High-yield low-temperature-resistant beta-galactosidase lactic acid bacteria and application thereof

Abstract

The invention relates to a high-yield low-temperature-resistant beta-galactosidase lactic acid bacterium and application thereof, and belongs to the technical field of microorganisms, the preservation number of screened high-yield low-temperature-resistant beta-galactosidase lactobacillus sakei J15 is CGMCC (China General Microbiological Culture Collection Center) NO.32056, and the screened high-yield low-temperature-resistant beta-galactosidase lactobacillus sakei J15 can produce high-yield lactose-degrading beta-galactosidase at low temperature and can hydrolyze lactose in milk at low temperature. Therefore, the lactobacillus sakei J15 disclosed by the invention can have beta-galactosidase with high enzymatic activity at a relatively low temperature, and can be directly applied to cow milk and dairy products stored at a low temperature. The method has important application value for reducing cost and saving energy, and can be applied to industrial production of lactose-free dairy products.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to high-yield low-temperature resistant beta-galactosidase lactic acid bacteria and applications thereof. Background Art

[0002] Human lactose malabsorption or intolerance is due to the inability to digest large amounts of lactose due to insufficient β-galactosidase activity. β-galactosidase hydrolyzes lactose into glucose and galactose and is essential for individuals with lactose intolerance. β-galactosidase is widely derived from microorganisms such as bacteria, yeast, and filamentous fungi. Generally, the optimal temperature for β-galactosidase isolated from lactic acid bacteria species is 40-65°C. Most lactic acid bacteria-derived β-galactosidases have an optimal temperature of 40°C for hydrolyzing lactose in milk. Although the activity of β-galactosidase screened from lactic acid bacteria is relatively high, its ability to hydrolyze lactose at high temperatures is relatively high.

[0003] The most commonly used β-galactosidase currently has a relatively high optimum reaction temperature and a relatively strict pH requirement, which results in problems such as high production costs and high energy consumption. In the lactose-free dairy product production industry, the ideal β-galactosidase is one that has high enzyme activity under low-temperature storage conditions for milk or dairy products and can effectively decompose lactose in milk.

[0004] Lactic acid bacteria can be used as a starter in various fermented foods, or can be added directly to dairy products. Using lactic acid bacteria with high production of low-temperature resistant β-galactosidase as a starter for dairy products can decompose lactose under low temperature conditions, reduce lactose content, and increase the content of lactic acid bacteria.

[0005] Therefore, how to screen out lactic acid bacteria that produce high-temperature-resistant β-galactosidase is of great significance. Summary of the invention

[0006] In view of the problems existing in the prior art, the object of the present invention is to design and provide a technical solution for high-yield low-temperature resistant β-galactosidase lactic acid bacteria and application thereof.

[0007] The present invention is specifically implemented through the following technical solutions:

[0008] The first aspect of the present invention provides a high-yield low-temperature resistant β-galactosidase lactic acid bacteria, which is Latilactobacillus sakei J15, and its preservation number is: CGMCC NO.32056.

[0009] The second aspect of the present invention provides the use of the above-mentioned high-yield low-temperature resistant β-galactosidase lactic acid bacteria in the production of β-galactosidase.

[0010] The third aspect of the present invention provides a method for producing β-galactosidase, which comprises inoculating the strain according to claim 1 into a fermentation medium for fermentation culture, and then isolating and obtaining the β-galactosidase.

[0011] Further, the fermentation medium in the method is:

[0012] MRS modified medium (peptone 1%, beef extract 1%, yeast powder 0.5%, K2HPO4 0.2%, diammonium citrate 0.2%, sodium acetate 0.5%, lactose 2%, Tween 80 0.1mL, MgSO4·7H2O 0.058%, MnSO4·4H2O 0.025%).

[0013] Furthermore, the fermentation culture conditions in the method are: culture at 30°C for 24 hours.

[0014] The sake lactobacillus (Latilactobacillus sakei J15) of the present invention can produce high-yield low-temperature resistant beta-galactosidase at low temperatures, and can also hydrolyze lactose in milk at low temperatures. Therefore, the sake lactobacillus (Latilactobacillus sakei J15) of the present invention can have high enzyme activity beta-galactosidase at relatively low temperatures, and can be directly applied to cow's milk and dairy products under low temperature storage. It has important application value for reducing costs and saving energy, and can be applied to the industrial production of lactose-free dairy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Phylogenetic tree of the screened strains;

[0016] Figure 2 The growth curves of the strains at different temperatures;

[0017] Figure 3 Shows the growth curves of the strains under different carbohydrates. DETAILED DESCRIPTION

[0018] The present invention is further described below with reference to the examples.

[0019] The culture medium and reagents used in the embodiments of the present invention are as follows:

[0020] Culture medium formula:

[0021] Culture medium for isolating β-galactosidase active lactic acid bacteria: peptone 1%, beef extract 1%, yeast powder 0.5%, K2HPO4 0.2%, diammonium citrate 0.2%, sodium acetate 0.5%, glucose 2%, Tween 80 0.1mL, MgSO4·7H2O 0.058%, MnSO4·4H2O 0.025%, agar 2%, X-gal (5-bromo-4-chloro-3-indole β-D-galactoside) 1%. In this article, the "%" in the culture medium means how many grams of a certain component are contained in 100mL of the culture medium.

