Method for separating and purifying polyphenol extract from sugarcane by-product molasses

Abstract

The invention discloses a preparation method for separating and purifying a polyphenol extract from cane molasses, which comprises the following steps: firstly, diluting the cane molasses to 10-15 times of a solid matter, adding pectinase, cellulase and xylanase for composite enzymolysis, centrifuging to take supernate, and sequentially filtering through a multi-stage gradient ultrafiltration membrane; then sequentially passing through AB-8 and XDA-1 macroporous resin chromatographic columns, and carrying out gradient elution by using ethanol; and finally, concentrating or spray-drying the collected eluent to obtain a cane molasses polyphenol concentrated solution or powder. According to the method disclosed by the invention, the problem of low extraction efficiency in the prior art is effectively solved by combining composite enzymolysis with membrane separation and macroporous resin adsorption technologies, the polyphenol purity can be remarkably improved, and a green and efficient preparation method is provided for high-value utilization of the cane molasses.

Description

Technical Field

[0001] This invention relates to the field of high-value utilization of by-products in the sugar industry and extraction of natural products, and particularly to a method for separating and purifying polyphenolic compounds from molasses, a by-product of sugarcane sugar production. Background Technology

[0002] Sugarcane is an important economic crop for sugar production. During the sugar industry, processes such as juicing, clarification, and crystallization generate a large number of byproducts, among which molasses is one of the largest, accounting for approximately 2-3% of the sugarcane's weight. Molasses is a brownish-black viscous liquid containing abundant polyphenolic compounds, polysaccharides, and minerals, among other active ingredients. However, currently, molasses is mainly used as low-value animal feed or fertilizer, with extremely low utilization of its high-value components, resulting in a huge waste of biomass resources.

[0003] Polyphenols are a class of secondary metabolites widely found in the plant kingdom, possessing various biological activities such as antioxidant, antibacterial, anti-inflammatory, antiviral, and antitumor effects, and have broad application prospects in food, medicine, and cosmetics. Traditional polyphenol extraction mainly relies on fruits, vegetables, tea, and traditional Chinese medicinal herbs; however, extracting polyphenols from these raw materials often faces problems such as limited resources, high costs, and low extraction efficiency. In contrast, sugarcane molasses, as a byproduct of the sugar industry, has a high content of polyphenols and possesses enormous development potential.

[0004] Currently, the application of polyphenol extraction from molasses is still in its early stages, and existing patented technologies (patent application numbers CN201380051377.3, CN 201410550872.5) have some shortcomings:

[0005] (1) Low pretreatment efficiency: Molasses has a complex composition and contains a large number of impurities such as polysaccharides, proteins, and minerals. Traditional physical adsorption methods are difficult to effectively destroy the molasses matrix structure, resulting in insufficient release of polyphenols and low extraction rate.

[0006] (2) Complex process and high cost: Existing extraction processes mostly use organic solvent extraction, vacuum concentration and other methods, which are not only complex to operate and energy-intensive, but also pose a risk of organic solvent residue, which limits their industrial application.

[0007] Therefore, developing an efficient method for extracting and preparing polyphenols from sugarcane molasses is of great significance for improving the utilization rate of molasses resources and expanding the sources of polyphenol raw materials. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a method for efficiently separating and purifying polyphenol extracts from sugarcane molasses. This method utilizes a combination of enzymatic hydrolysis, membrane separation, and macroporous resin technology to solve the problems of low extraction efficiency and high cost in existing technologies.

[0009] To achieve the above objectives, the present invention first discloses a method for separating and purifying polyphenol extracts from sugarcane by-product molasses, comprising the following steps:

[0010] (1) Pretreatment: Dilute the sugarcane molasses, add a compound enzyme for hydrolysis, centrifuge and take the supernatant. The compound enzyme is composed of pectinase, cellulase and xylanase.

[0011] (2) Membrane separation: The supernatant obtained in step (1) is filtered through a gradient ultrafiltration membrane with a molecular weight cutoff of 2 to 200 kDa.

[0012] (3) Dynamic adsorption and elution: The supernatant obtained after filtration in step (2) is passed through AB-8 and XDA-1 macroporous resin chromatography columns in sequence and eluted by ethanol gradient.

