A method for preparing a lactobacillus plantarum fermented milk

Abstract

The present application belongs to the technical field of dairy product manufacturing, and specifically discloses a preparation method of Lactobacillus plantarum fermented milk, a composite fermented milk or a fermented product obtained by the preparation method, a composition, and use of the composition in preparation of the composite fermented milk or the fermented product.

Description

Technical Field

[0001] This invention relates to the field of dairy manufacturing technology, and in particular to a method for preparing Lactobacillus plantarum fermented milk, a compound fermented milk or fermented product obtained according to the preparation method, a composition and the use of the composition in the preparation of compound fermented milk or fermented product. Background Technology

[0002] Fermented milk is a food made by fermenting milk. Common probiotic strains used in fermented yogurt include Streptococcus thermophilus and Lactobacillus bulgaricus subsp. *Lactobacillus plantarum*, listed in the edible bacteria directory, is often used to ferment plant-based products. However, because *Lactobacillus plantarum* cannot directly utilize the nutrients in milk to sustain its growth, there are relatively few commercially available fermented milk products made solely from *Lactobacillus plantarum*.

[0003] Urolithin (Uro) has significant antioxidant, anti-inflammatory, anticancer, and antibacterial effects. Currently, Uro is found to be produced by intestinal flora and is a natural metabolite of ellagitannins (ETs), a class of compounds found in pomegranates and some other fruits and nuts. Utilizing bio-fermentation to convert ETs in pomegranates into Uro to develop Uro-rich foods offers advantages such as simple processing, convenient consumption, and safety. Chinese invention patent ZL202211404309.8 has reported that *Lactobacillus plantarum* CCFM1291 can metabolize dietary ellagitannins to produce urolithin A. This *Lactobacillus plantarum* and its fermented products have functions such as slowing aging, prolonging life, and improving animal mobility, exhibiting significant probiotic effects. However, because this strain is not a commonly used fermenting agent, it has not been used in dairy products. Currently, there is a lack of functional fermented milk products on the market that slow aging.

[0004] Currently, research on single-strain fermentation of *Lactobacillus plantarum* and its application in blended fruit products is limited, particularly regarding the growth of *Lactobacillus plantarum* in milk and the transformation of bioactive substances in fruits. This has become a technical bottleneck in the industrialization of blended fruit and *Lactobacillus plantarum* fermented milk products. On the one hand, *Lactobacillus plantarum* exhibits slow growth, slow acid production, and low viable cell counts in dairy products; on the other hand, the addition of pomegranate powder presents challenges in the proliferation, fermentation, and transformation of *Lactobacillus plantarum* in milk.

[0005] Therefore, this invention, through the addition of nitrogen source, optimization of strain activity, and adjustment of the amount of pomegranate powder added, prepares a plant-based fermented milk with high viable bacteria count and rich in urolithin-like anti-aging components, which has broad market and practical significance. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, this invention provides a method for preparing a *Lactobacillus plantarum* fermented milk containing pomegranate powder. By adding an appropriate amount of nitrogen source to the milk, determining the optimal activation time and inoculum quantity, *Lactobacillus plantarum* rapidly proliferates in the milk and rapidly produces acid. Furthermore, by adding pomegranate powder to the *Lactobacillus plantarum* fermented milk, optimal conversion is determined, and other active ingredients are produced. Thus, a compound fermented milk or fermented product is obtained according to the described preparation method, along with a composition used in the preparation process and its application in the preparation of the compound fermented milk or fermented product.

[0007] Preparation method

[0008] In a first aspect, the present invention provides a method for preparing Lactobacillus plantarum fermented milk, comprising the following steps:

[0009] (1) Ingredients: The raw milk powder, fermentation aid and optional ellagitannin-rich substances of the fermented milk are stirred evenly to obtain a liquid.

[0010] (2) High temperature sterilization: The liquid obtained in step (1) is preheated and heated, kept at the temperature for sterilization, and then cooled to room temperature to obtain sterilized and cooled liquid.

[0011] (3) Inoculation and fermentation: Inoculate the sterilized and cooled liquid obtained in step (2) with Lactobacillus plantarum to obtain compound fermented milk.

[0012] The term "optional" in the above text means that the component may or may not be present. For example, "the raw material components of the fermented milk, whole milk powder, fermentation aid and optional pomegranate freeze-dried powder" means "the raw material components of the fermented milk, whole milk powder and fermentation aid" or "the raw material components of the fermented milk, whole milk powder, fermentation aid and pomegranate freeze-dried powder".

[0013] In some embodiments, the fermentation aid described in step (1) is selected from one or more of the following: yeast extract and peptone.

[0014] In some embodiments, the yeast extract is selected from one or more of the following: yeast extract FM803, yeast extract FP103, and yeast extract FM985.

[0015] In some embodiments, the yeast extract is selected from one or more of the following: yeast extract FM803 and yeast extract FP103.

[0016] In some embodiments, the yeast extract is selected from yeast extract FM803.

