CN115644430A - Milk protein deep-hydrolyzed infant formula containing breast milk oligosaccharide LNnT - Google Patents

Milk protein deep-hydrolyzed infant formula containing breast milk oligosaccharide LNnT Download PDF

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CN115644430A
CN115644430A CN202211528079.6A CN202211528079A CN115644430A CN 115644430 A CN115644430 A CN 115644430A CN 202211528079 A CN202211528079 A CN 202211528079A CN 115644430 A CN115644430 A CN 115644430A
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lnnt
protein
formula
milk protein
milk
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李奋昕
李艳杰
关尚玮
孔小宇
刘彪
李放
王逸伦
闫雅璐
段素芳
司徒文佑
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Inner Mongolia Yili Industrial Group Co Ltd
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Abstract

The invention provides a milk protein deep hydrolysis infant formula containing breast milk oligosaccharide LNnT. Specifically, the invention provides a milk protein deep hydrolysis formula food, which contains lactose-N-neotetraose, wherein the total content of the lactose-N-neotetraose in the milk protein deep hydrolysis formula food is 21.4-857.2mg/100g based on the total dry matter of the milk protein deep hydrolysis formula food; the total protein content in the milk protein deep hydrolysis formula food is 12-18 g/100g, the total protein comprises hydrolyzed milk protein, and the protein with the molecular weight distribution of less than 1000dal accounts for more than 70% of the total protein. The invention also provides a preparation method and related application of the milk protein deep hydrolysis formula food.

Description

Milk protein deep-hydrolyzed infant formula containing breast milk oligosaccharide LNnT
Technical Field
The invention relates to a hypoallergenic formula food, in particular to a hypoallergenic milk protein deep hydrolysis infant formula food containing breast milk oligosaccharide LNnT, a preparation method and related applications thereof, and belongs to the technical field of special medical hypoallergenic foods for infants.
Background
In recent years, the incidence of food allergy (FH/FA) has been on the rise year by year and has become a focus of research. Among infant food allergies, cow milk protein and egg allergies are the most common. Avoidance therapy is currently used as a guideline opinion for cows' milk protein allergic children. In the process, a milk protein deep hydrolysis formulation (eHF) was used for diagnosis and treatment, and a milk protein partial (moderate) hydrolysis formulation (pHF) and a whole protein formulation were used for home reintroduction experiments. However, clinical studies show that long-term consumption of the deeply hydrolyzed formula avoids the need for a whole protein diet, which can lead to a certain degree of growth and development slowness in infants. Moreover, the long-term use of hydrolyzed formulas imposes an economic burden on the infant's family. Clinical data indicate that milk protein allergy populations, when tested for reintroduction, have a higher proportion of tolerance than whole protein formulations, and thus earlier transitions from extensively hydrolyzed to extensively hydrolyzed formulations. It is significant that the infant can improve the symptoms as soon as possible by the milk protein deep hydrolysis formula and then change from the milk protein deep hydrolysis formula to the whole protein formula as soon as possible.
Compared with the infants fed by breast milk, the infants not fed by breast milk have poor intestinal tract development. Microbial flora in the intestinal tract of the infant can be metabolized to generate Short Chain Fatty Acids (SCFA), and the SCFA can regulate various physiological functions of the organism and play an important role in regulating the health of the microenvironment of the intestinal tract. For example, acetic acid is an important source of host energy. While branched-chain short-chain fatty acids (BCFA) in the intestinal tract, such as isobutyric acid and isovaleric acid, which are produced by the metabolism of branched-chain amino acids such as valine, leucine, and isoleucine by the intestinal flora, are products of bacterial fermentation after undigested proteins and polypeptides reach the colon, mainly resulting from shedding of dietary or mucosal cells, and thus, the reduction of isobutyric acid and isovaleric acid can be regarded as a shift from protein fermentation to fiber fermentation, which is considered as a positive effect. Some studies report lower levels of isobutyric acid and isovaleric acid as measured in feces from whole breast-fed infants compared to those not receiving breast-feeding; milk protein allergic infants have higher concentrations and ratios of fecal branched short chain fatty acids than healthy infants.
In addition to the metabolism of the microbial flora in the infant's intestinal tract to produce short-chain fatty acids, harmful flora can invade the inside of the intestinal mucosa during the degradation process of the intestinal mucosa, mucopolysaccharides can be rapidly degraded into thiosulfate and free sulfate through an intermediate reaction, and finally toxic gas hydrogen sulfide is produced. In an inflammatory response, intestinal homeostasis is disrupted and thiosulfate can be oxidized to tetrathionate and promote further invasion by harmful bacteria.
Therefore, in the field of infant formula, there is a need for solutions to improve intestinal micro-health and to alleviate intestinal discomfort.
Disclosure of Invention
The invention aims to provide hypoallergenic lactoprotein deep hydrolysis formula food capable of improving intestinal microenvironment health.
The invention also aims to provide a preparation method of the hypoallergenic milk protein deep hydrolysis formula food.
The invention also aims to provide application of the hypoallergenic milk protein deep hydrolysis formula food.
The inventor of the present application finds in research that breast milk oligosaccharide lactose-N-neotetraose (LNnT) is beneficial to improving intestinal microenvironment health, particularly beneficial to improving infant intestinal microenvironment health, and can be specifically shown in the following steps: improving the content of acetic acid in the intestinal tract; reducing the production of intestinal isobutyric acid and/or isovaleric acid; reducing the production of intestinal hydrogen sulfide; and/or improving the ability of a subject to fight against an infection by an enteropathogenic bacterium, such as ETEC. Furthermore, the invention provides the hypoallergenic lactoprotein deep hydrolysis formula food capable of improving the intestinal microenvironment health by taking the breast milk oligosaccharide containing the lacto-N-neotetraose as a formula raw material.
Specifically, in one aspect, the invention provides a milk protein deep hydrolysis formula food, which contains lacto-N-neotetraose, wherein the total content of the lacto-N-neotetraose in the milk protein deep hydrolysis formula food is 21.4-857.2mg/100g based on the total dry matter of the milk protein deep hydrolysis formula food; the total protein content in the milk protein deep hydrolysis formula food is 12-18 g/100g, the total protein comprises hydrolyzed milk protein, and the protein with the molecular weight distribution of less than 1000dal accounts for more than 70% of the total protein.
According to a specific embodiment of the invention, the total content of lacto-N-neotetraose in the milk protein extensively hydrolyzed formula of the present invention is 42.8-428.6mg/100g.
According to a particular embodiment of the invention, the milk protein extensively hydrolyzed formula of the invention has a fat content of 15 to 29g/100g and a carbohydrate content of 50 to 58g/100g, based on the total dry matter of the milk protein extensively hydrolyzed formula.
According to a particular embodiment of the invention, the milk protein extensively hydrolysed formula of the invention is an infant formula powder. In some embodiments of the invention, the present invention provides a formula suitable for milk protein allergy high risk infants and infants with gastrointestinal dysfunction at the age of 0-1 year old, the formula comprising the breast milk oligosaccharide LNnT, and the formula is helpful for improving intestinal micro-health, relieving intestinal discomfort and improving the self-defense of pathogenic bacteria such as ETEC infection. In the milk protein deep hydrolysis formula food of the present invention, the total protein includes hydrolyzed milk protein, and the protein having a molecular weight distribution of 1000dal or less accounts for 70% or more, preferably 80% or more of the total protein, and has low allergenicity.
According to a specific embodiment of the invention, the milk protein deep hydrolysis formula food provided by the invention originally comprises one or more of hydrolyzed whey protein powder, hydrolyzed casein powder, hydrolyzed milk protein powder and hydrolyzed milk fat globule membrane protein.
