FIELD OF THE INVENTION The present invention relates to processes for improving the characteristics of the meat quality in products, and also a protein hydrolysate made from a fish material.

In general, the present invention relates to a use of a protein hydrolysate from fish for injection into fish or fish meat to improve the characteristics of the fish meat.

BACKGROUND AND PRIOR ART In connection with this project, a preliminary search was carried out in the patent literature to find applications of protein hydrolysate.

JP-A-57125648 describes a brine containing protein hydrolysate that can be injected into meat to increase the ability to retain water, and also to improve the taste.

EP-B1-797927 describes a protein hydrolysate that can be injected into raw meat.

US-A-6303162 describes injection of brine that contains a protein hydrolysate into meat.

From the publications given above, use of a protein material or a hydrolyse protein material, i. e. a protein hydrolysate, made from animal meat or a plant material for injection into animal meat, is known.

However, the present invention relates to the use of a protein hydrolysate from fish, by injection of this material into meat from animals or fish.

Large amounts of fish raw material get lost, or are not greatly utilised in the processing of fish today. From the whole fresh fish and to the finished products fresh fillet and salted/smoked fillet, the extent of utilisation, in the form of fraction of edible goods from the process, is about 50%. Thus, there is a large

potential for increasing the fraction of consumer product by better utilisation of the by-products.

The present invention has shown that it is possible to produce a protein hydrolysate from fish by-products which can be added to the fish fillets. A better utilisation of the raw materials is achieved, at the same time as the quality parameters for the fish fillets are maintained or improved, and also with the storage capacity of the product being maintained or improved.

Thus the present invention relates to a process to improve and/or maintain the lightness and colour characteristics of a fish fillet, where a protein hydrolysate is added to the fish fillet. The protein hydrolysate is preferably made from a protein-containing fish material. Furthermore, it is preferred that the protein hydrolysate is made from bone off-cuts from the fish processing industry, such as the bone off-cuts from salmon off-cuts.

It is especially preferred that the protein hydrolysate is made in accordance with the processing steps described in example 1. A preferred aspect of the invention uses fish fillet from salmon. In another aspect of the invention, the mentioned fish fillet is subjected to a smoking process or another form of wanted processing after the addition of protein hydrolysate.

Furthermore, the present invention relates to a process to improve the keeping quality and freshness of a fish fillet, where a protein hydrolysate is added to the fish fillet.

A further aspect of the invention relates to a process to reduce the development of rancidity in a fish fillet, where a protein hydrolysate is added to the fish fillet.

For these procedures (improve the keeping quality and/or reduce rancidity) it is preferred that the protein hydrolysate is made from a protein-containing fish material, for example in the form of bone off-cuts from the fish processing industry. One embodiment uses off-cuts from salmon. For example, the protein hydrolysate can be made in accordance with the processing steps described in example 1. Furthermore, the fish fillet can be subjected to a smoking process, or another wanted processing, after addition of the hydrolysate.

The present invention also relates to a protein hydrolysate, characterised in that it is made from fish by an enzymatic hydrolysis of a fish material, whereupon the water-soluble, protein-containing fraction is separated by decanting, and where the protein hydrolysate is thereafter preferably concentrated.

It is preferred that the fish material is off-cuts from the fish industry, and can contain off-cuts with bone fractions. Salmon can be used as the fish material.

The invention also relates to a procedure for manufacture of a protein hydrolysate from fish, where water, having a temperature of 40-80 °C, preferably about 60 °C, is added to a fish material at a fish: water ratio of 1.0 : 0.6 to 1.0 : 1.0 based on weight: weight, and where the mixture is stirred where the

temperature being kept at a temperature level suitable to achieve satisfactory enzymatic activity.

Protamex is preferably used as the enzyme material, and the mixture is preferably kept at a temperature interval of 50-60 °C for at least 30 minutes, more preferably at a temperature interval of 56-57 °C for at least 30 minutes, preferably at least 45 minutes.

