阅读说明：本技术 一种β-半乳糖苷酶的制备方法及其应用 (Preparation method and application of beta-galactosidase ) 是由 魏远安 曾宪维 谢志龙 杨新球 陈子健 浣金刚 于 2020-07-28 设计创作，主要内容包括：本发明属于生物化工技术领域,公开了一种β-半乳糖苷酶的制备方法,包括以下步骤：(1)将环状芽孢杆菌接种至LB培养基中培养活化,然后接种至种子罐中培养,得到种子培养液；(2)将种子培养液加入含有发酵培养基的发酵罐中发酵,得到环状芽孢杆菌发酵液；(3)将环状芽孢杆菌发酵液过滤或离心,去除菌体,得到β-半乳糖苷酶液；该发酵培养基含有乳糖、半乳糖、植物蛋白胨、玉米粉、酵母提取物、磷酸盐、碳酸盐和水。该方法的发酵时间短,缩短了生产周期,产生的酶液的比活力高。本发明还提供不使用交联剂制备β-半乳糖苷酶的固定化酶的方法,制得的固定化酶稳定性好,可连续使用264h以上,用该固定化酶制备半乳糖的成本大大降低。(The invention belongs to the technical field of biochemical engineering, and discloses a preparation method of beta-galactosidase, which comprises the following steps: (1) inoculating the bacillus circulans into an LB culture medium for culture activation, and then inoculating into a seeding tank for culture to obtain a seed culture solution; (2) adding the seed culture solution into a fermentation tank containing a fermentation medium for fermentation to obtain a bacillus circulans fermentation solution; (3) filtering or centrifuging the bacillus circulans fermentation liquor, and removing thalli to obtain beta-galactosidase liquor; the fermentation medium contains lactose, galactose, phytone, corn flour, yeast extract, phosphate, carbonate and water. The method has short fermentation time, shortened production period, and high specific activity of the enzyme solution. The invention also provides a method for preparing the immobilized enzyme of the beta-galactosidase without using a cross-linking agent, the prepared immobilized enzyme has good stability and can be continuously used for more than 264 hours, and the cost for preparing the galactose by using the immobilized enzyme is greatly reduced.)

1. A preparation method of beta-galactosidase is characterized by comprising the following steps:

(1) inoculating the bacillus circulans into an LB culture medium for culture activation, and then inoculating into a seeding tank for culture to obtain a seed culture solution;

(2) adding the seed culture solution prepared in the step (1) into a fermentation tank containing a fermentation medium for fermentation to obtain a bacillus circulans fermentation solution;

(3) filtering or centrifuging the bacillus circulans fermentation liquor obtained in the step (2), and removing thalli to obtain beta-galactosidase liquor;

the fermentation medium contains lactose, galactose, phytone, corn flour, yeast extract, phosphate, carbonate and water.

2. The method according to claim 1, wherein the Bacillus circulans used in step (1) is Bacillus circulans CCTCC NO: m2015424, preserved in China center for type culture Collection.

3. The process according to claim 1, wherein the fermentation medium in the step (2) contains lactose 5-30g/L, galactose 5-10g/L, phytone 10-30g/L, corn flour 2-5g/L, yeast extract 2-4g/L, phosphate 2-4g/L, carbonate 1-2g/L, and water in balance.

4. The method according to claim 1, wherein the fermentation conditions in the fermenter in the step (2) are fermentation at pH 5.5-8.0 for 24-35 h.

5. The method according to claim 4, wherein the fermentation conditions in the fermenter in the step (2) are 0 to 8 hours of fermentation, pH 6.5 to 7.0; the pH after 8h is 7.2-7.6.

6. A preparation method of immobilized beta-galactosidase enzyme is characterized by comprising the following steps:

preparing a beta-galactosidase solution according to the preparation method of any one of claims 1 to 5;

mixing ion exchange resin with the enzyme solution for reaction to obtain the immobilized beta-galactosidase enzyme.

7. The method according to claim 6, wherein the ion exchange resin contains a tertiary amine group; the dosage ratio of the ion exchange resin to the beta-galactosidase solution is 1 g: (70-140) U.

8. The method according to claim 6, wherein the reaction is carried out under a condition of 4 to 20ms/cm in electric conductivity.

9. A preparation method of galactooligosaccharide is characterized by comprising the following steps:

preparing an immobilized beta-galactosidase enzyme according to the production method of any one of claims 6 to 8; and mixing the immobilized enzyme and lactose, and stirring for reaction to obtain the galactooligosaccharide.

