Preparation method of lactoferrin-polyphenol nanocomposite with intestinal immune function

Abstract

The invention discloses a preparation method of a lactoferrin-polyphenol nano compound with an intestinal immune function. The method comprises the following steps: firstly, moderately hydrolyzing lactoferrin under the action of compound protease, and optimizing a protein molecular structure; then, compounding with water-soluble polyphenol (such as chlorogenic acid or epicatechin) by utilizing a metal ion chelating technology, and inducing lactoferrin to be bridged and aggregated by Ca to form a compound; and then, carrying out ultrasonic homogenization treatment, and further controlling the particle size to enable the particle size to be distributed in a range of 100-300 nm. The obtained compound is subjected to nanofiltration concentration and electrostatic spray drying to obtain a shell-core nano compound with a stable structure, and nitrogen-filled packaging is adopted to prevent oxidation. The lactoferrin-polyphenol nano-composite prepared by the invention not only has good stability and intestinal immunity enhancement effect, but also has remarkable antibacterial activity, and can be widely applied to functional foods and nutritional health-care products.

Description

Technical Field

[0001] The present invention relates to the field of food nutrition and functional factors, and specifically to a lactoferrin-polyphenol nanocomposite with intestinal immune function, a preparation method and an application thereof. Background Art

[0002] Lactoferrin is a multifunctional protein that exists naturally in breast milk. It is widely used in food, health products and medicine because of its biological activities such as antibacterial, antiviral, anti-inflammatory and immunomodulatory.

[0003] However, there are a series of technical defects in the practical application of lactoferrin, which limit its biological activity and stability, especially during processing and storage.

[0004] First, lactoferrin is susceptible to enzymatic hydrolysis, pH and temperature changes, and is prone to degradation under high temperature or acidic conditions, resulting in a decrease in activity.

[0005] Secondly, lactoferrin molecules are large and difficult to pass through the intestinal barrier, which affects the absorption and utilization of its immune active components in the human body.

[0006] In addition, lactoferrin itself has a high positive charge, which makes it easy to form large-particle aggregates when combined with polyphenol compounds, which is not conducive to the formation of a stable nanocomposite structure.

[0007] The above problems not only affect the interaction between lactoferrin and polyphenols, but also lead to the reduced dispersibility and stability of the nanocomplex in the intestine, thus limiting its biological activity. Summary of the invention

[0008] Technical problem to be solved by the present invention: The purpose of the present invention is to solve the deficiencies in the prior art, to provide a lactoferrin-polyphenol nanocomposite and a preparation method thereof that protects the activity of lactoferrin and significantly enhances its intestinal immune function, and to propose an application of the lactoferrin-polyphenol nanocomposite.

[0009] The technical solution of the present invention: The present invention discloses a method for preparing a lactoferrin-polyphenol nanocomposite with intestinal immune function, comprising the following steps: S1. Lactoferrin hydrolysis; S2. Inducing the formation of lactoferrin-polyphenol complexes by metal ion chelation technology, adding water-soluble polyphenol compounds to the lactoferrin hydrolyzate, and then adding calcium chloride, using Ca2+ to induce negatively charged lactoferrin to bridge and aggregate, and encapsulate the polyphenol compounds in the aqueous phase; S3. Ultrasonic homogenization treatment induces structural deconstruction and reconstruction of lactoferrin-polyphenol complex; S4. Nanofiltration concentration and polarized spray drying.

[0010] Furthermore, in the step S1, the lactoferrin hydrolysis includes moderately hydrolyzing the lactoferrin using a combination of endo-enzymes such as papain, bromelain, and ginger protease, the amount of composite protease added is 2000-3000U / g, the hydrolysis pH is 6.5-8.5, the hydrolysis temperature is 50-65°C, the hydrolysis time is 2-4h, and the ratio of any two proteases is 2:1-1:2.

[0011] Furthermore, in step S2, under the condition of pH 4-8, water-soluble polyphenol compounds such as chlorogenic acid are added to the lactoferrin hydrolyzate obtained in step S1, and then 0.1mM-0.5mM calcium chloride is added to use Ca2+ to induce negatively charged lactoferrin to bridge and aggregate, and encapsulate polyphenol compounds such as chlorogenic acid in the aqueous phase.

[0012] Furthermore, in the step S3, the ultrasonic treatment power is 30-60 Hz, the treatment time is 5-15 minutes, and after the ultrasonic treatment, the size of the lactoferrin-polyphenol nanocomposite is reduced from 10-100 μm to 100-300 nm, and is stably dispersed.

