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Factors affecting the nitrate content of foods Bodhiphala, Tewee 1969

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FACTORS AFFECTING THE NITRATE CONTENT OF FOODS by TEWEE BODHIPHALA BSc. in Pharmacy, University of Medical Sciences, Bangkok, Thailand 1959 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Food Science Faculty of A g r i c u l t u r a l Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s thes. is f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f F o o d Science The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date June 23, 1969 ( i ) ABSTRACT The n i t r a t e c o n t e n t o f some f o o d s was d e t e r m i n e d . Canned f o o d , b a b y f o o d , f r o z e n f o o d , and f r e s h v e g e t a b l e s w e re a n a l y z e d . Among them s p i n a c h and b e e t w e r e f o u n d t o h a v e t h e h i g h e s t n i t r a t e - n i t r o g e n c o n t e n t and f r o z e n f o o d h a d h i g h e r n i t r a t e - n i t r o g e n c o n t e n t t h a n o t h e r f o o d p r o d u c t s a n a l y z e d . N i t r a t e - n i t r o g e n o f f o o d i s p a r t i a l l y t r a n s f e r r e d t o t h e l i q u i d p o r t i o n w h e n e v e r t h e f o o d c o n s i s t s o f any l i q u i d . The amount o f n i t r a t e i n t h e l i q u i d p o r t i o n seemed, t o be h i g h e r t h a n i n t h e s o l i d p o r t i o n e x c e p t i n b e a n w h i c h has a p r o t e c t i v e s u r f a c e l a y e r as a f a c t o r o f l o w e r i n g t h e n i t r a t e f o u n d i n t h e l i q u i d p o r t i o n . The r a t e o f n i t r a t e t r a n s f e r r i n g f r o m s o l i d p o r t i o n t o l i q u i d p o r t i o n was n o t t h e same f o r a l l f o o d s and was n o t t h e same e v e n f r o m d i f f e r e n t p a r t s o f t h e same p l a n t . N i t r a t e - n i t r o g e n was n o t d e s t r o y e d b y c o o k i n g . E v e n a f t e r p o u r i n g o f f c o o k i n g w a t e r , some n i t r a t e - n i t r o g e n s t i l l r e m a i n e d i n t h e s o l i d p o r t i o n . The d i s t r i b u t i o n o f n i t r a t e among d i f f e r e n t p l a n t p a r t s was n o t u n i f o r m : b e a n l e a v e s , b e e t r o o t a n d s p i n a c h p e t i o l e s w e r e f o u n d t o h a v e h i g h e r n i t r a t e t h a n o t h e r p a r t s . N i t r o g e n f e r t i l i z a t i o n r e a d i l y i n c r e a s e d n i t r a t e - n i t r o g e n c o n t e n t i n s p i n a c h . The s o d i u m s a l i c y l a t e m e t hod was f o u n d t o be t h e most r e l i a b l e m e t hod f o r n i t r a t e d e t e r m i n a t i o n among d i f f e r e n t v. methods u s e d i n t h i s s t u d y . The d e t e r m i n a t i o n m i g h t be a f f e c t e d b y many f a c t o r s o c c u r i n g d u r i n g t h e p r o c e d u r e o f a n a l y s i s s u c h as p r o c e d u r e o f e x t r a c t i o n and t h e s p e c t r o -p h o t o m e t e r b l a n k s u s e d . O x i d i s i n g a g e n t s , arid r e d u c i n g ( i i ) agents do not seem to affect the analysis but pH v a r i a t i o n and sucrose which might occur in food probably are factors affecting apparent n i t r a t e content. Cooking did quickly destroy spinach n i t r a t e reductase enzyme a c t i v i t y . This means that n i t r i t e w i l l not be found after cooking unless the enzyme is regenerated, or unless there is microbial a c t i v i t y . ( i i i ) TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES v i ACKNOWLEDGEMENTS • ix INTRODUCTION 1 LITERATURE REVIEW 1. NITRATE CONTENT OF FOODS 3 2. METHOD OF ANALYSIS a) Sodium s a l i c y l a t e method 9 b) Rapid determination of n i t r a t e and 9 n i t r i t e i n plant material c) Phenol-disulfonic acid method 10 d) Nitrate ion electrode method 10 e) Some other methods of n i t r a t e 11 determination 3. NITRATE RELATIONSHIP TO FERTILIZER PRACTICE 12 4. NITRATE REDUCTASE 17 MATERIALS AND METHODS 1. SAMPLES FOR NITRATE DETERMINATION 20 2. METHODS OF ANALYSIS a) Sodium s a l i c y l a t e method 21 a b) Rapid.determination of n i t r a t e and n i t r i t e 22 in plant material c) Phenol-disulfonic acid method 24 d) Nitrate ion electrode method 26 3. FACTORS AFFECTING ANALYSIS 27 4. PLANT STUDIES a) Comparison of bean with spinach 28 (iv) Page b) Var ia t ions in d i f f erent plant parts 28 c) Ef fec t of nutr ient status 28 d) Ni tra te reductase studies 28 RESULTS AND DISCUSSION 1. NITRATE CONTENT OF FOODS a) Canned food 31 b) Strained baby food 31 c) Frozen food 33 d) Fresh vegetables 36 2. RECOVERY STUDY a) Sodium s a l i c y l a t e method 38 b) Rapid determination of n i t r a t e and 38 n i t r i t e in plant mater ia l c) Phenol -d i su l fonic acid method 40 d) Ni tra te ion electrode method 40 3. FACTORS AFFECTING ANALYSIS a) Time of extract ion 45 b) Repeated extract ion 48 c) C l a r i f i c a t i o n procedure and the use 48 of spectrophotometer blanks d) Reducing agents 53 e) Oxidis ing agent 55 f) Buffer solut ions 58 g) pH and t o t a l a c i d i t y in spinach 58 h) pH in beet 63 i ) Sucrose 66 j) Blanching 66 4. PLANT STUDIES a) Comparison of spinach and bean 68 (v) Page b) Variat ions i n d i f f erent plant parts 71 c) Ef fect of nutr ient status 73 d) N i tra te reductase studies 75 SUMMARY 7 8 LITERATURE CITED 81 (vi) LIST OF TABLES Table Page 1. The d i s t r i b u t i o n of n i t r a t e - n i t r o g e n between s o l i d 32 and l i q u i d portions of canned food obtained from Vancouver markets i n 1968. 2. The n i t r a t e - n i t r o g e n content of baby food obtained 34 from Vancouver markets in 1968. 3. The d i s t r i b u t i o n of n i t r a t e - n i t r o g e n between s o l i d 35 and l i q u i d portions of frozen food obtained from Vancouver markets i n 1968, 4. The n i t r a t e - n i t r o g e n content of fresh 37 vegetables obtained from d i f f erent sources. 5. The recovery of potassium n i t r a t e added to vegetables 39 using the sodium s a l i c y l a t e method of ana lys i s . 6. The recovery of potassium n i t r a t e added to spinach 41 using the rapid method of determination of n i t r a t e i n plant m a t e r i a l . 7. The recovery of potassium n i t r a t e added to 42 vegetables by the phenoLdisulfonic acid method. 8. The recovery of potassium n i t r a t e added to spinach 44 using the n i t r a t e ion electrode method. 9. The v a r i a t i o n of n i t r a t e - n i t r o g e n content i n 46 so lu t ion with b o i l i n g time. 10. The v a r i a t i o n of n i t r a t e - n i t r o g e n content i n spinach 47 with b o i l i n g time. 11. The v a r i a t i o n of n i t r a t e - n i t r o g e n content from the 49 successive residues of spinach ex trac t ion . (v i i ) Table Page 12. The v a r i a t i o n of n i t r a t e - n i t r o g e n using d i f f erent 51 procedures of c l a r i f i c a t i o n of spinach extract . 13. The recovery of n i t r a t e - n i t r o g e n when d i f f erent 52 blanks were used and with n i t ra tes added to d i f f erent species at d i f f erent times. 14. The effect of reducing agents on n i t r a t e - n i t r o g e n 54 determination. 15. The effect of oxa l i c acid on n i t r a t e - n i t r o g e n 56 determination in spinach. 16. The ef fect of an ox id i s ing agent on n i t r a t e - n i t r o g e n 57 determination in spinach. 17. The v a r i a t i o n of n i t r a t e - n i t r o g e n content in buffer 59 solut ions with b o i l i n g . 18. The v a r i a t i o n of pH and t o t a l a c i d i t y of spinach 61 during blanching. 19. The v a r i a t i o n of pH and the n i t r a t e - n i t r o g e n 62 content of spinach during blanching. 20. The v a r i a t i o n of pH and the n i t r a t e - n i t r o g e n content 64 of beet during blanching. 21. The comparison of n i t r a t e - n i t r o g e n recoveries from 67 water and sucrose so lut ions . 22. The v a r i a t i o n of n i t r a t e - n i t r o g e n t rans ferr ing from 69 s o l i d to l i q u i d port ion during blanching of vegetables. 23. The v a r i a t i o n of n i t r a t e - n i t r o g e n i n d i f f erent plant 72 part s . ( v i i i ) Table Page 24. N i t ra te -n i t rogen content i n d i f f erent parts of 74 spinach with and without n i t r a t e f e r t i l i z a t i o n . 25. The effect of blanching time on the n i t r a t e 77 reductase a c t i v i t y of spinach. (ix) ACKNOWLEDGEMENTS The writer would l i k e to express her appreciation to Dr. D.P. Ormrod who directed this research and who provided encouragement and advice. Thanks are also extended to the members of the research committee for use of f a c i l i t i e s and advice on the preparation of this thesis. Dr. J.F. Richards Dr. S. Nakai INTRODUCTION The presence and amounts of n i t ra tes in foods has been noted and tabulated by Richardson (1907) and Wilson (1949). N i tra te is sometimes used i n the manufacture of cheese to stop microbia l growth at a predetermined l eve l of fermentation by l i b e r a t i o n of n i t r i t e , which also serves as a preservat ive . Ni trate is also used in some other food products to prevent b a c t e r i a l invasion and/or for the purpose of maintaining a red color in the curing of meats. Ni trates are natural constituents of plant mater ia l since they are the main source of nitrogen required for growth. The roots of most plants absorb nitrogen from the s o i l in the form of n i t r a t e . It must be reduced to ammonia before i t can be incorporated into the nitrogenous compounds of the p lant . It is assumed that the f i r s t step i n n i t r a t e reduction is the conversion of n i t r a t e to n i t r i t e (Devl in , 1966). The most s i g n i f i c a n t trend is the increased use of nitrogen containing f e r t i l i z e r s which, in general , r e su l t in increased n i t r a t e content of plant materials which are used as food. Publ ic concern has been aroused over the poss ible health hazard of high leve ls of n i t r a t e s found in some foods, and on the use of these foods in infant d i e t s . The term "nitrate t o x i c i t y " as commonly used, is ac tua l ly " n i t r i t e t ox i c i ty" and is produced fol lowing the reduction of n i t r a t e to n i t r i t e wi th in the g a s t r o i n t e s t i n a l t rac t or before ingest ion . Both n i t r a t e and n i t r i t e , being highly water so luble , f ree ly traverse the g a s t r o i n t e s t i n a l wal l into the blood stream. N i t r i t e , but not n i t r a t e , oxidizes the ferrous i ron of the 2 red blood pigment hemoglobin and causes methemoglobinemia (Wright and Davison, 1964). Ni trates can also in ter fere with normal iodine metabolism of the thyro id gland and resu l t in a reduction in the l i v e r storage of vitamin A (Bloomfield and Welsch, 1961). Water supplies contaminated with n i t r a t e have contributed to , or been e n t i r e l y responsible f o r , poisoning of humans and animals (Knotek and Schmidt, 1964). Fresh vegetables and some food products are found to have n i t r a t e in concentrations s u f f i c i e n t to cause poisoning, p a r t i c u l a r l y with in fants . Widely d i f f e r i n g values for n i t r a t e content of i n d i v i d u a l vegetables and food products are reported. Probably many factors both in terna l and external contribute to this v a r i a t i o n . The purposes of this study were to obtain information on (1) the n i t r a t e content of foods, (2) the effects of various factors on n i t r a t e l e v e l , and (3) the recovery of added n i t r a t e i n food. 3 LITERATURE REVIEW 1. NITRATE .CONTENT OF FOODS Public health authorities are becoming increasingly aware, of the nitrate content of water supplies. The major source of nitrate contamination in water appears to be animal and human wastes (Wright and Davison, 1964). Nitrate concentrations are higher in dug and shallow wells than in dr i l l e d wells and are higher in wells with broken casings and poor covers. Such wells have been shown to vary markedly in nitrate content from day to day. High nitrate content of the water supply has been respon-sible for many infant deaths and constitutes the major human health hazard of nitrate toxicity. Water containing 60 ppm nitrate-nitrogen is considered hazardous for use with infants and small children (Kilgore ejt a l , 1963). The presence of excessive nitrate in drinking water used to make infant formulas was noted. Five cases are said to have resulted in death (Anonymous, 1950). After this experience a maximum safety limit of 10 ppm of nitrate-nitrogen in water was set by Il l i n o i s authorities. Vegetables and some other foods may contain a rather large percentage of nitrate. Cereal seeds may be disregarded, since any danger from them is negligible (Gilbert et ^ , 1946). Nitrates in considerable quantities in vegetables have been reported by several earlier investigators. Wilson (1943) determined the nitrate content in the sap of many plants growing with abundant moisture and points out that "The nitrate 4 content o f such v e g e t a b l e s as b e e t s , b r o c c o l i , cabbage, c a u l i f l o w e r , l e t t u c e , e t c . , suggests these foods may be t o x i c at t imes to humans . " Many l e a f y v e g e t a b l e s are known to accumulate l a r g e q u a n t i t i e s o f n i t r a t e and n i t r a t e content s have been r e p o r t e d by s e v e r a l r e s e a r c h w o r k e r s . Numerous f r e s h green v e g e t a b l e s grown a t I t h a c a , New York c o n t a i n e d h i g h n i t r a t e - over 1,000 ppm o f n i t r a t e was found i n b e e t s , b r o c c o l i , cabbage, c a u l i f l o w e r , c e l e r y and head l e t t u c e and l e s s than 1,000 ppm i n c a r r o t s , cucumber, c u r l y l e t t u c e and muskmelon. Rhubarb c o n t a i n e d as much as 5,400 ppm (Anonymous, 1950) . C o n s i d e r a b l e v a r i a t i o n was found among samples , as would be expected o f p l a n t s from d i f f e r e n t s o i l s and c u l t u r a l p r a c t i c e s . Only tomatoes were found to c o n t a i n no n i t r a t e . I t i s p o s s i b l e t h a t on such a d i e t a p e r s o n , as a v e g e t a r i a n , might e a s i l y consume the e q u i v a l e n t o f one to two grams and p o s s i b l y more, o f s a l t p e t e r (potass ium n i t r a t e ) d a i l y , even w i t h p o u r i n g o f f the water d u r i n g the p r e p a r a t i o n f o r the t a b l e o f some o f these f o o d s . The presence o f n i t r a t e s i s a p o s s i b i l i t y i n V a r i o u s b o d i l y s e c r e t i o n s , p a r t i c u l a r l y m i l k ( R i c h a r d s o n , 1907) . However, the m i l k o f cows fed 114 ppm n i t r a t e - n i t r o g e n i n t h e i r water supp ly d i d not c o n t a i n n i t r a t e . There i s the p o s s i b i l i t y o f n i t r a t e a c c u m u l a t i o n i n the t i s s u e s o f animals (Wright and D a v i s o n , 1964) . N i t r a t e content o f bo th m i l k and meat from animals t h a t were fed 100 or 150 mg. n i t r a t e -n i t r o g e n per k i l o g r a m per day f o r 6 to 12 months b e f o r e s l a u g h t e r was h i g h e r than c o n t r o l . 