CN116023515A

CN116023515A - Preparation method and application of corn starch sugar residue resistant starch

Info

Publication number: CN116023515A
Application number: CN202310049433.5A
Authority: CN
Inventors: 熊何健; 李晓柯; 马英; 何传波
Original assignee: Jimei University
Current assignee: Jimei University
Priority date: 2023-02-01
Filing date: 2023-02-01
Publication date: 2023-04-28
Anticipated expiration: 2043-02-01
Also published as: CN116023515B

Abstract

The invention discloses a preparation method and application of corn starch sugar residue resistant starch, and belongs to the technical field of functional foods. The preparation method of the corn starch sugar residue resistant starch mainly comprises the steps of repeatedly freezing and thawing the settled corn starch sugar residue at low temperature, adding 5-15 times of water, performing colloid mill water washing treatment, centrifuging to obtain precipitate, and drying to obtain the corn starch sugar residue resistant starch. According to the invention, through researching physicochemical properties and structural characteristics of the resistant starch for preparing the corn starch sugar residue, digestibility in a gastrointestinal tract static digestion model and in-vitro proliferation activity of bifidobacterium adolescentis and bifidobacterium animalis, prebiotic activity of the resistant starch is discussed, and high-value utilization of the corn starch sugar residue is promoted.

Description

Preparation method and application of corn starch sugar residue resistant starch

Technical Field

The invention belongs to the technical field of functional foods, and particularly relates to a preparation method and application of corn starch sugar residue resistant starch.

Background

With the increasing demand of people for saccharides and the continuous development of enzyme preparation engineering, the starch sugar industry is rapidly developed. The starch sugar residues are processing byproducts of the starch sugar industry, the annual yield is huge, no suitable high-value utilization way exists at present, and a large amount of sugar residues are stored in a simple open-air accumulation mode, so that huge environmental protection pressure is caused. In addition, the sugar slag contains high moisture and rich carbohydrate, and is easy to be fermented and utilized by anaerobic microorganisms in the accumulation and storage process to generate metabolic products such as ethanol and the like, so that certain potential safety hazards are caused, and therefore, the research on the efficient utilization of the sugar slag has practical significance.

Resistant starch has a variety of physiological functions. At present, the resistance increasing technology in China needs to be improved, the technology yield is low, an industrial production channel is not formed yet, research hot spots stop to improve the technology efficiency, resistant starch products still depend on import, and good sources of resistant starch are lacking in China. The abundant resistant starch resources in the corn starch sugar residues are utilized, so that the resource gap can be made up, the problem that byproducts in the starch sugar industry are difficult to utilize is solved, and a new research idea is provided for the high-value utilization of the sugar residues.

The type 3 resistant starch has high safety, a denser crystal structure and higher thermal stability, and is attracting attention in the field of resistant starch preparation. The 3-type resistant starch obtained by different preparation methods has different physicochemical properties, and can be applied to different fields such as food additives, nano particles, prebiotics and the like according to the needs.

In the starch sugar processing process, corn starch is hydrolyzed by a double-enzyme method to obtain sugar liquid after high-temperature gelatinization, and residual starch residues are often sold in a simple feed form, so that great waste of resources is caused.

Disclosure of Invention

The invention aims to provide a preparation method and application of corn starch sugar residue resistant starch, wherein a product obtained by washing and centrifugally drying settled corn starch sugar residue is defined as corn starch sugar residue resistant starch, and the prebiotic activity of the corn starch sugar residue resistant starch is studied through researching the physical and chemical properties and structural characteristics of the corn starch sugar residue resistant starch, the digestibility of the corn starch sugar residue resistant starch in a gastrointestinal static digestion model and the in-vitro proliferation activity of bifidobacterium adolescentis and bifidobacterium animalis, so that the high-value utilization of the corn starch sugar residue is promoted.

In order to achieve the above purpose, the present invention proposes the following technical scheme:

a method for preparing corn starch sugar residue resistant starch, comprising the following steps: freezing and thawing the settled corn starch sugar residue at low temperature repeatedly, adding 5-15 times of water, colloid milling, washing with water, centrifuging to obtain precipitate, and drying to obtain the corn starch sugar residue resistant starch.

Further, the freezing parameters are: the temperature is between minus 60 ℃ and minus 18 ℃ and the time is between 24 hours and 72 hours.

Further, the thawing parameters are: the temperature is 0-16 ℃.

Further, the low temperature freezing and thawing repetition times are 2-3 times.

Further, the colloid mill washing time is 2-10min; the centrifugal rotating speed is 4000-5000rpm, and the time is 15-30min; the drying temperature is 50-85 ℃ and the drying time is 3-24h.

The invention also provides the corn starch sugar residue resistant starch prepared by the preparation method.

The invention also provides application of the corn starch sugar residue resistant starch in preparation of the hypoglycemic food.

The invention also provides application of the corn starch sugar residue resistant starch in preparing prebiotic food.

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

(1) The content of carbohydrate in the CRS prepared by the invention is 82.55%, which is similar to that of natural starch, and the CRS contains 51.76% of resistant starch, the content of resistant starch is maintained at 42.6% after high-temperature treatment, the content of resistant starch is obviously higher than that of HS, the CRS has stronger gelatinization resistance, and the water separation rate after 48 hours of freezing is as high as 83.39%. The CRS is a high ordered small molecule block polymer formed by a C-shaped crystal structure, the particles are larger, the particle size distribution is uneven, the polymerization degree is 56, the starch chain is short, the solubility of the CRS is high, the expansion force is small, the short-range order degree of the CRS is obviously higher than that of natural starch NS and HS, the crystallinity is 20.68%, the compact structure causes difficult thermal decomposition, the thermal melting temperature and the heat absorption are higher than those of NS and HS, and the high thermal stability performance is realized.

(2) In the in vitro simulated digestion process, the hydrolysis rate of the CRS in gastric juice is 21.67%, the short-range order of the CRS is obviously increased to 6.68 under the action of gastric acid, the hydrolysis of an amorphous area promotes the proportion of a crystallization area to be obviously increased to 43.29%, the structure tends to be highly ordered after the CRS is acted by gastric juice, the CRS continuously enters intestinal juice for digestion, the digestion rate is not increased along with the digestion time after about 9% of reducing sugar is quickly released, and the method is different from the two-stage digestion of natural starch in small intestine, and further indicates that the internal structure of the CRS is stable, the sensitivity to enzymes is reduced, the CRS is difficult to degrade by amylase, and most of the CRS can enter colon and is subjected to probiotics glycolysis.

(3) The in vitro fermentation bifidobacterium of CRS-2 with the carbon source concentration of 2% keeps the highest activity, the bifidobacterium adolescentis and BB-12 proliferate 405 times and 22.5 times of the original colony number respectively, the pH of the fermentation liquor is most obviously reduced, which proves that the bifidobacterium can utilize resistant starch to meet the requirement of self proliferation, wherein the bifidobacterium adolescentis can efficiently select and utilize CRS-2, the proliferation effect is 1.9 times of GLU, BB-12 prefers the utilization of monosaccharide, the GLU culture effect is superior to CRS-2, and the CRS is the effective metagen of the bifidobacterium.

