JPH01312997A - Combination method for producing selected monosaccharide from corn grain coat by hydrolysis - Google Patents

Abstract

PURPOSE: To readily produce the subject monosaccharide in a large amount without requiring pretreatment of delignification by allowing a cellulolytic enzyme to act on one part or more of an acid hydrolyzate obtained by hydrolyzing corn kernel hulls by a strong acid.

CONSTITUTION: An acid hydrolyzate consisting of a liquid part comprising monosaccharides containing D-glucose, D-xylose and L-arabinose, and a solid part is obtained by hydrolyzing corn kernel hulls by a strong acid such as H₂SO₄ of 0.5-15 wt.% acid concentration at 80-110°C. The solid part is separated, and reacted by adding a cellulase thereto in pH3-6 at 25-55°C to provide the hydrolyzate consisting essentially of D-glucose. If necessary, the corn kernel hulls is hydrolyzed by a cellulolytic enzyme after mixing and treating the corn kernel hulls with a strong base solution such as NaOH of 0.5-5 wt.%. Further, one or more parts of the obtained hydrolyzate are hydrolyzed by a strong acid, and the monosaccharides are recovered from the hydrolyzate.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Monosaccharides in general have a wide range of commercial uses, and glucose in particular has found a variety of uses.

In addition to being used as the main substrate for the fermentation medium for ethanol production, glucose is also isomerized to flag-1--s, which are widely used as sweeteners. It is especially used in confectionery products.

The main source of glucose is cellulose, a polysaccharide of cellobiose, which is a disaccharide in which β-D-glucose is linked (1→4).In the United States, corn cobs are produced in large quantities as waste. Corn cobs are a major source of cellulose because of its availability. However, the use of cob is not always advantageous because it contains lignin. Cobs are a type of lignocellulose, in which cellulose is enclosed in a matrix of amorphous lignin and hemicellulose. Pretreatment of lignocellulose is necessary to break down the lignin matrix and facilitate the hydrolysis of cellulose.
The process as a whole is also geared towards removing lignin from lignocellulose. The disadvantages of delignification pretreatment are exacerbated by the relatively low value of lignin and the need to dispose of the chemical waste products resulting from the delignification process.

In the context of glucose production, it would be extremely beneficial to develop more effective and cheaper delignification methods or to find essentially lignin-free sources of cellulose. It is desirable that such a cellulose source be available in large quantities, and if the by-products associated with matacellulose production have some commercial value, the cellulose source can be used effectively with few waste disposal problems. . We found the source of cellulose in corn husk, a waste product from the corn grinding process. The husk of a corn kernel contains little or no lignin.

Therefore, the husk of corn grains can be hydrolyzed without delignification pretreatment, thereby obtaining a mixture containing primarily D-glucose, D-xylose and L-arabinose. D-xylose and L
- Arabinose is a monosaccharide that is a pentose, each of which has uses as a component of certain microbial culture or fermentation media, and O-xylose is also used for staining and rolling. , all monosaccharides obtained from hydrolysis have commercial uses and are equipped with economically advantageous conditions.

The theme of hydrolyzing the husk of corn kernels into a mixture of monosaccharides by recognizing the benefits derived from a rich source of cellulose that does not require delignification pretreatment to manually manipulate cellulose with hydrolyzing agents. Several variations were found. One is acid hydrolysis at elevated temperatures followed by enzymatic hydrolysis to obtain maximum yields of glucose and total monosaccharides. A second variant is to perform acid hydrolysis at low temperatures to obtain a solution containing primarily monosaccharides from hemicellulose, followed by enzymatic hydrolysis to break down the cellulose to glucose.

A third variant is to hydrolyze the cellulose with enzymes after pretreatment with a moderate base to obtain a solution in which the monosaccharides are virtually exclusively glucose, followed by hydrolysis of the hemicellulose component by acid treatment. . Each variant has the advantage of making the theme particularly harmonious, recommending its use according to market and processor requirements. Each variant also
It has unique characteristics that were discovered during the development of a method to use the husks of corn kernels as a raw material.

