Group Title: Citrus Station mimeo report - Florida Citrus Experiment Station ; CES 65-4
Title: Thiofurfural as an important factor in the development of off-flavor in canned orange juice during storage
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Title: Thiofurfural as an important factor in the development of off-flavor in canned orange juice during storage
Series Title: Citrus Station mimeo report
Physical Description: 7 leaves : ill. ; 28 cm.
Language: English
Creator: Blair, J. S
Citrus Experiment Station (Lake Alfred, Fla.)
Florida Citrus Commission
Publisher: Florida Citrus Experiment Station :
Florida Citrus Commission
Place of Publication: Lake Alfred FL
Publication Date: 1964
 Subjects
Subject: Canned orange juice -- Quality -- Florida   ( lcsh )
Orange juice -- Flavor and odor -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Includes bibliographical references (leaf 7).
Statement of Responsibility: J.S. Blair.
General Note: Caption title.
General Note: "October 6, 1964."
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Bibliographic ID: UF00072425
Volume ID: VID00001
Source Institution: University of Florida
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Resource Identifier: oclc - 75969310

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Citrus Station Mimeo Report CES 65-4
October 6, 1964


Thiofurfural as an Important Factor in the Development of Off-Flavor
in Canned Orange Juice During Storagel

J. S. Blair2
Florida Citrus Commission
Lake Alfred, Florida


Twelve years ago the author and co-workers reported (3) results of an in-
vestigation of possible causes of flavor deterioration which take place during
storage of canned orange juice. In that study, Valencia orange juice experi-
mentally canned at the Florida Citrus Experiment Station, in 1947 and again in
1948, was in each case placed for 400 days in controlled storage at 450, 700,
and 950F. In the American Can Company laboratory, infrared spectrophotometric
examination of the oil phases, distilled at atmospheric pressure from these
stored samples, indicated that certain acid-catalyzed hydration-dehydration
("ACHD") reactions had taken place in the terpenic constituents of the oil.
These appeared to be much less extensive in 700F. than in 950F. storage. Com-
parison with known samples indicated that these changes had produced terpenic
hydrocarbons, alcohols, and ethers not present, or present in much lower con-
centration, in the oil distilled from samples stored continuously at 450F.
These changes were offered as explanatory of a large part of the "terebinthine"
off-flavor particularly characteristic of the samples stored at 950F.

The samples stored continuously at 700F. exhibited a pronounced off-flavor
which seemed to be different in character from that found in the 950F. stored
samples. For want of a better descriptive term, the word "stale" was applied
to this flavor effect. Some evidence was offered in support of the idea that
this effect was in part due to an increase in the content of steam-volatile
free fatty acids, dissolved in the oil phase. But the statement was made that
"certain sulfur compounds" were also involved in this flavor effect. Mr. J. E.
Masters carried out careful work, using the sodium fusion method for detecting
organically-combined sulfur or nitrogen and found that sulfur compounds were
present while nitrogen compounds were absent in the distilled oil phase. In
the earlier paper (3) it was not intended to convey the idea that the "stale"
flavor components were characteristic of canned orange juice stored only at
700F, Certainly these components must be present in still higher concentration
after 950F. storage, but here their reception is interfered with by the pres-
ence of "ACHD" off-flavor components.

1 This paper is based upon research initiated by the American Can Company, but
was completed through assistance from the Florida Citrus Commission and the
Florida Citrus Experiment Station.

2 Previously employed by the Research Division of the American Can Company,
Barrington, Illinois; partially employed during 1963 and 1964 by the Florida
Citrus Commission following his retirement.
Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/6/64 JSB








-2-


While doing this work, the distilled oil phase was shaken with dilute
sodium hydroxide solution. This treatment effected a great improvement in
the odor of the oil. When the canned juice, from which the oil was derived,
had been stored at a temperature never higher than 700F., it seemed that most
of the agreeable odor of orange oil was restored. The phrase, "freshening of
the odor", was used by several observers to indicate an impression that the
sodium hydroxide treatment had removed some component or components which
interfered with olfactory perception of the fragrant components still actually
present. However, there were also other comments to the effect that an un-
treated portion of the distilled oil, as directly compared with a treated
portion of the same oil, could be called almost "skunky" in odor. The nature
of this malodor was strongly reminiscent of an olfactory impression obtained
in earlier work with canned products, other than citrus juices, in which the
matter of interest was the presence of furfural. There had been need for an
analytical procedure for the detection of furfural. The pentacyanoammono-
ferroate method, general for unsaturated aldehydes, but as we have found
especially sensitive for furfural, was chosen. The method is described in
every edition of Feigl's monograph on "Spot Tests". The ferroate reagent,
prepared by the interaction of sodium nitroprusside and ammonia, gives a
strong blue color with furfural but only in the presence of sulfide. Actu-
ally, in this case the test is for thiofurfural, and in making the test the
"skunky" odor of thiofurfural is very perceptible.

