Khóa luận tốt nghiệp Công nghệ sinh học: Evaluate the antimicrobial and antioxidant activities of some Lacticaseibacillus rhamnosus and Streptococcus thermophilus strains

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESEVALUATE THE ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF SOME Lacticaseibacillus rhamn

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

EVALUATE THE ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF SOME Lacticaseibacillus rhamnosus AND

Streptococcus thermophilus STRAINS.

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

First of all, I would like to express my sincere thanks to my two supervisors Assoc.Prof Dr DOUGLAS SHYU and Assoc Prof Dr NGUYEN BAO QUOC for theirpatience and wisdom, and for helping me conduct my thesis in a detailed and analyticalway This accomplishment would not have been possible without their guidance and the

opportunity they provided me to undertake this project in BT306 lab and TEEP program

I would like to express my profound gratitude for their invaluable advice and patiencethroughout this journey The experience of working with them and the other labmembers has been truly enriching

I would also like to extend my deepest gratitude to my labmates in BT306 (NPUST

— Taiwan) and RIBE 304 (NLU - Vietnam) Their unwavering support, insightfulthoughts, and inspiring ideas played a crucial role in my research I consider myselffortunate to have such wonderful friends and colleagues by my side throughout thisjourney

Finally, I cannot express enough gratitude to my family Their unconditionalsupport and encouragement have been my anchor during the challenging times Theybelieved in me, even when I doubted myself, and for that, I am eternally grateful

I am deeply thankful for everything and it's been a privilege learning under theirmentorship

CONEIRMATION AND COMMITMENT

My name is Truong Ngoc Hai Yen, student ID: 19126242, class: DH19SHD(Phone number: 0797783156, email: 19126242@st.hcmuaf.edu.vn), of Biotechnology,Nong Lam University Ho Chi Minh city This is my undergraduate thesis which wasconducted by myself I hereby declare that, all the information in this document has beenobtained and presented in accordance with academic rules and ethical conduct I takefull responsibility in front of the committee for these commitments

Thu Duc city, February 28", 2024

Student’s signature

Truong Ngoc Hai Yen

hị

Lactic acid bacteria (LAB) are one of the most significant groups of probioticorganisms, which are frequently used in a variety of fields including medical treatment,food industry, and livestock production These bacteria have attracted scientificattention due to their probiotic, antimicrobial and antioxidant properties since they were

first discovered by Louis Pasteur in 1857 In this research, the study was conducted to

screen the antimicroorganism and antioxidant activities of 8 strains of LAB including 2species: L rhamnosus (THH-2, B9, 38) and S thermophilus (Jt1, Jt4, Jt5, Jt9, Jt12)

strains The antimicrobial tests, using agar-diffusion, were carried out against various

pathogens like Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923,Salmonella enterica, Enterococcus faecium, Klebsiella pneumoniae ATCC 13883,Bacillus cereus, and Candida albicans Results showed no inhibitory activity againstthese 7 pathogens Regarding antioxidant properties, several methods including DPPH

scavenging, superoxide scavenging, and hydroxyl radical scavenging were conducted

using cell-free supernatant (CFS) and intact cells (ICs) extract The results showed thatall of the target strains possessed antioxidant activity as determined by those methodswith both types of extracts, in with the cell-free supernatant displayed higher activitythan the intact cell, and S thermophilus strains showed higher activities in eliminatingsuperoxide and hydroxyl radical when compared to LZ rhamnosus strains In addition,antibiotic susceptibility test was also performed, LZ rhamnosus was obsered to beresistant to vancomycin (3/3), chloramphenicol (3/3), kanamycin (2/3), and ampicillin(1/3), whereas, S thermophilus was resistant to kanamycin (5/5), and chloramphenicol(4/5), all 8 studied strains showed low susceptibility to sulfamethoxazole (MIC higherthan 256 pg/mL) The findings of this study suggest that the antioxidant characteristics

of the studied strains mainly exist outside the cell and can be effectively harnessed byidentifying them in the culture supernatant, in with CFSs of S thermophilus Jt9 wassuggested as promise sources against oxidative stress and warrants further investigation

Keyword: Lacticaseibacillus rhamnosus, Streptococcus thermophilus, lactic-acidbacteria, antimicrobial activities, antioxidant, antibiotic

1H

TÓM TẮT

Vi khuẩn lactic (LAB) là một trong những nhóm probiotic quan trọng nhất, thường

được sử dụng trong nhiều lĩnh vực bao gồm điều trị y tế, ngành công nghiệp thực pham

và chăn nuôi Những vi khuân này đã thu hút sự chú ý của giới khoa học đo tính chất probiotic, kháng khuẩn và chống oxi hóa của chúng từ khi được Louis Pasteur khám pha

ra lần đầu tiên vào năm 1857 Trong nghiên cứu này, việc nghiên cứu được tiên hành dé

sàng lọc hoạt động kháng khuẩn, kháng nam và chống oxi hóa của 8 chủng LAB bao

gồm 2 loài: 7 rhamnosus (THH-2, B9, 38) và S thermophilus (Jt1, Jt4, Jt5, Jt9, Jt12).

