Front Cover
 Table of Contents
 Preparation of concentrates, chilled...
 How contaminating organisms are...
 Sources and kinds of microorganisms...
 Survival of microorganisms in single-strength...
 Plant clean-up practices
 Use of microorganisms in the utilization...
 Literature cited

Group Title: Bulletin - University of Florida. Agricultural Experiment Station - no. 618
Title: Microbiology of citrus fruit processing
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00027748/00001
 Material Information
Title: Microbiology of citrus fruit processing
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: 62 p. : ill. ; 23 cm.
Language: English
Creator: Patrick, Roger
Hill, Elmer C
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1959
Subject: Citrus fruits -- Processing -- Florida   ( lcsh )
Fruit processing plants -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
Bibliography: Bibliography: p. 53-62.
Statement of Responsibility: Roger Patrick and Elmer C. Hill.
General Note: Cover title.
General Note: "A contribution from the Citrus Experiment Station, in cooperation with the Florida Citrus Commission"--T.p.
Funding: Bulletin (University of Florida. Agricultural Experiment Station) ;
 Record Information
Bibliographic ID: UF00027748
Volume ID: VID00001
Source Institution: Marston Science Library, George A. Smathers Libraries, University of Florida
Holding Location: Florida Agricultural Experiment Station, Florida Cooperative Extension Service, Florida Department of Agriculture and Consumer Services, and the Engineering and Industrial Experiment Station; Institute for Food and Agricultural Services (IFAS), University of Florida
Rights Management: All rights reserved, Board of Trustees of the University of Florida
Resource Identifier: aleph - 000927055
oclc - 18302187
notis - AEN7758

Table of Contents
    Front Cover
        Page 1
    Table of Contents
        Page 2
        Page 3
        Page 4
    Preparation of concentrates, chilled juice, canned and chilled sections
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
    How contaminating organisms are found
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
    Sources and kinds of microorganisms encountered in fruit processsing plants
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
    Survival of microorganisms in single-strength and concentrated citrus juices
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
    Plant clean-up practices
        Page 49
    Use of microorganisms in the utilization of citrus fruit waste materials
        Page 50
        Page 51
        Page 52
    Literature cited
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
Full Text


The publications in this collection do
not reflect current scientific knowledge
or recommendations. These texts
represent the historic publishing
record of the Institute for Food and
Agricultural Sciences and should be
used only to trace the historic work of
the Institute and its staff. Current IFAS
research may be found on the
Electronic Data Information Source

site maintained by the Florida
Cooperative Extension Service.

Copyright 2005, Board of Trustees, University
of Florida

December 1959

J. R. BECKENBACH, Director

(A Contribution from the Citrus Experiment Station,
in Cooperation with the Florida Citrus Commission)




Single copies free to Florida residents upon request to

Bulletin 618


INTRODUCTION ..................

CHILLED SECTIONS .................... ---- ... .....

Media ----- -----.. -----.............------
Direct Microscopic Method ....................-...
Chemical Methods ....-............... ----


--- -----................. ...--. .---- --- 3

.-- 5

-------------- 12
. --- -------- 12
----.... ---- 24
-.-.----- --- 25

PROCESSING PLANTS ........--..........-.....-------------- -----. 26
Exterior Contamination of Citrus Fruits ....-.......-.--.----------- 26
Washing and Storage of Fresh Fruit ......----.---... -------------.----- 28
Scale Insects and Fruit Flies ...-..................... ---- ------ ------ 29
Interior Contamination and Damaged Fruit ................ ------.----- 29

CITRUS JUICES --.............................-- ---. ---- --- ------------ 34
Effect of Storage on Survival of General Contaminants ..-.................. 34
Effect of Heat on Survival of General Contaminants ........----....----...--- 41
Effect of Storage on Survival of Enteric and Pathogenic Bacteria ... 44
Effect of Chemical Preservatives on Survival of Contaminants ....-... 47

PLANT CLEAN-UP PRACTICES ..................-- --------- -- ------------ 49

WASTE MATERIALS ......-............- ...- ..----------- 50
Molasses and Feed Yeast --...............--........ .. --- -- --- ---- 50

Alcohol ................ ---- .. ...-- .
Lactic A cid .................................. ....- -
Wine and Alcoholic Beverages -.............------
Vinegar ..................................... -
W aste Disposal ................-- ............... -
Combustible Gas ........--..............--..
Vitamin Supplements ........-.................... --

.. --... .~...---. -- 51
.. -.....---....-- -- 51
...-..-..- ----. -- -.. -- 51
---.. -... .........-- .--. 51
..-..-.....----....-------......-- 52
.............-- --.. .... 52
...---....- ---------.. .... 53

E...E....I...E.. -......--- .....- 53

LITERATURE CITED ------_---_---.

Microbiology of Citrus Fruit Processing


Emphasis on the utilization of citrus fruits grown in Florida
has shifted from the fresh fruit market to processed fruit pro-
ducts. According to Shuler and Townsend (114), Florida pro-
duced 116,400,000 boxes of citrus fruits during the 1957-58
season, which is 69 percent of the total citrus fruit production
for the United States. They indicate the number of boxes as
Oranges -......-.............. ............. 82,500,000 boxes
Grapefruit ..........-................-........ 31,100,000 boxes
Tangerines ....-...............-....-.. 2,100,000 boxes
Limes ................. ............- 200,000 boxes
Others ......................... ...... ..... 500,000 boxes

Cold weather decreased production considerably below the
135,600,000 boxes harvested the previous season.
A larger volume of fruit is anticipated as non-bearing acreage
matures. It was estimated that, as of June 1958, there were

1 Bacteriologist, Citrus Experiment Station, Lake Alfred, and Assistant
Bacteriologist, Florida Citrus Commission, Lake Alfred.

Fig. 1.-A large Florida citrus products plant as seen from the air.
(Courtesy Snively Groves.)

Florida Agricultural Experiment Stations

106,600 acres of non-bearing citrus trees planted in Florida (114).
There were 542,200 acres of bearing groves in Florida; this con-
stitutes 66 percent of the total acreage in the United States.
The principal processed citrus fruit products of commerce
are: canned single-strength juice, chilled single-strength juice,
canned fruit sections, chilled fruit sections, processed concen-
trated juice and frozen concentrated juice. Larger volumes of
canned or frozen blends of both citrus juices and of citrus juices
blended with other fruit juices are also processed.
The following figures were listed in the Annual Report of
the Florida Department of Agriculture (32) and by Shuler and
Townsend (114) for the number of boxes for the different kinds
of citrus fruits that were used in some processed citrus products.
Commercial processing plants used during the 1957-58 season
69 percent of the Florida citrus crop (32).

Utilization of Florida Citrus Fruits in Some Processed Products.

Oranges Grapefruit Tangerine

Frozen ....--.......---..~....... 43,810,000* 3,685,173t 141,5841
Processed ...........-............... 770,000*
Canned .--............-..... ...... | -- 3,024,395
Chilled I....-- 360,840 569,735
Chilled juices ............... 6,044,414 173,421
Canned and
blended juices -- 12,743,250 8,965,224 206,889

57,151,000 gallons of frozen orange concentrate and 1,149,000 gallons of processed
orange concentrate were produced.
t Also includes processed concentrate and frozen blended concentrate.
$ Also includes processed concentrate.
Also includes canned sections.

The methods of preparing, storing, marketing and distri-
buting these processed products successfully and plant sanitizing
procedures require an understanding of the microbiology in-
volved. It is our purpose to collect into this bulletin the infor-
mation relative to the subject of microbiology of citrus fruit

Microbiology of Citrus Fruit Processing

Commercial pasteurization of canned citrus products, such
as single-strength juices, prevents the occurrence of microbio-
logical problems in connection with such products. However,
deterioration and spoilage of frozen and processed concentrates,
chilled juices and chilled sections may occur because of the
growth of microorganisms. Therefore, some brief comments
will be made concerning the commercial production of these pro-
The general procedure for preparing fruit for storage prior
to processing has been reviewed in the literature by Beisel (11),
Brokaw (17), Madsen and D'Ercole (68), Kaufman and Camp-
bell (58) and Murdock and Brokaw (81, 82). It consists in gen-
eral of the following operations: receiving the fruit at the plant
and making the decision to accept or reject, grading for sound-
ness and maturity, removing leaves and twigs and sometimes
washing the fruit before storing.

Processed concentrated orange juice concentrated by heat and
packed hot (125) was one of the early concentrates of commerce.
The product did not achieve wide acceptance. Due to the heat
used in preparation, the color was usually not bright orange,
and the concentrate darkened during storage and transportation.
It did serve a particularly worthwhile purpose during World
War II, as it was used to supplement the diets of our besieged
allies. Production dropped from 1,882,000 gallons in 1942-43
to 240,000 as soon as the emergency was over (32). The method
of preparation and handling was improved, and during the 1957-

Fig. 2.-A young orange grove in central Florida.

Florida Agricultural Experiment Stations

58 season the production of processed concentrate was 1,149,081
gallons. Further growth in this part of the citrus fruit industry
was stifled by the popular acceptance of frozen concentrated
orange juice.
Frozen orange concentrate was first produced in commercial
quantities in the 1945-46 season; 226,000 gallons were prepared.
Since that time production has increased, and in the 1957-58
season 58,299,647 gallons of orange concentrate were produced.
Frozen grapefruit concentrate was first produced commercially
in the 1948-49 season with 116,123 gallons. In the 1957-58 sea-
son 3,330,301 gallons were produced (32).

Fruit Received

Ir1L I Evaporators 'u"u' 8 Canning a
Heat Tank Freezing
Fig. 3.-Flow diagram depicting the manufacture of frozen
citrus concentrate.

The juice was evaporated under vacuum at 1200F., causing
considerable damage to the flavor. After some experimentation
(125) and at the direction of L. G. MacDowell, the temperature
was lowered to 60-80 F., a concentrate of high density (55-600
Brix) was prepared and cutback to 42 Brix with fresh juice
to restore the fresh fruit flavor. Commercially it is then slush-
frozen, canned, frozen solid and stored in cases at 0 F. or

Microbiology of Citrus Fruit Processing

below. During recent years some processors expose the juice
to heat treatment before it enters the evaporators (58, 61);
others use steam-injection to stabilize citrus juices as indicated
by Keller et al (60). Such heat treatment inactivates the pectic
enzymes and destroys most of the non-spore forming bacteria
that might cause off-flavors in the juice during the early stages
of evaporation.

Fig. 4.-Water is removed from citrus juice in these evaporators at the
rate of 35,000,000 pounds per hour. (Courtesy Florida Citrus Canners

Florida Agricultural Experiment Stations

A stepwise freeze method has been developed (69, 120)
whereby the volume of water in the juice is reduced to make
citrus fruit concentrate. The juice is frozen to a slush in cans,
similar to those used in making 100-pound cakes of artificial ice.
The ice crystals are separated from the liquor; the liquor is re-
frozen and again separated. Such a process was in operation in
Florida for a short time; the product was very acceptable when
investigated for flavor and microbial content. The maximum
count in concentrated juice from the blending tank was 22,800
per ml of reconstituted juice. Economic and other reasons have
prevented the extensive use of this process.

For a long time many persons have tried to distribute chilled,
fluid orange juice to the doors of customers. A historical review
of this venture has been given by Hamrick (37, 38, 39). Lack
of sanitation and refrigeration certainly contributed to the fail-
ures, despite the fact that the failures have been attributed to

Fig. 5.-Extraction of juice at a large citrus plant.
(Courtesy Pasco Packing Co.)

Microbiology of Citrus Fruit Processing

other causes. Camp and Stahl (20) found that by using clean
utensils they could prepare juice and store it at 320 F. for 1
week without loss of flavor. Shrader and Johnson (113) and
Murdock and Brokaw (82) emphasized the importance of using
refrigeration technology to preserve orange juice. They urged
that the plant be installed without traps or dead ends, and with
all equipment as accessible for cleaning as in a dairy plant. The
use of sound fruit for this type of product has been recognized
by some investigators as having a distinct benefit (30, 81, 97,
Under the present standards issued by the United States
Department of Agriculture, chilled orange juice may be produced
from freshly extracted whole orange juice which may or may
not be stabilized with heat or other acceptable means; it may
be produced from frozen concentrated orange juice or from frozen

Fig. 6.-Refrigerated blending tanks in which concentrate and cutback
juice are mixed to the right proportion. (Courtesy Stokely-Bordo.)

