CN110923274B

CN110923274B - Method for preparing fermentation medium by using soybean meal hydrolysate

Info

Publication number: CN110923274B
Application number: CN201911308330.6A
Authority: CN
Inventors: 毛松举; 王小平; 胥九兵; 赵杰; 尤学波; 杨雪
Original assignee: Hulunbeier Northeast Fufeng Biotechnologies Co ltd
Current assignee: Hulunbeier Northeast Fufeng Biotechnologies Co ltd
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2022-05-31
Anticipated expiration: 2039-12-18
Also published as: CN110923274A

Abstract

The invention belongs to the technical field of biology, and discloses a method for preparing a fermentation culture by using a soybean meal hydrolysateA method of nutrient base comprising the steps of: taking fermentation medium raw materials, and preparing according to the following concentrations: 50-100g/L of glucose, 300g/L of soybean meal hydrolysate, K₂HPO₄1‑3g/L，MgSO₄·7H₂O 30‑80mg/L，MnSO₄·H₂O 2‑5mg/L，FeSO₄·7H₂O 2‑5mg/L，VB₁8-15mg/L, biotin 5-10 mug/L; stirring the raw materials uniformly, adjusting pH to 6-7, sterilizing, and naturally cooling to obtain the fermentation culture medium. The method has strong operability, is simple and feasible, and can greatly reduce the fermentation cost.

Description

Method for preparing fermentation medium by using bean pulp hydrolysate

Technical Field

The invention belongs to the technical field of biology, and relates to a method for preparing a fermentation medium by using a soybean meal hydrolysate.

Background

With the gradual development of the amino acid industry, a microbial fermentation method becomes the most promising production technology for producing amino acid, but the defect of high cost of a fermentation medium exists in the preparation process of microbial fermentation broth. For example, patent technology "CN 201510168444, a process for preparing granular threonine" performs seed culture and fermentation culture on brevibacterium flavum, wherein a nitrogen source adopts yeast extract, corn steep liquor and the like, so that the fermentation effect is good, the yield of threonine is high, but the price of the yeast extract is high, the large-scale fermentation dosage is large, and how to reduce or replace the yeast extract is a technical problem that needs to be solved. The applicant has conducted extensive research on amino acid fermentation technology and aims to reduce fermentation cost, and in the patent technology 'a method for replacing yeast powder with bean pulp enzymolysis liquid in threonine production' the method for replacing yeast powder with bean pulp enzymolysis liquid adopts a plurality of microorganisms for mixed enzymolysis of bean pulp to replace yeast cream as a nitrogen source, so that the fermentation efficiency is high, the cost is reduced, but the existing process is complex, once microbial pollution is caused, production stagnation is caused, and the risk is high.

Disclosure of Invention

In order to overcome the defects of the fermentation culture medium in the prior art, the invention applies the bean pulp hydrolysate to the fermentation culture medium on the basis of the 'process for hydrolyzing soybean meal' in the earlier patent technology, and aims to provide the method for preparing the fermentation culture medium by utilizing the bean pulp hydrolysate.

The invention is realized by the following technical scheme:

the method for preparing the fermentation medium by using the bean pulp hydrolysate comprises the following steps: taking fermentation medium raw materials, and preparing according to the following concentrations: 50-100g/L of glucose, 300g/L of soybean meal hydrolysate, K₂HPO₄ 1-3g/L，MgSO₄·7H₂O 30-80mg/L，MnSO₄·H₂O 2-5mg/L，FeSO₄·7H₂O 2-5mg/L，VB₁8-15mg/L, biotin 5-10 mug/L; stirring the raw materials uniformly, adjusting pH to 6-7, sterilizing, and naturally cooling to obtain the fermentation culture medium.

Further, the method comprises the steps of: taking fermentation medium raw materials, preparing according to the following concentration, glucose 80g/L, soybean meal hydrolysate 200g/L, K₂HPO₄ 2g/L，MgSO₄·7H₂O 50mg/L，MnSO₄·H₂O 3mg/L，FeSO₄·7H₂O 3mg/L，VB₁10mg/L, biotin 7 mu g/L; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation culture medium.

