阅读说明：本技术 一种葡糖淀粉酶的生产方法 (Production method of glucoamylase ) 是由 冯玉枚 马良 向斌 曾佳 于 2020-12-29 设计创作，主要内容包括：本发明涉及一种葡糖淀粉酶的生产方法,属于微生物发酵工程领域,葡糖淀粉酶的生产过程中采用槐糖补料来补充发酵液中的碳源,其槐糖中三糖的浓度为：总糖47g/100ml-65g/100ml；还原糖40g/100ml-55 g/100ml；葡萄糖38g/100ml-50 g/100ml,采用槐糖补料过程中,控制发酵液中的还原糖浓度0.7-1.2g/100ml,槐糖的补料速率控制在3.5g/L/h-7.0 g/L/h。本发明根据菌种的生长代谢特性流加槐糖,速率控制在3.5-7.0g/L/h(以总糖浓度计),既能促进黑曲霉的生长代谢,又可以诱导菌体提高葡糖淀粉酶产率效率,提升发酵水平。(The invention relates to a production method of glucoamylase, belonging to the field of microbial fermentation engineering, wherein a carbon source in fermentation liquor is supplemented by adopting sophorose supplementary materials in the production process of glucoamylase, and the concentration of trisaccharide in sophorose is as follows: total sugar 47g/100ml-65g/100 ml; reducing sugar 40g/100ml-55g/100 ml; 38g/100ml-50g/100ml of glucose, and in the feeding process of sophorose, the concentration of reducing sugar in the fermentation liquor is controlled to be 0.7-1.2g/100ml, and the feeding rate of sophorose is controlled to be 3.5g/L/h-7.0 g/L/h. The invention adds sophorose according to the growth metabolism characteristic of the strain, the speed is controlled at 3.5-7.0g/L/h (based on the total sugar concentration), the growth metabolism of Aspergillus niger can be promoted, the thallus can be induced to improve the yield efficiency of glucoamylase, and the fermentation level is improved.)

1. A production method of glucoamylase is characterized in that a sophorose feed is adopted to supplement a carbon source in a fermentation liquor in the production process of the glucoamylase, and the concentration of trisaccharide in sophorose is as follows: total sugar 47g/100ml-65g/100 ml; reducing sugar 40g/100ml-55g/100 ml; glucose 38g/100ml-50g/100 ml.

2. The method of claim 1, wherein: in the feeding process of sophorose, the concentration of reducing sugar in the fermentation liquor is controlled to be 0.7-1.2g/100ml, and the feeding rate of sophorose is controlled to be 3.5g/L/h-7.0 g/L/h.

3. The method of claim 2, wherein: in the production process of glucoamylase, dissolved oxygen is controlled to be not less than 20%.

4. The method of claim 3, wherein: the sophorose supplementing process is specifically operated as follows: when dextrin in a glucoamylase fermentation medium is exhausted, the glucose supplementation rate is increased to 2.0g/L/h, the glucose supplementation rate is increased to 7.0g/L/h within 10-15h after glucose supplementation, the content of reducing sugar in fermentation liquor is higher than 1.2g/100ml or the dissolved oxygen is lower than 20% after 90-120h, the glucose is reduced to 3.5g/L/h in a gradient manner, and the concentration of the reducing sugar in the fermentation liquor is maintained to be 0.7-1.2g/100 ml.

5. The method of claim 1, wherein: the method specifically comprises the following steps:

activating strains: inoculating the colony of glucoamylase producing strain to an activation culture medium, and culturing for 2.5-3.5d to obtain an activation culture solution;

② first-stage seed culture: inoculating 500ml-800ml of the activated culture solution obtained in the step I into 100L of seed culture medium, and culturing for 50h-80h to obtain seed culture solution;

③ Secondary culture of seeds: expanding the seed culture solution obtained in the second step to 1500L of a second-level seed culture medium, and culturing for 30-45 h to obtain a second-level seed solution; (ii) a

Fermentation culture of glucoamylase: inoculating the second-stage seed liquid obtained in the third step to 10m of a fermentation medium for cultivating for 140h-170 h.

