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Methods in
Molecular Biology 1887

Makoto Kanauchi Editor

Lactic Acid
Bacteria
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 Lactic Acid Bacteria

Methods and Protocols

Edited by

Makoto Kanauchi
Department of Food Management, Miyagi University, Sendai, Miyagi, Japan
Editor
Makoto Kanauchi
Department of Food Management
Miyagi University
Sendai, Miyagi, Japan

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8906-5 ISBN 978-1-4939-8907-2 (eBook)
https://doi.org/10.1007/978-1-4939-8907-2
Library of Congress Control Number: 2018960875

© Springer Science+Business Media, LLC, part of Springer Nature 2019

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Preface

Lactic acid bacteria have traditionally been used in food production as important fermenta-
tive bacteria. They have been used to prepare dairy products such as yogurt and cheese,
alcoholic beverages such as wine and whisky, and seasonings such as soy sauce and fish
products.
In 1908, Metchnikoff reported that lactic acid bacteria in yogurt improve intestinal
conditions. As a result of that benefit, lifespan extension can be expected as an influence from
the bacteria. Research into lactic acid bacteria for probiotic use advanced after Metchnikoff’s
reports. Generally, hundreds of species, 100 trillion cells of bacteria, are present as intestinal
flora in human intestines. Many researchers have reported on the role of bacteria as a
component of the bacterial flora in human intestines. Recently, the bacteria have been
used in medicine as an intestinal medicine. Moreover, immunobiotics using lactic acid
bacteria have been investigated recently by many scholars. Consequently, lactic acid bacteria
are used widely for human life. Many scholars hope to know more about how professional
researchers evaluate the various lactic acid bacteria functions.
This volume of the Methods in Molecular Biology series provides a collection of protocols
for numerous experimental approaches used by the authors for lactic acid bacteria research,
such as the isolation of lactic acid, along with applications of lactic acid for food production
and healthy function, in 16 chapters divided into three parts. All authors have contributed in
the format used in the Methods in Molecular Biology series. In these explanations, the
Materials sections list all the chemicals, reagents, buffers, and other materials used for the
protocols. Furthermore, detailed descriptions of every protocol are provided in the Methods
section. They are expected to lead to the successful completion of each method. Some
emergent difficulties or techniques for each protocol are presented in the Notes section.
In Part I, we explain bacteria metabolism, methods of isolating lactic acid bacteria from
natural substances, and bacteriocin as antibacterial substances produced by lactic acid
bacteria. Bacteriocin is a noteworthy substance that is recognized throughout the world
for its use as an antibacterial substance in food. Therefore, methods of selecting a lactic acid
bacteria strain to produce bacteriocin and evaluation of bacteriocin produced from the
bacteria are described. Furthermore, lactic acid bacteria produces lactic acid from sugar,
but the bacteria are also in a stress condition. As a result of the stress response to acid,
internal pH changes considerably. Actually, internal pH is an important indication for the
food industry and microbiology using the bacteria. Therefore, methods of assaying pH are
also described in this section.
Secondly, we present an application for the food industry in Part II. An author provides
methods for counting microorganisms such as lactic acid bacteria, yeast, and mold in yogurt,
and methods for quality analysis or texture. Furthermore, the polysaccharide produced from
lactic acid bacteria is mentioned successively in three chapters. The polysaccharide not only
has a relation to yogurt texture but also has a role in improving immunity. The authors
provide evaluation methods and analytical methods for the polysaccharide and the produc-
tion of lactic acid bacteria. Furthermore, in food production, the growth of lactic acid
bacteria is known to spoil the quality of food. Detection of spoilage of lactic acid bacteria
in beer using PCR method is demonstrated by the authors.

v
vi Preface

Finally, beneficial effects of lactic acid bacteria are presented in Part III. The authors
describe methods for the evaluation of detoxification by biosorption of heavy metals by
lactic acid bacteria, production of immunobiotics by the bacteria, adhesion of the bacteria to
intestinal mucosa, and neutralization of lipopolysaccharides (LPS) by the bacteria. Many
lactic acid bacteria have healthy function after oral ingestion as prebiotics. We hope to apply
the results to many research efforts in the domains of food science and health science.
I would like to acknowledge all authors for kindly contributing their chapters. We are
especially grateful to the Series Editor Dr. John Walker and the Editor of Springer Protocols,
David C. Casey, for their assistance, and to the information technology department for
providing the requisite framework, which greatly enhanced the compilation of the book
chapters.

Sendai, Japan Makoto Kanauchi

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I IDENTIFICATION AND METABOLISMS OF LAB

1 Isolation and Identification of Lactic Acid Bacteria from
Environmental Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Akihito Endo, Yasuhiro Tanizawa, and Masanori Arita
2 Basic Antibacterial Assay to Screen for Bacteriocinogenic Lactic
Acid Bacteria and to Elementarily Characterize Their Bacteriocins . . . . . . . . . . . . 15
Kensuke Arakawa
3 Assaying D-Body Amino Acids as D-Alanine and Amino Acid Racemase
(AARase) Activity Using NADH Oxidoreduction Enzymic System. . . . . . . . . . . . 23
Natsuki Matsumoto and Makoto Kanauchi
4 Intracellular pH Determination for the Study of Acid Tolerance of
Lactic Acid Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Hiromu Kudo and Yasuko Sasaki

PART II APPLICATIONS OF FOOD INDUSTRIES OF LABS

5 Yogurt Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Seiji Nagaoka
6 Purification, Rheological Characterization, and Visualization of
Viscous, Neutral, Hetero-exopolysaccharide Produced by Lactic
Acid Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
S. Ikeda, D. Kondoh, N. P. D. Aryantini, T. Urashima, and K. Fukuda
7 Structural Analysis of Exopolysaccharides from Lactic Acid Bacteria . . . . . . . . . . . 67
Gerrit J. Gerwig
8 Preparation of Exopolysaccharide Synthesized by Lactic Acid Bacteria . . . . . . . . . 85
Junko Nishimura
9 PCR Analysis Methods for Detection and Identification
of Beer-Spoilage Lactic Acid Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
S. Asano, M. Shimokawa, and K. Suzuki
10 Isolation of Lactic Acid Bacteria Eliminating Trimethylamine (TMA)
for Application to Fishery Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Satoshi Mohri and Makoto Kanauchi
11 Screening the Lactic Acid Bacteria converting Hydroxy Fatty Acid
from Unsaturated Fatty Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Makoto Kanauchi

vii
viii Contents

PART III HEALTHY FUNCTIONS OF LABS

12 Screening and Characterization of Immunobiotic Lactic Acid

Bacteria with Porcine Immunoassay Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Valeria Garcia-Castillo, Leonardo Albarracin, Haruki Kitazawa,
and Julio Villena
13 Biosorption of Heavy Metals by Lactic Acid Bacteria for Detoxification. . . . . . . . 145
Hideki Kinoshita
14 Adhesion of Lactobacillus to Intestinal Mucin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Keita Nishiyama and Takao Mukai
15 Eliminating Lipopolysaccharide (LPS) Using Lactic Acid Bacteria (LAB)
and a Fraction of its LPS-Elimination Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Makoto Kanauchi, Ayaka Kondo, and Kyoko Asami
16 Cloning and Expression of Lipopolysaccharide Elimination Protein (LEP)
in Lactic Acid Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Kyoko Asami and Makoto Kanauchi

