B NNG NGHIP V PHT TRIN NNG THN VIN C IN NNG NGHIP V CNG NGH SAU THU HOCH -----------------

BO CO CHUYN

TI NGHIN CU CNG NGH SN XUT THC PHM CHC NNG T U TNG LN MEN DNG CHO NGI BNH TIU NG

Ch nhim ti: Ths. Nguyn Th Hng Tr C quan ch tr: Vin C in nng nghip v Cng ngh sau thu hoch Thi gian thc hin: 1/2010 12/2010

H Ni, 2010

CHUYN THC HIN NM 2010 Ni dung 1: Phn lp, tuyn chn cc chng nm mc c hot tnh km hm glucosidaza v nghin cu to ch phm ging mc khi ng cho ln men u tng. Chuyn 1: Nghin cu tng quan, xc nh ni dung v phng php nghin cu. Chuyn 2: Phn lp cc chng nm mc ln men u tng to cht km hm glucosidaza. Chuyn 3: Tuyn chn chng nm mc c kh nng ln men to cht km hm glucosidaza v nghin cu c im hnh thi ca cc chng nm mc tuyn chn Chuyn 4: nh tn chng nm mc tuyn chn (n mc loi). Chuyn 5: Nghin xu xc nh mt s yu t thch hp cho qu trnh nui cy to ch phm nm mc cho ln men u tng to cht km hm - glucosidaza. Ni dung 2: Nghin cu quy trnh cng ngh ln men u tng to cht km hm - glucosidaza. Chuyn 6: Nghin cu nh hng ca cc loi u tng n kh nng to cht km hm -glucosidaza ca chng nm mc tuyn chn. Chuyn 7: Kho st kh nng ln men to cht km hm - glucosidaza ca chng nm mc la chn trn mi trng u tng. Chuyn 8: Nghin cu nh hng ca cc yu t cng ngh ti qu trnh ln men b mt to cht km hm - glucosidaza ca nm mc.

B NNG NGHIP V PHT TRIN NNG THN VIN C IN NNG NGHIP V CNG NGH SAU THU HOCH ------&-------

BO CO CHUYN

Chuyn 1 Nghin cu tng quan, xc nh ni dung v phng

php nghin cu
Thuc ti: Nghin cu cng ngh sn xut thc phm chc nng t u
tng ln men dng cho ngi bnh tiu

Ch nhim ti: Ths. Nguyn Th Hng Tr Trng nhm thc hin: Ths. Nguyn Th Hng Tr Thi gian thc hin: 1/2010 - 3/2010 Kinh ph thc hin: 10.000.000 ng
H Ni, 2010

BO CO KT QU THC HIN CHUYN

Chuyn 1: Nghin cu tng quan, xc nh ni dung v phng php nghin cu Ch nhim ti: Ths. Nguyn Thi Hng Tr Trng nhm thc hin: Ths. Nguyn Thi Hng Tr Thi gian thc hin: 1/2010 - 3/2010 Kinh ph thc hin: 10.000.000 ng I. TNG QUAN NGHIN CU I.1. Vi nt v thc phm chc nng Thut ng thc phm chc nng (functional food) ch nhng ch phm thc phm cha cc cht c hot tnh sinh hc cao, c dc tnh t ng vt, thc vt, vi sinh vt, mt s cht tng hp ging cc hot cht t nhin nh vitamin, hng liu v.v. c tnh cht thit dng gn ging thuc, nhng khng thay th thuc iu tr. Thc phm chc nng ang c cc chuyn gia nh gi l mt xu th dinh dng ca th k 21, p ng mt phn quan trng v nhu cu dinh dng v sc khe con ngi trong cuc sng hin i. Thc phm chc nng c phn thnh nhiu nhm, ph thuc tc dng ca sn phm nh thc phm chc nng cho ngi c ng huyt cao, thc phm chc nng cho ngi c cholesterol trong mu cao, thc phm chc nng ci thin tiu hoTuy nhin, bt c h thng phn loi no th thc phm chc nng c tc dng gim ng huyt cng c quan tm v xp thnh mt nhm chnh. Thc phm chc nng hin nay v cng phong ph v a dng, t t nhin ln men truyn thng ti cc sn phm do con ngi ch bin, nhng ch yu tp trung vo ba hnh thc sau y: - Thc phm chc nng c ngun gc t nhin: bao gm cc loi ng vt, thc vt, vi sinh vt m trong bn thn n c cha cc cht chc nng vi hm lng ln, c tc dng tch cc i vi c th nh c rt cha beta - caroten c kh nng lm sng mt, chng lo ho, ngn nga bnh ung th, ch xanh cha catechin, rong, to

