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The influence of variety and maturity on organic acids and related constituents in the highbush blueberry… Vance, Bayne Ferrier 1964

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THE INFLUENCE OF VARIETY AND MATURITY ON ORGANIC ACIDS AND RELATED CONSTITUENTS IN THE HIGHBUSH BLUEBERRY (Vaccinlum corymbosuin, L.)  by BAYNE FERRIER VANCE B. S. A . , The University of B r i t i s h Columbia, 1963.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the D i v i s i o n of Plant Science We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1964.  iii  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study.  I further agree that permission for ex-  tensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  It is  understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Division of Plant Science The University of British Columbia, Vancouver 8, Canada  ii  ABSTRACT  The possibility of evaluating the quality of blueberry fruit in chemical terms rather than by color or organoleptic methods was considered. The fruit of three varieties of commercial highbush blueberries at four stages of physiological maturity was analysed for total solids, water-insoluble solids, soluble solids, tltratable acids, total acids, volatile acids, reducing sugars, total sugars and sugar-acid ratios. Differences among varieties and stages of physiological maturity were sought by use of the above measurements supplemented by a study of the individual organic acid patterns of the f r u i t . Meaningful differences were evident among the four stages of physiological maturity for a l l measurements except volatile acids and total solids.  Varietal differences were evident from measurements  of the pH, soluble solids, sugar-acid ratios, and a number of the individual organic acids.  iv  ACKNOWLEDGEMENTS  The author wishes to express his appreciation and thanks for the guidance and assistance provided by the committee chairman Dr. A. J . Renney, Division of Plant Science, in the preparation of this thesis. Thanks are also extended to the committee members for their interest and assistance: Dr. V. C. Brink,  Division of Plant Science  Dr. G. W. Eaton,  Division of Plant Science  Dr. G . H. Harris,  Division of Plant Science  Dr. D. P. Ormrod,  Division of Plant Science.  Appreciation is also extended to the following individuals for their interest and assistance: Mr. I. Derics,  Laboratory Technician, Division of Plant Science  Dr. W. D. Kitts,  Division of Animal Science  Mr. G. R. Thorpe,  District Horticulturist, New Westminster, B. C.  Mr. G. 0. Twiss,  President, B. C. Blueberry Co-operative Association, Richmond, B. C.  Mrs. E. Whelan.  V  TABLE OF CONTENTS PAGE  INTRODUCTION  1  LITERATURE REVIEW  3  CHEMISTRY OF FRUITS  3  CHEMISTRY OF THE BLUEBERRY  6  ORGANIC ACIDS IN PLANTS  11  ORGANIC ACID METABOLISM  12  ORGANIC ACIDS AS FLAVOR COMPONENTS  15  PRACTICAL APPLICATION OF ORGANIC ACID- ANALYSIS IN FRUITS  17  ORGANIC ACIDS OF THE FAMILY ERICACEAE  IB  ANALYTICAL METHODS  20  MATERIAL AND METHODS  23  VARIETAL DESCRIPTION  23  EXPERIMENTAL DESIGN  24  EXPERIMENTAL  25  a. )  T o t a l solids  25  b. )  Water Insoluble s o l i d s  27  c. )  Soluble s o l i d s  28  d. )  pH  29  e. )  T i t r a t a b l e acids  29  f. )  T o t a l acids  30  vi  PAGE g. )  Volatile acids  30  h. )  Reducing sugars  32  i. )  Total sugars  32  j.)  Sugar-acid ratio  33  k.)  Organic acids  33  RESULTS AND DISCUSSION  40  a. )  Total solids  40  b. )  Water insoluble solids  43  c. )  Soluble solids  45  d. )  pH  49  e. )  Titratable acids  51  f. )  Total acids  55  g. )  Volatile acids  55  h. )  Reducing sugars  58  i. )  Total sugars  58  j.)  Sugar-acid ratio  64  k.)  Organic acids  67  CONCLUSIONS  77  LITERATURE CITED  80  ADDENDA  89  APPENDIX  90  vii  LIST OF TABLES TABLE I.  PAGE Harvesting dates of the fruit of three varieties of highbush blueberries at four stages of physiological maturity  II.  26  Percent total solids of the fruit of three highbush blueberry varieties at four stages of physiological maturity  III.  41  Percent water insoluble solids of the fruit of three highbush blueberry varieties at four stages of physiological maturity  IV.  44  Percent soluble solids of the fruit of three highbush blueberry varieties at four stages of physiological maturity  V.  46  pH content of the fruit of three highbush blueberry varieties as influenced by stage of physiological maturity  VI.  50  Percent titratable acids (as c i t r i c ) of the fruit of three highbush blueberry varieties at four stages of physiological maturity  .........  52  viii  TABLE VII.  PAGE Percent total acids (as c i t r i c ) of the fruit of three highbush blueberry varieties at four stages of physiological maturity  VIII.  56  Percent volatile acids (as acetic) present i n the fruit of three highbush blueberry varieties at four stages of physiological maturity  IX.  57  Percent reducing sugars of the fruit of three highbush blueberry varieties at four stages of physiological maturity  X.  59  Percent total sugar of the fruit of three highbush blueberry varieties at four stages of physiological maturity  XI.  60  Sugar-acid ratios of the fruit of three highbush blueberry varieties at four stages of physiological maturity  XII.  65  Acids of the highbush blueberry in order of elution from the anion exchange resin with corresponding Rj. x 100 values for a 1-butanolO N formic acid, 1:1 solvent system  68  ix  TABLE XIII.  PAGE The organic acids of the fruit of the highbush blueberry variety Weymouth at four stages of physiological maturity  XIV.  69  The organic acids of the fruit of the highbush blueberry variety Rancocas at four stages of physiological maturity  XV.  70  The organic acids o f the f r u i t of the highbush blueberry variety Weymouth at four stages of p h y s i o l o g i c a l maturity  71  X  LIST OF FIGURES FIGURE I.  PAGE Steam d i s t i l l a t i o n apparatus used for volatile acid determination  II.  Automatic fraction collector and resin column used for i n i t i a l separation of organic acids  III.  36  Apparatus used for descending paper chromatography  IV.  31  38  The influence of maturity on the percent soluble solids content of three varieties of highbush blueberries  V.  47  The influence of maturity on the percent titratable acid content of three highbush blueberry varieties  VI.  53  The influence of maturity on the percent total sugar content of the fruit of three highbush blueberry varieties  VII.  62  The influence of maturity on the sugar-acid ratio of the fruit of three highbush blueberry varieties  66  - 1 -  INTRODUCTION  The cultivation of the northern highbush blueberry (Vaccinium corymbosum, L.) began in 1921 when F. V. Coville crossed selections from the wild with other species (65). Between 1921 and 1939 he introduced eighteen varieties.  Today, practically a l l of  the present commercial acreage consists of these eighteen varieties or their progeny from succeeding crosses. The highbush blueberry is an economically important horticultural crop in British Columbia.  During the past six years the  commercial acreage has more than doubled to over one thousand acres (19).  At the present time there are more than twenty-five  varieties  listed as suitable planting stock by the British Columbia Department of Agriculture (75).  There are a number of factors to be considered  when choosing blueberry varieties; these include ripening date, growth habit, size of f r u i t , disease resistance, yield, and flavor. These characteristics a l l vary considerably between varieties, but in the present study special attention is given to flavor. To date very l i t t l e research has been done on quality factors in the blueberry; although one might suspect the characteristic tartness of many varieties to be attributed to a high organic acid content.  - 2-  The occurrence of a variety of free acids and their salts in plant tissues has become a well established fact in plant physiology.  One group of these acids, commonly referred to as plant acids  or organic acids, is distinguishable by i t s wide distribution and physiological functions.  These compounds are generally non-nitro-  genous, aliphatic, and non-volatile in nature. There is both academic interest and practical importance in the study of the individual organic acids of fruits such as the blueberry.  Organic acids are known to participate actively in fruit  metabolism (73), and also affect the flavor of fruit and fruit products (59, 6 2 ) .  In addition, chemical analysis of plants for  such compounds as the organic acids is becoming a valuable tool in plant taxonomy.  One authority feels that chemistry may ultimately  prove to be a better basis for classification than any of the present methods which are based primarily on morphology (72). The objective of the present study was to examine the i n fluence of maturity and variety on the organic acid content and certain associated chemical measurements in the fruit of the highbush blueberry.  - 3-  LITERATURE REVIEW  CHEMISTRY OF FRUITS  From a botanical point of view a fruit i s the ripened pericarp or flesh of the ovule together with the enclosed seeds and associated parts.  The flesh of the fruit may be dry or succulent  and edible or inedible.  However, to the pomologist the term is  restricted to the edible, succulent, development of one or more of the f l o r a l parts and does not include those botanical fruits classically treated as 'vegetables' such as the tomato, bean, and others. Fruits serve chiefly as a source of sugars, organic acids, vitamins, and flavors.  Protein and l i p i d substances occur in con-  siderable amounts only in the seeds which are rarely eaten or, as in berries, pass through the digestive tract with l i t t l e change (80). The chemistry of f r u i t s , like that of the entire plant, i s extremely dynamic.  A l l fruits undergo a number of distinct changes  in their chemistry from the time of embryo formation to maturity. Workers have established that both the titratable and total acidity increase as growth advances, attaining a maximum about the time ripening begins, and then decrease.  This sequence of events i s  apparent in the apple ( l ) , orange (67), grape (58), and the black currant (30).  Accompanying these changes in acidity is an increase  - 4 -  i n the soluble carbohydrate content of the f r u i t .  It has long been  i  known that the amount of sugar increases with f r u i t ripening. Thompson and Whittier (74) i n 1912 found an increase i n sugar content during ripening of apples, pears, peaches, grapes, tomatoes, strawberries, and persimmons.  In addition they found sucrose to be  the chief sugar of peaches, plums, and sweet potatoes; fructose to be the chief sugar of apples, pears, quince, and watermelons; and glucose to be the predominant sugar of the banana, strawberry,  grape,  tomato, and persimmon. In addition to the differences i n the chemistry of f r u i t s attributed to maturity there are a number of other factors that may result i n considerable v a r i a t i o n i n the sugar and acid content of fruit.  These include v a r i e t y , n u t r i t i o n , and climate.  F r u i t s grown  i n a cold and rainy climate tend to be more sour than those grown under warmer conditions.  Nitsch (58), i n a review a r t i c l e on the  physiology of f r u i t growth, c i t e s a study on grapes where the acid content was shown to decrease to half i t s value during a dry, sunny period.  Nitsch proposes that at higher temperatures organic acids  are metabolised in the r e s p i r a t i o n processes, whereas, at low temperatures they are accumulated.  The temperature necessary to induce  the r e s p i r a t i o n of malic acid i s lower than the one for t a r t a r i c acid.  Accordingly, f r u i t s r i c h i n t a r t a r i c acid (grapes) require  higher temperatures f o r ripening i n comparison with f r u i t s that are r i c h i n malic acid (apples).  C i t r i c acid needs a s t i l l higher tempe-  rature to be metabolised, so that fruits which contain this acid remain acid when mature (currants, citrus fruits, etc.). A number of researchers have demonstrated an increase in the sugar content of fruits resulting from f e r t i l i z e r application. Hopkins and Oourlay (35) were among the many to report an increase of soluble carbohydrates in fruit due to f e r t i l i z e r treatment.  This  i s , in effect, not a direct result of the f e r t i l i z e r , but an i n direct effect due to the increase in the production of photosynthates by the foliage. According to Nitsch (58) the source of both the sugars and organic acids of the fruit is in the leaves.  This can be inferred  from the fact that the acidity in apples i s higher in the center of the fruit than in the peripheral tissues (27)j this has also been shown to be true in studies on grapes (26).  Tompkins (77) suggests  that at least part of the acids are formed in the fruit from incomplete oxidation of carbohydrates in respiration.  Krotkov et a l (44)  suggest a close relationship between carbohydrates and acid metabolism in fruit attached to the tree, but conclude that the relation i s not a simple one.  They suggest that possibly the acids found in  the fruit arise from both the foliage and the fruit i t s e l f .  There  i s no c r i t i c a l evidence on this point, but i t is perhaps significant that an appreciable increase in the tltratable acidity has never been observed once the fruit has been detached from the tree.  Al-  though a small increase In titratable acidity may occur in the f i r s t  day or two a f t e r picking of immature f r u i t s Bennet-Clark (8),  (36).  i n a review a r t i c l e on the organic acids  of p l a n t s , points out the high increase i n organic acid production i n plants receiving n i t r a t e f e r t i l i z a t i o n as compared to those r e c e i v i n g ammonia.  