ABSTRACT SOME PHYSIOLOGICAL RESPONSES OF EXCISED ASPARAGUS SHOOTS TO WATER, TEMPERATURES AND ATMOSPHERES OF OXYGEN AND CARBON DIOXIDE by Everett C. Lougheed This work concerns the investigation of the storage of excised shoots (Spears) of asparagus (Asparagus offic~ inalis L.) in atmOSpheres modified in oxygen and carbon dioxide content, and elucidation of the reasons for the up- take of water that has been reported to occur under such atmOSpheres. The levels of oxygen used varied from 5 to 20% and carbon dioxide from 3 to 30%. AtmOSpheres were quickly established by flushing with prepared mixtures of gases or by purging oxygen to the desired level with nitrogen and adding carbon dioxide quantitatively by diSplacement of water. AtmOSpheres were maintained by flushing with desired mixtures or addition by quantitative displacement of water. All gases were obtained from commercial cylinders. In those chambers in which zero carbon dioxide was desired, the car- bon dioxide produced by reSpiration was absorbed in slaked lime (Ca(OH)2). The reSponse criteria studied were: tenderometer readings, changes in fresh weight (interpreted as water up~ take), pH, titratable acidity, per cent soluble solids, Everett C. Lougheed fiber and crude fiber content, per cent dry matter, and cook— ing quality. The modified levels of oxygen were ineffective and carbon dioxide highly effective on the physiological re- sponses recorded. Levels of carbon dioxide as low as 3%, when applied at BSOF and 85 to 90% relative humidity, in— creased tenderness, gains in weight (water uptake), and pH, and there was an upward trend in per cent soluble solids after correction for water uptake. The optimum range of carbon dioxide was 12 to 15%. The 30% level of carbon diox— ide increased the pH and per cent soluble solids more than 15% carbon dioxide; whereas 15% had a greater effect on water uptake and tenderness. There was no measurable effect of carbon dioxide on the pH or titratable acidity of the aSparagus frozen at -200F after carbon dioxide treatment, or on dry matter after correction for water uptake. The effects of 15% carbon dioxide on freshly harvest— ed aSparagus standing in water were evident in 2 days at 35°F, and peak reSponse was evident in 8 to 12 days. This concentration of carbon dioxide was effective in producing tenderness and increasing pH and water uptake of asparagus previously stored at 600F and 50% relative humidity for 1 day. Although the effect of the atmOSphere was considerable for longer pre-storage times, the net effect was lessened by losses during pre—storage. The tenderness and water content of the carbon dioxide-treated asparagus remained greater Everett C. Lougheed than the air—stored aSparagus when both were subsequently held for l to 6 days in air at 600F and 50% relative humid- ity. The higher pH of the carbon dioxide-treated asparagus decreased to a level similar to that of the aSparagus stored in air during this post-storage period. There was less effect of carbon dioxide at 45 and 550F than at 350. When the asparagus was not standing in water, atmospheres containing 15% carbon dioxide had only a slight tenderizing effect and increased the pH only slightly. A carbon dioxide concentration of 30% increased the pH even if water was not present. When water uptake was prevented by Carbowax, used as an external osmotic agent, the pH of asparagus treated with 12% carbon dioxide did not increase as much as it did when free access to water was permitted. It was shown that only a minor portion of the tender- izing effect can be attributed directly to uptake of water. The increased turgidity with water uptake decreased the force necessary to shear through the Spears. The uptake of water is attributed to a higher diffusion pressure deficit caused by some hydrolysis of substrate. The higher osmotic concentration within the asparagus was measured as a higher per cent soluble solids than the controls after correction for water uptake, and by a higher diffusion pressure deficit as measured by osmotic solutions of Carbowax 1000. The up- take of water under modified atmospheres was independent of temperatures between 35 and 55°F, and of treatments with Everett C. Lougheed Né—benzyladenine, maleic hydrazide, and 2, 4—dinitrophenolate at 35°F. However the 3 respiration inhibitors may not have been effective because of the low basal rate of metabolism. Water uptake in air was dependent on temperature, but in- dependent of the presence of the inhibitors at 35°F. It is believed that the major portion of the tender- izing effect of carbon dioxide is due to some dissolution of intercellular materials. This dissolution led to less fi— brous material in the aSparagus treated with carbon dioxide as analyzed by a blendor method, although the crude fiber content did not change. Although there was some effect of the carbon dioxide on asparagus stored without water, the effect was increased markedly when the asparagus was stored standing in water. The tenderness obtained by treatment with carbon clioxide was retained in the cooked aSparagus. This aSpar— Elgus, however, lacked flavor. Modified atmospheres containing carbon dioxide offer FNDssibilities as supplements to low temperature storage. FWor best results the aSparagus must be stored standing in Vvater and the atmOSpheres must be established quickly after llarvest. SOME PHYSIOLOGICAL RESPONSES OF EXCISED ASPARAGUS SHOOTS TO WATER, TEMPERATURES AND ATMOSPHERES ‘op OXYGEN AND‘CARBON DIOXIDE By Everett C. Lougheed A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of 'UDOCTOR OF PHILOSOPHY Department of Horticulture 1964 ACKNOWLEDGEMENTS To give proper thanks for all assistance received in this work would take a voice much more articulate than the author's, for help and encouragement, other than financial, are often so subtle as to defy definition. To the chairman of the guidance committee, Dr. D. H. Dewey, who directed the work and the writing of this thesis, the author's sincere appreciation. The members of the com~ mittee, Drs. D. R. Dilley, C. L. Hamner, P. Markakis, and C. J. Pollard were always co-operative and helpful when assistance was needed. Also thanks are due to Dr. R. R. Dedolph, and Professor E. W. Franklin, Department of Horti- culture, Ontario Agricultural College who assisted in the planning, and to Dr. R. L. Carolus who replaced Dr. Hamner for the final defense of the thesis. The author is grateful to the Ontario Department of ‘Agriculture for the leave granted from August, 1962 to 4August, 1964 to work on this thesis and for financial assist— Eince. For a Research Scholarship for 1963 the author is in- <1ebted to the Agricultural Institute of Canada. ii TABLE OF CONTENTS INTRODUCTION REVIEW OF LITERATURE GENERAL METHODS AND MATERIALS RESULTS Water Induction and Tenderness Temperature and AtmOSphere N6-Benzyladenine and Atmosphere AtmOSphere, Dry Matter, Fiber, Crude Fiber,. and Cooking Quality Oxygen and Carbon Dioxide Levels Pre- -Storage Condition and Atmosphere Shelf Life and Atmosphere Storage Period and Atmosphere Atmosphere and Fiber Content . Maleic Hydrazide and AtmOSphere Sodium 2 ,4- -Dinitrophenolate and Atmosphere External Osmotic Agent and Atmosphere--I External Osmotic Agent and AtmOSphere—-II GENERAL DISCUSSION AND CONCLUSIONS 'SUMMARY LITERATURE CITED (UPPENDIX iii Page 18 28 32 42 55 62 70 77 84 91 96 99 104 112 119 129 132 137 Table LIST OF TABLES IN TEXT The effects upon aSparagus of water induction by a vacuum of 25 inches of mercury for periods of 20, 40, and 60 minutes Per cent water loss from open cans over a period of a week at 3 temperatures and 2 atmOSpheres Temperature and precipitation the day before harvest The effects upon aSparagus of storage for l we k with the butts in water at 35, 45, and 55 F in air and 15% oxygen and carbon dioxide The effects upon asparagus of storage for 1 week with the butts in water at 35,45 and 55°F in air and 15% oxygen plus 15% carbon dioxide . . . . . . . . . . . . . . The effects upon asparagus of storage for 8 days at 350 F under modified atmOSpheres containing carbon dioxideésuperimposed upon treatments of water and N -benzyladenine The effects upon asparagus of storage for8 days at 350 F under modified atmOSpheres containing carbon dioxide superimposed Upon treatments of water and N —benzy1adenine The effects upon aSparagus of storage for 8 days at 35°F under modified atmOSpheres containing carbon dioxide superimposed upon treatments of water and N -benzyladenine The effects of atmOSpheres of air and 15% oxygen plus 15% carbon dioxide on the composition and tenderness of aSparagus stored at 35°F for 1 week with the butts in water iv Page 30 33 34 35 37 45 47 49 57 Table 10. 11. 12. 13. 14. 15. 16. 17. The effects of various levels of oxygen and carbon dioxide in a factorial arrangement on asparagus stored for 1 week at 35°F with the butts in water . . . . . . . The effects of atmOSpheres of air and 15% oxygen plus 15% carbon dioxide on asparagus stored at 35°F for 1 week with the butts in water, and subsequent effects of storage in air without water for l to 6 days at 60°F and 50% relative humidity . . . . . The effects of time in storage and atmospheres of air and 20% oxygen plus 15% carbon dioxide on asparagus stored at 35°F with the butts in water The effects of time in storage and atmOSpheres of oxygen and carbon dioxide in a factorial arrangement on the development of crude fiber and fiber in aSparagus stored at 35°F with the butts in water The effects of maleic hydrazide and atmOSpheres of air and 15% oxygen plus 15% carbon dioxide on asparagus stored at 35°F for 1 week with the butts in water The effects of atmOSpheres on aSparagus stored for 1 week at 35°F with the butts in a solution of sodium 2,4- -dinitrophenolate The effects of angSpheres on asparagus stored for 1 week at 35 F with the butts in solutions of Carbowax 1000 . . The effects of a modified atmOSphere of 9% oxygen and 12% carbon dioxide on aSparagus stored for 1 week at 35°F with the butts in solutions of Carbowax 1000 Page 64 79 86 93 97 101 107 113 LIST OF ILLUSTRATIONS Figure Page 1. The effects of atmOSpheric levels of carbon dioxide on aSparagus stored for 1 week at 35°F with the butts in water . . . . . . . . . 66 2. The influence upon asparagus of pre-storage for 0, l, 2, 3, and 4 days in air at 60°F and 50% relative humidity without water, on subsequent effects of storage for 1 week at 35°F in air and'15% oxygen plus 15% carbon dioxide (15-15) with the butts in water . . . 73 3. Changes in asparagus held at 60°F and 50% relative humidity in air without water for 1 to 6 days subsequent to storage in air and 15% oxygen plus 15% carbon dioxide (15—15) for 1 week at 35°F with the butts in water . . . . . . . . . . . . . . . . . . . 81 4. The effects of time in storage and atmospheres of air and 20% oxygen plus 15% ca bon dioxide (20—15) on aSparagus stored at 35 F with the butts in water . . . . . . . . . . . . . . . . 88 5. The effects of atmOSphege upon aSparagus stored for 1 week at 35 F with the butts in solutions of Carbowax 1000 . . . . . . . . 109 6. The effects of a modified atmosphere of 9% oxygen and 12% carbon dioxide upon aSparagus stored for 1 week at 35°F with the butts in solutions of Carbowax 1000 . . . . . . . . . . 116 vi Table A-1. LIST OF TABLES IN APPENDIX A summary of the analyses of variance for the effects upon asparagus of water induction by a vacuum of 25 inches of mercury for periods of 20, 40, and 60 minutes . . . . . . . . . . . z A summary of the analyses of variance for the effects upon aSparagus of storage for 1 week with the butts in water at 35, 45, and 55°F in air and 15% oxygen plus 15% carbon dioxide . . . . . . . . . . A summary of the analyses of variance for the effects Bpon aSparagus of storage for 8 days at 35 F in modified atmOSpheres containing carbon dioxide superimposed on treatments of water and N -benzyladenine A summary of the analyses of variance of the effects of atmOSpheres of air and 15% oxygen plus 15% carbon dioxide on the composition and tenderngss of asparagus stored for 1 week at 35 F with the butts in water A summary of the analyses of variance for the effects of various levels of oxygen and carbon dioxide upon aSparagus stored with the butts in water for 1 week at 35°F A summary of the analyses of variance for the effects of time in storage at 60°F and 50% relative humidity in air upon asparagus previously stored with the butts in water for 1 week at 35°F in air and 15% oxygen plus 15% carbon dioxide vii Page 138 139 140 142 143 144 Table A-lO. A-ll. A summary of the analyses of variance for the effects of time in storage and atmOSpheres of air and 20% oxygen plus 15% carbon dioxide on asgaragus stored with the butts in water at 35 F A summary of the analyses of variance of the effects of time in storage and atmos- pheres of oxygen plus carbon dioxide on the deve10pment of fiber and crude fiber in aSparagus stored at 35°F with the butts in water . . . . . . . . . . . . . A summary of the analyses of variance of the effects of atmosgheres on asparagus stored for 1 week at 35 F with the butts in a solution of sodium 2,4-dinitrophenolate (DNP) A summary of the analyses of variance of the effects of atmosgheres on aSparagus stored for 1 week at 35 F with the butts in solutions of Carbowax 1000 A summary of the analyses of variance of the effects of a modified atmosphere of 9% oxygen plus 12% carbon dioxide upon asparagus stored for 1 week at 35°F with the butts in solutions of Carbowax 1000 viii Page 145 146 147 148 149 INTRODUCTION Asparagus, ASparagus officinalis L. (Thompson and Kelly, 1957) is a perennial vegetable grown for its green or blanched (white) shoots which arise in the Spring from overwintering croWns. These shoots are harvested by cutting or snapping while still less than 15 inches high and are therefore succulent tissues. The entire growing period of individual shoots or Spears above ground, prior to harvest, may be less than 48 hours. The source of energy for the growth of the Spears is the reserves in the fleshy roots. After the harvest sea- son the shoots are allowed to grow, with the photosynthate produced in the ”fern” or foliage being translocated to the fleshy roots to provide a source of energy for the ensuing season. : Asparagus Spears are eaten as the fresh, canned, or frozen product. Because of relatively high costs of produc- tion, perishability, and difficulties in storage of the fresh plant parts, asparagus is a luxury food. ASparagus . Spears are immature and succulent, reSpire and transpire rapidly, and develop fiber and become tougher in storage (Bisson, Jones, and Robbins, 1926; Morse, 1917). Loss of quality through development of fiber and loss of flavor through respiratory losses which occur rapidly at high tem— peratures, are usually retarded by storage at 32°F, and net loss of moisture through storage at high relative humidities with the basal portions of the Spears standing in water or on a moist water—absorbent pad. Maximum duration of storage is 3 to 4 weeks (Wright, Rose, and Whiteman, 1954). Because quality of aSparagus is retained for such a Short time, methods of commercial CA (controlled or modified atmosphere) storage used for apples, by which the product produces its own atmospheres through respiration and exter— nal control of gaseous environment, are not suitable. By the time that a Suitable atmosphere is established the stor- age season might be almost over and the aSparagus already in poor condition. However, artificial means may be employed to estab- lish proper levels of carbon dioxide and oxygen within 1 day and could make the storage of aSparagus commercially feasible, provided that the results in quality preservation would warrant the added expense. Previous