Infant yoghurt easy to digest and low in gastrointestinal irritation and preparation method thereof

Abstract

The invention discloses easy-to-digest infant yoghurt with low gastrointestinal irritation and a preparation method thereof, and belongs to the technical field of food processing. The invention provides yoghurt which is easy to digest and low in gastrointestinal tract irritation and is prepared by adopting infant strain fermentation. Firstly, a protein hydrolysis type strain and an acid-producing type strain are modified, an acid-resistant nano armor and a weight-increasing armor are worn on the protein hydrolysis type strain and the acid-producing type strain respectively, so that the protein hydrolysis type strain has excellent acid resistance, and an anaerobic acid-producing strain has high density and is attached to the bottom of a fermentation tank, and anaerobic proliferation fermentation is facilitated; the preparation method comprises the following steps: heating raw milk to induce polymerization, cooling, adding the two bacteria, and carrying out intermittent stirring fermentation to obtain the infant edible yoghurt rich in medium and long peptides, namely SLAMAASNG and NKALALNT, which are easy to digest, weak in gastrointestinal irritation and low in bitter taste.

Description

Technical Field

[0001] This invention relates to easily digestible infant yogurt with low gastrointestinal irritation and its preparation method, belonging to the field of food processing technology. Background Technology

[0002] Infants under 36 months of age have an underdeveloped digestive system, insufficient gastric acid secretion, weak intestinal barrier function, and an immature immune system, making them far less tolerant to food than adults. Yogurt, as a fermented dairy product, is considered a suitable complementary food for infants due to its rich content of low-irritant hydrolyzed peptides and post-biotics. However, existing infant yogurts still have the following technical challenges: (1) High risk of gastrointestinal irritation. The fermentation strains of ordinary yogurt are mostly non-infant-specific strains such as Streptococcus thermophilus and Lactobacillus bulgaricus, which are not included in my country's "List of Strains that can be Used in Infant Foods". Their metabolites may disrupt the balance of intestinal flora in infants, causing diarrhea and bloating. Some products add sucrose and salt to adjust the flavor, increasing the metabolic burden on infants. (2) Low digestion and absorption efficiency. The average molecular weight of casein in cow's milk is as high as 23-25 ​​kDa. If it is not fully hydrolyzed, infants will not have sufficient digestive enzyme secretion, making it difficult to decompose and absorb, which may lead to indigestion. The residual lactose content is high, and infants have weak lactase activity, which can easily cause symptoms of lactose intolerance such as bloating and diarrhea. (3) Limited co-fermentation by two strains. Some strains that produce hydrolytic proteases have poor acid tolerance. As fermentation proceeds, the pH decreases, which limits the growth and proliferation of the strains and reduces the hydrolysis efficiency. (4) Difficult anaerobic fermentation process. Some acid-producing infant strains are strict anaerobic bacteria, making oxygen control during fermentation technically difficult and increasing fermentation costs.

[0003] Therefore, how to obtain an easily digestible and low-gastrointestinal-irritation infant yogurt and its preparation method under low-cost conditions has become a research hotspot and challenge. Summary of the Invention

[0004] To address the drawbacks of existing commercially available yogurts, which are mostly fermented using strains not suitable for infants and young children, and whose high molecular weight and low degree of hydrolysis can cause strong gastrointestinal irritation to infants and young children, this invention aims to provide a yogurt fermented using infant-grade strains that is easily digestible and has low gastrointestinal irritation. Firstly, the protein-hydrolyzing strain and the acid-producing strain are modified by adding acid-resistant nano-armor and weight-increasing armor, respectively. This gives the protein-hydrolyzing strain excellent acid resistance, while the anaerobic acid-producing strain adheres at a high density to the bottom of the fermentation tank, facilitating anaerobic proliferation and fermentation. Raw milk is heated to induce polymerization, and after cooling, the two strains are added for intermittent stirring fermentation. This yields an infant-grade yogurt rich in two easily digestible, low-gastrointestinal-irritation, and low-bitterness medium- and long-term peptides: SLAMAASNG (Ser-Leu-Ala-Met-Ala-Ala-Ser-Asn-Gly) and NKALALNT (Asn-Lys-Ala-Leu-Ala-Leu-Asn-Thr).

