Infant formula milk powder rich in breast milk oligosaccharide and having anti-inflammatory effect and preparation method of infant formula milk powder

Abstract

The invention discloses infant formula milk powder rich in breast milk oligosaccharide and having an anti-inflammatory effect and a preparation method of the infant formula milk powder rich in breast milk oligosaccharide, the infant formula milk powder rich in breast milk oligosaccharide and having the anti-inflammatory effect comprises LNB and LNT II, the content of the LNB is 2.5 g / 130g of milk powder, the content of the LNT II is 2.5 g / 130g of milk powder, or the infant formula milk powder rich in breast milk oligosaccharide and having the anti-inflammatory effect comprises the LNT II, the content of the LNT II is 5g / 130g of milk powder. The anti-inflammatory effect is relatively good, and the intestinal flora and the intestinal barrier can be regulated.

Description

Technical Field

[0001] The invention belongs to the technical field of infant formula milk powder, and particularly relates to infant formula milk powder rich in human milk oligosaccharides and having anti-inflammatory effects and a preparation method thereof. Background Art

[0002] Inflammation is an innate immune response. Normal inflammatory responses play an important role in pathogen attack, mechanical injury, and tissue healing. However, excessive inflammation can lead to various diseases, such as oxidative stress, inflammatory bowel disease, cardiovascular disease, metabolic syndrome, and cancer. Macrophages are the first line of defense against external threats in the human body and play an important role in immunity and inflammation through nonspecific recognition and phagocytosis of antigens. Lipopolysaccharide (LPS) is an endotoxin extracted from the outer membrane of Gram-negative bacteria that can cause inflammation. It binds to Toll-like receptor 4 (TLR4) and initiates the myeloid differentiation primary response protein 88 (MyD88) signaling pathway, which in turn activates macrophages through key inflammatory signaling pathways such as nuclear factor κB (NF-κB) transcription factors. Activation of NF-κB leads to the transcription of genes related to inflammatory mediators, such as nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and cytokines [such as interleukin-1β (IL-1β), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α)]. Subsequently, a large amount of nitric oxide (NO) and reactive oxygen species (ROS) are synthesized in the cells, and pro-inflammatory cytokines are released.

[0003] Human milk oligosaccharides (HMOs) are the most abundant solid components in human milk besides lactose and lipids. It is reported that nearly 200 HMOs have been discovered, mainly composed of five monosaccharide groups, including glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), fucose (Fuc) and sialic acid (Sa). Studies have shown that HMOs have a variety of physiological functions, such as preventing pathogen adhesion, regulating the composition of intestinal microbiota, improving the gastrointestinal barrier, antiviral activity, promoting immune system maturation, etc., and have potential support for the immune system, brain development and cognitive function. For example, 2'-fucosyllactose, 2-fucosyllactose, lactose-N-tetraose, lactose-N-undecasaccharide, 3'-sialyllactose and 6'-sialyllactose contribute to anti-inflammatory responses. It has been reported that a mixture of 2'-fucosyllactose and 6'-sialyllactose can exert anti-inflammatory effects by regulating lipopolysaccharide (LPS)-induced intestinal inflammation in suckling mice and the production of microbiota and metabolites in HT-29 epithelial cells. Another study found that 2'-fucosyllactose and 3-fucosyllactose prevent colitis mainly by enhancing intestinal barrier function and promoting anti-inflammatory responses. A variety of HMOs have been approved as generally recognized as safe (GRAS) substances by the US Food and Drug Administration (FDA), including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosuronidase (DiFL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and lacto-N-triose (LNT II), 6'-sialyllactose (6'-SL).

[0004] Lactose-N-disaccharide (Galβ1-3GlcNAc, LNB) and lactose-N-trisaccharide (GlcNAcβ1-3Galβ1-4Glc, LNTII) are classified as HMOs and can currently be obtained by fermentation or enzymatic synthesis. It is reported that LNB can promote the growth of bifidobacteria, regulate immune responses, and improve allergies. LNT II has antibacterial activity, can regulate immune function, improve lactose intolerance, and promote intestinal microbial colonization. Currently, both LNB and LNT II can be obtained by microbial fermentation, chemical synthesis and other methods. Since the composition and nutritional design of infant formula should mimic breast milk as much as possible to provide complete nutrition for infants, LNB and LNT II may be good candidates if applied to infant formula, but the details need further exploration. Summary of the invention

[0005] The first technical problem to be solved by the present application is to provide an infant formula milk powder rich in human milk oligosaccharides and having anti-inflammatory effects.

[0006] The second technical problem to be solved by the present application is to provide a method for preparing the above-mentioned infant formula milk powder.

[0007] The technical solution adopted by the present invention to solve the above-mentioned first technical problem is: an infant formula milk powder rich in human milk oligosaccharides and having anti-inflammatory effects, characterized in that: it comprises LNB and LNT II, ​​with a content of 2.5g LNB / 130g milk powder and 2.5g LNT II / 130g milk powder;

[0008] Alternatively, LNT II is included, with the content of 5g LNT II / 130g milk powder.

[0009] In order to facilitate storage, the moisture content of the infant formula milk powder is less than 4% and meets national standards.

[0010] To reduce lipid oxidation, the surface oil mass of the infant formula is less than 6.5%.

[0011] The technical solution adopted by the present invention to solve the above second technical problem is: a method for preparing the above infant formula milk powder rich in human milk oligosaccharides and having anti-inflammatory effects, characterized in that it comprises the following steps:

[0012] Mix whey protein, lactose and pure water and stir until dissolved; then let stand for hydration; then add melted oil phase material and continue stirring; then shear, homogenize and spray dry;

[0013] Add vitamins and minerals through dry mixing;

[0014] The LNB and LNT II corresponding to the infant formula milk powder, or the corresponding LNT II, ​​are added by dry mixing.

[0015] Since LNB and LNT II are thermally unstable and their biological activity will be destroyed under high temperature conditions, LNB and LNT II are added by dry mixing after spray drying to maintain their biological activity.

[0016] Compared with the prior art, the advantages of the present invention are: by adding LNB and LNT II to milk powder, or adding LNT II and setting a suitable content, the milk powder is 1. safe and non-cytotoxic, and is beneficial to promoting the phagocytosis of macrophages; 2. has an anti-inflammatory effect and is beneficial to improving immunity, and after being digested by intestinal digestive juice, it still has an anti-inflammatory effect; 3. is beneficial to regulating intestinal flora and regulating intestinal barrier; 4. has good solubility and the corresponding emulsion is stable.

[0017] Regarding the preparation method, adding LNB and LNT II or adding LNT II at an appropriate step is beneficial to maintaining the biological activities of LNB and LNT II. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1-1 Figure 2 shows the effects of GOS, LNB, LNT II, ​​GOS+LNB and their combination on (A) RAW264.7 macrophage viability (B) NO production (C) cell phagocytosis (sample incubation for 12 h) (D) cell phagocytosis (sample incubation for 24 h). Three independent experiments were performed in each group, and the bar graphs and error graphs represent the mean ± standard deviation, respectively.

[0019] Figure 1-2 The effects of GOS, LNB, LNT II, ​​GOS+LNB and their combination on (A) NO production and (B) iNOS gene expression level (C) ROS production and (D) COX-2 gene expression level (E) fluorescence images (magnification × 100). Three independent experiments were performed in each group, and the bar graphs and error graphs represent the mean ± standard deviation, respectively.

[0020] Figure 1-3 The effect of GOS, LNB, LNT II, ​​GOS+LNB, GOS+LNT II and LNB+LNT II on the mRNA expression of RAW264.7 cells. (A) Heat map of all mRNA expression in this study, the color is represented by log10 (relative gene expression); (B) IL-1β; (C) IL-6; (D) TNF-α; (E) TNF-β; (F) TGFBR1; (G) TLR4; (H) MyD88; (I) NF-κB. Three independent experiments were performed in each group, and the bar graphs and error graphs represent the mean ± standard deviation, respectively.

[0021] Figure 2-1 The graph shows the effects of GOS, LNB, LNT II, ​​GOS+LNB and their combination on (A) body weight, (B) food intake and (C) organ indexes of SD rats.

[0022] Figure 2-2 This is a graph showing the effects of GOS, LNB, LNT II, ​​GOS+LNB and their combination on the plasma biochemical indexes of SD rats.

[0023] Figure 2-3 Figure 2 shows the content of fecal short-chain fatty acids of GOS, LNB, LNT II, ​​GOS+LNB and their combination (A) on day 24. The changing trends of short-chain fatty acids from day 0, 8, 15 and 24 of (B) Control, (C) GOS, (D) LNB, (E) LNT II, ​​(F) GOS+LNB, (G) GOS+LNT II and (H) LNB+LNTII.

