FOOD JOHN R. MATCHETT Bureau of Agricultural and Industrial Chemistry, Washington 25, D . C.
T
HIS rcview covers the period froin about December 1949 t o Soveinber 1950. I t is a srqucl to the review of methods of' food analysis by Oscr (132) for tliv prriod Sovember 1948 t o Xovt~mher19-19.
tusis of an instrilmcnt for rapid moisture drtrrminution over u moisture range varying froni ne:ir dryness t o saturation. T h e rrwlte for apple and potato tissues showed t h a t energy ahsorption varied linwrly n i t h thc- :iiiiouiit of water in the tissues.
MOISTURE
PROTEINS AND AMINO ACIDS
Accurate and rapid methods for moisture in foods continue to attract the attention of analysts. 'The hope of finding one nirthod :rpplicahle t o all foods rem:iins rrniote, although a techniqur u&ig t,he Sicco-Rapid a p p i r a t w ir said to tie app1icaI)le t o a large variety of foods ( 2 2 ) . T h e Karl Fischer reagent has proved t o be of value in det'ermination of moisture in such foods as cereals, starches, dehydrated vegetables, egg powder, and sirup miscs. -1method employing azeotropic distillation aiid subsequent, determination of mater in the distillate b y titration with the Karl Fischer reagent has been applied t o oils containing as little as 0.00270 water (1.54). h simplified method. for standardization of the reagent has been proposed which avoids the preparation of atisolutely :inhydrous methanol (153). Methods and equipment usctl for the determination of nioisture have been the subject of :I conferencr (2) and of a review paper (68). Ilefcrencc methods suitable for caliliration of empirical or indirect moisture methods for dehydrated foods were critically reviewed and arguments presented t o show that' the equilibrium vapor pressure of wat,er may be a better index of stability of such foods than the moisture content (210). A simplified method, developed for moisture in whole grain.;, involves heating in air for 1G to 18 hours a t 130" C. (243). A mechanical device, consisting of a torsion balance combined wit'h a drying element for nieasuring the nioisture in flour, has lieeii wported (40). Determinations cnn be completed in 5 miriutw with a n accuracy of about 0.1 to 0.2%. Results of moisture dctcrmination b y thc Association of 0ffici:iI hgricultural Cheniipts air- aiid vacuum-oven prowdures on 10 samples of flour and flour niises were compared b y 11 analj-hts iii five Ia1)oratorier (2*97), Good agreement was obtainrd b y analysts within a given laboratory, b u t c o n d ( m b l e discrepancies were found among different laboratories. Deviations were less in samples run Ily the air-oven method than by the vacuum-oven method. h mc~ttiodfor determination of moisture in potato starch has heen reported (184) which involves giving the material a prelitninarjdrying in a freely ventilated o w n a t 130" C., followed hy desiccation beside phosphorus prntoside in vacuo a t 130" C. T h e determination requires about an hour. hir- and vacuum-oven mcthods were employed t o determine moisture in Polutions of sucrose, glucose, and fructose of differcnt acidities (220). Extent of decomposition and required drying timm were established for various temperatures (from 60" t o 98' C.) when drying with various additives such as pumice, sand, ('elite, or paper pulp. X rapid and simple procedure, employing ralciuni carbide, has been developed for moisture in fresh or frozen sweet corn (29;). Results agree with the vacuum-oven method within * 1%. Of particular value is the fact t h a t t h e method can be used b y the quality control group in processing plants. Instruments are now fairly widely used for rapid moisture determination in certain foods. I n general, such instruments utilize variations in the electrical properties of hygroscopic materials, and are useful only when the moisture content is not more than about 25Y0. A recent paper (268) appears t o establish thr feasibility of usr of nuclear absorption phenomena as the
Methods of estimating total nitrogen of proteins liy the Kjeldah1 procedure continue t o he investigated and the most import a n t unsolved piohlrma concerned x-ith this metho4 have heen n-ell sunirnarized (91). X procedure for the lijeldahl determination of nitrogen in nicotinic acid and tryptophan has been investigated in collaboration 11-ith 16 independent laboratories (298). T h e method eniployed appears t o he satisfactory and applicable t o easily decomposed as wvell as refractory compounds. I t has bcen observed t h a t the rate of digeqtion of nicotinic acid was roughly doubled for each 10" C. rise in boiling temperature (230). Iniportance of the concwitration of potassium sillfate in the digeqtion mixture has lieen stressetl (150, 26f7). Improved (lcsigns for digcation ant1 tlist~ill:ition equipment have been reported (@, 9.3, [ T I ) . 11inor improvements have b r r n suggested for various ni:icro, micro, and uitrariiicr~-Ii~r1~1ahl procedurcs (38, 10lj 1,?1, 13.9). A rapid method for estimating protein in co~vs'milk depends on determining ammonia liberated in alkaline solution, due largely t o the glutamine and asparagine content of milk proteins i.96). T h e biuret reaction has been uscd for the colorimetric estinintion of proteinc: in fish (27.3). Dilute alkali (0.1 t o O.2yOsodium hydroxide) has been reconimended for the extraction of protein from plants (9.1). T h e alkali has no apparent harmful effert on the constituent ainino acids. Fish proteins h n v ~liren isolated by dilute salt solutions tc,ni has lven developed for the elcctrophoretic separation of the Iaio\vii protein coniponents in egg white, including thcs rasily p r e r i p i t a t d ovomucin (5.7). Simplo, convenient,, and inexpensive techniques have been elaborated for thr. clectrophoretic separation of amino acids, peptides, and proteins on filter paper (3.5, 47! 18.3) and agar jelly (4). Location of the positions of the components is donr by ninhydrin, Pauly re:igrnt, or various dyw. Chromatographic methods for the separation and estirnatioti of proteins are still largely in the developmental stage. IIoTvever, it appears likely that this field will receive greater emphasis during the nest few years, hecause chromatographic techniques xrr rapid and convenient and d o not necessarily require espensive equipment and highly trained technicians. Three major components in egg m-hite proteins have been separated on a cation eschange column (174), the resolved fractions being identified b y refractometric optical measurements on the effluent immediately above the ion eschange column. Both one- and two-dimensional filter paper techniques have. been employed for the chromatography of other proteins ( 2 0 2 ) . T h e status of chromatographic methods for the determination of amino acids has recently been reviewed ( 1 5 5 ) . T h e precision of quantitative starch anti filter paper chromatography procedures was estimated a t :3 and 5 to 1570, respectively. Separation of amino acids on ion exchange columns has been &died (28, 86, 204, 234, I % ) , and chromatographic procedures have heen elahorated for thc separation of the dinitrophenyl derivatives of the amino acids on filter paper ( 2 2 , 226) and columnc: of chlorinated rubber (108). Superior sepamtions arc rlnimrd for the latter