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Contributors

LEONARDO ALBARRACIN  Laboratory of Immunobiotechnology, Reference Centre for

Lactobacilli (CERELA-CONICET), Tucuman, Argentina; Food and Feed Immunology
Group, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan;
Laboratory of Computing Science, Faculty of Exact Sciences and Technology, Tucuman
University, Tucuman, Argentina
KENSUKE ARAKAWA  Graduate School of Environmental and Life Science, Okayama
University, Okayama, Japan
MASANORI ARITA  Center for Information Biology, National Institute of Genetics, Mishima,
Shizuoka Prefecture, Japan; RIKEN, Center for Sustainable Resource Science, Kanagawa,
Japan
N. P. D. ARYANTINI  Department of Life and Food Sciences, Obihiro University of
Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan
KYOKO ASAMI  Department of Food Management, Miyagi University, Sendai, Miyagi, Japan
S. ASANO  Research Laboratories for Alcohol Beverages, Department of Fermentation and
Microbiology Technology, Asahi Breweries, Ltd., Ibaraki, Japan
AKIHITO ENDO  Department of Food, Aroma and Cosmetic Chemistry, Tokyo University of
Agriculture, Hokkaido, Japan
K. FUKUDA  Department of Life and Food Sciences, Obihiro University of Agriculture and
Veterinary Medicine, Obihiro, Hokkaido, Japan; Department of Agriculture and Animal
Science, Research Center for Global Agromedicine, Obihiro, Hokkaido, Japan
VALERIA GARCIA-CASTILLO  Laboratory of Bacterial Pathogenicity, Faculty of Biological
Sciences, University of Concepcion, Concepcion, Chile; Laboratory of Immunobiotechnology,
Reference Centre for Lactobacilli (CERELA-CONICET), Tucuman, Argentina; Food
and Feed Immunology Group, Graduate School of Agricultural Science, Tohoku University,
Sendai, Japan
GERRIT J. GERWIG  Microbial Physiology, Groningen Biomolecular Sciences and
Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands
S. IKEDA  Department of Food Science, University of Wisconsin-Madison, Madison, WI, USA
MAKOTO KANAUCHI  Department of Food Management, Miyagi University, Sendai, Miyagi,
Japan
HIDEKI KINOSHITA  Laboratory of Food Biochemistry, Department of Bioscience, School of
Agriculture, Tokai University, Kumamoto, Japan
HARUKI KITAZAWA  Food and Feed Immunology Group, Graduate School of Agricultural
Science, Tohoku University, Sendai, Japan; International Education and Research Center
for Food and Agricultural Immunology (CFAI), Graduate School of Agricultural Science,
Tohoku University, Sendai, Japan
AYAKA KONDO  Department of Food Management, Miyagi University, Sendai, Miyagi,
Japan
D. KONDOH  Division of Basic Veterinary Medicine, Obihiro University of Agriculture and
Veterinary Medicine, Obihiro, Hokkaido, Japan
HIROMU KUDO  Meiji University, Kanagawa, Japan
NATSUKI MATSUMOTO  Department of Food Management, Miyagi University, Sendai,
Miyagi, Japan

ix
x Contributors

SATOSHI MOHRI  Department of Food Management, Miyagi University, Sendai, Miyagi,

Japan
TAKAO MUKAI  Department of Animal Science, School of Veterinary Medicine, Kitasato
University, Towada, Aomori, Japan
SEIJI NAGAOKA  Fermented Milk Development Department, Food Development Laboratories,
R&D Division, Meiji Co., Ltd., Hachiouji, Tokyo, Japan
JUNKO NISHIMURA  Department of Life and Environmental Science, Faculty of Engineering,
Hachinohe Institute of Technology, Hachinohe, Aomori, Japan
KEITA NISHIYAMA  Department of Microbiology, School of Pharmacy, Kitasato University,
Tokyo, Japan
YASUKO SASAKI  Meiji University, Kanagawa, Japan
M. SHIMOKAWA  Research Laboratories for Alcohol Beverages, Department of Fermentation
and Microbiology Technology, Asahi Breweries, Ltd., Ibaraki, Japan
K. SUZUKI  Research Laboratories for Alcohol Beverages, Department of Fermentation and
Microbiology Technology, Asahi Breweries, Ltd., Ibaraki, Japan
YASUHIRO TANIZAWA  Center for Information Biology, National Institute of Genetics,
Mishima, Shizuoka Prefecture, Japan
T. URASHIMA  Department of Life and Food Sciences, Obihiro University of Agriculture and
Veterinary Medicine, Obihiro, Hokkaido, Japan
JULIO VILLENA  Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli
(CERELA-CONICET), Tucuman, Argentina; Food and Feed Immunology Group,
Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
 Part I

Identification and Metabolisms of LAB

 Chapter 1

Isolation and Identification of Lactic Acid Bacteria

from Environmental Samples
Akihito Endo, Yasuhiro Tanizawa, and Masanori Arita

Abstract
Isolation of lactic acid bacteria (LAB) is the first and crucial step to study possible roles of LAB in the
environment, especially in food fermentation. This is also important to use the organisms for further
application. LAB are diverse bacterial group and have diverse growth characteristics. Culture condition of
LAB is thus varied, and selection of a suitable culture medium is essential for the purposes. Identification is
also an important step, since certain desirable and undesirable characteristics are shared within species.
Identification was classically carried out by phenotypic characteristics but is usually performed by DNA
sequence-based approaches. 16S rRNA gene sequencing is generally used for identification, and sequencing
of housekeeping genes is used when needed. In addition, identification based on whole-genome sequence
similarities is becoming common. Here we describe isolation and identification of LAB briefly.

Key words Lactic acid bacteria, Isolation, Culture medium, Identification, 16S rRNA gene,
Housekeeping genes, Whole-genome sequence similarities

1 Introduction

Lactic acid bacteria (LAB) are a diverse group of bacteria which

phylogenetically belongs to the order Lactobacillales. This diverse
order includes 6 families, over 30 genera and over 300 species.
These microorganisms produce lactate as the main end products
from metabolism of glucose, and certain species also produce etha-
nol, CO2, and acetate. Lactate metabolism is not common in the
organisms. Bifidobacteria are not members of the order Lactoba-
cillales, and the organisms are thus not included in this chapter.
LAB can be seen in wide variety of habitats, including gastrointes-
tinal tracts, vaginal tracts, oral cavities of animals, fermented foods,
silages, plant surfaces, and composts. Certain species originated
from clinical samples. Because of the huge varieties, culture condi-
tion of LAB is varied. LAB require rich nutrients for growth, e.g.,
carbohydrates, amino acids, vitamins, minerals, and sometimes fatty
acids and peptides. They usually lack most part of TCA cycle and

Makoto Kanauchi (ed.), Lactic Acid Bacteria: Methods and Protocols, Methods in Molecular Biology, vol. 1887,
https://doi.org/10.1007/978-1-4939-8907-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

3
4 Akihito Endo et al.

quinone/ubiquinone biosynthesis systems, meaning that they do

not conduct respiration. Oxygen thus does not usually support
their growth, and they prefer anaerobic conditions rather than
aerobic conditions for growth. LAB generally inhabit in nature
with other microbes, including molds, yeasts, aerobes, and anae-
robes. Several chemicals, including antibiotics, are thus usually used
for a selective isolation of LAB from environmental samples. Iden-
tification is essential after the isolation of LAB. Identification is
usually conducted by phylogenetic analysis based on 16S rRNA
gene sequences, whereas species in certain LAB groups, e.g., Lac-
tobacillus plantarum group, Lactobacillus casei group, and Entero-
coccus faecium group, are known to share high sequence similarities
of 16S rRNA gene within the groups. Housekeeping genes are
alternative markers for an accurate identification of such LAB
groups. In addition, identification based on whole-genome
sequence similarities, e.g., all nucleotide identity (ANI) and in silico
DNA-DNA hybridization (DDH), is becoming common in recent
years.

