Streptococcus thermophilus ST65, culture method and application of streptococcus thermophilus ST65, fermented milk and preparation method of fermented milk

Abstract

The invention relates to streptococcus thermophilus ST65, a culture method and application of the streptococcus thermophilus ST65, fermented milk and a preparation method of the fermented milk, and belongs to the technical field of microbial fermentation. The invention provides streptococcus thermophilus ST65. The streptococcus thermophilus ST65 provided by the invention has excellent acid-producing and aroma-producing characteristics, and meanwhile, the yield of exopolysaccharides is moderate. When the streptococcus thermophilus ST65 provided by the invention is applied to the preparation of a fermented milk product, the post-acidification of the fermented milk is weak; the yield of acetaldehyde and butanedione in the fermented milk is high, and the fermented milk has the characteristic of good flavor; meanwhile, the streptococcus thermophilus ST65 also has moderate exopolysaccharide yield, so that the fermented milk has the characteristics of short curding time, good flavor, thick and fine taste, no roughness, no blurring and no wiredrawing, the texture of the fermented milk product is remarkably improved, and the sensory experience of the product is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to Streptococcus thermophilus ST65 and a culture method and application thereof, fermented milk and a preparation method thereof. Background Art

[0002] Starter culture is the core ingredient of fermented dairy products. The characteristics of the bacteria in the starter culture determine the fermentation time, texture and flavor of the fermented milk. Among them, the acid production characteristics of the bacteria include acid production during the fermentation process and post-acid production (i.e., acid production during the shelf life), which is an important indicator for measuring the production performance of lactic acid bacteria. Strong acid production during the fermentation process can shorten the fermentation time and improve equipment utilization, while weak post-acid effect during the post-acidification process can maintain a relatively stable acidity, maintain a high number of live bacteria and a good sweet-sour ratio in the fermented milk; Exopolysaccharides (EPS) are substances secreted outside the cells by some lactic acid bacteria during the fermentation process, which can improve the viscosity, texture and taste of fermented dairy products, and can also reduce the use of other additives that improve product texture, such as pectin, agar, starch, etc. However, the lactic acid bacteria in the prior art either produce little extracellular polysaccharides or produce too much extracellular polysaccharides which do not match the acid production characteristics, resulting in the yogurt being too thick and sticky, affecting the product quality. There are not many strains that have both moderate extracellular polysaccharide production capabilities and excellent acid production characteristics. Therefore, a thermophilic Streptococcus with excellent acid and aroma production characteristics and moderate extracellular polysaccharide production was screened out and applied to the preparation of fermented milk, which effectively shortened the fermentation time, increased the viscosity of the product and gave the product a good flavor, and had important application value. Summary of the invention

[0003] The purpose of the present invention is to provide Streptococcus thermophilus ST65 and its culture method and application, fermented milk and its preparation method. The Streptococcus thermophilus ST65 of the present invention can have a moderate extracellular polysaccharide production while having excellent acid and aroma production characteristics, so that the fermented milk produced is of good quality.

[0004] The present invention provides a strain of Streptococcus thermophilus ST65, wherein the Streptococcus thermophilus ST65 is deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms, the preservation time is October 11, 2024, and the preservation number is CGMCC No.32180.

[0005] The present invention also provides a method for culturing the thermophilic streptococcus ST65 described in the above technical solution, comprising the following steps: inoculating the thermophilic streptococcus ST65 into a culture medium for culturing to obtain the thermophilic streptococcus ST65.

[0006] Preferably, the culture medium includes M17 culture medium; the culture temperature is 37° C. to 42° C.; and the pH of the culture medium is 6.5 to 7.2.

[0007] The present invention also provides the use of the thermophilic streptococcus ST65 described in the above technical solution or the thermophilic streptococcus ST65 cultured by the culture method described in the above technical solution in the preparation of fermentation products;

[0008] The fermented product includes fermented milk.

[0009] The present invention also provides the use of the thermophilic streptococcus ST65 described in the above technical solution or the thermophilic streptococcus ST65 cultured by the culture method described in the above technical solution in any one or more of the effects shown in ① to ⑤:

[0010] ① Improve acid production characteristics;

[0011] ②Increase acetaldehyde content;

[0012] ③Increase the content of diacetyl;

[0013] ④Increase the content of extracellular polysaccharides;

[0014] ⑤Improve texture.

[0015] The present invention also provides a fermented milk, wherein the fermentation strain of the fermented milk comprises thermophilic Streptococcus ST65.

[0016] The present invention also provides a method for preparing the fermented milk described in the above technical solution, comprising the following steps:

[0017] Anaerobic culture of Streptococcus thermophilus ST65 to obtain a seed solution of Streptococcus thermophilus ST65;

[0018] The seed liquid of Streptococcus thermophilus ST65 is inoculated into the milk to be fermented for fermentation and culture to obtain the fermented milk.

[0019] Preferably, the milk to be fermented includes fresh milk and / or reconstituted milk.

[0020] Preferably, the inoculation amount is 1% to 3% of the mass of the milk to be fermented.

[0021] Preferably, the fermentation culture time is 5h to 12h; the fermentation culture temperature is 37°C to 45°C.

