Complex microbial inoculant with low lactulose utilization rate and application of complex microbial inoculant in preparation of yoghourt

Abstract

The invention relates to the technical field of food production and processing, in particular to a complex microbial inoculant with a low lactulose utilization rate and application of the complex microbial inoculant in preparation of yoghurt, the complex microbial inoculant comprises Streptococcus thermophilus 217-228 and Lactobacillus plantarum 217-229, the lactose content in the yoghurt is reduced, the lactose hydrolysis rate can reach 69.08%, the lactulose content is kept at a high level, and the complex microbial inoculant has the advantages that the lactulose utilization rate is high, and the lactulose utilization rate is high. And the yoghurt is suitable for people with lactose intolerance, retains the nutritional value of the yoghurt, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a composite bacterial agent with low lactulose utilization rate and application thereof in preparing yogurt, belonging to the technical field of food production and processing. Background Art

[0002] Lactose is a disaccharide found in mammalian milk, consisting of glucose and galactose. The digestion and absorption of lactose requires lactase on the brush border of the small intestine to break it down into monosaccharides before it can be absorbed and utilized by the human body. Lactose intolerance means that due to insufficient lactase activity, lactose cannot be completely digested and absorbed, which ferments in the intestine to produce gas, causing symptoms such as bloating and diarrhea.

[0003] Lactulose (β-D-galactosyl-1,4-D-fructose), also known as isomerized lactose and galactosyl fructose, is the product of the isomerization of the reducing end glucose of lactose to fructose. Lactulose does not exist naturally in raw milk, and different treatment methods produce different lactulose contents. Solid lactulose is in the form of white powder, easily soluble in water, and sweeter than lactose. It is low in calories and highly safe, can improve constipation, enhance the body's immunity, and reduce the level of blood toxins. It can be used as a functional sweetener in beverages, candies, jams, jellies, ice cream and other industries. The production methods of lactulose include chemical method, β-galactosidase method, diastereomerase method, etc. The extraction process is complicated and the price is relatively high. Cellobiose diastereomerase (CE enzyme, EC5.1.3.11) was first identified in white Ruminococcus albus. In order to solve the problem of high lactose content and weak functionality of yogurt in the prior art, the principle that cellobiose epimerase can directly isomerize lactose to generate lactulose is used. The enzyme is added during the pasteurization process, and the high temperature environment of pasteurization is used to isomerize part of the lactose to generate functional lactulose and epilactose, thereby reducing the lactose content in the final yogurt, which is suitable for patients with lactose intolerance. For example, Chinese patent document CN115191484A discloses a low-lactose yogurt containing prebiotics and a preparation method. During the fermentation process of yogurt, part of the lactose is converted into lactulose and epilactose, so that the finished yogurt contains prebiotic ingredients while meeting the low lactose standard. Patent CN100426975A provides a lactulose liquid milk and milk powder and a preparation method thereof, and the product can be used to prevent constipation, increase physical fitness and improve intestinal comprehensive health. At present, research has found that cellobiose epimerases from various lactic acid bacteria such as extremely thermophilic cellulose degrading bacteria, thermophilic bacteria, and thermophilic tennis bacteria can produce lactulose.

[0004] Yogurt is a fermented beverage obtained by microbial fermentation. The most commonly used fermentation agent is a mixture of thermophilic streptococci (str.thermophilus) and bulgaricus lactobacillus (L.bulgaricus). As a fermented dairy product, yogurt has high nutritional value and health benefits. During the yogurt fermentation process, lactic acid bacteria use lactose in milk as a carbon source to grow and reproduce, thereby reducing the lactose content, which is beneficial to patients with lactose intolerance. However, if lactulose is present, lactic acid bacteria will also use part of the lactulose to ferment and grow during the fermentation process, increase the acidification value, reduce the lactulose content, and reduce the nutritional value of yogurt. Therefore, it is of great practical significance to screen a strain that uses lactose but not lactulose to keep the lactulose content at a high level. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a composite bacterial agent with low lactulose utilization rate and application thereof in the preparation of yogurt. The strain provided by the present invention can efficiently utilize lactose and has a low lactulose utilization rate, and can reduce the lactose content and maintain the lactulose content at a high level during the preparation of yogurt.

