NL8301261A - METHOD FOR CONVERTING POLYSACCHARIDES - Google Patents

Description

f. *

Ref.ï Case R 299 (R)

Unilever N.V. in Rotterdam

Short designation: Process for converting polysaccharides

The invention relates to a process for converting polysaccharides, more particularly seed galactomannans. The polysaccharides can be converted into an isolated or uninsulated form or in the form of seed endosperma particles of a number of Legumi nosae, in particular of guar seed. Guar is Cyamop-sis tetragonolobus and is usually processed to obtain guar gum, which contains guaran as the main polysaccharide component. Guaran is known to have a galactomannan structure with a backbone of 1-4 linked beta-D-manno-pyranoyl moieties to which alpha-D-galactopyranosyl moieties are attached. Usually it contains 35-45 wt. % of galactose units and 65-55% of mannose units. It is believed to have a molecular weight ranging from 16 to 22 x 10 ^ as deduced from an intrinsic viscosity range of 12-16 dl / g as reported in Carbohydrate Research (Elsevier Scientific Publishing Company) , 1982, Volume 107, 20 biz. 17-32. Guaran, guar gum and guar seed are abundantly available, but in some applications their properties are less than the properties of another gum commonly used industrially, n.1. locust bean gum, which is obtained from locust bean (carob seeds) This gum, however, contains predominantly a galactomannan containing 20-25 wt% galactose units and 80-75 wt% mannose units. Carob kernel flour has more favorable properties, such as gelling, especially in combination with other polysaccharides, than guar gum.

Locust bean gum is becoming more expensive and scarce, due to poor harvests and generally no new planting of the locust bean tree. Therefore, efforts have been made to improve the guarand 8301261 * - * R 299 (R) 2 properties by removing some of the galactose units from the mannan backbone. A known method for the enzymatic hydrolysis of a part of the main chain gelactose-5 units is described in US-A-4 332 894 (Whistler), in which the treatment of guar gum or guaran solutions, in particular a 1% by weight aqueous solution containing the enzyme alpha-D-galactosidase is reported.

10

An earlier description of the enzymatic removal of galactosidase side chains from galactomannan, including from guar, is found in Phytochemistry (Pergamon Press), 1975, Vol. 11, pp. 1187-1194, in particular p. 1191 -1192, Table 3 and Figure 2. It also shows that it is necessary that the alpha-galactosidase enzyme is sufficiently free from contaminating chain-splitting enzyme (beta-mannanase). The experimental part of this publication (p. 1193, right column) clearly shows that 0.1 wt% solutions of galactomannan were treated with alpha-galactosidase enzymes.

Another more general description of the galactose-removed guaran preparation, with improved synergistic properties similar to that of locust bean gum, can be found in Abstracts of Xth International Carbohydrate Symposium, Sydney. Australia, TH, July 16, 1980.

30

For all of the methods described above and for all previously published methods of enzymatic modification of galactomannans known to the Applicant, it was generally accepted practice to incubate enzyme with the polysaccharide in a solution at concentrations up to 1% by weight to convert a homogeneous reaction mixture.

8301 261 R 299 (RJ, * 3

Finally, there is the dissertation by Charles Wesley Baker of Purdue University of 1973 describing the treatment of guaran solution with enzyme. On page 100, the treatment of 100 mg / l aqueous guaran solution is reported. Since non-depolymerized guaran does not form a solution at this concentration, but a thick paste, this description, which does not constitute an afterthrowable disclosure, is disregarded. Either the stated concentration has been incorrectly stated, or the guaran had been depolymerized before it was dissolved that it can no longer be considered as a representative starting material or it was material which had not been prepared by the careful methods of normal polysaccharide chemistry 15 hear. The latter possibility is very likely, because this dissertation on page 139 seems to confirm that the guaran used was depolymerized, since Table 6 lists an intrinsic viscosity for guaran of 6.12 dl / g, while the expected values for 20 non- depolymerized material is 2 to 3 times higher than this value.