6. The method of claim 5, wherein: the method comprises the following culture media:

activating a culture medium: contains 330g of dextrin 220-40 g, 20-40g of corn steep liquor, 20-40g of ammonium sulfate and pH4.0-4.5 in every 1L of water;

seed culture medium: contains 330g of dextrin 190-5.0, 20-40g of corn steep liquor, 20-40g of ammonium sulfate in 1L of water;

secondary seed culture medium: 360g of dextrin-containing 170-40 g of corn steep liquor, 20-40g of ammonium sulfate and 4.5-5.0 of pH4 in 1L of water;

glucoamylase fermentation medium: 300g of dextrin 130-containing water, 40-70g of corn steep liquor, 20-40g of ammonium sulfate, 5-20 g of soybean meal powder and pH4.5-5.0 in 1L of water.

7. The method according to any one of claims 1 to 6, wherein: the glucoamylase-producing strain is Aspergillus niger.

Technical Field

The invention relates to the technical field of building materials, in particular to an industrial production method of glucoamylase.

Background

Glucoamylase is an extracellular enzyme secreted by microorganisms and having exonuclease activity, which hydrolyzes the non-reducing terminal alpha-1, 4 glycosidic bond in carbohydrates such as starch, dextrin, glycogen, etc., and releases beta-D-glucose. The starch saccharifying agent is used as a starch saccharifying agent in a large amount in the fermentation industry, is in huge demand and is one of the most important industrial enzyme preparations. The carbon source is an important nutrient element for the growth and metabolism of the thallus and is also a main component element of glucoamylase molecules, and the aspergillus niger is one of the main strains for industrial production of the glucoamylase. In the production process of saccharifying enzyme produced by aspergillus niger, different carbon sources can cause the phenomena of large biomass and low enzyme activity in fermentation, unreasonable sugar supplement rate can often cause the fluctuation of reducing sugar and dissolved oxygen in the fermentation process to cause abnormal metabolism, the thalli are aged in advance, and the time for efficiently producing the enzyme is finished in advance. Therefore, sugar can directly influence the growth and metabolism of microorganisms, and the thalli have different requirements on sugar consumption in different physiological periods. Therefore, exploring a high-quality carbon source matrix and a reasonable sugar control standard in the process is an important work in improving the fermentation process of Aspergillus niger.

Disclosure of Invention

In order to solve the technical problems, the invention provides a production method for efficiently producing glucoamylase by aspergillus niger.

The technical scheme adopted by the invention is that,

a production method of glucoamylase is characterized in that a sophorose feed is adopted to supplement a carbon source in a fermentation liquor in the production process of the glucoamylase, and the concentration of trisaccharide in sophorose is as follows: total sugar 47g/100ml-65g/100 ml; reducing sugar 40g/100ml-55g/100 ml; glucose 38g/100ml-50g/100 ml.

Preferably, in the sophorose feeding process, the concentration of reducing sugar in the fermentation liquor is controlled to be 0.7-1.2g/100ml, and the feeding rate of sophorose is controlled to be 3.5g/L/h-7.0 g/L/h.

Further preferably, the dissolved oxygen is controlled to not less than 20% during the production of glucoamylase.

Still more preferably, the sophorose feeding process is specifically operated as follows: when dextrin in a glucoamylase fermentation medium is exhausted, the glucose supplementation rate is increased to 2.0g/L/h, the glucose supplementation rate is increased to 7.0g/L/h within 10-15h after the glucose supplementation, the rate is adjusted once every 12h according to the volume, the concentration of reducing sugar in a fermentation liquid in the process is stabilized at 0.7-1.2g/100ml, the content of reducing sugar in the fermentation liquid is higher than 1.2g/100ml after 90-120h or the dissolved oxygen is lower than 20%, the sugar is reduced to 3.5g/L/h in a gradient manner, and the concentration of reducing sugar in the fermentation liquid is maintained at 0.7-1.2g/100 ml. And the sugar supplementing rate is maintained to be 7.0g/L/h before fermentation for 90-120h, the enzyme production activity of the thalli is rapidly improved, after 90-120h, the sugar is reduced to 3.5g/L/h in a gradient manner according to the content of dissolved oxygen and reducing sugar in fermentation liquor higher than 1.2g/100ml, the metabolic environment of the thalli is stabilized, and the sugar repression effect in the metabolism of the thalli is reduced.

