CN110250319B - Preparation of novel corynebacterium glutamicum waste residue microecological preparation - Google Patents

Preparation of novel corynebacterium glutamicum waste residue microecological preparation Download PDF

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CN110250319B
CN110250319B CN201910617201.9A CN201910617201A CN110250319B CN 110250319 B CN110250319 B CN 110250319B CN 201910617201 A CN201910617201 A CN 201910617201A CN 110250319 B CN110250319 B CN 110250319B
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倪莉
陈丽
王扬
宋畅
张晨
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Abstract

The invention utilizes bacillus licheniformis, bacillus subtilis and saccharomyces boulardii relay to perform semi-solid fermentation on corynebacterium glutamicum waste residues, optimizes the variety and the addition amount of exogenous nutrients, and optimizes the initial inoculation proportion and the fermentation days of mixed bacteria relay fermentation, and prepares a novel bacterial residue microecological preparation with high viable count. The fermentation process comprises the following steps: inoculating Bacillus licheniformis for 24h, inoculating Bacillus subtilis for 24h, finally inoculating yeast, fermenting for 48h, wherein the inoculum concentration is 10%, and the initial viable count is 10 5 CFU/mL. The novel Corynebacterium glutamicum waste residue microecological preparation has high viable count reaching 6 multiplied by 10 8 CFU/mL; on the other hand, the microecological preparation has rich enzyme systems and functional metabolites such as polypeptides and the like.

Description

Preparation of novel corynebacterium glutamicum waste residue microecological preparation
Technical Field
The invention relates to the field of preparation of microecological preparations, in particular to a novel high-viable-count Corynebacterium glutamicum bacterial dreg microecological preparation which is prepared by utilizing Bacillus licheniformis, bacillus subtilis and Saccharomyces boulardii relay to perform solid-state fermentation on Corynebacterium glutamicum bacterial dreg, optimizing the variety and the addition amount of an exogenous additive, and the initial inoculation proportion and the fermentation days of mixed bacteria relay fermentation.
Background
Corynebacterium glutamicum dregs are a byproduct from monosodium glutamate processing. Because the cost is low and the protein content is high, most of the corynebacterium glutamicum strains recovered by monosodium glutamate factories are directly used as animal feed additives, the crude protein content of the corynebacterium glutamicum strains is 50-75%, the nucleic acid content is 6.53%, the crude fat content is 11.36%, the ash content is 3.03%, and the corynebacterium glutamicum strains contain nutrient substances necessary for the growth of microorganisms and can be used as a probiotic fermentation culture medium. The production of the probiotic feed by using waste biomass fermentation is a current research hotspot, and the feed is beneficial to controlling pathogens, increasing the digestibility of nutrient substances and improving the health condition of animals after being fermented by probiotics, does not have the problem of antibiotic residue and has great development potential. The single-strain microecological preparation has single function, and the product of the composite microbial inoculum can not only increase the diversity of probiotic strains, but also generate abundant enzyme systems and unknown growth factors, thereby improving the application effect. The mixed fermentation is divided into simultaneous fermentation and relay fermentation, wherein the simultaneous fermentation is to simultaneously inoculate a plurality of bacteria at the initial stage of fermentation, the relay fermentation utilizes different strains with different substrate adaptability, and inoculates different strains at different time periods of fermentation. Meanwhile, because the nutrient components in the corynebacterium glutamicum residues are unbalanced and part of nutrients are lost, and the growth of probiotics is not facilitated, the utilization rate of the corynebacterium glutamicum residues is improved by adding exogenous nutrient substances. The method for preparing the novel microecological preparation by mixed bacteria relay fermentation of the corynebacterium glutamicum bacterial residues has the advantages that the solid culture medium is optimized, relay fermentation is carried out according to the characteristics of the bacteria, the proliferation of probiotics can be promoted in the whole fermentation period, the viable count is high, and an enzyme system and unknown growth factors are enriched. The invention has obvious production and application values.