22 1
ANALYTICAL CHEMISTRY
222 technique. Most or the amino acids niay be regenerated from their dinitrophenyl derivatives by treatment with 2 S sulfuric acid containing a few drops of hydrogen peroxide or by heating in a sealed tube with saturated barium hydroxide (118). Separation of all of the naturally occurring amino acids from each other on one-dimensional filter paper chromatograms using buffered, water-immiscible solvents and buffer-impregnat'ed filter paper strips has been successful (108). This is particularly significant because i t provides the basis for rapid and convenient quantitaprotive estimations of amino acids in protein hydrolyzates. portional divider (Partogrid) for rapid determination of Rj values 011 one-dimensional filter paper chromatograms (partograms) has been suggested (166). Ot,her methods have been reported recently for the quantitative determination of amino acids b y filter paper chromatography ( 1 5 , 2 77'). Khile their applications t o foods have been limited, i t is likely t h a t greater use of these techniques will be made once reliability has been evaluated. T h e principal applications of filter paper chroniatography t o tood analysis have been of a qualitative nature. A new aniino acid, yaminobutyric acid, has been found in extracts of beet root (187), yeast ( I @ ) , and apple pulp (82). 8-Alanine has been reported in extracts of apple pulp (82). Glut'athione has been identified in an alcoholic extract of peanut kernels ( 1 4 7 ) . S u merous free amino acids have been reported in citrus juices ( I D ) , and extracts of apple, pear, prune, apricot, and avocado ( 8 % ) . Rockland and Dunn's rapid test t,ube chromatography technique has been modified for t'he estimation of monosodium glutamate and vegetable protein hydrolyzate in soups (157). Chemical methods for amino acids have recently been reviewed (131). Studies on the determination of amino acids b y a n ingenious isotope derivative technique have been continued (89), and the radioisotope carrier technique has been employed for the determination of alanine, glycine, and proline in e-,p-, and ?-caseins (66). Whole casein was shown t o contain less than 0.1% hydroxyproline. The isotope techniques are not likely t o be used for routine determinations of amino acids in proteins and foods because of the great number of manipulations and special equipment required. Peri-naphthindan-2,3,4-trionehydrate has been used in a titrimetric ammonia method for the estimation of amino acids (124). -4 reinvestigation of Pope and Stevens' method for determining amino acids b y means of iodonietric titration of their soluble copper salts has led t o an improved method using washed copper phosphate (164). B y its use 14 of 19 naturally occurring amino acids were determined with a comparatively high degree of accuracy. Aspartic acid in protein hydrolyzates has been determined by conversion of 6he aspartic acid t o fumaric acid with dimethyl sulfate, followed by colorimetric estimation of fumaric acid (67). Cysteine and cystine have been determined by argentometric amperometric titration (97). A colorimetric method employing pdimet~hylaminobenzaldehyde has been reported for the estiniation of hydroxyproline (128). The procedure requires only about 1 t o 2% as much protein a s has been necessary with previous methods. Methionine has been determined in a number of grains and legumes by a modified l i e c a r t h y and Sullivan sodium nitroprusside procedure ( 159). p-Dimethylaminobenzaldehyde has been used for the colorimetric estimation of tryptophan in unhydrolyzed proteins (17'5). This reagent has been used in the past for det,ermination of tryptophan in alkali-digested proteins. Values obtained for the tryptophan content of unhydrolyzed casein a n d egg albumin were considerably higher t h a n those found in the alkaline hydrolyzates. Glutathione has been determined polarographically in citrus juices and in extracts of potato and guava ( 3 3 ) . Results have been published of a collaborative study by 12 independent laboratories, on the microbiological assay of 16 amino acids in a synthetic test mixture and in hydrolyzates of whole egg, A\
egg albumin, casein, wheat glutcii, and peanut flour (160). T h e absolute mean deviation reported by nine laboratories for the analysis of the synthetic mixture varied between 6.; and 11.3%, \vhile the absolute mean deviation for individual amino acids varied between 0.4% (glutamic acid) and 5.6% (arginine). It has been observed (151) t'hat Leuconostoc inesenteroides P-60 and L. citrocorum 8081 are capable of utilizing peptides released by in vitro enzymatic hydrolysis of proteins. The use of these organisms in determining aniino acids in unhydrolyzed or partially hydrolyzed biological materials does not seem advisable. Improved procedures have been elaborated for the microbiological assay of cystine with L. /wsenferoides P-60 ( 2 5 )and tryptophan with Streptococcus jaecalis (117). A procedure for the microbiological assay of L-, D-, and uL-methionine has been suggested (24). DL-hlethionirie can be assayed with either L. w a binosus or Lactobacillus j e r m n t i , while L-methionine can be determined with L. niesenteroides P-60. u-Wethionine may hc estimated b y difference. METALLIC IONS