studies have shown that atmOSpheres of carbon dioxide may be useful ad- juncts in the storage of aSparagus (Barker and Morris, 1937; Carolus, Lipton, and Apple, 1953; Franklin g£_31., 1960, 1961; Lipton, 1960). The purpose of this thesis was twofold; firstly, to evaluate the worth of modified atmosphere storage as applied to aSparagus, and secondly, to investigate the reasons for the uptake of water and increased tenderness which had been found by Franklin et a1. (1960, 1961). REVIEW OF LITERATURE The tenderometer was established as being suitable for measurements of tenderness of aSparagus by Jenkins and Lee (1940). For raw asparagus they set 150 psi as being suitable as a line dividing fancy and standard grade based on tenderness alone. They used 4-inch tip sections of the Spears and filled the hopper. The Size of the Spear did not affect the tenderometer reading. Carolus §£_31. (1953) also used the tenderometer to measure the tenderness of both raw and cooked Spears. An average reading for raw Spears was 127 psi; for cooked Spears, 51 psi. They used 4 Spears per reading with the 4-inch sec- tions taken from just below the tip. The disadvantage in using the tenderometer is that the tissue is completely macerated and is no longer suitable for other analyses. When the hopper is completely filled 8 to 18 Spears may be used for one reading. Although other methods have been used for measuring tenderness such as a modified fruit pressure tester (Kramer g£_gl., 1949) and the shear-press (Wiley g£_31., 1956), the tenderometer appeared to be adequateznuieminently suitable for this work. The tenderness of aSparagus has usually been related ‘\ to fiber content. Jenkins and Lee (1940) found a signifi- cant correlation of 0.729:0.029 between tenderometer reading and crude fiber content. Kramer §£_31. (1949) using the blendor method of fiber analysis (Smith and Kramer, 1947), and an original pressure tester, found that the pressure varied directly with fiber content. Samples of larger stalks gave higher pressure readings than smaller stalks of the same level of fiber content. Scott and Kramer (1949) found that pressure readings of aSparagus stored in air increased slightly with time in storage and with increase in storage temperature. The fiber content, as measured by blendor method, seemed to decrease with time and storage temperature. Carolus g£_gl. (1953) and Brody and Francis (1957) have reported similarly that at high temperatures or high respiration rates, fiber "dis- appears." These results are at variance with the results of Bisson ££_31. (1926), Lipton (1958), and Morse (1917) who found fiber increased under these conditions. Scott and Kramer (1949) attributed the variation in results to method of analyzing for fiber. The material obtained by the official method (AOAC, 1960) should probably be designated as crude fiber; whereas fiber should be reserved for the material which is estimated by the blendor method.1 Although, as stated by Scott and 1This distinction is used arbitrarily in this thesis to distinguish between the two methods of analysis. L/Kramer (1949), the blendor method may give a better estima- tion of "organoleptic fibrousness" than the official method, the latter gives a better estimation of physiological changes within the spears as far as the synthesis or degradation of crude fiber is concerned. L The level of crude fiber has been found to increase with time in storage and with increasing temperature (Bisson §£_31,, 1926; Morse, 1917), although reports have indicated that the amount of fiber may decrease under the same condi— tions (Carolus g£_31., 1953; Scott and Kramer, 1949). The' increase in fiber is attributed to utilization of sugars in lignification in the pericycle1 and the vascular bundles (Bisson g£_gl., 1926; Brennan, 1958; Lipton, 1958). L Brennan (1958) found that asparagus Spears stored with cut ends immersed in water showed a more mature degree of development of the pericyclic fiber ring, but a lower per- centage of thick-walled tracheary elements per bundle. There was the suggestion that there was a slightly larger number of thick—walled cells in Spears stored dry as compared to those ,Stored in water. Wiley g£_gl. (1956) found that fiber con- tent as measured by the blendor method actually decreased when aSparagus was stored standing in water. Although these 1Brennan (1958) discusses the use of this term which is anatomically incorrect because aSparagus is a monocot. However, the term pericycle is used in this thesis in con- formity with previous work. authors evidently did not attempt to correct for weight changes due to water uptake, the per cent total solids de- creased very little, implying that this was a real decrease of fiber. Brennan (1958) attributed some of this decrease in water to stem elongation. Because the fiber content de- creases towards the tip of Spears, sampling for fiber at a Specific distanCe from the tip would give a small reduction because of stem elongation. Because aSparagus is a succulent crop the prevention of water loss is important to retard the parallel loss of quality. To prevent this water loss, aSparagus is often stored or shipped with the butts on pads of wetted water- absorbent material or stored standing in water. Scott and [\Kramer (1949) have given the following results for aSparagus stored standing in water. ASparagus may gain weight up to 20% or more (with snapped asparagus, 8 days in storage, tem- perature 32°F). Snapped aSparagus gained more weight than cut aSparaguS when placed in water, and lost less moisture under conditions favoring loss. The uptake of water in- creased with temperature; more at 40° than at 32°F; that at 50° being similar to 40°, and at 70° there was a rapid up- take-~some 14% after 1 day, but after 8 days there was a loss of 21.6%. This loss at higher temperatures was regarded as due to blocking of the conducting vessels at the cut sur- faces. At 35°F an increase in weight of 6.5% has been re— corded with cut, trimmed aSparagus as the average of 2 weeks of storage (Franklin g£_gl., 1960). Other workers have found that water uptake is less at 33° than 41°F or other. higher temperatures (Bisson §£_31,, 1926). Overall these results would indicate that water uptake is dependent on respiration. The maximum water uptake at all temperatures usually takes place in the first 24 hours (Bisson ££_31,, 1926; Scott and Kramer, 1949). Franklin §£_31. (1960) advanced the theory that the uptake of water by itself might tenderize asparagus as meas- ured by the tenderometer. They alluded to ”criSping" let- tuce and other leafy vegetables in a household refrigerator or the very fact that aSparagus is stored with the butts in water. By artificial induction of water by vacuum, they were unable to Show this tenderizing effect (Franklin g£_31., 1961). This failure was attributed to variations in tender- ness within the aSparagus and inability to induce sufficient water uptake by the asparagus. Morse (1917) showed that this tenderizing effect of water uptake could be detected although only by subjective measurement. He stated that aSparagus stored standing in water became more "brittle" with uptake of water. However, Brennan (1958) could not find differences between pressure readings of turgid and flaccid aSparagus. Wiley g£_gl. (1956).as discussed before, found lower pressure readings in aSparagus standing in water because the fiber content actual- ly decreased. Johnson (1947) has described the excess of water in plants (in the intercellular spaces) as water congestion. The pH of asparagus stored in air has been found to decrease with time in storage (Schweigart and Kellner, 1938). This change would appear to be part of the general aging process (Jacobs, 1951). When water is taken up by asparagus the per cent soluble solids decrease (Franklin g£_gl., 1960; Scott and Kramer, 1949). Because soluble solids are primarily con- cerned with the measurement of sugars, any reaction that affects the sugar concentration will also change the per cent soluble solids. Thus respiration losses with time in storage or with high temperature tend to decrease the per cent soluble solids. Sugars may also be lost, as measured by per cent soluble solids, upon their conversion to fiber (Morse, 1917). Modified atmospheres containing carbon dioxide have been noted as causing tenderness, as measured by the tender- ometer, of both raw and cooked asparagus (Carolus g£_al., 1953). These workers were investigating the effect of pack- aging on asparagus. They suggested that the tenderizing effect might have been due to lack of oxygen rather than a buildup of carbon dioxide although the oxygen levels in the packages were not measured. The authors noted this effect 10 at 350 and 55°F, and found more noticeable results in snapped than cut aSparagus. Lipton (1960) used the fibrometer, which measures the length of the Spear tender enough to be cut by a weighted wire, and found that at a temperature of 37.5°F the length of the spear that was tender after 7 days in storage was greater under various modified atmOSpheres than the controls. The effect was still apparent, although less uniformly so, after storage for 2 more days at 59°F. At 50°F the effects were not as conclusive although significant differences were noted. Franklin g£_al. (1960) found very marked effects of carbon dioxide on tenderness of aSparagus when the aSparagus was stored standing in water.1 Although the overall effect of oxygen was slight compared to carbon dioxide there was a significant lowering of the tenderometer readings with oxy— gen concentrations lower than that of air down to 5%. The temperature for these experiments was 35°F and the modified atmospheres varied from 5 to 15% oxygen with carbon dioxide concentrations from 0 to 15%. The duration of storage was 7 to 14 days. Thornton (1931) on the other hand, has observed toughening of asparagus in some modified atmospheres. 1 . . . This term is used synonymously With the less elegant but more expressive term "butts in water.” 11 Fifty per cent carbon dioxide caused toughening, described as "flabby tough shoot that failed to break with a snap as did the controls" at 0,4,10, and 15°C. However, with the method used, the oxygen levels must have varied considerably. It is possible that some of the apparent effect of carbon dioxide was really oxygen deficiency. The carbon dioxide concentrations were also much higher than those used by the other investigators. Thornton noted that increasing the temperature or the carbon dioxide concentration caused more injury. The cause of the tenderizing or toughening effect of modified atmOSpheres has not been well defined. Carolus st 31. (1953) stated that the increase in tenderness may be due to breakdown of fiber, and indeed they found that fiber levels decreased with increasing carbon dioxide levels. This fiber was, however, measured by the blendor method and this cannot be interpreted as meaning crude fiber. Franklin g£_31. (1960) argued that the breakdown of crude fiber as such did not likely occur, and the effect resulting in ten- derness may only have been due to some softening of the tis- sues causing easier disruption of the cells by a shearing force. Franklin §£_31. (1960) also regarded some of this tenderizing effect that they found as being due to increased water uptake which occurred at high concentrations of carbon dioxide. However, they were unable to substantiate that 12 this was the effect except by inference. Furthermore, because it is evident that carbon diox- ide has a tenderizing effect on asparagus not stored standing in water (Carolus ££_gl,, 1953; Lipton, 1960), Franklin g£_gl. (1960) postulated 2 effects; one effect on "dry” aSparagus which tenderized aSparagus to some extent, and the other effect which was much more evident, due to water uptake plus carbon dioxide. This latter effect could be partially due to water itself and also to its effects on the reactions causing tenderness with "dry" aSparagus. The relationship of quality to water uptake and storage of aSparagus standing in water has been reported by several authors. Thornton (1931) stored aSparagus in water and under modified atmOSpheres. However, the only result he cited was the observation that more injury occurred when aSparagus was stored in contact with moist Sphagnum moss and in water. Barker and Morris (1937) found that aSparaguS stored with the butts in water in air gained in weight and had a fresher appearance than that stored dry, but the butts rotted upon removal. Franklin §£_gl. (1960) found that increasing carbon dioxide levels up to 15% increased the gains in weight of aSparaguS stored with the butts in water. They postulated 3 possible ways in which carbon dioxide might cause increased water uptake to account for the gains in weight: (1) In- creased diffusion pressure deficit (DPD) as a result of . 13 hydrolysis of cellular breakdown, (2) Changes in permeability of cellular membranes. (3) Changes in pH affecting the growth of bacteria which might clog the conducting vessels of the xylem. They decided that the most likely cause was in- creased hydrolysis or cellular breakdown, although no def- inite proof was given. Galston and Purves (1960) refer to the work of Bogen (1951) who argued that the permeability of a membrane, as long as it remains similarly semipermeable, cannot effect the amount of water taken up. Although per— meability can effect the time to equilibrium the amount of water taken up will still depend on the DPD. Glinka and Reinhold (1962) showed that carbon dioxide bubbled through a solution containing disks of tissue would affect the per- meability of plant cell membranes. If the concentration of carbon dioxide were sufficiently high, 80% or over, and the exposure prolonged over 30 minutes, water was lost from the tissues. It would appear that the membranes had lost their semipermeable properties. The bacterial growth theory of diminishing water uptake has not been investigated, although Lipton (1960) has suggested that carbon dioxide aids in control of bacterial soft rot. The pH of plant parts stored in carbon dioxide would be expected to decrease with the formation of carbonic acid upon the dissolving of carbon dioxide in the water within a l4 plant. Willaman and Beaumont (1928) even considered this fact so well established as to say "that the acidity of the tissue fluid is increased by the accumulation of carbon di- oxide is well known, and does not need a specific illustra— tion." However, it has been shown that carbon dioxide in the atmOSphere increases the pH of plant sap. This effect has been noted by Franklin ££_31. (1960, 1961) for aSparagus and by Fife and Frampton (1935), and Thornton (1933b, 1937) for several plant parts including excised asparagus spears. Magness and Diehl (1924) also noted that the acidity of apple fruits decreases with increasing levels of carbon diox- ide. Franklin g£_31, (1961) found decreases in titratable acidity of aSparagus Spears under atmospheres containing carbon dioxide. However, the treatments with the least acid- ity were those causing the greatest water uptake, so this effect noted may be due to dilution. Thornton (1937) showed that this pH increase in aSparagus could occur very quickly, "a definite increase . with only a 20-minute treatment." Thornton thought the pH increase that he noted indicated an accumulation of end products of associated metabolic processes. Fife and Frampton (1935) found that the pH increase in sugar beet leaves was coincident generally with the deamidation of aSparagine and glutamine and the formation of ammonia. Under high concentrations of carbon dioxide, 15 ammonia nitrogen was found to have been split off from other soluble nitrogenous compounds as well as the amino acid amides. This change occurred in the light as well as the dark and did not take place in macerated or plasmolyzed tissue. The juice expressed from plants, whether treated previously with carbon dioxide or not, underwent a pH de- crease under high carbon dioxide concentrations. If the carbon dioxide concentration was too high (up to 100%) mak- ing the oxygen tension low, organic acids formed in respira- tion nullified the effect of increased pH through formation of OH- and the pH decreased. Fife and Ferguson (1941) cor- roborated the findings of Fife and Frampton (1935). Thornton (1937) showed that this pH increase would take place at temperatures of 2, l7, and 27°C at carbon di- oxide concentrations of 30 and 60%. The change caused by carbon dioxide was reversed when the treated material was allowed to stand in air. The per cent soluble solids of asparagus stored in water under high levels of carbon dioxide is considerably less than controls in air (Franklin §£_31., 1960). However, this is only a reflection of uptake of water attributed to high levels of carbon dioxide. As such, the per cent sol- uble solids means little because there was no correction for water uptake. The per cent soluble solids may also be ex- pected to decrease less under carbon dioxide because the rate of reSpiration is diminished by carbon dioxide (Thornton, 1933a). 