CN115644431A

CN115644431A - Formula food containing breast milk oligosaccharide for premature infant and preparation method and application thereof

Info

Publication number: CN115644431A
Application number: CN202211705101.XA
Authority: CN
Inventors: 李奋昕; 李艳杰; 关尚玮; 孔小宇; 刘彪; 李放; 王逸伦; 段素芳; 司徒文佑
Original assignee: Inner Mongolia Yili Industrial Group Co Ltd
Current assignee: Inner Mongolia Yili Industrial Group Co Ltd
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2023-01-31
Anticipated expiration: 2042-12-29
Also published as: CN115644431B

Abstract

The invention provides a formula food for premature infants containing breast milk oligosaccharide, and a preparation method and application thereof. Specifically, the invention provides a preterm infant formula comprising breast milk oligosaccharides including lacto-N-neotetraose, wherein the total content of lacto-N-neotetraose in the preterm infant formula is 21.4-857.2mg/100g based on the total dry matter of the preterm infant formula; and the total protein content in the formula food for the premature infant is 12-18g/100 g, the fat content is 15-29g/100 g, and the carbohydrate content is 50-58g/100 g. The invention also provides a preparation method and related application of the formula food for premature infants.

Description

Formula food containing breast milk oligosaccharide for premature infant and preparation method and application thereof

Technical Field

The invention relates to a formula food for premature infants, in particular to a formula food for premature infants containing breast milk oligosaccharide, a preparation method and related application thereof, belonging to the technical field of special medical lactose intolerance foods for infants.

Background

Most premature infants, especially ELBWI and/or VLBWI, have the defects of neonatal pulmonary hyaloplasmia, arterial duct patency, chronic lung disease and the like after, unstable vital signs, the need of ventilator assistance treatment and liquid intake limitation, and the possibility of repeated feeding intolerance and necrotizing intestinal colitis and the like, and the immaturity of the self conditions limits the breast feeding of the premature infants during hospitalization. The risk of allergy in preterm infants over term was high, and a questionnaire was included with 138 milk-allergic infants and 101 allergy-free healthy infants to evaluate the risk factors for milk allergy in infants up to 1 year of age, in which the risk of milk protein allergy in preterm infants was 2.5 times higher than in term infants. Early exposure to milk protein increased the risk of allergy, a prospective, case-controlled study with a total of 359 high-atopic risk infants, of which 279 in the intervention group (diet + environmental intervention) and 80 in the non-intervention group; the non-intervention group received breast feeding, normal milk protein formula feeding, or weaning diet without environmental advice. The main observation indicators are anthropometric indicators and allergic disease manifestations, with increased risk of gastrointestinal disease + urticaria, atopic dermatitis, and general allergic symptoms in infants exposed to the common milk protein formula at week 1 after birth.

On the other hand, infants who do not receive breast feeding have an intestinal development that is less robust than breast-fed infants. 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, 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 from shedding of dietary or mucosal cells, and therefore, the reduction of isobutyric acid and isovaleric acid can be regarded as a positive effect by switching from protein fermentation to fiber fermentation. 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 attack by harmful bacteria.

Therefore, in the field of preterm infant formulas, solutions are needed that are hypo-sensitive and improve intestinal micro-health.

Disclosure of Invention

It is an object of the present invention to provide a preterm infant formula that improves intestinal microenvironment health.

It is another object of the present invention to provide a method for preparing the preterm infant formula.

It is another object of the present invention to provide the use of said preterm infant formula.

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 intestinal tracts; reducing the production of intestinal isobutyric acid and/or isovaleric acid; reducing the production of intestinal hydrogen sulfide; and/or increasing the ability of an individual to fight infection by an enteropathogenic bacterium, such as ETEC. Furthermore, the present invention provides a preterm infant formula that improves intestinal microenvironment health by adding breast milk oligosaccharides including lacto-N-neotetraose to the preterm infant formula.

