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M E T H O D S OF S A M P L I N G V A R I O U S MZA.T I N G R E D I E N T S W I T H R E S P E C T TO A C C U R A C Y , P R E C I S I O N AND L I M I T A . T I O N S

OF R A . P I 0 M Z T H O D S TO D E T Z R M I N E M O I S T U R E , FA.T AND P R O T E I N

W . A . LANDHANN”

T E X A S A & M U N I V E R S I T Y

C O L L E G E S T A T I O N , T E X A S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

In the processing of meat f o r sausage manufacture one of the chief problems has always been obtaining an accurate analysis of the r a w ingredi- en t s so tha t t he composition and qua l i ty of the f i n a l product may be assured. Modern packing house pract ices include extensive qua l i ty control which has resu l ted i n uniform, r e l i ab le products f o r the customer, and, hopefully, more economical u t i l i z a t i o n of ingredients.

Good qua l i ty control requires a knowledge of sampling procedures and rapid methods of analysis which give accurate r e su l t s , pa r t i cu la r ly s ince meat i s so heterogeneous i n composition. Poor sampling prac t ices w i l l give r e s u l t s which are j u s t as useless o r unrel iable as those obtained by inaccurate methods of ana lys i s . selves are of ten inaccurate because the analyst confuses precision with ac- curacy. *nY methods may be precise, and s t i l l be inaccurate. That is, an ana lys t w i l l be able t o repeat h i s r e s u l t s qu i t e closely, but these r e su l t s w i l l not be the ac tua l t r u e value f o r t h e amount of the substance being determined. col laborat ive s tud ies show the need f o r revision o r replacement.

A s w i l l be shown later, the methods them-

Hence, some of the o ld “stand-by” methods, when examined by

J u s t as important is a study of sampling procedures, which should Perhaps more of ten than be done before deciding on a pa r t i cu la r procedure.

one would care t o think, t h i s s t e p of the qua l i ty control process i s en t i re - l y neglected.

What i s the ideal s i t ua t ion f o r accurate qua l i ty control? It would be, I think, a simple procedure f o r sampling (preferably from a homogeneous l o t of material), coupled w i t h a rapid method of analysis re- quir ing only a f e w minutes t o complete, thus avoiding shut-down o r delay on t h e processing l i n e . be included i n the spec i f ica t ions . A t present, none of the procedures commonly used can qual i fy i n a l l respects, and it i s doubtful if any ever w i l l meet a l l t he conditions of i dea l i t y .

A non-destructive type of analysis should perhaps also

L e t us examine some of t he sampling procedures used f o r meat trimmings, or meat ingredients. Several standardized sampling procedures

‘Professor, Meats Chemistry, Animal Science Department, Texas A&M Uni- ve r s i ty . College Stat ion, Texas. Paper read by R. L. Hostetler.

79.

f o r meat a r e published i n the Of f i c i a l Methods --- of the AOAC' and i n Labora- - t o ry Methods --- of the Meat Industry'. These sampling procedures cons is t of grinding the e n t i r e sample through a p l a t e containing holes l/S'' i n diam- e t e r , by means of an e l e c t r i c gr inder f i t t e d with a device f o r halving the sample. One half t he sample i s then thoroughly mixed and re-ground, again dividing the sample i n h a l f . "he process is repeated u n t i l a convenient s i z e sample (4-16 02.) is obtained, which is placed i n closed j a r s and re f r igera ted u n t i l analyzed. The e n t i r e procedure is carr ied out i n the cold t o minimize evaporative losses of moisture.

While t h i s process is f eas ib l e f o r samples of moderate s i ze , it

For ex- becomes qu i t e d i f f i c u l t t o sample l o t s of 500 lbs . o r more i n t h i s manner. How does one then obtain an adequate o r "representative" sample? ample, 500 l b . tubs of pork trimmings may be sampled by taking 2"-cores a t random throughout the tub. The minimum number of such cores should be nine t o g e t a s t a t i s t i c a l l y j u s t i f i a b l e estimate of the ac tua l mean value of the composition of the contents of the tub. If the process is such t h a t t he t r i m i n g s can be minced before sampling, it would be preferable t o re- move small samples a t random f r o m t i m e t o time from the moving stream of minced mixture. Frozen meat, usually packed i n 1CO l b . boxes can be sampled i n a similar way by taking 6 d r i l l i n g s a t random from each box. In a re f r igera ted railway car, boxes can be sampled by removing the neces- sary number a t random while they are being unloaded. Each box may then be sampled by d r i l l i n g s .

Any procedure such a s t h i s should be checked t o see i f adequate sampling is being obtained. r ep l i ca t e samplings, and comparing the r e s u l t s by means of a hierarchal o r nested design, which may be found i n any book on s t a t i s t i c s . give information a s t o the source of e r ror , and a l so w i l l indicate whether the number of boxes, the number of samples wi th in boxes, o r number of analyses must be increased t o achieve maximum accuracy. Once t h i s i s done, it is usual ly not necessary t o repeat such a study unless d i f f i c u l t y is again encountered.

