A milk fat globule membrane for regulating the surface components of Lactobacillus casei and its application

Abstract

This invention discloses the application of milk fat globule membranes in improving the tolerance of *Lactobacillus casei*, regulating the surface components of *Lactobacillus casei*, or preparing protective agents for *Lactobacillus casei*. Milk fat globule membranes can regulate protein expression in *Lactobacillus casei* to enhance its performance. Its characteristics include: promoting the synthesis of terpenoids, improving the antioxidant and antibacterial capabilities of *Lactobacillus casei* CGMCC NO.15956 and influencing changes in its surface properties; promoting the synthesis of cell wall teichoic acid and lipoteichoic acid, which helps maintain cell wall integrity, improves environmental tolerance, and affects the surface properties of lactic acid bacteria; and reducing the surface hydrophobicity of *Lactobacillus casei* CGMCC NO.15956. Based on these characteristics, milk fat globule membranes can protect *Lactobacillus casei* under oxidative stress, improve its survival rate, and reduce cell membrane damage. Therefore, this milk fat globule membrane can be used for encapsulation and protection of lactic acid bacteria.

Description

Technical Field

[0001] This invention relates to a milk fat globule membrane that regulates the surface components of Lactobacillus casei and its applications, belonging to the fields of food and medicine. Background Technology

[0002] Lactic acid bacteria are a general term for a group of Gram-positive, facultative or anaerobic bacteria that can utilize carbohydrates (mainly glucose or lactose) to produce lactic acid. They are non-spore-forming, non-motile, catalase-negative, and appear as rods, short rods, or spherical shapes. Lactic acid bacteria are widely found in fermented products, dairy products, the intestines of humans and animals, plants, and soil. Currently, lactic acid bacteria can be roughly divided into 23 genera, such as *Lactobacillus*, *Lactococcus*, *Bacillus*, *Streptococcus*, and *Bifidobacterium*. Lactic acid bacteria have rich probiotic functions, such as alleviating oxidative stress, delaying aging, maintaining gut microbiota balance, inhibiting pathogens, regulating immunity, and preventing cancer.

[0003] Milk fat globule membrane (MFGM) originates from the alveoli of the mammary gland. Triglycerides are synthesized on the rough endoplasmic reticulum of mammary epithelial cells, existing as milk fat microparticles and ultimately forming the outer lipid bilayer membrane on the surface of milk fat globules. MFGM is rich in phospholipids, glycosphingolipids, and proteins (mainly glycoproteins). The composition of MFGM varies across different studies, primarily due to differences in milk microbial content, lactation period, season, and extraction method. MFGM has numerous nutritional functions, such as promoting infant neural and intestinal development, enhancing immunity, preventing cancer, and exhibiting antibacterial and antiviral effects. Reports indicate that MFGM can enhance the growth activity of lactic acid bacteria, affect their adhesion ability, act as an encapsulating material to release lactic acid bacteria in the intestine, promote mucosal immunity, and act on the gut-brain axis to improve cognitive function. Furthermore, lactic acid bacteria have an affinity for MFGM and exhibit a tendency to attach to it, preferentially adhering to MFGM in the matrix. However, the underlying mechanisms of the interaction between lactic acid bacteria and milk fat globule membranes have not been fully elucidated.

[0004] Some scholars have proposed that the interaction between the milk fat globule membrane and lactic acid bacteria is an adhesion phenomenon primarily driven by bacterial surface characteristics. Firstly, there is non-specific adhesion, dominated by reversible interactions such as passive adsorption, electrostatic interactions, hydrophobic interactions, and van der Waals forces. Surface hydrophobicity is an important physicochemical property of bacteria, and its strength depends mainly on the number of polar groups on the bacterial surface, related to surface adhesins such as lipoteichoic acid, capsules, pili, and surface proteins (S-layer proteins). Secondly, there are irreversible, specific interactions, jointly accomplished by adhesins on the microbial surface and specific receptors on the cell surface or in mucus, involving adhesion factors, complementary receptors, and surface appendages. Therefore, elucidating the influence of the milk fat globule membrane on the surface components of lactic acid bacteria is crucial for discussing the potential mechanisms of their interaction. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of milk fat globule membrane in improving the tolerance of Lactobacillus casei, or in regulating the surface components of Lactobacillus casei, or in preparing a protective agent for Lactobacillus casei.

[0006] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the application of milk fat globule membrane in improving the tolerance of Lactobacillus casei or in regulating the surface components of Lactobacillus casei or in preparing a protective agent for Lactobacillus casei.

[0007] The milk fat globule membrane is derived from cow's milk.

[0008] The Lactobacillus casei mentioned is Lactobacillus casei CGMCC NO.15956, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.15956, deposit address in Beijing, China, and deposit date of June 19, 2018.

[0009] The present invention also includes the effects of milk fat globule membrane on the adhesion ability, hydrophobicity, self-polymerization and surface properties of Lactobacillus casei.

[0010] The surface components of *Lactobacillus casei* include one or more of terpenoids, teichoic acid, and lipoteichoic acid. The milk fat globule membrane upregulates the expression of glycosyltransferases involved in teichoic acid synthesis, promotes the synthesis of teichoic acid and lipoteichoic acid, maintains cell wall integrity, and can enhance environmental tolerance. It is also used as a protective agent for the surface charged properties of *Lactobacillus casei* CGMCC NO.15956.

[0011] The applications include increasing the expression of enzymes involved in the terpene synthesis pathway or upregulating the expression of glycosyltransferases involved in teichoic acid synthesis. This includes its application as a protective agent in *Lactobacillus casei* CGMCC NO.15956, which enhances the expression of enzymes involved in the terpene backbone biosynthesis pathway.

[0012] Among them, the application of milk fat globule membrane in a protective agent that downregulates the expression of surface-related proteins and affects the surface properties and adhesion ability of Lactobacillus casei CGMCC NO.15956.

[0013] The application also includes improving the tolerance of Lactobacillus casei under oxygen stress conditions.

[0014] The oxygen stress condition is a treatment with an H2O2 concentration of 8 mM for 3 hours.

