AU6742500A

AU6742500A - Rapid method for measuring complex carbohydrates in mammalian tissue

Info

Publication number: AU6742500A
Application number: AU67425/00A
Authority: AU
Inventors: Alan Leedham Hart; John West; Owen Archibald Young
Original assignee: AgResearch Ltd; New Zealand Meat Board
Current assignee: AgResearch Ltd
Priority date: 1999-08-17
Filing date: 2000-08-17
Publication date: 2001-03-13
Anticipated expiration: 2020-08-17
Also published as: BR0013395A; CA2382155A1; AR025303A1; EP1210451A4; WO2001012844A1; AU779415C; EP1210451A1; AU779415B2

Description

WO 01/12844 PCT/NZOO/00159 TITLE: RAPID METHOD FOR MEASURING COMPLEX CARBOHYDRATES IN MAMMALIAN TISSUE TECHNICAL FIELD 5 The present invention relates to a rapid method for measuring complex carbohydrates, particularly glycogen, in mammalian tissue. More particularly the present invention relates to the rapid measurement of glycogen in non-living mammalian tissue. BACKGROUND ART 10 There are presently a number of methods of measuring complex carbohydrates, and particularly glycogen, in mammalian tissue. The discussion of these follows. The relevance of the measurement of glycogen includes, for example, the ability to use the results of glycogen measurement as a determination of the ultimate pH of meat. This in tum is a direct measure of many of the qualities of meat. 15 There are a number of known methods of measuring ultimate pH in meat: including use of liquid nitrogen in a freeze/thaw process and the use of a pH electrode for pH determination. There are a number of variations of this method, also. However the maintenance and use of liquid nitrogen in the quantities needed for the measurement on a continuous series of carcasses reveals hazards for the work environment. Also there is some doubt as to the 20 accuracy and consistency of such measurement methods.

