Heterologous lactoferrin-carbohydrate covalent complex and preparation method thereof

Abstract

The invention relates to a heterologous lactoferrin-carbohydrate covalent complex and a preparation method thereof. The heterologous lactoferrin is subjected to carbohydrate chemical modification through a wet Maillard reaction. The covalent complex prepared by adopting the method has the advantages that the oxidation resistance is enhanced, and the dispersion stability of an aqueous solution, the surface hydrophilicity and the thermal stability are obviously enhanced. In addition, it is proved that the plant-derived recombinant human lactoferrin-carbohydrate covalent complex shows a structure and a property change trend similar to those of a bovine-derived lactoferrin-carbohydrate covalent complex. After the heterologous lactoferrin is covalently modified by the saccharide, the heterologous lactoferrin can exert a better effect, and the overall stability of the heterologous lactoferrin is enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of food ingredients and food processing, and in particular to a heterologous lactoferrin-saccharide compound covalent complex and a preparation method thereof. Background Art

[0002] Lactoferrin (LF), as a key immunoglobulin in milk proteins, constitutes a core component of the mammalian immune system. It plays an important role in promoting iron absorption, immune regulation, antibacterial, antiviral, antioxidant, anti-infection, and promoting the growth of bifidobacteria. Lactoferrin has been widely used in dairy products, especially infant formula. Under current technical conditions, the extraction process of heterologous lactoferrin, such as bovine lactoferrin (bLF), has matured, but its yield is relatively low; at the same time, human lactoferrin (hLF) in human milk, although rich in content, is difficult to popularize due to cost issues. In view of this, the research and development of plant-derived recombinant human lactoferrin (OsrhLF) may provide a new way to break through the existing limitations, and its development has potential scientific value and market prospects. However, the safety and safety of plant-derived recombinant human lactoferrin (OsrhLF) have not been fully studied and verified. In addition, considering the high sensitivity of LF to environmental factors, this has limited its stability and application range in industrial production to a certain extent.

[0003] However, covalent modification with carbohydrate compounds may provide an effective strategy to enhance the overall stability of heterologous lactoferrin.

[0004] Carbohydrates are the most abundant class of organic compounds in organisms and the main source of metabolic energy for plants and animals that feed on plants. Carbohydrates have good thickening and water-holding properties and can be introduced into proteins through the Maillard reaction to improve their functional properties. In food, proteins and peptides are often used as emulsifiers, while carbohydrates are often used as stabilizers or thickeners. Therefore, the effect of combining the two by certain means is greater than that of each alone.

[0005] The Maillard reaction is a non-enzymatic browning reaction that is widely present in food processing and biological systems, involving the reaction between amino compounds (such as proteins) and carbonyl compounds (such as carbohydrates). Through the Maillard reaction, bLF and OsrhLF can be combined with carbohydrate compounds to form innovative new bioactive complexes. At present, there are relatively few studies on the formation of covalent complexes between lactoferrin and carbohydrate compounds through the Maillard reaction. The reaction process is affected by factors such as reaction time and environmental conditions. Problems that may be encountered include the control of reaction conditions, uncertainty in the interaction between sugar molecules and proteins, and possible nonspecific binding. These problems may make it difficult to precisely control the grafting degree, structure and functional properties of the complex. In addition, glycosylation modification may affect the natural structure and function of lactoferrin, such as emulsification, foaming ability and antioxidant properties. Summary of the invention

[0006] The present invention provides a heterologous lactoferrin-sugar compound covalent complex and a preparation method thereof. The heterologous lactoferrin is subjected to glycochemical modification by wet Maillard reaction, and the obtained covalent complex has enhanced antioxidant capacity, and the dispersion stability of aqueous solution, surface hydrophilicity and thermal stability are significantly enhanced. It is also proved that the OsrhLF-sugar compound shows a structural and property change trend similar to that of the bLF-sugar compound. It is shown that after the sugar covalently modifies LF, LF can exert a better effect and enhance its overall stability; and the plant-derived recombinant human lactoferrin can be used for production, and has broad application prospects in the fields of food, health products, medicine, etc.

[0007] The solution of the present invention to solve the above technical problem is as follows: a heterologous lactoferrin-saccharide compound covalent complex, the raw materials of the covalent complex include heterologous lactoferrin and saccharide compound, wherein the mass ratio of the heterologous lactoferrin to the saccharide compound is 3:2-5.

[0008] Preferably, the heterologous lactoferrin is bovine lactoferrin or plant-derived recombinant human lactoferrin; and the carbohydrate compound is one of sodium alginate, maltodextrin, pectin and lactose.

[0009] Preferably, the heterologous lactoferrin is plant-derived recombinant human lactoferrin, and the carbohydrate compound is maltodextrin; the mass ratio of the heterologous lactoferrin to the carbohydrate compound is 3:4.