[Summary of the invention] The purpose of the present invention is to develop a method for producing D-glucose, D-xylose, and L-arabinose from substances that do not require any pretreatment for delignification and can be easily obtained in a multi-electrode manner. It's about doing. One embodiment involves hydrolyzing the outer husks of corn grains with acid at elevated temperatures, and then hydrolyzing the hydrolyzate with enzymes. In a more particular embodiment, acid hydrolysis is conducted at a temperature of about 85 to about 110<0>C. In other embodiments, the husks of corn kernels are
Hydrolysis with acid at temperatures below 5° C. yields a mixture of pentoses that can be recovered separately, followed by hydrolysis with enzymes to yield glucose from the unhydrolyzed cellulose.

In yet another embodiment, corn grain husks are pretreated with a moderate base and then subjected to enzymatic hydrolysis to yield glucose substantially exclusively as the monosaccharide, followed by hemicellulose and unreacted cellulose components. It performs acid hydrolysis of

[The detailed description of the invention is based on several discoveries. One of the fundamental discoveries from the corn kernel wet milling industry is that the outer husk of corn kernels contains little lignin. Typical analysis shows that the husk of a corn kernel is about 20% starch, about 30% cellulose, and
It contains about 30% hemicellulose, about 10% protein, and has a lignin content of less than 5%. Thus, corn grain hulls behave differently than typical lignocelluloses in that they do not require delignification to hydrolyze cellulose and hemicellulose components. The second discovery is that in acid hydrolysis of corn kernel husks, the yield of glucose is temperature dependent, but the yield of pentoses of D-xylose and di-arabinose is.

It doesn't change much. This allows a hitherto unrecognized control of the amount of hydrolyzate. It has also been discovered that pretreatment with a relatively mild base disrupts the crystallinity of the cellulose in the corn kernel husk to an extent that makes it susceptible to enzymatic hydrolysis. The combination of these discoveries provides several hydrolysis methods described herein.

One of the notable features of the invention is its flexibility, in that each method of the invention has a degree of freedom of choice that allows its practitioner to take into account market conditions; A notable feature is that, depending on the selection, the monosaccharide hydrolysis products can be separated into hexose and pentose streams without the need for any special separation steps other than simple filtration.

A first method of the invention is illustrated in FIG. In this process, corn kernel hulls are continuously hydrolyzed with strong acids at temperatures ranging from about 80 to about 110°C, the acid hydrolyzate is then hydrolyzed with cellulose-degrading enzymes, and the enzymatic hydrolyzate is recovered.

Strong acids that can be used in the first step of acid hydrolysis include:

Sulfuric acid, hydrochloric acid, phosphoric acid, hydrofluoric acid, trifluoroacetic acid, trichloroacetic acid and the like are included. The particular characteristics of the strong acid used are not important unless the acid is an oxidizing acid under the reaction conditions. Typical acid concentrations are about 0.5 to about 15% by weight
, more typically in the range of 3 to IO weight percent.

Hydrolysis with strong acids is carried out at temperatures ranging from about 80°C to about 110°C. The maximum temperature is regulated because if it exceeds about 110°C, the hydrolyzate will noticeably deteriorate.

The minimum temperature is regulated because glucose yield is temperature dependent and too low a temperature requires an unusually long reaction time to achieve maximum gelcol production. Approximately 95
A temperature range of ˜105° C. is optimal.

At least a portion of the reaction product from acid hydrolysis is then treated with a cellulolytic enzyme.

Unreacted cellulose is completely hydrolyzed.

The enzyme sessurase is heated at a temperature of about 25 to about 55°C, preferably about 35°C.
It is usually used between ~50°C. Generally, the mixture is buffered to a ρ0 of about 3 to about 6, more typically 4.0 to about 5.
Maintained at 0. Once the enzymatic hydrolysis is complete, the resulting hydrolyzate is collected and processed according to the requirements of the H'ta. That is, glucose can be separated from pentoses and the monosaccharides separated from other components or perhaps from the total hydrolyzate can be utilized without further processing.