2 Materials

2.1 Culture Media Major culture media for isolation of LAB are de Man, Rogosa, and
and Dilution Buffer Sharpe (MRS) medium, Lactobacillus selection (LBS) medium, and
M17 medium. These media are commercially available in several
producers. Moreover, acidic tomato/grape medium and fructophi-
lic LAB isolation medium are also used for isolation of specific LAB
species. These media usually contain rich nutrients but not anti-
biotics for selective isolation of LAB. Supplement of 10 mg/L of
sodium azide and 10 mg/L of cycloheximide is useful to suppress
growth of aerobes and fungi, respectively. Calcium carbonate
(5 g/L) supplemented into the agar medium is also useful for
visible distinction between LAB and other microbes, since clear
zones are formed surrounding colonies because of their acid pro-
duction. Anaerobic culturing by using a gas-generating kit or
anaerobic work station is usually helpful for isolation of LAB.
1. De Man, Rogosa, and Sharpe (MRS) medium: Weigh 52 g of
the MRS medium powder (Oxoid), put 1 L of water, mix
thoroughly, and weigh 15 g of agar (see Note 1). Autoclave at
121
C for 15 min prior to use. The medium is available in
several producers, and recipe for the medium varies between
producers.
2. Lactobacillus selection (LBS) medium: Weigh 84 g of the LBS
agar powder (Beckton Dickinson), put 1 L of water, and mix
thoroughly (see Note 2). Put 1.32 mL of glacial acetic acid, and
heat it until agar completely dissolves. Autoclave is not needed
for this medium.
 Isolation and Identification of LAB 5

3. M17 medium: Weigh 48.25 g of the M17 medium powder

(Beckton Dickinson), put 950 mL of water, mix thoroughly,
and heat it to completely dissolve the powder (see Note 3).
Autoclave at 121
C for 15 min, cool to 50
C, and add 50 mL
of sterile 10% lactose solution (optional).
4. Acidic tomato/grape broth medium: Weigh 10 g of glucose,
5 g of yeast extract, 10 g of peptone, 0.2 g of MgSO4·7H2O,
and 0.05 g of MnSO4·4H2O, and put 750 mL of water and
250 mL of grape/tomato juice (see Note 4). Set pH at 4.8 with
HCl. Autoclave at 121
C for 15 min prior to use.
5. Fructophilic LAB (FLAB) isolation medium: Basal media usu-
ally used for FLAB isolation medium are MRS, GYP, and FYP.
For preparation of GYP and FYP media, weigh 10 g of glu-
cose/fructose, 10 g of yeast extract, 5 g of Polypeptone, 2 g of
sodium acetate, 0.5 g of Tween 80, 0.2 g of MgSO4·7H2O,
0.01 g of MnSO4·4H2O, 0.01 g of FeSO4·7H2O, and 0.01 g
of NaCl [1]. Set pH at 6.8 with NaOH. If MRS or GYP
medium is used, supplementation of 10 g/L of fructose or
pyruvate is needed (see Note 5). Autoclave at 121
C for
15 min prior to use.
6. Saline: Weigh 8.5 g of NaCl and make up to 1 L with water. If
needed (usually for anaerobic bacteria), put 0.5 g of cysteine-
HCl. Autoclave at 121
C for 15 min prior to use.

2.2 DNA Isolation For genetical identification of LAB isolates, DNA isolation is the
first and crucial step. Several kits are available for isolation of DNA
from bacterial cells. Moreover, bacterial DNA can be isolated with
general techniques combined with physical cell disruption and
ethanol precipitation. Glass beads (approx. 0.1 mm in diameter)
and a beating machine are used for the cell disruption. The detailed
method is described below. Prepare all solutions using ultrapure
water or similar grade water and analytical grade reagents.
1. Extraction buffer: 100 mM Tris-40 mM EDTA·2Na buffer at
pH 9.0. Weigh 12.1 g of Tris and 14.9 g of EDTA·2Na,
dissolve it with 900 mL of water, and set pH at 9.0 with HCl.
Make up to 1 L with water. Autoclave at 121
C for 15 min
prior to use. Store at 4
C.
2. 3 M Sodium acetate solution: Weigh 246 g of sodium acetate
and dissolve it with 700 mL of water. Make up to 1 L with
water. Store at 4
C.
3. TE buffer: 10 mM Tris-1 mM EDTA buffer at pH 8.0. Weigh
1.2 g of Tris and 0.4 g of EDTA·2Na, dissolve it with 950 mL
of water, and set pH at 8.0 with HCl. Make up to 1 L with
water. Autoclave at 121
C for 15 min prior to use. Store at
4
C.
6 Akihito Endo et al.

4. Sodium dodecyl sulfate (SDS) solution: 10% (w/v) SDS solu-

tion. Weigh 10 g of SDS and make up with 100 mL with water.
Store at room temperature.
5. Benzyl chloride: Store at room temperature.
6. Isopropanol: Store at 4
C.
7. 70% Ethanol: Mix 700 mL of ethanol and 300 mL of water.
Store at 4
C.
8. Glass beads: 100 mg glass beads with 0.1 mm in diameter.
Weigh 100 mg of glass beads (0.1 mm in diameter) and sterilize
at 180
C for 30 min.
9. Cell crusher: FastPrep-24 (MP Biomedicals).
10. Spectrophotometer.

2.3 PCR, 1. Commercial PCR kit: Store at 20
C. The kit usually contains
Electrophoresis, DNA polymerase, PCR reaction buffer, and dNTP mixture.
Purification, 2. Primers used for amplification and sequencing of 16S rRNA
and Sequencing gene: 8F (50 -AGAGTTTGATCMTGGCTCAG-30 ), 15R (50 -A
AGGAGGTGATCCARCCGCA-30 ), 930F (50 -GCACAAGCG
GTGGAGCATGTGG-30 ), 520R (50 -ACCGCGGCTGCTGG
C-30 ), 800R (50 -CAGGACTACCAGGGTATCTAAT-30 ), and
1100R (50 -AGGGTTGCGCTCGTTG-30 ).
3. Primers used for amplification and sequencing of housekeeping
genes: recEXT-f (50 -GGCTATGAAACAAATTGAAAAA
CAATWYGGNAARGG-30 ) and recEXT-r (50 -TGTT
0
TAAACGGTGGAGCAACTTTRTTYTTNAC-3 ) for recA
gene; pheS-21-F (5’-CAYCCNGCHCGYGAYATGC-30 ),
pheS-22-R (50 -CCWARVCCRAARGCAAARCC-30 ), and
pheS-23-R (50 -GGRTGRACCATVCCNGCHCC-30 ) for pheS
gene; and rpoA-21-F (50 -ATGATYGARTTTGAAAAACC-30 ),
rpoA-23-R (50 -ACHGTRTTRATDCCDGCRCG-30 ), and
rpoA-22-R (50 -ACYTTVATCATNTCWGVYTC-30 ) for rpoA
gene.
4. TAE buffer: Weigh 4.8 g of Tris, 1.1 mL of acetic acid, and
0.074 g of EDTA·2Na, and dissolve it with 900 mL of water.
Make up to 1 L with water.
5. 1% Agarose gel: Weigh 1 g of agarose and put 100 mL of TAE
buffer. Heat it with microwave until agarose completely melts.
Cast agarose solution on a gel maker.
6. Commercial purification kit for PCR products.
7. BigDye Terminator Cycle Sequencing kit (Thermo Fisher Sci-
entific): Store at 20
C.
8. 3 M Sodium acetate: Prepare as described above.
9. 99% Ethanol: Store at room temperature.
 Isolation and Identification of LAB 7

10. 70% Ethanol: Prepare as described above. Store at room

temperature.
11. PCR machine.
12. Electrophoresis machine.
13. DNA sequencer: Applied Biosystems model 3130 (Thermo
Fisher Scientific).

