Protein composite material suitable for infants and application thereof

Abstract

The invention discloses a protein composite material suitable for infants and application thereof, and relates to the field of biological products. According to the invention, the active protein composite material entrapped with probiotics is constructed, so that the probiotics can be effectively protected from the influence of gastrointestinal tract extreme environment, and the stability and bioavailability of the probiotics are ensured. The composite material LF and OPN play a role similar to prebiotics and are nutrients of probiotics, and compared with monomeric protein, when the composite material LF and the probiotics are combined for use, the reproductive capacity and the vitality of the probiotics can be remarkably improved, and the synergistic interaction effect is achieved; the digestion characteristics of the protein composite material for protecting the probiotics are determined on the basis of the infant gastrointestinal dynamic digestion model, and the method is more suitable for infant audiences. The invention researches that the LF and OPN protein composite material coats the probiotics to improve the treatment potential of the probiotics, and the research content aims at the inflammatory intestinal diseases which are easy to occur in infants and conforms to the main functions of the infants.

Description

Technical Field

[0001] The present invention relates to the field of biological products. More specifically, the present invention relates to a protein composite material suitable for infants and its applications. Background Art

[0002] Breast milk is the most ideal source of nutrition for the growth and development of infants and young children. It is also rich in immunoglobulins and other substances that play an important role in preventing diseases. Lactoferrin (LF) and osteopontin (OPN) are important bioactive proteins in milk. The contents of LF and OPN are relatively high in colostrum, and their concentrations gradually decrease as the lactation period extends. This content characteristic indicates that LF and OPN play important roles in the early development stage of infants and young children. Studies have found that LF can completely resist the digestion of the gastrointestinal tract of infants and young children, and OPN can partially resist gastrointestinal digestion and reach the intestine to exert related bioactive functions, such as promoting intestinal growth and development, participating in immune regulation, slowing down the occurrence of inflammation, and inhibiting the growth of harmful bacteria. The isoelectric point of LF is about 8, which is an alkaline protein, and the isoelectric point of OPN is about 3.6, which is an acidic protein. The two proteins carry opposite charges in milk and can interact through van der Waals forces.

[0003] A large number of microbial flora coexist in the intestine, and these intestinal flora can be divided into beneficial bacteria and harmful bacteria. Among them, beneficial bacteria, also known as probiotics, are a class of active microorganisms colonized in the intestine that are beneficial to the body. Probiotics have been proven to have the effect of improving the intestinal environment. Probiotics can enhance the barrier function of the host intestine, reduce the permeability of the intestinal mucosa to bacteria, reduce the occurrence of pathogen translocation, and competitively inhibit the growth of pathogens. The main probiotics in the intestine of newborns are Bifidobacterium and Lactobacillus. Although probiotics have many effects, there are great difficulties in the use of probiotics, which limits their effective application. Most probiotics are mainly anaerobic bacteria and facultative anaerobic bacteria, and they do not form spores during the growth and reproduction process, and have poor stress resistance. At the same time, due to the influence of environmental temperature and other factors during storage, the activity of the flora is also easily severely damaged. In addition, after probiotics are ingested orally and enter the human body, they must pass through the digestive tract and attach to the surface of the intestinal mucosa after reaching the intestine to exert their effects. Most probiotics will die due to the action of gastric acid, choline, etc. in digestive juice before colonizing and exerting their effects, resulting in a sharp decrease in the flora that can finally colonize in the intestine, seriously affecting the efficacy of probiotics. Probiotics usually need to be coated and protected to reach the target site to exert their effects.