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 full-wave scan of amylose and amylopectin;

FIG. 2 is an amylose standard curve;

FIG. 3 is an iodine absorption curve of a starch sample;

FIG. 4 is a graph of particle size distribution of starch samples;

FIG. 5 is an infrared spectrum of a starch sample;

FIG. 6 is an X-ray diffraction pattern of a starch sample;

FIG. 7 is a graph showing thermodynamic properties of a starch sample;

FIG. 8 is a scanning electron micrograph (A. Times.2000. Times.B. Times.5000 times.) of a starch sample;

FIG. 9 is a glucose standard curve;

FIG. 10 is a graph of gastrointestinal hydrolysis of a starch sample;

FIG. 11 is a model of starch hydrolysis kinetics, wherein (a) is an NS hydrolysis kinetics curve and (b) is an HS hydrolysis kinetics curve;

FIG. 12 is an infrared spectrum of the digestion product; wherein, CRS-1 and CRS-2 in (A) are respectively products of CRS after digestion by stomach and intestine, and HS-1 and HS-2 in (B) are respectively products of HS after digestion by stomach and intestine;

FIG. 13 is an X-ray diffraction pattern of the digested product; wherein, CRS-1 and CRS-2 in (A) are respectively products of CRS after digestion by stomach and intestine, and HS-1 and HS-2 in (B) are respectively products of HS after digestion by stomach and intestine;

FIG. 14 is a graph showing the effect of different carbon source concentrations on bifidobacterium proliferation; wherein A is the influence on the proliferation of bifidobacterium adolescentis, and B is the influence on the proliferation of bifidobacterium animalis BB-12;

FIG. 15 is a graph showing the effect of different carbon sources on the bifidobacterium growth process; wherein A is the influence of a carbon source on proliferation of bifidobacterium adolescentis, and B is the influence of the carbon source on proliferation of bifidobacterium animalis BB-12.

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 are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The corn starch sugar residue used in the examples of the present invention was provided by Xiamen double bridge Co. Common corn starch, purchased from yellow dragon food industry limited; high amylose corn starch, purchased from henna, makinda trade limited. Common corn starch and high amylose corn starch are collectively referred to as native starch in the examples below.

The sedimentation type corn starch sugar slag provided by the invention specifically comprises the following components: in the process of producing starch sugar (maltose, glucose, fructose syrup, etc.) by using corn starch, a precipitated residue byproduct is produced at the saccharification completion stage.

Example 1

Preparation of corn starch sugar residue resistant starch (CRS): freezing the settled corn starch sugar residue at-18deg.C for 72h, thawing at 4deg.C, repeatedly freezing and thawing for 3 times, adding 15 times volume (V/W) of water, colloid milling, washing with water for 5min, centrifuging (4000 rpm,30 min), collecting precipitate, and drying (55deg.C for 24 h) to obtain corn starch sugar residue resistant starch.

The common corn starch (NS) and commercial resistant starch-high amylose corn starch (HS) are used as controls, and the physicochemical properties and the structural differences of the three starches are compared through component detection and analysis such as a Fourier transform infrared spectrometer, an X-ray diffractometer, a differential scanning calorimeter, a scanning electron microscope and the like, so that basis is provided for further research on the digestibility of CRS, the activity of prebiotics and the like.

A. Physical and chemical properties and structure research of corn starch sugar residue resistant starch:

1. experimental methods and content

The main reagents used in this experiment were all commercially available.

1. Analysis of basic ingredients of raw materials

Determination of total sugar content reference spectrophotometry for determination of total sugar in feed (DB 12/T847-2018); measurement of moisture content referring to "measurement of moisture in food safety national Standard food" (GB 5009.3-2016); determination of fat content reference "determination of fat in food safety national Standard food" (GB 5009.6-2016); measurement of protein content is described in "measurement of protein in food safety national Standard food" (GB 5009.5-2016); determination of ash content reference "determination of ash in food safety national Standard food" (GB 5009.4-2016).

2. Determination of resistant starch content

100+ -5 mg of starch sample was weighed to prepare 10% starch milk, and the 10% starch milk was gelatinized by heating at 90℃for 30min. After cooling to room temperature, adding a freshly prepared mixed solution of alpha-pancreatic amylase (120U) and glycosidase (12U) into a sample tube, placing the sample tube in a constant-temperature oscillating water bath at 37 ℃ for 16 hours, adding absolute ethyl alcohol to terminate enzymolysis, washing for multiple times, and centrifuging to obtain a precipitate. The precipitate was dissolved in 2mol/L KOH solution under ice bath conditions, sodium acetate buffer (ph=3.8) containing glycosidase (330U) was added to the tube, and the solution was subjected to enzymatic hydrolysis in a water bath at 50 ℃ for 30min, and the glucose content released in the supernatant was measured using a glucose kit, with the resistant starch content being glucose content×0.9.

3. Amylose content determination

In order to accurately determine the amylose content and eliminate the influence of amylopectin, a dual-wavelength method is adopted for determination.

3.1 standard stock solution of amylose and amylopectin

Accurately weighing 100mg of standard substance in a 50mL volumetric flask, adding 10mL of 1mol/L potassium hydroxide solution, taking out after the sample is completely dissolved in hot water, cooling to room temperature and fixing the volume to prepare 2mg/mL standard solution.

3.2 determination of measurement wavelength and reference wavelength

1mL of standard solution of amylose and 5mL of standard solution of amylopectin are respectively measured and added into 25mL of distilled water, the pH value of the solution is adjusted to 3.0, 0.5mL of reagent for preparing iodine is added, the volume is fixed to 50mL, and the solution is kept stand for 25min at room temperature. Scanning the whole wavelength on a dual wavelength spectrophotometer using distilled water as a blank, and determining the measurement wavelength lambda of amylose by a mapping method as shown in FIG. 1 ₁ =585 nm and reference wavelength λ ₂ ＝476nm。

3.3 establishment of amylose Standard Curve

Measuring 0.3, 0.5, 0.7, 0.9, 1.1 and 1.3mL of amylose standard solution in a 50mL volumetric flask, adding 25mL of distilled water, adjusting the pH of the solution to 3.0, adding 0.5mL of iodine reagent, fixing the volume to a scale, and standing for 25min. Taking distilled water as a blank, measuring absorbance at 476 and 585nm and recording as A _λ1 、A _λ2 It was found that Δastraight=a _λ2 -A _λ1 . An amylose standard curve (see FIG. 2) was drawn with the amylose concentration (mg/mL) as the abscissa and the absorbance difference as the ordinate. Amylose standard curve regression equation is y= 0.36557x-0.05462, r ² 0.9986, it shows a good fit.

3.4 determination of starch samples

Weighing 100mg of dehydrated and defatted sample in a 50mL volumetric flask, adding 10mL of 1mol/L potassium hydroxide solution, completely dissolving in hot water, and fixing the volume to scale to prepare 2mg/mL sampleAnd (3) liquid. Accurately measuring 1mL of starch sample clear liquid, adding the clear liquid into 25mL of distilled water, adjusting the pH of the solution to 3.0, adding 0.5mL of iodine reagent, fixing the volume of the solution to 50mL, and standing for 25min. The absorbance of the sample solution at 476,585 nm was measured with a dual wavelength spectrophotometer and recorded as A _λ1 、A _λ2 Measured DeltaA _Sample ＝A _λ2 -A _λ1 Sample quantification was performed using an amylose standard curve.

4. Determination of iodine absorption Curve and average polymerization degree

Accurately weighing 20mg of starch sample in a 50mL volumetric flask, adding a small amount of ethanol to infiltrate the sample, and adding 1mL of 2mol/L potassium hydroxide solution. To the starch sample after complete dissolution and dispersion, 10mL of distilled water was added and the pH of the solution was adjusted to 6.5 and the volume was set to scale. 10mL of sample solution is measured in a 100mL volumetric flask, 80mL of distilled water and 2mL of the iodine preparation reagent are added, and the volume is fixed to the scale. An iodine absorption curve was obtained by scanning at 500-800nm using an ultraviolet spectrophotometer. The average degree of polymerization is calculated by the following formula:

Wherein lambda is _max Represents the maximum absorption peak wavelength (nm) of the sample occurring in the scan region, and DP represents the average degree of polymerization of the starch sample.

