Subculture lactic acid bacteria starter, yogurt and preparation method thereof

Abstract

The present application relates to the technical field of dairy product processing, in particular to a subculture lactic acid bacteria starter and yogurt prepared by the lactic acid bacteria starter and a preparation method thereof. The subculture lactic acid bacteria starter is prepared by Streptococcus thermophilus LSE-2018DH003-G3, LSE-2018DH003-G9, Lactobacillus delbrueckii subsp. Bulgaricus LSR-L-L1 and LSR-L-L4, and the application of the subculture lactic acid bacteria starter in yogurt. The yogurt process steps are as follows: single-strain fermentation intermediate preparation; compound strain, fermentation agent preparation; yogurt base preparation; yogurt production. The fermentation agent prepared by the above process has fast acid production and good aroma production; the prepared yogurt has fine organization, good stringing, low post-acid and high viable count; the high cost problem caused by direct injection of bacteria is maximized, which saves more than 4.6 million yuan for the company since 2019.

Description

Technical Field

[0001] This invention relates to the field of dairy processing technology, specifically to a generation of lactic acid bacteria starter culture, yogurt prepared using the lactic acid bacteria starter culture, and a method for preparing the same. Background Technology

[0002] A starter culture is a microbial preparation containing at least one type of microorganism at a high concentration, which, when added to raw materials, can accelerate and control the production of fermented foods. Lactic acid bacteria starter cultures are considered the "heart" of fermented dairy products and are a core component in the production of high-quality fermented milk. When added to raw materials in the correct proportions, lactic acid bacteria starter cultures produce lactic acid, rapidly acidifying the milk and generating metabolites such as organic acids, aromatic compounds, and extracellular polysaccharides. These metabolites impart the unique flavor and texture to fermented milk and contribute to its specific structure.

[0003] With the improvement of living standards, the awareness and purchasing power of fermented dairy products among different consumer groups have been increasing year by year, and the output of fermented dairy products has been rising rapidly at an average annual rate of 25%. Against this backdrop, the demand for high-quality starter cultures in the dairy industry has also increased significantly. Since Chr. Hansen of Denmark successfully developed a direct-inoculation lactic acid bacteria starter culture in 1988, foreign giants such as Chr. Hansen, Danisco, and DSM now hold over 90% of the Chinese lactic acid bacteria starter culture market share. While domestic brands such as Meihua, Vermont, and Yiran are emerging, their market share remains less than 10%. In recent years, lactic acid bacteria starter cultures have gradually penetrated more food sectors, and domestic researchers have strengthened their research on them. The teams of Hou Baochao, Lu Jiacheng, Chen Shixian, Wang Fan, and Feng Lili have conducted research and evaluation on aspects such as the selection of probiotics in starter cultures, comparison of flavor substances with direct-inoculation starter cultures, fermentation state, time, fermentation characteristics, optimal fermentation ratio after strain compounding, post-acidity control, and viable cell count. However, there is still a significant technological gap compared to imported starter cultures, and there are relatively few commercial starter cultures with independent intellectual property rights. Given the current lack of high-quality starter cultures in China, it is crucial to screen for low-cost lactic acid bacteria starter cultures with strong bacterial activity, excellent fermentation flavor, and weak post-acidification, and to develop them into probiotic starter cultures with independent intellectual property rights, which has significant economic and social value.

[0004] An improved method for using a yogurt culture strain is disclosed in the prior art (Patent Application No.: 200710114505.0, Publication No.: CN 101176484A). This patent uses two strains of Lactobacillus delbrueckii subsp. bulgaricus and one strain of Streptococcus thermophilus, combined in a ratio of 1:0.5-1.5:1-4 to prepare a starter culture. The starter culture is inoculated into the substrate at an inoculum rate of 3%, and after storage at 4-8℃, the viable cell count reaches 10 on the tenth day. 8CFU / mL. The 3% inoculum content in this patent increases the tank cost required for producing the starter culture. The mixture of low-protein product substrate and high-protein starter culture results in a grainy texture. Using a combination of two Bacillus strains and one bacterial coccus to produce the starter culture increases the risk of bacteriophage infection. Furthermore, the use of fructooligosaccharides and isomaltooligosaccharides in the substrate accelerates post-acidification, making it difficult to control post-acidification at room temperature and significantly increasing the overall cold chain cost.

[0005] This invention addresses the need for yogurt starter cultures, using *Streptococcus thermophilus* and *Lactobacillus delbrueckii* subsp. bulgaricus as research subjects. A database of fermentation-related characteristics of lactic acid bacteria strains was established, and a yogurt starter culture with independent intellectual property rights was developed. The starter culture was evaluated based on its acid production, aroma production capacity, textural characteristics, activity during fermentation, and activity during storage, as well as post-acidification at 28°C. A subculture lactic acid bacteria starter culture was developed, and the diacetyl content in the produced starter culture was measured to be 22.8-24.6 ug / kg, the extracellular polysaccharide content to be 226.5-248.3 mg / L, and the viable cell count to be 7.3-8.1 × 10⁻⁶. 8 CFU / mL. Yogurt indicators: inoculum size 0.6-0.8%, fermentation time 4-4.5 h, water separation rate ≤0.45% after 21 days at 4℃, acidity limit 94-102°T after 21 days of incubation at 28℃, viable count 4.5-8.0 × 10⁻⁶. 8 cfu / mL. An example is Huangshi Laisier Dairy's small yogurt product, with an annual output of over 12,000 tons, saving the company more than 1.5 million yuan annually.

[0006] The purpose of this invention is to provide a generation of lactic acid bacteria starter, yogurt, and a method for preparing the same.

[0007] A subculture lactic acid bacteria starter culture is composed of the following strains inoculum: Streptococcus thermophilus LSE-2018DH003-G3, Streptococcus thermophilus LSE-2018DH003-G9, Lactobacillus delbrueckii subsp. Bulgaricus LSR-L-L1, and Lactobacillus delbrueckii subsp. Bulgaricus LSR-L-L4;

[0008] Among them, the Streptococcus thermophilus LSE-2018DH003-G3 has the accession number CGMCC NO.21841, the Streptococcus thermophilus LSE-2018DH003-G9 has the accession number CGMCC NO.21842, the Lactobacillus delbrueckii bulgaricus LSR-L-L1 has the accession number CGMCC NO.14750, and the Lactobacillus delbrueckii bulgaricus LSR-L-L4 has the accession number CGMCC NO.14752;

[0009] Preferably, the inoculum consists of 8-15 parts of Streptococcus thermophilus LSE-2018DH003-G3, 8-15 parts of Streptococcus thermophilus LSE-2018DH003-G9, 0.5-1.5 parts of Lactobacillus delbrueckii bulgaricus LSR-L-L1, and 0.5-1.5 parts of Lactobacillus delbrueckii bulgaricus LSR-L-L4.

