O'NIVERSllY OFRAWAfI UBRARt

IDENTIFICATION OF SOME MICROORGANISMS ISOLATED

FROM POI AND CHARACTERIZATION OF THE BIOLOGICAL

PROPERTIES OF ONE LACTIC ACID BACTERIA

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE

UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

FOOD SCIENCE

DEC 2003

By

Lijun He

Thesis Committee:

Alvin S. Huang, ChairpersonAurora A. Saulo

C. Alan Titchenal

© Copyright 2003By

Lijun He

111

Abstract

Lactic acid producing microorganisms from poi and the biological

properties of one lactic acid bacteria are studied in this work. Based on

phenotypical analysis, five bacterial strains isolated from fresh poi are identified

as Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactococcus lactis lactis,

Leuconostoc citreum and Leuconostoc lactis individually. The gas-producing

bacterial strain first identified as Lactobacillus acidophilus is finally identified as

Weissella confusa by combining the results of phenotypical analysis and

genotypical analysis. Acid tolerance of Weisselfa confusa is examined by

exposing the bacterial cells to MRS broth at different pH with/without adaptation.

When exposed to acid stress, the bacterial strain can survive at pH 3.70 with a

decreased growth rate. No bacteria are detected at pH 3.0. However, the survival

rate of the bacterial cells at pH 3.0 increases by adapting the cells at pH 4.46.

Results suggest that the bacteria can grow in acidic condition and the acid

tolerance is adaptable. The effect of the taro base on the growth of Weissella

confusa is investigated by examining the growth of the bacteria in four kinds of

taro/sucrose mixture (10% taro, 3% taro, 3% taro plus 1.3% sucrose, 3% taro

plus 3.2 sucrose). All four media supported the fast growth of the bacteria during

the incubation period and the pH of the taro base dropped fast, ranging from 3.39

to 3.73 after three-day incubation. The growth curves of W. confusa in

taro/sucrose base (3% taro plus 1.3%sucrose) and acid/heat treated poi were

established with the bacterial count and the pH of the sample every day. The

v

bacteria grew rapidly in both of the medium. These preliminary results seem to

indicate that a taro-based fermented food is feasibly produced by monoculture

with W. confusa as starter culture. In addition, a different pH change was

observed in these two taro-based media. These results suggest that the

acid/heat treatment and solid content had some effect on the pH change of the

taro medium.

Based on genotypical analysis, the bacterial strain isolated from sour poi

is identified as Lactobacillus p/antarum and the yeast-like microorganism isolated

from sour poi is identified as Candida tropicalis. Candida trapicalis can ferment

glucose, fructose, sucrose, and starch. Their existence may explain the acid

production at the late stage of the fermentation and the consumption of starch.

This work helps to elucidate the fermentation process of poi and improve

the production by identifying pure cultures of starters. In addition, this work

suggests Weisselfa confusa may be a potential bacterial strain in food

fermentation.

vi

Acknowledgements

I would like to thank Dr. Alvin S. Huang for being my advisor and giving

me the opportunity to work on this wonderful project focusing on food

fermentation in poi. I appreciate his valuable guidance and encouragement,

without which I would not have been able to successfully complete my Master of

Science degree.

I am grateful to the rest of my committee members Dr. Aurora A. Saulo

and Dr. C. Alan Titchenal. Their guidance and suggestions have proven

invaluable. I would also like to thank Dr. Halina M. Zaleski and Dr. Michael A.

Dunn for their assistance and advice throughout my study in this department.

I would like to acknowledge the US Army Research Center at Natick for

their generous financial support to this project. I would like to thank Honolulu Poi

Company for providing poi and taro corm in the study. I would like to thank

Silliker, Inc. Research Center for their technical support in the identification of

microorganisms. I also would like to thank all my lab mates for their help and

cooperation during this study.

Finally, my deep gratitude is given to my best friend Lei Zuo who inspires

me to complete my work, and also my family who have given me their

unwavering support and love at all times. I also want to thank all my good friends,

who have kept me calm throughout this project and who are available to help me

whenever I need assistance.

iv

Five bacterial strains from fresh poi are identified as Lactobacillus

acidophilus. Lactobacillus delbrueckii, Lactococcus lactis lactis, Leuconostoc

citreum, and Leuconostoc lactis based on phenotypical analysis. The bacterial

strain first identified as Lactobacillus acidophilus is finally identified as Weissella

confusa by combining the results of phenotypical analysis and genotypical

analysis. Weissella confusa can grow in acidic condition and the acid tolerance is

adaptable. Taro bases are made from taro corm or fresh poi. All the taro bases

support rapid growth of the bacteria, resulting in a rapid decrease in the pH of the

media. Based on genotypical analysis, Lactobacillus plantarum and Candida

tropicalis are isolated and identified from sour poi. This work suggests Weissella

confusa is a major bacterial strain involved in the fermentation of poi and it is

feasible to produce taro-based fermented food with the monoculture of Weissella

confusa.

Table of contents

Acknowledgements iv

Abstract v

List of Tables x

List of Figures xi

List of Appendix xii

Chapter 1: literature Review 1

Food Fermentation 1

Definition " '" " 1

Fermentation methods 1

Benefits 2

Lactic Acid Fermentation 2

Characteristics '" '" 2

Benefits 3

Examples 3

Lactic Acid Bacteria 5

. Characteristics 6

Taxonomy , 7

Identification method...........................•......... , 7

Selection 10

Application , 13

Food fermentation 14

vii

Food preservation 14

Probiotics '" " , 17

Research Objective 18

Chapter 2: Materials and Methods .21

Introduction 21

Preparation of bacteria isolates 21

Phenotypic characterization .22

Genotypic characterization 24

Preparation of taro bases 25

Inoculation and measurement ofthe growth of the bacteria 27

Acid stress test.. 28

Examination on preliminary characteristics of yeast-like colony 29

Chapter 3: Results and Discussion 31

Screening bacterial strains from fresh poi. 31

Phenotypic characterization 31

Genotypic characterization of the first bacterial strains 35

The biological properties of of WeisseJla confusa 39

The effect of pH on the growth of WeisseJla confusa 38

Acid tolerance response .41

Effect of taro based medium on the growth of Weissella

Confusa 44

Growth curves of WeisseJla confusa in taro/sucrose mixture and

acid/heat treated poi. .46

viii

Genotypic characterization of a lactic acid bacterial strain isolated from

sour poL 50

Genotypic characterization of one yeast-like colony isolated from sour

poi 53

Conclusion 55

Appendix: '" 57

Literature cited 69

IX

LIST OF TABLES

Table

1. Fermentation ways of major lactic acid bacteria in

fermented plant products 8

2. Comparison of the biochemical characteristics and

culture identification of the five lactic acid bacteria 32

3. Summary of the results of charbohydrate fermentation of

the five bacteria 34

4. Percent genetic difference between the first bacterial

strain and representative strains 36

5. Fermentation characteristics of the bacteria isolated in this

study compared with representative Weissefla confusa

and Lactobacillus acidophilus ATCC 4356 37

6. Percent genetic difference between the isolated bacterial

strain from sour poi and representative strains 52

7. Percent genetic difference between the yeast-like strain

and representative strains 54

x

LIST OF FIGURES

Figure Page

1. The Effect of pH on the growth of Weissella confusa .40

2. Survival of unadapted and acid-adapted cells of

Weissella confusa strains during pH challenge (pH 3) .43

3. Effect of different taro base on the growth of

Weissella confusa and the pH of the taro base .45

4. Values of pH and logarithm numbers of Weissella confusa

during fermentation in taro base treated with heat.. .47

5. Values of pH and logarithm numbers of Weissella confusa

during fermentation in taro base treated with acid and heat.. ....48

xi

LIST OF APPENDIX

Number

A. The USDA's nutritional makeup of poi. 57

B. The results oOhe first LAB strain isolated from poi. 59

C. The results of the second LAB strain isolated from poi. 61

D. The results of the third LAB strain isolated from poi. 63

E. The results of the fourth LAB strain isolated from poi. 65

F. The results of the fifth LAB strain isolated from poi. 67

xii

CHAPTER 1LITERATURE REVIEW

Food fermentation

Definition

Food fermentation is a process in which raw materials are converted to

fermented foods by the growth and metabolic activities of desirable

microorganisms. Common materials used in fermentation include milk, meat,

fish, vegetables, fruits, cereal grains, seeds, and beans. Generally, fermented

foods are composed of unused components of the raw materials and the

microbial by-products (Ray, 2001a).

Fermentation methods

Foods can be fermented in three different ways: natural fermentation (use

microflora in the raw materials as starter cultures), back slopping (use successful

products as starter cultures), and controlled fermentation (use pure culture as

starter cultures). The disadvantages of natural fermentation and back slopping

include inconsistent characteristics over a long period of time and high chances

of product failure due to the growth of undesirable flora and foodborne diseases

by pathogens. In controlled fermentation, pure cultures of single or mixed strains

or species of microorganisms are used as the starter culture. The products from

controlled fermentation are consistent and predictable. Generally, there is less

chance of product failure and foodborne diseases. However, in some cases, due

1

to the lack of growth of desirable secondary flora, the product may not have

certain delicate flavor characteristics (Ray, 2001 b).

Benefits

Fermentation plays an important role in food processing, including

enrichment of the human dietary through production of different flavors, aromas

and textures in food; preservation of a large amount of food; conversion of food

substrates biologically with vitamins, protein, essential amino acids and essential

fatty acids; detoxification; and low requirement in energy cost (Steinkraus, 2002).

Due to its healthful and natural characteristics, the consumption of

fermented foods has become popular since the 1970's. The fermented foods

available for general consumption include yogurt, buttermilk, fermented

sausages, kefir (a refreshing probiotic cultured-milk beverage), and kumiss (a

Russian and Mongolian fermented milk beverage) (Ray and Daeschel, 1992). My

project will focus on lactic acid fermentations.

Lactic acid fermentation

Characteristics

Lactic acid fermentation is a process in which the fermentable sugars are

converted to lactic acid by microorganisms including Leuconostoc, Lactobacillus,

Lactococcus, Pediocccus, Streptococcus, Bifidobacterium, and so on

(Steinkraus, 2002).

2

Benefits

Lactic acid fermentation probably is the simplest and safest way to

preserve food (Steinkraus, 2002). Fermentation has been widely used by

humans since biblical times. Lactic acid fermented foods enrich the human diet

with a wide variety of flavors, aromas, and textures, and play an important role in

feeding the world's population. Fermentation is also a low-cost and energy­

efficient method of food processing (Steinkraus, 1983a).

Lactic acid fermentation is an effective way to preserve food and has a

good safety record. The substrates used in lactic acid fermentation include

vegetables, fruit, milk, meat, cereal, and others. Lactic acid fermented foods are

widely present in different social cultures, such as German sauerkraut (fermented

cabbage), Korean kim-chi (fermented vegetables with a seasoning mixture

mainly consisting of salt, red pepper powder, garlic, ginger, green onion and

radish), Mexican pozol (fermented maize), Nigerian gari (fermented cassava),

and Middle-Eastern yogurt (fermented milk) (Steinkraus, 2002).

Examples

One of the most well known lactic acid fermented foods is yogurt.

Generally, the composition, flavor and texture of fermented products depend

upon the nature of fermenting organisms, the type of substrate and the

manufacturing process. Yogurt is fermented from milk by a mixed starter culture

consisting of Streptococcus thermophilus and Lactobacillus delbrueckii. The two

species grow symbiotically in milk. Streptococcus thermophilus provide an

3

anaerobic condition, formic acid, and CO2 to stimulate the growth of Lactobacillus

delbruekii. The bacteria.metabolize lactose to produce lactic acid. The

fermentation is temperature-controlled so as to reduce the pH to 4.6 or lower,

giving yogurt a sour flavor (Tamime and Deeth, 1980, Ray, 2001c).

Some other lactic acid fermented foods have been studied in detail.

Sourdough bread is an important fermented food from cereal. Lactobacillus

sanfranciscensis, L. reuteri and L. pontis have been isolated from traditional and

modern fermentations of rye and wheat (Vogel et aI., 1999). A classic lactic acid

vegetable fermentation, sauerkraut, is prepared from fresh cabbage that

shredded and mechanically pressed to expel the juice, providing fermentable

sugars and other nutrients for microbial growth. Anaerobic condition is obtained

though an tight packaging. Salt at 2.25% (glmlx100%) is added to stimulate the

growth of certain lactic acid bacteria. Cabbage is fermented by the combination

of Leuconostoc mesenteroides, Lactobacillus brevis and Lactobacillus plantarum

(Ray, 2001d; Ballcock and Azam-AIi, 1998).