[0022] The present invention provides Streptococcus thermophilus ST65. The thermophilus ST65 of the present invention has excellent acid and aroma production characteristics and a moderate extracellular polysaccharide production. The thermophilus ST65 provided by the present invention is applied to the preparation of fermented milk products, which not only has weak post-acidification, but also can make the acetaldehyde and diacetyl in the fermented milk have high yields and good flavor; at the same time, the thermophilus ST65 of the present invention also has a moderate extracellular polysaccharide production, which can make the fermented milk have the characteristics of short coagulation time, good flavor, thick and delicate taste, no roughness, no stickiness, and no stringiness, which significantly improves the texture of the fermented milk product and enhances the sensory experience of the product.

[0023] Biological deposit information

[0024] Streptococcus thermophilus ST65 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, referred to as CGMCC, and the deposit date is October 11, 2024. The address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32180. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The standard curve diagram of diacetyl provided by the present invention;

[0027] Figure 2 The relative molecular weight distribution diagram of ST65 extracellular polysaccharide provided by the present invention; wherein A and B are standard molecular weight sample test result diagrams; 1 is the standard measurement result of Mw=708kDa; 2 is the standard measurement result of Mw=200kDa; 3 is the standard measurement result of Mw=47.1kDa; 4 is the standard measurement result of Mw=9.6kDa; 5 is the standard measurement result of Mw=344kDa; 6 is the standard measurement result of Mw=21.1kDa; 7 is the standard measurement result of Mw=5.9kDa; 8 is the standard measurement result of Mw=0.667kDa; C is the test result diagram of thermophilic Streptococcus ST65;

[0028] Figure 3The present invention provides a graph showing the results of determination of the monosaccharide composition of the extracellular polysaccharide of Streptococcus thermophilus ST65; wherein, 1 is the determination result of the standard trehalose; 2 is the determination result of the standard fucose; 3 is the determination result of the standard rhamnose; 4 is the determination result of the standard galactosamine; 5 is the determination result of the standard arabinose; 6 is the determination result of the standard glucosamine; 7 is the determination result of the standard galactoside; 8 is the determination result of the standard glucose; 9 is the determination result of the standard xylose; 10 is the determination result of the standard mannose; and 11 is the determination result of the standard fructose. DETAILED DESCRIPTION

[0029] The present invention provides a strain of Streptococcus thermophilus ST65, which is deposited in the General Microbiology Center of China National Committee for the Preservation of Microbiological Cultures, with a preservation date of October 11, 2024 and a preservation number of CGMCC No.32180. The thermophilus ST65 colony of the present invention is round and milky white, and can have moderate extracellular polysaccharide production while having excellent acid and aroma production characteristics. The thermophilus ST65 provided by the present invention is applied to the preparation of fermented milk products, which not only weakens the acidification of fermented milk, but also makes the acetaldehyde and diacetyl in the fermented milk high, and has the characteristics of good flavor; at the same time, the thermophilus ST65 of the present invention also has moderate extracellular polysaccharide production, which can make the fermented milk have the characteristics of short coagulation time, good flavor, thick and delicate taste, and no stickiness, which significantly improves the texture of the fermented milk product and enhances the sensory experience of the product. The sequence of the 16s rDNA fragment of Streptococcus thermophilus ST65 of the present invention is shown in SEQ ID NO.1:

[0030]

[0031] The present invention also provides a method for culturing thermophilic streptococcus ST65 described in the above technical solution, comprising the following steps: inoculating thermophilic streptococcus ST65 into a culture medium for culturing to obtain thermophilic streptococcus ST65. In a specific embodiment, the thermophilic streptococcus ST65 obtained by culturing can be a culture obtained by culturing thermophilic streptococcus ST65 in a culture medium.

[0032] In a specific embodiment, the culture medium includes M17 culture medium; the culture temperature is 37° C. to 42° C.; and the pH of the culture medium is 6.5 to 7.2.

[0033] The present invention also provides the use of the thermophilic streptococcus ST65 described in the above technical solution or the thermophilic streptococcus ST65 cultured by the culture method described in the above technical solution in the preparation of fermented products; the fermented products include fermented milk.

[0034] The present invention also provides the use of the thermophilic streptococcus ST65 described in the above technical solution or the thermophilic streptococcus ST65 cultured by the culture method described in the above technical solution in any one or more of the effects shown in ① to ⑤:

[0035] ① Improve acid production characteristics;

[0036] ②Increase acetaldehyde content;

[0037] ③Increase the content of diacetyl;

[0038] ④Increase the content of extracellular polysaccharides;

[0039] ⑤Improve texture.

[0040] In a specific embodiment, the improved acid production characteristics include weak post-acidification. In a specific embodiment, the post-acidification refers to that the fermentation agent strain continues to produce acid during the storage stage of the fermented milk. In a specific embodiment, the fermented milk storage conditions include 0-10°C.

[0041] The present invention also provides a fermented milk, wherein the fermented strain of the fermented milk includes thermophilic streptococcus ST65. In a specific embodiment, the fermented strain of the fermented milk may include thermophilic streptococcus ST65 and other fermented strains. In a specific embodiment, the other fermented strains may be Lactobacillus bulgaricus LB42. In a specific embodiment, the thermophilic streptococcus ST65 may cooperate with Lactobacillus bulgaricus LB42 to achieve a synergistic effect.