[0006] The technical solution of the present invention is as follows:

[0007] A strain of Streptococcus thermophilus 217-228 was deposited in the General Microbiology Center of China Culture Collection Administration on October 21, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32290.

[0008] A strain of Lactobacillus plantarum (Lactobacillus plantarum) 217-229 was deposited in the General Microbiology Center of China Culture Collection Administration on October 21, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32289.

[0009] A composite bacterial agent comprises the above-mentioned thermophilic streptococci 217-228 and plant lactobacillus 217-229.

[0010] A method for preparing a composite bacterial agent, comprising the following specific steps: (1) inoculating thermophilic Streptococcus 217-228 into an activation medium for activation and culture, and then inoculating the obtained activated strain into a liquid medium for expansion and culture until OD 600 =1.8~2.2; (2) inoculating plant lactobacillus 217-229 into activation medium for activation culture, and then inoculating the obtained activated strain into liquid medium for expansion culture until OD 600=7.8-8.2; (3) mixing the thermophilic Streptococcus 217-228 obtained in step (1) and the plant lactobacillus 217-229 obtained in step (2) in a volume ratio of 1.5-2.5:0.5-1.5 to prepare a composite bacterial agent.

[0011] Preferably, according to the present invention, the activation culture medium and the liquid culture medium in step (1) are both LM17 culture medium, and the activation culture and expansion culture conditions are both static culture at 40-45°C.

[0012] Preferably, according to the present invention, the activation culture medium and the liquid culture medium in step (2) are both MRS culture medium, and the activation culture and expansion culture conditions are both static culture at 35-40°C.

[0013] The application of the composite bacterial agent in the preparation of low-lactose and high-lactulose yogurt.

[0014] The preparation method of the low-lactose high-lactulose yogurt comprises the following specific steps: adding cellobiose epimerase to raw milk to obtain sweet milk, and then inoculating the composite bacterial agent into the sweet milk at a volume ratio of 3-5% to ferment to obtain low-lactose high-lactulose yogurt.

[0015] Preferably according to the present invention, the cellobiose epimerase can convert lactose in raw milk into lactulose, and its encoding gene sequence is shown in SEQ ID NO.3.

[0016] According to the preferred embodiment of the present invention, the amount of cellobiose epimerase added is 2×10 -9 ~1.8×10 -8 kat / mL, reaction conditions are 60-90℃, pH 6.0-9.0, and reaction time is 30-180min.

[0017] Preferably according to the present invention, the fermentation condition is a constant temperature fermentation at 40-45° C. for 6-9 hours.

[0018] A probiotic with low lactulose utilization rate, characterized by comprising the above-mentioned Lactobacillus plantarum 217-229.

[0019] The preparation method of the above-mentioned probiotic agent is characterized by the following steps: inoculating plant lactobacillus 217-229 into an activation medium for activation and culturing, and then inoculating the obtained activated strain into a liquid medium for expansion and culturing until OD 600 =7.8~8.2, then you get the probiotic.

[0020] Preferably according to the present invention, the activation culture medium and the liquid culture medium are both MRS culture medium; and the activation culture and expansion culture conditions are both static culture at 35-40°C.

[0021] Application of the above probiotics in fermentation preparation or purification of lactulose products.

[0022] Beneficial effects:

[0023] The composite bacterial agent provided by the present invention can efficiently utilize lactose and has a low utilization rate of lactulose. In the process of preparing yogurt, it can not only reduce the lactose content, but also keep the lactulose content at a high level, retain the nutritional value of yogurt to the greatest extent, and cooperate with cellobiose epimerase to hydrolyze lactose up to 69.08%, which is very suitable for people with lactose intolerance. The probiotic agent provided by the present invention has a low utilization rate of lactulose. In the fermentation process of a product containing lactulose, the lactulose content can be kept at a high level, which helps to improve the nutritional value of the product and makes it have a greater advantage in the field of functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The growth of different thermophilic Streptococci cultured in different carbon sources for 24 hours.

