IV. HASIL DAN PEMBAHASAN
5.2. SARAN
Berdasarkan hasil penelitian perlu dilakukan identifikasi komponen antibakteri yang berperan dalam penghambatan bakteri EPEC K1.1. Selain itu, perlu dilakukan uji jumlah bakteri probiotik yang ada di dalam yogurt agar diketahui jumlahnya. Kajian aktivitas antidiare juga dapat diperdalam dengan pengujian mekanisme penempelan bakteri probiotik dan EPEC pada saluran pencernaan menggunakan model sel Caco-2.
DAFTAR PUSTAKA
Adisasmito W. 2007. Faktor Risiko Diare Pada Bayi Dan Balita Di Indonesia: Systematic Review Penelitian Akademik Bidang Kesehatan Masyarakat. Makara Kesehatan, Vol. 11, No. 1: 1-10. Afset JE, Bevanger L, Romundstad P, dan Bergh K. 2004. Association of atypical enteropathogenic
Escherichia coli (EPEC) with prolonged diarrhea. J Med Microbiol. 53, 1137-1144.
Alderbeth I, Cerquetti M, Poilane I, Wold AE, dan Collignon A. 2000. Mechanisms of colonisation and colonisation resistance of the digestive tract. Microbial Ecology of Health and Disease, 12, 223-239
Almatsier S. 2005. Prinsip Dasar Ilmu Gizi. PT Gramedia Pustaka Utama, Jakarta.
Angus F, S Smart, dan C Shortt. 2005. Prebiotic Ingridients with Emphasis on Galacto- oligosaccharides and Fructo-oligosaccarides dalam Probiotic Dairy Products. Tamime, A.Y. 2005. Blackwell Publishing Ltd, United Kingdom.
[AOAC] Association of Official Analytical Chemist. 1995. Official Method of the Association of Official Agriculture Chemist, Washington DC.
Arief II, RRA Maheswari, dan T Suryati. 2005. Karakteristik dan Nilai Gizi Protein Daging Sapi Dark Firm Dry (DFD) yang difermentasi oleh Lactobacillus plantarum yang diisolasi dari Daging Sapi. Laporan Penelitian Hibah Bersaing XIII/I.LPPM-IPB.
Arief II, RRA Maheswari, dan H Nuraini. 2008. Aktivitas Antimikroba Bakteri Asam Laktat yang Diisolasi dari Daging Sapi. Makalah Seminar Hasil Penelitian Departemen IPTP Fak. Peternakan IPB.
Boehm G dan Moro G. 2008. Structural and Functional Aspects of Prebiotics Used in Infant Nutrition. The Journal of Nutrition Influence of Diet on Infection and Allergy in Infants. J. Nutr. 138: 1818S–1828S.
Bourlioux P, Koletzko B, Guarner F, dan Braesco V. 2002. The intestine and its microflora are partners for the protection ot the host : report on the Danone symposium “The intelligent Intestine”, held in Paris, June 14, 2002. Am J Clin Nutr 2003; 78: 675-83.
Bowen R. 2006. Pathophysiology of Diarrhea. http://arbl.cvmbs.colostate.edu/ hbooks/pathphys/digestion/smallgut/diarrhea.html. [11 November 2010]
[BSN] Badan Standarisasi Nasional. 2009. SNI 2981: Yogurt. ICS 67.100.10.
Candela M, Seibold G, Vitali B, Lachenmaier S, Eikmanns BJ, Brigidi P. 2005. Real-time PCR quantification of bacteria adhesion to Caco-2 cells: competition between bifidobacteria and enterophatogens. Res Microbiol 156:887-95.
Celli J, Deng W, dan Finlay BB. 2000. Enteropathogenic Escherichia coli (EPEC) attachment to epithelial cells: exploiting the host cell cytoskeleton from the outside. Cell Microbiol. 2(1), 1-9. Chandan RC. 2006. Manufacturing Yogurt and Fermented Milks. Blackwell Publishing Asia.
Chen LC. 1983. Diarrhea and Malnutrition : Interaction, Mechanisms, and Interventions. The United Nations University Plenum Press: New York.
Clarke SC, Haigh RD, Freestone PP, dan Williams PH. 2002. Enteropathogenic Escherichia coli infection: history and clinical aspects. Br J Biomed Sci 59, 123–127.