In particular, in one aspect, the present invention provides a preterm infant formula comprising breast milk oligosaccharides including lacto-N-neotetraose in a total amount of 21.4 to 857.2mg/100g of lacto-N-neotetraose in the preterm infant formula based on total dry matter of the preterm infant formula; and the total protein content in the formula food for the premature infant is 12-18g/100 g, the fat content is 15-29g/100 g, and the carbohydrate content is 50-58g/100 g.

According to a particular embodiment of the invention, the total content of lacto-N-neotetraose in the preterm formula of the invention is from 42.8 to 428.6mg/100g.

According to a particular embodiment of the invention, in the preterm infant formula according to the invention, the total protein comprises hydrolysed milk protein, having a degree of hydrolysis between 8 and 23, and a molecular weight distribution of 3000dal or less representing more than 85%. Partially hydrolyzed proteins may have low sensitivity.

According to a specific embodiment of the present invention, in the formula for preterm infants, the raw material for providing total protein 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 fat-providing raw material in the preterm formula of the invention comprises a milk fat-containing base material (such as bovine milk or an isolated fraction from bovine milk), optionally also comprising vegetable oil and/or OPO structural fat. The vegetable oil may comprise one or more of sunflower oil, corn oil, soybean oil, canola oil, coconut oil, palm oil, walnut oil, preferably sunflower oil, corn oil and soybean oil, and is added to provide the product with a fat component, linoleic acid and alpha-linolenic acid. In addition, the raw material for providing the fat can also selectively comprise raw material OPO structure fat added for providing 1,3-dioleate-2-palmitic acid triglyceride. Because the OPO structural fat raw materials sold in the market at present have different purities, namely the content of the active ingredient 1,3-dioleate-2-palmitic acid triglyceride is different and is usually about 40% -70%, in the invention, in order to distinguish the active ingredient 1,3-dioleate-2-palmitic acid triglyceride and the raw materials thereof, the term "1,3-dioleate-2-palmitic acid triglyceride" is adopted when describing the active ingredient, and the term "OPO structural fat" is adopted when describing the food raw materials for providing the active ingredient 1,3-dioleate-2-palmitic acid triglyceride. The specific addition amount of the OPO structural fat can be converted according to the content requirement of 1,3-dioleate-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: 0 to 150 parts by weight of sunflower seed oil; 0 to 40 parts by weight of corn oil; 0 to 80 parts by weight of soybean oil; 0 to 140 parts by weight of OPO structural grease.

According to a particular embodiment of the invention, in the preterm formula of the invention, the carbohydrate providing raw material 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 food of the present invention, the raw material for providing the carbohydrate may include, in addition to the base material containing lactose, raw materials of lactose, an emulsifier and a starch substance to be hydrolyzed or gelatinized. Preferably, the raw materials comprise, based on 1000 parts by weight of the formula powder: 0-580 parts of lactose and 0-580 parts of non-lactose substances. The specific amount of lactose added can be adjusted within the stated range so that the preterm formula containing the breast milk oligosaccharide LNnT of the invention has a carbohydrate content of 50 to 58g/100g.

According to a particular embodiment of the invention, in the preterm formula of the invention, the starting material providing total protein comprises one or more of whey protein powder, casein powder, milk protein powder, milk fat globule membrane protein based.

According to a specific embodiment of the invention, the preterm infant formula powder containing the breast milk oligosaccharide LNnT of the invention further comprises one or more of proper DHA, ARA, nucleotide, lactoferrin, probiotics and the like, and further comprises compound nutrients comprising calcium powder, vitamins and minerals. Preferably, the preterm infant 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 to 24 parts by weight of 2' -fucosyllactose; 7 to 50 parts by weight of compound nutrient containing calcium powder, vitamins and minerals.

In the formula food for the premature infant 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 nutrient 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 to 5800 mu gRE

Vitamin D:25 to 70 mu g

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 to 70mg of alpha-TE

Nicotinamide: 10000 to 41550 mu g

Folic acid: 350 to 920 mu g

Biotin: 70 to 245 mu g

Pantothenic acid: 7100 to 25230 μ g

Inositol: 0-250mg

L-carnitine: 0-60mg.

