Infant formula milk powder rich in milk fat globule membrane protein, phospholipids and oligosaccharides and preparation method therefor

Abstract

An infant formula milk powder is rich in milk fat globule membrane protein, phospholipids, and oligosaccharides. A preparation method includes using raw cow milk as raw material, cleaning and pre-sterilizing raw cow milk, adding MFGM-rich whey protein powder, -lactalbumin powder, galactooligosaccharides, polyfructoses and other ingredients into the pre-sterilized raw cow milk, and performing pre-sterilization, homogenization, sterilization, concentration, and spray drying. By means of formula adjustment, the contents of biologically active substances having special functional components such as MFGM-protein, lactoferrin, -lactalbumin, total galactooligosaccharide, total polyfructose, sialic acid, total phospholipid, sphingomyelin, lecithin, phosphatidylserine, phosphatidylethanolamines, phosphatidylinositol, ganglioside, triglyceride and diglyceride in the infant formula milk powder are increased, thereby facilitating the colonization of probiotics in the intestinal microbiota of an infant, especially significantly enriching lactic acid bacteria in an intestinal tract, while reducing unclassified bacterial family and other miscellaneous bacteria.

Claims

1. An infant formula milk powder rich in milk fat globule membrane protein, phospholipids and oligosaccharides, prepared by using raw cow milk as raw material, added with milk fat globule membrane (MFGM)-rich whey protein powder, -lactalbumin powder, galactooligosaccharides and polyfructoses; wherein contents of functional active ingredients per 100 grams of the infant formula milk powder are: 0.146-0.438 g total MFGM-protein, 0.22-0.35 g total -lactalbumin, 0.2-0.6 g total lactoferrin, 0.1-0.3 g immunoglobulin IgG, 0.025-0.075 g lactadherin, 0.035-0.105 g Mucin short variant S1 (MUC1/Mucin1), 0.015-0.4 g total galactooligosaccharides, 0.001-0.003 g total polyfructoses, 0.05-0.15 g sialic acid, 0.175-0.525 g total phospholipid, 0.04-0.12 g sphingomyelin, 0.005-0.015 g ganglioside, 0.06-0.19 g lecithin, 0.04-0.14 g phosphatidylethanolamine, 0.02-0.06 g phosphatidylinositol, 0.007-0.021 g phosphatidylserine, 0.0174-0.0371 g diglyceride, and 0.0311-0.0598 g triglyceride.

2. The infant formula milk powder according to claim 1, wherein the contents of functional active ingredients per 100 grams of the infant formula milk powder are: 0.438 g total MFGM-protein, 0.35 g total a-lactalbumin, 0.35 g total lactoferrin, 0.172 g immunoglobulin IgG, 0.075 g lactadherin, 0.105 g MUC1/Mucin1, 0.4 g total galactooligosaccharide, 0.003 g total polyfructose, 0.15 g sialic acid, 0.525 g total phospholipid, 0.12 g sphingomyelin, 0.015 g ganglioside, 0.19 g lecithin, 0.14 g phosphatidylethanolamine, 0.06 g phosphatidylinositol, 0.021 g phosphatidylserine, 0.0371 g diglyceride, and 0.0598 g triglyceride.

3. The infant formula milk powder according to claim 1, wherein a bovine colostrum is added, which the bovine colostrum is made into 15-16 kilograms of bovine colostrum powder from 100 L fresh milk.

4. The infant formula milk powder according to claim 1, wherein 60 kg of the MFGM whey protein powder, 10 kg of the -lactalbumin powder, 106 kg of the galactooligosaccharides, and 4.33 kg of the polyfructoses are added into per 2000 L raw cow milk.

5. The infant formula milk powder according to claim 1, wherein the infant formula milk powder is prepared by: using raw cow milk as raw material, cleaning and pre-sterilizing raw cow milk at 85 C.-88 C. for 30s, adding MFGM-rich whey protein powder, -lactalbumin powder, galactooligosaccharides, polyfructoses and other ingredients into the pre-sterilized raw cow milk, and performing pre-sterilization at 85 C.-88 C. for 30 s, homogenization at 15 mPa, sterilization at 93 C.-95 C. for 15 s, concentration, and spray drying with an inlet air temperature of 150 C.-160 C., and an outlet air temperature of 85 C.-90 C.

6. A method for preparing the infant formula milk powder according to claim 1, comprising: using raw cow milk as raw material, cleaning and pre-sterilizing raw cow milk, adding MFGM whey protein powder, -lactalbumin powder, galactooligosaccharides, polyfructoses and other ingredients into the pre-sterilized raw cow milk, and performing pre-sterilization, homogenization, sterilization, concentration, and spray drying.

