EP2041578A1

EP2041578A1 - Method for qualitative detection of added proteins in foods

Info

Publication number: EP2041578A1
Application number: EP07785818A
Authority: EP
Inventors: Detlef Timmermann; Meinhard Behrens; Willpeter Doderer
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-07-03
Filing date: 2007-06-13
Publication date: 2009-04-01
Also published as: WO2008003388A1; DE102006030976A1

Abstract

According to the invention, a method is disclosed for the qualitative detection of added proteins in foods, in particular added protein hydrolysates or blood plasma in meat and meat products, wherein the foods are separated at the sample preparation stage into precipitate and fluid phases, the method being characterized in that at least one part of the fluid phase and/or at least one part of the precipitate phase is hydrolysed, and the released amino acids obtained in this manner are characterized qualitatively and quantitatively, the sample contents of these released fluid-phase and/or precipitate-phase amino acids being compared with similar sample contents originating from the corresponding foods which have not been treated with protein hydrolysates and/or blood plasma.

Description

Method for the qualitative detection of added protein substances in food

The invention relates to a method for the qualitative detection of added protein substances in foods, in particular of added protein hydrolysates or blood plasma in meat and meat products, wherein the foods are separated in the sample preparation in precipitate and fluid phase.

Food chemistry is a branch of applied chemistry that focuses on the composition, function of food and their components. The monitoring of manufacturers, trade and transport of foodstuffs and the proof of possible deception and misleading of the consumer are priorities of this area. Qualitative and quantitative analyzes of foods for authorized or prohibited food additives, residues of pesticides, fertilizers, solvents, antibiotics, hormones and heavy metals have been well known for many years.

The globalization of interstate commerce, which also affects the food industry to a great extent, requires reliable and reproducible analytical methods. Analytical needs exist for the entire chain from the manufacturer, the want to check its raw materials, about the trade that wants as many and reliable quality parameters when purchasing its products, to the consumer who wants to buy quality and authentic products.

Protein-containing additives in the form of high molecular weight meat powders or protein hydrolysates have been around for over 30 years and are originally from Asia. They must not be used according to the meat regulation in meat products. Excluded are vegetable proteins, gelatin for well-defined meat products and spices, which must comply with their definition of total nitrogen content and amino acid nitrogen content. When using such additives, a declaration in the list of ingredients is mandatory in any case. The use of blood plasma is also permitted only for a few and well-defined meat products, with the restriction of use being precisely regulated in the Guidelines of the Meat Ordinance. Approved products must also declare the use of blood plasma in the ingredients list.

Inadmissible additions of protein hydrolysates and blood plasma as well as declaration violations affect many products of the meat and sausage sector. By using protein hydrolysates, water can be additionally introduced into the product without the protein-water ratio shifting completely. Especially with sausages, the nitrogen balance can be shifted upwards, so that it is possible to reduce the proportion of value-determining muscle meat. Blood plasma, which naturally has a high water-binding capacity, also enables exceptional water inputs into a wide range of -products. Thus, for example, the production losses can be reduced or even overcompensated by cooking in cooked ham and other cooked cured products, that is to say that the end products are heavier than the starting products before cooking.

DE 195 16 077 C1 discloses a method for the qualitative detection of added protein hydrolysates in food, especially of added animal and vegetable protein hydrolysates in meat and meat products, which is characterized in that the food is processed to free amino acids containing samples and in the processed samples existing free amino acids are determined qualitatively and quantitatively, wherein the determined sample contents of free amino acids are compared with such sample contents, which come from the corresponding, not with protein hydrolysates treated food.

With the help of such a method, it is indeed possible to make already partially specific statements about additions of protein hydrolysates in meat and meat products, but these statements are incomplete in not a few cases, so that there is still a need for further and more accurate analysis in this Area exists.

From the state of the art is thus until today no highly reliable and broadband applied analysis method for the determination of added animal and vegetable protein hydrolysates in Lebens-. mittein, especially meat and meat products known. Also, the detection of pig or bovine blood plasma is also not possible.

The problem on which the invention is based thus results in a method for the qualitative analysis of the presence of added protein hydrolysates and of blood plasma in foods, which detects a large number of such supplements in a safe and broadband manner.

This problem is solved according to the invention by a method according to claim 1.

