CN115896139A - Beta-galactosidase and application thereof - Google Patents
Beta-galactosidase and application thereof Download PDFInfo
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- CN115896139A CN115896139A CN202210813084.5A CN202210813084A CN115896139A CN 115896139 A CN115896139 A CN 115896139A CN 202210813084 A CN202210813084 A CN 202210813084A CN 115896139 A CN115896139 A CN 115896139A
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- galactosidase
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- trehalose
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Abstract
The invention relates to beta-galactosidase and application thereof, wherein the beta-galactosidase is derived from Lactobacillus fermentum9-4, the gene of the beta-galactosidase comprises a gene for coding an alpha subunit and a gene for coding a beta subunit, the nucleotide sequence of the gene for coding the alpha subunit is shown as SEQ ID No.1, and the nucleotide sequence of the gene for coding the beta subunit is shown as SEQ ID No. 2. The beta-galactosidase coded by the beta-galactosidase gene consists of an alpha subunit and a beta subunit, wherein the amino acid sequence of the alpha subunit of the beta-galactosidase is shown as SEQ ID NO.3, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 4. The beta-galactosidase of the invention has high transglycosidic activity, can be applied to the enzymatic production of galactosyl oligosaccharide, and particularly the application of the beta-galactosidase in the synthesis of galactosyl trehalose.
Description
Technical Field
The invention relates to beta-galactosidase and application thereof, in particular to beta-galactosidase derived from lactobacillus fermentum9-4 and application thereof in synthesizing galacto-oligosaccharide and galactosyl trehalose.
Background
Beta-galactosidase is a kind of characteristic which can hydrolyze beta-D- (1-4) glycosidic bond of beta-D-galactoside compounds, and is commonly used for hydrolyzing lactose to produce galactose and glucose. The enzyme is a functional enzyme widely existing in microorganisms, plants and animal organs, and is commonly used for dairy product processing. In addition to this, their transglycosidic properties are also commonly used in the industrial production of galactosyl oligosaccharides.
Galacto-oligosaccharides GOS are one of the most common galacto-oligosaccharides naturally present in animal whey and milk, and are the only oligosaccharides that can be utilized by 8 large beneficial bacteria such as bifidobacterium, lactobacillus, etc. in the intestine of a human body. The galacto-oligosaccharide has low sweetness which is only 20-40% of sucrose, low calorific value, better heat resistance and acid resistance, higher stability in the digestive tract of a human body and higher application value. The method for catalytically synthesizing galacto-oligosaccharide by beta-galactosidase has the advantages of simple reaction path, no need of other auxiliary factors, lower cost of lactose as a substrate, simple reaction by-products and the like, thereby arousing wide interest of people.
Galactosyl trehalose GTO is a trehalose derivative, wherein one or more galactose is connected with one trehalose to form an oligosaccharide, and the galactose and the trehalose are mainly connected by beta- (1-4) and beta- (1-6) glycosidic bonds. Galactosyl trehalose as a derivative of trehalose has resistance to hydrolysis of trehalase due to the change of the chemical structure of trehalose, is not easily decomposed into glucose in human intestinal tracts, has the characteristics of a low-calorie sweetener, retains the functional characteristics of trehalose, and is a novel oligosaccharide with great development potential. Galactosyl trehalose shows significantly enhanced properties in terms of hygroscopicity, caries resistance, promotion of growth of bifidobacteria and cryoprotective effect, compared with trehalose.
The synthesis method of galactosyl trehalose includes chemical synthesis method and enzyme synthesis method. In chemical synthesis methods, it is currently difficult to achieve high efficiency of chemical synthesis of trehalose derivatives due to the difficulty of obtaining C2 symmetry or achieving strict control of the α - (1, 1) - α isomer, and although some pure chemical methods have been developed for the de-symmetrization of trehalose or for the manipulation of regioselective hydroxyl groups, these methods are generally long, inefficient and low yielding. In addition, the heavy metal catalyst and chemical reagent used in the chemical synthesis have toxic action, which is not beneficial to the development and production of food. The galactosyl trehalose is synthesized by an enzyme method, and particularly by using the enzyme from GRAS bacteria, the method is more environment-friendly, high in efficiency and simple in by-product.
