CN110144312B

CN110144312B - Bacillus thermophilus for producing maltotetraamylase and application thereof

Info

Publication number: CN110144312B
Application number: CN201910440212.4A
Authority: CN
Inventors: 吴敬; 宿玲恰; 杨亚楠
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-05-24
Filing date: 2019-05-24
Publication date: 2020-06-09
Anticipated expiration: 2039-05-24
Also published as: CN110144312A

Abstract

The invention discloses bacillus thermophilus and application thereof, and belongs to the technical field of fermentation engineering. The invention obtains a thermophilic bacillus for expressing maltotetraamylase, which synthesizes maltotetraose by taking maltodextrin as a substrate and strain fermentation liquor as a catalyst. The invention takes maltodextrin with the final concentration of 20g/100mL as a substrate, the addition amount of the maltotetraamylase fermentation liquor is 40U/g maltodextrin, the enzyme reaction is carried out at the temperature of 45 ℃ and the pH value of 7.5, the reaction time is 18h, and the conversion rate of maltotetraose can reach 65.7%.

Description

Bacillus thermophilus for producing maltotetraamylase and application thereof

Technical Field

The invention relates to thermophilic bacillus for producing maltogenic tetra-amylase and application thereof, belonging to the technical field of microorganisms.

Background

The maltotetraose is a linear chain maltooligosaccharide formed by connecting 4 α -D glucose groups by α -1,4 glycosidic bonds, is a functional food, has low sweetness which is 20 percent of that of cane sugar and high viscosity which is 2-5 times that of cane sugar, can replace food with high sweetness and high heat, has good moisture retention, strong thickening effect, acid resistance, heat resistance and easy formation of glossy involucra, can be applied to the field of papermaking, is easy to digest and absorb, has low osmotic pressure and can promote a human body to take Ca²⁺Absorption; can also inhibit enterotoxin-secreting clostridium perfringens, is helpful for improving intestinal health and enhancing human immunity, and can be applied in the fields of food and medical treatment.

At present, our country has less research on the method for preparing maltotetraose. In 2013, Qianying et al, Jenerac corporation, explored the reaction pH, temperature, substrate concentration and synergistic effect with pullulanase of a novel maltotetraamylase obtained by transforming Pseudomonas saccharophila, firstly explored the reaction pH to find that the optimum pH is 5.2-5.8, then explored the reaction temperature to find that the optimum temperature is 60 ℃, and on the basis of investigating the influence of a substrate liquefaction DE value, the lower the DE value is, the higher the conversion rate is, then investigated the influence of synergistic effect with pullulanase, found that the conversion rate is increased along with the increase of the pullulanase enzyme addition amount, and finally explored the substrate concentration to find that the optimum substrate concentration is 20%, and the conversion rate of finally generating maltotetraose can reach 54%, which is the highest level reported at present.

The maltotetraamylase (EC 3.2.1.60) is one of exo-amylase, which can specifically and sequentially cut the 4 th α -1,4 glycosidic bond from the non-reducing end of starch to generate malto-oligosaccharide mainly comprising maltotetraose.

After baking, crystallisation of amylopectin occurs in the starch granules, resulting in an increase in the crumb hardness. The crystallization depends on the length of the side chain, the longer the side chain, the larger the crystallization. Maltotetraamylase enzymes cleave the side chains of amylopectin, which reduces crystallization and thus increases resistance to staleness. Effectively solves the problems of easy aging and retrogradation of starch foods and prolongs the storage life.

Disclosure of Invention

The first purpose of the invention is to provide a strain of Bacillus thermophilus (Bacillus sp.) WSH-D7.11 which is preserved in China center for type culture collection in 2019, 4 and 25 months, wherein the strain preservation number is CCTCC M2019294, the strain is classified and named as Bacillus sp.WSH-D7.11, and the preservation address is Wuhan university in China.

The second object of the present invention is to provide a microbial preparation containing the above Bacillus thermophilus WSH-D7.11.

In one embodiment of the invention, the microbial preparation contains viable count more than or equal to 10⁷CFU/g thermophilic Bacillus cells.

