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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 Br i t i sh Columbia, 1963. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE in the Division of Plant Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1964. i i i In presenting this thesis in partial fulfilment of the re-quirements 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 i i 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 blue-berries 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 fruit . Meaningful differences were evident among the four stages of physio-logical 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. ) Total solids 25 b. ) Water Insoluble solids 27 c. ) Soluble solids 28 d. ) pH 29 e. ) Titratable acids 29 f. ) Total acids 30 v i 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 v i i LIST OF TABLES TABLE PAGE I. Harvesting dates of the fruit of three varieties of highbush blueberries at four stages of physiological maturity 26 II. Percent total solids of the fruit of three highbush blueberry varieties at four stages of physiological maturity 41 III. Percent water insoluble solids of the fruit of three highbush blueberry varieties at four stages of physiological maturity 44 IV. Percent soluble solids of the fruit of three highbush blueberry varieties at four stages of physiological maturity 46 V. pH content of the fruit of three highbush blue-berry varieties as influenced by stage of physiological maturity 50 VI. Percent titratable acids (as citric) of the fruit of three highbush blueberry varieties at four stages of physiological maturity . . . . . . . . . 52 v i i i TABLE PAGE VII. Percent total acids (as citric) of the fruit of three highbush blueberry varieties at four stages of physiological maturity 56 VIII. Percent volatile acids (as acetic) present in the fruit of three highbush blueberry varieties at four stages of physiological maturity 57 IX. Percent reducing sugars of the fruit of three highbush blueberry varieties at four stages of physiological maturity 59 X. Percent total sugar of the fruit of three high-bush blueberry varieties at four stages of physiological maturity 60 XI. Sugar-acid ratios of the fruit of three highbush blueberry varieties at four stages of physio-logical maturity 65 XII. Acids of the highbush blueberry in order of elution from the anion exchange resin with cor-responding Rj. x 100 values for a 1-butanolO N formic acid, 1:1 solvent system 68 i x TABLE PAGE XIII. The organic acids of the fruit of the highbush blueberry variety Weymouth at four stages of physiological maturity 69 XIV. The organic acids of the fruit of the highbush blueberry variety Rancocas at four stages of physiological maturity 70 XV. The organic acids of the f ru i t of the highbush blueberry variety Weymouth at four stages of physiological maturity 71 X LIST OF FIGURES FIGURE PAGE I. Steam distil lation apparatus used for volatile acid determination 31 II. Automatic fraction collector and resin column used for in i t i a l separation of organic acids 36 III. Apparatus used for descending paper chromatography 38 IV. The influence of maturity on the percent soluble solids content of three varieties of highbush blueberries 47 V. The influence of maturity on the percent titratable acid content of three highbush blue-berry varieties 53 VI. The influence of maturity on the percent total sugar content of the fruit of three highbush blueberry varieties 62 VII. 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 horti-cultural 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 fruit , 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 fac-tors 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 physio-logy. One group of these acids, commonly referred to as plant acids or organic acids, is distinguishable by its 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, 62). 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 in-fluence of maturity and variety on the organic acid content and certain associated chemical measurements in the fruit of the high-bush blueberry. - 3 -LITERATURE REVIEW CHEMISTRY OF FRUITS From a botanical point of view a fruit is the ripened peri-carp 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 floral parts and does not include those botanical fruits classi-cally 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 ipid 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 fruits, like that of the entire plant, is 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 is apparent in the apple ( l ) , orange (67), grape (58), and the black currant (30). Accompanying these changes in acidity is an increase - 4 -in 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 ru i t ripening. Thompson and Whittier (74) in 1912 found an increase in sugar con-tent 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 in the chemistry of f ru i ts attributed to maturity there are a number of other factors that may result in considerable variation in the sugar and acid content of f r u i t . These include variety, nutr i t ion, and climate. Fruits