A functional probiotic Lactobacillus sakei probio-65 from kimchi for atopic eczemadermatitis
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1 A functional probiotic Lactobacillus sakei probio-65 from kimchi for atopic eczemadermatitis syndrome Yong-Ha, Park PL/Director/Professor National R&BD Program of Personalized Medicine Institute of Personalized Medicine School of Biotechnology Yeungnam University, Republic of Korea
2 Atopic dermatitis Atopic dermatitis is an inflammatory, non-contagious skin disorder. The skin of a patient with atopic dermatitis reacts abnormally and easily to irritants, food, and environmental allergens and becomes red, flaky and very itchy. It also becomes vulnerable to surface infections caused by bacteria.
3 Atopic dermatitis Clinical appearance of atopic dermatitis lesions in patients. A. Mild atopic dermatitis appears as a localized area of skin inflammation with erythema and dryness. In a child + parent, B. Acute skin inflammation exacerbated by infection in a child with atopic dermatitis, C. Chronic skin inflammation with lichenification in an adult with adult dermatitis (S. Sehra et.al., An Bras Dermatol. 2008). A functional probiotic f Lactobacillus sakei probio-65 rom kimchi for atopic eczema-dermatitis syndrome
4 Factors related to atopic dermatitis Mite Pollen Food Air contamination Environmental factors a AD patient of parent - Appearance 60% for child Two AD patients of parent - Appearance 80% for child Genetic factors Atopic dermatitis Ig E IL-4 IL-2 TNF-α Immunological factors Microbial factors Staphylococcus aureus Fungi Yeast
5 Immunological mechanisms involved in atopic dermatitis. Initiation of the atopic response in AD involves the uptake and processing of allergens by LCs after penetration through the compromised epithelial barrier. The activated LCs then migrate to skin draining LN and with IL-4 promote Th2 differentiation. Keratinocytes produce chemokines including CCL17, CCL27, CCL11, CCL26. Chemokines CCL17 and CCL27 recruit Th2 cells to the skin whereas CCL11 and CCL26 recruit eosinophils. TSLP activates DCs to prime naive T helper cells to Th2 cells and promote Th2 memory. The potentiation of the inflammatory response involves the complication of AD with bacterial infections. Staphylococcal exotoxins activate LCs and IDECs to produce inflammatory mediators including IL-1β and TNF-α. The mediators lead to increased expression of chemokines which recruit T cells. Th2 cytokines in AD decrease AMPs making lesions susceptible to infection. PAF receptor can also bind to LTA in addition to PAF. LTA activated IDECs migrate to draining lymph nodes of skin to promote Th1 responses. FLG; EDC gene filaggrin, TSLP; thymic stromal lymphopoietin, IDEC; inflammatory dendritic epidermal cell, PAF; platelet-activating factor (S. Sehra et.al., An Bras Dermatol. 2008).
6 Probiotics in allergic diseases Intestinal microbiota are the greatest microbial exposure through out life and it is an important stimulus for both intestinal and immune development. Recent studies in humans support the importance of the intestinal microbiota in immune development, and have found a relationship with the development of allergic diseases. Intestinal microbiota of infants are more amenable to manipulation using probiotic supplementation than intestinal microbiota of adults
7 Source of isolation-kimchi An indigenous Korean food Kimchi is an unique fermented side dish, indispensable for any Korean meal, and therefore, it has always been the most representative Korean food. There are more than one hundred varieties of kimchi. Kimchi is rich in different types of Lactobacillus Recently lots of new species of bacteria and many functional probiotics were isolated from Kimchi
8 Population Dynamics of Lactic Acid Bacteria during Fermentation of Kimchi Lactobacillus sakei ( ) Leuconostoc species ( ) Weissella koreensis ( ). Total viable cell number ( ) Total acidity ( ). 김장김치내의미생물군집변화 (J Microbiology ) 8
9 Monitoring Population Dynamics of Lactic Acid Bacteria during Fermentation of Kimchi using Genome-Probing Microarrays K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 Appl. Environ. Microbiol (12):
