FI103583B - Enzymatically prepared hydrolyzate of a water-soluble carboxymethyl cellulose - Google Patents

Description

103583

HYDROLYZATE, ENZYMATICALLY PREPARED FROM WATER SOLUBLE CARBOXYMETHYL CELLULOSE

ENZYMATISKT FRAMSTÄLLT HYDROLYSAT AV EN VATTENLÖSLIG CARBOXI-METHYL CELLULOSA

The present invention relates to a novel enzymatically prepared water-soluble carboxymethylcellulose (CMC) hydrolyzate and processes for its preparation.

Cellulose derivatives such as carboxymethylcellulose, methylcellulose, methylethylcellulose, hydroxypropylmethylcellulose and hydroxypropylcellulose are calorie-free (non-metabolised in human or human intestinal tract), odorless, tasteless, water-soluble or water-soluble suspended. These cellulose derivatives may act as thickeners, binders, stabilizers, suspending agents or flow regulators. They form thin films that are resistant to oils, greases and organic solvents. They dissolve rapidly in cold and hot water and are physiologically inert. These functions make them useful in a wide range of applications in the food, pharmaceutical, cosmetic, paper, and other industries.

· · ·.: · In such use, the cleavage of cellulose derivatives: is generally considered undesirable and should be avoided.

: Cellulolytic and viscosity reducing enzymes have been deliberately avoided in the past.

The enzymatic hydrolysis of cellulose derivatives has been previously studied by indexing synergism studies between enzyme combinations, a possible model of substituent distribution; , the effect of various substituents on enzymatic hydrolysis and the like. Such studies have been published; in Chouchon et al., Biotech. Bioeng., 26, pp. 988-991 (1984); Henrissat et al. al., Biotechnology, 103583 2

Butter. 3, pp. 722-726 (1985); Chetkarov et. et al., Monatshefte Fur Chemie. Butter. 116, pages 1433-45 (1985); Chetkarov et.

et al., Monatshef te Fur Chemie. Butter. 117, pp. 1021-1026 (1986); Wirick, J. Polvm. Sci., Part A-1, Vol. 6, pp. 1195-1974 (1968); Bhattachar j ee, J. Polvm. Sci. , Part C, Vol. 36, pp. 509-521 (1971). Reducing edges of chain lengths have also been studied. Almin et al. al., Arch. Biochem. 124, 129 (1968); Ghose, Biotech. Bioenq., Vol. 11, page 239 (1969).

The present invention relates to an enzymatically prepared hydrolyzate of water-soluble carboxymethylcellulose and to a process for the preparation of a hydrolyzate. We have found that the properties of water-soluble carboxymethylcellulose can be enhanced by enzymatic hydrolysis to lower molecular weight polymers or oligomer mixtures. These enzymatic hydrolyzates and their fractions (from total hydrolyzate mixtures of oligomers to further separated oligomer mixtures of different lengths of chains) are more useful in many applications in the food, pharmaceutical, paper, cosmetic and non-textile hydrolysis industry than in with a higher molecular weight.

The present invention relates to a novel water-soluble carboxymethylcellulose hydrolyzate prepared from water-soluble enzymes, as well as fractions thereof.

»· ·« «« • ♦

The hydrolyzate is prepared from water-soluble carboxymethyl # ... φ cellulose. The hydrolyzate can be prepared with a variety of modified cellulolytic enzymes and non-modified cellulolytic enzymes. we, with the most popular sources of enzymes being strains of Trichoderma • '· reesei and Aspergillus.

Enzymatically prepared hydrolyzate and its fractions have many uses in the food, paper, pharmaceutical and cosmetic industries, but they are particularly useful as low calorie or low calorie substitutes for a large number of foods.

According to the invention, an enzymatically prepared hydrolyzate of water-soluble carboxymethylcellulose comprising a cellulose derivative hydrolyzed with an enzyme preparation is obtained to give an oligomeric mixture having an average degree of polymerization in the range of 3 to 300 and a molecular weight of 600 to 100,000, e.g. is less than 20 mPa.s. The cellulose derivative of the invention is selected from water soluble carboxymethyl cellulose. The enzyme preparation is usually selected from the group of cellulases, modified cellulases and mixtures thereof.