[0017] In some embodiments, the peptone is soybean peptone or pea protein hydrolysate.

[0018] In some embodiments, the ellagitannin-rich substance in step (1) is selected from one or more of the following: fruits (e.g., pomegranate, strawberry, blackberry, raspberry), nuts (e.g., walnut, almond) and seeds, and their derivatives; preferably, the derivative is selected from pomegranate freeze-dried powder and pomegranate juice; preferably, the derivative is pomegranate freeze-dried powder.

[0019] In some embodiments, the raw milk powder in step (1) is selected from one or more of the following: whole milk powder, raw milk, and other milk powders other than skim milk, such as whey protein powder and milk protein concentrate.

[0020] In some embodiments, the content of the raw material component described in step (1) meets one or more of the following criteria:

[0021] (1) Raw milk powder: 10-30 parts by weight; preferably, 15-20 parts by weight; more preferably, 15 parts by weight;

[0022] (2) Fermentation aid: 0.001 to 1 part by weight; preferably, 0.01 to 1 part by weight; preferably, 0.5 to 1 part by weight; more preferably, 0.5 part by weight;

[0023] (3) A substance rich in ellagitannins: 2.5 to 15 parts by weight; preferably, 7.5 to 15 parts by weight; more preferably, 7.5 parts by weight.

[0024] In some embodiments, the content of the raw material component described in step (1) meets one or more of the following criteria:

[0025] (1) Whole milk powder: 10-30 parts by weight; preferably, 15-20 parts by weight; more preferably, 15 parts by weight;

[0026] (2) Fermentation aid: 0.001 to 1 part by weight; preferably, 0.01 to 1 part by weight; preferably, 0.5 to 1 part by weight; more preferably, 0.5 part by weight;

[0027] (3) Pomegranate freeze-dried powder: 2.5 to 15 parts by weight; preferably, 7.5 to 15 parts by weight; more preferably, 7.5 parts by weight.

[0028] In some embodiments, step (1) further includes adjusting the initial pH of the feed solution to 6.4±1.0, preferably 6.4±0.5 or 6.4±0.1.

[0029] In some embodiments, the heat preservation and sterilization described in step (2) is carried out under the following conditions: 80-150℃, preferably 90-100℃, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100℃; for a duration of 1-40 min, preferably 5-30 min, for example 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 min.

[0030] In some embodiments, the fermentation in step (3) is carried out at 0-42°C, preferably 30-37°C, preferably 35°C or 37°C, until the pH value is 4.6±1.0, preferably 4.6±0.5 or 4.6±0.1, to obtain a compound fermented milk.

[0031] In some embodiments, the *Lactobacillus plantarum* in step (3) needs to be activated before inoculation; preferably, the activation time is 8h to 18h, for example 8, 9, 10, 11, 12, 13, 14, 15 or 16h; more preferably, the activation time is 12h.

[0032] In some embodiments, the inoculation amount of *Lactobacillus plantarum* in step (3) is 1% to 12%; preferably, the inoculation amount is 8% to 12%, for example 8%, 9%, 10%, 11% or 12%; more preferably, the inoculation amount is 8%.

[0033] In some embodiments, the *Lactobacillus plantarum* is *Lactobacillus plantarum* CCFM1291 (accession number GDMCCNo: 62802).

[0034] Compound fermented milk or fermented products

[0035] Secondly, the present invention provides a compound fermented milk or [other product] prepared according to the preparation method of the first aspect. Fermentation Products.

[0036] In some embodiments, the compound fermented milk or Fermentation The product further contains urolithiasis-like substances, as well as vitamins, amino acids and their derivatives, bioactive peptides or other active ingredients;

[0037] Preferably, the urolithin-like substances include: urolithin M5, urolithin C, or urolithin A;

[0038] Preferably, the vitamins include: dehydroascorbic acid, thiamine, or vitamin K1;

[0039] Preferably, the amino acid and its derivatives include L-tryptophan or 5-hydroxytryptamine;

[0040] Preferably, the bioactive peptide comprises: arginyl-γ-glutamic acid or lysyl-valine;

[0041] Preferably, the other active ingredients include: pyrazole, zidovudine, ethionamide, carnitine, 1-methylnicotinamide, p-aminobenzoic acid, panthioethylamine, 3,3'-diindolylmethane, dihydrocoumarin, cis-aconitine, citralic acid, N-acetylneuraminic acid, succinic acid, 4-hydroxybenzaldehyde, leucocarbamate, glycerophosphate choline, isopropylcatechol, indolelactic acid, Pipecolic acid, and 1-Methyluric acid.

[0042] In some embodiments, the compound fermented milk is a coagulated compound fermented milk, uniformly brown in color, with the characteristic odor of Lactobacillus plantarum fermented milk, and also has the distinctive taste and aroma of pomegranate. The product has a fine and uniform texture, with a small amount of whey separation.