Crane JK, Naeher TM, Shulgina I, Zhu C dan Boedeker EC. 2007. Effect of Zinc in Enteropathogenic Escherichia coli Infection. Infection and Immunity. Vol. 75, No. 12 p. 5974–5984
Cummings JH, Macfarlane GT, dan Englyst HN. 2001. Prebiotic digestion and fermentation. Am J Clin Nutr 73, 415S–420S.
[DEPKES] Departemen Kesehatan Republik Indonesia. 2008. Profil Kesehatan Indonesia 2007. Departemen Kesehatan Republik Indonesia, Jakarta.
Delzenne NM, Cani PD, dan Neyrinck AM. 2007. Modulation of glucagon-like peptide-1 and energy metabolism by inulin and oligofructose: experimental data. Journal Nutrition, 137, 2580S- 2551S.
De Roos NM dan Katan MB. 2000. Effect of probiotic bacteria on diarrhea, lipid metabolism, and carcinogenesis: a review of papers published between 1988 and 1998. Am J Clin Nutr, 71:405- 11
[FAO] Food and Agriculture Organization. 2001. Health and Nutritional Properties of Probiotic in Food including Powder Milk with Live Lactic Bacteria. Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Bacteria.
[FAO] Food and Agriculture Organization. 2002. Guidelines for the evaluation of probiotics in food. Report of Joint FAO/WHO Working Group on drafting Guidelines for the evaluation of probiotics in food. London Ontario, Canada.
[FAO] Food and Agriculture Organization. 2007. FAO technical meeting on prebiotics. AGNS-FAO, Italy.
Fardiaz S. 1989. Mikrobiologi Pangan. Dirjen Pendidikan Tinggi, Depdikbud, PAU PG, IPB, Bogor. Fooks LJ dan Gibson GR. 2002. Probiotics as modulators of the gut flora. Br J Nutr, 88, S39-S49. Forchielli ML, Walzer AW. 2005. The role of gut-associated lymphoid tissues and mucosal defence.
Br J Nutr 93, S41–S48.
Franck A. 2002. Technological functionality of inulin and oligofructose. Br J Nutr, 87, S287-S291. Franck A. dan LD Leenher. 2005. Inulin dalam Polysaccharides and Polyamides in the Food Industry
Volume I. Steinbuchel A. dan SK Rhee (eds.). Wiley VCH, Weinheim.
Freter M. 1992. Factors aVecting the microecology of the gut dalam In vitro analysis of probiotic strain combinations to inhibit pathogen adhesion to human intestinal mucus. Collado MC, Meriluoto J, dan Salminen S. 2006. Food Res Int 40 (2007) 629–636.
Galdeano MC dan Perdigon G. 2006. The Probiotic Bacterium Lactobacillus casei Induces Activation of the Gut Mucosal Immune System through Innate Immunity. Clin Vac Immun, p. 219-226. Gibson GR, Probert HM, Van Loo J, Rastall RA, dan Roberfroid MB. 2004. Dietary modulation of
the human colonic microbiota: updating the concept of prebiotics. Nutr Res Rev 17, 259–275. Gill HS. 2003. Probiotics to enhance anti-infective defenses in the gastrointestinal tract. Best Pract
Res Clin Gastroenterol, 17, 755-773.
Gill HS dan Guarner F. 2004. Probiotics and Human Health : a clinical prespective. Postgrad Med J, 80, 516-526.
Gmeiner M, Kneifel W, Kulbe KD, Wouters R, De Boever P, Nollet L, et al. 2000. Influence of a synbiotic mixture consisting of Lactobacillus achidophilus 74-2 and a fructooligosaccharide preparation on the microbial ecology sustained in a simulation of the human intestinal microbial ecosystem (SHIME reactor). App Microbiol Biotechnol, 53, 219-223.
Gomes TAT, Irino K, Girão DM, Girão VBC, Guth BEC, Vaz TMI, et al. 2004. Emerging Enteropathogenic Escherichia coli Strains?. Emerg Infect Dis. Vol. 10, No. 10.
