WO2021114694A1 - Method for preparing arrowhead resistant starch by ultrasound synergistic pullulanase - Google Patents

Abstract

Provided is a method for preparing arrowhead resistant starch by ultrasound synergistic pullulanase, wherein the method comprises the following steps: (1) cleaning and arranging the arrowhead; (2) extracting starch from the arrowhead; (3) preparing the resistant starch; (4) performing enzymolysis in an ultrasonic field, and freeze-drying to obtain the arrowhead resistant starch. In the preparation process of the arrowhead resistant starch by the pullulanase method, the method accelerates the movement of solvent molecules through the cavitation and mechanical vibration of the ultrasonic wave, and solves the problem of low enzymatic hydrolysis reaction efficiency in the preparation process of the resistant starch by the enzymatic method.

Description

Ultrasound synergized with pullulanase to prepare sage-resistant starch method Technical field

The present invention relates to the technical field of food processing, and particularly refers to a kind of Aquilaria starch as a raw material, which adopts ultrasonic and pullulan enzymatic treatment to prepare Astragalus resistant starch.

Background technique

Cigu is a perennial, perennial shallow-water herb of Ciguchiaceae in the Alisma family. It is an important aquatic vegetable in China. It is planted in various parts of China, mainly distributed in the Yangtze River Basin and other provinces to the south, with a cultivation area of about 20,000 hm ² per year. As a seasonal vegetable with high moisture content, Cigu is not easy to store. At present, Ci aunts are generally eaten directly as vegetables, with little processing. Cigu has a bitter taste and the market price is low. Therefore, Cigu's deep processing plays a very important role in improving Cigu's economic value and market competitiveness. Cigu is a low-fat, high-carbohydrate food. The starch content of Cigu is about 50% of the dry matter. Among them, 30.10% amylose is a good raw material for preparing resistant starch.

In recent years, people are concerned about health, and resistant starch has also become a new research object. Resistant Starch (RS) is defined as starch that cannot be absorbed by the small intestine of healthy humans, but can be fermented by the microbial flora in the large intestine. Resistant starch has a variety of physiological functions: Resistant starch participates in blood sugar regulation, weight control, lipid metabolism, relieves gastrointestinal diseases, and promotes the absorption of minerals and vitamins. Resistant starch has good physical and chemical properties for food processing. Resistant starch is in the form of white powder and has no peculiar smell. Adding an appropriate amount will not affect the sensory evaluation of food. Therefore, resistant starch has a wide range of applications in the food industry. Resistant starch has good heat resistance and high gelatinization temperature, which is convenient for promotion in food processing. Resistant starch has weak water holding capacity and is suitable to be added to low-humidity bakery foods, and is easy to control in production; resistant starch can be As a substrate for probiotics and bacteria preservatives, it can promote the growth of probiotics and ensure an appropriate amount; resistant starch has excellent physiological functions, and is widely used in the development of health foods in health foods.

Resistant starches are divided into four categories: physically embedded starch (RS1), resistant starch granules (RS2), retrograded starch (RS3), and chemically modified starch (RS4). The structure of RS1 and RS2 is easy to be destroyed after heating and gelatinization, so that the resistance to amylase disappears, and it is difficult to be used in the actual production process. Both RS3 and RS4 can be prepared in large quantities by processing native starch, but the chemical reagents added during the preparation of RS4 will affect food safety. Since the retrograded resistant starch RS3 is formed by recrystallization in the cooling process after starch gelatinization, it will not cause food safety problems, and the preparation process is easy to control, so it is the most promising.

The preparation methods of RS3 mainly include biochemical method and physical method. Common biochemical methods include acid debranching and enzyme debranching. The physical methods include heat treatment, extrusion treatment, microwave radiation, and ultrasonic treatment. Chemical modification is quick and simple, but it is usually not environmentally friendly, and it is easy to produce chemical residues in the product. Enzymes are sensitive to temperature, pressure, pH, and salt ions, and it is often difficult to control enzyme treatment. Enzymatic debranching often uses pullulanase, isoamylase and other debranching enzymes, and currently pullulanase is commonly used. Pullulanase, as a debranching enzyme, is also a kind of isoamylase, which can hydrolyze the α-1,6 glycosidic bonds in pullulan, pullulan and polysaccharides in an endoscopic manner. But the enzymolysis time is long. Relevant studies have shown that the yield and efficiency of resistant starch prepared by using physical or biochemical methods alone is low. Guo Xing et al. studied the enzymatic method to prepare cassava resistant starch content of 14.52%. After the starch paste is enzymatically treated and cooled to room temperature, ultrasonic treatment is added, and the content of cassava resistant starch increases to 19.19% (Guo Xing, Wen Qibiao. Enzymatic treatment and ultrasonic treatment against the formation of enzymatic starch[J].Modern Food Technology , 2007(01): 8-10.). Li Weiqing et al. researched that the resistant starch produced by ultrasound was 12.42%. On the basis of ultrasonic treatment, the technology of enzymatically hydrolyzing resistant starch was optimized, and the content of resistant starch was 18.57% (Li Baoyu. Purple potato resistant starch) Preparation, properties and research on the proliferation effect of bifidobacteria[D]. Fujian Agriculture and Forestry University, 2015.).