The third object of the present invention is to provide a method for producing maltotetraose, which comprises synthesizing maltotetraose using maltodextrin as a substrate and using the fermentation broth obtained by fermentation of Bacillus thermophilus WSH-D7.11 according to claim 1 as a catalyst.

In one embodiment of the invention, the reaction conditions of the method are that maltodextrin with the final concentration of 15-30g/100mL is used as a substrate, the enzyme adding amount of maltotetraamylase is 10-50U/g maltodextrin, the enzyme reaction is carried out at the pH of 6-9 and the temperature of 40-60 ℃, and the reaction period is 2-24 h.

In one embodiment of the invention, the reaction conditions of the process are that maltodextrin with a final concentration of 20g/100mL is used as a substrate, the enzyme addition of maltotetraamylase is 40U/g maltodextrin, and the enzyme reaction is carried out at pH 7.5 and a temperature of 45 ℃ for 18 h.

In one embodiment of the invention, the maltodextrin has a DE value of 5 to 7.

The fourth object of the present invention is to provide the use of the above Bacillus thermophilus WSH-D7.11 or the above microbial agent for preparing food containing maltotetraamylase.

The fifth purpose of the invention is to provide the application of the bacillus thermophilus WSH-D7.11 or the microbial agent in the production of the malt four-pond amylase.

The sixth purpose of the invention is to provide the application of the bacillus thermophilus WSH-D7.11 or the microbial agent in preparing the intestinal disease drugs.

The seventh purpose of the invention is to provide the application of the bacillus thermophilus WSH-D7.11 or the microbial agent in prolonging the shelf life of the bread, wherein the fermentation liquor obtained by adding the bacillus thermophilus WSH-D7.11 in the bread making process is fermented.

In one embodiment of the invention, the preparation conditions used are that all raw materials are stirred into a dough according to the bread preparation formula, a strain fermentation broth of maltotetraamylase is added, proofed for 60min at a temperature of 38 ℃ and a relative humidity of 85%, and baked for 30min at a firing temperature of 130 ℃ and a firing temperature of 190 ℃.

In one embodiment of the invention, the bread making formula comprises 190g of strong flour, 60g of pure milk, 20g of sugar, 2g of salt, 2 eggs, 4g of yeast and 10g of butter.

The invention has the advantages of

The invention provides a thermophilic bacillus for expressing maltotetraamylase, which takes maltodextrin with the final concentration of 15-30g/100mL as a substrate, the addition of the maltotetraamylase fermentation liquor is 10-50U/g maltodextrin, the enzymatic reaction is carried out at the temperature of 40-60 ℃ and the pH value is 6-9, the reaction period is 2-24h, and the yield of maltotetraose can reach 65.7 percent at most. The maltotetraose prepared by the maltotetraamylase has the advantages of wide reaction temperature range, low energy consumption and low material cost, can still obtain high substrate conversion rate under higher substrate concentration, can still normally react at a high temperature of 45 ℃, can avoid the phenomenon that a reaction system is easy to be infected with bacteria when a saccharide substance is used as a substrate, and lays a foundation for industrial amplification production.

According to the bread making formula, all the raw materials are stirred into a dough, 200ppm of malt glucoamylase fermentation liquor is added, the dough is proofed for 60min at the temperature of 38 ℃ and the relative humidity of 85%, the dough is baked for 30min at the temperature of 130 ℃ and 190 ℃ at the temperature of 190 ℃, the full texture parameters of the bread are obviously optimized, the problems that starch foods are easy to age and regenerate and the like are effectively solved, and the purpose of prolonging the shelf life is achieved.

Biological material preservation

A thermophilic Bacillus (Bacillus sp.) is classified and named as Bacillus sp WSH-D7.11, is preserved in China center for type culture collection in 2019, 4 and 25 months, and has the preservation number of CCTCC M2019294, and the preservation address is China, Wuhan university.

Drawings

FIG. 1 effect of initial pH of the enzymatic reaction on maltotetraose production;

FIG. 2 effect of enzyme reaction temperature on maltotetraose production;

FIG. 3 effect of enzyme addition on maltotetraose production;

FIG. 4 effect of substrate concentration of enzyme reaction on maltotetraose production;

FIG. 5 effect of enzyme reaction time on maltotetraose production;

FIG. 6 effect of addition of fermentation broth of strain on the overall texture of bread.

