Table Of ContentHEAT INDUCED INHIBITORY AGENTS OBTAINED
PROM PROCESSED FRUITS AND VEGETABLES
A DISSERTATION
Presented In P artial F u lfillm en t of the Requirements
for the Degree Doctor of Philosophy in the
Graduate School of the Ohio State U niversity
By
DOROTHY CULLER WILSON, B.A., M.Sc.
The Ohio State U niversity
1952
Approved by:
Adviser
i
ACKNOWLEDGMENTS
In recognition of their valuable help during th is
in v estig atio n , the author wishes to express her appreci
ation and acknowledgment:
To Dr. H.D. Brown, Department of H o rticu ltu re, for
his kind guidance, advice and constant counsel through-
out th is in v e stig a tio n .
To Dr. W.A. Gould, Department of H o rticu ltu re, for
supervising the processing of some of the samples p re
pared for th is study.
To Donald E. W ilson, Department of H o rticu ltu re , for
his suggestions and assistance and for drawing trie charts
used In th is m anuscript.
To Dr. H.H. W elser, Department of B acteriology, for
his constructive c ritic ism and advice in the preparation
of th is d iss e rta tio n .
£08702
ii
TABLE OF CONTENTS
I. INTRODUCTION............................................. 1
I I . REVIEW OF LITERATURE........................................................................ 5
A. P lant a n tib io tic s ........................................................... 5
1. Surveys completed in the search of a n ti
b io tic s from p lan ts............................................................... 5
B. A n tibio tic agents found in plant e x tra c ts that
have been c ry sta lliz e d and/or id e n tifie d ................. 10
C. P lant re sista n c e and growth in h ib ito ry agents . 40
D. The effect of some organic compounds on micro
organism growth ............................. k-S
E. Changes in p lan t and p lan t products th a t might
enhance or re ta rd the growth of food spoilage
organisms ......................................................................................... 53
F. Treatment of p lan t pathogens and processed
foods w ith a n tib io tic s ..........................; ............................ 55
I I I . METHODS..................................................................................................... 57
A. Methods fo r determ ining the presence of toxic
substances In d is tilla te s from f r u its and veg
etables and eth er products ............................................... 57
1. Method of preparing fra c tio n a l d is tilla te s
from f r u its and vegetables ........................................ 57
b. Method fo r determ ining in h ib ito ry a c t
iv ity in d is tille d fra c tio n s ............................ 59
2. Methods u tiliz e d in an attem pt to p u rify
and id e n tify substances responsible for
In h ib ito ry action in d is tilla te s ......................... 69
3. Method o f determ ining the effe c t of sucrose
so lu tio n s 1 heated for varying tim es and
tem peratures, on sev eral microorganisms . . . . 73
Method fo r determ ining the e ffe c t of pH
value on the in h ib ito ry action of heated
sucrose so lu tio n s ............................................................ 103
5. Method fo r determ ining the e ffe c t of
fu rfu ra l on the growth of o rg an ism s............... I l l
6. Measurement of fu rfu ra l and hydroxymethy1-
fu rfu ra l in heated sucrose so lu tio n s ............... 112
B. Method of determ ining in h ib ito ry ac tio n in
fre sh and processed f ru its and vegetables ............ 116
1. P reparation of sa m p le .................................................. 116
b. Method of te stin g fo r in h ib ito ry actio n .120
2. Method fo r determ ining the effeo t of d il
ution and ether ex tra ctio n on raw and pro
cessed f r u its and vegetables which produced
b ao te rlo id a l a c t i o n ..........................................................133
i i i
C. Method of determining the effects of processing
times and temperatures and varying concentrations
of added chemical substances in tomato Juice on
the growth of B. thermoacidurans and L. lyco-
o e r slc i....................................................................................... l4 l
IV.