5 Infants fed with dr ied milk where water contains a high concentration of n i t ra tes become i l l with methemoglobinemia (Knotek and Schmidt, 1964). Dried buttermilk contains microbes of l a c t i c fermentation Streptococcus l a c t i s , producing the a n t i b i o t i c n i s i n . This prevents the growth of the sporulat ing microbes of B a c i l l u s s u b t i l i s . B. subt i1 is usual ly occurs in dr ied milk and is capable of reducing n i t ra te s to n i t r i t e s . Canadian baby foods, as determined by Kamm et_ a l (1965) , contained n i t r a t e higher than those reported for New York State . For example, Canadian spinach contained 1,074-1,668 ppm of n i t r a t e and beets 634-2,165 compared to New York State which were 616-833 and 333-750 ppm of n i t r a t e re spec t ive ly . Among s tra ined baby foods (Anonymous, 1950) spinach and beets were e spec ia l ly high whereas tomato, peas and squash had no n i t r a t e . N i tra te content of vegetable products i n 1964, as reported by Jackson e_t a l (1967) , was not considered unusually h igh , beets being about 2,200 ppm, co l lards about 2,700 ppm and turnip greens about 1,500 ppm. The d i s t r i b u t i o n of n i t r a t e between s o l i d and l i q u i d portions of canned products was reported i n 1907 and 1964. With the exception of mixed vegetables, sweet corn and sweet peas, the n i t r a t e content was s l i g h t l y higher i n the l i q u i d port ion than the drained s o l i d s . Since n i t r a t e sa l t s are water so luble , some equi l ibr ium of n i t r a t e between the w e l l -drained port ion and the mother l iquor would be expected, e spec ia l ly i n canned leafy vegetables. The s ize and shape of 6 mixed vegetables, sweet corn and sweet peas, which expose less surface area to the l i q u i d than leafy vegetables, apparently are contr ibut ing factors in the lower values found in the l i q u i d port ion of these vegetables (Kilgore et_ a l , 1963) . There was no destruct ion of n i t ra tes due to cooking. It would be expected that the amount of n i t r a t e removed from the vegetable to the cooking l iquor would increase with the amount of cooking water and depend on the cooking time. (Kilgore e_t a l , 1963) . Juices of green vegetables and quick frozen foods, for example, spinach, rhubarb, ce lery and several other vegetables were frequently high i n n i t r a t e . Fresh-frozen spinach ju ice contained 6,600 ppm or 1% potassium n i t r a t e on a dry basis (Anonymous, 19 50). The quantity of n i t r a t e is least with a meat diet and greatest with an exc lus ive ly vegetable d i e t . Richardson (1907) had observed that cut beef cooked with fresh vegetables produced the color of nitrohemochromogen, ind ica t ing the presence of n i t r i t e s the same as cooking with sa l tpeter or n i t r i t e so lu t ion . Many prepared meat products such as corned beef, weiners, bologna and sausages have both n i t r a t e and n i t r i t e added to preserve color and retard spoi lage . The Meat Inspection D i v i s i o n of the U.S . Department of Agr i cu l ture (1960) establ ished the l e v e l of n i t r a t e ion in the f in i shed product to not exceed a maximum of 200 ppm or 60 ppm n i t r a t e - n i t r o g e n . Ni tra te is a potent d i u r e t i c and the kidney is an important organ for removal of n i t r a t e from the body. Some n i t r a t e can, however, be recycled from the blood into the 7 g a s t r o i n t e s t i n a l t rac t by s a l i v a r y and g a s t r o i n t e s t i n a l secretions. . N i t r i t e is found only in small concentrations in l i v i n g plant t i s sue . The toxin normally consumed, n i t r a t e , is reduced to n i t r i t e somewhere wi th in the animal body. 'The reduct ion ,o f n i t r a t e within the gas t ro in te s t ina l t rac t is a t t r ibuted so le ly to micro-organisms. Each ion of n i t r a t e that is found is a possible source of n i t r i t e that is about ten times more toxic than the n i t r a t e from which i t came. N i t r i t e , but not n i t r a t e , oxidizes the ferrous i ron of the red blood pigment c a l l e d hemoglobin to f e r r i c i r o n , producing a modified brown colored pigment c a l l e d methemoglobin which is incapable of transport ing or re leas ing oxygen to the body t i s sues . Animals so affected are said to be suf fer ing from methemoglobinemia. The hemoglobin of the newborn human and of the bovine fetus is more eas i l y oxidized to methemoglobin by n i t r i t e than that of the respective adul t . Cyanosis had been observed i n human infants ('blue babies') for a number of years . Cyanosis i s a dusky b l u i s h or purp l i sh d i s c o l o r a t i o n of skin or mucous membranes due to de f i c i ent oxygenation of the blood. Comly (1945) has described cyanosis in infants that could be corre lated with the n i t r a t e content of wel l water used to make the formula for the babies . This cyanosis was due to methemoglobinemia and i t s development was a t t r ibuted to reduction of the n i t r a t e to n i t r i t e i n the g a s t r o i n t e s t i n a l t rac t of the infant with d iarrhea . Dietary n i t r a t e interferes with vitamin A and carotene metabolism. The destruct ion of carotene was observed during an a r t i f i c i a l fermentation of corn s i lage and was more rapid 8 when n i t r a t e was added. High intakes of n i t ra te s produce i r r i t a t i o n of the g a s t r o i n t e s t i n a l t r a c t , nausea, vomitting and acute g a s t r o e n t e r i t i s , muscular weakness, bloody s too l s , i r r e g u l a r pulse , convulsions, co l lapse , and albuminous and poss ible bloody u r i n e . Sollmann (1948) has reported that 1 gm. of potassium n i t r a t e is a safe s ingle dose for humans. Five gm. per day (as the inorganic n i t r a t e sa l t ) is dangerous for adults (Steyn, 1960). According to Steyn (1960), infants are 100 times more suscept ible than adul t s . Previously Wilson (1949) claimed from theore t i ca l considerations that a toxic quantity of n i t r a t e for adults would be ingested from 3.9 oz. of rhubarb or about 2.3 oz. of spinach, turnips or c e r t a i n other vegetables. It has been found by Simon (1966) that the n i t r a t e content of fresh spinach var ied between 40 and 2,100 mg./kg. He recommended that spinach should not be given to infants in the f i r s t 3 months of l i f e , for at this age n i t r a t e can be reduced in the upper parts of the g a s t r o i n t e s t i n a l t r a c t , and because of the lowered diaphorase a c t i v i t y there is then increased s u s c e p t i b i l i t y to methemoglobinemia. The n i t r i t e is formed by b a c t e r i a l reduction of the n i t r a t e in the spinach eaten. From 1959-1965 there were cases of n i t r i t e poisoning reported i n Germany in in fants . Simon (1966) stated that infant feeding must not contain more than 300 mg. n i t r a t e per kg. Forages over 0.34 to 0.45% n i t r a t e - n i t r o g e n should be considered p o t e n t i a l l y tox ic for l ives tock and should be mixed 9 with safer feeds p r i o r to use. The d ie t should be adequate i n carbohydrate and p r o t e i n , and animals should not be permitted to get overly hungry. Abortions w i l l probably occur i n ca t t l e which were fed a higher proport ion of n i t r a t e (Wright and Davison, 1964). 2. METHOD OF ANALYSIS a) Sodium s a l i c y l a t e method Mtiller and Widemann (1955) compared four methods of n i t r a t e determination in water. They were the diphenylamine-sulphuric a c i d , bruc in - su lphur ic a c i d , sodium s a l i c y l a t e and pheno l -d i su l fon ic acid methods. The f i r s t two methods were not sa t i s fac tory for photometric determination. It was impossible to compare colors because of the high color development which needed too much d i l u t i o n . Among the methods tested, the bruc in - su lphur ic acid method gave more sa t i s fac tory resul ts than the diphenylamine-sulphuric acid method. There was no comparison in accuracy between these two and the la s t two methods which use o p t i c a l machines. The l a t t e r two methods were idea l with a high response s tra ight curve, which may + + prec i s e ly detect - 0.25 to - 0.5 mg. of n i t r a t e per l i t r e . The interferences were such that the sodium s a l i c y l a t e method was better than the phenol -d i su l fon ic ac id method. b) Rapid determination of n i t r a t e and n i t r i t e in plant  mater ia l This is the modif icat ion of Nelson et_.al, (1954). It is wel l adapted for measuring day-to-day changes in n i t r a t e or n i t r i t e , or for other appl icat ions in which the basic plant mater ia l remains e s s e n t i a l l y constant. It i s a d i a z o t i z a t i o n 10 of s u l f a n i l i c ac id and coupling with 1-naphthylamine to form a red dye. N i tra te must be reduced to n i t r i t e . The water used i n this work should be as free from copper as is poss ible and should contain less than 0.002 ppm of heavy metals. Samples containing less n i t r a t e than 10 ppm cannot usual ly be analyzed with accuracy because the t o t a l quantity of plant material must be kept low to avoid interference with the analys i s . c) Phenol -d i su l fonic ac id method This method was suggested by Barnett (1954) for n i t r a t e determination i n s i l a g e . The samples are extracted on a steam bath after dry ing . Phenol -d i su l fonic ac id is added and the. co lor is developed with ammonia s o l u t i o n . The author noted that the accuracy with added n i t r a t e showed a recovery of from 98-103%. At the same time i t was noted that various precautions must be taken i f m a t e r i a l , for example sugars, is carbonized during the treatment with the phenol -d i su l fon ic acid reagent and there is l i t t l e doubt that some of the high n i t r a t e contents recorded i n the l i t e r a t u r e owe t h e i r magnitude to this cause. d) Ni tra te ion electrode method Since the phenol -d i su l fon ic acid technique requires expensive reagents, Mayers and Paul (1968) suggested a n i t r a t e ion electrode method for n i t r a t e - n i t r o g e n determination. The technique is s i m i l a r to pH measurement. It i s claimed to be s p e c i f i c for the n i t r a t e i o n , and operates by the development of a po tent ia l across a th in layer of water-immiscible ion exchanger. These workers had d i f f i c u l t y in obtaining complete recovery of added n i t r a t e in s o i l s by this method. The recovery was less than 90% because of the lower s e n s i t i v i t y of the instrument at high n i t r a t e l e v e l s . They had an average . of 97% recovery by the pheno l -d i su l fon ic ac id method. The speed of the electrode analys is i s p a r t i c u l a r l y a t t r a c t i v e for s o i l t e s t i n g . e) Some other methods of n i t r a t e determination Kamm et a_l, (1965) mentioned a new method for the determination of n i t r i t e and n i t r a t e i n foods which would accurately determine concentration as low as 1 ppro-; 1-Naphthylamine is d iazot ized by n i t r i t e and coupled with excess amine to give 4-(1-naphthylazo)-1-naphthylamine. N i t ra te i s quant i ta t i ve ly reduced by passage through a cadmium column and determined as n i t r i t e , n i t r i t e passes through the column unal tered. Therefore, in samples containing both forms of n i trogen , n i t r a t e i s determined by d i f f erence . For co lor development the so lu t ion is placed i n the dark for two hours. At 5% sample i n water recoveries of 69% or better were obtained for a l l samples. At 25% sample i n water, only a few samples gave s a t i s f a c t o r y r e s u l t s . NumerojusO chemical methods for determination of n i t r a t e have been described i n the l i t e r a t u r e . None of them, however, have been very sa t i s fac tory for determination of n i t ra tes in s o i l and plant extracts because of the large amounts of i n t e r f e r i n g substances these extracts may conta in . Usua l ly , n i t r a t e is determined af ter chemical reduction to n i t r i t e . However, the usual reducing reagents are not s u f f i c i e n t l y s p e c i f i c , and i t is normally d i f f i c u l t to obtain quant i tat ive reduction of n i t r a t e to n i t r i t e . Lowe and Hamilton (1967) 12 mentioned a method u t i l i z i n g soybean nodule bacteroids for reduction of n i t r a t e to n i t r i t e . It was sens i t ive to as l i t t l e as 0.01 ug. of n i t r a t e - n i t r o g e n per ml. in the sample s o l u t i o n . Recovery was about 99-101%. 