THE INFLUENCE- OF VARSGUS FACTORS ON
THE INDUCTEON OF BTTTERNESS TN STORED

CARROTS BY ETHYLENE AND TTS DETECTION
BY FLUORESCENCE

i Thesis Tor the Degree of M. 5
MICHIGAN STATE UNIVERSITY

James Ernest Ells
1958

 

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THE INFLUENCE OF VARIOUS FACTORS ON THE INDUCTION OF
BITTERNESS IN STORED CARROTS BY ETHYLENE AND ITS

DETECTION BY FLUORESCENCE

By

JAMES ERNEST ELLS

AN ABSTRACT

Submitted to the College of Agriculture. Michigan State University
of Agriculture and Applied Science in partial fulfillment
of the requirements for the degree of

MASTER OF SCIENCE

Department of Horticulture

1958

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JAMES ERNEST ELLS ABSTRACT - l

The development of bitterness in stored carrots has become an econ-
omic problem of considerable importance in recent years, chiefly in carrots
grown for processing. The presence of ethylene in the atmosphere caused
non-bitter carrots to develop a bitter flavor after a few months in cold stor-
age.

Three tests (organoleptic, fluorescent, and spectrophotometric) were
used to determine the degree of bitterness. The organoleptic tests were
made on raw carrots which were rated on a scale of 0 to 4, with 0 being non-
bitter and 4 being highly bitter. Fluorescence determinations were made
by examining the cross sectional cut surfaces of a carrot root, one-half inch
below the crown with an ultra-violet lamp having a 2537 A filter. The rat-
ing was on a 0 to 4 basis with no fluorescence having a rating of 0 and an
intense yellow-green fluorescent speckling in the phloem denoting a 4 rating.
The spectrophotometric rating was arrived at on the basis of the absorption
of ultra-violet light at 240 mp, 265 mp, and 290 mp, by a solvent used to
extract the bitter principle (3 -methyl-6-methoxy-8-hydroxy-3, 4—dihydro-
isocoumarin) from the carrot roots. These tests have been successfully
correlated with one another.

Storage experiments involving variations in the variety and color

JAMES ERNEST ELLS ABSTRACT - 2

of carrots, soil type and temperature, and the storage atmosphere and tem-
perature, were conducted on carrots stored in sealed 55-gallon drums held
at four different temperatures. These drums received a continuous supply
of air, which amounted to one complete change of air per day. Variations

in the atmosphere were produced by injecting ethylene gas, apple emanations,
and automobile exhaust into the air line supplying the drums. Carrot sam-
ples were removed from these drums periodically for testing.

Ethylene, whether as a pure gas, or as a constituent in apple emana-
tions or automobile exhaust gas, produced a bitter flavor, fluorescence, and
a quantity of the bitter principle in carrots, which was significantly higher
in every case than the control carrots.

The quantity of ethylene present in the storage atmosphere and the
length of the storage period was significantly related to the degree of bitter-
ness induced in carrots.

The flesh color of the carrot had an apparent effect upon the induction
of bitterness by ethylene, with orange carrots becoming most bitter, yellow
carrots intermediately bitter, and white carrots least bitter, indicating that
if the precursor to carotene is involved in the bitter principle, some of this
precursor is present in white carrots.

Physiological age of the carrot was shown to be a factor in the degree

JAMES ERNEST ELLS ABSTRACT - 3

of bitterness induced by ethylene as determined by organoleptic, fluorescent,
and spectrophotometric tests, with mature carrots attaining a higher degree
of bitterness than either immature or overmature carrots.

In conclusion, ethylene as a pure gas or as a constituent in other
gases, was found to be effective in inducing bitterneSs, fluorescence, and
the bitter principle (3 —methy1-6-methoxy—8-hydroxy-3, 4-dihydroisocoumarin)
in stored carrots. Although all carrots responded to the ethylene treat-
ment, the degree of bitterness induced was influenced by the quantity of
ethylene, the time in storage, the flesh color, and the physiological age

of the carrots.

THE INFLUENCE OF VARIOUS FACTORS ON THE INDUCTION OF
BITTERNESS IN STORED CARROTS BY ETHYLENE AND ITS

DETECTION BY FLUORESCENCE

By

JAMES ERNEST ELLS

A THESIS

Submitted to the College of Agriculture, Michigan State University
of Agriculture and Applied Science in partial fulfillment
of the requirements for the degree of

MASTER OF SCIENCE

Department of Horticulture

1958

ACKNOWLEDGEMENT

The author wishes to express his sincere appreciation for the
time, encouragement and guidance so freely given by Dr. R. L. Carolus,
Dr. H. M. Sell, and Dr. D. H. Dewey, and to Dr. E. H. Lucas for his
help with the manuscript, and also to extend his thanks to others who
have helped during the course of this work. Among these, special thanks
to Arleigh Dodson of the Agriculture Chemistry Department for spectro-
photometric analyses and ethylene determinations; to Philip Coleman for
photographic help; to Dr. P. M. Bessey for his assistance in setting up
the first experiment; to Dr. J. M. F 0th for his committee services;

and to Wilhelmus Clerx for his laboratory assistance.

TABLE OF CONTENTS

Page
INTRODUCTION ...................... 1
REVIEW OF LITERATURE . . . .............. 2
Bitterness in Crops . . . . . ............ 2
Bitterness in Carrots (Isocoumarin Type) ........ 3
Factors Influencing Bitterness in Carrots ..... . . . 5
Changes in Carrot Composition During Storage ..... 6
Influence of Apple Emanations and Ethylene on Stored
Produce .............. . . . . . 7
Fluorescence .................... 10
GENERAL METHODS . -, .................. 11
Storage Apparatus .................. 11
Analytical Procedure ................. l3
STUDIES OF THE INFLUENCE OF CULTURAL AND STORAGE
PRACTICES ON THE DEVELOPMENT OF BITTERNESS IN CAR-
ROTS .......................... 16
Methods and Procedure ........... . . . . 17
Results .................. . . . . 18
Discussion........ ......... 24

CONTENTS CONT'D

Page
THE INFLUENCE OF VARIETY ON THE DEVELOPMENT OF

BITTERNESS IN STORED CARROTS . . . . ........ 26
Methods and Procedure . . . . ........... 26
Results . . . ................... 26
Discussion . ................... 29

THE INFLUENCE OF SOIL TEMPERATURE ON THE DEVELOP-

MENT OF BITTERNESS IN STORED CARROTS. . . . . . . . 30
Methods and Procedure ................ 30
Results and Discussion ................ 31

THE INFLUENCE OF STORAGE TEMPERATURE, AREA OF PRO-

DUCTION, AND SOURCE OF ETHYLENE ON BITTERNESS DE-

VELOPMENT ....................... 33
Methods and Procedure ................ 33
Results ....................... 34
Discussion ..... . ............ . . . 40

THE EFFECTS OF ETHYLENE ON WHITE, YELLOW AND ORANGE
CARROTS FROM WISCONSIN . . . . . . . . . . . . . . . . 49
Discussion ........ . ............ 49
THE EFFECTS OF ETHYLENE GAS UPON THE RESPIRATION OF

CARROTS IN STORAGE ................... 51

CONTENTS CONT'D

Page

Methods and Procedure ............... 51
Results. . . ............... . . . . 51
Discussion ........... . . . . ..... 53
GENERAL DISCUSSION ....... . . . . ........ 54
SUMMARY AND CONCLUSION . . . . ........... 56

LITERATURE CITED ............ ' ....... 59

INTRODUCTION

Producers and processors of carrots have recently observed that
the roots frequently become bitter during storage. In a few cases carrots
scheduled for processing were found to be bitter and were used in soups,
where the off-flavor was masked or in extreme cases the carrots were dumped.
Since such practices inevitably result in economic loss, investigations were
undertaken to determine the cause of this bitterness and to find means of pre-
venting its occurrence.

Considerable preliminary work on this problem has already been
done. The bitter principle has been identified as 3-methyl-6-methoxy-8-
hydroxy-3, 4-dihydroisocoumarin (37). Methods for its detection and quanti—
tative procedures for measuring it have been devised. Several possible
causes for the trouble have been explored with varying degrees of success.

One of the more promising approaches.has been from the standpoint
of atmospheric conditions prevailing in carrot storages. Bessey (4) observed
a relationship between the degree of bitterness and the presence of apples
in storage. To verify these observations, a study was undertaken to deter-
mine the influence of a number of environmental factors and variety on the

development of bitterness in carrots when subjected to various storage en-

Vironments.

REVIEW OF LITERATURE

Bitterness in Crops

Bitterness is a quality which is desirable in certain beverages, such
as coffee, cocoa and beer, but is quite undesirable in most horticultural crops.
Truscott (39) has reported an instance of bitterness in celery, which was so
severe that the crop was unmarketable. However, only one person in three
could detect this bitterness which was described as hot and peppery, and an
exaggeration of the normal celery flavor. Since it disappeared in storage,
it was regarded as a temporary phenomenon. Enslin (11) observed that bitter-
ness in cucumbers was traced to an enzyme, elaterase and occurred in thirty-

three different species in the Cucurbitaceae family. Higby (l 9) has identi-

 

fied isolimonin in navel orange juice which develops bitterness upon exposure

to air or pasteurization. He also refers to narigin, the bitter glycoside in

grapefruit skin, and limonin, the bitter substance on orange seeds.
Off-flavors have been reported by Himreimer (20) and Gilpin (14)

in root crops grown on soils which had been treated with either benezene

hexachloride (B. H. C.) or lindane. Lean (23) reported off-flavor in carrots

caused by aster yellows, and Yamaguchi _e_t_ _a_l_. (44) reported another off—

flavor in carrots associated with green "shoulders".

