CN114947131B - Ultrasonic preparation technology of lotus root RS5 type resistant starch with blood sugar reducing effect - Google Patents

Ultrasonic preparation technology of lotus root RS5 type resistant starch with blood sugar reducing effect Download PDF

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CN114947131B
CN114947131B CN202210560621.XA CN202210560621A CN114947131B CN 114947131 B CN114947131 B CN 114947131B CN 202210560621 A CN202210560621 A CN 202210560621A CN 114947131 B CN114947131 B CN 114947131B
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starch
lotus root
ultrasonic
fatty acid
resistant starch
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CN114947131A (en
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任晓锋
汤佳琳
梁秋芳
周成伟
马海乐
林滨凤
董燚帆
刘宝玲
袁茸
刘宇轩
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Zhongke Zhigu International Pharmaceutical Biotechnology Guangdong Co ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
    • A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention discloses an ultrasonic preparation technology of lotus root RS5 type resistant starch with a blood sugar reducing effect, and belongs to the technical field of starch-fatty acid compound preparation. Provides a green physical processing method for preparing the lotus root starch-fatty acid compound, and researches the influence of ultrasonic preparation technology on physicochemical property, structural property and digestion property of the lotus root starch-fatty acid compound. Based on the above, a type II diabetes mouse model is established, and the in vivo blood sugar reducing effect of lotus root starch-fatty acid compound serving as RS5 type resistant starch is further studied. The ultrasonic technology is used in the preparation process of the lotus root starch-fatty acid compound, so that the complexation index of the lotus root starch-fatty acid compound is effectively improved. The lotus root starch-myristic acid compound prepared by the invention has the effect of reducing blood sugar in type II diabetic mice.

Description

Ultrasonic preparation technology of lotus root RS5 type resistant starch with blood sugar reducing effect
Technical Field
The invention belongs to the technical field of preparation of starch-fatty acid compounds, and particularly relates to a preparation method and application of lotus root RS5 type resistant starch with a blood sugar reducing effect.
Background
Lotus root (Nelumbo nucifera gaertn), also called lotus root, is a perennial plant of the Nymphaeaceae family, has a cultivation history of over 3000 years in our country, and the planting industry is mainly distributed in areas such as Hubei and Jiangsu. The lotus root is one of the most widely eaten vegetables in China, contains rich starch, protein, vitamins, iron, calcium, alkaloids and other substances beneficial to human health, has higher medicinal value, has the effects of clearing heat and cooling blood, stopping bleeding and dissolving stasis, strengthening spleen and tonifying qi, maintaining beauty and keeping young and the like, and is a traditional medicine and food dual-purpose plant. However, in the current processing process of lotus roots, the lotus roots are mainly peeled and then vacuum-packed to serve as vegetable clean vegetable outlets or juice is squeezed and prepared into lotus root juice, and the deep processing technology of the lotus roots is lacking. The lotus root starch (Lotus root starch, LRS) is called lotus root starch for short, is a main nutrition component of lotus root, has high nutrition value, and has the effects of nourishing stomach, nourishing yin, preventing hyperlipidemia and the like. A great deal of researches show that the starch rich in the lotus root can be developed into a novel functional food, and the added value of the lotus root is improved.
Starch-fatty acid complex (RS 5) is a novel resistant starch, the formation principle of which is that fatty acid as a guest molecule enters the amylose hydrophobic helical cavity and combines with host molecule to form amylose-fatty acid complex due to hydrogen bonding. The RS 5-type resistant starch is prepared by various methods, such as dimethylsulfoxide method, hydrothermal method, extrusion cooking method, microwave treatment, ultrasonic treatment, etc., and starch particles are destroyed by different means, and amylose is combined with free fatty acid to form a complex. The hydrothermal preparation method can degrade starch molecular chains to generate more amylose, and the starch swells in a heating state, so that the starch molecules stretch, and fatty acid is more beneficial to entering amylose cavities. The cavitation effect and mechanical action of the ultrasonic wave helps to produce more amylose, promotes the interaction of amylose with fatty acids, and increases the dispersibility of fatty acids in the starch system. Kang Xuemin promote the compounding of amylose and fatty acid by ultrasonic treatment, and find that the compounding capacity of liquid fatty acid and amylose is higher than that of solid fatty acid, and the compounding degree of fatty acid is: decanoic acid > octanoic acid > lauric acid > myristic acid, the delta H of the compound after ultrasonic treatment is obviously higher than that of the compound without ultrasonic treatment, which indicates that the compound (KANG X,LIU P,GAO W,et al.Preparation of starch-lipid complex by ultrasonication and its film forming capacity[J].Food Hydrocolloids,2020,99:105340.).Raza with higher heat stability is formed by ultrasonic treatment, the arrowhead starch-linoleic acid compound is prepared by adopting 20/40kHz double-frequency ultrasonic treatment, the complexation index is more than 65 percent, the complexation index is highest at 40min and 60min of ultrasonic treatment, 83.4 percent and 81.26 percent respectively, and the compound prepared by ultrasonic treatment has higher digestion resistance (RAZA H,LIANG Q,AMEER K,et al.Dual-frequency power ultrasound effects on the complexing index,physicochemical properties,and digestion mechanism of arrowhead starch-lipid complexes[J].Ultrasonics Sonochemistry,2022,84:105978.).
Ultrasonic waves are used as a physical processing mode, and cavitation effect and mechanical effect can be generated. Ultrasound may cause the release of gelatinized starch molecules, promoting amylose and then the complexation of amylose with fatty acids. Although the ultrasonic technology is widely applied to the field of starch modification, no research on ultrasonic preparation of lotus root starch-fatty acid compound is seen at present, and no research on physiological effects of lotus root starch-fatty acid compound on blood sugar reduction and the like of type II diabetes is seen.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a green physical processing mode for preparing the lotus root starch-fatty acid compound, and research the influence of ultrasonic preparation technology on the physicochemical property, structural property and digestion property of the lotus root starch-fatty acid compound. Based on the above, a type II diabetes mouse model is established, and the in vivo blood sugar reducing effect of lotus root starch-fatty acid compound serving as RS5 type resistant starch is further studied.
The first object of the invention is to provide an ultrasonic preparation method of a lotus root starch-fatty acid compound, which takes lotus root starch as a raw material to prepare the lotus root starch-fatty acid compound under different ultrasonic conditions, and comprises the following steps:
(1) Weighing lotus root starch to prepare starch milk with the mass concentration of 5% -10%, adding different fatty acids according to the mass ratio of starch to fatty acid of 100:1-10:1, and magnetically stirring and compounding for 10-60min at 70-95 ℃.
(2) The compounded sample is rapidly placed in a multi-mode frequency ultrasonic device, the ultrasonic mode is synchronous ultrasonic, the ultrasonic intermittent ratio is 10s/5s, the ultrasonic frequency is single frequency 20kHz, 40kHz and 60kHz, the double frequency 20/40kHz, 20/60kHz, 40/60kHz, the triple frequency 20/40/60kHz, the ultrasonic power density is 20-100W/L, and the ultrasonic treatment time is 5-30min;
(3) Centrifuging the sample for 10min (4000 r/min), washing with 50% ethanol solution for three times, centrifuging (4000 r/min,10 min) to obtain precipitate, drying at 40deg.C for 12 hr, pulverizing, and sieving to obtain lotus root RS5 resistant starch.
The concentration of the lotus root starch milk in the step (1) is preferably 10%, the fatty acid is lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid, the fatty acid is preferably myristic acid, and the mass ratio of the lotus root starch to the fatty acid is preferably 20:1.