[0013] (4) Concentration and drying: Collect the ethanol eluent obtained in step (3) and concentrate it to obtain a concentrated sugarcane molasses polyphenol solution, or obtain sugarcane molasses polyphenol powder by spray drying.

[0014] In one embodiment of the present invention, in step (1), the sugarcane molasses is diluted to 10 to 15 times the solids (water is added until the solids content is 1 / 10 to 15 of the original), the amount of pectinase added is 0.8 to 1.5% of the molasses mass, the amount of cellulase added is 0.2 to 0.5% of the molasses mass, and the amount of xylanase added is 0.5 to 1.0% of the molasses mass.

[0015] In one embodiment of the present invention, in step (1), the complex enzyme hydrolysis refers to enzymatic hydrolysis at 50-55°C for 2-4 hours. After the enzymatic hydrolysis is completed, the temperature is heated to above 98°C and maintained for 2-5 minutes to inactivate the enzyme. The mixture is then centrifuged at 5000 rpm for 15 minutes, and the supernatant is collected for later use.

[0016] In one embodiment of the present invention, in step (2), the three-stage or higher gradient ultrafiltration membrane filtration refers to filtration through a three-stage gradient ultrafiltration membrane with molecular weight cutoffs of 80-120 kDa, 25-35 kDa, and 2-10 kDa, or filtration through a four-stage gradient ultrafiltration membrane with molecular weight cutoffs of 80-120 kDa, 25-35 kDa, 8-12 kDa, and 2-5 kDa, respectively.

[0017] Preferably, in step (2), the supernatant obtained in step (1) is sequentially filtered through multi-stage gradient ultrafiltration membranes with molecular weight cutoffs of 100kDa, 30kDa, 10kDa and 3kDa to remove macromolecular impurities and small molecule impurities.

[0018] In one embodiment of the present invention, in step (3), the enzymatic hydrolysis supernatant of sugarcane molasses is first subjected to an adsorption and elution process through AB-8 macroporous resin. The gradient elution conditions are: elution with pure water for 1-2 BV, elution with 20% ethanol for 2-3 BV, and a flow rate of 3-4 BV / h; then, elution with 60% ethanol for 3-4 BV, and a flow rate of 1.0-1.5 BV / h, and the eluent is collected.

[0019] In one embodiment of the present invention, in step (3), the eluent obtained by eluting the supernatant of sugarcane molasses enzymatic hydrolysis with AB-8 macroporous resin is combined and concentrated (concentrated to 50-60% of the original eluent volume), and then subjected to adsorption and elution process with XDA-1 macroporous resin. The gradient elution conditions are as follows: first, elute with 20% ethanol for 1-2 BV at a flow rate of 3-4 BV / h; then, elute with 80% ethanol for 3-4 BV at a flow rate of 1.0-1.5 BV / h, and collect the 80% ethanol eluent.

[0020] In one embodiment of the present invention, in step (4), the column chromatography eluents of the collected sugarcane molasses polyphenols are combined and then vacuum concentrated at a temperature of 50-55°C until the water content is <20%, thus producing a sugarcane molasses polyphenol concentrate.

[0021] In step (4) of one embodiment of the present invention, the concentrated sugarcane molasses polyphenol eluent is dried into powder using a spray dryer for traditional Chinese medicine extracts (outlet air temperature 80-90°C) to obtain sugarcane molasses polyphenol extract powder.

[0022] The present invention also discloses a concentrated liquid of sugarcane molasses polyphenols or a powder of sugarcane molasses polyphenols prepared according to the above method.

[0023] The present invention also discloses the application of the above-mentioned molasses polyphenol concentrate or molasses polyphenol extract powder in the food and pharmaceutical fields.

[0024] In one embodiment of the present invention, the food includes a low-GI food, and the medicine includes a blood sugar-lowering drug.

[0025] Beneficial effects:

[0026] (1) The present invention adopts a composite enzymatic hydrolysis technology to effectively destroy the structure of molasses polysaccharide, release bound polyphenols, and improve the polyphenol yield; multi-level gradient ultrafiltration membrane separation combined with dynamic adsorption and elution of macroporous resin realizes the removal of macromolecular impurities, greatly improving the purity of the product; and the whole process avoids the use of harmful organic solvents, making it green and environmentally friendly.