[0015] The concentration of the milk fat globule membrane is 0.5-15 mg / mL.

[0016] The application includes using milk fat globule membranes to repair cell membrane damage in Lactobacillus casei CGMCC NO.15956.

[0017] The concentration of the milk fat globule membrane is 0.5-5 mg / mL.

[0018] Among them, the application of milk fat globule membrane in a matrix that exhibits excellent adhesion to Lactobacillus casei CGMCC NO.15956.

[0019] Among them, surface-associated proteins play an important role in the adhesion of Lactobacillus casei CGMCC NO.15956 to the milk fat globule membrane.

[0020] This invention extracts milk fat globule membranes from cow's milk and investigates their effects on the surface components of *Lactobacillus casei*. Milk fat globule membranes can maintain cell wall integrity and influence the surface properties and adhesion ability of lactic acid bacteria by regulating terpene skeleton biosynthesis, teichoic acid synthesis, and surface protein expression. For oxidatively damaged *Lactobacillus casei*, milk fat globule membranes enhance teichoic acid biosynthesis and influence surface protein expression, strengthening the bacterial physical barrier and thus alleviating oxidative damage, improving the strain's tolerance to oxygen stress, protecting *Lactobacillus casei* under oxygen stress, increasing its survival rate, and reducing cell membrane damage. The milk fat globule membrane affects the surface components of *Lactobacillus casei*, and surface proteins mediate the adhesion between the milk fat globule membrane and *Lactobacillus casei*, thereby obtaining *Lactobacillus casei* with excellent performance and tolerance to environmental stress.

[0021] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The effects of the milk fat globule membrane on the surface components of *Lactobacillus casei* mainly involve the biosynthesis of terpenoid skeletons, the synthesis of teichoic acid, and the expression of surface proteins. The milk fat globule membrane can increase the expression of related enzymes in the terpenoid skeleton biosynthesis pathway, which is beneficial to the synthesis of terpenoid compounds and may enhance the antioxidant and antibacterial capabilities of *Lactobacillus casei*, as well as affect changes in its surface properties. The milk fat globule membrane can upregulate the expression of glycosyltransferases in teichoic acid synthesis, which may promote the synthesis of cell wall teichoic acid and lipoteichoic acid, maintain cell wall integrity, improve environmental tolerance, and affect the surface charge properties of *Lactobacillus casei*. The milk fat globule membrane can downregulate the expression of surface-related proteins, affecting the surface properties and adhesion ability of *Lactobacillus casei*. Furthermore, the milk fat globule membrane in this invention can improve the survival rate of *Lactobacillus casei* under oxygen stress, reduce cell membrane damage, promote the biosynthesis of teichoic acid, affect surface protein expression, repair cell wall damage, maintain cell morphology, and improve the adhesion ability of lactic acid bacteria to mucins. Attached Figure Description

[0022] Figure 1 Effects of MFGM on terpene skeleton biosynthesis in Lactobacillus casei CGMCC NO.15956;

[0023] Figure 2 The effect of MFGM on teichoic acid synthesis in Lactobacillus casei CGMCC NO.15956;

[0024] Figure 3 Effects of MFGM on surface protein expression in Lactobacillus casei CGMCC NO.15956;

[0025] Figure 4 Effect of MFGM on the survival rate of Lactobacillus casei CGMCC NO.15956 under oxygen stress;

[0026] Figure 5 Effects of MFGM on LDH of Lactobacillus casei CGMCC NO.15956 under oxygen stress;

[0027] Figure 6 Effects of MFGM on MDA activity of Lactobacillus casei CGMCC NO.15956 under oxygen stress;

[0028] Figure 7 Differentially expressed proteins related to teichoic acid metabolism;

[0029] Figure 8 Surface protein-associated differential proteins;

[0030] Figure 9 Adhesion of Lactobacillus casei CGMCC NO.15956 to different milk matrices;

[0031] Figure 10 The effect of MFGM on the hydrophobicity of Lactobacillus casei CGMCC NO.15956;

[0032] Figure 11 The effect of MFGM on the self-polymerization of Lactobacillus casei CGMCC NO.15956;

[0033] Figure 12 Effect of MFGM on the adhesion ability of Lactobacillus casei CGMCC NO.15956;

[0034] Figure 13 Characterization of the effect of MFGM on the surface properties of Lactobacillus casei CGMCC NO.15956;

[0035] Figure 14 Electrophoresis image of surface proteins from Lactobacillus casei CGMCC NO.15956;

[0036] Figure 15 Adhesion of MFGM to surface proteins before and after LiCl removal;

[0037] Figure 16 The protective effects of different proteins on Lactobacillus casei CGMCC NO.15956. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0039] Example 1: Differential protein expression analysis of Lactobacillus casei CGMCC NO.15956 by milk fat globule membrane

[0040] 1. Extraction of milk fat globule membrane

[0041] Milk (from Inner Mongolia Mengniu Dairy) was placed in centrifuge tubes. Centrifuge parameters were set as follows: temperature 4℃, speed 5000×g, centrifugation time 15 min. After centrifugation, the supernatant milk fat was collected. 3-5 times the volume of PBS (pH 7.4) washing solution was added to the centrifuge tubes. The tubes were placed in a 39℃ water bath for 30 min with gentle agitation. After the water bath, centrifugation was performed at 4℃, speed 5000×g, and time 15 min. This process was repeated to wash the milk fat until no soluble protein remained in the washing solution after centrifugation. Finally, the tubes were washed once with distilled water to obtain milk fat globules (MFG). The MFG globules were placed at -20℃ for 2 h, followed by a 40℃ water bath for 30 min, and then sonicated for 30 min to disrupt the MFG membrane. Centrifugation was then performed at 4℃, speed 5000×g, and time 30 min. Immediately after centrifugation, the tubes were placed at -20℃. After freezing, the supernatant fat layer was removed, and the resulting solution was MFGM. This solution was then freeze-dried for later use.

