CN114540425B - Method for producing fungus water-soluble red pigment by wood powder mixed solid state fermentation - Google Patents
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Abstract
A method for producing fungal water-soluble red pigment by wood powder mixed solid state fermentation comprises the following steps: (1) pre-treating wood flour; (2) activating the strain; (3) preparing a wood powder mixed culture medium; (4) Inoculating the bacterial cake to the wood powder mixed culture medium center in the step (3), and culturing and fermenting in a constant temperature and humidity incubator; (5) Cutting the fermented culture medium into small pieces, pouring the small pieces into a container, adding distilled water according to the liquid-to-material ratio of 30:1, sealing, performing water bath ultrasonic extraction, filtering, bottling, and freeze-drying to obtain water-soluble red pigment powder. The method is low in cost, pollution-free, and efficient and stable in production of the water-soluble red pigment.
Description
Technical Field
The invention relates to the technical field of biological color production and biological pigment extraction, in particular to a method for producing fungal water-soluble red pigment by utilizing wood powder mixed solid fermentation.
Background
There are many methods for optimizing the medium to increase the yield of the pigment fungi pigment, including solid fermentation by controlling carbon source, nitrogen source, carbon-nitrogen ratio, trace element, PH, temperature, fermentation time, etc., and liquid fermentation by controlling the size of the fermenter, inoculum size, rotation speed of the shaker, etc., in addition to the above conditions, and they have in common that the carbon-nitrogen source added is a substance extracted or synthesized in chemical industry, and the production of these substances requires a large amount of energy. Therefore, how to obtain high-efficiency pigment production with low energy consumption is a new research direction. The current research using inexpensive industrial and agricultural byproducts as a medium substrate has the following problems: (1) The selection of the types of the cheap matrix materials is not carried out according to the habit of the strains, but pursues low price; (2) The types of the low-cost matrix materials in one culture medium are single, and the low-cost matrix materials are not mixed and added according to the characteristics of different materials. In addition, the color optimization method of the SHL-1 fungus of the pyriform false-shell family is not reported, so the method for efficiently and stably producing the water-soluble red pigment by the strain has great significance for the application of the pigment.
Disclosure of Invention
The invention aims to provide a method for producing fungal water-soluble haematochrome by utilizing wood powder mixed solid fermentation, which can rapidly obtain a large amount of natural water-soluble haematochrome with the yield of up to 3.09g/L. The implementation process of the method is simple and easy to control, the used materials are natural and green, and the by-products can eliminate the influence on the environment and ecology by sterilizing.
In order to solve the technical problems, the technical scheme of the invention is as follows: the strain used was a fungus of the family Pyricularia pseudohousing (Arthroinium phaeospermum) SHL-1, which was deposited in China center for type culture Collection, 12 months and 17 days in 2018, with accession number: cctccc No: m2018900, deposit address: wuhan city Wuchang Lopa nationality university of Wuhan at mountain and mountain.
A method for producing fungal water-soluble red pigment by wood powder mixed solid state fermentation comprises the following steps:
(1) Cutting paulownia and bagasse into pieces with length and width of 1-2cm and thickness of 1-2mm, drying in a baking oven at 102 ℃ to constant weight, crushing the pieces, sieving with a 100-mesh sieve, placing the sieved pieces of wood powder into a storage tank, performing solid sterilization for 40min at 121 ℃ and 0.1MPa with an autoclave, and cooling to obtain pretreated wood powder;
(2) Activating strain, picking SHL-1 fungus blocks of Pseudomonas pyriform (Arthroinium phaeospermum) of Pyricularia, inoculating to PDA culture medium, sealing, culturing in dark in constant temperature and humidity incubator at 20+ -2deg.C for 14 days, filling mycelium into 90mm culture dish, and placing the culture dish into refrigerator at 4deg.C to obtain experimental strain;
(3) Respectively weighing paulownia powder and bagasse powder according to a certain concentration and proportion, adding the paulownia powder and the bagasse powder into a conical flask, pouring the conical flask into a PDA culture medium, uniformly mixing, sealing, sterilizing the mixture for 20min at 121 ℃ under 0.1MPa by using an autoclave, cooling the mixture to 80 ℃, transferring the uniformly shaken culture medium into a 90mm culture dish according to the amount of 20 mL/dish by using a sterile injector in an ultra-clean workbench, and cooling the mixture for 30min to obtain a wood powder mixed culture medium;
(4) Punching the strain in the step (2) into a bacterial cake with the diameter of 6mm by using a puncher in an ultra-clean workbench, inoculating the bacterial cake to the center of the wood powder mixed culture medium in the step (3) by using an inoculating needle, sealing, and placing the bacterial cake in a constant temperature and humidity incubator with the temperature of 20+/-2 ℃ for dark culture and fermentation for 14 days;
(5) Cutting the culture medium after fermentation in the step (4) into 1cm pieces 2 Small pieces, poured into a beaker, and distilled water was used as follows: distilled water is added into the culture medium with the liquid-to-material ratio of 3:1, after the preservative film is sealed, the beaker is placed into an ultrasonic cleaner to be extracted for 30min at 60 ℃, the ultrasonic cleaner is used for extracting twice, then a filter film with the thickness of 0.22 mu m is used for filtering, bottling is carried out, the mixture is placed into a refrigerator with the temperature of minus 60 ℃ for freezing for 24h, and then continuous freeze drying is carried out for 36h, thus obtaining the water-soluble red pigment powder.