2) Mineral one, per gram of mineral one:

iron: 20 to 110mg

Zinc: 23 to 90mg

Copper: 2000 to 4180. Mu.g

Iodine: 500 to 995 mu g

Selenium: 0 to 200. Mu.g

Manganese: 0 to 579. Mu.g.

3) Mineral two, per gram of mineral two:

sodium: 40 to 100mg

Potassium: 200 to 500mg.

4) Mineral three, per gram of mineral three:

calcium: 200 to 500mg

Phosphorus: 75 to 300mg.

5) Compounding magnesium chloride, wherein each gram of magnesium chloride is packaged as follows:

magnesium: 80 to 170mg.

6) Choline chloride per gram of choline chloride packet:

choline: 300 to 950mg.

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 preterm infant formula powder containing the breast milk oligosaccharide LNnT, the addition amount of compound nutrients is 7 to 52 parts by weight, wherein a compound vitamin nutrition package is preferably 2~4 parts by weight, a mineral first nutrition package is preferably 0.5 to 3 parts by weight, a mineral second nutrition package is preferably 2 to 16 parts by weight, a mineral third nutrition package is preferably 0.5 to 20 parts by weight, magnesium chloride is 0 to 3.5 parts by weight, choline chloride is 0 to 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 sulfates occur in ionic form in aqueous solution, as oxygen in oxidation reactionsThe agent induces oxidation of the vitamin, which destroys the vitamin structure. 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 particular embodiment of the invention, in the preterm infant formula containing LNnT according to the invention, the probiotic is bifidobacterium. Preferably, the amount of the bifidobacterium added is 0.1 to 0.2 weight part based on 1000 weight parts of the formula; further 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. More preferably, the 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 preterm formula of the present invention comprises the following raw materials:

80 to 180 parts by weight of hydrolyzed whey protein powder;

0 to 580 parts by weight of lactose;

0 to 580 parts by weight of solid corn syrup;

0 to 150 parts by weight of sunflower seed oil;

0 to 40 parts by weight of corn oil;

20 to 80 parts by weight of soybean oil;

0 to 140 parts of OPO structural grease;

0.428 to 4.286 parts by weight of lactose-N-neotetraose (LNnT);

0 to 24 parts by weight of 2' -fucosyllactose;

compound nutrients comprising calcium powder, vitamins and minerals, 7 to 50 parts by weight;

2 to 15 parts by weight of DHA;

3 to 22 parts by weight of ARA;

0.1 to 0.2 weight portion of bifidobacterium.

It can be understood that in the formula of the premature infant 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 preterm formula containing the breast milk oligosaccharide LNnT, the specifications of the product performance which are not specified or listed are implemented according to the national standards of infant formula or modified milk powder and the regulations of the relevant standards and regulations.

In the formula food for the premature infant containing the breast milk oligosaccharide LNnT, all raw materials are commercially available and selected to meet 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.

In another aspect, the present invention further provides a method for preparing the formula powder of the breast milk oligosaccharide LNnT for premature infants, wherein the preparation process mainly comprises: preparing materials, homogenizing, concentrating, sterilizing, spray drying, and dry mixing to obtain the final product. Specifically, the method for preparing the low-sensitivity premature infant formula powder containing the breast milk oligosaccharide LNnT comprises the following steps:

the formula food for premature infants is prepared by mixing lactose-N-neotetraose with other raw materials in the formula food for premature infants by adopting a wet or dry production process.

According to a particular embodiment of the invention, the process for the preparation of the preterm infant formula containing the breast milk oligosaccharide LNnT according to the invention comprises:

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: and (3) preserving the mixed oil in an oil storage tank for heat storage at the temperature of 40-50 ℃ for less than 12 hours to prevent the fat from being oxidized.

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: respectively adding calcium powder, mineral substances, vitamins and the like, respectively dissolving the calcium powder, the mineral substances, the vitamins and the like with 100-200kg of purified water, and then filling the mixture into a wet mixing cylinder, wherein 100kg of purified water is used for flushing an adding tank and a pipeline after each time of adding.

