WO2003013264A1

WO2003013264A1 - Hypoallergenic chocolate

Info

Publication number: WO2003013264A1
Application number: PCT/EP2002/007291
Authority: WO
Inventors: Rodolphe Fritsche; Carl Erik Hansen; Marcel Alexandre Juillerat; Sunil Kochhar
Original assignee: Societe des Produits Nestle SA; Nestle SA
Current assignee: Societe des Produits Nestle SA; Nestle SA
Priority date: 2001-08-01
Filing date: 2002-07-02
Publication date: 2003-02-20
Anticipated expiration: 2004-02-01
Also published as: GB0118820D0; PE20030266A1

Abstract

The present invention relates to a chocolate with reduced allergenicity comprising a hypoallergenic cocoa liquor or powder prepared by treating the cocoa liquor or powder with a protease to provide cocoa having an antigenicity lower than 10mg cocoa antigen protein/g protein as determined by reaction with polyclonal guinea pig anti-cocoa protein antiserum and to a hypoallergenic chocolate comprising cocoa powder and hypoallergenic milk powder. Or enzyme treated cocoa liquor or powder and hypoallergenic milk powder wherein the cocoa liquor or powder was treated with a protease.

Description

HYPOALLERGENIC CHOCOLATE

Field of the Invention

The present invention is directed to hypoallergenic chocolate containing cocoa with reduced allergenicity and milk proteins/peptides. As used herein, the term chocolate also includes chocolate materials generally referred to as compound and ice cream coatings.

Background to the Invention

Chocolate contains protein allergens which can cause symptoms ranging from gastrointestinal disturbances, skin reactions and asthma to severe allergic reactions, such as immediate allergic anaphylaxis. Although these symptoms are not highly f equent in the general population, it is desirable to produce a hypoallergenic (HA) cocoa and chocolate which does not result in such allergic responses for this group of consumers.

US 4,963,372 is directed to a hypoallergenic chocolate containing no cocoa liquor, cocoa powder or milk powder, the only protein source being protein hydrolysate and/or amino acids. Thus, there are no cocoa proteins present in this chocolate to trigger an allergic reaction. WO 82/00132 is directed to foodstuffs, in particular roast, ground coffee, with lower allergenicity and thrombogenicity prepared by removing water-soluble glycoproteins by isolelectric precipitation. WO 95/03708 is directed to a hypoallergenic chocolate containing at least partially defatted cocoa powder rendered substantially hypoallergenic by treatment with a supercritical fluid, such as carbon dioxide, and mixed with fat, sweeteners and an optional dairy component. US 4,078,093 is directed to a hypoallergenic chocolate prepared using cocoa powder which has been treated, by a prolonged two-step heat treatment, claiming denaturation of the protein allergens which cause chocolate allergies.

Thus, it is an object of the present invention to provide cocoa with reduced allergenicity which can be used in the manufacture of hypoallergenic chocolate.

Summary of the Invention

According to one aspect, the present invention provides a chocolate with reduced allergenicity comprising a hypoallergenic cocoa liquor or powder prepared by treating the cocoa liquor or powder with a protease to provide cocoa having an antigenicity lower than lOmg cocoa antigen protein/g protein as determined by reaction with polyclonal guinea pig anti-cocoa protein antiserum.

According to a further aspect the present invention relates to a hypoallergenic chocolate comprising cocoa powder and hypoallergenic milk powder.

A still further aspect relates to a hypoallergenic chocolate comprising enzyme treated cocoa liquor or powder and hypoallergenic milk powder wherein the cocoa liquor or powder was treated with a protease.

Detailed description of the invention

A preferred embodiment according to the present invention is a hypoallergenic dark chocolate comprising approximately 30-70% by weight enzymatically treated cocoa liquor or 3-20 %

cocoa powder. The chocolate may also comprise approximately 0-60% by weight sucrose, approximately 0.01-0.5% by weight lecithin, approximately 0.01-0.05% by weight methyl- vanillin, wherein the cocoa liquor or powder was treated with a protease. Cocoa butter is adjusted accordingly to control fluidity. All percentages are based on the total weight of the

chocolate.