The enzyme preparation is a cellulase or a modified cellulase (it is primarily modified to eliminate or prevent the formation of saccharide-producing enzymes in a cellulase preparation, e.g. by genetically modifying the microorganism from which the cellulase preparation is made), suitably produced by a microorganism selected from Trichid. and Penicillium. Most preferably, the cellulase preparation is derived from Trichoderma reesei from which at least one beta-glucosidase and cellobiohydrolase activity has been removed. Enzyme-ready; · You preferably contain endo-1,4-beta-glucanase.

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The invention also relates to a process for preparing an oligomer mixture from water-soluble carboxymethyl cellulose, comprising the steps of: · selecting a water-soluble carboxymethyl cellulose ...; selection of a cellulolytic agent comprising an enzyme which hydrolyses selected carboxymethylcellulose to a mixture of oligomers having an average degree of polymerization in the range of 3 to 100 and a molecular weight in the range of 500 to 100,000; and reacting the selected cellulolytic agent with the selected carboxymethylcellulose at a temperature and for a time sufficient to produce the oligomer mixture. The cellulose derivative is suitably selected from the group consisting of water-soluble carboxymethyl cellulose 103583 4

The step of selecting the cellulolytic agent typically includes a microorganism which produces the selection of the cellulolytic agent and the production of the cellulolytic agent from the microorganism culture. The cellulolytic agent produced by the microorganism can be purified to remove enzymes that could react with the cellulose derivative to produce saccharides. The microorganism is suitably selected from the group: Trichoderma. Aspergillus and Penicillium.

Alternatively, to prevent hydrolysis of water-soluble carboxymethylcellulose to saccharides, the selected microorganism may be treated by modifying the microorganism's genes so that production of the saccharide-forming enzymes is inhibited by the genes.

In addition, the invention contemplates the removal of all or part, typically 50 wt.%, Of selected fat in foods, and replacement of the removed fat with an oligomer blend produced according to the invention; and / or removing about 40% of the selected carbohydrate in the foodstuff and replacing the removed carbohydrate with the oligomer mixture produced according to the invention.

. The invention also contemplates the separation of • · ::: oligomer blends produced according to the invention into various average molecular weight oligomer fractions by removal of about 50 wt.% Of selected food fat and the fat removed by the stake in one or more fractions; and / or by separating the oligomeric · · · mixture produced according to the invention into oligomeric fractions of different molecular weights, removing *. about 40% by weight of selected carbohydrate in the food; and replacing the removed carbohydrate with one or more fractions.

Brief description of the pictures.

5, 103583

Figure 1 shows a molecular weight distribution pattern of carboxymethylcellulose and its hydrolyzate as described in Example 2 (i) herein;

Figure 2 shows a molecular weight distribution pattern of carboxymethylcellulose and its hydrolyzate as described in Example 3 herein;

Figure 3 shows the molecular weight distribution pattern of selected fractions of carboxymethylcellulose hydrolyzate as described in Example 3 herein.

This invention relates to a water-soluble carboxymethylcellulose hydrolyzate and fractions thereof. Hydrolyzates are characterized by having an average degree of polymerisation (PD) in the range of 3 to 300 and a molecular weight in the range of 500 to 100,000.

Preparation of the Enzyme The enzymes used in this invention are various food-grade cellulase preparations. They can be prepared from many different microorganisms such as Trichoderma. Aspergillus and Penicillium etc. The selected microorganism strain is cultivated by conventional means on a medium containing nutrients such that cellulase is formed; the microorganism is separated from the culture medium, the culture media are harvested and concentrated and dried in the usual manner. These · '· *: enzymes may be used as such or in mixtures and may be modified in a variety of ways known to one of ordinary skill in the art. The most popular enzyme preparation is obtained from Trichoderma reese. preparations from which the beta-glucosidase and / or the cello · · · have been chromatographically removed, or biohydrolase activity. Beta-glucosidase and or cellobiohydrolase activity is removed from the selected cellulase preparation to prevent cleavage of the cellulose derivative. into cellobiose and / or glucose. Genetic alteration of a suitable enzyme-producing microorganism can be enhanced by irradiation or mutagenic chemical agents (or by recombinant DNA inactivation by genetic means) to inhibit the production of beta-glucosidase and cellobiohydrolase by the microorganism. Cellulase preparations suitable for use herein include, for example, commercially available cellulase preparations under the tradename Econase CE series produced by Alko Ltd., Helsinki; Finland.

