Method for preparing beta-galactosidase-producing lactic acid bacteria permeabilized cells and galactooligosaccharide-rich yoghurt

Abstract

The invention discloses a method for preparing beta-galactosidase-producing lactic acid bacteria permeabilized cells and galactooligosaccharide-rich yogurt, which comprises the following steps: (1) inoculating a lactic acid bacteria strain capable of producing beta-galactosidase into an MRS culture medium taking lactose as a carbon source to obtain fermentation liquor; (2) carrying out low-temperature refrigerated centrifugation on the fermentation liquor obtained in the step (1), collecting wet thalli to obtain bacterial sludge, and washing the thalli by using a sterilized phosphate buffer solution; and adding sterile water to resuspend the thalli, pre-freezing, and transferring into a vacuum freeze dryer. And (3) redissolving the freeze-dried bacterial powder sample in the step (2) by using a sterilized phosphate buffer solution. The invention provides a simple and efficient lactic acid bacteria permeabilized cell preparation method, the prepared beta-galactosidase whole-cell catalyst can be directly added into food matrixes such as cow milk for in-situ hydrolysis of lactose and synthesis of GOS, and a new way is provided for in-situ preparation of GOS-rich yogurt.

Description

Technical Field

[0001] The invention relates to the field of enzyme preparation and application, and in particular to a method for preparing permeabilized lactic acid bacteria cells producing beta-galactosidase and galacto-oligosaccharide-rich milk. Background Art

[0002] β-galactosidase (EC3.2.1.23) is a glycoside hydrolase, also known as lactase, which has hydrolysis and transgalactosylation activities, can hydrolyze lactose and synthesize GOS. Currently, commercial β-galactosidase is mainly derived from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans, and the enzyme solution is obtained by conventional separation, extraction and purification methods. The purified enzyme solution can be directly used for lactose hydrolysis and conversion or used for lactose hydrolysis and conversion after immobilization treatment, but traditional separation and purification increases the cost of enzymes, limiting the application of β-galactosidase. Recent studies have found that β-galactosidase from lactic acid bacteria has unique advantages in the field of hydrolyzing lactose to synthesize GOS, such as the GOS product prepared by it has a higher probiotic index, and the strain itself has a GRAS status and good safety. However, β-galactosidase from lactic acid bacteria is mainly an intracellular enzyme, and the application process still requires the separation and extraction of the enzyme by physical or chemical methods. By preparing permeabilized cells, the cumbersome enzyme separation, extraction and purification steps can be avoided, while improving the activity and stability of the enzyme. Patent CN 102994589 A uses anhydrous ethanol to permeabilize Kluyveromyces lactis, and prepares GOS by adding the permeabilized cells to a lactose solution for cultivation. In addition, some studies have reported that β-galactosidase-producing Kluyveromyces lactis permeabilized cells can be prepared by using chloroform followed by freeze-drying (Rodriguez-Colinas et al., J. Agric. Food Chem., 2011), and permeabilized cells of Lactobacillus plantarum MCC2156 can be obtained by treating with 0.1% SDS and chloroform and used for lactose hydrolysis and GOS synthesis (Gobinath et al., Biotechnology Letters, 2014). However, the above permeabilization methods use organic solvents such as ethanol or chloroform, which limits the application of the whole-cell catalysts prepared by them in food processing. Patent CN 1737132 uses a repeated freeze-thaw method to treat Bacillus circulans ATCC 31382, Aspergillus oryzae ATCC 20423 and Candida pseudotropicalis ATCC46764 to prepare permeabilized cells with β-galactosidase activity and use them for the synthesis of GOS. Patent CN 102851220 uses high-pressure homogenization and ultrasound to extract β-galactosidase from Kluyveromyces fragilis, but the above methods are not suitable for industrial scale-up.