5. Determination of solubility and swelling degree

1.0g of a starch sample was dissolved in 10mL of distilled water to prepare a starch suspension with a mass fraction of 10%. The dispersed samples were shaken in a water bath at 50, 60, 70, 80, 90℃for 30min. After cooling to room temperature, the starch solution was centrifuged (3000 r/min,15 min) to collect the supernatant, which was transferred to a beaker of known constant weight and dried in an oven at 105 ℃. The solubility and swelling degree were calculated according to the following formula:

wherein m is ₁ The constant weight and g of the supernatant after drying are shown; m is m ₂ G represents the mass of the sediment after centrifugation; m represents the mass of the starch sample, g.

6. Determination of freeze-thaw stability

Accurately weighing 1.0g of starch sample in a centrifuge tube, adding pure water to prepare a 6% starch suspension, heating the system in a boiling water bath for 30min, stirring once every 5min, cooling to room temperature after gelatinization, centrifuging (3000 rmp,10 min), discarding supernatant, directly weighing, freezing at-20 ℃, taking out the centrifuge every 24h, repeating freeze thawing for 5 times, and calculating the water extraction rate according to the following formula:

Wherein m is ₀ G represents the mass of the centrifuge tube; m is m ₁ G, representing the mass of the centrifuge tube and the colloid in the tube; m is m ₂ The mass of the centrifuge tube and the sediment are shown in g.

7. Determination of particle size distribution

And measuring the particle size distribution of the starch sample by using a laser particle sizer, and controlling the optical concentration of the sample in a sample cell to reach 20% -30% during measurement.

8. Determination of starch short-range order

And (3) determining the double helix order degree of the starch sample by adopting a Fourier transform infrared spectrometer. Mixing the starch sample with the dried potassium bromide according to the proportion of 1:100, grinding into fine powder in an agate mortar, and tabletting by using a tabletting machine. At 400-4000cm ^-1 Scanning in the wave number range with a resolution of 4cm ^-1 Scanning is performed 32 times to obtain an infrared spectrogram of the starch sample. Analysis of spectra using OMNIC software, 800-1200cm ^-1 After the spectrum in the wave number range is converted into an absorbance form and output, deconvolution processing is used to set the peak width to 38cm ^-1 Enhancement factor 1.9.

9. Determination of starch long-range order

The crystallinity of the starch samples was determined using an X-ray diffractometer. The X-ray diffractometer was run at 40kV and 40mA using Cu-ka filtration. The scattering angle (2 theta) is 5-40 deg, the scanning speed is 4 deg/min, and the step size is 0.02. Peak-splitting fitting was performed on the diffraction patterns using the jack 6.0 software to calculate the relative crystallinity of the samples.

10. Thermal characterization of starch

The thermal properties of the starch samples were analyzed using a differential scanning calorimeter. A starch sample was mixed with distilled water in an aluminum crucible at a ratio of 1:3, mixing evenly. Using an empty crucible as a blank control, gradually heating the blank control at a temperature ranging from 25 ℃ to 150 ℃ at a speed of 10 ℃/min, recording a heat flow change curve, and analyzing an initial gelatinization temperature (T) according to a heat flow diagram ₀ ) Peak gelatinization temperature (Tp), end gelatinization temperature (Tc), gelatinization enthalpy value (Δh).

11. Analysis of surface particle morphology of starch

The morphology of the surface particles of the starch samples was observed using a scanning electron microscope. And (3) adhering the dried starch sample to conductive adhesive, then performing metal spraying treatment, and observing and storing an electron microscope image under different magnification factors for analysis.

12. Analysis of surface particle morphology of starch

13. Data processing

Experimental data are expressed as mean ± standard deviation, three sets of replicates were set for each experiment, and the significance analysis was performed using SPSS22.0 software, origin9.0 software, to process the data profile.

2. Results and discussion

1. Analysis of raw Material base Components and starch composition

The basic components of the three starch raw materials are shown in table 1. All three starches have carbohydrates as the main component, with only small amounts of protein and fat present. The fat and protein content in corn starch sugar residues is higher than that of common corn starch and high-amylose corn starch, and is probably due to the enrichment of components in the sugar manufacturing process. The content of carbohydrate in corn starch sugar residue is 82.55%, and the content of carbohydrate is not significantly different from that of commercial starch.

TABLE 1 basic ingredients of raw materials

Note that: the table is a comparative analysis between different starches, and the mean of the same parameter is followed by different letters to indicate that there is a significant difference (P < 0.05).

The starch composition analysis of the three materials is shown in Table 2. The CRS amylose content is 31.42%, slightly higher than NS, and the resistant starch content is up to 51.76%, indicating that the CRS forms a highly ordered structure with amylase hydrolysis resistance during production, NS contains a small amount of resistant starch, and HS is a commercial type 2 resistant starch with a content of up to 58.85%. After high-temperature gelatinization, NS is almost completely hydrolyzed, the content of HS resistant starch is reduced by about 20%, the content of CRS resistant starch is obviously higher than that of NS and HS, and the starch has high digestion resistance, so that the starch is a good gelatinization resistant starch material.

Table 2 starch composition of raw materials

In the sugar manufacturing process, corn starch generates shorter amylose by enzyme digestion of glycosidic bond, heating gelatinization promotes the escape of the amylose, and in the cooling process, the amylose is mutually close to form a double-helix structure, and the amylose is combined to form a crystallization area which is difficult to enzymolysis under the action of hydrogen bond and Van der Waals force, so that the CRS contains rich 3-type resistant starch.

In vitro small intestine digestion experiments, amylose content can generally affect starch hydrolysis rate. Amylose is more resistant to enzymolysis than amylopectin because of stronger hydrogen bonding between amylose, so HS is often used as a commercial type 2 resistant starch. Experiments show that HS is partially gelatinized under the action of water heat, so that the enzymolysis resistance is obviously reduced. Starch binding to amylase is one of the major factors affecting starch digestion, and low levels of gelatinization may still disrupt the physical barrier of the starch, promoting amylase invasion and thus speeding up enzymatic hydrolysis. The CRS has obvious anti-gelatinization effect, and has better application performance than HS in a food processing system with higher water content.

2. Iodine absorption curve and average degree of polymerization

The colored complex formed between starch and iodine reflects the information of the ratio, chain length, etc. between amylose and amylopectin. The starch wraps iodine molecules in a spiral structure through Van der Waals force, and the compound presents different colors due to different lengths of glycoside chains. The maximum absorption peak of blue complex formed by amylose and iodine is in the range of 600-640nm, while the maximum absorption peak of purple complex formed by amylopectin and iodine is in the range of 520-560 nm.

The iodine absorption curve of the starch samples is shown in figure 3. The maximum absorption peaks of NS and HS are 597 and 599nm respectively, and the wavelength is between the absorption peaks of amylose and amylopectin, which indicates that the starch is a mixture composed of amylose and amylopectin. The CRS cleaves the molecular chain by amylase and glycosidase, and the short amylose reacts with iodine to appear purple, resulting in a shift of the maximum absorption peak toward the amylopectin absorption wavelength. The binding capacity of starch to iodine is related to the amylose content, with high amounts of amylose in HS yielding a stronger iodine uptake capacity, while CRS binding to iodine yields slightly higher absorbance than NS.

The average degree of polymerization of the starch samples is shown in Table 3. As can be seen from the table, CRS is a small molecule oligomer with an average degree of polymerization of 56, significantly lower than native starch. CRS is a small molecule formed by cracking natural corn starch and a product of the small molecule and the natural corn starch polymerized with each other under the action of high temperature and enzyme digestion.