[0043] Composition

[0044] Thirdly, the present invention provides a composition comprising the following components: a fermentation aid and optionally a substance rich in ellagitannins, optionally further comprising raw milk powder.

[0045] In some embodiments, the fermentation aid is selected from one or more of the following: yeast extract and peptone;

[0046] Preferably, the yeast extract is selected from one or more of the following: yeast extract FM803, yeast extract FP103, and yeast extract FM985;

[0047] More preferably, the yeast extract is selected from one or more of the following: yeast extract FM803 and yeast extract FP103;

[0048] More preferably, the yeast extract is selected from yeast extract FM803;

[0049] In some embodiments, the peptone is soybean peptone or pea protein hydrolysate.

[0050] In some embodiments, the substance rich in ellagitannins is selected from one or more of the following: fruits (e.g., pomegranate, strawberry, blackberry, raspberry), nuts (e.g., walnut, almond) and seeds, and their derivatives;

[0051] Preferably, the derivative is selected from pomegranate freeze-dried powder and pomegranate juice;

[0052] Preferably, the derivative is lyophilized pomegranate powder.

[0053] In some embodiments, the raw milk powder is selected from one or more of the following: whole milk powder, raw milk, and other milk powders other than skim milk, such as whey protein powder and milk protein concentrate.

[0054] In some embodiments, the content of the component meets one or more of the following criteria:

[0055] (1) Raw milk powder: 10-30 parts by weight; preferably, 15-20 parts by weight; more preferably, 15 parts by weight;

[0056] (2) Fermentation aid: 0.001 to 1 part by weight; preferably, 0.01 to 1 part by weight; preferably, 0.5 to 1 part by weight; more preferably, 0.5 part by weight;

[0057] (3) A substance rich in ellagitannins: 2.5 to 15 parts by weight; preferably, 7.5 to 15 parts by weight; more preferably, 7.5 parts by weight.

[0058] In some embodiments, the content of the component meets one or more of the following criteria:

[0059] (1) Whole milk powder: 10-30 parts by weight; preferably, 15-20 parts by weight; more preferably, 15 parts by weight;

[0060] (2) Fermentation aid: 0.001 to 1 part by weight; preferably, 0.01 to 1 part by weight; preferably, 0.5 to 1 part by weight; more preferably, 0.5 part by weight;

[0061] (3) Pomegranate freeze-dried powder: 2.5 to 15 parts by weight; preferably, 7.5 to 15 parts by weight; more preferably, 7.5 parts by weight.

[0062] use

[0063] Fourthly, the present invention provides the composition of the third aspect for preparing the compound fermented milk of the second aspect of the present invention or Fermentation Uses in the product.

[0064] Beneficial effects of the invention

[0065] Compared with the prior art, the present invention has the following advantages and effects:

[0066] (1) This invention solves the problem that Lactobacillus plantarum hardly proliferates in milk and introduces it into fermented milk, which makes it have broad application prospects.

[0067] (2) This invention clarifies the optimal inoculation amount of Lactobacillus plantarum in milk and the activation time of seed liquid, which shortens the fermentation time of Lactobacillus plantarum and solves the problem of long fermentation time of Lactobacillus plantarum.

[0068] (3) The present invention adds compound pomegranate powder to plant fermented milk, and urolithiasis-like substances and active ingredients are detected in fermented milk. The probiotic function of this compound pomegranate fermented milk is greatly improved compared with ordinary fermented milk products. Attached Figure Description

[0069] Figure 1 pH changes in Lactobacillus plantarum fermented milk.

[0070] Figure 2 Growth curve of Lactobacillus plantarum in milk.

[0071] Figure 3 Screening for the optimal nitrogen source for Lactobacillus plantarum.

[0072] Figure 4 pH changes in Lactobacillus plantarum fermented milk with added nitrogen source.

[0073] Figure 5 Growth curve of Lactobacillus plantarum in milk with nitrogen source addition.

[0074] Figure 6 Growth curve of Lactobacillus plantarum in seed culture.

[0075] Figure 7 The effect of inoculation time of Lactobacillus plantarum on its fermentation activity.

[0076] Figure 8 The effect of Lactobacillus plantarum inoculation amount on pH changes in fermented milk.

[0077] Figure 9 The effect of the amount of *Lactobacillus plantarum* inoculated on the viable count of fermented milk.

[0078] Figure 10 pH changes in Lactobacillus plantarum fermented milk with different amounts of pomegranate powder added.

[0079] Figure 11 Changes in the number of viable Lactobacillus plantarum bacteria in milk with different amounts of pomegranate powder added.

[0080] Figure 12 Changes in water-holding capacity with different amounts of pomegranate powder added.

[0081] Figure 13 Changes in ETs content with different amounts of pomegranate powder added.

[0082] Figure 14Changes in UM5 content with different amounts of pomegranate powder added.

[0083] Figure 15 Volcano diagram before and after fermentation with 7.5% pomegranate added.

[0084] Figure 16 PCA plot of differential metabolites with 7.5% pomegranate addition.