[0003] At present, GOS is mainly prepared by enzyme catalysis using lactose as a substrate. There is still a lot of lactose, glucose and galactose remaining in the product, which needs to be further separated and purified by column chromatography before it can be added to infant formula or other dairy products, which further increases the production and application cost of GOS. Cow's milk contains 4-5% lactose. Directly catalyzing the in-situ hydrolysis of lactose in cow's milk by β-galactosidase and synthesizing GOS has become an alternative solution for separating and purifying GOS. For example, invention patent CN 102232417 adds lactase to pasteurized milk for enzyme treatment to produce sterilized milk with a GOS content of 0.2-1.2%. Invention patent CN 102369998 A uses lactase from Aspergillus, Saccharomyces and Kluyveromyces to directly treat whole milk, and performs enzyme treatment at a temperature of 50°C to obtain 0.1-5.19g / 100g of GOS. Duraiswamy Gobinath et al. used SDS to permeabilize Lactobacillus plantarum. When the lactose concentration was 40% (w / v), the GOS yield was as high as 34% (w / w) after 12 hours of permeabilization. Wang et al. found that β-galactosidase-producing Streptococcus thermophilus cells treated with nisin can be used as whole-cell catalysts to efficiently hydrolyze lactose, but did not test whether it can synthesize GOS (Applied Microbiology and Biotechnology, 2020).

[0004] Invention patent CN 114258950 A uses Nurica lactase to add to heat-treated concentrated milk, stir at low temperature for 15-30 minutes, and stand for 18-26 hours to produce GOS, and then add starter Lactobacillus bulgaricus and thermophilic Streptococcus, Bifidobacterium bifidum, Bifidobacterium longum and other probiotics to ferment yogurt to prepare GOS-containing yogurt. However, there is no report on the use of permeabilized lactic acid bacteria cells prepared by freeze-drying technology as whole cell catalysts to directly hydrolyze lactose in milk to synthesize GOS. Summary of the invention

[0005] In view of this, the present invention provides a method for preparing permeabilized lactic acid bacteria cells producing β-galactosidase and galacto-oligosaccharide-rich milk.

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

[0007] A method for preparing permeabilized lactic acid bacteria cells producing β-galactosidase and galacto-oligosaccharide-rich milk, characterized by comprising the following steps:

[0008] (1) inoculating a lactic acid bacteria strain capable of producing β-galactosidase into an MRS medium with lactose as a carbon source, culturing to the late exponential growth phase, and obtaining a fermentation broth;

[0009] (2) The fermentation liquid obtained in (1) is subjected to low-temperature freezing centrifugation, and then the wet cells are collected to obtain bacterial sludge, and the cells are washed with sterile phosphate buffer to remove residual culture medium; sterile water is added to resuspend the cells, and the cells are transferred to a vacuum freeze dryer after pre-freezing, and the temperature is set to -30°C, -20°C, and -10°C in sequence under vacuum conditions, each for 1 hour; then the temperature is set to 0°C, 5°C, 10°C, 15°C, 20°C, and 25°C, each for 3 hours, to complete the vacuum freeze drying process;

[0010] (3) re-dissolving the freeze-dried bacterial powder sample in (2) with sterile phosphate buffer to obtain a lactic acid bacteria permeabilized cell suspension having β-galactosidase activity.

[0011] Preferably, in step (1), the culture temperature is 35-38°C, the oscillation frequency is 200-250 r / min, and the culture time is 12-24 h.

[0012] Preferably, in step (2), the low temperature is 4°C.

[0013] Preferably, in step (2) and step (3), the pH of the sterile phosphate buffer is 7.0.

[0014] Preferably, in step (2), the pre-freezing temperature is -80°C.

[0015] Preferably, in step (2), the vacuum condition is <1.0 Pa.

[0016] Preferably, in step (2), the vacuum freeze-drying process takes a total of 37 hours to complete.

[0017] Preferably, in step (3), the reconstitution ratio of the sterile phosphate buffer to the freeze-dried bacterial powder sample is 1 g:6 mL.