3. NITRATE RELATIONSHIP TO FERTILIZER PRACTICE Nearly a l l of the nitrogen needed by plants for t h e i r growth is taken from the s o i l i n the form of n i t r a t e s . A smaller quantity may be absorbed as ammonium. It is l o g i c a l to in fer that , e spec ia l ly in the e a r l i e r stages of growth, s i g n i f i c a n t amounts of n i t ra tes should be found in the p lant . As the plant matures, smaller quant i t ies of n i t ra tes should occur; however, in c e r t a i n crops, p a r t i c u l a r l y beets, consid-erable quant i t ies occur in the f u l l y matured p lants . This is more astonishing in the case of f r u i t s because during the r ipening process, the n i t ra tes must be in contact with many reducing substances (Richardson, 1907). The accumulation of n i t r a t e thus implies that the rate of a s s imi la t ion has not kept pace with the rate of uptake. In some crops the n i t r a t e content has been shown to be p o s i t i v e l y associated with ult imate y i e l d , and t issue tes t ing of these crops has been advocated as a guide to optimum f e r t i l i z a t i o n . Plant to plant v a r i a t i o n might be enormous. Ni tra te w i l l not accumulate within a plant unless the external medium can furnish i t at a rate faster than the- rate of a s s i m i l a t i o n . Numerous experiments might be c i t ed in which an increase in external nutr ient nitrogen has effected-an increase in n i t r a t e accumulation. In some cases abnormal accumulation has been associated with heavy dressing of animal 13 manure. Clean fal lowing had been shown to increase the supply of ava i lab le n i t r a t e and thereby increase the n i t r a t e content of p lants . Timing of nitrogen f e r t i l i z a t i o n has been shown to have.amarked influence on the n i t r a t e content of pasture grasses, appl icat ions soon before harvest tending to increase accumulation. Plants growing i n low n i t r a t e s o i l might under spec ia l conditions accumulate n i t r a t e to dangerous l e v e l s . (Wright and Davison, 1964). Environmental factors inc luding those which stimulate microbia l conversion influence n i t r a t e uptake. Ni trate is taken up more r e a d i l y from solut ions prepared with potassium n i t r a t e than from those prepared with calcium or sodium n i t r a t e . Wright and Davison (1964) mentioned that increases i n the l e v e l of potassium in the so lut ion promote an accumulation of n i t r a t e in oats and i n corn when n i t r a t e is also present at a high l e v e l . The accumulation of n i t r a t e might be l imi t ed by 'balancing' a high l e v e l of ava i lab le nitrogen in the s o i l with heavy appl icat ions of other plant n u t r i e n t s . Nitrogen and potassium can accumulate wi th in the plant to levels much above those needed for maximum growth. Potassium and phosphorous may encourage accumulation of n i t r a t e . Peterson (1968) reported that the importance of the N factor is greater than the importance of P and K in n i t r a t e accumulation. It is conceivable that a r e l a t i o n between P or K and n i t r a t e - n i t r o g e n could occur i f P or K were l i m i t i n g plant growth, and that addit ions of P and K caused a growth response which resulted in d i l u t i o n of the N taken up by the p lant . The effect of the P and K would be i n d i r e c t , a l b e i t present, with the major 14 influence being the N content of the s o i l . The factors that had a major influence on the concentration of n i t r a t e i n the forage studied by Crawford e_t al_ (1961), were: species , part of p lant , stage of maturity of the p lant , l e v e l of nitrogen f e r t i l i z a t i o n , and l i g h t i n t e n s i t y . The most s i g n i f i c a n t was the increased use of nitrogen containing f e r t i l i z e r s , which, i n general , resul ted i n increased n i t r a t e content of plant material as mentioned by Balks and Plate (1955), G i l b e r t et a l (1946), Hanway and Englehorn (1958), Kretschmer (1958), Whitehead and Moxon (1952) and P h i l l i p s (1968). High amounts of n i t r a t e in spinach can be a t tr ibuted to excessive use of nitrogen f e r t i l i z e r . The optimal amount of f e r t i l i z e r use should be about 80 kg. of nitrogen per hectare, but frequently in prac t i ce in agr icu l ture th is amount is great ly exceeded. The experience gained i n cases of n i t r i t e poisoning of infants from eating spinach has led to the conclusion that too much f e r t i l i z e r should not be used for growing spinach (Simon, 1966). Schuphan (1965) found 450 mg. ni trate /100 g. dry weight in a sample of spinach from a f i e l d under normal c u l t i v a t i o n , but 3,482 mg. ni trate /100 g. dry weight from a f i e l d treated with four times the normal amount of nitrogenous f e r t i l i z e r . Crawford e_t al_, (1961) found that the concentration of n i t r a t e in oat plants in the f i e l d increased almost l i n e a r l y as the l e v e l of ni trogen f e r t i l i z e r was increased up to 200 pounds of ni trogen per acre. Increasing levels of nitrogen f e r t i l i z a t i o n increased the n i t r a t e content of weeds, wheat, timothy, smooth bromegrass and orchard grass. 15 Percentage recovery in the crop of N appl ied i n the f i e l d at various leve ls was influenced by both the i n i t i a l s o i l n i t r a t e content and amount of added N (Peterson and Attoe , 1965). P r a c t i c a l l y a l l of the t o t a l ava i lab le N (added N + i n i t i a l n i t r a t e - n i t r o g e n + N mineral ized by the s o i l ) not removed by the crop was present i n the s o i l as n i t r a t e at harvest . It is frequently assumed that the port ion of f e r t i l i z e r N added to the s o i l and not recovered i n the harvested port ion of a crop is retained by the roots and s o i l micro-organisms or lo s t by leaching or as gas. Any res idua l f e r t i l i z e r N exists l arge ly in the n i t r a t e form and the amounts of th is form present i n the p r o f i l e to a depth of 21 inches at the beginning of the growing season accounted for as much as 90% of the v a r i a t i o n in N uptake by oats. There was a s imi lar c o r r e l a t i o n with corn. These f indings indicate that the i n i t i a l content of n i t r a t e - n i t r o g e n is often of considerable importance in determining the amounts of f e r t i l i z e r N required to a t t a i n given leve ls of ava i lab le N and i n c a l c u l a t i n g recovery of N by the crops. N i t ra te accumulation i n s ta lks or stems of corn, sorghum and soybean plants was studied by Hanway and Englehorn (1958). Legumes in ro ta t ion or app l i ca t ion of manure or nitrogen f e r t i l i z e r increased the n i t r a t e content of the p lants . The amount of n i t r a t e in plants depended upon the stage of maturity , the degree of drought i n j u r y , and the a v a i l a b i l i t y of nitrogen in the s o i l . N i t ra te content was decreased, but not el iminated by e n s i l i n g corn p lant s . The n i t r a t e content of several organs of plants has been 16 reported [Crawford ejt a l , (1961); Hanway, (1962); and Whitehead and Moxon, (1952)]. General ly , the stem has the highest n i t r a t e concentration followed by roots > leaves y f l o r a l par t s . The basal port ion of the stem and the lowest leaves tend to have a higher n i t r a t e concentration than the top stem port ion and highest leaves. Crawford e_t a l (1961) showed that these differences occurred i n oats over a wide range of ava i lab le N. They also showed that when the ava i lable N was replenished d a i l y , the n i t r a t e concentration of oats d id not decrease with maturity as has been normally reported. H i s t o r i c a l l y , n i t r a t e poisoning of c a t t l e has been l a b e l l e d corn s ta lk poisoning (May.o, 1895) and oat hay poisoning (Bradley et a l , 1940). Excessive amounts of n i t r a t e - n i t r o g e n in forage consumed by l ives tock may cause serious problems. The s p e c i f i c effects of the ingest ion of toxic amounts of n i t r a t e by animals was c l e a r l y described by Davidson e_t a l (1941) and has been reviewed by Wright and Davison (1964). Some of the winter annuals have been known to cause ca t t l e deaths as a re su l t of excess n i t r a t e (Kretschmer, 1958). Scattered reports have indicated an increase i n recent years i n the deaths of ruminant animals from n i t r a t e i n forage (Crawford ejt''al_, 1961). Toxic leve ls of n i t ra te s (more than 2,000 ppm) were found by Lawrence et a l (1967) during the period June 5 to July 17 in samples harvested from grass which had been f e r t i l i z e d with 300 and 375 k g . / h a . of N f e r t i l i z e r i n the spr ing . It is suggested that f e r t i l i z e r rates in excess of 225 k g . / h a . N (200 l b . / a c . N ) may re su l t i n toxic levels of n i t r a t e in intermediate wheatgrass. 17 4. NITRATE REDUCTASE The major source of nitrogen for most higher plants and many micro-organisms is n i t r a t e . The metabolic pathway in the reduction of n i t r a t e and i t s a s s imi la t ion to ammonia by higher plants was suggested to be NO^" > N0 2~ > H 2 N 2 0 2 > HONH2 > NH 3 by Hageman ejt a l (1962). Ni tra te reductase catalyzes the f i r s t step and is a flavin-dependent molybdo-p r o t e i n , which accepts electrons from reduced pyr id ine nucleot ides . The p u r i f i c a t i o n and propert ies of this enzyme which catalyzes the .reduct ion of n i t r a t e to n i t r i t e by the o x i d i a t i o n of TPNH or DPNH ( t r i - or di-phosphopyridine nucleot ides , reduced form), from soybean leaves and other higher plant species was described by Evans and Nason (1953). The n i t r a t e reduction is probably catalyzed by enzymes from the sap or from contaminating micro-organisms. As enzymes are disrupted and d i l u t e d during ex trac t ion , they may be rap id ly oxidized unless protected. Cysteine is used for th is purpose as mentioned by Hageman and Waygood (1959). Use of cysteine has also been reported for the extract ion and p u r i f i c a t i o n for n i t r a t e reductase by Nicholas and Nason (1955) . T r i s ( t r i s [hydroxymethyl] aminomethane) is also used for i s o l a t i n g and p u r i f i c a t i o n of the enzyme at optimum n i t r a t e reductase a c t i v i t y of pH 7.4. Inorganic phosphate as potassium phosphate buffer was found to be essent ia l for maximum enzymatic a c t i v i t y with a 30% increase when phosphate is used. Ni tra te reductase i so la ted from young wheat embryos shows a s p e c i f i c and absolute dependence on added DPNH (Spencer, 1959). No n i t r i t e was 18 formed i n the absence of added DPNH. Keis ter et_ a l (1960) reported the highest concentration of DPNH used was 3.3 x -4 10 M i n t h e i r experiments which may be i n s u f f i c i e n t to give a measurable rate of reaction* In further experiments _ 3 with higher concentrations of DPNH (approximately 10 M) i t was poss ible to measure a slow u t i l i z a t i o n of DPNH. Ni tra te reduction is inducible in higher plants by n i t r a t e with greater induction at higher n i t r a t e l e v e l s . ( A f r i d i and Hewitt, 1963). Ni trate induction of n i t r a t e reductase a c t i v i t y was also studied by F e r r a r i and Varner (1969). Ni tra te reductase is normally assayed by measuring n i t r i t e product ion. It is a common f inding that while n i t r a t e accumulation may occur in plants for several reasons, n i t r i t e seldom reaches appreciable concentrations and normal leve ls are low compared with n i t r a t e . Klepper and Hageman (1969) mentioned in t h e i r paper that n i t r a t e reductase was induced i n young leaves of apple seedl ings . The l e v e l of n i t r a t e reductase was highest i n the l ea f t issue although the n i t r a t e content of the roots was much higher than that of the leaves. Hageman and Flesher (1960) determined the effect of l i g h t and n i t r a t e supply on the a c t i v i t y of n i t r a t e reductase in corn. The n i t r a t e reductase a c t i v i t y decreased roughly in proport ion to the amount of shading. Both l i g h t and n i t r a t e are necessary for the formation of n i t r a t e reductase in quant i t ies required by the plant for normal growth. The complications of enzyme i n a c t i v a t i o n by use of heat was studied by Esselen and Anderson (1956). The thermal 19 res is tance of enzymes, p a r t i c u l a r l y peroxidase, varies considerably even among v a r i e t i e s of the same vegetables. The enzymes may regenerate and develop a c t i v i t y on storage even though no a c t i v i t y can be demonstrated immediately after thermal processing (Joffe and B a l l , 1962 ; Vetter e_t a_L, 1959; Wilder , 1962; Zouei l and Esse len, 1958). The thermal destruct ion and regeneration of enzymes in green bean and spinach puree was also studied by Resende e_t al_ (1969). Information on the heat s t a b i l i t y of the enzyme is necessary to assess i t s poss ible effect during process ing. I f spinach with a high n i t r a t e content is prepared and then stored at normal temperature, a large amount of n i t r i t e can develop by b a c t e r i a l reduction of n i t r a t e (Simon, 1966). 