Further preferably, the method comprises the following culture media:

activating a culture medium: contains 330g of dextrin 220-40 g, 20-40g of corn steep liquor, 20-40g of ammonium sulfate and pH4.0-4.5 in every 1L of water;

seed culture medium: contains 330g of dextrin 190-5.0, 20-40g of corn steep liquor, 20-40g of ammonium sulfate in 1L of water;

secondary seed culture medium: 360g of dextrin-containing 170-40 g of corn steep liquor, 20-40g of ammonium sulfate and 4.5-5.0 of pH4 in 1L of water;

glucoamylase fermentation medium: 300g of dextrin-containing 130-70 g of corn steep liquor, 20-40g of ammonium sulfate, 5-20 g of soybean meal powder and pH4.5-5.0 in 1L of water;

the supplementary material is 47g/100ml-65g/100ml of total sugar; reducing sugar 40g/100ml-55g/100 ml; glucose 38g/100ml-50g/100ml self-made sophorose feed.

Still further preferably, the method specifically comprises the steps of:

activating strains: inoculating the colony of glucoamylase producing strain to an activation culture medium, and culturing for 2.5-3.5d to obtain an activation culture solution;

② first-stage seed culture: inoculating 500ml-800ml of the activated culture solution obtained in the step I into 100L of seed culture medium, and culturing for 50h-80h to obtain seed culture solution;

③ Secondary culture of seeds: expanding the seed culture solution obtained in the second step to 1500L of a second-level seed culture medium, and culturing for 30-45 h to obtain a second-level seed solution; (ii) a

Starch of glucoseFermenting and culturing the powder enzyme: inoculating the second-stage seed liquid obtained in the third step to 10m³Culturing in glucoamylase fermentation medium for 140-170 h.

Further preferably, the glucoamylase producing strain is aspergillus niger. The glucoamylase has a large demand on saccharide, and the selection of supplementary materials and the control of saccharide consumption in the process are important for the growth of the glucoamylase in order to prolong the active growth period of the glucoamylase. Therefore, the concentrated sophorose is selected, and the sugar supplement rate is reasonably controlled by combining the metabolic indexes on the tank so as to achieve the maximization of the enzyme production of the thalli. In the metabolic process, obvious growth inflection points appear when the thalli are cultured for 90-120h, and enough dissolved oxygen and supplementary materials are needed to be supplied before the enzyme production peak period is 90-120h, so that the activity of the thalli is ensured. After 90-120h, the thalli begin to age quickly, residual sugar is accumulated in the metabolic process, dissolved oxygen is reduced, and the enzyme activity is increased and reduced. In order to prolong the growth period of the thalli, the supplement needs to be reduced in time and the dissolved oxygen needs to be stabilized.

The invention has the following effects:

1. the invention adopts multi-stage amplification production to reach the highest fermentation output value, and the enzyme activity of glucoamylase amplification production can reach 60000IU/ml by optimizing the carbon source substrate and the process feeding process.

2. Through screening different types of carbon sources, experiments show that the sophorose is more beneficial to aspergillus niger to produce glucoamylase, can better promote the growth of thalli at the early stage and enable the thalli to rapidly enter a metabolic enzyme production period, and obviously improves the amplification of process enzyme activity. It is possible that the proportion of trisaccharides (total sugar, reducing sugar and glucose) in the sophorose has an effect of promoting enzyme production in the thallus process, the proportion of the reducing sugar in the sophorose can induce the thallus to produce glucoamylase, and a large amount of glucose can also ensure the growth and metabolism of the thallus.

