Glycophosphocholine probiotic combination for enhancing the same in dairy products and use thereof

Abstract

The application provides a probiotic combination for increasing glycerophosphocholine in dairy products and application thereof, which is composed of lactobacillus delbrueckii subsp. bulgaricus, streptococcus thermophilus, lactobacillus plantarum and lactobacillus fermentum. The probiotic combination can effectively increase the glycerophosphocholine content after fresh milk is fermented.

Description

Technical Field

[0001] This invention relates to a probiotic combination for enhancing the glycerophosphate choline content in dairy products and its application. Background Technology

[0002] Yogurt is a fermented milk product with a sweet and sour taste. It is made from milk that has been pasteurized, then beneficial bacteria are added, it is fermented, and finally cooled and packaged. Yogurt products on the market are divided into set yogurt and stirred yogurt.

[0003] Driven by the trend of functionalizing food, the global functional food market is projected to reach US$275.77 billion by 2025, with a compound annual growth rate of 7.9%. The domestic functional food market is also booming, with a clear functionalization trend emerging in the food and beverage market. Consumers' functional demands for yogurt are constantly upgrading. Beyond basic functions like regulating the digestive system, boosting immunity, and increasing appetite, more precise and diverse demands, such as calming the nerves, aiding sleep, and promoting beauty and health, are beginning to permeate fermented milk consumption.

[0004] L-α-glycerophosphate choline, also known as L-α-GPC, has the molecular formula C8H. 20 NO6P, molecular weight: 257.22. The most important function of GPC lies in the choline it produces, a water-soluble B vitamin that plays a vital role in the brain and nervous system. Studies show that GPC plays a crucial role in the production of certain hormones and neurotransmitters such as acetylcholine and human growth hormone, thereby supporting brain and nervous system function. It also plays an important role in lipid metabolism, neuroprotection, and cell signaling, and is essential for maintaining normal bodily functions.

[0005] Currently, there are two main methods for preparing L-α-glycerophosphate choline:

[0006] (1) Chemical synthesis method: This is the main production method of glycerol phosphoric acid choline. First, prepare raw materials glycerol, phosphoric acid and choline, mix them in a certain proportion, and then prepare glycerol phosphoric acid choline through condensation reaction. Another method is to dissolve lecithin in an alcohol solvent, add an inorganic base or organic base to carry out a hydrolysis reaction to obtain a reaction solution, filter and adjust the acidity to obtain glycerol phosphoric acid choline. Patent: A method for preparing L-α-glycerol phosphoric acid choline - CN201310241997.5 This invention relates to a method for preparing L-α-glycerol phosphoric acid choline, including: first, using (R)-epoxychloropropane and benzyl alcohol to condense under the action of a strong base to prepare (S)-benzyl glycidyl ether, then reacting (S)-benzyl glycidyl ether with chlorophosphocholine to obtain L-α-chloroglycerol phosphoric acid choline benzyl ether; then removing the benzyl protecting group of the obtained L-α-chloroglycerol phosphoric acid choline benzyl ether through a Pd / C hydrogenation reaction, and finally purifying to obtain L-α-GPC;

[0007] Patent: A Method for Preparing Glycerophosphate Choline - CN202110392453.3 discloses a method for preparing glycerophosphate choline, comprising the following steps: Step 1: Dissolving lecithin in an alcohol solvent, then adding an inorganic or organic base for hydrolysis to obtain a reaction solution, filtering, adding acid to the filtrate to adjust the pH value, and evaporating the alcohol solvent under reduced pressure to obtain compound (I); Step 2: Dissolving compound (I) in water and adding an extraction solvent for extraction to obtain compound (II); Step 3: Decolorizing compound (II) with activated carbon in an aqueous phase, then directly stirring with a mixed anion and cation exchange resin, evaporating to dryness, and adding a recrystallization solvent for recrystallization to obtain purified glycerophosphate choline. This invention uses a chemical hydrolysis method, using an inorganic weak base to hydrolyze lecithin in an alcohol solvent.

[0008] (2) Enzymatic hydrolysis: Using specific enzymes to decompose complex biomolecules containing choline (such as phosphoric acid) to obtain glycerophosphate choline.