Sử dụng phương pháp thạch khuếch tán để thí nghiệm kháng nhiều loại tác nhân nhưEscherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Salmonellaenterica, Enterococcus faecium, Klebsiella pneumoniae ATCC 13883, Bacillus cereus,

va Candida albicans Kết quả cho thay không có hoạt động ức chế đối với 7 loại tác nhân này Đối với tính chất chống oxi hóa, một số phương pháp bao gồm bắt giữ DPPH,

gốc superoxide và gốc hydroxyl đã được tiễn hành sử dụng mau dịch nồi (CFS) va dịch

tế bào nguyên (ICs) Kết quả cho thấy tất cả các chủng mục tiêu đều có hoạt động chống oxi hóa được xác định bởi những phương pháp này với cả hai loại chiết xuất, trong đó dịch nồi hiển thị hoạt động cao hơn tế bào nguyên, và chung S thermophilus hién thi hoạt động cao hon trong việc loại bỏ gốc superoxide và gốc hydroxyl so với chủng L.rhamnosus Ngoài ra, thử nghiệm độ nhạy cảm với kháng sinh cũng được thực hiện, 7.rhamnosus được quan sát có khả năng khang với vancomycin (3/3), chloramphenicol

(3/3), kanamycin (2/3), va ampicillin (1/3), trong khi đó, S thermophilus kháng với

kanamycin (5/5), va chloramphenicol (4/5), tat cả 8 chủng được nghiên cứu cho thay độ nhạy thấp với sulfamethoxazole (MIC > 256 pg/mL) Những phat hiện của nghiên cứu này gợi ý rằng các đặc tính chống oxi hóa của các chủng được nghiên cứu chủ yếu tồn tại bên ngoài tế bào và có thê được khai thác hiệu quả bằng cách xác định chúng trong dịch nồi nuôi cấy, trong đó CFSs của S thermophilus Jt9 được đề xuất là nguồn tiềm năng chống lại căng thăng oxi hóa và đáng được nghiên cứu thêm.

Từ khóa: Lacticaseibacillus rhamnosus, Streptococcus thermophilus, vi khuẩn lactic, hoạt động khang khuẩn, chống oxi hóa, khang sinh.

IV

TABLE OF CONTENTS

Page

ACKNOWLEDGMUENIS vss ssnsssssssnssasassssnceess ESiS.G68.5GS:3M913238448eSEESaag93S8Sa§SEE4018iC883,3ã8gã23.00-836 i

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2.4 Introduction of reactive OXYGEN SD€CI€S - - 0 22 22 222 S2 2 1221121151111 111 1 re 8

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CHAPTER 3 MATERIALS AND METHODS 5-72 S-c+csscserserrrsrrrrrrree 14

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3.2 Materials and equipment 8 148.2 1s WE CLO OTS ATISTIG toss ses ss s6 1á hồ HH5 H6 senza nu SS: E514g838930538.05590883555848ESu.7020134958.18010138850.001688 143.2.2 00.6 0 143.2.3 Required tools, equipments and chemicaÌS - ¿552552 ++2£++£+s>zeesexsss 15

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3.3.1 Preparation of L rhamnosus and S thermophilus stra1ms - 153.3.2 Preparation of pathogenic ImICTOOTØATIISINS 5 2 25222222 *+2£22E£+zE£zezzeczx 15

3.3.3 Cell-free supernatants (CFSs) preparatIOI - - + +++<++x++e+seererrrrerrrree 163.34, Intact cells: (ICs) DEGIDMESIOH ssssavsosczttasbeioiostsittgidtoglsstGsttoasiitilststBiBE4loiriogigs4e 16323.5 Agar Gitius one tho so: csssxssisssssecLsrse15220256051E88863833495%38160630018645u08135gi601E6340123014i083 17

3.3.6 Antibiotic susceptibility testing method - - 5+ 5+2 s+xs+svxseeererrrrrrrrree 17

3.3.7 Determination of the DPPH radical scavenging effect -5+-s<<+ 183.3.8 Superoxide anion scavenging aCfIVIẨY - - -c- c- + ss xxx nh ng ngư 193.3.9 Hydroxyl radical scavenging aC{IVIẨY - c2 221 2312211211221 21151 21 1 re 193.3210: SafIsHo:atalysistHiGNO(SiocessssesrenasosibndiiriooodgusientddioinsiibditSoifui9c500gi8080gi0u020g60004:nil 20

CHAPTER 4 RESULTS AND DISCUSSION -Ặ 2e 21

4.1 Antibiotic susceptibility testing 1077 21

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CHAPTER 5 CONCLUSIONS AND SUGGESTIONS -cc2ccccccecce 35

5.1 CONCLUSIONS sccercunamenenecemmnsesew aces eee 35

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vii

LIST OF ABBREVIATIONS

A : Ampicillin

AA : Ascorbic acid

ANOVA : Analysis of Variance

ATCC : American type culture selection

atm : Standard atmosphere

BHA : Butylated Hydroxyanisole

EFSA : European Food Safety Authority

et al : and others

GPx : Glutathione peroxidase

GR : Glutathione reductase

GRAS : Generally recognized as safe

HRS : Hydroxyl radical scavenging

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ICs : Intact cells

K : Kanamycin

LA : Lactic Acid

LAB : Lactid acid bacteria

MBC : Minimal Bactericidal Concentration

MIC : Minimal Inhibitory Concentration

MRS : De Man, Rogosa, and Sharpe medium

ROS : Reactive oxygen species

SOD : Superoxide dismutase

LIST OF TABLES

Page

Table 3.1 Microbialogical breakpoints categorising bacteria as resistant 18

Table 4.1 Minimum inhibitory concentration (MIC) values - 55+ 21Table 4.2 Minimum bactericidal concentration (MBC) values - +- 22

Antimicrobial subtances of LAB .- - c5 St Sx St ssrsrksrrsrrrrrrrree 3

Proposed modes of action of probiotic LAB antioxidants 4Colony morphology and cell morphology of L rhamnosus . 5Colony morphology of S thermophilus Jt1, Jt4, Jt5, Jt9 Jt12 796-wells plate with MIC experiment of S thermophilus Jt\ after 24 hours

st Si ee ie creel en es ee eto 22Inhibition growth of pathogens by L rhamnosus, cccceeceeeeeeeeee teens 25