Bitterness in Carrots (Isocoumarin Type)

The carrot bitterness with which this study is concerned was re-
ported by Truscott (39) in 1953. He described two types of bitterness. The
first flavor was a peppery taste which imparted a painful burning sensation
in the mouth and disappeared upon processing. The other flavor was a strong,
spicy-hot, flavor which also left a burning sensation in the mouth, and this
flavor was not destroyed by processing. In 1955, Yamaguchi e_t_ a_l_. (44)
reported a bitterness in carrots not due to aster yellows, green shoulders
or weed control chemicals. This bitterness which developed in storage,
was found in 10 to 90 per cent of the carrot roots. Atkins (2), Sondheimer
gt a_l_. (36), and Dodson gt a_l_. (9) have experienced a taste sensation in stored
carrots similar to that described by Truscott (39) and Yamaguchi gt .Ei' (44).

In a more recent publication Yamaguchi _e_t _ai. (44) found a differ-
ence in the alpha-carotene content between lots of bitter and non-bitter carrots.
Petroleum ether extracts of the chromatograms of magnesium oxide and
"Hy- flo Super Cel"1 indicated that the bitter fraction was strongly adsorbed
on the top portion of the column. Phillips (28) extracted the bitter fraction
from the top of the column with methanol (spectrograde) and determined the

bitter substance quantitatively with a model DK—2 Beckman spectrophoto-

1A product marketed by John's Mansville Corporation.

Concurrently, Sondheimer (36) found that a petroleum ether extract
of a sample of pureed carrots of known bitterness prior to canning, produced
a bell-shaped absorption curve when measured with a Beckman spectrophoto-
meter. The minimum absorption values were 240 mu and 290 mp, and the
maximum absorption value was 265 mp. The following formula was used to
express these results:

(optical optical , )
Opitcal (density 290 mu + density 240 mp)

density 265 mu - 2 x 40 m1.
Weight of sample (grams)

 

 

A positive correlation was noted between the rating determined by this for-
mula and that found by taste. He found that the phloem contained more of
the bitter principle than the xylem, and that this analytical procedure worked
equally well on fresh or processed carrots.

In a later publication, Sondheimer (37) reported the formula for the
bitter principle as C1 11-11204, molecular weight 208, melting point 76°C, and
named the compound 3-methyl-6-methoxy-8-hydroxy-3, 4-dihydroisocoumarin,
which in this paper has been abbreviated to "isocoumarin". From this data
he calculated the factor 2. 2, which when multiplied by the reading at 265 mu
gave the weight of isocoumarin in milligrams per 100 grams of sample.

Steam distillation of the bitter carrots yielded a distillate of tiny

oil droplets having a bitter taste. Isocoumarin could not be detected in the dis-

tillate by chemical analysis. However, isocoumarin was easily detected in
the residue. Moreover, crystalling isocoumarin was only slightly volatile
with steam (37). This supports Truscott's original contention that bitter-
ness is derived from two compounds.

According to Sondheimer (37) isocoumarin placed directly on the
tongue gave no bitter taste, but the taste panel found that a . 01 per cent aque-
ous solution was bitter. Further tests showed that when . 02 per cent iso-
coumarin was added to the steam distillate of non-bitter carrots, the tasting
panel could not distinguish it from the steam distillate of bitter carrots.

Dodson e_t a_l_. (9) extracted 100 grams of the principle from 14 bushels
of bitter carrots with acetone. This crystalline compound produced an ultra
violet absorption curve on the spectrophotometer similar to the one described
by Sondheimer. The colorless platelets were soluble in chloroform, ethyl

ether, water, and methanol.

Factors Influencing Bitterness in Carrots

Truscott (39) found that carrots lost their bitter flavor when held at
room temperature, or when they were allowed to sprout in storage. Yamaguchi
_e_t El: (44) stored carrots at 32°, 50° and 77° F, and found no significant differ-
ence in the occurrence of bitterness. However, Bessey (4) concluded that

carrots became more bitter at temperatures higher than 32° F.

U]

Neither Atkins (2) nor Bessey (4) found any varietal difference per-
taining to bitterness, although Yamaguchi _et__a_1_. (44) found that the progeny
of bitter carrots were more bitter than the progeny of non-bitter carrots.
Atkins and Sayre (3) observed that minor elements lessened the degree of
bitterness, and also that muck and sandy soils produced carrots more sus-
ceptible to bitterness. Neither Bessey (4) nor Yamaguchi _et a_l1 (44) could
find any significant influence due to soil type; and Yamaguchi gt a]: (44) and
Atkins (2) concluded from irrigation trials that soil moisture was not a fac-
tor in predisposing carrots to bitterness.

Bessey (4) in harvesting and handling-studies was unable to relate
rough handling to bitterness development, and indicated that early planted
carrots developed lower levels of bitterness than the later planted carrots
when placed in storage. Both Atkins (2) and Bessey (4) observed that im-
mature carrots attained less bitterness in storage than did mature carrots.

In storage studies, Bessey (4) found no correlation between oxygen
and carbon dioxide levels and bitterness; however, he obtained evidence

that indicated that apple emanations caused bitterness.

Changes in Carrot Composition During Storage
During the first 30 days of storage, Brown (6) found an increase in

carotene content of carrots on a dry weight basis. The carrots remained at

this higher level for the next 60 days. Platenius (29) observed that the mois-
ture in stored carrots at 32°, 35°, 40° and 50°F increased slightly in all lots
except those held at 32° F. He noted that sucrose was converted to reducing
sugars and the latter substances were reconverted to sucrose, and that the
conversions were accelerated by an increase in temperature.

Appleman (1) showed that the rate of respiration of fresh carrots
rapidly declined with the age of the roots. Once in storage, the respiration
rate was never as great as at the time of harvest, and there was no indication
of a sharp rise of carbon dioxide in storage (climacteric). Wright _e_t a_l_. (43)
showed that the respiration rate of carrots was three times as great as
apples. Werner (41) found reducing sugars to increase in carrot varieties
during storage, while Rygg (34) and Brown (6) observed an increase in caro-
tenoid content. Newhall (26) reports an increase in water of some lots when

the relative humidity was maintained above 94 per cent.

Influence of Apple Emanations and Ethylene on Stored Produce

Bessey (4) suggested apple emanations played a role in the develop-
ment of the bitter principle in carrots. A review of the literature reveals
that apples produce a vapor which delay abnormal sprouting in potatoes (13),
and that apples, pears, peaches, tomatoes, and bananas yield a gas which

stimulates ripening and produces a critical rise in respiratory activity of im-

mature fruit (13). Nelson (24) concluded that ethylene was the active ingredient
in apple emanations and measured its evolution (substantiated by Hanson and
Christensen (17) in 1939). Nelson (25) in another study showed that ethylene
from the apple prior to its climacteric is consumed in the ripening process.
Smock (35) noted the stimulating effect of one lot of apples upon another.

Rood (33) observed that apple emanations as well as ethylene caused brown
spot injury on lettuce and was most effective in producing injury at 44° F of
firm heads.

Ethylene is an unsaturated hydrocarbon gas, non-poisonous, has a
faint sweetish odor with a boiling point of -103. 9° C, and a specific gravity of
0. 975. It is soluble in water to the extent of 25. 6 cc per 100 grams of water
at 0°C (30). Denny (8) showed that kerosene heaters produced ethylene which
unduly ripened citrus while in transit to northern markets. Crocker (30)
tested 28 gases and found that ethylene, propylene, acetylene, butylene, and
carbon monoxide produced epinasty in tomato seedlings; but that ethylene was
500 to 500, 000 times more effective than the other four gases. Englis and
Dykins (30) concluded that ethylene had no effect upon pure enzymes and that
it acted directly on living protoplasm. Williamson (42) observed that ethy-
lene caused epinasty, yellow coloration, and abscission of leaves, flowers.
and fruits, and that tissue infected with certain pathogens showed a marked

stimulation in ethylene production. He cited the black spot of rose and the

shot hole of cherry as examples. In 1951, Hall (16) stated that the only re-
quirement for the production of ethylene is a fermentable substrate under
aerobic conditions. He believed that sugar was the original ethylene produc-
ing substance; however, he suggests that a complex sequence of intermediates
catalyzed by the necessary enzyme systems is required to produce ethylene.
In fruit ripening, he suggested that ethylene functioned autocatalytically, thus

accelerating its own production. Young e_t a_l_. (45) proved that Penicillium

 

digitalum evolved ethylene. In 1953, Curtis (7) showed that as little as 1 ppm
of ethylene damaged dormant nursery stock of apple and pear. The damage
occurred seven times as rapidly at 55°F as at 35° F. In one case the source
of ethylene was an apple room adjacent to a well insulated wall.

