A method for producing astaxanthin using lignocellulosic raw material

Abstract

The present application relates to a method for producing astaxanthin by using lignocellulosic raw materials, which comprises: sequentially carrying out steam explosion treatment, enzymatic hydrolysis treatment and fermentation treatment on the lignocellulosic raw materials to produce astaxanthin; and the fermentation bacteria used in the fermentation treatment include red Phaffia and a gene engineered Escherichia coli for producing pyruvic acid. The method for producing astaxanthin according to the present application is characterized in that after the lignocellulosic raw materials are subjected to steam explosion treatment, the hemicellulose is partially degraded, which is beneficial to subsequent enzymatic catalysis or microbial utilization, then enzymatic hydrolysis is carried out to form a high-concentration fermentable sugar solution mixed with five-carbon sugars and six-carbon sugars, and finally fermentation treatment is carried out by using the fermentable sugar as a carbon source. In order to improve the astaxanthin synthesis capacity of the red Phaffia, the present application creatively adopts a co-fermentation mode of the red Phaffia and the Escherichia coli, and uses the pyruvic acid synthesized by the Escherichia coli as a promoter to strengthen the astaxanthin synthesis of the red Phaffia, so that the astaxanthin yield is significantly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological fermentation, and relates to a method for producing astaxanthin from lignocellulosic raw materials, in particular to a method for producing astaxanthin from lignocellulosic raw materials by co-fermentation of Phaffia rhodozyma and Escherichia coli genetically engineered bacteria. BACKGROUND

[0002] Lignocellulosic agricultural and forestry wastes are an important renewable resource, which can be converted into bulk chemicals such as ethanol, butanol, succinic acid, etc. by chemical methods, biological methods, etc. However, due to the high conversion cost, it is difficult to realize industrial application so far. Selecting a high value-added product can improve the economy of lignocellulosic biomass resource conversion and utilization.

[0003] Astaxanthin is a ketone type carotenoid, a red solid powder, insoluble in water, soluble in organic solvents. It widely exists in the biological world, especially in aquatic animals such as shrimps, crabs, fish and birds, which plays a role in coloring. Astaxanthin has extremely strong antioxidant capacity, can remove nitrogen dioxide, sulfide, disulfide, etc., and can also reduce lipid peroxidation, effectively inhibit lipid peroxidation caused by free radicals. In addition, it has many physiological effects such as inhibiting tumor occurrence, enhancing immunity, removing free radicals in the body, etc. It has good therapeutic effect on skin cancer caused by ultraviolet rays, and also has preventive and therapeutic effect on eye diseases caused by diabetes, and has broad application prospects in health care products, medicine, cosmetics, food additives and aquaculture, etc.

[0004] Due to the structural characteristics of lignocellulose itself, microorganisms are difficult to directly convert lignocellulose, and it needs to be pretreated. At present, the pretreatment technology includes various methods, such as dilute acid method, hydrothermal method, steam explosion method, grinding method, lime method, etc. Steam explosion method is a relatively in-depth and effective method. During the steam explosion process, hemicellulose is partially degraded, which is beneficial to the subsequent enzyme catalysis or microbial utilization.

[0005] Phaffia rhodozyma is an important microorganism for synthesizing astaxanthin, which has been widely used in the field of feed and other fields. As a tetracyclic astaxanthin, it needs a long synthesis path in the body of Phaffia rhodozyma, which causes the yield and productivity of astaxanthin to be very low. Researchers have improved the performance of microorganisms in synthesizing astaxanthin to some extent through physical, chemical mutagenesis, domestication and biological engineering methods. It is found that adding astaxanthin precursors such as lycopene is an effective method to improve the yield of astaxanthin. However, if these precursor substances are directly added, the production cost of astaxanthin will be significantly increased. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application aims to provide a method for producing astaxanthin by using lignocellulose raw materials, and in particular to provide a method for producing astaxanthin by using lignocellulose raw materials through co-fermentation of Phaffia rhodozyma and Escherichia coli genetic engineering bacteria.

[0007] To achieve the object of the present application, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a method for producing astaxanthin by using lignocellulose raw materials, which comprises: sequentially performing steam explosion treatment, enzymatic hydrolysis treatment and fermentation treatment on the lignocellulose raw materials to produce astaxanthin; and the fermentation bacteria used in the fermentation treatment comprise Phaffia rhodozyma and Escherichia coli genetic engineering bacteria producing pyruvic acid.