In the method according to the invention, the foodstuffs, in particular meat and meat products, are separated into precipitate and fluid phase during sample preparation. This is done, for example, and in particular by comminuting a sample and homogenization with water, the homogenate is mixed with sulfosalicylic, so that high molecular weight proteins within a certain time at a certain temperature (for example, 30 minutes at + 4 ° C in the refrigerator) precipitate and subsequently the sample is filtered with a paper filter, leaving the precipitated proteins as filter residue as the precipitate phase, while the filtrate is present as such as a fluid phase. The fluid phase contains free amino acids and di- to oligopeptides, wherein according to the invention at least a portion of the fluid phase is hydrolyzed or can be, for example and preferably by means of 6N HCl, so that ultimately only free and released amino acids in the fluid Phase are present. The released amino acids obtained in this way are determined qualitatively and quantitatively, the determined sample contents of these fluid-released amino acids being compared with those sample contents which originate from the corresponding foods not treated with protein hydrolysates and / or blood plasma. Moreover, it is also possible that the released amino acids previously obtained as free amino acids as well as by the hydrolysis, which are now free in the fluid phase, in the sum of qualified tatively and quantitatively, wherein to analyze to what extent and how much amino acids have been released by the hydrolysis of the fluid phase, this can be done by means of a counter-control of a part of not yet subjected to the hydrolysis fluid phase, to the corresponding difference of the individual To recognize amino acids, which are present and in what amount of the amino acids released by the hydrolysis and which free amino acids and at what level were present in the fluid phase from the beginning.

According to the invention, at least part of the precipitate phase can be hydrolyzed and the released amino acids obtained in this way can be determined qualitatively and quantitatively, the determined sample contents of these precipitate-released amino acids being compared with those sample contents obtained from the corresponding protein hydrolyzates and / or blood plasma-derived foods.

Furthermore, it is advantageous to determine qualitatively and quantitatively the free amino acids originating from at least part of the fluid phase, the determined sample contents of these free amino acids being compared with those sample contents which are from the corresponding ones not treated with protein hydrolysates and / or blood plasma Food for more information about the food.

Among other things, essential to the invention is the fact that, in contrast to the method indicated in DE 195 16 077 C1, the fluid phase or precipitate phase is hydrolyzed to the di- to oligopeptides still in the fluid phase or precipitate phase break into their individual amino acids to provide this as a further basis for the later correlation. let the change in the addition of protein hydrolysates in the hands to keep.

With respect to correlative evaluations, it could be found that, when adding animal protein hydrolyzate, at least one of the following amino acids changed the concentration in the hydrolysed fluid phase compared to an untreated sample, the amino acids being aspartic acid, Threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, proline, hydroxyproline and hydroxylysine.

The same applies in an advantageous manner, as found in practice and thus proven that in the investigation of added vegetable protein hydrolyzate in the hydrolyzed fluid phase, at least one of the following amino acids is taken into account, thus their concentration changes: aspartic acid, threonine, serine, glutamic acid, Glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine and proline.

Moreover, it is advantageous since found and thus proven in practice that when investigating for added animal protein hydrolyzate in the precipitate phase at least one of the following amino acids is taken into account: hydroxyproline and hydroxylysine.

It could moreover be found that when pure vegetable protein hydrolyzate was added to the food in the precipitate phase, there were no significant changes in amino acid concentrations.

However, it is advantageous because this could be found and thus proven in practice that in the investigation of added blood plasma in the Fluid phase hydroxylysine is considered, as this indicates an indication of the addition of blood plasma in the food.

Due to the partially summary but diverse type and concentration changes of the individual amino acids can be made by combining correlation analysis of the individual amino acids and their concentrations corresponding conclusions by comparing untreated with untreated reference samples and deliberately treated samples finally at least qualitative statements, which then in a corresponding unknown Food sample via the previously known correlation rules quasi-retro-analytically statements in qualitative terms about purity or addition of protein hydrolysates - both animal and vegetable origin - allow.