3. The invention selects the supplementary material components which are suitable for the growth of the thalli, optimizes the fine control of the supplementary material in the process, controls the sugar consumption in the process and coordinates with the metabolic parameters, the control rate of the sugar consumption in the process and the change of the reducing sugar are reference values, and stabilizes the concentration of the reducing sugar in the fermentation liquor, namely controls the highest metabolic rate of the thalli. Increasing sugar supplement rate to 7.0g/L x h before fermenting for 90-120h, and rapidly increasing enzyme production activity of thallus after 90-120h according to dissolved oxygen and fermentation liquidThe content of the medium reducing sugar is properly reduced to 3.5g/L/h, the metabolic environment of the thallus is stabilized, and the sugar repression effect in the thallus metabolism is reduced. Therefore, the optimized process is controlled to be 26m³The average tank-releasing enzyme activity of the glucoamylase produced by the tank can reach 53458 IU/ml.

4. The glucoamylase producing strain is Aspergillus niger, the requirement of the strain on a carbon source is high, a proper carbon source substance is selected, especially a proper trisaccharide proportion in the carbon source can promote the growth of biomass and enzyme production efficiency in an equal proportion, and self-prepared high-concentration sophorose can efficiently stimulate the strain to metabolize and produce enzyme through the verification of a liquid fermentation level.

5. After self-prepared sophorose is used as a carbon source, the supplementation amount of the sophorose in the process is reasonably controlled by combining the dissolved oxygen parameter in the process and the accumulation amount of reducing sugar in fermentation liquor, a better process sugar control rate curve is obtained, and the amplification verification of the activity of the tank-releasing enzyme and the activity of the process enzyme is carried out.

Drawings

FIG. 1 is a process validation process of the present invention;

FIG. 2 metabolic parameter profile of experiment 2 in example 2;

FIG. 3 example 3 is a graph of sugar supplementation rate versus reducing sugars in fermentation broth;

FIG. 4 example 3 feed each component chromatogram.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited to the scope of the examples. These examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

Example 1

A method of fermenting a glucoamylase, the method comprising the steps of:

activating strains: inoculating a bacterial colony of a glucoamylase production strain into an activation culture medium, culturing at 34 ℃, culturing for 3d, and culturing on a shaking machine at 300rpm to obtain an activation culture solution;

the activation medium contains 270g of dextrin, 30g of corn steep liquor, 30g of ammonium sulfate and pH4.0-4.5 in 1L of water.

② first-stage seed culture: inoculating 700ml of the activated culture solution obtained in the step I into 100L of seed culture medium, culturing at 34 ℃ for 700h to obtain a primary seed culture solution;

the seed culture medium is 1L water containing 260g dextrin, 30g corn steep liquor, 30g ammonium sulfate and pH4.5-5.0.

③ Secondary culture of seeds: expanding the seed liquid obtained in the step two to 1500L of seed culture medium for culture, culturing at 34 ℃, and culturing for 40h to obtain secondary seed liquid.

The secondary seed culture medium is 1L water containing 250g of dextrin, 30g of corn steep liquor, 30g of ammonium sulfate and pH4.5-5.0.

Fermentation culture of glucoamylase: inoculating the seed liquid obtained in the step three to 10m³Culturing in fermentation medium at 34 deg.C for 160h, wherein the glucoamylase fermentation medium is 1L water containing dextrin 220g, corn steep liquor 60g, ammonium sulfate 30g, and soybean meal 15 g.

Ammonia water supplement: the fermentation process is an acid production process, and ammonia water is added to control the pH within the required range of 4.7-4.9.

Before optimizing the production method of the glucoamylase, the third step needs to be carried out, in the fermentation culture process of the glucoamylase, a carbon source which is most beneficial to the growth and enzyme production of thalli is screened from different carbon source matrixes to feed, the concentration of reducing sugar in fermentation liquor is controlled to be 0.7-1.2g/100ml, and the specific feeding process is as follows: the sugar-supplementing rate is 2.0g/L/h, the sugar-supplementing rate per hour is increased by 0.5g/L/h, the sugar-supplementing rate is increased to 5.5g/L/h within 10-15h, the set 5.5g/L/h is maintained before fermentation for 90-120h, and the sugar consumption in the middle and later stages is adjusted according to the thallus metabolism condition after fermentation for 90-120 h. The carbon sources for screening are shown in Table 1 below; as can be seen from Table 1, the most suitable carbon source is sophorose.

