Application of bifidobacterium and breast milk oligosaccharide composition in infant formula milk powder

Abstract

The invention belongs to the field of food application, and relates to application of a bifidobacterium and breast milk oligosaccharide composition in infant formula milk powder. According to the process, 2 '-fucosyllactose is taken as a carbon source, the 2'-fucosyllactose can be applied to infant food bifidobacterium through national approval, a growth curve, a pH value, acid resistance, cholate resistance, surface hydrophobicity and self-coagulation capacity are taken as indexes, and a combination and a proportion with the best synergistic effect are selected and applied to infant formula milk powder. The infant formula milk powder is closer to breast milk, the supplement effect of prebiotics and probiotics is fully played, and a thought is provided for preparation of the novel infant formula milk powder.

Description

Technical Field

[0001] The present invention belongs to the field of infant formula milk powder, and is about the application of bifidobacterium and human milk oligosaccharide composition in the preparation of infant formula milk powder. Background Art

[0002] Human milk oligosaccharides (HMOs) are complex oligosaccharides with diverse structures in human milk. Their content in human milk is second only to lactose and lipids, and they are the third largest nutrient in human milk. They play a key role in the health and growth and development of infants and young children. 2'-fucosyllactose (2'-FL) has the highest content among the more than 200 HMOs in human milk, accounting for about 30%. It has biological functions such as regulating the intestinal microecology of infants, preventing pathogens from adhering to the intestines, immunomodulatory effects, and promoting brain nerve development. 21% of mothers do not have fucosyltransferase 2, a key enzyme for the synthesis of 2'-FL in the mammary gland. Their breast milk does not contain or contains only a small amount of 2'-FL and other α-1,2-fucosylated HMOs. Therefore, it is very important to add 2'-FL to infant formula.

[0003] Bifidobacterium is a recognized safe probiotic that colonizes the human gastrointestinal tract during infancy and is the dominant genus in the intestines of breastfed infants. It has beneficial effects such as improving constipation, enhancing immunity, and maintaining intestinal balance. Bifidobacteria metabolize 2'-FL, which produces more diverse metabolites and abundant short-chain fatty acids compared to glucose, lowers intestinal pH, further creates an acidic intestinal environment, inhibits the growth and colonization of harmful bacteria, and thus regulates the composition of infant intestinal flora. The 2'-FL metabolic process is strain-specific, and different bifidobacteria use 2'-FL in different mechanisms, which ultimately leads to different utilization capabilities.

[0004] As of February 2023, the bifidobacteria approved by the state for use in infant foods include Bifidobacterium animalis lactis subspecies Bb-12, HN019 and Bi-07, Bifidobacterium breve M-16V, Bifidobacterium longum infantis subspecies R0033 and BB536, and Bifidobacterium bifidum R0071. The National Health Commission has again solicited opinions on 2”-fucosyllactose as a “new variety of food nutrient fortifier” and determined that the application range of 2-fucosyllactose in modulated milk powder (only for children’s milk powder), infant formula, formula for older infants and toddlers, and formula for special medical purposes is 0.7-2.4g / L (based on the ready-to-eat state, the powdered product increases the usage according to the multiple of preparation). However, for the bifidobacteria approved by the state for use in infant foods that have a good synergistic effect with 2'-FL, no one has studied it yet, nor has the optimal amount of 2'-FL been determined. Summary of the invention

[0005] This study is based on the fact that the process of bifidobacterium metabolizing 2'-FL is strain-specific, thus providing the following inventions:

[0006] One of the purposes of the present invention is to provide a bifidobacterium and 2'-FL composition with good synergistic effect and good probiotic function.

[0007] A second object of the present invention is to provide an infant formula milk powder containing human milk oligosaccharides and bifidobacteria.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A composition containing human milk oligosaccharides and probiotics with good prebiotic function, wherein the human milk oligosaccharides are 2'-fucosyllactose, and the probiotics are selected from bifidobacteria approved by the state for use in infant food; the ratio of the human milk oligosaccharides to the probiotics is 1.9 g / L:10 9 cfu / g.