grown in a cold and rainy climate tend to be more sour than those grown under warmer conditions. Nitsch (58), in a review ar t ic le on the physiology of f ru i t growth, cites 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 respiration processes, whereas, at low tempe-ratures they are accumulated. The temperature necessary to induce the respiration of malic acid i s lower than the one for tartar ic acid. Accordingly, f ru i t s r ich in tartaric acid (grapes) require higher temperatures for ripening in comparison with f ru i ts that are r ich in 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 ferti l izer application. Hopkins and Oourlay (35) were among the many to report an increase of soluble carbohydrates in fruit due to ferti l izer treatment. This i s , in effect, not a direct result of the fert i l izer, but an in-direct effect due to the increase in the production of photo-synthates 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 is 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 incom-plete oxidation of carbohydrates in respiration. Krotkov et a l (44) suggest a close relationship between carbohydrates and acid metabo-lism in fruit attached to the tree, but conclude that the relation is not a simple one. They suggest that possibly the acids found in the fruit arise from both the foliage and the fruit itself . There is no cr i t ica 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. A l -though a small increase In titratable acidity may occur in the first day or two after picking of immature f ru i ts (36). Bennet-Clark (8), in a review ar t i c le on the organic acids of plants, points out the high increase in organic acid production in plants receiving nitrate f e r t i l i za t ion as compared to those re -ceiving ammonia. The hypothesis i s that the mineral cations carried into the plant with the nitrate anion are received by the organic acid anion and remain after nitrate reduction. CHEMISTRY OF THE BLUEBERRY In comparison with other f ru i ts the blueberry is a com-parative newcomer to hort icultural science and as a result i t s l i t e -rature i s meager and incomplete. This is part icular ly true of i t s l i terature pertaining to the chemistry of the blueberry. The f i r s t recorded attempt to analyze blueberry f ru i ts was conducted by Atwater in 1906 (3). He proposed the following chemi-ca l composition for the blueberry: solids 11.6%, protein 0.1%, fat 3.0%, nitrogen free extract 13.5%, f iber 3.2%, and ash 0.4,1. In 1928 Chatfield and McLaughlin (21), in a revised edition of Atwater's bu l l e t in , reported 0.67 moles of acid expressed as an-hydrous c i t r i c acid and a sugar content of 12.4£ for fresh blue-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 83.4 gm. Carbohydrate 15.1 gm. Fat 0.6 gm. Protein 0.6 gm. Calcium 16.0 mg. Phosphorus 13.0 mg. Iron 0.8 mg. Vitamin A 280 I.U. Vitamin C 5-18 rag. Thiamin (.02 mg.) Riboflavin (.02 mg.) Niacin (.30 mg.) (The bracketed figures have never been verified). Chandler and Highlands (20) studying the fruit of the low-bush 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 blue-berry (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 vari-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 defi-nite negative relationship between berry size and acidity. In ad-dition Uhe showed the application of various fertil izers did not significantly influence the sugar or acid content of the fruit . - 8 -Ballinger et a l (addenda) reported the range of soluble solids in highbush blueberry f ru i t to vary from approximately ten to eighteen percent; and that variations in soluble solids may be related to the nitrogen-carbohydrate relationship of the plant or to the amount of f ru i t on the bush. Their results indicated that an excessively high nitrogen content, or a large y ie ld of f ru i t resulted in a decrease in soluble solids content of the blueberry f r u i t . One objective of chemical studies of the blueberry has been to investigate the poss ib i l i t y of using chemical tests as a basis for a harvesting index. Woodruff and Dewey (82), on studies with the Jersey variety, evaluated the use of sugar-acid ratios as a means of ascertaining the degree of ripeness. They found a highly signif icant correlation between the potential 3helf- l i fe and the sugar-acid rat io of the f ru i t measured at the time of harvesting. Poor holding quality was found to be associated with high sugar-acid rat ios. They found a signif icant correlation between pH and t i t ratable ac idi ty and also between the soluble sol id and the total sugar content. Under f i e l d conditions the authors recommended the use of pH and percent soluble solids measurements in place of t i t r a -table ac id i ty and tota l sugars for the estimation of the sugar-acid rat ios . In another experiment the authors found i t possible to d i s -tinguish between ripe and over-ripe berries on a basis of the sugar-acid ra t io . Rubel and Jersey berries with a sugar-acid rat io of - 9 -twelve or less should be classed as unripe, whereas a sugar-acid rat io greater than