In the method according to the invention, the foods to be examined are processed in the manner according to the invention via the corresponding separation and hydrolyzing steps to free and released amino acids containing samples, which are then subjected to a corresponding amino acid determination to the free and the released amino acids and their contents determine. The sample preparation as well as the amino acid determination proceed according to conventional methods. For example, the foodstuffs are first accurately weighed, diluted with distilled water, homogenized and then the fluid phase as well as the precipitate phase with the free or released amino acids contained ion exchange-chromatographically separated and after color reaction with ninhydrin or ortho-phthalene - Dialdehyde detected. This process is usually automated. Of course it is also conceivable that the amino acids z.Bsp. Phenylsenföl, FMOC, dabsyl chloride or Dansylclorid, derivatized and subjected to liquid chromatography, in particular a HPLC (High Performance Liquid Chromatography) with reversed-phase characteristic. The chromatograms obtained are evaluated both qualitatively and quantitatively. The amounts of the free or released amino acids obtained from the individual samples, which are obtained from foods treated with protein hydrolysates, are compared with those sample quantities which originate from corresponding untreated foods, to which no protein hydrolysates have been added. If some amino acid contents exceed the values determined from the corresponding, untreated food samples, this is at least an indication of an inadmissible addition of protein hydrolyzate, but not yet a proof, since the species occurring in the amino acids are always subject to a certain fluctuation range. However, when comparing the determined contents of the individual amino acids, it was surprisingly found that individual amino acid contents remain approximately constant despite the addition of protein hydrolysates in foods, in particular meat and meat products, preferably serine and aspartic acid in the case of protein hydrolyzate supplements of animal origin, while other amino acid contents are significant alter, preferably glycine and alanine. This surprising finding thus allows at least the direct comparison of the occurring in a natural range of fluctuation, but in Eiweißhydrolysatzusatz significantly changing amino acid content with empirically determined values to draw conclusions on possible additives in the form of protein hydrolysates or allowed, if without protein Hydrolyzate staggered corresponding foods are available, even the comparison of the determined sample contents in Eiweißhydrolysatzugäbe were quantitatively significantly changing amino acids with sample contents of these amino acids derived from not treated with protein hydrolysates foods. Due to the significance of the rating used for the assessment, it is possible to make a reliable statement about supplemental protein hydrolyzate.

Due to the fact that certain amino acid contents change significantly during addition of protein hydrolyzate of animal origin, while others remain virtually unchanged, it is expedient, for an addition of protein hydrolysates of animal origin, sample contents of free as well as released amino acids which increase with the addition of free or Released amino acids, which remain virtually unchanged by the addition, to make the comparison in relation. In particular, glycine and alanine significantly change in protein hydrolysates of animal origin while serine and aspartic acid remain nearly constant. The quotients are formed in such a way that a strongly varying amino acid content upon addition is divided by a virtually independent amino acid content, whereby the reciprocal value of such a quotient can also be used. The quotient formation makes it possible to identify samples conspicuously treated with animal protein hydrolysates whose amino acid contents are still within the natural fluctuation range despite inadmissible additions and thus also that these limiting cases are reliably recorded.

Since the addition of vegetable protein hydrolysates increases almost all levels of free amino acids. men, it is appropriate that for an addition of vegetable protein hydrolysates sample contents of substantially all free amino acids are used to carry out the comparison. This is not in contradiction to the fact that in the investigation on added vegetable protein hydrolyzate in the hydrolyzed fluid phase at least one of the following amino acids is taken into account, which are: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, Valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine and proline. The same applies in connection with the examination of samples for added animal protein hydrolyzate in the hydrolyzed fluid phase, which are the following potential amino acids, which are to be considered: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, Leucine, tyrosine, phenylalanine, lysine, arginine, proline, hydroxyproline and hydroxylysine.

When testing for added animal protein hydrolyzate in the precipitate phase, the amino acids hydroxyproline and hydroxylysine, at least one of them, should advantageously be taken into account. The same is not possible in the investigation on added vegetable protein hydrolyzate in the precipitate phase due to the lack of clarity with respect to individual amino acids.

In this connection, as an indicator of the addition of a possible blood plasma, it is considered that the changes in the amino acid hydroxylysine are taken into account in the fluid phase, in which case a test with a correspondingly specific antibody must be carried out for the final verifying determination. In order to increase the reproducibility of the results of the method according to the invention, it is expedient that the free amino acids contained in the food and the amino acids still to be released from the di- to oligopeptides are separated from the proteins contained in the foods during sample preparation, so that the proteins can not interfere with the amino acid determination, the resulting precipitate phase according to the invention then even with appropriate treatment in relation to the then released amino acids can provide even more valuable information.

Advantageously, the proteins contained in the food are first precipitated from the food before separation from the free amino acids or the di- to oligopeptides. It is also advantageous that the precipitated proteins are filtered off from them before the determination of the free amino acids or of the amino acids released from the fluid phase in order not to induce disturbances, and it is also advantageous in this context since in practice Proves that prior to filtration, the proteins and the free amino acids or di- to oligopeptides are subjected to a centrifugation.

Finally, it has been proven in practice that the hydrolysis of both the fluid phase and the precipitate phase is accomplished by the addition of HCl, preferably 6N-HCl.

The detection method according to the invention is economically interesting, since no additional equipment specially developed for the method is necessary, so that the devices located in a laboratory park can be used.

The shown in the drawings and in the tables te example to explain the invention in more detail.

Show it:

FIG. 1 is a schematic flow diagram of the amino acid analysis according to the invention;

Fig. 2 + 3 - respective amino acid chromatograms of samples without or with protein hydrolyzate additive,

Tab. 1-10 - the respective amino acid concentrations without or with respective additives (x a mean value, see standard deviation).

