Directly thrown low-temperature yogurt starter and preparation method and application thereof

Abstract

The present application relates to the technical field of microbial preparation, in particular to a direct-injection type low-temperature yogurt starter, and a preparation method and application thereof.The direct-injection type low-temperature yogurt starter comprises Lactobacillus plantarum JL01 ( Lactiplantibacillus plantarum ) bacterial powder and Paracaseicoccus JL02 ( Lacticaseibacillus paracasei ) bacterial powder; the mass ratio of the Lactobacillus plantarum JL01 bacterial powder and the Paracaseicoccus JL02 bacterial powder is 1:0.25-4.The Lactobacillus plantarum JL01 bacterial powder and the Paracaseicoccus JL02 bacterial powder are compounded as a composite starter, so that the yogurt can be fermented at low temperature, and the yogurt can be prepared from various animal milk and plant milk.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a direct-inoculation low-temperature yogurt starter, its preparation method, and its application. Background Technology

[0002] Yogurt is a unique flavored fermented dairy product made from dairy products through fermentation with lactic acid bacteria. It not only retains the nutritional components of dairy products, but also breaks down proteins, lactose, and lipids during fermentation, making them easier for the body to digest and absorb. It also has functional properties such as regulating intestinal flora. Most commonly used yogurt starter cultures are made from a combination of Streptococcus thermophilus and other lactobacilli, typically fermented at around 40℃. Numerous studies have shown that at low temperatures, bacterial strains develop different metabolic pathways and produce different metabolites, thereby improving the flavor, quality, and safety of the fermented product. Therefore, to meet the demand for low-temperature fermented yogurt products, there is an urgent need to develop a yogurt starter culture that is suitable for low-temperature fermentation and has broad applicability.

[0003] Direct-inoculation starter cultures refer to starter cultures that can be directly inoculated without activation or subculturing, and are prepared by mixing one or more bacterial powders and their adjuvants. The starter culture directly affects the quality of yogurt products and production costs. The quality of the starter culture is influenced by the strain itself and the production process. It requires high-density cultivation under suitable culture media and conditions, followed by processes such as centrifugation, addition of freeze-drying protectants, and freeze-drying. The composition of the culture medium is crucial in starter culture production.

[0004] Milk is highly susceptible to microbial contamination and spoilage during processing, distribution, and sales, resulting in significant amounts of waste milk. Furthermore, with increasingly stringent food safety standards, large quantities of raw milk that do not meet these standards cannot be effectively utilized. Milk contains a large amount of protein and is an ideal source of high-quality nitrogen for culture media. However, lactic acid bacteria cannot directly utilize large exogenous protein molecules; they must break them down into smaller peptides or amino acids through their own protein hydrolysis system. This leads to low utilization rates and slow growth. Therefore, to increase efficiency and conserve resources, the dairy industry urgently needs to treat waste milk for use in the production of yogurt starter cultures to achieve efficient and comprehensive utilization. Summary of the Invention

[0005] The purpose of this invention is to address the problem of low comprehensive utilization of large quantities of waste milk by providing a direct-inoculation low-temperature yogurt starter, which is a highly active direct-inoculation yogurt starter. This direct-inoculation low-temperature yogurt starter utilizes the preserved strain *Lactobacillus plantarum* JL01 (… Lactiplantibacillus plantarum ) and Lactobacillus paracasei JL02 ( Lacticaseibacillus paracaseiThis allows the prepared yogurt starter to be suitable for low-temperature fermentation of various dairy products to produce yogurt, offering wide applicability and improving the flavor, quality, and safety of fermented products. Furthermore, its direct-inoculation preparation makes it more convenient and faster to use.

[0006] To achieve the above-mentioned objectives, the specific technical solution of the present invention is as follows:

[0007] A direct-inoculation low-temperature yogurt starter, comprising Lactobacillus plantarum JL01 ( Lactiplantibacillus plantarum ) Bacterial powder and Lactobacillus paracasei JL02 ( Lacticaseibacillus paracasei The bacterial powder; wherein the mass ratio of Lactobacillus plantarum JL01 bacterial powder and Lactobacillus paracasei JL02 bacterial powder is 1:0.25-4.

[0008] Furthermore, in the aforementioned direct-inoculation low-temperature yogurt starter, the *Lactobacillus plantarum* JL01 ( Lactiplantibacillus plantarum It was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO: M 20241692, and the address of the depository is Wuhan University, Wuhan, China.

[0009] Furthermore, in the yogurt starter, the *Lactobacillus paracasei* JL02 ( Lacticaseibacillus paracasei It was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO: M 20241693, and the address of the depository is Wuhan University, Wuhan, China.

[0010] To better prepare direct-inoculation low-temperature yogurt starter, this application also provides a method for preparing the above-mentioned yogurt starter, including the following steps:

[0011] 1) Preparation of Lactobacillus plantarum JL01 bacterial powder;

[0012] The *Lactobacillus plantarum* strain JL01 was inoculated into MRS medium at a volume ratio of 1-2% to obtain a primary seed culture. Further subculturing and amplification were performed to obtain a secondary seed culture. The secondary seed culture was then inoculated into the fermentation medium containing milk peptone as described in section 5 and cultured. Fermentation aids (antioxidant: ascorbic acid 1-2 g / L; osmotic pressure protectant: betaine 0.5-1.0 g / L, glycerol 1-3 g / L; defoamer: polyether defoamer 0.5-1 g / L) were added, and the culture temperature was controlled at 35℃, with a constant pH of 6. After culturing for 12-16 h, 20 g / L glucose and milk peptone were added every hour for a total of 3 times, and the culture was repeated to obtain the fermentation broth. The fermentation broth was centrifuged, mixed with a lyophilization protectant (preferably a lyophilization protectant containing milk peptone), and lyophilized to obtain *Lactobacillus plantarum* JL01 bacterial powder.

[0013] 2) Preparation of Lactobacillus paracasei JL02 bacterial powder;

[0014] Lactobacillus paracasei JL02 strain was inoculated into MRS medium at a volume ratio of 1-2% to obtain a primary seed culture. Further subculturing and scale-up culture yielded a secondary seed culture. Fermentation aids (antioxidant: ascorbic acid 1-2 g / L; osmotic protectant: betaine 0.5-1.0 g / L, glycerol 1-3 g / L; defoamer: polyether defoamer 0.5-1 g / L) were added, and the culture temperature was controlled at 37℃, with a constant pH of 7. After culturing for 12-16 h, 20 g / L glucose and milk peptone were added every 2 hours for a total of 3 times, followed by further culture to obtain the fermentation broth. The fermentation broth was centrifuged, mixed with a lyophilization protectant (preferably a lyophilization protectant containing milk peptone), and lyophilized to obtain Lactobacillus paracasei JL02 bacterial powder.