Although the enzyme synthesis can produce galactosyl trehalose with high efficiency, the research on the production of galactosyl trehalose is less, and the related characteristics of the production of galactosyl trehalose by using beta-galactosidase derived from lactic acid bacteria are not reported. The method for synthesizing the galactosyl trehalose by the enzyme method can effectively improve the production efficiency of the galactosyl trehalose so as to improve the application of the galactosyl trehalose in food production.
Disclosure of Invention
The invention aims to provide beta-galactosidase and application thereof in producing galacto-oligosaccharides and galactosyl trehalose.
In order to achieve the purpose, the technical scheme of the invention is as follows: a gene of beta-galactosidase which can be used for producing galactosyltransferase, is derived from Lactobacillus fermentum9-4, and consists of a gene for coding an alpha subunit and a gene for coding a beta subunit, wherein the nucleotide sequence of the gene for coding the alpha subunit is shown in SEQ ID NO.1, and the nucleotide sequence of the gene for coding the beta subunit is shown in SEQ ID NO. 2.
The beta-galactosidase coded by the gene consists of an alpha subunit and a beta subunit, wherein the amino acid sequence of the alpha subunit of the beta-galactosidase is shown as SEQ ID No.3, and the amino acid sequence of the beta subunit is shown as SEQ ID No. 4.
The preparation method of the beta-galactosidase comprises the step of introducing the nucleic acid molecule into escherichia coli BL21 (DE 3) for induction expression to obtain the beta-galactosidase.
The optimum temperature of the beta-galactosidase is 45 ℃, the optimum pH value is 7.0, the specific activity of hydrolyzed oNPG is 197.42U/mg, and the beta-galactosidase has transglycosidic activity.
The method for producing galactosyl trehalose and galactooligosaccharides by using the beta-galactosidase comprises the following steps:
a. providing a mixture comprising: a galactose donor comprising a galactosyl group bound to a leaving group, the galactose donor being lactose, a galactose acceptor, the galactosyl acceptor being trehalose or lactose;
b. said beta-galactosidase, which enzyme has transglycosidic activity, said enzyme being contacted with the mixture;
c. incubating the mixture and enzyme, adding the enzyme of step b to the donor and acceptor mixture of step a to catalyze the transglycosylation reaction, under the following reaction conditions: adding enzyme amount: 10U/mL, reaction time 12h, reaction temperature: preparing a mixture of the galactosyl trehalose or galacto-oligosaccharide and the substrate at 30 ℃.
d. If the reaction acceptor is trehalose, adding 20U/mL trehalase into the product mixture, and reacting at 40 ℃ for 12h to remove the remaining trehalose from the substrate;
e. passing through a 200 mesh silica gel column 30X 400mm, eluting with n-butanol: isopropyl alcohol: acetic acid: water =7, as eluent, 4;
f. the sample eluted in step e was subjected to TLC detection using 10 μ L of capillary spotting with activated silica gel aluminum plate, n-butanol: isopropyl alcohol: acetic acid: developing in a chromatographic cylinder by using water = 7;
g. and (3) putting the sample into a rotary evaporation instrument, performing rotary evaporation concentration at 60 ℃ and 60rpm to 10mL to remove organic reagents, and freeze-drying the concentrated sample to obtain solid powder, wherein the solid powder is the obtained pure galactosyl trehalose or galacto-oligosaccharide.
The leaving group of the galactosyl donor of step a is a glycosyl group.
The content of the galactosyl donor in the step a is not less than 25 percent (W/V), and the content of the galactosyl acceptor is not less than 25 percent (W/V).
The application of the beta-galactosidase in the synthesis of galactosyl trehalose.
The beta-galactosidase is applied to the enzymatic production of galactosyl oligosaccharide.
Compared with the prior art, the invention has the following advantages and effects:
(1) The galactosyl trehalose is prepared by using beta-galactosidase from lactobacillus fermentum9-4 for the first time.
(2) The method for synthesizing the galactosyl trehalose by the enzyme method is established by utilizing the beta-galactosidase from the lactobacillus fermentum9-4, so that the production efficiency of the galactosyl trehalose is effectively improved, and the method is suitable for the requirements of the galactosyl trehalose on food production.
(3) Firstly, the method proposes that the specific trehalase is used for hydrolyzing the trehalose substrates mixed in the galactosyl trehalose products, improves the purification efficiency and is beneficial to reducing the industrial production cost.