The following samples were prepared for the experiments explained below:

1. Chicken breast fillet (fresh meat) without and with the addition of 1% and 3% of animal protein hydrolyzate (poultry)

2. Chicken breast fillet (fresh meat) without and with the addition of 1% and 3% vegetable protein hydrolyzate (soy).

3. Ham without and with the addition of 1% and 3% animal protein hydrolyzate (pig).

4. Boiled ham without and with the addition of 1% and 3% vegetable protein hydrolyzate (soy).

5. Boiled ham without and with the addition of 1% and 3% dried blood plasma (pig).

6. Boiled sausage without and with the addition of 1% and 3% dried blood plasma (bovine).

The various protein substances were mixed with water in the ratio 1: 4.

In this procedure, the food samples are processed and analyzed in a specific manner.

The sample is crushed with a Moulinette or similar nert. Then, weigh 5 grams of crushed sample material to the nearest lmg with 35 ml distilled water. homogenized in a beaker using an Ultra Turrax. For protein precipitation, 10 ml of 105% sulphosalicylic acid are added to the sample and mixed. To precipitate high molecular weight proteins, the sample is incubated for 30 minutes at 4 ⁰ C in the refrigerator. Subsequently, the sample is filtered with a paper-folded filter. In principle, two fractions are formed, namely a fraction with medium to high molecular weight proteins (called precipitate, since larger proteins precipitate on addition of sulfosalicylic acid) and another fraction in which free amino acids, dipeptides and oligopeptides are present. This fraction is called the NPN fraction. This NPN fraction is to some extent doubly analyzed in the present process. On the one hand, the per se free amino acids are determined in their concentration, on the other hand, all other nitrogen components are decomposed by acid hydrolysis (6N HCI addition) into free or released amino acids. The precipitate fraction is also decomposed by acid hydrolysis into released or free amino acids. Overall, therefore, three determinations of the concentration of free or released amino acids take place. Two determinations from the NPN fraction and one from the precipitate faction. The principle of amino acid analysis is shown schematically in Figure 1.

The determination of the concentration of the respective amino acids is ideally carried out with the aid of an amino acid analyzer or else by means of HPLC (High Performance Liquid Chromatography). The resulting amino acid chromatograms are evaluated both qualitatively and quantitatively. Figure 2 shows an amino acid chromatogram of a prepared sample without protein hydrolyzate additive. The individual peaks are assigned the respective amino acids. The individual peak areas correspond to the respective amino acid amounts. These are compared directly with a prepared sample with protein hydrolyzate additive (Figure 3).

The mean values given in the tables and their variances were determined in each case with n = 6 values using the two-factorial analysis of variance.

The samples from Batch 1 listed in the Tables have not been mixed with protein hydrolyzate. The values given for these batches show a natural fluctuation range of the individual amino acid concentration and serve as comparison parameters for an evaluation with regard to an addition of protein hydrolyzate.

Table 1 shows the amino acid concentration of hydrolyzed NPN in mg / 100g chicken breast fillet (fresh meat) without (lot 1), with 1% (lot 2) and with 3% (lot 3) of animal protein hydrolysis kit (poultry). It can be seen that even a protein hydrolyzate addition of 1% leads to a significant change in the amino acid concentration. The mean comparisons show that the amino acids asparagine acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, proline, hydroxyproline and hydroxylysine from batches 2 and 3 with animal protein hydrolyzate additive Compared to batch 1 (without animal protein hydrolyzate supplement) have significantly increased concentrations.

The amino acid concentration of hydrolysed precipitate (Table 2) shows in the averaging only a significant increase in the concentrations of the amino acids hydroxyproline and hydroxylysine. Both amino acids are reliable indicators of protein hydrolyzate supplements of animal origin for this fraction.

With an addition of 1% and 3% vegetable protein hydrolyzate (soy), Table 3 shows a significant increase in the concentrations of the amino acids aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine in the mean value comparisons of the hydrolyzed NPN , Leucine, tyrosine, phenylalanine, lysine, arginine and proline.

No changes in amino acid concentrations were noted with the hydrolyzed precipitate (Table 4).

Table 5 shows the amino acid concentration of hydrolyzed NPN in mg / 100g cooked ham without additive

(Lot 1), with 1% (lot 2) and with 3% (lot 3) of animal protein hydrolyzate supplement (pig). Even here, a small addition of protein hydrolyzate of 1% leads to a significant change in the amino acid concentrations. The averages show that the amino acids aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, proline, hydroxyproline and hydroxylysine from batches 2 and 3 with animal protein hydrolyzate additive compared to Batch 1

(without animal Einweißhydrolysatzusatz) have significantly increased concentrations.

