CN115708561B - Functional corn flour and efficient preparation method and application thereof - Google Patents

Abstract

A functional corn flour and its high-efficiency preparation method and application are provided. The invention belongs to the field of functional food processing. The invention aims to solve the technical problems of low efficiency and serious loss of active ingredients in the existing wet pulverizing process. The method comprises the following steps: firstly, adding water into corn kernels to carry out microwave cooking; then carrying out enzymolysis on cellulase; adding alkaline protease and quercetin, and soaking under ultrasonic assistance; then dissolving procyanidine in water, spraying the procyanidine on the surface of soaked corn, and finally vacuum drying to obtain the functional corn powder. The corn flour is prepared by reasonably combining and linking through a plurality of technologies, so that the corn flour milling efficiency is effectively improved, the prepared corn flour is rich in two polyphenol bioactive components of quercetin and procyanidine, has high resistant starch content and antioxidant activity, the optimized process also improves the chemical stability and biological accessibility of the active components, and meanwhile, the corn flour has good gelatinization and anti-aging characteristics.

Description

Functional corn flour and efficient preparation method and application thereof

Technical Field

The invention belongs to the field of functional food processing, and specifically relates to functional corn flour and an efficient preparation method and application thereof.

Background technique

With the diversification of people's dietary structure and the increasing number of hypertension, obesity, cardiovascular diseases, etc. caused by the unreasonable dietary structure such as the refinement of staple food and large-scale meat consumption, the demand for coarse grains such as corn is becoming more and more widespread. Corn is the world's largest food crop and is widely planted all over the world. In 2021, my country's annual corn production reached 270 million tons, making it the world's second largest corn producer. Corn has high nutritional value and medicinal value. It contains about 70% starch and 8%-14% protein. It is rich in nutrients such as vitamins, carotenoids and dietary fiber. It has the effects of beauty, eyesight, prevention of coronary heart disease and hypertension. The protein matrix in corn is a large and complex protein molecule formed by the combination of different protein subunits in gluten through disulfide bonds. Alcohol-soluble proteins exist in globules in the gluten matrix, and starch granules are embedded in the gluten matrix. Due to the unique combination of starch and protein in corn flour, starch gelatinization is limited during the food cooking process, which affects the processing and edible quality of corn food and is not conducive to the development and utilization of corn as a food raw material.

In the development of corn food products, grinding and milling is the basis for deep processing of corn kernels for food, and it is also a crucial pre-processing link. At present, there are three main methods for corn milling: dry method, semi-dry method and wet method. Different processing methods have an important influence on the processing effect. Corn flour prepared by dry method has good water absorption characteristics, but the processing quality is poor, the taste is rough, and it is easy to age; corn flour prepared by wet method has less damaged starch, better processing characteristics and edible quality; and the quality of corn flour prepared by semi-dry method is between dry method and wet method. However, the processing time required for wet milling of corn is long, usually requiring soaking for about 36-48 hours, which seriously reduces the milling efficiency and increases the production cost.

At present, the corn soaking process includes fermentation, enzyme and ultrasound. The fermentation soaking process conditions and process are relatively complex and difficult to control in production, so this method is not widely used in the corn flour production industry. The enzyme soaking process shortens the soaking time, reduces environmental pollution and improves production efficiency. The proteases used at home and abroad mainly include acid proteases, neutral proteases, alkaline proteases, bromelain and papain. In addition, in order to break the cell wall structure in corn kernels, wall-breaking enzymes such as cellulase, pectinase and xylanase are also used. The soaking methods include mixed soaking of acid and enzyme, step-by-step soaking of wall-breaking enzymes and proteases, simultaneous soaking after compounding of wall-breaking enzymes and proteases, and simultaneous soaking after compounding of several proteases. Ultrasonic-assisted technology has become a conventional technology for producing green, economical alternative foods and natural products. Ultrasonic cavitation shortens the soaking time and separates the protein from the starch in the corn, resulting in sufficient gelatinization of the starch during the processing of the corn flour and the formation of a good network structure of the protein, thereby improving the processing characteristics of the corn flour. However, during the wet soaking process, some bioactive ingredients in the corn will be lost with the soaking water, reducing the functional properties of the corn flour.

Summary of the invention

The purpose of the present invention is to solve the technical problems of low efficiency and serious loss of active ingredients in the existing wet milling process, and to provide a functional corn flour and an efficient preparation method and application thereof.

One of the purposes of the present invention is to provide an efficient preparation method of functional corn flour, which is carried out according to the following steps:

Step 1: adding water to corn kernels for microwave cooking, and then grinding at a low speed to obtain a mixed liquid;

Step 2: adjusting the pH value of the mixed solution to 5, and then adding cellulase for enzymolysis to obtain an enzymolysis solution;

Step 3: adjusting the pH value of the enzymatic hydrolysate to 9, then adding alkaline protease and quercetin, soaking under the assistance of ultrasound, and then filtering and draining to obtain modified corn particles;

Step 4: dissolve proanthocyanidins in water to obtain a proanthocyanidin solution, which is then sprayed on the surface of the modified corn particles and mechanically mixed. When the water content of the modified corn particles reaches 35-40%, the pH value is adjusted to neutral, and then high-speed grinding is performed. After vacuum drying, functional corn flour is obtained.