16 At 25°C the reSpiration rate of aSparagus was re— tarded at concentrations of carbon dioxide as low as 3%. .With increasing levels of carbon dioxide the average decrease in rate of oxygen uptake increased, with a maximum decrease of 35 to 43% with 60 to 70% carbon dioxide. A significant decrease was noted with 10% carbon dioxide (Thornton, 1933a). Maleic hydrazide (MH) has been Shown to retard res— piration of aSparagus slightly at 70°F. AS well, the maleic hydrazide appeared to make the aSparagus somewhat more tender as measured by a modified Balauf pressure tester. The MH treatment decreased the change in weight (mostly water up- take) to a small extent after 12 hours at room temperature, and there was a slight increase in Spear elongation. The effects of other inhibitors were also studied. Those inhib— itors which increased respiration increased the tenderness of aSparagus. One inhibitor, benzothiozol—Z—oxyacetic acid (Regulant 3201) which increased respiration to the greatest extent, decreased resistance to cut the most, and caused greatest gains in weight of the Spears standing in water. These authors (Brody and Francis, 1957) concluded, as had Carolus §£_31. (1953), that increased respiration may in- crease tenderness of aSparagus. Brody and Francis thought this was because of demand of substrate for respiration, leaving less available for lignification. N°—benzyladenine (N°-BA) at a concentration of 5 5 x 10' M has been shown to lower the reSpiration rate of 17 asparagus 16% at 21°C (Dedolph, Wittwer, and Tuli, 1961). This effect may be due to the influence of N°~BA upon the glycolytic pathway (Tuli and Wittwer, 1964). As well as inhibiting reSpiration to some extent, N°—BA or other phytokinins1 are known to affect protein synthesis or protein breakdown (Sugiura, Umemura, and Oota, 1962; Osborne, 1962). Because these chemicals are not readily translocated they must be applied in the area in which reactions are desired (Thimann and Laloraya, 1960). 2,4-dinitrophenol (DNP) is a respiration inhibitor that has been used in the study of galactose uptake by bacte- ria (Osborne, McLelland, and Horecker, 1961) and in studies of energy relations in living organisms because it is an agent uncoupling oxidation and phOSphorylation (Cooper and Lehninger, 1957). DNP may increase oxygen uptake, the amount of increase depending on the age of the tissue (Macdonald and DeKock, 1958), and pH of the solution (Beevers, 1953). The response for oxygen uptake changed from zero at pH 8, reached its peak at pH 5, and the effect was inhibitory at pH 3. For optimum inhibition the pH should be about 4. The active concentration ranges from 3 x 10'5 (Cooper and Lehninger, 1957) to 1 x 10’3M (Macdonald and DeKock, 1958; Stenlid, 1949). lThis nomenclature was suggested by Dedolph, Wittwer, and Maclean. Letter to the editors. Scientific American 208:13. 1963. 18 Water stress has been shown to affect metabolic reactions. Spoehr (1919) showed that in a Species of cacti desiccation favored an accumulation of polysaccharides and a decrease of monosaccharides. Opposed to this, later work indicated that in leaves of non—succulents, desiccation favored starch dissolution and the formation of sugars (Spoehr and Milner, 1939). The utilization of sucrose in synthesis in green peas has been found to decrease with de— creasing water content (Wager, 1954). The results of Petrie and Wood (1938) indicated that the protein content of leaves decreased with lessened amino acid and water content. The authors were unable to deter- mine whether this decrease was due to decreased synthesis or increased hydrolysis. Klotz (1958) interpreted some of the effects due to variation in water content as being a product of variations in protein hydration. Chen, Kessler, and Monselise (1964) found that upon increasing water stress there was a 3-phase change in protein level in citrus leaves; an increase at the beginning of dehydration, a decrease at medium hydration, and a slight increase again at extreme de-. hydration conditions. ASparagine and glutamine maintained a low and constant level during the developing water stress, while aSpartic and glutamic acid followed the 3-phase reac— tions established for protein. 19 Galston and Purves (1960) indicated that sucrose is not an ideal osmotic agent because it may be taken up and metabolized. Mannitol may also be assimilated and cause abnormal metabolism (Bailey and Setterfield, 1957; Trip, Nelson, and Krotkov, 1963). Polyethylene glycols, prepared as "Carbowaxes" by Union Carbide, have been suggested as osmotic agents because they are virtually inert. Because Carbowaxes are not homogenous their osmotic pressures cannot be calculated from their molecular weight. Jackson (1962) has given the osmotic pressures for several concentrations of Carbowax 1000. He also indicated that Carbowaxes might not be suitable for osmotic studies of delicate tissues. GENERAL METHODS AND MATERIALS Chambers framed with 2 x 2 inch white pine and of outside dimensions of 31 x 23 x 14 inches, covered with laminated aluminum plus Mylar film,1 were employed as atmos— pheric chambers inside a room at controlled temperatures. Access to the chambers was provided by means of a 6-inch square opening cut into the center of the clear film cover- ing the top. The opening was closed with an overlapping square of film sealed in place with pressure-sensitive tape. Light was excluded by a covering of black paper. Steel chambers of outside dimensions of 40 x 17.5 x 15 inches were also employed. A vision plate of 1/4 inch clear plastic was fitted completely across one end. Light was excluded as above for the film covered chambers. Zero per cent carbon dioxide was attained by plac— ing approximately 1/8 pound of slaked lime (Ca (OH)2) inside each chamber to absorb the carbon dioxide produced in res— piration. For experiments conducted in 1962, the chambers were flushed to the desired level of oxygen with nitrogen. Carbon dioxide was then added quantitatively by diSplacement of 1Zero Perm vapor barrier, Alumiseal Corp. 20 21 water. The excess carbon dioxide produced through respira- tion was flushed out to the desired level by mixtures of oxygen at the desired level plus nitrogen to make a total of 100%. Oxygen was added quantitatively by displacement of water. Those atmOSpheres designated as 20% oxygen represent- ing normal air were flushed as often as necessary with com- pressed air to maintain the carbon dioxide level. Those chambers in which 20% oxygen and 0% carbon dioxide was de- sired were vented to air to restore the desired oxygen con— centration and the carbon dioxide was absorbed in lime. An atmOSphere of 9% oxygen plus 12% carbon dioxide was employed for all tests in 1963. The chambers were flushed to the desired level with this mixture from prepared cylinders, both initially and as the levels of oxygen and carbon dioxide varied from those desired. The controls at 20% oxygen and 0% carbon dioxide were vented to air to re- store the levels of oxygen and the carbon dioxide was ab— sorbed in lime as described before. Atmospheres were tested with an Orsat type analyzer at least once every 2 days, and usually every day. Atmos- pheres were maintained at plus or minus 1.5% of the desired level at all times and usually plus or minus 1.0% for both oxygen and carbon dioxide. The atmospheres listed in the text and tables are referred to as in the following example: 10-15 represents 10% oxygen plus 15% carbon dioxide, with the remaining 75% being almost all nitrogen. 22 Two control atmOSpheres were used in most experiments; a control in storage air (Air), and another in a chamber at essentially the same level of oxygen and 0% carbon dioxide designated as 20-0. The duplicate control was used to eliminate relative humidity as a factor affecting the crite— ria studied. Although the control in the-chamber is desig- nated as 20-0, the oxygen level was usually above the 20% that the designation implies, averaging 20.5%. In almost all cases no differences were found between the controls for the criteria studied. In grouped statistical comparisons the two controls, Air and 20-0, are grouped as Controls. When a humidity higher than that produced by tran- spiration was desired, moist Sphagnum moss was placed in the bottoms of the chambers and kept moistened. The RH under such conditions was estimated as more than 95% because con— densation occurred on the inner surfaces of the chambers. Without the moss the RH, as measured by a hygrometer, was between 85 and 90%. The aSparagus was stored with butts in approximately 250 m1 of tap water, initially 2 to 2-1/2 inches deep, in "tin" cans. The method of using the tenderometer to measure tex- ture was essentially that used by Carolus g£_31. (1953), except that the hopper was filled for each measurement in- stead of using 4 Spears. Four—inch sections of Spears, excluding the tip portion, were used. The number of spears 23 necessary to fill the hopper varied from 8 to 18 with the average number being 12. Average sized spears were selected whenever possible. The juice expressed from the tenderometer was used for measurements of pH and per cent soluble solids in 1962. Measurements of pH were made with a Beckman Zero— matic pH meter and glass electrode. A combination electrode was used in the work in 1963. Because the tenderometer was not used in 1963 the juice was expressed by hand from sec- tions of Spears equivalent to those used in the tenderometer in previous work. The sections of the Spears were ground in a blendor before pressing through 2 layers of cheesecloth. The statistical analyses were performed on the hydrogen ion values equivalent to the pH. An Abbe type refractometer was used in 1962 and a hand refractometer in 1963 for obtaining the per cent sol- uble solids. The juice as obtained above for pH was used in these estimations. In most cases the per cent soluble solids readings were corrected for changes in weight as if all were due to change in water content. These values are designated as "corrected" or "correct" in the tables and figures. If not corrected there is no designation. The readings from the hand refractometer, when corrected for change in weight are considered somewhat unreliable. The measurements of fiber and crude fiber, dry matter and titratable acidity were corrected for change in water 24 content as described for soluble solids. Fiber analyses were performed by two methods; the official (AOAC, 1960) method, and the blendor method (Smith and Kramer, 1947). By AOAC procedures the fresh material was dried for 24 hours at 65°C, ground in a Wiley mill through a 20 mesh screen and stored in glass jars. This material was used for fiber analyses as well as estimations of dry weight. The analytical method, although essentially official, was modified as follows: Ether extraction was not performed. This may make the results somewhat high (Joslyn, 1950). Un- bleached cotton was used in the first filtering and alundum crucibles for the second. Analyses by the blendor method were performed accord- ing to the procedures outlined by Smith and Kramer (1947). The asparagus samples were frozen at minus 20°F immediately after storage treatment and stored at that temperature for 3 weeks before analyses. The frozen samples were cooked in a steam kettle for 20 minutes in boiling water before blend- ing. Screens (30 mesh) were eSpecially made to fit into the weighing pan of a Mettler balance. Titratable acidity was obtained from samples frozen at minus 20°F immediately after storage treatment and stored at that temperature until analyses 4 weeks later. The frozen samples were thawed, blended, heated to boiling, cooled and titrated to pH 8.1 with 0.1 N sodium hydroxide. 25 A Beckmann Zeromatic pH meter with combination electrode and thermocompensator was used. The pH values at the beginning of titrations were also recorded. The titratable acidity is expressed as grams of hydrated citric acid per 100 grams fresh material. After storage the samples were wiped dry before weighing to the nearest 0.5 grams. The difference in weight before and after treatments is expressed as a per cent of the original fresh weight. In the experiment Specifically designated to study dry weights, sections of Spears equivalent to those used in the tenderometer were Split lengthwise; one section was used for fiber analyses by the blendor method, and the other sec— tion used for dry weight estimations and crude fiber measure— ments by the official method. Material was initially dried as for fiber analyses. Aliquots were further dried at 135°C for 2 hours to estimate dry weights. In the experiment in 1961 entire Spears were Split longitudinally and one—half of each Spear used for fiber analyses and the other half for dry weight estimations. No critical organoleptic evaluation of the aSparaguS was made except after cooking. The appearance of the raw asparagus was cursorily checked after treatment and differ- ences between the controls and treated material noted. Most of the quality evaluations made in this thesis are based on completely objective measurements. 