The use of combined starters is common in lactic acid fermentation. All

natural fermentation use combined starters. The combined starters can stimulate

the growth of each other and achieve the desired properties quickly. However,

combined starters may Cause problems in managing industrial-scale production,

especially in standardizing the product. Compared with the fermentation with

combined starters, monoculture fermentation has some advantages. Some

research results indicated that monoculture fermentation is cost-effective

(Marshall and Tamine, 1997). Another study showed that a single starter may

4

give the final product a favorable sensory evaluation (Beasley, Tuorila and Saris,

2003). These results suggest that single starter culture is generally a feasible

option for the food industry.

Lactic acid bacteria play an essential role in the preservation and

production of wholesome food. Lactic acid-producing starter cultures have been

isolated and identified iii many traditional fermented foods. For example, lactic

acid bacteria were proved to be the major microorganisms in Mexican pozol

(Ampe et ai, 1999). Lactic acid bacteria have been isolated from the Nigerian

traditional fermented foods such as fufu (fermented yam, plantain, or cassava),

burukutu (fermented sorghum), and ogi-baba (fermented corn or brown millet

grains). Lactic acid bacteria also have been isolated from Congolese retted

cassava and Benin maize sourdough (Sanni at ai, 2002). Lactic acid bacteria are

even the starters of a Ugandan traditional fermented beverage bushera made

from sorghum or millet (Muyanja et ai, 2003).

In order to obtain new starters with beneficial characteristics, intensive

research on the selection and characterization of lactic acid-producing starter

cultures was conducted in the past decades. A brief review of lactic acid bacteria

is presented below.

Lactic acid bacteria

lactic acid bacteria are a diverse group of bacteria united by a

constellation of morphological, metabolic, and physiological characteristics,

5

which produce lactic acid as the major end product during the fermentation of

carbohydrates.

Lactic acid bacteria are widely distributed in nature. The natural reservoir

of lactic acid bacteria is the green plant material. Lactic acid bacteria are

introduced into animals when they consume the green plants. Not only present in

various nutrient-rich food products such as milk, meat and vegetables, these

bacteria also are normal flora of the mouth, intestine, and vagina of mammals

(Salminen and Wright, 1993).

Characteristics

The typical lactic acid bacteria are rods or cocci, short chains or pairs.

They are gram-positive, non-spore forming, catalase-negative, devoid of

cytochromes, facultative anaerobes, nutritionally fastidious, and able to produce

relatively large amounts of lactic acid as the major end product of metabolism

(Salminen and Wright, 1993). There are no functional heme-linked electron

transport systems or cytochromes in the bacteria. Lactic acid bacteria obtain

metabolic energy by substrate-level phosphorylation while oxidizing

carbohydrates and they lack a functional Krebs cycle (Jay, 1996a).

Lactic acid bacteria can be divided into two groups: the homofermenters

and the heterofermenters. Homofermenters produce mainly lactic acid via the

glycolytic (EMP, Embden-Meyerhof pathway) pathway, while heterofermenters

produce lactic acid plus ethanol, acetate and carbon dioxide, via the 6­

phosphogluconate/phosphoketolase pathway. The glycolytic pathway is used by

6

all lactic acid bacteria except leuconostocs, group III lactobacilli, oenococci and

weissella (Battcock and Azam-AIi, 1998). Table 1 lists the types of fermentation

produced by major lactic acid bacteria in fermented plant products.

Taxonomv

The classification of lactic acid bacteria is a loosely defined concept.

Generally, lactic acid bacteria include Lactococcus, Leuconostoc, Pediococcus,

streptococcus, Lactobacillus, Enterococcus, Aerococcus, Vagococcus,

Tetragenococcus, Camobacterium and Weissella (Salminen and Wright, 1993;

Jay, 1996a).

Identification methods

Traditional identification methods are based on physiological

characteristics, biochemical characteristics, and some morphology observations.

They are useful and indispensable in leaming the characteristics of the target

bacterium. However, these identification methods are limited in discrimination

and accuracy. Ambiguous results can lead to misidentification.

Many. changes have taken place in the taxonomy of bacteria in the past

decade. Many of the new taxa have been created as a result of the employment

of molecular genetic tools alone, or in combination with some of the more

traditional methods. In addition to the phenotypic characteristics, characteristics

that can be used to identify lactic acid bacteria include DNA homology, base

composition, 23S, 16S, and 58 rRNA sequence similarities, cell wall composition

7

Table 1. Fermentation ways of major lactic acid bacteria in fermented plant

products.

(From Beuchat et aI., 1995).

Homofermenter Facultative Obligate heterofermenterhomofermenter

Enterococcus faecium Lactobacillus Lactobacillus brevisbavaricus

Enterococcus faecalis Lactobacillus casei Lactobacillus buchneri

Lactobacillus Lactobacillus Lactobacillus cellobiosusacidophi/us coryniformis

Lactobacillus lactis Lactobacillus curvatus Weissella confusus

Lactobacillus delbrueckii Lactobacillus Lactobacillus coprophilusplantarum

Lactobacillus Lactobacillus sake Lactobacillus fermentatumleichmannii

Lactobacillus salivarius Lactobacillussanfrancffico

Streptococcus bovis Leuconostoc dextranicum

Streptococcus Leuconostoc mesenteroidesthermophi/us

Pediococcus acidi/actici Leuconostocparamesenteroides

Pedicoccus damnosus .

Pediococcuspentocacus

8

analysis, whole-cell protein fingerprinting, and fatty acid analysis (Jay, 1996b).

The DNA fingerprint technique is the ideal supplementary information in definitive

identification. The genetic fingerprinting techniques used for typing

microorganisms include PFGE (pulsed field gel electrophoresis), Rep-PCR

(repetitive sequence-based polymerase chain reaction), ribotyping, MLST (multi

locus sequencing typing), minisatellites, DNA chips, and AFLP -an acronym for

selective amplification of restriction enzyme fragments (Pouwels and Simons,

2003).

Taxonomic information can be obtained from RNA sequence. The 16S

rRNA subunit is highly conserved and is considered to be an excellent

chronometer of bacteria over time. By use of reverse transcriptase, 168 rRNA

can be sequenced to produce long stretches of genetic code, which allow for the

determination of precise phylogenetic relationships (Jay, 1996b). In all,

comparisons among 16S rRNA sequences are powerful and accurate techniques

for determining the phylogenetic relationships of microorganisms (Woese, 1987).

Some methods were developed based on PCR (polymerase chain

reaction) with species-specific primers derived from the 16S rRNA gene

sequence. PCR amplifications with group-specific or species-specific 16S rONA

primers and ARDRA (Amplified ribosomal DNA and restriction analysis) method

have been used to identify lactobacilli (Roy et aI., 2001). Papadelli et al. (2003)

develop species-specific PCR and DNA hybridization to rapidly detect and

identify Streptococcus macedonicus. This method further confirmed that S.

9

waius, a novel Streptococcus species, actually is phylogenetically identical to S.

macedonicus.

With the development of taxonomic study at the molecular level,

reclassification and new establishment have occurred. For example, the L.

acidophilus complex has been divided into L. acidophilus, L. crispatus, L.

amy/ovorus, L. gallinarum, L. gasser! and L. johnsonii at the species level

(Fujisawa et aI., 1992). With the use of RAPD (randomly amplified polymorphic

DNA) analyses, L. ga/linarum, the genomic sizes of which were estimated by

PFGE (pulse-field gel electrophoresis), was first introduced into characterized

Lactobacillus acidophilus complex, (Roy et aI., 2000). In 1993. a new lactic acid­

producing genus was established as Weissella based on the 165 rRNA

sequence analysis (Collins et aI., 1993). Molecular techniques have affected the

taxonomy of lactic acid bacteria as more identification of lactic acid bacteria is

based on the phenotypic analysis and the analysis of 165 rRNA.

Selection

The usage of commercial starter cultures helps to control the

manufacture of fermented food and obtain the desired identity, purity and quality

in the fermented product. The principles of hazard analysis critical control point

(HACCP) are being used in food production to assure stable high quality

production procedures (European food and feed cultures association.

http://www.effca.com).

10

Starter cultures of fermented foods are obtained by both genetic design

and from screening wild strains in nature. Genetic design can make desired

products. Research related to the genetics of lactic acid bacteria provides the

possibility of constructing new powerful starters. However, the time and cost

spent on obtaining legal approval, and subsequently developing a new market,

limit the application of genetically engineered strains (Egon Bech Hansen, 2002).

As a result, traditional screening is still widely used to obtain commercially

valuable strains for food fermentation.

Isolation and identification are the first steps to selecting wild strains with

potentially commercial value. Widely used methods to study microbial

communities in fermented foods are based on the statistical analysis of

information obtained from isolation and identification (Manolopoulou et ai., 2003).

Some other researchers use denaturing gradient gel electrophoresis (DGGE)

and traditional culture-depending methods for examining the bacterial community

of traditional cassava starch fermentation with a better result (Miambi, et ai.,

2003).

The main concerns in food fermentation are spoilage, foodborne

diseases, attractive flavor, taste and appearance, and health benefit. Lactic acid

bacteria contribute much to flavor production and food preservation. Lactic acid

bacteria have a long history of safe use. Some lactic acid bacteria were proved to

be probiolics with health benefit. Consequently, lactic acid bacteria are one of the

most widely studied starter cultures in the food industry.

11

The usage of bacteriocin-producing strains as starter cultures or

protective co- cultures is one of the possible ways to improve food safety. The

target bacteriocins usually have a wide antimicrobial spectrum. They are heat­

stable, pH stable within a wide pH range, and sensitive to proteolytic enzymes.

Some bacteriocin-producing strains have been identified to have potential to

improve the safety of the fermented product and could be potential commercial

strains. These identified bacteria strains include human isolate LactobacuJlus

acidophilus LF221 (Bogovic-Matijasic et aI., 1998; Rogelj et aI., 2002), nisin­

producing Lactococcus lactic WNC 20 strain isolated from nham - a traditional

Thai fermented sausage (Noonpakdee et aI., 2003).

The study on the survival ability and mechanisms of starter cultures is

one of the hot topics in the food industry. Starter bacteria are constantly

challenged by environmental stresses. Only the strains that survive against these

stresses can greatly improve the process of food fermentation. Tolerance against

acidic stress is one of the standards to screen useful starter cultures. Lactic acid

bacteria can produce lactic acid during fermentation, which means that they are

frequently challenged by acid stress. Lactic acid bacteria must develop some

mechanisms to survive and exert their effect on food. For example, the lactic acid

bacteria that carry out the malolactic fermentation play an important role in

modern commercial wine production. The bacteria must overcome the stress of

low pH, high ethanol content and the low temperature of the cellars during

fermentation. The acid-resistant mutant Leuconostoc oenos LoV8413 was

isolated and identified, which may be used for development of acid-resistant

12

starter cultures in wine (Drici-Cachon et aI., 1996). Another example of stress

response is related to Lactobacillus acidophilus LA1-1 , which is capable of

displaying adaptive response to stress and was suggested to have commercial

value (Kim et aI., 2001).

Another consideration in screening lactic acid bacterial strains is their

potential to be used as probiotics. Probiotics are viable bacterial cell preparation

or foods containing viable bacterial culture or components of bacterial cells that

have beneficial effects on the health of the host. The widely known

microorganisms used as probiotics include bifidobacterium and lactobacillus (Lee

et aI., 1999). The bacteria which can colonize in the gastrointestinal tract (GI)

and adjust the ecology of GI system can be seen as probiotics. Recent studies

on Lactobacillus acidophilus CRL 639 have shown that the strain is low-pH­

inducible acid tolerant and has an antagonistic effect on Helicobacter pylori in

Vitro (Lorca et al.. 2001; Lorca and Font de Valdez, 2001). These studies

suggested the potential usage of this bacteria strain as a probiotic. In conclusion.

bacteriocins, stress tolerance, and probiotics are the properties commonly used

to select commercial bacteria strains.

