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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.  i n Pharmacy, U n i v e r s i t y of Medical Bangkok, T h a i l a n d 1959  Sciences,  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f Food Science F a c u l t y o f A g r i c u l t u r a l Sciences  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA June, 1969  In p r e s e n t i n g an a d v a n c e d the  this  degree  Library shall  I further for  agree  scholarly  by  his  of  this  written  thesis  in p a r t i a l  fulfilment  of  at  University  of  Columbia,  the  make  that  it  permission  purposes  may be  representatives. thes.is  for  It  for  of  Food  Science  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  June  23,  1969  by  the  understood  gain  Columbia  for  extensive  granted  is  financial  available  permission.  Department  Date  freely  British  shall  Head o f  be  requirements  reference copying  that  not  the  of  I agree and this  or  allowed  without  that  Study. thesis  my D e p a r t m e n t  copying  for  or  publication my  (i)  ABSTRACT The n i t r a t e food,  baby  analyzed. highest  content  o f some f o o d s  food,  frozen food,  and f r e s h v e g e t a b l e s  Among  them  and beet  spinach  nitrate-nitrogen  content  content  than  Nitrate-nitrogen  of food  i s partially  p o r t i o n whenever  the food  the  solid  layer  i n the l i q u i d p o r t i o n except  as a f a c t o r  portion. liquid  other  food  i n bean which  transferred to the l i q u i d  found  transferring  by cooking.  distribution  was n o t u n i f o r m : found  from  still  i n the liquid solid  portion to  a n d was n o t t h e  pouring  remained  nitrate  root  than  o f f cooking  i n the s o l i d  among d i f f e r e n t  bean l e a v e s , beet  readily  than i n  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 Even a f t e r  of nitrate  t o have h i g h e r  fertilization  The amount  has a p r o t e c t i v e s u r f a c e  the nitrate  parts  some n i t r a t e - n i t r o g e n  The  analyzed.  p o r t i o n seemed, t o be h i g h e r  of lowering  different  t o have t h e had higher  products  p o r t i o n w a s n o t t h e same f o r a l l f o o d s  was n o t d e s t r o y e d  were  found  Canned  were  c o n s i s t s o f any l i q u i d .  The r a t e o f n i t r a t e  same e v e n f r o m  water,  were  and f r o z e n f o o d  nitrate-nitrogen  of n i t r a t e  was d e t e r m i n e d .  plant  parts  and spinach  other  petioles  parts.  increased nitrate-nitrogen  portion.  Nitrogen  content i n  spinach. The reliable  sodium  salicylate  method  m e t h o d was f o u n d  fornitrate  t o be t h e most  determination  among  different  v.  methods  used  i n this  affected  b y many  analysis  such  study.  factors  occuring  as p r o c e d u r e  photometer blanks  used.  The d e t e r m i n a t i o n m i g h t during  of extraction  be  the procedure  of  and t h e s p e c t r o -  O x i d i s i n g agents,  arid  reducing  (ii)  agents do not seem to a f f e c t  the a n a l y s i s but pH v a r i a t i o n and  sucrose which might occur i n food probably are f a c t o r s apparent  n i t r a t e content.  Cooking d i d q u i c k l y destroy spinach  n i t r a t e reductase enzyme a c t i v i t y . will  affecting  T h i s means that  not be found a f t e r cooking unless the enzyme i s  regenerated, or unless there i s m i c r o b i a l a c t i v i t y .  nitrite  (iii)  TABLE OF CONTENTS Page ABSTRACT  i  LIST OF TABLES  vi  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 n i t r i t e i n plant material  9  c) P h e n o l - d i s u l f o n i c  a c i d method  10  d) N i t r a t e i o n e l e c t r o d e method  10  e) Some other methods of n i t r a t e determination  11  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 in plant material  22  c) P h e n o l - d i s u l f o n i c  24  a c i d method  d) N i t r a t e i o n e l e c t r o d e method 3. FACTORS AFFECTING ANALYSIS  26 27  4. PLANT STUDIES a) Comparison of bean with spinach  28  (iv) Page b) V a r i a t i o n s i n d i f f e r e n t  plant parts  28  c) E f f e c t of n u t r i e n t status  28  d) N i t r a t e reductase  28  studies  RESULTS AND DISCUSSION 1.  NITRATE CONTENT OF FOODS a) Canned food  31  b)  S t r a i n e d baby food  31  c)  Frozen food  33  d) Fresh vegetables 2.  36  RECOVERY STUDY a)  Sodium s a l i c y l a t e  method  b) Rapid d e t e r m i n a t i o n of n i t r a t e n i t r i t e in plant material c)  method  38 40 40  FACTORS AFFECTING ANALYSIS a) Time of e x t r a c t i o n  45  b) Repeated e x t r a c t i o n  48  c)  48  C l a r i f i c a t i o n procedure and the use of spectrophotometer blanks  d) Reducing agents  53  e)  O x i d i s i n g agent  55  f)  Buffer solutions  58  g) pH and t o t a l  4.  and  P h e n o l - d i s u l f o n i c a c i d method  d) N i t r a t e ion e l e c t r o d e 3.  38  a c i d i t y i n spinach  58  h) pH i n beet  63  i)  Sucrose  66  j)  Blanching  66  PLANT STUDIES a)  Comparison of spinach and bean  68  (v) Page b) V a r i a t i o n s i n d i f f e r e n t  plant parts  71  c) E f f e c t of n u t r i e n t s t a t u s  73  d) N i t r a t e reductase  75  studies  SUMMARY  78  LITERATURE CITED  81  (vi)  LIST OF TABLES Table 1.  Page 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 p o r t i o n s of canned food obtained from Vancouver markets i n 2.  1968.  The n i t r a t e - n i t r o g e n content of baby food obtained from Vancouver markets i n  3.  34  1968.  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 p o r t i o n s of f r o z e n food obtained from Vancouver markets i n 4.  1968,  The n i t r a t e - n i t r o g e n content of f r e s h vegetables obtained from d i f f e r e n t  5.  sources.  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  6.  37  method of  The recovery of potassium n i t r a t e  39  analysis.  added to spinach  using the r a p i d method of determination of  41  nitrate  in plant material. 7.  The recovery of potassium n i t r a t e  added to  42  vegetables by the p h e n o L d i s u l f o n i c a c i d method. 8.  The recovery of potassium n i t r a t e u s i n g the n i t r a t e  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  11.  46  time.  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 with b o i l i n g  44  ion e l e c t r o d e method.  s o l u t i o n with b o i l i n g 10.  added to spinach  47  time.  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 s u c c e s s i v e r e s i d u e s of spinach  extraction.  49  (vii)  Table 12.  Page 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 u s i n g d i f f e r e n t procedures  13.  of c l a r i f i c a t i o n of spinach  different 14.  extract.  The recovery of n i t r a t e - n i t r o g e n when d i f f e r e n t blanks were used and with n i t r a t e s species at d i f f e r e n t  The e f f e c t  51  52  added to  times.  of reducing agents on n i t r a t e - n i t r o g e n  54  determination. 15.  The e f f e c t  of o x a l i c  determination 16.  The e f f e c t  56  in spinach.  of an o x i d i s i n g  determination 17.  a c i d on n i t r a t e - n i t r o g e n  agent on n i t r a t e - n i t r o g e n  57  in spinach.  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 b u f f e r  59  solutions 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 b l a n c h i n g . 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 d u r i n g b l a n c h i n g . 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  from  67  of beet d u r i n g b l a n c h i n g . 21.  The comparison of n i t r a t e - n i t r o g e n water and sucrose  22.  recoveries  solutions.  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 r a n s f e r r i n g from  69  s o l i d to l i q u i d p o r t i o n d u r i n g b l a n c h i n g 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 parts.  in different  plant  72  (viii)  Table 24.  Page N i t r a t e - n i t r o g e n content i n d i f f e r e n t spinach with and without n i t r a t e  25.  The e f f e c t reductase  p a r t s of  fertilization.  of b l a n c h i n g time on the n i t r a t e a c t i v i t y of  spinach.  74  77  (ix)  ACKNOWLEDGEMENTS The w r i t e r would l i k e to express  her a p p r e c i a t i o n to  Dr. D.P. Ormrod who d i r e c t e d t h i s r e s e a r c h and who p r o v i d e d encouragement and a d v i c e . Thanks are a l s o extended to the members o f the r e s e a r c h committee f o r use of f a c i l i t i e s of t h i s  and advice on the p r e p a r a t i o n  thesis. Dr. J.F.  Richards  Dr. S. Nakai  INTRODUCTION The presence  and amounts of n i t r a t e s  i n foods  has  been  noted and t a b u l a t e d by Richardson (1907) and Wilson (1949). Nitrate is  sometimes used i n the manufacture of cheese  stop m i c r o b i a l growth at a predetermined l e v e l  of  to  fermentation  by l i b e r a t i o n of n i t r i t e , which a l s o serves as a p r e s e r v a t i v e . Nitrate  is  bacterial red  a l s o used i n some other food products to  prevent  i n v a s i o n and/or f o r the purpose of m a i n t a i n i n g a  c o l o r i n the c u r i n g of meats. Nitrates  are n a t u r a l c o n s t i t u e n t s of p l a n t m a t e r i a l  since  they are the main source of n i t r o g e n r e q u i r e d f o r growth. roots of  of most p l a n t s  nitrate.  absorb n i t r o g e n from the s o i l  is  assumed that  the f i r s t  significant  trend is  the  fertilizers  which, i n g e n e r a l ,  it  can be  compounds of the p l a n t .  step i n n i t r a t e  c o n v e r s i o n of n i t r a t e to n i t r i t e  content  i n the form  It must be reduced to ammonia before  i n c o r p o r a t e d i n t o the nitrogenous  reduction is  ( D e v l i n , 1966).  the  The most  result  i n increased  nitrate  food.  P u b l i c concern has been aroused over the p o s s i b l e hazard of high l e v e l s of n i t r a t e s  is  actually "nitrite  nitrate  t r a c t or before  toxicity"  the g a s t r o i n t e s t i n a l  Nitrite,  but not n i t r a t e ,  to  nitrite  ingestion.  and n i t r i t e , being h i g h l y water s o l u b l e ,  traverse  and  The term " n i t r a t e  i s produced f o l l o w i n g the r e d u c t i o n of n i t r a t e  w i t h i n the g a s t r o i n t e s t i n a l  health  found i n some foods,  in infant diets.  t o x i c i t y " as commonly used, and  It  i n c r e a s e d use of n i t r o g e n c o n t a i n i n g  of p l a n t m a t e r i a l s which are used as  on the use of these foods  The  Both  freely  w a l l i n t o the blood stream.  oxidizes  the f e r r o u s  i r o n of  the  2  red b l o o d pigment hemoglobin and causes (Wright and Davison, 1964).  Nitrates  methemoglobinemia  can a l s o  interfere  with normal i o d i n e metabolism of the t h y r o i d gland and r e s u l t i n a r e d u c t i o n i n the l i v e r storage of v i t a m i n A (Bloomfield and Welsch, 1961). Water s u p p l i e s to,  contaminated with n i t r a t e have c o n t r i b u t e d  or been e n t i r e l y r e s p o n s i b l e  animals  f o r , p o i s o n i n g of humans and  (Knotek and Schmidt, 1964).  Fresh vegetables and some  food products are found to have n i t r a t e sufficient  in  to cause p o i s o n i n g , p a r t i c u l a r l y with  Widely d i f f e r i n g values  f o r n i t r a t e content  vegetables and food products are r e p o r t e d . factors  the n i t r a t e  factors i n food.  infants. of i n d i v i d u a l  Probably many  both i n t e r n a l and e x t e r n a l c o n t r i b u t e to t h i s  The purposes (1)  concentrations  of t h i s  content  on n i t r a t e  study were to o b t a i n i n f o r m a t i o n on  of foods,  level,  variation.  and (3)  (2)  the e f f e c t s  of v a r i o u s  the recovery of added n i t r a t e  3  LITERATURE REVIEW 1. NITRATE .CONTENT OF FOODS Public health authorities are becoming increasingly aware, of the n i t r a t e content of water supplies.  The major source of  n i t r a t e contamination i n water appears to be animal and human wastes (Wright and Davison, 1964).  Nitrate concentrations are  higher i n dug and shallow wells than i n d r 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 i n n i t r a t e content from day to day. High n i t r a t e content of the water supply has been respons i b l e for many infant deaths and constitutes the major human health hazard of n i t r a t e t o x i c i t y .  Water containing 60 ppm  nitrate-nitrogen i s considered hazardous  for use with infants  and small children (Kilgore ejt a l , 1963).  The presence of  excessive n i t r a t e i n drinking water used to make infant formulas was noted.  Five cases are said to have resulted i n  death (Anonymous, 1950).  After this experience a maximum  safety l i m i t of 10 ppm of nitrate-nitrogen i n water was set by I l l i n o i s authorities. Vegetables and some other foods may contain a rather large percentage of n i t r a t e .  Cereal seeds may be disregarded, since  any danger from them i s negligible (Gilbert et ^ , 1946). Nitrates i n considerable quantities i n vegetables have been reported by several e a r l i e r investigators.  Wilson (1943)  determined the n i t r a t e content i n the sap of many plants growing with abundant moisture and points out that "The n i t r a t e  4  content  of  such v e g e t a b l e s  cauliflower, toxic  at  lettuce,  times  to  Many l e a f y  vegetables  of n i t r a t e  by s e v e r a l  research  Ithaca,  ppm o f n i t r a t e cauliflower, in  carrots,  was  is  for  soils  found to  of  that  consume  the m i l k  of  cows  possibility  of n i t r a t e  from animals  higher  - over  the  cabbage, t h a n 1,000  w o u l d be  Rhubarb  expected  equivalent  the water  of  Only  as  of  a  one  (potassium  d u r i n g the  to  nitrate)  preparation  foods. is  a possibility  fed  than c o n t r o l .  1907).  ppm n i t r a t e - n i t r o g e n  Nitrate were  i n Various  (Richardson,  accumulation i n the  that  ppm  Considerable  a person,  saltpeter  f e d 114  1964).  1,000  nitrate.  n i t r o g e n p e r k i l o g r a m p e r day f o r was  as  d i d not c o n t a i n n i t r a t e .  and D a v i s o n ,  slaughter  vegetables  and muskmelon.  p a r t i c u l a r l y milk  supply  and meat  of  these  of n i t r a t e s  t h e i r water  (Wright  green  and l e s s  on s u c h a d i e t  more,  some o f  secretions,  However,  fresh  reported  and c u l t u r a l p r a c t i c e s .  c o n t a i n no  easily  The p r e s e n c e  large  have b e e n  broccoli,  f o u n d among s a m p l e s ,  might  table  may be  5,400 ppm (Anonymous, 1 9 5 0 ) .  