Disclosure of Invention
The invention aims to provide a preparation method of a novel corynebacterium glutamicum bacterial residue microecological preparation.
In order to realize the purpose, the invention adopts the following technical scheme:
a preparation method of a novel micro-ecological preparation of corynebacterium glutamicum waste residue utilizes mixed probiotics to relay and ferment the corynebacterium glutamicum waste residue to prepare the novel micro-ecological preparation, and specifically comprises the following steps:
(1) Preparation of a corynebacterium glutamicum dreg semisolid culture medium: taking 1000g of corynebacterium glutamicum residues, adding exogenous nutrients, adding deionized water to a constant volume of 1000mL, and naturally adjusting the pH value; sterilizing at 121 deg.C for 20min;
(2) Preparing probiotic seed liquid: respectively centrifuging the cultured seed liquid to remove culture medium, adding 30ml physiological saline, washing for three times, and respectively diluting the three seed liquids with physiological saline by adopting a blood cell counting method until the viable count is 10 5 CFU/mL；
(3) Mixed fermentation: inoculating the diluted bacillus subtilis seed solution obtained in the step (2) into the culture medium prepared in the step (1), and fermenting for 24 hours at 30 ℃; then inoculating diluted bacillus licheniformis seed liquid, and fermenting for 24h at 30 ℃; then inoculating the diluted saccharomyces boulardii seed liquid, and fermenting for 48h at 30 ℃.
The method for adding the exogenous nutrient in the step (1) comprises the following steps: every 1000g of corynebacterium glutamicum dregs are added with 22.33g of monosodium glutamate, 9.72g of ammonium sulfate, 42.50g of anhydrous glucose, 9.00g of monopotassium phosphate, 0.21g of manganese sulfate monohydrate, 2.10g of magnesium sulfate heptahydrate and 55.00g of sodium alginate.
The preparation method of the culture medium of the seed liquid in the step (2) comprises the following steps:
(1) Preparing a bacillus subtilis seed liquid culture medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding deionized water to reach the constant volume of 1000mL, adjusting the pH value to be =7.2 by using 5mol/L NaOH, and sterilizing at 121 ℃ for 20min;
(2) Preparing a bacillus licheniformis seed liquid culture medium: adding deionized water into 3g of beef extract, 10g of peptone and 5g of sodium chloride to reach the constant volume of 1000mL, adjusting the pH value to be =7.4 by using 5mol/L NaOH, and sterilizing at 121 ℃ for 20min;
(3) Preparing a liquid culture medium for the saccharomyces boulardii seeds: 10g of yeast extract, 20g of peptone and 20g of glucose, adding deionized water to the volume of 1000mL, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20min.
The culture method of the three seed solutions in the step (2) comprises the following steps: inoculating loop on slant culture medium of Bacillus subtilis, bacillus licheniformis and Saccharomyces boulardii, respectively, inoculating into corresponding 30ml seed liquid culture medium, and culturing at 30 deg.C and 220rpm for 12h, 12h and 18h respectively.
The inoculation amounts of the bacillus subtilis seed solution, the bacillus licheniformis seed solution and the saccharomyces boulardii seed solution in the step (3) are all 10% v/w.
The invention has the following beneficial effects:
1. the Corynebacterium glutamicum dregs are edible residues, and have high safety.
2. Compared with the traditional mixed bacteria fermentation mode, the relay fermentation is utilized to produce the microecological preparation, so that the viable count of the probiotics can be obviously improved.
3. The probiotics can secrete enzyme to hydrolyze protein to produce active peptide, improve the quality of the microecological preparation, and promote the health of livestock.
4. Integrates three different probiotics and products thereof such as enzyme systems and active peptides, directly promotes the health of livestock, solves the problem of Corynebacterium glutamicum residue, and realizes the effective utilization of resources.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
Example 1
The preparation method of the novel corynebacterium glutamicum waste residue microecological preparation specifically comprises the following steps:
(1) Preparation of probiotic seed liquid
1) Preparing a bacillus subtilis seed liquid culture medium: in 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, deionized water was added to the mixture to a volume of 1000mL, the mixture was adjusted to pH =7.2 with 5mol/L NaOH (about 0.2 mL), and the mixture was sterilized at 121 ℃ for 20min.