According to a particular embodiment of the invention, the milk protein extensively hydrolyzed formula of the invention comprises a fat providing material comprising, in addition to a milk fat containing base material (e.g., bovine milk or an isolated fraction from bovine milk), vegetable oil and/or OPO structural fat. The vegetable oil can comprise one or more of MCT oil, sunflower oil, corn oil, soybean oil, low erucic acid rapeseed oil, coconut oil, palm oil and walnut oil, preferably sunflower oil, corn oil and soybean oil, and the vegetable oil is added to provide fat component, linoleic acid and alpha-linolenic acid for the product. In addition, the raw material for providing the fat may optionally include a raw material OPO structural fat added for providing the 1, 3-dioleoyl-2-palmitic acid triglyceride. Because the raw materials of OPO structure fat sold in the market at present have different purities, namely the content of the 1, 3-dioleate-2-palmitic acid triglyceride serving as the active ingredient is different and is usually about 40-70%, in the invention, in order to distinguish the 1, 3-dioleate-2-palmitic acid triglyceride serving as the active ingredient from the raw materials thereof, the term 1, 3-dioleate-2-palmitic acid triglyceride is adopted when describing the active ingredient, and the commonly known OPO structure fat is adopted when describing the food raw materials for providing the 1, 3-dioleate-2-palmitic acid triglyceride serving as the active ingredient. The specific addition amount of the OPO structural fat can be converted according to the content requirement of the 1, 3-dioleoyl-2-palmitic acid triglyceride in the milk powder product and the purity of the OPO structural fat raw material. More preferably, the milk oligosaccharide LNnT-containing formula comprises the following raw materials by weight based on 1000 parts of milk powder: MCT oil 0-110 weight portions and sunflower seed oil 0-150 weight portions; 0-40 parts of corn oil; 0-80 parts of soybean oil; 0 to 140 parts by weight of OPO structural grease.
According to a particular embodiment of the invention, in the milk protein extensively hydrolyzed formula of the invention, the raw material providing the carbohydrates is derived from lactose and non-lactose derived material comprising one or more of pregelatinized starch, maltodextrin, corn syrup solids, glucose syrup. That is, in the formula of the present invention, the raw material for providing carbohydrate may include lactose as a raw material and a starch-based material that is pre-hydrolyzed and gelatinized, in addition to the base material (e.g., milk) containing lactose. Preferably, the raw materials comprise, based on 1000 parts by weight of the formula powder: 0 to 580 parts of lactose and 0 to 580 parts of non-lactose substances. The specific amount of lactose added can be adjusted within the stated range so that the protein extensively hydrolyzed formula of the invention containing the breast milk oligosaccharide LNnT has a carbohydrate content of 50-58 g/100g.
According to a particular embodiment of the invention, the dairy protein extensively hydrolyzed formula of the invention further comprises one or more of DHA, ARA, nucleotides, lactoferrin, nutrients, probiotics. Preferably, the protein deep-hydrolysis formula powder containing the breast milk oligosaccharide LNnT comprises the following raw materials in parts by weight based on 1000 parts by weight: 8-15 parts of DHA and 14-28 parts of ARA; 0 to 0.7 weight portion of lactoferrin; 0-24 parts of 2' -fucosyllactose; 7 to 50 weight portions of compound nutrient containing calcium powder, vitamin and mineral substance.
In the protein deep hydrolysis formula food containing the breast milk oligosaccharide LNnT, the nutrients are the combination of the nutrient components meeting the national standard, and different addition amounts are used according to different formulas. According to the formula food, any one or any combination of the following compound nutrient components can be selectively adopted if the nutrient is added according to the needs. Preferably, the compound nutrient at least comprises compound vitamins, calcium powder and a mineral substance nutrition bag, and the dosage of each component is as follows:
1) Compounding vitamins, wherein each gram of the compounding vitamins comprises the following components:
taurine: 140 to 340mg
Vitamin A: 1700-5800 mu gRE
Vitamin D: 25-70 mug
Vitamin B 1 ：2000～6800μg
Vitamin B 2 ：3000～6900μg
Vitamin B 6 ：1700～4000μg
Vitamin B 12 ：8～20μg
Vitamin K 1 ：200～700μg
Vitamin C:0 to 700mg
Vitamin E: 10-70 mg of alpha-TE
Nicotinamide: 10000-41550 mu g
Folic acid: 350-920 mu g
Biotin: 70 to 245 mu g
Pantothenic acid: 7100-25230 mu g
Inositol: 0-250mg
L-carnitine: 0-60mg
2) Mineral two per gram:
sodium: 40-100 mg
Potassium: 200-500 mg
2) Mineral three, per gram mineral two:
calcium: 200-500 mg
Phosphorus: 75-300 mg
3) Mineral one, per gram of mineral one:
iron: 20-110 mg
Zinc: 23 to 90mg
Copper: 2000-4180 ug
Iodine: 500-995. Mu.g
Selenium: 0 to 200 mu g
Manganese: 0 to 579 mu g
4) Compounding magnesium chloride, wherein each gram of magnesium chloride is packaged:
magnesium: 80-170 mg
5) Choline chloride per gram of the choline chloride bag
Choline: 300-950 mg
The base material of the compound nutrient is preferably lactose, solid corn syrup or L-sodium ascorbate. Based on 1000 parts by weight of the milk protein deep hydrolysis formula powder containing the breast milk oligosaccharide LNnT, the addition amount of compound nutrients is 7-52 parts by weight, wherein the compound vitamin nutrition package is preferably 2-4 parts by weight, the mineral two nutrition package is preferably 2-16 parts by weight, the mineral three nutrition package is preferably 0.5-20 parts by weight, the mineral one nutrition package is preferably 0.5-3 parts by weight, magnesium chloride is 0-3.5 parts by weight, choline chloride is 0-4.5 parts by weight, and the base material of each nutrition package is preferably lactose or sodium L-ascorbate.
The compound materials used to provide each nutrient in the nutrient pack may interact. For example, sulfates can accelerate the oxidative destruction of vitamins, reducing their availability. Since sulfate is present in the form of ions in aqueous solution, it acts as an oxidizing agent in an oxidation reaction to induce oxidation of vitamins and destroy the structure of vitamins. The trace elements have different abilities in oxidation-reduction reaction, and the activities of copper, zinc and iron are the strongest, and the activities of manganese and selenium are the second order. The B vitamins and vitamin C are susceptible to copper ion, vitamin B 2 Is susceptible to iron ions.
To ensure the utilization efficiency of nutrients, the invention selects a stable nutrient formulation, such as: the vitamin A is retinyl acetate, and the retinol contains 1 hydroxyl and 5 double bonds and is very easy to oxidize, but the stability of the retinol is greatly improved in the form of acetate; vitamin E is selected from tocopherol acetate, tocopherol is also very unstable, but the stability of tocopherol acetate is improved a lot; vitamin B 1 Selecting thiamine nitrate, wherein the thiamine nitrate is more stable than thiamine hydrochloride in the existence form of thiamine; the vitamin C is L-sodium ascorbate.
The content of each component of the compound nutrient is the additive amount for strengthening the nutrient substance, and does not include the content of the nutrient components in other raw materials of the milk powder.
According to a specific embodiment of the present invention, in the protein-extensively hydrolyzed formula of the present invention, the probiotic is bifidobacterium. Preferably, the bifidobacterium is added in an amount of 0.1 to 0.2 parts by weight based on 1000 parts by weight of the protein deep hydrolysis formula powder containing breast milk oligosaccharide; still more preferably 0.18 to 0.2 parts by weight. More preferably, the bifidobacterium powder contains 3 x 10 bifidobacteria per weight part 10 Above CFU. Preference is given toThe probiotic is selected from: one or more of Bifidobacterium animalis subsp lactis BB-12, bifidobacterium infantis YLGB-1496, bifidobacterium animalis subsp lactis HN019 and Bifidobacterium lactis BL-99.
According to a preferred embodiment of the present invention, the formula of the present invention comprises the following raw materials:
Figure BDA0003973844070000061
it can be understood that in the milk protein deep hydrolysis formula food containing the breast milk oligosaccharide LNnT, the specific dosage of each raw material is determined by adjusting on the premise of meeting the index requirement of the formula milk powder product. In the milk protein deep hydrolysis formula food containing the breast milk oligosaccharide LNnT, product performance indexes which are not described or listed in detail are implemented according to the national standards of infant formula food or modified milk powder and the regulations of related standards and regulations.