[0024] Figure 2-4Graph showing the effects of GOS, LNB, LNT II, ​​GOS+LNB and their combination on the relative expression levels of (A) lipid absorption and transport related genes and (B) intestinal tight junction protein genes.

[0025] Figure 2-5 Graph showing the effects of GOS, LNB, LNT II and their combination on (A) jejunal villus length (B) jejunal crypt depth (C) jejunal villus length to crypt depth ratio (D) and colonic crypt depth in SD rats.

[0026] Figure 2-6 This is a graph showing the effects of GOS, LNB, LNT II and their combination on the intestinal flora of SD rats (A) OTU (B) Xiangnan index (C) Chao1 index (D) Simpson index (E) ACE index.

[0027] Figure 2-7 Figure 3 Non-metric multidimensional scaling (NMDS) plots showing the microbiome differences of (A) GOS, LNB and LNT II groups (B) GOS+LNB, GOS+LNT II and LNB+LNT II groups (C) GOS, LNB and GOS+LNB groups (D) GOS, LNT II and GOS+LNT II groups (E) LNB, LNT II and LNB+LNT II groups at the OTU level based on binary-jaccard metric distance.

[0028] Figure 2-8 Graph showing the effects of GOS, LNB, LNT II and their combination on the composition of the intestinal flora of SD rats at the (A) phylum level and (B) genus level.

[0029] Figure 2-9 This is the linear discriminant effect size (LEfSe) analysis graph, and the LDA value was set to >4, P < 0.05.

[0030] Figure 2-10 A correlation network diagram was drawn for the first 80 genera in this study. In the figure, the spheres represent species, the size of the spheres represents abundance, and the color of the spheres represents the different phylum levels to which the species belong; the lines represent the correlation between two species, the thickness of the line represents the strength of the correlation, and the color of the line represents positive correlation (red) or negative correlation (green).

[0031] Figure 3-1 (A) Emulsion particle size potential diagram; (B) Emulsion particle size distribution diagram; (C) Reconstituted emulsion particle size potential diagram; (D) Reconstituted emulsion particle size distribution diagram.

[0032] Figure 3-2 (A) milk powder glass transition temperature diagram; (B) milk powder crystallization diagram; (C) milk powder morphology diagram;

[0033] Figure 3-3 This is a graph showing the effect of the reconstituted emulsion digestion products on the anti-inflammatory activity of Caco2. DETAILED DESCRIPTION

[0034] Example 1 Study on the anti-inflammatory activity of GOS, LNB, LNT II and their combination in vitro

[0035] In order to screen the best oligosaccharide or combination with anti-inflammatory effect through in vitro experiments, the LPS-induced Raw 264.7 macrophage inflammation model was used to explore whether different components GOS, LNB, LNT II, ​​GOS+LNB (w / w=1 / 1), GOS+LNT II (w / w=1 / 1), and LNB+LNT II (w / w=1 / 1) have anti-inflammatory activity, and to clarify the pathways that may be involved. After pretreatment with GOS, LNB, LNT II and their complexes in RAW264.7 cells, the content of NO, the content of ROS, phagocytosis, and the gene expression of enzymes such as iNOS and COX-2, the expression of inflammatory factor genes such as IL-6, IL-1β, TNF-α, TNF-β, and TGFBR1 in LPS-induced inflammation were determined. The expression of genes related to inflammatory pathways such as TLR4, MyDD8, and NF-κB was also detected.

[0036] 1.1 Experimental methods

[0037] 1.1.1 Cell recovery

[0038] Complete medium is composed of DMEM, 10% fetal bovine serum, and 1% penicillin-streptomycin solution. Take out the frozen macrophage cell line from -80℃, thaw the cell fluid in a 37℃ water bath, transfer the thawed macrophages in the cryopreservation tube to a 15mL centrifuge tube, centrifuge (1200rpm, 3min) to remove the old medium, add fresh medium, repeatedly blow to resuspend the cells, transfer to a culture dish, place in a cell culture incubator (37℃, 5% CO2) for incubation, and replace with fresh complete medium after 24h for continued culture. The revived cells need to be passaged three times, and the cells are used for subsequent experiments after stable growth.

[0039] 1.1.2 Cell passaging

[0040] In a 60mm cell culture dish, pour out the culture medium, add 1mL PBS to wash twice, add 2mL complete culture medium and blow evenly to make the cells fall off the wall. Take 500μL, transfer to a new culture dish, add 5mL culture medium, blow evenly, suck out the bubbles and throw them away. The culture medium is DMEM + double antibody, preheat before use; passaging can be one to three or one to four, depending on the cell density.

[0041] 1.1.3 Cell cryopreservation

[0042] The cells were washed twice with PBS, and the cells were blown up with complete culture medium and transferred into a 15 mL centrifuge tube. The tube was centrifuged (1200 rpm, 3 min), the culture medium was discarded, 1 mL of freezing solution was added, the tube was blown evenly and transferred into a freezing tube, and the tube was quickly placed in a -80 refrigerator for freezing.

[0043] 1.1.4 Cytotoxicity assay

[0044] GOS was purchased from Guangdong Xinjinshan (Yunfu) Biotechnology Co., Ltd., China. LNB and LNT II were purchased from Henglu Biotechnology Co., Ltd. (Qingdao, China).

[0045] GOS group, LNB group, LNT II group, GOS+LNB group, GOS+LNT II group, and LNB+LNT II group were set up, and the concentrations of each group were respectively configured to be 25, 50, 100, 250, 500, 1000, 1500, and 2000 μg / mL.

[0046] 40 mg GOS, 40 mg LNB, and 40 mg LNT II were weighed separately and dissolved in 4 mL complete culture medium to prepare a mother solution with an original concentration of 10 mg / mL. The mother solution was sterilized by passing through a 0.22 μm membrane and diluted with complete culture medium to 25-2000 μg / mL.

[0047] The specific dilution method is:

[0048] GOS: 2000 μg / mL: 100 μL GOS stock solution + 400 μL complete culture medium;

[0049] LNB: 2000 μg / mL: 100 μL LNB stock solution + 400 μL complete culture medium;

[0050] LNT II: 2000 μg / mL: 100 μL LNT II stock solution + 400 μL complete culture medium;

[0051] GOS+LNB: 2000μg / mL: 50μL GOS stock solution + 50μL LNB stock solution + 400μL complete culture medium;

[0052] GOS+LNT II: 2000 μg / mL: 50 μL GOS stock solution + 50 μL LNT II stock solution + 400 μL complete culture medium;

[0053] LNB+LNT II: 2000 μg / mL: 50 μL LNB stock solution + 50 μL LNT II stock solution + 400 μL complete culture medium.

[0054] The cytotoxic effects of GOS, LNB, LNT II and their combination on the viability of RAW264.7 macrophages were determined using CCK8. The cell viability of the control group was 100%. Briefly, RAW264.7 macrophages were cultured at 5.5×10 5 The cells were cultured at a density of 100 / mL in a 96-well culture plate and cultured overnight.

[0055] Discard the old culture medium, add 100 μL of 25, 50, 100, 250, 500, 1000, 1500, 2000 μg / mL of GOS group, LNB group, LNT II group, GOS+LNB group, GOS+LNT II group, LNB+LNT II group samples (samples are dissolved and diluted with complete culture medium), and continue to culture for 24 hours. The complete culture medium without any sample is used as the blank control group, and 1 μg / mL LPS is used as the positive control. After the incubation, add 10 μL CCK-8 to each well, incubate for 25-30 minutes, and measure the absorbance of each well at a wavelength of 450 nm by an enzyme reader. The effect of each sample on the activity of macrophages is shown by the relative cell viability. (If the culture medium turns yellow, it is necessary to replace the new culture medium and continue to add CCK8)

[0056] Relative cell viability = (OD2-OD0) / (OD1-OD0)

[0057] Where: OD0——absorbance of blank cell-free control;

[0058] OD1——absorbance of blank cell control;

[0059] OD2 – absorbance of each sample.

[0060] 1.1.5 NO determination

[0061] Stably cultured RAW264.7 macrophages were cultured at 5.5×10 5 Cells were inoculated into 96-well plates at a density of 1 μg / mL and cultured in complete medium for 24 h. The complete medium was aspirated and treated with samples of different concentrations (cytotoxic concentration) for 12 h (the samples were dissolved and diluted with DMEM). LPS (1 μg / mL) was added and incubated with the samples for 12 h, and then the cell supernatant was collected. NO production was determined using a NO detection kit according to the manufacturer's instructions, and the OD value was measured at a wavelength of 540 nm.