Because iron is a n important constitutent of foods and is included in some form in the enrichment of cereals, flour, and bread, a rapid and accurate method for its determination is of interest. Values t,hat check well with the A.O.A.C. method have been obtained by a t,echnique involving the muffle ignition of the sample with magnesium nitrate (178). This ashing -method causes no loss of iron, and possible interference by pyrophosphate is overcome by the presence of considerable quantities of magnesium, and by acid hydrolysis. Iodine determinations of foodstuffs are of value in a n y study of simple goiter. A quick colorimetric method involving the use of sodium nitrate and sulfuric acid has been suggested (17'6). Quant,itative values for sodium, potassium, and calcium iii human and cows' milk have been obtained by using flame spectroscopy techniques (90). The method is said t o be accurate to tvithin 5%. a p p l e slices niay be treated in a calcium chloride bath prior t o canning, for the purpose of increasing their firmness. 4 rapid control method for maint,aining the concentration of calcium chloride in the b a t h has been devised by modifying t,he soap method for measuring the hardness of water (193). Sodium chloride determinations in meat extracts and yeast hydrolyzates are important control measures. A rapid technique, requiring about 30 minutes, consists of deproteinizing the food with Carrez reagent and determining chloride in the protein-free filtrate with Volhard reagent ( 6 1 ) . I n the determination of magnesium by the use of thiazole yellow, errors due t o lack of consistency in the degree of dispersion of the red colored coordination complex seem t o have been circumvented by a new approach t o the problem (85). An accurately measured quantity of thiazole yellow was added t o the niagnesium solution and excess dye removed from the reaction mixture with n-butyl alcohol. .4 calibrat,ion curve is prepared relating quant,ities of magnesium directly with color intensity of the nbutyl alcohol solution of the excess dye. The method is applicable t o milk, plant tissue, soil extracts, and blood serum. Colorimetric methods have been described for the dcterniination of copper in the ash of food products ( 6 4 , 6 9 ) . One procedure ( 6 9 ) uses 1,8-dehydroxy-3-methylol anthraquinone, and in the other ( 6 4 ) pyridine and salicylic acid are utilized. FATS
All methods for measuring the iodine number of oils require long absorption times varying from 30 minutes t o 2 hours. For rapid control work of continuous plant operation, this is too long. A recently published modification of the Rosenniund-Kuhnhenn method should be of interest t o those concerned with plant operation (6). Iodine numbers can be obtained in 1 minute Tvith all ordinary nonconjugated fats and oils. The method scems t o
V O L U M E 23, NO, 2, F E B R U A R Y 1 9 5 1 agree well with the Hanus met'hod except in the case of t u n g oil. Agreement was also obtained with the Wijs method in the case of oils of loiv iodine number, b u t values were slight,ly higher in the east' of oils having iodine numbers of more than 100. .\ centrifugal method for determining fat in cacao products has tieen suggested ( 9 4 ) . A rather extt,nrive review with 74 references dealing with cholesterol determination will be of interest t o those working in this field ( 6 9 ) . ENZYMES
The r o k of phoepholipides a s they affect the consistency of foods, and their susceptibility t o probable taste changes brought :illout by oxidation and rancidity, is ne11 known. A method of tlrtretiiig cxiizyniatic breakdown of phospholipides, based on the fact t h a t serine aiid ethanolamine alone of the basic components of phospholipides liberate ammoiii:i on oxidation with periodate, h:is t)een rclported (168). One niethod of expressing the activity 0 1 papain i p 11)-the Balls-Hoover milk clotting units. A more recent inethod is baaed upon the liberation of tyrosine from a standard solution o i ovalbumin (61). Thv papain unit, is defined as that, amount of enzj-nie which iri 10 minutes a t 36" C. liberatcs :ill the tyrosint. from 1 nig. of dry c'gg d b u n i i n . A photometric iiiiihydrin method for measurement (if proteolysis has also been rt,ported (165). One of the first phosphatase tests for distinguishing pasteurized t'l~oiii rail- niilk was presented in 1934. Since then the test has I w ~improved, i and in 194T it became known as the Sanders and h g e r phosphatase test. More recently (116) a new technique h:ts been developed for this tc.st. The method depends on the formation of red phenolphthalein color from enzymic cleavage of plirnolphthalein diphosphate (sodium salt) a t p H 9 t o 10 in the Iwserice of a little chloroform with illcubation a t 3T" t o 38" C. IC no red color is visible after 24 hours, phosphatase is absent. CARBOHYDRiTES
Iu the determination of reducing sugars in plant materials, the alcohol-cstracted sugars in solution are usually clarified with neut u 1 lead acetate t o remove nonsugar reducing substances, and t.xct'ss lead is removed with disodium phosphate. h study ( 1 0 )of tlic. alcoholic extract of some 29 fruits a n d vegetables showed t h a t iii xliiiost half of theni deterniination of reducing sugars, by the .\.O.-\,C. copper microniethod and the Hassid ferricyanide mieroiiit.thod, could be carried out without removal of alcohol a n d without ariy form of clarification. The other half required treatment of' the alcoholic extract with a selected decolorizing carbon only. A modified Coalstad electronietric titrution method for deterniiiiiiig the end point in the Lane arid Eynon procedure has been rc,ported (6). Platinum \vas substit,uted for copper in one of the t.lt,ctrodes. A carbonate-buffered cupritartrat,e reagent permits tiit. determination of reducing sugars to a n accuracy of a few tciiths of lY0 ( 7 2 ) . The method is applicable t o the detcrmination of reducing sugars in refined sucrose'. A method useful for the measurement of lactose in bread ( 2 7 ) :md a picric acid procedure applicable for both lactose and sucrose in dairy products were reported (140). The Bertrand met,hod has treen modified for tlc~teriiiinationof 0.8 t o 40 mg. of sugar per sample of plant estracts (103). The usual time-consuming chemical methods for determining levulose have been superseded by a pohrographic technique (194), and the polarograph has also beeri used to measure the surface-active impurities in sugar (186). A complete report of handling, sampling, classification, a n d :iiialysis of raw sugar was preserited before t,he International Commission for 1-niform ;\lethods of Sugar Analysis in 1949 (84). An optimum procedure for preparing sucrose liquor and sirup Cor color meacurements has been proposed (116). Equations :inti a nomograph relating the reducing power and values for tIout)le polarization of sugar solutioris have facilitated the ap-