[0018] A method for in situ preparation of galacto-oligosaccharide-rich milk by using permeabilized lactic acid bacteria cells with β-galactosidase activity, comprising the following steps:

[0019] (4): adding the lactic acid bacteria permeabilized cell suspension having β-galactosidase activity obtained in step (3) to sterilized 12% (w / v) whole fat reconstituted milk to catalyze the lactose reaction,

[0020] The reconstituted milk is then incubated with enzyme treatment;

[0021] (5): Add commercial starter Lactobacillus bulgaricus and Streptococcus thermophilus at a ratio of 1 g / L to the enzyme-treated reconstituted milk obtained in step (4), ferment at 40-45° C. until the pH reaches 4.5-5.0, then stop fermenting, and refrigerate the fermented yogurt at 2-6° C. for post-ripening to obtain yogurt with a galacto-oligosaccharide content of 6 g / kg.

[0022] Preferably, in step (4), the reaction conditions for catalyzing the lactose reaction are: an enzyme amount of 1 to 10 U / mL, a reaction time of 1 to 5 h, and a reaction temperature of 40 to 60°C.

[0023] Compared with the prior art, the present invention has achieved the following technical effects:

[0024] The present invention provides a simple and efficient method for preparing permeabilized lactic acid bacteria cells. The prepared β-galactosidase whole-cell catalyst can be directly added into food matrices such as milk to carry out in-situ hydrolysis of lactose and synthesis of GOS, thus providing a new approach for in-situ preparation of GOS-rich yogurt. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a flow chart of a method for preparing permeabilized lactic acid bacteria cells producing β-galactosidase and galacto-oligosaccharide-rich milk. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0027] The plant lactobacillus (Lactobacillus plantarum) YLBGNL-S7 used in the following examples comes from the Food Microbiology and Biotechnology Research Center of Shihezi University and has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 2018397 and a deposit date of July 10, 2018. Lactobacillus kefiri (Lactobacillus kefiri) L6 comes from the Food Microbiology and Biotechnology Research Center of Shihezi University and has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20191030 and a deposit date of December 9, 2019. Pediococcus acidilactici (Pediococcus acidilactici) Y1 comes from the Food Microbiology and Biotechnology Research Center of Shihezi University and has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M20232025 and a deposit date of October 25, 2023.

[0028] Example 1: Cultivation of β-galactosidase-producing cells

[0029] A single colony of β-galactosidase-producing lactic acid bacteria was picked and inoculated into 20 mL of MRS liquid medium with lactose as the carbon source, and cultured at 37°C and 2000 r / min for 12 h to obtain a seed solution;

[0030] The activated seed liquid was inoculated into MRS medium with lactose as carbon source at a ratio of 2% (v / v), and fermented at 37°C and 200 r / min for 18 h to obtain fermentation liquid;

[0031] After the culture was completed, the cells were centrifuged at 8000 r / min for 15 min to harvest the β-galactosidase-producing bacteria.

[0032] The intracellular transglycosyl β-galactosidase producing bacteria are preferably Lactobacillus plantarum YLBGNL-S7, Lactobacillus kefiri L6, and Pediococcus acidilactici Y1.

[0033] The MRS liquid culture medium with lactose as the carbon source is preferably: 10 g / L peptone; 10 g / L beef extract; 5 g / L yeast extract; 10 g / L lactose; 1 ml / L Tween 80; 2 g / L K2HPO4; 5 g / L sodium acetate; 2 g / L diammonium citrate; 0.58 g / L MgSO4·7H2O; 0.25 g / L MnSO4·7H2O; 15 g / L agar; and sterilized at 115°C for 20 min.