2) Preparing a bacillus licheniformis seed liquid culture medium: 3g of beef extract, 10g of peptone and 5g of sodium chloride, adding deionized water to reach volume of 1000mL, adjusting pH to be 7.4 by using 5mol/L NaOH (about 0.2 mL), and sterilizing at 121 ℃ for 20min.
3) Preparing a liquid culture medium for the saccharomyces boulardii seeds: 10g of yeast extract, 20g of peptone and 20g of glucose, adding deionized water to the volume of 1000mL, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20min.
4) Inoculating the strain residue of Corynebacterium glutamicum to slant culture medium of Bacillus subtilis, bacillus licheniformis and Saccharomyces boulardii, inoculating to seed solution (30 ml conical flask), and culturing at 30 deg.C and 220rpm for 12h, 12h and 18h respectively.
5) The cultured seed liquid (viable count is about 10) 11 CFU/mL) to remove culture medium, adding 30mL of normal saline, washing for three times, and diluting the seed liquid by a blood cell counting method until the viable count is about 10 5 CFU/mL。
(2) Preparing a solid culture medium of the corynebacterium glutamicum dregs: accurately weighing 1kg of corynebacterium glutamicum waste residue, adding 22.33g of monosodium glutamate, 9.72g of ammonium sulfate, 42.50g of anhydrous glucose, 9.00g of monopotassium phosphate, 0.21g of manganese sulfate monohydrate, 2.10g of magnesium sulfate heptahydrate and 55.00g of sodium alginate, adding deionized water to a constant volume of 1000mL, and keeping the pH value at natural level; sterilizing at 121 deg.C for 20min;
(3) Mixed bacterium relay fermentation: inoculating the bacillus subtilis seed diluent into a corynebacterium glutamicum dreg solid culture medium, wherein the inoculation amount is 10% (v/w), fermenting for 24 hours at 30 ℃, then inoculating bacillus licheniformis with the inoculation amount of 10% (v/w), fermenting for 24 hours at 30 ℃, and finally fermenting for 48 hours at 30 ℃ with the inoculation amount of 10% (v/w) of blattella barnacii, wherein the fermentation period is 4 days in total.
The polypeptide content of the corynebacterium glutamicum dregs before and after fermentation is shown in table 1, and the table 1 shows that the polypeptide content in the dregs after fermentation is obviously improved, so that the fermented microecological preparation has rich enzyme systems and functional metabolites such as polypeptides, the residue of waste residues in the monosodium glutamate industry is solved, and the effective utilization of resources is realized.
TABLE 1 data relating to the polypeptide content of C.glutamicum residues before and after fermentation
Figure DEST_PATH_IMAGE002
Comparative example 1 media optimization
Optimizing a solid-state Corynebacterium glutamicum bacterial residue culture medium, culturing probiotics through relay fermentation, and finally determining the number of viable probiotics and the content of polypeptide before and after fermentation.
The experimental method is the same as that of example 1, and only the formulation of the semi-solid culture medium of the Corynebacterium glutamicum bacterial residues is different.
1. Non-optimized medium formulation: taking 1000g of corynebacterium glutamicum residues, adding deionized water to a constant volume of 1000mL, and naturally adjusting the pH value; sterilizing at 121 deg.C for 20min;
2. the optimized corynebacterium glutamicum dreg semisolid culture medium formula comprises the following components in parts by weight: taking 1000g of corynebacterium glutamicum dregs, adding 22.33g of monosodium glutamate, 9.72g of ammonium sulfate, 42.50g of anhydrous glucose, 9.00g of monopotassium phosphate, 0.21g of manganese sulfate monohydrate, 2.10g of magnesium sulfate heptahydrate and 55.00g of sodium alginate, adding deionized water to reach a constant volume of 1000mL and keeping the pH value natural; sterilizing at 121 deg.C for 20min;
the results are shown in table 2, in the un-optimized bacteria residue culture medium, the viable count of bacillus subtilis, bacillus licheniformis and saccharomyces boulardii is obviously lower than that of the optimized culture medium, and the optimized culture medium formula is better suitable for strain growth.