Florida Agricultural Experiment Stations

single-strength orange juice, or any combination of these pro-
The juice may be packed in milk bottles or wax cartons and
shipped in refrigerator trucks up to 1,200 miles (125). Wright
(146) described a system for the handling of fresh orange juice
without heat treatment or excessive refrigeration. The freshly
extracted juice flows over a 1/32-inch stainless steel sheet under
3 mercury vapor lamps of 3,000 watts and 15 amperes each. The
juice is cooled quickly to 330 F. and then goes to a cold-walled
tank where nitrogen is passed through to remove the entrained
oxygen. The juice is then transported in an atmosphere of nit-
rogen in insulated stainless steel tank trucks lined with plastic
or glass. Juice treated in this way has been hauled 1,450 miles
without reported loss of flavor.
One operator loads slush-frozen juice into sterilized stainless
steel tanks built in the holds of a ship, about 200,000 gallons,
and transports by sea to New York. The entire trip requires
about 60 hours (37). Refrigerated railroad cars are also used
for the bulk shipment of orange juice.
When bulk shipments of orange juice reach their destina-
tion, they must be repacked for distribution to the consumer.
The handling of a food product such as orange juice needs to be
done sanitarily and at a low temperature. Purko et al (105)
investigated chilled juice that had apparently not been stabilized
and found the product contaminated with strains of bacteria,
some of which would grow and deteriorate the juice if low tem-

Fig. 7.-Chilled orange juice is shipped to the North in this huge tanker.
(Courtesy Tropicana Products, Inc.)

Microbiology of Citrus Fruit Processing

peratures were not maintained. A good review pertaining to
chilled orange juice may be read in the paper prepared by Wen-
zel et al (135).
Fruit to be canned or packed as chilled sections are selected
after grading for size and washing. The peel is removed and
the sectionizing is done by hand. Canned sections are packed
in tins and air is removed by heat or vacuum. The cans then
are sealed and heated to distroy the microorganisms (125).
Sections to be distributed as a chilled product are usually
packed in glass containers, sealed and chilled quickly and then
placed in cold storage.
These chilled products are handled extensively by human
hands during their preparation and the utmost care and sanita-
tion is necessary. The knowledge gained from the study of
microbiology has helped to decrease loss due to spoilage.

Fig. 8.-Sectionizing and filling jars in the preparation of citrus salad.
(Courtesy Snively Groves, Inc.)
i -- -- m a

Florida Agricultural Experiment Stations

The discussion thus far has been dealing with the preparation
of citrus products of commerce. Next, it might be advisable to
review the means by which the organisms contaminating these
products are found. The bacteriological laboratory in each plant
usually has a record of special groups of organisms that may
cause loss of the product and also a record of those organisms
that may not grow in the product but remain viable and serve
prognostic purposes. General sanitation, the effectiveness of
the clean-up and the quality of the fruit used are all reflected
in the results gathered by the persons in the control laboratory
using a few or several of the methods that are to be discussed.

Most quality-control directors use standard techniques to
gather some of the information related to their duties; these
procedures are well known and will not be discussed here. Di-
rectors do not agree upon a single medium or even a group of
media to be employed in this part of their work, but many of
them are in agreement that certain media used continuously,
singly or in combination, give a reasonable index of the condi-
tions in the processing plant under their supervision. CAtrol
laboratories usually employ a standard medium for detecting
general contamination, such as tryptone glucose beef extract
agar (pH' 6.6-7.0) for the determination of those organisms
which may or may not grow in citrus fruit juice, but do show
the effectiveness of the clean-up and the general sanitation of
the plant. Other media used for this purpose are: tryptone
glucose yeast extract agar, pH 6.8, and dextrose tryptone agar,
pH 6.7. These are standard media and their formulae may be
found in manuals and laboratory handbooks. In conjunction
with these media, other media are used to find more select groups.
The microorganisms usually sought with these selective media
are: yeast, lactic acid bacteria and slime and gum-forming bac-
teria (Lactobacillus and Leuconostoc). These groups are com-
monly troublesome in the process or in the package if held at
inadequate storage temperatures before the product is consumed.
Other selective media are those employed to show the presence
of the coli-aerogenes group and the enterococcus bacteria.
The high count of general contamination is usually a warn-
ing sign, but does not indicate that spoilage organisms are pres-

Microbiology of Citrus Fruit Processing

ent nor does it indicate the kinds of spoilage organisms that may
be building up. Hays (42, 43) compared tomato serum agar
and Lindegren's agar, when isolating the causal organisms from
fermenting orange concentrate. He found the count was from
10- to 100-fold higher on tomato serum agar than on Lindegren's
agar. He found that the spoilage was caused by members of the
genus Lactobacillus. Lindegren's agar is formulated for yeast
cultivation and does not contain the nutrients necessary for the
growth of Lactobacilli. He found that by substituting orange
serum for tomato serum, the organisms grew better. Orange
serum agar may be purchased in the dehydrated form (121).
Peptonized milk agar with 1 percent dextrose and 0.022 g/100
ml brom cresol green has been used successfully by Patrick (98)
to detect these lactobacilli-like organisms. The colonies are
small and clearly marked with yellow.
Orange serum agar and McClesky's agar (84) have been
used widely to detect slime and gum-forming bacteria in con-
centrated orange juice. Streaks made from colonies on pepton-
ized milk agar, orange serum agar and McClesky's agar pro-
duced slime and gum colonies in 66 percent of the trials. Patrick
(98) used McClesky's agar (84) and peptonized milk agar with
dextrose and brom cresol green to learn of the contamination in
burst and soft fruit.

Fig. 9.-Highest quality citrus products are made possible through
continuous laboratory inspection. (Courtesy Adams Packing Co.)

Florida Agricultural Experiment Stations

McClesky's agar supported a growth of 43,000 per ml and
peptonized milk showed 54,000 organisms per ml of juice. Mur-
dock et al (74) evaluated 18 media for the growth of 6 isolated
strains of Leuconostoc, 10 strains of Lactobacillus and 4 strains
of Candida (yeast). Since these isolated strains are capable
of growing in citrus juices and concentrates, it was hoped that
by comparing their growth on these media, a medium would be
found that would support a good growth and at the same time
give a reasonable index of their presence. Orange serum, pH
5.4, supported the highest count for this combination of yeast
and bacteria. They concluded that this medium was suitable
for the detection of these organisms that frequently cause de-
terioration in orange juice and concentrates.
Vaughn and Emard (127) reported 0.1 percent sorbic acid in
a liver infusion medium favored the growth of Lactobacillus and
Leuconostoc. They found that most catalase positive bacteria
were inhibited. Hays and Riester (43) found that orange serum
agar containing 0.13 percent sorbic acid was a good medium for
evaluating lactic acid bacteria in citrus products. Berry et al
(7) have reported a rapid method for the presumptive identifica-
tion of bacteria associated with off-flavor in orange concentrate.
This is a simplified method for segregation of 3 genera, Lacto-
bacillus, Leuconostoc and Acetobacter, isolated from off-flavor
orange concentrate. The method is based on the cell morphology,
gram and catalase reactions, relations to oxygen and growth in
sucrose-gelatin. Acetic acid bacteria were determined qualita-
tively by means of ethanol oxidation in media at pH 4.1.
Hill and Faville (47) used 10 media to compare the bacteria
to yeast ratio with the intention of finding a medium that would
serve for obtaining a total count of microorganisms in citrus
juices. These investigators recognized also that no single me-
dium will support the growth of all types of microorganisms
equally well. They decided that 8 of the 10 media could be em-
ployed satisfactorily, viz., tryptone glucose extract agar, Difco
pH 7.0; Lindegren's yeast agar No. 1, pH 5.8; tomato serum
agar, pH 6.5; Sabouraud's dextrose agar, Difco pH 5.6; dextrose/
tryptone yeast extract agar, pH 7.0; dextrose tryptone agay,
Difco pH 7.0; dextrose agar, Difco pH 7.3; and Lindegren's yeast
agar No. 1, pH 5.8. A similar study was made by Patrick (97)
while making plant sanitary surveys (Table 1). Specimens were
collected at 6 different places in each of the 13 plants during a
season, and plated on 4 media used by the control laboratories

Microbiology of Citrus Fruit Processing

in this citrus area. Some laboratories used 1 medium; others
recognized the fact that select nutrients must be present and
used 2 media for routine examination.


Media Listed in Order of Value
Pack | Numbers per ml of reconstituted juice
1 2 3 4 5 6

1 1 2,500 1,700 2,900 2,200 2,500 2 ppl
2 193,800 18,000 30,700 183,000 64,600 1500
3 237,000 197,000 141,000 161,000 1 ppl 6 ppl
4 534,000 387,000 385,000 234,000 1 ppl 3 ppl
5 366,000 358,000 315,000 218,000 1 ppl 4 ppl
6 213,000 190,000 16,600 143,000 2 ppl 4 ppl
7 262,000 249,000 165,000 181,000 2 ppl 5 ppl
8 5,000 4,900 3,500 4,000 1 ppl 6 ppl
9 13,350 9,670 9,600 5,850 1 ppl 3 ppl
10 9,150 8,950 7,870 5,450 1 ppl 4 ppl
11 5,300 4,750 4,000 3,370 2 ppl 4 ppl
12 6,550 6,200 4,150 4,525 2 ppl 5 ppl

Dpl = Per plate
1. Lindegren's agar
2. Peptonized milk agar
3. Orange serum agar
4. McClesky's agar
5. Acid sucrose agar
6. Dextrose tryptone agar

Table 2 shows the results of this investigation. It will be
noted that media used for general plate counts did not receive
comparable ratings; dextrose tryptone agar rated higher than
glucose tryptone extract agar. The other 2 media are primarily
for the isolation and cultivation of yeast and mold; Lindegren's
agar No. 1 rated higher in these tests than Sabouraud's agar.
The media usually employed for the enumeration of yeast
and mold are: Lindegren's agar, Sabouraud's agar, wort agar
and potato dextrose agar. It must be remembered that there
are yeasts in association with the citrus fruit industry that do
not grow well, if at all, on these media. There are osmophilic
yeasts (osmoduric), those yeasts that can grow or require a
concentration of soluble solids above that normally found in
single-strength juice, and, hyperacidophylic yeasts, those that
can grow or require more than the usual amount of acid found
in single-strength orange juice.

Florida Agricultural Experiment Stations


Media Specimens Examined and Rank of Medium *
1 2 1 3 4 5 1 6

agar, No. 1 .......... 2 (14) 2 (19) 4 (24) 1 (21) 3 (22) 1 (15)
tryptone agar -. 1 (24) 1 (19) 2 (24) 2 (21) 1 (22) 2 (15)
Tryptone glucose
extract agar -.. 3 (24) 4 (19) 1 (24) 3 (21) 2 (22) 3 (15)
Sabouraud's agar .. 4 (22) 3 (17) 3 (22) 4 (19) 4 (20) 4 (14)
Note: Numbers of tests made and the rank achieved for each inoculated medium, based on
numbers of colonies produced, are indicated; for example, 2 (14): 2 being the rank
given the medium, 14 the number of tests made.
* Specimens examined:
1. Unwashed peel
2. Washed peel
8. Peel at extractors
4. Juice to finishers
5. Juice or reconstituted juice from blending tank
6. Reconstituted juice

Some good examples reported in the literature of osmophilic
yeasts in concentrated orange juice will be reviewed. Smith
and Gillmore (118) reported swelled cans of 420 Brix due to
yeast activity when they were incubated at 100.40 F. for 48
hours or longer. Recca and Mrak (107) isolated and identified
6 pieces of Zygosaccharomyces that grew rapidly in 650 Brix
medium and 12 other strains representing 6 genera that grew
slowly in 650 Brix medium. Ingram (55, 56) found an osmoph-
ilic yeast, Zygosaccharomyces, capable of growing in 650 Brix
orange concentrate at 40 F. Later a strain of the same genus
was found that was able to ferment orange juice at pH 3.0 con-
taining 70 percent sugar. Bissett et al (15), while working
with 2-, .4- and 6-fold concentrated orange juice, observed that
swells developed in cans due to yeast growth when the concen-
trates had been heated no higher than 1500 F.
Patrick (96) reported that 2 cans out of 12 commercial 420
Brix orange concentrates developed swells while in 42 storage.
The yeast count in each case was not high when plated with
Lindegren's agar; the counts were 5 per plate and 7,600 per ml
of juice. These concentrates also yielded low yeast counts when
plated on Sabouraud's agars, but yielded higher counts when
plated on 10 percent sucrose, acid-yeast agar, pH 3.5. The
sample showing 5 per plate yielded colonies too numerous to
count when plated on acid sucrose medium, 1 ml of 1:10,000
dilution. The other sample showed a slight increase, from 7,600

Microbiology of Citrus Fruit Processing

per ml to 8,700 per ml. When examining some 4-fold lime con-
centrate that showed signs of fermentation in 4 days at room
temperature, Patrick found there were 491,400 yeasts per ml
of reconstituted juice when the specimen was plated on acid dex-
trose agar, pH 3.5; 8,500 on wort agar, pH 5.0; and 50,400 on
potato dextrose agar, pH 6.4, when each medium was inoculated
from the same diluted specimen.
Ingram (55) isolated yeasts from concentrated orange juice
using a 50 percent dextrose, 1 percent citric acid and 1 percent
peptone in a medium at pH 3.0. It was also learned that if these
cultures were transferred annually and stored at 320 F. they
would retain the characteristics first observed, but they would
lose them if kept on ordinary media.
Since coliform bacteria have been recognized in citrus juices
and concentrates and regarded as indicators of filth-contamina-
tion, it is important to discuss at this point some media and other

Fig. 10.-Florida chilled orange juice in supermarket coolers.