20 MATERIALS AND METHODS 1. SAMPLES FOR NITRATE DETERMINATION Samples of canned food, baby food, frozen food and fresh vegetable were obtained from markets in the Vancouver area. Some vegetables were also grown in the greenhouse. In the greenhouse experiments 6 plants of beets cv Detro i t Dark Red, 6 plants of spinach cv Long Standing Bloomdale or 3 plants of green bush beans (snap bean) cv Tender crop were s tarted in each 6-inch p l a s t i c pot. Spinach and beet were thinned to 4 plants per pot 10 days l a t e r and bean was thinned to 1 plant per pot 14 days l a t e r . a) Canned food was opened and drained to separate the l i q u i d and s o l i d port ion for ana lys i s . The l i q u i d port ion was used for determination of n i t r a t e content d i r e c t l y by the sodium-sal icy late method as recommended by Muller and Widemann (1955). The s o l i d port ion was blended to make a puree with water i n the r a t i o one uni t s o l i d to three units water. An a l iquot was used i n analysis as for the l i q u i d p o r t i o n . b) Strained baby food was d i r e c t l y used for analysis since i t was already a puree. c) Frozen food was thawed, drained and separated into l i q u i d and s o l i d portions for analysis as for canned food. d) Fresh vegetables obtained from markets were washed and b lo t ted dry with paper towels then cut into small pieces and blended with water in the r a t i o of one uni t s o l i d to four units water. e) Some vegetables grown i n the greenhouse were harvested at the time of marketable maturi ty . This was about 5 weeks for spinach leaves , 6 weeks' for beet roots and 8 weeks for green 21 beans. They were washed, d r i e d , cut and then analyzed for n i t r a t e content as for the market samples of fresh vegetables. 2. METHODS OF ANALYSIS a) Sodium s a l i c y l a t e method This was the method most frequently used. Ten gm. of the puree are mixed with not more than 90 ml. water, 0.5 ml. of 5% CuSO^ s o l u t i o n , 1 gm. of Ca(0H) 2 ? MgCO^ mixture powder (made of 1 part Ca(0H) 2 and 2 parts MgCO^ by weight), heated on a steam bath for one hour and s t i r r e d occas iona l ly . This is t ransferred to a 100 ml . volumetric f l a s k , cooled, d i l u t e d to volume and mixed. The contents of the volumetric f lask are f i l t e r e d through Whatman #4 f i l t e r paper. To an a l iquot of 1 ml . f i l t r a t e , 1 ml . of 0.5% sodium s a l i c y l a t e is added and the mixture is evaporated to dryness on a steam bath. After coo l ing , 1 ml. concentrated s u l f u r i c acid is added and this is allowed to stand for 10 minutes. Six ml. of d i s t i l l e d water are added, the mixture cooled and made a lka l ine by adding 7 ml. of 30% sodium hydroxide. A yellow co lora t ion indicates the presence of n i t r a t e . This mixture is made up to a volume of 100 ml . The absorbance is determined using the Beckman DU Spectrophotometer a,t wavelength of 420 mu. The concentration of n i t ra te -n i t rogen is determined from a standard curve. The standard curve is prepared by reading absorbance of d i f f erent concentrations of standard potassium n i t r a t e s o l u t i o n . The standard so lut ion is made by d i s so lv ing 0.01805 gm. of potassium n i t r a t e in 100 ml. of water. One ml. of this 22 standard so lut ion is equivalent to 0.025 mg. of n i t r a t e -nitrogen . Al iquots of 0, 0.4, 0.8, 1.2, 1.6 and 2 ml. are evaporated to dryness on a steam bath with 1 ml. of 0.5% sodium s a l i c y l a t e , cooled and 1 ml. concentrated s u l f u r i c ac id added. The rest of the procedure was the same as that for samples of plant mater ia l . These a l iquots would contain amounts of 0, 0.01, 0.02, 0.03, 0.04 and 0.05 mg. of n i t r a t e - n i t r o g e n re spec t ive ly . b) Rapid determination of n i t r a t e and n i t r i t e i n  plant material This is the method recommended by Woolley et a l (1960). Plant material is blended with water i n the r a t i o of 1:5. One ml. of the f i l t r a t e is added to 9 ml . of 20% acet ic acid s o l u t i o n . By the use of a measuring scoop, 0.8 gm. of a powder described l a t e r is added. The sample is then shaken for 15 seconds and s i m i l a r l y shaken two times more at 3 minute i n t e r v a l s . Then the sample is centrifuged for 3 minutes at 1,000 G. The supernatant so lut ion is poured through a small loose plug of b o r o s i l i c a t e glass wool. The l i g h t absorbance is measured at a wavelength of 5 20 mp. on the Beckman DU Spectrophotometer, and the amount of n i t r a t e ca lcu lated from a standard curve. This method can determine both n i t r a t e and n i t r i t e contents i n food, with a s l i g h t modi f i ca t ion . For the determination of n i t r a t e i n the absence of n i t r i t e and n i t r i t e i n the absence of n i t r a t e , the fol lowing reagents are used: (a) 20% acet ic ac id so lut ion containing 0.2 ppm of copper as copper s u l f a t e . 23 (b) powder described by Nelson e_t al_ (1954) : 100 gm. of barium sul fate 75 gm. of c i t r i c acid 10 gm. of manganous sul fate dihydrate 4 gm. of s u l f a n i l i c acid 2 gm. of powdered zinc 2 gm. of 1-naphthylamine For the determination of n i t r i t e in the presence of n i t r a t e the fol lowing reagents are used: (a) 20% acet ic ac id so lut ion (No copper) (b) powder mixture of 100 gm. of barium sul fate 75 gm. of c i t r i c acid 4 gm. of s u l f a n i l i c ac id 2 gm. of 1-naphthylamine The determination of n i t r a t e i n the presence of n i t r i t e requires two runs, one with reagents for n i t r a t e and the other with reagents for n i t r i t e . The water should be as free from copper as poss ib l e , so demineralized water is used. Further , the e l e c t r i c a l conduct iv i ty of the water should never be higher than can be accounted for by disso lved carbon dioxide i n equi l ibr ium with the atmosphere. The standard curve for n i t r a t e - n i t r o g e n is made by using al iquots of 0, 0.2, 0.4, "0.6, 0.8, 1.0 ml. of standard n i t r a t e s o l u t i o n . Standard nitra ; te so lut ion is made by d i s so lv ing 0.01805 gm. potassium n i t r a t e in 100 ml. of water. One ml . i s equivalent to 0.025 mg. n i t r a t e - n i t r o g e n . These al iquots 24 would be equivalent to 0, 0.005, 0.01, 0.015, 0.02, 0.025, mg. n i t r a t e - n i t r o g e n re spec t ive ly . The standard curve of n i t r i t e - n i t r o g e n is made s i m i l a r l y by using al iquots of standard n i t r i t e so lut ion of 0.0015188 gm. potassium n i t r i t e i n one l i t r e of water. One ml. is equivalent to 0.00025 mg. n i t r i t e - n i t r o g e n . This d i l u t i o n was done because higher concentration of n i t r i t e so lut ion pro-duced too dark a c o l o r . The color formed by this method is cherry -red . c) Phenol -d i su l fonic ac id method This method was described by Barnett (1954) for the determination of n i t r a t e i n s i l a g e . Procedure F i f t y gm. (or less i f i t contains higher n i t r a t e ) of plant materials or vegetables are cut into small pieces and macerated with water to make 200 gm. F i f t y gm. of this mixture is ref luxed by b o i l i n g for 5 m i n . , f i l t e r e d and washed u n t i l no n i t r a t e - n i t r o g e n is found i n the residue by tes t ing with a powder mixture described by Nelson e_t al_ (1954) . This is then made to 100 ml. Each 5 ml. of this f i l t r a t e , which would be j-j^j- x ^200*^ = IT s a m P l e , is combined with 5 ml. Ag2S0^ 5 ml . of lead subacetate, and 5 ml. alumina cream for which the t o t a l volume becomes 20 ml. The mixture is then shaken for 15 min. This is f i l t e r e d through Whatman # 50 f i l t e r paper. Each 5 ml . of the above f i l t r a t e , which would be 2"7J x F = T2~ ° ^ s a m P l e > is evaporated to dryness. Af ter . coo l ing , 1 ml. of pheno l -d i su l fon ic acid reagent is added, with almost con-tinuous s t i r r i n g for 10 min. with a glass rod. This is transferred 25 to a graduated test-tube with 20 ml. water. To the contents of the,test-tube, 10 ml. of 10% ammonia and 10 ml. of water are added, shaking after each addition. This is f i l t e r e d through Whatman # 50 and the yellow color development is read at wavelength 420 mu on the Beckman DU Spectrophotometer and compared with that obtained from a standard. A blank is prepared using 5 ml. of d i s t i l l e d water instead of the sample extract and the instrument zero is set with water. The standard curve is prepared by using standard potassium n i t r a t e solution. Reagents 1. Saturated s i l v e r sulphate solution This is made by dissolving 1.41 gm. of s i l v e r sulphate i n 100 ml. hot water and storing in an amber bot t l e , since the s o l u b i l i t y of s i l v e r sulphate i n hot water is 1.41 gm. to 100 ml. and in cold water is 0.51 gm. 2. Ammonium solution (10% W/V NH^) This is made by d i l u t i n g concentrated ammonium hydroxide i n water to make the s p e c i f i c gravity to 0.9575. 3. Phenol-disulfonic acid reagent This is prepared by dissolving 25 gm. of pure phenol i n 150 ml. of concentrated nitrogen-free sulphuric acid. To the mixture is added 70 ml. of fuming sulphuric acid. The flask and i t s contents are heated for 2 hours on a b o i l i n g water bath. 4. Lead subacetate solution Two hundred and f i f t y gm. of lead acetate is added to 750 ml. of water and 160 gm. of litharge (lead oxide). 26 The mixture is placed in a bot t l e and shaken on a mechanical shaker for 24 hours. The mixture is thereafter f i l t e r e d and the volume of f i l t r a t e made up to 1,000 ml. with d i s t i l l e d water. 5. Alumina cream A saturated so lut ion of potassium aluminum sulphate is f i r s t prepared by d i s so lv ing 12 gm. of potassium aluminium sulphate i n 100 ml. water since i t s s o l u b i l i t y is 11 gm. i n 100 ml . water. Concentrated ammonia is added with s t i r r i n g . When the mixture smells of ammonia, the addi t ion is stopped and more saturated potassium aluminium sulphate solu-t ion is added u n t i l an ac id react ion to litmus is just obtained. Shaking for an hour after preparat ion is conductive to the s t a b i l i t y of the cream as found by Barnett (1954). 6. Standard n i t r a t e so lut ion 0.072 gm. of pure potassium n i t r a t e i s d issolved i n 100 ml . of water. One ml . of this so lut ion contains 0.1 mg. n i t r a t e - n i t r o g e n . d) Ni tra te ion electrode method This is claimed to be s p e c i f i c for the n i t r a t e ion , and operates by the development of a po tent ia l across a th in layer of water-immiscible ion exchanger. The apparatus consists of a n i t r a t e ion e lectrode , a calomel reference e lectrode, and a pH meter with an expanded m i l l i v o l t sca le . The technique is s i m i l a r to pH measurement. Spinach was cut into small pieces and blended with water. The electrodes were immersed, the mixture was s t i r r e d by a magnetic s t i r r e r , and the potent ia l i n m i l l i v o l t s read within 27 15 to 30 sec. af ter d r i f t had ceased and the reading compared with a standard curve. A c a l i b r a t i o n curve was obtained using standard n i t r a t e so lut ions . The curve was prepared on semi-logarithmic paper with electrode potent ia l on the l i n e a r ax i s , and n i t r a t e concentration on the logarithmic ax i s . Recovery studies were conducted by adding potassium n i t r a t e so lu t ion to the samples used and n i t r a t e - n i t r o g e n was determined by methods noted above. 3. FACTORS AFFECTING ANALYSIS Di f ferent extract ion procedures for samples were selected in order to compare the v a r i a t i o n of n i t r a t e - n i t r o g e n contents i n samples. Absorbance measurements of color development were made against d i f f erent blanks. Reducing agent and ox id i s ing agent were selected to add to the plant extract for study of t h e i r effects on n i t r a t e contents. Buffer solutions were also used to study the effect of pH on n i t r a t e in food. The effect of pH of the sample extract ions were determined. In the case of foods containing sucrose, the sugar may affect the n i t r a t e determination therefore sucrose and n i t r a t e mixture were analyzed for n i t r a t e - n i t r o g e n . Ni tra te content in food probably var ied because of the food process ing . The effect of blanching of vegetables was therefore studied i n this research. Samples were blanched in b o i l i n g water for d i f f erent times, for example 0, 1, 3 , 5 , 10, and 20 minutes, and drained for separation of l i q u i d and s o l i d portions for n i t r a t e - n i t r o g e n determination. The s o l i d port ion was blended with some water and n i t r a t e - n i t r o g e n content was 28 determined by the sodium s a l i c y l a t e method the same as for the l i q u i d portion. Nitrate solution was also added to blanching water and nitrate-nitrogen recovery was also studied for d i f f e r e n t blanching times. 