Fenning (13) demonstrated that auto exhaust produced some unsatur-
ated hydrocarbons, but did not identify them. Rahrbaugh (32) noted that auto—
mobile exhaust induced epinasty in pea seedlings, and that 25 ml. of motor
exhaust produced the same effect as 0. 05 ml. of ethylene. Further work by
Crocker, Zimmerman and Hitchcock (30) found automobile exhaust to contain
9. 3 percent carbon monoxide. Rahrbaugh (32) reasoned that exhaust gas con-
tained ethylene since the epinastic response was too great to be caused by
carbon monoxide alone.

The influence of ethylene and other Storage conditions on the post-
harvest physiology of fruits and vegetables has received excellent treatment

in three review articles; Biale (5) 1950, Pentzer and Heinze (27) 1954, and

IO.

Ulrich (40) 1958. However, the influence of ethylene on Stored carrots is not

mentioned.

Fluorescence

Fluorescence refers to the property of substances to emit visible light
when excited with invisible radiation. The invisible radiation is generally pro-
duced by ultra—violet light, which was discovered by Ritter (22) in 1801. Flu-
orescence is generally defined as invisible light with a wave length between 40
and 4000 A. Radley (31) cites examples where fluorescence had found practi-
cal application in the field of agriculture in distinguishing between barley seeds,
between rye grass seedlings, in determining composition of animal feeds, in
determining the amount of coumarin present in sweet clover, in detecting
bruises on fruit, and to determine infection.

Bessey (4) noted that bitter carrots had a yellow— green fluorescence
and the intensity was related to the degree of bitterness and to the isocoumarin
content of the root. Radley (31) states that a number of three-carbon compounds
show a greenish fluorescence in alkaline solution. Strain (38) observed a flu-
orescence above the beta carotene band in the adsorption column. The substance
in this band has not yet been isolated into crystalline form. Zechmeister and
Sandoval (47) reported that petroleum ether was a good solvent for extracting
many fluorescent substances from plant tissue. The extracted substances could
be measured quantitatively with a Beckman spectrophotometer. According to

Goodwin and Kavanaugh (16) vitamin A has a yellow or green fluorescence.

11.

GENERAL METHODS

Storage Apparatus

The carrots were placed in mesh bags, in 55-‘ gallon air tight drums.
fitted with removable heads, and placed in cold storage. The removable heads
were equipped with inlet and outlet tubes, connected to a compressed air source,
and regulated so that a rubber tube attached to the inlet tube of each drum con-
veyed 144 ml. of air per minute, or a complete change every 24 hours, through
a metering bottle to the drum.

To produce apple emanations, a 16-liter, wide mouthed bottle filled
with apples of various seasonal varieties and fitted with a gas tight lid having
inlet and outlet hoses attached to the drum (Figure 1). Air was passed into
the bottle and then through a sodium hydroxide wash bottle to remove carbon
dioxide, and then into the drum stored in the cooler. The exhaust from the
drum was passed through an outlet hose, and bubbled through a water seal.

The control and the ethylene drums received their air directly from
the metering bottles, the only difference being that a removable section of
hose with a volume of 42 ml. was filled with ethylene gas and inserted into _
the air flow-line every 48 hours. One such charge of ethylene provided a
temporary maximum of 200 ppm of ethylene gas in the atmosphere of the drum.
Since 55 gallons of air were passing through the drum each day, the ethylene

was soon dissipated and had to be readministered.

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13.

Analytical Procedure

At each sampling a representative bag of carrots was removed, and
small segments were taken out of several carrots and tasted. An organo-
leptic rating for the sample was designated on the basis of a 0 to 4 scale with
0 being non-bitter and 4 being extremely bitter.

A fluorescent rating was made on the same carrots by slicing a half
inch of the crown off of each carrot and examining it in a dark room under a
ultra-violet lamp with a 2537 A filter1 for fluorescence. Each root was assig-
ned a value on the basis of a 0 to 4 scale, with 0 being non-fluorescent, and 4
being highly fluorescent. The values for each root were averaged. The threshold
for bitterness by fluorescence was approximately 1. 5. The type of fluores-
cence associated with bitterness is shown in Figure 2. In this figure is shown
the three enlarged photographs of a normal and bitter carrot in cross section.
The lower photograph reveals the difference between the carrots under the
2537 A lamp. The speckled fluorescent pattern in the phloem is typical of
bitter carrots, with this particular carrot having a fluorescence intensity
rating of 4.

The spectrophotometric determinations were made using a longi-
tudinally cut quarter from each carrot of the sample. These quarters were
pureed in a Waring Blendor with an amount of water equal to their weight. A

five- gram sample was weighed into a 50 ml. ground glass stoppered, Erlen-

"Mineral Light" model SL 2537 short wave. Ultra-Violet Products, Inc.,
South Pasadena, California.

Figure 2. Fluorescence of cross sections of normal and bitter
carrot roots. (Normal on left, bitter on right).

A. Under photoflood reflector larnp.
B. Under ultra violet light 3650 A wave length.
C. Under ultra violet light 2537 A wave length.

(X1 filter used with all lights)

 

 

 

1
meyer flask and shaken with 40 ml. of "Skelly-solve B" three times for

15 seconds each, to extract the bitter principle. The supernatant was de-
canted into a spectrophotometer cell and absorption determined with a DK-2
Beckman spectrophotometer. The ultra violet light was passed through the
cell at wave-lengths of 240 mu, 265 mp, and 290 mp. The absorption of
light at these wave lengths was recorded on a graph, and the data was
interpreted with the aid of Sondheimer's formula to estimate the quantity
of isocoumarin. This procedure is a modification of Sondheimer's method
(34). The spectrophotometric determination of isocoumarin ranged from
0. 00 for non-bitter carrots to 17. 00 for highly bitter carrots, with the
taste threshold for bitterness evaluated at 0. 75. These results are more
exact than those obtained by either the organoleptic or fluorescent methods

of analysis, which are subjective in nature.

——_————..—-—_-

1
Skelly-solve, a product marketed by the Skelly Oil Company.

16.

STUDIES OF THE INFLUENCE OF CULTURAL AND STORAGE PRACTICES
ON THE DEVELOPMENT OF BITTERNESS IN CARROTS

Many of the original suspected causes of bitterness, such as nutri-
tion and spray chemicals often having been explored were eliminated as pos-
sible causes by several workers; however, there still remained some ques-
tion as to the influence of planting date, time of harvest, and soil type.

Bessey (4) observed that the soil types were of little consequence
in predisposing carrots to bitterness, while Atkins (2) believed that muck and
sandy soils produced carrots more inclined to bitterness than upland soils.

In regard to harvest dates, Atkins (2) showed that earlier harvested carrots
became more bitter than later harvested carrots, which was confirmed by
Bessey (4). In addition, Bessey found that earlier planted carrots became
more bitter in storage than those that were planted later.

As work on bitterness progressed, it became apparent that post-
harvest handling influenced the development of bitterness. Bessey (4) ob-
served that the bitterness developed in carrots stored with apples, and thought
it was due to ethylene emanations from the fruit.

The purpose of this experiment was to test the influence of apple
emanations and ethylene on the development of bitterness in carrots, of dif-

ferent maturities, that had been grown on different soils.

17.

Methods and Procedure

Through the courtesy of the Gerber Products Company, Long Type
Chantenay carrots were furnished for the experiment. Samples were taken
from two muck fields and from a mineral soil planting. One of the muck
plots was seeded on April 10 and designated "early", the other muck plot was
planted on May 1 and designated "late", and the mineral plot also planted on
May 1, was designated "mineral". Carrots from these three plots were
harvested at different times and subjected to various storage conditions.

The first harvest was made on August 8 from the two muck areas.
At time of harvest, there was no organoleptic, fluorescent, of spectrophoto-
metric evidence of bitterness. Five 20-root lots of carrots from each of
two fields were placed in storage.

The carrots were sampled at weekly intervals during storage for
bitterness by organoleptic, fluorescent, and spectrophotometric determina-
tions. Estimations were made on six, 20-root samples at weekly intervals
for five weeks.

A second harvest was made from the same plots on September 6,
and in addition, carrots from the mineral loam were included. Seven lots
from each of the three fields were placed in each drum. This time, however,
a fourth drum was added, which was given only an initial charge of 42 ml. of

ethylene. This treatment is referred to as "ethylene prime".

18.

The third and final harvest was made from these plots on October 11.
The carrots were not bitter at time of harvest. A bushel of carrots from each
plot was also stored at this time in a pit at the horticultural farm as an addi-
tional control for the refrigerated carrots that were in the drums. A sample
of carrots from the "late" muck was _packed in ice at the time of harvest, and
placed in the ethylene drum because it had been suggested that carrots might
not become bitter when the respiratory activity was retarded from the time
of harvest.

The carrots which were held in pit storage were dug on December 3,
and found to be non-bitter, and non-fluorescent. They were then divided
into two lots and placed in the control and ethylene drums for 10 days at

which time they were again observed for fluorescence.

Results

Table I A. B and C show the influence of pre-harvest environment
and storage under various conditions on the development of bitterness as in-
dicated by organoleptic, fluorescent, and spectrophotometric tests. The
figures in Table I A are averages of carrots from both muck fields and the
mineral field.