[0005] The present invention provides a protein peptide, wherein the protein peptide is a SLAMAASNG or NKALALNT polypeptide, and the amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0006] This invention provides a method for preparing yogurt suitable for infants and young children, the method comprising the following steps: (1) Two strains were cultured. *Lactobacillus fermentum* CECT 5716 and *Bifidobacterium longum* subsp. *bifidobacterium* BB536 were activated and cultured in MRS broth at 37℃ for 24 hours anaerobically, and passaged twice. The strains were collected by centrifugation at 2000-3000×g for 5-10 min, washed twice with deionized water, and resuspended in physiological saline (0.9% NaCl) to adjust the concentration to 10. 8 -10 9 CFU / mL.

[0007] (2) Preparation of CECT 5716 acid-resistant armor. Take a certain amount of bacterial suspension containing approximately 5 × 10⁻⁶... 7 -5×10 8 Add 100-150 μL of PBS solution containing 10-20 mg / mL EGCG to the CFU strain and gently vortex for 1 minute. Slowly add 100-150 μL of PBS solution containing 5 mg / mL FeCl3 while continuously vortexing. Finally, add 5 mL of PBS (10 mM, pH 7.4) and continue vortexing for 3 minutes to complete the reaction.

[0008] (3) Preparation of weight-enhancing armor. Cassava starch or corn starch was dispersed in water, and the pH was adjusted to 10-11. Sodium tripolyphosphate was added, with an addition amount of approximately 0.1%-0.5% of the dry starch basis. It was slowly dripped into sesame oil under high-speed shear at 1500-3000 rpm, dispersing into micron-sized droplets. The mixture was placed in a 50-60℃ water bath and gently stirred at 100-150 rpm for 2-5 hours. The upper oil phase was discarded, and residual oil was removed by repeated washing with ethanol. Finally, it was washed with deionized water and freeze-dried to obtain dry microsphere powder. 100-140 mg of the microsphere powder was then mixed with 5×10... 7 -5×10 8 Mix the CFU bacterial suspension and vortex for 1-2 minutes to complete the reaction.

[0009] Adjust the pH of 10-20 L of raw milk to 6.5-7.5, heat at 80-90℃ for 30-50 min to induce micropolymerization, cool to 35-40℃, add two modified bacterial strains, stir intermittently every 10 min, and ferment for 12-24 h to obtain yogurt.

[0010] This invention provides a method for preparing yogurt, wherein the yogurt contains the aforementioned protein peptide SLAMAASNG or NKALALNT polypeptide, and the method includes the following steps: (1) Preparation of bacterial suspension The fermentation broths of Lactobacillus fermentum CECT 5716 and Bifidobacterium longum subsp. BB536 were centrifuged to collect the bacterial strains, and then resuspended in physiological saline to prepare Lactobacillus fermentum CECT 5716 and Bifidobacterium longum subsp. BB536 bacterial suspensions, respectively.