High concentrations of certain polysaccharides (i.e., 30% by weight) have sometimes been used in processes involving enzymatic modification, especially enzymatic modification of starch. However, such methods described in the literature involve the enzymatic degradation of the main chain of the polysaccharide, with a very marked reduction in viscosity and destruction of gelling properties. Ultimately, a homogeneous solution is then obtained.

However, such enzymatic conversions at higher concentrations are the exception rather than the rule, because homogeneous conditions were required.

8301261 'R 299 (R) 4

In the present case, partial enzymatic hydrolysis, of about half, of the galactose from galactomannan containing 20-45, preferably 35-45% by weight of galactose, leads to the creation of more synergistic regions along the mannan. chain and therefore to more structure formation and gelation after the hydrolysis.

It has now been found that a thick aqueous paste or slurry containing 2-50 wt% galactomannan can be incubated with a practically specific alpha-galactosidase enzyme preparation to lower the galactose content of the galactomannan from a value between 35 and 45 wt.%, preferably a value between 10 and 27 wt.%. The synergistic properties of the galactomannan 15 are thereby significantly improved.

Preferably, the aqueous galactomannan paste used as starting material contains from 5 to 30% by weight of galactomannan, more in particular from 8 to 20% by weight. The modified galactomannan preferably contains between 13 and 25% by weight, and particularly preferably between 5 and 19%, galactose. The latter appears to be a new substance with very good gelling properties.

According to the present invention, the galactoman nanoplastic preparation is incubated with the gelactosidase enzyme under conditions such that the enzyme removes only part of the galactose units from the mannan main chain, while the main chain does not or hardly depolymerize .

This is accomplished in a practical manner by kneading the paste, usually at a temperature between 0 and 70 ° C, preferably between 20 and 40 ° C, either with the enzyme or with the enzyme added after hydration. If desired, enzyme powder can also be mixed with a dry preparation of the polysaccharide for hydration.

8301261 R 299 (R) ï 5

Incubation is continued until the galactose content of polysaccharide has reached the desired level. This usually takes from 12 to 48 hours.

The pH of the paste should be within the active range of the enzyme, usually between 4 and 6. The addition of a small amount of acid or buffer solution may be desirable.

The enzyme preparation should exhibit a substantially specific α-pha-galactosidase activity and at most only a low beta-mannanase activity. Therefore, after incubation, the galactomannan should have a molecular weight not less than two thirds of the original value. Suitable enzymes can be of vegetable origin (e.g. from lucerne, coffee beans or guar seed) or they can be obtained from bacterial or fungal cultures (e.g. Aspergillus niger).

20

After removing a portion, e.g. half of the galactose units of the galactomannan, the enzyme is deactivated, for example, by heat treatment. Although it may be desirable to purify the modified galactomannan to remove free galactose and enzyme material therefrom, e.g. by dissolving and precipitating the modified galactomannan, this is usually not necessary and the product as such can be used for various applications where the gelling properties are important. In particular, it can be used in combination with other polysaccharides such as agar, agarose, carrageenan and xanthan, taking advantage of the synergistic interaction with these substances.

8301261 * 'R 299 (R) 6

The theological properties of the modified guaranes obtained according to the invention, in particular those containing 15 to 19% by weight of galactose, are such that they are valuable ingredients for foods, such as gelled desserts, "mousses" and ice cream , and also for certain cosmetic preparations. For efficient processing it is in some cases recommended to prepare intimate or fairly homogeneous mixtures of at least one emulsifier and / or other polysaccharide with modified guar in advance. These mixtures may contain an amount of emulsifier at least equal to the weight of the modified guar and any other gums. The combinations are conveniently prepared from solutions or melts, using methods such as spray drying, spray cooling or drum drying.

Trial 1

Effect of purified alpha-galactosidase on de-polymerized guar galactomannan (guaran beta-mannase border-galactomannan) _

Target:

This test was conducted to determine the effect of the substrate concentration on the rate of release of D-galactose from guar galactomannan by alpha-galactosidase enzyme. In these experiments, partially depolymerized guar galactomannan (guar beta-mannanase border-galactomannan) was used to minimize viscosity effects.