Regardless of the date of harvest (A, B, or C), the averages of

carrots subjected to apple emanations, ethylene gas. and"ethylene prime" were

TABLE I

The Influence of Age and Storage Treatment on Development of Bitterness in
Chantenay Carrots Stored at 32°F

A. Early Harvest

 

 

 

 

 

Weeks in
Storage Control Apple Ethylene Average
Organoleptic Rating of Bitternessa
1 0. 0 1. 0 2. 0 0. 7
2 0. 0 2. 0 2.0 1. 3
3 l. 0 3. 0 3. 0 2. 3
4 0. 0 l. 0 2. 0 2. 0
5 0. 0 1. 5 l. 0 0. 8
Ave. 0.2 1.7 2.0 l. 3
Fluorescence Ratinggof Bitternessb
1 0. 0 0. 0 0. 5 0. 2
2 0. 5 1. 0 2. 0 l. 2
3 1. 0 2. 0 2. 0 1. 7
4 0. 5 3. 0 3. 5 2. 3
5 0. 5 2. 0 2. 0 1. 5
Ave. 0. 5 1. 6 2. 0 l. 4
Spectrophotometric Rating of Bitterness (Isocoumarin)C
l . 045 . 075 . 135 . 087
2 . 250 . 690 1. 390 . 780
3 . 735 2. 055 l. 820 1. 537
4 . 380 2. 575 5. 330 2. 762
5 . 495 7. 040 5. 760 4. 428
Ave. . 383 2. 487 2. 885 1. 919
Treatment Storage T X S
L. S. D. 5% 1% 5% 1% 5% 1%
Organoleptic . 65 . 98 . 84 1. 26 - -
Fluorescence . 41 . 60 . 53 . 77 . . 92 -
Spectrophotometric 1. 02 1. 48 1. 31 1. 90 2. 29 3. 31

 

aRating ona 04 basis; taste threshold: 1

1)Rating on a 0-4 basis; taste threshold: 1. 5
CRatings have ranged from 0. 0-17. 0; taste threshold: 0. 75

TABLE I (Continued)

B. Intermediate Harvest

 

Weeks in

 

 

 

Control Apple Ethylene Ethylene Prime Average
Storage
Orga_noleptic Rating of Bitternessa

l 0. 0 0. 6 1. 0 0. 0 0. 4

2 0. 0 1. 0 1. 7 1. 3 1. 0

3 0. 3 2. 0 2. 7 1. 3 1. 6

4 0. 0 2. 3 2 7 1. 3 1. 6

5 0. 0 1. 6 l. 3 0. 3 0. 8

6 0. 3 2. 6 2. 7 2. 0 1. 9

7 0. 0 2. 6 2. 0 2. 0 1. 7

8 0. 0 2. 3 1. 7 2. 0 1. 5

Ave. 0.1 1.9 2.0 1.3 1.3
5-week ave. 0. 1 1. 5 l. 8
Fluorescence Rating of Bitternessb

1 0. l7 1. 17 l. 17 1. 83 l. 09

2 0. 00 2. 00 2. 33 2. 33 l. 67

3 0. 00 2. 50 3. 00 2. 17 1. 92

4 0. 00 3. 67 3. 67 2. 00 2 34

5 0. 00 2. 00 2 67 l. 67 1. 59

6 0. 00 2. 67 3 33 2. 67 2. 17

7 . 0.10 2. 43 2. 10 2. 37 1. 75

8 0. 07 2. 80 2. 73. l. 90 1. 88

Ave. 0. 04 2. 41 2. 63 2. 12 l. 80

S-week ave. 1. 03 2. 27 2 57

OONOMnb-mwr—

‘ c
Spectrophotometric Rating of Bitterness (Isocoumarin)

 

. 073
. 390
. 510
. 700
. 087
. 180
. 120
. 150

Ave. . 277
5-week ave. . 352

L. S. D.
Organoleptic
Fluorescence

Spectrophotometric 1. 19

.380'

1.883
6.707
4.800
5.457
5.580
7.143
8.523
5.059
3.845

Treatment

1%

. 6
..34
1. 60

. 613
2. 483
6. 583
5. 337

10. 410

7. 653
7. 177
8. 930
6. 148
5. 085

5

l

. 919
2. 173
2. 510
4. 383
2. 857
3. 460
2. 757
3. 357
2. 802
Storage

% 1%

. 6 . 8

..36 . 48

. 68 2. 27

.496
1.732
4.077
3.805
4.703
4.218
4.299
5.240
3.322

. 99
4. 53

 

a, b,c

See footnotes Table I A.

20.

TABLE I (Continued)

C. Late Harvest

 

 

Weeks in

d
Ethylene Iced Average

 

 

 

 

Storage Control Apple Ethylene
Organoleptic Rating of Bitternessa
1 0. 0 0. 7 0. 3 1. 0 0. 3
2 0. 0 1. 3 1. 3 1. 0 0. 9
3 0. 0 2. 7 1. 3 0. 0 1. 3
4 0. 7 3. 0 2. 7 2. 0 1. 9
Ave. 0.2 1. 9 1.4 1.0 1.2
Fluorescence Rating of Bitternessb
1 0. 00 1. 51 0. 98 1. 07 0. 83
2 0. 00 2. 53 2. 07 1. 64 1. 53
3 0. 00 2. 34 2. 15 0. 85 1. 50
4 0. 44 2. 78 2. 58 1. 80 1. 93
5 0. l4 2. 88 2. 73 1. 94 1. 92
6 0. 27 2. 95 2. 67 l. 93 1. 96
Ave. 0. 14 2. 50 2. 20 1. 54 1. 61
5-week ave. 0. ll 2. 41 2. 10
Spectrophotometric Rating of Bitterness (Isocoumarin)C
1 . 440 . 913 . 517 . 702 . 623
2 . 440 1. 647 1. 813 . 976 1. 301
3 . 387 3. 733 3. 170 1. 560 2. 430
4 . 217 4. 383 4. 143 2. 370 2. 581
5 . 490 4. 430 3. 603 1. 440 2. 841
6 . 367 5. 560 3. 867 2. 200 3. 265
Ave. . 390 3. 444 2. 852 1. 541 2. 229
5-week ave. . 394 3. 021 2. 649
Treatment Storage T X S
L. S. D. 5% 1% 5% 1% 5% 1%
Organoleptic . 60 . 85 . 70 . 97 - -
Fluorescence . 21 . 29 . 30 . 41 - -
Spectrophotometric . 41 . 56 . 58 . 78 1. 01 1. 37

a,b

.—

’ CSee footnote Table I A.

C1Ethylene iced carrots are not included in statistics.

21.

22.

TABLE II

Analysis of Variance Summary for the Influence of Age and Storage Treatment
on Development of Bitterness in Chantenay Carrots

 

 

 

‘ _ Or ganoleptic Fluorescence Spectrophotom etric
Factor D‘ R Variance . Variance Variance
A Harvest
Source 1 l. 10 . 27 8. 53
Treat. 2 6. 75’” 5. 99W 178. 15’”
Store. T 4 l. 60* 3. 79W 176. 09* *
S x T 2 . 35 . 37 5. 62
T x ST 8 . 20 . 67* 50. 49*
SxST 4 .13 .06 13.46
Error 8 . 35 . 16 9. 78
Source 2 3. 00M . 26 39. 23
Treat. 2 17. 67" 33. 90M 2342. 40’”
Store. T 7 3. 14’” 1. 75W 327. 83W
S x T 4 . 83 . 26 61. 22
T x ST 14 . 52 . 66M 18. 86
S x ST 14 . 43 . 19 106. 18*
Error 28 . 52 . 19 40. 45
Factor D. F Organoleptic D. F. Fluorescence D. F. Spectrophotometric
Variance Varlance Variance
Source 2 . 58 2 33. 03M 2 1. 56
Treat. 2 9. 75’” 2 275. 97M 2 472. 29’”
Store. T 3 5. 07M 5 17. 75*” 5 97. 17’”
S x T 4 . 84 4 10. 76’” 4 10. 22*
T x ST 6 . 83 10 2. 14 10 29. 17M
SxST 6 .22 10 1.73 10 1.89
Error 12 . 46 20 . 92 20 3. 47

 

*Significant to the 5% level.

MSignificant to the 1% level.

23.

all significantly more bitter than the controls under all three methods of
analysis (organoleptic, fluorescence, and spectrophotometric).

Organoleptically, the average of the first sample from storage was

significantly more bitter than the first sample in harvests B and C; while in
harvest A there was no significant difference between the first sample and the
last sample. Using fluorescent and spectrophotometric analysis in all har-
vests (A, B and C), the averages of the last samples were significantly more
bitter than the averages of the first samples.

The spectrophotometric analysis under all harvests (A, B and C)
show figures which are significantly more bitter for treatment x storage.
while fluorescence analysis under harvests A and C show figures which are
Significantly more bitter than others. Organoleptic estimations show no
Significance for treatment x storage.

In terms of isocoumarin, on the five—week averages for treatments,
C arrots which were harvested in September (B) became considerably more
bitter than those harvested in a more immature (A) or more mature (C)

C 0ndition.

It is also observed that "ethylene prime" carrots did not become as
bitter as those receiving ethylene and appleemanatiohs continuously, but
In Ore bitter than the controls. In the third harvest, the iced carrots were

Observed to lag behind the non-iced in the development of isocoumarin.

24.

As indicated in Table II, the organoleptic estimations for source were
significant for harvest B, and the fluorescence estimations for source were
s ignificant in harvests A and C.