In the step (1), the lotus root starch and the fatty acid are preferably compounded for 20min at the temperature of 95 ℃.
The ultrasonic frequency in the step (2) is preferably 20/40kHz of double-frequency ultrasonic.
The ultrasonic power density in the step (2) is preferably 20W/L.
The ultrasonic treatment time in the step (2) is preferably 20min.
The second purpose of the invention is to use the lotus root starch-fatty acid compound for mice with type II diabetes and explore the influence of the lotus root starch-fatty acid compound on physiological effects such as blood sugar reduction.
In the embodiment of the invention, the application comprises the application of taking lotus root starch-myristic acid compound (U-LRS-MA) to assist in improving the weight, blood sugar, blood fat, liver metabolism, intestinal health and the like of a type II diabetes mouse, but does not relate to a treatment method of diseases.
The lotus root starch-fatty acid compound can be used as a functional food, and can be prepared into a functional food with the effect of reducing blood sugar or a meal replacement food for stabilizing postprandial blood sugar, controlling weight and regulating lipid metabolism.
The lotus root starch-myristic acid compound (U-LRS-MA) is a V-shaped compound, wherein the content of resistant starch is 68.20%.
The invention has the beneficial effects that:
(1) According to the invention, an ultrasonic technology is used in the preparation process of the lotus root starch-fatty acid compound, so that the complexing index of the lotus root starch-fatty acid compound is effectively improved, the complex index of the lotus root starch-fatty acid compound is improved by 17.46% compared with that of the lotus root starch-fatty acid compound which is not ultrasonically treated for 2.93% under the ultrasonic conditions of 20/40kHz, 20W/L ultrasonic power density, 20min ultrasonic treatment time and 10s/5s ultrasonic intermittent ratio after the lotus root starch and the myristic acid are compounded for 20min at 95 ℃.
(2) The structure of the lotus root starch-myristic acid compound (U-LRS-MA) prepared by ultrasonic assistance is changed. The content of resistant starch is increased from 34.58% to 68.21%, the final hydrolysis rate is reduced from 69.84% to 31.58%, and the modified starch has good enzymolysis resistance and good application prospect in food processing.
(3) The lotus root starch-myristic acid compound (U-LRS-MA) prepared by ultrasonic assistance has the effect of reducing blood sugar of type II diabetes mice. Compared with the mice in the model group, the lotus root RS5 type resistant starch is raised, the weight of the mice with diabetes is increased, the organ index is reduced, the Fasting Blood Glucose (FBG) is reduced by 23.01 percent and 50.21 percent, and the Glycosylated Serum Protein (GSP) is not significantly different from the normal group (NC). The intake of the lotus root RS5 type resistant starch can also regulate the blood lipid level, reduce the risk of cardiovascular diseases, relieve the liver injury condition of the diabetic mice, improve the concentration of Short Chain Fatty Acids (SCFAs), especially the concentration of acetic acid and butyric acid, of the diabetic mice, and help to restore the intestinal balance of the diabetic mice, wherein the blood glucose reducing effect of RS5-5% is optimal. The effect of adding 5% of lotus root RS5 type resistant starch (RS 5-5%) is more improved, compared with a model group (MC), TC, TG and LDL-c of RS5-5% are respectively reduced by 44.33%, 34.85% and 40.75%, and HDL-c concentration is increased by 24.43%; ALP, ALT and AST were reduced by 22.14%, 83.96% and 27.98%, respectively.
(4) The invention widens the application range of lotus root starch, provides an important theoretical basis for deep processing of lotus root, provides a theoretical basis for application of ultrasonic technology in preparation of resistant starch, provides theoretical guidance for application of lotus root resistant starch in hypoglycemic food, and has important academic value and application prospect.
Drawings
FIG. 1 is a block diagram of a multi-mode ultrasound device;
FIG. 2 is a short chain fatty acid standard curve;
FIG. 3 is the effect of fatty acid species on lotus root RS 5-type resistant starch complexation index in example 1;
FIG. 4 is the effect of compounding time on the RS 5-type resistant starch complexation index of lotus roots in example 1;
FIG. 5 is the effect of compounding temperature on the RS 5-type resistant starch complexation index of lotus roots in example 1;
FIG. 6 is the effect of myristic acid addition on the RS 5-type resistant starch complexation index of lotus roots in example 1;
FIG. 7 is the effect of different ultrasound conditions (frequencies) on the RS 5-type resistant starch complexation index of lotus roots in example 2;
FIG. 8 is the effect of different ultrasound conditions (power density) on the RS 5-type resistant starch complexation index of lotus roots in example 2;
FIG. 9 is the effect of different ultrasound conditions (ultrasound time) on the RS 5-type resistant starch complexation index of lotus roots in example 2;
FIG. 10 is a surface topography of lotus root starch and RS5 type resistant starch of examples 3 and 4,
(A：LRS-500×，a：LRS-5000×，B：LRS-MA-500×，b：LRS-MA-5000×，C：U-LRS-MA-500×，c：U-LRS-MA-5000×)；
FIG. 11 is XRD patterns of lotus root starch and RS5 type resistant starch of examples 3 and 4;
FIG. 12 is a Fourier transform attenuated total reflection infrared spectrum of lotus root starch and RS 5-type resistant starch of examples 3 and 4;
FIG. 13 is an in vitro digestion and kinetic fitting curve (a-in vitro digestion; b-kinetic fitting) of lotus root starch and RS 5-type resistant starch of examples 3 and 4;
FIG. 14 is the effect of lotus root RS5 resistant starch on diabetic mice body weight;
Note that: NC-normal group; MC-model sets; * Meaning that * represents P <0.05, ** represents P <0.01, and *** represents P <0.001 compared to the model group; # Meaning that # represents P <0.05, ## represents P <0.01, and ### represents P <0.001 compared to the normal group.
FIG. 15 is the effect of lotus root RS 5-resistant starch on glycosylated serum protein of diabetic mice;
Note that: NC-normal group; MC-model sets; * Meaning that * represents P <0.05, ** represents P <0.01, and *** represents P <0.001 compared to the model group; # Meaning that # represents P <0.05, ## represents P <0.01, and ### represents P <0.001 compared to the normal group.
FIG. 16 shows the effect of lotus root RS5 resistant starch on diabetic mice TC (A), TG (B), LDL-C (C) and HDL-C (D);
Note that: NC-normal group; MC-model sets; * Meaning that * represents P <0.05, ** represents P <0.01, and *** represents P <0.001 compared to the model group; # Meaning that # represents P <0.05, ## represents P <0.01, and ### represents P <0.001 compared to the normal group.
FIG. 17 shows the effect of lotus root RS5 resistant starch on ALP (A), ALT (B), AST (C) in diabetic mice;
Note that: NC-normal group; MC-model sets; * Meaning that * represents P <0.05, ** represents P <0.01, and *** represents P <0.001 compared to the model group; # Meaning that # represents P <0.05, ## represents P <0.01, and ### represents P <0.001 compared to the normal group.
FIG. 18 is the effect of lotus root RS5 resistant starch on liver tissue sections of diabetic mice;
FIG. 19 shows the effect of lotus root RS 5-resistant starch on SCFAs content (a-total acid content, b-acetic acid content, c-propionic acid content, d-isobutyric acid content, e-butyric acid content, f-isovaleric acid content) in diabetic mice.
Note that: NC-normal group; MC-model sets; * Meaning that * represents P <0.05, ** represents P <0.01, and *** represents P <0.001 compared to the model group; # Meaning that # represents P <0.05, ## represents P <0.01, and ### represents P <0.001 compared to the normal group.
