A Pediococcus strain producing transglycosidic active β-galactosidase and its application

Abstract

The present invention provides a Pediococcus strain producing transglycoside active β-galactosidase and its application, belonging to the field of microbial technology. The Pediococcus strain of the present invention is Pediococcus sp. Y1, which is classified and named as Pediococcus sp. Y1 and deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20232025. The Pediococcus sp. Y1 of the present invention is cultured in a fermentation medium to obtain a crude enzyme liquid containing transglycoside active β-galactosidase, and the crude enzyme liquid catalyzes lactose to prepare oligosaccharides of a specific structure, and the oligosaccharides contain a higher proportion of transfer trisaccharides.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Pediococcus strain producing transglycoside active beta-galactosidase and application thereof. Background Art

[0002] Galacto-oligosaccharides (GOS) are functional oligosaccharides composed of 2-10 galactose units connected by glycosidic bonds such as β-(1,3), β-(1,4), and β-(1,6) with glucose or galactose as reducing ends. GOS was first discovered in animal milk and is a natural prebiotic oligosaccharide. GOS can promote the colonization of probiotics such as intestinal bifidobacteria and lactobacilli in the intestine, and its functional properties in infant formula have been widely recognized. GOS also has prebiotic properties such as inhibiting the attachment of pathogenic bacteria to the colon epithelium, lowering serum cholesterol and blood pressure, preventing colon cancer, and enhancing immunity. In addition, the sweetness of GOS is only 0.3 to 0.6 times that of sucrose, has good water solubility and palatability, and has high stability under high temperature and low pH conditions, so it has important application value in the food industry.

[0003] At present, due to its special and complex structure, the access to GOS is still very limited. Oligosaccharides with different glycosidic bond types and polymerization degrees also have different probiotic properties. The content of GOS naturally present in food is generally very small, and it is mainly synthesized by enzymatic methods. It is mainly prepared by catalyzing the lactose reaction with β-galactosidase (EC 3.2.1.23) with transglycosidic activity. β-galactosidase can transfer galactose residues with lactose as a substrate to synthesize GOS with different polymerization degrees and glycosidic bond types. The yield, polymerization degree and β-glycosidic bond type of GOS mainly depend on the source of β-galactosidase and the transglycosidic conditions used in the reaction process (initial lactose concentration, enzyme addition amount, reaction temperature, reaction time, pH, etc.). Therefore, screening high transglycosidic activity β-galactosidase from different microbial sources is of great significance for the efficient synthesis of GOS with specific polymerization degrees and glycosidic bonds.

[0004] At present, commercial β-galactosidase mainly comes from many strains, such as Aspergillus oryzae, Bacillus circulans, Kluyveromyces lactis, Bifidobacterium bifidum, Lactobacillus plantarum, Pediococcus, etc. Comparative genomic studies on different strains of Pediococcus acidilactici have shown that the metabolic characteristics of Pediococcus acidilactici at the strain level are closely related to its living environment, especially some carbohydrate hydrolase (CAZy) genes have great differences between different strains of Pediococcus acidilactici. It is speculated that the enzymatic properties of β-galactosidase produced by different Pediococcus strains are quite different, and thus the yield and composition of GOS synthesized by β-galactosidase are also quite different. At present, there are no relevant reports on the yield and composition of GOS synthesized by β-galactosidase from different strains. Summary of the invention

[0005] The purpose of the present invention is to enrich the sources of transglycosidic β-galactosidase-producing strains and lay a foundation for further developing and applying the β-galactosidase produced by the strain to efficiently synthesize GOS. The present invention provides a Pediococcus strain producing transglycosidic β-galactosidase and its application. The β-galactosidase prepared by the Pediococcus sp. Y1 can catalyze the synthesis of oligosaccharide products mainly composed of transdisaccharides and transtrisaccharides using lactose as a substrate.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The invention provides a Pediococcus strain capable of producing transglycosidically active β-galactosidase. The Pediococcus strain is Pediococcus sp. Y1. The Pediococcus sp. Y1 is classified and named as Pediococcus sp. Y1, and is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20232025 and a deposit date of October 25, 2023.

[0008] The present invention provides the use of the Pediococcus Y1 in preparing transglycoside active beta-galactosidase.