Ultrasound is a physical processing method with a frequency higher than the threshold of human hearing. In recent years, ultrasound has aroused widespread interest in basic food research and commercial applications. Ultrasonic treatment has shown beneficial effects in food processing and preservation, including higher product yield, shortened processing time, reduced operation and maintenance costs, improved quality, and reduced pathogens. Ultrasound not only improves the quality and safety of food, but also provides an opportunity to create new products with unique properties. The application of ultrasound in starch systems is mostly a liquid-solid two-phase system with water as the medium. The sound energy of ultrasound cannot be absorbed by molecules, so it is converted. A chemically usable form is achieved through cavitation. Ultrasonic waves produce ultrasonic cavitation in the solution and produce microbubbles. When the microbubbles burst, high energy is released and converted into high pressure and high temperature, producing physical and chemical effects. The physical effects include strong micro-jets, shear forces, shock waves generated by bubble bursts, and sound waves. The chemical effect is caused by free radicals, such as hydroxide (OH) and hydrogen (H) radicals, generated by the decomposition of water molecules in cavitation caused by bubble collapse. The cavitation effect produces strong local shear, high temperature, and free radicals, which reduces the viscosity of starch paste and breaks the C-C bonds in starch molecules.

Both the physical method and the biochemical method are used to reduce the viscosity of the starch system after gelatinization, increase the ratio of amylose, and increase the concentration of suitable chain-length starch chains in the system. Therefore, in the actual production process, the physical method and the biochemical method are often used in combination to improve the production efficiency of resistant starch. Studies have shown that the ultrasonic wave combined with enzymatic method is better for improving the yield of resistant starch and shortening the time of enzymatic hydrolysis than the enzymatic method for preparing resistant starch. Hu found that ultrasonic water bath and α-amylase act on mung bean starch at the same time, which is easier for starch retrogradation (Hu AJ, Li Q, Zheng J, Yang L and Qin ZP, Study on structure of hydroxypropyl tapioca starch phosphate ester prepared by ultrasound.Cereals Oils 1:13-15 (2012)). Lu et al. found that pullulanase and ultrasonic debranching treatment produced a synergistic effect, increased the number of linear chains in pea starch, and effectively increased the content of SDS and RS (Lu Zhan-Hui, Belanger Nicholas, Donner Elizabeth, etc.). Debranching of pea starch using pullulanase and ultrasonication synergistically to enhance slowly digestible and resistant starch[J].Food Chemistry:S0308814618310884-.). At present, the research of ultrasonic-enzymatic preparation of resistant starch mainly focuses on the step-by-step processing. There are few reports on the simultaneous use of ultrasound and enzymatic hydrolysis of starch. There is no report on the preparation of RS3 type resistant starch from Cigu by ultrasonic and enzymatic method. You Manjie et al. researched that the content of resistant starch prepared by acid method-microwave method was 10.99%, and the yield of resistant starch was not high. (You Manjie, Liu Xin, Zhao Lichao, et al. Study on the preparation of Cigu resistant starch by acid hydrolysis-microwave method[J].Food Industry Science and Technology,2009(12):262-264.).

The present invention introduces advanced multi-mode ultrasonic technology. It is hoped that ultrasonic and pullulanase will synergistically hydrolyze the starch molecules of Ciguchi. The process is accompanied by degradation and enzymatic hydrolysis of starch molecules. Ultrasound degrades starch while also promoting enzymatic hydrolysis. In order to solve the problems of low enzymolysis reaction efficiency, long enzymolysis time, and large enzyme consumption in the process of preparing resistant starch by the enzymatic method, the yield of resistant starch can be improved.

Summary of the invention

In order to solve the above-mentioned problems, on the basis of the pullulan enzymatic preparation of Cigu resistant starch, ultrasonic and enzymatic treatment of Cigu starch was carried out to study the effect on the yield of Cigu resistant starch.

The method of the present invention for preparing the resistant starch with ultrasonic and pullulanase is carried out according to the following steps:

(1) Wash the fresh auntie, peel, remove the tail and cut into pieces, and weigh. Add equal volume of distilled water at 4°C, beat in a juicer, and filter the beaten slurry through a sample sieve (200 mesh sieve→300 mesh sieve) to remove fibers;

(2) Put the filtrate in a beaker and let it stand overnight, remove the supernatant, wash the precipitate with 0.2% NaOH solution to remove protein, adjust to pH=7 with a 0.1mol/L hydrochloric acid solution, and place it in 250ml centrifugation Centrifuge in the cup, discard the supernatant, scrape off the non-white impurities on the remaining solid surface, and then repeatedly wash with deionized water until all the impurities are removed;

(3) Drying at 50°C, pulverizing, passing through a 100-mesh sieve, and finally preparing Cigu starch, encapsulating, and storing in a desiccator.

