Group Title: Citrus Station mimeo report - Florida Citrus Experiment Station ; 55-1.
Title: The diacetyl test and its application in citrus concentrate quality control
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 Material Information
Title: The diacetyl test and its application in citrus concentrate quality control
Series Title: Citrus Station mimeo report
Physical Description: 4 leaves : ill. ; 28 cm.
Language: English
Creator: Hill, E. C
Wenzel, F. W
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: 1954
Subject: Concentrated fruit juices -- Preservation -- Florida   ( lcsh )
Citrus juices -- Preservation -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
Bibliography: Includes bibliographical references (leaf 7).
Statement of Responsibility: E.C. Hill and F.W. Wenzel.
General Note: Caption title.
General Note: "October 12, 1954."
 Record Information
Bibliographic ID: UF00072360
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 74323312

Full Text

Citrus Station Mimeo Report 55-1
Oct. 12, 1954

The Diacetyl Test and Its Application in Citrus Concentrate
Quality Control
E. C. Hill and F. W. Wenzel

Chemical tests for diacetyl and acetylmethylcarbinol as a means for detect-
ing and preventing spoilage in citrus concentrates due to lactic acid bacteria
have been previously discussed (1), (2), (3), (4), (5), and (6). The colorimet-
ric methods described are rapid, simple and reliable. The tests, although quan-
titative in a sense, were designed primarily as a means of determining the accum-
ulation during processing of metabolic products of bacteria producing the off-
taste and spoilage described as "buttermilk".

During the 1953-54 season, the diacetyl test was used to some extent by 15
of the 23 citrus concentrate plants operating in Florida. Of these, 11 plants
used the test as a routine procedure for detecting and preventing "buttermilk"
spoilage of citrus juices during evaporation.

The work done in the past at this Station on diacetyl tests has been in the
nature of experimental work and the data reported has come from pilot plant runs.
During the 1953-54 season, we were fortunate in being able to collect data at a
commercial concentrate plant during a period when the plant was experiencing in-
creased microbiological activity progressing to spoilage. Diacetyl tests were
run on the evaporator feed, first, second, third, and fourth stages of the evap-
orator, the pulpy add-back juice and the canned concentrate. Emphasis was placed
on the second stage and readings were made every 2 hours. Readings on the other
check points were made less frequently.

To obtain comparable data the diacetyl test procedure used was identical
with that previously used. It was as follows:

25 ml. of distillate obtained from 300 ml. of reconstituted juice
(12Brix + 0.20)
10 ml. of distillate
5 ml. of 5% alpha-naphthol in ethyl alcohol
2 ml. of 40% KOH + 0.3% creatine

Shake 15 seconds.
Stand 10 minutes.
Shake 15 seconds.
Read on electrophotometer nulled against blank of distilled water
and reagents prepared at the same time as the sample.

Sampling was begun shortly before a general cleanup was scheduled. The
first reading on the distillate from the second stage of the evaporator indicated
8.8 ppm diacetyl (Figure 1). This, of course, is very high. However, in 4 1/2
hours, the diacetyl content in the distillate from the second stage had risen to
19.7 ppm. Processing was discontinued and the plant thoroughly cleaned. The
following day the diacetyl content ranged between 1.2 and 2.2 ppm in the dis-
tillate from the second stage. The second day after cleanup the distillate

Florida Citrus Experiment Station and
Florida Citrus Commission, Lake Alfred, Fla.
532 10/12/54 ECH

contained from 1.8 to 2.8 ppm diacetyl. The third day, 64 hours after cleanup,
the diacetyl content in the distillate from the second stage contained 12.8 ppm
diacetyl; this increased rapidly, reaching 28.7 ppm diacetyl in 3 hours. From
the data collected, it appeared that the rapid increases in diacetyl content in
the second stage of the evaporator began below the 8 ppm level.

After the plant was thoroughly cleaned and evaporation was again begun,
diacetyl tests were run on an around-the-clock basis on juice from the second
stage of the evaporator every 2 hours (Figure 2). Tests were also made on the
evaporator feed and the fourth stage concentrate every four hours, as well as
on the canned product. Plate counts on orange serum agar were made from juice
from the second stage of the evaporator and the finished canned product. When
the diacetyl content began to rise, readings were made at hourly intervals. There
was a gradual rise in the diacetyl content after 45 hours of operation which in-
creased to over 5 ppm by the 63rd hour. After the diacetyl content had passed
the 5 ppm level, it then rapidly increased to 14.6 ppm in 9.5 hours.

Figure 3 is another example of the diacetyl test results on the juice from
the second stage of this commercial evaporator. Here again is demonstrated the
very rapid increase in diacetyl and acetylmethylcarbinol content after the 5 ppm
level is reached.

The proper application of the diacetyl test for the concentrate plant from
which the previous data has been obtained is demonstrated in Figure 4. When the
diacetyl test on samples from the second stage of the evaporator indicated more
than 5 ppm, evaporation was discontinued. The plant was then thoroughly cleaned
and operations were resumed at a low diacetyl level.

