CN112831086A - Preparation method and application of special solid strain for liquid fermentation culture - Google Patents
Preparation method and application of special solid strain for liquid fermentation culture Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a special solid strain for liquid fermentation culture, a colloid matrix for preparing the solid strain, a preparation method of the colloid matrix, and a method for liquid fermentation by using the special solid strain. After the fermentation is finished, the colloid substrate can be melted in the fermentation liquor by heating, and the colloid substance does not need to be separated from the fermentation liquor. In addition, the colloid matrix is in a loose and porous structure obtained by a method of slowing after freezing, has the characteristics of ventilation and strong water absorption, can store a large amount of seed liquid, nutrient substances and fresh air in the colloid matrix, contains nutrient substances required by the growth of strains such as protein, cellulose and the like, is favorable for the rapid germination of the strains, and shortens the culture period; the loose and porous colloid matrix can greatly increase the effective surface area in unit volume, a large amount of fresh hyphae can be germinated and stored on the surface and inside of the matrix, the activity of the strain is improved, and the strain can be rapidly propagated in a liquid culture medium after inoculation.
Description
Technical Field
The invention belongs to the field of biotechnology fermentation, and particularly relates to a special solid strain for liquid fermentation culture and a preparation method thereof, a colloid matrix for preparing the solid strain and a preparation method thereof, and a method for liquid fermentation by using the special solid strain.
Background
The strain is a root base in the field of biological fermentation, and the strain production technology is the most key technical support for the development of the industry. The quality, purity and activity of the strain determine the quality of the product, and the yield is high or low.
In recent years, the products of food, health products, medicines, daily chemicals and cosmetics are more and more produced by adopting an industrial fermentation method, and the currently adopted strains in the industrial fermentation are liquid strains or solid strains. Although the liquid strain has high activity and convenient inoculation, the production process is complicated, the investment is large, the requirements on technology and equipment are strict, various indexes in the strain culture process are uncontrollable, the risk is high, the strain is easy to contaminate and age, and the storage and the transportation are inconvenient; the solid strain mainly takes solid matters such as grains, sawdust and the like as culture mediums, although the strain is propagated and carried, the strain activity is low, the growth period is long, the pipeline is easy to block when the culture medium after inoculation and culture is discharged from culture equipment, the strain is difficult to separate from fermentation liquor, the strain is easy to infect mixed bacteria, great inconvenience is brought to production, and the application of the strain is limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention firstly provides a solid strain colloid matrix which is prepared by taking a colloid substance as a raw material and carrying out the processes of dissolving, shaping, freezing, slowing, dehydrating, drying, sterilizing and the like.
Wherein the colloidal substance is one or more of agar, xanthan gum, carrageenan, sodium alginate, sodium carboxymethylcellulose, gelatin, pectin and other colloidal substances.
Wherein the dissolving is to add water into the colloidal substance, heat and dissolve the colloidal substance, and uniformly mix the colloidal substance and the water to prepare 1-6% of colloidal solution.
Wherein the shaping is to inject the colloid solution into a mould for cooling and prepare small particles with the length or the particle diameter of 2-20 mm.
The small particles during shaping are in various shapes such as blocks, spheres, hemispheres or strips.
Wherein the freezing is to freeze the shaped colloid particles and fully freeze the colloid particles; the freezing is preferably performed at a temperature below-18 ℃.
Wherein the tempering is to thaw the fully frozen colloidal particles at room temperature.
Wherein, the dehydration is to carry out preliminary dehydration by adopting methods such as physical extrusion or centrifugation and the like after the colloid particles are completely thawed.
Wherein the drying is to place the colloid particles in drying equipment and fully dry the colloid particles within the temperature range of 40-85 ℃.
The sterilization is to place the solid colloidal particle matrix in a culture container, the loading capacity is 1/5-1/3 of the container volume, the container is sealed by a cotton plug, a silica gel plug or a breathable film, and the solid colloidal particle matrix is sterilized at 121 ℃ for 40-90 min.
The invention also provides a method for preparing the special solid strain by using the solid strain colloidal matrix, which comprises the following steps:
1) preparing a seed solution: inoculating a strain mother liquor with the strain concentration of 60-90% into a liquid culture medium suitable for the strain according to the inoculation amount of 5-30% in an aseptic environment, and uniformly mixing;
2) inoculation: uniformly inoculating the seed solution into the colloidal matrix according to the inoculation amount of 2-10 mL per gram of the colloidal matrix in an aseptic environment, wherein the seed solution after inoculation culture can be completely absorbed by the colloidal matrix;
3) culturing: and (3) placing the inoculated colloidal matrix in an environment suitable for the strain to culture until the surface of each colloidal matrix particle is full of a layer of cultured thalli, and finishing the preparation of the solid strain.
In the above method, the culture time in step 3) is preferably 3 to 20 days.