As shown in Figure 1, the reaction products from acid hydrolysis can be separated into a liquid part and a solid part. Separation can be accomplished by conventional means such as membrane separation or simple filtration. Filtration separation is preferred and if this is carried out it is important that the filter cake is not allowed to dry until its next use.

The monosaccharides in the liquid portion are primarily D-glucose, D-xylose and L-arabinose, and this liquid portion can be separately processed to isolate and purify it into one or more monosaccharide components. can. On the other hand, the solid portion is subjected to enzymatic hydrolysis. A hydrolyzate is recovered from this, which mainly contains D-glucose.

The solid separation step described above is preferred for maximizing glucose yield, i.e. after hydrolysis with one strong acid, the total glucose yield is higher when liquid and solid are separated than when they are not separated. Also, this separation is effectively D
- Enzyme hydrolyzate containing only glucose is produced as a single soluble monosaccharide, facilitating purification.

The second method of the invention, the flow of which is shown in FIG. 2, is similar to the sequential acid and enzymatic hydrolysis method, except that the acid hydrolysis is performed at a temperature below about 80°C. Acid hydrolysis of corn kernel husks at relatively low temperatures produces a pentose-rich mixture, thus providing selectivity not available at higher temperatures. As mentioned above, the second
In the method, the acid hydrolysis is carried out at a temperature lower than about 80°C, preferably from about 35 to about 70°C, more preferably from about 40 to about 6°C.
It is carried out in the range of 0°C. The acids and their concentrations that can be used are the same as described above.

In addition, treating the acid hydrolyzate with a cellulolytic enzyme is also the same as before.

As shown in Figure 2, the acid hydrolyzate can be separated into a liquid part and a solid part. As already mentioned, this separation can be carried out by conventional means such as filtration or membrane separation, but simple filtration is preferred. The liquid portion is mainly composed of monosaccharides, D-xylose and L-arabinose, and contains some soluble polysaccharides. The solid portion is resuspended and subjected to hydrolysis by cellulolytic enzymes. The obtained hydrolyzate mainly contains D-glucose as a single monosaccharide.

This solids separation provides two product streams. The liquid part obtained from the separation of the acid hydrolyzate mainly contains pentoses and some soluble polysaccharides, whereas the enzymatic hydrolyzate contains mono-MII
R mainly contains glucose. This therefore has the advantage, on the one hand, of allowing a facile separation between pentoses and, on the other hand, of yielding D-glucose. However, it must be understood that if a solid separation step is carried out, the total glucose yield will be reduced, possibly due to some polysaccharides being dissolved in the liquid part of the separated hydrolyzate stream.

In another method, shown in Figure 3, corn kernel husks are mixed with a dilute FJ solution of a strong base at a temperature of about 10 to about 40°C, then first hydrolyzed with cellulolytic enzymes and then hydrated with a strong acid. It is decomposed and the hydrolyzate is recovered. The purpose of pretreatment with a base is to reduce the crystallinity of cellulose and make it easier to hydrolyze with enzymes.

This base pretreatment is exceptionally mild compared to similar treatments for lignocellulosic materials. The individual properties of the bases are not important.

Alkali metal hydroxides and carbonates are preferably used. Sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, and mixtures thereof are present in an amount of about 0.5 to about 5%1 usually about 0.5 to about 5%.
An example of a suitable base is used at a concentration of about 3%. Pretreatment with the base is carried out at a temperature of about 10 DEG to about 40 DEG C., usually between about 15 DEG and about 30 DEG C., for a period of time sufficient to achieve the stated purpose. The pretreatment time with the base varies depending on the contact efficiency of the corn kernel hull with the base, temperature, base concentration, etc., but is typically in the range of about 15 to about 180 minutes.

