AU2019100970A4

AU2019100970A4 - Composition containing lactoferrin and sialic acid, preparation method and use thereof

Info

Publication number: AU2019100970A4
Application number: AU2019100970A
Authority: AU
Inventors: Yanyu Yang; Zhenglai Zhou
Original assignee: Unipharm Healthy Manufacturing Co Ltd
Current assignee: Unipharm Healthy Manufacturing Co Ltd
Priority date: 2019-08-29
Filing date: 2019-08-29
Publication date: 2019-10-03
Anticipated expiration: 2027-08-29

Abstract

The present invention relates to an infant formula or nutritional supplement composition comprising lactoferrin and sialic acid in a weight ratio of between 5:1 and 50:1. The composition may further comprise 0.5 to 2 parts by weight of a cationic colloid, which may be selected from one or more of: modified starch, gellan gum, locust bean gum, konjac gum, dextrin, carrageenan, xanthan gum, pectin, microcrystals, cellulose, propylene glycol alginate, gelatin, agar, guar gum, and sodium alginate.

Description

TITLE

Composition containing lactoferrin and sialic acid, preparation method and use thereof

FIELD OF THE INVENTION

The present invention relates to a composition, a process for its preparation and use, in particular a composition comprising lactoferrin and sialic acid, a process for its preparation and use. It belongs to the technical field of composition and processing technology.

BACKGROUND OF THE INVENTION

Sialic acid, N-acetylneuraminic acid, has the molecular formula C11H19NO9. N-acetylneuraminic acid is the most widely distributed in nature, mainly glycoproteins, glycolipids or animal cell membranes or secretions. In a glycoprotein or a glycolipid, since the keto group at the 2-position forms a glycosidic bond, it is located at the non-reducing end of the sugar side chain, and its anionic charge or a unique chemical structure makes it have various physiological specificities. In the natural world, from the virus to the animal, the enzyme which is hydrolyzed to remove the terminal N-acetylneuraminic acid (neuraminidase) has been identified by the treatment of this enzyme. For example, this sugar residue constitutes a binding site of an influenza virus of a red blood cell membrane, and plays a major role in the mutual recognition of plasma glycoproteins and hepatocytes or the recognition mechanism of lymphocytes to the circulation pathway.

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2019100970 29 Aug 2019

Although the biosynthetic pathway is somewhat different in animals and bacteria, it is subjected to N-acetylmannosamine or its 6-phosphate, which is catalyzed by a specific pyrophosphatase to react with CTP to form CMP-N-acetylneuraminic acid. After that, the sugar chain is incorporated by a glycosyl transfer reaction.

Studies have shown that sialic acid has important biological functions in the growth and development of infants, including: improving the memory and intelligence of infants; anti-recognition; antiviral effects; inhibiting leukocyte adhesion and anti-inflammatory effects. However, infant formula containing sialic acid produced in the existing industry has a large loss of sialic acid during production, transportation, and storage. Studies have shown that sialic acid has important biological functions in the growth and development of infants, including: improving the memory and intelligence of infants; anti-recognition; antiviral effects; inhibiting leukocyte adhesion and anti-inflammatory effects. However, there is no industrial production of infant formula containing sialic acid, and there is no complete formula and process.

Sialic acid is a kind of natural saccharide compound widely present in biological systems. It is an essential nutrient for human brain cognitive development and has important biological functions such as cell recognition, survival, reproduction, biofilm flow, and endocytosis. Wait. The supply of exogenous sialic acid can increase the concentration of gangliosides in the brain of animals and increase the learning ability of animals, especially cognitive levels. Studies have shown that mouse cubs have obstacles in cognition and behavior when sialic acid intake is insufficient, and cognitive ability is significantly improved after sialic acid supplementation is sufficient; small consumption of

2019100970 29 Aug 2019 sialic acid formula containing high dose is small Pigs have significantly better learning and memory skills than pigs who consume regular formula. Therefore, the researchers believe that early sialic acid intake after birth can lay the foundation for long-term cognitive development.

In clinical studies, Dida et al. confirmed by double-blind trial that supplementation of high sialic acid milk to infants aged 0-6 months can increase serum ganglioside levels and promote cognitive development. The current study found that milk-source sialic acid is high in casein glycomacropeptide (CGMP) produced during the process of rennet agglutination. In addition to providing a large amount of sialic acid to the human body, this bioactive peptide has the function of promoting the proliferation of bifidobacteria, inhibiting the infection of viruses and toxins, suppressing allergic reactions, and serving as a nutritious food for patients with phenylketonuria (PKU). And so on.

In addition, breast milk is rich in nutrients, such as lactoferrin, which is beneficial to enhance the resistance of infants and young children.

However, there are many breast milk shortages in reality, which requires the selection of other products to help increase the immunity of infants and young children. At present, the most common formula on the market is formula milk powder, there are many brands, such as Nestle, Abbott, Dutch Nuoyouneng, etc., and according to different needs, there are a variety of functional infant formula milk powder, and meet the growth cycle of infants of different ages. The basic nutrients needed. However, most of the formula milk powder on the market lacks some beneficial ingredients in breast milk, and it is difficult to truly improve the immunity of infants and young children and promote the

2019100970 29 Aug 2019 growth and development. Therefore, many companies and scholars are committed to developing a formula that is closer to the nutritional components of breast milk to improve the immunity of infants and young children.

