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Halophilic organisms in sufu, Chinese cheese
Pao, Shi-Chiang, Ph.D.
The Ohio State University, 1994
UMI 300 N. Zeeb Rd. Ann Arbor, M I 48106 Halophilic Organisms in Sufu, Chinese Cheese
DISSERTATION
Presented in Partial Fulfillment of the Requirement For the degree Doctor of Philosophy in the
Graduate School of The Ohio State University
by Shi-Chiang Pao, M.S.
The Ohio State University 1 9 9 4
Dissertation Committee: Approved by
Dr. W.J. Harper
Dr. M.E. Mangino , A d v is e r
Dr. M.K. Ndife Food Science & Nutrition
Dr. E.R. Richter Graduate Program To My Parents and Family
ii ACKNOWLEDGMENTS
I wish to express my sincere appreciation to Dr. W.J. Harper, my adviser, and Dr. D,J. Dzurec, my former adviser, for their guidance and support throughout the course of my graduate studies. I would also like to thank Dr. M.E. Mangino, Dr. E.R. Richter and Dr. M.K. Ndife for serving on my examination committee, and their valuable suggestions. Thanks go to the other faculty, staff and students of the Department of Food Science & Technology for their assistance and encouragement. Special thanks to my parents, brother and sister for their love and support throughout my educational career. Gratitude is expressed to all my friends, especially my room-mates for their help, paryer and encouragement. Finally, my deepest appreciation goes to God for my life in Him, and I acknowledge Jesus as my Lord and Savior, VITA
May 6, 1962 Born, Taipei, Taiwan R.O.C.
1984 Dietician Trainee, Chinese Medical Center
Taipei, Taiwan R.O.C.
1985 B.S., Chinese Culture University
Taipei, Taiwan R.O.C.
1985-1987 Officer, 2nd Lieutenant.
Chinese marine Corps, Taiwan R.O.C. 1989 Quality Control Technician
Miceli Dairy Products Co., Cleveland, Ohio 1989-1991 Graduate Research Assistant
The Ohio State University, Columbus, Ohio 1992-1993 Research Techanician
Silliker Laboratories of Ohio, Inc.. Ohio
1994-Present Graduate Research Assistant
The Ohio State University, Columbus, Ohio
iv PUBLICATIONS
P a o , S. & W.J. Harper, 1994, "Microbiological quality of
commercial sufu with various brine composition", IFT Annual
Meeting Abstracts, Paper No. 73-11.
Pao, S. & W.J. Harper, 1994, "Isolation and characterization of
moderate halophiles from commercial sufu with various brine composition", IFT Annual Meeting Abstracts, Paper No. 59B-12.
Lia, O.K., S. Pao & W.J. Harper, 1994, "Effect of tofu processing
residue on the development of physical and sensory properties of cookies", IFT Annual Meeting Abstracts, Paper No. 36D-6.
Pao, S. & D.J. Dzurec, 1990, "Spoilage & pathogenic microorganism quality of sufu, Chinese cheese", IFT Annual Meeting Abstracts. Paper No. 714.
FIELD OF STUDY
Major Fields: Food Science and Nutrition
v TABLE OF CONTENTS Page
DEDICATION i i
ACKNOWLEDGEMENTS i i i VITA i v
LIST OF TABLES...... v i i i
LIST OF FIGURES...... x i
INTRODUCTION...... 1
CHAPTER I: Microbiological Quality of Commerical Sufu with
Various Brine Composition ...... 3
1.1 Literature Review ...... 3
1.2 Plan of Study ...... 16 1.3 Materials and Methods ...... 1 7
1.4 Results ...... 2 4
1.5 Discussion ...... 3 6
1.6 Conclusions ...... 3 9
CHAPTER If: Isolation & Characterization of Moderate Halophiles from Commercial Sufu with Various Brine Composition ...... 41
2.1 Literature Review ...... 41
2.2 Plan of Study ...... 5 3
2.3 Materials and Methods ...... 5 4 vi 2.4 Results ...... 64
2.5 Discussion ...... 76
2.6 Conclusions ...... 81
CHAPTER III: Combination Effects of NaCI & Ethanol on the Growth
& Survival of Halotolerant & Moderately Halophilic
Cocci...... 8 3
3.1 Literature Review ...... 83
3.2 Plan of Study ...... 100
3.3 Materials and Methods ...... 101
3.4 Results ...... 104
3.5 Discussion ...... 126
3.6 Conclusions ...... 131
3.7 Additional Data ...... 133 CHAPTER IV: Sensory Evaluation on Sufu Produced with the
Addition of Pediococcus halophilus ...... 142 4.1 Literature Review ...... 142
4.2 Plan of Study ...... 147
4.3 Materials and Methods ...... 148
4.4 Results ...... 161
4.5 Discussion ...... 171
4.6 Conclusions ...... 176 4.7 Additional Data ...... 178
GENERAL DISCUSSION...... 182
REFERENCE...... 184 LIST OF TABLES Page 1.1 Various alcoholic saline solutions for brining and aging
of sufu ...... 1 4
1.2 Comparison of brine composition of commercial sufu 25
1.3 Comparison of microbiological quality of commercial
sufu...... 2 8
2.1 Inhibitory effect of ethanol on the growth of
microorganisms on solid medium ...... 4 4
2.2 Approximate minium Aw for growth of microorganisms... 4 6
2.3 Salt responses of different microorganisms ...... 51
2.4 Media & conditions for identification of spoilage yeasts.. 60
2.5 Salient properties of yeasts ...... 61
2.6 Population of moderate halophilies in commercial sufu... 65
2.7 Salt response of moderate halophilies isolated from sufu...... 6 7
2.8 Differential characteristics of Gram<+) halophilic
isolates...... 6 9 2.9 Differential characteristics of Gram(-) halophilic
isolates ...... 6 9
2.10 Characteristics of Pediococcus isolates as compared to
those Pediococcus halophifus (ATCC 33315)...... 70 2.11 Characteristics of yeast isolates as compared to those viii o f Saccharomyces rouxi (NRRL Y2547) ...... 74 3.1 Aspects of cellular metabolism that are affected by
ethanol 8 5
3.2 Responses of microorganisms to low Aw thought to be part
of osmo-regulatron 9 0
3.3 The major types of compatible solutes accumulated by
different groups of microorganisms ...... 9 2 3.4 Growth of Marinococcus haiophilus in m edium con tain in g
NaCI & ethanol ...... 133
3.5 Growth of Micrococcus halobius in medium containing
NaCI & ethanol ...... 134
3.6 Growth of Paracoccus halodenitrificans in m edium
containing NaCI & ethanol ...... 135 3.7 Growth of Pediococcus halophilius in medium containing
NaCI & ethanol ...... 136
3.8 Growth of Staphylococcus aureus in medium containing NaCI & ethanol ...... 137
3.9 Survival of S. aureus, cultured orginally in TSB, in medium
containing NaCI & ethanol ...... 138
3 .1 0 S urvival of S. aureus, cultured orginally in TSB with
10% NaCI, in medium containing NaCI & ethanol... 139
3.11 Survival of P. haiophilus, cultured orginally in TSB with
5% NaCI, in medium containing NaCI & ethanol 140 3.12 Survival of P. haiophilus, cultured orginally in TSB with
15% NaCI, in medium containing NaCI & ethanol... 141
ix 4.1 Some microbial food conversions ...... 143
4.2 Time table for inoculation of P. haiophilus...... 150 4.3 Panel prescreening questionnaire form ...... 152 4.4 Panel prescreening triangle test form ...... 154
4.5 Descriptive terms and definations ...... 156 4.6 Evaluation form for the sensory characteristics of
...... sufu. 158
4.7 Intensity of sufu sensory attributes as judged by nine
trained panelists ...... 162
4.8 Statistical significance of the differences (creaminess &
rancid taste) between samples ...... 165 4.9 Preference of sufu sensory qualities as ranked by nine
trained panelists ...... 166
4.10 Statistical significance of the differences (overall
preference, creaminess preference & rancid taste preference) between samples ...... 166
4.11 Growth and survival of P. haiophilus in the sufu preparation...... 168
4.12 Growth and survival of non-halophilic bacteria in sufu
preparation...... 168
4.13 Raw data of intensity evaluation ...... 178
4.14 Raw data of preference test ...... 181
x LIST OF FIGURES
Page
1.1 Flow chart for sufu production ...... 9
1.2 Flow chart for regular tofu production ...... 1 1 1.3 Standard curve for sodium concentration ...... 2 0
1.4 Concentration of NaCI & ethanol in commercial sufu 2 7
1.5 Microbiological profile of commerical sufu ...... 2 9
1.6 Relationship between ethanol & moderate halophile in
commercial sufu ...... 3 0
1.7 Relationship between (NaCI+ethanol)% & lactic acid
bacteria in com m ercial sufu ...... 3 1
1.8 Relationship between pH values & total aerobic
organism s in com m ercial sufu ...... 3 3
1.9 Relationship between lactic acid bacteria and yeasts &
molds in commercial sufu ...... 3 4
1.10 Relationship between total aerobic organisms and
yeasts & molds in commercial sufu ...... 3 5
2.1 A schematic diagram of the combined influence of pH &
Aw on microbial growth ...... 4 7
2.2 Microbes grouped according to response to salt ...... 50
2.3 Dicotomic key for the identification of moderately xi halophilic eubacteria 5 9 2.4 Moderate halophiles in the commercial sufu ...... 6 6 2.5 The relationship between P. haiophilus & moderate
halophiles in com m ercial sufu ...... 7 2 2.6 The relationship between P. haiophilus & eth a n o l in
commercial sufu ...... 7 3
2.7 Ethanol tolerance of moderate halophilic cocci ...... 7 5
3.1 Model showing interactions of hexanol and ethanol with a membrane 8 7 3.2 Differences in the surface hydrophobicity of H. elongata
after growth in different NaCI concentrations 9 3
3.3 Three types of results with combinations of antimicrobial
agents tested by the agar diffusion technique 96
3.4 Three types of results with combinations of
antimicrobics ...... 9 7 3.5 Rate of killing with antimicrobial agents singly and in
combination ...... 9 8
3.6 Effect of salt on the growth of Marino, haiophilus 105
3.7 Effect of salt on the growth of Micro, halobius...... 105
3.8 Effect of salt on the growth of Para, halodentrificans. .. 105
3.9 Effect of salt on the growth of Pedio. haiophilus...... 106
3.10 Effect of salt on the growth of Staphyio aureus...... 106 3.11 Effect of ethanol on the growth of Marino, haiophilus.... 108 3.12 Effect of ethanol on the growth of Micro halobius 108 3.13 Effect of ethanol on the growth of xij Para, haiodenitrificans...... 108 3.14 Effect of ethanol on the growth of Pedio. haiophilus 110 3.15 Effect of ethanol on the growth of Staphylo. aureus 110 3.16 Combination effects on the growth of M arino. haiophilus.. 111 3.17 Combination effects on the growth of Micro, halobius 111 3.18 Combination effects on the growth of Para, haiodenitrificans...... 111 3.19 Growth of P. haiophilus with 3% ethanol and various
amounts of NaCI ...... 113
3 .2 0 Growth of P. haiophilus with 6% ethanol and various amounts of NaCI ...... 114 3.21 Growth of S. aureus with 3% ethanol and various amounts of NaCI ...... 115 3.22 Growth of S. aureus with 6% ethanol and various amounts of NaCI ...... 116
3.23 Growth of S. aureus with 9% ethanol and various
amounts of NaCI ...... 117 3.24 Independent effects of NaCI & ethanol on the survival of
P. haiophilus...... 119
3.25 Independent effects of NaCI & ethanol on the survival of S. aureus...... 120 3.26 Effects of NaCI & ethanol on the survival of S. aureus (culture: 0% NaCI) ...... 121 3.27 Effects of NaCI & ethanol on the survival of S. aureus (culture: 10% NaCI)...... 122
xiii 3.28 Effects of NaCI & ethanol on the survival of P. haiophilus
(culture: 5% NaCI) ...... 123
3.29 Effects of NaCI & ethanol on the survival of P. haiophilus
(culture: 15% NaCI) ...... 124
4.1 Sensory profile of sufu samples made with and without
the addition of P. haiophilus...... 163
4.2 Protein pattern of sufu samples analyzed by SDS-PAGE... 169
xiv INTRODUCTION
Sufu, also referred to as Tou-fu-ru in Mandarin, Tau-zu in
Taiwanese and Fu-ju in Cantonese is a creamy product made from
cubes of soybean curd (tofu) by the action of mold. This product is
a traditional fermented food and is widely consumed by the Asian as an appetizer.
The general, procedure for making sufu consists of the
following steps : preparation of tofu, preparation of molded tofu,
brining, and aging. During the brining and aging steps, salt and
wine are added to flavor, as welf as to preserve the final product.
However, to date, the actual brine composition of commercial sufu
has not been reported. Thus far, the effectiveness of the alcoholic saline solution on the inhibition of the growth of microorganisms has not been throughly investigated. Little or no knowledge of the relationship between brine composition and microbiological and sensory quality of sufu has been established.
The goals of the research were: (1) to investigate the relationship between brine composition and microbiological quality of commercial sufu, (2) to study the effectiveness of alcoholic saline solutions on the growth and survival of 1 microorganisms occurred in sufu, (3) to study the possible role of halophilic organisms in sufu flavor development. Data from this research may be useful in discerning the formulation of sufu brine, and in proposing guidelines to help insure the manufacture of high quality sufu. CHAPTER I
MICROBIOLOGICAL QUALITY OF COMMERCIAL SUFU WITH VARIOUS BRINE COMPOSITION
1.1 LITERATURE REVIEW
Chinese Cheese, Sufu
Sufu, also named tou-fu-ru or Chinese cheese, is a highly flavored soft, cheese-type product. It is made from cubes of soybean curd (tofu) by overgrowing them with a mold of the g e n u s Actinomucor, Mucor, or R h iz o p u s and further fermenting the cubes in brine (Su, 1986; Shurtleff and Aoyagi, 1979a.).
Although sufu is the name which first appeared (Wai, 1929) and is most commonly used in the literature, the product is domestically called toe-fu-ru in Mandarin, tau-zu in Taiwanese, and fu-ju in Cantonese. In the Western world, sufu has been 3 4
referred to as Chinese cheese or fermented tofu by some
researchers and "bean cake" by Chinese grocers (Wang &
Hesseltine, 1970).
Sufu is sold in nearly every Chinese grocery and is widely
consumed by Chinese people as an appetizer (Su, 1986; Shurtleff & Aoyagi, 1979a.). This traditional Oriental food is
manufactured not only in large quantities in the regions of
Chekiang and Kiangsu Provinces in Mainland China (Wai, 1929),
but also it is commercially and domestically produced in Hong
Kong, Indonesia, Japan, Philippine, Taiwan, Thailand, Vietnam and
the United States of America. In 1982, the annual production of
sufu in Taiwan was approximately 12,000 metric tons and the
annual consumption was approximately 660 g per person (Su, 1 9 8 6 ).
Sufu is marketed as either yellowish-white or red blocks of 2 to 4 cm square, and 1 to 2.5 cm in thickness (Wai. 1929,
1968). The cubes are usually bottled in brine, and stored and marketed at room temperature. Sufu is consumed directly or in combination with rice or noodles for any meal of the day. In addition to the regular white-yellowish sufu, various types of sufu have been developed by adding different ingredients to the brine solution. For examples (Wang & Hesseltine, 1970; Shurtleff & Aoyagi, 1979a.): "red sufu (hon-fang)" is made by adding red fermented rice and soy mash; "rose sufu" is made by adding rose essence; "hot sufu" is made by adding hot pepper ; and 5
"drunk sufu" is made by adding rice mash or wine to the brine.
The added ingredients provide color, special flavor, and desired
arom a.
According to Wai (1968), the manufacture of soybean curd
was started during the era of the Hang dynasty. In the "Pen Ts'ao
Kang Mu" or Chinese Materia Medica of 1596, compiled by Li Shi-
chin, it was inferred that soybean curd was invented by Liu An (179 B.C. - 122 B.C.), king of Wainan. However, it is not known
when the manufacture of sufu actually began. The Food
Encyclopedia, written by Wang Su-Hsiung (1861 A.D.) of the
Ch'ing Dynasty, described this product as follows: "Hardened tofu
is difficult to digest, and it is not good for children, old people
or patients. Sufu, which is prepared from tofu, and which is
better when aged, is very good for patients." According to this,
Wai (1968) stated, "We may presume to say that soybean cheese had been sold long before the Ch’ing Dynasty."
Microorganisms Used in Sufu Fermentation
A variety of fungi or molds are used in the sufu
fermentation,. These include: Actinomucor efegans, Mucor
hiemalis, Mucor silvaticus, a n d Mucor subtilissimus (Wai, 1968;
Wang, 1967; Su, 1986). Each of them can be used for fermentation to yield satisfactory sufu. However, Actinomucor
e f e g a n s is described as the most preferred. This species produces more effective proteolytic enzymes than other species 6 of M uco r and is the one commonly used in commercial operations
(Wai, 1968). Molds used in sufu fermentation should have
characteristics as follows (Wang & Hesseltine, 1970):
(1) Produce enzyme systems having high proteolytic and
lipolytic activity to act upon proteins and lipids in tofu.
(2) Produce a white or light yellowish mycelium mat to ensure
that the product has an attractive appearance. (3) The texture of the mycelial mat, which forms over the
surface of tofu cubes, should be dense and thick in order to serve as an envelope preventing any distortion in cube
shape.
(4) Do not produce any disagreeable odor or astringent taste. (5) Do not produce mycotoxin.
Wai (1968) isolated a strain of Actinomucor eiegans fro m
three sufu factories in Taipei, Taiwan. He described its
morphological characteristics as :
(1) Length of sporangiophore: 0.5 ~2 cm (2) Diameter of sporangia: 35 - 84 p
(3) Diameter of columella: (18.2 x 22.4) - (39.2x 40.6) p
(4) Diameter of spore: 6.3 - (11.2 x 12.6) p (5) Color: white to light gray The species can be easily recognized by its stolon and
rhizoid formation and by the verticillate branching (Benjamin & Hesseltine, 1957). Growth conditions for maximum protease
production by this mold species were investigated by Wang et al. (1974). The results show that the optimal conditions for maximum enzyme production of Actinomucor mucor grown on wheat bran were 50 - 63 % moisture and 20°C for 3 days. Since this mold is fast growing and requires high moisture for growth
and enzyme synthesis, the danger of contamination by toxin-
producing fungi such as Aspergillus flavus a n d Aspergillus o c h r a c e u s, which require low moisture leveis (calculated at 33%) for toxin synthesis, would be minimal (Wang et al., 1974). Other application of A, e le g a n s is in the fermentation process of metiauza, which is a fermentation product of the solid waste
material (okara) from the manufacture of tofu (Beuchat, 1987; Kronenberg & Hang, 1984, 1985).
Jong and Yuan (1985) isolated a new species from a starter
culture used in the manufacture of sufu, and described this new species as Actinomucor taiwanensis. Compared with A. elegans, A. taiwanensis differs principally on the basis of sporangiospore size (Jong & Yuan, 1985). In A. elegans sporangiospores are 5 to 8p in diameter as against 7 to 15p. in A . taiwanensis. In addition, the maximum growth temperature is 32°C for A. e le g a n s and 37°C for A. taiwanensis. 8
Preparation of Sufu
Preparation of sufu involves four main steps (Su, 1980, 1986; Reddy & Salunkhe, 1989): preparation of tofu, preparation of Pehtze {tofu freshly over-grown with the fungus), salting, and aging (Figure 1.1).
Preparation of tofu
First, soybeans of selected quality are washed with water to remove dust and dirt (Skurray et at., 1980) Next, the
soybeans are soaked in about 3 times their weight of water. Some have suggested soaking the beans for about 4 to 5 hours at room temperature (Hesseltine, 1985). However, Shurtleff & Aoyagi (1979b.) states that the beans need to be soaked 8 to 10 hours in summer, or for 16 to 20 hours in winter. This soaking softens the cellular structure of the beans prior to grinding them. The proper soaking time is the period that allows the
beans to absorb 1.2 times their weight of water and increase about 2.4 times in volume (Shurtleff and Aoyagi, 1979b ). Both the recovery of tofu solids and the flavor may be affected by the soaking time, soaking temperature and the type of the selected soybeans (Shurtleff and Aoyagi, 1979b.; Tsai et al.. 1981; Johnson et al., 1984; & Skurray et al., 1980). Just before grinding, the soaked beans should be rinsed several times to reduce high surface bacteria contamination. The beans are then ground with added water to a milky slurry in a stone mill. For 9
C o ag u lan t Actinomucor D re s s in g M u c o r o r m ix tu r e R h izo p u s
M illin g
S oy m ilk
Soy c u rd
P re s s in g
T o fu (3 kg)
Fermentation
V a s h in g
P ro c e s s in g I Aging I S u fu (1 -9 k g )
Figure 1.1 Flow chart for sufu production*
* From Reddy U Salunkhe (1 989) 10
this step, a water to bean ratio of 10 : 1 is suitable (Beddows & Wong, 1987; Su, 1986). Next, the milky slurry is boiled for about 7 to 20 minutes and filtered through cheese cloth to separate the soymilk from the residue. The heat treatment (1) inactivates soybean trypsin inhibitors which interfere with the utilization
of the protein in the human; (2) reduces the undesirable raw or beany flavors; (3) increases shelf-life and keeping quality of the final product by killing micro-organisms formerly residing on the soybeans; (4) and facilitates extraction of soymilk from the slurry (Chien & Snyder, 1983; Omosaiye et al., 1978; Shurtleff & Aoyagi, 1979b.). A typical procedure for tofu manufacture is sho w n in Figure 1.2.
Preparation of pehtze
Pehtze is a name given to the small cubes of tofu covered by a growth of grayish hairlike mycelium of molds belonging to the genus Actinomucor, Mucor or Rhizopus (Su, 1986). In order to
prepare pehtze, tofu is cut into cubes of about 2 to 4 cm in length and 1 to 2.5 cm in thickness. Traditionally, inoculation is achieved simply by placing tofu cubes on the rice straw. In this procedure tofu cubes are very likely to be contaminated by undesirable microorganisms. Therefore, in modern factories, tofu cubes are heated in an oven at 100° C for 15 minutes before inoculation (Wai, 1964; Su, 1980). The heat treatment helps to inactivate contaminating organisms and dehydrates the surface Clean Soybeans
Wash & soak <- W ater 8 - 1 2 h r I D ra in — W ater I G rind 4- - W ater
Cook slurry, 10:1 = waterbeans Defoam er
Extract soymilk 6-8* solids — Okare
C oagulate <- Coagulant 7 0 - 8 5 ° c
Remove sypernatant Whey
Press curd to form tofu Whey
0.05i to 0.20 psi, 15-20 min.
Cut tofu; cool in water 5 °c ,60-90 min.
Package & refrigerate 2 - 4 0 c
Figure 1.2 Flow chert for regular tofu production
* From shurtleff & Aoyagi (1979b.) 12
of tofu cubes, making them less susceptible to bacterial spoilage. According to Chou et al. (1988), the moisture content of the tofu cubes is reduced to about 65% during heat treatment. Hesseltine (1965) proposed a modified heat treatment procedure. In his procedure, tofu cubes are immersed in an acidic saline solution (6% NaCI and 2.5% citric acid in water) before being heated in a hot air oven. The soaking procedure reduces the pH of the surface of the small tofu cubes. This modified pretreatment aids in preventing the growth of undesirable contaminating bacteria while allowing mold growth. After cooling to room temperature, the cubes are then placed on trays, inoculated with a pure culture of an appropriate fungus. They are then incubated at 12 to 20°C for 3 to 7 days, which allows the mold mycelium to grow enough to cover the surface of the tofu (Su, 1980, 1986; Wai, 1968).