When the above investigation (3) was terminated, the general concept
arrived at was that hydrogen sulfide, which is present even in fresh orange
juice (11) but is considerably augmented by heating and storage (9), tends to
react as is well known (4, 5, 10), with aldehydes to produce thioaldehydes.
Such thioaldehydes differ considerably in odor from the aldehydes themselves
and in a way which would certainly be considered malodorous in a citrus pro-
duct. However, preliminary evidence had indicated that furfural was the
particular aldehyde which was of most concern in this problem. It was
desirable to publish a second paper presenting this evidence, but it was
felt that some further confirmation was needed, and other work intervened. It
was not until 1963 that the support of the Florida Citrus Commission together
with the interest and cooperation of the processed products group at the Florida
Citrus Experiment Station made it possible to resume this investigation and seek
confirmatory evidence. Results presented in this report are largely qualitative
but nevertheless quite persuasive in regard to the concept just described. Fur-
fural appears to be essentially absent in fresh juice (1, 12) but develops in
canned juice, principally during storage. A quantitative study of the rates at
which furfural develops during storage of commercially canned orange juice at
various temperatures is in progress at the Citrus Experiment Station.


Experimental Procedure and Results

Tests for furfural. Two tests were used in the qualitative analyses for
furfural in aqueous solutions obtained from various sources during studies,
which were carried out at the Citrus Experiment Station in 1963. The ferroate

Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400- 10/6/64 JSB








-3-


test, as previously mentioned, gives a blue color and the benzidine test (14) a
reddish or "magenta" color. Both tests were applied in M/10 citrate buffer, pH
3.5, rather than in dilute acetic acid normally used for these tests. The two
tests were found to be of about equal sensitivity for furfural, detecting with
ease 2 micromoles or 192 micrograms in 10 ml. aqueous solution. Both tests
seem quite specific for furfural; the only other aldehyde giving colors, say
even 20% as strong as those given by furfural, is hexadienal, which is a very
unlikely natural component in citrus. Citral gives very feeble colors indeed,
and does not interfere significantly,

Recovery of furfural from canned orange juice. When 1500 ml. of canned
Valencia orange juice after 10 months storage at 800F. was distilled, after
adding sufficient NaOH to raise the pH level to about pH 5.5 to prevent form-
ation of furfural during boiling, five successive 10 ml. portions of aqueous
distillate each gave a barely perceptible but nevertheless positive test for
furfural.

When 1500 ml. of canned Hamlin orange juice after 39 months storage at
800F. was distilled under the same conditions, and five 10 ml. portions of
aqueous distillate were collected for the benzidine test, alternating with five
10 ml. portions for the ferroate test, both tests gave very strongly positive
results for the presence of furfural, and in each series the five tubes showed
equality of color, showing that much furfural doubtless remained in the distill-
ing flask.

Even though two different varieties of orange were involved in this com-
parison, it seems a safe conclusion that the main cause of the difference in
results was the difference in duration of storage at 800F. In view of the
quantitative study which is now in progress, only these qualitative statements
will be made at present.

Synthesis of thiofurfural. Thiofurfural was prepared under nitrogen in
a specially designed apparatus, the details of which will not be discussed here.
Measured amounts of furfural and sodium sulfide were mixed in a citrate buffer
at pH 3.5. Sufficient time was allowed for reaction (40 min.) and a sample was
then withdrawn. This gave a very strong ferroate test for thiofurfural and
exhibited a strong, characteristic "skunky" odor.

The system was kept under nitrogen and two days later this same reaction
mixture gave a negative test for thiofurfural, although the "skunky" odor was
still strong. Also, 35 days after the synthesis, the odor was weak, vaguely un-
pleasant or stale, and could hardly be called "skunky". However, when the mix-
ture was again heated and stirred, a strong "skunky" odor reoccurred and a
strong ferroate test was again obtained. These changes are attributable to
slow polymerization of thiofurfural at room temperature with subsequent depoly-
merization by heat as further discussed below.

