Streptococcus thermophilus and application thereof
By isolating high-vibrant lactase-based Streptococcus thermophilus from yak yogurt, the problem of low hydrolysis rate of the existing lactase hydrolysis method is solved, and the efficient hydrolysis of lactose is achieved, which increases the sweetness and the demand for pure natural foods are improved, and production is reduced. cost.
Patent Information
- Application Number
- CN202411604460.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-12
- Publication Date
- 2025-02-18
AI Technical Summary
The existing lactase hydrolysis method is relatively low when preparing low-lactose milk or fermented milk, and cannot fully meet the ideal sweetness requirements. At the same time, the process of industrial production and purification of lactase is complex and costly.
A novel high-vibrant lactase-producing Streptococcus thermophilus was isolated from yak yogurt, and was classified as Streptococcus salivary thermophilus subsp.thermophilus, and the collection number is CGMCC No. 31799. The lactase activity of this strain can reach more than 150U/mL, which can efficiently hydrolyze lactose and prepare fermented milk without added sucrose and sweetener.
It achieves efficient hydrolysis of lactose, improves the sweetness of fermented milk, meets consumers' demand for pure natural foods, and reduces the amount of lactase added and reduces production costs.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of fermentation strains, and in particular to a thermophilic streptococcus and application thereof. Background Art
[0002] Lactose is the main sugar in milk and is less sweet than sucrose. Lactase can hydrolyze lactose into sweeter and more easily absorbed monosaccharides - glucose and galactose, also known as β-D-galactosidase. At the same time, lactase also has the transfer effect of galactosidase, and can use lactose to synthesize oligosaccharides. Lactase is currently widely used in the field of dairy processing to prepare lactose-free or low-lactose products, solving the problem that people with lactose intolerance cannot normally consume dairy products. Lactase is widely present in animals, plants and microorganisms, among which lactase from microorganisms is the most widely studied. At present, lactase from microorganisms has a high industrial application value.
[0003] The commonly used lactase in industry mainly comes from yeast, mold and thermophilic bacteria. The enzymatic properties of microbial β-galactosidase from different sources are very different. Especially for strains in extreme environments (such as thermophilic bacteria, psychrophilic bacteria, etc.), the lactase they produce has special excellent properties. Most of the lactase used in industry comes from molds (Aspergillus spp.) and Kluyveromyces spp.). With the research on β-galactosidase, lactase from lactic acid bacteria has received widespread attention. As probiotics, lactic acid bacteria play an important role in human health and are widely used in the food industry. They are also hot strains for studying the source of lactase.
[0004] At present, the lactase hydrolysis method is often used in the industry to prepare low-lactose milk or fermented milk, that is, using medium-high temperature enzymes to hydrolyze for 2-4 hours at 30-40°C. However, due to the limitation of lactase activity, the hydrolysis rate of most lactases is low, the degree of hydrolysis is not complete, and the product cannot reach the ideal sweetness requirement; the process of industrial production and purification of lactase is cumbersome and costly.
[0005] At the same time, consumers now pay great attention to the safety and health of food, and the demand for pure natural food is increasing. There are many zero-sugar fermented milk products on the market, and some products have added sugar substitutes - sweeteners, such as aspartame, stevioside, erythritol, sucralose, etc. Although it can provide a certain sweetness, sweeteners will bring a certain discomfort to consumers, and sweeteners are food additives and do not meet the requirements of pure natural food. The other part is to not add any sugar source substances, use ordinary lactase to hydrolyze lactose to achieve the production of pure natural food, but the degree of hydrolysis is not complete, resulting in the acidity of the product becoming more and more obvious during the shelf life, which brings an unpleasant experience to consumers.
[0006] Therefore, developing a strain that produces high-activity lactase and can be used to prepare fermented milk without sucrose or sweeteners is of great significance to the development of the dairy industry. Summary of the invention
[0007] The purpose of the present invention is to provide a thermophilic streptococcus and application thereof in view of the problems existing in the prior art.