The following recipe for milk chocolate using enzymatically treated cocoa liquor or powder is as follows approximately 40-55% by weight sugar, approximately 10-18% by weight cocoa liquor, approximately 10-20% by weight skimmed milk powder, approximately 0.01%-0.5% lecithin by weight and approximately 0.01-0.1% by weight vanillin. Cocoa butter is adjusted accordingly. The milk powder may be hypoallergenic milk powder and is preferably whey based.

This invention is also applicable to HA chocolate compound coatings, such as milk chocolate compound coating comprising crystal sugar (25-60%), confectionery coating fat (24-40%), HA milk powder (5-30%), enzymatically treated cocoa powder (3-20%), vanillin (0.01-0.1%) and soya lecithin (0.01-0.5%), wherein the cocoa powder was treated with a protease.

It has surprisingly been found that cocoa powder without any treatment is already hypoallergenic. This is contrary to the teachings in WO 95/03708 and US 4,078,093 which teaches that a special treatment is required to produce hypoallergenic chocolate. Thus, it is now possible to make a completely hypoallergenic milk chocolate or compound chocolate using cocoa powder or protease treated cocoa liquor and hypoallergenic milk powder. Preferably, the milk powder is whey based.

According to an embodiment of this aspect of the invention milk chocolate comprising cocoa powder and HA milk can be made using equivalent quantities of cocoa powder and HA milk powder to obtain , approximately 40-55% by weight sugar, approximately 10-18% by weight cocoa liquor, approximately 10-20% by weight skimmed milk powder, approximately 0.01%- 0.5% lecithin by weight and approximately 0.01-0.1% by weight vanillin.

This invention is also applicable to HA chocolate compound coatings, such as milk chocolate compound coating comprising crystal sugar (25-60%), confectionery coating fat (24-40%), HA milk powder (5-30%), cocoa powder (3-20%), vanillin (0.01-0.1%) and soya lecithin (0.01-0.5%), wherein the cocoa liquor or powder was treated with a protease.

The invention is illustrated by the following examples.

Preparative Examples: Cocoa material

i) Cocoa liquor preparation West African Amelonado cocoa beans were fermented for 0 to 7 days. The fermented and unfermented beans were sun dried and hand-peeled. Cocoa nibs were roasted at 130 °C for 45 minutes. Unroasted and roasted nibs were ground to liquor by conventional methods.

ii) Cocoa seed protein preparation Dried cocoa beans were passed through a bean crusher followed by a winnower. 20-30g of cocoa nibs were milled for a few seconds. The nib powder was passed through a 0.8mm sieve and kept at 4 °C. 300 mg of cocoa crude powder (CCP) was suspended in 3 ml of extraction buffer (100 mM sodium phosphate, pH 8 containing 1 % (w/v) SDS) and sonicated for 30 seconds at full speed (10 mm probe at medium setting and full power). The suspension was cooled on ice for 15 to 30 minutes and centrifuged at 15,000 rpm at 4°C for 15 min. The clear

supernatant was removed, passed through a 0.22 μm filter disc and kept at 4 °C until utilized. This preparation was used for the allergenicity tests.

iii) Cocoa Acetone Powder preparation

Cocoa acetone powder was prepared from non-defatted cocoa beans according to Noigt et al (1993) Food Chem 47: 145-151. lOg of cocoa nib powder produced in (ii) was suspended in 200 ml 80 % (v/v) aqueous acetone and stirred for 1 hour at 4°C. The suspension was centrifuged at 15,000 rpm for 15 min at 4°C. The residue was extracted 5 times with 200 ml 80 % (v/v) aqueous acetone followed by 3 times washing with 100 % acetone. The cocoa acetone powder was dried under reduced pressure.

iv) Cocoa liquor enzymatic treatment

Flavourzyme 1000L (Fungal endo- and exo-protease mixture (pH range 4-8)) was dissolved in water at 50°C and added to melted cocoa liquor produced in (i). The reaction was carried out with 25 to 80% moisture at 50°C. The liquor was incubated in temperature controlled reactors and pasteurised at 90 °C for 10 min, and dried directly to less than 5% moisture with or without vacuum at 70°C. If liquor-roasting was required, the samples were roasted at 125°C for 30 min.