3 Methods

Isolation of LAB, DNA extraction, and sequencing-based identifi-

cation, including 16S rRNA gene, housekeeping genes, and whole-
genome sequences, are introduced here.

3.1 Isolation of LAB 1. Environmental samples are serially diluted with saline and
spread onto appropriate agar medium listed in Subheading 2.
2. The media are incubated at 30 or 37
C under anaerobic/
aerobic conditions for 48 to 72 h. Incubation temperatures at
25 and 42
C are used for isolation of mesophilic LAB and
thermophilic LAB, respectively. Longer incubation hour (up to
120 h) is needed for isolation of specific slowly growing LAB,
e.g., wine LAB O. oeni.
3. Colonies on agar media can be cultured in liquid broth which is
the same medium used for isolation but exclusive of agar.

3.2 DNA Isolation 1. Cultured cells are harvested (10,000  g, 5 min) in plastic
tubes, discard supernatant, and suspend in a solution contain-
ing 250 μL of extraction buffer, 50 μL of 10% SDS solution,
and 150 μL of benzyl chloride. One hundred milligrams of
glass beads (0.1 mm in diameter) is added to the suspension,
and the mixture is beaten at maximum speed for 2 min in a
beating machine (model FastPrep-24, MP Biomedicals).
2. One hundred fifty microliters of 3 M sodium acetate solution is
added to the beaten samples, and the samples are cooled on ice
for 15 min.
3. The samples are centrifuged (15,000  g, 10 min), and the
resultant supernatant is transferred to a new tube.
4. Four hundred fifty microliters of isopropanol is added to the
supernatant. The samples are mixed and centrifuged
(15,000  g, 15 min).
5. Supernatant is discarded and 70% ethanol is added to the
samples. The samples are centrifuged (15,000  g, 5 min).
6. Supernatant is removed carefully and the samples are dried.
The resultant DNA is dissolved in 50 μL of TE buffer, and
8 Akihito Endo et al.
3.3 Amplification
and Sequencing of 16S
rRNA Gene
3.4 Amplification
and Sequencing
of Housekeeping
Genes
concentration and quality of DNA are determined by spectro-
photometry. DNA can be diluted with TE buffer at a concen-
tration of 10 ng/μL.
1. PCR reaction mixture comprises 10 pmol of each primer, PCR
reaction buffer, 2-mM MgCl2, 0.2 mM each dNTP, 1.25 U of
Taq DNA polymerase, and 10 ng of the isolated DNA. 8F and
15R primers are used for this PCR. PCR program consisted of
35 cycles of 94
C for 30 s, 55
C for 30 s, and 72
C for 90 s
with the last extension at 72
C for 2 min. Amplification can be
confirmed by agarose gel (1%) electrophoresis in TAE buffer.
2. The PCR products are purified by commercial PCR purifica-
tion kits according to the manufacturer’s instruction. The pur-
ified DNA is used for cycle sequencing.
3. Reaction mixture for cycle sequencing comprises 1 μL of Big-
Dye Terminator Cycle Sequencing kit, 2 μL of 5  sequencing
buffer, 1 μL of primer (1.6 pmol), 5 μL of deionized water, and
1 μL of the purified DNA. PCR program consisted of 25 cycles
of 96
C for 10 s, 50
C for 5 s, and 60
C for 4 min. Primers
used for amplification, i.e., 8F and 15R, can be used in this
step. If needed, primers 930F, 520R, 800R, and 1100R are
helpful for determination of nearly whole 16S rRNA gene
sequencing.
4. The products have to be purified for further analysis. The
products are transferred into sterile tubes, added with 1 μL of
3 M sodium acetate and 25 μL of 99% ethanol, and kept for
15 min at room temperature (RT).
5. Centrifuge (15,000  g, 15 min, RT), discard supernatant, and
add 125 μL of 70% ethanol.
6. Centrifuge (15,000  g, 5 min, RT), discard supernatant, and
dry up.
7. Samples can be analyzed in a DNA sequencer (Applied Biosys-
tems model 3130) according to the manufacturer’s
instructions.
8. BLAST analysis is helpful for determination of sequence simi-
larities between the isolates and sequences of known bacterial
species deposited in database. NCBI database (https://blast.
ncbi.nlm.nih.gov/Blast.cgi) provides this valuable service.
Sequence similarities over 99% are generally acceptable values
for species identification [2] (see Note 6).
1. 16S rRNA gene sequence similarity is not always sufficient for
species discrimination. Housekeeping genes are more variable
and therefore have a greater degree of resolution. They are thus
recommended to use for identification and description of novel
species [3]. Among the housekeeping genes, recA, pheS, and
 Isolation and Identification of LAB 9

rpoA are widely used as complementary phylogenetic markers

for taxonomy of LAB. Here thus introduces amplification and
sequencing of the three housekeeping genes (see Note 7).
(a) For amplification of recA gene, PCR reaction mixture com-
prises 25 pmol of each primer, PCR reaction buffer, 2-mM
MgCl2, 0.1 mM each dNTP, 2 U of Taq DNA polymerase,
and 10 ng of the isolated DNA. Primers recEXT-f and
recEXT-r are used for this PCR [4]. PCR program consisted
of initial denaturation at 94
C for 5 min; 35 cycles of 94
C
for 45 s, 45
C for 120 s, and 72
C for 105 s; and the last
extension at 72
C for 7 min. Amplification can be con-
firmed by agarose gel (1%) electrophoresis.
(b) For amplification of pheS gene, PCR reaction mixture
comprises 25 pmol of each primer, PCR reaction buffer,
2-mM MgCl2, 0.2 mM each dNTP, 1.25 U of Taq DNA
polymerase, and 10 ng of the isolated DNA. Primers
pheS-21-F and pheS-22-R are used for this PCR, and a
primer pheS-23-R can be replaced with pheS-22-R when
amplification is not seen by using primers pheS-21-F and
pheS-22-R [5]. PCR program consisted of initial denatur-
ation at 95
C for 5 min; 3 cycles of 95
C for 60 s, 46
C
for 135 s, and 72
C for 75 s; 30 cycles of 95
C for 35 s,
46
C for 75 s, and 72
C for 75 s; and the last extension at
72
C for 7 min. Amplification can be confirmed by
agarose gel (1%) electrophoresis.
(c) For amplification of rpoA gene, PCR reaction mixture
comprises 25 pmol of each primer, PCR reaction buffer,
2-mM MgCl2, 0.2 mM each dNTP, 1.25 U of Taq DNA
polymerase, and 10 ng of the isolated DNA. Primers
rpoA-21-F and rpoA-23-R are used for this PCR, and a
primer rpoA-22-R can be replaced with rpoA-23-R when
amplification is not seen by using primers rpoA-21-F and
rpoA-23-R [5]. PCR program consisted of initial denatur-
ation at 95
C for 5 min; 3 cycles of 95
C for 60 s, 46
C
for 135 s, and 72
C for 75 s; 30 cycles of 95
C for 35 s,
46
C (or 42
C) for 75 s, and 72
C for 75 s; and the last
extension at 72
C for 7 min. Amplification can be con-
firmed by agarose gel (1%) electrophoresis.
2. Purification of the PCR products, cycle sequencing, purifica-
tion of cycle sequencing products, analysis with a DNA
sequencer, and BLAST analysis can be carried out by the
method as described above, except that primers for cycle
sequencing are those used for PCR. Threshold for species
identification is somewhat unclear when housekeeping genes
are used as markers. Strains classified in the same species usually
share over 95–97% sequence similarities based on these two
genes (see Note 6).
10 Akihito Endo et al.