[0085] Figure 17 Thermographs before and after fermentation with 7.5% pomegranate powder added.

[0086] Figure 18 Changes in the relative content of potential active substances in compound pomegranate fermented milk.

[0087] Figure 19 pH changes of Lactobacillus plantarum CCFM 1291 in the scale-up experiment of compound pomegranate fermented milk process.

[0088] Figure 20 Changes in the viable count of Lactobacillus plantarum CCFM 1291 in the scale-up experiment of compound pomegranate fermented milk process. Detailed Implementation

[0089] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0090] The pomegranate powder used in the following examples was purchased from Navitas. The yeast extracts FM803 (total nitrogen ≥10.0%, amino nitrogen ≥5.0%), FP103 (total nitrogen ≥13.0%, amino nitrogen ≥2.5%, peptone content ≥20.0%), and FM985 (total nitrogen ≥10.0%, amino nitrogen ≥3.5%) were all purchased from Angel Yeast. The Lactobacillus plantarum CCFM 1291 used in the following examples is described in the patent application text ZL202211404309.8, accession number GDMCC No: 62802.

[0091] Yeast extract powder FM803 is a light yellow powder that is easily soluble in water. It is rich in amino acids, polypeptides, B vitamins, nucleotides, trace elements, etc., and has a high content of free amino acids. Its physicochemical properties are as follows:

[0092]

[0093] Yeast extract FP103 is a light yellow powder that is easily soluble in water at low concentrations; it is granulated to prevent dust generation; it is rich in peptides, mainly small peptides and oligopeptides, with a balanced amino acid ratio; it does not carry the risks associated with traditional animal and plant peptones, such as pathogenicity, cultural taboos, or genetic modification; its physicochemical properties are as follows:

[0094]

[0095] Yeast extract powder FM985 is a light yellow powder; it is easily soluble in water and rich in amino acids, polypeptides, B vitamins, nucleotides, trace elements, etc.; its physicochemical properties are as follows:

[0096]

[0097] The culture media involved in the following examples are as follows:

[0098] MRS solid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.05 g / L, Tween 80 1 mL / L, agar powder 15 g / L, cysteine ​​0.5 g / L.

[0099] MRS liquid culture medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.05 g / L, Tween 80 1 mL / L, cysteine ​​0.5 g / L.

[0100] The detection methods involved in the following embodiments are as follows:

[0101] pH test method: Weigh 5 mL of fermented milk and measure the pH value directly using a pH meter.

[0102] OD 600 Determination: The activated bacterial solution was inoculated into MRS medium and placed in an incubator at 37℃. The growth of the strain was measured every 2 hours.

[0103] Method for detecting viable counts of Lactobacillus plantarum: After thoroughly shaking the fermented milk, add 0.5 mL of the solution to 4.5 mL of physiological saline, shake well, and then perform serial dilution. Record the viable counts in the fermented pomegranate juice using the plate pour method. For specific procedures, refer to GB4789.35-2023 "National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination" to determine the viable counts of lactic acid bacteria in fermented milk.

[0104] High-performance liquid chromatography (HPLC) method for the detection of punicin and urolithin M5 (UM5):

[0105] Thaw the fermented milk and vortex the sample thoroughly. Add fermented milk to acidified methanol (30 mL methanol to 200 μL concentrated hydrochloric acid) at a ratio of 1:4, and vortex until homogeneous. Centrifuge at 5000 g for 10 min and collect the supernatant. Concentrate and dry. Redissolve in 200 μL methanol. Let stand overnight at 4°C, then centrifuge at 12000 rpm for 20 min and collect the supernatant. Add the supernatant to the detection bottle for UPLC analysis.

[0106] Chromatographic conditions: ACQUITY was used. HSS T3 (2.1×100mm, 1.7μm) column (Waters, Milford, MA, USA), flow rate 0.3 mL / min, column temperature 40℃, injection volume 0.8 μL. Mobile phase: A 0.1% trifluoroacetic acid in water, B methanol; gradient elution program: 0–1.95 min, 5%–18% B; 1.95–4.72 min, 18%–28% B; 4.72–6.11 min, 28%–50% B; 6.11–7.50 min, 50%–90% B; 7.50–8.06 min, 90%–5% B; 8.06–9.17 min, 5%–5% B.

[0107] Methods for detecting non-target metabolomics:

[0108] Thaw the fermented milk at 4°C. After thawing, vortex the sample for 1 min to mix thoroughly. Accurately transfer an appropriate amount of sample into a 2 mL centrifuge tube. Add 400 μL of methanol solution and vortex for 1 min. Centrifuge at 12,000 rpm at 4°C for 10 min. Collect all supernatant and transfer it to a new 2 mL centrifuge tube. Concentrate and dry the sample. Accurately add 150 μL of 2-chloro-L-phenylalanine (4 ppm) solution prepared with 80% methanol and water to reconstitute the sample. Filter the supernatant through a 0.22 μm membrane. Add the filtrate to the detection bottle for LC-MS detection.

