Sugarcane molasses extract with effect of reducing blood sugar as well as preparation method and application of cane molasses extract

Abstract

The invention discloses a cane molasses extract with a blood sugar reducing effect as well as a preparation method and application thereof, and belongs to the field of functional foods. According to the invention, cane molasses is subjected to dissolution, centrifugation, ultrafiltration impurity removal, desalination and nanofiltration concentration, and is mixed with calcium chloride, so that the cane molasses extract with the effect of reducing blood sugar is prepared. Experimental results prove that the cane molasses extract prepared by the invention has the characteristics of low glycemic index (GI), carbohydrate digestive enzyme inhibition, high temperature resistance, long-term storage resistance and the like, can also effectively repair pancreas and liver injury and regulate intestinal flora, and has a comprehensive hypoglycemic effect. The food with low glycemic index is prepared on the basis of the high sucrose carrier for the first time, and the food has sweet taste and the function of reducing blood sugar. The invention provides a new raw material scheme for the preparation of dietary supplement food and hypoglycemic drugs for hyperglycemia people, and has prominent practical application value and wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of functional foods, and in particular to a sugarcane molasses extract with hypoglycemic effects, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a chronic disease characterized by hyperglycemia, caused by absolute or relative insulin deficiency and impaired insulin utilization. In recent years, the incidence of diabetes has been increasing year by year. Diabetic patients need to strictly limit their sugar intake and often consume a certain amount of sugar substitutes to control blood sugar levels. However, currently used sugar substitutes (such as artificial sweeteners) are controversial in terms of safety and lack physiological repair functions. Molasses is a byproduct of sugarcane sugar production, containing high levels of phenolic and flavonoid compounds, which have antioxidant, immunomodulatory, antibacterial, and anticancer physiological activities. However, existing natural molasses has a high glycemic index (GI > 80) due to its high sucrose content, which cannot meet the needs of diabetic patients. The bioactivity of sugarcane polyphenols has not been systematically applied in molasses extracts, and the polyphenols in molasses easily lose their activity after high temperatures or long-term storage.

[0003] Therefore, there is an urgent need to provide a sugar substitute product with a low glycemic index and a longer duration of polyphenol activity. Summary of the Invention

[0004] The purpose of this invention is to provide a sugarcane molasses extract with hypoglycemic effects, its preparation method, and its application, in order to solve the problems existing in the prior art. This invention produces a sugarcane molasses extract with hypoglycemic effects, which has both sweetness and the function of lowering blood sugar, providing a new raw material solution for the preparation of dietary supplements and hypoglycemic drugs for people with high blood sugar.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing a sugarcane molasses extract with hypoglycemic effects, comprising the following steps:

[0007] Take sugarcane molasses, dilute it, centrifuge to collect the supernatant, remove impurities by ultrafiltration, desalinate by electrodialysis, concentrate by nanofiltration to obtain the enriched solution;

[0008] The enriched solution is mixed with calcium chloride to obtain the sugarcane molasses extract.

[0009] Furthermore, the ultrafiltration impurity removal is performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove impurities.

[0010] Furthermore, the nanofiltration concentration involves using a nanofiltration membrane with a molecular weight cutoff of 300 Da to remove large molecular sugars and impurities, concentrating the supernatant to 20%-25% of its volume.

[0011] Optionally, the nanofiltration concentration is operated at a pressure of 1.0-1.4 MPa and a pH of 4.5-5.5.

[0012] The present invention also provides a sugarcane molasses extract obtained according to the above preparation method.

[0013] The present invention also provides the application of the above-mentioned sugarcane molasses extract in the preparation of health food products that help maintain healthy blood sugar levels.

[0014] The present invention also provides a health food that helps maintain healthy blood sugar levels, with the above-mentioned sugarcane molasses extract as the main active ingredient.

[0015] The present invention also provides the application of the above-mentioned sugarcane molasses extract in the preparation of a drug for lowering blood sugar.

[0016] The present invention also provides a drug for lowering blood sugar, with the above-mentioned sugarcane molasses extract as the main active ingredient.