[0010] Preferably, there are 12 portions of Streptococcus thermophilus LSE-2018DH003-G3 inoculation solution, 12 portions of Streptococcus thermophilus LSE-2018DH003-G9 inoculation solution, 1 portion of Lactobacillus delbrueckii bulgaricus LSR-L-L1 inoculation solution, and 1 portion of Lactobacillus delbrueckii bulgaricus LSR-L-L4 inoculation solution.

[0011] This invention also provides a method for preparing a subculture lactic acid bacteria starter culture, comprising the following steps:

[0012] 1) Preparation of single-strain fermentation intermediate: After the strain has been cultured to the 4th to 8th generation, the single strain is inoculated into a sterilized skim milk bottle with a protein content of 3.2% to 3.4% at a volume ratio of 2% to 3%. The fermentation intermediate is carried out at 40 to 44°C. The final acidity of cocci is 84 to 92°T, and the final acidity of bacilli is 82 to 90°T.

[0013] 2) Preparation of fermentation agent by compounding strains: The intermediate agent fermented in step 1) is compounded according to the strain inoculation amount of 1-3% by volume, and inoculated into skim milk medium with protein content of 3.0-3.2%. Fermentation is carried out at 40-44℃ for 6-7 hours, and the final acidity is 88-96 degrees.

[0014] The present invention also provides yogurt prepared using the aforementioned subculture lactic acid bacteria starter, made from the following raw and auxiliary materials in weight percentages:

[0015] Lactic acid bacteria starter 0.6-0.8%, raw milk protein content after purification 2.8-3.0%, 0.5-1.5% agar, 0.5-1.5% starch, 0.005-0.015% sea salt, 0.5-1.5% stabilizer, 0.5-1.5% white sugar;

[0016] Preferably, the ingredients are: 0.7% lactic acid bacteria starter, 2.9% protein content of purified raw milk, 1% agar, 1% starch, 0.01% sea salt, 1% stabilizer, and 1% white sugar.

[0017] The present invention also provides a method for preparing the yogurt, comprising the following steps:

[0018] 1) Preparation of yogurt base: After purification, the raw milk protein content is 2.8-2.9%. Heat it to 60-70℃, add 0.5-1.5 parts agar, 0.5-1.5 parts starch, 0.01-0.03 parts sea salt, 0.5-1.5 parts stabilizer, and 0.5-1.5 parts white sugar. Mix with the remaining ingredients, test the physicochemical properties, adjust the volume, homogenize, pasteurize, and cool for later use.

[0019] 2) Yogurt production: Inoculate 0.6-0.8% of the starter culture into the base material in step 1), ferment at 40-44℃ for 4-4.5 hours, with an endpoint acidity of 62-66°T, ferment, break the emulsion, ripen, fill, conduct microbial testing, and track post-acidity.

[0020] This invention utilizes four registered and preserved strains from the Royal Laisier Dairy Strain Resource Bank: *Streptococcus thermophilus* LSE-2018DH003-G3 and LSE-2018DH003-G9, and *Lactobacillus delbrueckii* bulgaricus strains LSR-L-L1 and LSR-L-L4. The preservation details are as follows:

[0021] Classification and nomenclature: Streptococcus thermophilus;

[0022] Latin name: (Streptococcus thermophilus);

[0023] Biological material (strain) of Ginseng: LSE-2018DH003-G3;

[0024] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0025] The abbreviation for the depository institution is CGMCC.

[0026] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0027] Deposit date: March 1, 2021;

[0028] Registered with the China National Collection Center (CGMCC) No. 21841.

[0029] Classification and nomenclature: Streptococcus thermophilus;

[0030] Latin name: (Streptococcus thermophilus);

[0031] Biological material (strain) of Ginseng: LSE-2018DH003-G9;

[0032] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0033] The abbreviation for the depository institution is CGMCC.

[0034] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0035] Deposit date: March 1, 2021;

[0036] Registered with the China National Collection Center (CGMCC) No. 21842.

[0037] Classification and nomenclature: Lactobacillus delbrueckii subsp. bulgaricus;

[0038] Latin name: (Lactobacillus delbrueckii subsp. Bulgaricus);

[0039] Biological material (strain) of ginseng: LSR-L-L1;

[0040] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0041] The abbreviation for the depository institution is CGMCC.

[0042] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0043] Date of deposit: September 26, 2017;

[0044] Registered with the Collection Center: CGMCC NO.14750.

[0045] Classification and nomenclature: Lactobacillus delbrueckii subsp. bulgaricus;

[0046] Latin name: (Lactobacillus delbrueckii subsp. Bulgaricus);

[0047] Biological material (strain) of ginseng: LSR-L-L4;

[0048] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0049] The abbreviation for the depository institution is CGMCC.

[0050] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0051] Date of preservation: September 26, 2017;

[0052] Registered with the China National Collection Center (CGMCC) No. 14752.

[0053] Compared with the prior art, the present invention has the following beneficial effects: all four strains of the present invention are registered and preserved strains with excellent fermentation characteristics, the specific characteristics of which are as follows:

[0054] 1. Adaptable to product processing conditions, the two cocci produce acid quickly, with low post-acidity, aroma, and stickiness; the two bacilli do not produce stickiness, produce good aroma, and have a high viable count; the four strains are stable after 4-8 generations of subculture; by controlling the cocci-bacilli ratio through strain compounding, the fineness, viscosity, and post-acidity of the product can be adjusted, resulting in a subculture lactic acid bacteria starter with independent intellectual property rights. The cocci-bacilli compounding ratio is LSE-2018DH003-G3+LSE-2018DH003-G9+LSR-L-L1+LSR-L-L4=1.2%+1.2%+0.1%+0.1%.

[0055] 2. Acid production is rapid during fermentation. The inoculum amount of the subculture lactic acid bacteria starter is 600-800g / ton, and the fermentation time is 4-4.5h. Compared with commercial direct-inoculation starter, the fermentation time is very similar.

[0056] 3. Low post-acidity and high viable cell count result in yogurt with excellent acidity and aroma, a delicate texture, and a post-acidity of 94-102°T in 10 batches of samples incubated at 28℃ for 21 days, with viable cell counts ranging from 1.5 to 8.0 × 10⁻⁶. 8 cfu / mL, greater than the national standard requirement of 10 6 cfu / mL.