It is further defined that the material-liquid ratio in step 1 is 1:3.

It is further defined that the power of the microwave cooking in step 1 is 400-600 W and the time is 6-10 min.

It is further defined that the rotation speed of the low-speed grinding in step 1 is 2500-3000 rpm.

It is further defined that the amount of cellulase added in step 2 is 0.5-1.5% of the mass of the mixed solution.

It is further defined that the temperature of the enzymatic hydrolysis in step 2 is 40-60°C and the time is 50-70 minutes.

It is further defined that the amount of alkaline protease added in step 3 is 1-3% of the mass of the enzymatic solution.

It is further defined that the concentration of quercetin in the enzymatic hydrolysate of step 3 is 2-4 mg/mL.

It is further defined that the ultrasonic power in step 3 is 100-300W.

It is further defined that the soaking temperature in step 3 is 50-70° C. and the soaking time is 3-5 hours.

It is further defined that the filtrate obtained by filtering after soaking in step 3 is refluxed into the enzymatic hydrolyzate for recycling.

It is further defined that the concentration of the proanthocyanidin solution in step 4 is 4-6 mg/mL.

It is further defined that the rotation speed of the high-speed grinding in step 4 is 16000-17000 rpm.

It is further defined that the vacuum degree of vacuum drying in step 4 is 0.04-0.08 MPa and the temperature is 60-70°C.

The second object of the present invention is to provide a functional corn flour prepared by the above method.

The third object of the present invention is to provide an application of the functional corn flour prepared by the above method, which is used for preparing functional corn food.

Compared with the prior art, the present invention has the following significant effects:

The present invention prepares corn flour by rationally combining and connecting multiple technologies. The prepared corn flour is not only rich in two polyphenolic bioactive ingredients, quercetin and proanthocyanidins, but also has an optimized preparation process that improves the chemical stability and bioaccessibility of the active ingredients. The corn flour has good gelatinization and anti-aging properties. The specific advantages are as follows:

1) The present invention prepares corn flour by rationally combining and connecting a plurality of technologies. First, corn kernels are subjected to microwave cooking to enable them to quickly absorb water, resulting in the softening and partial destruction of the corn cortex, pre-gelatinization of starch, and partial denaturation of protein, which is beneficial to the subsequent enzymatic hydrolysis of cellulase, so that the fiber in the corn cortex is further degraded, and part of the insoluble dietary fiber is converted into soluble dietary fiber, which is beneficial to the subsequent soaking treatment.

2) The present invention adjusts the pH of the enzymatic solution to alkaline, adds alkaline protease and quercetin in sequence, and then performs protease wet soaking with the assistance of ultrasonic cavitation. Ultrasonic cavitation effectively interrupts the force between starch and protein, destroys the starch-protein composite structure, greatly promotes the separation of starch and protein, shortens the soaking time, and improves the soaking efficiency. In addition, quercetin is added under alkaline conditions to promote it to form non-covalent and covalent complexes with starch and protein in corn respectively through interaction, which greatly improves the functionality of corn flour and can provide raw material source guarantee for the development of new functional corn food. And the soaking liquid can also be recycled for the next batch of corn soaking, which not only saves the consumption of water and enzymes, saves the amount of protease and quercetin, but also reduces the loss of nutrients in corn, and ensures the corn soaking efficiency and soaking effect. Greatly reduces the cost, and realizes green and environmentally friendly sustainable production.

3) The present invention uses an aqueous solution of proanthocyanidins to adjust the moisture of the drained modified corn particles, and then adopts a semi-dry method to prepare flour, thereby avoiding the shortcomings of the dry method, such as poor quality of flour preparation and long time consumption of wet method, and ensures good processing quality of corn flour while efficiently preparing flour. The vacuum drying is then used to promote the formation of starch-polyphenol complexes, thereby increasing the content of resistant starch.

4) The method of the present invention shortens the soaking time of the traditional wet milling process from 36 hours to 5-6 hours, significantly improves the efficiency of corn milling, and has the characteristics of being green and safe, short production time and high product added value. The prepared corn flour is rich in two polyphenolic bioactive ingredients, quercetin and proanthocyanidins, and the resistant starch content and antioxidant activity of the corn flour are increased. The complex formed by the starch and protein in the corn and the quercetin and proanthocyanidins improves the chemical stability and biological accessibility of the active ingredients. At the same time, the corn flour has good gelatinization and anti-aging properties, and can be used in the research and development of functional corn foods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a bar graph showing the starch-polyphenol composite rate in the functional corn flours prepared in Examples 1-3 and Comparative Examples 1-3;

