CN114885989B

CN114885989B - Laccase cross-linked beet pectin-soybean oil body composite emulsion and preparation method and application thereof

Info

Publication number: CN114885989B
Application number: CN202210642975.9A
Authority: CN
Inventors: 钱珊珊; 侯俊财; 赵旭
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2023-06-02
Anticipated expiration: 2042-06-08
Also published as: CN114885989A

Abstract

The invention discloses laccase cross-linked beet pectin-soybean oil body composite emulsion and a preparation method and application thereof, and belongs to the technical field of oil processing. The method comprises the following steps: (1) extracting soybean oil bodies; (2) Preparing beet pectin solution with the concentration of 0.1wt% or 0.075wt%, adding the soybean oil body, adding laccase, stirring at high speed to obtain emulsion, standing, and regulating pH of the emulsion to 7-8 to obtain the laccase cross-linked beet pectin-soybean oil body composite emulsion. Compared with emulsion without laccase beet pectin, the composite emulsion prepared by the invention retains more liquid drops and obviously reduces the release amount of free fatty acid after simulated gastrointestinal digestion, and proves that the composite emulsion has slower digestion rate, is easy to generate satiety and has important value in food processing application.

Description

Laccase cross-linked beet pectin-soybean oil body composite emulsion and preparation method and application thereof

Technical Field

The invention relates to the technical field of grease processing, in particular to laccase cross-linked beet pectin-soybean grease body composite emulsion, and a preparation method and application thereof.

Background

Soybean (soybean) is one of the most important oil crops and is also an important source of protein and grease. The soybean protein contains essential amino acids for human body, and can reduce incidence rate of cardiovascular and cerebrovascular diseases. Unsaturated fatty acids in soy fat have beneficial nutritional properties that make them ideal ingredients in food, health and pharmaceutical products. The soybean also contains a large amount of phospholipids, and is a structure of amphiphilic molecule groups with hydrophilic groups and hydrophobic groups. The soybean oil body consists of a phospholipid monolayer and a central lipid surrounded by amphipathic oleosins. The oleosin may protect the body of the grease against environmental stresses such as moisture changes, temperature fluctuations, and the effects of oxidizing agents. In addition, soybean contains tocopherol with strong oxidation resistance and soybean polypeptide capable of reducing blood pressure. With innovative processing of soybean products, soybeans are popular with consumers, so that the soybean industry is continuously developed at a high speed. In the food processing process, the soybean oil bodies are easily influenced by mineral elements, thermal circulation and acidity regulation, the stability of oil body systems is reduced, the flocculation condition of the oil bodies is common, and the product range of the soybean oil bodies in the food, medical and pharmaceutical industries is seriously influenced.

Currently, some researchers improve the physical stability of the grease body by altering the interfacial composition of the droplets with polysaccharides. Matsuyama et al found that xanthan molecules could interact with lipid surface protein molecules to produce hybrid conjugates with improved surface activity. Furthermore, wang found that gum arabic adsorbed on the surface proteins of the oil body can stabilize the oil-water interface at different NaCl concentrations. Su et al studied the effect of the anionic polysaccharide sodium alginate on the stability of soybean oil emulsion under different environmental conditions (including NaCl, pH and freeze-thaw cycles) by analyzing particle charge, particle size and distribution. The result shows that the stability of the emulsion of the natural oil body can be obviously improved by coating the oil body with the polysaccharide sodium alginate. Mohammadi investigated the antioxidant activity of olive leaf extracts encapsulated by nanoemulsions in soybean oil liposomes. The nano-coated olive leaf extract can better control peroxide number than the uncoated olive leaf extract through peroxide number, TBARS number and rancidity thermal stability tests. However, the encapsulated olive leaf extract has a lower thermal stability due to the phenolic compounds being blocked in the dispersed emulsion droplets. Wu et al have found that after three freeze-thaw cycles of a carrageenan coated soy oil body emulsion having pH values of 3 and 7 in the presence of sucrose, the droplets aggregate very little. These results indicate that the carrageenan-coated soy oil body emulsion has higher stability than the uncoated emulsion.

Soybean varieties are numerous and are distinguished by content mainly due to two major categories, namely high fat and high protein soybeans, in the current stage, there is considerable research on fat bodies, but less research on fat bodies than the two soybean varieties. In addition, with the improvement of the living standard of people, consumers pay attention to not only the taste and flavor of the grease products, but also the health level, so that the development of a healthy grease product which has higher stability and can ensure low digestion speed is not necessary.

Disclosure of Invention

The invention aims to provide laccase cross-linked beet pectin-soybean oil body composite emulsion, and a preparation method and application thereof, so as to solve the problems of the prior art.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a preparation method of laccase cross-linked beet pectin-soybean oil body composite emulsion, which comprises the following steps:

(1) Extracting soybean oil: soaking soybean seeds in water, adding NaCl mixed solution into the soaked seeds, grinding, filtering and centrifuging, collecting upper emulsion, dispersing the upper emulsion in buffer solution, centrifuging, and collecting upper oil emulsion; heating the soybean oil body to obtain the soybean oil body;

(2) Preparing beet pectin solution with the concentration of 0.1wt% or 0.075wt%, adding the soybean oil body, adding laccase, stirring at high speed to obtain emulsion, standing, and regulating pH of the emulsion to 6-8 to obtain the laccase cross-linked beet pectin-soybean oil body composite emulsion.

Further, in step (1), the volume ratio of soybean seeds to water is 1:5; the soaking temperature is 4-5 ℃ and the soaking time is 18-22h.

Further, in the step (1), the NaCl mixed solution consists of 50mmol/L Tris-HCl, 0.4mol/L sucrose and 0.5mol/L NaCl, and the mass volume ratio of the soaked seeds to the NaCl mixed solution is 1:5.

further, in the step (2), the soybean oil body is added in an amount of 10v% of the beet pectin solution.

Further, in step (2), the soybean oil bodies include high-oil soybean oil bodies (HOSOB) and high-protein soybean oil bodies (HPSOB).

Further, the high-oil soybean oil body was added to a sugar beet pectin solution having a concentration of 0.1wt%, and the high-protein soybean oil body was added to a sugar beet pectin solution having a concentration of 0.075 wt%.

Further, in step (2), the pH is adjusted to 4.5 prior to adding the laccase; the addition amount of laccase is 0.4-0.5U.

Further, in the step (2), the high-speed stirring is 10000rpm/min for 10min; the standing time is 24 hours.

The invention also provides laccase cross-linked beet pectin-soybean oil body composite emulsion prepared by the preparation method.

The invention also provides application of the laccase cross-linked beet pectin-soybean oil body composite emulsion in preparing an oil product.

