Phân lập và tuyển chọn vi khuẩn lactic có khả năng kháng vi sinh vật gây bội nhiễm mụn trứng cá do sử dụng sản phẩm chứa corticoid tt tiếng anh

MINISTRY EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY MAJOR: MICROBIOLOGY CODE: 9420107 DUONG THI BICH ISOLATION AND SELECTION OF LACTIC ACID BACTERIA INHIBITING THE PROLIFERATIO

MINISTRY EDUCATION AND TRAINING

CAN THO UNIVERSITY

SUMMARY MAJOR: MICROBIOLOGY

CODE: 9420107

DUONG THI BICH

ISOLATION AND SELECTION OF LACTIC ACID BACTERIA INHIBITING THE PROLIFERATION OF ACNE-CAUSING BACTERIA USING TOPICAL

CORTICOSTEROID

2018

THESIS FULFILLED

AT CANTHO UNIVERSITY

First Supervisor: Assoc, Prof Nguyen Van Ba

Secondary Supervisor: Assoc, Prof Huynh Van Ba

Thesis was defended at basis valuattion comittee Location: Thesis Defend Room, 2nd Floor–

CanTho University Rectorate

at 14 h 00 12/7/2018

Oponent person No 1: Dr Mai Nguyet Thu Hong Oponent person No 2: Assoc Prof Duong Xuan Chu

For referencing:

Learning Resource Center, Can Tho University

Viet Nam National Library

Literature

1 Duong Thi Bich, Nguyen Van Ba, Huynh Van Ba, 2014

Presence of Propionibacterium acnes, Staphylococcus

aureus, Staphylococcus epidermidis in patients with acne

using topical corticosteroids Journal of 108-Clinical Medicine and Pharmacy, 1 9 (5): 65-70

2 Duong Thi Bich, Nguyen Van Ba, Huynh Van Ba, 2015

Isolation and selection off lactic acid bacteria inhibited

capacity of Propionibacterium acnes, Staphylococcus

aureus, Staphylococcus epidermidis in acne patients using

topical VietNam Medical Journal, 427 (1): 41-45

3 Duong Thi Bich, Nguyen Van Ba, Huynh Van Ba, 2016

Inhibited ability of lactic acid bacteria isolated from skin on acne-causing bacteria VietNam Medical Journal, 448 (1): 23-27

4 Nguyen Kim Đong, Duong Thi Bich, Nguyen Chi Toan,

2017 The Estimation method of S-acid lactic the fermentation products Aloe vera gel by High-Performance Liquid Chromatography Jounal of Natrura Science, 126 (1B/2017)

Chapter 1 INTRODUCTION

The necessary of dissertation

.The acne is diseases of sebum folliculitis popular

in skin, especially in sebum-increasing skin like facial, back and chest areas Many researches indicated that acnes relate to four main factors including: the increase sebum production, abnormal follicular keratinization,

abundant of Propionibacterium acnes (P acnes), and

production of inflammation Besides, unsuitable cosmetic – consuming problems, especially topical corticoid, impacted seriously on skin such as: more serious acnes, serious skin problems, decrease of collagen, and increase of superinfection bacteria Nowadays, the treatments of acnes are being applied by many methods including antibiotics, hormones, and retinoids However, those methods have still had some problems such as resistance of superinfection microorganisms or side effects on consumers For this reason, finding out the new methods for inhibiting superinfection microorganisms on acnes have been necessary in order to manage and treat the acnes

Purpose of dissertation

Selection of lactic acid bacteria inhibiting the proliferation of acne-causing bacteria using topical corticoid The introduces a bio-competitive approach to acne skin care

Research contents

(1) Evaluating the presences of (P acnes,

Staphyloccocus aureus (S aureus), Staphylococcus epidermidis (S epidermidis), Demodex) in patients with

acne using topical corticoid (2) Surveying the

antibiotic resistances of acne-causing bacteria and selection the indicator bacteria (IB) for further study (3) Isolation and selection of lactic acid bacteria inhibiting IB (4) Surveying the bioactivities of selected lactic acid bacteria and screening for applying them in

skin care (5) Using Aloe vera gel as carier substrate to

ferment and apply on skin care

New contributions of dissertation

- Determination of popular acne-causing bacteria using topical corticoid and evaluation of antibiotic resistance of them

- Looking for the lactic acid bacteria for the capacity of inhibiting the growth of three kinds of acne-causing bacteria These lactic acid bacteria can be produce the antioxidant and moisturizing compounds

- First step to establish the method to ferment

Aloe vera gel with L plantarum 05SL3 for-skin care

Chapter 2 LITERATURE REVIEW

2.1 Acnes overview

Acne is skin inflammation, popular in juvenility ages with clinical manifestations including whiteheads, blackheads, pustules (Medical Ministry , 2015) Acnes usually relate to endogenous and exogenous factors such as the increase sebum productiona, abnormal follicular keratinization, abundant of

