阅读说明：本技术 一种亚麻籽蛋白/亚麻籽胶/多酚复合凝聚包埋体系的制备方法 (Preparation method of linseed protein/linseed gum/polyphenol complex coacervation embedding system ) 是由 禹晓 邓乾春 相启森 黄沙沙 聂成镇 朱莹莹 翟娅菲 申瑞玲 于 2019-09-19 设计创作，主要内容包括：本发明公开了一种亚麻籽蛋白/亚麻籽胶/多酚复合凝聚包埋体系的制备方法。本发明将亚麻籽萌动耦合微波预处理,强化亚麻籽多酚的内源性合成和溶出效率,再分别提取富含酚类化合物的亚麻籽蛋白和亚麻籽胶,并对亚麻籽蛋白进行空气压等离子体射流处理,最后基于静电自组装构建能够运载亚麻籽油的亚麻籽蛋白/亚麻籽胶/多酚复合凝聚体系。利用本发明获得的亚麻籽蛋白/亚麻籽胶/多酚复合凝聚体系能够显著提高亚麻籽油、紫苏籽油、鱼油和藻油的物理稳定性和氧化稳定性。本发明作为一种递送体系包埋材料和健康食品原料具有极大的应用前景。(The invention discloses a preparation method of a linseed protein/linseed glue/polyphenol complex coacervation embedding system. According to the method, flax seed germination is coupled with microwave pretreatment, endogenous synthesis and dissolution efficiency of flax seed polyphenol are enhanced, flax seed protein and flax seed gum rich in phenolic compounds are respectively extracted, the flax seed protein is subjected to air pressure plasma jet treatment, and finally a flax seed protein/flax seed gum/polyphenol composite coagulation system capable of carrying flax seed oil is constructed based on electrostatic self-assembly. The linseed protein/linseed gum/polyphenol complex coacervation system obtained by the method can obviously improve the physical stability and oxidation stability of linseed oil, perilla seed oil, fish oil and algae oil. The invention has great application prospect as a delivery system embedding material and a health food raw material.)

1. A preparation method of a linseed protein/linseed gum/polyphenol complex coacervation embedding system is characterized by comprising the following specific steps:

(1) pretreatment of linseed raw material: cleaning and removing impurities from the flaxseeds harvested in the same year to obtain flaxseeds with complete, full and uniform seeds;

(2) germinating flaxseeds: soaking flaxseeds in weak alkaline electrolyzed functional water for 0.5-1 h, draining the soaked flaxseeds, uniformly spreading the flaxseeds in a seedling tray, and then placing the seedling tray in a constant-temperature constant-humidity incubator to germinate for 3-4 days; after germination is finished, eluting the germinated flaxseeds with deionized water to remove alkaline electrolyzed functional water attached to the surfaces of the germinated flaxseeds;

(3) and (3) low-temperature freeze drying: pre-freezing germinated flaxseeds for 1.5-2 h at-20 ℃, and then carrying out vacuum freeze-drying treatment for 18-20 h at the freezing temperature of-20 to-18 ℃ and under the vacuum degree of 0.12-0.14 mbar to ensure that the final moisture content of the germinated flaxseeds is 16-20%;

(4) microwave pretreatment: placing the freeze-dried germinated flaxseeds in a culture dish with the diameter of 10cm, sealing the mouth with a preservative film, and performing microwave pretreatment for 3-5 min under the condition of 650-700 w;

(5) extracting flaxseed gum: adding germinated flaxseeds subjected to microwave treatment into deionized water (m/v:1: 8-1: 10), mechanically stirring and extracting for 3-5 h at the conditions of 50-60 ℃ and 800-1000 rpm/min, filtering, continuously extracting for 2-3 times, combining extracting solutions, adding equal volume of absolute ethyl alcohol to precipitate flaxseed gum, centrifuging 10-15 min supernatant at 5000g of room temperature, and performing vacuum freeze drying treatment on the obtained precipitate for 10-12h at the conditions of-20-18 ℃ and 0.12-0.14 mbar vacuum degree to obtain flaxseed gum rich in flaxseed polyphenol;