seventeen indicates over-ripeness. Woodruff et al (83) on studies of blueberry f ru i t ripening demonstrated that changes in t i tratable ac id i ty were more apparent than changes in 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 in the tota l sugar content were found to be re lat ive ly small, and the fact that the majority of the sugar content was present prior to the time of red coloration suggests that sugars have limited value as a f i e ld test for maturity. These authors report a signif icant decrease in the t i tratable ac id i ty for the Jersey variety after the appearance of red coloration. From a high of 9.03% at the time of i n i t i a l red coloration the t i t ratable ac id i ty decreased to 1.15% on a dry weight basis twenty days later . In comparison with the Rubel variety the t i t ratable acid content of mature Jersey berries was found to be considerably lower at a l l stages of f ru i t development. There were however no appreciable differences in tota l sugar content at matu-r i t y (Jersey 12.7% and Rubel 12.6%). A positive l inear relationship of the sugar-acid rat ios was evident for both var iet ies . The authors recommended the use of t i t ratable acid measurements as a harvesting index. It was generally observed that pa latabi l i ty improved with an increased sugar-acid ra t io . - 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 fruit . 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 high-bush 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 fruit; and de-creases in the titratable acid content and the keeping quality of the fruit . The component sugars of the highbush blueberry have not yet been studied. Barker et al (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 fruit . Physiological maturity, therefore, appears to be expedited by an increased number of fertilizations. Kimura and Mizuno (42), using paper chromatographic tech-niques, 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 ic , 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 tr ic 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 flavo-noids, lignin, phenolic glucosides, etc. - 12 -Of the large number of organic acids that have been iso-lated 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 ists eighty-two 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 metabo-lism of plants. It is now impossible to consider organic acids in plant tissues without taking into account almost every aspect of meta-bolism. 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 is in the tricarboxylic acid cycle. The bulk of the acids present in fruit are of the same nature as those operating as inter-mediates 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 al (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 result ing energy was not dissipated but was used in the formation of high energy phosphate bonds in adenosine triphosphate. Other workers were able to show the presence of the cycle in tissue of other seedlings, but i t has never been demonstrated in the mature plant. The question of the general occurrence of the cycle in the mature plant remains unanswered. Some authorities (14) suggest that the tr icarboxyl ic acid cycle of the seedling might be discarded or that certain tissues in 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 in the mitochondria, whereas the accumulation of organic acids occurs in 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 in the vacuoles of i t s ce l l s . This i s exemplified by the high content of c i t r i c acid in the lemon and malic acid in the apple. Kitsch (58), in his review ar t ic le on the physiology of f ru i t growth, cites the two prevalent theories that account for this buildup of acids. The f i r s t of these assumes the presence of blocking mechanisms or inhibitors at specif ic points in the cycle to be the cause of a buildup of one metabolite. The second theory postulates that the acids are formed by the f ixat ion 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 re-action. 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 tempe-ratures 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 oxi-dative. Since the dry regions in which succulents flourish tend to have a marked f a l l in night temperature, i t is l ikely 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 ce 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 phos-phate, 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 evi-dence 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 con-centration 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 cel 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. Interconversion of extracyclic acids is also known to occur. 