giu axt amin v vitamin c tc dng gim bo, gim hm lng cholesterol trong mu. - Thc phm chc nng l sn phm ca qu trnh ch bin, khng c cht b sung nh cc sn phm t sa (sa chua, pho mt) kch thch tiu ho, iu ho huyt p, tng kh nng min dch. Trong qu trnh ln men, ngoi vic to ra cc peptit chc nng th mt s lng ln cc vi khun lactic cng l yu t quan trng to nn chc nng sinh hc ca sn phm. - Thc phm qua ch bin v b sung cc cht chc nng. y l thnh qu ca cng ngh ch bin hin i vi hng lot cc sn phm chc nng nh nc cam c b sung canxi nhm gim nguy c long xng, cc thc phm c b sung vitamin E c tc dng dng tng cng kh nng sinh dc, lm p da I.2. Thc phm chc nng t u tng ln men Cy u tng hay cn gi l u nnh c tn khoa hc l Glycine max, thuc h u (Fabaceae). Vit Nam, u tng l cy cng nghip, cy thc phm ngn ngy c gi tr dinh dng cao dng lm thc phm cho ngi v thc n chn nui. Chnh v vy u tng ang l mt trong 10 chng trnh u tin pht trin ca ngnh nng nghip nc ta. Trong ht u tng c cc thnh phn ho hc sau: Protein (40%), lipit (12-25%), gluxit (10 - 15%); c cc mui khong Ca, Fe, Mg, P, K, Na, S; cc vitamin A, B1, B2, D, E, F; cc enzym, sp, nha, cellulo. u tng c coi l mt ngun cung cp protein hon chnh v cha mt lng ng k cc axit amin khng thay th cn thit cho c th nh: isoleucin, leucin, lysin, methionin, phenylalanin, tryptophan, valin. Sn phm t cy u tng c s dng rt a dng nh dng trc tip ht th hoc ch bin thnh u ph, p thnh du u nnh, gi u tng, sa u nnh... Trn th gii, ngoi cc sn phm t u tng nh trn th u tng ln men c s dng rt ph bin di cc dng thc phm nh natto, miso (Nht Bn), sufu (Trung Quc), tempeh (Indonesia), Doenjang (Hn Quc). u tng ln men khng nhng gia tng gi tr dinh dng m cn gim rt nhiu cc cht khng dinh dng nh