[0009] The invention provides the use of the Pediococcus Y1 in preparing galacto-oligosaccharides.

[0010] The present invention also provides a method for preparing transglycoside-producing β-galactosidase by using the Pediococcus Y1, comprising the following steps: inoculating the Pediococcus Y1 into a seed culture medium for activation culture, inoculating the activated Pediococcus Y1 into a fermentation culture medium for fermentation to obtain a fermentation liquid, centrifuging the fermentation liquid to collect wet bacteria, preparing a bacterial suspension with a phosphate buffer, ultrasonically crushing, and centrifuging to collect a supernatant to obtain a crude enzyme liquid containing the transglycoside-producing β-galactosidase.

[0011] The present invention also provides a method for preparing transglycoside-producing β-galactosidase by using the Pediococcus Y1, comprising the following steps: inoculating the Pediococcus Y1 into a seed culture medium for activation culture, inoculating the activated Pediococcus Y1 into a fermentation culture medium for fermentation to obtain a fermentation liquid, centrifuging the fermentation liquid to collect wet bacteria, preparing a bacterial suspension with a phosphate buffer, adding lysozyme for incubation, ultrasonically crushing, and centrifuging to collect a supernatant to obtain a crude enzyme liquid containing the transglycoside-producing β-galactosidase.

[0012] Preferably, the seed culture medium and fermentation culture medium are: peptone 10g / L, beef extract powder 10g / L, yeast extract powder 5g / L, lactose 20g / L, K2HPO45 g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, MgSO4·7H2O 0.58g / L, Tween-80 1mL / L, MnSO40.25 g / L, pH (6.2-6.4), sterilized at 121°C for 15min.

[0013] The present invention also provides a method for producing oligogalactose by using the crude enzyme solution obtained by the method for preparing transglycoside-producing β-galactosidase with the Pediococcus Y1, comprising the following steps: adding the obtained crude β-galactosidase enzyme solution to a lactose solution to catalyze the transglycoside reaction, thereby obtaining an enzyme reaction solution containing oligogalactose.

[0014] Preferably, the lactose solution is a lactose solution prepared in a pH 7.0 phosphate buffer with a mass concentration of 75 to 525 mg / mL.

[0015] Preferably, the reaction conditions include: adding 3-5 U / mL of enzyme to 150 μL of lactose solution, the reaction temperature is 40-65° C., and the reaction time is 2-48 h.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The Pediococcus sp. Y1 strain disclosed in the present invention is derived from cheese and has the characteristics of good safety (GRAS, QPS), high proliferation rate, good genetic stability, easy cultivation, low cost, etc. In a fermentation medium composed of lactose as a carbon source, nitrogen sources such as yeast extract powder, peptone and beef extract powder, and inorganic salts, beta-galactosidase is fermented and produced. After fermentation, enzyme activity is detected, and the enzyme activity in the fermentation liquid can reach 14.52U / mL. The prepared beta-galactosidase crude enzyme solution is added to a 30% (w / v) lactose solution prepared with a phosphate buffer solution with a pH of 7.0, and the catalytic reaction is carried out at 50°C for 28 hours. The GOS yield can reach up to 38.4% (w / w), and the lactose hydrolysis rate reaches about 69.1% (w / w), wherein the transfer disaccharide and transfer trisaccharide are 15.6% (w / w) and 18.3% (w / w), respectively. At present, there is no report on the yield and composition of GOS synthesized by β-galactosidase from Pediococcus genus using lactose as substrate. Therefore, the Pediococcus sp. Y1 separated and sieved from cheese samples disclosed in the present invention has obvious advantages due to its higher transglycosidation activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Phylogenetic analysis of Pediococcus sp. Y1 producing transglycosidically active β-galactosidase.

[0019] Figure 2 The effect of initial lactose concentration on the synthesis of GOS was analyzed by TLC. Where: S is standard sugar (lactose, galactose, glucose mixture), 1-8: correspond to the transglycosidation reaction products with initial lactose concentrations of 75, 150, 225, 300, 337, 375, 450, 525 mg / mL for 12 hours.