Another component we are concerned with, lactoferrin (LF), also known as lactotransferrin (LTF), is a multifunctional glycoprotein in transferrin. Lactoferrin is a globular glycoprotein with a molecular weight ranging from 76 to 80 kDa and widely present in various exudates such as milk, saliva, tears, and snivel. Lactoferrin is also present in metaphase neutrophils and is secreted by some acinar cells. Lactoferrin can be extracted from milk or obtained using recombinant DNA techniques. Human colostrum has the highest lactoferrin concentration, followed by human milk, with the lowest concentration in milk (150 mg/l).

Lactoferrin is one of the components of the human immune system. It has antibacterial activity (antibacterial, antifungal) and is part of the innate defense, mainly in the mucosa. Lactoferrin protects infants from pathogens such as bacteria. Lactoferrin also interacts with DNA and RNA, polysaccharides and heparin and exhibits certain physiological functions in these receptor-ligand complexes.

Lactoferrin is involved in the transmembrane transport of iron and controls the balance and secretion of iron ions in the blood. It is found in the milk, plasma and neutrophils of humans and other mammals and is the secretion of mammals such as saliva, gall bladder, tears and pancreas, the main proteins in the composition. The concentration of lactoferrin in milk varies from 7 g/L in human colostrum to 1 g/L in mature milk.

2019100970 29 Aug 2019

X-ray diffraction indicates that lactoferrin is a polypeptide chain comprising about

700 amino acids and two homologous globular domains: N-ring and C-loop. The N-loop corresponds to the 1-33rd amino acid residue in the peptide chain, and the C-loop corresponds to the 345-692 amino acid residues, and the two domains are joined at both ends by a short-chain alpha-helix. Each loop consists of two subdomains (subdomains): N1, N2 and C1, C2 and contains an iron binding site and a glycosylation site. The degree of glycosylation of lactoferrin may vary, so the molecular weight of lactoferrin varies from 76 to 80 kDa. The stability of lactoferrin is related to its degree of glycosylation.

Lactoferrin is a basic protein with an isoelectric point of 8.7, which exists in two forms: holo-lactoferrin rich in iron form and apo-lactoferrin without iron form. The two have different tertiary structures: apo-lactoferrin is characterized by an open N-ring and a closed C-ring, while the holo-lactoferrin rings are closed. Each lactoferrin molecule reversibly binds two irons, zinc, copper or other metal ions. The two binding sites are located in two protein globular domains, respectively. Each ion binds to six ligands: four from the ligand in the peptide chain (2 tyrosine residues, 1 histidine residue and 1 aspartate residue) and 2 carbonates or Bicarbonate ion.

Lactoferrin forms a red complex with iron; its affinity for iron is 300 times higher than that of transferrin. Affinity increases in weakly acidic media. When inflammation occurs, this property facilitates the transfer of iron from transferrin to lactoferrin because inflammation often causes a decrease in the pH value of the tissue due to the accumulation of lactic acid and other organic acids. The concentration of ferric iron in human milk is about 10% to 30% (100% means that all lactoferrin molecules contain 2

2019100970 29 Aug 2019 iron atoms). It is shown that lactoferrin not only involves the transport of iron, zinc, and copper ions, but also regulates the absorption of these ions. The presence of zinc and copper ions in the solution does not affect the iron binding capacity of lactoferrin and may even increase.

Lactoferrin exists in different forms of polymer in plasma and exudates: monomeric to tetrameric. Lactoferrin is more likely to be present in the body and in the form of a polymer, especially when the concentration is high. Studies have found that tetramers are the predominant form of lactoferrin under physiological conditions.

The oligomeric form of lactoferrin depends mainly on the concentration of the protein, while the formation of the poly-polymeric form is strongly influenced by Ca²⁺. In the presence of Ca²⁺, when the concentration is 10“ ⁹ ~ 10“ ¹⁰M, lactoferrin mainly exists as a monomer; but when the concentration is increased to 10“ ⁹ ~ 10“ ¹⁰M, the monomer will be converted into a tetramer form. In general, the concentration of lactoferrin in the blood is between the monomer and tetramer transition concentrations, so that lactoferrin has a polymerized state of monomer and tetramer in the blood. Many of the functional properties of lactoferrin depend on its oligomeric form. For example, lactoferrin monomers bind tightly to DNA, while tetramer forms do not.

Lactoferrin can alter the spatial conformation of the virus, causing the virus to lose its ability to bind to human host cell receptors. Lactoferrin forms a protective layer on the surface of the receptor, preventing the adhesion of viruses and human cells.

According to research in recent years, exogenous supplemental lactoferrin can play a very good role in antiviral. In addition, a large number of academic papers have shown

2019100970 29 Aug 2019 that sialic acid can act as a viral inhibitor. Therefore, lactoferrin combined with sialic acid is a good antiviral agent.

The combination of lactoferrin and sialic acid helps to strengthen immunity and make babies grow healthier.

A product of a lactoferrin composition for promoting brain development is disclosed in US Pat. No. 8, 852,658 B2, which also mentions that sialic acid is an important component of known human milk polysaccharides and nervous tissues. However, the article does not pay enough attention to industrial production, and only discloses some information such as usage.

JP2009542226A explicitly mentions that sialic acid and lactoferrin can be used in formula milk powder. The patent involves various ingredients, but does not clarify the proportional relationship between the two, but focuses on the combination of sialic acid and lactoferric iron. The amount of protein used did not notice the problem of uniform mixing of trace elements and various physical reactions in actual production.

