A method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate

Abstract

The invention discloses a method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate, which belongs to the technical field of xylose production, and comprises the following steps: gramineous plants are hydrolyzed with dilute acid to obtain xylose hydrolysate; the xylose hydrolysate flows through a granular activated carbon adsorption column to obtain a xylose decolorizing solution; an alcohol eluent is passed into the granular activated carbon adsorption column to obtain an alcohol eluent; the alcohol eluent is subjected to rectification and concentration, decolorization, filtration, and chromatographic separation steps to obtain a B material rich in ferulic acid and a C material rich in p-coumaric acid; the pH of the B material and the C material is adjusted to 9-10, rectification and concentration, and acid precipitation to obtain high-purity ferulic acid and p-coumaric acid respectively; an alkali eluent is passed into the granular activated carbon adsorption column after alcohol elution to obtain an alkali eluent. The present invention, on the basis of not changing the xylose production process, utilizes renewable granular activated carbon to separate ferulic acid and p-coumaric acid, obtains high-purity ferulic acid and p-coumaric acid at low cost, has a good separation effect, and reduces the discharge of wastewater.

Description

Technical Field

[0001] The invention relates to the technical field of xylose production, and in particular to a method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate. Background Art

[0002] Xylose is mainly prepared by extracting hemicellulose from gramineous plants. It is widely used in food, medicine and other fields, mainly for the production of xylitol. China is a major producer and exporter of xylose and xylitol, and the corresponding production technology is mature and stable. The xylose production process is: dilute acid hydrolysis-neutralization-decolorization-purification-concentration-crystallization-finished product. The traditional decolorization process uses powdered activated carbon to remove non-sugar organic matter in the hydrolyzate, including pigments, lignin, proteins and other substances. However, powdered activated carbon cannot be reused, the cost is very high, and the adsorbed substances cannot be recycled.

[0003] The hydroxycinnamic acids in plant cell walls are mainly ferulic acid (4-hydroxy-3-methoxycinnamic acid, Ferulic acid, FA) and p-coumaric acid (p-hydroxycinnamic acid, p -Coumaric acid, p -CA), which is mainly combined with lignin and hemicellulose through ester bonds or ether bonds. Ferulic acid and p-coumaric acid are natural antioxidants with antioxidant and free radical scavenging effects. They are increasingly widely used in the fields of food, medicine and cosmetics. For example, they can be used as food additives and food preservatives, as well as in medicine for the prevention and treatment of cardiovascular and cerebrovascular diseases and as cosmetic additives to inhibit the formation of melanin and resist ultraviolet radiation.

[0004] At present, ferulic acid and p-coumaric acid are mostly extracted by alkaline hydrolysis using wheat bran as raw materials. The raw materials are limited, the products are single, and the scale is limited. The cost is high, which limits their application prospects. In the dilute acid hydrolysis process of grass plants, as hemicellulose degrades, these two hydroxycinnamic acids also peel off from the main body of lignocellulose and dissolve in the hydrolyzate together with xylose. There is also a considerable concentration of protein hydrolyzate in the xylose hydrolyzate. Ferulic acid, p-coumaric acid and protein hydrolyzate in the hydrolyzate are not fully recovered, resulting in waste.

[0005] Chinese patent CN101337881B discloses a method for preparing ferulic acid, p-coumaric acid and pentosan, and introduces the technology of using dilute alkali solution of different concentrations for low temperature hydrolysis, then separating pentosan, and extracting p-coumaric acid and ferulic acid respectively. It is known that the current pentosan market is not yet mature, and ferulic acid and p-coumaric acid are difficult to achieve separate hydrolysis under alkaline conditions, so the downstream separation is very difficult. Chinese patent CN112707812B discloses a method for separating ferulic acid and p-coumaric acid in xylose hydrolyzate using chromatographic technology, and uses chromatographic technology to achieve sugar acid separation, but the separation efficiency is low and the cost is high.

[0006] In view of the above problems in the prior art, the present invention combines many years of design and use experience in related fields to design a method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate to overcome the above defects. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate, which adopts granular activated carbon fixed bed adsorption decolorization technology to realize the extraction of ferulic acid, p-coumaric acid and protein hydrolysate in xylose hydrolysate, and separates and purifies with the help of chromatographic separation, membrane separation and acid precipitation, isoelectric precipitation and other technologies, so as to obtain ferulic acid, p-coumaric acid and protein hydrolysate at low cost and high efficiency.

[0008] In order to achieve the above object, the technical scheme adopted by the present invention is as follows: a method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate, comprising the following steps:

[0009] Step 1, dilute acid hydrolysis: the grass plant is subjected to dilute acid hydrolysis and solid-liquid separation to obtain a xylose hydrolyzate, wherein the refractive index of the xylose hydrolyzate is 1-10%;

[0010] Step 2, activated carbon adsorption: the xylose hydrolyzate obtained in step 1 flows through a granular activated carbon adsorption column to obtain a xylose decolorized solution until the granular activated carbon adsorption reaches saturation;

[0011] Step 3, alcohol elution: passing an alcohol eluent into the saturated granular activated carbon adsorption column in step 2 to obtain an alcohol eluate, until the transmittance of the alcohol eluate at 420 nm is 75-95%, and collecting the alcohol eluate;

[0012] Step 4, separation of ferulic acid and p-coumaric acid: the alcohol eluate collected in step 3 is concentrated by distillation to obtain a hydroxycinnamic acid concentrate, and the pH of the hydroxycinnamic acid concentrate is adjusted to 5-7, and then subjected to decolorization, filtration, and chromatographic separation steps to obtain a B material rich in ferulic acid and a C material rich in p-coumaric acid, wherein the eluent used for the chromatographic separation is an alcohol aqueous solution with a mass fraction of 40-99%;

[0013] Step 5, purification: after adjusting the pH of material B and material C in step 4 to 9-10, respectively, distillation concentration, acid precipitation, centrifugation, and drying are performed to obtain ferulic acid with a purity of ≥99.0% and p-coumaric acid with a purity of ≥99.0%, respectively;

[0014] Step 6, alkaline elution: introducing an alkaline eluent into the granular activated carbon adsorption column after the alcohol elution in step 3 to obtain an alkaline eluent until the transmittance of the alkaline eluent at 420 nm is greater than 95%, and collecting the alkaline eluent;

[0015] Step 7, separation of protein hydrolysate: The alkaline eluate collected in step 6 is concentrated by evaporation and de-alkalized by electrodialysis to obtain an electrodialysis fresh water solution, and an inorganic acid is added dropwise to the electrodialysis fresh water solution until the pH value is 2.5-4.0. After standing for 2-24 hours, the crude protein hydrolysate is obtained by centrifugation and drying.