'ml tie ~ bi w

Florida Agricultural Experiment Stations

means that have been suggested to determine their existence
and significance. The standard method for the examination of
water and sewage (3) defines the coliform group as "all aerobic
and facultative anaerobic, gram negative, non-spore forming
bacilli which ferment lactose with gas formation." Whether
these organisms are of any significance when found in citrus
products is still debatable, but because the numbers present in
water and milk have a definite bearing on public acceptance of
quality as applied to health, naturally coliforms would be sought
in citrus fruit products and the methods already in use would be
applied. When coliform bacteria are found in drinking water,
they are regarded with suspicion. If Escherichia coli or coli-like
organisms are encountered, they are regarded as indicators of
contamination with filth. Wolford (140) regarded the coliforms
found in single-strength juice stored at -100 F. as significant
since they were not found in juice from sound fruit prepared
in a clean plant.
Wolford and Berry (138, 139) placed varying amounts of
diluted orange juice into standard lactose broth and also format
ricinoleate broth. If gas formed during the incubation period,
streak inoculations were made on eosin methylene blue agar
(E.M.B.) and colonies were selected, purified and studied later
or they were reinoculated into standard lactose broth to make
the completed coliform test. If gas formed in the reinoculated
tubes, the test was considered positive. Patrick (95, 99) inocu-
lated E.M.B. agar plates with dilutions of the specimen and then
inoculated select purified colonies into standard lactose broth
to make the completed test. He also reported (98) that select
coli-like colonies from E.M.B. plates were inoculated into Eijk-
man's lactose medium and standard lactose broth; if no gas
appeared in Eijkman's medium, it was assumed that E. coli was
not present.
The search for coliform bacteria in water involves the use
of "coliform index" or "most probable number" (M.P.N.) as a
standard procedure (3). According to Levine (63), the index
(10 percent positive lactose broth tubes inoculated with 10 ml
of sample) "is to be considered an index of attainability brought
about by engineering skill rather than an index of safety." The
failure to recognize this condition has led to much confusion
when attempts are made to apply the present standards for
drinking water to other beverages, foods and some conditions
in our environment. Levine cites some illustrations: mechan-

Microbiology of Citrus Fruit Processing

ically operated swimming pools generally are permitted a "most
probable number" (M.P.N.) of one per 100 ml; bathing beaches,
50 per ml or even 1,000 per 100 ml may be permitted. The
standard for soft shell clams is a M.P.N. of less than 230. When
this was found not applicable economically, the M.P.N. was
raised to 2,400 per 100 ml. To quote Levine (63): "The fact
that present drinking water standards are designed for waters
which are or can be subjected to special treatments is all too
often disregarded or not appreciated, and the tendency to project
this rigid standard into the sanitary quality of frozen fruit
juices, untreated waters or even treated waters in some localities
(tropical and subtropical) is leading to much confusion and seri-
ous conflict in opinions." This does not mean, however, that dis-
regard for good sanitation in processing plants should replace
our present system, but rather we should develop a better under-
standing of the significant coliforms (128, 129), if they are
present, as well as other bacteria in citrus products (3, 6, 124).
Levine (64) and Kaplan and Appleman (59) have suggested
that the determination of enterococci in food, water and orange
juice be included in the routine tests to learn the extent of fecal
pollution. Winter and Sandholzer (136, 137) have made a com-
parison of counts of enterococci and coliforms from 13 different
sources on 3 different media. They thought that enterococci
might serve as a more specific and reliable index of fecal pollu-
tion than coliform bacteria. The results are given in Table 7.
Beisel and Troy (9) and Njoku and Skinner (93) utilized the
boric acid medium of Vaughn and Levine (126) in a more con-
centrated form in their study of a suitable medium for the de-
tection of coliform bacteria. The boric acid nutrient solution
was prepared as follows:
Proteose peptone (Difco) .................. 10.0 grams
Lactose (Difco) ...... .............. .... 5.0 grams
K2HPO4 anhydrous .............. .... 12.2 grams
KH2PO4 ....... -.................. 4.0 grams
Boric acid ..... ...................... 3.25 grams
Distilled water ............................. 1,000 ml
The fruit concentrate and medium were combined as follows:
25 ml of 42 Brix concentrate were blended with 75 ml of sterile
boric acid medium adjusted to pH 7.0. They found that this
medium gave less false positive tests than when an equivalent
amount of concentrate was added to standard lactose broth.
However, none of the positive presumptive tests with either
medium gave confirmative tests on E.M.B. agar plates.

20 Florida Agricultural Experiment Stations

Faville and Hill (28) compared 4 presumptive liquid media
to evaluate their efficiency in suppressing gas formation by
yeasts and thus aid in the detection of E. coli. They inoculated
standard lactose, brilliant green lactose bile, lauryl tryptose,
and boric acid lactose (Vaughn and Levine) media each with
50 strains of yeast and 16 strains of E. coli. These media, with
the exception of the lauryl tryptose, were also inoculated with
48 samples of orange concentrate. The media were unsatisfac-
tory for the presumptive test. The essential findings are pre-
sented in Tables 3, 4 and 5. Boric acid medium suppressed the
growth of gas-forming yeasts and gave a slight inhibition of
E. coli at 98.6' F. (370 C.) and 109.40 F. (430 C.). The other
media except standard lactose were better inhibitors of yeast
at 109.40 F. (430 C.) than at 98.60 F. (370 C.). Standard lactose
broth and brilliant green lactose bile did not support a good
growth of E. coli at 109.40 F.

SVaughn- Brilliant
Standard Levine Green
Lactose Boric Acid Lactose
Broth Medium Bile
37C. I 43-C. I 3/C. 43C. j 37C. 43C.

Total number of samples.. 48 48 48 48 48 48
Number of positive
presumptive tests -.....-.. 48 33 34 14 37 9
Number of positive
tests confirmed -... ...... 8 4 0 0 3 2
Percentage of total
tests confirming ............ 16.7 8.3 0 0 6.2 4.2

Wolford (141, 142) compared boric acid lactose broth with
standard lactose broth for the isolation of E. coli from citrus
products. The inoculations were made from serial dilutions of
orange juice and positive presumptive tests were confirmed by
streaking on Levine's E.M.B. agar using Gram's stain, lactose
fermentation, citrate utilization, the methyl red test and Voges-
Proskauer reaction. He concluded that since large numbers of
E. coli were recovered when boric acid broth was inoculated, the
medium was superior to standard lactose for isolation purposes.
The results are listed in Table 6. This investigation involved

Microbiology of Citrus Fruit Processing


Hours Incubation

Total strains ..........
Number growing ...
Percent growing .....
Number gassing ....-
Percent gassing ....-


24 48


24 48


24 48

SBoric Acid

24 48


50 50 50 50 50 50 50
S36 42 23 25 22 23 6
72.0 84.0 46.0 50.0 44.0 46.0 12.0
10 12 3 12 0 3 0
20.0 24.0 6.0 24.0 0 6.0 0

E. coli

Total strains ..-....- ... 16 16 16 16 16 16 16
Number growing -- 16 16 16 16 16 16 11
Percent growing ... 100.0 100.0 100.0 100.0 100.0 100.0 68.8
Number gassing ......... 16 16 16 16 16 16 11
Percent gassing ......... 100.0 100.0 100.0 100.0 100.0 100.0 68.8



Hours Incubation

Total strains ...............
Number growing ........
Percent growing ..........
Number gassing .........
Percent gassing .....-...


24 48


24 48


24 48


50 50
13 3
26.0 6.0
9 0
18.0 0

E. coli

Boric Acid

24 48


Total strains ........- | 16
Number growing .- 16
Percent growing -....... 100.0
Number gassing ......... 10
Percent gassing .. 62.5

-16-16 -16 I -i
16 16 16 16 16 16 16
16 16 16 16 16 16 16
100.0 100.0 100.0 100.0 100.0 100.0 100.0
12 5 7 14 14 14 14
75.0 31.25 43.75 87.5 87.5 87.5 87.5
F i

Florida Agricultural Experiment Stations

3,372 tubes of each medium, but it is not clear how many samples
of the source material were examined. This may be one reason
for the lack of conformity with the report of Faville and Hill

[ Buffered Boric Acid Standard Lactose
Lactose at 43 C. at 350 C.

1. Number of tests ............. 3372 3372
2. Negative presumptives -. | 2688 1235
3. Positive presumptives ...... 684 (20.3%) 2137 (63.7%)
(a) E. coli .................... 315 180
(b) Intermediates ...... 21 62
(c) Aerobacter .......... 174 625
(d) Citrate negative .. 37 23
(e) False positives ... 137 1247

Martinez and Appleman (71) also checked persumptive media
in order to decrease or eliminate false positive tests because of
lactose-fermenting yeasts. They compared standard lactose,
brilliant green bile and lauryl sulfate tryptose media by inocu-
lating simultaneously with 62 samples of concentrated orange
juice. They found that brilliant green lactose bile and lauryl
sulfate tryptose did not inhibit gas formation (false positives)
when 10 ml inoculations were used, but these media were effec-
tive when smaller amounts of juice were used for inoculations.
A boric acid medium similar to Difco E-C medium and Difco
lauryl tryptose containing Duponal flakes instead of lauryl sul-
fate has been proposed by Hays (41, 42), but a proper evaluation
cannot be given at this time. Wolford (142) concluded that the
attaching of great significance to the presumptive results shows
a disregard for the chemical nature of the product tested.
It would be of great advantage if a reliable presumptive me-
dium could be found, in that confirmed and completed tests re-
quire long incubation periods before much information con-
cerning the coliform group may be had. It is not necessary for
the industrial routine technician to identify the species present,
but occasionally for the sake of information and clarity groups
of select cultures have been observed for biochemical and physi-
cal characteristics (65, 99, 139, 140).

Microbiology of Citrus Fruit Processing 23


No. of Enterococci Coliforms
Samples per ml or g per ml or g

Human feces ..---..- i 9 140,000 11,000,000
Raw sewage ............ 17 8,200 631,000
Polluted Waters A* ... 3 603 30
Polluted Waters B** 15 17 165
Cow feces ............... .... 5 5,600 110,000
Horse feces ..--............... 6 201,000 110,000
Sheep feces ........... .. 4 101,000 110,000
Pig feces ...... ........ 3 1,880,000 11,000,000
Raccoon feces ............ 3 3,230,000 110,000,000
Skunk feces ........... 2 2,100,000 11,000,000
Opossum feces .......... 2 1,100,000 11,000,000
Virgin soil ..................... 5 0 0
Pasture soil ................. 5 6 4

Samples of sea water collected at point where sewage entered water.
** Samples of sea water collected at least 50 yards from shore.