[0009] The method for producing astaxanthin according to the present application can partially degrade hemicellulose after steam explosion treatment of the lignocellulose raw materials, which is beneficial to subsequent enzymatic catalysis or microbial utilization, then performs enzymatic hydrolysis on the treated product to form a high-concentration fermentable sugar solution mixed with five-carbon sugars and six-carbon sugars, and finally performs fermentation treatment by using the fermentable sugar as a carbon source. In order to improve the astaxanthin synthesis capacity of Phaffia rhodozyma, the present application creatively adopts a co-fermentation mode of Phaffia rhodozyma and Escherichia coli genetic engineering bacteria producing pyruvic acid, and uses the pyruvic acid synthesized by Escherichia coli as a promoter to strengthen the astaxanthin synthesis of Phaffia rhodozyma, thereby significantly improving the astaxanthin yield, and providing an effective method for synthesizing astaxanthin by using renewable resources.

[0010] Preferably, the inoculation amount of the Phaffia rhodozyma in the fermentation system is 3-10%, for example, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and other specific point values within the numerical range can also be selected, which will not be repeated here. Preferably, 5-7%.

[0011] Preferably, the inoculation amount of the Escherichia coli genetic engineering bacteria in the fermentation system is 0.5-5%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, and other specific point values within the numerical range can also be selected, which will not be repeated here. Preferably, 1-2%.

[0012] In order to make the pyruvic acid produced by Escherichia coli fully strengthen the fermentation production of astaxanthin by Phaffia rhodozyma, while avoiding the competition for carbon sources between Escherichia coli and Phaffia rhodozyma, the present application preferably satisfies the above numerical range for the inoculation amount of the two bacteria.

[0013] Preferably, the inoculation time of the Escherichia coli genetic engineering bacteria is 24-72h after the inoculation of Phaffia rhodozyma, for example, 24h, 36h, 48h, 60h, 72h, and other specific point values within the numerical range can also be selected, which will not be repeated here.

[0014] Preferably, the fermentation system has a pH of 5.5-7.0, such as 5.5, 6.0, 6.5, 7.0, etc.

[0015] Preferably, the fermentation system has a temperature of 14-35℃, such as 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 32℃, 34℃, etc.; and a fermentation time of 30-120h, such as 30h, 45h, 60h, 75h, 90h, 105h, 120h, etc.

[0016] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0017] In order to fully strengthen the production of astaxanthin by Rhodotorula rubra using pyruvic acid produced by Escherichia coli, and avoid the competition for carbon sources between Escherichia coli and Rhodotorula rubra, the present application preferably uses a low-temperature and slightly acidic environment suitable for the growth of Rhodotorula rubra, and delays the inoculation time of Escherichia coli, and inoculates Escherichia coli after Rhodotorula rubra enters the logarithmic phase.

[0018] The above fermentation parameters cooperate with each other to achieve better fermentation effect, which is conducive to obtaining astaxanthin with higher yield.

[0019] Inorganic salts and trace elements also need to be added to the fermentation system.

[0020] The inorganic salts may be, for example, KH2PO4, KCl, MgSO4·7H2O, etc.; and the trace elements may be, for example, FeCl3·6H2O, CoCl2·6H2O, CuCl2·2H2O, ZnCl2, Na2MoO4·2H2O, H3BO3, MnCl2·4H2O, etc.

[0021] Preferably, the initial fermentable sugar concentration in the fermentation system is 10-60g / L or more, such as 10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L, 40g / L, 45g / L, 50g / L, 55g / L, 60g / L, etc. Other specific point values in the above numerical range can be selected, which will not be repeated here. Preferably, 25-35g / L. During the fermentation process, sugar solution is supplemented according to the residual sugar amount in the fermentation tank, and when the residual sugar amount is less than 2.0g / L, sugar solution is added to maintain the sugar concentration in the fermentation tank at more than 5g / L. The pH value of the fermentation broth is adjusted by adding ammonia water.

[0022] Preferably, ammonium salt is added to the steam explosion treatment system.