even w i t h p o u r i n g o f f  the  bodily  Numerous  curly lettuce  two grams and p o s s i b l y daily,  foods  accumulate  contents  and head l e t t u c e  possible  vegetarian,  known t o  found i n b e e t s ,  from d i f f e r e n t  It  these  cabbage,  New Y o r k c o n t a i n e d h i g h n i t r a t e  much as  tomatoes were  are  and n i t r a t e  cucumber,  v a r i a t i o n was  broccoli,  suggests  workers.  celery  c o n t a i n e d as  plants  etc.,  beets,  humans."  quantities  grown a t  as  There i s tissues  content 100  6 to  or  of  of both  150 mg.  12 months  in the animals  milk  nitrate-  before  5  Infants  fed with d r i e d m i l k where water contains  c o n c e n t r a t i o n of n i t r a t e s  become i l l with  (Knotek and Schmidt, 1964). microbes of l a c t i c  methemoglobinemia  Dried buttermilk  fermentation Streptococcus  producing the a n t i b i o t i c n i s i n .  contains lactis,  This prevents  the  of the s p o r u l a t i n g microbes of B a c i l l u s s u b t i l i s . u s u a l l y occurs i n d r i e d milk and i s nitrates  to  a high  growth B.  subti1is  capable of r e d u c i n g  nitrites.  Canadian baby foods,  as determined by Kamm et_ a l  (1965) ,  contained n i t r a t e higher than those r e p o r t e d f o r New York State.  For example,  of n i t r a t e  Canadian spinach contained 1,074-1,668 ppm  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 Among s t r a i n e d baby foods were e s p e c i a l l y  (Anonymous, 1950)  respectively.  spinach and beets  high whereas tomato, peas and squash had no  nitrate. N i t r a t e content  of vegetable products i n 1964,  r e p o r t e d by Jackson e_t a l  as  (1967) , was not considered u n u s u a l l y  h i g h , beets being about 2,200 ppm, c o l l a r d s about 2,700 ppm and t u r n i p 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 p o r t i o n s of canned products was r e p o r t e d i n 1907 and 1964. With the exception  of mixed v e g e t a b l e s ,  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 p o r t i o n than the d r a i n e d s o l i d s . water s o l u b l e ,  Since n i t r a t e  salts  some e q u i l i b r i u m of n i t r a t e between the  d r a i n e d p o r t i o n and the mother l i q u o r would be especially  liquid  i n canned l e a f y v e g e t a b l e s .  are well-  expected,  The s i z e and shape of  6  mixed v e g e t a b l e s , surface  sweet corn and sweet peas, which expose l e s s  area to the l i q u i d than l e a f y v e g e t a b l e s ,  are c o n t r i b u t i n g f a c t o r s  i n the lower values  l i q u i d p o r t i o n of these vegetables  that  found i n the  ( K i l g o r e et_ a l , 1963) .  There was no d e s t r u c t i o n of n i t r a t e s would be expected  apparently  due to cooking.  It  the amount of n i t r a t e removed from the  vegetable to the cooking l i q u o r would i n c r e a s e with the amount of cooking water and depend on the cooking time.  ( K i l g o r e e_t a l ,  1963) . Juices example,  of green vegetables and quick f r o z e n foods,  s p i n a c h , rhubarb, c e l e r y and s e v e r a l  were f r e q u e n t l y high i n n i t r a t e .  for  other vegetables  F r e s h - f r o z e n spinach  juice  contained 6,600 ppm or 1% potassium n i t r a t e on a dry b a s i s (Anonymous, 19 50). The q u a n t i t y of n i t r a t e g r e a t e s t with an e x c l u s i v e l y had observed that cut beef  is  vegetable d i e t .  Richardson (1907)  cooked with f r e s h vegetables produced  the c o l o r of nitrohemochromogen, nitrites  l e a s t with a meat d i e t and  i n d i c a t i n g the presence  the same as cooking with s a l t p e t e r  or n i t r i t e  Many prepared meat products such as corned beef,  of  solution.  weiners,  bologna and sausages have both n i t r a t e and n i t r i t e added to preserve  c o l o r and r e t a r d s p o i l a g e .  The Meat I n s p e c t i o n  of the U . S . Department of A g r i c u l t u r e (1960) e s t a b l i s h e d level  of n i t r a t e  the  ion i n the f i n i s h e d product to not exceed a  maximum of 200 ppm Nitrate is  Division  or 60 ppm n i t r a t e - n i t r o g e n .  a potent  d i u r e t i c and the kidney i s  important organ f o r removal of n i t r a t e n i t r a t e can, however,  an  from the body.  be r e c y c l e d from the blood i n t o  Some the  7  gastrointestinal secretions..  t r a c t by s a l i v a r y and  N i t r i t e is  l i v i n g plant tissue.  gastrointestinal  found only i n small c o n c e n t r a t i o n s  in  The t o x i n normally consumed, n i t r a t e ,  is  reduced to n i t r i t e somewhere w i t h i n the animal body. 'The r e d u c t i o n , o f n i t r a t e w i t h i n the g a s t r o i n t e s t i n a l attributed solely that  is  found i s  to micro-organisms. a possible  ten times more t o x i c Nitrite,  but not n i t r a t e ,  Each ion of  source of n i t r i t e  than the n i t r a t e oxidizes  tract  nitrate  that  is  from which i t  the f e r r o u s  is  about  came.  i r o n 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 c o l o r e d pigment c a l l e d methemoglobin which is  incapable of t r a n s p o r t i n g or r e l e a s i n g  tissues.  Animals so a f f e c t e d  oxygen to the body  are s a i d to be s u f f e r i n g from  methemoglobinemia.  The hemoglobin of the newborn human and  of the bovine fetus  i s more e a s i l y  by n i t r i t e  o x i d i z e d to  than that of the r e s p e c t i v e  methemoglobin  adult.  Cyanosis had been observed i n human i n f a n t s f o r a number of y e a r s .  Cyanosis i s  in infants  of the b l o o d .  deficient  Comly (1945) has d e s c r i b e d  that c o u l d be c o r r e l a t e d with the n i t r a t e  cyanosis content  of w e l l water used to make the formula f o r the b a b i e s . cyanosis  was due to methemoglobinemia and i t s  a t t r i b u t e d to r e d u c t i o n of the n i t r a t e gastrointestinal  t r a c t of the  Dietary n i t r a t e metabolism.  babies')  a dusky b l u i s h or p u r p l i s h  d i s c o l o r a t i o n of s k i n or mucous membranes due to oxygenation  ('blue  This  development  to n i t r i t e  was  i n the  infant with d i a r r h e a .  interferes  with v i t a m i n A and carotene  The d e s t r u c t i o n of carotene was observed during  an a r t i f i c i a l fermentation of corn s i l a g e and was more r a p i d  8  when n i t r a t e was added. High intakes  of n i t r a t e s  produce i r r i t a t i o n of  gastrointestinal  t r a c t , nausea,  gastroenteritis,  muscular weakness, bloody s t o o l s ,  pulse,  convulsions,  collapse,  the  v o m i t t i n g and acute irregular  and albuminous and p o s s i b l e  bloody u r i n e . Sollmann (1948) has r e p o r t e d that nitrate (as  is  a safe s i n g l e  dose f o r humans.  the i n o r g a n i c n i t r a t e s a l t )  (Steyn,  1960).  is  (1960),  be ingested spinach,  that  from 3.9  adults  infants  are  100  theoretical  a t o x i c q u a n t i t y of n i t r a t e oz.  of rhubarb or about 2.3  t u r n i p s or c e r t a i n other  f o r a d u l t s would oz.  of  vegetables.  It has been found by Simon (1966) that content  for  than a d u l t s .  P r e v i o u s l y Wilson (1949) claimed from considerations  potassium  Five gm. per day  dangerous  According to Steyn  times more s u s c e p t i b l e  1 gm. of  the  nitrate  of f r e s h spinach v a r i e d between 40 and 2,100  mg./kg.  He recommended that spinach should not be given to i n f a n t s the f i r s t  3 months of l i f e ,  f o r at t h i s  age n i t r a t e can be  reduced i n the upper p a r t s of the g a s t r o i n t e s t i n a l because of the lowered diaphorase a c t i v i t y increased s u s c e p t i b i l i t y is  there  to methemoglobinemia.  formed by b a c t e r i a l r e d u c t i o n of the n i t r a t e  eaten.  t r a c t , and  is  then  The n i t r i t e i n the  From 1959-1965 there were cases of n i t r i t e  r e p o r t e d i n Germany i n i n f a n t s .  in  spinach  poisoning  Simon (1966) s t a t e d  that  i n f a n t feeding must not c o n t a i n more than 300 mg. n i t r a t e per k g . Forages over 0.34 considered p o t e n t i a l l y  to 0.45% n i t r a t e - n i t r o g e n should be toxic  for livestock  and should be mixed  9 with s a f e r  feeds p r i o r to use.  The d i e t  should be  adequate  i n carbohydrate and p r o t e i n , and animals should not be p e r m i t t e d to get  o v e r l y hungry.  A b o r t i o n s w i l l probably  occur i n c a t t l e which were fed a higher p r o p o r t i o n of (Wright and Davison,  nitrate  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 nitrate  d e t e r m i n a t i o n i n water.  They were the  diphenylamine-  s u l p h u r i c a c i d , b r u c i n - s u l p h u r i c a c i d , sodium s a l i c y l a t e phenol-disulfonic satisfactory  a c i d methods.  The f i r s t  two methods were not  f o r photometric d e t e r m i n a t i o n .  It was  impossible  to compare c o l o r s because of the high c o l o r development needed too much d i l u t i o n .  Among the methods t e s t e d ,  b r u c i n - s u l p h u r i c a c i d method gave more s a t i s f a c t o r y than the d i p h e n y l a m i n e - s u l p h u r i c a c i d method.  results  There was no  The l a t t e r  were i d e a l with a high response s t r a i g h t +  precisely  two  two methods  c u r v e , which may  +  d e t e c t - 0.25  The i n t e r f e r e n c e s  which  the  comparison i n accuracy between these two and the l a s t methods which use o p t i c a l machines.  and  to - 0.5  were such that  mg. of n i t r a t e per  litre.  the sodium s a l i c y l a t e  was b e t t e r than the p h e n o l - d i s u l f o n i c b) Rapid d e t e r m i n a t i o n of n i t r a t e  method  a c i d method. and n i t r i t e  in plant  material This i s It  is well  or n i t r i t e ,  the m o d i f i c a t i o n of Nelson et_.al,  (1954).  adapted f o r measuring day-to-day changes i n n i t r a t e or f o r other a p p l i c a t i o n s  m a t e r i a l remains e s s e n t i a l l y c o n s t a n t .  i n which the b a s i c It  is  plant  a diazotization  10  of s u l f a n i l i c a c i d and c o u p l i n g with 1-naphthylamine a red dye.  N i t r a t e must be reduced to n i t r i t e .  i n t h i s work should be as f r e e  to  form  The water  used  from copper as i s p o s s i b l e  should c o n t a i n l e s s than 0.002 ppm of heavy Samples c o n t a i n i n g l e s s n i t r a t e  metals.  than 10 ppm cannot  be analyzed with accuracy because the t o t a l  and  usually  q u a n t i t y of p l a n t  m a t e r i a l must be kept low to avoid i n t e r f e r e n c e w i t h  the  analys i s . c)  P h e n o l - d i s u l f o n i c a c i d method This method was suggested by Barnett  determination i n s i l a g e . bath a f t e r color is  drying.  The samples  developed w i t h ammonia s o l u t i o n .  98-103%.  added and the.  The author noted  showed a recovery of  must be taken i f m a t e r i a l , f o r example s u g a r s , d u r i n g the treatment with the p h e n o l - d i s u l f o n i c is  little  is  precautions  carbonized  acid  reagent  doubt that some of the high n i t r a t e  recorded i n the l i t e r a t u r e owe t h e i r magnitude to t h i s d) N i t r a t e ion e l e c t r o d e  ion e l e c t r o d e technique specific  is  Mayers and Paul  cause.  a c i d technique  requires  (1968) suggested a n i t r a t e  method f o r n i t r a t e - n i t r o g e n d e t e r m i n a t i o n . s i m i l a r to pH measurement.  f o r the n i t r a t e  of a p o t e n t i a l exchanger.  contents  method  Since the p h e n o l - d i s u l f o n i c expensive r e a g e n t s ,  across  It i s  i o n , and operates by the  These workers had d i f f i c u l t y  The  claimed to be development  a t h i n l a y e r of w a t e r - i m m i s c i b l e  recovery of added n i t r a t e  that  from  At the same time i t was noted that v a r i o u s  and there  nitrate  are e x t r a c t e d on a steam  Phenol-disulfonic acid is  the accuracy with added n i t r a t e  (1954) f o r  in obtaining  i n s o i l s by t h i s method.  ion complete  The  recovery was l e s s than 90% because of the lower of the instrument at high n i t r a t e l e v e l s .  sensitivity  They had an average  of 97% recovery by the p h e n o l - d i s u l f o n i c a c i d method. speed of the e l e c t r o d e soil  analysis  .  The  is p a r t i c u l a r l y attractive  for  testing. e)  Some other methods of n i t r a t e  determination  Kamm et a_l, (1965) mentioned a new method f o r d e t e r m i n a t i o n of n i t r i t e and n i t r a t e  the  i n foods which would  a c c u r a t e l y determine c o n c e n t r a t i o n as low as 1 ppro-; 1-Naphthylamine is  d i a z o t i z e d by n i t r i t e and coupled w i t h excess amine to  4-(1-naphthylazo)-1-naphthylamine.  Nitrate is  give  quantitatively  reduced by passage through a cadmium column and determined as nitrite,  n i t r i t e passes through the column u n a l t e r e d .  i n samples  c o n t a i n i n g both forms of n i t r o g e n , n i t r a t e  determined by d i f f e r e n c e .  For c o l o r development  i s p l a c e d i n the dark f o r two h o u r s . recoveries  the  is solution  At 5% sample i n water  of 69% or b e t t e r were obtained f o r a l l  At 25% sample i n water,  Therefore,  only a few samples  gave  samples.  satisfactory  results. NumerojusO chemical methods f o r d e t e r m i n a t i o n of have been d e s c r i b e d i n the l i t e r a t u r e . have been very s a t i s f a c t o r y soil  and p l a n t e x t r a c t s  is  None of them,  determined a f t e r  and i t  may c o n t a i n .  chemical r e d u c t i o n to  to n i t r i t e .  Usually, nitrite.  sufficiently  i s normally d i f f i c u l t to o b t a i n  r e d u c t i o n of n i t r a t e  in  because of the l a r g e amounts of  However, the u s u a l reducing reagents are not specific,  however,  f o r d e t e r m i n a t i o n of n i t r a t e s  i n t e r f e r i n g substances these e x t r a c t s nitrate  nitrate  quantitative  Lowe and Hamilton (1967)  12  mentioned a method u t i l i z i n g soybean nodule b a c t e r o i d s r e d u c t i o n of n i t r a t e to n i t r i t e . little  as 0.01  solution.  It was s e n s i t i v e to  sample  Recovery was about 99-101%. TO FERTILIZER PRACTICE  Nearly a l l of the n i t r o g e n needed by p l a n t s growth i s  taken from the s o i l  to i n f e r t h a t , significant  especially  o c c u r ; however,  their  It i s  A logical  i n the e a r l i e r stages of growth,  amounts of n i t r a t e s  As the p l a n t matures,  for  i n the form of n i t r a t e s .  