CN108148767B - Preparation method of Aspergillus niger seed spore microbial inoculum - Google Patents

Abstract

The invention discloses a preparation method of Aspergillus niger seed spore microbial inoculum, which comprises the following steps: (1) culturing seed spores in a corncob culture medium; (2) preparing a seed spore microbial inoculum by adopting a liquid-wax separation method; (3) inoculating the seed spore microbial inoculum prepared in the step (2) into a bran koji culture container for culture and koji preparation. The method solves the limitation and instability of eggplant bottle seed sources, greatly reduces the production amount of the seed sources, reduces the operation steps of inoculation in the starter propagation process, avoids the occurrence of bacterial contamination, greatly saves the labor cost of enterprises at the same time, and creates better economic benefit for the enterprises.

Description

Preparation method of Aspergillus niger seed spore microbial inoculum

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a preparation method of a seed spore microbial inoculum in an Aspergillus niger starter propagation process.

Background

Bran koji is prepared on the basis of citric acid production, and the existing method for preparing koji by citric acid production mainly comprises two methods, namely bran is used for culturing in a glass triangular flask, the method comprises the steps of washing the bran with water, washing off flour in the bran, removing part of water in the bran to ensure that the bran contains about 50 percent of water, loosely filling the bran into 2000ml triangular flasks with about 35 grams per flask, inoculating aspergillus niger spores in a solanum torvum bottle after sterilization for culturing for 7-10 days, and connecting 50-60 triangular flasks to each eggplant bottle for use after the eggplant bottle is mature and qualified by spot inspection. When in use, about 150 bottles of bran koji are required to be connected into each seed tank, and the starter making method is basically used by the current citric acid production enterprises, but the method has low bran koji inspection rate, more operators and high operation technical requirements. Secondly, corncob particles are used as carriers, nutrient substances required by growth of Aspergillus niger are added, the culture is carried out in a large container, each seed tank only needs to be accessed with 3-4 culture units, the method greatly reduces the workload and the number of operators, and because the number is small, the bran koji for production can be tested by 100%, and the reliability of the bran koji is ensured.

The method is characterized in that an eggplant bottle is used as a seed source, the limitation is that the eggplant bottle is cultured on a slant, the area is limited, and the number of spores is small, so that one eggplant bottle corresponds to one bran koji culture container. The inoculation method has the disadvantages that the eggplant bottles are large in manufacturing amount and complicated in inoculation operation, and the quantity and quality of the spores scraped from each eggplant bottle are different to a certain extent, so that the bran koji quality is fluctuated to a certain extent. Therefore, the problem of seed spores is solved, the quality of the bran koji is ensured, and the problem to be solved by enterprises is urgently needed.

Disclosure of Invention

Aiming at the problems in the prior art, the applicant of the invention provides a preparation method of an Aspergillus niger seed spore microbial inoculum. The method solves the limitation and instability of eggplant bottle seed sources, greatly reduces the production amount of the seed sources, reduces the operation steps of inoculation in the starter propagation process, avoids the occurrence of bacterial contamination, greatly saves the labor cost of enterprises at the same time, and creates better economic benefit for the enterprises.

The technical scheme of the invention is as follows:

a preparation method of Aspergillus niger seed spore microbial inoculum comprises the following steps:

(1) culturing seed spores in a corncob culture medium;

(2) preparing a seed spore microbial inoculum by adopting a liquid-wax separation method;

(3) inoculating the seed spore microbial inoculum prepared in the step (2) into a bran koji culture container for culture and koji preparation.

The corncob culture medium in the step (1) consists of corncob particles and nutrient solution required by growth of Aspergillus niger; the corncob particles are prepared by selecting clean corncobs with good water absorbability as raw materials, crushing the corncobs by a crusher, and sieving the crushed corncobs to obtain particles with the diameter of 5-10 mm; the nutrient solution comprises the following components: 0-800 parts of corn slurry powder and glucose, 0-120 parts of ammonium sulfate and 0-6 parts of soybean meal enzymolysis filtrate by volume, wherein the soybean meal enzymolysis filtrate by volume is 1L of soybean meal enzymolysis filtrate corresponding to each gram of the corn slurry powder, the glucose and the ammonium sulfate;

the soybean meal enzymolysis filtrate is prepared by the following method:

crushing soybean meal with protein content of 46%, and sieving with a 40-mesh sieve;

adding water to prepare slurry, wherein the weight ratio of the soybean meal to the water is 1: 6;

regulating the pH value to 6.5 by using 40% sodium hydroxide, and then heating to 60 ℃;

adding alkaline protease powder, wherein the addition amount of the enzyme powder is 2000 enzyme activity units per gram of soybean meal powder, and carrying out heat preservation enzymolysis for 3 hours;

fifthly, heating the enzymolysis liquid to 100 ℃ after enzymolysis, filtering while the liquid is hot, and adding water to adjust the solid content of the filtrate to 10% for later use.