In the milk protein deep hydrolysis formula food containing the breast milk oligosaccharide LNnT, all raw materials can be obtained commercially, and the selection of all raw materials meets the requirements of relevant standards, wherein the breast milk oligosaccharide LNnT meets the requirements of the invention. In addition, the compound nutrient can also be compounded by itself. "compounding" is used herein for convenience only and does not mean that the components of the formulation must be mixed together prior to use. All raw materials are added and used on the premise of meeting relevant regulations.
On the other hand, the invention also provides a method for preparing the milk protein deep hydrolysis formula powder of the breast milk oligosaccharide LNnT, and the preparation process mainly comprises the following steps: preparing materials, homogenizing, concentrating, sterilizing, spray drying, and dry mixing to obtain the final product. Specifically, the method for preparing the low-sensitivity milk protein deep hydrolysis formula powder containing the breast milk oligosaccharide LNnT comprises the following steps:
and mixing the parent lactose-N-neotetraose with other raw materials in the protein deep hydrolysis formula food by adopting a wet or dry production process to prepare the protein deep hydrolysis formula food.
According to a specific embodiment of the invention, the method for preparing the deep hydrolyzed formula powder containing the breast milk oligosaccharide LNnT comprises the following steps:
1) Adding powder: various powder raw materials are metered according to the formula and then uniformly added into a powder preparation tank through an air conveying system for storage.
2) Vacuum powder absorption: various powder raw materials in the powder mixing tank are sucked into the vacuum mixing tank through a vacuum system.
3) Dissolving and oil blending: the grease specified in the formula is put into an oil dissolving room according to the formula requirement, the temperature of the oil dissolving room is kept between 50 and 90 ℃, and after the oil is dissolved, the oil is pumped into a mixed oil storage tank through an oil pump and a flowmeter according to the formula proportion requirement.
4) And (3) mixed oil storage: the mixed oil is stored in an oil storage tank in a heat-insulating way at the temperature of 40-50 ℃ for less than 12 hours to prevent fat oxidation.
5) Weighing: the mixed oil is pumped into a mixing tank through an oil pump according to the formula requirement.
6) Dissolving and adding nutrients: calcium powder, mineral substances, vitamins and the like are added respectively, 100-200 kg of purified water is used for dissolving respectively, then the mixture is thrown into a wet mixing cylinder, and after each time of beating, the adding tank and the pipeline are flushed by 100kg of purified water.
7) Breast milk oligosaccharide LNnT solubilizing addition: and (3) dissolving the breast milk oligosaccharide LNnT raw material by using part of the mixed material liquid in the step (6), and adding the dissolved material liquid into a mixing tank to obtain the mixed material liquid containing the breast milk oligosaccharide.
8) And (3) filtering: filtering the mixed feed liquid by a filter screen to remove physical impurities possibly brought in the raw materials.
9) Homogenizing: homogenizing the mixed material liquid by a homogenizer, homogenizing the modified starch and the vegetable oil, then adding other substances and nutrients for secondary homogenization, wherein the primary pressure of the homogenization is 105 +/-5 bar, and the primary pressure is 32 +/-3 bar, and mechanically processing the fat globules to disperse the fat globules into uniform fat globules.
10 Cooling and storage: and (3) feeding the homogenized material liquid into a plate heat exchanger for cooling: cooling to below 20 ℃, temporarily storing in a pre-storage cylinder, entering the next procedure within 6 hours, and starting the stirrer according to the set requirement.
11 Concentration and sterilization: double-effect concentration is used during production, the sterilization temperature is more than or equal to 83 ℃, and the sterilization time is 25 seconds. The discharged material concentration is 48-52% dry matter.
12 Concentrated milk storage, pre-heating filtration, spray drying: the concentrated milk is temporarily stored in a concentrated milk balancing tank. Preheating to 60-70 deg.C by scraper preheater, filtering the preheated material by filter with 1mm aperture, pumping into drying tower by high pressure pump, spray drying, and agglomerating the fine powder at the tower top or fluidized bed as required. Air inlet temperature: 165-180 ℃, the exhaust temperature is 75-90 ℃, the high-pressure pump pressure is 160-210 bar, and the tower negative pressure is-4 to-2 mbar.
13 Fluidized bed drying and cooling: the powder from the drying tower is dried for the second time by the fluidized bed (the first stage) and then cooled to 25-30 ℃ by the fluidized bed (the second stage). Meanwhile, the breast milk oligosaccharide is mixed with a carrier and then heated to 60-65 ℃, and is uniformly dispersed on the surface of the powder under the action of compressed air, so that the powder particles are agglomerated to increase the granularity and instant solubility of the powder particles.
14 Subpackaging: and (3) weighing, bagging and subpackaging the DHA, the ARA lactoferrin and the bifidobacterium by powder workshop personnel according to the formula requirements.
15 Dry blending): and uniformly mixing the weighed DHA, ARA, lactoferrin, bifidobacterium and milk powder in a dry mixer.
16 Sieving powder: the granularity of the milk powder is uniform through the vibrating screen, and the powder slag is discarded.
17 Powder discharge: and (4) receiving the powder by using a sterilized powder collecting box, and conveying the powder to a powder feeding room from a powder discharging room.
18 Powdering: pouring the milk powder into a powder storage tank on a large and small packing machine according to the packing requirement.
19 Packaging: and (5) filling nitrogen for packaging by an automatic packaging machine of 800 g. The oxygen content is lower than 1% when charging nitrogen. The oxygen content of the 900 g iron can automatic nitrogen-filled package is lower than 5 percent.
20 ) boxing: and (4) filling the packaged small bags into a paper box, adding a powder spoon, and sealing by using a box sealing machine.
21 Inspection of finished products: and sampling and inspecting the packaged product according to an inspection plan.
22 Storage in warehouse: and warehousing and storing the qualified product at normal temperature with the humidity less than or equal to 65 percent.
On the other hand, the invention also provides application of the milk protein deep hydrolysis formula food in preparing foods for improving intestinal microenvironment health and increasing product tolerance. According to a specific embodiment of the invention, the improving the intestinal microenvironment health comprises: improving the content of acetic acid in intestinal tracts; reducing the amount of intestinal isobutyric acid and/or isovaleric acid; reducing the amount of intestinal hydrogen sulfide; and/or increasing the ability of an individual to fight infection by an enteropathogenic bacterium, such as ETEC.
In summary, the invention provides a milk protein deep hydrolysis formula food containing breast milk oligosaccharide LNnT and a preparation method and application thereof, and the formula food is beneficial to improving the intestinal health of people with high risk of milk protein allergy and gastrointestinal function discomfort, especially infants, and especially promoting the intestinal microenvironment health. In addition, the milk protein deep hydrolysis formula food containing the breast milk oligosaccharide LNnT can increase the product tolerance, shorten the using time of the hydrolysis formula for infants and use the whole protein formula as soon as possible.
Drawings
FIG. 1 shows the results of a small batch fermentation of individual HMO monomers to produce acetic acid in a simulated infant intestinal environment.
Figure 2 shows the results of a small batch fermentation of individual HMO monomers to produce acetic acid as a percentage of total short chain fatty acids in a simulated infant gut environment.
Fig. 3 shows the results of modeling the intestinal environment of infants with LNnT and four HMO monomers producing isobutyric acid.
Fig. 4 shows results of small batch fermentation of LNnT versus other HMO monomers to produce isobutyric acid as a percentage of total short chain fatty acids in a simulated infant gut environment.
Fig. 5 shows the results of LNnT versus other HMOs small batch fermentations to produce isovaleric acid in a simulated infant gut environment.
Fig. 6 shows the results of simulating the production of hydrogen sulfide as a percentage of total gas production by LNnT and four HMO monomers in an infant gut environment.
Figure 7 is a graph of the percentage of total acid produced by combined small-batch fermentation of LNnT with probiotics in a simulated infant gut environment.
Figure 8 is a graph of results of a small batch fermentation of LNnT in combination with probiotics to produce isovaleric acid in a simulated infant gut environment.
Figure 9 is a graph of the results of a small batch fermentation of LNnT in combination with probiotics to produce total short chain fatty acids in a simulated infant gut environment.