[0013] Furthermore, in step S4, an electrostatic spray drying device is used to further induce the lactoferrin-polyphenol nanocomposite to undergo structural re-beats with different charged micro-bends, forming a microstructure step in which the polar head faces inward and the non-polar head faces outward, thereby forming a shell-core encapsulation structure for the water-soluble polyphenol.

[0014] Furthermore, the spray drying air inlet temperature is 90-100°C, and the air outlet temperature is 40-50°C.

[0015] Furthermore, after step S4, the method further includes nitrogen filling and packaging the lactoferrin-polyphenol nanocomposite.

[0016] Furthermore, in step S1, the purity of the lactoferrin is greater than 80%.

[0017] The present invention also discloses a lactoferrin-polyphenol nanocomposite with intestinal immune function, wherein the complex is formed by combining lactoferrin and water-soluble polyphenol through metal ion chelation, has a particle size of 100-300 nm, has a shell-core structure, and exhibits enhanced intestinal immune function and antibacterial activity. The present invention also discloses an application of a lactoferrin-polyphenol nanocomposite with intestinal immune function. The lactoferrin-polyphenol nanocomposite prepared based on the preparation method of the lactoferrin-polyphenol nanocomposite with intestinal immune function, or based on the lactoferrin-polyphenol nanocomposite, can enhance the intestinal immune function by stimulating intestinal epithelial cells to secrete immune factors.

[0018] The beneficial effects of the present invention compared with the prior art are as follows: By optimizing the enzymatic hydrolysis conditions, introducing metal ion chelation technology and ultrasonic homogenization treatment, the stability and bioavailability of the complex are effectively improved. The technical solution of the present invention not only protects the activity of lactoferrin, but also significantly enhances its intestinal immune function, and has important application value.

[0019] The present invention systematically optimizes the hydrolysis conditions of lactoferrin (such as enzyme combination, pH, temperature and time to achieve moderate hydrolysis) and the chelation process (pH value and calcium ion concentration), thereby ensuring that lactoferrin successfully combines with polyphenols to form a stable nanocomposite while retaining its biological activity. This optimization not only avoids excessive damage to the structure of lactoferrin, but also improves the activity and structural stability of the complex, making the product suitable for long-term storage and a variety of functional application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a relative molecular mass distribution diagram of the lactoferrin hydrolysate in the present invention; Figure 2 is the intrinsic fluorescence spectrum of lactoferrin and its polyphenol complex at 298 K in the present invention; Figure 3 is the average particle size of lactoferrin and its polyphenol complex in the present invention; Figure 4 is the average particle size change of the lactoferrin-polyphenol complex after ultrasound in the present invention; Figure 5 The effect of spray drying on the polyphenol retention rate of the lactoferrin-polyphenol complex in the present invention; Figure 6 The effect of the lactoferrin-polyphenol complex of the present invention on the expression of cellular immune factors; Figure 7 This is the effect of the lactoferrin-polyphenol complex in the present invention on the Thl / Th2 differentiation balance of intestinal cells. DETAILED DESCRIPTION

[0021] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0022] The present invention provides a method for preparing a lactoferrin-polyphenol nanocomposite with intestinal immune function, which comprises five steps: moderate hydrolysis of lactoferrin, metal ion chelation technology-induced compounding, deconstruction and reconstruction of the polyphenol nanocomposite structure, nanofiltration concentration and polarized spray drying.

[0023] 1. Raw material selection and pretreatment The lactoferrin raw material used in the present invention is commercially available or laboratory-independently separated lactoferrin, with a purity of more than 80%, ensuring the quality and activity of lactoferrin. The polyphenols used are water-soluble chlorogenic acid and epicatechin, which have good antioxidant and immunomodulatory activities.

[0024] 2. Lactoferrin hydrolysis Objective: To moderately hydrolyze lactoferrin to enhance its structural stability and avoid the formation of large aggregates in subsequent steps.

[0025] step: Dissolve lactoferrin in deionized water and adjust the lactoferrin concentration to 5% (w / v). Add composite protease (papain, bromelain, ginger protease) at an enzyme dosage of 2500 U / g lactoferrin.

[0026] Hydrolysis conditions: pH: 7.5 Temperature: 55℃ Hydrolysis time: 3 hours Enzyme ratio: papain: bromelain: ginger enzyme = 2:1:1 Experimental data: The hydrolysis degree of lactoferrin after hydrolysis was 58.60%. The HPLC test results showed that the molecular weight distribution of lactoferrin after hydrolysis was stable and the structure was intact.