[0004] However, most of the existing probiotic coating materials are targeted at the adult population. For the infant population, whose gastrointestinal environment is significantly different from that of adults, there are very few choices of coating materials, and probiotic coating materials suitable for infants are urgently needed to be developed. In addition, the infant intestinal tract and immune system are not yet fully developed, and are prone to inflammatory bowel diseases, etc., which seriously affect growth and health. In the case of intestinal diseases, the intestinal environment is extremely harsh, with oxidative stress, intestinal environment disorders, etc., which seriously affect probiotic colonization. At present, most of the existing probiotic coating technologies only play a protective role through physical barrier methods, and the effect of such coating materials is very limited. Therefore, it is very important to explore safe and efficient active coating materials suitable for the infant population. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a protein composite material suitable for infants and its application. The present invention provides the following technical solutions:

[0006] In the first aspect of the present invention, a protein composite material suitable for infants is provided, and the protein composite material comprises lactoferrin and osteopontin.

[0007] In a preferred embodiment, the mass ratio of lactoferrin in lactoferrin and osteopontin is 0.1 - 0.6.

[0008] Preferably, when the mass ratio of lactoferrin in lactoferrin and osteopontin is 0.5, that is, when the ratio of LF and OPN proteins is 1:1, the turbidity reaches the maximum, the colloid formed by the two proteins is the most significant, and the coating effect on probiotics is the best.

[0009] In a preferred embodiment, the use concentration of the protein composite material is 10 mg / mL.

[0010] In a preferred embodiment, the protein composite material is a nutritional supplement, an encapsulating agent, and / or complementary food.

[0011] In the second aspect of the present invention, an application of the protein composite material in preparing a probiotic active coating material suitable for infants is provided.

[0012] In one embodiment, the probiotic active coating material refers to: (a) a material that can improve the resistance of probiotics to gastrointestinal digestion; or (b) a material with a high targeting delivery efficiency in the gastrointestinal tract.

[0013] Preferably, the age of the infant is 0 - 12 months after birth, that is, an infant within 12 months.

[0014] Preferably, the probiotic is Bifidobacterium (BB).

[0015] Preferably, during application, the probiotic active coating material is used orally.

[0016] The present invention has the following outstanding features and remarkable progress compared with the prior art:

[0017] 1) The present invention constructs an active protein composite material encapsulating probiotics, which can effectively protect probiotics from the influence of the extreme gastrointestinal environment and ensure their stability and bioavailability.

[0018] 2) The composite materials LF and OPN involved in the present invention are bioactive components in breast milk, which themselves have positive effects on promoting the development of infants' gastrointestinal tract, inhibiting inflammation, and regulating immunity;

[0019] 3) The composite materials LF and OPN involved in the present invention play the role of prebiotics and are the "nutrients" of probiotics. Compared with monomeric proteins, when combined with probiotics, they can significantly improve the reproductive ability and vitality of probiotics, playing a synergistic effect.

[0020] 4) The present invention determines the digestive characteristics of the protein composite material protecting probiotics based on the infant gastrointestinal dynamic digestion model, which is more suitable for the infant audience.

[0021] 5) The present invention studies the improvement of the therapeutic potential of probiotics by coating probiotics with the LF and OPN protein composite material. The research content targets the inflammatory bowel diseases that are prone to occur in infants, which is in line with its main function. Description of the Drawings

[0022] Figure 1 is the turbidity of the mixture of LF and OPN at different protein ratios at pH 4;

[0023] Figure 2 is the appearance of the protein composite material;

[0024] Figure 3 is the microstructure of the protein composite material;

[0025] Figure 4 is the in vivo tracing of the protein composite material coating probiotics (Bifidobacterium); Detailed Embodiments

[0026] Example 1 Construction of the LF and OPN Protein Composite Material (LO)

[0027] The protein powders used were commercially available protein powders on the market, lactoferrin protein powder (1043NVD, Friesland Campina, Netherlands) and osteopontin protein powder (B470239, Arla Foods Ingredients, Denmark).