3.5 Whole-Genome Whole-genome relatedness of 70% experimentally determined

Sequence Similarities using DDH has long been a gold standard in microbial species
delineation. Recently, the use of whole-genome-based metrics
such as ANI has been spreading as a replacement for traditional
DDH technique. ANI represents mean nucleotide identity in a
pair-wise sequence alignment between two genomes. The ANI
value of 95% approximately corresponds to the DDH value of
70% and is now being recognized as a cutoff line to demarcate
species [6, 7]. In most cases, the interspecies ANI is below 85%,
much lower than the widely used threshold, even between species
difficult to distinguish by 16S rRNA gene sequences such as species
in L. casei group and L. plantarum group [8]. There are several
implementations of ANI. Among them, ANIb is the most standard
method, in which one of the genomes in a pair (query) is cut into
fragments, typically about 1000 nt in length, and they are aligned
to the reference genome using the BLASTN algorithm. ANIb is
calculated as the mean identity of all BLASTN matches that showed
more than 30% overall sequence identity over an alignable region of
at least 70% of their length [6]. Similarly, ANIm uses MUMmer for
sequence alignment [7], and more robust and faster methods such
as OrthoANI and OrthoANIu have also been proposed recently
[9]. Apart from ANI, Genome Blast Distance Phylogeny (GBDP) is
another in silico metrics for whole-genome relatedness. It can be
calculated using Genome-to-Genome Distance Calculator
(GGDC, http://ggdc.dsmz.de), which also estimates in silico
DDH with confidence intervals [10]. The species threshold of in
silico DDH is 70%, same as the wet-lab DDH.
To calculate ANI or in silico DDH, assembled draft or com-
plete genome sequences are used. For complete genomes consist-
ing of multiple replicons or draft genomes, all of the sequences
should be bundled in a single file (multi-FASTA format).
For valid identification, comparison against the genome from a
type strain is desirable. Now, large amount of genomic data are
deposited in the public sequence databases. The NCBI Assembly
database is of use to obtain reference genomes. Genomes from type
strains can be searched by typing keywords like ‘(Lactobacillus
plantarum) AND “assembly from type material”[From Type
Material]’ in the search form. Genome sequence files in a FASTA
format (the one with a file suffix “genomic.fna.gz”) should be
downloaded from the NCBI FTP server and be unarchived.
Several ways to calculate ANI using online tools are introduced
below.

3.5.1 ANI Calculator ANI calculator (http://enve-omics.ce.gatech.edu/ani/) is oper-

ated by a research group that originally proposed ANI [6]. ANI
can be calculated by uploading a query genome and a reference
genome in the submission form. A job name and an e-mail address
 Isolation and Identification of LAB 11

can be specified optionally. Then, press “Submit” button to start

calculation. Notification will be sent when the job is completed if an
e-mail address is provided. Of note, ANI values are less reliable
when the number of aligned fragments is not sufficient. For exam-
ple, genomes from different species that share similar mobile ele-
ment might exhibit higher ANI than expected.

3.5.2 JSpeciesWS JSpeciesWS (http://jspecies.ribohost.com/jspeciesws/ [11]) can

accommodate two or more genomes (up to 15). ANI can be
calculated in an all-against-all manner between each pair of the
genomes or can be calculated against each of reference genomes if
one genome is specified as a query. This will be useful in a proposal
of new taxa, in which comparison to all species of close relatives is
required. In addition to uploading local files, genome data can be
imported from its associated genome database GenomesDB. In
addition to ANIb, it can calculate ANIm and correlation coeffi-
cients of tetranucleotide frequencies (Tetra), which is an alignment-
free metrics for genome relatedness. The stand-alone version JSpe-
cies is also available for download and can be run on a local
computer [7].

3.5.3 DFAST DDBJ Fast Annotation and Submission Tool (DFAST [8]) is origi-
nally developed by the authors to assist prokaryotic genome anno-
tation and data submission to DNA Data Bank of Japan (DDBJ). It
also provides a function to assess quality of genomes and taxonomic
identification using ANI (https://dfast.nig.ac.jp/analysis/assess
ment/, currently only available for Lactobacillus and Pediococcus).
DFAST is equipped with reference genomes, thus uploading of
reference genomes is not required. All users have to do is upload
a query genome. By default, ANI is calculated against all of the
reference genomes. The target genomes can be barrowed down by
specifying organism groups to reduce running time. In addition,
DFAST reports completeness and contamination values of the
query genome as calculated using CheckM [12].

4 Notes

1. MRS medium is designed for isolation of general LAB [13] and

is still used as a gold standard for isolation of LAB from various
environmental samples. Antibiotics, e.g., sodium azide
(10 mg/L) and cycloheximide (10 mg/L), are helpful for
selective isolation of LAB. Supplement of calcium carbonate
(5 g/L) is helpful to distinguish between acid producers
(mainly LAB) and others.
2. LBS medium is designed for selective isolation of lactobacilli
from oral and stool samples [14]. The medium contains large
12 Akihito Endo et al.

amounts of sodium acetate (25 g/L) which inhibits the growth

of many microorganisms, including bacteria and molds. Acetic
acid is also added to the medium (1.32 mL/L) to lower pH,
which results in growth inhibition of nontarget microorgan-
isms. Because of these chemicals, autoclaving is not needed
before use. Addition of tomato juice to the medium is helpful
to isolate specific lactobacilli (Lactobacillus acidophilus).
3. M17 medium is usually used for isolation of Lactococcus lactis
and Streptococcus thermophilus from dairy products, including
yogurt and cheese [15]. The medium contains 1.9% (w/v)
disodium-β-glycerophosphate for buffer action, and the chem-
ical also helps for selective isolation of S. thermophilus from
yogurt by suppression of Lactobacillus delbrueckii subsp.
bulgaricus.
4. Acidic tomato broth medium and acidic grape medium are
used for isolation of wine LAB, especially a malolactic ferment-
ing Oenococcus oeni [16, 17]. The organism is acidophilic and
requires a specific growth factor. Tomato or grape juice pro-
vides the growth factor. Anaerobic culturing is essential for
isolation of O. oeni. Antibiotics, e.g., sodium azide (10 mg/
L) and cycloheximide (10 mg/L), are helpful for selective
isolation of LAB.
5. FLAB, including Fructobacillus spp., Lactobacillus kunkeei, and
Lactobacillus apinorum, share specific growth characteristics:
preference of fructose than glucose as a growth substrate and
requirement of external electron acceptor(s) for glucose
metabolism. Oxygen, pyruvate, and fructose are usually used
as the electron acceptors. These mean, unlike to other LAB,
aerobic culturing dramatically enhances growth of these inter-
esting microorganisms. Aerobic culturing usually produces
maximum biomass. Supplement of sodium azide and cyclohex-
imide is helpful for selective isolation of FLAB.
6. As described, certain LAB species share over 99% sequence
similarities based on 16S rRNA gene sequences, and thus iden-
tification should be carefully conducted. L. plantarum group
species, i.e., L. plantarum, L. pentosus, and L. paraplantarum,
share over 99.5% sequence similarities based on 16S rRNA
gene but share 83–87% similarities based on recA gene
sequences [18]. Fructobacillus tropaeoli possesses 99.2 and
85.6% sequence similarities with Fructobacillus pseudoficulneus
based on 16S rRNA gene and recA, respectively [19].
7. Once a draft or complete genome is determined, nucleotide
sequences for housekeeping genes such as pheS, rpoA, and recA
are easily obtained by using standard genome annotation pipe-
lines like DFAST and RAST [20] (http://rast.nmpdr.org).
However, it is not easy to extract a full-length 16S rRNA