[0010] The method for preparing the heterologous lactoferrin-saccharide compound covalent complex as described above comprises the following steps:

[0011] 1) dissolving heterologous lactoferrin in distilled water, stirring with magnetic force at room temperature, and hydrating overnight to prepare a heterologous lactoferrin aqueous solution;

[0012] 2) dissolving the carbohydrate compound in the heterologous lactoferrin aqueous solution, stirring magnetically at room temperature, and hydrating overnight to obtain the heterologous lactoferrin-carbohydrate compound aqueous solution;

[0013] 3) adjusting the pH of the heterologous lactoferrin-saccharide compound aqueous solution and incubating;

[0014] 4) The incubated heterologous lactoferrin-saccharide compound aqueous solution is freeze-dried to obtain a heterologous lactoferrin-saccharide compound covalent complex.

[0015] Preferably, in step 1), the stirring time is 2 hours and the hydration temperature is 4°C.

[0016] Preferably, in step 1), the concentration of the aqueous solution of heterologous lactoferrin is 3 g / L. The solution with this concentration is used to prevent flocculation when heated with sugar compounds.

[0017] Preferably, in the step 2), the mass ratio of heterologous lactoferrin to saccharide in the heterologous lactoferrin-saccharide aqueous solution is 3: 4. Exceeding this ratio will result in the heterologous lactoferrin and saccharide failing to react effectively, thereby changing the solubility and thermal stability of the product.

[0018] Preferably, in step 3), the pH of the heterologous lactoferrin-saccharide compound aqueous solution is adjusted to 11.

[0019] Preferably, in step 3), the incubation temperature is 75° C. and the incubation time is 6 h.

[0020] The use of the heterologous lactoferrin-saccharide compound covalent complex as described above in the preparation of food ingredients.

[0021] The beneficial effects of the present invention are as follows: the present invention performs sugar chemical modification on heterologous lactoferrin by wet Maillard reaction. The covalent complex prepared by the method has enhanced antioxidant capacity, and the dispersion stability of aqueous solution, surface hydrophilicity and thermal stability are significantly enhanced. It is also proved that the OsrhLF-sugar covalent complex shows a structural and property change trend similar to that of the bLF-sugar covalent complex. It is shown that after the heterologous lactoferrin is covalently modified with sugars, the heterologous lactoferrin can exert a better effect and enhance its overall stability; the present invention is simple to operate, the conditions are mild and controllable, and it has an objective prospect for large-scale production.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0024] Figure 1 This is a structural diagram of the early stage of Maillard reaction between heterologous lactoferrin and carbohydrate compounds of the present invention;

[0025] Figure 2 A diagram showing the degree of Maillard reaction of heterologous lactoferrin-saccharide covalent complex reaction provided in Examples 1-8; A showing the color change of Examples 1-8 and the corresponding bLF solution and OsrhLF solution before and after heating; B showing the free amino content of Examples 1-8 and H-bLF solution and H-OsrhLF solution; C showing the spectral characteristics of the colorless intermediate product and the brown final product of Examples 1-8, bLF solution and OsrhlF solution before and after heating;

[0026] Figure 3 The SDS-PAGE images of the heterologous lactoferrin-saccharide compounds provided in Examples 1-8; A is the protein bands of bLF, H-bLF, Example 1, Example 2, Example 3, and Example 4 (corresponding to numbers 1-6, respectively); B is the protein bands of OsrhLF, H-OsrhLF, Example 5, Example 6, Example 7, and Example 8 (corresponding to numbers 7-12, respectively).

[0027] Figure 4 Circular dichroism spectra of Examples 1-8 and Control Groups 1-4;

[0028] Figure 5 A is a fluorescence spectrum analysis diagram of Examples 1-8 and Control Groups 1-4; A is a fluorescence spectrum diagram of Examples 1-4 and bLF and H-bLF; B is a fluorescence spectrum diagram of Examples 5-8 and OsrhLF and H-OsrhLF;

[0029] Figure 6 A is a Fourier transform infrared spectrum analysis diagram of Examples 1-8 and Control Groups 1-4; A is a Fourier transform infrared spectrum diagram of Examples 1-4 and bLF and H-bLF; B is a Fourier transform infrared spectrum diagram of Examples 5-8 and OsrhLF and H-OsrhLF;

[0030] Figure 7A is a graph of particle size and potential analysis of Examples 1-8 and Control Groups 1-4; A is a graph of particle size distribution of Examples 1-4 and bLF and H-bLF; B is a graph of particle size distribution of Examples 5-8 and OsrhLF and H-OsrhLF; C is a graph of average particle size of Examples 1-8 and bLF, H-bLF, OsrhLF and H-OsrhLF; D is a graph of Zeta potential of Examples 1-8 and bLF, H-bLF, OsrhLF and H-OsrhLF;