[0011] (2) Preparation of CECT 5716 acid-resistant armor The *Lactobacillus fermentum* CECT 5716 bacterial suspension prepared in step (1) was added to a PBS solution containing 10-20 mg / mL EGCG. The inoculation concentration was 5 × 10⁻⁶. 7 ~5×10 8 After vortexing for 1-2 min, add PBS solution containing 3-7 mg / mL FeCl3 and vortex for 1-2 min. Finally, add PBS solution and vortex for 0.5-1 min to complete the reaction and obtain a mixture containing Lactobacillus fermentum CECT 5716. (3) Prepare BB5366 heavy armor Disperse cassava starch in water, adjust the pH to 10-11, add sodium tripolyphosphate at a concentration of 0.1%-0.5% of the dry starch. Slowly add sesame oil at a high-speed shear rate of 1500-3000 rpm, with the amount of sesame oil being 1-2% of the dry starch. After the sesame oil is added, place the reaction system in a 50-60℃ water bath and gently stir at 100-150 rpm for 2-5 hours. Pour off the upper oil phase, wash repeatedly with ethanol to remove residual oil, and finally wash with deionized water. Freeze-dry to obtain dry microsphere powder. Mix the obtained microsphere powder with the BB5366 bacterial suspension prepared in step (1), with a bacterial concentration of 5×10⁻⁶. 7 ~5×10 8 CFU, after vortexing for 1-2 minutes, the reaction is completed, and a mixture containing Bifidobacterium longum subsp. BB5366 is obtained; (4) Adjust the pH of raw milk to 6.5~7.5 and heat at 80~90℃ for 30~50 min to obtain the milk system; (5) Add the mixture containing Lactobacillus fermentum CECT5716 obtained in step (2) and the mixture containing Bifidobacterium longum subsp. BB5366 obtained in step (3) to the milk system obtained in step (4). The amount added is 0.2-0.5% of the milk mass. The concentration of Lactobacillus fermentum CECT5716 or Bifidobacterium longum subsp. BB5366 is 1×10⁻⁶. 3 ~1×10 4 Yogurt can be obtained by intermittent stirring every 10 to 20 minutes at 35 to 40°C for 12 to 24 hours.

[0012] The present invention also provides yogurt prepared by the above method.

[0013] This invention also provides the application of the above-mentioned protein peptides SLAMAASNG or NKALALNT in the preparation of food, pharmaceuticals, health products or nutritional products.

[0014] In one embodiment of the present invention, the food, medicine, health product or nutritional product may also contain a derivative of protein peptide, wherein the derivative of protein peptide refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain group, amino terminus or carbonyl terminus of the protein peptide.

[0015] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0016] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0017] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0018] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0019] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0020] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0021] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages.

[0022] In one embodiment of the present invention, the food also includes special dietary foods.

[0023] In one embodiment of the present invention, the nutritional products include medical nutritional products, nutritional products formulated for special populations, and nutritional supplements.

[0024] The present invention also provides a food, medicine, health product or nutritional product containing the above-mentioned SLAMAASNG or NKALALNT polypeptide.

[0025] In one embodiment of the present invention, the food, medicine, health product or nutritional product may also contain a derivative of protein peptide, wherein the derivative of protein peptide refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain group, amino terminus or carbonyl terminus of the protein peptide.

[0026] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0027] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0028] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0029] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0030] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0031] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0032] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages.

[0033] In one embodiment of the present invention, the food also includes special dietary foods; the health products also contain acceptable excipients.

[0034] The present invention also provides an expression vector or recombinant microorganism in which the above-mentioned protein peptide is expressed.

[0035] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0036] In one embodiment of the present invention, the recombinant microorganism is a bacterium or a fungus.

[0037] Beneficial effects (1) Reduce the digestive burden of yogurt and alleviate adverse intestinal irritation. This invention obtains high-abundance SLAMAASNG and NKALALNT polypeptides through heated polymerization followed by CECT5716 fermentation and hydrolysis. Both polypeptides have the qualities of being easily recognized by intestinal peptidases, having high PepT1 affinity, low bitter peptide content, and low gastrointestinal irritation.

[0038] (2) Co-incubating CECT 5716 with EGCG and FeCl3 improves fermentation efficiency. This increases the resistance of CECT 5716 to low pH environments, allowing it to ferment simultaneously with BB536, shortening the yogurt production cycle, and also increasing the gastric acid resistance of CECT 5716, thereby improving intestinal colonization rate.

[0039] (3) Improve the anaerobic fermentation environment of BB536 and reduce the difficulty of fermentation technology. BB536 is modified into a three-phase composite by starch, sodium trimetaphosphate and oil, so that it adheres to the bottom of the fermentation tank, which can realize an anaerobic environment and is conducive to its reproduction and fermentation.

