WO2005070223A1

WO2005070223A1 - Methods for packing a soy milk product

Info

Publication number: WO2005070223A1
Application number: PCT/DK2005/000037
Authority: WO
Inventors: Hong Zhi Huang; Jian Guo Tang
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2004-01-23
Filing date: 2005-01-21
Publication date: 2005-08-04
Anticipated expiration: 2006-07-23

Abstract

The present invention relates to methods for packaging soy milk products comprising the step of adding to the soy milk product an enzyme composition comprising an oxidoreductase. The invention also relates to soy milk products obtainable by the methods of the present invention.

Description

METHODS FOR PACKAGING A SOY MILK PRODUCT

Field of the Invention The present invention relates to methods for packaging a soy milk product.

Background Soy milk products, including tofu, are widely used by the food service sector, catering, institutions as well as by private households, especially in China and Japan. However, the shelf-life is only few days as an off-flavour/off-taste may develop during storage as a result of oxidative processes, e.g. biotic and/or abiotic oxidative processes. It is an object of the present disclosure to provide improved methods for packaging a soy product in such a way that the shelf-life is significantly increased. US 4,929,451 discloses a method for eliminating disagreeable odours from soy milk during production.

Summary of the Invention The present invention relates to a method for a packaging a soy milk product, comprising: a) obtaining a soy milk product, b) adding to the soy milk product an enzyme composition comprising an effective amount of an oxidoreductase enzyme; and, c) packaging the soy milk product to produce a packaged soy milk product. The invention also relates a packaged soy milk product obtainable by the above method.