Figure 10 is a graph of the results of a small batch fermentation of LNnT in combination with probiotics to produce hydrogen sulfide as a percentage of total gas production in a simulated infant gut environment.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the technical aspects of the present invention with reference to specific examples, which are intended to illustrate the present invention and not to limit the scope of the present invention.
Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. In the examples, each breast milk oligosaccharide material was from the supplier Jennewein, and the content of breast milk oligosaccharides was determined by a method conventional in the art. The operating conditions not specified in detail in the examples were carried out according to the usual procedures in the art.
Example 1
This example provides a milk protein extensively hydrolyzed formula containing the breast milk oligosaccharide LNnT (1000 kg prepared):
120 kg of hydrolyzed whey protein powder, 115 kg of lactose, 100kg of modified starch, 335kg of solid corn syrup, 120 kg of high-oleic acid sunflower seed oil, 40 kg of corn oil, 50 kg of soybean oil, 80 kg of OPO structural fat, 0.428kg of LNnT, 38 kg of compound nutrient, 9 kg of DHA, 18 kg of ARA, 0.1 kg of bifidobacterium and 0.65kg of nucleotide.
The compound nutrient comprises about 3.0 kg of compound vitamin nutrient package, about 2.0 kg of choline chloride nutrient package, about 12 kg of calcium powder nutrient package, 16kg of sodium potassium nutrient package, about 2 kg of mineral nutrient package and about 3.0 kg of magnesium chloride nutrient package, and the base material of each nutrient package is solid corn syrup.
The specific preparation process of the milk protein deep hydrolysis formula powder containing the breast milk oligosaccharide LNnT comprises the following steps:
1) Adding powder: various powder raw materials are metered according to the formula and then uniformly added into a powder preparation tank through an air conveying system for storage.
2) Vacuum powder suction: various powder raw materials in the powder mixing tank are sucked into the vacuum mixing tank through a vacuum system.
3) Dissolving and preparing oil: the grease specified in the formula is put into the oil-dissolving chamber according to the formula requirement, the temperature of the oil-dissolving chamber is kept between 50 and 90 ℃, and after the oil is dissolved, the oil is pumped into a mixed oil storage tank through an oil pump and a flowmeter according to the formula proportion requirement.
4) And (3) mixed oil storage: the mixed oil is stored in an oil storage tank in a heat-insulating way at the temperature of 40-50 ℃ for less than 12 hours to prevent fat oxidation.
5) Weighing: and pumping the mixed oil into a mixing tank through an oil pump according to the formula requirement.
6) Dissolving and adding nutrients: calcium powder, mineral substances, vitamins and the like are added respectively, 100-200 kg of purified water is used for dissolving respectively, then the mixture is thrown into a wet mixing cylinder, and after each time of beating, the adding tank and the pipeline are flushed by 100kg of purified water.
7) Breast milk oligosaccharide LNnT solubilizing addition: and (4) dissolving the breast milk oligosaccharide LNnT by using part of the mixed feed liquid in the step (6), and adding the dissolved solution into a mixing tank to obtain the mixed feed liquid containing the breast milk oligosaccharide LNnT.
8) And (3) filtering: filtering the mixed feed liquid by a filter screen to remove physical impurities possibly brought in the raw materials.
9) Homogenizing: homogenizing the mixed material liquid by a homogenizer, homogenizing the modified starch and the vegetable oil, then adding other substances and nutrients for secondary homogenization, wherein the primary pressure of the homogenization is 105 +/-5 bar, and the primary pressure is 32 +/-3 bar, and mechanically processing the fat globules to disperse the fat globules into uniform fat globules.
10 Cooling and storage: and (3) feeding the homogenized material liquid into a plate heat exchanger for cooling: cooling to below 20 ℃, temporarily storing in a pre-storage cylinder, entering the next procedure within 6 hours, and starting the stirrer according to the set requirement.
11 Concentration and sterilization: double-effect concentration is adopted during production, the sterilization temperature is more than or equal to 83 ℃, and the sterilization time is 25 seconds. The discharged material concentration is 48-52% dry matter.
12 Concentrated milk storage, pre-heating filtration, spray drying: the concentrated milk is temporarily stored in a concentrated milk balancing tank. Preheating to 60-70 deg.C by scraper preheater, filtering the preheated material by filter with 1mm pore diameter, pumping into drying tower by high pressure pump, spray drying, and agglomerating fine powder at the tower top or fluidized bed as required. Air inlet temperature: 165-180 ℃, the exhaust temperature is 75-90 ℃, the high-pressure pump pressure is 160-210 bar, and the tower negative pressure is-4 to-2 mbar.
13 Fluidized bed drying and cooling: the powder from the drying tower is dried for the second time by the fluidized bed (the first stage) and then cooled to 25-30 ℃ by the fluidized bed (the second stage). Meanwhile, the breast milk oligosaccharide is mixed with a carrier and then heated to 60-65 ℃, and the mixture is uniformly dispersed on the surface of the powder under the action of compressed air, so that the powder particles are agglomerated to increase the granularity and the instant solubility of the powder particles.
14 Subpackaging: and (3) weighing, bagging and subpackaging DHA, ARA lactoferrin and bifidobacterium by powder making workshop personnel according to the formula requirements.
15 Dry blending): and uniformly mixing the weighed DHA, ARA, lactoferrin, bifidobacterium and milk powder in a dry mixer.
16 Sieving powder: the granularity of the milk powder is uniform through the vibrating screen, and the powder residue is discarded.
17 Powder discharge: and (4) receiving the powder by using a sterilized powder collecting box, and conveying the powder to a powder feeding room from a powder discharging room.
18 Powdering: pouring the milk powder into a powder storage tank on a large and small packaging machine according to the packaging requirements.
19 Packaging: and (5) filling nitrogen for packaging by an automatic packaging machine of 800 g. The oxygen content is lower than 1% when charging nitrogen. The oxygen content of the 900 g iron can in the automatic nitrogen-filled package is lower than 5 percent.
20 ) boxing: and (4) filling the packaged small bags into a paper box, adding a powder spoon, and sealing by using a box sealing machine.
21 Inspection of finished products: and sampling and inspecting the packaged product according to an inspection plan.
22 ) warehousing and storing: and warehousing and storing the qualified product, wherein the storage is required to be carried out at normal temperature, and the humidity is less than or equal to 65%.
Wherein the product contains 12g/100g of protein, 29g/100g of fat, 55g/100g of carbohydrate and 42.8mg/100g of lactose-N-neotetraose.
Example 2
This example provides a milk protein extensively hydrolyzed formula powder (1000 kg prepared) containing the breast milk oligosaccharide LNnT:
180 kg of hydrolyzed whey protein powder, 150 kg of modified starch, 350 kg of lactose, 100kg of high oleic acid sunflower seed oil, 20 kg of corn oil, 50 kg of soybean oil, 110 kg of OPO structural fat, 1 kg of breast milk oligosaccharide LNnT, 38 kg of compound nutrient, 3.5 kg of DHA, 7.2 kg of ARA, 0.1 kg of bifidobacterium and 0.65kg of nucleotide.
The compound nutrient comprises about 3.0 kg of compound vitamin nutrient package, about 2.0 kg of choline chloride nutrient package, about 12 kg of calcium powder nutrient package, 16kg of sodium potassium nutrient package, about 2 kg of mineral nutrient package and about 3.0 kg of magnesium chloride nutrient package, and the base material of each nutrient package is solid corn syrup.
The product preparation process was as in example 1.
Wherein the product contains 14.4g/100g of protein, 28g/100g of fat, 50g/100g of carbohydrate and 100mg/100g of lactose-N-neotetraose.
Example 3
This example provides a milk protein extensively hydrolyzed formula powder (1000 kg prepared) containing the breast milk oligosaccharide LNnT:
140 kg of hydrolyzed whey protein powder, 120 kg of modified starch, 410 kg of solid corn syrup, 30 kg of MCT grease, 30 kg of high-oleic acid sunflower seed oil, 20 kg of corn oil, 70 kg of soybean oil, 140 kg of OPO structural fat, 1.5 kg of breast milk oligosaccharide LNnT, 38 kg of compound nutrient, 8.5 kg of DHA, 18 kg of ARA, 0.2 kg of bifidobacterium and 0.65kg of nucleotide.