TABLE 3 average degree of polymerization of starch samples

3. Solubility and swelling analysis

When starch is heated under the condition of excessive water, amylose in starch particles is heated to escape, the crystal structure of the starch particles is damaged due to the rise of the temperature, water molecules penetrate into the particles to be combined with the starch through hydrogen bonds to cause the starch particles to swell, in the process, the starch solubility reflects the capacity of the amylose molecules in the starch particles to escape and dissolve in water, and the swelling degree reflects the water absorption degree of the starch at different temperatures. The action between starch and water molecules is mainly influenced by the factors of the molecular weight, the chain length of branched chains, the content of amylose and the like of the starch, and the hydration is different due to different compositions of the starch.

The degree of hydrolysis and the degree of swelling of the starch samples at different temperatures are shown in tables 4 and 5. The solubility of the CRS is obviously higher than that of the natural starch, the influence of the temperature increase on the CRS is small, when the heating temperature is low, free water is easy to infiltrate into a starch damaged structure, so that the swelling power of the CRS is obviously higher than that of the natural starch, the solubility and the swelling power of the NS and HS samples are increased along with the gradual increase of the heating temperature, when the heating temperature reaches over 70 ℃, the swelling degree of the NS is obviously higher than that of the CRS, the NS is obviously heated and expanded, when the temperature is increased to 90 ℃, the NS is completely gelatinized, forms starch colloid with water molecules, no amylose is dissolved out, the swelling degree reaches the maximum value, and the solubility is reduced to 0.

Under the low temperature condition, because of the limitation of hydrogen bonds and semi-crystalline structures formed in starch molecules, water molecules are difficult to permeate into starch particles, along with the rising of heating temperature, the structures of the starch particles gradually loose and collapse, so that a large amount of amylose is released and dissolved, the damage of the starch structures promotes the increase of the exposed hydrogen bonds in a crystallization area, and water molecules are extremely easy to enter the starch particles to generate hydration, so that the solubility and the swelling power of natural starch are increased, in the process, the amylose inhibits the swelling of the starch particles, so that the solubility and the swelling power of HS samples are lower, and CRS cuts off molecular chains through double enzymolysis, so that a large amount of short amylose is generated, and small molecular sugars such as maltose, glucose and the like reduce the molecular weight in the starch, so that the solubility is high at high temperature and the swelling power is low.

Table 4 solubility of starch samples

TABLE 5 swelling degree of starch samples

4. Analysis of freeze-thaw stability

The freeze-thawing stability is one of important properties of starch, starch is associated with water molecules through hydrogen bonds at high temperature to form starch gel, when starch colloid is frozen, water molecules are converted into ice crystals at low temperature, the phenomenon of syneresis of the gel easily occurs when the temperature rises, and the water separation rate of the starch is high, so that the freeze-thawing stability is poor.

The results in table 6 show that all starch samples remained stable in water evolution after 48h freeze thawing, higher in CRS and HS water evolution rates of 83.39%, 76.70%, respectively, while NS showed low water evolution rates. In the freezing process, the water evolution of the starch gel is mainly related to amylose retrogradation due to the enhancement of the interaction between starch molecular chains, and under the low-temperature condition, the amylose is combined through hydrogen bonds, so that the action between water molecules and starch chains is weakened, and the water molecules are easy to separate out from the colloid, so that the water evolution rate is related to the content of the amylose. The solubility and swelling degree analysis show that CRS and HS are difficult to gelatinize below 100 ℃, starch gel cannot be formed, and starch samples only show partial component dissolution and slight expansion, so that acting force between starch and water molecules is weak, water separation is easy to occur, and freeze thawing stability is poor. The NS forms gel with water molecules at high temperature, when treated at low temperature, amylose retrogrades to cause water separation, and the water separation rate is low, which indicates that the NS has better freeze-thawing stability and is suitable for developing frozen products in the food industry.

TABLE 6 freeze-thaw stability of starch samples

5. Particle size distribution analysis

The size of the starch granule affects the efficiency of binding to amylase and thus the digestion characteristics of starch. The particle size distribution profiles of the three starch samples are shown in figure 4.

The grain size figures of the starch show normal distribution, the grain size distribution of different starch samples is obviously different, the grain size distribution of the CRS is wider, the grain size distribution of the CRS obtained by a high-temperature enzyme cutting process is uneven, the grain size distribution of the natural starch is centralized, the average grain size of the CRS is 65.07 mu m, the average grain sizes of NS and HS are respectively 11.76 mu m and 10.10 mu m, the grain size of the CRS is far more than that of the natural starch, and the grain size is probably due to the fact that branched amylopectin dissolves out in the formation process of the resistant starch, and a larger network space structure is formed with amylose molecules. The NS average particle size was observed to be slightly larger than HS, consistent with scanning electron microscope results.

6. Short range order analysis

Information about the helicity, molecular chain conformation and the like of the starch can be obtained through Fourier transform infrared spectrum and is used for monitoring the change of the short-range order degree of the sample. The infrared scanning results of the three starches are shown in FIG. 5, at 3100-3700cm ^-1 The spectrum in the range is related to the O-H stretching vibration in hydrogen bonds, where CRS and HS are absorbedThe peak is stronger due to the hydrogen bonding interactions of more amylose molecules inside the starch. 800-1200cm ^-1 The spectrum at this point is often considered as the fingerprint region of starch, which is extremely sensitive to short range order changes. 995cm ^-1 The absorption peak at this point is related to the degree of hydration of the starch crystallization zone, 1022cm ^-1 The absorption peak at which is related to the stretching vibration of the amorphous region, and 1047cm ^-1 The intensity of the absorption peak at this point is influenced by the proportion of the starch crystallization area. 1047/1022cm ^-1 And 995/1022cm ^-1 Is used to describe the Degree of Order (DO) and the degree of helicity (DD) of the starch, respectively.

The results of the order and helicity measurements of the three starch samples are shown in Table 7. CRS shows the highest degree of order, corn starch undergoes molecular chain fragment rearrangement after high-temperature complex enzyme action, and a local highly ordered structure appears. In native starch, the double helix built up of amylopectin side chains forms crystalline regions, with amylose interpenetration in the amorphous regions, and with relatively low amylopectin content in the HS, the DO and DD values are lower than NS. From the results, the DO and DD values are not exactly equal, indicating that not all duplex is involved in crystal formation and that the short-range order is mainly affected by the duplex content in the starch.

TABLE 7 degree of order and helicity of starch samples

7. Long range order analysis

The X-ray diffraction results of the three starch samples are shown in fig. 6.

According to the "two-phase" hypothesis, in the diffraction pattern, the crystals exhibit sharp strong peaks, while the amorphous phase exhibits a diffuse peak shape. The sharp diffraction peaks of NS occurring near diffraction angles of 15.2 °, 17.3 ° and 23.0 ° are characteristic peaks of the a-type crystal. Diffraction curves of HS have Jiang Yanshe peaks around 5.5 °, 17.2 ° and 20.0 °, showing a b+v crystal structure, and diffraction peaks around 20.0 °, indicating that part of amylose forms a complex with lipid, showing a V crystal structure. The CRS diffraction curve shows strong diffraction peaks near the 2 theta of 16.9 degrees, 22.2 degrees and 26.5 degrees, which indicates that the CRS crystal type is C-type, the CRS is converted from A-type crystal to B-type crystal in the high-temperature composite enzymolysis process, the phenomenon is possibly related to the increase of the long chain proportion in starch by enzymolysis, the appearance of B-type crystal structure indicates the improvement of the enzymolysis resistance of the CRS, and the new diffraction peak at 26.5 degrees is observed to be possibly due to the formation of a semi-crystalline gel network structure between amylose in the starch cooling process, so that new crystal is generated.