The paulownia in the step (1) is paulownia rim material produced from the lotus in the east of the Shandong province, and the bagasse is white bagasse left after the sugar and the sugarcane rind are squeezed from black sugarcane produced in Guangxi province.
The PDA culture medium used in the step (2) is used by mixing PDA powder with deionized water according to the proportion of 46.1g/L, boiling, and sterilizing at high temperature.
In the step (3), the concentration of wood powder in the culture medium is 3-5 mg/mL, the concentration ratio of paulownia powder to bagasse powder is 3mg/mL+3 mg/mL-5 mg/mL+5mg/mL, and the wood powder mixed culture medium is uniformly mixed until the wood powder is completely suspended in the PDA culture medium before sterilization.
And (4) burning the puncher and the inoculating needle with an alcohol lamp to be red, cooling to normal temperature, and inoculating the mycelia on one surface of the fungus cake downwards during inoculation, wherein the whole process is carried out in an ultra-clean workbench.
And (3) cooling the pigment extract in the step (5), filtering, and filling the pigment extract in 20mL sample bottles when freezing, wherein the concentration of the pigment solution in each bottle is not more than 10mL.
The ultra-clean workbench is used after ultraviolet sterilization for 30min, and the operation process is performed around the ignition alcohol lamp.
Compared with the existing fungal pigment production technology, the invention has the advantages that:
(1) In the application of the material, the natural green biomass material is used as an additive of the culture medium, so that the environment and ecology cannot be damaged, and the utilization value and the way of agricultural byproducts can be increased;
(2) Screening tree species capable of promoting the growth of the pear spore pseudomonas (Arthroinium phaeospermum) SHL-1 and pigment yield through experiments before wood powder mixed culture; the minimum wood powder addition amount affecting the growth of the pear spore pseudomonas (Arthroinium phaeospermum) SHL-1 and the pigment yield is determined through the experiment of the wood powder addition amount, and a targeted reference is provided for wood powder mixed culture;
(3) The biomass wood powder of different types and sources is subjected to targeted mixed addition culture, so that the unique performance of each wood powder can be fully exerted in one culture medium, under the coordination action of different wood powder, the unit biomass of the Shl-1 of the Pyricularia graminis fungus (Arthroinium phaeospermum) can be rapidly increased, and the pigment yield of the SHL-1 of the Pyricularia graminis fungus (Arthroinium phaeospermum) can be further improved, and the yield of the water-soluble red pigment of the SHL-1 of the Pyricularia graminis fungus (Arthroinium phaeospermum) can be effectively and stably improved in a certain fermentation period.
Detailed Description
The technical scheme of the invention is further described below by examples. The materials and instruments used in the examples are all commercially available.