Guerrant RL, Van Gilder T, Steiner TS, Thielman NM, dan Slutsker L, et al. 2001. Practice Guidelines for the Management of Infectious Diarrhea. Clinical Infectious Diseases, 32, 331- 50. Infect Dis Soc Ame.
Gupta V dan Garg R. 2009. Probiotics. Ind J Med Microbiol, 27(3):202-9.
Harish K dan Varghese T. 2006. Probiotic in humans-evidence based review. Cal Med J 4 (4):e3. Hariyadi RT, N Anjaya, Suliantari, L Nuraida, dan B Satiawiharja. 2001. Penuntun Praktikum
Teknologi Fermentasi. Jurusan Teknologi Pangan dan Gizi, Institut Pertanian Bogor, Bogor. Heyman M dan Menard S. 2002. Probiotic microorganism: How they affect intestinal
pathophysiology. Cell Mol Life Sci. Vol. 59, pp 001-015.
Hooper LV, Midtvedt T dan Gordon JL. 2002. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Ann Rev Nutr, 22, 283-307.
Indratiningsih W, S Salasia, dan E Wahyuni. 2004. Produksi yogurt shiitake (Yohsitake) sebagai pangan kesehatan berbasis susu. J Teknol Ind Pangan Vol. XV (1): 54-60.
Isolauri E, Sutas Y, Kankaapaa P, Arvilommi H, dan Salminen S. 2001. Probiotics: Effect on Immunity. Am J Clin Nutr, 73, 444S-450S.
Jack RW, Tagg JR, Ray B. 1995. Bacteriocins of gram-positive bacteria dalam Probiotics: a critical review of their potential role as antihypertensives, immune modulators, hypocholesterolemics, and perimenopausal treatments. Liong Min-Tze. 2007. Nutr Rev vol. 65, 7: 316-328.
Janda JM dan Abbot SL. 2006. The Enterobacteria (eds.). ASM Press, Washington, USA. Jay JM. 2000. Modern Food Microbiology 6th Edition. Maryland, Aspen Publishers, Inc.
Jenie BSL. 2003. Pangan Fungsional Penyusun Flora Usus yang Menguntungkan. Makalah pada Seminar Sehari Keseimbangan Flora Usus Bagi Kesehatan dan Kebugaran, Bogor.
Kaper JB. 1996. Defining EPEC dalam Association of atypical enteropathogenic Escherichia coli (EPEC) with prolonged diarrhea. Afset JE, Bevanger L, Romundstad P, dan Bergh K. 2004. J Med Microbiol. 53, 1137-1144.
Kaplan H dan Hutkins RW. 2000. Fermentation of Fructooligosaccharides by Lactic Acid Bacteria and Bifidobacteria. App Environ Microbiol, p. 2682–2684 Vol. 66, No. 6.
King D. 2009. Study Guide Histology of the Gastrointestinal System. School of Medicine. http://www.siumed.edu/~dking2/erg/giguide.htm. [11 Oktober 2010]
Kiernan JA. 1990. Histological and Histochemichal Methods: Theory & Practice. Oxford: Pergamon press.
Kullisaar T, Songisepp E, Mikelsaar M, Zilmer K, Vihalemm T, dan Zilmer M. 2003. Antioxidative probiotic fermented goats’ milk decreases oxidative stress-mediated atherogenicity in human. Br J Nutr, 90, 449-456.
Lambert J dan Hull R. 1996. Upper gastrointestinal disease and probiotics dalam Probiotic Dairy Products. Tamime, A.Y. 2005. Blackwell Publishing Ltd, United Kingdom.
La Torre L, Tamime AY, dan Muir DD. 2003. Rheology and sensory profiling of set-type fermented milks made with different commercial probiotic and yogurt starter cultures. Int J Dairy Technol, 56, 163-170.
Lee YK, Lim CY, Teng WL, Ouwehand AC, Tuomola EM, dan Salminen S. 2000. Quantitative Approach in the Study of Adhesion of Lactic Acid Bacteria to Intestinal Cells and Their Competition with Enterobacteria. App Environm Microbiol. p. 3692–3697 Vol. 66, No. 9. Link-Amster H, Rochat F, Saudan KY, Mignot O, Aeschlimann JM. 1994. Modulation of a spesific
humoral immune response and changes in intestinal flora mediated through fermented milk intake dalam Effect of probiotic bacteria on diarrhea, lipid metabolism, and carcinogenesis: a review of papers published between 1988 and 1998. De Roos NM dan Katan MB. 2000. Am J Clin Nutr, 71:405-11.