The amino acid concentrations of hydrolysed precipitate (Table 6) show significant increases only in the case of the amino acids hydroxyproline and hydroxylysine in the averaging comparisons, just as with the addition of animal hydolyte with poultry origin the concentrations.

With the addition of 1% and 3% vegetable protein hydrolyzate (soy), Table 7 shows a significant increase in the concentrations of the amino acids aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine in the averaging of NPN , Isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine and proline.

No changes in amino acid concentrations were noted for the hydrolyzed precipitate (Table 8).

The differences in the values between the amino acid concentrations of the hydrolyzed chicken breast fillet NPN and boiled vegetable protein supplement (soya) are due to varying degrees of hydrolysis in the production of protein hydrolyzate (see Tables 3 and 7).

Just as protein hydrolyzates of animal and vegetable origin tend to differ in their amino acid spectra, the protein additive blood plasma also leaves some traces. In particular, the amino acid hydroxylysine is a good indicator of the presence of blood plasma in the sample. Tables 9 and 10 show that at the free amino acid concentrations, the amino acid hydroxylysine is significantly increased in the samples with a dry blood plasma addition of 1% and 3%.

The detection method according to the invention is economically very interesting. The problem is international. In addition, the affected meat and convenience area is huge and steadily growing, so there is a large sample potential. Advantageously, the detection method does not require any apparatuses specially developed for the method and equipment. Many laboratories already have adequate equipment due to other analytical methods. If not, the corresponding equipment components can easily be obtained from the laboratory retailer. The detection method can thus be established relatively quickly as routine analysis.

Claims

claims

1. A method for the qualitative detection of added protein substances in food, especially of added protein hydrolysates or blood plasma in meat and meat products, the food in the sample preparation in precipitate and fluid phases are separated, characterized in that at least a portion of the fluid phase and / or at least a portion of the precipitate phase is hydrolyzed and the released amino acids obtained in this way are determined qualitatively and quantitatively, the determined sample contents of these amino acids released in fluid phase and / or precipitate phase being compared with those sample contents obtained from the corresponding , not with protein hydrolysates and / or blood plasma-treated foods.

2. The method according to claim 1, characterized in that the originating from at least a portion of the fluid phase free amino acids are determined qualitatively and quantitatively, wherein the determined sample contents of these free amino acids are compared with those sample contents from the corresponding, not with protein hydrolysates and / or blood plasma-derived foods.

3. The method according to any one of claims 1 to 2, characterized in that for an addition of protein hydrolysates of animal origin sample contents free Amino acids that increase with the addition are correlated with sample contents of free amino acids that remain virtually unchanged by the addition to perform the comparison.

4. The method according to any one of claims 1 to 3, characterized in that sample contents of substantially all free amino acids are used to carry out the comparison for an addition of vegetable protein hydrolysates.

5. The method according to any one of claims 1 to 4, characterized in that in the sample preparation, the free amino acids contained in the food are separated from the proteins contained in the food.

6. The method according to any one of claims 1 to 5, characterized in that the proteins contained in the food before separation from the free amino acids are first precipitated from the food.

7. The method according to any one of claims 1 to 6, characterized in that the precipitated proteins are filtered off before determining the free amino acids of these.

8. The method according to any one of claims 1 to 7, characterized in that prior to filtering the proteins and the free amino acids are subjected to centrifugation.

9. The method according to any one of claims 1 to 8, characterized in that the food by means would be separated from suifosalicylic acid in precipitate and fluid phases.

10. The method according to any one of claims 1 to 9, characterized in that the hydrolysis of the fluid phase is accomplished by the addition of HCl.

11. The method according to any one of claims 1 to 10, characterized in that the hydrolysis of the precipitate phase is accomplished by adding HCl.

12. The method according to any one of claims 1 to 11, characterized in that in the investigation of added animal protein hydrolyzate in the hydrolyzed fluid phase at least one of the following amino acids is considered: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, Isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, proline, hydroxyproline and hydroxylysine.

13. The method according to any one of claims 1 to 12, characterized in that in the investigation of added vegetable protein hydrolyzate in the hydrolyzed fluid phase at least one of the following amino acids is considered: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, Isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, proline.

14. The method according to any one of claims 1 to 13, characterized in that at least one of the following amino acids is considered in the investigation on added animal protein hydrolyzate in the Präzipi- tatphase: hydroxyproline, hydroxylysine.

15. The method according to any one of claims 1 to 14, characterized in that is considered in the investigation of added blood plasma in the fluid phase hydroxylysine.