[0025] Figure 2 This is the growth curve of Streptococcus thermophilus 217-228 on different carbon sources, where ■ is lactose and ● is lactulose.

[0026] Figure 3 The growth of different Lactobacillus plantarum strains cultured in different carbon sources for 24 hours.

[0027] Figure 4 This is the growth curve of Lactobacillus plantarum 217-229 on different carbon sources, where ■ is lactose and ● is lactulose.

[0028] Figure 5 The figure is the SDS-PAGE electrophoresis diagram of cellobiose epimerase, where M is a marker; 1 is a whole cell protein sample; 2 is an intracellular protein sample; 3 is an extracellular protein sample; and 4 is a purified protein sample.

[0029] Figure 6 A: Effect of temperature on the activity of cellobiose isomerase; B: Stability of cellobiose isomerase at 65℃(●), 70℃(■), 75℃(▲), 80℃(▼); C: Effect of pH on the activity of cellobiose isomerase; D: Stability of cellobiose isomerase at pH6.0(●), pH7.0(■), pH8.0(▲), pH9.0(▼).

[0030] Figure 7 A: Lactose conversion rate under optimal conditions with different enzyme concentrations; B: Line graph of lactose conversion rate changing over time under optimal enzyme concentration and reaction conditions.

[0031] Figure 8This is the curve of lactulose concentration changing with the fermentation time of thermophilic Streptococcus; among them, ■ represents thermophilic Streptococcus 217-115, and ● represents thermophilic Streptococcus 217-228.

[0032] Fig. 9 This is the curve of lactulose concentration changing with Lactobacillus plantarum fermentation time; wherein ■ represents Lactobacillus plantarum H3, and ● represents Lactobacillus plantarum 217-229. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with embodiments, but the scope of protection of the present invention is not limited thereto.

[0034] The contents not described in detail in the examples are all based on the conventional techniques in the art, and the experimental materials and reagents not described in detail are all common commercial products. Among them, the genome extraction kit, product purification kit and homologous recombination enzyme were purchased from Vazyme Co., Ltd. (Nanjing, China), and the plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd. (Beijing, China); lactose, glucose and lactulose were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Caldicellulosiruptor bescii was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number DSM 6725, and the bacterial solution concentration was 2.16×10 10 CFU / mL.

[0035] Example 1

[0036] Screening of Streptococcus thermophilus

[0037] Take 3cm of naturally fermented kimchi, vegetables, and soil 2 Cut into pieces, incubate at 42℃ for 24h in 100mL LM17 medium to activate the strain, shake well and dilute gradually, take 100μL and spread on LM17 solid medium containing bromocresol purple and incubate for 24h, pick a single colony and incubate at 42℃ for 24h in 5mL LM17 medium, measure the strain OD 600 The 16S rDNA gene sequence was used for sequencing, and a total of 31 thermophilic Streptococcus strains were screened. Subsequently, the 31 strains were inoculated into 5 mL LM17 medium and LM17 medium modified with lactulose at a volume ratio of 2% and cultured at 42°C for 24 h. The OD of the strains was determined. 600 Values, such as Figure 1 As shown, strain OD 600 The values ​​can reflect the utilization of lactose and lactulose by the strains, among which strains 217-228 are target strains that can efficiently utilize lactose but have low utilization of lactulose. The 16S rDNA sequencing results thereof are shown in SEQ ID NO.1.

[0038] The thermophilic Streptococcus thermophilus obtained by the above screening was named Streptococcus thermophilus 217-228, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32290.

[0039] Thermophilic Streptococcus 217-228 were cultured in LM17 medium and LM17 medium modified with lactulose, incubated at 42°C for 24 h, and samples were taken every 2 h to measure OD 600 Value, draw a growth curve, such as Figure 2 As shown, S. thermophilus 217-228 showed the ability to utilize lactose efficiently and lactulose inefficiently, which was consistent with the expected behavior.