The data from this plant indicated that after the juice in the second stage
of the evaporator had passed the 5 ppm diacetyl level, an operational interruption
for cleanup was indicated, otherwise bacterial metabolic products would accumu-
late at an extreme rate with a rapid progression to spoilage. At this plant
emphasis was placed on the second stage of the evaporator and, generally speaking,
the stages in which the juice is maintained at from 180 to 250Brix afford the
.optimum conditions for the growth of lactic acid organisms. Routine diacetyl
tests should also be run on the pulpy juice add-back and good control practice
necessitates infrequent but periodic check of the canned, finished product.
Diacetyl test check points will vary to some extent from plant to plant and at
the discretion of the plant personnel upon whom the responsibility falls for
maintaining a quality product.

The diacetyl tests, being used in commercial plants, vary in procedure from
one plant to another as do check points and sampling frequency. Since the
diacetyl tests is primarily used to aid in preventing spoilage while the concen-
trate is being produced, any method or procedure which produces results satis-
factory to the quality control personnel of any one plant is of course accomplish-
ing the purpose for which it is intended.

Isopropyl alcohol, because of its accessibility, has in many plants been
substituted for ethyl alcohol as a solvent for a-naphthol. When isopropyl alco-
hol is used as a solvent, a different red color is produced by the diacetyl

Florida Citrus Experiment Station and
Florida Citrus Commission, Lake Alfred, Fla.
532a 10/12/54 ECH


reaction. Figure 5 shows spectral curves of the 2 colors produced by the diacetyl
reaction when using the 2 alcohols as a-naphthol solvents. When isopropyl alco-
hol was used the color reaction produced a curve which had its peak absorption at
535 millimicrons. The color reaction produced when ethyl alcohol was used as a
solvent for a-naphthol had its peak at 545 millimicrons. Figure 5 also shows the
transmission characteristic of the Fisher 525 filter used in obtaining all pre-
vious data on the diacetyl test at this Station. As indicated by the graph, the
least absorption or highest transmission of light was between 520 and 525 millimi-
crons. This filter would favor the color produced when isopropyl alcohol was used
as a solvent and would therefore give higher readings with this color.

Another variable in the diacetyl test is the standing time permitted between
the addition of reagents and the time at which the readings are made. Figure 6
shows the reaction of diacetyl over a period of one hour. Two curves are shown,
one in which the a-naphthol reagent used was ethyl alcohol, the other using iso-
propyl. The color develops very rapidly, reaching maximum intensity in one
minute or less in the case of the ethyl alcohol solvent, and five minutes with
isopropyl alcohol as a solvent. After reaching a maximum intensity there is a
steady decrease in color, the most rapid occurring in the reaction using ethyl
alcohol as the a-naphthol solvent.

The development of color is slower when acetylmethylcarbinol is the reactant.
Intensity of color development for acetylmethylcarbinol over a period of from 1
to 60 minutes at 1, 3, and 5 ppm levels is shown in Figure 7. The dotted lines
represent the test using ethyl alcohol as the solvent for a-naphthol while the
solid lines represent the test using isopropyl alcohol. At the 5 ppm acetyl-
methylcarbinol level, the maximum color intensity was reached at 10 minutes for
the test using ethyl alcohol as a solvent for a-naphthol, while the test using
isopropyl alcohol reached maximum in 25 minutes. Although the maximum color
development was slower with acetylmethylcarbinol than with diacetyl, the decrease
in intensity was also slower.

Calibration curves using ether-washed acetylmethylcarbinol at reaction times
of 10 and 30 minutes with a-naphthol in ethyl and isopropyl alcohol are repre-
sented in Figure 8. When isopropyl alcohol is used as the solvent for a-naphthol
the greatest slope occurs at a reaction time of 30 minutes. If ethyl alcohol is
used the greatest slope occurs at a reaction time of 10 minutes. Although there
is considerable difference between the 4 curves it would appear that either of
the 4 procedures would give satisfactory sensitivity for quality control labora-

The diacetyl test will be the greatest aid to the technician who understands
the limitations and variables involved and who has become sufficiently familiar
with the test in relation to his plant to properly interpret the results obtained.
The test is flexible,and possible variations in procedure are almost unlimited.
Although any method may be changed and improved there would be little virtue in
doing so in this case unless the changes resulted in greatly reducing the time
required to perform the test or still further simplify it; the sensitivity pro-
vided by most of the procedures being used is quite adequate.

The data collected at commercial plants confirms the experimental pilot

Florida Citrus Experiment Station and
Florida Citrus Commission, Lake Alfred, Fla.
532b 10/12/54 ECH

plant work previously reported, in that, once the diacetyl and acetylmethylcarbi-
nol content reached a certain level the increase was no longer gradual but became
very rapid. That point was around 5-6 ppm in the plant reported upon. Although
this particular point would vary from plant to plant and with testing procedures,
the general trends should remain the same. It is likely that if this increased
microbiological activity has not progressed too far, prompt action iray be taken
to delay a general cleanup by such measures as improvement of the fruit grading,
"burping" and flushing of the offending evaporator, the introduction of high acid
juice and spot cleaning of equipment such as holding tanks, extractors, or fin-
ishers. The best action, however, is a thorough general cleanup.