FIG2 is an infrared spectra of starch-polyphenol complexes in functional corn flours prepared in Examples 1-3 and Comparative Examples 1-3;

FIG3 is a bar graph showing the free amino and free thiol contents of the protein-polyphenol complex in the functional corn flours prepared in Examples 1-3 and Comparative Examples 1-3;

FIG4 is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis diagram of the protein-polyphenol complex in the functional corn flour prepared in Examples 1-3 and Comparative Examples 1-3;

FIG5 is a bar graph showing the resistant starch content and antioxidant activity of the functional corn flours obtained in Examples 1-3 and Comparative Examples 1-3 after digestion;

FIG6 is a bar graph showing the bioaccessibility of active ingredients in functional corn flours obtained in Examples 1-3 and Comparative Examples 1-3 after digestion;

FIG7 is a bar graph showing the chemical stability of active ingredients in the functional corn flours prepared in Examples 1-3 and Comparative Examples 1-3 under heat treatment at 65° C.;

FIG8 is a bar graph showing the chemical stability of active ingredients in the functional corn flours prepared in Examples 1-3 and Comparative Examples 1-3 under heat treatment at 85° C.;

FIG9 is a bar graph showing the chemical stability of the active ingredients in the functional corn flours prepared in Examples 1-3 and Comparative Examples 1-3 under heat treatment conditions at 100° C.

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained through commercial channels by those skilled in the art.

The terms "comprising," "including," "having," "containing," or any other variation thereof, as used in the following examples, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

When equivalent, concentration or other value or parameter is represented by the range limited by range, preferred range or a series of upper preferred value and lower preferred value, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether the scope is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the scope is intended to include its end value and all integers and fractions within the scope. In the present application specification and claims, range limitation can be combined and/or interchanged, if these ranges are not otherwise stated, include all sub-ranges contained therein.

The indefinite articles "a" and "an" before the elements or components of the present invention have no limitation on the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the quantity obviously refers to the singular form only.

Example 1: The efficient preparation method of a functional corn flour of this example is carried out according to the following steps:

Step 1: corn kernels were added with water (solid-liquid ratio was 1:3) and microwaved at 500 W for 8 min, and then ground at 2800 rpm for 20 s to obtain a mixed solution;

Step 2: adjusting the pH value of the mixed solution to 5, then adding 1% of the mass of the mixed solution of cellulase, and performing enzymolysis at 50° C. for 60 minutes to obtain an enzymolysis solution;

Step 3: The pH value of the enzymatic hydrolysate is adjusted to 9, and then alkaline protease and quercetin accounting for 2% of the mass of the enzymatic hydrolysate are added, and the concentration of quercetin in the enzymatic hydrolysate is 3 mg/mL. The enzymatic hydrolysate is soaked at 60° C. for 4 hours under 200W ultrasonic assistance, and then filtered. The filtrate is refluxed into the enzymatic hydrolysate for recycling, and the precipitate is drained to obtain modified corn particles;

Step 4: dissolve proanthocyanidins in water to obtain a proanthocyanidin solution with a concentration of 5 mg/mL, spray it on the surface of the modified corn particles, and perform mechanical mixing. When the water content of the modified corn particles reaches 38%, adjust the pH value to neutral, and then perform high-speed grinding at 16800 rpm. Then, vacuum dry the wet corn flour under the conditions of a vacuum degree of 0.06 MPa and a temperature of 65°C. After drying, pass through a 100-mesh sieve to obtain functional corn flour. After measurement, the quercetin content in the obtained corn flour is 9 mg/g, and the proanthocyanidin content is 5 mg/g.

Example 2: The efficient preparation method of a functional corn flour of this example is carried out according to the following steps:

Step 1: Add water to corn kernels (solid-liquid ratio is 1:3) and microwave cook for 9 min at 450 W, then grind at 2800 rpm for 20 s to obtain a mixed solution;

Step 2: adjusting the pH value of the mixed solution to 5, then adding 0.5% of cellulase by weight of the mixed solution, and performing enzymolysis at 55° C. for 65 minutes to obtain an enzymolysis solution;

Step 3: The pH value of the enzymatic hydrolysate is adjusted to 9, and then alkaline protease and quercetin accounting for 1.5% of the mass of the enzymatic hydrolysate are added, and the concentration of quercetin in the enzymatic hydrolysate is 2.5 mg/mL. The enzymatic hydrolysate is soaked at 65° C. for 3.5 hours under 250W ultrasonic assistance, and then filtered. The filtrate is refluxed into the enzymatic hydrolysate for recycling, and the precipitate is drained to obtain modified corn particles;

Step 4: dissolve proanthocyanidins in water to obtain a proanthocyanidin solution with a concentration of 5.5 mg/mL, spray it on the surface of the modified corn particles, and perform mechanical mixing. When the water content of the modified corn particles reaches 38%, adjust the pH value to neutral, and then perform high-speed grinding at 16800 rpm. Then, vacuum dry the wet corn flour under the conditions of a vacuum degree of 0.05 MPa and a temperature of 70°C. After drying, pass through a 100-mesh sieve to obtain functional corn flour. After measurement, the quercetin content in the obtained corn flour is 7.2 mg/g, and the proanthocyanidin content is 5.5 mg/g.