[0042] 2. Protein Sample Preparation

[0043] Lactobacillus casei CGMCC NO.15956 (stored in glycerol at -80℃) was streaked onto MRS solid medium and cultured at 37℃ for 48 h. Single colonies were obtained and inoculated into MRS liquid medium, and activated by incubation at 37℃ for 18 h. The twice-activated Lactobacillus casei CGMCC NO.15956 was mixed (crystal concentration approximately 1×10⁹ CFU / mL) and inoculated into MRS liquid medium at a ratio of 1.0% (v / v). The culture was incubated at 37℃ until the stationary phase. The cells were collected by centrifugation at 3000×g for 10 min at 4℃, and resuspended in MRS medium and MRS medium containing 2.5 mg / mL MFGM for 3 h. The culture supernatant was removed and the cells were collected. The prepared cells were flash-frozen in liquid nitrogen for later use. 300 μg of sample was taken for enzymatic digestion as follows: 300 μg of sample was added to dithiothreitol (DTT) to a concentration of 100 mM, incubated in a boiling water bath for 5 min, and cooled to room temperature. Add 200 μL L UAbuffer (8 M Urea, 150 mM Tris-HCl, pH = 8.0), mix well, transfer to a 10 kDa ultrafiltration centrifuge tube, and centrifuge at 12000 × g for 15 min. Add 200 μL L UAbuffer, centrifuge at 12000 × g for 15 min, and discard the filtrate. Add 100 μL IAA (50 mM IAA in UA), shake at 600 rpm for 1 min, incubate at room temperature in the dark for 30 min, and centrifuge at 12000 × g for 10 min. Add 100 μL UA buffer, centrifuge at 12000 × g for 10 min, and repeat twice. Add 100 μL NH4HCO3 buffer, centrifuge at 14000 × g for 10 min, and repeat twice. Add 60 μL Trypsin buffer (6 μg Trypsin in 40 μL NH4HCO3 buffer), shake at 600 rpm for 1 min, and incubate at 37℃ for 16–18 h. Replace with a new collection tube, centrifuge at 12000×g for 10 min, collect the filtrate, add an appropriate amount of 0.1% trifluoroacetic acid solution to reconstitute, desalt using a desalting centrifuge column, and collect the peptides.

[0044] 3. TMT peptide labeling and peptide fractionation

[0045] Take 100 μg of the peptide obtained in step 2 above and label it according to the Thermo Fisher TMT Labeling Kit instructions. Labeling channel: controll(TMT) 10 -126 marker), control2(TMT) 10 -127N marking), control3 (TMT) 10 -127C marking); M1(TMT) 10-131N marking), M2(TMT) 10 -131C marking), M3 (TMT) 10 -131N label). Equal volumes of each labeled peptide were mixed, and the dried peptides were fractionated using a high-pH reversed-phase column. The samples were then collected and pooled, and each peptide fraction was dried and reconstituted with 0.1% FA for LC-MS analysis.

[0046] 4. LC-MS / MS analysis

[0047] A suitable amount of peptide was taken from each sample for chromatographic separation using a nano-flow rate Easy nLC 1200 chromatography system (Thermo Scientific). Buffer solutions: Solution A was a 0.1% aqueous formic acid solution, and Solution B was a mixture of 0.1% formic acid, acetonitrile, and water (acetonitrile being 95%). 100% of Solution A was used to equilibrate the column. The sample was fed into a Trap Column (100 μm × 20 mm, 5 μm, C18) (Thermo Scientific, Acclaim PepMap C18). In the chromatographic analysis column (75μm×150mm, 3μm, C18), sample gradient separation was performed at a flow rate of 300 nl / min. The elution program is shown in Table 3-3. After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer. The analysis time was 90 min, detection mode: positive ion, precursor ion scan range: 400-1800 m / z, first-stage mass spectrometry resolution: 60,000@m / z 200, first-stage maximum IT: 50 ms. Peptide secondary mass spectrometry analysis was performed using the following method: after each full scan, secondary mass spectra of the 20 highest intensity precursor ions were acquired (MS2 scan). Secondary mass spectrometry resolution: 45,000@m / z 200, maximum intensity (MT) of 50ms, MS2 activation type: HCD, isolation window: 1.6m / z, normalized collision energy: 32.

[0048] 5. Proteomics data analysis

[0049] The final LC-MS / MS raw RAW files were imported into the Sequest HT search engine in Proteome Discoverer software (version 2.4, Thermo Scientific) for database retrieval. The database used for the database search was uniprot-Lactobacillus casei

[1582] -27636-20221103.fasta, which was obtained from the protein database at https: / / www.uniprot.org / taxonomy / 1582, with protein entry number 27636 and download date of November 2022. The protein abundance levels of each sample were compared. Proteins with a fold change in expression >1.2 or <1 / 1.2 and a p-value <0.05 were considered differentially expressed proteins. The functional metabolic pathways of differentially expressed proteins were enriched using the GO, COG, and KEGG databases.

[0050] 6. Data Processing and Analysis

[0051] GraphPad Prism 8.0 software was used for plotting, and SPSS 26 was used for statistical analysis. The experimental data of each group are expressed as mean ± standard deviation (Mean ± SD, n = 3). Different letters indicate significant differences (P < 0.05).

[0052] 7. Results and Analysis

[0053] 7.1 Biosynthesis of Terpenoid Skeleton

[0054] TMT analysis identified 476 significantly differentially expressed proteins. Compared to bacteria under normal conditions, 207 differentially expressed proteins were upregulated and 269 were downregulated. The number of downregulated proteins was greater than the number of upregulated proteins, but the fold change of upregulated proteins was higher than that of downregulated proteins. Analysis of metabolic pathways significantly enriched by these differentially expressed proteins revealed that the proteins primarily affecting the surface components of *Lactobacillus casei* CGMCC NO. 15956 included terpene backbone biosynthesis, teichoic acid synthesis, and surface protein expression.

