Group Title: Citrus Station mimeo report - Florida Citrus Experiment Station ; 58-1
Title: Enterococcus-like organisms in citrus juices
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 Material Information
Title: Enterococcus-like organisms in citrus juices
Series Title: Citrus Station mimeo report
Physical Description: 7 leaves : ; 28 cm.
Language: English
Creator: Patrick, Roger
Hill, E. C
Citrus Experiment Station (Lake Alfred, Fla.)
Florida Citrus Commission
Publisher: Florida Citrus Experiment Station :
Florida Citrus Commission
Place of Publication: Lake Alfred FL
Publication Date: 1957
 Subjects
Subject: Citrus juices -- Microbiology -- Florida   ( lcsh )
Citrus juices -- Composition -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Includes bibliographical references (leaf 7).
Statement of Responsibility: Roger Patrick and E.C. Hill.
General Note: Caption title.
General Note: "September 26, 1957."
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Bibliographic ID: UF00072386
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 74906629

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Citrus Station Mimeo Report 58-1
September 26, 1957

Enterococcus-like Organisms in Citrus Juices
Roger Patrick and E. C. Hill

There is no concrete evidence of a health hazard due to the presence of
streptococci or possibly enterococci in citrus juices or frozen citrus concen-
trates. Nevertheless, since such bacteria have been found in citrus products
(5 8, 9) and since the enteric organism, S. faecalis, has been suggested (1, 9,
10) as being of sanitary significance in foods, it was deemed advisable to
investigate the nature of the enterococcus-like bacteria that sometimes are found
in citrus juices or concentrates.

Sherman (12) reviewed and discussed the characteristics of the streptococci.
They are gram-positive, usually spherical or ovoid, and non-capsulated bacteria.
The enterococci, one of four groups within the genus Streptococcus, are normal
intestinal inhabitants that are not pathogenic. Bergeyts Manual (2) gives the
reactions of this enteric group, including that they will grow at 450C. in 6.5%
NaCI, tolerate 0.1% methylene blue, survive 620C. for 30 minutes and grow at pH
9.6.

The purpose of this paper is to present the results obtained concerning the
characteristics of streptococci and enterococcus-like organisms isolated by the
authors from citrus juices and concentrates; also to review briefly, information
about these bacteria that has been published in the literature. Thus, such
information will be available to citrus processors should any questions be raised
in the future by public health officials concerning the occurrence and signifi-
cance of these microorganisms in citrus products.

Review of literature. Kaplan and Appleman (8) isolated 71 strains of
enterococci from 42 cans of frozen orange concentrate obtained from 3 processing
plants. All of them survived 600C. for 30 minutes, were able to grow at pH 9.6,
and in the presence of 0.1% methylene blue. They grew at 45.50C. and in the
presence of 6.5% NaCI. Gelatin was liquified by 63 of the cultures, which they
designated as .. liQuefaciens; the remaining 8 were classified as .S. faecalis.
The reactions of these 8 organisms when inoculated into milk were not given,
although Bergeyts Manual (2) considers such reactions distinctive also. Larkin
et al. (9) also found enterococci in citrus concentrates. Farraro and Appleman
(5) examined cans from 9 brands of frozen orange concentrate and found all
samples positive for enterococci; they suggested the need of further study to
explain the uniform presence of these bacteria in this product.

Allen and Fabian (1) reported that S. faecalis remained viable longer than
E. coli when inoculated into acid foods, such as orange juice, pH 3.5, and held
at 3000. for 7 days. They did not consider their presence in moderate numbers
as a sure sign of dangerous contamination, but rather of possible contamination,
and suggested that a search for the cause should be made. Other investigators
have observed the hardiness of enterococci in acid foods. Hahn and Appleman (6)
reported that a strain of S. faecalis survived 72 hours at -170C. in orange
concentrate; however, enteric rods could not be recovered after 24 hours. In

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
814 c 9/26/57 RP







-2-


another study they (7) found that S. faecalis was destroyed when the peel oil
content of orange concentrate was increased to 1000 ppm. It was also observed
that when concentrate was stored at 40C., the bactericidal action of citric
acid was greater than H01 when added at comparable hydrogen ion concentrations.
The acid exerted a more toxic effect than was indicated by the degree of dis-
sociation.