[0040] The steps of 16S rDNA gene sequencing are as follows: extracting DNA of the screened strain using a kit, and performing PCR amplification using universal primers for bacterial 16S rDNA gene;

[0041] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0042] 1492R:5'-GGTTACCTTGTTACGACTT-3'

[0043] The 16S rDNA gene sequence amplification product of the strain was entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained 16S rDNA sequence was subjected to BLAST analysis with the existing sequences in the NCBI database to confirm the strain species.

[0044] The formula of the LM17 medium is (1 L): 5.0 g soytone, 2.5 g peptone, 2.5 g casein peptone, 2.5 g yeast extract powder, 5.0 g beef extract powder, 0.5 g sodium ascorbate, 19.0 g sodium β-glycerophosphate, 0.25 g magnesium sulfate, 10 g lactose, and dilute to 1 L with deionized water;

[0045] The lactulose-modified LM17 culture medium is prepared by replacing 10.0 g / L lactose with 10.0 g / L lactulose on the basis of the original culture medium.

[0046] Example 2

[0047] Screening of Lactobacillus plantarum

[0048] Take 1 mL of naturally fermented dairy products in 100 mL MRS medium, incubate at 37 ° C for 24 h to activate the strain, shake well and dilute in gradients, take 100 μL and spread on MRS solid medium containing bromocresol purple and incubate for 24 h, pick a single colony and incubate at 37 ° C for 24 h in 5 mL MRS medium, and measure the strain OD 600 The 16S rDNA gene sequence was used for sequencing. The sequencing steps were the same as those in Example 1. A total of 6 strains of Lactobacillus plantarum were screened out. Subsequently, the 6 strains were inoculated at a volume ratio of 2% in 5 mL of MRS medium modified with lactose and lactulose and cultured at 37°C for 24 h. The strain OD was measured. 600 Values, such as Figure 3 As shown, strain OD 600 The value can reflect the utilization of lactose and lactulose by the strain. As can be seen from the figure, strain H4 has a low utilization rate of lactulose, but its utilization rate of lactose is also low. The strain has poor growth potential and is difficult to ferment. Among them, strains 217-229 are target strains that can efficiently utilize lactose but have a low utilization rate of lactulose. The 16S rDNA sequencing results are shown in SEQ ID NO.2.

[0049] The Lactobacillus plantarum obtained by the above screening was named Lactobacillus plantarum 217-229, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on October 21, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32289.

[0050] The strains 217-229 were cultured in MRS medium modified with lactose and lactulose at 37°C for 24 h, and samples were taken every 2 h to measure OD 600 Value, draw a growth curve, such as Figure 4 Lactobacillus plantarum 217-229 showed the ability to utilize lactose efficiently and lactulose in a low efficiency, which was consistent with the expected behavior.

[0051] Among them, the formula of the above-mentioned MRS culture medium is (1L): peptone 10.0g, beef extract powder 10.0g, yeast extract powder 5.0g, sodium acetate 3.02g, K2HPO4 1.16g, MgSO40.05g, triammonium citrate 2.0g, MnSO4 0.03g, Tween 801.0mL, glucose 20g, and deionized water is made up to 1L.

[0052] The lactose-modified MRS medium is prepared by replacing 20 g / L glucose with 20.0 g / L lactose on the basis of the original medium;

[0053] The lactulose-modified MRS culture medium is prepared by replacing 20 g / L glucose with 20.0 g / L lactulose on the basis of the original culture medium.