[0057] 4. The starter culture contains 22.8-24.6 ug / mL of diacetyl and 226.5-248.3 mg / L of extracellular polysaccharide, which enhances the sour aroma and maintains the yogurt's texture. The yogurt has a water separation rate of ≤0.45% after 21 days, ensuring a high-quality taste.

[0058] 5. The intermediate agent and production fermentation agent have a simple preparation process that is safe and environmentally friendly. Since 2019, it has saved the company more than 4.6 million yuan in direct economic benefits. Attached Figure Description

[0059] Figure 1 The growth curves for the four strains are shown.

[0060] Figure 2 The acid production curves for the four strains are shown.

[0061] Figure 3 This is a standard curve for diacetyl.

[0062] Figure 4 This is the standard curve for extracellular polysaccharides.

[0063] Figure 5 Growth curves for co-culture of LSE-2018DH003-G3, LSE-2018DH003-G9, LSR-L-L1, and LSR-L-L4.

[0064] The present invention will be further illustrated below with reference to embodiments, but is not limited to these embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0065] Example 1

[0066] Preparation of Subgeneration Lactic Acid Bacteria Starter Culture

[0067] Intermediate preparation for strain activation and rejuvenation: Four strains from the Huangshi Laisier Strain Resource Bank were used: *Streptococcus thermophilus* LSE-2018DH003-G3 (CGMCC NO.21841), *Streptococcus thermophilus* LSE-2018DH003-G9 (CGMCC NO.21842), *Lactobacillus delbrueckii* bulgaricus LSR-L-L1 (CGMCC NO.14750), and *Lactobacillus delbrueckii* bulgaricus LSR-L-L4 (CGMCC NO.14752). The cocci were activated using TJA, and the bacilli were activated using MRS. Rejuvenation was achieved by adding appropriate amounts of sterilized milk, carrot juice, and tomato juice to the culture medium, ensuring a viable bacterial count of ≥10⁻⁶ bacilli. 9 cfu / mL, cocci ≥10 8 CFU / mL, rejuvenation completed, and genetic stability of the strain was tested. The strain was cultured to the 6th generation. At a volume ratio of 2.5%, a single strain was inoculated into a bottle of sterilized skim milk with a protein content of 3.3%. The intermediate fermentation was carried out in a constant temperature incubator at 42℃. The final acidity of cocci was 87°T, and the final acidity of bacilli was 86°T. The microbial properties were tested and the strain was ready for use.

[0068] The skim milk culture medium consisted of: skim milk powder dissolved in double-distilled water at 50°C to obtain an intermediate skim milk (3.5%) and a starter culture (3.1%). The mixture was hydrated for 30 minutes and then fermented at 0.70 × 10⁻⁶. 5 Sterilize with Pa for 15 minutes and set aside for use.

[0069] Physiological saline: Dissolve 8.5g NaCl in 1000mL of water, dispense into test tubes, 9mL each, 1.05×10 5 Sterilize at 15 min (Pa) and set aside for use.

[0070] MRS liquid medium: Dissolve 20g glucose, 10g casein peptone, 10g beef extract, 5g yeast extract, 5g anhydrous sodium acetate, 2g triammonium citrate, 2g dipotassium hydrogen phosphate, 1mL Tween-80, 0.25g manganese sulfate, and 0.58g magnesium sulfate in double-distilled water and bring the volume to 1000mL. Accurately weigh the magnesium sulfate and manganese sulfate, dissolve them separately, and then mix them with the remaining ingredients. The pH should be 6.2-6.4. MRS solid medium: MRS liquid medium + 1.5% agar powder, 1.05×10⁻⁶ 5 Sterilize at 15 min (Pa) and set aside for use.

[0071] TJA medium: Combine 50 mL tomato juice, 5 g yeast extract, 10 g beef extract, 20 g lactose, 2 g glucose, 2 g dipotassium hydrogen phosphate, 1 g Tween-80, and 5 g anhydrous sodium acetate. Add double-distilled water to a final volume of 1000 mL and adjust the pH to 6.8 ± 0.2. TJA solid medium: TJA liquid medium + 1.5% agar powder, 1.05 × 10⁻⁶ 5 Sterilize at 15 min (Pa) and set aside for use.

[0072] Preparation of starter culture by compounding strains: The intermediate agent fermented in step (1) was compounded at a volume ratio of 2% of the strain inoculation amount, using 12 parts of Streptococcus thermophilus LSE-2018DH003-G3 inoculation solution, 12 parts of Streptococcus thermophilus LSE-2018DH003-G9 inoculation solution, 1 part of Lactobacillus delbrueckii bulgaricus LSR-L-L1 inoculation solution, and 1 part of Lactobacillus delbrueckii bulgaricus LSR-L-L4 inoculation solution. These were inoculated into sterilized skim milk medium with a protein content of 3.1%, and fermented at 42℃ for 6.5 hours. The final acidity was 90 degrees Celsius. Microbial analysis was then performed, and the mixture was ready for use.

[0073] The shelf life of the generation-type probiotic starter was determined under 4℃ refrigeration conditions. Its condition was checked daily, and the viable cell count was tested. A small sample of the substrate was inoculated with 0.7% of the starter culture for fermentation, and the fermentation status, acidity, and viable cell count were compared. After 8 days, the fermentation time was extended to 4.8 hours. The viable cell count showed little change, and the incubated sample showed no change. Therefore, the shelf life of the generation-type probiotic starter culture was determined to be 7 days.

[0074] The intermediate lactic acid bacteria preparation in Example 1: after 14 hours of coccal fermentation, the acidity was between 84-92°T; after 8 hours of bacillus fermentation, the acidity stabilized at 82-90°T, and the viable count was greater than 10 in both cases. 8 cfu / mL; limiting acidity determination was performed on 4 strains as follows: Figure 2The four bacterial strains, LSE-2018DH003-G3 and LSE-2018DH003-G9, have limiting acidities of 114 and 115°T, respectively, while LSR-L-L1 and LSR-L-L4 have limiting acidities of 160 and 156°T, respectively. After fermentation with an intermediate agent, these four strains were combined in a specific ratio and fermented using a coccidial symbiotic relationship. The resulting starter culture was produced after 6-7 hours of fermentation, achieving an acidity of 82-90°C and a viable cell count of 6.9-8.1 × 10⁻⁶. 8 cfu / mL, shelf life is 7 days.