The invention discloses the following technical effects:

according to the invention, the laccase cross-links the beet pectin-HOSOB and the beet pectin HPSOB, the addition of the laccase enables the beet pectin to be adsorbed fast and fully cover the surface of the proteolized liposome, the negative charge amount is increased, the zeta-potential is obviously improved, and meanwhile, the beet pectin molecules are cross-linked, so that the particle size of the soybean oil body composite emulsion is obviously reduced (P < 0.05) due to structural rearrangement. The increase in zeta potential also produces stronger repulsive and steric hindrance such that the surface hydrophobicity is improved, resulting in a more stable and uniform emulsion. And the pH value of the emulsion is regulated and controlled to be between 6 and 8, so that the long-term stable storage of the laccase cross-linked beet pectin-soybean oil body composite emulsion is easier. Finally, the result of in vitro digestion simulation experiment of the emulsion shows that after the oil body emulsion added with laccase and beet pectin is digested by stomach and intestinal tracts, compared with the emulsion without laccase and beet pectin, more liquid drops are reserved, the oil body can be better protected, the release amount of free fatty acid is obviously reduced, and the composite emulsion has slower digestion rate, is easy to generate satiety, and has important value in food processing application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the effect of varying concentrations of beet pectin on the stability of a soy fat body emulsion;

FIG. 2 shows the measurement of absorbance of sugar beet pectin;

FIG. 3 shows the results of a laccase activity assay;

FIG. 4 is a graph showing the effect of laccase cross-linked beet pectin on the soybean oil body emulsion potential;

FIG. 5 is a graph showing the effect of laccase cross-linked beet pectin on the average particle size of soybean oil lipid emulsion;

FIG. 6 is an effect of laccase cross-linked beet pectin on the surface hydrophobicity of a soy oil body emulsion;

FIG. 7 is a graph showing the effect of laccase cross-linked beet pectin on the emulsifying activity of a soybean oil body emulsion;

FIG. 8 is the effect of laccase cross-linked beet pectin on emulsion stability of soybean oil body emulsion;

FIG. 9 is an effect of laccase cross-linked beet pectin on soybean oil body emulsion stability;

FIG. 10 is a graph showing the effect of laccase cross-linked beet pectin on the peroxide number of a soybean oil body emulsion;

FIG. 11 is a graph showing the effect of pH on zeta potential of laccase cross-linked beet pectin-soybean oil body composite emulsion;

FIG. 12 is a graph showing the effect of pH on particle size of laccase cross-linked beet pectin-soybean oil body composite emulsion;

FIG. 13 is a graph showing the effect of pH on stability of laccase cross-linked beet pectin-soybean oil body composite emulsion;

FIG. 14 is a graph showing the effect of pH on the peroxide value of laccase cross-linked beet pectin-soybean oil body composite emulsion;

FIG. 15 is a graph showing the effect of laccase cross-linked beet pectin on zeta potential of soybean oil bodies following in vitro digestion;

FIG. 16 is a graph showing the effect of laccase cross-linked beet pectin on particle size of soybean oil body following in vitro digestion;

FIG. 17 is a simulated gastric digestion ultra-high resolution microscopy image of laccase cross-linked beet pectin on soybean oil bodies;

FIG. 18 is an ultra-high resolution microscopy image of laccase cross-linked beet pectin on simulated intestinal digestion of soybean oil bodies;

FIG. 19 is a graph showing the effect of laccase cross-linked beet pectin on the release of free fatty acids after in vitro digestion of soybean oil bodies.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

Materials, instruments and reagents used in the present invention are commercially available unless otherwise specified; the experimental methods used, unless otherwise specified, are all routine in the art.

1 Experimental method

1.1 preparation of Soy oil body

Extraction of fat bodies was studied with reference to Tzen et al and Ding et al. The soybean seeds are soaked for 20 hours (4-5 ℃) with distilled water according to the volume ratio of 1:5. Mixing with solution (50 mmol/L Tris-HCl,0.4mol/L sucrose, 0.5mol/L NaCl, pH 7.5,1:5 w/v), grinding, filtering, centrifuging for 30min (10000 rpm/min,4 ℃ C.), repeating twice; after collecting the top emulsion, it was dispersed in Tris-HCl buffer (50 mmol/L Tris-HCl, pH 7.5,1:5 w/v) and centrifuged; repeating for three times, and obtaining the upper emulsion as the grease body. Heating the fat emulsion in water bath at 80deg.C for 20min, cooling, and adding 0.01% NaN ₃ And is placed in a refrigerator at 4 ℃ for standby.

1.2 measurement of essential Components of fat body

1.2.1 determination of fat content

Reference is made to the Soxhlet extraction method in GB 5009.6-2016.

1.2.2.2 determination of protein content

Reference is made to the combustion process in GB 5009.5-2016.

1.2.2.3 determination of moisture content

Reference is made to the direct drying process in GB 5009.3-2016.

1.2.2.4 calculation of the extraction yield of fat and oil

Wherein:

w1-seed weight (g); w2-fat body weight (g).

1.3 Effect of different concentrations of sugar beet pectin on the characteristics of the Soy oil body emulsion

1.3.1 preparation of sugar beet pectin-fat body composite emulsion

Preparing beet pectin solutions with concentration of 0.025%, 0.05%, 0.075%, 0.1%, 0.15%, and 0.2%, heating, stirring to accelerate dissolution, cooling, adding a certain amount of high-oil soybean oil lipid (HOSOB) (10%) or high-protein soybean oil lipid (HPSOB) cream (10%), adjusting pH to 4.5, dispersing at 10000rpm/min for 30min, standing at room temperature for 24 hr, adjusting pH to 7, and sealing.

1.3.2 determination of zeta potential and average particle size

Both zeta potential and average particle size measurements are referred to the Zhao et al test.

Zeta potential measurement: the volume fraction of the diluted emulsion (PBS dilution) was about 1:1000, as measured by a potentiometric analyzer.

Measurement of particle size: the diluted emulsion (PBS diluted) was measured by a laser particle sizer after reaching the shade. Refractive index: 1.08.

1.3.3 measurement of surface hydrophobicity

The surface hydrophobicity (H0) of the grease was measured by the method of reference Boyer. Samples were diluted in gradient 0.25%, 0.50%, 1.00%, 1.50% and 2.00% (w/v). ANS (100. Mu.L, 8 mmol/L) was added to 10mL of the sample and mixed well, protected from light for 15min. The excitation and emission wavelengths were 390nm and 470nm, respectively. The surface hydrophobicity index is the slope of the regression curve of fluorescence intensity versus protein solution mass.

1.3.4 determination of the emulsion Properties

Referring to the Pearce et al test, 20. Mu.L of the emulsion was mixed with 4.98mL SDS (0.1%). Measuring absorbance A0 at 500nm wavelength, standing for 30min, measuring absorbance A30, taking SDS solution as blank, and calculating according to the formula:

EAI-emulsifying Activity index, m2/g

ESI-emulsion stability index

N-dilution factor, get 250

C-protein mass concentration in aqueous protein solution before emulsion formation, g/mL

-volume fraction of oil phase in emulsion

1.3.5 determination of flocculation stability of emulsion

10mL of the sample was placed in a sample bottle, and the sample was left at room temperature for 0d and 14d, and then observed.