[0028] 1. Protein ratio screening

[0029] To determine the optimal ratio of LF and OPN when they combine under the acidic conditions of infant gastric juice, LF and OPN powders were separately dissolved in deionized water to prepare protein solutions with a concentration of 10 mg / mL, and their protein concentrations were measured by the BCA method. 1 M HCl was used to adjust the solutions to the acidity of infant gastric juice, pH 4. According to Table 1 shown below, LF and OPN were mixed in a 96-well plate according to the following ratios, and the absorbance value at a wavelength of 600 nm was detected using a multifunctional microplate reader.

[0030] Table 1 LF and OPN ratio table

[0031]

[0032]

[0033] The results are as Figure 1 shown. As the ratio of LF / (LF + OPN) gradually increases, the turbidity of the solution gradually increases. When the ratio of LF / (LF + OPN) is 0.1 - 0.6, its turbidity is relatively high, obvious interaction occurs, and the turbidity reaches the maximum at 0.5; as the protein ratio continues to increase, the turbidity of the solution gradually decreases. Thus, under the condition of pH 4, when the ratio of LF to (LF + OPN) protein is 0.1 - 0.6, the interaction between the two proteins is obvious, with the potential to coat probiotics, and the best ratio is 0.5.

[0034] 2. Gel structure characterization

[0035] (1) Appearance of protein gel: The 10 mg / mL LF and 10 mg / mL OPN protein solutions were separately adjusted to pH 4, and then 5 mL of each protein solution was taken and mixed evenly. The protein mixture was centrifuged at 3500 rpm for 10 minutes, and the appearance of the gel was observed and photographed.

[0036] The appearance of the LF and OPN gels is as Figure 2 shown. The upper layer is a clear and transparent solution, and the lower layer forms a reddish-brown condensate, indicating that the two proteins interact to cause liquid-liquid phase separation and form a gel.

[0037] (2) Microstructure: Scanning electron microscopy was used to detect the structural characterization of the LF-OPN system. The centrifuged protein gel was placed in a -80 °C refrigerator and frozen overnight. The next day, it was put into a pre-frozen dryer for freeze-drying to obtain a solid sample. The microscopic structure and morphology of the protein gel were observed using a scanning electron microscope at a scanning voltage of 3 kV, and the images were scanned at a magnification of 1000×.

[0038] The results of the electron microscopy are as Figure 3As shown, its microstructure is a loose and porous network structure with uniform pore sizes and evenly distributed positions, which conforms to the microscopic structural characteristics of hydrogels. In addition, the pore diameter is about 5 μm and the length of BB is about 8 μm, which enables the bacteria to be encapsulated in the composite material and not easily escape through the pores.

[0039] Example 2 Coating Application of Protein Composite Material on Probiotics

[0040] 1. Coating of probiotics:

[0041] Since the LF-OPN(LO) composite material spontaneously complex-condenses in an acidic gastric environment, the LF-OPN protein mixture can be directly mixed with Bifidobacterium bifidum (BB). The number of BB was measured using a McFarland turbidimeter. Take 2 mL of LF-OPN (1:1, 1 mg / mL) protein mixture and mix it with 2×10 9 CFU of BB to obtain the low-concentration coating group (BB-LO1); 2 mL of LF-OPN (1:1, 10 mg / mL) protein mixture and 2×10 9 CFU of BB were mixed to obtain the high-concentration coating group (BB-LO10); 2×10 9 CFU of BB were placed in 2 mL of 1×PBS solution as the uncoated probiotic control group.

[0042] 2. In vitro simulated digestion:

[0043] Take 2 mL of BB, BB-LO1, and BB-LO10 respectively, add an appropriate amount of 1 M HCl to adjust the pH to 4, then add 2% pepsin-HCl solution, incubate at 37 °C in the dark at 120 rpm for 30 min, collect the digestive juice, dilute it, and plate count.