Example 1
This example is another example of the method for producing fungal water-soluble red pigment using wood flour mixed solid state fermentation according to the present invention, comprising the steps of:
(1) Cutting paulownia and bagasse into small blocks by a knife, putting the small blocks into a baking oven at 102 ℃ to be dried to constant weight, then crushing the wood blocks by a crusher, screening the wood blocks by a 100-mesh sieve, putting the screened wood powder into a 200mL storage tank, then performing solid sterilization for 40min at 121 ℃ and 0.1MPa by an autoclave, and cooling to obtain pretreated biomass material powder;
(2) The strain preserved in the test tube in the laboratory is shoveled into small blocks by an inoculating shovel, inoculated in a newly manufactured PDA culture medium, sealed and placed in a constant temperature and humidity incubator at 20+/-2 ℃ for dark culture for 14 days, the mycelium is fully distributed in a culture dish with 90mm, and the culture dish is placed in a refrigerator at 4 ℃ to be used as an experimental strain;
(3) Respectively weighing 400mg of paulownia wood powder according to a certain concentration and proportion, adding 300mg of bagasse powder into a 250mL conical flask, pouring 100mL of new PDA culture medium into the conical flask, uniformly mixing, sealing, sterilizing the mixture for 20min by using a high-pressure sterilizing pot at 121 ℃ and 0.1MPa, cooling the mixture to 80 ℃, transferring the uniformly shaken culture medium into a 90mm culture dish by using a 30mL sterile injector in an ultra-clean workbench according to the amount of 20 mL/dish, and cooling for 30min to obtain a wood powder mixed culture medium;
(4) Beating the activated strain in step (2) into a bacterial cake with the diameter of 6mm by using a sterilized puncher in an ultra-clean workbench, inoculating the bacterial cake to the center of the wood powder mixed culture medium in step (3) by using a sterilized inoculating needle, performing the whole process in the ultra-clean workbench, ensuring sterile operation, sealing a culture dish, and placing the culture dish in a constant temperature and humidity incubator with the temperature of 20+/-2 ℃ for dark culture and fermentation for 14 days;
(5) Cutting the culture medium after fermentation in step (4) into small pieces by a surgical knife, pouring the small pieces into a 300mL beaker, adding distilled water according to the liquid-to-material ratio of 30:1, sealing by a preservative film, placing the beaker into an ultrasonic cleaning machine, ultrasonically extracting at 60 ℃ for 30min, extracting twice, cooling pigment solution, and filtering by a 0.22 mu m filter membrane. And subpackaging the filtrate into 20mL sample bottles, filling 10mL of the filtrate into each bottle, freezing the bottles in a refrigerator at the temperature of minus 60 ℃ for 24 hours, and then continuously freeze-drying the bottles for 36 hours to obtain water-soluble red pigment powder, wherein the pigment yield is shown in the table I.
Example 2
This example is an example of the method for producing fungal water-soluble red pigment using wood flour mixed solid state fermentation according to the present invention, comprising the steps of:
(1) Cutting paulownia and bagasse into small pieces by a hacking knife, putting into a baking oven at 102 ℃ to be dried to constant weight, crushing wood chips by a crusher, screening with a 100-mesh sieve, putting the screened wood powder into a 250mL storage tank, performing solid sterilization for 40min at 121 ℃ and 0.1MPa by an autoclave, and cooling to obtain pretreated wood powder;
(2) The strain preserved in the test tube in the laboratory is shoveled into small blocks by an inoculating shovel, inoculated in a newly manufactured PDA culture medium, sealed and placed in a constant temperature and humidity incubator at 20+/-2 ℃ for dark culture for 14 days, the mycelium is fully distributed in a culture dish with 90mm, and the culture dish is placed in a refrigerator at 4 ℃ to be used as an experimental strain;
(3) Respectively weighing 500mg of paulownia wood powder according to a certain concentration and proportion, adding 400mg of bagasse powder into a 250mL conical flask, pouring 100mL of new PDA culture medium into the conical flask, uniformly mixing, sealing, sterilizing the mixture for 20min by using a high-pressure sterilizing pot at 121 ℃ and 0.1MPa, cooling the mixture to 80 ℃, transferring the uniformly shaken culture medium into a 90mm culture dish by using a 30mL sterile injector in an ultra-clean workbench according to the amount of 20 mL/dish, and cooling for 30min to obtain a wood powder mixed culture medium;
(4) Beating the activated bacterial species of the pseudomonas pyriform fungi (Arthroinium phaeospermum) SHL-1 in the step (2) into a bacterial cake with the diameter of 6mm by using a sterilized puncher in an ultra-clean workbench, inoculating the bacterial cake to the wood powder mixed culture medium center in the step (3) by using a sterilized inoculating needle, performing the whole process in the ultra-clean workbench, ensuring sterile operation, sealing a culture dish, and placing the culture dish in a constant temperature and humidity incubator with the temperature of 20+/-2 ℃ for dark culture and fermentation for 14 days;
(5) Cutting the culture medium after fermentation in step (4) into small pieces by a surgical knife, pouring the small pieces into a 500mL beaker, adding distilled water according to the liquid-to-material ratio of 30:1, sealing a preservative film, placing the beaker into an ultrasonic cleaning machine, ultrasonically extracting at 60 ℃ for 30min, extracting twice, cooling pigment solution, and filtering with a 0.22 mu m filter membrane. And subpackaging the filtrate into 20mL sample bottles, filling 10mL of the filtrate into each bottle, freezing the bottles in a refrigerator at the temperature of minus 60 ℃ for 24 hours, and then continuously freeze-drying the bottles for 36 hours to obtain water-soluble red pigment powder, wherein the pigment yield is shown in the table I.
