FIELD OF THE INVENTION

The present invention relates to a method for reducing the serum separation/syneresis in a pectin containing agueous mass by the use of certain enzymes, and enzyme compositions therefore.

BACKGROUND OF THE INVENTION

Dicotyledonous plants comprise some of the major crops cultured by man, such as beans, peas, beets as well as most other fruits and vegetables.

The primary cell wall and middle lamella of these plants usually have a high content of pectic substances. The pectic matrix consists of smooth regions of polygalacturonic acids and of rhamnogalacturonan.

The polygalacturonic acid areas are usually highly esterified by methoxyl groups. Side groups consisting of araban, galactan and arabinogalactan are attached to the rhamnogalacturonan residues. The presence and distribution of the carboxy-methyl groups in pectin significantly alters its solubility and physico- chemical and gel forming properties.

This is of . major importance both in the plant cell wall, where the pectic matrix is a key regulator of porosity and passive diffusion of macromolecules but also in industrial pro- cessing of pectin and pectin containing plant material.

Pectinesterase (PE) , EC 3.1.1.11 (Enzyme Nomenclature 1992, Academic Press, Inc., 1992) , hydrolyses the ester linkage between methanol and galacturonic acid in esterified pectin. PE is found both in plants and in microorganisms. That is, PE offers a possibility of completely controlling the properties of pectin by altering the degree of esterification (DE) .

Thus, an endogenous highly methoxylated content of pectin (HM pectin) in various fruits and vegetables can enzymatically be modified to a low methoxylated pectin by pectinesterase. In com- bination with the natural content of calcium ions this is suffi¬ cient for an in si tu gelation or an in si tu thickening to take place if the PE is substantially free from depolymerizing enzyme

activities (Calesnik, E. J. et al 1950, Arch , of Biochem. , 29,

432-440 . Meurens, M. , 1978, Rev. Ferment . Ind . Aliment . , 33 , 95-

104) , since the presence of such activities will substantially break down the pectin. It is a well known problem that several products that are fully or partially vegetable or fruit based, such as ketchup, mustard and jam, "weep" or separate on standing. The processes used result in an indelicate appearance of the product.

Furthermore use of the product on e . g. bread or rolls results in the bread or roll getting soaked and disintegrate. Similarly other products such as fruit soups and yoghurts separate and develop an unattractive appearance.

It is an object of this invention to provide a method of causing a decrease of the serum separation in an aqueous mass containing methoxylated pectin without the need for addition of stabilising agents, such as externally added hydrocolloids, modified starch etc.

SUMMARY OF THE INVENTION The present invention relates to a method for reducing the serum separation of an agueous mass containing pectin, comprising the steps of : a) providing an aqueous mass substantially free from pectin depolymerising enzymes. b) adding an effective amount of pectinesterase, and c) incubating said mass in the presence of divalent cations.

Surprisingly it has been found that no other stabilising agent is needed in relation to the method according to the invention.

Hereby the method according to the invention offers the unique feature that the serum separation can be reduced or even avoided without the addition of any further external agents, whereby the only external agent being added is an effective amount of pectinesterase, and optionally divalent ions, such as alkaline earth ions, especially calcium ions.

Thus, for the first time it has been proven possible to utilise deesterified endogenous pectin in itself for a direct reduction of serum separation.

DETAILED DESCRIPTION OF THE INVENTION

As indicated the present invention relates to a method for reducing the serum separation of an aqueous mass containing pectin, comprising the steps of : a) providing an aqueous mass substantially free from pectin depolymerising enzymes. b) adding an effective amount of pectinesterase, and c) incubating said mass in the presence of divalent cations.

According to one embodiment of the invention the pectin may be endogenous HM.

In an embodiment of the method according to the invention the pectinesterase used is derived from a fungus of the genus Aspergillus, preferably A. japonicus, (S. Ishii et al . , 1979, Journal of Food Science 44, p 611-614), A . aculeatus , A . niger (EP 0 388 593 Al) , A . awamori (EP 0 388 593 Al) , or the genera FXisarium. Sclerotonia, or Penicillium, (Kikkoman: DE 2843351; US 4,200,694) . These pectinesterases exhibit a relatively low pH optimum, corresponding to the relatively low pH optimum of many fruits. The pectinesterase to be used in the invention should preferably be substantially free from pectin depolymerizing enzymes, such as pectinlyases or polygalacturonases. In case these activities are present in substantial amounts the pectin will be degraded. Such enzymes are obtainable by using a host system for the expression of the enzyme which does not produce any pectin depolymerizing enzymes (WO 94/25575) .

The activity of the pectinesterase is indicated in Pectin Esterase Units (PEU) defined as the amount of enzyme which under standardised conditions hydrolyses 1 mmol carboxyl groups per minute. A folder describing the Novo Nordisk assay ABT-SM- 0005.02.1 is available upon request.

According to the invention the pectin should be present in the aqueous mass in an amount from 0. l%w/w to 10%w/w preferably from 0.2%w/w to 1.0%w/w.