[0023] In order to further improve the degradation efficiency of hemicellulose, the prior art generally adds a certain amount of sulfuric acid in the steam explosion process, but the addition of sulfuric acid often produces more fermentation inhibitors, such as acetic acid and furfural, etc., which has a great influence on the subsequent fermentation. Therefore, the present application creatively uses ammonium salt as an additive, which reduces the pH of the lignocellulose steam explosion system by virtue of its weak acidity, improves the degradation efficiency of hemicellulose in the steam explosion process, improves the biodegradability of lignocellulose, and at the same time reduces the generation of fermentation inhibitors; in addition, the ammonium salt remaining in the system can provide nitrogen source for the subsequent fermentation link, thereby reducing the cost of the fermentation link.

[0024] Preferably, the ammonium salt includes any one or a combination of at least two of ammonium sulfate, ammonium chloride or ammonium phosphate.

[0025] Preferably, the mass ratio of the ammonium salt to the lignocellulose raw material is (0.1-10):100, for example 0.1:100, 1:100, 2:100, 4:100, 6:100, 8:100, 10:100, etc., preferably (2-4):100.

[0026] When the mass ratio of the ammonium salt to the lignocellulose raw material meets the above numerical range, the effect of improving the degradation efficiency of hemicellulose in the steam explosion process, improving the biodegradability of lignocellulose, and at the same time reducing the generation of fermentation inhibitors is more significant.

[0027] Preferably, the solid content in the steam explosion treatment system is 60-90%, for example 60%, 70%, 75%, 80%, 90%, etc., and further preferably 70-75%.

[0028] Preferably, the pressure of the steam explosion treatment is 0.8-2.0 MPa, for example 0.8 MPa, 1.0 MPa, 1.2 MPa, 1.4 MPa, 1.5 MPa, 1.6 MPa, 2.0 MPa, etc., and the duration is 1-10 min, for example 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 8 min, 10 min, etc.

[0029] Other specific point values of the above numerical ranges can be selected, which will not be repeated here.

[0030] The above steam explosion treatment parameters cooperate with each other, which can make the steam explosion effect better, and is conducive to obtaining higher yield of astaxanthin.

[0031] Preferably, the enzyme treatment uses a complex enzyme, and the complex enzyme includes any one or a combination of at least two of cellulase and hemicellulase.

[0032] Preferably, the cellulase includes any one or a combination of at least two of β-glucosidase, endoglucanase, exoglucanase or pectinase.

[0033] Preferably, the hemicellulase comprises xylanase and / or laccase.

[0034] Preferably, the complex enzyme comprises beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase.

[0035] When the complex enzyme used in the method of the present application comprises beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase at the same time, the enzymatic effect is better, and the concentration of the obtained fermentable sugar solution is higher.

[0036] Preferably, the ratio of filter paper activity, beta-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the complex enzyme is (3-5):(1-2):(0.5-1):(1-2):(0.5-1).

[0037] When the complex enzyme used in the method of the present application comprises beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase at the same time, the enzymatic effect is better, and the concentration of the obtained fermentable sugar solution is higher.

[0038] The specific point value in the above (3-5) can be 3, 3.5, 4, 4.5, 5, etc.

[0039] The specific point value in the above (1-2) can be 1, 1.2, 1.5, 1.8, 2, etc.

[0040] The specific point value in the above (0.5-1) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0041] Other specific point values within the above numerical ranges can be selected, which will not be described one by one here.

[0042] Preferably, the solid content in the enzymatic treatment system is 15-30%, for example, 15%, 16%, 18%, 20%, 22%, 23%, 24%, 26%, 28%, 30%, etc.

[0043] Preferably, the temperature of the enzymatic treatment is 40-60℃, for example, 40℃, 42℃, 45℃, 47℃, 50℃, 52℃, 55℃, 58℃, 60℃, etc., and the time of the enzymatic treatment is 20-40h, for example, 20h, 25h, 30h, 35h, 40h, etc.

[0044] Other specific point values within the above numerical ranges can be selected, which will not be described one by one here.

[0045] The above enzymatic treatment parameters cooperate with each other, which can make the enzymatic effect better, and is conducive to obtaining higher yield of astaxanthin.

[0046] In the present application, the lignocellulosic raw material includes any one or a combination of at least two of straw, rice husk, corn cob, sawdust, wood chips, branches or sugarcane residue.

[0047] Preferably, the lignocellulosic raw material is crop waste.