Salting and aging
The freshly prepared pehtze is salted in a number of different ways (Hesseltine, 1985; Su, 1980, 1986). Commonly, the pehtze is placed in an earthen jar and covered with salt. After 3-4 days, pehtze is taken out and rinsed wtth water for further processing. At this point, the salted pehtze cubes are added, together with a dressing mixture and salt water (about 10 to 20% NaCI), into glass jars. After being sealed and stored for three to six months, sufu is ready for consumption. 13
In another conventional method, pehtze is immersed in an
alcoholic saline solution containing about 12% sodium chloride
and 10% ethanol (Wang, 1983) and aged at 20°C for one to three
months. The resultant product is sufu. Wai (1968) studied the
brining and aging by immersing the pehtze into various alcoholic
saline solutions (Table 1.1). The results showed that the
following solutions helped preserve pehtze both at room
temperature and at 10° C for six months (Wai, 1968): (a) Kaoliang wine to make up 10% ethyl alcohol content plus benzaldehyde
0.01% and NaCI 2%; (b) Kaoliang wine to make up 10% ethyl
alcohol content plus acetic acid 1% and NaCI 4%; (c) Kaoliang
wine to make up 10% ethyl alcohol content plus 5% NaCI
Microbiological Quality of Sufu
Tofu is a highly hydrated protein curd containing more than
80% water. It is quite perishable, even under refrigeration.
During tofu and pehtze preparation, contamination from processing equipment and human hand is unavoidable. The modified pretreatment described earlier is designed by Hesseltine (1965) to reduce the amount of undesirable microorganisms. Nevertheless, according to Pao (1989), microorganisms (such as Peudomonas fluorescens and
Staphylococcus aureus) can survive this pretreatment. During the salting and aging steps of sufu ripening, ethanol and sodium chloride solution are added to retard the growth of undesirable Table I I Various alcoholic saline solutions for brining & aging of sufu*
( 1) Benzoic acid 0 1% plus ethyl acetate 0 05%
(2 ) Acetic acid 2% plus ethyl acetate 0.05%
(3 ) Propionic acid 1 % plus ethyl acetate 0.05%
(4 ) Benzaldehyde 0.01% plus ethyl acetate 0.05%
(5 ) Orange peel oil 1 % plus etheyl acetate 0 05%
(6 ) Acetic acid I % plus NaCI 2% & kaoliang vine to make up 5% ethyl alcohol content
(7 ) Kaoliang vine to make up 5% ethyl alcohol content plus NaCI 2%, anise 0Q1%& red papper
(8 ) Kaoliang vine to make up 10% ethyl alcohil content plus benzaldehyde 0 01 % & NaCI 2%
(9 ) Kaoliang vine to moke up 10% ethyl alcohol content plus acetic acid !% &. NaCI 4%
(10) Kaoliange vine to make up 10% ethyl alcohol content plus NaCI 5%
(11) ethyl alcohol 10% plus NaCI 5%
* From Wei (1960) 15
microorganisms (Wang & Hesseltine, 1970). However, the
effectiveness of these alcoholic saline solutions on the
inhibition of the growth of microorganisms has not been
reported. To date, there has been no comprehensive scientific
emphasis on the microbiological quality of commercial sufu.
Brine composition had been briefly discussed earlier (Wai,
1986), but, the actual brine composition of commercial sufu has
not been reported. Little or no knowledge of the relationship between brine composition and microbiological quality has been established. 16
1.2 PLAN OF STUDY
This study was directed to investigate the relationship between brine composition and microbiological quality of commercial sufu. 17 1.3 MATERIALS AND METHODS
Collection of Samples
Sixteen samples of ten brands of sufu were purchased at
three local retail stores in Columbus, OH. They were made in
Mainland China, Hong Kong, San Francisco and Taiwan, and were
displayed either at room temperature or under refrigeration in
the local stores. In each of these three stores, all available
brands of sufu were obtained. Thus, some brands of sufu were
purchased in duplicate or triplicate. AJI chemical and
microbiological analyses were begun within two days of
purchase. Before tests, all samples were maintained at the same
temperature as they were in the local stores.
Chemical Measurements
Brine solution of each sample was analyzed for ethanol
concentration, sodium chloride concentration and pH. The
methods and materials used for these chemical measurements were as follows.
Ethanol in brine
Ethanol concentrations in brine were determined according to the method described by Caputi & Mooney (1983). Analyses were performed on a Hewlett Packard Model 5890A gas chromatograph, equipped with flame ionization detector, 18 integrator, heated on-colume injector, and 6 ft x 2 mm id glass column packed with 0.2% Carbowax 1500 on 80 to 100 mesh Carbopack C. The packed column was conditioned at 275°C for 15 hours with a nitrogen flow rate of 30 ml/min. The operating conditions were as follows: nitrogen flow rate, 15 ml/min; injection port and detector temperatures, 175°C; oven temperature, 105°C. To determine ethanol in brine, 0.2% (v/v) 2-propanol, and 10% ethanol was used as the internal standard and ethanol standard, respectively. The operating procedures were as follow: dilute ethanol standard solution 1:10 with
internal standard solution, inject three 1.0 pL aliquots and determine average peak (RR'), dilute sample 1:10 with internal standard solution, inject 1,0 pL, and determine response ratio (RR).
%ethanol = (RR x %ethanol in standard)/P?PT
Salt in brine
Sodium concentrations of brine were determined by a Perkin Elmer Model 360 atomic absorption spectrophotometer. The operating procedures described in the manufacturer's manual (Perkin-Elmer, 1973) were followed with modification according to J.A.C.A.C ( Heckman, 1967; Meuron, 1963). The operating conditions were as follows: wavelength, 589 nm; slit setting, 0,7 nm; light source, hallow cathode lamp for Na+; flame, air- 19
acetylene. To prepare standard solutions, dry reagent grade NaCI
at 100°C overnight and make 2.5422 g to 1 liter with deionized
H 2O. Dilute 10 ml of this solution to 100 ml and further dilute 1, 2, 4, 6, 8, and 10 ml of diluted solution to 100 ml to make
standard solution containing, respectively, 1, 2, 4, 6. 8 and 10 ppm Na. Store standard solutions in cleaned and dried
polyethylene bottles. To measure NaCI% in brine, 1 ml brine was
first diluted in 99 ml deionized H 2O and homogenized in a blender for 2 min. To a 10 ml aliquot of diluted brine in a 100 ml volumetric flask, 50 ml of 24% Trichloroacetic acid (TCA) solution was added and diluted to volume with deionized water.
Samples were shaken at five minute intervals for 30 minutes and
then filtered. Appropriate dilutions were made with deionized
water for the measurement. A standard curve (Figure 1.3) was prepared from the standard solutions and concentration of sodium in the sample was obtained from the standard curve.
OH Of sufu The pH values of sufu samples were measured at 25° C with a Fisher Scientific Accumet pH meter.
Microbiological Analyses
Each sample was analyzed for total aerobic plate counts, lactic acid bacterial counts, yeasts & molds, sporeformers, coliforms and coagulase-positive staphylococci according to Absorbance (%) iue . Sadr cre o sdu concentration sodium for curve Standard 1.3 Figure 100 20 0 - 40 60 - 60 0 - 80 - 0 .45 921B R2 0.999 RA2 5.5425 - 9.241 + Bx 2 netain (p.g/1) oncentration C 4 1 1
2 20 21
methods outlined in Compendium of Methods for the
Microbiological Examination of Foods (1976). In addition, each
sample was also tested for moderate halophiles and extreme halophiles according to Ventosa (1988), and Bergev's Manual of Systematic Bacteriology (1984). Briefly, these methods were as fo llo w s :
Io ta ! aerobic Plate counts
Appropriate dilutions were pour plated in duplicate in plate count agar (Difco) and incubated at 35°C for 48 hours.
Yeasts and molds
Appropriate dilutions were surface plated in duplicate on potato dextrose agar (Difco) acidified to pH 3.5 with 10% sterilized tartaric acid. Plates were incubated at 25°C for 5 days.
Sporeformers
Portions (10 ml, 1 ml and 0.1 ml) of the sufu homogenate were added to three different 100 ml flasks of melted plate count agar and heat-shocked in a water bath at 80°C for 10 mm. After heat-shocking, the mixture from each flask was distributed among five petri plates, which were then incubated at 35°C for 48 hours. 22
C o n fo rm s
A solid media method was used that permitted enumeration of injured coliforms. Appropriate dilutions were surface plated in triplicate onto Trypticase soy agar (BBL) and incubated at room temperature for 1 hours to allow metabolic recovery to occur. Violet red bile agar (Difco) was then added in an overlay,
and the plates were incubated at 35°C for 24 hours. Presumptive coliform colonies were inoculated into brilliant green bile broth
(BBL). Those that produced gas within 48 hours at 35°C were counted as confirmed coliforms.
Slaohvlocucci
A recovery method for staphylococci was used in which appropriate dilutions were made in Tryticase soy broth (BBL) and incubated at 35°C for 2 hours. Dilutions were surface plated in triplicate onto Baird-Parker agar (Difco) enriched with egg yolk.
Presumptive staphylococcal colonies were transferred into 0.5 ml reconstituted EDTA coagulase plasma (Difco), incubated at 37°C, and examined hourly for 4 hours for clot formation.
Coagulase (+) colonies were reported as Staphyiococcu aureus
Moderate halophiles
The medium employed was as follows (%. w/v): NaCI. 17.8; CaCI2 2H20, 0.036; KCI, 0.2; NaHC03. 0.006; NaBr, 0 023;
FeCl3-6H20, trace; Proteose-Peptone no. 3 (Difco), 0.5; yeast 23
extract (Difco), 1; glucose, 0.1; Bacto-Agar (Difco), 2. Final pH
is 7.2, adjusted with 1N KOH. The samples were surface plated in duplicate on the selective medium (either directly or diluted in sterile 10% salt solution) and incubated at 35°C for 7 to 10 days in sealed plastic bags.
Extreme halophiles
Appropriate dilutions were surface plated in duplicate on nutrient agar (Difco) containing 25% (w/v) NaCI. The plates are wrapped in plastic bags to prevent drying and are incubated at 35°C for 7 to 15 days. Growth is indicated by development of a red color, and isolation can be achieved by suspending the growth in 25% NaCI and streaking suitable agar media.
Statistical Anysis
The Spearman Rank Correlation Coefficient was used for the analysis of correlation of data (Neter et al., 1989).
Statistical significance was accepted if the probability value was less than or equal to 0.05 for the correlation. 24 1.4 RESULTS
The relationship between brine composition and the
microbiological quality of commercial sufu was investigated. Sixteen samples of ten brands {brand A, B, C, D, E, F, G, H, I, J) of
sufu were tested. Gas chromatograph and atomic absorption
techniques were applied to measure the concentrations of
ethanol and sodium chloride, respectively, in brine. Total
aerobic organisms, coliforms, staphylococci, lactic acid bacteria, sporeformers, yeasts & molds, moderate and extreme
halophiles in sufu, were determined by plate count methods. pH
levels were also measured.
Brine Composition of Commercial sufu
Results of chemical analyses of brine are presented in
Table 1.2. Ethanol concentrations ranged from 0.87 to 21.42%.
Most brands (70%; brand A, B, C, E, F, G and J) had brines with an
average ethanol content between 10 and 20%. Variation was seen among samples of same brand. The greatest variation was seen among three samples of brand F, in which ethanol% ranged among 20.27 to 9.97%. Sodium chloride concentrations of brines ranged from 9.89 to 22.96% with an average of 13.99%.
The greatest difference among samples of the same brand was found for brand A samples (4.68%. In addition, only two brands
(brand D and I) of samples had sodium chloride content higher 25 Table 1.2 Comparison of brine composition of commercial sufu
Sample* Source NaCl% Ethanol% pH
(Brine) (Producer)
Mean1 SD2 Mean3 SD Mean1 SD
A- t Taiwan 14.57 0.20 16.09 0.07 5.56 0.01
A-2 9.89 0.20 13.58 0.12 5.75 0.06
B - 1 Taiwan 10.99 0.20 12.53 0.02 5.81 0.01
B~2 11.13 0.39 13.99 0.05 5.59 0.03
C-1 Taiwan 13.05 0.39 13.11 0.04 5.34 0.00
C-2 12.23 0.00 11 90 0.03 5.28 0.00
D - 1 Taiwan 22.96 0.00 0.37 0.05 5.30 0.00
E-1 China 12.92 0.20 11.01 0.04 5.95 0.02
F - l Hong Kong 11.95 0.00 18.02 0.04 5.77 0.03
F-2 12.78 0.00 20.27 0.10 5.45 0.00
F-3 14.71 0.40 9.97 0.05 6.12 0.00
G - 1 California 10.72 0.19 15.59 0.06 5.91 0.04
H - 2 China 13.33 0.00 21.42 0.16 5.20 0.02
H-3 14.02 0.20 21.34 0.18 5.15 0.00
I-2 Taiwan 17.32 0.T9 5.01 0.04 4.96 0.00
J - 2 Hong Kong 13.19 0.20 15,05 0.14 6.26 0.00
* Samples (brand A to J) were obtained from store 1. 2, and 3.
1 Data represent the average of two traits.
2 Standard Deviation
3 Data represent the average of three trails 26
than 15%. Total ethanol% + NaCI% ranged from 22.32 to 35.36%.
The pH values were between 4.95 and 6.27. The relationships between ethanol%, NaCI% and pH values are shown in Figure 1.4. No correlation was found between NaCI%, ethanol% and pH.
Microbiological Quality of Commercial Sufu
Table 1.3 shows the log cell number of various
microorganisms from commercial sufu. High levels of aerobic
plate counts (> 105 cfu/g) and spore counts (> 104 cfu/g) were observed in 80% of all the brands. In addition, 60% of the brands had yeasts & molds counts greater than 105 cfu/g and lactic acid bacterial counts greater than 104 cfu/g. Halophiles were found in most samples tested, and high levels of moderate halophiles (> 105 cfu/g) were found in 60% of all brands of samples. Staphylococci and coliforms were not found in any sample tested. The relationships of various organisms in commercial sufu are shown in Figure 1.5.
Relationship between Brine Composition & Microbiological
Quality of the Ten Brands of Commercial Sufu
Results of this study indicate that ethanol concentrations of sufu brines were negatively correlated (r=-0.81, P=0.003) with the levels of moderate halophiles in sufu (Figure 1.6). In addition, negative correlation (Figure 1.7) was found between total ethanol% + NaCI% and lactic acid bacteria counts (r=-0.72, Figure 1.4 Concentration of NaCI & ethanol in commercial sufu sufu commercial in ethanol & NaCI of Concentration 1.4 Figure Concentration (%) Concentration (%) a) < < EhnUal % (EthanoUNaCl) (Ethanol*NaCtr/. (Ethanol*NaCtr/. Dt arne acrig o ape ad brands) and samples to according arranged (Data a Q o o II f l I Ifcl E G I J I H G F E D c 0 al% ■ £:hanoif%) ■ NaCl<%) I aI i%) NaCI Brands Samples tao :% Ethanol il 1J1 i 27 Table 1.3 Comparison of microbiological quality of commercial sufu
I 2 3 4 5 SUFU APC Spore Lactic YAM M.H. EH.
Mean* so7 Mean SD Mean SD Mean SD Mean SD Mean SO
A* 1 5.20 0.01 2.78 0.02 4 72 0.02 3.1 0 0.06 1.00 0.00 0.00 0.00
A-2 5.76 0.00 5.18 0.1 7 2.00 0.00 5.18 0.03 4.70 0.03 0.00 0.00
6-1 7.04 0.04 6.13 0.1 9 4.16 0.00 6.26 0.01 5.48 0.1 2 0.00 0.00
B-2 6.50 0.01 4.09 0.02 5.19 0.01 4.61 0.1 0 5.48 0.04 0.00 0.00
C-1 4.42 0.12 4.03 0.05 1.00 0.00 3.16 0.01 3.36 0.10 0.00 0.00
C-2 3.28 0.09 0.95 0.97 3.49 0.23 2.57 0.02 5.80 0.06 0.00 0.00
D-i 5.85 0.02 4.54 0.21 5.26 0.01 5.23 0.00 6.23 0.04 0.00 0.00
E-1 6.83 0.01 6.10 0.04 4.46 0.06 3.86 0.08 5.8S 0.31 0.00 0.00
F-1 4.30 0.05 2.60 0.13 2.30 0.01 2.46 0.01 0.00 0.00 1.90 0.01
F-2 6.52 0.07 5.78 0.06 1.00 0.12 3.30 0.1 4 1,00 0.05 0.00 0.00
F-3 6.41 0.29 5.30 0.40 1.48 0.1 5 5.00 0.20 3.60 0.02 0.00 0.00
G - 1 8.48 0.00 2.70 0.0 3 6.00 0.00 6.70 0.04 0.00 0.00 0.00 0.00
H-2 3.02 0.00 2.04 0.03 1.00 0.02 2.94 0.00 2.04 0.00 0.00 0.00
H-3 2.99 0.12 2.00 0.01 1.40 0.00 2.36 0.00 2.15 0.10 2.43 0.62
1-2 5.15 0.23 4.61 0.1 6 4.48 0.02 4.00 0.09 6.40 0.29 1.30 0.08
J-2 6.35 0.00 5.78 0.27 1.30 0.01 5.1 0 0,03 6.08 0.03 0.00 0.00
Total aerobic plates counts (log cfu/g) Sporeformers (log cfu/g) Lactic acid bacteria counts (log cfu/g) Yeasts & molds counts (log cfu/g) Moderate halophiles (log cfu/g) Etreme halophiles (log cfu/g) Data represent average of two trails Standard deviation iue . Mcoilgcl rfl o te omril Sufu Commercial the of Profile Microbiological 1.5 Figure Log (cfu/g) a d ran B □ Y&M l E m Lactic . h 29 iue . Rltosi bten tao & drt hlpie in halophiles oderate m & ethanol between Relationship 1.6 Figure Brand Concentration(%) o ril sufu ercial m com Ethanol ■ Log (cfu/g)Log 30 Brand iue . Rltosi bten NC+tao) lci acid lactic & (NaCI+ethanol)% between Relationship 1.7 Figure 0
Concentration(%) 20 atra n omeca sufu ercial comm in bacteria 40 I Ethanol +NaCI
Log(cfu/g) Acid Bacteria Lactic
31 32
P=0.019). Positive correlation {Figure 1.8) was found between pH and aerobic plate counts (r-0.76, P-0.011). Moreover (Figure
1.9 and 1.10), yeasts & molds counts were positively correlated with both lactic acid bacteria counts (r=0.76, P =0.011) and aerobic plate counts (r=0.75, P-0.013). No correlation was found between NaCI% and any group of microorganism tested in this study. iue . Rltosi bten H ttl eoi ogns in s organism aerobic total & pH between Relationship 1.8 Figure
Brand 5 0 H Level pH o cal sufu l rcia e m com 0 5 10 5 0 10 Log(cfu/g) Aerobic ■ Counts Plate
33 34
~a c £5 □ Y&M CD
10 Log (cfu/g)
Figure 1.9 Relationship between lactic acid bacteria and yeasts & molds in commercial sufu 35
H
C
F
E
1 -O □ Y&M c A * i—nj J 00
10 Log (cfu/g)
Figure 1.10 Relationship between total aerobic organisms and
yeasts & molds in commercial sufu 36 1.5 DISCUSSION
Brine Composition of Commercial Sufu
Since there is no standard processing method or defined
taste for commercial sufu, it is not unexpected that quite
irregular salt% and ethanol% combinations of brine were found in this study. In addition, large variation of salt or ethanol concentrations among samples from the some brand is an indication that some factories had poor quality controll and
produced inconsistent product.
Microbiological Quality of Commercial Sufu
In this present study, high levels of aerobic plate counts and spore counts were observed in a majority of the samples. This indicates either that spoiled (or highly contaminated) tofu were usedfor sufu manufacture, or that poor processing
practices (such as, inappropriate handling or poor sanitation)
were involved. To produce sufu with lower microbial counts, it
will be necessary to reduce initial loads by using fresh tofu to
make pehtze, and by improving sanitation and processing
techniques. Result shows no coliforms or staphylococci were found in any sample. However, this result does not provide
enough information to say that pathogenic organisms were not
involved in the fermentation process of commercial sufu, that toxins were not produced. Coliforms or staphylococci (or other 37
pathogens) could have grown to high levels (thus producing toxins ) and had been killed during the salting and aging process. More toxilogical study on the commercial sufu would be required to assess this potential safety hazzard. Positive correlation was found between between pH level
and total aerobic plate counts, and founded between yeasts & molds counts and both aerobic plate counts and lactic acid bacteria counts. These relationships further suggest that organisms introduced to the tofu or pehtze were able to grow, during the preparation processes. Thee variations in microbial
counts found among different brand as well, as different samples in the same brand .would be expected, since many environmental variables that may affect the final bacterial counts were involved in the sufu fermentation.
Chemical Composition and Microbiological Quality of the Sufu
Larsen (1962) defined nonhalophiles as those that grow best in media containing lower than 2% sodium chloride, and
slight, moderate and extreme halophifes as those that grow best in media containing 2 to 5%, 5 to 20%, and 20 to 30% sodium chloride, respectively. Since sodium chloride concentrations of the brine ranged from 9.89 to 22.96%, and only one out sixteen of samples had a NaCI% higher than 20%, the salt concentration of most commercial sufu is suitable for the growth of moderate halophiles. This is why high levels of moderate halophiles were 38
found in six out of ten brands of samples, but, only low levels of extreme halophiles were found in three of all sample tested. No correlation was found between NaCI concentrations and any group of microorganism tested in this study. However, negative correlations were found between ethanol concentrations and population of moderate halophiles, and between total ethanol% + NaCI% and lactic acid bacteria counts. These data suggest that the population of moderate halophiles in commercial sufu is not determined by the concentration of sodium salt in the brine. Alcohol, instead of salt, appears to be an important inhibition factor of the growth of halophiles. Furthermore, the total ethanol% + NaCI% of the brine wsas shown to be a factor that may negatively influence the survival of lactic acid bacteria at the time of salting and aging. Many nonhalophilic microorganisms (such as sporeformers and yeasts & molds) are capable of surviving through the brine and aging process. Although significant correlation was not found between levels of these organisms (except lactic acid bacteria) and brine composition, some specific relationship still can be noted. For example, samples of brand H which were observed to have the highest amount of ethanol in brine had only low levels of total aerobic organisms were founded in them. 39 1.6 CONCLUSIONS
The relationship between microbiological quality and brine composition of commercial sufu (fermented tofu) was investigated. Microflora in sixteen samples of ten brands of sufu
were determined by plate count methods. Gas chromatography and
atomic absorption techniques were applied to measure ethanol and NaCI, respectively, in brine.
Ethanol and NaCI concentrations ranged from 0.87 to 21.42% and 9.89 to 22.96%, respectively. Total ethanol% + NaCI% ranged from 22.59 to 35.36%. pH values were between 4.96 and 6.26. High levels of aerobic plate counts (> 105 cfu/g) and spore counts (> 104 cfu/g) were observed in 80% of all brands. In addition, 60% of all brands of samples had yeasts & molds counts greater than
10s cfu/g and lactic acid bacterial counts greater than 1 04 cfu/g.
Halophiles were found in most sample tested, and, high levels of moderate halophiles (>105 cfu/g) were found in 60% of all brands of samples. Staphylococci and coliforms were not found in any sample tested.
Negative correlations were found between ethanol concentrations and levels of moderate halophiles (r=-0.8i.