s m a l l e r q u a n t i t y may be absorbed as ammonium.  should be found i n the  smaller quantities  of n i t r a t e s  occur i n the f u l l y matured p l a n t s .  i s more a s t o n i s h i n g ripening process,  considThis  i n the case of f r u i t s because during  the n i t r a t e s  reducing substances  plant.  should  in c e r t a i n crops, p a r t i c u l a r l y beets,  erable quantities  of  as  ug. of n i t r a t e - n i t r o g e n per ml. i n the  3. NITRATE RELATIONSHIP  The  for  the  must be i n contact with many  (Richardson, 1907).  accumulation of n i t r a t e thus  implies  that  the  rate  a s s i m i l a t i o n has not kept pace with the r a t e of uptake.  some crops the n i t r a t e content associated  has been shown to be  w i t h u l t i m a t e y i e l d , and t i s s u e t e s t i n g  In  positively of these  crops has been advocated as a guide to optimum f e r t i l i z a t i o n . P l a n t to p l a n t v a r i a t i o n might be enormous. N i t r a t e w i l l not accumulate w i t h i n a p l a n t unless e x t e r n a l medium can f u r n i s h i t of  assimilation.  an i n c r e a s e increase  at a r a t e f a s t e r  Numerous experiments  accumulation has been a s s o c i a t e d  than the- r a t e  might be c i t e d i n which  i n e x t e r n a l n u t r i e n t n i t r o g e n has  i n n i t r a t e accumulation.  the  effected-an  In some cases abnormal with heavy d r e s s i n g of animal  13  manure.  Clean f a l l o w i n g had been shown to i n c r e a s e  of a v a i l a b l e n i t r a t e and thereby i n c r e a s e of p l a n t s .  the  the n i t r a t e  content  Timing of n i t r o g e n f e r t i l i z a t i o n has been shown to  have.amarked i n f l u e n c e applications  on the n i t r a t e  content  of pasture  soon before h a r v e s t tending to i n c r e a s e  s o i l might under s p e c i a l  conditions  to dangerous  accumulate n i t r a t e  levels.  (Wright and  1964).  Environmental f a c t o r s  i n c l u d i n g those which  microbial conversion influence  n i t r a t e uptake.  taken up more r e a d i l y from s o l u t i o n s nitrate  grasses,  accumulation.  P l a n t s growing i n low n i t r a t e  Davison,  supply  stimulate Nitrate  is  prepared with potassium  than from those prepared with c a l c i u m or sodium n i t r a t e .  Wright and Davison (1964) mentioned that  increases  i n the  of potassium i n the s o l u t i o n promote an accumulation of i n oats and i n corn when n i t r a t e  is  a l s o present  level  nitrate  at a high  level.  The accumulation of n i t r a t e might be l i m i t e d by ' b a l a n c i n g ' a high l e v e l  of a v a i l a b l e n i t r o g e n i n the s o i l with heavy  applications  of other p l a n t n u t r i e n t s .  N i t r o g e n and potassium  can accumulate w i t h i n the p l a n t to l e v e l s much above needed f o r maximum growth. encourage that  Potassium and phosphorous may  accumulation of n i t r a t e .  Peterson  the importance of the N f a c t o r i s  (1968) r e p o r t e d  g r e a t e r than the  importance of P and K i n n i t r a t e accumulation. conceivable  those  It  is  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 p l a n t growth, and that additions  of P and K caused a growth response which  i n d i l u t i o n of the N taken up by the p l a n t . P and K would be i n d i r e c t , a l b e i t p r e s e n t ,  resulted  The e f f e c t  of  with the major  the  14  i n f l u e n c e being the N content The f a c t o r s of n i t r a t e were: level  of the  soil.  that had a major i n f l u e n c e  on the  concentration  i n the forage s t u d i e d by Crawford e_t al_ (1961),  s p e c i e s , p a r t of p l a n t ,  stage of m a t u r i t y of the  of n i t r o g e n f e r t i l i z a t i o n ,  and l i g h t  intensity.  most s i g n i f i c a n t was the i n c r e a s e d use of n i t r o g e n fertilizers, content  which, i n g e n e r a l ,  plant, The  containing  resulted i n increased  nitrate  of p l a n t m a t e r i a l as mentioned by Balks and P l a t e  (1955), G i l b e r t et a l Kretschmer  (1946), Hanway and Englehorn (1958),  (1958), Whitehead and Moxon (1952) and P h i l l i p s  High amounts of n i t r a t e  i n spinach can be a t t r i b u t e d  e x c e s s i v e use of n i t r o g e n f e r t i l i z e r .  (1968). to  The optimal amount of  f e r t i l i z e r use should be about 80 k g . of n i t r o g e n per  hectare,  but f r e q u e n t l y  is  in practice in agriculture this  g r e a t l y exceeded.  The experience  p o i s o n i n g of i n f a n t s  amount  gained i n cases of  from e a t i n g spinach has l e d to  nitrite the  c o n c l u s i o n that too much f e r t i l i z e r should not be used growing spinach  (Simon,  for  1966).  Schuphan (1965) found 450 mg. n i t r a t e / 1 0 0  g. dry weight  i n 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. n i t r a t e / 1 0 0  g.  dry weight from a f i e l d  with four times the normal amount of nitrogenous  fertilizer.  Crawford e_t al_, (1961) found that the c o n c e n t r a t i o n of i n oat p l a n t s level  i n the f i e l d i n c r e a s e d almost  treated  nitrate  l i n e a r l y as  the  of n i t r o g e n f e r t i l i z e r was i n c r e a s e d up to 200 pounds of  n i t r o g e n per a c r e .  I n c r e a s i n g l e v e l s of n i t r o g e n  i n c r e a s e d the n i t r a t e bromegrass  content  and orchard g r a s s .  fertilization  of weeds, wheat, timothy,  smooth  15  Percentage  recovery i n the crop of N a p p l i e d i n the  at v a r i o u s l e v e l s was i n f l u e n c e d by both the i n i t i a l n i t r a t e content  and amount of added N (Peterson  P r a c t i c a l l y a l l of the t o t a l  initial  n i t r a t e - n i t r o g e n + N m i n e r a l i z e d by the s o i l )  harvest.  It  is  soil  and A t t o e ,  1965).  removed by the crop was present  a v a i l a b l e N (added N +  i n the s o i l  f r e q u e n t l y assumed that  not  as n i t r a t e  at  the p o r t i o n 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 p o r t i o n of a crop i s  r e t a i n e d by the roots  organisms or l o s t by l e a c h i n g or as gas. f e r t i l i z e r N exists largely of t h i s  form present  harvested  and s o i l m i c r o Any r e s i d u a l  i n the n i t r a t e form and the  i n the p r o f i l e  field  amounts  to a depth of 21 inches  at  the beginning of the growing season accounted f o r as much as 90% of the v a r i a t i o n i n N uptake by o a t s . c o r r e l a t i o n with c o r n . content  These f i n d i n g s  of n i t r a t e - n i t r o g e n i s  often  There was a s i m i l a r  indicate  that the  of c o n s i d e r a b l e  initial  importance  i n determining the amounts of f e r t i l i z e r N r e q u i r e d to  attain  given l e v e l s of a v a i l a b l e N and i n c a l c u l a t i n g recovery of N by the  crops.  N i t r a t e accumulation i n s t a l k s  or stems of c o r n ,  sorghum  and soybean p l a n t s was s t u d i e d by Hanway and Englehorn (1958). Legumes i n r o t a t i o n or a p p l i c a t i o n of manure or n i t r o g e n fertilizer  i n c r e a s e d the n i t r a t e  amount of n i t r a t e  in plants  of the p l a n t s .  The  depended upon the stage of m a t u r i t y ,  the degree of drought i n j u r y , i n the s o i l .  content  and the a v a i l a b i l i t y of  N i t r a t e content was decreased,  but not  nitrogen eliminated  by e n s i l i n g corn p l a n t s . The n i t r a t e  content  of s e v e r a l  organs of p l a n t s has been  16  reported  [Crawford ejt a l ,  and Moxon,  (1952)].  (1961); Hanway, (1962); and Whitehead  G e n e r a l l y , the stem has the  highest  n i t r a t e c o n c e n t r a t i o n followed by roots > leaves y  floral  parts.  The b a s a l p o r t i o n of the stem and the lowest leaves tend to have a h i g h e r n i t r a t e c o n c e n t r a t i o n than the top stem p o r t i o n and h i g h e s t  leaves.  differences  o c c u r r e d i n oats over a wide range of a v a i l a b l e N.  They also  Crawford e_t a l  (1961) showed that  these  showed that when the a v a i l a b l e N was r e p l e n i s h e d d a i l y ,  the n i t r a t e c o n c e n t r a t i o n of oats d i d not decrease w i t h m a t u r i t y as has been normally r e p o r t e d . Historically,  n i t r a t e p o i s o n i n g of c a t t l e  corn s t a l k p o i s o n i n g et a l , 1940).  (May.o, 1895)  has been  labelled  and oat hay p o i s o n i n g  (Bradley  E x c e s s i v e amounts of n i t r a t e - n i t r o g e n i n forage  consumed by l i v e s t o c k may cause s e r i o u s effects  problems.  The s p e c i f i c  of the i n g e s t i o n of t o x i c amounts of n i t r a t e by animals  was c l e a r l y d e s c r i b e d by Davidson e_t a l  (1941) and has been  reviewed by Wright and Davison (1964).  Some of the w i n t e r  annuals have been known to cause c a t t l e  deaths  excess n i t r a t e  (Kretschmer, 1958).  i n d i c a t e d an i n c r e a s e  l e v e l s of n i t r a t e s et a l  S c a t t e r e d r e p o r t s have  i n recent years  animals from n i t r a t e i n forage  as a r e s u l t of  i n the deaths  of ruminant  (Crawford ejt''al_, 1961).  Toxic  (more than 2,000 ppm) were found by Lawrence  (1967) during the p e r i o d June 5 to J u l y 17 i n samples  harvested from grass which had been f e r t i l i z e d w i t h 300 and 375 k g . / h a . of N f e r t i l i z e r i n the s p r i n g . that f e r t i l i z e r r a t e s may r e s u l t  in toxic  It  is  suggested  i n excess of 225 k g . / h a . N (200  l e v e l s of n i t r a t e i n intermediate  lb./ac.N) wheatgrass.  17  4.  NITRATE REDUCTASE The major source of n i t r o g e n f o r most higher p l a n t s and  many micro-organisms i s n i t r a t e . r e d u c t i o n of n i t r a t e and i t s  The metabolic pathway i n the  a s s i m i l a t i o n to ammonia by higher  p l a n t s was suggested to be NO^" HONH  >  catalyzes  the f i r s t  2  >  NH by Hageman ejt a l  >  2  (1962).  3  step and i s  N0 ~  Nitrate  a flavin-dependent  p r o t e i n , which accepts e l e c t r o n s  H N 0 2  2  2  >  reductase molybdo-  from reduced p y r i d i n e  nucleotides. The p u r i f i c a t i o n and p r o p e r t i e s catalyzes of  t h e . r e d u c t i o n of n i t r a t e  of t h i s  enzyme which  to n i t r i t e by the  TPNH or DPNH ( t r i - or d i - p h o s p h o p y r i d i n e  oxidiation  nucleotides,  reduced f o r m ) , from soybean leaves and other higher p l a n t s p e c i e s was d e s c r i b e d by Evans and Nason (1953).  The n i t r a t e  r e d u c t i o n i s probably c a t a l y z e d by enzymes from the sap or from contaminating micro-organisms.  As enzymes are d i s r u p t e d  and d i l u t e d d u r i n g e x t r a c t i o n ,  they may be r a p i d l y o x i d i z e d  unless p r o t e c t e d .  used f o r t h i s purpose  Cysteine i s  mentioned by Hageman and Waygood (1959).  as  Use of c y s t e i n e has  a l s o been r e p o r t e d f o r the e x t r a c t i o n and p u r i f i c a t i o n f o r n i t r a t e reductase by N i c h o l a s and Nason (1955) . [hydroxymethyl]  aminomethane)  is  Inorganic phosphate  b u f f e r was found to be e s s e n t i a l  reductase  as potassium  i s used.  Nitrate  from young wheat embryos shows a s p e c i f i c  dependence  phosphate  f o r maximum enzymatic  w i t h a 30% i n c r e a s e when phosphate isolated  (tris  a l s o used f o r 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 a c t i v i t y of pH 7.4.  Tris  on added DPNH (Spencer,  1959).  activity  reductase  and  absolute  No n i t r i t e was  18  formed i n the absence of added DPNH.  K e i s t e r et_ a l  (1960)  r e p o r t e d the h i g h e s t c o n c e n t r a t i o n 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  give a measurable r a t e of r e a c t i o n *  In f u r t h e r  to  experiments _3  with higher c o n c e n t r a t i o n s i t was p o s s i b l e  of DPNH (approximately 10  to measure a slow u t i l i z a t i o n of DPNH.  Nitrate reduction is n i t r a t e with greater (Afridi  and H e w i t t ,  reductase (1969).  i n d u c i b l e i n higher p l a n t s by  i n d u c t i o n at higher n i t r a t e 1963).  N i t r a t e i n d u c t i o n of  a c t i v i t y was a l s o s t u d i e d by F e r r a r i N i t r a t e reductase  n i t r i t e production.  It  is  levels. nitrate  and Varner  i s normally assayed by measuring a common f i n d i n g that while  accumulation may occur i n p l a n t s seldom reaches  M)  for several  appreciable concentrations  reasons,  and normal  nitrate nitrite  levels  are low compared with n i t r a t e . Klepper and Hageman (1969) mentioned i n t h e i r paper nitrate  reductase was induced i n young leaves of  seedlings. leaf  The l e v e l  of n i t r a t e  t i s s u e although the n i t r a t e  higher than that of the  corn.  reductase was h i g h e s t i n the content  of the roots was much  (1960) determined the e f f e c t  of  supply on the a c t i v i t y of n i t r a t e reductase  The n i t r a t e reductase  are necessary  light in  a c t i v i t y decreased roughly i n  p r o p o r t i o n to the amount of shading.  quantities  apple  leaves.  Hageman and F l e s h e r and n i t r a t e  that  Both l i g h t and n i t r a t e  f o r the formation of n i t r a t e reductase  in  r e q u i r e d by the p l a n t f o r normal growth.  The c o m p l i c a t i o n s  of enzyme i n a c t i v a t i o n by use of heat  was s t u d i e d by E s s e l e n and Anderson (1956).  The thermal  19  resistance  of enzymes,  p a r t i c u l a r l y peroxidase,  c o n s i d e r a b l y even among v a r i e t i e s The enzymes may regenerate  varies  of the same v e g e t a b l e s .  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 thermal p r o c e s s i n g Wilder,  1962;  (Joffe  and B a l l ,  Z o u e i l and E s s e l e n ,  1962 ; V e t t e r e_t a_L, 1959;  1958).  d e s t r u c t i o n and r e g e n e r a t i o n of enzymes spinach puree was also  The thermal i n green bean and  s t u d i e d by Resende e_t al_ (1969).  Information on the heat s t a b i l i t y of the enzyme necessary If  to assess i t s  after  possible  effect  spinach with a h i g h n i t r a t e content  is  during p r o c e s s i n g . is prepared and then  s t o r e d at normal temperature, a l a r g e amount of n i t r i t e can develop by b a c t e r i a l r e d u c t i o n 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,  vegetable were obtained from markets  frozen food and f r e s h  i n the Vancouver a r e a .  