[0034] Example 2: Preparation of crude enzyme solution and permeabilized cells

[0035] Wash the wet cells obtained after freeze centrifugation with pH 7.0 phosphate buffer for 3-5 times to remove the residual culture medium. Redissolve the wet cells with sterilized phosphate buffer solution to prepare a bacterial suspension. Add 1 mg / mL lysozyme to the bacterial suspension, incubate at 37°C for 3 hours, and then use an ultrasonic disruptor to low-temperature disrupt the bacterial suspension. The supernatant after freeze centrifugation of the bacterial suspension is the crude β-galactosidase enzyme solution;

[0036] Wash the wet cells obtained after freeze centrifugation with pH 7.0 phosphate buffer for 3-5 times to remove the residual culture medium. Add sterile water to resuspend the cells, pre-freeze at -80°C and transfer to a vacuum freeze dryer for freeze drying. After freeze drying, highly active β-galactosidase freeze-dried powder is obtained;

[0037] The conditions for the ultrasonic disruptor to perform low-temperature disruption of the bacterial suspension are preferably on for 2 seconds and off for 4 seconds, with continuous ultrasound for 15 minutes.

[0038] The freeze-drying conditions are preferably pre-cooled at -50°C for 3 hours, vacuumed at -40°C, and after the vacuum degree reaches 1.0 Pa, the temperatures are set to: -30°C, -20°C, -10°C for 1 hour each, and then the temperatures are set to 0°C, 5°C, 10°C, 15°C, 20°C, 25°C for 3 hours in sequence, and the vacuum freeze-drying process is completed in 37 hours in total.

[0039] Example 3: Detection of β-galactosidase activity in crude enzyme solution and permeabilized cells

[0040] Take 20 μL of appropriately diluted crude enzyme solution and freeze-dried powder and react with 80 μL of 0.1M phosphate buffer (pH 7.0) prepared with o-nitrobenzene-β-D-galactoside (ONPG) solution at 50°C for 10 min. After the reaction, add 100 μL of 1 mol / L sodium carbonate solution to terminate the reaction. Use an enzyme reader to measure the absorbance at a wavelength of 420 nm, and calculate the enzyme activity using the ONPG standard curve.

[0041] The results showed that the activity of crude enzyme solution extracted by ultrasonic crushing of Lactobacillus plantarum YLBGNL-S7 could reach 2400±12.6U / g wet bacteria, the apparent β-galactosidase activity of permeabilized cells could reach 2274±14.5U / g wet bacteria, and the relative extraction rate could reach 94.8%, as shown in Table 1.

[0042] The enzyme activity of the crude enzyme solution extracted by ultrasonic disruption of Lactobacillus kefir L6 can reach 363±7.25U / g wet bacteria, and the corresponding apparent β-galactosidase activity of permeabilized cells can reach 308±8.55U / g wet bacteria, and the relative extraction rate can reach 85%, as shown in Table 1.

[0043] The enzyme activity of the crude enzyme solution extracted by ultrasonic disruption of Pediococcus acidilactici Y1 can reach 139±3.26U / g wet bacteria, and the corresponding apparent β-galactosidase activity of permeabilized cells can reach 104±2.83U / g wet bacteria, and the relative extraction rate can reach 75%, as shown in Table 1.

[0044] Table 1: Apparent β-galactosidase activity of crude enzyme solution extracted by ultrasonication and permeabilized cells of different strains

[0045]

[0046] Example 4: Effect of different enzyme treatment temperatures on GOS content in yogurt

[0047] 1 mL of 12% (w / v) sterilized reconstituted milk was used as the matrix, and the YLBGNL-S7 permeabilized cells (3U / mL) obtained in Example 3 were added to the reconstituted milk, and the mixture was incubated in a constant temperature water bath at 40, 45, 50, 55, and 60° C. for 3 h to obtain enzyme-treated reconstituted milk containing GOS. Commercial starter (Lactobacillus bulgaricus and Streptococcus thermophilus) was added at a ratio of 1 g / L, stirred evenly, and packaged. When the mixture was fermented at 42° C. to pH 4.7, the fermentation was stopped, and the fermented yogurt was refrigerated at 4° C. for post-ripening to obtain the fermented yogurt rich in GOS.