v) Cocoa powder enzymatic treatment Commercially available cocoa powder was treated as 20% suspension in 80% water using 1% enzyme for 24 hours at 45°C. After incubation the suspension was heat inactivated for 10 min at 90 °C and dried e g by lyophilisation. Protein concentrations of the cocoa extracts was determined according to the protocol by Lowry et al. (1951) J. Biol. Chem 193:265-275 as modified by Bio-Rad (DC protein assay kit). Bovine serum albumin was used as a protein standard. Determination of free primary amino groups by fluorescence was carried out according to the protocols of Udenfriend et al. Science 1973, 178, 871-872. Cocoa was extracted using 50% acetic acid (3g/30 ml, 1 min sonication, 1 min Polytron homogenization, centrifugation 10 min 10.000 g, filtration on 0.2 μm membrane). Degree of hydrolysis was defined as the ratio between the primary amino groups for the sample and the total amino groups following 6 N HC1 hydrolysis.

Example 1

Analysis of cocoa antigenicity by ELISA inhibition

All the cocoa samples of above preparative examples were treated by SDS and sonication as described in preparative example (ii).

Residual cocoa antigenicity was determined by ELISA inhibition wherein antigen (cocoa, milk) is determined by its inhibition capacity on a standardized antigen-antibody system using a polyclonal guinea pig anti-cocoa protein antiserum. Full cocoa extract was used for immunizing guinea pigs in order to have the totality of anti-cocoa antibodies which are polyclonal and contains antibodies to all cocoa proteins. Wells of microtitration plates were coated with 150μl of a standard cocoa protein powder (as prepared in preparative example (ii)) (acetone powder 3/21 unfermented, SDS treated) at 50μg/ml in carbonate-bicarbonate buffer and incubated 24 hours at 4°C. Plates were washed 4 times in a PBS-Tween buffer and free reacting sites were blocked by adding 200 μl/well of fish gelatine (0.5 % in PBS-Tween). Plates were incubated 1 hour at room temperature (RT) and washed again 4 times in PBS- Tween.

In separate tubes, 1 part of standard cocoa protein powder dilution as prepared in preparative example (ii) or the enzymatically treated cocoa of preparative examples (iv) and (v) were incubated for 1 hour at RT with 1 part of guinea pig anti-cocoa protein antibody (diluted 1 :2000) After incubation, lOOμl of this inhibition mixture was added to the above coated and blocked microtitration wells and incubated 2 hours at RT. Plates were washed 4 times in PBS- Tween. A rabbit anti-guinea pig peroxidase labelled conjugate (0.1 ml of a 1 :2000 dilution) was then added, plates incubated 1 hour at RT and washed 4 times in PBS-Tween. The chromogenic substrate (0 1 ml o-phenylene-diamine) was added. After 15 minutes incubation, optical density is read at 492 nm on an ELISA plate reader. The allergenicity of cocoa liquor was examined and the results are shown in Tables 1 and 2.

Table 1 shows that autolysis as well as treatment with the enzyme, Flavourzyme, reduces efficiently cocoa antigenicity by a factor of 30. Fermentation and roasting reduce also antigenicity 2-4 times. Final residual cocoa antigenicity after these processes is around 1% of the initial values (1000 mg/g)

Table 1

Cocoa protein antigenicity (determined by ELISA Inhibition)

Results are expressed as mg cocoa antigen protein / g protein

Table 2 shows surprisingly that the antigenicity of cocoa powder is low and could be further reduced by enzymatic treatments to a level of 2 mg cocoa antigen/g protein Table 2

Enzymatic treatment of cocoa powder with different enzymes Cocoa powder: Gerkens Non-alcalinized, 10-12% fat

Example 2

Enzymatic treatment of different types of cocoa powder

Aqueous suspensions (80% water) of various types of commercially available cocoa powders (Gerkens or Bensdorp) were subjected to enzymatic treatment using 1% Flavourzyme at 45°C for 24 h, and followed by heat-inactivation at 90°C for 10 min followed by lyophihzation The results are shown in table 3 Table 3

Enzymatic treatment of different types of cocoa powder, cocoa antigenicity expressed in mg

cocoa antigen / g protein

It was suφrisingly found that many types of cocoa powder have a low allergenicity without treatment Example 3:

Preparation of chocolate using enzymatically treated cocoa

Dark chocolate was made according to the following recipe: 38% enzymatically treated cocoa liquor as prepared in preparative example (iv) 50% sucrose, 12% cocoa butter, 0.3% lecithin, 0.02% methyl-vanillin.