The starting materials

According to the invention, the hydrolyzate of the cellulose derivative and its fractions can be prepared by enzymatic hydrolysis of the soluble cellulose derivative. Popular cellulose derivatives for use herein are water soluble carboxymethyl celluloses.

Standard method for the preparation of hydrolyzate

Water-soluble carboxymethylcellulose hydrolysates are prepared from water-soluble carboxymethylcellulose as defined above by enzymatic hydrolysis using a cellulosic preparation containing endo-1,4-beta-glucanase as the only active hydrolytic agent, with only a negligible amount of saccharide and saccharide. which are absorbed from the human intestine (e.g., glucose) or · or hydrolyzed by intestinal bacteria (e.g., cellulose, biopsy). On the other hand, the average polymerization degree (PD) of the oligomers formed in such hydrolysis is less than 300, and thus the viscosity of the hydrolyzate solutions is significantly reduced compared to the viscosity of the non-hydrolyzed aqueous carboxymethylcellulose solutions.

i i i

Specific conditions that are suitable and a specific time sufficient to ensure the desired hydrolysis can be readily determined for each selected cellulose derivative and for each selected enzyme preparation.

7 103583

The water-soluble carboxymethyl celluloses used as starting materials in the present invention are per se calorie-free. Since the hydrolysis of the invention does not produce significant amounts of metabolizable sugars, the hydrolyzates of the invention with improved properties are particularly useful as low-calorie substitutes in foods.

The water-soluble carboxymethylcellulose hydrolyzate produced according to the invention and its fractions can be used, for example, as new low-calorie low-fat substances or as fillers. These hydrolyzates can be used to replace fat in a variety of foods such as bakery products, butter frosting and puddings. At least 50% of the fat can be replaced by these hydrolyzates, but the most preferred amount is 40% at least when carboxymethyl hydrolyzates are used. The amount that can be replaced depends on the intended use. The nutritional composition and food quality of the new product should improve or remain unchanged.

The hydrolyzate of the water soluble CMC produced according to the present invention and its fractions can also be used as novel low calorie excipients. These hydrolyzates can be used to replace carbohydrates such as sugar in various baking products or other foods. Replaceable; the amount of carbohydrate depends on the use and the average chain length of the hydrolyzate • · ·, ·: oligomer. By conventional t *% :: methods, the hydrolyzate mixture can be further separated into oligomeric *: * · fractions having different average chain lengths. The viscosity of the different fractions varies with the average chain length I · · of the oligomers contained in the fractions. Depending on the particular food use, the invention • · · investigates the selection of a fraction of a hydrolyzate mixture having a · viscosity (average chain length) most appropriate. · A particular food use. The selection of a particular fraction having average chain length and the amount of such fraction to be used for a particular foodstuff, may vary according to the fat or carbohydrate to be replaced 103583 8, it was found that the higher the absolute amount of substitute desired for a particular foodstuff, the lower the viscous fraction (average molecular weight) of the oligomer mixture should be used.

The invention will be explained in more detail in the following embodiments:

Example 1 - Preparation of Cellulase

Beta-glucosidase activity was removed by ion exchanger chromatography from a cellulase preparation, Econase CE, Alko OY, Helsinki, Finland, produced from Trichoderma reesei. The cellulase preparation (column A, Table 1) was passed through a cation exchange column (S-Sepharose FF, Pharmacia, LKB Biotechnology AB, Uppsala, Sweden) and equilibrated with 50 mM sodium acetate pH 3.8 as equilibration buffer. Unbound protein (including oligomer-producing endoglucanases) was washed off with equilibration buffer (column B, Table 1). Beta-glucosidase activity remained bound to the column and could be separately extracted with 1 M NaCl.