[0015] 3) Mix the prepared Lactobacillus plantarum JL01 powder and Lactobacillus paracasei JL02 powder at a mass ratio of 1:0.25-4.

[0016] Furthermore, the viable count of *Lactobacillus plantarum* JL01 bacterial powder obtained reached 1.71 × 10⁻⁶. 12 CFU / g, viable count still greater than 1×10⁻⁶ after storage at 4℃ for 90 days. 12 CFU / g.

[0017] Furthermore, the viable count of the *Lactobacillus paracasei* JL02 bacterial powder reached 1.21 × 10⁻⁶. 12 CFU / g, viable count still greater than 1×10⁻⁶ after storage at 4℃ for 90 days. 12 CFU / g.

[0018] To better prepare *Lactobacillus plantarum* JL01 and *Lactobacillus paracasei* JL02 bacterial powders, milk peptone was added to the fermentation medium. The preparation of milk peptone also addresses the problem of low utilization of waste milk.

[0019] A method for preparing milk peptone using milk includes the following steps:

[0020] 1) Extraction of crude protein from milk:

[0021] Using milk as raw material, the pH was adjusted to 4.6-4.8 with 2-4 mol / L hydrochloric acid, and then the mixture was centrifuged to collect the crude milk protein.

[0022] The milk mentioned is either fresh milk or waste milk; the waste milk includes, but is not limited to, any one or more of the following: milk that has exceeded its shelf life or raw milk that does not meet food standards.

[0023] 2) Protease hydrolysis:

[0024] Collect crude milk protein and mix it with pure water, then add an appropriate amount of protease for hydrolysis to obtain protease hydrolysate;

[0025] Preferably, the specific operation of step 2) is as follows: milk crude protein and pure water are mixed in a mass ratio of 1:2; a compound protease is selected and added at a mass of 6‰ of the total mass of milk crude protein and pure water; the hydrolysis conditions are: pH 7.0 and hydrolysis time 9 h; through this step, the degree of hydrolysis of the protease hydrolysate can be 36.03%.

[0026] 3) Ultrasound-assisted protein hydrolysis:

[0027] The protein hydrolysate obtained above was treated in an ultrasonic generator at 100-150 W for 0.5-1 h to obtain a milk protein hydrolysate with a high degree of hydrolysis; after treatment, the degree of hydrolysis of milk protein increased to 38.71%.

[0028] 4) Preparation of milk peptone from hydrolysate:

[0029] The above-mentioned milk protein hydrolysate was treated in a water bath at 90-100°C for 0.5-1 h to inactivate the enzymes and further hydrolyze the protein, resulting in a degree of hydrolysis of 39.19%. The mixture was centrifuged again, and the supernatant was collected. The supernatant was then spray-dried (inlet air 160-190°C, outlet air 70-90°C) to obtain milk peptone.

[0030] The milk peptone prepared by the above method passed the tests for transparency, alkaline precipitation, pH, amino nitrogen, total nitrogen, loss on drying, residue on ignition, and bacteriological examination. Among these tests, the amino nitrogen, total nitrogen, and total protein contents were higher than those of other commercially available peptones, and the molecular weight distribution was better than that of commercially available peptones, indicating that it is more conducive to microbial growth as a nitrogen source for microorganisms.

[0031] In addition, the milk peptone obtained above has a higher proportion of cysteine ​​(antioxidant) and aspartic acid, glutamic acid and proline (osmolar protectants) in its amino acid composition. Therefore, the milk peptone of the present invention has great potential in alleviating oxidative stress of strains and improving osmolar resistance.

[0032] Furthermore, the milk peptone obtained above achieved a DPPH scavenging rate of 77.32%, and its antioxidant capacity was significantly higher than that of other peptones. This indicates that milk peptone contains more amino acids and peptides with antioxidant capacity, which can alleviate the oxidative stress and oxidative damage of high-density cultured strains, thereby benefiting the growth of facultative anaerobic and anaerobic fermentation strains.

[0033] To further utilize milk peptone as a nitrogen source in culture media in a more efficient manner, this invention proposes to apply milk peptone prepared by the above methods or combinations of method steps in culture media.

[0034] Furthermore, a fermentation medium containing milk peptone is provided, wherein the milk peptone obtained by the above method replaces the peptone in the MRS medium, and the remaining components are the same as those in the MRS medium.

[0035] More preferably, the amount of milk peptone added to the fermentation medium is 5-10 g / L.

[0036] For example: Lactobacillus plantarum JL01 ( Lactiplantibacillus plantarum The fermentation medium was prepared with the following proportions: milk peptone 10 g / L, beef extract 10 g / L, yeast powder 7.5 g / L, glucose 30 g / L, manganese sulfate 0.5 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, Tween 80 1.0 g / L, and magnesium sulfate 0.58 g / L.

[0037] Lactobacillus paracasei JL02 ( Lacticaseibacillus paracasei The fermentation medium was prepared with the following proportions: milk peptone 5 g / L, beef extract 10 g / L, yeast powder 2.5 g / L, glucose 30 g / L, manganese sulfate 0.5 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, Tween 80 1.0 g / L, and magnesium sulfate 0.58 g / L.

[0038] To better utilize milk peptone, the present invention also provides the application of milk peptone obtained by any of the methods described above in a freeze-drying protectant.

[0039] Preferably, a milk peptone lyophilization protectant comprises the following components in weight percentage:

[0040] Trehalose 10-20%, milk peptone 5-10%, glycerol 1-3%, the balance being sterile distilled water, the total mass percentage is 100%.

[0041] Compared with existing technologies, the beneficial effects of this invention are:

[0042] (i) The milk peptone prepared by this invention has transparency, alkaline precipitation, pH, amino nitrogen, total nitrogen, loss on drying, residue on ignition, and bacteriological test results all within the specified range and are qualified, and can replace the peptone in MRS culture medium. In addition, the amino nitrogen, total nitrogen, and total protein content are all higher than other commercially available peptones, indicating higher quality.

[0043] (II) The milk peptone prepared in this invention, through amino acid composition analysis, shows that the cysteine ​​content is significantly higher than that of other commercially available peptones. Numerous studies have shown that cysteine ​​can act as an antioxidant in the culture medium, alleviating the oxidative stress response of high-density cultured strains, thereby benefiting the growth of facultative anaerobic and anaerobic fermentation strains. In addition, aspartic acid, glutamic acid, and proline can act as osmotic pressure protectants in the culture medium, showing great potential in improving the osmotic pressure tolerance of strains.