[0048] The method of the present application can improve the economy of lignocellulosic biomass resource conversion and utilization.

[0049] Preferably, the lignocellulosic raw material is further subjected to a crushing treatment before the steam explosion treatment.

[0050] Preferably, the particle size after the crushing treatment is 1-50mm, such as 1mm, 5mm, 10mm, 20mm, 30mm, 40mm, 50mm, etc., and other specific point values in this range can be selected, which will not be repeated here. Further preferably, 10-20mm.

[0051] As a preferred technical solution of the present application, the method for producing astaxanthin using lignocellulosic raw material includes:

[0052] (1) crushing the lignocellulosic raw material and then performing steam explosion treatment, wherein an ammonium salt is added to the steam explosion treatment system, and the mass ratio of the ammonium salt to the lignocellulosic raw material is (0.1-10):100;

[0053] (2) then performing enzymatic hydrolysis treatment using a composite enzyme, wherein the composite enzyme includes beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase;

[0054] (3) then performing co-fermentation treatment using Rhodotorula rubra and a gene engineered Escherichia coli strain producing pyruvate, wherein the inoculation amount of the Rhodotorula rubra in the fermentation system is 3-10%, the inoculation amount of the gene engineered Escherichia coli strain in the fermentation system is 0.5-5%, the inoculation time of the gene engineered Escherichia coli strain is 24-72h after the inoculation of the Rhodotorula rubra, the pH of the fermentation treatment system is 5.5-7.0, the fermentation treatment temperature is 14-35℃, and the fermentation treatment time is 30-120h.

[0055] Compared with the prior art, the present application has the following beneficial effects:

[0056] In one aspect, in order to improve the ability of Phaffia rhodozyma to synthesize astaxanthin, the present application adopts the co-culturing method of Phaffia rhodozyma and the genetically engineered Escherichia coli producing pyruvic acid. In order to provide pyruvic acid and not compete with Phaffia rhodozyma for carbon source, the low temperature and acidic environment suitable for the growth of Phaffia rhodozyma is adopted, and the inoculation time of Escherichia coli is postponed, that is, Escherichia coli is inoculated after Phaffia rhodozyma enters the logarithmic phase, and the appropriate inoculation amount of strains is adopted.

[0057] On the other hand, the present application uses ammonium salt as an additive, utilizes its weak acidity to reduce the pH of the steam explosion system of lignocellulose, improve the degradation efficiency of hemicellulose in the steam explosion process, improve the biodegradability of lignocellulose, and reduce the generation of fermentation inhibitors. In addition, the ammonium salt remaining in the system can provide nitrogen source for the subsequent fermentation link, thereby reducing the cost of the fermentation link. DETAILED DESCRIPTION

[0058] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with the preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0059] The Escherichia coli involved in the following examples is a genetically engineered Escherichia coli producing pyruvic acid, which is Escherichia coli YP02 strain with the preservation number of CGMCC No. 12463, preserved by China General Microbiological Culture Collection Center, on May 18, 2016, and located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing.

[0060] The Phaffia rhodozyma involved in the following examples is purchased from China Industrial Microbial Culture Collection Management Center, with the preservation number of CICC 33064, preserved on January 29, 2015, and located at No. 6, Yard 6, Jiuxianqiao Middle Road, Chaoyang District, Beijing.

[0061] The formula of the seed culture medium involved in the following examples is as follows: carbon source: 1% glucose; ammonium chloride: 2 g / L; macro elements (mmol / L): (NH4)2HPO419.92, NH4H2PO47.56, KCl 2.00, NH4Cl 7.50, MgSO4·7H2O 1.50; micro elements (μmol / L): FeCl3·6H2O 8.88, CoCl2·6H2O 1.26, CuCl2·2H2O 0.88, ZnCl2 2.20, Na2MoO4·2H2O 1.24, H3BO3 1.21, MnCl2·4H2O 2.50.

[0062] The fermentation medium used in the following examples is as follows: carbon source: hydrolyzed sugar mother liquor; macro elements (mmol / L): KH2PO47.56, KCl 2.00, MgSO4·7H2O 1.50; micro elements (μmol / L): FeCl3·6H2O 8.88, CoCl2·6H2O 1.26, CuCl2·2H2O 0.88, ZnCl2 2.20, Na2MoO4·2H2O 1.24, H3BO3 1.21, MnCl2·4H2O 2.50.