[0031] Figure 8 Surface hydrophobicity index diagrams of Examples 1-8 and Control Groups 1-4;

[0032] Fig. 9 A is a differential scanning calorimetry (DSC) graph of Examples 1-8 and Control Groups 1-4; A is a DSC graph of Examples 1-4 and bLF and H-bLF; B is a DSC graph of Examples 5-8 and OsrhLF and H-OsrhLF;

[0033] Fig.10 A is a graph showing the antioxidant activity of Examples 1-8 and Control Groups 1-4; A is a graph showing the DPPH radical scavenging activity of Examples 1-8 and bLF, H-bLF, OsrhLF, and H-OsrhLF; B is a graph showing the ABTS radical scavenging activity of Examples 1-8 and Control Groups bLF, H-bLF, OsrhLF, and H-OsrhLF; C is a graph showing the FRAP reducing ability of Examples 1-8 and Control Groups bLF, H-bLF, OsrhLF, and H-OsrhLF. DETAILED DESCRIPTION

[0034] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0035] The working principle of the present invention is: Figure 1 As shown in the figure, heterologous lactoferrin undergoes Maillard reaction with carbohydrate compounds to form covalent complexes. During the process, disaccharides (such as lactose Lac) and oligosaccharides (such as maltodextrin Mal and pectin Pec) participate in the reaction to generate high-polymerization products. These high-polymerization products not only enhance the thermal stability of heterologous lactoferrin, but also significantly improve its antioxidant capacity, which is crucial for improving the nutritional value of food and extending the shelf life. At the same time, high-polymerization polysaccharides such as sodium alginate SA participate in the reaction by forming Schiff base intermediates in the middle of the reaction, further enhancing the stability and functionality of LF.

[0036] Under the conditions of wet heat treatment, the amino acid residues of heterologous lactoferrin react with the aldehyde groups in the carbohydrate molecules to form a stable CN covalent bond. The formation of this covalent bond not only changes the secondary and tertiary structures of heterologous lactoferrin, but also effectively prevents thermal aggregation that may occur during high-temperature treatment. The results show that the aqueous dispersion stability of heterologous lactoferrin has been significantly improved, which means that during food processing, heterologous lactoferrin is less likely to precipitate or aggregate, thereby maintaining the uniformity and taste of the food. In addition, the increased surface hydrophilicity of heterologous lactoferrin helps its dissolution and dispersion in an aqueous environment, while the improved thermal stability ensures the activity and functionality of heterologous lactoferrin during food processing and storage.

[0037] The sources of raw materials used in the following examples are as follows:

[0038] 1. Bovine lactoferrin (bLF) with a purity of ≥95% was sourced from Shanghai Yuanye Biotechnology Co., Ltd.

[0039] 2. Recombinant human lactoferrin from Oryza sativa (OsrhLF) with a purity of ≥95% was obtained from Wuhan Kangjian Biotechnology Co., Ltd.

[0040] 3. Sodium alginate (SA) was sourced from Shanghai Aladdin Reagent Co., Ltd.

[0041] 4. Lactose (Lactosum, Lac) was sourced from Shanghai MacLean Biochemical Technology Co., Ltd.

[0042] 5. Maltodextrin (Mal) was sourced from Shanghai Rowan Chemical Technology Co., Ltd.

[0043] 6. Pectin (Pectin, Pec) comes from Meilun Biotechnology Co., Ltd.

[0044] Example 1

[0045] This embodiment provides a bovine lactoferrin-sodium alginate covalent complex (H-bLF-SA), and the preparation steps are as follows:

[0046] 1) Weigh bovine lactoferrin and sodium alginate powder (SA) according to the mass ratio; dissolve bovine lactoferrin in distilled water to obtain a 3 g / L bovine lactoferrin solution, stir magnetically at room temperature for 2 h, and hydrate at 4°C overnight to obtain a bLF solution.

[0047] 2) Sodium alginate powder was added to the bLF solution, and the mixture was magnetically inverted at room temperature for 2 h and then hydrated at 4°C overnight to obtain a bLF-SA solution.

[0048] 3) The pH of the bLF-SA solution obtained in step 2) was adjusted to 11.0, and a photo was taken to record the appearance of the solution at this time.

[0049] 4) Incubate the bLF-SA solution obtained in step 3) at 75° C. for 6 h, and take photos to record the appearance of the solution.

[0050] 5) The sample in 4) is freeze-dried to obtain a bovine lactoferrin-sodium alginate covalent complex (H-bLF-SA).

[0051] Example 2

[0052] This embodiment provides a bovine lactoferrin-maltodextrin covalent complex (H-bLF-Mal), and its preparation steps are basically the same as those in Example 1, except that in step 2), sodium alginate (SA) is replaced by maltodextrin (Mal).