The invention also relates to the use of a protein hydrolysate as an additive to fish meat or fish forcemeat, to increase the keeping quality of the mentioned fish meat or fish forcemeat, and an application as an additive to fish meat or fish forcemeat to prevent oxidative conversion of components in the mentioned fish meat or fish forcemeat, where addition of the protein hydrolysate replaces or reduces addition of conventional antioxidants to fish meat or fish forcemeat.

EXPERIMENTAL PART Example 1 Manufacture of protein hydrolysate from fish Fish bone off-cuts from processing of salmon were kept on ice, with a plastic foil between the ice and the bones, for one to five days before enzymatic hydrolysis. Water at 60 °C was added to about 100 kg bone off-cuts and two stirrer blades were rotated at a speed of 200 rpm to mix the water and the fish bones. The temperature of the mixture was around 35-45 °C at the start.

A proteolytic enzyme mixture (Protamex, available from Novozymes Ltd, Denmark) was added to the mixture at a concentration of 1/1000 of the weight of the off-cuts, and the hydrolysis was allowed to run for 2 hours with continuous stirring. The mixture was heated, and the incubation comprised an optimal period of 45 minutes where the temperature of the mixture was 56-57 °C. The reaction mixture was deactivated by addition of steam (10 minutes to 95 °C). Afterwards, the mixture separated into three fractions; heavy insoluble components at the bottom, a watery phase with dissolved proteins (mid-fraction) and a fat fraction at the top. The protein concentration in the water fraction was about 6% (by weight) dependent on the rotational speed of the stirring during the hydrolysis. The protein hydrolysate was concentrated in an APV-evaporator to a protein concentration of about 40% (by weight).

Example 2 Application of the protein hydrolysate in smoked salmon The hydrolysate was injected, together with brine, to salmon fillets for smoking by application of a Fomaco automatic injection machine of the type FGM 16/64F. The composition of the mixtures used in the injection is given in the results section.

Example 3 Determination of colour characteristics Industrial colour of the muscle surface was determined by using a Minolta Chromameter, CR-200 (Minolta Camera Co. Ltd. , Osaka, Japan). The detector was placed on the dorsal and the ventral side of the central line in the muscle surface of the fillet, and carried out the measurements in L*a*b* mode, and mean values and standard deviation were determined. The colour was determined by carrying out measurements on five fillets from each combination.

Example 4 Keeping quality pH pH in a fresh muscle was measured in a 1:1 mixture of muscle mixture and 0.15 M KCI pa, while pH of conserved muscle was measured in a 1: 5 by weight ratio of muscle mixture and water. A PHM 80 Radiometer (Copenhagen, Denmark) with a glass electrode was used and 3-6 parallels of each sample were used to calculate a mean value.

TBARS TBARS (Thiobarbituric Acid Reactive Substances) is one of the most common chemical methods to determine the oxidation level in fish muscle. These values were determined in an aqueous trichloroacetic acid extract according to the methods given in Witte et al., 1970 and Dulavik et al., 1998, both of which are incorporated here by reference. Three parallel applications were carried out for each group of muscle sample.

Example 5 Testing of sensory characteristics (among them smell, colour, firmness, taste) Sensory tests were carried out on a smoked, marinated salmon product injected with different concentrations of protein hydrolysate made from salmon off-cuts.

Relative sensory characteristics of the different test products with regard to a total of 16 different test parameters were given by experienced test people according to a scale from 1 to 10.

RESULTS Colour For a series of fish species it is desirable to be able to produce a product with good colour characteristics, and it especially important for salmon to achieve or maintain the characteristic red colour. Therefore, colour analyses were taken of fillets injected with protein hydrolysate. The results are shown in the figures 1-6.

Also added to all the samples were an 18% sodium chloride solution and addition of this alone (i. e. without protein hydrolysate) functioned as a control.

Figure 1 shows the instrumental redness (a*) at the fillet surface during 1-42 days cold storage and after 8 months storage in frozen condition of vacuum packed smoked salmon fillets. Each bar represents a mean value of 10 individual colour measurements on a total of 5 fillets, n=5 in each test group.