[0062] 1.1.6 Determination of ROS

[0063] The cells were cultured in 96-well plates (5.5 × 10 5Cells were pretreated with 100, 500 μg / mL oligosaccharides for 12 h after cell attachment, and LPS was added for another 12 h. Cells were then exposed to 50 μL DCFH-DA for 30 min and then rinsed three times with PBS. Fluorescence was measured at 480 / 530 nm using a microplate reader.

[0064] 1.1.7 Phagocytosis assay

[0065] RAW264.7 macrophages were cultured at 5.5 × 10 5 The concentration of cells / mL was inoculated into 96-well culture plates, 100 μL per well, and the cell culture medium was replaced after 24 hours. The experimental group [adding oligosaccharides (100, 250, 500 μg / mL)], LPS group (1 μg / mL LPS), and blank control group were set up with 6 duplicate wells. After 12 hours, the supernatant was discarded, washed twice with PBS, 100 μL neutral red dye (1 mg / mL) was added, and placed in a 37°C, 5% CO2 incubator for 1 hour, then taken out, washed twice with PBS, and 150 μL cell lysate was added to each well. At the same time, 150 μL cell lysate was used as a blank control. Continue to be placed in a 37°C, 5% CO2 incubator for culture, and the absorbance value (OD value) was detected at 540nm on an ELISA reader after 4 hours. The cell phagocytosis index was calculated according to the following formula: phagocytosis index = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. Preparation of cell lysis solution: aspirate 0.9 mL of glacial acetic acid and dilute to 15 mL with ultrapure water to prepare a 1 mol / L glacial acetic acid solution, and mix the prepared glacial acetic acid solution with anhydrous ethanol in a ratio of 1:1 to prepare a cell lysis solution.

[0066] 1.1.8 QPCR

[0067] RAW264.7 macrophages were counted at 5.5×10 5 The cells were inoculated at a density of 1 μg / mL in a 24-well plate, with 1 mL per well. The cells were cultured with complete medium for 24 hours, the complete medium was aspirated, and samples of different concentrations (100, 500) were added for 12 hours (the samples were dissolved and diluted with DMEM), LPS (1 μg / mL) was added and incubated with the samples for 12 hours, and then the cells were collected.

[0068] Do the following:

[0069] A. Wash the cells twice with PBS, lyse the cells with lysis buffer, and extract RNA according to the reagent instructions;

[0070] B. According to the instructions of the reverse transcription kit, transcribe the total RNA into cDNA;

[0071] C. Using cDNA as template, the expression levels of target genes IL-1β, IL-6, TNF-α, TNF-β, TGFBR1, COX-2, and iNOS mRNA were detected. Six parallel tests were set up for each sample test group, including

[0072] HPRT was an internal reference gene.

[0073] The primers were synthesized by Youkang Biotechnology Co., Ltd. and the sequences are shown in Table 1-1 below.

[0074] Table 1-1 Primer sequences used in cell experiments

[0075]

[0076] 1.1.9 Data Analysis

[0077] Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS software (version 25.0), using one-way ANOVA test and Duncan multiple comparisons. Significant differences were set at P < 0.05, and different letters represent significant differences between groups. One-way ANOVA test and Turkey post hoc test were used to compare whether the sample treatment group was significantly different from the control group or LPS group. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. Images were drawn using software such as Origin 2021 and Adobe Illustrator 2020.

[0078] 1.2 Experimental Results

[0079] 1.2.1 Effects of GOS, LNB, LNT II and their combination on cell viability and phagocytic ability

[0080] The cells were treated with oligosaccharides (GOS, LNB and LNT II) or their combinations (GOS+LNB, GOS+LNT II, ​​LNB+LNT II) at a concentration of 25-2000 μg / mL for 24 hours. Figure 1-1 As shown in A, the three samples and their combinations had no toxic effects on macrophages, and with the increase of concentration, there was a trend of promoting cell proliferation, which confirmed that GOS, LNB, LNTII and their combination were biosafe within the concentration range of 25-2000 μg / mL and could be used to explore the anti-inflammatory effects on RAW 264.7 macrophages.

[0081] We investigated whether these oligosaccharides and their combinations could effectively inhibit NO within a concentration range that was not cytotoxic (25–2000 μg / mL). Figure 1-1As shown in B, GOS, LNB, LNT II and their combination can significantly inhibit LPS-induced NO release, and most of them have the best effect at 500 μg / mL. Therefore, we screened 500 μg / mL as the high-dose group and 100 μg / mL as the low-dose group for subsequent studies.

[0082] Macrophages are important immune cells in the human body, and their phagocytic activity is one of the ways they exert their immune function. In previous studies, some HMOs have been shown to resist infection by external pathogens by enhancing the phagocytic ability of macrophages. For example, 6'-sialyllactose increases the inflammatory response and phagocytic response to Staphylococcus aureus infection. In another study, it was reported that sialyllactose can effectively promote the phagocytosis of Pseudomonas aeruginosa K by human THP-1 macrophage-like cells, thereby preventing bacterial infection. Figure 1-1 As shown in C, after 12 h of treatment with GOS, LNB, LNT II and their combination, the phagocytosis of the 100 μg / mL LNT II group was enhanced. When the cells were treated for 24 h, the phagocytosis of all groups (except the LPS group and the control group) increased compared with that at 12 h, especially in the 500 μg / mL GOS group ( Figure 1-1 D) The results of the study indicate that GOS, LNB, LNT II and their mixture may be effective ingredients with immunomodulatory activity.

[0083] 1.2.2 Effects of GOS, LNB, LNT II, ​​and their combination on NO generation, ROS release, iNOS mRNA expression, and COX-2 expression

[0084] like Figure 1-2 As shown in A, LPS significantly increased NO production in RAW 264.7 macrophages compared with the control group. However, oligosaccharide treatment alleviated this increase. This may be because LPS can upregulate the expression of iNOS in cells and promote the production and release of NO, while oligosaccharides can inhibit these biochemical processes, thereby exerting an anti-inflammatory effect. Therefore, we studied the expression level of iNOS and found that the expression of iNOS and the content of NO showed similar trends ( Figure 1-2B). GOS, LNB, and LNT II significantly inhibited the increase in LPS-induced iNOS mRNA expression and NO release in a dose-dependent manner (500 μg / mL was more effective than 100 μg / mL), with 500 μg / mL LNT II having the most significant inhibitory effect. Among the oligosaccharide combination groups, LNB+LNT II had the best inhibitory effect on NO and iNOS. These results indicate that GOS, LNB, LNT II, ​​and their combination can downregulate iNOS expression, inhibit NO production, and exert anti-inflammatory effects, especially under the conditions of 500 μg / mL LNT II and LNB+LNT II.

[0085] ROS are key mediators that regulate various physiological functions, including immune responses. However, excessive ROS can produce toxic effects, exacerbate inflammatory responses, and induce tissue damage. Therefore, we investigated whether individual oligosaccharides or their combinations can scavenge ROS, restore ROS balance, limit inflammatory responses, and protect the host from damage under LPS-induced proinflammatory conditions. We also detected the mRNA expression level of COX-2, a proinflammatory enzyme and a regulatory gene for ROS. After LPS stimulation, the fluorescence intensity increased significantly ( Figure 1-2 C and E). In contrast, after treatment with GOS, LNB, LNT II, ​​or their combination (100 and 500 μg / mL), the fluorescence intensity gradually decreased. GOS and LNT II (500 μg / mL) had the lowest fluorescence intensity, and the fluorescence microscopy images also reflected a similar ROS reduction trend ( Figure 1-2 E). Figure 1-2 As shown in C and D, high doses of GOS, LNB, LNT II and their combination can effectively reduce the expression level of COX-2. The powerful ability of GOS, LNB, LNT II and their compound preparations to reduce ROS in LPS-stimulated RAW 264.7 macrophages may be related to their inhibition of the expression of proinflammatory enzymes such as COX-2.

[0086] 1.2.3 Effects of GOS, LNB, LNT II and their combination on the expression of pro-inflammatory, anti-inflammatory cytokines and TLR4 / MyD88 / NF-κB signaling pathway-related mRNA in LPS-induced RAW264.7 macrophages

[0087] IL-1β, IL-6, TNF-α, and TNF-β are important proinflammatory cytokines, while TGFBR1 is an important anti-inflammatory factor in the immune and inflammatory systems. IL-6, in particular, plays an important role in experimentally induced autoimmune diseases, and elevated IL-6 levels have also been observed in chronic inflammation. TNF-α was originally considered to be cytotoxic to tumor cells and is a potent mediator of inflammation and many normal physiological functions. The effects of GOS, LNB, LNT II, ​​and their combination on the mRNA expression of these genes were evaluated using RT-qPCR. Figure 1-3 As shown in the figure, LPS significantly increased the levels of all four pro-inflammatory cytokines and decreased the levels of anti-inflammatory factors compared with the control group (P<0.05). Specifically, after LPS treatment (1 μg / mL), the expression levels of IL-1β, IL-6, TNF-α, TNF-β, and TGFBR1 were approximately 1400, 60, 10, 120, and 0.6 times that of the control group, respectively.