Chinese patent application CN108029768A discloses an infant formula containing lactoferrin and probiotics, which simultaneously adds probiotics and lactoferrin to infant formula, and regulates the intestinal flora of infants and young children through probiotics. Lactoferrin protects against the invasion of external microorganisms. However, a professor at the Children's Hospital Medical Center in Cincinnati, Ohio, and his research team demonstrated that red blood cells (CD71+ cells) expressing CD71 receptors can suppress the immune response in 6-day-old mice. They also found that CD71+ cells in human cord blood also have unique immunosuppressive properties, but CD71+ cells in

2019100970 29 Aug 2019 adult mice do not have these characteristics, suggesting that immunosuppressive properties are restricted to newborns. The researchers also pointed out that although the immune system of newborns is inhibited, the growth of probiotics can be improved. It can be seen that the probiotics of newborns are sufficient to achieve self-sufficiency, and the addition of new probiotics is likely to cause their own probiotics to breed and disorder, which has a negative effect on the growth of newborns.

CN109007037A A composition for enhancing infant immunity and a product thereof, comprising 30-60 parts by weight of milk powder, 0.05-2 parts of lactoferrin, 5-30 parts of concentrated whey protein, 5 parts by weight -15 parts of prebiotic, 20-50 parts of desalted whey powder, 1-5 parts of lactalbumin, 0.1-2 parts of N-acetylneuraminic acid and 0.003-0.05 parts of folic acid compound. However, according to this ratio, lactoferrin and N-acetylneuraminic acid are not the best use of the human body. The key is the loss in the digestive tract of the body. At the same time, the molecular distance between the two is too far to be used by the human body at the same time.

The current problem is that lactoferrin and sialic acid supplemented at the same time, and there is no stable composition that specifically maintains the two components, because both are very good nutritional supplements, and the current products are simultaneously Most of the products supplemented with two supplements are concentrated on infant formula, which causes great loss in the production process, and there is an unreasonable absorption in the human body. Another important problem is that the mixing of the two is difficult to be particularly uniform due to factors such as particle size and charge. Since the components used in the trace are very important, they

2019100970 29 Aug 2019 are very unfavorable for the uniformity of the final product.

BRIEF SUMMARY OF THE INVENTION

The object of the present invention is to get a composition comprising lactoferrin and sialic acid, a preparation method and use thereof, a composition or a product containing the composition, and a mixture of the composition in the actual production process in the presence of the corresponding colloid uniformity problem, while keeping the two components unbroken, can maintain the uniformity and stability of the product, so that the final product can not only meet the nutritional needs of the human body, but also has high utilization rate.

Problem research:

In order to improve the mixing uniformity of the final product and to maintain the original function to the maximum extent, we have carefully studied the production process. After improvement, we not only made great progress in these two aspects, but also improved the taste of mixed product.

Generally speaking, the dry mixing process is the most commonly used, but due to the different particle sizes of the components, different charges and different viscosities, unevenness and agglomeration may occur during the mixing process. Dry blending is a process widely used in a variety of industries, such as pharmaceutical manufacturing and food and animal feed production. Mixing is an important step in the food industry to produce products with constant properties and to obtain uniformity of different properties such as color, texture, taste and concentration. In the most common nutritional fortified

2019100970 29 Aug 2019 milk powder, the raw material of the dry blending method is a large package base powder, and the base powder refers to milk or goat milk and processed products thereof (whey powder, whey protein powder, Skim milk powder, whole milk powder, etc.) as the main raw material, adding semi-finished products of infant formula milk powder produced by wet process with or without nutrients and other excipients, so in order to prevent the outer packaging during the mixing process In case of pollution and infection of materials, at this stage, the outer packaging of the raw materials should be vacuumed and smear, and the inner packaging should be vacuumed and sterilized before being sent to the next process.

In the pretreatment process, the specific operation is as follows: the first inspection of the qualified large-package base powder is carried out step by step, the first vacuuming, the first vacuuming, the second vacuuming, and then sent to the tunnel for sterilization and transmission; The raw materials such as various additives and nutrients that have passed the inspection are vacuumed and sent to the sterilization tunnel for sterilization and transportation. The next step is mixing and packaging. Among them, the problem of mixing is the uniformity.

In the process of mixing powdered foods, the operation of two or more components should enable uniform distribution of individual particles of different materials present, especially for some of components, such as sialic acid. The amount of sialic acid added to the milk powder is small, and the small amount means that it is difficult to mix uniformly. And the pH value of sialic acid is about 1.8-2.3 (2% solution), which is very acid in terms of taste. If it is not mixed evenly, it will greatly affect the mouthfeel of the final product.

2019100970 29 Aug 2019

Whether the mixing is evenly affected by many factors, including:

Particle size: The particle size of the powder directly affects the fluidity of the powder.

The smaller the particles, the larger the contact area and the greater the intermolecular force, which in turn leads to agglomeration between the powders. The smaller the particles, the higher the surface moisture content, the higher the surface tension, the more the powder will be condensed and the less likely to flow;

The water content of the powder

The greater the water content of the powder, the less fluid the powder will be due to the formation of the liquid bridge and the capillary forces between the particles. The liquid bridge will develop into a solid bridge under improper temperature control: solidification.

Powder composition

Many powder materials contain more or less amorphous glassy components, such as amorphous sugar and maltodextrin. The glass transition temperature is very important because amorphous components may be sticky.

Powder density and tapped density

The density of the powder depends on many aspects, such as the nature of the particles themselves, the intermolecular forces (van der Waals forces), whether they have charge and charge, the glycoprotein and water content, the particle diameter, and so on. The density of the powder must be in a range to ensure a high production quality.