Detailed Description of the Invention The soy milk product of the present invention comprises soy milk as such, as well as processed soy milk products, such as a tofu product. A soy milk product suitable for use in the method of the invention may be obtained by soaking a volume of soy beans in water, e.g. for 24 to 48 hours, and milling the beans in water, e.g. in 8 times the volume of water. The bean slurry may be filtrated to remove bean residues before being boiled and cooled down to yield soy milk. The soy milk may have a Brix about 7.5, and a pH about 6.9. A processed soy milk product for use in the method of the invention, such as tofu, may be produced from soy milk in a process comprising the following steps; denaturation of the soy milk proteins and coagulation to a curd, e.g. at 70-90°C in the presence of cat-ions, e.g. CaSO₄, and/or MgCI₂. The protein curd may then be drained and pressed to remove excess water (tofu juice), and cooled down to yield tofu. Tofu may comprise 80-90% of water and 10-20% dry solids. The tofu curd may be cut into pieces, e.g. cubes, of desired size and packaged. Usually, and preferred for the invention, the tofu is packaged with addition of 20% volume of tofu juice around the tofu pieces. The packaging is preferably performed as an air tight packaging, such as in an air tight container, such as in a sealed plastic box and/or in sealed layer of plastic folio, and more preferably as a vacuum packaging. Where the soy milk product is soy milk as such the oxidoreductase enzyme is preferably added to the soy milk by applying an enzyme composition comprising an oxidoreductase enzyme directly to the soy milk. Where the soy milk product is a processed soy milk product such as tofu, the oxidoreductase enzyme is preferably added to the tofu by applying an enzyme composition comprising an oxidoreductase enzyme to the tofu juice which preferably accompanies the tofu during and following packaging. As described above a packaged soy milk product, such as a packaged tofu product may develop an off-flavour/off-taste during storage. By adding to the soy milk product before packaging an enzyme composition comprising an oxidoreductase enzyme the off-flavour/off- taste can be reduced. Without being bound by theory it is proposed that the beneficial effect is due that the amount of oxygen present in the packaged soy milk product is reduced. Oxidative processes, hereunder processes involved in microbial growth, which would otherwise give rise to volatile compounds coursing off-flavour/off-taste are thereby inhibited. The oxidoreductase enzyme may be any oxidoreductase enzyme selected from the list consisting of; glucose oxidase, galactose oxidase, hexose oxidase, carbohydrate oxidase, pyranose oxidase, amino acid oxidase, and laccase. Preferably the oxidoreductase enzyme is a glucose oxidase. In a preferred embodiment the enzyme composition further comprises a catalase. Glucose oxidase (EC1.1.3.4) catalyzes the reaction beta-D-glucose + O₂ <=> D-glucono-1 ,5-lactone + H₂O₂. Catalase (EC1.11.1.6) catalyzes the reaction H₂O₂ <=> ¹/₂ O₂ + H₂O. The over-all result is the consummation of ¹ O2 for every molecule of glucose oxidized. Glucose may be added as a substrate for the oxidoreductase, particularly in the amount of 0.005% to 10%, more particularly 0.01 to 2.5% (w/w), or most particular in the amount of 0.05 to 0.5 % (w/w). In the methods of the present invention, any glucose oxidase may be used which possesses suitable enzyme activity in an appropriate pH and temperature range. It is preferable that the enzymes are active over broad pH and temperature ranges. In a preferred embodiment, the enzymes have a pH optimum in the range of about 3 to about 10. In a more preferred embodiment, the enzyme(s) has a pH optimum in the range of about 4.5 to about 8.5. In another preferred embodiment, the enzymes have a temperature optimum in the range of about 0°C to about 100°C, more preferably in the range of 2°C to 50°C, and most preferably in the range of 5°C to 25°C. Following packaging the soy milk product is preferably stored, e.g. for at least 5 days, for at least 10 day, for at least 15 day, or even for at least 20 days, and preferably the storage is performed under conditions, e.g. in respect of temperature, which allows the added oxidoreductase to react with the appropriate substrate. Thus the soy milk product is preferably stored at a temperature at least for a part of the storage period is at leas 0°C, such as at least 2°C, at least 4°C, at least 6°C, at least 8°C, at least 10°C, at least 15°C, or even at least 20°C. The term "effective amount" is defined herein as an amount of one or more enzymes that is sufficient for providing a measurable effect on at least one property of interest of the soy milk product. In this case the property of interest is defined herein as a reduction of off- flavour determined as described in Example 2. The source of the enzymes is not critical for use in the methods of the present invention for improving one or more properties of interest of the soy milk product. Accordingly, the enzymes may be obtained from any source such as a plant, micro organism, or animal. The enzymes are preferably obtained from a microbial source, such as a bacterium or a fungus, e.g., a filamentous fungus or yeast and may be obtained by techniques conventionally used in the art. In a preferred embodiment, the enzymes are obtained from a bacterial source. For example, the enzymes may be obtained from an Acetobacter, Acinetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Comamonas, Clostridium, Gluconobacter, Halobacterium, Mycobacterium, Rhizobium, Salmonella, Serratia, Streptomyces, E. coli, Pseudomonas, Wolinella, or methylotrophic bacterium strain. In a more preferred embodiment, the enzymes are obtained from an Acetobacter aceti, Alcaligenes faecalis, Arthrobacter oxidans, Azotobacter vinelandii, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus anitratum, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Comamonas testosteroni, Clostridum tyrobutyricum, Gluconobacter dioxyaceticus, Gluconobacter liquefaciens, Gluconobacter suboxydans, Halobacterium cutirubrum, Mycobacterium convolutum, Rhizobium melioti, Salmonella typhimu um, Serratia marcescens, Streptomyces lividans, Streptomyces murinus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, or Wolinella succinogens strain. In another preferred embodiment, the enzymes are obtained from a fungal source. For example, the enzymes may be obtained from a yeast strain such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain; or from a filamentous fungal strain such as an Acremonium, Aspergillus, Aureobasidium, Chrysosporium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Monilia, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Schizophyllum, Sclerotium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain. In another more preferred embodiment, the enzymes are obtained from an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lignorum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Monilia sitophila, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysporum, Polyporus pinsitus, Polyporus versicolour, Sclerotium rolfsii, Sporotrichum thermophile, Trichoderma citrinoviride, Trichoderma hamatum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma polysporum, Trichoderma reesei, Trichoderma saturnisporum, or Trichoderma viride strain. The enzymes may be obtained from the organism in question by any suitable technique and in particular by use of recombinant DNA techniques known in the art (c.f. Sambrook, J. et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, USA). The use of recombinant DNA techniques generally comprises cultivation of a host cell transformed with a recombinant DNA vector, consisting of the product gene of interest inserted between an appropriate promoter and terminator, in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture. The DNA sequence may be of genomic, cDNA or synthetic origin or any mixture of these, and may be isolated or synthesized in accordance with methods known in the art. The enzyme may also be obtained from its naturally occurring source, such as a plant or organism, or relevant part thereof. A suitable glucose oxidase (EC 1.1.3.4) may be a fungal glucose oxidase, in particular from a strain of Aspergillus, such as an Aspergillus niger glucose oxidase, or a glucose oxidase from a strain of Cladosporium sp. in particular from Cladosporium oxysporum. In the methods of the present invention, the enzymes may be obtained from commercial suppliers, preferably from Novozymes A/S. Commercially available glucose oxidases useful in the present invention are GLUZYME™ 2.500 BG, GLUZYME™ 10000 BG and GLUZYME™ MONO 10000 BG, available from Novozymes A S, Denmark). Other commercially available glucose oxidases useful in the present invention are FERMIZYME™ GO 10.000 and FERMIZYME™ GO 1500 available from DSM, HYDERASE™ 15 and HYDERASE™ HC available from Amano or OXYGO® available from Genencor Int., USA. A suitable laccase (EC 1.10.3.-) may be a fungal laccase, in particular from a strain of Myceliopthora. A suitable carbohydrate oxidase (EC 1.1.3.-) may be a fungal carbohydrate oxidase, in particular from a strain of Michrodochium, and most particularly from a strain of Michrodochium nivale. A suitable catalase (EC1.11.1.6) may be a fungal glucose oxidase, in particular from a strain of Aspergillus, such as an Aspergillus niger glucose oxidaseor from a strain of