(4) Accurately weigh the Aquilaria starch in an Erlenmeyer flask to prepare a starch suspension (4%, m/m), and place it in an autoclave for high temperature treatment at 121°C for 20 minutes.

(5) Put it in a 50℃ constant temperature water bath, adjust the pH value to 5.0 with 0.05mol/L HCl solution, add pullulanase, while applying ultrasonic treatment, perform debranching treatment to prepare Cigu resistant starch, keep 50℃ water bath Conditions for enzymatic hydrolysis.

(6) Cool to room temperature, then pour the sample into a petri dish, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator.

The addition amount of pullulanase described in step (5) is 6 npun/g (starch)-58 npun/g (starch); preferably, the addition amount of enzyme is 32 npun/g (starch).

Wherein, the enzymolysis time of pullulanase described in step (5) is 8h-40h; preferably, the enzymolysis time is 24h.

Wherein the specific parameters of the ultrasound effect in step (5) are ultrasound time 5min-40min, preferably ultrasound time 10min; ultrasound power 60W-300W, preferably ultrasound power 240W; ultrasound frequency 20kHz, 40kHz, 60kHz, 20/60kHz, 40/60kHz, preferably ultrasonic frequency 60kHz; ultrasonic intermittent ratio 1:8 (ultrasonic 5-40s, intermittent 5s); preferably ultrasonic intermittent ratio is 20s/5s.

The beneficial effects of the present invention are:

(1) The physical method used in the present invention is ultrasonic treatment. Ultrasonic treatment has shown beneficial effects in food processing and preservation, including higher product yield, shortened processing time, reduced operation and maintenance costs, etc. It is a new physical method of starch modification.

(2) The present invention uses a multi-mode ultrasonic treatment technology in the preparation process of the pullulan enzymatic method to prepare Cigu resistant starch. Ultrasound promotes the enzymatic hydrolysis of pullulanase, in order to solve the problem of low enzymatic hydrolysis reaction efficiency in the process of preparing resistant starch by enzymatic method.

(3) The present invention uses Cigu starch as a raw material to prepare Cigu resistant starch. Because resistant starch has multiple physiological functions and good food processing characteristics, it is beneficial to the comprehensive utilization of Cigu starch and increases its added value.

Description of the drawings

Figure 1 is the structure diagram of the multi-mode ultrasonic biological treatment equipment of the present invention, in which 1, 2, 3 are ultrasonic vibration plates, 4 is a liquid container, 5 is a water bath, 6 is a temperature probe, 7 is a circulating pump, and 8 is a computer Program controller, 9, 10, 11 are ultrasonic controllers.

Figure 2 is the XRD diffraction pattern of Cigu resistant starch. From top to bottom, the ultrasound-assisted preparation of Cigu resistant starch (CS), the non-ultrasound-assisted preparation of Cigu resistant starch (WCS) and native starch (YDF) are respectively X-ray diffraction patterns of three samples.

Figure 3 shows the scanning electron micrographs of three samples of raw starch (YDF), WCS prepared without ultrasound assistance, and CS resistant starch (CS) prepared with ultrasound assistance, A: YDF x1000; B: YDF x8000 ; C: WCS x1000; D: WCS x8000; E: CS x1000; F: CS x8000.

Detailed ways

Unless otherwise specified, the terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be described in further detail below in conjunction with specific embodiments and with reference to data. It should be understood that these examples are only for exemplifying the present invention, rather than limiting the scope of the present invention in any way.

Figure 1 is the multi-mode ultrasonic biological treatment equipment of the present invention. The equipment is equipped with a computer program controller 8, which can set ultrasonic working parameters (ultrasonic power density, frequency, pulse working time, intermittent time and total treatment time) to be controlled separately Three ultrasonic controllers 9, 10, 11, respectively connected to three different frequency ultrasonic vibration plates 1, 2, 3, can achieve single frequency / two frequency / three frequency ultrasonic treatment; put the solution to be processed into the liquid Single-frequency/dual-frequency/multi-frequency ultrasonic treatment is performed in the device 4, and the circulating pump 7 is started to circulate the solution. The automatic control of the temperature of the solution is realized by the water bath 5 and the temperature probe 6.