223 proximate determination of mixtures of a n y two known reducing sugars and sucrose (152). The method is uscful for analysis of chocolate, sugar sirups, and the like. A general qualitative test for carbohydrates (41 ) and several qualitative tests for fructose or other ketohexoses have been presented (53, 66, fl4,203). Disches' carbazole method was modified t o permit differentiation between galacturonic and glucuronic acids (44). Sugars did not interfere in the test. -4qualitative scheme for the identification of sucrose, glucose, fructose, maltose, and lactose in macro amounts, based upon solubility differences and specific color reactions, was presented (189). This method may be useful for occasional qualitative identifications. Triphenyltetrazolium chloride dissolves in water t o form a colorless solution. After being reduced, this salt forms triphenylformazaii, Lvhich is a red, water-insoluble compound. Reduced triphenyltetrazolium chloride has been used for the quantitative colorimetric determination of lactose in milk and for glucose and fructose in honey (113). Microdeterniination of sugars appliciible t o the study of spots on paper chromatograms involves preparing paper strips in duplicate. Location of sugar spots on one is deterniiried by the Partridge or Horrocks method. Corresponding parts of the other strip are cut out and t,he sugar is extracted from each individually with methanol, the methanol is removed by evaporation, and the sugar in the residue is determined colorimetrically by a modification of Partridge's aniline phthalate niethod ( 1 4 ) . The method is suitable for pentoses and aldohesOW but not ketoses. Recent reviews of paper chromatography of the carbohydrates (34, 196) criiphasize the possibilities of extending this technique t o the food field. I t need no longer tie ncbcessarg- for food chemists t o express their reducing values as glucose without having definite knowledge of the identity of the sugars in their material. Techniques have been presented t h a t may a l l o ~ a tenfold extension of the quantities now separable by paper chroniatography (126, 203). The properties of 22 filter papers were tested for use in chromatography ( 9 8 ) . At present most of the known sugars and many of their derivatives have been separated by paper chromatography, eluted from the paper, and analyzed. An improvement iii elution techniquc allows simple, rapid, and quantitative removal of the separated sugars from the developed chromatogram ( 1 0 2 ) . It has recently been shon-n t h a t the best separation of sugars occurs when paper chromatogranis are run a t 3 i " C . ( 8 0 ) . Several interesting experiments are offered, one of which is the separation of a- a n d p-methyl rhamnosides. Other workers have presented improvements in procedures developed for their particular needs ( 8 , 16). F o r instance, paper chromatography has been employed t o separate the radioactive products formed during photosynthesis in Cl402,and for the separation and identification of carboxylic acid and phosphate esters ( 8 ) . A multiple development in one direction permitted the resolution of a homologous series of oligosaccharides up t o a degree of polymerization of about 10 glucose units (86). Qualitative paper chromatography has been used t o identify glucose, fructose, and sucrose in lemon, orange, and grapefruit juices (107). The presence of sucrose in lemon juice (about 0.05yo)was demonstrated but was undetected by chemical analy' Sugar beet juice has been analyzed quantitatively for rafse by paper chromatography (1, 4 2 ) . This would have been a n enormous task b y a n y other method. The use of paper chromatography for the separation and quantitative analysis of the sugar-phosphate est,ers has been reported (31 ). The chromatography of the sugars on charcoal using dilute alcohol was reported (188), and this work should be of interest t o anyone using charcoal t o remove "nonsugar" reducing substances. X procedure was reported (106) in which starch was extracted from sugar-free vegetable material n-ith cold dilute perchloric acid, aiid both starch and amylose were determined colorimetrithrone Tvas used in a like manner for the quant,itative animal glj-cogen (167). These procedures all offer the
224
ANALYTICAL CHEMISTRY
advantage t h a t no previous acid hydro1 0 1 th(3st. polyaacchilrides is newssary, because hydrolysis antl volar development in the strong sulfuric acid occu~'a t thr. same time. A note on thta spwificity of the anthrone rwction ( 161 ) rwiews the requii,rmerits for the anthrone-sugar-sulfuric. acid rea(-tioii. .\ mt,thod for the approximate drLtrrniiiiatioii of pectin in raw beet, sugar WBP presentrd (192). The inel hod c d l s for saponification of the pectin, dist~illatioiiof thr. niethyl iilcohol, oxidation of the alcohol with chromic acid, and back-titration with thiosulfati.. A method of measuring the jellying po~veri ) f pwtiri is hasect o t i the sagging of a piece of jelly unrl(3r its o\vn w i g h t (.$.'). >leasurcnirntP of gel strengths by using the I,uc~r.;-T.orhiii~ilcl"pwtinomc,tcr" (AB, 7.4)h a w heen reported. wr-IntIw
The qwetrophotoriit~tricmethod for vitaiiiiri .Inow a p p t w s in the I:. S. Pharmacopeia ( 1 4 2 ) ,and calls for a1isorl)ancies of the unmponifiahlr extract at wive lengths 310 nip, 325 inp, and 334 mp. I t rtquiros that the mtio o f the cwlorimrtrir (untimony trirhloridc) t o the spectrophotonirtrir assay valur~sshall fall within the spwified limits of 1.0 t o 1.3 ( 1 8 0 ) . A method for tho d(>tcrmiriation of vitamin A in t h r p r c w n c t ~c i f intcrfcring su1Jstancw has been reported (109)involving the u w of 3 1 j a n d 338.5 mp as refermcc. points rat,her than 310 anti 340 mp for correrting for ohtraneoul; mattcxr absorption hy the Morton anti Stubbs mcsthotl. Study of a h t t e r mt+hod for vitamin A in f(w1s w:is continued. and a t,echniqur in which 30% acetone in hr>saricwas used for extraction of vitamin h from standard sampks followd by chromatography on magnesium oxidr and subwqucnt determinations x i t h antimony trichloride gam' results w1iic.h corrcslatcd fairly wc~ll with the potrncies of the standards (162). .I 1)iological niethod of vitaniin r\ assay Ivhich perhaps is more ( ~ fundanicnnt:tl f iritcxrest than of practical application, at least a t present, is based on the