Further, the bean pulp hydrolysate is prepared by the following process:

crushing the bean pulp, placing the crushed bean pulp in a reaction kettle, adding corn steep liquor and glutamic acid residue, adding citric acid to prepare suspension with the solid content of 30-40%, and homogenizing by using a high-pressure homogenizer to refine the particle size; heating to 90 deg.C, and hydrolyzing with ultrasound for 5-20 min; continuing to hydrolyze for 5-7h, then performing microwave-assisted hydrolysis for 2-4min, stopping microwave, cooling to 45 ℃, adding ammonia water, adjusting pH to 2.5-3.5, then adding acid protease, performing enzymolysis for 6-9h, inactivating enzyme at 95 ℃ for 3min, then filtering, discharging residue, then adding activated carbon for decolorization, and filtering to remove activated carbon to obtain a soybean meal hydrolysate.

Preferably, the adding amount of the corn steep liquor accounts for 30-50% of the weight of the soybean meal, and the adding amount of the glutamic acid residue accounts for 20-40% of the weight of the soybean meal.

Preferably, the concentration of the citric acid is 1-2 mol/L.

Preferably, the parameters of the homogenization treatment are as follows: the pressure is 30MPa, the temperature is 32 ℃, and the time is 10-20 s.

Preferably, the ultrasonic frequency is 20kHz, and the microwave power is 300W.

Preferably, the addition amount of the acid protease is 500U/L.

The invention also claims a fermentation medium prepared according to any one of the above methods.

Preferably, the fermentation medium is used for microbial fermentation to produce glutamic acid.

Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:

the method for hydrolyzing the protein by acid and enzyme by using the physical method has the characteristics of quick and thorough hydrolysis, low cost, small investment, wide application and the like;

according to the invention, the glutamic acid residue and the corn steep liquor are added into the soybean meal, and the three proteins can be jointly hydrolyzed by an optimized process, so that the nutrition in the hydrolysate is more balanced and comprehensive, and the fermentation effect is better compared with that of a single hydrolysate;

according to the invention, by means of a soybean meal hydrolysis technology, the corn steep liquor and the glutamic acid residues are matched with the soybean meal, and the macromolecular protein which is difficult to utilize by microorganisms in the corn steep liquor and the glutamic acid residues is hydrolyzed to free amino nitrogen and micromolecular substances which are easy to utilize by thalli, so that the advantages of the raw materials are effectively utilized and used as specific nutrient components of a fermentation medium, the phenomenon that more foams are generated in the fermentation process is reduced, the effective utilization rate of the fermentation tank capacity is increased, and the fermentation contamination rate is reduced;

the method adopts a mode of hydrolyzing various proteins by weak acid and protease simultaneously and adopts various modes of high-pressure homogenization, ultrasound and microwave, so that the leaching rate of the proteins is improved, the hydrolysis degree is greatly improved, the hydrolysis condition is mild, and amino acid components cannot be damaged; homogenizing with a high-pressure homogenizer to refine the particle size, so that the protein particles reach 1-10 μm level, and the hydrolysis contact area is increased; the ultrasonic treatment with proper frequency can not only improve the protein extraction rate, but also destroy protein chains, improve the protein solubility and the hydrophilicity, and is beneficial to overcoming the defect of incomplete weak acid hydrolysis; the microwave with certain power enables peptide molecules to vibrate in an electromagnetic field, so that the structure of partial polypeptide which cannot be hydrolyzed by citric acid is loosened, sulfydryl is destroyed, and the internal structure is changed, thereby being beneficial to further enzymolysis.

Drawings

FIG. 1: the effect of different acid types on the hydrolysis rate;

FIG. 2: the influence of different hydrolysis temperatures on the hydrolysis rate;

FIG. 3: the influence of microwave time on the hydrolysis rate;