[0048] The sugar composition and content of GOS yogurt prepared in situ under different incubation temperature conditions were determined by high performance liquid chromatography. The specific method is:

[0049] The fermented yogurt rich in GOS was diluted with triple distilled water to a sugar concentration of 1%, filtered with a 0.22 μm filter membrane, and then analyzed by HPLC injection. The injection volume was 10 μL, the chromatographic column was Hi-Plex Na Column 30 mm × 7.7 mm (Agilent), the mobile phase was triple distilled water, the flow rate was 0.25 mL / min, the column temperature was 80 °C, and the injection volume was 10 μL. The liquid phase results of the standard sample are shown in Figure 1 As shown in Table 2, the sugar composition and content of GOS-rich yogurt under different temperature incubation conditions are determined as shown in Table 2:

[0050] Table 2: Effect of enzyme treatment temperature on GOS content and residual lactose mass fraction in yogurt

[0051]

[0052]

[0053] The results in Table 2 show that as the temperature continues to rise from 40 to 50°C, the GOS content increases significantly, and the GOS content is the highest at 50°C, which is 6.68 g / kg. Thereafter, as the temperature continues to rise, the GOS content decreases significantly, and the GOS content at 60°C is only 2.06 g / kg.

[0054] Example 5: Effect of different enzyme treatment times on GOS content in yogurt

[0055] The YLBGNL-S7 permeabilized cells obtained in Example 3 were added to 1 mL of sterilized 12% (w / v) reconstituted milk, so that the enzyme concentration in the reaction system was 3U / mL, and incubated at 50°C for 1, 2, 3, 4, and 5 hours, respectively, to obtain enzyme-treated reconstituted milk containing GOS. Commercial starter (Lactobacillus bulgaricus and Streptococcusthermophilus) was added at a ratio of 1 g / L, stirred evenly and packaged, and fermented at 42°C until pH 4.7, then the fermentation was stopped, and the fermented yogurt was refrigerated at 4°C for post-ripening to obtain the fermented yogurt rich in GOS. The sugar composition and content of GOS yogurt prepared in situ under different temperature incubation conditions were determined by high performance liquid chromatography. The specific method is:

[0056] The GOS-rich fermented yogurt was diluted with triple distilled water to a sugar concentration of 1%, filtered with a 0.22 μm filter membrane, and then analyzed by HPLC injection, with an injection volume of 10 μL, a chromatographic column of Hi-Plex Na Column 30 mm × 7.7 mm (Agilent), a mobile phase of triple distilled water, a flow rate of 0.25 ml / min, a column temperature of 80°C, and an injection volume of 10 μL. The sugar composition and content of GOS-rich yogurt under different enzyme treatment time conditions are shown in Table 3:

[0057] Table 3: Effect of enzyme incubation time on GOS content and lactose residue in yogurt

[0058]

[0059]

[0060] The results showed that the GOS content increased significantly during the enzyme treatment time of 1h to 3h, and the GOS content was the highest at 3h, which was 6.21g / kg. After that, as the enzyme treatment time increased, the GOS content slowly decreased, and the GOS content at 5h was 5.09g / kg.

[0061] Example 6: Effect of different enzyme addition amounts on GOS content in yogurt

[0062] Using 1 mL of 12% (w / v) sterilized whole-fat reconstituted milk solution as the substrate, add the YLBGNL-S7 permeabilized cells obtained in Example 3 with apparent enzyme activity at concentrations of 1, 3, 5, 8, and 10 U / mL to the reconstituted milk solution, and at 50°C, the enzyme-treated reconstituted milk containing GOS is prepared. Add commercial starter (Lactobacillus bulgaricus and Streptococcus thermophilus) at a ratio of 1 g / L, stir evenly and package, and ferment at 42°C until pH 4.7, stop fermentation, and refrigerate the fermented yogurt at 4°C to obtain the fermented yogurt rich in GOS. The sugar composition and content of GOS yogurt prepared in situ under incubation conditions at different temperatures were determined by high performance liquid chromatography. The specific method is:

[0063] The GOS-rich fermented yogurt was diluted with triple distilled water to a sugar concentration of 1%, filtered with a 0.22μm filter membrane, and then analyzed by HPLC injection, with an injection volume of 10μL, a chromatographic column of Hi-Plex Na Column 30mm×7.7mm (Agilent), a mobile phase of triple distilled water, a flow rate of 0.25mL / min, a column temperature of 80°C, and an injection volume of 10μL. The sugar composition and content of GOS-rich yogurt under different enzyme addition conditions are shown in Table 4:

[0064] Table 4: Effect of enzyme addition amount on GOS content and residual lactose mass fraction in yogurt

[0065]

[0066] In Table 4, the results show that the GOS content increased significantly when the enzyme dosage increased from 1U / mL to 3U / mL, and the GOS content was the highest at 3U / mL, which was 6.84g / kg. After that, with the increase of enzyme dosage, the GOS content slowly decreased, and the GOS content was 3.16g / kg when the enzyme dosage was 10U / mL.

[0067] Example 7: GOS content in permeabilized cell-treated yogurt under optimal conditions

[0068] 1 mL of 12% (w / v) sterilized reconstituted milk solution was used as the substrate, and 3 U / mL of YLBGNL-S7 permeabilized cells obtained in Example 3 were added to the reconstituted milk solution, and the enzyme-treated reconstituted milk containing GOS was prepared at 50°C. Commercial starter (Lactobacillus bulgaricus and Streptococcus thermophilus) was added at a ratio of 1 g / L, stirred evenly and packaged, and fermented at 42°C until pH 4.7, then the fermentation was stopped, and the fermented yogurt was refrigerated at 4°C for ripening to obtain the GOS-rich fermented yogurt. The sugar composition and content of the GOS-rich yogurt obtained after fermentation and the control fermented milk were determined by high performance liquid chromatography. The specific method is:

[0069] The GOS-rich fermented yogurt was diluted with triple distilled water to a sugar concentration of 1%, filtered with a 0.22 μm filter membrane, and then analyzed by HPLC injection, with an injection volume of 10 μL, a chromatographic column of Hi-Plex Na Column 30 mm × 7.7 mm (Agilent), a mobile phase of triple distilled water, a flow rate of 0.25 mL / min, a column temperature of 80°C, and an injection volume of 10 μL. The sugar composition and content of GOS-rich yogurt under different enzyme treatment time conditions are shown in Table 5:

[0070] Table 5: Effects of permeabilized cell treatment fermentation and conventional fermentation on GOS content and residual lactose mass fraction in yogurt

[0071]

[0072] As shown in Table 5, the GOS content in the fermented yogurt treated with permeabilized cells can reach 6.84 g / kg, of which the transferred disaccharides and transferred trisaccharides are 2.48 g / kg and 3.36 g / kg, respectively, the oligosaccharides above the transferred trisaccharides account for 1.00 g / kg, and the residual lactose content is 7.19 g / kg. The GOS content in the fermented yogurt without enzyme treatment is only 0.17 g / kg, and the residual lactose is 37.36 g / kg.

[0073] Example 8: pH and TA values ​​of GOS-enriched yogurt and control yogurt at different fermentation times

[0074] Using 1 mL of 12% (w / v) sterilized reconstituted milk solution as a substrate, 3 U / mL of YLBGNL-S7 permeabilized cells obtained in Example 3 were added to the reconstituted milk solution, and the mixture was incubated at 50° C. for 3 h to obtain enzyme-treated reconstituted milk with GOS.

[0075] The commercial starter (Lactobacillus bulgaricus and Streptococcus thermophilus) was added to the enzyme-treated reconstituted milk containing GOS at a ratio of 1 g / L, stirred evenly and packaged, and fermented at 42°C until pH 4.7, then the fermentation was stopped, and the fermented yogurt was refrigerated at 4°C for ripening to obtain the fermented yogurt rich in GOS. The results are shown in Tables 6 and 7:

[0076] Table 6: Changes in pH and titratable acidity during fermentation of GOS-enriched yogurt

[0077]

[0078] Table 7: Changes in pH and titratable acidity during fermentation of control yogurt

[0079]

[0080] It can be seen from Tables 6 and 7 that the pH value of the fermented yogurt after enzyme treatment is slightly lower than that of the control yogurt, and the coagulation time is shortened.