[0008] In order to achieve the above object, the technical solution adopted by the present invention is:
[0009] A thermophilic streptococcus, characterized in that it is classified and named Streptococcus salivarius subsp. thermophilus; it is deposited in the China General Microbiological Culture Collection Center, the preservation time is September 2, 2024, and the preservation number is CGMCC No.31799.
[0010] The invention separates a novel thermophilic streptococcus producing high-activity lactase from yak yogurt. The lactase activity of the thermophilic streptococcus can reach above 150 U / mL, and lactose can be hydrolyzed into glucose and galactose with higher sweetness. The fermented milk prepared by using the thermophilic streptococcus can realize the non-addition of sweeteners and sucrose, which can meet the consumer's requirements for the ideal sweetness of the fermented milk and the demand for pure natural food. At the same time, the addition amount of lactase is reduced in industry, which further saves the cost and is convenient for popularization and application.
[0011] The present invention also protects the culture method of the thermophilic streptococcus.
[0012] The above-mentioned culture method of thermophilic streptococcus comprises the following steps:
[0013] Streptococcus thermophilus was inoculated into M17 medium for culture;
[0014] Preferably, the culture medium comprises sodium citrate at a mass concentration of 2‰-7‰, the culture temperature is 35-45°C, and the culture pH is 6.5-7.5.
[0015] Another object of the present invention is to provide the application of the thermophilic streptococcus.
[0016] The application of the thermophilic streptococcus in the preparation of high-activity lactase products and / or fermented dairy products.
[0017] A method for preparing fermented milk using the thermophilic streptococcus comprises the following steps:
[0018] The seed culture solution of the thermophilic streptococcus is inoculated into the milk product to be fermented, and fermented to obtain fermented milk.
[0019] Furthermore, the seed culture solution of Streptococcus thermophilus is prepared by the following steps:
[0020] Step 1, activating the thermophilic Streptococcus using a slant culture medium;
[0021] Step 2: placing the activated thermophilic streptococcus in a liquid culture medium for culturing to obtain a seed culture solution of the thermophilic streptococcus.
[0022] Furthermore, in step 1, the slant culture medium includes M17 culture medium, the pH is 6.5-7.5, the activation temperature is 35-45° C., and the activation time is 24-72 h.
[0023] Furthermore, in step 2, the liquid culture medium is cultured at a temperature of 35-45° C. and the culture time is 16-24 h.
[0024] Furthermore, the milk product to be fermented includes pasteurized milk that has been subjected to standardization, hydration, materialization and homogenization treatments.
[0025] Furthermore, during the preparation of fermented milk, the amount of seed culture solution added accounts for 3% to 5% of the volume of the fermented milk product.
[0026] Preferably, the fermented dairy product comprises sodium citrate in a mass concentration of 2‰-7‰.
[0027] Furthermore, during the preparation of fermented milk, acid-producing bacteria are inoculated, and the acid-producing bacteria include at least one of Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus helveticus; the inoculation amount of the acid-producing bacteria is 1×10 3 ~1×10 6 cfu / mL.
[0028] Furthermore, during the preparation of fermented milk, the fermentation temperature is 35-42°C, the fermentation time is 4-8h, and the acidity at the end of the fermentation is 70-75°T.
[0029] The present invention also provides a probiotic bacterial preparation comprising the thermophilic streptococcus.
[0030] A probiotic preparation comprises the live bacteria of the thermophilic streptococcus and / or its fermentation product.
[0031] A freeze-dried bacterial powder is a powder preparation prepared by vacuum freeze-drying a mixture of the thermophilic streptococcus and a cryoprotectant; the cryoprotectant includes a carbohydrate protectant and a protein protectant;
[0032] The mass ratio of the carbohydrate protective agent to the protein protective agent is 1:1-3;
[0033] The total mass concentration of the cryoprotectant in the freeze-dried bacterial powder is 100-300 g / L.
[0034] The present invention provides a freeze-dried bacterial powder, which is a powder preparation prepared by vacuum freeze-drying a mixture of the thermophilic streptococcus and a cryoprotectant. By controlling the raw material selection and proportion of the cryoprotectant, the survival rate of the thermophilic streptococcus in the prepared freeze-dried bacterial powder can reach more than 94%, and the number of live bacteria in the bacterial powder can reach 5.0×10 11 cfu / mL or above, which greatly improves the use value of the strain and lays a foundation for its industrial application.