FIG. 4: the influence of different enzyme types and enzyme activities on the hydrolysis rate.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A process for hydrolyzing soybean meal, comprising the steps of:

crushing bean pulp, placing the crushed bean pulp into a reaction kettle, adding 50 wt% of corn steep liquor and 25 wt% of glutamic acid residues (the emergence of the glutamic acid residues can be specifically referred to as the influence of the glutamic acid residues on the growth and development and blood biochemical indexes of Qinchuan beef cattle, 2017,44 (8): 2295-; heating to 90 ℃, and carrying out ultrasonic-assisted hydrolysis for 10min at an ultrasonic frequency of 20 kHz; continuing to hydrolyze for 6 hours, then performing microwave-assisted hydrolysis for 3 minutes with the microwave power of 300W, stopping microwave, cooling to 45 ℃, adding ammonia water, adjusting the pH to 3, then adding acid protease (the enzyme activity is 5 ten thousand U/g), the addition amount is 500U/L, performing enzymolysis for 6 hours, deactivating enzyme at 95 ℃ for 3 minutes, then filtering with a diatomite filter to remove residues, then adding 1% by weight of active carbon to decolor for 30 minutes, and filtering to remove the active carbon to obtain the soybean meal hydrolysate for fermentation.

Example 2

A process for hydrolyzing soybean meal, comprising the steps of:

crushing bean pulp, placing the crushed bean pulp in a reaction kettle, adding corn steep liquor accounting for 40% of the weight of the bean pulp and 30% of glutamic acid residue in parts by weight, adding 2mol/L citric acid at a constant speed to prepare suspension with the solid content of 40%, and homogenizing by using a high-pressure homogenizer (the pressure is 30MPa, the temperature is 32 ℃, and the time is 15 s) to refine the particle size and control the particle size to be 1-10 microns; heating to 90 ℃, and carrying out ultrasonic-assisted hydrolysis for 10min at an ultrasonic frequency of 20 kHz; continuing to hydrolyze for 5 hours, then performing microwave-assisted hydrolysis for 3 minutes with the microwave power of 300W, stopping microwave, cooling to 45 ℃, adding ammonia water, adjusting the pH to 2.5, then adding acid protease (with the enzyme activity of 5 ten thousand U/g) with the addition of 500U/L, performing enzymolysis for 7 hours, inactivating enzyme at 95 ℃ for 3 minutes, then filtering with a diatomite filter to remove residues, then adding 1% by weight of active carbon to decolorize for 30 minutes, and filtering to remove the active carbon to obtain the soybean meal hydrolysate for fermentation.

Example 3

Detection of hydrolysis Rate

1. Determination of the Total Nitrogen content

Kjeldahl method

The principle is that proteins contain nitrogen organic compounds. The food is heated and digested together with sulfuric acid and a catalyst to decompose protein, and the decomposed ammonia is combined with sulfuric acid to produce ammonium sulfate. Then alkalizing and distilling to free ammonia, absorbing with boric acid, titrating with sulfuric acid or hydrochloric acid standard solution, and multiplying the consumption of acid by a conversion coefficient to obtain the protein content.

Calculating the formula:

the nitrogen content (%) in the sample was =0.014 × (a-B) × 100%;

a- - - -the number of ml of HCl solution used for titrating a sample;

b- -ml of HCl solution used for titration of the blank;

protein content (%) in the sample, = nitrogen content in the sample/0.16;

determination of amino nitrogen content

The principle and the detection method of the formaldehyde titration method for determination are as follows:

the principle is as follows:

amino acid nitrogen is also known as ammoniacal nitrogen. The amino acid contains acidic-COOH and also contains basic-NH₂They interact to make the amino acids neutral inner salts. When adding formaldehyde solution, -NH₂In combination with formaldehyde, the basicity disappears and the-COOH becomes acidic, and the end point can be determined by titration with a sodium hydroxide standard solution and an acidimeter.

The detection method comprises the following steps:

sucking 5.0ml of sample, placing the sample in a 100ml volumetric flask, adding water to the scale mark, uniformly mixing, sucking 20.0ml of sample, placing the sample in a 200ml beaker, and adding 60ml of water. The magnetic stirrer was started and titrated with 0.05N sodium hydroxide standard solution to acidimeter indicated PH = 8.2.

Add 10ml of formaldehyde solution and mix well, titrate to PH =9.2 with 0.05N sodium hydroxide standard solution, and count the number of milliliters of 0.05N sodium hydroxide standard solution consumed.

Meanwhile, 80ml of water is taken, the pH is adjusted to 8.2 by using 0.05N sodium hydroxide standard solution, 10.0ml of formaldehyde solution is added, and the pH is adjusted to 9.2 by using 0.05N sodium hydroxide standard solution, so that a reagent blank test is carried out.