[0042] The present invention also provides a method for preparing fermented milk described in the above technical scheme, comprising the following steps: anaerobically culturing thermophilic streptococcus ST65 to obtain thermophilic streptococcus ST65 seed liquid; inoculating the thermophilic streptococcus ST65 seed liquid into the milk to be fermented, fermenting and culturing, and obtaining fermented milk.

[0043] The present invention anaerobically cultures thermophilic streptococcus ST65 to obtain a seed liquid culture of thermophilic streptococcus ST65. In a specific embodiment, the culture medium used for the anaerobic culture may be an M17 liquid culture medium. In a specific embodiment, the pH of the M17 liquid culture medium may be 6.5 to 7.2. In a specific embodiment, the culture temperature of the anaerobic culture is 37° C. to 42° C. In a specific embodiment, before the anaerobic culture is performed, it also includes activating the thermophilic streptococcus ST65 using an activation culture medium. In a specific embodiment, the culture medium used for the activation culture may be an M17 culture medium. In a specific embodiment, the activation culture time is 24h to 72h. In a specific embodiment, the activation culture temperature is 37° C. to 45° C.

[0044] After obtaining the thermophilic Streptococcus ST65 seed liquid culture fluid, the present invention inoculates the thermophilic Streptococcus ST65 seed liquid into the fermented milk for fermentation and culture to obtain fermented milk. In a specific embodiment, the fermented milk includes fresh milk and / or reconstituted milk. In a specific embodiment, the inoculation amount of the inoculation can be 1% to 3% of the mass of the fermented milk. In a specific embodiment, the titrated acidity of the fermented milk is 70°T; the fermentation culture temperature can be 37°C to 45°C.

[0045] To further illustrate the present invention, the thermophilic Streptococcus thermophilus ST65 and its culture method and application, fermented milk and its preparation method provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Acquisition of Streptococcus thermophilus ST65 strain:

[0047] Take 5g of homemade yogurt from Xinjiang, add it to a sterile bottle containing 45mL of normal saline, stir and mix, and then dilute it with normal saline to a suitable concentration (10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ), oscillate and mix. Take 100 μL of the mixed sample solution, apply it to M17 medium (Qingdao Haibo Biological) plate, and culture it anaerobically at 37°C for 48 hours. Select colonies with lactic acid bacteria characteristics, repeatedly draw three lines on the plate, obtain pure strains, and store them at -80°C.

[0048] Screening of strains with better acid production characteristics: After the strains are activated for 2 generations, they are inoculated with 2% (volume fraction) in 100mL skim milk medium (Anjia high calcium skim milk powder, concentration 10%), and cultured at 37°C until the skim milk coagulates. The coagulation state and time are observed and recorded. The acidity of the obtained yogurt is measured at different shelf lives. Select strains with moderate acid production ability and weak post-acidification during the fermentation process.

[0049] Screening of aroma-producing strains: The strains with better acid-producing characteristics screened out above were inoculated with 2% (volume fraction) in 100 mL of skim milk culture medium (Anjia high-calcium skim milk powder, concentration 10%), and cultured at 37°C until the skim milk coagulated. The acetaldehyde and diacetyl contents were determined to screen for aroma-producing strains.

[0050] Screening of extracellular polysaccharide-producing strains: The strains with good acid-producing characteristics and strong aroma-producing properties screened above were inoculated into 100 mL of reconstituted milk medium at a volume fraction of 2%, and fermented in a water bath at 43°C for 5 to 6 hours. The extracellular polysaccharide content was determined by the sulfuric acid-phenol method.

[0051] The extracellular polysaccharide content produced by ST65 is 346.03 mg / L. The curdling state and time are observed and recorded. The evaluation indexes of the obtained yogurt mainly include tissue state, string length, viscosity and taste. A strain with better acid production characteristics, strong aroma production and moderate extracellular polysaccharide production is selected, which can prepare fermented milk with thick and delicate taste, not rough, not sticky, and not stringy.

[0052] Identification of ST65 strain: 16S rDNA molecular biological identification was performed on the screened thermophilic Streptococcus ST65. 1 μL of ST65 glycerol bacteria was taken, mixed with sterile water, and bacterial universal primers were added to perform colony PCR amplification, and its gene sequence was determined to identify it as thermophilic Streptococcus.

[0053] Culture and storage method: ST65 was inoculated into M17 medium, cultured at pH = 6.8, 37°C, and stored frozen at -80°C.

[0054] Example 1

[0055] This embodiment provides a fermented milk with excellent acid production characteristics, strong aroma production and moderate extracellular polysaccharide content, and the preparation method thereof is as follows:

[0056] (1) Streptococcus thermophilus ST65 was activated using M17 medium with an inoculum size of 1×10 6 CFU / mL, activation conditions: pH = 6.8, 37°C, 48h.

[0057] (2) The thermophilic Streptococcus ST65 activated in step (1) was inoculated into M17 medium at an inoculum size of 1×106 CFU / mL, anaerobically cultured at 37°C for 16 h to obtain bacterial solution. Centrifuged at 8000 rpm for 10 min to collect the bacteria, added an equal volume of physiological saline to resuspend and obtain seed culture solution.