Guaran beta-mannanase border-galactomannan (GMLG, 0.2 ml 0.2-10 wt%) in sodium acetate buffer (0.1M, pH 4.5) was incubated with guar seed-35 alpha for 5 min at 40eC galactosidase II (see BV McCleary (1983) Phytochemistry 22, pp. 649-658), 13 n cat on p-nitrophenyl alpha-D-galactopyranoside. The reaction was stopped and 83 0 1 2 6 1 7 R 299 (R)% the increase in the amount of reducing sugar was noted as a measure of the degree of hydrolysis. The results are shown in Table I and it clearly shows that in order for the enzyme to act at maximum rate, a substrate concentration of more than 10% by weight (100ml / ml) is required.

Similar results were obtained when an alpha-galactosidase from Aspergillus niger was used.

Increasing the substrate concentration from 0.2 to 10% by weight resulted in an almost ten-fold increase in activity.

TABLE I 15

Substrate Concentration Efficacy of the _ (wt%) _ enzyme (% of maximum) 0.2 4.4 0.5 9.5 20 1.0 14.5 2.5 30.5 5.0 71.5 10.0 100.0 This Table provides data on the optimal substrate concentration to obtain maximum hydrolysis without the likely modifying effects of an excessively high viscosity. The effect of the viscosity will become more apparent in the other examples to be given herein, in which naturally occurring guar galactomannan was used.

Examples 1 and 2 100 g of guam flour was extracted with boiling aqueous ethanol (80% v / v) and the vacuum dried flour was added to 3 1 of ice cold water with rapid mixing 830 1 26 1 R 299 (R) 8 in a Kenwood mixing device. The pasta was allowed to stand for 2 hours, after which it was (batchwise) mixed in a Waring Blender. The paste was heated to 60 ° C and remixed using the Kenwood mixer.

5

Samples were taken from the above paste (465 g, containing 15 g of guar flour, ie 3.2% w / v), which were treated as follows: 10 A. To the paste sample was added: guar alpha galactosidase II (3ml, 720n cat on p-nitrophenyl alpha-D-galactopyranoside) plus sodium acetate buffer (2.40ml, 2M, pH 4.5), and this mixture was incubated in the mixing bowl at 37 ° C for 24 hours a Farinograph. The paste was mixed for 15 minutes at 2 hour intervals for the first 8 hours.

The reaction was stopped by heating the reaction mixture to 80 ° C, after which polysaccharide was recovered by alcohol precipitation. The precipitate was washed with ethanol, acetone and ether and dried under vacuum.

A sample of the dried material was dissolved in water and centrifuged, and galactomannan polysaccharide precipitated with alcohol. The purified polysaccharide had a galactose / mannose ratio of 25:75 and a specific viscosity of 17.6 dl / g (cf. the original galacto-mannan value of 17.7 dl / g, and a galactose / mannose ratio of 40:60).

B. To guar paste (465 g, 3.2 w / v% as mentioned under A), guar alpha-galactosidase II (2 ml, 480 35 n cat) plus sodium acetate buffer (3 ml, 2M, pH 4.5) and water (41 ml) added. The paste was mixed thoroughly with a spatula and at 40 ° C for 48 hours

8301261 9 incubated without further mixing. The reaction was stopped by treating the paste at 100 ° C for 10 min, and the polysaccharide (as well as a sample of purified galactomannan) was prepared as described above. The purified galactomannan showed a galactose / mannose ratio of 24:76 and a specific viscosity of 17.6 dl / g).

Example 3 Ethanol-washed pieces of guar (40 g) were left at 40 ° C for 4 hours and then at 60 ° C for 2 hours in distilled water to absorb. The material was then mixed in a Parinograph mixing bowl while an additional 100 ml of water was added.

Acetate buffer (4 ml, 2M, pH 4.5) plus guar alpha-galac tosidase II (6 ml, 1440 n cat) plus 17 ml of water were added with mixing. The concentration of guar-20 pieces was 5.5% w / v. The paste was mixed for 5 minutes at a time for the following 8 hour period and left overnight in the Farinograph mixing bowl without stirring (at 37 ° C). The paste was mixed again and incubated for an additional 24 hours (total incubation time 48 hours). The galactomannan in an aliquot ("aliquot") of this paste was purified and the remaining paste was treated with alcohol and dried by solvent exchange. The purified galactomannan showed a galactose / manno-30se ratio of 23.5: 76.5 and a specific viscosity of 14.0 dl / g.