7. The method for preparing the infant formula milk powder according to claim 6, wherein the pre-sterilizing is performed at 85 C.-88 C. for 30 s, the homogenization is performed at 15 mPa, the sterilization is performed at 93 C.-95 C. for 15s, and the spray drying is performed at an inlet air temperature of 150 C.-160 C., and an outlet air temperature of 85 C.-90 C.

8. A method for promoting the enrichment of probiotics in the intestinal tract of an infant or for promoting the immunity of an infant, comprising administration of the infant formula milk powder according to claim 1 to the infant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1E show the composition of the fecal flora of the newborn piglet on the eighth day of Example 1 of the present disclosure: wherein panel FIG. 1A shows diversity (Sobs index); panel FIG. 1B shows Shannon index; panel FIG. 1C shows -diversity of PCoA based on unweighted Unifrac distance; panel FIG. 1D shows differential microbial composition based on Wilconxon rank sum test; and panel FIG. 1E shows genus-level Linear Sex Analysis Effect Size (LefSe) analysis, Linear Discriminant Analysis (LDA) score >4; *P<0.05; **P<0.01.

(2) FIGS. 2A-2E show the composition of the fecal flora of the newborn piglet on the twenty-first day of Example 1 of the present disclosure: wherein panel FIG. 2A shows diversity (Sobs index); panel FIG. 2B shows Shannon index; panel FIG. 2C shows -diversity of PCoA based on unweighted Unifrac distance; panel FIG. 2D shows differential microbial composition based on Wilconxon rank sum test; and panel FIG. 2E shows genus-level Linear Sex Analysis Effect Size (LefSe) analysis, Linear Discriminant Analysis (LDA) score >4; *P<0.05; **P<0.01.

(3) FIGS. 3A and 3B show the functional status of the intestinal flora of the newborn piglet of Example 1 of the present disclosure: wherein panel FIG. 3A is the differential abundance of Kyoto Encyclopedia of Genes and Genomes (KEGG) on the eighth day; and panel FIG. 3B is the differential abundance of Kyoto Encyclopedia of Genes and Genomes (KEGG) on the twenty-first day.

(4) FIGS. 4A-4G show the influence of expression of intestinal barrier-related genes and intestinal permeability of the twenty-first day newborn piglets in Example 1 of the present disclosure:

(5) Expression of intestinal barrier-related genes in ileal mucosa (FIGS. 4A-4C) and colonic mucosa (FIGS. 4D-4F); and expression of plasma DAO levels (FIG. 4G).

(6) FIGS. 5A and 5B show the effect of piglet fecal SCFAs concentration and intestinal GPRs gene of Example 1 of the present disclosure: wherein panel A shows the difference in the concentration of short-chain fatty acids in piglet feces, and panel B shows the expression of receptor genes.

(7) FIG. 6 shows a production flow chart of the present disclosure of Example 1 of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

(8) In order to make the objectives, technical solutions, and advantages of this disclosure clearer, the technical solutions of this disclosure will be described clearly and completely below by reference to the drawings. It is apparent that the described embodiments are part of the embodiments of this disclosure, but not exhaustive. Based on embodiments of this disclosure, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of this disclosure. Unless otherwise expressly stated, throughout the specification and claims, the term comprising or a variation thereof such as including or contain is construed as including the stated element or component, without excluding other elements or other components.

Examples 1: Comparison of Active Ingredients in the Infant Formula Milk Powder and Breast Milk and Comparative Examples. Refer to Table 2.

Comparative Example: CN106359604A

(9) TABLE-US-00002 TABLE 2 This Comparative Breast Functional Active disclosure Example Milk Ingredients Unit Per 100 g Per 100 g Per 100 g Total MFGM-protein g 0.438 0.251 0.1 Total -lactalbumin g 0.35 2-3 Total lactoferrin g 0.35 0.0344 1-2 Immunoglobulin IgG g 0.172 0.172 0.7-2 Lactadherin g 0.075 0.043 0.093 MUC1/Mucin1 g 0.105 0.0602 0.73 Total g 0.4 0.89 2.21 galactooligosaccharide Total polyfructose g 0.003 0.1 Sialic acid g 0.15 0.171 0.39 Total phospholipid g 0.525 0.301 1.71 Sphingomyelin g 0.12 0.0688 0.675 Sphingomyelin g 0.015 0.0086 0.003 Lecithin g 0.19 0.324 Phosphatidylethanolamine g 0.14 0.576 Phosphatidylinositol g 0.06 0.0489 Phosphatidylserine g 0.021 0.08 Diglyceride g 0.0371 3.183629 Triglyceride g 0.0598 386.5839

(10) It may be seen from the above comparison that the active ingredients of the formula milk powder of the present disclosure are more comprehensively close to breast milk. Particularly, the ingredients such as total -lactalbumin, lecithin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, diglyceride, triglyceride are not present in CN106359604A. The amount of total lactoferrin was nearly 10 times higher than that in the comparative example, indicating reduced gap from breast milk.