[0088] Compared with the LPS group, GOS, LNB, LNT II and their combination significantly inhibited the secretion of proinflammatory cytokines and increased the expression of anti-inflammatory cytokines. Among the single oligosaccharides, LNT II (500 μg / mL) showed obvious advantages over GOS in inhibiting the mRNA expression levels of IL-1β, IL-6, TNF-α and TNF-β. Among the oligosaccharide combinations, LNB+LNT II (500 μg / mL) showed the best inhibitory effect on IL-1β, IL-6 and TNF-β, and the most obvious promoting effect on TGFBR1. These findings suggest that LNB and LNB+LNT II can stimulate strong anti-inflammatory activity by reducing the expression of proinflammatory cytokines and increasing the expression of anti-inflammatory cytokines. It has been reported that HMOs with different molecular weights, structures and polymerization degrees produce different biological properties. For example, 2'-Fucosyllactose can competitively bind to mucosal surface receptors, thereby inhibiting the binding and infection of intestinal pathogens. In this study, LNT II and LNB+LNT II showed superior anti-inflammatory ability compared with other groups. We speculate that the superior anti-inflammatory effect of LNT II over GOS may be because LNT II contains N-acetylglucosamine, which can effectively reduce the secretion of pro-inflammatory cytokines stimulated by viral infection. Compared with LNB, LNT II has a stronger anti-inflammatory effect, which may be due to its larger molecular weight and higher degree of polymerization. Oligosaccharides with different structures have different activation effects on macrophages, and the specific biological properties of these oligosaccharides need further study.

[0089] To gain insight into the molecular mechanisms underlying the immunomodulatory effects of GOS, LNB, LNT II, ​​and their combination, we investigated several signaling pathways that are closely associated with cellular immune processes, particularly the TLR4, MyD88, and NF-κB pathways. Our results showed that both LNT II and the combination of LNB and LNT II could effectively reduce LPS-induced overexpression of TLR4, MyD88, and NF-κB in a dose-dependent manner ( Figure 1-3 GI). Therefore, we preliminarily believe that the regulation of TLR4 / MyD88 / NF-κB signaling pathway by LNT II and LNB+LNT II is a mechanism to alleviate inflammatory symptoms. This effect may be related to LNB and LNTII rather than GOS, which have N-acetylglucosamine terminals and may interact with the N-terminal extracellular domain of TLR.

[0090] 1.3 Summary

[0091] In summary, through the above cell experiments, we found that LNT II and LNB+LNT II groups had the best in vitro anti-inflammatory effects. LNT II and LNB+LNT II can effectively inhibit NO release, ROS levels, pro-inflammatory enzymes and cytokine mRNA expression, while enhancing phagocytosis and anti-inflammatory factors. At the same time, LNT II can promote the production of short-chain fatty acids, significantly increase the expression of colonic lipid absorption and transport genes, promote the development of jejunal villi, and improve the ability to absorb substances. The LNB+LNT II group can significantly increase the expression of genes related to tight junction proteins and intestinal epithelial cell proliferation and differentiation, and plays an important role in maintaining intestinal homeostasis and intestinal barrier. The specific comparative analysis is as follows.

[0092] When attacked by LPS, macrophages are activated and release various proinflammatory factors and cytokines, and excessive release can lead to extensive tissue damage and pathological changes. Our results showed that 500 μg / mL of LNT II and LNB+LNTII used in the study significantly inhibited the secretion of NO and ROS induced by LPS, inhibited the expression of proinflammatory enzymes iNOS and COX-2, inhibited the mRNA expression levels of proinflammatory factors IL-1β, IL-6, TNF-α and TNF-β, promoted the mRNA expression level of anti-inflammatory factor TGFBR1, and increased the phagocytic activity of macrophages.

[0093] In addition, the results of cytotoxicity experiments showed that the LPS and oligosaccharide doses used in this study had no significant cytotoxic effects, indicating that the anti-inflammatory effect of oligosaccharides was not the result of poor cell viability. However, which targets may be affected by GOS, LNB, LNT II and their combinations and play an important role in the anti-inflammatory effect remains to be studied.

[0094] We found that LNT II and LNB+LNT II attenuated the LPS-induced mRNA expression levels of TLR4, MyD88, and NF-κB, indicating that LNT II and LNB+LNT II inhibited the TLR4 / MyD88 / NF-κB signaling pathway, thereby inhibiting the LPS-induced release of NO and ROS. Studies have reported that different molecular weights, structures, and polymerization degrees of HMOs will produce different biological properties. For example, sialylated HMOs can stimulate T cell maturation and promote the transformation of T cell responses into more balanced Th1 / Th2 cytokine production and low-level immunity. Some fucosylated HMOs can stimulate macrophage activity. Therefore, we speculate that the anti-inflammatory effect of LNB and LNT II is better than that of GOS because LNT II contains N-acetylglucosamine. The anti-inflammatory effect of LNT II is better than that of LNB, which may be due to the larger molecular weight and polymerization degree of LNT II. Oligosaccharides with different structures show different activation effects on macrophages, and the specific biological characteristics still need further study.

[0095] Example 2. Regulation of early intestinal flora and intestinal barrier by GOS, LNB, LNT II and their combination

[0096] In order to screen out the best oligosaccharides or combinations for promoting intestinal tight junction proteins and promoting the growth of beneficial bacteria through in vivo experiments, different experimental groups GOS, LNB, LNT II, ​​GOS+LNB (m / m=1 / 1), GOS+LNT II (m / m=1 / 1), LNB+LNT II (m / m=1 / 1) were set up, as follows.

[0097] 2.1 Experimental methods

[0098] 2.1.1 Animal Experiment Design

[0099] After adaptive feeding, 70 21-day-old SD rats were randomly divided into 7 groups according to the formula, with 10 rats in each group and 3 to 4 rats per cage. GOS was purchased from Guangdong Xinjinshan (Yunfu) Biotechnology Co., Ltd., China. LNB and LNT II were from Henglu Biotechnology Co., Ltd. (Qingdao, China).

[0100] Table 2-1 Animal experiment grouping scheme

[0101]

[0102] The diets of the experimental animals are shown in Table 1.

[0103] Based on 5g oligosaccharides per liter of infant formula (the content of oligosaccharides in breast milk is 5-25g / L), the average intake of infants is about 150mL formula milk / kg BW / day, and the content of oligosaccharides ingested is 750mg / kg BW. Therefore, the animal experiment design selected 750mg / kg BW as the oral dose. The ethics number of the animal experiment plan is: 12202, which was approved by the Ethics Committee of Ningbo University. After the rats arrived at the animal experiment center, they were adaptively fed for 3 days. According to the divided groups, they were gavaged with the corresponding oligosaccharides or oligosaccharide combinations at a dose of 750mg / kg BW and a volume of 1mL for 24 consecutive days. Feces were collected before and every 1 week after gavage for microbial composition and short-chain fatty acid analysis, and intestinal flora and metabolic differences were compared. After feeding, blood was collected from the eyeballs, plasma was collected, and plasma components were analyzed. The colon and jejunum were collected for pathological sections and analysis of intestinal tight junction proteins and lipid absorption genes.

[0104] 2.1.2 Determination of rat body weight growth rate, average food consumption and organ index

[0105] Starting from the day the rats were separated into cages, the body weight of each rat and the remaining amount and amount of food added in each cage were weighed every 3 days, and the average food consumption and organ index were calculated according to the calculation formula:

[0106]

[0107] 2.1.3 Plasma biochemical analysis

[0108] The whole blood collected in the experiment was placed in a 1.5 mL anticoagulant tube, centrifuged at 5000 rpm for 3 min, and the supernatant (plasma) was placed in a new anticoagulant tube. Six rat plasma samples were selected from each group, and the rat plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), glucose (GLU), urea (UREA), creatinine (CREA), triglyceride (TG), total cholesterol (Cholesterol, CHO), high density lipoprotein (HDL), low density lipoprotein (LDL), uric acid (UA) and other indicators were analyzed using an automatic biochemical analyzer.

[0109] 2.1.4 Histopathological observation of jejunum and colon

[0110] About 1 cm of rat colon tissue was taken and fixed in 10% neutral formalin fixative (6 rat tissue samples were selected in each group), and sent to Hangzhou Hawk Biotechnology Co., Ltd. for paraffin embedding, sectioning, hematoxylin and eosin staining and digital slice scanner observation.