The invention also provides a method for liquid fermentation by using the special solid strain, which comprises the following steps:
1) scattering the special solid strain in an aseptic environment, and inoculating the special solid strain to fermentation equipment for liquid fermentation;
2) after fermentation is completed, the fermentation liquor is heated in a fermentation device to melt the strain colloid matrix.
In the method for carrying out liquid fermentation, the inoculation amount refers to the mass ratio of the solid strain colloidal matrix to the liquid culture medium, and the proportion is 0.005-0.5%; the heating refers to that the temperature of the fermentation liquor reaches more than 95 ℃ and is kept for more than 1 minute.
According to the invention, a solid strain colloid matrix is prepared by taking a colloid substance as a raw material, and then is inoculated and cultured to prepare a solid strain for liquid fermentation, after the fermentation is finished, the colloid matrix can be melted in a fermentation liquid by heating, so that the purpose of removing the original solid matrix tissue form of the strain is achieved, and the fermentation liquid does not block an equipment pipeline when being discharged; the inoculation amount of the solid strain is very small, and the concentration of the colloidal substance in the fermentation liquor is not more than 0.5 percent, so that the viscosity of the fermentation product is not obviously influenced, and the colloidal substance is not required to be separated from the fermentation liquor; the colloid substances are all made of food-grade materials, are safe and non-toxic, and have no influence on the use and processing of the final fermentation product; in addition, the colloid matrix is in a loose and porous structure obtained by a method of slowing after freezing, has the characteristics of ventilation and strong water absorption, can store a large amount of seed liquid, nutrient substances and fresh air in the colloid matrix, contains nutrient substances required by the growth of strains such as protein, cellulose and the like, is favorable for the rapid germination of the strains, and shortens the culture period; the loose and porous colloid matrix can greatly increase the effective surface area in unit volume, and can germinate and store a large amount of fresh hyphae on the surface and in the matrix, so that the activity of the strains is improved, and the strains can be rapidly propagated in a liquid culture medium after inoculation. The solid strain of the invention has simple production process, small investment, strong stability, difficult contamination and aging, and convenient storage and transportation.
Drawings
FIG. 1 is a comparison of colloidal particles before and after freeze-tempering.
FIG. 2 is a graph comparing the fermentation effect of the special solid bacterial strain and the traditional solid bacterial strain of millet.
Detailed Description
In order to better explain the present invention, some specific embodiments will be provided hereinafter, it should be noted that the described specific embodiments are only for illustrating the present invention and should not be construed as limiting the present invention, and any changes and modifications that can be conceived by those skilled in the art should be within the scope covered by the present invention.
Examples 1-4 preparation of solid seed gum base
The proportions of colloidal material in examples 1-4 are shown in the following table:
| proportion of colloidal substance (percentage by weight) | |
| Example 1 | Agar 70%, xanthan gum 20%, carrageenan 5%, and sodium alginate 5% |
| Example 2 | 50 percent of agar, 20 percent of carrageenan and 30 percent of carboxymethyl cellulose |
| Example 3 | Xanthan gum 60%, gelatin 20%, pectin 20% |
| Example 4 | Agar 100% |
Mixing the colloid powder, adding water, heating to dissolve, and mixing to obtain 4% colloid solution; injecting the colloid solution into a mold, cooling and shaping to prepare small blocky particles with the particle size of about 10 mm; freezing the shaped colloid particles at-18 deg.C, completely freezing, taking out, and thawing at room temperature; after completely unfreezing, performing preliminary dehydration by adopting a centrifugal method, and discharging water in the colloid particles; putting the colloid particles into an oven, and fully drying at 50 ℃; the solid colloidal particle matrix was placed in a glass screw bottle, loaded with 1/4 container volumes, sealed with a cotton plug and sterilized at 121 ℃ for 60 min. And preparing the solid strain colloid matrix.
The attached figure 1 of the specification is a comparison graph of the colloidal particles before and after freezing-moderation in the above example 4, wherein the left side (experiment) is a strain colloidal matrix obtained by freezing-moderation technology treatment, and the inside of the strain colloidal matrix is loose and porous, so that the strain colloidal matrix has the characteristics of ventilation, air permeability and strong water absorption, and a large amount of seed solution, nutrient substances and fresh air can be stored in the strain colloidal matrix, thereby facilitating the rapid germination of strains and shortening the culture period. The loose and porous colloid matrix can greatly increase the effective surface area in unit volume, and can germinate and store a large amount of fresh hyphae on the surface and in the matrix, so that the activity of the strains is improved, and the strains can be rapidly propagated in a liquid culture medium after inoculation. The right (control) seed gum substrate was not treated by freeze-tempering technique, and had a compact texture, which was inferior to the former in its structure as a seed substrate.