Pit-stored carrots were found non-bitter and non-fluorescent after a
month in storage. After treatment in the drums for ten days it was revealed
that these carrots which were placed in the ethylene drum became fluorescent,
with the mineral carrots having as estimation of 1. 6, the late planted muck
carrots having an estimation of 0. 9, and the early planted muck carrots hav-

in g a rating of 0. 5. The controls remained practically non-fluorescent with

a rating of 0. 1 for both the early and late and 0. 0 for the mineral.

Discussion

A statistical analysis of the data indicated that neither soil type nor
time of planting had a significant effect on the development of isocoumarin
according to the spectrophotometric rating, which is the most exact quantitative
te St. The organoleptic and fluorescent estimations showed highly significant
reSults for source in three cases, indicating that soil type and time of planting
made a difference; however, it must be remembered that these are only sub-
jeetive methods. The statistical analysis of the data also indicate that ethylene,
Whether in the form of apple emanations, or as a pure gas, promoted bitter-
ness, fluorescence, and isocoumarin development in carrots. The pit-stored

ca~1’:rots provided further evidence of this phenomenon when they became bitter

after subjection to ethylene.

25.

The fact that "ethylene prime" carrots were significantly less bitter than
the ethylene and apple emanation treated carrots, and more bitter than the con-
trol, suggests that the degree of bitterness may be related to the quantity of
ethylene present.

The higher values of ethylene treated carrots under all three methods of
analysis as compared with the apple emanation treated carrots from the early

and intermediate harvests on the basis of the five-week average, and the rever-
sal of this tendency in the late harvest, suggests that the more mature apples
used in the later harvest produced a larger quantity of ethylene than was being
adrninistered to the ethylene drum.

The figures Show that the iced carrots lagged behind noneiced carrots in
production of isocoumarin suggesting thatthe icing slowed down the metabolism
0f the carrots which, in turn, slowed down the production of the bitter principle.

The fact that both the spectrophotometric and fluorescent ratings for the
Ethylene treatment on a five-week average are higher for B than for A or C sug-
gests that there is a physiological age when carrots are most susceptible to the
development of isocoumarin. The reason for using the five-week average was to
put A, B and C on a comparable basis. The reason for considering the ethylene
trefitment and not the apple emanation treatment was because the ethylene was
aclrministered in like quantities to all harvests, whereas the amount of ethylene
em anation for apples varied with the variety and maturity of the apples used.

The fact that there was no statistical difference in spectrophotometric measurements

be‘ZWeen planting dates (source) was probably because plantings were too close together.

26.

THE INFLUENCE OF VARIETY ON THE DEVELOPMENT OF BITTERNESS
IN STORED CARROTS
Although neither Bessey (4) nor Atkins (2) found a difference in varie-

tal susceptibility of bitterness, six varieties were subjected to storage studies

i nvolving ethylene.

Methods and Procedure

Arrangements were made with the Ferry Morse Company of Detroit
to procure the following varieties, grown on muck: Touchon, Long Type
Chantenay, Red Core Chantenay, Irnperator, St. Valery, and Gold Pak. On
September 25, the varieties were harvested and a sample of each was exam-
ined for fluorescence. None was found. The remainder of each variety was
divided into four lots and 24 mesh bags were placed into two 55- gallon drums
at 34°F. Both the "ethylene prime" and the control drum received 144 ml. of
air per minute with the ethylene prime drum receiving a charge of 42 ml. of

ethylene gas at the start of the treatment.

Re Sl—llts

After three weeks in storage, a sample was analyzed and although
the taste was not affected by the ethylene treatment, the appearance of fluores-
CenCe was quite apparent. After six weeks when the second and final sample

Was taken, a bitter flavor had developed in these carrots. Table III shows

TABLE III

The Influence of Variety on the Development of Bitterness in Stored Carrots as
Determined by Organoleptic, Fluorescent and Spectrophotometric Ratings

 

 

 

 

 

 

 

at 34° F
Days to Control Ethylene
Variety Maturity 3 wk.- 6 wk. Ave. 3 wk. 6 wk. Ave.

Organoleptic Rating of Bitterness
Touchon 68 0. 0 0. 0 0. 0 0. 0 2. 0 1. 0
L. T. Chantenay 70 0. 0 1. 0 0. 5 0. 0 1. 0 0. 5
R. C. Chantenay 77 0. 0 0. 0 0. 0 0. 0 2. 0 1. 0
Imperator 77 0. 0 0. 0 0. 0 0. 0 1. 0 0. 5
Gold.Pak 77 0. 0 0. 0 0. 0 0. 0 l. 0 0. 5
St. Valery 85 0. 0 0. 0 0. 0 0. 0 1. 0 0. 5
Ave. 0. 0 0. 1 0. 1 0. 0 1. 3 O. 7

Fluorescent Rating of Bitterness
Touchon 68 0. 0 ‘ 0. 0 0. 0 2. 0 l. 8 1. 9
L. T. Chantenay 70 0. 0 0. 0 0. 0 2. 0 1. 3 1. 7
R. C. Chantenay 77 0. 0 0. 0 0. 0 2. 0 1. 6 1. 8
Imperator 77 0. 0 0. 0 0. 0 2. 0 0. 9 l. 5
Gold Pak 77 0.0 0.0 0.0 1.0 1.1 1.1
St. Valery 85 0. 0 0. 0 0. 0 1. 0 0. 7 1. 7
Ave. 0.0 0.0 0.0 1.7 1.1 1.4

Spectrophotometric Ratingof Bitterness
Touchon 68 . 100 ’. 042 . 071 . 837 2. 110 1. 474
L. T. Chantenay 70 . 075 . 071 . 073 . 622 l. 330 1. 475
R. C. Chantenay 77 . 204 . 270 . 237 . 331 l. 870 1. 101
Imperator 77 . 125 . 090 . 158 . 678 1. 370 1. 024
Gold Pak 77 .149 . 196 .173 .702 1.540 1.121
St. Valery 85 . 055 .033 . 044 . 140 . 270 . 205
Ave. . 093 . 084 . 126 . 552 1. 415 l. 067

Treatment Storage Time T. x S. T.

L. S. D. 5% 1% 5% 1% 5% 1%
Organoleptic . 511 . 613 l. 37 1. 51 . 786 -
Fluorescent . 357 . 427 — - - -
Spectrophotometric . 414 . 649 1. 45 — — -

__

27.

28.

the results of these findings, with the varieties listed in the order of maturity.
Table IV indicates the variance summary for the influence of variety of bitter-

ness development.

TABLE Iv

Analysis of Variance Summary for the Influence of Variety on the Development
of Bitterness in Carrots

 

Organoleptic Fluorescent Spectrophotom etric

 

Factor D‘ F' Variance Variance Variance
Variety 5 . 08 . 146 . 204
Treatment 1 2. 05* 12. 586W 4. 498’”
Storage Treat. l 3. 38’” . 286 l. 115*
V x T 5 . 14 . 165 . 159
VxST 1 .07 .047 .066
T x S T 1 2. 03* . 286 . 621
Error 5 . 14 . 068 . 157

 

*Significant to 5% level.
“Significant to 1% level.

29.

Discussion

Carrots which gave a reading of less than 0. 75 were not considered
bitter; therefore, there is apparently good agreement between organoleptic
and spectrophotometric ratings. The fluorescence rating of ethylene treated
carrots in the first sample is shown to be higher than the rating of the second
sample. This is probably due to the fact that the method of rating carrots
for fluorescence was changed in the period between the two samplings. The
rating scale remained the same; however, the carrots in the first sample
were rated by estimating the amount of fluorescence of a particular lot and
assigning a number to it. In the second lot, a value was assigned to each
carrot in the lot, and their average value became the value for the lot. Had
the individual root analysis been used on both the first and second lot, it is
believed that the first lot would have been lower than the second.

Although the varieties all responded to the "ethylene prime" treat-
ment, it is noted that the values for Touchon were higher than the values for
St. Valery. While all varieties were planted on the same day, Touchon was
more nearly mature than St. Valery on the basis of days to maturity. The
difference in bitterness ratings may, therefore, be due to maturity rather

than variety.

30.

THE INFLUENCE OF SOIL TEMPERATURE ON THE DEVE LOPMENT
OF BITTERNESS IN STORED CARROTS
Since carrots appear to vary in their degree of bitterness from year
to year (4), it was thought that soil temperature during growth might influence

the development of isocoumarin in stored carrots.

Methods and Procedure

On July 15, seeds of the variety Nantes, Red Core Chantenay, and Im-
perator were planted in a muck-loam soil mixture, in 48 2-gallon, glazed
crocks. They remained outside until September 5, when they were moved to
a greenhouse, placed in temperature tanks, and thinned to 12 carrots in each
crock. There were 12 crocks for each of four tanks, with the water in these
tanks regulated at 50°, 60°, 70° and 80°F. Three harvests were planned so
that physiological age as well as variety and temperature could be included
in the study of bitterness development. At each harvest, four carrots were
taken from each crock, two of these carrots going into a control drum, and
two into a drum which received only an initial charge of 42 ml. of ethylene
gas (ethylene prime). The harvests were taken on October 8, November 23.
and December 20, and the carrots were stored for 21, 24, and 38 days, re-

spectively.

31.