Since enterococci have been isolated from citrus juice (34,
59, 104), the citation by Winter and Sandholzer (136) giving
a recommended procedure for detecting the presence of entero-
cocci as a routine check for water analysis should be included
here. See Table 7 for comparative results. The details and
formulae should be consulted in the original publication. In
another leaflet (137), they described the following glucose-yeast
agar medium combined with sodium azide-penicillin medium of
Sherman and White, pH 7.4-7.6.
Yeast extract ........... 0.5 percent
Tryptone ..........--.. 0.5 percent
Glucose ........................ 0.5 percent
Sodium azide ........... 0.03 percent
Methylene blue .......... 0.001 percent
Agar ........................... 1.5 percent
Penicillin .................... 650 Oxford units per liter

The penicillin is added just before the plates are poured.
Reinbold et al (108) have given a selective medium for the
isolation and identification of the enterococcus group. The meth-
od is based on the ability of the enterococci to utilize sodium
citrate as a source of carbon and to convert ditetrazolium chlor-
ide to blue diformizan in the presence of 0.01 percent sodium
azide. The medium consists of the following: yeast extract
(Difco) 1.0 percent; trypticase (B.B.L.) 1.0 percent; sodium ci-
trate 2.0 percent; and agar 1.5 percent. The medium is ad-
justed to pH 7.0. When melted for plating, add 1 ml of 0.1 per-

Florida Agricultural Experiment Stations

cent sterile aqueous ditetrazolium chloride and 1 ml of a 10
percent sterile aqueous solution of sodium azide per 100 ml of
medium. The inoculated plates are stratified with more of the
same medium after solidification has taken place. The plates
are incubated at 98.50 F. for 72 hours.
Patrick (104) inoculated streptococcus organisms from citrus
products into Bacto-enterococcus confirmatory medium, Difco.
If the cultures grew, they were subjected to other biochemical
tests to identify them more completely. Lake et al (62) have
presented a scheme for the identification of S. faecium and S.
lurans; they do not ferment melizitose, alphamethyl glucoside,
glycerol anaerobicallyy), sorbitol and citrate. They do not re-
duce tetrazolium in a special glucose agar nor tolerate 0.04 per-
cent tellurite, and do ferment melibiose and require folic acid
for growth. The two strains are thought to be varieties of 1
species (62). Litsky et at (66) used glucose azide broth as a
presumptive medium and ethyl violet broth as a confirmatory
medium for detecting enterococci, as they had found ethyl violet
was selective for gram-negative bacteria. Slanetz et al (115)
have improved the procedures using membrane filters to deter-
mine the numbers of enterococci in water and sewage; these
may be adaptable to citrus juices.

The use of culture methods to show the extent of contamina-
tion has its advantages, but it is time consuming. Because of
the volume of production, the control laboratory is reminded
constantly of the need for means and methods that will give
results quickly. Much time has been given to the application
of direct microscopic count to citrus juices and concentrates.
Methods have been developed and put to use with modification
to suit the individual. There is a wide disagreement between
counts derived from direct examination and those from culture-
plate methods (43). Usually, with a continuous operation in a
plant the number of organisms that may be seen easily in a pre-
pared slide increases, but many develop simultaneously that are
not seen so easily. The latter forms cause great concern, because
the organisms responsible for "off-flavor" are almost certain to
be in this group. The rise in numbers as seen with the aid of
the microscope is taken as a warning of spoilage. There are
strains of bacteria involved that are almost undetectable when
mixed with citrus juice and it is very possible, because of that

Microbiology of Citrus Fruit Processing

and in spite of very intensified inspection, that spoilage does
occur in the process.
A discussion of the use of stained slides in plant routine
checks was presented briefly by Hays and Riester (43). Mur-
dock and Brokaw (81) believe that the stained slide routine is
valuable for the control of quality in citrus concentrates.
A staining method that was among the first to give some sat-
isfactory results was the method developed by North for use in
the dairy industry (92). When the aniline oil methylene blue
stain was used to determine bacteria in milk, higher counts were
reported than when plain methylene blue stains were employed.
Many advantages have been realized with its use in the citrus
An improvement in staining technique for direct microscopic
examination was made by Hill et al (50). Five ml of reconsti-
tuted juice and 5 ml of a 0.075 percent aqueous solution of crys-
tal violet are mixed in a test tube. The tube is stoppered and
shaken, 0.01 ml of the juice-dye mixture is transferred to 1
sq cm area on a clean glass slide and spread evenly with a straight
nichrome wire. The slide is dried with a heat lamp and the sur-
face is coated with a plastic spray (Spraint) (Krylon). The
plastic coating is clear and produces a slide that is permanent
and may be filed for future reference; there is no need for fixing
solutions, destaining or washing.
Rowell (110) found the slide-plate method for counting viable
organisms in orange juice to be advantageous. A petri dish is
prepared with an amount of agar medium and after it has con-
gealed, a sterile microscope slide is placed on the surface. Over
the slide is poured a known amount of orange serum agar mixed
with a known quantity of diluted orange juice. The preparation
is incubated 7 hours at 860 F. and the slide is cut out of the plate
and the agar on the surface is trimmed to about 2.5 x 2.5 cm.
It is then dried on the slide with an infrared lamp and stained
with Loeffler's methylene blue stain, washed in alkaline rinse
water and covered with a glass cover. The colonies are dark
blue on a light blue background. Spreader colonies do not inter-
fere and only 1 dilution is necessary for counts ranging from
10,000 to 10,000,000 per ml.

The detection of potential spoilage contamination by chemi-
cal means has been given considerable thought. Hill et al (50,

Florida Agricultural Experiment Stations

51) have reported a method, modified from an earlier applied
method, that involves the detection of diacetyl in the distillate
from orange juice before off-flavors occur. The test is colorime-
tric and they used the reagents of the Voges-Proskauer test.
The amount of diacetyl present may be determined by making
a calibration curve, using known amounts of diacetyl in water
and determining the intensity of the color with an electropho-
tometer. Modification of this method has been submitted by
Beisel et al (14) and Byer (19).
A means for measuring the dissolved oxygen used correlated
with the number of yeasts inoculated into sterile juice was re-
ported by Henry et al (46). They were able to show also the effect
of chance contamination in "off-flavor" line-samples that might
not be brought to the attention of the investigator through the
use of unsuitable media.

A citrus processing plant that operates under rigid rules of
sanitation uses fruit with a minimum of microbial contamina-
tion (30). The successful manufacture of products of good
quality begins with a critical enforcement of sanitary regulations
in the early part of the process. Rigid sanitation throughout the
plant-process is almost futile if the fruit used has a high mi-
crobial contamination.
Fruit from the grove will carry the contamination common
to that area and season. Teunisson and Hall (123) made a gen-
eral observation of some cultures common to citrus processing
plants and found them to be the usual contaminants of soil, un-
treated surface water and dusty air. The degree of contamina-
tion on the fruit exterior will vary also if the fruit has been on
the ground or received an infestation from air-borne dust. Smith
and Gall (117) made a survey of microorganisms in Norfolk fine
sand from Pineapple orange groves in 2 separate areas: 5 plots
in 1 area were sampled during March, April and May; in the
other area 5 plots were sampled during April and May. The
average numbers of organisms per gram of soil are given in
Table 8. The authors did not mention the presence of yeast in
the soil samples, but the soil beneath the tree may be infested
with yeast because of the deterioration of fallen fruit. Due to

Microbiology of Citrus Fruit Processing

seasonal conditions in the grove, the fruit may arrive at the
processing plant with a heavy contamination on the exterior
surfaces, some of which will be of sanitary significance.


Bacteria and
1,800,000 |:

-Z :


Bacteria and
Actinomyces I Mold
1,700,000 10,000
1,950,000 26,000
2,300,000 11,000
3,050,000 12,000
700,000 25,000
2,450,000 7,000
1,700,000 6,000
3,350,000 10,000
2,200,000 8,000
1,150,000 16,000

Bacteria and

Fig. 11.-Unloading oranges into temporary storage bins.
(Courtesy Minute Maid Corporation.)



I 6,000


1 -...--
2 ......
3 ....
4 ......

1 ......
2 ......
3 ......
4 ......
5 ......

;~-~1~~~'U"L~'~ j

Florida Agricultural Experiment Stations

The citrus fruit is washed before the juice is extracted. Gen-
erally, the fruit is sprayed with water, moistened with detergent
while passing over brush-rolls, passed over brush-rolls under
water spray and finally rinsed with chlorine water before going
to the juice extractors. Some operators believe it is not good
practice to store washed fruit in bins (17) because of subsequent
spoilage, even though sodium pentachlorophenol or borax has
been used in the wash water.
Beisel et al (10) reported on the effectiveness of a fruit-
washing plan where the detergent was replenished intermittently
rather than continuous for a shift. As a result, the fruit came
from the washer with a low infection of microorganisms adher-
ing to the peel. Before washing, the peel carried 1,000,000 or-
ganisms per sq cm; after washing, the number had been reduced
to 38,000 per sq cm. A series of tests were made by Patrick
(97) on peel of washed and unwashed fruit in commercial juice
processing plants. The highest population found in any 1 test
on 24 unwashed peel samples was 210,000, with an average of
22,476 per sq cm. Nineteen tests on washed peel gave a maxi-
mum figure for any 1 test of 18,880, with an average figure of
2,595 per sq cm. Ten of the 19 tests gave less than 10 micro-
organisms per area, thus indicating the thoroughness with which
some operators wash fruit. Murdock and Brokaw (81) present
graphically the decrease in contamination on fruit surfaces after
Fig. 12.-Cannery bulk fruit bins being loaded with oranges.
(Courtesy Pasco Packing Co.)

Microbiology of Citrus Fruit Processing

Washing should remove not only dirt but also scale insects.
Detergents tend to loosen the scales from the peel so they may
be removed with sprayed water and the friction of the brush-
rolls. Scale insects proved to be a source of coliform contamina-
tion (98) and also contributed bacteria and yeast on an average
of 320,000 per medium size fruit. Slime and gum-forming or-
ganisms, similar to Leuconostoc, averaging 220,000 per fruit,
were also associated with scale-infested surfaces.
When trees have been infested with aphids, scale or white-
flies that excrete honeydew, sooty mold (Capnodium citri) will
develop on the surface of leaves and fruits (109, 122). This
membrane of black mycelia may be removed by the detergents,
forceful water sprays and brush-rolls employed in the wash
units of most fruit-processing plants. Improperly washed fruit
can be a source of contamination.
Fruit flies (Drosophila sp.) are very effective carriers of con-
tamination, because of the desire, if not the necessity, to feed
on fermenting fruit. Because of their size, they can invade the
slightly open parts in the equipment where juice is being ex-
tracted and finished. Recca (107) isolated 9 strains of yeast
representing 4 genera from the intestinal tract of fruit flies.
Six of the 9 strains were also isolated from the surface of fruit.
Patrick (96) found fruit flies to be carriers of coliform bacteria
that fermented standard lactose broth at 37.50 C. but failed to
ferment Eijkman's lactose broth at 45-46 C. Simmon's citrate
agar inoculated with fruit fly suspension produced 2,300 colonies
per fly. Dextrose tryptone agar gave a colony count of 118,000
per fly, consisting mostly of bacteria; while wort agar yielded a
colony count of 156,000 per fly, consisting mostly of yeast. These
organisms may pass through the intestinal tract of fruit flies
undamaged and many of them grow when deposited in orange
juice or other suitable media.

Packinghouse rejects collect at the packinghouses where
fresh fruit is boxed for the market. Juice from this fruit may
be used in the processing plants, and it may also be a source of
undesirable contamination. The fruit may be handled many
times, and often roughly, before it is placed in the bins at the
processing plant. The amount of damage done to this fruit de-
pends upon the stage of maturity, the kind of fruit and the care

Florida Agricultural Experiment Stations

in handling during the hauling, sorting, loading and unloading.
Sometimes the fruit is left in the truck and exposed to the sun
and rain for hours before it is unloaded and stored for use. Such
exposures and delays are not conducive to the production of
low-count juice. Fruit that has been mishandled is a potential
source of undesirable contamination to a processing plant (143).
Burst fruit is attractive to fruit flies and because of the con-
tamination carried by them, burst fruit with fly infestations
should not be used. Juice from such fruit has been sampled for
presence of microorganisms (96). Dextrose tryptone agar inocu-
lated with juice from burst fruit produced 6,300 colonies per ml,
mostly bacteria. Sabouraud's agar produced 11,000 yeasts and
bacteria per ml. McClesky's agar yielded slime and gum-forming
bacteria, resembling Leuconostoc, to the amount of 43,000 per ml;
rod forms, resembling Lactobacilli, grew 54,000 colonies per ml

Fig. 13.-Cleaning oranges with soap on a brush-washer.
(Courtesy Snively Groves, Inc.)


i"4W -.X..S

Microbiology of Citrus Fruit Processing

of juice. A sample of juice from over-mature but acceptable
fruit, taken from the grove undamaged, was plated. This juice
contained less than 5 organisms per ml. Murdock et al (77) found
that burst and otherwise unsound fruit carried contamination
significant in the production of off-flavor. Burst fruit should
not be used and undamaged fruit with surface soiled with juice
from burst fruit should be washed to protect the process from
undesirable contamination.

Fig. 14.-Oranges receiving a germicidal rinse.