[0075] Example 2

[0076] Preparation of Subgeneration Lactic Acid Bacteria Starter Culture

[0077] Intermediate preparation for strain activation and rejuvenation: Four strains from the Huangshi Laisier Strain Resource Bank were used: *Streptococcus thermophilus* LSE-2018DH003-G3 (CGMCC NO.21841), *Streptococcus thermophilus* LSE-2018DH003-G9 (CGMCC NO.21842), *Lactobacillus delbrueckii* bulgaricus LSR-L-L1 (CGMCC NO.14750), and *Lactobacillus delbrueckii* bulgaricus LSR-L-L4 (CGMCC NO.14752). The cocci were activated using TJA, and the bacilli were activated using MRS. Rejuvenation was achieved by adding appropriate amounts of sterilized milk, carrot juice, and tomato juice to the culture medium, ensuring a viable bacterial count of ≥10⁻⁶ bacilli. 9 cfu / mL, cocci ≥10 8 CFU / mL, rejuvenation completed, and genetic stability of the strain was tested. The strain was cultured to the 4th generation. At a volume ratio of 2%, a single strain was inoculated into a bottle of sterilized skim milk with a protein content of 3.2%. The intermediate fermentation was carried out in a constant temperature incubator at 40℃. The final acidity of cocci was 84°T, and the final acidity of bacilli was 82°T. The microbial properties were tested and the strain was ready for use.

[0078] The skim milk culture medium consisted of: skim milk powder dissolved in double-distilled water at 45°C to obtain 3.5% skim milk intermediate and 3.0% starter culture medium; hydration for 30 min; and 0.70 × 10⁻⁶ ppm. 5 Sterilize with Pa for 15 minutes and set aside for use.

[0079] Physiological saline: Dissolve 8.5g NaCl in 1000mL of water, dispense into test tubes, 9mL each, 1.05×10 5 Sterilize at 15 min (Pa) and set aside for use.

[0080] MRS liquid medium: Dissolve 20g glucose, 10g casein peptone, 10g beef extract, 5g yeast extract, 5g anhydrous sodium acetate, 2g triammonium citrate, 2g dipotassium hydrogen phosphate, 1mL Tween-80, 0.25g manganese sulfate, and 0.58g magnesium sulfate in double-distilled water and bring the volume to 1000mL. Accurately weigh the magnesium sulfate and manganese sulfate, dissolve them separately, and then mix them with the remaining ingredients. The pH should be 6.2. MRS solid medium: MRS liquid medium + 1.5% agar powder, 1.05×10⁻⁶ 5 Sterilize at 15 min (Pa) and set aside for use.

[0081] TJA medium: Combine 50 mL tomato juice, 5 g yeast extract, 10 g beef extract, 20 g lactose, 2 g glucose, 2 g dipotassium hydrogen phosphate, 1 g Tween-80, and 5 g anhydrous sodium acetate. Add double-distilled water to a final volume of 1000 mL and adjust the pH to 6.8 ± 0.2. TJA solid medium: TJA liquid medium + 1.5% agar powder, 1.05 × 10⁻⁶ 5 Sterilize at 15 min (Pa) and set aside for use.

[0082] Preparation of fermentation agent by compounding strains: The intermediate agent fermented in step (1) was compounded at a volume ratio of 1% of the strain inoculation amount, consisting of 8 parts of Streptococcus thermophilus LSE-2018DH003-G3 inoculation solution, 8 parts of Streptococcus thermophilus LSE-2018DH003-G9 inoculation solution, 0.5 parts of Lactobacillus delbrueckii bulgaricus LSR-L-L1 inoculation solution, and 0.5 parts of Lactobacillus delbrueckii bulgaricus LSR-L-L4 inoculation solution. The mixture was inoculated into sterilized skim milk medium with a protein content of 3.0–3.2%, and fermented at 40°C for 6 hours. The final acidity was 88 degrees Celsius. Microbiological testing was then performed, and the mixture was ready for use.

[0083] The shelf life of the generation-type probiotic starter was determined under 4℃ refrigeration conditions. Its condition was checked daily, and the viable cell count was measured. A small sample of the substrate was inoculated with 0.6% of the starter culture for fermentation, and the fermentation status, acidity, and viable cell count were compared. After 8 days, the fermentation time was extended to 4.8 hours. The viable cell count showed little change, and the incubated sample showed no change. Therefore, the shelf life of the generation-type probiotic starter culture was determined to be 7 days.

[0084] Example 3

[0085] Preparation of Subgeneration Lactic Acid Bacteria Starter Culture

[0086] Intermediate preparation for strain activation and rejuvenation: Four strains from the Huangshi Laisier Strain Resource Bank were used: *Streptococcus thermophilus* LSE-2018DH003-G3 (CGMCC NO.21841), *Streptococcus thermophilus* LSE-2018DH003-G9 (CGMCC NO.21842), *Lactobacillus delbrueckii* bulgaricus LSR-L-L1 (CGMCC NO.14750), and *Lactobacillus delbrueckii* bulgaricus LSR-L-L4 (CGMCC NO.14752). The cocci were activated using TJA, and the bacilli were activated using MRS. Rejuvenation was achieved by adding appropriate amounts of sterilized milk, carrot juice, and tomato juice to the culture medium, ensuring a viable bacterial count of ≥10⁻⁶ bacilli. 9 cfu / mL, cocci ≥10 8 CFU / mL, rejuvenation completed, genetic stability test of strain conducted, strain cultured to 8 generations, single strain inoculated into a bottle of sterilized skim milk with 3.4% protein content at a volume ratio of 3%, fermentation intermediate in a 44℃ constant temperature incubator, the final acidity of cocci was 92°T, the final acidity of bacilli was 90°T, and its microbial properties were tested, ready for use;

[0087] The skim milk culture medium consisted of skim milk powder dissolved in double-distilled water at 55°C to obtain an intermediate skim milk (3.6%) and a starter culture (3.2%). The mixture was hydrated for 30 minutes and then fermented at 0.70 × 10⁻⁶. 5 Sterilize with Pa for 15 minutes and set aside for use.

[0088] Physiological saline: Dissolve 8.5g NaCl in 1000mL of water, dispense into test tubes, 9mL each, 1.05×10 5 Sterilize at 15 min (Pa) and set aside for use.

[0089] MRS liquid medium: Dissolve 20g glucose, 10g casein peptone, 10g beef extract, 5g yeast extract, 5g anhydrous sodium acetate, 2g triammonium citrate, 2g dipotassium hydrogen phosphate, 1mL Tween-80, 0.25g manganese sulfate, and 0.58g magnesium sulfate in double-distilled water and bring the volume to 1000mL. Accurately weigh the magnesium sulfate and manganese sulfate, dissolve them separately, and then mix them with the remaining ingredients. The pH should be 6.4. MRS solid medium: MRS liquid medium + 1.5% agar powder, 1.05×10⁻⁶ 5 Sterilize at 15 min (Pa) and set aside for use.