[0016] Preferably, in step 1, the mass ratio of the grass plant to the dilute acid is 1:8-1:10, and the hydrolysis conditions are 100° C.-130° C. for 1 h-4 h;

[0017] In step 1, the grass plant is any one of corn cobs, bagasse, bamboo, and wheat straw, or a combination of several of them, and the dilute acid is any one of hydrochloric acid, sulfuric acid, and acetic acid with a mass fraction of 1-1.5%.

[0018] Preferably, the xylose decolorizing solution in step 2 is deacidified by electrodialysis to obtain a xylose neutralized solution with a pH of 2.5-3.5, and the xylose neutralized solution is subjected to membrane concentration, ion exchange, multi-stage concentration, crystallization, centrifugation, and drying steps to obtain xylose crystals;

[0019] After the ion exchange, the solution conductivity is ≤100 μS / cm, and the transmittance at 420 nm is ≥60%;

[0020] The ion exchange includes cation exchange resin and anion exchange resin.

[0021] Preferably, when the transmittance of the xylose decolorizing solution at 420 nm in step 2 is 40-90%, the granular activated carbon adsorption reaches saturation;

[0022] The granular activated carbon after alkaline elution in step six is ​​recycled according to steps two, three and six.

[0023] Preferably, when the pH of the xylose decolorization liquid obtained by repeating step 2 after the alkaline elution in step six is ​​≥6.0, the xylose decolorization liquid is refluxed to the xylose hydrolyzate raw material tank; when the pH is <6.0, the xylose decolorization liquid is subjected to electrodialysis deacidification, membrane concentration, ion exchange, multi-stage concentration, crystallization, centrifugation, and drying processes to obtain xylose crystals.

[0024] Preferably, the alcohol eluent in step three is an alcohol aqueous solution with a mass fraction of 40-99%, and the alcohol aqueous solution is selected from any one or a combination of methanol aqueous solution, ethanol aqueous solution, propanol aqueous solution, and isopropanol aqueous solution.

[0025] Preferably, the system temperature of the chromatographic separation in step 4 is 35-80° C., and the system pressure is 0.2-0.54 MPa.

[0026] Preferably, in step 5, a sodium hydroxide solution with a mass fraction of 10-20% is added to material B and material C respectively to adjust the pH to 9-10.

[0027] Preferably, in step 5, material B is distilled and concentrated to a mass fraction of ferulic acid of 30-45%, and material C is distilled and concentrated to a mass fraction of p-coumaric acid of 30-46%;

[0028] Acid precipitation in step 5: Add 5-10% hydrochloric acid by mass to the B material and C material after distillation and concentration, respectively, until the pH is 2.5-4.0 and stand for 2-24 hours to precipitate ferulic acid crystals and p-coumaric acid crystals, respectively.

[0029] Preferably, the alkaline eluent in step six is ​​an alkaline solution with a mass fraction of 1-4%, and the alkaline solution is selected from any one of sodium hydroxide solution, potassium hydroxide solution, and calcium hydroxide solution, or a combination of several thereof.

[0030] Preferably, the pH of the electrodialysis fresh water in step seven is 10.0-10.5;

[0031] The mass fraction of the inorganic acid in step seven is 5-20%, and the inorganic acid is selected from any one of hydrochloric acid, sulfuric acid, and acetic acid or a combination of several thereof.

[0032] The invention is beneficial in that:

[0033] 1. The present invention uses acid hydrolysis to prepare ferulic acid and p-coumaric acid without changing the xylose production process, separates ferulic acid, p-coumaric acid and protein hydrolysate through recyclable granular activated carbon, and obtains high-purity ferulic acid and p-coumaric acid at low cost by using the acid precipitation principle. The purity of ferulic acid and p-coumaric acid is ≥99.0%, the separation effect is good, the discharge of wastewater is reduced, and the production value of plant straw is improved.

[0034] 2. The dual solvent elution of granular activated carbon in the present invention can achieve efficient separation of hydroxycinnamic acid (i.e., ferulic acid and p-coumaric acid) and protein hydrolysate. The operation is simple, and near-zero emission of the process is achieved. At the same time, conditions are created for recovering the protein hydrolysate.

[0035] 3. Based on the traditional xylose production process, the present invention only needs to add a small amount of core equipment to achieve high-purity production of ferulic acid and p-coumaric acid, with low raw material cost, simple process route and strong operability, high product purity, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The present invention is a schematic flow diagram of a method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate. DETAILED DESCRIPTION

[0037] In order to facilitate understanding by those skilled in the art, the present invention is further described below in conjunction with specific embodiments.