[0009] Patent: A Method for Preparing Glycerylphosphocholine (GPC) by Phosphatase Hydrolysis - CN201010248594.X. This invention discloses a method for preparing glycerylphosphocholine (GPC) by enzymatic hydrolysis, belonging to the field of lipid development and application technology. The method includes the following steps: using powdered phosphoric acid, alcohol-soluble phosphoric acid, and high-purity PC as raw materials, glycerylphosphocholine (GPC) is prepared by phosphatase hydrolysis of phosphorylphosphocholine (PC) in an aqueous phase; decolorization with activated carbon to obtain an aqueous solution of GPC, which is then converted to an alcohol phase and purified using cationic resin adsorption and anionic adsorption methods to obtain a high-purity aqueous solution of GPC; low-temperature, reduced-pressure rotary evaporation yields a colorless and transparent L-α-GPC solution with a chemical purity of 98.8%.

[0010] Patent: A Method for Separating and Purifying Glycerophosphate Choline by Silica Glycol Column Chromatography - CN201110004065.X. This invention discloses a method for separating and purifying glycerophosphate choline (L-α-GPC) using silica gel column chromatography, belonging to the field of lipid development and application technology. The method includes the following steps: using an enzymatic hydrolysis reaction solution as raw material, Ca2+ and Cl- are first removed using an ion exchange resin, then the aqueous phase is converted to an alcohol phase. L-α-GPC, GPE, LPC, and other byproducts are separated by silica gel column chromatography. Activated carbon is used for decolorization, and vacuum concentration is performed to remove water, yielding a colorless and transparent product. Alternatively, using an alcoholysis reaction solution as raw material, direct separation is performed using a silica gel column, Na+ is removed using a cation exchange resin, activated carbon is used for decolorization, and vacuum concentration is performed to remove water, yielding the product. Product indicators: Chemical purity above 99.6%. All patents utilize chemical synthesis and enzymatic hydrolysis methods to obtain glycerophosphate choline.

[0011] Currently, there is no relevant technology for producing glycerophosphate choline through bio-fermentation, and nutritional supplements in the form of additives are increasingly resisted by consumers, failing to meet the national standard definition of yogurt. Therefore, developing a probiotic combination fermentation agent to enhance the glycerophosphate choline content and flavor of yogurt has broad application prospects. Summary of the Invention

[0012] To address the above problems, this invention provides a combination of probiotics that enhance the glycerophosphate choline content in dairy products and its application.

[0013] The objective of this invention is achieved through the following solution: a probiotic combination that enhances the glycerophosphate choline content of dairy products.

[0014] Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. Bulgarian )inm25-LB, Streptococcus thermophilus ( Streptococcus salivarius subsp. thermophilic inm25-ST, Lactobacillus plantarum ( Lactiplantibacillus plantarum E680, Lactobacillus fermentum ( Limosilactobacillus leaven Composed of inm25;

[0015] Lactobacillus fermentum ( Lactobacillus fermentum The strain is inm25, with accession number CGMCC No. 14612, accession date September 13, 2017, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] Lactobacillus plantarum ( Lactobacillus plantarum strain E680, with accession number CGMCC No. 14217, accession date June 2, 2017, depositary institution: China General Microbiological Culture Collection Center, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing;

[0017] thermophilic streptococci ( Streptococcus thermophilus The strain is inm25-ST, with accession number CGMCC No. 15446, accession date March 12, 2018, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp.bulgaricus The strain is inm25-LB, with accession number CGMCC No.15445, accession date March 12, 2018, deposited at China General Microbiological Culture Collection Center, address No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0019] Furthermore, the weight fractions were Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus )inm25-LB: Streptococcus thermophilus ( Streptococcus salivarius subsp. thermophilic inm25-ST: Lactobacillus plantarum ( Lactiplantibacillus plantarum E680: Lactobacillus fermentum ( Limosilactobacillus fermentum )inm25 is 3~5:10~100:2~5:3~60.

[0020] An application of a probiotic combination for enhancing glycerophosphate choline in dairy products to increase the glycerophosphate choline content of fresh milk after fermentation.

[0021] Advantages of the present invention: (1) The probiotic combination can effectively increase the content of glycerophosphate choline produced after fermentation of fresh milk.