Experimental material: Cigu purchased from Kaiyuan Tourism Supermarket of Jiangsu University

Pullulanase was purchased from sigma company in the United States (enzyme activity: 1000npun/ml)

The Aquila starch of the present invention is obtained from Aquila:

(1) Wash the fresh auntie, peel, remove the tail and cut into pieces, and weigh (about 5000g). Add equal volume of distilled water at 4°C, beat in a juicer, and filter the beaten slurry through a sample sieve (200 mesh sieve→300 mesh sieve) to remove fibers;

(2) Put the filtrate in a beaker and let it stand overnight, remove the supernatant, wash the precipitate with 0.2% NaOH solution to remove protein, adjust to pH=7 with a 0.1mol/L hydrochloric acid solution, and place it in 250ml centrifugation Centrifuge in the cup, discard the supernatant, scrape off the non-white impurities on the remaining solid surface, and then repeatedly wash with deionized water until all the impurities are removed;

(3) Dry, dry at 50°C, pulverize, and pass through a 100-mesh sieve to finally obtain Cigu starch, encapsulate, and store in a desiccator to obtain 300g of dried Cigu starch, which is native starch (YDF).

Example 1: Screening of the amount of pullulanase for preparing the resistant starch pullulanase

(1) Accurately weigh 2g of Cigu starch in a 100ml Erlenmeyer flask to prepare a starch suspension (4%, m/m), and place it in an autoclave at 121°C for 20 minutes.

(2) Put it in a 50°C constant temperature water bath, adjust the pH value with 0.05mol/L HCl solution, add pullulanase, the amount of which is shown in Table 1, and perform debranching treatment at 50°C in a water bath for 24 hours.

(3) Cool to room temperature, then pour the sample into a petri dish, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator. Refer to the national standard NY-T 2638-2014 to determine the content of Cigu resistant starch.

See Table 1 for the yield of resistant starch of Cigu. With the increase of the amount of pullulanase, the yield of resistant starch shows a trend of first increasing and then stabilizing. When the amount of pullulanase is 32npun/g (starch) , Continue to increase the amount of enzyme added, the yield of resistant starch remains stable, and the change is not significant. The enzyme addition amount is 32npun/g (starch), and the yield of resistant starch is 21.36% (compared to Agu starch, the content of resistant starch is increased by 16%.), which is used for subsequent test condition screening.

Table 1: The effect of different enzymes on the yield of resistant starch

Enzyme addition amount (npun/g (starch))	0	6	19	32	45	58
Resistant starch yield (%)	5.36	16.42	20.32	21.36	21.47	22.24

Example 2: Screening of enzymatic hydrolysis time for preparation of Cigu resistant amylase by pullulan enzymatic method

(1) Accurately weigh 2g of Amygdalus starch in a 100ml Erlenmeyer flask to prepare a starch suspension (4%, m/m), and place it in an autoclave at 121°C for 20 minutes.

(2) Put it in a 50℃ constant temperature water bath, adjust the pH value with 0.05mol/L HCl solution, add 32npun/g (starch) of pullulanase, debranching treatment under 50℃ water bath, enzyme See Table 2 for solution time.

(3) Cool to room temperature, then pour the sample into a petri dish, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator. Refer to the national standard NY-T 2638-2014 to determine the content of Cigu resistant starch.

See Table 2 for the yield of resistant starch of Ciguin. With the increase of pullulan enzyme hydrolysis time, the yield of resistant starch shows a trend of increasing first and then stable. After pullulanase enzymatic hydrolysis for 24 hours, continue to increase enzymatic hydrolysis. Over time, the yield of resistant starch remained stable and did not change significantly. The enzymatic hydrolysis time was selected to be 24h, and the yield of resistant starch was 21.29% (compared to Agu starch, the content of resistant starch increased by 15.93%.), which was used for the screening of subsequent test conditions.

Table 2: The effect of different enzymatic hydrolysis time on the yield of resistant starch

Enzymolysis time (h)	0	8	16	twenty four	32	40
Resistant starch (%)	5.36	18.89	20.15	21.29	21.96	22.10

Example 3: Ultrasound and Pullulan Enzyme Method for Preparation of Sugu Resistant Starch Ultrasound Time Screening

(1) Accurately weigh 2g of Aquilaria starch into a 100ml Erlenmeyer flask, prepare a starch suspension (4%, m/m), and place it in an autoclave at 121°C for 20 minutes.

(2) Put it in a 50℃ constant temperature water bath, adjust the pH to 5.0 with 0.05mol/L HCl solution, add 32npun/g (starch) of pullulanase, under the action of ultrasound, carry out debranching treatment, keep the 50℃ water bath Conditions, continue enzymatic hydrolysis to 24h. Among them, the ultrasonic frequency (60KHZ), the ultrasonic period is 30s/5s, the ultrasonic power is 180w, and the ultrasonic time is shown in Table 3.

(3) Cool to room temperature, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator. Refer to the national standard NY-T 2638-2014 to determine the content of Cigu resistant starch

See Table 3 for the yield of resistant starch of Cigu. As the ultrasound time increases, the yield of resistant starch shows a trend of first increasing and then decreasing. When the ultrasound time is 10 min, the yield of resistant starch is the highest 23.89% (compared to the pullulan enzyme treatment, the content of resistant starch is increased by 2.6%.), the ultrasound time is 10 min, which is used for the screening of subsequent test conditions .