Application

Lactic acid fermentation is one of the oldest forms of biopreservation.

Lactic acid bacteria are one kind of the starter cultures that are responsible for

the fermentation process. Now lactic acid bacteria have been used extensively in

13

the food industry. Generally, lactic acid bacteria can be used in food

fermentation, food preservation and probiotics.

Food fermentation

Fermentation plays an important role in enriching the diet of humans with

desirable flavor, aroma, and texture. Fermentation process involves the

production of preservative products, such as organic acids, alcohol and carbon

dioxide. Fermentation also is responsible for the unique identity and the sensory

attributes of fermented food. Some desirable products include the flavor

compounds diacetyl, acetaldehyde, as well as compounds which may have

positive health implications such as vitamins, antioxidants and bioactive peptides

(Ross et aI., 2002).

In modern large-scale production, defined lactic acid bacterial strains can

be used to produce desirable foods that have dramatically improved the quality

and consistency of the raw materials. The commonly used lactic acid producing

genera include Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, and

Streptococcus.

Since lactic acid fermentation has been discussed in the former section,

now we will focus on food preservation and probiotics.

Food Preservation

The most ancient lactic acid fermentation probably was the product of

fermented sour milk, in which the low pH inhibited other undesirable

14

microorganisms. Other foods widely preserved by lactic acid fermentation around

the world include fruit, vegetable and vegetable! fish! shrimp mixtures.

Some research suggests that lactic acid bacteria were able to improve

the safety of minimally processed fruit and vegetables (Breidt and Fleming,

1997). Also, some lactic acid bacteria strains were able to preserve the ready-to­

use vegetables because of their inhibition against Aeromonas hydrophila, Listeria

monocytogenes, Salmonella typhimurium and Staphylococcus (Vescovo et aI.,

1996). These studies indicate the potential application of lactic acid bacteria as

biopreservatives.

The study on the mechanisms of antimicrobial activity revealed that lactic

acid bacteria preserved foods by the production of organic acids, hydrogen

peroxide, diacetyle, and bacteriocins.

Organic acids can reduce the pH of the environment, in which the growth

of many pathogenic and spoilage microorganisms is inhibited. The antimicrobial

mechanisms include interfering with the maintenance of cell membrane potential,

inhibiting active transport, reducing intracellular pH and inhibiting metabolic

functions (Ross et aI., 2002).

Hydrogen peroxide is produced by many of the lactic acid

microorganisms. This chemical was first used in combination with heat to

produce sterile milk. The antimicrobial effect is based on its reaction with other

components to form inhibitory substances.

Diacetyl (2,3-butanedione, biacetyl) is the compound responsible for the

characteristic aroma and flavor of butter. It is also an antimicrobial compound.

15

The antimicrobial activity depends on dosage. One study suggested that the

inhibitory level for yeasts and gram-negative bacteria is 200 I-l9/ml, while the

inhibitory level for non-lactic gram-positive bacteria is 300 I-l9/ml. The

concentration of less than 350 I-l9!ml can inhibit lactic acid bacteria (Daeschel,

1989; Vandenbergh, 1993).

Bacteriocins are proteins or peptides that are inhibitory to other bacteria,

encompassing metabolic end products, antibiotic-like substances, and

bactericidal proteins. Jack et al. (1995) extended the concept of bacteriocins to

encompass extracellularly released primary or modified products of bacterial

ribosomal synthesis. In all, bacteriocins are heterogeneous antagonists varying in

molecular weight, biochemical properties, sensitive host range, and mode of

action. Many bacteriocins produced by lactic acid bacteria are active against

food-borne pathogens such as Listeria monocytogenes, Clostridium perfringens,

Bacillus cereus, and Staphylococcus aureus. The inhibitory spectrum of

bacteriocins can be narrow or wide (Klaenhammer, 1988). Most lactic acid

bacteria are able to produce bacteriocins. The bacteriocin-producing lactic acid

bacteria include Lactobacillus, Streptococcus, Leuconostocs, Pediococcus, and

others (Klaenhammer, 1988). Recently, more bacteriocin-producing lactic acid

bacterial strains, such as Lactobacillus acidophilus LF221 (Bogovic-Matijasic et

aI., 1998), Lactococcus lactis FS92 (Mao et aI., 2001), Lactococcus lactis NK24

(Lee and Paik, 2001), were isolated and identified.

Since lactic acid bacteria have antibacterial activity and are GRAS

(generally regarded as safe) bacteria, the possible usage of the broad-spectrum

16

bacteriocins from laclic acid bacteria or direct usage of laclic acid bacterial

cultures as natural food preservatives is of increasing interest in the food

industry. Nisin, the well-characterized bacteriocin produced by Lactococcus lactis

lactis, has been accepted and approved in more than 50 countries including the

US and the EC (European Community) countries. Nisin has now been widely

used in the production of cheese, milk, desserts, yogurt, fermented beverages,

meat, fish, and canned foods (Vandenbergh, 1993; Soomro et aI., 2002). With

natural, safe, and antimicrobial characteristics, lactic acid bacteria or some

metabolic end products are good options for food preservation.

Probiotics

Animals, including humans, evolved with the normal flora of

microorganisms that exist in the intestinal tract. The normal flora protects us

against the invasion of other microorganisms. However, this protection can be

destroyed by unbalanced diet, antibiotic therapy and stress (Mattila-Sandholm et

al., 1999).

Probiotics are viable bacterial cell preparations or foods containing viable

bacterial cultures or components of bacterial cells that have beneficial effects on

the health of the host. The most thoroughly studied probiolics are bifidobacterium

and lactobacillus (Lee et aI., 1999).

Probiotics have a number of potential benefits, including antimicrobial

properties. antimutagenic properties, anticarcinogenic properties, immune

system stimulation, reduction in serum cholesterol concentration, reduction in

17

allergy, aid in lactose digestion, and adjuvant function (Tannock, 1999; Shah,

2001).

Probiotics are probably safe even at high dose level. With the injection of

probiotic LAB strains Lactobaillus rhamnosus HN001 (DR20), Lb. acidophifus

HN017, and Bifidobacterium factis HN019 (DR10), no toxic effect was detected in

mice. Consequently, such probiotics are likely to be safe for human use (Zhou et

al.,2000).

The effect of probiotics can be affected by administration. Oral

administration of probiotic Lactobacilli may be therapeutic. One study reported

the ability of Lactobacillus pfantarum 299v to colonize in the intestinal tract of

children with HIV and to elicit specific systemic immune response after oral

supplementation (Cunningham-Rundles et aI., 2000).

In general, studies of probiotics focus on the beneficial effect of probiotics

on human health. Lactobacillus is one of the most Widely studied genera with

beneficial properties. A large number of Lactobacillus strains are classified as

probiotics. One well-known example is Lactobacillus acidophilus NCFM, which

has proved to be probiotics and has industrial applications (Sanders and

Klaenhammer, 2001).

Research objectives

Poi is a traditional staple food in Hawaii. II tastes somewhat like fresh,

pure yogurt if it is left in a cool place for 2-3 days before consumption (Begley et

aI., 1981).

18

Traditionally, the marvelous physical development of the Hawaiian is

attributed to poi (Allen and Allen, 1933). It is an excellent carbohydrate source

with 27 grams of carbohydrates based on 100 grams portion of edible sample

(USDA, 2001). The starch in poi is composed of small granules and is extremely

digestible. In Hawaii, poi commonly was used for infants suffering from

malnutrition and food allergies. The effect of poi on the prevention of allergic

disease in potentially allergic infants has been demonstrated (Roth et aI., 1967).

Some people claimed that poi actually promotes gastrointestinal healing

(http://www.thepoicompany.com). However, there is no peer-reviewed research

to support the gastrointestinal healing function of poi.

The purposes of the study of natural food fermentations are to improve

food safety, improve nutritional value, improve production methods and reduce

production costs (Steinkraus, 1983b). Poi is a naturally fermented food. Its sour

taste results from metabolism of lactic acid bacteria, such as Lactobacillus and

streptococcus (Allen and Allen, 1933; Huang et aI., 1994). However, little is

known about the definite starter cultures of poi and their characteristics.

This study is an attempt to screen potentially industrial strains that can be

used in the fermentation of taro base by investigating the starter cultures of poi

and exploring the characteristics of starter cultures.

The objectives of this study are to

1. Isolate and identify microorganisms in poi.

2. Prepare taro base for inoculation.

19

3. Examine the biological properties of the indigenous bacteria, including

growth in the taro base. and acid tolerance.

The eventual purpose of this project is to screen for bacterial strains and

improve the standardization of poi production and food safety by switching the

natural fermentation process to the controlled inoculation process.

20

CHAPTER 2MATERIALS AND METHODS

Introduction

MRS agar (Man, Rogosa and Sharpe agar) was chosen to isolate lactic

acid bacteria from poi in this study because the agar is a selective medium which

contains growth factors, such as polysorbate, acetate, magnesium, and

manganese, for lactobacillus species (http://www.merck.de). The isolated lactic

acid bacteria were first identified with traditional phenotypic tests, including

morphological observation, biochemical tests, and physiological tests. However,

phenotypic tests alone are insufficient to identify microorganisms accurately. The

sequence of 16S rRNA is one of the most effective indexes to define the

taxonomic status. The genotypic test by comparing 16S rRNA sequence was

introduced to positively identify the microorganisms in this project.

Acid tolerance and growth properties of the fermentation process also

were investigated to examine the possible commercial usage of the identified

bacterial strains in this project.

Preparation of bacterial isolates

Fresh poi samples were collected directly from the processing line of a

poi factory (Honolulu Poi Company, Honolulu, Hawaii) and immediately brought

back to the campus of University of Hawaii at Manoa. The unrefrigerated fresh

poi samples were homogenized and serially diluted into appropriate

concentrations with sterilized water at a ratio of 1:10. The MRS agar (Difco) was

21

used for inoculation. Indigenous lactic acid bacteria in poi were screened with the

spread-plate technique. A diluted sample of 0.1 ml was spread over the surface of

MRS agar with a sterilized, L-shaped bent rod. Lactic acid microorganisms were

then incubated at 300e for 24 hours.

The isolated bacteria were purified by randomly sampling single colonies.

A single bacterial colony was mixed with 1 ml of sterilized water and the resultant

bacterial suspension was streaked on MRS agar plates. The plates were

incubated at 300e for 24 hours. After incubation, the cultures could be used for

further testing, or be shipped, or be maintained at 4°e until next usage. The

bacterial isolates were subcultured once a week following the procedure of

culture transfer techniques. A loopful of culture was inoculated on MRS agar

plate by drawing the culture lightly over the surface in a straight or zigzag line.

The plates were incubated at 300e for 24 hours.

Phenotypic characterization

The bacterial isolates were examined by phenotypic tests, including

morphological observations, Gram-stain, biochemical tests, and physiological

tests.

Macroscopic and microscopic methods were used to observe the

morphological characteristics. The colony morphology was observed with

macroscopic methods. The colony characteristics, including size, shape, color,

surface texture, and elevation, were recorded. The colony was mixed with 0.85%

saline on the slide. The 100x phase objective microscope was used to observe

22

cell morphology. Gram stain was performed with 40x phase objective to focus

and 100x phase oil immersion objective.

Biochemical tests used in this project were the catalase test and the

oxidase test. Catalase is the enzyme that degrades hydrogen peroxide into water

and oxygen. The catalase test was performed with a 2% hydrogen peroxide

solution. The result was recorded as positive if there were bubbles in the mixture.

Oxidase plays an important role in the action of the electron transport system

during aerobic respiration. Oxidase is responsible for the formation of H20 or

H20 2 from oxygen (02), Oxidase reagent, N,N,N,N-Tetramethyl-p­

phenylenediamine -2HCL was used in the oxidase test. The isolated

microorganism was mixed with the oxidase reagent. The production of a dark

purple color within 30 seconds was recorded as positive.