Results and Discussion

Although two analyses of each harvest were planned, there were not
enough good carrots to make two tests from the last harvest; therefore, only
the results of the first analysis for each harvest appears in Table V. The
quantity of carrots in each sample was too small to obtain a spectrophoto-
metric reading. However, fluorescence observations were made, and a
representative sample was tasted and found to agree with the fluorescence
evaluations. The data in Table V indicate the fluorescence ratings and the
average weights for the roots. "Ethylene prime" carrots were more fluor-
escent than the controls, and temperature influenced the weight of the
roots. Nantes variety grew best at 50° F, while Imperator and Red Core
Chantenay grew best at 60° F. Temperature had no statistically significant
effect upon the development of bitterness.

This experiment showed that ethylene was effective in producing
fluorescence and bitterness in carrots. The more mature carrots in the
last harvest were also more fluorescent than carrots in earlier harvests

With the exception of Red Core Chantenay grown at 50° F.

TABLE V

The Influence of Soil Temperature on the Development of Root Weight
and Bitterness in Stored Carrots

 

 

Fluorescence Rating

 

Temper- Variety Ave. Wt- Control Ethylene Prime

 

 

ature (Gm S) Harvests Harve sts

 

 

2 3 Ave. 1 2 3 Ave.

y...

 

50° Nantes 30. 4 0. 0 0. 0 0. 0 0. 0 1. 3 0. 8 2. 8 1. 6
Chantenay 31. 5 0. 0 0. 0 0. 0 0. 0 3. 0 2. 5 2. 3 2. 9
Imperator 22. l 0. 0 0. 0 0. 0 0. 0 1. 8 1. 0 2. 1 1. 6
Average 28. 0 0.0 0.0 0.0 0.0 2.0 1.4 2.4 1. 9

60° Nantes 28. 3 0. 0 0. 2 0. 0 0. 0 l. 0 2. 5 3. 3 2. 3
Chantenay 41. 7 0. 0 0. 0 0. 0 0. 0 1. 3 2. 2 3. 3 2. 3
Imperator 30. 0 0. 0 0. 0 0. 0 0. 0 0. 0 2. 5 2. 3 1. 6
Average 33. 3 0. 0 0. 1 0. 0 0. 0 0. 8 2. 4 3. 0 2. 1

70° Nantes 24. 6 0. 2 0. 0 0. 1 0. l 2. 3 0. 7 2. 6 1. 9
Chantenay 34. 8 0. 0 0. 0 0. 0 0. 0 0. 8 2. 0 2. 4 1. 7
Imperator 17.2 0.0 0.0 0.0 0.0 1.2 0.0 2.5 1.2 -
Average 25. 5 0. 1 0. 0 0. 0 0. 0 1. 4 0. 9 2. 5 1. 6

80° Nantes 21. 6 0. 0 0. 0 0. 0 0. 0 2. 3 1. 5 3. 2 2. 3
Chantenay 22. 3 0. 0 0. 0 0. 0 0. 0 0. 8 2. 5 1. 7 1. 7
Imperator 13. 8 0. 0 0. 0 0. 0 0. 0 2. 5 2. 2 2. 7 2. 4
Average 19.2 0.0 0.0 0.0 0.0 1. 9 2.1 2.5 2.2

Ave. for treatment 0. 0 2. 0
Treatment
L. S. D. 5% 1%

.37 .50

 

33.

THE INFLUENCE OF STORAGE TEMPERATURE, AREA OF PRODUCTION.
AND SOURCE OF ETHYLENE ON BITTERNESS DEVELOPMENT

Ethylene treatment produced bitterness in different varieties of
carrots grown at different soil temperatures, in carrots grown on different
soils, and in roots of different physiological ages.

This experiment was to determine whether bitterness would occur at
different storage temperatures. Stored carrots were also treated with auto-
mobile exhaust gas and compared with ethylene treated roots to determine
whether the ethylene in the exhaust gas was sufficient to cause bitterness
(33). In some storages in which bitter carrots were found, gasoline lift

trucks had been used.

Methods and Procedure

On January 17, Imperator carrots from California, and Nantes and
Long Type Chantenay carrots from Michigan were purchased. The Impera-
tor carrots were harvested immaturely approximately three weeks prior to
treatment. The Chantenay carrots were overmature, the Nantes carrots
were mature at harvest, and both had been in storage for approximately three
months prior to treatment. Samples of these carrots were tasted and exam-
ined for fluorescence prior to subjecting them to treatment, and no evidence

of bitterness or fluorescence was found.

34.

Three storages at temperatures of 32°, 42° and 52°F were available
for the experiment. Eight drums, each containing seven bags of 16 carrots
from each of the three sources were used. A control and ethylene drum were
placed in each storage, and in addition, the 32° F storage contained carrots
in drums receiving apple emanations and automobile exhaust gas.

A 16-liter jug of apples was placed in the 32°F storage and a gas
analysis of the apple emanation indicated that ethylene was being evolvedl.
The drum receiving the exhaust treatment was taken to the delivery door
entrance twice a week and connected to the exhaust pipe of a running truck
engine for five minutes. The drum was returned to the storage and re-
connected to the air line.

Duplicate, eight-root samples were taken from each drum every 10

days for spectrophotometric and fluorescence determinations.

Results

Since there was no significant difference between duplicate samples,
the values shown in Table VI A and B are averages of the duplicates. The
spectrophotometric rating for treatment and storage period at 32° F for the
three varieties of carrots are given in Table VIA. In less than ten days
after storage all treatments resulted in significant increases in the isocou-
marin content of Imperator and Chantenay carrots. On the basis of the averages

Analysis made by Arleigh Dodson, Department of Agricultural Chemistry,
Michigan State University.

TABLE VI

The Influence of Storage, Temperature, Area of Production, and Source of
Ethylene on Bitterness Development of Carrots in Storage

A. Spectrophotometric Ratings (Temperature 32° F)

 

Days in

 

 

 

 

Control Apple Ethylene Exhaust Average
Storage .
Imperator - Source: California
10 . 081 . 741 . 582 . 772 . 544
20 . 056 . 408 . 563 . 337 . 341
30 . 098 1. 598 1. 364 . 950 1. 003
40 . 127 2. 420 . 894 1. 020 1. 115
50 . 072 l. 948 1. 470 . 710 1. 050
90 .190 2.372 1. 906 .819 1. 322
Average” . 087 1. 423 . 975 . 758 . 811
Long Type Chantenay - Source: Michigan
10 . 000 . 267 . 801 . 692 . 440
20 . 179 . 640 . 530 . 441 . 304
30 . 198 . 842 . 920 . 810 . 693
40 . 172 l. 646 1. 284 . 886 . 997
50 . 157 l. 886 1. 230 . 606 . 970
90 .313 1. 312 1.502 .780 . 977
Average" . 141 1. 056 . 953 . 687 . 681
Nantes - Source: Michigan
10 . 291 2. 495 1. 955 2. 145 1. 722
20 . 631 4. 140 3. 665 1. 390 2. 457
30 . 701 3. 046 5. 600 2. 186 2. 883
40 . 812 3. 800 2. 766 2. 542 2. 480
50 . 646 3. 676 3. 950 3. 052 2. 831
90 1. 157 6. 740 3. 900 2. 860 3. 664
Average* . 616 3. 431 3. 587 2. 263 2. 475
Ave. all var. . 281 1. 970 1. 838 l. 236 1. 322
Treatment Time in Storage T x S
L. S. D. 5% 1% 5% 1% 5% 1%
Imperator . 283 . 397 . 317 . 444 . 633 . 887
Chantenay . 181 . 254 . 202 . 283 . 404 . 567
Nantes 1. 174 1. 646 - - - -

 

*Statistical evaluations and averages do not include the values for 90
days in storage.

TABLE VI (Continued)

B. Fluorescence Rating (Temperature 32° F)

 

Days in

 

 

 

 

Storage Control Apple Ethylene Exhaust Average
Imperator - Source: California
10 0.0 1.3 1.0 1.3 0.9
20 0.0 1.8 1.1 1.1 1.0
30 0.0 1.8 1.6 1.3 1.2
40 0. 0 3. 3 2. 5 3. 5 2. 3
50 0.0 2.5 1. 9 0.5 1.2
90 0. 0 3. 2 3. 9 2. 4 2. 4
Average" 0. 0 2. 1 l. 1. 5
Correlation with spectrophotometric rating: r . 800
Long Tge Chantenay - Source: Michigan
10 0. 0 0. 3 0. 7 0. 5 0. 4
20 0. 0 0. 0 0. 5 0. 1 0. 2
30 0. 1 0. 6 0. 7 0. 9 0. 6
40 0. 1 0. 9 0. 6 1.1 0. 7
50 0.0 1.2 0.8 0.0 0.5
90 0. 8 0. 6 1. 0 1. 5 1. 0
Average* 0. 0 0. 0. 7 0. 5
Correlation with spectrophotometric rating: I . 827
Nantes - Source: Michigan
10 0.1 1.7 1.9 0.9 1.2
20 . 0.6 1.3 1.5 0.3 0.9
30 0.7 2.1 1.7 1.1 1.4
40 1.1 3.1 2.7 3.1 2.5
50 0. 8 3. 0 2. 9 2. 1 2. 2
90 2. 3 3. 9 3. 6 1. 7 2. 9
Average” 1. 7 2. 2 2. 1 1. 5
Ave. all var. 0. 2 1. 1. 1. 2
Correlation with spectrophotometric rating: r . 442
Treatment Storage
L. S. D. 5% 1% 5% 1%
Imperator . 25 - - -
Chantenay . 44 . 62 - -
Nantes . 57 . 80 . 64 . 90

 

*Averages do not include the values for 90 days in storage.