1.3.6 determination of peroxide number

Referring to titration in GB5009.227-2016, 1mL of emulsion was taken and 5mL of isooctane was added: isopropanol (volume ratio 2:1), after mixing well, centrifuging, taking 1mL of supernatant, adding 20 μl of ferrous chloride, 20 μl of potassium thiocyanate solution and 5mL of methanol: n-butanol (volume ratio 2:1), measured in the dark for 20min at 510nm, the Peroxide Value (PV) was calculated as follows:

PV＝(0.5×A×k×n)/(55.86×2×m)

absorbance of A-sample

Slope of k-Fe3+ marked curve

m-content of fat in the sample being weighed

n-volume fraction of supernatant aspirated

1.4 Effect of laccase Cross-Linked sugar beet pectin on the characteristics of Soybean oil body emulsions

1.4.1 determination of ferulic acid absorbance

An appropriate amount of beet pectin was diluted to 1% and a buffer solution without pectin was used as blank. And measuring the absorption spectrum of beet pectin with the wavelength of 200-400 nm.

1.4.2 determination of laccase Activity

Varying amounts of laccase (0-0.5U) were added to the beet pectin (0.075% and 0.1%) samples. The absorbance within 3000s was measured at 325nm at 25 ℃.

1.4.3 sample pretreatment

Preparing beet pectin solution containing 0.1% and 0.075%, heating and stirring to accelerate dissolution, adding HOSOB cream (10% of beet pectin solution volume) or HPSOB cream (10%), adjusting pH to 4.5, adding laccase, and stirring with high-speed disperser at 10000rpm/min for 10min to obtain uniform emulsion. And standing at room temperature for 24 hours, regulating the pH of the emulsion to 7, and sealing and preserving.

1.4.4 determination of zeta potential and average particle size

The method is the same as 1.3.2

1.4.5 determination of surface hydrophobicity

The method is the same as 1.3.3

1.4.6 determination of the emulsion Properties

The method is the same as 1.3.4

1.4.7 determination of flocculation stability of emulsion

The method is the same as 1.3.5

Determination of the peroxide number of 1.4.8

The method is the same as 1.3.6

1.5 influence of environmental factors on stability of laccase-crosslinked beet pectin-soybean oil body composite emulsion

1.5.1 sample pretreatment

Preparing beet pectin solution containing 0.1% and 0.075%, heating and stirring to accelerate dissolution, adding HOSOB cream (10%) or HPSOB cream (10%), adding laccase, and stirring with high speed disperser at 10000rpm/min for 10min to obtain uniform emulsion.

Controlling the pH: samples were adjusted to pH 2.5,6,7,8, respectively, and periodically sampled for measurement.

1.5.2 determination of zeta potential and average particle size

The method is the same as 1.3.2

1.5.3 determination of flocculation stability of emulsion

The method is the same as 1.3.5

Determination of the peroxide number of 1.5.4

The method is the same as 1.3.6

1.6 Effect of laccase Cross-Linked sugar beet pectin on Soybean oil body simulation in vitro digestion Properties

1.6.1 preparation of emulsions

And preparing a beet pectin-grease body composite emulsion and a beet pectin-grease body composite emulsion added with laccase respectively, wherein the grease body emulsion without the beet pectin is used as a control.

1.6.2 preparation of simulated digestive juice

A simulated digestive juice was prepared according to the static in vitro digestion method of Chen et al, and modified.

Simulating saliva: 1.594g/L sodium chloride, 0.328g/L ammonium nitrate, 0.636g/L potassium dihydrogen phosphate, 0.202g/L potassium chloride, 0.308g/L trisodium citrate dihydrate, 0.198g/L urea, 0.146g/L sodium lactate, 30g/L mucin, and adjusting the pH to 6.8.

Simulating gastric juice: 2g/L sodium chloride, 0.82g/L potassium chloride, 0.27g/L sodium dihydrogen phosphate, 0.0176g/L ascorbic acid, 0.65g/L glucose, 3.2g/L pepsin, and adjusting pH to 2.0.

Simulation of intestinal juice: 218.7g/L sodium chloride, 36.7g/L calcium chloride, 1g/L bovine serum albumin, 3.4g/L sodium bicarbonate, 24g/L pancreatin, 24g/L lipase, 54g/L bile salt, and adjusting the pH to 7.0.

1.6.3 simulation of oral digestion

The prepared emulsion was mixed with simulated saliva 1:1 and digested for 5min on a constant temperature shaker (100 rpm/min) at 37 ℃.

1.6.4 simulate gastric digestion

The simulated oral cavity digestive juice is mixed with the simulated gastric juice in a ratio of 1:1, and digested for 1h by a constant temperature shaking table (100 rpm/min) at 37 ℃.

1.6.5 simulated intestinal digestion

The simulated gastric digest was rapidly pH 7.0 adjusted with 2M sodium hydroxide, mixed with simulated intestinal fluid 1:3, digested with a constant temperature shaker (100 rpm/min) at 37℃for 2h, and sampled periodically for free fatty acid determination.

1.6.6 determination of free fatty acids

The Free Fatty Acids (FFA) released in intestinal digestion (every 20min up to 120 min) were quantified using the pH-stat method with reference to the method of Chen et al. Since the change in pH during digestion is due to release of FFA, the quantification of FFA is determined by calculating the amount of NaOH to neutralize the pH of the digestive fluid. The percent release formula for FFA:

FFA(％)＝100×((V_NaOH×M_NaOH×M_Lipid)/(2×W_Lipid))

V NaOH-NaOH volume at pH 7.0 (mL)

M NaOH-molar concentration of NaOH (M)

M Lipid-average molecular weight of soybean oil (g/mol)

W Lipid-total mass of Soybean oil (g)

Ultra-high resolution microscopic observation of grease body after 1.6.7 digestion

The digested fat body structure was observed with a GE Delta Vision OMX SR ultra-high resolution microscope. A2 mL dilution was added with 80. Mu.L of staining solution containing 0.02% Nile Red and 0.1% Nile Blue A, and the mixture was mixed and sampled on a glass slide. The Ar/K and He/Ne dual-channel laser modes are adopted for observation at excitation wavelengths of 488nm and 630nm, and AcquireSR is adopted for image acquisition.

1.6.8 data statistical analysis

The data obtained were subjected to analysis of variance (ANOVA) with SPSS Statistics 20 (SPSS inc., chicago, IL, USA); significant differences were examined by multiple ranges of Duncan (P <0.05 indicates significant differences, P >0.05 indicates insignificant differences); mapping was performed using Origin 2017 (OriginLab Corporation, northampton, mass., USA).

2 results and analysis

2.1 Effect of beet pectin on the physicochemical stability of the Soy oil body emulsion

2.1.1 essential composition of soybean oil body

The basic compositions of HOSOB and HPSOB and their extraction rates are shown in Table 1. From the table, the extraction yield of HPSOB (5.85±0.06%) was significantly lower than HOSOB (8.24±0.09%) (P < 0.05), which is attributable to the higher fat content of HOSOB.

In addition, as can be seen from table 1, there was a significant difference in fat content, protein content and moisture content between HPSOB and HOSOB (P < 0.05). The fat contents of HPSOB and HOSOB were (30.38.+ -. 0.47)% and (42.76.+ -. 0.44)% (P < 0.05), respectively. The protein content and the moisture content of HPSOB are (8.99+ -0.02)% and (59.24 + -0.27)%, respectively, which are significantly higher than HOSOB (P < 0.05).