[0055] Terpenes, also known as isoprene compounds, are a large class of natural products composed of isoprene (C5) units. There are two biosynthetic pathways: the mevalonate pathway (MVA) and the non-mevalonate pathway (MEP / DOXP). For example... Figure 1Incubation with MFGM can enhance the expression of HMG-CoA synthase (HMGS), mevalonate kinase (M5K), and CDP-ME kinase (CMK) in the terpene backbone biosynthesis of *Lactobacillus casei* CGMCC NO.15956, thus promoting the synthesis of terpenoids. A large number of terpenoids possess antioxidant and anti-inflammatory physiological functions and are also important antibacterial substances. They can disrupt the cell wall and cell membrane of pathogenic bacteria, affecting metabolism, or bind to proteins on the cell surface, preventing nutrient absorption and thus inhibiting growth, leading to the death of pathogenic microorganisms. Furthermore, terpenoids are hydrophobic hydrocarbons, and their secretion into the extracellular space may cause changes in surface properties. In summary, MFGM may enhance the antioxidant and antibacterial capabilities of *Lactobacillus casei* CGMCC NO.15956 and influence its surface properties.

[0056] 7.2 Synthesis of Teichoic Acid

[0057] Teichoic acids (TAs) are a class of anionic polymers composed of repeating aldose phosphate units found in the cell walls of Gram-positive bacteria. Based on their binding sites, teichoic acids can be divided into two categories: wall teichoic acids (WTAs), which are covalently bound to cell wall peptidoglycans; and lipid teichoic acids (LTAs), which are anchored to the cell membrane. Teichoic acids participate in maintaining cell mechanical strength, cell adhesion to the host, resistance to antimicrobial agents, tolerance to environmental stimuli, biofilm formation, cell division, and other biological processes. Figure 2 Upregulation of glycosyltransferase (TagE) and peptidoglycan teichoic acid transferase (TagRUV) expression in the lipoteichoic acid synthesis pathway favors lipoteichoic acid synthesis. Studies have shown that lipoteichoic acid helps regulate bacterial surface charge and hydrophobicity, thereby affecting the binding of extracellular molecules and playing a role in protecting bacteria from various stresses. MFGM promotes the upregulation of glycosyltransferase (LafA, LafB) expression in Lactobacillus casei CGMCC NO.15956. In the lipoteichoic acid synthesis pathway, LafA and LafB catalyze the formation of Gal-Glc-DAG from DAG, which is then further catalyzed by lipophosphoryl phosphate synthase (LtaS) to synthesize the polyglycerol phosphate backbone, promoting the synthesis of Type I LTA. Due to the presence of low isoelectric point lipoteichoic acid, the bacterial surface is negatively charged, giving the lactic acid bacteria cell wall anionic properties. Therefore, non-specific adhesion between bacterial cells and recipient cells can occur through electrostatic interactions.

[0058] 7.3 Surface proteins

[0059] During their growth, lactic acid bacteria secrete proteins outside their cells or bind to the cell surface, performing various functions; these proteins are collectively called lactic acid bacteria cell surface proteins. These surface proteins perform multiple functions, such as maintaining cell morphology, competitively inhibiting the adhesion sites of pathogens in the gastrointestinal tract, and regulating immune responses. Furthermore, these surface proteins interact with host receptors during the adhesion process of lactic acid bacteria. Figure 3 As shown, MFGM significantly upregulated the expression of surface proteins LPXTG motif and extracellular matrix proteins of *Lactobacillus casei* CGMCC NO.15956, while differentially downregulated the expression of cell wall surface anchor family proteins, conserved extracellular matrix proteins, pyruvate kinase (Pyk), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cysteine ​​synthase (CYS), fructose-6-phosphate isomerase (Pgi), lactate dehydrogenase (Ldh), 2,3-diphosphoglycerate-dependent phosphoglycerate mutase (GpmA), pyruvate dehydrogenase E1 subunit α (PdhA), and D-3-phosphoglycerate dehydrogenase (PGDH). In general, MFGM downregulated most of the surface proteins of *Lactobacillus casei* CGMCC NO.15956, therefore MFGM may lead to a decrease in the surface hydrophobicity of *Lactobacillus casei* CGMCC NO.15956, which is consistent with the measured decrease in hydrophobicity. Glycolytic enzymes participate in glycolysis within bacterial cells and, as part-time proteins, are located on the bacterial surface as adhesins. They specifically bind to human intestinal epithelial cells or extracellular matrix components such as laminin, collagen, and mucin, promoting the adhesion and colonization of probiotics in the gut. The addition of MFGM significantly decreased glycolytic enzyme expression, which may have led to a decrease in the adhesion ability of *Lactobacillus casei* CGMCC NO.15956. This is consistent with the measured decrease in the adhesion ability of *Lactobacillus casei* CGMCC NO.15956 to mucins and collagen after the removal of surface proteins.

[0060] Example 2: Protective effect of milk fat globule membrane on Lactobacillus casei under H2O2 treatment

[0061] 1. Effect of milk fat globule membrane on the survival rate of Lactobacillus casei CGMCC NO.15956 after oxidative damage

[0062] Lactobacillus casei CGMCC NO.15956 was activated according to the method in Example 1. The Lactobacillus casei CGMCC NO.15956 activated twice was mixed (bacterial concentration approximately 1 × 10⁻⁶). 9CFU / mL was inoculated into MRS liquid medium at a ratio of 1.0% (v / v) and incubated at 37°C until the stationary phase. The cells were then collected by centrifugation at 3000×g for 10 min at 4°C, washed with pre-cooled sterile physiological saline, and collected again. The obtained bacterial cells were resuspended in MRS broth containing 0.5, 1, 2.5, 5, 10, and 15 mg / mL milk fat globule membranes and 8 mM H2O2. The bacterial suspensions were incubated statically at 37°C for 3 h. Bacterial suspensions were collected every 1 h, serially diluted with physiological saline, and plate counts were performed according to GB 4789.35-2016.