degeneration of nerve tissue of the central wrvous system of rats in vitamin A deficiency (50). Such lesions are pwsent t o varying degrees in rats that arc still grolving :ind froin cstoriial appearancc are in good condition. Abnormalitic's, tic~ttic~ttd by histological tJxaminations taken as the basis of csvduiition of the prophylactic assay, which covers an 8-rveek prrind, intlicatrd that the procedurta was at lrnst as accurate as tho 3-n.ec~kcurativcb growth test. A collal)orative study by the Association of Official Agricultural ('liemists on the, determination of carotene. in alfalfa has been continued ( 1 4 4 ) . Oiie hundred and ten laboratoric>s h a r e participated in the. study of the chromatographic procedurc: n-ith a final rc.ronimentlatior1 that overnight extraction at room temperature be used as an alternat,ive t o refluxing for 1 hour, t h a t a mor(' uniform adsorhciit be sought, and a procedure for purification of solvents be devised. The use of a mixture of equal partsof toluene, (,thy1 alcohol, and ethyl acetate for giving it complete extraction ( i f rarotenoids from feeds n'as suggested ( 3 2 ) . The chromatographic separation of carotene from other pigments was similar t o that of the Association of Official Agricultural Chemists. This 1)rocedure avoids saponification, as docs another for carrots, spinach, and red pepper (60) where carotenoids were extracted and chromatographed directly. h method for estimating carotene aud anatto in the presence of dimethylaminoazobenzene b y means of paper chromatography has been suggested (43). A rapid two-stage chromatographic method applicable t o the determination of carotene in dried peas has been reported (66). Considerable research has been undertaken on assay methods for vitaniin U,,, and in the United States a recent collaborative study spoiisored by the C. S. Pharmacopeia Xnti-;lnemia Board has been the basis for adoption of an official procedure. Reports on microbiological techniques seem t o hxve been limited t o Lactobacillus leichinannii ATCC 4797 ( 1 1 , 138, 163, 172, 180) and strain 313 (172). Media containing crystalline amino acids a s a nitrogen snnrce and adsorbed tomato juice filtrate (158)and fu-
inaiic acid (181)a s growth stimulants have been st.udied. AIicrobiological assay met,hodsapplird t o alfalfa will give misleading rrsults, bcrause there are present factors other than vitamin H12 t o which I,. leichmarmii respond (11). I t appears likely t h a t t,hese factors may consist of the naturally occurring desoxyrihosidrs, whirl1 have been shown t o respond t o the H12 assay. ('hromatographic niethotls have. been proposed for the separatioii ( i f compounds PO iiig vitaniin H12 activity (95,201). Hutyl ;il(diol was suital ily for the separation of B12 from tAithor. wtIt,iiiiic.-dt.sosyribosenucleositie or thymidine. Phenol W A S iiot suita1)lfl for separation (96). Condition6 have been definml for stpirating R,, and €312, from misturw of these two subst:tii(w (2001). The rrduccd forms of HI,, c x l l t ~ lH12&and B12b, are k i i i i i v i i to tie identical. Rat (148) antl chick ( 2 8 ) assay int:thods for B12 have beeii I Y X ti. Tcehniques for preparing B12-depletedchicks for use are itietl (13). A colorimetric method is based on t,hr col(ir of a )lytic, protluct of HI? after prcfrrential extraction t o separates it f'rorn impurities ( 5 2 ) . Rrsults tiw in ngreement with thow (11)t : i i r i c s t l tiy mirrobiological niclthoda. I'ul)lish~~i data on recent iinprovcsnients in t,he estimatioii OT vittiniin E i t i ~lacking except for one report based on the spwti'ophoti)metric drtc~rminatioiiof a-tocopherol in the presence of Ttiicophcrol (25'9). The problem of determining tocophtwls is ~ ~ i n i p l i c ~by ~ t ct,he d plurality of tocopherols having different :iotivitiex, by tht. interfcrerice of other inactive compounds, :tiid by t h c s iiistatiility of tocopherols during manipulation. Pantothcnol and pantothenates assumr importance as nieinliers of thv vitamin B compltlx, and a rapid colorimetric method has IIWJI tlrscribcd for estimating pantothenates in tablets and ampnules (200). ripplication of the nitithod t o food products is not inviitioned by the authors. Efforts have' been made t o use' diff'ertbntial assays of free and total pantothenic acid with 1,.ambinostis iic3fo1,c~and after suitable enzymcx treatment as a guide in attvinpts to coricentrat,e the bound forms from yeast and livrr ( 12? ). .4 number of colorimetric tcc*hiiiquesfor choline inviil us(' of Reinrrkc's salt have 1m.n suggested. Recent iii(2iits iiicludr a gravimetric inethocl utilizing the same rc~:iKf~nt (133). When this method is uscd in assaying folic acid-cwntaining liver fractions, falsely high results are obtained (3). This is due t o the fact that such fractions contain a basic suhstancr which precipitates with animoniuni reirieckate. The "liver" leiricbckate is, however, insolublc~in acc'torie, while choline winwk:itr is ~ o l u b l ethus , offering a meam of separation (3). A spectrophotometric method for vitamin C has t)(b(,ii ilvscvitird ( 11 1 ) and methods for vitaniiri C in the presence offt~ri~oiir salts have been investigated (20, 81 ), particularly from the, standpoint of accurate vitamin C tlt~tcrmiliationsin canned f o o d s . llearis of stabilizing thc standard solution L f 2,6-dichloro iridophenol are suggested (81) and thv use of a sodium ii hydrochloric acid buffer solution or of a 10% acetic acid-0.17, oxalic acid solution as a n extracting medium obviates difficultit+ t h e t o the presence of iron or copper (20). A new book on vitamin analytical techniques, presenting the physical, chemical, microbiological, and animal assay methods oinployed,should be of interest t o t,hose n-orking in this field ( 7 0 ) . COLOR, ODOR, AXD TASTE
.1method giving approximate color values of tomato paste iiivolvee a color indexobtained by measuring the color of extracts of the paste xvith a photoelectric colorimeter instead of by reflectance measurements (38). The method may have broad applicatioii t o many kinds of foods such as citrus products, avocados, dates, and peppers. Another method consists of determining the color with a Hunter color-difference meter and then interpretilig specifications on a special chromaticity diagram (204). Uecausr the procedures are objective and unrelated t o the ability of the operator t o match or evaluate colors, the personal factor is eliininated.