(11) The active ingredients in Table 2 were prepared according to the following method through wet processing process, as shown in FIG. 6. The raw cow milk, used as raw material, was cleaned and pre-sterilized. After that, 60 kg MFGM whey protein powder, 10 kg -lactalbumin powder, 106 kg galactooligosaccharides, and 4.33 kg polyfructoses were added to 2000 L of the pre-sterilized raw milk, which was then homogenized, sterilized, concentrated and dried.

(12) The above method were conducted at the following main process parameters: the pre-sterilization (85 C.-88 C., 30 s); homogenization 15 mPa; sterilization (93 C.-95 C., 15 s); spray drying (inlet air temperature 150 C.-160 C., and outlet air temperature 85 C.-90 C.).

(13) The testing of the nutritional indicators, physical and chemical indicators, microbial indicators and sensory indicators of the products described in this disclosure is carried out in strict accordance with the testing items and testing methods specified in the National Standards on Food Safety of P. R. China (GB-10765-2010).

Example 2: Evaluation of the Influence of the Product on Intestinal Microecology Based on the Piglet Model

(14) 1. Test Method

(15) 1.1 Establishment of Piglet-Based Model and Sampling

(16) 16 piglets (1.530.04 kg) from different litters (one piglet per litter) were randomly assigned to the control group fed normal saline (referred to as CON group) and the experimental group, which were fed galactooligosaccharides, MFGM whey protein powder, -lactalbumin powder, galactooligosaccharides, polyfructose (abbreviated as GMF group, Example 1 of this disclosure). From the first day to the seventh day after birth, the piglets in the GMF group were administered 5 mL GMF solution (1 g/kg body weight) every day, and the piglets in the CON group were fed the same amount of normal saline. The piglets consumed sow milk and water normally throughout the lactation period. Commercial feed was added from the eighth day postpartum. Health status was monitored daily, and body weights were recorded on day 21. On day 21, 5 piglets (approximately the average weight per group) were selected, and piglet blood samples were taken from the jugular vein. Plasma was collected after centrifugation at 3000 g for 10 min at 4 C. Then, feces were collected and quick-frozen in liquid nitrogen for analysis of microbial composition. After the piglets were euthanized, duodenum, jejunum, and ileum specimens were fixed in 10% phosphate-buffered formalin for morphological evaluation. The mucous membranes of the midcolumns and midcolumns were rapidly obtained and frozen in liquid nitrogen for gene expression characterization. All samples were stored at 80 C. until further analysis.

(17) 1.2 Establishment of Piglet-Based Model and Sample Detection

(18) 1.2.1 Piglet Plasma Sample Testing

(19) The contents of diamine oxidase (DAO) and immunoglobulins (including IgA, IgG and IgM) in piglet plasma were determined by ELISA.

(20) 1.2.2 Detection of Piglet Intestinal Microecology

(21) Intestinal samples were removed from 10% phosphate-buffered formalin, dehydrated through graded ethanol series (70%-100%), then cleared with xylene, and embedded in paraffin Serial sections (5 m thick) were taken. By using an imaging microscope, at least 15 intact and well-oriented villi and the associated crypt magnification for each fragment were measured. Villus height was measured from the villus tip to the villus-crypt junction, and crypt depth was defined as the invagination depth between adjacent villi.

(22) 1.2.3 High-Throughput 16S rRNA Sequencing for Piglet Faces

(23) The V3-V4 region of the 16S rRNA gene was amplified with primers and purified with Axy PrepDNA gel extraction kit. Then, the purified PCR products were pooled in equimolar amounts and sequenced on the platform.

(24) 1.2.4 Determination of Short Chain Fatty Acids in Piglet Feces

(25) Quantitative analysis was performed for short-chain fatty acids including acetate, propionate, and butyrate in piglet fecal samples by ion chromatography.

(26) 2. Results

(27) 2.1 Effects of GMF on Growth and Development of Piglets

(28) 2.1.1 Effect of GMF on Piglet Body Weight

(29) As shown in Table 3, body weight of the piglets in the GMF group significantly increased on day 8 and day 14 (P<0.05) compared with the piglets in the CON group. In addition, the average daily gain on days 1-8, 1-21 and the whole period (day 1-21) in the GMF group were significantly increased (P<0.05).