The preparation method of the rice core culture medium comprises the following steps:

taking 0-800g of corn steep liquor powder and glucose, 0-120g of ammonium sulfate and 0-6L of soybean meal enzymolysis filtrate, adding water for dissolving, fixing the volume to 6L after dissolving, uniformly mixing 6L of nutrient solution into 8kg of corncobs, placing the corncobs in a low-temperature drying box for drying after the corncobs completely adsorb the nutrient solution, wherein the temperature does not exceed 60 ℃, and obtaining a culture medium finished product after drying.

The method for culturing the provenance spores in the step (1) comprises the following steps: placing corncob culture medium into a triangular flask, adding water according to the weight ratio of the culture medium to the water of 1:1.2-1.6, sterilizing, cooling to 35 ℃, selecting a ring spore with an inoculating needle for inoculation, placing in a 35 ℃ culture chamber for static culture for 7-14 days, and after the culture is finished, the water content of seed source is lower than 20%.

The method for preparing the seed source spore microbial inoculum by the liquid-wax separation method in the step (2) comprises the following steps: and (3) mixing the sterilized liquid paraffin according to the weight-volume ratio of the seed bran koji to the liquid paraffin of 1: 10, pouring the mixture into seed source triangular flask bran koji, after uniform mixing, enabling spores to fall off and dissolve in paraffin, filtering the mixture by using two layers of sterile gauze, and removing culture medium particles to obtain spore suspension, namely the seed source spore fungicide.

The method for preparing the yeast in the step (3) comprises the following steps:

filling a corncob finished product culture medium into a bran koji culture container, wherein the container is provided with an air inlet and an air outlet, adding water according to the weight ratio of the culture medium to the water of 1: 1.6-1.8, after the finished corncob completely absorbs water, putting the finished corncob into a sterilization pot to sterilize at 121 ℃ for 90min, and cooling for later use after sterilization;

and secondly, connecting the seed spore microbial inoculum with a sterile injector for injection inoculation under the sterile condition, connecting each spore suspension with a plurality of bran koji culture containers, and placing the inoculated containers in a culture room at 28-32 ℃ for ventilation culture.

The preparation and yeast-making process of the seed spore microbial inoculum comprises the following steps:

triangular flask seed source → sterile liquid wax is added → spore suspension is obtained by filtration → large bran koji is inoculated by injection

The beneficial technical effects of the invention are as follows:

the invention well solves the defect of eggplant bottle seed sources in the starter propagation process, the spore fungicide is applied to the existing production, and the qualification rate of the applied large bran koji is not lower than that of the existing large bran koji taking the second-generation eggplant bottles as seed sources. The application of the spore fungicide can effectively reduce the workload of operators, and the eggplant bottle is not used, so that the inoculation link is greatly simplified, and the number of people can be reduced by 3-5 people.

The spore microbial inoculum of the invention takes triangular flask culture as a spore source, aspergillus niger spores are separated by medium or equipment, spores prepared by each triangular flask can correspond to a plurality of barrels of large bran koji, and bran koji fluctuation caused by inconsistency of seed source quality and quantity is reduced to a certain extent. Meanwhile, in the starter propagation process, the sporophyte is directly injected into the big bran koji barrel, so that the complicated step of pouring sterile water into a second-generation eggplant bottle and then scraping spores is omitted, the workload is reduced, the opportunity of bacterial contamination can be reduced, and the fewer steps are realized, and the fewer opportunities of bacterial contamination are reduced.

Detailed Description

The present invention will be described in detail with reference to examples.

Example 1

A preparation method of Aspergillus niger seed spore microbial inoculum comprises the following steps:

(1) culturing seed spores in a corncob culture medium;

the selection of the corncob carrier and the preparation of the soybean meal hydrolysate are as described above, and the preparation of the nutrient solution is as follows; taking 800g of corn steep liquor powder and glucose respectively, adding 120g of ammonium sulfate, mixing and dissolving in water, and fixing the volume to 6L after dissolving. Uniformly mixing 6L of nutrient solution into 8kg of corncobs, and placing the corncobs in a low-temperature drying box for drying after the corncobs completely adsorb the nutrient solution, wherein the temperature is not more than 60 ℃. And drying to obtain a finished culture medium product.