TABLE 1

Example 2

On the basis of example 1, after selecting sophorose as a carbon source with higher enzyme production, the upper limit of different feeding rates was changed, and the specific scheme is shown in table 2. Before optimizing the production method of glucoamylase, the preparation needs to be carried out, the sugar supplement starting speed is 2.0g/L/h, the sugar supplement speed per hour is increased by 0.5g/L/h, the speed is increased to the set highest speed within 10-15h, the set highest sugar supplement speed is maintained before fermentation for 90-120h, the sugar consumption in the middle and later periods is adjusted according to the metabolism condition of thalli after 90-120h, and the lower limit of the sugar supplement speed is 3.5 g/L/h. And optimizing an optimal process feeding process by combining parameters of dissolved oxygen and reducing sugar on the tank through process enzyme activity amplification and tank release level, and verifying the enzyme activity level on the tank. As can be seen from Table 2, the highest rate of sugar supplementation was 7.0g/L/h, and the highest enzyme activity level was obtained.

TABLE 2

Wherein, the aspergillus niger glucoamylase production process uses sophorose as a supplementary material, the upper limit of sugar supplement is set to be 7.0g/L/h, the lower limit of sugar reduction in the middle and later stages is set to be 3.5g/L/h, and thalli can fully utilize the supplementary material for growth, metabolism and enzyme production. Experiment 2 specific metabolic parameters are shown in figure 2. In the experiment 2, the fermentation time is 165h, and the tank-releasing enzyme activity reaches 57262 IU/ml.

Example 3

A method of fermenting a glucoamylase, the method comprising the steps of:

activating strains: inoculating a bacterial colony of a glucoamylase production strain into an activation culture medium, culturing at 34 ℃, culturing for 3d, and culturing on a shaking machine at 300rpm to obtain an activation culture solution;

the activation medium contains 270g of dextrin, 30g of corn steep liquor, 30g of ammonium sulfate and pH4.0-4.5 in 1L of water.

② first-stage seed culture: inoculating 700ml of the activated culture solution obtained in the step I into 100L of seed culture medium, culturing at 34 ℃ for 700h to obtain a primary seed culture solution;

the seed culture medium is 1L water containing 260g dextrin, 30g corn steep liquor, 30g ammonium sulfate and pH4.5-5.0.

③ Secondary culture of seeds: expanding the seed liquid obtained in the step two to 1500L of seed culture medium for culture, culturing at 34 ℃, and culturing for 40h to obtain secondary seed liquid.

The secondary seed culture medium is 1L water containing 250g of dextrin, 30g of corn steep liquor, 30g of ammonium sulfate and pH4.5-5.0.

Fermentation culture of glucoamylase: inoculating the seed liquid obtained in the step three to 10m³Culturing at 34 deg.C in fermentation medium containing 1L water containing 220g of dextrin, 60g of corn steep liquor, 30g of ammonium sulfate, and 15g of soybean meal for 160 h.

During the fermentation, supplemented sophorose is used, the concentration of which is 61.8g/100ml of total sugar, 54.1g/100ml of reducing sugar and 43.2g/100ml of glucose. Supplementing 2.0g/L/h (calculated by total sugar) of sophorose when fermenting for 43h, if the dissolved oxygen fluctuates between 20% and 40%, the sugar supplementing rate increases by 0.5g/L/h per hour, the upper limit is 7.0g/L/h, the dissolved oxygen is controlled to be more than 20%, and the reducing sugar is controlled to be 0.7-1.2g/100ml, if the dissolved oxygen decreases or the reducing sugar content increases, the sugar supplementing rate is reduced to maintain the content of the dissolved oxygen and the reducing sugar, the experiment 57h reaches the upper limit of sugar supplementing, the upper limit of sugar supplementing is maintained in the process to be 7.0g/L/h, when the dissolved oxygen and the reducing sugar are deviated from the maintained parameters during 94h-100h, the sugar is reduced, and the lower limit is 3.5g/100 ml.

Ammonia water supplement: the fermentation process is an acid production process, and ammonia water is added to control the pH within the required range of 4.7-4.9. The glucoamylase activity of the crude enzyme solution obtained by 167h of glucoamylase fermentation is 59192 IU/ml.