P=0.003). Positive correlation was found between pH and aerobic plate counts (r=0.76, P=0.011). In addition, yeasts & molds counts were positively correlated with both lactic acid bacteria counts (r-0.76, P-0.011) and aerobic plate counts (r=0.75, P=0.013). No 40 correlation was found between NaCI%. ethanol% and pH. No correlation was found between NaCI% and any group of microorganism tested in this study. Data from this research may be useful in discerning the quality of commercial sufu and in proposing guidelines to help insure the manufacture of high quality sufu. CHAPTER II
ISOLATION AND CHARACTERIZATION OF MODERATE HALOPHILES FROM COMMERCIAL SUFU WITH VARIOUS BRINE COMPOSITION
2.1 LITERATURE REVIEW
Ethanol as a Food Preservative
It is well known in the medical field that ethanol (ethyl alcohol) at high concentrations is an effective disinfectant. Similarly, the preservative function of lower levels of alcohol that are generated during the preparation of fermented foods and drinks is well recognized. Furthermore, the use of wine as a preservative also is not a new idea. Since ancient time, the
Chinese learned to use the combination of wine and salt to preserve highly perishable food such as sufu (fermented tofu). 41 42 Research conducted later by scientists further disclosed that ethanol can increase the antimicrobial effectiveness of other
substances such as sorbic acid in wine (Banwart. 1989). Nevertheless, it is only recently that the benefit of the deliberate addition of low concentrations of ethanol as a mean of prolonging the self-life of packaged foods has been recognized by scientists (Banwart, 1989; Seiler & Russell, 1991).
Ethanol is bactericidal, but not sporicidal (Banwart, 1989; Seiler & Russell, 1991). It has poor penetrating power and may readily be inactivated by organic matter (Banwart, 1989). The antimicrobial effect of an ethanol solution is generally determined by its concentration. At a concentration of 95% or greater, vegetative cells tend to be resistant, whereas at 60 to 70% most microorganisms are killed in less than a minute (Seiler and Russell, 1991). Solutions containing less than 30% ethanol are rarely biocidal in the short term; Gram-negative bacteria tend to be destroyed more rapidly than are Gram- positive organisms (Seiler and Russell, 1991) Concentrations of ethanol above 15% result in immediate inactivation of most vegetative organisms; whereas most bacteria exhibit a dose* dependent inhibition of growth over the range from 1 to 10% ethanol (Ingram. 1984). Some organisms are exceptionally tolerant to higher alcohol concentrations, notably the lactobacilli, including L. homohiachii and L. heterohiachit, the causative spoilage agents of sake (Seiler and Russell. 1991). 43
Variation in ethanol tolerance between different microorganisms has been demonstrated by Yamashita et al (1975), and the results in tabular form {Table 2.1) can be found in a recent review by Seiler and Russell (1991).
Salt as a Food Preservative Salt (NaCI) has long been used as a food preservative. The preservation function of salt is primarily based on the fact that, at high concentrations, salt lowers the water activity (Aw) of food and exerts a drying effect on both food and microorganisms (Jay, 1992; Davidson et al., 1983). The Aw is an index of the availability of water for chemical reactions and microbial
growth, and may be defined as a ratio (Banwart, 1989):
2. Aw = P/Po = Equilibrium relative humidity/100
As expressed by this formula. Aw is the ratio of the vapor
pressure above a material or solution (P) and that of pure water
(Po) under the same condition. By the addition of solutes, the concentration of pure water is decreased and the rate of escape
from the surface is diminished (Banwart. 1989). Therefore, the Aw of a food may be measured by the equilibrium relative humidity (ERH). All microorganisms exhibit a maximum, optimum, and minimum Aw for growth. However, for the consideration of food preservation, the minimum Aw of food is 44
Table 2.1 Inhibitory effect of ethanol on the growth of micro organisms on solid medium*
Species Ethanol content of medium (vol%)
4 8 1 2
Bacteria Escherichia coli . Bacillus su tit tits . Staphylococcus aureus +■ - Sarcina iutea - Aerobacter aerogenes + - SerraTia marcescens • Pseudo mom as fluorescens + . Salmonella typhimurium + - Brevibacter ammoniagenes +■ - Micrococcus epidermidis + + - Streptococcus faecahs +■ + Lactobacillus plantarum ■h + - Lactobacillus sake +■ + Yeast Torulopsis utilis + - Candida albicans Mycotorula japonica + - Endomycopsis fibuliger • Endomyces selsu -- Ptchta membranaefactens - Saccharomyces rouxii - Fungi Aspergillus niger - Pentcillium chrysogenum -- Rhizopus javanicus * - Mucor plumbeus ±_ - Monifia formosa - Tnchoderma vinde - Dematium pullulans - - Actinomyces Strepiomyces aibus *- Nocardta gardnen
From Serler & Russell (199T) G ro w th No growth 45
of most interest. The minimum Aw for the growth of various microorganisms are shown in Table 2.2. Generally speaking, for growth, molds require a lower Aw than yeasts, and yeasts require a lower Aw than bacteria. According to Yoshii (1979). the optimum water activities of P. ha/ophifus, a halotolerant organism, are 0.99 to 0.94, which correspond to 5 to 10% (w/v) salt content. The lowest water activity in which P. hatophilus can grow is 0.808, which corresponds to 24% (w/v) salt content.
While salt continues to have value as a preservative, it is now applied more commonly, according to Davidson et al. (1983), in combination with other antimicrobials (such as organic acids) or preservation techniques (such as heat treatment). For example, pasteurized cured meats are not heated sufficiently to inactivate all the spores, however the surviving spores are unable to germinate and outgrow in the presence of the curing salts (Gould & Jones, 1989). Hence, the microbiological stability and safety of these meats are impressively high. Another good example is stabilizing intermediate moisture foods (IMF) by the incorporation of an antimycotic agent (such as sorbic acid) tc control the osmotolerant flora (Gould & Jones, 1989). Figure 2.' is a schematic diagram of the combined influence of pH and Aw on microbial growth (Hocking, 1991). We may conclude that some microorganisms can grow over a wide range of salt concentration. Their presence in salted food is not simply or directly determined by salt content or Aw value 46
Table 2.2 Approximate minimum Aw for growth of microorganism*
Organism Minimum Aw Organism Minimum Aw
Most spoilage bacteria 0.90-0.91 Staphylocpccus albus 0.88-0 92 Acinetobacter 0 95-0.98 S. aureus 0 83-0.92 Amromonas 0.95-0.98 Streptococcus 0 92-0 98 Bacillus 0.90-0.99 Vibrio parahaemolyticus 0.94-0.98 B. cereus 0.92-0.95 Halophilic bacteria 0,75 Clostridium botulinum 0,90-0.98 Most yeasts 0.87-0.94 Type A 0.93-0.95 Osmophilic yeasts 0.60-0 78 Type B 0.93-0.98 Most molds 0,70-0 80 Type E 0.94-0.97 Xeropbilic molds 0.60-0.70 C. pertringens 0.93-0.97 Aspergillus 0.68-0 88 Corynebactenum 0.95-0.98 A. fiavus 0.78-0 9 Enterobacter 0.95-0.98 A. halophilicus 0.68 Escherichia coti 0.94-0.97 A. mgar 0 80-0.84 Flavobacterium 0 95-9.98 Mucor 0.80-0.93 Lactobacillus 0.90-0.98 PeniciSiium 0,78-0.90 Leuconostoc 0 96-0 98 Rhodotoruta 0.89-0.92 Micrococcus 0.90-0.95 Saccharamyces caravistae 0.90-0.94 Pseudo. Huore scans 0.94-0.97 S. rouxii 0 62-0 81 Salmonella 0.93-0.96 Xeromyces bisporus 0 60-0 61
From Banwart (1989) iue . A ceai darm f h cmie ifune f H & pH of influence combined the of diagram schematic A 2.1 Figure Water Activity 0.7 0.8 0.9 1.0 3 Fo Hcig (1931) Hocking From * w n irba growth* microbial on Aw sas molds & Ysaats 4 Lactobacilli H p 5 ^ osomlng porsform S * >y c a y l t> t*p S aiophilic H 6 s rj aureua scruj bacteria bacteria 7 47 48
of the food. Depending upon the nature of these organisms and
their involvement in the food preparation, these microorganisms may or may not be desirable in the food.
Halotolerant and Halophilic Microorganisms Quite a number of definitions of these two terms has been given in the literature, based on the growth response to NaCI
(Rodriguez-Valera, 1988). In general, halophiles are defined as
those "salt-loving" microorganisms that require a high concentration of salt for optimal growth. Halotolerant
organisms are defined as those which grow best without significant amounts of NaCI in their media, but can also grow in high salt environment. These microorganisms can grow or
survive over a wide range of salt concentrations, including
highly salted food. For example, an extreme halophilic archaeobacterium (halobacterium) has been isolated from fermented fish sauce, which has a concentration of 4.4 to 5.1 M
NaCI (Thongthai et al.. 1992). This organism growing optimally at salt concentration between 3.5 and 5 M, and is common in crude solar salt (Grant & Larsen, 1989). In addition, moderate halophiles has been isolated from salted fermented food, such as miso paste. Some of these halophiles. such as Pediococcus h a io p h H u s and Pediococcus acidilactici, play an important beneficial role in the fermentation of miso; whereas high levels of salt tolerant Bacillus and Clostridium found during the 49
preparation process, are considered as harmful m miso (Reddy &
Salunkhe, 1989). Distinctions between various kinds of halophiles are made on the basis of their salt requirement as well as salt tolerance (Larsen, 1986; Vreeland. 1987). Larsen (1962) defined
nonhalophiles as those that grow best in media containing less
than 2% NaCI, and slight, moderate, and extreme halophiles as those that grow best in media containing 2 to 5%, 5 to 20%, and
20 to 30% NaCI, respectively. Kushner (978) expanded Larsen's
definition, adding "borderline extreme halophiles". "facultative halophiles" and "salt-tolerant bacteria". Based on this expansion, nonhalophiles. slight, moderate, borderline-extreme,
and extreme halophiles were defined as those that "grow best" in
less than 0.2 M (-1%) salt, 0.2 to 0.5 M (-1 to 3%) salt, 0.5 to 2.5
M (-3 to 15%) salt, 1.5 to 4.0 M (~9 to 23%) salt, and 2.5 to 5.2 M
(saturated) salt (~15 to 32%). respectively. Those moderate
halophiles able to grow in <0.1 M salt were termed "facultative
halophiles". Those that tolerate high salt concentrations but do not require them for growth are called halotolerants organisms Larsen (1986) discussed the distinction between "tolerance for
salt", and "requirement for salt". Two figures (Fig. 2.2) were
constructed to present this view. Recently, Kushner (1993)
presented a modified scheme (Table 2.3) which better described salt responses of different microorganisms in the medium that 50
Tolerance
Extreme
Moderate i 6 Slight No
10 20 30
NaCI (%)
B. Requirement
No
: A S •« * . / ' -J « / V / t t / Moderate 4 Extreme 3 • »✓ o ** * X ;• » / v O . * i g • - * / •• I?« i / ^ \ • Z * / \ • ,* * / \ - ' ^ / \ ) / / ; 1 Slight / l 10 20 30
NaC! (%)
Figure 2.2 Microbes grouped according to response to salt’
* From Larsen (1988) 51
Table 2.3 Salt response of different microorganisms*
Category Salt concentration (M)
Minimum Optimal Maximal
Nonhalophiles < 0.2 0.3-1 .0
Slight halophiles 0.2 0 . 2 - 0.5 2.0
Modertate halophilles 0 .4 0.5-2.0 3.0-3.5
Borderline extreme 2.0 3.0-4.0 5.2 halophiles
Extreme halophiles 2.0 3.0-4.0 5 2
Halotolerant Nonhalophiles, which can tolerate high salt concentrations extremely halotolerant cells can grow in 2 5 M NaCI
Facultative halophiles Require ca. 0.5 M NaCI only under cenatn conditions
From Kusher (1993) 52
supports the most rapid growth under optimal salt
concentrations.
Microbiological Aspect of Commercial Sufu The relationship between brine composition and
microbiological quality of commercial sufu was investigated earlier (Chapter 1). The actual brine composition of commercial sufu and the effectiveness of these alcoholic saline solutions on the growth inhibition of microorganisms had been examined and discussed in the Chapter 1. Since sufu is not a sterile product, it was of interest to investigate the microorganisms presented in the commercial products, especially those moderate halophiles that may exhibit growth in the salting and aging process of sufu. This present work reports on the isolation and characterization of the moderate halophiles found in the commercial sufu. Results of this study are important for later investigation of the possible participation of moderate halophiles in the sufu fermentation. 53 2.2 PLAN OF STUDY
This study was directed to the isolation and characterization of moderate halophiles from commercial sufu with various brine compositions. 54 2.3 MATERIALS AND METHODS
Collection of Samples
Sixteen samples of ten brands of sufu were purchased at three local retail stores in Columbus. OH. According to earlier
investigations (Chapter 1), the ethanol and NaCI concentrations of brine samples ranged from 0.87 to 21.42% and 9.89 to 22.96%,
respectively. Total ethanol% + NaCI% ranged from 22.32 to 35.36%. pH values were between 4.96 and 6.26.
Enumeration and Isolation of Halophiles
For the enumeration of moderately halophilic
microorganisms, the following medium, according to Ventosa (1988), was employed (%. w/v): NaCI, 17.8; CaCl2'2H20 , 0.036; KCl, 0.2g; NaHC03, 0.006g; NaBr, 0.023g; FeCI3 6H20, trace;
Proteose-Peptone No. 3 (Difco), 0.5g; yeast extract (Difco), ig; glucose, 0.1g; Bacto-Agar (Difco), 2g. Final pH is 7.2. adjusted with 1N KOH. The samples were surface plated in duplicate on the medium (either directly or diluted in buffered sterile 10% salts solution) and incubated at 35°C aerobically m sealed containers and anaerobically in jars with the Gas Pak Anaerobic System (BBL). The appearance of growth was checked and counted after 7 and 14 days of incubation.
To isolate moderate halophiles, a culture selection strategy, previously discussed by Vreeland (1993). was used 55
with some modification. For samples of each brand, all
morphologically distinct colonies that exceeded 102 cfu/g, were
selected in duplicate for identification. Pure cultures were
obtained after several times of streaking or picking. To isolate
halophilic fungi, Malt Yeast Agar (MYA) containing 18% NaCI was
used. The samples were plated in the same manner, and
incubated aerobically at 25°C in sealed containers for up to 14 days. Colonies grown on anaerobic plates were not selected for this study. However, general colony and cell morphology were observed and compared with those aerobes.
Maintenance of Isolates
The isolates were maintained on agar slants with 10%
(w/v) marine salts (%, w/v), according to Ventosa et al. (1983):
NaCI, 8.1; MgCI2, 0.7; MgS04, 0.96; CaCl 2 , 0.036; KCl, 0.2;
N a H C 0 3 , 0.006; NaBr, 0.0026g (Rodriguez-Valera et al., 1980) supplemented with 0.5% (w/v) Proteose-Peptone no. 3 (Difco),
1% (w/v) yeast extract (Difco) and 0.1% (w/v) glucose.
Halophilic fungi were maintained on Malt Yeast Agar (MYA) containing 10% NaCI.
Characterization of Bacterial Isolates
Characteristics of isolates were determined based on morphological, physiological and biochemical tests that made in duplicate. Unless otherwise indicated, incubation was at 35°C 56
and the pH of all media was adjusted to 7.0 with KOH and
contained 10% (w/v) marine salts as above.
Physiological tests
Aerobic growth at various salt concentrations was determined on solid medium with total marine salts at 0.5, 2, 5,
10, 15, 20 or 25% (w/v). These media were supplemented with
nutrients as in the maintenance medium. The concentration in which visible growth appeared first was considered optimal; growth was considered positive when it was visible after 14 days of incubation. The ability to grow anaerobically was evaluated on streaked plates of the same medium incubated in jars with the GasPak Anaerobic System (BBL). The appearance of growth was checked after 14 days of incubation. The pH range over which growth occurred was determined in a similar way, as for salt range, on media in which the pH was adjusted to
4.4, 5.5, 6.5, 7.5, 8.5 with HCI or KOH. The pH was read or adjusted after sterilization. The temperature range was also determined as above by incubation at 5, 25, 35, or 45°C. The result was considered positive if there was visible growth after 7 days, except for 5°C, at which the incubation time was 3 w e e ks 57
Morphological studies
Isolates obtained from the maintenance medium with
optimum salt concentrations were used for morphological studies. Motility, cell shape and cell size were determined in
wet mounts. Colony morphology, size, and production of
pigments were examined on maintenance medium after 5 days of
incubation. Gram staining was performed according to a
modified method by Dussault (1955).
Biochemical tests
Catalase activity was determined by adding a drop of 3%
(v/v) H2O 2 directly to the colonies on the plates. Oxidase
production was detected by the method of Kovacs (1956). Acid production from glucose, sucrose, lactose, mannitol, inositol,
maltose and galactose was detected in the basal medium: 10%
(w/v) marine salts, 1% (w/v) Peptone P (Oxoid), 0.5% (w/v)
yeast extract (Difco) and 0.001% (w/v) phenol red.
Carbohydrates were sterilized by filtration and added to the
previously autoclaved basal medium at a final concentration of 1% (w/v).
Identification of Bacterial Isolates
The results of above tests were analyzed by the identification keys of the Bergey's Manual of Determinative 58 Bacteriology (1994) and Ventosa (1988). Figure 2.3 is a simple
identification scheme taken from Ventosa (1988).
Identification of Yeast Isolates Procedure for yeast identification described by Pitt &
Hocking (1985) was used for this study. For each isolates, a 3 to 7 day culture obtained from the maintenance medium was streaked on various plates as described in Table 2.4, and checked for growth after 3 and 7 days of incubation at 25°C. Colony color, size and shape were also determined. Approximate cell shape, cell size, position of budding, and the presence or absence, type and number of ascospores was examined in wet mounts. A list of salient properties on various media are listed in Table 2.5. The results of above tests were analyzed by the identification keys to spoilage yeasts by Pitt and Hocking (1985).
Growth of Halophiles in Alcoholic Medium Test organisms
Cultures of Marinococcus halophilus (ATCC 27964),
Micrococcus halobius (ATCC 21727), Paracoccus hafodenitrificans (ATCC 13511), Pediococcus halophilus (ATCC
33315), Sporosarcina halophila (ATCC 35676) were used as test organism s. Gram Negative Gram Positive
Motile cocci Non motile cocci Strictly Anaerobic Aerobic or Facultatively Anaerobe
Sporelorming Non-sporelorming
Sporetoimtng Nonsporetormmg Aerobic Facult Anaerobic
V V Sporarcm a Marinococcus Micrococcus Cmvwt Bod* Spwoth#i*»
(A)
Mow* Non mow*
Peirtnc PoUr w FlagaKi LilerW flag***
CtettndHiw H*tob*d*roid** H*to*nMiobium PiiMOCCut DvWyi HitonoKWi fl*wobecl*nwn Viboo Sfxmcnui
(B|
Figure 2.3 Dicotomic key for the identification at genus level of Gram-positive (A) and Gram-negative (B) moderatly halophilic heterotrophic eubacteria*
m From Ventoas (1988) CD 60
Table 2.4 Media & conditions for identification of spoilage yeasts*
Medium Purpose
Czapek agar Assessing ability to utilise nitrate as a sole nitrogen source
Malt Extract Agar (MEA) Colony morphology
MEA + 0.5% acetic acid Assessing preservative resistance
MEA at 37°C Assessing growth at elevated temperatures
Malt Yeast 50% Glucose Agar Growth at reduced Aw in the presence of high carbohydreate (MY50G) levels
Matt yeast 10% salt 12% Growth at reduced Aw in the presence of NaCI glucose Agar (MY 10-t2>
From Pitt & Hocking (1985) Incubation is at 25°C unless specified. Inspection should be at 3 and 7 days after incubation. 61
Table 2.5 Slient properties of yeasts*
Species Ceil Colon. Color CZ 37°C MEA+ MY MY length size MEA 0.5% 50G 10 acetic
B. intermedius 4.5-7 1.5-2 white 0 ♦ 0 w 0
C. k nisei 3-25 5-8 white w + + 0 0
D. hansenu 2.5-4 2.5-4 white w 0 0 w +
K. apiculata 3.5-6 2-4 white + / - 0 0 0 0
P. membranae. 4-6 3-4 white w v w +■ 0 0
R. qlutinis 4.5-5 5-10 red + / - 0 0 0 0
$. bail it 5-8 2-3 white 0 0 + + 0
S. bisporus 3.5-7 2-3 white 0 0 0 + 0
S. cerevtsiaa 5-12 2.5-4 white w + w 0 0
S. rouxii 5-7 2-3 white 0 0 0 + +
Sch. p o m b e 5-7 1.5-2 white w + + w 0
7. holmii 4 -5 1-2 white 0 0 0 0 0
From > 7 t7 I T Hocking ~(T985) w weak vw very weak + >1mm diam in 7 days 62
Media preparation
The Trypticase Soy Broth (BBL) supplemented with NaCl was used as the basat medium. Filter sterilized ethanol was
added to the autoclaved basal medium to achieve final concentrations of 10% NaCl and various amount (0, 3, 6 and 9%) of ethanol.
Inoculation
Individually, colonies of each strain ere taken from a 2 to
5 day old culture on maintenance agar, and were suspended in
sterile 5% NaCl saline solution. After the cell population was estimated by direct microscope count, a proper amount of the suspension was inoculated to each 60 ml of growth medium (in screw capped 200ml glass bottles) to reach a final concentration of 104 to 105 cfu/ml. This inoculation size was
later confirmed by plate count.
G row th
Cells were grown in bottles inclined in a 30°C incubator with shaking. Growth was estimated turbidimetrically at 660nm by use of a spectrophotometer (Spectronic 20, Bausch &
Lomb) against an uninoculated blank. 63
Statistical Analysas
The Spearman Rank Correlation Coefficient was used for the analysis of correlation of data (Neter et al., 1989).
Statistical significance was accepted if the probability value was less than or equal to 0.05 for the correlation. 6 4 2.4 RESULTS
Isolation and Observation Halophilic microorganisms were isolated from nine out of ten brands of commercial sufu with various brine concentrations
(10 to 23% of NaCl, as previously determined). The levels of moderate halophiles in these products are presented in Table 2.6
and Figure 2.4. A strong positive correlation was found between
aerobic moderate halophilic counts and anaerobic halophilic
counts (r=0.794, P=0.006). The presence of sesame oil in the
product was read from the label or visually detected, and recorded. Sesame oil was found in five of the ten brands of sufu collected for this study. Apparently, the presence of sesame oil
was not correlated with the presence and the level of either total
moderate halophiles or any group of isolates that had similar
morphological characteristic.