Taking 2 2000ml triangular bottles, filling 80g of corncob culture medium into each bottle, adding 128ml of water, sterilizing for 30min at 121 ℃, cooling to 35 ℃ after sterilization, selecting a ring spore with an inoculating needle for inoculation, placing in a 35 ℃ culture chamber for static culture for 9 days, wherein the water content is 16.3 percent and 18.0 percent respectively after culture.

(2) Preparing a seed spore microbial inoculum by adopting a liquid-wax separation method;

pouring 800ml of sterilized liquid paraffin into each bottle, mixing uniformly, filtering by using two layers of sterile gauze, removing culture medium particles to obtain spore suspensions of 490ml and 510ml respectively, taking a small amount of spores from each bottle, performing shake flask inspection, and using the spores as seed spores after the spore suspensions are qualified.

(3) Inoculating the seed spore microbial inoculum prepared in the step (2) into a bran koji culture container for culture and koji preparation.

Filling 1.9kg of corncob finished product culture medium into a 25L bran koji culture container, adding 3.4L of water, placing the finished corncob into a sterilization pot to sterilize for 90min at 121 ℃ after the finished corncob completely absorbs water, and cooling for later use after sterilization;

② 8ml of each bran koji culture container is injected and inoculated, 61 and 63 bran koji containers can be respectively connected, and 124 containers are jointly used for culture and koji making, and after the koji making is finished, the qualified rate of the bran koji is tested by shaking the bottle. The results of koji-making with spore molds and eggplant bottles are shown in Table 1.

Example 2

A preparation method of Aspergillus niger seed spore microbial inoculum comprises the following steps:

(1) culturing seed spores in a corncob culture medium;

the selection of the corncob carrier and the preparation of the soybean meal hydrolysate are as described above, and the preparation of the nutrient solution is as follows; taking 200g of corn milk powder and glucose respectively, 60g of ammonium sulfate and 3.4L of soybean meal hydrolysate, adding water, mixing, dissolving, and fixing the volume to 6L. Uniformly mixing 6L of nutrient solution into 8kg of corncobs, and placing the corncobs in a low-temperature drying box for drying after the corncobs completely adsorb the nutrient solution, wherein the temperature is not more than 60 ℃. And drying to obtain a finished culture medium product.

Taking 2 2000ml triangular bottles, filling 100g of corncob culture medium into each bottle, adding 120ml of water, sterilizing at 121 ℃ for 30min, cooling to 35 ℃ after sterilization, selecting a ring spore with an inoculating needle for inoculation, placing in a 35 ℃ culture chamber for static culture for 10 days, wherein the water content is 11.5% and 12.3% respectively after culture.

(2) Preparing a seed spore microbial inoculum by adopting a liquid-wax separation method;

pouring 1000ml of sterilized liquid paraffin into each bottle, mixing uniformly, filtering with two layers of sterile gauze, removing culture medium particles to obtain 680ml and 690ml spore suspensions respectively, taking a small amount of spores from each bottle, performing shake flask inspection, and using the spores as seed spores after the spore suspension is qualified.

(3) Inoculating the seed spore microbial inoculum prepared in the step (2) into a bran koji culture container for culture and koji preparation.

Filling 2.0kg of corncob finished product culture medium into a 25L mouldy bran culture container, adding 3.5L of water, placing the finished corncob into a sterilization pot to sterilize for 90min at 121 ℃ after the finished corncob completely absorbs water, and cooling for later use after sterilization;

② 8ml of each bran koji culture container is injected and inoculated, 85 and 86 bran koji containers can be respectively connected, 171 containers are jointly used for culture and koji making, and the qualified rate of the bran koji is tested by shaking the bottle after the koji making is finished. The results of koji-making with spore molds and eggplant bottles are shown in Table 1.

Example 3

A preparation method of Aspergillus niger seed spore microbial inoculum comprises the following steps:

(1) culturing seed spores in a corncob culture medium;

the selection of the corncob carrier and the preparation of the soybean meal hydrolysate are as described above, and the preparation of the nutrient solution is as follows; taking 400g of corn milk powder and glucose respectively, 120g of ammonium sulfate and 1L of soybean meal hydrolysate, adding water, mixing, dissolving, and fixing the volume to 6L. Uniformly mixing 6L of nutrient solution into 8kg of corncobs, and placing the corncobs in a low-temperature drying box for drying after the corncobs completely adsorb the nutrient solution, wherein the temperature is not more than 60 ℃. And drying to obtain a finished culture medium product.