This can be simply done by making several

Th i s w i l l

An i n t e re s t ing approach t o the accurate analysis of the percent f a t i n 500 lbs . of trimmings has been suggested by H. 0. H a r t l e ~ . ~ contents of the tub a re spread out on a t ab le and separated by eye- estimate in to three groups - high fat , medium fat , and low f a t . Each group is weighed t o determine the t o t a l bulk weight i n each "stratum." Three samples from each group a r e chosen a t random, mixed together, and the f a t determined f o r each group.

The

The t o t a l weight of fa t i n each stratum

'Official Methods of Analysis - A.O.A.C., Ninth Edition, Association of

'Laboratory Methods of the Meat Industry.

'Hartley, H. 0. - Personal Communication.

Of f i c i a l Agricul tural Chemists, Washington, D. C ., 1960. American Meat I n s t i t u t e ,

Chicago, 1957.

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is calculated, and the values f o r a l l s t r a t a a r e sumed. f a t i n the t o t a l sample (sum of bulk weights of the groups) can be de- termined. The variance of the determination i s of the order of 2% and the accuracy achieved through the sum of the weighted means i s considerably bet- t e r than with non-s t ra t i f ied sampling methods. However, somewhat more ef- f o r t and time a r e required t o carry out such a procedure.

Thus the per cent

In summary, whi le many and varied sampling techniques a r e t o be found, any which a r e used should be adequately t e s t ed t o be sure they a r e r e l i a b l e and t h a t they provide representat ive samples. The r e s u l t s of such a study, properly car r ied out , w i l l save time and money i n reducing t o a minimum the number of samples t o be analyzed f o r rout ine qua l i ty control .

Now what about rapid methods and t h e i r l imi ta t ions? Meat proces- sors a r e in te res ted primarily i n the moisture, f a t , and pro te in content of t h e i r raw mater ia ls and f in i shed products. mind a r e f inding more o r less general use. rad ia t ion f o r moisture, Babcock-type o r conductivity analyses f o r f a t , and dye-binding methods f o r proteins give sa t i s f ac to ry r e su l t s , but usua l ly must be correlated with standard methods by means of regression curves.

Several methods which come to Ealances employing infra-red

The Cotlove chloride analyzer, o r a recent potentiometric t i t r a t i o n method4 have been shown t o give excel lent r e s u l t s f o r s a l t . A l l of these serve t h e i r purpose i n rapid qua l i ty control , but have not been studied c r i t i c a l l y enough t o e s t ab l i sh them as standard methods.

In making comparisons of rapid methods with standard methods, it should be rea l ized t h a t standard o f f i c i a l methods have many short-comings, and a re subject t o many of the same l imi ta t ions a s the newer rapid methods. One of the d i f f i c u l t i e s i n comparing r e s u l t s of a new method with a standard method is t h a t t he inherent e r rors of the standard method a re not c l ea r ly known. The AOAC has had f o r a number of years a continuing pro- gram of col laborat ive s tud ies of methods t o obtain t h i s information and i s continually revis ing i ts o f f i c i a l methods as r e s u l t s of these s tudies become avai lable . The American k a t I n s t i t u t e Foundation has a l so con- ducted a similar study of meat methods, with s i x t y laborator ies p a r t i c i - pating.

Results of these method s tudies have been qu i t e informative, espec ia l ly when examined by a technique described by Youden5, 6 , 7. the r e su l t s a r e p lo t ted on a two-dimensional s c a t t e r diagram, where the X-axis and Y - a x i s have the same sca le .

Briefly,

The values ( s ing le determinations)

4Krzeminski, L. F., Bartal , A. and Landmann, W. A., J. Food Science, 30,

%ouden, W. J., Anal. Chem., - 32, 23A (1960).

‘Youden, W. J . , And. Qual i ty Control, 15, - 24 (1959).

52 (1965).

7Youden, W. J., J. O f f . Agr. Chem., I 45, 169 (1962).

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from two d i f f e ren t sau.iples of s imilar mater ia ls are p lo t t ed f o r each laboratory, X being the r e s u l t of t h e f i rs t determination and Y t h a t of t h e second. The consensus of a l l values i s considered t h e t r u e value and through t h i s point t h e diagram i s divided i n t o four quadrants. The bias-free estimate of t h e standard deviation of a l l po in ts i s calculated and two c i r c l e s a re drawn, using the concensus point a s t h e centroid. The smaller c i r c l e has a radius of 1.17s, and the la rger , 2.45s. Accord- ing t o p robab i l i t i e s based on c i r cu la r normal d is t r ibu t ion , 95% of the poin ts should l i e within the l a rge r c i r c l e and c lus t e r around the centroid. pe r s i s t en t b ias , t h e poin ts would l i e along a 45O l i n e through the centroid, and i n the upper r i g h t and lower l e f t quadrants. s u l t s (high for one sample, low for another) would place t h e points i n t h e upper l e f t and lower r i g h t quadrants. these Youden p l o t s t o some method s tudies a r e shown i n the following s l ides .

If per fec t precis ion exis ted, but i f each laboratory had

B r a t i c re-

Examples of t h e appl icat ion of

Slide 1 - Moisture Determination. The r e s u l t s show individual laboratory bias, and some serious e r r a t i c r e s u l t s . pr imari ly from f a i l u r e t o follow the drying conditions exactly. temperatures and t i m e of heating were of ten not the same for t h e two samples, and were the primary causes of the e r r a t i c r e su l t s .