Figure 2 shows instrumental yellowness (b*) at the fillet surface during 1-42 days cold storage and after 8 months storage in frozen condition of vacuum packed smoked salmon fillets. Each bar represents a mean value of 10 individual colour measurements on a total of 5 fillets, n=5 in each test group.

Figure 3 shows instrumental lightness (L*) at the fillet surface during 1-42 days cold storage and after 8 months storage in frozen condition of vacuum packed smoked salmon fillets. Each bar represents a mean value of 10 individual colour measurements on a total of 5 fillets, n=5 in each test group.

Figure 4 shows instrumental redness (a*) in transversal cuts through the fillet during 1-42 days cold storage and after 8 months storage in frozen condition of vacuum packed smoked salmon fillets. Each bar represents a mean value of 10 individual colour measurements on a total of 5 fillets, n=5 in each test group.

Figure 5 shows instrumental yellowness (b*) in transversal cuts through the fillet during 1-42 days cold storage and after 8 months storage in frozen condition of vacuum packed smoked salmon fillets. Each bar represents a mean value of 10 individual colour measurements on a total of 5 fillets, n=5 in each test group.

Figure 6 shows instrumental lightness (a*) in transversal cuts through the fillet during 1-42 days cold storage and after 8 months storage in frozen condition of vacuum packed smoked salmon fillets. Each bar represents a mean value of 10 individual colour measurements on a total of 5 fillets, n=5 in each test group.

Surprisingly, it was found that application of the protein hydrolysate gave improved colour characteristics for the salmon fillets. The red colour of the fish was especially improved (figure 1), but the lightness of the fish fillet was also improved. Furthermore, in storage the colour remained for a longer time with the addition of protein hydrolysate than for corresponding control tests without protein hydrolysate.

Thus, an aspect of the present invention is a procedure where a protein hydrolysate is applied to improve and/or maintain the colour characteristics and lightness characteristics of the fish fillet.

Keeping quality It is especially important to produce a fillet product with good keeping quality. pH in fillet muscle is used as a parameter to determine the freshness of the fish and breakdown products from bacterial growth will in general lead to a dramatic change in pH.

Figure 7 shows pH values in muscle of smoked salmon fillets, vacuum-packed and cold stored for 6 weeks at 4 °C and stored frozen for 8 months at-30 °C.

Each bar is a mean of 5 fillets, each determined in three parallels.

From the results shown in fig. 7 it can be seen that increasing concentrations of protein hydrolysate has a favourable effect on pH, and thus the fish freshness after different storage times. This is surprising, and constitutes an important aspect of the present invention.

Reduced rancidity Figure 8 shows TBARS values in smoked fish fillets injected with different marinades at the start, after 3 and 6 weeks cold storage and at 8 months storage in frozen condition.

It can be seen in fig. 8 that the rancidity in the fat determined with the TBARS method was significantly lower in products injected with hydrolysate. An increase in TBARS values of about 0.5 to 1.6 units should have been expected due to addition of hydrolysate, instead reductions in TBARS values were measured in all fillets with added hydrolysate.

This test shows surprisingly that the protein hydrolysate reduces the rancidity, and it is expected that addition of protein hydrolysate can replace, or reduce, the use of anti-oxidants as an additive to prevent oxidation.

Sensory characteristics (among them smell, colour, firmness, taste) It is important that a product, to which a protein hydrolysate from fish is added, does not get undesirable characteristics, such as, for example, changed taste or smell. Therefore, a thorough investigation has been carried out where different sensory characteristics have been tested by a test panel of 7-8 trained persons.

The results are given in tables 1 and 2 and it surprisingly appears that parameters such as smell and taste are not significantly changed.

It is especially unexpected that there was no development of a rancid and"old" taste at concentrations of protein hydrolysate below 20% protein.