(6) extracting linseed protein: carrying out vacuum freeze drying treatment on the degummed germinated flaxseeds for 10-12h at the freezing temperature of-20 to-18 ℃ and the vacuum degree of 0.12 to 0.14mbar, crushing, adding the crushed germinated flaxseeds into a NaOH solution with the pH value of 9 to 10, magnetically stirring and extracting for 3-5 h at the speed of 400 to 600rpm/min, filtering, continuously extracting for 2-3 times, and combining the extracting solutions; adjusting the pH value of the extracting solution to 4.0-5.0 by using a 1MHCl solution to precipitate the linseed protein, centrifuging for 10-15 min at room temperature of 4000 plus 5000g to obtain the linseed protein rich in the linseed polyphenol, redissolving by using deionized water, adjusting the pH value of the solution to 6.8-7.0 by using a 0.5M diluted HCl solution, and carrying out vacuum freeze drying treatment for 18-20 h under the conditions of the freezing temperature of-20 to-18 ℃ and the vacuum degree of 0.12-0.14 mbar to obtain the linseed protein;

(7) respectively preparing the obtained flaxseed gum and flaxseed protein into dispersions with mass concentrations of 0.25% and 1% by taking deionized water as a dispersing agent, magnetically stirring and dissolving for 4-5 h at the rotating speed of 400-600 rpm/min, and then standing for 10-12h in a refrigerator at 4 ℃ to fully dissolve the flaxseed gum and the flaxseed protein;

(8) flax seed protein plasma jet treatment: carrying out air pressure plasma jet treatment on the dissolved linseed protein solution in the step (7) for 5-10 s;

(9) complex coacervation embedding system: and (3) adjusting the pH value of the linseed protein solution treated by the plasma to 3.0-3.5, dropwise adding the linseed gum solution fully dissolved in the step (7) in the magnetic stirring process, and continuously magnetically stirring for 2-3 hours to obtain the linseed protein/linseed gum/polyphenol complex coacervation embedding system.

2. The method for preparing the linseed protein/linseed gum/polyphenol complex coacervation embedding system according to claim 1, wherein the preparation parameters of the weakly alkaline electrolyzed functional water in the step (2) are as follows: the electrolyte is a calcium chloride solution with the mass fraction of 0.8-1.0%, and the electrolytic voltage is 8-10V; the pH value of the weakly alkaline electrolyzed functional water is 9.45-9.55, and the ORP value is 145-165 mV.

3. The method for preparing a linseed protein/linseed gum/polyphenol complex coacervation embedding system according to claim 1, wherein the specification of the seedling raising tray in the step (2) is 34cm long by 25cm wide; in order to ensure the germination rate of flaxseeds, the spreading amount of the flaxseeds on each seedling raising tray is 30-35 g, and 100-150 mL of alkaline electrolyzed functional water is sprayed every 8-10 hours during germination so as to promote the synthesis of endogenous phenolic compounds in the germination process of the flaxseeds.

4. The method for preparing a linseed protein/flaxseed gum/polyphenol complex coacervation encapsulation system according to claim 1, wherein the moisture content of the germinated flaxseeds in step (3) is maintained at 18-20% after low-temperature freeze drying, and residual moisture in the germinated flaxseeds during subsequent microwave pretreatment can be used as a medium for absorbing microwave energy.

5. The preparation method of the flax seed protein/flax seed gum/polyphenol complex coacervation embedding system according to claim 1, wherein the microwave pretreatment power in step (4) is 650-700 w, and the microwave time is 3-5 min.

6. The preparation method of the flax seed protein/flax seed gum/polyphenol complex coacervation embedding system according to claim 1, wherein in step (8), the discharge power of the air pressure plasma jet is 700-750 w, the distance between the air pressure plasma jet and the liquid surface is 30-35 mm, and the air pressure plasma treatment time is 5-10 s.