(30) TABLE-US-00003 TABLE 3 Effect of MFGM and LF on piglet body weight CON GMF P value Weight (kg) Day 8 2.33 0.08 2.62 0.08 0.022 Day 21 5.68 0.145 6.18 0.129 0.024 Average daily weight gain (g) Day 1-8 116.43 9.08 149.39 10.62 0.041 Day 1-21 197.86 6.66 221.56 5.54 0.019
2.1.2 Effect of GMF on IgG Concentration in Piglet Plasma

(31) As can be seen in Table 4, the IgG concentration in piglet plasma on day 21 was significantly increased after GMF feeding (p<0.05), while other parameters such as Glu, IgA, IGM, TG, HDL-c and LDL-c were not different. Butyrophilin (BTN), mucin (MUC), xanthine oxidoreductase (XOR), lactadherin (MFG-E8) and fatty acid binding protein (FABP) have different biochemical properties. Previous studies have shown that GMF supplementation in formula has growth-promoting effects on neonatal health and intestinal maturation in infants and animals, as indicated by the elevated plasma IgG levels in the present study.

(32) TABLE-US-00004 TABLE 4 Effect of MFGM and LF on piglet plasma on day 21 CON GMF P value GLU (mmol/L) 7.33 0.46 6.36 0.07 0.128 IgA (g/L) 0.85 0.06 0.92 0.05 0.379 IgG (g/L) 7.08 0.34 9.20 0.30 0.001 IgM (g/L) 1.02 0.06 0.90 0.09 0.257 TG (mmol/L) 1.18 0.08 1.11 0.11 0.658 HDL-C (mmol/L) 1.37 0.12 1.39 0.08 0.910 LDL-C (mmol/L) 2.08 0.09 2.20 0.21 0.650
2.1.3 Effects of GMF on Intestinal Morphological Tract Development of Piglets

(33) To determine the intestinal morphological development of the piglets, the villus height and crypt depth of the piglets were determined (Table 5). The results showed that the villus height of the duodenum and ileum of those in the GMF group could be significantly increased, while the depth of duodenal crypts was reduced (p<0.05).

(34) TABLE-US-00005 TABLE 5 Effect of MFGM on piglet intestinal microecology on day 21 CON GMF P value Villus height (m) Duodenum 416.70 18.09 480.45 19.20 0.042 Jejunum 423.50 22.58 399.00 18.86 0.429 Ileum 342.78 33.80 444.68 19.04 0.030 Crypt depth (m) Duodenum 184.92 13.69 112.93 5.35 0.001 Jejunum 135.43 10.27 107.07 9.71 0.080 Ileum 102.95 5.78 111.33 9.30 0.466 Villus height/crypt depth Duodenum 2.32 0.24 4.63 0.52 0.004 Jejunum 3.21 0.31 3.62 0.25 0.336 Ileum 3.71 0.52 4.47 0.52 0.332
2.2 Effects of GMF on Intestinal Flora of Piglets

(35) In order to study the differences of early microflora between CON and GMF piglets, 16S rRNA high-throughput sequencing technology was used to evaluate their microbial diversity, composition and differences.

(36) 2.2.1 Flora Composition of Piglets

(37) The fecal flora of piglets on day 8 is shown in FIG. 1. The -diversity index analysis showed that the diversity (Sobs) index of the GMF group decreased significantly (P<0.05) (FIG. 1A), while the Shannon index did not change (FIG. 1B). The -diversity, PCoA analysis showed significant differences between CON group and GMF group (FIG. 1C). From the perspective of flora composition, the differential flora of piglets showed that GMF could significantly enrich Lactobacillus and reduce unclassified bacteria (P<0.05) (FIG. 1D). Linear discriminant analysis effect size (LEfSe) analysis also confirmed a significant increase in Lactobacillus in piglets in the GMF group (FIG. 1E).

(38) The fecal flora of piglets on day 21 is shown in FIG. 2. For -diversity, FIG. 2A and FIG. 2B showed that the diversity (Sobs) index and Shannon index of the GMF group increased significantly. For -diversity, PCoA analysis showed that the CON group was significantly different from the GMF groups (FIG. 2C). The flora differences were shown at the genus level (FIG. 2D) the Bacteroides, Enterococcus, Christensenella, and Robusia showed an increasing trend, while members of the genus Eubacterium showed a decrease trend. The linear discriminant analysis effect size (LEfSe) analysis showed (FIG. 2E) a significant upward trend in the GMF group for Bacteroides, Enterococcus, Robustia, g_ruminant_UCG-002, g_Christensen_R-7_group, g_Marvinburi Antiaceae, g_CHKCI001 and unclassified bacteria.