[0058] (3) The seed culture solution obtained in step (2) is inoculated into reconstituted milk at an inoculation rate of 2%, and anaerobically fermented at 43°C until the titrated acidity reaches 70°T, thereby obtaining fermented milk with good acid production characteristics, strong aroma production and moderate extracellular polysaccharide production.

[0059] Example 2

[0060] This embodiment provides a fermented milk with excellent acid production characteristics, strong aroma production and moderate extracellular polysaccharide content, and the preparation method thereof is as follows:

[0061] (1) Streptococcus thermophilus ST65 was activated using M17 medium with an inoculum size of 1×10 6 CFU / mL, activation conditions: pH = 7.2, 39°C, 54h.

[0062] (2) The thermophilic Streptococcus ST65 activated in step (1) was inoculated into M17 medium at an inoculum size of 1×10 6 CFU / mL, anaerobically cultured at 39°C for 18h to obtain bacterial solution. Collect the bacteria by centrifugation at 7000rpm for 10min, add an equal volume of physiological saline to resuspend, and obtain seed culture solution.

[0063] (3) The seed culture solution obtained in step (2) is inoculated into fresh milk at an inoculum amount of 2%, and anaerobically fermented at 37°C until the titrated acidity reaches 70°T, thereby obtaining fermented milk with good acid production characteristics, strong aroma production and moderate extracellular polysaccharide production.

[0064] Example 3

[0065] This embodiment provides a fermented milk with excellent acid production characteristics, strong aroma production and moderate extracellular polysaccharide content, and the preparation method thereof is as follows:

[0066] (1) Streptococcus thermophilus ST65 was activated using M17 medium with an inoculum size of 1×10 6 CFU / mL, activation conditions: pH=6.5, 42°C, 24h.

[0067] (2) The thermophilic Streptococcus ST65 activated in step (1) was inoculated into M17 medium at an inoculum size of 1×10 6 CFU / mL, and cultured anaerobically at 37°C for 16 hours to obtain bacterial liquid. The bacteria were collected by centrifugation at 8000 rpm for 10 minutes, and an equal volume of physiological saline was added to resuspend them to obtain seed culture liquid.

[0068] (3) The seed culture solution obtained in step (2) is inoculated into the reconstituted milk at an inoculation rate of 2%, and anaerobically fermented at 45° C. until the titrated acidity reaches 70° C., thereby obtaining fermented milk with good acid production characteristics, strong aroma production and moderate extracellular polysaccharide production.

[0069] Example 4

[0070] The present embodiment provides a fermented milk with excellent acid production characteristics, strong aroma production and moderate extracellular polysaccharide content. The preparation method thereof is different from that of Example 1 in that after the seed culture solution is inoculated into the reconstituted milk at an inoculum amount of 2%, Lactobacillus bulgaricus LB42 (the deposit number of Lactobacillus bulgaricus subspecies LB42 is CGMCC No.15751, the deposit date is 2018.05.11, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. This strain is donated by Weikang Probiotics (Suzhou) Co., Ltd.) is inoculated with 1.0×10 5 The inoculum amount of CFU / mL was inoculated into the reconstituted milk, and the other steps were referred to Example 1.

[0071] Comparative Example 1

[0072] This comparative example provides a fermented milk, which differs from Example 1 only in that "Streptococcus thermophilus ST65" is replaced by "Streptococcus thermophilus I ( Express CN, purchased from Chr. Hansen)", and its preparation method refers to Example 1.

[0073] Comparative Example 2

[0074] This comparative example provides a fermented milk, which differs from Example 1 only in that "Streptococcus thermophilus ST65" is replaced by "Streptococcus thermophilus II producing exopolysaccharide (YF-L822, purchased from Chr. Hansen)", and its preparation method refers to Example 1.

[0075] Comparative Example 3

[0076] This comparative example provides a fermented milk, which differs from Example 1 only in that "Streptococcus thermophilus ST65" is replaced by "Streptococcus thermophilus III producing exopolysaccharide (Yo-Mix 883, purchased from Danisco)", and its preparation method refers to Example 1.

[0077] Comparative Example 4

[0078] This comparative example provides a fermented milk, which differs from Example 1 only in that "Streptococcus thermophilus ST65" is replaced by "Streptococcus thermophilus IV with high extracellular polysaccharide production (Yo-Mix 800, purchased from Danisco)", and its preparation method refers to Example 1.

[0079] Comparative Example 5

[0080] This comparative example provides a fermented milk, which is different from comparative example 1 only in that 1% by mass of agar (10000561, purchased from Sinopharm Chemical Reagent Co., Ltd.) is added to the fermented milk, and the other steps refer to comparative example 1.

[0081] Comparative Example 6

[0082] This comparative example provides a fermented milk, which is different from comparative example 1 only in that 1% by mass of pectin (XW01900069507, purchased from Sinopharm Chemical Reagent Co., Ltd.) is added to the fermented milk, and the other steps refer to comparative example 1.

[0083] Comparative Example 7

[0084] This comparative example provides a fermented milk, which is different from Example 1 only in that the fermentation temperature is 36° C., and the other steps refer to Example 1.

[0085] Comparative Example 8

[0086] This comparative example provides a fermented milk, which is different from Example 1 only in that the fermentation temperature is 46° C., and the other steps refer to Example 1.

[0087] Test Example 1

[0088] Determination of acidity and pH of fermented milk.