In further embodiments of the invention it may be advantageous to add sugar, acetic acid and/or salt in amounts from 0-60%w/w, 0-10%w/w, and 0-10%w/w, respectively.

The method of the invention will normally be carried out at a pH value from 2.5 to 7.5, preferably from pH 3 to pH 5, and for a time period for the incubation step from 5 to 60 minutes, preferably from 10 seconds to 30 minutes.

The incubation step will normally be performed at a tempera¬ ture from 10 to 60°C, preferably from 15 to 50°C.

According to the invention the PE is added in an amount of from 4 to 400 PEU, preferably from 9 to 135 PEU, better from 25 to 110 PEU, still better from 35 to 90 PEU, and more preferably from 45 to 70 PEU/kg VSS (Vegetable Soluble Solids, VSS is measured refractometrically as % Brix) .

According to the invention the method is designed for use with a pertinacious mass, especially food, and especially products such as tomato juice, tomato slurry, tomato paste or salsa or ketchup.

The method of the invention has been found to be advantageous with products wherein the pectin containing aqueous mass is derived from broccoli, pepper, mustard, apples, tomatoes oranges, lemons, grapes, lime, pears, carrots, peas, cauliflower, and berries, such as blackcurrant, blue-berries, strawberries, and raspberries.

Products obtained by the process of the invention are such as jam, marmalade, jelly, juice, paste, soup, dressing, sauce, condiment, ketchup, salsa, chutney, pudding, mousse, or other deserts.

MATERIALS AND METHODS Materials: Hot break tomato paste

Pectinesterase prepared according to WO 94/25575 Brookfield Viscometer, DVII

DUR Refractometer, Schmidt and Haensch, Germany Blotter test paper (Bridge & Company)

Methodβ: Viscosity Measurement :

The viscosity was measured by Spindel C, specification no. 93. Measurements at shear rate 2.5 rpm and 20 rpm were carried out.

Measurement of vegetable soluble solids (VSS) :

A drop of the test solution is applied to the sample well in the DUR refractometer.

The instrument indicates directly the solids content in % Brix.

Blotter Test:

To quantify the serum separation from the solid content the serum separation evaluation procedure of using a blotter test paper was used (Gould W.A. , et al 1992, Tomato Production Processing & Technology 3rd edition CTI Publications) .

7.0 g of ketchup was weighted out in a metal cylinder (dia 38 mm) placed in the centre of the blotter paper. After 7 min the serum separation was measured at the four rules. The measurement is given as an average of the four figures. A low value indicates a low degree of syneresis/serum separ¬ ation.

EXAMPLES

EXAMPLE 1

Hot break tomato paste was diluted from 22.5% VSS to 8.5% VSS and homogenised at 300 bar. Afterwards, eight samples of 455 g were weighed out in 1000 ml containers, the temperature was adjusted to 40°C. 8 x 25 ml enzyme solutions of increasing PE concentrations were added to the above mentioned substrates, the final enzyme protein concentrations were as follows: 0, 17, 34, 59, 68, 85,

102, 136 PEU/kg VSS. The samples were then incubated for 30 min at 40°C.

After the enzyme treatment a ketchup was prepared by adding 300 g of brine to each of the samples. The brine consists of sugar, salt and acetic acid (Skott, W. P. Die Industrielle Obst- und Gemύseverwertung, 1970 55 229-234) . The prepared ketchup were then heat treated, 88°C in 3 min. The samples were then cooled in an ice bath and finally placed in the refrigerator until analysis could take place, The results from the analysis is shown in Table I

Table I

Pectinesterase Blotter Test Viscosity Viscosity Concentration mm 2.5 rpm 20 rpm PEU/kg VSS cP cP

0 20.7 8305 1455

17 18.2 9027 1543

34 14.4 9527 1660

59 9.7 8745 1595

68 5.8 22900 3330

85 10.3 31965 4250

102 14.2 35505 4780

136 25.0 45415 8340

From the Blotter test results it is clearly seen that the syneresis or serum separation can be controlled by adjusting the concentration of pectinesterase. Not surprisingly, also the viscosity depends on the enzyme concentration. It is noticed that it is possible at the same time to obtain both a reduction of the syneresis and a viscosity increase.

REFERENCES CITED IN THE SPECIFICATION

1. Calesnik, E.J. et al 1950, Arch , of Biochem. , 29 432-440.

2. Meurens, M. , 1978, Rev. Ferment . Ind. Aliment . , 33 95-104)

5 3. Gould W.A. , et al 1992, Tomato Production Processing & Technology, 3rd edition CTI Publications

4. S. Ishii et al . , 1979, Journal of Food Science 44 611-614

5. EP 0 388 593 Al

6. DE 2843351 10 7. US 4,200,694

8. WO 94/25575

9. Skott, W. P. Die Industrielle Obst- und Gemύseverwertung, 1970 55 229-234

10. Enzyme Nomenclature 1992, Academic Press, Inc., 1992 15 11. Novo Nordisk assay ABT-SM-0005.02.1