Detailed Description
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated. The invention will be described in further detail below in connection with specific examples and with reference to the data. It should be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
FIG. 1 is a diagram showing a multi-mode ultrasonic apparatus according to the present invention, which is provided with a computer program controller for controlling three ultrasonic controllers respectively for setting ultrasonic operation parameters (ultrasonic power density, frequency, pulse operation time, intermittent time and total treatment time), and is connected with three ultrasonic vibration plates of different frequencies respectively, so that single frequency/two frequencies/three frequencies ultrasonic treatment can be realized; and (3) putting the sample to be treated into a liquid container for single-frequency/double-frequency/multi-frequency ultrasonic treatment, and starting a circulating pump to circulate the solution. The automatic control of the solution temperature is realized through a water bath kettle and a temperature probe.
In the following examples, the Chinese names, english names or abbreviations of the following nouns may be used, but whether the Chinese names, english names or abbreviations are used, they represent a compound or a pharmaceutical or an agent. Specifically as shown in table 1:
Table 1 Chinese and English control abbreviation table
The lotus root starch used in the invention can be purchased from commercial lotus root starch. The preparation can also be carried out by the following method.
(1) Rapidly cleaning fresh lotus roots, removing sand, peeling, removing heads and tails, cutting into blocks, and soaking in a mixed solution of 1% NaCl and 0.2% NaHSO 3 to prevent browning;
(2) Cleaning lotus root, cutting into small pieces, and weighing. Adding a proper amount of distilled water (liquid-to-material ratio=1:6 (mL/g)), pulping in a juicer, and sequentially filtering the prepared slurry through a sample sieve (200-300 mesh sieve) to obtain filter residues;
(3) The residue was soaked in 0.05% NaOH solution for 40min to remove protein. Filtering, and collecting filtrate; repeatedly adding distilled water into the filter residue, cleaning and filtering twice, and collecting the filtrate; the filtrate was placed in a beaker and allowed to stand overnight, the supernatant was discarded, and then distilled water was added for dissolution. Adjusting pH to 7 with 0.1mol/L hydrochloric acid solution, centrifuging in a 250ml centrifugal cup, removing supernatant, scraping off non-white impurities on the surface of the residual solid, and repeatedly cleaning with deionized water until the impurities are completely removed;
(4) Drying at 50deg.C, pulverizing, sieving with 100 mesh sieve, and making into lotus root starch, packaging, and storing in dryer.
1. Preparation technology and performance characterization of lotus root starch-fatty acid compound
(1) The lotus root starch-fatty acid complex complexation index determination method and the calculation formula prepared in the embodiment of the invention are as follows: 100mg of lotus root starch-fatty acid compound is weighed, 1mL of absolute ethyl alcohol is added to wet a sample, 9mLNaOH solution (1 mol/L) is added to stir evenly, and then boiling water is used for 10min. After cooling to room temperature, the volume was set to 100mL. Taking 5mL of solution, adding 50mL of distilled water, sequentially adding 1mL of acetic acid solution (1 mol/L) and 1mL of iodine solution (2 g KI, 1.3g I 2 constant volume to 100 mL), constant volume to 100mL, developing for 10min, and measuring the absorbance at 620nm by using an ultraviolet spectrophotometer. The blank is lotus root starch, and the Composite Index (CI) is calculated according to the following formula.
Wherein: a 1 -absorbance of sample; a 0 -light absorption value of lotus root starch.
(2) The method for measuring the surface morphology of the lotus root starch and the lotus root RS5 resistant starch prepared in the embodiment of the invention comprises the following steps: uniformly spreading the dried sample on a conductive station adhered with conductive adhesive, performing metal spraying treatment on the sample under vacuum condition, and observing the surface morphology of the sample by adopting an S-3400N type variable vacuum tungsten filament scanning electron microscope under the acceleration voltage of 15 kV.
(3) The X-ray diffraction determination method of lotus root starch and lotus root RS5 type resistant starch prepared in the embodiment of the invention comprises the following steps: the crystal type of the sample was determined using powder X-ray diffractometry. Scanning range is 5-40 degrees, scanning speed is: 5 DEG/min.
(4) The Fourier transform attenuated total reflection infrared spectrometry method of lotus root starch and lotus root RS5 type resistant starch prepared in the embodiment of the invention comprises the following steps: measurements were made using a fourier transform infrared spectrometer equipped with a diamond ATR sampling attachment, and the ATR crystals were washed with ethanol prior to each measurement. And in the measurement process, the dried sample to be measured is placed in a Fourier transform attenuated total reflection infrared spectrometer, and the sample is contacted with a diamond ATR top plate for scanning measurement. Taking air as background blank, scanning the infrared spectrogram with the wavelength of 400-4000cm -1 and the resolution of 4cm -1 for 64 times.
(5) The lotus root starch prepared in the embodiment of the invention has the in-vitro digestion characteristic determination method and calculation formula of lotus root RS5 resistant starch: 200mg of the sample was accurately weighed and added to 10mL of sodium acetate solution (0.1M, pH=5.2) and equilibrated at 37℃for 1h. Then 5mL of the mixed enzyme solution (200U/mL. Alpha. -amylase, 160U/mL amyloglucosidase) was added and in vitro digestion was performed at 37 ℃. 500. Mu.L was sampled at 20min and 120min, respectively, and the enzymatic reaction was stopped by adding four volumes of absolute ethanol, and centrifuged at 4000r/min for 10min. Taking a proper amount of supernatant, measuring the glucose content by a DNS method, and calculating the contents of fast-digestion starch (RDS), slow-digestion starch (SDS) and Resistant Starch (RS) according to the following formulas.
RS(％)＝100-RDS-SDS
Wherein: g20-glucose content measured after 20min of sample enzymolysis; g120-glucose content measured after the sample is subjected to enzymolysis for 120 min; t S -sample weight, mg.
(6) The enzymolysis kinetics determination method of lotus root starch and lotus root RS5 resistant starch prepared in the embodiment of the invention comprises the following steps: 200mg of the sample was accurately weighed and added to 10mL of sodium acetate solution (0.1M, pH=5.2) and equilibrated at 37℃for 1h. Then 5mL of the mixed enzyme solution (200U/mL. Alpha. -amylase, 160U/mL amyloglucosidase) was added and in vitro digestion was performed at 37 ℃. Kinetics of enzymatic hydrolysis of starch in vitro: taking the glucose content of the samples at the time points of 10, 20, 30, 40, 60, 90, 120, 180 and 240min respectively, measuring the glucose content by adopting a DNS method, drawing an in-vitro digestion curve of the starch and performing first-order dynamics fitting.
Ct(％)＝C∞(1-e-kt)
Wherein: HR-hydrolysis rate,%; t S -sample weight, mg; g-glucose content released by the sample at different times, mg; c t -starch hydrolysis rate at time t,%; c ∞ -starch hydrolysis rate at the time of stopping the reaction,%; k-digestion kinetic constant; t-enzymolysis time, min.
2. Blood sugar reducing efficacy research of lotus root resistant starch on diabetic mice
(1) The preparation of the lotus root RS5 resistant starch is carried out according to the optimal process of a medium ultrasonic wave assisted hydrothermal method: the mass ratio of the myristic acid to the lotus root starch is 20:1, the mixture is compounded for 20 minutes at 95 ℃ and then subjected to ultrasonic treatment at the ultrasonic frequency of 20/40kHz, the ultrasonic power density of 20W/L and the ultrasonic treatment time of 20 minutes, and the RS5 type resistant starch is prepared under the ultrasonic parameters of the ultrasonic intermittent ratio of 10s/5s, wherein 17.46% of lotus root starch-myristic acid compound is contained.