[0020] Figure 3 The effect of reaction temperature on the synthesis of GOS was analyzed by TLC. S is a standard sugar (lactose, galactose, glucose mixture), 1-6: correspond to the transglycosidation reaction products at reaction temperatures of 40, 45, 50, 55, 60, 65°C for 12h.

[0021] Figure 4 The effect of reaction time on the synthesis of GOS was analyzed by TLC. Where: S is standard sugar (lactose, galactose, glucose mixture), 1-12: corresponding to the transglycosidic reaction products at reaction time of 2, 4, 6, 8, 12, 16, 20, 24, 28, 32, 40, 48h.

[0022] Figure 5 HPLC analysis of galacto-oligosaccharides synthesized by transglycosidic β-galactosidase produced by Pediococcus Y1.

[0023] Biological Deposit Description

[0024] Pediococcus Y1, classified as Pediococcus sp.Y1, with the Latin name Pediococcus sp.Y1, was deposited in the China Center for Type Culture Collection (CCTCC) on October 25, 2023, located at Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M 20232025. DETAILED DESCRIPTION

[0025] The present invention provides a Pediococcus strain producing transglycosidic active β-galactosidase, wherein the Pediococcus strain is Pediococcus sp. Y1, and the Pediococcus sp. Y1 is classified and named as Pediococcus sp. Y1, and is deposited in the China Center for Type Culture Collection, with a deposit number of: CCTCC NO: M 20232025, and a deposit date of October 25, 2023. The Pediococcus sp. Y1 of the present invention is isolated from the traditional handmade cheese of ethnic minorities in Yili, Xinjiang. The Pediococcus sp. Y1 of the present invention has the following biological characteristics: Gram-positive cocci, no spore formation, 1-2 mm circular colonies formed on MRS culture medium, neat edges, and milky white surface; can ferment lactose, galactose, and glucose, but cannot ferment sucrose, maltose, xylose, raffinose, mannitol, and sorbitol; starch hydrolysis test, gelatin hydrolysis test, catalase test, and hydrogen sulfide production test are all negative.

[0026] In the present invention, the nucleotide sequence length of 16SrRNA of the transglycosidic active β-galactosidase strain Y1 involved in the present invention is 1572bp. Through homology comparison analysis by NCBI-blast, the strain Y1 was screened to be classified into one branch with Pediococcus acidilactici and Pediococcus pentosaceus, so the strain Y1 was tentatively designated as Pediococcus sp. Y1.

[0027] The present invention provides the use of the Pediococcus Y1 in the preparation of transglycoside-active β-galactosidase. The activity of the transglycoside-active β-galactosidase obtained by fermentation of the Pediococcus Y1 of the present invention is as high as 14.52 U / mL.

[0028] The present invention provides the use of the Pediococcus Y1 in the preparation of galacto-oligosaccharides. The crude enzyme liquid containing transglycoside-producing β-galactosidase obtained by fermentation of the Pediococcus Y1 of the present invention is used to catalyze lactose to obtain galacto-oligosaccharides with a specific structure, wherein the galacto-oligosaccharides are mainly composed of transfer disaccharides and transfer trisaccharides, which are 15.6% (w / w) and 18.3% (w / w) respectively, and the mass fraction of galacto-oligosaccharides above the transfer trisaccharides is 4.5% (w / w).

[0029] As an implementation method, the present invention provides a method for preparing β-galactosidase with transglycoside activity by using the Pediococcus Y1, comprising the following steps: inoculating Pediococcus Y1 into a seed culture medium for activation culture, inoculating the activated Pediococcus Y1 into a fermentation culture medium for fermentation to obtain a fermentation broth, centrifuging the fermentation broth to collect wet bacteria, preparing a bacterial suspension with a phosphate buffer, ultrasonically crushing, and centrifuging to collect the supernatant, which is a crude enzyme solution containing β-galactosidase with transglycoside activity.