Example 3
This example is another example of the method for producing fungal water-soluble red pigment using wood flour mixed solid state fermentation according to the present invention, comprising the steps of:
(1) Cutting paulownia and bagasse into small blocks by a knife, putting the small blocks into a baking oven at 102 ℃ to be dried to constant weight, then crushing the wood blocks by a crusher, screening the crushed wood blocks by a 100-mesh sieve, putting the screened wood powder into a 200mL storage tank, then performing solid sterilization for 40min at 121 ℃ and 0.1MPa by an autoclave, and cooling to obtain pretreated wood powder;
(2) The strain preserved in the test tube in the laboratory is shoveled into small blocks by an inoculating shovel, inoculated in a newly manufactured PDA culture medium, sealed and placed in a constant temperature and humidity incubator at 20+/-2 ℃ for dark culture for 14 days, the mycelium is fully distributed in a culture dish with 90mm, and the culture dish is placed in a refrigerator at 4 ℃ to be used as an experimental strain;
(3) Respectively weighing 400mg of paulownia wood powder according to a certain concentration and proportion, adding 500mg of bagasse powder into a 250mL conical flask, pouring 100mL of new PDA culture medium into the conical flask, uniformly mixing, sealing, sterilizing the mixture for 20min by using a high-pressure sterilizing pot at 121 ℃ and 0.1MPa, cooling the mixture to 80 ℃, transferring the uniformly shaken culture medium into a 90mm culture dish by using a 30mL sterile injector in an ultra-clean workbench according to the amount of 20 mL/dish, and cooling for 30min to obtain a wood powder mixed culture medium;
(4) Beating the activated strain in step (2) into a bacterial cake with the diameter of 6mm by using a sterilized puncher in an ultra-clean workbench, inoculating the bacterial cake to the center of the wood powder mixed culture medium in step (3) by using a sterilized inoculating needle, performing the whole process in the ultra-clean workbench, ensuring sterile operation, sealing a culture dish, and placing the culture dish in a constant temperature and humidity incubator with the temperature of 20+/-2 ℃ for dark culture and fermentation for 14 days;
(5) Cutting the culture medium after fermentation in step (4) into small pieces by a surgical knife, pouring the small pieces into a 300mL beaker, adding distilled water according to the liquid-to-material ratio of 30:1, sealing by a preservative film, placing the beaker into an ultrasonic cleaning machine, ultrasonically extracting at 60 ℃ for 30min, extracting twice, cooling pigment solution, and filtering by a 0.22 mu m filter membrane. And subpackaging the filtrate into 20mL sample bottles, filling 10mL of the filtrate into each bottle, freezing the bottles in a refrigerator at the temperature of minus 60 ℃ for 24 hours, and then continuously freeze-drying the bottles for 36 hours to obtain water-soluble red pigment powder, wherein the pigment yield is shown in the table I.
As can be seen from Table one, when only one wood flour was added to PDA, bagasse was added to PDA to promote the growth of fungi, paulownia was able to increase the yield of the fungi, and the pigment yield of the two control groups was smaller than that of the blank group, indicating that adding one wood flour alone did not have an advantage in increasing the pigment yield. However, when we add wood flour in the proportions claimed, the wood flour works well synergistically for fungi, not only increasing the diameter of the fungi but also increasing the pigment yield of the fungi, thereby increasing pigment yield. As can be seen from Table I, the pigment yield of all examples was greater than that of the blank, wherein when bagasse was added at 3mg/mL and paulownia was added at 4mg/mL, and the experiment according to the claims was conducted, the pigment yield was 3.09g/L, which is 2 times that of the blank. The method for producing the fungal water-soluble red pigment by utilizing the wood powder mixed solid fermentation is described to have obvious effect on improving pigment yield of the SHL-1 fungus of the Pogostemon palustraceae.
The above are only some examples of the present invention and are not intended to limit the present invention in any way. Many possible variations and modifications of the disclosed method, or equivalent embodiments for making equivalent changes, will occur to those skilled in the art, without departing from the spirit and scope of the invention.
