WO1990014438A1

WO1990014438A1 - Method for determining nutritional value of meat

Info

Publication number: WO1990014438A1
Application number: PCT/US1990/002177
Authority: WO
Inventors: Melville A. Thomas
Original assignee: Thomas Melville A
Priority date: 1989-05-22
Filing date: 1990-04-20
Publication date: 1990-11-29
Also published as: AU5643690A

Abstract

The protein content of meat can be established by determining its cholesterol 7 alpha-hydroxylase level and then relating this level to a reference standard. The method can also be employed to detect the presence of chemical residues and biological residues in the meat.

Description

METHOD FOR DETERMINING NUTRITIONAL

VALUE OF MEAT

BACKGROUND OF THE INVENTION

1. Field Of The Invention This invention generally relates to methods for assaying for the nutritional value of meat based upon its relationship with protein content. This invention also relates to the detection of harmful levels of certain chemical residues such as pesticides, herbicides and antibiotics in meat products.

DESCRIPTION OF THE PRIOR ART

(a) Meat Processing Literature. The meat processing industry has long recognized the value of the ability to rapidly determine, and consequently label, the protein content of meat. Such determinations are especially helpful in assuring uniform quality and cost characteristics between different batches of meat. One of the most accurate and widely used methods for determining protein content is the procedure known as Kjeldahl analysis. However, because of the laboratory skills and time required to conduct a Kjeldahl analysis, not to mention the fact that such analysis uses flammable and otherwise dangerous chemicals, the test has not been widely employed in meat plant quality control and cost control operations. This lack of acceptance is particularly true in the context of large meat processing facilities utilizing computerized batch component/cost programming procedures. Such high speed processes require rapid as well as accurate means for determining the nutritional values of various meat products while they are in process.

Consequently, the meat processing industry has turned to other protein content test techniques or related methods which are better suited for use in large scale, computerized meat processing plants. For example, many meat processing plants employ various colorimetric techniques to make protein percentage determinations. Such techniques usually employ reactions between acid orange 12, a monoazo dye, Colour Index Constitution Number 15970 and the protein components of meat which form certain insoluble complexes. These insoluble complexes are then detected and/or colorimetrically evaluated. For example, Acid Change Dye 12, which is the brightest orange in aqueous solution, reacts with meat protein to form certain insoluble complexes. These complexes are removed by filtration and the free non-bound dye concentration is measured colormetrically by difference. The dye bound to the protein is then determined by difference and the dye binding capacity of the meat sample calculated (e.g. by determination of mg. dye bound per mg. of protein). Analysis of duplicate meat samples by other test procedures such as Kjeldahl analysis, are utilized to chart a standard curve relating free dye concentrations to mg. of protein in the sample. This procedure is carried out for each type of meat product for which rapid, accurate colorimetric determination of the protein content is required for on-line production control. The Assoc. Anal. Chem., has adopted this procedure, see Journal of Food Science, Vol. 36, P. 509 (1971). However, sensitivities to certain time and temperature parameters which are inherent in these colorimetric tests, tend to produce inaccuracies that are often unacceptable for many present day, high speed, computerized batch formulations of processed meats.

One variation in the Acid Orange 12 tests developed in response to the need for greater speed and accuracy demanded by computerized processes is described in U.S. Patent 3,936,271 (the "271 patent"). The 271 patent teaches that in order to insure reaction of Acid Orange 12 with the protein of the meat sample, the sample must be macerated in the presence of an aqueous solution of a weak organic acid, so as to emulsify the meat protein on the acid side of the isoelectric point. This step further facilitates binding of the acid dye with the protein. The preferred solution is a 0.1m reagent grade aqueous citric acid solution. However, the minimum end point temperature at which the disclosed emulsification- acidification is accomplished remains highly significant, if not crucial, to the accuracy of this particular test. Hence, a protein determination process which would not be so temperature, sensitive would tend to produce more consistent and accurate results.