trypsin v chymotrypsin. l l do v sao n cc thc phm t u tng ln men khng b chng bng, y hi nh n cc sn phm t u tng khng ln men. Nhng nghin cu trong nhiu nm tr li y cho thy u tng c rt nhiu cc hot cht sinh hc qu v gi tr ca chng c tng ln rt nhiu khi tri qua qu trnh ln men nh vi sinh vt. V d nh thu nhn dch sa u tng ny mm giu isoflavon c hot tnh sinh hc cao (daidzein, genistein) v vitamin E; axit gamma aminobutyric (GABA); peptit c kh nng km hm enzym chuyn ha angiotensin... Gn y, cc nh khoa hc cn pht hin ra u tng ln men cn rt giu cht km hm - glucosidase. I.3. C s khoa hc ca vic s dng cht km hm glucosidaza iu tr bnh tiu ng. Bnh i tho ng cn gi l bnh tiu ng, l mt bnh do ri lon chuyn ha cacbonhydrat khi hoc mn insulin ca tuyn ty b thiu hay gim tc ng trong c th, biu hin bng mc ng trong mu cao hn 7 mmol/l. Hin nay t l t vong ca bnh tiu ng xp th ba sau ung bu, tim - mch mu no. Theo T chc WHO, nm 1985 trn th gii c tnh c khong 30 triu ngi mc bnh tiu ng. Sau 10 nm (nm 1994) con s ny ln n 110 triu ngi. Nm 2000 c trn 180 triu ngi v con s ny d bo s tng xp x 370 triu ngi vo nm 2030 [8]. Bnh nhn tiu ng c phn thnh hai nhm: Tiu ng typ 1 hay cn gi l tiu ng ph thuc insulin: do tuyn ty khng hot ng to ra insulin cn bng ng huyt nn phi tim hoc ung insulin. Tiu ng typ 2 hay cn gi l tiu ng khng l thuc insulin: chim t l ch yu ca bnh tiu ng. C th ngi bnh vn tit ra insulin nhng khng khin ng khng c a vo t bo m ch lu hnh trong mu. Mun iu tr bnh ny ngi ta ch cn p dng mt ch n ung hp l l kim ch c bnh. Cc thuc cha bnh tiu ng c chia lm 6 nhm gm: biguanides, sulphonylurea, glinidines, thiazolidinediones, cht km hm dipeptidyl peptidase IV v cht km hm glucosidaza (AGIs). Hin nay, mt lot cc cht km hm enzym -

glucosidaza c s dng iu tr bnh tiu ng nh voglibose (U.S. Pat. No. 6,200,958), acarbose (U.S. Pat. No. 5,643,874), miglitol (U.S. Pat. No. 4,639,436). Tinh bt
(nhiu phn t glucose)

Mch mu

Hnh 1.1. nh hng ca cht km hm - glucosidaza n qu trnh trao i ng trong c th Cc nghin cu trn th gii lm sng t c s khoa hc ca vic s dng cht km hm -glucosidaza iu tr bnh tiu ng nh sau: tinh bt hoc ng (sucrose) l mt cht dinh dng quan trng cn c gi l carbohydrate. Cu to ca n gm hai hay nhiu monosaccharide (glucose hoc fructose). c th ngi hp th c, carbohydrate s phn ct hon ton thnh cc monosaccharide nh h thng enzym tiu ha. Di tc dng ca amylase, tinh bt b phn ct thnh maltose. Sucrose v maltose l cc disaccharide. Di tc dng ca - glucosidaza chng s b phn ct thnh cc monosaccharide ti rut non. AGIs c th km hm -glucosidaza nn ngn cn s to thnh monosaccharide t disaccharide lm cho qu trnh tng hp insulin din ra d dng hn [28]. Cc kt qu nghin cu cho thy, cht km hm -glucosidaza c tc dng gia tng nng peptit - 1 dng glucagon (GLP-1) v peptit-2 dng glucagon (GLP-2) trong huyt tng. GLP-1 v GLP-2 l cc hormon c sinh ra t t bo L ni tit