10 Population Dynamics of Lactic Acid Bacteria during Fermentation of Kimchi 10
11 New Species of Lactic Acid Bacteria isolated from Kimchi 11
12 New Functional Species of Lactic Acid Bacteria isolated from Kimchi Name Strain No. Year Functions Lactobacillus hilgardii GABA Production Lactobacillus sakei Probio Anti tumor Lactobacillus sakei Probio Anti corona viral activity Leuconostoc mesenteroides Probio Anti corona viral activity Lactobacillus sakei Probio Anti allergy Lactobacillus acidophilus KFRI Anti Cancer activity 12
13 Experiment Strategy Construct a lactic acid bacterial culture collection from Kimchi Screening Tolerance in gastric and bile juice Activity of macrophage and production of Nitric Oxide (NO) Activity of mast cells and production of histamine Selection of Characterization 16S rdna Sequencing Antibacterial activity Antibiotic sensitivity In vivo Test using mouse model Visual evaluation IgE and IL-4 analysis Clinical trial in children SCORing of Atopic Dermatitis (SCORAD) score CCL 17 and CCL 27 analysis
14 In vitro test using mouse macrophage and mast cell Probio 65 Whole cell lysate Cytoplasmic fraction Cell wall Mouse macrophage RAW264.7 Mouse mast cell RBL-2H3 Assay Production of Nitric oxide Assay Production of histamine
15 Characterization of Lactobacillus sakei Probio 65 SEM of Lactobacillus sakei probio 65
16 Antibacterial activity of Lactobacillus sakei Probio 65 Pathogenic microorganisms Clear zone (mm) Escherichia coli KCTC Salmonella typhimurium SG 31 Salmonella gallinarium ST 28 Staphylococcus aureus KCTC Enterobacter cloacae KCTC Staphylococcus epidermidis KCTC Klebsiella pneumonia KCTC Shigella flexneri KCTC Citrobacter freundii KCTC Bacillus megaterium KCTC Against Staphylococcus aureus HSAc Paper disc: 400 ul of supernatant of Lact. sakei probio 65
17 Ex vivo Screening System Foxp3-GFP Knock-in mice 6~8 weeks Mesenteric Lymphocyte Co-culture With probiotics for 72hrs Foxp3 IL 10 production IL 12 production Foxp3+ population IL 10/IL 12 ratio IL-10 : Anti-inflammatory cytokine IL-12 : Pro-inflammatory cytokine Foxp : TF of Regulatory T cell IL-10 / IL-12 B A C A : 1 st candidate B,C : 2 nd candidate
18 Analysis of biomarkers from mesenteric lymphocytes after co-culture with L. sakei probio65 Foxp3 + cells Interleukin-10 Interleukin-12 The ratio of IL-10 to IL-12 INF-γ IL-17 PBS(phosphate buffered saline), Test-live(L. sakei probio65 live bacteria), Test-dead(L. sakei probio65 dead bacteria), Test-cont(supplement)
19 In vivo test using mouse model Chemical allergen DNCB Feed Determination Visual evaluation IgE, IL-4 Feed with live Lact. sakei probio 65 DNCB: 1-chloro 2, 4-dinitrobenzene
20 IgE and IL-4 levels in murine serum by L. sakei Probio 65 A B Change in (A) IgE level in serum from DNCB-induced mice. (B) IL-4 levels in splenocytes from DNCB-induced mice. NC, negative control; PC, positive control; T, test group supplemented with L. sakei probio 65. *P.05 compared with NC and PC by Duncan s multiple range test
21 Recovery of damaged skin by L. sakei Probio 65 Starting of the experiment End of the experiment Group Allergen Day NC PC DNCB T DNCB NC : negative control PC : positive control T : test mouse group supplemented with L. sakei probio 65 in feed.
22 Human clinical trials Based on double blind, Placebo study Children: aged 2 to 10 years with light atopic symptoms and allergy Dosage: 5 x 10 9 C. F. U./2 g, 2 time/d Experimental period: 12 weeks At enrollment, the levels of total serum IgE, eosinophil cationic protein, and specific IgE to common allergens in the area were determined. Clinical evaluation Probiotics group Freeze-dried L.sakei (microcrystalline cellulose as carrier) 2g X 2/day Placebo group Microcrystalline cellulose- 2g X 2/day Standardized scoring system, SCORing of Atopic Dermatitis (SCORAD) was applied (Mild (SCORAD score <25), Moderate (SCORAD score, 25-50), Severe (SCORAD score >50) Chemokines in the serum CCL 17 and CCL 27 were measured Statistical analysis were performed using SPSS software.