4. PLANT STUDIES a) Comparison of bean with spinach Bean and spinach were used for nitrate-nitrogen content determination with the same blanching time. Both were blanched and drained and n i t r a t e content determined i n both l i q u i d and s o l i d portions. b) Variations i n d i f f e r e n t plant parts Bean, beet and spinach were grown in the greenhouse. Spinach was harvested at marketable maturity and cut into young and old p e t i o l e s , young and old leaves, f l o r a l parts and stem. Nitrate content was determined by the soidum s a l i c y l a t e method. Beets were harvested and cut into root, stalk and leaves for ni t r a t e determination. Bean plants were divided into leaves, young and old pods, p e t i o l e , and stem and n i t r a t e determined. c) Effect of nutrient status Six plants of spinach cv Long Standing Bloomdale were started i n each p l a s t i c pot i n greenhouse and watered with 100 ml. of nutrient solution on alternate days. The nutrient solution was 5 ml. 1M KNO^ and 5 ml. l M C a f N O j ^ in water to make a l i t r e . Plants were thinned to four plants per pot two weeks l a t e r . At the 37th day, they were harvested for analysis. d) Nitrate reductase studies Spinach was grown i n a growth chamber at 16 hrs. 29 day p h o t o p e r i o d and 20°C t e m p e r a t u r e and w a t e r e d w i t h 100 ml. o f t h e a b o v e - n o t e d n u t r i e n t s o l u t i o n e v e r y d a y . P l a n t s were h a r v e s t e d 3.weeks l a t e r f o r n i t r a t e r e d u c t a s e d e t e r m i n a t i o n . P r o c e d u r e S p i n a c h l e a v e s were removed f r o m d i f f e r e n t p l a n t s and c o m p o s i t e d t o f o r m a sa m p l e . L e a v e s , a f t e r r e m o v a l f r o m p l a n t s , were immersed i m m e d i a t e l y i n c o l d (2°C) d e i o n i z e d w a t e r and. c a r r i e d t o t h e l a b o r a t o r y . The d e r i b b e d l e a v e s were b l o t t e d d r y , w e i g h e d , c u t i n t o s m a l l p i e c e s and b l e n d e d w i t h a V i r t i s "23" m i c r o b l e n d o r a t maximum s p e e d f o r 2 m i n u t e s w i t h H m l . o f c o l d (2°C) g r i n d i n g medium f o r e a c h gram o f t i s s u e . The b l e n d i n g f l a s k was k e p t i n an i c e b a t h . The homogertate was p r e s s e d t h r o u g h c h e e s e c l o t h and c e h t r i f u g e d i n a c o l d room f o r 15 m i n u t e s a t 20 ,000 G.. The s u p e r n a t a n t l i q u i d was d e c a n t e d t h r o u g h g l a s s w o o l and a s s a y e d . The homogenates and e x t r a c t s were k e p t c o l d (3-5°C) t h r o u g h o u t . The a s s a y s were c o m p l e t e d w i t h i n 2 t o 3 h o u r s a f t e r s a m p l i n g . The n i t r a t e r e d u c t a s e was measured by a m o d i f i c a t i o n o f t h e method d e s c r i b e d by Evans and Nason ( 1 9 5 3 ) . The a s s a y m i x t u r e c o n t a i n e d ' 1.0 m l . o f p o t a s s i u m p h o s p h a t e , 0.2 m l . o f KNO^, 0.5 m l . DPNH, 0.2 m l . o f enzyme e x t r a c t , and d e i o n i z e d w a t e r . t o b r i n g t h e volume t o 2.0 m l . The m i x t u r e was i n c u b a t e d a t 27°C f o r 15 m i n u t e s and t h e r e a c t i o n s t o p p e d by a d d i n g 1 m l . o f s u l f a n i l a m i d e r e a g e n t . N ^ d n a p t h y l ) e t h y l e n e d i a m i n e h y d r o c h l o r i d e r e a g e n t , (1 ml.) was added and t h e c o n t e n t s mixed by i n v e r t i n g t h e t u b e s . The c o l o r was a l l o w e d t o d e v e l o p f o r 5 m i n u t e s b e f o r e c e n t r i -f u g i n g a t 1,500 G f o r 10 m i n u t e s t o remove t h e t u r b i d i t y . The a b s o r b a n c y was d e t e r m i n e d by r e a d i n g e a c h sample a g a i n s t i t s 30 own blank ( a l l reagents except for DPNH) in the Beckman DU Spectrophotometer at 540 mu. The a c t i v i t y of the n i t r a t e reductase was expressed as u^M KNC^ as indicated by a standard curve. The standard curve was prepared by using 0.2 ml. of d i f f erent concentration of potassium n i t r i t e so lut ion in place of enzyme extract . Reagents 1. Grinding medium containing 0.1 M T r i s (Tris hydroxymethyl aminomethane), 0.01 M cyste ine , and 0.003 M EDTA (ethylenediaminetetraacetic acid) at a pH of between 7.3 and 7.8 (adjusted with HC1). 2. 0.1 M potassium phosphate buf f er . 3. 0.1 M KN0 3 . _ 3 4. 1.36 x 10 M DPNH (diphosphopyridine nucleotides reduction form) 5. 1% W/V sulfani lamide in 1.5 N HC1. 6. N - ( l napthyl) ethylene diamine hydrochloride 0.02% W/V. 31 RESULTS AND DISCUSSION 1. NITRATE CONTENT OF FOODS a) Canned Food Table 1 shows the d i s t r i b u t i o n of n i t r a t e between s o l i d and l i q u i d portions of canned products. The n i t r a t e content was frequently higher in the l i q u i d port ion than in the drained s o l i d s . Since n i t r a t e sa l t s are water so luble , much of the n i t r a t e ex i s t ing i n the s o l i d port ion of canned food would disso lve in the l i q u i d por t ion . This would ra i se the n i t r a t e content i n the l i q u i d p o r t i o n . In the case of spinach, n i t r a t e in the l i q u i d port ion was subs tant ia l l y higher than i n the s o l i d . Ni trate content was lower in the l i q u i d port ion than i n the s o l i d port ion i n bean which exposes less surface area to the l i q u i d than spinach l ea f . Apricot and peaches also have lower n i t r a t e content i n the l i q u i d port ion than i n the s o l i d p o r t i o n . Among canned vegetable foods, n i t r a t e content was highest i n spinach. According to Wilson (1949), 1 gm. of potassium n i t r a t e ingested at a s ingle meal may be t o x i c . Consuming 17 ounces of only s o l i d port ion or 10.7 ounces of l i q u i d port ion of this spinach sample would therefore poss ib ly be a toxic quantity of n i t r a t e . b) Strained baby food Ni trates themselves are r e l a t i v e l y non-toxic constituents of foods. Since n i t r i t e s are the toxic p r i n c i p l e at lower intake levels i t follows that the n i t r a t e content of food is an index of the amount of n i t r i t e which may be formed. Thus n i t ra tes represent a po tent ia l hazard. Publ ic concern has 32 Table 1 The d i s t r i b u t i o n of n i t r a t e - n i t r o g e n between s o l i d and l i q u i d portions of canned food obtained from Vancouver markets in 1968. N i t ra te -n i t rogen in mg./gm. Canned food S o l i d L i q u i d 1* 2 Aver. 1 2 Aver. Beans .102 .078 .090 .086 .054 .070 Beets (whole) .200 .180 .190 . 233 .188 . 211 Peas .053 .043 .048 .090 .086 .088 Spinach . 268 .284 .276 .411 .429 .420 Apricot (halved) .465 .435 .450 .460 .432 .446 Peaches (s l iced) .495 .417 .456 . 236 . 258 . 247 * Each sample was from a separate container obtained.at a d i f f erent time. 33 been aroused over the poss ible health hazard of high leve ls of n i t ra tes found in some foods, p a r t i c u l a r l y i n the use of these foods in infant d i e t s . Some baby foods were analyzed for n i t r a t e as shown in Table 2. Among samples of baby food analyzed in th is experiment, one sample of beet contained the highest amount of n i t r a t e - n i t r o g e n . The n i t r a t e - n i t r o g e n content in canned food was found to be highest in spinach and second highest in beet, but spinach is not found alone for baby food. The beet baby food sample studied here was found to have 0.350 mg./ gm. n i t r a t e - n i t r o g e n and should be considered unsafe in infant feeding. According to the amount of n i t r a t e in spinach for infant feeding suggested by Simon (1966) there must not be more than 0.300 mg./gm. This is because of the danger of poss ible microbia l a c t i v i t y i n storage, for n i t r i t e in toxic amounts can be formed i f there is a high n i t r a t e content. If the same condit ion happens with other foods, even the peas and carrot sample for which n i t r a t e - n i t r o g e n was 0.070 mg./gm. (equivalent to n i t r a t e more than 0.300 mg./gm.) must be considered to be toxic for infant feeding, c) Frozen food Some frozen foods were selected for determination of n i t r a t e - n i t r o g e n content. It was found that n i t r a t e -nitrogen content of frozen food was s l i g h t l y higher than in e i ther canned food or baby food. Table 3 shows the content of n i t r a t e - n i t r o g e n in frozen food. The n i t r a t e - n i t r o g e n content of the l i q u i d port ion of frozen spinach was higher than that of the s o l i d port ion after thawing. This is comparable with 34 Table 2 The n i t r a t e - n i t r o g e n content of baby food obtained from Vancouver markets in 1968. N i tra te -n i t rogen in mg /gm Food 1* 2 aver. Vegetables -Bean .163 .125 .144 Beet .350 .150 .250 Carrot .075 .083 .079 Corn (mixed with mi lk , and other; ingredients) .041 .038 .040 Peas .055 .024 .040 Peas and carrot .070 .070 .070 Spinach (mixed with mi lk , and othe ingredients) r . .113 .062 .088 Squash . 200 .138 . 169 Mixed vegetables .046 .043 .045 F r u i t -Applesauce .253 .231 .242 Apricot .185 . 201 .193 Peach .195 .185 .190 Plum .123 . .117 .120 *Each sample was from a separate container obtained at a d i f f erent time. 35 Table 3 The d i s t r i b u t i o n of n i t r a t e - n i t r o g e n between s o l i d and l i q u i d portions of frozen food obtained from Vancouver markets in 1968. Foods Ni tra te -n i t rogen in mg./gm. Whole port ion So l id port ion Liqu id port ion ! * 2 Aver. 1 2 Aver. 1 2 Aver. Bean .150 .190 .170 .140 .186 .163 .040 .055 .048 Peas .075 .090 .083 - - - - - -Spinach . 260 . 304 . 282 .240 . 280 .260 .300 .303 .302 Each sample was from a separate container obtained at a d i f f erent time. 36 canned spinach. The shape and size of bean which exposes less surface area to the l i q u i d is probably a contr ibut ing factor i n the lower amount of n i t r a t e - n i t r o g e n in the l i q u i d port ion of frozen bean as i t was i n canned food. Frozen peas d id not have much l i q u i d phase after thawing and there was i n s u f f i c i e n t l i q u i d avai lable for n i t r a t e ana lys i s . The n i t r a t e - n i t r o g e n content of frozen food was higher compared to both baby food and canned food. This would be p a r t l y due to migration of n i t r a t e into the added l i q u i d i n canned food. Storage under r e f r i g e r a t i o n may also be the cause of higher n i t r a t e - n i t r o g e n content i n food. The n i t r a t e - n i t r o g e n content of frozen spinach was found to be higher for the duration of storage under r e f r i g e r a t i o n for 8-11 days than fresh spinach, by P h i l l i p s (1968). This increase could be due to oxidat ion of amino n i trogen. N i t r i t e -nitrogen was also found to increase under re f r igera ted storage, probably because of microbia l a c t i v i t y . The conditions of s tor ing food should therefore be considered in keeping the amount of n i t r a t e - n i t r o g e n in food as low as p o s s i b l e . , d) Fresh vegetables Some fresh vegetables from markets were analyzed for n i t r a t e - n i t r o g e n content. Some vegetables were grown in the greenhouse and also analyzed for n i t r a t e - n i t r o g e n content. Table 4 shows the n i t r a t e content of vegetables from the market and from the greenhouse. The vegetables i n this case, were grown in the greenhouse with no nutr ient so lut ion or any f e r t i l i z e r . Most of the n i t r a t e - n i t r o g e n contents of vegetables obtained from the market were higher than those from the 37 Table 4 The n i t r a t e - n i t r o g e n content of fresh vegetables obtained from d i f f erent sources. Foods Ni tra te -n i t rogen in mg ./gm. 1* 2 3 4 5 • 6 7 Aver. Vegetables obtained from the market Bean .138 .070 - - - - - .104 Spinach .165 .135 .248 .098 - - - . 162 Vegetables obtained from the green-house Beet root . 212 . 198 - - - - - .205 Bean .043 .053 .016 .014 - - - .032 Spinach .027 .033 .073 .078 .038 .065 .127 .068 *Each sample was obtained at a d i f f erent time. 38 greenhouse. This d i f f e r e n c e i n n i t r a t e - n i t r o g e n content probably was due to d i f f e r e n t v a r i e t i e s of vegetables and to the p o s s i b i l i t y that market vegetables were grown wi t h some n i t r a t e f e r t i l i z e r . 2. RECOVERY STUDY D i f f e r e n t methods of n i t r a t e - n i t r o g e n determination were used f o r the recovery study. a) Sodium s a l i c y l a t e method This was.the method most f r e q u e n t l y used. The standard curve prepared was e x c e l l e n t compared to other methods stud i e d . One standard curve was prepared w i t h higher n i t r a t e -n i t r o g e n concentration while another was prepared f o r lower concentration i n order to provide a reference f o r wide v a r i a t i o n of n i t r a t e - n i t r o g e n content i n foods. Potassium n i t r a t e s o l u t i o n was added to spinach and bean f o r recovery studies as shown i n Table 5. This method gave the recoveries of n i t r a t e - n i t r o g e n i n food i n the range of 104.0 to 107.0%. The recovery of n i t r a t e - n i t r o g e n a f t e r adding potassium n i t r a t e s o l u t i o n to spinach was more v a r i a b l e than i n bean. b) Rapid de 'termination of n i t r a t e and n i t r i t e i n  p l a n t m a t e r i a l This method was used f o r comparison w i t h other methods of n i t r a t e - n i t r o g e n determination. The standard curve had to be prepared three times i n order to get a s t r a i g h t l i n e . The problem was probably caused by the powder described by Nelson e_t a l (1954) which would not mix r e a d i l y . This f a c t o r might cause v a r i a t i o n i n c o l o r development and a f f e c t the 39 Table 5 The recovery of potassium n i t r a t e added to vegetables using the sodium s a l i c y l a t e method of ana lys i s . N i tra te -n i t rogen in mg./gm. Average % recovery Food Found in sample Total found Added 1* 2 Aver. 1 2 Aver. Spinach 2.025 1.975 2.000 1.000 3 . 250 2.890 3.070 107.0 Bean .114 . 130 .122 . 200 .326 .334 .330 104.0 Laboratory dupl i ca te . 40 r e s u l t s . The spinach leaves used for the recovery study by th is method did not give a sa t i s fac tory recovery as shown in Table 6. In fac t , less n i t r a t e was found in one sample after n i t r a t e addit ion than before. The determination of n i t r a t e content in food for this research was therefore not done by this method. There was no measurable n i t r i t e in the spinach leaves. c) Phenol -d i su l fonic acid method The phenol -d i su l fon ic ac id technique requires expensive reagents, and extracts must be f i l t e r e d and evaporated to dryness before reagents for color development can be added (Mayers and Paul , 1968). Standard solutions of potassium n i t r a t e give a very s tra ight standard curve by this method. A recovery study was done with plant materials containing d i f f erent amounts of n i t r a t e . Table 7 shows the v a r i a t i o n in recoveries found. It was found that bean samples which contained lower n i t r a t e had poor recovery of added n i t r a t e and spinach samples high in n i t r a t e gave an exaggerated recovery, but for a sample of spinach lower in n i t r a t e the recovery was found to average 69.0%. A sui table range of n i t r a t e content in samples which can be analyzed by this method should be estimated and a sui table d i l u t i o n made f i r s t . d) Ni trate ion electrode method This method was suggested by Mayer and Paul (1968) for the determination of n i t r a t e in s o i l . The standard curve was prepared on semi-logarithmic paper with molari ty logarithmic and mV reading l i n e a r . The recovery was studied with one uni t 41 Table 6 The recovery of potassium n i t r a t e added to spinach using the rapid method of determination of n i t r a t e i n plant mater ia l . N i tra te -n i t rogen in mg./gm. Food Found in sample ..Added Tota l found 1* 2 Aver. 1 2 : Aver. Spinach 1.125 1.161 1.143 .025 .795 1. 