[0054] Example 3

[0055] Purification of cellobiose epimerase

[0056] Caldicellulosiruptor bescii was inoculated into LB liquid medium at a volume ratio of 2%, and cultured overnight at 37°C and 200 rpm before collection. The gene was extracted using a genomic kit, and the gene sequence is shown in SEQ ID NO.3. The gene of cellobiose epimerase was amplified using universal primers:

[0057] CE-NdeⅠ-F:5'-GCCTGGTGCCGCGCGGCAGCCATATGGATATCACTAAGTTCAA-3'

[0058] CE-XhoⅠ-R:5'-CAGTGGTGGTGGTGGTGGTGCTCGAGACCAACACGCTTGATGATTT-3'

[0059] The obtained cellobiose epimerase gene and vector pET-22b(+) were digested with restriction endonucleases NdeⅠ and XhoⅠ, respectively, and purified with Product purification Kit (Vazyme, 115 Nanjing, China), and then the two fragments were connected with T4 DNA ligase. The connected product was then transformed into Escherichia coli XL1-Blue competent cells to obtain recombinant strains. The recombinant strains were screened on LB solid medium containing ampicillin, and the recombinant plasmids were extracted and sequenced by Biotech Bioengineering Solabio Co., Ltd (Shanghai, China). The verified plasmids were then transformed into Escherichia coli BL21 (DE3).

[0060] The pET-22b-CE vector was introduced into Escherichia coli BL21 (DE3) to obtain the recombinant strain E. coli BL21 (DE3) / pET-22b-CE, and IPTG was added at a final concentration of 0.5 mM for 24 hours at 16°C. The induced bacterial liquid was collected, centrifuged at 10,000 rpm for 5 min, washed twice with PBS buffer (pH 7.0), and resuspended in 20 mmol L -1The E. coli cells were lysed by ultrasonication at an intensity of 400 wt and a frequency of 5 s / 5 s for 99 cycles. The whole cell lysate was centrifuged at 4 °C and 12,000 rpm for 15 min, the supernatant was collected, filtered through a 0.22 μm membrane, and purified by Ni column.

[0061] The purification process includes adding the following solutions in sequence: 5 mL of binding buffer, filtering to obtain a supernatant sample; washing with 5 mL of washing buffer, collecting the eluate; dialyzing overnight in a phosphate buffer at pH 7.4 to remove imidazole ions and obtain purified cellobiose isomerase.

[0062] The components of the binding buffer are: 10 mmol L -1 Phosphate buffer, 500 mmol L -1 NaCl, 25mmolL -1 Imidazole, solvent water, pH 7.4.

[0063] Washing buffer composition: 10 mmol L -1 Phosphate buffer, 500 mmol L -1 NaCl, 50 mmol L -1 Imidazole, solvent water, pH 7.4.

[0064] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze whole cells, intracellular, Ni column flow-through fractions, and protein samples. The samples were added with SDS-PAGE sample buffer and boiled for 5 minutes. SDS-PAGE was performed using 50 g / L stacking gel and 120 g / L separating gel to separate protein bands, followed by staining with Coomassie Brilliant Blue R-250 (Sangon, Shanghai, China). The expressed cellobiose epimerase was observed in the host bacteria, with a size of approximately 43 kDa. After purification, the obtained protein bands were as shown in Figure 2. Figure 5 shown.

[0065] The SDS-PAGE sample buffer composition is: 0.25 mol L -1 Tris-HCl buffer, pH 6.8; 0.5 vol / vol glycerol, 100 g / L SDS, 0.05 vol / vol 2-mercaptoethanol, 0.5 g / L bromophenol blue, solvent water.

[0066] Example 4

[0067] Enzymatic Characterization of Purified Cellobiose Epimerase

[0068] Using lactose as substrate, the activity of purified cellobiose epimerase was determined under different temperature conditions. 100 μL 250 g / L lactose, 100 μL 5.0 g / L cellobiose epimerase and 800 μL 12.5 mmol L -1 The enzyme assay reaction was prepared by mixing with phosphate buffer (pH 7.0) to obtain 1 mL of reaction system, and the temperature stability of the enzyme was determined at 65°C, 70°C, 75°C, and 80°C for different reaction times. Different buffers were replaced with the above contents, and the reaction was carried out in citric acid buffer (10 mmol L -1 , pH 5.0-6.0), phosphate buffer (10 mmol L -1 , pH 6.5-8.0), Tris-HCl buffer (10 mmol L -1 , pH 8.5-9.0), sodium bicarbonate-sodium hydroxide buffer (10 mmol L -1 , pH 9.0-11.0) for 60 min to determine the pH stability of the enzyme, and the residual activity was determined by incubating the cellobiose isomerase at different pH values ​​(6.0, 7.0, 8.0 and 9.0) for different periods of time. In each experiment, the highest activity was considered as 100% and the relative activity was calculated.