The hypothesis i s that the mineral cations c a r r i e d  into the plant with the n i t r a t e anion are received by the organic acid anion and remain after n i t r a t e reduction.  CHEMISTRY OF THE BLUEBERRY In comparison with other f r u i t s the blueberry i s a comparative newcomer to h o r t i c u l t u r a l science and as a r e s u l t i t s rature i s meager and incomplete.  lite-  This i s p a r t i c u l a r l y true of i t s  l i t e r a t u r e pertaining to the chemistry of the blueberry. The f i r s t recorded attempt to analyze blueberry f r u i t s was conducted by Atwater i n 1906 (3).  He proposed the following chemi-  c a l composition f o r the blueberry: s o l i d s 11.6%, protein 0.1%, 3.0%,  nitrogen free extract 13.5%, f i b e r 3.2%,  and ash 0.4,1.  fat In  1928 C h a t f i e l d and McLaughlin (21), i n a revised e d i t i o n of Atwater's b u l l e t i n , reported 0.67 moles of acid expressed as anhydrous c i t r i c acid and a sugar content of 12.4£ f o r fresh b l u e berries. The Canada Department of Health and Welfare (16) l i s t s the  - 7 -  the chemical constituents of one hundred grams of fresh or frozen blueberries as follows: Water Carbohydrate Fat Protein Calcium Phosphorus Iron Vitamin A Vitamin C Thiamin Riboflavin Niacin  83.4 gm. 15.1 gm. 0.6 gm. 0.6 gm. 16.0 mg. 13.0 mg. 0.8 mg. 280 I.U. 5-18 rag. (.02 mg.) (.02 mg.) (.30 mg.)  (The bracketed figures have never been verified). Chandler and Highlands (20) studying the fruit of the lowbush blueberry reported a moisture content of 90%, a hydrogen ion concentration of 3.5, and a reducing sugar content of 9.85%. Both vitamins A and C have been reported in the fresh blueberry (53).  A study of the anthocyanin pigments in the blueberry  was conducted by Suomalainen and Keranen (71). Uhe (78), in studies with mature fruit of the Jersey v a r i ety, has shown a definite positive relationship between berry size and sugar content.  He found that the smaller berries had an average  soluble solids content of 10.5^ in comparison with the largest berries which gave an average reading of 15.0%. There was a definite negative relationship between berry size and acidity.  In ad-  dition Uhe showed the application of various f e r t i l i z e r s did not significantly influence the sugar or acid content of the f r u i t .  - 8 -  Ballinger et a l (addenda) reported the range of soluble s o l i d s i n highbush blueberry f r u i t to vary from approximately ten to eighteen percent; and that variations i n soluble solids may be related to the nitrogen-carbohydrate r e l a t i o n s h i p of the plant or to the amount of f r u i t on the bush.  Their results indicated that  an excessively high nitrogen content, or a large y i e l d of f r u i t resulted i n a decrease i n soluble s o l i d s content of the blueberry fruit. One objective of chemical studies of the blueberry has been to investigate the p o s s i b i l i t y of using chemical tests as a basis for a harvesting index. with the Jersey v a r i e t y ,  Woodruff and Dewey (82), on studies  evaluated the use of sugar-acid r a t i o s as  a means of ascertaining the degree of ripeness.  They found a highly  s i g n i f i c a n t c o r r e l a t i o n between the p o t e n t i a l 3 h e l f - l i f e and the sugar-acid r a t i o of the f r u i t measured at the time of harvesting. Poor holding q u a l i t y was found to be associated with high sugaracid ratios.  They found a s i g n i f i c a n t correlation between pH and  t i t r a t a b l e a c i d i t y and also between the soluble s o l i d and the t o t a l sugar content.  Under f i e l d conditions the authors recommended the  use of pH and percent soluble s o l i d s measurements i n place of t i t r a table a c i d i t y and t o t a l sugars for the estimation of the sugar-acid ratios.  In another experiment the authors found i t possible to d i s -  tinguish between r i p e and over-ripe berries on a basis of the sugaracid ratio.  Rubel and Jersey berries with a sugar-acid r a t i o of  - 9 -  twelve or l e s s should be classed as unripe, whereas a sugar-acid r a t i o greater than seventeen indicates over-ripeness. Woodruff et a l (83)  on studies of blueberry f r u i t ripening  demonstrated that changes i n t i t r a t a b l e  a c i d i t y were more apparent  than changes i n any other constituent during the ripening of the blueberry f r u i t , and suggested the use of acid measurements as a harvesting index.  On a fresh weight basis the changes i n the t o t a l  sugar content were found to be r e l a t i v e l y small, and the fact that the majority of the sugar content was present p r i o r to the time of red coloration suggests that sugars have limited value as a f i e l d test f o r maturity. the t i t r a t a b l e  These authors report a s i g n i f i c a n t decrease i n  a c i d i t y for the Jersey v a r i e t y after the appearance  of red c o l o r a t i o n .  From a high of 9.03% at the time of i n i t i a l red  coloration the t i t r a t a b l e a c i d i t y decreased to 1.15% on a dry weight basis twenty days l a t e r .  In comparison with the Rubel v a r i e t y the  t i t r a t a b l e acid content of mature Jersey berries was found to be considerably lower at a l l stages of f r u i t development.  There were  however no appreciable differences i n t o t a l sugar content at matur i t y (Jersey 12.7% and Rubel 12.6%).  A positive l i n e a r relationship  of the sugar-acid r a t i o s was evident f o r both v a r i e t i e s .  The authors  recommended the use of t i t r a t a b l e acid measurements as a harvesting index.  It was generally observed that p a l a t a b i l i t y improved with an  increased sugar-acid r a t i o .  - 10 -  Bowers and Dewey (12) analyzed the total sugar and total acid content of the Jersey and Rubel varieties.  They found a posi-  tive linear correlation in the sugar-acid ratio with time for a period of twenty days after the appearance of red coloration in the f r u i t .  Rubel berries were found to have a higher total sugar  and titratable acid content than Jersey berries at comparable stages of ripeness. Kushman and Ballinger (addenda), in studies with the highbush blueberry variety Wolcott, found that the interval between successive harvests had a profound effect on the quality of the fruit.  Lengthening the harvest intervals to nine or twelve days  caused an increase in sugars, pH, and the size of the f r u i t ; and decreases in the titratable acid content and the keeping quality of the f r u i t . The component sugars of the highbush blueberry have not yet been studied.  Barker et a l (7) on preliminary studies of the  sugars of the lowbush blueberry reported an increase of reducing sugars associated with increasing physiological age.  Trace amounts  of sucrose appeared only as the berries approached maturity and were never found In association with the green f r u i t .  Physiological  maturity, therefore, appears to be expedited by an increased number of fertilizations. Kimura and Mizuno (42), using paper chromatographic techniques, succeeded in showing the presence of xylose, fructose, galac-  - 11 -  tose, and two unidentified sugars in studies with Vacclnium uliginosum (whortleberry), a close relative of the blueberry.  ORGANIC ACIDS IN PLANTS The common organic acids are colorless substances which are usually soluble in organic solvents as well as in water.  In compari-  son with mineral acids organic acids are usually classified as weakly acidic. The presence of acidic compounds in plant material has been known since the time of the Greeks and Romans when tartar was used as a drug and as an emetic.  By the latter half of the eighteenth century  malic, c i t r i c , and oxalic acids had been isolated and identified  (11).  At the present time well over one hundred different organic acids have been reported in various plant species.  The organic acids  of plants have been shown to occur in a number of forms.  Many exist  in the free state, such as malic in apples, c i t r i c in lemons, and tartaric in grapes.  Others occur as metallic salts, the best example  being oxalic acid which is found as the very insoluble calcium salt. S t i l l others are found as esters.  Neish (56) in a review article on  the metabolic pathways of aromatic compounds cites shikimic and quinic acids as precursors for secondary growth substances such as the flavonoids, lignin, phenolic glucosides,  etc.  - 12 -  Of the large number of organic acids that have been isolated from plant material the aliphatic acids are quantitatively the most important.  There are however a considerable number of aromatic  acids commonly found in plant tissues.  Buch (13) has compiled a  bibliography of the organic acids in higher plants and l i s t s eightytwo of those of most common occurrence with their structural formulae and the family, genus, and species in which they have been identified.  ORGANIC ACID METABOLISM The organic acids occupy a central position in the metabolism of plants.  It is now impossible to consider organic acids in  plant tissues without taking into account almost every aspect of metabolism.  The close metabolic relationship of organic acids to fats,  carbohydrates, and proteins emphasizes their key role in plants. Perhaps the most notable function and occurrence of organic acids i s in the tricarboxylic acid cycle.  The bulk of the acids  present in fruit are of the same nature as those operating as intermediates in cycle.  This cycle releases energy and interrelates fat,  carbohydrate, and protein metabolism.  By analogy with animals, the  probable functioning of such a cycle in plants had been proposed.  The  presence of certain enzymes and certain acids of the cycle had been demonstrated in particular plants, but the complete system of enzymes and acids was not shown in a single plant tissue until 1951 when Millerd et a l (55) showed the entire cycle to be present in mung bean  - 13 -  seedlings.  Mitochondria recovered from the seedlings were able to  oxidize the Intermediates of the cycle.  The oxidation of pyruvic acid  to carbon dioxide and water was also demonstrated, and the r e s u l t i n g energy was not dissipated but was used i n the formation of high energy phosphate bonds i n adenosine triphosphate.  Other workers were able to  show the presence of the cycle i n tissue of other seedlings, but i t has never been demonstrated i n the mature p l a n t . The question of the general occurrence of the cycle in the mature plant remains unanswered.  Some authorities (14) suggest that  the t r i c a r b o x y l i c acid cycle of the seedling might be discarded or that certain tissues i n the mature plant have such a cycle whereas others do not. The reactions of the c i t r i c acid cycle take place i n the mitochondria, whereas the accumulation of organic acids occurs i n the vacuoles.  There i s as yet no explanation as to how and why a plant  organ often accumulates just one acid from the cycle i n the vacuoles of i t s c e l l s .  This i s exemplified by the high content of c i t r i c  i n the lemon and malic acid i n the apple.  acid  Kitsch (58), i n his review  a r t i c l e on the physiology of f r u i t growth, c i t e s the two prevalent theories that account f o r t h i s buildup of acids.  The f i r s t of these  assumes the presence of blocking mechanisms or i n h i b i t o r s at s p e c i f i c points i n the cycle to be the cause of a buildup of one metabolite. The second theory postulates that the acids are formed by the f i x a t i o n of carbon dioxide.  In experiments with tagged carbon dioxide Thurlow  - 14 -  and Bonner (76) showed that carbon dioxide is fixed by the leaves to produce malic acid in a reaction similar to the Wood and Workman reaction. There are a number of other cyclic processes involving organic acids.  One of these, the glyoxalate cycle has been shown to  occur in a number of higher plants (17, 49).  This cycle provides a  by-pass between iso-citrate and malate, a route for the synthesis of glyoxalate, and is thought to be the pathway for oxalate formation. The metabolism of organic acids in succulent plants is also of interest.  Succulents are a morphological rather than a taxonomic  grouping - having leaves or photosynthetic stems consisting of thickened spongy tissue.  Many plants are capable of accumulating  large concentrations of acid in their foliage.  With the majority of  plants these acids, once formed, are relatively 3table and remain intact except under adverse conditions such as starvation.  The suc-  culents on the other hand, are characterized by a diurnal fluctuation in acid content where acids are formed primarily during the night and disappear during the day.  Ransom and Thomas (61) in a review of this  phenomenon report the probable origins of these acids is generally assumed to be associated with starch degradation.  Coupled with this  is the discovery by Wood and Werkman (81) of carbon dioxide fixation in the synthesis of oxaloacetic acid from pyruvic acid.  There is no  simple relation between starch and organic acids, since at low temperatures workers have shown that there is insufficient starch consumed  - 15 -  to account for the acids formed.  It has been suggested that at low  temperatures acids are formed primarily by carbon dioxide fixation, whereas at the higher temperatures their formation may be mainly oxidative.  Since the dry regions in which succulents flourish tend to  have a marked f a l l in night temperature, i t is l i k e l y that in nature their acids are formed predominantly by carbon dioxide fixation. The organic acids present in plant tissue exist, to some degree, as salts in the c e l l vacuoles.  These salts are thought to act  as buffers in maintaining a suitable acid-base balance in the plant. Thus, the organic acids provide the anions for a portion of the mineral cations absorbed by the plant.  