TABLE 1 basic composition and extraction yield of soybean oil

Note that: lower case letter differences represent significant differences (P < 0.05); letter identical indicates that the difference is not significant (P > 0.05).

2.1.2 Effect of sugar beet pectin on the zeta potential of the Soy oil body emulsion

Different concentrations of sugar beet pectin have an effect on the zeta potential of the soybean oil body emulsion. The addition of beet pectin can significantly raise the zeta-potential of the HOSOB and HPSOB emulsions. The zeta potential of the HOSOB emulsion of the blank control group was-20.19.+ -. 0.83mV, the HPSOB emulsion was-21.31.+ -. 0.55mV, and both had no significant effect (P > 0.05). When the concentration of the added beet pectin is 0.05%, the zeta-potentials of the HOSOB emulsion and the HPSOB emulsion are respectively-31.44+/-0.63 mV and-28.13+/-0.54 mV, which are obviously higher than that of the fat body emulsion of the blank control group (P < 0.05) and are obviously different (P < 0.05); the zeta-potential of the HOSOB and HPSOB emulsions essentially showed increasing levels with increasing sugar beet pectin concentration. When added at 0.2%, the zeta potential of the HOSOB emulsion was-35.74.+ -. 0.42mV, which is significantly higher than that of the HPSOB emulsion (-32.41.+ -. 0.32 mV) (P < 0.05).

2.1.3 Effect of sugar beet pectin on surface hydrophobicity of Soy oil body emulsion

Different concentrations of sugar beet pectin have an effect on the surface hydrophobicity (H0) of the soy oil body emulsion. The surface hydrophobicity of the soybean oil fat emulsion can be obviously improved by adding the beet pectin, and when 0.025 percent of beet pectin is added, the surface hydrophobicity of the HOSOB emulsion and the HPSOB emulsion are 484.80 +/-8.65 and 454.25+/-17.07 respectively, which are obviously higher than that of the fat body emulsion (334.80 +/-15.70 and 318.17 +/-12.94) of a blank control group (P < 0.05). As the sugar beet pectin concentration increases, the surface hydrophobicity of the soy oil body emulsion increases significantly (P < 0.05).

2.1.4 Effect of sugar beet pectin on the average particle size of the Soybean oil lipid emulsion

The average particle size of the soybean oil body emulsion is reduced by adding the beet pectin, when the beet pectin is added in an amount of 0.025%, the average particle sizes of the HOSOB emulsion and the HPSOB emulsion are respectively 1.68+/-0.12 mu m and 2.18+/-0.15 mu m, which are obviously lower than that of the oil body emulsion (7.26+/-0.20 mu m and 8.04+/-0.13 mu m) of a blank control group (P < 0.05), and the average particle sizes of the HOSOB emulsion and the HPSOB emulsion are obviously different (P < 0.05). The average particle size of both HOSOB and HPSOB emulsions became progressively larger with increasing concentration, and when 0.2% of beet pectin was added, the average particle size of HOSOB and HPSOB emulsions was 7.34.+ -. 0.25 μm and 8.17.+ -. 0.13 μm, with no significant difference (P > 0.05) from the fat body emulsion of the blank.

2.1.5 Effect of sugar beet pectin on the emulsion Properties of the Soy oil body emulsion

The emulsion properties of the soybean oil body emulsion are affected by the sugar beet pectin with different concentrations. The emulsion activity of the soybean oil fat emulsion can be obviously improved by adding beet pectin, and when 0.025 percent of beet pectin is added, the emulsion activities of HOSOB and HPSOB emulsion are respectively 13.58+/-0.11 m2/g and 10.33+/-0.18 m2/g which are obviously higher than that of the fat body emulsion (12.51+/-0.33 m2/g and 9.14+/-0.23 m 2/g) of a blank control group (P < 0.05). The HOSOB emulsion reached a maximum of EAI (15.67.+ -. 0.41m 2/g) when 0.1% sugar beet pectin was added. The HPSOB emulsion reached a maximum of EAI (11.83.+ -. 0.24m 2/g) at 0.075% addition.

The sugar beet pectin with different concentrations has influence on the emulsion stability of the soybean oil body emulsion. The emulsion stability of the soybean oil fat emulsion can be obviously improved by adding the beet pectin, when 0.025 percent of beet pectin is added, the emulsion stability of the HOSOB emulsion and the HPSOB emulsion is 123.86 +/-2.86 and 119.30 +/-3.30 respectively, and the emulsion stability is obviously higher than that of the soybean oil fat emulsion (112.67 +/-3.44 and 102.46 +/-2.80) of a blank control group (P < 0.05). As the polysaccharide concentration increases, the emulsion stability of the soybean oil body emulsion tends to increase and then decrease. The HOSOB emulsion reached a maximum ESI (161.60.+ -. 3.46) when 0.1% sugar beet pectin was added. The ESI reached a maximum value (144.24.+ -. 3.31) at 0.075% addition of HPSOB emulsion. At the same addition level, the emulsion stability of the HOSOB emulsion is significantly higher than that of HPSOB (P < 0.05).

2.1.6 Effect of sugar beet pectin on the stability of the Soy oil body emulsion

The effect of varying concentrations of sugar beet pectin on the stability of the soy fat body emulsion is shown in figure 1. As can be seen, both HOSOB and HPSOB emulsions flocculate to varying degrees with the storage time. After 15d of storage at room temperature, the flocculation of the two soybean fat body emulsions without the beet pectin is quite obvious, the flocculation degree of the fat body emulsion with the beet pectin is lower, and the flocculation phenomenon is weakened and then enhanced with the increase of the addition amount of the beet pectin.

2.1.7 Effect of sugar beet pectin on the peroxide value of the Soy oil body emulsion

Sugar beet pectin at different concentrations has an effect on the peroxide value of the soybean oil body emulsion. In 0-15d, the peroxide values of the HOSOB and HPSOB emulsions tended to increase and then decrease. The oxidation degree of the fat body emulsion to which the beet pectin is added is smaller than that of the fat body emulsion to which the beet pectin is not added as a whole.

The peroxide value of HOSOB emulsion of the blank group reaches the maximum value of 15.43+/-0.38 mmol/kg at 9d, and the peroxide value of HOSOB emulsion added with 0.025%, 0.05%, 0.075%, 0.1%, 0.15% and 0.2% beet pectin is also remarkably lower than that of HOSOB emulsion (P < 0.05) of the blank group at 9d, namely, the peroxide value reaches the maximum value of 10.63+/-0.53, 11.54+/-0.27, 10.36+/-0.32, 9.07+/-0.08, 10.88+/-0.39 and 11.41+/-0.40 mmol/kg.

The peroxide value of the HPSOB emulsion added with 0.025%, 0.05%, 0.075%, 0.1%, 0.15%, 0.2% beet pectin reaches the maximum value of 17.47+ -0.42, 16.06+ -0.23, 15.38+ -0.21, 16.07+ -0.37, 16.39+ -0.18, 16.52 + -0.32 mmol/kg at 9d, which is significantly lower than that of the HPSOB emulsion (18.98+ -0.25 mmol/kg) of the blank control group (P < 0.05).