nm trong vng ngoi bin ca rut non v rut kt. GLP-1 kch thch s tit insulin ph thuc glucoza v s sinh si t bo beta. GLP-2 ci thin cu trc v chc nng ca rut non bng cch tng cng cu trc ca lng nhung v tng hot tnh vn chuyn v enzym. GLP-2 lm gim s vim rut. GLP-1 v GLP-2 c to ra khi c s kch thch thn kinh, hormon hoc cc yu t dinh dng. Cht km hm glucosidaza l mt cht km hm -glucosidaza t nhin c kh nng ngn cn s ph hy cacbonhydrat nn ko di thi gian c mt ca cacbonhydrat trong rut. Do vy mt lng ln cacbonhydrat s n vng ngoi bin ca rut non v tng tc vi t bo L lm tng s tit GLP-1 v GLP-2. Ngi ta cho rng khi c mt glucoza ngoi bin rut non, glucoza s b ln men to cc axit bo chui ngn kch thch s sn xut v tit GLP-1 v GLP-2 hoc qu trnh vn chuyn glucoza ph thuc vo Na khi qua mng t bo L s kch thch s tit GLP-1 v GLP-2. Nng GLP-1 trong huyt tng tng s thc y qu trnh kim sot glycemic v cn tr s c mt glucoza trong mu. I.4. Cht km hm glucosidaza (AGIs) t vi sinh vt AGIs c th c to ra bng nhiu con ng khc nhau nh: chit xut t thc vt (t hoa Punica granatum, 1-deoxynojirimicin hay DNJ t l du), tng hp bng con ng ha hc (sulfonamit v cc dn xut ca xanthon) hoc bng con ng sinh hc nh cc vi sinh vt. Tuy nhin, sn xut AGIs quy m ln bng con ng chit xut t nhin rt tn km v khng n gin, mt khc AGIs c tng hp theo con ng ha hc li c nhc im l gy mt s tc dng ph nh ri lon chc nng gan v cc triu chng au d dy cho ngi s dng. Do vy vic sn xut AGIs bng vi sinh vt ang thu ht s quan tm nghin cu ca cc nh khoa hc cng nh cc nh sn xut v chng s dng an ton, khng c cc tc dng ph i vi c th sng. Hin nay mt s chng vi sinh vt nh: Aspergillus oryzae, Streptomyces, Actinoplanes v Flavobacterium saccharophilium, Saccharomyces cerevisiae ... c nghin cu l rt thch hp cho sinh tng hp AGIs [23, 33]. Mt s AGIs ph bin nh: acarbose - mt pseudo-tetrasaccharite c thu nhn t canh trng ln men

Actinoplanes spp. SE-50. Validamycin A t Streptomyces hygroscopicus var. limoneus. Adiposin, trestatin B, cyclophellitol v CKD-711 c ly t Streptomyces calvus TM521, Streptomyces dimorphogenes v Phellinus spp [33]. Tuy nhin, AGIs t A. oryzae l hng nghin cu mi. I.5. A. oryzae v ln men b mt I.5.1. Dinh dng ca A. oryzae A. oryzae thuc chi Aspergillus, h Trichocomaceae, b Eurotiales, lp Eurotiomycetes, ngnh Ascomycota v thuc gii nm (Kitamoto, 2002). Bo t ca A. oryzae c mu vng hoa cau, khng cha c t aflatoxin. A. oryzae tit ra mi trng cc enzym thy phn nh cellulase, pectinase, xylanase v hemicellulase khi sng trn mi trng c ngun c cht tng ng cellulose, pectin, xylan v hemicellulose nn A. oryzae c ng dng rt rng ri trong qu trnh sn xut, ch bin n v trong cng nghip sn xut cc enzym. Nht Bn A. oryzae c s dng trong qu trnh sn xut thc n nh x du, ru sak v trong cng nghip sn xut enzym. Vit Nam A. oryzae c s dng ch yu trong qu trnh lm tng. Nm 2001, b gen di truyn ca A. oryzae c phn tch v gii m. H gene gm 8 nhim sc th vi 12 ngn gene v 37 triu cp base (Galagan et al., 2005, Machida et al., 2005) (Hnh 1.2).

Hnh 1.2. Cu trc b gen ca A. oryzae (Machida et al., 2005)