[0063]

[0064] In the formula: N1: number of viable bacteria after treatment; N0: number of viable bacteria in normal MRS broth medium. 2. Effect of milk fat globule membrane on cell membrane damage of Lactobacillus casei CGMCC NO.15956

[0065] Lactobacillus casei CGMCC NO.15956 cultured to the logarithmic growth phase (bacterial concentration approximately 1×10⁻⁶) 9 The cells were centrifuged at 3000×g for 10 min at 4°C, washed three times with sterile PBS (0.1 mol / L, pH 7.4), and resuspended in PBS solution. Cell disruption was performed using a cell disruptor, and the supernatant was collected after centrifugation. The lactate dehydrogenase (LDH) content and malondialdehyde (MDA) activity were measured using a kit. Protein content was determined using a BCA kit.

[0066] 3. Protein Sample Preparation

[0067] The method is the same as in Example 1.

[0068] 4. TMT Peptide Labeling and Peptide Segregation

[0069] The method is the same as in Example 1. The labeled channels are: control1 (TMT10-126 label), control2 (TMT10-127N label), control3 (TMT10-127C label); H2O2-1 (TMT10-128N label), H2O2-2 (TMT10-128C label), H2O2-3 (TMT10-129N label); H2O2-M1 (TMT10-129C label), H2O2-M2 (TMT10-130N label); H2O2-M3 (TMT10-130C label).

[0070] 5. LC-MS / MS Analysis

[0071] The method is the same as in Example 1.

[0072] 6. Proteomics Data Analysis

[0073] The method is the same as in Example 1.

[0074] 7 Data Processing and Analysis

[0075] Plotting was performed using Origin 8.0 and GraphPad Prism 8.0 software, and statistical analysis was conducted using SPSS 26. The experimental data of each group are expressed as mean ± standard deviation (Mean ± SD, n = 3). Different letters indicate significant differences (P < 0.05).

[0076] 8. Results and Analysis

[0077] 8.1 Effects of different concentrations of milk fat globule membrane on *Lactobacillus casei* CGMCC NO.15956 under oxygen stress

[0078] Preliminary experiments selected a stress duration of 3 hours and an H2O2 concentration of 8 mM as the oxygen stress model conditions for *Lactobacillus casei* CGMCC NO.15956. During the oxygen stress process, 0.5, 1, 2.5, 5, 10, and 15 mg / mL of MFGM were added. The experimental results are as follows... Figure 4 As shown, after the addition of MFGM, the survival rate of Lactobacillus casei CGMCC NO.15956 under oxygen stress showed a gradual increasing trend compared with the model group. When the MFGM concentration was 2.5 mg / mL, the survival rate of Lactobacillus casei CGMCC NO.15956 under oxygen stress reached 70.42±0.91%, and when the MFGM concentration was greater than 2.5 mg / mL, the survival rate did not change significantly (P>0.05).

[0079] from Figure 5 As can be seen, LDH activity in the culture medium significantly increased after oxygen stress (P < 0.05), indicating that the cell membrane of *Lactobacillus casei* CGMCC NO.15956 was damaged under these conditions, proving the successful establishment of the model. After adding different concentrations of MFGM, the activity of lactate dehydrogenase in the supernatant of *Lactobacillus casei* CGMCC NO.15956 culture medium gradually decreased, indicating that MFGM has a certain protective effect on the cell membrane during oxygen stress. When the MFGM concentration was greater than 1 mg / mL, the lactate dehydrogenase activity did not change significantly (P > 0.05), and when the concentration was greater than 2.5 mg / mL, there was no significant difference in lactate dehydrogenase activity compared to normal conditions (P > 0.05). Therefore, the protective concentration of MFGM is 2.5 mg / mL. MDA is a product of cell membrane lipid peroxidation and has cytotoxicity. Excessive accumulation in the cell can damage the biomembrane structure and accelerate oxidative damage. Figure 6Compared with the model group, the intracellular MDA content of Lactobacillus casei CGMCC NO.15956 was significantly decreased after MFGM treatment (P < 0.05). Therefore, MFGM can effectively regulate the excessive accumulation of intracellular MDA in Lactobacillus casei CGMCC NO.15956, thereby protecting the biofilm structure of Lactobacillus casei CGMCC NO.15956 under oxygen stress.

[0080] 8.2 Differentially Expressed Protein Analysis

[0081] Compared with the control group (Control), the stress group (H2O2) showed 92 significantly upregulated proteins and 206 significantly downregulated proteins, with more downregulated proteins than upregulated proteins, indicating that oxygen stress reduces protein expression in *Lactobacillus casei* CGMCC NO. 15956 to some extent. After the addition of milk fat globule membrane, compared with the stress group (H2O2), the milk fat globule membrane protection group (H2O2_M) showed 336 significantly upregulated proteins and 221 significantly downregulated proteins, with more upregulated proteins than downregulated proteins.

[0082] 8.3 Effect of milk fat globule membrane on the expression of proteins related to teichoic acid biosynthesis in Lactobacillus casei CGMCC NO.15956

[0083] Teichoic acid is an important component of Gram-positive bacteria, playing a crucial role in bacterial resistance to antibiotics, cell permeability to metabolites, cell tolerance to internal and external stresses, and bacterial-mammal cell interactions. For example... Figure 7 Proteomic analysis revealed that, compared to the control group, the expression of D-alanine activator (DltC) and transcription factor (TF) was downregulated in the stress group. Upon addition of MFGM, the expression of D-alanine activator (DltC), glycosyltransferase (LafB), lipophosphate synthase (LtaS), poly(glycerol phosphate) α-glucosyltransferase (TagE), and transcription factor (TF) was upregulated. MFGM can stimulate the biosynthesis of lipoteichoic acid and teichoic acid in *Lactobacillus casei* CGMCC NO.15956, thereby repairing cell wall damage, maintaining cell morphology, and improving tolerance to oxygen stress.