Larkin et al. (10) inoculated orange concentrate with S. liquefaciens and
S. faecalis, held it in storage for 147 days at -100F. and found that these
organisms remained viable and the numbers constant. This concentrate had also
been fortified with 4% citric acid, resulting in a pH of 3.0.

Detection of enterococcus-like organisms in citrus juices and frozen citrus
concentrates. While examining in 1954 various samples of orange juice, it was
observed that streptococci were usually present in juice that was extracted from
cold-damaged fruit. Also at times such organisms were found in juice from either
overmature fruit or that which had been bruised and mishandled. This led to the
examination during the past four years of samples of freshly extracted orange
juice, commercial frozen orange concentrate and commercial frozen grapefruit
concentrate so that the occurrence of streptococci in these products could be
determined, as well as the number of these streptococci that could be classified
in the enterococcus group.

The 34 samples of orange juice, that were examined, were extracted in the
pilot plant from fruit damaged in varying degrees by cold weather. The 212
commercial frozen orange concentrates and 99 commercial frozen grapefruit con-
centrates that were used in this investigation were obtained semi-monthly during
the 1956-57 citrus season from 24 processing plants. During this season,
although there was not a major freeze, cold weather did occur in some of the
citrus producing areas with the result that some of the crop was damaged and
such fruit was available for processing.

Since it was desired to determine the occurrence of not only streptococci,
but also coliforms, these organisms were isolated after inoculating 20 ml. of
lauryl tryptose broth with 10 ml. of a 1:10 dilution of either raw or reconsti-
tuted juice and incubating the tubes at 35-37oC. for 48 hours. If streptococci
grew in this medium and were evident upon microscopic examination, then trans-
plants were made to a presumptive medium for enterococci since the identification
of all streptococci in the citrus juices was not of primary interest. The pre-
sumptive medium used for this screening purpose was an enterococci agar (4) that
had the following composition:

Tryptone 5 grams Sodium chloride 65 grams
Yeast extract 5 grams Methylene blue 0.01 grams
Dextrose 5 grams Agar 15 grams
Sodium azide 0.4 grams
This medium will have a pH of 8.0 and is incubated at 450C. for 48 hours.


Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
814d 9/26/57 RP










Cultures of streptococci that grew on this presumptive agar were then sub-
jected to a confirmatory test for enterococci (4) in the following manner. A
broth medium was made of the above formula and just before using, 65 units of
penicillin were added to each 100 ml. of broth. Enough of this broth was added
to each inoculated presumptive slant to cover approximately one-half of the
surface. Incubations were made at 450C. If coccus-shaped, gram positive or-
ganisms grew at 450C. in this medium, containing 6.5% NaCI, and were catalase
negative, they were considered confirmed enterococci.

Streptococci were isolated from 24 (71%) of the 34 samples of orange juice
that were extracted in the pilot plant from 4 lots of freeze-damaged fruit;
also, from 191 (90%) of the 212 samples of frozen orange concentrate and from
29 (29%) of the 99 frozen grapefruit concentrates. Thus, 244 samples (71%) from
a total of the 345 citrus juices or concentrates examined were found to contain
streptococci. Of these 244 cultures, 26 grew on the enterococci presumptive
medium and only 13, or 5%, of the total number of streptococci isolated, survived
the confirmatory test for enterococci.

Classification of enterococcus-like organisms. For differentiation into
species biochemical and other tests, as indicated in Tables 1 and 2, were made
on the 13 cultures that were confirmed as organisms of the enterococcus group.
These cultures grew at 450C. in the presence of 6.5% NaCI, were catalase nega-
tive, and were considered to be gram-positive. They produced ammonia in 4% pep-
tone water, pH 7.0, at 450C., and also grew in the same medium adjusted to pH
9.6. They grew in milk with 0.1% methylene blue at 3700. and 4500., respectively.
Growth occurred in lactose broth with 10% bile and gave a methyl-red reaction
(pH 4.0-4.5) in lactose and glucose broths. All of these reactions are positive
for the enterococcus group. However, when the cultures were heated at 6200. for
30 minutes, seven of them failed to survive, which is an irregularity for the
enterococcus group. These seven cultures grew at pH 9.6 at 370C., liquified
gelatin and fermented sorbitol. Inoculated tubes of litmus milk reacted in
uniform agreement; the dye was reduced, peptonization began within 24 hours and
digestion of the weakly coagulated solids continued almost to completion. Milk
with 0.1% methylene blue did not show any coagulation. When these reactions for
the seven organisms, which did not survive at 620C. for 30 minutes, are compared
with those of S. liquefaciens listed in Tables 1 and 2, the similarity is
readily recognized. The proteolyzed milk was not bitter to taste as is usual
S.liquefaciens.