[0030] As another possible implementation method, the present invention provides a method for preparing transglycosidic active β-galactosidase by Pediococcus Y1, which comprises the following steps: inoculating Pediococcus Y1 into a seed culture medium for activation culture, inoculating the activated Pediococcus Y1 into a fermentation culture medium for fermentation to obtain a fermentation broth, centrifuging the fermentation broth to collect wet bacteria, using a phosphate buffer to prepare a bacterial suspension, adding lysozyme for incubation, ultrasonically crushing, and centrifuging to collect the supernatant, which is a crude enzyme solution containing transglycosidic active β-galactosidase. The addition of lysozyme for incubation in the present invention can make the bacteria more thoroughly crushed, increase the release of β-galactosidase, and thus improve the enzyme activity.

[0031] In the present invention, the seed culture medium and the fermentation culture medium are: peptone 10g / L, beef extract powder 10g / L, yeast extract powder 5g / L, lactose 20g / L, K2HPO45 g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, MgSO4·7H2O0.58g / L, Tween-80 1mL / L, MnSO40.25g / L, pH (6.2-6.4), sterilized at 121°C for 15min.

[0032] In the present invention, the seed culture conditions involved are: activating the strain by shaking and culturing Pediococcus sp. Y1 at 30-38° C. and a rotation speed of 100-250 rpm for 12-24 hours; the fermentation conditions involved are: inoculating the activated Pediococcus sp. Y1 into a fermentation medium, with an inoculation amount of 1%-10% of the fermentation medium, and fermenting and culturing at 30-38° C. and a shaking rotation speed of 100-300 rpm for 12-24 hours to obtain a fermentation liquid.

[0033] In the present invention, the obtained fermentation broth is subjected to freeze centrifugation to collect wet bacteria, a bacterial suspension is prepared with a 0.1M phosphate buffer solution at pH 7.0, and then the bacteria are crushed in an ice bath at 0-4°C by ultrasonic crushing. The suspension obtained after crushing is the crude β-galactosidase enzyme solution.

[0034] The present invention provides a method for producing oligogalactose using a crude enzyme solution obtained by preparing a method for producing transglycoside-active β-galactosidase using the Pediococcus Y1, comprising the following steps: adding the obtained β-galactosidase crude enzyme solution to a lactose solution to catalyze the transglycoside reaction, thereby obtaining an enzyme reaction solution containing oligogalactose. The lactose solution of the present invention is a lactose solution having a mass concentration of 75 to 525 mg / mL prepared in a pH 7.0 phosphate buffer, preferably a lactose solution of 100 to 400 mg / mL. The reaction conditions of the present invention include: adding 3 to 5 U / mL of enzyme to 150 μL of lactose solution, a reaction temperature of 40 to 65° C., and a reaction time of 2 to 48 hours.

[0035] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0036] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1 Isolation and Screening of Pediococcus sp. Y1

[0038] (1) Take out the preserved traditional handmade cheese sample of Xinjiang Yili ethnic minorities in the clean bench, place it in 45 mL of sterilized saline, mix it evenly, and then use sterile saline test tubes to dilute it to 10 -5 , 100 μL of the dilution was evenly spread on the MRS solid medium containing X-gal and lactose as the carbon source, and placed at 37°C for constant temperature culture for 24 to 48 hours. Single colonies that can hydrolyze X-gal and are blue were picked, and streaked under the same conditions, and streaked three times. After obtaining pure culture, it was preserved on a slant and glycerol.

[0039] Among them, MRS medium (plate): agar powder 20g / L, peptone 10g / L, beef extract powder 10g / L, yeast extract powder 5g / L, lactose 20g / L, K2HPO45 g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, MgSO4·7H2O 0.58g / L, Tween-80 1mL / L, MnSO40.25 g / L, pH (6.2~6.4), sterilization at 121℃ for 15min.

[0040] (2) Pick a single blue colony and culture it in MRS medium at 37°C, 200 rpm for 18 h. Then, centrifuge the fermentation broth at 4°C (8000 rpm, 10 min), wash it three times with pH 7.0, 0.1 mol / L phosphate buffer, add lysozyme with a final concentration of 1 mg / mL, incubate it at 37°C for 2.5 h, ultrasonically disrupt it under ice bath conditions (work for 3 s and stop for 6 s, power 125 W, time 15 min), centrifuge it at 12000 rpm for 20 min, and the supernatant is the crude enzyme solution.