[0069] The method for determining the enzymatic activity of the cellobiose epimerase is as follows: 1.0 mL of the enzyme solution is mixed with 1.0 mL of a 50 g / L lactose solution, incubated at 75°C for 30 min, and the reaction is terminated in a boiling water bath for 10 min. One unit of cellobiose epimerase activity is defined as the enzyme catalyzing the production of 1 μmol of lactulose per second. The enzymatic activity of cellobiose epimerase is calculated as follows:

[0070] Cellobiose epimerase activity (kat / g):

[0071] Where: c represents the concentration of lactulose in the measurement solution (mol L -1 ), 10 represents the total volume of the reaction reagent (mL), D is the dilution ratio, 1 represents the volume of the enzyme solution involved in the reaction (mL), V represents the volume of the dissolved enzyme powder (mL), m represents the concentration of cellobiose diastereomerase in the measurement solution (g / L), 30 represents the reaction time (min), 1.67×10 -8 Indicates the conversion from U to kat, 1U = 1.67 × 10 -8 kat.

[0072] like Figure 6 As shown in Figure 2A, the maximum enzyme activity was observed at 75°C, and the catalytic activity exceeded 90% in the range of 65°C to 70°C, but a significant decrease in activity was observed above 75°C. Figure 6As shown in Figure 1-B, the temperature stability curve of cellobiose isomerase shows that the enzyme remains stable at 65-70°C for 4 hours with a relative activity greater than 90%. However, after incubation at 65°C and 70°C for 24 hours, the relative activity drops to 78% and 61%, respectively. Figure 6 -C. Figure 6 -D, the activity and stability of the enzyme were further studied in the pH range of 6.0-10.0, and the maximum activity was observed at pH 7; even after incubation for 24 hours at pH 6.0 and 7.0, the relative activity of the enzyme was still higher than 78% and 74%, respectively. In summary, the optimal temperature of the enzyme is 75°C and the optimal pH is 6.5.

[0073] Example 5

[0074] Preparation of sweet milk containing cellobiose epimerase

[0075] Different amounts of cellobiose isomerase were added to the raw milk system to make the final enzyme content 2×10 -9 ~1.8×10 -8 kat / mL, and then reacted at the optimum temperature of 75℃ for 1h, 1.5h, 2h, 2.5h, and 3h after mixing. Three parallel experiments were set for each enzyme addition amount. Raw milk without cellobiose isomerase was used as a blank control, and sweet milk was obtained after cellobiose isomerase reaction. HPLC was used to determine the content of lactose and lactulose to determine the optimal enzyme dosage of cellobiose isomerase required for preparing sweet milk.

[0076] The above lactose and lactulose determination steps are as follows: the mixture is centrifuged at 10000 rpm for 10 min, and the supernatant is filtered through a 0.22 μm filter membrane to obtain an HPLC sample. The raw milk and sweet milk samples are pretreated: 5 mL of 45 mmol L -1 The sulfuric acid solution was shaken with a vortexer for 1 min. The resulting mixture was centrifuged at 6000 rpm for 20 min, and the supernatant was filtered with a 0.22 μm filter membrane to obtain an HPLC sample.

[0077] The determination of carbohydrates in samples was performed using a Shimadzu superior LC-20A HPLC system, which consisted of a RID-10A detector, an LC-20AT HPLC pump, and a SIL-20A autosampler. The chromatographic column was a glycosyl paktm column (6.5×300 mm), 85°C, the injection volume was 20 μL, and the mobile phase was water (flow rate was 0.2 mL / min); different concentrations of lactose (10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L) and lactulose (5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L) were used to draw calibration curves.