[0017] Furthermore, it also includes pharmaceutically acceptable excipients.

[0018] The present invention discloses the following technical effects:

[0019] This invention produces a sugarcane molasses extract with hypoglycemic effects by dissolving, centrifuging, ultrafiltration for impurity removal, desalting, and nanofiltration for concentration, followed by mixing with calcium chloride. Experimental results demonstrate that the sugarcane molasses extract prepared by this invention has a low glycemic index (GI), inhibits carbohydrate digestive enzymes, effectively repairs pancreatic and liver damage, regulates intestinal flora, and exhibits comprehensive hypoglycemic effects. This invention is the first to prepare a low glycemic index food based on a high-sucrose carrier, possessing both sweetness and hypoglycemic function; moreover, the polyphenols in this sugarcane molasses extract are not deactivated by high temperatures or long-term storage. This invention provides a new raw material solution for the preparation of dietary supplements and hypoglycemic drugs for people with hyperglycemia, possessing outstanding practical application value and broad application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 A schematic diagram of the hypoglycemic pathway mechanism of sugarcane molasses extract;

[0022] Figure 2 Figure showing the experimental results of regulating blood glucose levels with sugarcane molasses extract;

[0023] Figure 3 Figure showing the experimental results of regulating glucose tolerance levels with sugarcane molasses extract;

[0024] Figure 4 Figure showing experimental results of sugarcane molasses extract improving pancreatic lesions;

[0025] Figure 5 Figure showing experimental results of sugarcane molasses extract improving liver disease;

[0026] Figure 6 Figure showing the experimental results of regulating gut microbiota with sugarcane molasses extract. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Sugarcane molasses is a thick, viscous liquid byproduct that remains after sugarcane juice has been refined and crystallized into sucrose. It cannot recrystallize and is therefore a high-sugar, high-calorie food. Its consumption is restricted, and it is generally used as animal feed, resulting in low added value.

[0033] The hypoglycemic pathway mechanism of the sugarcane molasses extract of this invention is as follows: Figure 1 As shown.

[0034] Example 1: Preparation of Sugarcane Molasses Extract

[0035] First, the sugarcane molasses is diluted with purified water to Brix 30°, centrifuged, and then filtered through an ultrafiltration membrane (10kDa) to remove impurities. Electrodialysis is used for desalination to ensure that the sodium content is below 120mg / 100g. Further concentration is achieved using a nanofiltration membrane. The ultrafiltration permeate is then treated through a 300Da nanofiltration membrane at an operating pressure of 1.0-1.4MPa and a pH of 4.5-5.5 to remove large-molecule sugars and impurities. The solution is concentrated to 20%-25% of its original volume to obtain a polyphenol-rich solution. 5% (w / w) of calcium chloride is added to the enriched solution to allow sugarcane polyphenols, calcium ions, and sucrose to form a eutectic.

[0036] Example 2: Human Experiment on the Glycemic Index of Sugarcane Molasses Extract

[0037] Preparation method of mixture of sucrose and sugarcane polyphenols:

[0038] The pressed bagasse was extracted with 80% ethanol by volume for polyphenol extraction at 45°C for 3 hours. After filtration to remove the bagasse, the mixture was concentrated at 70°C for 2 hours to remove the ethanol and then freeze-dried to obtain a sugarcane polyphenol extract. 1000g of sucrose was dissolved in water, and 10g of the sugarcane polyphenol extract was added. The mixture was stirred thoroughly for 1 hour and then freeze-dried to obtain a mixture of sucrose and sugarcane polyphenols.

[0039] Twelve healthy adult volunteers (half male and half female) were selected for the test. The volunteers were aged 23-33 years, with a mean age of 26.50 ± 4.12 years and a mean body mass index (BMI) of 20.72 ± 1.15 kg / m². 2 The participants were light manual laborers with no history of diabetes, other metabolic diseases, digestive system diseases, endocrine system diseases, or mental illnesses. The glycemic index (GI) test was conducted according to WS / T 652-2019, "Methods for Determination of Glycemic Index of Foods." All experiments related to this invention were approved by the ethics committee, and informed consent was obtained from the volunteers.