The bias arose here Oven

Slide 2 - Protein Determination. (AOAC) This "old f a i t h f u l , " t h e Kjeldahl nitrogen method, shows 30% of the labora tor ies outside the c i r c l e which should contain 95% of the poin ts . only s l i g h t l y improved when spec i f ic d i rec t ions were given regarding length of t i m e of digestion, amount and kind of ca t a lys t . deviations from the prescribed method, some of which a re not consis tent within a laboratory, a r e indicated here.

This d i s t r ibu t ion was

Serious

Slide 3 - Fat . This diagram shows r e s u l t s of more than one method, and some of t h e b i a s (points c lose t o the 45' l i n e ) i s due t o the method difference. However, a l l methods appear t o give cornparable r e s u l t s , since the methods were not s ign i f icant ly d i f f e ren t . The f a u l t again i s pr imari ly the individual b i a s .

Slide 4.- S a l t . The method appears t o 'ce sa t i s fac tory , although These labora tor ies were asked t o check t h e i r pro-

The c lus t e r s of po in ts occur because values were rounded off when some r e s u l t s show bias. cedures. reported.

Slide 5 - N i t r i t e . The c l a s s i c example of a thoroughly un- sa t i s f ac to ry method subject t o errors due t o individual handling and treatment of sample, as w e l l as t o individual bias.

I n closing, these s tudies show convincingly t h a t t he most serious l imi t a t ion of a l l rethods i s the des i re of the analyst t o be a non-conform- ing c rea t ive individual. The point I wish t o make, however, i s simply t h a t i n using any method - rap id o r standard - one mst be constant ly aware of t he var ia t ions which e x i s t . To keep these differences t o a minimum, it

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i s extremely important t o use a re l iab le , t es ted sampling procedure and t o follow the specified direct ions of the ana ly t ica l method a s exactly a s possible. samples, would seem t o be i n order.

Frequent checks on laboratory procedures, using known

ow

k.0 55.0 55.2 55.4 55.6 55.8 56.0 56.2 56.b 56.6 56.8 57.0 57.2 57.4 57.6 57.0 ' ' 1 ' ' 1 1 ' ' ' '

Slide 1

Results of moisture determina- t i on on two samples of a l l meat sausage, using AOAC method 23.003 (a) . Inner c i r c l e 1.17 std. dev., outer c i r c l e 2.45 s t d . dev .

! Slide 2

Protein determination on two samples of a l l meat sausage, ac- cording t o method AMI-A4a54, a Ueldahl procedure specifying HgO as ca ta lys t , as in AOAC method 2.036.

Slide 3

Y.2

91.8

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t i 29.0-

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Fat determination on two samples of all meat sausage. Methods indicated in text.

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Salt determination on two samples of all meat sausage, using method AMI-A6a54.

n

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f ' , t I I I I I 6 2.7 2.8 2.9 3.0 3.1 Y.2 3.3 3.4

P r C a t U t ia 4 1 . b

Slide 5

Results of determinations of sodium nitrite on two samples of all meat sausage. Range of 1.17 std. dev. shown by circle is much too large for a reliable method.

z ..

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84.

I n closing, these studies show convincingly t h a t the most serious l imi ta t ion of a l l methods i s the des i re of the analyst t o be a non-conforming creat ive individual. The point I wish t o make, however, i s simply tha t i n using any method - rapid or standard - one mst be constantly aware of the var ia t ions which exis t . To keep these differences t o a mini- mum, it i s extremely important t o use a re l iab le , t e s t ed sampling procedure and t o follow the specified direct ions of the ana ly t ica l method a s ex- a c t l y a s possible. Frequent checks on laboratory procedures, using known s q l e s , would seem t o be i n order.

DR. ROBERT SAFFLE: Thank you, Bob. We appreciate the presenta- t i o n of D r . Landmann's on the methods and some of the l imi ta t ions which we might expect. considerably more research work. Cur t h i r d speaker has been with us a t l e a s t once and I believe two times. George Brissey graduated from U.C.L.A. with an A.B. i n Fhilosophy and Chemistry i n 1933; he then joined S w i f t & Company i n t h e i r Control Laboratory; he 's been with Swift ever since except f o r a short s t i n t with Uncle Sam. He was t ransferred t o the R & D Center i n Chicago in 1944 and f o r several years, he w a s Associate Director of Research, coordinating the Neat Research a c t i v i t y f o r Swift & Company. Since 1963, he has been Assistant General Ifanager of the Qual i ty Assurance Department i n the General Office of Swift & Coxripany i n Chicago. which he w i l l cover i s the "Specific Needs f o r Research Related t o S t a t i s t i c a l Control i n Yanufacturing and Nhrketing Processed Neats". agreement, i n se t t ing up the program, he decided t o take a specif ic method concerned with M.I.D. t o shcw how s t a t i s t i c control was used i n working out a specif ic nethod.

It ce r t a in ly points out an area t h a t needs

The area

By