RESULTS............................................................................................ 59
A. 1. R esults of the e ffe c ts of d is tilla te s
obtained from fru its and vegetables on
aeveral bacteria . . ................................................ 59
2. R esults on the effect of sucrose and amino
acid d is tilla te s ons everal microorganisms. 70
3. R esults of the effects of heated sucrose
solutions on severalm icroorganisms . . . . . . . 75
4. Results of the effect of pHv alue on the
inhibitory action of heateds ucroses olutions 109
5. R esults of the effect of furfural on the
growth of microorganisms ........................................... 112
esu lts of the quantative measurement of
furfural and hydroxymethylfurfural* in
heated sucrose solutions ........................................... 116
B. 1 . Results on the study of lngibltory action
in fresh and processed fru its and vegetables 121
2. R esults on the effect of d ilution and
ether extraction on raw and processed
fru its and vegetables which produced
b actericid al aotion ............................................................ 139
C. R esults of the effect of altered composition
and processing procedures on the growth of
B. thermoacldurans and L. lycop ersici in
tomato J u ic e ........................................................................ 144
V. DISCUSSION OF RESULTS .......................................................... I 54
VI. CONCLUSIONS............................................................................................. I65
VII. BIBLIOGRAPHY.......................................................................................... 167
IV
No. INDEX TO CHARTS Page
1. The e ffe c t of heated sucrose so lu tio n s on 3. e lllp -
soldeuB. (24 h rs. incubation, 0 .5 ml te st so lu tio n }. . . g l
2. The e ffe c t of heated sucrose so lu tion s on 9. e llip -
soldeus. (4g h rs. incubation, 0 . 5ml te s t so lu tio n )....3 3
3 . The e ffe c t of heated sucrose solu tion s on S. e lllp -
soldeus. (24 h rs. Incubation, 1 ml te s t so lu tio n }. . . . . 3 5
4. The e ffe c t of h ea ted sucrose so lu tio n s on S’., e l ljJBz _
so ld eu s. (4g h rs . in cu b atio n , 1 ml te s t solution]). . . . . 37
5. The e ffe c t of h eated sucrose so lu tio n s on L .ly co -
p e r s lc l. (24 h rs. in cu b atio n , 0.5 ml te s t s o lu tio n )...3 9
6. The e ffe c t of h eated sucrose so lu tio n s on L ly co-
p e rs ic i (24 h rs. In cu b atio n , 1 ml te s t s o lu tio n ). . . . . . 91
7. The e ffe c t of h eated sucrose so lu tio n s on A. a c e tic
(24 h rs. in cu b atio n , 1 ml te s t s o lu tio n ).....................................93
g. The e f f e c t of h e a te d sucrose so lu tio n s on A. a c e tl.
(4g h rs. in cu b atio n , 1 ml te s t s o lu tio n ) .............................. 95
9. The e ffe c t of heated sucrose solutions on B .su b tllis
(24 hrs. incubation, 1 ml te st so lu tio n )............................ 97
10. The e ffe c t of heated sucrose solutions on L. mesent-
eroides. (24 hrs. incubation, 1 ml te st so lu tio n )•••• 99
11. The e ffe o t of heated sucrose solutions on B. thermo-
aoidurans .(4g hrs. incubation, 1 ml te st solu tion ) ...1 0 1
12. The effeo t of heated suorose solutions on L.plantarum
(4g hrs. Incubation, 1 ml te st s o lu t io n ) ....... 103
13. The e ffe c t of heated suorose solu tion s on E. c o ll
(24 hrs. incubation, 1 ml te st so lu tio n )........................... 105
14.The effeo t of heated sucrose solutions on M.pyogenes
var. aureus. (24 h rs. incubation, 1 ml te st so ln T .... 10J
15* Comparison of tu rb idity readings of organisms grown
in media containing 1 ml of 50 B rix solution heated
to 250°F for 3 hrs. to organisms grown in media v ith
1 ml of 50° Brix solu tion . ( Both media adjusted to
the eame pH ) ........................................................................................ 114
HEAT INDUCED INHIBITORY AGENTS
OBTAINED FROM PROCESSED FRUITS .RID VEGETaBLuS
Introduction
The dramatic success of p en icillin has motivated scien
tis t s throughout the world to search for other compounds that
may prove successful in retarding or controlling the growth
of detrimental micro and macroorganisms. The majority of the
work on antibiotics is directed toward finding new or better
chemotherapeutic agents to serve in the control of diseases
in humans and animals* However, in recent years, the work on
an tib iotics has been extended to include the control of plant
pathogens and microorganism spoilage in processed foods. Plant
pathogen antibiotic researches are directed toward finding the
basic biochemical mechanisms responsible for the normal defense
which a plant has against disease causing organisms to which
it is susceptible. Tne control of processed food spoilage
organisms has been attempted by the use of antibiotics iso
lated from bacteria and molds as well as the use of inherent
constituents of the product that are beneficial in controlling
growth of the organism*
A ntibiotic substances have been found in bacteria, Actino
myces . yeast, molds, Basidiomvcetes. algae, lichens, and higher
plants* A variety of definitions have been assigned to the
term antibiotic due to the variety and quantity of compounds
found in these diversified field s. Baron (IS) has defined an
antibiotic as a member of the group of substances generally
1
2
referred to as an tisep tics, disinfectants, bactericides, or
fungicides. Waksman (224) states that an antibiotic is a
substance oroduced by microorganisms which inhibit or destroy
other microorganisms. Schaffer (193) defines antibiotic as
an organic chemical, produced by higher plants and animals
or by microorganism, that selectively checks or destroys the
growth of organisms. The definition preferred by th is author
is that of B&iton (1£ ) who defines an antibiotic as a proouct
of metabolism which w ill in small amounts retard or destroy
living organisms.