Some vegetables were a l s o grown i n the greenhouse. greenhouse experiments 6 plants  (snap bean) cv Tender crop were s t a r t e d  each 6 - i n c h p l a s t i c  pot.  Spinach and beet were thinned  Canned food was opened and d r a i n e d to separate  used f o r d e t e r m i n a t i o n of n i t r a t e sodium-salicylate  water  in to  later.  l i q u i d and s o l i d p o r t i o n f o r a n a l y s i s .  (1955).  the  The l i q u i d p o r t i o n was  content d i r e c t l y by the  method as recommended by M u l l e r and Widemann  The s o l i d p o r t i o n was blended to make a puree with  i n the r a t i o one u n i t s o l i d to three u n i t s water.  a l i q u o t was used i n a n a l y s i s b)  of  per pot 10 days l a t e r and bean was thinned to 1 p l a n t  per pot 14 days a)  of beets cv D e t r o i t Dark Red,  of spinach cv Long Standing Bloomdale or 3 p l a n t s  green bush beans  4 plants  6 plants  In the  as f o r the  An  liquid portion.  S t r a i n e d baby food was d i r e c t l y used f o r a n a l y s i s  since  i t was already a puree. c) and  Frozen food was thawed,  s o l i d portions  for a n a l y s i s  d r a i n e d and separated  into  liquid  as f o r canned food.  d) Fresh vegetables obtained from markets were washed and blotted  dry with paper towels then cut i n t o small p i e c e s and  blended with water  i n the r a t i o of one u n i 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 m a t u r i t y . spinach l e a v e s ,  This was about 5 weeks f o r  6 weeks' f o r beet roots  and 8 weeks f o r  green  21  beans.  They were washed,  n i t r a t e content  dried,  cut and then analyzed f o r  as f o r the market samples  of f r e s h v e g e t a b l e s .  2. METHODS OF ANALYSIS 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. of ml.  Ten gm.  the puree are mixed with not more than 90 m l . water, of 5% CuSO^ s o l u t i o n ,  powder  1 gm. of Ca(0H)  (made of 1 p a r t Ca(0H)  2  ? MgCO^ mixture  and 2 p a r t s MgCO^ by  2  heated on a steam bath f o r one hour and s t i r r e d This i s  0.5  weight),  occasionally.  t r a n s f e r r e d to a 100 m l . v o l u m e t r i c f l a s k ,  cooled,  d i l u t e d to volume and mixed. The  contents of the v o l u m e t r i c f l a s k  Whatman #4 f i l t e r paper.  To an a l i q u o t of 1 m l .  1 m l . of 0.5% sodium s a l i c y l a t e evaporated to dryness  is  through  filtrate,  added and the mixture  on a steam b a t h .  concentrated s u l f u r i c a c i d i s stand f o r 10 minutes.  are f i l t e r e d  After cooling,  added and t h i s  is  allowed  is  1 ml. to  Six m l . of d i s t i l l e d water are added,  the mixture cooled and made a l k a l i n e by adding 7 m l . of 30% sodium h y d r o x i d e . A yellow  coloration indicates  the presence  This mixture i s made up to a volume of 100 m l . is  of  nitrate.  The absorbance  determined using the Beckman DU Spectrophotometer  wavelength  of 420 mu.  a,t  The c o n c e n t r a t i o n of n i t r a t e - n i t r o g e n  is  determined from a standard c u r v e . The different solution. gm.  standard curve i s prepared by reading absorbance of concentrations  of standard potassium  The standard s o l u t i o n  of potassium n i t r a t e  nitrate  i s made by d i s s o l v i n g  i n 100 m l . of water.  0.01805  One m l . of  this  22  standard s o l u t i o n  is  equivalent  to 0.025 mg. of  nitrate-  nitrogen . A l i q u o t s of 0, to dryness  0.4,  0.8,  1.2,  1.6  and 2 m l . are  on a steam bath with 1 m l . of 0.5% sodium  cooled and 1 m l . concentrated s u l f u r i c a c i d added. the procedure was the same as that f o r samples These a l i q u o t s would c o n t a i n amounts of 0, 0.04  evaporated  and 0.05  mg. of n i t r a t e - n i t r o g e n  salicylate, The r e s t  of  of p l a n t m a t e r i a l .  0.01,  0.02,  0.03,  respectively.  b) Rapid d e t e r m i n a t i o n of n i t r a t e and n i t r i t e  in  plant material This i s  the method recommended by Woolley et a l  P l a n t m a t e r i a l i s blended with water i n the r a t i o of 1:5. ml.  of the f i l t r a t e  solution.  is  added to 9 m l . of 20% a c e t i c  By the use of a measuring scoop,  described l a t e r  is  0.8  c e n t r i f u g e d f o r 3 minutes  at 1,000  G.  The l i g h t  absorbance  borosilicate  i s measured at a wavelength and the amount  c a l c u l a t e d from a standard c u r v e .  This method can determine both n i t r a t e and n i t r i t e i n f o o d , with a s l i g h t m o d i f i c a t i o n . nitrate  i n the absence of n i t r i t e  of n i t r a t e , (a)  sample  The supernatant  of 5 20 mp. on the Beckman DU Spectrophotometer, of n i t r a t e  gm. of a powder  Then the  i s poured through a small loose plug of  g l a s s wool.  acid  then shaken f o r 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 .  solution  One  added.  The sample i s  is  (1960).  For the determination of  and n i t r i t e  i n the  absence  the f o l l o w i n g reagents are used:  20% a c e t i c  a c i d s o l u t i o n c o n t a i n i n g 0.2  copper as copper  sulfate.  contents  ppm of  23  (b) powder d e s c r i b e d by Nelson e_t al_ (1954) : 100 gm. of barium  sulfate  75 gm. of c i t r i c a c i d 10 gm. of manganous s u l f a t e  dihydrate  4 gm. of s u l f a n i l i c a c i d 2 gm. of powdered z i n c 2 gm. of  1-naphthylamine  For the d e t e r m i n a t i o n of n i t r i t e i n the presence  of  n i t r a t e the f o l l o w i n g reagents are used: (a)  20% a c e t i c  acid solution  (No copper)  (b) powder mixture of 100 gm. of barium  sulfate  75 gm. of c i t r i c a c i d 4 gm. of s u l f a n i l i c a c i d 2 gm. of  1-naphthylamine  The d e t e r m i n a t i o n of n i t r a t e i n the presence  of  nitrite  r e q u i r e s two r u n s , one with reagents f o r n i t r a t e and the  other  with reagents f o r n i t r i t e . The water should be as f r e e d e m i n e r a l i z e d water i s used.  from copper as p o s s i b l e ,  Further,  the  so  electrical  c o n d u c t i v i t y of the water should never be h i g h e r than can be accounted f o r by d i s s o l v e d carbon d i o x i d e i n e q u i l i b r i u m with the  atmosphere. The standard curve f o r n i t r a t e - n i t r o g e n i s made by u s i n g  aliquots solution.  of 0,  0.2,  0.4, "0.6,  0.8,  1.0 m l . of standard n i t r a t e  Standard n i t r a t e s o l u t i o n i s made by d i s s o l v i n g ;  0.01805 gm. potassium n i t r a t e i n 100 m l . of water.  One m l .  is  aliquots  e q u i v a l e n t to 0.025 mg. n i t r a t e - n i t r o g e n .  These  24  would be e q u i v a l e n t nitrate-nitrogen  to 0,  0.005, 0.01,  0.015,  0.02,  respectively.  The standard curve of n i t r i t e - n i t r o g e n s i m i l a r l y by u s i n g a l i q u o t s  of standard n i t r i t e  0.0015188 gm. potassium n i t r i t e is  equivalent  0.025, mg.  i n one l i t r e  solution  of water.  to 0.00025 mg. n i t r i t e - n i t r o g e n .  was done because higher c o n c e n t r a t i o n  i s made  This  of n i t r i t e  of One m l .  dilution  solution  duced too dark a c o l o r . The c o l o r formed by t h i s method  pro-  is  cherry-red. c)  Phenol-disulfonic  a c i d method  This method was d e s c r i b e d by Barnett d e t e r m i n a t i o n of n i t r a t e  (1954) f o r  the  in silage.  Procedure Fifty  gm.  of p l a n t m a t e r i a l s  (or l e s s i f  it  contains  nitrate)  or vegetables are cut i n t o small p i e c e s  and macerated with water to make 200 gm. mixture i s  higher  F i f t y gm. of  r e f l u x e d by b o i l i n g f o r 5 m i n . , f i l t e r e d  u n t i l no n i t r a t e - n i t r o g e n  is  this  and washed  found i n the r e s i d u e by t e s t i n g  w i t h a powder mixture d e s c r i b e d by Nelson e_t al_ (1954) . is  then made to 100 m l . Each 5 m l . of t h i s f i l t r a t e ,  ^200*^  IT  =  s a m  Ple,  5 m l . of l e a d s u b a c e t a t e ,  15 min. This i s  filtered  is  which would be j-j^j-  The mixture i s  F  =  T2~  °^  s a m  Ple>  1 m l . of p h e n o l - d i s u l f o n i c tinuous  then shaken  through Whatman # 50 f i l t e r  Each 5 m l . of the above f i l t r a t e , x  is  x  combined with 5 m l . Ag2S0^  and 5 m l . alumina cream f o r which  t o t a l volume becomes 20 m l .  2"7J  This  for  paper.  which would be  evaporated to dryness.  a c i d reagent i s  the  After. cooling,  added, with almost  s t i r r i n g f o r 10 min. with a g l a s s r o d . This i s  con-  transferred  25  to a graduated  t e s t - t u b e w i t h 20 ml. water.  To the contents  of t h e , t e s t - t u b e , 10 ml. of 10% ammonia and 10 ml. o f water are added, shaking a f t e r each a d d i t i o n .  This i s f i l t e r e d  through Whatman # 50 and the yellow c o l o r development i s read at wavelength 420 mu on the Beckman DU Spectrophotometer  and  compared with that obtained from a standard. A blank i s prepared u s i n g 5 ml. o f d i s t i l l e d water i n s t e a d of the sample e x t r a c t and the instrument  zero  i s s e t with  water.  The standard curve i s prepared by u s i n g standard  nitrate  solution.  potassium  Reagents 1. Saturated s i l v e r sulphate  solution  This i s made by d i s s o l v i n g 1.41 gm. of s i l v e r sulphate i n 100 ml. hot water and s t o r i n g i n an amber b o t t l e , s i n c e the s o l u b i l i t y o f s i l v e r sulphate i n hot water i s 1.41 gm. to 100 ml. and i n c o l d water i s 0.51 gm. 2. Ammonium s o l u t i o n  (10% W/V NH^)  This i s 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 g r a v i t y to 0.9575. 3. P h e n o l - d i s u l f o n i c a c i d  reagent  This i s prepared by d i s s o l v i n g 25 gm. of pure phenol  i n 150 ml. o f concentrated n i t r o g e n - f r e e s u l p h u r i c a c i d .  To the mixture  i s added 70 ml. of fuming s u l p h u r i c a c i d .  f l a s k and i t s contents are heated  The  f o r 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 l e a d acetate i s added to 750 ml. of water and 160 gm. of l i t h a r g e  (lead o x i d e ) .  26  The mixture i s p l a c e d i n a b o t t l e shaker f o r 24 h o u r s . the volume of f i l t r a t e  and shaken on a mechanical  The mixture i s  thereafter  made up to 1,000  f i l t e r e d and  m l . with  distilled  water. 5. Alumina cream A s a t u r a t e d s o l u t i o n of potassium aluminum sulphate  is  first  aluminium sulphate  prepared by d i s s o l v i n g i n 100 m l . water s i n c e  11 gm. i n 100 m l . water. stirring.  12 gm. of its  potassium  solubility  Concentrated ammonia is  added with  When the mixture smells of ammonia, the a d d i t i o n  stopped and more s a t u r a t e d potassium aluminium sulphate tion is  added u n t i l an a c i d r e a c t i o n to litmus  Shaking f o r an hour a f t e r p r e p a r a t i o n i s stability  is  Standard n i t r a t e  soluobtained.  to  the  (1954).  solution  0.072 gm. of pure potassium n i t r a t e i n 100 m l . of water.  just  conductive  of the cream as found by Barnett 6.  is  is  One m l . of t h i s  is  dissolved  s o l u t i o n contains  0.1 mg.  nitrate-nitrogen. d) N i t r a t e ion e l e c t r o d e This  is  method  claimed to be s p e c i f i c  and operates by the development l a y e r of w a t e r - i m m i s c i b l e of a n i t r a t e  of a p o t e n t i a l  ion exchanger.  ion e l e c t r o d e ,  f o r the n i t r a t e across  a thin  The apparatus  a calomel reference  a pH meter with an expanded m i l l i v o l t s c a l e .  ion,  consists  electrode,  The technique  and is  s i m i l a r to pH measurement. Spinach was cut i n t o small p i e c e s and blended with water. The e l e c t r o d e s were immersed, the mixture was s t i r r e d by a magnetic  s t i r r e r , and the p o t e n t i a l  i n m i l l i v o l t s read w i t h i n  27  15 to 30 sec.  after  d r i f t had ceased and the reading compared  w i t h a standard c u r v e . A c a l i b r a t i o n curve was obtained u s i n g standard n i t r a t e solutions.  The curve was prepared on s e m i - l o g a r i t h m i c paper  with e l e c t r o d e  potential  on the l i n e a r a x i s ,  c o n c e n t r a t i o n on the l o g a r i t h m i c  and n i t r a t e  axis.  Recovery s t u d i e s were conducted by adding potassium nitrate  s o l u t i o n 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 Different  e x t r a c t i o n procedures f o r samples were s e l e c t e d  i n 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 in  samples.  made a g a i n s t  Absorbance measurements different  blanks.  of c o l o r development were  Reducing agent and o x i d i s i n g  agent were s e l e c t e d to add to the p l a n t e x t r a c t their effects  on n i t r a t e c o n t e n t s .  used to study the e f f e c t  pH of the sample e x t r a c t i o n s  of  foods  containing sucrose,  determination therefore  f o r study of  Buffer solutions  of pH on n i t r a t e  of  i n food.  were determined.  the sugar may a f f e c t  sucrose  contents  were a l s o The e f f e c t  In the the  case  nitrate  and n i t r a t e mixture were  analyzed f o r n i t r a t e - n i t r o g e n . N i t r a t e content food p r o c e s s i n g . therefore  i n food probably v a r i e d because of  The e f f e c t  studied i n this  and  20 minutes,  portions  of b l a n c h i n g of vegetables was  research.  b o i l i n g water f o r d i f f e r e n t  the  times,  Samples were blanched i n f o r example 0,  1,  3,5,  10,  and d r a i n e d f o r s e p a r a t i o n of l i q u i d and s o l i d  for n i t r a t e - n i t r o g e n determination.  The s o l i d p o r t i o n  was blended w i t h 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 f o r  the l i q u i d p o r t i o n .  N i t r a t e s o l u t i o n was a l s o added to  b l a n c h i n g water and n i t r a t e - n i t r o g e n recovery was a l s o s t u d i e d 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 f o r n i t r a t e - n i t r o g e n content d e t e r m i n a t i o n with the same b l a n c h i n g time. were blanched  Both  and d r a i n e d 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 p o r t i o n s . b) V a r i a t i o n s i n d i f f e r e n t p l a n t p a r t s Bean, beet and spinach were grown i n the greenhouse. Spinach was harvested at marketable  m a t u r i t y and cut i n t o young  and o l d p e t i o l e s , young and o l d l e a v e s , f l o r a l p a r t s and stem. N i t r a t e content was determined  by the soidum s a l i c y l a t e method.  Beets were h a r v e s t e d and c u t i n t o r o o t , s t a l k and leaves f o r n i t r a t e determination.  Bean p l a n t s were d i v i d e d i n t o l e a v e s ,  young and o l d pods, p e t i o l e , and stem and n i t r a t e  determined.  