Table 1 Sensory comparison of four marinated products of smoked salmon fillets at start. Mean values estimated for each sensory characteristic, resulting from ANOVA and Tukey tests. Tests which have the same letter are not significantly different at an alpha level 0.05. N=8 SensorycharacteristiciVl Signifi Contro 1 8% l 12%. ; ; D 20% 18% Protein +,'Protein + Protein + 'NaCI 18% NåCI 18% NaCI 18% NaCI 1. Smell of smoke ns 5. 1a 4. 9a 5. 6a 5. 5a 1. Smell of smoke ns 0. 6a 0. 6a 0. 6a 0. 8a 2. Rancid smell f" 3. Old/differing smefl ns 0. 9a 0. 5a 0. 5a 0. 8a f 3. Old/dlffering smell '4.''Loss of moisture. 4. LQSS Of mOIStUre ns 4. 5a 5. 3a 5. Oa 4. 6a 5. Red colour ; ; * 2. 7a 2. Ob 2. 2ab 2. 2ab 6. Yellow colour 6. 4a 7. 1a 6. 3a 6. 4a 7. Transversat cut * 5. Oab 4. Ob 4. 4ab 5. 2a 8. Firmness ns 4. 7a 4. 5a 4. 6a 4. 4a 9. Taste of smoke lO. Saftta ;, '"' a 3. 6a 1 0. Salt tasto, ns 4. 1 a 3. 9a 3. 9a 3. 6a ns 1. 2a 0. 7a 0. 9a 1. 3a ns 1. 3a 1. 0a 0. 9a 0. 7a 1. Rancid taste . <... ns 5. 6a 6. 0a 5. 9a 5. 7a ., 12. Rancid taste ns 5. 6a 6. Oa 5. 9a 5. 9a 14. Chewing resistance 15. Stickiness ns 3. 6a 3. 9a 3. 6a 3. 6a 15. Sttckmess 16. Aftertaste ns 4. 7a 4. 5a 4. 4a 4. 5a 16. Aftertaste ; D Symbols : *** : p<0. 001, ** : p<0. 01, * : p<0, 05 ns : not significant (p>0. 05)

Table 2 Sensory comparison of four marinated products of smoked salmon fillets at start. Mean values estimated for each sensory characteristic, resulting from ANOVA and Tukey tests. Tests which have the same letter are not significantly different at an alpha level 0.05. N=7 Sensory characteristic Signifi Centre) 8% 1 12% 20% cance 18% Protein + Protein + Protein + NaC) 18% NaCt 18% NaC) 18% Nazi. ns 5 5a 5. 3a 5. 4a 4. 8a 1. 4 Smell ofismoke ; : T ; E, e I 2. Rancid smell ns 0. 5a 0. 5a 0. 4a 0. 3a 2. Rancid smell 3. Old/differingEsmell ns 0. 4a 0. 6a 0. 7a 0. 9a ns 3. Oa 3. 3a 3. 3a 3. 9a 4* Loss of moisture p * 4. 8c 5. 1bc 5. 8ab 6. Oa 5. Red calour * 2. 3a 2. 1ab 1. 8b 1. 9ab 6. Yellow colour ns 6. 6a 6. 6a 7. 1a 7. 2a 7. Transverse Vcut ; t nus 4 1a 3. 8a 4. 5a 4. 7a X 8-Firmness ns 5. 2a 4. 9a 5. 3a 5. Oa 9. Tas'te of smoke ns 4. 6a 4. 3a 4. 3a 4. 1 a 110. Salt taste ns 0. 9a 0. 9a 0. 7a 0. 8a 11. Old/difFering, taste. 12. Rancid taste, ns 0. 4a 0. 6a 0. 4a 0. 4a 12. Rancid-taste ns 6. 9a 6. 8a 7. 1 a 7. Oa 13. Juicy ns 3 2a 3. 4a 3. 3a 3. 4a 14. Chewing resistance 15. Stickiness ns 4. 3a 4. 3a 4. 1a 4. 1a 15. Stickiness 16. Aftertaste ns 5. 1 a 4. 6a 5. Oa 4. 7a Symbols : *** : p<0. 001, ** : p<0. 01, * : p<0, 05 ns : not significant (p>0. 05)