Salt Response of Isolates Growth response of a total of 32 isolates in media of different salt concentrations were examined. The range of salt concentration allowing growth is described in Table 2.7. most isolates (81%) appeared to be typical moderate halophiles
Optimal growth of these moderate halophiles was occurred at NaCl concentrations between 5 and 10%. 65
Table 2.6 Population of moderate halophiles in commercial sufu
B rand Moderate Halophiles(logio cfu/g)
Aerobic Counts AnaerobicCounts Mean1 SD2 Mean SD
A* 4.40 2.62 4.40 1.48 B* 5.48 0.00 5.53 0.11 O 5.80 1.72 5.49 2.46 D 6.32 0.00 6.36 0.00 E 5.85 0.00 6.86 0.00 F 3.13 1.86 3.20 1.81 G* 0.00 0.00 0.00 0.00 H 2.01 0.07 1.90 0.16 I* 6.40 0.00 6.36 0.00 J 6.01 0.00 3.78 0.00
Sample containing sesame oil Data represent average of two trails of tests on single or replicate samples Standard deviation of aerobic or anaerobic counts (log cfu/g) iue . Mdrt hlpie i te omril sufu commercial the in halophiles Moderate 2.4 Figure Moderate Halophiles (log cfu/g) 0 4H 2-\ 6 1 - - A B C D E F G HJ I II II II I Vs V | 1 T 1 I T T I I I I I I and n ra B I I V V II h | t i r i 1 lii In | E9 Anaerobic Coui Anaerobic E9 ■ Aerobic Count Aerobic 66
67
Table 2.7 Salt response of moderate halophilies isolated from sufu
Isolate** Sait content (%) required for growth Minium Optimuin Maximum
0 1 - A 0.3 2.0-5.0 1*0 02-A * 0.5 2.0-5.0 .*5.0
02 - A 0.5 5.0 20.0 04-A* 0.5 5.0 20.0
05-B 0.5 5.0-10.0 20. 06-B* 0.5 5.0-10.0 20.0
07-B 0.5 10.0 20 0 0S-B* 0.5 10.0 20.0
09-C 0.5 5.0 20.0 10*C* 0.5 5.0 20.0
I I'D 0.5 5.0 20.0 12-D* 0.5 5.0 20.0
1 3-D 0.5 10.0 20.0 14-D* 0.5 I 0.0 20.0
I 5-E 0.5 SO 20.0 16-E* 0.5 5.0 20.0
1 7-F 0.5 2.0 15.0 U-F* 0.5 2.0-5.0 15.0
19-F 0.3 10.0 20.0 I0-F* 0.5 10.0 20.0
2I-H 5.0 10.0 20.0 22-H* 3.0 10.0 20.0
23-H 0.J 2.0 15.0 34.H* 0.5 2.0 15.0
23-1 0.5 5.0 20.0 26-1* 0.5 5.0 20.0
27-1 0.5 2.0 15 0 2«-l* 0,5 2.0 15.0
29-1 0.5 3.0 25,0 30-1- 0.3 5.0 25.0
3 l-J 0.5 10.0 25.0 32-J’ 0.3 5.0-10.0 23.0
* isolates (01 to 32) were isolates from sufu samples (A to -1). On the same plate, a similar colony was isolated. That means 02-A* is the duplicated isolation of 01 - A 68 Characteristics of Bacterial Isolates Morphological and biochemical character of these isolates were examined and are presented in Table 2.8 and 2.9. The results of these tests were analyzed by the identification keys of the Bergey's Manual of Determinative Bacteriology (1994) and Ventosa (1988). Most isolates (69% of total) were identified as Gram- postive cocci and belong to genus Pediococcus or Micrococcus. The cocci were present in all brands of samples that had moderate halophiles. A total of 25% of the isolates appeared to be Gram- negative rods and belonged to the genus of Vibro o r Flavobacterium. No halophilic sporeformer was found in levels higher than 102 cfu/g in sufu. Some gray colonies of strict anaerobic halophiles were also found in this study, and they are exclusively straight rods when observed under microscope. These cultures (sporeformers and strict anaerobes) together with some other moderate halophiles were not identified, because their populations in sufu were not greater than 1Q2 cfu/g. High populations of moderate halophiles {=> 10s cfu/g) were found in six brands of sufu and they were exclusively Gram-positive nonsporeforming cocci. The predominate halophiles isolated from five of this six brands of sufu (Brand B, C, D, E & I) were further identified as Pediococcus haiophilus (Table 2.10). Other isolates were not identified to genus level, because their occurance were not as abundant or frequent. Based on statistic analyses, the level of P. haiophilus in different brands of sufu was correlated with 69
Table 2.8 Differential characteristics of Gram (+) halophilic iso la te s
Isolates 0 1 -A. 02-A. 17-F, 1B-F, 03-A, 04-A, 05-8. 06-B, 23-H, 24-H. 27-1, 28-1. 09-C, 10-C, 11-D. 12-D, 3 1 -J. 32-J 15-E, 16-E. 25-1, 26-1 .
Cell Cocci (Clusters, tetrads) Cocci (Tetrads, pairs) Catalase + -
Oxidase + +
Spore-formed - -
M o tility - -
Aerobic grwoth + +
Anaerobic growth - +
Genus* Micrococcus Pediococcus
* Isolates were identified to genus levels
Table 2 . 9 Differential characteristics of Gram (-) halophilic is o la te s
Isolates 07-8, 0S-B, 13-0, 14-D. 21 -H. 22-H 19-F.20-F
Cell Curved rods Rods Pigmentation White to cream Yellow Catalase + + Oxidase Spore-formed *- Aerobic grwoth + Anaerobica growth ■
Genus* Vibro Fla vobacterium.
* Isolates were identified to genus levels 70
Table 2.10 Characteristics of Pediococcus isolates as compared to those of Pediococcus haiophilus {ATCC 33315)
Characteristic os-a. oa-a*. os-b. os-b*. oe-c. ATCC33315 10-C *. 11-D, 1 2 -D \ 25-1, 26*1*
Morphological studies Colony diameter (mm) <0.5mm <0.5 mm color Clear to gray Clear to gray shape Round, slight covex Round, slight covex Gram stain + + Cell shape cocci (singles, cocci (singles, pairs, tetrads) pairs, tetrads) Cell diameter (pm) 0 . 6- 0.8 0 . 6- 0.8 Endospore M o tility Physiological tests Oxidase Catalase Aerobic growth Anaerobic growth Growth at 10°C 25°C 45°C Growth at pH 4.5 pH 5.5 pH 6.5 Growth with 0.5% NaCl 20% NaCl 25% NaCl Acid production From Fructose Galactose Glucose Lactose Maltose Mannitol Mannose Raffinose Sorbitol (•>2 Surcose (•+■)3 Xylose
Positive reaction for 05-B, 06*B, 09-C, 10-C, Positive reaction lor 09-C, 10-C,11-D, 12-D, 25-I. 26-I. Negative reaction lor 03-A, 04-A. 71
both aerobic moderate halophiles (r-0.769, P-0.009) and anaerobic moderate halophiles (r-0.907, P-0.000). Figure 2.5 shows the
differences between levels of these counts. In addition, the relationship was determined between brine composition and the predominate halophiles in commercial sufu (Figure 2.6). A very strong negative correlation was found between the levels of P. haiophilus and concentrations of ethanol0/© (r—0.957, P-0.0005).
Characteristics of Fungal Isolates Only low levels (-103 cfu/g) of halophilic fungi were found in two of all samples. These fungi were able to grow in both of the media used for the isolation of fungi. Under the microscope, both isolates were yeastlike. The results of morphological and physiological tests are summarized in Table 2.11. According to the identification keys described by Pitt & Hocking (1985), the two isolates resemble the xerophilic yeast Saccharomyces rouxii.
Ethanol Tolerance of Haloterant and Halophilic Organisms To understand the ethanol tolerance of moderate halophilic and halotolerant cocci, the growth responses of seven cultures were examined in media containing various amounts of ethanol. The result (Figure 2.7) shows that P. haiophilus exhibited a high tolerance, and growth of this cocci was found in medium containing 6% ethanol. iue . Te eainhp ewe P hipiu & moderate & haiophilus P. between relationship The 2.5 Figure
Log (cfu/g) - f 0-“ 2 - 3 - 4 1 - 5 8 7 - 7 - - ABCDEFGHIJ aohls n omril sufu commercial in halophiles Brands Anaerobic M.H Anaerobic Aerobic M.H Aerobic . haiophilus P.
72
iue . Rltosi bten . aohls ehnl in ethanol & haiophilus P. between Relationship 2.6 Figure a haiophilus (log cfu/g) - 4 5- O omrcl sufu commerical E C ands d n ra B B F A J P.haiophilus G H - 15 - -20 25 -2 30
Ethanol% 73 74
Table 2.11 Characteristics of yeast isolates as compared to those Of Saccharomyces rouxi (NRRL Y2547)
Ctaa racteris tics# Isolates {29-1. 30-1*) NRRL Y2547
Morphological studies (on Melt Yeast Agar) Colony diameter (mm) 2-3mm on MEA 2-3mm on MEA color White White shape Round Round Cell diameter (jxm) 4 - 7 4 - 7 shape Spheroidal, Spheroidal, multipolar budding multipolar budding
Physiological tests Growth on: Malt Yeast 50% Glucose Agar 4* Malt Yeast 10% Salt 12% Glucose Agar +
# Incubation is at 25°C. Inspection should be at 3 and 7 days after incubation. + 1mm diam or more in 7 days. Ethanol (%)
Figure 2.7 Ethanol tolerance of moderate halophilic cocci 76 2.5 DISCUSSION
Enumeration and Isolation
Halophilic microorganisms were isolated for the first time from commercial sufu. The strong positive correlation found in between aerobic and anaerobic moderate halophiles indicates that facultative microorganisms, most likely, are dominant in these products. This was evidenced by the high levels (>105 cfu/g) of these moderate halophiles whichwere found in 44% of all samples tested. This provided the basis for study on the characteristics of these halophiles and later investigation (Chapter 4) on their
involvements in sufu fermentation.
Characteristic of the Isolates
In this investigation, morphological and physiological
characteristic of these isolates were examined. The majority of the isolates were identified as Gram-positive cocci. This is not a surprise, since the resistance of Gram-positive bacteria to
ethanol is relatively higher than that of Gram-negative bacteria {Seiler & Russell. 1991). Also, most if not all, Gram-postive moderate halophiles are cocci (Ventosa, 1988; Ramos-
Cormenzana, 1993). These Gram-positive cocci were identified as either Pediococcus or Micrococcus. Eight isolates appeared to be Gram-negative rods, and they are Vibro and Ffavobacterium. The two rods were found in levels lower than 104 cfu/g in sufu. 77 None of the halophilic organisms isolated from this study were
considered to be a human pathogen. Cocci ( Pediococcus and Micrococcus) found in this study are common in many fermented foods. The most abundant moderate halophile found in the commercial sufu was identified as Pediococcus haiophilus. an important bacterium for soy sauce and miso fermentation. The
differences in the results of some biochemical tests, obtained with the predominate isolates we studied, compared with the
ATCC culture, may be due to unstable properties of the organisms. Since the level of micrococci was generally low in the commercial sufu, the identification work on those isolates was not pursued. Yeasts were only found in two samples. According to the
identification keys described by Pitt and Hocking (1985), this
isolates resemble the xerophilic yeast Saccharomyces rouxii. This yeast is considered as the most osmo-tolerant yeast, and
responsible for many food (such as dried fruits and juice concentrates) spoilage outbreaks (Splittstoesser, 1991). Nevertheless, Sacch. rouxii is well reconganized as a desirable microorganism for miso and soy sauce fermentation (Reddy
&Safunkhe, 1989). In order to confirm the identification results of this study, more biochemical tests would need to be conducted. 78 Factors Associated with P. haiophilus A much stronger correlation was found in between the
levels of P. haiophilus and anaerobic moderate halophiles (r=0.907), when it was compared with the correlation in between P. haiophilus and aerobic moderate halophiles (r-0.769). This
result suggests that P. haiophilus. a facultative anaerobe, is even
more predominate among moderate halophilic anaerobes. A strong
negative correlation was found in between the levels of P.
h a io p h ilu s in sufu and the ethanol content of brine. This
indicates that the amount of ethanol in the brine, instead of salt, was the major factor that determines the growth and survival of
P. haiophilus in the commercial sufu. This information may be useful for discerning the proper amount of ethanol needed for
sufu manufacture.
Moderate Halophiles in Sufu Fermentation
This study has shown that the predominate halophilic
bacteria isolated from commercial sufu, made in various countries, are similar to the flora involved in soy sauce or miso
fermentation reported from Japan and Malaysia (Yokotsuka, 1991;
Ho et al., 1984). This similarity is most likely determined by the
high salt content and fermentation substrates of these products.
In miso fermentation, the primary role of P. haiophilus. a lactic
acid producer, is to lower mash pH to below 5.0, thereby stimulating the growth of Sacch. rouxii which could produce 79
ethanol and other flavor compounds (Yokotsuka, 1991). In sufu,
however, the role of P. haiophilus is not clear. More study is
needed to understand if the involvement of P. haiophilus is desirable.
The predominance of P. haiophilus in these products may be explained by 1) its tolerance to high salt content; 2) its flexibility of growing aerobically and anaerobically; 3) its ability to produce inhibition factors, such as organic acids, against the growth of others; 4) its adaptability to a wide pH range of growth medium, and 5) its resistance to low levels of ethanol. The relatively high ethanol tolerance of P. haiophilus was also demonstrated in this study. This result agrees with Seiler and Russell (1991).
G ro w th of Sacch. rouxii is an important part of fermentation process for many oriental fermented soybean products. High levels ofSacch. rouxii is usually found at the later stage of soy sauce or miso fermentation (Reddy &Salunkhe, 1989;
Yokotsuka, 1991). In this study, however, no significant levels of
S acch , ro u xii were found in commercial sufu tested. This difference is possibly related to the oxygen availability of these products. In both soy sauce and miso fermentation, aeration procedures are required to maintain aerobic condition for the growth of this yeast (Yokotsuka, 1991). Contradictorily, not only are aeration procedures not involved in sufu fermentation, many sufu manufactures add sesame oil in sufu brine, and age the 80 product in airtight bottles. The package and the thin layer of oil
suspended on the top of the brine, may serve as a barrier against
oxygen migration. Thereby, the available oxygen in the brine
decreases while the populations of halophilic bacteria increase. In the oxygen diminished environment, the growth of aerobic
organisms is less likely. This may explain why little or no halophilic fungi were found in this study. Since P. haiophilus is
facultative anaerobic, highly tolerant to salt and ethanol, the finding of this microorganism in high populations in commercial
sufu is reasonable.
In conclusion, various kinds of moderate halophiles were found in commercial sufu. The presence and the level of the
predominate organism, P. haiophilus, was directly associated with the content of ethanol in brine. While the roles of these moderate halophiles remain unclear, their involvement in the fermentation is certain. Since no moderate halophile has been reconginized as human pathogen, the presence of these moderate halophiles in sufu should not raise a safety issue, but rather a quality concern. It is worthwhile to point out that while the common halophilic microorganisms presented in commercial sufu product have been isolated and characterized in this study A much richer microflora may exist during earlier stages of the sufu fermentation, and this remains to be investigated. 81 2.6 CONCLUSIONS
Halophilic microorganisms were isolated from commercial
sufu (fermented tofu) with various brine compositions. A total of 32 isolates were characterized and identified to the genus level. The range of salt concentration allowing growth was determined for each isolate, and 81% of the isolates appeared to
be typical moderate halophiles. Most isolates (69% of total) were identified as Gram-positive cocci and belong to genus Pediococcus or Micrococcus. In addition, 25% of isolates
appeared to be Gram-positive rods and belong to the genus Vibro or Flavobacterium. High populations of moderate halophiles (>10s cfu/g) were found in seven of ten brands of the samples.
They were exclusively Gram-positive non-sporeforming cocci.
The predominate halophile isolated from six of seven brands of samples was further identified as Pediococcus haiophilus. In addition, a strong negative correlation was found between the levels of P. haiophilus in sufu and the ethanol content of the brine (r*-0.957, P=0.000). Finally, low levels of halophilic yeast were found in a few samples. The presence and the levels of these halophiles in the product can be reasoned by the composition of sufu brine as well as the process involved in sufu fermentation.
Results of this study suggest that moderately halophiles. such as P. haiophilus, may play an important role during the 82 salting and aging process of sufu. Brine composition, especially ethanol%, appears to influence the growth of halophiles in sufu. Although some of the common halophilic microorganisms presented in commercial sufu product have been isolated and characterized in this study, a much richer microflora may exist during earlier stages of the sufu fermentation, and remains to be investigated. CHAPTER III
COMBINATION EFFECTS OF ETHANOL & SODIUM CHLORIDE ON THE GROWTH & SURVIVAL OF HALOTOLERANT & MODERATELY HALOPHILIC COCCI
3.1 LITERATURE REVIEW
Effects of Ethanol on Microorganisms
Ethanol is a ubiquitous small molecule that may be produced chemically as a part of petroleum utilization, or biochemically as a product of microbial fermentation It has long been employed both as a food preservative, or as a disinfectant against the growth and survival of microorganisms.
The use of ethanol as an antimicrobial agent m food had been summenzed in the Chapter 2. Therefore, the following review is
83 8 4 solely to discuss research on the antimicrobial mode of action of ethanol.
Although there may well be distinctions in mode of action that are related to the amount of ethanol to which the spoilage organisms are exposed, it is believed that the basic actions of ethanol on both eukaryotic and prokaryotic organisms share the same general principles (Ingram & Buttke, 1984; Seiler &
Russell, 1991). According to the review article by Ingram & Buttke (1984), these actions appear to be dominated by the physicochemical properties of ethanol rather than involving a specific receptor. Hence, all hydrophobic and electrostatic interactions in the cytosolic and envelope components of cells can potentially be affected (Ingram & Buttke, 1984). The aspects of cellular metabolism which are affected by ethanol have been summerized and listed by Seiler & Russell (1991). The authors also concluded that the other effects of ethanol listed in Table 3.1 are consequent upon the disruption of normal membrane structure and function by ethanol. This means that the primary site of action was connected with the cell membrane, either through a direct effect on the membrane, or indirectly from im pairment of the biosynthesis of a membrane component (Seiler & Rusell, 1991). Therefore, to understand the antimicrobial properties of ethanol, all major interactions between ethanol and cell membrane should be noted. 85
Table 3.1 Aspects of cellular metabolism that are affected by e th a n o l*
Morphology Glycolytic enzymes Peptidoglytic biosynthesis DNA biosynthesis RNA biosynthesis Protein biuosynthesis Protein secretion Fatty acid biosynthesis Phospholipid biosunthesis Membrabe (possive) permeability Solute uptake/ion pumping systems
* This list (taken from Seiler & Russell. 1991) is a compilation and not all the effects have necessarily been observed in every microorgnaims studied. 8 6
The first thing which needs to be mentioned is the fact
that the toxicity of alcohols is directly related to their chain length and hydrophobicity. Ethanol, a short chain hydrocarbon, is very polar and partitions poorly into the membrane. In contrast (Figure 3.1), the hydrocarbon tail of hexanol favors its concentration within the membrane. According to Ingram &
Buttke (1984), longer-chain alcohols, up to a chain length of around 10 carbon atoms, are much more potent inhibitors than are the shorter-chain alcohols. However, longer fatty alcohols
(over 10 carbon atoms in length) are relatively ineffective as inhibitors. For organisms such as E. coli, the growth toxicity roughly doubles with each additional aliphatic carbon atom up to
octanol (Ingram & Buttke, 1984). Another important interaction is the ethanol-induced
changes in membrane lipid composition. According to studies
with Lactobacillus heterohiochii (Uchida, 1975a & 1975b),
Escherichia coli (Ingram, 1976), Tetrahymena pyrifirmis (Nandini-kinshore et a!.. 1979), Mycobacterium smegmatis (Taneja & Khuller, 1980), and a yeast, Saccharomyces cerevisiae
(Beaven et a)., 1982), clearly lipid composition of cell membrane
can be significantly influenced by the presence of ethanol in
their growth medium. Since the early study by Uchida (1975a), this adaptive response is considered as a possible biochemical basis for tolerance to alcohol. In general, an increase in alcohol concentration resulted in a significant increase in membrane 87
4 ^ 4
Hexanol Ethanol
Figure 3.1 Model showing interactions of hexanol & ethanol with a membrane
From Ingram & Buttke (1984) 8 8
lipid unsaturation. According to Casey & Ingledew (1986) and
Carey & Ingram (1983), the functionally important part of the adaptative changes to the presence of ethanol was a tendency towards increased chain length (although in favor of
unsaturates), which would offset the disruptive effects of ethanol. By this view, the actual net effect of ethanol on plasma membranes is a decrease in membrane integrity (i.e., physical disruption of membrane integrity and structure).
Recently, Seiler & Russell (1991) explained the ethanol- induced fatty acid changes in terms of the "Lipid shape theory".
According to this theory, the balance of size and shape of the head-group and the fatty acyl chains determine wether it is a bilayer (lamellar) or non-bilayer (hexagonal) phase. By partitioning in the surface of the membrane, the ethanol would alter the balance of head-group and acyl chain sizes. This change would favor the formation of a non-bilayer hexagonal phase, which would disrupt normal membrane packing and cause gross leakage ions and other solutes. However, this effect could be counteracted by an alteration in the acyl chains.
Microbial Response to Sodium Chloride
Sodium chloride, commonly termed (table) salt, has been used since ancient time to flavor and preserves a variety of meat, fish, and vegetables. The preservation function of salt is mainly based on the fact that salt lowers the water activity 89
(Aw) of food as described earlier (Chapter 2). In this chapter,
the discuss will focus on the mechanisms of halotolerance in
microorganisms.
The basic mechanisms of microbial response to low water
activity environment had been summarized (Table 3.2) by Leistner & Russell (1991). According to the authors, the
accumulation of intracellular-compatible solutions is the most
important response among all others. Most common food solutes (such as sodium chloride,
sucrose, glucose) do not readily penetrate the cell membrane
(Gould, 1989). When the extracellular food solute concentration
is highly increased, the cell membrane will shrink from the wall
due to passive flow of the water. Such cells cannot remain viable for long, because proper turgor pressure is probably
needed for wall growth and thus cell division (Leistner &
Russell, 1991). However, all organisms tolerate some levels of
reduction in Aw, and some of them have very effective
mechanisms for water regain. The basic water regain
mechanism operated by cells that are able to overcome the
initial loss involves raising the quantities of cytoplasmic solute(s), and thus encourage the flow of water back into the
cell (Gould, 1989). Whether these solutes are synthesized
intracellularly or taken up from the extracellular medium, they
must not prevent the proper functioning of cytoplasmic enzymes.
That's why they are commonly described as "compatible solutes". 9 0
Table 3.2 Response of microorganisms to low Aw thought to be part of osmo-regulation**
Response Function
Accumulation of compatible solutes Maintenance of cellular water relations
Decreased content of MOO*# Osmotic control of periplasm
Changed outer membrane protein Regulation of ionVsolute permeability composition#
Altered membrean lipid composition Maintenance of lipid bilayer phase
From Leistner & Rusetl (1991J Membrane-derived oligosaccharides # Only in Gram-negative bacteria 91
The major types of compatible solutes accumulated by different groups of microorganisms is listed in Table 3.3. Another important halotolerant mechanism is that the cell membrane lipid changes in response to low Aw. According to
Vreeland (1987), the bacteria studied so far have all tended to alter their phospholipid patterns in favor of more negatively charged species when adapted to higher levels of NaCI. It has been suggested that this change in charge might be useful in preventing the formation of non-bilayer lipid phases. According to Leistner & Russell (1991), it is hypothesized that preservative solutes exert their antimicrobial action at least partly by their action in disrupting membranes through the formation of non-bilayer lipid phases. Therefore, those microorganisms that are able to grow in conditions of low Aw must have some mechanism for preventing the formation of non bilayer phases. This mechanism involves altering membrane lipid composition by the increase in proportion of anionic lipid(s) (Leistner & Russell, 1991). Moreover, changes in the hydrophobic-hydrophilic cell surface character of cells in response to NaCI had been demonstrated by Hart & Vreeland
(1988). The results suggest that when Halomonas efongata (a moderate halophiles) adapts to different NaCI concentrations, it alters the affinity of its outermost cell surface to water (Figure 3.2). 92
Table 3.3 The major types of compatible solutes accumulated different group of micro-organisms*
Microorganism Compatible solutes
Eubacteria K+, amino aids#
Yeast & fungi (moulds) K+, polyols##
From Leistner & Rusell (1991) # Mainly proline and betaines, together with glutamate, glutamine. T- amionbutyrate and alanine, in different bacterial species. # # Including glucerol, trehalose, arabitol and mannitol in different species. 9 3
100
o x> 60
■£ 40
0 0.05 0.37 1.37 2.0 2.5 3.4 NaCI (M)
Figure 3. 2 Differences in the surface hydrophobicity of log-phase cells of H. elongate after growth in various NaCI conc.*
From Hart & Vreeland (1988) 94
Combination Effects of Preservatives Clearly the use of anitmicrobial agents in combination can be advantageous, principally by allowing the less extreme use of any single treatment, with consequent improvement in product quality, or sometimes with the identification of new product
opportunities (Gould & Jones, 1989). Generally speaking, when
two antimicrobials are used in combination, three results (additive, synergism, or antagonism) may occur. The definitions of these terms (additive, synergism, or antagonism) has been given by Barry (1976) and described as follows:
(1) Additive effect occurs when the effect observed with the
combination is equal to the sum of the effects observed with the two compounds tested separately, or equal to that of the most active compound in the combination.