[0109] Chromatographic conditions: using ACQUITY HSS T3 (2.1×100mm, 1.8μm) (Waters, Milford, MA, USA) column, flow rate 0.3 mL / min, column temperature 40℃, injection volume 2 μL. Positive ion mode, mobile phase 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2), gradient elution program: 0–1 min, 10% B2; 1–5 min, 10%–98% B2; 5–6.5 min, 98% B2; 6.5–6.6 min, 98%–10% B2; 6.6–8 min, 10% B2. In negative ion mode, the mobile phase consisted of acetonitrile (B3) and 5 mM ammonium formate water (A3), with the gradient elution program as follows: 0–1 min, 10% B3; 1–5 min, 10%–98% B3; 5–6.5 min, 98% B3; 6.5–6.6 min, 98%–10% B3; 6.6–8 min, 10% B3.

[0110] Mass spectrometry conditions: Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, USA), electrospray ionization (ESI) source, and positive and negative ion modes were used for data acquisition. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 alb, and the auxiliary gas was 10 alb. The capillary temperature was 325 °C. A first-stage full scan was performed at a resolution of 70,000 m / z, with a first-stage ion scan range of 100–1000 m / z. Second-stage fragmentation was performed using an HCD with a collision energy of 30 eV and a second-stage resolution of 17,500 m / z. The first three ions acquired were fragmented, and unnecessary MS / MS information was removed using dynamic exclusion.

[0111] Example 1: Fermentation characteristics of Lactobacillus plantarum CCFM 1291 in milk

[0112] Preparation of seed culture: Lactobacillus plantarum CCFM 1291 was streaked onto MRS solid medium and cultured at 37℃ for 48 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 18 h to activate the culture. The activation was repeated for two generations to obtain the activated culture.

[0113] Preparation of Lactobacillus plantarum fermented milk: 15% whole milk powder was stirred evenly, sterilized at 95℃ for 20 minutes, and after cooling to room temperature, inoculated with Lactobacillus plantarum CCFM 1291. During fermentation, samples of the fermented milk were taken every 12 hours to test the pH value (results are shown in [link to results]). Figure 1 ), viable count of lactic acid bacteria (see test results) Figure 2 ).

[0114] Depend on Figure 1It can be seen that after 72 hours, fermented milk with Lactobacillus plantarum CCFM 1291 can only reduce the pH of the milk to 6.08.

[0115] Depend on Figure 2 It can be seen that *Lactobacillus plantarum* CCFM 1291 proliferates slowly in milk, with the viable count only increasing to 1-2 × 10⁻⁶ within 24 hours. 8 CFU / mL. The viable bacterial count rapidly decreased to the initial bacterial count after 24 hours.

[0116] Example 2: Screening of the optimal nitrogen source and dosage for Lactobacillus plantarum CCFM 1291

[0117] Preparation of seed solution: as described in Example 1.

[0118] Preparation of nitrogen source selection media: The content of peptone, beef extract, and yeast extract in MRS medium was reduced to 1 g / L, and nitrogen source was selected as the sole nitrogen source. The activated seed culture was inoculated into different nitrogen source media. After 8 hours, samples were taken to detect the OD values ​​of *Lactobacillus plantarum* CCFM 1291 in different nitrogen source media to determine the promoting effect of different nitrogen sources on the growth and proliferation of *Lactobacillus plantarum* (results are shown in [link to results]). Figure 3 ).

[0119] After determining the optimal nitrogen source, the optimal nitrogen source was added to the milk in a gradient. Samples of the fermented milk were taken every 12 hours, and the pH value of the fermented milk was measured (results are shown in [link to results]). Figure 4 ), viable count of lactic acid bacteria (see test results) Figure 5 ).

[0120] Depend on Figure 3 It can be seen that, compared with other nitrogen sources, yeast extract powder FM803 has the best effect on promoting the growth and proliferation of Lactobacillus plantarum within 8 hours.

[0121] Depend on Figure 4 It can be seen that with the addition of gradient yeast extract FM803, an addition of 5 g / L has reduced the pH to 4.76 within 12 hours.

[0122] Depend on Figure 5 It can be seen that the addition of 5g / L yeast extract FM803 enables *Lactobacillus plantarum* to multiply to 10^10 in milk within 12 hours. 9 CFU / mL. Furthermore, adding excessive amounts of yeast extract does not increase the viable count of *Lactobacillus plantarum*.

[0123] Based on the screening of the optimal nitrogen source and the determination of the optimal addition amount, the fermentation aid was obtained as 5g / L yeast extract powder FM803.

[0124] Example 3: Optimal inoculation time and optimal inoculum quantity of seed culture

[0125] Preparation of seed solution: as described in Example 1.