[0044] Table 2 In vitro simulated digestion of protein composite material-coated probiotics (Bifidobacterium bifidum)

[0045]

[0046] The results are shown in Table 2. There were no surviving colonies in the BB group after in vitro simulated gastric digestion. Probiotics still survived after in vitro digestion of the composite material group, and more bacteria survived in the high-concentration coating group than in the low-concentration coating group. This indicates that the protein composite material has a good protective effect against gastric digestion for BB, and the 10 mg / mL composite material shows a better probiotic protection effect compared to the low-concentration composite material.

[0047] 3. In vivo tracing:

[0048] Take 45 μg of CY5-SE (MCE, 146368-14-1) fluorescent dye and 2×10 9CFU BB was mixed in 1 mL of 1×PBS solution, incubated at 37 °C for 30 min, and free dye was removed after washing with PBS. The stained BB (1×10 9 CFU) was intragastrically administered to 15-day-old C57BL / 6J mouse pups. The mice were anesthetized at 0 h and 16 h after intragastric administration, and ventral in vivo imaging tracing was performed.

[0049] The results are as Figure 4 shown. At 0 h, obvious fluorescence signals were presented in the gastrointestinal tracts of mice in each group. At 16 h, the fluorescence signals were significantly weakened, and the fluorescence signals in the control group and LO-1 group completely disappeared. Only the LO-10 group still had fluorescence signals, indicating that after 16 h of in vivo digestion, more Bifidobacterium in the LO-10 group remained in the body. Thus, it can be seen that the protein composite material (10 mg / mL) can effectively protect BB against gastrointestinal digestion in vivo and increase its colonization efficiency in the intestine.

[0050] 4. The situation of the protein composite material promoting the growth of probiotics

[0051] A total of 4 groups were set, the BB group, the LF plus BB group (BB-LF), the OPN plus BB group (BB-OPN), and the composite protein plus BB group (BB-LO). 2×10 8 CFU BB was added to each well of a 96-well plate and mixed with the corresponding monomeric protein or composite protein. The volume of the solution in each well was adjusted to 200 μL with BB growth medium, and the final protein concentration was 10 mg / mL. Subsequently, the 96-well plate was placed in a microplate reader stored in an anaerobic workstation and gently shaken at 100 rpm. The absorbance value of each well at OD 600 nm was monitored and recorded every hour for a total of 12 h.

[0052] Table 3 The effect of the protein composite material on promoting the growth of probiotics (Bifidobacterium)

[0053]

[0054] As shown in the results of Table 3, with the increase of time, BB grew rapidly and began to level off after 8 hours. There was no significant difference in the growth of bacteria between the BB group and the BB-LF10 group all the time; at 4.5 h, the growth of Bifidobacterium in the BB-OPN group and the BB-LO group was significantly increased compared with that in the BB group and the BB-LF group; starting from the 6th h, the BB-LO group was significantly increased compared with the BB-OPN group. This shows that the protein composite material has a significant effect on promoting the growth of Bifidobacterium, and its effect is better than that of monomeric protein.

Claims

1. A protein composite material suitable for infants, characterized in that: The protein composite material comprises lactoferrin and osteopontin.

2. The protein composite material suitable for infants according to claim 1, characterized in that: The mass ratio of lactoferrin to osteopontin is 0.1-0.

6.

3. The protein composite material suitable for infants according to claim 2, characterized in that: The mass ratio of lactoferrin to osteopontin is 0.

5.

4. The protein composite material suitable for infants according to claim 2 or 3, characterized in that: The protein composite material is used at a concentration of 1 to 10 mg / mL.

5. The protein composite material suitable for infants according to claim 1, characterized in that: The protein composite material is a nutritional supplement, an encapsulating agent and / or a complementary food.

6. Use of the protein composite material according to any one of claims 1 to 5 in the preparation of probiotic active coating materials suitable for infants.

7. The use according to claim 6, characterized in that: The infant age is 0-12 months after birth.

8. The use according to claim 6, characterized in that: The probiotics are Bifidobacterium.

9. The use according to claim 6, characterized in that: When used, the probiotic active coating material is used orally.

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