(2) Synergistic effect occurs when the effect observed with a combination is greater than the sum of the effects observed with the two compounds independently.
(3) Antagonistic effect occurs when the combination is less effective than the most active drug in the combination.
While combinations of antimicrobials are commonly used in the food industry, research on the effectiveness of such combinations is quite limited. Therefore, interactions among many commonly used antimicrobial agents are poorly understood. More studies need to be done in this area in order to provide a 95 better understanding. The combination effects of two
antimicrobial agents can be studied by various methods (such as agar diffusion techniques, broth dilution techniques, and comparison of killing rates), and have been discussed by Barry (1976) and Davidson & Parish (1989). Figure 3.3, 3.4 & 3.5 shows
that the possible results of these tests.
Combination Effects of Ethanol and NaCI
Ethanol and sodium chloride are commonly used as food preservatives against spoilage or toxin production by undesirable microorganisms. However, the combination effect of this two preservative is not well understood. According the study by
Shapero et al. (1978), in foods with reduced Aw, low concentrations of ethanol have a specific anitmicrobial effect. The authors further stated that as the Aw is lowered, the amount
of ethanol required to have a complete inhibitory effect on growth of S. aureus is decreased, showing synergism, as has been
found also with glycols and potasium sorbate. Since only one
organism (S. aureus) was tested under a single incubation temperature (37°C), the usefulness of the results is rather limited. In our earlier studies (Chapter 1 & 2), the findings of
moderate halophiles in commercial sufu, and its correlation with
brine composition was discussed. Results of these studies indicate that some halophiles seem to be more resistant to sufu brine, and frequently exist in the commercial sufu. Therefore, to 96
Additive Synergism Antagonism
Figure 3.3 Three types of bateriostatic results with combinations of antimicrobial agents tested by the agar diffusion technique*
From Barry (1976) iue . Tre ye o rsls o te iia inhibitory minimal the (on results of types Three 3.4 Figure
Antimicrobic B (jag/mt) rm .. opih (d) 1972. (Ed.). Hoeprich, P.D. From s d Hre Rw n. Hgrtw, d, . 203. p. Md., Hagerstown, Inc., Row & Harper 1sted. ocnrto) ih obntos f antimicrobics* of combinations with concentration) niirbc (fig/mll A Antimicrobic Additive Synergism Antagonism netos diseases, infectious
97 Figure 3.5 Rate of killing with antimicrobial agents singly and and singly agents antimicrobial with killing of Rate 3.5 Figure
Log (cfu/ml) 4 1 4 12 8 4 0 12 8 4 0 Drugs A+B Drugs * From A.L. Barry & L.D. Sabath. 1974. Manual of Clinical Clinical of Manual 1974. Sabath. L.D. & Barry A.L. From * ahntn DC, . 434. p. D.C., Washington, n combination* in irbooy 2 E. ., ente EH Salig J.P. & Spaulding E.H. Lennette, E.H, Ed. 2d Microbiology. Truant (Eds ). Amercian Society for Microbiology, Microbiology, for Society Amercian ). (Eds Truant No drug No Drug B Drug Drag A Drag or o Incunation of Hours rg C+D Drugs rg D Drug No drug No Drag C Drag 4 8 4 0 rg E-t-F Drugs rg F Drug rg E Drag No drug No 12 98 99 gain a better understanding on the preservative effect of alcoholic brine, more study is needed on the combination effects of alcoholic saline solution on the growth and survival of halotolerants and moderate halophiles. 3.2 PLAN OF STUDY 100
This study was directed to investigate the combination effects of salt and ethanol on the growth and survival of moderate halophilic and halotolerant cocci. 101
3.3 MATERIALS AND METHODS
C u ltu re s
Cultures of Marinococcus halophilus (ATCC 27964),
Micrococcus haiobius (ATCC 21727), Paracoccus
haiodenitrificans (ATCC 13511), Pediococcus hafophilus (ATCC
33315), and Staphylococcus aureus (R245) (a culture obtained from the Silliker Laboratories of Ohio, Inc.) were used as test organisms. These cultures were maintained on agar slants with
10% marine salts as described earlier (Chapter 2). Among the test organisms, Marinococcus halophilus, Micrococcus haiobius, a n d Paracoccus haiodenitrificans are typical moderate halophilic organisms; whereas Pediococcus halophilus is considered either as a halotolerant or as a moderate halophile.
In addition, Staphylococcus aureus is perhaps the best known halotolerant bacteria.
Media Preparation
Trypticase Soy Broth (BBL) with supplemented with variuos concentrations of NaCI was used as basal medium.
Filter sterilized ethanol was added to autoclaved basal medium to achieve the desired final concentrations of NaCI and ethanol Individually, colonies of each strain are taken from a 2 to 5 day old culture on maintenance agar, and were suspended in sterile 5% NaCI saline solution. After the cell population was estimated by direct microscope count, a proper amount of the suspension was inoculated to each 60 ml of growth medium (in screw capped 200 ml glass bottles) to give a final concentration of 104 to 105 cfu/ml. This inoculation size was later confirmed by plate counts. After inoculation, cells were grown in bottles inclined in a 30°C incubator with shaking. Appromixmatly 5 ml of culture was removed from the bottles for the measurement of growth, at each designated time. Growth was estimated turbidimetrically at 660nm by use of a spectrophotometer (Spectronic 20, Bausch & Lomb) against uninoculated blanks.
Survival Assay To determine the effects of various combinations of ethanol and salt on survival, colonies of S. aureus and P. halophilus were taken from a 2 and 5 day old culture, respectively, and inoculated to TSB with various amounts of NaCI and allowed to grow for 2 and 4 days (respectively). After total cell population of each culture was estimated by direct microscope count, these cultures were adjusted with original TSB NaCI medium to the desired cell populations (about 106 cell/ml). Individually, portions (0.1 ml) of these fresh cultures were then added to sterilized test tubes containing 9.9 ml of TSB supplemented with proper amounts of salt and ethanol. Incubation was continued for 3 days at 22°C, and 0.2 ml of cultures 103 were removed each time for celt population measurement by plate count methods as described in Chapter one.
Data Interpolation
Growth was not considered as significant unless an absorbance reading at 660nm was greater than 0.1. A significant delayed growth was defined as the occurance of growth only after 10 days of incubation. A significant inhibition effect was recorded either if no significant growth was observed at all, or if a significant delayed growth was observed.
For the survival assay, a significant killing effect was recorded if a minimum of 2 logio (cfu/ml) reduction was found within 72 houres of incubation. The difference between two survival responses was analyzed by Paired T-test (Velleman, 1989).
Statistical significance was accepted if the probability value was less than or equal to 0.05 for the difference. 104 3.4 RESULTS
The effects of salt and ethanol on the growth and survival of halotolerants and moderate halophiles were investigated. Five different cocci were used as test organisms in this study. All cultures, except Paracoccus haiodenitrificans, selected for this study were Gram-positive cocci, since they represent the most ethanol resistant group (Chapter 2). Growth assays were
conducted to all five test organisms; whereas survival assays were applied to two organisms that shown greater ethanol tolerance, based on the results of the growth assays. Data obtained from this study are presented in detail in Tables 3.4 to
3.12 of the Additional Data.
Effects of Salt on Growth
Growth responses of five cultures in Trypticase Soy Broth
(TSB) with various amounts of salt were examined. The results are presented in Figure 3.6 to 3.10. Data in Figure 3.6, 3.7 and 3.8 show that Marinococcus halophilus, Micrococcus haiobius, and Paracoccus haiodenitrificans shared a similar pattern of salt response. They all failed to grow in TSB, but were capable to grow in TSB supplemented with 5 to 20% of NaCI. Optimal growth of Marino, halophilus. Micro, haiobius, and Para, haiodenitrificans occurred in TSB with 5%, 10%, and 5% of NaCI, respectively. 105
E c 0.8 <0o 0% NaCI * 0.6 - 5% NsCI 1 0% NaCI 15% NaCI 20% NaCI 0.2-
0.0 0 5 10 15 20 2 5 30 35 Day a Figure 3.6 Effect of salt on the growth of Marinococcus halophilus 1.4 - ce 1.2- (0o 1.0- «0 0% NaCI - 0.8 - 5% NaCI o 0.6- 10% NaCI o 15% NaCI 0.4 - j= 20% NaCI o* 0.2- O 0.0 ■* 0 5 10 15 20 25 30 35 Days Figure 3.7 Effect of salt on the growth of Micrococcus haiobius 1.2
0.8- 0% NaCI 5% NaCI 0 6 - a T 0% NaCI 15% NaCI 0 0.4 -C 20% NaCI 1o 0,2
0.0 Oi ..I 11 o . | M 0 S 10 is 20 25 30 35 Days Figure 3.8 Effect of salt on the growth of Para, haiodenitrificans 106
■o— o% NaCI
-•------5 % N a C I -• ----- 10% N aCI ■*------15% NaCI ■m 20% NaCI
0 5 10 IS 20 25 30 35 D ays
Figure 3.9 Effect of salt on the growth of Pediococcus hatophtius
o 0.8 - to to -0- 0% NaCI 0.6 5% NaCI a 10% NaCI o 0.4 - 15% NaCI 20% NaCI * 0.2- o
3 0.0 0 5 10 15 20 25 30 35 Days Figure 3.10 Effect of salt on the growth of Staphyio. aureus 107 Both Pediococcus halophilus and Staphylococcus aureus w e re able to grow well in TSB (Figure 3.9 & 3.10). Optimal growth of
P. halophilus and S. aureus were observed in TSB with and without an addition of 5% NaCI, respectively. Notablely, a
significant delayed growth of P. halophilus was observed in TSB
supplemented with 20% NaCI. Moreover, unlike other cultures tested, S. aureus failed to grow in TSB supplemented with 20% of NaCI.
Effects of Ethanol on Growth
Growth responses of five cultures in TSB with various
amounts of ethanol were also examined. The results are presented in Figure 3.11 to 15. Clearly, there was a negative
impact of ethanol on both growth rates and maximum cell yields
of all organisms tested in this study, in general, the inhibition effects of ethanol increased whenever a higher concentration
was used in this study, until complete growth inhibition was
reached. Data in Figure 3.11, 3.12 & 3.13 indicate that
Marinococcus halophilus, Micrococcus haiobius, and P aracoccus haiodenitrificans were able to grow in TSB supplemented with optimal amounts of NaCI and 3% ethanol. However, they all failed to grow in TSB with an addition of equal to or more than
6% ethanol, under optimal salt conditions. In addition, the 108
E c 0 8
0.6 0% Ethanol O 2% Ethanol o' 0.4 6. 9 612% Ethanol
* e O 0.0 -V 0 5 1 0 1 5 2 0 ZS J O 3 5 D ays Figure 3.11 Effect of ethanol on the growth of Marino, halophilus
1.4 1.2 IS(0 0% Ethanol O.B - O 3% Ethanol o 0.6 6. 9 & 12%. Ethanol 0.4 - * o 0.2- O 0 5 10 15 20 25 30 35 D ays
Figure 3.12 Effect of ethanol on the growth of Micro, haiobius
1.0- o.e- C% Ethanol 0.6 - a 3% Ethanol 6 S. 9 6 12% Ethanol
* 0.2- blo C3 o.o h 1 ...... ■ 0 5 10 15 20 25 30 35 D a y *
Figure 3.13 Effect of ethanol on the growth of Para, haiodenitrificans 109 growth of Micrococcus haiobius in this medium was significantly
delayed. Both P. halophilus and S. aureus were able to grow in TSB with
6% ethanol and optimal amounts of NaCI (Figure 3.14 & 3.15). However, the growth of P. halophilus in this medium was
significantly delayed. In addition, with a delayed growth in TSB with 9% ethanol, S. aureus exhibited a superior adaptability to e th a n o l.
Combination Effects of Salt and Ethanol on Growth
The combination effects of NaCI and ethanol on the growth of all test organisms was also examined. Data in Figure 3.16 & 17 show that the growth of Marinococcus halophilus a n d Paracoccus haiodenitrificans were not significantly inhibited when 3% ethanol was used in combination with low level of salt (5% NaCI), or when high level of salt (20% NaCI) was added alone.
However, significant inhibition effects on M arino. halophilus an d
Para, haiodenitrificans occurred when 3% ethanol was added together with equal to or greater than 10 and 15% of NaCI, respectively. Data in Figure 18 shows that significant inhibition effects on the growth of Micrococcus haiobius were observed whenever 3% of ethanol were added. When this addition (3% of ethanol) was in combination with 5 or 10% of NaCI, delayed growth occurred. However, when it was in combination with 15 110
0.6
0.5 cE
0.4 - -o- a 0% Ethanol b 0.3 3% Ethanol o 6% Ethanol 0.2 9 6 12% Ethanol * o O 0.1
0.0 4 0 s 10 15 20 25 30 35 Days Figure 3.14 Effect of ethanol on the growth of Pedio. halophilus
0.8-
0% Ethanol 0.6 3% Ethanol C3 6% Ethanol 6 0 .4- 9% Ethanol * 12% Ethanol k_o o 0.2-
0-0 05 1 0 15 20 2S 30 35
Days Figure 3.15 Effect of salt on the growth of Staphylo. aureus 111
0.8
5% NaCI 10% NaCI 1 S% NaCI 0.4 20% NaCI o o' 5% NaCI A 3 % Ethanol 1 0 % NaCI A 3% Ethanol 0.2 - 15% NaCI A 3% Ethanol o* 20% NaCI A 3% Ethanol 0.0 0 S 10 15 20 25 30 35 Days Figure 3.16 Combination effects on growth of Marino, halophifus
E c 1.0 5% NaCI 10% NaCI 0.8- 15% NaCI 0.6 20% NaCI o 5% NaCI A 3 % Ethanol 0 0.4 10% NaCI A 3% Eihanol 1 15% NaCI A 3% Ethanol o 0.2 20% NaCI A 3% Eihanol tf 0.0 H 0 5 10 15 20 25 30 35 Days Figure 3.17 Combination effect on growth of Micro, hafobius 1.2
1.0 5% NaCI 0.8 10% NaCI 15% NaCI 0.6- 20% NaCI o b 5% NaCI A 3% Ethanol 0 4 10% NaCI A 3% Ethanol * 15% NaCI A 3% Ethanol o 0.2 20% NaCI A 3% Ethanol 0.0 0 5 10 15 20 25 30 35 Days Figure 3.18 Combination effect on growth of Para, ha/odemtnficans 112
or 20% of NaCI, no significant growth was detected within 30 days of incubation.
Data in Figure 19 & 20 indicate that significant inhibition
effects on the growth of P. halophilus occurred when 3 and 6% of
ethanol were added in combination with equal to or more then 15
and 10% of NaCI, respectively. Notably no significant inhibition
occurred when these levels of ethanol and NaCI were used alone, except a delayed growth was observed when 20% NaCI was added. No growth in 30 days of incubation was found when 6% ethanol
was used in combination with equal to or more then 10% of NaCI. In addition, a delayed growth was further inhibited when 3% of
ethanol was included in TSB with 20% NaCI.
No significant growth of S. aureus was observed when 3, 6 and 9% of ethanol were added in combination with equal to or
more than 15, 10 and 5% of NaCI, respectively (Figure 21, 22 & 23). Moreover, no significant inhibition effect was noted when
these levels of ethanol and NaCI were used alone, except a delayed growth was observed when 9% ethanol was added. In addition, a delayed growth was found when 6% of ethanol was included in TSB with 5% NaCI.
Independent Effects of Salt & Ethanol on Survival
Effects of salt and ethanol on the survival of P. halophilus a n d S. aureus (two relatively high ethanol tolerant organisms) were examined. Before the survival tests, each of the organisms iue 3. Figure
Growth (O.D. at 660 nm) Gorwth (O.D. at 660 nm) Grewth (0 D „ #M nwj Growth (O.D. at 660 nm) 19 G rowth of of rowth G 19 - M f ■ f i M 0-0M 0.2- 0.3 0.4 0.5 0.0 0.1 02 0.3 0.4 0.5 0.0 o.t 0.2 0.3 0.4 0.0 0.5 0.1 0.2 0.3 0.4 0.5 5 0 1 10 5 0 5 0 5 0 5 0 35 30 25 20 15 10 5 0 vros mut f NaCi of amount various & 0 0 t 5 0 5 0 35 30 25 20 15 to 5 1 1 2 2 3 35 30 25 20 15 10 5 eiccu halophilus Pediococcus Days s y a D s y a D 0 5 0 35 30 25 20 t 3 Ethanol 3% ith w 20% NaCi & 3% Ethanol 3% & NaCi 20% 20% NaCi 20% 3% Ethanol 3% None 3% Ethanol 3% 15% N a C Ii 3% Ethanol 3% Ii C a N 15% NaCi 15% Nona 3% Ethanol 3% 10% NaCi 5 3% Ethanol 3% 5 NaCi 10% NaCi 10% Nona 5% NaCi 5 3% Ethanol 3% 5 NaCi 5% Ethanol 3% 5% NaCi 5% Nona
113 114
0.5
0.3 Nona 5% NaCi o 0.2 6% Ethanol o 5% NaCi & 6% Ethanol 0.1 -e * o 0.0 0 5 10 15 20 25 30 35 O Days ¥ 0.5
0.4
0.3 - Nona 10% NaCi 0.2 6% Ethanol 10% NaCi A 6% Ethanol 0.1 * 2 0.0 o 0 5 10 ’5 ac 25 30 35 Days ? 0.5
0 4
0.3 - None 15% NaCi o 0.2 6% Ethanol o 15% NaCi & 6% Ethanol 0.1 o* 0.0 0 5 10 15 20 25 30 35 Days £ c 0.5
0.4
0.3 None 20% NaCi O 0.2 6% Ethanol d 20% NaCi 4 6% Ethanol
« o 0.0 a 0 5 10 1i 20 25 30 35
Figure 3.20 Growth of Pediococcus halophilus with 6% Ethanol & various amount of NaCi Figure
Growth (O.D. at 660 nm) Growth (O.D. at 660 nm) Growth (0.0. at 660 nm) 3.21 Growth ot ot Growth 3.21 0.0 0 2 - 0.4 - 0.4 0.6- o.a- 0.0 0.0 0.2- 0.2 0.4- 0.4 0.6 ■ 0.6 0.8 ■ 0.8 i.o ■
4 A 0
vros mut NaCi f o amount various & S0 5 Days ays D 1 0 1 0 tpyoocs aureus Staphylococcus 15 15 20 ■ w 10% NaCi & 3% Ethanol 3% & NaCi 10% ■w ■ 3% Ethanol 3% ■ None ■ — 10-/. NaCi 10-/. — %NC 3 Ethanol 3% 4 NaCi 5% Ethanol 3% 3% Ethanol 3% 15% NaCi & 3% Etranol 3% & NaCi 15% 5% NaCi 5% None Nona 55%NaCI t 3 Ethanol 3% ith w
115 Figure 3.22 Growth of of Growth 3.22 Figure
Growth (0.0, at 660 nm) Growth (O.D. a| 660 nm) Growth (O.D. at 660 nm) 0.0 0.8 - 0.0 0.2 0.4 0,2 ■ 0.6 0 4 0.6 0.8 0.8 0
4 0 0 vros mon o NaCi of ount am various & 5 5 5 Daya Days 1 0 10 1 0 tpyoocs aureus Staphylococcus 1 5 1 5 t 5 t 20 20 20 6% Ethanol 6% 6% Ethanol 6% 5.NC 8 % Ethanol 6% 8 NaCi 15V. Nona 15% NaCi 15% Ethanol 6V. 10V.8 NaCi Nona 10% NaCi 10% 5% NaCi 5% % ai 6 Eihanol 6% & NaCi 5% 6% Ethanol 6% Nona ih % Ethanol 6% with
116 iue .3 rwh of Growth 3.23 Figure
Growth (O.D. al 660 nm) Growth (O.D. at 660 nm) Growth (O.D. at 660 nm) 0.0 0.2 0.4 - 0.4 0.6 0.8 1.0 0.0 ♦ 0.2- 0.4 0.6 0.8- 0.0 0.2 0.4 0.6 08 0 & various amount of NaCi of various amount & 0 10 0 W J ff W 5 1 0 s s ays D tpyoocs aureus Staphylococcus 1 5 10 Daya 15 20 20 20 — a — 15% NaC NaC I 15% — — a ■ 15% NaCi 6 9% Ethanol 9% 6 NaCi 15% ■ ■ a 10% NaCi 6 9% Ethanol 9% 6 NaCi 10% Ethanol 9% ■aNaCi 10% ■a Nona -a -®— ■ 9% Ethanol 9% ■ Nona — ih % Ethanol 9% with 5% NaC! 6 9% Eihanol 9% 6 NaC! 5% 9% Ethanol. 9% NaCi 5%
117 118
was cultured in TSB with two levels of NaCi. Data in Figure 24 &
25 show that significant killing only occurred when the P. h a lo p h ilu s or S. aureus were inoculated in TSB with 20% ethanol. No other level of NaCi or ethanol (when used singly) was able to
cause a significant reduction of cell counts in this study. The results also indicate that there is a significant difference
(P<0.05) between independent killing effects of 20% ethanol on P.
h a lo p h ilus cultured originally in TSB with 5 and 15% of NaCi. A much higher killing rate was observed on the P. halophilus
cultured originally in TSB with a high concentration of NaCi. This difference, however, was not statistically significant in the case of S. au reu s cultured originally in TSB with 0 and 10% NaCi.