26 ASparagus of the Viking variety was used for the 1961 and 1962 tests. It was harvested by cutting every 2 to 3 days, depending on the growing conditions. The material was then sorted and trimmed to lengths of from 5 to 7 inches with a minimum diameter just below the tip of 1/4 inch. ,The asparagus was washed in cool running water to remove the sand which might have damaged the tenderometer or refractom— eter, and usually stored overnight at 32°F with the butts in water before modified atmosphere storage. In 1963 the asparagus was mainly the Martha Washing- ton variety and was harvested by field snapping. Spears were washed in cool running water, wiped dry, weighed and stored immediately in the designated modified atmospheres. Spears less than 1/4 inch in diameter and those over-mature or injured were discarded at the time of washing. Other aSparagus was purchased from a market gardener. It was the Viking variety and was harvested by cutting. This material was handled as above except it was not trimmed but was placed in storage as cut in harvesting. Statistical analyses were performed by standard analysis of variance procedures (Snedecor, 1956). Trend comparisons were usually used and Duncan's multiple range test (Duncan, 1955) at the 5% probability level when it was required. The comparisons were broken down to single de- grees of freedom only if there was a possibility of signif- icance at the 5% level and only significant comparisons are 27 reported for other than main effects. The mean squares for the analyses of variance are reported in the Appendix and the tables are given numbers prefixed by the letter A. Significance is denoted by * for Significance at the 5% level, and ** at the 1% level. All values in figures and tables are means. The number of items represented by the mean may be deduced from the text or the analysis of variance tables. RESULTS Water Induction and Tenderness1 It was postulated by Franklin g£_31. (1960) that one of the ways in which modified atmospheres might tender- ize aSparagus was directly through uptake of water. This water supposedly could result in firm, turgid tissue which would shear or cut more easily. Morse (1917) showed that uptake of water resulted in more brittle aSparaguS although the fiber content increased in storage. Attempts have been made to measure the contribution of water content to the tenderness of aSparagus with negative results (Franklin gt 31., 1961). These failures were attributed to poor sampling technique and over-mature asparagus. This experiment was designed to remedy these inadequacies. Methods and Materials Individual bunches of similar aSparagus were wrapped in cheesecloth and submerged in water in a cooking retort. The retort was evacuated to approximately 25 inches of 1A Short review of the literature containing the most pertinent references and the immediate aim are pre- sented before each experiment. 28 29 mercury and vacuum was maintained for periods of 20, 40, and 60 minutes. There were 6 samples per treatment and 2 replicates representing harvest dates of May 17 and 20, 1962. The experiment was designed as a randomized complete block with the variation in the 6 samples being used as sampling error. Resu1ts and Discussion A summary of the analyses of variance is given in Table A—1,1 and the means are presented in Table 1. The difference in tenderness between 0 time and the others was significant, with no difference in the other com- parisons. It was not realistic to compare the gains in weight for 0 time, necessarily 0, with the gains in weight for the other time intervals. This statistical comparison was not made. The gain in weight for the 20—minute treatment was significantly less than for the 40— and 60—minute treatments. There was no difference between the 40- and 60—minute treat- ments. After these water induction treatments, areas of a dull, glazed, water—soaked appearance were prominent on the surface of the spears, suggesting water congestion as de- scribed by Johnson (1947). 1For all tables the prefix A indicates Appendix, without prefix indicates text. 30 Table 1. The effects upon aSparagus of water induction by a vacuum of 25 inches of mercury for periods of 20, 40, and 60 minutes w Gain in Per Cent Soluble Periods Tenderometer Weight a Solids (min.) (p51) (% fr. wt.) ' (correct.)° 0 155.3’ ----- 6.09 6.09 20 146.7 11.72 4.96 5.52 40 147.4 15.53 4.80 5.54 60 145.0 14.85 4.78 5.50 20 + 40 + 60 146.5 ————— C 4.85 5.52 40 + 60 146.4 15.09 4.79 5.51 'fi aPercent of original fresh weight and is thus designated in all tables.'v“ bWithout designation means values not corrected for change in water content; such designation 15 used in all figures and tables. ' cThis value not needed as no comparison made. The uptake of water decreased the tenderometer read- ings, implying an increase in tenderness of the asparagus. This increase in tenderness, however, was not marked and probably would not be noticed by the consumer, especially because there was considerable variation within the samples. Although Brennan (1958) was unable to find such differences as reported here, his basis for assuming flaccid or turgid state was previous storage condition. 31 The increase in tenderness due to water uptake by vacuum treatment was not nearly as great as in aSparaguS treated with modified atmosphere. But the uptake of water was much less, amounting to 12 to 15% as compared to 20 to 30% under a modified atmOSphere of 15% oxygen and 15% carbon dioxide imposed at 35°F for 1 week (Franklin g£_gl., 1960, 1961). The added water under vacuum probably replaced the gases in the intercellular Spaces to a great extent, while under the modified atmOSpheres the water was likely taken into the cells. The effect of the location of the water upon relative tenderness, or its effect upon tenderness after cooking is not known. It is likely that this water would have little effect after cooking because there would be excess water present in the cooking process. The mean of the readings for soluble solids for 0 time, not corrected and corrected for water uptake, was Significantly larger than the means of the other times. The other comparisons were not significant. However, these dif- ferences have little meaning here except implying dilution of the soluble materials. It is not likely that physiolog- ical differences of any importance could have occurred in the short times of treatment. The differences that remained after correction for gain in weight only signify that con— siderable leaching of soluble materials took place during the treatments. 32 Temperature and Atmosphere All of the work done to date in these investigations of the effects of carbon dioxide plus water (Franklin.g£_gl., 1960, 1961) was at a storage temperature of 35°F. If some of the tenderizing effect of modified atmOSpheres is through metabolic changes, temperature could be expected to have an effect. Scott and Kramer (1949) have Shown that water up— take or loss in aSparagus is affected by temperature. The fiber content can also be expected to increase at higher temperatures (Bisson g£_gl,, 1926; Morse, 1917), although decreases have also been recorded (Carolus §£_31., 1953; Scott and Kramer, 1949). The latter decreases, however, were measured using the blendor method of analysis (Smith and Kramer, 1947) and were not, therefore, measurements of crude fiber. Methods and Materials Temperatures of 35, 45, and 55°F, covering the range frequently encountered in commercial storages, were selected. Temperature cabinets within a room at 35°F were used for the 45 and 55°F conditions. Unfortunately there was insufficient Space in the cabinets for 2 storage chambers; thus the con~ trol lots were necessarily in the open air. Sphagnum moss, saturated in water, was used in the chambers and cabinets to increase the relative humidity (RH). The RH in the chambers 33 was probably over 90%, while that of the air in the cabinets was considerably less. Some measure of the difference in RH was obtained by placing cans containing 2 inches of water in each treatment of the first replicate (Table 2). The atmos— pheres were 15% oxygen and 15% carbon dioxide (15-15). Table 2. Per cent water loss from open cans over a period of a week at 3 temperatures and 2 atmospheres Temperatures (0F) AtmOSpheres 35 g 45 55 Air 2.3 12.1 15.4 15-15 1.6 2.1 2.9 There were 2 replicates representing harvest dates of May 7 and 17, 1962. The storage period was 1 week. Four samples were selected per treatment. The spears of one sam- ple were cut to a length of 14 cm for measurements of in- crease in length. Because this aSparagus gained more weight, leading to the possibility of an abnormal distribution, only 3 samples were used in the analyses of variance. The exper— iment was set up as a split plot with temperatures the main plot and atmOSpheres the sub-plot. There was no sampling error for the analysis of increases in length because there was only 1 sample per treatment. 34 Results and Discussion The variability between replicates (harvest dates) reflects the growing conditions prior to harvest (Table 3). Table 3. Temperature and precipitation the day before harvest , Harvest Max. Temp. Min. Temp. YPrecip. Replicate date (°F) (°F) (inches) 1 May 7, 1962 66 37 Trace 2 May 17, 1962 87 61 Nil. The tenderometer readings were higher and the gains in weight lower in the second replicate (Table 5). With essentially no rainfall it is assumed that this was due to the different temperatures during growth. A summary of the analyses of variance is presented in Table A-2. The interactions are given in Table 4 and the overall means in Table 5. Tenderometer Overall, the tenderometer readings increased with temperature as Scott and Kramer (1949) had found. But, as the Significant interaction Shows, there was little effect of the temperatures in air, and the only large difference was at 35°F under the modified atmosphere. There was no differ- ence between readings at 45 and 55°F. The readings at the higher temperatures were only slightly lower under the modified atmOSpheres. Table 4. The effects upon aSparagus of storage forol week with the butts in water at 35, 45, and 55 F in air and 15% oxygen and carbon dioxide. Temperatures (°F) Atmospheres 35 45 + 55 Tenderometer Air 146.0 150.2 (psi) 15-15 118.8 146.6 Gain in weight Air 7.65 13.38 (% fr. wt.) 15-15 21.80 18.09 Increase in Air 1.18 2.00 length (cm) 15-15 2.44 2 27 35 45 55 pH Air 6 10 5 82 5 71 15-15 6 81 6 57 6 27 Fiber Air 0 0197 0 0177 0.0320 (% fr. wt., 15-15 0.0157 0 0137 0.0273 correct.) Gain in Weight As Franklin g£_gl, (1960, 1961) found, the gains in weight appeared to be the inverse of the tenderometer read- ings. The asparagus with the lowest tenderometer readings had the higheSt gains in weight when the effects of the atmOSphere and air storage are compared. The air-stored 36 aSparagus gained approximately one-half that of the atmos— phere-treated aSparagus with a corresponding reduction in tenderometer reading from 148.8 to 137.3 psi. Although the aSparagus stored at 45 and 55° gained more weight (took up more water) the tenderometer readings were higher than at 35°. Thus, in this case, the water could not have had a direct effect on tenderness. Possibly the development of fiber at the higher temperatures nullified any effect of water uptake. The interaction of air vs. 15-15 x 35 vs. 45 + 55 was significant. At 35° almost 3 times as much water uptake occurred under the 15-15 atmOSphere as in air. In air, the water uptake at the 2 higher temperatures combined was ap— proximately twice as great at 35°, although the gain at 45 + 55, under the 15-15 atmOSphere was considerably less (approx- imately 4%) than the gain at 35°F. Assuming more loss by transpiration in air at the higher temperatures (see Table 2), the gain at 45 and 55° may have been up to 3 times that at 35°. Although the RH under the modified atmosphere was con— siderably closer at the different temperatures, it is likely that the gains in weight, after correction for somewhat greater tranSpiration at the higher temperatures, were at 1east that at 35°. 37 .mnowuomnouofi on» How v oHan uooncH .o:am> oawufia o>m£ memos omonp mcofiuomwoucfi endowmacwflm mum: moon“ omamoomo .uonfim How ooN>Hmcm opmowaaow oco >Hcon .ommuOPm owowon mosHm> v mo cmozm uuuuuu mo.m 00.0 we.m oe.mH o.mea new: osmo.o oo.a no.6 me.m oo.aa e.~ma m .oom ...... oa.m no.6 mm.m oa.oH s.ema a .ooa goo: H nopm< owao.o mm.m mm.o os.m mm.0H n.5ma manna ammo.o mu.a mw.m om.m ov.HH w.wva uw< mononamosp< IIIIII va.m no.0 om.m <5.ma m.wea mm + we homo.o om.m Ho.m Hm.m ow.ma w.oma mm undo.o mo.H mo.o ov.m oo.ma w.mva we undo.o Hw.a mm.c ew.m mo.va ov.mma mm Amov unnumuomsofi nnnnnn nun: oa.o no.0 nunuu «.mmu new: owemo.o ----_ ma.o ow.m ..... o.oma m .oom ...... ---- om.o mm.o ----- w.mma H .oom Hmcamduo m . . A.uoouuoo AEov mm A.poouuoov A.u3.uw RV Awmmv ..»3 .HM $v newcoq GM moflaow vnwflog wouoeo o a o a wonfim owmouocH uhwwdwww cw comm woocoh ‘- oUHXOHU conumo $md mafia com>xo $ma one new ad mo mm one no mm pm noun: :4 wagon 6:» Eva: xooz H wow ommuoum mo mswmumamm com: mmoomwo onH .m oHan 38 The increase in water uptake in air as caused by higher temperatures was similar to that obtained by Scott and Kramer (1949), and suggests water uptake was affected by reSpiration rate. However, under the 15-15 atmOSphere, res- piration induced water uptake did not occur as the uptake was independent of temperature. Although carbon dioxide may inhibit reSpiration in asparagus (Thornton, 1933a), such inhibition, if it occurred had no effect on water uptake. Per Cent Soluble Solids (Corrected) The trends of decrease of soluble solids with time and with increasing temperature may be attributed to respir- atory losses. The differences were not statistically sig- nificant, probably due to lack of precision in the experimen~ tal design. The per cent soluble solids, after correction for water uptake, under the 15-15 atmosphere was greater than in asparagus stored in air. The readings of soluble solids were corrected for gains in weight with the assumption that all the gains were due to water uptake. The readings as measured directly by the refractometer only furnish proof that the gains in weight are by water uptake alone, that is, lower per cent soluble solids with higher water uptake (Franklinwg£_gl., 1960, 1961). But, if adequate correction is made for water uptake, the per cent soluble solids may yield information as to hydrolysis or other catabolic reactions causing higher 39 soluble solids. This correction could easily be misleading. If carbon dioxide is inhibiting respiration to any extent the per cent soluble solids under atmOSpheres containing carbon dioxide would be higher for this reason alone, than a normal atmosphere containing 20% oxygen and no carbon dioxide, after comparative storage conditions. This is true only if there is no water uptake. The greater water uptake under modified atmospheres masks the respiration effect if one occurs. The per cent soluble solids, pH and fiber estima- tions were made from that section of the Spears used in the tenderometer, that is, from just below the tip to 4 inches down the stem. The gains in weight (water uptake) were calculated from the weights of the whole Spears. Only if it can be assumed that the 4-inch section is an average sec— tion can the correction be justified. Downes §£_gl, (1962) and Culpepper and Moon (1935) showed that the apical sections of asparagus Spears are somewhat lower in water content, that is, higher in total solids and dry matter than the basal por- tions. Probably the intermediate 4—inch sections used in these tests represent average portions of the Spears. A mechanism for the increased uptake of water may also be suggested on the basis of the values for per cent soluble solids. The Significantly higher corrected per cent soluble solids under the modified atmospheres indicates the development of a higher diffusion pressure deficit (DPD). 40 g The pH values for the asparagus stored in the 15—15 atmosphere were significantly higher than in air. The dif— ferences in pH between temperatures, and the interaction of air vs. 15-15 x 35 vs. 45 + 55 were significant. The pH of the asparagus from air and the 15-15 atmOSphere decreased with the increase in temperature (Table 4), the decreases in the modified atmOSphere being slightly greater. These de- creases were the result of the normal aging processes in stored asparagus (Schweigart and Kellner, 1938) which were accelerated at the higher temperatures. At these higher temperatures the aging processes overcame the effect of the 15-15 atmOSphere, evident at 35°F where aging was retarded by low temperature. Increase in Length The interaction of air vs. 15-15 x 35 vs. 45 + 55 was significant. In air the increase in length at the higher temperatures was considerably greater than at 35°F; under the 15—15 atmosphere the increase in length was con— siderably less at the higher temperatures than at 35°. These results bear a remarkable similarity to those for water up— take. It would seem that the transpirational losses at the higher temperatures were important not only in gains in weight (effective or net water uptake) but also in ex- tension growth. 