[0040] (4) Secreting extracellular polysaccharides to improve the smooth texture of yogurt. During fermentation, BB536 secretes linear extracellular polysaccharides (EPS) with a specific molecular weight. EPS can bind with casein micelles in milk to form a uniform network support structure and prevent excessive aggregation of casein to form coarse curd lumps. EPS itself has the characteristics of being viscous but not sticky, which can fill the gaps between curds and make the yogurt texture more delicate and smooth, similar to a natural thickener, but without the heaviness of artificial additives, and it is also conducive to achieving a clean ingredient list. Attached Figure Description

[0041] Figure 1 Protein digestibility in yogurt.

[0042] Figure 2 : Determination of the viscosity of yogurt.

[0043] Figure 3 : The content of extracellular polysaccharides in yogurt.

[0044] Figure 4 The number of live bacteria in yogurt.

[0045] Figure 5 The relative abundance of two peptides in yogurt.

[0046] Figure 6 Content of bitter peptides in yogurt.

[0047] Figure 7 Content of CECT5716 in feces.

[0048] Figure 8 : Degradation rate of peptides.

[0049] Figure 9 IC50 concentration of PepT1 affinity.

[0050] Figure 10 Diameter of the inhibition zone.

[0051] Figure 11 : Antioxidant IC50 concentration. Detailed Implementation

[0052] The *Lactobacillus fermentum* CECT 5716 and *Bifidobacterium longum* subsp. BB536 used in the following examples were purchased from Mingzhou Biotechnology Co., Ltd.; the MRS broth culture medium used was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0053] The detection methods involved in the following embodiments are as follows: Detection of protein digestibility The in vitro digestion model INFOOGEST 2.0 was used to digest the hydrolyzed protein samples, and the digestibility was determined by Kjeldahl nitrogen determination.

[0054] Viscosity detection The viscosity of the sample was measured using a rotational viscometer with an LV-4 rotor at a speed of 30 rpm to determine whether yogurt was formed (the viscosity of stirred yogurt should not be lower than 1000 mPa·s).

[0055] Detection of extracellular polysaccharide content Trichloroacetic acid was added to the sample to remove proteins. After centrifugation, the supernatant was collected and precipitated with alcohol. After reconstitution, the sample was dialyzed to obtain polysaccharides. The content of extracellular polysaccharides was detected by the phenol-sulfuric acid method.

[0056] Detection of total bacterial viability in yogurt The yogurt was diluted, spread on MRS plates, and anaerobically cultured. The bacterial count was used to determine the bacterial content.

[0057] Detection of peptide abundance The relative abundance of specific peptides, i.e., the response value of liquid chromatography-tandem mass spectrometry, was identified using an ultra-high performance liquid chromatography-tandem mass spectrometer and PEAKS Studio.

[0058] Detection of bitter peptide content Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect bitter hydrophobic peptides, and the relative content of bitter peptides was indirectly characterized by calculating the integral area of ​​the hydrophobic region in the elution curve.

[0059] Detection of CECT 5716 content in feces Mice were administered yogurt by gavage for 7 consecutive days, with 10g of yogurt per kilogram of mouse body weight per day. On the seventh day, mouse feces were collected, homogenized, and total DNA was extracted. The bacterial content was quantified by qPCR to evaluate the gastric acid resistance of CECT 5716 and its ability to enhance intestinal colonization.

[0060] Example 1: Preparation of Yogurt The specific steps are as follows: (1) Cultivate two strains Lactobacillus fermentum CECT 5716 and Bifidobacterium longum subsp. BB536 were inoculated into MRS broth medium and cultured anaerobically at 37°C for 24 hours. After two subcultures under the above conditions, fermentation broths of Lactobacillus fermentum CECT 5716 and Bifidobacterium longum subsp. BB536 were prepared, respectively. The obtained fermentation broth was centrifuged at 2000 rpm for 5 min to collect the bacterial strain, washed twice with deionized water, and resuspended in physiological saline (0.9% NaCl) to adjust the concentration to 5 × 10⁻⁶. 8 CFU / mL, respectively, were used to prepare Lactobacillus fermentum CECT 5716 bacterial suspension and Bifidobacterium longum subsp. BB536 bacterial suspension.