[0040] Twelve subjects were randomly divided into three groups, adjusted for gender and age. Subjects fasted from 8:00 PM the night before the test until the following morning, when fasting blood glucose was measured using the fingertip capillary method. Afterwards, they consumed sucrose (50g of carbohydrates), a mixture of sucrose and sugarcane polyphenols (50g of carbohydrates), and sugarcane molasses extract (50g of carbohydrates), respectively. Blood glucose levels were measured using the fingertip capillary method at 15, 30, 45, 60, 90, and 120 minutes after each food intake. Blood glucose levels after consuming 50g of glucose were used as a control. The testing interval between the different foods was greater than 72 hours.

[0041] Calculate the area under the glycemic response curve for each subject to the two foods, and calculate the glycemic index (GI) using the following formula:

[0042]

[0043] Among them, AUC S Area under the blood glucose response curve after a meal for the food being tested; AUC G The area under the glucose response curve after a glucose meal is denoted as .

[0044] The results are shown in Table 1. The experimental results show that the sugarcane molasses extract prepared in Example 1 has the lowest GI value of 38.8. The sugarcane molasses extract prepared in this invention is a low-GI food, suitable for diabetics, and is significantly better than ordinary sucrose and a mixture of sucrose and sugarcane polyphenols.

[0045] Table 1. Statistical results of glycemic index of different foods

[0046] food sucrose Mixture of sucrose and sugarcane polyphenols Sugarcane molasses extract GI value 65.7 58.6 38.8

[0047] Example 3 In vitro digestibility analysis

[0048] Equal masses of sucrose, a mixture of sucrose and sugarcane polyphenols, and sugarcane molasses extract were weighed and added to an equal volume of water. The pH was adjusted to 5.2, and 5% sucrase was added. The solution was then placed in a shaking incubator at 37°C and equilibrated for 20 min. Samples (0.1 mL) were taken at time points of 0, 20, 40, 60, 120, and 180 min. 0.9 mL of potassium dihydrogen phosphate buffer was added, and the sample was then placed in boiling water to inactivate the enzyme. The glucose content was determined using the 3,5-dinitrosalicylic acid (DNS) method.

[0049] The formula for calculating the hydrolysis rate is as follows:

[0050] Hydrolysis rate (%) = 0.9 × G t / TS;

[0051] Among them, G tThe values ​​represent the glucose content (mg) in the system at 0, 20, 40, 60, 120, and 180 min. TS represents the sucrose content in the sample.

[0052] The results are shown in Table 2. It can be seen that sucrose in the three samples had different hydrolysis rates at different time points, and the sugarcane molasses extract had the lowest rate, which was only 38% at 180 min, a decrease of 69% compared to sucrose and 58% compared to the mixture of sucrose and sugarcane polyphenols. This result indicates that the polyphenols in the sugarcane molasses extract prepared in this invention effectively slow down the hydrolysis process of sucrose.

[0053] Table 2 Statistical results of sucrose hydrolysis rate in different foods

[0054]

[0055]

[0056] Example 4: Polyphenol activity stability in sugarcane molasses extract

[0057] Equal masses of sucrose, a mixture of sucrose and cane polyphenols, and cane molasses extract were weighed and added to an equal volume of water. After boiling for 5 hours and cooling, 4 times the volume of anhydrous ethanol was added to a concentration of 80% for polyphenol extraction. The mixture was stirred for 24 hours, then concentrated and freeze-dried. The inhibitory effect of polyphenols on sucrase in different products was then detected. Equal masses of sucrose, a mixture of sucrose and cane polyphenols, and cane molasses extract were weighed and stored at room temperature in the dark for one year. 4 times the volume of anhydrous ethanol was added to a concentration of 80% for polyphenol extraction. The mixture was stirred for 24 hours, then concentrated and freeze-dried. The inhibitory effect of polyphenols on sucrase in different products was then detected.