Florida Citrus Experiment Station and
Florida Citrus Commission, Lake Alfred, Fla.
532c 10/12/54 ECH

Fig. 1




- 5
I ,

64 70 0

10 20 30 40 50
Processing Time Eours

60 70




Fig. 3

Fig. 4

15 j

0 10 20

30 40 50

Processing Time Hours

~'~~~---- -- ^- ^----

S y. ^ V ^ ''' --l-; ---- ^"- -lIy'w ^--.A
0 10 20 30 40 50 0 10
Processing Time Hours




~L-L-i~---L-I.-L~,~,~-_-L---i 1 r

~ j; __

..I--- _.~


0 Fig. 2
O 5


a 10 -- --...-._ -.- _


0 10 20 30 40 50 60 70
Processing Time Hours


1 Isopropyl alcohol
2 Ethyl alcohol
3 Green filter
5, 525

o /




Fig. 5









450 475 500 525 550

Diacetyl Reaction Time Fig 6
1 naphthol in Isopropyl
2 naphthol in Ethyl



o 5C




Fig. 7
Acetylmethylcarbinol Reaction Time
filter 525 B
1 i- naphthol in Isopropyl alcohol
2 naphthol in Ethyl alcohol

/5 ppm
Cc /

S // -.- -

,, / 3 ppm

"""------- 2

1. ppm

Fig. 8

1 t naphthol in
30 min.
2 naphthol in
10 min.

Isopropyl /

Isopropyl/ 2
/ ,1
*L > 1

Y 1 <-l- naphthol in Ethyl
alcohol 10 min.
2 .- naphthol in Ethyl
alcohol 30 min.

2 A3 c 4 6 7etyl
ppm Acetylmethylcarbinol



c __

10 20 30 40 50 60

Literature Cited

1. Beisel, Gordon C., Dean,R. Willard, Kitchel, Robert L, Rowell, Kenneth M.,
Nagel, Charles W. and Vaughn, Reese H. Sources and detection of Voges-
Proskauer reactants in California valencia orange juice. Paper presented
at fourteenth annual meeting of the Institute of Food Technologist, July 1,
1954, Los Angeles, Calif.

2. Byer, Ellis M. Visual detection of either diacetyl or acetyl-methyl-carbinol
in frozen concentrated orange juice. Food Technol., 8, 173 (1954).

3. Hill, E. C. and Wenzel, F. W. Use of diacetyl test for detection of micro-
biological spoilage in citrus juices. Report presented at Fourth Annual
Citrus Processors Meeting, Lake Alfred, Fla. October 6, 1953.

4. Hill, E. C., Wenzel, F. W. and Barreto, A. Colorimetric method for detection
of microbiological spoilage in citrus juices. Food Technol., 8, 168 (1954).

5. Hill, E. C., Wenzbl, F. W. and Barreto, A. Comparison of various methods
for detection of microorganisms which produce off-flavors in orange concen-
trate. Report presented at Third Annual Citrus Processors Meeting, Lake
Alfred, Fla. October 1, 1952.

6. Wenzel, F. W., Hill, E. C., and Barreto, A. Detection of acetylmethyl-
carbinol or diacetyl in orange concentrate as an indication of the growth
of certain bacteria which produce off-flavors. Report to citrus processors
at Citrus Experiment Station, Lake Alfred, Fla. May 15, 1952.

Florida Citrus Experiment Station and
Florida Citrus Commission, Lake Alfred, Fla.
532d 10/12/54 ECH

Cathode Ray Irradiation of Orange Concentrate

J. Allen Brent

Orange concentrate of 420 Brix was irradiated by cathode rays using a one

million volt generator at dose levels of 500,000, 1,000,000, 2,000,000, and

5,000,000 roentgens. The samples were treated in a thin film in polyethylene

tubes at temperatures of frozen, 380 to 400, and 1000F. The samples were analyzed

for taste, pectinesterase activity, cloud stability, bacteriological activity,

and ascorbic acid. All irradiated samples were judged inferior to the control,

the degree of off-flavor being proportional to the dose level and the tempera-

ture of the sample during irradiation.

Inhibition of pectinesterase varied from about 2 to 75%, being proportional

to temperature and dose level at 2,000,000 roentgens or higher. Storage stability

at 400F. was not increased appreciably at levels up to 2,000,000 and was decreased

by irradiation at 5,000,000 roentgens. Ascorbic acid retention varied from 98

to 84% on samples irradiated frozen. All irradiated samples were found to be

bacteriologically sterile.

Florida Citrus Experiment Station and
Florida Citrus Commission, Lake Alfred, Fla.

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