WO 01/12844 PCT/NZOO/00159 2 Methods of Measurement of Glycogen or Metabolites in Meat Samples The iodine method: The principle of this method is that glycogen will react with a mixture of iodide, iodine and calcium chloride, forming an amber pigment in acid solution that has a linear absorption at least over a small, specified range. The glycogen is extracted from the 5 meat with perchloric acid that is then filtered and centrifuged to recover a solution of glycogen which is reacted with the iodine. The extraction can also be by liquid nitrogen, potassium hydroxide, ethanol and ammonium chloride. However, methods of extraction and then assay are time-consuming and employ aggressive chemical reagents. 10 Hydrolysis of glycogen with enzymes: The principle of this method is that glycogen hydrolyses to glucose, after which standard methods of measurement of free glucose may be used. The amyloglucosidase method of Dreiling et al (Meat Science, Vol 20, p. 167) is one such method, although other enzymes may be used. A muscle sample is homogenised with 15 perchloric acid, and centrifuged, The supernatant, containing dissolved glycogen, is neutralised. Amyloglucosidase is added, converting the glycogen to glucose, for measurement. The first part of the method takes approximately 30 minutes at 37 0 C. However there are some instances with the processing of meat in which a test of some 30 minutes or more is too long a time to wait for the test results, and some reagents are 20 aggressive. It is an object of the present invention to provide a rapid method for glycogen measurement in mammalian tissues, and in particular non-living tissue. It is a further object of the present WO 01/12844 PCT/NZOO/00159 3 invention that in addition to the provision of a rapid accurate measurement of glycogen in tissue, the method also utilise mild or non-aggressive reagents. Whilst the prior art and uses of glycogen measurement have been described with reference to a determination of the ultimate pH in meat, it will be appreciated that this method is not 5 limited thereto. For example, low glycogen levels in the liver of bobby calves are an indicator of inadequate feeding before slaughter. Thus such rapid method of assessment of these levels could assist in ensuring good animal welfare. It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice. 10 Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only. DISCLOSURE OF INVENTION For the purposes of this specification, the term "rapid" is used to refer to times of less than 15 30 minutes and, more preferably, significantly less than 30 minutes According to one aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, said method comprising the steps of: (a) extracting a sample of tissue to be tested; 20 (b) forming a homogenous slurry of the sample with an aqueous solution; (c) adding sufficient hydrolysing enzyme for ensuring complete hydrolysis of WO 01/12844 PCT/NZOO/00159 4 glycogen in the slurry; and (d) measuring the concentration of glucose in the slurry. Advantageously, said complex carbohydrate is glycogen. Advantageously, said aqueous solvent is water. Optionally said solvent may include at least one agent intended to 5 standardise ionic conditions and/or facilitate the steps of the above method. Preferably the formation of the homogenous slurry is effected with a high or a low speed homogeniser, or with an ultrasonic apparatus. According to another aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, substantially as described above, 10 wherein steps (b) and (c) are performed simultaneously. According to another aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, substantially as described above, wherein steps (c) and (d) are performed simultaneously. Advantageously, the hydrolysing enzyme may be any enzyme, or combination of enzymes, L 5 capable of hydrolysis of glycogen to glucose. According to a still further aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, substantially as described above, in which said hydrolysing enzyme is selected from the group: amyloglucosidase; a-amylase; a-glucosidase, and a combination thereof. 0 According to another aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, substantially as described above, WO 01/12844 PCT/NZOO/00159 5 wherein the amyloglucosidase added in step (c) is in a form selected from: a powder; a liquid suspension; and a solution. According to another aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, substantially as described above, in 5 either embodiment, wherein said method further includes a step (e): measuring the concentration of lactate in the sample. According to another aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, substantially as described above, wherein steps (d) and (e) are performed simultaneously. o According to another aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, substantially as described above, wherein steps (c), (d) and (e) are performed simultaneously. The measurement of both metabolites gives a good post-mortem estimate of the concentration of glycogen present in tissue at the time of death, no matter when the 5 measurement is made. Advantageously, the method is conducted at approximately room temperature, although it may be conducted at a temperature in the range 0*C to 100 C. Measurement of Glucose 0 There are a range of methods for measuring glucose. For the present invention the most useful are those adapted from known technologies to measure glucose in blood. These are WO 01/12844 PCT/NZ00/00159 6 usually based on the generation of hydrogen peroxide in stoichiometric proportion to glucose, as catalysed by glucose oxidase. Therefore, according to a still further aspect of the present invention there is provided a rapid method of measuring complex carbohydrates in mammalian tissue, substantially as described 5 above, and in which glycogen levels are measured and in which said measurement of the concentration of glucose is achieved by the construction of sensors incorporating said hydrolysing enzyme and glucose oxidase. Measurement of Lactate 10 There are a range of methods for measuring lactate, including the standard NADH-linked method and those based on the generation of hydrogen peroxide in stoichiometric proportion to lactate, as catalysed by lactate oxidase. This latter compound can be incorporated into any sensor which may already incorporate glucose oxidase. 15 BRIEF DESCRIPTION OF THE DRAWINGS Further aspects of the present invention will become apparent from the following example, which is given by way of example only, and with reference to the accompanying drawings in which: Figure 1 is a graph of glucose concentration and glucometer reading of a glucose 20 sample in acetate buffer; Figure 2 is a graph of the glucometer reading for glucose and concentration of glucose WO 01/12844 PCT/NZOO/00159 7 added to meat slurry samples; Figure 3 is a graph of the kinetics of glucose formation from glycogen in acetate buffer in the present of amyloglucosidase; Figure 4 is a graph of glucometer readings and the concentration of glucose added to 5 a meat/acetate buffer slurry; Figure 5 is a graph of glucose value and glycogen added to a post rigor meat slurry, at 5 minutes incubation at 55 'C; Figure 6 is a graph of muscle glycogen concentration in post slaughter samples, using the method of measurement of the present invention; 10 Figure 7 is a graph showing the kinetics of glycogen loss, pH fall and lactate increase in a bovine muscle sampled after slaughter; and Figure 8 is a graph of the repeatability of glycogen determination by the method of the present invention. 