The contamination in the interior of fruit offers a big chal-
lenge to the fruit processor (81) and supplies a condition most
difficult to control, because exterior symptoms are not always
visible to give warning. Small injured or infected spots on the
peel may not be seen as the fruit travels on the inspection belt,
but the interior of the fruit may be far from acceptable.
It has been pointed out (25, 40, 48, 94) that plant pathogenic
microorganisms grow in the acid tissue of citrus fruits. Wolford
et al (138, 143) found that juice prepared from "soft rot" infected

Florida Agricultural Experiment Stations

Valencia oranges contained a microbial population approxi-
mately 2,500 times more concentrated than juice prepared from
sound fruit. He also found that coliform bacteria were more
numerous in juice from "soft rot" infected fruit. He isolated
46 coliform cultures for study. Most of the cultures were charac-
teristic of Aerobacter; 3 cultures were Escherichia. Patrick (99)
made a study of 217 coliform cultures from orange concentrates
and damaged oranges. Only those coliform colonies were selected
that produced a metallic sheen on eosin methylene blue agar
(E.M.B.) thus resembling Escherichia coli. Among these cul-
tures, the Aerobacter aerogenes types were represented by 41 cul-
tures, the intermediate types by 37 cultures and the E. coli types
by 139 cultures. Soft fruit, due to bruises, and fruit of poor
quality generally contribute high contamination of yeast, mold
and bacteria (97). Slime and gum-forming bacteria were com-
mon contaminants in juice from over-mature fruit that was
drying out (98).
A common source of mold in commercially extracted citrus
juice is black rot, which frequently affects early varieties (49,
109, 116, 122). The disease begins at the stylar end, spreads
along the core and usually does not show external symptoms of
damage. The causal fungus (Alternaria citri) has been ob-
served in Hamlin, Jaffa, Parson Brown, Pineapple, Ruby Blood
and Valencia oranges and tangerines. A widespread infection of
Jaffa oranges recently caused considerable trouble in concen-
trate plants in Florida. Hill (49) made an investigation of
Jaffa oranges collected from an infested grove. There was a
visible deterioration in 54 percent of the oranges picked in Oc-
tober, with 43 percent showing Alternaria infestation according
to the isolation tests. The incidence of infestation had decreased
among the fruits inspected in January, partly because of the
early drop of infected fruits, and also possibly because of in-
creased individual fruit resistance and the climatic change to a
dryer season.
Aspergillus niger has been observed (94) developing in the
stylar end of calamondin fruits while on the tree. It is generally
considered that the ports of entry for these fungi are in varieties
of fruit with weak stylar tissue, common to some varieties, or
through injuries. A. niger has been observed by Patrick in Lue
Gim Gong oranges following a severe hurricane. The fungus
probably gained entrance into the fruit through thorn or twig
punctures. The interiors of most of the oranges were entirely

Microbiology of Citrus Fruit Processing



h, 7r;-"r




Fig. 15.-Black rot or black heart in an orange caused by Alternaria fungi.

, r,_



., .t'- ;, :it

Florida Agricultural Experiment Stations

black due to the growth of the fungus. The mold count was not
determined in the extracted juice, but by restricting the growth
of mold on inoculated plates, the yeast count in association with
this condition was observed to be 333,200 per ml of juice. There
were tiny specks in the peel showing previous injury that would
pass almost unnoticed. There was a high correlation between
fruits with injury specks and black interiors. Oranges without
noticeable injuries yielded an extracted juice with 3,000 organ-
isms per ml.
Hill and Faville (31) inoculated mature oranges on the tree
with cultures of acid-tolerant bacteria that had been isolated
previously from orange juice. The oranges were collected and
examined periodically over a period of 5 weeks. The exterior
of the fruits did not give much, if any, indication of the deteri-
orating tissue. The organisms grew in the juice-bearing tissue
and produced deterioration. High counts were maintained in
the fruit over a period of 5 weeks. It has been reported by Fa-
ville and Hill (30) that the use of fruit with a citric acid content
below 0.6 percent generally produced a juice with a high count,
above 50,000 per ml, while juice from fruit with a citric acid
content above 0.6 percent produced a juice with low counts. The
use of over-mature fruit with low acidity will often give a high-
count juice.
Wolford (143, 144) investigated the sources of coliforms in
frozen orange concentrate and found that a direct contamination
by handling during production was not indicated. Sufficient
coliforms were isolated from aseptically harvested fruit to show
they were normally occurring flora in groves. Packinghouse
wash tanks were the principal sources of the organisms in frozen
orange juice. E. coli was isolated from oranges delivered to con-
centrate plants. When found in the concentrate, it indicated
the condition of the fruit used rather than the sanitary condition
in the plants. Coliform positives were found to be less than
10/100 ml.

In spite of the fact that citrus juices contain relatively large
amounts of citric acid, which is bacteriostatic for many microbial
species, experience has taught that many types of microorgan-

Microbiology of Citrus Fruit Processing

isms survive or grow in citrus products and contribute to spoil-
age of such products. If these organisms are capable of growth
after passing through the process, and the product is not stored
properly, spoilage or sufficient change of flavor may take place to
make the product undesirable.
In the early development of the citrus products industry,
when chilled juice was thought to be the product that would be
in great demand (20, 113), the investigators were interested in
knowing what organisms would live in juice and how they could
be eliminated without destroying the fresh-fruit flavor. It had
been demonstrated (20) that, by selecting the fruit and using
good sanitation, a juice could be bottled without heating and
be kept at 320 F. for a week or more without loss of flavor.
Other investigators (113) observed that Escherichia coli, Lacto-
bacillus acidophilus and Bacillus subtilis failed to grow in orange
juice at any temperature. The death rate was rapid at 98.60
F., slower at 770 F. and still slower at 10.40 F. They used E. coli
as a guide to learn the possible behavior of the pathogenic bac-
teria; strains of pathogens were not used because of the diffi-
culty in detecting them as survivors. They found strains of
yeast which grew in orange juice slowly at 00 F. and rapidly at
32 F., and which, when stored at 5 F., died-off in 6 months
from an initial number of "several thousand" to "a few hun-
dred"; also, the total bacteria count dropped during this time
in storage.

Fig. 16.-Supermarket freezer containing cans of Florida citrus concentrate.

...... ---.-.- -. .

""-'-'-- ~ Q "\^.^,i," BrLliSSS afi --flli

Florida Agricultural Experiment Stations

Observations made of contamination in commercial packs of
concentrate revealed facts not in conformity with laboratory
packs. One report (26) covered the observations of commercial
packs of orange, tangerine and grapefruit-orange concentrates
that showed an initial count of 105,000, 102,000 and 13,000 micro-
organisms per ml, respectively. About 88 percent of the mi-
croorganisms were destroyed at all temperatures when the cans
were stored at 75, 45, 25, 20, 15, 10, 5, 0, -8 and -20 F. for
30 days. Seventy-five percent were destroyed in 7 days at 20
and 250 F.; and 75 percent in 15 days at 15 and 450 F. It was
concluded by these investigators that because of the rapid re-
duction in numbers possible microorganisms were not respon-
sible for any changes in the juice during storage.
Observations on the death rate of organisms in citrus con-
centrates were made at intervals for 6 months by Moore et al
(72). Three orange concentrates were prepared-Hamlin, Pine-
apple and Valencia; Dancy tangerine; and 2 of grapefruit-Dun-
can and Marsh Seedless. The concentrates stored at 40 F.
showed an immediate reduction in total count and leveling off in
about 2 months. At -8 F. the reduction in total count was
slow, with a gradual decrease over the entire 6 months, at which
time there was a reduction of 80 percent. The reduction of num-
bers in concentrate stored at 100 F. was a little more rapid for
the first 4 months and after 6 months there was a decrease of
84 percent. At 200 F. the reduction was rapid for the first 3
months (95 percent) and remained at that level for the remain-
ing 3 months. Concentrates stored at 320 F. and 400 F. showed
a reduction of 91 percent after 5 weeks and 4 weeks, respec-
tively; and after 6 months the numbers had been reduced 96
percent. Correlation between death rate and the concentration
of acid was not determined.
A decrease in numbers (96) has been noted in 13 samples of
commercial 420 Brix orange concentrate stored at 0 and 420 F.
after 3 and 4 months. The numbers were reduced to negligible
amounts in most of the samples stored at 0. The same observa-
tion was true for samples stored at 42 F., except for 3 speci-
mens. Two cans showed swelling after storage for 143 days and
130 days, respectively; the other specimen showed no signs of
fermentation after 121 days, but did contain a yeast count of
8,300 per ml.
Patrick and Huggart (101) observed a rapid decline in the
number of organisms during the first month when four- and six-

Microbiology of Citrus Fruit Processing

fold concentrates were stored at -8, 20 and 40 F. The rate
of decline was slower at -8 F. than at higher temperatures.
The decrease in counts in all the samples ranged from 74 percent
in 1 pack stored at -8 to 90 percent in products stored at high-
er temperatures. Storage of the concentrates at 600 F. was not
satisfactory because of fermentation, clarification and gelatin.

Fig. 17.-Orange serum agar plate inoculated with Leuconostoc mesente-
roides, one of the organisms responsible for "buttermilk" spoilage in citrus

Wolford (145) noticed that the total count in 4 commercial
packs of orange concentrate was reduced rapidly during the first
6 weeks at -10' F., the percentage of cells destroyed ranged
from 23 to 63. By using a special medium, potato dextrose agar,
pH 5.6, that was more favorable to the growth of yeast, the de-
cline showed a percentage spread of 21 to 54. After 11 months
samples stored at -10 F. showed a 36 percent survival, and

Florida Agricultural Experiment Stations

those stored at -950 F. showed a 90 percent survival, while
samples that had been warmed at intervals to 20 and 40 F.
averaged about 2 percent survival. Coliforms were practically
eliminated from the samples subjected to varying time and
temperature conditions.
Growth of 3 different types of organisms implicated in orange
juice spoilage, Leuconostoc sp., Lactobacillus plantarum var.
mobilis and Zygosaccharomyces vini, was reported by Berry
et al (8). These organisms decreased in growth rate with a
decrease in temperature below their optimum of 860 F. An
increase in the concentration of soluble solids from the optimums
of 12 and 200 Brix resulted in a decrease in growth rate. Leu-
conostoc and Lactobacillus strains did not grow at 400 F. regard-
less of the degree Brix, and did not grow in 420 Brix regardless
of the temperature. Yeasts grew in all concentrations through
420 Brix at 400 F.
Murdock and DuBois (79) reported the survival of Leuconos-
toe, Lactobacillus and yeast in 42, 58.5 and 700 Brix concentrated
orange juice stored at 0, 15 and 400 F. The death rate increased
generally with the temperature and with the concentration,
except with yeast in 42' Brix, which grew at 40' F., but died
slowly at 0 F. and rapidly at 150 F. When these organisms were
observed in 12, 20, 30, 42, 58.5 and 700 Brix at 750 F., bacterial
growth occurred in all concentrations through 300 Brix while
yeasts grew in concentrations through 58.5 Brix. In 700 Brix
concentrate all strains died, Leuconostoc dying more rapidly than
the other test organisms.
Rushing et al (111) studied the survival and growth of mi-
croorganisms in citrus concentrates and found that no cans of
concentrated juice of 500 Brix or higher swelled in 350 F. stor-
age, but at all concentrations below 700 Brix swells were ob-
served at 50 and 600 F. Yeasts were the main spoilage agents.
Numbers decreased as concentration and storage time increased.
Slime and gum-forming bacteria decreased more rapidly at 50
and 60 than at 350 F.
According to Barretto (5), the mean generation time of Leu-
conostoc mesenteroides was 83.2 minutes in 30 Brix juice and
54.3 in 100 Brix juice during the logarithmic growth phase. An-
other illustration may be cited in the investigation of Curl et al
(23). A 650 Brix concentrate was stored at 400 F. for 3, 6,
12 months without evidence of spoilage, but when placed at 800
F., the containers swelled in 2 days. The yeast responsible for

Microbiology of Citrus Fruit Processing

the fermentation remained viable at the lower temperature in
spite of the concentration and acidity, and grew when tempera-
ture became more suitable. Concentration is not always an in-
hibitory agent to growth. An osmophilic yeast that fermented
concentrated orange juice (650 Brix) was reported by Ingram
(55) to be Zygosaccharomyces sp.; he also reported (56) a
Zygosaccharomyces sp. that grew in a dextrose solution up to
70 percent concentration and a pH of 3.0. The optimum for its
growth, however, was 30 percent dextrose and pH 4.5. Eight
species of the genus Zygosaccharomyces were identified by Recca
(107) from among 92 yeast cultures isolated from fruits, juices,
concentrates and other sources in citrus processing plants located
in southern California. Two cultures from the 92 isolations
failed to grow in a 65 Brix sugar solution. Thirteen other
species representing 9 genera grew in a 650 Brix dextrose solu-
tion. None of the cultures grew in 750 Brix medium, but all of
the cultures were able to grow in a 350 Brix medium.
Bacteria, inoculated artificially into oranges on the tree and
in the laboratory, were observed by Hill and Faville (48) to
grow and produce signs of deterioration. Three cultures were
used: Aerobacter aerogenes, Xanthomonas sp. "A" and Achrom-
obacter sp. in 10 oranges each. After 5 weeks A. aerogenes was
observed to be growing at pH 3.7 to 4.3 and had increased in
numbers from less than 1,000 per ml to a range of 75,000 to
9,400,000; Xanthomonas sp. "A" from less than 1,000 per ml to
a range of 11,000 to 157,000,000 per ml; Achromobacter sp. in-
creased from 10 to 500 per ml initially to 20 to 71,000 per ml.
Barretto (5), working with acid-tolerant organisms found, when
checking growth rates in orange juice at 60, 70 and 800 F., that
L. plantarum, L. brevis and Achromobacter sp. grew best at 800
F. in 100 Brix juice. Also, the optimum concentration and tem-
perature for L. mesenteroides was 10 Brix at 70 F. Rushing
et al (112) repeated the work on Lactobacillus brevis, L. plan-
tarum var. mobilis, Leuconostoc detranicum and L. mesente-
roides. L. mesenteroides was sensitive to pH, and growth or
decline was observed at pH 3.4 in both 12 and 180 Brix, at pH
3.6 in 180 Brix and at pH 3.8 in 420 Brix. L. brevis failed to
grow at pH 3.8 in 420 Brix. The most rapid growth observed
was L. mesenteroides at pH 4.0 in 120 Brix.
Faville, Hill and Parish (29) inoculated E. coli, Achromobac-
ter sp., Leuconostoc mesenteroides and Rodotorula sp. into 10.50
Brix orange juice and concentrated orange juice (420 Brix).