Example 3: The efficient preparation method of a functional corn flour of this example is carried out according to the following steps:

Step 1: Add water to corn kernels (solid-liquid ratio is 1:3) and microwave cook for 7 min at 550 W, then grind at 2800 rpm for 20 s to obtain a mixed solution;

Step 2: adjusting the pH value of the mixed solution to 5, then adding 1.5% of cellulase by weight of the mixed solution, and performing enzymolysis at 50° C. for 55 minutes to obtain an enzymolysis solution;

Step 3: The pH value of the enzymatic hydrolysate is adjusted to 9, and then alkaline protease and quercetin accounting for 2.5% of the mass of the enzymatic hydrolysate are added, and the concentration of quercetin in the enzymatic hydrolysate is 3.5 mg/mL. The enzymatic hydrolysate is soaked at 55° C. for 4.5 hours under 150W ultrasonic assistance, and then filtered. The filtrate is refluxed into the enzymatic hydrolysate for recycling, and the precipitate is drained to obtain modified corn particles;

Step 4: dissolve proanthocyanidins in water to obtain a proanthocyanidin solution with a concentration of 4.5 mg/mL, spray it on the surface of the modified corn particles, and perform mechanical mixing. When the water content of the modified corn particles reaches 38%, adjust the pH value to neutral, and then perform high-speed grinding at 16800 rpm. Then, vacuum dry the wet corn flour under the conditions of a vacuum degree of 0.07 MPa and a temperature of 60°C. After drying, pass through a 100-mesh sieve to obtain functional corn flour. After measurement, the quercetin content in the obtained corn flour is 8.4 mg/g, and the proanthocyanidin content is 4.5 mg/g.

Comparative Example 1: Corn flour was prepared by dry method. The specific process was as follows: corn grits were prepared by peeling and degerming the screened corn kernels using an efficient peeling and milling machine, quercetin and proanthocyanidins were added, and then the corn flour was crushed in a high-speed universal grinder to obtain corn flour and passed through a 100-mesh sieve. The final contents of quercetin and proanthocyanidins in the dry corn flour were 9 mg/g and 5 mg/g, respectively.

Comparative Example 2: Corn flour was prepared by semi-dry method. The specific process is as follows: corn grits were prepared by peeling and degerming the screened corn kernels using an efficient peeling and milling machine, distilled water containing quercetin and proanthocyanidins was added to the corn grits, the moisture content of the corn grits was adjusted to 38%, and then the corn grits were crushed in a high-speed universal grinder to obtain corn flour, which was placed in a drying oven and dried at 40°C and passed through a 100-mesh sieve. The final contents of quercetin and proanthocyanidins in the semi-dry corn flour were 9 mg/g and 5 mg/g, respectively.

Comparative Example 3: Corn flour was prepared by wet method. The specific process was as follows: the screened corn kernels were placed in a beaker and distilled water was added, the mass ratio of corn to distilled water was 1:3, then quercetin and proanthocyanidins were added, soaked for 36 hours and then ground in a colloid mill, the corn slurry obtained after grinding was filtered to remove most of the water to obtain wet corn flour, placed in a drying oven and dried at 40°C and passed through a 100 mesh sieve, the final contents of quercetin and proanthocyanidins in the wet corn flour were 9 mg/g and 5 mg/g, respectively.

Detection test:

(I) Test methods

1. Determination of corn starch-polyphenol complex rate

Corn flour with added polyphenols was mixed with water at a solid-liquid ratio of 1:10, and then 1% neutral protease was added for enzymatic hydrolysis for 1h to remove protein, and the starch precipitate was taken after centrifugation. The starch precipitate was then prepared into a 10% dispersion, heated at 95°C for 10min to gelatinize it, 1mL of gelatinized sample was added to 5mL of distilled water, vortexed for 2min and centrifuged at 4000rpm for 15min. 500μL of supernatant was added to 15mL of distilled water and 2mL of iodine solution, and the test tube was flipped about 10 times. The absorbance of the solution was measured at 620nm using a spectrophotometer. The absorbance of corn flour without polyphenols was used as a blank sample. The starch-polyphenol composite rate was calculated by the following formula:

Where: CI is the recombination rate; _A0 is the absorbance of corn flour without polyphenols added; _A1 is the absorbance of corn flour with polyphenols added.

2. Infrared spectroscopic determination of corn starch-polyphenol complex

The structure and functional group changes of starch-polyphenol complex in corn flour were determined and analyzed by infrared spectrometer. Functional corn flour was mixed with water at a solid-liquid ratio of 1:10, and then 1% neutral protease was added for enzymatic hydrolysis for 1 hour to remove protein. After centrifugation, the starch precipitate was taken and freeze-dried. The freeze-dried sample was mixed with potassium bromide and ground, and pressed into small discs. The wave number scanning range was 500-4000cm ^-1 , the resolution during the scanning process was 4cm ^-1 , and air was used as the background for scanning, and finally the infrared spectrum curve was obtained.