[0053] Example 3

[0054] This embodiment provides a bovine lactoferrin-pectin covalent complex (H-bLF-Pec), and its preparation steps are basically the same as those in Example 1, except that in step 2), sodium alginate (SA) is replaced by pectin (Pec).

[0055] Example 4

[0056] This embodiment provides a bovine lactoferrin-lactose covalent complex (H-bLF-Lac), and its preparation steps are basically the same as those in Example 1, except that in step 2), sodium alginate (SA) is replaced by lactose (Lac).

[0057] Example 5

[0058] This embodiment provides a plant-derived recombinant human lactoferrin-sodium alginate covalent complex (H-OsrhLF-SA), and the preparation steps are as follows:

[0059] 1) Weigh plant-derived recombinant human lactoferrin and sodium alginate powder (SA) in a mass ratio of 3:4; dissolve plant-derived recombinant human lactoferrin in distilled water to prepare a 3 g / L plant-derived recombinant human lactoferrin solution, stir magnetically at room temperature for 2 h, and hydrate at 4°C overnight to obtain an OsrhLF solution.

[0060] 2) Sodium alginate powder was added to the OsrhLF solution, and the mixture was magnetically inverted at room temperature for 2 hours and then hydrated at 4°C overnight to obtain an OsrhLF-SA mixed solution.

[0061] 3) The pH of the OsrhLF-SA mixed solution obtained in step 2) was adjusted to 11.0, and a photo was taken to record the appearance of the solution at this time.

[0062] 4) The OsrhLF-SA mixed solution obtained in step 3) was incubated at 75° C. for 6 h, and a photo was taken to record the appearance of the solution.

[0063] 5) The sample in step 4) is freeze-dried to obtain a plant-derived recombinant human lactoferrin-sodium alginate covalent complex (H-OsrhLF-SA).

[0064] Example 6

[0065] This embodiment provides a plant-derived recombinant human lactoferrin-maltodextrin covalent complex (H-OsrhLF-Mal), and its preparation steps are basically the same as those in Example 5, except that in step 2), sodium alginate (SA) is replaced by maltodextrin (Mal).

[0066] Example 7

[0067] This embodiment provides a plant-derived recombinant human lactoferrin-pectin covalent complex (H-OsrhLF-Pec), and its preparation steps are basically the same as those in Example 5, except that in step 2), sodium alginate (SA) is replaced with pectin (Pec).

[0068] Example 8

[0069] This embodiment provides a plant-derived recombinant human lactoferrin-lactose covalent complex (H-OsrhLF-Lac), and its preparation steps are basically the same as those in Example 5, except that in step 2), sodium alginate (SA) is replaced by lactose (Lac).

[0070] The following control groups were set up:

[0071] Control group 1 (abbreviated as bLF): bovine lactoferrin solution with a concentration of 3 g / L;

[0072] Control group 2 (abbreviated as OsrhLF): recombinant human lactoferrin solution with a concentration of 3 g / L;

[0073] Control group 3 (H-bLF): 3 g / L bovine lactoferrin solution heated at 75°C for 6 h;

[0074] Control group 4 (H-OsrhLF for short): a bovine lactoferrin solution with a concentration of 3 g / L heated at 75°C for 6 hours.

[0075] The above control group and examples are characterized as follows:

[0076] 1. Verification of the binding of heterologous LF-saccharide covalent complexes

[0077] 1. Changes in free amino content and Maillard browning degree

[0078] like Figure 2 As shown in A, the color and transparency of OsrhLF solution, bLF solution, OsrhLF-SA solution, and bLF-SA solution did not change significantly before and after the incubation reaction. However, OsrhLF-Mal solution, bLF-Mal solution, OsrhLF-Lac solution, and bLF-Lac solution changed from the original colorless and transparent solution to a yellow transparent solution after the reaction, while OsrhLF-Pec solution and bLF-Pec solution changed from colorless to yellow with turbidity. Such changes intuitively highlight the dynamic process of the Maillard reaction. Under continuous heating, the Maillard reaction rate of OsrhLF, bLF and Mal, Pec, and Lac was significantly accelerated compared with SA, and the reaction was advanced to the later stage. This phenomenon can be attributed to the difference in the structure of polysaccharides and disaccharides, and the precipitation of OsrhLF-Pec solution and bLF-Pec solution may be due to the intermolecular repulsion caused by the cleavage and demethylation of the glycosidic bond of Pec under pH 11.