A composition of the present invention, wherein

A composition comprising lactoferrin and sialic acid according to the present invention, wherein the composition comprises, but is not limited to, lactoferrin and sialic

2019100970 29 Aug 2019 acid, and the ratio of the two components in parts by weight is:

(5-50): 1;

Preferably the parts by weight are (20-40): 1;

Most preferably 30:1;

The above composition further comprises 0.5-2 parts by weight of a cationic colloid;

The above colloids are modified starch, gellan gum, locust bean gum, konjac gum, dextrin, carrageenan, xanthan gum, pectin, propylene glycol alginate, gelatin, agar, guar gum, sodium alginate. One or more; wherein carrageenan is preferably used, the specific reason will be clearly described below.

The above colloid is preferably a combination of carrageenan, guar gum, propylene glycol alginate, and the ratio of the three preferred combination colloids is (1-2): (1-2): (1-2);

The product of the invention has high dependence on the cationic glue, and the inventors have made several colloidal compositions. The inventors have tested the two combinations, and the effect is worse than the combination of three to four kinds of colloids. The following four combinations are not exhaustive, but there are several situations that are unsatisfactory. After careful screening by the inventors, the preferred choices are as follows:

The ratio by weight of xanthan gum, locust bean gum, konjac gum and carrageenan is (1-2): (1-2): (1-2): (1-2);

The ratio by weight of dextrin, carrageenan, xanthan gum and sodium alginate is (1-2): (1-2): (1-2): (1-2);

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The ratio by weight of carrageenan, locust bean gum, konjac gum and guar gum is (1-2): (1-2): (1-2): (1-2);

The ratio by weight of carrageenan, xanthan gum, pectin and guar gum is (1-2): (1-2): (1-2): (1-2).

First set the temperature of the heatable three-dimensional mixer to 50-60°C. After 5-20 minutes, wait until the temperature distribution in the mixer is even. Add sialic acid and lactoferrin to the mixer and mix for 10-20 minutes. Heat to 65°C, add a part of the colloid, mix for 20-30 minutes, then add the remaining colloid, continue mixing for 20-30 minutes, then reduce the temperature to 40-45°C, then mix for 10-20 minutes, then wait for a while Reducing the temperature to room temperature, thereby obtaining the sialic acid and lactoferrin compositions of the present invention.

Other powder excipients may be further added, mixed for another 30-40 minutes, and the mixing is over. Get products such as milk powder, fortified nutrients.

It is foreseeable that in the process of mixing, there will be a certain proportion of protein molecules at 60-65°C, the chain of its folded structure will unfold, but the proportion of protein is very low, so this loss is negligible, and when the temperature At the time of the decline, a part of the protein is reversible and will return to the original structure; and at this temperature, most of the functional groups of the protein molecule can still maintain the original function.

In addition, colloid particles protect them from heat by encapsulating the protein molecules inside.

Preferably, the weight of the previously added colloid is preferably from one third to

2019100970 29 Aug 2019 one half of the total weight, and after many trials, the dispersion of the final product is optimal in this ratio range.

Wherein, a more preferred mixture of sialic acid and lactoferrin is added in a batchwise manner, that is, half of the weight of sialic acid is first added, and after completely mixing with lactoferrin, the time ranges from about 10 to 30 minutes, and then The remaining half by weight of sialic acid was added before the addition of the colloid and mixing was continued for about 5-10 minutes. The advantage of this is that it further solves the problem of the taste of sialic acid mentioned above. After adding half of the mixture and mixing evenly, add half of the mixture, and the uniformity of the mixture should be continuously increased to about 3-5. %.

Mixing two small amounts of raw materials in advance helps to help the final powder mix evenly, providing the opportunity to combine sialic acid and lactoferrin molecules. At 50-60 temperatures, sialic acid and lactoferrin are not destroyed. However, the molecular motion will be strengthened, and the mixing rate and bonding rate will be improved;

Here we emphasize that the above-mentioned colloids, the most commonly used in actual production, are the preferred colloidal composition and weight ratio we mentioned, namely: carrageenan, guar gum, propylene glycol alginate combination, more selective The production use ratio is 1:1:1. Of course, using the ratio (1-2) as above: (1-2): (1-2) is also effective.

In the present invention, the best choice of carrageenan is: K-type carrageenan (also known as kappa-carrageenan, also known as carrageenan) and type I carrageenan (iota carrageenan) because they are only soluble in temperatures of 40 to 70 Celsius between

2019100970 29 Aug 2019 hot water and hot milk. Moreover, they only gel in the presence of certain cations. This means that we can produce two types of milk powders with different textures by using excipients with cations (eg Ca, Mg).

In addition, one of the most important properties that distinguish carrageenan from other hydrocolloids is its ability to interact with milk proteins. The high reactivity of carrageenan with milk is due to the strong electrostatic interaction between the negatively charged ester sulfate groups in the carrageenan molecule and the strong positive charge of the bovine cheese protein micelles. Another form of interaction is through the linkage established between the ester sulfate group of carrageenan and the carboxylic acid residue of the amino acid constituting the protein. The reactivity with proteins depends on many factors such as the concentration of carrageenan, the type of protein, temperature, pH value and isoelectric point of the protein. The ability of carrageenan to complex with milk proteins combined with its hydrogel properties makes it an effective ingredient in stable and gelled dairy products.

Quality requirements for carrageenan: 80% by particle size distribution through 200 mesh, moisture content below 15%, pH value 7.0-10.0.

The other two colloids should also be 80% through 200 mesh and moisture content below 15%.