[0089] Acid production characteristics are an important indicator to measure the production performance of lactic acid bacteria, which usually includes two stages: acid production during the fermentation process and acidification after the shelf life. Acid production during the fermentation process refers to the process of fermented milk production and preparation, in which the starter strain uses lactose to produce acidic substances. In this process, the starter strain has a strong acid production ability, which can shorten the fermentation time and improve equipment utilization. As the pH value of the fermented milk decreases, the surface activity of the protein molecules decreases to form tiny sub-colloidal molecular clusters. The colloidal molecules aggregate into a uniform hydrated protein network structure to produce coagulation. Post-acidification refers to the fact that the starter strain continues to produce acid when the fermented milk is stored at 0-10°C. Strong post-acidification will cause the acidity to continue to increase during the shelf life and seriously affect the sweet and sour taste of the product.

[0090] Determination method: According to GB 5413.34 Determination of acidity of milk and dairy products, titration acidity determination is carried out, acidity unit: °T; at the same time, the pH of fermented milk is determined with a pH meter.

[0091] As can be seen from Table 1-1, in the embodiment, whether it is fresh milk fermentation or reconstituted milk fermentation, the acidity can reach 70°T within 5h to 12h. Compared with the thermophilic streptococcus in the prior art, the thermophilic streptococcus ST65 provided by the present invention has a relatively moderate acid production rate and can meet the needs of commercialization. The fermented milk acidity of Example 4 takes a shorter time to reach 70°T, indicating that the addition of Lactobacillus bulgaricus LB42 in the fermented milk can cooperate with the thermophilic streptococcus ST65 provided by the present invention to enhance synergy and facilitate the fermentation process.

[0092] The post-acidification of the fermented milk provided by Examples 1 to 4, Comparative Examples 1 to 4, Comparative Examples 7 and Comparative Examples 8 was measured, and the results are shown in Table 1-2. It can be seen from the results that the acidity of the fermented milk of Examples 1 to 3, Comparative Examples 7 and Comparative Examples 8 only increased by 4 to 5°T during the 21-day shelf life; the fermented milk of Example 4 increased by 8°T because of the addition of Lactobacillus bulgaricus, and Lactobacillus bulgaricus had a strong ability to produce acid after the shelf life; the fermented milk of Comparative Examples 1 to 3 increased by 10 to 12°T during the 21-day shelf life, which was significantly higher than that of Examples 1 to 3, and the acidity of Comparative Example 4 increased by 7°T, which was slightly higher than that of Examples 1 to 3, indicating that the fermented milk fermented with ST65 had a significant advantage in weak post-acidification.

[0093] Table 1-1 Titrated acidity, pH and fermentation time of different fermented milks

[0094]

[0095]

[0096] Table 1-2 Post-acidification test results of different fermented milks

[0097]

[0098] Test Example 2

[0099] Determination of acetaldehyde and diacetyl content in fermented milk.

[0100] Determination method of acetaldehyde:

[0101] Take 20 mL of the fermented milk to be tested and mix it with an equal volume of 8% trichloroacetic acid, let it stand and then centrifuge it (centrifugation conditions: 7500 rpm, 10 min), and then filter the supernatant with filter paper as a sample. At the same time, use sterile fresh milk as a control.

[0102] (1) Preparation of reagents

[0103] First, weigh 4g potassium iodide and place it in a 100mL brown volumetric flask, add an appropriate amount of water to dissolve (as little as possible to facilitate the dissolution of iodine later), then add 1.27g iodine (potassium iodide must be dissolved before iodine dissolves, otherwise iodine will not dissolve) and mix well. After the iodine is completely dissolved, add water to make the volume to 100mL, shake well, and prepare a 0.1mol / L iodine standard solution. Then accurately measure 10.0mL of 0.1mol / L iodine standard solution in a 100mL brown volumetric flask, dilute to the scale with distilled water, shake well, and prepare a 0.01mol / L iodine standard solution. Then accurately measure 10.0mL of 0.01mol / L iodine standard solution in a 100mL brown volumetric flask, dilute to the scale with distilled water, shake well, and prepare a 0.001mol / L iodine standard solution for sample titration. The above solutions are prepared now.

[0104] (2) Sample measurement

[0105] Take 5mL of 1% NaHSO3 solution and place it in a 250mL conical flask, add 25mL of the treated sample supernatant, mix well and place at room temperature for 1h, then add 1mL of 1% starch solution, titrate with 0.1mol / L iodine solution until it is close to light blue-purple, then titrate with 0.01mol / L iodine solution until it is light blue-purple, and if the light blue-purple does not fade within 30s, the above titrations are not counted. Then add 20mL of 1mol / L NaHCO3, shake and mix thoroughly to make the solution blue-purple disappear, and finally titrate with 0.001mol / L iodine solution to the blue-purple endpoint, record the volume of standard iodine solution consumed, do two parallels for each sample, and do a blank test at the same time.

[0106] Acetaldehyde content (mg / L) = (V1-V2) × 0.022 × C × 106 / 25

[0107] Where V2─volume of iodine standard solution consumed in titration blank (mL);

[0108] V1─ Volume of iodine standard solution consumed by titrating sample (mL);

[0109] C─Concentration of 1 / 2 iodine standard solution (mol / L);

[0110] 25─ Sample weight (mL);

[0111] 0.022─Basic chemical unit of acetaldehyde reaction (g).