[0111] 2.1.5 Analysis of fecal short-chain fatty acids

[0112] Feces were collected on days 0, 7, 14, and 24 of gavage, frozen quickly, and stored at -80°C. Approximately 700 mg of fecal samples were diluted in 3 mL of saturated sodium chloride, allowed to stand for 30 min, and then homogenized until no obvious lumps were present. Then, it was placed on ice and acidified with 120 μL of sulfuric acid (concentrated sulfuric acid: water = 1:10, v:v). After uniform mixing, 4.5 mL of n-hexane was added to extract short-chain fatty acids, followed by vortexing for 30 s and standing for 30 min. The samples were then centrifuged (4°C, 10,000 rpm, 20 min). The supernatant was transferred to a 10 mL centrifuge tube containing 1 g of anhydrous sodium sulfate and centrifuged again under the same conditions to remove residual water. After centrifugation, the supernatant was transferred to a gas phase injection bottle and stored at -20°C or -80°C until gas chromatography analysis. Short-chain fatty acids were determined using a gas chromatograph (Shimadzu, Japan, GC-2030AF, 230V) and a standard curve was drawn. The short-chain fatty acid content in each gram of fresh feces was calculated based on the standard curve. The calculation formula is as follows:

[0113]

[0114] Wherein, B is the content of short-chain fatty acids in the stool sample (mg / g); A is the peak area of ​​the sample into the standard curve. The calculated result (μg / mL); 4.5 is the volume of n-hexane (mL); m is the mass of stool (g); 1000 is the conversion factor.

[0115] 2.1.6 Analysis of intestinal flora structure

[0116] Feces of 6 rats in each group were taken for intestinal flora structure analysis. After extracting the total DNA of the fecal samples, a certain proportion of quantitative reference sequences (spike-in DNA) were added. Specific primers with barcodes were synthesized according to the full-length primer sequences, and the products were amplified by PCR, purified, quantified and homogenized to form sequencing libraries (SMRT Bell). The constructed libraries were first quality controlled, and then qualified libraries were sequenced using PacBio Sequel. Alpha diversity was calculated using Chao1, Ace, Shannon, Simpson, Coverage, and PD_whole_tree using QIIME2 2020.6 software. Beta diversity was calculated using non-metric multidimensional scaling (NMDS) on the Bray-Curtis distance matrix.

[0117] 2.1.7 Colon RT-qPCR

[0118] Total RNA was extracted from colon tissue using an RNA extraction kit (TransGen, China) according to the manufacturer's instructions. The concentration and purity of the extracted RNA were then measured using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA). RNA was reverse transcribed into cDNA using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2 (AccurateBiology, China). The levels of ZO-1, ZO-2, Cdx1, Cdx2, Muc2, GPR43, GPR109a, MCT, SMCT, and FABP4 were determined using a real-time quantitative PCR system (Thermo Fisher Scientific, USA) and PerfectStart Green qPCR SuperMix Kit (TransGen, China). The primer sequences used are shown in Table 2-2, and the gene expression levels were measured using 2 -ΔΔCt Method calculation.

[0119] Table 2-2 Primer sequences used in animal experiments

[0120]

[0121] 2.1.8 Data Analysis

[0122] Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS software (version 25.0), using one-way ANOVA test and Duncan multiple comparisons, with significant differences set at P < 0.05. Images were drawn using software such as Origin 2021 and Adobe Illustrator 2020.

[0123] 2.2 Experimental Results and Conclusions

[0124] 2.2.1 Effects of GOS, LNB, LNT II and their combination on body weight growth rate, food consumption and organ indexes in rats

[0125] Figure 2-1 The changes in rat body weight (A), average food consumption (B) and organ index (C) under 7 different intervention conditions are shown. After random cage feeding, the body weight and food consumption of rats were proportional to time, and there was no significant difference between the groups, indicating that the intervention of GOS, LNB, LNT II and their combination would not affect the normal growth of rats, or produce adverse effects such as obesity and weight loss. There was no significant difference in kidney and spleen indexes between the groups, indicating that GOS, LNB, LNT II and their combination (all six groups) would not cause adverse effects such as edema, hyperplasia or organ atrophy of organs such as kidney and spleen. This shows that prebiotic oligosaccharides may only improve the health status of animals, not growth performance. Hepatocytes take up SCFAs to promote gluconeogenesis, and 50-70% of acetic acid is absorbed in the liver. The liver index of the GOS, LNT II, ​​and LNB+LNT II groups increased significantly, indicating that the rat liver metabolism is more vigorous and the function is more complete.

[0126] 2.2.2 Effects of GOS, LNB, LNT II and their combination on rat plasma biochemistry

[0127] Twelve plasma biochemical indices of rats were measured ( Figure 2-2 ). ALT and AST play an important role in transamination, protein synthesis and catabolism. The increase in the activity of these enzymes can promote the metabolism of amino acids in plasma. TG and GLU can reflect the body's lipid metabolism and sugar metabolism respectively, and the TP content can reflect the body's protein metabolism. TG and TP (except LNB+LNT II) in the oligosaccharide group increased significantly, which may be due to the body's improved utilization efficiency of lipids and proteins; GLU content increased significantly (except GOS), indicating that the body improved sugar metabolism. The results showed that the intervention of GOS, LNB, LNT II and their combination (all six groups) can maintain blood sugar levels, promote cholesterol transport to the liver, and will not cause a decline in liver and kidney function in rats.

[0128] 2.2.3 Effects of GOS, LNB, LNT II and their combination on short-chain fatty acids in rat feces

[0129] Short-chain fatty acids (SCFAs) are products of intestinal microbiota fermentation of dietary polysaccharides. They are fatty acids with less than 6 carbon atoms, including acetic acid (C2), propionic acid (C3), and butyric acid (C4). The most abundant phyla, Bacteroidetes and Firmicutes, are both involved in polysaccharide degradation, so SCFAs can be considered the most abundant microbiota metabolites in the intestine. SCFAs have been shown to reduce drug-induced intestinal toxicity, such as the anti-tumor drug 5-fluorouracil and the vasodilator drug nitroglycerin. SCFAs that are not metabolized by cells can also reach various parts of the body through the blood circulation. Although the content of these SCFAs involved in the circulation is relatively small, they can act as ligands to affect signaling pathways such as mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB), and participate in regulating physiological activities. As an important microbiota metabolite, SCFAs also play an important anti-inflammatory role in the intestine. It is reported that the content of SCFA in the intestine is significantly negatively correlated with proinflammatory factors TNF-α, IL-1β, IFN-γ, IL-6, and myeloperoxidase, and significantly positively correlated with the inflammatory inhibitor IL-10. In addition, SCFA can enhance intestinal barrier function by upregulating the expression and distribution of tight junction proteins and protecting intestinal epithelial cells. For example, propionic acid and butyric acid promote the production of mucin MCU2 by stimulating goblet cells and regulating the MAPK pathway, respectively. Butyric acid increases the expression of tight junction proteins Claudin-3 and Claudin-4 by activating Akt / mTOR-mediated protein synthesis. Butyric acid can also enhance tight junctions by increasing the expression of synaptopodin and tight junction proteins CLDN4, CLDN5, and CLDN11. Propionic acid can enhance the speed and persistence of intestinal epithelial cell migration, which is beneficial to the recovery of intestinal damage. In vitro studies have also shown that SCFA can reduce lipopolysaccharide-induced intestinal barrier dysfunction by increasing transepithelial resistance and the expression of Claudin-1 and adjusting the distribution of tight junction protein ZO-1. Therefore, this study used gas chromatography to analyze the content of short-chain fatty acids in rat feces during the feeding period ( Figure 2-3 ).

[0130] Numerous in vitro studies have shown that the fermentation of acidic HMOs such as 3′-SL and 6′-SL and 2′-FL can increase the concentration of SCFAs and lactic acid, and can regulate the abundance of intestinal flora during the fermentation process. Kong et al. tested the fermentability of 3-FL and LNT II through in vitro fermentation experiments. The results showed that both 3-FL and LNT II increased the concentration of acetic acid and lactic acid, 3-FL promoted the growth of Bacteroides and Enterococci, and LNT II promoted the proliferation of Bifidobacterium and Collinsella. Figure 2-3As shown in the figure, LNT II and GOS+LNB can promote the production of short-chain fatty acids. The fecal short-chain fatty acids in the LNB, GOS+LNT II, ​​and LNB+LNT II groups were lower than those in the control group, which may be due to the upregulation of lipid absorption genes, thereby promoting the absorption of short-chain fatty acids in the colon.