[0063] The β-glucosidase used in the following examples is purchased from Sigma Company, model number G0395; the endoglucanase is purchased from Sigma Company, model number E2164; the exoglucanase is purchased from Arladin Company, model number D298997; the pectinase is purchased from Arladin Company, model number P128776; the xylanase is purchased from Sigma Company, model number X3876; the laccase is purchased from Arladin Company, model number L304691.

[0064] The astaxanthin in the fermentation broth obtained in the following examples is analyzed by the following method: the cells in the fermentation broth are recovered by centrifugation and washed twice with distilled water. The yeast cell wall is treated with 3M HCl and boiled. The yeast solution is centrifuged and the astaxanthin is extracted with acetone. The content of astaxanthin is determined by HPLC equipped with a UV-vis detector. The detection column is a C18 column (4.6 mm x 250 mm), acetonitrile: ultrapure water (95:5) is used as the mobile phase, and the flow rate is 1 mL / min.

[0065] Example 1

[0066] This example provides a method for generating astaxanthin using corn stalks, as follows:

[0067] (1) The corn stalks are crushed to a particle size of 20 mm; the corn stalk particles are mixed with water to a moisture content of 30%, ammonium salt is added at a ratio of 1:100 of ammonium sulfate to corn stalks, and stirred uniformly; the steam explosion machine is loaded for steam explosion treatment, and the treatment conditions are set as follows: pressure 1.8 MPa, holding time 2.4 min, and the steam-exploded corn stalks are collected;

[0068] (2) The pretreated corn stalks are subjected to enzymatic hydrolysis by a complex enzyme, which is a mixture of β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, and the ratio of filter paper enzyme activity, β-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the complex enzyme is 4:2:1:2:1. First, the complex enzyme with filter paper enzyme activity of 20 FPU / g is added, and the enzymatic hydrolysis is carried out at 50°C for 36 h. After the end of the enzymatic hydrolysis, the hydrolysis sugar mother liquor is obtained through sedimentation and filtration, and the concentrations of monosaccharides such as glucose and xylose in the solution are determined. It is determined that the glucose concentration in the solution reaches 95 g / L, and the xylose concentration reaches 35 g / L, which completely meets the requirements of the fermentation of astaxanthin;

[0069] (3) The astaxanthin is produced by co-culturing Escherichia coli and Phaffia rhodozyma;

[0070] (3.1) Seed culture: 100 mL of seed culture medium is placed in a 250 mL triangular flask, and the seed culture medium is sterilized at 115°C for 30 min. After cooling, Escherichia coli and Phaffia rhodozyma are inoculated into the seed culture medium at an inoculation amount of 1% (v / v), and the seed liquid is obtained by culturing at pH = 6.0, 26°C and 200 rpm for 30 h;

[0071] (3.2) Fermentation culture: the volume of the fermentation medium in a 5L fermentation tank is 3L, and the fermentation medium is sterilized at 121°C for 20 min. The hydrolysis sugar mother liquor obtained in step (2) is added to make the monosaccharide concentration in the fermentation tank 5%. The Phaffia rhodozyma seed liquid is inoculated into the fermentation medium at an inoculation amount of 5% (v / v), and the astaxanthin fermentation liquid is obtained by culturing at pH = 6.0, 20°C and 200 rpm for 24 h, and then adding the Escherichia coli seed liquid (inoculation amount 1%) and culturing for 72 h. During the culture, the pH of the fermentation system is adjusted by using ammonia water to maintain at about 6.0.

[0072] The components in the fermentation liquid are quantitatively analyzed by using a high-performance liquid chromatograph, and it is detected that the astaxanthin yield is 0.2 mg / g of bacterial body.