Cladosporium sp. in particular from Cladosporium oxysporum. The catalase may be present as a side activity of the glucose oxidase composition. In the methods of the present invention, such an enzyme composition may be obtained from commercial suppliers, preferably from Novozymes A/S. Commercially available glucose oxidases compositions comprising an appropriate catalase side activity useful in the present invention are

GLUZYME™ 2.500 BG, GLUZYME™ 10000 BG and GLUZYME™ MONO 10000 BG, available from Novozymes A/S, Denmark. The treatment of the soy milk product with the one or more enzymes necessarily involves contacting the soy milk product with the enzyme(s) under suitable conditions.

Accordingly, the enzyme treatment may be performed by contacting the soy milk product with the one or more enzymes in an enzyme composition. The enzyme composition may comprise a single enzyme component, e.g., a mono-component enzyme composition, or a mixture of two or more enzymes. Thus, the enzymes to be used in the methods of the present invention may be in any form suitable for the use in question, e.g., in the form of a dry powder, agglomerated powder, or granulate, in particular a non-dusting granulate, a liquid, in particular a stabilized liquid, or a protected enzyme. The enzymes may me diluted and/or dissolved in an appropriate solvent, preferably water, before being applied to the soy milk product. In terms of enzyme activity, the appropriate dosage of a given enzyme will depend on the enzyme in question. The skilled person may determine a suitable enzyme unit dosage on the basis of methods known in the art. In the methods of the present invention the effective amount of the glucose oxidase is about 0.001 g to about 200 g enzyme protein per kg soy milk product, more preferably about 0.01 g to about 20 g per kg soy milk product, even more preferably about 0.1 g to about 2 g per kg soy milk product, and most preferably about 0.2 g per kg soy milk product. The glucose oxidase is present in the solution in the process water in an amount effective for reducing off-flavor in the finished product, particularly 1 to 20000 GODU per kg soy milk product, particularly 10 to 15000 GODU per kg soy milk product, particularly 50 to 10000 GODU per kg soy milk product, particularly 500 to 7500 GODU per kg soy milk product or more particularly 1000 to 5000 GODU per kg soy milk product, or most particularly around 2000 GODU per kg soy milk product. One GODU (Glucose Oxidase Units) is the amount of glucose oxidase enzyme, which under standard conditions (i.e. pH 5.6, 30°C, 20 min. incubation time, acetate buffer, and glucose 16.2 g/l as substrate) forms 1 micromol of hydrogen peroxide per minute. The packaged soy milk product, e.g. a packaged processed soy milk product such as packaged tofu product, may be consumed as it is following opening and removal of the packaging material, or it may be subjected to a thermal treatment before or after opening of the package before being consumed, e.g. by using the soy milk product in a process for production of a meal, comprising subjecting the tofu to elevated temperatures. Preferably the packaged soy milk product is subjected to a treatment either before or after opening and removal of the packaging material, e.g. a thermal treatment, leading to the inactivation of the added enzymes before consumption. It is understood that any of the embodiments described herein may be combined to produce a packaged soy milk product. The invention also relates to packaged soy milk product obtainable by the methods of the present invention. The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Materials and methods The enzyme compositions used or suitable for use were commercial oxidoreductases: GLUZYME™ 10000 BG, a glucose oxidase preparation from fermentation of Aspergillus niger, and available from Novozymes A/S. FLAVOURSTAR™, a laccase derived from Myceliophthora thermophila, and available from Novozymes A/S. Packed Tofu with juice (20% w/w) produced by Beijing No.2 Soy Processing Plant was bought from a Chinese supermarket. Dissolved oxygen was measured using a CyberScan DO/Temperature Meter. Inhibition of microbial growth was assayed by dilution plating onto LB-plate and manual microbial colony counting. Sensory analysis is carried out by a panel of eight judges. The flavour/taste of a vacuum packaged soy milk product produced according to the method of the invention is rated and compared to a conventional product using a scale from 0 to 15, where 0 referred to least off-flavour/off-taste (fresh taste) and 15 to most off-flavour/off-taste. Prior to the sensory analysis a small training is conducted comparing freshly produced soy milk products (5 days of storage) with the reference applied in the evaluation (3 weeks of storage). Tofu samples of 0.5 kg are out portioned in small cubs with lids and served at room temperature (about 20°C). The samples are tested in triplicates served in random order. The results are given as mean value of the eight judges.