Detailed Description

(I) HPLC detection of enzymatic conversion products

Heating the sample after the enzyme reaction for 20min to inactivate the enzyme, centrifuging at 12000r/min for 10min, taking the supernatant, appropriately diluting with deionized water, coprecipitating with acetonitrile, standing for 2h, centrifuging at 12000r/min for 10min, taking the supernatant, and passing the supernatant through a membrane for later use. The HPLC detection conditions are as follows: agilent 1200HPLC chromatograph, Agilent autosampler, Column 4.6mm × 250mm × 5 μm Sbylonis Amino Column; an agent difference detector; the mobile phase is acetonitrile: ultrasonic degassing with water (70:30) for 20min at a flow rate of 0.8 mL/min; the column temperature was 35 ℃.

(II) determination of bread texture

Slicing the baked bread cooled at room temperature for 2h, slicing into 10mm uniform slices with a slicer, selecting two middle slices, overlapping, measuring the whole texture of the bread with a physical property analyzer in TPA mode, and repeating 3 times for each sample to obtain an average value. Setting parameters: the probe model P/36, the speed before the test is 3.0mm/s, the test speed is 1.0mm/s, the speed after the test is 3.0mm/s, the compression degree is 50%, the induction force is 5g, and the compression time interval is 1 s.

(III) formula of culture medium

LB seed culture medium comprises 5g/L yeast powder, 10g/L peptone 1%, and 10g/L sodium chloride.

The TB fermentation medium comprises 24g/L yeast powder, 12g/L peptone, 5g/L glycerol and 12.54g/L K₂HPO₄，2.31g/LKH₂PO₄。

Maltotetraose amylolytic enzyme screening plate: 1% of soluble starch and 1% of agar powder.

Beef extract peptone slant culture medium: 3g/L beef extract, 10g/L peptone, 5g/L NaCl, 15-25g/L agar powder and pH 7.4-7.6.

(IV) definition and determination method of enzyme activity

Definition of enzyme activity: the amount of enzyme required to produce 1. mu. mol of maltotetraose per minute was defined as 1 enzyme activity unit (U).

The method for measuring the enzyme activity comprises the following steps: the measurement system is 15 mL: 1mL of 1% soluble starch was used as a substrate, and 0.9mL of a buffer (50mM Na) was added₂HPO₄Citric acid, pH 7.0), adding 1.0mL buffer solution into blank control, keeping temperature in 55 deg.C water bath for 10min, adding 0.1mL enzyme solution, reacting for 10min, adding 3mL DNS solution, boiling water bath for 7min, cooling with ice water, diluting to 15mL, and adjusting volume at OD₅₄₀The absorbance values were measured as follows.

EXAMPLE 1 screening of strains

Collecting soil from different places (refuse dump, starch factory, vegetable market, food factory, grain processing factory, hotel, etc.) in different areas, mixing 2g of soil sample with 9mL of sterile water, adding glass beads, shaking, standing for a moment, adding 5mL of supernatant into LB culture medium, and carrying out shake culture at 25-37 ℃ and 200r/min for 2-3 d. Adding 10mL of culture solution into another fresh LB culture medium to continue shaking culture, repeating the operation twice, coating the solution on an LB solid plate, performing inverted culture at 25-37 ℃ for 2-3d, observing a single colony, and screening strains with good growth conditions.

The single colony is selected and inoculated into a high-flux shallow hole plate filled with LB seed culture medium and is cultured overnight at 37 ℃ under the shaking of 200 r/min. Then inoculating the liquid strain into a high-flux deep-hole plate filled with a TB fermentation culture medium according to the inoculation amount of 5 percent, and placing the plate at the temperature of 26-33 ℃ for shaking culture at 200r/min for 2-3 d. Centrifuging the culture solution at 12000rpm for 5min, collecting supernatant, and diluting 0.1mL of the supernatant in gradient (10)^-4，10^-5，10^-6) Spread on a maltotetraose starch hydrolase screening plate containing a plate separation medium, cultured in an incubator at 50 ℃ for 2 to 3 hours, then measured for D/D (hydrolysis loop diameter/colony diameter), and the plate with the larger D/D value is selected for further screening.