[0061] (2) Preparation of CECT 5716 acid-resistant armor Take 100 μL of the Lactobacillus fermentum CECT 5716 suspension prepared in step (1), containing approximately 5 × 10⁻⁶ cells / mL. 7 CFU strain was added to 150 μL of PBS solution containing 10 mg / mL EGCG (epigallocatechin gallate). After gentle vortexing (at 200 rpm) for 1 minute, 150 μL of PBS solution containing 5 mg / mL FeCl3 was slowly added dropwise while continuously vortexing (at 200 rpm) for 2 minutes. Finally, 5 mL of PBS (10 mM, pH 7.4) was added, and the mixture was vortexed (at 200 rpm) for 3 minutes to complete the reaction, yielding a mixture containing Lactobacillus fermentum CECT 5716.

[0062] (3) Prepare BB5366 heavy armor Cassava starch was dispersed in water, and the pH was adjusted to 10. Sodium tripolyphosphate was added at a concentration of 0.1% of the dry starch. Sesame oil (2% of the dry starch) was slowly added dropwise under high-speed shear at 3000 rpm. After the sesame oil was added, the reaction system was placed in a 50°C water bath and gently stirred at 150 rpm for 5 hours. The upper oil phase was discarded, and the residual oil was removed by washing with ethanol several times. Finally, the mixture was washed with deionized water and freeze-dried to obtain dry microsphere powder. 140 mg of the obtained microsphere powder was mixed with 100 μL of the BB5366 bacterial suspension prepared in step (1), containing approximately 5 × 10⁻⁶ mg / L. 7 The CFU strains were mixed and vortexed (at 200 rpm) for 2 min to complete the reaction, resulting in a mixture containing Bifidobacterium longum subsp. BB5366.

[0063] (4) Adjust the pH of 10 L of raw milk to 7.5, heat at 90℃ for 40 min, and cool down to 35℃ to obtain the milk system.

[0064] (5) Add the modified CECT 5716 and BB5366 to the milk system obtained in step (4) above, that is: add the mixture containing Lactobacillus fermentum CECT 5716 obtained in step (2) and the mixture containing Bifidobacterium longum subsp. longum BB5366 obtained in step (3). The amount added is based on the bacterial concentration of Lactobacillus fermentum CECT 5716 or Bifidobacterium longum subsp. longum BB5366, and the bacterial concentration added is 1×10. 4 With a concentration of CFU / L, intermittent stirring every 10 minutes, and fermentation at 35-40℃ for 24 hours, yogurt can be obtained.

[0065] Example 2: Preparation of Yogurt The specific implementation is the same as in Example 1, except that the tapioca starch in step (3) is changed to corn starch.

[0066] Comparative Example 1: The specific implementation method is the same as in Example 1, except that the adjustment step (4) is to adjust the pH of 10 L of raw milk to 7.5 to obtain a milk system; and to prepare yogurt according to the method in Example 1.

[0067] Comparative Example 2: The specific implementation method is the same as in Example 1, except that step (2) is omitted, and the modified CECT 5716 in step (4) is adjusted to the CECT 5716 bacterial suspension prepared in step (1), and the bacterial concentration added is: 10 4 CFU / L; Yogurt was prepared according to the method in Example 1.

[0068] Comparative Example 3: The specific implementation method is the same as in Example 1, except that step (3) is omitted, and the modified BB536 in step (4) is adjusted to the BB536 bacterial suspension prepared in step (1), with the added bacterial concentration being: 10 4 CFU / L; Yogurt was prepared according to the method in Example 1. Comparative Example 4: The specific implementation method is the same as in Example 1, except that step (3) is omitted, and the strain in step (4) is adjusted to be only the modified CECT 5716 (single-strain fermentation), with a strain concentration of 10. 4 CFU / L; Yogurt was prepared according to the method in Example 1.

