rapid method for the determination of meat in food products

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RAPID METHOD FOR THE DETERMINATION OF MEAT IN FOOD PRODUCTS G. R. SKURRAY,lk V. A. LYSAGHT School of Food Sciences, Hawkesbury Agricultural College, Richmond, New South Wales 2753, Australia (Received: 25 March, 1977) ABSTRACT A method for the determination of meat infoodproducts is described based on the determination of protein-bound 3-methylhistidine. The method involved high pressure liquid chromatography and reduced the elution time of previous methods from 7 h to 10 min. The accuracy of the method was increased by using electronic integration. The method was used to determine the meat content of nine meat pies and three hamburger patties. INTRODUCTION It has been established that the amino acid 3-methylhistidine occurs in the myofibrillar tissue of animals (Johnson et al., 1967). Hibbert & Lawrie (1972) suggested the use of 3-methylhistidine as a possible indicator of meat protein content because it was heat stable and was thought to be present at similar levels in animal tissue. A significant correlation was found between the 3-methylhistidine content and the percentage of meat in mixtures of beef and soya protein that had been sterilised in cans. Further studies by Rangeley & Lawrie (1976) showed that there was a similar concentration of 3-methylhistidine in lamb and beef muscle but the concentrations in pork were higher and varied markedly between samples. This variation was found to be due to a dipeptide containing 3-methylhistidine in pork and could be removed by washing the sample with distilled water. The method of Rangeley & Lawrie (1976) involved experimental errors of the order of 10 ~ involving calculation of chromatographic peak areas. Furthermore, the elution time was of the order of 7-10 h. High pressure liquid chromatography offers the advantages of speed, low running costs and accurate electronic integration of peak areas. 111 Fd. Chem. (3) (1978)---O Applied Science Publishers Ltd, England, 1978 Printed in Great Britain

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Page 1: Rapid method for the determination of meat in food products

RAPID METHOD FOR THE DETERMINATION OF MEAT IN FOOD PRODUCTS

G. R. SKURRAY ,lk V. A. LYSAGHT

School of Food Sciences, Hawkesbury Agricultural College, Richmond, New South Wales 2753, Australia

(Received: 25 March, 1977)

ABSTRACT

A method for the determination of meat infoodproducts is described based on the determination of protein-bound 3-methylhistidine. The method involved high pressure liquid chromatography and reduced the elution time of previous methods from 7 h to 10 min. The accuracy of the method was increased by using electronic integration. The method was used to determine the meat content of nine meat pies and three hamburger patties.

INTRODUCTION

It has been established that the amino acid 3-methylhistidine occurs in the myofibrillar tissue of animals (Johnson et al., 1967). Hibbert & Lawrie (1972) suggested the use of 3-methylhistidine as a possible indicator of meat protein content because it was heat stable and was thought to be present at similar levels in animal tissue. A significant correlation was found between the 3-methylhistidine content and the percentage of meat in mixtures of beef and soya protein that had been sterilised in cans. Further studies by Rangeley & Lawrie (1976) showed that there was a similar concentration of 3-methylhistidine in lamb and beef muscle but the concentrations in pork were higher and varied markedly between samples. This variation was found to be due to a dipeptide containing 3-methylhistidine in pork and could be removed by washing the sample with distilled water.

The method of Rangeley & Lawrie (1976) involved experimental errors of the order of 10 ~ involving calculation of chromatographic peak areas. Furthermore, the elution time was of the order of 7-10 h. High pressure liquid chromatography offers the advantages of speed, low running costs and accurate electronic integration of peak areas.

111 Fd. Chem. (3) (1978)---O Applied Science Publishers Ltd, England, 1978 Printed in Great Britain

Page 2: Rapid method for the determination of meat in food products

112 G.R. SKURRAY, V. A. LYSAGHT

The aim of the present study was to use high pressure liquid chromatography to determine the 3-methylhistidine content of meat and meat products.

MATERIALS AND METHODS

Source of materials All meat samples were prepared from longissimus dorsi muscle of freshly killed

beef or lamb. Meat products were purchased fresh or frozen from local supermarkets and L-3-methylhistidine was obtained from Sigma Chemicals.

Chemical analysis The meat products we.re prepared according to the methods used by Rangeley &

Lawrie (1976). The nitrogen content of the samples was determined by the method described by Pearson (1962). Only the filling portion of meat pies was analysed. Samples were hydrolysed by placing 10 g in 450 ml of6t~ HCI and refluxing for 22 h. The hydrolysate was filtered through sintered glass and evaporated to dryness twice using a rotary evaporator. The hydrolysate was made up to 25ml with 5 % NaHCO 3. After 10min, freshly prepared 1-fluoro-2,4-dinitrobenzene (FDNB) solution (1 ml of FDNB dissolved in 20 ml ethanol) was added. The mixture was stirred and allowed to stand for 18 h and then the ethanol was evaporated on a steam bath. The remaining mixture was extracted twice with 20 ml of diethyl ether to remove DNP derivatives of non-basic amino acids. A 10/~1 aliquot of the mixture was applied to a spherisorb silica (5/am) column (8 x 250 mm) and eluted with degassed, distilled water. A flow rate of 0.5 ml/min was obtained using a Spectra Physics 3500 B high pressure liquid chromatograph with a 250nm ultra-violet detector. Peak areas were determined by an electronic integrator (Linear Corporation). Standard solutions of DNP derivatives of 3-methylhistidine (20-100 mg/litre) were used.