API Rapid CH Strip system (API System, Plainfield, NY) was used to

investigate other physiological characteristics of the isolated bacteria. The

characteristics are the fermentability in various carbohydrate sources, the ability

to grow at high temperature, the hemolytic properties, and gas production. The

bacterial suspension was centrifuged at 8,000-10,000rpm for 20 minutes after

being incubated in MRS broth for 2 days at 30°C. The supernatant was discarded

and the pellet was resuspended in 10ml of sterile 0.85% saline. The bacterial

suspension was centrifuged in the same condition as stated above and the

supematant was discarded. The pellet was resuspended in 2ml of sterile 0.85%

saline. A total of one milliliter of this saline solution was divided into three

sections to inoculate MRS broth, MRS agar plate and TSA (Trypticase soy agar)

23

/5% blood agar plates individually. The bacteria-inoculated MRS agar plate was

incubated at 45°C for 48 hours to examine the bacteria's ability to grow at high

temperature. The bacteria-inoculated MRS broth was overlayed with paraffin wax

to test the growth and gas production of the isolated bacteria. The TSAl5% blood

agar plate was used to test the hemolytic properties of the bacteria. Both MRS

broth and TSAl5% blood plate were incubated at 30°C for 48 hours. Another 1ml

of resultant saline solution was used for the CH (carbohydrate) strip test. The test

was performed at 30°C for 4 days. The results were analyzed by the Vitek ATB

Expression System (Version 4.0). The Vitek ATB Expression System was

established based on "Bergey's Manual of Systematic Bacteriology" Volume 2,

1986 (Sneath, 1986).

Genotypic characterization

The genotypic characterization was performed by comparing 16S rRNA

sequence. Genomic DNA was isolated from purified bacterial colonies. The 16S

rRNA gene was amplified by PCR from the DNA sequence. The DNA sequences

corresponding to E. coli positions 005 and 1540, and 005 and 531 were used as

full-length package primer and 500bp package primer individually. Amplification

products were purified with Microcon 100 (Millipore) molecular weight cut-off

membranes. Running a portion of the products on an agarose gel was used to

check the quality and quantity of the purified product. Cycle sequencing of the

168 rRNA amplification products was performed with AmpliTaq FS DNA

polymerase and dRhodamine dye terminators. A 8ephadex G-50 spin column

24

was used to remove excess dye-labeled terminators from the reaction. The

resultant samples were ready for further test.

The samples were electrophoresed on an ABI Prism 377 DNA

Sequencer. Applied Biosystems DNA editing and assembly software were used

to analyze data. Sequence comparisons were carried out with the MicroSeq

software (Applied Biosystems)

The samples could be maintained for further usage after they were

centrifuged, dried under vacuum and frozen at -20°C. Once resuspended in a

solution of formamide/blue dextran/EDTA and denatured, the samples were

ready to follow the procedure stated above for further study.

Preparation of taro bases

The taro base treated with only heat was prepared from taro corms. Taro

corms were taken from Honolulu Poi Company. After being washed and stript

thoroughly, the outside layer of the taro corm was cut off carefully. The inside of

the corm was weighed and mixed with sterilized water at the ratio of 1:1 in the

blender. The taro mixture was sampled to measure the solid concentration. The

procedure used to exam the solid concentration was described above. The rest

of the taro mixture was heated until the boiling point while being stirred

continuously. The taro mixture was cooled to room temperature and used as taro

base.

The taro base was diluted with sterile water at the ratio of 1:2. The diluted

taro base was separated into three groups. Sucrose was supplemented into the

25

taro base at the concentration of 0, 1.3g/100g, and 3.2g/100g individually. The

taro bases were heated until the boiling point while continuously being stirred.

After cooled to room temperature, the resultant taro base was ready for further

usage.

The acid/heat treated taro base was prepared with commercial poi

obtained from the Honolulu Poi Company. The solid concentration was examined

to determine further dilution factors. The obtained fresh poi was sampled and

heated in an oven at 100°C overnight. The solid concentration was obtained by

calculating the weight difference between the samples before and after heating.

The acid treatment was performed with a combination of sorbic acid and lactic

acid. Sorbic acid was added to fresh poi until the final concentration reached 0.1 g

/100g (weight ratio). A lactic acid solution of 85% concentration subsequently

was added to the resultant taro paste until the pH decreased to approximately

3.0. Sterilized water was added into the treated taro paste to reach a final solid

concentration of 15%. After an overnight acidic treatment, the taro paste was

placed into water bath at BOaC for 2 hours and then cooled to room temperature.

A 6N NaOH solution was used to adjust the pH of the taro paste to be higher

than 6,0. The resultant taro paste was used as taro base.

The resultant taro bases were sampled and tested for the viability of

microorganisms with MRS agar and Simplate system. The taro base was

sampled and diluted at the ratio of 1:1O. A sample suspension of 0.1 ml was

inoculated and spread on the MRS agar. After a 24-hour incubation at 37°C, the

colonies were counted. Using the Simplate system, a single vial of Butterfield's

26

Buffer Concentrate was poured into a stomacher bag and 223mI of sterile

distilled water was added. After the bag was agitated for about 30 seconds, the

resultant buffer was mix€d completely with 25mg of sample. The sample

suspension was then added into the vial with a different medium for the

examination of TPC (total plate count), Coliform & E. coli, and Yeast & Mold

individually. The entire contents of a single test medium vial were poured on the

center platform of the base plates. Air bubbles were removed and the excess

medium was poured off. The Simplate used to detect the TPC was kepI in an

inverted position (transparent lid down) in the incubator at 35°C for 24 hours. The

wells that changed color from blue to pink after 24-hour incubation were recorded

as positive. The Simplate to detect Coliforms &E. coli was kept in an inverted

position (transparent lid down) in the incubator at 35°C for 24 hours. The wells

with a color different from the original yellow were recorded as positive in the

total coliform count. The wells with fluorescence under U.V. light were recorded

as positive in the E. coli count. The Simplate used to detect the Yeast &Mold

was kept (transparent lid up) in the incubator at 30°C for 48 hours. The wells with

fluoresce under the U.V. light were recorded as positive.

The taro base with negative results was used as the medium for further

inoculation of the bacteria isolated from poi.

Inoculation and Measurement of the growth of the bacteria

The parameters used to monitor the growth of bacteria were bacterial

counts and the pH change of the taro base. The bacteria used for inoculation

27

were grown for 24 hours on MRS agar. The bacterial cultures were inoculated

into a prepared taro base and the initial concentration of bacteria reached a

range of 104 to 106 CFU/ml. Then the cultures were incubated at room

temperature for a period of 5 to 6 days. Two samples were taken out each day:

one for a pH measure and another for a bacteria count. The pH was measured

directly with Orion 230 Aplus pH meter (Orion Research, MA, USA). The samples

used for bacterial counts were appropriately diluted so that the plate count would

be in the readable range of less than 103 CFU/ml. 0.1 ml of the diluted sample

was spread onto MRS agar. The plates were incubated at 30°C for 24 hours and

the numbers of bacteria colonies were counted.

Acid stress test

The acid stress test was conducted in a series of MRS broth, the pH of

which was adjusted to 6.10, 4.46, 3.70, 3.00, and 2.16 respectively. The pH of

the MRS broth was adjusted with the addition of 6.0N hydrochloric acid solution.

The bacterial culture used in acid test was grown on MRS agar at 30°C for 24

hours. A small aliquot of the prepared culture was added to 50ml MRS broth at

various pH values with an initial concentration of 105 CFU/ml. After incubation at

different acidity levels for 24 hours, the bacterial counts were measured on MRS

agar plates following the procedure described in section of the "Inoculation and

measurement of growth of the bacteria". Based on the results, the sublethal pH

and lethal pH were determined.

28

The acid adaptability of the bacteria was examined by challenging

adapted bacterial cells with the MRS broth at lethal pH. The protocol was as

follows. The bacteria were adapted in the MRS broth at sublethal pH and

incubated at 30°C for 24 hours. The adapted bacterial cells were diluted to

approximate concentration. 0.1 ml of bacteria suspension was inoculated into a

fresh MRS broth at lethal pH. The initial inoculation concentration of the bacteria

in the taro base was at the level of 105 CFU/ml. After a 24-hour incubation, the

sample was diluted appropriately and bacterial colonies were counted. The

survival rate was calculated by NINo (N is the CFU/ml after 24-hour incubation

and No is the CFUlml at zero time). The nonadapted bacteria cells were used in

the control group. The nonadapted bacteria cells were challenged directly with

the MRS broth at lethal pH. The challenging procedure is the same as above.

Examination of preliminary characteristics of a yeast-like colony

A yeast-like colony was first isolated from fresh poi after incubation on

MRS agar at 30°C for 24 hours. The cells were sub-cultured on potato dextrose

agar (PDA) at 25°C for 24 hours. The colony morphology and cell morphology

were examined. The methods have been described in the section on "Phenotypic

characterization" in this study.

Fructose and sucrose fermentation were investigated in this test. The

isolate was inOculated into assimilation tubes containing fructose or sucrose

broth. The culture was incubated at 2S"C for 24 hours.

29

The method used to identify the yeast-like colony has been described in

the section on "Genotypic characterization" in this study.

30

CHAPTER 3RESULTS and DISCUSSION

Screening Bacterial Strains from Fresh Poi

Starter cultures can be obtained by genetically designing new bacterial

strains with useful biological characteristics.or screening from wild strains in

nature. The traditional screening by isolating and identifying microorganisms from

fermented food was used in this study. Several microorganism strains were

isolated and identified from the poi obtained from Honolulu Poi Company.

Phenotypic identification

Phenotypic characterization was employed to offer preliminary

identification of microorganisms. A total of five lactic acid bacteria were identified

from the samples isolated form fresh poi. The five lactic acid bacteria were

Lactobacillus acidophilus, Lactobacillus delbruckii, Lactococcus lactis lactis,

Leuconostoc citreum, and Leuconostoc tactis. The results used for identification

were listed in the Table 2 and Table 3. Table 2 shows the biochemical

characteristics and the culture identifications of the five lactic acid bacteria. All

the cells of the five bacterial strains were cocccobacilli, gram-positive, catalase

negative, and oxidase negative, which all are the typical characteristics of lactic

acid bacteria. In addition, the five bacterial strains have their own unique

characteristics. The bacterial strain identified as Leuconostoc citreum showed no

lysis of blood cells. All the other four bacterial strains could imcompletely lyse

blood cells. The bacterial strain identified as Lactobacillus acidophilus was the

31

Table 2. Comparison of the biochemical characteristics and culture

identification of the five lactic acid bacteria

"+"; Positive; "-"; Negative

Lactobacillus Lactobacillus Lactococeus Leuconostoc Leuconostocacidonhiius delbrueckii lactis lactis citreum lactis

Colony Round, Smooth, Smooth, Smooth, Smooth,morphology raised, shiny, tiny, tiny, pinpoint, pinpoint,

and white translucent, translucent, and andand creamy and creamy translucent translucentwhite white

Cell Cocccobacilli Coccobacilli coccobacil1i coccobacilli coccobacillimornholoov-Gram + + + + +reactionCatalase - - - - -Oxidase - - - - -Hemolysis a a a I'Y a.Growth at + + - + +45°CGas + - - - -

I nroduction

32

only one that produced gas. Except the bacterial strain identified as Lactococcus

lactis lactis, all the other four bacterial strains could grow at 45°C. Table 3

summarizes the carbohydrate fermentability of these five bacteria. All these

species can make use of glucose, fructose, sucrose, D-mannose, and maltose.

None of them could use starch as the carbohydrate source.

Four of the five identified lactic acid bacteria could grow at the

temperature of 45°C, indicating that most of the starter cultures could grow at

relatively high temperature. The existence of Lactococcus lactis lactis, which

cannot grow at 45°C, suggests that the taro corms probably are not heated

evenly during poi production. These results are helpful in understanding the

source of starter cultures in natural fermentation process of poi.

Starch is the major component in taro. Since none of isolated bacteria

were able to consume starch, there must be some other mechanisms in poi that

can degrade starch. All the identified bacterial strains utilize glucose, fructose,

and sucrose as the carbon sources. These results partly explain the fast

consumption of sucrose and the change in content of fructose and glucose in poi

production observed in the previous study (Huang et aI., 1994), suggesting that

these sugars are the major carbon sources for growth. These results give some

clues in choosing useful starter cultures and preparation of taro base in the future

application.

Poi fermentation is a process to produce gas. Out of the five identified

strains, L. acidophilus did produce gas and therefore could be one of the most

prevailing strains in poi.