In some tissues the anions  of the organic acids serve to hold the majority of the mineral cations present.  Sinclair and Eny ( 6 6 ) showed that 51% to 13% of the cations  present in the fruits of orange and grapefruit were bound with phosphate, sulfate and other inorganic anions. The relation of organic acids to nitrogen metabolism in the plant is of considerable importance.  Transamination is an important  link between organic acids and nitrogen metabolism, although the evidence for these pathways is somewhat scarce.  McVicar and Burris (48)  in studies with the tomato plant, postulate the conversion of alpha ketoglutarate to glutamate.  Although i t has never been verified this  reaction and that of oxaloacetate to aspartate are generally thought to occur in plant tissues.  - 16 -  Nitrogen nutrition has a profound effect on the organic acid content of plants.  Bonner (9) has shown that plants fertilized with  nitrogen In the form of ammonia contain a substantially lower concentration of acids than do the same plants fertilized with nitrate nitrogen.  This increased synthesis of acids is thought to result from  the increased amount of mineral cations which remain in the c e l l after the reduction of the nitrate anion. Another aspect of the metabolism of the organic acids is the interconversion among acids.  Prior to the discovery of the tricarbo-  xylic acid cycle many studies were undertaken that showed the conversion of citrate to malate.  With the advent of the tricarboxylic acid cycle  the metabolic pathways involved were explained. extracyclic acids is also known to occur.  Interconversion of  Metabolic pathways for the  interconversion of shikimic and quinic acids are known (63). The organic acids also play a role in photosynthesis.  Both  glycolic and glyceric acid are known to be amongst the early products of photosynthesis and thus serve as precursors for the formation of many other compounds (70).  ORGANIC ACIDS AS FLAVOR COMPONENTS Technically the flavor of a substance is measured by i t s effect on our senses of taste, smell, and touch. tion of flavor is synonomous with taste.  The popular concep-  Taste i t s e l f is comprised of  - 17 -  the four components of bitter, salty, sweet, and sour.  The salt and  sour tastes differ from the bitter and sweet in that the latter are usually non-ionic (54). In plant tissues sweetness i s furnished by the sugars and to a very small extent by amino acids.  Sourness is associated with the  hydrogen ion, although i t depends not only on the ion concentration but also on the total acid present (34). Although i t alters the taste the addition of sugar to fruit changes neither the hydrogen ion concentration nor the total acidity. Pangborn (59), through the use of taste panel techniques, has shown that equal concentrations of organic acids vary in sourness. With studies on c i t r i c , l a c t i c , tartartic, and acetic acids c i t r i c acid was found to be the most sour of the four components.  PRACTICAL APPLICATION OF ORGANIC ACID ANALYSIS IN FRUITS The practical application of organic acid analysis has not been f u l l y studied and no definite applications are presently employed in commercial agricultural practices. A number of workers have shown that fruits and fruit products can be identified by characterization and identification of their component organic acids through the use of chromatographic techniques. Jorysch et a l (39) have used organic acid analysis as a means of identifying fruit juices; through this technique they have also been  - 18 -  able to detect the adulteration of juices.  In studies with black rasp-  berry juice changes in the acid pattern were noted with the addition of grape or apple juices. Kenworthy and Harris (41), working with apples, showed that varietal differences may be detected by analysis of organic acids. Clements (23) reports measurable differences in the c i t r i c and malic acid content of the orange varieties Washington Navel and Valencia. Markakis et a l (50) report a distinction between Rubel and Jersey blueberries of the same physiological age in that the former contain an appreciably greater amount of c i t r i c acid. Swain (72) makes note of the fact that with the ever i n creasing refinement in methods of plant analysis i t may become possible to classify plants on a chemotaxonomic basis; organic acid analysis would be important here.  ORGANIC ACIDS OF THE FAMILY ERICACEAE Considerable effort has gone into the identification of i n dividual organic acids in members of the plant kingdom.  The following  is a summary of the findings to date in the Ericaceae family. In 1905 Mason (51) found benzoic acid to be present in cranberries.  Griebel (29) in 1910 reported the presence of benzoic acid  in cranberries and whortleberries; Merriam and Fellers (53) in 1936 found traces of this acid in fruit of wild highbush blueberry.  - 19 -  Later studies on the acid of the cultivated highbush blueberry make no mention of the presence of benzoic acid (50). The presence of chlorogenic acid in this family was f i r s t discovered by Gorter (29) in Vaccinium lucidum. thought to be ubiquitous in higher plants.  Citric acid is  Nelson (57) was among  the f i r s t to identify c i t r i c acid in the fruit of the blueberry.  His  work showed c i t r i c acid and malic acids to be the predominant acids in this fruitj the absence of i s o - c i t r i c acid was also confirmed. Mehlitz and Matzik (52) found that the freshly pressed juices of many fruits, including the blueberry, contained acetic and formic acids.  They were able also to show the.presence of salicyclic  acid in the fresh blueberry. Glycolic acid has been identified in the leaves of Arbutus anedo and in the flowering twig of Erica multlflora (6).  Kaiser (40)  found lactic acid and oxalic acid to be present in the whortleberry. The presence of quinic acid i s characteristic of the genus Vaccinium. Lebedev and Linquist (46) were the f i r s t to find i t in the f r u i t ; in the studies with the whortleberry they estimated i t to be one of the main acids present. The occurrence of succinic acid in the huckleberry (Vaccinium myrtillus) was shown by Ramstad (60).  Tartaric acid has been isolated  in trace amounts from the fruit of the huckleberry by Harris and  - 20 -  Thrams (31).  Kohman (43) found the oxalic acid content of blueberries  to be 0.026,1 on a fresh weight basis. Kiimiro and Mizuno (42) using ion exchange methods found the fruit of the whortleberry to contain malic, c i t r i c , quinic, and gluconic acids.  Quinic acid was estimated to account for approximately  sixty percent of the total acid content. The f i r s t detailed study of the organic acids of the highbush blueberry was conducted by Markakis et a l (50) on the varieties Rubel and Jersey.  Glutamic, aspartic, quinic, galacturonic, glyceric,  glycolic, succinic, glucuronic, citramalic, malic, c i t r i c , malonic, chlorogenic, caffeic, oxalic and phosphoric acids were tentatively identified and quantitatively estimated.  On an equivalent basis, more  malic, chlorogenic and phosphoric and less c i t r i c and quinic acids were present in the ripe berries than in the unripe berries.  The ripe  Rubel berries contained more c i t r i c acid than Jersey berries of the same physiological age.  The concentration of phosphoric acid showed  essentially no change in the two stages of maturity studied, whereas the concentration of most of the other acids decreased considerably.  ANALYTICAL METHODS Measurements of the chemical composition of fruits are normally made by standard techniques recognized and accredited by such bodies as the Association of Official Agricultural Chemists or govern-  - 21 -  ment research agencies.  Thus, while the history of the analytical  methods employed in the project for the determination of reducing sugars, total sugars, total acidity, titratable acidity, volatile acidity, soluble solids, total solids, etc. is of interest i t is beyond the scope of this work.  Standardized procedures are avail-  able for a l l the aforementioned methods.  On the other hand no  technique for the combined determination of the individual organic acids has yet been accepted by the Association of the Official Agricultural Chemist3. Quantitative work on the biochemistry of organic acids has long been hindered by lack of techniques suitable for the determination of those organic acids present in trace amounts.  The older  methods of organic acid determination are based on the qualitative separation of the individual components either by fractional precipitation methods of the lead salts or by fractional d i s t i l l a t i o n of the esterifiod acid fraction (10).  The main disadvantage of  fractional precipitation methods is that the precipitates commonly include phosphate, sulfate, some carbohydrate, and much nitrogenous material ( l l ) .  Hartman and H i l l i g (33) report that tannins and  pectins are precipitated with lead acetate and often cause some degree of error in acid analysis, although in the loose definition of the term they can be classed as acidic constituents.  In addition,  with fractional precipitation methods the minor plant acids are usually overlooked.  - 22 -  Determination of plant acids by fractional d i s t i l l a t i o n of the methyl or ethyl esters has also been used widely.  The acidified  fraction is esterified with diazomethane or by heating in alcoholic hydrochloric acid and the resulting mixture of esters d i s t i l l e d under reduced pressure.  If a sufficient quantity of material is available,  i t is possible to separate esters of the individual acids in this manner.  But as an analytical method fractional d i s t i l l a t i o n is  cumbersome and requires large amounts of material and is difficult to put on a quantitative basis (10). In 1944 Consden et a l (24) described the use of paper chromatography as a means for the separation of organic compounds.  In 1946  a method for the separation of organic acids by partition paper chromatography was f i r s t proposed by Isherwood (38).  Refinements of this  technique by Lugg and Overell (47) resulted in the practical application of the method for the determination of the organic acids in natural tissues.  With the new methods organic acid analysis became  more precise and i t was possible to Isolate and characterize the acids of minor occurrence. Preliminary separation of non-amino acids by the use of ion exchange resins was f i r s t described by Busch et a l (15) in 1952. Fractional separation of organic acids was achieved by the use of resin column chromatography and elution solvents.  MATERIALS AND METHODS  VARIETAL DESCRIPTION The highbush blueberry varieties examined in this study were Weymouth, Rancocas, and Jersey.  These are among the most common  of those grown in the Fraser Valley. The Weymouth variety was introduced in 1936, originating from a cross of June x Cabot (65).  It is an early maturing variety  and i 3 the most extensively planted of the earlier varieties in British Columbia.  Darrow (25) estimated the Weymouth variety to  comprise approximately ten percent of the total U. S. acreage in 1961.  The Weymouth bush grows upright and is classed as below  average in vigor.  The fruit is of large size, lacking aroma, and  usually of poor dessert quality (25). Rancocas is a mid-season maturing variety resulting from a highbush backcross (Rubel x a selection from a lowbush-highbush cross) f i r s t introduced in 1926.  The bush grows upright with medium  vigor producing small, slightly aromatic fruit of good quality (5). The Jersey variety was introduced in 1928.  It produces a  late maturing berry of medium size lacking aroma yet of f a i r l y good dessert quality (65).  It i s the most extensively planted variety  - 24 -  in the U. S. and in 1961 comprised over thirty percent of the total acreage (25).  EXPERIMENTAL DESIGN The samples used in the investigation were obtained from the plantation of G. 0. Twiss, 1380 Blundell Rd., Richmond, B. C. from the 1964 crop. A l l berries were taken from mature bushes.  The bushes of  the Weymouth and Rancocas varieties were seven years old and those of the Jersey variety were twelve years old. as a 3 x 4 factorial;  The experiment was patterned  the three varieties being harvested at four  stages of physiological maturity with a l l treatments being replicated twice in a completely randomized design. The four stages of physiological maturity were selected using a modification of a classification originally described by Barker et al (7) on studies of the lowbush blueberry.  For the purposes of this  investigation the fruit was harvested at four distinct stages of physiological maturity based on size and color: small green (less than 1 cm. in diameter), large green (greater than 1 cm. in diameter), reddish (greater than 1 cm. in diameter), and mature blue (greater than 1 cm. in diameter).  Harvesting was conducted when i t was felt  that a majority of the berries on the bushes had reached a stage of maturity applicable to one of the preceding classifications.  This  procedure was followed for a l l samples with the exception of the  - 25 -  Jersey mature blue f r u i t which was harvested at a somewhat e a r l i e r stage to f a c i l i t a t e completion of the experiment i n the time a v a i l able.  The harvesting dates are given i n Table I. Each sample consisted of two i n d i v i d u a l r e p l i c a t i o n s con-  s i s t i n g of approximately s i x hundred grams of berries each, picked at random from one hundred bushes.  After p i c k i n g , the samples were  sealed i n polyethelene containers and refrigerated at -10°C.  EXPERIMENTAL The samples were removed from storage and allowed to p a r t i a l l y thaw for one-half hour at room temperature.  