[0131] 2.2.4 Effects of GOS, LNB, LNT II and their combination on colonic lipid absorption genes and intestinal barrier in rats

[0132] A small amount of SCFAs exist in the intestine in a bound form and can directly cross the epithelial barrier. However, most of them exist in an ionic state and require specialized transporters for absorption. The passage of SCFAs across the intestinal epithelial mucosa involves active transport mediated by two receptors: monocarboxylate transporter (MCT) and sodium-coupled monocarboxylate transporter (SMCT) receptors. Both MCT and SMCT are highly expressed in colonocytes and throughout the gastrointestinal tract (including the small intestine and cecum). G protein-coupled receptors (GPR43, GPR109a) are short-chain fatty acid receptors that can mediate short-chain fatty acids through the MAPK signaling pathway, play an important role in regulating physiological processes such as fat formation and leukocyte function in the body, and in the absorption of nutrients in the animal intestine.

[0133] The animal intestine is not only an important place for digestion and absorption of nutrients, but also an innate barrier to maintain the stability of the body's internal environment. The intestinal barrier includes mechanical barriers, chemical barriers, microbial barriers and immune barriers, and its integrity is closely related to intestinal function and body health. Among them, tight junction proteins have multiple functions. They can not only maintain the epithelial barrier function, hinder the invasion of toxic macromolecules and microorganisms, but also selectively regulate the entry of small molecules and ions into the body; at the same time, tight junction proteins are also involved in the regulation of gene transcription, cell proliferation and differentiation status. There are many proteins involved in the formation of tight junctions of intestinal epithelial cells, among which claudin, occlaudin and occludin (ZO) family proteins are the most important tight junction proteins, and are also typical members of the tight junction protein family. They play an important role in maintaining the difference between the substances on both sides of the epithelial cells and maintaining cell polarity. ZO-1 and ZO-2 are related to the maintenance and regulation of the palisade and barrier function of the intestinal epithelium, and participate in important processes such as cell substance transport and maintaining epithelial polarity. The Cdx gene belongs to the caudal homeobox gene family, which contains three homologous genes, namely Cdx1, Cdx2 and Cdx4. Among them, Cdx1 and Cdx2 genes play an important role in the proliferation and differentiation of intestinal epithelial cells during the normal development of organisms. Mucin Muc2 is a chemical barrier that covers the surface of the digestive tract to form a mucus layer, which plays a role in isolating pathogens and protecting digestive tract epithelial cells. Mucin in the colon can wrap food residues to prevent intestinal epithelial cells from being scratched.

[0134] The relationship between barrier dysfunction and intestinal dysbiosis suggests that there is an interaction between the intestinal barrier and specific intestinal commensal bacteria and pathogenic microorganisms. Probiotics can promote intestinal barrier function by ligand-mediated stimulation of the expression of several toll-like receptors in intestinal epithelial cells. For example, bacteria-mediated toll-like receptor 2 can stimulate and regulate the expression and localization of specific intestinal epithelial tight junction proteins. In addition, prebiotics can have additional direct effects on intestinal barrier function through mechanisms independent of the intestinal microbiota, such as promoting the significant upregulation and relocation of tight junction proteins between intestinal epithelial cells, thereby enhancing intestinal barrier function. Studies have shown that in vitro fermentation of inulin can lead to the upregulation of tight junction genes such as Occludin, Claudin-3 and ZO-1. Supplementation of FOS can relocate tight junction proteins such as ZO-1 and Occludin to the vicinity of tight junctions. In addition, both in vivo and in vitro studies have demonstrated that GOS can upregulate the expression of ZO-1, Occludin and Claudin-1 genes under LPS-stimulated conditions.

[0135] The results of this study show that oligosaccharide treatment has a very significant effect on the relative expression of each tight junction protein gene ( Figure 2-4 ). Among them, LNT II can significantly increase the expression of genes for colonic lipid absorption (FABP4) and transport (GPR43, GPR109a), thereby promoting the absorption and transport of short-chain fatty acids in the colon. The LNB+LNT II group can significantly increase the expression of tight junction proteins (ZO-1, ZO-2) and genes related to intestinal epithelial cell proliferation and differentiation (Cdx1 and Cdx2), playing an important role in maintaining intestinal homeostasis and intestinal barrier. These regulatory effects may regulate the development of the rat intestine by directly acting on target tissues and gene regulatory genes, or by regulating the intestinal flora. It may also regulate the mutual restraint and influence between the intestinal flora and the intestinal barrier, thereby regulating the metabolism of the rat body.

[0136] 2.2.5 Effects of GOS, LNB, LNT II and their combination on the structural composition of rat intestine

[0137] The height of the villi, the width of the villi, the depth of the crypts, and V / C of the small intestine are important indicators of nutrient digestion and absorption in the intestine of animals. The intestinal villi are the main mucosal structure of the small intestine. The longer the villi, the larger the absorption surface area and the more sufficient the absorption of nutrients. The deeper the crypts, the villi can be updated as needed to cope with the normal shedding or inflammation of pathogens. V / C can reflect the functional state of the small intestine. The larger the value, the stronger the absorption capacity. The increase in V / C is directly related to the increase in epithelial cell turnover. Studies have found that GOS can promote the growth and proliferation of intestinal organs and is beneficial to the integrity of the intestinal barrier. This experimental study showed that the jejunum and colon of rats had normal histological morphology and intact mucosal structure, indicating that GOS, LNB, LNT II and their combination treatments would not have adverse effects on the rat intestine. Except for the GOS+LNB group, the other sample treatment groups were able to promote the development of jejunal villi and improve the absorption capacity of substances compared with the blank group ( Figure 2-5 ).

[0138] 2.2.6 Effects of GOS, LNB, LNT II and their combination on the composition of rat intestinal flora

[0139] In order to analyze the effects of GOS, LNB, LNT II and their combination on the intestinal microbial community of early-life rats, this study used 16S rRNA high-throughput sequencing to quantitatively analyze the intestinal flora and evaluate its microbial diversity, composition and differences. Operational taxonomic unit (OUT) was used to cluster sequences with a similarity of ≥97%, that is, one OTU represented one species. Figure 2-6 As shown in A, there were 54, 9, 319, 21, 25, 73, and 14 independent OTUs in the Control, GOS, LNB, GOS+LNB, GOS+LNT II, ​​and LNB+LNT II groups, respectively, and 806 common OTUs among the seven groups. The Shannon index, Simpson index, ACE index, and Chao1 index were calculated for the observed OTUs to evaluate the α-species diversity of the community ( Figure 2-6 BE). Shannon index and Simpson index are both important indicators for measuring species richness and uniformity in a community, and are positively correlated with species and distribution uniformity. Chao1 is positively correlated with species. The larger the ACE value, the more real species the sample has. As shown in the figure, after 24 days of feeding in seven different feeding groups, there was no significant difference in the Simpson index, Chao1 index, and ACE index of SD rats. The GOS+LNT II group had a higher Shannon index than the GOS group and the GOS+LNB group, indicating that GOS+LNT II can improve the richness and uniformity of the intestinal flora.

[0140] Non-Metric Multi-Dimensional Scaling (NMDS) is a method used to compare and analyze species diversity among groups. As shown, the differences among the three groups were significantly different (p<0.05) ( Figure 2-7 ).

[0141] At the phylum level, all groups had similar taxonomic communities, dominated by Firmicutes and Bacteroidetes ( Figure 2-8 A). Firmicutes is the largest microbial community in the intestinal flora, including most beneficial bacteria such as lactic acid bacteria and Ruminococcus; Bacteroidetes is an important microbial component in the intestine, which mainly uses polysaccharides as an energy source and produces short-chain fatty acids such as acetic acid, propionic acid, butyric acid and valeric acid after fermentation. Both play an important role in protecting the digestive system from damage by intestinal pathogens. This study is consistent with the results of GOS research on intestinal flora, revealing the dominant position of Firmicutes and Bacteroidetes microorganisms in the life process.

[0142] At the genus level, it is mainly composed of unclassified_Muribaculaceae, Lactobacillus, Prevotella, Lachnospiraceae_NK4A136_group, Ruminococcus, UCG-005, unclassified_Lachnospiraceae, etc. ( Figure 2-8 B).

[0143] To verify the differential microorganisms that were significantly different at the species level, we performed linear discriminant effect size (LEfSe) analysis ( Figure 2-9). The LDA value distribution histogram showed that the LNB group was enriched in Bifidobacteri ales and Bifidobacteriaccae. Studies have shown that LNB is a core component of human milk oligosaccharides and can promote the growth of bifidobacteria in infant feces in in vitro fermentation experiments. Previous studies have shown that HMOs can selectively stimulate the growth of beneficial bacteria, mainly Bifidobacterium. Among many microbial species, Bifidobacterium is the main consumer of HMOs in the infant intestinal microbiome. In addition, only a few Bacteroides and Lactobacillus were found to be able to degrade HMOs. However, only Bifidoba cterium and a few Bacteroides can fully utilize HMO as the main energy source, and Lactoba cillus has the weakest HMO utilization ability. The GOS+LNB group is enriched in Prevotellacea and Prevotella. Prevotella contains enzymes that play an important role in mucus protein degradation, which may lead to increased intestinal permeability. It has been confirmed that it has the enzymes and gene clusters necessary for fermentation and utilization of complex polysaccharides. Studies have found that African children who eat a low-fat, high-fiber diet have abundant intestinal Prevotella and high levels of short-chain fatty acids.