10. The preparation method according to claim 9, wherein the amount ratio of immobilized beta-galactosidase to lactose is 3000-25000U of immobilized beta-galactosidase per kilogram of lactose; the lactose is selected from at least one of food grade lactose, pharmaceutical grade lactose or whey filtrate powder.

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a preparation method and application of beta-galactosidase.

Background

Galacto-oligosaccharides (GOS) are a class of naturally occurring oligosaccharides, typically having a molecular structure in which 1-7 galactosyl groups are attached to a galactose or glucose molecule. It is found in trace amounts in animal milk and in higher amounts in breast milk. The galacto-oligosaccharide has strong stability, even under the acidic condition, is not digested by human digestive enzyme, is rarely digested and absorbed by human small intestine, has good bifidobacterium proliferation activity, inhibits the growth of harmful pathogenic bacteria and putrefying bacteria, promotes the absorption of calcium, magnesium and potassium, and can achieve the effect of relaxing bowel due to the property of water-soluble dietary fiber. Therefore, galactooligosaccharides are widely used in the food field, for example, galactooligosaccharides are added to infant formulas as a nutritional supplement.

The galacto-oligosaccharide is prepared from lactose (food grade lactose, pharmaceutical grade lactose, organic lactose, whey filtrate powder, whey liquid, etc.) as raw material by catalyzing with beta-galactosidase (EC 3.2.1.23). Beta-galactosidase is widely found in nature, and is contained in fruits such as apple and apricot, and is also available as microorganisms such as bacteria, yeast, and mold. Because the beta-galactosidase obtained from the microorganism has high yield, short production period and low production cost, the microorganism is often used for large-scale production of the beta-galactosidase.

Galactooligosaccharides are generally produced by a method in which free cells and an enzyme solution are directly used for lactose or lactose is subjected to symbiotic fermentation with microorganisms. However, these methods introduce impurities such as free cells or proteins, even secondary metabolites, and require complicated separation and purification processes, and the utilization rate of the enzyme is not high, which is not favorable for clean production and cost control. In order to solve the problem, the enzyme production performance of beta-galactosidase and the use method of the beta-galactosidase need to be optimized. The enzyme is immobilized and can be reused, which becomes the solution direction for large-scale production of galactooligosaccharides. However, while it is often necessary to use glutaraldehyde or the like as a cross-linking agent for the immobilized enzyme carrier in the process of immobilizing the enzyme, the use of a cross-linking agent is sometimes limited, for example, the use of glutaraldehyde is limited in the process of producing a nutrition enhancer oligosaccharide. In addition, the method of symbiotic fermentation of lactose and microorganisms is often limited, and genetically modified strains cannot be used particularly for infant formula foods, organic foods and the like. In addition, in the prior art, the usage of immobilized beta-galactosidase is only 8 batches, i.e. the number of times of cyclic usage is only 8 (patent application No. 201480049114.3), and the utilization rate of the enzyme is not high. In the prior art, the fermentation period for directly producing beta-galactosidase by microbial fermentation is long, and the fermentation process is complex.

Therefore, it is desirable to provide a new method for preparing beta-galactosidase immobilized enzyme, and the immobilized enzyme prepared by the method has good stability, can be recycled for many times, and improves the utilization rate of the enzyme. In addition, it is also desirable to provide a novel method for preparing beta-galactosidase, which can shorten the fermentation period and simplify the fermentation process, and the immobilized enzyme prepared from the enzyme solution produced by fermentation by the process has better service performance.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, one of the objectives of the present invention is to provide a method for producing beta-galactosidase by using bacillus circulans (bacillus circulans), which can simplify the fermentation process and shorten the fermentation production period.

The invention also aims to provide a preparation method of the immobilized beta-galactosidase enzyme, which does not need to use a cross-linking agent, avoids the influence of the residual cross-linking agent on the food safety, and has good stability and the number of times of recycling exceeds 10.

The invention also aims to provide a preparation method of galacto-oligosaccharide, which is characterized in that the immobilized enzyme of the beta-galactosidase prepared by the invention catalyzes lactose reaction to produce galacto-oligosaccharide.

A preparation method of beta-galactosidase comprises the following steps:

(1) inoculating the bacillus circulans into an LB culture medium for culture activation, and then inoculating into a seeding tank for culture to obtain a seed culture solution;

(2) adding the seed culture solution prepared in the step (1) into a fermentation tank containing a fermentation medium for fermentation to obtain a bacillus circulans fermentation solution;

(3) filtering or centrifuging the bacillus circulans fermentation liquor obtained in the step (2), and removing thalli to obtain beta-galactosidase liquor;

the fermentation medium contains lactose, galactose, phytone, corn flour, yeast extract, phosphate, carbonate and water.