[0027] (2) In this invention, pectinase, xylanase and cellulase are compounded in a certain proportion, and the compounded enzymes are used to enzymatically hydrolyze molasses. The compound enzymes can more effectively destroy the polysaccharide structure in molasses and promote the release of polyphenols, thereby further increasing the polyphenol content in the extract.

[0028] (3) In this invention, a large molecular weight (80-120kDa) ultrafiltration membrane is first selected for filtration, and then a multi-stage gradient filtration is performed by combining an ultrafiltration membrane with a decreasing molecular weight gradient. This can effectively prevent large molecular weight molecules from clogging the pores of the ultrafiltration membrane, while effectively retaining low molecular weight polyphenols and further increasing the polyphenol content.

[0029] (4) The total polyphenol content (determined by the Folin-Ciocalteu method) in the final concentrate obtained by extracting polyphenols from molasses is as high as 15.74 mg GAE / g (on a dry basis), which is much higher than the polyphenol content extracted by existing methods. Furthermore, the polyphenols extracted by the method of the present invention have a better blood sugar lowering effect and can be used to prepare low-GI foods. Attached Figure Description

[0030] Figure 1 The blood glucose curve for test subject #1;

[0031] Figure 2 The blood glucose curve for test subject #2;

[0032] Figure 3 The blood glucose curve for test subject #3;

[0033] Figure 4 The blood glucose curve for test subject number 4;

[0034] Figure 5 The blood glucose curve for test subject number 5;

[0035] Figure 6 The blood glucose curve for test subject number 6;

[0036] Figure 7 The blood glucose curve for test subject number 7;

[0037] Figure 8 The blood glucose curve for test subject number 8;

[0038] Figure 9 The blood glucose curve for test subject number 9;

[0039] Figure 10 The blood glucose curve for test subject number 10. Detailed Implementation

[0040] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to existing technology.

[0041] Test methods

[0042] 1. Polyphenol content determination method: The Folin-Ciocalteu method was used for determination. The total phenol content is expressed as milligrams of gallic acid equivalent per gram of dry sample (mg GAE / g DW).

[0043] 2. Bread Making Method: Combine high-gluten flour, granulated sugar, yeast, and salt in a dough mixer and mix well. Then add water and eggs, mixing slowly at first, then quickly until the gluten is mostly formed. Next, add butter and mix slowly at first, then quickly until the dough can form a thin, even film. Remove the dough and let it rest at room temperature for 30 minutes. Divide the dough into several 150g portions, round them, and let them rest for 15 minutes. Proof in a proofing box with 80±5% humidity and 36±2℃ for 60 minutes. Finally, bake in an electric oven at 180℃ (top heat) and 200℃ (bottom heat) for 25 minutes.

[0044] 3. Blood Glucose Testing Method: Following the method outlined in WS / T 652-2019, a total of 10 participants were tested (5 males and 5 females). The experiment was conducted over four days. The blood glucose levels of the control group were measured and averaged on the first two days. On the third day, blood glucose levels were measured in the group using commercially available sugarcane extract, and on the fourth day, blood glucose levels were measured in the group using sugarcane molasses polyphenols. The testing procedure on each day involved first measuring fasting blood glucose, followed by consuming either 130g of bread and 250mL of water, or 50g of glucose and 250mL of water (carbohydrate content ≈ 50g), completed within 5-10 minutes. Postprandial blood glucose was then measured at 15 minutes, 30 minutes, 60 minutes, and 120 minutes. The blood glucose meter used was a Roche EasyPad.

[0045] Example 1

[0046] A method for separating and purifying polyphenol extracts from sugarcane by-product molasses includes the following steps:

[0047] Take 80g of sugarcane molasses, dilute it with water to a solids volume of 12 times, add 0.96g of pectinase (1.2% of molasses mass), 0.24g of cellulase (0.3% of molasses mass), and 0.48g of xylanase (0.6% of molasses mass), and enzymatically hydrolyze it at 53℃ for 3 hours. After enzymatic hydrolysis, heat to 98℃ and hold for 3 minutes to inactivate the enzymes, then centrifuge at 5000rpm for 15 minutes and collect the supernatant for later use.