(39) 2.2.2 Flora Functions of Piglets

(40) To further explore the functional profile of bacterial communities, we performed a phylogenetic study of the communities through PICRUSt using the KEGG database. As shown in FIG. 3A, on day 8, GMF increased glycolysis/gluconeogenesis, glycerolipid metabolism, MAPK signaling pathway, endophagy, flavonoid biosynthesis, and caffeine metabolism significantly, while genes related to porphyrin and chlorophyll metabolism and nitrogen metabolism were decreased. As shown in FIG. 3B, on day 21, GMF intervention significantly enriched methane metabolism, arginine and proline metabolism, oxidative phosphorylation, biosynthesis of phenylalanine, tyrosine and tryptophan, butyrate metabolism, lipid biosynthetic proteins, propionate metabolism, degradation of valine, leucine and isoleucine, -alanine metabolism, phenylalanine metabolism, tryptophan metabolism, RNA polymerase, degradation of limonene and pinene, but genes linked to other ion-coupled transporters and other transporters decreased.

(41) 2.3 Effects of GMF on Intestinal Functions of Piglets

(42) 2.3.1 Intestinal Barrier Functions of Piglets

(43) To clarify intestinal barrier function and intestinal permeability, the expression of intestinal barrier-related genes in mucosa (ileum and colon) and plasma DAO levels were detected. Ileal tight junction proteins (E-Cadherin, ZO-1) (FIG. 4A), mucins (mucin-1, mucin-2, mucin-4) (FIG. 4B), IL-22 (FIG. 4C) gene expression was significantly increased in the GMF group (P<0.05). Similarly, the gene expression of tight junction proteins (Occludin expression, connexin-1 and ZO-1) (FIG. 4D), mucin-20 (FIG. 4E) and cytokines (TNF- and IL-1) (FIG. 4F) was significantly elevated in the GMF group (P<0.05). Plasma DAO levels were significantly decreased in the GMF group (P<0.05) (FIG. 4G).

(44) 2.3.2 Concentration of SCFAs and Receptor Gene Expression in Feces of Piglets

(45) Concentrations of SCFAs in piglet feces and their receptor gene expression in the intestinal tract. The results showed that the concentrations of acetate, propionate and butyrate in the GMF group were all significantly higher than those in the CON group (p<0.05) (FIG. 5A). Furthermore, the gene expression of GPR41 in the colonic mucosa (FIG. 5B) in the GMF group increased. The increased short-chain fatty acid concentration and receptor thereof (GPR41) in the GMF group promoted propionic acid metabolism, regulated probiotic colonization and short-chain fatty acid metabolism, and finally activated enterocytes and enhanced intestinal barrier function.

(46) In summary, the piglet model experiments showed that oral administration of GMF (galactooligosaccharides, -lactalbumin powder, MFGM protein whey powder and polyfructoses) in piglets could significantly improve growth performance and reduce plasma IgG levels. The probiotic colonization (Lactobacillus, Enterococcus and Robusia) produced one week after birth promoted the production of short-chain fatty acids, and enhanced intestinal barrier function by increasing the expression of genes tight junctions (Occludin protein and ZO-1), mucins (Mucin-2 and Mucin-4) and cytokines (IL-1 and IL-22), thus improving piglet growth performance throughout the neonatal period. Our findings suggest that galactooligosaccharides, -lactalbumin powder, milk fat globule membrane protein and polyfructose play very important roles in regulating the intestinal microbiome in early infancy.

(47) Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of this disclosure, but not to limit thereto. Although this disclosure has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand modifications made to the technical solutions described in the foregoing embodiments, or equivalent replacements of some technical features thereof are possible, without making the essence of the corresponding technical solutions depart from the spirit and scope of the technical solutions of the embodiments of this disclosure.

INDUSTRIAL APPLICABILITY

(48) The infant formula milk powder rich in milk fat globule membrane protein, phospholipids and oligosaccharides of this disclosure is prepared by: using raw cow milk as raw material, added with MFGM-rich whey protein powder, -lactalbumin powder, galactooligosaccharides and polyfructoses, contents of biologically active substances with special functional ingredients such as MFGM-protein, lactoferrin (LF), -lactalbumin (-La), and the like are increased through formula adjustment, so as to promote the colonization of probiotics in the intestinal flora of infants, especially to significantly enrich the lactic acid bacteria in the intestinal tract, while reducing the unclassified bacteria and other miscellaneous bacteria, thereby increasing the content of immune factors in the intestinal tract and reducing the incidence of intestinal diseases. The formula is suitable for the development of formula milk powder for infants and older infants.