[0010] In a preferred embodiment of the present invention, the probiotics include: a combination of Bifidobacterium animalis subsp. lactis Bb-12, Bifidobacterium bifidum R0071, Bifidobacterium longum subsp. infantis R0033, Bifidobacterium animalis subsp. lactis HN019, Bifidobacterium animalis subsp. lactis Bi-07, Bifidobacterium longum subsp. longum BB536, and Bifidobacterium breve M-16V.

[0011] In a preferred embodiment of the present invention, the probiotics include one or any combination of Bifidobacterium bifidum R0071, Bifidobacterium longum subsp. infantis R0033 and Bifidobacterium breve M-16V.

[0012] In a preferred embodiment of the present invention, the probiotics are a combination of Bifidobacterium bifidum R0071 and Bifidobacterium breve M-16V.

[0013] In a preferred embodiment of the present invention, the probiotics include lactobacillus and bifidobacterium, and the weight ratio of the human milk oligosaccharide to bifidobacterium is 1:4.

[0014] The composition of the present invention comprises specific human milk oligosaccharide 2'-FL and specific probiotics Bifidobacterium bifidum R0071 and Bifidobacterium breve M-16V, which cooperate with each other to work synergistically and have more live bacteria and probiotic functions.

[0015] The second object of the present invention is achieved by adopting the following technical solution:

[0016] An infant formula milk powder containing human milk oligosaccharides and bifidobacteria comprises a composition of the purpose, wherein the composition accounts for 5% of the infant formula milk powder.

[0017] After activation and cultivation, the probiotics Bifidobacterium bifidum R0071 and Bifidobacterium breve M-16V were collected by centrifugation, resuspended in sterile water, mixed with maltodextrin sterilized by ultraviolet for more than 30 minutes, vacuum freeze-dried and crushed into probiotic powder. The two kinds of powders were dissolved again, gradient diluted and plated for counting, and the content of live bacteria in various powders was recorded. The two kinds of powders were prepared in the above order in a ratio of 3:1 to prepare composite probiotic powder, and the probiotic content in the composite probiotic powder was 10 9 CFU / g.

[0018] Skim milk powder 40g, whole milk powder 25g, desalted whey powder 25g, peanut oil 4g, probiotics 4g, 2'-FL 1g / 100g (1.9g / L), vitamin A 559.0μg, vitamin D 7.8μg, vitamin E 8.2mg, vitamin K1 53.0μg, vitamin B1640.0μg, vitamin B2 1190.0μg, vitamin B6 448.0μg, vitamin B12 2.3μg, vitamin C 96.2mg, niacin 3980.0μg, folic acid 79.5μg, niacin 3980.0μg, folic acid 79.5μg, pantothenic acid 3790.0μg, biotin 24.2μg, choline 126.0mg, calcium 419.0mg, iron 5.5mg, zinc 5.4mg, sodium 174.0mg, magnesium 52.5mg, copper 398.0μg, manganese 326.0μg, phosphorus 278.0mg, iodine 74.5μg, chlorine 334.0mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1-2 This is the growth curve of seven strains of Bifidobacterium when 2'-FL is used as the carbon source in Example 1 of the present invention.

[0020] Figure 3-4 The pH values ​​of seven strains of Bifidobacterium when 2'-FL is used as the carbon source in Example 1 of the present invention.

[0021] Figure 5 The figure shows the acid resistance of seven strains of Bifidobacterium when 2'-FL is used as the carbon source in Example 1 of the present invention.

[0022] Figure 6 The bile salt tolerance of seven strains of Bifidobacterium in Example 1 of the present invention when 2'-FL is used as the carbon source.

[0023] Figure 7 The surface hydrophobicity of seven strains of Bifidobacterium when 2'-FL is used as the carbon source in Example 1 of the present invention.