275 1.035 *Laboratory dup l i ca te . 42 Table 7 The recovery of potassium n i t r a t e added to vegetables by the phenol -d i su l fon ic acid method. Food Ni t ra te -n i t rogen in mg./gm. Found in sample - Added Tota l found Average % recovery 1* 2 Aver. 1 2 Aver. Bean .0 22 .023 .023 .080 .039 .029 .034 13. 8 ** Spinach "•' 11.410 11.520 11.465 .400 14.510 14.610 14.560 773.8 Spinach .682 . 576 .629 .800 1.306 1.056 1.181 69.0 Laboratory dup l i ca te . *30 gm. of spinach used in 200 ml. water compared to 10 gm. used i n the other sample. 43 of the puree of spinach leaves with s ix ty units water. The average of the recoveries was only 42.0% (Table 8). Mayer and Paul (1968) found that the instrument is less sens i t ive at high n i t r a t e l e v e l s . The recoveries found here are rather low, but might be the re su l t of the high levels of n i t r a t e i n the spinach used or of poss ible problems inherent in the. electrometer used. 44 Table 8 The recovery of potassium n i t r a t e added to spinach using the n i t r a t e ion electrode method. Food Ni t ra te -n i t rogen i n mg./gm; Average % recovery Found in sample • Added Total found 1* 2 : Aver • 1 2 Aver. Spinach . 588 . 806 .69 7 .600 .806 1.09 2 .949 42.00 Laboratory dupl i ca te . 45 3. FACTORS AFFECTING ANALYSIS a) Time of extract ion Ni tra te solut ions of the same concentration were b o i l e d for d i f f erent lengths of time and n i t r a t e contents were found to be higher i n the so lut ion with longer time of b o i l i n g as shown i n Table 9. Almost the same resu l t s occurred on the extract ion of plant materials as shown i n Table 10. Recovery of added n i t r a t e was low with no blanching and increas with blanching up to 5 min. The apparent increase i n n i t r a t e would be at least p a r t l y due to decreased volume of so lut ion with increased b o i l i n g time. Volumes were not measured. The reason for the v a r i a b i l i t y between samples and between experi -ments (see, for example, Table 5) is not c l e a r . The n i t r a t e content of the t issue may i t s e l f be a fac tor . Vapour was. co l l ec ted with a condenser at the time of extract ion of plant materials such as bean, beet, and spinach. Ni tra te was not detectable in these condensates. This might suggest that i n b o i l i n g or extract ing materials containing n i t r a t e a l l n i t r a t e w i l l be concentrated i n the remaining materials af ter some water has been evaporated. The concentration would be higher according to the length of time in b o i l i n g or ex trac t ing . The amount of n i t r a t e detected tended to decrease when b o i l i n g time with spinach was too long. With b o i l i n g times of 10 and 20 min. the n i t r a t e detected was var iab le and lower than at shorter times of b o i l i n g as shown in Table 10. This condit ion was also found with turnip greens, c o l l a r d s , and beets as studied by Ki lgore et al_ (1963). 46 Table 9 The v a r i a t i o n of n i t r a t e - n i t r o g e n content in so lut ion with b o i l i n g time. Detai l s Time of b o i l i n g min. N i t ra te -n i t rogen in mg./gm. Average % recovery 1* 2 Aver. Potassium 0 .190 .178 .184 92.00 n i t r a t e at 1 .187 .195 .191 95.50 0.200 mg./gm. 3 .190 .198 .194 97. 00 added to 5 .197 . 203 . 200 100.00 water 10 . 206 .220 . 213 106.50 20 .279 .253 . 266 133.00 *Separate samples on d i f f erent days. 47 Table 10 The v a r i a t i o n of n i t r a t e - n i t r o g e n content in spinach with b o i l i n g time. Detai l s Time of b o i l i n g i n min. Ni tra te mg./gm. -ni trogen in Average % recovery 1* 2 Aver. Spinach 0 .248 .092 .170 Spinach with 0 .593 .660 .627 57.07 0.800 mg./gm. 1 .847 .950 .899 92.57 of n i t r a t e - 3 1.030 .950 .990 102.50 nitrogen added 5 1.080 .980 1.030 107.50 10 .740 .802 . .771 75.13 20 .950 .910 .930 95.00 *Separate samples on d i f f erent days. 48 b) Repeated extraction It was found in the previous studies that plant material with longer extraction time had more nitrate-nitrogen, so a repeated extraction of plant material was carried out. Since the method of extraction by Muller and Widemann (1955) used only the f i l t r a t e of the extraction for n i t r a t e determination, the residue and f i l t e r paper might contain some n i t r a t e . Each residue with f i l t e r paper was extracted again and analyzed for ni t r a t e content. Both spinach leaves and spinach leaves with some ni t r a t e solution added before extraction were used for this study. There was found to be some ni t r a t e l e f t over in successive residues with decreasing amounts as shown in Table 11. It might be explained that spinach tissue s t i l l retains some ni t r a t e after extractions and the tissue would release some of this n i t r a t e in the repeated extraction. I f t h i s i s not so, the ni t r a t e detected in the residue might be formed i n some way. It means in either case that n i t r a t e s t i l l exists i n spinach tissue even after cooking and draining water. c) C l a r i f i c a t i o n procedure and the use of spectrophotometer blanks As noted, the f i l t r a t e from the extraction of plant material was used for ni t r a t e determination by the sodium s a l i c y l a t e method in this research. Extraction without f i l t e r i n g was also investigated for comparison of the apparent n i t r a t e content. The extracted l i q u i d from spinach was not clear and produced a f i n a l mixture on which i t was impossible to read absorbance with the spectrophotometer. The f i n a l 49 Table 11 The v a r i a t i o n of n i t r a t e - n i t r o g e n content from the successive residues of spinach ex trac t ion . Detai l s Residues extracted Nitrate-•nitrogen in mg./gm. Average % n i t r a t e -nitrogen l e f t over on residue 1* 2 Aver. Spinach 1.920 1. 750 1. 835 1st .250 .210 . 230 12.51 + 2nd .100 .100 .100 5.48 3rd .100 .070 . 085 4.63 4th .100 .120 .110 6 .05 Spinach 2.700 2 . 660 2.680 with 1 mg./ 1st .380 .320 .350 13. 05 gm. n i t r a t e - 2nd .120 .110 .115 4.47 nitrogen 3rd .100 .100 .100 3.73 added. 4th .110 .100 .105 3.92 l a b o r a t o r y dupl ica tes . •••Compared to the f i r s t ex trac t ion . 50 so lut ion was therefore centrifuged at 1,000 G. for 3 min. after the color had been developed and the supernatant was used for the reading. The v a r i a b i l i t y of the resu l t s using th is technique is shown in Table 12. There was not very much dif ference i n resul t s between the two procedures of c l a r i f i c a t i o n . Both spinach l ea f extract and spinach l ea f extract with some n i t ra te added were s tudied. The recoveries after adding some n i t r a t e in the extract ion was rather poor by the centr i fugat ion procedure, so the f i l t r a t i o n procedure was selected for the n i t r a t e determination i n this research. Di f ferent blanks were also invest igated for t h e i r effect on the r e s u l t s . D i s t i l l e d water was usual ly used as the blank. The extract before adding reagents for color development was also t r i e d as the blank. Bean produced a turb id extract even after centr i fugat ion which could not be used for a blank for beans containing low n i t r a t e . When some n i t r a t e was added to bean, such a turb id extract could.be used as the blank, but i t resu l ted i n lower values as shown i n Table 13. Undiluted extract should not be used for a blank in the case of bean. It should be noted that the d i l u t i o n of th i s bean extract used i n actual n i t r a t e determinations was 1:100. The extract of spinach was c l earer than bean but the resu l t s using i t s own extract as a blank were s t i l l lower than that using d i s t i l l e d water as a blank. The resul t s were also d i f f erent when the points of adding n i t r a t e were v a r i e d . When adding n i t r a t e to bean before heating for ex trac t ion , more n i t r a t e could be recovered as i t was found that heating apparently made the t issue release n i t r a t e . The n i t ra te detected from the bean sample with n i t r a t e added after heating was lower perhaps because the t issue Table 12 The v a r i a t i o n of n i t r a t e - n i t r o g e n content using d i f ferent procedures of c l a r i f i c a t i o n of spinach extract . Procedure Ni tra te -n i t rogen in mg./gm. Average % recovery Found in sample Total found af ter adding n i t r a t e - n i t r o g e n 1 mg./gm. 1* 2 Aver. 1 2 Aver. F i l t r a t i o n Centr i fugat ion 2.025 2.0 25 1.975 1.975 2.000 2.000 3.250 2.665 2 .890 2.600 3.070 2.633 107.00 63. 25 l a b o r a t o r y dupl icates . 52 Table 15 The recovery of n i t r a t e - n i t r o g e n when d i f f erent blanks were used and with n i t ra tes added to d i f f erent species at d i f f erent times. Deta i l s N i t ra te -n i t rogen found in mg./gm. ,Average % 1* 2 Aver. recovery Bean samples water as blank .114 .116 .155 -extract as blank - - - -adding n i trate - ; ni trogen 0.2 mg./gm. before heating water as blank .326 .314 .320 102.50 extract as blank .204 .190 .197 -after heating water as blank .304 .292 .298 91.50 extract as blank .180 .174 .•17 7 -Spinach samples water as blank 1.650 1.680 1.665 -extract as blank 1.540 1.570 1.555 -adding n i t r a t e -nitrogen .1 mg./gm. before heating water as blank 2 .430 2.520 2.475 80.50 extract as blank 2.500 2.500 2.500 94.50 af ter heating water as blank 2.630 2.670 2.650 98.50 extract as blank 2.550 2 .600 2.575 102.00 l a b o r a t o r y dupl ica tes . 53 apparently ti e d up some of the added n i t r a t e . This did not happen with spinach. The recovery of n i t r a t e added after the tissue had already been heated was s l i g h t l y higher than that added before heating. This indicated the v a r i a t i o n of tissue properties among d i f f e r e n t species of plants. Some reagents such as CuSO^ solution and Ca(OH)2-MgCO\ mixture used i n the extraction were also investigated for their e f f e c t on the v a r i a b i l i t y of the r e s u l t s . These reagents were heated alone without plant materials using the same procedure as extracting with plant material. The color development was measured and interpreted i n terms of nitrate-nitrogen content. These reagents produced only clear solution observed v i s u a l l y after reagents for color development were added. The solution produced color as nitrate-nitrogen only equivalent to 0.001 mg. r in the same volume of color solution usually used i n the experiments for n i t r a t e determination. So i t could be said that there was no effect of these reagents i n i n t e r f e r i n g with the nitrate-nitrogen r e s u l t s , d) Reducing agents Ascorbic acid and oxalic acid are supposed to be present i n plant tissue such as spinach leaves. They are reducing agents and might cause some v a r i a t i o n i n the n i t r a t e determination. Different amounts of ascorbic acid and oxalic acid were added to n i t r a t e solution and the recovery of n i t r a t e was determined as shown in Table 14. Only small variations of n i t r a t e recoveries occurred when various amounts of oxalic acid and ascorbic acid were added to n i t r a t e solution. It might be concluded that there was no effect of these reducing 54 Table 14 The ef fect of reducing agents on n i t r a t e - n i t r o g e n determination. Deta i l s M l . of acid added Ni tra te -n i t rogen found in mg./ml. n i t r a t e so lu t ion Average % recovery 1* 2 Aver. 0.0 25 mg./ml. 0 .026 . 027 .027 106.00 oxa l i c ac id was added to 1 ml. 0.2 .026 .026 .026 104.00 n i t r a t e so lu t ion containing n i t - • 0.4 .026 .026 .026 104.00 rate -n i trogen 0.0 25 mg./ml. 0.6 .025 .026 .026 102.00 0.8 .026 .026 .026 104.00 1.0 .026 .025 .026 102.00 0.025 mg./ml. 0 .025 .025 .025 100.00 ascorbic acid was added to 1 0.2 .025 .025 .025 100.00 ml . n i t r a t e so lu t ion con- 0.4 .025 .027 .026 104.00 ta in ing n i t r a t e -nitrogen 0.025 0.6 .025 .025 .025 100.00 mg./ml. 0.8 .026 .026 .026 104.00 1.0 .024 .024 .024 96.00 *Laboratory dupl i ca tes . 55 agents on n i t r a t e determination, at least at the concentration used i n the present study. Oxal ic ac id was also added to spinach l ea f extract i n this study. It did not have very much effect on the r e s u l t s . Addi t ion of oxa l i c acid is compared with n i t r a t e detect ion from spinach l ea f extract without added oxa l ic ac id as shown in Table 15. This should be considered as the same resul t s as for reducing agents added to n i t r a t e so lu t ion which had almost no effect on n i t r a t e determination. Ascorbic acid is known to be very unstable . The addi t ion of ascorbic acid to plant extract probably does not need to be s tudied . e) Oxid i s ing agent Since reducing agents had been invest igated for any effect on analysis for n i t r a t e , the effect of ox id i s ing agent should also be considered. Hydrogen peroxide was selected as an ox id i s ing agent which might exis t i n plant t issue na tura l ly or which might be contacted l a t e r during process ing. Hydrogen peroxide was added to spinach puree both before and af ter the procedure of ex trac t ion , i n order to f ind the effect of an o x i d i s i n g agent on the determination of n i t r a t e content. Spinach samples used were grown i n the growth chamber with watering every day with n i t r a t e s o l u t i o n . Table 16 shows the resul ts of this inves t i ga t ion . The resul t s were almost the same as reducing agent effect in which seemingly there was a- small increase i n the n i t r a t e - n i t r o g e n content in the ana lys i s . An increase in n i t r a t e - n i t r o g e n content by the use of ox id i s ing agent occurred i n some samples. As shown i n the tab le , only 56 Table 15 The effect of oxa l i c acid on n i t r a t e - n i t r o g e n . determination in spinach. Food Ni tra te -n i t rogen in mg./gm. Average % recovery Found i n sample Total found after adding oxa l i c acid (1 mg./gm.) 1* 2 Aver. 1 2 Aver. Spinach 1.900 2.060 1.980 2.060 2.100 2.080 105.18 Laboratory dupl icates . Table 16 The effect of an oxidising agent on nitrate-nitrogen determination in spinach. Details Nitrate-nitrogen i n mg./gm. Found before adding H 20 2 Aver, Total found after adding H2°2 Aver. Average % change Adding 0.1%' H 20 2 before heating after heating Adding 1.0% H 20 2 before heating after heating Adding 2 mg. of ni t r a t e -nitrogen and H 20 2 after heating 1. 