[0126] Seed culture obtained after two generations of activation was inoculated into MRS liquid medium. OD values ​​were measured every 2 hours to determine the growth characteristics of *Lactobacillus plantarum* (results are shown in [link to results]). Figure 6 ).Depend on Figure 6 It can be seen that Lactobacillus plantarum CCFM 1291 reached the end of the logarithmic phase in the seed culture within 14 hours.

[0127] Three time points were selected: 12 hours into the logarithmic phase, 14 hours into the end of the logarithmic phase, and 16 hours into the stationary phase. A 2% inoculum was added to the fermented milk. Samples were taken from the fermented milk 8 hours later to test the pH value and the viable count of lactic acid bacteria (results are shown in [link to results]). Figure 7 ),Depend on Figure 7 It can be seen that the initial viable cell count of *Lactobacillus plantarum* was lowest at 12 hours, and the viable cell count increased to the maximum after 8 hours of fermentation, with the pH decreasing most significantly. Therefore, the optimal activation time for the seed culture is 12 hours.

[0128] The obtained seed culture was inoculated into the fermented milk at inoculation rates of 1%, 2%, 4%, 8%, and 12%. Samples of the fermented milk were taken every 4 hours to test its pH value (results are shown in [link to results]). Figure 8 ), viable count of lactic acid bacteria (see test results) Figure 9 ).

[0129] Depend on Figure 8 It can be seen that when the inoculum concentration reaches 8%, the pH value of Lactobacillus plantarum fermented milk decreases from 6.36 to 4.41 after 8 hours.

[0130] Depend on Figure 9 It can be seen that the viable bacterial count of 8% inoculation amount is the same as that of 12% inoculation amount after 8 hours, and it multiplies to 2×10⁻⁶ after 8 hours. 9 CFU / mL. Therefore, the optimal inoculum concentration is 8%.

[0131] Example 4: Growth characteristics and water-holding capacity of Lactobacillus plantarum CCFM 1291 in compound pomegranate fermented milk

[0132] Preparation of compound pomegranate fermented milk: 15% whole milk powder, 0.5% yeast extract FM803, and pomegranate powder in different proportions were stirred evenly to adjust the initial pH to 6.4±0.1. After high-temperature sterilization at 95℃ for 20 minutes, the liquid was obtained after cooling.

[0133] The seed culture was prepared as in Example 1. Two generations of activated *Lactobacillus plantarum* were inoculated into the culture medium. Samples of the compound pomegranate fermented milk were taken every 4 hours to test the pH value (results are shown in [link to sample]). Figure 10 ), viable count of lactic acid bacteria (see test results) Figure 11Once the pH of the compound pomegranate fermented milk decreased to 4.5-4.7, samples were taken to test its water-holding capacity (test results are shown in [link to test results]). Figure 12 ).

[0134] Depend on Figure 10 and Figure 11 It can be seen that compared with the absence of pomegranate powder, the number of viable bacteria increased faster and the pH decreased faster with the addition of pomegranate powder, indicating that the addition of pomegranate powder has a promoting effect on the growth of Lactobacillus plantarum in milk.

[0135] Depend on Figure 12 It can be seen that the addition of pomegranate powder increases the water-holding capacity of fermented milk, thereby making the fermented milk structure more compact and reducing whey separation in compound pomegranate fermented milk.

[0136] Example 5: Transformation characteristics of Lactobacillus plantarum CCFM 1291 with the addition of graded pomegranate powder

[0137] Prepare compound pomegranate fermented milk according to Examples 1-4. When the pH of the compound pomegranate fermented milk drops to 4.5-4.7, take samples for high-performance liquid chromatography (HPLC) detection (see results below). Figure 13 , Figure 14 ).

[0138] Depend on Figure 13 It can be seen that increasing the amount of pomegranate powder added increases the content of pomegranate glycosides, and the conversion rate reaches its highest when the amount of pomegranate powder added is 7.5%.

[0139] Depend on Figure 14 It can be seen that when the amount of pomegranate powder added reaches 7.5%, the content of urolithin M5 reaches its highest level.

[0140] Therefore, the optimal amount of pomegranate powder added is 7.5%.

[0141] Example 6: Detection of active substances in compound pomegranate fermented milk

[0142] According to Example 5, a compound pomegranate fermented milk was obtained. 7.5% of the compound pomegranate fermented milk was subjected to non-target detection (detection results are shown in...). Figure 15 , Figure 16 , Figure 17 (UCM stands for unfermented pomegranate milk, FCM stands for fermented compound pomegranate milk)).

[0143] Depend on Figure 15 It was found that 133 differentially metabolites were detected in the compound fermented pomegranate milk, including 9 fatty acids, 28 carboxylic acids and their derivatives, 13 organic oxygenates, 6 phenolic compounds, 6 benzene and substituted derivatives, 4 indole and their derivatives, and 67 other compounds; among them, the content of 85 substances increased and the content of 48 substances decreased.

[0144] Depend on Figure 16 It can be seen that the compound pomegranate milk separated from the second component after fermentation. From the second component, it can be found that the compound pomegranate milk fermented with Lactobacillus plantarum CCFM 1291 separated (PCA2: 34.1%).