[0081] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing permeabilized cells of lactic acid bacteria producing β-galactosidase, characterized in that: The following steps are involved: (1) inoculating a lactic acid bacteria strain capable of producing β-galactosidase into an MRS medium with lactose as a carbon source, culturing to the late exponential growth phase, and obtaining a fermentation broth; (2) The fermentation liquid obtained in (1) is subjected to low-temperature freezing centrifugation, and then the wet cells are collected to obtain bacterial sludge, and the cells are washed with sterile phosphate buffer to remove residual culture medium; sterile water is added to resuspend the cells, and the cells are transferred to a vacuum freeze dryer after pre-freezing, and the temperature is set to -30°C, -20°C, and -10°C in sequence under vacuum conditions, each for 1 hour; then the temperature is set to 0°C, 5°C, 10°C, 15°C, 20°C, and 25°C, each for 3 hours, to complete the vacuum freeze drying process; (3) re-dissolving the freeze-dried bacterial powder sample in (2) with sterile phosphate buffer to obtain a lactic acid bacteria permeabilized cell suspension having β-galactosidase activity.

2. The method for preparing permeabilized cells of lactic acid bacteria producing β-galactosidase according to claim 1, characterized in that: In the step (1), the culture temperature is 35-38°C, the oscillation frequency is 200-250 r / min, and the culture time is 12-24 h.

3. The method for preparing permeabilized cells of lactic acid bacteria producing β-galactosidase according to claim 1, characterized in that: In the step (2), the low temperature is 4°C.

4. The method for preparing permeabilized cells of lactic acid bacteria producing β-galactosidase according to claim 1, characterized in that: In the steps (2) and (3), the pH of the sterile phosphate buffer is 7.

0.

5. The method for preparing permeabilized cells of lactic acid bacteria producing β-galactosidase according to claim 1, characterized in that: In the step (2), the pre-freezing temperature is -80°C.

6. The method for preparing permeabilized cells of lactic acid bacteria producing β-galactosidase according to claim 1, characterized in that: In the step (2), the vacuum condition is <1.0 Pa.

7. The method for preparing permeabilized cells of lactic acid bacteria producing β-galactosidase according to claim 1, characterized in that: In the step (2), the vacuum freeze-drying process takes a total of 37 hours to complete.

8. The method for preparing permeabilized cells of lactic acid bacteria producing β-galactosidase according to claim 1, characterized in that: In the step (3), the reconstitution ratio of the sterile phosphate buffer to the freeze-dried bacterial powder sample is 1 g:6 mL.

9. A method for preparing oligogalactose-rich milk in situ using permeabilized lactic acid bacteria cells having β-galactosidase activity, characterized in that: The following steps are involved: (4): adding the lactic acid bacteria permeabilized cell suspension having β-galactosidase activity obtained in step (3) to sterilized 12% (w / v) whole fat reconstituted milk to catalyze the lactose reaction, The reconstituted milk is then incubated with enzyme treatment; (5): Add commercial starter Lactobacillus bulgaricus and Streptococcus thermophilus at a ratio of 1 g / L to the enzyme-treated reconstituted milk obtained in step (4), ferment at 40-45° C. until the pH reaches 4.5-5.0, then stop fermenting, and refrigerate the fermented yogurt at 2-6° C. for post-ripening to obtain yogurt with a galacto-oligosaccharide content of 6 g / kg.

10. The method for preparing galacto-oligosaccharide-rich milk in situ using permeabilized lactic acid bacteria cells having β-galactosidase activity according to claim 9, characterized in that: In the step (4), the reaction conditions for catalyzing the lactose reaction are: an enzyme amount of 1 to 10 U / mL, a reaction time of 1 to 5 hours, and a reaction temperature of 40 to 60°C.

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