Cloudy fruit juices and methods for making same

Details Cloudy fruit juices and methods for making same Cloudy fruit juices and methods for making same

1999-07-19

2002-12-10

Hendricks, Keith (Department: 1761)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

Reexamination Certificate

active

06492159

ABSTRACT:

FIELD OF THE INVENTION
The instant invention relates to cloudy juices and methods for making same. The invention is particularly concerned with cloudy juices from pomaceous fruit. The invention is further concerned with cell wall—cloud—material obtainable by a method according to the invention and uses thereof. The invention also concerns enzyme preparations which can suitably be used for the preparation of cloud material and/or juice containing said cloud material.
BACKGROUND OF THE INVENTION
Liquefaction is a process which is frequently used for the manufacturing of clear apple juice. The process aims at a complete degradation of cell walls using cell wall degrading enzymes. This process can be realized by treating apples with an appropriate pectolytic enzyme, for instance pectin lyase (PL), in combination with a crude cellulase preparation, containing several endoglucanases, cellobiohydrolase (CBH) and several other activities.
A process for making a cloudy apple juice has been described in Vincken et al. (1996, PhD Thesis,
Enzymic Modification of Cellulase-Xyloglucan Networks
, Wageningen Agricultural University, The Netherlands, Chapter 9) by partial degradation of apple cell walls. This yields a suspension of eroded apple cells which is mainly composed of cellulose and xyloglucan. It is unknown why these cellulose-xyloglucan complexes do not precipitate like cellulose particles do. It is thought that the xyloglucan molecules play a role in stabilization.
By this process Vincken et al. have been able to produce a cloudy apple juice on a small scale, using pectin lyase and small amounts of a crude cellulase preparation and ripe apples. The use of ripe apples is thought to be important in this process, because these contain large amounts of xyloglucan endotransglycosylase (XET) . It is thought that a small amount of xyloglucanase is required to trigger the depolymerizing action of XET. The XET then weakens the apple cell wall, facilitating juice release but leaving the major part of the cellulose-xyloglucan network intact. A too large dose of cellulase destroys the cloud.
From an industrial point of view, the process above has two important drawbacks. Firstly, the enzyme formulation used in this process apparently contains components that are detrimental for cloud stability. Secondly, the balance between fungal glucarases and endogenous XET, is very important in order to obtain a cloudy juice. However, this balance is difficult to manage in practice as it requires knowledge of the endogenous XET-levels. No methods are known to determine such levels in an industrial context and the method is difficult to reproduce, even on a small scale. A further disadvantage, associated with the use of ripe apples, resides in their susceptibility to fungal attack. Fungi, such as Penicillium produce mycotoxins, which end up in the juice.
Another method for making a cloudy apple juice is disclosed in WO 95/34223. The method herein disclosed involves the use of enzymes that attack the hairy regions of the pectin backbone, such as rhamnogalacturonase (RGase), rhamnogalacturonan acerylescerase (RGAE), galactanase, and combinations thereof. A strong warning is expressed against the use of homogalacturonan degrading enzymes as they would destroy cloud stability (vide page 10, Experiment 4, page 6, 11, 13 to 16).
It is an object of the invention to provide a reproducible method for making cloudy apple juice which can be practiced on an industrial scale.
SUMMARY OF THE INVENTION
It was surprisingly found that a cloudy apple juice is obtained when a method is used comprising the step of treating apple material with a mixture of enzymes comprising a homogalacturonan depolymerizing activity, such as pectin lyase (EC 4.2.2.10), and an enzyme with endoglucanase activity (EC 3.-2.1.4), in the absence of enzymes which reduce the cloud volume by more than 25%, as determined by a method which comprises:
(a) suspending 5 mg of a cloud material
obtainable by homogenizing 1 g of apple pieces of
Malus malus
L. cv. Jonagold, after removal of the peel and core, using a Braun Kitchen machine (MX32, Frankfurt, Germany; 5 mm blade), incubating at 40° C. under continuous head-over-tail mixing at 150 rpm in 3 mL of a 200 mM NaOAc buffer (pH 4) containing 0.01% (w/v) NaN3, 1% ascorbic acid, 50 mU pectin lyase and an amount of endoglucanase with an equivalent of 25 mU of xyloglucanase activity,
 in 3 ml of a 25 mM NaOAc buffer (pH 5), containing about 0.01% (w/v) NaN3,
(b) Incubating the cloud suspension so obtained for 1 h at about 40° C., in the presence of the said enzyme, during which the cloud suspension and enzyme is mixed head-over-tail,
(c) allowing the cloud to settle at about 40° C. for 24 hours, and
(d) determining the effect of the enzyme on cloud stability by calculating the difference between the cloud volume or the cloud suspension obtained by following the above steps in the absence of the said enzyme and subtracting the volume obtained of the cloud suspension obtained by following the above steps in the presence of the enzyme.
By the use of this test, enzyme cocktails in which undesirable enzymes are absent have been developed. The enzyme preparations are characterised by the presence of an endoglucanase having CMCase activity, preferably a CMCase activity to xyloglucanase activity ratio of at least 0.7, and an enzyme having homogalacturonan depolymerizing activity such as a combination of pectin esterase and polygalacturonase, preferably pectin lyase.
In principle, there is no upper limit to the ratio (endoI in Table 1 has a CMCase/XGase ratio of about 114 and performs very well in a process for making a cloudy juice according to the invention; the CMCase activity is by far the most important in the partial apple liquefaction of apple material, but it cannot be concluded that XGase activity is absolutely redundant).
By using a method according to the invention, enzymes of the crude cellulase preparation which destroy cloud stability can be identified. Accordingly, a preferred embodiment of the invention is a process which is essentially free from cellobiohydrolase activity (EC 3.2.1.91).


REFERENCES:
patent: 94/14953 (1994-07-01), None
Vincken et al., Ph.D. “Zyloglucan Endotransglycosylase Activity in Apples In Ripening-Related: Implications for Fruit Juice Processing”, Thesis, Enzymic Modification of Cellulase-Xyloglucan Networks. Chpt. 9, Jan. 1996.
Nakamura S. et al., “Cloning and Sequencing of a cDNA for Poplar Endo-1,4-&bgr;-Glucanse”, Plant Cell Physiol. 36, 1229-1235, Jan. 1995.
Beldman et al., “Purification , Characterization and Comparison of all Detectable Endoglucanases, Exoglucanases and &bgr;-glucosidases”, Eur. J. Biochem. 146, 301-308, Jan. 1985.
Ito et al., “Transformation of Intact Yeast Cells Treated with Alkali Cations”, J. Bact. 153, 163-168 Jan. 1983.
Somogyi “Notes on Sugar Determination”, J. Biol. Chem. 195, 19-23, Jan. 1952.
Vincken et al., “The Effect of Xyloglucans on the Degradation of Cell-Wall-Embedded Cellulose by the Combined Action of Cellobiohydrolase and Endoglucanases . . . ”, Plant. Physiol. 104, 99-107, Jan. 1994.

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