c) E f f e c t of n u t r i e n t s t a t u s Six p l a n t s of spinach cv Long Standing Bloomdale were s t a r t e d i n each p l a s t i c pot i n greenhouse and watered with 100 ml. of n u t r i e n t s o l u t i o n on a l t e r n a t e days. s o l u t i o n was 5 ml. 1M KNO^ make a l i t r e . weeks l a t e r .  The n u t r i e n t  and 5 ml. l M C a f N O j ^  i n water to  P l a n t s were thinned to four p l a n t s per pot two At the 37th day, they were harvested f o r a n a l y s i s .  d) N i t r a t e reductase s t u d i e s Spinach was grown i n a growth chamber at 16 h r s .  29  day  photo  period  ml.  o f the above-noted  were h a r v e s t e d  and 20°C  temperature  nutrient  3.weeks  later  and watered  solution  with  everyday.  f o rnitrate  100  Plants  reductase determination.  Procedure Spinach composited were  removed  from  a sample.  Leaves,  after  t o form  immersed  carried  l e a v e s were  immediately  to the laboratory.  dry,  weighed,  "23"  microblendor  cold  (2°C) g r i n d i n g  blending pressed 15  kept  cold  the  medium  method  speed  f o r each  wool  after  nitrate  described  t o 2.0 m l .  minutes  by Evans  reagent.  N^d  color  fuging  The m i x t u r e  was  stopped  t o develop  a t 1,500 G f o r 10 m i n u t e s  absorbancy  was d e t e r m i n e d  Virtis H ml. o f  The was  room f o r  decanted  and e x t r a c t s  were  completed  by a m o d i f i c a t i o n  mixture  0.2 m l . o f KNO^,  0.5 m l .  water.to  i n c u b a t e d a t 27°C  by a d d i n g  of  The a s s a y  and d e i o n i z e d  1 ml. o f  bring the  f o r 15 sulfanilamide  hydrochloride reagent,  and t h e c o n t e n t s mixed  was a l l o w e d  with  was  (1953).  phosphate,  napthyl) ethylenediamine  ( 1 m l . ) was a d d e d The  The a s s a y s  and Nason  extract,  and t h e r e a c t i o n  a  i n a cold  r e d u c t a s e was m e a s u r e d  DPNH, 0.2 m l . o f e n z y m e  blotted  The homogertate  The homogenates  plants,  water and.  of tissue.  liquid  and  sampling.  c o n t a i n e d ' 1.0 m l . o f p o t a s s i u m  volume  gram  The s u p e r n a t a n t  (3-5°C) throughout.  from  with  f o r 2 minutes  i n an i c e b a t h .  and assayed.  plants  l e a v e s were  c h e e s e c l o t h and c e h t r i f u g e d  2 t o 3 hours The  (2°C) deionized  The d e r i b b e d  a t maximum  a t 20 ,000 G..  glass  removal  s m a l l p i e c e s and blended  was k e p t  through  through  within  cut into  flask  minutes  were  i n cold  different  by i n v e r t i n g  f o r 5 minutes t o remove  by r e a d i n g each  before  the tubes. centri-  the turbidity. sample  The  against i t s  30  own blank ( a l l  reagents except f o r DPNH) i n the Beckman DU  Spectrophotometer  at 540 mu.  reductase was expressed  The a c t i v i t y of the  nitrate  as ^uM KNC^ as i n d i c a t e d by a standard  curve. The standard curve was prepared by u s i n g 0.2 m l . of different  c o n c e n t r a t i o n of potassium n i t r i t e  p l a c e of enzyme  solution  in  extract.  Reagents 1.  G r i n d i n g medium c o n t a i n i n g 0.1 M T r i s  hydroxymethyl aminomethane),  0.01  EDTA ( e t h y l e n e d i a m i n e t e t r a a c e t i c 7.3  and 7.8  M cysteine, acid)  (Tris  and 0.003 M  at a pH of between  (adjusted w i t h HC1).  2.  0.1 M potassium phosphate  3.  0.1 M KN0 .  buffer.  3  _3  4.  1.36  x 10  M DPNH (diphosphopyridine  nucleotides  r e d u c t i o n form) 5.  1% W/V s u l f a n i l a m i d e  6. N - ( l napthyl) 0.02% W/V.  i n 1.5  N HC1.  ethylene diamine h y d r o c h l o r i d e  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 p o r t i o n s of canned p r o d u c t s .  The n i t r a t e  content was f r e q u e n t l y higher i n the l i q u i d p o r t i o n than i n the d r a i n e d s o l i d s .  Since n i t r a t e  s a l t s are water  soluble,  much of the n i t r a t e  existing  food would d i s s o l v e  i n the l i q u i d p o r t i o n .  This would  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  i n the  than i n the s o l i d .  i n the s o l i d p o r t i o n of canned raise  l i q u i d p o r t i o n was s u b s t a n t i a l l y  N i t r a t e content was lower i n the  higher  liquid  p o r t i o n than i n the s o l i d p o r t i o n i n bean which exposes l e s s surface  area to the l i q u i d than spinach l e a f .  peaches a l s o have lower n i t r a t e content than i n the s o l i d p o r t i o n .  A p r i c o t and  i n the l i q u i d p o r t i o n  Among canned vegetable  foods,  n i t r a t e content was h i g h e s t i n s p i n a c h . A c c o r d i n g to Wilson (1949), 1 gm. of nitrate  ingested  at a s i n g l e  meal may be t o x i c .  17 ounces of only s o l i d p o r t i o n or 10.7 p o r t i o n of t h i s  Consuming  ounces of  spinach sample would t h e r e f o r e  a t o x i c q u a n t i t y of  potassium  liquid  p o s s i b l y be  nitrate.  b) S t r a i n e d baby food Nitrates  themselves are r e l a t i v e l y  c o n s t i t u e n t s of foods. at lower intake l e v e l s food i s  Since n i t r i t e s it  follows  non-toxic  are the t o x i c p r i n c i p l e  that the n i t r a t e content  of  an index of the amount of n i t r i t e which may be formed.  Thus n i t r a t e s  represent  a p o t e n t i a l hazard.  P u b l i c 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 p o r t i o n s of canned food obtained from Vancouver markets i n  1968.  N i t r a t e - n i t r o g e n i n mg./gm. Canned food  Solid  Liquid  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 (sliced)  .495  .417  .456  . 236  . 258  . 247  * Each sample was from a separate d i f f e r e n t time.  container obtained.at  a  33  been aroused over the p o s s i b l e nitrates foods  found i n some foods,  in infant diets.  nitrate  p a r t i c u l a r l y i n the use of these  Some baby foods were analyzed f o r  as shown i n Table Among samples  experiment,  h e a l t h hazard of high l e v e l s of  2.  of baby food analyzed i n  this  one sample of beet contained the h i g h e s t 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  i n canned  food was found to be h i g h e s t i n spinach and second h i g h e s t b e e t , but spinach is  not found alone  f o r baby f o o d .  in  The  beet baby food sample s t u d i e d 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 feeding. infant  According to the amount of n i t r a t e  feeding  i n spinach f o r  suggested by Simon (1966) there must not be  more than 0.300 mg./gm. possible  in infant  This i s because of the danger of  microbial activity  amounts can be formed i f  i n storage,  there  is  for n i t r i t e  a high n i t r a t e  the same c o n d i t i o n happens with other foods,  in  toxic  content.  If  even the peas and  c a r r o t sample f o r 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  to be t o x i c  for infant  c)  considered  feeding,  Frozen food Some f r o z e n foods were s e l e c t e d f o r d e t e r m i n a t i o n  of n i t r a t e - n i t r o g e n content. n i t r o g e n content either  It was found that  of frozen food was s l i g h t l y higher than i n  canned food or baby food.  nitrate-nitrogen  nitrate-  i n f r o z e n food.  Table 3 shows the content 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 p o r t i o n of f r o z e n spinach was higher than that of the s o l i d p o r t i o n a f t e r  thawing.  This i s  of  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 t r a t e - n i t r o g e n i n mg /gm Food 1*  2  aver.  Bean  .163  .125  .144  Beet  .350  .150  .250  Carrot  .075  .083  .079  Corn (mixed with m i l k , and o t h e r ; ingredients)  .041  .038  .040  Peas  .055  .024  .040  Peas and c a r r o t  .070  .070  .070  .113  .062  .088  Squash  . 200  .138  . 169  Mixed vegetables  .046  .043  .045  Applesauce  .253  .231  .242  Apricot  .185  . 201  .193  Peach  .195  .185  .190  Plum  .123 .  .117  .120  Vegetables  -  Spinach (mixed with m i l k , and othe r . ingredients)  Fruit  -  *Each sample was from a separate d i f f e r e n t time.  c o n t a i n e r obtained at a  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 p o r t i o n s from Vancouver markets  in  of f r o z e n food  obtained  1968.  N i t r a t e - n i t r o g e n i n mg./gm. Whole p o r t i o n  Foods  Solid portion  Liquid portion  !*  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  Each sample was from a separate d i f f e r e n t time.  . 280  .260  .300  .303  c o n t a i n e r obtained at a  .302  36  canned s p i n a c h . l e s s surface factor  i n the  The shape and s i z e of bean which exposes  area to the l i q u i d i s probably a c o n t r i b u t i n g lower amount of n i t r a t e - n i t r o g e n  i n the  p o r t i o n of f r o z e n bean as i t was i n canned food. peas d i d not have much l i q u i d phase a f t e r was i n s u f f i c i e n t  l i q u i d available  The n i t r a t e - n i t r o g e n  Frozen  thawing and there  for n i t r a t e  analysis.  content of frozen food was  higher compared to both baby food and canned food. would be p a r t l y due to m i g r a t i o n of n i t r a t e l i q u i d i n canned food.  This  i n t o the added  Storage under r e f r i g e r a t i o n may a l s o  be the cause of higher 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  liquid  content i n food.  content of frozen spinach was found to  be higher f o r the d u r a t i o n of storage under r e f r i g e r a t i o n 8-11  days than f r e s h s p i n a c h , by P h i l l i p s  increase  (1968).  This  could be due to o x i d a t i o n of amino n i t r o g e n .  n i t r o g e n was a l s o found to increase  under r e f r i g e r a t e d  probably because of m i c r o b i a l a c t i v i t y . s t o r i n g food should t h e r e f o r e amount of n i t r a t e - n i t r o g e n  Nitritestorage,  The c o n d i t i o n s  be considered  i n food as low as  for  i n keeping  of the  possible.,  d) Fresh vegetables Some f r e s h vegetables from markets were analyzed nitrate-nitrogen  content.  Some vegetables were grown i n  greenhouse and a l s o analyzed f o r n i t r a t e - n i t r o g e n  for  the  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 t h i s case, were  grown i n the greenhouse with no n u t r i e n t fertilizer.  Most of the n i t r a t e - n i t r o g e n  s o l u t i o n or any 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 f r e s h vegetables obtained from d i f f e r e n t  sources.  N i t r a t e - n i t r o g e n i n mg ./gm. Foods 1*  2  3  4  5 •  6  7  Aver.  Bean  .138  .070  -  -  -  -  -  .104  Spinach  .165  .135  -  -  -  . 162  Beet root  . 212  . 198  -  -  -  .205  Bean  .043  .053  .016  .014  -  -  -  .032  Spinach  .027  .033  .073  .078  Vegetables obtained from the market  .248  .098  Vegetables obtained from the greenhouse  -  -  .038  *Each sample was obtained at a d i f f e r e n t  .065  time.  .127  .068  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  p r o b a b l y was  due  content  to d i f f e r e n t v a r i e t i e s of v e g e t a b l e s and to  the p o s s i b i l i t y t h a t market v e g e t a b l e s were grown w i t h some nitrate  fertilizer.  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 d e t e r m i n a t i o n were used f o r the r e c o v e r y s t u d y . a) Sodium s a l i c y l a t e method T h i s was.the method most f r e q u e n t l y used. s t a n d a r d curve p r e p a r e d was studied.  One  The  e x c e l l e n t compared t o o t h e r methods  s t a n d a r d curve was  prepared w i t h higher n i t r a t e -  n i t r o g e n c o n c e n t r a t i o n w h i l e another was  p r e p a r e d f o r lower  c o n c e n t r a t i o n i n o r d e r to p r o v i d e a r e f e r e n c e f o r wide v a r i a t i o n o f n i t r a t e - n i t r o g e n content i n f o o d s . Potassium n i t r a t e s o l u t i o n was  added to s p i n a c h and  bean f o r r e c o v e r y s t u d i e s as shown i n Table 5.  T h i s method  gave the r e c o v e r i e s o f 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 r e c o v e r y of n i t r a t e - n i t r o g e n a f t e r  adding p o t a s s i u m n i t r a t e s o l u t i o n to s p i n a c h was  more v a r i a b l e  than i n bean. b) Rapid de 'termination o f n i t r a t e and n i t r i t e i n plant material T h i s method was  used f o r comparison  methods o f n i t r a t e - n i t r o g e n d e t e r m i n a t i o n .  with other  The s t a n d a r d  curve  had t o be p r e p a r e d t h r e e times i n o r d e r to get a s t r a i g h t The problem was  line.  p r o b a b l y caused by the powder d e s c r i b e d by  N e l s o n 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 u s i n g the sodium s a l i c y l a t e  method of  analysis.  N i t r a t e - n i t r o g e n i n mg./gm. Found i n sample  Food  Total  Average % recovery  found  Added 1*  Spinach  2  Aver.  1  2  Aver.  2.025 1.975 2.000 1.000 3 . 250 2.890 3.070  Bean  Laboratory  .114  . 130  duplicate.  .122  . 200  .326  .334  .330  107.0 104.0  40  results. The spinach leaves used f o r the recovery study by method d i d not give a s a t i s f a c t o r y Table 6.  In f a c t ,  recovery as shown i n  l e s s n i t r a t e was found i n one sample  nitrate  a d d i t i o n than b e f o r e .  content  i n food f o r t h i s  t h i s method.  this  The determination of  r e s e a r c h was t h e r e f o r e  There was no measurable n i t r i t e  after  nitrate  not done by  i n the  spinach  leaves. c)  P h e n o l - d i s u l f o n i c a c i d method The p h e n o l - d i s u l f o n i c  expensive reagents,  a c i d technique  and e x t r a c t s  requires  must be f i l t e r e d and evaporated  to dryness before  reagents f o r c o l o r development  (Mayers and P a u l ,  1968).  Standard s o l u t i o n s straight  nitrate.  of potassium n i t r a t e give a very  standard curve by t h i s  done with p l a n t m a t e r i a l s  can be added  method.  A recovery study was  containing different  amounts of  Table 7 shows the v a r i a t i o n i n r e c o v e r i e s  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 n i t r a t e gave an exaggerated spinach lower i n n i t r a t e 69.0%.  A suitable  and spinach samples  r e c o v e r y , but f o r a sample of  the recovery was found to  range of n i t r a t e  content  d i l u t i o n made  average  i n samples which  can be analyzed by t h i s method should be estimated suitable  high i n  and a  first.  d) N i t r a t e ion e l e c t r o d e  method  This method was suggested by Mayer and Paul f o r the d e t e r m i n a t i o n of n i t r a t e  in s o i l .  (1968)  The standard curve  was prepared on s e m i - l o g a r i t h m i c paper with m o l a r i t y l o g a r i t h m i c and mV reading l i n e a r .  