Claims (7)
1. A method for producing fungal water-soluble red pigment by wood flour mixed solid state fermentation, which is characterized by comprising the following steps:
(1) Cutting paulownia and bagasse into pieces with length and width of 1-2cm and thickness of 1-2mm, drying in a baking oven at 102 ℃ to constant weight, crushing the pieces, sieving with a 100-mesh sieve, placing the sieved pieces of wood powder into a storage tank, performing solid sterilization for 40min at 121 ℃ and 0.1MPa with an autoclave, and cooling to obtain pretreated wood powder;
(2) Activating a strain, picking up SHL-1 fungus blocks of the Pseudomonas pyriform (Arthroinium phaeospermum) of the Pyricularia, inoculating the fungus blocks into a PDA culture medium, sealing, placing the culture medium in a constant temperature and humidity incubator at 20+/-2 ℃ for dark culture for 14 days, placing the culture dish into a refrigerator at 4 ℃ after hypha is fully covered with a culture dish to serve as an experimental strain, and preserving the strain in a China center for type culture collection at 12 months and 17 days in 2018, wherein the preservation number is: cctccc No: m2018900, deposit address: wuhan university of Wuhan and Jia of Wuchang in Wuhan city;
(3) Respectively weighing paulownia powder and bagasse powder according to a certain concentration and proportion, adding the paulownia powder and the bagasse powder into a conical flask, pouring the conical flask into a PDA culture medium, uniformly mixing, sealing, sterilizing the mixture for 20min at 121 ℃ under 0.1MPa by using an autoclave, cooling the mixture to 80 ℃, transferring the uniformly shaken culture medium into a 90mm culture dish according to the amount of 20 mL/dish by using a sterile injector in an ultra-clean workbench, and cooling the mixture for 30min to obtain a wood powder mixed culture medium;
(4) Punching the strain in the step (2) into a bacterial cake with the diameter of 6mm by using a puncher in an ultra-clean workbench, inoculating the bacterial cake to the center of the wood powder mixed culture medium in the step (3) by using an inoculating needle, sealing, and placing the bacterial cake in a constant temperature and humidity incubator with the temperature of 20+/-2 ℃ for dark culture and fermentation for 14 days;
(5) Cutting the culture medium after fermentation in the step (4) into 1cm pieces 2 Small pieces, poured into a beaker, and distilled water was used as follows: distilled water is added into the culture medium with the liquid-to-material ratio of 3:1, after the preservative film is sealed, the beaker is placed into an ultrasonic cleaner to be extracted for 30min at 60 ℃, the ultrasonic cleaner is used for extracting twice, then a filter film with the thickness of 0.22 mu m is used for filtering, bottling is carried out, the mixture is placed into a refrigerator with the temperature of minus 60 ℃ for freezing for 24h, and then continuous freeze drying is carried out for 36h, thus obtaining the water-soluble red pigment powder.
2. The method for producing fungal water-soluble red pigment according to claim 1, wherein in step (1), paulownia is an edge material of paulownia produced from eastern lotus, and bagasse is white bagasse left after pressing sugar and sugarcane rind from black sugarcane produced in guangxi.
3. The method for producing fungal water-soluble red pigment according to claim 1, wherein the PDA powder is mixed with deionized water and boiled in a ratio of 46.1g/L in the PDA medium used in the step (2) and sterilized at high temperature.
4. The method for producing fungal water-soluble red pigment by using wood powder mixed solid state fermentation according to claim 1, wherein in the step (3), the concentration of the wood powder in a culture medium is 3-5 mg/mL, the concentration ratio of paulownia powder to bagasse powder is 3mg/mL+3 mg/mL-5 mg/mL+5mg/mL, and the wood powder mixed culture medium is uniformly mixed until the wood powder is completely suspended in a PDA culture medium before sterilization.
5. The method for producing fungal water-soluble red pigment by wood flour mixed solid state fermentation according to claim 1, wherein in the step (4), the puncher and the inoculating needle are used after being burnt by an alcohol lamp and cooled to normal temperature, and the inoculating process is carried out in an ultra-clean workbench in the whole course by inoculating the mycelia-carrying surface of the fungus cake downwards.
6. The method for producing fungal water-soluble red pigment according to claim 1, wherein the pigment extract in step (5) is filtered after being cooled, and the pigment solution is contained in 20mL sample bottles at the time of freezing, wherein the concentration of the pigment solution in each bottle is not more than 10mL.
7. The method for producing fungal water-soluble red pigment according to claim 2, wherein said ultra-clean bench is used after ultraviolet sterilization for 30 min.
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| CN110106091A (en) * | 2019-03-13 | 2019-08-09 | 广西大学 | A method of timber is dyed using Li Baojia shell section SHL-1 fungi |
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