[0069] Comparative Example 5: The specific implementation method is the same as in Example 1, except that step (2) is omitted, and the strain in step (4) is adjusted to be the modified BB536 single-strain fermentation, with a strain concentration of 10. 4 CFU / L; Yogurt was prepared according to the method in Example 1.

[0070] Example 3: Experimental Results The performance of the yogurts obtained in Example 1 and Comparative Examples 1-5 was tested respectively; (1) The yogurts obtained in Example 1 and Comparative Examples 1-5 were digested using the INFOGEST 2.0 in vitro digestion model to digest the hydrolyzed protein samples, and the digestibility was determined by Kjeldahl nitrogen determination. (2) The viscosity of the yogurts obtained in Example 1 and Comparative Examples 1 to 5 was measured using a rotational viscometer with an LV-4 rotor at a speed of 30 rpm. (3) The yogurt samples obtained in Example 1 and Comparative Examples 1-5 were respectively subjected to the addition of trichloroacetic acid to remove protein, centrifuged and the supernatant was collected and subjected to alcohol precipitation, reconstituted and dialyzed to obtain polysaccharides, and the extracellular polysaccharide content was detected by the phenol-sulfuric acid method. (4) The relative abundance of specific peptides was identified using an ultra-high performance liquid chromatography-tandem mass spectrometer and PEAKS Studio. (5) The yogurt obtained in Example 1 and Comparative Examples 1-5 was diluted, spread on MRS plates, anaerobic cultured, and the bacterial strain content was determined by counting method; (6) Trichloroacetic acid was added to the yogurt samples obtained in Example 1 and Comparative Examples 1-5 to remove protein, and salt and small molecule sugars were removed by desalting column. The bitter hydrophobic peptides were detected by reversed-phase high performance liquid chromatography. The relative content of bitter peptides was indirectly characterized by calculating the integral area of ​​the hydrophobic region in the elution curve. (7) Animal experiments were conducted using the yogurts obtained in Example 1 and Comparative Examples 1-5 respectively: Mice were administered yogurt by gavage for 7 consecutive days, with 10g of yogurt per kilogram of mouse body weight per day. On the seventh day, mouse feces were collected, homogenized, and total DNA was extracted. The bacterial content was quantified by qPCR to evaluate the gastric acid resistance of CECT 5716.

[0071] The results are shown in Table 1 below: Table 1: Performance characterization at the fermentation endpoint

[0072] The results show: In Comparative Example 1, the abundance of two peptides was low, resulting in low protein digestibility, and there was also a high content of bitter peptides.

[0073] In Comparative Example 2, the number of live bacteria in the yogurt was low, and the protein was not hydrolyzed, resulting in low protein digestibility and poor intestinal colonization ability of CECT5716.

[0074] In Comparative Example 3, fewer BB536 cells survived, resulting in less acid production and less extracellular polysaccharide production, which prevented the formation of a sufficiently viscous yogurt texture.

[0075] In Comparative Example 4, the two types of peptides had low abundance, low protein digestibility, and insufficient viscosity to form a yogurt texture, while having high content of bitter peptides.

[0076] In Comparative Example 5, the protein digestibility was low and the abundance of the two peptides was low.

[0077] Example 4: Study on peptide properties The protein of this invention contains SLAMAASNG and NKALALNT peptides. These two peptides are chemically synthesized, and their performance is tested. The specific methods are as follows: 1. Easily recognized by intestinal peptidase The brush border membrane sacs of the small intestine of mice were extracted and prepared into a suspension (concentration: 0.5 mg / mL, based on total protein). 1 mg / mL of SLAMAASNG peptide aqueous solution and NKALALNT peptide aqueous solution were added respectively, and the suspensions were incubated at 37℃ for 15 min. The degradation rate of the peptides was detected by liquid chromatography-mass spectrometry. The results show: SLAMAASNG peptide (S peptide) was 75% converted into dipeptides and tripeptides within 15 min, indicating that it is easily digested by intestinal enzymes.