41 Per Cent Fiber (Corrected) The fiber content at 55°F was greater than at 35 or 45°F suggesting the results of Bisson gt_31. (1926) and Morse (1917) for crude fiber, although the fiber content was slightly less at 45 than at 35°F. There was also a decrease in fiber under the modified atmOSphere as Carolus g£_gl. (1953) had found. This decrease was consistent at the 3 temperatures, although the fiber content at 45° under both air and modified atmOSphere was less than at 35°F. The average fiber content decreased with time in storage as has been reported before by Scott and Kramer (1949). However, here the overall loss may be attributed to the effect of the modified atmOSphere. Scott and Kramer (1949) believed that the blendor method of fiber analysis was better correlated to organolep- tic quality than the official method, that is, gave a better indication of fibrousness. Although the latter may be true, how much of the tenderizing effect is due to tissue or cel- lular breakdown is not known. Because the blendor method is based on physical separation, any factor changing the inter- cellular structure can be expected to decrease the yield of fiber. This intercellular breakdown is thought to occur under modified atmOSpheres (Franklin §£_31,, 1960). The relationship between fiber content and tender- ometer reading was relatively consistent (Table 5), with low fiber content at low tenderometer readings. At 45°, however, 42 despite the lower fiber content, the tenderometer readings were higher than at 35°F. N°-Benzyladenine and AtmOSphere The water uptake that occurs under modified atmos— pheres could be related to respiration directly through :active water uptake, or indirectly by passive uptake through reactions causing changes in osmotic concentration. Dedolph §£_gl. (1961) and Tuli and Wittwer (1964) have shown that N°-benzyladenine (N°-BA)1 retards the res— piration of aSparagus and thereby, elongation and weight losses. Phytokinins haVe also been shown to retard protein breakdown or possibly increase protein synthesis in treated tissue (Sagiura §£_gl., 1962; Osborne, 1962). Fife and Frampton (1935) and Fife and Ferguson (1941) attributed the pH increase in sugar beet leaf tissue treated with carbon dioxide, to reactions beginning with the breakdown of pol— ypeptides or deamidation of amino acids. Possibly N°-BA could have an effect on these breakdown reactions. This experiment was designed to determine the inter- relationships of water, modified atmOSphere, and N°~BA on asparagus stored at 35°F. 1Supplied as Verdan, courtesy of the Shell Oil Company. 43 Methods and Materials There were 2 replicates representing harvest dates of May 21 and 30, 1962. Five atmOSpheres were used: air; 20% oxygen plus 0% carbon dioxide; 15% oxygen plus 0% carbon dioxide; 15% oxygen plus 15% carbon dioxide; and 15% oxygen plus 30% carbon dioxide. Asparagus was stored with and with— out the butts in water, and with and without N°~BA treatment. The N°~BA was applied as a 60-second dip in a 10 ppm solu— tion before storage. The asparagus designated as non- treated was dipped in water for the same length of time. The storage duration was 8 days at a temperature of 35°F. The experiment was designed as a Split plot with the atmos- pheres as the main plot and the other treatments in a facto— rial arrangement as the sub-plots. Results and Discussion A summary of the analyses of variance is presented in Table A—3. The significant interactions are given in Tables 6 and 7 and the overall means in Table 8. The two control atmOSpheres, air and 20—0 are grouped as controls, and the 3 atmospheres of 15-0, 15-15, and 15-30 as modified atmospheres. Tenderometer The interactions of water x controls vs. modified atmOSpheres and water x 15-0 vs. 15-15 + 15-30 were signif- icant. 44 Although atmOSpheres of 15 and 30% carbon dioxide tenderized aSparagus when no water was present, the effect was enhanced with water. Even the aSparagus in air and the 20-0 atmosphere with water was somewhat more tender than without water. This further substantiates the hypothesis postulated previously (Franklin g£_31., 1960, 1961; also Table 1) that water alone could cause increased tenderness of aSparagus. In this respect water uptake again was related to tenderometer readings (see changes in weight). The effect of the 15-0 atmosphere was similar to that of the controls, partially confirming the findings of Franklin g£_gl. (1961) that oxygen concentrations less than that of normal air have little effect on tenderness of aSparagus. The N°~BA had no effect on tenderness. Changes in Weight The interactions of water x controls vs. modified atmOSpheres, water x 15-15 vs. 15-30, and water x 15-0 vs. 15-15 + 15-30 were significant. In all cases when an atmOSphere containing carbon dioxide was employed the asparagus gained approximately twice as much weight when water was present, and lost Slight— ly less when water was absent, than the controls or the 15-0 atmosphere. There was considerably less gain in weight when water was present and Slightly more loss when water was 45 Table 6. The effects upon aSparagus of storage for 8 days at 35°F under modified atmospheres containing carbon dioxide superimposed upon treatments of water and N -benzyladenine Tenderometer (psi) a Modifiedb Controls AtmOSphere 15-0 15-15 + 15-30 + Water 148.1 129.1 149.0 119.2 — Water 153.0 148.2 152.0 146.2 Change in Weight (% of fr. wt.) 15-15 Modified + Controls Atmosphere 15-0 15-30 15-15 15-30 + Water 8.40 14.83 7.63 18.87 23.67 14.40 - Water -3.77 -3.08 —3.26 -2.99 -2.71 —3.16 pH Immediately After Storage Air 20—0 15-0 15-15 + 15-30 15-15 15-30 + Water 5.94 6.00 5.97 6.72 6.77 6.67 — Water 5.90 5.82 5.80 6.39 6.26 6.60 + N°-BA° — N°~BA + Water 6.12 6.17 — Water 6.01 5.97 I ,Per Cent Soluble Solids (Corrected) + Water - Water 6 6 6 6 AtmOSpheres N -BA - N -BA + N ~BA — N ~BA 15—0 4.62 4.74 5.02 4.89 15-0 + 15-30 5.56 5.40 5.25 5.52 aControls designates air + 20-0. b . . . Mod1f1ed atmOSpheres; deS1gnates 15-0 + 15—15-t15—30. cN°~benzyladenine at 10 ppm. 46 absent, in the 15—30 than the 15-15 atmosphere. In this case possibly the carbon dioxide concentration was high enough to damage the tissues or alter the permeability of the membranes (Glinka and Reinhold, 1962). There was essen- tially no difference in the 3 atmospheres free of carbon dioxide, that is, air, 20-0, and 15-0. It should be emphasized that the mean values for atmOSpheric treatments include treatments with and without the butts in water so that the losses in weight when water was absent tended to nullify the increases when water was present. Obviously water was necessary for gain in weight through water uptake. The N6 —BA had no effect on changes in weight. It is evident from this experiment that carbon dioxide enhances gain in weight when water is present for free uptake. The optimum concentration of carbon dioxide was less than 30%, and from previous work, Franklin g£_gl. (1960), may be even less than 15%. There is also the impli- cation that carbon dioxide may aid in preventing water loss when water is not available for uptake. However, the small differences observed might be partly due to some non—photo- synthetic carbon dioxide fixation (Allentoff §£_gl., 1954), or a decrease in respiration caused by carbon dioxide (Thornton, 1933a). Table 7. The effects upon asparagus of storage for 8 days at 35°F under modified atmOSpheres containing carbon dioxide superimposed upon treatments of water and N9-benzyladenine Per Cent Soluble Solids (Corrected) AtmOSphere + N°-BAa - N°~BA Air 4.89 4.86 20—0 4.70 5.04 Increase in Length (cm) Controls AtmosPhereC 15-0 15-15 + 15-30 + Water 1.05 2.05 1.18 2.52 - Water 0.40 0.56 0.39 0.64 Fiber (% of fr. wt., Corrected) _ Modified A1r 20-0 Controls. AtmOSphere + Water 0.0172 0.0092 0.0132 0.0078 - Water 0.0066 0.0082 0.0074 0.0108 pH After Storage at -20°F 15—15 15-15 + + g - 15-0 15-301_ 15-0 15~30 + Water 6.57 6.70 _ + N9—BA 6.57 6.51 — Water 6.57 6.49 - N9-BA 6.57 6-67 a 6 . ' N -benzyladen1ne at 10 ppm. °Controls designates air plus 20-0. cDesignates 15-0 + 15-15 + 15-30. 48 Per Cent Soluble Solids (Corrected) The overall per cent soluble solids decreased with time, undoubtedly due to reSpiratory losses of substrate. The difference in soluble solids between replicates can be attributed to the differences of the original samples, which were consistent throughout the experiment and are reflected in the replicate means. It must be stressed here that these readings were corrected for weight changes so that a Significant difference between water treatments would not occur unless some factor other than actual water content is having some influence. The interaction of water x N°-BA x 15-0 vs. 15-15 plus 15-30 was significant. A higher per cent soluble solids resulted from the 2 atmOSpheres containing carbon dioxide compared to that containing only 15% oxygen and no carbon dioxide. When water was present and the treatment of N°-BA imposed the per cent soluble solids was lower under the 15-0 atmOSphere than in the same atmosphere without N°-BA, while under the 15-15 plus 15-30 atmOSpheres the per cent sol- uble solids was higher with N°—BA than without. Without water these differences were reversed. This could be inter- preted that in the presence of water N°-BA caused either more breakdown or lower respiratory activity, which resulted in higher soluble solids under high concentrations of carbon dioxide. When water was absent, N°-BA had the opposite effect. 49 #009 am UoMOHm one moomu pm nououm mammflp co woumefiumo >pflvfiom manmumnuflw 0cm m0 .EQQ.0H pm ocficowma>wconn0z .ommuoum whomon monam> m mo :moEu .UoN>Hm:m Hfipns ounpmwomsop n .psmwoz Emowm mo madam 00H won @000 oawufio oopmu0>s mo mamum .>pflvflom oflnmumuufifim 00.0 00HH.0 0000.0 wo.H 00.0 00.0 00.N v.mva :00: 00.0 0HHH.0 oaa0.0 00.0 00.0 00.0 mm.m m.oea m .000 00.0 0HmH.0 mu00.0 RH.H 00.0 0m.m em.m 0.0ea a .000 0000 w uopm< 00.0 00HH.0 0000.0 00.H 00.0 ma.0 00.m 0.00H a 50.0 00HH.0 0000.0 ma.H 00.0 00.0 mm.m w.HOH + 00HH.0 0000.0 00.0 00.0 NH.0 00.0: H.00H : 00.0 mNHH.0 00H0.0 >0.H 0H.0 00.0 mH.mH 0.0MH + noun: H0.0 0NNH.0 0000.0 pm.H 00.0 00.0 H0.m H.00H 0mn0H 00.0 m>0H.0 N000.0 00.H 00.0 00.0 0m.u m.ama 0HI0H 00.0 ~0HH.0 00H0.0 00.0 00.0 mm.v m0.H 0.00H 0:0H 00.0 00NH.0 0000.0 00.0 00.0 00.0 00.0 0.00H 0n0m H0.0 meHH.0 0HH0.0 00.0 m0.0 00.0 00.0 H.HOH wfi< monoanOEu< 00.0 00vH.0 N000.0 III: 00.0 00.0 nun: 0.nva :00: 00.0 meaa.0 0000.0 ---- 00.0 mm.m ---- 0.004 m .000 00.0 00ea.0 0000.0 ---- 04.0 00.0 ---- 0.004 a .000 Handmauo o . . owNHOum A.poouuoov A.poouuoo A800 mm A.poouuoov A.u3.wm $0 Awmmv woum< >pfinflo< .p3.uw $0 camcoa mvflaow unwfloz wopoeouo . o a 0 ca 1 A10 a. 0:. 000: 5 000000 «0 0.0.0 000000 0:00.. ommouucn ocficovma>nconu 2 can woumS Mo mucoswmouu Goa: nomomswuomnm oofixowv conumo mcfindmucoo monoma :mosum Uofiwfiuos noon: m00m pm m>mc 0 wow ommHOum mo mammummmm com: muoowwo 028 .w oHan 50 The interaction of N°~BA x air vs. 20-0 was signif- icant. All of this effect is due to the difference in 20-0 plus N°~BA and 20-0 less N°-BA, the higher per cent soluble solids when N°—BA was absent. A higher per cent soluble solids because of lowered respiration rate, as caused by N°~BA, does not apply here as it may in the previous inter- action. Possibly some effect of water stress has been active in these interactions as water stress is known to result in abnormal metabolism (Chen ££_gl., 1964; Petrie and Wood, 1938; Spoehr, 1919; Spoehr and Milner, 1939; Wager, 1954). The interactions of N°-BA and water stress suggest fields for further investigation. In any case, the per cent soluble solids generally was higher in atmosphere containing carbon dioxide than in an atmOSphere of similar oxygen concentration without carbon dioxide, or in an atmosphere of air. It was impossible to ascertain reSpiratory losses during this investigation and thereby correct for their effect on soluble solids. The higher per cent soluble solids under the modified atmOSpheres were due to less loss or an increase with atmospheric treatment as compared to the controls. AtmOSpheres containing carbon dioxide are known to depress respiration of aSparagus (Thornton, 1933a) so as to retard losses. According to the work of Fife and Framp— ton (1935) some substrate breakdown could also enter into this increase of soluble solids. This increase is not 51 considered as due to hydrolysis of starch as might occur in potato tubers under modified atmospheres (Denny and Thornton, 1941) as there is little starch in asparagus Spears (Jacobs, 1951). 25 The pH data were statistically analyzed as hydrogen ion concentration and the differences which appear in Tables 6 and 7 were considerably larger when expressed as hydrogen ion concentration. The comparison of controls vs.modified atmOSpheres was significant showing the general effect of the modified atmOSpheres in increasing pH. No consideration need be given to the other main effects because interactions contain- ing them were significant. The differences contained in the interaction of water x air vs. 20—0 were small, although larger as hydrogen ion concentration, and may indicate sampling error. The sig- nificance was due to the lower pH in the 20-0 atmOSphere when the butts of the aSparagus were not in water. The other significant interactions of water x atmos— pheres generally indicate the necessity for the presence of water for increases in pH under a low concentration (15%) of carbon dioxide. At the 30% level of carbon dioxide the pres- ence of water was unnecessary. 