[0048] The supernatant was sequentially centrifuged through multi-stage gradient ultrafiltration membranes with molecular weight cutoffs of 100 kDa, 30 kDa, 10 kDa, and 3 kDa to remove large molecular impurities. The filtered supernatant was then passed through an AB-8 macroporous resin column, eluted with pure water for 1.5 BV, followed by 2.5 BV elution with 20% ethanol (flow rate 3.5 BV / h), and then eluted with 60% ethanol for 4 BV (flow rate 1.2 BV / h). The 60% ethanol eluent was collected. The AB-8 column eluent was concentrated (to 50% of its original volume) and then subjected to XDA-1 macroporous resin column adsorption. Elution was first performed with 20% ethanol for 1.5 BV (flow rate 3.5 BV / h), followed by 80% ethanol for 4 BV (flow rate 1.2 BV / h). The 80% ethanol eluent was collected.

[0049] The collected eluents were combined and concentrated in a vacuum concentrator (50°C) to a water content of 16.8%, yielding 15.2 g of sugarcane molasses polyphenol concentrate. The total polyphenol content (determined by the Folin-Ciocalteu method) reached 15.74 mg GAE / g (on a dry basis).

[0050] Example 2

[0051] A method for separating and purifying polyphenol extracts from sugarcane by-product molasses includes the following steps:

[0052] Take 100g of sugarcane molasses, dilute it with water to a volume 10 times its original volume, add 1.2g of pectinase (1.2% of molasses mass), 0.2g of cellulase (0.2% of molasses mass), and 0.5g of xylanase (0.5% of molasses mass), and hydrolyze at 50℃ for 2.5h. After hydrolysis, heat to above 98℃ and hold for 2min to inactivate the enzymes, then centrifuge at 5000rpm for 15min and collect the supernatant for later use.

[0053] The supernatant was sequentially centrifuged and filtered through a multi-stage gradient ultrafiltration membrane with molecular weight cutoffs of 100 kDa, 30 kDa, 10 kDa, and 3 kDa. The filtered supernatant was then passed through an AB-8 macroporous resin column, eluted with pure water for 1 BV, followed by 2 BV elution with 20% ethanol (flow rate 3 BV / h), and then eluted with 60% ethanol for 5 BV (flow rate 1.0 BV / h). The 60% ethanol eluent was collected. The AB-8 eluent was then concentrated (to 50% of its original volume) and subjected to XDA-1 macroporous resin column adsorption. Elution was then performed with 20% ethanol for 1 BV (flow rate 3 BV / h), followed by 80% ethanol for 5 BV (flow rate 1.0 BV / h). The 80% ethanol eluent was collected.

[0054] The collected eluents were combined and concentrated in a vacuum concentrator (50°C) to a water content of 19.8%, yielding 20.5g of sugarcane molasses polyphenol concentrate. The total polyphenol content (determined by the Folin-Ciocalteu method) reached 15.06mg GAE / g (on a dry basis).

[0055] Comparative Example 1

[0056] The difference between Comparative Example 1 and Example 1 is that the enzymatic hydrolysis step is omitted, and Comparative Example 1 only uses an AB-8 macroporous resin chromatography column to elute the filtrate.

[0057] Take 80g of sugarcane molasses, dilute it 15 times with water directly, and perform ultrafiltration membrane separation without enzymatic hydrolysis (pass it through ultrafiltration membranes with molecular weight cutoffs of 100kDa, 30kDa, 10kDa, and 3kDa in sequence).

[0058] The filtrate was first passed through an AB-8 macroporous resin chromatography column, eluted with pure water for 2 BV, then eluted with 20% ethanol for another 2 BV (flow rate 3 BV / h), and then eluted with 60% ethanol for 5 BV (flow rate 1.0 BV / h). The 60% ethanol eluent was collected.

[0059] The collected eluents were combined and concentrated under vacuum to a water content of 25.8%, yielding 31.7 g of concentrated sugarcane molasses polyphenols. The total polyphenol content (determined by the Folin-Ciocalteu method) reached 7.62 mg GAE / g (on a dry basis).

[0060] Comparative Example 2

[0061] The difference between Comparative Example 1 and Example 1 is that the enzymatic hydrolysis step is omitted.

[0062] A method for separating and purifying polyphenol extracts from sugarcane by-product molasses includes the following steps:

[0063] Take 80g of sugarcane molasses, dilute it 15 times with water directly, and perform ultrafiltration membrane separation without enzymatic hydrolysis (pass it through ultrafiltration membranes with molecular weight cutoffs of 100kDa, 30kDa, 10kDa, and 3kDa in sequence).