[0027] Control group: under the same conditions, no compound enzyme treatment was added and lactoferrin maintained its original structure.

[0028] like Figure 1As shown in the figure, after being hydrolyzed by a composite enzyme, lactoferrin is mainly degraded into small peptide fragments with a molecular weight of less than 5000 Da, accounting for 61.25%, followed by 500-5000 Da hydrolysis fragments with a content of 24.06%, and a small amount (14.69%) of hydrolysis fragments >5000 Da. The molecular weight of protein hydrolysates is usually closely related to their biological activity. Studies have shown that polypeptides with a molecular weight between 500-1000 Da have high antioxidant activity and immune activity, as well as good transdermal, moisture absorption and moisturizing properties. Such polypeptides are more easily absorbed and utilized by the human body than intact protein macromolecules. The molecular weight distribution results of this patent show that lactoferrin has a high content of 500-1000 Da digestion product fragments after being hydrolyzed by a composite enzyme. It is speculated that composite enzyme hydrolysis increases the immunomodulatory activity of lactoferrin.

[0029] 3. Non-covalent complexation of polyphenols Purpose: Through Ca² + Chelation technology induces lactoferrin and polyphenols to form a stable nanocomplex.

[0030] step: The hydrolyzed lactoferrin solution was adjusted to pH 6.0, and water-soluble chlorogenic acid and epicatechin were added to make the polyphenol concentration reach 20 mg / g lactoferrin. Subsequently, calcium chloride solution was added dropwise, Ca² + The concentration was controlled at 0.3 mM and stirred thoroughly.

[0031] Experimental data: After the chelation reaction, the fluorescence intensity of the complex measured by intrinsic fluorescence spectroscopy was significantly reduced, indicating that polyphenols were successfully embedded in the structure of lactoferrin. The particle size analysis results showed that the average particle size of the complex was about 150 nm and was evenly distributed.

[0032] Control group: No polyphenols and calcium chloride were added, and the next step was directly performed. The results showed that no stable complex was formed.

[0033] Intrinsic fluorescence spectroscopy can reflect the structural changes of proteins and their binding to molecules, and has become an effective technique to study the changes in the polar microenvironment after protein-polyphenol interactions.

[0034] like Figure 2 As shown in the figure, when water-soluble chlorogenic acid and epicatechin were added at a concentration of 20 mg / g, the fluorescence peak of the lactoferrin-polyphenol complex showed an obvious red shift, which indicated that there might be hydrogen bonding interactions between lactoferrin and polyphenols in the hydrophobic cavity of the protein.

[0035] In addition, the fluorescence intensity of the lactoferrin-chlorogenic acid complex was significantly lower than that of the lactoferrin-epicatechin complex, indicating that the addition of chlorogenic acid led to the unfolding of the lactoferrin structure and the exposure of more tryptophan and tyrosine groups compared with epicatechin. This indicates that the binding affinity of chlorogenic acid to lactoferrin is higher than that of epicatechin.

[0036] Protein aggregation has a significant impact on protein solubility and other functional properties, and protein aggregation is usually assessed by characterizing protein particle size. Figure 3 In the experiment, the particle size of lactoferrin-polyphenol complex was significantly increased compared with that of lactoferrin (P<0.05), indicating that lactoferrin interacted with chlorogenic acid and epicatechin to form a nanocomplex.

[0037] 4. Ultrasonic homogenization of lactoferrin-polyphenol nanocomplexes Objective: To further deconstruct and reconstruct lactoferrin-polyphenol complexes by ultrasonic treatment to obtain stable nanostructures.

[0038] step: The chelated lactoferrin polyphenol complex was placed in an ultrasonic device for treatment, the ultrasonic power was set to 45 Hz, the treatment time was 10 minutes, and the temperature was controlled below 25°C during the ultrasonic treatment.

[0039] Experimental data: After ultrasonic treatment, the particle size of the complex decreased from 150 nm before chelation to 120 nm, and the complex was uniformly dispersed in the solution.

[0040] Transmission electron microscopy (TEM) observation showed that the composite was in the shape of a regular sphere with uniform distribution.

[0041] Control group: The particle size of the complex without ultrasonic treatment was larger, the dispersion was uneven, and the precipitation rate was higher.