Inhibition growth of pathogens by supernatant of S thermophilus 25

Inhibition growth of pathogens by suspension of S thermophilus 26DPPH free radical scavenging activity (0) .e:ccscceseseeeeseeeceeeeeseeeeeeeees 2796-wells plate with DPPH scavenging by ICs of L rhamnosus strains .27Superoxide anion scavenging activity (9) -ccscssccssrsresrrrrres 2996-wells plate with superoxide anion scavenging by CEFSs 29Hydroxyl scavenging activity (%) eceeceeceeeeeeeeeeeeeteeeeeeteteeetettteteene dLFigure 4.10 96-wells plate with hydroxyl scavenging by CFSs of S thermophilus

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XI

CHAPTER 1 INTRODUCTION

1.1 Overview

Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylatedhydroxy-anisole (BHA), and n-propyl gallate (PG) exhibit strong antioxidant againstvarious oxidation systems; however, their use in food also have adverse effects such asliver damage and carcinogenic effects, which led to be restricted or prohibited in certaincountries (Osuntoki, 2010) Besides that, after the discovered revolution in the “golden

era”, the main problems of chemotherapy were possessed when some excitingcompounds (such as tetracyclines, cephalosporins, aminoglycosides, and macrolides)are in danger of losing their efficacy because of the increase in microbial resistance

(Mayers, 2009) which led to the overuse of antimicrobial products and the growingemergence of various resistant pathogens Thus, in recent years, it becomes veryimportant to find safer and natural antioxidants as well as antimicrobials using biologicalresources to replace synthetic products

The availability of probiotic microorganisms as natural antioxidants andantimicrobials has also been investigated Besides the long history of consumptionwhich proves the safety of consuming lactic acid bacteria (LAB), lactic cultures havebeen reported to make up a large interest in the antioxidant effects as well as

antimicrobial activities Their beneficial effects have possessed their role in

health-promoting, against diseases and make these microorganisms desirable for use in dairyproductions and other food products Antioxidant properties of LAB and other beneficialeffects of these bacteria species have proved the potential on eliminating oxidative stressand possessing inhibit pathogens activity (Ji et a/., 2015)

Among LAB group, Lacticaseibacillus rhamnosus strains and Streptococcusthermophilus strains have attracted attention for their potential probiotic effects inhuman health They are recognized as one of the important members of the healthyhuman microbiota (Naaber ef a/., 1998), which have ability on antimicrobial effects;ability to endure gastrointestinal stresses, acid tolerance, bile tolerance; protection of theepithelial barrier; and enhancement of immune response (Mathipa-Mdakane andThantsha, 2022)

1.2 Objective

The objective of this thesis 1s to screen and evaluate the antioxidant andantimicrobial activity (including antibacterial and antifungal) of someLacticaseibacillus rhamnosus and Streptococcus thermophilus strains In addition,antibiotic susceptibility also be determined

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction of lactic acid bacteria

2.1.1 Overview

Lactic acid bacteria (LAB) include the genera Leuconostoc, Lactococcus,Lactobacillus, Pediococcus, Enterococcus, Streptococcus, Vagococcus, Aerococcus,

Carnobacterium, Tetragenococcus, Oenococcus and Weissella (Zheng et al., 2020)

They are gram-positive microorganisms known as the main safe industrial-scaleproducers of lactic acid (LA)

The lactic acid bacteria (LAB) are a heterogeneous group of gram positive,

non-spore-forming bacteria with a rod-shaped or coccoid morphology Lactic acid is the

predominant end-product when LAB engage in hexose fermentation, and it is due to the

preservative properties of lactic acid that has for many centuries rendered this group ofbacteria applicable in food and feed fermentations, in particular for the production ofdairy products (Mahony and Sinderen, 2014)

organic acids (lactic, citric, acetic, fumaric, and malic)

Figure 2.1 Antimicrobials substances of LAB (Sofia et.al., 2020)

produce lactic acid as a byproduct of carbohydrate fermentation, which helps in

preserving food and providing a characteristic flavor

Apart from their role in food fermentation, lactic acid bacteria have attracted

attention for their potential health benefits These bacteria are recognized for theirprobiotic properties, which promote gut health and enhance the immune system.Additionally, they exhibit antimicrobial and antioxidant properties Organic acids

(lactic, citric, acetic, fumaric) and malic acid, as well as hydrogen peroxide and

bacteriocins, are some of the antimicrobial substances produced by lactic acid bacteria.Enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione

peroxidase (GPx) are part of the defense system in LAB species Moreover, a

non-enzymatic defense mechanism against various reactive oxygen species (ROS) has alsobeen documented

Scavenging of Free Radicals Regulating of Enzymes

DPPH Peroxyl radical Produce antioxidant enzymes

ABTS Hydrogen peroxide Regulate host antioxidant enzymes Superoxide anion Nitroso Reduce ROS producing enzymes

Hydroxy radical

Figure 2.2 Proposed modes of action of probiotic LAB antioxidants Based on the

study of (Feng and Wang, 2020)

2.2 Introduction of Lacticaseibacillus rhamnosus

2.2.1 Overview and taxonomy

Lacticaseibacillus rhamnosus was previously known as Lb casei subsp.rhamnosus, etymologically pertaining to rhamnose, is a bacterium that belongs to the