[0022] MRS broth medium: peptone 1%, beef extract 1%, yeast powder 0.5%, K2HPO4 0.2%, diammonium citrate 0.2%, sodium acetate 0.5%, glucose 2%, Tween 80 0.1mL, MgSO4·7H2O 0.058%, MnSO4·4H2O 0.025%. The "%" in the medium herein means how many grams of a certain component are contained in 100mL of the medium.

[0023] Phosphate buffer: NaH2PO4 0.265%, Na2HPO4 0.825%. In this article, the "%" in the culture medium means how many grams of a certain component are contained in 100 mL of water.

[0024] Example 1: Preliminary screening of lactic acid bacteria with high β-galactosidase production

[0025] (1) Preliminary screening of lactic acid bacteria in fermented foods

[0026] Select yogurt fermented food, take 100 μL yogurt and add 900 μL 0.85% NaCl solution to dilute to different concentrations (10 -2 ~10 -4 ), spread on MRS solid medium with X-gal added, mark it, and observe and select colonies after culturing at 30°C for 24 hours. As a result, 15 separate colonies were selected on the medium and numbered 1 to 15.

[0027] (2) Rescreening of high-yielding β-galactosidase lactic acid bacteria

[0028] The strain was cultured in MRS broth at 30°C for 24h, centrifuged (7000×g, 15min, 4°C), resuspended in 50mM phosphate buffer (pH 7.0), and broken by ultrasonic treatment. The supernatant was the crude enzyme. The specific activity of β-galactosidase was determined using ONPG (o-nitrophenyl-β-D-pyranogalactoside) as a substrate. The crude enzyme (0.1mL) was mixed with 1mL of substrate (phosphate buffer containing 2mMONPG, pH 7.0) and measured at 405nm using a UV spectrophotometer. One unit of activity is defined as the amount of enzyme required to produce 1μmol o-nitrophenol per minute.

[0029] As shown in Table 1, the β-galactosidase activity of 15 initially screened strains was measured, and it was found that the enzyme activity of strain 15 was the highest, reaching 1.85 mU / mL. The 16s rDNA sequence of the strain was determined to identify the strain.

[0030] Table 1 Comparison of enzyme activities of 15 strains producing β-galactosidase

[0031]

[0032]

[0033] Example 2: Identification of high-yielding β-galactosidase lactic acid bacteria

[0034] The strain No. 15 obtained by screening was identified by 16s rRNA sequencing. The sequencing sequence results were compared with the homologous sequences in the NCBI database by BLAST to determine the species of the strain. The species can be identified when the homology exceeds 99%. The phylogenetic tree was constructed using the near-joining method in the software MEGA-X for strains with higher homology.

[0035] Results: The 16s rRNA sequencing results of strain No. 15 were as follows.

[0036] The gene sequence of 16s rRNA of screening strain 15 (as shown in SEQ ID NO.1):

[0037] CTCTCGTTTAGATTGAAGGAGCTTGCTCCTGATTGATAAACATTTGAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTAAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAAACCTAACACCGCATGGTGTAGGGTTGAAAGATGGTTTCGGCTATCACTTTAGGATGGACCCGCGGTGCATTAGTTAGTTGGTGAGGTAAAGGCTCACCAAGACCGTGATGCATAGCCGACCTGAGAGGGTAATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTGGAGAAGAATGTATCTGATAGTAACTGATCAGGTAGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGGTAACCCTTCGGGGAGCCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGA

[0038] As shown in Table 2, the screening strain No. 15 was Latilactobacillus sakei, which had 100.00% homology with the standard strain Latilactobacillus sakei ZFM229.

[0039] Table 2 Identification results of strains with high β-galactosidase activity

[0040]

[0041] like Figure 1 As shown, the evolutionary distance between the screened strains Latilactobacillus sakei J15 and Latilactobacillus sakei ZFM229 and Lactiplantibacillus plantarum LG138 was the smallest.

[0042] At the same time, the strain Latilactobacillus sakei J15 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the storage address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The storage name is Lactobacillus sakei J15, the storage time is September 24, 2024, and the strain storage number is CGMCC No.32056.

[0043] Example 3: Growth curves of Latilactobacillus sakei J15 at different temperatures and β-galactosidase activity

[0044] The strains were cultured at 5, 10, 15, 20, 25, 30, and 37°C for 60 h, and the growth curves were measured at 600 nm at 3, 6, 9, 12, 24, 36, 48, and 60 h, respectively. Figure 2 At the same time, the specific enzyme activity of β-galactosidase was determined at different temperatures. The strain was cultured at 30°C for 18 h, and the crude enzyme solution was prepared and measured at 5, 10, 15, and 30°C.

[0045] Test results: Latilactobacillus sakei J15 strain grew better at 25℃ and 30℃, entered the logarithmic growth phase after 3 hours, and the bacteria increased rapidly; it reached the stable growth period at 24-60 hours, when the number of live bacteria remained relatively stable; while at other temperatures (5, 10, 15, 20 and 37℃), it entered the logarithmic growth phase after 9 hours of growth, especially at 5, 10, 15℃, it was still in the logarithmic growth phase until 60 hours.