gene sequence from a draft genome reconstructed from short-
read sequencing data. Bacterial genomes usually harbor multi-
ple copies of rRNA genes, and such repetitive regions are
difficult to reconstruct by de novo assembly.
References
1. Endo A, Futagawa-Endo Y, Dicks LM (2009)
Isolation and characterization of fructophilic
lactic acid bacteria from fructose-rich niches.
Syst Appl Microbiol 32:593–600
2. Stackebrandt E, Ebers J (2006) Taxonomic
parameters revisited: tarnished gold standards.
Microbiol Today 33:152–155
3. Mattarelli P, Holzapfel W, Franz CM, Endo A,
Felis GE, Hammes W, Pot B, Dicks L, Della-
glio F (2014) Recommended minimal stan-
dards for description of new taxa of the
genera Bifidobacterium, Lactobacillus and
related genera. Int J Syst Evol Microbiol
64:1434–1451
4. Felis GE, Dellaglio F, Mizzi L, Torriani S
(2001) Comparative sequence analysis of a
recA gene fragment brings new evidence for a
change in the taxonomy of the Lactobacillus
casei group. Int J Syst Evol Microbiol
51:2113–2117
5. Naser SM, Dawyndt P, Hoste B, Gevers D,
Vandemeulebroecke K, Cleenwerck I,
Vancanneyt M, Swings J (2007) Identification
of lactobacilli by pheS and rpoA gene sequence
analyses. Int J Syst Evol Microbiol
57:2777–2789
6. Goris J, Konstantinidis KT, Klappenbach JA,
Coenye T, Vandamme P, Tiedje JM (2007)
DNA-DNA hybridization values and their rela-
tionship to whole-genome sequence similari-
ties. Int J Syst Evol Microbiol 57:81–91
7. Richter M, Rossello-Mora R (2009) Shifting
the genomic gold standard for the prokaryotic
species definition. Proc Natl Acad Sci U S A
106:19126–19131
8. Tanizawa Y, Fujisawa T, Kaminuma E,
Nakamura Y, Arita M (2016) DFAST and
DAGA: web-based integrated genome annota-
tion tools and resources. Biosci Microbiota
Food Health 35:173–184
9. Yoon SH, Ha SM, Lim J, Kwon S, Chun J
(2017) A large-scale evaluation of algorithms
to calculate average nucleotide identity. Anto-
nie Van Leeuwenhoek 110:1281–1286
10. Meier-Kolthoff JP, Auch AF, Klenk HP, Goker
M (2013) Genome sequence-based species
delimitation with confidence intervals and
improved distance functions. BMC Bioinfor-
matics 14:60
Isolation and Identification of LAB 13
11. Richter M, Rossello-Mora R, Oliver
Glockner F, Peplies J (2016) JSpeciesWS: a
web server for prokaryotic species circumscrip-
tion based on pairwise genome comparison.
Bioinformatics 32:929–931
12. Parks DH, Imelfort M, Skennerton CT,
Hugenholtz P, Tyson GW (2015) CheckM:
assessing the quality of microbial genomes
recovered from isolates, single cells, and meta-
genomes. Genome Res 25:1043–1055
13. De Man JC, Rogosa M, Sharpe ME (1960) A
medium for the cultivation of lactobacilli. J
Appl Bacteriol 23:130–135
14. Rogosa M, Mitchell JA, Wiseman RF (1951) A
selective medium for the isolation and enumer-
ation of oral and fecal lactobacilli. J Bacteriol
62:132–133
15. Terzaghi BE, Sandine WE (1975) Improved
medium for lactic streptococci and their bacter-
iophages. Appl Microbiol 29:807–813
16. Dicks LMT, Van Vuuren HJJ, Dellaglio F
(1990) Taxonomy of Leuconostoc Species,
Particularly Leuconostoc oenos, as Revealed
by Numerical Analysis of Total Soluble Cell
Protein Patterns, DNA Base Compositions,
and DNA-DNA Hybridizations. Int J Syst
Evol Microbiol 40:83–91
17. Garvie EI (1967) Leuconostoc oenos sp.nov. J
Gen Microbiol 48:431–438
18. Torriani S, Felis GE, Dellaglio F (2001) Differ-
entiation of Lactobacillus plantarum,
L. pentosus, and L. paraplantarum by recA
gene sequence analysis and multiplex PCR
assay with recA gene-derived primers. Appl
Environ Microbiol 67:3450–3454
19. Endo A, Irisawa T, Futagawa-Endo Y,
Sonomoto K, Itoh K, Takano K, Okada S,
Dicks LM (2011) Fructobacillus tropaeoli
sp. nov., a fructophilic lactic acid bacterium
isolated from a flower. Int J Syst Evol Microbiol
61:898–902
20. Overbeek R, Olson R, Pusch GD, Olsen GJ,
Davis JJ, Disz T, Edwards RA, Gerdes S,
Parrello B, Shukla M, Vonstein V, Wattam
AR, Xia F, Stevens R (2014) The SEED and
the Rapid Annotation of microbial genomes
using Subsystems Technology (RAST). Nucleic
Acids Res 42:D206–D214
 Chapter 2

Basic Antibacterial Assay to Screen for Bacteriocinogenic

Lactic Acid Bacteria and to Elementarily Characterize Their
Bacteriocins
Kensuke Arakawa

Abstract
Bacteriocins ribosomally produced by lactic acid bacteria are antibacterial peptides expected to be used as a
safe biopreservative and a fermentation controller in food industry. The modified agar-well diffusion
method is most frequently used for antibacterial activity assay to screen for potentially bacteriocin-
producing strains and to elementarily characterize their bacteriocins and the relatives. Here, I describe
procedure of the modified agar-well diffusion assay in the details.

Key words Lactic acid bacteria, Bacteriocin, Bacteriocinogenics, Antibacterial activity assay, Agar-well
diffusion method

1 Introduction

Bacteriocins are antibacterial peptides ribosomally biosynthesized

by some bacteria including lactic acid bacteria (LAB). Up to date,
many LAB bacteriocins have been discovered, and some of them
have been deeply characterized in structure, genetics, antibacterial
intensity and spectrum, mode of action, secretion and immunity
mechanisms, and so on. On the basis of such characteristics, LAB
bacteriocins are broadly classified into two groups [1]: Class I,
lanthionine and the other modified/unusual amino acids-
containing bacteriocins (named as lanthibiotics), and Class II,
non-lanthibiotic bacteriocins consisting of usual proteinogenic
amino acids. Class II is further divided into four subgroups: Class
IIa, pediocin-like listericidal bacteriocins; Class IIb, synergistically
acting two-component bacteriocins; Class IIc, head-to-tail circular
bacteriocins; and Class IId, the other Class II bacteriocins. Most
LAB bacteriocins have much stronger antibacterial activity particu-
larly against Gram-positive bacteria than any other LAB metabolites
such as organic acid including lactate and acetate, hydrogen

Makoto Kanauchi (ed.), Lactic Acid Bacteria: Methods and Protocols, Methods in Molecular Biology, vol. 1887,
https://doi.org/10.1007/978-1-4939-8907-2_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