Drawings
FIG. 1 is a SDS-PAGE pattern of beta-galactosidase. Lane 1 in FIG. 1 is a crude enzyme; lane 2 shows the purified enzyme.
FIG. 2 is a graph showing the effect of temperature on β -galactosidase. The optimal temperature diagram for a LacLM in fig. 2; b is a temperature tolerance profile for LacLM.
FIG. 3 is a graph showing the effect of pH on β -galactosidase. FIG. 3 is the pH optimum of A LacLM; b is the pH tolerance profile of LacLM.
FIG. 4 shows the product galactooligosaccharide by TLC and HPLC analysis. In FIG. 4, A is a TLC chart, lactose as a substrate in lane 1 and a product in lane 2; and B is an HPLC chart.
FIG. 5 shows the TLC analysis of galactosyl trehalose produced by the reaction. Lane 1 shows the substrates lactose and trehalose; lane 2 is the product.
Figure 6 is a TLC analysis of purified galactosyl trehalose. Lane 1 is the product of the enzymolysis of trehalase; 2. lane 3 is the product purified on a silica gel column.
FIG. 7 is a one-dimensional nuclear magnetic resonance of galactosyl trehaloseAnd (4) vibration spectrum. In FIG. 7, A and B are 1 H, spectrogram; C. d is 1 And C, spectrum.
FIG. 8 shows two-dimensional NMR spectra of galactosyl trehalose. A in FIG. 8 is a H-H COSY spectrum; b is HSQC spectrogram; c is HMBC spectrogram.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to examples.
The present example provides a method for preparing beta-galactosidase and producing galactosyl trehalose, which utilizes beta-galactosidase with transglycosylation activity to react with lactose and trehalose mixture, wherein the lactose is used as a donor to provide galactosyl, and the trehalose is used as an acceptor to combine with the galactosyl.
Example 1
Preparation and purification of beta-galactosidase
(1) Adopting the codon preference of escherichia coli genome, carrying out codon optimization on beta-galactosidase gene from Lactobacillus fermentum9-4, and respectively showing the gene sequences of a beta-galactosidase large subunit LacL and a small subunit LacM as a sequence 3 and a sequence 4 in a sequence table after optimization; the optimized gene is entrusted to Shanghai biological engineering Co., ltd for chemical synthesis;
(2) The optimized and synthesized gene is integrated into an Escherichia coli expression vector pET-22b (+) and transformed into a competent cell BL21 (DE 3). The strain was spread and inoculated on ampicillin-resistant LB plates to a final concentration of 100. Mu.g/mL, and cultured overnight at 37 ℃;
(3) Selecting single colony, inoculating into finger-shaped bottle containing LBA liquid culture medium, shake culturing at 37 deg.C and 220rpm for 4 hr to make OD 600 =1.0; transferring the whole liquid as seed solution into 800mL LBA liquid culture medium, performing amplification culture at 37 deg.C and 220rpm for 4 hr to make OD 600 =0.4-0.6, adding IPTG inducer with final concentration of 0.3mM, and culturing at 37 ℃ and 220rpm for 4h;
(4) And (3) centrifuging the bacterial liquid for inducing expression for 4h at 4 ℃ and 5000rpm for 30min, pouring out the culture medium, and collecting thalli precipitates. And the somatic cells were resuspended in 50mM phosphate buffer pH = 7.4; adding 1% of 10% Tritiox-100 to the resuspension solution, and then sonicating the cells. The working parameters of the ultrasonic cell disruptor were set as follows: the ultrasonic power is 40%, the ultrasonic working time is 2s, the intermittence time is 2s, the ultrasonic crushing is 45min, the crushed liquid is centrifuged for 30min at the temperature of 4 ℃ and the rpm of 12000rpm, the cell debris is discarded, the supernatant fluid is the crude enzyme liquid of the beta-galactosidase, and the crude enzyme liquid is stored in a refrigerator at the temperature of-20 ℃ after passing through a filter membrane of 0.22 mu m.
(5) Purifying the crude enzyme solution of beta-galactosidase by using an AKTA protein purification instrument and a nickel ion metal chelate chromatography method; the crude enzyme is filtered by a 0.22 mu m filter, mixed with a binding buffer solution, loaded, and sequentially subjected to gradient elution by an elution buffer solution containing 20-500mM imidazole, and an eluent corresponding to the peak appearance is collected and subjected to SDS-PAGE verification.