[0079] The A 294 and A 420 represent the spectral characteristics of the colorless intermediate and brown final product in the Maillard reaction, respectively, while A 294 / A 420 The ratio reflects the degree of polymerization. Figure 2 As shown in C, OsrhLF solution, bLF solution, H-OsrhLF, H-bLF group did not show significant changes. In contrast, the covalent complexes formed in Examples 1-8 294 With A 420 Both aspects show an upward trend, indicating that the intermediate and late products of the Maillard reaction have been formed, but the final stage of the reaction has not yet been reached. 294 / A 420 Ratio analysis further showed that the H-OsrhLF-Pec, H-bLF-Pec, H-OsrhLF-Mal, H-bLF-Mal and H-OsrhLF-Lac, H-bLF-Lac groups showed higher polymerization degrees than those of the H-OsrhLF-SA, H-bLF-SA groups, while the high polymerization degree of the H-OsrhLF-Pec, H-bLF-Pec groups might have been misjudged due to the increased turbidity.

[0080] The free amino group content was further determined by the OPA method using a microplate reader to verify the extent of the condensation reaction between heterologous lactoferrin and sugar compounds in the Maillard reaction, such as Figure 2As shown in B, compared with OsrhLF solution and bLF solution (LF group), the free amino content of H-OsrhLF solution and H-bLF solution (H-LF group) after incubation reaction was significantly decreased; compared with H-OsrhLF solution and H-bLF solution (H-LF group), the free amino content of H-OsrhLF-Mal, H-bLF-Mal (H-LF-Mal group) and H-OsrhLF-Lac, H-bLF-Lac (H-LF-Lac group) was significantly reduced, while there was no significant change in H-OsrhLF-SA, H-bLF-SA (H-LF-SA group) and H-OsrhLF-Pec, H-bLF-Pec (H-LF-Pec group). The results revealed that bLF and OsrhLF underwent protein aggregation after heat treatment, and the addition of the four sugars prompted the aldehyde groups of these sugars to react with the ε-amino group of the lysine residues in the side chain of LF or the α-amino group of the terminal amino acid to form a CN covalent bond, which led to a decrease in the content of free amino groups. It is worth noting that Mal and Lac have simpler structures than SA and Pec and have a faster reaction rate, resulting in a more significant amount of binding to the amino groups of bLF and OsrhLF.

[0081] 2. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

[0082] like Figure 3 As shown in A, compared with bLF, the main band of H-bLF (80 kDa) showed a tailing phenomenon, accompanied by the appearance of a degradation band, and most of the proteins gathered at the top of the separation gel; compared with H-bLF, the band distribution of H-bLF-SA remained consistent, while the main bands of H-bLF-Mal and H-bLF-Lac were lighter in color, and H-bLF-Pec showed a similar pattern to bLF, but there were significant differences. In addition, as Figure 3 As shown in B, compared with OsrhLF, the main band (80 kDa) of H-OsrhLF was lighter in color, and the protein was mainly concentrated near the loading well; compared with H-OsrhLF, H-OsrhLF-SA, H-OsrhLF-Mal and H-OsrhLF-Lac all showed high molecular weight bands (over 180 kDa), among which H-OsrhLF-Mal showed enrichment near the main band, while H-OsrhLF-Pec maintained a high similarity with OsrhLF.

[0083] These observations reveal the partial degradation of LF after heat treatment and the structural changes caused by it, which lead to reduced stability of the solution system and more prone to protein coagulation under the action of reducing agents, manifested as tailing and accumulation of electrophoretic bands. Compared with bLF, OsrhLF showed more significant protein degradation after long-term heat treatment. In addition, the appearance of high molecular weight bands indicates that bLF and OsrhLF formed polymers through Maillard reaction after the addition of sugars, among which Pec had a significantly lower polymerization effect on LF than SA, Mal and Lac.

[0084] 2. Effects of carbohydrate compounds on the structure and micromorphology of heterologous lactoferrin

[0085] 1. Circular dichroism analysis

[0086] like Figure 4 As shown, compared with OsrhLF solution and bLF solution, the ellipticity of the CD spectrum peak of the H-OsrhLF solution, H-bLF solution and the covalent complex provided in Examples 1-8 after wet heat treatment in the wavelength range of 208nm to 220nm decreased. This phenomenon indicates that the secondary structure of OsrhLF and bLF has changed under wet heat treatment and the covalent action of sugars. Further, according to the secondary structure content data listed in Table 1, the proportion of irregular coil structures of H-bLF and H-OsrhLF has increased compared with the original OsrhLF and bLF. On the basis of H-bLF, the α-helix content of H-bLF-Mal has increased, while the β-folding content has decreased; on the contrary, the α-helix content of H-bLF-SA, H-bLF-Pec and H-bLF-Lac has decreased, and the β-folding content has increased. Compared with H-OsrhLF, H-OsrhLF-SA showed a decrease in α-helix content and an increase in β-sheet content, while H-OsrhLF-Mal, H-OsrhLF-Pec, and H-OsrhLF-Lac showed the opposite trend. These findings suggest that the molecular order of OsrhLF and bLF was destroyed during the Maillard reaction, and the covalent binding with sugars led to the reorganization of the secondary structure of OsrhLF and bLF. In general, SA, Mal, and Lac were more effective in protecting the stability of the LF molecular structure than Pec.

