Probiotic in Indonesia : Local issue Indigenous Probiotic Research
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1 Probiotic in Indonesia : Local issue Indigenous Probiotic Research Ingrid S. Surono SEAMEO TROPMED RCCN University of Indonesia
2 DADIH, TRADITIONAL FERMENTED MILK OF WEST SUMATRA
3 Dadih, Indonesian traditional fermented raw buffalo milk of West Sumatra No heat application on the buffalo milk
4 5 potential dadih strains Lactobacillus plantarum strains IS Lactobacillus plantarum IS Enterococcus faecium strains IS Enterococcus faecium strains IS Enterococcus faecium strains IS-16183
5 Screening studies Viability Adhesion Pathogen inhibition and displacement Immune modulation Detoxification
6 Viability In vivo Sprague Dawley rats and Balb/c mouse L. plantarum IS and IS-20506
7 3D Contour Plot (LACTIC-indeks-1.STA 5v*1810c) Distance Weighted Least Squares C3 C2 VAR3 C1 C0 A0 A1 A2 A above VAR1 Fig 1. Effect on probiotic suplementation on faecal lactic bacteria of Sprague Dawley rats A0 :control; A1 : L.casei Shirota; A2 : L.plantarum IS-10506; A3 : L. plantarum IS-20506
8 Fig 2. Interaction between kind of probiotic and treatment period on Faecal coliform A0 :control; A1 : L.casei Shirota; A2 : L.plantarum IS-10506; A3 : L. plantarum IS-20506
9 Balb/c mice, control, IS dan LGG Rerata jumlah bakteri asam laktat inhibitor TLR2 inhibitor TLR4 tidak ada inhibitor Rerata 1, Jumlah bakteri Coliform Jumlah bakteri E Coli PROBIOTIK LGG PROBIOTIK LIS TANPA PROBIOTIK Kelompok Perlakuan 1.0E-4 PROBIOTIK LGG PROBIOTIK LIS Kelompok Perlakuan TANPA PROBIOTIK A B Fig 3. Bacteriological profile Lactobacillus rhamnosus GG (LGG) group, Lactobacillus plantarum IS (LIS) group in Balb/c induced alergen ovalbumin. A. mean of total fecal lactic acid bacteria afer treatment. LGG and LIS group significantly higher viable count compared to control, at p=0,010 dan p=0,03., respectievely B. Fecal Coliform and Eschericia coli of Balb/c mouse LGG and LIS group has no significant different as compared to control group
10 Viability in Indonesian elderly Enterococcus faecium IS Table 1. Effect of dietary milk and nonfermented milk containing strain IS on fecal coliforms and lactic acid bacteria in elderly subjects Treatments Number of coliforms (log cfu/g) Number of LAB (log cfu/g) Before milk intake (d 0) 5.79 b 6.69 b After milk intake (d 21) 5.91 b 6.49 b After milk containing strain IS intake (d 42) a,b Means in the same column with different superscript letters differ (P<0.05) 6.79 a 7.36 a
11 Adhesion properties of 5 dadih s strains
12 Table 2. Inhibition of pathogen adhesion by LAB strains isolated from dadih fermented milk. Results are shown as average % adhesion inhibition ± SD. % adhesion inhibition 1 Strains Bacteroides vulgatus DSM 1447 Clostridium histolyticum DSM 627 Escherichia coli K2 Salmonella typhimurium ATCC Staphylococcus aureus DSM IS ±11.5* 27.5±9.5* 34.7±10.5* 22.8±2.0* 38.9±6.8* IS ±11.9* 29.5±6.5* 29.5±1.5* 27.2±5.3* 40.1±8.4* IS ±8.8* 29.7±9.4* 34.0±2.8* 24.5±6.3* 37.9±6.3* IS ±0.7* 27.6±8.7* 35.5±0.9* 22.0±0.0* 29.5±7.8* IS ±1.9* 25.5±5.3* 32.7±2.2* 37.6±14.6* 39.4±8.1* 1 Changes in the adhesion of pathogens in the absence of LAB (control) were assigned a value of 0%. * Significantly different from control 0% (p < 0.05).