[0144] In this study, the Spearman algorithm was used to visualize the correlation between species with the top 80 P values ​​at the genus level (correlation coefficient threshold: 0.1, P < 0.05). A positive correlation indicates that the abundance changes of the two species are in the same direction, and a negative correlation indicates that the abundance changes of the two species are in opposite directions ( Figure 2-10). The results showed that a total of 53 bacterial genera were significantly correlated, mainly belonging to the Firmicutes phylum. There was a significant positive correlation between Lactobacillus (No.: 49) and Limosilactobacillus (No.: 31), that is, as the abundance of Lactobacillus increased, the abundance of Limosilactobacillus had a significant increasing trend. Similarly, Lachnoclostrid um (No.: 20) was significantly positively correlated with Lachnospiraceae-NK4A136-group (No.: 45). Studies have shown that Tibetan tea polysaccharides can promote the growth of microorganisms such as Lachnoclostridium, Bacteroides, and Bifidobacterium, while promoting the production of short-chain fatty acids. The reduction of SCFAs-producing bacteria (including Lachnospiraceae_NK4A136_group) in mice may be a potential trigger for colonic dysbiosis. These significantly correlated differential species all belong to the phylum Firmicutes. The results of this study indicate that the differential species of the above-mentioned phylum Firmicutes can promote each other's growth, especially when beneficial bacteria are dominant, which can promote the growth of beneficial bacteria in the same phylum and have a positive impact on the health of the body.

[0145] 2.3 Summary

[0146] (1) LNB can regulate intestinal health by increasing the absolute abundance of beneficial intestinal bacteria (such as Lactobacillus and Bifidobacterium).

[0147] (2) LNT II can promote the production of short-chain fatty acids and significantly increase the expression of colon lipid absorption (FABP4) and transport (GPR43, GPR109a) genes, thereby promoting the absorption and transport of short-chain fatty acids in the colon. Compared with the blank group, the LNT II-treated group can promote the development of jejunal villi and improve the ability to absorb substances.

[0148] (3) The LNB+LNT II group significantly increased the expression of tight junction proteins (ZO-1, ZO-2) and genes related to intestinal epithelial cell proliferation and differentiation (Cdx1 and Cdx2), which play an important role in maintaining intestinal homeostasis and intestinal barrier. Compared with the blank group, the LNB+LNTII treatment group promoted the development of jejunal villi and improved the ability to absorb substances.

[0149] These regulatory effects may regulate the development of the rat intestine by directly acting on the target tissues and gene regulatory gene expression, or by regulating the intestinal flora. They may also regulate the mutual restraint and influence between the intestinal flora and the intestinal barrier, thereby regulating the metabolism of the rat body.

[0150] Example 3 Preparation and characterization of milk powder and in vitro anti-inflammatory activity study

[0151] Based on the results of previous in vitro and in vivo experiments, the best oligosaccharides or combinations were selected, infant formula was produced, and in vitro simulated digestion was carried out to explore whether infant formula rich in oligosaccharides still has anti-inflammatory and other effects after gastrointestinal digestion.

[0152] That is, according to the in vitro and in vivo experiments of Examples 1 and 2, the LNT II group and the LNB+LNTII group, which have obvious anti-inflammatory effects and can promote intestinal absorption, are selected, and the formula milk powder is designed according to the formula of the LNT II group and the LNB+LNTII group (for the conversion method, please refer to the introduction in Example 2 above for details), and the experiment of Example 3 is carried out. Specifically, the formula of the LNT II group is: 5g LNT II / 130g milk powder; the formula of the LNB+LNTII group is: 2.5g LNB / 130g milk powder, 2.5g LNT II / 130g milk powder.

[0153] 3.1 Experimental methods

[0154] 3.1.1 Milk powder preparation

[0155] Milk powder preparation method: whey protein, lactose and pure water are mixed and stirred magnetically (55±2℃) until dissolved, and then placed at 4℃ overnight (12h) for hydration. Then microwave-melted oil phase materials (1,3-dioleyl-2-2 palmitic acid triglyceride, sunflower oil, high oleic sunflower oil, human milk fat, coconut oil, linseed oil) are added to the water phase, and heating and stirring are continued (55±2℃, 30 minutes). Then shearing (8000rpm, 1min), homogenization (150±5bar, 3 times) and spray drying (inlet temperature 180℃, outlet temperature 90℃, 5bar) are immediately performed. Vitamins, minerals, LNT II or LNB and LNT II corresponding to the above-mentioned infant formula milk powder are added by dry mixing. That is, two oligosaccharide milk powders were prepared, the first one was 5g LNT II / 130g milk powder; the second one was 2.5g LNB / 130g milk powder, 2.5g LNT II / 130g milk powder. Control milk powder: the method is the same as above, but without oligosaccharides, that is, without LNT II and LNB.

[0156] For the sake of ease of description, the three milk powders above are respectively recorded as LNT II group, LNB+LNT II group, and Control group.

[0157] 3.1.2 Characterization of milk powder emulsion

[0158] The preparation method of milk emulsion for characterization was similar to the preparation method of milk powder in 3.1.1 above, but the addition position of oligosaccharides, vitamins and minerals was adjusted to add them during the homogenization step. Then 13 g of milk powder was dissolved in 90 ml of water for characterization experiments.

[0159] 3.1.2.1 Determination of particle size and zeta potential

[0160] The average particle size and zeta potential of the emulsion and reconstituted emulsion were measured at room temperature using a nanoparticle sizer (Malvern Instruments Ltd, ZS-90, UK). The samples were appropriately diluted 100 times with pure water and equilibrated at 25°C for 10 minutes to avoid multiple scattering.

[0161] 3.1.2.2 Determination of moisture content and water activity

[0162] Weigh 2.0-2.5 g of sample and measure the moisture content using a rapid moisture meter (Sartorious, MA 150, Germany). The water activity (aw) is measured using a water activity tester.

[0163] 3.1.2.3 Determination of surface oil content

[0164] 3 g of the spray-dried powder was gently mixed with 15 mL of petroleum ether to remove the surface oil in the powder. The collected petroleum ether containing the surface oil was then evaporated at room temperature in a fume hood and weighed to calculate the surface oil content.

[0165] 3.1.2.4 Determination of thermal properties of milk powder

[0166] The thermal properties of the spray-dried powder were analyzed using a thermal analyzer (Swiss Mettler-Toledo Group, DSC1 / 200 / T45). About 10 mg of the powder was sealed in a sealed aluminum pan, heated from 25°C to 85°C (5°C / min) under a nitrogen flow rate of 25 mL / min, and then cooled to 5°C (50°C / min).

[0167] 3.1.2.5 Determination of lactose crystallinity

[0168] The lactose crystallinity of the sample was determined using an X-ray diffractometer (Rigaku Ultima IV, Rigaku Corporation, Japan). 10 mg of the powdered sample was placed in a sample container and then scanned from 6° to 55° at a speed of 1° / min to determine the diffraction intensity of the crystalline part. The diffraction intensity spectrum was analyzed using MDI Jade 6.0 software.

[0169] 3.1.2.6 Scanning electron microscope observation of powder surface microstructure

[0170] The surface morphology of the powders was analyzed using a scanning electron microscope (Sigma 300, Carl Zeiss Group, Germany) equipped with a tungsten filament. The accelerating voltage was set to 3 kV (for morphology analysis) and 15 kV (for spectral analysis), and the detector was SE2 to obtain images of different magnifications to observe the surface morphology of the powders.

[0171] 3.1.3 In vitro simulated digestion of reconstituted emulsions

[0172] The first milk powder, the second milk powder and the control group milk powder prepared in 3.1.1 above were dissolved in water, specifically, 13 grams of milk powder was dissolved in 90 ml of water to prepare 3 reconstituted emulsions, and the following in vitro simulated digestion was performed respectively. For the specific components of simulated saliva, simulated gastric juice and simulated intestinal juice, please refer to the table below. Oral phase: Take 10 mL of reconstituted emulsion and 10 mL of simulated saliva (containing 30 mg of mucin and 50 mg of α-amylase) and mix them evenly, and adjust the pH to 6.8. The mixed solution was stirred continuously at 100 rpm for 10 minutes at 37°C to obtain oral digestive fluid.