[0073] Example 2

[0074] The present embodiment provides a method for generating astaxanthin by using poplar sawdust, as follows:

[0075] (1) The poplar sawdust is crushed to a particle size of 40 mm; the poplar sawdust particles are mixed with water to a water content of 40%, ammonium salt is added at a ratio of 3:100 of ammonium chloride to poplar sawdust, and stirred uniformly; the poplar sawdust is loaded into a steam explosion machine for steam explosion treatment, and the treatment conditions are set as follows: pressure 2.0 MPa, and holding time 3.0 min; the poplar sawdust after steam explosion is collected;

[0076] (2) The pretreated poplar sawdust is subjected to enzymatic hydrolysis by a complex enzyme, which is a mixture of β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, and the ratio of filter paper enzyme activity, β-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the complex enzyme is 5:2:1:2:1. First, the complex enzyme with filter paper enzyme activity of 20 FPU / g is added, and the enzymatic hydrolysis is carried out at 50°C for 48 h. After the end of the enzymatic hydrolysis, the hydrolysis sugar mother liquor is obtained through sedimentation and filtration, and the concentrations of monosaccharides such as glucose and xylose in the solution are determined. It is determined that the glucose concentration in the solution reaches 88 g / L, and the xylose concentration reaches 32 g / L, which completely meets the requirement of fermentation of astaxanthin;

[0077] (3) The astaxanthin is produced by co-culturing of E. coli and Phaffia rhodozyma;

[0078] (3.1) Seed culture: 100 mL of seed culture medium is placed in a 250 mL triangular flask, and the seed culture medium is sterilized at 115°C for 30 min. After cooling, E. coli and Phaffia rhodozyma are inoculated into the seed culture medium at an inoculation amount of 2% (v / v), and the seed liquid is obtained by culturing at pH = 6.5, 24°C and 200 rpm for 36 h;

[0079] (3.2) Fermentation culture: the volume of fermentation medium in a 5L fermentation tank is 3L, and the fermentation medium is sterilized at 121°C for 20 min. The hydrolysis sugar mother liquor obtained in step (2) is added to make the monosaccharide concentration in the fermentation tank 5%. The Phaffia rhodozyma seed liquid is inoculated into the fermentation medium at an inoculation amount of 8% (v / v), and the astaxanthin fermentation liquid is obtained by culturing at pH = 6.5, 22°C and 200 rpm for 24 h, and then adding the E. coli seed liquid (inoculation amount 0.5%) and culturing for 84 h. During the culture, the pH of the fermentation system is adjusted by using ammonia water to maintain at about 6.5;

[0080] The components in the fermentation liquid are quantitatively analyzed by using a high-performance liquid chromatograph, and it is detected that the astaxanthin yield is 0.25 mg / g of bacterial body.

[0081] Example 3

[0082] The present embodiment provides a method for generating astaxanthin by using bagasse, as follows:

[0083] (1) The bagasse is crushed to a particle size of 5 mm; the bagasse particles are mixed with water to a water content of 45%, ammonium salt is added at a ratio of 5:100 of ammonium sulfate to bagasse, and stirred uniformly; the bagasse is loaded into a steam explosion machine for steam explosion treatment, and the treatment conditions are set as follows: pressure 1.6 MPa, and holding time 5 min, and the bagasse after steam explosion is collected;

[0084] (2) the pretreated bagasse is subjected to enzymolysis by a complex enzyme, the complex enzyme being a mixture of β-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, the ratio of filter paper enzyme activity, β-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the complex enzyme being 5:1:1:2:1; first, the complex enzyme with filter paper enzyme activity of 25 FPU / g is added, the enzymolysis temperature is 50°C, and the enzymolysis is performed for 36 h; after the end of the enzymolysis, the hydrolysis sugar mother liquor is obtained through sedimentation and filtration, and the concentrations of monosaccharides such as glucose and xylose in the solution are determined; through determination, the glucose concentration in the solution reaches 97 g / L, and the xylose concentration reaches 35 g / L, and the sugar concentration completely meets the requirement for fermentation of astaxanthin;

[0085] (3) astaxanthin is produced by co-culturing of E. coli and Phaffia rhodozyma;

[0086] (3.1) seed culture: 100 mL of seed culture medium is placed in a 250 mL triangular flask, and sterilization is performed at 115°C for 30 min. After cooling, E. coli and Phaffia rhodozyma are inoculated into the seed culture medium at an inoculation amount of 5% (v / v), and seed liquid is obtained by culturing at pH=6.0, 22°C and 200 rpm for 40 h;

[0087] (3.2) fermentation culture: the volume of fermentation medium in a 5L fermentation tank is 3L, and sterilization is performed at 121°C for 20 min; the hydrolysis sugar mother liquor obtained in step (2) is added to make the monosaccharide concentration in the fermentation tank 5.5%; the seed liquid of Phaffia rhodozyma is inoculated into the fermentation medium at an inoculation amount of 10% (v / v), and after culturing at pH=6.0, 22°C and 200 rpm for 24 h, the seed liquid of E. coli is added (inoculation amount 2%), and after culturing for 72 h, astaxanthin fermentation liquid is obtained. During the culturing, ammonia water is used to adjust the pH of the fermentation system, so that the pH is maintained at about 6.0.