[0038] Step 1, dilute acid hydrolysis: The grass plant is subjected to dilute acid hydrolysis and solid-liquid separation to obtain a xylose hydrolyzate, and the refractive index of the xylose hydrolyzate is 1-10%;

[0039] Step 2, activated carbon adsorption: the xylose hydrolyzate obtained in step 1 flows through a granular activated carbon adsorption column to obtain a xylose decolorized solution until the granular activated carbon is saturated with adsorption, and hydroxycinnamic acid (i.e., ferulic acid and p-coumaric acid) and protein hydrolyzate are adsorbed on the granular activated carbon;

[0040] Step 3: Alcohol elution: Add alcohol eluent to the saturated granular activated carbon adsorption column in step 2 to obtain an alcohol eluate until the transmittance of the alcohol eluate at 420 nm (OD 420 ) is 75-95%, and the alcohol eluate is collected;

[0041] Step 4, separation of ferulic acid and p-coumaric acid: the alcohol eluate collected in step 3 is concentrated by distillation to obtain a hydroxycinnamic acid concentrate, the pH of the hydroxycinnamic acid concentrate is adjusted to 5-7, and then subjected to decolorization, filtration, and chromatographic separation steps to obtain a B material rich in ferulic acid and a C material rich in p-coumaric acid, and the eluent used for chromatographic separation is an alcohol aqueous solution with a mass fraction of 40-99%;

[0042] Step 5, purification: after adjusting the pH of material B and material C in step 4 to 9-10, respectively carry out rectification and concentration, acid precipitation, centrifugation, and drying to obtain ferulic acid with a purity of ≥99.0% and p-coumaric acid with a purity of ≥99.0%;

[0043] Step 6: Alkaline elution: Add an alkaline eluent into the granular activated carbon adsorption column after the alcohol elution in step 3 to obtain an alkaline eluent until the transmittance (OD 420 )>95%, collect the alkaline eluate;

[0044] Step 7, separation of protein hydrolysate: The alkaline eluate collected in step 6 is concentrated by evaporation and de-alkalized by electrodialysis to obtain an electrodialysis fresh water solution, and an inorganic acid is added dropwise to the electrodialysis fresh water solution until the pH value is 2.5-4.0. After standing for 2-24 hours, the crude protein hydrolysate is obtained by centrifugation and drying.

[0045] The hydroxycinnamic acid mentioned in the present invention is ferulic acid and p-coumaric acid. The present invention effectively utilizes the waste liquid generated by xylose hydrolysis, obtains xylose hydrolyzate by dilute acid hydrolysis of gramineous plants, and uses granular activated carbon to adsorb hydroxycinnamic acid and protein hydrolyzate in the xylose hydrolyzate without affecting the xylose production process. Then, the granular activated carbon is respectively eluted with alcohol and eluted with alkali based on the different solubility of hydroxycinnamic acid and protein hydrolyzate in the eluent, so as to achieve efficient separation of hydroxycinnamic acid and protein hydrolyzate, and the operation is simple, so as to achieve near-zero emission of the process, and at the same time, create conditions for recovering the protein hydrolyzate therein.

[0046] The present invention separates and purifies by means of chromatographic separation, membrane separation and acid precipitation, isoelectric precipitation and other technologies, and obtains ferulic acid, p-coumaric acid and protein hydrolysate respectively. It is possible to obtain high-purity ferulic acid and p-coumaric acid at low cost, and the alcohol eluent and the alkaline eluent in the production process can be recycled by distillation to reduce waste liquid discharge, and the separated protein hydrolysate crude product can be used as an organic nitrogen source for fermented feed in animal husbandry. The process route is simple and highly operable, and the product purity is high, which is suitable for large-scale industrial production.

[0047] In the present invention, the mass ratio of the gramineous plant to the dilute acid in step 1 is 1:8-1:10, the hydrolysis conditions are 100℃-130℃ for 1h-4h, the refractive index of the xylose hydrolyzate is 1-10%, the gramineous plant is any one or a combination of corn cobs, bagasse, bamboo, and wheat straw, and the dilute acid is any one of hydrochloric acid, sulfuric acid, and acetic acid with a mass fraction of 1-1.5%. In step 2, the flow rate of the xylose hydrolyzate into the granular activated carbon is 0.25-1.0BV / h; when the transmittance (OD 420 ) is 40-90%, it is considered that the adsorption of the granular activated carbon has reached saturation. At this time, the introduction of xylose hydrolyzate into the granular activated carbon is stopped, and the granular activated carbon is sequentially subjected to alcohol elution desorption in step three and alkali elution desorption in step six.

[0048] The xylose decolorized liquid in step 2 is deacidified by electrodialysis to obtain a xylose neutralized liquid with a pH of 2.5-3.5; the xylose neutralized liquid is concentrated by membrane to obtain a membrane concentrated liquid, and the solid content of the membrane concentrated liquid is 12%-24%; the membrane concentrated liquid is subjected to an ion exchange process, using cation exchange resin and anion exchange resin to make the conductivity of the membrane concentrated liquid ≤100μS / cm and the transmittance ≥60%, the cation exchange resin is preferably 001*7 resin, and the anion exchange resin is preferably D301 resin, and then concentrated by multi-stage evaporation to a solid content of ≥85% in the solution, and then enters a crystallizer to be cooled to a temperature of ≤35°C, xylose crystals are precipitated, and centrifugal drying is performed, and the cooling rate in the crystallizer is preferably 0.5-2.0°C / h.

[0049] In step 3, the alcohol eluent is preferably an alcohol aqueous solution with a mass fraction of 40-99%, and the alcohol aqueous solution is selected from any one or a combination of methanol aqueous solution, ethanol aqueous solution, propanol aqueous solution, and isopropanol aqueous solution. The alcohol eluent introduction rate is preferably 0.25-1.0 BV / h.

[0050] The total mass fraction of ferulic acid and p-coumaric acid in the hydroxycinnamic acid concentrate obtained in step 4 is preferably 15-45%. The hydroxycinnamic acid concentrate is decolorized by an activated carbon decolorization column, filtered and then chromatographically separated. The system temperature of the chromatographic separation device is 35-80° C., the system pressure is 0.2-0.54 MPa, the ferulic acid content in material B is ≥90%, and the p-coumaric acid content in material C is ≥80%.