V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1
225
1)egrc.e of nonenzymatic Iironxiiig of some dehydrated vcgcst:igr o f decomposition of the raw material from which the taIi1t.s diiring storagr has ~ ) C T I I tiir:tsui,ed (73) as the differrncc tx,canticd product was prepared. t \ v ( ~ s nopt,ical density v:ilurs of c~sttxrtedsoluble color matcria1.q Shaking samples of potato flakes, flour, and starch flour with citloroforni in ordrr t o determine sand has been found t o be unf r o i i i s:inqiles before anti :iftcar atoixgc. tnce spectrophot oiiicter :itid photoelectric reflec-tomrtrr der~c:ndable(186). A better procedure makes use of hydrolysis of h a w bwn usc.tl t o study and determine t h r t:stc'nt ni~~:~sut~cirients t h r starch with hydrochloric acid, arid this is even preferable to t o \c.Iiich original n:tt~ur:tlcolor is preserved during proct'ssinp :ind :ishing (186). .sulisc~clu(~nt storage of crrt:iin foods (60, 112). llcthods of detecting Cysticercus-roritarniriatcd mrat huve .\ pliot80clectricmr~tlliodfor : L P S ~ S P ~ flour II~ color :is :in indicat o r 1)ct~tlsuggested (100). c icerci representing the 1:itval forins of fioiit, gra,de comes from 1GiigI:ind (SS). .In apparatus h i t + IHY~IIof Taenia soliu~n(pork tLtpen-orm of m a n ) and ?'ueuiu~hyt~chu.s t i ( ~ \ - [ ~ l t r p for c ~ i this, the nmliiigs of which :ire indepc,ii(l('iit 0 1 t lit' saginatirs (t:ipe\vorm of m a n ) , either dead or alivc, give :t pink c,iT(*c.t of natural or artifici:il b1v:icIiing. fluorcwcsnce on illumination with ultraviolet of sprcific \vavt: l'c~i~inarientglass color st:iiidartls h:iw been devclopcd 1'ot. usr lciigth. Thr Iiropcrty of luminescence has also I ) r r n upplied for i t i :I simple color c'onip:it~:itor for qxdiiig the color of r n : i p l c ~ rirriy) clrtcction of impurities in o t h r r foods such as fruit juices, vinegar. ( I ! / 1. vcxgetable oils, butter, and flours (182). T I i ( , (ahernistry of H:ivor 1i:ia I ) ( Y I ~ thc, subject of ii r r u ~ i i trc'\.ic.\\The possible prrsertcc of coliform organisms i n citrus juicca itas ( 1 ;,i 1, ; t i i d flavor p r o f i k ~lt:tv(* II(YII discussed in anothc~i~ tvrc.iit t )cic-omeof incrrasing importancc during the past year, and apparrn,t 1y sntisf:tct,ory methods remain still t o 1x3 worked out, :is i i p I)ulilic:ition (25). .A r:ithcbr mitnprehctiaive study of odors i + also o1 iiilibrvat, in this conitcction ( 2 7 9 ) . One of several suggrstioii+ p l i c b d t o thmc commodities. It has been rcportrd (199) tlint varif o i , iiiiprovement in fl;tvor c.v:tlu:itioii procedure has becbn :I scoring ous organisms meeting the drfinition for colifornis prrsistcd in ,*)'htctii devised t o diffri~c~liti:ite brtwc~enintensity of :ibnorm:ii 01' f t , o z r n conccntratrd orange juicc fot, 43 rvrcks. LIost w r e Aerouiitl(~sir:tt)lcattributes :itit1 pi,rfert,c~l intensity of typiral or drsit,I ):ir ter rpr rics, but some resembling Esclzer ich iu coli \v('I'c f ou rid. 111 has been found t 0 1"'";tliic~:ittributes of food ( 7 8 ) . The 111a survey made of citrus proccsssirig plants in Florida during tht, villct producible results rel:itrtl t o position of thc tcst i n i i t r 1!)48-49 soason, no E. coli \vas found in any of t h r saniplrs studied iiuls. .Artother prowdurc. Ixisrd on paired a n d triangle rliffcrrncc~ (2:YBi. This paper poilit. out t h r in1port:ince of the nietli:i usrtl. trsts rmphasizes t h e us(! of "~v:inn-up" samples, rcfc~rc~ncc~ st:in(I; ~ r d *rin,ses, ~ and ccintt.01 of t.hc c1u:intity judged and of tlic' tiinis 3IISCELLANEOUS iiitcarv:il Lietween jutlgitig ( i f ' sittiipl~s(1,$1). AIrt hods h:ivc brrn mggrstrd for measuring the firmntw of red .\inong investig:itions ol iiictlio(ls applicable to q x v i f c f'ootl:: is tlic tl(:vc:lopment of :I pi~occduivfor cv:iluating oils (22,3)- i i i ra\v : r i i d 'I'hr tirtc,ction of egg in s:ihtl crram is based on a colorimtstric nic:tlioti of drtc~rtniningchoiin? derived from rgg Itbcithin by acid c : l l l l l c Y l peas (99). hydi,olysis :tiid conv(~rsionof thc. choline into its pink rcincckatr .i study undertnkcti t o tlctcrtnine the i't':ison for rmttic i.