Combination Effects of Salt and Ethanol on Survival
Data in Figure 26 & 27 show that significant killing effects
on S. a u re u s occurred whenever NaCi and ethanol was used in combination. This is also true with P. h a lo p h ilu s (Figure 28 & 29), except when 10% NaCi and 10% ethanol was used in combination. No significant killing effect on S. aureus w a s detected when 10% ethanol, 10% NaCi and 20% NaCi were used singly. Also, the differences between the survival response in
TSB and the survival responses in TSB with the addition of 10% ethanol, 10% NaCi or 20% NaCi was not statistically significant. However, when 10% ethanol was used in combination with either F ig u re 3.24 Independent effects of NaCi & ethanol on the survival survival the on ethanol & NaCi of effects Independent 3.24 re u ig F ~3 o o U E
Cell counts (log clu/ml) 1 2 3 4 5 2 3 6 1 4 S 7 8 6 7 8 9 9 of of 0 0 b Culur: 5 Ci aC N 15% re: ltu u C (b) doocs al l s ilu h p lo ha ediococcus P Cutr: % NaCi 5% ulturo: C ) A < 20 20 H o u rs u o H Hours 0 4 0 4 080 8 60 0 6 80 2 0 % Ethanol % 0 2 2 0 % NaCi % 0 2 10% Ethanol 10% 10% NaCi 10% Nona 20% Ethanol 20% 20% NaCi 20% 10% Ethanol 10% TO% NaCi TO% Nona
119 Figure 3.25 Independent effects of NaCi & ethanol on the survival survival the on ethanol & NaCi of effects Independent 3.25 Figure
C*H counts (tog cfu/ml) Coll counts (log ctu/ml) of of 2 3 4 5 6 8 7 9 20 40 60 60 6 0 6 0 4 0 2 0 tpyoocs aureus Staphylococcus -r 20 40 60 80 8 0 6 0 4 0 2 0 b Clue 1% NaCi 10% Culture: (b) £) utr: % NaCi 0% Cuttur#: (£.) rs u o H rs u o H e Non# ■+ -e -■ ■■ 0 Ethanol 10% # ----- # Ethanol ■#■■■—■ 10% ■m • • ------Non# o ------20% NaCi 20% ------0 Ethanol 20% 20% NaCi 20% 0%NaCt C a N % 10 2 0 % Ethanol % 0 2 10 % NaCi % 10 120 Figure 3 .2 6 Effects of NaCJ & ethanof on the survival o f f o survival the on ethanof & NaCJ of Effects 6 .2 3 Figure o o Call counts (log clu/ml) Cell counts (log cfu/ml) Call counts (log cfu/mlj aureus 10 10 TO 2 to 3 1 4 5 a 6 7 9 3 2 4 1 5 6 0 a 7 9 0 0 0 Cutr: %NaCI) 0% ulture: (C 0 Hoi?, 20 0 4 „ 0 2 rs u o H
0 6 60 0 6 080 8 60 0 8 80 0 8 -D- ■+ -■ P Nono — "P e -e ■* ~ None ~ P ------10% NaCi A 10% Ethanol 10% A NaCi 10% to% NaC) to% 10% NaCJ & 20% Ethanol 20% & NaCJ 10% T 0% NaC) T 0% 20% NaC) NaC) 20% 20% Ethanol 20% I NaC 20% 0 Ethanol 20% None 20% NaC) & 10% Ethanol 10% & NaC) 20% 20% NaC) 20% Neno 10% Eihanol 10% 1 0% Ethanol 0% & 20% Ethanol 20% Staphyto. 121
F igure 3.27 Effects of NaCi & ethanol on on ethanol & NaCi of Effects 3.27 igure F
II counts (log cfu/ml) coun(s efu/ml) ***** counts (log cfu/ml) Cell counts (log cfu/m l) 10 3 2 4 10 1 5 6 7 8 9 10 2 4 10 3 6 7 8 5 1 9 2 4 3 6 1 5 7 8 9 0 aureus 0 0 20 Clue 10%NaCI) (Culture: 20 20 20 rs u o H rs u o H 060 40 Hours 40 rs u o H 40 0 6 0804060 0 6 80 80 80 the survival of of survival the 0 ai 2% Ethane) 20% & NaCi 20% 0 Ethanol 20% NaCi 20% e n o N 0 ai 1% Ethanol 10% & NaCi 20% None 0 NaCi 20% None 0 Ethanol 20% 0 ai 2% Ethanol 20% & NaCi 10% 0 Ethanol 10% 0 NaCi 10% None 0 Ethanol 10% 0 NC & 0 Ethanol 10% & NaCi 10% NaC I 10%
Staphyto. 122 Figure 3.28 Effects of NaCi & ethanol on the survival of of survival the on ethanol & NaCi of Effects 3.28 Figure
Cell counts (log cfu/ml) ^ counts (log cfu/ml) C*M «*««** (log cfu/ml) C«H co«"«* Clofl cfu/nl) 2 4 3 1 5 6 7 8 0 2 4 6 a 2 3 4 1 5 6 8 7 0 halophilus 0 0 0 0 3 60 0 4 0 2 I 1 — * 1 ’ —I 0 8 0 6 0 4 0 2 0 3 0 6 0 4 0 2 rs u o H Hours rs u o H rs u o H Cutr: %NaCI) 5% ulture: (C o None -o o 0 ai 0 Ethanol 20% 4 NaCi 20% ■o— ■« -Q------• 1 0% None NaCJ «•— ■a » 0 ai 0 Ethanol 20% 4 NaCi 10% ■» 2 0 % Ethanol % 0 2 0 Ethanol 20% NaCi % 0 2 ai 0 Ethanol 10% 4 NaCi % 0 2 NaCi % 0 2 None 0 Ethanol 10% None % NC 4 10 Ethanol 1 0% 4 NaCi 10% 0 Ethanol 10% NaCi 0% l
Pedio.
123 124
6 "a 8 o 7 ©s» 6 None M 5 10% NaCi A - 10% Ethanol 3 : 10% NaCi & 10% Ethanol
2 - « 1 — i— — i— —I- 20 40 60 80 Hours
fi o None 10% NaCi 20% Ethanol 3 10% NaCi & 20%. Ethanol o
Hours
8 O 7 a t 6 None o 5 20% NaCi M 4 10% Ethanol e 3 3 20% NaCi & 1 0% Ethanol o 2 1 T <-> 20 40 60 80
Hours
8 u 7 at o 6 None 5 20% NaCi 4 20% Ethanol 3 20% NaCi & 20% Ethanol 9 0 2 V 1 "3 v 20 40 60 80 Figure 3.2S Effects of NaCi & ethanol on the survival of Pedio. halophiius (Culture: 15%NaCI) 125
10 or 20% NaCi, significant killing effects appeared.
Statistically speaking, the difference between the survival response in TSB and the survival response in TSB with the addition 10% ethanol in combination with either 10 or 20% NaCi was significant (P<0.05). A similar result happened to P. h a lo p h ilu s when 10% ethanol was used in combination with 20%
NaCi. Therefore, the killing effect of NaCi and ethanol on S. a u re u s and P. halophilus may be considered as synergistic with these special combinations. The results also indicate that this synergistic effect on S au reu s was significantly greater (P<0.05) when a higher level of salt (20% NaCi) was used in combination with 10% of ethanol. In addition, to both test organisms, additive effects occurred when 20% ethanol was used together with either 10 or 20% of NaCi. Notably, the combination effects, that were produced by using 20% NaCi with 10 or 20% ethanol, were significantly greater (P<0.05) when the P. halophilus w a s originally cultured in TSB with higher level of salt (15% NaCi). However, no significant difference was observed in the case of S. aureus. 126 3.5 DISCUSSION
Effects of Salt on Growth and Survival Results of growth and survival assays reveal that NaCi alone is not an effective preservative against the growth and
survival of halotolerant bacteria and moderate halophiles. Not only it is incapable of killing halotolerants and moderate halophiles, but the addition of low level of NaCi (5%) in the medium can actually induce the growth of moderate halophiles. These results fully agree with the previous literature {Chapter
1 )- Although 20% NaCi is not enough to cause significant killing effect on S. a u re u s , it is good to know that the growth of S.
a u r e u s is significantly inhibited by this level (20%) of NaCi. This result indicates that, if food manufactures can ensure an even distribution of 20% (or more) of NaCi in the food materials, these materials may be exempted from the risk of the growth of S. a u re u s , even if low levels of contamination from the environment are sometimes unavoidable. This information, is in agreement with Troller (1987), and is especially useful as brining or salting process is involved in food preparation.
Effects of Ethanol on Growth & Survival
Results of growth assays also indicate that the growth of most moderate halophiles tested in this study were inhibited by 127 the addition of 9% of ethanol. Reduction of growth rate (and
sometimes delayed growth) is a general ethanol response of halotolerants and moderate halophiles, when ethanol is added at levels lower than that can cause a complete growth inhibition, in this study. Relatively speaking. S. aureus and P. halophilus show greater tolerance to ethanol than the other test organisms. This result makes them become good challenge microorganisms for growth or survival studies in high ethanol and salt environments.
Compared with Shapero et al. (1978) and Ballesteros et al. (1992), S. au reu s tested this study had a higher ethanol
tolerance, and was capable to grow (with a delayed growth) and survive in TSB with 9% and 10% of ethanol, respectively. This result, however, is not contradictary with those earlier studies,
since growth and survival assays of this study were conducted at 30 and 22°C, respectively, instead of a much elevated temperature (37°C) used in both of the previous studies. Since S.
a u re u s , a halotolerant human pathogen, can grow and survive in
relatively high ethanol environments, it is particularly
important to know how this organism may response to ethanol when used in combination with salt.
Combination Effects of Salt & Ethanol on Growth & Survival Result of this study suggested that inhibition effects on both growth and survival of halotolerants and moderate halophiles can be greatly enhanced when proper amounts of NaCi 128
and ethanol are used in combination. It is not unexpected that the level of ethanol and NaCi (used in combination) required for a complete growth inhibition is quite lower than that needed for
an effective killing. Results of growth assays also show that P. halophilus and S. a u re u s have higher tolerance (to the combination effect) than other organisms tested. They can grow
in medium with 6% ethanol plus 5% NaCi, as well as 3% ethanol plus 10% NaCi in 3 weeks. Thus, these two organisms should be of special concern in food containing these ranges of NaCi and ethanol. Data from this study also indicated that significant inhibition effects (against all organisms tested) can be achieved when 3, 6 and 9% of ethanol are used in combination with 15, 10 and 5% of NaCi, respectively. Therefore, from a microbiological standpoint, a minium of 10 days' shelf-life can be predicted when foods are preserved with such levels of ethanol and NaCi at room temperature. Nevertheless, it is necessary to conduct proper experiment for each type of food, in order to know the shelf-life acurately.
Based on survival data, synergistic effects were found in various combinations, for both organisms tested. These results agree Shapero et al. (1978) who also observed a synergistic effect of ethanol and reduced Aw on the growth and survival of S. aureus. Results of this study also show that when excess amounts of ethanol were added these synergistic effects were reduced to additive effects. A simple explanation for the 129 changes is that the killing enhancement contributed by NaCi may be masked by a much greater killing ability of high levels of ethanol alone. Result of this study also indicates that the
amount of NaCi and ethanol required for a synergism is depended
on the target organism. In this study, a difference of the needed
combinations for a synergistic effect was demonstrated in between of S. au reu s and P. h a lo p h ilu. s
It is good to know that S. a u re u s can be effectively killed in medium containing 10% NaCi and 10% ethanol. This information
can be useful whenever alcoholic brine is to be used in food
preparation. The fact that P. h a lo p h ilu s (but not S. aureus) can
survive in medium containing 10% NaCi and 10% ethanol may also
help us to understand why P. h a lo p h ilu s was found so frequently in the commercial sufu studied earlier (Chapter 2). Notably, no other test organism in this study could survive through such a difficult environment. Moreover, It is commonly known that when a sublethal amount of antimicrobial agent(s) is used, a delayed growth may occurr. This type of growth was also demonstrated in several cases in this study. This means that any of these survived organisms, in this study, may cause a delayed growth in some time after our last experiment measurement.
The differences of the inhibition effects of ethanol, observed in between of the tests performed on P. h a lo p h ilu s cultured in TSB supplemented with 5 and 15% NaCi, was significant. This result indicates that cells of P. h a lo p h ilu s, 130 originally cultivated from TSB with 15% NaCi, appeared to be a
better target for the killing by ethanol. A possible reason for
these differences is that a lower cell surface hydrophobicity was first formed on the surface of the cells cultured in high salt environment. Subsquently, this change could increase the celt membrane permeability of ethanol. Thus the destructive ability of ethanol was enhance. If this assumption is indeed true, the synergistic and additive effects discussed above may also be explain by this reason. Some more study on this phenomenon is needed in order to prepare a good model to explain the combination effect of NaCi and ethanol on a cellular basis. 131 3.6 CONCLUSIONS
Growth and survival assays were conducted to analyze the
inhibition effects of NaCi and ethanol on halotoferant and moderately halophilic cocci. Results of this study revel that NaCi
alone is not an effective preservative against halotolerants and moderate halophiles. Ten percent of ethanol may be serve as a good preservative to prevent the growth, but not survival, of all the halotolerants and moderate halophiles tested in this study. Among five organisms tested, S. aureus and P. halophlius a p p e a re d to have greater adaptability to ethanol, and were able to grow in medium containing 9 and 6% of ethanol, respectively. S. aureus, however, was the only organisms which failed to grow in medium containing 20% of NaCi. From a microbiological standpoint, a minium of 10 days shelf-life can be predicted when 3, 6 and 9% ethanol are used in combination with 15, 10 and 5% NaCi, respectively, in foods that are stored at room temperature.
Synergistic and additive effects, produced by the combinations of ethanol and NaCi, against the survival of P. h a lo p h ilus and S. aureus were also observed. No antagonistic effect was found when ethanol and NaCi were used in combination. Compared with S. aureus, P. halophilus had greater resistance to the combination effects of NaCi and ethanol, and it survived in medium containing 10% NaCi and 10% ethanol. 132 Finally, the cells of P. halophilus, originally cultivated from TSB with higher level (15%) of NaCi, appeared to be more susceptible to ethanol. This suggests that the synergistic and additive killing effects of NaCi and ethanol on organisms could be caused by the lowered cell surface hydrophobicity that was induced by the high NaCi environment. Subsquently, an increased cell membrane permeability of ethanol might be responsible for the synergistic killing effects observed. 133
3.7 ADDITIONAL DATA
Table 3.4 Growth of Marinococcus halophilus in medium containing NaCi & ethanol
Time (Days) 0 2 4 6 10 18 30
M e a n of Growth (O.D. at 660 nm)"
Elhanol(%) 0 0.00 0,00 0.00 0.00 0.00 0.00 0.00 0.00 (0.00)* (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
5 0 0 .00 0.03 0.19 0.30 0.57 0.75 0.66 (0.00) (0.00) (0.00) (0.01) (0.01) (0.00) (0.02)
10 0 0 .00 0.01 o.oa 0.27 0.46 0.58 0.50 (0.00) (0,00) (0 0 1 ) (0.03) (0.01) (0.00) (0.02)
15 0 0 .00 0.05 0.11 0.14 0.36 0.50 0.50 (0.00) (0.00) (0.01) (0.01) (0.05) (0.05) (0.02)
20 0 0 .00 0.00 0,00 0.03 0.23 0.50 0.54 (0.00) (0.00) (0,00) (0.01) (0.04) (0.05) (0.00)
5 3 0 .00 0.00 0.00 0.02 0.24 0.45 0.39 (0.00) (0.00) (0.00) (0.01) (0.01) (0.00) (0.03)
10 3 0 .00 0.00 0.00 0.01 0.03 0.09 0.13 (0.00) (0.00) (0.00) (0.01) (0.01) (0.01) (0.01)
15 3 0 .00 0.02 0.02 0.00 0 01 0.05 0.09 (0.00) (0.01) (0.01) (0.00) (0.00) (0.00) (0.01)
20 3 0 .00 0.03 0.02 0.01 0.01 0.01 0.07 (0.00) (0.01) (0.01) (0.00) (0.00) (0.00) (0.01)
5 -2 0 6 -1 2 0,00 0,00 0.00 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) (0,00) (0.00) (0.00) (0.00)
Data represent the average of two experimental analyses Standard deviation 134
Table 3.5 Growth of Micrococcus halobius in medium containing NaCi & ethanol
Time (Days) 0 2 4 6 10 1B 30
Mean of Growth (O.D. at 660 nm )"
NaCKft) £ lh a n o im 0 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (0.00)* (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
s 0 0 .00 0.12 0.41 0.43 0.51 1.00 0.5B (0.00) (0.04) (0.02) (0.02) (0.01) (0,09) (0.05)
10 0 0 .00 0.19 0.47 0.49 0.60 1.05 0.90 (0.00) (0.02) (0.01) (0.03) (0.01) (0.02) (0.00)
15 0 0 .00 0.65 0.31 0.40 0.55 0.72 1.00 (0.00) (0,05) (0.04) (0.01) (0.04) (0.02) (0.02)
20 0 0 .00 0.00 0.02 0.44 0.50 0.78 0.78 (0.00) (0.00) (0.01) (0.04) (0.01) (0.05) (0.11)
5 3 0 .00 0.01 0.00 0.00 0.00 0.29 0.50 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.01)
10 3 0 .00 0.02 0.01 0.01 0.00 0.18 0.66 (0.00) (0.00) (0.00) (0.00) (0.00) (0.01) (0.07)
15 3 0 .00 0.00 0.00 0.00 0.00 0.00 0.08 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.02)
20 3 0 .00 0,03 0.02 0.01 0.01 0 01 0.03 (0.00) (0.01) (0.01) (0.00) (0.00) (0.00) (0.01)
5-20 6-12 0.00 0.00 0,00 0.00 0.00 0.00 0.00 (0.00) (0.00) (0,00) (0.00) (0.00) (0.00) (0.00)
** Data represent the average of two experimental analyses * Standard deviation 135
Table 3.6 Growth of Paracoccus halodenitrificans in medium containing NaCI & ethanol
Time (Days) 0 2 4 6 10 18
Mean of Growth (O.D. at 660 nm)" NaCIOfc) ■ \i*fa\ 0 0 0.00 0.00 0.00 0.00 0.00 0.00 (o .o o r (0.00) (0.00) (0.00) (0.00) (0.00)
s 0 0 .00 0.57 0.95 0.97 1.00 1.05 (0,00) (0.05) (0.02) (0.03) ■(0.07) (0.03)
10 0 0 .00 0.1S 0.95 1.00 1.00 1.02 (0.00) (0.02) (0.01) (0.07) (0.01) (0.02)
15 0 0 .00 0.04 0.54 0.85 0.97 088 (0.00) (0.00) (0.05) (0.02) (0.04) (0.08)
20 0 0 .00 0.01 0.00 0.06 o.ao 0.70 (0.00) <0 01) (0.00) (0,00) (0.06) (0.05)
5 3 0 .00 0.12 0.60 0.60 0.64 0.66 (0.00) (0.00) (0.03) (0.03) (0.02) (0.02)
10 3 0 .00 0.03 0.05 0.45 0.41 0.39 (0.00) (0.00) (0.00) (0.00) (0.02) (0.06)
15 3 0 .00 0.00 0.00 0.00 0.00 0.21 (0.00) (0.00) (0.00) (0.00) (0.00) (0.07)
20 3 0 .00 0.03 0.02 0.01 0.01 0.01 (0.00) (0.01) (0.01) (0.00) (0.00) (0.00)
5-20 6-12 0.00 0.00 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
Data represent the average of two experimental analyses Standard deviation 136
Table 3.7 Growth of Pediococcus halophilus in medium containing NaCI & ethanol
Time (Days) 0 2 4 6 10 18 30
Mean of Growth (O.D. at 660 nm)**
NaCI(%l Elhanoif%l
0 0 0.00 0.01 0.15 -J 0.42 0.42 0.38
(0 0 0 )- (0,00) (0.03) o b o (0.01) (0.04) (0,10)
5 0 0 .00 0.05 0.42 0.42 0.41 0.43 0.44 (0.00) (0.02) (0.01) (0.00) (0.01) (0.00) (0.02)
10 0 0 .00 0.03 0.42 0.39 0.37 0.35 0.35 (0.00) (0.00) (0.11) (0.03) (0,02) (0,00) (0.02)
15 0 0 .00 0.02 0.09 0.19 0.23 0.19 0.20 (0.00) (0.00) (0.03) (0.01) (0.00) (0.05) (0 03)
20 0 0 .00 0.00 0.00 0.01 0.00 0 12 0.17 (o.oo) (0.00) (0.00) (0.00) (0.00) (0.03) (0.02)
0 3 0 .00 0.00 0.00 0.18 0.27 0.28 0.23 (0.00) (0.00) <0.00) (0.01) (0.01) (0.03) (0 03)
0 6 0 .00 0.00 0.00 0.00 0.08 0,19 0.29 (0.00) (0.00) (0.00) (0.00) (0 01 > (0.03) (0.01)
5 3 0 .00 0.01 0.16 0.24 0,24 0.24 0.39 (0.00) (0.00) <0 00) (0.01) (001) (0.00) (0.04)
5 6 0 .00 0.00 0.02 0.02 0.04 0.18 0.18 (0.00) (0.00) (0.00) (0.00) <0.01} (0 00) (0.02)
to 3 0 00 0.00 0.17 0.10 0.11 0.14 0.12 (0.00) (0.00) (0.05) (0.01) (0.00) (0.01) (0.01)
10 6 0 .00 0.01 0.02 0.05 0.01 0.00 0.02 (0.00) (0,00) (0.00) (0.01) (0.00) (0.00) (0.01)
15 3 0 .00 0.00 0.01 0.04 0.05 0.06 0 11 (0.00) (0.01) (0.01) (0.00) (0,00) (0.00) (0 01)
15 6 0 .00 0.02 0.01 0 01 0.00 0.01 0.01 (0.00) (0,01) (0.00) (0.00) (0.00) (0.00) (0 00)
20 3 0 .00 0.00 0.00 0.00 0 00 0.00 0.09 (0.00) (0.00) (0.00) (0.00) (0.00) (0 00) (0 02)
20 6 0 .00 0.00 0.00 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
0-20 9-12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) (0.00) (0.00) (0 00) (0.00)
Data represent the average of two experimental analyses Standard deviation 137 Table 3.8 Growth of Staphylococcus aureus in medium containing NaCI & ethanol
Time (Days)*** 1 2 3 4 6 10 18
Mean of Growth (O.D. at 660 nm)"
NaCK%) EthanoW%) 0 0 0.58 0.68 0.77 0.79 0.90 0.72 0.56 < 0.02)* (0.00) (0.05) (0.02) (0.00) (0.01) (0.04) 5 0 0.50 0.72 0.75 0.58 0.51 0.50 0.52 (0.01) (0.00) (0.02) (0.00) (0.01) (0.02) (0.05) 10 0 0 .09 0.18 0.44 0.47 0.41 0.43 0.47 (0.00) (0,00) (0.00) (0.02) (0.01) (0.02) (0.01) 15 0 0 .03 0.01 0.11 0.17 0.29 0.34 0.39 (0.01) (0.00) (0.04) (0.01) (0.00) (0.00) (0.02) 20 0 0 .00 0.02 0.01 0.01 0.01 0.05 0.02 (0.00) (0.01) (0.01) (0.00) (0.00) (0.02) (0.01) 0 3 0 .14 0.28 0.53 0.55 0.70 0.75 0.50 (0.03) (0.01) (0.11) (0.07) (0.02) (0.02) (0.10) 0 € 0 .03 0.11 0.40 0.51 0.53 0.57 0.54 (0.00) (0.02) (0.00) (0.00) (0 0 ) (0.01) (0.04) 0 9 0 .00 0,00 0.00 0.01 0.00 0.75 0.22 (0.00) (0.00) (0,00) (0.00) (0.00) (0.21) (0.09) 5 3 0 .02 0.30 0.51 0.54 0.53 0.64 0.73 (0.01) (0.01) (0.03) (0.00) (0.04) (0.10) (0.02) 5 6 0.00 0.00 0.03 0.02 0.08 0.09 0.24 (0 .0 0 ) (0.00) (0.00) (0.00) (0.02) (0.01) (0.06) 5 9 0 .00 0.00 0.00 0.00 0.00 0.01 0.00 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 10 3 0 .01 0.07 0.08 0.14 0.15 0.24 0.36 (0.00) (0.01) (0.01) (0.04) (0.03) (0.08) (0.05) 10 6 0 .00 0.01 0.04 0.00 0.04 0.01 0.01 (0.00) (0,00) (0.00) (0.00) (0.01) (0.00) (0.00) 10 9 0 .00 0.01 0.00 0.00 0.01 0.01 0.01 (0.00) (0.01) (0.00) (0.00) (0.00) (0.00) (0.00) 15 3 0 .00 0.08 0.02 0.04 0.04 0.01 0.09 (0.00) (0.03) (0.01) (0.01) (0,00) (0.00) (0.02) 15 6 0 .00 o.cfi 0.01 0.03 0.02 0.00 0.01 (0.00) (0.00) (0.00) (0.01) (0.01) (0.00) (0.00) 15 9 0.00 0.00 0,05 0.00 0.00 0,00 0.00 (0 .0 0 ) (0.00) (0.03) (0,00) (0.00) (0.00) (0 00) 20 0-90 0.00 0.00 0.00 0.00 0.00 0.00 0,00 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 0 -2 0 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) (0,00) (0.00) (0.00) (0.00)
*** Reading at o day were o.oo for all combinations Data represent the average of two experimental analyses Standard deviation 138
Table 3.9 Survival of Staphylococcus aureus, cultured orginally in TSB, in medium containing NaCI & ethanol
Time (Hours) 0 0.5 2 6 24 48 72
Mean of Growth (logio cfu/ml)**
NaCI(%) Ethanolf%t 0 o 6.30 6.50 6.90 7,30 7.60 8.00 9.04 ( 0 .0 0 ) ’ (0.00) (0.03) {0.02) (0.02) (0.11) (0.05)
10 0 6.30 6.38 6.36 6.99 6.30 7.70 9.00 (0.00) (0-05) (0.00) (0.02) (0.01) (0.02) (0.21)
20 0 6.30 6.40 6.11 5.90 5.70 5.00 6 64 {0.00) (0.10) (0.01) (0.03) (0.00) (0.02) (0.02)
0 10 6.30 6.73 6.53 6.95 6.66 5.90 7.08 (0.00) (0.11) (0.00) (0,03) (0.00) (0.02) (0.10)
0 20 6.30 6.20 5.60 5.70 0.00 0.00 0 00 (0.00) (0.00) (0.01) (0.15) (0.00) (0.00) (0.00)
10 10 6.30 6.48 6.30 6.30 3.90 2.70 2.48 (0.00) (0.04) (0.03) (0.00) (0.30) (0.10) (0.02)
10 20 6.30 5.00 3.48 2.66 0.00 0.00 0.00 (0 .0 0 ) (0.00) (0.03) (0.07) {0.00) (0.00) (0.00)
20 10 6.30 6.38 6.15 4.95 1.70 0,00 0.00 (0,00) (0.06) (0.01) (0.00) (0.12) (0.00) (0.00)
20 20 6.30 4.08 3.90 2.30 0.00 0.00 0.00 (0.00) (0.02) (0.00) (0.04) (0.00) (0.00) (0.00)