The purification results are shown in figure 1: marker;1: coarse LacLM enzyme; 2: lacLM purified enzyme. It can be seen that two bands appear in 35-48kDa and 63-75kDa of purified LacLM, because LacLM has two subunits of LacL and LacM, and its theoretical molecular weights are 72.26kDa and 35.25kDa, respectively, and its apparent molecular weight corresponds to theoretical molecular weight.
Example 2
Determination of the enzymatic Properties of beta-galactosidase
(1) Optimum temperature and temperature tolerance of beta-galactosidase
Optimum temperature: mixing 10 μ L of the enzyme solution with 90 μ L of 0.02M oNPG with pH =7.4, reacting at 30-65 deg.C with a gradient of 5 deg.C for 10min, and immediately adding 100 μ L of ice-cold 1M NaCO 3 Stopping the reaction by the solution, measuring the light absorption value at 420nm, calculating the enzyme activity, calculating the relative enzyme activity of each measurement group by taking the highest enzyme activity of the measurement result as 100%, and setting 3 parallel enzymes at each temperature. As shown in FIG. 2A, the optimum temperature of LacLM was 45 ℃.
Temperature resistance: 10 mu L of properly diluted enzyme solution is respectively kept at 20-60 ℃ for 1h, a gradient is set at every 5 ℃, the enzyme solution is mixed with 90 mu L of 0.02M oNPG with pH =7.4, the enzyme activity is measured after the reaction is carried out for 10min at 45 ℃, the enzyme activity of the untreated enzyme is taken as 100%, the relative enzyme activity of each group is calculated, and 3 parallel enzymes are set at each temperature. The results are shown in fig. 2B, the enzyme activity of LacLM can be stably maintained at more than 98% at 20-30 ℃, the enzyme activity rapidly decreases at 30-50 ℃, and the LacLM is completely inactivated until 55 ℃.
(2) Optimum pH and pH tolerance of beta-galactosidase
Optimum pH: 0.02M of oNPG solution with pH values of 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5 and 11 is prepared respectively, 10 mu L of properly diluted enzyme solution is taken to be fully mixed with 90 mu L of oNPG solution with different pH values, the enzyme activity is measured after the reaction is carried out for 10min at 45 ℃, the relative enzyme activity of each measurement group is calculated by taking the highest enzyme activity of the measurement result as 100 percent, and 3 parallels are set for each pH value. As shown in FIG. 3A, lacLM was found to have an optimum pH of 7.0, and to maintain 80% or more of the enzyme activity in the reaction at pH6.0 to 7.5.
pH tolerance: respectively preparing buffer solutions with different pH values, respectively diluting the enzyme solution with the buffer solutions, storing at 4 ℃ for 1h, then taking 10 mu L of diluted enzyme solution, reacting at 45 ℃ for 10min, and then measuring the enzyme activity, taking the enzyme activity of untreated enzyme as 100%, calculating the relative enzyme activity of each measurement group, and setting each pH value to be 3 parallels. As shown in FIG. 3B, lacLM can maintain over 50% of its activity at pH5.5-9.5, and has a wide range of pH tolerance. Can maintain more than 80% of activity in the pH range of 6.0-9.0.
Example 3
Method for producing galacto-oligosaccharide by beta-galactosidase enzyme method
Adding beta-galactosidase to 50% (W/V) lactose to perform catalytic reaction, wherein the reaction conditions are as follows: adding enzyme amount: 10U/mL, reaction temperature: 30 ℃, reaction time: 12h, a reaction mixture containing galacto-oligosaccharides was obtained.
The reaction product was diluted moderately and checked by TLC, 10 μ L was spotted by capillary on an activated silica gel aluminum plate, and the mixture was diluted with n-butanol: isopropyl alcohol: acetic acid: and (3) developing in a chromatographic cylinder by using water =7 as a developing agent, performing chromatography to a position 1cm away from the top end of the silica gel plate, taking out and airing, uniformly spraying 20% concentrated sulfuric acid, airing, and baking in an oven at 105 ℃ for 10min for color development. As a result, as shown in FIG. 4A, three spots of galactooligosaccharides, GOS1, GOS2 and GOS3, were clearly seen in lane 1, which is the substrate lactose, and lane 2, which is the product.