Lactobacillaceae family (Corsetti, 2011) These bacteria are common in traditional

fermented foods, animals, human, and natural habitats

According to studies of Felis, 2007 and Zheng et al., 2020, Lacticaseibacillus

rhamnosus 1s classified as a bacterium within the domain of Bacteria It is further

categorized within the phylum Firmicutes, class Bacilli, under the order Lactobacillales.This bacterrum belongs to the family Lactobacillaceae and is grouped within the genusLacticaseibacillus Its full scientific name 1s Lacticaseibacillus rhamnosus

Reported as nonpathogenic and probiotic strains, these beneficial bacteria strains

are believed to offer various health advantages, such as combating intestinal pathogens

and eliminate oxidative free radial Specifically, L rhamnosus has been deemed safe by

the US Food and Drug Administration with its “Generally Recognized As Safe (GRAS)”status and included in the “Qualified Presumption of Safety (QPS)” list by the

“European Food Safety Authority” (EFSA) (Pino et al., 2022)

2.2.2 Characteristics

L rhamnosus LR-L31 L rhamnosus LR-L34 L rhamnosus LR-L35

Colony morphology

Gram stain

Lacticaseibacillus rhamnosus is a gram-positive anaerobic bacteria, non-motile,

non-sporulating, acidophilic and facultative heterofermentative bacterium Cells havethe form of rods, singly or in short chains As they are mesophile bacteria, they could

grow on MRS or M17 medium after incubating at the optimum temperature at 37°C for

24 - 48 hours (Boonma ef a/., 2014)

2.2.3 Studies related to Lacticaseibacillus rhamnosus.

Selected strains belonging to L rhamnosus species are extensively used asprobiotics in food formulations, health and functional foods based on their well-documented for its probiotic activities and clinical benefits (Kalliomaki et a/., 2001).For instance, the beneficial effects of L rhamnosus GG (Gorbach-Goldin) have beenstudy for 2 decades (Saxelin ef al., 1995; Vanderhoof and Mitmesser, 2010), which hadbeen identified as the potential probiotic strain due to its resistance ability to acid andbile, as well as the well growth characteristics and the effects of adherence of severalpathogens that allow it to survive and persist within the gastrointestinal tract (de Champs

et al., 2003; Doron et al., 2005; Forestier ef al., 2001) L rhamnosus CA15 (DSM33960) was detected as broad spectrum of antimicrobial activity, which the high effectspossessed against Candida albicans ATCC10231, Candida krusei ATCC 14243,Gardnerella vaginalis ATCC 14018, and Streptococcus agalactiae DSM 2134 (Pino etal., 2022) In addition, in the same study, Lacticaseibacillus rhamnosus CA15 (DSM33960) showed the ability to inhibit the adhesion as well as to displayed all the testedpathogens with percentages higher than 50% L rhamnosus CJNU 0519, was shown to

produce the bacteriocin rhamnocin 519, which displayed a narrow antimicrobial

spectrum that included S aureus and strongly inhibited L monocytogenes

The antioxidative effects using L rhamnosus strains also reported L rhamnosusCA15 (DSM 33960) with similar DPPH radical scavenging activity at a percentageabout 80%, was detected using different concentrations of the strain culture filtrate,while this strain was able to scavenge superoxide anion in a dose-dependent manner,reaching about 80% at a concentration of 3 mg/l

L rhamnosus CA15 (DSM 33960) strain classified as a high hydrogen peroxideproducer, high concentration of organic acid production including lactic acid (799.200mmol/L), propionic (98.54 mmol/L), succinic (98.48 mmol/L), butyric (39.61 mmol/L)and acetic acids (18.31 mmol/L) (Pino et al., 2022)

2.3 Introduction of Streptococcus thermophilus

2.3.1 Overview and taxonomy

Streptococcus thermophilus is a gram-positive bacterium, motile, endospores forming It is also classified as a lactic acid bacterium, which is commonlyfound in fermented milk products and is generally used in the production of yogurt

non-6

According to Bergey’s classification system, Streptococcus thermophilus isclassified as a bacterium within the domain of Bacteria It is further categorized withinthe phylum Bacillota, class Bacilli, under the order Lactobacillales This bacteriumbelongs to the family Streptococcaceae and is grouped within the genus Streptococcus.Its full scientific name is Streptococcus thermophilus.

It is recognized as a fermentative facultative anaerobe, belonging to the viridansgroup The genus Streptococcus includes several pathogenic species, such as S

pneumoniae and S pyogenes, but food industries consider S thermophilus as pathogenic and potential probiotic bacteria

non-2.3.2 Characteristics

Like most lactic acid bacteria, Streptococcus thermophilus is non-spore-forming,

non-motile, catalase-negative, facultatively anaerobic and metabolically fermentative

Microscopically, the cell of S thermophilus were circular in shape (0.7 - 0.9 pm in

diameter) and commonly occurring in pairs or chains when grown in liquid media

S thermophilus Jt1 S thermophilus Jt4 S thermophilus Jt5

—

Figure 2.4 Colony morphology of S thermophilus Jt1, Jt4, Jt5, Jt9 Jt12

Although its name would appear to indicate that S thermophilus has a high

optimum growth temperature, S thermophilus has an optimal growth temperature as

7

mesophilic range of 35 - 42°C S thermophilus is negative for cytochrome, oxidase,

catalase testing, and positive for alpha-hemolytic activity The colony characters of S

thermophilus can be described as creamy white, circular, irregular, convex with thecolony size around 0.5 - 3.0 mm (Guevarra, 2016)

2.3.3 Studies related to Streptococcus thermophilus strains

S thermophilus has been considered as produces various of important propertieswhich being used as production of lactase, an enzyme that converts lactose (milk sugar)into a simple sugar, which helps people who are lactose intolerant of milk digest Theprevious study has proved that the consumption of this microbe has facilitated toalleviate symptoms of lactose intolerance and other gastrointestinal problems (Sharma

et al., 2014) With this unique feature of this strain, it has been added to several healthsupplements and become the potential strains in application