[0046] Although the optimum growth temperature of the strain is 25-30°C, the β-galactosidase activity of the strain at 15°C is the highest, reaching 789.31 mU / mg (Table 3). At 5°C, the strain still maintains a certain enzyme activity (41.08 mU / mg).

[0047] This indicates that Latilactobacillus sakei J15 strain has higher β-galactosidase activity at low temperature.

[0048] Table 3 β-galactosidase activity of Latilactobacillus sakei J15 at different temperatures

[0049]

[0050] Example 4: Ability of Latilactobacillus sakei J15 to utilize 49 carbohydrates

[0051] API 50CH was used to determine the ability of Latilactobacillus sakei J15 to utilize carbohydrates. The strain was activated by culturing in MRS medium for 24 hours, and two McFarland turbidity suspensions were prepared, and then the suspensions were added to 50 microbiological tubes on the reagent strip. When the bacteria can utilize different substances in the substrate as the only carbon source, if the bacteria can utilize or grow, the color changes, which is indicated by positive (+), and the color changes slightly, which is indicated by (±). If the bacteria cannot utilize the carbon source, the color does not change, which is indicated by negative (-).

[0052] Measurement results: The results showed that Latilactobacillus sakei J15 was able to utilize 16 kinds of carbohydrates (Table 4), namely, L-arabinose, D-ribose, galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, arbutin, esculin, salicin, D-maltose, D-lactose, D-melanobiose, cellulose, D-trehalose, and D-toluose.

[0053] Table 4 Fermentation capacity of Latilactobacillus sakei J15 on different carbohydrates and their derivatives

[0054]

[0055] Example 5: Growth curve and enzyme activity of Latilactobacillus sakei J15 in medium containing different carbohydrates

[0056] The strains were cultured in glucose, arabinose, galactose, fructose, sucrose, lactose, and maltose for 36 h, and the growth curves were measured at 600 nm at 3, 6, 9, 12, 24, and 36 h, respectively. Figure 3 ), and at the same time, the β-galactosidase specific enzyme activity was determined (Table 5).

[0057] Test results: Figure 3 As shown, Latilactobacillus sakei J15 strain has a relatively obvious logarithmic growth phase in the medium supplemented with different carbohydrates, except arabinose, in the growth environment of other carbon sources. After 24 hours of cultivation, the OD value reached above 1.0.

[0058] As shown in Table 5, it can be seen that the specific enzyme activity of β-galactosidase of the strain after being cultured in the medium with lactose as the carbon source is much higher than that in other carbon source growth environments, and the specific enzyme activity is as high as 1555.68mU / mg. This shows that Latilactobacillus sakeiJ15 can effectively decompose lactose. Table 5 Specific enzyme activity of β-galactosidase of Latilactobacillus sakei J15 after growth in different carbohydrates

[0059]

[0060] Example 6: A method for producing β-galactosidase

[0061] The high-yield cold-resistant β-galactosidase-resistant Lactobacillus sakei J15 was activated in MRS medium and inoculated into 100 mL of MRS modified medium (1% peptone, 1% beef extract, 0.5% yeast powder, 0.2% K2HPO4, 0.2% diammonium citrate, 0.5% sodium acetate, 2% lactose, 0.1 mL Tween 80, 0.058% MgSO4·7H2O, 0.025% MnSO4·4H2O) and cultured at 30°C for 24 hours. The expanded cultured bacterial liquid was centrifuged at a centrifugal speed of 8000g for 10 minutes, washed three times with sterile water, centrifuged to obtain wet cells, and an appropriate amount of 50 mM phosphate buffer (pH 7.0) was added. The cells were broken by ultrasonic treatment, and the ultrasonic extraction time was 20 minutes, the temperature was 4°C, and the power was 40W, and the supernatant was collected. The results showed that the enzyme yield was 19.36 mg / L and the enzyme activity was 30.11 mU / mL.

Claims

1. A high-yield low-temperature resistant β-galactosidase lactic acid bacteria, which is Latilactobacillus sakeiJ15, and its preservation number is: CGMCC NO.32056.

2. Use of the low temperature high-yield β-galactosidase lactic acid bacteria as claimed in claim 1 in producing β-galactosidase.

3. A method for producing low-temperature resistant β-galactosidase, characterized in that: The method comprises inoculating the strain as claimed in claim 1 into a fermentation medium for fermentation culture, and then isolating and obtaining the β-galactosidase.

4. A method for producing low-temperature resistant β-galactosidase according to claim 3, characterized in that: The fermentation medium is: MRS modified medium: peptone 1%, beef extract 1%, yeast powder 0.5%, K2HPO4 0.2%, diammonium citrate 0.2%, sodium acetate 0.5%, lactose 2%, Tween 80 0.1mL, MgSO4·7H2O0.058%, MnSO4·4H2O0.025%.

5. A method for producing low-temperature resistant β-galactosidase according to claim 3, characterized in that: The fermentation culture conditions are: Incubate at 30°C for 24 h.

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