7) Human milk oligosaccharide LNnT solubilization addition: and (4) dissolving the human milk oligosaccharide LNnT raw material by using part of the mixed feed liquid in the step (6), and adding the dissolved material into a mixing tank to obtain the mixed feed liquid containing the human 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 with a first-stage pressure of 105 + -5 bar and a first-stage pressure of 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 Concentrated 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 discharging concentration is 48-52% of 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 ℃ by a scraper preheater, filtering the preheated material by a filter with the aperture of 1mm, pumping the filtered material into a drying tower by a high-pressure pump for spray drying, and agglomerating fine powder on the tower top or a fluidized bed as required. Air inlet temperature: 165 to 180 ℃, the air exhaust temperature is 75 to 90 ℃, the high-pressure pump pressure is 160 to 210bar, and the tower negative pressure is-4 to-2 mbar.

13 Fluidized bed drying and cooling: and (3) drying the powder discharged from the drying tower for the second time by a fluidized bed (first stage), and cooling to 25-30 ℃ by a fluidized bed (second stage). Meanwhile, the human 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 particle size and the instant solubility of the powder particles are increased by agglomeration.

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 at normal temperature with the humidity less than or equal to 65 percent.

In another aspect, the invention also provides the use of the preterm formula in the preparation of a food product for improving gut 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 the intestinal tract; 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 formula food for premature infants containing breast milk oligosaccharide LNnT, a preparation method and an application thereof.

Drawings

FIG. 1 shows the results of a small fermentation run of individual HMO monomers to produce acetic acid in a simulated infant intestinal environment.

Figure 2 shows the results of simulating small batch fermentation of individual HMO monomers in an infant gut environment to produce acetic acid as a percentage of total short chain fatty acids.

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 combined small batch fermentation of LNnT with probiotics to produce isovaleric acid as a percentage of total acid 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. The hydrolyzed whey protein powder used in each example had a degree of hydrolysis of 8-23 and a molecular weight distribution of 85% or more below 3000 dal.

Example 1

This example provides a preterm infant formula containing the human milk oligosaccharide LNnT (1000 kg prepared):

80 kg of hydrolyzed whey protein powder, 115 kg of lactose, 435kg of solid corn syrup, 130 kg of high-oleic acid sunflower seed oil, 50 kg of corn oil, 60 kg of soybean oil, 90 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 formula powder of the premature infant containing the human milk oligosaccharide LNnT comprises the following steps:

2) Vacuum powder suction: various powder raw materials in the powder mixing tank are sucked into the vacuum mixing tank through a vacuum system.

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: respectively adding calcium powder, mineral substances, vitamins and the like, respectively dissolving the calcium powder, the mineral substances, the vitamins and the like in 100-200kg of purified water, and then filling the mixture into a wet mixing cylinder, wherein after each time, a filling tank and a pipeline are flushed by 100kg of purified water.

7) Human milk oligosaccharide LNnT solubilization addition: and (3) dissolving the human milk oligosaccharide LNnT by using part of the mixed feed liquid in the step (6), and adding the dissolved human milk oligosaccharide LNnT into a mixing tank to obtain the mixed feed liquid containing the human milk oligosaccharide LNnT.

9) Homogenizing: homogenizing the mixed material liquid with a homogenizer at a first-stage pressure of 105 + -5 bar and a first-stage pressure of 32 + -3 bar, and mechanically processing the fat globules to disperse them into uniform fat globules.

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 discharging concentration is 48-52% of dry matter.

Wherein the product contains 6.4g/100g of protein, 33g/100g of fat, 55g/100g of carbohydrate and 42.8mg/100g of lactose-N-neotetraose.

Example 2

180 kg of hydrolyzed whey protein powder, 500 kg of lactose, 100kg of high oleic acid sunflower oil, 20 kg of corn oil, 50 kg of soybean oil, 110 kg of OPO structural fat, 1 kg of human 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 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

140 kg of hydrolyzed whey protein powder, 530 kg of solid corn syrup, 60 kg of high oleic acid sunflower oil, 20 kg of corn oil, 70 kg of soybean oil, 140 kg of OPO structural fat, 1.5 kg of human 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 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

120 kg of hydrolyzed whey protein powder, 155 kg of lactose, 395 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 structural fat, 2 kg of human milk oligosaccharide LNnT, 38 kg of compound nutrient, 9.5 kg of DHA, 19.4 kg of ARA, 0.1 kg of bifidobacterium and 0.65kg of nucleotide.