15 BEST MODES FOR CARRYING OUT THE INVENTION Chemicals, Equipment and Meat. Amyloglucosidase from the fungus Aspergillus niger, in powder form, was added to meat slurries as a liquid suspension or solution. In suspension, 25 mg amyloglucosidase was dissolved in 5 ml of 3.2M ammonium sulphate and adjusted to pH 6.0 with ammonia. This 20 particular solvent is known to be one in which the enzyme is stable. As an alternative, a clear solution can be obtained by the use of 25 mg of powder, dissolved in 5 ml of 0.2M sodium WO 01/12844 PCT/NZOO/00159 8 acetate at a pH of 4.5. 0.-D-Glucose of a standard analytical grade was used. Glucose measurement was made with an Esprit glucometer (Bayer). Test sensors used in the Esprit were from Bayer New Zealand Limited. These were used for one reading only then discarded. 5 The meat samples tested were obtained from the longissimus lumborum muscle of a beef carcass, obtained from a butcher. Pre-rigor muscle, variously gluteus medius, semitendinosus and longissimus lumborum, was obtained from an abattoir. These muscles were dissected from unstimulated carcasses approximately 25 minutes after slaughter and tested very shortly thereafter. The muscles were held at room temperature while measurements were made. 10 Glycogen Test Procedure The test medium for all experiments was 0.2M sodium acetate buffer at a pH of 4.4 and at a temperature of 55"C. In the experiments the muscle or meat (in samples of accurately known weight, but approximately 1 g) was homogenised in 5 ml of buffer, with a high speed 15 Polytron shearing head. This was usually set at 25,000 rpm. Alternatively a lower speed homogeniser may be used, if so desired. The homogeniser may be a stainless steel paddle like blade rotating at 2000 rpm in a steel cup within an interior shaped like a standard domestic Waring blender. After homogenisation the enzyme solution was added. The volume of this solution was 20 usually 200 p1, containing 1 mg of amyloglucosidase. The mixture was briefly shaken, then held at 55"C. Small aliquots, of approximately 20 pl, were withdrawn at intervals with disposable pipettes, WO 01/12844 PCT/NZOO/00159 9 and spotted onto plastic film. The glucometer sensor sampled these drops and returned a meter reading for glucose concentration in 30 seconds. As a control, tests were also carried out with a range of glucose and glycogen concentrations in an acetate buffer to which no meat or meat samples had been added. 5 Lactate Measurement In one range of experiments lactate concentration was also measured. At various times after slaughter, a crude aliquot of the slurry, containing homogenised muscle and amyloglucosidase was centrifuged in a micro-centrifuge (at 10, 000 rpm for 30 seconds). 0 The clear supernatant was recovered and analysed for lactate concentration by the NADH linked method. Results The results from the glucometer were in mg of glucose/di. These results are tabulated in Fig. 5 1 of the attached drawings. It is noted that the relation was linear but that the readings were approximately double the glucose concentration in fact present and did not pass through the origin. The exact reason for the approximate doubling of the readings is not known. However it is understood that this might relate to the characteristics of glucose in blood. a-D-glucose as 0 a laboratory chemical dissolved in acetate at a pH of 4.5 may require a different calibration from that in blood. However, the failure of the straight line to pass through the origin WO 01/12844 PCT/NZOO/00159 10 suggests that the acetate medium affected the sensor performance. Example 1 Various samples of meat (the samples being as described above) between 0.90 and 1.16 g 5 were homogenised in the high speed homogeniser in 5 ml of acetate buffer containing up to 16 mg of added glucose. This addition rate translates to approximately 267 mg/dl, assuming the density of meat is about 1 g/ml. As can be seen from Fig. 2 of the attached drawings the relationship, the relationship between added glucose and the average meter reading is linear. The positive value of detected glucose 10 with zero added glucose may be explained by the small quantities of glucose left over from glycolysis in the meat samples. Example 2 The method of Example 1 was repeated over a range of samples and concentrations in which 1.5 various quantities of glycogen (between 0 - 14 mg) were added to 5 ml of acetate buffer and the reactions were started by amyloglucosidase addition. The results are as set out in Fig. 3 of the attached drawings, in which can be seen the glucose values peaked and declined slightly. The reason for the decline is not understood but it is possible that this may result from a contaminating activity in the enzyme preparation or from 20 isomerisation reactions of glucose liberated from glycogen. The data tabulated in Fig. 3 suggest that at even the highest concentrations of glycogen, WO 01/12844 PCT/NZOO/00159 11 around 40 activity units of amyloglucosidase, are sufficient to fully hydrolyse the glycogen within approximately 5 minutes. Example 3 5 The experiment the results of which are tabulated in Fig. 3 was repeated in the presence of rigor meat. Meat samples ranged from between 0.97 - 1.07 g in six tests. The results are as set out in Fig. 4 of the attached drawings. Individual data points were abstracted to plot glycogen added against meter readings for 5 minutes after the amyloglucosidase addition. The results of this are shown in Fig. 5. A 10 quadratic equation was fitted, but the shape was close to a straight line. Example 4 Samples of gluteus medius and a semitendinosus muscle were removed from pre-rigor meat, cut to samples in the size between 0.95 - 1.05 g, and homogenised in 5 ml of acetate buffer. 15 This slurry was then treated with amyloglucosidase. The tests began 1.3 hours after slaughter, first with the semitendinosus muscle and at that time the muscle contained only 3.8 mg glycogen /g. By 2 hours the glycogen level declined to 1 mg/g. In contrast, gluteus medius muscle sample contained 13.1 mg/g at 2.3 hours post slaughter, and the value declined steadily with time. The results are as shown in Fig. 6. 20 This experiment was repeated using longissimus lumborum muscle from another animal. This muscle is frequently used as an indicator of high pH condition. Lactate concentrations WO 01/12844 PCT/NZOO/00159 12 were also measured. The above tests as described were carried out and the results are as shown in Fig. 7 of the attached drawings. In this method the measurement of lactate was as per the standard NADH-linked method rather than the use of a sensor based on lactate oxidase. 5 Example 5 The robustness of the preferred embodiment of the test of the present invention was further determined by the following: meter readings were recorded in which triplicate aliquots of glycogen were added to three replicate rigor meat samples weighing 1.0 +/- 0.05 g. The 10 slurry was treated in the same manner as described above for Example 2. The results of this are set out in Fig. 8 of the attached drawings. Whilst the Examples given above to show the best method of performing the invention are all with reference to meat samples (beef) it would be appreciated by those skilled in the arts that other tissue samples may be equally treated in like manner to produce glycogen 15 measurement as a result. Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof. Throughout this specification and the claims which follow, unless the context requires 20 otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