[0088] High-performance liquid chromatography is used to quantitatively analyze the components in the fermentation liquid, and through detection, the astaxanthin yield is 0.32 mg / g of bacteria.

[0089] Example 4

[0090] The application provides a method for generating astaxanthin by using bagasse, and the difference between the method and example 3 is only that the process of step (3.2) is different: the volume of fermentation medium in a 5L fermentation tank is 3L, and sterilization is performed at 121°C for 20 min; the hydrolysis sugar mother liquor obtained in step (2) is added to make the monosaccharide concentration in the fermentation tank 5.5%; the seed liquid of Phaffia rhodozyma is inoculated into the fermentation medium at an inoculation amount of 10% (v / v), and after culturing at pH=6.0, 22°C and 200 rpm for 96 h. During the culturing, ammonia water is used to adjust the pH of the fermentation system, so that the pH is maintained at about 6.0. Other operations remain unchanged.

[0091] The components in the fermentation broth were quantitatively analyzed by using a high performance liquid chromatograph, and the astaxanthin yield was 0.23 mg / g of bacteria.

[0092] Example 5

[0093] The present application provides a method for generating astaxanthin by using bagasse, which is different from the method of Example 3 only in that the inoculation amount of the E. coli seed liquid in step (3.2) is 8%, and other operations remain unchanged.

[0094] The components in the fermentation broth were quantitatively analyzed by using a high performance liquid chromatograph, and the astaxanthin yield was 0.19 mg / g of bacteria.

[0095] Example 6

[0096] The present application provides a method for generating astaxanthin by using bagasse, which is different from the method of Example 3 only in that the culture conditions in step (3.2) are changed from pH = 6.0, 22℃ and 200 rpm for 24 h to pH = 7.5, 37℃ and 200 rpm for 24 h, and other operations remain unchanged.

[0097] The components in the fermentation broth were quantitatively analyzed by using a high performance liquid chromatograph, and the astaxanthin yield was 0.25 mg / g of bacteria.

[0098] Example 7

[0099] The present application provides a method for generating astaxanthin by using bagasse, which is different from the method of Example 3 only in that the fermentation process in step (3.2) is different: the volume of the fermentation medium in a 5L fermentation tank is 3L, which is sterilized at 121℃ for 20 min, and the hydrolyzed sugar mother liquor obtained in step (2) is added to make the monosaccharide concentration in the fermentation tank 5.5%. The red Phaffia yeast seed liquid is inoculated into the fermentation medium at a inoculation amount of 10% (v / v) and the E. coli seed liquid (inoculation amount 2%) at the same time, and the fermentation broth is cultured at pH = 6.0, 22℃ and 200 rpm for 96 h to obtain an astaxanthin fermentation broth. During the culture, the pH of the fermentation system is adjusted by using ammonia water to maintain at about 6.0. Other operations remain unchanged.

[0100] The components in the fermentation broth were quantitatively analyzed by using a high performance liquid chromatograph, and the astaxanthin yield was 0.09 mg / g of bacteria.

[0101] Example 8

[0102] The application provides a method for producing astaxanthin by using bagasse, which is different from the method of embodiment 3 only in that the ammonium salt is added in the ratio of 5:100 of ammonium sulfate to bagasse in step (1) is replaced by adding 5% sulfuric acid in the ratio of 1:100 of sulfuric acid to bagasse. Other operations remain unchanged.

[0103] The components in the fermentation liquid are quantitatively analyzed by using a high-performance liquid chromatograph, and the astaxanthin yield is 0.21 mg / g of bacteria by detection.

[0104] The applicant declares that the application illustrates a method for producing astaxanthin by using lignocellulose raw materials through the above embodiments, but the application is not limited to the above embodiments, that is, it does not mean that the application must rely on the above embodiments to be implemented. It should be understood by those skilled in the art that any improvement on the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the application.