Taking 2 2000ml triangular bottles, filling 66g of corncob culture medium into each bottle, adding 100ml of water, sterilizing at 121 ℃ for 30min, cooling to 35 ℃ after sterilization, selecting a ring spore with an inoculating needle for inoculation, placing in a 35 ℃ culture chamber for static culture for 13 days, wherein the water content is 7.5 percent and 8.0 percent respectively after culture.

(2) Preparing a seed spore microbial inoculum by adopting a liquid-wax separation method;

and 660ml of sterilized liquid paraffin is poured into each bottle, after uniform mixing, two layers of sterile gauze are used for filtering, culture medium particles are removed to obtain spore suspensions of 410ml and 430ml respectively, a small amount of spores are taken from each bottle for shake flask inspection, and the spores are used as seed spores after the spore suspensions are qualified.

(3) Inoculating the seed spore microbial inoculum prepared in the step (2) into a bran koji culture container for culture and koji preparation.

Filling 2.5kg of corncob finished product culture medium into a 25L mouldy bran culture container, adding 4L of water, placing the finished corncob into a sterilization pot to sterilize for 90min at 121 ℃ after the finished corncob completely absorbs water, and cooling for later use after sterilization;

② 8ml of each bran koji culture container is injected and inoculated, 51 and 53 bran koji containers can be respectively connected, 104 containers are jointly used for culture and koji making, and the qualified rate of the bran koji is tested by shaking the bottle after the koji making is finished. The results of koji-making with spore molds and eggplant bottles are shown in Table 1.

Example 4

A preparation method of Aspergillus niger seed spore microbial inoculum comprises the following steps:

(1) culturing seed spores in a corncob culture medium;

the selection of the corncob carrier and the preparation of the soybean meal hydrolysate are as described above, and the preparation of the nutrient solution is as follows; taking 6L of soybean meal hydrolysate. Uniformly mixing 6L of soybean meal hydrolysate into 8kg of corncobs, and placing the corncobs in a low-temperature drying box for drying after the corncobs completely adsorb nutrient solution, wherein the temperature is not more than 60 ℃. And drying to obtain a finished culture medium product.

Taking 2 2000ml triangular bottles, filling 66g of corncob culture medium into each bottle, adding 92ml of water, sterilizing for 30min at 121 ℃, cooling to 35 ℃ after sterilization, selecting a ring spore with an inoculating needle for inoculation, placing in a 35 ℃ culture chamber for static culture for 7 days, wherein the water content is 15.3 percent and 14.0 percent respectively after culture.

(2) Preparing a seed spore microbial inoculum by adopting a liquid-wax separation method;

and 660ml of sterilized liquid paraffin is poured into each bottle, after uniform mixing, two layers of sterile gauze are used for filtering, culture medium particles are removed to obtain 425ml and 440ml of spore suspension respectively, a small amount of spores are taken from each bottle for shake flask inspection, and the spores are used as seed spores after the spore suspension is qualified.

(3) Inoculating the seed spore microbial inoculum prepared in the step (2) into a bran koji culture container for culture and koji preparation.

Filling 2.4kg of corncob finished product culture medium into a 25L bran koji culture container, adding 4.3L of water, placing the finished corncob into a sterilization pot to sterilize for 90min at 121 ℃ after the finished corncob completely absorbs water, and cooling for later use after sterilization;

② 8ml of each bran koji culture container is injected and inoculated with 53 and 55 bran koji containers respectively, a total of 108 containers are inoculated for culture and koji making, and after the koji making is finished, the qualified rate of the bran koji is tested by shaking the bottle. The results of koji-making with spore molds and eggplant bottles are shown in Table 1.

Comparative example:

filling 2.4kg of corncob finished product culture medium into a 25L bran koji culture container, adding 4.3L of water, placing the finished corncob into a sterilization pot to sterilize for 90min at 121 ℃ after the finished corncob completely absorbs water, and cooling for later use after sterilization. Respectively pouring sterile water into 100 eggplant bottles, scraping Aspergillus niger spores in the eggplant bottles by using a sterile needle to form spore suspension in each eggplant bottle, then pouring into bran koji culture containers, connecting one bran koji culture container to each eggplant bottle, and placing the inoculated containers in a culture chamber at 30 ℃ for ventilation culture and koji making. And after the koji making, shaking the bottle to test the bran koji qualification rate. The results of koji-making with spore molds and eggplant bottles are shown in Table 1.