Additionally, it is known to produce a range of antimicrobial factors, such asmetabolic byproducts, antibiotic-like substances, and bactericidal proteins known asbacteriocins Bacteriocin produced by S thermophilus has reported as the effective bio-

preservative ingredient into model food systems by showing the inhibit control of

pathogenic and spoilage microorganisms These components help in preventing varioustypes of infections caused by pathogenic microbes S thermophilus and some intestinalpathogenic organisms including Klebsiella sp., Pseudomonas sp., Salmonella sp., and

E coli (Silva et al., 2018)

Some classical yogurt bacteria S thermophilus inhibit peroxidation of lipidsthrough scavenging the reactive oxygen radicals, such as hydroxyl radical, or hydrogenperoxide (Lin and Yen, 1999)

2.4 Introduction of reactive oxygen species

Free radicals or reactive oxygen species (ROS) is a class of highly reactive moleculeswhich 1s capable of independent existence, containing at least one oxygen atom and one ormore unpaired electron They can be free radicals, atoms, and molecules with an unpairedelectron in their outer shell, and because of their chemical structure, ROS are unstable, highlyreactive species and highly toxic ROS includes hydroxyl radical (OH), hydroperoxy] radical(HOO»), superoxide anion free radicals (O2.), singlet oxygen, as well as free nitrogen radicals.Under physiological conditions, small quantities of ROS are formed during cell processes,such as aerobic respiration or inflammatory processes, mainly in hepatocytes and

8

macrophages Reactive oxygen species are primarily signaling molecules In addition, theyinduce cell differentiation and apoptosis, thus contributing to the natural ageing process Theyalso participate in muscle contractions, regulation of vascular tone, and determine bactericidaland bacteriostatic activity Increased production of free radicals is caused by excessiveexposure to UV radiation, long-term stress conditions, intense physical exercise, improperdiet and use of stimulants Under physiological conditions, there is a balance between the

generation and removal of free radicals from the body (Jakubezyk ef al., 2020)

Reactive oxygen species (ROS) can cause damage to the basic building blocks ofthe cell including DNA, protein and lipids DNA damage can occur in the form of doublestranded breaks

During evolution, most living organisms possessed enzymatic defense (superoxidedismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR)) and non-enzymatic antioxidant defenses (glutathione, thioredeoxin, Vitamin C, Vitamin E), andrepair systems to protect them against ROS (Wang ef al., 2017) However, thesenaturally antioxidant systems are generally not enough to against oxidative stress, whichlead to the need of antioxidant additives using substance that delay or prevent the

damage of ROS to human body Several synthetic antioxidants, including butylated

hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), have been widely used,their safety has recently been questioned due to liver damage and carcinogenicity Theseissues led those synthetic addictive to be restricted or prohibited in certain countries,and increase the attention of finding safer and more effective antioxidants from bio-resources such as plant extract and microorganisms

2.5 Introduction of some pathogenic microorganisms

2.5.1 Salmonella enterica

Salmonella sp is a gram-negative bacteria that 1s regconized as one of the mostimportant bacteria genus belonging to the family Enterobacteriaceae

According to Bergey’s classification system, Sa/monella enterica 1s classified as a

bactertum within the domain of Bacteria It is further categorized within the phylumProteobacteria, class Gamma Proteobacteria, under the order Enterobacteriales Thisbactertum belongs to the family Enterobacteriaceae and is grouped within the genusSalmonella Its full scientific name is Salmonella enterica

Salmonella is a motile bacteria and non-forming spore In food, Salmonella ispresent in meat, eggs, vegetables, butter, and dairy products Food that is contaminatedwith Salmonella or other harmful germs usually looks, tastes, and smells normal.Furthermore, food is not the only way Salmonella infects people, they also spreadthrough contaminated water, the environment, other people, and animals They arepresent in the intestines of animals such as pigs, cows, poultry, turtles, snakes, etc.2.5.2 Escherichia coli

Escherichia coli (E.coli) is a gram-negative bacterium that is considered the mostnumerous aerobic commensal inhabitant of the large intestine It belongs to theEnterobacteriaceae family

According to Bergey’s classification system, Escherichia coli is classified as a

bacterium within the domain of Bacteria It is further categorized within the phylumProteobacteria, class Gamma Proteobacteria, under the order Enterobacteriales Thisbactertum belongs to the family Enterobacteriaceae and is grouped within the genusEscherichia Its full scientific name 1s Escherichia coli

E.coli 1s commonly found in the lower intestine of warm-blooded organisms Most

E.coli strains are harmless However, some strains such as Shiga toxin-producing E.coli

can cause diarrhea, infection in people’s tract, and and severe serious food poisoning It

is usually transmitted to humans through contaminated food, such as raw orundercooked products, contaminated vegetables, yogurt, and cheese made from rawmilk Consumption of contaminated water and other food can also lead to infection, aswell as cross-contamination during the preparation of products

2.5.3 Staphylococcus aureus

Staphylococcus aureus (S.aureus) is a gram-positive cocci, that belongs to the

facultative anaerobes group, does not have flagella, can not move, and is a non-forming

spore bacteria

According to Bergey’s classification system, Staphylococcus aureus 1s classified

as a bacterium within the domain of Bacteria It 1s further categorized within the phylumFirmicutes, class Bacilli, under the order Bacillales This bactertum belongs to thefamily Staphylococcaceae and is grouped within the genus Staphylococcus Its speciesname is aureus, thus forming its full scientific name as Staphylococcus aureus