The base-pretreated material is subjected to hydrolysis using cellulolytic enzymes under the conditions described above without being filtered. The enzymatic hydrolyzate is then hydrolyzed with strong acids as described above to obtain the final product stream.

Like the first and second methods, this third method also includes the step of separating the enzyme hydrolyzate into a liquid portion and a fixed portion. The liquid portion obtained by such processes as membrane separation or simple filtration contains essentially D-glucose as the single monosaccharide and some soluble polysaccharides. Therefore, using this separation step it is possible to obtain a relatively pure monoglucose stream.

The solid portion is hydrolyzed with strong acid to give an acid hydrolyzate containing primarily D-glucose, D-xylose and L-arabinose. However, because some polysaccharides are dissolved in the liquid part of the separation process, the total yield of monosaccharides is less than if the enzymatic hydrolyzate was not subjected to separation, and this is not recommended if maximizing yield is important. Separation is not a good idea.

The following examples merely illustrate the invention and do not limit it.

[Examples] Example 1 15 g of corn kernel hulls obtained as waste from a major corn grinding company were mixed with 200 ml of 7% sulfuric acid at varying temperatures for 5.5 to 6.0 hours.Then, the reaction mixture was cooled. The filter cake was washed with approximately 100 ml of water. The filter cake was approximately 25% by weight of the starting material. This filter cake was analyzed by HPLC.
Glucose, xylose and arabinose were analyzed and the results shown in Table 1 were obtained.

(Left below) Table 1: Effect of temperature on acid hydrolysis Time, hr 5.5 6 6
5.5 Temperature, 'C100857060 Hydrolyzate production Hydrolyzate weight % Clucose 29.8 24.9 12.3 3
．． 3 xylose 14,8 13,6 14.4
8.3 Arabinose 10.3 9.0 10.3
11.6 The above results show that the extent of glucose production is temperature dependent. Between 70 and 100<0>C, the amount of pheasant and arabinose appears to be unaffected by temperature. At temperatures of 60° C. and below, the yield of xylose decreases.

Example 2 Corn kernel hulls were hydrolyzed with 7% sulfuric acid at 100° C. for 5.5 hours. In experiment A, the acid hydrolyzate was filtered, the filter cake was washed with washes added to the filtrate, dried, redispersed in acetate buffer (0.1 molar, pH 4.5), and 0.4
% cellulase at 45° C. for 24 hours. In experiment B, the acid hydrolyzate was filtered, the filter cake was washed with washes added to the filtrate, and the filter cake was then redispersed in acetate buffer without drying. This dispersion was enzymatically hydrolyzed as in Experiment A. In Experiment C, instead of filtering the acid hydrolyzate, the pH was adjusted to 4.5, then acetate buffer was added as described above, and the mixture was treated with enzymes. The experimental results are shown in Table 2.

(Margins below) Hayashi #A It is shown that.

Example 3 14.9 g of dried corn kernel husks and 7% sulfuric acid 2
The mixture with 00+ml was heated at 60° C. for 5.5 hours.

Filter the cooled mixture and leave a filter cake of approx.
Washed with resistant water. The wash liquor and filtrate were mixed and analyzed and found to contain 2.5 g of glucose based on the weight of the initial husk.
5%, xylose 8.4%, arabinol 1000
% was included.

The filter cake was dispersed in approximately 250+1 of distilled water to adjust its Pl+ to 4.8. After adding P.8 acetate buffer (1M, 30+ml) and 1.2g of cellulase, the volume was adjusted to 300ml. The mixture was kept at 45° C. for 24 hours while being shaken, and then filtered to obtain a filter cake corresponding to 9.8% of the weight of the rind. Analysis of the filtrate revealed that it contained 17.4% glucose based on initial husk weight and 1% other monosaccharides.
Example 4 A sample of corn kernel husks was mixed with a 1.5% sodium hydroxide solution (75 ml of base per 5 g of husks) at ambient temperature for about 90 minutes. This mixture was adjusted to pH 4.5,
At this pH, acetate buffer was added to a concentration of 0.1 molar and the mixture was heated at 45°C for 24 hours at 0.4% (V/
V) was treated with cellulase. In Experiment A, the enzyme hydrolyzate was filtered and the filter cake obtained by washing with the washing liquid added to the filtrate was acid-hydrolyzed at 100° C. in the same manner as in Example 3.