[0064] The filtrate was first passed through an AB-8 macroporous resin column, eluted with pure water for 2 BV, then with 20% ethanol for another 2 BV (flow rate 3 BV / h), followed by 4 BV elution with 60% ethanol (flow rate 1.0 BV / h). The ethanol eluent was collected. The AB-8 eluent was then concentrated (to 50% of its original volume) and passed through an XDA-1 macroporous resin column for adsorption. First, 2 BV was eluted with 20% ethanol (flow rate 3 BV / h), then 5 BV was eluted with 80% ethanol (flow rate 1.0 BV / h). The 80% ethanol eluent was collected.

[0065] The collected eluents were combined and concentrated under vacuum to a water content of 22.5%, yielding 23.6 g of concentrated sugarcane molasses polyphenols. The total polyphenol content (determined by the Folin-Ciocalteu method) reached 8.11 mg GAE / g (on a dry basis).

[0066] Comparative Example 3

[0067] The difference between Comparative Example 3 and Example 2 is that the amount of compound enzyme added is different.

[0068] A method for separating and purifying polyphenol extracts from sugarcane by-product molasses includes the following steps:

[0069] Take 100g of sugarcane molasses, dilute it with water to a volume 10 times its original volume, add 0.5g of pectinase (0.5% of molasses mass), 0.1g of cellulase (0.1% of molasses mass), and 0.2g of xylanase (0.2% of molasses mass), and enzymatically hydrolyze at 50℃ for 2.5h. After enzymatic hydrolysis, heat to above 98℃ and hold for 2min to inactivate the enzymes, then centrifuge at 5000rpm for 15min and collect the supernatant for later use.

[0070] The supernatant was sequentially centrifuged and filtered through a multi-stage gradient ultrafiltration membrane with molecular weight cutoffs of 100 kDa, 30 kDa, 10 kDa, and 3 kDa. The filtered supernatant was then passed through an AB-8 macroporous resin column, eluted with pure water for 1 BV, followed by 2 BV elution with 20% ethanol (flow rate 3 BV / h), and then eluted with 60% ethanol for 5 BV (flow rate 1.0 BV / h). The 60% ethanol eluent was collected. The AB-8 eluent was then concentrated (to 50% of its original volume) and subjected to adsorption on an XDA-1 macroporous resin column. Elution was then performed with 20% ethanol for 1 BV (flow rate 3 BV / h), followed by 80% ethanol for 5 BV (flow rate 1.0 BV / h). The 80% ethanol eluent was collected.

[0071] The collected eluents were combined and concentrated in a vacuum concentrator (50°C) to a water content of 20.6%, yielding 27.6 g of sugarcane molasses polyphenol concentrate. The total polyphenol content (determined by the Folin-Ciocalteu method) reached 11.2 mg GAE / g (on a dry basis).

[0072] Comparative Example 4

[0073] The difference between Comparative Example 4 and Example 1 is that the proportion of the compound enzyme added is different.

[0074] Take 80g of sugarcane molasses, dilute it with water to a solids volume of 12 times, add 0.96g of pectinase (0.5% of molasses mass), 0.24g of cellulase (0.3% of molasses mass), and 0.48g of xylanase (1.2% of molasses mass), and enzymatically hydrolyze it at 53℃ for 3 hours. After enzymatic hydrolysis, heat to 98℃ and hold for 3 minutes to inactivate the enzymes, then centrifuge at 5000rpm for 15 minutes and collect the supernatant for later use.

[0075] The supernatant was sequentially centrifuged through multi-stage gradient ultrafiltration membranes with molecular weight cutoffs of 100 kDa, 30 kDa, 10 kDa, and 3 kDa to remove large molecular impurities. The filtered supernatant was then passed through an AB-8 macroporous resin column, eluted with pure water for 1.5 BV, followed by 2.5 BV elution with 20% ethanol (flow rate 3.5 BV / h), and then eluted with 60% ethanol for 4 BV (flow rate 1.2 BV / h). The 60% ethanol eluent was collected. The AB-8 column eluent was concentrated (to 50% of its original volume) and then subjected to XDA-1 macroporous resin column adsorption. Elution was first performed with 20% ethanol for 1.5 BV (flow rate 3.5 BV / h), followed by 80% ethanol for 4 BV (flow rate 1.2 BV / h). The 80% ethanol eluent was collected.