In summary, the addition of beet pectin significantly increases the zeta potential (P < 0.05) of both soybean oil bodies and significantly reduces the particle size (P < 0.05) of the oil bodies. The addition of beet pectin can significantly improve the surface hydrophobicity, emulsifying property and flocculation phenomenon (P < 0.05) of the fat body emulsion, and significantly reduce the oxidation degree of the fat body emulsion within 0-15 d. By combining the above data, it was determined that the optimum amount of HOSOB added was 0.1% of beet pectin and that the optimum amount of HPSOB added was 0.075% of beet pectin.

2.2 Effect of laccase Cross-Linked sugar beet pectin on the characteristics of Soybean oil body emulsions

2.2.1 determination of absorbance of beet pectin

The absorbance of beet pectin is measured as shown in FIG. 2. It can be seen that the maximum of one absorption spectrum of beet pectin is observed at one wavelength range (320-330 nm) due to the presence of ferulic acid. Thus, we measured the cross-linking ability of laccase to pectin at this wavelength of 325 nm.

2.2.2 determination of laccase Activity

The laccase activity was determined as shown in FIG. 3.

From panel a, the absorbance of the 0.1% sugar beet pectin solution did not change significantly in the absence of laccase. When laccase is added to a 0.1% sugar beet pectin solution, the absorbance decreases over time, due to enzyme-catalyzed cross-linking of ferulic acid groups. The absorbance drops sharply within the first 0-500s and then drops gradually. The slope of initial relative absorbance (a (t)/a (0)) over time increased with increasing laccase concentration, so we used 0.5U in the rest of the experiment.

From panel B, there was no significant change in absorbance of the 0.075% sugar beet pectin solution without laccase. When laccase is added to the 0.075% sugar beet pectin solution, the absorbance decreases over time, due to enzyme-catalyzed cross-linking of ferulic acid groups. The absorbance drops sharply within the first 0-500s and then drops gradually. The slope of initial relative absorbance (a (t)/a (0)) over time increases with increasing laccase concentration. But between 0.4U and 0.5U, the reaction rate did not change much, so we used 0.4U in the rest of the experiment.

2.2.3 Effect of laccase-crosslinked sugar beet pectin on the zeta potential of soybean oil body emulsions

The effect of laccase cross-linked beet pectin on the soybean oil body emulsion potential is shown in FIG. 4. From the figure, laccase cross-linked beet pectin can raise zeta potential of soybean oil liposome composite emulsion. The zeta potential of the HOSOB emulsion of the blank control group was-19.27.+ -. 0.92mV, the HPSOB emulsion was-18.43.+ -. 0.61mV, and both had no significant effect (P > 0.05). The zeta potential of the HOSOB emulsion after laccase addition was-19.49.+ -. 0.56mV, the HPSOB emulsion was-18.27.+ -. 0.44mV, and both had no significant effect (P > 0.05). The zeta potential of HOSOB added with beet pectin was-32.83+ -0.83 mV, and that of HPSOB emulsion was-30.41+ -0.79 mV, which were both significantly higher than that of the fat body emulsion of the blank control group (P < 0.05), and also significantly different (P < 0.05). The zeta-potentials of the laccase cross-linked beet pectin HOSOB composite emulsion and the laccase cross-linked beet pectin HPSOB composite emulsion are-41.36+/-0.94 mV and-40.99+/-0.83 mV respectively, which are obviously higher than that of the grease emulsion added with beet pectin (P < 0.05), and the zeta-potentials are not obviously different (P > 0.05).

2.2.4 Effect of laccase-crosslinked sugar beet pectin on the average particle size of Soybean oil lipid emulsion

The effect of laccase cross-linked beet pectin on the average particle size of the soybean oil lipid emulsion is shown in FIG. 5. From the graph, laccase cross-linked beet pectin can reduce the average particle size of soybean oil-fat body composite emulsion, the average particle size of HOSOB emulsion of blank control group is 7.04+/-0.28 μm, HPSOB emulsion is 7.84+/-0.29 μm, and both have significant effect (P < 0.05). The average particle size of the HOSOB emulsion after laccase addition was 7.21+ -0.30 μm, the HPSOB emulsion was 7.96+ -0.27 μm, and neither was significantly higher than the fat body emulsion of the blank (P > 0.05), but the two were significantly different (P < 0.05). The average particle size of HOSOB added with beet pectin is 4.03+ -0.24 μm, the average particle size of HPSOB emulsion is 4.59+ -0.19 μm, which are significantly higher than that of fat body emulsion of blank group (P < 0.05), and the two are also significantly different (P < 0.05). The average particle sizes of the laccase cross-linked beet pectin HOSOB composite emulsion and the laccase cross-linked beet pectin HPSOB composite emulsion are respectively 2.61+/-0.11 mu m and 2.89+/-0.12 mu m, which are obviously higher than that of the grease emulsion added with beet pectin (P < 0.05), and the average particle sizes are obviously different (P < 0.05).

2.2.5 Effect of laccase Cross-Linked sugar beet pectin on surface hydrophobicity of Soybean oil body emulsion

The effect of laccase cross-linked beet pectin on the surface hydrophobicity (H0) of the soybean oil body emulsion is shown in FIG. 6. From the graph, laccase cross-linked beet pectin can improve the surface hydrophobicity of soybean oil liposome composite emulsion, the surface hydrophobicity of HOSOB emulsion of a blank control group is 346.27 +/-13.13, the surface hydrophobicity of HPSOB emulsion is 322.21 +/-11.24, and the two have obvious influence (P < 0.05). The surface hydrophobicity of the HOSOB emulsion after laccase addition is 335.42 +/-15.62, the HPSOB emulsion is 312.62 +/-10.27, and the surface hydrophobicity of the HOSOB emulsion is not obviously higher than that of the fat body emulsion (P > 0.05) of the blank control group, and the surface hydrophobicity of the HOSOB emulsion and the HPSOB emulsion are obviously different (P < 0.05). The surface hydrophobicity of HOSOB added with beet pectin is 564.46 +/-14.83, the surface hydrophobicity of HPSOB emulsion is 534.74 +/-12.29, which are obviously higher than that of fat body emulsion (P < 0.05) of a blank control group, and the two are obviously different (P < 0.05). The surface hydrophobicity of the laccase cross-linked beet pectin HOSOB composite emulsion and the laccase cross-linked beet pectin HPSOB composite emulsion are 678.85 +/-23.84 and 668.56 +/-21.47 respectively, which are obviously higher than that of the oil body emulsion added with beet pectin (P < 0.05), and have no obvious difference (P > 0.05).