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S.aureus are capable of producing endotoxin enterotoxin, a toxin implicated incases of staphylococcal food poisoning Staphylococci are tolerant in solutions with saltconcentrations up to 12% and sugar concentrations up to 50%, even Enterotoxinproduced by S aureus can still be active after being boiled for 30 minutes (Bhunia,2008) Therefore, they have the potential to cause poisoning on processed foods such assalted meat, cream cakes, cheeses, heat-processed dishes, etc., and especially on

uncooked food

2.5.4 Candida albicans

Candida albicans 1s a popular pathogenic yeast It is common fungus that livesskin and inside the body, including the mouth, throat, gut and vagina Although theynormally live in a body with small amounts that couldn’t cause problems, Candida cancause infections if 1t grows out of control or enters deep into the body such as the kidney,heart, or brain

According to Bergey’s classification system in 2000, Candida albicans is

classified within the domain of Eukaryota It is further categorized within the phylum

Fungi, class Saccharomycetes, under the order Saccharomycetales This yeast belongs

to the family Saccharomycetaceae and is grouped within the genus Candida Its speciesname is albicans, thus forming its full scientific name as Candida albicans

2.5.5 Enterococcus faecium

Enterococcus faecium is a gram-positive, coccal shaped, facultative anaerobicbactertum that can occur in pairs or chains Its natural habitat includes thegastrointestinal tract, oral cavity, and vaginal tract of a wide variety of animals Thecolonies that are produced appear wet and have an average size of 1 - 2 mm È faeciumcan survive for long periods of time inside hospital, sewage, and soil It has the ability

to proliferate in basic, acidic, isotonic, or hypertonic environments with temperatures

ranging from 10 to45°C (Mourand ef a/., 2014)

According to Bergey’s classification system in 2000, Enterococcus faecium is

classified as a bacterium within the domain of Bacteria It 1s further categorized withinthe phylum Bacillota, class Bacilli, under the order Lactobacillales This bacteriumbelongs to the family Enterococcaceae and is grouped within the genus Enterococcus.Its species name is faecium, thus forming its full scientific name as Enterococcusfaecium

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Enterococci are recorded as part of the normal intestinal flora Among theEnterococci, Enterococcus faecalis and Enterococcus faecium are considered as themain causative agents of infection in humans In the 1970, they emerged as a leadingcause of hospital-acquired infections (Gilmore ef a/., 2013) Enterococcus faecalis and

E faecium cause a variety of infections, including endocarditis, urinary tract infections,prostatitis, intra-abdominal infection, cellulitis, and wound infection as well asconcurrent bacteremia Enterococci do not produce toxins like staphylococci andstreptococci, but their virulence comes from other properties like durability, structure,and antibiotic resistance (Miller et a/., 2016) In the past two decades, EF faecium hasrapidly evolved as a worldwide nosocomial pathogen by successfully adapting toconditions in a nosocomial setting and acquiring resistance against glycopeptides (Top

et al., 2008)

2.5.6 Klebsiella pneumoniae

According to Bergey’s classification system, K/ebsiella pneumoniae 1s classified

as a bacterium within the domain of Bacteria It is further categorized within the phylum

Pseudomonadota, class Gammaproteobacteria, under the order Enterobacterales This

bactertum belongs to the family Enterobacteriaceae and is grouped within the genusKlebsiella Its species name is pneumoniae, thus forming its full scientific name asKlebsiella pneumoniae

Klebsiella pneumoniae is an important gram-negative opportunistic pathogen thatcauses a variety of infectious diseases, including urinary tract infections, bacteremia,pneumonia, and liver abscesses In 1882, Carl Friedlander first described Klebsiellapneumoniae as an encapsulated bacillus after isolating the bactertum from the lungs ofthose who had died from pneumonia Klebsiella pneumoniae is a gram-negative,nonmotile, encapsulated, rod-shaped bacillus present within the nasopharynx andgastrointestinal tract which found in the environment and has been associated with

pneumonia in patient populations with alcohol use disorder or diabetes mellitus The

bacterrum typically colonizes human mucosal surfaces of the oropharynx andgastrointestinal (GI) tract Once the bacterrum enters the body, it can display highdegrees of virulence and antibiotic resistance (Ronning ef a/., 2019) Pneumonia caused

by K pneumoniae can be broken down into community-acquired or hospital-acquired

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pneumonia Although community-acquired pneumonia is a fairly common diagnosis,

infection with K pneumoniae is uncommon

2.5.7 Bacillus cereus

Bacillus cereus is a toxin-producing facultatively anaerobic gram-positive bacterium.The bacteria are commonly found in the environment and can contaminate food It canquickly multiply at room temperature with an abundantly present preformed toxin Somestrains of B cereus cause food poisoning, while some strains are reported as beneficial tothe animal's gut microbiome

Per Bergey's classification system, Bacillus cereus is a Bacteria, belonging to phylum

Bacillota, class Bacilli, order Bacillales, family Bacillaceae, and genus Bacillus Thisreflects its cellular structure, life functions, and certain shared traits with similar bacteria.Its full scientific name is Bacillus cereus

There are two types of B cereus food poisoning The first type, caused by an emetic

toxin, results in vomiting, while the second type, caused by enterotoxins, gives diarrhoea

In a small number of cases both types of symptoms are recorded, probably due to production

of both types of toxins There has been some debate about whether or not the enterotoxin

can be performed in foods, and cause an intoxication (Granum and Lund, 1997)

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CHAPTER 3 MATERIALS AND METHODS