[0087] Table 1 Secondary structure content of Examples 1-8 and corresponding control groups

[0088]

[0089] 2. Fluorescence spectroscopy analysis

[0090] like Figure 5As shown in A, bLF exhibits a significant fluorescence emission peak at 334nm. In contrast, the fluorescence intensity of H-bLF after heat treatment is increased, while the fluorescence intensity of the covalent complex formed by bLF and sugars shows a downward trend, especially for bLF modified with Mal and Lac, whose fluorescence intensity decreases significantly. Compared with Examples 1-4 (bLF group), Examples 5-8 (OsrhLF group) show similar fluorescence intensity change patterns, but overall the OsrhLF group (such as Figure 5 The fluorescence intensity of OsrhLF group (shown in B) is slightly lower than that of bLF group. This difference may be due to the difference in tryptophan content in the OsrhLF structure, resulting in a lower fluorescence quantum yield.

[0091] The above characterization results show that the tryptophan amino groups on the side chains of bLF and OsrhLF participate in the Maillard reaction, and the interaction between Mal and Lac and tryptophan is stronger. In addition, after heat treatment, the maximum emission wavelength of LF shifts from 334nm to 339nm, showing a red shift phenomenon. This indicates that the structure of LF has changed, the exposure of tryptophan residues has reduced the hydrophobicity of its microenvironment, and the influence of Mal and Lac on the structure of LF is particularly prominent.

[0092] 3. Fourier infrared spectroscopy analysis

[0093] like Figure 6 As shown, the infrared absorption of proteins mainly occurs in the amide A band (3250-3300 cm -1 , NH stretching vibration), amide I band (1600-1700cm -1 , C=O stretching vibration) and amide II band (1450-1550 cm -1 , NH bending and CN stretching), while the amide I band and amide II band are closely related to the secondary structure of the protein. When functional group modification occurs at the molecular level through the Maillard reaction, FTIR shows the appearance and disappearance of the bands, as well as the changes in the intensity and position of the bands.

[0094] The main absorption peak of bLF is at 3282.79 cm -1 , 1659.93cm -1 and 1541.33cm -1 The main absorption peak of OsrhLF is at 3285.86 cm -1 , 1659.93cm -1 and 1541.33cm -1 Compared with bLF and OsrhLF, the band intensities of H-bLF and H-OsrhLF changed; compared with H-bLF and H-OsrhLF, the covalent compounds provided in Examples 1-8 produced different degrees of shifts in the positions of amide A, amide I and amide II bands, and at 1000 cm-1 New IR bands appeared nearby. The changes in the characteristic peaks of each group of heterologous lactoferrin indicate that the amino and carboxyl groups of heterologous lactoferrin participated in the covalent reaction and changed its secondary structure, which is consistent with the results of CD; the appearance of new spectral bands may be caused by CN stretching vibration, indicating the formation of Schiff base. Overall, during the wet heat treatment, bLF, OsrhLF and the four sugar compounds all underwent Maillard reaction. In addition, compared with bLF, the sugar-covalently modified OsrhLF showed similar structural vibrations to bLF, which indirectly illustrates the future application potential of OsrhLF.

[0095] 4. Particle size and potential

[0096] The particle size of each embodiment and the control group was tested by a nanoparticle size potential analyzer. Figure 7 As shown in A and 7B, the particle size distribution of OsrhLF, bLF, H-OsrhLF, H-bLF, H-OsrhLF-SA, and H-bLF-SA showed bimodal or multimodal characteristics, and particles larger than 1000 nm were detected in H-OsrhLF-SA and H-bLF-SA. In contrast, the particle size distribution of H-OsrhLF-Mal, H-bLF-Mal, H-OsrhLF-Pec, H-bLF-Pec and H-OsrhLF-Lac and H-bLF-Lac showed a single peak, among which the distribution width of H-OsrhLF-Pec and H-bLF-Pec was wider.

[0097] Further analysis of the average particle size, such as Figure 7 As shown in Figure C, the average particle sizes of bLF and OsrhLF are 288.9 nm and 151.7 nm, respectively, and the average particle sizes of H-OsrhLF and H-bLF are significantly reduced after heat treatment. Compared with H-OsrhLF and H-bLF, the average particle sizes of H-OsrhLF-SA, H-bLF-SA, H-OsrhLF-Pec, and H-bLF-Pec increased, while those of H-OsrhLF-Mal, H-bLF-Mal, H-OsrhLF-Lac, and H-bLF-Lac decreased significantly. These data indicate that the dispersibility of bLF and OsrhLF in aqueous solution is not uniform, and the covalent modification of Mal, Lac, and Pec helps to enhance the stability of OsrhLF and bLF solutions. On the contrary, SA, due to its inherent viscosity, is easy to form larger particles with OsrhLF and bLF, thereby reducing the uniformity of the solution.