[0112] Determination of diacetyl content:

[0113] (1) Drawing of diacetyl standard curve

[0114] Take 15μL of diacetyl and dissolve it in distilled water until it reaches 500mL. Accurately measure 0.0mL, 2.0mL, 4.0mL, 6.0mL, 8.0mL, and 10.0mL of diacetyl standard solution, and then add distilled water to make up to 10mL respectively. Take 5mL of the above standard solutions of different concentrations and put them into 12 test tubes placed side by side. Add an equal volume of 8% trichloroacetic acid and mix well. The concentrations of diacetyl are 0mg / L, 3mg / L, 6mg / L, 9mg / L, 12mg / L, and 15mg / L respectively. Add 0.5mL of 1% o-phenylenediamine solution to the front test tubes and 0.5mL of water to the back test tubes. After fully mixing, let it react in the dark for 30min. After the reaction is complete, add 2.0mL of 4.0mol / L hydrochloric acid to terminate the reaction. After shaking well, use the back test tubes as blank controls and measure the absorbance value at a wavelength of 335nm using an ultraviolet spectrophotometer. Draw a standard curve with diacetyl concentration as the horizontal axis and absorbance value as the vertical axis, such as Figure 1 shown.

[0115] (2) Determination of sample content

[0116] Take 20mL of the fermented milk supernatant after protein precipitation with trichloroacetic acid, add equal volumes to two test tubes, add 0.5mL of 1% o-phenylenediamine solution to test tube No. 1, add 0.5mL of water to test tube No. 2, mix thoroughly and react in the dark for 30min, then add 2.0mL of 4.0mol / L hydrochloric acid to the two test tubes to terminate the reaction, mix well and use test tube No. 2 as the control solution, use an ultraviolet spectrophotometer to measure the absorbance at a wavelength of 335nm, and calculate the diacetyl content according to the standard curve. If the diacetyl content in the sample is high and the measured absorbance exceeds 1.0, the sample needs to be appropriately diluted with 4% trichloroacetic acid, and then the diacetyl content is determined according to the above method.

[0117] Table 2 Determination results of acetaldehyde and diacetyl in different fermented milks

[0118]

[0119] The fermented milk provided by Examples 1 to 4, Comparative Examples 1 to 4, and Comparative Examples 7 and Comparative Examples 8 was tested for acetaldehyde and diacetyl content, and the results are shown in Table 2. It can be seen that the content of acetaldehyde and diacetyl of the flavor substances produced by the examples is significantly higher than that of the comparative examples, indicating that compared with the thermophilic streptococcus in the prior art, the fermented milk prepared by the thermophilic streptococcus ST65 provided by the present invention has high acetaldehyde and diacetyl content. Among them, the acetaldehyde and diacetyl content of the fermented milk of Example 4 is significantly higher than that of the fermented milk of Example 1, indicating that the addition of Lactobacillus bulgaricus LB42 to the fermented milk can cooperate with the thermophilic streptococcus ST65 provided by the present invention to promote the production of more flavor substances during the fermentation process.

[0120] Test Example 3

[0121] Viscosity and stability testing.

[0122] Viscosity measurement: After fermentation for 5-6 hours, the fermented milk was placed in a refrigerator overnight, and the viscosity of the fermented milk at about 10°C was measured the next day. The viscosity was measured using a proRheo viscometer, No. 2 rotor, 10s interval, and 64rpm.

[0123] The viscosity and stability of the fermented milk provided by Examples 1 to 4 and Comparative Examples 1 to 8 were measured during the shelf life (refrigerated at 0 to 10°C for 21 days), and the results are shown in Table 3. The results show that compared with each comparative example, the initial viscosity of the fermented milk provided by Examples 1 to 4 is worse than that of Comparative Examples 1 to 3, and Comparative Examples 5 to 8 are not as good as Comparative Example 4, but the viscosity change during the shelf life is less than that of all comparative examples, indicating that the thermophilic Streptococcus ST65 of the present invention can significantly increase the viscosity of the fermented milk and improve its stability when used to prepare fermented milk, and its effect of improving the viscosity and stability of the product is more significant than that of the thermophilic Streptococcus in the prior art, and the additional addition of stabilizers such as pectin and agar can be avoided. Among them, the viscosity of the fermented milk of Example 4 is slightly larger than that of Example 1, and the stability is better, indicating that the addition of Lactobacillus bulgaricus LB42 in the fermented milk can cooperate with the thermophilic Streptococcus ST65 provided by the present invention, synergistically enhance the effect, improve the texture, and further improve the stability of the product.

[0124] Table 3 Viscosity measurement results of different fermented milks after different days of storage

[0125]

[0126] Test Example 4

[0127] Sensory evaluation.

[0128] 10 professional tasters who are familiar with the evaluation standards of fermented milk and fully understand the sensory characteristics were hired to score the fermented milk stored at low temperature for 24 hours. The sensory characteristic scores are shown in Table 4-1. The average is taken as the score of each individual item to calculate the sensory evaluation results of different fermented milks, as shown in Table 4-2.