[0042] like Figure 4 As shown in Figure 3, the particle size of lactoferrin-polyphenol complexes was significantly reduced after ultrasonic treatment, because the cavitation-induced microfluidic effect can enhance molecular mass transfer and increase the collision frequency between lactoferrin and polyphenol molecules, thereby promoting the formation of smaller and more uniform complexes.

[0043] 5. Nanofiltration concentration and electrostatic spray drying Objective: To increase the concentration of the composite and optimize the structure retention during spray drying.

[0044] step: First, nanofiltration concentration was performed for 40 minutes, and the concentration of lactoferrin polyphenol complex was increased to 20%. After concentration, the complex was dried using an electrostatic spray drying device, and the spray drying inlet temperature was controlled at 95°C and the outlet temperature was controlled at 45°C.

[0045] Experimental data: The dried lactoferrin polyphenol composite powder showed a microscopic core-shell structure. The particle size analysis results showed that the powder particle size after spray drying was about 150 nm, and the particle size distribution was uniform. The dried composite had good stability, and the antioxidant activity retention rate of polyphenols under storage conditions reached 90%.

[0046] Control group: conventional spray drying (without electrostatic treatment) was used. The results showed that the particle size of the composite was larger after drying, and the polyphenol activity was significantly lost.

[0047] 6. Nitrogen filling packaging Purpose: To prevent oxidation and degradation of the compound during storage by nitrogen-filled packaging.

[0048] step: The dried lactoferrin-polyphenol nanocomposite powder is placed in a nitrogen-filled packaging bag, and then sealed and stored after being filled with nitrogen.

[0049] Experimental data: After three months of storage, accelerated aging tests showed that the structure of the complex in the nitrogen-filled packaging group was intact and the polyphenol oxidation rate was less than 5%, while the control group (without nitrogen filling) showed a polyphenol oxidation rate of up to 20%.

[0050] Characteristic analysis 1. Particle size and morphology analysis: The particle size of the composite was measured by transmission electron microscopy (TEM) and dynamic light scattering. The results showed that the final composite particle size was in the range of 120-150 nm and was uniformly spherical.

[0051] 2. Stability test: The composite powder was stored at 40°C for 14 days, and the results of the accelerated aging test showed that the composite had good dispersibility, no obvious increase in particle size, and high stability.

[0052] 3. Biological activity test: Intestinal immune function: Through in vitro cell experiments, the secretion of immune factors by intestinal epithelial cells treated with the complex was significantly increased.

[0053] In the complex immune response process, IL-10, as a multifunctional cytokine, especially as B cell stimulating factor-2, can induce activated B cells to produce IgA. As key regulatory molecules of the immune system, immune cytokines can not only regulate the activity of immune cells and promote their proliferation, but also coordinate the balance of various immune responses. They are often used as an important indicator to evaluate the strength of the body's immune function.

[0054] like Figure 3-6 As shown in the figure, the IL-10 content in the model group was significantly lower than that in the normal group (P<0.05). This result revealed that the inflammatory cell model was successfully established. Compared with the model group, the IL-10 content in the lactoferrin-polyphenol complex group was significantly increased (P<0.05), indicating that the lactoferrin-polyphenol complex can relieve inflammation by upregulating IL-10 expression.

[0055] The balance of Thl / Th2 cell differentiation is crucial to the normal immune function of the body, and its imbalance is closely related to the occurrence of many diseases. Taking the Th1 / Th2 ratio as a balance indicator, the effect of lactoferrin-polyphenol complex on the balance of Th1 / Th2 cytokine secretion in inflammatory intestinal cells was further explored. Figure 7 The results showed that compared with the normal group, the Th1 / Th2 ratio of the model group increased significantly (P<0.001), indicating that Th1 in intestinal cells was overexpressed, which may be related to the enhancement of intestinal inflammatory response. Compared with the model group, the Th1 / Th2 of the lactoferrin-polyphenol complex was significantly reduced (P<0.01). This shows that the lactoferrin-polyphenol complex has an immunomodulatory effect and can repair the Th1 / Th2 imbalance by regulating the secretion of cytokines, thereby enhancing intestinal immune function and improving intestinal immune homeostasis.

[0056] Summary of results The invention discloses a method for preparing a lactoferrin-polyphenol nanocomposite with intestinal immune function, which realizes effective compounding of lactoferrin and polyphenol through reasonable hydrolysis, polyphenol chelation, ultrasonic treatment, nanofiltration concentration and electrostatic spray drying to form a nanocomposite with stable structure. The experimental results show that the nanocomposite has significant intestinal immune function and antibacterial effect, and has good stability during storage.