In order to better quantitatively compare the method of the present invention with the conventional mixing method in the prior art, the inventors tested 15 sets of comparative data, and the products of the conventional mixed mode by the method of heating mixing and stepwise addition of the present invention. The range of uniformity improvement is

2019100970 29 Aug 2019 between 9.6-11.2%;

Mixing uniformity and weighing accuracy, system stability are called the three performance indicators of the batching system. The uniformity of the batch material not only affects the performance index of the subsequent process equipment, but also directly relates to the quality of the final product. Especially in the infant formula industry, the uniformity of the ingredients directly affects the nutritional balance. If the ingredients are mixed unevenly, it may cause poisoning.

Following the idea of this method, the inventors further found that direct cooling after mixing produces a slight agglomeration phenomenon, which will have a certain impact on the use of the product. After further experiments, the inventor has obtained a favorable effect to improve the problem. The way of mixing and cooling down at the same time is perfect.

The above composition is used in infant formula milk powder.

The above composition is used in a nutritional supplement powder.

The composition described above is used in nutritional supplement tablets.

Overall effect:

In the case of the same mixing time, the mixing rate is increased by nearly 50%; the walling rate of the overall powder is reduced by about 80%, the waste is reduced by about 58%; the fluidity of the whole powder is increased by 65%, and the production efficiency is increased by 45%. Left and right; improved heat resistance of lactoferrin and sialic acid (protected to some extent when sealing the bag)

Adding colloid, the mouthfeel is improved, and does not affect digestion and

2019100970 29 Aug 2019 absorption;

Long-term research and experiments by the inventors are based on the following mechanism: the addition of colloidal powder is to form a coating system that combines the binding molecules of sialic acid and lactoferrin with colloidal molecules through hydrogen bonds, van der Waals forces and other non-covalent bonds. And the physical viscosity is combined to form a mixed macromolecular particle. Increasing the temperature is to increase molecular vibration, increase the success rate of the package, and reduce the water content. It has been verified by experiments that it is feasible to test based on this mechanism and it is effective.

Mixed macromolecules after wrapping:

1. The overall particle surface is smoother and more suitable for post-packing;

2. Due to the wrapping, the van der Waals force between the molecules is reduced, and the force of hydrogen bonding is also reduced;

3. Reduced the strong charge of sialic acid and lactoferrin and adjuvant powders;

4. The entire heating process increases the water evaporation rate and reduces the water content, thus reducing the liquid bridge and capillary forces, reducing the caking strengh, thus reducing the cohesive force;

5. The wrapping system can protect the lactoferrin and sialic acid from being destroyed by heat sealing to a certain extent;

Research and analysis:

The inventors have found that the colloid is charged, and in the presence of a charge, the composition of the present invention can be very well combined in proportion to the

2019100970 29 Aug 2019 parts by weight, and at the same time, the loss in the mixed production process is very small due to the presence of the colloid;

The reason for the colloidal attraction is that the particles present in the colloid are exactly the micelles, and the colloid is composed of micelles. The micelle is composed of a rubber core, an adsorption layer and a diffusion layer. The rubber core is agglomerated by many molecules or other particles. It has strong adsorption capacity, and there is an electric double layer, that is, an adsorption layer and a diffusion layer, on the periphery of the rubber core. In layman's terms, the colloid nucleus adsorbs ions with a certain charge, forming colloidal particles, and the charged colloidal particles can further adsorb oppositely charged ions. The ion layer in the colloidal particles is called the adsorption layer, and the ion layer adsorbed by the colloidal particles is called the diffusion layer. Since the colloidal particles have a large surface area, the adsorption capacity is strong, and the adsorbed ions and its tightly bound are difficult to separate, so that the charged colloidal particles in the colloid can be stably present. The ability of the colloidal particles to adsorb oppositely charged ions is relatively small, and the adsorbed ions are easily separated. The micelles are electrically neutral. Therefore, the rubber particles are charged, while the colloid is electrically neutral. Description: The charging of the rubber particles can be either adsorption or ionization.

The second key point of the present invention is that after long-term research by the inventors, it was found that 20-50 sialic acid polymers can bind to a point on a lactoferrin, and under the action of a charged colloid, the combination is very stable. The combination of physical states not only does not affect the absorption of the human body,

2019100970 29 Aug 2019 but also protects the sialic acid and lactoferrin from being destroyed during processing. This is a very important invention of the present invention, and the inventor has gone through this concept. Several trials were carried out and the mixed products of each test were sent for inspection. According to the loss of the two components, which colloid and processing method were better, it was concluded that the sialic acid and lactoferrin were present due to the presence of colloidal charges. The combination of proteins is very good, which is beneficial to the human body, while protecting the two from damage and increasing the end use rate.

Another advantage of the present invention is that the two compositions are used in advance in the presence of a colloid to form a composition for separate use, which facilitates dispersion in the product at the time of use, which is advantageous for the stability of the produced product and avoids use due to uneven mixing. Uneven distribution of nutrients occurs.

Another innovation of the present invention is the proven step-by-step cooling mixing. After many experiments by the inventors, it is found that if the temperature is lowered after mixing, if the temperature is lowered too fast, the problem of agglomeration will occur, and there is a mixture. The problem of unevenness, the problem caused by sudden cooling, can be solved well by the stepwise cooling method of the present invention.

We should understand that the above mixing method is a mixed way of intention, because the inventor cannot test all the temperatures and check the mixing uniformity in an exhaustive manner, but the inventors have already cooled down most of the

2019100970 29 Aug 2019 conventional methods according to the conventional method. The method has been tested, and the time and temperature range of the method disclosed in the present invention are all optimal ranges.