Propionibacterium acnes (P acnes), and productin of

inflammation (Tahir, 2010)

2.2 Superinfection bacteria on acnes

Many researchers relating superinfection bacteria on

acnes indicated that P acnes, S aureus, S epidermidis,

and Demodex on acnes accounted for 32%, 45%, 49%,

and 75,5%, respectively (Dolenc-Voljc et al., 2005;

Dhillon and Varshney, 2013)

P acnes is one of skin microbiome, about 105

-106 cfu/cm2 on sebaceous glands areas such as face, head, and back, besides about 102 cfu/cm2 on others P

acnes can produce enzymes damaging host cells

including sialidase, neuraminidase, lipase, hyaluronan as well as attract macrophages and dendritic cells stimulating inflammatory responses (Bru¨ggemann, 2010)

S aureus is skin and mucosa microbiome

developing well on hand, chest, abdomen, and nose

Healthy humans containing S aureus are about 10-35% usually and 20-75% unusually, respectively S aureus

can cause diseases by producing staphylokinase and A

protein inhibiting C3b, C1q and IgG so on S aureus

can cause diseases with clinical manifestations such as pustules, abscesses, skin scabs

S epidermidis is skin microbiome inhibiting

inflammatory responses via activities of lipoteichoic

acid (LTA) S epidermidis is an opportunistic bacteria

on acnes with 6,8-49% (Dhillon and Varshney, 2013)

S epidermidis can cause diseases via producing

exopolysaccharides, and then forming biological membranes inhibiting attacks of host’s macrophages and antibacterial factors, decreasing growth and mutation of antibiotics – resistant genes, producing poly-γ-glutamic acid, PNAG/PIA and PSMs protecting bacteria cells from host’s innate immunity

Demodex is the mite – sizing parasites among

arthropods in hair follicles located near the noses,

eyebrows, heads, and eyelids Demodex causes diseases

via making hair follicle lesions, blockage of pores

causing inflammations, and create opportunities for

other invasive pathogens like P acnes và S aureus

(Tchernev, 2011) Some clinical manifestations include

hair follicles, itching, redness, rough skin, pustules 2.3 Topical corticoid impacts on acne

Corticoid’s full name is corticosteroid, a compound owning bioactivities similar as hormone steroids secreted from the adrenal glands with anti-inflammatory and anti-allergy activities However, the over-uses of corticosteroid also have many side effects such as atrophy, thinning of skin, redness or stretch marks, vasodilatation, collagen depletion, loss of pigments, severe acnes, skin lesions and opportunities

for pathogens such as S aureus, P acnes and Demodex (Klein et al., 2001)

2.4 Overview of lactic acid bacteria and their applications in skin care

The term of lactic acid bacteria (LAB) refers to the group of bacteria that are capable of fermenting carbohydrates to form lactic acid LABs have some characteristics such as Gram - positive; non-spore; anaerobics or micro - aerobics; rod or sphere forms; negative catalase test, oxidase and gelatinase

LABs often grow with nutrient-rich media such as development on a variety of foods (milk, meat, vegetables), and some are members of the oral, intestinal and vaginal microbiota In the process of metabolism, LABs can inhibit other microorganisms by nutrient competition or producing products such as lactic acid; acetic acid; hydrogen peroxide; carbon dioxide; bacteriocin; diacetyl; and so on

LABs are beneficial bacteria and have been widely applied in a huge of fields including livestock, fisheries, and food processing Recently, LABs have

been widely applied in medicine such as preventing diarrhea, improving lactose tolerance, stimulating immunity system, preventing allergies, reducing cases

of colon cancer, and improving diseases related to cardiovascular

In addition, LABs can be used to caring and protecting skin through immune stimulating features, inhibition of superinfective bacteria on the skin, decrease of melanin formation, skin anti-aging,

elasticity and hydration (Chen et al., 2006; Tsai et al.,

Chapter 3 METHODOLOGY

3.1 Evaluating the presences of (P acnes, S

aureus, S epidermidis, Demodex) in patients with

acne using topica corticoid

The purpose is to identify common superinfection factors in acnes using topical corticoids for subsequent researches

Survey period from March - 2013 to October – 2014: Interviews collected informations from acne patients and sampling specimens for microbiological examination

Screening of corticoids in cosmetics by thin-layer chromatography with standard dexamethasone acetate and chemical methods

3.2 Survey of antibiotic resistances of causing bacteria and selection the indicator bacteria

acne-Using Kirby-Bauer agar plating method with, climdamycin, erythromycin, doxycycline, oxacillin, and tetracycline, trimethoprim/sulfamethoxazole Selected strains with high prevalent and high resistance to antibiotics were tested, extracted DNA, and identified

by sequencing for bacterial indicator

3.3 Isolation and selection of LAB strains inhibiting bacterial indicator

a) Isolating LAB

rice washing water, powdered making waste , tofu whey, shaving skin… samples were collected to ferment during 1-2 days Using MRS agar medium with CaCO3 1% for isolating microorganisms The bacteria were selected (contain lactic acid dissolving CaCO3) for morphological and biochemical tests ( Gram stain, catalase, oxidase, gelatinase testings) LABs were cultured in liquid MRS with glycerol 20% at -40°C for coservation

b) Investigating the inhibited ability of IB

1) Well diffusion agar method

Using well diffusion agar method was the replacement of centrifuged bacteria borth by bacterial suspension The inhibition of indicators was determined

by measuring aseptic distances excluding the diameter

6, 12, 24, and 30 hours with formula

With:

N = Number of bacteria in 1 mL of medium

C = total number of colonies counted on plates

ni = Number of plate count in dilution i

di = dilution factor i

3 4 Survey some biological properties of selected LABs applied in skin care

a Testing for bacteriocin production

Using spot method wthi Bacillus coagulans JCM

2257T; Micrococcus leteus NBRC 12708; S aureus ATCC 12600T; Pediococus pentosacus JCM 5885 were

IB

b Antioxidant activity

The 24 hours - cultured broth of LAB in MRS medium was centrifuged to remove cells (Dn-Lp), then diluted to 30%, 50%, 70% and 100% Assay of antioxidant activity with DPPH (2.2- Diphenyl-1-

picrylhydrazyl) was tested, vitamin C as a control Reaction mixtures were measured by Genesys 10S UV-Vis spectrometer at 517 nm Antioxidant activity (%) (average of 3 measurements) was calculated by the formula:

100)(

ODc

ODt ODc

HTCO

Trong đó:

ODc: Optical density of DPPH and ethanol

ODt: Optical density of DPPH and LAB

c Moisturizing ability

LAB - cultured broth was filtered to remove cells,

and then diluted into 10%, 50% and 100% 1 mL was added on 2 mL tubes and incubated at 50 oC, humidity 60% during 96 hours Each test concentration was repeated 3 times, control was glyceril 5%, 10% and water The results were calculated by the following formula:

d The activity of lactic acid production

LAB strains cultured in liquid MRS were centrifuged removing cells Lactic acid production was determined by high performance chromatography (HPLC) (using US HPLC-1210 and RSpak SH-1011 column) at 75oC, HClO4 3mM as the mobile phase, flow rate 0.6 mL/min and 2 mL of sample volume

Lactic acid was determined by the BF-5 biosensor (Japan)

e Screening on antibiotic resistance for acne treatment

Using Kirby-Bauer agar plating method with, climdamycin, erythromycin, doxycycline, oxacillin, and

cost-a Survey growing time: Bacteria were cultured in liquid MRS, counted by colony counting method after

2, 6, 12, 24, 30 hours

b Survey on the use of carbon sources: LABs

were cultured on TSA with 20% of glucose, lactose, saccharose, and starch The growth of bacteria at 37 °C during 24 hours were observed

c Screening on medium compositions and conditions of biomass

1) Ability using sugar

The experiment was designed according Table 3-1

Bảng 3.1: Composition medium of LAB

The experiment consisted of 3 tests, 1 factor and 3 replicate Control was MRS cultured broth The surveying criteria were bacterial biomass after culturing

24 hours

2) Screening on the ratio of medium components

The experiment was designed according Table

3) Screening on cultured conditions

The experimental design consisted of two factors including temperature and pH, three levels of replication, three replications (Table 3.3) The surveying criteria were bacterial biomass after culturing

a Sceening the ratio of composition

Fermentation ingredients included FOB aloe vera gel, vinamilk condensed milk and LAB The three-factor, three-level, two-replication tests were performed

at room temperature (25-30 °C) (Table 3.4) Target monitoring: pH, LAB, bacterial and sensory (smell, color, product status)

Bảng 3.4: Ratio of composition fermenting

Aloe vera Gel (%) 50 30 20

Sweetened condensed milk (%) 1 2 3

b Screening storage time and temperature

Storage conditions including temperature and time were screened The temperatures were investigated at three levels of 15 °C, 25 °C and 30 °C, three repeated times Monitoring indicators: pH, LAB, bacterial infection, and sensory

Chapter 4 RESULTS AND DISCUSSION

4.1 Evaluating the presences of P acnes, S.aureus, S

epidermidis, Demodex in patients with acne using

topical corticoid

During surveying period from March 2013 to October 2014 at Can Tho Dermatology Hospital with the total of volunteer patients was 148 The results showed that disease was popular among 19-25 year-olds The patiens used topical corticoid were more popular than others (Corticoid – causing acnes were 53.4%, other products were 39.9%)

The acnes infecting P acnes, S epidermidis, S

aureus, and Demodex were 56.8%, 89.9%, 60.8%, and

8.9%, respectively The most common bacterial species

were S epidermidis, P acnes and S aureus accounting

for 45.6% (P <0.001) (Figure 4.1) As a result, abuse of corticoids were given rise to many microorganisms, making acne worse