52 The failure of the pH to increase under the 15-15 and 20—0 atmospheres might be the result of some water stress when water was not freely available. Such stress has been noted as causing changes or reversals of reactions in plant tissues (Chen §£_31., 1964; Petrie and Wood, 1938; Spoehr, 1919; Spoehr and Milner, 1939; Wager, 1954). How— ever a greater stress would be expected in air than in the 20—0 atmOSphere because of the higher tranSpiration rate in air, yet the pH was higher in the air treatment. The pos— tulated water stress effect on pH will be discussed further in the Carbowax experiments (see below). The 30% carbon dioxide concentration was evidently high enough to overcome any possible effect of water stress because the pH was essentially the same whether water was present or not. The differences in the interaction of water x N°—BA were again small. The differences in pH are so slight as to make a valid physiological interpretation impossible, al- though the statistical inference is plain. Increase in Length The increase in length appears to be a reflection of uptake or loss of water as measured by a gain or loss of weight. The interactions of water x controls vs. modified atmOSpheres, and water x 15—0 vs. 15—15 plus 15—30 were 53 Significant. Under the modified atmospheres when water was present, there was approximately twice as much elongation as in the controls; when water was not present, although there was still more elongation under the modified atmospheres than in the atmospheres containing only oxygen, the differ— ence was substantially less. There is the suggestion from Tables 6 and 7 that this slightly greater elongation may have been due to less water loss rather than a larger gain. Extension growth occurred although there was some weight loss in the lots without water. Fiber (Per Cent of Fresh Weight, Cgrrected) The fiber content of the second replicate was higher than that of the first replicate. The tenderometer readings of the second replicate were also somewhat higher, suggest- ing the relationship of fiber to tenderometer readings found by Jenkins and Lee (1940). The interaction of water x air vs. 20—0 and the interaction of water x controls vs. modified atmospheres were significant. The data in Table 7, representing these interactions tend to Show that fiber develops in asparagus stored with the butts in water in an atmosphere of approximately 20% oxygen, while under the modified atmOSpheres containing car- bon dioxide, the reverse was true. In the controls the re- sults are similar to those of Brennan (1958) who found a 54 more mature degree of development of the pericyclic fiber ring when water was present. Under the modified atmospheres the total per cent fiber was higher when water was absent. The average percentage of fiber did not decrease with storage in water as Wiley et al. (1956) found (Table 8). pH After Freezer Storage These analyses were performed on aSparagus quick frozen after treatment and stored at -20°F until analyzed. There was little difference among the pH readings. The pH of the untreated aSparagus, even without storage, increased until it was almost equal to that of the material that had been stored in the modified atmospheres. It had been noted that the pH of the juice expressed from asparagus, whether previously stored under modified atmospheres or not, increased in a few hours at room temperature so that the lots from asparagus treated with carbon dioxide became al- most indistinguishable on the basis of pH. Possibly this in- crease is due to the same reactions that cause increase under modified atmOSpheres, that is, the formation of ammonia. The significant interaction of water x 15-0 vs. 15-15 + 15-30 shows that a small part of the effect of the storage previously in modified atmospheres had been retained in the frozen material. The pH of the aSparagus stored in water under carbon dioxide had a pH approximately 0.2 units higher than that stored without water, while the aSparagus previously stored in the 15-0 atmosphere had the same pH 55 with and without water treatment. The effect of the N6 -BA treatments was small. In general the significant interaction of N°-BA x 15-0 vs. 15-15 + 15-30, like the similar significant interaction in the values for fresh aSparagus, really indicates little, because of the small differences in pH. Titratable Acidity After Freezer Storage (Corrected)7 Therewere no significant differences between the measurements of titratable acidity. These measurements reflected pH readings. AtmOSphere, Dry Matter, Fiber, Crude Fiber and Cooking Quality Previous results for per cent dry matter have been confusing, mostly weight losses after a week of storage be- ing reported but sometimes small gains in dry weight (Frank- lin g£_al., 1960). The blendor method of fiber analysis (Smith and Kramer, 1947) also had given results that were not clear (see previous experiment). This experiment was designed primarily to study dry matter and fiber changes in asparagus stored in modified atmospheres. It was hoped that the use of carefully selected samples before storage would overcome errors in sampling due to variations in aSparagus. A small post-storage cooking trial was also performed. 56 Methods and Materials Five carefully randomized and selected samples were used for each treatment before and after storage. There were 2 replicates representing harvest dates of May 22 and 28, 1962. Two atmospheres were used: 15% oxygen and 15% carbon dioxide (15-15), and a control in storage air. The duration of storage was 1 week at 35°F. Samples were se— lected before and after storage to provide evidence that the results previously reported were being duplicated here. These latter results were not analyzed statistically. Dry matter and per cent fiber and crude fiber were estimated as describediJI"General Methods and Materials.” For the cooking trial there were only 4 samples per treat- ment, and only after storage values were considered. The aSparagus was cooked for 6 minutes in boiling water. Gen— eral observations were made of the.color and flavor of the cooked aSparagus. Texture was meaSured with the tender— ometer. Results and Discussion A summary of the analyses of variance is presented in Table A—4, and the means and significant interaction are given in Table 9. 57 Table 9. The effects of atmospheres of air and 15% oxygen plus 15% carbon dioxide on the composition and tenderness of aSparagus stored at 35°F for 1 week with the butts in water Tender- Gain in Per Cent ometer Weight Soluble (psi) (% fr.wt.) pH Solids Originaia 146.2 ---- 6.05 5.67 After 1 week Air 153.8 7.02 6.00 4.50 15-15 120.2 19.80 6.80 4.50 Dry Tender- Matter Crude Fiber Fiber ometer (% fr.wt., (% fr. wt.) (% fr.wt., (cooked, correct.) _'-.. (correct.)correct:) psi) AtmOSpheres 1 Air 6.812 0.6902 0.7136 0.0180 107.2 15~15 6.737 0.6312 0.6871 0.0136 50.2 Before storage 7.201 0.7098 0.7098 0.0168 ————— After storage 6.346 0.6116 0.6910 0.0149 ----- Fiber (% fr. wt., correct.)b AtmOSpheres Before Storage After Storage Air 0.0133 0.0223 15-15 . 0.0203 0.0069 aMean of 4 values before storage. bSub-table represents the interaction of before storage vs. after storage x air vs. 15-15, significant at the 5% level. 58 Per Cent Dry Matter (Corrected) The percentages of dry matter not corrected for water uptake were considered of little value because they would merely measure dilution by water uptake and no other physiological reSponse (Franklin §£_21., 1960). It was assumed that all the gains in fresh weight were due to water uptake, and corrections were made accordingly. There were no significant differences among any of the sources of variation. The only large difference, al- though not statistically Significant, was between the per- centages of dry matter before and after storage. This dif- ference, if real, may only have been due to respiratory losses with time. From this it may be deduced that non-photosynthetic carbon dioxide fixation must play a very small or no part in the increases in weight under atmospheres containing carbon dioxide. There also appears to be no indication that the modified atmosphere depressed the respiration rate to a noticeable extent. However, retardation of reSpiration would be difficult to measure at 35°F because of the low basal rate occurring at this temperature. Crude Fiber (Official Method) and FIBE?_TBlendor Method) The interaction for fiber measured by the blendor method (corrected) of before storage vs. after storage x air vs. 15-15 was significant. Apparently the fiber content 59 increased during the week of storage in air, while in the 15-15 atmOSphere the fiber content decreased. These results are similar to those found by Carolus 3£_gl. (1953). The 2 means for before storage each represent 10 measurements; 5 for each of the 2 replicates, of supposedly similar aSpar- agus within each replicate. The 2 means should be Similar yet there is considerable discrepancy. An average value of the means (0.0168) might be more reliable. In any case, even using this value, there would appear to be a decrease in fiber or fibrous material under the 15-15 atmosphere. There was no indication that fiber decreased with time in storage as Scott and Kramer (1949) had found. The statistically significant differences in crude fiber (before vs. after storage x air vs. 15—15) disappeared after the values were corrected for water uptake. Crude fiber, although a complex of materials includ— ing cellulose, hemicellulose, lignin, and pentosans is not readily broken down by normal physiological processes in plants. Although cellulases have been at least tentatively identified in plant tissues (Kristiannson, 1950; Singh, Mathur and Mehta, 1938; Sterling, 1961), for the most part they are not considered as being active at low temperature. It appears then, that the major increase in tenderness of aSparagus that cannot be attributed to water content, is not due to breakdown of crude fiber. Therefore it must be asso- Ciated with some dissolution of intercellular materials. 60 This dissolution may have led to the lower fiber measurements by the blendor method, as reported here and by Carolus g£_gl. (1953) under modified atmOSphereS. Because this method relies on strictly physical separation less fiber may have been left on the screens after washing, subsequent to treat- ments with carbon dioxide. Cooking Trial If the tenderizing effect of the modified atmospheres is to be of any practical importance it must be capable of translation into terms of cooking quality. The tenderometer readings of the asparagus from the 15-15 treatment were significantly lower than those of the asparagus stored in air. The values of the aSparagus from the 15—15 treatment were approximately one—half that of the material stored in air. One of the theories of the tender- izing effect of modified atmOSpheres on raw aSparagus was that the firmness or turgidity of the carbon dioxide-treated asparagus resulted in lower tenderometer readings. This turgidity cannot be a factor in asparagus cooked in water because of the excess water present which would permeate the tissue. Therefore the tenderness in the aSparagus must have been due to some breakdown of fibrous materials or dissolving of cell-cementing substances. The treated aSparagus after cooking had a green color darker than fresh aSparaguS. This observation had 61 been made before by Thornton (1931) and Carolus ££_31. (1953). To two feminine observers this color was too dark to be char- acteristic of aSparagus. The main criticism of the carbon dioxide-treated aSparagus was lack of flavor. There was also the criticism that the treated aSpar- agus was too "mushy." All samples were cooked Simultaneous- ly for the same length of time. Somewhat lesser cooking time would have been adequate for the carbon dioxide-treated aSparaguS. The cooking water was an amber color at the end of the cooking period. Carolus g£_gl. (1953) also reported finding this color. There is the possibility that the ap- pearance of this pigment in the cooking water was related to the appearance of the dark green color of the cooked aspar— agus. General Results of Treatment Table 9 shows that the atmOSphere containing carbon dioxide had its usual effect, lower tenderometer readings, higher pH values and uptake of water than the control. The per cent soluble solids (as presented here not corrected for water uptake) in the treated and air—stored aSparagus was the same. If the readings were corrected for uptake of water the treated aSparagus would have a per cent soluble solids content approximately one-fifth higher. This is argument for hydrolysis or lower rate of respiration under carbon dioxide. 62 Oxygen and Carbon Dioxide Levels Franklin g£_gl. (1960) studied the effects upon aSparaguS stored with the butts in water of levels of 0, 5, 10, and 15% carbon dioxide and 5, 10, and 15% oxygen in a factorial arrangement. Oxygen levels lower than that of air decreased tenderometer readings and pH slightly but had lit- tle effect on gains in weight or per cent soluble solids. However, the same oxygen concentrations in a later exper- iment (Franklin g£_gl,, 1961) at the same concentrations of carbon dioxide, had little effect on any of the reSponse criteria studied. Increasing carbon dioxide levels was more effective than decreasing oxygen concentrations. Five per cent carbon dioxide resulted in slightly lower tenderometer readings, higher pH values and water uptake, and lower per cent sol- uble solids (not corrected for water uptake) than the con- trols. Carbon dioxide levels of 10 and 15% increased these effects with 15% causing the greatest change. A previous experiment (Tables 6, 7, and 8) estab— lished that 15 and 30% carbon dioxide gave almost similar results. Thirty per cent was less effective in tenderizing aSparagus and inducing water uptake, but more effective in causing higher pH and per cent soluble solids. Therefore, it was decided to investigate the effects of carbon dioxide concentrations somewhat lower than 15%. 63 Materials and Methods Samples of asparagus harvested on June 5 and 7, 1962 (2 replicates) were stored for 1 week at 35°F in air and at oxygen levels of 10 and 20% plus carbon dioxide levels of 0, 3, 6, 9, 12 and 15% in a factorial arrangement. Duplicate readings were taken for all responses other than gains in weight. Results and Discussion A summary of the analyses of variance is presented in Table A-5, and the results are given in Table 10 and Figure l. Tenderometer There was no response to oxygen levels. The compar- ison of air vs. all other treatments was significant. Be— cause "all other treatments" includes so many different com- binations of oxygen and carbon dioxide the comparison is of limited value except as an indication of the general effect of the modified atmOSpheres. A Significant linear response of tenderometer readings to carbon dioxide was obtained, the highest tenderometer reading being at the lowest carbon diox- ide level. The lowest tenderometer reading was 110.9 under 15% carbon dioxide, although the reading at 12%, 115.2 psi, was not much greater. .64 Table 10. The effects of various levels of oxygen and carbon dioxide in a factorial arrangement on aSparagus stored for 1 week at 35° F with the butts in water Per Cent Gain in Soluble Tenderometer Weight Solids (psi) (% fr.wt.) pH (correct.) Originala Rep. 1 145.0 ----- 6.25 5.30 Rep. 2 142.5 ----- 6.00 5.50 Mean 143.8 ----- 6.11 5.40 After 1 week Rep. 1 129.6 11.90 6.35 4.62 Rep. 2 132.8 13.81 6.32 4.93 Mean . 131.2 12.86 6.33 4.78 Atmospheres Air 147.0 4.75 6.00 4.61 All others 129.8 13.53 6.38 4.79 Oxygen (%) 10 128.8 14.05 6.39 4.82 20 131.0 13.01 6:36 4.76 Carbon dioxide (%) 0 144.6 5.45 6.04 4.66 3 141.8 8.05 6.16 4.55 6 137.4 12.82 6.42 4.67 9 129.2 17.22 6.64 4.69 12 115.2 18.80 6.77 5.12 15 110.9 18.82 6.85 5.04 aMean of 2 values before storage. 65 .wopms ea muons 0:» and: 000m «0 x003 A How concum mammummmm no 000x000 eonueo mo mao>oH ofiuonomosum mo muoomwo 009 .H ousmwm 66 ..9 o. .01.... -1 o. . g 3 4‘3 30" " O .2 o 8”. I z o O n. O .1 -< O: . .... ° ‘8 8:“!: o 0 do; . ...x .3. -+ C . 0 . . 3‘ 8 b O E *0 .30.... 1 : gx : "O 0.:xho .1 . o ’13: l l 1 1 _l_ l 1 J _1 l L l l l l 1 1k acfioqvnq—om ooooogo 150000060000 2233‘2—9 Hd (god) Josomoupuu ooa‘o. l ..x.. q . ...O/:. 4‘2 .01.. .- C I! K .0 . . ..x. ... dao o .x. . —4 C O D b O O . o D 0. HQ. .001 . .— C O 0 o. C \ l .9/* 0 4n 0 ”00 -‘ ...“... ~o ... -4 ¥ 1 1 l l 1 1 J L 1 l 1 1 '4 V. «.I 0. e e «r. N q o n o o o no In no 0 v v «t v N "' ' (potoouoo) (woo and) ”nos .IQMOS W09 ”d mum «mg a! mo Figure 1 l5 l2 Por cont Carbon Oioxido Por cont Carbon Dioxid. 67 As Figure 1 shows the readings overlap considerably. Pos~ sibly 12% carbon dioxide would be almost as efficient as 15% in tenderizing aSparagus. Gain in Weight The comparisons of replicate 1 vs. replicate 2, and air vs. all other treatments were significant. .The differ- ence between replicates indicates the difference between the harvest dates and variability in sampling. The comparison of air vs. all other treatments includes too many atmOSpheric combinations to yield information as to a particular one. Most of the sums of squares for the effect of carbon dioxide was in the linear regression of carbon dioxide on gain in weight, although the quadratic regression was also significant. The latter effect was due to the levelling off of the curve at the 12 to 15% range of carbon dioxide. This curve would run parallel to a line that might be drawn to represent the tenderness of the asparagus as affected by carbon dioxide. That is, the reverse of tenderometer read- ings. Thus the postulated effect of gain in weight (water uptake) on tenderness of asparagus (Franklin g£_gl., 1960). The cubic regression was also significant. This implies that at a sufficiently high concentration of carbon dioxide the gains in weight would begin to decrease. This confirms the results of the previOus experiment where the gains in weight in 30% carbon dioxide were considerably less than in 15%. 