Half the sample  (approximately 300 gm.) was placed i n a Waring blendor, the remaining h a l f being returned to the freezer. The sample was homogenized at high speed for f i v e minutes or u n t i l such time as a s l u r r y was formed. were used i n a l l determinations.  Portions of t h i s s l u r r y  The s l u r r y was s t i r r e d w e l l p r i o r  to removal of each p o r t i o n . The following i s an outline of the methods of analysis employed i n t h i s study:  a.) (80).  Total s o l i d s .  The method used was that described by Winton  Approximately 20 gm. of the blended sample was accurately  weighed i n a tared covered metal dish and dried at 70°C. f o r at least  Table I Harvesting dates of the fruit of three varieties of highbush blueberries at four stages of physiological maturity.  Small green  Large green  Reddish  Mature blue  Weymouth  June 23  July 10  July 21  July 26  Rancocas  June 23  July 21  July 31  August 6  Jersey  June 23  July 21  August 6  August 13  - 27 -  six hours under reduced pressure in a current of air (about two bubbles per second) which had been dried by passage through sulfuric acid.  After drying the cover was replaced and the dish and contents  were allowed to cool in a desiccator and weighed to a constant weight. Duplicate determinations were made and the percent total solids was calculated as follows: Percent total solids „, Dry weight of blueberries Welgh't of sample  b.)  Water insoluble solids.  Following the method outlined by  Ruck (64) a weighed sample of the slurry was boiled with water to extract soluble material.  The water insoluble material was collected  on a previously dried, weighed f i l t e r paper and washed with hot water. The insoluble solids were then oven dried and weighed. Duplicate 25 gm. samples of the blended material were weighed to the nearest .01 gm. on a torsion balance.  Each duplicate  was then transferred to a 400 ml. beaker with hot water and diluted with additional hot water to about 200 ml. boiled gently for fifteen to twenty minutes.  The mixture was then One of the duplicate  samples was then transferred to a 250 ml. volumetric flask, cooled and made up to volume at 2 0 ° C , (the f i l t r a t e from this was used later for acid determinations).  The sample from the volumetric flask and  that from the beaker were then filtered separately through No. 4 Whatman paper that had been previously washed with hot d i s t i l l e d water, oven dried for two hours at 1 0 0 ° C , cooled in a desiccator  - 28 -  and weighed in a covered weighing dish.  The i n i t i a l filtrate from  the volumetric flask sample was set aside for acid analysis and the insoluble material in both f i l t e r papers was washed with 800 ml. of hot water.  The f i l t e r papers and contents were transferred to their  original weighing dishes and dried overnight at 8 0 ° - 9 0 ° C , cooled in a desiccator and weighed.  The recommended drying temperature of  100° - 105°C was discontinued when charring of pilot samples was noted.  The percent water-insoluble solids was calculated as follows:  Percent water insoluble solids » (Weight of dry insoluble material)(4).  c.)  Soluble solids.  Soluble solids were estimated by the pro-  cedure of Ruck (64) using a refractometer equipped with a percent sugar scale. Representative samples of the slurry were placed on the refractometer prisms and read directly.  If the consistency of the  slurry was such that a direct reading was not possible, a portion of the slurry was pressed through No. 4 Whatman f i l t e r paper.  If a cor-  rection for temperature was necessary, correction factors given in the A. 0. A. C. Methods of Analysis (3) were used.  Percent soluble  solids were estimated by the following equation: Percent soluble solids *> (Percent solids by refractometer)(l00-a) where a « percent water insoluble solids.  - 29 -  d. )  pH. Following the procedure outlined by Ruck (64) the  effective acidity was measured from direct readings on a pH meter that had previously been standardized with buffer solutions of pH 4.0 and 7.0.  A 50 to 75 gm. portion of the slurry was placed in a  250 ml. beaker and 50 ml. of d i s t i l l e d water added and the reading made on the pH meter.  Duplicate determinations were made on each  sample.  e. )  Titratable acids.  The titratable acid content was deter-  mined using a modification of the method outlined by Ruck (64) for estimation of total acidity.  The distinction between total and  titratable acids i s that the former are estimated after passage of the acid solution through a cation exchange resin (79). Titratable acidity was determined by titrating an aliquot of the water extract with standardized sodium hydroxide to pH 8.1 using a pH meter. A 25 ml. aliquot of the filtrate from the previous waterinsoluble solids determination was pipetted into a 250 ml. beaker and approximately 100 ml. of distilled water were added.  The mix-  ture was then titrated with 0.980 N NaOH to pH 8.1 using a pH meter. The amount of base required was recorded and from this figure the percent titratable acid was estimated and expressed as c i t r i c acid. Measurements were taken on duplicate samples and the percent t i t r a table acids was calculated from the following equation:  - 30 -  Percent titratable acid  f. )  =  1 T5"  (equivalent weight of acld)(N of NaQH)(titer) (weight of sample)  Total acids.  The percent total acidity was determined by  the use of a method outlined by Whiting (79).  A 25 ml. pipetted  aliquot of the f i l t r a t e from the previous water-insoluble  solids  determination was passed through a 30 cm. x .8 cm. column of Dowex 50W x 4 (50-100 mesh) cation exchange resin in the hydrogen form; the column was then washed with d i s t i l l e d water to remove any acids remaining.  The effluent was collected in a 250 ml. beaker and the  total acid was estimated by the same procedure as described for the determination of titratable acids.  g. )  Volatile acids.  The percent volatile acids was determined  by a modification of the method described by Ruck (64).  Volatile  acids were steam d i s t i l l e d from the sample and titrated with standardized sodium hydroxide.  The acids were calculated and expressed  on a percent basis as acetic. The apparatus used is shown in Figure I.  About 200 ml. of  d i s t i l l e d water were boiled in the steam source apparatus with the connecting tube open to facilitate the removal of the dissolved carbon dioxide and to saturate that portion of the apparatus with steam.  A continuous passage of cold water through the condensor is  required.  A two gm. sample of the slurry was accurately weighed and  - 31 -  Figure I. Steam d i s t i l l a t i o n apparatus used for volatile acid determination.  - 32 -  with the aid of a minimum amount of d i s t i l l e d water i t was transferred to the steam d i s t i l l a t i o n flask.  The steam hose was then connected  and the volatile acids were d i s t i l l e d over through the condensor and into a 250 ml. beaker containing d i s t i l l e d water.  Distillation was  continued for five minutes and the condensate was then titrated with 0.0245 N NaOH to pH 8.1 using a pH meter.  Preliminary tests indicated  that after five minutes of vigorous d i s t i l l a t i o n no acids came over. The total volatile acids were expressed as percent acetic and calculated as follows: Percent volatile acids  h. )  -  (equivalent weight of acid)(N of NaOH)(titer) (weight of sample/  Reducing sugars.  The percent reducing sugars was determined  by the method of Lane and Eynon (45) where invert sugar reduces the copper of Fehling's solution to cuprous oxide, a red insoluble precipitate.  The volume of the sugar solution from the sample required  to completely reduce a measured volume of Fehling's solution being determined by titration with methylene blue as an indicator.  Calcu-  lations were based on the factors computed by the A. 0. A. C. for use with this method (2).  i. )  Total sugars.  The percent total sugars was determined by  inversion of the sucrose present in the fruit by acid hydrolysis.  - 33 -  After inversion was completed the method described for reducing sugars was used.  j.)  Sugar-acid ratio.  The sugar-acid ratio for each sample was  calculated by dividing the percentage of total sugars by the percent titratable acidity.  k.)  Organic acids.  The organic acids of the highbush blueberry  were determined using a number of the modifications of Markakis et a l (50) on a technique originally developed by Busch et al (15) for the investigation of the acids of the c i t r i c acid cycle using anion exchange chromatography. 25 gm. of an equal mixture of the blended samples from the two replicates was transferred into 100 ml. of boiling water and allowed to boil gently for ten minutes with occasional stirring.  The  hot slurry was removed from the heat source and 2 ml. of 1 N n i t r i c acid were added (32) and the mixture was allowed to cool at room temperature.  After cooling 100 ml. of 35% ethyl alcohol were added  and the mixture was left to stand overnight.  The precipitated pectins  and alcohol insoluble solids were then removed by f i l t r a t i o n through Whatman No. 4 paper.  The acid solution was then concentrated to  approximately 50 ml. by evaporation in a stream of cold a i r . The concentrate was then passed through a 30 x 8 cm. column of Dowex 50W cation exchange resin in the hydrogen formj the eluted  - 34 -  acids were received in a 100 ml. volumetric flask.  The column was  washed with d i s t i l l e d water and the receiving flask was made up to volume and stored in the refrigerator. Dowex 1-X8 acetate (50-100 mesh) was used for fractionating the acid mixture on a resin column 35 cm. long and 1.0 cm. in diameter.  Commercial Dowex 1-X8 chloride was converted to Dowex 1-X8  acetate by washing the column with 1 N sodium acetate until a negative test for the chloride ion was obtained by testing the effluent with 1 N silver nitrate.  Excess sodium acetate was displaced by  0.1 N acetic acid. Aliquotes of the acid mixture corresponding to a total acid content of 1 to 1.5 meq. were used for fractionation.  Normally this  consisted of 10 ml. for the small green and large green samples and 20 ml. for the reddish and mature blue samples.  After the sample  had been applied to the column, 15 ml. of d i s t i l l e d water were passed through prior to the addition of the concentration gradient elution system.  A 250 ml. separatory funnel was connected to the top of the  column with Teflon tubing and served as an eluant reservoir.  Acetic  acid was passed through the column in a steadily increasing concentration.  The eluting solutions consisted of 50 ml. of 0.75 N  acetic acid followed by 75 ml. of 1.5 N acetic aced, 125 ml. of 3.0 N acetic acid and finally 250 ml. of 6.0 N acetic acid. rate was approximately 1 ml. per minute.  The column flow  - 35 -  Fifty-one fractions of 10 ml. each were collected in test tubes on an automatic fraction collector (Figure I I ) .  The fractions  were transferred to 50 ml. beakers and evaporated to dryness in a stream of air at room temperature.  The residues were redissolved in  2 ml. of 10% ethyl alcohol and returned to their original test tubes. A 1 ml. aliquot of this was placed in a 250 ml. beaker containing 100 ml. of d i s t i l l e d water and its acid content was quantitatively determined by titration with .0245 N sodium hydroxide to pH 8.1 using a pH meter. Portions of the remaining 1 ml. were used for paper chromatography.  The fractions were spotted 2.5cm. apart on 57 x 7.5 cm.  sheets of Whatman No. 1 f i l t e r paper.  The spotted papers were i r r i -  gated using one dimensional, descending method; the solvent system consisting of the upper phase of a mixture of 1-butanol and 3 N formic acid, 50:50 by volume.  It was found by experimentation that the Rf  values of the acids varied considerably with the age of the solvent. Acid chromatographed with solvent that had been mixed the previous day produced considerably higher R|. values.  One might expect this to be  due to esterification of the acid and alcohol components.  Solvent  used immediately after preparation gave poor resolution of these acids. This was possibly due to the greater solubility of the acids in the aqueous portion which had not been completely removed.  To elimiate  this variability the solvent for each sample was prepared two to four hours prior to U3e and i t was allowed to reach equilibrium before use.  - 36 -  Figure II. Automatic fraction collector and resin column used for i n i t i a l separation of organic acids.  - 37 -  The lower phase of the solvent mixture was used for vapor equilibrium. The chromatograms were run in glass chromatocabs (Figure III) in a controlled temperature chamber; the temperature was maintained at 20 - 1°C. After twelve to sixteen hours of irrigation the papers were removed from the chromatocabs and air dried.  After drying the papers  were dipped in a solution of acridine, O.lfi in 99.51 ethyl alcohol (68), and air dried.  The acids appeared as yellow spots on a white  to pale yellow background.  Under ultra-violet radiation the acids  appeared as intensely greenish-yellow flourescent spots. For identification twenty-one known acids were passed through the column in small groups and fractionated, titrated, and paper chromatographed in the same manner as the samples.  Standards were dis-  solved in d i s t i l l e d water and in some cases a ten percent solution of iso-propanol was used for those acids not readily soluble in water. From these data i t was possible to tentatively identify and quantitatively estimate the acids present in the samples. Oxalic acid was not separable by the chromatographic techniques used and oxalate determination was attempted by the method outlined by Winton (80).  