[0078]

[0079] c is the concentration of lactulose in the sample solution calculated from the standard curve (unit: mg / L); c 0 is the concentration of lactulose in the blank calculated from the standard curve (unit: mg / L); V is the volume of the fixed volume (unit: mL); m is the sample weight (unit: mL); and f is the dilution factor.

[0080] Cellobiose epimerase can break down lactose into epiliac and lactulose. Figure 7 -A shows that when 1.60×10 -8 kat / mL of cellobiose epimerase, the maximum conversion rate of lactulose was 44.16%, and the conversion rate did not change significantly with the further increase of enzyme concentration. Next, in order to determine the optimal reaction time, the reaction was carried out at 1.60×10 -8 kat / mL cellobiose isomerase, the reaction time was extended, such as Figure 7 -B, the conversion rate reached the maximum conversion rate of 54.18% after 60 min. In summary, the optimal addition amount of cellobiose isomerase is 1.60×10 -8 kat / mL, and the optimal reaction time was 60min.

[0081] Example 6

[0082] Thermophilic Streptococcus 217-228 was inoculated into LM17 medium at a volume ratio of 2% and placed at 42°C for 24 h for activation culture. The activated strain was then expanded to OD 600 =2.0; inoculated into a 20 g / L lactulose solution at a volume ratio of 2% and incubated at 42°C for 24 h, sampling was performed every 2 h, and the lactulose content was determined by HPLC. The detection method was the same as in Example 5, and the 20 g / L lactulose solution was used as a blank control. The results are shown in Figure 8 As shown, the utilization rate of lactulose by thermophilic Streptococcus 217-228 was low, and the lactulose retention rate was 86.1% after 24 hours of fermentation, while the lactulose retention rate of another thermophilic Streptococcus 217-155 screened was only 41% after 24 hours of fermentation.

[0083] Example 7

[0084] Lactobacillus plantarum 217-229 was inoculated into MRS medium at a volume ratio of 2% and then cultured at 37°C for 24 h for activation. The activated strain was then expanded to OD 600=8.0; inoculated into a 20 g / L lactulose solution at a volume ratio of 2% and incubated at 37°C for 24 h, sampling was performed every 2 h, and the lactulose content was determined by HPLC. The detection method was the same as in Example 5, and the 20 g / L lactulose solution was used as a blank control. The results are shown in Fig. 9 As shown, the utilization rate of lactulose by Lactobacillus plantarum 217-229 was low, and the lactulose retention rate was 90.4% after 24 hours of fermentation, while the lactulose consumption of another strain of Lactobacillus plantarum H3 was significantly increased after 24 hours of fermentation.

[0085] Example 8

[0086] Preparation of yogurt / sweet yogurt

[0087] Take 100 mL of the above raw milk and the sweet milk obtained after the cellobiose epimerase treatment under the optimal conditions, respectively put them into 100 mL plastic containers, and heat them in a boiling water bath for 10 min to inactivate the cellobiose epimerase, wherein the raw milk without the cellobiose epimerase is used as a blank control. Add 1.5 mL of plant lactobacillus 217-229 obtained by culturing in Example 7 to the above two groups of containers, respectively, and ferment at 42°C for 8 hours, the raw milk is fermented into yogurt, and the sweet milk is fermented into sweet yogurt. Move the yogurt and sweet yogurt into a 4°C refrigerator, and after 12 hours, measure the lactose and lactulose contents according to the method of Example 5.

[0088] The concentrations of lactose and lactulose in the system before and after fermentation with Lactobacillus plantarum are shown in Table 1. Before fermentation, the lactose content in sweet milk was 21.68 g / L and the lactulose content was 8.72 g / L. The contents of lactose and lactulose in the fermented sweet yogurt were 12.32 g / L and 7.84 g / L, respectively. After fermentation with Lactobacillus plantarum 217-229, the lactose content decreased by 43.17% and the lactulose content decreased by only 10.09%. Before fermentation, the lactose content of raw milk was 50.00 g / L. In the sweet yogurt made after fermentation, the lactose content decreased by 73.36%.