33

Table 3. Summary of the results of charbohydrate fermentation of the five

bacteria

Lactobacillus Lactobacillus Lactococcus Leuconostoc Leuconostocacidophilus delbrueckii lactis lactis citreum lactis

Control - - - - -N- + - + + -AcetvlalucosamineAmygdalin . - - + -L-Arabinose . - - + -Arbutin - - + + -Cellobiose - - + + -Esculin - - + + -D-Fructose + + + + +Galactose + - + - -B-Gentiobiose - - + + -D-Glucose + + + + +Lactose - - - - +Maltose + + + + +Mannitol - - + - -D-Mannose + + + + +Melibiose - - - - +a-Methyl D- - - - + -olucosideD-Raffinose - - - - +Ribose - - + - -Salicin - - + + -Starch - - - - -Sucrose + + + + +Trehalose - - + + -D-Turanose - - - + +D-Xylose + - + + -

34

Based on the results of phenotypic analysis, the bacterial strain first

identified as Lactobacillus acidophilus was further identified with genotypic

identification.

Genotypic identification of the taraet bacterial strain

Lactobacillus species are commonly present in human fecal samples.

Some lactobacillus species have received considerable attention due to their

putative healthy properties. Lactobacillus acidophilus is one of these well-known

probiotics.

The interesting bacterial strain identified as Lactobacillus acidophilus by

phenotypic characterization was further identified with the genotypic

characterization. Based on the analysis of 16S rRNA, the bacterial strain first

identified as Lactobacillus acidophilus was later identified as Weissella confusa.

The results are present in Table 4, which lists the first ten percentage of genetic

differences in 16S rRNA sequence between the tested bacterial strain and the

representative bacterial strains in the MICROSEQTM database. The percentage

of 16S rRNA sequence difference ranges from 0 to 13.88%. Based on this

comparison, the tested bacterial sample was identified as a 100% match to

Weissella confusa.

In Table 5, the carbohydrate fermentation pattern of the isolated bacterial

strain was compared to that of the Weissella confusa representative strain

(Facklam, et ai, 1989; Olano, et aI., 2001) and that of Lactobacillus acidophilus

35

Table 4. Percent genetic difference between the first bacterial strain and the

representative strains

Percent genetic difference from sample SDecies0.00 Weissella confusa3.02 Weissella viridescens4.09 Weissella minor5.86 Weissella kandleri6.25 Weissella Daramesenteroides8.17 Weissella halotolerans13.10 Lactobacillus vaainalis13.10 Lactobacillus reuteri13.64 Lactobacillus oris13.88 Lactobacillus suebicus

36

Table 5. Fermentation characteristics of the bacteria isolated in this study

compared with representative Weissella confusa and Lactobacillus

acidophilus ATCC 4356

+, growth; ., no growth.

Fermentation Weissella confuse Lactobacillus Our resultcharacteristics (from literature) acidophilus ATCC

4356 "(from literature)Arabinose - N/A -Fructose + N/A +Galactose N/A + +Glucose + N/A +Lactose - + -Maltose + + +Mannitol - . -Rhamnose . - -Salicin + + -Sorbitol N/A - -Sucrose + + +Trehalose - + -

37

ATCC 4356 (Sanders and Klaenhammer, 2001). Based on the comparison, the

carbohydrate fermentation pattems of these three bacteria were similar.

The API rapid CH system relies heavily on carbohydrate fermentation

patterns. The analysis system of the API Rapid CHL Strip system used in this

study was established based on "Bergey's Manual of Systematic Bacteriology"

volume 2, 1986 (Sneath, 1986). However, Weisella were established as one

genus of lactic acid bacteria in 1993 based on the 16S rRNA sequence data

(Collins et ai., 1993). Therefore the database of the analysis system doesn't

contain the genus Weissella.

In addition, the results of the characterization also show that the bacterial

strain isolated from poi is heterofermentative with the gas production. However,

Lactobacillus acidophiJus was homofermenter and WeisseJla confusa was

obligate heterofermenter (Beuchat, L. R., 1995). This comparison further

suggested that the isolated bacteria most possibly are Weissella confusa.

The 16S rRNA subunit is highly conserved and is considered to be an

excellent chronometer of bacteria over time. Therefore the information of 16S

rRNA allows for the determination of precise phynogenetic relationships (Jay,

1996b). Based on all the analysis, the isolated bacterial strain finally is identified

as Weissella confusa.

There have been nine species in the genus Weissella, including

WeisseJla confusa. Weissella haJotoJerans, Weissella hellenica, Weissella

kandJeri, Weisse/Ja minor, Weisse/Ja paramesenteroides, Weisse/Ja

thailandensis. Weissella viridescens, and Weissella cibaria sp. nov (Bjorkroth et

38

aI., 2002). WeisselJa strains are distributed in nature and in a variety of fermented

food. Compared with other WeisseJla species, WeisseJla confusa was more

frequently isolated and identified from fermented food. WeisseJla confusa had

been isolated from boza (Hanicoglu and Karapinar, 1997), som-fak (Paludan­

Muller et aI., 1999), tapai (Bjorkroth et aI., 2002), togwa (Mugula et aI., 2003) and

bushera (Muyanja et aI., 2003). Till now, there is no further study on WeisseJla

confusa, such as its growth, its response to acidity, and its significance in

fermented food and human health (Facklam et aI., 1989; Collins et aI., 1993;

Christine et aI., 1999; Arrel et al., 2001, K. Johanna Bjorkroth et ai, 2002;). The

purification and identification of the bacterial strain WeisseJla confusa in this

project provide the possibility to study the biological properties of the bacteria

and its significance in food fermentation.

The Biological Properties of Weissella confusa

The effect of pH on the growth of Weissella confusa

The effect of pH on the growth of WeisseJla confusa was examined in the

MRS broth acidified with 6.0N hydrochloric acid. After being adjusted to a specific

pH, the broth was inoculated by W. confusa at a concentration of 1.8x1 05 CFU/ml

and then incubated at 30°C for 24 hours. Fig. 1 shows the effect of pH on the

growth of W. confusa after incubation. The bacteria grew rapidly at pH 6.10 and

the population increased from 105CFU/ml to 1010 CFU/ml after 24 hours. The

39

Fig. 1. The Effect of pH on the growth of Weissella confusa

12

o~..-6.1 4.46 3.7

pH

40

3 2.16

growth rate decreased when the pH of the broth was either 4.47 or 3.7. At the pH

level of 3.0 or 2.16, the bacterial count dropped to zero after the incubation.

These results indicate that these bacteria would be able to grow in sour poi,

which generally have a pH ranges between 3.9 and 4.5.

Lactic acid bacteria tend to be able to grow in the relatively acidic

condition due to a continuous accumulation of laelic acid produced during

growth. However, there are different lower pH limits for growth because of the

various capability of acid tolerance. For example, the known probiotic genus

Bifidobacterium usually doesn't grow at pH below 4.5 and one lactic acid baeleria

Pediococcus acidilactici can grow at pH 3.8 (Ray, 2000e). In comparison with

these known lactic acid bacteria, the isolated W. confusa is able to grow in a

more acidic media. Although there are a number of recent studies on the

mechanism of acid tolerance response of bacteria, the mechanism of acid

tolerance response of W. confusa has not been studied. The acid adaptability of

W confusa was explored in the next study on its acid tolerance response.

Acid tolerance response

In response to stressful stimulation, many microorganisms develop an

adaptive stress response which increases their ability to survive an extreme

condition, such as a high acid environment (Foster, 1999). Acid tolerance

response (ATR) is the process that a brief exposure of cells to mild acidic

41

environment enables them to survive subsequent exposure to a more acidic

environment (Ray, 2000f).

In this acid tolerance response the pH value of 4.46 was chosen as the

sublethal pH and the pH value of 3.0 was chosen as the lethal pH. Both were

based on the previous results. The sublethal pH is an acidic condition that the

tested cells can grow although the growth rate decreases. The lethal pH is the

one that the tested cells can't grow without adaptation.

The results of the survival rate of Weissella confusa with or without

adaptation are displayed in Fig. 2. With an initial concentration of 5.3 x 105

CFU/ml, the adapted cells had a survival count of 3.7x104• For the control group

without adaptation, the cells have only 10 viable cells detected with ihe initial

concentration of 3.2 x 105 CFU/ml. The survival rate of the isolated bacteria was

increased by adaptation when the cells were exposed to the challenge of pH 3.0.

The results indicated that the bacteria could be adapted by the sublethal

environment and became more acid resistant.

Acid tolerance response has been studied in a number of lactic acid

bacteria. There are different identified mechanisms. Some studies show that the

acid tolerance is consistent with the production of acid shock protein (Drici­

Cachon et aI., 1996; Lorca et aI., 2001). Arginine catabolism was proved to

contribute to acid tolerance in some other lactic acid bacteria (Rollan et aI.,

2003).

This is the first exploration about the acid tolerance of Weissellaconfusa.

The result suggests that the Weissella confusa strain may be a valuable starter

42

Fig. 2. Survival of unadapted and acid-adapted cells of Weissella confusa

strains during pH challenge (pH 3.00)

o

1-1-

.flfiii -2>.~

:IIII

'0 -3E

~..~-4

-5nonadapted cells adapted cells

_logarithm of survival rate

43

culture with the characteristics of acid tolerance and may play an important role

in food fermentation.

Effect of taro based medium on the growth of Weissella confusa

The effect of taro medium on the growth of W. confusa was studied.

There are two taro solid content levels (10% and 3%) used for comparison.

Sucrose was used as the complementary component in the 3% solid content taro

paste. The levels of sucrose are 0%, 1.3%, and 3.2% respectively. The cell

population and the change in pH were used to measure the growth of W.

confusa. In Figure 3, the bacterial counts and the pH of the medium after

incubation for three days are compared. With the initial pH of 5.60, the pH of

medium decreased fast and the pH values of the four taro mediums were

practically identical, ranging from 3.39 to 3.73 after the incubation. However, the

pH generally dropped from 6.40 to pH 4.13 after 3 days in a natural fermentation

of poi (Huang et ai., 1994). Also the solid concentration of naturally fermented poi

is around 20%, greater than the 3% and 10% used in this study.

All four media supported the fast growth of the bacteria during the

incubation period. The bacterial counts in the media with the solid concentration

of 10% increased from approximately 1x 105 to above 1x 109• At the solid

concentration of 3%, the medium with the addition of sucrose at the level of

1.3g/1 DOg gave the best result and the bacterial count reached 1x 1011• As

indicated in Huang's study (1994), sucrose in taro may be the preferred

carbohydrate source and therefore a proper amount of sucrose would be needed

44

Fig. 3. Effect of different taro base on the growth of Weissella confusa and

the pH of the taro base

12

10

8

6

4

2

0

v§i"

-tJ0

...~rJ"o

• cell population (Log CFUlg

45

.pH values

in a taro-based medium for a sustainable fermentation. The results of three-day

fermentation seem to indicate that there is an optimal amount of sucrose addition

and 1.3g/100g is the best level among the tested three levels.

Growth curves of Weissella confusa in taro/sucrose mixture and acid/heat

treated poi

The growth curves of W. confusa in taro/sucrose base and acid/heat

treated poi were established. The taro/sucrose mixture with 3% solid content of

taro and 1.3% added sucrose was chosen to test the growth curve of W. confusa.

Fig. 4 exhibits the growth curve of the bacteria along with its pH change in the

taro/sucrose mixture during a five-day period. With an initial concentration of

1.7x1 05 CFUlml, W. confusa grew rapidly in the first three days, increasing to

1011 CFU/ml and the pH dropped to 3.39 at the third day. At the end of the fifth

day, the bacterial number reached to 1010 CFU/ml. Fig. 5 shows the growth curve

of W. confusa and its pH change in the acid/heat treated poi. The initial

concentration of bacterial cells was 9x1 03 CFU/g. The bacterial cells also grew

fast and reached above 1011 CFUlg after three-day incubation. At the same time

the pH level decreased to around 5.25.

The above results are the first attempts to inoculate the lactic acid

bacteria W. confusa in taro/sucrose mixture or acid/heat treated poi. W. confusa

used in the study was isolated from naturally fermented poi. The bacteria grew

vigorously in both taro bases. The relatively high bacterial counts were

maintained for up to 7 days in the acid/heat treated poi. These preliminary results

46

Fig. 4. Values of pH and logarithm numbers of Weissella confusa during

fermentation in taro-base treated with heat

12 6

10 5-m-::::)II.CJ 8 4m0

...I-c 3%0 Q.;;

C'll'5Q.