[0035] Furthermore, the carbohydrate protective agent includes at least one of trehalose and sorbitol, and the protein protective agent includes at least one of whey protein and skimmed milk powder; preferably, the carbohydrate protective agent is trehalose, and the protein protective agent is skimmed milk powder.
[0036] Furthermore, the freeze-dried bacterial powder also includes ascorbic acid and / or sodium citrate; in the freeze-dried bacterial powder, the mass concentration of ascorbic acid is 10-30 g / L, and / or the mass concentration of sodium citrate is 2-7 g / L.
[0037] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:
[0038] 1. The present invention separates a new type of thermophilic streptococcus that produces high-activity lactase from yak yogurt. The lactase activity of the thermophilic streptococcus can reach more than 150U / mL, and lactose can be hydrolyzed into glucose and galactose with higher sweetness. The fermented milk prepared by using the thermophilic streptococcus can achieve no addition of sweeteners and sucrose, which can meet consumers' requirements for the ideal sweetness of fermented milk and the demand for pure natural food. At the same time, the amount of lactase added is reduced in industry, which further saves costs and is convenient for promotion and application.
[0039] 2. The present invention provides a freeze-dried bacterial powder, which is a powder preparation prepared by vacuum freeze-drying a mixture of the above-mentioned thermophilic streptococcus and a cryoprotectant. By controlling the raw material selection and proportion of the cryoprotectant, the survival rate of thermophilic streptococcus in the prepared freeze-dried bacterial powder can reach more than 94%, and the number of viable bacteria in the bacterial powder can reach 5.0×10 11 cfu / mL or above, which greatly improves the use value of the strain and lays a foundation for its industrial application. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Figure 1 The results of SDS-PAGE detection of lactase expression and purity.
[0041] Figure 2 This is the detection diagram of HPAEC on hydrolysis products.
[0042] Figure 3 This is the Swissmodel structure diagram of lactase.
[0043] Figure 4 This is a graph showing the results of lactase activity testing of sucrose-free fermented milk prepared in Examples and Comparative Examples.
[0044] Figure 5 This is a data graph showing the survival rate test of the lyophilized protective agent in Example 7.
[0045] Figure 6 This is a data graph showing the survival rate test of the lyophilized protective agent in Example 8.
[0046] Figure 7 This is a data graph showing the survival rate test of the lyophilized protective agent in Example 9. DETAILED DESCRIPTION
[0047] The present invention will be described in detail below in conjunction with the accompanying drawings.
[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0049] Example 1
[0050] This preparation example provides a thermophilic streptococcus that produces high-activity lactase, and the culture and storage method thereof is as follows:
[0051] (1) A thermophilic Streptococcus with high-activity lactase was isolated from yak yogurt; the strain was classified and named Streptococcus salivarius subsp. thermophilus; and was deposited in the China General Microbiological Culture Collection Center on September 2, 2024, with the accession number CGMCC No. 31799.
[0052] (2) The thermophilic Streptococcus producing high-activity lactase obtained in step (1) is inoculated into M17 culture medium for culture and preservation.
[0053] (3) The culture temperature of step (2) is 35°C and the pH is 7.
[0054] The thermophilic Streptococcus obtained by screening was subjected to 16S rDNA molecular biological identification. A single colony was picked, mixed with sterile water, and bacterial universal primers were added to perform colony PCR amplification and determine its gene sequence. Its 16S rDNA base sequence is as follows:
[0055]
[0056] Studies have found that the thermophilic streptococcus provided by the present invention has the excellent characteristic of producing high-activity lactase. As a lactic acid bacteria species, the thermophilic streptococcus studies the lactase produced by it, which not only ensures the safety of the lactase, but also improves the industrial application value of the strain, reduces the complex process of industrial production and purification of lactase and the amount of lactase added, saves costs, and has great significance in the industrial production of food.