(mlts i i i judgiiig sulfitcd footls shovwl that tiisting one saniplr of n u e h r t l (56). Thtb old Stroheckcr method hits been inodificd for the t i c s pt:itor*s containing 12 to 100 p ~ ) . n i of' . sulfur diosidr t l u l i c d th(* tcrrnin:ition of ry(' Hour in mixtures with whcat flour (6S), and 3 :tcuity of the judges for dctccting subsequent sulfitcd sztniplrs i:ihr:ttory prcicrdurcS for evaluating curd-producing c:ip:icity of ( 1 ; I . The authors rrcominend cv:ilu:tting only one sulfitcd Samsoyliritn products hiis twen described (106). Curd volume is :in ~ i l t ,per judging session until :I i i i o i ' t ' reliable tecliniquo is tlewl:iwurat(' iridtss t o tlic per writ of soluble protein in soy flours op"ti. X sprct,romrtric nicthod for estimating solids iri tomato prodSotne of the difficulticbs iiivolvcd in judging flavor of I'ootls t h i i i ucts h a p hcrri proposrd ( 1 7 7 ) ,the rrsults having additional valuc e:\ii lx! identified by color diffctviices are illust,rated i n :i report ot i n that thc>y?:in be u s r d :is :I critrrion o f tht. tirgrc:e of ripencsa of :in c.v:ilu:ction of spicrs. qpicr oils, and ol~~orcsins ( 196). t hc t o n i a t o ~ sused. .\ rapid mc~thoclfor tl(~tc~rminntion of crude fiixr has l)ri*n IIXSPOILA(;E A V I ) COh'rAMlNATlOR p o r t c d ( 7 1 1 . >I:itrri:tis of' high fat, contc!nt must first tw d(.futtrti l l e t h o d s of detcrmiriiitg inarct counts in foods haw: brvn t lir t o xvoicl rrt'or t h a t n q y I)(, ciiusrd by foaming. suiiject, of three rcc.ciit 1i:iprrs (63, 170, 181), involving :in inil't,op>-lenc~glycol is usctl i n mrtny food preparations ;tnd particuprovc~iiic~rit over th(2 o l t l c ~ r WiIdin:~nmethod, c s t r w t i o n 0 1 thcx 1:tt.ly i t i imit:~tion v:iiiilla rstracts. The usual separation of pro'luct with castor oil folio\\-cd l>y gasoline, or prcfrrriitial propyk~iirglycol by tiiwct distil1:~tionhas been found unsatisfacstuiiiing which 1c:ivc.x uncli:tngrd t h c i i i t fragments so that t h r y and charring of sugar ivheti present. tory brc:turc~of poor rrco :ipiic:Lr in their natur:il colot~. 1 ' 3 txrtioii mc~thotlPhave n found equally uns:itisfactory. A .\lethods of the Sation:il Clic'eee Institute and the .issoci:itioii prorrtiurc dcpc~ndingupon the isolation of propylene glycol from c i i 0ffici:tl Agricu1tur:tl ('lic*niiats 1'01. deterinining rst t ~ : i n c ~ i u c \wiilln c1tr:tc.t ti>- c*odistill:ttion with a n organic solvent such as niattcbr iri cheesc h:ivr l h v ~ i i i i i o t l i f i r t l lor use with Sivisi: pt,oduvts 11cpt:inr has t x ~ nrcsportcd ( 6 2 ) . The method c:m be used ivheri ( Xj. propyk~iicglycol occurs :done or in the presenccl of glycrrol. The conventional Sc~Iic~c:htc~~~-li:~ller rolorimetric tnetliocl fot, The A.O..\.C. l'o\vcr-Chrstnut a n d Baily-Andrew nicthods for D1)T is not applicablc i l l tlics pt'rsc~nccof more than t,raccss of f a t . cxffviiic. in C O ~ ~ C Y:tiid ! tea are time-con,suniing. .I semimicroIt hiis Ibeen found ( 3 7 ) t h t Cclitc impregnated with sulfut~ic~ mcthocl hiis liren suggested requiring only 15 t o 25% of the time :rritl-i'uining sulfuric acid, :itid slurrirtl with carbon ttstrachloride try for the A.O,.I,(!. method (18). An improved method will Iiold fats within :t clir~~iiiatogr;iI,hic column, while DI>T will for di~t(~rniiiiirig throt~rorninc!in cocoa products is also of ititerest IJ:W through with the c a h o i i tcxtr:tchloride. From 90 t o 1007, of' (7s). 