The compound nutrients comprise 3.0 kg of compound vitamin nutrient package, 2.0 kg of choline chloride nutrient package, 12 kg of calcium powder nutrient package, 16kg of sodium potassium nutrient package, 2 kg of mineral nutrient package and 3.0 kg of magnesium chloride nutrient package, and the base material of each nutrient package is solid corn syrup.
The product preparation process was as in example 1.
Wherein the product contains 11.2g/100g of protein, 29g/100g of fat, 53g/100g of carbohydrate and 150mg/100g of lactose-N-neotetraose powder.
Example 4
This example provides a milk protein extensively hydrolyzed formula containing the breast milk oligosaccharide LNnT (1000 kg prepared):
120 kg of hydrolyzed whey protein powder, 115 kg of lactose, 80 kg of modified starch, 355 kg of solid corn syrup, 120 kg of high-oleic acid sunflower seed oil, 40 kg of corn oil, 50 kg of soybean oil, 80 kg of OPO (o) structural fat, 2 kg of breast milk oligosaccharide LNnT, 38 kg of compound nutrient, 9.5 kg of DHA (docosahexaenoic acid), 19.4 kg of ARA (alpha-linolenic acid), 0.1 kg of bifidobacterium and 0.65kg of nucleotide.
The compound nutrient comprises about 3.0 kg of compound vitamin nutrient package, about 2.0 kg of choline chloride nutrient package, about 12 kg of calcium powder nutrient package, 16kg of sodium potassium nutrient package, about 2 kg of mineral nutrient package and about 3.0 kg of magnesium chloride nutrient package, and the base material of each nutrient package is solid corn syrup.
The product preparation process was as in example 1.
Wherein the product contains 9.6g/100g of protein, 29g/100g of fat, 55g/100g of carbohydrate and 200mg/100g of lactose-N-neotetraose.
Example 5
This example provides a milk protein extensively hydrolyzed formula powder (1000 kg prepared) containing the breast milk oligosaccharide LNnT:
180 kg of hydrolyzed whey protein powder, 380 kg of lactose, 120 kg of modified starch, 50 kg of MCT grease, 40 kg of high oleic acid sunflower seed oil, 10 kg of corn oil, 50 kg of soybean oil, 110 kg of OPO structural fat, 2.5 kg of breast milk oligosaccharide LNnT, 38 kg of compound nutrient, 3.8 kg of DHA, 7.8 kg of ARA, 0.1 kg of bifidobacterium and 0.65kg of nucleotide.
The compound nutrient comprises about 3.0 kg of compound vitamin nutrient package, about 2.0 kg of choline chloride nutrient package, about 12 kg of calcium powder nutrient package, 16kg of sodium potassium nutrient package, about 2 kg of mineral nutrient package and about 3.0 kg of magnesium chloride nutrient package, and the base material of each nutrient package is solid corn syrup.
The product preparation process is as in example 1.
Wherein the product contains 14.4g/100g of protein, 26g/100g of fat, 50g/100g of carbohydrate and 250mg/100g of lactose-N-neotetraose.
Example 6
This example provides a milk protein extensively hydrolyzed formula powder (1000 kg prepared) containing the breast milk oligosaccharide LNnT:
140 kg of hydrolyzed whey protein powder, 150 kg of modified starch, 350 kg of solid corn syrup, 60 kg of high-oleic acid sunflower seed oil, 20 kg of corn oil, 70 kg of soybean oil, 140 kg of OPO structural fat, 3 kg of breast milk oligosaccharide LNnT, 38 kg of compound nutrient, 9.5 kg of DHA, 19.5 kg of ARA, 0.2 kg of bifidobacterium and 0.65kg of nucleotide.
The compound nutrient comprises about 3.0 kg of compound vitamin nutrient package, about 2.0 kg of choline chloride nutrient package, about 12 kg of calcium powder nutrient package, 16kg of sodium potassium nutrient package, about 2 kg of mineral nutrient package and about 3.0 kg of magnesium chloride nutrient package, and the base material of each nutrient package is solid corn syrup.
The product preparation process is as in example 1.
Wherein the product contains 11.2g/100g of protein, 29g/100g of fat, 53g/100g of carbohydrate and 300mg/100g of lactose-N-neotetraose.
Example 7
This example provides a milk protein extensively hydrolyzed formula containing the breast milk oligosaccharide LNnT (1000 kg prepared):
120 kg of hydrolyzed whey protein powder, 100kg of lactose, 80 kg of modified starch, 370 kg of solid corn syrup, 90 kg of MCT grease, 10 kg of high-oleic acid sunflower seed oil, 40 kg of corn oil, 50 kg of soybean oil, 80 kg of OPO structural fat, 10.5 kg of breast milk oligosaccharide composition, 38 kg of compound nutrient, 7 kg of DHA, 14 kg of ARA, 0.1 kg of bifidobacterium and 0.65kg of nucleotide.
The compound nutrient comprises about 3.0 kg of compound vitamin nutrient package, about 2.0 kg of choline chloride nutrient package, about 12 kg of calcium powder nutrient package, 16kg of sodium potassium nutrient package, about 2 kg of mineral nutrient package and about 3.0 kg of magnesium chloride nutrient package, and the base material of each nutrient package is solid corn syrup.
The product preparation process was as in example 1.
The protein content is 9.6g/100g, the fat content is 27g/100g, and the carbohydrate content is 55g/100g. 2' -fucosyllactose in the product: 700mg/100g. The amount of lacto-N-neotetraose in the product is; 350mg/100g.
Example 8
This example provides a milk protein extensively hydrolyzed formula powder (1000 kg prepared) containing the breast milk oligosaccharide LNnT:
200kg of hydrolyzed whey protein powder, 400 kg of lactose, 100kg of modified starch, 90 kg of high oleic acid sunflower seed oil, 10 kg of corn oil, 50 kg of soybean oil, 100kg of OPO structure fat, 12.686 kg of breast milk oligosaccharide composition, 38 kg of compound nutrient, 3 kg of DHA, 6kg of ARA, 0.1 kg of bifidobacterium and 0.65kg of nucleotide.
The compound nutrient comprises about 3.0 kg of compound vitamin nutrient package, about 2.0 kg of choline chloride nutrient package, about 12 kg of calcium powder nutrient package, 16kg of sodium potassium nutrient package, about 2 kg of mineral nutrient package and about 3.0 kg of magnesium chloride nutrient package, and the base material of each nutrient package is solid corn syrup.
The product preparation process was as in example 1.
The protein content is 16g/100g, the fat content is 25g/100g, and the carbohydrate content is 50g/100g. 2' -fucosyllactose in the product: 840mg/100g. The amount of lacto-N-neotetraose in the product is; 428.6mg/100g.
Experiment I for regulating acid production and gas production of intestinal tract by breast milk oligosaccharide LNnT
In the invention, the air pressure, gas components and the content of short-chain fatty acid of a product after fermentation are measured by simulating a fermentation experiment in an infant intestinal environment, and the effect of regulating and controlling intestinal acid production and gas production by breast milk oligosaccharide LNnT is investigated.
Collecting samples: stool samples of infants fed with 3-6 months old breast milk or formula powder were selected. Collecting one oral swab, one fresh breast milk and one corresponding infant feces of each mother in the breast feeding group during the month of the month; collecting one part of excrement of each infant in the formula powder artificial feeding group. Fresh feces were obtained from donors, transported to the laboratory in ice bags over 4 hours, fermented, and the pressure, gas composition, short chain fatty acids of the fermentation product were measured.
1. Preparation of culture medium
(1) Preparing YCFA anaerobic basic culture medium, and subpackaging 30ml of the YCFA anaerobic basic culture medium into anaerobic penicillin bottles with the total volume of 50ml for later use.