800 1.800 IV 800 1.800 3.880 2.000 2.000 2: 000 2 .000 3 .100 1.900 1.900 1.900 1.900 3 .490 1.980 1.9-00 1.950 1.980 4.000 1.900 1.900 2.190 2.080 3.880 ^Laboratory duplicates 1.940 1.900 2.070 2.030 3.940 102.50 100.28 108.92 107.00 114.13 58 one sample with added n i t r a t e so lut ion showed a change of 114.13% which was a l i t t l e higher than other samples. An ox id i s ing agent might also be discounted as an i n t e r f e r i n g agent in n i t r a t e determination. f) Buffer solutions Buffer solut ions were used for studies of effects of pH on n i t r a t e determination. The same amount of n i t r a t e was added to buffer solutions at d i f f erent pH values, these were b o i l e d for d i f f erent times and n i t r a t e contents determined. The pH of the so lut ion was measured after adding n i t r a t e so lu t ion and b o i l i n g . The resul t s are shown in Table 17. Among three d i f f erent pH values n i t r a t e - n i t r o g e n was found to be highest i n the lowest pH 5.4 and lowest i n the middle pH 6.66. It was found that with longer b o i l i n g , pH and volume of buffer so lu t ion usual ly decreased because of the evaporation of some of the l i q u i d . It was found before by Ki lgore e_t al_ (1963) that no n i t r a t e is l o s t during cooking. Since the volume of buffer so lut ion was decreased on b o i l i n g , n i t r a t e content found should have increased, but d id only at pH 5.4 g) pH and t o t a l a c i d i t y i n spinach Because some v a r i a t i o n of apparent n i t r a t e - n i t r o g e n was caused by pH, the pH and t o t a l a c i d i t y of spinach were studied to f ind i f any interference might be occurring in the n i t r a t e determination i n spinach. Spinach leaves were blanched for d i f f erent lengths of time, drained and both l i q u i d and s o l i d portions used as separate samples for measuring pH and t o t a l a c i d i t y . The s o l i d port ion was blended and made to 100 ml. with d i s t i l l e d water, then f i l t e r e d . Total a c i d i t y was 59 Table 17 The v a r i a t i o n of nitrate-nitrogen content in buffer solutions with b o i l i n g . pH of buffer solution B o i l i n g time -min. pH Volume i n ml. Average % recovery of n i t r a t e -nitrogen 1* 2 Aver. 1 2 Aver. 7.20 0 7 . 20 7 . 20 7.20 100 100 100 91 1 7.15 7.15 7.15 90 88 89 91 3 7.20 7.10 7.15 88 82 85 80 5 7.00 6.90 6.95 90 90 90 70 10 6.90 6.60 6.85 90 86 88 82 20 7.00 7 .10 7 . 05 88 86 87 90 6.66 0 6.66 6.66 6.66 100 100 100 110 1 6.64 6.66 6.65 94 96 95 89 3 6.65 6.65 6.65 95 95 95 83 5 6.65 6.65 6.65 96 94 95 88 10 6.64 6.66 6.65 95 95 95 55 20 6.64 6.66 6.65 95 95 95 87 5.40 0 5.40 5.40 5.40 100 100 100 108 1 5.40 5.40 5.45 93 99 96 119 3 5.45 5.45 5.45 95 95 95 125 5 5.40 5.40 5.40 97 93 95 120 10 5.45 5.35 5.40 98 94 96 125 20 5.40 5.40 5.40 93 93 93 124 Conducted on d i f f e r e n t days. 60 determined by t i t r a t i o n with O.iNNaOH to pH 8.1 and ca lculated as oxa l i c a c i d . Table 18 shows the v a r i a t i o n of pH and t o t a l a c i d i t y of spinach during blanching. The pH of the l i q u i d portions gradually decreased with increased time of blanching. The pH of the s o l i d portions increased at f i r s t and f i n a l l y decreased but was s t i l l higher than the pH of the l i q u i d port ions . Tota l a c i d i t y of the l i q u i d portions increased with increasing time of blanching and then decreased a l i t t l e at the 20 min. blanching time, while s o l i d portions decreased general ly as the blanching time increased. As noted i n Table 17, the apparent n i t r a t e - n i t r o g e n contents in buffer solut ions at d i f f erent pH values were d i f f e r e n t . Table 18 shows the effect of blanching on pH of spinach. pH changes might therefore affect the resul t s of n i t r a t e determination in spinach. Another experiment was c a r r i e d out to determine the re la t ionsh ip of n i t r a t e content, pH and blanching time of spinach as shown i n Table 19. The n i t r a t e - n i t r o g e n content was found to be higher i n the l i q u i d port ion than i n the s o l i d port ion the same as the d i s t r i b u t i o n of n i t r a t e between s o l i d and l i q u i d portions of canned products as noted by Jackson et a l (1967). Ni trate increased in l i q u i d port ion with increased blanching time as was noted by Ki lgore et a l (1963) . The increase of the n i t r a t e - n i t r o g e n content in the l iquor according to increased blanching time was found to be i r r e g u l a r and the t o t a l amount of n i t r a t e - n i t r o g e n in l i q u i d and s o l i d portions sometimes was higher and sometimes was lower than the amount found i n the uncooked sample. This might be p a r t l y the effect of pH v a r i a t i o n during blanching as 61 Table 18 The v a r i a t i o n of pH and t o t a l a c i d i t y of spinach during blanching. Deta i l s Blanching time -min. So l id port ion L i q u i d port ion 1* 2 Aver 1 2 Aver. PH 0 6.25 6.30 6.28 - _ _ 1 6.60 6.60 6.60 6.50 6.55 6.53 3 6.60 6.10 6.35 6.45 6.50 6.48 5 6.55 6.65 6.60 6.55 6.55 6.55 10 6.55 6.65 6.60 6.55 6.45 6.50 20 6.55 6.55 6.55 6.45 6.45 6.45 % t o t a l a c i d i t y 0 2.34 1.67 2.01 - - -1 1.33 . 1.00 1.17 1.33 1.33 1.33 3 0.67 1.00 0.84 1.00 1.50 1.25 5 1.00 0.83 0.92 1.67 1.33 1.50 10 1.00 0.83 0.92 2.00 1.67 1.84 20 0.67 0.67 0.67 1.67 1.50 1. 59 *Separate samples on d i f f erent days. Table 19 The v a r i a t i o n of pH and the n i t r a t e - n i t r o g e n content of spinach during blanching. (10 gm. of spinach leaves used for each sample) Blanching So l id port ion pH Aver. Mg. of n i trate-nitrogen per sample Aver L iqu id port ion ph Aver. Mg. of n i trate -nitrogen per sample 6.45 6.45 6.55 6.60 6.55 6.50 6.45 6.45 6.45 6.60 6.65 6.60 6.45 6.45 6.50 6.60 6.60 6.55 0.37 .14 . 20 .13 .19 .05 0.33 .12 .16 .13 .17 .09 .35 .13 .18 .13 .18 .07 6.45 6.50 6.45 6.45 6.40 6.45 6.46 6.50 6.35 6.30 6.45 6.48 6.48 6.40 6.35 .27 .40 .17 .36 .36 .23 .36 .23 .34 . 28 ^Laboratory dupl icates . 63 was noted before . The nonuniform samples might be another cause of the var ia t ions which were noted by Wright and Davison (1964). They found that n i t r a t e is not uniformly dis tr ibuted. throughout the various plant t i s sues . Spinach samples used here were co l l ec ted from many d i f f erent leaves, so the v a r i a t i o n of n i t r a t e amount among samples must have occurred to some extent. It was d i f f i c u l t to d i s t r i b u t e leaves to make uniform samples which would have about the same n i t r a t e content. Ki lgore et a l (1963) suggested the organizat ion of samples only to minimize the var ia t ions and not to el iminate v a r i a b i l i t y , h) pH i n beet Beet was also studied as for spinach. The root of beet is usual ly used as food but some people eat beet leaves as w e l l , so beet leaves and stem were also inves t igated . Beet p lant s , grown i n the greenhouse and harvested at 40 days of age, were divided into 3 parts : leaves, stem and root . Each was blanched and drained to obtain separate samples of l i q u i d and s o l i d por t ions , i t s pH and n i t r a t e - n i t r o g e n content was recorded as shown in Table 20. The pH values of a l l l i q u i d portions were more var iab le than of s o l i d portions with blanching for d i f f erent lengths of time. N i t ra te -n i t rogen was also transferred to the l i q u i d port ion in a l l three parts of beet and increased the n i t r a t e content in the l i q u i d portions with increased blanching time. The n i t r a t e - n i t r o g e n content of the s o l i d port ion was i r r e g u l a r , p a r t i c u l a r l y i n the l eaf . N i tra te became higher at 20 min. time of blanching after i t had been decreasing at shorter times. This might be due to Table 20 The v a r i a t i o n of pH and the n i t r a t e - n i t r o g e n content of beet during blanching. So l id port ion Liquid port ion Blanching time -min. pH Mg. of n i t r a t e -nitrogen per s amp1e pH Mg. of n i t r a t e -nitrogen per s amp1e 1* 2 Aver. 1 '2 : Aver. 1 2 ; Aver. 1 ; 2 ; Aver. Leaves 20 gm. 0 6.00 6.00 6.00 .780 .820 .800 - - - • - - -1 5.80 5.90 5.85 1.000 1.400 1.200 6.20 6.10 6.15 .540 .460 .500 3 5.85 5.85 5.85 1.010. 0.930 .970 6.00 5.90 5.95 .640 .620 .630 5 5.85 5.7 5 5.80 .820 .900 .860 6.00 6.00 6.00 .690 .670 .680 10 5.75 5.85 5.85 .650 .610 .630 5.85 5.85 5.85 1.000 1.000 1.000 20 5.80 5.90 5.85 1.150 1.350 1. 250 5.85 5.8 5 5.85 1.100 1.060 1.080 Stem 10 gm. 0 5.80 5.90 5. 85 .800 .840 .820 - - - - - -Cont'd Table 20 Cont'd S o l i d port ion L i q u i d port ion Blanching time pH Ag. of n i t r a t e -l i trogen per ; amnl e pH 4g. of n i t r a t e -l i t rogen per 5 amp1e -mm. 1* 2 Aver. 1 2 Aver. 1 2 Aver. 1 2 Aver. Stem 10 gm. 1 5.75 5.65 5.70 .800 .800 .800 6 . 25 6.35 6.3C .450 .490 .470 3 5.70 5.70 5.70 .480 . 520 .500 6.15 6.15 6 .1£ .770 .730 .750 5 5.70 5.70 5.70 .640 .660 .650 6.05 6.05 6.0 5 .460 .440 .450 10 5.70 5.70 5.70 .750 .710 .730 6.00 5.90 5.95 .570 . 530 . 550 20 5.75 5.65 5.70 .600 . 580 . 590 6.00 5.90 5.95 1.000 .940 .970 Root 10 gm. 0 6.20 6.20 6.20 1.100 .900 1.000 - - -1 6. 20 6.10 6.15 .900 .740 .820 6.35 6.25 6.30 1. 200 1.160 1.180 3- 6.35 6.35 6.35 .650 .690 .670 6.25 6.25 6.25 1.160 1.040 1.100 5 6.35 6.35 6.35 .650 .670 .660 6.25 6.25 6.25 .700 .660 .680 10 6.35 6.15 6.25 .720 .690 . 710 6.20 6.30 6.25 .400 . 260 .330 20 6.25 6.25 6.25 .750 . 770 .760 6.30 6.20 6.25 1.700 1.680 1.690 ^Separate samples on d i f f erent days. 66 the succulence of the leaves which would be able to r e t a i n n i t r a t e - n i t r o g e n content after soaking longer at longer blanching times. The occurrence of these var iab le resul ts might be also explained as for spinach for which i t d id not. seem to be poss ible to make up uniform samples to obtain the same amount of n i t r a t e - n i t r o g e n in samples before blanching, i ) Sucrose Some canned food and baby food samples were found to have sugar i n the range of 8.2 - 27.2%. Some fresh and frozen vegetables might also contain some sugar. Sugar was found to be carbonized during the treatment with the reagent used i n the n i t r a t e determination by the phenol -d i su l fon ic ac id method and erroneous information on the n i t r a t e determination of the product containing sugar might be obtained (Barnett , 1954). Sugar might affect n i t r a t e deter-mination by the sodium s a l i c y l a t e method as w e l l . Table 21 shows the comparison of n i t r a t e - n i t r o g e n recovered from water and sucrose so lut ion which had been added to the same amount of potassium n i t r a t e s o l u t i o n . The recovery from sucrose so lu t ion was higher than 100% and higher than recovery from water. This showed that sugar did affect the n i t r a t e determination r e s u l t s , and the degree of affect depended on the concentration of sugar present i n the sample, j) Blanching Some food obtained from plants might be cooked before being ready for the tab le . The food-constituents can poss ibly be changed by such cooking procedure. The effect of blanching and cooking of some vegetables was observed. Some of the 67 Table 21 The comparison of n i t r a t e - n i t r o g e n recoveries from water and sucrose so lu t ions . Mg. n i t r a t e - n i t r o g e n per Detai l s Found Average % recovery Added 1* 2 Aver. Water .020 .021 .021 .021 105.0 .100 .100 .100 . .100 100.0 .200 .175 .190 .183 91.3 Sucrose 11.25% .020 .086 .069 .078 390.0 Sucrose 10%"" .100 .120 .115 .118 118.0 <-.200 .180 .190 .185 92.5 Sucrose 15% 0 .063 .060 .062 Sucrose 10% 0 .025 .030 .028 Sucrose 5% 0 .030 .028 .0 29 Sucrose 1% 0 .015 .016 .016 ^Laboratory dupl ica tes . 68 n i t ra tes of most vegetables selected for this study transferred from the vegetable to the cooking water during blanching. The amount of transfer depended on species , shape and s ize of vegetables, and the time used in cooking. The increase of the n i t r a t e - n i t r o g e n content in the l iquor with time of blanching was i r r e g u l a r as was noted before i n the study of pH and t o t a l a c i d i t y in spinach. This was also found i n other vegetables s tudied. The i r r e g u l a r i t y of the n i t r a t e t rans ferr ing might be the re su l t of some causes found before. They were the d i f f erent pH of cooking mixtures, the sugar content, the natural v a r i a b i l i t y of the samples, the time of cooking and so on. The fact found by Ki lgore ejt a l (1963) was that n i t r a t e was not los t by cooking. Even with pouring of f cooking water, some n i t r a t e s t i l l exists in food. The time of cooking should be found which minimizes the amount of n i t r a t e i n food. This means that some other propert ies and behavior of food should be known and considered at the same time to f ind a sui table i n d i v i d u a l condit ion for cooking each food. The fol lowing Table 22 i l l u s t r a t e s the v a r i a t i o n of n i t r a t e - n i t r o g e n t rans ferr ing from vegetables to cooking water at d i f f erent times of blanching. In some the t o t a l amount of n i t r a t e - n i t r o g e n in s o l i d port ion and l i q u i d port ion is found to be higher than in the uncooked sample. In spinach there is a marked increase i n t o t a l apparent n i t r a t e - n i t r o g e n with blanching, but th is does not occur in bean. 4. PLANT STUDIES a) Comparison of spinach and bean Table 22 shows the comparison of n i t r a t e - n i t r o g e n Table 2 2 The v a r i a t i o n of n i t r a t e - n i t r o g e n trans ferr ing from s o l i d to l i q u i d port ion during blanching of vegetables. Mg... of n i t r a t e - n i t r o g e n per sample Detai l s Blanching time So l id port ion L i q u i d port ion Average - • mm. 1* 2 Aver. 1 2 Aver. t o t a l Spinach 50 gm. 0 10. 