[0024] Figure 8 The self-aggregation ability of seven strains of Bifidobacterium when 2'-FL is used as the carbon source in Example 1 of the present invention.

[0026] Fig. 9 The growth curves of different proportions of different bifidobacterium combinations with 2'-FL as the carbon source in Example 2 of the present invention are shown in FIG.

[0027] Figure 10-12 The number of colonies of different proportions of different bifidobacterium combinations when 2'-FL is used as the carbon source in Example 2 of the present invention is

[0028] Fig.13 The effect of different 2'-FL addition amounts on the viable colony count of combined Bifidobacterium in Example 3 of the present invention

[0029] Fig.14 The effect of different 2'-FL addition amounts on the pH of combined Bifidobacterium in Example 3 of the present invention DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically noted, the raw materials used in the embodiments of the present invention can be obtained commercially: If not specifically noted, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0031] The inventors of this case have proved through specific experiments that the probiotic-prebiotic composition of the present invention has good synergistic effects.

[0032] The experimental methods and test substances used in each embodiment and control are as follows:

[0033] Experimental preparation steps. Activation and cultivation of test strains: Bifidobacterium in glycerol tubes was inoculated into MRS medium at 3% (v / v), cultured anaerobically at 37°C for 48 hours, single bacteria were selected for anaerobic culture at 37°C for 18-20 hours, streaked on solid plates for 3 subcultures, and inoculated into culture medium with different carbon sources for subculture.

[0034] Example 1 Bifidobacterium animalis subsp. lactis Bb-12, Bifidobacterium bifidum R0071, Bifidobacterium longum subsp. infantis R0033, Bifidobacterium animalis subsp. lactis HN019, Bifidobacterium animalis subsp. lactis Bi-07, Bifidobacterium longum subsp. longum BB536, Bifidobacterium breve M-16V utilize 2-fucosyllactose, comprising the following steps:

[0035] (1) Growth curve. Bifidobacterium was inoculated into culture medium with 2'-FL and GLU as carbon sources and cultured anaerobically at 37°C for 60 h. Samples were taken every 4 h and OD was measured. 600 nm absorbance value.

[0036] (2) pH value. Bifidobacterium was inoculated in culture medium containing 2'-FL and GLU as carbon sources and cultured anaerobically at 37°C for 60 h. Samples were taken every 4 h to measure pH value.

[0037] (3) Sugar utilization and consumption rate. The 2'-FL content in the bifidobacterium culture medium was determined by HPLC. Take 5 ml of the fermentation broth, centrifuge at 12000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm membrane, and detect it by HPLC. HPLC detection conditions: differential refractive index detector; chromatographic column is RezexROA-organic acid H (8%) (henomenex, USA), column temperature is 50°C; mobile phase is 5 mM sulfuric acid solution, flow rate is 0.6 m / min; injection volume is 10 μL, detector temperature is 35°C.

[0038] Table 1 Sugar consumption rate of seven strains of Bifidobacterium when 2'-FL was used as carbon source

[0039] (4) Acid resistance. Sterilized 12M HCl solution was added to the culture medium with 2'-FL and GLU as the sole carbon source, and the pH was adjusted to 3.0. Bifidobacterium was inoculated into the culture medium, and the culture medium without acid treatment was used as a control. The culture medium was placed in a 37°C constant temperature incubator for anaerobic culture for 2 hours and then the plate was plated for counting.

[0040] (5) Bile salt tolerance. Bifidobacterium was inoculated into a culture medium containing 0.3% ox bile salt and 2'-FL and GLU as the sole carbon source, respectively, and the corresponding culture medium without ox bile salt was used as a control. The culture medium was placed in a 37°C constant temperature incubator for anaerobic culture for 4 hours and then the plates were plated and counted.