[0041] (3) Take 20 μL of crude enzyme solution and 80 μL of ONPG solution prepared with 0.1 mol / L phosphate buffer (pH 7.0) and react at 37°C for 10 min. After the reaction, add 100 μL of 1 mol / L sodium carbonate solution to terminate the reaction. Use a microplate reader to measure the absorbance at a wavelength of 420 nm, calculate the enzyme activity, and screen out strains with high β-galactosidase activity. The control group uses 20 μL of inactivated crude enzyme solution to participate in the reaction under the same conditions. The enzyme activity unit is defined as: under certain conditions, the amount of enzyme that catalyzes the hydrolysis of ONPG to produce 1 μmol of o-nitrophenol (ONP) in 1 min is 1 enzyme activity unit (U).

[0042] (4) Based on the strains with high β-galactosidase activity screened out in step (3), 50 μL of the crude enzyme solution of step (2) of the corresponding strain was reacted with 150 μL of 300 mg / mL lactose solution, reacted at 40°C for 12 hours, and terminated by treating at 100°C for 10 minutes. Thin layer chromatography (TLC) was performed on the reaction products of different strains. The sample was diluted to 1% with anhydrous ethanol, and the diluted sample was spotted onto a heat-activated thin layer silica gel plate by capillary tube, and developed with n-butanol-ethanol-water (5:3:2, V / V) as the developing agent. The color developer (0.5% (w / v) of 3,5-dihydroxytoluene dissolved in 20% sulfuric acid) was sprayed on the TLC plate and baked in an oven at 110°C for 10 minutes for color development. According to the size of the corresponding oligosaccharide product spots on the TLC plate, strains C1, C3, Y1, Y2, F1 and F2 that produced transglycosylation active β-galactosidase were screened.

[0043] Example 2 Identification of strains producing transglycosidic β-galactosidase

[0044] The strain Y1 obtained by screening in the above Example 1 was identified.

[0045] (1) Morphological observation and physiological and biochemical tests

[0046] The Y1 strain was inoculated on MRS solid medium and cultured at 37℃ for 24h-48h. The strain Y1 formed round colonies on the MRS medium with neat edges, smooth surface and milky white color; the bacteria were spherical and Gram staining was positive. Physiological and biochemical experiments of strain Y1 showed that the strain could ferment lactose, galactose and glucose, but could not ferment sucrose, maltose, xylose, raffinose, mannitol and sorbitol. The starch hydrolysis test, gelatin hydrolysis test, catalase test and hydrogen sulfide production test were all negative.

[0047] (2) Cloning and sequence analysis of 16S rRNA

[0048] The genomic DNA of strain Y1 was extracted, and PCR amplification was performed using universal primers 27f and 1492r of bacterial 16S rRNA gene. The PCR results were identified by agarose gel electrophoresis. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing using universal primers 27f and 1492r. The nucleotide sequence of 16S rRNA is shown in SEQ ID No.1. The sequencing results were analyzed by homology comparison from NCBI-blast. The comparison results showed that the 16S rRNA gene sequence of strain Y1 and the 16S rRNA gene sequence of multiple Pediococcus strains were 99% similar. A phylogenetic tree was established based on the sequences of related species in the database. The evolutionary distance was calculated using the neighbor-joining method. The p-distance and Kimura-2 parameter dual-parameter method was used in MEGA 7.0 software. The Bootstrap method was used to evaluate the stability of the evolutionary tree branch clustering. The results were repeated 1000 times to obtain the species classification of enzyme-producing strains. Figure 1 As shown, the screened strain Y1 was classified into one branch with Pediococcus acidilactici and Pediococcus pentosaceus. Therefore, strain Y1 was tentatively designated as Pediococcus sp. Y1.

[0049] Example 3 Method for producing β-galactosidase by fermentation using the strain Pediococcus sp. Y1 producing transglycosidic β-galactosidase

[0050] The specific steps are as follows:

[0051] (1) Seed culture

[0052] Seed culture medium: peptone 10 g / L, beef extract powder 10 g / L, yeast extract powder 5 g / L, lactose 20 g / L, K2HPO4 5 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, MgSO4·7H2O 0.58 g / L, Tween-80 1 mL / L, MnSO4 0.25 g / L, pH (6.2~6.4), sterilization at 121℃ for 15 min.