7. The preparation method of the linseed protein/linseed gum/polyphenol complex coacervation embedding system according to claim 1, wherein in the step (9), the volume ratio of the linseed protein to the linseed gum is 3: 1-5: 1, and the magnetic stirring speed is 120-150 rmp/min, so that the linseed protein and the linseed gum can form the linseed protein/linseed gum/polyphenol complex coacervation embedding system based on electrostatic complexation.

Technical Field

The invention relates to the technical field of food processing, in particular to a preparation method of a linseed protein/linseed gum/polyphenol complex coacervation embedding system.

Background

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs), including alpha-linolenic acid (ALA) of plant origin and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of marine origin, have been reported to reduce risk factors for metabolic diseases, such as obesity, type 2 diabetes, cardiovascular and cerebrovascular diseases, and the like. Based on the results of dietary nutrition survey of residents in China, the difference between the actual intake of ALA, EPA and DHA in the diet and the recommended intake still exists. The low water solubility of n-3 PUFA-rich functional lipids, typically linseed oil, perilla seed oil, fish oil, algal oil, and the like, limits their use as functional food materials in liquid foods such as dairy products, beverages, and the like to some extent. Therefore, improving the bioavailability of ALA, EPA and DHA by changing the intake mode of linseed oil and the like so as to reduce the effective intake amount of ALA, EPA and DHA to the maximum extent and further expand the application range of the ALA, EPA and DHA in the field of food is an important way for effectively solving the serious deficiency of n-3PUFA intake.

The nanoemulsion delivery system provides the possibility of improving the bioavailability of n-3PUFA due to the ability to alter the digestion, absorption and metabolism of the lipid active components carried thereby. Among other things, multilayer oil-in-water emulsions based on electrostatic self-assembly can improve the interfacial limitations of conventional emulsion systems. The emulsion prepared by the method has better physical stability in the aspects of external stress factors such as ionic strength, pH value and temperature. Complex coacervation using electrostatic self-assembly has been investigated to prepare protein-polysaccharide stabilized linseed oil emulsion systems. Flaxseed is rich in functional proteins and in flaxseed gum components in addition to being the main source of the plant-derived n-3 fatty acid ALA. Studies have shown that flaxseed protein has a higher surface charge and smaller emulsion droplet size than soy protein, which to some extent depends on the presence of flaxseed gum with flaxseed protein. Furthermore, the amino acid profile of linseed protein is nutritionally desirable and is considered nutritionally equivalent to other oilseed proteins such as soy. Flaxseed gum is also a vegetable polymer that can be used as an emulsifier for the construction of emulsion gel systems. Meanwhile, the flaxseed gum is also an anionic heteropolysaccharide and water-soluble dietary fiber, has a certain improvement effect on the health of organisms, particularly intestinal tracts, but is only applied to a food system as a food ingredient at present. The electrostatic complexation based on the linseed protein and the linseed gum can be used for constructing an n-3 PUFA-rich emulsion system for carrying linseed oil and the like from the technical aspect or the nutritional aspect.

However, the oxidation stability of the n-3 PUFA-rich emulsion such as linseed oil and the like applied to food systems such as milk beverages and the like and the lipid oxidation in the gastrointestinal tract digestive absorption process after oral administration are also key factors influencing the bioavailability of ALA, EPA and DHA in the emulsion system. Therefore, how to construct a high-physical and oxidation-stability linseed protein/linseed gum/polyphenol complex coacervation embedding system based on targeted enrichment of endogenous antioxidant components in linseed, which is used for carrying n-3 PUFA-rich functional lipids such as linseed oil, is a technical problem that needs to be solved urgently by the technical staff in the field.