3. Determination of free amino and free thiol groups of zein-polyphenol complex

Functional corn flour was mixed with water at a solid-liquid ratio of 1:10, and then 1% α-amylase was added for enzymatic hydrolysis for 2 h to remove starch. After centrifugation, the protein precipitate was obtained and freeze-dried, and the free amino group and free thiol group of the freeze-dried sample were determined.

The free amino group content was determined by the o-phthalaldehyde method (OPA). 80 mg of OPA was dissolved in 2 mL of 95% ethanol and mixed with 50 mL of 10 mM sodium tetraborate buffer (pH 9.7), 5 mL of 20% (w/w) SDS and 200 μL of β-mercaptoethanol. After thorough mixing, the mixture was diluted to 100 mL with distilled water to prepare the OPA reagent. 200 μL of the sample solution (2 mg/mL) was reacted with 4 mL of the OPA reagent at room temperature for 5 min, and then the absorbance at 340 nm (A ₃₄₀ ) was determined by a _UV -visible spectrophotometer. Distilled water was used as a blank sample to analyze the free amino group content.

The free thiol content was determined by Ellman's reagent. A DTNB solution with a mass concentration of 4 mg/mL was prepared with Tris-Gly buffer (0.086 mol/LTris, 0.09 mol/L glycine, 0.04 mol/L EDTA, 8 mol/L urea, pH 8), namely Ellman's reagent. 15 mg of sample was dissolved in 5 mL of Tris-glycine buffer, 50 μL of Ellman's reagent was added, and the mixture was incubated at 25°C for 1 hour, centrifuged at 5,000 × g for 10 minutes, and the absorbance of the supernatant was measured at 412 nm (A ₄₁₂ ). An equal amount of distilled water was used instead of the sample as a blank control, and the free thiol content was analyzed according to A ₄₁₂ .

4. Determination of Zein-Polyphenol Complex by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Functional corn flour was mixed with water at a ratio of 1:10, and then 1% α-amylase was added for enzymatic hydrolysis for 2 hours to remove starch. After centrifugation, the protein precipitate was taken and freeze-dried. The concentrations of separation gel and concentration gel were 12% and 4%, respectively, and the sample protein concentration was 2 mg/mL. The sample was dissolved in a buffer (pH 6.80.5M Tris-HCl and glycerol, SDS, β-mercaptoethanol and bromophenol), then heated at 95°C for 5 minutes, and the sample volume was 15uL. After the electrophoresis, the protein was stained with Coomassie Brilliant Blue R250. The SDS-PAGE gel electrophoresis was analyzed using Image Lab Software 3.0.

5. In vitro digestion experiment

A 500 mg corn flour sample was soaked in 50 mL of simulated gastric fluid (2 g NaCl and 7 mL 37% HCl dissolved in 1000 mL distilled water, adjusted to pH 1.2) containing 0.1% pepsin. The mixture was then shaken at 100 rpm at 37°C for 2 h to simulate gastric digestion. Subsequently, the gastric digested sample was transferred to a simulated intestinal fluid (6.8 g KH ₂ PO ₄ dissolved in 250 mL distilled water, mixed with 190 mL 0.2 M NaOH, fixed to 1000 mL with distilled water, adjusted to pH 7.5) containing 1% pancreatin, 0.5% amyloglucosidase, and 0.5% bile salts, and shaken for another 4 h to simulate intestinal digestion under the same conditions. The sample was placed in an ice bath for 30 min to terminate the digestion reaction.

6. Determination of resistant starch content

After starch digestion, it is divided into three categories according to its bioavailability: rapidly digestible starch (RDS) refers to starch that can be quickly digested and absorbed in the small intestine within 20 minutes; slowly digestible starch (SDS) refers to starch that can be completely absorbed in the small intestine within 20-120 minutes but the absorption rate is relatively slow; resistant starch (RS) refers to starch that is not digested and absorbed by the human small intestine within 120 minutes. The content of glucose (FG) is determined by the DNS method. The calculation formula is as follows:

Wherein: FG is the amount of free glucose before enzymatic hydrolysis (mg); _G20 is the glucose content after 20 minutes of enzymatic hydrolysis (mg); _G120 is the glucose content after 120 minutes of enzymatic hydrolysis (mg); TS is the total starch content of the system (mg).