Calculating the hydrolysis rate:

hydrolysis ratio = total amount of hydrolyzed amino acids state/nitrogen content of raw material × 100.

Effect of one, different acid types on hydrolysis

Different acids are selected, including hydrochloric acid, citric acid and acetic acid, and because too high acid concentration often causes too much volatilization of the acid, the volatilized acid can cause severe corrosion of equipment, alkali liquor consumed during neutralization is increased, and finally the salt content of the product is too high to facilitate fermentation production of microorganisms; therefore, the acid concentration is 1.5mol/L, hydrolysis is carried out for 6h, the solid content of the content is 30-40%, a high-pressure homogenizer is adopted for homogenization treatment, ultrasonic treatment is adopted, as shown in figure 1, different acid types have large influence on the hydrolysis rate, the worst is acetic acid which is only 27.8%, the difference between citric acid and hydrochloric acid is not large and is respectively 36.7% and 38.1%, but the hydrochloric acid belongs to strong acid, not only can damage amino acids such as tryptophan and the like, but also can easily corrode equipment, and therefore, the citric acid is more suitable for being selected.

Secondly, determining the hydrolysis temperature of citric acid

Since temperature had a greater effect on acid hydrolysis, the effect of temperature on hydrolysis rate was studied. As shown in FIG. 2, the rate of formation of amino nitrogen increases with the increase in temperature when 1.5mol/L citric acid is added and hydrolysis is carried out at different hydrolysis temperatures for 6 hours, but the increase in amino nitrogen gradually decreases when the temperature reaches 90 ℃ because decarboxylation of amino acids and Maillard reaction of amino acids and aldehydes occur relatively easily at 90 ℃ or higher, and these reactions consume amino acids in the hydrolysate.

And thirdly, the influence of microwave time on the hydrolysis rate.

After homogenization, ultrasonic hydrolysis and citric acid hydrolysis, a plurality of groups of controls are adopted to verify the influence of microwave time on the hydrolysis rate, and the microwave enables peptide molecules to vibrate in an electromagnetic field, so that part of polypeptide structures which cannot be hydrolyzed by the citric acid become loose, sulfydryl is destroyed, and the internal structure is changed, thereby being beneficial to further enzymolysis; as shown in FIG. 3, the microwave time is controlled to be 3min, and increasing the microwave time has no influence on the hydrolysis rate, but may damage the amino acid structure, resulting in the decrease of the amino acid nutritive value.

And fourthly, influence of different enzyme types on the hydrolysis rate.

Homogenizing, performing ultrasonic treatment, performing citric acid hydrolysis, performing microwave treatment, and performing enzymolysis with different enzymes including trypsin, acid protease, alkaline protease and papain at a suitable temperature, as shown in FIG. 4, with the increase of enzyme activity, the hydrolysis rate is improved, the enzyme activity required by the acid protease group reaching the highest hydrolysis point is the least, the comprehensive cost and hydrolysis rate are considered, and the acid protease is selected for hydrolysis most suitably.

Example 4

The method for preparing the fermentation medium by using the bean pulp hydrolysate comprises the following steps:

the fermentation medium raw material was prepared at a concentration of 80g/L glucose, 200g/L soybean meal hydrolysate (prepared as in example 1), K₂HPO₄ 2g/L，MgSO₄·7H₂O 50mg/L，MnSO₄·H₂O 3mg/L，FeSO₄·7H₂O 3mg/L，VB₁10mg/L, biotin 7 mu g/L; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation culture medium.

Yeast extract as nitrogen source medium conventional in the art: 80g/L glucose, 20g/L yeast extract, K₂HPO₄2g/L，MgSO₄·7H₂O 50mg/L，MnSO₄·H₂O 3mg/L，FeSO₄·7H₂O 3 mg/L，VB₁10mg/L, biotin 7. mu.g/L.

Half of the yeast extract was replaced with soybean meal hydrolysate: 80g/L glucose, 10g/L yeast extract, 100g/L dregs hydrolysate and K₂HPO₄ 2g/L，MgSO₄·7H₂O 50mg/L，MnSO₄·H₂O 3mg/L，FeSO₄·7H₂O 3 mg/L，VB₁10mg/L, biotin 7. mu.g/L.