15
16 Kensuke Arakawa

peroxide, acetaldehyde, diacetyl, and reuterin. Then, the bacterio-

cins are heat-stable and comparatively pH tolerant and have nar-
row-/broad-spectrum antibacterial activity against food spoilage
and pathogenic Gram-positive bacteria such as Bacillus cereus, Lis-
teria monocytogenes, and Staphylococcus aureus. In addition, LAB
bacteriocins are generally recognized to be safe, because their pro-
ducers, namely, LAB, are familiar with human throughout long
history and their peptide backbones are easy to be digested and
absorbed in the human gastrointestinal tract. Such capable LAB
bacteriocins are expected to be used as a safe and effective biopre-
servative and a fermentation controller instead of conventional
artificial additives in food industry; and therefore many trials for
food application of LAB bacteriocins have been performed and
reported until now. Recently, it is also considered to be the almost
truth that bacteriocins produced by enteric LAB would contribute
to human and animal health by acting as a probiotic factor to kill
pathogens and to maintain microbiota balance in the gastrointesti-
nal tract [2].
In general bacteriocin research, the first step is principally to
screen for a potential bacteriocin producer (a bacteriocino-
genic strain) from LAB collection. In the screening with antibacter-
ial activity assay, the agar-well diffusion method [3] is most
frequently employed after some modifications. Occasionally, the
cross streak method [4], the spot-colony overlay method [5],
spot-on-lawn method [6], paper disk method [7], and microplate
method [8] would be also employed for the screening. However,
some of these methods could be affected by antibacterial activity of
organic acids including lactate and acetate and bacteriophages. In
the modified agar-well diffusion assay, acidic effect is eliminated by
neutralizing test samples such as cell-free culture supernatant (CFS)
before use. Moreover, effect of bacteriophages is distinguishably
reduced in the modified method, because hard agar used there for
propagation of a bacteriocin-indicator makes it difficult for bacter-
iophages to diffuse inside. Perhaps from these reasons, the modified
agar-well diffusion method is most frequently used for bacteriocin
activity assay.
After screening for potential bacteriocinogenics, elementary
characterization of them and their bacteriocins (or bacteriocin-
like inhibitory substances; BLIS) are normally performed as the
second step. For example, relationship between bacterial growth
of and bacteriocin/BLIS production from a producer is investi-
gated, and then heat/storage stability, pH tolerance, proteolytic
susceptibility, and antibacterial spectrum of the bacteriocin/BLIS
are evaluated. Also for these characterizations, the modified agar-
well diffusion assay is mainly used with further modifications as
shown in later sections. After that, advanced characterization is
carried out if needed.
 Antibacterial Assay of Bacteriocins 17

In this chapter, I present procedure of antibacterial activity

assay using the modified agar-well diffusion method, which is the
most basic and important technique in bacteriocin research.

2 Materials

All reagents without any indications are purchased from Nacalai

Tesque (Kyoto, Japan) or FUJIFILM Wako Pure Chemical Corpo-
ration (Osaka, Japan).

2.1 Bacterial Strains 1. Lis. monocytogenes VTU 206 (see Note 1): Distributed from
Department of Veterinary Public Health, Faculty of Agricul-
ture, University of Tokyo (Tokyo, Japan).
2. Lactobacillus delbrueckii subsp. bulgaricus JCM 1002T (see
Note 1): Distributed from Microbe Division (Japan Collection
of Microorganisms; JCM) in RIKEN BioResource Research
Center (RIKEN BRC; Ibaraki, Japan).
3. Lactobacillus sakei subsp. sakei JCM 1157T (see Note 1):
Distributed from JCM.
4. Lactococcus lactis subsp. lactis NBRC 12007 (see Note 2):
Distributed from NITE Biological Resource Center (NBRC)
in National Institute of Technology and Evaluation (NITE;
Chiba, Japan). Formerly named as IFO 12007 and NCDO
497.
5. Lc. lactis subsp. lactis JCM 7638 (see Note 2): Distributed
from JCM. Formerly named as IO-1.
6. Lc. lactis subsp. lactis NBRC 100933T (see Note 2):
Distributed from NBRC.
7. Lactobacillus gasseri JCM 11046 (see Note 3): Distributed
from JCM. Same as strain LA158.
8. Lb. gasseri JCM 11657 (see Note 3): Distributed from JCM.
Same as strain LA39.
9. Lb. gasseri JCM 1131T (see Note 3): Distributed from JCM.
10. Leuconostoc mesenteroides subsp. mesenteroides 406 and 213M0
(see Note 4): These strains were isolated and stocked in my
laboratory.
11. Leu. mesenteroides subsp. mesenteroides NBRC 100496T (see
Note 4): Distributed from NBRC.

2.2 Bacterial 1. De Man, Rogosa, and Sharpe (MRS) broth (see Note 5): MRS
Medium broth powder (Oxoid, Hampshire, UK) is completely dissolved
in distilled water appropriately. After dispensing 5 mL of the
solution to each tube, the medium is sterilized using an auto-
clave at 121  C for 15 min.
18 Kensuke Arakawa

2. Tryptone, yeast extract, lactose, and glucose (TYLG) broth (see

Note 5) [9]: 1.0 g of tryptone (Bectone, Dickinson and Com-
pany; BD, Franklin Lakes, NJ), 0.5 g of yeast extract (BD),
0.5 g of lactose monohydrate, 0.5 g of D-glucose, 0.01 g of L-
cysteine hydrochloride monohydrate, and 0.01 g of Tween
80 are completely mixed and dissolved in 100 mL of distilled
water, and then the solution is adjusted to pH 6.8–7.0 with
1–6 N NaOH (see Subheading 2.3, item 1). After dispensing
5 mL of the solution to each tube, the medium is sterilized
using an autoclave at 121  C for 15 min.
3. GM17 broth (see Note 5): M17 broth powder (Oxoid) is
completely dissolved in distilled water appropriately. After dis-
pensing 4.5 mL of the solution to each tube, the medium is
sterilized using an autoclave at 121  C for 15 min. 10 % (w/v)
lactose and 10 % (w/v) D-glucose solutions are also prepared
and autoclaved under the same condition. 250 μL of both
sugar solutions are aseptically added to the M17 broth tubes.
4. MRS agar (see Note 6): MRS agar powder (Oxoid) is mixed in
distilled water appropriately, and completely dissolved with
boiling. After that, the agar solution is sterilized using an
autoclave at 121  C for 15 min. Before use, it is kept at
55–60 C in a water bath to avoid solidification.
5. Standard Plate Count (SPC) agar (see Note 6): SPC agar
powder (Nissui Pharmaceutical, Tokyo, Japan) is mixed in
distilled water appropriately and completely dissolved with
boiling. After that, the agar solution is sterilized using an
autoclave at 121  C for 15 min. Before use, it is kept at
55–60  C in a water bath to avoid solidification.

2.3 Reagent Solution 1. 1–6 N NaOH: 6 N NaOH solution is diluted with distilled
water appropriately.
2. 1–6 N HCl: HCl reagent (12 N) is diluted with distilled water
appropriately.
3. Sterile physiological saline (0.85% NaCl solution): NaCl is
mixed and dissolved in distilled water appropriately. After dis-
solving, the solution is sterilized using an autoclave at 121  C
for 15 min.
4. Enzyme solutions [9]: Catalase and protease solutions are used
for enzymatic susceptibility tests of a bacteriocin/BLIS. Each
enzyme is dissolved in and diluted with distilled water to the
final concentration at 1 U/mg (see Note 7).
 Antibacterial Assay of Bacteriocins 19

3 Methods

Operation for bacterial inoculation, agar plate preparation, well

formation, sample dilution, and sample injection are aseptically
performed in a clean bench.