7. In vitro Antioxidant Activity Assay

The antioxidant activity of corn flour samples after digestion was evaluated by DPPH free radical scavenging ability. Samples were collected after simulated gastrointestinal digestion, and 1.0 mL of 0.5 mg/mL sample was mixed with 2.0 mL of 0.2 mM DPPH-ethanol solution. The mixture was reacted in the dark for 30 minutes, and the absorbance was measured at 517 nm after the reaction. The DPPH distilled water solution was used as the blank. The DPPH free radical scavenging ability was calculated as follows:

8. Determination of bioaccessibility of active ingredients

After the simulated gastrointestinal digestion of corn flour samples was completed, they were centrifuged at 4000g for 40min at 4°C. The collected supernatant represents the "mixed micelle" part of the intestinal fluid, which contains bioavailable active ingredients. Quercetin and proanthocyanidins in the mixed micelles were extracted with anhydrous ethanol, vortexed and centrifuged at 4000g for 20min. The absorbance of the supernatant was then measured at wavelengths of 373nm and 546nm using an ultraviolet spectrophotometer, and the contents of quercetin and proanthocyanidins were obtained by substituting into the standard curve for calculating the bioaccessibility of the active ingredients after simulated gastrointestinal digestion. Bioaccessibility is expressed as the ratio of the content of the active ingredient in the micelle to the content of the active ingredient in the sample before simulated digestion.

9. Determination of chemical stability of active ingredients

The functional corn flour samples were placed in a water bath at 65, 85 and 100°C for 2 hours, and then the contents of quercetin and proanthocyanidins in the corn flour after heat treatment at different temperatures were measured. The retention rate of the active ingredient was expressed as the ratio of the active ingredient content in the sample after heat treatment to the initial active ingredient content in the sample before heat treatment.

10. Determination of corn flour gelatinization characteristics

3.5g corn flour sample and 25mL distilled water were added to the aluminum box of the rapid viscosity analyzer (RVA), and the rotating paddle was calibrated first, and then the sample and water were fully stirred with the rotating paddle, and then installed on the RVA tester for testing. The sample was kept at 50℃ for 1min, then heated to 95℃ at a rate of 4℃/min, kept at 95℃ for 5min, and then dropped to 50℃ at the same rate and kept for 5min to form corn paste and analyze the gelatinization characteristics of the sample.

11. Determination of aging characteristics of corn flour

The gelatinized corn flour sample was placed in a small culture dish, sealed with plastic wrap, and refrigerated at 4°C for 7 days to allow the sample to form a gel. The gelled sample was tested for texture. The TPA measurement mode was selected, and a cylindrical metal probe P/0.5 was used. Test conditions: pre-test rate 1.0mm/s, test rate 2.0mm/s, post-test rate 2.0mm/s, test distance 10.0mm, compression degree 40%, interval between two compressions 2s, trigger force 5g, and the aged hardness (g) of the sample was analyzed.

(II) Results and analysis

1. Characterization of corn starch-polyphenol complex

The starch-polyphenol complex rate refers to the degree of complexation between starch and polyphenols. Figure 1 is a bar graph of the starch-polyphenol complex rate in functional corn flour. The starch-polyphenol complex rate in the corn flour of Comparative Example 1 is very low. Since there is no water involved in the dry flour production, the starch and polyphenols cannot be effectively in contact, resulting in a low complex rate. The corn flours of Comparative Examples 2 and 3 gradually increased the starch-polyphenol complex rate, which may be related to the participation of water and the longer reaction time. Compared with the three comparative examples, the corn flours of the three examples significantly increased the starch-polyphenol complex rate, and Example 1 had the largest starch-polyphenol complex rate, which greatly promoted the interaction and combination of starch and polyphenols.

The essence of infrared absorption spectrum formation is rotation-vibration energy level transition, and the absorption peak of the molecule and the intensity and position of the peak can be determined according to the vibration of the molecule. Figure 2 is an infrared spectrum of starch-polyphenol complex in functional corn flour. All samples have a broadband between 3000-3500cm ^-1 , which is caused by the stretching vibration and absorption of the hydrogen bond group (OH). The peak at 2927cm ^-1 is caused by the antisymmetric stretching vibration of _CH2 , and the band at 1649cm ^-1 is attributed to the stretching vibration of COO-. In addition, there are many absorption peaks at 500-1800cm ^-1 , which are mostly caused by some double bond carbon, ester bond and ether bond vibration. All absorption peak intensities of starch-polyphenol complex in corn flour of comparative examples 1-3 are low, while embodiments 1-3 significantly increase the infrared spectrum absorption peak intensity of starch-polyphenol complex, especially the absorption peak of starch-polyphenol complex in embodiment 1 is the strongest. It is worth noting that the absorption peak in the wavelength range of 3000-3500 cm ^-1 becomes wider and the peak intensity is greater, which indicates that starch and polyphenols are non-covalently bound through hydrogen bonds. The starch-polyphenol complex of Example 1 has stronger absorption peaks at wavelengths of 576, 930, 1020 and 1155 cm ^-1 , indicating that starch and polyphenols have stronger interactions, resulting in changes in starch structure. In addition, the complex of starch and polyphenols does not produce new absorption peaks, indicating that no new covalent bonds are formed. Therefore, starch and polyphenols in functional corn flour form non-covalent complexes through non-covalent interactions.

