Y HOC VIET NAM THANG - SO 2/2014 DANH GlA DOC TINH CAP CUA CHE PHAM SYNBIOTIC VA KHA NANG SINH B A O TIT CUA B. SUBTILIS HU58 Ho Anh Son*, Nguyin Linh Toan*, Nguyen Trong Tai*", Nguyen Thi Mai Phuffng*** T O M TAT Muc tieu nghien ciru la danh gia doc tfnh cap ciia che pham synbiotic va kha nang tang sinh bao ty B. subtilis HU58. Ket qua nghien ciru cho thay khong co sy thay doi cong thyc mau, chffe nang gan than va hinh anh giai phau benh gan than cua chuot uong synbiotic. So lUOng B. subtilis HU58 tang sinh manh khoang 48 gicJ roi giam dan sau uong vi khuan. Tu" khoa: acute toxicity, synbiotic, mice, B. subtilis HU58 SUMMARY EVALUATE ACUTE T O X I C I T Y OF S Y N B I O T I C AND PROLIFERATIVE A B I L I T Y OF B. S U B T I L I S H U The research objective was to assess the acute toxicity of synbiotic preparations and proliferative ability of B. subtilis HU58. The study results showed no change in. Our purpose to evaluate the acute toxicity of synbiotic and proliferative ability of B. subtilis HU58. The results showed there were un change in blood cell counts, kidney and liver function and anatomical images of mice livers and kidneys. The number of B. subtilis HU58 proliferated rapidly within 48 hours and then decrease gradually after days of B. subtilis HU58 oral administrabon. I. DAT VAN DE Tren the gidi, phan Idn eac san pham thffc pham bo sung dffdc san xuat d dang probiotie hay prebiotic rieng re. Viee ket hdp hai chat ciing mpt san pham se cd gia tri sff dung cao hdn nhieu va tien Idi cho ngffdi tieu diing nen dffdc gpi la synbiotic. Cae dac tinh chii yeu ciia synbiotic la kha nang khang khuan, tang cffdng loai bd vi khuan gay benh va tang dap ffng mien djch khang nguyen dac hieu va khong dac hieu; tang kha nang thffc bao; chdng ung thff, chong ia chay; chdng dj ffng; ngan chan hdi chffng bang xffdng; giam chat beo huyet va dffdng mau. Nhd vay, synbiotic * Hoc vien Quan y '* 'oai Hpc Y Vinh '** Vien Cong nghe Smh hoc Phan bien khoa hoc: TS. Can Van Mao thyc s y la san pham cd nhieu hffa hen. San pha'm synbiotic (P&P) sff dung ngjiien ciry tao tff viec phoi trpn giffa dan xuat tff cam gao vdi bao tff ciia chung vi sinh vat sinh bao tii" B. subtilis HU58. Oe cd ed sd cho cac nghien cffu tiep va dffa dffpc san pham thj trffdng, can kiem soat tfnh an toan ciia che pham, vay muc tieu eiia chiing toi la danh gia dpc tfnh cOa che pham (neu cd) va kha nang sinh bao tff sau dpng vat thffc nghiem udng che pham nay. (Day ia mpt phan san pham ciia de tai "Nghien cffu san xuat che pham synbiotic tff vi khuan sinh bao tff va cam gao" Ma so: KC04.TN01/11-15). II. OOI TUOMG VA PHU'aNG PHAP NGHIEN CU\J . . Ddi tu'dng nghien cu'u Chuot nhat trang ban dpng vat Hpc vien Quan y eung cap, khong phan biet gidng, khoe manh, dat tieu ehuan thi nghiem, can nang 20,0 ± 2,0 g. Chuot dffdc nuoi dieu kien nhiet dp 24±2°C, anh sang t y nhien, nydc va thffc an dffde nau ehfn, bao dam theo nhu cau. Qua trinh nudi dffdc bat dau trffdc tien hanh thf nghiem ft nhat ngay. . . Phu'dng phap nghien cu'u 2.2.1. Nghien cu'u dgc tinh cap Opng vat dffdc chfa cac 16, moi 16 10 cor, Chuot eiing 16 