Data represent the average of two experimental analyses Standard deviation 139
Table 3.10 Survival of Staphylococcus aureus, cultured orginally in TSB with 10% NaCI, in medium containing NaCI & ethanol
Time (Hours) 0 0.5 2 6 24 48 '2
Mean of Growth (logic cfu/ml)*’
NaCH%) 0 0 6.30 6.54 6.95 7,00 7.30 7.70 8.75 (0.00)* <0.14 ) (0.13) (0.03) (0.02) (0.10) (0.15)
10 0 6.30 6.30 6.65 7.00 7.00 8.20 8.20 (0.00) (0.04) (0.41) (0.12) (0.01) (0.02) (0.07)
20 0 6.30 6.15 6.48 6.46 5.78 5,85 5.90 (0.00) (0.00) (0.04) (0.03) (0.01) (0.12) (0.02)
0 10 6.30 6.38 6.72 7.00 6.90 6.95 6.57 (0.00) (0.01) (0.00) (0.00) (0.01) (0.02) (0.1 1)
0 20 6.30 6.00 6.00 5.48 1.70 0.00 0,00 (0.00) (0.00) (0.02) (0.05) (0.10) (0.00) (0.00)
10 10 6.30 6.08 6.62 7.00 5.60 4,72 2.48 (0.00) (0.04) (0.13) (0.02) (0.22) (0.01) (0.01)
10 20 6.30 4.34 2.00 1.48 0.00 1.00 0.00 (0.00) (0.03) (0.03) (0.02) (0.00) (0.02) 10.00)
20 10 6.30 6.08 6.40 5.90 3.30 2.81 1 48 (0.00) (0.02) (0.01) (0.10) (0.10) (0.01) (0.02)
20 20 6.30 4.70 2.81 1.60 0.00 0.00 0.00 (0.00) (0.00) (0.01) (0.02) (0.00) (0.00) (0.00)
Data represent the average of two experimental analyses Standard deviation 140
Table 3.11 Survival of Pediococcus hatophilus. cultured orginally in TS8 with 5°A NaCI. in medium containing NaCI & ethanol
Time (Hours) 0 0.5 2 6 24 40 72
Mean of Growth (logio cfu/ml)"
NaClt%) Ethanolf%) 0 0 5.70 5.58 5.70 5.70 5.61 6.30 6.71 <0-00)* (0.03) (0.02) (0.02) (0.00) (0.01) (0.15)
10 0 5.70 5.60 5.61 5.60 5.43 5.50 7.15 (0.00) (0.01) (0.01) (0.02) (0.01) (0.05) (0.07)
20 0 5.70 5.59 5.43 5.30 5.71 6.00 5.90 (0.00) (0.03) (0.01) (0.03) (0.10) (0.04) (0.02)
0 10 5.70 538 5.30 5.32 5.49 5.23 5.00 (0.00) (0.10) (0.01) (0.03) (0.20) (0.02) (0.03)
0 20 5.70 5,30 4.95 4.48 4.T5 3.30 1,00 (0.00) (0.01) (0.01) (0.05) (0 02) (0.00) (0 01)
10 10 5.70 5.62 5.48 5.49 5.34 5.40 5.34 (0.00) (0.04) (0.03) (0.00) (0.30) (0.11) (0.07)
10 20 5.70 5.11 4.91 2.90 1.00 0.00 0.00 (0 .0 0 ) (0.04) <0.07) (0.02) (0.01) (0.00) (0 00)
20 10 5.70 5.48 5.00 4.15 2.04 0.00 0.00 (0.00) (0.02) (0.04) (0.01) (0.09) (0,00) (0.00)
20 20 5.70 3.60 3.30 2.99 1.60 0.00 0.00 (0,00) (0.06) (0.02) (0.10) (0.03) (000) (0 00)
Data represent the average of two experimental analyses Standard deviation 141
Table 3.12 Survival of Pediococcus halophifus, cultured orginally in TSB with 15% NaCI. in medium containing NaCI & ethanol
Time (Hours) 0 0.5 2 6 24 40 72
Mean of Growth (logio cfu/ml)**
N a C i m E th a n o lf% ) 0 o 5.70 5.47 5.34 5.60 5.95 6.95 6.95 (0 .0 0 )- (0.01) (0.02) (0.00) (0.04) (0.00) (0.01)
10 0 5.70 5.52 5.62 5.57 6.04 6.48 7.04 (0.00) (0.03) (0.01) (0.03) (0.03) (0.06) (0.03)
20 0 5.70 5.85 5.46 5.49 5.96 5.77 5.96 (0.00) (0.05) (0.02) (0.00) (0.09) (0.05) (0.02)
0 10 5.70 5 83 5.72 5.63 5.54 5.45 4.96 (0.00) (0.11) (0.03) (0.01) (0.12) (0.07) (0.01)
0 20 5.70 4.28 3,48 2.11 0.00 0.00 0.00 (0.00) (0.01) (0.06) (0.11) (0.00) (0.00) (0.00)
10 10 5.70 5.74 5 43 5,48 5-28 5.26 4.96 (0.00) (0.03) (0.00) (0.02) (0.00) (0.09) (0.01)
10 20 5.70 3.60 1.30 1.00 0.00 0.00 0.00 (0.00) (0.07) (0.1 1) (0.03) (0.00) (0.00) (0.00)
20 10 5.70 4.28 4.30 1.95 0.00 0.00 0.00 (0.00) (0.08) (0.01) (0 07) (0.00) (0.00) (0.00)
20 20 5,70 1.78 0.00 0.00 0.00 0.00 0.00 (0.00) (0.04) (0.00) (0 00) (0.00) (0.00) (0.00)
Data represent the average of two experimental analyses Standard deviation CHAPTER IV
SENSORY EVALUATION ON SUFU PRODUCED WITH THE ADDITION OF PEDIOCOCCUS HALOPHILUS
4.1 LITERATURE REVIEW
Pediococcus hafophUus in Food The pediococci are a group of lactic acid bacteria, and, they are harmless bacteria in general and have not been implicated in food poisoning outbreaks (Raccach, 1987). Some of the species of
Pediococcus are useful microorganisms, and are involved (either naturally or intentionally) in food processing. Table 4.1 is a list of common useful microorganisms, and some related food conversions (Banwart, 1989). Clearly, species of Pedicoccus, along with other lactic acid bacteria, are responsible for wide variety kinds of food fermentation. 142 143
Table 4.1 Some microbial food conversions*
Microorganisms S u b s tra te s P ro d u cts
Lactic acid bacteria, species of Red meat Sausages Leuconostoc, Lactobacillus, Cabbage Sauerkraut Pediococcus and/or Cucumbers Pickles Streptococcus Olives Olives Vanilla beans Vanilla Cream Sour cream, cultured butter Milk Cultured milk, yogurt. Ripened & unripened cheese
Propionibactarium Unripened cheese cheese Brevibacterium linens Peniciltium roqueforti P. camemberti
Lactic acid bacteria with yeast Taro Poi Flour (dough) Sour dough bread, pancakes Ginger plant Ginger beer Beans Verm icelli Rice, black gram Idli Soybeans, wheat Shogu (soy sauce), miso Kaffir corn Kaffir corn beer
Yeasts Malt Beer Fruit Wine Molasses Rum Grain mash Whiskey Flour (dough) Bread
Yeasts with Acetobacter Sugar, fruit, potatoes, Vinegar or Gluconobacter honeym maltm grain, alcohol
Halophilic bacteria Fish Nuoc-mam-ngapi
Bacillus subtilis Soybeans Natto
Actinomucor elegans, Mucor Soybean curd Sufu
Rhizopus oligosporus Soybeans Tempeh. tempe
From Banwart (1989) 144 Pediococcus halophiius, also named as soy pediococcus. is a salt-tolerant, homofermentative, lactic acid bacterium (Kanbe & Uchida, 1987). Although this organism had been frequently isolated from many fermented foods (Chapter 2), it exists in the natural environment, including soybean seeds (Kanekar, et al.,
1992). According to Yoshii (1979), the optimum water activities for growth of P. halophiius are 0.99 to 0.94, which correspond to 5 to 10% (w/v) salt content; the lowest water activity in which P. h a lo p h iiu s can grow is 0.808, which corresponds to 24% (w/v) salt content. This bacterium can grow in a pH range of 5.5 to 9.0 and temperature range of 20 to 42 °C, and, its optimum growth occurs at 25 to 30 °C (Fukushima, 1989). Other important characteristics of this organism have been described previously in Chapter 2.
Pediococcus halophiius in Sufu Since sufu is not a sterile product, it was of interest to investigate the microorganisms present in the commercial products, especially those moderate halophiles that may exhibit growth in the salting and aging process of sufu. In Chapter one of this study, I reported the finding of high levels of moderate halophiles in the commercial sufu. Results in Chapter two further indicate that the predominate halophilic organism, in most brands of the sufu, was P. halophiius. To answer why P. halophiius (and not other moderate halophiles) is the dominate organism in sufu, the investigation was extended to the growth and survival 145
behaviors of a group of moderately halophilic and halotolerant organisms. The results of the investigation (Chapter 3) show that P. halophiius has high resistance to the combination effect of ethanol and salt, and this may explain its domination in sufu and other salted food. In conclusion, all above studies indicate that this organism may play an important role during the salting and aging process of sufu.
Sensory Properties of Sufu Soybeans, which have a relatively low price and high nutritional value, are used in a variety of forms as part of human diet. Fermented soybean foods, such as miso, tempeh, soy sauce and soybean paste are common dietary items in the Orient. These products, rich in proteins and amino acids, are used primarily as flavoring agents that add flavor to generally bland and low- protein rice diets (Vaidehi & Kadam, 1989). Likewise, the unique flavor and nutritional value of sufu greatly enhance its popularity among Asian. According to Wang & Hesseltine (1970), in addition to nutrients, the flavor of sufu makes the diet more edible by stimulating the appetite, and contributing nutritional value to people who may otherwise not consume enough nutrients. It is generally believed that the flavor and aroma of sufu mature as the aging process progresses (Reddy & Salunkhe, 1989; Shurtleff & Aoyagi, 1979). According to Reddy & Salunkhe (1989), during the first 10 days of aging, the mold enzymes act on 146 substrates such as proteins and lipids to generate various hydrolyzed products. These hydrolyzed products provide the principal constituents for the development of mild and characteristic flavors in sufu (Reddy & Salunkhe, 1989). Many factors may affect the flavor and aroma of the product. For instance, various kinds of wine, spices, rice mash, or sesame oil are sometimes added with the brine to provide additional flavors to sufu (Chapterl).
Previous research on the fermentation process of sufu has mainly concentrated on the chemical changes that occur (Reddy & Safumkhe, 1989). However, little or no work has been published on either the involvement of halophilic organisms in sufu processing or the sensory quality of matured sufu. Therefore, it is important to study and compare the sensory quality of sufu produced with and without the addition of halophilic bacterial inoculum. 147 4.2 PLAN OF STUDY
This study was directed to investigate the effect of Pediococcus haiophiius on the sensory quality of sufu. 148 4.3 MATERIALS AND METHODS
Culture organisms
Actinomucor taiwanensis (ATCC 52360) and Pediococcus halophiius (ATCC 33315) were used for this study. A .
taiwanensis was maintained on Potato Dextrose Agar (PDA). For
cultivation of spores, the organism was inoculated on PDA, and incubated at 25°C for 7 days. The culture of P. halophiius was
maintained on (and harvested from) an agar slant with 10% marine salts as described previously (Chapter 2).
Sample preparation
First, 2000 g of Vinton 81 soybeans (Steyer Seeds, Inc.,
Tiffin, Ohio) were washed and soaked in 6,000 mi sterile water at 10° C. After 20 hours, the soaked beans (wet weight 5,000 g) were ground in a Hobart Mixer/Grinder (Model A 100T). The ground beans were then mixed with 13,500 ml sterile water in a steam jacketed kettle, heated to boiling, and allowed to remain at 100° C for 20 minutes. After this heat treatment, the mixture was filtered through cheese cloth. About 10,000 g of soymilk were obtained. After these samples were cooled to 70°- 75° C, 35 g of (C aS Q ^^O (in 200 ml hot sterile water) was gently added and mixed with the soymilk. The resulting curd was coagulated protein. The coagulum was then pressed in sterilized tofu forming boxes for an hour. About 2,500 g tofu was obtained, 149
with approximately 75% moisture. The curd was cut into small
cubes of 2 x 2 x 1 cm. The cubes were then placed in a hot air
oven for surface drying for 15 minutes at 100° C. After the cubes
were cooled to 40° - 45° C, A. taiwanensis was inoculated onto
the surfaces of the cubes (approximately 102 organisms per gram of tofu). The cubes were then incubated at 22° C. After three days, these molded cubes were placed in sterilized 300ml glass
jars (six cubes per jar) and covered with 30 g of sterilized NaCI. After three days of salting, sterilized water and ethanol were
added to each jar to allow complete immersion of the tofu cubes.
The final concentration of ethanol and salt content in each jar is
10% and 12%, respectively. The cubes were then aged at 22° C for one m onth.
To test the effect of P. halophiius on the sensory quality of sufu, a culture of P. h a lo p h iiu s was inoculated (approximately 103 organisms per gram of tofu) onto the surface of sample cubes
at three different stages. Sufu sample #1 was prepared with the
addition of P. halophiius at the same time as A. taiwanensis was
inoculated. Sufu sample #2 was prepared with the inoculation of
P. h a lo p h iiu s at the same time as NaCI was added into the jar. Sufu sample #3 was prepared with the inoculation of P .
h a lo p h iiu s at the same time as water and ethanol were added into
the jar. A control sample was prepared without the inoculation of P. halophiius. Table 4.2 is the time table for the inoculation of P. h a lo p h iiu s. 150
Table 4.2 Time table for inoculation of P. h a lo p h iiu s
Time Inoculation Fermentation stage
Day 0* Sample #1 Begining of fermentation Day 3 Sample #2 Begining of salting Day 6 Sample #3 Begining of aging Day 36 End of sufu preparation
* The day that fresh tofu was made 151
Microbial Analyses The plate count methods for estimating fungi and moderate
halophilic organisms, as described previously (Chapter 1), were
used for the microbiological analyses. For enumeration of the
total non-halophilic aerobic bacteria in the samples, a modified standard plate count method was used by adding cycloheximide
(0.1 g of cycloheximide / ml. of ethanol / liter of SPC media) (Sigma) to prevent the growth of mold.
Sensory Evaluation
This descriptive analysis was accomplished with the help of a selected and trained sensory panel. The nine panelists were
selected from fifteen Asian students based on prescreening
flavor tests and questionnaires results. The methods of screening, training, testing and data analysis procedures described by Lyon et al. (1992) were followed with modification,
and are described as follows:
Panel selection
The initial screening was based on questionnaire results that were filled out by fifteen Asian students or staff in the
Ohio State University. Table 4.3 is an example of the questionnaire form. Only healthy Asians who like soybean products and were familiar with the taste of sufu were selected 152
Table 4.3 Panel prescreening questionaire form
Your Name ______
Please answer the following questions (circle the right answer):
1. Are you an Asian student or staff in O.S.U.?
Yes No
2. Do you have any illness that affects your senses, especially taste and smell?
Yes No
3. Do you take any medications that affect your senses, especially taste and smell?
Yes No
4. Do you smoke or chew tobacco (or similar stuff)?
Yes No
5. Do you have dentures, diabetes, oral disease, sinus infection or allegeriec?
Yes No
6. Did you receive any training on sensory evaluation in the past?
Yes No
7. Did you serve as a sensory panelist before7
Yes No
8. Do you dislike soybean products (such as soybean milk, tofu, or sufu)?
Yes No
9. Do you familiar with the taste of sufu (fermented tofu)?
Yes No 153
as the panelists for this study. Previous training or experience
was highly desirable, but not essential.
Two sets of triangle tests were used to select the candidates who are most perceptive to differences in two of the major flavors (salty and acid) existing in sufu For testing,
panelists were seated in individual testing booths in a well- designed sensory laboratory. When testing, each panelist was
presented with three coded samples, two of which were alike and
one of which was different. The panelist was asked to select the different sample from two sets of test samples. The solutions used for set 1 and set 2 of this screening test were two levels of
sodium chloride (1.5 and 3.0%) and two levels of acetic acid (0.01
and 0.02%), respectively. The order of serving was carefully controlled in a random manner. Table 4.4 is an example of flavor
test form used for this study. Candidates who passed the initial questionnaire screening, and correctly answered both sets of the triangle test were accepted as the sensory evaluation trainees.
Panel training
Two sessions (an hour for each) of training were made in the sensory laboratory. During training, the panel was seated around a table for discussion. All descriptive terms were generated and determined in the training meetings. The training steps were as follows: 154
Table 4.4 Panel prescreening triangle test form
Your Nam e ______
You will be given two sets of three samples. Please taste the samples in the order presented and circle the number of the odd (different) sample in each sample set.
1. Which one is the different sample among the 1st set samples? #261 #059 #726
2. Which one is the different sample among the 2nd set samples? #347 #825 #411
Thank you very much for your help! 155 (1) Word generation . Assessors evaluated two different
commercial sufu samples, and wrote down as many terms as they could to describe the sensory characteristics fully.
(2) Discussions. Assessors agreed on what terms were relevant and should be used for the study. The definitions for each term were also described, discussed and agreed by the panel. The
descriptors and definition established by the panel is listed in Table 4.5.
(3) Use of intensity scales. Assessors were familiarized with
the use of the full range of a scale and tested on their ability to use a scale in a consistent manner.
(4) G e n e ra / instruction. Assessors received practical instruction
on the test procedure (e.g. to spit, instead of swallow), the format of the test.
(5) Practice the test. A simple sensory profile was drawn up and
tested with two different commercial sufu samples.
(6) Feed-back. Feed-back was given to the panel and further discussions were held to rectify any problems that arose from the practice test.
Testing procedure
Panelists were instructed not to swallow any of the samples and were provided with spit cups for this purpose. All panel sessions were conducted in a sensory panel room equipped with partitioned booths and fluorescent lights. Pure water at 156
Table 4.5 Descriptive terms and definitions
Term Definition
Color intensity Darkness of the sample on the surface Wine (aroma) Ethanol smell Rancid (odor) Aged oil smell
Creaminess Smoothness and mouthcoatmg Firmness Amount of force required to compress partially or fully Salty Salty taste Sour Acid taste Rancid Aged oil taste Bean paste miso or fermented soybean taste Sweet Sweet taste 157
approximately 20°C and white bread (or apples) were supplied to
clear the assessors' palate and diminish any carryover between samples. At each test session, each panelist received four different samples in random order. They were asked to analyze
the appearance, odor, texture and taste of the samples. Table 4.6 is an example of the evaluation form.
Data analyses
All intensity scores of the descriptive analysis were first
measured and quantified from 0 to 12. Then, one-way analysis of
variance (ANOVA) was done to determine the significance of the treatment variable on the sensory attributes of sufu. Mean scores for each attribute, which were significantly affected by the treatment variable, were further subjected to multiple paired t-test to determine the differences between the samples.
To analyze if one sample was preferred to the other, paired
Wilcoxon signed rank tests were conducted. The relationship
between the preference score on individual attribute (that was affected by the treatments) and both the overall preference score and attributes' intensity scores were determined by Spearman's rank correlation analysis.
The principle and methods of the statistical analyses used for this study were according to O'Mahony (1986) and Lyon et al. (1992). Statistical significance was accepted if the probability 158 Table 4.6 Evaluation form for the sensory characteristic of sufu
Name______Date______
For descnptive analysis, please place a vertical line across the horizontal >>ne at me pomt which best describes that attribute m the sample. and mark the sample number on the line you drawn For the preference test, please rank the sample and it's attributes according to your perteranee Mark *1* if you like it extremely; mark2 *’ if you like it; mark *3* it you neither like nor dislike if. mark"4 il you dislike it. mark 'S' it you dislike n extremely.
Overall preference #924____ #581____ *102____ #744 ___
APPEARANCE
Color intensity weak strong
Preference #924____ #581____ #102____ #744___
o o o n
Wine (aroma) weak strong
Preference #924____ #581____ # t0 2____ #744 ___
Ranctd (odor) weak Strong
Preference #924____ #581____ #102____ #744___
TEXTURE
Creaminess Slight extreme
Preference #924____ #581____ #102____ #744___
Firmness slight extreme
Preference #924____ #581 ____ #102____ #744
t a s t e
Sally weak strong
Preference #924____ #581____ #102____ #744 ___
Sour weak strong
Preference #924____ # 5 8 f ____ #102 #744 ___
Rancid (taste) weak strong
Preference #924____ #581____ #102____ #744.
Bean paste weak strong
Preference #924____ #581 ____ #102____ *7 4 4
Sweet (aner-taste) slight extreme
Preference #924____ #581____ #102____ #744 ___ 159 value was less than or equal to 0.05 for the ANOVA, difference,
preference and correlation.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SDS-PAGE was used for determining the degree of protein
degradation. The procedure used was adopted from the Bio-Rad Instruction Manual described as follows:
Reagents (1) Bio-Rad Mini-Protean li gel: 12% single percentage gel.
(2) Sample buffer : mix 4 ml of 0.5M Tris-Hcl (pH 6.8), 3.2 ml of
glycerol, 6.4 ml of 10% SDS, 1.6 ml of 2-8 mercaptoethanol, and
0.8 ml of 0.05% bromophenol blue with 16 ml of distilled water. (3) Running buffer (5X): mix 9 g of Tris base, 43.2 g of glycine, 3 g
of SDS with distilled water (to have a total volume of 600 ml).
(4) Staining solution: 0.1% Coomassie blue R-250 in fixation (40%
methanol, 10% acetic acid).
(5) Destaining solution: 10% methaniol/7.5% acetic acid.
(6) Sample solution: dilute and homogenize 1 g of sample with 9 ml of distilled water.
Erocedure
(1) Add 250 jul of sample solution and 1 ml of sample buffer in dispossable culture tube. Heated the mixed solution at 95°C for 5 min in a water bath. 160
(2) Dilute 70 ml of running buffer (5X) with 280 ml of distilled w a te r.
(3) Assemble the mini-Protein II unit with the gel.
(4) Add approximately 115 ml of running buffer to the upper chamber. Fill until the buffer reach the level halfway between the short and long plates. (5) Pour the remainder of the buffer into the lower buffer cham ber.
(6) Load 10 pi of sample prepared samples into the wells under the electrode running buffer with a syringe.
(7) Run the electrophoresis at 200 volts (constant voltage) for approximately 30 min. (8) Remove the gel.
(9) Stain the gel in staining solution for 45 min. (10) Destain the gel for 3 hours.