[0044] (III) The milk peptones prepared by this invention have a molecular weight of less than 5000, and the proportion of molecular weights less than 500 Da reaches 67.54%. The smaller the molecular weight, the easier it is to be decomposed and utilized by microorganisms. The molecular weight distribution of the peptones is better than that of commercially available peptones, indicating that they are easier to be decomposed and utilized by microorganisms as a nitrogen source for microorganisms.

[0045] (iv) The milk peptone prepared by this invention has a DPPH scavenging rate of 77.32% and an antioxidant capacity that is significantly higher than other peptones. This indicates that the milk peptone contains more amino acids, peptides and other substances with antioxidant capacity, which can alleviate the oxidative stress and oxidative damage of high-density cultured strains, thereby benefiting the growth of facultative anaerobic and anaerobic fermentation strains.

[0046] (v) Using the above-mentioned milk peptone as a nitrogen source and freeze-drying protectant, the biomass of the fermentation broth was significantly increased, and the number of live bacteria in the bacterial powder increased by more than 10 times, with obvious effects.

[0047] (vi) If waste milk is used to prepare milk peptone, and the processed milk peptone is then used in fermentation culture medium, the amount of waste milk used can be increased, thus achieving efficient and comprehensive utilization of waste milk.

[0048] (vii) Compared with commercially available starter cultures, the direct-inoculation low-temperature yogurt starter culture prepared by this invention is more suitable for low-temperature fermentation of yogurt (20~25℃) and has a wide range of applications. Pure cow's milk, skim milk, high-calcium milk, reconstituted cow's milk, goat's milk, plant-based soy milk, and plant-based coconut milk can all be used as fermentation substrates.

[0049] (viii) Compared with commercially available starter cultures, the direct-inoculation low-temperature yogurt starter culture prepared by this invention has better storage stability. After being stored at 25℃ for 90 days, the survival rate reached 85.3%, and the viable count reached 1.23 × 10⁻⁶. 12 CFU / g. Attached Figure Description

[0050] Figure 1 This is a bar chart showing the degree of hydrolysis of milk crude protein after adding different types of proteases in Example 1;

[0051] Figure 2The graph shows the antioxidant capacity test results of milk peptone prepared for this application (hydrolyzed with different types of proteases) and other commercially available peptones.

[0052] Figure 3 Figure showing the effect of different pH values ​​on the growth of Lactobacillus plantarum JL01;

[0053] Figure 4 Figure showing the effect of different pH values ​​on the growth of Lactobacillus paracasei JL02;

[0054] Figure 5 The growth curves of Lactobacillus plantarum JL01 at different culture times;

[0055] Figure 6 The growth curves of Lactobacillus paracasei JL02 at different culture times;

[0056] Figure 7 The response surface and contour lines of the interaction between the freeze-drying protectant and the freeze-drying protectant. Detailed Implementation

[0057] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0058] Any feature disclosed in this specification (including the claims and abstract) may be replaced by other equivalent or similar features, unless specifically stated otherwise. That is, unless specifically stated otherwise, each feature is merely one example of a series of equivalent or similar features.

[0059] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0060] Unless otherwise specified, % in this application refers to the percentage content by mass.

[0061] The Lactobacillus plantarum JL01 involved in this application ( Lactiplantibacillus plantarum It was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO: M 20241692, and the address of the depository is Wuhan University, Wuhan, China.

[0062] The involved Lactobacillus paracasei JL02 ( Lacticaseibacillus paracasei It was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO: M 20241693, and the address of the depository is Wuhan University, Wuhan, China.

[0063] Lactobacillus plantarum JL01, screened from traditional yak milk from Aba, Sichuan, possesses at least one of the following characteristics:

[0064] Lactobacillus plantarum JL01 can efficiently convert tributyric acid esters into butyric acid, and the butyric acid content can be controlled and adjusted by changing the tributyric acid ester content, thus meeting the application requirements of various functional foods. For example, when the tributyric acid ester content is 20 g / L, the butyric acid yield of Lactobacillus plantarum JL01 can reach 120.92 ± 1.45 mg / kg.

[0065] *Lactobacillus plantarum* JL01 exhibits strong acid resistance; for example, after 4 hours of culture at pH 2.0, its survival rate reaches 90.01%. *Lactobacillus plantarum* JL01 also demonstrates strong tolerance to gastric and intestinal fluids; its survival rate in gastric fluid reaches 97.00%, and in intestinal fluid, it reaches 98.37%. Furthermore, *Lactobacillus plantarum* JL01 exhibits strong bile salt tolerance; after 3 hours of culture in MRS medium containing 0.2 g / 100 mL bile salts, the viable count remains at 5.012 × 10⁻⁶. 7 CFU / mL. *Lactobacillus plantarum* JL01 exhibits strong self-aggregation; its self-aggregation capacity reaches 55.02%. For example, its coagulation with *Listeria monocytogenes* is 41.17%, and with *Escherichia coli* it is 39.25%; thus indicating that this strain has strong gastrointestinal adhesion and colonization capabilities. *Lactobacillus plantarum* JL01 can grow well at relatively low temperatures; after culturing at 25℃ for 24 h, its OD... 620nm The value reached 3.69.

[0066] Lactobacillus paracasei JL02 was screened from traditionally naturally fermented milk in Xinjiang and possesses at least one of the following characteristics:

[0067] 1) It has good acid resistance;

[0068] 2) It has good bile tolerance;

[0069] 3) It has the ability to produce high levels of extracellular polysaccharides;

[0070] 4) It has good ability to grow at low temperatures.

[0071] Furthermore, the *Lactobacillus paracasei* JL02 ( Lacticaseibacillus paracasei The strain produces acid rapidly, increasing the acidity in yak milk from 40.3% to 40.3% within 6–14 hours. o T increased to 97.6 o The acidity level tends to stabilize after 14 hours; it can survive in the acidic environment of the stomach, with a survival rate of up to 89.4%.

[0072] Furthermore, the aforementioned *Lactobacillus paracasei* JL02 ( Lacticaseibacillus paracaseiThe strain was cultured in MRS medium containing 0.1 g / 100 mL ox bile salts for 3 h, and the viable count of the strain remained at 10. 7 CFU / mL or higher.

[0073] Furthermore, the aforementioned *Lactobacillus paracasei* JL02 ( Lacticaseibacillus paracasei It has a high capacity for producing extracellular polysaccharides, up to a maximum of 380.25 mg / L.