Florida Agricultural Experiment Stations

E. coli showed a definite decrease in both juice and concentrate
during the first hour at 860 F. and a gradual decrease during the
7 hours, but not complete destruction. Achromobacter sp. died
off rapidly in the first hour and was completely destroyed in 6
hours in concentrate, while in orange juice there was a gradual
increase (10-fold) in numbers during the 7-hour period. L.
mesenteroides died off rapidly during the second and third hours
in concentrate; then gradually, but not completely, during the
remainder of the 7-hour incubation. In orange juice the death
rate was just about equal to the growth rate. Rodotorula just
about maintained its numbers in concentrate, but showed a defi-
nite increase after 3 hours in single-strength juice. Similar spe-
cimens of 420 Brix concentrates were inoculated by Faville et al
(31) and incubated at 1040 F. E. coli died in 15 minutes, L.
mesenteroides in 3 hours and Achromobacter sp. in 5 hours.
Similar inoculations in 420 Brix concentrate were incubated at
37.4, 86 and 1040 F. and observed for 7 hours. E. coli showed
a rapid decline in the first 30 minutes at 37.4 and at 860 F.,
with a more rapid decline at the higher temperature. The de-
cline was much more gradual after the first hour and the culture
was still surviving in large numbers after 7 hours. L. mesente-
roides died off rapidly in the first 3 hours. It was completely
destroyed in that time at 1040 F. There was a gradual decline
throughout the observation and many cells were surviving after
7 hours at 86 F. The numbers remained the same in inocula-
tions made at 37.40 F. throughout the 7-hour period. Achromo-
bacter sp. showed a rapid decline with complete destruction at
1040 F. and 860 F. in 5 and 6 hours, respectively. Rodotorula
showed a slight decline at 104 and 37.40 F. throughout the 7-
hour period, while at 860 F. there was no apparent change in
numbers during the observation.
At the Citrus Experiment Station we have observed the mi-
crobiological changes in unstabilized chilled orange juice. The
total count, as indicated by plating on orange serum agar, pH
5.7, stored at 400 F., increased from 79,000 to 99,000 per ml in
Hamlin orange juice held in storage 21 days in wax cartons;
in Valencia orange juice the count increased from 39,500 to
1,000,000 per ml in 21 days. There was a decrease in count from
3,600 to 2,500 per ml in chilled Pineapple orange juice after 14
days of storage. Valencia orange concentrate, reconstituted,
cooled and stored at 400 F. for 23 days, increased in count from
400 to 39,200 per ml. Organisms usually increased in total

Microbiology of Citrus Fruit Processing

count in unheated juice during storage, and decreased slowly in
heat stabilized juice to 500 or less at the close of the storage
period. Some strains of yeast will grow at 400 F. in single-
strength juice and may be responsible for off-flavors that may
develop in the pack.
Acidity alone does not limit microlife. There is less destruc-
tion of microorganisms in sweetened juice stored at 00 F. than
in unsweetened juice (85). However, the number surviving
decreases with the increase of concentration of the solids except
for those microbial types that may be classed as osmophilic (27,
55, 56, 107). McAllister (83) isolated Xanthomonas sp., Ach-
romobacter and yeast most frequently from commercial packs
of concentrated orange juice. He found that yeast survived in
largest numbers during long storage periods. He reported a
new Bacterium sp. that grew in a 40 percent sugar solution.
When grown in 2.5 percent glucose, this species lowered the pH
to 3.7 and required 3.25 ml of 0.1 N NaOH to neutralize the
acidity in 5 ml of solution. Yeasts isolated from citrus products
(107) were found to grow at low pH levels; over half of the 57
cultures studied grew at pH 1.5 and practically all of them grew
at pH 2.0. One culture of Torulopsis failed to grow at pH 3.0,
2.0 and 1.5.
Concentration and acidity combined are not a restrictor of
growth in some instances. The senior author has observed a
concentrated 4-fold Persian lime juice show active fermentation
at room temperature. The initial count in the single-strength
juice was 4,800 yeasts per ml. In 4-fold concentrate the initial
count was 5,500 per ml of reconstituted juice. The sealed con-
tainers of concentrate blew open 4 days later. The total acid in
the 4-fold concentrate was 25.2 percent, as anhydrous citric acid.
Acidophilic yeasts were encountered in citrus products by
Recca and Mrak (107) ; they isolated 92 cultures comprising 35
species. They reported that 54 of the cultures isolated grew at
pH 1.5; 38 at pH 2.0; 40 at pH 2.5; and 91 at pH 3.0, thus veri-
fying the belief that yeasts in general can grow in media having
a low pH.

When single-strength juice was given a heat exposure of a
few seconds, time not designated, at 185 to 1900 F., Nolte and
Von Loesecke (89) noted the organisms that remained viable
were not the types that would grow in canned citrus juice and

Florida Agricultural Experiment Stations

cause spoilage. While making heat treatment applicable to the
present development of the citrus concentrate industry, it was
shown by Murdock et al (76) that yeast had a higher thermal
resistance in single-strength juice and concentrate than was ex-
hibited by Leuconostoc sp. or Lactobacillus sp. Also, yeast
had a higher thermal resistance in concentrate than in single-
strength juice, possibly due to the sugar content. These investi-
gators suggested that operators of juice concentrate plants
should apply the heat treatment to single-strength rather than
to concentrate.
In our own investigations (102), we found that Leuconostoc
plantarum in single-strength juice at pH 3.6, 3.8 and 4.0 was
almost completely destroyed by a heating time of 12 seconds at
1650 F. Leuconostoc mesenteroides at the same pH values with
a heating time of 12 seconds at 1550 F. was completely destroyed,
except at pH 4.0. At that pH the effective temperature-time
exposure was 1650 F. for 12 seconds. When an unidentified
mixed culture, common to grove-run fruit, was mixed into the
juice at pH 3.8 and 4.0, 1900 F. for 12 seconds was not sufficient
to give 100 percent destruction of the contamination present.
Enterococci were killed at 1650 F. at pH 4.1 in the heating sec-
tion of a plate-type heat exchanger in about 14 seconds. The
organisms in 2-, 3- and 4-fold concentrates (103), prepared from
the grove-run fruit or with an unidentified mixed culture added
at pH 3.9, were not killed when exposed at 1850 F. for 12 seconds.
At pH of 3.8 there was no survival of organisms in 3-fold con-
centrate at 185 F. after 12 seconds. The numbers surviving
in 2-fold and 4-fold concentrates may not be considered signifi-
cant, as the numbers growing on orange serum agar were low
and an appreciable number of these would most likely not cause
spoilage under favorable conditions.
When Bissett et al (16) heated sweetened and unsweetened
lime juice for 5 seconds at temperatures ranging from 120 to
2000 F. at intervals of 100 F., most of the organisms were de-
stroyed at 1500 F. or above. The sweetened juice was prepared
by taking 8.8' Brix unsweetened lime juice and adding sugar to
450 Brix. Pasteurizing the lime juices at 170 to 2000 F.,
followed by storage at 350 F., was satisfactory for handling both
products. Bissett et al (15) also found that by heating 2-, 4-
and 6-fold orange concentrates at 140 F., the product could be
stored at 350 F. without spoilage. Products processed at 1600
F. or above were not subject to spoilage when stored at 35 and

Microbiology of Citrus Fruit Processing

800 F. Six-fold concentrates heated to 1300 F. did not ferment
in storage. They feel that these figures are minimum and warn
that even though "products processed at 1600 F. or above were
not subject to cans swelling in either 350 F. or 80 F. storage,
it is believed that somewhat higher temperatures should be
used in practice to provide a margin of safety."
The survival of coliform bacteria in single-strength frozen
orange juice for 2 to 43 weeks at -100 F. was observed by
Wolford (140). There was a rapid drop of total number in the
first five weeks, then the decline became less, indicating a mix-
ture of sensitive and resistant organisms in the original con-
tamination. The decline of coliform bacteria in these packs
of frozen single-strength orange juice stored at -10 F. did
not follow that of the total count. Those organisms resembling
E. coli were recovered most frequently in juice prepared from
soft fruit; they remained viable during the entire storage period
of 43 weeks. Two hundred and thirty-six cultures of coliform
bacteria were isolated for more intensive study. Forty-six of
these cultures resembled E. coli; 20 were intermediate forms,
E. freundii; and 170 resembled Aerobacter.
Off-flavor in concentrated orange juice that was caused by
microbial growth has been discussed by a few investigators.
Hays and Riester (43) found bacteria capable of growing in 35'
Brix orange concentrate and producing an off-flavor, or "butter-
milk flavor," in the product in the evaporators of a concentrate
plant. They believe the spoilage could be controlled by rapid
heating and cooling of the juice before it enters the evaporators.
They observed that 10 seconds at 1600 F. was sufficient to kill
these organisms. Similar conclusions were given by Murdock
et al (75) and Patrick and Hill (102, 103). Murdock et al (75)
reported that some strains of Leuconostoc and Lactobacillus
produced an off-flavor in 20 Brix juice after 2 days. They also
found that the Lactobacillus strains grew faster than Leucon-
ostoc in 200 Brix orange juice. A routine check for the develop-
ment of diacetyl in the processing system is recommended by
Hill and Wenzel (51) and Murdock and Brokaw (81).
In the commercial processing of citrus products, the diacetyl
and direct microscopic slide tests are repeated simultaneously
every 2 to 3 hours. The combined routine checks require less
than an hour to perform. Samples are usually taken from the
evaporator-feed juice and the concentrates at the 20 or 300 Brix
stages. The results are plotted on graph paper that shows par-

Florida Agricultural Experiment Stations

allel build-up of microorganisms and increase or diacetyl-acetoin
resulting from bacterial metabolism.

The fact that concentration and low-temperature storage do
not insure destruction of microorganisms generally in unheated
citrus fruit products raises the possibility of the survival of
pathogenic organisms, particularly those of fecal origin. The
products may be polluted through agencies such as insects, per-
sonnel, wash water and rodents. Pathogens of fecal origin are
not so easily detected as some of their non-pathogenic associates.
Coliforms and specifically E. coli have been regarded as indica-
tors of fecal pollution in water and food. Recently enterococci
have been given prominence as indicators of fecal contamination.
Shrader and Johnson (113) observed the behavior of E.
coli in frozen orange juice held at various temperatures and
found that the death rate decreased as temperatures were de-
creased; E. coli failed to survive longer than 2 weeks in frozen
juice. Wolford (140) isolated from frozen orange juice viable
coliforms that resembled E. coli after 43 weeks at -100 F.
According to Faville et al (29), E. coli showed a significant de-
crease in 10.50 Brix orange juice at 860 F. during a period of 7
hours. In concentrate, 420 Brix, the count was reduced from
10,000,000 to less than 1,000 per ml under similar conditions.
When incubated at 37.40 F., the numbers decreased from 100,-
000,000 to 1,000,000 per ml in 7 hours. At 86 and 104 F. the
reduction in numbers was very rapid, and at the latter tempera-
ture E. coli could not be found after 15 minutes. Patrick (103)
inoculated E. coli into orange juice at room temperature and was
unable to recover it after 4 days. When the concentration was
increased to 310 Brix, E. coli could not be found after 48 hours.
Grapefruit sections, inoculated and frozen, were free of viable
cells after 8 days. Allen and Fabian (1) found that E. coli var.
communis was viable in orange juice, pH 3.5, after 5 days at
860 F. Dack (24) reported E. coli of fecal origin viable after
12 months in orange concentrate stored at 0 F.; coliforms nat-
ural to the product and E. coli added experimentally decreased
rapidly at 850 F. Smith and Gillmore (118) inoculated E. coli
into 420 Brix orange concentrate and found it was viable for 4
hours at 98.50 F. but could not be re-isolated after 8 hours' incu-
bation at 98.50 F.