[0076] The collected eluents were combined and concentrated to a water content of 18.2% using a vacuum concentrator (50℃) to obtain 16.3g of sugarcane molasses polyphenol concentrate, with a total polyphenol content (determined by the Folin-Ciocalteu method) of 11.4mg GAE / g.

[0077] Comparative Example 5

[0078] The difference between Comparative Example 5 and Example 1 is that xylanase and cellulase are omitted.

[0079] Take 80g of sugarcane molasses, dilute it with water to a solid volume of 12 times, add 0.96g of pectinase (1.2% of the molasses mass), and enzymatically hydrolyze it at 53℃ for 3 hours. After enzymatic hydrolysis, heat to 98℃ and hold for 3 minutes to inactivate the enzyme, then centrifuge at 5000rpm for 15 minutes and collect the supernatant for later use.

[0080] The supernatant was sequentially centrifuged through multi-stage gradient ultrafiltration membranes with molecular weight cutoffs of 100 kDa, 30 kDa, 10 kDa, and 3 kDa to remove large molecular impurities. The filtered supernatant was then passed through an AB-8 macroporous resin column, eluted with pure water for 1.5 BV, followed by 2.5 BV elution with 20% ethanol (flow rate 3.5 BV / h), and then eluted with 60% ethanol for 4 BV (flow rate 1.2 BV / h). The 60% ethanol eluent was collected. The AB-8 column eluent was concentrated (to 50% of its original volume) and then subjected to XDA-1 macroporous resin column adsorption. Elution was first performed with 20% ethanol for 1.5 BV (flow rate 3.5 BV / h), followed by 80% ethanol for 4 BV (flow rate 1.2 BV / h). The 80% ethanol eluent was collected.

[0081] The collected eluents were combined and concentrated to a water content of 15.6% using a vacuum concentrator (50°C) to obtain 14.7g of sugarcane molasses polyphenol concentrate, with a total polyphenol content (determined by the Folin-Ciocalteu method) of 8.81mg GAE / g.

[0082] Comparative Example 6

[0083] The difference between Comparative Example 6 and Example 1 is that a two-stage gradient ultrafiltration membrane is selected for ultrafiltration.

[0084] A method for separating and purifying polyphenol extracts from sugarcane by-product molasses includes the following steps:

[0085] Take 80g of sugarcane molasses, dilute it with water to a solids volume of 12 times, add 0.96g of pectinase (1.2% of molasses mass), 0.24g of cellulase (0.3% of molasses mass), and 0.48g of xylanase (0.6% of molasses mass), and enzymatically hydrolyze it at 53℃ for 3 hours. After enzymatic hydrolysis, heat to 98℃ and hold for 3 minutes to inactivate the enzymes, then centrifuge at 5000rpm for 15 minutes and collect the supernatant for later use.

[0086] The supernatant was centrifuged and filtered sequentially through a two-stage gradient ultrafiltration membrane with molecular weight cutoffs of 100 kDa and 30 kDa to remove macromolecular impurities. The filtered supernatant was then passed through an AB-8 macroporous resin column, eluted with pure water for 1.5 BV, followed by 2.5 BV elution with 20% ethanol (flow rate 3.5 BV / h), and then eluted with 60% ethanol for 4 BV (flow rate 1.2 BV / h). The 60% ethanol eluent was collected. The AB-8 column eluent was concentrated (the volume of the concentrated eluent was 50% of the original volume) and then subjected to adsorption on an XDA-1 macroporous resin column. Elution was first performed with 20% ethanol for 1.5 BV (flow rate 3.5 BV / h), followed by 80% ethanol for 4 BV (flow rate 1.2 BV / h). The 80% ethanol eluent was collected.

[0087] The collected eluents were combined and concentrated to a water content of 21.6% using a vacuum concentrator (50°C) to obtain 29.1g of sugarcane molasses polyphenol concentrate. The total polyphenol content (determined by the Folin-Ciocalteu method) reached 9.33mg GAE / g (on a dry basis).

[0088] Comparative Example 7

[0089] The difference between Comparative Example 7 and Example 1 is that the molecular weight is different during ultrafiltration.