In summary then, it could be said that all of the foregoing colorimetric methods for determining protein percentages in meats contain drawbacks in terms of their time requirements, accuracy, technical skill requirements, turn around times and temperature sensitivities. Consequently, the need still remains for more simple, rapid and accurate tests for meat nutrition characteristics, particularly those carried out in the context of large scale, computerized meat processing plants.

(b) Medical Literature. It has long been recognized that cholesterol 7 alpha-hydroxylase is linked to: (1) biosynthesis of bile acids, (2) regulation of absorbic acid, (3) regulation of plasma lipoprotein levels and (4) tri-glyceride and lipoprotein metabolism.

SϋMMARY OF THE INVENTION

This invention generally involves the use of cholesterol 7 alpha-hydroxylase to empirically determine the nutritional value of meat products by correlation of the 7 alpha-hydroxylase concentration and protein content of the meat. That is to say that it is generally accepted that higher nutritional values are associated with higher protein content in meat products and applicant has discovered that the cholesterol 7 alpha-hydroxylase concentration of a meat sample correlates with the protein concentration, and hence nutritional value of the meat sample. Applicant has also discovered that there is also a very useful correlation between the chilesterol 7 alpha-hydroxylase and the presence and/or concentration of certain chemical residues often found in meat. The process whereby nutritional value and/or residue level determinations are made, begins with dissolving a meat to be tested in a basic solution. A wide variety of chemical compounds, hereinafter more fully described, having a basic pH can be employed.

The basic solutions employed in the practice of this invention should be capable of dissolving the meat sample without destroying its proteins chemical structure. Preferably basic solutions capable of getting a meat sample into a liquid form, without the need for centrifugation steps are preferred. This permits test personnel to proceed directly from the dissolution to extraction. Representative basic solutions which can be used in the practice of this invention would include, but not be limited to, potassium hydroxide, and most pre-ferably 28% potassium hydroxide, ethanolic potassium hydroxide mixtures. Although this process is capable of being used on meat tissue in general (fowl, fish, beef, lamb, etc.) it is particularly well suited to testing the protein content of beef products. Thereafter, the level of cholesterol 7 alpha-hydroxylase in such a basic solution is determined. Preferably this determination is made by mass ion-monitoring techniques. The level of cholesterol 7 alpha-hydroxylase in the basic solution is then related to a standard reference to arrive at a nutritional value, or arrive at a contamination level. Use of various reference standard comparative techniques well known to the art can be employed. By way of example only, use of an analysis of duplicate meat samples by other test procedures such as Kjeldahl analysis, color chart comparisons, etc. may be used to produce standard curves relating cholesterol 7 alpha- hydroxylase concentration levels to protein content (e.g., to mg of protein contained in a sample portion of the meat). Most preferably these relationships will be determined, and acted upon, by computerized methods known to the art. Analogous test can be designed for specific chemical residues.

The levels of cholesterol 7 alpha-hydroxylase used in the practice of this invention are best measured by various mass ion-monitoring techniques. As previously noted, the most preferred of these techniques will include gas chromatography-mass spectrometry (and most preferably those using cholestane as the internal standard) gas chromatography, column chromatography, paper chromatography, and liquid chromatography. The most preferred of these tests also will be computer controlled chromatography systems. It should also be noted that many of the most commonly used assays techniques for measurement of cholesterol 7 alpha hydroxylase levels in man also can be employed in the practice of this invention. For example, Kienle, M.G., et al, Evaluation of Enxyme Activities by Gas Chromatography 267-276 (1984), 267-276 discloses various methods for detection and evaluation of its metabolic activities which are largely based upon selected ion-monitoring methods. The relative advantages and disadvantages of these known procedures are discussed by Fears, R. and Sabine, Jr., in: Cholesterol 7 alpha-hydroxylase (7 alpha- Monooxygenase) CRC Press, Inc., 1987 (44-47) and these two references are incorporated into this patent disclosure by reference.