13 1 Changes in the adhesion of pre-adhered pathogens following the addition of buffer without LAB strains (control) were assigned a value of 0%. * Significantly different from control (p<0.05) Table 3. Displacement of pathogens by LAB strains isolated from dadih fermented milk. Results are shown as average % pathogen displaced ± SD Strains Bacteroide svulgatus DSM 1447 Clostridium histolyticum DSM 627 % displacement pathogen 1 Escherichia coli K2 Salmonella typhimuriu m ATCC Staphylococc us aureus DSM IS ±9.8* 21.7±1.5* 63.8±1.5* 52.3±9.3* 31.0±6.2* IS ±4.9* 22.5±1.9* 61.7±2.7* 61.5±3.1* 20.3±7.9* IS ±8.5* 18.4±4.2* 62.3±0.9* 57.3±15.3* 16.4±7.3 * IS ±5.4* 20.3±8.9* 59.9±2.7* 57.9±9.0* 18.3±8.8* IS ±9.4* 19.3±2.9* 64.6±8.8* 53.3±3.3* 21.5±5.4*
14 Fig 4. Adherence of pathogen strains (B) to human intestinal mucus. Results were expressed average % adhesion to mucus ±SD.
15 Table 4. Adhesion to hydrocarbons of viable and nonviable probiotic and pathogenic strains as measured using the BATH test. Results are expressed as a mean ± standard deviation (SD) (n 4). Pathogen strains % adhesion to hydrocarbons Live (mean±sd) Non-viable (mean±sd) Bacteroides vulgatus DSM ±13.9* 75.6±13.6 Clostridium histolyticum DSM ±1.8* 61.6±4.6 Clostridium difficile DSM ± ±15.2 Escherichia coli K2 32.7±14.5* 70.1±13.8 Enterobacter sakazakii ATCC ±19.7* 78.4±17.5 Staphylococcus aureus DSM ± ±11.34 Probiotic strains Live (mean±sd) % adhesion to hydrocarbons Non-viable (mean±sd) Enterococcus faecium IS Enterococcus faecium IS Enterococcus faecium IS Lactobacillus plantarum IS Lactobacillus plantarum IS ± ± ±10.0* 76.3± ± ± ± ± ± ±8.4 *) significant differences (p<0.05) between hydrophobicity percentages achieved with live and dead cells.
16 Table 5. Percentages of autoaggregation for pathogen (A) and probiotic (B) strains from dadih strains at different times. Control time= 0h. A) Pathogen strains % autoaggregation (20 C) % autoaggregation (37 C) 2h 16 h 20 h 24h 2h 16 h 20 h 24h Bacteroides vulgatus DSM ± ± ± ± ± ± ± ±5.0 Clostridium histolyticum DSM ± ±8.8 * 31.7±5.8 * 63.1±6.9 * 0.2± ± ±2.5 * 58.6±6.2 * Clostridium difficile DSM ± ± ± ± ± ± ± ±5.0 Escherichia coli K2 2.5± ± ± ± ± ± ± ±3.8 Enterobacter sakazakii ATCC ± ± ± ± ± ± ± ±6.0 Staphylococcus aureus DSM ±6.1 * 28.7±1.0 * 29.4±3.3 * 62.4±5.0 * 11.3±0.5 * 31.3±3.7 * 38.3±5.5 * 58.4±6.0 * Probiotics strains 2h % autoaggregation (20 C) 16 h 20 h 24h 2h % autoaggregation (37 C) 16 h 20 h 24h Enterococcus faecium IS ± ± ± ± ± ± ± ±9.0 Enterococcus faecium IS ± ± ± ± ± ± ± ±8.0 Enterococcus faecium IS ±0.5* 1.4±0.5* 14.2± ± ± ± ± ±5.5 Lactobacillus plantarum IS ± ± ± ± ± ± ± ±6.3 Lactobacillus plantarum IS ±0.5* 3.8±2.0* 7.2±2.4* 26.7± ±0.5 * 22.8± ± ±10.0 *) Significant differences among all strains tested in each time
17 BATH (%) Autoaggregation ( % after 20 h incubation) Probiotic strains BATH (mean±sd) Aggregation (mean±sd) Enterococcus faecium IS ± ±5.0 Enterococcus faecium IS ± ±2.3 Enterococcus faecium IS ± ±2.5 Lactobacillus plantarum IS ± ±3.0 Lactobacillus plantarum IS ± ±2.4 Fig 5. Autoaggregation index of the probiotic bacteria isolated from dadih as a function of adhesion of xylene after incubation at room temperature (20 C). Values are the average from at least three experiments.