[0074] Furthermore, the aforementioned *Lactobacillus paracasei* JL02 ( Lacticaseibacillus paracasei Incubate at 25℃ for 24 h, OD 620nm The value reached 3.21.

[0075] Example 1:

[0076] Preparation of milk peptone:

[0077] 1. Extraction of crude protein from milk

[0078] Adjust the pH of waste milk (milk expired 30 days ago) to 4.6-4.8 (isoelectric point) using 2 mol / L hydrochloric acid. Use a peristaltic pump to pump the solution into the feed inlet of a tubular centrifuge at a flow rate of 3 L / min. Stop centrifugation when no liquid is discharged from the outlet. Open the inner chamber of the tubular centrifuge and collect the crude milk protein.

[0079] 2. Proteolytic enzyme digestion

[0080] The above-mentioned crude milk protein was mixed with pure water at a mass ratio of 1:2-4, and different proteases (trypsin, neutral protease, papain, alkaline protease, and complex protease) were added at a mass ratio of 4‰. Hydrolysis was carried out for 6 hours. The proteases were screened using the degree of protein hydrolysis as an indicator. The degree of protein hydrolysis (%) was calculated as amino nitrogen / total nitrogen × 100. Total nitrogen content was determined using the Kjeldahl method (GB 5009.5—2016), and amino nitrogen was determined using the formaldehyde titration method (GB 5009.235—2016). A three-factor, three-level orthogonal experiment was then designed to determine the final proteolytic conditions based on the hydrolysis feed-to-liquid ratio, enzyme dosage, and hydrolysis time (as shown in Table 1).

[0081] Table 1: Three-factor, three-level experimental design and results of milk enzymatic hydrolysis conditions

[0082]

[0083] Depend on Figure 1 As shown in Table 1, the final enzymatic hydrolysis was performed by adding 6‰ complex protease. According to the instructions, the temperature was controlled at 50℃, pH 7.0, milk crude protein to pure water ratio was 1:2 (W / W), and hydrolysis was carried out for 9 hours. The results showed that the degree of protein hydrolysis reached 36.03% under these conditions.

[0084] 3. Ultrasound-assisted protein hydrolysis

[0085] The treated protein hydrolysate was placed in an ultrasonic generator with a power of 120 W and treated for 1 hour. After treatment, the degree of hydrolysis of milk protein increased to 38.71%.

[0086] 4. Preparation of milk peptone from hydrolysate

[0087] The milk protein hydrolysate, which had undergone enzymatic hydrolysis and ultrasonic treatment, was then subjected to a boiling water bath for 30 minutes to inactivate the enzymes and further hydrolyze the protein, ultimately achieving a degree of hydrolysis of 39.19%. Subsequently, a peristaltic pump was used to pump the hydrolysate at a flow rate of 3 L / min into the inlet of a tubular centrifuge, and the supernatant was collected at the outlet. The supernatant was then spray-dried (inlet air 180°C, outlet air 90°C) to obtain milk peptone.

[0088] Experiment 1: Comparative Evaluation of Milk Peptone

[0089] 1. Comparative Evaluation of Basic Physicochemical Properties and Microbiological Testing

[0090] According to the physicochemical and bacteriological testing methods and standards for peptone in the Chinese Standards for Quality Control of Major Raw and Auxiliary Materials for Biological Products (2000 Edition), the milk peptone prepared in Example 1 was compared and evaluated with commercially available peptone, tryptone, soy peptone, and casein peptone from common brands.

[0091] Table 2: Physicochemical Properties and Microbiological Testing of Peptone

[0092]

[0093] As shown in Table 2, the transparency, alkaline precipitation, pH, amino nitrogen, total nitrogen, loss on drying, residue on ignition, and bacteriological test results of the milk peptone prepared by this invention are all within the specified range and are qualified. The amino nitrogen, total nitrogen, and total protein contents are all higher than those of other commercially available peptones, indicating higher quality.

[0094] 2. Amino acid composition analysis

[0095] Take 10 mg of peptone sample in a test tube, add 10 mL of 6 mol / L HCl solution, and place in an oven at 110℃ for hydrolysis for 24 h. After hydrolysis, filter the hydrolysate, take 0.5 mL of the filtered hydrolysate, evaporate to dryness under reduced pressure, and make up to 1 mL. Filter through a 0.22 μm filter membrane and analyze using an automated amino acid analyzer. Specific results are shown in Table 3.

[0096] Table 3: Amino acid composition analysis of peptone

[0097]

[0098] As shown in Table 3, the cysteine ​​content of the milk peptone prepared in this invention is significantly higher than that of other commercially available peptones. Numerous studies have shown that cysteine ​​can act as an antioxidant in the culture medium, alleviating oxidative stress in high-density cultured strains, thus benefiting the growth of facultative and anaerobic fermentation strains. Furthermore, aspartic acid, glutamic acid, and proline have been reported in the literature to act as osmotic pressure protectants, entering cells as compatible solutes to mitigate the adverse effects of increased osmotic pressure on strain growth during the later stages of fermentation. In conclusion, based on the analysis of amino acid composition, the milk peptone of this invention has great potential in alleviating oxidative stress in strains and improving their osmotic pressure tolerance.

[0099] 3. Molecular weight comparison

[0100] Different molecular weight peptide standard solutions (0.1% by mass) were prepared using the mobile phase, filtered through a 0.45 μm microporous membrane, and injected to obtain chromatograms of the series of standards. Molecular calibration curves were obtained by plotting the logarithm of molecular weight (lgMw) against retention time or by performing linear regression. 10 mg of sample was weighed into a 10 mL volumetric flask, diluted to the mark with the mobile phase, filtered through a microporous membrane, and then injected. Chromatographic conditions: Column: TSKgel 2000 SWXL 300 mm × 7.8 mm; Flow rate: 0.5 mL / min; Injection volume: 20 μL; Detection wavelength: UV 220 nm; Column temperature: 30℃; Mobile phase: acetonitrile / water / trifluoroacetic acid, 45 / 55 / 0.1 (V / V). Specific results are shown in Table 4.

[0101] Table 4: Molecular weight distribution of peptone

[0102]

[0103] As shown in Table 4, the milk peptones prepared by this invention have molecular weights of less than 5000, and the proportion of molecular weights less than 500 Da reaches 67.54%. The smaller the molecular weight, the easier it is for microorganisms to decompose and utilize them. The molecular weight distribution of the peptones is better than that of commercially available peptones, indicating that they are easier for microorganisms to decompose and utilize them as a nitrogen source for microorganisms.

