sodium sulfite.

5. The method according to claim 1, characterized in that The added amount of the activator is 0.1-1 mg / g bacterial mud.

6. The method according to claim 5, characterized in that The activator is added in the form of an aqueous solution, and the concentration of the activator solution is 1-10wt%.

7. The method according to claim 1, characterized in that The specific operation method of step (2) is: first add the hydrolase, and keep it warm at 35°C-50°C for the first time, then add the activator, and keep it warm at 52°C-70°C for the second time; Or, add the hydrolase and the activator at the same time, keep the temperature at 35°C-50°C for the first time, and then raise the temperature to 52°C-70°C for the second time; Alternatively, the activator is first added, and the mixture is kept warm for the first time at 52°C-70°C, then the temperature is lowered to 35°C-50°C, a hydrolase is added, and the mixture is kept warm for the second time at 35°C-50°C.

8. The method according to claim 1, characterized in that The temperature and time of the two insulations in step (2) are preferably 35°C-42°C for 2-10 h and 55°C-65°C for 6 h-14 h, respectively.

9. The method according to claim 1, characterized in that: The fermentation residue for producing long-chain dibasic acids in step (1) is the residue produced by aerobic fermentation of Candida tropicalis to produce long-chain dibasic acids.

10. The method according to claim 1, characterized in that The separation in step (1) is centrifugal separation or flocculation separation; the centrifugal separation is to centrifuge at a centrifugal speed of 2000rpm-8000rpm for 3-10min, separate and discard the supernatant to obtain bacterial sludge; the flocculation separation is to let the fermentation liquid stand for 2h-4h, remove residual alkanes, use 1%-5% high viscosity chitosan and 1%-3% sodium alginate for flocculation, discard the supernatant, and obtain bacterial sludge.

11. The method according to claim 1, characterized in that: In step (3), the treated bacterial residue is used for fermentation to produce long-chain dibasic acids by fermentation of tropical yeast with alkanes.

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