2. Characterization of Zein-Polyphenol Complex

Under aerobic alkaline conditions, polyphenols are easily oxidized to form quinones. When in contact with proteins, they are susceptible to attack by nucleophilic groups (amino and thiol groups, etc.) on the proteins and undergo further covalent complex reactions, which can lead to a reduction in the free amino and free thiol content of the proteins. Figure 3 is a bar graph of the free amino and free thiol content of the protein-polyphenol complex in functional corn flour. The protein-polyphenol complexes in the three comparative corn flours have higher free amino and free thiol groups. Since the wet milling process increases the contact area between corn protein and polyphenols in the water system, the rate of the complex reaction is accelerated, resulting in the corn flour prepared in comparative example 3 significantly reducing the free amino and free thiol groups of the protein-polyphenol complex. Compared with comparative examples 1-3, examples 1-3 further reduce the free amino and free thiol groups of the protein-polyphenol complex. SDS was used to determine the free amino group, which can destroy non-covalent bonds. Therefore, the reduction of free amino groups proves that the protein and polyphenols are covalently bound. When determining the free sulfhydryl group, 8 mol/L urea was used, which can inhibit the conversion of sulfhydryl groups into disulfide bonds. Therefore, the reduction of free sulfhydryl groups also proves that the polyphenols are covalently bound to the free sulfhydryl groups of proteins. The quinone formed by the oxidation of polyphenols can not only react with the free amino group to form a C-N covalent bond, but also react with the sulfhydryl group of cysteine to form a C-S covalent bond. In addition, the protein-polyphenol complex in the corn flour of Example 1 has the lowest free amino group and free sulfhydryl group, indicating that more protein-polyphenol covalent bonds are formed in the corn flour of Example 1.

The covalent binding between protein and polyphenol can be verified by SDS-PAGE. Figure 4 is an SDS-PAGE diagram of the protein-polyphenol complex in functional corn flour. Marker is the standard protein molecular weight, and the moving distance of the protein molecule in electrophoresis is mainly determined by the relative molecular mass of the protein. The protein-polyphenol complex of all sample corn flours shows high molecular weight aggregates at a position greater than 140kDa. Since the loading buffer solution containing SDS and β-mercaptoethanol is used in SDS-PAGE electrophoresis to disconnect the non-covalent interactions and covalent disulfide bonds in the protein-polyphenol complex, the presence of the high molecular weight aggregate band indicates that C-N and/or C-S covalent bonds are formed between the protein and the polyphenol, thereby generating a protein-polyphenol covalent complex. The protein-polyphenol complex in the corn flour of Comparative Examples 1-3 shows a faint high molecular weight band, while the high molecular weight aggregate band of Examples 1-3 is darker in color, indicating that the corn flour prepared in the examples effectively increases the covalent bonds between the protein and the polyphenol. In addition, the high molecular weight band of the protein-polyphenol complex in the corn flour of Example 1 is the darkest in color, indicating that there are the most covalent bonds between the protein and the polyphenols, and the covalent bonding between the two is the most obvious, forming a more stable protein-polyphenol covalent complex, which has a positive effect on improving the stability and bioaccessibility of polyphenols and protecting the antioxidant activity.

3. Resistant starch content, antioxidant activity and bioaccessibility of active ingredients

FIG5 is a bar graph showing the resistant starch content and antioxidant activity of functional corn flour after digestion, and FIG6 is a bar graph showing the bioaccessibility of active ingredients after digestion of functional corn flour.

As can be seen from Figure 5, the resistant starch content of the corn flours of the three comparative examples is relatively low, while the resistant starch content of the corn flours of the three examples is significantly increased, and Example 1 has the highest resistant starch content. Resistant starch has multiple physiological functions such as lowering blood sugar, preventing colon cancer and obesity, and plays an important role in regulating blood sugar, blood lipids and digestive function. In addition, the DPPH free radical scavenging ability of the corn flours of all examples is significantly higher than that of all comparative examples, especially Example 1 has the highest DPPH free radical scavenging ability, showing the strongest antioxidant activity.

As can be seen from Figure 6, quercetin and proanthocyanidins have lower bioaccessibility in the comparative example, both below 35%, and proanthocyanidins have higher bioaccessibility than quercetin. Compared with the comparative example, all embodiments improve the bioaccessibility of the active ingredient, whether it is quercetin or proanthocyanidins, and Example 1 has the highest bioaccessibility, which may be related to its improved chemical stability of the active ingredient. Higher bioaccessibility will lead to higher bioavailability, indicating that the body has a higher absorption efficiency for nutrients or active ingredients. In addition, all embodiments change the way the active ingredient is bioavailable, from higher proanthocyanidin bioaccessibility to higher quercetin bioaccessibility, resulting in more expensive quercetin (200 yuan/g) being easier for the body to absorb than relatively cheap proanthocyanidins (150 yuan/g), which can effectively increase the added value of the product. Therefore, the corn flour prepared by the present invention has higher resistant starch content, stronger antioxidant activity and higher bioaccessibility, and can be applied to the development of starch-based functional foods.