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 its effect on our senses of taste, smell, and touch. The popular concep-tion of flavor is synonomous with taste. Taste itself 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 is 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 con-centration 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 , lactic, tartartic, and acetic acids c i tr ic 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 fully studied and no definite applications are presently employed in commercial agricultural practices. A number of workers have shown that fruits and fruit pro-ducts can be identified by characterization and identification of their component organic acids through the use of chromatographic techniques. Jorysch et al (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 tr ic and malic acid content of the orange varieties Washington Navel and Valencia. Markakis et al (50) report a distinction between Rubel and Jersey blue-berries of the same physiological age in that the former contain an appreciably greater amount of c i tr ic acid. Swain (72) makes note of the fact that with the ever in-creasing refinement in methods of plant analysis i t may become pos-sible to classify plants on a chemotaxonomic basis; organic acid ana-lysis would be important here. ORGANIC ACIDS OF THE FAMILY ERICACEAE Considerable effort has gone into the identification of in -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 cran-berries. 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 first discovered by Gorter (29) in Vaccinium lucidum. Citric acid is thought to be ubiquitous in higher plants. Nelson (57) was among the f irst to identify c i tr ic acid in the fruit of the blueberry. His work showed citric acid and malic acids to be the predominant acids in this fruitj the absence of iso-citric 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 is characteristic of the genus Vaccinium. Lebedev and Linquist (46) were the first to find i t in the fruit; 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 glu-conic acids. Quinic acid was estimated to account for approximately sixty percent of the total acid content. The f irst detailed study of the organic acids of the high-bush blueberry was conducted by Markakis et al (50) on the varieties Rubel and Jersey. Glutamic, aspartic, quinic, galacturonic, glyceric, glycolic, succinic, glucuronic, citramalic, malic, c i tr ic , malonic, chlorogenic, caffeic, oxalic and phosphoric acids were tentatively identified and quantitatively estimated. On an equivalent basis, more malic, chlorogenic and phosphoric and less citric and quinic acids were present in the ripe berries than in the unripe berries. The ripe Rubel berries contained more citric 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 determina-tion 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 pre-cipitation methods of the lead salts or by fractional distil lation 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 Hi l l ig (33) report that tannins and pectins are precipitated with lead acetate and often cause some de-gree 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 distillation 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 disti l led 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 distil lation is cumbersome and requires large amounts of material and is difficult to put on a quantitative basis (10). In 1944 Consden et al (24) described the use of paper chroma-tography as a means for the separation of organic compounds. In 1946 a method for the separation of organic acids by partition paper chro-matography was first proposed by Isherwood (38). Refinements of this technique by Lugg and Overell (47) resulted in the practical appli-cation 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 irst described by Busch et al (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 i3 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 irst 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 fairly good dessert quality (65). It is 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. The experiment was patterned as a 3 x 4 factorial; 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 phy-siological 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 ru i t which was harvested at a somewhat ear l ier stage to fac i l i t a te completion of the experiment in the time ava i l -able. The harvesting dates are given in Table I. Each sample consisted of two individual replications con-s ist ing of approximately six hundred grams of berries each, picked at random from one hundred bushes. After picking, the samples were sealed in polyethelene containers and refrigerated at -10°C. EXPERIMENTAL The samples were removed from storage and allowed to pa r t i -a l l y thaw for one-half hour at room temperature. Half the sample (approximately 300 gm.) was placed in a Waring blendor, the remaining half being returned to the freezer. The sample was homogenized at high speed for f ive minutes or un t i l such time as a s lurry was formed. Portions of this s lurry were used in a l l determinations. The slurry was st i r red well prior to removal of each portion. The following i s an outline of the methods of analysis employed in this study: a.) Total so l ids . The method used was that described by Winton (80). Approximately 20 gm. of the blended sample was accurately weighed in a tared covered metal dish and dried at 70°C. for 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 ex-tract soluble material. The water insoluble material was collected on a previously dried, weighed f i l ter 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. The mixture was then boiled gently for fifteen to twenty minutes. 