[0051] Specifically, when the pH of the hydroxycinnamic acid concentrate is acidic, ferulic acid and p-coumaric acid are precipitated together to obtain a mixture, and ferulic acid and p-coumaric acid cannot be obtained separately; when the pH is alkaline, ferulic acid and p-coumaric acid are both present in the form of organic acid salts, but the separation degree of the two organic acid salts in the chromatography is small. The pH of the hydroxycinnamic acid concentrate is adjusted to 5-7, and ferulic acid and p-coumaric acid are separated under elution with an alcohol aqueous solution with a mass fraction of 40-99%, thereby obtaining a B material rich in ferulic acid and a C material rich in p-coumaric acid.

[0052] In step 5 of the present invention, a sodium hydroxide solution with a mass fraction of 10-20% is added to the material B and the material C respectively, the pH is adjusted to 9-10, and then the material B is rectified and concentrated to a mass fraction of 30-45% of ferulic acid and a mass fraction of 30-46% of p-coumaric acid in the material C, and the top fraction can be recovered as an alcohol eluent; the acid precipitation in step 5: hydrochloric acid with a mass fraction of 5-10% is added dropwise to the material B and the material C after the rectification and concentration to a pH of 2.5-4.0, and the mixture is allowed to stand for 2-24 hours to precipitate ferulic acid crystals and p-coumaric acid crystals respectively, and high-purity ferulic acid and p-coumaric acid are obtained by centrifugal drying, and the purity of both is ≥99.0%.

[0053] In step five of the present invention, sodium hydroxide solution is added to convert the ferulic acid in material B and p-coumaric acid in material C into corresponding organic sodium salts, alcohol is removed from material B and material C by distillation and concentration, and hydrochloric acid is added to neutralize the sodium salts of ferulic acid and p-coumaric acid by utilizing the low water solubility of ferulic acid and p-coumaric acid, so that high-purity ferulic acid and p-coumaric acid are precipitated.

[0054] In step 6 of the present invention, the flow rate of the alkaline eluent is 0.25-1.0 BV / h, the alkaline eluent is an alkaline solution with a mass fraction of 1-4%, and the alkaline solution is preferably any one or a combination of sodium hydroxide solution, potassium hydroxide solution, and calcium hydroxide solution. And the granular activated carbon after alkaline elution can be recycled according to step 2, step 3, and step 6, so as to obtain xylose decolorization liquid, alcohol eluent and alkaline eluent.

[0055] When the granular activated carbon after alkali elution repeats step 2 to obtain xylose decolorization liquid, when the pH of the xylose decolorization liquid is ≥6.0, the obtained xylose decolorization liquid is refluxed to the xylose hydrolyzate raw material tank, and when the pH of the xylose decolorization liquid is <6.0, the xylose decolorization liquid is subjected to electrodialysis deacidification, membrane concentration, ion exchange, multi-stage concentration, crystallization, centrifugation, and drying processes to obtain xylose.

[0056] Specifically, the granular activated carbon is regenerative and can be recycled, thereby reducing the production cost of ferulic acid and p-coumaric acid and reducing waste. In the process of decolorizing the xylose hydrolyzate with the granular activated carbon after alkali elution in step 2, the pH value of the xylose decolorization liquid obtained at the beginning is alkaline. In order to avoid the influence of pH on subsequent elution, the xylose decolorization liquid with alkaline pH value needs to be refluxed to the hydrolyzate raw material tank until the pH of the xylose decolorization liquid is less than 6.0. When the pH is less than 6.0, the xylose decolorization liquid stops refluxing, and then the electrodialysis deacidification process and other processes are carried out to obtain xylose.

[0057] In step seven, the alkaline eluent is concentrated by evaporation to a solid content of 20-30%, and the electrodialysis is performed to remove the alkali until the pH of the electrodialysis fresh water is 10.0-10.5. The alkaline solution recovered by electrodialysis can be used as an alkaline eluent. The mass fraction of the inorganic acid in step seven is 5-20%, and the inorganic acid is selected from any one or a combination of hydrochloric acid, sulfuric acid, and acetic acid.

[0058] In step 7 of the present invention, the inorganic base in the alkaline eluent is recovered by electrodialysis, so that the pH of the alkaline eluent is reduced to 10.0-10.5. The isoelectric precipitation property of the protein is then used to adjust the pH to 2.5-4.0, so that the protein hydrolysate is precipitated, and the protein hydrolysate is obtained by centrifugation and drying. The protein hydrolysate can be used as an organic nitrogen source for fermented feed in animal husbandry. In the present invention, the solid content is determined by Abbe refractometer, and ferulic acid and p-coumaric acid are quantitatively detected by high performance liquid chromatography.

[0059] The specific embodiments are as follows

[0060] Chromatographic conditions for phenolic acid content detection:

[0061] Chromatographic column: Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm);

[0062] Mobile phase: 1% glacial acetic acid (phase A)-methanol (phase B), elution conditions: 28% methanol for 0-28 min, 95% methanol for 28-40 min, 28% methanol for 40-45 min;

[0063] Flow rate: 0.8 mL / min;

[0064] Column temperature: 40°C;

[0065] Detector: UVD 320nm. Example 1

[0066] Step 1, dilute acid hydrolysis: the washed corn cobs were mixed with 1.25% hydrochloric acid at a mass ratio of 1:8, and hydrolyzed at 100°C for 4 hours. During the hydrolysis process, exhaust gas was intermittently discharged, and xylose hydrolyzate was collected by solid-liquid separation. The refractive index of the xylose hydrolyzate was 1%.