Liong Min-Tze. 2007. Probiotics: a critical review of their potential role as antihypertensives, immune modulators, hypocholesterolemics, and perimenopausal treatments. Nutr Rev vol. 65, 7, 316- 328.
Lisal JS. 2005. Konsep Probiotik dan Prebiotik untuk Modulasi Mikrobiota Usus Besar. J Med Nus Vol. 26 No.4.
van Loo J, Coussement P, De Leenheer L, Hoebregs H, Dan Smits G. 1995. On the presence of inulin and oligofructose as natural ingredients in the Western diet dalam Probiotic Dairy Products. Tamime AY. 2005. Blackwell Publishing Ltd, United Kingdom.
Lu L dan Walker WA. 2001. Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium. Am J Clin Nutr 73(suppl):1124S–30S.
Manatsathit S, Dupont HL, Farthing MJG, et al. 2002. Guideline for the Management of acute diarrhea in adults. J Gastroenterol Hepatol 17, S54-S71.
Manning TS, Gibson GR. 2004. Prebiotics. Best Practice and Research Clinical Gastroenterology. 18(2): 287-298
Masco L, Ventura M, Zink R, Huys G, dan Swings J. 2004. Polyphasic taxonomic analysis of Bifidobacterium animalis and Bifidobacterium lactis reveals relatedness at the subspecies level: reclassification Bifidobacterium animalis as Bifidobacterium animalis subsp. animalis subsp. nov. and Bifidobacterium lactis as Bifidobacterium animalis subsp. lactis subsp. nov. Int J Sys Evolution Microbiol, 54, 1137-1143.
Mattila-Sandholm T, Myllarinen P, Crittenden R, Mogensen G, Fonden R, Saarela M, 2002. Technological challenges for future probiotic foods. Int Dairy J 12, 173–182.
Meydani SN dan Ha WK. 2000. Immunologic effect of yogurt. Am J Clin Nutr, 71, 861-872.
Michail S dan Abernathy F. 2002. Lactobacillus plantarum Reduces the In Vitro Secretory Response of Intestinal Epithelial Cells to Enteropathogenic Escherichia coli Infection. J Ped Gastroenterol Nutr. 35,350–35.
Miyazaki Y, Kamiya S, Hanawa T, Fukuda M, Kawakami H, Takahashi H, Yokota H. 2010. Effect of probiotic bacterial strains of Lactobacillus, Bifidobacterium, and Enterococcus on enteroaggregative Escherichia coli. J Infect Chemother, 16:10–18.
Moat AG, Foster JW, dan Spector MP. 2002. Microbial Physiology. 4th Ed. A Willey Interscience Publication, John Willey and sons, New York.
Molis C, Flourie B, Ouarne F, Gailing MF, Lartigue S, Guibert A, et al. 1996. Digestion, excretion, and energy value of fructooligosaccharides in healthy humans dalam prebiotics: present and future in food science and technology. Wang, Y. 2009. Food Res Int, 42, 8-12.
Muchtadi D. 1993. Teknik Evaluasi Nilai Gizi Protein. Program Pasca Sarjana. Institut Pertanian Bogor, Bogor.
Naidu AS dan RA Clemens. 2000. Probiotics dalam Natural Food Antimicrobial Systems. Naidu, A.S. (ed). CRC Press, LLC.
Nougayrède JP, Fernandes PJ dan Donnenberg MS. 2003. Adhesion of enteropathogenic Escherichia coli to host cells. Cell Microbiol 5, 359–372.
Oliviera MN dan Damin MR. 2003. Efeito do teor de solidos e da concentrcao de sacarose na acidificaca, fermeza e viabilidade de bacterias do iogurte e probioticas em leite fermentado dalam Probiotic Dairy Products. Tamime, A.Y. 2005. Blackwell Publishing Ltd, United Kingdom.