The procedure involved precipitation of  oxalate as i t s calcium salt, dissolving i t in hydrochloric acid, and back titrating with standardized sodium hydroxide. The data for a l l measurements with the exception of the  - 38 -  Figure III. Apparatus used for descending paper chromatography.  - 39 -  organic acids was subjected to statistical analysis by methods outlined by Steel and Torrie (69).  The variation due to varieties,  stages of physiological maturity, and sampling methods was determined.  In addition, the variation due to analytical technique  was estimated for the percent titratable acids in an effort to determine i f differences could be attributed to this cause.  - 40 -  RESULTS AND DISCUSSION  A l l data reported for these experiments are expressed on a fresh weight basis unless otherwise stated.  a.)  Total solids.  There was a'gradual increase in the percent  total solids content associated with fruit development of Rancocas and Jersey fruit up to the reddish stage at which time a slight drop was recorded (Table II).  The earlier maturing Weymouth variety showed a  continuous increase in solids content during the later maturity stages examined (Table II). The total solids content determined for the mature blue fruits of a l l three varieties was considerably less than that of the 17.6/? reported by Atwater (3). Chandler and Highlands (20), in studies of the lowbush blueberry, reported a moisture content of 90% which can be interpreted as a total solids content of 10$.  The values obtained under the con-  ditions of these experiments were in a l l cases considerably higher t than their figure. :  Statistical analysis of the data showed that there were no significant differences in the percent total solids content among the three varieties or the four harvest dates.  There was however, a  highly significant interaction between varieties and stages of maturity.  - 41 -  Table II Percent total solids of the fruit of three highbush blueberry varieties at four stages of physiological maturity.1  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  11.94 11.29 12.99 13.71  12.30 11.81 12.71 13.77  12.12 11.59 12.80 13.74  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  14.61 14.96 16.20 15.81  13.70 14.62 15.67 15.75  14.05 14.76 15.94 15.78  12.58 13.68 14.09 13.87  12.43 13.65 14.14 13.91  12.51 13.67 14.12 13.89  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means: Weymouth Rancocas Jersey Stages of maturity means: Small green Large green Reddish Mature blue  Variety - n.s.  12.57 15.17 13.54 12.93 13.34 14.30 14.47  Maturity - n.s.  Variety x Maturity - •**  "'"A detailed example of the statistical methods used for this data is presented in the Appendix.  - 42 -  This indicates that the effect of variety on total solids content depends on the stage of physiological maturity selected, or alternatively the effect of physiological maturity on the solids content may depend on the variety selected.  This was true in a number of instances,  the most obvious being at the reddish stage where the Rancocas variety was found to have a total solids content of 15.94!^ in comparison with the Weymouth variety's value of 12.80./?. With the exception of the Weymouth variety the highest moisture content was found in the small green f r u i t .  This variability in  the solids content of the Weymouth fruit at the two green stages may have been due to temporary climatic factors at the time of harvesting. An increase in transpiration associated with increased temperatures and a decreasing moisture supply could possibly account for the decreasing moisture content of the maturing f r u i t .  The increase  in soluble solids evident for a l l varieties is a more likely explanation.  The general increase in total solids is substantiated by the  fact that as a general rule the maturing process is associated with a decreasing moisture content in plants. The slight increase in the moisture content noted in the mature blue fruit of the Jersey and Rancocas varieties could perhaps be due to temporary weather conditions or to one or more of a number of metabolic processes in the f r u i t .  Woodruff et a l (83), on studies  with the Jersey variety, also noted a decrease in a number of the  - 43 -  solid constituent after the appearance of red coloration.  Their  work showed a significant decrease in lignin, lipides and waxes, and cellulose on fruit harvested after the appearance of red coloration. There were some differences in the total solids content among the three varieties.  At a l l four stages of physiological matu-  r i t y the percent total solids of the Rancocas variety was considerably greater than that of the other two varieties - although not s i g n i f i cantly so. The use of percent total solids as an indication of maturity or for varietal identification does not appear to be feasible based on the results of this study.  b.)  Water-insoluble solids.  The trend in the water-insoluble  solids content followed that of the total solids content (Table III). A progressive increase through the small green and large green stages was followed by an evident decline with the appearance of red coloration. There was no significant variation in the water-insoluble solids content between the varieties or maturity stages examined. There was however, a significant interaction between varieties and maturity stages.  This was evident in that fruit of the Rancocas  variety at the small green and large green stages contained a considerably greater amount of water-insoluble solids that fruit of either of the other two varieties at the same stages.  - 44 -  Table III Percent water insoluble solids of the fruit of three highbush blueberry varieties at four stages of physiological maturity.  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  5.16 5.08 5.59 5.08  5.12 5.15 5.37 5.30  5.14 5.12 5.48 5.19  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  6.65 6.75 4.99 4.61  6.41 6.68 5.14 4.74  6.53 6.72 5.07 4.68  4.78 5.29 4.89 4.68  4.67 5.35 4.95 4.71  4.71 5.32 4.92 4.70  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means:  Weymouth Rancocas Jersey  Stages of maturity means: Small green Large green Reddish Mature blue  Variety - n.s.  5.23 5.75 4.92 5.47 5.72 5.14 4.85  Maturity - n.s.  Variety x Maturity -  - 45 -  The results of Woodruff et a l (83) discussed under total solids are confirmed by the observations made in the present study. The practical use of a percent water-insoluble solids determination for measuring maturity or for varietal identification does not however preclude the possibility of using measurements of one or more of the constituents of the water-insoluble solids content for such a purpose. Woodruff et a l have shown a highly significant difference in lignin content and significant differences in a number of other constituents in blueberry fruit harvested at stages between reddish and overmature blue.  Their observations were taken on the Jersey  variety and as yet no published information i s available on the content of other varieties.  Further studies on other varieties are  certainly required before any conclusions on this application can be reached.  c.)  Soluble solids.  Table IV outlines the results obtained  for the percent soluble solids.  Statistical analysis showed a  significant difference in the soluble solids content between varieties; the Jersey variety showing a higher percentage of soluble solids at a l l but one picking stage.  These results are graphically  illustrated in Figure IV. There was also a highly significant difference in the  - 46 -  Table IV Percent soluble solids of the fruit of three highbush blue berry varieties at four stages of physiological maturity.  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  5.74 5.96 9.95 10.75  5.81 6.04 9.95 10.78  5.78 6.00 9.95 10.77  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  5.55 8.20 10.66 11.50  5.61 8.20 10.73 11.42  5.58 8.20 10.70 11.46  6.53 7.91 11.48 13.10  6.45 8.02 11.49 13.17  6.49 7.97 11.49 13.14  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means:  Weymouth Rancocas Jersey  Stages of maturity means: Small green Large green Reddish Mature blue  Variety - *  8.12 8.98 9.77 5.95 7.39 10.71 11.79  Maturity - **  Variety x Maturity - **  - 47 -  Figure IV. The influence of maturity on the percent soluble solids content of three varieties of highbush blueberries.  - 48 -  soluble solids content between stages of maturity.  In a l l three  varieties the percent soluble solids showed a progressive increase over the four maturity stages examined. Bowers and Dewey (12) in studies with the Jersey variety, reported a soluble solids content of 13.6% eight days after the appearance of red coloration, which corresponds f a i r l y closely to the values obtained in this investigation.  The use of soluble solids  as an index of maturity appears to be worthy of consideration for the grower.  Under-ripe fruit could be readily detected by this method  providing a suitable standard was established for each variety.  How-  ever, i t i s perhaps more important for the grower to avoid placing overmature fruit on the fresh market.  As this study did not include  measurements on over-mature fruit i t is not possible to assess the value of soluble solids measurements for this purpose. The highly significant interaction makes i t difficult to assess the differences among the soluble solids contents of the three varieties.  However, overall significant differences among varieties  were shown. Soluble solids measurements provide a rough estimation of the sugar, organic acid, and free amino acid content of tissues. Results presented later in this discussion indicate that s i g n i f i cant differences were not evident between varieties on the basis of titratable or total acid content nor for reducing or total sugar con-  - 49 -  tent.  If the interaction effect was constant the varietal differ-  ences indicated by the soluble solids content may possibly be a t t r i buted to differences in the free amino acid content. There did not appear to be any definite relationship between the percent total solids and soluble solids except for the fact that both showed a positive relationship with maturation of the fruit with the exception of the slight decrease in total solids content noted in the previous discussion of total solids.  d.)  pH. The pH measurements taken on the samples are recorded  in Table V. There was a highly significant increase in the pH value which was directly related to the stages of physiological maturity. Highly significant differences were also apparent between the three varieties.  On the basis of the pH measurements the early maturing  variety Weymouth would appear to have the most acidic flavor, assuming that in this case the hydrogen ion concentration is the best index of sourness (34) and that the type of acids present are similar in a l l three varieties. The significant increase in the pH values past the green stage was as one might expect, correlated with the decrease in acid content.  Again the highly significant interaction does not permit  a true test of the differences between variety and stages of maturity means.  The significant correlation reported by Woodruff and Dewey  (82) was also apparent in the results of this study.  - 50 -  Table V pH content of the fruit of three highbush blueberry varieties as influenced by stage of physiological maturity.  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  3.10 3.05 3.20 3.50  3.05 3.10 3.20 3.55  3.08 3.08 3.20 3.53  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  3.25 3.30 3.30 3.60  3.30 3.30 3.35 3.55  3.28 3.30 3.33 3.58  3.35 3.25 3.60 3.75  3.30 3.25 3.60 3.70  3.33 3.25 3.60 3.73  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means: Weymouth Rancocas Jersey Stages of maturity means: Small green Large green Reddish Mature blue  Variety - **  Maturity -  3.22 3.37 3.47 3.22 3.21 3.30 3.61  Variety x Maturity - * *  - 51 -  The pH values of the early maturing Weymouth fruit and the mid-season Rancocas fruit at the mature blue stage corresponds to the figure of 3.5 reported by Chandler and Highlands ( 2 0 ) .  The ripe fruit  of the Jersey variety however showed a considerably higher value of 3.73.  e.)  Titratable acids.  The titratable acid content of the fruit  is shown in Table VI. There was a highly significant decrease in the acid content of the fruit of a l l three varieties after the appearance of red coloration.  This trend follows that reported by workers on  other fruits ( l , 30, 58, 67) who have found that the maximum amount of acid exists in most fruits prior to the final stages of ripening. This decrease may be related to temperature for as Nitsch (58) points out the oxidation of acids occurs at higher temperatures, lower temperatures being conducive to acid accumulation. Figure V illustrates the changes in the acid content of the three varieties over the four harvesting stages.  Statistical analysis  showed no significant difference in the acid content between the three varieties.  There was however a highly significant interaction be-  tween stages of maturity and varieties.  One possible portion of this  interaction may have been at the mature blue stage of the Rancocas variety where a considerably higher acid content was found in comparison with the other two varieties.  It was interesting to note that  - 52 -  Table VI Percent titratable acids (as c i t r i c ) of the fruit of three highbush blueberry varieties at four stages of physiological maturity.  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  2.58 3.06 1.89 1.07  2.47 3.11 1.94 1.03  2.53 3.09 1.92 1.05  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  2.25 3.42 1.74 1.17  2.14 3.35 1.74 1.18  2.20 3.39 1.74 1.18  2.51 3.16 1.74 1.03  2.54 3.02 1.72 1.00  2.53 3.09 1.73 1.02  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means:  Weymouth Rancocas Jersey  Stages of maturity means: Small green Large green Reddish Mature blue  Variety - n.s.  2.14 2.12 2.09 2.42 3.19 1.80 1.08  Maturity - **  Variety x Maturity - *»•  Figure V. The influence of maturity on the percent titratable acid content of three highbush blueberry varieties.  - 54 -  at the mature blue stage the Rancocas variety had the highest acid content; this did not however correspond to the lowest pH value. The significant relationship between titratable acidity and pH reported by Woodruff and Dewey (82) was not apparent in the present study.  The highly significant differences in the pH values  between the varieties was for some reason not duplicated in the titratable acid content.  This may possibly be due to a larger amount  of interaction in the titratable acid data which may have masked the varietal differences, or to the fact that a considerable amount of the acid of the fruit is not present in a dissociated form. The results show that i t is possible to distinguish between immature and mature fruit by the use of titratable acid analysis. Statistical measurements were taken to determine i f the variation due to the experimental procedures in the laboratory was large enough to influence the interpretation of results.  By the  statistical methods used i t was possible to compare the variation between the sub-samples used in the laboratory analysis with that between f i e l d replicates.  The results indicated that variation between  field replications was significantly greater than that between laboratory sub-samples.  The magnitude of the variation due to laboratory tech-  nique accounted for less than 0.50% of the total varation.  This  would indicate that the variation due to laboratory technique did not interfere with the interpretation of the differences due to  - 55 -  experimental units.  It was assumed that the laboratory techique for  the other measurements followed a similar pattern.  f. )  Total acids.  In a l l samples tested the total acid content  was found to be greater than that for titratable acids(Tables VI, VII).  This i s probably because a portion of the acids are present as  insoluble salts and are therefore not measurable by direct titration methods.  The amount of the acids held as salts did not appear to  vary to any considerable extent between varieties.  However, there  was a considerable difference in the amount of acid present in the salt form at the different stages of maturity for a given variety. For example in the Rancocas berries at the large green stage 420 mg. of acid/100 gm. of fresh fruit was shown to be in the salt form, whereas at the mature blue stage only 150 mg. of acid/100 gm. of fresh fruit was present in the salt form.  This would seem to i n d i -  cate that the amount of acid occurring in the salt form is dependent on the total acid content of the fruit and not on the stage of fruit development.  One might also suspect the nutritional status of the  plant to be a factor here.  g. )  Volatile acids.  There were no significant differences in  the volatile acid content of the three varieties nor between the four stages of maturity (Table VIII).  A highly significant interaction  was noted between the varieties and maturity stages.  - 56 -  Table VII Percent total acids (as c i t r i c ) of the fruit of three highbush blueberry varieties at four stages of physiological maturity.  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  2.69 3.43 1.95 1.24  2.66 3.57 1.98 1.15  2.68 3.50 1.97 1.20  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  2.36 3.77 1.94 1.32  2.32 3.78 1.95 1.33  2.34 3.78 1.95 1.33  2.76 3.43 1.93 1.18  2.88 3.43 1.98 1.16  2.82 3.43 1.96 1.17  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means:  Weymouth Rancocas Jersey  2.33 2.35 2.34  Stages of maturity means: Small green Large green Reddish Mature blue  Variety - n.s.  Maturity -  2.61 3.57 1.95 1.23  Variety x Maturity - **  Table VIII Percent volatile acids (as acetic) present i n the fruit of three highbush blueberry varieties at four stages of physiological maturity.  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  .029 .037 .029 .011  .029 .029 .029 .011  .029 .033 .033 .011  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  .044 .044 .029 .029  .044 .040 .029 .029  .044 .042 .029 .029  .029 .029 .022 .015  .037 .029 .026 .015  .033 .029 .024 .015  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means:  Weymouth Rancocas Jersey  Stages of maturity means: Small green Large green Reddish Mature blue  Variety - n.s.  .026 .036 .025 .035 .035 .027 .018  Maturity - n.s.  Variety x Maturity -  - 58 -  The volatile acid content was not included in the titratable or total acid content.  The methods used for determination of t i t r a -  table and total acids employed the use of heat which resulted in the loss of the volatile constituents.  The volatile acids present were  calculated as acetic - although formic, propionic, and possibly others are present in blueberry f r u i t .  h. )  Reducing sugars.  The reducing sugar content of the fruit  examined i s presented in Table IX.  Statistical analysis of the data  indicated that there were highly significant differences in the reducing sugar content among the four stages of physiological maturity. No significant differences were found in the reducing sugar content of the three varieties. As one would expect there was a definite increase in the reducing sugar content of the fruit with ripening.  The highly signi-  ficant interaction indicates that at a given stage of maturity the reducing sugar content of the varieties may differ considerably. At the mature blue stage the Jersey variety had a considerably greater reducing sugar content than either of the other two varieties.  Thus,  as with percent soluble solids, i t may be possible to differentiate between ripe and unripe f r u i t .  i. )  Total sugars.  The results obtained for the total sugar  content of the fruit are presented in Table X.  - 59 -  Table IX Percent reducing sugars of the fruit of three highbush blueberry varieties at four stages of physiological maturity.  Rep. I  Rep. II  Mean va]  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  3.47 4.63 8.58 9.52  3.41 4.92 9.18 9.66  3.44 4.77 8.88 9.59  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  3.61 5.05 9.19 10.61  3.91 5.38 8.91 10.93  3.76 5.22 9.06 10.73  4.37 5.12 10.71 11.82  4.64 4.85 10.50 12.41  4.51 4.99 10.61 12.22  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means:  Weymouth Rancocas Jersey  Stages of maturity means: Small green Large green Reddish Mature blue  Variety - n.s.  6.80 7.20 8.05 3.90 4.99 9.51 10.83  Maturity - **  Variety x Maturity - **  - 60 -  Table X Percent total sugar of the fruit of three highbush blueberry varieties at four stages of physiological maturity.  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  3.54 4.93 8.86 9.86  3.50 5.34 9.52 9.79  3.52 5.15 9.19 9.83  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  3.75 5.47 9.35 10.81  4.06 5.63 9.30 11.12  3.92 5.55 9.33 10.97  4.67 5.19 11.02 12.88  4.75 5.22 10.80 13.15  4.71 5.21 10.91 13.02  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means:  Weymouth Rancocas Jersey  Stages of maturity means: Small green Large green Reddish Mature blue  Variety - n.s.  6.92 7.44 8.46 4.05 5.30 9.81 11.27  Maturity -  Variety x Maturity -  - 61 -  As was the case with reducing sugars there was a highly significant increase in the total sugar content of a l l three varieties with increasing maturity.  This trend is illustrated in .  Figure VI. The differences in the sugar content of the varieties were not significant, indicating that under the conditions of this study the varieties were similar in sugar content. However, the highly significant interaction would seem to indicate that measurable differences did exist between the varieties at a given stage of maturity.  For example, the late season variety  Jersey had a considerably greater sugar content than either of the other varieties at the mature blue stage. The results obtained for the total sugar content of the fruit examined during this investigation tend to substantiate the work of Uhe (78) who found that berries picked late in the season contained less acid and more sugar than those harvested at the beginning of the season.  The results for the mature blue fruit harvested  at the beginning of the season.  The results for the mature blue fruit  harvested during the present study follow this trend.  But in this  instance i t would appear that there is a tendency towards a higher sugar and lower acid content for the later maturing varieties, although this trend may or may not be significant.  -  62 -  Figure VI. The influence of maturity on the percent total sugar content of the fruit of three highbush blueberry varieties.  - 63 -  From these results one may postulate that the late maturing varieties contain appreciably more sugar than the earlier varieties. This might have been due to the increased temperatures experienced during the ripening period or perhaps a result of the longer photosynthetic period received by the later maturing fruits. There was a direct relationship between the decrease in acids and the increase in sugars.  The role of organic acids in the  metabolism of fruit i s s t i l l not known.  It may be that the organic  acids in the respiration process are converted to sugars by a reversal of the glycolytic processes. Bowers and Dewey (12), in studies with the Rubel and Jersey varieties, observed that f u l l ripeness of the fruit was attained eight to twelve days after the appearance of red coloration in the skin.  During this eight to twelve day period the maximum sugar and  minimum acid levels were reached.  This indicates that in the present  study harvest dates for the mature blue fruit were nearly ideal for optimum measurements of sugars and acids (Table 1).  This fact  suggests the use of time interval measurements as an index of maturity.  However, Bailey (4) has reported that the variability in esti-  mation of the development time from bloom to maturity for blueberry fruit is too variable within any given variety to be of any practical use as an indication of maturity.  - 64 -  Bowers and Dewey (12) also found a total sugar content of 10.3% in the Jersey variety at eight days sifter red coloration. The figure of 13.02^ reported in this study was considerably higher. The use of sugar measurements as an indication of maturity was found to be quite practical.  It was not possible to differenti-  ate between varieties on a total sugar content basis.  The low-re-  ducing sugar content of the fruit examined may be a result of the high acid content.  Any non-reducing sugars formed, such as sucrose,  are probably subjected to acid hydrolysis and converted to reducing sugars.  j.)  Sugar-acid ratio.  The results of the sugar-acid ratios of  the three varieties and maturity stages are presented in Table XI. Statistical analysis showed that significant and highly significant differences existed between the sugar-acid ratios of the varieties and the stages of maturity respectively.  There was a pro-  nounced increase in the sugar-acid ratio of the fruit from the green stages to the reddish stage of development; and a similar increase between the reddish and mature blue stages. Figure VII illustrates the very slight increase in the sugar-acid ratio between the small green and large green stages followed by the marked increase in the later stages of maturity.  - 65 -  Table XI Sugar-acid ratios of the fruit of three highbush blueberry varieties at four stages of physiological maturity.  Rep. I  Rep. II  Mean value  Weymouth Weymouth Weymouth Weymouth  small green large green reddish mature blue  1.37 1.61 4.63 9.21  1.42 1.72 4.91 9.51  1.40 1.67 4.80 9.36  Rancocas Rancocas Rancocas Rancocas  small green large green reddish mature blue  1.67 1.60 5.37 9.24  1.90 1.68 5-34 9.43  1.79 1.64 5.36 9.34  1.86 1.64 6.34 12.50  1.87 1.73 6.29 13.15  1.87 1.69 6.32 12.88  Jersey Jersey Jersey Jersey  small green large green reddish mature blue  Variety means:  Weymouth Rancocas Jersey  Stages of maturity means: Small green Large green Reddish Mature blue  Variety - *  Maturity -  4.30 4.53 5.67 1.68 1.69 5.49 10.51  Variety x Maturity - n.s.  - 66 -  Figure VII. The influence of maturity on the sugar-acid ratio of the fruit of three highbush blueberry varieties.  - 67 -  From these data i t was possible to show that mature fruit of Jersey variety had a significantly higher sugar-acid ratio in comparison with the other two varieties.  Thus, i t may be possible to  distinguish between varieties on this basis. Blueberry fruit of high sugar content may not be too desirable in some instances.  A high sugar content associated with a  low acid content may produce a bland fruit lacking the characteristic tartness desirable in blueberry varieties. The scope of the present investigation did not include evaluation of the quality of the fruit studied but rather attempted to determine i f differences could be detected which might be used for future investigations where some sort of standard could be employed.  k.)  Organic acids.  Table XII shows the R^ x 100 values of the  standard acids used in this investigation and the approximate fraction number in which these acids were located after passage through the anion exchange resin.  With this information i t was possible to sepa-  rate and tentatively identify the acids present in the f r u i t .  A num-  ber of the acids were not completely separable by fractionation and as a result appeared as pairs in the paper chromatograms. Results for these are grouped and expressed as one.  The results of the  organic acid analysis of the three varieties at the four stages of fruit development are presented in Tables XIII, XIV, and XV.  - 60 -  Table XII  Acids of the highbush blueberry in order of elution from the anion exchange resin with corresponding Rf x 100 values for a 1-butanol:3N formic acid, 1:1 solvent system.  Approximate fraction number  Rf x 100 value  Aspartic  3  12.0  Glutamic  3  17.5  Shikimic  4  38.0  Quinic  5  23.5  Galacturonic  9  9.0  Glyceric  12  42.5  Glycolic  12  57.5  Malic  15  51.5  Citric  17  46.5  Chlorogenic  24  69.5  Caffeic  24  78.0  Phosphoric  33  25.5  - 69 -  Table XIII The organic acids of the fruit of the highbush blueberryvariety Weymouth at four stages of physiological maturity. (All figures expressed as meq./lOO gm. fresh f r u i t ) .  Small green  Large green  Reddish  Mature blue  Aspartic and glutamic  0.60  0.78  0.41  Shikimic  0.05  0.05  0.05  Quinic  1.96  3.14  1.85  0.85  Galacturonic  0.62  0.78  0.45  0.15  Glyceric and glycolic  0.40  0.42  0.30  0.12  Malic  3.70  4.15  2.05  1.10  Citric  25.88  30.78  16.98  11.03  Chlorogenic and caffeic  1.05  1.27  1.00  0.39  Phosphoric  0.64  0.69  0.30  0.18  Unknowns  0.20  3.28  1.76  0.88  0.16  -  Total  35.10  45.34  25.15  14.86  Total by titration  38.81  50.00  28.00  17.20  - 70 -  Table XIV The organic acids of the f r u i t of the highbush blueberry variety Rancocas at four stages of physiological maturity. ( A l l figures expressed as meq./lOO gm. fresh f r u i t ) .  Small green  Large green  Reddish  Mature blue  Aspartic and glutamic  0.46  0.87  0.36  0.21  Shikimic  0.60  0.71  0.33  0.25  Quinic  1.04  3.66  2.01  0.77  Galacturonic  0.45  0.72  0.40  0.12  Glyceric and glycolic  0.42  0.58  0.27  0.14  Malic  1.20  3.96  2.38  0.98  Citric  21.36  35.31  13.33  9.41  Chlorogenic and caffeic  0.80  1.45  0.95  0.50  Phosphoric  0.62  0.74  0.24  0.16  Unknowns  1.00  4.61  3.39  2.68  Total  27.95  49.61  23.66  15.22  Total by titration  33.40  54.02  27.75  18.93  - 71 -  Table XV The organic acids of the fruit of the highbush blueberryvariety Jersey at four stages of physiological maturity. (All figures expressed as meq./lOO gm. fresh f r u i t ) .  Small green  Large green  Reddish  Mature blue  Aspartic and glutamic  0.48  0.71  0.30  0.15  Shikimic  0.10  0.12  0.05  0.05  Quinic  1.05  3.26  1.97,  0.64  Galacturonic  0.28  0.59  0.37  0.20  Glyceric and glycolic  0.27  0.38  0.25  Malic  1.08  3.70  2.48  1.24  Citric  26.32  31.85  17.32  10.46  Chlorogenic and caffeic  0.59  1.35  1.09  0.61  Phosphoric  0.86  0.83  0.44  0.23  Unknowns  1.04  3.47  2.17  1.26  Total  32.07  46.26  26.44  14.92  Total by titration  42.00  49.00  28.10  16.78  - 72 -  The predominant acid at a l l stages of fruit development appeared to be c i t r i c . Markakis et a l (50).  This was also reported by Nelson (57) and In a l l samples c i t r i c acid comprised more than  60% of the total acids determined, and at the small green stage of the Rancocas variety i t accounted for 11 fo of the acids determined. There did not seem to be any apparent differences between varieties in the c i t r i c acid content as was reported by Markakis et a l .  They  found ripe berries of the Rubel variety contained more c i t r i c acid than those of the Jersey variety of the same age.  The present re-  sults might be explained by the fact that no significant varietal differences were noted for either titratable or total acids.  There  was however a substantial decrease in the actual amount of c i t r i c acid present in fruit during the later stages of development in a l l three varieties. Nelson (57) was not able to show the presence of i s o - c i t r i c acid in blueberry f r u i t .  Markakis et a l (50), using techniques simi-  l a r to those of the present study, supplemented by s i l i c a gel chromatography verified Nelson's finding.  The presence or absence of  i s o - c i t r i c acid in the present study was not confirmed.  Carles et a l  (18) were unable to differentiate between c i t r i c and i s o - c i t r i c acid using paper chromatographic methods employing a butanol-formic acid solvent system. Malic acid was the acid of second largest amounts. finding agrees with the results of Markakis et a l (50).  This  Here again  - 73 -  there were no apparent varietal differences; but there was in each variety a definite decrease in malic acid after the appearance of red coloration in the skin. Quinic acid was found to occur extensively; at the large green stage of the Rancocas variety i t accounted for approximately 7.'5/£ of the total acid content determined.  But the amount estimated  was notably less than that reported by Kimura and Mizuno (42).  These  workers reported that the quinic acid content of Vaccinium uliginosum, a close relative of the highbush blueberry, comprised over sixty percent of the total acid content of the f r u i t .  The close botanical  relationship between these species would lead one to expect a somewhat similar organic acid pattern. Chlorogenic and caffeic acids were not separable by the techniques used.  These acids occurred in a l l three varieties; and  closely followed the pattern of the majority of the acid components. They increased through the small and large green stages and subsequently decreased through the reddish and mature blue stages.  The  rate of decline for these two acids at the reddish stage was not as apparent as i t was for the other acids.  This would seem to indicate  that these two acids, or at least one of them, increased during the later stages of ripening and only began to diminish during the very final stages of fruit development.  The content of these two acids  was similar among the three varieties examined.  - 74 -  It was of interest to note the appearance of aspartic and glutamic acids.  These two amino acids, which cannot be s t r i c l y  classed as organic acids in the usual sense of the term, were also reported by Markakis et a l (50).  Cleaver et a l (22) note that these  are the only common amino acids that are absorbed by anion exchange resins.  This would explain their appearance in the fractions. Both of these acids occurred in the early fractions and no  attempt was made to separate them.  At a l l stages of maturity for  the three varieties these acids constituted approximately 1.5% of the total acid content determined. Shikimic acid was found to be present in a l l treatments except the mature blue stage of the Weymouth variety.  The acid  occurred in only trace amounts in the Weymouth and Jersey varieties, but in the Rancocas variety i t was present in amounts of up to 1.6% of the total acids determined.  Thus i t appears i t may be possible  to differentiate between some varieties from an estimation of this acid. Galacturonic, glyceric, glycolic, and phosphoric acids were found to occur in a l l three varieties at a l l stages of maturity in small amounts.  The pattern of a l l these acids followed that of the  overall acid content showing no appreciable difference among varieties and decreasing with the ripening process.  - 75 -  Throughout the experiments unknown acid spots appeared on the chromatograms.  While in many cases the Rf x 100 values of these  unknowns did correspond with those of known acids their appearance was not consistent and in most cases the fraction number did not match that of any of the standard acids employed.  Some of these  spots may have been artifacts of sugar breakdown.  While most of  the sugars should have been washed through the anion exchange resin, some may have remained.  Hulme (37) reported that strongly basic  anion exchange resins, similar to the ones used i n the present experiments, are capable of breaking down sugar molecules producing artifacts which may interfere with acid determinations. Oxalic acid was not separably by the chromatographic techniques employed and an attempt to estimate the oxalic acid content of the samples by the method described by Winton (80) was not successful.  Considerable variation between measurements of the same  sample showed the unsuitability of this procedure; perhaps refinements in technique might improve this situation.  Winton points out  that solutions with a c i t r i c acid content of more than one percent should be avoided.  The high c i t r i c acid content of the samples  tested, even after suitable dilution, may have been the reason for the inconsistency of the results.  According to the results of  Kohman (43) one would expect the oxalic acid content of the mature berries to be approximately 0.55 meq./lOO gm. of fresh f r u i t .  - 76 -  The overall results correspond f a i r l y well with those of Markakis et a l (50), although they were able to show the presence of succinic, glucuronic, malonic, and citramalic acids.  These acids  were not identifiable in the present study; occasionally spots were noted that had Rf x 100 values corresponding to those of succinic acido  The above authors' results showed a varietal difference in  the amounts of c i t r i c acid present between the Rubel and Jersey varieties; whereas in this study no varietal differences were evident on the basis of c i t r i c acid content for the three varieties examined. There were no major differences in the type of acids reported by Markakis et al (50) and those reported here, nor in the total acid content; there was also close agreement in the actual amounts of the acids present.  In ripe Jersey fruit the above  authors reported the c i t r i c acid content of the fruit to account for 11.6% of the total acids whereas in the present study a value of 10% was found.  Their figures show malic acid to constitute 1,2%  of the acid content of the ripe fruit compared to the 8.3$ determined in the present study.  - 77 -  CONCLUSIONS  The results of this study showed that i t was possible to detect varietal differences in the fruit of commerial highbush blueberries by the use of chemical measurements.  Differences due to the  physiological state of fruit development were also apparent. Much of the dissimilarity was obviously masked by the large amount of interaction present between the varieties and stages of fruit maturity.  Despite this high interaction meaningful differences  were evident among stages of maturity in the soluble solids, t i t r a table acid, total acid, pH, total sugars, reducing sugars, and sugaracid ratios of the f r u i t .  These results indicate that the use of  chemical measurements as an index of maturity i s certainly feasible. In a l l probability the sugar-acid ratio is the best chemical index of maturity from a practical point of view.  It is unique in that i t  i s directly related to a l l the above mentioned measurements.  If  suitable standards were established for each variety, and assuming the reported correlation between sugars and acids is v a l i d , sugaracid ratios could be used as an important criterion of quality i n the blueberry f r u i t .  Over-mature fruit was not examined in this i n -  vestigation but other workers have shown the practicability of sugaracid ratios for the identification of over-mature f r u i t .  - 78 -  Varietal differences were evident from measurements of the pH, soluble solids, and sugar-acid ratios.  At the mature blue stage  the late-season variety Jersey was found to have the highest sugaracid ratio, and the highest soluble solids and sugar content.  There  was an apparent trend towards increased sugar content in the varieties maturing later in the season. The highly significant difference in the pH measurements of varieties was not apparent in the titratable and total acid data. This indicated a difference in the amount of dissociated acid and i n directly showed a difference in undissociated acid content among varieties.  The fact that each of the varieties examined reaches the mature  blue stage at different times during the season might lead one to expect differences in their chemical composition.  Conversely, one  might expect marked similarities in the chemical composition of the three varieties because of their common genetical background. Organic acid analysis showed c i t r i c to be the predominant acid i n blueberries.  Malic, quinic, and the acid pair chlorogenic  and caffeic, were the most prevalent of the minor acids identified. Aspartic, glutamic, shikimic, glacturonic, glyceric, glycolic, oxalic, and phosphoric acids were also tentatively identified and quantitatively estimated.  Iso-citric, benzoic, succinic, glucuronic, malonic,  and citramalic acids which had previously been reported as occurring in the blueberry were not detected in the present study.  - 79 -  The majority of the acid constituents behaved similarly increasing during the earlier stages of fruit development and decreasing with the appearance of red coloration.  The most notable  exception to this trend was the acid pair chlorogenic and caffeic which, for the three varieties studied, increased in relation to the other acids continuously until the very final stages of maturity. The relatively high shikimic acid content of the Rancocas variety may provide a basis for varietal distinction. It may not be possible to extrapolate the trends reported here to other varieties, or even to the same varieties grown under different conditions.  However, i t has been shown that quality evalu-  ation of blueberry fruit by chemical methods may be possible i f suitable organoleptic and chemical standards can be established.  Blue-  berry fruit of uniform quality w i l l undoubtedly create wider consumer acceptance.  - 80 -  LITERATURE CITED  Archibold, H. K. 1932. Chemical studies in the physiology of apples. XII. Ripening processes in the apple and the relation of time of gathering to the chemical changes l n cold storage. Annals of Botany. 46:407T459. Association of O f f i c i a l Agriculture Chemists. 1960. O f f i c i a l methods of analysis. Ninth edition. 832 pp. Washington. Atwater, W. 0. 1902. Principles of nutrition and nutritive value of food. U. S. Department of Agriculture, Farmer's Bulletin 142. 