Preferably, the Bacillus circulans used in step (1) is Bacillus circulans (Bacillus circulans) purchased from American Type Culture Collection (ATCC) under No. 31382; further preferably, the bacillus circulans used in the step (1) is bacillus circulans CCTCC NO: m2015424, preserved in China center for type culture Collection. Bacillus circulans CCTCC NO: m2015424 was induced by Bacillus circulans ATCC No.31382 with UV light and lithium chloride.

Preferably, the LB medium in step (1) contains 8-15g/L peptone, 2-8g/L yeast extract and 2-8g/L sodium chloride.

Further preferably, the LB medium in step (1) contains 10-12g/L peptone, 4-6g/L yeast extract and 4-6g/L sodium chloride.

Most preferably, the LB medium in step (1) contains about 10g/L peptone, about 5g/L yeast extract and about 5g/L sodium chloride.

Preferably, the activation of the culture in step (1) is carried out at 36 to 38 ℃ for 16 to 24 hours.

Preferably, the seeding into the seed tank in step (1) is cultured at 36-38 ℃ for 5-7 hours.

Preferably, after the activation of the culture in the step (1) and before the inoculation, the method further comprises the following steps: adding the culture activated bacillus circulans into 20-500mL LB culture medium for shake culture for 5-8 hours, and then inoculating.

Preferably, the corn flour is corn steep liquor dry powder.

Preferably, the fermentation medium in the step (2) contains 5-30g/L of lactose, 5-10g/L of galactose, 10-30g/L of phytone, 2-5g/L of corn steep liquor dry powder, 2-4g/L of yeast extract, 2-4g/L of phosphate, 1-2g/L of carbonate and the balance of water.

Preferably, the fermentation medium also contains an antifoaming agent, and the content is 0.5-2 g/L.

Preferably, the fermentation medium contains lactose in an amount of 8-15 g/L.

Preferably, the fermentation conditions in the fermentation tank in the step (2) are fermentation at pH 5.5-8.0 for 24-35 h.

Preferably, the fermentation conditions in the fermentation tank in the step (2) are that the fermentation temperature is 36-38 ℃, the pH is 6.0-7.6, the aeration (air) amount is 80-160L/min, and the stirring speed is 120-220 rpm.

Further preferably, the fermentation conditions in the fermentation tank in the step (2) are 0-8h of fermentation, and the pH value is 6.5-7.0; the pH value is 7.2-7.6 after 8 hours; even more preferably, the pH after 8h is between 7.3 and 7.5.

Preferably, the filtration in the step (3) is microfiltration or ceramic membrane filtration, and the microorganisms in the fermentation liquor can be removed through microfiltration, meanwhile, the introduction of mixed bacteria in the operation process can be reduced, and the cleanness and sanitation of the enzyme liquor are ensured.

Preferably, the filtration in step (3) is performed using a microfiltration membrane, and more preferably, the microfiltration membrane is a 0.2-0.8 μm microfiltration membrane.

Preferably, the centrifugation in step (3) is performed by using a disc centrifuge.

Preferably, after the beta-galactosidase solution obtained in the step (3) is obtained, the method further comprises a concentration treatment, wherein the concentration treatment is to salt out and purify the obtained beta-galactosidase solution by using 10-35% (mass fraction) of ammonium sulfate or sodium chloride, and then concentrate the beta-galactosidase solution by using a 10-100KD membrane. The treatment can ensure that the electric conductivity of the beta-galactosidase solution is 4-20ms/cm and the enzyme activity is 80-120U/mL.

Preferably, the phosphate is at least one selected from the group consisting of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, calcium hydrogen phosphate, calcium pyrophosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acid pyrophosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and sodium pyrophosphate.

Preferably, the carbonate is at least one of sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate.

Preferably, the plant peptone is soybean peptone.

Preferably, the antifoaming agent is selected from at least one of calcium, aluminum, magnesium soaps or fatty alcohols of lauric acid, palmitic acid, fatty glycerides, stearic acid and palmitic acid.

The invention also relates to a preparation method of the immobilized beta-galactosidase, which comprises the following steps:

mixing the ion exchange resin with the beta-galactosidase solution for reaction to obtain the immobilized beta-galactosidase enzyme.

Preferably, the ion exchange resin contains tertiary amine groups.

Preferably, the ion exchange resin is a styrene type weak base anion exchange resin; further preferably, the ion exchange resin is a macroporous styrene weak base anion exchange resin with tertiary amino functional groups.