[0053] Seed culture conditions: Pediococcus sp. Y1 was cultured at 37°C and 200 rpm for 15 h to activate the strain;

[0054] (2) Fermentation culture

[0055] Fermentation medium: peptone 10 g / L, beef extract powder 10 g / L, yeast extract powder 5 g / L, lactose 20 g / L, K2HPO4 5 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, MgSO4·7H2O 0.58 g / L, Tween-80 1 mL / L, MnSO4 0.25 g / L, pH (6.2~6.4), sterilization at 121℃ for 15 min.

[0056] Fermentation conditions: The Pediococcus sp. Y1 activated in step (1) was inoculated into a fermentation medium at an inoculation amount of 2% (volume percentage) of the fermentation medium, and fermented and cultured at 37° C. and 200 rpm for 18 h to produce β-galactosidase.

[0057] (3) Post-fermentation treatment

[0058] The fermentation liquid obtained in step (2) was subjected to refrigerated centrifugation to collect wet cells, and a bacterial suspension was prepared with a 0.1M phosphate buffer of pH 7.0, and ultrasonically broken under ice bath conditions (working for 3s and stopping for 6s, power 125W, time 15min), and refrigerated centrifuged at 12000rpm for 20min, and the supernatant was the crude enzyme solution. 20μL of the crude enzyme solution was reacted with 80μL of 0.1mol / L phosphate buffer (pH 7.0) prepared o-nitrobenzene-β-D-galactoside (ONPG) solution at 37°C for 10min, and 100μL of 1mol / L sodium carbonate solution was added after the reaction to terminate the reaction, and the absorbance at a wavelength of 420nm was measured using an ELISA instrument, and the enzyme activity was calculated by the ONPG standard curve method. The enzyme activity was detected to be 6.95U / mL.

[0059] Example 4 Method for producing β-galactosidase by fermentation using the strain Pediococcus sp. Y1 producing transglycosidic β-galactosidase

[0060] The specific steps are as follows:

[0061] (1) Seed culture

[0062] The seed culture medium is the same as that in Example 3.

[0063] Seed culture conditions: Pediococcus sp. Y1 was cultured at 37°C and 200 rpm for 15 h to activate the strain;

[0064] (2) Fermentation culture

[0065] The fermentation medium was the same as in Example 3.

[0066] Fermentation conditions: The Pediococcus sp. Y1 activated in step (1) was inoculated into a fermentation medium at an inoculation amount of 2% of the fermentation medium, and fermented and cultured at 37° C. and 200 rpm for 18 h to produce β-galactosidase.

[0067] (3) Post-fermentation treatment

[0068] The fermentation broth obtained in step (2) was subjected to refrigerated centrifugation to collect wet bacteria, and a bacterial suspension was prepared with a 0.1 M phosphate buffer solution at pH 7.0. Lysozyme at a final concentration of 1 mg / mL was added and incubated at 37°C for 2.5 h. Ultrasonic disruption was performed under ice bath conditions (working for 3 s and stopping for 6 s, power 125 W, time 15 min), and refrigerated centrifugation was performed at 12000 rpm for 20 min. The supernatant was the crude enzyme solution.

[0069] Take 20μL crude enzyme solution and 80μL 0.1mol / L phosphate buffer (pH 7.0) prepared ONPG solution and react at 37℃ for 10min. After the reaction, add 100μL 1mol / L sodium carbonate solution to terminate the reaction. Use microplate reader to measure the absorbance at 420nm wavelength, and calculate the enzyme activity by ONPG standard curve method. The enzyme activity was detected to be 14.52U / mL.

[0070] Example 5 Method for producing galacto-oligosaccharides using crude β-galactosidase produced by fermentation of Pediococcus sp. Y1

[0071] 1. Effect of crude β-galactosidase solution on the catalytic reaction of different initial lactose concentrations

[0072] The specific steps include:

[0073] (1) The crude enzyme solution obtained in Example 4 was added with an enzyme amount of 4.54 U / mL (75, 150, 225, 300, 337, 375, 450, 525 mg / mL, pH 7) in different initial lactose concentration solutions and incubated at 40° C. for 12 h.