The results of the relative crystallinity calculations for the three starch samples are shown in table 8. The crystallization area in the starch is mainly influenced by the amylopectin, the content of the amylopectin is high, the number of double helix formed by the side chain is increased, and the proportion of the crystallization area in the starch is increased, so that more amylopectin is contained in NS to form a more compact crystallization structure, and the proportion of the crystallization area of HS is far smaller than that of other samples. The relative crystallinity of CRS was 20.68%, indicating that the starch recovered an ordered structure during cooling, and was rearranged to form a duplex from amylose, and stacked to form a crystalline structure. The starch relative crystallinity calculation result is inconsistent with the short range order result, the CRS short range order is higher, but the relative crystallinity is lower than NS, and may be that microcrystalline or amorphous phase exists in the crystallization area during starch rearrangement, so that imperfect crystals are formed.

TABLE 8 relative crystallinity of starch samples

HS has low crystallinity but high resistance to enzymolysis, which may be related to crystal type and particle morphology, and type a crystals are very susceptible to enzymatic hydrolysis due to differences in crystal arrangement, while type B polymorphs have slow digestion rate, which may be one of the reasons for high resistance of HS, so the relative crystallinity of starch does not directly reflect its anti-digestion properties.

8. Thermal characterization

The results of the thermal properties of the starch samples are shown in FIG. 7. There is a significant difference in thermodynamic properties between different starches, and a symmetrical peak of NS was observed around 64.4 ℃, which is the gelatinization endotherm of starch, associated with the heated depolymerization of the duplex. The HS and the CRS have no thermal transition phenomenon below 100 ℃, when the temperature is continuously increased to 134.3 ℃, 137.3 ℃ and 138.3 ℃, strong endothermic peaks, namely phase change endothermic peaks, appear in the NS, the HS and the CRS successively, and the temperature required by the internal microcrystal of the CRS for being heated and destroyed is highest, so that the CRS structure is more stable than that of the natural starch, and the crystal structure of the CRS needs to be destroyed by higher heat.

The enthalpy values corresponding to the phase transition endothermic peaks of the CRS, the NS and the HS are respectively 11.04J/g, 10.57J/g and 10.47J/g, which are consistent with the short-range order degree result of starch, and the heat consumption of opening the double helix is higher when the ordering degree of the CRS is higher than that of other samples, which reflects the fact that the enthalpy value measured by a differential scanning calorimeter is related to the ordered structure of the double helix of starch and is not the damage of long-range crystals, so that the resistant starch has good heat stability and is difficult to melt at high temperature.

9. Surface particle morphology analysis

The particle morphology of the three starch samples is shown in figure 8. Natural starch exhibits a distinct granular structure, NS particles are mostly prismatic and polygonal, a small fraction is circular, the particles are large, and tiny holes are observed on the surface of the particles, which have been shown to be channels towards the center of the starch, associated with rapid hydrolysis of the starch. HS particles are mostly in the shape of a rod or a silk rod, the particles are smaller, and the surface is smoother. The number of granular structures in the form of rods or filaments increases with the apparent amylose content of the starch. The CRS has completely lost particle morphology, is in a block-shaped, flake-shaped or other structure, has larger particle size, rough surface and numerous ravines, is more compact than natural starch, and the formation of the structure can be related to rearrangement of amylose among the amylose and hydrolysis of surface starch by amylase in the retrogradation process, and the special lamellar structure of the CRS can be one of reasons for generating enzymolysis resistance. Resistant starch has been reported to increase bifidobacteria survival in the reverse environment of the gastrointestinal tract, possibly in relation to the formation of ravines on the surface of resistant starch.

Because starch granules are totally different in structure, the enzyme hydrolysis mode is also different, the NS surface provides a channel for enzyme to invade the starch, and amylase preferentially hydrolyzes an amorphous region after entering the center of the granule, so that a rapid hydrolysis mode from inside to outside is formed, and a slow hydrolysis mode from outside to inside is distinguished from HS and CRS.

B. In vitro digestion and degradation research of corn starch sugar residue resistant starch;

1. experimental method

According to the invention, CRS (example 1), NS and HS are taken as research objects, a starch hydrolysis curve is obtained through a standardized static digestion model, a slope log diagram (logarithm ofslope, LOS) is used for analyzing the starch hydrolysis process, and the structural characteristics of digested products are analyzed, so that a basis is provided for the digestion resistance of the CRS.

Sodium cholate (> 98.0%); the reagents used were all analytically pure.

1. In vitro digestion of starch

1.1 preparation of digestive electrolyte

A gastric-intestinal two-section digestion model is constructed, various salt ion solutions are prepared according to the table 9, the mixed electrolyte is adjusted to the corresponding pH value by using a 6mol/L hydrochloric acid solution, then the volume is fixed to 400mL, and the electrolyte is placed at the temperature of minus 20 ℃ for standby.

TABLE 9 preparation of electrolyte solutions

Note that: "-" means no addition.

1.2 preparation of simulated gastrointestinal fluids

Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) are prepared from the electrolyte solution, fresh enzyme solution and CaCl containing stabilizing factors ₂ The solution is formed.

CaCl ₂ Preparing a solution: 2.205g of calcium chloride dihydrate is weighed, the volume is fixed to 50mL, a stock solution of 0.3mol/L is prepared, and the stock solution is placed at the temperature of minus 20 ℃ for standby.

Bile juice: bile acid salt is used for simulating bile juice, 3.45g of sodium cholate is accurately weighed and dissolved in 50mL of SIF electrolyte solution, and the solution is placed at the temperature of minus 20 ℃ for standby.

Preparation of SGF: 0.8g pepsin (250U/mg) was dissolved in 8mL SGF electrolyte, and 25. Mu.L CaCl was added to freshly prepared enzyme solution ₂ The solution and 37.5mL electrolyte are mixed uniformly, the pH value of the system is adjusted to 3.0 by using 1mol/L hydrochloric acid solution, the volume is fixed to 50mL, and the solution is heated at 37 ℃, so that the reagent needs to be prepared at present.

Preparation of SIF: 0.8g of porcine pancreatic alpha amylase (50U/mg) was dissolved in 25mL of SIF electrolyte solution, and 12.5mL of simulated bile juice and 0.2mL of CaCl were sequentially added to the enzyme solution ₂ The pH of the mixed system is adjusted to 7.0 by using 1mol/L sodium hydroxide solution to reach 100mL, the mixed system is placed at 37 ℃ for heating, and the reagent needs to be prepared on site.

1.3 preparation of starch samples

Starch-containing foods are often consumed after maturation, so that the gelatinization step is completed before the in vitro digestion procedure begins, with minor modifications to the standardized procedure to accommodate the sample type. Accurately weighing 1.65g of starch sample in a conical flask, adding 10mL of distilled water, sealing with aluminum foil paper to prevent evaporation of water, placing in a water bath at 90 ℃ for 30min, cooling the system in a water bath at 37 ℃ after starch is fully gelatinized, and inhibiting retrogradation of starch.

1.4 stomach digestion

After the starch sample had cooled to room temperature, the digestion procedure was started. To simulate human gastrointestinal peristalsis, digestion was completed in a water bath at 37℃and at a shaking rate of 100 rmp. 10mL of warm SGF solution is taken and added into a sample bottle, enzymolysis time is 1h, sampling is carried out at 30 min and 60min respectively, the sampling tube is subjected to enzyme deactivation in water bath at 90 ℃ for 5min, supernatant is obtained through centrifugation, and the amount of glucose released by starch digestion is measured later.