[0084] 8.4 Effect of milk fat globule membrane on surface proteins of Lactobacillus casei CGMCC NO.15956

[0085] like Figure 8In the stress group, the expression of chaperone proteins GroEL, DnaK, GroES, Hsp60, GrpE, Hsp, DnaJ and extracellular matrix proteins were significantly upregulated (P < 0.05), and the expression of glycolysis-related proteins was also upregulated, but the difference was not significant (P > 0.05). After the addition of MFGM, the expression of surface chaperone proteins Hsp, DnaK, GroEL, GroES, GrpE, HslO, elongation factors Tuf and FusA, and part-time proteins enolase (Eno), glyceraldehyde-3-phosphate dehydrogenase (GADPH), phosphoglycerate kinase (Pgk), pyruvate kinase (Pyk), triose phosphate isomerase (TipA), lactate dehydrogenase (Ldh), glutamine synthase (GS), and pyruvate carboxylase (PC) in Lactobacillus casei CGMCC NO.15956 was significantly downregulated (P < 0.05), while the expression of cell surface protein, extracellular matrix protein, and LPTTG motif protein was significantly upregulated (P < 0.05).

[0086] Chaperone proteins participate in various fundamental cellular processes, including protein folding, peptide translocation, degradation of misfolded proteins, and refolding of damaged proteins after cellular exposure to stress. Significant upregulation of molecular chaperone protein expression in the stress group facilitated protein repair in *Lactobacillus casei* CGMCC NO.15956 under oxygen stress, enhancing its oxygen stress resistance. Conversely, significant downregulation of molecular chaperone proteins in *Lactobacillus casei* CGMCC NO.15956 under MFGM-mediated oxygen stress indicated that MFGM intervention may reduce protein damage under oxygen stress in *Lactobacillus casei* CGMCC NO.15956. Glycolytic enzymes participate in glycolysis intracellularly in bacteria and, as part-time proteins, localize on the bacterial surface as adhesins, specifically binding to human intestinal epithelial cells or extracellular matrix components such as laminin, collagen, and mucin, promoting the adhesion and colonization of probiotics in the gut. The expression of glycolytic enzymes decreased after the addition of MFGM, possibly because the oxygen stress experienced by *Lactobacillus casei* CGMCC NO.15956 was reduced under the protection of the milk fat globule membrane, leading to a relative decrease in carbohydrate metabolism. Therefore, the expression of glycolytic enzymes, which are part-time proteins, on the surface of *Lactobacillus casei* CGMCC NO.15956 requires further investigation. LPXTG motif protein is a widely distributed bacterial surface protein that participates in bacterial binding to mucins, enhancing bacterial adaptability to the environment. The addition of MFGM significantly increased the expression of LPXTG motif protein, which is beneficial for improving the adaptability of *Lactobacillus casei* CGMCC NO.15956 to oxygen stress and may also enhance the adhesion ability of lactic acid bacteria. In summary, oxygen stress leads to an increase in molecular chaperone proteins in *Lactobacillus casei* CGMCC NO.15956, promotes the repair of damaged proteins, and their expression significantly decreases after MFGM protection. In addition, the milk fat globule membrane may enhance the adhesion ability of lactic acid bacteria to mucins and their adaptability to stress environments by increasing the expression of surface proteins, extracellular matrix proteins, and LPXTG motif proteins.

[0087] Example 3: Effect of MFGM on Lactobacillus casei CGMCC NO.15956

[0088] 1. Determination of adhesion of Lactobacillus casei CGMCC NO.15956 to different milk matrix components

[0089] MFGM, milk fat globules (MFG), cow's milk, and skim milk were freeze-dried into solid powders and a solution of 5 mg / mL (m / v) was prepared using sterile PBS (0.01 mol / L, pH = 7.4). 2 mL of this solution was added to a 12-well plate and fixed, then incubated overnight at 4°C. The wells were washed twice with sterile PBS to remove unbound matrix. The bacterial concentration was adjusted to 10⁻⁶. 6The cfu / mL concentration was measured, and viable bacteria were counted. After adding 2 mL of bacterial culture, the mixture was incubated at 37°C for 2 h, and the wells were washed 5 times with sterile PBS to remove unbound bacteria. 1 mL of 0.05% (v / v) Triton X-100 was added and the mixture was treated at 37°C for 20 min to release the adhered *Lactobacillus casei* CGMCC NO.15956, followed by a wash with 1 mL of sterile PBS. Viable bacteria were counted according to GB 4789.35-2016. The adhesion rate was calculated using the formula shown in Figure 2. Treatment of *Lactobacillus casei* with 0.05% (v / v) Triton X-100 at 37°C for 30 min did not affect its viability.

[0090]

[0091] In the formula: N1 represents the number of viable bacteria adhering to the substrate; N0 represents the number of viable bacteria in the bacterial solution before adhesion.

[0092] 2. Effect of milk fat globule membrane on the surface properties of Lactobacillus casei CGMCC NO.15956

[0093] 2.1 Adhesion ability

[0094] Under aseptic conditions, the adhesion ability of *Lactobacillus casei* CGMCC NO.15956 to mucin and collagen was determined in polystyrene 12-well plates. Bovine serum albumin (BSA) at a concentration of 5 mg / mL, mucin, and collagen were prepared using sterile PBS (0.1 mol / L, pH 7.4). 2 mL of each solution was added to the 12-well plate for fixation and incubated overnight at 4°C. The adhesion assay was performed using the same method as in Example 3, which determined the adhesion of *Lactobacillus casei* CGMCC NO.15956 to different milk matrix components.

[0095] 2.2 Hydrophobicity

[0096] Determination of OD of resuspended bacterial suspension 600 The value is denoted as A0. Then, 1 mL each of xylene (a nonpolar solvent), ethyl acetate (an electron donor), and chloroform (an electron acceptor) is mixed with 3 mL of the bacterial suspension. The mixture is allowed to stand at room temperature for 10 min, then mixed again and allowed to stand for 20 min before the OD value in the aqueous phase is measured. 600 Value, denoted as A1, is used to calculate the hydrophobicity of Lactobacillus casei CGMCC NO.15956:

[0097]

[0098] 2.3 Self-cohesion ability

[0099] Determination of OD of resuspended bacterial suspension 600 The value is denoted as A0. Then, 4 mL of bacterial suspension is added to each test tube, and the tubes are incubated at room temperature for 1, 2, and 3 hours respectively. Then, 1 mL of the supernatant solution is taken from each tube and its OD value is measured.600 The value was repeated 3 times, and the average value was recorded as A2. The autoagglutination ability of Lactobacillus casei CGMCC NO.15956 was calculated:

[0100]

[0101] 2.4. Observation of changes in surface properties of Lactobacillus casei CGMCC NO.15956 using infrared spectroscopy

[0102] Collect the cultured bacterial cells and resuspend them in MRS medium containing 5 mg / mL milk fat globule membrane. After standing for 3 hours, centrifuge at 8000×g for 10 minutes, wash three times with ddH2O, and then adjust the OD value of the bacterial suspension in ddH2O. 600 The bacterial powder was obtained by vacuum freeze-drying to a concentration of 0.5. Under an infrared lamp, 1-2 mg of the sample was mixed with potassium bromide (KBr) powder at a ratio of 100:1 (w:w). The mixture was thoroughly ground in a quartz grinder, pressed into tablets using a mold, and the infrared spectra of each sample were measured using a Fourier transform infrared spectrometer (FTIR spectrometer) at a detection wavelength of 4000 cm⁻¹. -1 -400cm -1 The resolution is 4cm. -1 The number of scans was 32. The results were analyzed using OMNIC software.

[0103] 2.5 Comparison of adhesion of Lactobacillus casei CGMCC NO.15956 to milk fat globule membrane before and after surface protein removal

[0104] The cultured bacterial suspension was inoculated into sterile MRS liquid medium at a 1% (v / v) inoculation rate and incubated statically at 37°C for 16 h. The cells were centrifuged at 6000×g for 10 min at 4°C, the supernatant was discarded, and the bacterial pellet was collected. The cells were washed with sterile PBS solution (0.1 mol / L, pH = 7.4), centrifuged at 6000×g for 10 min, and this process was repeated three times. The cells were then collected. A 5 mol / L LiCl solution was prepared and added to the cells. The mixture was incubated in an ice-water bath. The pH of the LiCl solution was 3, the treatment time was 30 min, and the extraction ratio was 1.5 g of wet bacterial weight: 3 mL of LiCl solution. The mixture was centrifuged at 8000×g for 20 min in a refrigerated centrifuge, and the supernatant was collected as the surface protein. The protein was dialyzed for 72 h and then freeze-dried for later use. The surface proteins of Lactobacillus casei CGMCC NO.15956 were removed by incubation with LiCl solution in ice water. After centrifugation, the bacterial cells were collected, which were the Lactobacillus casei CGMCC NO.15956 with the surface proteins removed. The adhesion test method was the same as the method for determining the adhesion of Lactobacillus casei CGMCC NO.15956 to different milk matrix components in Example 3.

[0105] 3. Data Processing and Analysis

[0106] GraphPad Prism 8.0 software was used for plotting, and SPSS 26 was used for statistical analysis. The experimental data of each group are expressed as mean ± standard deviation (Mean ± SD, n = 3). Different letters indicate significant differences (P < 0.05).

[0107] 4. Results and Analysis

[0108] 4.1 Determination of the adhesion ability of Lactobacillus casei CGMCC NO.15956 to milk fat globule membrane

[0109] from Figure 9 The results show that *Lactobacillus casei* CGMCC NO.15956 exhibits a certain degree of adhesion to the milk matrix. The adhesion effects with MFGM, milk fat globules (MFG), and cow's milk were not significantly different (P>0.05), with adhesion rates of 69.80±5.56%, 72.31±1.98%, and 66.89±6.22%, respectively. However, all were significantly higher than those of skim milk (49.77±5.60%) (P<0.05). This suggests that the adhesion effect of *Lactobacillus casei* CGMCC NO.15956 to cow's milk may be due to the effect of the milk fat globule membrane. Further investigation is needed regarding the adhesion sites of MFGM on *Lactobacillus casei* CGMCC NO.15956. Therefore, MFGM has significant potential for the production and application of *Lactobacillus casei* CGMCC NO.15956.

[0110] 4.2 Changes in the surface hydrophobicity and self-aggregation of Lactobacillus casei CGMCC NO.15956 after treatment with milk fat globule membrane

[0111] Depend on Figure 10 As shown, MFGM treatment significantly decreased the affinity of *Lactobacillus casei* CGMCC NO.15956 for xylene (P < 0.05), indicating that MFGM reduces the surface hydrophobicity of *Lactobacillus casei* CGMCC NO.15956. MFGM significantly increased the affinity of *Lactobacillus casei* CGMCC NO.15956 for chloroform and ethyl acetate, enhancing both the electrochemical and electronegative abilities of the cell surface. However, the affinity for chloroform was significantly higher than that for ethyl acetate, indicating that the electrochemical ability of the treated *Lactobacillus casei* CGMCC NO.15956 surface was higher than its electronegative ability. Therefore, MFGM made the surface of *Lactobacillus casei* CGMCC NO.15956 non-acidic and electronegative. Figure 11 As shown, after MFGM treatment, the self-aggregation ability of *Lactobacillus casei* CGMCC NO.15956 did not change significantly (P>0.05), indicating that MFGM had no significant effect on the self-aggregation ability of *Lactobacillus casei* CGMCC NO.15956. In summary, the enhanced hydrophobicity and electronegativity of the *Lactobacillus casei* CGMCC NO.15956 surface after MFGM treatment may be related to the surface adhesion of *Lactobacillus casei* CGMCC NO.15956.

[0112] 4.3 Changes in the adhesion ability of Lactobacillus casei CGMCC NO.15956 to mucin and collagen after treatment with milk fat globule membrane.

[0113] Changes in the hydrophobicity of lactic acid bacteria surfaces may affect their ability to adhere to mucins and collagen. For example... Figure 12 After treatment with MFGM, the adhesion abilities of *Lactobacillus casei* CGMCC NO.15956 to mucin and collagen decreased to 69.54±0.36% and 70.20±1.00%, respectively, representing decreases of 1.85% and 7.22% compared to before treatment. This indicates that MFGM significantly reduced the adhesion ability of the bacteria to collagen. This may be because MFGM affects the hydrophobicity of the lactic acid bacteria surface, making the surface of *Lactobacillus casei* CGMCC NO.15956 hydrophilic, thereby reducing its non-specific adhesion ability to mucin and collagen. Alternatively, MFGM adhering to the surface of *Lactobacillus casei* CGMCC NO.15956 may occupy the adhesion sites of mucin and collagen, thus reducing the adhesion ability of *Lactobacillus casei* CGMCC NO.15956 to mucin and collagen.