The reaction product was diluted moderately and filtered through a 0.22 μm filter for HPLC detection, column: aminex HPX-87C, bio-Rad,300 mm. Times.7.8 mm; mobile phase: ultrapure water; column temperature: 75 ℃; a detector: a differential refractive display; flow rate: 0.6mL/min; sample injection amount: 10 μ L. As a result, as shown in FIG. 4B, two galactooligosaccharide peaks were observed, and the peak-off times were 7.848min and 9.009min, respectively, which were not detected due to the low content of GOS3.
Example 4
Production, separation and identification of galactosyl trehalose
(1) Production of galactosyl trehalose by beta-galactosidase enzyme method
Preparing 25% (W/V) lactose and 25% (W/V) trehalose as reaction substrates by using 0.05M phosphate buffer solution with pH =7.4, adding 10U/mL of enzyme, reacting at 30 ℃ for 12h, boiling for inactivation, centrifuging at 13000rpm at room temperature for 10min, and obtaining the supernatant as a product mixture. The product mixture obtained in the above step was diluted appropriately and subjected to TLC detection, 10 μ L of which was spotted by capillary on an activated silica gel aluminum plate, and the mixture was diluted with n-butanol: isopropyl alcohol: acetic acid: and (2) developing the gel in a chromatographic cylinder by using water =7. As a result, as shown in FIG. 5, the substrates lactose and trehalose are shown in lane 1, and the products are shown in lane 2, and two spots of transglycosylation products, transglycosylation product 1 and transglycosylation product 2, respectively, can be clearly seen.
(2) Separation and purification of galactosyl trehalose
A method for the isolation of galactosyl trehalose from a product mixture comprising the steps of:
a. trehalase hydrolysis: adding commercial trehalase into the reaction supernatant to adjust the concentration to 20U/mL, reacting at 40 deg.C for 12h, and completely hydrolyzing trehalose into glucose.
b. Silica gel column chromatography: silica gel 200 mesh was packed into a 30 × 400mm column, purified with n-butanol: isopropyl alcohol: acetic acid: and (2) taking water =7.
C, TLC: the sample eluted in step b was analyzed by TLC and as shown in FIG. 6, a single spot was seen in lanes 2 and 3 as transglycoside product 2.
d. Concentrating and drying: and putting the sample into a rotary evaporator, performing rotary evaporation concentration at 60 ℃ and 60rpm to 10mL to remove the organic reagent, performing freeze drying on the concentrated sample to obtain solid powder, and storing the solid powder in a drying dish.
(3) Structural identification of galactosyl trehalose
Dissolving the dried sample in D 2 NMR spectroscopy in O, using a nuclear magnetic detector model Bruker AVANCE NEO 500 (NMR) 1 H 500MHz， 1 C125 MHz), the detection temperature is 27 ℃, and one-dimensional nuclear magnetism is carried out 1 H spectrum, 1 C spectrum, two-dimensional nuclear magnetism NOESY (H-H), COSY (H-H), HSQC (H-C), HMBC (H-C) detection. One-dimensional nuclear magnetic spectrum is shown in fig. 7 (a, B: 1 spectrum H, C, D: 1 spectrum C), which was subjected to integration, peak labeling and multiplex analysis, the substance consisted mainly of three monosaccharides (two alpha-sugars and one beta-sugar), and the two alpha-sugars were preliminarily judged as trehalose and the beta-sugar as beta galactose. The two-dimensional nuclear magnetic spectrum is shown in FIG. 8, wherein A in FIG. 8 is H-H COSY spectrogram; b is HSQC spectrogram; c is HMBC spectrogram. The structure of the substance is deduced to be beta-Gal (1-6) -alpha-Glc- (1-1) -alpha-Glc by combining one-dimensional nuclear magnetic spectrum, namely galactosyl trehalose connected by beta, 1-6 glycosidic bonds.
The above examples demonstrate that the method can be used for producing galacto-oligosaccharides and galactosyl trehalose by using the beta-galactosidase, and separating the pure galactosyl trehalose.
The present invention is not limited to the above-described technical solutions, and any modifications of the present invention, including various simple modifications to the technical solutions, fall within the scope of the present invention.