18 Bacteroides vulgatus Clostridium histolyticum % Coaggregation % coaggregation IS IS IS IS IS IS IS IS IS IS Staphylococcus aureus Enterobacter sakazakii % Coaggregation % Coaggregation IS IS IS IS IS IS IS IS IS IS Fig 6. Coaggregation abilities of probiotic strains with pathogens after 2h incubation at 20ºC expressed as percentages. Values are the average ±SD from at least three experiments.
19 Clostridium difficile Escherichia coli % Coaggregation % Coaggregation IS IS IS IS IS IS IS IS IS IS Fig 6. Coaggregation abilities of probiotic strains with pathogens after 2h incubation at 20ºC expressed as percentages. Values are the average ±SD from at least three experiments.
20 Immune modulation In vivo studies (Sprague Dawley rats and Balb/c mouse)
21 Effect of Probiotic on faecal siga in Sprague Dawley Rat FAECAL ANAEROBIC BACTERIA Delta OD days 0-7 days 0-10 days A1B0 A2B0 A3B0 Fig 7. Effect of Probiotic on faecal siga in rats A1 : L. casei Shirota; A2 : L. plantarum IS-10506; A3 : L plantarum IS-20506
22 E. faecium IS in Young children (under 5 years) Serum IgA siga saliva Serum IgG Nutritional status
23 IgG serum total konsentrasi IgG (mg/ml) Awal Akhir kontrol probiotik Delta IgG serum probiotic group (0.94 mg/ml) > control group (0.569 mg/ml) kenaikan konsentrasi total IgG serum (mg/ml) normal underweight underweight berat The highest increment in normal nutritional status children
24 IgA serum total based on nutritional status Delta IgA serum total (mg/ml) normal plasebo Placebo undernourished Nutritional Status pro Probiotic tik Severe undernourished IgA serum normal > undernourished> severe undernourished E. faecium IS effective in increasing IgA serum total in undernourished children
25 IgA SALIVA TOTAL Concentration (mg/ml) IgA Saliva Total ** hari ke-0 ** placebo Treatment ** ** probiotik Probiotic hari ke-90 Day 0 Day 90 **Significant at α = 0.05
26 siga SALIVA TOTAL BASED ON NUTRITIONAL STATUS Delta IgA saliva total (mg/ml) Normal normal Undernourished kurang sangat Severe kurang undernourished Nutritional Status Status Gizi Placebo plasebo Probiotic probiotik siga undernourished > severe undernourished > normal E. faecium IS more effective in increasing siga in undernourished and severe undernourished children
27 Body weight (linear regression) Body weight (kg) y2 = 0.105x R 2 = y1 = x R 2 = Minggu ke- placebo probiotik Linear (placebo) Linear (probiotik) Slope (b) = PBB, b2 (probiotic) = 1.5 b1 (placebo) Increase body weight in probiotic group tends to be higher than placebo, 1.5 times
28 persen (%) Probiotik Kontrol Fig 12. Percentage of children with improved nutritional status after probiotic treatment Table 3. Percentage of children with improved nutritional status Nutritional status improvement % subject who improved nutritional status Control Probiotic SevereUnderweight to underweight Severe Underweight to normal Underweight to normal
29 Detoxification Bio elimination of cyanobacterial toxin Microcystin-LR (MC-LR) Metabolically active removal of MC-LR