125 7.875 9 .000 - - - 9.000 1 3. 360 11.500 7.430 12.150 14.000 13.075 20.505 —- 3 3. 150 5.000 4v325 16.000 10.500 13 . 250 17 .575 5 2. 800 5.500 4.150 11.500 13.500 12 .500 16.650 10 .3. 250 3. 750 3.500 10.000 12.000 11.000 14.500 20 2. 500 3.000 2. 750 8.250 13.000 10.625 13.37 5 Bean 10 0 gm. 0 13. 500 6.750 10.125 - • - - 10.125 1 7. 500 5.000 6.250 5.850 2.700 4.275 10.525 3 7. 750 3.500 5. 625 8.130 2.700 5.415 . 11.040 5 6. 600 3.400 5.000 7.000 3.250 5.125 10.125 10 7. 000 4.500 5.750 7.000 3.000 5.000 10.750 20 8. 250 4.250 6. 250 8.500 3.500 6.000 12.250 Water with n i t r a t e -nitrogen added 40 mg. per 0 1 3 - - -3 8 . 0 0 0 37",. 4 0 0 38.000 35.600 39.000 39 .600 36.800 38.200 38.800 36.800 38.,l200 38.800 sample - 5 - - - 38.400 40.600 39.500 39.500 Cont'd Table 22 - Cont'd Detai l s Blanching time mm. Mg. of n i t r a t e -ni trogen per sample S o l i d port ion L Lquid port ion Average t o t a l 1* 2 Aver. 1 2 Aver. Water, etc . 10 - - - 41 . 200 44.000 42.600 42.600 .20 - - - 55 .800 50.600 53.200 53.200 Spinach 50 gm. 0 L2 . "00 4.900 8.650 - - - 8. 650 Spinach was 0 29.650 33.000 31.325 - - - 31.325 added with 1 5.750 7.500 6.625 36 .600 40 .000 38.300 44.925 n i t r a t e -nitrogen 3 9.500 ' 7.500 8.500 42 .000 40.000 41.000 49.500 40 mg. per 5 10.000 10.000 10 .000 44 .000 39 .000 41. 500 51.500 sample 10 7 .000 7. 500 7 .250 30 .000 32.600 31.300 38.550 20 10 .500 8 . 500 9.500 37 .000 37.000 37.000 46.500 Separate samples on d i f f erent days. 71 content of spinach and bean. Even 100 gm. of bean sample contained less n i t r a t e than 50 gm. of spinach sample af ter b lanching. The n i t r a t e transferred from the plant t issue of spinach to the cooking water was more than of bean. The shape and s ize of plant are probably the causes of v a r i a t i o n i n n i t r a t e t rans fer . The bean has a protect ive epidermal layer and exposes less surface/area than spinach to cooking water. Blanching time affected n i t r a t e t rans ferr ing from spinach more than from bean. Ni tra te content i n s o l i d and l i q u i d portions of bean were almost equivalent , while n i t r a t e in the l i q u i d port ion of spinach was much higher than in the s o l i d p o r t i o n . b) Var ia t ions in d i f f erent plant parts Crawford et a l (1961), Hanway (1962) and Whitehead and Moxon (1952) determined the var ia t ions in n i t r a t e contents of several p lant organs. General ly , the stem has the highest n i t r a t e concentration followed by roots , leaves and then f l o r a l p a r t s . The basal port ion of the stem and the lowest leaves tend to have a higher n i t r a t e concentration than the top stem port ion and highest leaves. Di f ferent parts of bean, beet and spinach were d i s t r i b u t e d into separate samples. The v a r i a t i o n of n i t r a t e in d i f f erent parts of bean, beet and spinach are shown i n Table 23. Spinach was found to have n i t r a t e concentration in d i f f erent parts i n agreement with the e a r l i e r workers, that is n i t r a t e i n stem was greater than i n leaves which was greater than in f l o r a l par t s . In contrast young leaves and young pet io les were found to have higher n i t r a t e than o ld ones. This might have 72 Table 23 The v a r i a t i o n of n i t r a t e - n i t r o g e n i n d i f f erent plant p a r t s . Mg. of n i t r a t e - n i t r o g e n per gm. Plant parts Bean Beet Spinach 1* 2 Aver. 1 2 Aver. 1 .2 Aver. Root - - .110 .090 .100 - - • • -Stalk or stem .030 .019 .025 .080 .084 .082 .088 .138 .113 Pet io les o ld .038 .029 .033 - - - .060 .058 .059 young .042 .030 .036 - - - .160 .120 .140 Leaves o ld .037 .040 .038 .039 .041 .040 .073 .038 .056 young .053 .039 .049 - - - .078 .065 .072 F l o r a l or pod old . 033 .042 .038 - - - - - -young .016 .014 .015 - .034 .044 .039 Laboratory dupl ica tes . 73 been due to the se l ec t ion of samples. Old leaves and pet io les used here were almost yellow and less n i t r a t e was found. It was the same with bean. Bean was found to have less n i t r a t e i n the stem than i n the leaves and f l o r a l p a r t s . The stem . ' -of bean was probably too tough to be extracted completely. Bean f l o r a l parts tend to have higher n i t r a t e in old pods than young pods which agrees with the l i t e r a t u r e . Beet n i t r a t e var ia t ions were d i f f erent from the l i t e r a t u r e on n i t r a t e in some plants i n that n i t r a t e concentration was higher in root than s ta lk and s ta lk higher than leaves. The n i t r a t e d i s t r i b u t i o n of a l l plants was not found to be the same, i t appeared to vary with species . c) Ef fect of nutr ient status It was noted by Brown and Smith (1966) that nitrogen f e r t i l i z a t i o n caused a s i g n i f i c a n t increase in n i t r a t e content in red radishes , ka le , mustard and turnip roots and tops. This was also found with the spinach studied here. Each pot of 4 spinach plants was watered with n i t r a t e so lu t ion from plant ing to harvest time with a t o t a l n i t r a t e -nitrogen appl ied of 305 mg. per pot. Table 24 shows the recovery of n i t r a t e - n i t r o g e n from this spinach compared to those grown without f e r t i l i z e r at the same time. N i t r a t e -nitrogen was found to be higher in the f e r t i l i z e d p lant s , but nitrogen f e r t i l i z e r added was not a l l recovered in the harvested spinach. Peterson and Attoe (1965) noted that the unrecovered f e r t i l i z e r was retained by the roots and s o i l micro-organisms or los t by leaching or as gas. N i t r a t e -nitrogen content found i n spinach with added nitrogen 74 Table 24 Ni tra te -n i t rogen content in d i f f erent parts of spinach, with and without nitrogen f e r t i l i z a t i o n . Parts of plant Mg of n i t r a t e - n i t r o g e n per gm. Average times increased by f e r t i l i z a t i o i No f e r t i l i z e r With f e r t i l i z e r+ - . : 1* 2 Aver. 1 2 Aver. Stem .086 .140 .113 2.531 2.303 2.467 21.08 Pet io le .204 .194 .199 3.271 3.129 3 . 200 16.08 Leaves .134 .120 .127 1.234 1.428 1.331 10.55 F l o r a l .042 .036 .039 . 721 .821 . 771 19 .99 l a b o r a t o r y dupl ica tes . + F e r t i l i z e r was made of 5 ml. of 1 M CafNO^),, and 5 ml. of 1 M KN03 in a l i t r e of water. T o t a l l y each pot of 4 plants received 305 nig. n i t r a t e - n i t r o g e n . 75 f e r t i l i z a t i o n was as much as 21.08 times higher in stem, 10.55 times i n leaves and 19.99 times in f l o r a l parts than i n u n f e r t i l i z e d p lants . This means that nitrogen f e r t i l i z a t i o n increased n i t r a t e accumulation in spinach, d) Ni tra te reductase studies As noted most plants absorb nitrogen from the s o i l i n the form of n i t r a t e and i t must be reduced to ammonia before i t may be incorporated into the nitrogeneous compounds of the p lan t . The f i r s t step in n i t r a t e reduction is the conversion of n i t r a t e to n i t r i t e . The n i t r a t e poisoning is p r i m a r i l y due to the t o x i c i t y of n i t r i t e . N i t r i t e may be formed i n food before ingest ion or wi th in the digest ive t r a c t . N i tra te i n food may be reduced by bac ter ia to n i t r i t e during storage. N i tra te reductase is an enzyme capable of cata lyz ing this reduction (Evans and Nason, 1953). N i tra te reductase was studied here to invest igate the poss ible n i t r i t e formation i n food, such as vegetables, which may reach consumption. That n i t r a t e induces the formation of n i t r a t e reductase a c t i v i t y i n barley was found by F e r r a r i and Varner (1969). Spinach grown i n the growth chamber with n i t r a t e so lu t ion appl ied was found to have some n i t r a t e reductase a c t i v i t y . The a c t i v i t y was determined by measuring n i t r i t e formation. The measurement was compared to the standard curve prepared from potassium n i t r i t e . The n i t r a t e reductase a c t i v i t y of spinach used i n this experiment was found to have the c a p a b i l i t y of producing potassium n i t r i t e at the rate of 536.67 uM per gm. of fresh spinach leaves. 76 Resende e_t a l (1969) had discussed the thermal destruct ion of enzymes i n spinach puree. In the present research spinach was blanched for several d i f f erent times and enzyme a c t i v i t y was determined. It was found that enzyme ac-. t i v i t y of spinach was completely inact ivated wi th in only 8 sec. The enzyme a c t i v i t y of spinach and i t s thermal effect are shown i n Table 25. Therefore blanching or cooking spinach for a short time w i l l stop enzyme a c t i v i t y which produces toxic n i t r i t e , but i t might be regenerated i n some way as noted by Resende et a l (1969). 77 Table 25 The effect of blanching time on the n i t r a t e reductase a c t i v i t y of spinach. Blanching time uM of KNC^ produced per gm. of fresh spinach leaves - sec. 1* 2 3 4 Average 0 420.00 726.67 453.33 546.67 536.67 2 106.67 106.67 53.33 46.67 •''78 .34 v -4 8 66.47 0 0 0 13.33 0 0 0 a.9_.9-5j 0 *Separate samples on d i f f erent days. 78 SUMMARY 1. Spinach and beet were found to have higher n i t r a t e -nitrogen content than other vegetables and f r u i t analyzed. Frozen food had higher n i t r a t e - n i t r o g e n content than canned or fresh foods. Any food cons i s t ing of some l i q u i d had n i t r a t e - n i t r o g e n i n the l i q u i d p o r t i o n . The l i q u i d port ion was higher i n n i t r a t e - n i t r o g e n than the s o l i d port ion except i n bean which has a protect ive surface layer . Fresh market vegetables were found to have higher n i t r a t e - n i t r o g e n content than those grown i n the greenhouse without f e r t i l i z e r . 2. Among d i f f erent methods of n i t r a t e ana lys i s , the sodium s a l i c y l a t e method was found to be the most r e l i a b l e . D i s t i l l e d water should be used as a spectrophotometer blank in . the determination of n i t r a t e by the sodium s a l i c y l a t e method. The recovery of n i t r a t e - n i t r o g e n after adding potassium n i t r a t e to food ranged from 104.0 to 107.0%. The recovery var ied among d i f f erent species and d i f f erent times of adding n i t r a t e . 3. Cooking and b o i l i n g food containing n i t r a t e caused an increase of n i t r a t e - n i t r o g e n content i n the remaining food after some water had been evaporated. No n i t r a t e - n i t r o g e n was found i n the condensate. The n i t r a t e content in the l i q u i d increased as the time of cooking or b o i l i n g increased. N i t r a t e -nitrogen content of spinach increased as cooking time increased up to 10 min . ; at 10 and 20 min. of cooking i t was decreased. 4. Spinach had much higher n i t r a t e - n i t r o g e n content than bean. The n i t r a t e d i s t r i b u t i o n from s o l i d port ion to l i q u i d port ion of spinach was also higher than bean. 79 5. N i t ra te -n i t rogen was not completely extracted from food containing n i t r a t e , from 3.73 to 13.051 of n i t r a t e -nitrogen was found i n the residue af ter ex trac t ion . 6. Reducing and ox id i s ing agents may be discounted as , i n t e r f e r i n g agents i n n i t r a t e determination. 7. The maximum n i t r a t e - n i t r o g e n recovery from buffer solut ions i n this study was found to be at pH 5.4. pH of spinach puree ranged from 6.28 to 6.60 and beet ranged from 5.70 to 6.35. 8. Sugar was found to affect n i t r a t e - n i t r o g e n determination by increasing the apparent n i t r a t e concentration i n proport ion with the concentration of sugar present in food. 9. Spinach grown with nitrogen f e r t i l i z e r was found to accumulate n i t r a t e - n i t r o g e n and d i s t r i b u t e i t in d i f f erent concentrations i n d i f f erent plant par t s . The rate of n i t r a t e accumulation and d i s t r i b u t i o n was not uniform among d i f f erent plant parts among species . 10. N i tra te reductase, the enzyme which reduces n i t r a t e to n i t r i t e , was measured i n spinach by determining n i t r i t e formation i n spinach. Ni trate reductase was most eas i ly found i n spinach f e r t i l i z e d with n i t r a t e s o l u t i o n . This enzyme was found to be inact ivated wi th in only 8 sec. by blanching. Therefore n i t r i t e might not be found after cooking, even though n i t r a t e remains af ter cooking even af ter pouring of f the cooking water. The remaining n i t r a t e might be reduced to n i t r i t e i n some way and cause poisoning. Nitrogen f e r t i l i z e r should be minimized i n growing plants used as food. The procedure of 80 preparation.of food should also be considered to minimize nitrate-nitrogen content of food for consumption. 81 LITERATURE CITED Anonymous 1950 Ni tra te in vegetables. N u t r i t i o n Rev. 8: 230-231. A f r i d i , M.M.R.K. and Hewitt, E . 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