48 pp. Washington. Bailey, J. S. 1947. Development time from bloom to maturity i n c u l t i vated blueberries. Proceedings of the American Society for Horticultural Science. 49:193-195. s  Baker, R. E., and Butterfield, H. M. 1951. Commercial bushberry growing i n California. California Agricultural Extension Service, Circular 169. 56 pp. Berkeley. Balansard, J. 1951. A study of the hepato-renal diuretics. V. The presence of glycolic acid i n various drugs used as diuretics. Medicine Tropicale. 11:638-639. (Abstract in Chemical Abstracts. 46:1716. 1952). Barker, W. G., Wood, F. A., and Collins, W. B. 1963. Sugar-levels i n f r u i t of the lowbush blueberry estimated at four physiological ages. Nature. 198:810-811. Bennet-Clark, T. A. 1937. Organic acids of plants. Annual Review of Biochemistry. 6:579-594.  81  (9)  -  Bonner, J . 1944. Effect of varying nutritional treatments on growth and rubber accumulation in guayule. Botanical Gazette. 105:352-364.  (10)  1950.  Plant biochemistry. New York.  537 pp.  Academic Press Inc.  (11)  Bonner, W. D . J r . 1946. Studies on the organic acids of plants. 103 pp. Thesis, Ph. D. California Institute of Technology, Pasadena.  (12)  Bowers, R. C , and Dewey, D. H. 1960. Relation of sugar-acid ratios to the ripening and deterioration of Jersey and Rubel blueberry fruits. Michigan Agricultural Experimental Station Quarterly Bulletin. 4 3 : 3 0 3 - 3 1 1 .  (13)  Buch, M. L . 1960. A bibliography of organic acids in higher plants. U. S. Department of Agriculture, Agricultural Handbook No. 164. 100 pp. Washington.  (14)  Burris, R . H. 1953. Organic acids in plant metabolism. of Plant Physiology. 4 : 9 1 - 1 1 4 .  (15)  Annual Review  Busch, H . , Hurlbert, R . B., and Potter, V . R . 1952. Anion-exchange chromatography of acids in the c i t r i c acid cycle. Journal of Biological Chemistry. 196:717-727.  (16)  Canada Department of Health and Welfare. 1958. Table of food values recommended for use in Canada. 286 pp. Queen s Printer. Ottawa. 1  (17)  Canvin, D. T . , and Beevers, H. J . 1961. Sucrose synthesis from acetate in the germinating castor bean: kinetics and pathway. Journal of Biological Chemistry. 236:988-995.  (18)  Carles, J . , Schneider, A . , and Lacoste, A. M. 1958. Table XXXVI. RQ values of organic acids. of Chromatography. 1:23 (appendix).  Journal  - 82 -  (19)  Carter, A. C. 1963. The small f r u i t , grape and nut industry in British Columbia for 1962 with an outlook for 1963. British Columbia Department of Agriculture Bulletin. 4 pp. Victoria.  (20)  Chandler, F. B . , and Highlands, M. E. 1950. Blueberry juice. Food Technology.  4:285-286.  (21)  Chatfield, C , and McLaughlin, L . I. 1928. Proximate composition of fresh fruits. U. S. Department of Agriculture Circular No. 50. 20 pp. Washington.  (22)  Cleaver, C. S., Hardy, R. A., and Cassidy, H. G. 1945. Chromatographic adsorption of amino acids on organic exchange resins. Journal of the American Chemical Society. 67:1343-1352.  (23)  Clements, R. L . 1964. Organic acids in citrus fruits. I. Varietal differences. Journal of Food Science. 29:276-280.  (24)  Consden, R., Gordon, A. H . , and Martin, A. J . P. 1944. Qualitative analysis of proteins: a partition chromatographic method using paper. Biochemical Journal. 38:224-232.  (25)  Darrow, G. M . , and Moore, J . N. 1962. Blueberry growing. U. S. Department of Agriculture Farmer's Bulletin No. 1951. 33 pp. Washington.  (26)  Gatet, L . 1939. Biochemical research on the ripening of fruits. Annales de Physiologie et de Physiochimie Biologique. 15:984-1064. (Abstract in Chemical Abstracts. 35:4798. 1941).  (27)  Gerber, C. 1897. Recherches sur l a maturation des fruits charnus. 279 pp. Thesis, Ph. D. University of Paris, Paris, France. (Abstract in Reviews of Science. 8:526-529. 1897).  - 83 -  (28)  Gorter, K. 1909. Distribution of chlorogenic acid in nature. Archiv der Pharmazie und Berichte der Deutschen Pharmazeutischen. 247:184-196. (Abstract in Chemical Abstracts. 3:2583. 1909).  (29)  Griebel, C. 1910. Chemical composition of cranberries and whortleberries. Zeitschrift fur Untersuchung der Nahrungs und Genussmittel Sowie der Gebrauchsgegenstande. 19:241-252. (Abstract in Chemical Society Journal. 98:4410. 1910).  (30)  Guillaume, A . , and Adnot, J . 1933. Variations in the c i t r i c acid and sugar contents of black currants according to the degree of ripeness. Annales des Falsifications et des Fraudes. 26:75-87. (Abstract in Chemical Abstracts. 27:2474. 1933).  (31)  Harris, C. H . , and Thrams, W.D. 1916. The fruit of Vaccinium corymbosum. 114:73.  (32)  (33)  Chemical News.  Hartman, B. G. 1943. The polybasic acids of fruits and fruit products. Journal of the Association of Official Agricultural Chemists. 26:444-462. 1934.  and H i l l i g , F. Acid constituents of food products. Journal of the Association of Official Agricultural Chemists. 17:522-531.  (34)  Harvey, R. B. 1920. The relation between the total acidity, the concentration of the hydrogen ion, and the taste of acid solutions. Journal of the American Chemical Society. 42:712.  (35)  Hopkins, E . F . , and Gourlay, J . L. 1930. The effect of nitrate applications on the soluble carbohydrate in apples. Proceedings of the American Society for Horticultural Science. 27:32-36.  (36)  Hulme, A. C. 1948. Studies in the nitrogen metabolism of the apple fruit. Changes in the nitrogen metabolism of the apple during the normal and ethylene induced climacteric rise in the rate of respiration. Biochemical Journal. 43:343-349.  - 84 -  (37) 1953.  An action of strongly basic anion-exchange resins and solutions containing sugar. Nature. 171:610.  (38)  Isherwood, F. A. 1946. The determination and isolation of the organic acids in f r u i t . Biochemical Journal. 40:688-695.  (39)  Jorysch, D . , Sarris, P . , and Marcus, S. 1962. Detection of organic acids in fruit j u i c e 3 by paper chromatography. Food Technology. 16:90-93.  (40)  Kaiser, H. 1925. Acids of the whortleberry and tamarind. Suddeutsche Apothekerzeitung. 65:48-49. (Abstract in Chemical Abstracts. 19:1149. 1925).  (41)  Kenworthy, A. L . , and Harris, N. 1960. Organic acids in apples as related to variety and source. Food Technology. 14:372-375.  (42)  Kimura, W., and Mizuno, M. 1959. Sugars and organic acids of the fruit of Vaccinium uliginosum. Journal for the Utilization BT Agricultural Products (Japan). 6:218-220.  (43)  Kohman, E. F. 1939. Oxalic acid in foods and i t s behavior and fate in the diet. Journal of Nutrition. 18:233-246.  (44)  Krotkov, G . , Wilson, D. G . , and Street, R. W. 1951. Acid metabolism of Mcintosh apples during their development on the tree and in cold storage. Canadian Journal of Botany. 29:79-90.  (45)  Lane, J . H . , and Eynon, L. 1923. Deteraination of reducing sugars by means of Fehling*s solution with methylene blue as internal indicator. Journal of the Society of Chemical Industry. 42:32-37.  (46)  Lebedev, A . , and Lindquist, E. 1933. The acids of the whortleberry. Zeitschrift fuer Lebensmittel-Untersuchung und Forschung. 65:476-477. (Abstract in Chemical Abstracts. 27:4598. 1933).  (47)  Lugg, J . W* H . , and Overell, B. T. 1947. Partition chromatography of organic acids on a paper sheet support. Nature. 160:87-88.  - 85 -  (48)  MacVicar, R., and Burris, R. H. 1948. Studies on the nitrogen metabolism in tomato with use of Isotopically labelled ammonium sulfate. Journal of Biological Chemistry. 176:511-515.  (49)  Marcus, A . , and Velasco, J . 1960. Enzymes of the glyoxylate cycle in germinating peanuts and castor beans. Journal of Biological Chemistry. 235:563-567.  (50)  Markakis, P . , Jarczyk, A . , and Krishna, S. P. 1963. Nonvolatile acids of blueberries. Agricultural and Food Chemistry. 11:9-11.  (51)  Mason, G. F . 1905. The occurrence of benzoic acid naturally in cranberries. Journal of the American Chemical Society. 27:613-614.  (52)  Mehlitz, A . , and Matzik, B. 1956. Volatile acids in fruit juices. Industrielle Obstund Gemueseverwertung. 41:227-229. (Abstract in Chemical Abstracts. 50:17241. 1956).  (53)  Merriam, 0. A . , and Fellers, C. R. 1936. Composition and nutritive studies on blueberries. Food Research. 1:501.  (54)  Meyer, L. H. 1960. Food Chemistry. 385 pp. Corporation. New York.  (55)  Millerd, A . , Bonner, J . , Axelrod, B . , and Bandurski, R. 1951. Oxidative and phosphorylative activity of plant mitochondria. Proceedings of the National Academy of Science, U. S. 37:855-862. (Abstract in Chemical Abstracts. 46:4060. 1952).  (56)  Neish, A. C. 1960. Biosynthetic pathways of aromatic compounds. Review of Plant Physiology. 11:55-80.  (57)  Nelson, E . K. 1927. Non-volatile acids of the pear, quince, apply, logan berry, blueberry, cranberry, lemon, and pomegranate. Journal of the American Chemical Society. 49:13001302.  Reinhold Publishing  Annual  - 86 -  (58)  Nitsch, J . P. 1953. The physiology of fruit growth. Plant Physiology. 4:199-236.  Annual Review of  (59)  Pangborn, R. M. 1963. Relative taste intensities of selected sugars and organic acids. Journal of Food Science. 28:726-733.  (60)  Ramstad. E. 1954. Chemical investigation of Vaccinium myrltillus. American Pharmaceutical Association Journal, Scientific Edition. 43:236-240. (Abstract in Chemical Abstracts. 48:7260. 1954).  (61)  Ransom, S. L . , and Thomas M. 1960. Crassulacean acid metabolism. Physiology. 11:81-110.  Annual Review of Plant  (62)  Rice, A. C , and Pederson, C. S. 1954. Chromatographic analysis of organic acids in canned tomato juice, including the identification of pyrrolidonecarboxylic acid. Food Research. 19:106-113.  (63)  Robinson, T. 1963. The organic constituents of higher plants. Burgess Publishing Company. Minneapolis.  (64)  Ruck, J . A. 1963. Chemical methods for analysis of fruit and vegetable products. Canada Department of Agriculture Publication 1154. 47 pp. Ottawa.  (65)  Shoemaker, J . S. 1955. Small fruit culture. Company. Toronto.  (66)  447 pp.  306 pp.  McGraw-Hill Book  Sinclair, W. B . , and Eny, D. M. 1946. Significance of the alkaline ash of citrus juices. Proceedings of the American Society for Horticultural Science. 47:119-112.  (67) 1944.  and Ramsey, R. C. Changes in the organic acid content of Valencia oranges during development. Botanical Gazette. 106:140-148.  - 87 -  (68)  Smith, I. 1960. Chromatographic and electrophoretic techniques. I. 617 pp. Interscience Publishers, Inc. New York.  (69)  Steele, R. G. D . , and Torrie, J . H. 1960. Principles and procedures of statistics. McGraw-Hill Book Company. Toronto.  (70)  S t i l l e r , M. 1962. The path of carbon in photosynthesis. of Plant Physiology. 13:151-171.  (71)  Suomalainen, H . , and Keranen, A. 1961. First anthocyanins appearing during the ripening of blueberries. Nature. 191:498-499.  (72)  Swain, T. 1963. Chemical plant taxonomy. New York.  (73)  Thimann, K. V . , and Bonner, W. D. 1950. Organic acid metabolism. Annual Review of Plant Physiology. 1:75-108.  (74)  Thompson, F . , and Whittier, A. C. 1912. Forms of sugar found in common plants. Proceedings of the American Society for Horticultural Science. 9:16-21.  (75)  Thorpe, G. R. 1959. Highbush blueberry culture in British Columbia. British Columbia Department of Agriculture Horticultural Circular No. 84. 11 pp. Victoria.  (76)  Thurlow, J . , and Bonner, J . 1948. Fixation of atmospheric carbon dioxide in the dark by leaves of Bryophyllum. Archives of Biochemistry. 19:509-511.  (77)  Tompkins, R. G. 1954. Unsolved problems in the preservation of food. The influence of cultural conditions on the quality and preservation of fruits and vegetables. Journal of the Science of Food and Agriculture. 5:161-167.  (78)  Uhe, G. 1957. Influence of certain factors on the acidity and sugar content of the Jersey blueberry. Economic Botany: 11:331-343.  543 pp.  481 pp.  Annual Review  Academic Press Inc.  - 88 -  (79)  Whiting, G. C. 1958. The non-volatile organic acids of some berry fruits. Journal of the Science of Food and Agriculture. 9:244-243.  (80)  Winton, A. L. 1958. The analysis of foods. Inc. New York.  999 pp.  John Wiley and Sons,  (81)  Wood, H. G . , and Werkman, C. H. 1936. The utilization of carbon dioxide in the dissimilation of glycerol by propionic acid bacteria. Biochemical Journal. 30:48-53.  (82)  Woodruff, R. S., and Dewey, D. H. 1959. A possible harvest index for Jersey blueberries based on the sugar and acid content of the f r u i t . Michigan Agricultural Experimental Station Quarterly Bulletin. 42:340-349.  (83)  , 1960.  , and Sell, H. M. Chemical changes of Jersey and Rubel blueberry fruit associated with ripening and deterioration. Proceedings of the American Society for Horticultural Science. 75:387-401.  - 89 -  ADDENDA  Balltnger, W. E . , B e l l , H. K . , and Kenworthy, A. L. 1958. Soluble solids in blueberry fruit in relation to yield and nitrogen content of fruiting-shoot leaves. Michigan Agricultural Experimental Station Quarterly Bulletin. 40:912-914. Kushman, L. J . , and Ballinger, W. E. 1963. Influence of season and harvest interval upon quality of Wolcott berries grown in eastern North Carolina. Proceedings of the American Society for Horticultural Science. 83:395-405.  - 90 -  APPENDIX  STATISTICAL METHODS EXAMPLE  Analysis of Variance Table f o r Percent T o t a l Solids Degrees of Freedom  Source  Sums of Squares  Total  41.18  23  Treatment  40.57  11  Varieties  1.76  2  1.14  0.24n.s.  Maturity stages  10.02  3  3.34  0  V x M  28.79  6  4.80  Error  0.61  12  0.05  varieties  Variance  1.14  0.24  ^maturity  3.34 4750"  0.70  F  4.80  94.12  T7&5  0735"  v x ra Where:  n.s. #  *0.5  ^  7 0  n.s. •Sf-S-  94.12  5.14  10.92  4.76  9.78  3.09  5.07  -  not s i g n i f i c a n t at 5% l e v e l of s i g n i f i c a n c e .  »  s i g n i f i c a n t at 5% l e v e l of s i g n i f i c a n c e .  *> s i g n i f i c a n t at  l e v e l of s i g n i f i c a n c e .  •0.1  

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