30 Cyanobacteria, blue-green algae typical for eutrophic water bodies toxicity: hepatotoxins (liver toxins), tumour promoters, inhibit protein phosphatases 1 and 2A, acute LD 50 (mouse, i.p.) ca µg/kg depending on the toxin variant, oral toxicity weaker (lethal doses ca times higher Microcystin-LR (6) D-Glu (iso) (7) N-methyldehydroAla COOH (5) Adda HN OCH 3 H 3 C O NH H CH 3 CH 3 H O N HN C N O CH 3 O N NH O (1) D-Ala CH 2 H 3 C O CH 3 HN CH 3 H N CH 3 O COOH (2) L-Leu NH 2 (4) L-Arg (3) D-erythro-β-methylAsp (iso) WHO guideline value: max. 1 µg/l microcystin-lr in drinking water
31 Fig MC-LR Removal percentage of the best two strains IS and IS-2050 compared to commercial Shirota strain (freeze dried cultures)
32 MC-LR removed (%) b c d Incubation time (h) c a d c d MC-LR removed (%) b d d Incubation time (h) a c e Fig. 5a Microcystin-LR removal percentage of viable (black), heat-treated (grey), and acid treated (white) cells of Lactobacillus plantarum IS-10506, 8.6x x cfu per assay at ph 7, 22 o C. Error bars show the SD of the mean of three experiments. Bars with different letter are significantly different at 95 % confidence level Fig. 5b Microcystin-LR removal percentage of viable (black), heat-treated (grey), and acid treated (white) cells of Lactobacillus plantarum IS-20506, 7.6x x cfu per assay at ph 7, 22 o C. Error bars show the SD of the mean of three experiments. Bars with different letter are significantly different at 95 % confidence level
33 MC-LR removed (%) MC-LR rem oved (% ) incubation time (h) incubation time (h) Fig. 6a Microcystin-LR removal percentages by viable cells of Lactobacillus plantarum IS at ph 7, White bars (22 o C) and Grey bars (37 o C). Error bars indicate confidence intervals at 95% level. Fig.6b Microcystin-LR removal percentages of viable cells of Lactobacillus plantarum IS at ph 7,White bars (22 o C) and Grey bars (37 o C). Error bars indicate confidence intervals at 95% level.
34 IS IS MC-LR removed (%) MC-LR removed (%) 9 60 log viable cells/ml log viable cells/ml Incubation time (h) Incubation time (h) 7 Figure 7a. MC-LR removed (%) and log viable cells ml -1 of IS strain at different glucose concentration during incubation at 37 C. Empty legends stand for MC-LR removed, and filled legend for log viable cells. Symbols (, ) 0% of glucose, (, ) to 1% of glucose, (, ) 2% of glucose and (, ) 3% of glucose. Figure 7b. MC-LR removed (%) and log viable cells ml -1 of IS strain at different glucose concentration during incubation at 37 C. Empty legends stand for MC-LR removed, and filled legend for log viable cells. Symbols (, ) 0% of glucose, (, ) to 1% of glucose, (, ) 2% of glucose and (, ) 3% of glucose.
35 MC-LR removed (%) E E E E E E+13 bacterial concentration (CFU ml -1 ) L. rhamnosus GG L. rhamnosus LC-705 B. longum 46 B. lactis 420 B. lactis Bb12 L. plantarum Lp-115 L. plantarum IS L. plantarum IS Figure 8. Effect of bacterial concentration on removal of MC-LR. Initial MC-LR concentration 100 μg L -1, incubation time 24 h, temperature 37 C, average ± SD, n=3.