2.2.6 Effect of laccase Cross-Linked sugar beet pectin on the emulsion Properties of Soybean oil body emulsions

The effect of laccase cross-linked beet pectin on the emulsification properties of the soy oil body emulsion is shown in figures 7 and 8. As shown in fig. 7, laccase cross-linked beet pectin can significantly improve the emulsification activity of soybean oil-liposome composite emulsion, the emulsification activity of the HOSOB emulsion of the blank group is 12.39±0.43, the emulsification activity of the hpsob emulsion is 10.03±0.32, and the two have significant effects (P < 0.05). The emulsion activity of the HOSOB emulsion after laccase addition was 12.55+ -0.32, the HPSOB emulsion was 10.09+ -0.25, which were not significantly higher than the fat body emulsion of the blank control group (P > 0.05), but were significantly different (P < 0.05). The emulsifying activity of HOSOB added with beet pectin is 15.76+ -0.42, the emulsifying activity of HPSOB emulsion is 14.16+ -0.32, which are both significantly higher than that of fat body emulsion of blank control group (P < 0.05), and the two are also significantly different (P < 0.05). The emulsion activities of the laccase cross-linked beet pectin HOSOB composite emulsion and the laccase cross-linked beet pectin HPSOB composite emulsion are 17.49+/-0.40 and 16.35+/-0.44 respectively, which are obviously higher than that of the oil body emulsion added with beet pectin (P < 0.05), and the emulsion activities are obviously different (P < 0.05).

The effect of laccase cross-linked beet pectin on the emulsion stability of the soybean oil body emulsion is shown in figure 8. From the figure, laccase cross-linked beet pectin can obviously improve the emulsion stability of the soybean oil liposome composite emulsion. The emulsion stability of the HOSOB emulsion after laccase addition was 117.42 + -3.76, the HPSOB emulsion was 98.85+ -3.12, and none of them was significantly higher than the fat body emulsion of the blank (115.26 + -4.01 and 97.15 + -2.77) (P > 0.05), but the two were significantly different (P < 0.05). The emulsion stability of HOSOB added with beet pectin is 159.85 + -3.35, the emulsion stability of HPSOB emulsion is 144.16 + -4.013, which are both significantly higher than that of fat body emulsion of blank group (P < 0.05), and the two are also significantly different (P < 0.05). The emulsion stability of the laccase cross-linked beet pectin HOSOB composite emulsion and the laccase cross-linked beet pectin HPSOB composite emulsion are 182.89 +/-3.69 and 174.71 +/-2.28 respectively, which are obviously higher than that of the oil body emulsion added with beet pectin (P < 0.05), and the emulsion stability are obviously different (P < 0.05).

2.2.7 Effect of laccase Cross-Linked sugar beet pectin on the stability of Soybean oil body emulsion

The effect of laccase cross-linked beet pectin on the stability of the soybean oil body emulsion is shown in figure 9. As can be seen from the graph, the HOSOB and HPSOB emulsions all had different degrees of aggregation and flocculation with prolonged storage time. After 15d storage at room temperature, the flocculation phenomenon of the oil body emulsion added with laccase (Lac) is not obviously different from that of the oil body emulsion without any additives, the soybean oil body emulsion added with beet pectin (SBP) shows slight milk out, and the flocculation degree of the oil body emulsion of laccase cross-linked beet pectin (Lac+SBP) is the lowest.

2.2.8 Effect of laccase Cross-Linked sugar beet pectin on the peroxide value of the Soy oil body emulsion

The effect of laccase cross-linked beet pectin on the peroxide number of the soybean oil body emulsion is shown in FIG. 10. From the graph, the peroxide value of the laccase cross-linked beet pectin-soybean oil body composite emulsion tends to increase and then decrease in 0-15 d. The oxidation degree of the laccase cross-linked beet pectin-soybean oil body composite emulsion is smaller than that of the soybean oil body emulsion added with beet pectin.

The peroxide value of HOSOB emulsion of the blank group reaches the maximum value of 15.27+/-0.40 mmol/kg at 9d, the peroxide value of HOSOB emulsion reaches the maximum value of 15.39+/-0.24 mmol/kg after laccase is added, the peroxide value of HOSOB emulsion of beet pectin is added reaches the maximum value of 9.04+/-0.095 mmol/kg at 9d, and the laccase cross-linked beet pectin HOSOB composite emulsion reaches the maximum value of 7.24+/-0.22 mmol/kg at 10d, which is obviously lower than that of HOSOB emulsion of beet pectin (P < 0.05).

The peroxide value of HPSOB emulsion of the blank group reaches the maximum value 19.06+/-0.26 mmol/kg at 9d, the peroxide value of HPSOB emulsion reaches the maximum value 18.86+/-0.21 mmol/kg after laccase is added, the peroxide value of HPSOB emulsion of beet pectin is added reaches the maximum value 14.40+/-0.23 mmol/kg at 9d, and the peroxide value of laccase cross-linked beet pectin HPSOB composite emulsion reaches the maximum value 11.68+/-0.36 mmol/kg at 9d, which is obviously lower than that of HPSOB emulsion of beet pectin (P < 0.05).

2.3 influence of pH on physicochemical properties of laccase-crosslinked beet pectin-soybean oil body composite emulsion

2.3.1 Effect of pH on zeta potential of laccase Cross-Linked sugar beet pectin-Soybean oil body Complex emulsion

The effect of pH on zeta potential of laccase cross-linked beet pectin-soybean oil body composite emulsion is shown in FIG. 11, with pH 7 as control. As can be seen from the graph, zeta-potentials of laccase cross-linked beet pectin-high oil soybean oil body composite emulsion under the conditions of pH 2.5 and pH 6 are respectively-24.66+/-0.53 mV and-35.20+/-0.99 mV, which are remarkably lower than those of a control group (-41.66+/-0.96 mV) (P < 0.05), and the potential of the composite emulsion under the condition of pH 8 is-42.41 +/-0.66 mV, which is not remarkably different from that of the control group (P > 0.05).

The zeta-potential of the laccase crosslinked beet pectin-high protein soybean oil body composite emulsion is-20.69+/-0.30 mV and-27.47+/-0.64 mV under the conditions of pH 2.5 and pH 6, which are obviously lower than that of a control group (-37.21 +/-0.86) (P < 0.05), and the potential of the composite emulsion is-36.45+/-0.49 mV under the condition of pH 8, which is not obviously different from that of the control group (P > 0.05).

2.3.2 influence of pH on particle size of laccase Cross-Linked sugar beet pectin-Soybean oil body composite emulsion

The effect of pH on particle size of laccase cross-linked beet pectin-soybean oil body composite emulsion is shown in FIG. 12, and pH 7 condition is used as control group. As can be seen from the graph, the particle sizes of laccase cross-linked beet pectin HOSOB composite emulsion under the conditions of pH 2.5 and pH 6 are respectively 19.61+/-0.95 mu m and 7.51+/-0.30 mu m, which are obviously lower than that of a control group (2.80+/-0.36 mu m) (P < 0.05), and the particle size of the composite emulsion under the condition of pH 8 is 3.19+/-0.12 mu m, which is not obviously different from that of the control group (P > 0.05).

The particle size of the laccase crosslinked beet pectin HPSOB composite emulsion is-23.45+/-1.03 mu m and 8.71+/-0.20 mu m under the conditions of pH 2.5 and pH 6, and is obviously lower than that of a control group (3.13+/-0.20 mu m) (P < 0.05), and the particle size of the composite emulsion under the condition of pH 8 is 4.11+/-0.12 mu m and has no obvious difference from that of the control group (P > 0.05).