[0105] The preferred embodiments of the application are described in detail above, but the application is not limited to the specific details in the above embodiments, and within the technical concept scope of the application, various simple modifications can be made to the technical solutions of the application, and these simple modifications all belong to the protection scope of the application.

[0106] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the application will not further describe various possible combination manners.

Claims

1. A method for producing astaxanthin using lignocellulosic raw material, characterized by, The method comprises: sequentially performing steam explosion treatment, enzymatic hydrolysis treatment and fermentation treatment on the lignocellulosic raw material to produce astaxanthin; the fermentation bacteria used in the fermentation treatment comprise red Phaffia and a gene engineered Escherichia coli for producing pyruvic acid; the gene engineered Escherichia coli for producing pyruvic acid is Escherichia coli YP02 strain with a preservation number of CGMCC No. 12463; The ammonium salt is added in the system of the steam explosion treatment; The enzymatic hydrolysis treatment uses a composite enzyme, and the composite enzyme is beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase; The inoculation amount of the red Phaffia in the fermentation system is 3-10%; The inoculation amount of the gene engineered Escherichia coli in the fermentation system is 0.5-5%; The inoculation time of the gene engineered Escherichia coli is 24-72 h after the inoculation of the red Phaffia; The pH of the system of the fermentation treatment is 5.5-7.0; The temperature of the fermentation treatment is 14-35 DEG C, and the time of the fermentation treatment is 30-120 h.

2. The method for producing astaxanthin using a lignocellulosic material according to claim 1, characterized by, The ammonium salt comprises any one or a combination of at least two of ammonium sulfate, ammonium chloride or ammonium phosphate.

3. The method for producing astaxanthin using a lignocellulosic material according to claim 1, wherein The mass ratio of the ammonium salt to the lignocellulosic raw material is (0.1-10):

100.

4. The method for producing astaxanthin using lignocellulosic raw material according to claim 3, characterized by, The mass ratio of the ammonium salt to the lignocellulosic raw material is (2-4):

100.

5. The method for producing astaxanthin using lignocellulosic feedstock according to claim 1, wherein The pressure of the steam explosion treatment is 0.8-2.0 MPa, and the duration is 1-10 min.

6. The method for producing astaxanthin using lignocellulosic feedstock according to claim 1, wherein, The ratio of filter paper enzyme activity, beta-glucosidase activity, pectinase activity, xylanase activity and laccase activity in the composite enzyme is (3-5):(1-2):(0.5-1):(1-2):(0.5-1).

7. The method for producing astaxanthin using lignocellulosic feedstock according to claim 1, wherein The temperature of the enzymatic hydrolysis treatment is 40-60 DEG C, and the time of the enzymatic hydrolysis treatment is 20-40 h.

8. The method for producing astaxanthin using lignocellulosic feedstock according to claim 1, wherein The lignocellulosic raw material comprises any one or a combination of at least two of straw, rice husk, corncob, sawdust, wood chips, branches or sugarcane residue.

9. The method for producing astaxanthin using lignocellulosic feedstock according to claim 1, wherein, The lignocellulosic raw material is crop waste.

10. The method for producing astaxanthin using lignocellulosic feedstock according to claim 1, wherein The lignocellulosic raw material is further subjected to a crushing treatment before the steam explosion treatment.

11. The method for producing astaxanthin using a lignocellulosic feedstock according to claim 10, wherein The particle size after the crushing treatment is 1-50 mm.

12. The method for producing astaxanthin using lignocellulosic feedstock according to claim 1, wherein The method comprises: (1) crushing the lignocellulosic raw material and then performing steam explosion treatment, wherein an ammonium salt is added in the system of the steam explosion treatment, and the mass ratio of the ammonium salt to the lignocellulosic raw material is (0.1-10):100; (2) then performing enzymatic hydrolysis treatment using a composite enzyme, wherein the composite enzyme is beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase; (3) then using red phaffia and pyruvate-producing escherichia coli genetically engineered bacteria for co-fermentation treatment, the inoculation amount of the red phaffia in the fermentation system is 3-10%, the inoculation amount of the escherichia coli genetically engineered bacteria in the fermentation system is 0.5-5%, the inoculation time of the escherichia coli genetically engineered bacteria is 24-72 h after the inoculation of the red phaffia, the pH of the fermentation treatment system is 5.5-7.0, the fermentation treatment temperature is 14-35 DEG C, and the time is 30-120 h.