Endo-1,4-beta-glucanase and beta-glucosidase activities were measured as described by Bailey & Nevalainen (1981): Enzyme Microb.

Technol. 3: 153-157.

• ♦ • · * • · · »» · ·

The relative enzymatic activity of the Econase preparations described before in Table 1 before and after the ion-exchange column passage appears to be a typical means of the invention for the preparation of an essential beta-glucosidase-free preparation of the oligomer hydrolyzate of the invention. sex.

Table 1 9 103583

Enzyme Relative enzyme activity (%)

A

before the ion-exchange ion-exchange method after the method

Beta-glucosidase 100 L endo-1,4-beta-glucanase 100 70

Although Table 1 shows the relative enzyme activities, the absolute amount of enzyme used in any given example is then given by the amount of enzyme activity used in the nano-catal (nkat) units according to the universal activity, which is the amount of enzyme per second of nanomolar. (In the context of this application, a hydrolyzate reaction product such as an oligomer or glucose which can reduce a substance such as dinitrosalicylic acid which is reduced by a hydrolyzate reaction product and is provided later.) Method Bailey et. et al., Enzyme Microb. Technol. Butter. 9, pages 153-157 describes how such enzyme activity assays can be made using glucose as a standard.

Example 2 • ♦ · • ♦ · · · ·

Carboxymethylcellulose hydrolyzate * ♦ (i) Hydrolysis with Trichoderma reesee-derived enzyme preparation 20 kg carboxymethylcellulose (CMC 7MFD type, cellulose: rubber, also commercially available Bl)

Hercules Chemical Company, 92507, Rueil-Malmaison Ceder, France; 7MFD described average viscosity, food grade sodium carboxymethyl cellulose (where 7 out of 10 103583 10 glucose units are substituted with carboxymethyl) was mixed with 320 L of water and the pH of the solution was adjusted to 5.5 with 15% phosphoric acid and the temperature raised to 40 ° C. 0.27 L of enzyme preparation having endo-1,4-beta-glucanase activity of 1,780,000 nkat, from which beta-glucosidase activity has been removed by chromatography (as described in Example 1), was added to the CMC solution. After 1 hour, an additional 20 kg of CMC was added to the solution. After hydrolysis for 23 hours, the enzyme was inactivated by heating (90 ° C, 15 min). Finally, the hydrolysis solution was concentrated as usual with an evaporator and spray-drying.

The product contained less than 2% by weight of glucose and salicylose. When the same hydrolysis was performed with the original cellulase enzyme preparation from Trichoderma reesees, the glucose and cellobiose formed was more than 5% by weight.

The molecular distribution pattern of carboxymethylcellulose 50 and its hydrolyzate pattern 60 is shown in Figure 3.

The molecular weight distribution model was determined as described in Example 2a.

(ii) Hydrolysis with Asperaillus and Penici11umum-Derived Enzyme Preparations The enzyme preparations selected were commercially available from Cellulase AP 3 (Amano Pharmaceutical Co., Ltd., Nagoya, Japan). prepared using a strain of Aspergillus and

Cellulase CP (Sturge Enzymes, North Yorkshire, England) was prepared using a Penicillium strain. Carboxymethyl cellulose hydrolyzates were prepared as in Example 2c (i) except that 30 g of CMC-7MFD was used in 1 L of water and the amount of enzyme added was 0.028 g of Cellulase AP 3 (with endo-1,4-beta-glucanase activity of 1350 nkat ) and 0.048 g Cellulase CP (with 1350 nkat endo-1,4-beta-glucanase activity). The viscosity and molecular weight distribution pattern of the hydrolyzate produced by each cellulase were similar (Fig. 3) to the hydrolyzate produced by the enzyme derived from Trichoderma reesee.

The viscosities of various cellulose derivatives and their hydrolysates, as described and prepared in Example 2, were measured using a Haake-Roto-visco viscometer with sensor systems NV (Karlsruhe, Germany) (Table 2). Viscosities were measured at 25 ° C in aqueous solutions. Table 2 shows the concentrations (by weight) of several solutions, all having the same viscosity.