(2) Mouse feeding and diabetes model establishment
The raising and management of the test animals follow the relevant regulations, and the whole test process is completed in the animal experiment center of Jiangsu university. The test animals were male ICR mice, 8-10 weeks old, purchased from Jiangsu university animal laboratory center. The temperature of the experiment room is controlled to be 18-23 ℃, the relative humidity is 45-55%, the experiment room is circulated around the clock for 12/12h, basic feed is fed, free drinking water and ingestion are carried out, and the experiment room is adaptively pre-fed for 1 week.
Diabetes model mice were induced by high sugar high fat diet in combination with Streptozotocin (STZ). After adaptive feeding, mice were weighed and assayed for fasting blood glucose, and then randomized into two groups: normal group (15) feeding common feed; diabetes group (75) fed with high sugar and high fat feed. After 6 weeks of feeding, STZ solution (35 mg/kg) was injected into the left lower abdominal cavity at a dose of body weight for three consecutive days, and the feeding with the high-sugar and high-fat feed was continued; normal groups of mice were injected with equal doses of citric acid-sodium citrate buffer and continued normal feed feeding. After 72 hours, the fasting is not forbidden for 12 hours, and the fasting blood sugar is measured by tail vein blood sampling, and the ratio is more than or equal to 11.1mmol/L, which indicates that the establishment of the diabetes mouse model is successful.
(3) Experimental grouping and administration
The normal 15 mice are taken as blank control groups, the rest mice with successful diabetes modeling are divided into 5 groups, 15 mice in each group, and the test period is 4 weeks. The specific groupings and feeding are shown in Table 2:
TABLE 2 grouping and feeding of mice
(4) Determination of the body weight of mice
The weight of the mice is called as initial weight when the mice are grouped; weighing once after molding; then weighing once a week; the end of the test (four weeks) is the final body weight.
(5) Mouse organ index determination
Mice were sacrificed by cervical removal after blood collection, liver, kidney, spleen were dissected rapidly, rinsed with normal saline, filter paper blotted dry and recorded, and organ index (Viscera index, VI) was calculated from the following formula:
Wherein: m 1 -wet weight of viscera, mg; m 2 -mouse body weight, mg.
(6) Mouse fasting blood glucose assay
The fasting blood glucose was measured in normal and diabetic mice after successful molding, and in four week experiments, the fasting blood glucose was measured once a week. Mice were required to be fasted (fasted without water) for 12 hours prior to fasting blood glucose measurement, tail vein was used to take blood, and a blood glucose meter was used to measure fasting blood glucose.
(7) Biochemical index determination of mouse blood collection and serum
After 4 weeks of lotus root resistant starch intervention experiments, after mice are fasted and not forbidden for 12 hours, eyeballs are taken out to take blood, after automatic coagulation, centrifugation is carried out for 15 minutes at 4 ℃ (3000 r/min), serum is separated, and the serum is preserved at-80 ℃.
Assaying glycated serum proteins (Glycated serum protein, GSP) in serum according to the procedure of the kit instructions; four blood lipid levels: triglycerides (TG), total cholesterol (Total cholesterol, TC), high density lipoprotein cholesterol (HIGH DENSITY lipoprotein cholesterol, HDL-c) and low density lipoprotein cholesterol (Low density lipoprotein cholesterol, LDL-c); liver function index: alkaline phosphatase (Alkaline phosphatase, ALP), glutamic pyruvic transaminase (ALANINE TRANSAMINASE, ALT) and glutamic oxaloacetic transaminase (ASPARTATE AMINOTRANSFERASE, AST).
(8) Mouse liver histopathological assay
Fresh livers of mice were fixed in 4% paraformaldehyde solution at 4℃and subjected to gradient ethanol dehydration, wax impregnation, embedding, and slicing in a microtome to give approximately 4 μm slices. Staining was performed with hematoxylin-eosin (HE), and the morphology of liver tissue was observed with a microscope.
(9) Determination of short-chain fatty acids
Sample pretreatment: 200mg of mouse feces is weighed, 1.0ml of 5% sulfuric acid solution is added, vortex oscillation is carried out for 2min, ice water bath ultrasonic treatment is carried out for 10min, vortex mixing is carried out for 1min again, centrifugation is carried out for 20min at 13000r/min at 4 ℃, and the supernatant is taken to pass through a 0.45 mu m water-based film and then gas phase detection is carried out.
Gas chromatography conditions: the gas chromatography GC-2010Plus is combined with a Flame Ion Detector (FID) for measurement, a chromatographic column is DB-FFAP (30 m multiplied by 0.53mm,1.00 mu m), the temperature of a sample inlet (SPL 1) is 250 ℃, the split ratio is 30:1, and the flow rate is 3.0mL/min; the detector (FID) temperature was 280℃and the flow rates of nitrogen, hydrogen and air were 30, 50 and 300mL/min, respectively; the sample loading was 1. Mu.L. Heating program: the initial temperature is 90 ℃, the temperature is kept for 2min, the temperature is raised to 165 ℃ at the speed of 25 ℃/min, and the temperature is kept for 3min; heating to 190 ℃ at a rate of 20 ℃/min, and keeping for 4min; then the temperature is raised to 210 ℃ at the speed of 25 ℃/min and the temperature is kept for 1min. Each sample was independently repeated three times and the Short Chain Fatty Acid (SCFAs) content in the mouse feces was calculated according to a standard curve.
Drawing a standard curve: short Chain Fatty Acids (SCFAs) were determined by external standard method, and standard curves were drawn for six short chain fatty acids (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid) at concentrations of 0, 0.5, 1, 2,3, 4mg/mL, respectively. The fatty acid standard curve results are shown in fig. 2, with R 2 for all standard curves greater than 0.99.
Example 1: preparation optimization (without ultrasonic treatment) of lotus root RS5 type resistant starch
(1) Influence of fatty acid species on lotus root RS5 resistant starch: 6g of lotus root starch is weighed to prepare 10% (m/m) starch milk, and 20:1 (starch: fatty acid, m/m) fatty acid (lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid) is dissolved in 40mL absolute ethanol, mixed, and magnetically stirred and compounded for 30min at 90 ℃. Naturally cooling the sample to room temperature after the compounding is finished, centrifuging at 4000r/min for 10min, washing with 50% ethanol solution for three times, centrifuging (at 4000r/min for 10 min) to obtain precipitate, drying at 40 ℃ for 12h, pulverizing, and sieving to obtain lotus root RS5 resistant starch.
Fig. 3 shows the effect of fatty acid on the RS 5-type resistant starch complexation index of lotus root, and shows that the degree of complexation between fatty acid and lotus root starch is high or low: myristic acid > palmitic acid > linoleic acid > stearic acid > oleic acid > lauric acid, the complexing degree of myristic acid and lotus root starch is highest, and the Complexing Index (CI) is 11.143%.
(2) Influence of the compounding time on lotus root RS5 resistant starch: 6g of lotus root starch is weighed to prepare 10% (m/m) starch milk, myristic acid of 20:1 (starch: fatty acid, m/m) is dissolved in 40mL absolute ethyl alcohol, and after mixing, the mixture is magnetically stirred and compounded for 10, 20, 30, 40, 50 and 60 minutes at 90 ℃. Naturally cooling the sample to room temperature after the compounding is finished, centrifuging at 4000r/min for 10min, washing with 50% ethanol solution for three times, centrifuging (at 4000r/min for 10 min) to obtain precipitate, drying at 40 ℃ for 12h, pulverizing, and sieving to obtain lotus root RS5 resistant starch.