dffdc danh so va nhot chung vao chuong. Trffdc cho chuot diing thudc, chuot bj bo ddi 12 gid. Sau tim kha nang hda tan ciia che pham, chiing tdi tim dffdc lieu P&P cao nhat cho| chuot uong la 15 g/kg can nang chuot, tffdng ffng viJ 0,2ml/10g the trpng. Cac lieu tiep theo dffdc tfnh giam so vdi lieu toi da % , tong sd cd nhom lieu dffdc sff dung. Sau cho diing thudc, chuot dffoc theo ddi, an va uong t y do. Thdi gian theo doi lien tuc 72 gid. Sd ehupt chet dddc dem theo nhdm. Cac chi tieu theo doi: Tinh trang toan than, long, tfnh chat phan (khudn, nat), thay doi hanh vi; chi so huyet hpc, chdc nang gan than doi vdi nhdm chuot diing lieu cao nhat. 2.2.2. Oem quan the vi khuan phin Tong so gom ^ chuot, 6iScic nuoi rieng tiftig eon. Moi ehupt dffdc cho uong mot lieu nhat, Y HOC VIET NAM THANG • SO 2/2014 10 CFU vi khuan dffdc hoa tan nffdc cat. Lay phan vao cac thdi diem khac (toi da sau ngay). Trffdc thu thap phan, day hop thu thap dffdc trai giay trang da dffdc say vo khuan. Chuot dffdc dffa vao timg hop rieng le. Cae mau phan rieng biet cua tffng ehupt dddc thu thap, danh so. Can 0,lg phan pha vdi Q,9m! dem PBS, sau dd dddc trpn (votex) ky de tan phan hoan toan va u tii am d 65° gid. Quan the vi khuan dffdc dem ngay. Mat vt khuan dddc xac dinh bang phffdng phap pha loang va cay trai tren dia moi tru'dng LB agar. Cac dTa thach sau eay dffdc u d 37°C 48 gid cho den cac khuan lac dffdc hinh hoan toan. III. KET QUA . . Ket qua danh gia dpc tinh cap 3.1.1. Tinh trang toan than Sau uong lieu nhat che pham synbyotic, nhdm lieu cao tffdng ffng vdi 15 g/kg the trpng va cac nhdm uong vdi lieu thap hdn, tat ea ehupt thf nghiem d cae 16 deu an uong binh thu'dng, tinh trang phan, quan sat hoat dpng ciia chuot thay chuot binh thffdng. Bang 3.1: So Iffdng chuot song sau udng lieu nhat che pham symbiotic P&P Nhom Liieu su' dung (g/kg TLCT/24h) 2,5 5,0 7,5 10,0 12,5 15,0 n 10 10 10 10 10 10 So lu'dng dong vat chet ' (sau'72h) 0 0 0 So lu'dng dong vat song (sau 72h) 10 10 10 10 10 10 Theo doi sau 72 gid, khong co chuot thi nghiem bi chit, phau thuat ehupt cho thiy hinh anh cac cd quan binh th&dng. 3.1.2. Sit bien doi cac chi sd huyet hgc Chuot tai' nhdm sff dung che pham synbiotic lieu eao nhat (15 g/kg) dffde lay mau, kiem tra sd Iffdng te bao rriau de so sanh vdi nhdm chffng khong uong che pham. Bang 3.2: Sd Iffdng te bao mau ngoai vi mau chuot Nhom uong Nhom chffng Synbiotic ( n = ) fn=10) p>0,05 Hong cau (T/l) 7,9± 0,26 p>0,05 Bach eau (G/l) 9,1± 3,3 620 ±65,3 p>0,05 Tieu cau (G/l) Sdiiiu dbang 3.2 cho thiy sdl&dng tebao mau ngoai vi cua nhom chuot udng Synbiotic lieu cao 15g/kg khong co s&khac biet dang ke vdi nhom ch&ng (p>0,05). So l&dng hong cau dao dpng quanh khoang G/i; sdl&dng bach cBu dao dpng quanh G/l va so l&dng tieu cau khoang trin 700 G/l. 3.1.3. Theo doi chu'c nSng gan than Bang 3.3: Nong dp ure, creatinin, SGOT va 5GPT mau ehupt Nhom chu'ng Nhom uong Synbiotic ( n = ) (n=10) 22,67±2,11 Ure (mg/dl) 0,55± 0,07 Creatinin (mg/dl) 32,5± 2,59 SGOT (U/l) SGPT(U/1) "1 ^7,6+3,56 | 35,5 + 8,92 Kit qua thu d&dc d bang 3.3 cho thiy ndng cdc chit ure, creatinin nhom ch&ng