36 On going research L. plantarum IS : HIV/AIDS patient Profile fecal microbiota Humoral immune system Cellular immune system
37 Published papers 1. Collado, M. C., Surono, I.S., Meriluoto, J., Salminen, S., Potential Probiotic Characteristics of Lactobacillus and Enterococcus Strains Isolated from Traditional Dadih Fermented Milk against Pathogen Intestinal Colonization. Journal of Food Protection Vol.70, Collado, M. C., Surono, I., Meriluoto, J., Salminen, S., Indigenous Dadih Lactic Acid Bacteria: Cell- Surface Properties and Interaction with Pathogen. Journal of Food Science, vol. 72, Surono, I.S., Collado, M.C., Salminen, S. and Meriluoto, J. Effect of glucose and incubation temperature on metabolically active Lactobacillus plantarum from dadih in removing microcystin-lr. Food and Chemical Toxicology (in communication) 4. Usman Pato, Ingrid S. Surono, Koesnandar, Akiyoshi Hosono, Hypo-cholesterolemic Effect of Indigenous Dadih Lactic Acid Bacteria by Deconjugation of Bile Salts. Asian-Aust. J. Anim. Sci., Vol. 17 No. 12 : Jorry Dharmawan, Ingrid S. Surono, Lee Yuan Kun, Adhesion Properties of Indigenous Dadih Lactic Acid Bacteria on Human Intestinal Mucosal Surface Asian-Aust. J. Anim. Sci., Vol. 19 No. 5 : Surono, I.S., In vitro Probiotic properties of indigenous dadih lactic bacteria. Asian-Aust. J. of Anim. Sci. 16, 5,
38 Financial Support Viability, and immuno modulatory properties : Ministry of Research and Technology ( ) Adhesion and detoxification properties : Academy of Finland decisions RC and RC
39 Prebiotic Research Roots and tubers as source of prebiotic : Sweet potato, bread fruit, taro, araroot
40 Government Regulation BADAN POM (Indonesian FDA) Probiotic as functional foods Claim of Nutrition fact : Enriched, fortified : contain probiotic bacteria more than minimum dose 10 6 cfu/day viable count until expired date Containing : probiotic more than minimum dose 10 7 cfu/day Rich in probiotic : more than minimum dose 10 8 cfu/day List of Probiotic bacteria?
41 Nutritient function claim : Probiotic (Lactobacillus and Bifidobacterium, expect L. bulgaricus) can help in maintaining intestinal function) Confirmation of the presence of probiotic bacteria? Bifidobacteria : can be produced in home industry?
42 Health benefit claim No health claim in Label and advertisement Label and advertisement for probiotic product should mention Genus, species, and strain, minimun viable counts at the end of expired date, information about amount of probiotic and its fisiological effect. Storage condition, contact number of producer Human studies on Indonesian people, minimum 20 subjects : Adequate amount?
43 Prebiotic as functional foods Enriched, fortified (at least 10% from daily value 10g/day) Containing : % from daily value (10g/day) per serving Rich in prebiotic, very good source : at least 20 % of DV per serving No nutrient function claims in label and advertisement No helath claim in label and advertisement
44 Consumer Perspective Lack of knowledge about probiotic and prebiotic : Street vendor sells probiotic product without refrigerator Street vendor sells yogurt and claim as yogurt probiotic Concern about the viable counts in probiotic product (real cfu/ml or g) Relatively expensive
45 Street vendor sells Home made yogurt and claim as yogurt probiotic Yogurt probiotic (Yogurt for health)
46 Producers Perspective Rigid government regulations for health claim : using defined commercial strain with scientific based evident, can not do health claim Need to educate customer to gain awareness on health benefit of probiotic
47 High Appreciation to Wonderful Team work
48
49 Thank You Very Much for your kind attention
Indigenous Probiotics and Immunological Effects Ingrid S Surono Food Technology Department, Faculty of Engineering-BINUS University,
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