The product preparation process is 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

180 kg of hydrolyzed whey protein powder, 500 kg of lactose, 90 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 human 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 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

140 kg of hydrolyzed whey protein powder, 500 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 human 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 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 300mg/100g of lactose-N-neotetraose.

Example 7

120 kg of hydrolyzed whey protein powder, 120 kg of lactose, 430kg of solid corn syrup, 100kg of high-oleic acid sunflower oil, 40 kg of corn oil, 50 kg of soybean oil, 80 kg of OPO structural fat, 10.5kg of human 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 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

200kg of hydrolyzed whey protein powder, 500 kg of lactose, 90 kg of high oleic acid sunflower oil, 10 kg of corn oil, 50 kg of soybean oil, 100kg of OPO structural fat, 8978 kg of human milk oligosaccharide composition, 8978 kg of zxft 8978 kg of compound nutrient, 3 kg of DHA, 6kg of ARA, 0.1 kg of bifidobacterium and 0.65kg of nucleotide.

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 O 0.09；

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 and an ETEC non-adding group, and the final concentration of added ETEC in the culture medium with added ETEC group is 10 ¹⁰ CFU/mL。

TABLE 1 fermentation conditions List

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 up 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 an anaerobic workstation, a 1mL syringe is matched with a No. 5 needle to suck 0.5mL of suspension (clear side in a pretreatment box), 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 37 ℃ constant temperature box 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 the instrument;

(2) adjusting the gas detection chamber to a certain vacuum level by using a vacuum generator through a gas distribution module;

(3) sucking the gas in the small bottle into a detection chamber of the instrument through a gas sampler, and adjusting the volume of the gas through a gas distribution module;

(4) detecting CO entering the gas detection chamber 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 into a 1.5ml centrifuge tube, adding 100 mu L of crotonic acid metaphosphoric acid solution, and freezing 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; sample inlet temperature: 250 ℃, sample size: 1.0 μ L, split ratio: (5:1); carrier gas: high purity nitrogen; flow rate: keeping the temperature at 2.5mL/min for 6.5min, and increasing the temperature 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 acetic acid generated by small-batch fermentation of various HMO monomers in simulated infant intestinal environment are shown in figure 1, and the significant difference and P value of the four HMO monomers and a control group are shown in table 2.

TABLE 2

It can be seen that LNnT increased acetic acid in the feces fermentation group of breast-fed and formula-fed infants, regardless of the presence or absence of ETEC-simulated diarrhea, compared to the four HMO monomers and the control group. 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

It can be seen that LNnT increased the proportion of acetic acid in the feces fermentation group of breast-fed and formula-fed infants, regardless of the presence or absence of ETEC-simulated diarrhea, compared to the four HMO monomers and the control group. 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 by 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

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 is superior to 3'-SL and LNT, and is comparable to 2' -FL and 3-FL in effect.

Results of the simulation of LNnT in the infant intestinal environment compared to the percentage of total short chain fatty acids produced by small batch fermentation of other HMO monomers with isobutyric acid are shown in fig. 4, and significant differences and P values are shown in table 5.

TABLE 5

It can be seen that in the feces fermentation group of breast milk and formula fed infants, the addition of HMO monomer may reduce the production of isobutyric 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, 3'-SL and LNT when ETEC exists or not, and the effect is equivalent to that of 2' -FL.

The results of the simulated infant intestinal environment in which LNnT was fermented in small batches 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

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

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 better than that of 3-FL, 3'-SL and LNT, and the effect is equivalent to that of 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 short-chain fatty acid content of the fermented product 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 combining 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 added or not added with ETEC. ETEC was added to a final concentration of 10 ¹⁰ CFU/mL。

TABLE 8 fermentation conditions List

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 the culture medium, the glycerol tube strain preserved in a refrigerator at the temperature of-80 ℃ is taken out, inoculated in an MRS culture medium for activation, and then the activated bacterium liquid is inoculated in the corresponding culture medium by an injector.