[0067] Step 2, activated carbon adsorption: The xylose hydrolyzate enters the bottom of the fixed bed filled with granular activated carbon and flows out from the top at a flow rate of 0.25 BV / h. The xylose decolorization liquid is obtained by the granular activated carbon. Hydroxycinnamic acid and protein hydrolysis products are adsorbed on the granular activated carbon. When the transmittance of the xylose decolorization liquid at 420 nm (OD 420 ) is 40%, the granular activated carbon adsorption reaches saturation, the fixed bed stops feeding, and the solution in the fixed bed is pushed out with compressed air;

[0068] Preparation of xylose: The xylose decolorized solution obtained in step 2 was deacidified by electrodialysis to obtain a xylose neutralized solution with a pH of 2.5. The xylose neutralized solution was concentrated by membrane (pressure-driven membrane) to obtain a membrane concentrate with a solid content of 12% (determined by Abbe refractometer); the membrane concentrate was subjected to an ion exchange process (001*7 resin and D301 resin) to make the solution conductivity 100μS / cm and the transmittance (OD 420 ) is 60%, and then it is concentrated by multi-stage evaporation to a solid content of 85% in the solution, and then enters the crystallizer to cool to 35°C, and centrifugally dry to obtain xylose crystals, and the cooling rate is 0.5°C / h;

[0069] Step 3: Alcohol elution: Add alcohol eluent into the saturated granular activated carbon fixed bed in step 2 to obtain an alcohol eluate. When the transmittance of the alcohol eluate (OD 420) is 75%, the elution is stopped, the solution in the fixed bed is pushed out with compressed air, and the alcohol eluent is collected. The flow rate of the alcohol eluent is 0.25 BV / h, and the alcohol eluent is a 40% by mass methanol aqueous solution;

[0070] Step 4, separation of ferulic acid and p-coumaric acid: the alcohol eluate obtained in step 3 is concentrated by a distillation tower to obtain a hydroxycinnamic acid concentrate, the mass fraction of hydroxycinnamic acid in the hydroxycinnamic acid concentrate is 15%, and then the pH of the hydroxycinnamic acid concentrate is adjusted to 5, and after passing through an activated carbon decolorization column and filtration, it enters a chromatographic separation device, the chromatographic filler is a C18 filler, the eluent is a 40% methanol aqueous solution by mass, the system temperature is 35°C, the system pressure is 0.2MPa, and material B and material C are separated, the purity of ferulic acid in material B is 90.2%, and the purity of p-coumaric acid in material C is 82.3%;

[0071] Step 5, purification: add 10% sodium hydroxide solution by mass to the material B and the material C in step 4, respectively, so that the pH of the material B and the material C are both 9, and then perform rectification and concentration respectively until the mass fraction of ferulic acid in the material B is 30%, and the mass fraction of p-coumaric acid in the material C is 34%, and add 5% hydrochloric acid by mass to the concentrated material B and the material C under stirring, adjust the pH to 2.5, and stand for 2 hours, respectively, precipitate ferulic acid crystals and p-coumaric acid crystals, and finally centrifuge and dry to obtain pure ferulic acid and p-coumaric acid, with a purity of 99.0% for ferulic acid and 99.3% for p-coumaric acid;

[0072] Step 6: Alkaline elution: Add an alkaline eluent into the fixed bed of granular activated carbon after alcohol elution in step 3 to obtain an alkaline eluent. When the transmittance of the alkaline eluent (OD 420 ) is 97%, stop eluting, use compressed air to push out the solution in the fixed bed, collect the alkaline eluent, the alkaline eluent flow rate is 0.25BV / h, and the alkaline eluent is 1% by mass sodium hydroxide solution;

[0073] Step 7, separation of protein hydrolysate: The alkaline eluate in step 6 is concentrated by evaporation to a solid content of 20%, and electrodialysis is performed to remove alkali to obtain an electrodialysis fresh water solution, the pH of the electrodialysis fresh water solution is 10.0, 5% by mass sulfuric acid is added dropwise to the electrodialysis fresh water solution until the pH is 2.5, and the protein hydrolysate is precipitated after standing for 2 hours, and the crude protein hydrolysate is obtained by centrifugation and drying. Example 2

[0074] Step 1, dilute acid hydrolysis: the washed bagasse is mixed with 1% sulfuric acid in a mass ratio of 1:9, and hydrolyzed at 125°C for 2 hours. During the hydrolysis process, exhaust gas is intermittently, and the xylose hydrolyzate is collected by solid-liquid separation. The refractive index of the hydrolyzate is 5%;

[0075] Step 2, activated carbon adsorption: The xylose hydrolyzate enters the bottom of the fixed bed filled with granular activated carbon and flows out from the top at a flow rate of 0.5 BV / h. The xylose decolorization liquid is obtained by the granular activated carbon. Hydroxycinnamic acid and protein hydrolysis products are adsorbed on the granular activated carbon. When the transmittance of the xylose decolorization liquid at 420 nm (OD 420 ) is 60%, the granular activated carbon adsorption reaches saturation, the fixed bed stops feeding, and the solution in the fixed bed is pushed out with compressed air;

[0076] Preparation of xylose: The xylose decolorized solution obtained in step 2 was subjected to electrodialysis deacidification to obtain a xylose neutralized solution with a pH of 3. The xylose neutralized solution entered the membrane concentration process and was concentrated by pressure-driven membrane to obtain a membrane concentrate with a solid content (determined by Abbe refractometer) of 20%. The membrane concentrate was subjected to an ion exchange process (001*7 resin and D301 resin) to make the solution conductivity 80μS / cm and the transmittance (OD 420 ) is 76%, and then it is concentrated by multi-stage evaporation to a solid content of 89% in the solution, and then enters the crystallizer to cool to 30°C, and centrifugally dry to obtain xylose crystals, with a cooling rate of 1.0°C / h;

[0077] Step 3: Alcohol elution: Add alcohol eluent into the saturated granular activated carbon fixed bed in step 2 to obtain an alcohol eluate. When the transmittance of the alcohol eluate (OD 420 ) is 86%, the elution is stopped, the solution in the fixed bed is pushed out with compressed air, and the alcohol eluent is collected. The flow rate of the alcohol eluent is 0.5 BV / h, and the alcohol eluent is a 50% by mass ethanol aqueous solution;