[0129] Table 4-1 Yogurt sensory scoring standards

[0130] project feature Sweet and sour ratio (10 points) The fermented milk has a moderate sweetness and sourness, and the sourness is not abrupt and not irritating Aroma (10 points) It has the typical fermented flavor of fermented milk, not bland Thickness (10 points) The product is thick and has a strong sense of substance when eaten Delicateness (10 points) No whey separation, no powdery feeling, no granular feeling Wire drawing (10 points) Fermented milk does not pull strings and does not stick to the mouth

[0131] Table 4-2 Sensory evaluation results of different fermented milks

[0132] Group Sweet and sour ratio aroma Thickness Fineness Wire drawing Example 1 8.2 8.1 8.3 8.1 7.6 Example 2 8.0 8.1 8.2 8.1 7.7 Example 3 8.2 8.2 8.4 8.2 7.6 Example 4 8.3 8.5 8.4 8.3 7.7 Comparative Example 1 5.5 6.7 2.4 2.6 2.0 Comparative Example 2 7.1 6.6 6.7 6.1 5.5 Comparative Example 3 7.2 6.4 7.5 6.1 5.7 Comparative Example 4 7.8 6.7 8.9 7.7 7.2 Comparative Example 5 5.4 6.7 4.7 3.0 2.1 Comparative Example 6 5.5 6.6 4.0 3.1 2.0 Comparative Example 7 7.9 7.7 7.5 7.6 7.0 Comparative Example 8 7.7 7.6 7.5 7.3 7.0

[0133] The results show that: compared with each comparative example, the fermented milk products provided in Examples 1 to 3 have a moderate sweet-sour ratio, a strong typical fermented milk aroma, a medium-thick product, a strong physical feel in the mouth, no whey precipitation, a fine texture, no stringiness, and a good sensory experience, indicating that the thermophilic Streptococcus ST65 of the present invention can significantly improve the product texture and enhance the sensory experience of the product when used to prepare fermented milk. The overall sensory score of the fermented milk of Example 4 is better than that of Example 1, indicating that the addition of Lactobacillus bulgaricus LB42 to the fermented milk can cooperate with the thermophilic Streptococcus ST65 provided by the present invention, synergistically enhance the effect, and facilitate the fermentation process. Although Comparative Example 4 measured a higher viscosity in the viscosity determination part of Test Example 3, the actual sensory evaluation results showed that Comparative Example 4 was too thick and sticky.

[0134] Test Example 5

[0135] Determination of extracellular polysaccharide content.

[0136] Determination method:

[0137] According to the inoculation amount of 2%, the cultured seed liquid is inoculated into fresh milk or reconstituted milk sterilized at 95°C for 20 minutes, and the fermentation is terminated when the acidity value is about 70°T. Add an appropriate amount of protease to the fermented milk, stir well, place it in an electric hot water bath and heat it to 55°C, enzymatically hydrolyze for 20 minutes, centrifuge at 4°C (7000rpm, 18min) to remove bacteria and protein, collect the supernatant, slowly add 95% ethanol with a volume twice that of the supernatant to precipitate polysaccharides, place it at 4°C overnight, centrifuge it the next day (5500rpm, 9min), discard the supernatant, collect the precipitate, add pure water to resuspend it until it is dissolved, and centrifuge it at 4°C (11500rpm, 20min) to collect the supernatant. Slowly add 95% ethanol in a volume 3 times that of the supernatant to precipitate, then centrifuge (5500rpm, 9min) to discard the supernatant, collect the filter mud, add pure water to resuspend until the filter mud is dissolved, and then centrifuge at 4°C (11500rpm, 20min), collect the supernatant, put it into a dialysis bag with a molecular weight cutoff of 7000Da and dialyze it with pure water for 2 days. Change the water every 2h during the dialysis process. After the dialysis is completed, centrifuge (11500rpm, 20min) to remove impurities, collect the supernatant and freeze-dry it to obtain crude extracellular polysaccharides. The content of extracellular polysaccharides was determined using the sulfuric acid-phenol method.

[0138] Table 5 Determination results of extracellular polysaccharides in different fermented milks

[0139]

[0140]

[0141] The extracellular polysaccharide content of the fermented milk provided by Examples 1 to 4 and Comparative Examples 1 to 8 was measured, and the results are shown in Table 5. The results show that the extracellular polysaccharide content of the fermented milk provided by Examples 1 to 3 is significantly higher than that of Comparative Examples 1 to 3 and Comparative Examples 5 to 8, indicating that the thermophilic Streptococcus ST65 of the present invention generates a high content of extracellular polysaccharides when used to prepare fermented milk. The extracellular polysaccharide content of the fermented milk of Example 4 is slightly higher than that of Example 1. The extracellular polysaccharide content of the fermented milk of Comparative Example 4 is significantly higher than that of the other fermentation treatment groups, but combined with the viscosity measurement and sensory evaluation results, it can be seen that although Comparative Example 4 produces more extracellular polysaccharides, the excessive extracellular polysaccharide production makes the viscosity of the fermented milk prepared by Comparative Example 4 too high, resulting in the prepared fermented milk being too sticky and thick, and the overall taste is not as good as the fermented milk prepared by thermophilic Streptococcus ST65 described in the present application.

[0142] Determination of relative molecular weight and monosaccharide composition of extracellular polysaccharides.