[0090] TJA medium: Combine 50 mL tomato juice, 5 g yeast extract, 10 g beef extract, 20 g lactose, 2 g glucose, 2 g dipotassium hydrogen phosphate, 1 g Tween-80, and 5 g anhydrous sodium acetate. Add double-distilled water to a final volume of 1000 mL and adjust the pH to 6.8 ± 0.2. TJA solid medium: TJA liquid medium + 1.5% agar powder, 1.05 × 10⁻⁶ 5Sterilize at 15 min (Pa) and set aside for use.

[0091] Preparation of fermentation agent by compounding strains: The intermediate agent fermented in step (1) was compounded at a volume ratio of 3% of the strain inoculation amount, using 15 parts of Streptococcus thermophilus LSE-2018DH003-G3 inoculation solution, 15 parts of Streptococcus thermophilus LSE-2018DH003-G9 inoculation solution, 1.5 parts of Lactobacillus delbrueckii bulgaricus LSR-L-L1 inoculation solution, and 1.5 parts of Lactobacillus delbrueckii bulgaricus LSR-L-L4 inoculation solution. The mixture was inoculated into sterilized skim milk medium with a protein content of 3.0–3.2%, and fermented at 44℃ for 7 hours. The final acidity was 96 degrees Celsius. Microbiological testing was then performed, and the mixture was ready for use.

[0092] The shelf life of the generation-type probiotic starter was determined under 4℃ refrigeration conditions. Its condition was checked daily, and the viable cell count was measured. A small sample of the substrate was inoculated with 0.8% of the starter culture for fermentation, and the fermentation status, acidity, and viable cell count were compared. After 8 days, the fermentation time was extended to 4.8 hours. The viable cell count showed little change, and the incubated sample showed no change. Therefore, the shelf life of the generation-type probiotic starter culture was determined to be 7 days.

[0093] Example 4

[0094] Viable cell count, growth curve, aroma production, and extracellular polysaccharide detection of subculture lactic acid bacteria starter

[0095] (1) The method for detecting viable bacteria is GB / T14699.1, and the culture medium for bacilli is MRS and the culture medium for cocci is TJA.

[0096] (2) The Finnish Bioscreen fully automated growth curve analyzer was used. It was turned on and preheated 30 minutes in advance. The honeycomb plate was sterilized with ultraviolet light for 30 minutes. The culture medium was used as a blank control group. Four strains were inoculated into liquid culture medium at a volume ratio of 2%. After shaking evenly, 200 μL of liquid culture medium was quickly inoculated into the honeycomb plate. Three parallel samples were set up for each group.

[0097] (3) The intermediate agent in Example 1 and the production starter were used to detect diacetyl in dairy products by o-phenylenediamine colorimetric method, referring to Shao Yadong's study on the characteristics of diacetyl production by lactic acid bacteria in traditional fermented dairy products.

[0098] (4) The intermediate agent and the production starter from Example 1 were used to detect extracellular polysaccharides using the phenol-sulfuric acid method;

[0099] In Example 4, the viable cell count of a single strain at generations 4-8 was detected, with cocci exceeding 10. 8 cfu / mL, bacteria greater than 10 9 cfu / mL. Growth curve as shown. Figure 1As shown, none of the four strains exhibited a lag phase after 24 hours. The lag phase for cocci was 0-2 hours, and for bacilli, it was 0-1 hour. Specifically, the logarithmic phase for strain LSR-L-L1 was 2-9 hours, for strain LSR-L-L4 it was 2-8 hours, and for the two cocci it was 3-7 hours. The standard curves for diacetyl and extracellular polysaccharides are shown below. Figure 3 , 4 The LSR-L-L1 and LSR-L-L4 strains showed good aroma production at diacetyl levels of 8.5-9.6 and 10.5-11.6 ug / mL, respectively. Both strains had low extracellular polysaccharide content and weak mucilage production, with extracellular polysaccharide values ​​ranging from 128.1 to 160.2 mg / L.

[0100] Subgeneration lactic acid bacteria starter culture, growth curve as shown Figure 5 The lag phase was 0-1 h, the logarithmic phase 2-10 h, the first stationary phase 11-26 h, the second logarithmic proliferation phase 27-32 h, the stationary phase 33-48 h, and no decline phase was observed after 48 h. Seven stability experiments were conducted. During fermentation, the starter culture fermented for 6-7 h, with an acidity of 82-90°T, diacetyl content of 22.8-24.6 ug / mL, and extracellular polysaccharide content of 226.5-248.3 mg / L. This data provides a basis for adjusting the fermentation time, product fineness, and acidity / aroma of subsequent products.

[0101] Example 5

[0102] Yogurt preparation using serial lactic acid bacteria starter

[0103] Yogurt base preparation: After cleaning the raw milk, the protein content of the raw milk is 2.7% (if the physicochemical properties are low, skim milk powder is added to increase the protein content). Heat the milk to 65°C and set aside. Add 1 part agar (dissolved in cold water before adding), 1 part starch, 0.02 parts sea salt, 1 part stabilizer, and 1 part white sugar (the stabilizer and 7% white sugar are first dry-mixed and dissolved in water at 75°C). Then mix with the remaining ingredients, test the physicochemical properties using FT120, make up the volume, homogenize, pasteurize, and cool for later use.

[0104] Yogurt production: 0.7% of the starter culture is inoculated into the base material in step 1), fermented at 42℃ for 4.3h, with an endpoint acidity of 64°T, followed by fermentation, demulsification, ripening, filling, microbial testing, and post-acidity tracking.

[0105] Post-acid tracking: Take 22 bottles of the filled product, place half in a 4℃ refrigerator and the other half in a 28℃ incubator for post-acid tracking, viable count detection and water separation rate.

[0106] In Example 5, the finished product contained 2.8-2.9% protein and 3.6-3.8% fat, with an inoculum size of 0.6-0.8%, a fermentation time of 4-4.5 hours, a fermentation acidity of 64±2°T, an acidity of 66-68°T after refrigeration, and an ultimate acidity of 94-102°T for 10 batches of samples incubated at 28°C for 21 days. The viable cell count was 1.5-8.0×10⁻⁶.8 With a cfu / mL concentration and a 21-day water separation rate of ≤0.45%, the product maintains a high quality taste while having a high number of live bacteria. An example is Huangshi Laisier Dairy's small yogurt product, which has an annual output of more than 12,000 tons. It uses a generational lactic acid bacteria starter, saving the company more than 1.5 million yuan in production costs every year.