4 20Q.

a;CJ

2 1

0 0

0 1 2 3 4 5

Days

-logarithm numbers of bacteria count

47

Fig. 5. Values of pH and logarithm numbers of Weissella confusa during

fermentation in taro-base treated with acid and heat

14 8

-!!J 12:J - 7LL(J 10en0 6.J 8-c J:0 a... 6.!! - 5~a.

40a.- - 4CD 2(J

o- - 3

0 1 2 3 4 5 6 7

days

-+-IogarithmnulTbers c:A bacteria count pH

48

seem to indicate that the isolated W. confusa would be a feasible starter culture

in making a taro-based fermented food with controlled fermentation. Although

commercial fermented food production usually uses a combination of more than

one bacterial strain in order to maximize the synergies among the strains,

Marshall and Tamine (1997) pointed out that monoculture is cost-effective in

managing industrial-scale production. In addition, Beasley et al. (2003) have

recently used a monoculture of Lactococcus /actis to produce a fermented

soymilk with promising sensory characteristics. The results here seem to indicate

that the monoculture of W. confusa in a taro base is rather feasible.

The solid content in the acid/heat treated poi was about 15% at the point

of inoculation, while the solid content in the taro/sucrose mixture was about

4.3%. The fact that both media supported the fast growth of W. confusa indicated

that there were sufficient nutrients even in the taro-based medium with low level

of solid content. This suggests that there would be a wide range of acceptable

taro-based medium to be used for fermentation with the lactic acid bacteria. The

flexibility would be important in formulating a taro-based fermented product, in

which taste, mouthfeel, and costs are all factors to be considered.

One noticeable difference between the taro/sucrose mixture and the

acid/heat treated poi was the changes of pH. After three days, the pH was 5.25 in

the acid/heat treated poi and 3.39 in taro/sucrose individually. The pH in the

acid/heat treated poi was much higher than the pH value around 4.10 in most

three-day sour poi products. The fresh poi was sterilized by a combination of acid

and heat treatment. The acidlheat treatment may have some effect on the pH

49

change of the taro medium. In addition, the solid concentration of the acid/heat

poi was 15% comparing with that of 4.3% in the taro/sucrose. The solid content

may also contribute to the buffer capacity of the acid/heat treated poi and explain

the difference between these two media.

Fresh poi is hard to handle. The sticky texture of poi renders poor heat

transfer so that sterilization could not be accomplished by heat alone. Lactic acid

alone is not enough to kill all the microorganisms in the poi. Therefore, fresh poi

was treated by a combination of acid and heat, which indicated a long time of

preparation. By comparison, the preparation of taro/sucrose mixture needed heat

alone and cost shorter time. The fast drop of pH in taro/sucrose mixture helps to

reduce the rate of contamination. The appropriate addition of sucrose would be

beneficial for improving the taste and promoting the growth. Given to all these

factors, the taro/sucrose mixture gives beiter result than acid/heat treated poi.

Some improvement to the taro/sucrose mixture is needed to obtain the desired

final product. Further study should be focused on the optimization of the taro­

based medium, based on the growth of the bacteria, pH change, effect in taste,

and cost factors.

Genotypic Characterization of a Lactic Acid Bacterial Strain

Isolated from Sour Poi

Some effort was given to the isolation and identification of

microorganisms from sour poi. One strain of bacteria was isolated from sour poi

50

at pH 3.84. Based on the preliminary observation, the bacteria are single or

paired cocobacilli, gram positive, catalase negative, and oxidase negative. The

colony is flat and translucent.

Table 6 shows the percentage of genetic differences in the 16S rRNA

sequence between the tested bacteria and the first ten representative strains in

the MICROSEQTM database. The percentages of 16S rRNA sequence difference

ranged from 0.27 to 10.96%. Based on this comparison, the tested bacterial

strains showed a 99.73% match to Lactobacillus plantarum. The bacterial strain

was most probably identified as Lactobacillus plantarum.

Lactobacilus plantarum is facultative homofermenter. Its growth will end at

a pH of 3.6 to 4.0 (Sneath et aI., 1986). Therefore, Lactobacilus plantarum

usually dominate at the late stages of fermentation (Oyewole and Odunfa, 1990;

Hounhouigan et aI., 1993). These studies may elucidate the reason why L.

pantarum can be isolated from sour poi.

Lactobacilus plantarum is a valuable inhabitant of the intestinal tract of.

human beings (Lund et aI., 2000). Lactobacilus plantarum is also widely

distributed in the fermenting plant materials, such as most vegetable

fermentations and natural cereal fermentations (Oyewole and Odunfa, 1990;

Olasupo er aI., 1997). Lactobacilus plantarum may be responsible for the acid

production, which inhibited the growth of some undesirable microorganisms. The

existence of Lactobacilus plantarum may suggest part of the healthy benefits of

poi.

51

Table 6 Percent genetic difference between the isolated bacterial strain

from sour poi and representative strains

Percent genetic difference from sample Soecies0.27 Lactobacillus Dlantarum0.36 Lactobacillus Dentosus8.89 Lactobacillus aJimentarius9.36 Lactobacillus collinoides9.51 Lactobacillus brevis10.07 Lactobacillus farciminis10.24 Lactobacillus hi/Clardii10.42 Lactobacillus Darabuchneri10.87 Lactobacillus rhamnosus10.96 Pediococcus Darvulus

52

Genotypic Characterization of One Yeast-like Colony Isolated

from Sour Poi

There was a preliminary study on a yeast-like colony isolated from a sour

poi product. Yeasts have been frequently observed in sour poi. Allen & Allen

(1933) has identified two yeast-like colonies, Mycoderma cerevisiac and Oidium

/actis, as starter cultures of traditional poi. In this study, the cells of the isolated

yeast-like colony are large, round and oval. The colonies on PDA are white,

opaque, round and raised. The yeast-like culture was able to ferment fructose

and sucrose.

Table 7 shows that the percentage of genetic difference in 168 rRNA

sequence between the sample and the first ten representative strains in the

MICR08EQTM database. The percentage of 168 rRNA sequence difference

ranges from 0 to 10.94%. Based on the comparison, the tested samples showed

a 100% match to Candida tropicalis.

Candida tropicalis is found among the normal human mucocutaneous flora

and in nutrient-rich environment. Candida tropicalis is one of the opportunistic

mycoses. It is known to cause infection, especially in immunocompromised or

predisposed patients. It is a major cause of endocarditis among patients with

heart damages or intravenous drug abuse. Also it leads to systemic disease in

patients with cancer, diabetes, or chronic alcoholism (Land & McCracken, 1991).

Generally, Candida tropica/is is gas production positive and acid

production positive (Larose et aI., 1976). However, it doesn't produce acetic acid.

53

Table 7. Percent genetic difference between the yeast-like strain and

representative strains

Percent genetic difference from sample Species

0.00 Candida tropicalis2.81 Candida maltosa4.38 Lodderomvces elonqisporus4.84 Candida lodderae5.94 Candida paraDsiJosis5.84 Candida osornensis7.19 Candida albicans10.00 Candida erqatensis10.66 Candida shehatae shehatae10.94 Candida sake

54

Candida tropicalis can make use of carbohydrates for growth, such as starch,

glucose, galactose, maltose, succinic acid, and others. (Barnett et ai., 2000)

The existence of Candida tropicafis partly explains the usage of starch.

Based on my study, the lactic acid bacteria isolated from poi cannot make use of

starch as a carbon source. However, starch is the major component of taro.

There must be some other microorganisms that degrade starch to provide

enough carbon sources for the growth of species in poi. Candida tropicalis may

be one of the starter cultures which are able to make use of starch.

As Candida tropicalis maybe cause opportunistic mycosis under some

condition, poi should not be suggested as a dietary aid to immunosuppressive

patients or immunodeficient patients.

Finally, the existence of Candida tropicalis shows that the present

processing method of poi potentially could cause some diseases if the conditions

are not well controlled. Based on these results, it is necessary to study the

microflora in poi and improve the safety of poi by changing the production from

natural fermentation to controlled fermentation.

Conclusion

Lactic acid bacteria are responsible for the production of poi. Base on the

phenotypic analysis, the lactic acid bacteria isolated from fresh poi in this study

are Lactobacillus acidophilus. Lactobacillus delbrucki/, Lactococcus factis factis,

Leuconostoc citreum, and Leuconostoc factis. One lactic acid bacteria isolated

55

from sour poi is Lactobacilus plantarum and one yeast-like colony isolated from

poi is Candida tfOpicalis based on genotypic analysis.

The lactic acid bacteria first identified as Lactobacillus acidophilus is

identified as Weissella confusa based on phenotypic analysis and genotypic

analysis. Weissella confusa can grow in the acidic condition and its acid

tolerance is adaptable. Sucrose doesn't inhibit the growth of the bacteria and the

taro/sucrose mixture supports the fast growth of the bacteria. The result suggests

that the bacteria would be a feasible starter culture in making a taro based

fermented food with controlled fermentation and it is feasible to produce taro­

based fermented food with the monoculture of W. confusa.

Based on these results, More characterizations of the biological

properties of Weissella confusa are necessary.

56

APPEDIXA

The USDA's nutritional makeup of Poi

NOB No' 11349.Value per

Sample Std.Nutrient Units 100 grams of

edible portionCount Error

Proxlmates·Water g 71.64 2Energy kcal 112 0Energy kj 469 0Protein Il 0.38 2

Total lipid (fat) g 0.14 1Carbohvdrate, by difference g 27.23 0

Fiber, total dietary g 0.4 0Ash g 0.61 1

MineralsCalcium, Ca mg 16 1

Iron, Fe mg 0.88 1Magnesium, Mg mg 24 1Phosphorus, P mg 39 1Potassium, K mg 183 2Sodium, Na mg 12 2

Zinc, Zn mg 0.22 0Copper, Cu mil 0.166 0

Manganese, Mn mg 0.370 0Selenium, Se mcg 0.7 0

VitaminsVitamin C, total ascorbic acid mg 4.0 0

Thiamin mg 0.130 0Riboflavin mg 0.040 0

Niacin mg 1.100 0Pantothenic acid mg 0.293 0

Vitamin B-6 mg 0.273 0Folate, total mcg 21 0

Folic acid mcg 0 0Folate, food mcg 21 0Folate, DFE MC!l-DFE 21 0

Vitamin B-12 mcg 0.00 0Vitamin A, IU IU 20 0Vitamin A, RE mC!l-RE 2 0

57

Vitamin E mg ATE I 0.180 I 0 ILipids

Fatty acids, total saturated g 0.029 04:0 g 0.000 06:0 g 0.000 08:0 g 0.000 010:0 g 0.000 012:0 g 0.000 014:0 g 0.000 016:0 g 0.025 018:0 g 0.004 0

Fatty acids, total monounsaturated g 0.011 016:1 undifferentiated g 0.000 018:1 undifferentiated g 0.011 0

20:1 g 0.000 022: 1 undifferentiated g 0.000 0

Fatty acids, total polyunsaturated g 0.058 018:2 undifferentiated g 0.040 018:3 undifferentiated g 0.018 0

18:4 g 0.000 020:4 undifferentiated l! 0.000 0

20:5 n-3 g 0.000 022:5 n-3 l! 0.000 022:6 n-3 g 0.000 0

Cholesterol mg 0 0USDA Nutrient Database for Standard Reference, Release 14 (July 2001)

58

APPEDIX B

The result of the first LAB strain isolated from poi

CULTURE 10 REPORT

Culture Condition Test ResultDeMan Rogosa Sharpe Colony Morphology Tiny, white, roundAgar 48 hous at 30°C colonies

Cell Morpholoav CoccobacilliCatalase NeaativeOxidase NegativeGram reaction Positive

DeMan Rogosa Sharpe Growth PositiveBroth with agar plug, 4 Gas Production Positivedays at 30°CBlood Agar Plate, 24 Hemolysis Alphahours at 30°CDeMan Rogosa Sharpe Growth at 45°C PositiveAgar, 4 days at 45°C

59

API RAPID CH STRIP RESULTS

Test Result Test ResultControl - Esculin -Glycerol - Salicin -Erythritol - Cellobiose -D-Arabinose - Maltose +L-Arabinose - Lactose -Ribose . Melibiose -D-Xylose + Sucrose +L-Xylose - Trehalose -Adonitol - Inuline -B·Methyl-xyloside - Melezitose -Galactose + D-Raffinose -D·Glucose + Starch -D·Fructose + Glycogen -D-Mannose + Xylitol -L-Sorbose - B-Gentiobiose -Rhamnose - D·Turanose -Dulcitol - D-Lyxose -Inositol . D-Tagatose -Mannitol - D-Fucose -Sorbitol - L-Fucose -a-Methyl D· - D·Arabitol -Mannosidea-Methyl D- - L·Arabitol -glucosideN- + Gluconate -AcetylglucosamineAmygdalin - 2-Keto·gluconate -Arbutin - 5-Keto·gluconate -

IDENTIFICATION

Most probable identification:Lactobacillus acidophilus

60

APPEDIX C

The result of the second LAB strain isolated from poi

CULTURE 10 REPORT

Culture Condition Test ResultDeMan Rogosa Sharpe Colony Morphology Smooth, translucent, tiny,Agar 48 hous at 30°C creamy white colonies

Cell Moroholoav Sinale caired coccobacilliCatalase NegativeOxidase NegativeGram reaction Positive

DeMan Rogosa Sharpe Growth PositiveBroth with agar plug, 4 Gas Production Negativedays at 30°CBlood Agar Plate, 24 Hemolysis Alphahours at 30°CDeMan Rogosa Sharpe Growth at 45°C PositiveAgar, 4 days at 45°C

61

API RAPID CH STRIP RESULTS

Test Result Test ResultControl - Esculin -Glycerol - Salicin - .