[0078] Step 4, separation of ferulic acid and p-coumaric acid: the alcohol eluate obtained in step 3 is concentrated by a distillation tower to obtain a hydroxycinnamic acid concentrate, the mass fraction of hydroxycinnamic acid in the hydroxycinnamic acid concentrate is 30%, and then the pH of the hydroxycinnamic acid concentrate is adjusted to 6, and after passing through an activated carbon decolorization column and filtration, it enters a chromatographic separation device, the chromatographic filler is a C18 filler, the eluent is a 50% ethanol aqueous solution by mass, the system temperature is 60°C, the system pressure is 0.32MPa, and material B and material C are separated, the purity of ferulic acid in material B is 93.2%, and the purity of p-coumaric acid in material C is 82.0%;

[0079] Step 5, purification: adding a 15% sodium hydroxide solution by mass to the material B and the material C in step 4, respectively, so that the pH of the material B and the material C are both 9.5, and then respectively distilling and concentrating until the mass fraction of ferulic acid in the material B is 40%, and the mass fraction of p-coumaric acid in the material C is 40%, and adding 8% hydrochloric acid by mass to the concentrated material B and the material C under stirring, respectively, adjusting the pH to 3.0 and standing for 10 hours, respectively precipitating ferulic acid crystals and p-coumaric acid crystals, and finally centrifuging and drying to obtain pure ferulic acid and p-coumaric acid, the purity of ferulic acid is 99.5%, and the purity of p-coumaric acid is 99.0%;

[0080] Step 6: Alkaline elution: Add an alkaline eluent into the fixed bed of granular activated carbon after alcohol elution in step 3 to obtain an alkaline eluent. When the transmittance of the alkaline eluent (OD 420 ) is 96%, stop eluting, use compressed air to push out the solution in the fixed bed, collect the alkaline eluent, the alkaline eluent flow rate is 0.5BV / h, and the alkaline eluent is a 3% by mass potassium hydroxide solution;

[0081] Step 7, separation of protein hydrolysate: The alkaline eluate in step 6 is concentrated by evaporation to a solid content of 25%, and electrodialysis is performed to remove alkali to obtain an electrodialysis fresh water solution, the pH of the electrodialysis fresh water solution is 10.3, 10% by mass hydrochloric acid is added dropwise to the electrodialysis fresh water solution until the pH is 3.0, and the protein hydrolysate is precipitated after standing for 10 hours, and the crude protein hydrolysate is obtained by centrifugation and drying. Example 3

[0082] Step 1, dilute acid hydrolysis: the washed corn cobs were mixed with 1.25% acetic acid in a mass ratio of 1:10, and hydrolyzed at 130°C for 1 hour. During the hydrolysis process, exhaust gas was intermittently discharged, and xylose hydrolyzate was collected by solid-liquid separation. The refractive index of the xylose hydrolyzate was 10%;

[0083] Step 2, activated carbon adsorption: The xylose hydrolyzate enters the bottom of the fixed bed filled with granular activated carbon and flows out from the top at a flow rate of 1.0 BV / h. The xylose decolorization liquid is obtained by the granular activated carbon. Hydroxycinnamic acid and protein hydrolysis products are adsorbed on the granular activated carbon. When the transmittance of the xylose decolorization liquid at 420 nm (OD 420 ) is 90%, the fixed bed stops feeding, and the solution in the fixed bed is pushed out with compressed air;

[0084] Preparation of xylose: The xylose decolorized solution obtained in step 2 was subjected to electrodialysis for deacidification to obtain a xylose neutralized solution with a pH of 3.5. The xylose neutralized solution was concentrated by membrane (pressure-driven membrane) to obtain a membrane concentrate with a solid content of 24% (determined by Abbe refractometer); the membrane concentrate was subjected to an ion exchange process (001*7 resin and D301 resin) to obtain a solution with a conductivity of 20 μS / cm and a transmittance (OD 420) is 78%, and then it is concentrated by multi-stage evaporation to a solid content of 95% in the solution, and then enters the crystallizer to cool to 20°C, and centrifugally dry to obtain xylose crystals, with a cooling rate of 2.0°C / h;

[0085] Step 3: Alcohol elution: Add alcohol eluent into the saturated granular activated carbon fixed bed in step 2 to obtain an alcohol eluate. When the transmittance of the alcohol eluate (OD 420 ) is 95%, the elution is stopped, the solution in the fixed bed is pushed out with compressed air, and the alcohol eluent is collected. The flow rate of the alcohol eluent is 1.0 BV / h, and the alcohol eluent is a methanol aqueous solution with a mass fraction of 99%;

[0086] Step 4, separation of ferulic acid and p-coumaric acid: the alcohol eluate obtained in step 3 is concentrated by a distillation tower to obtain a hydroxycinnamic acid concentrate, the mass fraction of hydroxycinnamic acid in the hydroxycinnamic acid concentrate is 45%, and then the pH of the hydroxycinnamic acid concentrate is adjusted to 7, decolorized by an activated carbon decolorization column, filtered and then enters a chromatographic separation device, the chromatographic filler is C18 filler, the eluent is a methanol aqueous solution with a mass fraction of 99%, the system temperature is 80°C, the system pressure is 0.54MPa, and material B and material C are separated, the purity of ferulic acid in material B is 91%, and the purity of p-coumaric acid in material C is 83%;

[0087] Step 5, purification: add 20% sodium hydroxide solution by mass to the material B and material C of step 4 respectively, so that the pH of material B and material C are both 10.0, and then perform rectification and concentration respectively until the mass fraction of ferulic acid in material B is 45%, and the mass fraction of p-coumaric acid in material C is 45%, and add 10% hydrochloric acid by mass to the concentrated material B and material C respectively under stirring, adjust the pH to 4.0 and stand for 24 hours respectively, precipitate ferulic acid crystals and p-coumaric acid crystals respectively, and finally centrifuge and dry to obtain pure ferulic acid and p-coumaric acid, with the purity of ferulic acid being 99.8% and the purity of p-coumaric acid being 99.7%;