TABLE 2 media optimization Process related data
Figure DEST_PATH_IMAGE004
Comparative example 2 fermentation Strain modification
Changing fermentation strains of a semisolid Corynebacterium glutamicum slag culture medium, culturing probiotics through relay fermentation, and finally measuring the number of viable probiotics before and after fermentation.
The experimental procedure was the same as in example 1, except that the fermentation strain was a semisolid C.glutamicum strain residue medium.
1. The fermentation strains of example 1 were Bacillus subtilis, bacillus licheniformis and Saccharomyces boulardii.
2. The fermentation strains of comparative example 2 were bacillus subtilis, aspergillus niger and bacillus licheniformis, lactobacillus paracasei.
TABLE 3 data relating to the optimization of the culture Medium
Figure DEST_PATH_IMAGE006
Comparative example 3 Simultaneous fermentation with Mixed bacteria
(1) Preparation of probiotic seed liquid
1) Preparing a bacillus subtilis seed liquid culture medium: in 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, deionized water was added to the mixture to a volume of 1000mL, the mixture was adjusted to pH =7.2 with 5mol/L NaOH (about 0.2 mL), and the mixture was sterilized at 121 ℃ for 20min.
2) Preparing a bacillus licheniformis seed liquid culture medium: 3g of beef extract, 10g of peptone and 5g of sodium chloride, adding deionized water to reach volume of 1000mL, adjusting pH to be 7.4 by using 5mol/L NaOH (about 0.2 mL), and sterilizing at 121 ℃ for 20min.
3) Preparing a liquid culture medium for the saccharomyces boulardii seeds: 10g of yeast extract, 20g of peptone and 20g of glucose, adding deionized water to the volume of 1000mL, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20min.
4) Inoculating the strain residue of Corynebacterium glutamicum to slant culture medium of Bacillus subtilis, bacillus licheniformis and Saccharomyces boulardii, inoculating to seed solution (30 ml conical flask), and culturing at 30 deg.C and 220rpm for 12h, 12h and 18h respectively.
5) Culturing the seed liquid (viable count about 10) 11 CFU/mL) to remove culture medium, adding 30mL of normal saline, washing for three times, and diluting the seed liquid by a blood cell counting method until the viable count is about 10 5 CFU/mL。
(2) Preparing a solid culture medium of the corynebacterium glutamicum dregs: accurately weighing 1kg of corynebacterium glutamicum waste residue, adding 22.33g of monosodium glutamate, 9.72g of ammonium sulfate, 42.50g of anhydrous glucose, 9.00g of monopotassium phosphate, 0.21g of manganese sulfate monohydrate, 2.10g of magnesium sulfate heptahydrate and 55.00g of sodium alginate, adding deionized water to a constant volume of 1000mL, and keeping the pH value at natural level; sterilizing at 121 deg.C for 20min;
(3) Simultaneous fermentation of mixed bacteria: inoculating the bacillus subtilis seed diluent, the bacillus licheniformis diluent and the saccharomyces boulardii diluent into a solid culture medium according to the inoculation amount of 10% (v/w), uniformly mixing, and fermenting for 4 days at 30 ℃.
The results of simultaneous fermentation of the mixed bacteria are shown in table 4, the simultaneous fermentation of the three bacteria is not beneficial to the growth and reproduction of the bacteria and the generation of secondary metabolites, and the number of the viable bacteria detectable in the relay fermentation system of the mixed bacteria is obviously higher than that of the simultaneous fermentation of the mixed bacteria, which shows that the relay fermentation performed according to the fermentation sequence of the strains of the invention can produce obvious effect on the growth of the bacteria.