Disclosure of Invention

In view of the above, the invention provides a preparation method of a linseed protein/linseed gum/polyphenol complex coacervation embedding system, which can significantly improve the physical and oxidation stability of the carried linseed oil and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

the preparation method of the linseed protein/linseed gum/polyphenol complex coacervation embedding system comprises the following specific steps:

(1) pretreatment of linseed raw material: cleaning and removing impurities from the flaxseeds harvested in the same year to obtain flaxseeds with complete, full and uniform seeds;

(2) germinating flaxseeds: soaking flaxseeds in weak alkaline electrolyzed functional water for 0.5-1 h, draining the soaked flaxseeds, uniformly spreading the flaxseeds in a seedling tray, and then placing the seedling tray in a constant-temperature constant-humidity incubator to germinate for 3-4 days; after germination is finished, eluting the germinated flaxseeds with deionized water to remove alkaline electrolyzed functional water attached to the surfaces of the germinated flaxseeds;

(3) and (3) low-temperature freeze drying: pre-freezing germinated flaxseeds for 1.5-2 h at-20 ℃, and then carrying out vacuum freeze-drying treatment for 18-20 h at the freezing temperature of-20 to-18 ℃ and under the vacuum degree of 0.12-0.14 mbar to ensure that the final moisture content of the germinated flaxseeds is 16-20%;

(4) microwave pretreatment: placing the freeze-dried germinated flaxseeds in a culture dish with the diameter of 10cm, sealing the mouth with a preservative film, and performing microwave pretreatment for 3-5 min under the condition of 650-700 w;

(5) extracting flaxseed gum: adding germinated flaxseeds subjected to microwave treatment into deionized water (m/v:1: 8-1: 10), mechanically stirring and extracting for 3-5 h at the conditions of 50-60 ℃ and 800-1000 rpm/min, filtering, continuously extracting for 2-3 times, combining extracting solutions, adding equal volume of absolute ethyl alcohol to precipitate flaxseed gum, centrifuging 10-15 min supernatant at 5000g of room temperature, and performing vacuum freeze drying treatment on the obtained precipitate for 10-12h at the conditions of-20-18 ℃ and 0.12-0.14 mbar vacuum degree to obtain flaxseed gum rich in flaxseed polyphenol;

(6) extracting linseed protein: carrying out vacuum freeze drying treatment on the degummed germinated flaxseeds for 10-12h at the freezing temperature of-20 to-18 ℃ and the vacuum degree of 0.12 to 0.14mbar, crushing, adding the crushed germinated flaxseeds into a NaOH solution with the pH value of 9 to 10, magnetically stirring and extracting for 3-5 h at the speed of 400 to 600rpm/min, filtering, continuously extracting for 2-3 times, and combining the extracting solutions; adjusting the pH value of the extracting solution to 4.0-5.0 by using a 1MHCl solution to precipitate the linseed protein, centrifuging for 10-15 min at room temperature of 4000 plus 5000g to obtain the linseed protein rich in the linseed polyphenol, redissolving by using deionized water, adjusting the pH value of the solution to 6.8-7.0 by using a 0.5M diluted HCl solution, and carrying out vacuum freeze drying treatment for 18-20 h under the conditions of the freezing temperature of-20 to-18 ℃ and the vacuum degree of 0.12-0.14 mbar to obtain the linseed protein;

(7) respectively preparing the obtained flaxseed gum and flaxseed protein into dispersions with mass concentrations of 0.25% and 1% by taking deionized water as a dispersing agent, magnetically stirring and dissolving for 4-5 h at the rotating speed of 400-600 rpm/min, and then standing for 10-12h in a refrigerator at 4 ℃ to fully dissolve the flaxseed gum and the flaxseed protein;

(8) flax seed protein plasma jet treatment: carrying out air pressure plasma jet treatment on the dissolved linseed protein solution in the step (7) for 5-10 s;

(9) complex coacervation embedding system: and (3) adjusting the pH value of the linseed protein solution treated by the plasma to 3.0-3.5, dropwise adding the linseed gum solution fully dissolved in the step (7) in the magnetic stirring process, and continuously magnetically stirring for 2-3 hours to obtain the linseed protein/linseed gum/polyphenol complex coacervation embedding system.