Erythritol - Cellobiose -D-Arabinose - Maltose +L-Arabinose - Lactose -Ribose - Melibiose -D-Xylose - Sucrose +L-Xvlose - Trehalose -Adonitol - Inuline -~·Methyl. - Melezitose -xylosideGalactose · D·Raffinose -D·Glucose + Starch -D·Fruciose + Glycogen -D-Mannose + Xylitol -L·Sorbose - B-Gentiobiose -Rhamnose · D·Turanose -Dulcitol - D-Lyxose -Inositol - D·Tagatose -Mannitol - D·Fucose -Sorbitol - L-Fucose -a-Methyl D· · D·Arabitol -Mannosidea-Methyl D· · L·Arabitol -glucosideN- - Gluconate -AcetvlalucosamineAmygdalin - 2·Keto-gluconate -Arbutin - 5-Keto-gluconate -

IDENTIFICATION

Most probable identification:Lactobacillus de/brueckii

62

APPEDIX 0

The result of the third LAB strain isolated from poi

CULTURE 10 REPORT

Culture Condition Test ResultDeMan Rogosa Sharpe Colony Morphology Smooth, translucent, tiny,Agar 48 hous at 30°C creamy white colonies

Cell MoroholoaY Sinale paired coccobacilliCatalase NeaativeOxidase NeaativeGram reaction Positive

DeMan Rogosa Sharpe Growth PositiveBroth with agar plug, 4 Gas Production Negativedays at 30°CBlood Agar Plate, 24 Hemolysis Alphahours at 30°CDeMan Rogosa Sharpe Growth at 45°C NegativeAaar, 4 days at 45°C

63

API RAPID CH STRIP RESULTS

Test Result Test ResultControl - Esculin +Glycerol - Salicin +Erythritol - Cellobiose +D·Arabinose - Maltose +L·Arabinose - Lactose -Ribose + Melibiose -D-Xylose + Sucrose +L·Xvlose - Trehalose +Adonitol - Inuline -B-Methvl-xvloside - Melezitose -Galactose + D-Raffinose -D-Glucose + Starch -D-Fructose + Glvcoaen -D-Mannose + Xylitol -L-Sorbose - 13-Gentiobiose +Rhamnose - D-Turanose -Dulcitol - D-Lyxose -Inositol - D-TaQatose -Mannitol + D-Fucose -Sorbitol - L-Fucose -lX-Methyl D- - D-Arabitol -Mannosidea-Methyl D- - L-Arabitol -glucosideN- + Gluconate -AcetylglucosamlneAmvadalin - 2-Keto-aluconate -Arbutin + 5-Keto-gluconate -

IDENTIFICATION

Most probable identification:Lactococcus lactis lact;s

64

APPEDIX E

The result of the fourth LAB strain isolated from poi

CULTURE 10 REPORT

Culture Condition Test ResultDeMan Rogosa Sharpe Colony Morphology Smooth, pinpoint,Agar 48 hous at 30°C translucent colonies

Cell Morphology Single paired coccobacilliCatalase NeQativeOxidase NeaativeGram reaction Positive

DeMan Rogosa Sharpe Growth PositiveBroth with agar plug, 4 Gas Production Negativedavs at 30°CBlood Agar Plate, 24 Hemolysis Negativehours at 30°CDeMan Rogosa Sharpe Growth at 45°C PositiveAoar, 4 days at 45°C

65

API RAPID CH STRIP RESULTS

Test Result Test ResultControl - Esculin +Glycerol - Salicin +Erythritol - Cellobiose +D-Arabinose - Maltose +L-Arabinose + Lactose -Ribose - Melibiose -D·Xylose + Sucrose +L-Xylose - Trehalose +Adonitol - Inuline -~-Methyl- - Melezitose -xvtosideGalactose - D-Raffinose -D-Glucose + Starch -D-Fructose + Glycogen -D-Mannose + Xvtitol -L-Sorbose - a-Gentiobiose +Rhamnose - D-Turanose +Dulcitol - . D-Lvxose -Inositol - D-Tagatose -Mannitol - D-Fucose -Sorbitol - L-Fucose -a-Methyl 0- - D-Arabitol -Mannosidea-Methyl D- + L-Arabitol -alucosideN- + Gluconate -AcetvlalucosamineAmygdalin + 2-Keto-aluconate -Arbutin + 5-Keto-aluconate -

IDENTIFICATION

Most probable identification:Leuconostoc citreum

66

APPEDIX F

The result of the fifth LAB strain isolated from poi

CULTURE 10 REPORT

Culture Condition Test ResultDeMan Rogosa Sharpe Colony Morphology Smooth, pinpoint,Agar 48 hous at 30°C translucent colonies

Cell MorDholoav Sinale Daired coccobacilliCatalase NeqaliveOxidase NeaativeGram reaction Positive

DeMan Rogosa Sharpe Growth PositiveBroth with agar plug, 4 . Gas Production Negativedays at 30°CBlood Agar Plate, 24 Hemolysis Alphahours at 30°CDeMan Rogosa Sharpe Growth at 45°C PositiveAaar, 4 days at 45°C

67

API RAPID CH STRIP RESULTS

Test Result Test ResultControl - Esculin -Glycerol - Salicin -Erythritol - Cellobiose -D-Arabinose - Maltose +L-Arabinose - Lactose +Ribose - Melibiose +D-Xv\ose - Sucrose +L-Xvlose - Trehalose -Adonitol - Inuline -~-Methyl- - Melezitose -xylosideGalactose - D-Raffinose +D-Glucose + Starch -D-Fructose + Glvcoaen -D-Mannose + Xylitol -L-Sorbose - 6-Gentiobiose -Rhamnose - D-Turanose +Dulcitol - D-Lyxose -Inositol - D-Tagatose -Mannitol - D-Fucose -Sorbitol - L-Fucose -a-Methyl D· - D-Arabitol -Mannosidea-Methyl D· - L-Arabitol -alucosideN- . Gluconate -AcetvlalucosamineAmygdalin - 2-Keto-aluconate -Arbutin - 5-Keto-gluconate -

IDENTIFICATION

Most probable identification:Leuconostoc lactis

68

LITERATURE CITED

Allen, O.N., Allen, EK, 1933. The manufacture of poi from taro in Hawaii: with

special emphasis upon its fermentation. Hawaii, Agri. Exp. Stn., Bull. No. 70.

Ampe, F., Omar, N. B., Moizan, C., Wacher, C and Guyot, J.-P., 1999.

Polyphasic study of the spatial distribution of microorganisms in Mexican

Pozol, a fermented maize dough, demonstrates the need for cultivation­

independent methods to investigate traditional fermentations. Applied and

Environmental Microbiology 65 (12),5464-5473.

Barnett, J.A., Payne, RW. &Yarrow, D., 2000. Yeasts: Characteristics and

identification, 3rd Ed., Cambridge University Press, pp. 270-271.

Battcock, M., Azam-AIi, S., 1998. Bacterial fermentation. In: Fermented Fruits

and Vegetables: A Global Perspective.

http://www.fao.org/docrep/x0560e/x0560e10.htm

Beasley, S., Tuorila, H., Saris, P.E.J., 2003. Fermented soymilk with a

monoculture of Lactococcus lactis, International Journal of Food Microbiology

81,159-162.

Begley, BW., Spielmann, H., Vieth, G.R, 1981. Poi consumption: consumption

of a traditional Staple in the Contemporary Era in Honolulu, Hawaii. Hawaii

Institute of Tropical Agriculture and Human Resources, College of Tropical

Agriculture and Human Resources, University of Hawaii, pp. 3-4.

Beuchat, L. R, 1995. Application of biotechnology to fermented foods. Food

Technology 49 (1), 97-99.

69

Bjorkroth, K. J., Schillinger, U., Geisen, R., Weiss, N., Hoste, B., Holzapfel, W.H.,

Korkeala, H.J. and Vandamme. P., 2002. Taxonomic study of Weissella

confusa and description of Weissella cibaria sp. nov, detected in food and

clinical samples. International Journal of Systermatic and Evolutionary

Microbiology 52,141-148.

Bogovic-Matijasic, B., Rogelj, I., Nes, I.F., Holo, H., 1998. Isolation and

characterization of two bacteriocins of Lactobacillus acidophilus LF221.

Applied Microbiology and Biotechnology 49, 606-612.

Breidt, F., Fleming, H. P., 1997. Using lactic acid bacteria to improve the

safety of minimally processed fruits and vegetables. Food Technology 51 (9),

44-49.

Collins, M.D.• Samelis, J., Metaxopoulos, J. and Wallbanks, S., 1993. Taxonomic

studies on some leuconostoc-like organisms from fermented sausages:

description of a new genus Weissella for the Leuconostoc paramesenteroides

group of species. Journal of Applied Bacteriology 75, 595-603.

Cunningham-Rundles, S., Ahrne, S., Bengmark, 5., 2000. Probiotics and

immune response, Am J Gastroenterol 95(1 Suppl), pp. 22-25.

Daeschel, M.A., 1989. Antimicrobial Substances from Lactic Acid Bacteria for

Use as Food Preservatives. Food Technology 42, 164-167.

Drici-Cachon, Z., Guzzo. J., Cavin. J.-F., Divies, C., 1996. Acid tolerance in

Leuconostoc oemos: Isolation and characterization of an acid-resistant

mutant. Applied Microbiology and Biotechnology 44,785-789.

70

European food and feed cultures association, http://W.JIIW.effca.com

Facklam, R., Hollis, D. and Collins, M.D., 1989. Identification of gram-positive

coccal and coccobacillary vancomycin-resistant bacteria. Journal of Clinical

Microbiology 27,724-730.

Foschino, R., Arrigoni, C., Picozzi, C., Mora, D., Galli, A, 2001. Phenotypic and

genotypic aspects of Lactobacillus samfranciscensis strains isolated from

sourdoughs in Italy. Food Microbiology 18, 277-285.

Foster, J.W., 1999. When protons attach: microbial strategies of acid adaptation.

Curro Opin. Microbiol. 2, 170-174.

Fujisawa, T., Benno, Y., Yaeshima, T., Mitsuoka, T., 1992. Taxonomic study of

the Lactobacillus acidophilus group, with recognition of Lactobacillus

gallinarum sp. Nov. and Lactobacillus johnsonii sp. Nov. and synonymy of

Lacobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of

Lactobacillus amylovorus (Nakamura 1981). Int. J. Syst. Bacteriol. 42, 487­

491.