Examples 5 to 8: preparation of special solid strain
Taking the solid strain colloidal matrix prepared in the embodiment 1-4 for standby; inoculating strain mother liquor with the strain concentration of 75% into a liquid culture medium suitable for the strain according to the inoculation amount of 10% in an aseptic environment, and uniformly mixing; uniformly inoculating the seed solution into the solid strain gum substrate prepared in the examples 1-4 according to the inoculation amount of 4mL per gram of gum substrate in an aseptic environment; placing the inoculated culture container in an environment of 25 ℃ for dark culture until the surface of each colloid matrix particle is full of a layer of cultured thalli, and preparing the special solid strain.
Examples 9 to 12: liquid fermentation with special solid strain
The special solid strains prepared in the examples 5-8 are scattered in an aseptic environment, inoculated in fermentation equipment for liquid fermentation, and the inoculum size is 0.05 percent (mass ratio of the solid strain colloidal matrix to the liquid culture medium); after the fermentation is finished, heating the fermentation liquor in a fermentation device to 100 ℃, keeping for 1.5 minutes to melt the colloid substrate, and discharging the fermentation liquor after proper cooling.
Description accompanying figure 2 is a graph comparing the fermentation effect of the specific solid spawn and the traditional solid spawn of millet in example 8. The special solid strain and the millet solid strain in the example 8 are respectively used for fermentation under the same mother strain, inoculation amount, fermentation equipment, fermentation medium and fermentation conditions, and samples are taken from the fermentation equipment for comparison after 150 hours of fermentation. The amount of thalli (globules) in the product fermented by using the solid strain in the embodiment 8 of the invention is obviously more than that of the product fermented by using the traditional millet solid strain, which shows that the solid strain has obvious advantages in the aspect of fermentation effect compared with the traditional solid strain.
Claims (10)
1. A solid strain colloidal substrate is characterized in that: the solid strain colloid matrix is prepared from colloid substances by the processes of dissolving, shaping, freezing, slowing, dehydrating, drying and sterilizing.
2. The solid seed gum substrate of claim 1, wherein: the colloidal substance is one or more of agar, xanthan gum, carrageenan, sodium alginate, sodium carboxymethylcellulose, gelatin, pectin and the like.
3. The solid seed gum substrate of claim 1, wherein:
wherein the dissolving is to add water into the colloidal substance, heat and dissolve the colloidal substance, and uniformly mix the colloidal substance and the water to prepare 1-6% of colloidal solution;
wherein the shaping is to inject the colloid solution into a mould for cooling to prepare small particles with the length or the particle diameter of 2-20 mm;
wherein the freezing is to freeze the shaped colloid particles and fully freeze the colloid particles;
wherein the tempering is to put the fully frozen colloidal particles into room temperature for thawing;
wherein, the dehydration is that after the colloid particles are completely thawed, the physical extrusion or centrifugation is adopted for preliminary dehydration;
wherein the drying is to place the colloidal particles in drying equipment and fully dry the colloidal particles at the temperature of 40-85 ℃;
the sterilization is to place the solid colloidal particle matrix in a culture container, the loading capacity is 1/5-1/3 of the container volume, the container is sealed by a cotton plug, a silica gel plug or a breathable film, and the solid colloidal particle matrix is sterilized at 121 ℃ for 40-90 min.
4. A solid seed gum substrate as claimed in claim 3, wherein: wherein the freezing is performed at a temperature below-18 ℃.
5. A solid seed gum substrate as claimed in claim 3, wherein: the shaped small particles are in various shapes such as blocks, spheres, hemispheres or strips.
6. A method of producing a specialized solid seed culture using a solid seed colloidal matrix as defined in any one of claims 1 to 5, wherein: the method comprises the following steps:
1) preparing a seed solution: inoculating a strain mother liquor with the strain concentration of 60-90% into a liquid culture medium suitable for the strain according to the inoculation amount of 5-30% in an aseptic environment, and uniformly mixing;
2) inoculation: uniformly inoculating the seed solution into the colloidal matrix in one of claims 1 to 3 in an aseptic environment according to the inoculation amount of 2 to 10mL per gram of the colloidal matrix, wherein the seed solution can be completely absorbed by the colloidal matrix after inoculation culture;
3) culturing: and (3) placing the inoculated colloidal matrix in a proper environment for culturing until the surface of each colloidal matrix particle is full of a layer of cultured thalli.
7. The method according to claim 6, wherein the culturing time in step 3) is 3 to 20 days.
8. A specific solid bacterial species prepared by the method of claim 6 or 7.
9. A method for liquid fermentation using the special solid bacterial strain of claim 7, which is characterized in that: the method comprises the following steps:
scattering the special solid strain in an aseptic environment, and inoculating the special solid strain to fermentation equipment for liquid fermentation;
after fermentation is completed, the fermentation liquor is heated in a fermentation device to melt the strain colloid matrix.
10. The method of claim 9, wherein: the inoculation amount refers to the mass ratio of the solid strain colloidal matrix to the liquid culture medium, and the proportion is 0.005-0.5%; the heating refers to that the temperature of the fermentation liquor reaches more than 95 ℃ and is kept for more than 1 minute.
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