[0107] Example 6

[0108] Yogurt preparation using serial lactic acid bacteria starter

[0109] Yogurt base preparation: After cleaning the raw milk, the protein content of the raw milk is 2.8% (if the physicochemical properties are low, skim milk powder is added to increase the protein content). Heat the milk to 60°C and set aside. Add 0.5 parts agar (dissolved in cold water before adding), 0.5 parts starch, 0.01 parts sea salt, 0.5 parts stabilizer, and 0.5 parts white sugar (the stabilizer and 7% white sugar are first dry-mixed and dissolved in water at 70°C). Then mix with the remaining ingredients, test the physicochemical properties using FT120, make up the volume, homogenize, pasteurize, and cool for later use.

[0110] Yogurt production: 0.6% of the starter culture is inoculated into the base material in step 1), fermented at 40℃ for 4 hours, with an endpoint acidity of 62°T, followed by fermentation, demulsification, ripening, filling, microbial testing, and post-acidity tracking.

[0111] Post-acid tracking: Take 22 bottles of the filled product, place half in a 4℃ refrigerator and the other half in a 28℃ incubator for post-acid tracking, viable count detection and water separation rate.

[0112] Example 7

[0113] Yogurt preparation using serial lactic acid bacteria starter

[0114] Yogurt base preparation: After purification, the raw milk protein content is 2.9% (if the physicochemical properties are low, skim milk powder is added to increase the protein content). Heat to 70℃ and set aside. Add 1.5 parts agar (dissolved in cold water before adding), 1.5 parts starch, 0.03 parts sea salt, 1.5 parts stabilizer, and 1.5 parts white sugar (the stabilizer and 7% white sugar are first dry-mixed and dissolved in water at 80℃). Then mix with the remaining ingredients, test the physicochemical properties using FT120, make up to volume, homogenize, pasteurize, and cool for later use.

[0115] Yogurt production: 0.8% of the starter culture is inoculated into the base material in step 1), fermented at 44℃ for 4.5 hours, with an endpoint acidity of 66°T, followed by fermentation, demulsification, ripening, filling, microbial testing, and post-acidity tracking.

[0116] Post-acid tracking: Take 22 bottles of the filled product, place half in a 4℃ refrigerator and the other half in a 28℃ incubator for post-acid tracking, viable count detection and water separation rate.

[0117] Comparative Example 1

[0118] Preparation and application of subculture lactic acid bacteria starter culture in yogurt

[0119] (1) Following the steps in Example 1, experiments were conducted using four strains in a ratio of 10:1, 11:1, 13:1, 14:1, 1:1, and 5:1. The results were compared with those of LSE-2018DH003-G3+LSE-2018DH003-G9+LSR-L-L1+LSR-L-L4 = 1.2%+1.2%+0.1%+0.1% (12:1) in Table 1 below.

[0120] Table 1: Fermentation time, acidity, diacetyl, and diacetyl detection for different compounding ratios.

[0121] 10:1 6.8 85 19.8 216.8 11:1 6.5 87 20.2 222.1 12:1 6.5 85 23.5 248.3 13:1 6.2 85 18.7 256.5 14:1 6.0 86 18.2 262.3 1:1 8.2 86 29.8 125.8 5:1 7.5 85 28.6 163.3

[0122] As shown in the table above: For producing starter culture, a ball-to-salicylate ratio of 10-14:1 results in a fermentation time of 6.8-6.0 hours, a fermentation acidity of 85-87°T, and the highest yield is achieved with a ball-to-salicylate ratio of 12:1. At a ball-to-salicylate ratio of 1:1, the fermentation time is extended, with an extracellular content of 125.8 mg / L and a diacetyl content of 29.8 ug / mL. At a ball-to-salicylate ratio of 5:1, the fermentation time is 7.5 hours, with an extracellular content of 163.3 mg / L and a diacetyl content of 28.6 ug / mL. Based on the fermentation time and diacetyl and extracellular polysaccharide content, the optimal fermentation ratio was selected as LSE-2018DH003-G3+LSE-2018DH003-G9+LSR-L-L1+LSR-L-L4 = 1.2%+1.2%+0.1%+0.1%.

[0123] (2) Yogurt was made using the steps in Example 5. The fermentation substrate was a generation of lactic acid bacteria starter culture. The fermentation acidity was set at 64±2°T. The fermentation time was compared. The acidity was measured after 21 days of incubation at 28°C. The water separation rate is shown in Table 2 below.

[0124] Table 2. Acidity and water separation rate of products after heat preservation for different proportions of fermentation substrate and fermentation time.

[0125]

[0126]

[0127] As shown in Table 2 above: LSE-2018DH003-G3+LSE-2018DH003-G9+LSR-L-L1+LSR-L-L4 = 1.2%+1.2%+0.1%+0.1%, fermentation time 4.2h, after 21 days of incubation at 28℃, the acidity was low, the limiting acidity was 102°T, and the water separation rate after 21 days was less than 0.45%. This subculture lactic acid bacteria starter produces acid quickly, has low acidity, and good water retention performance.

[0128] Comparative Example 2

[0129] Comparison of fermentation with generation-type lactic acid bacteria starter and commercial direct-inoculation strains

[0130] Commercial strains selected: Danisco, Chr. Hansen, and DSM, all of which are stirred strains. The inoculation dosage was inoculated according to the instructions. The substrate was a small yogurt substrate. The fermentation time, acidity, water separation rate, and post-acidity were compared with the fermentation substrate of the subgeneration lactic acid bacteria starter of this invention. The product's fineness, viscosity, sourness, aroma, and overall taste were scored. The results are shown in Tables 3 and 4 below.

[0131] Table 3 Fermentation time, acidity, water separation rate, and post-acidification

[0132] Danisco 5.0 64 0.48 108 Chr. Hansen 4.8 64 0.45 106 DSM 5.0 64 0.52 108 Subgeneration fermentation agent 4.5 64 0.45 102

[0133] As shown in Table 3 above: the fermentation time of the four starter cultures for yogurt was 4.5-5.0 hours, the water separation rate after 21 days was 0.45-0.52%, and the acidity after 21 days of heat preservation did not exceed 108°T. Among them, the subculture starter culture had the shortest fermentation time and the lowest acidity after heat preservation.

[0134] Table 4: Product smoothness, viscosity, acidity, aroma, and overall taste.