The six remaining cultures, that survived 6200. for 30 minutes, failed to
grow at pH 9.6 when incubated at 370C., which may be an irregularity of some
significance. They neither liquified gelatin nor fermented sorbitol. When inocu-
lated into litmus, they readily reduced the indicator, produced acid, and formed
a firm coagulum; coagulation was not observed when inoculated in milk with 0.1%
methylene blue. A glance at Tables 1 and 2 will show the similarity of the
characteristics as observed with those known for S. faecalis. All of these
cultures grew more readily at 4500C. than at 370C., which is the optimum tempera-
ture for growth of bacteria in the enterococcus group; none of them grew pro-
fusely on the surfaces of solid media. These two characteristics may be just
irregularities of little significance. Coagulation of milk is a distinctive

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
814 e 9/26/57 RP










Table 1. Some


cultural characteristics of the streptococcus-like organisms that survived the enterococcus-
confirmatory test.


4% Peptone 1 0.1% Methylene Ulrich Litmus
Culture Catalase Gram's water_ a Gelatin Sorbitol blue milk milk milk
numbers stain pH 7.0 pIH 9.6 pH 9.6 Z 620C. at 370C. at 370c.
at 450C. at 45C. lat 37C. 30 min. at 370C. at 450C.

2-33 + + + + + RP RP RP RPC
2-39 + + + + + + RP R RP RPC
2-56 + + + + + + RP RP RP RPC
3- 7 + + + + + + RP RP RP RPC
6-34 + + + + + RP RP RP RPC
6-35 + + + + + + RP RP RP RPC
6-43 + + + + + + RP RP RP RPC
S.
liquifaciens + + + + + R RACP
2-60 + + -c + RO RO RO RAC
3-24b + + + -c + RO R0 RO RAG
3-25b + + + c + RO RO RO RAG
3-26b + + + RO RO RO RAC
3-27b + + -c + RO RO RO RAC
3-66 + + -c + RO RO RO RAC
S.
faecalis + + + R RAC


RP
Key: RPC
RAC
RO


Reduced,
Reduced,
Reduced,
Reduced,


proteoy zed
proteolyzod, coagulated
acidified, coagulated
oxidized


Ammonia production from
Cultures isolated from z


4% peptone water.
*aw juice extracted from


frozen fruit at this Station.
c Failed to grow.


Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
814 9/26/57 RP








Table 2. Some cultural characteristics of tho streptcaccous-like organisms that survived the enterococcus-
confirmatory test

Culture Starch Inulin 10%l Hippuric Glucose Glycerine Esculin Mannit Raffinose Salicin Sucrose Lactose Trehalose
numbers bile acid methyl red+1 1 __ ____ methyl red+
2-33 + + + + + + + + +
2-39 + + + + + + + + +
2-56 + + + + + + + + +
3-7 + + + + + + + + +
6-34 + + + + + + + +
6-35 + + + + + + + +
6-43 + ++ + + + + +
S.
liquifaciens + + + + + + + + + +
2-60 + + + + + + +
3-24b + + + + + + + + +
3-25b + + + + + + + +
3-26b + + + + + -+ + + +
3-27b + + + + + + + +
3-66 + + + + + + + +
S. faecalis 7 + 7 + + + T + 7 +


Cultures isolated from raw juice extracted from frozen fruit at this Station

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
814a 9/26/57-RP







-4-


characteristic for enterococci. However, milk with 0.1% methylene blue should
not be used in place of litmus milk, since methylene blue exerts a bacterio-
static action on some enterococci.