[0143] Method for determination of relative molecular weight of extracellular polysaccharide:

[0144] Relative molecular weight was determined by high performance liquid exclusion chromatography using a refractive index detector (RI) (Agilent 1260, DEAC 908338). 1 mg EPS-ST65 was dissolved in 1 mL 0.1 M NaNO 3, filtered with a 0.22 μm microporous membrane filter, then injected into the chromatographic column and eluted with distilled water at a flow rate of 0.6 mL / min. The relative molecular weight was calculated using a calibration curve obtained using various standards (Mw = 708, 344, 200, 47.1, 21.1, 9.6, 5.9 and 0.667 kDa).

[0145] The relative molecular weight distribution of exopolysaccharide of Streptococcus thermophilus ST65 was obtained as follows Figure 2 As shown, it can be seen that the relative molecular weight of the exopolysaccharide of Streptococcus thermophilus ST65 is 3020 kDa, which is larger than the molecular weight of most exopolysaccharides produced by Streptococcus thermophilus that have been reported.

[0146] Method for determination of monosaccharide composition of extracellular polysaccharides:

[0147] The monosaccharide composition of the polysaccharide was analyzed by ion chromatography. The ion chromatography analysis conditions were as follows: flowability A (50 mM sodium hydroxide solution), B (1 M sodium acetate solution), C (200 mM sodium hydroxide solution), D (water), chromatographic column: Thermo Fisher ion chromatography column PA100, detector: amperometric detector.

[0148] Accurately weigh 5 mg of freeze-dried polysaccharide and place it in a 5 mL magnetic stirring vial, add 500 μL of 12 M sulfuric acid, and magnetically stir at room temperature for 0.5 h. Then dilute the sulfuric acid to 2 M with pure water, tighten the bottle cap, and heat it in a 100 °C water bath for 2 h. After cooling, transfer it to a 100 mL volumetric flask and make up the volume. It can be directly filtered and injected for testing. At the same time, prepare standard substances (trehalose, fucose, rhamnose, galactosamine, arabinose, glucosamine, galactose, glucose, xylose, mannose and fructose are used as standards) and dissolve them in pure water and dilute them to the appropriate concentration as standard controls.

[0149] The monosaccharide composition of the exopolysaccharide of Streptococcus thermophilus ST65 was determined by Figure 3 It can be seen that the exopolysaccharide produced by Streptococcus thermophilus ST65 is composed of glucose and galactose, with a molar ratio of 1:1.

[0150] In summary, the thermophilic streptococcus ST65 provided by the present invention has excellent acid and aroma production characteristics and moderate extracellular polysaccharide production. It is applied to the preparation of fermented milk products, significantly improving the content of flavor substances acetaldehyde and diacetyl, giving the product a medium viscosity, improving the product texture, and improving product stability, thereby avoiding the additional addition of stabilizers such as pectin and agar. The fermented milk prepared by ST65 has a moderate sweet-sour ratio, medium thickness, a strong sense of the entrance, a fine texture, no drawing, no sticking, and a better sensory experience. In addition, further adding Lactobacillus bulgaricus LB42 to the fermented milk can cooperate with the thermophilic streptococcus ST65 provided by the present invention, synergistically enhance the effect, further improve the product stability, improve the texture of the fermented milk product, and enhance the sensory experience of the product.

[0151] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Streptococcus thermophilus ST65, characterized in that: The thermophilic Streptococcus ST65 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 11, 2024, and the deposit number is CGMCC No.32180.

2. The method for cultivating thermophilic streptococcus ST65 according to claim 1, comprising the steps of: The thermophilus Streptococcus ST65 is inoculated into a culture medium for culturing to obtain the thermophilus Streptococcus ST65.

3. The culture method according to claim 2, characterized in that The culture medium includes M17 culture medium; the culture temperature is 37° C. to 42° C.; and the pH of the culture medium is 6.5 to 7.

2.

4. Use of the thermophilic Streptococcus ST65 according to claim 1 or the thermophilic Streptococcus ST65 cultured by the culture method according to claim 2 or 3 in the preparation of fermentation products; The fermented product includes fermented milk.

5. Use of the thermophilic streptococcus ST65 according to claim 1 or the thermophilic streptococcus ST65 cultured by the culture method according to claim 2 or 3 in any one or more of the effects shown in ① to ⑤: ① Improve acid production characteristics; ②Increase acetaldehyde content; ③Increase the content of diacetyl; ④Increase the content of extracellular polysaccharides; ⑤Improve texture.

6. A fermented milk, characterized in that: The fermentation strain of the fermented milk includes Streptococcus thermophilus ST65.

7. The method for preparing the fermented milk according to claim 6, comprising the following steps: Anaerobic culture of Streptococcus thermophilus ST65 to obtain a seed solution of Streptococcus thermophilus ST65; The seed liquid of Streptococcus thermophilus ST65 is inoculated into the milk to be fermented for fermentation and culture to obtain the fermented milk.

8. The method for preparing fermented milk according to claim 7, characterized in that: The milk to be fermented includes fresh milk and / or reconstituted milk.

9. The method for preparing fermented milk according to claim 7, characterized in that: The inoculation amount is 1% to 3% of the mass of the milk to be fermented.

10. The method for preparing fermented milk according to claim 7, characterized in that: The fermentation culture time is 5h to 12h; the fermentation culture temperature is 37°C to 45°C.

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