Distillation of stored canned juice and treatment of the distilled oil
phase with AgN03. Fifteen hundred milliliters of canned Valencia orange juice
which had been in 80F. storage for eight months was distilled in the apparatus
previously described (3), which enabled the oil to be conveniently collected

Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida. 400-10/6/64 MSB








-4-


for examination. This juice had been deliberately packed with a high oil con-
tent, 0.054% by the usual Clevenger method. The oil was divided into two equal
portions, each in small vials covered with black paper. Four drops of 3% AgNO3
solution were added to one vial, and four drops water to the other. The vials
were closed with ground-glass stoppers and then shaken. Then they were submitted
to six observers with no knowledge of the samples. All observers noted a marked
difference in odor, in favor of the AgNO3-treated sample, although only three
used the word "skunky" to describe the odor of the untreated sample. The other
three observers merely said, essentially, that the untreated sample smelled as
would be expected of oil from warm-stored canned juice, and that the treated
oils smelled better than this.

Fourteen days later the experiment was repeated. The distilled oil phase
was divided into three portions, two of which were treated with AgNO3 and the
third shaken with water only. Three expert observers had no difficulty, in
this triangle test, in singling out the untreated sample as relatively malodorous.

Addition of synthetic thiofurfural in the distillation of limonene with
water. Using the apparatus described previously (3), 1500 ml. of water, 2 ml.
of limonene and 8 ml. of the synthetic thiofurfural mixture, previously prepared,
were heated to boiling and the steam distilled limonene collected. One portion
of limonene was treated with AgNO3 and the other with water only. Four out of
five observers preferred the treated sample, using such words as "skunky" for
the untreated sample and "fresher" for the treated one.

Mechanism of furfural formation. Comparison of ascorbic acid and D-xylose
as progenitors of furfural. That furfural is derivable from ascorbic acid when
heated in M/10 citrate buffer, pH 3.5, and not at all from D-xylose under the
same conditions, was shown by an experiment in which furfural was tested for by
use of benzidine. Solutions of ascorbic acid and of D-xylose, both 0.020 molar,
were prepared in this buffer solution, and from these, by dilution with the
buffer, 0.001 molar solutions were also obtained. Ten-milliliter portions of
these four solutions were placed in test-tubes and heated by immersion in a
boiling water bath for 2 hours. Benzidine reagent was added to each cooled tube.
All of the xylose systems and an unheated ascorbic acid system remained complete-
ly colorless. The heated ascorbic acid systems all developed a yellow or tan
color. Maximum color seemed to be reached in 60 minutes.


Discussion of Results

The aqueous phase of distillate from canned Valencia and Hamlin orange
juices, which had been stored at 800F. for 10 and 39 months, respectively, were
found to contain furfural when qualitatively analyzed using either the ferroate
or benzidine tests. This confirmed preliminary evidence, previously obtained by
the author, that furfural was involved in changes that cause off-flavors which
developed in canned orange juice during storage. Also, Kirchner and Miller
(12) in large-scale work found 5.21 mg. of furfural per kilogram of canned
Valencia orange juice after storage for three years at "ordinary temperature".
However, they reported only a trace in fresh or freshly-canned juice.


Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/6/64 JSB











Hydrogen sulfide has been reported (11) to be in fresh orange juice.
Jansen and Jang (9) found that it is considerably augmented by heating and
storage.

Aldehydes react (4, 5, 10) with hydrogen sulfide to produce thioaldehydes,
Synthetic thiofurfural was prepared, as reported above, by the reaction of
furfural and sodium sulfide in a citrate buffered solution of pH 3.5. The
chemical and physical properties of thiofurfural were quite thoroughly investi-
gated by Baumann and Fromm (2) and have not been investigated more recently ex-
cept by Meintz and Wilkinson (15). They confirmed the results of Baumann and
Fromm (2) in regard to certain physical properties. These findings are in
accord with the generalization by Campaigne (5) that "it is highly questionable
whether a monomeric aliphatic thial has ever been prepared". From the inter-
action of sulfide with furfural at low temperature, Baumann and Fromm (2) re-
port the formation of two different trimers, but under other conditions they
report the formation of polymericc thiofurfural" of the "astonishingly high"
molecular weight of more than 2000. When thiofurfural was synthesized by the
author, the oily substance formed was very insoluble in water and the mixture
at first gave a strong blue color with ferroate reagent, but on standing lost
this property. Feigl remarks that the ferroate test is given only by the
"monomeric" thioaldehydes initially present. The intense "characteristic"
malodor also disappears, but only on more prolonged standing, being eventually
replaced by an odor described in the records of the present work as "slightly
stale, vaguely unpleasant, not skunky". However, upon then heating this
system, the "skunky" odor reappears, and the ferroate test again becomes
strongly positive.