Ouwehand A dan Conway P. Specificity of spent culture fluids of Lactobacillus spp. to inhibit adhesion of enteropathogenic fimbriated Escherichia coli cells. Microbiol Ecol Health Dis 1996;9:239–46 dalam Pathologic and physiologic interactions of bacteria with the
gastrointestinal epithelium. Lu L dan Walker WA. 2001. Am J Clin Nutr 73(suppl):1124S– 30S.
Oyetayo VO. 2004. Performance of rats orogastrically dosed with faecal strains of Lactobacillus acidophillus and challenged with Escherichia coli, Af J Biotechnol. Vol 3. No. 8, pp 409-411. Parassol N, Freitas M, Thoreux K, Dalmasso G, Bourdet-Sicard R, Rampal P. 2005. Lactobacillus
casei DN-114 001 inhibits the increase in paracellular permeability of enteropathogenic Escherichia coli-infected T84 cells. Res Microbiol, 156, 256–262.
Perdigon G, Rachid M, De Budeguer MV, dan Valdez JC. 1994. Effect of yogurt feeding on the small and large intestine associated lymphoid cells in mice dalam Yogurt and Immunity. van de Water J dan Naiyanetr P. 2008. Handbook of Fermented Functional Foods. Second edition. CRC Press, Taylor & Francis Group, Boca Raton.
Rasic JL, Kurmann JA. 1983. Bifidobacteria and their role dalam Review : Yogurt as probiotic carrier food. Lourens-Hattingh A dan Viljoen BC. 2001. Int Dairy J 11:1–17.
Rautava S, Ruuskanen O, Ouwehand A, Salminen S, Isolauri E. 2004. The hygiene hypothesis of atopic disease- an extended version. J Pediatr Gastroenterol Nutr , 38, 378–88.
Reid G, Jass J, Sebulsky MT, McCormick JK. 2003. Potensial uses of probiotics in clinical practice. Clin Microbiol Rev, 4, 658-672.
Roberfroid MB. 1997. Health benefits of non-digestible oligossacharides dalam Probiotic Dairy Products. Tamime, A.Y. 2005. Blackwell Publishing Ltd, United Kingdom.
Rolfe RD. The role of probiotic cultures in the control of gastrointestinal health. 2000. J Nutr, 130, 396S-402S.
Sahaja. 2008. DOTE Anatomy Topics: The anatomy, histology and development of the small intestine. University of Debrecen. http://anatomytopics. wordpress.com/2008/12/20/20-the- anatomy-histology-and-development-of-the-small-intestine/ [11 Oktober 2010]
Salminen S, Bouley C, Boutron-Ruault MC, Cummings JH, Franck A, Gibson G R, Isolauri E, Moreau MC, Roberfroid MB, dan Rowland IR. 1998. Functional food science and gastrointestinal physiology and function dalam Probiotic Dairy Products. Tamime AY. 2005. Blackwell Publishing Ltd, United Kingdom.
Salminen S, Von Wright A, dan Ouwehand A. 2004. Lactic Acid Bacteria. Marcel Dekker, Inc, New York.
Samuelson DA. 2007. Textbook of veterinary histology. Elseiver Inc, St Louis, Missouri.
Santoso. 2009. Susu dan Yogurt Kedelai. Seri Teknologi Pangan Populer (Teori dan Praktek). Laboratorium Kimia Pangan, Faperta, UWG.
Sartor RB. 2004. Therapeutic manipulation of the enteric microflora in inflammatory bowel disease: Antibiotics, probiotics, and prebiotics. Gastroenterol, 126, 1620-1633.
Sasaki M, Bosman BW, Tan PS. 1995. Comparison of proteolytic activities in various lactobacilli. J Dairy Res 62 (4): 601-610.
Saulnier DMA, Molenaar D, de Vos WM, Gibson GR, dan Kolida S. 2007. Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays. Appl Environ Microbial, 73, 1753-1765.
Saulnier DMA, Spinler JK, Gibson GR dan Versalovic J. 2009. Mechanisms of probiosis and prebiosis: considerations for enhanced functional foods. Curr Op Biotechnol, 20, 135-141. Schmid K, RC Scholathauer, U Friedrich, C Staudt, J Apajalahti, dan EB Hansen. 2006. Development
of Probiotics Food Ingredients dalam Probiotics in Food Safety and Human Health. Goktepe, Juneja, dan Ahmedna (eds.). CRC Press-Taylor and Francis Group, Florida.