The methods by which the levels of cholesterol 7 alpha-hydroxylase in the basic solution can be determined can vary considerably. Ion-moitoring techniques are of course highly preferred; and of these techniques, gas chromatography mass spectrometry, using cholestane as internal standard, is the most preferred technique. However, gas chromatography, HPLC chromatography and/or various computer controlled chromatography systems can also be employed.

Again, the reference standard or curve to which the level of cholesterol 7 alpha-hydroxylase in the basic solution is compared can be established by various known methods such as Kjeldahl analysis, colorimetric determinations of the percentage of protein in meats, etc. However, because of the acknowledged accuracy of Kjeldahl analysis, reference curves established by it are a preferred reference curve to which the cholesterol 7 alpha-hydroxylase cholesterol level vs. protein concentration can be made. Similarly, cholesterol 7 alpha-hydroxylase concentration to chemical residue levels can be established by various appropriate test procedures for residues such as those listed in Table V of this patent disclosure, are well known to the art.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The samples used in the herein disclosed process can be prepared for analysis by various procedures which are within the scope of this patent disclosure. However, one particularly preferred method of preparation runs as follows: one gram of meat tissue sample is extracted in a preliminary extraction with a basic solution such as 5 molar potassium hydroxide. By way of example, one gram of tissue can be dissolved in about one gram (or one amount) of 5m potassium hydroxide. This material is then vortexed and incubated at 60 degrees C. for about 10 minutes to allow precipitation of the tissue. A C-18 extraction column is prepared for use in this test as follows. First the column should be washed one time in an extraction solution of the type known to this art. One particularly useful extraction solution can be prepared by piping 10.0 ml of Sequanal grade (Pierce Chemical Company) into a 100 ml Class "A" volumetric flask containing approximately 900 ml of HPLC grade methanol. It is then diluted to the mark with methanol. Such a solution will usually remain stable for approximately 6 months. Volumes of 100 ml or less may be stored at room temperature in the working area. These volumes should be stabilized for approximately one week. In any case, the column should be then washed twice with methanol, three times with water, and once with acetonitrile. All common volumes are employed.

After the column is washed, 200 microliters of a buffer for liquid chromotography of the type known to this art is added to the prepared column. One particularly useful buffer was prepared by Applicant as follows: 2000 ml of deionized or HPLC grade water was measured into a suitable vessel. Thereafter 239 mcl of reagent grade or better phosphoric acid was piped into the vessel. The two substances were then mixed and filtered through a 0.2 mem nylon 66 membrane filter. The resultant composition was then stabilized for one month at room temperature and then for 3 months at 5-20 degrees C., or until turbid. To the one amount of the sample from step one, that is to the sample prepared with the 5m potassium hydroxide, there was added two microliters of the internal standard, at a concentration of one milligram percent. A wash extraction step then was carried out. It involved washing the extraction column, containing the sample, in the following manner. First, it is washed once with the buffer, once with water, and once with 500 microliters of acetonitrile.

The compound was then eluted off the column. This is preferably done by eluting from the extraction column one time, with 500 microliters of methanol to give a total column of 2,000 microliters. The resulting solution was then evaporated gently, and reconstituted with methanol. It was then ready for analysis. This is most preferably done by known ionmonitoring techniques.

Computer Considerations

It is well known in this art that the various kinds of chromatographic systems may use a dedicated low-cost computer to control all analyzer functions and perform all data reductions. For example, a computer can monitor each individual chromatographic detector, perform integration of peak areas, correct for zero offset and drift, identify components from elution times, apply response factors, and compute the composition of the sample by normalization of peak areas or by comparison with calibration standards for select components. Partial or complete stream analysis may be printed out on a teletypewriter, continuously, or on demand, in accordance with program format. The computer also may simultaneously provide analog trend output to conventional recorders for closed-loop control and communicate analysis results to a larger supervisory computer (e.g., for material balance calculations or for direct digital control). High and low level alarms for select components may also be set by the computer. The computer program may also include sub-routines for detecting and alarming in case of mal-function failure in the chromatograph analyzers.