[0145] Depend on Figure 17 The clustering of three distinct biological replicates indicates good homogeneity and data reliability among them. Heatmap analysis before and after fermentation reveals enrichment of alcohols, acids, ketones, and amino acid metabolites after fermentation with *Lactobacillus plantarum* CCFM 1291. Notably, the content of various vitamins significantly increased after fermentation. The substances in the compound pomegranate milk after fermentation with *Lactobacillus plantarum* CCFM 1291 show significant differences. In summary, both cluster analysis and principal component analysis demonstrate that the fermentation metabolites of the compound pomegranate milk produce significant differences.

[0146] Depend on Figure 18 It was found that the relative content of vitamins was significantly increased among the elevated substances; potentially active amino acids and their derivatives, L-tryptophan and serotonin, were also detected; in addition, several bioactive peptides, arginyl-γ-glutamic acid and lysyl-valine, were also detected. Figure 18 Some or all of the listed substances have potential anti-inflammatory, antioxidant, anti-infective and anti-aging effects.

[0147] Example 7: Growth characteristics of Lactobacillus plantarum CCFM 1291 in a scale-up experiment of compound pomegranate fermented milk process

[0148] Preparation of compound pomegranate fermented milk: 80% raw milk, 1g / L pea protein hydrolysate, and 20% pomegranate juice were stirred evenly to adjust the initial pH to 6.4±0.1. After high-temperature sterilization at 95℃ for 5 minutes, the liquid was obtained after cooling.

[0149] The seed culture was prepared as in Example 1. Two generations of activated *Lactobacillus plantarum* were inoculated into the culture medium. Samples of the compound pomegranate fermented milk were taken every 4 hours to test the pH value (results are shown in [link to sample]). Figure 19 ), viable count of lactic acid bacteria (see test results) Figure 20 ).

[0150] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing Lactobacillus plantarum fermented milk, comprising the following steps: (1) Ingredients: The raw material components of the fermented milk, namely raw milk powder, fermentation aid and optional substances rich in ellagitannins, are stirred evenly to obtain a liquid. (2) High temperature sterilization: The liquid obtained in step (1) is preheated and heated, kept at the temperature for sterilization, and then cooled to room temperature to obtain sterilized and cooled liquid. (3) Inoculation and fermentation: The sterilized and cooled liquid obtained in step (2) is inoculated with Lactobacillus plantarum for fermentation to obtain compound fermented milk.

2. The preparation method according to claim 1, wherein, The fermentation aid mentioned in step (1) is selected from one or more of the following: yeast extract and peptone; Preferably, the yeast extract is selected from one or more of the following: yeast extract FM803, yeast extract FP103, and yeast extract FM985; More preferably, the yeast extract is selected from one or more of the following: yeast extract FM803 and yeast extract FP103; More preferably, the yeast extract is selected from yeast extract FM803; or Preferably, the peptone is soybean peptone or pea protein hydrolysate.

3. The preparation method according to claim 1, wherein, The ellagitannin-rich substance mentioned in step (1) is selected from one or more of the following: fruits (e.g., pomegranate, strawberry, blackberry, raspberry), nuts (e.g., walnut, almond) and seeds, and their derivatives; Preferably, the derivative is selected from pomegranate freeze-dried powder and pomegranate juice; Preferably, the derivative is lyophilized pomegranate powder.

4. The preparation method according to claim 1, wherein, The raw milk powder mentioned in step (1) is selected from one or more of the following: whole milk powder, raw milk, and other milk powders other than skim milk, such as whey protein powder and milk protein concentrate.

5. The preparation method according to any one of claims 1-4, wherein, The content of the raw material components mentioned in step (1) meets one or more of the following criteria: (1) Raw milk powder: 10-30 parts by weight; preferably, 15-20 parts by weight; more preferably, 15 parts by weight; (2) Fermentation aid: 0.001 to 1 part by weight; preferably, 0.01 to 1 part by weight; preferably, 0.5 to 1 part by weight; more preferably, 0.5 part by weight; (3) A substance rich in ellagitannins: 2.5 to 15 parts by weight; preferably, 7.5 to 15 parts by weight; more preferably, 7.5 parts by weight. Preferably, the content of the raw material components in step (1) meets one or more of the following criteria: (1) Whole milk powder: 10-30 parts by weight; preferably, 15-20 parts by weight; more preferably, 15 parts by weight; (2) Fermentation aid: 0.001 to 1 part by weight; preferably, 0.01 to 1 part by weight; preferably, 0.5 to 1 part by weight; more preferably, 0.5 part by weight; (3) Pomegranate freeze-dried powder: 2.5 to 15 parts by weight; preferably, 7.5 to 15 parts by weight; more preferably, 7.5 parts by weight.

6. The preparation method according to any one of claims 1-5, wherein, Step (1) further includes adjusting the initial pH of the feed solution to 6.4±1.0, preferably 6.4±0.5 or 6.4±0.