Fig. 4 shows the effect of the complexing time on the RS 5-type resistant starch complexing index of lotus root, and shows that the complexing index increases as the complexing time increases, myristic acid complexes with amylose sufficiently to form a complex. When the compounding time is 20min, the complexation index of the lotus root starch and the myristic acid reaches 14.72%, and the amylose is continuously dissolved out due to heating and stirring in the compounding process and combined with the fatty acid, and the heating can enlarge the double helix diameter of the amylose in starch molecules, so that the fatty acid can conveniently enter a helical cavity, and an amylose-fatty acid compound is formed. After the complexing time exceeds 20min, the complexing degree of myristic acid and amylose is rather decreased. When the compounding time was 60min, the complexation index was reduced by 4.44%. Therefore, the formation of the amylose-myristic acid complex is most favored when the complexing time is 20 min.
(3) Influence of the compounding temperature on lotus root RS5 type resistant starch: 6g of lotus root starch is weighed to prepare 10% (m/m) starch milk, myristic acid of 20:1 (starch: fatty acid, m/m) is dissolved in 40mL absolute ethyl alcohol, and after mixing, the mixture is magnetically stirred and compounded for 20min at 70, 75, 80, 85, 90 and 95 ℃. Naturally cooling the sample to room temperature after the compounding is finished, centrifuging at 4000r/min for 10min, washing with 50% ethanol solution for three times, centrifuging (at 4000r/min for 10 min) to obtain precipitate, drying at 40 ℃ for 12h, pulverizing, and sieving to obtain lotus root RS5 resistant starch.
Fig. 5 shows the effect of the compounding temperature on the RS 5-type resistant starch complexation index of lotus root, and it can be seen from the figure that the complexation index of lotus root starch and myristic acid is higher as the compounding temperature is increased. The CI was 15.20% maximum at a compounding temperature of 95 ℃. The RS5 type complex is formed due to hydrophobic interaction between amylose and fatty acid, which is an endothermic process, and heating favors the formation of the complex between amylose and fatty acid, so that the higher the complexing temperature, the more favoring the formation of the complex. And as the temperature rises, starch particles absorb water and expand, the original crystal structure is destroyed, the gelatinization degree is deepened, and more amylose overflows from the crystal structure; and an increase in temperature will cause the amylopectin side chains to break, forming more amylose, contributing to the formation of the amylose-fatty acid complex. Therefore, selecting a complexing temperature of 95 ℃ enables the formation of more amylose-myristic acid complexes.
(4) Influence of fatty acid addition on lotus root RS 5-type resistant starch: 6g of lotus root starch is weighed to prepare 10% (m/m) starch milk, and 100:1, 50:1, 40:1, 30:1, 20:1 and 10:1 (starch: fatty acid, m/m) myristic acid is dissolved in 40mL absolute ethyl alcohol and mixed, and then magnetically stirred and compounded for 20min at 95 ℃. Naturally cooling the sample to room temperature after the compounding is finished, centrifuging at 4000r/min for 10min, washing with 50% ethanol solution for three times, centrifuging (at 4000r/min for 10 min) to obtain precipitate, drying at 40 ℃ for 12h, pulverizing, and sieving to obtain lotus root RS5 resistant starch.
Fig. 6 shows the effect of the amount of myristic acid added on the complexation index of lotus root RS 5-type resistant starch, and shows that the complexation index of amylose and myristic acid increases and decreases with increasing myristic acid content, and the complexation index is the highest at 20:1, and is 14.46%. At lower myristic acid ligand concentrations, amylose is more of a double helix structure that tends to remain stable; with the presence of more ligand, the ligand will enter the amylose single-helix cavity, forming an amylose-fatty acid complex; however, excessive fatty acid complex is in solution, and due to steric hindrance, the uncoordinated myristic acid may be trapped in the gaps of the helix and cannot be completely complexed with amylose, so that the complexing index of the complex is reduced. Therefore, when the amount of myristic acid added is 20:1 (starch: myristic acid, m/m), the ratio of amylose to myristic acid is the highest.
Example 2: optimization of RS 5-type resistant starch of lotus root prepared by ultrasonic assistance
Weighing 6g of lotus root starch to prepare 10% (m/m) starch milk, adding 20:1 (starch: fatty acid, m/m) myristic acid, and magnetically stirring and compounding at 95 ℃ for 20min. The compounded sample is rapidly placed in a multimode frequency ultrasonic device, the ultrasonic mode is synchronous ultrasonic, and the ultrasonic intermittent ratio is 10s/5s. The effect of the ultrasonic power density (20, 40, 60, 80, 100W/L) and the ultrasonic treatment time (5, 10, 15, 20, 25, 30 min) on the RS5 type resistant starch composite index of lotus roots at different ultrasonic frequencies (20, 40, 60, 20/40, 20/60, 40/60, 20/40/60 kHz) was investigated. Centrifuging the sample for 10min (4000 r/min), washing with 50% ethanol solution for three times, centrifuging (4000 r/min,10 min) to obtain precipitate, drying at 40deg.C for 12 hr, pulverizing, and sieving to obtain lotus root RS5 resistant starch.
Ultrasonic treatment affects the structural and functional characteristics of starch, and its influencing factors include ultrasonic frequency, ultrasonic intensity, ultrasonic time, etc. FIGS. 7-9 are the effect of different ultrasound conditions on the RS 5-type resistant starch complexation index of lotus roots. The cavitation effect of the ultrasonic wave can reduce the molecular chain length of the starch, reduce the interaction force between starch particles, and compared with single-frequency ultrasonic, the double-frequency ultrasonic energy can generate more cavitation energy, so that bubbles collapse rapidly, and the increase of the amylose content is facilitated. Thus, as shown in FIG. 7, the amylose-fatty acid complex was significantly increased with the double frequency ultrasound of 20/40kHz, and the complexation index was 16.82%, compared to the non-ultrasound. The sonochemical effect is improved by the ultrasonic intensity, and the increase of the ultrasonic intensity can lead to the increase of the sonochemical effect, so that more amylose is promoted to overflow from the expanded starch particles; excessive ultrasonic intensity can cause scattering of sound waves onto the walls of the sample cavity, reduced energy in the reaction medium, abnormal growth of bubbles, and reduced cavitation effects. As shown in FIG. 8, when the ultrasonic power density was 20W/L, the complexation degree of lotus root starch and myristic acid was highest, which was improved by 2.20% compared with that without ultrasonic treatment. As shown in fig. 9, the complexation index first increases with increasing sonication time, and the highest complexation index is 17.46% when the sonication time is 20 min. As the sonication time increases, more of the swollen starch particles are broken down, amylose overflows and the dispersibility of fatty acids increases, promoting complexation between amylose and fatty acids; however, the ultrasonic treatment time is prolonged to be more than 20min, which may result in a decrease in complexation index, possibly because the treatment time is too long, and the shearing action and mechanical action of ultrasonic waves may cause excessive shearing of starch chains to form amylose with different starch chain lengths, which is rather unfavorable for the formation of amylose-fatty acid complexes.
The results demonstrate that ultrasonic treatment can enhance the binding of lotus root starch to myristic acid, forming more amylose-fatty acid complexes. Starch is gelatinized under the action of water heat and is combined with fatty acid to form an amylose-fatty acid compound, but the damage degree of the pure water heat to the starch is low, the ultrasonic cavitation effect can induce the disintegration of the expanded granular starch, more amylose is cracked, the water discharge in the spiral structure is promoted, a left spiral cavity which is easier for hydrophobic groups such as fatty acid to enter is formed, and stable RS5 resistant starch is formed. Under different ultrasound conditions, the differences in complex formation may have different dispersibility due to myristic acid under cavitation effects of ultrasound, and ultrasound may also improve the dispersibility of the ligands in the gelatinized starch suspension. Therefore, the ultrasonic treatment of the sample is favorable for forming the lotus root starch-myristic acid compound, and more lotus root starch-myristic acid compound can be formed under the ultrasonic conditions of 20/40kHz ultrasonic frequency, 20W/L ultrasonic power density, 20min ultrasonic treatment time and 10s/5s ultrasonic intermittent ratio.