Examples 4 and 5

Guar pieces were ground to pass through a 22 mm screen and dried with boiling ethanol. The flour (30 g) in water (180 ml) was heated in a boiling water bath for 10 min and then stored at 40 ° C for 20 hours at 8301261 R 299 (R) 10. The heterogeneous paste was divided into two equal batches and treated as follows: 5 A.

One pasta batch (105 g) was placed in the mixing bowl of a Farinograph and the following solutions were added: guar alpha-galactosidase II (2 ml; 480 n cat); sodium acetate buffer (3 ml, 2M, pH 4.5); and water (10 ml). The final concentration of flour in water was 15% w / v. The paste was mixed and incubated at 40 ° C for 48 hours.

B.

The second pasta batch was treated in the same manner as the first, except that the amount of water added was 55 ml, compared to 5 ml for batch A). The final concentration of this paste was 10% w / v. The paste was mixed and incubated at 40 ° C for 48 hours.

At the incubation, the reactions were terminated by incubating the pastes at 100 ° C for 10 min. A sample of each paste was removed and galacto-mannan extracted to be analyzed. The mass of the sample was freeze-dried. The purified gelactomannans (samples A and B) both had a galactose / mannose ratio of 27:73 and a specific viscosity of 19.8 dl / g.

30

Example 6

The effect of beta-mannanase contamination in alpha-galactosidase preparations on the properties of alpha-galactosidase-modified guar galactomannans 35

Guar pastes were prepared as described in Example 1. Guar flour paste (3.2 w / v%, 155 ml) 8301261 R 299 (R) 11 was incubated with guar alpha-galactosidase II at 37 ° C for 20 hours. (0.5 ml, 240 n cat) plus sodium acetate buffer (5 ml, 0-250 n cat). The reaction was stopped by treating the pastes at 100 ° C for 10 min, and the galactomannan was purified and dried by solvent exchange (ethanol, acetone, ether) and drying under vacuum. The amounts of beta-mannanase used were cat (sample A)? 0.025 n cat (sample B); 0.25 n cat (sample C); and 2.5 n cat (sample D). The activity of the beta mannanase was standardized using locust bean galactomannan (0.28 w / v%) in a sodium acetate buffer (0.1M, pH 4.5) as a substrate.

The galactose / mannose ratio of each of the samples 15 was 19:81 and the specific viscosity was: 16.54 dl / g (A); 16.21 dl / g (B); 14.34 dl / g (C), * and 4.79 dl / g (D). These results demonstrate that at a high substrate concentration, a galactomannan with a galactose / ratio approximately equal to that of John's bread-galactomannan and with equivalent to slightly better viscosities can be prepared from guar galactomannan using guar alpha galactosidase which is contaminated to at least 0.1% by beta mannanase.

1% beta-mannanase contamination (sample D) caused significant depolymerization.

Example 7

Guar meal (1 kg) was extracted with boiling aqueous ethanol (80 w / v%) for 10 min, removing free liquid by suction. The flour was fed to the mixing bowl of a Hobart Mixer containing 10 liters of ice cold water. The mixer was set to rotational speed 1 and the flour was added slowly to limit the formation of clumps to a minimum of 35. As the pasta started to thicken, a further amount (10 L) of ice cold water was added in portions. The pasta was mixed for an additional 10 min at 8301261 V, R 299 (R) 12 with the mixer set to setting 3, then the pasta was left overnight to allow complete hydration. The paste was further mixed (setting 3), adding guar alpha-galactosidase II (20 micro-5 cat) in a sodium acetate buffer (pH 4.8; 100 ml) with thorough mixing. The contents bowl was sealed with a plastic film and the contents incubated at 37 ° C for 48 hours. The reaction was stopped by heating the pasta to 80 ° C using a steam jet, after which the pasta was freeze dried. The guar flour thus treated contained guaran with a galactose / mannose ratio of 18:82.