[0090] Take 80g of sugarcane molasses, dilute it with water to a solids volume of 12 times, add 0.96g of pectinase (1.2% of molasses mass), 0.24g of cellulase (0.3% of molasses mass), and 0.48g of xylanase (0.6% of molasses mass), and enzymatically hydrolyze it at 53℃ for 3 hours. After enzymatic hydrolysis, heat to 98℃ and hold for 3 minutes to inactivate the enzymes, then centrifuge at 5000rpm for 15 minutes and collect the supernatant for later use.

[0091] The supernatant was sequentially centrifuged and filtered through a three-stage gradient ultrafiltration membrane with molecular weight cutoffs of 30 kDa, 10 kDa, and 3 kDa to remove large molecular impurities. The filtered supernatant was then passed through an AB-8 macroporous resin column, eluted with pure water for 1.5 BV, followed by 2.5 BV elution with 20% ethanol (flow rate 3.5 BV / h), and then eluted with 60% ethanol for 4 BV (flow rate 1.2 BV / h). The 60% ethanol eluent was collected. The AB-8 column eluent was concentrated (the volume of the concentrated eluent was 50% of the original volume) and then subjected to adsorption on an XDA-1 macroporous resin column. Elution was first performed with 20% ethanol for 1.5 BV (flow rate 3.5 BV / h), followed by 80% ethanol for 4 BV (flow rate 1.2 BV / h). The 80% ethanol eluent was collected.

[0092] The collected eluents were combined and concentrated to a water content of 12.8% using a vacuum concentrator (50°C) to obtain 13.2g of sugarcane molasses polyphenol concentrate. The total polyphenol content (determined by the Folin-Ciocalteu method) reached 8.03mg GAE / g (on a dry basis).

[0093] Comparative Example 8

[0094] Commercially available sugarcane polyphenol extract is a viscous liquid with a water content of 17.8% and a total polyphenol content (determined by the Folin-Ciocalteu method) of 12.80 mg GAE / g (on a dry basis).

[0095] The experimental data from the comparative examples and the comparative examples show that the method of the present invention, through the combination of compound enzymatic hydrolysis pretreatment and multi-stage column chromatography desorption, significantly increases the polyphenol content in the polyphenol extract. Compared with the comparative example without enzymatic hydrolysis, the polyphenol content is increased by nearly 100%, which verifies the effectiveness and superiority of the process of the present invention.

[0096] In this invention, the enzymatic hydrolysis process significantly affects the final polyphenol extraction efficiency. Specifically, the choice of enzyme and the ratio of enzymes in the compound directly influence the polyphenol content in the final extract. Experimental data from Examples 3-5 and Comparative Examples 3-5 show that if only a single enzyme is added, the polyphenol content in the extract is only about half that of the examples. Adding a small amount of the compound enzyme also significantly reduces the polyphenol content. Furthermore, the ratio of pectinase, xylanase, and cellulase also has a significant impact. Comparative Example 4 increased the amount of xylanase while reducing the amounts of the other two enzymes, resulting in a low polyphenol content in the final extract, only 11.40 mg GAE / g (dry basis).

[0097] The filtration after enzymatic hydrolysis also has a significant impact on the polyphenol content in the final extract. As can be seen from the experimental data of the examples and comparative examples 6-7, if only ultrafiltration membranes with larger molecular weights are used for filtration, more polyphenols will be filtered out, resulting in a decrease in polyphenol content. If ultrafiltration membranes with smaller molecular weights (below 50 kDa) are used for filtration first, large molecular weight impurities will clog the pores of the ultrafiltration membrane, resulting in a decrease in the polyphenol content in the supernatant.

[0098] Furthermore, the sugarcane polyphenol extract prepared in Example 1 and the commercially available sugarcane polyphenol extract in Comparative Example 8 were selected as samples. The two sugarcane polyphenol extracts were added to bread, and their glycemic index was tested. Three groups were set up for the test, and the main ingredients were as follows: (1) Control group: 50g glucose; (2) Bread ingredients of commercially available sugarcane extract group: 1000g high gluten flour, 100g white sugar, 12g salt, 10g yeast, 100g egg, 500g water, 120g butter, 14g commercially available sugarcane extract; (3) Bread ingredients of sugarcane molasses polyphenol extract group: 1000g high gluten flour, 100g white sugar, 12g salt, 10g yeast, 100g egg, 500g water, 120g butter, 14g sugarcane molasses polyphenols. The high-gluten flour used is Yihai Kerry Jinshan bread flour, which has a carbohydrate content of about 70%. Therefore, the carbohydrate content of the flour in the formula is about 700g. The amount of commercially available sugarcane extract and sugarcane molasses polyphenol extract added is 2% of 700g.