Table 3: The effect of different ultrasound time on the yield of Cigu resistant starch

Ultrasound time (min)	0	5	10	20	30	40
Resistant starch (%)	21.29	21.94	23.89	22.87	22.46	20.85

Example 4: Ultrasound and Pullulan Enzyme Method for the Screening of Ultrasonic Power for the Preparation of Sugu resistant Starch

(1) Accurately weigh 2g of Aquilaria starch into a 100ml Erlenmeyer flask, prepare a starch suspension (4%, m/m), and place it in an autoclave at 121°C for 20 minutes.

(2) Put it in a 50℃ constant temperature water bath, adjust the pH to 5.0 with 0.05mol/L HCl solution, add 32npun/g (starch) of pullulanase, under the action of ultrasound, carry out debranching treatment, keep the 50℃ water bath Conditions, continue enzymatic hydrolysis to 24h. The ultrasonic frequency (60kHz), the ultrasonic period is 30s/5s, the ultrasonic time is 10min, and the ultrasonic power is shown in Table 3.

(3) Cool to room temperature, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator. Refer to the national standard NY-T 2638-2014 to determine the content of Cigu resistant starch

See Table 4 for the yield of resistant starch of Cigu. With the increase of ultrasonic power, the yield of resistant starch showed a trend of first increasing and then decreasing. When the ultrasonic power is 240W, the yield rate of resistant starch is the highest 24.94% (compared to the pullulan enzymatic treatment, the resistant starch content is increased by 3.65%.), the ultrasonic power is selected as 240W, which is used for the screening of subsequent test conditions .

Table 4: The influence of different ultrasonic power on the yield of Cigu resistant starch

Ultrasonic power W	0	60	120	180	240	300
Resistant starch yield%	21.29	21.41	22.27	23.20	24.94	22.47

Example 5: Screening of Ultrasonic Frequency for Preparation of Cigu Resistant Starch by Ultrasound and Pullulan Enzyme Method

(1) Accurately weigh 2g of Amygdalus starch into a 100ml Erlenmeyer flask, prepare a starch suspension (4%, m/m), and place it in an autoclave at 121°C for 20 minutes.

(2) Put it in a 50℃ constant temperature water bath, adjust the pH value to 5.0 with 0.05mol/L HCl solution, add 32npun/g (starch) of pullulanase, perform debranching treatment under the action of ultrasound, and keep the 50℃ water bath Conditions, continue enzymatic hydrolysis to 24h, in which the ultrasonic power (240w), the ultrasonic period is 30s/5s, the ultrasonic time is 10min, and the ultrasonic frequency is shown in Table 5.

(3) Cool to room temperature, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator. Refer to the national standard NY-T 2638-2014 to determine the content of Cigu resistant starch

See Table 5 for the yield of resistant starch of Cigu. As the ultrasound time increases, the yield of resistant starch shows a trend of first increasing and then decreasing. When the ultrasonic frequency was 60kHz, the yield of the resistant starch was the highest 23.89% (compared to the pullulan enzyme treatment, the resistant starch content increased by 2.2%.), the ultrasonic frequency was selected as 60kHz for the subsequent screening of experimental conditions.

Table 5: The influence of different ultrasonic frequencies on the yield of A. vulgaris resistant starch

Ultrasonic frequency (kHz)	0	20	40	60	20/60	40/60
Resistant starch yield (%)	21.29	22.14	23.09	23.49	22.50	21.62

Example 6: Screening of ultrasonic intermittent ratio for preparation of Sugu resistant starch by ultrasonic and pullulan enzyme method

The parameters of the ultrasonic intermittent ratio experiment are shown in Table 6.

(1) Accurately weigh 2g of Amygdalus starch in a 100ml Erlenmeyer flask, prepare a starch suspension (4%, m/m), and place it in an autoclave at 121°C for 20 minutes.

(2) Put it in a 50℃ constant temperature water bath, adjust the pH to 5.0 with 0.05mol/L HCl solution, add 32npun/g (starch) of pullulanase, under the action of ultrasound, carry out debranching treatment, keep the 50℃ water bath Conditions, continue enzymatic hydrolysis to 24h, in which the ultrasonic power is 240W, the ultrasonic frequency is 60kHz, the ultrasonic time is 10min, and the ultrasonic intermittent ratio is shown in Table 6.

(3) Cool to room temperature, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator. Refer to the national standard NY-T 2638-2014 to determine the content of Cigu resistant starch

The yield of resistant starch of Cigu is shown in Table 6. As the ultrasonic intermittent ratio increases, the yield of resistant starch shows a trend of first increasing and then decreasing. When the ultrasonic intermittent ratio is 20s/5s, the yield of resistant starch is the highest 25.80% (compared to the pullulan enzyme treatment, the content of resistant starch is increased by 4.51%.), the ultrasonic frequency is selected as 60kHz for the follow-up Screening of test conditions.