3.1 Time and place of study

This study is conducted from 3rd August 2023 to 31‘, December 2023, which tookplace at BT306 - Functional Genomic Laboratory, Department of Biological Science

and Technology, National Pingtung University of Science and Technology (NPUST),

Ping Tung County, Taiwan

3.2 Materials and equipments

All of these strains are available in BT306 - Functional Genomic Laboratory,Department of Biological Science and Technology, NPUST

3.2.2 Culture media

M17 medium (BioLife) was be used to culture both Lacticaseibacillus rhamnosus

and Streptococcus thermophilus strains

Suspend 42.250 grams M17 broth in 1000 mL distilled water Dispense it intotubes or flasks or as desired For M17 agar medium, prepare with same proportion butadd in 15 grams agar for culture Dispense it into sterilize plates Sterilize by autoclaving

at 121°C, 1 atm for 20 minutes

Bacterial pathogenics were grown in nutrient medium broth (HIMEDIA) Suspend

13.000 grams in 1000 mL purified water For nutrient agar medium, prepared the same

proportion but add in 15 grams agar Sterilize by autoclaving at 121°C, 1 atm for 20minutes

Yeast pathogens such as Candia albica were grown in sabouraud dextrose medium(LQ129V, HIMEDIA) Suspend 30.000 grams in 1000 mL distilled water for agar

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medium, prepare with same proportion but add in 15 grams agar Sterilize byautoclaving at 121°C, 1 atm for 20 minutes.

Stored all the prepared medium at 4°C in a tightly closed container Checking thecontaminated situation before using

3.2.3 Required tools, equipments and chemicals

Required tools including: Petri dishes, 96-wells plates, test tubes, Durham tubes,

erlenmeyer flask, transfer micropipettes, innoculation loop, cell spreader, Bunsen

burner, sterilized gloves

Required equipments including: Laminar airflow cabinet, centrifugation,incubator, microplate reader (BioTek), autoclave sterilizer, digital scale, vortex mixer,laboratory sample storage cabinets

Required chemicals including: L(+)-Ascorbic acid puriss (SIGMA-ALDRICH),Butylated hydroxytoluene / 2,6-di-tert-butyl-p-cresol (Fist chemical works), DPPHstock solution (SIGMA), ferrous sulfate FeSO4.7H20 (SHIMAKU’S PURECHEMICAL, pyrogallol (SIGMA), phosphate buffer solution (PBS) ( 10 mM, pH 7.4:

137 mM NaCl, 2.7 mM KCl, 10 mM Na›HPOa, 2 mM KH2POs), agar (GCS), brilliant

blue (KOCH-LIGHT), ethanol (Honeywell), amphotericin B (Cyrusbioscience),

hydrogen peroxide 30% (PanReac), antibiotic (SIGMA), anaerobic pack (MGC)

3.3 Methods

3.3.1 Preparation of L rhamnosus and S thermophilus strains

Culture stocks including Lacticaseibacillus rhamnosus (THH-2, B9, 38) andStreptococcus thermophilus (Jt1, Jt4, Jt5, Jt9, Jt12) were obtained from the culturecollection of BT306 - Functional Genomic Laboratory, Department of BiologicalScience and Technology, NPUST Briefly, all strains were preserved and stored in 20%glycerol at -80°C

The target strains were streaked onto M17 agar and were incubated at 37°C for 48

hours The single colony of each strain were inoculated into M17 medium broth and

grown at 37°C with shaking overnight OD measurement at 600 nm and CFU calculatorformula were used to determine and adjust bacteria concentration

3.3.2 Preparation of pathogenic microorganisms

Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Salmonellaenterica, Enterococcus faecium, Klebsiella pneumoniae ATCC 13883, Bacillus cereus,

lệ)

Candida albicans were used as_ pathogenic microorganisms Pathogenicmicroorganisms as stock culture collection used in this thesis are available at the BT306laboratory Bacteria pathogenic were cultured into the nutrient medium, and yeastpathogenic bacteria were cultured into the sabouraud medium Briefly, each pathogenwas streaked onto respectively medium agar and incubated at 37°C for overnight Thesingle colony of each strain was inoculated into the suitable medium broth and grown

at 37°C with shaking overnight to be ready to use in experiments

3.3.3 Cell-free supernatants (CFSs) preparation

Preparing the cell-free supernatant (CFS) by using the LZ rhamnosus and S

thermophilus strains cultured at 37°C overnight with M17 medium broth under aerobicand anaerobic condition, respectively Then adjusted strains into concentration of 102

CFU/mL to centrifuge at 8000 xg for 5 minutes, and collected supernatant Collectedsupernatant was used in agar-diffusion method, while diluted supernatant in deionizedwater to concentration at 10% (v/v) was be used in antioxidant evaluating

The prepared CFSs were then used on the same day of analysis Immediate use ofthe prepared CFSs is essential to maintain and prevent any changes or deterioration thatcould occur over time

3.3.4 Intact cells (ICs) preparation

After L rhamnosus and S thermophilus strains had been incubated overnight at aprecisely maintained temperature of 37°C, they were harvested using a centrifuge set at

4000 xg for a period of 10 minutes This ensured that the cells were separated effectivelyfrom the surrounding medium The collected cell pellets were then thoroughly washedthree times with phosphate buffer solution (PBS) to clean the cells and remove anyresidual medium or other substances that might interfere with the subsequent steps or

measurements Total cell number were adjusted to 108 CFU/mL at the concentration of

intact cell samples

The prepared intact cells were then used on the same day Immediate use of theprepared cells is essential to maintain their viability and prevent any changes ordeterioration that could occur over time