[0057] In summary, the present invention innovatively constructs a stable lactoferrin-polyphenol nanocomposite structure by combining metal ion chelation technology and ultrasonic homogenization. This method not only effectively solves the problem of easy aggregation after lactoferrin and polyphenol are combined, but also improves the dispersibility and bioavailability of the complex in the intestine, making the active ingredients easier to be absorbed by the intestine, and significantly enhancing the intestinal immune function of the product.

[0058] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above specific embodiments. The above specific embodiments and the description in the specification are only for further illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of the present invention to be protected is defined by the claims and their equivalents.

Claims

1. A method for preparing a lactoferrin-polyphenol nanocomposite with intestinal immune function, characterized in that: The following steps are involved: S1. Lactoferrin hydrolysis; S2. Inducing the formation of lactoferrin-polyphenol complexes by metal ion chelation technology, adding water-soluble polyphenol compounds to the lactoferrin hydrolyzate, and then adding calcium chloride, using Ca2+ to induce negatively charged lactoferrin to bridge and aggregate, and encapsulate the polyphenol compounds in the aqueous phase; S3. Ultrasonic homogenization treatment induces structural deconstruction and reconstruction of lactoferrin-polyphenol complex; S4. Nanofiltration concentration and polarized spray drying.

2. The method for preparing the lactoferrin-polyphenol nanocomposite with intestinal immune function according to claim 1, characterized in that: In the step S1, the lactoferrin hydrolysis includes using a combination of endo-enzymes such as papain, bromelain, ginger protease, etc. to moderately hydrolyze the lactoferrin, the added amount of the composite protease is 2000-3000U / g, the hydrolysis pH is 6.5-8.5, the hydrolysis temperature is 50-65°C, the hydrolysis time is 2-4h, and the ratio of any two proteases is 2:1-1:

2.

3. The method for preparing the lactoferrin-polyphenol nanocomposite with intestinal immune function according to claim 1, characterized in that: In step S2, under the condition of pH 4-8, water-soluble polyphenol compounds such as chlorogenic acid are added to the lactoferrin hydrolyzate obtained in step S1, and then 0.1 mM-0.5 mM calcium chloride is added to use Ca2+ to induce negatively charged lactoferrin to bridge and aggregate, and encapsulate polyphenol compounds such as chlorogenic acid in the aqueous phase.

4. The method for preparing the lactoferrin-polyphenol nanocomposite with intestinal immune function according to claim 1, characterized in that: In the step S3, the ultrasonic treatment power is 30-60 Hz, and the treatment time is 5-15 minutes. After the ultrasonic treatment, the size of the lactoferrin-polyphenol nanocomposite is reduced from 10-100 μm to 100-300 nm, and is stably dispersed.

5. The method for preparing the lactoferrin-polyphenol nanocomposite with intestinal immune function according to claim 1, characterized in that: In step S4, an electrostatic spray drying device is used to further induce the lactoferrin-polyphenol nanocomposite to undergo structural re-beats with different charged micro-bends, forming a microstructure with the polar head facing inward and the non-polar head facing outward, thereby forming a shell-core encapsulation structure for the water-soluble polyphenol.

6. The method for preparing the lactoferrin-polyphenol nanocomposite with intestinal immune function according to claim 5, characterized in that: The spray drying air inlet temperature is 90-100℃, and the air outlet temperature is 40-50℃.

7. The method for preparing the lactoferrin-polyphenol nanocomposite with intestinal immune function according to claim 1, characterized in that: After step S4, the method further includes nitrogen filling and packaging the lactoferrin-polyphenol nanocomposite.

8. The method for preparing the lactoferrin-polyphenol nanocomposite with intestinal immune function according to claim 1, characterized in that: In the step S1, the purity of the lactoferrin is greater than 80%.

9. A lactoferrin-polyphenol nanocomposite with intestinal immune function, characterized in that: The complex is formed by combining lactoferrin and water-soluble polyphenols through metal ion chelation, has a particle size of 100-300 nm, has a shell-core structure, and exhibits enhanced intestinal immune function and antibacterial activity.

10. An application of a lactoferrin-polyphenol nanocomposite with intestinal immune function, characterized in that: The lactoferrin-polyphenol nanocomposite prepared by the method for preparing the lactoferrin-polyphenol nanocomposite with intestinal immune function according to any one of claims 1 to 8, or the lactoferrin-polyphenol nanocomposite with intestinal immune function according to claim 9 can enhance intestinal immune function by stimulating intestinal epithelial cells to secrete immune factors.

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