Table 2

Concentrations of cellulose derivatives and their corresponding hydrolyzates in a solution having a viscosity of 20 mPa.s (milli-Pascals-second) (milli-Pascals per second) at 25 ° C.

Cellulose derivative Concentration (by weight

Carboxymethylcellulose 2%

Carboxymethylcellulose-20% hydrolyzate

As the data in Table 2 show, the hydrolyzate of the cellulose derivative in aqueous solution has substantially less viscosity than the weight by weight of the same amount of cellulose derivative itself and can be incorporated into food in substantially greater amounts as fat or sugar substitute without the composition, volume, density or the like of the food is compromised.

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III

Example 3 - Fractionation of Carboxymethyl Cellulose Hydrolyzate: Carboxymethyl cellulose hydrolyzate was prepared as. Example 2c, except that CMC 7LDF, a low viscosity, food grade 103583 12 cellulose gum with 7 out of 10 glucose units substituted with carboxymethyl and available under the tradename Blanose, Hercules Chemical Co., France, was used as the raw material. 1.6 kg of CMC was used in 8 L of water and that the amount of enzyme added was 13.2 ml with 87,000 nkat endo-1,4-beta-glucanase activity. 5 ml of hydrolyzate (0.5 g of dry substance) was fractionated into three more fractions by gel permeation chromatography (Pharmacia K 26/100 column, Sephacryl S-200 gel, Pharmacia LKB Biotechnology AB, S-75182 Uppsala, Sweden). Distilled water was used as eluent, the flow rate was 14 ml / h, the fractionation process lasted 45 hours and the fractions collected at 0.5 hour intervals were combined into three fractions (18h - 26h, 90, 26h - 32h, 100 and 32h - 38h). curve 110 figure 5). The molecular weight distributions of carboxymethylcellulose, curve 70, carboxymethylcellulose hydrolyzate, curve 80, and three additional fractions, curves 90, 100, and 110, were determined by HPLC as described in Example 2.

Example 4 - Evaluation of a low fat material according to the invention in Madeira cake

A standard Madeira cake was baked by mixing 200g of high-quality cake flour, 250g of sugar-caster, 130g of high-quality fat, 16g of skimmed milk powder, 3g ·. · · Salt, 12g of baking powder, 180g of water and 176g of eggs:: (rendered). 180 g of the mixture was measured in greased dishes and baked at 170 ° C for 30 min. Madeira cake and in which 40% of the fat was replaced (eg 130g fat was reduced by 40% to 78g) 1) carboxymethylcellulose hydrolyzate (low fat according to the invention), 2) carboxymethylcellulose «« «With lozenge or 3) potato maltodextrin (a conventional low-fat substance), was compared to standard Madeira cakes: and with each other.

Table 3 13 103583

meager-fat

Content of substance replaced

The amount of fat tested in the yield product (%) of tea tested (p%) of the results

Standard --- --- Good volume, we got a cake with a clear texture. Easy-to-eat, mouth-watering food

Same as 40.0 2.2 Good Volume, Standard, Cake Carboxime Cake with Clear Styling Cellulose Composition, Loss Hydro The standard for eating edible lysate was fat, but not as good as fat.

Same as 40.0 0.3 Lower - Standard, Volume carboxymetric style cellulose was obtained, Loss Hydro Edible was lysate poor, The cake was used as a clearly greasy gummy and none: · · · • · ·

Same as 40.0 0.3 Good volume,: Y: standard, a cake of potato with a clear maltodex composition, triin was obtained. Eating was noted for fat near: van substitute standard and keena carboxymethyl; ··; cellulose hydrolyzate. Again, a little worse.

103583 14

Example 5 - Evaluation of a low fat substance of the invention in butter icing

Standard butter frosting was prepared by mixing 179 g butter (non-salted), 225 g powdered sugar and 96 g water. Butter spreads in which 30% fat was replaced (i.e., 179 g butter was reduced by 30% to 125.3 g) 1) carboxymethyl cellulose hydrolyzate (low fat material according to the invention), 2) carboxymethyl cellulose or 3) potato maltodextrin, compared butter frosting and each other.