68 The level of oxygen had no effect on the gains in weight. pg The comparison of air vs. others was significant, although again this comparison yields little information because the others includes so many different atmOSpheric combinations. The level of oxygen had no effect. The linear and quadratic regressions of carbon diox— ide level on pH were significant. As Figure 1 shows the effect could be considered linear up to 12% carbon dioxide and then the response became quadratic. The peak reSponse was again in the range of 12 to 15% carbon dioxide. Per Centw§oluble Solids (Corrected) The difference between replicates, which was present in the samples taken before storage, only indicates the variability in the aSparagus of the 2 harvest dates. There was no response of soluble solids to oxygen level. Inspection of Figure 1 explains why the linear-and cubic regressions of carbon dioxide level on soluble solids 'were significant. Carbon dioxide levels from 0 to 9% gave a linear response and little real effect within this range. However, at 12% carbon dioxide the per cent soluble solids increased, and then decreased again at 15% carbon dioxide to yield the cubic regression. 69 General Because an atmOSphere containing 12 to 15% carbon dioxide seemed most effective, an atmOSphere of 9% oxygen and 12% carbon dioxide was used in 1963. Twelve per cent was chosen instead of 15% because it would be more easily maintained in a large storage room. This level is also within the limits of atmosphere generators (Dewey and Pflug, 1963). Because any atmOSphere used on such a perishable crop as aSparagus must be imposed as soon as possible after harvest, this is of Special consideration. Too much dete— rioration would occur in the time necessary for the crop to "generate" its own atmosphere. The 9% oxygen was chosen becauSe ventilation could be used to control the atmOSphere and a level lower than that in air might have an effect additive to that of carbon dioxide (Franklin et al., 1960). 70 Pre-Storage Condition and Atmosphere Brief investigations (Franklin gt_gl., 1961) had shown that aSparagus that was not freshly harvested, al— though apparently in good saleable condition, was not favor- ably affected by storage in modified atmOSpheres. This experiment was designed to ascertain the effect of post- harvest aging upon the value of carbon dioxide as a tender- izing medium. Methods and Materials Freshly cut aSparagus was divided into samples of approximately 15 spears each. Two samples were analyzed immediately and 2 were put into storage at 35°F, one in air and the other in an atmOSphere of 15% oxygen plus 15% carbon dioxide (15-15). The remaining samples were placed in a storage at 60°F and 50% relative humidity (RH). On each of 4 consecutive days following harvest 4 samples were taken from the latter storage. Two of these samples were analyzed immediately to give a baseline value for that storage period, and the other-2 samples were placed in storage at 35°F, one in air and the other in the 15-15 atmOSphere as above. Each pair of samples was analyzed after a week. During the pre-storage period the asparagus was kept dry; the butts were placed in water for the week of storage. There were 2 replicates representing harvest dates of May 24 and June 1, 1962. 71 Results and Discussion Statistical analyses were not performed on this data because a meaningful overall analysis was impossible. The data are presented in Figure 2. Tenderometer The modified atmospheres had a tenderizing effect after all the pre-storage durations, but decreased in effect as the pre-storage duration was lengthened on the basis of final pressure readings. Actually the effect remained rela— tively constant, at least for the first 3 days of pre-stor- age, but the initial readings were so high after the pre- storage treatment (prior to storage at 35°F), that the aSparagus never recovered its initial tenderness. The great- est increase in tenderness, based on the pre-storage condi- tion, occurred after 1 day of pre-storage. After the week in air at 35°F, the aSparagus sub- sequent to pre—storage treatments of 0 and 1 days, became tougher; the aSparagus undergoing pre-storage treatments of 2, 3, and 4 days became more tender. The gains in weight, below; and the earlier experiment, Table 1., indicate that some of this effect may have been due to water uptake. The evidence presented in this experiment shows that carbon dioxide treatment plus water will tenderize old aspar- agus considerably although never restoring it to its original texture. For greatest effect the carbon dioxide must be ap— plied to fresh asparagus or to aSparagus within 1 day of harvest. Figure 2. 72 The influence upon aSparagus of pre-storage for 0, 1, 2, 3, and 4 days in air at 60°F and 50% relative humidity without water, on subsequent effects of storage for 1 week at 35°F in air and 15% oxygen plus 15% carbon dioxide with the butts in water. -—— After storage for 1 week in 15% oxygen plus 15% carbon dioxide (15-15). ....After storage for 1 week in air. °-1__ At end of pre—storage period. 73 Day. Tenderometer o —————————— ‘1. I ____________ .1 2 ----------- 4 3 ---------- :1 4 _________ 1 L 1 1 l 4 L J :20 :30 I40 :50 I60 no :60 psi Change in Fresh Weight 0 i: _____________ I f’ ------------ 2 E- ----------- 3 P ------------- 4 t' ————————————————— 11571 1 1 1 n. 1 1 1, 1 1 J, 1 J -l8 «5 -l2 -9 -6 -3 0 3 6 9 :2 l5 l8 2: Per cent pH 0 i" _________________ ' i' __________________ 2 i' _________________ 3 t2. ------------------- 4 i- __________ l l l l J l 1 l l l l l J 5.6 5.7 6.6 5.9 60 6| 6.2 63 6.4 65 66 6.7 6.8 pH Soluble Solids o -------------- ‘1 l ‘‘‘‘‘‘‘ '1 2 ------------ 1 3 ------------ 1! 4 ————————————————— I L, 1. 1 1 1, 1 1 1 11, 11 1 1‘ 1 J 3.2 3.4 3.6 3.6 4.0 42 4.4 4.6 4.6 so 52 5.4 56 58 Per cent Figure 2 74 Qhange in weight The gains in weight paralleled the increases in ten- derness. With the aSparagus having the pre-storage treat- ments of O and 1 day the modified atmOSpheres resulted in a much higher uptake of water than did air. With the aspar- agus from the 2, 3, and 4 days of pre-storage the effect of the atmOSpheres, although still greater than that of air, was less different with increasing storage time. The uptake in air after all pre-storage treatments was considerably greater than for O pre—storage time. The uptake of water under the modified atmosphere, beginning when the aSparagus was placed in the storage at 35°F, was similar for the pre—storage treatments of 1, 2, and 3 days and for the fresh asparagus with 0 time in pre- storage. The greatest uptake was after 4 days of pre-stor- age. The greater uptake after the pre-storage stress may be attributed to the water deficit of the tissues partially dried at 60°F and 50% RH. Despite these large overall increases in water up- take, the effective gain in weight, that is,above zero loss or gain, was never as much as for fresh aSparagus because of losses during pre-storage treatments. 2! The pH of the aSparagus decreased with increasing time in the pre-storage treatment. This general trend was noted by Schweigart and Kellner (1938). 75 The pH of all samples was greatly increased by stor- age in modified atmOSpheres and usually slightly decreased by storage in_air, regardless of the previous pre-storage treatment. The only exception to the slight decrease with storage in air was in the sample having 3 days of pre— storage. Except for the asparagus having the 4-day pre- storage treatment, there were similar increases in pH due to modified atmOSpheres after all pro-storage times. .HOWever, because the original pH at the end of the pre-storage peri— ods of l, 2, and 3 days was lower, the overall effect of the modified atmospheres plus pre-storage treatment was to pro- duce a lower pH than the asparagus with 0 time in pre—stor- age plus the modified atmOSphere. The pH of the aSparagus undergoing the 4-day pre-storage treatment did not follow the trend of decrease with pre-storage, and the increase in pH in the modified atmosphere was only approximately half that of the aSparagus from the other pre-storage treatments. This may have been due to excessive water stress. Previous— ly, Table 6, it was shown that the pH increase under modi- fied atmOSpheres was less when the asparagus was under water stress or in a semi~dry condition. 76 Per Qent Soluble Solids No attempt was made to correct these values for water uptake as was usually done. The relatively high tem- perature, 60°F, may have resulted in considerable respirato— ry losses which would make the corrected values less valid than those at 35°F. The values for per cent soluble solids were incon— sistent. The unexpected decrease in soluble solids after 1 day of pre-storage was probably because of a more rapid loss of sugars through respiration than of water through transpi- ration. According to Bisson gt_gl. (1926) the most rapid loss of sugars is during the first 24 hours after harvest. After this sudden initial change, the loss of water in the pre—storage treatments had the effect of increasing the per cent soluble solids even above the initial reading. By the fourth day the per cent soluble solids was again some- what lower than the original readings. Possibly this was because respiration began to play a larger part in weight loss than respiration. However, sampling error may have been considerable. ~After the 1 week of storage subsequent to 0 pre- storage, the average per cent soluble solids for the aSpar— agus from the modified atmosphere and air was the same. -Because there was a greater uptake of water under the mod- ified atmosphere this really implies a higher percentage of soluble solids under the modified atm05phere if correc— tion were made for water uptake. Generally, after all the 77 other prewtreatments plus the storage at 35°F, the per cent soluble solids was lower in the modified atmospheres than in air. The exception was asparagus that had undergone the pre- storage treatment for 1 day. The decreases of soluble solids with time in storage at 35°F can be attributed mainly to dilution through uptake of water, but some respiratory losses must have occurred too. Shelf Life and AtmOSpheres AtmOSpheres containing 15% carbon dioxide were effective in tenderizing both freshly harvested and stored aSparagus (Figure 2). This experiment was designed to dis— cover if the tenderizing effect was only transient or re- tained at higher storage temperatures after treatment. Methods and Materials There were 2 replicates representing harvest dates of May 7 and 17, 1962. The freshly harvested aSparagus was stored for 1 week, standing in water at 35°F. The atmos— phcres imposed were 15% oxygen plus 15% carbon dioxide (15-15), and a control in storage air. After storage for 1 week a sample from each atmosphere was analyzed. The remain- ing samples from each treatment were removed from the water at this time and placed in air at 60°F and 50% RH, chosen to simulate poor storage conditions for quality retention. Each day thereafter for 6 days, 1 sample from each of the 2 atmos— pheres was analyzed. 78 Results and Discussion Because respiratory losses of dry matter might have been considerable at the high temperature (60°F) no attempt was made to correct readings for water loss. In any case the interest is in relative change not absolute values. The statistical analyses were performed only on the values of the 6 days of post-storage treatment because it is the retention of the values already obtained through previ- ous storage in the atmOSpheres that is important. .In the figures the values at the end of the storage period at 35°F are given except for weight loss. It was impossible to represent the differences at 0 time adequately because the losses represent a percentage, while the other quality criteria are absolute measurements. The changes during the week in storage at 35°F are given in Table 11 to verify that previous results attri— butable to atmOSpheres were duplicated. Table 11 also shows the overall means for the 6 days of post-storage treatment for the atmOSpheres and replicates. The changes with time in the post-storage treatment are presented in Figure 3. A summary of the statistical analyses is given in Table A—6. Table 11. 79 The effects of atmospheres of air and 15% ox ygen plus 15% carbon dioxide on aSparagus stored at 35°F for 1 week with the butts in water, and subsequent effects of storage in air without water for 1 to 6 days at 60 F and 50% relative humidity After Storage for 1 Week at 35°F in AtmOSpheres of: Originala Air 15-15 Tenderometer (psi) 132.0 147.5 112.5 Gain in weight(% fr. wt.) ————— 6.85 21.20 pH 6.18 6.09 6.80 Per cent soluble solids 6.25 4.95 4.75 After Storage for l to 6 Days at 60°F and 50% Relative Humidity Replicates Atmospheres 1 2 Air 15—15 Tenderometer (psi) 138.0 162.0 159.7 140.3 Loss in weight (% fr.wt.) 16.24 14.13 15.95 14.42 pH 5.83 5.84 5.78 5.89 Per cent soluble solids 5.47 4.49 5.26 4.70 f aMean of 8 values, 4 for each replicate prior to storage. bMean of the 6 days of post-storage treatment. 80 .uowmz :M mausn 6:» spa: momm on 2663 H com AmH-mHv moaxomo nonaau end mafia cmmsxo $md one man GM oMMNOum o» uconvomDSm m>mo o o» H wow nova: psonufis mam ca xpfiowesn o>fiumaou Rom cum mooo Hm paw: mammumamm aw momcmnu .m madman 81 Alt ' ' n n 7 9; F I) n a n V ' of cones enqngos woo 10d 9 ‘0 l —o l l l o 1 I, -10 _o \/- \ a } .... W l l 1’ a . q” ‘0' \ 3 8 \ \ \ i -N -N l I l k _- __ x \ \ \ \ \ \ \ ..o ‘0 1 1 1 1 1 1 1 1\ L 1 1 1 1x 0 0 O O O V 3 '3 2 3 z 9 s g 8 8 1’ 2 a nod) 10501110109110 1 (woo 104) N5!" "flu u! eon ‘A 82 Tenderometer The difference between replicates was significant, the mean pressure reading of the second replicate being 24 psi more than the first. The second replicate was harvested at the same time as that of the second replicate of the tem- perature study and illustrates the effect of weather just previous to harvest on quality(Tab1e 3). The weather dur- ing growth of the second replicate was very hot and dry after a previous cold period, resulting in tough aSparagus. The difference between replicates was also evident in the per cent soluble solids, the second replicate having lower values. These differences are not important in this study. The difference between air and 15-15 was significant, showing that the asparagus that had been stored in the mod- ified atmosphere retained its tenderness during the post- storage treatment. The difference that was present after the week of storage diminished with time, except after 5 days, when the difference between the aSparagus previously stored in air and 15-15 was approximately the same as at the end of the week of storage. Weight‘poss The linear regression of days in post-storage treat— ment against weight losses was significant, meaning that with increasing time there was more weight lost. The dif- ference between atmOSpheres was not significant; implying that rate of weight loss, which was mostly water, was not 83 affected by the previous high water uptake under the mod- ified atmOSphere. This suggests that the permeability of the cellular membranes was not altered as Glinka and Rein- hold (1962) found with sunflower hypocotyl sections treated with carbon dioxide in solution. This also implies that the water taken up under the influence of carbon dioxide is in the cells and is held by the cells against drying forces. Pl! The difference between the pH of the asparagus from the air- and carbon dioxide-treated asparagus was significant. The pH of the carbon dioxide-treated aSparagus was consist- ently slightly higher. The