Schiller LR. 2000. Diarrhea, Medical Clinics of North America dalam Diare Akut Infeksius pada Dewasa. Zein U. 2004. Fakultas Kedokteran, Universitas Sumatera Utara.
Shah NP. 2000. Probiotic bacteria : Selective enumeration and survival in dairy foods. J Dairy Sci, 83, 894-907.
Sharma AK, Mohan P, dan Nayak BB. 2005. Probiotic : Making a comeback. Department of Pharmacology Armed Forces Medical College, Pune.
Siegumfeldt H, Rechninger BK, Jacobsen M. 2000. Dynamic changes of intracellular pH in individual lactic acid bacterium cells in response to a rapid drop in extracellular pH. App Environm Microbiol. 66:2330-2335.
Silvia. 2002. Pembuatan Yogurt Kedelai (Soygurt) dengan Menggunakan Kultur Campuran Bifidobacterium bifidum dan Streptococcus thermophilus. Skripsi. Fakultas Teknologi Pertanian. Insitut Pertanian Bogor, Bogor.
Slonczewski JL dan Foster JW. Microbiology Evolving Science. http://www.microbwiki.com/small_intestine. [22 Februari 2010]
Surono IS. 2004. Probiotik Susu Fermentasi dan Kesehatan. YPMMI, Jakarta.
Tamime AY. 2005. Probiotic Dairy Products. Blackwell Publishing Ltd, United Kingdom.
Tamime AY, Skriver A, dan Nilsson LE. 2005. Starter cultures. Fermented Milks. Blackwell Publishing, Oxford.
Teitelbaum JE dan Walker WA. 2002. Nutritional impact of pre- and probiotics as protective gastrointestinal organisms. Ann Rev Nutr 22, 107–138.
Trabulsi LR, Keller R, dan Tardelli Gomes TA. 2002. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis 8, 508–513.
Tungland BC. 2003. Fructooligosacharides and other fructans: structures and occurrence, production, regulatory aspect, food applications and nutritional health significance dalam Probiotic Dairy Products. Tamime, A.Y. 2005. Blackwell Publishing Ltd, United Kingdom.
Walker WA. 2008. Mechanisms of action of probiotics. Clin Infect Dis 46, S87–S91.
Wang Y. 2009. Prebiotics: Present and future in food science and technology. Food Res Int 42:8-12 Winarno FG. 2003. Mikroflora Usus Bagi Kesehatan dan Kebugaran. Makalah Sehari Keseimbangan
Flora Usus Bagi Kesehatan dan Kebugaran. Bogor, 15 Februari 2003. Jurusan Teknologi Pangan dan Gizi, Fakultas Teknologi Pertanian, Institut Pertanian Bogor, Bogor.
Yan F dan Polk DB. 2006. Mechanisms of Probiotic Regulation of Host Homeostatis. Nutrition and Health : Probiotics in Pediatric Medicine. Humana Press. Totowa.
Zakaria F. 2003. Aspek Biokimia dan Gizi Pangan Fungsional Prebiotik. Makalah Sehari Keseimbangan Flora Usus Bagi Kesehatan dan Kebugaran. Bogor, 15 Februari 2003. Jurusan Teknologi Pangan dan Gizi, Fakultas Teknologi Pertanian, Institut Pertanian Bogor, Bogor. Zein U. 2004. Diare Akut Infeksius pada Dewasa. Fakultas Kedokteran, Universitas Sumatera Utara. Zhang G dan Ghosh S. 2001. Toll-like receptor-mediated NF-kappaB activation: a phylogenetically
conserved paradigm in innate immunity. J Clin Invest 107,13–9.
Zoumpopoulou G, B Foligne, K Christodoulou, C Grangette, B Pot dan E Tsakalidou. 2008. Lactobacillus fermentum ACA-DC 179 displays prebiotic potential in vitro and protects againts trinitrobenzene sulfonic acid (TNBS)- induced colitis and Salmonella infection in urine models. Int J Food Microbiol 121: 18-19
Zubillaga M, Weill R, Postaire E, Goldman C, Caro R, Boccio J. 2001. Effect of probiotics and functional foods and their use in different diseases. Nutr Res 21: 569-579.