Theo kha phn loi ca Klich: A. oryzae c c im pht trin khc nhau khi nui cy trn cc mi trng nh czapek, czapek yeast agar (CYA25), czapek yeast agar with 20% sucrose (CY20S), malt extract agar (MEA), rt him loi sinh hch nm. Quan st qua knh hin vi vt knh 10 v 40 thy c im hnh thi ca A. oryzae nh: 1 hoc 2 hng th bnh, bng nh gi c hnh qu l, hnh chy hoc hnh cu..., ng knh t (8) 22-50 (90) m; cung sinh bo t nhn hoc sn si, kch thc t 500-2500 (5000) m. Bo t trn hnh cu n hnh trng, nhn n c gai nh, ng knh (3,5) 4-8,5 (10) m. A. oryzae l sinh vt dinh dng ha nng hu c, chng ch c kh nng thu nhn nng lng t mi trng bn ngoi nh qu trnh oxy ha hiu kh hoc qu trnh ln men k kh cc cht hu c ngoi bo. A. oryzae l loi hiu kh hon ton v c kiu cacbon d dng thuc loi hoi sinh, c ngha l c kh nng phn gii xc sinh vt, s dng cc cht hu c lm cht dinh dng. * Ngun cacbon A. oryzae c th s dng ngun thc n cacbon khc nhau v khng c nhng i hi nghim ngt i vi mt loi thc n cacbon. Nhiu loi nm mc cn c kh nng ng ho c cc hp cht hu c rt bn vng hoc rt c i vi nhiu sinh vt khc (cc hp cht n-alcal, ancaloid, phenol, nhiu cht khng sinh...). i vi ngun thc n hu c phc tp nh: xilan, tinh bt, xenlulo, kitin... trc ht A. oryzae phi sinh ra cc enzym phn hu cc hp cht ny thnh cc hp cht n phn t sau mi ng ho c chng. * Ngun nit Cc loi A. oryzae khc nhau c th c nhu cu khc nhau i vi ngun thc n nit. A. oryzae thng c kh nng s dng c ngun nit v c v hu c. * Cc nguyn t khong Bng cc phng php phn tch quang ph, ngi ta xc nh c tt c cc cht khong c trong t bo nm mc. V cc nguyn t i lng, c th k n: S, P, K, Ca, Mg, Fe, chng thng chim t vi phn nghn n vi phn trm trng

lng kh. Cc nguyn t vi lng c Mn, Mo, Zn, Cu, Co, Ni, B. Hm lng cc nguyn t khong trong t bo nm mc khng nhng thay i tu thuc v iu kin nui cy, thnh phn mi trng, thi gian nui cy. I.5.2. Mt s yu t nh hng n qu ln men b mt * Nhit Nhit ng vai tr ch yu trong qu trnh sinh trng ca si nm, n cng tc ng n vic sn sinh bo t v s ny mm ca chng. Mi loi nm mc c khong nhit sinh trng v pht trin nht nh. Phn ln nm mc pht trin trong khong 15 - 320C v sinh trng tt nht khong 25 - 300C. * m Mi qu trnh trao i cht ca t bo vi sinh vt u xy ra trong mi trng nc. Tuy nhin nhu cu v nc i vi tng loi, tng chng v thi k pht trin khc nhau c khc nhau. Trong nhng nhn t tc ng vo s pht trin ca nm mc, m c vai tr rt ln. m ca mi trng cng nh m tng i ca khng kh l yu t quan trng quyt nh s sinh trng ca h si nm v s sn sinh bo t. * thong kh A. oryzae hon ton hiu kh v ch pht trin c trong iu kin thong kh. Mt lng nh kh cacbon l cn thit cho bo t Aspergillus ny mm nhng nu nng ny qu cao li km hm s pht trin ca chng. Khi nui cy nm mc bng phng php b mt cn ch ti dy khi mi trng m bo thong kh cn thit cho s pht trin ca nm mc. * pH mi trng pH ca mi trng cng nh hng n s pht trin ca A. oryzae. Phn nhiu A. oryzae pht trin trong phm vi pH t 4 - 8 nhng mt s nm mc c kh nng chu c nhng c cht c pH axit hn hoc kim hn. Tuy nhin khi dng phng php nui cy b mt, pH mi trng t nh hng n qu trnh sinh trng v pht trin ca nm mc v mi trng rn c tnh m cao.