We are now confronted by the necessary assumption that the flavor effect
of thiofurfural in stored canned orange juice, tasted at room temperature, must
be for the most part an effect of the polymerized material, not predominantly
the "skunky" odor of relatively unpolymerized thioaldehyde. Furthermore, we
must take into account the evidence of Kipnis and co-workers (10) that thio-
aldehydes are reducible to mercaptans, and possibly the evidence of Cairns and
co-workers (4) that even without reduction, malodorous disulfides can be formed.
In the present work, synthetic thiofurfural, partly polymerized, was added to
citrate buffer dispersions of limonene and this mixture distilled. The distilled
limonene phase reproduced the observations with the distilled oil phase from
stored canned juice very well as to a "skunky" odor removable from the oil by
silver nitrate. But it is difficult to add synthetic polymeric thiofurfural to
a model system, for tasting at room temperature, with any expectation of repro-
ducing the same physical and all the chemical circumstances as those present in
stored canned orange juice.

During the 1963 study in Florida it was found that neither n-hexanal (which
gives no color with the reagent), nor 2-hexenal, 2-octenal, 2-nonenal, nor
citral (which give relatively very weak colors), gave with dilute sulfide
solution, in pH 3.5 citrate buffer, any odor so outstandingly perciptible above
the hydrogen sulfide odor as that given by furfural. All of the aldehydes were
used in roughly the same, very low concentration. The other thioaldehydes seemed,
at most, to be weakly onion-like; thiofurfural was unique in exhibiting a "skunky"
attribute.

Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida. 400-10/6/64 JSB










It. had been previously observed by Blair and co-workers that the odor of
the oil, recovered when stored canned orange juice was distilled, was much im-
proved when it was treated with AgNO3; also, that the "skunky" aroma was removed.
These observations were confirmed by the results, as reported above, obtained
during this investigation.

It is essential to the present postulate to think of thiofurfural as slowly
forming during storage of canned orange juice and dissolving in the discrete oil
phase as fast as it is formed. The source of the furfural is not from hydroly-
sis of pentose, as might at first be thought because of the fact that in the
presence of hot strong mineral acids, pentoses do yield furfural (17). Nor does
the furfural come from hexoses, in spite of the fact that oxidative "browning"
of hexoses does yield a small amount of furfural (16). Under the anaerobic and
relatively weakly acid conditions, and moderate temperatures during the storage
of canned juice, there is strong evidence that the sole source of furfural is
the decarboxylation of ascorbic acid. This reaction has been studied by Lamden
and Harris (13), by Huelin (8), and recently by Cier et. al. (6).

In Figure 1 the author presents a plausible explanation of this situation.
It is based on the well-established fact that sugars are capable of existence
in different molecular structures, varying in stability. We may suppose that
when pentose xylosee) first appears in solution as the primary product of the
acidic hydration and decarboxylation of ascorbic acid, the pentose is in a
labile form. Molecules having this structure can indeed be dehydrated to form
furfural, but such molecules are simultaneously rearranging by a competitive
reaction to give the pyranose ring structure of xylose, which sugar authori-
ties represent to be relatively unreactive. Solid xylose, whether of the D-
structure available to us, or of the L-structure used by Cier et. al. (6)
consists mostly of the stable cyclic form, and hence did not yield appreciable
amounts of furfural at pH 3.5 in our case or even at pH 2.6 as used by Cier
et al. (6). If this speculation is right, then any factor, such as any possi-
ble catalyst, which could affect the rate of rearrangement of the sugar mole-
cule in solution, could affect the yield of furfural from ascorbic acid in a
moderately acid solution. Coggiola (7) has announced very recently the
discovery that a pure solution of ascorbic acid, heated in an atmosphere of
carbon dioxide, yielded 2,5-dihydro-2-furoic acid. He noted that this compound
conforms in composition to the empirical formula C5H603, which empirically is
a hydrate of furfural.