[0078] NKALALNT peptide (N-peptide) was converted into dipeptides and tripeptides by 76% within 15 min, indicating that it is easily digested by intestinal enzymes.

[0079] 2. PepT1 affinity Caco-2 cells were cultured on Transwell plates to form a dense monolayer. SLAMAASNG and NKALALNT peptides were hydrolyzed by intestinal peptidase according to the method in step 1. Glycyl-sarcosine (Gly-Sar) was added to the hydrolysate at an amount of 0.8 mM. The peptide concentration required to inhibit 50% Gly-Sar transport was then determined.

[0080] The results show: The IC50 of SLAMAASNG peptide (S peptide) is 0.42 mM and that of NKALALNT peptide (N peptide) is 0.47 mM, indicating that low concentrations can occupy the Gly-Sar transport channel, demonstrating strong affinity and rapid absorption.

[0081] 3. Antibacterial properties Using Escherichia coli and Staphylococcus aureus as antibacterial targets, the antibacterial effects of the two peptides were evaluated using the inhibition zone detection method.

[0082] The specific method is as follows: First, the bacterial suspensions of *Escherichia coli* and *Staphylococcus aureus* were adjusted to a McFarland turbidity of 0.5 (OD600 approximately 0.08–0.10) and evenly spread on agar plates. Iso-sized wells were prepared using a sterile punch. Then, different concentrations of the test peptide solution and control samples were added to the wells. After pre-diffusion at 4°C, the plates were incubated at 37°C for 18–24 hours. Finally, the antibacterial efficacy of the two peptides against representative Gram-negative and Gram-positive strains was quantitatively evaluated by measuring and comparing the diameter of the inhibition zones.

[0083] The results show: SLAMAASNG peptide (S peptide) showed inhibition zone diameters of 13.2 mm and 14.1 mm against Escherichia coli and Staphylococcus aureus, respectively. The inhibition zone diameters of NKALALNT peptide (N-peptide) against Escherichia coli and Staphylococcus aureus were 14.9 mm and 18.1 mm, respectively.

[0084] This indicates that SLAMAASNG peptide and NKALALNT peptide both have high antibacterial activity against Escherichia coli and Staphylococcus aureus.

[0085] 4. Antioxidant function The antioxidant capacity of peptides was detected using DPPH and ABTS kits purchased from Nanjing Jiancheng Biotechnology Research Institute.

[0086] The specific method is as follows: The medium-long peptides SLAMAASNG or NKALALNT were prepared into test solutions with concentration gradients of 0.1, 0.2, 0.4, 0.8, 1.0, 1.5, and 2.0 mg / mL using deionized water. Whey protein served as the control group, and test solutions were prepared at concentrations of 1.0, 5.0, 10.0, 15.0, 20.0, and 25.0 mg / mL. Perform the tests according to the instructions for the DPPH and ABTS kits, plot the concentration-scavenging rate curves, and calculate the IC50 value using nonlinear regression. The results show: The SLAMAASNG peptide (S peptide) prepared in this invention has IC50 values ​​of 0.72 mg / mL for DPPH and 0.58 mg / mL for ABTS. The IC50 values ​​of NKALALNT peptide (N-peptide) against DPPH and ABTS were 0.53 mg / mL and 0.61 mg / mL, respectively, indicating good antioxidant capacity.