I.6. Tnh hnh nghin cu v th nghim cht km hm glucosidaza t u tng ln men ngoi nc Kaisa v cng s nghin cu tm ra cc loi thc phm ngn chn bnh tiu ng. H kim tra hn 100 loi cht chit t thc phm vi mc ch km hm cc enzym tiu ho v cui cng h tm thy mt loi cht chit t u tng ln men bng cc chng Aspergillus sp. c kh nng km hm - glucosidaza. Loi u tng ln men ny gi l touchi v do vy cht chit cn c gi l cht chit touchi (TEtouchi extract). Touchi l mt loi u tng ln men truyn thng bng chng nm mc A. oryzae khng sinh c t, an ton vi con ngi v ng vt kinh t - l thc phm truyn thng rt c yu thch ca ngi Trung Quc, sau c a vo Nht Bn v c s dng lm thc phm hn 3000 nm hai nc ny [13]. Cng ty Nippon ca Nht Bn - mt Cng ty ni ting chuyn cung cp cc cht b sung cho sn xut thc phm chc nng sn xut c AGIs t u tng ln men. Trong phn gii thiu ca mnh, Nippon dn hng lot cc nghin cu khoa hc ca Fujita chng minh v hiu qu v tnh an ton ca AGIs c chit t u tng ln men [29]. Vo thng 6 nm 2008, cht chit touchi c cp php vo th trng Chu u, mt th trng ni ting v s qun l nghim ngt cc loi sn phm thc phm. Sn phm ny nhn c s nh gi cao ca U ban c vn Thc phm v Ch bin Hi ng chung chu u. Hin nay, cht chit touchi c s dng rng ri khng ch cc nc Nht Bn, Trung Quc m c cc nc Chu u. Trung Quc c hng trm cng ty sn xut cht chit u tng ln men cung cp cho ngnh ch bin thc phm chc nng. V mt iu quan trng l hot tnh km hm enzym glucosidaza ch c tm thy u tng ln men bi cc chng nm mc Aspergillus sp m khng c trong u tng ln men bng chng Bacillus subtilis natto to ra nato, mt mn n truyn thng ni ting ca Nht Bn. Jing Chen v cng s [23] nghin cu cng ngh sn xut AGIs t chng nm mc A. oryzae, Actinomucor elegans v Rhizopus arrhizus quy m phng th nghim.

Nhm tc gi cho rng s hnh thnh AGIs khng ph nhiu vo nguyn liu u tng m li b nh hng bi chng vi sinh vt cng nh cc yu t cng ngh. Khi ln men trn cc loi u tng khc nhau, hot tnh km hm -glucosidaza khng c s khc bit ng k. ng thi, nng mui NaCl trong qu trnh chn u tng ln men cng quyt nh n s hnh thnh AGIs nhiu hay t. Nng mui t 1012,5% lm gim kh nng hnh thnh AGIs mt cch ng k (gn 50%). Tc gi cho rng iu ny l nguyn nhn dn n s khc bit v hot tnh km hm glucosidaza 31 loi u tng ln men c thu thp c ngun gc khc nhau Trung Quc. Kh nng km hm -glucosidaza trong qu trnh ln men u tng khng nhng ph thuc vo chng vi sinh vt la chn m cn chu nh hng ca nhiu yu t nh thi gian nui cy, m c cht, pH ban u... Bi vy vic ti u ha qu trnh nui cy c ngha cho vic sn xut AGIs. Cng ngh chit sut v thu hi AGIs l mt cng ngh quan trng trong sn xut. Kawabata v cng s [24] nghin cu thm d mt s dung mi chit AGIs t u tng ln men, tuy nhin hiu sut tch chit cn ph thuc vo t l dung mi/ c cht, nhit v thi gian, kt qu cho thy rng khi chit bng nc, cn hoc hoc hn hp cn:nc t l 1:5 hoc 5:1 cho hot tnh km hm -glucosidaza cao, nhit v thi gian chit l 400C - 600C trong 12 - 20 gi hoc 1000C-1400C trong 1-3 gi. Tc gi cng nghin cu mt s phng php c c v tinh sch sn phm. c c sn phm tc gi s dng mt s dung mi hu c c cc nh methanol, ethanol, propanol, ethyl axetate, hexanv s dng cc phng php nh sc k ct, sc k trao i ion, sc k lng cao p tinh sch sn phm. c rt nhiu cng trnh nghin cu chng minh AGIs t u tng ln men l an ton qua cc th nghim trn chut v ngi. N c s dng trc ba n cn tr qu trnh thu phn cacbonhydrat trong rut non. Cc kt qu nghin cu ca Hiroyuki Fujita v cng s [18] cho thy cht chit t u tng ln men bi A. oryzae c tc dng km hm -glucosidase nhng li khng km hm cc enzym tiu ha