1)I)T :idded t o butter oil \vas rcseovcred with this method. Congrcssion:il itctioti i r i rispealing the t a s on oieoni:ug:trine -1 colorimetric method for estimating trimethylamine is ~f i t i ni:tdr it evident that somc niothod n.ould have to be found to diftcn'st in fitih decomposition (@), and the presence of sucrinic acid ferentiate bct\vcen butter :inti margarine. A test has bren sug1i:ia 1)ren tshown t o br :tn intlrs of drcomposition in tuna (7;). grsted (54) t o diffcrentiate b u t t r r from margarine, based on the I~Lstensivedecomposition c~spcrinic~iits have becn carried out on "critical temperature o f dissolution" (CTD test) first enunciated cr('ani and d a t a presentctl on butyric and water-insolublr acids hy Vnlenta over 55 years ago. I n the proposed test a mist,urc of (V'I.1) in cream itnd I)utter ( 7 5 ) . Individual volatilc acids :i.c ethyl and isoamyl alcohols of specified strength is used as a soltiviclence of decomposition have been applied t o canned turi;i vent of the oil from the butter or margarine. Upon heating and ( 7 6 j s t h r results indicating th:tt thew :icids arc' a good indcs of t l r r stirring thc inisturc t ) c c o m r s clcar and honiogencous. T h r mix-
ANALYTICAL CHEMISTRY
226 ture is then cooled while being stirred and the temperature is noted tit the first discernible h r b i d i t y . The magnitude of temperature spread at xhich but,ter and margarine become turbid was significant enough, even in the hands of different analysts, to distinguish between the two commodities. The toxic factor derived from "agenized!' (nitrogen trichloridetreated) flour has been investigated by several laboratories ( 9 , 26, 119, 249,150, 269). The sulfoxiniine of methionine has been tentatively identified as the t,oxic factor (119, 169). Use of nitrogen trichloride for flour bleaching is no longer permitted in the Zjnited States. LITERATURE CITED
Albion, N . , and Gross, D., Analyst, 75, 454 (1950). Anon., Chon. Age, 62, 231 (1950). Auerbach, 11.C., J . Am. Pharm. dssoc., 39, 480 (1950). Baertich, E., Pharmapie, 5, 206 (1950). Beham, G. H., aiid Klee, Leo, J . I m . Oil C h e m i s t s ' Soc., 27, 127 (1950). Bell, E. V., and Graham, J. C., Intc~ru,S u g a r J . , 52, 90 (1950). Bennett, E. H., Cereal C h e m . , 27, 222 (1950). Benson, I k s h a m , .J. H . , Calviii, AI., Goodale, T. C., Haas, ., aiid Stepka, JY. J., J . z4m, Chein. Soc., 72, 1710 (1950). Bentley, H., NcDermott, E., Pace, J., JVhitehead, ,J., and N o ran, T., S a t u r e , 165, 150 (1950). Bevenue, .I.,and Washauer, B., J . Assoc. O.i%c. l y r . C'hcmiats, 33, 122 (1950). Bickoff, E. A I . , Livingston, .I.L., aiid diiell, AI. S., Arch. Biochcm., 28, 242 (1950). Biserte, G., and Osteux, R., C'orupt. rocd., 230, 1404 (1950). Black, D. J. G., Getty, J., Coates, AI. E., Harrison, G . F.,and Kon. 3. K.. Biochmn. J . . 46. viii il950). Blass, J . , Macheboeuf, AI.,'and Xfiiiez, G., BULL.soc. chini. b i d . , 32, 130 (1950). Block, H. ,J., FoodInds.. 22, 88 (1950). I3oaas. __ L.. Cuendet. L. S..Ehreiithal. I.. Koch. It.. and Smith, F., S a t u r e , 166, 520 (1950). Boggs, b l . M.,and \Yard, .I.C., Food Techno(.. 4, 282 (1950). lI.,eds., "Partition Chroma11.G., J . A m . Ctirvi. S u r . . 72, 229 (1950). tography." Biochemical Society Symposia No. 3, Chap. 5, p. (150) lieiner. L., Xlisani, F.. and Keiss. P., A r c h . Biochem., 25, 447 52, Cambridge UniT-ersity Press, 1950. (1950). (197) Williams. Sidney, J . d4ssoc. Ofic. A g r . Chemists, 33, 174 (1950). (151) Reisen, W.H., arid El\-ehjcni, C . .\.. -4rch. Biocheni., 25, 335 (198) Willits, C . O., and Ogg, C. L., I h i d . , 33, 179 (1950). (1950). (199) Wolford, E. R.. Food Technol., 4, 241 (1950). (152) Itheinlander, -4.H.. dizalvst, 75, 444 (1950). (200) Wollish, E. G.. and Schmall, hI., -4s.~~. CHEM., 22, 1033 (1950). (153) Ricciuti, C., and IT illits, C. O., .I.Assoc. Ofic. A g r . C'hemists, (201) Woodruff, H. B., and Foster, J. C., J . Biol. Chem., 183, 569 33,469 (1950). (1950). (154) Roberts, I?. M., and Levin. H.. - 4 ~ 4CHEM.. ~ . 21, 1553 (1949). (202) Wynn, V.. and Rogers, G., dicstmlian J . Sci. Research, B3,124 (155) Rockland, L. B.. Abstracts of Papers, 117th Meeting A x . (1950). CHEM.Sor., Division of Agricultural and Food Chemistry, p. (203) Yanofsky, C., Wasserman, E., and Bonner, D. hl., Science, 111, 5A, 1950. 61 (1950). (156) Rockland, L. R.. and Dunn, AI. S., S c i e n c c , 111, 332 (1950). (204) Younkin, 8. G., Food Technol., 4, 350 (1950). (157) Rockland, L. B., Vnderwood, J . C . , and Beavens, E. -4.. Calif. Citrograph, 35,490 (1950). (158) Rose. W.Q., Food Techno!., 4, 230 (1950). RECEIVED DecembPr 20, 1050.