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 filtrate 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 dist i l led water, oven dried for two hours at 1 0 0 ° C , cooled in a desiccator - 28 -and weighed in a covered weighing dish. The in i t ia l filtrate from the volumetric flask sample was set aside for acid analysis and the insoluble material in both f i l ter papers was washed with 800 ml. of hot water. The f i l ter papers and contents were transferred to their original weighing dishes and dried overnight at 80° - 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 re-fractometer 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 ter 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 disti l led 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 is 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 water-insoluble 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 tr ic acid. Measurements were taken on duplicate samples and the percent t i tra-table acids was calculated from the following equation: - 30 -Percent titratable = 1 (equivalent weight of acld)(N of NaQH)(titer) acid T5" (weight of sample) f. ) 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 fi ltrate 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 disti l led 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 disti l led from the sample and titrated with stand-ardized 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 dist i l led 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 distillation apparatus used for volatile acid determination. - 32 -with the aid of a minimum amount of disti l led water i t was transferred to the steam distil lation flask. The steam hose was then connected and the volatile acids were disti l led over through the condensor and into a 250 ml. beaker containing disti l led 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 distil lation no acids came over. The total volatile acids were expressed as percent acetic and calculated as follows: Percent volatile - (equivalent weight of acid)(N of NaOH)(titer) acids (weight of sample/ h. ) 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 pre-cipitate. 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 al (50) on a technique originally developed by Busch et al (15) for the investigation of the acids of the citric acid cycle using anion ex-change 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 nitric 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 filtration through Whatman No. 4 paper. The acid solution was then concentrated to approximately 50 ml. by evaporation in a stream of cold air. 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 disti l led 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 dia-meter. Commercial Dowex 1-X8 chloride was converted to Dowex 1-X8 acetate by washing the column with 1 N sodium acetate until a nega-tive 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 disti l led 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 con-centration. 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. The column flow rate was approximately 1 ml. per minute. - 35 -Fifty-one fractions of 10 ml. each were collected in test tubes on an automatic fraction collector (Figure II). 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 disti l led 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 chroma-tography. The fractions were spotted 2.5cm. apart on 57 x 7.5 cm. sheets of Whatman No. 1 f i l ter 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 chro-matographed in the same manner as the samples. Standards were dis-solved in disti l led 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 it was possible to tentatively identify and quantita-tively estimate the acids present in the samples. Oxalic acid was not separable by the chromatographic tech-niques used and oxalate determination was attempted by the method outlined by Winton (80). The procedure involved precipitation of oxalate as its 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 out-lined by Steel and Torrie (69). The variation due to varieties, stages of physiological maturity, and sampling methods was deter-mined. 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 blue-berry, 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 blue-berry varieties at four stages of physiological maturity.1 Rep. I Rep. II Mean value Weymouth small green 11.94 12.30 12.12 Weymouth large green 11.29 11.81 11.59 Weymouth reddish 12.99 12.71 12.80 Weymouth mature blue 13.71 13.77 13.74 Rancocas small green 14.61 13.70 14.05 Rancocas large green 14.96 14.62 14.76 Rancocas reddish 16.20 15.67 15.94 Rancocas mature blue 15.81 15.75 15.78 Jersey small green 12.58 12.43 12.51 Jersey large green 13.68 13.65 13.67 Jersey reddish 14.09 14.14 14.12 Jersey mature blue 13.87 13.91 13.89 Variety means: Weymouth 12.57 Rancocas 15.17 Jersey 13.54 Stages of maturity means: Small green 12.93 Large green 13.34 Reddish 14.30 Mature blue 14.47 Variety - n.s. 