Table 6: The effect of different ultrasonic intermittent ratios on the yield of resistant starch of Cigu

Ultrasonic interval ratio (s/5s)	0	5	10	20	30	40
Resistant starch yield (%)	21.29	21.88	22.93	25.80	22.62	21.95

.

Structural Characterization and Characteristic Analysis of Experimental Cases of Cigu Resistant Starch

A: Sample preparation: Sugu resistant starch (CS) prepared by ultrasound

(1) Accurately weigh 2g of Cigu starch in a 100ml Erlenmeyer flask to prepare a starch suspension (4%, m/m), and place it in an autoclave at 121°C for 20 minutes.

(2) Put it in a 50℃ constant temperature water bath, adjust the pH to 5.0 with 0.05mol/L HCl solution, add 32npun/g (starch) of pullulanase, under the action of ultrasound, carry out debranching treatment, keep the 50℃ water bath Conditions, continue enzymatic hydrolysis to 24h, where the ultrasonic-assisted enzymatic hydrolysis conditions are: ultrasonic time 10min, ultrasonic power 240W, ultrasonic frequency 60KHZ, ultrasonic intermittent ratio 20s/5s.

(3) Cool to room temperature, then pour the sample into a petri dish, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator. Refer to the national standard NY-T 2638-2014 to determine the content of Cigu resistant starch. The content of resistant starch is 25.8%. This sample is used for subsequent structural characterization and characteristic analysis.

B: Sample preparation: WCS resistant starch prepared without ultrasound assistance

(1) Accurately weigh 2g of Cigu starch in a 100ml Erlenmeyer flask to prepare a starch suspension (4%, m/m), and place it in an autoclave at 121°C for 20 minutes.

(2) Put it in a 50℃ constant temperature water bath, adjust the pH value to 5.0 with 0.05mol/L HCl solution, add 32npun/g (starch) of pullulanase, maintain 50℃ water bath conditions, and enzymatically hydrolyze for 24h. (No ultrasonic treatment)

(3) Cool to room temperature, then pour the sample into a petri dish, age at 4°C for 24 hours, freeze-dry for 48 hours, grind, package, and store in a desiccator. Refer to the national standard NY-T 2638-2014 to determine the content of Cigu resistant starch. The content of resistant starch is 21.29%. This sample is used for subsequent structural characterization and characteristic analysis.

Among them, in Figure 2-3 and Table 7-8, the three samples of Sugu resistant starch (CS) prepared with ultrasound assistance, Sugu resistant starch (WCS) and native starch (YDF) prepared without ultrasound assistance, refer to the above Samples prepared by different methods.

(1) Thermal characteristics analysis

Experimental conditions: accurately weigh 2.5 mg of starch sample in an aluminum sample pan with a one-ten thousand balance, add 7.5 microliters of deionized water, seal the sample pan and equilibrate at room temperature for 1 hour, put it into the sample holder in the instrument, and seal it with The empty aluminum box is used as a reference. Scan range: 30～150℃; scan rate: 10℃/min; atmosphere: high purity nitrogen, flow rate: 40mL/min; record the scan and calculate the start temperature To, peak temperature Tp, end temperature Tc and Tc on the endothermic curve Range temperature Tr.

Thermal characteristics (DSC) gelatinization parameters are affected by the molecular structure of amylopectin, the ratio of amylose to amylopectin, the ratio of crystalline to amorphous or a combination thereof. In this study, the DSC curves of three samples of raw starch (YDF), Sugu resistant starch (WCS) prepared without ultrasound assistance, and Sugu resistant starch (CS) prepared with ultrasound assistance were determined. Among them, the T of YDF, WCS and CS starch ₀ (initial gelatinization temperature), T _P (peak gelatinization temperature), T _C (end gelatinization temperature), T _C -T ₀ (transition temperature range), and ΔH (gelatinization enthalpy) are shown in Table 13. The gelatinization temperature reflects the stability of the crystal. T _{0 is} related to the melting temperature of the weakest crystallization in starch granules, and T _C is related to the melting temperature of high perfect crystallization. It can be seen from Table 7 that for _{the changes of T 0} and T _C , YDF>CS>WCS. In the process of starch samples processed by WCS and CS, the original crystals were destroyed, resulting in too short linear molecules, which regenerated and rearranged to form a crystal structure with low stability. _{The T C} -T _{0 of the} ultrasonic-treated starch samples was the smallest, followed by the WCS-treated starch samples. The transition temperature is affected by the molecular structure of the crystal region. Decreasing the value of T _C -T ₀ reduces the diversity of RS3 crystals and improves the integrity of the helical ordered structure. The reduction in the transformation temperature range of the treated starch sample indicates that its uniformity is improved and the crystallites are complete. The enhancement of the perfection of the crystallites is the reason for the enhancement of the anti-digestibility of starch. The gelatinization enthalpy (ΔH) mainly reflects the loss of double helix sequence in starch granules rather than the loss of crystallization. Starches containing a lot of amylose require more energy to break the intermolecular bonds in the crystallization zone. On the contrary, amylopectin is generally distributed in the amorphous region, and its crystal size is small and easy to melt. The ΔH of CS-treated starch samples was the largest, followed by WCS-treated starch samples. During the processing of WCS and CS samples, the content of linear starch with suitable chain length increases, which is conducive to the formation of double helix structure, and ΔH increases. The increase in ΔH can be explained by hydrogen bonds and other intermolecular forces that increase the order and stability of the double helix structure. The phase transition temperature and endothermic enthalpy increase with the steady increase of crystallinity and double helix structure. The increase in T ₀ , T _P , T _C and ΔH and _{the decrease in T C} -T ₀ of the CS-treated sample are compared with the WCS-treated sample to reflect that the crystals of the CS-treated sample have a higher degree of perfection.