Example 1 To a 25g sample of tofu juice (pH6.9), 0.025g glucose and 0.025g glucose oxidase (GLUZYME™ 10000 BG) were added. The solution was incubated at room temperature with stirred and dissolved oxygen was measured. The results are shown in Table 1. The juice was deletion plated onto LB plates and the colonies counted after 5 days. The results showed a significant reduction of colonies on the plates representing samples from the juice treated with glucose oxidase (data not shown).

Example 2 Glucose oxidase (GLUZYME™ 10000 BG) is added to tofu juice in a concentration of 0.2 g/L and 1.6 g Dextrose/L. The tofu is cut into cubes of an appropriate size and the tofu cubes are vacuum-packed with approx. 20% of tofu juice. The vacuum-packaged tofu is stored for three weeks in a refrigerator at about 4°C until the sensory analysis is performed. The addition of glucose oxidase clearly reduces the amount of off-flavour/off-taste compared to the reference, and hereby improves the taste of the tofu following storage.

Example 3 A laccase preparation originating from Myceliopthora is tested as described in Example 2. FLAVOURSTAR™ is added to tofu juice in a concentration of 1 g/L or 0.2 g/L. The improvement of taste and reduction of off-flavour note is comparable to the effect obtained with glucose oxidase in example 1.

Example 4 A carbohydrate oxidase from Microdochium nivale (Gerlachia oryzae) is tested as described in Example 2. Carbohydrate oxidase is added to tofu juice in a concentration of 5- 80 mg enzyme protein/L. The improvement of taste and reduction of off-flavour note is comparable to the effect obtained with glucose oxidase in example 1.

Claims

1. A method for a packaging a soy milk product, comprising: a. obtaining a soy milk product, b. adding to the soy milk product an enzyme composition comprising an effective amount of an oxidoreductase enzyme; and, c. packaging the soy milk product to produce a packaged soy milk product.

2. The method of claim 1 wherein the soy milk product is a processed soy milk product such as tofu.

3. The method of claims 1-2, wherein the oxidoreductase enzyme is an enzyme selected from the list consisting of; glucose oxidase, galactose oxidase, hexose oxidase, carbohydrate oxidase, pyranose oxidase, amino acid oxidase, and laccase

4. The method of claims 1-3, wherein the glucose oxidase is derived from Aspergillus sp., preferably from Aspergillus oryzae or Aspergillus niger, or from Bacillus sp., preferably from Bacillus licheniformis.

5. The method of claims 1-4, wherein further an effective amount of a catalase enzyme is present in the enzyme composition.

6. The method of claims 1-5, wherein the packaging is performed as a vacuum packaging.

7. A packaged soy milk product obtainable by the method of any of claims 1-6.