36.

37.

of the five sampling periods, all treatments resulted in marked increases in
the spectrophotometric readings for the Nantes variety. Figures 3 and 4 in-
dicate that the isocoumarin or fluorescent content of Imperator and Chantenay
carrots increased more slowly during storage than Nantes variety. After

ten days in storage, the Nantes variety reached an average isocoumarin level
which was higher than either of the other varieties.

The average for all varieties shows the spectrophotometric readings
highest for apple emanations, lower for ethylene, and lowest for exhaust.
These differences are probably due to the concentration of ethylene in the
drums during the course of the experiment.

The fluorescent ratings of treatments at various storage periods for
the same three varieties of carrots are given in Table VI B. All treatments
increased fluorescence in all three varieties of carrots. Only Nantes showed
a significant increase in fluorescence for continued storage after ten days.

A correlation between spectrophotometric and fluorescent ratings were found
for all varieties.

In Figures 5, 6, 7 and 8 are Shown the comparative fluorescence
effect of ultra-violet light of wavelengths of 3650 A with the Imperator and
Nantes carrots. Figures 5 and 6 show cross sections of Imperator and
Nantes carrots which have been treated with ethylene, apple emanations

and engine exhaust fumes, and photographed under three types of light. The

38.

Figure 3. Spectrophotometric ratings of isocoumarin in three varieties of

 

carrots under four storage treatments at 32° F.

 

 

 

 

 

 

 

 

 

 

 

 

 

Imperator
Rating
2 _\| "____..-- apple
/” “"""-_’ . . "" ethylene
II" . . ' ' o O . O
l 4 f" 2-2-LL": .
Erica-7'” ’ \ .,__ __ _ _._— — —- - ° exhaust
\ /
’ _Er v.‘ —: control
10 20 30 40 50 90
Days in Storage
Chantenay
2 T __._ ~
I. ~~~~~~~~~ w_ _ . apple
“a .o . “ ‘ '0 ethylene
1 i. . 9;- - , . exhaust
‘\.\-‘." ’7", \‘I— —-""""" f.- .. ————
_ § _ / . . ~ control
.’ \D
1 20 36 D 50 so
Days in Storage
Nantes
7 .
I: apple
I /
6 j //
/
. /
. /
o /
5 J . o //
o I/ I
41 /.\.‘. .. ot/c/Io -. . s ...... ..o
// .\\ ,"’._~?.., ethylene
/ ' \ \ // z o
34 ’3 ‘./ ' °' .—__.__._.____. -
’//'.,' :;/’ 7 ‘ exhaust
o .’ /'
2 T; /’
\../
1 1 ' control
/'—'H/.\./
10 26 3o 45 56 96

Days in Storage

39.

Figure 4. Fluorescent ratings of isocoumarin in three varieties of
carrots under four storage treatments at 32° F.

 

 

 

Imperator
Rating
4 ‘ , . ethylene
/.\ . . ' .

3 , //°\‘ .L; , —— -—"° apple

If 3..“ ‘.”_:°".' /° exhaust
2 ._ - - - ./7 ' \0. . I

P ’ ’ ' O " \ / /
l q‘ fi-‘o"‘/’. \ I /
\ , /
e f 7' e - control
10 20 30 40 50 90
Days in Storage

2 . Chantenay

exhaust

ethylene
control

apple

 

 

 

Days in Storage

Nantes

4 ‘ -—"' apple

ethylene

control

exhaust

 

 

 

Days in Storage

40.

lower photographs were taken under 2537 A ultra-violet light, which was used
exclusively for determining the fluorescence rating of carrots. The rating
for each carrot increased as the intensity of the fluorescent speckling in its
phloem. Figures 7 and 8 illustrate external fluorescence of Imperator and
Nantes carrots also treated with ethylene, apple emanations and exhaust
fumes, and photographed under three sources of light. It is noted that Im-
perator has only three fluorescent specks under 2537 A ultra-violet light.
while Nantes carrots are quite fluorescent under all treatments. Although
this external fluorescence is not used as a criterion of bitterness, it is
indicative of high intensity of speckled phloem fluorescence.

The influence of storage temperature upon the development of fluores-
cence and isocourmarin under control and ethylene treatment as determined
by spectrophotometric and fluorescent ratings are given in Table VIII, Since
Chantenay carrots deteriorated in 42° and 52° F storage, they were not in-
cluded in the statistical analysis. The analysis of the data indicated that tem-

perature was not a significant factor in the development of isocoumarin.

Discussion

On the basis of the results obtained, storage temperature in the range
from 32° to 52°F did not differentially influence the development of bitter-
ness. Carrots from each of the three sources all became bitter when treated

with ethylene regardless of the temperature.

TABLE VII

41.

Analysis of Variance Summary for the Influence of Treatment on the Develop-
ment of Isocoumarin in Different Varieties of Stored. Carrots at 32° F as
Determined by Spectrophotometric and Fluorescent Methods

 

 

 

 

 

Variance
Factor D' F' Imperator Chantenay Nantes
Spectrophotometric Analysis
Replication 1 4, 601 257, 282 53, 144
Treatment 3 3, 096, 795’Ml 1, 443, 344’” 18, 839, 233W
Storage Period 4 956, 689‘” 767, 750’” 1, 723, 432
T x SP 12 316, 129* 270. 803’” 1.239. 814
Error 19 101, 908 74, 448 1, 091, 586
Fluorescence Analysis
Treatment 3 425. 6** 40. 0* 268. 3’”
Storage 4 132. 3* 16. 5 63. 1*
Error 12 32. 2 10. 3 l7. 3

 

*Significant at 5% level.
MSignificant at 1% level.

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43.

This experiment is in agreement with previous work and demonstrates
that auto exhaust fumes which contain ethylene, can induce bitterness in car-
rots. It also indicated the possibility that the development of isocoumarin in
carrots is a function of the quantity of ethylene and of time.

The fact that Nantes carrots responded more to treatment than either
the Imperator or the Long Type Chantenay suggests that the effect is probably
related to maturity. The Nantes carrots were grown to full maturity for
fresh market, harvested and pit-stored until shipped to market in January.
The Imperator carrots from California were harvested and packaged im-
mature for the fresh market a week prior to being purchased on the market,
while the Chantenay carrots were planted as early as weather permitted in
the spring, and grown as long as the weather permitted in the fall, so that
maximum yield was produced. The Chantenay carrots were probably over-
mature at time of harvest, while the Imperator carrots were probably too
immature to respond to ethylene with as high a production of isocoumarin as
produced by the Nantes carrots.

The treatment did not have identical effects on the three varieties,
even though they were stored in the same drum. Chantenay had the highest
bitterness rating under apple emanations, with both methods of analysis.
Nantes was shown to be equally affected by ethylene and apple emanations.

Ethylene may produce metabolic changes that cause bitterness.

44.

therefore the quantity of ethylene should be a factor in determining the amount
of bitterness produced. It is possible that the quantity of ethylene adminis-
tered exceeded the quantity of ethylene given off by the apples during the
first few weeks of storage, and later the situation reversed itself. Linking
this with the relative maturity1 of the carrots, the Chantenay variety might
have reached the end of their receptive period when the ethylene adminis-
trations were highest, and therefore became more bitter under ethylene
treatment. The Nantes might have been nearing the end of their receptive
period as ethylene in the apple emanations were surpassing the ethylene
administrations. The Imperator carrots were quite immature, and retained
their susceptibility to ethylene after the apple emanations of ethylene sur-
passed the administered ethylene and, therefore, they showed higher ratings

with apple emanations.

A mature orange type carrot was considered to be one which has
grown for more than 60 days, is orange to the tap root, and has stumped
at the junction of the tap root and fleshy root.

An immature orange type carrot is one which has grown for less
than 80 days, has no shoulders or color change to clearly define the junction
of the root with the tap root.

An overmature orange type carrot is one which has grown for over
100 days.

Figure 5. Fluorescence of cross sections of Chantenay carrot
roots. (Photographs of the same carrots taken under '
different lights).

A. Photoflood reflector lamp
B. Ultra-violet light 3650 A. wavelength
C. Ultra-violet light 2537 A wavelength

Vertical rows from left to right:

Fluorescence intensity No. 4 rating
Fluorescence intensity No. 3 rating
Fluorescence intensity No. 2 rating
Fluorescence intensity No. 1 rating
Fluorescence intensity No. 0 rating (single carrot section)

Horizontal rows from top to bottom:
Ethylene treated carrots

Apple emanation treated carrots
Gasoline engine exhaust treated carrots

 

 

 

Figure 6. Fluorescence of cross section of Imperator carrot
roots. (Photographs of the same carrots taken under
different lights).

A. Photoflood reflector lamp
B. Ultra-violet light 3650 A wavelength
C. Ultra-violet light 2537 A wavelength

Vertical rows from left to right:

Fluorescence intensity of No. 4 rating
Fluorescence intensity of No. 3 rating
Fluorescence intensity of No. 2 rating
Fluorescence intensity of No. 1 rating
Fluorescence intensity of No. 0 rating (single carrot section)

Horizontal rows from top to bottom:
Ethylene treated carrots

Apple emanation treated carrots
Gasoline engine exhaust treated carrots

46.