khong cd s& khac biit dang ke vdi nhom uong chi pham synbiotic. nSng than dn dinh sau chuot uong lieu nhit synbiotic 15 g/kg. Tudng ttf, nong dp hai enzyme ia SGOT va SGPT cua gan khong co s& khac che pham va nhom chuhg. p>0,05 p>0,05 p>0,05 | p>0,05 . mau ngoai vi cua chuot Dieu cho thiy ch&c biet giO^ nhom s& dung Y HOC VIET NAM THANG - SO 2/2014 WOTA 2/ Hinh anh vl the cau true gan 3.1.4. Hinh anh vi the gan than Hinh 1: Hinh anh vl the cau true than ' • . - ; ^ - Z-. ,' •w •,cv i T&dng tu' hinh anh vi the ciu true gan (Hinh 2) cho thiy ciu true cua gan vdi cac tieu thuy ro. Hinh anh vi thi cau true than (Hinh 1) cho thay Khoang c&a co ranh gidi ro. Trong khoang c&a co ciu true than ro vung vo va vung tuy. Vung vo cac dng mat, tieu quan mat, dpng mach va tinh vdi cac cau than gom cac mao mach xung huyet. mach gan. Co si/xam nhiem ft te bao lympho va Bao Bowman mong, khoang Bowman t& do. Sat bach cau toan. Cac ti bao xip be gom vdi cau than la Macula densa, cac ong l&dn gan hai hang tebao canh cac xoang mach. Rai raced va l&dn xa d&dc lot bieu mo khoi vudng. Vung cac te bao Kulfer va te bao npi mo lot long. vdi cac dng gop. Khe than co cac mach mau xung huyet A: 3.2. Danh gia kha nang tang so Iffdng quan the vi khuan B. subtilis HU58 rupt Hinh 3.3. Sff thay doi so Iffdng bao tff vi khuan B. subtilis HU58 theo thdi gian 24 il 4S Thcri oian Kit qua thu d&dc d hinh 3.3 cho thiy d thdi diim tr&dc cho chuot uong chi pham, sdl&dng bao t&B subtilis HU58 dat m&c Iff CFU/g, mat dp d&dc xem la mat dp ban dau vdn co (base line) gii Sau du^ vi khuan dang bao t& vao cd the chuot qua d&dng uong, sd Iwdng quan the vi khuan lin m&c 5xl(f° CFU/g, cao hdn ca l&dng bao t&dwa vao ban dau. IV. BAN LUAN 4,1. Sff dung che pham P&P khong gay doc San pham synbyotic cd phan la chat xd tff cam gao va vi khuan Bacillus deu da dffdc chffng minh an toan qua trinh sff dung nhff mpt thyc pham chye nang. Cac khia canh an toan Y HQC VIET NAM THANG - SO 2/2014 duOc quan tam chfnh la chung vi khuan dddc bo sung vao che pham co an toan hay khong. Cac nha nghien ciru da eong bd che pham synbiotic chira Bacillus khdng gay hai eho cd the. Sorokulova (2007) cdng bo cac ket qua cho thay sff dung che pham chffa Bacilus kh6ng gay tan mau, vi khuan cung khong gay cac chat dpc to, cae gen dpc nhff bceTva cytK khong tim thay. Cac tac gia cung khdng phat hien dddc lieu gay chet tren chuot. Cac nghien cffu tru'dng dien de tim dpc tfnh ban cap ciia che pham cung khong phat hien ton thffdng d cae cd quan, mo. Cong bd mdi day ciia Sulemankhil va cpng sy (2012) cung cho rang cac vi khuan thye pham chffe nang la an toan. Nghien cffu eho thay sff dung vi khuan lactobacillus cd tac dung chong viem, lam giam cytokine IL-8, dong thdi khdng phat hien dffdc bat thffdng tren ehupt udng lien tue vdng 28 ngay. Khdng eo sy bien doi nao ve sff tan mau hay cae chat sinh hpc khac cd the. . . Kha nang tang so lu'dng quan t h e vl khuan B. subtilis H U So Iffdng bao tff thai qua phan chuot cao hdn so Iffdng dffdc dffa vao cd the giai thfch la bao tff da QiftSc mam rupt sau phat trien va lai tai tao bao tff. Neu rupt dpng vat