It should be noted that the various features described in the foregoing detailed description may be combined in any suitable manner without contradiction, and various possible combinations will not be further described in order to avoid unnecessary repetition.
In addition, various different embodiments of the present invention can be arbitrarily combined, and the same should be regarded as the disclosure of the present invention as long as the idea of the present invention is not violated.
Claims (8)
1. A gene for β -galactosidase which can be used for the production of galactosyltransferase from Lactobacillus fermentum9-4, characterized in that: the gene of the beta-galactosidase consists of a gene for coding an alpha subunit and a gene for coding a beta subunit, wherein the nucleotide sequence of the gene for coding the alpha subunit is shown as SEQ ID NO.1, and the nucleotide sequence of the gene for coding the beta subunit is shown as SEQ ID NO. 2.
2. The beta-galactosidase encoded by the beta-galactosidase gene of claim 1, consisting of an alpha subunit and a beta subunit, wherein: the amino acid sequence of the alpha subunit of the enzyme is shown in SEQ ID NO.3, and the amino acid sequence of the beta subunit is shown in SEQ ID NO. 4.
3. The method for preparing beta-galactosidase according to claim 2, wherein the beta-galactosidase is obtained by introducing the nucleic acid molecule into Escherichia coli BL21 (DE 3) for induction expression.
4. The beta-galactosidase according to claim 2, wherein the optimum temperature is 45 ℃, the optimum pH is 7.0, the specific activity of hydrolyzed oNPG is 197.42U/mg, and the enzyme has transglycosidic activity.
5. The method for producing galactosyl trehalose and galactooligosaccharides using the beta-galactosidase according to claim 2, comprising the steps of:
a. providing a mixture comprising: a galactose donor comprising a galactosyl group bound to a leaving group, the galactose donor being lactose, a galactose acceptor, the galactosyl acceptor being trehalose or lactose;
b. said beta-galactosidase, which enzyme has transglycosidic activity, said enzyme being contacted with the mixture;
c. incubating the mixture and enzyme, adding the enzyme of step b to the donor and acceptor mixture of step a to catalyze the transglycosylation reaction, under the following reaction conditions: adding enzyme amount: 10U/mL, reaction time 12h, reaction temperature: preparing a mixture of the galactosyl trehalose or galacto-oligosaccharide and a substrate at 30 ℃;
d. if the reaction acceptor is trehalose, adding 20U/mL trehalase into the product mixture to react at 40 ℃ for 12h to remove the remaining trehalose from the substrate;
e. passing through a 200 mesh silica gel column 30X 400mm, eluting with n-butanol: isopropyl alcohol: acetic acid: water =7, as eluent, 4;
f. the sample eluted in step e was subjected to TLC detection using capillary spotting of 10 μ L on activated silica gel aluminium plate, n-butanol: isopropyl alcohol: acetic acid: developing in a chromatographic cylinder by using water = 7;
g. and putting the sample into a rotary evaporator, performing rotary evaporation concentration at 60 ℃ and 60rpm to 10mL to remove the organic reagent, and freeze-drying the concentrated sample to obtain solid powder, wherein the solid powder is the obtained pure galactosyl trehalose or galacto-oligosaccharide.
6. The method of claim 4, wherein the galactosyl donor content of step a is not less than 25% (W/V) and the galactosyl acceptor content is not less than 25% (W/V).
7. Use of the beta-galactosidase according to any one of claims 2 and 3 for the synthesis of galactosyltransferase.
8. Use of the beta-galactosidase according to any one of claims 2 and 3 in the enzymatic production of galactosyl oligosaccharides.
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| CN105177085A (en) * | 2015-08-13 | 2015-12-23 | 山东大学 | Application of regioselectivity-specific transglycosylation alpha-galactosidase |
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| JP2021087390A (en) * | 2019-12-04 | 2021-06-10 | 合同酒精株式会社 | β-galactosidase |
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| CN105177085A (en) * | 2015-08-13 | 2015-12-23 | 山东大学 | Application of regioselectivity-specific transglycosylation alpha-galactosidase |
| CN110684685A (en) * | 2019-10-14 | 2020-01-14 | 广西大学 | Lactobacillus fermentum 9-4 and application thereof |
| JP2021087390A (en) * | 2019-12-04 | 2021-06-10 | 合同酒精株式会社 | β-galactosidase |
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