Methodology

(Protein Level). In order to relate the cholesterol 7 alpha- hydroxylase levels to the protein content, as determined by Kjeldahl analysis, various cuts of beef from selected animals previously fed a prepared diet, e.g., New York Strip, Top Sirloin, Rib Eye, Tenderloin Beef Burgers, Breakfast Steak, and Chopped Beef were analyzed for protein content of the tissue. Representative blood samples in the respective animals also were analyzed for the same component. Activities for the cholesterol 7 alpha hydroxylase were measured in both the blood and the tissue. The results are presented in Tables I, II, III and IV which are found at the end of these specifications. It should also be noted that the values shown in Tables I, II and III were analyzed by a statistical program. Table IV gives the number of determination measurements, the mean, the standard deviation and the coefficient of variance percentage (CV%) for the data shown in Columns A through G. Calorie content was done by a bomb colorimetry wherein the samples washed and the heat generated measured. Fat content in the tissue also was measured by sohxlet method or equivalent.

Residue Detection. Chemical residue assay (s) also were conducted on both the tissue and on the blood of the animal. Chemical residue in the context of meat normally implies the presence of certain foreign chemical substances such as:

1. Antibiotics

(a) Aminoglycosides, e.g., gentamicin (b) Avermectins - Ivermectin

(c) Chloramphenicol

(d) Tetracyclines

2. Anthelmentic Invermectins

(a) Albendazole

(b) Fembendazole

3. Hormone/Steroid Compounds & Combinations

(a) Progesterone Preparations

(b) Estradiol Preparations

(c) Diethyl Stilbestrol (DES)

4. Sulfonamide

(a) Sulfamethizine

(b) Sulfathiazole

(c) Sulfamerazine

5. Chemical Contaminants

(a) Organophosphate

(b) Organochloride By way of further qualitative and quantitative examples, the most commonly encountered residues in meat products generally fall into the following classes of compounds at the levels of sensitivity indicated.

MRS (Multiple Residue Screen) 1000

TEST ID CHEMICAL RESIDUE SENSITIVITY

5.001 Clorinated Hydrocarbon Pesticides

Aldrin 20ppb

BHC 10ppb

Chlordane 150ppb

Dieldrin 10ppb

DDT 40ppb

DDE 20ppb

TDE (DDD) 40ppb

Endrin 30ppb

Heptachlor 10ppb

Heptachlor epoxide 10ppb

Lindane 10ppb Methoxychlor 150ppb

Toxaphene 500ppb

PCBs 300ppb

HCB 10ppb

Nonachlor 50ppb

Mirex 40ppb

Strobane 500ppb

5.006 Organophosphate Pesticides

Dioxathion 100ppb

Diazinon 100ppb

Methyl Parathion 100ppb

Fanitrothion 100ppb

Malathion 100ppb

Ethyl Parathion 100ppb

Ruelene 100ppb

Gardona 100ppb

Ethion 100ppb

Trithion 100ppb

Coumaphos 100ppb

5.015 Zeronal (Ralgro) 1ppb

5.200 Trenbolone 1ppb TEST ID CHEMICAL RESIDUE SENSITIVITY

5.040 Melegesterol Acetate (MGA) 10ppb

5.018 Sulfonamides

Sulfamethazine 70ppb Sulfadimethoxine 70ppb Sulfabromomethazine 70ppb MRS (Multiple Residue Screen) 1000

5.018 Sulfonamides (Continued)

Sulfamethoxypyridazine 70ppb Sulfaethoxypyridazine 70ppb Sulfachloropyridazine 70ppb

5.022 Chloramphenicol 30ppb

5.031 Tetracycline Residue

Oxytetracycline 50ppb Chlortetracycline 50ppb

Tetracycline 50ppb

5.034 Benzimidazoles

Albendazole 25ppb

Fenbendazole 25ppb Mebendazole 25ppb

Oxfenbendazole 25ppb

5.035 Ivermectin 8ppb 5.048 Penicillin G 10ppb

Again, detection of the above noted chemical residues, can be carried out by any of the following test procedures.