Comparative example 1

On the basis of example 3, the feed parameters were varied: during the fermentation, supplemented sophorose is used, the concentration of which is 61.8g/100ml of total sugar, 54.1g/100ml of reducing sugar and 43.2g/100ml of glucose. Supplementing 2.0g/L/h (calculated by total sugar) of sophorose when fermenting for 45h, if the dissolved oxygen fluctuates between 20% and 40%, the sugar supplementing rate increases by 0.5g/L/h per hour, the upper limit is 5.0g/L/h, the dissolved oxygen is controlled to be more than 20%, and the reducing sugar is controlled to be 0.3-0.6g/100ml, if the dissolved oxygen decreases or the reducing sugar content increases, the sugar supplementing rate is reduced to maintain the content of the dissolved oxygen and the reducing sugar, the experiment 51h reaches the upper limit of sugar supplementing, the upper limit of sugar supplementing is maintained in the process, when the dissolved oxygen and the reducing sugar are deviated in 137h, the sugar is reduced, and the lower limit is 3.5g/100 ml.

Ammonia water supplement: the fermentation process is an acid production process, and ammonia water is added to control the pH within the required range of 4.7-4.9. The glucoamylase activity of the crude enzyme solution obtained by fermenting the glucoamylase for 165h is 46727 IU/ml.

Comparative example 2

On the basis of example 3, the feed parameters were varied: during the fermentation, supplemented sophorose is used, the concentration of which is 61.8g/100ml of total sugar, 54.1g/100ml of reducing sugar and 43.2g/100ml of glucose. Supplementing 2.0g/L/h (calculated by total sugar) of sophorose when fermenting for 45h, if the dissolved oxygen fluctuates between 20% and 40%, the sugar supplementing rate increases by 0.5g/L/h per hour, the upper limit is 7.0g/L/h, the dissolved oxygen is controlled to be more than 20%, and the reducing sugar is 1.3-1.5mg/100ml, if the dissolved oxygen decreases or the reducing sugar content increases, the sugar supplementing rate is reduced to maintain the content of the dissolved oxygen and the reducing sugar, 55h of the experiment reaches the upper limit of sugar supplementing, the upper limit of sugar supplementing is maintained in the process, when the dissolved oxygen and the reducing sugar are deviated from the maintained parameters within 93h-99h, the sugar is reduced, and the lower limit is 3.5g/100 ml.

Ammonia water supplement: the fermentation process is an acid production process, and ammonia water is added to control the pH within the required range of 4.7-4.9. The glucoamylase activity of the crude enzyme solution obtained by fermenting the crude enzyme solution for 165h by the glucoamylase is 47692 IU/ml.

Comparative example 3

On the basis of example 3, the feed parameters were varied: a self-prepared 65% glucose solution with the concentration of total sugar 68g/100ml, reducing sugar 65g/100ml and glucose 64g/100ml is used in the fermentation process. Glucose is supplemented by 2.0g/L/h (calculated by total sugar) when the fermentation is carried out for 45h, if the dissolved oxygen fluctuates between 20% and 40%, the sugar supplementing rate increases by 0.5g/L/h per hour, the upper limit is g/L/h, the dissolved oxygen is controlled to be more than 20%, the reducing sugar is controlled to be 0.7-1.2g/100ml, if the dissolved oxygen is reduced or the reducing sugar content is increased, the sugar supplementing rate is reduced to maintain the content of the dissolved oxygen and the reducing sugar, the experiment 54h reaches the upper limit of sugar supplementing, the upper limit of sugar supplementing is maintained in the process, the parameters of the dissolved oxygen and the reducing sugar maintained in the culture process are deviated, the sugar is reduced, and the lower limit is 3.5g/100 ml. Ammonia water supplement: the fermentation process is an acid production process, and ammonia water is added to control the pH within the required range of 4.7-4.9. The glucoamylase activity of the crude enzyme solution obtained by fermenting 163h with glucoamylase was 38946 IU/ml.

Comparative example 4

On the basis of example 4, no feed was performed.

The glucoamylase activity of the crude enzyme solution obtained by fermenting the glucoamylase for 44h is 18714 IU/ml.