[0058] Determination of sucrase inhibitory activity: Weigh a certain amount of sugarcane polyphenol extract and prepare the required concentration with 0.1 mol / L phosphate buffer solution. Pipette 0.5 mL of the sample solution into a test tube, add 0.5 mL of 56 mmol / L sucrose solution as substrate, shake well, and incubate in a water bath at 37℃ for 5 min. Then add 0.5 mL of sucrase solution and react at 37℃ for 20 min. Finally, place the test tube in boiling water to inactivate the enzyme for 5 min. After cooling, use a glucose assay kit to detect the absorbance of the sample at 505 nm, and record it as A1. Set up a sample control group (sample + buffer + substrate), with absorbance recorded as A2; a blank group (buffer + enzyme solution + substrate), with absorbance recorded as A3; and a blank control group (buffer + buffer + substrate), with absorbance recorded as A4. The inhibition rate of the sample against sucrase is calculated using the following formula:

[0059] Sucrase inhibition rate (%) = [1 - (A1 - A2) / (A3 - A4)] × 100;

[0060] Table 3. Statistical results of sucrase inhibition rate in different foods

[0061]

[0062] As shown in Table 3, the sugarcane molasses extract of the present invention retains 98% of its activity after boiling for 5 hours and 95% of its activity after being stored naturally in the dark for 1 year, thus solving the problems of unstable heating of active substances in sugarcane and loss of activity after long-term storage.

[0063] Example 5: Animal experiment on the hypoglycemic effect of sugarcane molasses extract

[0064] 1. Experimental Methods

[0065] db / db diabetic mice were randomly divided into a model group and a sugarcane molasses extract intervention group, with 10 mice in each group. Sugarcane molasses extract was dissolved in physiological saline and administered by gavage at a dose of 500 mg / kg, designated as the ESM group. Ten normal mice were selected as the control group. Mice in the control and model groups were administered the same amount of physiological saline by gavage, designated as the Control and Model groups, respectively. This gavage treatment lasted for 8 weeks. After 8 weeks, mice were weighed, and fresh feces were collected and placed in sterile centrifuge tubes. The feces were then frozen in liquid nitrogen and immediately stored at -80°C for later use. Mice were euthanized by cervical dislocation. After euthanasia, the mice were placed abdomen-up on a dissection table, and 75% ethanol solution was applied to the abdomen. The abdominal cavity was opened, and organs were quickly removed, including the pancreas and liver tissue, which were immediately weighed. The collected tissue samples were placed in sterile culture dishes, rinsed twice with physiological saline, and then placed in sterile centrifuge tubes. The collected tissues were frozen in liquid nitrogen and immediately stored at -80°C for later use.

[0066] 1.1 Measurement of fasting blood glucose level

[0067] Eight weeks later, the mice in each group were fasted overnight for 12 hours, with free access to water during this period. Fasting blood glucose (FBG) levels were measured using a blood glucose meter by collecting blood from the tail vein.

[0068] 1.2 Oral glucose tolerance test

[0069] After 8 weeks of drug administration, mice in the normal group and model group were fasted for 12 hours, and their blood glucose was measured at 0 min. Then, they were administered 1 g / kgbw glucose solution by gavage, and their blood glucose levels were measured at 30, 60, 90 and 120 min. The values ​​were read and oral glucose tolerance (OGTT) curves were plotted, and the area under the corresponding curve (AUC) was calculated.

[0070] 1.3 Histopathological Analysis

[0071] Mouse liver and pancreas tissues were fixed in 4% paraformaldehyde for at least 24 hours. The tissues were trimmed and placed in a dehydration box. The tissues were then sequentially immersed in a gradient of 75-100% alcohols for dehydration. The tissues were then encapsulated in paraffin wax and embedded in an embedding machine. The molten wax was placed in an embedding frame and cooled at -20°C. After the paraffin solidified, the tissues were removed and trimmed. Sections were prepared using a paraffin microtome, with a thickness of 4 μm. The sections were fixed on glass slides, dried at 60°C, and then stored at room temperature.