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 alterna-tively 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 mois-ture content was found in the small green fruit . 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 tempe-ratures and a decreasing moisture supply could possibly account for the decreasing moisture content of the maturing fruit . The increase in soluble solids evident for a l l varieties is a more likely expla-nation. 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 fruit . Woodruff et al (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-rity the percent total solids of the Rancocas variety was considerably greater than that of the other two varieties - although not signifi-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 con-siderably 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 small green 5.16 5.12 5.14 Weymouth large green 5.08 5.15 5.12 Weymouth reddish 5.59 5.37 5.48 Weymouth mature blue 5.08 5.30 5.19 Rancocas small green 6.65 6.41 6.53 Rancocas large green 6.75 6.68 6.72 Rancocas reddish 4.99 5.14 5.07 Rancocas mature blue 4.61 4.74 4.68 Jersey small green 4.78 4.67 4.71 Jersey large green 5.29 5.35 5.32 Jersey reddish 4.89 4.95 4.92 Jersey mature blue 4.68 4.71 4.70 Variety means: Weymouth 5.23 Rancocas 5.75 Jersey 4.92 Stages of maturity means: Small green 5.47 Large green 5.72 Reddish 5.14 Mature blue 4.85 Variety - n.s. 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 how-ever 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 al 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 is available on the con-tent 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 varie-ties; 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 small green 5.74 5.81 5.78 Weymouth large green 5.96 6.04 6.00 Weymouth reddish 9.95 9.95 9.95 Weymouth mature blue 10.75 10.78 10.77 Rancocas small green 5.55 5.61 5.58 Rancocas large green 8.20 8.20 8.20 Rancocas reddish 10.66 10.73 10.70 Rancocas mature blue 11.50 11.42 11.46 Jersey small green 6.53 6.45 6.49 Jersey large green 7.91 8.02 7.97 Jersey reddish 11.48 11.49 11.49 Jersey mature blue 13.10 13.17 13.14 Variety means: Weymouth 8.12 Rancocas 8.98 Jersey 9.77 Stages of maturity means: Small green 5.95 Large green 7.39 Reddish 10.71 Mature blue 11.79 Variety - * 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 fair ly 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 is 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 signifi-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 at tr 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, as-suming 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 small green 3.10 3.05 3.08 Weymouth large green 3.05 3.10 3.08 Weymouth reddish 3.20 3.20 3.20 Weymouth mature blue 3.50 3.55 3.53 Rancocas small green 3.25 3.30 3.28 Rancocas large green 3.30 3.30 3.30 Rancocas reddish 3.30 3.35 3.33 Rancocas mature blue 3.60 3.55 3.58 Jersey small green 3.35 3.30 3.33 Jersey large green 3.25 3.25 3.25 Jersey reddish 3.60 3.60 3.60 Jersey mature blue 3.75 3.70 3.73 Variety means: Weymouth 3.22 Rancocas 3.37 Jersey 3.47 Stages of maturity means: Small green 3.22 Large green 3.21 Reddish 3.30 Mature blue 3.61 Variety - ** Maturity - 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 (20). 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 tempe-ratures 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 compa-rison with the other two varieties. It was interesting to note that - 52 -Table VI Percent titratable acids (as citric) of the fruit of three highbush blueberry varieties at four stages of physiological maturity. Rep. I Rep. II Mean value Weymouth small green Weymouth large green Weymouth reddish Weymouth mature blue Rancocas small green Rancocas large green Rancocas reddish Rancocas mature blue Jersey small green Jersey large green Jersey reddish Jersey mature blue 2.58 2.47 2.53 3.06 3.11 3.09 1.89 1.94 1.92 1.07 1.03 1.05 2.25 2.14 2.20 3.42 3.35 3.39 1.74 1.74 1.74 1.17 1.18 1.18 2.51 2.54 2.53 3.16 3.02 3.09 1.74 1.72 1.73 1.03 1.00 1.02 Variety means: Weymouth 2.14 Rancocas 2.12 Jersey 2.09 Stages of maturity means: Small green 2.42 Large green 3.19 Reddish 1.80 Mature blue 1.08 Variety - n.s. 