[0098] The Zeta potential values ​​of each embodiment and the control group were tested by a nanoparticle size potential analyzer. Figure 7As shown in Figure D, the Zeta potential values ​​of bLF and OsrhLF are 19.6 mV and 5.2 mV, respectively. After heat treatment, the charge properties of H-OsrhLF and H-bLF changed from positive to negative, and this change may be related to the high pH treatment conditions. The surface charge of OsrhLF is lower than that of bLF, which may be due to the lower isoelectric point of OsrhLF than that of bLF. Compared with H-OsrhLF and H-bLF, the absolute values ​​of the Zeta potential of H-OsrhLF-SA, H-bLF-SA and H-OsrhLF-Pec and H-bLF-Pec increased significantly, indicating that after the negatively charged SA and Pec covalently bind to LF, the potential value of their solutions is higher than -30 mV, thereby significantly improving the stability of the bLF and OsrhLF solution systems. The increase in net negative charge of H-OsrhLF-Mal, H-bLF-Mal, H-OsrhLF-Lac, and H-bLF-Lac suggests that the covalent binding of Mal and Lac to heterologous lactoferrin reduces the positively charged amino acid groups in heterologous lactoferrin. It is worth noting that although the particle size and potential results of the H-LF-Pec solution showed good stability, it is not suitable as a long-term stable system from the perspective of overall stability due to its high turbidity.

[0099] 3. Analysis of changes in physicochemical properties and antioxidant activity

[0100] 1. Surface hydrophobicity

[0101] The hydrophobicity of each embodiment and the control group was tested by ANS method using a fluorescence spectrometer. Figure 8 As shown, compared with the LF group (OsrhLF, bLF), the surface hydrophobic index of the H-LF group (H-OsrhLF, H-bLF) was increased; compared with the H-LF group (H-OsrhLF, H-bLF), the surface hydrophobic index of the covalent complex provided in Examples 1-8 was reduced, and the H-LF-Mal group (H-OsrhLF-Mal, H-bLF-Mal) and the H-LF-Lac group (H-OsrhLF-Lac, H-bLF-Lac) were the most significant. This result shows that in the Maillard reaction, the hydrophobic groups of OsrhLF and bLF are exposed and covalently bonded with hydrophilic sugars, so that the hydrogen bonding ability of the sugar hydroxyl group changes the surface hydrophobicity and leads to an increase in surface hydrophilicity. Among them, the H-LF-Mal group and the H-LF-Lac group show better improvement effects due to their excellent solubility, and this phenomenon has also been reported in other literature. In addition, compared with the bLF group (Examples 1-4), the OsrhLF group (Examples 5-8) showed better surface hydrophilicity.

[0102] 2. Thermal stability

[0103] like Fig. 9 As shown in Figures A and 9B, the thermal denaturation temperatures of bLF and OsrhLF are 80.0°C and 74.4°C, respectively. Compared with bLF and OsrhLF, the DSC curves of the covalent complexes provided in Examples 1-8 show similar endothermic peaks, but their thermal denaturation temperatures are increased. The thermal denaturation temperatures of the covalent complexes provided in Examples 1-4 are increased by 17.5 to 48.9°C, while the thermal denaturation temperatures of the covalent complexes provided in Examples 1-4 are increased by 18.1 to 58.4°C. In particular, the thermal denaturation temperatures of H-OsrhLF-Mal, H-bLF-Mal, H-OsrhLF-Lac, and H-bLF-Lac are the highest among all carbohydrate covalent complexes, indicating that the covalent modification of carbohydrates significantly enhances the thermal stability of bLF and OsrhLF, among which the effects of SA, Mal, and Lac are particularly significant.

[0104] 3. Antioxidant Activity

[0105] Through a variety of antioxidant methods (DPPH free radical scavenging rate, ABTS + The antioxidant capacity of heterologous lactoferrin and its covalent complexes with carbohydrates was evaluated by free radical scavenging rate and FRAP method. Fig.10 As shown in A, the DPPH radical scavenging activity of the H-LF group (H-OsrhLF, H-bLF) decreased compared with the LF group (OsrhLF, bLF). However, compared with the H-LF group, the DPPH scavenging activity of the H-LF-SA group (H-OsrhLF-SA, H-bLF-SA), H-LF-Mal (H-OsrhLF-Mal, H-bLF-Mal) and H-LF-Lac (H-OsrhLF-Lac, H-bLF-Lac) was significantly improved, especially the activity of H-LF-Pec (H-OsrhLF-Pec, H-bLF-Pec) was the most significantly improved.