 

Figure 7. Fluorescence of Imperator and Chantenay carrots under

ethylene treatment (photographs of two groups of carrots).

A and D - Under photoflood reflector lamp

B and E - Under ultra-violet light 3650 A wavelength
C and F - Under ultra-violet light 2537 A wavelength

The carrots on the left are Imperator (A, B and C), and
the carrots on the right are Chantenay (D, E and F). The
carrots in each photograph are arranged from left to right
according to their cross sectional fluorescence rating, with
No. 4 rating on the left to No. 1 rating on the right.

Note external fluorescence in Chantenay carrots (F) as
compared to lrnperator carrots (C). which show only three
tiny specks as indicated by arrows.

 

Figure 8. Fluorescence of Chantenay carrots under apple emana-
tions and gasoline engine exhaust treatment (photographs
of two groups of carrots).

A and D - Under photoflood reflector lamp
B and E - Under ultra-violet light 3650 A wavelength
C and F - Under ultra-violet light 2537 A wavelength

The carrots on the left were treated with apple emanations
(A, B and C), while the carrots on the right were treated with
gasoline engine exhaust (D, E and F). The carrots in each
photograph are arranged from left to right according to their
cross sectional fluorescence rating, with No. ,4 on the left,
and No. 1 on the right.

 

 

 

 

49.

THE EFFECTS OF ETHYLENE ON WHITE, YELLOW AND ORANGE
CARROTS FROM WISCONSIN

On November 9 a sample of white, yellow and orange fleshed carrots
were obtained from Wisconsin. These carrots were all examined for fluores-
cence with the yellow and white carrots revealing no fluorescence, while
some of the orange carrots revealed fluorescence of two types. The first
type was the speckled isocoumarin type, located in the phloem, and the
other type consisted of glowing areas located in the core.

After a month of ethylene treatment the yellow carrots. Yellow A
(Yellow Belgium selection A-1630-5 DSCD) and Yellow B (Yellow Belgium
University of Wisconsin), and the white carrots showed the following spectro~

photometric values:

Control Ethylene
White . 098 2. 13
Yellow A . 000 3. 12
Yellow B . 123 4. 15

The orange carrots exhibited a strong fluorescence, indicating a high iso-

coum arin content.

Discussion
Because carotene content increases in storage (5), it has been sug-

gested that the bitter principle is formed from some precursor of carotene.

This experiment with yellow and white carrots indicates that this is
not the case since white carrots developed an isocoumarin content of con-
siderable magnitude. However, as the yellow carrots developed more iso-
coumarin than white roots, carotene may have a role in the development
of high isocoumarin values.

The fact that the orange carrots which showed a core fluorescence
were not bitter, indicates that isocoumarin was not causing the fluorescence.
When they were treated with ethylene, they developed the isocoumarin—type

fluorescence in addition to the core fluorescence.

51.

THE EFFECTS OF ETHYLENE GAS UPON THE RESPIRATION OF
CARROTS IN STORAGE
Since ethylene was presumed to influence some metabolic processes
in carrots, it was suggested that it might effect the respiration rate. The
atmosphere of the control and ethylene drums were sampled for carbon di-
oxide to determine if there was any increase in respiration due to the addi-

tion of ethylene.

Methods and Procedure
An Orsat gas analyzer was used to determine the carbon dioxide
in the drums which contained the Chantenay carrots under storage treat-
ment. The analyzer was inserted into the air line to the drum, and three
samples were removed. The first two were expelled, and the third was analy-

zed for carbon dioxide.

Results

Figure 9 shows the results of periodic analysis of the carbon dioxide
contents of the control and ethylene drums during a six-week period. The
graph indicates a generally declining quantity of carbon dioxide until Septem-
ber 6, when fresh carrots were added. The carrots in both the control and
ethylene drums responded by an increase in carbon dioxide accumulation;

however, the carbon dioxide of the ethylene drum increased until September

53.

16, when it reached a high of 3. 4 per cent, compared to a high of 1. 4 per cent
for the control on that same date. In both cases the carbon dioxide concen-
tration fell off until October 5, when more carrots were put into the drums

for treatment, after which it started to rise again.

Discussion

This experiment was conducted to check the respiration of the carrots
which were undergoing storage treatment. The results were so striking that
carbon dioxide concentrations were measured in the apple emanation drum
and the ethylene prime and control drums. These results further indicated
that ethylene stimulated the evolution of carbon dioxide.

Since the Orsat analyzer is not designed to make respiration deter-
minations, it was decided to forego these samplings and conduct an experi-
ment on the effects of ethylene on carrot respiration at a later date with the
proper equipment. The attempt which was made, using the proper respira-
tion equipment was unsuccessful; therefore, the previous results have been
presented to indicate the effects of ethylene upon the respiration of carrots.

The immediate rise of carbon dioxide concentration in the control
drum is due to the high respiration of the warm roots. As the roots are
cooled, their respiration and carbon dioxide evolution decreased. It further
decreased as carrot samples were removed. When the next lot of carrots

was added, only a few samples of the original lot remained.

54.

GENERAL DISCUSSION

This study has demonstrated that ethylene in the storage atmosphere
will cause rapid metabolic changes to take place in the roots of carrots that
result in the production of bitterness of the isocoumarin type. This effect
takes place in less than ten days, and may be brought about not only by
ethylene gas, but also by emanations from apples and gasoline engine ex-
haust fumes. The incipient development of this type of bitterness in carrot
roots is readily detected by the fluorescence of the bitter product of this
metabolic activity in the phloem tissue adjacent to the cambium. At this
stage the concentration of isocoumarin is too low to be readily detected
organoleptically and the roots are still edible. The high degree of corre-
lation between the isocoumarin content in the root and its fluorescent value
introduces a rapid method of detection of bitterness in carrots. F luores-
cent measurements may be utilized in detecting carrots that will become
bitter before they are organoleptically bitter.

There is evidence that immature or overmature carrots do not
develop isocoumarin as rapidly, or to as high a content as that found in
mature carrots. In immature carrots perhaps the substrate required for
the development of isocoumarin may be partially limiting; in overmature

carrots the low respiratory rate and the possible loss or conversion of

some of the precursor of isocoumarin reduces the rate of development and
total quantity of the bitter principle formed. Therefore, in fresh market
carrots, which are harvested immature and consumed shortly after harvest.
bitterness is not likely to be a problem. Carrots that are harvested at

full maturity for subsequent processing should be stored in an atmosphere
free of ethylene and processed at once if any fluorescence is detected.

The variety, root color, soil temperature or type, storage tem-
perature, or area of production, seemed to have little bearing upon the
qualitative development of bitterness in storage after ethylene was injected
into the storage atmosphere.

Whether or not ethylene enters into the synthesis of the bitter
principle or the carotene and the isocoumarin precursors are the same
compound remains to be demonstrated.

The association of ethylene with these biochemical and physical
phenomena has not been heretofore reported in the literature for any crop
and may possibly find use in evaluation of the influence of ethylene on

other products.

56.

SUMMARY AND CONCLUSION

Ethylene has been demonstrated to be effective in producing bitter-
ness, fluorescence, and isocoumarin as determined by organoleptic, ultra-
violet, and spectrophotometric tests, in all carrots subjected to ethylene
treatment. The carrots used in these tests included carrots of three dif-
ferent colors (orange, yellow, and white), of nine different varieties, of
three physiological ages (immature, mature, and overmature), produced
on two major soil types (loam and muck), in two states (Michigan and
California), grown at four soil temperatures (50°, 60°, 70° and 80° F),
planted and harvested during four different months, and stored at five
different temperatures (32°, 34°, 42° and 52° F).

The ethylene was administered in two ways to the storage atmos-
phere, which was receiving one complete change of air each day. The
first method was to apply ethylene to a concentration of 200 ppm every
48 hours, and the second method was to apply an initial atmosphere of
200 ppm and allow it to be dissipated without being replenished. Both
treatments were effective in producing bitterness, fluorescence, and
isocoumarin in carrots, although the second method was not as effective
as the first, leading to the assumption that the degree of bitterness is

related to the quantity of ethylene.

57.

Two other treatments consisted of apple emanation and automobile
exhaust fumes, both of which contain ethylene. These treatments were
also effective in producing bitterness, fluorescence and isocoumarin in
carrots to a lesser or greater extent than the ethylene treatments. The
degree to which these phenomena were expressed is believed to be contin-
gent on the amount of ethylene contained in these gases.

Of the variable factors among the carrot samples (color, variety,
physiological age, soil temperature and type, planting and harvesting time.
area of production, and storage temperature) only color and physiological
age had any modifying effect on the development of bitterness, fluorescence,
and isocoumarin. These factors were not statistically proven, however,
possibly because the experiment was not designed to specifically test these
factors.

The white carrots did not become as bitter as the yellow ones, nor
the yellow carrots as bit ter as the orange, indicating that, if the precursor
to carotene is involved in the formation of the bitter principle, as has been
suggested, this precursor is also present to a limited degree in white carrots.

Physiological age of the root seemed important since mature carrots
became much more bitter and fluorescent and contained more isocoumarin

after identical treatment than either the immature or overmature carrots.

This phenomenon was noted in three experiments.
Further experiments will be required to determine whether similar
results can be obtained in successive years, and to statistically test the

observations regarding physiological age and color.

58.

3.

6.

9.

10.

11.

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