(11) Wash and store the gel in 7% acetic acid solution. 161 4.4 RESULTS
Terminology Development A word list was first developed by allowing each panelist
to list any term that he/she felt would describe the individual
character notes perceived. At this point, the discussion was
generally on the four basic sensory categories (e.g., appearance, aroma, texture, taste), and the objective was to list as many potential descriptors as possible. Then, they were required to
examine and discuss carefully the word list, confirm the most relevant ones, delete irrelevant ones and add missing words if
any. Also, the panel defined the meaning of each term. The list
of word and definition that finalized in Table 4.5 was given to the panelists for reference during the sensory tests. Thus, a total of ten attributes w£.i evaluated for their intensity in the
samples. Preference tests on the overall preference and preference on the ten attributes were also conducted at the same tim e .
Evaluation of Attributes
Of the ten attributes used to describe the sensory quality of the four different sufu, eight showed no significant differences between samples tested (Table 4.7 & Figure 4.1). However, significant differences were found between both creaminess and rancidity (taste) of the samples. The differences (creaminess 162
Table 4.7 Intensity of sufu sensory attributes as judged by nine trained panelists
Sample Control______#_L * 2 F-rano P-ualue Mean SO Mean SO Mean SO Mean SO
Color Intensity 2.97* 0.61 3-Ma i .08 3.33* 0.86 3.28* 1.00 0.105 0.957
Wine aroma 4.49a 0.67 4.37 0.29a S.67a 0.03 S.91a 0-66 1.099 0.371
Rancid odor 2.23a -- 3.44a -- 3.07* -- 3.543 .. 0 3S7 0 473
Creaminess S. 12* 1-41 7.19b 0.26 7.77b 0.77 5.80* 0.02 4.321 0.01 1
Firm ness 5.34* 0.50 3.82* 0.64 4.36* 0.61 4.67a 0.45 0.980 0.4 IS
S a lty taste 7.33* 0.28 7.64* 0.68 8.16* 0.53 8.29* 0.44 0.672 0.576
Sour taste 2.54* 0.23 3.23* 0.37 2.9* 0.71 3.17* 0.12 0.142 0.934
Rancid taste 4.24* 0.14 2.40b 0.06 4.49* 0.50 4.71a 0.13 3.291 0.033
Bean paste 5.22* 0.77 5.16* 0.03 6.4S* 0.32 6.45* 0.26 2.153 0.113
Sw eet after-taste 5.26* 0.43 4.57* 0.07 6.23* 0.70 5.65* 0.17 1.732 0.181
Raw socres are recorded in Additional Data (Table 4,13) Data show the average of single or duplicate performance (mean scores) of nine judges. Score ranged from 0 (not present) to 1 2 (extrem e). Standard deviation of the mean of two trails. a. b Same letter superscnpts in the same row indicate no significance at p <.0.05 level Color intensity
Sweet taste Wine aroma
Bean paste Rancid odor Control Sample #1 Rancid taste Creaminess Sample #2 Sample #3
Firmess
Salty
Figure 4.1 Sensory profile of sufu samples made with and without
the addition of P. halophilus 164 and rancid taste) between each sample were further analyzed by
T-test (Table 4.8). The mean values of creaminess scores of sample #1 and #2 were significantly higher then that of sample #3 and control sample. Whereas the mean rancidity (taste) intensity of sample #1 was significantly lower than that of ail other samples.
Evaluation of Preferences The significant differences on overall preference, creaminess and rancid taste were rated and evaluated (Table 4.9 & 4.10). According to overall preference scores, sample #2 was more preferred by the panel when compared with control sample
and sample #3. However, no significant difference was found between sample #1 and rest of the samples. Data also indicate that the creaminess detected in sample #1 and #2 were similarly preferred by the panel when they are compared with the control sample and sample #3. When the preference of rancidity (taste) in the samples was tested, sample #1 was more preferred than control sample and sample #3. However the differences between sample #2 and rest of samples were not statistically significant (p>0.05).
Relationship between Attributes and Preference Positive correlation was found between the mean values of preference on creaminess and both the degree of creaminess 165
Table 4.8 Statistical significance of the differences (creaminess & rancid taste) between samples
S3mples/(P~value) Creammess Rancid(taste)
Sample #1 & Sample 02 0 .2 4 1 * 0 .0 0 2 Sample 01 & Sample 03 0 .0 0 3 0 .0 0 2 Sample #1 & Control 0 .0 0 4 0.00 1 Sample 02 & Sample 03 0.006 0 .7 1 0 * Sample #2 & Control 0.003 0 .5 3 8 * Sample 03 & Control 0.331* 0 .2 1 8 *
No significance (p < 0 .0 5 ) difference 166
Table 4.9 Preference of sufu sensory qualities as ranked by nine trained panelists
Sample Control______#1______#2______«3. Mean Median SO Mean Median SD Mean Median SO Mean M edian SC
O v e ra ll P re fe re n c e 3.22 3.5* 0.71 3.1 T 3.0ab 0.00 2.SS 2.5b 0.71 3.33 3.S3 0.71
C ream iness 3.33 3.5* 0.71 2.22 2.0b 0.00 2.11 2.0b 0.00 3.1 1 3.0* 0.00
Rancid 3.10 3.0b 0.00 2.11 2.0* 0.00 2.67 3.0^0.00 3.22 3.0b 0.00 taste
* Raw scores are recorded in Appendix It Mean Data represent the average of duplicated performance (mean ranks)of nine judges. Score ranged from I (like it extremely} to 5 (dislike it extremely). Median Data represent the average of duplicated performance (median ranks) of nine judges. SD Standard diviation o f the median of rtvo trails. a, b Same letter superscnpts in the same row indicate no significance at p <.0.05 level. ab No significance (p <^0.05) difference when compared with other samples.
Table 4.10 Statistical significance of the difference (overall preference, creaminess preference & rancid taste preference) between samples
(P-value)
Samples Overall Creaminess Rancid (taste)
Sample #1 & Sample <*2 0.054- 0.390* 0.062* Sample *1 & Sample #3 0.377- 0.015 0.022 Sample #1 & Control 0.400- 0.009 0.023 Sample M2 & Sample *f3 0.014 0.014 0.058* Sample M2 & Control 0.034 0.010 0.088* Sample #3 & Control 0.300* 0.264* 0.337-
No significance (p <0.05) difference 167 (p<0.001) and the median of overail preference (p=0.05) on the samples. No correlation was found between rancidity (either intensity or preference) and the overall preference of the sam ples.
Microbial Analyses
The growth of Actinomucor taiwanensis and Pediococcus h a lo p h ilu s were constantly checked. After the inoculation at zero day, growth of A. taiwanensis on all sample took place quickly. Dense mycelial mats of A . taiwanensis were able to fully cover the sample cubes after three days of incubation. Culture of P. halophilus was surfaced inoculated on Sample #1 at the same time as the mold was added. Sample #2 and #3 were inoculated with P. h a lo p h ilu s at the third and sixth days of the process. Data in Table 4.11 shows the growth and survival of this halophtle during the fermentation. Total non-halophilic aerobic bacteria in the samples were also measured. Counts are present in Table 4.12. Under macroscopic and microscopic observations, these cells exclusively resembled B a c illu s ,
Protein Pattern
Figure 4.2 is the result of SDS-PAGE analysis on the laboratory prepared samples and a Brand E (Chapter 1} commercial sufu sample. Column A, B, C and D show the degrees 168
Table 4.11 growth and survival of P. halophilus in the sufu sampJes
Sample Control______#_2______<* 3 (Unit: log cfu/g) M e a n * SD** M ean SD Mean SD M ean SD
Day 0 <2.00 0.00 3.21 0.02
Day 3 <2.00 0.00 4.60 0.71 3.20 0.00
Day 6 <2.00 0.00 7.42 0.S3 7.10 0.06 3.22 0.01
Day 9 <2.00 0.00 4.02 0.08 3.25 0.15 <2.00 0.00
Oay 30 <2.00 0.00 <2.00 0.00 <2.00 0.00 <2.00 0.00
Data represent the average of duplicated tests. Standard diviatwn
Table 4 12 Growth and survival of non-halophilic bacteria in the sufu samples
Sample Control______# 2______*L3______(Unit: log cfu/g) M e a n * SD** Mean SD Mean SD M ean SD
Day 0 <2.00 0.00
Cay 3 5.60 0.09 5.48 0.21
Day 6 5.14 0.11 5.20 0.23 5.06 0.04
Day 30 3.03 0.08 3.21 0.12 3.77 0.36
Data represent the average of duplicated tests. Standard divtattcn 1 69
Colum n* A b CDEFGH
Various sufu samples were loaded on top of different column: Control sample taken after salting Column A S a m p le W\ taken after salting Column B Sample #2 taken after salting Column C Sample #3 taken after salting Column D Commerical sufu sample Column E Control sample taken after aging Column F Sample #1 taken after aging Column G Sample #2 taken after aging Column H
Figure 4.2 Protein pattern of sufu samples analyzed by SDS-PAGE 170 of protein degradation on the control sample, sample #1, sample
#2 and sample #3, respectively, after salting process. Column F, G and H show the difference of proteolysis on the control sample, sample #1 and sample #2, respectively, after one month of aging. Column E presents the protein pattern of a commercial sufu. Apparently, proteins in the control sample was less degraded than the proteins in sample #1 and sample #2, after salting or aging process. After aging for one month, the protein pattern of sample #1 appeared to be more similar to the commercial sufu tested. 171 4.5 DISCUSSION
Result of microbial tests indicate that proper growth of
both mold and halophiles took place in the experiment samples. This is important before the effect of P. h a lo p h ilu s on sufu can
be meaningfully evaluated. It was not unexpected that no growth of P. halophilus was found in sample #3, since 10% of ethanol was added at the same time as the organism was inoculated.
This result agree with the result of early study in the Chapter three. In Chapter 2, high population of P. halophilus were able to be recovered in the commercial sufu with salt and ethanol content more than 12% and 10%, respectively. However, result of this study shows the inoculated P. h a lo p h ilu s failed to survive 30 days in the product. Two possible reasons can explain this difference. First, wild strains of P. halophilus may have greater resistance to the alcoholic brine. This characteristic may be established environmentally or genetically. Secondly, in the commercial sufu, instead of pure ethanol, various types of wine and ingredients were mixed into the brine. The organic compounds, in the wine or ingredients, may inactivate the killing by ethanol (Banwart, 1989).
High levels of non-halophilic aerobic bacteria were found after three days of fermentation. It is Possible that these bacteria originated from organisms (such as B a c illu s spores) that survived through the soymilk processing, or were 172
accidentally introduced at the time of tofu processing. Since the
populations of these non-halophilic bacteria were not high (less than 106 cfu/g), the tofu was not considered as spoiled (Dotson
et at., 1977). The growth of these non-halophilic bacteria was very rapid at first three days of the fermentation. However, no
growth was observed once the process of salting started.
Apparently, the growth of P. halophilus (which happened mainly
during the process of salting) had little or no effect on the
growth of these non-halophilic bacteria. This data agree with the result in the Chapter one which indicates no correlation
between the counts of total aerobic organisms and moderate
halophiles in the commercial sufu. Samples inoculated with P. halophilus at or prior to salting
stage were found to be significantly different from both samples without addition of P. halophilus, and with the inoculation after salting stage. A higher intensity of creaminess was found in the samples inoculated with P. ha lo p h ilu s prior to or at salting
stage. This result suggests that the P. halophilus inoculated may enhance the protein degradation during the sufu processing. The
result of protein staining in this study not only confirmed this
suggestion, but also indicates that the greater enhancement occurred the earlier the organism was introduced.
Moreover, the intensity of rancidity (taste) of the samples was lower, when this organism was inoculated prior to salting stage (the same time as mold was inoculated). Several possible 173
reason may explain this difference: (1) P. halophilus may utilize
or convert products of lipid oxidation; and (2) the growth P. h a lo p h ilus may inhibit the lipid oxidation by reducing the oxygen availability. Nevertheless, more experiments are needed in
order to really understand the cause of this difference.
Generally speaking, the results of sensory evaluation indicate that P. halophilus can significantly affect sensory quality of sufu, and earlier inoculation of this organism allows a greater
performance of this organism in the fermentation.
The positive correlation between the preference of creaminess and both the intensity of creaminess and the overall preference suggests that the creaminess introduced by the addition of P. halophilus is recognizable and recommendable.
Moreover, the increases of creaminess may increase the overall sensory quality of sufu. Although no correlation was found in between rancidity (either intensity or preference) and the overall preference of the samples, the sample with lowest level of rancid taste was the most preferred sample based on the results of both overall preference and preference on rancid taste.
This means the difference of rancid taste might have some impact on the panel's judgment on overall preference.
Results also indicated that no significant difference of color intensity, wine aroma intensity, rancid odor, firmness, salty taste, sour taste, bean paste taste and sweetafter taste was found between the samples. This does not mean the 174
intensities of these attributes were not changed at all. For
example, the rancid odor in the samples may be different, however, it may covered by the strong aroma of ethanol. The
changes of the intensities could be indistinguishable by the panel, yet, were able to influence the panel’s judgment on the overall preference. More chemical and physical study are needed
to analyze the relationship between the overall preference and
the chemical and physical characteristics of the samples.
Although the results of this study suggest that P. h a lo p h ilu s can positively affect sensory quality of sufu under controlled experimental condition, it is not certain, however, whether and when sufu producers should add this organism into commercial sufu processing. Since the commercial sufu processing methods, brine composition, and original microbiological quality of tofu used for the fermentation may be very different, the use of P. h a lo p h ilu s may give different results for different manufacturers. (Chapter 1). These factors may potentially have impact on the sensory maturation of sufu and must be considered before a good understanding can be achieved.
In Chapter 2, results of isolation and characterization studies of commercial sufu indicate that high population of P. h a lo p h ilus was existed in most brands of sufu tested. Data of this study further suggests that the involvement of P. halo ph ilu s in sufu fermentation can be beneficial for the development of the 175
desired flavor. Therefore, it is important to develop a new sufu
processing procedure with the addition of pure culture of P.
h a lo p h ilus at early stage of sufu fermentation. The advantage of this new processing is two folds. First, the proper inoculation size can be pre-determined, this may allow the growth of P. h a lo p h ilus under quality control. Secondly, since the ability of P. halophilus to metabolize organic acids differs from strain to strain (Kanbe & Uchida, 1982), this new method may recruit only suitable strains of P. h a lo p h ilu s for the fermentation.
Nevertheless, to be certain about these potential benefits, this new processing method needs to be further developed and evaluated in real sufu factories. 176 4.6 CONCLUSIONS
A sensory panel was selected and trained to analyze the effect of adding a pure culture of Pediococcus halophilus, during three stages of sufu processing, on the sensory quality of
sufu. After descriptive terms were generated and defined by the panel, quantitative descriptive analysis was performed to develop a descriptive profile for each sufu. Preference tests on the overall sensory quality and each specific attribute were
also performed. The results of these preference tests were analyzed, in conjunction with the descriptive profiles, to evaluate the impacts of the differences that introduced by the additions of P. halophilus. Samples inoculated with P. halophilus at or prior to salting stage were found to be significantly different from either sample without addition of P. halophilus, or with the inoculation after salting stage. Higher intensity of creaminess, and lower intensity of rancidity (taste) was found in the sam ples inoculated with P. h a lo p h ilu s at or prior to salting stage. Furthermore, result of SDS-PAGE study confirmed that the higher creaminess found in the samples with P. h a lo p h liu s was caused by greater protein degradation. No significant difference of color intensity, wine aroma intensity, rancid odor, firmness, saltiness, sourness, bean paste and sweet after taste was found between all samples. The intensity of creaminess 177
was positively correlated with the preference of the attribute of creaminess. In addition, the total preference score was
correlated with both the preferences of creaminess and rancidity (taste).
Data from this study suggest further study on the positive role of P. h a lo p h ilu s in sufu maturation. Investigations on the physical and chemical quality of sufu made with P. halophilus, and the interactions between microorganisms that naturally existed in sufu fermentation are especially needed. Table 4.13 Raw data of intensity evaluation (' Tral nambar) 4a Treatment Tudge Color-\' Color-2 Wine-1 Wine-2 Rancid (odot) N
1 1 > 1 4.3 6.9 4.6 2.4 5.9 o 2 1 2 4.1 S.3 8.3 4.6 4 o 1 3 3 0.1 3 2.5 5.1 5 H 4 1 4 3 2.1 3.7 1.2 1.7 S ] O S 0,2 5.9 4.2 9.1 6.8 6 1 6 2.6 1.9 3.6 0.2 4.2 1 7 7 0.9 1.3 4.2 5.8 1.3 8 1 8 1 .6 2.8 8 8 0.6 9 o 1 9 4.4 5,9 2.1 1 .1 1.5 > 10 2 1 2.9 6.6 7.4 7.7 3.1 H 11 2 2 4.5 6 6.4 7.8 2.5 > 12 2 3 2.3 2 3.1 3.1 6.1 13 2 4 3 2.4 4.3 3.3 2.1 14 2 5 1 .7 2.9 9.1 8.1 S.1 15 2 6 3.3 2.7 4.1 8.5 1.7 16 2 7 2.3 2.1 6.4 2.4 1.9 17 2 a 1.8 2.8 6.1 6.1 2 1 18 2 9 2.7 5.9 4.3 3.8 3 19 3 1 S.S 5 2.3 6.8 5 20 3 2 4 6.8 7 8.3 5 21 3 3 1.5 3.8 7 7 7 8 22 3 4 3 2.1 5.8 4.3 2.8 23 3 5 1.7 5.6 6.1 8.7 6.1 24 3 6 1.4 3.7 8.9 9.9 1.4 25 3 7 3 1.3 5.3 4.6 1.3 26 3 8 1.8 1 .7 4.9 4.9 1 .5 27 3 9 1.3 5.9 1.7 2.9 1 28 4 1 3.5 6.3 3.4 3.3 4.1 29 4 2 4.3 5.6 9 6.3 3.2 30 4 3 0.6 1 3.7 2.6 2.6 31 4 4 3 3 0.5 0.3 0.6 32 4 5 17 2.6 7.6 8, 7 3.3 33 4 6 2 1.4 7.7 4.4 1.1 34 4 7 1 .4 1.3 ■3.1 3.5 1.3 35 4 8 1.8 3.5 6.4 6.4 2.1 16 4 9 4.5 5.9 3,3 0.7 1 .8 CD Cfeaminess-1 Creaminess-2 Firmness-1 Firminess-2 Salty-1 Salty-? Sour H a 1 6.7 a 5.1 6.3 7.1 8.1 8.2 3.5 2 S.9 7.8 7.9 6.1 8 5,6 8.2 3 5.3 5 5.8 4.1 7.5 8.3 4.1 * 4 7.8 2.8 1.1 3.8 7.3 4.5 6.1 5 5.1 7.7 2.9 2.1 7.6 5.2 2.1 6 9.9 10 0.1 1.4 1 1.3 10.8 3 a 7 6.8 6.4 4 1.8 o 8.9 7.0 1.3 9 8 8.9 7.1 6.2 2.5 7.7 8.9 0.3 *+ 9 10 1 1.2 4.2 1 .4 6.7 5.2 0.5 10 9 6 8.6 7.2 8.4 6.8 4.5 2.3 9 11 6.7 4.9 6.1 9.3 4.7 7.9 8. 1 • 12 a 6.8 4 5.3 3.3 7.3 8.2 3.3 13 9.6 6.6 7 2.6 7.3 4.5 4 14 B. 1 7.8 6.8 1.2 9.3 7.6 4.4 IS 8.8 9.1 3 3 10.9 10 1 .9 16 8.9 7.8 3.1 5.1 9.6 9.1 1.1 1? 9.9 5.9 1 .8 2.1 10.1 9.4 0.3 18 10 10.3 2.8 0.4 10.8 8.9 0.5 19 4.9 2.3 4 9.1 5.3 9.3 5.9 20 4.2 7.8 5.8 3.9 8.8 7.4 7,2 21 4.1 5.8 5.5 5.1 6.7 9. B 3.5 22 4.9 5.5 1 .9 1.6 6.4 5.8 4 23 3.5 3.6 4 3 1.4 8.7 8.4 3.5 24 7.3 7.4 3.8 7.2 11 10.8 2 3 2S 5 8 5.3 4.6 3.2 10.1 5.7 1 3 26 7 6 5 7.5 5.5 9.6 8.3 0.3 27 10 9.4 7.5 2 2 10.8 6.3 0.5 26 4.8 3.7 5.2 8 9.3 3.3 2 9 29 5 5 7,5 6.4 8.5 5.5 6.6 7.8 30 3.7 1.7 5.9 6.6 4.7 9 1 6 31 6 1.6 3.4 5.5 5.9 5.8 5.7 32 6,2 5.5 3.4 2.4 0.2 6.2 2 33 7.3 6.1 0.8 5.7 10.5 1 1.2 0.8 34 3 3 2.8 7.5 6.4 7.9 4 1.3 3S 8 2 6.5 8.5 4.2 5 10 0.3 36 10 1.7 3.8 3.9 10.8 8.1 0.5 ' nI
Treatment Judge Overall-1 Overall-2 Creaminess-1 (ieaminess-2 Rancid-1 Rancid- 1 1 t 2 2 t 2 1 3 1 3 4 1 4 5 1 5 6 1 G 7 1 7 e 1 6 9 1 9 to 2 1 11 2 2 12 2 3 13 2 4 14 2 5 15 2 f> 16 2 7 17 2 8 16 2 9 19 3 t 20 3 2 21 1 3 22 3 4 23 3 5 24 3 6 25 3 1 26 3 6 27 3 9 26 4 1 29 4 2 30 4 3 31 4 4 32 4 5 33 4 6 34 4 7 35 4 B 36 4 9 GENERAL DISCUSSION
For the first time the relationship between brine composition and microbiological quality of commercial sufu (Chapter 1), the characteristics of moderate halophiles isolated from the commercial sufu (Chapter 2), the effectiveness of alcoholic saline solutions on the growth and survival of halotolerants and moderate halophiles (Chapter 3), and the effect of halophilic organisms on the sufu flavor development (Chapter 4) have been investigated. The results of these studies provide several new understandings on the commercial sufu, as well as the microorganisms present in the sufu. First, high levels of non-halophilic aerobic sporeformers are present in most brands of commercial sufu tested. They are most likely introduced at the time of tofu processing and multiplied in the early stage (before salting) of the fermentation. Therefore, to lower the level of sporeformers in sufu, it will be necessary to reduce initial loads by using fresh tofu to make pehtze, and by improving sanitation and processing techniques. Furthermore, high populations of Pediococcus halophiles were found in most brands of sufu. The data suggest that moderately halophiles, such as P. halophilus, may play an important role during the salting and aging process of sufu. Moreover, strong negative correlation was found between the levels of P. halophilus in sufu and 182 183 ethanol content of brine. This result further indicates that brine composition, especially ethanol%, appears to influence the growth and/or survival of halophiles in the sufu fermentation. At this stage of study, however, the effect of sufu brine on the growth and survival of P. halophilus, and the influences of this organism on sufu remained unclear. Thus, further studies were made. Synergistic and additive effects, produced by the combinations of ethanol and NaCI, against the growth and survival of P. halophilus and S. aureus were observed. This indicates that both the ethanol and salt content in sufu brine are important factors that may affect the quality as well as safety of the product. Compared with S. aureus, P. h a lo p h ilu s had greater resistance to the combination effects of NaCI and ethanol, and it survived in medium containing 10% NaCI and 10% ethanol for at least three days. This result explains why high level of P. halophilus was so frequently found in the commercial sufu. Finally, the role of P. h a lo p h ilu s in sufu maturation was analyzed by sensory evaluation. In generally, the addition of P. h a lo p h ilu s at early stages of the fermentation helped the overall sensory quality (such as creaminess) development of sufu. This result shows that the presence of P. halophilus in sufu fermentation appeart to be desirable. Overall, this investigation discovered (1) a relationship between brine composition and microbiological quality of sufu, and (2) the presence and importance of P. h a lo p h ilu s in sufu fermentation. Further study should focus on the possible roles of P. h a lo p h ilu sin sufu fermentation, and the possibility (and advantages) of using this organism as part of a starter culture. REFERENCE
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