TABLE 4 fermentation Process related data
Figure DEST_PATH_IMAGE008
The relevant data show that the number of live bacteria obtained by fermenting the probiotics by using the optimized corynebacterium glutamicum culture medium is far larger than that of the non-optimized corynebacterium glutamicum dreg culture medium, and each probiotic is improved by about 1.5 times. Compared with the mixed fermentation, the inoculation mode of the relay fermentation has more advantages, and each strain has strong multiplication capacity in different stages. The result shows that the viable count obtained by culturing probiotics through relay fermentation is improved by about 1.2 times than that obtained by simultaneous fermentation of mixed bacteria. The content of the polypeptide after fermentation reaches 22.4 +/-0.8 percent.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (2)

1. A preparation method of a novel Corynebacterium glutamicum waste residue microecological preparation is characterized by comprising the following steps: the method for preparing the novel microecological preparation by utilizing the mixed probiotics to relay and ferment the corynebacterium glutamicum waste residues specifically comprises the following steps:
(1) Preparation of a corynebacterium glutamicum dreg culture medium: taking 1000g of corynebacterium glutamicum dregs, adding exogenous nutrients, adding deionized water to reach the constant volume of 1000mL, and naturally adjusting the pH value; sterilizing at 121 deg.C for 20min;
(2) Preparing probiotic seed liquid: respectively centrifuging the cultured Bacillus subtilis seed solution, bacillus licheniformis seed solution, and Saccharomyces boulardii seed solution to remove cultureAfter culturing, 30ml of normal saline is added for washing for three times, and the three seed solutions are respectively diluted by normal saline by adopting a blood cell counting method until the viable count is 10 5 CFU/mL；
(3) Mixed fermentation: inoculating the diluted bacillus subtilis seed solution obtained in the step (2) into the culture medium prepared in the step (1), and fermenting for 24 hours at 30 ℃; then inoculating diluted bacillus licheniformis seed liquid, and fermenting for 24h at 30 ℃; inoculating diluted Saccharomyces boulardii seed solution, and fermenting at 30 deg.C for 48 hr;
the added exogenous nutrients in the step (1) are as follows: adding 22.33g of monosodium glutamate, 9.72g of ammonium sulfate, 42.50g of anhydrous glucose, 9.00g of monopotassium phosphate, 0.21g of manganese sulfate monohydrate, 2.10g of magnesium sulfate heptahydrate and 55.00g of sodium alginate into every 1000g of corynebacterium glutamicum dregs;
the preparation method of the culture medium of the seed liquid in the step (2) comprises the following steps:
1) Preparing a bacillus subtilis seed liquid culture medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding deionized water to reach the constant volume of 1000mL, adjusting the pH value to be =7.2 by using 5mol/L NaOH, and sterilizing at 121 ℃ for 20min;
2) Preparing a bacillus licheniformis seed liquid culture medium: adding deionized water into 3g of beef extract, 10g of peptone and 5g of sodium chloride to reach the constant volume of 1000mL, adjusting the pH value to be =7.4 by using 5mol/L NaOH, and sterilizing at 121 ℃ for 20min;
3) Preparing a liquid culture medium of the saccharomyces boulardii seeds: adding deionized water to the yeast extract 10g, peptone 20g and glucose 20g, metering the volume to 1000mL, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20min;
the culture method of the three seed solutions in the step (2) comprises the following steps: inoculating loop on slant culture medium of Bacillus subtilis, bacillus licheniformis and Saccharomyces boulardii, respectively, inoculating into corresponding 30ml seed liquid culture medium, and culturing at 30 deg.C and 220rpm for 12h, 12h and 18h respectively.
2. The method for preparing a novel Corynebacterium glutamicum waste residue microecological preparation according to claim 1, wherein the microorganism is selected from the group consisting of: the inoculation amounts of the bacillus subtilis seed solution, the bacillus licheniformis seed solution and the blakedil yeast seed solution in the step (3) are all 10% v/w.
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