TABLE 1

The implementation example shows that the yield of the seed spore fungicide of the invention is superior to that of the existing eggplant bottle seed in koji making, and the table shows that the spore fungicide and the eggplant bottle are basically consistent in the germ contamination rate of the bran koji, but the spore fungicide is obviously better than that of the eggplant bottle in the variation rate of the bran koji, because the spore is scraped off by a sterile needle when the eggplant bottle is used for preparing suspension, a large amount of hyphae are also scraped off, and the excessive hyphae can be visually observed under a microscope, which is a cause of the bran koji variation, and the preparation process of the spore fungicide avoids the problem. Meanwhile, in the aspect of seed source production, the seed source production amount of spore fungicide is reduced by more than 98% in comparison with that of eggplant bottles for every hundred seed sources required by the bran koji containers, so that the workload of operators is greatly reduced, and the seed sources are more finely controlled due to small quantity, and the production level is favorably improved.

Claims (4)

1. A preparation method of Aspergillus niger seed spore microbial inoculum is characterized by comprising the following steps:

(1) culturing seed spores in a corncob culture medium;

filling a corncob culture medium into a triangular flask, adding water according to the weight ratio of the culture medium to the water of 1:1.2-1.6, sterilizing, cooling to 35 ℃, selecting a ring spore with an inoculating needle for inoculation, placing in a 35 ℃ culture chamber for static culture for 7-14 days, and keeping the seed source water content below 20% after the culture is finished;

(2) preparing a seed spore microbial inoculum by adopting a liquid-wax separation method;

the method for preparing the seed source spore microbial inoculum by the liquid-wax separation method comprises the following steps: and (3) mixing the sterilized liquid paraffin according to the weight-volume ratio of the seed bran koji to the liquid paraffin of 1: 10, pouring the mixture into seed source triangular flask bran koji, uniformly mixing, then, enabling spores to fall off and dissolve in paraffin, filtering by using two layers of sterile gauze, and removing culture medium particles to obtain spore suspension, namely seed source spore fungicide;

(3) inoculating the seed spore microbial inoculum prepared in the step (2) into a bran koji culture container for culture and koji preparation.

2. The method according to claim 1, wherein the corncob medium in step (1) consists of corncob particles and a nutrient solution required for growth of Aspergillus niger; the corncob particles are prepared by selecting clean corncobs with good water absorbability as raw materials, crushing the corncobs by a crusher, and sieving the crushed corncobs to obtain particles with the diameter of 5-10 mm; the nutrient solution comprises the following components: 800g of corn starch and 800g of glucose respectively, 60-120g of ammonium sulfate and 0-6L of soybean meal enzymolysis filtrate;

the soybean meal enzymolysis filtrate is prepared by the following method:

crushing soybean meal with protein content of 46%, and sieving with a 40-mesh sieve;

adding water to prepare slurry, wherein the weight ratio of the soybean meal to the water is 1: 6;

regulating the pH value to 6.5 by using 40% sodium hydroxide, and then heating to 60 ℃;

adding alkaline protease powder, wherein the addition amount of the enzyme powder is 2000 enzyme activity units per gram of soybean meal powder, and carrying out heat preservation enzymolysis for 3 hours;

fifthly, heating the enzymolysis liquid to 100 ℃ after enzymolysis, filtering while the liquid is hot, and adding water to adjust the solid content of the filtrate to 10% for later use.

3. The method of claim 1, wherein the corncob medium is prepared by the method comprising: taking 800g of corn steep liquor powder and 800g of glucose respectively, 60-120g of ammonium sulfate and 0-6L of soybean meal enzymolysis filtrate, adding water for dissolving, fixing the volume to 6L after dissolving, uniformly mixing 6L of nutrient solution into 8kg of corncobs, placing the corncobs in a low-temperature drying box for drying after the corncobs completely adsorb the nutrient solution, wherein the temperature does not exceed 60 ℃, and obtaining a culture medium finished product after drying.

4. The method according to claim 1, wherein the koji-making in step (3) is:

filling a corncob finished product culture medium into a bran koji culture container, wherein the container is provided with an air inlet and an air outlet, adding water according to the weight ratio of the culture medium to the water of 1: 1.6-1.8, after the finished corncob completely absorbs water, putting the finished corncob into a sterilization pot to sterilize at 121 ℃ for 90min, and cooling for later use after sterilization;

and secondly, connecting the seed spore microbial inoculum with a sterile injector for injection inoculation under the sterile condition, connecting each spore suspension with a plurality of bran koji culture containers, and placing the inoculated containers in a culture room at 28-32 ℃ for ventilation culture.