Table 7 DSC parameters of Cigu resistant starch

(2) X-ray diffraction analysis

Experimental conditions: X-ray diffraction measurement conditions: Cu target is selected as characteristic ray; measuring angle range is 5°～30°; scanning speed is 5°/min. The difference in sample moisture content does not affect the X-ray diffraction pattern.

XRD is more sensitive to the long-term crystalline structure of starch granules composed of repeating double helix units. The X-ray diffraction pattern may depend on the source of starch as well as environmental growth conditions and processing conditions. According to the XRD pattern, starch crystallization can be divided into A type, B type and C type at present. The A-type crystal structure is composed of short chains, usually found in cereal starches, while fruit and tuber starches are B-type structures, which are composed of long chains. The C-type crystal structure is a combination of A-type and B-type structures, usually found in leguminous starch. The X-ray diffraction patterns and relative crystallinity of three samples of native starch (YDF), WCS without ultrasound assistance and CS resistant starch (CS) prepared with ultrasound assistance are shown in Figure 2 and Table 8. . The XRD diffraction pattern of YDF has diffraction fronts at diffraction angles of 15°, 17°, 18° and 23°, which is a typical type A crystal. WCS and CS treatments significantly changed the diffraction pattern of the sample. Diffraction peaks appeared at diffraction angles of 15°, 17°, 19°, 22° and 24°, and the crystal type changed from type A to type B. The glutinous rice starch paste (10%, w/w) was debranched by pullulanase and then regenerated at 25°C to obtain short-chain amylose crystals with a B-type X-ray pattern. The difference in crystallinity of starch samples can be attributed to the following aspects: crystal size, number of crystal regions (affected by the content of amylopectin and branch chain length), and the orientation of the double helix in the crystal regions. The degree of double helix interaction. The structure of starch molecules is divided into microcrystalline, subcrystalline and amorphous regions. Relative crystallinity is defined as the ratio of the area of the crystalline region to the area of the crystalline region plus the microcrystalline region. The relative crystallinity reflects the proportion of dense crystal regions. The crystallization area is difficult to be hydrolyzed by enzymes. Therefore, relative crystallinity can be used to measure starch resistance. The higher the relative crystallinity, the stronger the resistance of starch to enzymes. The relative crystallinity is the ratio of the crystalline area to the total diffracted area. It can be seen from the table that the relative crystallinity of YDF, WCS and CS samples are 54.7%, 52.4% and 55.6%, respectively. The crystallinity of the WCS sample is lower than that of the YDF sample, indicating that the interaction between the double helix in the YDF crystalline region is stronger, and the orientation of the crystal grains to the X-ray beam is better. The experimental results are consistent with those reported in the literature. The relative crystallinity of the CS sample is higher than that of the WCS and YDF samples, which means that the double helix strands are reoriented in a tighter manner, providing a more stable crystalline structure. Ultrasound cooperates with pullulanase to act on amygdalus starch molecules, and its debranching degree is higher. At the same time, due to the cavitation effect of ultrasound, the rupture of the chain is caused, which gives the molecular chain a higher chance of arrangement and aggregation, so that it can recombine into a double helix, thereby forming a completely crystalline structure. The increase in relative crystallinity may be due to the enhanced binding between starch chains and the disrupted double helix rearrangement in the crystallization region, which leads to an increase in crystal integrity or the formation of new crystals. The level of crystallinity is always lower than double helix, which shows that not all helix ordered starch molecules are arranged into crystals.

Table 8 XRD pattern parameters of Cigu resistant starch

(3) Scanning electron microscope

Scanning electron microscopy analysis: Fix the double-sided tape on the stage of the scanning electron microscope, dip a toothpick into a little starch sample and apply it evenly on the double-sided tape and press it gently. Then use the ear-washing ball to blow off the excess starch, place the stage in the gold-plated instrument, and use the ion sputtering coater to plate the starch sample with gold film. After 20 minutes, take out the stage and place it in the scanning electron microscope. Observe the granular morphology of starch under 8000 times and 8000 times).