Example 4:

Preparation of chocolate using cocoa powder and HA milk

Milk chocolate was made according to the following recipe 6% cocoa powder (Gerkens 10-12% fat)

19% hypoallergenic milk powder,

50% sucrose,

25% cocoa butter,

0.3% lecithin, 0.02% vanillin

The antigenicity of the chocolate made in accordance with examples 3 and 4 were evaluated and the results are shown in table 4.

Alternatively, the milk chocolate may be made from enzymatically treated cocoa liquor made in accordance with preparative example (iv) and hypoallergenic milk powder. Table 4

Residual antigenicities in chocolate, as determined by ELISA inhibition, and expressed in mg antigen /g protein

Results in table 4 show first that cocoa specific antigenicity was strongly reduced in dark HA chocolate (2 5 mg cocoa antigen/g protein) as compared to commercial dark chocolate (188 mg cocoa antigen/g protein) This residual cocoa antigenicity is therefore below 10 mg / g protein, which validates its hypoallergenic claim This confirms that enzymatic treatment of cocoa liquor in dark HA chocolate is able to reduce strongly cocoa antigenicity Residual cocoa specific antigenicity in the HA milk chocolate is strongly reduced (residual value4.3) This is due to the fact that cocoa powder used for preparing HA milk chocolate is already

hypoallergenic BLG (β-lactoglobulin) is an important allergenic protein of cow's milk and is

used here as a representative molecule for allergenicity evaluation of milk One can see that BLG specific allergenicity is in the hypoallergenic range (0 4 mg BLG/g protein) This is due

to the fact that HA milk powder was used for producing HA milk chocolate In summary, table 4 shows that both dark HA chocolate and milk HA chocolate preparations are hypoallergenic (HA) for cocoa and milk protein antigens.

Claims

Claims:

1. A chocolate with reduced allergenicity comprising a hypoallergenic cocoa liquor or powder prepared by treating the cocoa liquor or powder with a protease to provide cocoa having an antigenicity lower than lOmg cocoa antigen protein/g protein as determined by reaction with polyclonal guinea pig anti-cocoa protein antiserum.

2. A chocolate as claimed in claim 1 which is milk chocolate, dark chocolate or compound chocolate.

3. A dark chocolate as claimed in claim 1 comprising approximately 30-70% by weight enzymatically treated cocoa liquor or 3-20% cocoa powder, based on the total weight of the chocolate.

4. A dark chocolate as claimed in claim 3 further comprising approximately 10-60% by weight sucrose.

5. A milk chocolate as claimed in claim 2 further comprising approximately 40-55% by weight sugar, approximately 10-18% by weight cocoa liquor, approximately 10-20% by weight skimmed milk powder, approximately 0.01%-0.5% lecithin by weight and approximately 0.01-0.1% by weight vanillin

6. A hypoallergenic chocolate comprising cocoa powder and hypoallergenic milk powder.

7. A hypoallergenic chocolate comprising enzyme treated cocoa liquor or powder and hypoallergenic milk powder wherein the cocoa liquor or powder was treated with a protease.

8. A chocolate as claimed in claim 6 or claim 7 wherein the hypoallergenic milk powder is whey based.

9. A chocolate as claimed in any of claims 6 to 8 which is milk chocolate or compound chocolate.

10. A milk chocolate as claimed in any of claims 6 to 8 comprising approximately 3-20% by weight cocoa powder, approximately 5-30% by weight hypoallergenic milk powder, approximately 35-60% by weight sucrose, approximately 0.01-0.5% by weight lecithin.