Microbiology of Citrus Fruit Processing

a. .^ ^ --'
4' 4

9 gi Al l *
? ft .A,

Fig. 18.-Photomicrographs of orange juice showing microorganisms of
importance to the citrus processing industry, a, Lactobacillus plantarum;
b, yeast; c, Leuconostoc mesenteroides.
Strains of Salmonella typhosa (causing paratyphoid A) in-
oculated into orange juice at pH 3.5 and stored at 24.80 F. sur-
vived 7 days, according to Beard and Cleary (6). They inocu-
lated the same strain into broth media at pH 3.5 and incubated
them at 99.5 F.; the culture was dead in 24 hours. They con-
cluded that the effect of hydrogen ions was inhibited appreciably
at low temperatures. The pathogens, S. typhosa and Shigella
paradysenteriae (an organism causing summer complaint among
infants), inoculated by Hahn and Appleman (35) into concen-
trated orange juice, failed to survive freezing for 24 hours. They
also found that Streptococcus faecalis showed a survival rate
of 1.58 percent after 72 hours at 1.4 F. Similar concentrate
was inoculated with fecal material containing 39,000 coliforms

Florida Agricultural Experiment Stations

and 11,000 S. faecalis and refrozen. Coliforms could not be re-
isolated after 24 hours, but S. faecalis was still viable after 3
days. They concluded that since the pathogens did not survive
freezing in the concentrate, there was little danger of infection
from these organisms.
Also, the inability to recover coliform organisms from orange
concentrate inoculated with fecal material would indicate that
coliforms reported in orange juice after long storage periods
were probably not of fecal origin. They suggested that S. fae-
calis would serve better as an index for fecal pollution in orange
juice than E. coli. This opinion is also given by Allen and Fabian
(1, 2), since they found that S. faecalis remained viable longer
in orange juice at pH 3.5 than did any strain of E. coli. Beard
and Cleary (6) observed an appreciable inhibition in the growth
of bacteria by acidity at low temperatures. In orange juice
stored at -4 F., the survival period varied from 50 hours for
streptococci to 170 hours for strains of E. typhosa and S. dysen-
teriae. They concluded that high acid food may carry infection,
even if such food is preserved by storage at 0 F. or below.
Kaplan and Appleman (59) isolated 76 strains of enterococci
from commercial packs of frozen orange concentrate, 42 cans
from 3 plants; 63 were identified as S. liquefaciens and 13 as S.
faecalis. These samples of concentrate had been held in storage
2 to 8 months. These investigators consider enterococci more
resistant to adverse environmental conditions than E. coli. They
have suggested enterococci as indicators of pollution in frozen
foods. Patrick and Hill (104) described 13 enterococcus-like
cultures that had been isolated from citrus concentrates; 7 cul-
tures resembled S. liquefaciens and 6 resembled S. faecalis.
Storage in concentrate at 0 or below did not devitalize these
organisms very noticeably; the survival time is not known defi-
nitely. The authors are inclined to believe that these organisms
are associated with over-mature and damaged fruit. Until more
is known about the natural habitat and sources of contamination,
the significance of enterococci in food cannot be evaluated prop-
erly, in that the organisms are commonly found in both soil and
the intestines of warm-blooded animals.
Hahn and Appleman (36) conducted an experiment to learn
the lethal effect of citrus peel oil on S. faecalis, E. coli, S. para-
dysenteriae and S. typhosa. The organisms except S. faecalis
died in the controls with no oil within 24 hours at 1.4 F. S.
faecalis was destroyed by 1,000 ppm of peel oil in 24 hours and

Microbiology of Citrus Fruit Processing

was adversely affected in a decreasing manner by 100 ppm and
10 ppm of peel oil; 1 ppm had no apparent effect on the culture.
A food preparation that is generally consumed without heat
treatment is usually suspected of being a good medium for food
poisoning. Clostridium botulinum types A and C and Staphylo-
coccus aureus did not produce toxins when inoculated into 42
Brix orange concentrate and incubated at 77 and 99.50 F., re-
spectively, according to Smith and Gillmore (118). These or-
ganisms are commonly associated with food poisoning, but they
failed to grow or produce toxins in orange concentrate. The
authors did not believe that frozen concentrate could cause the
illnesses referred to as botulism and food poisoning.
McAllister (83) isolated 8 cultures of Micrococcus consisting
of 3 species from concentrated orange juice. Since they did not
form toxin, he believed these organisms to be of little signifi-
cance. The cultures were identified as Micrococcus luteus, M.
conglomeratus and M. candidus; the organisms are common con-
taminants of water, air and dust. Vaughn et al (129) reviewed
the literature on the significance of microorganisms in frozen
citrus products and concluded that there were no known health
hazards of bacterial origin.

Unpasteurized citrus products not kept in cold storage are
almost certain to contain some organisms that will cause spoil-
age. Chemical preservatives may be added, if approved by the
Federal Food and Drug Administration and State and local au-
thorities, but must be declared on the label. Sodium benzoate
is often used as a preservative in fruit products. Rahn and Conn
(106) reported that undissociated sodium benzoate at a con-
centration of 25 ppm in an acid medium suppressed the growth
of 9 strains of yeast experimentally. Fountain preparations and
fruit-drink bases have been known to ferment because of the
conditions under which they were stored. An orange beverage
base with 1 percent of sodium benzoate added did not harbor
spoilage organisms (27), vegetative cells were destroyed in 1
minute and the ascospores of Saccharomyces cerevisiae were
killed within 1 hour.
The investigators believed the lethal effect was due to the
concentration of sodium benzoate combined with the effect of
low pH. The toxicity of sodium benzoate increased as the pH

Florida Agricultural Experiment Stations

increased. Rahn and Conn (106) reported that undissociated
salicylic acid prevented the growth of 9 strains of yeast in an
acid medium at 4 ppm concentration.
Sulfur dioxide has been used for many years as a fumigant
and preservative; in the days of "Magic and Spells" certain pro-
fessionals, when dealing with misunderstood conditions, handled
sulfur with ceremony and mystery to maintain prestige.
According to Rahn and Conn (106), sulfur dioxide in water
dissociates into ineffective SOs ions and effective HSO0 ions.
The latter at 10 ppm in an acid medium inhibit the multiplication
of E. co'i but not yeast; 70 ppm killed E. coli but did not kill a
strain of wine yeast. The undissociated H2SO3 inhibited wine
yeast in 0.4 ppm concentration and killed the cells at 7 ppm.
Ingram (54) found the viability of yeast in 4 to 40 percent dex-
trose solution containing 0 to 400 ppm of SO2 to correlate with
the amount of free SO2. A similar but less exact correlation was
found in orange juice of comparable SO2 concentrations. Free
SO2 was less effective in killing yeast in fruit juice than in dex-
trose solutions. Feigenbaum and Israelashvili (33) reported
that the amount of SO2 combined with citrus juice increased with
decreasing pH and with increasing glucose concentration. By
adding 1.5 g of citric acid, 1.5 g of glucose and 24 mg of sodium
sulfite into 20 g of citrus concentrate, 96 percent of SO2 was
bound. The addition of arabinose increased the amount of
bound SO2. The dilution of the concentrate affected the amount
of bound SO2 only slightly. Ingram (53) observed that the dis-
tillable SO2 in 65 Brix Florida concentrated orange juice dimin-
ished more rapidly when stored above 68 F. He believed this
was due to diffusion and oxidation. The loss was rapid in closed
vessels and he was inclined to believe that unknown causes were
also contributors to the disappearance of SO2. Cruess (22) re-
ported that SO2 in fruit juice inhibited the growth of wild yeasts
and vinegar bacteria (acetic acid bacteria), but permitted Sac-
charomyces ellipsoideus to develop. Chace et al (21) commented
on the use of sulfur dioxide as a preservative in citrus juice
for beverage bases. The juice is treated with 1,200 ppm of sul-
fur dioxide and allowed to stand until the clear juice can be re-
moved. The analysis then showed sulfur dioxide in 800 to 900
ppm; by the time the juice reached the bottler, the amount was
reduced greatly.
Morgan et al (73) investigated the use of carbon dioxide, 120
pounds per square inch; sodium benzoate, 0.1 percent; and

Microbiology of Citrus Fruit Processing

sodium metabisulfite, to provide 250 ppm of S02 in orange juice
and concentrate. The containers were stored at 40, 60 and 70'
F. After 43 days at 400 F., the count in single-strength juice in
CO2 atmosphere was 500 per ml; with sodium benzoate, 1,000 per
ml; and in SO2, 3,000 per ml. At 60 F. the count increased con-
siderably during 43 days; with CO2 the count was 123,000;
sodium benzoate, 520,000; and SO2, 22,100 per ml. At 700 F.
in an atmosphere of CO2 the count was 30,000 per ml; in sodium
benzoate, less than 100; and in SO2, 17,000 per ml. The 620
Brix concentrate that had been treated similarly decreased in
count during the storage period.
Patrick and Atkins (100) reported that tangerine sherbet
base fermented in sealed 6-ounce cans in 3 days when held at
800 F., but remained unfermented 13 days at 800 F. when 0.025
percent sorbic acid was added. Sealed cans containing 0.05 per-
cent sorbic acid stored at 80 F. did not ferment during the stor-
age period of 84 days. Lower concentration of sorbic acid was
more toxic to slime and gum-forming bacteria than to yeasts.

The extent of operation between clean-up periods and the
extensiveness of the clean-up depend upon many factors, such
as variety and maturity of fruit, climatic conditions and labora-
tory findings. The routine clean-up practices have been reviewed
in various publications (11, 12, 13, 17, 30, 43, 78, 82). In some
plants there is a general cleaning once each shift; in other plants,
this general cleaning is supplemented by a special cleaning of
holding tanks and lines twice each shift; in still other plants the
practice is to clean thoroughly once each 24 hours. There is no
uniformity among operators on the frequency of clean-up jobs.
A chlorine residual of 5 ppm in the water supply normally is
adequate to prevent growth in water lines. Many plants use
water with 10 to 25 ppm of chlorine for clean-up (119). Whether
the operators use quaternary salts or chlorine seems to make
little difference to the end result (71). Murdock and Brokaw
(81) recommended 2 thorough clean-up periods per week with
intermittent clean-up every 4 hours using water containing 25
ppm of chlorine from the plant service water supply to flush ex-
tractors, juice-lines, finishers and fresh juice tanks. Equipment
that handles extracted juice and pulp is not cleaned easily.
Some operators believe that water from a hose at normal
city water pressure (about 40 pounds) does not clean quickly

Florida Agricultural Experiment Stations

nor thoroughly enough. Because of this, high-pressure water
guns are used, operating at 150 to 400 pounds per square inch
gauge (p.s.i.g.). Murdock and Brokaw (80) have constructed
portable jet cleaners that make the clean-up operations simpler
and more effective. Other operators use a cool water rinse,
then a detergent rinse, followed by steam and then a chlorine
rinse. It is important that juice extractors be rinsed after each
shut-down of 15- or 20-minutes duration because of the growth
of microorganisms in the juice film on the equipment (11, 12,
13). Recently Walkley and Wilson (134) determined the tox-
icity of an aqueous solution of dioctyl-dimethyl-ammonium to
yeast in 30 percent orange juice and 12 percent sucrose at stated
pH levels. The most effective destruction of cells was reported
at pH 3.5 after short exposures. There was 100 percent killing
of yeast in 1:100 dilution in 5 minutes; also at 1:1,000 in 15
minutes. The results for exposures at pH 3.3 and 3.2 were not
given. This compound might prove of value in plant sanitation.
Murdock and Brokaw (81) presented by means of a graph
the increase of surface contamination as washed fruit traveled
over conveyors that were not kept clean. Much emphasis is
placed on the control and eradication from processing plants of
microorganisms that cause spoilage in citrus products, and also
on the control of organisms that may indicate or cause suspicion
of pollution with filth (17, 18, 63, 64, 99, 104, 141, 142).