2.3.3 influence of pH on stability of laccase-crosslinked beet pectin-soybean oil body composite emulsion

The influence of pH on the stability of laccase crosslinked beet pectin-soybean oil body composite emulsion is shown in FIG. 13, pH 7 is taken as a control group, and the flocculation phenomenon of the soybean oil body composite emulsion with pH 2.5 is obvious after the soybean oil body composite emulsion is stored for 15 days at room temperature, but the flocculation phenomenon difference of the composite emulsion is not obvious compared with the control group at pH 6 and pH 8.

2.3.4 influence of pH on peroxide value of laccase-crosslinked beet pectin-soybean oil body composite emulsion

The effect of pH on the peroxide value of laccase cross-linked beet pectin-soybean oil body composite emulsion is shown in FIG. 14. The pH 7 condition was used as a control. From the graph, the peroxide value of the laccase cross-linked beet pectin-soybean oil body composite emulsion tends to be increased and then decreased in 0-15 d.

As is clear from FIG. A, the pH 2.5, pH 6, pH 8 and the control group reached the maximum values at 11d, respectively 13.32.+ -. 0.30mmol/kg, 8.87.+ -. 0.20mmol/kg, 7.79.+ -. 0.14mmol/kg and 7.25.+ -. 0.22mmol/kg.

As seen from FIG. B, the pH 2.5, pH 6 and the control group reached the maximum values at 9d, respectively, of 16.45.+ -. 0.40mmol/kg, 12.85.+ -. 0.17mmol/kg and 11.67.+ -. 0.36mmol/kg. The HPSOB composite emulsion at pH 8 reaches the maximum value of 10.74+/-0.35 mmol/kg at 11 d.

In the above, the pH of the laccase cross-linked beet pectin-soybean oil body composite emulsion is adjusted to 6-8, which is more beneficial to the stability and preservation of the emulsion.

2.4 Effect of laccase Cross-Linked sugar beet pectin on Soybean oil body simulation in vitro digestion Properties

2.4.1 Effect of laccase crosslinked sugar beet pectin on Soy oil body simulation on zeta potential after in vitro digestion

The effect of laccase cross-linked beet pectin on zeta potential of soybean oil bodies after simulated in vitro digestion is shown in fig. 15, and graph a shows zeta potential of oil body composite emulsion after simulated gastric juice digestion. As can be seen from the graph, the HOSOB and HPSOB composite emulsions without beet pectin are positively charged on the surfaces after gastric digestion, the zeta-potentials are respectively 12.20+/-0.19 mV and 9.36+/-0.26 mV, and the zeta-potentials of the HOSOB and HPSOB composite emulsions after the beet pectin is added are respectively positive to negative, are respectively-20.02+/-0.45 mV and-15.91+/-0.16 mV, and are remarkably higher than those of the HOSOB and HPSOB composite emulsions without the beet pectin (P < 0.05). The zeta-potential of laccase cross-linked beet pectin HOSOB and HPSOB composite emulsion is-25.76+ -0.19 mV and-19.40+ -0.17 mV, respectively, which is significantly higher than that of beet pectin alone (P < 0.05).

Panel B shows the zeta potential of the simulated intestinal fluid after digestion of the fat mass. As can be seen from the graph, the zeta-potentials of the HOSOB and HPSOB composite emulsions without added beet pectin after intestinal digestion were-8.27.+ -. 0.33mV and-6.43.+ -. 0.11mV, respectively, and the zeta-potentials of the HOSOB and HPSOB composite emulsions after added beet pectin were-11.33.+ -. 0.46mV and-10.41.+ -. 0.39mV, respectively, which were significantly higher than those without added (P < 0.05). The zeta-potential of laccase cross-linked beet pectin HOSOB and HPSOB composite emulsion is-14.36+ -0.44 mV and-12.69+ -0.33 mV, respectively, which is significantly higher than that of beet pectin alone (P < 0.05).

2.4.2 Effect of laccase Cross-Linked sugar beet pectin on Soybean oil body simulation in vitro particle size after digestion

The effect of laccase cross-linked beet pectin on particle size of soybean oil body after simulated in vitro digestion is shown in fig. 16, and fig. a shows particle size of oil body composite emulsion after simulated gastric juice digestion. As is clear from the graph, the particle sizes of the HOSOB and HPSOB composite emulsions without beet pectin after gastric digestion were 0.46.+ -. 0.05. Mu.m, and 0.63.+ -. 0.01. Mu.m, respectively. The particle size of HOSOB and HPSOB composite emulsion after adding beet pectin is 0.65+ -0.03 μm and 0.83+ -0.03 μm respectively, which is significantly higher than that of the non-added (P < 0.05), and the particle size of laccase cross-linked beet pectin HOSOB and HPSOB composite emulsion is 0.83+ -0.01 μm and 0.97+ -0.04 μm respectively, which is significantly higher than that of the added beet pectin alone (P < 0.05).

Panel B shows the particle size of the fat body composite emulsion after simulated intestinal fluid digestion. As is clear from the graph, the particle sizes of the HOSOB and HPSOB composite emulsions without beet pectin after intestinal digestion were 0.11.+ -. 0.00. Mu.m, and 0.15.+ -. 0.00. Mu.m, respectively. The particle size of HOSOB and HPSOB composite emulsion after adding beet pectin is 0.21+ -0.01 μm and 0.24+ -0.01 μm respectively, which is significantly higher than that of non-added (P < 0.05), and the particle size of laccase cross-linked beet pectin HOSOB and HPSOB composite emulsion is 0.31+ -0.01 μm and 0.33+ -0.00 μm respectively, which is significantly higher than that of beet pectin only added (P < 0.05).

2.4.3 laccase Cross-Linked beet pectin on Soy oil body simulation microstructure after in vitro digestion

The microstructure of laccase cross-linked beet pectin after in vitro digestion of soybean oil body is shown in fig. 17 and 18. Fig. 17 shows an ultra-high resolution microscopy image of a soybean oil-liposome composite emulsion after gastric digestion, wherein red represents the liposome surface proteins and green the lipids in the droplets. As can be seen from the graph, compared with the fat body without added beet pectin, the beet pectin HOSOB composite emulsion and the beet pectin HPSOB composite emulsion can retain more liquid drops after being digested by simulated stomach for 1 hour; compared with the grease body without laccase, the laccase cross-linked beet pectin HOSOB composite emulsion and the laccase cross-linked beet pectin HPSOB composite emulsion retain more liquid drops, and most lipid liquid drops are still wrapped in protein, so that the structure is not destroyed. Wherein, the liquid drops of the laccase cross-linked beet pectin HOSOB composite emulsion are denser than the liquid drops of the laccase cross-linked beet pectin HPSOB composite emulsion. In conclusion, laccase cross-linked beet pectin-soybean oil bodies can better protect the oil bodies in the stomach digestion process.

Fig. 18 shows an ultra-high resolution microscopy image of a soybean oil-liposome composite emulsion after intestinal digestion, wherein red represents the liposome surface proteins and green the lipids in the droplets. From the graph, the number of grease body fluid drops is obviously reduced after the HOSOB composite emulsion and the HPSOB composite emulsion are digested for 2 hours in the intestinal tract. The laccase cross-linked beet pectin HOSOB complex emulsion and the laccase cross-linked beet pectin HPSOB complex emulsion retain more droplets than the lipid body without laccase added, which is similar to the gastric digestion results.