large differences in pH imme- diately after the week of storage became very small after the first day at the higher temperature in air. This re— versal of pH as caused by carbon dioxide has been recorded by Fife and Frampton (1935). During the remaining 5 days at 60°F the pH of the asparagus from both treatments changed only very slightly after this reversal. Per Qent Soluble Solids The differences between replicates and between at- mospheres were significant. The difference between repli- cates implies the difference in growing conditions as dis- cussed for tenderometer readings. The per-cent soluble solids of the aSparagus from the 15-15 atmOSphere was con- sistently lower than that that had been stored in air. This 84 was due to dilution by water in the modified atmOSpheres. As discussed before it was impossible to correct adequately for this dilution because of respiratory losses at 60°F. Storage Period and AtmOSphere Thornton (1937) found that at 100C or higher temper- atures, in atmospheres of 50 to 70% carbon dioxide plus 20% oxygen, the pH of asparagus increased 0.1 unit in 20 minutes and 0.4 units in 5 hours. If the other reactions bringing about tenderness, gains in weight, and changes in per cent soluble solids are related to pH change they must occur quickly too. This experiment was initiated to study the effect of carbon dioxide on aSparagus held for less than a week, the shortest period previously used, and for longer periods of time. Methods and Materials The modified atmosphere used was 20% oxygen or slightly higher, and 15% carbon dioxide (20-15). The 20% oxygen was chosen because the chambers were Opened momentar- ily every 2 days to remove samples. .When the chambers were opened the oxygen level was restored to that desired. Pre— vious work (Franklin g£_gl., 1961; also Table 10) had indi— cated that levels of oxygen between 5 and 20% had little effect on any of the response criteria measured. The con- trol lots were stored in air. 85 There were 2 replicates representing harvest dates of May 18 and June 1, 1964. The 10 sampling times (thus storage periods) were at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days. The temperature of storage was 35°F and the aSpar— agus was stored with the butts in water. The experiment was designed as a Split plot with atmOSpheres the main plot and storage periods as sub-plots. Result and Discussion A summary of the analyses of variance is presented in Table A-7, and the means are given in Table 12. The interactions are presented in graphic form in Figure 4. Tenderometer The tenderometer readings of the second replicate or harvest were significantly lower than those of the first replicate. The interactions of storage periods (linear and quadratic) x air vs. 20-15 were significant. Lines repre- senting the effect of the atmospheres can best be pictured as straight lines together at the origin, in this case 147.5 psi, and diverging with time is storage. There was a tend- ency, as the significant quadratic interaction showed, for a levelling off of these effects with time. 86 Table 12. The effects of time in storage and atmospheres of air and 20% oxygen plus 15% carbon dioxide on aSparagus stored at 35°F with the butts in water M-m- .. Per Cent Gain in Soluble Tenderometer Weight Solids (psi) (% fr. wt.) pH (correct.) Originala Rep. 1 154.8 ----- 6.00 5.50 Rep. 2 139.5 ----- 6.00 5.55 Mean 147.5 ----- 6.00 5.52 After storage Rep. 1 148.4 11.92 6.18 5.13 Rep. 2 133.2 11 09 6.11 4.66 Mean 140.6 11 50 6 14 4.80 AtmOSpheres Air 154.4 4.26 5.94 4.70 20-15 127.2 18.75 6.54 5.08 Storage periods (days) 0 147.5 ----- 6.00 5.52 2 143.2 6.57 6.12 5.33 4 151.2 9.78 6.18 5.23 6 141.0 13.92 6.26 . 5.22 8 138.2 12.88 6.19 4.76 10 134.5 13.52 6.24 4.68 12 133.0 12.95 6.28 4.58 14 143.2 13.45 6.05 4.54 16 138.8 15.80 6.15 4.58 18 137.2 16.15 6.04 4.50 aMean of 4 values before storage. 87 65» saw: m com>xo $om O .uopmz ad mupan mm um Umqum mammummmm no Amanomv oofixOfio conumo Rma mafia one mam mo monoanOEum ocm ommuOHm ca mafia mo muoowwo 65H .v ousmfim 88 o ’ '72 .\ .1- / / \ ( 42 , u! \\ s \ i .1! < a! / o \\ r o - '53 / I, N a 6 \ o s- ‘ c: \‘ ‘0 J ‘0 \\ x «o / 4'9 \ ,/ \\ // I \ -v ( “V \ \ \ \ \\ «N I) d" \ I \ \\ LllllllllLlll‘Lk l L l l 5 o o co Q q u q o o o o o _ N «5 o o o o n 2 2 S 1’ ‘1' — no (god) 10101110109110; ° ‘2 '9 ‘9 - \\ c - " \/8 N > ‘3 g / 9° ( 9. O \ s o o ) 0° I co L\ 19 \\\ \\ V \\ V \\ \\ N N O 10 L 1 1 1 1 1 1 L 1 1 1 1 1 9 9. '. N 9 9 '9. ". N. '0 O '2 9 '0 ° ooooovvvv “‘ “(wooucn (pesoeuoo) 'PQIOS .IQ"I°S W” I'd 111010111 118013 01 0109 Figure 4 89 Although none of this aSparagus was really tough, the difference between the aSparagus stored in air and under the 20—15 atmOSphere was marked. Considering 150 psi as the upper limit of fancy grade aSparagus (Jenkins and Lee, 1940), all of the asparagus treated with carbon dioxide was of fancy quality, and all that treated with air less than fancy, after the second day of storage. The effect of tenderizing under carbon dioxide was pronounced even in the 2-day stor- age period, the difference due to the atmOSphere being 11 psi. -From these results it would appear that carbon di- oxide plus water treatment would be useful in retaining ten- derness, or obtaining tenderness in aSparagus that was of borderline quality because of lack of tenderness. Such a treatment could be effective in 2 days at 35°F. Gain in Weight The 3 interactions of duration of storage (linear, quadratic, and cubic) x air vs. 20-15 were significant, with the bulk of the sums of squares in the interaction with the linear regression. The gains in weight were rapid in storage, reaching the levelling off point of 4.30% at the end of 2 days in air, whereas in the 20—15 atmOSphere the levelling off point came at the end of 6 days at a gain of 22.65%. The linear in— crease with time in both atmospheres up to the levelling off point indicates the linear regression, the levelling off the 9O quadratic regression. The cubic regression may indicate the increase in gain in weight in the modified atmOSpheres from the 14th to the 18th day. This last gain was about 5% and there was no indication of levelling off at the end of 18 days. Again as in previous experiments the gains in weight (water uptake) were the reverse of tenderometer readings or paralleled a line representing tenderness. p33 The interaction of storage period (linear) x air vs. 20-15 was significant . The pH of the samples that had been in air showed a linear decrease with time in storage. This decrease was found previously in these experiments, and is thought to be the results of the normal aging processes of the tissues (Schweigart and Kellner, 1938). The pH of the asparagus in the 20—15 atmOSphere increased markedly with time in a lin- ear fashion up to 10 days, after which the pH levelled off with only slight changes (probably sampling error) at later storage periods. The overall effect is expressed in the significant quadratic regression of storage period on pH. Per Cent Soluble Solids (Corrected) The per cent soluble solids in the first replicate was significantly higher than the second, although this dif- ference was not present in the original estimations before storage. 91 The soluble solids content of the aSparagus from the 20-15 atmOSphere was significantly higher than that of the aSparagus in air. This difference is similar to the differ— ences found in all of this work. The linear decrease of soluble solids with time was significant. The lines representing the decrease of soluble solids with time run parallel for the 2 atmOSpheric condi- tions, with the atmOSphere containing carbon dioxide having a persistently higher per cent soluble solids, except after 12 days when the levels were essentially the same. The lin- ear decrease with time was due to respiratory losses over the long storage period. Atmosphere and Fiber Content Carolus §£_gl. (1953) found that there was a de- crease in fiber content as measured by the blendor method, under modified atmospheres. These results were reaffirmed by results reported here (see Table 9). However, there was no change in crude fiber content after the values were cor- rected for water uptake. These analyses were conducted to further investigate earlier findings. 92 Methods and Materials Fiber and crude fiber analyses were performed on aSparagus of the 1961 season1 that had been stored at var- ious atmospheres as described below. Frozen samples were analyzed by the blendor method 2 weeks after atmOSpheric treatment. Dried samples were analyzed for crude fiber by the official AOAC method (See section uHm x mm .m> we tmmoo.o ton.vo mo.o kno.an tvm.mHHH H mHimH.-m> uHm x mm + me .m> mm N .mOEpm x .QEoH «Hwoo.H tt¢b.NdOH *twb.m t*NH.ome kmh.wnHH H mHan .m> uH<. . 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Source of 142 A summary of the analyses of variance of the effects of atmospheres of air and 15% oxygen plus 15% carbon dioxide on the composition and tenderness of aSparagus stored for 1 week at 35°F with the butts in water. 4 Dry Matter Crude Fibera Deg. of (% fr. wt., (% fr.wt.) Fiber b (%jfr.wt., Variation Freedom correct.) correct.) (correct.) Replicate 1 0.5934 0.70 0.82 6.16 Treatment 3 Before vs. after storage 1 7.3051 9.62** 0.35 0.38 Air vs. 15-15 1 0.0544 3.47* 0.70 1.98 Before vs. after x air vs. 15—15 1 0.0000 1.31 0.02 13.11* Error 3 1.9048 0.23 0.21 1.02 Sampling error 32 0.1873 0.85 0.92 1.39 Total 39 Source of Degrees of Tenderometer Readings Variation Freedom After Cooking (psi) Replicate V .1 900.25 Air vs. 15-15 1 12966.00** Error 1 132.25 Sampling error 12 309.54 Total 15 aValues x 10. b Values x 100. Table A-5. 143 A summary of the analyses of variance for the effects of various levels of oxygen and carbon dioxide upon asparagusostored with the butts in water for 1 week at 35 F. Per Cent Gain in Soluble Source of Deg. of Tend. Weight Solids _mVariation Freedom A (psi) (% fr.wt) pH (correct.) Replicate 1 132.48 23.66 2.07 1.1881 Treatment 12 Air vs. others 1 1085.46** 142.29** 124.47** 0.1202 Oxygen levels 1 58.52 6.51 0.81 0.0588 Carbon dioxide levels 5 Linear 1 751l.79** 614.42** 351.63 1.4853** Quadratic 1 202.52 31.39** 36.73** 0.1817 Cubic 1 107.80 14.99* 0.86 0.2228* Remainder 2 99.75 1.16 1.58 0.1340 Oxygen x carbon dioxide 5 Oxygen x carbon dioxide (linear) 1 284.29 0.32 0.00 0.0070 Oxygen x carbon di- oxide(quadratic) 1 115.00 2.15 0.77 0.1467 Oxygen x carbon dioxide (cubic) 1 0.18 2.05 0.00 0.0000 Oxygen x carbon dioxide (re- mainder) 2 77.88 1.68 0.25 0.0332 Error 12 83.44 1.86 1.51 0.0425 Sub-total 25 Sampling error 26 33.29 ----- 1.35 0.0205 Total 51 144 Table A-6. A summary of the analyses of variance for the effects of time in storage at 60°F and 50% rela- tive humidity in air upon asparagus previously stored with the butts in water for 1 week at 35°F in air and 15% oxygen plus 15% carbon dioxide _- j— Tender- Loss in Per Cent Source of Deg. of ometer Weight Soluble Variation Freedom (psi) (% fr.wt.) pH Solids Replicate 1 3456.0** 26.88 0.08 5.70** Total treatment 11 Air vs. 15-15 1 2242.6** 14.10 71.17* 1.87* Days of post- storage treat- ment 5 100.1 225.58 2.27 0.09 Lineara 1 1112.01** Air vs. 15-15 x days of post- storage treat- ment 5 . 43.4 17.28 8.61 0.06 Error 11 99.8 90.98 14.32 0.12 Total 23 ‘7‘..— 3Broken down into single degrees of freedom only if there is a possibility of significance. .145 Table A-7. A summary of the analyses of variance for the effects of time in storage and atmOSpheres of air and 20% oxygen plus 15% carbon dioxide on aSparagus stored with the butts in water at 35 0F . . Per Cent Tender- Gain 1n Soluble Source of Deg. of ometer Weight Solids Variation Freedom (psi) 0% fr.wt.) pH (correct.) Replicate 1 2340.90* 6.81 13.93 2.2137** Air vs. 20-15(A)a 1 7398.40* 2101.05** 758.38 1.4326* Error (a) 1 12.10 3.54 10.35 0.0002 Sub-plot total 3 Storage periods 9 Linear (B) 1 444.51** 619.24** 0.07 4.9374** Quadratic (C) 1 128.03 139.30** 67.02** 0.0180 Cubic (D) 1 19.50 91.47** 4.44 0.0733 Remainder (E) 6 94.64 3.24 4.02 0.0617 Air vs. 20-15 x storage periods 9 A x B 1 999.94** 326.71** 117.10** 0.0042 A x C 1 346.11* 71.43** 1.37 0.2505 A x D 1 36.55 31.86** 10.36 0.0623 A x E 6 26.58 2.65 4.46 0.0928 Error (b) 18 50.67 4.54 2.40 0.0769 Total 39‘ aLetters in parentheses, (A), indicate a comparison as used in the interactions. 146 Table A-8. A summary of the analyses of variance of the effects of time in storage and atmOSphereS of oxygen plus carbon dioxide on the development of fiber and crude fiber in aSparagus stored at 35 0F with the butts in water. W Crude Fiber Fiber Source of Deg. of (% fr. wt) (% fr.wt., Variation Freedom (correct.) correct.) Replicate l 0.05 0.03 1.94* Total treatment 13 Air vs. 20-0 1 0.20 0.09 0.06 Controls vs. others 1 2.03** 0.04 0.84 Others 11 Oxygen levels 2 0.02 0.15 0.30 Carbon dioxide levels 3 Linear 1 4.03** 0.15 Quadratic 1 0.01 1.50* Cubic 1 0.01 0.03 a Oxygen x carbon dioxide 6 0.04 0.30 0.49 Error (a) 13 0.19 0.20 0.41 Sub-plot total 27 Storage period 1 1.19 0.15 0.01 Storage period x treatment 13 0.00 0.30 0.51 Error (b) 14 0.36 0.25 0.56 Total 55 f v—w aBreakdown of the sums of squares was performed down to single degrees of freedom only if there was a possibility of there being significance at the 5% level. If there was no chance of significance the mean squares are not reported here. 147 Table A-9. A summary of the analyses of variance of the effects of oatmospheres on aSparagus stored for 1 week at 35° F with the butts in a solution of sodium 2,4- dinitrophenolate (DNP) Per Cent Gain in Soluble Source of Deg. of Weight Solids Variation Freedom (% fr.wt.) pH (correct.) Replicate 1 20.41 11.47 3.08* Atmospheres 2 0.22a Air vs. 20-0 1 0.05 48.81 Controls vs. 9—12 1 1660.84** 810.61* Error (a) 2 3.98 30.43 0.14 Sub-plot total 5 DNP 1 52.29 4.77 1.21 DNP x atmOSphere 2 6.70 18.67 0.82 Error (b) 3 9.25 13.15 1.94 Sampling error 36 1.33 2.69 0.07 Total 47 aBreakdown of the sums of squares was single degrees of freedom only if there was a there being significance at the 5% level. If performed down to possibility of there was no significance the mean Square is not reported here. 148 Table A-lO. A summary of the analyses of variance of the effects ofoatmospheres on aSparagus stored for 1 week at 35 F with the butts in solutions of Carbowax 1000 Per Cent Change Soluble Source of Deg. of in Weight Solids Variation Freedom (% fr. wt.) pH (correct.) Replicate 1 74.66 62.71 0.98 AtmOSpheres 2 Air vs. 20—0 (A)a 1 126.86* 32.68 0.23 Controls vs. 9-12 (B) 1 317.07* 461.87* 1.73 Error (a) 2 6.55 4.76 0.39 Sub-plot total 5 Carbowax conc. 6 Linear (C) 1 1752.47** 24.60** 0.32 Quadratic (D) 1 419.11** 39.27** 0.00 Cubic (E) 1 195.53** 12.32** 0.44* Remainder (F) l 26.91** 1.50 0.17 Carbowax x atmos. 12 A x C l 7.30** 0.59 0.29 A x D 1 3.40* 0.08 0.02 A x E 1 1.26 0.20 0.44* A x F 3 0.09 6.91** 0.06 B x C 1 79.66** 2.12 0.00 B x D l 97.32** 0.51 0.00 B x E 1 41.17** 0.06 0.03 B x F 3 8.79** 7.42** 0.14 Error (b) 18 0.70 0.69 0.08 Total 41 aLetters in parentheses used in the interactions. (A) indicate a comparison as 149 Table A-ll. A summary of the analyses of variance of the effects of a modified atmOSphere of 9% oxygen plus 12% carbon dioxide upon asparagus stored for 1 week at 35 0F with the butts in solutions of Carbowax 1000 Source of Degrees of Change in Weighta Variation Freedom (% fr. wt.) pH Replicate 1 0.26 19.07 Carbowax conc. 6b 477.75** 6.41 Error 6 0.37 4.75 Sub-total 13 Sampling error 14 0.45 1.33 Total 27 aCoded by adding 4 to all values. Instead of using trend comparisons, comparison was made by using Duncan's test (1955) and values are given in Table 17, text. ”a.“ . . LIV ‘. 4..- nHu PM... r9“ - 4.116% HICHIGRN STRTE UNIV. LIaRnRIEs ‘lIWWWWIWWW“IIWlHW 31293102225341