In Experiment B, the enzymatic hydrolyzate was not filtered and all of it was subjected to acid hydrolysis as described above. The experimental results are shown in Table 3, where the monosaccharides are given in weight percent based on the hulls used.

(Left below) The results in Table 3 show that when corn grain husks treated with a mild base are hydrolyzed with enzymes, relatively pure curcose can be obtained. It also shows that to maximize monosaccharide production, enzyme-treated material should not be filtered before acid hydrolysis. Furthermore, other data show that if the filtrate is only treated with enzymes, the yield of glucose is reduced from 342 to 15% without filtration, so that the material initially treated with base is less susceptible to hydrolysis by It also indicates that it should not be filtered beforehand.

[Brief explanation of the drawing]

FIG. 1 shows a flowchart in which corn kernel husks are hydrolyzed with a strong acid at a temperature of 80° C. or higher, followed by an optional separation step and then hydrolyzed with an enzyme. FIG. 2 shows a flowchart in which corn kernel husks are hydrolyzed with a strong acid at a temperature of 80° C. or lower, followed by an optional separation step and then hydrolyzed with an enzyme. FIG. 3 shows a flowchart in which corn kernel husks are pretreated with a mild base and then first hydrolyzed with an enzyme, followed by an optional separation step and then hydrolyzed with an acid.

Claims (1)

[Claims] 1. The husk of corn grains is hydrolyzed with a strong acid under conditions that produce an acid hydrolyzate, and at least a portion of the acid hydrolyzate is further hydrolyzed with a cellulolytic enzyme. 1. A method for hydrolyzing corn grain husks, comprising contacting with a corn grain husk and then recovering an enzymatic hydrolyzate. 2. The conditions used in the above hydrolysis step are 80-1
2. The method of claim 1, comprising a temperature of 10<0>C. 3. Separate the acid hydrolyzate into a liquid part whose monosaccharides are mainly D-glucose, D-xylose, and L-arabinose, and a solid part, and hydrolyze the solid part with an enzyme to mainly contain D-glucose. 3. The method of claim 2, further comprising producing an enzymatic hydrolyzate. 4. The method of claim 3, wherein the acid hydrolysis step is carried out at a temperature of 35 to 80C. 5. The acid hydrolyzate contains monosaccharides mainly D-xylose and L-
Separated into a liquid part, which is arabinose, and a solid part,
5. The method of claim 4, further comprising enzymatically hydrolyzing the solid portion to produce an enzymatic hydrolyzate containing primarily D-glucose. 6. The enzyme is cellulase, and the enzyme reaction is about 25 to about 5
2. The method of claim 1, carried out at a temperature of 5<0>C and a pH of about 3 to about 6. 7. Mix the husks of corn kernels with a dilute solution of a strong base for about 1 hour.
A hydrolyzate obtained by mixing at a temperature of 0 to about 40°C, hydrolyzing the base-treated hull with a cellulolytic enzyme, and then acid-hydrolyzing at least a part of the enzymatic hydrolyzate with a strong acid. A method for hydrolyzing corn grain husks comprising recovering. 8. Separate the enzymatic hydrolyzate into a liquid part whose monosaccharide is mainly D-glucose and a large part, and acid hydrolyze the solid part to mainly produce D-glucose, D-xylose and L-arabinose. 8. The method of claim 7, further comprising producing an acid hydrolyzate containing: 9. The method of claim 7, wherein the enzyme is a cellulase and the enzymatic hydrolysis is carried out at a temperature of about 25 to about 55°C and a pH of about 3 to about 6.