[0057] The activity of lactase can reach 110-156U / mL, which can effectively hydrolyze lactose in milk, greatly reducing the lactose content in milk. This can alleviate the symptoms of consumers' lactose intolerance to a certain extent, allowing yogurt to achieve a delicious sweetness and sourness without adding additives, thus satisfying consumers' demand for pure natural food.
[0058] The characteristics of the lactase are determined by further experiments on the lactase produced by the thermophilic streptococcus provided by the present invention: extraction, separation, purification, sequencing, and structural verification.
[0059] The lactase purification method is as follows: the thermophilic streptococcus producing high-activity lactase is cultured in M17 medium with a pH of 6.8, an activation temperature of 42°C, and an activation time of 48h. The activated thermophilic streptococcus producing high-activity lactase is placed in a liquid culture medium for culture, and the culture temperature in the liquid culture medium is 42°C and the culture time is 24h. Centrifuge at 5000rpm for 10min, then resuspend in 25mL of 20mM phosphate buffer with a pH of 7.0, and then add PMSF to a final concentration of 0.1mM. Ultrasonic disruption is used, 200W, on for 3s, off for 6s, and lasts for 8min. Then centrifuge at 4°C and 12000rpm for 50min, and the supernatant obtained is the crude enzyme solution of lactase.
[0060] Load 10mL of Ni-sephrose into two chromatography columns, and then balance with 20-25mM phosphate buffer, add 1M imidazole to the crude enzyme solution, and rotate and bind with Ni-sephrose at 4-6℃ for 60min. Take out the chromatography column, recover the flow-through, elute with pH 7.0 20mM phosphate buffer containing 20mM imidazole to remove impurities, elute the target protein into the EP tube with elution buffer, and finally dialyze to remove imidazole. The SDS-PAGE method was used to detect the expression and purity of lactase (M: Marker 1: bacterial disruption solution 2: crude enzyme solution 3: purified lactase), and the results are as follows: The protein is well expressed and has a high purity, with a molecular weight of approximately 110kDa. The test results are as follows Figure 1 shown.
[0061] Purified lactase was used to degrade β-1,3 / 1,4 / 1,6-galactobiose, and the hydrolyzate was detected by HPAEC. The 7 groups from top to bottom are: Gal-β-1,6-Gal+lactase; Gal-β-1,4-Gal+lactase; Gal-β-1,3-Gal+lactase; Gal-β-1,6-Gal; Gal-β-1,4-Gal; Gal-β-1,3-Gal; Gal. The results are as follows Figure 2 As shown, the purified lactase can hydrolyze β-1,6-galactobiose, and the only product is galactose. It has no degradation effect on β-1,3 / 1,4-galactobiose, indicating that the lactase is β-1,6-galactosidase.
[0062] The amino acid sequencing of the purified lactase was performed by mass spectrometry, and the results were as follows: QVLKDFYKTEFDETDLNFINVPGHLELQGFGSPQYVNTQYMKLRQSLNGKWKIHYAQNTNQVLKDFYKTEFDETDLNFINVPGHLELQGFGSPQYVNTQYPWDGKEFLRPPQVPQES。
[0063] The amino acid sequence of the lactase was compared with the NCBI database, and it was found that the lactase of the present application had the highest similarity with WP011226267.1 and the lowest similarity with WP048790041.1. Through sequence comparison, we found that the amino acid sequence had high similarity in different strains, which may be conservative.
[0064] The basic physical and chemical properties of lactase were analyzed using an online analysis website (https: / / web.expasy.org / protparam / ). The results are as follows:
[0065] Sequence length: 1044 amino acids; molecular weight: 119.0 kDa; isoelectric point: 4.83; extinction coefficient: 207350 M - 1 cm -1 (280nm water solvent); half-life: 0.8h (mammalian reticulocytes), 10min (yeast cells), 10h (Escherichia coli); instability coefficient 2: 33.53 shows that the protein has high stability; average hydrophilicity coefficient: -0.476 The protein is a hydrophilic protein.