[0041] (6) Surface hydrophobicity. Bifidobacterium was inoculated into culture medium with 2'-FL and GLU as the sole carbon source, respectively, and cultured at 37°C under anaerobic conditions for 24 hours. The culture was centrifuged (4°C, 6000r, 5min), and the supernatant was discarded to obtain a precipitate. The bacterial sludge was washed twice with sterile PBS, and then suspended in a sterile PBS solution and mixed thoroughly. The concentration of the bacterial suspension was adjusted so that the absorbance (OD600nm) reached 0.4±0.02 (A0). Subsequently, 800μL of the bacterial suspension and 160μL of xylene were successively aspirated, shaken and mixed, and allowed to stand (25°C, 1h). The supernatant was discarded, the aqueous phase was aspirated, and A1 was measured at 600nm.

[0042] Surface hydrophobicity (%) = (1-A1 / A0) × 100%

[0043] (7) Self-aggregation ability. Bifidobacterium was inoculated into culture medium with 2'-FL and GLU as the sole carbon source, respectively, and cultured at 37°C under anaerobic conditions for 24 hours. Centrifugation was performed (4°C, 6000r, 5min), and the supernatant was discarded to obtain a precipitate. The bacterial sludge was washed twice with sterile PBS, and then suspended in a sterile PBS solution and mixed thoroughly. The concentration of the bacterial suspension was adjusted so that the absorbance (OD600nm) reached 0.35±0.02 (A0). 800μL of the bacterial suspension was vortexed for 20s and allowed to stand (37°C, 2h). The supernatant was aspirated and A1 was measured at 600nm.

[0044] Self-aggregation ability (%) = (1-A1 / A0) × 100%

[0045] Example 2 Bifidobacterium breve M-16V-Bifidobacterium longum infantis subsp. R0033, Bifidobacterium breve M-16V-Bifidobacterium bifidum R0071, Bifidobacterium breve M-16V: Bifidobacterium longum infantis subsp. R0033 use 2-fucosyllactose in different proportions, and the bifidobacterium mixing ratio is 1:1; 1:2; 2:1; 1:3; 3:1. The following steps are included:

[0046] (1) Growth curve Bifidobacterium was inoculated in a culture medium with 2'-FL as the carbon source and cultured anaerobically at 37°C for 60 h. Samples were taken every 4 h and the OD was measured. 600 nm absorbance value.

[0047] (2) pH value Bifidobacterium was inoculated into a culture medium containing 2'-FL as a carbon source and cultured anaerobically at 37°C for 60 h. Samples were taken every 4 h to measure the pH value.

[0048] (3) When the number of live colonies reaches the end of the logarithmic phase, dilution plates are plated.

[0049] Example 3 Effect of adding 0.7, 1.0, 1.3, 1.6, 1.9, 2.2, 2.4 g / L 2-fucosyllactose on the combination of Bifidobacterium breve M-16V and Bifidobacterium longum infantis subsp. R0071 = 3:1. The following steps are included:

[0050] (1) Colony viability The bifidobacteria in the glycerol tube were inoculated into skim milk medium at 3% (v / v), cultured anaerobically at 37°C for 24 h, and diluted and plated when the culture reached the end of the logarithmic phase.

[0051] (2) pH Bifidobacterium was inoculated into skim milk medium with 2'-FL as the carbon source and cultured anaerobically at 37°C for 24 h, and the pH value was measured.

[0052] Example 4 Preparation of an infant formula milk powder containing 2-fucosyllactose and bifidobacteria

[0053] (1) After activating and culturing the probiotics Bifidobacterium bifidum R0071 and Bifidobacterium breve M-16V, the cells were collected by centrifugation, resuspended in sterile water, mixed with maltodextrin sterilized by ultraviolet light for more than 30 minutes, vacuum freeze-dried and crushed into probiotic powder. The two kinds of bacterial powder were dissolved again, gradient diluted and plated for counting, and the content of live bacteria in each kind of bacterial powder was recorded. The two kinds of bacterial powder were prepared in the above order in a ratio of 3:1 to prepare a composite probiotic powder, and the probiotic content in the composite probiotic powder was 10 9 CFU / g.