The formula of the YCFA anaerobic basal medium is as follows (g/L): tryptone 10, yeast extract 2.5, L-cysteine hydrochloride 1, naCl 0.9, caCl 2 ·6H 2 O 0.009，KH 2 PO 4 0.45，K 2 HPO 4 0.45，MgSO 4 ·7H 2 O0.09；
Also comprises the following components: 1mL of resazurin (1 mg/mL), 2mL of heme (5 mg/mL) and 200 mu L of vitamin I solution;
wherein the vitamin I solution comprises (mg/mL): biotin (VH) 0.05, cobalamin (VB 12) 0.05, p-aminobenzoic acid 0.15, folic acid 0.25, pyridoxamine (VB 6) 0.75.
(2) The culture medium required by the embodiment of the invention is prepared.
Before fermentation experiments, breast milk oligosaccharides (prebiotics) are added to the YCFA anaerobic basal medium as required to form the culture medium required by the embodiment of the invention. The final concentration of each breast milk oligosaccharide added in the embodiment of the invention in the culture medium is 4 per mill.
The added breast milk oligosaccharides (prebiotics) involved in the fermentation experiments are shown in table 1. Wherein each culture medium is divided into an ETEC adding group or an ETEC not adding group, wherein the final concentration of added ETEC in the culture medium with added ETEC group is 10 10 CFU/mL。
TABLE 1 list of fermentation conditions
Figure BDA0003973844070000161
2. In vitro fermentation
(1) Preparation of samples before fermentation:
accurately weighing 0.800 +/-0.010 g of fresh excrement, putting the fresh excrement into one side of a stirring spoon of an excrement pretreatment box, and calculating and supplementing PBS buffer solution with corresponding volume according to the mass-volume ratio of 10%. Vortex for about 5-10 minutes, break the fecal debris well, and mix well with PBS buffer to prepare a uniform 10% fecal suspension (w/v). Standing the feces pretreatment box on a table, and filtering by two layers of filter screens to obtain turbid liquid for later use.
(2) Inoculation: in the anaerobic workstation, 0.5mL of suspension (clear side in the pretreatment box) is sucked by a 1mL syringe and a No. 5 needle, and a butyl rubber plug of a penicillin bottle is punctured and a culture medium is injected.
Wherein, inoculation and dynamic sampling are completed in an anaerobic workstation, and 5 biological replicates are respectively arranged in each culture medium of a breast feeding group and an artificial feeding group.
And (4) subpackaging the residual excrement original sample and the excrement turbid liquid according to the requirement, marking and freezing for other detection. After thawing the frozen fecal sample within 30 minutes, it was gently mixed with the culture medium, added to the batch fermentation medium as the initial culture material, and the solution was continuously mixed to maintain the desired degree of mixing uniformity. Because the thawing time was consistent, the initial bacterial composition was similar for each group.
(3) Fermentation:
if gas production analysis is needed, before fermentation, the pressure of the penicillin bottle is detected and recorded by a barometer for 0 hour of fermentation. Then placing the penicillin bottle in a constant temperature box at 37 ℃ for standing culture for 24 hours without disturbance.
After the culture is finished, the small bottle is taken out, is not opened, and is directly frozen at-20 ℃ for detection.
3. Gas detection
The fermentation vial was taken out, the pressure at the end of fermentation (24 hours) was detected and recorded with a barometer, and the gas composition was detected with a gas analyzer (HL-QT 01, hailu biotechnology limited, han).
Specifically, the instrument consists of a gas sampler, valve module, vacuum generator, and gas detection chamber that integrates a plurality of gas sensors. The gas distribution module controls the amount of gas introduced into the gas detection chamber by means of a vacuum generator. The detection steps are as follows: (1) detecting gas in the blank culture medium, and calibrating an instrument; (2) adjusting the gas detection chamber to a certain vacuum level by using a vacuum generator through a gas distribution module; (3) the gas in the small bottle is sucked into a detection chamber of the instrument through a gas sampler, and the volume of the gas is adjusted through a gas distribution module; (4) detecting CO entering the gas detection chamber by using corresponding gas sensors respectively 2 ，H 2 ，CH 4 ，H 2 S is 4 gases in total;
(5) the gas ratio was calculated by preset software.
4. Short chain fatty acid detection
Short chain fatty acid concentrations, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, were determined using a gas chromatograph (9720, fuli, zhejiang, inc.). The method comprises the following specific steps:
(1) Preparing before sample introduction: using a sterile needle to suck 500 mu L of fermentation liquid, placing the fermentation liquid into a 1.5ml centrifuge tube, adding 100 mu L of crotonic acid metaphosphoric acid solution, and freezing the solution at-30 ℃ for 24h. After thawing, centrifugation was carried out at 10000rpm at 4 ℃ for 3min, and the supernatant was collected and filtered through a 0.22 μm filter (Millipore), and 100. Mu.L of the sample extract was taken out and put into a vial for gas phase sample, and the vial was closed with a cap to remove air bubbles, and then subjected to sample analysis.
(2) The gas chromatography instrument conditions were as follows: a chromatographic column: agilent FFAP 30m × 0.25mm × 0.25 μm; column temperature: heating to 180 deg.C at 75 deg.C/min for 1min, heating to 220 deg.C at 50 deg.C/min for 1min; a sample inlet: temperature: 250 ℃, sample size: 1.0 μ L, split ratio: (5; carrier gas: high purity nitrogen; flow rate: 2.5mL/min for 6.5min,2.8mL/min2 rising to 2.8mL/min for 2min; a detector: FID; temperature: 250 ℃; tail blowing: 20mL/min; hydrogen gas: 30mL/min; air: 300mL/min.
(3) And (4) carrying out quantitative determination by using a peak area internal standard method, and automatically calculating by using software built in a workstation according to a standard curve equation internal standard method.
5. Results of efficacy investigation experiments
The detection results of the small-batch fermentation of various HMO monomers to generate acetic acid in the simulated infant intestinal environment are shown in figure 1, and the significant difference and P value of LNnT and four HMO monomers and a control group are shown in table 2.
TABLE 2
Whether there is a significant difference P value
Lnnt blank vs **** <0.0001
2’-FL vs.LNnT * 0.0147
3-FL vs.LNnT **** <0.0001
LNnT vs.LNT **** <0.0001
LNnT vs.3’-SL **** <0.0001
It can be seen that LNnT increased acetic acid in the feces fermentation group of breast-fed and formula-fed infants, compared to the four HMO monomers and control group, regardless of whether ETEC was added to simulate diarrhea. The effect of LNnT is more remarkable and is better than that of other HMO monomers.
Results simulating small batch fermentation of individual HMO monomers in the infant intestinal environment to produce acetic acid as a percentage of total short chain fatty acids are shown in fig. 2, and significant differences and P-values for lnnt and four HMO monomers and controls are shown in table 3.
TABLE 3
Whether there is a significant difference P value
Lnnt blank vs **** <0.0001
2’-FL vs.LNnT ** 0.0045
3-FL vs.LNnT **** <0.0001
LNnT vs.LNT **** <0.0001
LNnT vs.3’-SL **** <0.0001
It can be seen that LNnT increased the proportion of acetic acid in the feces fermentation group of breast-fed and formula-fed infants, compared to the four HMO monomers and control group, regardless of whether ETEC was added to simulate diarrhea. The effect of LNnT is more remarkable and is better than that of other HMO monomers.
The results of simulating the production of isobutyric acid from LNnT and four HMO monomers in the infant intestinal environment are shown in fig. 3, and the significant differences and P values are shown in table 4.
TABLE 4
Figure BDA0003973844070000181
It can be seen that in the formula fed infant fecal fermentation group, whether or not ETEC was added to simulate diarrhea, the addition of HMO monomer reduced the production of isobutyric acid compared to the blank. The effect of adding LNnT is remarkable, and is better than 3-FL, 3'-SL and LNT when ETEC is not available, and the effect is equivalent to that of 2' -FL; in the presence of ETEC, LNnT was superior to 3'-SL and LNT, and was comparable to 2' -FL and 3-FL.
Results of simulating LNnT in the infant intestinal environment versus small batch fermentation of other HMO monomers to produce isobutyric acid as a percentage of total short chain fatty acids are shown in fig. 4, with significant differences and P values in table 5.