[0135] Danisco 4.8 4.9 4.6 4.6 4.2 Chr. Hansen 4.8 4.8 4.7 4.7 4.3 DSM 4.8 4.8 4.7 4.5 4.2 Subgeneration fermentation agent 4.8 4.6 4.8 4.9 4.6

[0136] As shown in Table 4 above: after fermentation, demulsification, and post-ripening, the products were tasted by 25 people. Four different starter cultures were evaluated. All products were smooth, fine, and had a stringy texture, scoring 4.8 points. Viscosity testing showed that the secondary starter culture had the lowest viscosity (4.6 points) compared to the other three. Tasting the sourness and aroma, the secondary starter culture had the best sourness and aroma, scoring higher than the other three. The final score was based on a comprehensive evaluation of the yogurt's consistency, viscosity, sourness, aroma, and aftertaste, with the average score from the 25 participants being taken. The secondary starter culture received the highest score of 4.6 points.

[0137] Results Analysis

[0138] The intermediate starter culture, production starter culture, and yogurt prepared by this invention are analyzed using the following methods: Acidity, viable cell count, diacetyl, extracellular polysaccharides, and whey separation rate of the yogurt are determined.

[0139] The acidity titration method shall conform to GB5009.239-2016;

[0140] In all examples, the viable cell count detection method used was GB / T14699.1, and the culture medium used was MRS and TJA solid culture medium.

[0141] Diacetyl detection: Refer to Shao Yadong's method.

[0142] Detection of extracellular polysaccharides: Phenol-sulfuric acid method

[0143] Yogurt whey separation rate test: Use a syringe to draw up the supernatant, filter it, weigh it, and calculate the whey separation rate.

[0144] The test results are as follows:

[0145] The four strains of this invention were selected for experiments from generations 4 to 8. The intermediate fermentation time, acidity, viable cell count, diacetyl, and extracellular polysaccharide values ​​are shown in Table 5 below:

[0146] Table 5. Fermentation time, acidity, viable cell count, diacetyl, and extracellular polysaccharide values ​​of intermediate agents.

[0147]

[0148] As shown in Table 5 above: A single strain was inoculated at a volume ratio of 2% for skim milk fermentation. The viable count of cocci reached 10⁻⁶. 8 cfu / mL, bacteria 10 9 CFU / mL. After 14 hours of coccal fermentation, the acidity was 84-92°T and the extracellular polysaccharide content was 128.1-160.2 mg / L. Based on the characteristics of the two strains' growth curves, the logarithmic phase was 2-8 hours and the stationary phase was long. After 8 hours of fermentation, the acidity was 82-90°T and the extracellular polysaccharide content was lower. Among them, the LSR-L-L4 strain produced better aroma with a diacetyl content of 10.5-11.6 ug / mL.

[0149] The production starter culture prepared in this invention is prepared by compounding the prepared intermediate agent. The inoculum amount is 2%, and the compounding ratio is LSE-2018DH003-G3+LSE-2018DH003-G9+LSR-L-L1+LSR-L-L4=1.2%+1.2%+0.1%+0.1%. The fermentation time, acidity, viable cell count, diacetyl, and extracellular polysaccharide content are shown in Table 6 below.

[0150] Table 6. Fermentation time, acidity, viable cell count, diacetyl, and extracellular polysaccharides of the starter culture.

[0151] 6.0 86 <![CDATA[6.9×10 8 ]]> 22.8 236.8 6.5 84 <![CDATA[7.3×10 8 ]]> 24.2 242.3 7.0 88 <![CDATA[8.1×10 8 ]]> 23.5 248.3 6.8 90 <![CDATA[7.5×10 8 ]]> 23.1 226.5 6.0 86 <![CDATA[6.4×10 8 ]]> 22.9 239.4 6.4 82 <![CDATA[8.1×10 8 ]]> 24.6 235.7 6.5 84 <![CDATA[7.4×10 8 ]]> 23.7 233.3

[0152] As shown in Table 6 above: After fermentation with intermediate agents, the four strains were compounded in proportion and produced a starter culture through mutualistic symbiosis with cocci. The fermentation time was 6-7 hours, the acidity was 82-90°T, the diacetyl content was 22.8-24.6ug / mL, and the extracellular polysaccharide content was 226.5-248.3mg / L.

[0153] The yogurt prepared according to this invention is prepared by inoculating the prepared starter culture at a mass ratio of 0.6-0.8% with the fermentation of a small yogurt base, followed by demulsification and post-ripening. The acidity and viable cell count at 4℃ for 21 days, the acidity and viable cell count of 10 batches of samples after incubation at 28℃, and the water separation rate of one batch after 21 days are shown in Tables 3, 4, and 5 below.

[0154] Table 7. Acidity and viable bacteria count at 4℃ for 21 days

[0155] Acidity °T 66 66 68 67 66 67 68 66 68 66 68 <![CDATA[Viable count 10 8 cfu / mL]]> 4.5 5.7 6.5 8.0 11 13 15 14.5 16.0 16.2 18.0

[0156] As shown in Table 7 above: Under refrigeration conditions, the acidity of the product changes little, remaining stable between 66-68°T for 21 days, with a viable bacteria count of 4.5-18.0 × 10⁻⁶. 8 cfu / mL.

[0157] Table 8. Acidity and viable bacterial counts after incubation at 28℃

[0158] Batch 1 Acidity °T 64 68 84 86 92 94 94 94 94 94 94 Batch 2 Acidity °T 64 68 82 85 90 96 96 94 96 96 96 Batch 3 Acidity °T 62 71 83 88 90 91 94 97 96 96 96 Batch 4 Acidity °T 63 72 84 88 92 91 96 97 96 96 96 Batch 5 Acidity °T 66 76 88 96 100 102 100 102 101 102 102 Batch 6 Acidity °T 62 69 85 94 96 98 96 96 97 97 96 Batch 7 Acidity °T 63 67 83 90 94 96 98 96 98 97 96 Batch 8 Acidity °T 66 72 86 86 88 89 90 92 94 98 100 Batch 9 Acidity °T 65 70 85 86 89 90 94 96 98 98 98 Batch 10 Acidity °T 63 69 83 87 90 92 95 98 98 98 98 <![CDATA[Viable count 10 8 cfu / mL]]> 4.5 6.7 8 7.2 6.5 5.2 4 3.2 2.2 2 1.5

[0159] As shown in Table 8 above: For samples kept at 28℃ for 21 days, the acidity changes showed that the limiting acidity of 10 batches of samples was between 94-102°T, with lower acidity later. The viable bacterial count of the samples kept at 28℃ for 21 days ranged from 1.5 to 8.0 × 10⁻⁶. 8 cfu / mL

[0160] Table 9. 21-day water separation rate

[0161] Water separation rate % 0.00 0.00 0.10 0.15 0.21 0.34 0.36 0.4 0.42 0.44 0.45

[0162] As shown in Table 9 above: the water separation rate of the 21-day sample is ≤0.45%, the water retention rate is good, there is basically no whey separation, and the texture is fine.