[0022] (2) The probiotic combination improves the aroma and taste of fermented milk products. There will be no off-flavor in the fermented milk after fermentation. It can be mixed with other food ingredients in the later stage, which preserves the nutrition and flavor of fermented milk and has no safety risks. Attached Figure Description

[0023] Figure 1 This study analyzed the glycerophosphate choline content produced by fermenting fresh milk with eight different probiotic strains in Example 1.

[0024] Figure 2 This is a mass spectrum.

[0025] Figure 3 This is the standard curve for glycerophosphate choline. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments:

[0027] Unless otherwise specified, the experimental methods used in the following implementation examples are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified.

[0028] Example 1, see attached document Figure 1-3 A probiotic blend that enhances glycerophosphate choline in dairy products.

[0029] Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. Bulgarian )inm25-LB, Streptococcus thermophilus ( Streptococcus salivarius subsp. thermophilic inm25-ST, Lactobacillus plantarum ( Lactiplantibacillus plantarum E680, Lactobacillus fermentum ( Limosilactobacillus leaven Composed of inm25;

[0030] Lactobacillus fermentum ( Lactobacillus fermentum The strain is inm25, with accession number CGMCC No. 14612, accession date September 13, 2017, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0031] Lactobacillus plantarum ( Lactobacillus plantarum strain E680, with accession number CGMCC No. 14217, accession date June 2, 2017, depositary institution: China General Microbiological Culture Collection Center, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing;

[0032] thermophilic streptococci ( Streptococcus thermophilus The strain is inm25-ST, with accession number CGMCC No. 15446, accession date March 12, 2018, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp.bulgaricus The strain is inm25-LB, with accession number CGMCC No.15445, accession date March 12, 2018, deposited at China General Microbiological Culture Collection Center, address No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0034] A probiotic blend for enhancing glycerophosphate choline in dairy products, comprising Lactobacillus delbrueckii subsp. bulgaricus by weight (… Lactobacillus delbrueckii subsp. Bulgarian )inm25-LB: Streptococcus thermophilus ( Streptococcus salivarius subsp. thermophilic inm25-ST: Lactobacillus plantarum ( Lactiplantibacillus plantarumE680: Lactobacillus fermentum ( Limosilactobacillus fermentum )inm25 is 3~5:10~100:2~5:3~60.

[0035] A probiotic blend that enhances glycerophosphate choline in dairy products, wherein the glycerophosphate choline is L-α-glycerophosphate choline.

[0036] The above four strains have been disclosed in the existing patent: 201910694197.6.

[0037] Experimental steps: 1. Initial screening of strains

[0038] Eight strains preserved in the laboratory were inoculated into MRS liquid medium at a 1% inoculum concentration and cultured at 37°C for 24 hours. Then, at a 1% inoculum concentration, they were inoculated into sterilized fresh milk along with *Lactobacillus delbrueckii* subsp. bulgaricus inm25-LB and *Streptococcus thermophilus* inm25-ST, and cultured statically at 37°C for 4–6 hours until the final bacterial count reached 10⁻⁶. 8 The concentration was cfu / mL. After ripening at 4°C for 6 hours, an equal volume of sterile water was added, the mixture was stirred, and centrifuged at 10,000 r / min for 10 min. The supernatant was obtained as the sample. After passing through a 0.22 μm aqueous filter membrane, the physicochemical properties were tested.

[0039] UPLC-MS / MS for the detection of glycerophosphate choline:

[0040] Sample preparation: Take 200 μL of sample, add 800 μL of acetonitrile as extraction buffer, vortex to mix, sonicate at low temperature for 30 min, centrifuge at 12000 r / min for 5 min, collect the supernatant and filter through a 0.22 μm organic filter membrane, place in a sample vial, and transfer to a UPLC-MS / MS platform for detection.

[0041] Liquid chromatography conditions: Column: HYPERSIL GOLD C18 column (3μm, 2.1mm*100mm); Column temperature: 35℃; Flow rate: 0.3mL / min; Injection volume: 1μL; Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Elution gradient: 0-2min, phase B = 10%; 2-6min, phase B = 10%-90%; 6-8min, phase B = 90%; 8-8.1min, phase B = 90%-10%; 8.1-10min, phase B = 10%.