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3.3.5 Agar-diffusion method

To analyze the performance of the antimicrobial activity, the cell-free supernatant(CFS) and the suspension of the target Ù rhamnosus and S thermophilus strains 1s used

to inhibit the growth of undesirable pathogenic microogarnisms

Using agar-diffusion assay to do the antimicrobial activitity analysis Preparingthe nutrient agar for bacterial indicators and sabouraud agar for yeast pathogenic,inoculate 100 wL indicator culture with the final concentration of about 108 CFU/ml.Well is bored into place and drop 100 uL of the CFSs and suspension (the overnightincubated bacteria) that have prepared before into the well M17 broth was used as thenegative control Using ampicilin, kanamycin and amphotericin B as the respectivelypositive control After 24 hours of incubation at 37°C, observed and mesured the size ofthe diameter inhibition zone If the inhibited activities were observed, then conductedthe MIC, MBC experiments to determine the effect concentration

3.3.6 Antibiotic susceptibility testing method

Using 96-wells dilution for testing antibiotic susceptibility Briefly, used 10

antibiotics including ampicillin, ciprofloxacin, vancomycin, erythromycin, cefotaxime,chloramphenicol, sulfamethoxazole, rifampicin, kanamycin, tetracycline with the

highest concentration at 256 uL/mL, diluted 2 times serially in the 96 wells plate to thelowest concentration at 0.125 wL/mL And then respectively add 100 uL of the targetbacterial solution to 100 uL diluted antibiotic in a 96 well plate Likewise, use M17broth as the negative control, whereas the wells which have the same proportions butwithout antibiotic was used as positive control Measured and recorded the OD value atwave-length at 600 nm at 0 hour and after 24 hours The assays were performed intriplicated The MIC values then compared with the data of Table 3.1 used for definition

of microbiological breakpoint as reported by European Food Safety Authority (EFSA)

(European Food Safety, 2008; Stefanska eí al., 2021)

After observed the MIC values, conducted MBC tested by spread plate method.Briefly, The MBC is measured by subculturing the broths used for MIC determinationfrom the MIC values to the highest concentration onto fresh M17 agar plates, then

incubated at 37°C for 48 hours The triplicated plates which did not observed any

bacteria growth were defined as MBC values, respectively

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Table 3.1 Microbialogical breakpoints categorising bacteria as resistant (ug/mL).

Bacteria strainsAntibiotic

3.3.7 Determination of the DPPH radical scavenging effect

The DPPH radical scavenging activity was determined with slightly modified(DUz et al., 2020) The DPPH method was based on the capture of the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical for antioxidants, producing a decrease in absorbance

at 517 nm The DPPH was prepared in methanol and adjusted into the ODs1 um value at0.7 + 0.02 The solution was homogenized and storage in a dark bottle, which was usedonly in the day of analysis In the dark, 100 uL of sample (cell-free supernatant or intactcell) and 200 uL of diluted solution (PBS or deionized water) were transferred and

mixed with 200 uL of radical DPPH The mixture was shaking incubated at 37°C in a

dark condition for 30 minutes It's important to note that during this process, the mixturemust be shaken to ensure proper mixing of the components

Likewise, control was prepared with these same proportions but replacing sample

as methanol Culture medium or PBS was used as a blind For this experiment, BHT atthe concentration 100 pg/mL was used as the positive control After 30 minutes, ODabsorbance 1s measured at 517 nm

This test was triplicated and then calculated of the DPPH radical scavengingactivity (%) of strains by the formula:

Scavenging Activity (%) = (1 — (As — Ab))/Ac x 100As: Absorbance of sample

Ab: Absorbance of blind sample

Ac: Absorbance of control sample

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3.3.8 Superoxide anion scavenging activity

The superoxide anion (O-) radical scavenging activity assay was conductedspectrophotometrically with pyrogallol autoxidation method (Adesulu-Dahunsi ef a/,2018) The principle behind this method is based on the production of pyrogallol-quinone, which is brown, through a reactive intermediate, the semiquinone radical, andthe ability of SOD to inhibit this reaction by radical dismutation (Liu ef al., 2013)

The mixture contained 30 uL of the samples (cell-free supernatants of intact

cells), 260 pL (50 mM) phosphate buffer at pH 8.2 and 9 hL of 3 mM pyrogallol(dissolved in 10 mM HCl) Likewise, control was prepared with these same proportionsbut without sample The mixtures were left to react at room temperature Then theabsorbance (at 325 nm) was taken at 0 min and 10 min

This test was triplicated and then calculated the superoxide scavenging activity(%) by the formula:

Scavenging Activity (%) = 1 — (A10 — A0)/(C10 — C0) x 100A10: Absorbance of the sample reaction at 10 min

AO: Absorbance of the sample reaction at 0 min

C10: Absorbance of the control reaction at 10 min

CÓ: Absorbance of the control reaction at 0 min

3.3.9 Hydroxyl radical scavenging activity

The hydroxyl radical scavenging (HRS) activity assay was conducted with the

slightly modified method (Adesulu-Dahunsi ef a/., 2018) Briefly, the mixture contained

200 pL samples (CFSs or ICs), 400 pL FeSO¿ (0.5 mM), 200 pL brilliant blue (0.435mM) The reaction was started when added 300 pL H202 (3.0% w/v) This mixture isthen incubated at a temperature of 37°C for a duration of 20 minutes It's important tonote that during this process, the mixture must be shaken to ensure proper mixing of the

components Likewise, prepare the mixture with same proportions but without sample

Control one was also same proportions but without both sample and Fenton reaction.Then the absorbance at 624 nm was measured

This test was triplicated and calculated the activity by the formula:

Scavenging activity (%) = (As — A0)/(A — AO) x 100As: Absorbance of the sample, A0: Absorbance of the mixture without sample

A: Absorbance of the mixture without sample and Fenton reaction

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