[0163] In summary, all four strains of this invention are registered and preserved strains with excellent fermentation characteristics, specifically as follows:

[0164] 1. Adaptable to product processing conditions, the two cocci produce acid quickly, with low post-acidity, aroma, and stickiness; the two bacilli do not produce stickiness, produce good aroma, and have a high viable count; the four strains are stable after 4-8 generations of subculture; by controlling the cocci-bacilli ratio through strain compounding, the fineness, viscosity, and post-acidity of the product can be adjusted, resulting in a subculture lactic acid bacteria starter with independent intellectual property rights. The cocci-bacilli compounding ratio is LSE-2018DH003-G3+LSE-2018DH003-G9+LSR-L-L1+LSR-L-L4=1.2%+1.2%+0.1%+0.1%.

[0165] 2. Acid production is rapid during fermentation. The inoculum amount of the subculture lactic acid bacteria starter is 600-800g / ton, and the fermentation time is 4-4.5h. Compared with commercial direct-inoculation starter, the fermentation time is very similar.

[0166] 3. Low post-acidity and high viable cell count result in yogurt with excellent acidity and aroma, a delicate texture, and a post-acidity of 94-102°T in 10 batches of samples incubated at 28℃ for 21 days, with viable cell counts ranging from 1.5 to 8.0 × 10⁻⁶. 8 cfu / mL, greater than the national standard requirement of 10 6 cfu / mL.

[0167] 4. The starter culture contains 22.8-24.6 ug / mL of diacetyl and 226.5-248.3 mg / L of extracellular polysaccharide, which enhances the sour aroma and maintains the yogurt's texture. The yogurt has a water separation rate of ≤0.45% after 21 days, ensuring a high-quality taste.

[0168] 5. The prepared yogurt has a delicate texture, good stringiness, low post-acidity, and high live bacteria count; it minimizes the high costs associated with direct inoculation of starter cultures, saving the company more than 4.6 million yuan since 2019.

[0169] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A subculture lactic acid bacteria starter culture, characterized in that, The inoculum consists of the following strains: Streptococcus thermophilus ( Streptococcus thermophilus LSE-2018DH003-G3, Streptococcus thermophilus ( Streptococcus thermophilus LSE-2018DH003-G9, Lactobacillus delbrueckii bulgaricus strain ( Lactobacillus delbrueckii subsp. Bulgaricus LSR-L-L1, Lactobacillus delbrueckii bulgaricus strain ( Lactobacillus delbrueckii subsp. Bulgaricus LSR-L-L4; Among them, the Streptococcus thermophilus LSE-2018DH003-G3 has the accession number CGMCC NO. 21841, the Streptococcus thermophilus LSE-2018DH003-G9 has the accession number CGMCC NO. 21842, the Lactobacillus delbrueckii bulgaricus LSR-L-L1 has the accession number CGMCC NO. 14750, and the Lactobacillus delbrueckii bulgaricus LSR-L-L4 has the accession number CGMCC NO. 14752; The starter culture consists of lactic acid bacteria inoculum in the following weight ratio: 8–15 portions of Streptococcus thermophilus LSE-2018DH003-G3 inoculation solution, 8–15 portions of Streptococcus thermophilus LSE-2018DH003-G9 inoculation solution, 0.5–1.5 portions of Lactobacillus delbrueckii bulgaricus LSR-L-L1 inoculation solution, and 0.5–1.5 portions of Lactobacillus delbrueckii bulgaricus LSR-L-L4 inoculation solution.

2. The subculture lactic acid bacteria starter culture as described in claim 1, characterized in that, It consists of a lactic acid bacteria inoculum in the following weight ratio: Twelve inoculum solutions of Streptococcus thermophilus LSE-2018DH003-G3, twelve inoculum solutions of Streptococcus thermophilus LSE-2018DH003-G9, one inoculum solution of Lactobacillus delbrueckii bulgaricus LSR-L-L1, and one inoculum solution of Lactobacillus delbrueckii bulgaricus LSR-L-L4.

3. A method for preparing a subculture lactic acid bacteria starter culture as described in any one of claims 1 to 2, characterized in that, Includes the following steps: 1) Preparation of single-strain fermentation intermediate: After the strain has been cultured to the 4th to 8th generation, the single strain is inoculated into a sterilized skim milk bottle with a protein content of 3.2% to 3.4% at a volume ratio of 2% to 3%. The fermentation intermediate is carried out at 40 to 44 °C. The final acidity of cocci is 84 to 92 °T, and the final acidity of bacilli is 82 to 90 °T. 2) Preparation of fermentation agent by compounding strains: The intermediate agent fermented in step 1) is compounded according to the strain inoculation amount of 1-3% by volume, and inoculated into skim milk medium with protein content of 3.0-3.2%. Fermentation is carried out at 40-44 ℃ for 6-7 h, and the final acidity is 88-96 degrees.

4. A yogurt prepared using the subculture lactic acid bacteria starter culture according to any one of claims 1 to 3, characterized in that, Made from the following raw and auxiliary materials by weight percentage: Lactic acid bacteria starter 0.6-0.8%, raw milk protein content after purification 2.8-3.0%, 0.5-1.5% agar, 0.5-1.5% starch, 0.005-0.015% sea salt, 0.5-1.5% stabilizer, 0.5-1.5% white sugar.

5. The yogurt as described in claim 4, characterized in that, Made from the following raw and auxiliary materials by weight percentage: The formula contains 0.7% lactic acid bacteria starter, 2.9% protein content of purified raw milk, 1% agar, 1% starch, 0.01% sea salt, 1% stabilizer, and 1% white sugar.

6. The method for preparing yogurt according to any one of claims 4 to 5, characterized in that, Includes the following steps: 1) Preparation of yogurt base: After purification, the raw milk protein content is 2.8-2.9%. Heat the milk to 60-70℃, add 0.5-1.5 parts agar, 0.5-1.5 parts starch, 0.01-0.03 parts sea salt, 0.5-1.5 parts stabilizer, and 0.5-1.5 parts white sugar. Mix with the remaining ingredients, test the physicochemical properties, adjust the volume, homogenize, pasteurize, and cool for later use. 2) Yogurt production: Inoculate 0.6-0.8% of the starter culture into the base material in step 1), ferment at 40-44℃ for 4-4.5 h, with an endpoint acidity of 62-66 °T, ferment, break the emulsion, ripen, fill, conduct microbial testing, and track post-acidity.

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