[0066] The swissmodel model was used to analyze the structure of lactase in combination with the lactase sequence. The results are as follows: Figure 3 shown.
[0067] Example 1 also discloses a method for preparing fermented milk using thermophilic streptococci that produce high-activity lactase, comprising the following steps:
[0068] (1) The thermophilic Streptococcus screened in Example 1 was activated using M17 medium with an inoculum size of 1×10 6 CFU / mL, activation conditions: pH 7.0, culture temperature 35℃, culture time 48h.
[0069] (2) The thermophilic Streptococcus activated in step (1) was inoculated into M17 medium at an inoculum size of 1×10 6 CFU / mL, cultured at 35°C for 24 hours to obtain bacterial solution. Centrifuged at 8000 rpm for 10 minutes to collect the bacteria, and resuspended with physiological saline of the same volume as the bacterial solution before centrifugation to obtain seed culture solution.
[0070] (3) The seed culture solution obtained in step (2) is inoculated with pasteurized milk at a volume ratio of 3%, and fermented at 42° C. for 5 hours to obtain fermented milk without added sucrose prepared by the thermophilic streptococcus that produces high-activity lactase.
[0071] Example 2
[0072] The present invention also discloses a method for preparing fermented milk by using thermophilic streptococci that produce high-activity lactase, comprising the following steps:
[0073] (1) The thermophilic Streptococcus screened in Example 1 was activated using M17 medium with an inoculum size of 1×10 6 CFU / mL, activation conditions: pH 6.8, culture temperature 40℃, culture time 44h.
[0074] (2) The thermophilic Streptococcus activated in step (1) was inoculated into M17 medium at an inoculum size of 1×10 6 CFU / mL, cultured at 39°C for 20h to obtain bacterial solution. Collect the bacteria by centrifugation at 7000rpm for 15min, and resuspend them in physiological saline with the same volume as the bacterial solution before centrifugation to obtain seed culture solution.
[0075] (3) The seed culture solution obtained in step (2) is inoculated with pasteurized milk at a volume ratio of 4%, sodium citrate with a mass concentration of 2‰ is added to the pasteurized milk, and fermented at 35°C for 8h to obtain fermented milk without added sucrose prepared by the thermophilic streptococcus producing high-activity lactase.
[0076] Example 3
[0077] The present invention also discloses a method for preparing fermented milk by using thermophilic streptococci that produce high-activity lactase, comprising the following steps:
[0078] (1) The thermophilic Streptococcus screened in Example 1 was activated using M17 culture medium with an inoculum size of 1×10 6 CFU / mL and activation conditions of pH 7.2, a culture temperature of 42° C., and a culture time of 40 h.
[0079] (2) The thermophilic Streptococcus activated in step (1) was inoculated into M17 medium at an inoculum size of 1×106 CFU / mL, and cultured at 42°C for 16 hours to obtain a bacterial solution. The bacterial cells were collected by centrifugation at 6000 rpm for 20 minutes, and resuspended with a volume of physiological saline equal to that of the bacterial solution before centrifugation to obtain a seed culture solution.
[0080] (3) The seed culture solution obtained in step (2) is inoculated with pasteurized milk at a volume ratio of 5%, sodium citrate with a mass concentration of 3‰ is added to the pasteurized milk, and fermented at 40°C for 6h to obtain fermented milk without added sucrose prepared by the thermophilic streptococcus producing high-activity lactase.
[0081] Example 4
[0082] The present invention also discloses a method for preparing fermented milk by using thermophilic streptococci that produce high-activity lactase, comprising the following steps:
[0083] (1) The thermophilic Streptococcus screened in Example 1 was activated using M17 medium with an inoculum size of 1×10 6 CFU / mL, activation conditions: pH 7.0, culture temperature 42°C, culture time 40h.
[0084] (2) The thermophilic Streptococcus activated in step (1) was inoculated into M17 medium at an inoculum size of 1×10 6 CFU / mL, cultured at 39°C for 18h to obtain bacterial solution. Collect the bacteria by centrifugation at 8000rpm for 10min, and resuspend them in physiological saline with the same volume as the bacterial solution before centrifugation to obtain seed culture solution.