Firstly, single colony is picked to carry out plate streaking for 3-4 times, and then is inoculated into a fermentation medium to carry out liquid oscillation culture, the culture temperature is 37 ℃, the rotating speed of a shaking table is 200rpm, and the culture time is 48 h. The total amount of 7 strains are obtained, the enzyme activity of fermentation liquor of each strain is shown in table 1, the strains with relatively high enzyme activity are selected for streak subculture, and HPLC detection is carried out on products of the strains which are converted into soluble starch, and finally, the maltotetraamylase producing strain which is stable in enzyme production and has high enzyme activity is obtained.

TABLE 1 enzymatic Activity of different strains

The strain is subjected to PCR amplification of 16S rDNA, recovery of PCR amplification fragments, T vector connection and screening of recombinant vectors, the verified recombinant vectors are sent to Shanghai Biotechnology Limited company for sequencing after agarose electrophoresis detection, and the sequencing result is logged in an NCBI website and is compared with the nucleotide sequence by BLAST to be identified as the bacillus thermophilus.

The purified bacterial strain is sent to a preservation organization for preservation, the preservation number is CCTCC M2019294, the bacterial strain is named as Bacillus sp WSH-D7.11, the preservation organization is the China center for type culture preservation, and the preservation address is the university of Wuhan, Lojiashan mountain, Wuhan city.

EXAMPLE 2 preparation of maltotetraose

Using 50mmol/L, pH 6.0.0, 7.0, 7.5, 8.0, 9.0 Na₂HPO₄Maltodextrin with a final concentration of 15% (W/V) prepared from citric acid buffer, 30U/g maltodextrin added in the fermentation broth of maltotetraamylase, 5U/g pullulanase added in the fermentation broth of maltotetraamylase, a constant temperature water bath shaker at 45 ℃ and 150rpm, and reacting for 12 h. The enzymatic conversion products were detected by HPLC.

The results show that the maltotetraose generated by enzyme catalysis has obvious change along with the increase of the pH, and the yield of the maltotetraose reaches 62.9% when the pH is 7.5. An excessively high pH leads to a decrease in the yield of maltotetraose (see FIG. 1).

EXAMPLE 3 preparation of maltotetraose

Using 50mmol/L, pH 7.5.5 Na₂HPO₄Maltodextrin with the final concentration of 15% (W/V) prepared by citric acid buffer solution, 30U/g maltodextrin added in the fermentation liquor of maltotetraamylase, 5U/g pullulanase added in the fermentation liquor of maltotetraamylase, constant temperature water bath shaking tables with the temperature of 40, 45, 50, 55 and 60 ℃ respectively, 150rpm and reaction for 12 hours. The enzymatic conversion products were detected by HPLC.

The results show that the temperature has a great influence on the yield of maltotetraose, and that the yield of maltotetraose reaches 63.2% when the temperature is 45 ℃. Thereafter, the temperature was further increased and the yield of maltotetraose began to decrease (see FIG. 2). When the reaction temperature is about 45 ℃, the temperature is higher, so that a reaction system containing a large amount of sugar is prevented from being influenced by bacterial pollution, the enzyme conversion efficiency is improved, the process is simplified, and the industrial process of preparing the maltotetraose is promoted.

EXAMPLE 4 preparation of maltotetraose

Using 50mmol/L, pH 7.5.5 Na₂HPO₄Maltodextrin with 15% (W/V) final concentration prepared by citric acid buffer solution, maltodextrin with 10, 20, 30, 40, 50U/g fermentation liquor of maltotetraamylase, pullulanase with 5U/g maltodextrin, constant temperature water bath shaker at 45 deg.C, 150rpm, and reaction for 12 h. The enzymatic conversion products were detected by HPLC.

The results showed that the yield of maltotetraose increased with increasing enzyme addition, and that the yield of maltotetraose reached 64.7% when the amount of maltotetraose fermentation broth added was 40U/g maltodextrin. After that, the enzyme addition amount was further increased, and the yield of maltotetraose was slightly decreased (see FIG. 3).