DESCRIPTION OF PREFERRED EMBODIMENT

The embodiments of the present invention are intended to be illustrative, and not to limit the scope of the present invention. Any slight difference in nutrient composition due to the difference in milk source should not be used as a reason for the inconsistency between the embodiment of the present invention and the specification, so the present invention aims to protect the purpose of adding nutrients, rather than to make the ratio particularly strict. The ground is limited to a specific value.

The equipment used in dry mixing production is as follows:

Transmission equipment, including powder conveyor, conveyor belt, conveyor chain, sealed transfer window, lifting freight elevator; ventilation equipment, including central air conditioning, air filter, ozone generator; pretreatment equipment, including vacuum platform, vacuum cleaner, tunnel sterilization Mixing equipment, including operating platform, shelf, three-dimensional mixer, dry powder mixing mixer; packaging equipment, automatic canning machine, capping machine, inkjet printer, operating platform; measuring equipment, electronic scale, air pressure gauge, automatic Measuring canning machine; storage equipment, shelves, ground support, forklift; sanitary equipment, tool disinfection cabinet, washing machine, work clothes disinfection cabinet, air shower, ozone generator, alcohol sprayer, vacuum cleaner, garbage bin, etc.; inspection equipment, analytical balance , oven, centrifuge, electric furnace, impurity filter,

2019100970 29 Aug 2019 protein measuring device, insoluble index stirrer, fume hood, dry and wet heat sterilizer, water bath, etc.

EMBODIMENT 1

The composition containing lactoferrin and sialic acid of the present invention is prepared as follows:

First set the temperature of the heatable three-dimensional mixer to 50°C. After 20 minutes, wait until the temperature distribution in the mixer is even, add sialic acid and lactoferrin to the mixer and mix for 10 minutes, and then heat to 65°C. (here set to heat with 100w power), add half the weight of the colloidal mixture, mix for 20 minutes, then add the remaining half of the weight of the colloid, continue mixing for 20 minutes, then reduce the temperature to 40°C, then mix for 10 minutes, then Stop the temperature control, naturally reduce the temperature to room temperature, add other powder excipients, mix for another 30 minutes, and the mixing is over.

The above colloid is a combination of carrageenan, guar gum, and propylene glycol alginate, and the ratio of the three preferred combination colloids is 1:1:1.

Lactoferrin and sialic acid, the ratio of the two components in terms of parts by weight: 30:1.

Embodiment 2

First set the temperature of the heatable three-dimensional mixer to 60°C. After 30 minutes, wait until the temperature distribution in the mixer is even, add sialic acid and

2019100970 29 Aug 2019 lactoferrin to the mixer, mix for 20 minutes, then heat to 65°C. (here set to heat with 10Ow power), add one-third of the total weight of the colloid, mix for 30 minutes, then add two-thirds of the total weight of the colloid, continue mixing for 30 minutes, then lower the temperature At 45°C, mix for another 20 minutes, reduce the temperature to room temperature, add other powder excipients, mix for another 40 minutes, and mix.

The above colloid is a combination of carrageenan, guar gum, and propylene glycol alginate, and the ratio of the three preferred combination colloids is 1:2:2.

Lactoferrin and sialic acid, the ratio of the two components in terms of parts by weight is: 50:1.

Embodiment 3

First set the temperature of the heatable three-dimensional mixer to 55°C. After 25 minutes, wait until the temperature distribution in the mixer is even, add sialic acid and lactoferrin to the mixer, and mix for 15 minutes, and then heat to 65°C. (here set to heat with 100w power), add a part of the colloid, mix for 25 minutes, then add the remaining colloid, continue mixing for 25 minutes, then reduce the temperature to 42°C, then mix for 15 minutes, reduce the temperature to room temperature, Add other powder excipients, mix for another 35 minutes, and mix.

The above colloid is a combination of carrageenan, guar gum, and propylene glycol alginate, and the ratio of the three preferred combination colloids is 2:1:1.

Lactoferrin and sialic acid, the ratio of the two components in parts by weight is: 5:1.

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Embodiment 4

First set the temperature of the heatable three-dimensional mixer to 50°C. After 30 minutes, wait until the temperature distribution in the mixer is even, add sialic acid and lactoferrin to the mixer, and mix for 20 minutes, and then heat to 65°C. (here set to heat with 10Ow power), add two-fifths of the weight of the colloid, mix for 20 minutes, then add the remaining three-fifths of the weight of the colloid, continue mixing for 30 minutes, then reduce the temperature to 40°C After mixing for another 20 minutes, the temperature was lowered to room temperature, other powder adjuvants were added, and the mixture was mixed for another 30 minutes, and the mixing was completed.

The above colloid is a combination of carrageenan, guar gum, and propylene glycol alginate. The ratio of the three preferred combination colloids is 1:2:1.

Lactoferrin and sialic acid, the ratio of the two components in terms of parts by weight: 20:1.

Embodiment 5

First set the temperature of the heatable three-dimensional mixer to 55°C. After 20 minutes, wait until the temperature distribution in the mixer is even, add sialic acid and lactoferrin to the mixer, and mix for 15 minutes, then heat to 65°C. (here set to heat with 100w power), add a part of the colloid, mix for 25 minutes, then add the remaining colloid,

2019100970 29 Aug 2019 continue mixing for 30 minutes, then reduce the temperature to 42°C, then mix for 12 minutes, reduce the temperature to room temperature, Add other powder excipients, mix for another 35 minutes, and mix.

The above colloid is a combination of carrageenan, guar gum, and propylene glycol alginate. The ratio of the three preferred combination colloids is 1:1:2.