[0104] 4. Antioxidant capacity

[0105] Weigh 1 g of each peptone sample into a 10 mL volumetric flask and dilute to volume to prepare a peptone aqueous solution. Mix 2 mL of each peptone sample aqueous solution with 2 mL of 0.2 mmol / L DPPH, react in the dark for 30 min, and measure the absorbance at 517 nm, denoted as As. Under the same conditions, replace the DPPH solution with anhydrous ethanol, denoted as Ab; under the same conditions, replace the sample with distilled water, denoted as Ac. Calculate the DPPH scavenging rate % = (1 - (As - Ab) / Ac) × 100.

[0106] Depend on Figure 2 It can be seen that the milk peptone prepared by this invention has a DPPH scavenging rate of 77.32% and an antioxidant capacity that is significantly higher than other peptones. This indicates that the milk peptone contains more antioxidant peptides and other antioxidant substances, which can alleviate the oxidative stress and oxidative damage of high-density cultured strains, thereby benefiting the growth of facultative anaerobic and anaerobic fermentation strains.

[0107] Example 2:

[0108] Culture medium preparation

[0109] 1. Fermentation compounding of Lactobacillus plantarum JL01 medium

[0110] Using *Lactobacillus plantarum* JL01 as the subject, and based on the universal lactic acid bacteria culture medium MRS, the peptone prepared in this invention was used to replace the peptone in the MRS culture medium. A four-factor, three-level orthogonal experiment was designed to combine the addition amounts of carbon and nitrogen sources with strong interaction in the culture medium, as shown in Table 5.

[0111] Table 5: Results of the 4-factor, 3-level orthogonal experimental design for Lactobacillus plantarum JL01 culture medium

[0112]

[0113] As shown in Table 5, the carbon and nitrogen sources in the culture medium were optimized through orthogonal experiments. The final culture medium for *Lactobacillus plantarum* JL01 was composed of 10 g / L milk peptone, 10 g / L beef extract, 7.5 g / L yeast extract, 30 g / L glucose, 0.25 g / L manganese sulfate, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 1.0 g / L Tween 80, and 0.58 g / L magnesium sulfate.

[0114] 2. Fermentation medium for Lactobacillus paracasei JL02

[0115] Using Lactobacillus paracasei JL02 as the subject, and MRS medium, a universal culture medium for lactic acid bacteria, as the basis, the peptone prepared in this invention was used to replace the peptone in the MRS medium. A four-factor, three-level orthogonal experiment was designed to combine the addition amounts of carbon and nitrogen sources with strong interaction in the culture medium, as shown in Table 6.

[0116] Table 6: Results of the 4-factor, 3-level orthogonal experimental design for *Lactobacillus paracasei* JL02 culture medium

[0117]

[0118] As shown in Table 6, the carbon and nitrogen sources in the culture medium were optimized through orthogonal experiments. The final culture medium for Lactobacillus paracasei JL02 was composed of 5 g / L milk peptone, 10 g / L beef extract, 2.5 g / L yeast extract, 30 g / L glucose, 0.25 g / L manganese sulfate, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 1.0 g / L Tween 80, and 0.58 g / L magnesium sulfate.

[0119] Example 3:

[0120] High-density culture of strains

[0121] 1. High-density culture of Lactobacillus plantarum JL01

[0122] Thaw the glycerol tubes containing the strain and inoculate them into MRS medium at a volume concentration of 0.5-2%. Incubate at 25-40°C for 10-30 h and subculture 1-3 times to obtain the primary seed culture. Inoculate the primary seed culture into MRS medium with an initial pH of 5-8 at a volume concentration of 0.5-2% and incubate at 25-40°C for 10-30 h to obtain the secondary seed culture.

[0123] The secondary seed culture of the strain was inoculated into the fermentation medium at an inoculum rate of 2% (v / v), and cultured for 24 h at different temperatures (30℃, 35℃, 37℃, 40℃, 45℃) and different medium pH values ​​(5.0, 6.0, 7.0, 8.0). The OD values ​​at different fermentation temperatures and medium pH values ​​were measured. 620 nm According to OD 620 nm Determine the culture temperature and initial pH of the culture medium. Figure 3 It can be seen that the suitable culture temperature for Lactobacillus plantarum JL01 is 35℃ and the pH of the culture medium is 6.

[0124] Prepare 30 L of the above-mentioned fermentation medium in a 50 L fermenter, and add fermentation aids (antioxidant: ascorbic acid 1-2 g / L; osmotic pressure protectant: betaine 0.5-1.0 g / L, glycerol 1-3 g / L; defoamer: polyether defoamer 0.5-1 g / L). After sterilization, inoculate by flame method, with an inoculation amount of 1-2% (v / v), stirring speed of 50-100 rpm / min. Use 12.5% ​​ammonia water as a neutralizing agent to control the pH of the fermentation broth to 6 during fermentation. Incubate at 35℃ for 12-16 h. Feed-in fermentation is carried out according to different feeding strategies, as shown in Table 7. (Where OD...) 620 nmAbsorbance determination: After diluting the fermentation broth by a certain factor, the absorbance value of the diluted solution at a wavelength of 620 nm was measured and recorded using a UV spectrophotometer; Viable cell count determination: Performed according to GB4789.35-2016 (National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination); Dry weight determination: A certain amount of fermentation broth was accurately taken into a centrifuge tube, centrifuged at 8000 r / min for 10 min, the supernatant was discarded, the precipitate was washed twice with distilled water, and the cells were dried in an oven to constant weight.

[0125] Table 7: Effects of different feeding strategies on the growth of Lactobacillus plantarum JL01

[0126]

[0127] As shown in Table 7, based on the biomass of the fermentation broth, the feeding strategy selected was Strategy 3, namely, during the logarithmic growth phase, with 20 g / L glucose and milk peptone added every hour for a total of 3 times. At this time, the highest OD value, cell dry weight, and viable cell count of the fermentation broth reached 18.65, 11.79 g / L, and 2.95 × 10⁻⁶ g / L, respectively. 10 CFU / mL.

[0128] 2. Lactobacillus paracasei JL02

[0129] Thaw the glycerol tubes containing the strain and inoculate them into MRS medium at a volume concentration of 0.5-2%. Incubate at 25-40°C for 10-30 h and subculture 1-3 times to obtain the primary seed culture. Inoculate the primary seed culture into MRS medium with an initial pH of 5-8 at a volume concentration of 0.5-2% and incubate at 25-40°C for 10-30 h to obtain the secondary seed culture.