The recovery was s t u d i e d with one u n i t  41  Table 6  The recovery of potassium n i t r a t e added to spinach u s i n g the r a p i d method of determination of  nitrate  in plant material.  N i t r a t e - n i t r o g e n i n mg./gm. Food  Found i n sample  Total  found  ..Added 1*  Spinach  *Laboratory  1.125  2  1.161  duplicate.  Aver.  1.143  .025  1  2  :  .795  1. 275  Aver.  1.035  42  Table 7  The recovery of potassium n i t r a t e  added to  vegetables by the p h e n o l - d i s u l f o n i c  a c i d method.  N i t r a t e - n i t r o g e n i n mg./gm. Found i n sample  Food  Total  - Added  Bean  1*  2  .0 22  .023  Aver.  .023  .080  Average % recovery  found  1  2  .039  .029  Aver.  .034  13. 8  Spinach "•' 11.410 11.520 11.465  .400 14.510 14.610 14.560  773.8  Spinach  .800  **  Laboratory  .682  . 576  .629  1.306  1.056  1.181  duplicate.  *30 gm. o f spinach used i n 200 m l . water compared to 10 gm. used i n the other sample.  69.0  43  of the puree of spinach leaves w i t h s i x t y u n i t s water. average and Paul  of the r e c o v e r i e s  was only 42.0%  (1968) found that  at high n i t r a t e  levels.  (Table 8 ) .  the instrument is  The r e c o v e r i e s  less  electrometer  used.  Mayer  sensitive  found here are r a t h e r  low, but might be the r e s u l t of the high l e v e l s of i n the spinach used or of p o s s i b l e  The  nitrate  problems inherent i n  the.  44  Table 8  The recovery of potassium n i t r a t e u s i n g the n i t r a t e  added to  spinach  ion e l e c t r o d e method.  N i t r a t e - n i t r o g e n i n mg./gm; Food  Found i n sample 1*  Spinach  Laboratory  . 588  2 : Aver  . 806  duplicate.  Total • Added  •  .69 7  found  .600  1  2  .806  1.09 2  Average % recovery  Aver.  .949  42.00  45  3. FACTORS AFFECTING ANALYSIS a) Time of  extraction  Nitrate solutions b o i l e d for d i f f e r e n t  of the same c o n c e n t r a t i o n were  lengths of time and n i t r a t e  contents  were found to be higher i n the s o l u t i o n with longer time of b o i l i n g as shown i n Table 9.  Almost the same r e s u l t s  on the e x t r a c t i o n of p l a n t m a t e r i a l s  occurred  as shown i n Table  10.  Recovery of added n i t r a t e was low with no b l a n c h i n g and i n c r e a s with b l a n c h i n g up to 5 min.  The apparent i n c r e a s e  in nitrate  would be at l e a s t p a r t l y due to decreased volume of w i t h i n c r e a s e d b o i l i n g time.  Volumes were not measured. The  reason f o r the v a r i a b i l i t y between samples ments  (see,  content  f o r example,  of the  solution  Table 5)  t i s s u e may i t s e l f  and between e x p e r i -  i s not c l e a r .  The n i t r a t e  be a f a c t o r .  Vapour was. c o l l e c t e d with a condenser at the of  e x t r a c t i o n of p l a n t m a t e r i a l s  spinach.  time  such as bean, b e e t , and  N i t r a t e was not d e t e c t a b l e i n these condensates.  This might suggest that  i n b o i l i n g or e x t r a c t i n g  materials  c o n t a i n i n g n i t r a t e a l l n i t r a t e w i l l be concentrated remaining m a t e r i a l s  after  i n the  some water has been evaporated.  c o n c e n t r a t i o n would be h i g h e r a c c o r d i n g to the length of i n b o i l i n g or The  The time  extracting. amount of n i t r a t e detected tended to decrease  when b o i l i n g time w i t h spinach was too l o n g .  With b o i l i n g  times of 10 and 20 min. the n i t r a t e detected was v a r i a b l e and lower than at s h o r t e r times of b o i l i n g as shown i n Table This c o n d i t i o n was a l s o found with t u r n i p greens, and beets as s t u d i e d by K i l g o r e et al_ (1963).  10.  collards,  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 s o l u t i o n with b o i l i n g  Details  Time of boiling min.  content i n  time.  Nitrate-nitrogen mg./gm. 1*  2  in Average % recovery Aver.  Potassium  0  .190  .178  .184  92.00  nitrate  1  .187  .195  .191  95.50  0.200 mg./gm.  3  .190  .198  .194  97. 00  added to  5  .197  . 203  . 200  100.00  10  . 206  .220  . 213  106.50  20  .279  .253  . 266  133.00  at  water  *Separate  samples on d i f f e r e n t  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 spinach with b o i l i n g  Time of boiling i n min.  Details  content i n  time.  Nitrate -nitrogen in mg./gm. 1*  2  Average % recovery  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  nitrate-  3  1.030  .950  .990  102.50  n i t r o g e n 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 e r e n t  days.  48  b) Repeated e x t r a c t i o n It was found  i n the previous s t u d i e s that p l a n t  m a t e r i a l with longer e x t r a c t i o n time had more n i t r a t e - n i t r o g e n , so a repeated e x t r a c t i o n o f p l a n t m a t e r i a l was c a r r i e d out. Since the method o f e x t r a c t i o n by M u l l e r and Widemann (1955) used only the f i l t r a t e of the e x t r a c t i o n f o r n i t r a t e d e t e r m i n a t i o n , the r e s i d u e and f i l t e r paper might c o n t a i n some nitrate.  Each r e s i d u e with f i l t e r paper was e x t r a c t e d again  and analyzed f o r n i t r a t e content.  Both spinach leaves and  spinach leaves with some n i t r a t e s o l u t i o n added before e x t r a c t i o n were used f o r t h i s study. some n i t r a t e l e f t over  i n s u c c e s s i v e r e s i d u e s with  amounts as shown i n Table 11. spinach t i s s u e s t i l l and  There was found to be decreasing  I t might be e x p l a i n e d t h a t  r e t a i n s some n i t r a t e a f t e r e x t r a c t i o n s  the t i s s u e would r e l e a s e some of t h i s n i t r a t e i n the  repeated e x t r a c t i o n .  Ifthis  i s not so, the n i t r a t e  i n the r e s i d u e might be formed i n some way. case that n i t r a t e s t i l l cooking  detected  I t means i n e i t h e r  e x i s t s i n spinach t i s s u e even a f t e r  and d r a i n i n g water.  c) C l a r i f i c a t i o n procedure spectrophotometer  and the use of  blanks  As noted, the f i l t r a t e  from the e x t r a c t i o n of p l a n t  m a t e r i a l was used f o r n i t r a t e determination by the sodium s a l i c y l a t e method i n t h i s r e s e a r c h .  E x t r a c t i o n without  f i l t e r i n g was a l s o i n v e s t i g a t e d f o r comparison of the apparent n i t r a t e content.  The e x t r a c t e d l i q u i d from spinach was not  c l e a r and produced a f i n a l mixture to  on which i t was impossible  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 the s u c c e s s i v e residues  Details  Residues extracted  of spinach  extraction.  N i t r a t e - •nitrogen i n mg./gm. 1*  Spinach  content from  2  Aver.  Average % nitratenitrogen l e f t over on residue  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  2.700  2 . 660  2.680  Spinach with 1 mg./  1st  .380  .320  .350  13. 05  gm.  2nd  .120  .110  .115  4.47  nitrogen  3rd  .100  .100  .100  3.73  added.  4th  .110  .100  .105  3.92  nitrate-  laboratory  duplicates.  •••Compared to the f i r s t  extraction.  +  50 s o l u t i o n was t h e r e f o r e  c e n t r i f u g e d at 1,000  G. f o r 3 min.  after  the c o l o r had been developed and the supernatant was used the r e a d i n g . is  The v a r i a b i l i t y of the r e s u l t s  shown i n Table 12.  results  between the  spinach l e a f  using this  added were s t u d i e d .  technique  There was not very much d i f f e r e n c e  two procedures of c l a r i f i c a t i o n .  extract  for  and spinach l e a f The r e c o v e r i e s  in  Both  e x t r a c t with some n i t r a t e  after  adding some n i t r a t e  i n the e x t r a c t i o n was r a t h e r poor by the c e n t r i f u g a t i o n procedure, so the f i l t r a t i o n procedure was s e l e c t e d f o r the determination i n this  research.  D i f f e r e n t blanks were a l s o i n v e s t i g a t e d effect  on the r e s u l t s .  the b l a n k .  The e x t r a c t before  even a f t e r  Bean produced a t u r b i d  c e n t r i f u g a t i o n which could not be used f o r a  such a t u r b i d e x t r a c t  but i t r e s u l t e d i n lower values extract  When some n i t r a t e was  c o u l d . b e used as the b l a n k ,  as shown i n Table 13. U n d i l u t e d  should not be used f o r a blank i n the case of bean.  It should be noted that  the d i l u t i o n of t h i s  i n a c t u a l n i t r a t e determinations was 1:100. spinach was c l e a r e r than bean but the r e s u l t s extract  their  adding reagents f o r c o l o r  blank f o r beans c o n t a i n i n g low n i t r a t e . added to bean,  for  D i s t i l l e d water was u s u a l l y used as  development was a l s o t r i e d as the b l a n k . extract  nitrate  as a blank were s t i l l  bean e x t r a c t  used  The e x t r a c t  of  using i t s  own  lower than that u s i n g  distilled  water as a b l a n k . The r e s u l t s  were a l s o d i f f e r e n t when the p o i n t s  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  h e a t i n g f o r e x t r a c t i o n , more n i t r a t e c o u l d be recovered as was found that h e a t i n g apparently made the t i s s u e nitrate. nitrate  The n i t r a t e  of  it  release  detected from the bean sample with  added a f t e r h e a t i n g was lower perhaps because the  tissue  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 procedures  content u s i n g  of c l a r i f i c a t i o n of spinach  different  extract.  N i t r a t e - n i t r o g e n i n mg./gm. Procedure  T o t a l found a f t e r adding n i t r a t e - n i t r o g e n 1 mg./gm. Average % recovery  Found i n sample 1*  2  Aver.  1  2  Aver.  Filtration  2.025  1.975  2.000  3.250  2 .890  3.070  107.00  Centrifugation  2.0 25  1.975  2.000  2.665  2.600  2.633  63. 25  laboratory  duplicates.  52  Table 15  The recovery of n i t r a t e - n i t r o g e n when blanks were used and with n i t r a t e s different  species at d i f f e r e n t  added to  times.  N i t r a t e - n i t r o g e n found i n mg./gm.  Details  different  1*  2  Aver.  .114  .116  .155  ,Average % recovery  Bean samples water as blank extract  as blank  -  -  -  -  adding n i t r a t e - ; n i t r o g e n 0.2 mg./gm. before  heating  water as blank  .326  .314  .320  102.50  extract  .204  .190  .197  -  water as blank  .304  .292  .298  extract  .180  .174  .•17 7  -  1.650  1.680  1.665  -  1.540  1.570  1.555  water as blank  2 .430  2.520  2.475  80.50  extract  2.500  2.500  2.500  94.50  water as blank  2.630  2.670  2.650  98.50  extract  2.550  2 .600  2.575  102.00  after  as blank  heating as blank  91.50  Spinach samples water as blank extract  as blank  -  adding n i t r a t e n i t r o g e n .1 mg./gm. before  after  heating as blank  heating as blank  laboratory  duplicates.  53  apparently t i e d up  some of the  happen w i t h s p i n a c h . t i s s u e had  already  The  This  This  of  results.  heated alone without p l a n t m a t e r i a l s as e x t r a c t i n g with p l a n t m a t e r i a l . measured and  interpreted  2  using The  for their  These reagents were the  same procedure  c o l o r development  i n terms of n i t r a t e - n i t r o g e n  These reagents produced only  tissue  Ca(OH) -MgCO\  e x t r a c t i o n were a l s o i n v e s t i g a t e d  the v a r i a b i l i t y of the  the  plants.  Some reagents such as CuSO^ s o l u t i o n and  e f f e c t on  not  s l i g h t l y higher than that  i n d i c a t e d the v a r i a t i o n of  among d i f f e r e n t species  mixture used i n the  did  recovery of n i t r a t e added a f t e r  been heated was  added before h e a t i n g . properties  added n i t r a t e .  was  content.  c l e a r s o l u t i o n observed v i s u a l l y  a f t e r reagents f o r c o l o r development were added. produced c o l o r as n i t r a t e - n i t r o g e n only  The  equivalent  solution  to 0.001  mg.  r  i n the  same volume of c o l o r s o l u t i o n u s u a l l y used i n the  experiments f o r n i t r a t e d e t e r m i n a t i o n . that there was  no  So  i t could be  said  e f f e c t of these reagents i n i n t e r f e r i n g with  the n i t r a t e - n i t r o g e n r e s u l t s , d) Reducing agents Ascorbic present i n p l a n t  a c i d and  t i s s u e such as spinach l e a v e s .  reducing agents and determination.  o x a l i c a c i d are supposed to  D i f f e r e n t amounts of a s c o r b i c  determined as shown i n Table 14.  n i t r a t e recoveries  are  might cause some v a r i a t i o n i n the n i t r a t e  a c i d were added to n i t r a t e s o l u t i o n and was  They  be  ascorbic  might be  concluded that there was  variations  of  amounts of o x a l i c  a c i d were added to n i t r a t e s o l u t i o n . no  oxalic  recovery of n i t r a t e  Only small  occurred when various  a c i d and  the  a c i d and  It  e f f e c t of these reducing  54  Table 14  The e f f e c t of reducing agents on n i t r a t e - n i t r o g e n determination.  Ml. of acid added  Details  0.0 25 m g . / m l . o x a l i c a c i d was added to 1 m l . nitrate solution containing n i t - • rate-nitrogen 0.0 25 m g . / m l .  0.025 m g . / m l . ascorbic acid was added to 1 ml. nitrate s o l u t i o n containing nitraten i t r o g e n 0.025 mg./ml.  *Laboratory  N i t r a t e - n i t r o g e n found i n mg./ml. n i t r a t e s o l u t i o n 1*  2  Aver.  Average % recovery  0  .026  . 027  .027  106.00  0.2  .026  .026  .026  104.00  0.4  .026  .026  .026  104.00  0.6  .025  .026  .026  102.00  0.8  .026  .026  .026  104.00  1.0  .026  .025  .026  102.00  0  .025  .025  .025  100.00  0.2  .025  .025  .025  100.00  0.4  .025  .027  .026  104.00  0.6  .025  .025  .025  100.00  0.8  .026  .026  .026  104.00  1.0  .024  .024  .024  96.00  duplicates.  55  agents on n i t r a t e d e t e r m i n a t i o n , used i n the present  at l e a s t at the  concentration  study.  O x a l i c a c i d was a l s o added to spinach l e a f in this  study.  It d i d not have very much e f f e c t  results.  A d d i t i o n of o x a l i c  detection  from spinach l e a f  as shown i n Table 15. results  a c i d is  on the  compared with  e x t r a c t without  extract  nitrate  added o x a l i c  acid  This should be considered as the same  as f o r reducing agents added to n i t r a t e s o l u t i o n which  had almost no e f f e c t  on n i t r a t e  determination.  A s c o r b i c a c i d i s known to be very u n s t a b l e .  The  a d d i t i o n of a s c o r b i c a c i d to p l a n t e x t r a c t probably does not need to be e)  studied.  