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 be-tween field 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 is 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 indi-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 citric) of the fruit of three high-bush blueberry varieties at four stages of physiological maturity. Rep. I Rep. II Mean value Weymouth small green Weymouth large green Weymouth reddish Weymouth mature blue Rancocas small green Rancocas large green Rancocas reddish Rancocas mature blue Jersey small green Jersey large green Jersey reddish Jersey mature blue 2.69 2.66 2.68 3.43 3.57 3.50 1.95 1.98 1.97 1.24 1.15 1.20 2.36 2.32 2.34 3.77 3.78 3.78 1.94 1.95 1.95 1.32 1.33 1.33 2.76 2.88 2.82 3.43 3.43 3.43 1.93 1.98 1.96 1.18 1.16 1.17 Variety means: Weymouth Rancocas Jersey 2.33 2.35 2.34 Stages of maturity means: Small green 2.61 Large green 3.57 Reddish 1.95 Mature blue 1.23 Variety - n.s. Maturity - Variety x Maturity - ** Table VIII Percent volatile acids (as acetic) present in the fruit of three highbush blueberry varieties at four stages of physiological maturity. Rep. I Rep. II Mean value Weymouth small green .029 .029 .029 Weymouth large green .037 .029 .033 Weymouth reddish .029 .029 .033 Weymouth mature blue .011 .011 .011 Rancocas small green .044 .044 .044 Rancocas large green .044 .040 .042 Rancocas reddish .029 .029 .029 Rancocas mature blue .029 .029 .029 Jersey small green .029 .037 .033 Jersey large green .029 .029 .029 Jersey reddish .022 .026 .024 Jersey mature blue .015 .015 .015 Variety means: Weymouth .026 Rancocas .036 Jersey .025 Stages of maturity means: Small green .035 Large green .035 Reddish .027 Mature blue .018 Variety - n.s. 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 ra -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 fruit . h. ) Reducing sugars. The reducing sugar content of the fruit examined is presented in Table IX. Statistical analysis of the data indicated that there were highly significant differences in the re-ducing 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 fruit . 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 small green 3.47 3.41 3.44 Weymouth large green 4.63 4.92 4.77 Weymouth reddish 8.58 9.18 8.88 Weymouth mature blue 9.52 9.66 9.59 Rancocas small green 3.61 3.91 3.76 Rancocas large green 5.05 5.38 5.22 Rancocas reddish 9.19 8.91 9.06 Rancocas mature blue 10.61 10.93 10.73 Jersey small green 4.37 4.64 4.51 Jersey large green 5.12 4.85 4.99 Jersey reddish 10.71 10.50 10.61 Jersey mature blue 11.82 12.41 12.22 Variety means: Weymouth Rancocas Jersey 6.80 7.20 8.05 Stages of maturity means: Small green 3.90 Large green 4.99 Reddish 9.51 Mature blue 10.83 Variety - n.s. Maturity - ** Variety x Maturity - ** - 60 -Table X Percent total sugar of the fruit of three highbush blue-berry varieties at four stages of physiological maturity. Rep. I Rep. II Mean value Weymouth small green Weymouth large green Weymouth reddish Weymouth mature blue Rancocas small green Rancocas large green Rancocas reddish Rancocas mature blue Jersey small green Jersey large green Jersey reddish Jersey mature blue 3.54 3.50 3.52 4.93 5.34 5.15 8.86 9.52 9.19 9.86 9.79 9.83 3.75 4.06 3.92 5.47 5.63 5.55 9.35 9.30 9.33 10.81 11.12 10.97 4.67 4.75 4.71 5.19 5.22 5.21 11.02 10.80 10.91 12.88 13.15 13.02 Variety means: Weymouth 6.92 Rancocas 7.44 Jersey 8.46 Stages of maturity means: Small green 4.05 Large green 5.30 Reddish 9.81 Mature blue 11.27 Variety - n.s. 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 varie-ties 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 be-ginning 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 photo-synthetic 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 is s t i l l not known. It may be that the organic acids in the respiration process are converted to sugars by a re-versal of the glycolytic processes. Bowers and Dewey (12), in studies with the Rubel and Jersey varieties, observed that fu 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 matu-ri ty . 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 small green 1.37 1.42 1.40 Weymouth large green 1.61 1.72 1.67 Weymouth reddish 4.63 4.91 4.80 Weymouth mature blue 9.21 9.51 9.36 Rancocas small green 1.67 1.90 1.79 Rancocas large green 1.60 1.68 1.64 Rancocas reddish 5.37 5-34 5.36 Rancocas mature blue 9.24 9.43 9.34 Jersey small green 1.86 1.87 1.87 Jersey large green 1.64 1.73 1.69 Jersey reddish 6.34 6.29 6.32 Jersey mature blue 12.50 13.15 12.88 Variety means: Weymouth Rancocas Jersey 4.30 4.53 5.67 Stages of maturity means: Small green 1.68 Large green 1.69 Reddish 5.49 Mature blue 10.51 Variety - * Maturity - Variety x Maturity - n.s. - 66 -Figure VII. The influence of maturity on the sugar-acid ratio of the fruit of three high-bush 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 com-parison 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 de-sirable 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 evalu-ation 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 fruit . 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 Rf x 100 value number 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 blueberry-variety Weymouth at four stages of physiological maturity. (All figures expressed as meq./lOO gm. fresh fruit) . Small Large Reddish Mature green green blue Aspartic and glutamic 0.60 0.78 0.41 0.16 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 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 fruit of the highbush blueberry variety Rancocas at four stages of physiological maturity. (All figures expressed as meq./lOO gm. fresh fruit) . Small Large Reddish Mature green green 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 blueberry-variety Jersey at four stages of physiological maturity. (All figures expressed as meq./lOO