As to the presence of hydrogen sulfide, it is of interest that Kirchner
and co-workers (11) found that while this substance could be carried out of
fresh orange juice by a current of nitrogen at room temperature, none could be
carried out from stored canned juice in this way. The indication, therefore,
is that in canned juice the hydrogen sulfide is entirely "bound" as non-
volatile compounds. However, upon heating, such compounds may in part vola-
tilize, and may also release some hydrogen sulfide. However, our postulate of
the role played by thiofurfural in canned orange juice is by no means dependent
solely on the characteristic nature of the odor released by heating and collect-
ed in the distilled oil phase. Also, into consideration must be taken the proof
that both hydrogen sulfide and furfural are formed during storage of canned
juice, and that these two components spontaneously interact in pH 3.5 citrate
buffer at room temperature. It is reasonable to say that it would be impossible
for thiofurfural not to be present, in a proportion governed largely by con-
ditions of storage.
Florida Citrus Commission and Florida Citrus Experiment Station,
Lake Alfred, Florida. 400 -10/6/64 JSB








Figure 2. Decarboxylation of Ascorbic Acid.


HOCH C

H2C CH
H2C CH


/o\
H CO

CO
CO


HOCH CHOH

I I
H2C CH-CHO + CO2
I I


Ascorbic Acid
C6H806


SPentose (reactive form)


C5H 005


HOCH -CHOH

C CHOH
H20 CHOH


/at pH


CH CH


II
CH

0'


I
C -CHO


I
0


CHOH


Pentose (stable form)1 Furfural


C5H402


Conversion of stabilized pentose to furfural requires boiling with strong mineral acid.


C5H1005


I


+H20
------/











References
1. Attaway, John A., Richard W. Wolford and George J. Edwards. Isolation and
identification of some volatile carbonyl compounds from orange essence,
J. Agr. Food Chem. 10, 102-4 (1962).

2. Baumann, E. and E. Fromm. The thioderivatives of furfural. Ber. 24,
1591-9 (1891).

3. Blair, J. S., Edith M. Godar, J, E. Masters and D, W. Riester. Exploratory
experiments to identify chemical reactions causing flavor deterioration
during storage of canned orange juice. I. Incompatibility of peel oil
constituents with the acid juice. Food Research 17, 235-60 (1952).

4. Cairns, T. L., G. L. Evans, A. W. Larchar and B. C. McKusick. gem-Dithiols.
J. Am. Chem. Soc. 74, 3982-9 (1952).

5. Campaign, E. Thiones and thials. Chem. Revs. 39, 4 (1946).

6. Cier, Andre, Claude Nofre, Bartholemy Drevon, and Anne Lefier. Study of
the degradation of ascorbic acid in an inert atmosphere. Bull. Soc. Chim.
France, 1959, 74-7.

7. Coggiola, I. M. 2,5-dihydro-2-furoic acid: a product of the anaerobic
decomposition of ascorbic acid. Nature 200, 954-5 (1963).

8. Huelin, F. E. Studies on the anaerobic decomposition of ascorbic acid.
Food Research 18, 633-9 (1955).

9. Jansen, Eugene F. and Rosie Jang. Cysteine and glutathione in orange
juice. Arch. Biochem. Biophys. 40, 359-63 (1952).

10. Kipnis, Frank, Isidore Levy and John Ornfelt. Mercaptans from aldehydes.
J. Am. Chem. Soc. 71, 2270 (1949).

11. Kirchner, J. G., R. G. Rice, J. M. Miller and G. J. Keller. The presence
of hydrogen sulfide in citrus juice. Arch. Biochem. 25, 231 (1950).

12. Kirchner, J. G., and John M. Miller. Volatile water-soluble and oil
constituents of Valencia orange juice. J. Agr. Food Chem. 5, 283-91
(1957).

13. Lamden, Merton P. and R. S. Harris. Browning of ascorbic acid in pure
solutions. Food Research 15, 79-89 (1950).

14. McCance, R. A. Rapid colorimetric method of determining pentoses. Biochem.
J. 20, 1111 (1926).

15. Meints, R. E., and J. A. Wilkinson. Reactions in liquid hydrogen sulfide.
V. Reaction with furfural. J. Am. Chem. Soc, 51, 803 (1929).

16. Natarajan, C. P., and G. MacKinney. Studies on the darkening of orange
juice. Food Technol. 3, 373-5 (1949).
17. Sinclair, Walton B. and E. T. Bartholomew. Methods for determining pentoses
as furfural in citrus fruits. Am. J. Botany 22. 829-42 (1935).




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