The citrus waste material that accumulates as the result of
processing citrus fruit for juice and concentrate has become a
source of by-products of considerable economic value (4, 44,
45, 70, 125). The discarded fruit, peel, screened pulp, rag and
seeds are the principal constituents of citrus pulp feed. When
lime-treated peel is being processed, quantities of press liquor
(peel juice or press water) run from the lime-treated peel in the
presses in the preparation for drying. It is estimated that there
are 3 gallons of press liquor from each box (90 pounds) of fruit.
Millions of gallons of press water are prepared annually. The
liquor contains 8 to 15 percent total dissolved solids, of which 45
percent is sugar. This liquor may be concentrated and sold
as feed molasses (44) ; sometimes it may be put over drying pulp
and sold as sweet feed or it may be used as diluted press liquor

Microbiology of Citrus Fruit Processing

or molasses to grow microorganisms to make other marketable
products and feed-supplements. Nolte and his co-workers (91)
grew Torula utilis, a wild yeast, in press liquor and gained an
average of 46 percent dry yeast based on the sugar content of
the juice. This species grew rapidly and produced very little
alcohol. Veldhuis and Gordon (130) used molasses diluted with
2 volumes of water in a continuous propagating system and re-
ported a recovery of 60 percent yeast, Torula utilis.

Alcohol may be produced from press liquor. Nolte et al (88)
experimented with the production of alcohol from press liquor,
using strains of yeast that were noted for high yields of alcohol.
Their data indicated that 25 gallons of press liquor were required
to yield 1 gallon of alcohol with 12.5 ounces of yeast as a by-prod-
uct that could be used in stock feed. During World War II an al-
cohol production plant was established locally and operated suc-
cessfully on diluted citrus press liquor or citrus molasses. The
citrus peel oil must be removed since it hampers fermentation.
The ethyl alcohol produced from this source was the same chem-
ically as that from other sources, but its utilization was limited
by law.
Lactic acid may be produced from press liquor, with good
yields, when inoculations of specific lactic acid-producing bac-
teria are used. The experiments of Nolte and Von Loesecke (90)
yielded from 61 to 84 percent lactic acid based on the sugar con-
verted. Lactic acid was produced in this area for a short time
on the pilot-plant scale, using citrus press liquor as a carbohy-
drate source.
Much work has been done and reported by Nolte (88), Chace
and his coworkers (21, 125), Joslyn and Marsh (57) and Hill
(52). Macfie (67) outlined the method for preparing orange
wine and suggested other fruit juices for the preparation of wine.
Wine making in the citrus producing area of Florida has not
become established extensively.

Vinegar may be made from citrus juice that contains 9 per-
cent or more sugar (125). This is effected by organisms of the

Florida Agricultural Experiment Stations

genus Acetobacter by different processes. The citrus juice is
first fermented and after the active fermentation has ceased,
the fluid is oxidized to acetic acid by any of the several systems
of aeration that favors the oxidative action by Acetobacter.
Robert R. McNary and Marshall H. Dougherty of the Florida
Citrus Commission prepared during the 1958-59 season orange,
grapefruit, tangerine and orange peel vinegars. Their efforts
have been directed toward developing methods for producing
good vinegar efficiently.

Veldhuis and Gordon (130) realized an 80 percent reduction
in biochemical oxygen demand (B.O.D.), because the sugar con-
tent of the press liquor had been reduced considerably during
the fermentation that resulted in building up high yeast-yields.
The yeast obtained may be used as stock feed supplements. The
reduction of 75 percent in the oxygen consumed value meant that
the spent liquor could be disposed of with less trouble than would
have been encountered with the unfermented liquor.
Von Loesecke et al (131, 132) and Wakefield (133) experi-
mented with different means for disposing of wastes from citrus
cannery effluents. They found that trickling filters gave the
best solution to the problem, whereas the use of pits, emptying
into large bodies of water or tide water rivers, was impractical.
A filter could be flooded intermittently over a period of 10 hours
at the rate of 1.5 million gallons per acre per day. The effluent
from their experimental filters showed a reduction in B.O.D.
of 88 percent. The cost of construction and the upkeep of such
a system was high and therefore this method of disposal was
not adopted by the industry. Von Loesecke (132) reviewed the
programs in the development of waste disposal, and reported
that fermentation in retention tanks was satisfactory, but not
feasible because of the lack of space. He reviewed briefly the
methods in use and their merits which are limited by the locality.
Marston (70) used aeration of plant waste in tanks inoculated
with yeast to reduce the B.O.D.

McNary, et al (86, 87) experimented with the waste liquors
from citrus processing plants and maintained a fermentation
that produced methane. They found that the production of
methane was stimulated when peel oil was removed. After yeast

Microbiology of Citrus Fruit Processing

fermentation, oil and alcohol were removed by aeration in the
first stage of the process. Then methane was produced in the
second stage by anaerobic bacterial fermentation. The method
showed promise as a means of producing methane and also dis-
posing of citrus plant wastes.

The dry excess sludge from the treatment of citrus waste
waters by the activated sludge process (133) has been found by
Dougherty and McNary (unpublished data 2) to contain signifi-
cant quantities of the "B" group of vitamins, thiamin, niacin,
riboflavin, pantothenic acid and vitamin B12, plus a high protein
value. The B12 value is high enough that the dried sludge could
be used as a B12 supplement in chicken feeds. Drum drying was
superior to oven drying in the preparation of this product. The
protein content of the dry sludge ranged from 28.9 to 43.8 per-
cent, which is high enough for a protein supplement in animal

1. ALLEN, C. H., and F. W. FABIAN. Comparison of Escherichia coli and
Streptococcus faecalis as a test organism to determine the sanitary
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207. 1954.
2. ALLEN, C. H., and F. W. FABIAN. Comparison of Escherichia coli and
Streptococcus faecalis as a test organism to determine the sani-
tary quality of food. Part II. Jour. of Milk and Food Technol.
17: 237-242. 1954.
examination of water and sewage. 10th Ed. 1955.
4. BAIER, WILLARD E. Citrus by-products and derivatives-An intro-
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5. BARRETTO, ALBERT, JR. Studies on the rate of growth of potential
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7. BERRY, J. M., J. F. FOLINAZZO, and D. I. MURDOCK. A rapid method
for the presumptive identification of bacteria which have been as-

2After this manuscript was assembled, the data referred to has been
published: Activated citrus sludge-Vitamin content and animal feed po-
tential. Sewage and Ind. Wastes 30: 1151-1155. 1958.

54 Florida Agricultural Experiment Stations

sociated with off-flavor and odors in concentrated orange juice.
Food Technol. 8: 70-72. 1954.
8. BERRY, J. M., L. D. WITTER, and J. F. FOLINAZzO. Growth character-
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9. BEISEL, C. G., and V. S. TROY. The Vaughn-Levine boric acid medium
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10. BEISEL, C. G., L. A. COURTNEY, and J. KINGSBERRY. Some observa-
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17. BROKAW, CHARLES H. The role of sanitation in quality control of
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19. BYER, ELLIS. Visual detection of either diacetyl or acetyl methyl
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173-174. 1954.

20. CAMP, A. F., and A. L. STAHL. Cold storage methods of handling
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364, 379. 1934.

21. CHANCE, E. M., H. W. VON LOESECKE, and J. L. HEID. Citrus fruit
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1-46. Nov., 1940. Revised Aug. 1942.

22. CRUESS, W. V. The effect of sulfurous acid on fermentation organ-
isms. Ind. and Eng. Chem. 4: 581-585. 1912.

Microbiology of Citrus Fruit Processing

M. K. VELDHUIS. Concentrated orange juice storage studies with
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24. DACK, GAIL M. Significance of enteric bacilli in foods. Am. Jour.
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25. DOIDGE, E. M. The origin and cause of citrus canker in South Africa.
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26. DUBoIS, C. W., and T. J. KEw. Storage temperature effects on frozen
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28. FAVILLE, L. W., and E. C. HILL. Relative efficiencies of several liquid
presumptive media used in the microbiological examination of
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29. FAVILLE, L. W., E. C. HILL, and E. C. PARISH. Survival of micro-
organisms in concentrated orange juice. Food Technol. 5: 33-36.
30. FAVILLE, L. W., and E. C. HILL. Incidence and significance of micro-
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31. FAVILLE, L. W., and E. C. HILL. Acid-tolerant bacteria in citrus
juices. Food Research 17:281-287. 1952.
32. Florida Department of Agriculture, Citrus and Vegetable Inspection
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34. FERRARO, F. M., and M. D. APPLEMAN. Microbiology of frozen orange
concentrates. IV. Further studies of Enterococci in frozen orange
concentrate. Applied Microbiol. 5: 300-303. 1957.
35. HAHN, SAUL S., and M. D. APPLEMAN. Microbiology of frozen orange
concentrate. I. Survival of enteric organisms in frozen orange
concentrate. Food Technol. 6: 156-157. 1952.
36. HAHN, SAUL S., and M. D. APPLEMAN. Microbiology of frozen orange
concentrate. II. Factors influencing the survival of microorgan-
isms in frozen orange concentrate. Food Technol. 6:165-167.
37. HAMRICK, DAVID O. Problems related to production and distribution
of cartoned orange juice. The Citrus Industry 38: 20-26. 1957.
38. HAMRICK, DAVID O. Problems related to production and distribution
of cartoned orange juice. The Citrus Industry 38: 29-31. 1957.
39. HAMRICK, DAVID O. Problems related to production and distribution
of cartoned orange juice. The Citrus Industry 38: 22-24. 1957.

Florida Agricultural Experiment Stations

40. HASSE, CLARA H. Pseudomonas citri. The cause of citrus canker.
Agr. Research 4: 97-100. 1915.
41. HAYS, G. L. Private communication to the chairman of the Institute
of Food Technologists committee for the investigation of micro-
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42. HAYS, G. L. The isolation, cultivation, and identification of organ-
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juice. Proc. Fla. State Hort. Soc. 64: 135-137. 1951.
43. HAYS, G. L., and D. W. REISTER. The control of "off odor" spoilage
in frozen concentrated orange juice. Food Technol. 6: 386-389.
44. HENDRICKSON, R. Florida citrus molasses. Clarification of citrus
press liquor. U. of Fla. Agr. Expt. Sta. Bul. 469. 1954.
45. HENDRICKSON, R., and J. W. KESTERSON. Citrus by-products of Flor-
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Agr. Expt. Sta. Bul. 487. 1951.
46. HENRY, R. E., N. H. STRODTZ, and D. I. MURDOCK. An exploratory
study of respiration rates of microorganisms in orange juice as a
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47. HILL, E. C., and L. W. FAVILLE. Comparison of plating media used
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48. HILL, E. C., and L. W. FAVILLE. Studies on the artificial infection of
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49. HILL, E. C. Microbiological examination of Jaffa oranges with sty-
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50. HILL, E. C., F. W. WENZEL, and A. BARRETO. Colorimetric method
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51. HILL, E. C., and F. W. WENZEL. The diacetyl test as an aid for qual-
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52. HILL, HALBERT P. Production of alcoholic beverages from citrus
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53. INGRAM, M. The germicidal effects of free. and confined sulfur di-
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54. INGRAM, M. Behavior of sulfur dioxide in concentrated orange juice.
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55. INGRAM, M. Fermentation of concentrated orange juice. Food Mfgr.
24: 77-81, 121-124. 1949. Bio. Abs. 24: 6914. 1950.

Microbiology of Citrus Fruit Processing

56. INGRAM, M. Osmophilic yeast from concentrated orange juice. Jour.
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57. JOSLYN, M. A., and G. L. MARSH. Suggestions for making orange
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58. KAUFMAN, C. W., and H. A. CAMPBELL. Some fundamental consider-
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59. KAPLAN, MILTON T., and MILO D. APPLEMAN. Microbiology of frozen
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67. MACFIE, GEORGE B., JR. Wines from tropical fruits. Proc. Fla. State
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68. MADSEN, H. S., and A. D. D'ERCOLE. Frozen concentrated citrus juices
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70. MARSTON, HAROLD. A citrus plant eliminates waste. Proc. Fla. State
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71. MARTINEZ, J., and M. D. APPLEMAN. Certain inaccuracies in the de-
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392-394. 1949.

Florida Agricultural Experiment Stations

changes in frozen concentrated citrus juices-preliminary report.
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Microbiology of Citrus Fruit Processing

Experimental treatment of citrus waste water. Food Technol. 5:
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87. MCNARY, ROBERT R., and RICHARD W. WOLFORD. Citrus processing
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Florida Agricultural Experiment Stations

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