TABLE 5
Whether there is a significant difference P value
Lnnt blank vs **** <0.0001
LNnT vs.2’-FL ns >0.9999
LNnT vs.3-FL **** <0.0001
LNnT vs.LNT **** <0.0001
LNnT vs.3’-SL **** <0.0001
It can be seen that in the feces fermentation group of breast milk and formula fed infants, whether or not ETEC was added to mimic diarrhea, the addition of HMO monomer may reduce the production of isobutyric acid compared to the blank. The effect of adding LNnT is remarkable, and is better than 3-FL, 3'-SL and LNT when ETEC exists or not, and the effect is equivalent to that of 2' -FL.
The results of the small-batch fermentation of LNnT in the environment of simulated infant intestinal tract compared with other HMOs to produce isovaleric acid are shown in FIG. 5, and the significant difference and P value are shown in Table 6.
TABLE 6
Figure BDA0003973844070000191
It can be seen that in the formula fed infant fecal fermentation group, the addition of HMO monomer may reduce the production of isovaleric acid compared to the blank, whether or not ETEC was added to simulate diarrhea. The effect of adding LNnT is remarkable, and is better than 3-FL and 3'-SL without ETEC, and the effect is equivalent to that of LNT and 2' -FL. LNnT is superior to 3' -SL in the presence of ETEC.
The results of simulating the percentage of hydrogen sulfide produced by LNnT and four HMO monomers in the total gas production in the infant intestinal environment are shown in fig. 6, and the significant difference and P values are shown in table 7.
TABLE 7
Whether there is a significant difference P value
Lnnt blank vs **** <0.0001
2’-FL vs.LNnT Whether or not 0.9992
3-FL vs.LNnT **** <0.0001
LNnT vs.LNT **** <0.0001
LNnT vs.3’-SL **** <0.0001
It can be seen that in the feces fermentation group of breast-fed and formula-fed infants, the addition of HMO monomer affected the production of hydrogen sulfide whether or not ETEC was added to simulate diarrhea. The effect of adding LNnT is remarkable and is superior to 3-FL, 3'-SL and LNT, and the effect is equivalent to 2' -FL.
Experiment II for regulating and controlling intestinal acid production and gas production by combining breast milk oligosaccharide LNnT and probiotics
According to the invention, the air pressure, gas components and the content of short-chain fatty acid of a product after fermentation are measured through a fermentation experiment under the environment of simulating the intestinal tract of an infant, and the effect of regulating and controlling the acid production and the gas production of the intestinal tract by the combination of breast milk oligosaccharide LNnT and probiotics is investigated.
The added probiotics and prebiotics involved in the experiment are shown in table 8. Wherein each culture medium is divided into two cases of adding or not adding ETEC. ETEC was added to a final concentration of 10 10 CFU/mL。
TABLE 8 fermentation conditions List
Figure BDA0003973844070000201
2. Strain activation and identification
Respectively taking bacterial strains BB12, YLGB-1496, HN019 and BL-99 bacterial powder, and preparing the bacterial strains to 10 percent in an anaerobic workstation 7 CFU/mL, using plate count method for detecting concentration. Before preparing culture medium, taking out the glycerol tube strain preserved in a refrigerator at-80 deg.C, inoculating in MRS culture medium for activation, and inoculating the activated bacteria liquid to corresponding culture medium with injectorIn the formula (I).
Taking bacterium powder of strain ETEC (ATCC 35401), and preparing the powder to 10 in an anaerobic workstation 10 CFU/mL, using plate count method for detecting concentration.
Other operations of the experimental method are basically combined with breast milk oligosaccharide LNnT and probiotics to regulate and control intestinal acid production and gas production effects.
The combination of LNnT and probiotics in small batch fermentation simulated the infant intestinal environment to produce isovaleric acid as a percentage of total acid, as shown in fig. 7.
It can be seen that the combination of LNnT with probiotics was superior to the blank and four probiotic alone acting groups in all experimental groups. And in the formula powder group, the combination of LNnT and probiotics produced isovaleric acid in a lower percentage of total acid than LNnT.
Results of small batch fermentation of LNnT in combination with probiotics to produce isovaleric acid in a simulated infant gut environment are shown in fig. 8. It can be seen that the combination of LNnT with probiotics outperformed the blank and the four probiotics alone in all experimental groups.
The significant differences and P-values between LNnT and probiotic combinations and LNnT are shown in table 9. As can be seen from table 9, the combination of LNnT and probiotic bacteria significantly reduced isovaleric acid (P < 0.0001) compared to LNnT, which represents a synergistic effect of the combination of LNnT and probiotic bacteria. There was no significant difference between LNnT and the combination of the four probiotics, respectively, with a greater tendency for LNnT + BL-99 to reduce isovaleric acid compared to LNnT + BB12 (P = 0.1254).
TABLE 9
Figure BDA0003973844070000211
Figure BDA0003973844070000221
The combination of LNnT and probiotic (LNnT + BL-99 works best, the effect of HN019 and YLGB-1496 plus escherichia coli) produced lower percentage of isovaleric acid in total acid than LNnT and probiotic alone.
The results of a small batch fermentation of LNnT in combination with probiotics in the infant intestinal environment were simulated to produce total short chain fatty acids as shown in fig. 9. It can be seen that the combination of LNnT with probiotics outperformed the blank and the four probiotics alone in all experimental groups.
Further comparisons between groups were made with combinations of LNnT and different probiotics and the results are shown in table 10.
TABLE 10
Whether there is a significant difference P value
LNnT+BB12
Breast milk-without ETEC-total acid vs. formula powder-without ETEC-total acid ** 0.0014
LNnT+YLGB-1496
Breast milk-without ETEC-total acid vs. formula powder-without ETEC-total acid * 0.0114
LNnT+HN019
Breast milk-without ETEC-total acid vs. formula powder-without ETEC-total acid *** 0.0004
LNnT+BL-99
Breast milk-without ETEC-total acid vs. formula powder-without ETEC-total acid **** <0.0001
Breast milk with ETEC total acid vs formula powder with ETEC total acid * 0.0229
It can be seen from table 10 that the four compositions produced higher total acid levels in the formula than the breast milk group (P < 0.05) in the absence of ETEC. Of these, LNnT and BL-99 in the presence of ETEC still produced more short chain fatty acids in the formula than in the breast milk group, suggesting that it contributes to a healthier intestinal environment (P = 0.0229).
Results of small batch fermentation of combinations of LNnT and probiotics to produce hydrogen sulfide as a percentage of total gas production in simulated infant intestinal environments are shown in fig. 10. It can be seen from figure 10 that LNnT in combination with probiotics outperformed the blank and the four probiotic alone acting groups in all experimental groups.
The results of further comparing the different groups are shown in table 11.
TABLE 11
Whether there is a significant difference P value
Breast milk _ without ETEC vs. breast milk _ with ETEC *** 0.0008
Breast milk-without ETEC vs. formula powder-without ETEC **** <0.0001
Breast milk with ETEC vs formula powder with ETEC *** 0.001
Formula powder _ without ETEC vs. formula powder _ with ETEC ns 0.6134
As can be seen from table 11, stool from formula-fed infants resulted in higher production of hydrogen sulfide in the gut compared to the breast-fed group with or without ETEC. The presence or absence of ETEC in the breast-feeding group produced a significant difference, and not in the formula group, indicating that ETEC was not a major factor in hydrogen sulfide production when formula was fed, and conversely, the presence of ETEC may result in more hydrogen sulfide gas production in breast-fed infants.
Further comparison of the group without ETEC against breast milk shows that the hydrogen sulfide produced by the combination of LNnT + BL-99 is lower than that of LNnT monomer (P < 0.05), and that LNnT + BB12 and LNnT + YLGB-1496 have no significant difference but significant trend, i.e. P <0.1, with LNnT, indicating that the combination of LNnT and probiotics can produce synergistic gain effect. See table 12 for comparative results.