1. Thin layer Chromatograph (TLC)

2. Gas Chromatography (GC)

3. Ultra Violet Scanning (UV Spec)

4. High performance Liquid Chromatography (HHC)

5. GC/Mass Spec (GC/MS)

6. Liquid Chromatography/Mass Spec (LC/MS) Further Testing Considerations

Several considerations are commonly used to define and/or respond to residue drugs and chemicals in food products such as meat. Analytical sensitivity of residue (s) by appropriate conventional laboratory methods, e.g., TLC/GC, HPLC, etc., are generally established. For example, a least detectable limit

(LDL) of less than (LT), say, 20 parts per billion

(ppb) may be established. Other considerations may be operative as well. For example, violative levels dictated by legal definition (e.g., Ford Safety Inspection Service of the United States Department of Agriculture have established various allowable levels in a given food).

In all cases where drugs and/or chemical residues were at the nonviolative level all determinations made by the herein disclosed methods were within ± 3.0 standard deviations of the meat value. In all cases where a violative level has been detected, protein concentrations vs the 7 alpha- hydroxylase levels fell outside of the acceptable statistical deviations from the meat. That is to say the violative levels of 50 ppb or greater corresponded to an abnormal protein and 7 alpha- hydroxylase con-centration as indicated by the tables.

Hence, this data clearly shows that a direct correlation between the protein concentration and the alpha 7 does exist. Therefor, from the foregoing description one skilled in this art can determine, by high speed computer processes, the percentage of protein of meat products. Those skilled in this art will also appreciate that, without departing from the spirit and scope of this patent disclosure, various changes and modifications of the non-critical aspects of this invention can be made.

Claims

This having disclosed my invention, I claim:

1. A method for determining the protein content of meat, said method comprising the sequential steps of:

dissolving a sample of meat whose nutritional value is to be determined in a basic solution;

determining the level of cholesterol 7 alpha-hydroxylase in the basic solution; and relating the level of cholesterol 7 alpha-hydroxylase in the basic solution to a standard reference to arrive at a protein content.

2. A method for determining the protein content of beef, said method comprising the sequential steps of:

dissolving a sample of beef whose nutritional value is to be determined in a potassium hydroxide solution;

determining the level of cholesterol 7 alpha-hydroxylase in the potassium hydroxide solution by isolation of the cholesterol 7 alpha-hydroxylase from the potassium hydroxide solution by procedures selected from the group consisting of TLC, gc, UVspec., HPLC, gc/ms and LC/MS and relating the level of cholesterol 7 alpha-hydroxylase in the potassium hydroxide solution to a standard reference to arrive at a nutritional value based upon protein content of the beef.

3. A method for determining the protein content of beef, said method comprising the sequential steps of:

dissolving a sample of beef whose nutritional value to be determined in a 5m potassium hydroxide solution;

determining the level of cholesterol 7 alpha-hydroxylase in the potassium hydroxide solution by isolation of the cholesterol 7 alpha-hydroxylase solution by liquid gas chromatography mass spectrometry; and

relating by computer added comparisons, the level of cholesterol 7 alpha-hydroxylase in the 5m potassium solution to a standard reference to arrive at a nutritional value based upon protein content of the beef sample.

4. A method for detecting the presence of residues in meat, said method comprising the sequential steps of:

dissolving a sample of meat suspected of containing contaminants in a basic solution;

determining the level of cholesterol 7 alpha-hydroxylase in the basic solution; and relating the level of cholesterol 7 alpha-hydroxylase in the basic solution to a contamination standard reference to arrive at a level of contamination.

5. A method for detecting the presence of residue selected from the group consisting of anti- biotics, antiparasitic agents, hormone/steroid compounds, sulfa drugs chemical contaminants and pollutants in beef method comprising the sequential steps of:

dissolving a sample of beef suspected of containing contaminants in a potassium hydroxide solution;

determining the level of cholesterol 7 alpha-hydroxylase in the potassium gydroxide.