Example 3: preparation of lotus root RS5 type resistant starch (LRS-MA): 6g of lotus root starch is weighed to prepare 10% (m/m) starch milk, myristic acid of 20:1 (starch: fatty acid, m/m) is dissolved in 40mL absolute ethyl alcohol, and after mixing, magnetic stirring and compounding are carried out for 20min at 95 ℃. Naturally cooling the sample to room temperature after the compounding is finished, centrifuging at 4000r/min for 10min, washing with 50% ethanol solution for three times, centrifuging (at 4000r/min for 10 min) to obtain precipitate, drying at 40 ℃ for 12h, pulverizing, and sieving to obtain lotus root RS5 type resistant starch (LRS-MA).
Example 4: preparation of lotus root RS 5-resistant starch (U-LRS-MA) with the assistance of ultrasonic waves: weighing 6g of lotus root starch to prepare 10% (m/m) starch milk, adding 20:1 (starch: fatty acid, m/m) myristic acid, and magnetically stirring and compounding at 95 ℃ for 20min. The compounded sample is rapidly placed in a multimode frequency ultrasonic device, the ultrasonic mode is synchronous ultrasonic, the ultrasonic intermittent ratio is 10s/5s, the ultrasonic frequency is 20/40kHz, the ultrasonic power density is 20W/L, and the ultrasonic treatment time is 20min. Centrifuging the sample for 10min (4000 r/min), washing with 50% ethanol solution for three times, centrifuging (4000 r/min,10 min) to obtain precipitate, drying at 40deg.C for 12 hr, pulverizing, and sieving to obtain lotus root RS 5-resistant starch (U-LRS-MA).
Experimental example 1: analysis of lotus root starch and lotus root RS5 type resistant starch surface morphology
The procedure of example 3 was followed, using the lotus root starch of this experimental example as a commercial product, to prepare a non-ultrasonic lotus root RS 5-type resistant starch (LRS-MA); the procedure of example 4 was followed, to provide an ultrasound-assisted preparation of lotus root RS 5-type resistant starch (U-LRS-MA).
The surface morphology of lotus root starch and lotus root RS5 resistant starch at the magnification of 500 x and 5000 x is shown in figure 10. The lotus root starch is round or elliptical in shape and smooth and flat in appearance. The lotus root starch has the advantages that the starch structure is destroyed at 95 ℃, amylose is dissolved out and myristic acid forms an amylose-myristic acid compound, the starch is changed in morphology and the particle size is obviously enlarged, and particles are aggregated to form a larger particle structure after ultrasound. The starch-fatty acid complex crystals exhibit an irregular shape, forming a parallel lamellar semi-crystalline polymer structure, and the parallel lamellar structure is positively correlated with the complex content. From figures b and c, more parallel lamellar structures appear in LRS-MA after ultrasound, demonstrating that ultrasound contributes to the formation of lotus root starch-myristic acid complex, consistent with earlier research results.
Experimental example 2: x-ray diffraction analysis of lotus root starch and lotus root RS5 resistant starch
The procedure of example 3 was followed, using the lotus root starch of this experimental example as a commercial product, to prepare a non-ultrasonic lotus root RS 5-type resistant starch (LRS-MA); the procedure of example 4 was followed, to provide an ultrasound-assisted preparation of lotus root RS 5-type resistant starch (U-LRS-MA).
X-ray diffraction can qualitatively demonstrate the formation of the V-complex and observe changes in its crystal structure. As can be seen from fig. 11, LRS has distinct characteristic peaks at 15 °,17 °, 18 °, 23 °, and is a typical a-type crystal structure. Amylose is usually type B crystals after cooling recrystallization, but due to the entry of guest molecules, amylose-guest molecule complexation competes with amylose recrystallization to form crystals of type V structure. After LRS and MA were heat compounded, two central diffraction peaks appeared at around 13 ° and 20 °, and a small diffraction peak appeared at 7.5 °, and the crystal structure of starch was changed from a-type to V-type, indicating the formation of amylose-fatty acid complex. The diffraction peaks of amylopectin did not appear at 17℃for LAS-MA and U-LAS-MA, indicating that the complexing of amylose with MA inhibited the recrystallization of amylopectin. Compared with U-LAS-MA, LAS-MA has a small diffraction peak near 21 degrees, and is a characteristic peak of uncomplexed MA. As shown, the diffraction peak of U-LRS-MA was higher and sharper than that of LRS-MA, further demonstrating that ultrasound can promote interactions between lotus root starch and myristic acid. Under the action of ultrasonic waves, the mechanical shearing force and cavitation effect of the ultrasonic waves act on the expanded starch particles to generate more amylose molecules, and the ultrasonic waves can improve the dispersibility of MA, thereby promoting the formation of amylose-fatty acid complexes.
Experimental example 3: fourier transform attenuated total reflection infrared spectrum analysis of lotus root starch and lotus root RS5 type resistant starch
The procedure of example 3 was followed, using the lotus root starch of this experimental example as a commercial product, to prepare a non-ultrasonic lotus root RS 5-type resistant starch (LRS-MA); the procedure of example 4 was followed, to provide an ultrasound-assisted preparation of lotus root RS 5-type resistant starch (U-LRS-MA).
The infrared spectrograms of lotus root starch and RS5 type resistant starch are shown in figure 12. The infrared beam of the fourier transform attenuated total reflection infrared apparatus can only penetrate to a particle depth of 2 μm, so ATR-FTIR only provides structural features near the surface of the starch particles. The characteristic peak of 1640cm -1 is H-O-H bending vibration on the amorphous region of starch, which is a typical characteristic peak of starch. The characteristic absorption peak at 2925cm -1 was attributed to the stretching vibration of the methylene group on the glucose ring, and the characteristic peaks of LRS-MA and U-LRS-MA were shifted to low frequency compared to LRS, indicating that the addition of MA promoted the interaction between methylene groups, further enhancing the hydrophobic interaction between LRS and MA. The absorption peaks of LRS-MA at around 1710cm -1 and 2850cm -1 are carbonyl stretching vibration peaks of fatty acids, which are probably caused by free myristic acid aggregation, indicating that the binding between starch and fatty acid is a hydrophobic interaction. U-LRS-MA lacks 1710cm -1 and 2850cm -1 characteristic peaks compared with LRS-MA, which indicates that more myristic acid is wrapped in the helix structure of amylose under the action of ultrasound, resulting in insignificant or even vanishing characteristic peaks.
Experimental example 4: in-vitro digestion characteristics and enzymolysis kinetics analysis of lotus root starch and lotus root RS5 resistant starch
The procedure of example 3 was followed, using the lotus root starch of this experimental example as a commercial product, to prepare a non-ultrasonic lotus root RS 5-type resistant starch (LRS-MA); the procedure of example 4 was followed, to provide an ultrasound-assisted preparation of lotus root RS 5-type resistant starch (U-LRS-MA).