gm. fresh fruit) . Small Large Reddish Mature green green 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 tr ic . This was also reported by Nelson (57) and Markakis et a l (50). In a l l samples citric 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 tr ic acid content as was reported by Markakis et a l . They found ripe berries of the Rubel variety contained more ci tr ic 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 tr ic 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 iso-citric acid in blueberry fruit . Markakis et al (50), using techniques simi-lar to those of the present study, supplemented by s i l ica gel chro-matography verified Nelson's finding. The presence or absence of iso-citric acid in the present study was not confirmed. Carles et al (18) were unable to differentiate between c i tr ic and iso-citric acid using paper chromatographic methods employing a butanol-formic acid solvent system. Malic acid was the acid of second largest amounts. This finding agrees with the results of Markakis et al (50). 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 per-cent of the total acid content of the fruit . The close botanical relationship between these species would lead one to expect a some-what 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 subse-quently 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 stricly classed as organic acids in the usual sense of the term, were also reported by Markakis et al (50). Cleaver et al (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 varie-ties 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 in the present ex-periments, are capable of breaking down sugar molecules producing artifacts which may interfere with acid determinations. Oxalic acid was not separably by the chromatographic tech-niques employed and an attempt to estimate the oxalic acid content of the samples by the method described by Winton (80) was not suc-cessful. Considerable variation between measurements of the same sample showed the unsuitability of this procedure; perhaps refine-ments in technique might improve this situation. Winton points out that solutions with a c i tr ic acid content of more than one percent should be avoided. The high c i tr ic 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 fruit . - 76 -The overall results correspond fairly 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 citric acid present between the Rubel and Jersey varieties; whereas in this study no varietal differences were evi-dent on the basis of c i tr ic acid content for the three varieties examined. There were no major differences in the type of acids re-ported 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 citric 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 blue-berries 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 ra -table acid, total acid, pH, total sugars, reducing sugars, and sugar-acid ratios of the fruit . These results indicate that the use of chemical measurements as an index of maturity is 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 is 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 valid, sugar-acid ratios could be used as an important criterion of quality in the blueberry fruit . Over-mature fruit was not examined in this in-vestigation but other workers have shown the practicability of sugar-acid ratios for the identification of over-mature fruit . - 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 sugar-acid 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 in-directly showed a difference in undissociated acid content among varie-ties. 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 citric to be the predominant acid in 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 quantita-tively 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 de-creasing 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 suit-able organoleptic and chemical standards can be established. Blue-berry fruit of uniform quality wil 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 ln cold storage. Annals of Botany. 46:407T459. Association of Official Agriculture Chemists. 1960. Official 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 in culti-vated blueberries. Proceedings of the American Society for Horticultural Science. 49:193-195. s Baker, R. E., and Butterfield, H. M. 1951. 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Proceedings of the American Society for Horticultural Science. 83:395-405. - 90 -APPENDIX STATISTICAL METHODS EXAMPLE Analysis of Variance Table for Percent Total Solids Source Total Treatment Varieties Maturity stages V x M Error Sums of Squares 41.18 40.57 1.76 10.02 28.79 0.61 Degrees of Freedom 23 11 2 3 6 12 Variance 1.14 0.24 n.s. 0 ^ 7 0 n . s . •Sf-S-3.34 4.80 94.12 0.05 *0.5 5.14 4.76 3.09 • 0 . 1 10.92 9.78 5.07 variet ies ^maturity F v x ra Where: n.s. # 1.14 T7&5 3.34 4750" 4.80 0735" 0.24 0.70 94.12 - not signif icant at 5% level of significance. » signif icant at 5% level of significance. *> signif icant at level of significance. 

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