Preferably, the dosage ratio of the ion exchange resin to the beta-galactosidase solution is 1 g: (70-140) U.

Preferably, the temperature of the reaction is 6-35 ℃; further preferably, the temperature of the reaction is 8-25 ℃; even more preferably, the temperature of the reaction is between 8 and 20 ℃.

Preferably, the reaction time is 16-24 h.

Preferably, the ion exchange resin is soaked in water for 2-5h before use to obtain the activated resin. This step is to keep the ion exchange resin wet and avoid the change of the ion exchange resin structure caused by over-drying of the ion exchange resin.

Preferably, the water is deionized water.

Preferably, the reaction is carried out under conditions of a conductance of 4-20ms/cm (i.e., the conductance of the β -galactosidase solution is adjusted to 4-20 ms/cm).

Most preferably, the invention provides a preparation method of immobilized beta-galactosidase enzyme, which comprises the following steps:

soaking the ion exchange resin in deionized water for 2-5h to obtain activated ion exchange resin, and mixing the ion exchange resin with beta-galactosidase solution according to the dosage ratio of 1 g: (70-140) U, stirring under the condition of electric conductance of 4-20ms/cm, and reacting for 16-24h to obtain the immobilized beta-galactosidase.

In another aspect, the present invention also relates to a method for preparing galactooligosaccharide, comprising the steps of:

mixing immobilized beta-galactosidase enzyme and lactose, stirring and reacting to obtain galacto-oligosaccharide.

Preferably, the dosage ratio of the immobilized beta-galactosidase to the lactose is that 3000-25000U of immobilized beta-galactosidase is added to each kilogram of lactose.

Preferably, the lactose is lactose solution, and the mass concentration of the lactose solution is 35-55%.

Preferably, the reaction temperature is 45-60 ℃, and the reaction time is 6-35 h.

Preferably, the lactose is selected from at least one of food grade lactose, pharmaceutical grade lactose or whey filter powder (food grade lactose, pharmaceutical grade lactose or whey filter powder are conventional commercially available products).

Compared with food-grade lactose and medical-grade lactose, the whey filtrate powder has higher salt content and contains more nutrients such as B vitamins, proteins and the like.

Compared with the prior art, the invention has the following beneficial effects:

(1) the method has the greatest advantages that the problems of the process and the cost for preparing the galactooligosaccharides in a large scale are solved, the fermentation time for fermenting and producing the beta-galactosidase is short due to the use of the fermentation culture medium, the production period is shortened, the fermentation mode is simple, the fed-batch induction is not needed, the enzyme activity is favorably improved in the later stage of fermentation in the prior art, the personnel burden and the equipment investment are increased due to batch ingredients, and the hidden danger of bacterial contamination exists.

(2) The strain used for producing the beta-galactosidase by fermentation is a non-genetically modified strain, is safe and reliable, and the enzyme liquid produced by the strain in the fermentation process has higher specific activity, so that the use amount of resin can be reduced during the production of immobilized enzyme, and better performance of converting lactose into GOS can be shown after immobilization.

(3) The preparation process of the immobilized beta-galactosidase enzyme does not need chemical reagents such as a cross-linking agent and the like, so that the influence of the residual cross-linking agent on the food safety is avoided, the prepared immobilized enzyme has good stability, can be recycled for more than 10 times or continuously used for more than 264 hours, and the most direct influence on the preparation of galactooligosaccharides is that the cost of using the enzyme is reduced.

Drawings

FIG. 1 is a graph showing the comparison of the enzyme activity and the fermentation time of the fermentation solutions in the three cases of the fermentation tank A, the fermentation tank B and the fermentation tank C in example 6.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Several concepts describing the present invention are described below.

Definition of beta-galactosidase enzyme activity: 1mL of a beta-galactosidase solution was added to 1mL of a 0.1M phosphate buffer (pH6.0) containing ONPG (i.e., o-nitrophenyl beta-D-galactopyranoside, which can be hydrolyzed by beta-galactosidase to galactose and yellow o-nitrophenol, and thus the activity of beta-galactosidase can be detected by a change in the color of the culture broth) at a concentration of 4mg/mL, and the reaction was stopped at 50 ℃ by adding 2mL of 10% (mass fraction) sodium carbonate solution, and the amount of hydrolysis of ONPG was calculated by measuring the o-nitrophenol content in the product. The amount of enzyme required to hydrolyze 1. mu.M ONPG per minute under these conditions is defined as one unit of enzyme activity (U).

Content determination standard of galactooligosaccharide: refer to AOAC office Method 2001.02 Determination of trans-Galacto-oligosaccharides (TGOS) in Selected food products.