Enzyme access to the interior of starch granules is a major factor affecting starch digestibility. In vitro digestion and dynamics fit curves of lotus root starch and RS5 type resistant starch are shown in fig. 13, the hydrolysis rate and the hydrolysis rate of LRS are higher than those of LRS-MA, which indicates that amylase is less likely to enter the inside of a complex after starch and fatty acid form the complex, and the fatty acid prevents the combination of amylase and starch, so that the enzymolysis resistance of starch is achieved. The final hydrolysis rates (C ∞) of LRS, LRS-MA and U-LRS-MA are 69.84%, 32.37% and 31.58%, respectively, and the minimum hydrolysis rate of U-LRS-MA is due to the cavitation effect of ultrasound acting on amylopectin debranching, and more amylose is generated to combine with myristic acid to form a compound; the ultrasonic energy promotes the opening of the amylose cavity, so that more binding sites are exposed, amylose is facilitated to enter the cavity, and a compound is formed under the action of hydrogen bonds; and the ultrasound is favorable for the dispersion of MA in the system, and can generate more amylose-myristic acid complexes to influence the enzymolysis property of starch.
As can be seen from Table 3, the reduced RDS content of U-LRS-MA, as compared to LRS-MA, is probably due to the fact that ultrasound acts mainly on the amorphous regions of starch, favoring the formation of more ordered structures by the starch, resulting in reduced RDS. The RS content of LRS, LRS-MA and U-LRS-MA was 34.58%, 67.03% and 68.20%, respectively, and the RS content was proportional to the amylose-myristic acid complex content. The RS content of U-LRS-MA is highest, and C ∞ is smallest because the resistant starch has a compact structure and fewer enzymolysis sites. Higher RS content can be more effective in preventing metabolic syndrome, including diseases such as insulin resistance, obesity, and diabetes.
TABLE 3 in vitro digestion parameters of lotus root starch and lotus root RS5 resistant starch
Note that: different lower case english letters in the same index in the table indicate significant differences (p < 0.05) between the same set of data.
Experimental example 5: influence of lotus root RS 5-type resistant starch on weight of diabetic mice
As can be seen from fig. 14, the body weights of the normal group (NC) and diabetic group mice remained substantially the same at the initial stage of the test, with no significant difference (P > 0.05). During the test period of resistant starch feeding, the weight of normal mice normally increases along with time, and the weight of diabetic mice is obviously reduced due to the influence of hyperglycemia and other complications, and is extremely obviously different from that of NC mice (P < 0.001), so that the weight-reducing symptoms of the diabetic mice are met. Compared with the model group (MC) mice, the weight of the mice with the lotus root RS5 type resistant starch diabetes is increased, the weight of the mice with the 5% lotus root RS5 type resistant starch (RS 5-5%) group is obviously increased (P < 0.05), and the weight is increased by 4.46%. The test result shows that the intake of the lotus root resistant starch is helpful for relieving the weight loss symptom of the diabetic mice, and the effect of adding 5% of lotus root RS5 type resistant starch is more remarkable.
Experimental example 6: influence of lotus root RS 5-type resistant starch on organ index of diabetic mice
The organ indexes of the mice in each test group are shown in Table 4. As can be seen from the table, liver, kidney, spleen index of the diabetic mice were significantly increased (P < 0.001) compared to the normal mice (NC), wherein liver, kidney, spleen index of the model Mice (MC) were increased by 58.79%, 50.45% and 52.82%, respectively, indicating that diabetes caused damage to the liver, kidney and spleen of the mice. Compared with the mice in the model group, the invention discovers that after the mice with diabetes take lotus root RS5 resistant starch, the liver index and the kidney index of the mice are extremely reduced (P is less than 0.001), and the liver index of the mice with diabetes is reduced by 16.59 percent. Compared with the model group, the spleen index of the mice with RS5-5% is obviously reduced by 7.71% (P < 0.05), and the spleen index of the mice with RS5-15% is not obviously different from that of the model group (P > 0.05), which proves that the intake of the lotus root RS5 type resistant starch has a great improvement on liver and kidney injury of diabetic mice, but has a slightly poorer improvement effect on spleen injury.
TABLE 4 influence of lotus root RS 5-resistant starch on organ index
Note that: NC-normal group; MC-model sets; data are expressed as mean ± standard deviation; * Meaning that * represents P <0.05, ** represents P <0.01, and *** represents P <0.001 compared to the model group; # Meaning that # represents P <0.05, ## represents P <0.01, and ### represents P <0.001 compared to the normal group.
Experimental example 7: influence of lotus root RS 5-type resistant starch on fasting blood glucose of diabetic mice
Fasting hyperglycemia is one of the typical symptoms of diabetic mice. The weekly fasting blood glucose values (FBG) of the mice of each test group are shown in table 5, and the FBG of the mice of the normal group (NC) was substantially between 5.0 and 7.0 during the test, belonging to the FBG value range of the normal mice. After molding, the FBG of mice in the diabetic group was greater than 11.1mmol/L, with no significant differences (P > 0.05) between groups. FBG in model group (MC) mice was always at high level and gradually increased from 19.70mmol/L to 32.60mmol/L during the trial, possibly due to the progressive exacerbation of the diabetic condition. In the first week of experiment, FBG and MC groups raised with RS5 resistant starch of lotus root were not significantly different (P > 0.05). After four-week feeding of lotus root RS5 type resistant starch, all intervention groups have extremely significant reduction (P < 0.001) of FBG values of diabetic mice, which indicates that ingestion of lotus root RS5 type resistant starch can relieve hyperglycemia characteristics of diabetic mice. From this, it can be seen that the RS5-5% group has a remarkable hypoglycemic effect compared with the MC group, and the FBG value thereof is reduced by 50.21%.
TABLE 5 Effect of lotus root resistant starch on fasting blood glucose
Note that: NC-normal group; MC-model sets; data are expressed as mean ± standard deviation; * Meaning that * represents P <0.05, ** represents P <0.01, and *** represents P <0.001 compared to the model group; # Meaning that # represents P <0.05, ## represents P <0.01, and ### represents P <0.001 compared to the normal group.
Experimental example 8: influence of lotus root RS 5-type resistant starch on glycosylated serum protein of diabetic mice
The principle of the glycosylated serum protein detection is that serum glucose can perform non-enzymatic saccharification reaction with the amino group at the N end of albumin and other serum protein molecules to form a high molecular ketoamine structure. Glycated Serum Proteins (GSPs) are effective in reflecting the glycemic control status of diabetic mice over the past four weeks (particularly the last 2 weeks), and are more effective in reflecting diabetic conditions than fasting blood glucose. As shown in FIG. 15, GSP values of the normal group (NC) and the model group (MC) were about 1.93mmol/L and 2.49mmol/L, respectively, and the glycated serum proteins of the model group (MC) were in a high-level state. Compared with a model group (MC), the GSP of the test group of diabetic mice which take lotus root resistant starch has extremely significant difference (P < 0.001), which proves that the intake of the lotus root resistant starch has obvious relieving effect on diabetes. The GSP of the RS5-5% group is 1.94mmol/L, and the GSP has no significant difference (P > 0.05) with the glycosylated serum protein level of the normal group (NC), which proves that the addition of the RS5 type resistant starch of 5% lotus root has better control effect on the blood sugar of diabetic mice in a short period.