1.5 intestinal digestion

Taking out the sample after stomach digestion, adjusting the pH of the digestion system to 7.0 by using a sodium hydroxide solution with the concentration of 1mol/L, then adding 20mL of warm SIF solution, sampling at 5, 10, 15, 20, 30, 60, 90, 120, 150, 180, 210, 240, 300 and 360min respectively for enzymolysis time period of 6h, inactivating enzyme in a water bath at 90 ℃ for 5min by using a sampling tube, centrifuging to obtain supernatant, and measuring the glucose released by starch digestion later.

1.6 enzymatic Condition optimized hydrolysis

From the small intestine digestion data obtained from the above experiments and the analysis of the pre-experiment results, it is known that the sample of HS has reached the slow hydrolysis stage when it is sampled, so that the enzyme conditions for processing the sample of HS are optimized to obtain the whole starch hydrolysis process. The specific digestion method is modified as follows: a sample of 2.5g starch was weighed for subsequent experiments.

2. Conversion of the hydrolysis product

The supernatant from the intestinal digestion stage collected is incubated with amyloglucosidase, causing the hydrolysis product to be converted from maltose to glucose. The specific operation is as follows: accurately sucking 100 mu L of the collected supernatant in a 5mL volumetric flask, adding a small amount of sodium acetate buffer solution (pH 4.5) containing amyloglucosidase (330U), and placing the system in a 37 ℃ water bath for incubation for 1h to ensure that the conversion of the product is completed. After incubation, all volumetric flasks are fixed to the scale, and 1mL of sample solution at different time points is taken out for subsequent experiments.

3. Determination of sugar content of hydrolysate

The amount of glucose released by starch hydrolysis was determined using DNS method.

3.1 preparation of Standard substance

Accurately weighing 100mg of glucose, dissolving in distilled water, and fixing the volume to 100mL to prepare a glucose standard solution of 1mg/mL for later use.

3.2 determination of standard curve

Accurately measuring 0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mL standard solution in 25mL volumetric flasks, adding 1.5mL LDNS reagent into each volumetric flask by using distilled water, placing the system in a boiling water bath for color development for 5min, taking out after color development is completed, and fixing the volume after cooling to room temperature. 200. Mu.L of standard solutions of different concentrations were pipetted into a 96-well plate and absorbance was measured at 540nm using an enzyme-labeled instrument. A standard curve was drawn with the glucose content as the abscissa and the absorbance as the ordinate (see fig. 9). The regression equation of the glucose standard curve is y= 0.39417x-0.0333, R ² 0.9968, it shows a good fit.

3.3 determination of samples

1mL of the hydrolysate in 3.2.2 was aspirated into 25mL volumetric flasks, 1mL of distilled water was added to each volumetric flask, followed by 1.5mL of LDNS color-developing agent, and the system was developed under boiling water bath conditions for 5min. 200 mu L of the sample solution with the fixed volume is taken in a 96-well plate, absorbance is measured at a wavelength of 540nm by using an enzyme-labeled instrument, the amount of sugar released by starch hydrolysis is calculated according to a glucose standard curve, and the starch hydrolysis amount is calculated as glucose amount multiplied by 0.9.

3.4 quantitative determination of starch digestion Components

To accurately quantify the different digestion components in starch and analyze the starch hydrolysis process, a first order kinetic model was prepared using a modified slope Log (LOS) method.

From previous studies, it was shown that the digestibility curve of starch can be fitted to a simple first order equation:

C _t ＝C _∞ (1-e ^-kt )(5)

wherein C is _t Represents the concentration of the reactant at time t, C _∞ Represents the concentration at which digestion reaches plateau, k being the quasi-first order rate constant.

For convenience of drawing, the above expression is often expressed in a corresponding logarithmic form:

the relationship of In (dc/dt) to time t is expressed as a linear dependence, the slope is-k, and the intercept on the y-axis is In (C _∞ k) A. The invention relates to a method for producing a fibre-reinforced plastic composite The specific drawing operation is as follows: from experimental data, the digestibility at different time points is selected to calculate the slope, which can be calculated as (C) assuming that the sampling time interval is sufficiently small ₂ -C ₁ )/(t ₂ -t ₁ ) The calculated slope is taken as the ordinate with the natural log value and the corresponding average time (t ₁ +t ₂ ) And/2 is an abscissa, a natural logarithmic graph of the slope is drawn, and analysis is performed based on actual digestion data, wherein the time point of reaching the digestion plateau is not within the range of the graph.

The discontinuity shown in the LOS linear graph reveals the process of starch digestion from fast to slow, and assists in calculating the digestion rate constants of different digestion components and different stages, so that the multi-scale digestion of starch can be more accurately described than the pseudo first-order rate constant obtained in a simple first-order equation.

3.5 structural Properties of digestion products

The method for determining the short-range order degree of the digestion product generated by hydrolysis is the same as the method for determining the long-range order degree.

3.6 data processing

Three sets of replicates were set up for each experiment, experimental data results were calculated using Excel, and data maps were processed using origin9.0 software.

2. Results and discussion

1. Digestion of starch

The hydrolysis curves generated by the three starch samples under the conditions of the gastrointestinal model are shown in figure 10. Different kinds of starch exhibit different digestion patterns. In the simulated gastric digestion phase, the hydrolysis rate of starch was not significant over time, with CRS being as high as 21.67% in the stomach, whereas only a small amount of starch in NS and HS was hydrolyzed, with degradation rates of 3.35% and 3.58%, respectively. Because the gastric juice does not contain amylase, the provided acidic environment can hydrolyze starch into short chain molecules, and the CRS prepared by the high temperature-enzymolysis debranching process contains small molecule linear starch, under the action of the gastric juice, the short amylose on the surface of the CRS is acidolyzed into small molecule sugar, and the damage of the surface structure promotes the release of the residual reducing sugar in the interior, so that the digestibility of the CRS in the gastric juice is higher. From the iodine absorption curve results, NS and HS have high polymerization degree, starch chain length, and natural starch is only degraded into short chain molecules in gastric juice, releasing a small amount of reducing sugar.

The starch samples showed significant differences in hydrolysis rate after entering the small intestine stage. The amount of reducing sugar produced by hydrolysis of NS and HS increases rapidly, wherein NS reaches the plateau phase after 2 hours of digestion in the small intestine and the rate of hydrolysis of HS remains stable after a digestion period of 5 hours. As shown by the thermal characteristic results of the starch, the NS is completely gelatinized through the pretreatment operation, the starch loses the growth ring structure and is converted into a disordered state, the amylase in the intestinal juice is contacted with the amylase, the rapid hydrolysis mode is started, the final hydrolysis rate reaches 88.29%, and the higher blood sugar reaction is caused. HS slightly swells after pretreatment, but still maintains the starch granule structure, amylase is hydrolyzed from the surface to the inside, the digestibility is low, the hydrolysis rate is slow, and the final hydrolysis rate reaches 60.87%. The CRS releases only a small amount of reducing sugar in intestinal juice, and the hydrolysis rate is basically kept constant after reaching 30%, so that the CRS has high enzymolysis resistance.

2. Starch digestion kinetics model

Digestion of samples in the small intestine is considered to be the focus of research in order to model starch hydrolysis. From the above results, it was shown that HS had reached the second digestion stage after 5min of hydrolysis in the small intestine, and that proper enzyme conditions were selected for digestion in order to obtain the overall hydrolysis profile for modeling. The results of the starch hydrolysis kinetic model are shown in fig. 11, and the CRS is hardly hydrolyzed in the intestinal tract, so that a kinetic digestion model cannot be constructed, and the in vivo enzyme hydrolysis resistance of the model is high. By monitoring the hydrolysis process of starch using a slope log, a clear break point is observed in the line graph, revealing that starch does not complete hydrolysis with a single rate constant, and that starch contains components with different enzyme sensitivities, is a powerful evidence for multi-scale digestion of starch.