[0074] (2) The enzyme reaction solution containing oligogalactose obtained in step (1) was diluted to 1% with anhydrous ethanol, and the diluted sample was spotted onto a heat-activated thin-layer silica gel plate using a capillary tube. The plate was developed with n-butanol-ethanol-water (5:3:2, V / V) as a developing agent. The color developer (3,5-dihydroxytoluene 0.5% (w / v) dissolved in 20% sulfuric acid) was sprayed onto the TLC plate and then baked in an oven at 110°C for 10 minutes for color development. Different sugars showed different colors. Compared with standard sugars, the results are shown in Table 1. Figure 2 , with the increase of initial lactose concentration, the yield of GOS increased first and then decreased. When the initial lactose concentration was 300 mg / mL, the yield of GOS was the highest.

[0075] 2. Effect of crude β-galactosidase solution on lactose catalysis at different temperatures

[0076] (1) The crude enzyme solution obtained in Example 4 was added to a 300 mg / mL lactose solution prepared with a phosphate buffer solution having a pH of 7.0 at an enzyme dosage of 4.54 U / mL for catalytic reaction. The reaction was incubated at different reaction temperatures (40, 45, 50, 55, 60, 65° C.) for 12 h to obtain an enzyme reaction solution containing galacto-oligosaccharides.

[0077] (2) The detection step of obtaining the enzyme reaction solution containing oligogalactose is the same as step 1 (2) of this embodiment. The results are shown in Figure 3 As the reaction temperature increased, the yield of GOS increased first and then decreased. At a reaction temperature of 50°C, the yield of GOS was the highest.

[0078] 3. Effect of crude β-galactosidase solution on lactose catalysis at different reaction times

[0079] (1) The crude enzyme solution obtained in Example 4 was added to a 300 mg / mL lactose solution prepared in a phosphate buffer solution with a pH of 7.0 at an enzyme dosage of 4.54 U / mL, and the catalytic reaction was carried out at 50° C. The reaction was incubated at different reaction times (2, 4, 6, 8, 12, 16, 20, 24, 28, 32, 40, 48 h) to obtain an enzyme reaction solution containing galacto-oligosaccharides.

[0080] (2) The detection step of obtaining the enzyme reaction solution containing oligogalactose is the same as step 1 (2) of this embodiment. The results are shown in Figure 4 GOS was synthesized within the initial 2 h of the reaction. As the reaction time increased, the yield of GOS gradually increased and reached the highest at 28 h.

[0081] Example 6 Determination of the yield and composition of GOS synthesized by β-galactosidase produced by fermentation of Pediococcus sp. Y1

[0082] The transglycosidation reaction product after 28 hours of reaction was diluted with triple distilled water to a final sugar concentration of 1g / 100mL, filtered with a 0.22μm filter membrane, and then loaded for analysis. The determination of the transferred disaccharides was analyzed using an Aps-2HYPERSIL (4.6mm×250mm) chromatographic column and a RID-10A differential detector. The mobile phase was 70% (v / v) acetonitrile / water solution. The flow rate and column temperature were set to 1mL / min and 40℃, respectively, and the injection volume was 10μL. The different GOS components were analyzed using a sugar analysis column Hi-PlexNa (300mm×7.7mm) chromatographic column and a RID-10A differential detector. The mobile phase was triple distilled water. The flow rate and column temperature were set to 0.2mL / min and 80℃, respectively, and the injection volume was 20μL. Combining the analysis results of Aps-2HYPERSIL and Hi-PlexNa columns, it can be determined that the yield of GOS synthesized from lactose catalyzed by β-galactosidase produced by Pediococcus sp. Y1 is 38.4% (w / w), and the GOS components are mainly transferred disaccharides and transferred trisaccharides, which are 15.6% (w / w) and 18.3% (w / w), respectively. The mass fraction of GOS above the transferred trisaccharide is 4.5% (w / w). Figure 5 , Figure 5 Among them, the retention times 14.423min and 15.182min are solvent peaks. The peak at retention time 44.556min is galactose, the peak at retention time 41.382min is glucose, 34.745min is disaccharide (lactose and transfer disaccharide are not separated), 29.536min is oligogalactotriose, and 9.200-27.274min are oligogalactose components above trisaccharide.