The meat content of the products was calculated from their 3-methylhistidine content relative to skeletal muscle.

RESULTS

The DN P derivative of 3-methylhistidine was separated from the other DN P amino acids by high pressure chromatography (Fig. 1). The 3-methylhistidine content of beef and lamb skeletal muscle was 5.4 and 5.2 (_+ 0-1) mg/gN respectively compared with the value of 6.0 (+0.7) mg/gN obtained by Rangeley & Lawrie (1976).

The protein and 3-methylhistidine content of the commercial samples of meat pies and hamburger patties varied markedly (see Table 1). There was no significant correlation between the protein content of the products and their meat content (r

Page 3: Rapid method for the determination of meat in food products

DETERMINATION OF MEAT IN FOOD PRODUCTS 113

Fig. 1.

IOC

Transmission

('/.) 5o

3- methyl histidine

I I i i "~ 4 6 8 I0

Time (mini

Chromatogram of acid hydrolysate of bovine skeletal muscle showing the separation ofDNP 3- methylhistidine.

- 0.26, p > 0.05) as calculated from their 3-methylhistidine content. The meat pie, sample 4, contained the highest amount of protein but the lowest calculated amount of meat.

DISCUSSION

The 3-methylhistidine contents of longissimus dorsi muscle of beef and lamb obtained by the method described are similar to those obtained by Rangeley & Lawrie (1976). However. high pressure liquid chromatography with electronic integration of peak areas resulted in a chromatographic time of 10 rain with an experimental error of 1.9 % compared with the method ofRangeley & Lawrie (1976) which hacl a chromatographic time of 7 h with an experimental error of 11.7 ~o.

The new method described involves the preparation of a DNP derivative of 3- methylhisfidine. This has the advantage of increasing the sensitivity of the method

Page 4: Rapid method for the determination of meat in food products

a

TABLE 1 PROTEIN, 3-METHYLHISTIDINE AND MEAT CONTENT (WET WEIGHT BASIS) OF MEAT

PRODUCTS

Sample Protein 3-Methylhistidine Meat (g %) (g/gN) (g/lO0 g)

Longissimus dorsi Beef 25-7 5.4 100

Lamb 23.4 5.2 100 Meat Pie

1 8.2 4.1 76 2 4-8 2-3 43 3 5-3 4.5 83 4 11-5 1.9 35 5 6.2 3.4 63 6 6.7 5-2 96 7 5.7 4.5 83 8 6-1 3.4 63 9 6.5 3.8 70

Hamburgers 1 2.5 1-3 24 2 2.3 1.1 20 3 2.8 1-6 30

Standard error of mean 0.08 0-06 0.83

due to the high extinction coefficient of the DNP derivative and the solubility of the DNP 3-methylhistidine in acid solution allows the separation of other DNP amino acids which would interfere with the chromatographic separation.

The lack of correlation between the protein content of the meat products and their meat content calculated from their 3-methylhistidine content suggests that significant amounts of non-skeletal muscle protein were used in their preparation. The hydroxyproline content of the samples would indicate whether the protein was connective tissue or vegetable protein (Skurray & Herbert, 1974) and this would have a significant bearing on the nutritional value of the products (Skurray & Osborne, 1976).

REFERENCES

HIBBERT, 1. & LAWRIE, R. A. (1972). Technical note: The identification of meat in food products. J. Fd. Tcchnol., 7, 333-5.

JOHNSON, P., HARRIS, C. I. & PERRY, S. V. (1967). 3-Methylhistidine in actin and other muscle proteins. Biochem. J., 10S, 361-9.

PEARSON, D. (1962). The chemical analysis of foods, 5th edn. London, Churchill. RANGELEY, W. R. D. & LAWRIE, R. A. (1976). Methylamino acids as indices in meat products. J. Fd.

Technol., 11, 143-59. SKURRAY, G. R. (1976). The nutritive value of non-skeletal meat tissue. Proc. AuNt. Nut. Soc. Conf.,

Sydney. SKUR~Y, G. R. & HERBERT, L. S. 0974). Batch dry rendering: Influence of raw materials and processing

conditions on meat meal quality. J. Sci. Fd. Agric., 25, I071-9. SKUPd~Y, G. R. & OSSO•NE, C. (1976). Nutritional value of soya protein and milk co-precipitates in

sausage products. J. Sci. Fd. Agric., 27, 175--80.