Come of the incentive to undertake plant antibiotic in
vestigations has been derived from medical treatments used
by native trib es, folklore of substances used to prevent d is
ease, and the success that has been ascribed to the use of
herbs in medicines in the civ ilized countries. The results
of the search of antibiotics in higher plants have not re
sulted in such outstanding success as some of the an tib iotics
produced by fungi and bacteria. None of the plant an tib iotics
have diown su fficien t promise to be beneficial as a therapeutic
*
age nt.
The fir st work on the use of antibiotics in food proc
essing was in itia ted as the result of the commercial produc
tion of p en icillin . Some of the antibiotics have been success
ful in destroying many food spoilage organisms per sej however,
when applied to the numerous variety of organisms encountered
in preserving foods, it would seem implausible to expect one
antibiotic to inhibit the growth of a ll of the se organisms
thereby eliminating the need for heat or cold preservation.
However, any new method which could be used to shorten the
processing time would consequently retain more of the natural
properties of the raw product.
Some of the advantages and disadvantages of the use of
antibiotics in processing foods as liste d by Morse (162) are:
advantages, (1 ) moderate costs, (2) shorter processing time,
(3 ) greater producing capacity, (4) higher quality, (5) higher
nutritive value, and (6 ) lower sellin g price. Disadvantages,
(1 ) incomplete protection (i.e . the in itia tio n of growth of
organisms over long periods of time), (2) organisms may be
come re sista n t to the an tib iotic, and (3 ) the effect of the
continued use of the inhibiting agent on human metabolism.
To the lis t of advantages the elim ination of holding foods
at refrig eratio n tem peratures might also be added.
Many canners have experienced spoilage in their products
even though the process tim es, tem peratures, and methods were
id en tical to previous years when no spoilage occurred. There
is a tendency to credit th is spoilage en tire ly to the increased
heat resistance of the organisms that p articu lar year. However,
two facto rs must be kept in mind whenever organisms abound;
(1) the increased resistan ce of the organism to the process
and (2) the lowered "resistan ce” of the product. This lowered
resistance could be caused by changes in the chemical con
stitu e n ts of the f r u it or vegetable due to varying environmental
conditions and to the chemical and physical differences of
v a rie tie s. Another consideration might include Irv in g ’s (110)
statem ent th a t an tib io tic substances may occur in plants in an
inactive form and become active only if the plant is properly
processed (chemically or physically).
This research was designed to find agents which are capa
ble of reducing processing tim es, tem peratures or both by in
h ib itin g the growth of spoilage microorganisms. This would
include any substance th at might be added to the product or
is inherent in the product whether a true an tib io tic agent or
an inhibitory compound.
Progress reported to date on the use of an tib io tics in
food preservation indicate th a t a short heat process is re
quired along with the addition of the a n tib io tic . A compound
that would f u l f i l l these requirements must necessarily possess
therm ostable c h a ra c te ristic s.
Surveys have been completed on the effect of some of the
b acterial and fungal a n tib io tic substances on food spoilage
organisms, and numerous experiments have been conducted on
the effect of many organic compounds on microorganisms. This
work was, th erefo re, lim ited to a survey of heat induced in
h ib ito ry agents produced by processing fru its and vegetables
«
(and ingredients used in these products) at various times and
tem peratures.
L iterature Review
A, Plant antibiotics*
1. Surveys completed in the search of antibiotics from plants.
The literature on plants used as therapeutic agents for
diseases is as old as the history of microorganisms. (107)*
Ancient Assyria used lotu s, o liv es, laurel, myrtle, asphodel,
and garlic as medicines. Dates, fig s , onions, lettuce, crocus,
and opium were used as remedies by the Egyptians; juices of
celery, parsley, asparagus, peppers, and cabbage by the Greeks
and Romans; and amber, musk, manna, cloves, peppers, rhubarb,
and nutmeg by the Arabians. (193)• The discovery of P en icillin
prompted many reserach workers to test the usefulness of these
remedies in modern day medicine.
Over 7,000 plant ex tracts have been examined for b acteri
cidal properties. (193) (12) (10) (36) (39) (79) (S3) (32)
(207) (97) (106) (149) (169) (173) (135) (139) (191) and (199).
Since there is no standard method of testin g for antibiotic
compounds and owing to the difference in environmental con
ditions of plant species collected throughout the world, some
variation in resu lts have been reported. In general, however,
there was agreement to the extent that most plants belonging
to the same fam ily resulted in sim ilar specific action and
potency.
Atkinson and Rainsford (12) examined 450 flow ering plants
which are native to A ustralia. The plants were tested by the
cylinder plate method against Staphylococcus aureus and B acillus