[0106] In addition, if Fig.10As shown in Figures B and 10C, the ABTS radical scavenging activity and FRAP reducing ability of the covalent complex also showed a similar trend to the DPPH scavenging activity. The difference in antioxidant activity may be related to the number of browning products or intermediates produced during the glycosylation process, and the melanoidins generated by the Maillard reaction have a strong correlation with the ABTS radical scavenging activity and advanced glycation end products due to their rich nonspecific hydroxyl scavenging activity. Therefore, the antioxidant capacity of bLF and OsrhLF after heat treatment may be reduced due to the damage of active groups, while the browning products produced by Mal and Lac in the Maillard reaction give them stronger antioxidant properties. The enhanced antioxidant activity of the H-LF-SA group may be related to the large number of intermediates in its reaction system, while the H-LF-Pec group showed weak antioxidant activity. In addition, the Maillard reaction products, as metal chelators, can bind to metal ions and prevent lipid peroxidation by reducing the concentration of transition metal ions. The FRAP test results further confirmed the potential role of LF-sugar covalent complexes in metal ion scavenging.

[0107] In summary, although the solution stability of plant-derived recombinant human lactoferrin (OsrhLF) after heat treatment alone is not as good as that of bovine lactoferrin (bLF), when it forms a covalent complex with carbohydrate compounds, it shows similar structure and properties to bLF-carbohydrate complex. This suggests that OsrhLF-carbohydrate complex can be used as an effective substitute for bLF in the food industry, especially in application scenarios that require improved thermal stability and enhanced antioxidant capacity.

[0108] It is particularly noteworthy that maltodextrin (Mal) and lactose (Lac) performed well in improving the physicochemical properties of LF, which may be related to their unique molecular structures and reaction characteristics.

[0109] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any ordinary technician in the industry can smoothly implement the present invention as shown in the drawings and described above. However, any equivalent changes, modifications and evolutions made by technicians familiar with the profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A heterologous lactoferrin-saccharide covalent complex, characterized in that: The raw materials of the covalent complex include heterologous lactoferrin and carbohydrate compounds; wherein the mass ratio of the heterologous lactoferrin to the carbohydrate compound is 3:2-5.

2. The heterologous lactoferrin-saccharide covalent complex according to claim 1, characterized in that: The heterologous lactoferrin is bovine lactoferrin or plant-derived recombinant human lactoferrin; the carbohydrate compound is one of sodium alginate, maltodextrin, pectin and lactose.

3. A heterologous lactoferrin-saccharide covalent complex according to claim 2, characterized in that: The mass ratio of the plant-derived recombinant human lactoferrin to maltodextrin is 3:

4.

4. A method for preparing the heterologous lactoferrin-saccharide covalent complex according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) dissolving heterologous lactoferrin in distilled water, stirring with magnetic force at room temperature, and hydrating overnight to prepare a heterologous lactoferrin aqueous solution; 2) dissolving the carbohydrate compound in the heterologous lactoferrin aqueous solution, stirring magnetically at room temperature, and hydrating overnight to obtain the heterologous lactoferrin-carbohydrate compound aqueous solution; 3) adjusting the pH of the heterologous lactoferrin-saccharide compound aqueous solution and incubating; 4) The incubated heterologous lactoferrin-saccharide compound aqueous solution is freeze-dried to obtain a heterologous lactoferrin-saccharide compound covalent complex.

5. The method for preparing a heterologous lactoferrin-saccharide covalent complex according to claim 4, characterized in that: In the step 1), the stirring time is 2 hours and the hydration temperature is 4°C.

6. A method for preparing a heterologous lactoferrin-saccharide covalent complex according to claim 4 or 5, characterized in that: In the step 1), the concentration of the heterologous lactoferrin aqueous solution is 3 g / L.

7. The method for preparing a heterologous lactoferrin-saccharide covalent complex according to claim 4, characterized in that: In the step 2), in the heterologous lactoferrin-saccharide compound aqueous solution, the mass ratio of the heterologous lactoferrin to the saccharide compound is 3:

4.

8. The method for preparing a heterologous lactoferrin-saccharide covalent complex according to claim 4, characterized in that: In the step 3), the pH of the heterologous lactoferrin-saccharide compound aqueous solution is adjusted to 11.

9. The method for preparing a heterologous lactoferrin-saccharide covalent complex according to claim 4, characterized in that: In step 3), the incubation temperature is 75° C. and the incubation time is 6 h.

10. Use of a heterologous lactoferrin-saccharide covalent complex according to claims 1-3 in preparing food ingredients.

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