[0087] 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 protein peptide, characterized in that, The protein peptide is a SLAMAASNG or NKALALNT polypeptide, with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A method for preparing yogurt, wherein the yogurt contains the protein peptide of claim 1, characterized in that, The method includes the following steps: (1) Preparation of bacterial suspension The fermentation broths of Lactobacillus fermentum CECT 5716 and Bifidobacterium longum subsp. BB536 were centrifuged to collect the bacterial strains, and then resuspended in physiological saline to prepare Lactobacillus fermentum CECT 5716 bacterial suspension and Bifidobacterium longum subsp. BB536 bacterial suspension, respectively. (2) Preparation of CECT 5716 acid-resistant armor The *Lactobacillus fermentum* CECT 5716 bacterial suspension prepared in step (1) was added to a PBS solution containing 10-20 mg / mL EGCG. The inoculation concentration was 5 × 10⁻⁶. 7 ~5×10 8 After vortexing for 1-2 min, add PBS solution containing 3-7 mg / mL FeCl3 and vortex for 1-2 min. Finally, add PBS solution and vortex for 0.5-3 min to complete the reaction and obtain a mixture containing Lactobacillus fermentum CECT 5716. (3) Prepare BB5366 heavy armor Disperse cassava starch in water, adjust the pH to 10-11, add sodium tripolyphosphate at a concentration of 0.1%-0.5% of the dry starch. Slowly add sesame oil at a high-speed shear rate of 1500-3000 rpm, with the amount of sesame oil being 1-2% of the dry starch. After the sesame oil is added, place the reaction system in a 50-60℃ water bath and gently stir at 100-150 rpm for 2-5 hours. Pour off the upper oil phase, wash repeatedly with ethanol to remove residual oil, and finally wash with deionized water. Freeze-dry to obtain dry microsphere powder. Mix the obtained microsphere powder with the BB5366 bacterial suspension prepared in step (1), with a bacterial concentration of 5×10⁻⁶. 7 ~5×10 8 CFU, after vortexing for 1-2 minutes, the reaction is completed, and a mixture containing Bifidobacterium longum subsp. BB5366 is obtained; (4) Adjust the pH of raw milk to 6.5~7.5 and heat at 80~90℃ for 30~50 min to obtain the milk system; (5) Add the mixture containing Lactobacillus fermentum CECT 5716 obtained in step (2) and the mixture containing Bifidobacterium longum subsp. BB5366 obtained in step (3) to the milk system obtained in step (4). The concentration of Lactobacillus fermentum CECT 5716 or Bifidobacterium longum subsp. BB5366 added is 1×10⁻⁶. 3 ~1×10 4 Yogurt can be obtained by intermittent stirring every 10 to 20 minutes at 35 to 40°C for 12 to 24 hours.

3. The yogurt prepared by the method of claim 2.

4. The use of the protein peptide according to claim 1 in the preparation of food, pharmaceuticals, health products or nutritional products.

5. The application according to claim 4, characterized in that, The food, medicine, health product or nutritional product may also contain derivatives of protein peptides. The derivatives of protein peptides refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain groups, amino terminus or carbonyl terminus of the protein peptide. Preferably, the food, medicine, health product, or nutritional product is one with antioxidant, easily digestible, low gastrointestinal irritation, and / or antibacterial properties.

6. The application according to claim 4, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field. Optionally, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition. Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form; Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions; Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection; Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

7. The application according to claim 4, characterized in that, The food products include, but are not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages. The food items mentioned also include special dietary foods.

8. The application according to claim 4, characterized in that, The nutritional products mentioned include medical nutritional products, nutritional products formulated for special populations, and nutritional supplements.

9. A food, medicine, health product, or nutritional product, characterized in that, The food, medicine, health product, or nutritional product contains the protein peptide described in claim 1; the food, medicine, health product, or nutritional product is a food, medicine, health product, or nutritional product with antioxidant, easily digestible, low gastrointestinal irritation, and / or antibacterial properties. Optionally, the food, medicine, health product or nutritional product may also contain derivatives of protein peptides. The derivatives of protein peptides refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain groups, amino terminus or carbonyl terminus of the protein peptide. Optionally, the drug may further contain pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field. Optionally, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition. Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form; Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions; Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection; Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers; Optionally, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; The food products also include special dietary foods; the health products also contain acceptable excipients.

10. An expression vector or recombinant microorganism, characterized in that, The expression vector or recombinant microorganism expresses the protein peptide of claim 1; Preferably, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors; Preferably, the recombinant microorganism is a bacterium or a fungus.

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