The inventor finds in earlier studies that moderate germination pretreatment can increase the content of polyphenols in flaxseed without significantly affecting the content, structure and function of flaxseed protein and flaxseed gum, accompanied by a significant increase in the in vitro antioxidant activity of flaxseed. This is mainly due to endogenous synthesis of SDG, SECO, free and bound phenolic acids in flax seeds induced by germination pre-treatment. Air pressure plasma jet treatment is being developed for the modification of proteins of plant or animal origin. Studies have shown that a moderate period of plasma treatment can improve the functional properties of plant or animal proteins such as foaming, emulsifying, etc. Our research also found that the air pressure plasma jet treatment for a short time significantly improves the emulsifying activity and stability of linseed protein, which would be expected to further improve the potential of linseed protein as an emulsifier for the construction of nano-emulsions. The secoisolariciresinol diglucoside mainly exists in the secondary cell wall of the linseed ectodermal bone cell and is a key component of the linseed endogenous antioxidant system. In mature flax seeds, secoisolariciresinol diglucoside is hardly present in free form, but covalently bound to 3-hydroxy-3-methyl-glutaric acid (HMGA) via an ester bond, and on average 4 HMGA crosslinks per 5 SDGs to form lignan oligomers. Other phenolic compounds, including Caffeic Acid Glycoside (CAG), p-coumaric acid glycoside (CouAG), Ferulic Acid Glycoside (FAG) and gossypol glycoside (HDG), participate in the formation of lignan macromolecules. Among these, HDG is directly linked to HMGA, whereas CAG, CouAG and FAG are cross-linked to SDG glycosides and determine the chain length and molecular weight of the lignan multimer. Due to the specific spatial distribution, existing form and molecular polarity characteristics of the secoisolariciresinol diglucoside macromolecules, the secoisolariciresinol diglucoside macromolecules with higher content coexist in the extraction process of the flaxseed gum and the flaxseed protein. Notably, we found that a short time air pressure plasma jet treatment, while improving the functional properties of flaxseed proteins, particularly the emulsifying activity and stability, induces a secoisolariciresinol diglucoside macromolecular "depolymerization" effect, thereby increasing the antioxidant activity of flaxseed proteins in vitro. On the basis, the inventor further researches to obtain a linseed protein/linseed gum/polyphenol complex coacervation embedding system;

compared with the prior art, the invention has the beneficial effects that:

(1) the germination pretreatment improves the total phenol and flavone contents of the flaxseed raw material: the flax seeds are soaked in alkaline electrolyzed functional water and then are germinated. The average content of total phenols of germinated flaxseed determined by adopting a forlin phenol colorimetric method reaches 1100mg/100g, is improved by 60% compared with that of ungerminated flaxseed (686mg/100g), and is improved by 26% compared with that of traditional deionized water germination (873mg/100 g).

(2) According to the invention, the microwave treatment of the germinated flaxseed improves the dissolution and release of the flaxseed phenolic compounds in the extraction process of flaxseed gum and flaxseed protein. The average content of total phenols in the linseed protein reaches 959mg/100g, which is increased by 25% compared with the linseed protein without microwave; the average content of total phenols in the flaxseed gum reaches 780mg/100g, which is improved by 19 percent compared with the flaxseed gum without microwave.

According to the method, the extracted linseed protein is treated by adopting air pressure plasma jet (5-10 s), so that the emulsifying activity and the emulsifying stability of the linseed protein are remarkably improved, and compared with the untreated linseed protein, the emulsifying activity is increased by 16-24%, and the emulsifying stability is increased by 17-22%. The total phenol content of the linseed protein treated by air pressure plasma jet (5-10 s) is 1033-1127 mg/100g, and compared with that of the linseed protein which is not treated, the total phenol content is increased by 8-18%. After being treated by air pressure plasma jet flow (5-10 s), the DPPH free radical scavenging activity in the linseed protein is 819-890 mu mol/100g, FRAP is 6884-7029 mu mol/100g, ABTS is 406-427 mmol/100g, and compared with the linseed protein which is not treated, the DPPH free radical scavenging activity is increased by 28-39%, 9-11% and 16-22%.

Further, the parameters for preparing the weak alkaline electrolyzed functional water in the step (2) are as follows: the electrolyte is a calcium chloride solution with the mass fraction of 0.8-1.0%, and the electrolytic voltage is 8-10V; the pH value of the weakly alkaline electrolyzed functional water is 9.45-9.55, and the ORP value is 145-165 mV.