[0085] (3) The seed culture solution obtained in step (2) is inoculated with pasteurized milk at a volume ratio of 3.5%, sodium citrate with a mass concentration of 5‰ is added to the pasteurized milk, and fermented at 39°C for 7h to obtain fermented milk without added sucrose prepared by the thermophilic streptococcus producing high-activity lactase.
[0086] Example 5
[0087] The present invention also discloses a method for preparing fermented milk by using thermophilic streptococci that produce high-activity lactase. The difference between the preparation method and Example 1 is that step (3) is changed to: the seed culture solution obtained in step (2) is inoculated with pasteurized milk at a volume ratio of 3%, and then Lactobacillus bulgaricus is inoculated at a volume ratio of 1.2×10 6 The inoculum amount of CFU / mL was inoculated into pasteurized milk, fermented at 42°C for 5h, and sodium citrate with a mass concentration of 6‰ was added to the pasteurized milk, so as to obtain fermented milk without added sucrose prepared by using the thermophilic streptococcus producing high-activity lactase.
[0088] Example 6
[0089] The present invention also discloses a method for preparing fermented milk by using thermophilic streptococci that produce high-activity lactase. The difference between the preparation method and Example 1 is that step (3) is changed to: the seed culture solution obtained in step (2) is inoculated with pasteurized milk at a volume ratio of 3%, and then Lactobacillus acidophilus is inoculated at a volume ratio of 1.2×10 6 The inoculum amount of CFU / mL was inoculated into pasteurized milk, fermented at 42°C for 5h, and sodium citrate with a mass concentration of 4‰ was added to the pasteurized milk to obtain fermented milk without added sucrose prepared by using the thermophilic streptococcus producing high-activity lactase.
[0090] Comparative Example 1
[0091] Comparative Example 1 provides a fermented milk, which differs from Example 1 only in that "the thermophilic Streptococcus used in Example 1" is replaced by "commercial starter Streptococcus thermophilus ST446 (purchased from Haoyue)", and its preparation method is the same as that of Example 1.
[0092] Comparative Example 2
[0093] Comparative Example 2 provides a fermented milk, which differs from Example 1 only in that "the thermophilic Streptococcus used in Example 1" is replaced by "commercial starter Streptococcus thermophilus ST447 (purchased from Haoyue)", and its preparation method is the same as that of Example 1.
[0094] Comparative Example 3
[0095] Comparative Example 3 provides a fermented milk, which differs from Example 1 only in that 4% by weight of sodium citrate is added to the pasteurized milk in step (3). The preparation method is the same as that of Example 1.
[0096] Comparative Example 4
[0097] Comparative Example 4 provides a fermented milk, which differs from Example 1 only in that the pH of the pasteurized milk in step (3) is adjusted with 0.1 mol / L HCL, which is consistent with the pH value in Comparative Example 3 (higher than the isoelectric point of protein), and its preparation method is the same as that of Example 1.
[0098] The lactase activity of the fermented milks of Examples 1-6 and Comparative Examples 1-4 was measured respectively. The lactase activity was tested by the ONP standard curve absorbance method. The test results are shown in the attached Figure 4 At the same time, the prepared yogurt was subjected to a taste evaluation, and the taste evaluation was mainly for sweetness, with 0 being no sweetness, 1 being weak, 2 being weak, 3 being moderate, 4 being sweet, and 5 being very sweet. The test results are shown in Table 1.