Sources of enterococcus-like organisms found in citrus juices. As pointed
out previously the occurrence of streptococci in freshly extracted orange juices
was noted chiefly when overmature fruit or fruit damaged because of cold weather
or otherwise were used. Therefore, it is believed that the condition of the
fruit, either in the grove or as it is received at the processing plant is a
major factor in this problem. Sherman (11) points out that j. faecalis and ..
liauefaciens-type of enterococci occur commonly on surfaces of plants; also that
none of the hemolytic, parasitic types of this enteric group have been isolated
from plant materials. As indicated in Tables 1 and 2, four cultures of
enterococcus-like organisms were isolated from orange juices that were extracted
from fruit that was known to have been damaged by low temperatures in the grove
and then left on the trees for about four months. The nine other cultures came
from samples of commercial frozen orange concentrates; these isolations were
made about 3, 4, and 7 months after the date when the first possible cold injury
to fruit could have occurred.

Concentrate stored in large containers to be used in the future as add-back
may be a source of streptococci if it was processed from damaged fruit. Like-
wise, unheated cut-back juice extracted from such fruit could also contain these
bacteria.

Murdock et al. (11) reported that a plate-type heat exchanger could be a
source of bacterial contamination in processing frozen concentrated orange juice,
In conducting heat treatment experiments in the Station pilot plant to determine
the amount of heat necessary for the destruction of streptococci in orange juices,
difficulties were caused by the accumulation of these organisms in the cooling
section of the plate-type heat exchanger. Commercial frozen citrus concentrates
may contain streptococci as a result of careless cleaning of heat exchangers
when damaged, overmature or mishandled fruit may be used. Batches of the same
juice from freeze-damaged Pineapple oranges, that contained streptococci, were
heated in a plate-type pasteurizer to 1650, 1750, 1850, 1950 and 2050F.; the
time required for the juice to pass through the regenerative, heating and cooling
sections of the heat exchanger was about 14 seconds. No streptococci survived
even the lowest heat-exposure.

During the examination of the commercial frozen concentrates, it was noted
that in a considerable number of instances, streptococci predominated and lactose-
fermenting bacteria were not viable after incubation of lauryl tryptose broth
that had been inoculated with 1:10 dilutions of reconstituted juices. When
unheated cut-back juices, known to have been extracted from cold-damaged fruit,
were used in experimental packs of orange concentrate, both streptococci and
lactose-fermenting bacteria were readily found in the lauryl tryptose broth.
These observations suggest the possibility that some of the commercial concen-
trate samples contained cut-back juice in which both of the above types of
bacteria had been destroyed by heating, but then this heated juice became con-
taminated again with streptococci that had previously accumulated in the cooling
section of the heat exchanger.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
84f 9/26/57 RP






-5-


Significance of enterococcus-like organisms in citrus juices and frozen
citrus concentrates. Because of the complexity of the problem, various investi-
gators (1, 3, 6, 12) have concluded that enterococci, and especially .. faecalis,
should not be used as an indicator of pollution in foods unless the validity of
this is established by further investigations. Vaughn et al. (12) in a review on
the significance of fecal streptococci as indicators of pollution, concluded
that because of their universal presence they probably are not good indicators
of sanitation. They found no evidence reported in the literature that fecal
streptococci would grow in frozen citrus products. Sherman (11) reiterated that
some of the species of enterococci are separated from each other by rather thin
and shaky boundaries. Dack (3) reminded us that "millions of cans of frozen
concentrated orange juice have been consumed without causing any known cases of
enteric infections,"

Data obtained concerning the occurrence and characteristics of streptococci
isolated from citrus juices and concentrates at this Station are in agreement
with observations reported by some other investigators (8, 5). However, after
consideration of this and other information reported on this subject, we are not
in agreement with Larkin et al. (9) who concluded that a few enterococci in
citrus concentrates indicate the possibility of other fecal organisms also being
present. Being in a citrus producing area where the history of fruit used for
experimental or commercial processing is more readily obtainable, it seems highly
improbable that the streptococci, including the enterococcus-like bacteria, that
have been isolated from citrus juices or frozen citrus concentrates are of any
public health significance. They are more likely a surviving contamination from
the area where the fruit was grown and the condition or quality of the fruit used
is reflected by their presence in these citrus products.