4. Chemical stability of active ingredients

Figures 7-9 are bar graphs of the chemical stability of active ingredients in functional corn flour under heat treatment conditions of 65°C, 85°C and 100°C. It can be seen that under the three heat treatment temperature conditions, the retention rate of active ingredients in all comparative examples is low, and the retention rate of active ingredients further decreases with the increase of heat treatment temperature. The retention rates of quercetin and proanthocyanidins in the comparative examples at 65, 85 and 100°C are 47%-53%, 37%-43% and 23%-34%, respectively. Compared with the comparative examples, the retention rates of active ingredients in all examples under the three heat treatment temperature conditions are significantly improved, reaching 78%-82%, 69%-73% and 58%-64%, respectively. The retention rates of quercetin and proanthocyanidins in Example 1 are the highest, showing the best chemical stability against high temperatures. Because quercetin and proanthocyanidin molecules form physical and chemical interactions with starch and protein in corn flour through specific groups, their structural stability is improved, and degradation is slowed down under the influence of the external environment, thereby improving their retention rate after heat treatment. Therefore, the functional corn flour prepared by the present invention can maintain good chemical stability of the active ingredients quercetin and proanthocyanidin therein during the heat processing process.

5. Gelatinization and aging characteristics

Table 1 shows the gelatinization characteristic parameters and aging hardness of functional corn flour. There are differences in the gelatinization characteristics of the three comparative examples, but the differences are not large. Compared with comparative example 1 (dry corn flour) and comparative example 2 (semi-dry corn flour), comparative example 3 (wet corn flour) has a higher gelatinization viscosity, which may be due to the fact that the wet processing makes the corn flour particle size smaller and the starch granules more intact, making the corn flour easier to gelatinize. However, the soaking time of wet-process corn flour is too long and the flour making efficiency is low. The flour making process (5-6h) of all embodiments significantly reduces the soaking time of the traditional wet flour making process (36h) and improves the corn flour making efficiency. The gelatinization characteristics of the corn flours of the three embodiments are better than those of comparative example 3, especially the corn flour of embodiment 1 has the highest gelatinization viscosity and shows the best gelatinization characteristics. In addition, compared with all comparative examples, the corn flours of all embodiments have lower aging hardness, and the aging hardness of the corn flour of embodiment 1 is the lowest, showing obvious anti-aging properties. Therefore, the functional corn flour prepared by the present invention has good processing quality.

Table 1 Gelatinization characteristic parameters and aging hardness of functional corn flour

Different letters in the same column indicate significant differences (p<0.05).

The above are only preferred specific embodiments of the present invention, which are all different implementations based on the overall concept of the present invention, and the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (5)

1. An efficient preparation method of functional corn flour, characterized in that the method is carried out according to the following steps: Step 1: adding water to corn kernels for microwave cooking, and then grinding at a low speed to obtain a mixed liquid; the material-liquid ratio is 1:3, the microwave cooking power is 400-600 W, the time is 6-10 min, and the low-speed grinding speed is 2500-3000 rpm; Step 2: adjusting the pH value of the mixed solution to 5, and then adding cellulase for enzymolysis, the amount of cellulase added is 0.5-1.5% of the mass of the mixed solution to obtain an enzymolysis solution; the enzymolysis temperature is 40-60 ° C, and the time is 50-70 min; Step 3: The pH value of the enzymatic hydrolysate is adjusted to 9, and then alkaline protease and quercetin are added, the amount of alkaline protease added is 1-3% of the mass of the enzymatic hydrolysate, and the concentration of quercetin in the enzymatic hydrolysate is 2-4 mg/mL, and the enzymatic hydrolysate is soaked under ultrasound assistance, and then filtered and drained to obtain modified corn particles; the ultrasonic power is 100-300 W, the soaking temperature is 50-70 ° C, and the time is 3-5 h; Step 4: Dissolve proanthocyanidins in water to obtain a proanthocyanidin solution with a concentration of 4-6 mg/mL, then spray it on the surface of the modified corn particles and perform mechanical mixing. When the water content of the modified corn particles reaches 35-40%, adjust the pH value to neutral, and then perform high-speed grinding at a speed of 16000-17000 rpm. After vacuum drying, functional corn flour is obtained. 2. The method according to claim 1 is characterized in that the filtrate obtained by filtering after soaking in step 3 is refluxed into the enzymatic hydrolyzate for recycling. 3. The method according to claim 1, characterized in that the vacuum degree of vacuum drying in step 4 is 0.04-0.08 MPa and the temperature is 60-70°C. 4. Functional corn flour prepared by the method according to any one of claims 1 to 3. 5. The functional corn flour prepared by the method according to any one of claims 1 to 3 is used for preparing functional corn food.