[0054] (2) Infant formula: skim milk powder 40g, whole milk powder 25g, desalted whey powder 25g, peanut oil 4g, probiotics 4g, 2'-FL 1g / 100g (1.9g / L), vitamin A 559.0μg, vitamin D 7.8μg, vitamin E 8.2mg, vitamin K1 53.0μg, vitamin B1640.0μg, vitamin B2 1190.0μg, vitamin B6 448.0μg, vitamin B122.3μg, vitamin C 96.2mg, niacin 3980.0μg, folic acid 79.5μg, niacin 3980.0μg, folic acid 79.5μg, pantothenic acid 3790.0μg, biotin 24.2μg, choline 126.0mg, calcium 419.0mg, iron 5.5mg, zinc 5.4mg, sodium 174.0mg, magnesium 52.5mg, copper 398.0μg, manganese 326.0μg, phosphorus 278.0mg, iodine 74.5μg, chlorine 334.0mg.

[0055] (3) Preparation of infant formula milk powder

[0056] The preparation process is: mixing ingredients (adding minerals, heat-stable vitamins, vegetable oils, etc.) → homogenization → cooling → sterilization, concentration → spray drying → dry mixing ingredients (adding oligosaccharides, probiotics and heat-stable vitamins, etc.) → packaging → finished product inspection

[0057] Homogenizing pressure: 15MPa; spray drying temperature: inlet 150℃, outlet 70℃, inlet 160℃, outlet 75-80℃, inlet 170℃, outlet 85℃-90℃.

[0058] The present invention illustrates a kind of infant formula milk powder containing human milk oligosaccharide and bifidobacterium provided by the present invention through the above-mentioned embodiment, but the above-mentioned is only the best embodiment of the present invention. It should be pointed out that the above embodiment is only used to illustrate the present invention, and is not used to limit the protection scope of the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements can also be made, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A nutritional composition comprising or consisting of: - human milk oligosaccharides; and -Probiotics. 2 . The nutritional composition according to claim 1 , wherein the human milk oligosaccharide is 2′-fucosyllactose.

3. The nutritional composition according to claim 1 or 2, wherein the probiotic is selected from one or more of Bifidobacterium animalis lactis subsp. Bb-12, Bifidobacterium bifidum R0071, Bifidobacterium longum infantis subsp. R0033, Bifidobacterium animalis lactis subsp. HN019, Bifidobacterium animalis lactis subsp. Bi-07, Bifidobacterium longum subsp. BB536, and Bifidobacterium breve M-16V; preferably Bifidobacterium bifidum R0071, Bifidobacterium longum infantis subsp. R0033, and Bifidobacterium breve M-16V, more preferably Bifidobacterium bifidum R0071 and Bifidobacterium breve M-16V.

4. The nutritional composition according to any one of claims 1 to 3, wherein the human milk oligosaccharides are provided in the form of natural sources, and / or synthetic sources, and / or bacterial fermentation sources.

5. The nutritional composition according to any one of claims 1 to 4, wherein the added amount of 2'-fucosyllactose is preferably 0.7-2.4 g / L, more preferably 1.9 g / L.

6. The nutritional composition according to any one of claims 1 to 5, wherein the ratio of Bifidobacterium bifidum R0071 to Bifidobacterium breve M-16V is 1:1, 1:2, 2:1, 1:3, 3:1, more preferably 3:

1.

7. The nutritional composition according to any one of claims 1 to 6, wherein the added amount of Bifidobacterium bifidum R0071-Bifidobacterium breve M-16V is 10 7 cfu / g.

8. An infant formula containing human milk oligosaccharides and probiotics, comprising the nutritional composition of claim 1, wherein the composition accounts for 5% of the infant formula, and further comprises skim milk powder, whole milk powder, desalted whey powder, peanut oil, vitamins, niacin, folic acid, pantothenic acid, biotin, choline, calcium, iron, zinc, sodium, magnesium, copper, manganese, phosphorus, iodine, and chlorine.

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