The starch digestion parameters calculated from the kinetic model are shown in table 10, where the digestion rate constants are expressed at the same enzyme activity level, in order to compare the different digestion patterns of the two starches. Good fitting of all line patterns was observed (r ² > 0.864), the data reliability is higher.

TABLE 10 hydrolysis kinetic parameters

Note that: ND is not detectable, and k is expressed as the alpha-amylase activity at 1600U.

As shown by DSC analysis results, at 90 ℃, the NS has the gelatinization effect, the starch loses the grain structure and is converted into a disordered state, and more alpha glucan chains are exposed to enzyme liquid, so that the sensitivity of amylase attack is improved. The results of the present invention show that NS is composed of 66.78% RDS component and 47.07% SDS component, and that the total digestion group is observedOver 100% because the LOS plot, while very sensitive to digestion phase, involves a numerical derivative of discrete rate data points, making it inherently inaccurate, rapidly digesting phase k ₁ Stage k of slower digestion ₂ The improvement by 4.8 times is probably due to the fact that the starch is not broken by chewing in the oral digestion stage, larger pills are formed, and amylase is infiltrated into the interior of the granules to become the speed limiting step.

HS initial digestion Rate constant (k) ₁ = 0.66043) a fast and severe digestion phase occurs, with a very short duration (5 min) of digestion, corresponding to digestion of about 18.18% of RDS in HS. Because the surfaces of the HS particles are not provided with holes, amylase is digested from outside to inside after being adsorbed, the digestion process can be regarded as a layer-by-layer digestion mechanism, so that the digestion rate constant is extremely low (k= 0.00383), the actual C is difficult to obtain in the period of up to 6 hours of digestion time without reaching the plateau _∞2 Content, this digestion pattern reflects the nature of HS as resistant starch.

The CRS releases 9% of reducing sugar in the small intestine, the hydrolysis rate is not increased with the extension of the digestion time, and the CRS is different from the digestion mode of natural starch, cannot be degraded by enzymes in the intestinal tract, and has high enzymolysis resistance.

3. Analysis of structural Properties of digestion products

The infrared scan of the digested products is shown in fig. 12, with no product residue after NS is digested from the gastrointestinal tract, so the focus of the study is on CRS and HS digested product characterization. The infrared spectrum of all digestion products is lower in the characteristic peak intensity of-OH and shifts to high wave numbers compared with the original starch, which shows that partial starch is hydrolyzed after digestion of gastric juice and intestinal juice, resulting in reduced hydrogen bonding in the interior of the granule. Furthermore, the digestion products were observed to be at a wavenumber of 995cm ^-1 The absorption peak is reduced, probably because the imperfect crystals present in the starch are destroyed during digestion. While the residue of pepsin and amylase results in a digestive product of 1600-1700cm ^-1 The characteristic absorption peak of protein amide in the wavenumber range is enhanced.

The results of the calculation of the degree of order and the degree of helicity of the digested product are shown in Table 11. The DO value and DD value of the digested CRS and HS are increased compared with those of the original starch, and the in vitro digestion model proves that the amorphous area of the starch is hydrolyzed, so that the starch order degree is increased. As can be seen from the results of fig. 10, the CRS is acidolyzed by the gastric juice, and the internal residual reducing sugar is released, so that the starch order is greatly improved after gastric digestion, the CRS structure tends to be highly ordered, and is hardly hydrolyzed in intestinal juice, which is consistent with the calculation results in table 11. Under the action of acidolysis of gastric juice, HS releases only a small amount of reducing sugar, which indicates that gastric juice has little damage to starch structure, DO and DD values of HS are observed to be increased compared with that of original starch, and the reasons are probably that the HS is subjected to pretreatment to cause starch annealing, starch chain mobility is increased, double helix movement and starch rearrangement are promoted, so that starch order is increased, when HS continues to enter intestinal juice for digestion, amylase in digestive juice attacks surface starch, hydrolysis of an amorphous region causes relatively increased ordered structure, and amylopectin content is less to cause that helicity generated by digestion products is far lower than CRS.

TABLE 11 degree of order and helicity of digestion products

The X-ray diffraction results of the digested products are shown in FIG. 13. Compared with the original starch X-ray diffraction pattern, the CRS digestion product has new diffraction peaks at diffraction angles of 5.5 degrees and 15.1 degrees, which indicates that the content of B-type crystals in the starch is increased, the enzymolysis resistance is enhanced compared with that of the original starch, and the product after HS is digested by intestinal juice has new diffraction peaks at diffraction angles of 22 degrees, which are considered as characteristic diffraction peaks of high-crystallinity B-type starch. The crystal structure of the digested product was unchanged from that of the original starch, indicating that the starch did not involve a transition in crystal type during digestion.

The results of calculation of the crystallinity of the digested products are shown in Table 12. The crystallinity of the product of the CRS after acidolysis by gastric juice is obviously increased compared with that of the original starch, and the product is not increased along with the digestion process, and the result is the same as the result obtained previously, the starch of the CRS after gastric digestion is obviously increased in order, the structure tends to be highly ordered, the proportion of a crystallization area is obviously increased, and further hydrolysis is difficult to occur in intestinal juice, so that the product can reach the large intestine for glycolysis. Under the action of gastric juice, only a small part of starch undergoes acidolysis, the crystallinity does not change obviously, and the proportion of a crystallization area is obviously increased after intestinal juice treatment, which indicates that amylase hydrolyzes an amorphous area on the surface of HS particles, and the proportion of the crystallization area is increased. The intensity of the diffraction peak of HS-2 was observed to decrease around 20 ° at the diffraction angle, indicating that hydrolysis of part of the lipid complex occurred during digestion.

TABLE 12 crystallinity of digested products

C. Prebiotic activity study of corn starch sugar residue resistant starch

In the invention, an in vitro digestion residue CRS-2 of CRS (CRS-2 is digested by adopting the gastrointestinal tract static digestion mode, a digestion product CRS-2 is collected, crushed and screened by a 100-mesh sieve) is taken as a study object, glucose (GLU) and standard prebiotics-fructo-oligosaccharides (FOS) are taken as contrast, and the capability of the CRS as potential prebiotics for proliferating bifidobacterium adolescentis and bifidobacterium animalis is explored, so that basis is provided for developing and applying the CRS in the field of prebiotics.

Raw materials: tween 80 (Oleica cdapprox.70%); magnesium sulfate (more than or equal to 99.0 percent); manganese sulfate (more than or equal to 99.0 percent); l-cysteine hydrochloride (. Gtoreq.98%). The reagents were all analytically pure.

1. Experimental methods and content

1. Preparation of culture medium

Accurately weighing 10g of tryptone, 8g of beef extract, 4g of yeast powder, 2g of dipotassium hydrogen phosphate, 1g of tween-80, 5g of sodium acetate, 2g of tri-ammonium citrate, 0.2g of magnesium sulfate and 0.04g of manganese sulfate, dissolving the nutrient components in distilled water, adjusting the pH value to 6.8+/-0.2, and then fixing the volume to 1L, and placing the mixture at the temperature of-20 ℃ for standby.

Basal medium: glucose was used as a fermentation carbon source, and the glucose concentration was controlled to 20mg/mL based on sugar-free MRS medium.

Improving a culture medium: based on sugar-free MRS culture medium, CRS-2 and FOS are respectively added as fermentation carbon sources, and the fermentation carbon sources are sterilized for standby.

2. Preparation of physiological saline

Accurately weighing 1.7g of sodium chloride, dissolving in distilled water, fixing the volume to 200mL, preparing physiological saline with the concentration of 0.85%, placing in 121 ℃ for autoclaving for 20min, and adding thermosensitive L-cysteine hydrochloride by using membrane filtration to ensure that the final concentration is 0.05% (m/v).