[0099] Table 1
[0100]
[0101] The results show that: Figure 4It can be seen that the lactase activity of the fermented milk prepared by the thermophilic streptococcus of the high-activity lactase of implementation 1-6 is significantly higher than the fermented milk of the commercial fermentation agent thermophilic streptococcus (comparative examples 1 and 2) selected on the market, and the enzyme activity of the fermented milk of the bacteria used alone in implementation 2 is lower than the enzyme activity after adding sodium citrate. It is also concluded from the oral evaluation of Table 1 that the taste sweetness of embodiment 1-6 is also significantly better than that of comparative example 1-4, and at the same time, comparative case 4 excludes the ph change caused by sodium citrate, which promotes the influence of the high-activity lactase produced by the bacteria. It can be concluded that sodium citrate can significantly promote the high-activity lactase produced by the bacteria. The fermented milk prepared by the thermophilic streptococcus of the high-activity lactase of the present invention can more completely hydrolyze lactose in milk, improve the content of glucose and galactose after lactose hydrolysis, and the sweetness is higher and easier to absorb, which is achieved on the basis of not adding sweeteners and sucrose to a certain extent, and achieves an ideal sweetness, meeting the needs of consumers for pure natural food. At the same time, the addition of lactase is reduced industrially, saving costs. In addition, in Examples 5 and 6, Lactobacillus bulgaricus and Lactobacillus acidophilus were added. Compared with Example 1, the two bacteria synergistically fermented and interacted with each other, which was more conducive to the hydrolysis of lactase by Streptococcus thermophilus, laying a foundation for the application of starter in industry.
[0102] Example 7
[0103] Example 7 discloses a method for preparing a strain preparation of thermophilic Streptococcus that produces high-activity lactase, comprising the following steps:
[0104] The thermophilic Streptococcus selected in Example 1 after activation for three generations was inoculated into M17 medium and cultured at 35°C until the temperature reached 10 8 When the number of viable bacteria is above CFU / mL, the bacterial cells are collected by centrifugation, and freeze-drying protective agent, trehalose, sorbitol, whey protein, and skimmed milk powder are added respectively. The total mass concentration of the protective agent is 200g / L.
[0105] Freeze-drying process: pre-freezing, control the temperature of the layer plate to -50℃ within 1 hour and keep it for 3 hours; primary drying, control the temperature of the layer plate to -30℃ within 1.5 hours and keep it for 25 hours; secondary drying, control the temperature of the layer plate to 25℃ within 1 hour and keep it for 20 hours.
[0106] The freeze-dried powder of Streptococcus thermophilus was obtained. The freeze-dried survival rate was as high as 82.5%. The test results are as follows: Figure 5 shown.
[0107] Example 8
[0108] Example 8 discloses a method for preparing a strain preparation of thermophilic Streptococcus that produces high-activity lactase, comprising the following steps:
[0109] (1) The thermophilic Streptococcus activated for three generations was inoculated into M17 medium and cultured at 37°C until the temperature reached 108 When the number of viable bacteria is above CFU / mL, the bacterial cells are collected by centrifugation, and freeze-drying protective agents are added respectively. The single sugar protective agent trehalose and protein skim milk powder with the best freeze-drying survival rate are selected from Example 7, and then a certain ratio of 1:1 and 3:1 is compounded. The total mass concentration of the protective agent is 200g / L.
[0110] (2) Freeze-drying process: Pre-freezing, control the temperature of the layer plate to -50°C within 1 hour and keep it for 3 hours; primary drying, control the temperature of the layer plate to -30°C within 1.5 hours and keep it for 25 hours; secondary drying, control the temperature of the layer plate to 25°C within 1 hour and keep it for 20 hours.
[0111] The freeze-dried powder of Streptococcus thermophilus was obtained. The freeze-dried survival rate was as high as 90.5%. The specific test results are as follows: Figure 6 shown.
[0112] Example 9
[0113] Example 9 discloses a method for preparing a strain preparation of thermophilic Streptococcus that produces high-activity lactase, comprising the following steps:
[0114] (1) The thermophilic Streptococcus activated for three generations was inoculated into M17 medium and cultured at 42°C until the temperature reached 10 8 When the viable bacterial count was above 400 CFU / mL, the bacterial cells were collected by centrifugation, and freeze-drying protective agents were added respectively. The composite protective agent with the best freeze-drying survival rate was selected from Example 8, with trehalose and skim milk powder in a ratio of 1:1, and then small molecule protective agent ascorbic acid 20 g / L ( Figure 7 Combination 1) and sodium citrate 6g / L ( Figure 7 Combination 2), the total mass concentration of the protective agent is 200g / L,
[0115] (2) Freeze-drying process: Pre-freezing, control the temperature of the layer plate to -50°C within 1 hour and keep it for 3 hours; primary drying, control the temperature of the layer plate to -30°C within 1.5 hours and keep it for 25 hours; secondary drying, control the temperature of the layer plate to 25°C within 1 hour and keep it for 20 hours.