[0130] The secondary seed culture of the strain was inoculated into the fermentation medium at an inoculum rate of 2% (v / v), and cultured for 24 h at different temperatures (30℃, 35℃, 37℃, 40℃, 45℃) and different medium pH values ​​(5.0, 6.0, 7.0, 8.0). The OD values ​​at different fermentation temperatures and medium pH values ​​were measured. 620 nm (Using the culture medium before inoculation as a blank), based on OD 620 nm Determine the culture temperature and initial pH of the culture medium. Figure 4 It can be seen that the suitable culture temperature for Lactobacillus paracasei JL02 is 37℃ and the pH of the culture medium is 7.

[0131] Prepare 30 L of the above-mentioned fermentation medium in a 50 L fermenter, and add fermentation aids (antioxidant: ascorbic acid 1~2 g / L; osmotic pressure protectant: betaine 0.5~1.0 g / L, glycerol 1~3 g / L; defoamer: polyether defoamer: 0.5~1 g / L). After sterilization, inoculate by flame method, with an inoculation amount of 1~2% (v / v), stirring speed of 50~100 rpm / min. Use 12.5% ​​ammonia water as a neutralizing agent to control the pH of the fermentation broth to 7 during fermentation. Incubate at 37℃ for 12~16 h. Feed batch fermentation is carried out according to different feeding strategies, as shown in Table 8.

[0132] Table 8: Effects of different feeding strategies on the growth of Lactobacillus paracasei JL02

[0133]

[0134] As shown in Table 8, based on the biomass of the fermentation broth, the feeding strategy selected was Strategy 4, namely the logarithmic growth phase, where 20 g / L glucose and milk peptone were added every 2 hours for a total of 3 times. At this time, the highest OD value, cell dry weight, and viable cell count of the fermentation broth reached 16.97, 10.91 g / L, and 1.92 × 10⁻⁶ g / L, respectively. 10 CFU / mL.

[0135] Experiment 2: High-density culture verification

[0136] 1. Validation of high-density culture of Lactobacillus plantarum JL01

[0137] The high-density culture method of *Lactobacillus plantarum* JL01 described in Example 3 was compared with the unoptimized basic MRS medium, and the cultures were incubated at a conventional temperature of 37°C. The OD of the fermentation broth was measured every 2 hours. 620 nm The viable cell count was measured, and growth curves were plotted with time on the horizontal axis and each measured index on the vertical axis to compare and verify the effect of high-density culture.

[0138] Depend on Figure 5 It can be seen that high-density culture of Lactobacillus plantarum JL01 OD 620 nm The viable bacterial counts reached 18.66 and 2.98 × 10⁻⁶, respectively. 10 The CFU / mL increased by 7.35 times and 9.09 times respectively, and the growth lag phase was shorter, while the growth logarithmic phase and growth stationary phase were longer, significantly improving the high-density culture effect of the fermentation broth.

[0139] 2. Validation of high-density culture of Lactobacillus paracasei JL02

[0140] The high-density culture method for *Lactobacillus paracasei* JL02 described in Example 3 was compared with that of unoptimized basal MRS medium, and the cultures were incubated at a conventional temperature of 37°C. The OD of the fermentation broth was measured every 2 hours.620 nm The viable cell count was measured, and growth curves were plotted with time on the horizontal axis and each measured index on the vertical axis to compare and verify the effect of high-density culture.

[0141] Depend on Figure 6 It can be seen that high-density culture of Lactobacillus paracasei JL02 OD 620 nm The viable bacterial counts reached 16.97 and 1.95 × 10⁻⁶, respectively. 10 The CFU / mL increased by 7.58 times and 7.89 times respectively, and the growth lag phase was shorter, while the logarithmic growth phase and the growth stationary phase were longer, significantly improving the high-density culture effect of the fermentation broth.

[0142] Example 4:

[0143] Preparation of Lactobacillus plantarum JL01 bacterial powder

[0144] 1. Centrifuge the fermentation broth.

[0145] The fermenter containing *Lactobacillus plantarum* JL01 cultured under the optimal high-density conditions described in Example 3 was connected to an air compressor and a refrigerated dryer to introduce a small amount of air, maintaining a certain pressure inside the fermenter (approximately 0.05 MPa). A tubular centrifuge was connected to the outlet, and the discharge flow rate was controlled at 2-5 L / min. The centrifuge was stopped after the fermentation broth was used up. The centrifuge chamber was opened, and the bacterial sludge was collected. The sludge was placed in the fermenter, and an equal mass of physiological saline was added. The pH was adjusted to 6.5-7.0 using 4 mol / L sodium hydroxide. The sludge was stirred and washed, then centrifuged again, and the sludge was collected.

[0146] 2. Freeze-dried mushroom sludge

[0147] The washed bacterial sludge and sterile freeze-drying protectant were mixed at a mass ratio of 1:1.5 as shown in Table 9, and allowed to stand for 15-45 minutes. The bacterial sludge mixture was poured into the freeze dryer tray, controlling the material thickness to be 0.5-1.5 cm. The in-situ freeze dryer was then started, and the following drying processes were performed: pre-freezing: temperature -20 to -50℃, time 1-3 h; sublimation drying: temperature -10 to 10℃, time 5-10 h; desorption drying: temperature 20-30℃, time 8-15 h. After freeze-drying, the mixture was pulverized and packaged under constant temperature, humidity, and sterile conditions, and stored at -18℃ for later use.

[0148] The viable bacterial count of samples with different freeze-drying preservatives was determined according to GB4789.35-2016 (National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination). Regression analysis and fitting were performed on the data in Table 9, as shown in Tables 10 and 12. Figure 7 Thus, a regression equation for the fitted experimental factors was obtained: Y = 1.23 + 0.07A + 0.22B + 0.012C + 0.050AB + 0.050AC - 0.040BC - 0.094A 2-0.089B 2 +0.001C 2 The experimental model P A value <0.01 indicates that high and low levels of each factor have a highly significant impact on the viable bacterial count in the response value. (Misfit term) P The value of 0.1493 > 0.05 indicates no significant impact, suggesting a good model fit and effective analysis and prediction. The negative coefficient of the quadratic term indicates the model has a maximum value. By solving for the maximum value of the regression equation, the optimal addition amounts for each factor were found to be 15.5% trehalose, 10.0% milk peptone, and 1.03% glycerol (the remainder being sterile distilled water). Under these conditions, the viable count of the bacterial powder was 1.71 × 10⁻⁶. 12 CFU / g, survival rate 91.92%, viable count still greater than 1×10 after storage at 4℃ for 90 days. 12 It has a high CFU / g count and high storage stability.