2.4.4 Effect of laccase-crosslinked sugar beet pectin on the in vitro digestion of Soy oil body-mimicking the free fatty acid Release

The effect of laccase cross-linked beet pectin on the release of free fatty acids after in vitro digestion of soybean oil body is shown in figure 19. From the graph, the release rate of the free fatty acid is faster in 0-40min and then gradually slows down in the simulated intestinal digestion process, and the release amount of the free fatty acid is smooth in 80-120 min.

From panel a, it can be seen that after simulated intestinal digestion for 120min, the release of free fatty acids from the HOSOB complex emulsion with added beet pectin was 22.82±0.48% significantly lower than that without added beet pectin (24.64±0.51%) (P < 0.05). The free fatty acid release amount of the HOSOB composite emulsion of laccase cross-linked beet pectin is 21.03+/-0.35%, which is significantly lower than that of laccase-free (P < 0.05).

From panel B, it can be seen that after simulated intestinal digestion for 120min, the release of free fatty acids from the HPSOB complex emulsion with added beet pectin was 20.69±0.50%, significantly lower than that without added beet pectin (22.24±0.53%) (P < 0.05). The free fatty acid release of HPSOB composite emulsion of laccase cross-linked beet pectin is 19.34+/-0.43%, which is significantly lower than that of laccase-free (P < 0.05).

Conclusion 3

After digestion by simulated gastric fluid, the pH of the emulsion is lower than the isoelectric point of protein (pH is about 4.3), so that the emulsion contains a large amount of H ⁺ The negative charge on the surface of the grease body is neutralized, and the zeta potential of the emulsion is changed from negative to positive. Because beet pectin belongs to anionic polysaccharide, the zeta-potential of emulsion after covalent crosslinking with the surface protein of the oil body still presents a negative value, and the zeta-potential (absolute value) of the soybean oil body composite emulsion after laccase is added is higher. After simulated intestinal juice digestion, the zeta potential of all samples was negative and the zeta potential (absolute value) of the fat body emulsion with added beet pectin was significantly higher than that of the fat body emulsion without added (P<0.05 The zeta potential (absolute value) of the added laccase beet pectin lipid body emulsion is significantly higher than that of the added laccase (P<0.05). The zeta potential (absolute value) of all samples was reduced compared to that before digestion. This is probably due to the fact that under strong acid conditions, milk is caused by pH change, enzymatic hydrolysis, etcThe liquid environment is abnormal, and the stability of the emulsion system is reduced.

Compared with the fat body composite emulsion without adding the beet pectin, the grain size of the fat body composite emulsion with the beet pectin is obviously increased (P < 0.05), and the grain size of the enzyme cross-linked beet pectin-soybean fat body composite emulsion prepared by the invention is obviously higher than that of the fat body composite emulsion without adding laccase (P < 0.05). This may be because beet pectin covalently crosslinks the surface proteins of the fat body to form a bilayer or multilayer protective coating that prevents pepsin from contacting the enzyme sites on the fat body surface. However, the surface protein of the grease without sugar beet pectin is hydrolyzed into small molecular polypeptide by pepsin, so that the network structure of the emulsion is damaged, and the emulsion is easily flocculated. After digestion, the particle size of all samples is further reduced because lipase enzymes hydrolyze triacylglycerides in the soybean oil bodies to form monoglycerides and free fatty acids, and the oil bodies with large particle sizes are more easily decomposed, resulting in smaller particle sizes. The fat body emulsion with the sugar beet pectin protective coating has a higher particle size than the non-added one, because the coating protects part of the fat body from hydrolysis.

Compared with the soybean oil body emulsion without the beet pectin, the soybean oil body emulsion with the beet pectin retains more liquid drops after being digested by stomach; the added laccase beet pectin grease body emulsion has more liquid drops than the added laccase, most of proteins still wrap the lipid, and the grease body structure is not destroyed. After intestinal digestion, the liquid drops can be further reduced, because various enzymes in intestinal juice can directly act on the lipid after the protein on the surface of the lipid is hydrolyzed; in addition, as the surface proteins of the grease body are destroyed, bile salts and other mineral ions in intestinal fluid can cause aggregation between oil drops, and the distribution of green spots can be seen in the figure. More red protein signals can be seen by the grease body added with laccase beet pectin, which indicates that the interaction of beet pectin and grease surface protein influences the digestion of protein, and the emulsion in the mode has good hydrolysis resistance stability.

Free fatty acid release rate is reduced when intestinal juice is digested, and contact of lipase and lipid is limited because released fatty acid can be accumulated on the surface of the grease body. Compared with the fat body emulsion without the beet pectin, the fatty acid release rate of the fat body emulsion with the beet pectin is obviously reduced, which is caused by that the beet pectin forms a double-layer or multi-layer protective coating on the surface of the liquid drops, the combination of pepsin and lipase with protein and lipid is reduced, and the release of fatty acid is slowed down. In addition, beet pectin can be combined with calcium ions in the emulsion to prevent the calcium ions from removing long-chain fatty acids accumulated on the surface of the grease body, so that the release of fatty acids is reduced. The reduced fat digestion rate may affect the physiological function of the gastrointestinal tract and may produce a feeling of satiety. Therefore, the laccase cross-linked beet pectin soybean oil lipid composite emulsion has wide application value.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. An application of laccase cross-linked beet pectin in reducing the free fatty acid release amount of soybean oil body composite emulsion is characterized in that,

the soybean oil body composite emulsion is laccase cross-linked beet pectin-soybean oil body composite emulsion, and the preparation method comprises the following steps:

(1) Extracting soybean oil: soaking soybean seeds in water, adding NaCl mixed solution into the soaked seeds, grinding, filtering and centrifuging, collecting upper emulsion, dispersing the upper emulsion in buffer solution, centrifuging, and collecting upper oil emulsion; heating the soybean oil body for 20min in a water bath kettle with the temperature of 80 ℃ to obtain the soybean oil body; the soybean oil body is a high-protein soybean oil body, and the protein content is 8.99+/-0.02%;

(2) Preparing beet pectin solution with the concentration of 0.075wt%, adding the soybean oil body, adding laccase, stirring at high speed to prepare emulsion, standing, and regulating the pH of the emulsion to 6-8 to obtain laccase crosslinked beet pectin-soybean oil body composite emulsion; before adding the laccase, adjusting the pH to 4.5; the addition amount of laccase is 0.4-0.5U;

The free fatty acid release amount is the free fatty acid release amount of the soybean oil body composite emulsion after gastric juice and intestinal juice digestion simulation.

2. The use according to claim 1, wherein in step (1) the volume ratio of soybean seeds to water is 1:5; the soaking temperature is 4-5 ℃ and the soaking time is 18-22h.

3. The use according to claim 1, wherein in step (1) the NaCl mixed solution consists of 50mmol/L Tris-HCl, 0.4mol/L sucrose and 0.5mol/L NaCl; the mass volume ratio of the soaked seeds to the NaCl mixed solution is 1:5.