[0088] Step 6: Alkaline elution: Add an alkaline eluent into the fixed bed of granular activated carbon after alcohol elution in step 3 to obtain an alkaline eluent. When the transmittance of the alkaline eluent (OD 420 ) is 99%, the elution is stopped, the solution in the fixed bed is pushed out with compressed air, and the alkaline eluent is collected. The alkaline eluent flow rate is 1.0 BV / h, the alkaline eluent is an alkaline solution with a mass fraction of 4%, and the mass ratio of sodium hydroxide to potassium hydroxide in the alkaline solution is 1:1;

[0089] Step 7, separation of protein hydrolysate: The alkaline eluate in step 6 is concentrated by evaporation to a solid content of 30%, and electrodialysis is performed to remove alkali to obtain an electrodialysis fresh water solution, the pH of the electrodialysis fresh water solution is 10.5, 20% by mass hydrochloric acid is added dropwise to the electrodialysis fresh water solution until the pH is 4.0, and the protein hydrolysate is precipitated after standing for 24 hours, and the crude protein hydrolysate is obtained by centrifugation and drying. Example 4

[0090] Step 1, dilute acid hydrolysis: the washed wheat straw is mixed with 1.5% hydrochloric acid in a mass ratio of 1:10, and hydrolyzed at 130°C for 1 hour. During the hydrolysis process, exhaust gas is intermittently, and xylose hydrolyzate is collected by solid-liquid separation. The refractive index of the xylose hydrolyzate is 10%;

[0091] Step 2, activated carbon adsorption: The granular activated carbon in the fixed bed is the granular activated carbon after alkaline elution in step 6 of Example 3. The xylose hydrolyzate enters the fixed bed filled with granular activated carbon from the bottom and flows out from the top. The flow rate is 1.0 BV / h. The xylose decolorization liquid is obtained through the granular activated carbon. Hydroxycinnamic acid and protein hydrolysis products are adsorbed on the granular activated carbon. When the transmittance of the xylose decolorization liquid at 420 nm (OD 420 ) is 90%, the granular activated carbon adsorption reaches saturation, the fixed bed stops feeding, and the solution in the fixed bed is pushed out with compressed air;

[0092] Preparation of xylose: The pH of the xylose decolorizing solution in step 2 was determined. When the pH of the xylose decolorizing solution was ≥6, it was refluxed into the xylose hydrolyzate raw material tank. When the pH was <6, the xylose decolorizing solution was deacidified by electrodialysis to obtain a xylose neutralized solution with a pH of 3.5. The xylose neutralized solution was concentrated by membrane to obtain a membrane concentrate with a solid content of 20% (determined by Abbe refractometer). The membrane concentrate was subjected to an ion exchange process (001*7 resin and D301 resin) to make the solution conductivity 50μS / cm and the transmittance (OD 420 ) is 78%, and then it is concentrated by multi-stage evaporation to a solid content of 95% in the solution, and then enters the crystallizer to cool to 20°C, and centrifugally dry to obtain xylose crystals, with a cooling rate of 2.0°C / h;

[0093] Step 3: Alcohol elution: Add alcohol eluent into the saturated granular activated carbon fixed bed in step 2 to obtain an alcohol eluate. When the transmittance of the alcohol eluate (OD 420 ) is 95%, the elution is stopped, the solution in the fixed bed is pushed out with compressed air, and the alcohol eluent is collected. The flow rate of the alcohol eluent is 1.0 BV / h, and the alcohol eluent is a propanol aqueous solution with a mass fraction of 99%;

[0094] Step 4, separation of ferulic acid and p-coumaric acid: the alcohol eluate obtained in step 3 is concentrated by a distillation tower to obtain a hydroxycinnamic acid concentrate, the mass fraction of hydroxycinnamic acid in the hydroxycinnamic acid concentrate is 45%, and then the pH of the hydroxycinnamic acid concentrate is adjusted to 7, decolorized by an activated carbon decolorization column, filtered and then enters a chromatographic separation device, the chromatographic filler is C18 filler, the eluent is a methanol aqueous solution with a mass fraction of 99%, the system temperature is 80°C, the system pressure is 0.54MPa, and material B and material C are separated, the purity of ferulic acid in material B is 93.1%, and the purity of p-coumaric acid in material C is 85.2%;

[0095] Step 5, purification: add 20% sodium hydroxide solution by mass to the material B and the material C in step 4, respectively, so that the pH of the material B and the material C are both 10.0, and then perform rectification and concentration respectively until the mass fraction of ferulic acid in the material B is 43%, and the mass fraction of p-coumaric acid in the material C is 46%, and add 10% hydrochloric acid by mass to the concentrated material B and the material C under stirring, adjust the pH to 4.0, and stand for 24 hours, respectively, precipitate ferulic acid crystals and p-coumaric acid crystals, and finally centrifuge and dry to obtain pure ferulic acid and p-coumaric acid, with a purity of 99.7% for ferulic acid and 99.7% for p-coumaric acid;

[0096] Step 6: Alkaline elution: Add an alkaline eluent into the fixed bed of granular activated carbon after alcohol elution in step 3 to obtain an alkaline eluent. When the transmittance of the alkaline eluent (OD 420 ) is 99%, the elution is stopped, the solution in the fixed bed is pushed out with compressed air, and the alkaline eluent is collected. The alkaline eluent flow rate is 1.0 BV / h, the alkaline eluent is an alkaline solution with a mass fraction of 4%, and the mass ratio of sodium hydroxide to potassium hydroxide in the alkaline solution is 1:1;

[0097] Step 7, separation of protein hydrolysate: The alkaline eluate in step 6 is concentrated by evaporation to a solid content of 30%, and electrodialysis is performed to remove alkali to obtain an electrodialysis fresh water solution, the pH of the electrodialysis fresh water solution is 10.5, 20% by mass hydrochloric acid is added dropwise to the electrodialysis fresh water solution until the pH is 4.0, and the protein hydrolysate is precipitated after standing for 24 hours, and the crude protein hydrolysate is obtained by centrifugation and drying.