Taking bacterium powder of strain ETEC (ATCC 35401), and preparing the bacterium 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 results of small batch fermentation of LNnT in combination with probiotics in simulated infant intestinal environment produced isovaleric acid as a percentage of total acids as shown in fig. 7.

It can be seen that the combination of LNnT with probiotics outperformed the blank and the 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 LNnT + BL-99 having a greater tendency to reduce isovaleric acid than LNnT + BB12 (P = 0.1254).

TABLE 9

The combination of LNnT and probiotic (LNnT + BL-99 works best) produces a 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 gut environment were simulated to produce total short chain fatty acids as shown in figure 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.

Watch 10

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.

Further comparison of the results for the different groups is shown in table 11.

TABLE 11

As can be seen from table 11, feces from formula-fed infants, with or without ETEC, resulted in higher intestinal production of hydrogen sulfide than in the breast-fed group. 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 combination of LNnT + BL-99 produces less hydrogen sulfide than LNnT monomer (P < 0.05), LNnT + BB12 and LNnT + YLGB-1496 have no significant difference with LNnT, but have a significant trend, i.e. P <0.1, indicating that the combination of LNnT and probiotics can produce synergistic gain effect. See table 12 for comparative results.

TABLE 12

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

In the ETEC-free formula powder group, hydrogen sulfide generated by LNnT + YLGB-1496 and LNnT + BL-99 is lower than that generated by LNnT (P < 0.05), and LNnT + HN019 and LNnT have no significant difference but have a significant trend, namely P <0.1, which shows that after the LNnT is combined with probiotics, the hydrogen sulfide can be synergistically enhanced and more reduced. The results of the comparison are shown in Table 14.

TABLE 14

Watch 15

Table 15 analysis shows that no significant difference in hydrogen sulfide production was observed under different fermentation conditions in the group of formulations with ETEC.

Claims

1. A preterm infant formula comprising breast milk oligosaccharides including lacto-N-neotetraose, wherein the total content of lacto-N-neotetraose in the preterm infant formula is from 21.4 to 857.2mg/100g, based on the total dry matter of the preterm infant formula; and the total protein content in the formula food for the premature infant is 12-18g/100 g, the fat content is 15-29g/100 g, and the carbohydrate content is 50-58g/100 g.

2. The preterm infant formula according to claim 1, wherein the total content of lacto-N-neotetraose in the preterm infant formula is from 42.8 to 428.6mg/100g.

3. The preterm infant formula of claim 1, wherein the total protein comprises hydrolyzed milk protein, the degree of hydrolysis is from 8 to 23, and the molecular weight distribution is 85% or more below 3000 dal.

4. The preterm infant formula of claim 3, wherein the material providing total protein comprises one or more of hydrolyzed whey protein powder, hydrolyzed casein powder, hydrolyzed milk protein powder, hydrolyzed milk fat globule membrane protein.

5. The preterm infant formula of claim 1, wherein the fat providing raw material comprises a base material comprising milk fat, further comprising vegetable oil and/or OPO structural fat;

the carbohydrate-providing raw material is derived from lactose and non-lactose derived materials including one or more of pregelatinized starch, maltodextrin, corn syrup solids, glucose syrup.

6. The preterm infant formula of claim 1, further comprising one or more of DHA, ARA, nucleotides, lactoferrin, nutrients, probiotics.

7. The preterm infant formula of any one of claims 1~6, wherein the preterm infant formula is for:

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

The capability of resisting pathogenic bacteria ETEC of an individual is improved.

8. A method of making the preterm infant formula of any one of claims 1~7, comprising:

9. Use of the preterm infant formula of any one of claims 1~7 in the preparation of a food product for improving gut microenvironment health.

10. The use of claim 9, wherein improving gut microenvironment health comprises:

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

The capability of resisting the pathogenic bacteria ETEC of the individual is improved.