[0116] The freeze-dried powder of Streptococcus thermophilus was obtained. The freeze-dried survival rate was as high as 94.8%, and the number of live bacteria in the powder was 5.4×10 11 CFU / mL; specific test results are as follows Figure 7 shown.
[0117] In some embodiments, the ratio of carbohydrate protective agent to protein protective agent in the cryoprotectant in Example 8 is adjusted to 1:2; the carbohydrate protective agent is replaced with sorbitol, and the protein protective agent is replaced with whey protein, and the mass ratio of the carbohydrate protective agent to the protein protective agent is adjusted to 1:1-3. The other parameters and preparation process are the same as those in Example 8. The freeze-dried bacterial powder of Streptococcus thermophilus obtained has a freeze-drying survival rate of more than 90%.
[0118] In some embodiments, the total mass concentration of the cryoprotectant in the freeze-dried bacterial powder is 100-300 g / L.
[0119] In some embodiments, in the freeze-dried bacterial powder, the mass concentration of ascorbic acid is 10-30 g / L, and / or the mass concentration of sodium citrate is 2-7 g / L, which can effectively further increase the number of viable bacteria.
[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A thermophilic streptococcus, characterized in that The classification name is Streptococcus salivarius subsp. thermophilus; it is deposited in the China General Microbiological Culture Collection Center on September 2, 2024, and the deposit number is CGMCC No.31799.
2. The method for cultivating thermophilic streptococcus as claimed in claim 1, wherein The following steps are involved: Streptococcus thermophilus was inoculated into M17 medium for culture; Preferably, the culture medium includes sodium citrate at a mass concentration of 2‰-7‰, the culture temperature is 35-45°C, and the culture pH is 6.5-7.
5.
3. Use of the thermophilic streptococcus according to claim 1 in the preparation of high-activity lactase products and / or fermented dairy products.
4. A method for preparing fermented milk using the thermophilic streptococcus according to claim 1, characterized in that: The following steps are involved: The thermophilic streptococcus is inoculated into the milk product to be fermented, and fermented to obtain fermented milk.
5. The method for preparing fermented milk by using thermophilic streptococcus according to claim 4, characterized in that: The fermented dairy product comprises sodium citrate in a mass concentration of 2‰-7‰.
6. The method for preparing fermented milk by using thermophilic streptococcus according to claim 4, characterized in that: The acid-producing bacteria were also inoculated, and the acid-producing bacteria included at least one of Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus helveticus. The inoculation amount of the acid-producing bacteria was 1×10 3 ~1×10 6 cfu / mL.
7. A probiotic agent, characterized in that: Comprising live bacteria of the thermophilic streptococcus according to claim 1 and / or its fermentation product.
8. A freeze-dried bacterial powder, characterized in that: A powder preparation prepared by vacuum freeze drying a mixture of the thermophilic Streptococcus as claimed in claim 1 and a cryoprotectant; Cryoprotectants include carbohydrate protectants and protein protectants; The mass ratio of the carbohydrate protective agent to the protein protective agent is 1:1-3; The total mass concentration of the cryoprotectant in the freeze-dried bacterial powder is 100-300 g / L.
9. The freeze-dried bacterial powder according to claim 8, characterized in that: The carbohydrate protective agent includes at least one of trehalose and sorbitol, and the protein protective agent includes at least one of whey protein and skimmed milk powder; preferably, the carbohydrate protective agent is trehalose, and the protein protective agent is skimmed milk powder.
10. The freeze-dried bacterial powder according to claim 8 or 9, characterized in that: The freeze-dried bacterial powder also includes ascorbic acid and / or sodium citrate; in the freeze-dried bacterial powder, the mass concentration of ascorbic acid is 10-30 g / L, and / or the mass concentration of sodium citrate is 2-7 g / L.