1.

7. The preparation method according to any one of claims 1-6, wherein, The heat preservation and sterilization described in step (2) is carried out under the following conditions: 80-150℃, preferably 90-100℃, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100℃; Continue for 1-40 minutes, preferably 5-30 minutes, such as 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 minutes.

8. The preparation method according to any one of claims 1-7, wherein, The fermentation described in step (3) is carried out at 0-42°C, preferably 30-37°C, and more preferably 35°C or 37°C, until the pH value is 4.6±1.0, preferably 4.6±0.5 or 4.6±0.1, to obtain a compound fermented milk.

9. The preparation method according to any one of claims 1-8, wherein, The *Lactobacillus plantarum* in step (3) needs to be activated before inoculation; preferably, the activation time is 8h to 18h, for example 8, 9, 10, 11, 12, 13, 14, 15 or 16h; more preferably, the activation time is 12h.

10. The preparation method according to any one of claims 1-9, wherein, The inoculation amount of *Lactobacillus plantarum* in step (3) is 1% to 12%; preferably, the inoculation amount is 8% to 12%, for example, 8%, 9%, 10%, 11% or 12%; more preferably, the inoculation amount is 8%.

11. The preparation method according to any one of claims 1-10, wherein, The Lactobacillus plantarum mentioned is Lactobacillus plantarum CCFM1291 (accession number GDMCC No: 62802).

12. The compound fermented milk or fermented product obtained by the preparation method according to any one of claims 1-11.

13. The compound fermented milk or fermented product of claim 12, further comprising urolithin-like substances, as well as vitamins, amino acids and their derivatives, bioactive peptides or other active ingredients; Preferably, the urolithin-like substance comprises: Urolithin M5, urolithin C, or urolithin A; Preferably, the vitamins include: dehydroascorbic acid, thiamine, or vitamin K1; Preferably, the amino acid and its derivatives include L-tryptophan or 5-hydroxytryptamine; Preferably, the bioactive peptide comprises: arginyl-γ-glutamic acid or lysyl-valine; Preferably, the other active ingredients include: pyrazole, zidovudine, ethionamide, carnitine, 1-methylnicotinamide, p-aminobenzoic acid, panthioethylamine, 3,3'-diindolylmethane, dihydrocoumarin, cis-aconitine, citralic acid, N-acetylneuraminic acid, succinic acid, 4-hydroxybenzaldehyde, leucocarbamate, glycerophosphate choline, isopropylcatechol, indolelactic acid, Pipecolic acid, and 1-Methyluric acid.

14. A composition comprising: a fermentation aid and optionally a substance rich in ellagitannins, optionally further comprising raw milk powder; The fermentation aid is selected from one or more of the following: yeast extract and peptone; Preferably, the yeast extract is selected from one or more of the following: yeast extract FM803, yeast extract FP103, and yeast extract FM985; More preferably, the yeast extract is selected from one or more of the following: yeast extract FM803 and yeast extract FP103; More preferably, the yeast extract is selected from yeast extract FM803; or Preferably, the peptone is soybean peptone or pea protein hydrolysate.

15. The composition of claim 14, wherein, The ellagitannin-rich substances are selected from one or more of the following: fruits (e.g., pomegranate, strawberry, blackberry, raspberry), nuts (e.g., walnut, almond) and seeds, and their derivatives; Preferably, the derivative is selected from pomegranate freeze-dried powder and pomegranate juice; Preferably, the derivative is lyophilized pomegranate powder.

16. The composition of claim 14 or 15, wherein, The raw milk powder is selected from one or more of the following: whole milk powder, raw milk, and other milk powders other than skim milk, such as whey protein powder and milk protein concentrate.

17. The composition according to any one of claims 14-16, wherein the content of the component conforms to one or more of the following: (1) Raw milk powder: 10-30 parts by weight; preferably, 15-20 parts by weight; more preferably, 15 parts by weight; (2) Fermentation aid: 0.001 to 1 part by weight; preferably, 0.01 to 1 part by weight; preferably, 0.5 to 1 part by weight; more preferably, 0.5 part by weight; (3) A substance rich in ellagitannins: 2.5 to 15 parts by weight; preferably, 7.5 to 15 parts by weight; more preferably, 7.5 parts by weight. Preferably, the content of the component meets one or more of the following criteria: (1) Whole milk powder: 10-30 parts by weight; preferably, 15-20 parts by weight; more preferably, 15 parts by weight; (2) Fermentation aid: 0.001 to 1 part by weight; preferably, 0.01 to 1 part by weight; preferably, 0.5 to 1 part by weight; more preferably, 0.5 part by weight; (3) Pomegranate freeze-dried powder: 2.5 to 15 parts by weight; preferably, 7.5 to 15 parts by weight; more preferably, 7.5 parts by weight.

18. Use of the composition according to any one of claims 14-17 in the preparation of the compound fermented milk or fermented product according to claim 12 or 13.

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