3.1 Preparation 1. LAB strains are precultivated twice in MRS (for lactobacilli)
of CFS Samples from and TYLG (or GM17; for LAB cocci) broths at each optimum
LAB Cultures temperature such as 25, 30, or 37  C for 24 h (see Note 5).
2. The LAB strains are cultivated under the same conditions as
above for the following CFS sample preparation (see Note 8).
3. Cultures are pH-adjusted normally to 7.0 using 1–6 N HCl
and 1–6 N NaOH and then centrifuged at 1600  g for 20 min
(see Notes 7, 9, and 10).
4. After centrifugation, the culture supernatants are filtered
through a 0.20 μm membrane (syringe filter; Sartorius, Göttin-
gen, Germany) to prepare CFS samples (see Notes 7 and 10).
5. If needed, CFS is aseptically diluted twofold with sterile physi-
ological saline using wells of a microtiter plate.

3.2 Preparation 1. Lis. monocytogenes VTU 206, Lb. delbrueckii subsp. bulgaricus
of Agar Plates JCM 1002T or Lb. sakei subsp. sakei JCM 1157T used as a
Containing indicator in the modified agar-well diffusion assay are preculti-
an Indicator Strain vated twice in TYLG (for VTU 206) and MRS (JCM 1002T
and Formation and JCM 1157T) broths at 30 (JCM 1157T) and 37  C (VTU
of Sample Wells 206 and JCM 1002T) for 24 h (see Note 5).
2. 50 μL of culture solution of an indicator strain and 20 mL of
SPC (for VTU 206) or MRS (for JCM 1002T and JCM 1157T)
agar solution at around 50–55  C are poured into a petri dish
and gently but well stirred before solidification (see Note 11).
3. After agar solidification, punch wells (at most 20) in an agar
plate using a metal cork-borer (6 mm in diameter) which is
flame-sterilized and cooled down before use (see Note 12).

3.3 Sample Injection 1. 50 μL of CFS samples and the twofold dilutions prepared in
into Wells above or bacteriocin solution are injected into a well.
and Incubation 2. The agar plate is incubated at 30 (JCM 1157T) or 37  C (VTU
of the Agar Plate 206 and JCM 1002T) for 24 h.

3.4 Evaluation of 1. After incubation, a clear zone without cell growth of the indi-
Antibacterial Activity cator around a well means presence of a bacteriocin/BLIS.
Antibacterial activity of a bacteriocin/BLIS is evaluated with
size (mm in diameter) of the clear zone (see Notes 13 and 14).
Moreover, antibacterial titer is determined by arbitrary units
(AU) which was defined as the reciprocal of the highest dilu-
tion inhibiting the growth of the indicator strain (see Note 15).
20 Kensuke Arakawa

4 Notes

1. Lis. monocytogenes VTU 206, Lb. delbrueckii subsp. bulgaricus

JCM 1002T, and Lb. sakei JCM 1157T are highly bacteriocin
sensitive and therefore used as an indicator strain on the anti-
bacterial assay [8–10].
2. Lc. lactis subsp. lactis NBRC 12007 and JCM 7638 produce
nisins A and Z, respectively [10]. Nisin is the most famous
Class I lanthibiotic bacteriocin and is already practically used
as biopreservatives in worldwide food industry. Nisin A reagent
(Sigma) is also launched onto the market. They are often used
for a positive control in the antibacterial assay. On the other
hand, Lc. lactis subsp. lactis NBRC 100933T, a non-bacteriocin
producer, is used as a negative control.
3. Lb. gasseri JCM 11046 and JCM 11657 produce gassericins T
and A, respectively [8]. Gassericin A is a representative Class IIc
circular bacteriocin. Gassericin T is a representative class IIb
two-component bacteriocin. They are used as a positive control
in the antibacterial assay, whereas Lb. gasseri JCM 1131T, a
non-bacteriocin producer, is used as a negative control in
the case.
4. Leu. mesenteroides subsp. mesenteroides 406 and 213M0 were
isolated from Mongolian traditional fermented milk, airag, in
my laboratory [9, 11]. They both produce an identical Class IIa
listericidal bacteriocin to mesentericin Y105 [12, 13] and
therefore can be used as a positive control in the antibacterial
assay. In the case, Leu. mesenteroides subsp. mesenteroides
NBRC 100496T, a non-bacteriocin producer, should be used
as a negative control.
5. MRS and BM17 broths are generally used to cultivate LAB
rods (lactobacilli) and LAB cocci, respectively. In my labora-
tory, TYLG broth is used to propagate LAB cocci and Lis.
monocytogenes [9].
6. In this procedure, two kinds of agar media are used to cultivate
indicator strains for the modified agar-well diffusion antibac-
terial activity assay. MRS agar is used for Lb. delbrueckii subsp.
bulgaricus JCM 1002T and Lb. sakei JCM 1157T [8, 10]. SPC
agar is used for Lis. monocytogenes VTU 206 [9, 11].
7. In the case of enzymatic susceptibility tests of a bacteriocin/
BLIS using catalase and several proteases [9], CFSs at pH 2.0,
7.0, 7.5, and 7.8 are prepared for reactions with pepsin
(37  C), catalase (25  C), trypsin (37  C), α-chymotrypsin
(25  C), and proteinase K (37  C), respectively. After each
reaction for 1 h, enzymes are inactivated with heating at
95  C for 10 min. Then, each enzymatically treated CFS is
 Antibacterial Assay of Bacteriocins 21

used for antibacterial activity assay. By decrease of antibacterial

activity, it can be judged whether the BLIS in CFS is a bacteri-
ocin with a peptide backbone or not.
8. In the case of analysis of relationship between bacterial growth
of and bacteriocin/BLIS production from a producer [9], the
strain is cultivated for 72 h. Each culture after incubation for
0, 2, 4, 8, 12, 18, 24, 48, and 72 h is collected and used to
evaluate the growth by measurement of culture pH, turbidity,
and viable cell count and to prepare CFS for antibacterial
activity assay.
9. In the case for determination active pH range of a bacteriocin/
BLIS [9], culture supernatant is pH-adjusted to 2, 3, 4, 5, 6, 7,
8, 9, 10, and 11 using 1–6 N HCl and 1–6 N NaOH. After pH
adjustment, the culture supernatant is centrifuged again and
followed by sterile-filtration to prepare CFS samples.
10. In the case of heat/storage-stability tests of a bacteriocin/BLIS
[9], CFSs at pH 4.5 and 7.0 are prepared. The CFSs are heated
at 65  C for 15–60 min or 99–121  C for 15 min or stored at
4  C for 7 days or 25  C for 1–7 days. After heating and
storage, all CFSs are used as samples to measure antibacterial
activity.
11. In the case for estimation of antibacterial spectrum of a bacte-
riocin/BLIS [9], various species bacteria such as other lactic
acid bacteria, food-spoilage bacteria, and foodborne pathogens
are used as tested strains instead of the indicator strains.
Growth media and conditions and inoculum ratio are depen-
dent on each tested strain.
12. It should not be over time (>1 h) to punch wells, because
indicator cells gradually grow. If you feel difficult to punch
wells only using a sterile cork-borer, I recommend that residual
agar gel in a well is removed using a needle-like tip tweezers
which is of course flame-sterilized and cooled down before use
as well as the cork-borer.
13. Size of clear zones formed are various among indicators even if
the same sample is injected. In addition, even if the same
indicator is used, the size is varied among growth media and
(pre)cultivation conditions of the indicator. Therefore when
you evaluate antibacterial activity of a bateriocin/BLIS, you
must use a bacterial medium and fixed culture conditions for an
indicator.
14. Size of clear zones formed do not correspond to the antibac-
terial titer, because bacteriocins/BLISs have different diffu-
sionability and permeability into agar gel. To determine
antibacterial titer, it is necessary to employ the sample CFS
dilution method.
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