khdng phai la moi trffdng phii hdp, bao tff sau dffa vao se bj thai theo dffdng phan va nhffng bao tff da mam se bj chet rupt. Mffc dp thay doi Idn ve so Iffdng hinh bao tff va sy cd m^t ciia bao t y tat ca cac mau phan chuot thf nghiem 6a eho thay rang rupt la mdi trddng thfch hdp nhff la ndi cff tru tff nhien cho no. Nghien ciru ciia Cutting va cpng sff tru'dc day CLJng da chffng minh dffdc B. subtilis ed the sinh trffdng, phat trien va tao bao tff dieu kien yem khf eua rupt gia. Do thj d hinh 3.3 cung cho thay so Iffdng vi khuan tri d mffc dp cao khoang 48 gid va bat dau giam dan theo thdi gian. Sau thi so Iffdng vi khuan da gl^m xuong tdi mffc ban dau. Nhff vay bao tff vi khuan B. subtilis HU58 da mam, phat trien va hinh bao tff ruot gia cua ehupt tru'dc dffdc thai ngoai mdi tru'dng qua phan. V. KET LUAN 1. Khong phat hien dffdc dpc tinh cap d lieu tdi da 15 g/kg the trpng sau chudt udng lieu nhat che pham Synbiotic ed nguon gdc tff cam gao. Chudt udng ehe pham Synbiotic vdi lieu nhat khdng lam thay ddi tinh trang toan than, so lu'dng te bao mau, chffe nang va hinh anh vi the gan than. 2. Bao tff vi khuan B. subtilis MUSS da mam, phat trien va hinh bao tff rupt ket ciia chuot trffdc dffdc thai ngoai moi trffdng qua phan. TAI LIEU T H A M KHAO 1. Collins MD, Gibson GR. Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut. Am J Cim Nutr. 1999 ;69{5):1052S-1057S. 2. GabrJella, C. and Cutting S., (2002), Bacillus Probiotic: Spore Germination in the Gastrointestinal Tract. Appl Environ Microbiol 68(5): 2344-2352. J. Hisanga, S., (1980), Studies on the germination of genus Bacillus spores in rabbit and canine intestines. J. Nagoya City Med. -^5S0C 30: 456-469. t . Hoa, TT., Due, LH., Isticato, R , Baccigalupi, L., Ricca, E., Van, PH. and Cutting, SM., (2001), The fate and dissemination of Bacillus subtilis spores in a murine model. Appl Environ Microbiothl: 3819-3823, 5. Irina B. Sorokulova, Iryna V. Pinchuk, Muriel Denayrolles, Irina G. Osipova, Jen M. Huang, Simon M. Cutting, Maria C. Urdaci. The Safety of Two Bacillus Probiotic Strains for Human Use. Dig Dis Sci (2008) 53: 954-963. 6. Le H. D., Huynh A. H „ Barbosa T.M.,. Henriques A.O., and Cutting S.M. (2004)Charactenzation of Bacillus Probiotics Available for Human Use. Applied and Environmental Microbiology, 70(4): 2161-2171. 7. Nguyen K. M. T, Nguyen Q. U., Huynh A. H., Le H. D., Tran T. H., Claudia R. S., Adriano . H., and Cutting S.M. (2006) The Intestinal Life Cycle of Bacillus subtilis and Close Relatives. Journal of Bacteriology. 188(7): 2692-2700. . OF SYNBIOTIC AND PROLIFERATIVE ABILITY OF B. SUBTILIS HU58 The research objective was to assess the acute toxicity of synbiotic preparations and proliferative ability of B. subtilis HU58. . uong synbiotic. So lUOng B. subtilis HU58 tang sinh manh trong khoang 48 gicJ roi giam dan sau 7 ngay uong vi khuan. Tu" khoa: acute toxicity, synbiotic, mice, B. subtilis HU58. livers and kidneys. The number of B. subtilis HU58 proliferated rapidly within 48 hours and then decrease gradually after 7 days of B. subtilis HU58 oral administrabon. I. DAT VAN DE Tren