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 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 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, (5): 65-70 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 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 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 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 acnecausing 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 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 105106 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 (Bruăggemann, 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 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 antiinflammatory 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., 2013) 2.5 Overview of Aloe vera and its applications in cosmetics Aloe vera grows in temperate and tropical areas Aloe vera gel contains 99% of water and other organic compounds including phenolics such as emodin aloe, aloe, aloein; organic acids such as saponins and terpenoids; polysaccharides such as arabinan, glucuronic acid, galactan, as well as many minerals, amino acids, vitamins With these ingredients, aloe vera gel is used to moisturize, slow down the skin aging process, be cool and anti-bacteria, anti-fungi Moreover, it can treat mild skin infections such as boils, cysts benign, inflammation (Nandal and Bhardway, 2012; Sanghi, 2015) Therefore, aloe vera is widely used in the cosmetic industry Chapter 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 acnecausing bacteria and selection the indicator bacteria 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 of well 2) Inoculating selected LAB into cultured broth containing IB The moving on mL of 2-2,5x105 CFU / mL of LABs was added into 20 mL of TSB medium containing 2-2,5x105 CFU/mL of indicating bacteria Results were defined by colony -counting method at 2, 6, 12, 24, and 30 hours with formula With: N = Number of bacteria in 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 T 2257 ; 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-17 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 tetracycline, trimethoprim/sulfamethoxazole 3.5 Fermenting aloe vera gel for skin care products 3.5.1 Surveying media and conditions biomass LAB culture The purpose of experiment was to find a costeffective alternative media of LAB cultured biomass providing for Aloe vera fermentation 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 Composition Whey tofu (%) Glucose (%) Sacarose (%) Lactose (%) KH2PO4 (%) (NH4)SO4 (%) MT 100 20 0 2 MT 100 20 2 MT 100 0 20 2 The experiment consisted of tests, factor and 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 32 Bảng 3.2: Ratio of LAB culture medium Composition Whey tofu (%) Carbohydrat (%) KH2PO4 (%) (NH4)SO4 (%) level 100 15 1,5 1,5 level 100 20 2 level Variabiity 100 25 2,5 2,5 0,5 0,5 The experiment consisted of three factors, three levels and two replications The surveying criteria were bacterial biomass after culturing 24 hours 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 24 hours Bảng 3.3: Conditions of growth of LAB T (oC) 30 37 45 pH 6,5 3 5,5 3 3 3.5.2 Fermenting aloe vera gel for skin care production a Sceening the ratio of composition Fermentation ingredients included FOB aloe vera gel, vinamilk condensed milk and LAB The threefactor, three-level, two-replication tests were performed 10 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 Composition Water (%) Aloe vera Gel (%) Sweetened condensed milk (%) LAB (% pH 25 50 75 50 30 20 3 6±0,5 6±0,5 6±0,5 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 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 yearolds 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 1 postive cocci bacilli negative negative positive color Colony Size (mm) Gram Cell Catalase Oxydase CaCO3 55 22 15 53 14 82 25 57 82 82 82 Ratio (%) 67,0 26,9 6,1 18,29 64,63 17,08 100 30,48 69,51 100 100 100 By well diffusion agar method, 25 of 82 LAB strains showed the inhibited ability to at least one indicator (Table 4.2) Bảng 4.2: Ration of LAB the inhibited ability IB IB n 25 12 P acnes 46Pa S.aureus 10Sa S epidermidis 09Se LAB Ratio (%) 44,64 21,48 14,28 The 05SL3 strain isolated from human skin without acne showed inhibited three indicators with mm of S aureus 10Sa and S epidermidis 09Se as well as 18 mm of P acnes 46Pa (Figure 4.2) a b c Hình 4.2: The ability IB of LAB 05SL3 (a: S aureus 10Sa; b: S epidermidis 09Se; c P acnes 46Pa) 13 LAB 05SL3 were inoculated into broth of IB, monitoring microbial density at 2, 6, 12, 24 and 30 hours Result showed that LAB 05SL3 was able to reduce 48,6% of S aureus 10Sa, 47,3% of S epidermidis 09Se, and 60,2% of P acnes 46Pa compared to control at 30h (P