O x i d i s i n g agent Since reducing agents had been i n v e s t i g a t e d  effect  on a n a l y s i s  for n i t r a t e ,  should a l s o be c o n s i d e r e d .  the e f f e c t  f o r any  of o x i d i s i n g agent  Hydrogen peroxide was s e l e c t e d as  an o x i d i s i n g agent which might e x i s t i n p l a n t t i s s u e n a t u r a l l y or which might be contacted  l a t e r during p r o c e s s i n g .  peroxide was added to spinach puree both before procedure of e x t r a c t i o n ,  samples  the  of an  content.  Spinach  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 . of t h i s  and a f t e r  i n order to f i n d the e f f e c t  o x i d i s i n g agent on the d e t e r m i n a t i o n of n i t r a t e  Hydrogen  investigation.  reducing agent e f f e c t  Table 16 shows the  The r e s u l t s  were almost the same as  i n which seemingly  increase  i n the n i t r a t e - n i t r o g e n content  increase  i n n i t r a t e - n i t r o g e n content  agent o c c u r r e d i n some samples.  results  there was a- small i n the a n a l y s i s .  by the use of  An  oxidising  As shown i n the t a b l e ,  only  56  Table 15  The e f f e c t  of o x a l i c a c i d on n i t r a t e - n i t r o g e n .  determination i n spinach.  N i t r a t e - n i t r o g e n i n mg./gm. Found i n sample  Food  1*  Spinach  1.900  Laboratory  2  2.060  duplicates.  Aver.  1.980  T o t a l found a f t e r adding o x a l i c a c i d (1 mg./gm.) 1  2.060  2  2.100  Average % recovery  Aver.  2.080  105.18  The e f f e c t o f an o x i d i s i n g  Table 16  in  Found before adding H 0 2  2  i n mg./gm. Total  found a f t e r adding H  2  2°2  Average % change  Aver.  Aver, Adding 0.1%' H 0  determination  spinach.  Nitrate-nitrogen Details  agent on n i t r a t e - n i t r o g e n  2  before heating  1. 800  2.000  1.900  1.980  1.900  1.940  102.50  after heating  1.800  2.000  1.900  1.9-00  1.900  1.900  100.28  before heating  IV 800  2:  000  1.900  1.950  2.190  2.070  108.92  after heating  1.800  2 .000  1.900  1.980  2.080  2.030  107.00  after heating  3.880  3 .100  3 .490  4.000  3.880  3.940  114.13  ^Laboratory  duplicates  Adding 1.0% H 0 2  2  Adding 2 mg. o f nitratenitrogen and H 0 2  2  58  one sample with added n i t r a t e s o l u t i o n showed a change of 114.13% which was a l i t t l e  h i g h e r than other samples.  An  o x i d i s i n g agent might a l s o be discounted as an i n t e r f e r i n g agent i n n i t r a t e f)  Buffer  determination. solutions  Buffer solutions  were used f o r s t u d i e s of  of pH on n i t r a t e d e t e r m i n a t i o n . was added to b u f f e r s o l u t i o n s were b o i l e d f o r d i f f e r e n t  The same amount of  at d i f f e r e n t  nitrate  pH v a l u e s ,  these  times and n i t r a t e contents determined.  The pH of the s o l u t i o n was measured a f t e r s o l u t i o n and b o i l i n g .  effects  The r e s u l t s  adding n i t r a t e  are shown i n Table  17.  Among three d i f f e r e n t pH values  n i t r a t e - n i t r o g e n was found to  be h i g h e s t 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 b u f f e r s o l u t i o n u s u a l l y decreased because of the of some of the l i q u i d . (1963) that no n i t r a t e  It was found before by K i l g o r e e_t al_ is  l o s t during c o o k i n g .  Since  volume of b u f f e r s o l u t i o n was decreased on b o i l i n g , content  the  nitrate  found should have i n c r e a s e d , but d i d only at pH 5.4  g) pH and t o t a l Because  a c i d i t y i n spinach  some v a r i a t i o n of apparent  was caused by pH, the pH and t o t a l s t u d i e d to f i n d  i f any i n t e r f e r e n c e  n i t r a t e determination i n spinach. for different  lengths of time,  acidity.  nitrate-nitrogen  a c i d i t y of spinach were might be o c c u r r i n g i n the Spinach leaves were blanched  d r a i n e d and both l i q u i d and  s o l i d p o r t i o n s used as separate total  evaporation  samples  f o r measuring pH and  The s o l i d p o r t i o n was blended and made to  100 m l . with d i s t i l l e d water,  then f i l t e r e d .  T o t a l a c i d i t y was  59  Table 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 i n b u f f e r s o l u t i o n s with b o i l i n g .  pH of buffer solution  7.20  6.66  5.40  Conducted  Boiling time -min.  1*  2  Average % recovery of nitrateAver. n i t r o g e n  Volume i n ml.  pH Aver.  1  2  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  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  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  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 as o x a 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 b l a n c h i n g . portions  and c a l c u l a t e d  The pH of the  liquid  g r a d u a l l y decreased w i t h i n c r e a s e d time of b l a n c h i n g .  The pH of the s o l i d p o r t i o n s  i n c r e a s e d 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 portions.  T o t a l a c i d i t y of the l i q u i d p o r t i o n s  liquid  i n c r e a s e d with  i n c r e a s i n g time of b l a n c h i n g and then decreased a l i t t l e the  20 min. b l a n c h i n g time, while s o l i d p o r t i o n s  g e n e r a l l y as the b l a n c h i n g time As noted i n Table 17, contents i n b u f f e r s o l u t i o n s different. spinach.  at  decreased  increased. the apparent  nitrate-nitrogen  at d i f f e r e n t pH values were  Table 18 shows the e f f e c t pH changes might t h e r e f o r e  n i t r a t e determination i n spinach.  of b l a n c h i n g on pH of affect  the r e s u l t s  Another experiment  c a r r i e d out to determine the r e l a t i o n s h i p of n i t r a t e  of  was content,  pH and b l a n c h i n g 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  liquid  p o r t i o n than i n the s o l i d p o r t i o n 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 p o r t i o n s as noted by Jackson et  al  (1967).  of canned products  N i t r a t e increased i n l i q u i d  p o r t i o n with i n c r e a s e d b l a n c h i n g time as was noted by K i l g o r e et a l  (1963) .  The i n c r e a s e  of the n i t r a t e - n i t r o g e n content  in  the l i q u o r a c c o r d i n g to i n c r e a s e d b l a n c h i n g time was found to be i r r e g u l a r and the t o t a l l i q u i d and s o l i d p o r t i o n s  amount of n i t r a t e - n i t r o g e n  in  sometimes was higher and sometimes  was lower than the amount found i n the uncooked sample. might be p a r t l y the e f f e c t  This  of pH v a r i a t i o n d u r i n g b l a n c h i n g 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.  Details  PH  % total  acidity  Blanching time -min.  Solid portion  Liquid portion  1*  2  Aver  1  2  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  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 e r e n t  days.  Aver.  _  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  Table 19  during b l a n c h i n g .  (10 gm. of spinach leaves used f o r each  Mg. of n i t r a t e n i t r o g e n per sample  pH  sample)  Liquid portion  Solid portion Blanching  spinach  Aver  Aver.  Mg. of n i t r a t e n i t r o g e n per sample  ph  Aver.  6.45  6.45  6.45  0.37  0.33  .35  6.45  6.45  6.45  .14  .12  .13  6.45  6.45  6.45  .27  .23  6.55  6.45  6.50  . 20  .16  .18  6.50  6.46  6.48  .40  .36  6.60  6.60  6.60  .13  .13  .13  6.45  6.50  6.48  .17  .23  6.55  6.65  6.60  .19  .17  .18  6.45  6.35  6.40  .36  .34  6.50  6.60  6.55  .05  .09  .07  6.40  6.30  6.35  .36  . 28  ^Laboratory  duplicates.  63  was noted b e f o r e .  The nonuniform samples might be another  cause of the v a r i a t i o n s which were noted by Wright and Davison (1964).  They found that n i t r a t e i s not u n i f o r m l y  d i s t r i b u t e d . t h r o u g h o u t the v a r i o u s p l a n t t i s s u e s . samples used here were c o l l e c t e d  Spinach  from many d i f f e r e n t  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 e x t e n t .  It was d i f f i c u l t to  distribute  leaves to make uniform samples which would have about same n i t r a t e c o n t e n t .  K i l g o r e et a l  the  (1963) suggested the  o r g a n i z a t i o n of samples only to minimize the v a r i a t i o n s and not to e l i m i n a t e v a r i a b i l i t y , h) pH i n beet Beet was also  s t u d i e d as f o r s p i n a c h .  beet i s u s u a l l y used as food but some people as w e l l ,  so beet leaves and stem were a l s o  Beet p l a n t s , of age,  grown i n the greenhouse  were d i v i d e d i n t o 3 p a r t s :  eat beet leaves investigated.  and h a r v e s t e d at 40 days leaves,  stem and r o o t .  Each was blanched and d r a i n e d to o b t a i n separate l i q u i d and s o l i d p o r t i o n s ,  its  The root of  samples  pH and n i t r a t e - n i t r o g e n  was recorded as shown i n Table 20.  The pH values  of  content  of a l l  liquid  p o r t i o n s were more v a r i a b l e than of s o l i d p o r t i o n s with blanching for d i f f e r e n t also  lengths  of time.  N i t r a t e - n i t r o g e n was  t r a n s f e r r e d to the l i q u i d p o r t i o n i n a l l three p a r t s of  beet and i n c r e a s e d the n i t r a t e content w i t h i n c r e a s e d b l a n c h i n g time.  i n the l i q u i d p o r t i o n s  The n i t r a t e - n i t r o g e n  of the s o l i d p o r t i o n was i r r e g u l a r ,  content  p a r t i c u l a r l y i n the  N i t r a t e became h i g h e r at 20 min. time of b l a n c h i n g a f t e r had been d e c r e a s i n g at s h o r t e r times.  leaf. it  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 d u r i n g blanching.  Liquid portion  Solid portion Blanching time -min.  Mg. of n i t r a t e n i t r o g e n per s amp1e  pH  Aver.  1  '2 : Aver.  Mg. of n i t r a t e n i t r o g e n per s amp1e  pH  1  2 ; Aver.  1 ;  2 ; Aver.  -  -  -  -  1*  2  0  6.00  6.00  6.00  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  5.80  5.90  5. 85  Leaves 20 gm. .780  .820  .800  - •  -  Stem 10 gm. 0  .800  .840  .820  -  -  -  -  -  Cont'd  Table 20 Cont'd  Liquid portion  Solid portion Ag. of n i t r a t e l i t r o g e n per ; amnl e  pH  Blanching time -mm.  4g. of n i t r a t e l i t r o g e n per 5 amp1e  pH  1  2  6.3C  .450  .490  .470  6.15  6.1£  .770  .730  .750  6.05  6.05  6.0 5  .460  .440  .450  .730  6.00  5.90  5.95  .570  . 530  . 550  . 590  6.00  5.90  5.95  1.000  .940  .970  -  -  Aver.  1  2  Aver.  Aver.  1*  2  Aver.  1  2  1  5.75  5.65  5.70  .800  .800  .800  6 . 25  6.35  3  5.70  5.70  5.70  .480  . 520  .500  6.15  5  5.70  5.70  5.70  .640  .660  .650  10  5.70  5.70  5.70  .750  .710  20  5.75  5.65  5.70  .600  . 580  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  Stem 10 gm.  Root 10 gm.  ^Separate samples on d i f f e r e n t  days.  -  1.680  1.690  66  the succulence  of the leaves which would be able to  n i t r a t e - n i t r o g e n content b l a n c h i n g times.  after  soaking longer at  retain  longer  The occurrence of these v a r i a b l e  results  might be a l s o e x p l a i n e d as f o r spinach f o r which i t d i d not. seem to be p o s s i b l e  to make up uniform samples  same amount of n i t r a t e - n i t r o g e n i)  to o b t a i n  the  i n samples before b l a n c h i n g ,  Sucrose Some canned food and baby food samples were found  to have sugar i n the range of 8.2  - 27.2%.  Some f r e s h and  frozen vegetables might a l s o c o n t a i n some sugar.  Sugar was  found to be carbonized during the treatment with the used i n the n i t r a t e d e t e r m i n a t i o n by the a c i d method and erroneous  reagent  phenol-disulfonic  i n f o r m a t i o n on the  nitrate  d e t e r m i n a t i o n of the product c o n t a i n i n g sugar might be obtained  (Barnett,  1954).  Sugar might a f f e c t  m i n a t i o n by the sodium s a l i c y l a t e  nitrate  method as w e l l .  deter-  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  s o l u t i o n 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 . sucrose  s o l u t i o n was higher than 100% and higher than recovery  from water.  This showed that sugar d i d a f f e c t  determination r e s u l t s ,  the  and the degree of a f f e c t  the c o n c e n t r a t i o n of sugar present j)  The recovery from  i n the  nitrate  depended on  sample,  Blanching Some food obtained from p l a n t s might be cooked before  being ready f o r the t a b l e .  The f o o d - c o n s t i t u e n t s  be changed by such cooking p r o c e d u r e .  The e f f e c t  and cooking of some vegetables was observed.  can p o s s i b l y of b l a n c h i n g  Some of  the  67  Table 21  The comparison of n i t r a t e - n i t r o g e n from water and sucrose  recoveries  solutions.  Mg. n i t r a t e - n i t r o g e n per Details  Average % recovery  Found Added  1*  2  Aver.  .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  Water  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  duplicates.  68  nitrates  of most vegetables s e l e c t e d f o r t h i s  study  t r a n s f e r r e d from the vegetable to the cooking water d u r i n g blanching.  The amount of t r a n s f e r depended on s p e c i e s ,  and s i z e of v e g e t a b l e s , increase  and the time used i n c o o k i n g .  of the n i t r a t e - n i t r o g e n content  shape The  i n the l i q u o r with  time of b l a n c h i n g 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  also  a c i d i t y in spinach.  found i n other vegetables s t u d i e d . nitrate  The i r r e g u l a r i t y of  t r a n s f e r r i n g might be the r e s u l t  found b e f o r e .  They were the d i f f e r e n t  the sugar c o n t e n t ,  T h i s was  of some causes  pH of cooking m i x t u r e s ,  the n a t u r a l v a r i a b i l i t y of the  the time of cooking and so on.  The f a c t  cooking water,  some n i t r a t e  samples,  found by K i l g o r e ejt a l  (1963) was that n i t r a t e was not l o s t by c o o k i n g . pouring o f f  the  still  Even with  e x i s t s i n 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 p r o p e r t i e s and  b e h a v i o r of food should be known and c o n s i d e r e d at the same time to f i n d a s u i t a b l e food.  i n d i v i d u a l c o n d i t i o n f o r cooking each  The f o l l o w i n g 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 r a n s f e r r i n g from vegetables to cooking water at d i f f e r e n t  times of b l a n c h i n g .  of n i t r a t e - n i t r o g e n  In some the t o t a l  i n s o l i d p o r t i o n and l i q u i d p o r t i o n  found to be higher than i n the uncooked sample. there  is  a marked i n c r e a s e  with b l a n c h i n g , but t h i s 4.  