[0098] It should be understood that the purpose of these embodiments is only to illustrate the present invention and is not intended to limit the protection scope of the present invention. In addition, it should also be understood that after reading the technical content of the present invention, those skilled in the art can make various changes, modifications and / or variations to the present invention, and all of these equivalent forms also fall within the protection scope defined by the claims attached to this application.

Claims

1. A method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate, characterized in that: The following steps are involved: Step 1, dilute acid hydrolysis: the grass plant is subjected to dilute acid hydrolysis and solid-liquid separation to obtain a xylose hydrolyzate, wherein the refractive index of the xylose hydrolyzate is 1-10%; Step 2, activated carbon adsorption: the xylose hydrolyzate obtained in step 1 flows through a granular activated carbon adsorption column to obtain a xylose decolorized solution until the granular activated carbon reaches an adsorption saturation state; Step 3, alcohol elution: Add an alcohol eluent into the granular activated carbon adsorption column that has reached adsorption saturation in step 2 to elute to obtain an alcohol eluate, until the transmittance of the alcohol eluate at 420 nm is 75-95%, and collect the alcohol eluate; Step 4, separation of ferulic acid and p-coumaric acid: the alcohol eluate collected in step 3 is concentrated by distillation to obtain a hydroxycinnamic acid concentrate, and the pH of the hydroxycinnamic acid concentrate is adjusted to 5-7, and then subjected to decolorization, filtration, and chromatographic separation steps to obtain a B material rich in ferulic acid and a C material rich in p-coumaric acid, wherein the eluent used for the chromatographic separation is an alcohol aqueous solution with a mass fraction of 40-99%; Step 5, purification: adjusting the pH of material B and material C in step 4 to 9-10 respectively, and then performing rectification and concentration, acid precipitation, centrifugation, and drying respectively to obtain ferulic acid with a purity of ≥99.0% and p-coumaric acid with a purity of ≥99.0% respectively; Step 6, alkaline elution: introducing an alkaline eluent into the granular activated carbon adsorption column after the alcohol elution in step 3 to obtain an alkaline eluent until the transmittance of the alkaline eluent at 420 nm is greater than 95%, and collecting the alkaline eluent; Step 7, separation of protein hydrolysate: The alkaline eluate collected in step 6 is concentrated by evaporation and de-alkalized by electrodialysis to obtain an electrodialysis fresh water solution, and an inorganic acid is added dropwise to the electrodialysis fresh water solution until the pH value is 2.5-4.

0. After standing for 2-24 hours, the solution is centrifuged and dried to obtain a crude protein hydrolysate.

2. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 1, characterized in that: In step 1, the mass ratio of the grass plant to the dilute acid is 1:8-1:10, and the hydrolysis condition is 100°C-130°C for 1h-4h; In step 1, the grass plant is any one of corn cobs, bagasse, bamboo, and wheat straw, or a combination of several of them, and the dilute acid is any one of hydrochloric acid, sulfuric acid, and acetic acid with a mass fraction of 1-1.5%.

3. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 1, characterized in that: The xylose decolorized liquid in step 2 is deacidified by electrodialysis to obtain a xylose neutralized liquid with a pH of 2.5-3.5, and the xylose neutralized liquid is subjected to membrane concentration, ion exchange, multi-stage concentration, crystallization, centrifugation, and drying steps to obtain xylose crystals; After the ion exchange, the solution conductivity is ≤100 μS / cm, and the transmittance at 420 nm is ≥60%; The ion exchange includes cation exchange resin and anion exchange resin.

4. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 1, characterized in that: When the transmittance of the xylose decolorizing solution at 420 nm in step 2 is 40-90%, the granular activated carbon adsorption reaches saturation; The granular activated carbon after the alkaline elution in step six is ​​recycled according to steps two, three and six.

5. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 4, characterized in that: When the pH of the xylose decolorizing solution obtained by repeating step 2 after the alkaline elution of the granular activated carbon in step 6 is ≥ 6.0, the xylose decolorizing solution is refluxed to the xylose hydrolyzate raw material tank; When the pH value is less than 6.0, the xylose decolorized liquid is subjected to the steps of electrodialysis deacidification, membrane concentration, ion exchange, multi-stage concentration, crystallization, centrifugation and drying to obtain xylose crystals.

6. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 1, characterized in that: The alcohol eluent in step 3 is an alcohol aqueous solution with a mass fraction of 40-99%, and the alcohol aqueous solution is selected from any one or a combination of methanol aqueous solution, ethanol aqueous solution, propanol aqueous solution, and isopropanol aqueous solution.

7. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 1, characterized in that: In step 5, 10-20% by mass of sodium hydroxide solution is added to material B and material C respectively to adjust the pH to 9-10.

8. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 1, characterized in that: In step 5, material B is distilled and concentrated to a mass fraction of ferulic acid of 30-45%, and material C is distilled and concentrated to a mass fraction of p-coumaric acid of 30-46%; Acid precipitation in step 5: Add 5-10% hydrochloric acid by mass to the B material and C material after distillation and concentration, respectively, until the pH is 2.5-4.0 and stand for 2-24 hours to precipitate ferulic acid crystals and p-coumaric acid crystals, respectively.

9. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 1, characterized in that: The alkaline eluent in step six is ​​an alkaline solution with a mass fraction of 1-4%, and the alkaline solution is selected from any one of sodium hydroxide solution, potassium hydroxide solution, and calcium hydroxide solution or a combination of several thereof.

10. The method for extracting hydroxycinnamic acid and protein hydrolysate from xylose hydrolysate according to claim 1, characterized in that: The pH of the electrodialysis fresh water in step 7 is 10.0-10.5; The mass fraction of the inorganic acid in step seven is 5-20%, and the inorganic acid is selected from any one of hydrochloric acid, sulfuric acid, and acetic acid or a combination of several thereof.

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