Adopt above-mentioned further beneficial effect to lie in: the alkalescent electrolyzed functional water has relatively high pH value and contains-OH and H₂、Ca²⁺Etc., having a certain reducibility. We speculate that the weakly alkaline electrolyzed functional water has a regulation effect on genes related to endogenous biosynthesis of a flax phenolic compound in the process of promoting flax seed germination, so that the effect of the weakly alkaline electrolyzed functional water is obviously superior to that of deionized water and tap water.

Further, the specification of the seedling raising tray in the step (2) is 34cm in length and 25cm in width; in order to ensure the germination rate of flaxseeds, the spreading amount of the flaxseeds on each seedling raising tray is 30-35 g, and 100-150 mL of alkaline electrolyzed functional water is sprayed every 8-10 hours during germination so as to promote the synthesis of endogenous phenolic compounds in the germination process of the flaxseeds.

Further, the moisture content of the germinated flaxseeds in the step (3) is still kept to be 18-20% after low-temperature freeze drying, and residual moisture in the germinated flaxseeds can be used as a medium for absorbing microwave energy during subsequent microwave pretreatment.

Further, the power of the microwave pretreatment in the step (4) is 650-700 w, and the microwave time is 3-5 min. On one hand, the microwave pretreatment can inhibit the activity of the lipase of the germinated flaxseeds; on the other hand, the microwave pretreatment can promote the dissolution and release of phenolic compounds coexisting with the flaxseed gum and the flaxseed protein in the subsequent extraction process of the flaxseed gum and the flaxseed protein

Adopt above-mentioned further beneficial effect to lie in: the microwave pretreatment can inhibit various enzymes such as lipoxidase and the like activated in the flax seed germination process, and avoid the adverse effects of the peroxidation of residual lipid coexisting with the flax seed protein in the extraction process on the emulsification characteristic and the oxidation stability of the flax seed protein. In addition, microwave pretreatment can increase the dissolution and release of phenolic compounds during the extraction of flaxseed gum and flaxseed protein, which may be mainly attributed to the fact that microwave treatment can induce the "depolymerization" effect of secoisolariciresinol diglucoside macromolecules, changes in molecular polarity, changes in phenolic acid morphology, and the like. In summary, the multiple effects synergistically induce targeted enrichment of phenolic compounds in extracted flaxseed protein and flaxseed gum.

Further, in the step (8), the discharge power of the air pressure plasma jet is 700-750 w, the distance between the air pressure plasma jet and the liquid level is 30-35 mm, and the air pressure plasma processing time is 5-10 s; the emulsifying activity and the emulsifying stability of the linseed protein can be improved by short-time air pressure plasma treatment; meanwhile, the treatment can further release phenolic compounds, and the antioxidant activity of the linseed protein is improved.

Adopt above-mentioned further beneficial effect to lie in: in an aqueous phase system, the primary and secondary active oxygen and active nitrogen compounds generated by short-time air pressure plasma treatment can change the microenvironment of hydrophobic/hydrophilic amino acid, thereby improving the emulsifying activity and the emulsifying stability of the treated linseed protein. More importantly, the air pressure plasma treatment in a short time induces the depolymerization effect of the lignan macromolecules, so that CAG, CoUG and FAG crosslinked with the SDG are released from side chains, and the antioxidant activity of the linseed protein is obviously increased. The processing time is critical to affect the emulsifying and antioxidant properties of the flaxseed protein. It was found that further extension of the treatment time resulted in a gradual decrease of the flax seed protein emulsifying activity, total phenolic content and antioxidant activity.

Further, in the step (9), the volume ratio of the flax seed protein to the flax seed gum is 3: 1-5: 1, and the magnetic stirring speed is 120-150 rmp/min, so that the flax seed protein and the flax seed gum can form a flax seed protein/flax seed gum/polyphenol complex coacervation embedding system based on electrostatic complexation.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.