[0042] Mass spectrometry conditions: Ion source: ESI (Turbo Spray); Polarity: Positive; Spray voltage: 5500V; Curtain gas: 30psi; Collision gas: 9psi; Nebulization temperature: 550℃; Scanning mode: MRM primary mass spectrometry scan range m / z 100~3000; secondary mass spectrometry scan range m / z 100~3000. Precursor ion: 258 m / z, Daughter ions: 104.1 m / z and 124.9 m / z (e.g., ...). Figure 2 (As shown). A standard curve for L-α-glycerophosphate choline was established: results are shown in […]. Figure 3 ;

[0043] Strains with high glycerophosphate choline content, namely inm25, E680, and inm28-LL, were selected for secondary screening.

[0044] 2. Secondary screening of strains

[0045] The probiotics inm25, E680, and inm28-LL selected in the initial screening were reactivated and cultured at a bacterial concentration of 10. 6 CFU / mL bacteria were inoculated into sterilized milk, fermented at 37℃ for 4-5 hours, and then the fermented milk was subjected to sensory evaluation.

[0046] Table 1 Sensory evaluation of fermented milk

[0047]

[0048] Note: Each indicator has a maximum score of 5 points, and the higher the score, the better the result.

[0049] As shown in Table 1, inm25 had the best sensory evaluation results, followed by E680. Therefore, these two strains were selected as target strains for further study.

[0050] Example 2: Detection of the genetic stability of Lactobacillus fermentum inm25 and Lactobacillus plantarum E680.

[0051] Lactobacillus fermentum inm25 and Lactobacillus plantarum E680 from Example 1 were passaged 10 times on MRS liquid medium. The 1st, 5th and 10th generation samples were inoculated into sterilized fresh milk, and the method was the same as in Example 1. The method for detecting glycerophosphate choline content was also the same as in Example 1 to determine its passage stability. Specific data are shown in Table 2.

[0052] Table 2. Results of the passaging stability test

[0053]

[0054] The results in Table 2 show that the fermentation performance of Lactobacillus fermentum inm25 and Lactobacillus plantarum E680 of different generations was normal, the glycerophosphate choline content was within 10%, the genetic stability of the strains was good, and they met the requirements for production and use.

[0055] Example 3: Preparation of fermented milk by different ratios of bacterial strains

[0056] When two strains of Lactobacillus fermentum inm25 and Lactobacillus plantarum E680 are mixed in a ratio of 1:1 to 1:1000, the total bacterial concentration is 1×10⁻⁶. 6 The concentration of cfu / mL was determined using inm28-LR as a control strain. Fermented milk was prepared with *Lactobacillus delbrueckii* subsp. bulgaricus inm25-LB and *Streptococcus thermophilus* inm25-ST. The fermentation temperature was 37℃, and the fermentation time was 5-6 h. The method and standards for preparing the fermented milk were in accordance with the national standard GB 19302-2025 "Fermented Milk". The method for detecting glycerophosphate choline was the same as in Example 1. The results are shown in Table 3.

[0057] Table 3. Production of glycerophosphate choline by strains with different proportions

[0058]

[0059] As shown in Table 3, the total acid content gradually increased with the increase of the proportion of E680, while the glycerophosphate choline and sensory evaluation remained stable. The glycerophosphate choline content reached its maximum of 52.46 ± 3.76 ug / L at a ratio of 1:10, and the sensory evaluation was the best. Therefore, the optimal ratio of inm25 to E680 is 1:10.

[0060] Example 4: Preparation of Fermented Milk by Combination of Microbial Strains

[0061] After mixing two strains, *Lactobacillus fermentum* inm25 and *Lactobacillus plantarum* E680, at a ratio of 1:10, the total bacterial concentration was 10. 4 -10 8 At CFU / mL, with inm28-LR as the control strain, fermented milk was prepared using *Lactobacillus delbrueckii* subsp. bulgaricus inm25-LB and *Streptococcus thermophilus* inm25-ST. The fermentation method followed the national standard GB 19302-2025 "Fermented Milk," and the method for detecting glycerophosphate choline was the same as in Example 1. The results are shown in Table 4.

[0062] Table 4. Glycerol Phosphocholine Content in Fermented Milk

[0063]

[0064] Table 5 Sensory evaluation results of fermented milk

[0065]

[0066] Note: Each assessment item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all scores.