[0114] 4.4 Characterization of the effect of milk fat globule membrane on the surface properties of Lactobacillus casei CGMCC NO.15956

[0115] The peak values ​​in each segment of the infrared spectrum correspond to specific components, such as: (3100-2800 cm⁻¹) -1 The peaks (1800-1500 cm⁻¹) are characteristic peaks generated by the extensional vibrations of the functional groups -CH₃, -CH₂-, and -CHO, and typically correspond to the fatty acid components of various membranes. -1 This corresponds to the characteristic peaks produced by amide I and amide II of proteins and peptides; (1500-1200 cm⁻¹) -1 This corresponds to the mixed region, containing stretching vibrations of proteins, fatty acids, and phosphate compounds; (1200-900cm) -1 The peaks ()) are characteristic peaks generated by the stretching vibrations of the functional groups C-OH, COC, and CC, corresponding to the polysaccharide region and mainly reflecting the composition of carbohydrates within the cell wall. For example... Figure 13 Significant differences were observed in the infrared absorption spectra of Lactobacillus casei CGMCC NO.15956 after treatment with MFGM, particularly in the (3100-2800 cm⁻¹) range. -1 (1800-800cm) -1 The presence of peaks similar to MFGM, distinct from normal bacterial cells, in the region indicates that MFGM can act on the surface of *Lactobacillus casei* CGMCC NO.15956 and affect the characterization of bacterial membrane fatty acids and surface proteins. Notably, peaks similar to MFGM, occurring in the 1800-1200 cm⁻¹ region of *Lactobacillus casei* CGMCC NO.15956, are observed. -1 The peaks showing differences in the region were relatively concentrated and the characteristic peak of MFGM appeared, indicating that MFGM may interact with Lactobacillus casei CGMCC NO.15956 through surface proteins.

[0116] 4.5 Effect of surface proteins on the adhesion of Lactobacillus casei CGMCC NO.15956 to milk fat globule membrane

[0117] from Figure 14 It can be seen that the surface proteins of Lactobacillus casei CGMCC NO.15956 were almost completely removed after one LiCl extraction. Therefore, it is believed that LiCl treatment can eliminate the influence of surface proteins on the adhesion ability of Lactobacillus casei. Figure 15It can be seen that the adhesion ability of *Lactobacillus casei* CGMCC NO.15956 was significantly reduced after the removal of surface proteins (P<0.05), with the adhesion rate decreasing from 72.93±0.57% to 62.51±1.19%. This indicates that the surface proteins of *Lactobacillus casei* CGMCC NO.15956 participate in the adhesion process between the bacteria and MFGM and play a significant role. Since peptidoglycan and teichoic acid in lactic acid bacteria may also mediate the adhesion of lactic acid bacteria, the adhesion between *Lactobacillus casei* CGMCC NO.15956 and MFGM was still observed after the removal of surface proteins.

[0118] Example 4: Protective effect of different proteins on Lactobacillus casei CGMCC 15956

[0119] Lactobacillus casei CGMCC NO.15956 was activated according to the method in Example 1. The Lactobacillus casei CGMCC NO.15956 activated twice was mixed (bacterial concentration approximately 1 × 10⁻⁶). 9 CFU / mL was inoculated into MRS liquid medium at a ratio of 1.0% (v / v) and incubated at 37°C until the stationary phase. The cells were then collected by centrifugation at 3000×g for 10 min at 4°C, washed with pre-cooled sterile physiological saline, and collected again. The obtained bacterial cells were resuspended in MRS broth containing 5 mg / mL (m / v) BSA, zein, mucin, MFGM, and casein, with an H2O2 concentration of 8 mM. The bacterial suspension was incubated at 37°C for 3 h, and the cells were collected. The broth was serially diluted with physiological saline, and plate counting was performed according to GB 4789.35-2016. The survival rate was calculated using the following formula:

[0120]

[0121] In the formula: N1: the number of viable bacteria after protection under tolerance treatment conditions; N0: the number of viable bacteria after H2O2 tolerance treatment.

[0122] The results are as follows Figure 16 As shown, after 3 hours of treatment, there was no significant difference in the survival rate of *Lactobacillus casei* CGMCC NO.15956 between the corn protein-added group and the model group. The addition of BSA (bovine serum albumin), mucin, milk fat globule membrane, and casein all significantly improved the survival rate of *Lactobacillus casei* CGMCC NO.15956 (P < 0.05). However, it is clear that the addition of milk fat globule membrane provided the best protection for *Lactobacillus casei* CGMCC NO.15956, significantly better than other proteins (P < 0.05). Therefore, compared with other proteins, milk fat globule membrane can better protect *Lactobacillus casei* CGMCC NO.15956 under stress.

Claims

1. The application of milk fat globule membrane in improving the tolerance of Lactobacillus casei under oxygen stress, wherein the surface components of Lactobacillus casei include one or more of terpenoids, celloteichoic acid, and lipoteichoic acid, and wherein Lactobacillus casei is Lactobacillus casei CGMCC NO.15956.

2. The application according to claim 1, characterized in that, The milk fat globule membrane is derived from cow's milk.

3. The application according to claim 1, characterized in that, The applications include increasing the expression of enzymes involved in the synthesis pathway of terpenoids or upregulating the expression of glycosyltransferases in teichoic acid synthesis.

4. The application according to claim 1, characterized in that, The oxygen stress condition was a treatment at an H2O2 concentration of 8 mM for 3 hours.

5. The application according to claim 1, characterized in that, The concentration of the milk fat globule membrane is 0.5-15 mg / mL.

6. The application according to claim 5, characterized in that, The concentration of the milk fat globule membrane is 0.5-5 mg / mL.

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