[0072] 1.4 Intestinal flora assay

[0073] For collected fecal samples, the precipitate was resuspended in 1 mL of phosphate buffer (0.1 mol / L). Total bacterial DNA was then extracted from fermentation samples at different culture time points according to the EZNAStool DNA kit instructions, and the V3-V4 hypervariable region of 16S rRNA was amplified by PCR (primers: 338F and 806R). The amplified PCR products were purified by agarose gel electrophoresis, and the target band was excised and recovered. Further purification was performed using the AxyPrep DNAGel Extraction kit, followed by quantification using a QuantiFluor™ fluorometer. Furthermore, the purified amplified products were mixed in equal volumes, ligated with sequencing adapters, and a sequencing library was constructed. Finally, all test samples were sequenced using an Illumina PE250 (OTUs were defined based on a 97% similarity threshold) and analyzed using QIIME.

[0074] 2. Experimental Results

[0075] 2.1 Hypoglycemic effect

[0076] like Figure 2 and Figure 3 As shown, compared with the model group, the fasting blood glucose of mice in the sugarcane molasses extract group decreased by 21.61% and glucose tolerance increased by 20.15%.

[0077] 2.2 Improve tissues and organs

[0078] like Figure 4 As shown, compared with the model group, the pancreatic index of mice in the sugarcane molasses extract group decreased by 27.54%;

[0079] The pancreatic islet cells of the model group mice were severely shrunken and deformed, while those of the mice in the sugarcane molasses extract group showed significant improvement.

[0080] like Figure 5 As shown, compared with the model group, the liver index of mice in the sugarcane molasses extract group decreased by 21.32%.

[0081] The livers of mice in the model group showed extensive inflammatory cell infiltration, as well as pathological phenomena such as cell necrosis, nuclear dissolution, and fatty degeneration. The sugarcane molasses extract group showed significant improvement.

[0082] In summary, sugarcane molasses extract can significantly reduce organ indices and improve organ tissue lesions.

[0083] 2.3 Regulating the gut microbiota

[0084] like Figure 6 As shown in Table 4, sugarcane molasses extract significantly regulated the intestinal flora disorder in mice, mainly manifested as a decrease in Firmicutes (F) abundance and an increase in Bacteroidetes (B) abundance, with an F / B ratio decrease of 90.29%.

[0085] Table 4 Results of intestinal flora detection in mice of each group

[0086] detection indicators Normal group NC Model group MC Sample group ESM Firmicutes (F) content (%) 65.2 77.27 63.91 Bacteroidetes (B) content (%) 12.31 2.43 20.24 F / B 5.29 32.45 3.16

[0087] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a sugarcane molasses extract with hypoglycemic effects, characterized in that, Includes the following steps: Take sugarcane molasses, dilute it, centrifuge to collect the supernatant, remove impurities by ultrafiltration, desalinate by electrodialysis, concentrate by nanofiltration to obtain the enriched solution; The enriched solution is mixed with calcium chloride to obtain the sugarcane molasses extract.

2. The preparation method according to claim 1, characterized in that, The ultrafiltration impurity removal process involves using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove impurities.

3. The preparation method according to claim 1, characterized in that, The nanofiltration concentration involves using a nanofiltration membrane with a molecular weight cutoff of 300 Da to remove large molecular sugars and impurities, concentrating the supernatant to 20%-25% of its volume.

4. The preparation method according to claim 3, characterized in that, The nanofiltration concentration operates at a pressure of 1.0-1.4 MPa and a pH of 4.5-5.

5.

5. A sugarcane molasses extract obtained by the preparation method according to any one of claims 1-4.

6. The use of the sugarcane molasses extract as described in claim 5 in the preparation of health foods that help maintain healthy blood sugar levels.

7. A health food product that helps maintain healthy blood sugar levels, characterized in that, The main active ingredient is the sugarcane molasses extract as described in claim 5.

8. The use of the sugarcane molasses extract as described in claim 5 in the preparation of a drug for lowering blood sugar.

9. A drug for lowering blood sugar, characterized in that, The main active ingredient is the sugarcane molasses extract as described in claim 5.

10. The medicament as claimed in claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.

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