3. Activation of bacterial species

Activation of bifidobacterium adolescentis: dissolving 10 capsule powders in sterilized basic culture medium, placing in an anaerobic tank, and standing at 37deg.C for culturing for 48 hr to complete strain activation, and activating strain of 2 generations for subsequent experiment.

Activation of bifidobacterium animalis BB-12: breaking glass tube filled with bacteria powder, sucking physiological saline with sterile straw to blow bacteria powder to uniformity, sucking bacteria liquid into basic culture medium, and culturing under the same condition as Bifidobacterium adolescentis.

4. Preparation of experimental bacterial liquid

Centrifuging the strain subjected to passage 2 times at a rotating speed of 3000rmp for 5min, removing the supernatant, adding physiological saline, blowing uniformly to remove metabolites adhered to the strain, centrifuging again to obtain strain precipitate, repeating the steps 2 times, and re-suspending the strain in 10mL physiological saline.

5. Resistant starch medium optimization

Based on sugar-free MRS culture medium, adding CRS digestion product as fermentation carbon source, controlling CRS-2 concentration to be 0%, 0.5%, 1%, 2%, 4% (m/v), and sterilizing the improved culture at 121deg.C for 20min. Accurately sucking 1mL of experimental bacterial liquid into 10mL of improved culture medium, placing the system into an anaerobic tank, standing and culturing for 48h under the anaerobic condition at 37 ℃, sampling and measuring the number of viable bacteria at 0 and 48h respectively, and monitoring the pH value change.

6. Growth curve of cell

CRS-2 medium with optimal proliferation effect in step 5 was prepared, and GLU and FOS medium with the same carbon source concentration were prepared as controls based on sugar-free MRS medium. Accurately sucking 1mL of experimental bacterial liquid into 10mL of culture medium, sampling and measuring the number of living bacteria at 0, 4, 8, 12 and 24 hours respectively, taking the culture time as an abscissa and the number of living bacteria as an ordinate, and drawing growth curves of the bacterial bodies in different carbon source culture mediums.

7. Viable count

Taking the physiological saline prepared in the step 2 as a diluent, sucking 900 mu L of physiological saline, adding the diluent into a sterile centrifuge tube containing 100 mu L of bacterial liquid, uniformly mixing, and carrying out 10 < -1 > -10 < -7 > dilution. And (3) coating 100 mu L of bacterial solutions with different dilutions on an MRS agar plate, wherein 2 plates are coated for each dilution as a parallel, performing anaerobic culture for 48 hours in an inverted manner, counting and counting the plates with colony numbers within a range of 30-300, and controlling the dilution coating time within 2 hours.

8. Determination of the pH value of the fermentation liquor

The fermentation broth was centrifuged (3000 rmp,10 min) and the supernatant was taken, and the pH of the supernatant was measured using a pH meter and recorded.

9 data processing

Three sets of replicates were set up for each experiment and the significance analysis was performed using SPSS22.0 software, origin9.0 software to process the data pattern.

2. Results and discussion

1. CRS-2 Medium optimization

The results of measuring the viable count of the two strains inoculated by the plate dilution method are shown in Table 13.

TABLE 13 viable count of stock solutions

The effect of different carbon source concentrations on the proliferation of bifidobacteria is shown in fig. 14, when the strains are fermented by using CRS-2 as a carbon source, the proliferation conditions of different strains are obviously different, the activity of the bifidobacteria is increased along with the increase of the carbon source concentration, when the carbon source concentration is 2%, the maximum viable count of both strains is maintained, the number of the adolescent bifidobacteria and BB-12 is respectively increased to 405 times and 22.5 times of the number of original bacterial colonies, at the moment, the carbon source concentration is continuously increased, the activity of the strains is reduced, and the possible reasons are that the osmotic pressure is increased due to excessive carbon source, the cells of the bacteria are broken due to dehydration, and the number of the viable bacteria is reduced along with the dehydration.

From FIG. 14, it can be observed that CRS-2 significantly stimulates the growth of bifidobacteria adolescentis, while BB-12 grows more gradually, indicating that the different species have different abilities to utilize resistant starch, probably due to the lack of RS-specific transport and hydrolysis systems in part of the bacterial cells.

The ability of the bacteria to adhere to the starch surface is considered to be a primary condition for fermentation. Anaerobic bacteria located in the intestinal tract are able to efficiently utilize undigested carbohydrates to produce short chain fatty acids, providing the necessary energy source for the microorganism and host. RS can be fermented by beneficial bacteria at the colon to produce short chain fatty acids such as acetate, butyrate and propionate, resulting in a decrease in the pH of the fermentation broth, and thus the pH of the fermentation broth is related to the metabolism of the bacteria, and changes in pH can reflect to some extent the proliferative capacity of different carbon source concentrations. The trend of pH value change along with the concentration of the carbon source is observed to be consistent with the number of bacterial colonies, when the activity of bacterial liquid is higher, the release of short chain fatty acid and organic acid is facilitated, so that the pH value of fermentation liquid is reduced, the activity of BB-12 bacterial liquid is lower than that of bifidobacterium adolescentis, but the pH value is reduced more remarkably, and the pH value is related to different acidification characteristics of bacterial strains.

2. Growth curve of cell

The effect of different carbohydrates as fermentation carbon sources on bifidobacterium growth is shown in figure 15. No obvious increase in colony count of bifidobacteria in all components 8h before culturing is observed, which indicates that the strain is in a growth lag phase, the culture time is prolonged continuously, the colony count is increased rapidly, and the strain grows into a log phase. The bifidobacteria of different species have different growth capacities, and the number of viable bacteria of the bifidobacteria adolescentis after fermentation is high, which is possibly related to the growth characteristics of the strains.

When the bifidobacterium adolescentis uses the CRS-2 as a fermentation carbon source, the growth rate of the thalli is higher than that of the GLU culture medium, the proliferation effect is 1.9 times that of the GLU culture medium, 66.71% of the FOS culture effect is achieved, and the RS has the capability of rapidly proliferating the bifidobacterium adolescentis, has relatively abundant sugar residue resources and low price, and can replace the FOS to become a new generation of prebiotics.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

1. The preparation method of the corn starch sugar residue resistant starch is characterized by comprising the following steps: repeatedly freezing and thawing the settled corn starch sugar residue at low temperature, adding 5-15 times of water, colloid milling, washing with water, centrifuging to obtain precipitate, and drying to obtain the corn starch sugar residue resistant starch.

2. The method for preparing corn starch sugar residue resistant starch according to claim 1, wherein the freezing parameters are: the temperature is between minus 60 ℃ and minus 18 ℃ and the time is between 24 hours and 72 hours.

3. The method for preparing corn starch sugar residue resistant starch according to claim 1, wherein the thawing parameters are: the temperature is 0-16 ℃.

4. The method for producing corn starch sugar residue resistant starch according to claim 1, wherein the number of times of freezing and thawing at low temperature is 2 to 3.

5. The method for preparing corn starch sugar residue resistant starch according to claim 1, wherein the colloid mill water washing time is 2-10min; the centrifugal rotating speed is 4000-5000rpm, and the time is 15-30min; the drying temperature is 50-85 ℃ and the drying time is 3-24h.

6. A corn starch sugar residue resistant starch prepared by the method for preparing a corn starch sugar residue resistant starch of claims 1-5.

7. Use of the corn starch sugar residue resistant starch of claim 6 for preparing a hypoglycemic food.

8. Use of the corn starch sugar residue resistant starch of claim 6 for the preparation of a prebiotic food.