[0149] Table 9: Response Surface Experiment Design and Results

[0150]

[0151] Table 10: Analysis of Variance of Response Surface Experiment Results

[0152]

[0153] Example 5:

[0154] Preparation of Lactobacillus paracasei JL02 bacterial powder:

[0155] The only difference between this embodiment and Example 4 is that the fermentation broth of *Lactobacillus paracasei* JL02 was selected as the experimental subject. Through regression analysis and fitting of the response surface methodology data, the optimal freeze-drying protectant was determined to be 14.1% trehalose, 8.2% milk peptone, and 1.92% glycerol (the remainder being sterile distilled water).

[0156] Under these conditions, the viable count of the bacterial powder was 1.21 × 10⁻⁶. 12 CFU / g, survival rate 90.22%, viable count still greater than 1×10 after storage at 4℃ for 90 days. 12 It has a high CFU / g count and high storage stability.

[0157] Experiment 3: Testing the effectiveness of the freeze-drying protectant in this application.

[0158] The same steps as in Examples 4 and 5 were used to prepare *Lactobacillus plantarum* JL01 powder and *Lactobacillus paracasei* JL02 powder, respectively. The only difference was that no freeze-drying protectant was added; all other steps were the same.

[0159] Experimental results showed that the viable cell counts of the final *Lactobacillus plantarum* JL01 and *Lactobacillus paracasei* JL02 bacterial powders were 9.72 × 10⁻⁶. 10 CFU / g and 7.20×10 10 The CFU / g indicates that the number of viable bacteria in the bacterial powder obtained by adding the freeze-drying protectant is increased by more than 10 times.

[0160] Example 6: Direct-inoculation low-temperature fermentation agent compounding

[0161] The *Lactobacillus plantarum* JL01 powder prepared in Example 4 and the *Lactobacillus paracasei* JL02 powder prepared in Example 5 were directly mixed in different mass ratios (0:1, 1:4, 2:3, 1:1, 3:2, 4:1, 1:0) without activation and set aside. Pure milk (protein content 3.0%) was used as the fermentation substrate, with 3% sucrose added. The mixture was homogenized at 20 MPa for 5 min using a high-pressure homogenizer, then pasteurized at 95℃ for 5 min. After cooling to room temperature, different proportions of yogurt starter were inoculated at an inoculation rate of 1‰ by mass. Fermentation was carried out at 25℃ for 24 h, followed by post-ripening at 4℃ for 12 h. The coagulation time was observed and recorded during fermentation, and 10 experienced tasters were invited to conduct sensory evaluations of the yogurt. The scoring criteria referred to the sensory requirements for fermented milk in GB 19302—2010 (National Food Safety Standard—Fermented Milk), with specific details shown in Table 11. The final scores were recorded and calculated.

[0162] Table 11: Sensory Evaluation Criteria for Yogurt

[0163]

[0164] Table 12: Effect of starter culture ratio on yogurt quality

[0165]

[0166] As shown in Table 12, considering both curdling time and sensory evaluation scores, the optimal fermentation effect is achieved when the direct-inoculation low-temperature yogurt starter is a mixture of *Lactobacillus plantarum* JL01 and *Lactobacillus paracasei* JL02 in a mass ratio of 3:2.

[0167] Example 7: Application of direct-inoculation low-temperature yogurt starter:

[0168] 1. Preparation of fermented emulsion

[0169] Using pure cow's milk (protein content 3.0%), skim milk, high-calcium milk, reconstituted milk (10~20% (w / w) milk powder), goat's milk, plant-based soy milk, and plant-based coconut milk as fermentation substrates, 2~8% sucrose or fructose syrup was added. The mixture was homogenized in a high-pressure homogenizer at 15~25 MPa for 5~10 min, then heated to 95℃ for pasteurization for 5 min, and cooled to room temperature for later use.

[0170] 2. Low-temperature fermentation

[0171] The direct-inoculation low-temperature yogurt starter prepared in Example 6 above (with a mass ratio of Lactobacillus plantarum JL01 powder to Lactobacillus paracasei JL02 powder of 3:2) was added to different emulsions at a mass ratio of 0.5 to 1‰, fermented at a fermentation temperature of 20 to 25°C for 15 to 25 h, and then placed in a refrigerator at 4°C for post-ripening for 6 to 12 h.

[0172] 3. Evaluation of Fermented Yogurt

[0173] Using the direct-inoculation low-temperature yogurt starter prepared above, yogurts prepared with different raw materials as fermentation substrates were tested and evaluated according to GB 19302—2010 (National Food Safety Standard - Fermented Milk) for sensory requirements, physicochemical indicators, and lactic acid bacteria count.

[0174] Table 13: Quality Evaluation of Low-Temperature Fermented Yogurt Made from Different Raw Materials

[0175]

[0176] Table 13 shows that the direct-inoculation low-temperature yogurt starter prepared in Example 6 (with a mass ratio of *Lactobacillus plantarum* JL01 powder to *Lactobacillus paracasei* JL02 powder of 3:2) was used as the fermentation substrate. Yogurts made from pure cow's milk (3.0% protein content), skim milk, high-calcium milk, reconstituted milk (10-20% (w / w) milk powder), goat's milk, plant-based soy milk, and plant-based coconut milk were evaluated according to the national standard GB 19302-2010. All sensory requirements were met, and the non-fat milk solids, protein, acidity, and lactic acid bacteria counts were all above the lower limit of the national standard, indicating that all products were qualified. This starter has a wide range of applications and can be used to produce qualified yogurt products from various animal and plant milks.

[0177] Experiment 4:

[0178] The application effects of the fermentation agent were compared using the same method as in Example 7, wherein:

[0179] Commercially available starter culture 1: Chuanxiu - Lactic acid bacteria yogurt starter culture powder;

[0180] Commercially available starter culture 2: Ubit-50 yogurt starter culture powder;

[0181] Commercially available starter culture 3: Baishengyou - Home Yogurt Starter Culture;

[0182] The yogurt starter of this application is the direct-inoculation low-temperature yogurt starter prepared in Example 6, wherein the mass ratio of *Lactobacillus plantarum* JL01 bacterial powder to *Lactobacillus paracasei* JL02 bacterial powder is 3:2.

[0183] All tests used pure milk as the fermentation raw material, and the remaining steps were the same as in Example 7. Low-temperature fermented yogurt was prepared, and the products were evaluated in accordance with the national standard GB 19302-2010.

[0184] Table 14: Quality Evaluation of Low-Temperature Fermented Yogurt with Different Starter Ingredients

[0185]

[0186] As shown in Table 14, when commercially available yogurt starter was used in the low-temperature fermentation of yogurt, all three groups of samples had indicators that did not meet the national standards and were therefore unqualified products. However, the direct-inoculation low-temperature yogurt starter in this application met the requirements.