1. A rapid method of measuring complex carbohydrates in mammalian tissue, said method comprising the steps of: (a) extracting a sample of tissue to be tested; 5 (b) forming a homogenous slurry of the sample with an aqueous solution; (c) adding sufficient hydrolysing enzyme for ensuring complete hydrolysis of glycogen in the slurry; and (d) measuring the concentration of glucose in the slurry. 10

2. A rapid method of measuring complex carbohydrates as claimed in claim 1 wherein said method is performed in less than 30 minutes.

3. A rapid method of measuring complex carbohydrates as claimed in either claim 1 or claim 2 wherein said complex carbohydrate is selected from glycogen, lactate and a 15 combination of these.

4. A rapid method of measuring complex carbohydrates as claimed in any one of the preceding claims wherein said aqueous solvent is water. 0

5. A rapid method of measuring complex carbohydrates as claimed in any one of the WO 01/12844 PCT/NZOO/00159 14 preceding claims wherein said aqueous solvent includes at least one agent to standardise ionic conditions obtaining for the method.

6. A rapid method of measuring complex carbohydrates as claimed in any one of the 5 preceding claims wherein the formation of the homogenous slurry is effected with apparatus selected from: a high speed homogeniser; a low speed homogeniser and an ultrasonic apparatus.

7. A rapid method of measuring complex carbohydrates as claimed in any one of the 10 preceding claims wherein said hydrolysing enzyme is selected from the group: amyloglucosidase; a-amylase; x-glucosidase, and a combination thereof.

8. A rapid method of measuring complex carbohydrates as claimed in claim 7 wherein said hydrolysing enzyme is amyloglucosidase which is in a form selected from: a powder; 15 a liquid suspension; and a solution.

9. A rapid method of measuring complex carbohydrates as claimed in any one of the preceding claims wherein said method further includes a step (e): measuring the concentration of lactate in the sample. 20

10. A rapid method of measuring complex carbohydrates as claimed in any one of the WO 01/12844 PCT/NZOO/00159 15 preceding claims wherein steps (b) and (c) are performed simultaneously.

11. A rapid method of measuring complex carbohydrates as claimed in any one of claims 1 to 9 wherein steps (c) and (d) are performed simultaneously. 5

12. A rapid method of measuring complex carbohydrates as claimed in claim 9 wherein steps (d) and (e) are performed simultaneously.

13. A rapid method of measuring complex carbohydrates as claimed in claim 9 wherein 10 steps (c) to (e) are performed simultaneously.

14. A rapid method of measuring complex carbohydrates as claimed in claim 9 wherein said measurement of lactate concentration is by the NADH-linked method. L 5

15. A rapid method of measuring complex carbohydrates as claimed in claim 9 wherein said measurement of lactate concentration is based on the generation of hydrogen peroxide in stoichiometric proportion to lactate, as catalysed by lactate oxidase.

16. A rapid method of measuring complex carbohydrates as claimed in any one of the 0 preceding claims wherein said glycogen measurement is effected by use of sensors which WO 01/12844 PCT/NZOO/00159 16 incorporate said hydrolysing enzyme and glucose oxidase.

17. A rapid method of measuring complex carbohydrates as claimed in claim 16 wherein said sensor further includes lactate oxidase. 5

18 A rapid method of measuring complex carbohydrates as claimed in any one of the preceding claims wherein said method is carried out at room temperature.

19. A rapid method of measuring complex carbohydrates as claimed in any one of the 10 preceding claims wherein said method is performed post-mortem, providing a measurement of concentrations of complex carbohydrates at the time of death.

20. A rapid method of measuring complex carbohydrates as claimed in claim 19 wherein said method is performed wherein said measurement is up to half an hour after slaughter. 15

21. Measurement of ultimate pH by use of the method of measuring complex carbohydrates as claimed in any one of the preceding claims wherein said tissue is meat.

22. Measurement of ultimate pH by use of the method of measuring complex 20 carbohydrates as claimed in any one of the preceding claims wherein said tissue is muscle. WO 01/12844 PCT/NZOO/00159 17

23. Measurement of ultimate pH by use of the method of measuring complex carbohydrates as claimed in claim 22 wherein said muscle is selected from: the longissimus lumborum; gluteus medius; semitendinosus and longissimus lumborum muscles.