Hanicoglu, 0., Karapinar, M., 1997. Microflora of boza, a traditional fermented

Turkish beverage. International Journal of Food Microbiology 35,271-274

Hansen, E.B., 2002. Commercial bacterial starter cultures for fermented foods of

the future. International Journal of Food Microbiology, 78, 119-131.

Hounhouigan, D.J., Nout, M.J.R., Nago, C.M., Houben, J.H., Rornbouts, F.M.,

1993. Characterization and frequency distribution of species of lactic acid

bacteria involved in the processing of rnawe, a fermented maize dough from

Benin. International Journal of Food Microbiology 18 (4),279·287.

71

Huang, A.S., Lam, S.Y., Nakayama, T.M., Lin, H., 1994. Microbiological and

chemical changes in poi stored at 20°C. Journal of Agricultural and Food

Chemistry 42, 45-48.

Jack, R.W., Tagg, J.R., Ray B., 1995. Bacteriocins of gram-positive bacteria.

Microbiological Review 59 (2), 171-200.

Jay, J.M., 1996a. Modern Food Microbiology, Fifth ed. Chapman & Hall Inc., New

York, pp. 132-133.

Jay, J.M., 1996b. Modern Food Microbiology, Fifth ed. Chapman & Hall Inc., New

York, pp. 14-15.

Jobin, M-P, Clavel, T., Carlin, F., Schmitt, P., 2002. Acid tolerance response is

low-pH and late-stationary growth phase inducible in Bacillus cereus TZ415.

International Journal of Food Microbiology 79,65-73.

Kim, W.S., Perl, L., Park, J.H., Tandianus, J.E., Dunn, N.W., 2001. Assessment

of stress response of the probiotic Lactobacillus acidophilus. Current

Microbiology 43, 346-350.

Klaenhammer, T.R. 1988. Bacteriocins of lactic acid bacteria. Biochimie 70,

337-349.

Land, G.A. and McCrachen, A.W., 1991. Fungal infections in the compromised

host. In: Arora, D.K., Ajello, L., Mukerji, K.G. (Ed.), Handbook of Applied

Mycology. Marcel Dekker, New York, pp.94-96

Larone, D.H., 1976. Medically Important Fungi: A guide to identification. Harper

and Row Publisher, Hagerstown, MD, pp. 49.

72

Larone, D.H., 1995. Medically Important Fungi: A guide to identification, 3rd Ed.,

ASM Press, Washington D.C., pp. 65-67.

Lee, N. -K., Paik, H. -D., 2001. Partial characterization of lacticin NK24, a newly

identified bacteriocin of lactococcus lactis NK24 isolated from Jeot-gal.

Food Microbiology 18, 17-24.

Lee, Y. -K., Nomoto, K., Salminen, S., Gorbach, S.L., 1999. Handbook of

Probiotics. Wiley Publisher, New York, pp.1; 19.

Lorca, G.L., Font de Valdez, G., 2001. A low-pH-inducible, stationary-phase acid

tolerance response in Lactobacillus acidophilus CRL 639. Current

Microbiology 42,21-25.

Lorca, G.L., Wadtrom, T., Font de Valdez, G., Ljungh, A., 2001. Lactobacillus

acidophilus autolysins inhibit Helicobacter pylori in Vitro. Current Microbiology

42,39-44.

Lund, B.M., Braid-Parker, T.C., Gould, GW., 1999. Microbiological Safety and

Quality of Food. KLUWER academic pUblishers, Vol I & II.

MRS Agar

http://service.merck.de/microbiologyltedisdata/prods/4973-1106600500.html

Manolopoulou, E, Sarantinopoulos, P., Zoidou, E, Aktypis, A., Moschopoulou,

E, Kandarakis, I. G., Anifantakis, EM., 2003. Evolution of microbial

populations during traditional Feta cheese manufacture and ripening.

International Journal of Food Microbiology 82, 153-161.

73

Mao, Y., Muriana, P. M., Cousin MA, 2001. Purification and transpositional

inactivation of Lacticin FS92, a broad-spectrum bacteriocin produced by

Lactococcus !aclis FS92. Food Microbiology 18, 165-175.

Marshall, V.M., Tamine, A.Y., 1997. Starter cultures employed in the

manufacture of biofermented milks. International Journal of Dairy Technology

50,35-41.

Maltila-Sandholm, T., Blum, S., Collins, J.K., Crittenden, R, deVos, W., Dunne,

C., Fondan, R, Grenov, G., Isolauri, E., Kiely, B., Marteau, P., Morelli, L.,

Ouwehand, A, Reniero, R, Saarela, M., Salminen, S., Saxelin, M., Schiffrin,

E., Shanahan, F., Vaughan, E., von Wright, A., 1999. Probiotics: towards

demonstrating efficacy. Trends Food Sci. Techno!. 10,393-399.

Miambi, E., Guyot, J.-P., Ampe, F., 2003. Identification, isolation and

quantification of representative bacteria from fermented cassava dough using

an integrated approach of culture-dependent and culture-independent

methods. International Journal of Food Microbiology 82 (2), 111-120.

Mugula, J.K., Nnko, SAM., Narvhus, J.A., Sorhaug, T., 2003. Microbiological

and fermentation characteristics of togwa, a Tanzanian fermented food.

International Journal of Food Microbiology 80,187-199.

Muyanja, C. M. B. K, Narvhus, J. A, Treimo, J., Langsrud, T., 2003. Isolation,

characterisation and identification of lactic acid bacteria from bushera: a

Ugandan traditional fermented beverage. International Journal of Food

Microbiology 80(3),201-210.

74

Noonpakdee, W., Santivarangkna, C., Jumriangrit, P., Sonomoto, K., Panyim, S.,

2003. Isolation of nisin-producing Lactococcus factis WNC 20 strain from

nham, a traditional Thai fermented sausage. Intemational Journal of Food

Microbiology 81, 137-145.

Olano, A., Chua, J., Schroeder, S., Minari, A., Salvia, M.L., Hall, G., 2001.

Weissella confusa (Basonym: Lactobacillus confuses) Bacteremia: a

Case Report. Journal of Clinical Microbiology 39 (4), 1604-1607.

Olasupo, NA, Olukoya, D.K., Odunfa, SA, 1997. Identification of Lactobacillus

species associated with selected African fermented foods. Z. Naturforsch 52,

105-108

Oyewole, O.B., Odunfa, SA, 1990. Characterisation of lactic acid bacteria in

cassava fermentation during fufu production. J. Appl. Bacteriol. 68,145-152.

Paludan-Muller, C., Huss, H.H., Gram, L.. 1999. Characterization of lactic acid

bacteria isolated from a thai low-salt fermented fish product and the roe of

garlic as substrate for fermentation. International Journal of Food Microbiology

46,219-229.

Papadelli. M., Manolopoulou, E., Kalantzopoulos, G., Tsakalidou, E., 2003. Rapid

detection and identification of Streptococcus macedonicus by species-specific

PCR and DNA hydridisation. International journal of Food Microbiology 81,

231-239.

Pouwels. D., Simons, G., 2003. Fingerprinting Microorganisms. Food

Technology 57(2}, 36-40.

75

Ralph, W., John, J., Tagg, R, Ray, B., 1995. Bacteriocins of gram-positive

bacteria. Microbiological Reviews 59(2), 171-200.

Ray, B., 2001a. Fundamental Food Microbiology, second ed. CRC Press LLC.

Boca Raton, Florida, pp. 110.

Ray, B., 2001b. Fundamental Food Microbiology, second ed. CRC Press LLC.

Boca Raton, Florida, pp. 165-166.

Ray, B., 2001c. Fundamental Food Microbiology, second ed. CRC Press LLC.

Boca Raton, Florida, pp. 170-171.

Ray, B., 2001d. Fundamental Food Microbiology, second ed. CRC Press LLC.

Boca Raton, Florida, pp. 184-185.

Ray, B., 2001e. Fundamental Food Microbiology, second ed. CRC Press LLC.

Boca Raton, Florida, pp. 111-115.

Ray, B., 2001f. Fundamental Food Microbiology, second ed. CRC Press LLC.

Boca Raton, Florida, pp. 91.

Ray, B., Daeschel, M., 1992. Food biopreservalives of microbial origin. CRC

Press, Inc. Boca Raton, Florida, pp. 17-21.

Rogelj, I. Bogovic-Matijasic, B., Canzek-Majhenic, A., Stojkovic, S., 2002. The

survival and persistence of Lactobacillus acidophilus LF221 in different

ecosystems. International Journal of Food Microbiology 76, 83-91.

Ross, R P., Morgan, S., Hill, C., 2002. Preservation and fermentation: past,

present and future. International Journal of Food Microbiology, 79, 3-16.

Roth, A., Worth, RM., Lichton, I.J., 1967. Use of poi in the prevention of allergic

disease in potentially allergic infants. Annals of Allergy 25, 501·506.

76

Roy, D., Ward, P., Vincent, D., Mondou, F., 2000. Molecular identification of

potentially probiotic lactobacilli. Current Microbiology 40, 40-46.

Roy, D., Sirois, S., Vincent, D., 2001. Molecular Discrimination of Lactobacilli

used as starter and probiotic cultures by amplified ribosomal DNA restriction

analysis. Current Microbiology 42, 282-289.

Salminen, S., Wright, AV., 1993. Lactic Acid Bacteria. Marcel Dekker, Inc.,

New York, pp. 1-4.

Sanders, M.E., Klaenhammer, T.R., 2001. Invited review: the scientific basis of

Lactobacillus acidophilus NCFM functionality as a probiotic. Journal of Dairy

Science 84, 319-331.

Sanni AI., Morton-Guyot J., Guyot J.P., 2002. New efficient amylase-producing

strains of Lactobacillus plantarum and L. fermentum isolated from different

Nigerian traditional fermented foods. International Journal of Food

Microbiology 72(1-2),53-62.

Shah, N.P., 2001. Functional foods from probiotics and prebiotics. Food

Technology 55(11),46-53.

Sneath, P.H.A. (Ed.), 1986. Bergey's Manual of Systematic Bacteriology, Vol. 2.

Williams &Wilkins, Baltimore.

Soomro, AH., Masud, T., Anwaar, K., 2002. Role of lactic acid bacteria (LAB) in

food preservation and human health- a review. Pakistan Journal of Nutrition 1

(1),20-24.

Steinkraus, K. H., 2002. Fermentations in Wortd Food Processing.

Comprehensive Reviews in Food Science and Food Safety 1, 23-32.

77

Steinkraus, K.H., 1983a. Lactic acid fermentation in the production of foods from

vegetables, cereals and legumes. Antonie Van Leeuwenhoek 49 (3), 337-348.

Steinkraus, K.H., 1983b. Handbook of Indigenous Fermented Foods

(Microbiology series, Vol. 9). Marcel Dekker, Inc., New York, pp. 620

Tamime A.Y., Deeth H.C., 1980. Yougurt: Technology and Biochemistry. Journal

of Food Protection 43(12),939-977.

Tannock, G.W., 1999. Probiotics-a critical review. Horizon Scientific Press,

Norfolk, England, pp. 1.

Tetteh, G.L., Beuchat, L.R, 2003. Exposure of Shigella f1exneri to acid stress

and heat shock enhances acid tolerance. Food Microbiology 20 (2), 179-185.

USDA Nutrient Database for Standard Reference.

http://www.nal.usda.gov/fnic/cgi-bin/listnut.pl

Vandenbergh, P.A., 1993. Lactic acid bacteria, their metabolic products and

interference with microbial growth. FEMS Microbiology Reviews 12,

221-238.

Vescovo, M., Torriani, S., Orsi, C., Macchiarolo, F., Scolari, G., 1996. Application

of antimicrobial-producing lactic acid bacteria to control pathogens in ready-to­

use vegetables. Journal of Applied Bacteriology 81, 113-119.

Vogel, RF., Knorr, R, Muller, M.R, et aI., 1999. Non- dairy lactic fermentations:

the cereal world, Antonie Van Leeuwenhoek 76 (1-4), 403-411.

Woese, C.R, 1987. Bacterial evolution. Microbiological Review 51, 221-271.

78

Zhou, J.S., Shu, Q., Rutherfurd, K.J., 2000. Safety assessment of potential

probiotic lactic acid bacterial strains Lactobacillus rhamnosus HN001, Lb.

acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice.

International Journal of Food Microbiology 56, 87-96.

79