khc nh amylase, protease, pepsin, trypsin, chymotrypsin hoc lipase. Hin nay ngi ta cha pht hin ra mt tc dng ph no v tiu ho nh: chng bng, au bng y hi, nn ma khi s dng sn phm ny. Cht chit touchi cn c tc dng iu ho s hot ng ca cc enzym tiu ha trong rut non nn n l mt sn phm an ton v c li cho tiu ho. Cc ch s v cn nng hay chiu cao khng thay i. Cht chit touchi c hm lng ng thp v c lng ln cc cht bo khng bo ha nh axit alpha-linolenic (axit bo omega-3) c tc dng h triglycerit mu v LDLcholesterol, y l 2 ch s lipit c hi hay tng nhng bnh nhn i ng. V th, y l mt tc dng c li cho bnh nhn tiu ng. Ngoi ra cht x cha trong sn phm u tng ln men s hnh thnh mt lp mng mng gip hp thu glucoza chm hn do lm chm tc tiu ha ti d dy v vic gn glucoza ti rut non. Ln men u tng bng chng A. oryzae ci thin cht lng dinh dng thc phm t u tng. Sau 48 gi ln men u tng bng chng A. oryzae loi c phn ln cht km hm tripsin, hm lng protein th tng v lng axit amin t nhin khng thay i so vi u tng sng v cc loi thc n t u tng thng thng khc. Hm lng cc mch peptit c nh (< 20 kDa) tng, khng cha cc mch peptit ln (> 60kDa) thay v 22,1% u tng thng thng. Hiroyuki Fujita v cng s [17] th nghim cht chit touchi trn chut liu 100 mg/kg v 500 mg/kg. Kt qu cho thy hm lng glucose trong mu gim so vi nhm i chng (n=10) sau 15 - 60 pht np sucrose. Hm lng glucose trong mu gim tng ng l 17.96 0,42 v 16,76 0,39 mmol/l khi so snh vi nhm chut i chng l 19,91 0,58 mmol/l. Nhm tc gi ny cng th nghim trn ngi bnh tiu ng [19]. xc nhn s an ton, 9 ngi khe mnh c ung 3g/ngy trong 12 tun. Kt qu l khng c s thay i v ng huyt v cc ch s ha sinh mu, cn nng nh gi hot tnh km hm -glucosidaza trong ch phm, 18 bnh nhn tiu ng typ 2 c th nghim vi liu 0,9 g/ngy (0,3g/ba n) trong 6 thng. Kt qu cho thy hm lng ng mu ban u l 9,31 0,71 mmol/l v HbA1c: 10,24 0,58%. Sau 6 thng th nghim, hm lng ng mu