Lactoferrin and sialic acid, the ratio of the two components in terms of parts by weight: 40:1.

Embodiment 6

The operation method is the same as that in the first embodiment, wherein the colloidal composition is: dextrin, carrageenan, xanthan gum, and sodium alginate in a weight ratio of 1:1:1:1.

Embodiment 7

The operation method is the same as in the second embodiment, wherein the colloidal composition is: carrageenan, xanthan gum, pectin, guar gum in a weight ratio of 1:1:1:1.

Embodiment 8

The operation method is the same as in the third embodiment, wherein the colloidal composition is: xanthan gum, locust bean gum, konjac gum, carrageenan in a weight ratio of 1:1:1:1.

Embodiment 9

The operation method is the same as in the fourth embodiment, wherein the colloidal composition is: carrageenan, locust bean gum, konjac gum, guar gum in a weight ratio of

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1:1:1:1.

Embodiment 10

The operation method is the same as that in Embodiment 5, wherein the colloidal composition is: carrageenan, xanthan gum, pectin, guar gum in a weight ratio of 1:1:1:1.

Analysis of the effect of the embodiment:

For the dispersion of the final product, it is necessary to understand the meaning of uniformity from two aspects: one is to find out whether the various parts of the powder (various powder contents) are uniform on the macro; the second is to examine the microscopic examination between various powder particles and particles, whether it is evenly dispersed.

Macroscopic uniformity: This is the uniformity of the first level. From the macroscopic requirements, the sampling test of the upper and lower parts, the left part and the right part of the container is required. The result must be that the proportions of the various powder formulas are the same, and the material dead angle cannot appear in the container. It is macroscopically uniform. For example, the mixing principle of the three-dimensional mixer and the V-type mixer is gravity diffusion type, and it is easy to achieve macroscopic uniformity, that is, the upper part and the lower part of the equipment container are uniform.

The macroscopically uniform test method is:

1. Take samples uniformly from the upper, middle, lower, left and right sides of the tank;

Second, the number of samples taken is relatively large, 50 grams -100 grams;

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3. The difference in the content of each powder in each sampling meets the inspection standard;

4. The direct difference between the two samples meets the test criteria.

Microscopic uniformity: This is the uniformity of the second level (requiring higher uniformity of uniformity), taking microscopic samples (the size of the sample size determines the high and low requirements of the inspection accuracy), and the various powders between the particles are uniform. It is diffusely distributed and conforms to the proportion of the powder formula, which is microscopic uniformity. Ordinary three-dimensional mixers are difficult to achieve uniform dispersion on the microscopic scale due to the lack of shearing means between the microscopic particles and the particles.

Only one level of uniformity is not truly uniform, and only two levels of uniformity are achieved at the same time to achieve practical standards. For example, the macroscopic parts of the test powder are basically uniform, and the microscopic sample test found that the particles clumped and did not fully diffuse, so the uniform standard was not reached.

The microscopically uniform test method is:

1. Increase the number of samples and the location of the samples;

Second, reduce the amount of sampling; the amount is as little as 1 gram or a few grams;

3. The content of each powder in the sample satisfies the inspection standard;

4. The various powder particles inside the sample show a uniform dispersion under the microscope, and there is no aggregation of the same kind of powder; microscopic

2019100970 29 Aug 2019 observation (microscopic particle uniformity detection) microscope is also a commonly used method for testing the uniformity of mixing. Intuitive and capable of checking the uniform dispersion of powder particles in the microscopic state;

Detection Indicator:

1. The bulk density was measured using a 100 mL graduated cylinder; the sample was free to fall into the cylinder up to about 50 mL, and the bulk density was calculated from the weight and volume of the powder in the cylinder, using the tap density measured using an automatic tap density analyzer. Measuring the true density by using a gas hydrometer;

2. Use an oven to measure moisture content;

3. Measuring the particle size distribution by using a particle size analyzer (mastersizer 3000E);

4. Evaluate powder flow capacity by using a powder rheometer;

5. Perform microscopic observation to analyze particle shape and surface.

After making a plurality of samples of different mixing modes, the measurements are performed in the above manner to obtain the method of the present invention. This is the result obtained with the support of the data, but it can be known that most of them are through simple qualitative comparison. Which method is obtained yields a better product, and data comparison cannot be obtained in a quantitative manner. That is to say, the above detection method is not the focus of the present invention, and is a method which can be grasped by a person of ordinary skill in the art.

Among them, regarding the measurement of particle size distribution is a very

2019100970 29 Aug 2019 important indicator. The sample cylinder automatically rotates at a certain angle within one cycle to ensure that the powder surface is horizontal, thus ensuring the measurement accuracy of the powder surface height. Ensure that the test accuracy error of the sample is <±1%; 100 times/min, 200 times/min, 250 times/min, 300 times/min. Three kinds of glass sample cylinders were arranged, and the volume was 25 mL, 50 mL, and 100 mL, respectively. The conclusion was that the method of grading cooling, temperature rising, pre-mixing, and partial addition were mixed, and the obtained particle size distribution effect was relatively optimal.

About the stability test:

We have done long-term stability tests (shelf life and room temperature samples) and accelerated tests (6 months), which fully meet the standard requirements. Store samples under different conditions, temperatures from 4 to 50°C, with different lighting conditions; evaluate products at different time intervals, typically 2, 4, 8 and 12 weeks, for microbial challenge testing; Stability, if the sample does not change substantially at high temperatures for 8 weeks, it should be stable for 1 year at room temperature.