amount  in total  apparent  is  In spinach  nitrate-nitrogen  does not occur i n bean.  PLANT STUDIES a) Comparison of spinach and bean Table 22 shows the comparison of  nitrate-nitrogen  Table  22  The v a r i a t i o n portion  of n i t r a t e - n i t r o g e n t r a n s f e r r i n g  during b l a n c h i n g  of  from s o l i d to  liquid  vegetables.  Mg... of n i t r a t e - n i t r o g e n per sample Details - •  Spinach  50 gm.  —-  Bean 10 0 gm.  Water with nitraten i t r o g e n added 40 mg. per sample -  Blanching time mm.  Solid 1*  portion  Liquid  portion  Average  2  Aver.  1  2  Aver.  -  -  -  total  9.000  0  10. 125  7.875  9 .000  1  3. 360  11.500  7.430  12.150  14.000  13.075  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  0  13. 500  6.750  10.125  -  • -  1  7. 500  5.000  6.250  5.850  3  7. 750  3.500  5. 625  5  6. 600  3.400  10  7. 000  20  8. 250  -  10.125  2.700  4.275  10.525  8.130  2.700  5.415 . 11.040  5.000  7.000  3.250  5.125  10.125  4.500  5.750  7.000  3.000  5.000  10.750  4.250  6. 250  8.500  3.500  6.000  12.250  38.000  35.600  36.800  36.800  37",. 4 0 0  39.000  38.200  38.,200  38.000  39 .600  38.800  38.800  38.400  40.600  39.500  39.500  0 1  -  -  -  3 5  -  20.505  -  -  l  Cont'd  Table 22 - Cont'd Mg. of Details  Water,  etc.  Blanching time mm.  n i t r a t e - n i t r o g e n per sample L Lquid p o r t i o n  Solid portion 1  2  Aver.  Average total  1*  2  Aver.  10  -  -  41 . 200  44.000  42.600  42.600  .20  -  -  -  55 .800  50.600  53.200  53.200  -  -  8. 650  -  31.325  Spinach 50 gm.  0  L2 . "00  4.900  8.650  -  Spinach was added with nitratenitrogen 40 mg. per sample  0  29.650  33.000  31.325  -  1  5.750  7.500  6.625  36 .600  40 .000  38.300  44.925  3  9.500  ' 7.500  8.500  42 .000  40.000  41.000  49.500  5  10.000  10.000  10 .000  44 .000  39 .000  41. 500  51.500  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 e r e n t  days.  71  content  of spinach and bean.  contained l e s s n i t r a t e blanching.  Even 100 gm. of bean sample  than 50 gm. of spinach sample  The n i t r a t e  after  t r a n s f e r r e d from the p l a n t t i s s u e of  spinach to the cooking water was more than of bean.  The  shape and s i z e of p l a n t are probably the causes of v a r i a t i o n in nitrate  transfer.  The bean has a p r o t e c t i v e  epidermal  l a y e r and exposes l e s s s u r f a c e / a r e a than spinach to cooking water.  B l a n c h i n g time a f f e c t e d  spinach more than from bean.  nitrate  t r a n s f e r r i n g from  N i t r a t e content  i n s o l i d and  l i q u i d p o r t i o n s of bean were almost e q u i v a l e n t , nitrate  i n the  while  l i q u i d p o r t i o n of spinach was much higher than  i n the s o l i d p o r t i o n . b) V a r i a t i o n s i n d i f f e r e n t Crawford et a l  plant  parts  (1961), Hanway (1962) and Whitehead  and Moxon (1952) determined the v a r i a t i o n s contents of s e v e r a l p l a n t organs. the h i g h e s t n i t r a t e and then f l o r a l  in nitrate  G e n e r a l l y , the stem has  c o n c e n t r a t i o n followed by r o o t s ,  parts.  The b a s a l p o r t i o n of the stem and the  lowest leaves tend to have a higher n i t r a t e  concentration  than the top stem p o r t i o n and h i g h e s t l e a v e s .  Different  of bean, beet and spinach were d i s t r i b u t e d i n t o samples.  The v a r i a t i o n of n i t r a t e  was g r e a t e r parts.  is  than i n leaves which was greater  In c o n t r a s t  p a r t s of Spinach was  concentration in different  agreement w i t h the e a r l i e r workers, that  parts  separate  in different  bean, beet and spinach are shown i n Table 23. found to have n i t r a t e  leaves  parts  nitrate than i n  in  i n stem floral  young leaves and young p e t i o l e s were  found to have higher n i t r a t e  than o l d ones.  T h i s 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 plant  in  different  parts.  Mg. of n i t r a t e - n i t r o g e n per gm. Plant parts  Bean 1*  2  Root  Spinach  Beet Aver.  1  2  Aver.  1  .2  Aver.  -  -  .110  .090  .100  -  .080  .084  .082  .088  .138  .113  - • •  -  S t a l k or stem  .030  .019  .025  Petioles  old  .038  .029  .033  -  -  -  .060  .058  .059  young  .042  .030  .036  -  -  -  .160  .120  .140  old  .037  .040  .038  .039  .041  .040  .073  .038  .056  young  .053  .039  .049  -  -  -  .078  .065  .072  . 033 .042  .038  -  -  -  -  -  -  .014  .015  .044  .039  Leaves  Floral  or pod old young  Laboratory  .016  duplicates.  -  .034  73  been due to the s e l e c t i o n  of samples.  used here were almost yellow was the same with bean.  Old leaves and p e t i o l e s  and l e s s n i t r a t e was found.  Bean was found to have l e s s  i n the stem than i n the leaves and f l o r a l  parts.  of bean was probably too tough to be e x t r a c t e d Bean f l o r a l  p a r t s tend to have higher n i t r a t e  young pods which agrees with the l i t e r a t u r e . v a r i a t i o n s were d i f f e r e n t some p l a n t s  nitrate  The stem . ' -  completely. i n o l d pods than Beet  nitrate  from the l i t e r a t u r e on n i t r a t e  The n i t r a t e  d i s t r i b u t i o n of a l l p l a n t s was not found to be the same, appeared to vary with  status  It was noted by Brown and Smith (1966) n i t r o g e n 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 n i t r a t e content and t o p s . Each  it  species.  c) E f f e c t of n u t r i e n t  here.  in  i n that n i t r a t e c o n c e n t r a t i o n was higher i n root  than s t a l k and s t a l k h i g h e r than l e a v e s .  roots  It  i n red r a d i s h e s ,  that  increase  in  k a l e , mustard and t u r n i p  This was a l s o found with the spinach  studied  pot of 4 spinach p l a n t s was watered with  nitrate  s o l u t i o n from p l a n t i n g to harvest  time with a t o t a l  n i t r o g e n a p p l i e d of 305 mg. per p o t . recovery of n i t r a t e - n i t r o g e n from t h i s those grown without  nitrate-  Table 24 shows the spinach compared to  f e r t i l i z e r at the same time.  Nitrate-  n i t r o g e n was found to be higher i n the f e r t i l i z e d  plants,  but n i t r o g e n f e r t i l i z e r added was not a l l recovered i n the harvested s p i n a c h .  Peterson and Attoe  (1965) noted that  unrecovered f e r t i l i z e r was r e t a i n e d by the roots micro-organisms or l o s t by l e a c h i n g or as gas. n i t r o g e n content  and s o i l Nitrate-  found i n spinach with added n i t r o g e n  the  74  Table 24  N i t r a t e - n i t r o g e n content i n d i f f e r e n t s p i n a c h , with and without n i t r o g e n  Mg  With f e r t i l i z e r - . +  1  2  :  Average times i n c r e a s e d by f ertilizatioi  Aver.  1*  2  Stem  .086  .140  .113  2.531  2.303 2.467  21.08  Petiole  .204  .194  .199  3.271  3.129  3 . 200  16.08  Leaves  .134  .120  .127  1.234  1.428  1.331  10.55  Floral  .042  .036  .039  laboratory +  Aver.  of  fertilization.  of n i t r a t e - n i t r o g e n per gm.  No f e r t i l i z e r  Parts of plant  parts  . 721  .821  . 771  19 .99  duplicates.  F e r t i l i z e r was made of 5 m l . of 1 M CafNO^),, and 5 m l . of 1 M KN0  3  received  i n a l i t r e of water.  T o t a l l y each pot of 4 p l a n t s  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 10.55  21.08  times i n leaves and 19.99  in unfertilized plants.  times higher i n stem,  times i n f l o r a l  parts  This means that n i t r o g e n  than  fertilization  i n c r e a s e d n i t r a t e accumulation i n s p i n a c h , d) N i t r a t e reductase  studies  As noted most p l a n t s i n the form of n i t r a t e before  absorb n i t r o g e n from the  and i t must be reduced to ammonia  i t may be i n c o r p o r a t e d i n t o the nitrogeneous  of the p l a n t .  The f i r s t  c o n v e r s i o n of n i t r a t e  compounds  step i n n i t r a t e r e d u c t i o n i s  to n i t r i t e .  p r i m a r i l y due to the t o x i c i t y formed i n food before  of n i t r i t e .  ingestion  this  N i t r a t e reductase  is  is  N i t r i t e may be  or w i t h i n the d i g e s t i v e  an enzyme capable of  r e d u c t i o n (Evans and Nason, 1953).  Nitrate  was s t u d i e d here to i n v e s t i g a t e the p o s s i b l e i n food,  the  The n i t r a t e p o i s o n i n g  N i t r a t e i n food may be reduced by b a c t e r i a to n i t r i t e storage.  soil  tract.  during  catalyzing  reductase  nitrite  formation  such as v e g e t a b l e s , which may reach consumption.  That n i t r a t e  induces  the formation of n i t r a t e reductase  i n b a r l e y was found by F e r r a r i and Varner  (1969).  activity  Spinach  grown i n the growth chamber with n i t r a t e s o l u t i o n a p p l i e d was found to have some n i t r a t e reductase  activity.  was determined by measuring n i t r i t e f o r m a t i o n .  The a c t i v i t y The measurement  was compared to the standard curve prepared from potassium nitrite. this  The n i t r a t e reductase  a c t i v i t y of spinach used i n  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 spinach l e a v e s .  at the r a t e of 536.67 uM per gm. of  fresh  76  Resende  e_t a l  (1969) had d i s c u s s e d  the thermal  d e s t r u c t i o n of enzymes i n spinach puree.  In the  present  r e s e a r c h spinach was blanched f o r s e v e r a l  different  times and  enzyme a c t i v i t y was determined.  It was found that  t i v i t y of spinach was completely  i n a c t i v a t e d w i t h i n only  8 sec.  The enzyme a c t i v i t y of spinach and i t s  are shown i n Table 25. f o r a short time w i l l nitrite,  al  (1969).  thermal  effect  Therefore b l a n c h i n g or cooking spinach stop enzyme a c t i v i t y which produces  but i t might be regenerated  Resende et  enzyme ac-.  toxic  i n some way as noted by  77  Table 25  The e f f e c t of b l a n c h i n g time on the reductase  B l a n c h i n g time - sec.  nitrate  a c t i v i t y of s p i n a c h .  uM of KNC^ produced per gm. of f r e s h spinach leaves 1*  2  4  3  Average  0  420.00  726.67  453.33  546.67  536.67  2  106.67  106.67  53.33  46.67  •''78 .34  4  66.47  0  13.33  8  *Separate samples  0  0  on d i f f e r e n t  0  days.  0 0  v-  a.9_.9-5j 0  78  SUMMARY 1.  Spinach and beet were found to have higher  n i t r o g e n content  than other vegetables and f r u i t  Frozen food had higher n i t r a t e - n i t r o g e n content or f r e s h foods.  Any food c o n s i s t i n g  nitrate-nitrogen  i n the l i q u i d p o r t i o n .  nitrate-  analyzed. than canned  of some l i q u i d had The l i q u i d p o r t i o n  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 p o r t i o n except i n bean which has a p r o t e c t i v e  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 than those grown i n the greenhouse 2.  Among d i f f e r e n t  sodium s a l i c y l a t e  methods  content  without  fertilizer.  of n i t r a t e  analysis,  method was found to be the most  the  reliable.  D i s t i l l e d water should be used as a spectrophotometer in.the  d e t e r m i n a t i o n of n i t r a t e by the sodium  method.  salicylate  The recovery of n i t r a t e - n i t r o g e n a f t e r  nitrate  to food ranged from 104.0  v a r i e d among d i f f e r e n t  to 107.0%.  s p e c i e s and d i f f e r e n t  blank  adding potassium  The recovery times of adding  nitrate. 3.  Cooking and b o i l i n g food c o n t a i n i n g n i t r a t e caused an  increase after  of n i t r a t e - n i t r o g e n content  some water had been evaporated.  found i n the condensate.  The n i t r a t e  i n the remaining food No n i t r a t e - n i t r o g e n was content  i n the  liquid  i n c r e a s e d as the time of cooking or b o i l i n g i n c r e a s e d . n i t r o g e n content  of spinach i n c r e a s e d as cooking time  up to 10 m i n . ; at 10 and 20 min. of cooking i t was 4. bean.  Nitrateincreased  decreased.  Spinach had much higher n i t r a t e - n i t r o g e n content 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 p o r t i o n to  p o r t i o n of spinach was a l s o higher than bean.  than  liquid  79  5.  N i t r a t e - n i t r o g e n was not completely  food c o n t a i n i n g n i t r a t e ,  from 3.73  n i t r o g e n was found i n the r e s i d u e 6.  Reducing and o x i d i s i n g  interfering 7.  agents i n n i t r a t e  after  from  nitrate-  extraction.  agents may be discounted  as ,  determination. recovery from b u f f e r  i n t h i s study was found to be at pH 5.4.  spinach puree ranged from 6.28 5.70  to 13.051 of  The maximum n i t r a t e - n i t r o g e n  solutions  extracted  to 6.60  to  6.35.  8.  Sugar was found to a f f e c t  pH of  and beet ranged from  nitrate-nitrogen  d e t e r m i n a t i o n by i n c r e a s i n g the apparent n i t r a t e  concentration  i n p r o p o r t i o n with the c o n c e n t r a t i o n of sugar present i n food. 9.  Spinach grown with n i t r o g e n 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  concentrations  plant parts.  in different  it  in  different  The r a t e of  accumulation and d i s t r i b u t i o n was not uniform among plant parts 10.  nitrate  different  among s p e c i e s .  Nitrate reductase,  the enzyme which reduces  to n i t r i t e , was measured i n spinach by determining formation i n s p i n a c h .  nitrate  nitrite  N i t r a t e reductase was most e a s i l y found  i n spinach f e r t i l i z e d with n i t r a t e  solution.  This enzyme was  found to be i n a c t i v a t e d w i t h i n only 8 sec.  by b l a n c h i n g .  Therefore n i t r i t e might not be found a f t e r  cooking,  nitrate  remains a f t e r  cooking  water.  cooking even a f t e r pouring o f f  even though the  The remaining n i t r a t e might be reduced to n i t r i t e way and cause p o i s o n i n g .  Nitrogen f e r t i l i z e r  minimized i n growing p l a n t s  used as f o o d .  i n some  should be  The procedure of  80  p r e p a r a t i o n . o f food should a l s o be considered to minimize n i t r a t e - n i t r o g e n content of food f o r consumption.  81  LITERATURE  CITED  Anonymous 1950  Nitrate in vegetables. N u t r i t i o n Rev. 8: 230-231.  Afridi,  M.M.R.K.  1964  Balks,  and H e w i t t , E . J .  The i n d u c i b l e formation and s t a b i l i t y reductase i n higher p l a n t s . J . of Exper. B o t . 15: 251-271.  of  nitrate  R. and P l a t e , E .  1955  Utersuchungen tiber den N i t r a t g e h a l t von F u t t e r p f l a n o z • Landwirt Forsch 7: 203-211.  Barnett, 1954  A.F.G. 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