[0067] The results in Tables 4 and 5 show that adding *Lactobacillus fermentum* inm25 and *Lactobacillus plantarum* E680 to fermented milk significantly increases the content of glycerophosphate choline in the fermented milk, and the higher the inoculum size, the higher the glycerophosphate choline content. However, excessively high or low inoculum concentrations affect the sourness, sweetness, and taste of the fermented milk. The optimal inoculum concentration is 1.0 × 10⁻⁶. 7 CFU / mL, this concentration can produce a sufficient amount of glycerophosphate choline and also improve the flavor and texture of fermented milk.

[0068] Example 5: Effect of different fermentation temperatures on glycerophosphate choline yield

[0069] The final concentration of Lactobacillus fermentum inm25 and Lactobacillus plantarum E680 was mixed to 10. 7 Fermented milk was prepared using Lactobacillus delbrueckii subsp. bulgaricus inm25-LB and Streptococcus thermophilus inm25-ST at a dose of CFU / mL, with inm28-LR as a control group. The preparation process followed the national standard GB 19302-2025 "Fermented Milk". The method for detecting glycerophosphate choline was the same as in Example 1. The fermentation temperatures were set at 37℃, 42℃, and 46℃, and the fermentation time was 5-6 hours. The results are recorded in Tables 6 and 7.

[0070] Table 6. Effects of different fermentation processes on glycerophosphate choline yield

[0071]

[0072] Table 7 Sensory evaluation results of yogurt prepared at different fermentation temperatures

[0073]

[0074] As shown in Tables 6 and 7, the addition of a mixed strain of *Lactobacillus fermentum* inm25 and *Lactobacillus plantarum* E680 during the fermentation of milk resulted in an increase in glycerophosphate choline content with rising temperature. However, no glycerophosphate choline was detected at 46℃, possibly because the excessively high fermentation temperature affected the growth and metabolism of the probiotics, thus preventing the production of glycerophosphate choline. Therefore, the optimal temperature for glycerophosphate choline production is 42℃. This concentration provides sufficient glycerophosphate choline while also improving the flavor and texture of the fermented milk.

[0075] Example 6: Different fermentation times

[0076] The final concentration of Lactobacillus fermentum inm25 and Lactobacillus plantarum E680 was mixed to 10.7 Fermented milk was prepared using Lactobacillus delbrueckii subsp. bulgaricus inm25-LB and Streptococcus thermophilus inm25-ST at a dose of CFU / mL, with inm28-LR as a control group. The preparation process followed the national standard GB 19302-2025 "Fermented Milk". The method for detecting glycerophosphate choline was the same as in Example 1. The fermentation times were set to 3.0, 4.0, and 5.0 h, respectively. The results are recorded in Tables 8 and 9.

[0077] Table 8. Effects of different fermentation processes on glycerophosphate choline yield

[0078]

[0079] Table 9 Sensory evaluation results of yogurt prepared at different fermentation times

[0080]

[0081] As shown in Tables 8 and 9, the glycerophosphate choline (GPC) content increased with time during the fermentation process of fermented milk after adding a mixture of *Lactobacillus fermentum* inm25 and *Lactobacillus plantarum* E680. However, when the temperature was increased to 5.0 h, the GPC content did not differ significantly from that at 5.0 h. This may be because the fermentation time reaches 4.0 h, after which the growth and metabolism of probiotics are affected, thus preventing the continued production of GPC. Therefore, the optimal time for GPC production is 4.0 h. This concentration provides sufficient GPC while also improving the flavor and texture of the fermented milk.

[0082] Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. Bulgarian )inm25-LB, Streptococcus thermophilus ( Streptococcus salivarius subsp. thermophilic inm25-ST, Lactobacillus plantarum ( Lactiplantibacillus plantarum E680, Lactobacillus fermentum ( Limosilactobacillus leaven Composed of inm25;

[0083] The gene sequence of *Lactobacillus delbrueckii* subsp. bulgaricus inm25-LB is as follows:

[0084] gagtttgatc ctggctcatg acgatcgctg gcggcgtgcc taatacatgc aagtcgagcg

[0085] agctgaattc aaagattcct tcggggtgat ttgttggatg ctagcggcgg atgggtgagt

[0086] aacacgtggg caatctgccc taaagactgg gataccactt ggaaacaggt gctaataccg

[0087] gataacaaca tgaatcgcat gattcaagtt tgaaaggcgg cgtaagctgt cactttagga

[0088] tgagcccgcg gcgcattagc tagttggtgg ggtaaaggcc taccaaggca atgatgcgta

[0089] gccgagttga gagactgatc ggccacattg ggactgagac acggcccaaa ctcctacggg

[0090] aggcagcagt agggaatctt ccacaatgga cgcaagtctg atggagcaac gccgcgtgag

[0091] tgaagaaggt tttcggatcg taaagctctg ttgttggtga agaaggatag aggcagtaac

[0092] tggtctttat ttgacggtaa tcaaccagaa agtcacggct aactacgtgc cagcagccgc

[0093] ggtaatacgt aggtggcaag cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg

[0094] aatgataagt ctgatgtgaa agcccacggc tcaaccgtgg aactgcatcg gaaactgtca

[0095] ttcttgagtg cagaagagga gagtggaatt ccatgtgtag cggtggaatg cgtagatata

[0096] tggaagaaca ccagtggcga aggcggctct ctggtctgca actgacgctg aggctcgaaa

[0097] gcatgggtag cgaacaggat tagataccct ggtagtccat gccgtaaacg atgagcgcta

[0098] ggtgttgggg actttccagt cctcagtgcc gcagcaaacg cattaagcgc tccgcctggg

[0099] gagtacgacc gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag

[0100] catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctgtgctac

[0101] acctagagat aggtggttcc cttcggggac gcagagacag gtggtgcatg gctgtcgtca

[0102] gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg tctttagttg

[0103] ccatcattaa gttgggcact ctaaagagac tgccggtgac aaaccggagg aaggtgggga

[0104] tgacgtcaag tcatcatgcc ccttatgacc tgggctacac acgtgctaca atgggcagta

[0105] caacgagaag cgaacccgcg agggtaagcg gatctcttaa agctgttctc agttcggact

[0106] gcaggctgca actcgcctgc acgaagctgg aatcgctagt aatcgcggat cagcacgccg

[0107] cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggaa gtctgcaatg

[0108] cccaaagtcg gtgggataac ctttatagga gtcagccgcc taaggcaggg cagatgactg

[0109] gggtgaagtc gtaacaaggt agccgt

[0110] The gene sequence of Streptococcus thermophilus inm25-ST is as follows:

[0111] cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtagaac gctgaagaga

[0112] ggagcttgct cttcttggat gagttgcgaa cgggtgagta acgcgtaggt aacctgcctt<e000286>

[0113] gtagcggggg ataactattg gaaacgatag ctaataccgc ataacaatgg atgacacatg

[0114] tcatttattt gaaaggggca attgttccac tacaagatgg acctgcgttg tattagctag

[0115] taggtgaggt aatggctcac ctaggcgacg atacatagcc gacctgagag ggtgatcggc

[0116] cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttcgg

[0117] caatgggggc aaccctgacc gagcaacgcc gcgtgagtga agaaggtttt cggatcgtaa

[0118] agctctgttg taagtcaaga acgggtgtga gagtggaaag ttcacactgt gacggtagct

[0119] taccagaaag ggacggctaa ctacgtgcca gcagccgcgg taatacgtag gtcccgagcg

[0120] ttgtccggat ttattgggcg taaagcgagc gcaggcggtt tgataagtct gaagttaaag

[0121] gctgtggctc aaccatagtt cgctttggaa actgtcaaac ttgagtgcag aaggggagag

[0122] tggaattcca tgtgtagcgg tgaaatgcgt agatatatgg aggaacaccg gtggcgaaag

[0123] cggctctctg gtctgtaact gacgctgagg ctcgaaagcg tggggagcga acaggattag

[0124] ataccctggt agtccacgcc gtaaacgatg agtgctaggt gttggatcct ttccgggatt

[0125] cagtgccgca gctaacgcat taagcactcc gcctggggag tacgaccgca aggttgaaac

[0126] tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac

[0127] gcgaagaacc ttaccaggtc ttgacatccc gatgctattt ctagagatag aaagttactt

[0128] cggtacatcg gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt

[0129] taagtcccgc aacgagcgca acccctattg ttagttgcca tcattcagtt gggcactcta

[0130] gcgagactgc cggtaataaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct

[0131] tatgacctgg gctacacacg tgctacaatg gttggtacaa cgagttgcga gtcggtgacg

[0132] gcgagctaat ctcttaaagc caatctcagt tcggattgta ggctgcaact cgcctacatg

[0133] aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt

[0134] gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt

[0135] ttggagccag ccgcctaagg tgggacagat gattggggtg aagtcgtaac aaggtaagcc

[0136] gt

[0137] The gene sequence of Lactobacillus fermentum inm25 is as follows:

[0138] caggatgaac gccggcggtg tgcctaatac atgcaagtcg aacgcgttgg cccaattgat

[0139] tgatggtgct tgcacctgat tgattttggt cgccaacgag tggcggacgg gtgagtaaca

[0140] cgtaggtaac ctgcccagaa gcgggggaca acatttggaa acagatgcta ataccgcata

[0141] acagcgttgt tcgcatgaac aacgcttaaa agatggcttc tcgctatcac ttctggatgg

[0142] acctgcggtg cattagcttg ttggtggggt aacggcctac caaggcgatg atgcatagcc

[0143] ​gagttgagag actgatcggc caaatggga ctgagacacg gcccatactc ctacgggagg

[0144] cagcagtagg gaatcttcca caatgggcgc aagcctgatg gagcaacacc gcgtgagtga

[0145] agaagggttt cggctcgtaa agctctgttg ttaaaaga acacgtatga gagtaactgt

[0146] tcatacgttg acggtatta accagaaagt cacggctaac tacgtgccag cagccgcggt

[0147] aatacgtagg tggcaagcgt tatccggatt tattgggcgt aaagagagtg caggcggttt

[0148] tctaagtctg atgtgaaagc cttcggctta accggagaag tgcatcggaa actggataac

[0149] ttgagtgcag aagagggtag tggaactcca tgtgtagcgg tggaatgcgt agatatatgg

[0150] aagaacacca gtggcgaagg cggctacctg gtctgcaact gacgctgaga ctcgaaagca

[0151] tgggtaggga acaggattag ataccctggt agtccatgcc gtaaacgatg agtgctaggt

[0152] gttggagggt ttccgccctt cagtgccgga gctaacgcat taagcactcc gcctggggag

[0153] tacgaccgca aggttgaaac tcaaaggaat tgacggggc ccgcacaagc ggtggagcat

[0154] gtggtttaat tcgaagctac gcgaagaacc ttaccaggtc ttgacatctt gcgccaaccc

[0155] tagagatagg gcgtttcctt cgggaacgca atgacaggtg gtgcatggtc gtcgtcagct

[0156] cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtta ctagttgcca

[0157] gcattaagtt gggcactcta gtgagactgc cggtgacaaa ccggaggaag gtggggacga

[0158] cgtcagatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gacggtacaa

[0159] cgagtcgcga actcgcgagg gcaagcaaat ctcttaaaac cgttctcagt tcggactgca

[0160] ggctgcaact cgcctgcacg aagtcggaat cgctagtaat cgcggatcag catgccgcgg

[0161] tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgagagtt tgtaacaccc

[0162] aaagtcggtg gggtaacctt ttaggagcca gccgcctaag gtgggacaga tgattagggt

[0163] gaagtcgtac

[0164] The gene sequence of Lactobacillus plantarum E680 is as follows:

[0165] tgcaagtcga acgaactctg gtattgattg gtgcttgcat catgatttac atttgagtga

[0166] gtggcgaact ggtgagtaac acgtgggaaa cctgcccaga agcgggggat aacacctgga

[0167] aacagatgct aataccgcat aaacttgg accgcatggt ccgagcttga aagatggctt

[0168] cggctatcac ttttggatgg tcccgcggcg tattagctag atggtggggt aacggctcac

[0169] catggcaatg atacgtagcc gacctgagag ggtaatcggc cacattggga ctgagacacg

[0170] gcccaaactc ctacgggagg cagcagtagg gaatcttcca caatggacga aagtctgatg

[0171] gagcaacgcc gcgtgagtga agaagggtttt cggctcgtaa aactctgttg ttaaagaaga

[0172] acatatctga gagtaactgt tcaggtattg acggtattta accagaaagc cacggctaac

[0173] tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggatt tattgggcgt

[0174] aaagcgagcg caggcggttt tttaagtctg atgtgaaagc cttcggctca accgaagaag

[0175] tgcatcggaa actgggaaac ttgagtgcag aagaggacag tggaactcca tgtgtagcgg

[0176] tgaaatgcgt agatatatgg aagaacacca gtggcgaagg cggctgtctg gtctgtaact

[0177] gacgctgagg ctcgaaagta tgggtagcaa acaggattag ataccctggt agtccatacc

[0178] gtaaacgatg aatgctaagt gttggagggt ttccgccctt cagtgctgca gctaacgcat

[0179] tagcattcc gcctggggag tacggccgca aggctgaaac tcaaaggaat tgacggggc

[0180] ccgcacaagc ggtggagcat gtggtttaat tcgaagctac gcgaagaacc ttaccaggtc

[0181] ttgacatact atgcaaatct aagagattag acgttccctt cggggacatg gatacaggtg

[0182] gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca

[0183] acccttatta tcagttgcca gcattaagtt gggcactctg gtgagactgc cggtgacaaa

[0184] ccgggagaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg

[0185] tgctacaatg gatggtacaa cgagttgcga actcgcgaga gtaagctaat ctcttaaagc

[0186] cattctcagt tcggattgta ggctgcaact cgcctacatg aagtcggaat cgctagtaat

[0187] cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac

[0188] catgagagtt tgtaacaccc aaagtcggtg gggtaaccta tgaggaacca

[0189] Based on the results of the above embodiments, the mixed preparation of *Lactobacillus delbrueckii* subsp. bulgaricus inm25-LB, *Streptococcus thermophilus* inm25-ST, *Lactobacillus fermentum* inm25, and *Lactobacillus plantarum* E680 provided by the present invention, when applied to fermented milk fermentation, can significantly increase the content of glycerophosphate choline in the relevant fermented milk without changing the existing process. At the same time, it can also improve the aroma, taste, and lipid-lowering function of fermented milk products, providing a theoretical basis for the development of functional fermented milk.

[0190] Without altering existing processes, this method utilizes *Lactobacillus delbrueckii* subsp. bulgaricus inm25-LB, *Streptococcus thermophilus* inm25-ST, *Lactobacillus fermentum* inm25, and *Lactobacillus plantarum* E680 to produce flavored fermented milk. This method enhances the nutritional value of fermented milk products, not only increasing the glycerophosphate-choline content but also improving the flavor and quality. Fermented milk products produced using this formulation do not suffer from changes to the overall fermentation process, improving both the taste and nutritional value of the fermented milk.

[0191] An application of a probiotic combination for enhancing glycerophosphate choline in dairy products to increase the glycerophosphate choline content of fresh milk after fermentation.

[0192] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, which fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention patent should be determined by the appended claims.

Claims

1. Use of a glycerophosphocholine boosting probiotic combination to increase the glycerophosphocholine content after fermentation of fresh cow milk, characterized in that Glycerophosphocholine boosting probiotic combination in dairy products Lactobacillus delbrueckii subsp. bulgaricus (Lb) Lactobacillus delbrueckii subsp. bulgaricus ) inm25-LB, Streptococcus thermophilus (St) Streptococcus salivarius subsp. thermophilus ) inm25-ST, Lactobacillus plantarum (Lp) Lactiplantibacillus plantarum ) E680, Lactobacillus fermentum (Lf) Limosilactobacillus fermentum ) inm25. Lactobacillus fermentum ( Lactobacillus fermentum The strain is inm25, with accession number CGMCC No. 14612, accession date September 13, 2017, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Lactobacillus plantarum (ATCC 8014) Lactobacillus plantarum ) E680, the strain preservation number is CGMCC No. 14217, the preservation date is June 2, 2017, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No. 1, Xibaixili, Chaoyang District, Beijing, China. Streptococcus thermophilus (ATCC 19258) Streptococcus thermophilus ) inm25-ST, the preservation number of the strain is CGMCC No. 15446, the preservation date is March 12, 2018, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No. 1, Xibaixili, Chaoyang District, Beijing, China. Lactobacillus delbrueckii subsp. bulgaricus (LB) Lactobacillus delbrueckii subsp.bulgaricus ) inm25-LB, the strain preservation number is CGMCC No. 15445, the preservation date is March 12, 2018, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No. 1, Xibaixili, Chaoyang District, Beijing.

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