[0173] Gastric phase: 20 mL of the above oral digestive fluid was mixed with an equal volume of simulated gastric fluid (SGF, containing 64 mg of pepsin), and the pH was adjusted to 2.0 ± 0.05 with HCl after mixing. The mixed solution was continuously stirred at 100 rpm at 37°C for 60 min to obtain a gastric phase mixed solution.

[0174] Small intestine stage: Take 20mL of the above-mentioned gastric phase mixed solution, adjust the pH to 7.0, add 2.6mL simulated intestinal fluid (SIF), 8mL bile salt solution (375mg of pig bile salt) and 2.6mL of pig pancreas (8mL containing 375mg of pancreatic enzyme) pancreatic enzyme solution. Use 0.1M NaOH to adjust the sample pH to 7.0±0.05, terminate gastric digestion, and obtain intestinal phase digestion fluid. In other words, simulated digestion is carried out in the above three stages, namely, oral phase, gastric phase, and intestinal phase to obtain the final intestinal phase digestion fluid. Then the intestinal phase digestion fluid is freeze-dried for 48h, the freeze-dried powder is collected, liquid nitrogen is added to grind it evenly, and it is placed in a -80℃ refrigerator until it is used to incubate the following Caco-2 cells. The freeze-dried powder of the intestinal phase digestion fluid is dissolved in complete culture medium, and Caco-2 cells are incubated after passing through a 0.22μm sterilization membrane.

[0175] Specific composition table of simulated saliva, simulated gastric juice and simulated intestinal juice

[0176]

[0177] 3.1.4 Effect of reconstituted emulsion digestion products on the anti-inflammatory activity of Caco2

[0178] 3.1.4.1 Cytotoxicity

[0179] Human colon adenocarcinoma cells (Caco-2 cells) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2 concentration and 90% humidity. The medium was changed every 2 days. During cell passage, cells were cultured at 1×10 6 The number of cells was inoculated into a 60 mm diameter culture dish and cultured until confluence.

[0180] CCK-8 (Cell Counting Kit-8) was used to determine the cytotoxicity and determine the appropriate sample concentration. Logarithmic phase cells were seeded into 96-well plates (5 × 10 4 Each well was incubated for 24 hours, and then 10 μL of CCK-8 solution was added to each well for 1 hour. The supernatant was aspirated and the absorbance of the supernatant was measured at 450 nm using an ELISA reader.

[0181] 3.4.1.2 Determination of inflammatory factors

[0182] Stably cultured Caco-2 cells were cultured at 5×10 5 Cells were inoculated into 96-well plates at a density of 100 cells / mL and cultured with complete medium for 24 hours. The complete medium was aspirated and treated with samples of different concentrations (cytotoxic concentration) for 12 hours (the samples were dissolved and diluted with DMEM), LPS (1 μg / mL) was added and incubated with the samples for 12 hours, and then the cell supernatant was collected. The production of TNF-α, IL-1β, and IL-10 was determined using Elisa kits according to the manufacturer's instructions.

[0183] 3.2 Experimental Results and Discussion

[0184] 3.2.1 Particle size and potential of emulsion and reconstituted emulsion

[0185] Generally speaking, smaller droplet size indicates higher stability, while a larger absolute zeta potential generally improves stability by increasing electrostatic repulsion. In unstable emulsions, insufficient electrostatic repulsion can lead to droplet flocculation and aggregation. Figure 3-1 AB showed that the Zeta potential of the emulsion was between -36 and -38 mV, and the average particle size was about 400-450 nm. It is worth noting that the particle size of the emulsion in the LNT II group was smaller and the absolute potential was larger than that in the control group, which may be due to the fact that the charged groups in LNT II enhanced the stability of the emulsion. The Zeta potential of the reconstituted emulsion was between -38 and -40 mV, which was slightly higher than the Zeta potential of the emulsion after homogenization ( Figure 3-1 C). Figure 3-1D shows that the particle size distribution of the reconstituted emulsion is similar to that of the homogenized formula milk, with an average particle size distribution between 400 and 500 nm. This result indicates that the three prepared formula milk powders have good solubility and can ensure the stability of the formula emulsion.

[0186] 3.2.2 Glass transition temperature, crystallinity and surface properties

[0187] The glass transition temperature (Tg) is the temperature at which amorphous lactose changes from a "metastable" glassy state to a rubbery state and is an important indicator of milk powder agglomeration. Figure 3-2 A shows that there is no significant difference in Tg between LNT II and the control group, and LNB+LNT II has the lowest Tg. This may be related to the fact that the lower the aw, the higher the Tg, which is consistent with the results of a previous study (Table 3-1). LNB has a certain water absorption capacity, and its presence may cause the aw value of milk powder to increase and the Tg value to decrease. Previous studies have shown that Tg has an important influence on the stability of milk powder; the lower the Tg, the greater the molecular fluidity of the milk powder, the lower the viscosity, which is conducive to the crystallization of lactose. For example, the LNB+LNT II group has the highest water content and aw, but the lowest solubility (Table 3-1), and obvious crystallization peaks appear at 10°, 19.1° (α-lactose monohydrate) and 20.1° (anhydrous α:β lactose crystals) ( Figure 3-2 B), but there was no significant difference in surface oil content. Figure 3-2 As shown in Figure C, there is no significant difference in the surface micromorphology of the three groups of spray-dried powder particles, but the LNB+LNT II formula powder looks rougher than the other two groups. The above results show that the low Tg of the LNB+LNT II spray-dried powder is conducive to agglomeration, while the higher surface roughness is due to the presence of lactose crystals. The lower Tg of LNB+LNT II increases the appearance of lactose crystals, making LNB+LNT II milk powder more unstable during storage than the other two milk powders.

[0188] Table 3-1 Moisture content, water activity and surface oil content of milk powder

[0189]

[0190] 3.2.3 Effect of reconstituted emulsion digestion products on the anti-inflammatory activity of Caco2

[0191] The intestinal digest was freeze-dried and the powder was collected. The cells were incubated with culture medium after reconstitution. High concentrations (1000, 1500 and 2000 μg / mL) of intestinal digest of the control group, LNT II group and LNB+LNT II group significantly inhibited cell viability; therefore, the concentration range of 25-500 μg / mL was selected for subsequent experiments ( Figure 3-3). LPS treatment significantly increased the inflammatory level of Caco-2 cells. Digestate pretreatment significantly reduced the secretion of TNF-α and IL-1β, while significantly promoting the release of IL-10 ( Figure 3-3 BD). It is noteworthy that the LNT II group significantly inhibited the secretion of TNF-α compared with the Control group, and the LNB+LNT II group had the most significant inhibitory effect on TNF-α. Anti-inflammatory activity was also observed in the intestinal digesta of the Control group without the addition of HMOs. This may be due to the hydrolysis of whey protein isolate (WPI) in the milk powder to form anti-inflammatory peptides, or the presence of functional lipids such as docosahexaenoic acid (DHA). The results of the study indicate that the bioactive oligosaccharides added to the milk powder retain their original anti-inflammatory activity and work synergistically with other active ingredients in the base powder to fight inflammation.

[0192] 3.3 Summary

[0193] 1) The milk powder in the LNT and LNB+LNT groups still had anti-inflammatory effects after in vitro simulated digestion treatment.

[0194] 2) In vitro simulated digestion and LPS-induced Caco-2 inflammation models showed that milk powder digests could inhibit the release of TNF-α and IL-1β, while promoting the production of IL-10. It is worth noting that LNB+LNT II had the most significant effect in inhibiting TNF-α. Oligosaccharide-rich milk powder was produced. The prepared formula milk powder has good physicochemical properties and immunoenhancing activity. This study will be helpful for the functional evaluation and product development of HMO.

Claims

1. An infant formula milk powder rich in human milk oligosaccharides and having anti-inflammatory effects, characterized in that: Includes LNB and LNTII, with a content of 2.5g LNB / 130g milk powder and 2.5g LNT II / 130g milk powder; Alternatively, LNT II is included, with the content of 5g LNT II / 130g milk powder.

2. The infant formula milk powder according to claim 1, characterized in that: The moisture content of the infant formula milk powder is less than 4% by mass.

3. The infant formula milk powder according to claim 1, characterized in that: The surface oil mass of the infant formula milk powder is less than 6.5%.

4. A method for preparing an infant formula milk powder rich in human milk oligosaccharides and having anti-inflammatory effects as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: Mix whey protein, lactose and pure water and stir until dissolved; then let stand for hydration; then add melted oil phase material and continue stirring; then shear, homogenize and spray dry; Add vitamins and minerals through dry mixing; The LNB and LNT II corresponding to the infant formula milk powder, or the corresponding LNT II, ​​are added by dry mixing.

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