[0083] From the above experiments, it can be seen that the Pediococcus sp. Y1 strain of the present invention can ferment and produce β-galactosidase, and the prepared β-galactosidase crude enzyme solution is added to a 300 mg / mL lactose solution prepared with a phosphate buffer having a pH of 7.0 to obtain oligosaccharides. In addition, during the transglycosidation experiment, the amount of enzyme added is only 3 to 5 U / mL, which is less than that of β-galactosidase from other strains. Based on the fact that β-galactosidase is currently expensive and difficult to apply in industry, the present invention uses less enzyme addition to make its industrial application value higher. At the same time, the yield of trisaccharides in the preparation of galacto-oligosaccharides by the present invention is improved. Compared with disaccharides and trisaccharides whose prebiotic effects are unknown, the galacto-oligosaccharides prepared by the Pediococcus sp. Y1 strain of the present invention have more significant prebiotic effects, especially when the target products are GOS with different compositions, the Pediococcus sp. Y1 strain of the present invention is the best choice for enriching trisaccharide products.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Pediococcus that produces transglycosidically active β-galactosidase ( Pediococcus sp.) Y1, characterized in that Deposited in China Center for Type Culture Collection, the deposit number is: CCTCC NO: M 20232025, and the deposit date is October 25, 2023; The transglycosidic active β-galactosidase produced by Pediococcus Y1 synthesizes galacto-oligosaccharide; the galacto-oligosaccharide contains trisaccharide components.

2. Use of Pediococcus Y1 as claimed in claim 1 in the preparation of transglycosidic active β-galactosidase.

3. Use of Pediococcus Y1 as claimed in claim 1 in the preparation of galacto-oligosaccharides.

4. A method for preparing transglycosidically active β-galactosidase using Pediococcus Y1 as claimed in any one of claims 1 to 3, characterized in that: The method comprises the following steps: inoculating Pediococcus Y1 into a seed culture medium for activation culture, inoculating the activated Pediococcus Y1 into a fermentation culture medium for fermentation to obtain a fermentation liquid, centrifuging the fermentation liquid to collect wet bacteria, using a phosphate buffer to prepare a bacterial suspension, ultrasonically crushing, and centrifuging to collect a supernatant to obtain a crude enzyme liquid containing beta-galactosidase with transglycoside activity.

5. A method for preparing transglycosidically active β-galactosidase using Pediococcus Y1 as claimed in any one of claims 1 to 3, characterized in that: The method comprises the following steps: inoculating Pediococcus Y1 into a seed culture medium for activation culture, inoculating the activated Pediococcus Y1 into a fermentation culture medium for fermentation to obtain a fermentation liquid, centrifuging the fermentation liquid to collect wet bacteria, using a phosphate buffer to prepare a bacterial suspension, adding lysozyme for incubation, ultrasonically breaking, and centrifuging to collect a supernatant to obtain a crude enzyme liquid containing beta-galactosidase with transglycoside activity.

6. The method according to claim 4 or 5, characterized in that The seed culture medium and fermentation culture medium are: peptone 10g / L, beef extract powder 10g / L, yeast extract powder 5g / L, lactose 20g / L, K2HPO4 5g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, MgSO4·7H2O 0.58g / L, Tween-80 1mL / L, MnSO4 0.25 g / L, pH 6.2-6.4, sterilization at 121℃ for 15 min.

7. A method for producing galacto-oligosaccharides using the crude enzyme solution obtained by the method according to any one of claims 4 to 6, characterized in that: The method comprises the following steps: adding the crude β-galactosidase solution obtained by the method according to any one of claims 4 to 6 into a lactose solution to catalyze the transglycoside reaction, thereby obtaining an enzyme reaction solution containing galacto-oligosaccharides.

8. The method according to claim 7, characterized in that The lactose solution is a lactose solution prepared with a pH 7.0 phosphate buffer and having a mass concentration of 75 to 525 mg / mL.

9. The method according to claim 7, characterized in that The reaction conditions include: adding 3-5 U / mL of enzyme to 150 μL of lactose solution, the reaction temperature is 40-65° C., and the reaction time is 2-48 hours.

Images