15 Example 8

Portions of ice cream were prepared with the following composition in a small scale pilot plant with a capacity of 40 1: 10% non-fat milk solids 20 10% fat 15% sucrose 0.3% emulsifier 0.2% stabilizer 64.5 % water.

25

As stabilizer, resp. used: (1) guar gum or (2) locust bean gum or (3) alpha-galactosidase modified guar gum from Example 7.

The ice cream was packaged in 0.5 L blocks and its melting properties were measured at 15 ° C.

The results are shown in Table II.

8301261 R 299 (R) '* 13

TABLE II

No. Stabilizer Galactose / Drip Vormannose Speed Hold.

5 ratio of ice to ice v. Stabil. (ml / h) after 4 h 1 Guar gum 40:60 60 poor 2 Carob 10 pits 23:77 23 good 3 Modified guar gum 18:82 27 very good 15

As shown in Table II, incorporating alpha-galactosidase-modified guar gum # instead of locust bean gum # with a galactose content equal to that of locust bean gum # led to obtain ice cream that exhibited the same or better properties in comparison with the ice cream containing locust bean gum as a stabilizer. The dripping rate of the ice cream containing alpha-galactosidase modified guar gum was slightly higher than that of the locust bean gum ice cream # but its dimensional stability after 4 hours at 15 ° C was noticeably better. The ice cream containing unmodified guar gum was noticeably worse # both in terms of dripping speed and shape retention than the ice cream products containing the other stabilizers.

These results demonstrate that the alpha-galactosidase-modified guar gum # prepared by incubation with enzyme in a thick paste # is at least equal to naturally occurring locust bean gum # in its functional properties.

8301261, R 299 (R) 14

Example 9

Mixtures of xanthan (0.5 w / v%) and galactomannan (1.0 w / v%) in water were prepared as follows: The ingredients were dispersed in water in a screw bottle 5 using a top-powered Atomix, autoclaved at 120 ° C for 5 min, remixed with the Atomix, centrifuged (3000 rpm, 2 min) to remove bubbles, heated in a water bath again to be melted, and finally poured into perspex shapes of approx. 1 1/4 cm and a depth of approx. 1.2 cm. The yield stress was measured on the gel plugs (?), At room temperature, after maturing for 24 hours using an Straton Materials Tester. Samples were compressed between parallel plates at a crosshead speed of 20 mm / min. was applied. When galactomannan was used: (1) guar gum or (2) locust bean gum or (3) alpha-galactosidase-modified guar gum from Example 3, or (4) alpha-20 galactosidase-modified guar gum from Example 7.

The results are reported in Table III

TABLE III

25 - «—--

No. Galactomannan Galactose / Fluid Mannose Tension Ratio (N) 30 1 Guar Gum 40:60 no gel formed 2 Locust bean gum 23:77 3.3 3 Modif. guar gum 23.5: 76.5 3.0 35 4 "" 18:82 5.3 8301261 15 R 299 (R)

The interaction between modified guar gum and xan-than is similar to the interaction between locust bean gum and xanthan, and is different from that between guar gum and xanthan, in which no gel is formed.

Similar results have been obtained with regard to the interaction between galactomannans and agarose.

830 1 26 1

Claims (7)

2. Process according to claim 1, characterized in that the aqueous slurry contains 8-20% by weight of galactomannan. 3. A method according to any one of claims 1 or 2, characterized in that the incubation of the galactomannan slurry is stopped when the galactose content of the galactomannan has decreased to a value between 10 and 27% by weight. 4. A method according to any one of claims 1 to 3, characterized in that the incubation is stopped at a galactose content of the galactomannan between 13 and 25% by weight. 5. A method according to any one of claims 1 to 4, characterized in that the incubation is stopped at a galactose content between 15 and 19% by weight. Process according to any one of claims 1 to 5, characterized in that the final average molecular weight of the galactomannan has a value which is more than two thirds of the starting value. 7. A galactomannan with a galactose content of between 15 and 19. 83 0126 1 R 299 (R) ~ r * * A synergistic mixture of polysaccharides consisting of a galactomannan according to claim 7 and a polysaccharide from the group consisting of xanthan, agar and agarose. 830 1 26 J