Component quantitative test

Sialic acid detection

Test samples: Examples 1-12

Sialic acid was detected by high performance liquid chromatography, using C18 (250mm x 4.6mm, 5um) column; detection conditions: column temperature 30 ° C; fluorescence detector excitation wavelength 373nm, emission wavelength 448nm; The flow rate was 1 mL/min; the injection volume was 10 μ L; the mobile phase was selected

2019100970 29 Aug 2019 to be methanol: acetonitrile: ultrapure water, and the ratio was 8:9:84.

2. Protein Detection

A method commonly used in the art is employed, and a commercially available kit is used in the present invention.

The results in Table 1 show that the product obtained by the method of the present invention is prepared into a prepared milk powder, and after testing for the final product, the index is 100 g of sialic acid, lactoferrin content and total protein content in the product, for detecting saliva Acid, lactoferrin, and total protein will not be destroyed when warming the mixture.

Table 1

	The amount of modified milk powder g/1 OOg
	Sialic acid	lactoferrin	total protein
embodimentl	0.33	1.10	85.11
embodiment2	0.34	1.10	84.62
embodiments	0.34	1.08	85.00
embodiment4	0.33	1.09	83.98
embodiments	0.32	1.11	84.92
embodiments	0.31	1.10	83.39
embodiment?	0.30	1.07	84.00
embodiments	0.33	1.08	85.00
embodiment9	0.34	1.10	84.61
embodimentl 0	0.33	1.11	83.70

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Sensory evaluation:

Table 2 is the scoring standard for milk powder uniformity and taste.

score	evaluation	Rating criteria
Tonicity	odor
10	Very good	Sinking time < 10 seconds; number of small white points < 10; no white spots on the cup wall; no clumps	The unique aroma of milk powder, the smell is naturally no bitter
8-9	good	11 seconds < sinking time < 20 seconds; a small amount of small white spots, fine particles; small white spots on the wall of the cup and the number of flakes < 10; 1 s briquettes < 5;	The unique aroma of milk powder is not strong, slightly bitter
6-7	common	21 seconds < sinking time < 30 seconds; a small amount of small white spots, small particles around the small white spots; a small amount of small white spots and flakes on the cup wall; 1 < agglomerate < 5;	The unique milky milk is not strong, slightly vegetable oily, slightly bitter
3-5	bad	31 seconds < sinking time < 40 seconds; a large number of small white spots and flakes, no significant difference between the middle and the periphery; 6 < agglomerate < 10;	Have other odors, bitter taste is heavier
<3	Very bad	41 seconds < sinking time; a large	The milk is not strong

number of small white spots and flakes, no significant difference between the middle and the surrounding; 11 < agglomerate; cup wall has a large number of small white spots and flakes do not fall

and there are other odors, and the bitterness is strong.

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According to the above criteria, the scores of Examples 1 to 12 of the present invention are as shown in Table 3 below.

Table 3 The score of the embodiment of the present invention (15-person evaluation group, the average score, the individual detail score is limited to the length is not attached, only the average score of two digits after the decimal point is attached)


Tonicity	odor
embodimentl	9.22	9.31
embodiment2	8.92	9.06
embodiments	9.03	9.05
embodiment4	9.31	9.26
embodiments	9.21	9.18
embodiments	8.79	8.92
embodiment?	8.61	8.63
embodiments	8.92	8.89
embodiments	8.58	8.61
embodimentl 0	8.68	8.72

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Claims

CLAIM

1. A composition comprising lactoferrin and sialic acid, characterized in that the composition comprises, but is not limited to, lactoferrin and sialic acid, and the ratio of the two components in parts by weight is:

(5-50): 1;

Preferably, the parts by weight are (20-40): 1;

Most preferably it is 30:1.
2. The composition of claim 1 wherein said composition further comprises from

0.5 to 2 parts by weight of cationic colloid, wherein the colloid is one or more in : modified starch, gellan gum, locust bean gum, konjac gum, dextrin, carrageenan, xanthan gum, pectin, microcrystals, cellulose, propylene glycol alginate, gelatin, agar, guar gum, and sodium alginate, wherein carrageenan is preferably used.
3. The composition according to claim 2, characterized in that the composition of the colloids, preferably the three colloids and the parts by weight are:

The combination of carrageenan, guar gum, and propylene glycol alginate, the ratio of the three preferred combinations of colloids is (1-2): (1-2): (1-2);

The ratio by weight of carrageenan, locust bean gum, konjac gum and guar gum is (1-2): (1-2): (1-2): (1-2);

The ratio by weight of carrageenan, xanthan gum, pectin and guar gum is (1-2): (1-2): (1-2): (1-2);

The ratio by weight of xanthan gum, locust bean gum, konjac gum and carrageenan is (1-2): (1-2): (1-2): (1-2);

i

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The ratio by weight of dextrin, carrageenan, xanthan gum, and sodium alginate is (1-2): (1-2): (1-2): (1-2).
4. A method of preparing a composition according to any one of claims 1 to 3, wherein:

First set the temperature of the heatable three-dimensional mixer to 50-60°C; after the temperature distribution in the mixer is uniform, add sialic acid and lactoferrin to the mixer, mix for 10-20 minutes, and heat to 65°C; add a part of the colloid, mix for 20-30 minutes, then add the remaining colloid, continue mixing for 20-30 minutes, then reduce the temperature to 40-45°C, mix for another 10-20 minutes, reduce the temperature to room temperature, add other powder accessories, mix for another 30-40 minutes, the mixing is over.
5. Use of a composition according to any one of claims 1 to 4, characterized in that the composition is used in infant formula, nutritional supplement powder, nutritional supplement tablet.