EXAMPLE 5 preparation of maltotetraose

Using 50mmol/L, pH 7.5.5 Na₂HPO₄Maltodextrin with final concentration of 15, 20, 25, 30% (W/V) and prepared from citric acid buffer solution, 40U/g maltodextrin and 5U/g pullulanase of maltodextrin were added, and the mixture was reacted for 12 hours in a constant temperature water bath shaker at 45 ℃ and 150 rpm. The enzymatic conversion products were detected by HPLC.

The results showed that the yield of maltotetraose reached 65.4% at a substrate concentration of 20% during the conversion of maltotetraamylase to maltodextrin (see FIG. 4).

EXAMPLE 6 preparation of maltotetraose

Using 50mmol/L, pH 7.5.5 Na₂HPO₄Maltodextrin with 20% (W/V) final concentration prepared by citric acid buffer, 40U/g maltodextrin added in the fermentation liquid of maltotetraamylase, 5U/g pullulanase added in the fermentation liquid of maltotetraamylase, a constant temperature water bath shaker at 45 ℃ and 150rpm, and reacting for 2, 4, 8, 12, 18 and 24 hours. The enzymatic conversion products were detected by HPLC.

The results showed that the yield of maltotetraose reached 65.7% at 18h of the enzymatic reaction and 64.2% at 24h of the enzymatic reaction, with a reduced conversion. During the enzyme reaction, the rate of the enzyme-catalyzed reaction gradually increases within 2h initially, and the rate of the catalyzed reaction gradually decreases within 2h to 18h (see FIG. 5).

Example 7 application of maltotetraamylase in baking

In the above bread making method, the parameters of TPA test (full texture) after the blank control, bread to which maltotetraamylase fermentation broth was added at 100ppm, 200ppm and 400ppm, respectively, was stored at room temperature for 7 days were examined, and the hardness, gel elasticity and chewiness of bread to which 200ppm of enzyme was added were significantly lower than those of the blank control and bread to which 100ppm of enzyme was added, similar to those of the blank control and bread to which 400ppm of enzyme was added, much better in elasticity than those of the other three, and similar to the blank control in cohesion and elasticity, and overall, the texture of bread to which 200ppm of enzyme was added was much better than that of bread to which 100ppm of enzyme was added, and similar to that of bread to which 400ppm of enzyme was added (see FIG. 6).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A strain of Bacillus thermophilus (Bacillus sp.) WSH-D7.11 is preserved in China center for type culture Collection in 2019, 4 and 25 months, with the preservation number of CCTCC NO: M2019294 and the preservation address of China, Wuhan university.

2. A microbial preparation comprising Bacillus thermophilus WSH-D7.11 according to claim 1.

3. The microbial agent according to claim 2, wherein the microbial agent contains viable count of 10 or more⁷CFU/g thermophilic Bacillus cells.

4. A process for producing maltotetraose, which comprises synthesizing maltotetraose using maltodextrin as a substrate and the fermentation broth obtained by fermentation with Bacillus thermophilus WSH-D7.11 according to claim 1 as a catalyst.

5. The process according to claim 4, wherein the reaction conditions are such that a final concentration of maltodextrin in the range of 15-30g/100mL is used as a substrate, the amount of enzyme added to the fermentation broth obtained by fermentation of Bacillus thermophilus WSH-D7.11 according to claim 1 is 10-50U/g maltodextrin, and the enzymatic reaction is carried out at a pH of 6-9 and a temperature of 40-60 ℃ for a reaction period of 2-24 hours.

6. The process according to claim 5, wherein the maltodextrin has a DE value of 5 to 7.

7. Use of a thermophilic bacillus sp WSH-D7.11 according to claim 1 or a microbial inoculant according to claim 2 or 3 for the preparation of a food product comprising maltotetraamylase.

8. Use of the Bacillus thermophilus WSH-D7.11 according to claim 1 or the microbial inoculant according to claim 2 or 3 for the production of maltotetraamylase.

9. Use of a thermophilic bacillus sp WSH-D7.11 according to claim 1 or a microbial agent according to claim 2 or 3 for extending the shelf life of bread.