TABLE 12
Whether there is a significant difference P value
LNnT vs.LNnT+BB12 ns 0.0516
LNnT vs.LNnT+YLGB-1496 ns 0.0516
LNnT vs.LNnT+HN019 ns 0.1113
LNnT vs.LNnT+BL-99 * 0.0304
LNnT+BB12vs.LNnT+YLGB-1496 ns >0.9999
LNnT+BB12vs.LNnT+HN019 ns 0.9803
LNnT+BB12vs.LNnT+BL-99 ns 0.9957
LNnT+YLGB-1496vs.LNnT+HN019 ns 0.9803
LNnT+YLGB-1496vs.LNnT+BL-99 ns 0.9957
LNnT+HN019vs.LNnT+BL-99 ns 0.8869
No significant difference was observed in hydrogen sulfide production under different fermentation conditions for the mother's milk ETEC group. The results of the comparison are shown in Table 13.
Watch 13
Whether there is a significant difference P value
LNnT vs.LNnT+BB12 ns 0.7561
LNnT vs.LNnT+YLGB-1496 ns 0.4407
LNnT vs.LNnT+HN019 ns 0.6746
LNnT vs.LNnT+BL-99 ns 0.5078
LNnT+BB12vs.LNnT+YLGB-1496 ns 0.9753
LNnT+BB12vs.LNnT+HN019 ns 0.9998
LNnT+BB12vs.LNnT+BL-99 ns 0.9855
LNnT+YLGB-1496vs.LNnT+HN019 ns 0.9937
LNnT+YLGB-1496vs.LNnT+BL-99 ns >0.9999
LNnT+HN019vs.LNnT+BL-99 ns 0.9969
In the ETEC-free formula powder group, LNnT + YLGB-1496 and LNnT + BL-99 produce less hydrogen sulfide than LNnT (P < 0.05), LNnT + BB12 and LNnT + HN019 do not have significant difference with LNnT, but have significant trend, namely P <0.1, which shows that the combination of LNnT and probiotics can synergically and more reduce the production of hydrogen sulfide. The results of the comparison are shown in Table 14.
TABLE 14
Whether there is a significant difference P value
LNnT vs.LNnT+BB12 ns 0.3447
LNnT vs.LNnT+YLGB-1496 * 0.0477
LNnT vs.LNnT+HN019 ns 0.0638
LNnT vs.LNnT+BL-99 * 0.0313
LNnT+BB12vs.LNnT+YLGB-1496 ns 0.3272
LNnT+BB12vs.LNnT+HN019 ns 0.4431
LNnT+BB12vs.LNnT+BL-99 ns 0.2013
LNnT+YLGB-1496vs.LNnT+HN019 ns 0.9987
LNnT+YLGB-1496vs.LNnT+BL-99 ns 0.9936
LNnT+HN019vs.LNnT+BL-99 ns 0.9596
Watch 15
Whether there is a significant difference P value
LNnT vs.LNnT+BB12 ns 0.5117
LNnT vs.LNnT+YLGB-1496 ns 0.1455
LNnT vs.LNnT+HN019 ns 0.1728
LNnT vs.LNnT+BL-99 ns 0.1586
LNnT+BB12vs.LNnT+YLGB-1496 ns 0.5939
LNnT+BB12vs.LNnT+HN019 ns 0.6786
LNnT+BB12vs.LNnT+BL-99 ns 0.6362
LNnT+YLGB-1496vs.LNnT+HN019 ns >0.9999
LNnT+YLGB-1496vs.LNnT+BL-99 ns >0.9999
LNnT+HN019vs.LNnT+BL-99 ns >0.9999
Table 15 analysis showed that no significant difference in hydrogen sulfide production was observed under different fermentation conditions in the ETEC group of formula powders.

Claims (10)

1. A milk protein deep hydrolysis formula food contains lactose-N-neotetraose, and the total content of the lactose-N-neotetraose in the milk protein deep hydrolysis formula food is 21.4-857.2mg/100g based on the total dry matter of the milk protein deep hydrolysis formula food; the total protein content in the milk protein deep hydrolysis formula food is 12-18 g/100g, the total protein comprises hydrolyzed milk protein, and the protein with the molecular weight distribution of less than 1000dal accounts for more than 70% of the total protein.
2. The milk protein deep hydrolysis formula of claim 1, wherein the total content of lacto-N-neotetraose in the milk protein deep hydrolysis formula is 42.8-428.6mg/100g.
3. A milk protein extensively hydrolyzed formula according to claim 1, wherein the milk protein extensively hydrolyzed formula has a fat content of 15 to 29g/100g and a carbohydrate content of 50 to 58g/100g, based on the total dry matter of the milk protein extensively hydrolyzed formula.
4. A milk protein extensively hydrolysed formula according to any one of claims 1 to 3, which is an infant formula powder.
5. The milk protein deep hydrolysis formula of any one of claims 1 to 4, wherein the protein providing source comprises one or more of hydrolyzed whey protein powder, hydrolyzed casein powder, hydrolyzed milk protein powder, and hydrolyzed milk fat globule membrane protein.
6. A milk protein deep hydrolysis formula according to any of claims 2 to 5, wherein the fat providing raw material comprises vegetable oil and/or OPO structural fat in addition to the base raw material comprising milk fat;
the carbohydrate-providing raw material is derived from lactose and non-lactose derived materials, the non-lactose derived materials including one or more of pregelatinized starch, maltodextrin, corn syrup solids, glucose syrup;
preferably, the milk protein deep hydrolysis formula further comprises one or more of DHA, ARA, nucleotides, lactoferrin, nutrients, probiotics;
more preferably, the probiotic is a bifidobacterium, more preferably selected from: one or more of Bifidobacterium animalis subsp lactis BB-12, bifidobacterium infantis YLGB-1496, bifidobacterium animalis subsp lactis HN019 and Bifidobacterium lactis BL-99.
7. A milk protein deep hydrolysis formula according to any of the claims 1 to 6, for use in:
improving the content of acetic acid in the intestinal tract;
reducing the amount of intestinal isobutyric acid and/or isovaleric acid;
reducing the amount of intestinal hydrogen sulfide; and/or
Improving the ability of the individual to resist infection by pathogenic bacteria such as ETEC.
8. A process for the preparation of a milk protein extensively hydrolyzed formula according to any one of claims 1 to 7, which comprises:
mixing the parent lactose-N-neotetraose with other raw materials in the protein deep hydrolysis formula food by adopting a wet method or dry method production process to prepare the protein deep hydrolysis formula food;
preferably, the raw materials for providing the total protein in the raw materials of the protein deep hydrolysis formula food comprise one or more of hydrolyzed whey protein powder, hydrolyzed casein powder, hydrolyzed milk protein powder and hydrolyzed milk fat globule membrane protein.
9. Use of a milk protein extensively hydrolysed formula according to any one of claims 1 to 7 in the manufacture of a food product for improving gut microenvironment health and increasing product tolerance.
10. The use of claim 9, wherein the improving gut microenvironment health comprises:
improving the content of acetic acid in intestinal tracts;
reducing the amount of intestinal isobutyric acid and/or isovaleric acid;
reducing the amount of intestinal hydrogen sulfide; and/or
Improving the ability of the individual to resist infection by pathogenic bacteria such as ETEC.
CN202211528079.6A 2022-11-30 2022-11-30 Milk protein deep-hydrolyzed infant formula containing breast milk oligosaccharide LNnT Pending CN115644430A (en)

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CN114223729A (en) * 2021-11-30 2022-03-25 内蒙古伊利实业集团股份有限公司 Application of bifidobacterium longum subsp. infantis and breast milk oligosaccharide composition in formula milk powder
CN114258961A (en) * 2021-11-30 2022-04-01 内蒙古伊利实业集团股份有限公司 Application of bifidobacterium lactis and breast milk oligosaccharide composition in infant formula food
CN114568526A (en) * 2020-11-30 2022-06-03 内蒙古伊利实业集团股份有限公司 Mother emulsified infant formula powder for improving intestinal microenvironment health and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106259953A (en) * 2015-06-01 2017-01-04 内蒙古伊利实业集团股份有限公司 Breast milk 0-12 month babies ' formula milk powder containing nucleotide and preparation method thereof
CN114568526A (en) * 2020-11-30 2022-06-03 内蒙古伊利实业集团股份有限公司 Mother emulsified infant formula powder for improving intestinal microenvironment health and application thereof
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