[0187] The above yogurt starter culture was further subjected to storage stability testing. The starter culture was placed in an environment of 25°C for 30 days, 60 days, and 90 days, and the survival rate (number of live bacteria / number of live bacteria before placement × 100%) was calculated.

[0188] Table 15: Evaluation of storage stability of fermentation agent

[0189]

[0190] As shown in Table 15, the direct-inoculation yogurt starter prepared in this application exhibits better storage stability than three commercially available yogurt starters. After being placed at 25℃ for 90 days, the survival rate was 85.3%, and the viable count reached 1.23 × 10⁻⁶. 12 CFU / g.

[0191] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

[0192] This background section is provided to generally present the context of the invention. The work of the currently named inventors, the work to the extent described in this background section, and aspects described in this section that did not constitute prior art at the time of application are neither expressly nor impliedly acknowledged as prior art to the invention.

Claims

1. A direct-inoculation low-temperature yogurt starter, characterized in that: Including Lactobacillus plantarum ( Lactiplantibacillus plantarum JL01 bacterial powder and Lactobacillus paracasei ( Lacticaseibacillus paracasei JL02 bacterial powder; Lactobacillus plantarum ( Lactiplantibacillus plantarum JL01 bacterial powder and Lactobacillus paracasei ( Lacticaseibacillus paracasei The mass ratio of JL02 bacterial powder is 1:0.25-4; The aforementioned Lactobacillus plantarum ( Lactiplantibacillus plantarum JL01 was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO: M 20241692, and the address of the depository is Wuhan University, Wuhan, China. The aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei JL02 was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO: M 20241693, and the address of the depository is Wuhan University, Wuhan, China.

2. The preparation method of the direct-inoculation low-temperature yogurt starter as described in claim 1, characterized in that... Includes the following steps: 1) Preparation of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum JL01 bacterial powder; Lactobacillus plantarum ( Lactiplantibacillus plantarum JL01 strain was inoculated into MRS medium at a volume ratio of 1-2% to obtain primary seed culture; further subculture and amplification culture were carried out to obtain secondary seed culture. The secondary seed culture of the strain was then inoculated into a fermentation medium containing milk peptone at a volume ratio of 1-3%, and fermentation aids were added. The culture temperature was controlled at 30-40℃, the pH was kept constant at 5-7, and the culture was carried out for 12-16 hours. Then, the culture was fed in batches to obtain the fermentation broth. The fermentation broth was centrifuged and freeze-dried to obtain *Lactobacillus plantarum* (Lactobacillus plantarum). Lactiplantibacillus plantarum JL01 bacterial powder; 2) Preparation of Lactobacillus paracasei ( Lacticaseibacillus paracasei JL02 bacterial powder; Lactobacillus paracasei ( Lacticaseibacillus paracasei JL02 strain was inoculated into MRS medium at a volume ratio of 1-2% to obtain primary seed culture; further subculture and amplification were carried out to obtain secondary seed culture. The secondary seed culture of the strain was then inoculated into a fermentation medium containing milk peptone at a volume ratio of 1-3%, and fermentation aids were added. The culture temperature was controlled at 30-40℃, the pH was kept constant at 5-7, and the culture was carried out for 12-16 hours. Then, the culture was fed in batches to obtain the fermentation broth. The fermentation broth was centrifuged and freeze-dried to obtain *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei JL02 bacterial powder; 3) Prepare Lactobacillus plantarum ( Lacticaseibacillus paracasei JL01 bacterial powder and Lactobacillus paracasei ( Lacticaseibacillus paracasei Mix JL02 bacterial powder in the specified proportions to obtain the final product.

3. The preparation method of the direct-inoculation low-temperature yogurt starter as described in claim 2, characterized in that: The aforementioned Lactobacillus plantarum ( Lacticaseibacillus paracasei The JL01 bacterial powder contains 1.71 × 10⁻⁶ live bacteria. 12 CFU / g, viable count still greater than 1×10⁻⁶ after storage at 4℃ for 90 days. 12 CFU / g.

4. The method for preparing the direct-inoculation low-temperature yogurt starter as described in claim 2, characterized in that: The aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei The JL02 bacterial powder contains 1.21 × 10⁻⁶ live bacteria. 12 CFU / g, viable count still greater than 1×10⁻⁶ after storage at 4℃ for 90 days. 12 CFU / g.

5. The method for preparing the direct-inoculation low-temperature yogurt starter as described in claim 2, characterized in that: The fermentation aids in step 1) include 1-2 g / L of the antioxidant ascorbic acid, 0.5-1.0 g / L of the osmotic pressure protectant betaine, and 1-3 g / L of glycerol; The defoaming agent polyether is 0.5~1 g / L; the fermentation aids in step 2) include antioxidant ascorbic acid 1~2 g / L; osmotic pressure protectant betaine 0.5~1.0 g / L, glycerol 1~3 g / L; and defoaming agent polyether 0.5~1 g / L.

6. The method for preparing a direct-inoculation low-temperature yogurt starter as described in any one of claims 2-5, characterized in that, The fermentation medium containing milk peptone is the fermentation medium in which milk peptone replaces the peptone in the MRS medium; the method for preparing the milk peptone is as follows: Using milk as raw material, the pH is adjusted to 4.6-4.8 with 2-4 mol / L hydrochloric acid, and the crude milk protein is collected by centrifugation using a tubular centrifuge. The collected crude milk protein is mixed with pure water at a mass ratio of 1:1-3, and then protease is added at a mass ratio of 2-6‰. The mixture is hydrolyzed at 30-50℃ and pH 5-8 for 3-9 hours to obtain a protease hydrolysate. This hydrolysate is then treated in a 100-150 W ultrasonic generator for 0.5-1 hours to obtain a milk protein hydrolysate with a high degree of protein hydrolysis. The milk protein hydrolysate is then subjected to a 90-100℃ water bath for 0.5-1 hours to inactivate the enzyme and further hydrolyze the protein. The hydrolysate is centrifuged, and the supernatant is collected. The supernatant is spray-dried to obtain milk peptone. The milk used is fresh milk or waste milk. The waste milk is any one or more of the following: milk past its shelf life or raw milk that does not meet edible standards.

7. The application of the direct-inoculation low-temperature yogurt starter as described in claim 1 in low-temperature yogurt fermentation.

8. The application as described in claim 7, characterized in that, The temperature for low-temperature yogurt fermentation is 20~25℃; the amount of yogurt starter added is 0.5~1wt‰.

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