The fermentation process comprises the following steps: inoculating Brevibacterium flavum GDK-9 into 50L full-automatic fermentation tank containing 30L fermentation medium for fermentation culture with inoculation concentration of OD_600nmFermenting for 48h when the fermentation time is 0.8, and collecting fermentation liquor; in the whole fermentation process, the fermentation temperature is controlled to be 35 ℃, the ventilation ratio is 1: 0.6, the stirring speed is 300r/min, the dissolved oxygen is maintained at 20 percent, and the fed-batch mass percentage is concentratedGlucose with a degree of 20% maintains residual sugar not less than 1.0%, and defoaming agent is fed in for defoaming.

After fermentation is finished, detecting the concentration of thalli and the concentration of glutamic acid in fermentation liquor; the fermentation conditions of the different nitrogen source media are shown in table 1:

TABLE 1

Group of	OD_600nm	Glutamic acid yield g/L in fermentation liquor
			Hydrolysate of soybean meal	54.9	139.4
Yeast extract	49.1	126.5
			Half of yeast extract is replaced by bean pulp hydrolysate	52.8	135.3

And (4) conclusion: as shown in the above table 1, compared with the conventional yeast extract, the soybean meal hydrolysate of the present invention is used as a nitrogen source, and both the fermentation thallus concentration and the glutamic acid yield are improved to a certain extent, which indicates that the soybean meal hydrolysate prepared by the method of hydrolyzing the soybean meal, the corn steep liquor and the glutamic acid residue of the present invention is more suitable for the absorption and the utilization of microorganisms, can greatly reduce the cost of producing amino acid on a large scale, creates income for enterprises,

the foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The method for preparing the glutamic acid fermentation medium by using the bean pulp hydrolysate comprises the following steps:

taking fermentation medium raw materials, and preparing according to the following concentrations: 50-100g/L of glucose, 300g/L of soybean meal hydrolysate, K₂HPO₄ 1-3g/L，MgSO₄·7H₂O 30-80mg/L，MnSO₄·H₂O 2-5mg/L，FeSO₄·7H₂O 2-5mg/L，VB₁8-15mg/L, biotin 5-10 mug/L; stirring the raw materials uniformly, adjusting pH to 6-7, sterilizing, and naturally cooling to obtain fermentation culture medium;

the bean pulp hydrolysate is prepared by the following process:

crushing the bean pulp, placing the crushed bean pulp in a reaction kettle, adding corn steep liquor and glutamic acid residue, adding citric acid to prepare suspension with the solid content of 30-40%, and homogenizing by using a high-pressure homogenizer to refine the particle size; heating to 90 deg.C, and hydrolyzing with ultrasound for 5-20 min; continuing to hydrolyze for 5-7h, then performing microwave-assisted hydrolysis for 2-4min, stopping microwave, cooling to 45 ℃, adding ammonia water, adjusting pH to 2.5-3.5, then adding acid protease, performing enzymolysis for 6-9h, inactivating enzyme for 3min at 95 ℃, then filtering, discharging residue, then adding activated carbon for decolorization, and filtering to remove activated carbon to obtain a soybean meal hydrolysate;

the adding amount of the corn steep liquor accounts for 30-50% of the weight of the soybean meal, and the adding amount of the glutamic acid residue accounts for 20-40% of the weight of the soybean meal;

the concentration of the citric acid is 1-2 mol/L;

the parameters of the homogenization treatment are as follows: the pressure is 30MPa, the temperature is 32 ℃, and the time is 10-20 s;

the ultrasonic frequency is 20kHz, and the microwave power is 300W;

the addition amount of the acid protease is 500U/L.

2. Method according to claim 1, characterized in that it comprises the following steps: taking fermentation medium raw materials, preparing according to the following concentration, glucose 80g/L, soybean meal hydrolysate 200g/L, K₂HPO₄ 2g/L，MgSO₄·7H₂O 50mg/L，MnSO₄·H₂O 3mg/L，FeSO₄·7H₂O 3mg/L，VB₁10mg/L, biotin 7 mu g/L; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation culture medium.