Scanning electron microscopy (SEM) was used to observe the morphological characteristics of three samples of raw starch (YDF), WCS prepared without ultrasound assistance, and CS resistant starch (CS) prepared with ultrasound assistance, and different processing methods were used. The morphological characteristics of the Agu starch samples were analyzed, and the results are shown in Figure 3. Scanning electron micrographs show the shape and surface microstructure of different samples. The scanning electron micrograph of the YDF sample showed that small particles of different sizes were oval or irregular, with a smooth surface, accompanied by some protrusions and depressions, which may be due to partial damage of the starch during the starch extraction and drying process. The particle morphology of WCS and CS samples changed significantly. The grain structure of YDF is destroyed, the grain morphology is rough, irregular, the structure is dense, and the size is different. The WCS and CS samples showed a blocky and dense structure, and the particle size increased compared to YDF. Some fragments can be observed on the surface of WCS and CS samples. There are some shallow layered bands and grooves on the surface of the WCS sample. There are many deep-layered bands, holes, and irregular structures on the surface of the CS sample. The formation of non-uniform cavities may be caused by the cavitation effect of ultrasound. The changes in the surface structure caused by ultrasound can be attributed to the formation of local hot spots when the bubbles burst, as well as the shear forces formed by micro-currents and shock waves. The dense and rigid structure of starch has high resistance to enzymes. The irregular flaky shape may be formed by the leaching of amylose, the destruction of the amylopectin crystallization area and the rearrangement of the starch chain. The apparent structure of the pea starch regenerated by autoclave enzymatic hydrolysis is similar to the irregular flaky cohesive structure, and the size is not uniform. The retrogradation of starch debranching and/or degradation results in the transformation of disordered linear starch molecules to a double helix structure, which enhances the interaction between starch chains and the rearrangement of the dissociated double helix, as well as the arrangement of the three-dimensional cohesive network. The short-range and long-range order structures have changed. This compact structure and large particle size may have low sensitivity to digestive enzymes.

Claims (4)

1. The method for preparing Sugu resistant starch by ultrasonic and pullulanase is characterized in that it is carried out according to the following steps:

   (1) Wash the fresh auntie, peel, remove the tail and cut into pieces, and weigh; add an equal volume of distilled water, beaten in a juicer, and filter the beaten pulp through a sieve to remove fibers;

   (2) Put the filtrate in a beaker and let it stand overnight, remove the supernatant, wash the precipitate with 0.2% NaOH solution to remove protein, adjust to pH=7 with a 0.1mol/L hydrochloric acid solution, and place it in 250ml centrifugation Centrifuge in the cup, discard the supernatant, scrape off the non-white impurities on the remaining solid surface, and then repeatedly wash with deionized water until all the impurities are removed;

   (3) Drying at 50°C, pulverizing, passing through a 100-mesh sieve, and finally preparing amygdalus starch, packaging, and storing in a desiccator;

   (4) Accurately weigh the amygdalus starch in an Erlenmeyer flask to prepare a starch suspension with a mass concentration of 4%, and place it in an autoclave at 121°C for 20 minutes;

   (5) Put it in a 50℃ constant temperature water bath, adjust the pH value to 5.0 with 0.05mol/L HCl solution, add pullulanase and apply ultrasonic treatment at the same time, perform debranching treatment to prepare Cigu resistant starch, keep it at 50℃ Enzymatic hydrolysis under water bath conditions; the addition amount of pullulanase is 6npun/g (starch)-58npun/g (starch); the enzymatic hydrolysis time of pullulanase is 8h-40h;

   The specific parameters of ultrasonic action are ultrasonic time 5min-40min, ultrasonic power 60W-300W, ultrasonic frequency 20kHz, 40kHz, 60kHz, 20/60kHz, 40/60kHz, ultrasonic intermittent ratio 1:8 (ultrasonic 5-40s, intermittent 5s);

   (6) After cooling to room temperature and drying, the resistant starch can be obtained.
2. The method according to claim 1, wherein the method for preparing the resistant starch with pullulanase in combination with ultrasound is characterized in that the added amount of pullulanase in step (5) is 32 npun/g (starch).
3. The method according to claim 1, wherein the method for preparing the resistant starch with ultrasonic and pullulanase is characterized in that the enzymatic hydrolysis time of the pullulanase in step (5) is 24h.
4. The method according to claim 1, wherein the ultrasound-assisted pullulanase preparation method is characterized in that the specific parameters of the ultrasound effect in step (5) are ultrasound time of 10 min; ultrasound power of 240 W; ultrasound frequency of 60 kHz ; The ultrasonic intermittent ratio is 20s/5s.

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