[0099] The blood glucose variation data of test subjects 1-10 are shown in Table 1, and the blood glucose generation curves of test subjects 1-10 are shown in Table 1. Figures 1-10As shown, the results indicate that the GI values ​​of the sugarcane molasses polyphenol group were all lower than those of commercially available sugarcane extracts, and 7 people achieved a low GI effect (GI value ≤ 55), indicating that sugarcane molasses polyphenols have a good effect in inhibiting postprandial blood glucose elevation.

[0100] The dough in the commercially available sugarcane extract group and the sugarcane molasses polyphenol group showed reduced gluten strength and a slightly darker crust color, but there was no significant difference. The taste and flavor were similar to the control group bread.

[0101] Table 1

[0102]

[0103] Note: * represents female.

[0104] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for separating and purifying polyphenol extracts from sugarcane by-product molasses, characterized in that, Includes the following steps: (1) Pretreatment: Dilute the sugarcane molasses, add a compound enzyme for hydrolysis, centrifuge and take the supernatant. The compound enzyme is composed of pectinase, cellulase and xylanase. (2) Membrane separation: The supernatant obtained in step (1) is filtered through a gradient ultrafiltration membrane with a molecular weight cutoff of 2 to 200 kDa. (3) Dynamic adsorption and elution: The supernatant obtained after filtration in step (2) is passed through AB-8 and XDA-1 macroporous resin chromatography columns in sequence and eluted by ethanol gradient. (4) Concentration and drying: Collect the ethanol eluent obtained in step (3) and concentrate it to obtain a concentrated sugarcane molasses polyphenol solution, or obtain sugarcane molasses polyphenol powder by spray drying.

2. The method according to claim 1, characterized in that, In step (1), the sugarcane molasses is diluted to 10 to 15 times the solids. The amount of pectinase added is 0.8 to 1.5% of the molasses mass, the amount of cellulase added is 0.2 to 0.5% of the molasses mass, and the amount of xylanase added is 0.5 to 1.0% of the molasses mass.

3. The method according to claim 1, characterized in that, In step (1), the hydrolysis temperature of the compound enzyme is 50-55℃ and the hydrolysis time is 2-4h.

4. The method according to claim 1, characterized in that, In step (2), the three-stage or higher gradient ultrafiltration membrane filtration includes sequential filtration through a four-stage gradient ultrafiltration membrane with molecular weight cutoffs of 80-120 kDa, 25-35 kDa, 8-12 kDa, and 2-5 kDa.

5. The method according to claim 1, characterized in that, In step (3), the supernatant is first passed through AB-8 macroporous resin for adsorption and elution. The gradient elution conditions are: elution with pure water for 1-2 BV, elution with 20% ethanol for 2-3 BV, and a flow rate of 3-4 BV / h; then elution with 60% ethanol for 3-4 BV, and a flow rate of 1.0-1.5 BV / h, and the eluent is collected.

6. The method according to claim 1, characterized in that, In step (3), the eluents after elution of the supernatant by AB-8 macroporous resin are combined and concentrated, and then adsorbed and eluted by XDA-1 macroporous resin. The gradient elution conditions are: first, elute with 20% ethanol for 1-2 BV at a flow rate of 3-4 BV / h; then, elute with 80% ethanol for 3-4 BV at a flow rate of 1.0-1.5 BV / h, and collect the 80% ethanol eluent.

7. The method according to claim 1, characterized in that, In step (4), the column chromatography eluents of the collected sugarcane molasses polyphenols are combined and then concentrated under vacuum at a temperature of 50-55°C until the water content is <20%, thus producing a concentrated sugarcane molasses polyphenol solution.

8. The method according to claim 7, characterized in that, In step (4), the concentrated sugarcane molasses polyphenol solution is dried into powder using a spray dryer to obtain sugarcane molasses polyphenol extract powder.

9. The concentrated sugarcane molasses polyphenol solution or the sugarcane molasses polyphenol extract powder prepared by any one of claims 1 to 8.

10. The application of the molasses polyphenol concentrate or molasses polyphenol extract powder according to claim 9 in the food and pharmaceutical fields.

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