23 Patient populations analyzed for efficacy of the probiotic Assessed for eligibility (n=107) Excluded (n=19) -failed to meet inclusion criteria (16) - refusal (3) Randomized and received supplement (n=88) Probiotic (n=45) Placebo (n=43) Follow up at week 12 (n=41) - refusal to ingest powder (1) - non-attendance (1) - poor compliance (2) Follow up at week 12 (n=34) - refusal to ingest powder (2) - non-attendance (2) - poor compliance (4) - other probiotic agent ingestion (1)
24 Changes in the SCORAD score after L. sakei Probio 65 treatment Probiotics group a (n=41) Placebo group a (n=34) P Vaue b Week 6 Total score 35.1 ( ) 37.9 ( ) 0.19 Extent and Intensity 26.8 ( ) 30.5 ( ) 0.07 Itch and Sleep loss 8.4 ( ) 7.3 ( ) 0.12 Week 12 Total score 28.8 ( ) 35.8 ( ) 0.01 Extent and Intensity 22.7 ( ) 27.8 ( ) 0.02 Itch and sleep loss 6.1( ) 7.9 ( ) 0.07 a Values are presented as mean (95% confidence interval) b By analysis of covariance
25 Improvement of the SCORAD score after the experiment
26 Correlation of the serum CCL 17 (A) and CCL 27(B) with SCORAD score among the 75 patients
27 Serum pre-treatment and post-treatment values of CCL 17& CCL 27 in the probiotic and placebo group Probiotic group Placebo group P value Log TARC/CCL 17 (pg/ml) Baseline 2.60 ( ) 2.50 ( ) 0.24 Week ( ) 2.48 ( ) 0.03 Log CTACK/CCL27 (pg/ml) Baseline 3.68 ( ) 3.63 ( ) 0.46 Week ( ) 3.62 ( ) 0.03 TARC- Thymus activation regulated chemokine CTACK- Cutaneous T-cell attracting chemokines Values are presented as mean (95% confidence interval)
28 Conclusions Lactobacillus sakei probio-65 isolated from Kimchi exhibited probiotic properties like High tolerance to gastric and bile juice. Good antibacterial activity against variety of pathogenic bacteria, especially Staphylococcus aureus. During in vitro analysis probio-65 exhibited immuno-stimulating properties like increased production of Nitric oxide and decreased Histamine production. In vivo analysis in mouse observed a fast recovery of the damaged skin induced allergy by DNCB along with decreased IgE and IL-4 levels. Supplementation of L. sakei in children with atopic dermatitis was associated with a substantial clinical improvement and a significant decrease in chemokine levels. In summary Lactobacillus sakei probio-65 is an ideal probiotic candidate for the treatment of atopic dermatitis. A functional probiotic from kimchi for atopic eczema-dermatitis syndrome
29 Patent/Publicationss Patents Korea : No. KR China : No. CN B USA : No. US11/ Europe : No. EP PCT : No. PCTKR Publications Chun Wook Park, Myoungsub Youn, Young-Mi Jung, Hongik Kim, Yoonhwa Jeong, Han-Ki Lee, Hye One Kim, Insun Lee, Soo Won Lee, Kook Hee Kang, and Yong-Ha Park New functional probiotic Lactobacillus sakei Probio 65 alleviates atopic symptoms in the mouse. J Med Food 11 (3), Sung-Il Woo, Ji-Yoon Kim, Yong-Ju Lee, Nam-Shik Kim, Youn-Soo Hahn Effect of Lactobacillus sakei supplementation in children with atopic eczema dermatitis syndrome. Ann Allergy Asthma Immunol. 104:
30 Commercialization Lactobacillus sakei Probio 65 is now commercially available as different products through, Dietary supplement Skin care Hand care ProBionic Techno valley Yongsandong 514, Yusung, Daejeon, Republic of Korea ( Tel:
31 Commercialization Amway Kimchi Ilpum Kimchi Kimchi Starter Jonggafood Seasoning Pulmuone Kimchi
32 Improvements in atopic dermatitis syndrome by Boyan-I After 10 days (used every two or three hours) After 7 days (used every two or three hours) After 10 days (used every two or three hours) After 10 days (used every two or three hours) After 1 day (used every two or three hour) After 2 months for hand herpes
33 Acknowledgements Yeungnam University Byeongchun Kim, Ph.D Chungbuk University Yoonsoo Han, M.D. Sungil Woo, M.D. Hanllym University Chanwook Park, M.D. Jinkyoung Kim, M.D. ProBionic Corp. Hongik Kim, Ph.D Insun Lee, Ph.D Han-Ki Lee, Ph.D Dankook University Yoonhwa Jeong, Ph.D Myoungsub Youn, M.Sc Korea Research Institute of Bioscience and Biotechnology Younghyo Jang, Ph.D., Minyoung Jeong, Ph.D., Gwangju Institute of Science and Technology Sin-Hyeog Im, Ph.D.,
34 A functional probiotic from kimchi for atopic eczema-dermatitis syndrome
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