Experimental example 9: influence of lotus root RS5 type resistant starch on blood fat of diabetic mice
Dyslipidemia is a characteristic of diabetes and results in increased concentrations of TC, TG and LDL-c and decreased concentrations of HDL-c in the blood. FIG. 16 shows the effects of lotus root resistant starch on Triglyceride (TG), total Cholesterol (TC), high density lipoprotein cholesterol (HDL-c) and low density lipoprotein cholesterol (LDL-c). Compared with Normal (NC) mice, model (MC) mice have increased TC, TG and LDL-c significance (P < 0.001) and decreased HDL-c significance (P < 0.001), indicating that Model (MC) mice have disturbed lipid metabolism and exhibit typical hyperlipidemia symptoms. Diabetes mice are fed with 4-week lotus root RS5 type resistant starch, TC, TG, LDL-c is reduced, HDL-c is increased, and the lotus root resistant starch is indicated to protect islet beta cells from lipid toxicity and improve blood lipid level. In comparison with model group (MC), RS5-5% decreased TC, TG and LDL-c by 44.33%, 34.85% and 40.75%, respectively, and HDL-c concentration increased by 24.43%. In both groups of diabetic mice fed RS5, RS5-5% had a very significant difference in blood lipid level (P < 0.01) from model group (MC), RS5-15% compared to model group (MC), TG did not have a significant difference (P > 0.05), TC, LDL-c and HDL-c were significantly different, indicating that the addition of 5% RS5 resistant starch was more beneficial to lipid metabolism in diabetic mice.
High levels of total cholesterol and low density lipoprotein cholesterol are major risk factors for cardiovascular disease, whereas high density lipoprotein cholesterol can reverse cholesterol transport, promote cholesterol metabolism, and contribute to reducing cardiovascular disease risk. As can be seen from fig. 16, the four blood lipid levels of the RS5-5% group all have very significant differences (P < 0.01) from the model group (MC), and are close to the blood lipid level of the normal group mice, which indicates that the addition of the RS 5-type resistant starch of 5% lotus root has the efficacy of reducing blood lipid in diabetic mice, and has a certain risk of reducing cardiovascular diseases in diabetic mice.
Experimental example 10: influence of lotus root RS 5-type resistant starch on liver function metabolism of diabetic mice
Alkaline phosphatase (ALP), glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) are abundant in liver, and any damage to liver cells can increase their contents in blood, and can be used for evaluating liver diseases. The ALP, ALT and AST levels in the serum of each group of mice can be shown in FIG. 17, and the results indicate that each diabetic group of mice has significantly increased ALP, ALT and AST (P < 0.05) over the serum of the normal group (NC) of mice, indicating that diabetes causes different degrees of damage to the liver of the mice. The feeding of the lotus root RS5 type resistant starch in 4 weeks of the diabetic mice reduces ALP, ALT and AST in serum, which proves that the intake of the lotus root resistant starch can relieve the liver injury condition of the diabetic mice. Compared with a model group (MC), ALP, ALT and AST in the RS5-5% group are reduced by 22.14%, 83.96% and 27.98%, the ALP, ALT and AST are remarkably different from MC, and the liver function metabolism level of the MC is close to that of NC, so that the liver injury of a diabetic mouse can be relieved by adding 5% of lotus root RS5 type resistant starch to feed the diabetic mouse, and the liver plays a metabolism regulation function consistent with the early liver index result.
Experimental example 11: influence of lotus root RS 5-type resistant starch on liver tissue of diabetic mice
Fig. 18 is a view of liver tissue sections of mice in each test group, and it is clear that the liver cells in the normal group (NC) are arranged in a regular manner, and the liver cell structures are distributed radially around the central vein. Liver cells of model group (MC) mice are not clear in structure, are arranged in disorder, and have denaturation and swelling; local cells appear necrotic, disintegrated or lysed; small numbers of cells were accompanied by steatosis, and fatty vacuoles were visible in the serum. The structure of the liver cells of the RS5-5% group is complete, and the phenomenon of cytomegaly hardly occurs. The RS5-15% of the liver cells showed slight degeneration and swelling, but slightly improved compared to the MC liver cells. Liver cell slice results prove that the resistant starch is helpful for relieving liver injury of diabetic mice. Wherein, the RS5-5% liver cells are closer to the normal liver cells, which indicates that the addition of 5% lotus root RS 5-type resistant starch has better relieving effect on the liver injury condition of the diabetic mice and is consistent with the liver function metabolic result.
Experimental example 12: influence of lotus root RS 5-type resistant starch on short chain fatty acid of diabetic mice
The T2DM patient may have dysbacteriosis of intestinal microbiota, and Short chain fatty acids (Short CHAIN FATTY ACIDS, SCFAs) are the most main microbial metabolites in the intestinal tract, and have functions of glucose homeostasis, inflammation, satiety and the like. FIG. 19 shows the effect of lotus root resistant starch on the content of SCFAs. Compared with the normal group (NC), the total acid content of SCFAs in the feces of the mice in the model group (MC) is reduced by 71.3%, wherein the contents of acetic acid, propionic acid, isobutyric acid, butyric acid and isovaleric acid are extremely remarkably reduced (P < 0.001), and the total acid content of SCFAs of the diabetic mice is remarkably improved (P < 0.001) after lotus root resistant starch is taken. SCFAs have effects of protecting intestinal health, reducing intestinal dysfunction, reducing incidence of intestinal cancer and colon cancer, and increasing resistance to constipation and diarrhea by inhibiting growth and reproduction of pathogenic bacteria. The contents of acetic acid, propionic acid, isobutyric acid, butyric acid and isovaleric acid in the feces of diabetic mice are all increased by the dietary dry prognosis of lotus root resistant starch. It shows that the lotus root resistant starch can be fermented into SCFAs by microorganisms in intestinal tracts, and the intestinal microbiota is regulated by diet intervention, so that the intestinal microbiota disturbance of diabetic mice is improved and the intestinal balance is maintained. Production of acetic and butyric acids can increase glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) secretion in the gut, thereby promoting insulin secretion and improving T2DM. Butyrate is an important factor in maintaining intestinal health and involved in immunomodulation and regulation of intestinal barrier function, and can improve insulin resistance, fasting hyperglycemia, and inhibit adipocyte inflammation. From the figure, the SCFAs content of the RS5-5% group is increased by 180.70%, the acetic acid content is increased by 251.54%, and the butyric acid content is increased by 181.64%, which indicates that the dietary intervention of adding 5% lotus root RS5 type resistant starch is more beneficial to maintaining the intestinal health of diabetic mice and recovering the intestinal balance.

Claims (2)

1. The ultrasonic preparation method of the lotus root starch-fatty acid compound is characterized by comprising the following steps of:
(1) Weighing lotus root starch to prepare starch milk with the mass concentration of 10%, adding myristic acid according to the mass ratio of starch to fatty acid of 20:1, and magnetically stirring and compounding for 20min at 95 ℃;
(2) The compounded sample is rapidly placed in a multimode frequency ultrasonic device, the ultrasonic mode is synchronous ultrasonic, the ultrasonic intermittent ratio is 10s/5s, the ultrasonic frequency is double-frequency 20/40 kHz, and the ultrasonic treatment time is 20 min; the ultrasonic power density is 20W/L;
(3) Centrifuging the sample after ultrasonic treatment at a speed of 4000r/min, washing with 50% ethanol solution for three times, centrifuging at a speed of 10min and a speed of 4000r/min, drying the obtained precipitate at 40deg.C at a speed of 12h, pulverizing, and sieving to obtain lotus root RS5 type resistant starch.
2. The lotus root starch-fatty acid compound prepared by the ultrasonic preparation method of the lotus root starch-fatty acid compound of claim 1 is a V-shaped compound, wherein the content of resistant starch is 68.20%.
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Title
Physicochemical properties and digestion mechanism of starch-linoleic acid complex induced by multi-frequency power ultrasound;Husnain Raza，et al.;Food Chemistry;第364卷;1-11 *
Studies on nutritional intervention of rice starch- oleic acid complex (resistant starch type V) in rats fed by high-fat diet;Bo Zheng，et al.;Carbohydrate Polymers;第246卷;1-10 *

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