[0089] Example 9

[0090] Preparation of yogurt / sweet yogurt

[0091] Since yogurt and sweet yogurt fermented by Lactobacillus plantarum alone cannot coagulate, other probiotics need to be added during the yogurt production process. The pretreatment method is the same as that in Example 8. 2 mL of thermophilic Streptococcus 217-228 obtained by culture in Example 6 and 1 mL of Lactobacillus plantarum 217-229 obtained by culture in Example 7 are added to the above two groups of containers respectively, and fermented at a constant temperature of 42° C. for 8 hours. The yogurt and sweet yogurt are moved into a 4° C. refrigerator, and the lactose and lactulose contents are determined according to the method of Example 5 after 12 hours.

[0092] The concentrations of lactose and lactulose in the system before and after co-fermentation with thermophilic streptococci and plant lactobacillus are shown in Table 1. The lactose content in sweet milk before fermentation was 21.68 g / L and the lactulose content was 8.72 g / L. The lactose and lactulose contents in the fermented sweet yogurt were 15.46 g / L and 6.62 g / L, respectively. After fermentation with thermophilic streptococci 217-228 and plant lactobacillus 217-229, the lactose content decreased by 28.69% and the lactulose content decreased by 24.08%. Before fermentation, the lactose content of raw milk was 50.00 g / L, and the lactose content in the sweet yogurt made after fermentation decreased by 69.08%.

[0093] Table 1. Concentrations of lactose and lactulose in raw milk and sweet milk before and after fermentation

[0094]

Claims

1. A strain of Streptococcus thermophilus 217-228 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32290.

2. A strain of Lactobacillus plantarum (Lactobacillus plantarum) 217-229 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32289.

3. A composite bacterial agent, characterized in that: The composite bacterial agent comprises the thermophilic streptococci 217-228 described in claim 1 and the plant lactobacillus 217-229 described in claim 2.

4. The method for preparing the composite bacterial agent according to claim 3, characterized in that: The steps are as follows: (1) inoculating thermophilic Streptococcus 217-228 into an activation medium for activation culture, and then inoculating the activated strain into a liquid medium for expansion culture until the OD 600 =1.8~2.2; (2) inoculating plant lactobacillus 217-229 into activation medium for activation culture, and then inoculating the obtained activated strain into liquid medium for expansion culture until OD 600 =7.8-8.2; (3) mixing the thermophilic Streptococcus 217-228 obtained in step (1) and the plant lactobacillus 217-229 obtained in step (2) in a volume ratio of 1.5-2.5:0.5-1.5 to prepare a composite bacterial agent.

5. The preparation method according to claim 4, characterized in that: The activation culture medium and liquid culture medium in step (1) are both LM17 culture medium, and the activation culture and expansion culture conditions are both static culture at 40-45°C.

6. The preparation method according to claim 4, characterized in that: The activation culture medium and liquid culture medium in step (2) are both MRS culture medium, and the activation culture and expansion culture conditions are both static culture at 35-40°C.

7. Use of the composite bacterial agent according to claim 3 in the preparation of low-lactose and high-lactulose yogurt.

8. The use according to claim 7, characterized in that: The following steps are involved: Cellobiose isomerase is added to raw milk to obtain sweet milk, and then the composite bacterial agent according to claim 2 is inoculated into the sweet milk at a volume ratio of 3-5% to ferment to obtain low-lactose and high-lactulose yogurt.

9. The use according to claim 8, characterized in that: The cellobiose epimerase can convert lactose in raw milk into lactulose, and its encoding gene sequence is shown in SEQ ID NO.3; the cellobiose epimerase is added in an amount of 2×10 -9 ~1.8×10 -8 kat / mL, reaction conditions are 60-90℃, pH 6.0-9.0, and reaction time is 30-180min.

10. The use according to claim 8, characterized in that: The fermentation condition is constant temperature fermentation at 40-45° C. for 6-9 hours.

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