SUMMARY

Streptococci, including some enteroccus-like organisms, were isolated from
244 samples of citrus juices and commercial frozen citrus concentrates; thus,
71% of the 345 samples analyzed for bacterial contamination contained streptococci.
Of these isolations 11% survived an enterococci presumptive test, but only 13
cultures or 5% survived a confirmatory test for enteric streptococci. Biochemical
reactions and other tests indicated that seven of these 13 cultures were similar
to S. liauifaciens; however, they showed some irregularities in that they failed
to survive 6200. for 30 minutes, did not coagulate milk in the presence of 0.1%
methylene blue, acid production was not observed in litmus milk and the pro-
teolyzed casein was not bitter. The remaining six cultures had characteristics
similar to S. faecalis, but showed two irregularities in that they did not coagu-
late milk in the presence of 0,1% methylene blue and failed to grow at pH 9.6
at 370C., which is reported as the optimum temperature for growth of this
organism. Four of the six cultures, similar to faecalis, were isolated from
juice extracted from cold-damaged fruit and the other two cultures from commercial
orange concentrate.



Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
814 g 9/26/57 RP







-6-

Streptococci were destroyed in orange juice, extracted from freeze-damaged
fruit, by heating to 1650F. in a plate-type heat exchanger, requiring 14 seconds
for the juice to pass through the regenerative, heating and cooling sections.
It was also observed that streptococci accumulated in the cooling section of the
heat exchanger, when freeze-damaged fruit was being processed, and thus became a
source of contamination of juice previously heated.

The occurrence of enterococcus-like organisms and other streptococci in
citrus juices is an indication of the condition or quality of the fruit used,
since such bacteria have been usually found in overmature fruit or that damaged
by cold weather or mishandling.

On the basis of information now available, it is highly improbable that the
enterococcus-like organisms that have been found in citrus juices or frozen
citrus concentrates are of any public.health significance.





-7-


LITERATURE CITED


1. Allen, C. H. and F. W. Fabian. Comparison of Escherichia coli and
Streptococcus faecalis as a test organism to determine the sanitary
quality of food. Part II. J. of Milk and Food Technol. 17: 237-
242. 1954.

2. Bergey's Manual of Determinative Bacteriology. 6th Ed., 1948. Williams
and Wilkins Company, Baltimore, Md.

3. Dack, G. M. Significance of enteric bacilli in foods. Am. J. Pub.
Health. 45: 1151-1155. 1955.

4. Difco manual of dehydrated culture media and reagents for microbiological
and clinical laboratory procedures. 9th Ed., 1953. Difco Labora-
tories, Detroit, Mich.

5. Farraro, Frank M. and Milo D. Appleman. Microbiology of frozen orange
concentrate. IV. Further studies of enterococci in frozen orange
concentrate. Bact. Proc. A 15, 27. 1956.

6. Hahn, Saul S. and M. D. Appleman. Microbiology of frozen orange concen-
trate. I. Survival of enteric organisms in frozen orange concentrate.
Food Technol. 6: 156-158. 1952.

7. Hahn, Saul S. and M. D. Appleman. Microbiology of frozen orange concen-
trate. II. Factors influencing the survival of microorganisms in
frozen orange concentrate. Food Technol. 6: 165-167. 1952.

8. Kaplan, Milton T. and Milo D. Appleman. Microbiology of frozen orange
concentrate. III. Studies of enterococci in frozen concentrated
orange juice. Food Technol. 6: 167-170. 1952.

9. Larkin, Edward P., Warren Litsky, and James E. Fuller. Fecal streptococci
in frozen foods. I. A bacteriological survey of some common frozen
foods. Applied Microbiol. 3: ,98-101. 1955.

10. Larking, Edward P., Warren Litsky, and James E. Fuller. Fecal streptococci
in frozen foods. III. Effect of freezing storage on E. coli, S.
faecalis and S. liquefaciens inoculated into orange juice concentrate.
Applied Microbiol. 3: 104-106. 1955.

11. Murdock, D. I., C. H. Brokaw and W. E. Allen. Plate type heat exchanger
as a source of bacterial contamination in processing frozen concen-
trated orange juice. Proc. Fla. State Hort. Soc. 67: 179-183. 1954;
Food Technol. 9: 187-189. 1955.

12. Sherman, James M. The streptococci. Bact. Rev. 1: 1-97. 1937.

13. Vaughn, Reese H., D. I. Murdock and C. H. Brokaw. Microorganisms of
significance in frozen citrus products. Food Technol. 11: 92-95.
1957.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
814b 9/26/57 RP




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