According to an experienced panel, the results were as described in Table 4.

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Table 4 15 103583

meager-fat

Content of substance replaced

The amount of fat tested in the yield product (%) of tea tested (p%) of the results

Standard --- --- Acceptable frosting was obtained. It was solid, soft in composition and held water very well. This was much better than butter frosting with carboxymethyl cellulose and potato maltodextrin

Same as 30.0 4.3 Very good standard, flammable carbox brew. Solid, styled cellulose soft, colossal hydrated lysate particularly good used waterproofing, fat grease It was better as a resin than standard frosting: Same as 30.0 0.5 Poor, no good * · standard, snug: * · * : carboxime frosting-; . style pulp. The system lozenge did not consider water to be a good substitute for fat • · · V; Same as 30.0 2.7 Again, poor, standard, unacceptable carboxymetric frosting was styled cellulose. Water Loss Unable to Stay In Racial Van Replacement: As 16 103583

Example 6-- Evaluation of a filler according to the invention in marzipan

The standard marzipan product was prepared by mixing 21.8 g of powdered sugar, 21.8 g of caster sugar, 43.7 g of almond powder, 0.8 g of vanilla flavor, 10.9 g of egg (lightly whipped) and 1 g of lemon juice each. 100 g per product. The mixture was spheronized, lightly kneaded, spun and cut. Marzipan, where 10% of the sugar was replaced by carboxymethylcellulose hydrolyzate (filler according to the invention), had a good almond flavor and was the same color as the standard marzipan product. It was also easy to rotate and cut. A significant percentage reduction in calories may require a higher percentage of sugar replacement. Suitable lower viscosity fractions of carboxymethylcellulose hydrolyzate may be used in amounts greater than 10% by weight, suitably as high as 40%, to obtain a normal composition.

It will now be apparent to the average skilled person that other embodiments, improvements, details and uses may be made in accordance with the foregoing document and spirit, and within the scope of the patent, limited only by the following claims written within the scope of the patent law, including the doctrine of equivalents.

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Claims (9)

1. A water-soluble enzymatically prepared hydrolyzate of a cellulose derivative, characterized in that it comprises an oligomer mixture having an average degree of polymerization in the range of 3 to 300 and a molecular weight of 500 to 100,000 and whose viscosity is less than 20 mPa. s at 25 ° C as measured by a Haake-Rotovisco viscometer with the sensor system NV at a concentration of 20% by weight and prepared by hydrolyzing a water-soluble carboxymethyl cellulose with an enzyme preparation having 1,4-beta-glucanase activity. The enzymatically prepared hydrolyzate according to claim 1, characterized in that the enzyme preparation is selected from a group of cellulases, modified cellulases and their mixtures. The enzymatically prepared hydrolyzate according to claim 1, characterized in that the enzyme preparation is a cellulase, a modified cellulase produced by microorganisms selected from the group Trichoderma, Aspergillus and Penicillium. 4. The enzymatically prepared hydrolyzate according to patent; Claim 1, characterized in that the enzyme preparation comprises a cellulase preparation, which is obtained from a ♦. Trichoderma reesei from which at least one beta-glucosidase and cellobiohydrolase activity has been removed. 103583 whose average degree of polymerization is in the range of 3-300 and molecular weight 500 - 100,000 and whose viscosity is less than 20 mPa. s in 25 ° C matt with Haake-Rotovisco viscometer with sensor system NV at a concentration of 20 wt%. The process according to claim 5, characterized in that the enzyme preparation has been produced by means of a microorganism culture. The process according to claim 6, characterized in that the enzyme preparation produced by the microorganism has been purified to remove enzymes which react with the cellulose derivative to form saccharides. The process according to claim 7, characterized in that the microorganism is Trichoderma. Aspergillus or Penicillium. The method according to claim 8, characterized in that the microorganism is processed to alter the genes of the microorganism so that the saccharide-producing enzymes are made incapable by means of the genes.