gim cn 8,61 0,66 mmol/l, HbA1c: 8,96 0,3%, ch s m mu v tng cholesterol, triglycerit cng gim, nhng ch tiu ha sinh khc nh GOT, GPT, urea, calcium. Hn na, enzym tiu ha khng b nh hng trong sut qu trnh th nghim. Trong mt cng b khc, Hiroyuki Fujita v cng s [18] th nghim trn 8 bnh nhn c nguy c mc bnh tiu ng v 4 bnh nhn tiu ng. Nhng i tng c nguy c mc tiu ng c cho ung cht chit touchi vi liu lng t 0,1 n 10,0 g trc khi n 75g sacarose. Kt qu cho thy s km hm hm lng glucose trong mu sau ba n c quan st thy sau 60 v 90 pht tiu th sacarose. nhm bnh nhn tiu ng c dng 0,3g cht chit touchi trc khi n 200g cm, cht chit touchi th hin tc dng gim ng huyt c ngha vi liu s dng thp nht l 0,3 g, ng huyt sau ba n gim c ngha ti thi im 60 pht v 120 pht sau khi n, so snh vi i chng khng ung cht chit touchi. C cc i tng c nguy c mc tiu ng v cc bnh nhn tiu ng u khng c bt c tc dng ph no nh au bng, bun nn, ph hoc y hi sau khi n cht chit touchi, khng quan st thy s bt thng trong mu hay cc ch s sinh ha. Do , tc gi khuyn co cht chit touchi c th c s dng n nh ng huyt ngi bnh tiu ng trong thi gian di. Chnh v nhng l do trn m C quan Qun l Thc phm v Dc phm ca M v Hi Bo v sc kho ca Nht Bn khuyn co s dng sn phm c cha cht AGIs t u tng ln men nh mt thc phm chc nng phng chng bnh tiu ng. I.7. Tnh hnh nghin cu v th nghim cht km hm glucosidaza t u tng ln men Vit Nam Vit Nam c khong 4,5 triu ngi mc bnh tiu ng trong c ti 65% ngi pht hin bnh mun. D tnh n nm 2025 c khong 7 triu ngi mc bnh tiu ng, trong i tho ng type 2 chim trn 90s Nhng nm gn y, trn th trng c rt nhiu loi thc phm chc nng, trong loi thc phm chc nng dng cho ngi bnh tiu ng c gi t hn trm nghn n vi triu nh: vin gip xc Tianshi, to xon Tianshi, oyster Plus, tinh du hoa anh tho, ng trng h tho

c by bn v qung co c kh nng cha khi bnh. Tuy nhin, do mi ph bin Vit Nam nn vic qun l loi sn phm ny cn nhiu bt cp. Thm vo , li dng s thiu hiu bit ca ngi tiu dng, mt s nh phn phi qung co qu tc dng khin nhiu ngi hiu sai v dng sai thc phm chc nng. Hin nay cha bnh tiu ng ngi ta thng dng cc nhm thuc l sulphonylurea v biguanidses vi mc ch lm tng hiu lc tc dng ca insulin, tng tnh thm mng t bo vi glucose. Ngoi ra, cc thuc dng km hm glucosidaza cng c s dng nhiu, nhng cc thuc ny ch yu thng c ch nh cho nhng ngi mc bnh, trong khi xu hng chung l tm cch phng bnh. Trong nhng nm gn y c mt s cng trnh nghin cu v sn xut thc phm chc nng cho ngi bnh tiu ng nh rau m tan, chanh leo ha tan c cha xylooligosaccharide (XOS) [9]; tr du ung lin, tr du ti lc cha 1deoxynojirimicin (DNJ) [2], ng chc nng fructooligosaccharide (FOS) t tht qu chui [7]. Cng ngh sn xut thc phm chc nng cha protein giu crom hu c t nm men Saccharomyces cerevisiae c tc dng phng chng bnh tiu ng typ 2 cng v ang c nghin cu [5]. Trng Hng Lan v cng s [3] xc nh c mt s nguyn liu giu cht chc nng (i mch, vng trng, mp ng) s dng cho sn xut thc n c tc dng lm gim hm lng ng trong mu. Cc nghin cu v u tng ln men mi ch tp trung vo to sn phm dinh dng natto (Nguyn Thy Chu v cng s) hoc to thc phm chc nng giu nattokinaza (Nguyn La Anh) hay to sn phm chc nng cha peptit chuyn ha angiotensin (ACEIP) [2]. Nguyn Vn Nguyn nghin cu cng ngh chit xut touchi t u tng bng phng php ln men vi sinh vt. Tuy nhin, nghin cu ny mi ch dng mc thm d cng ngh chit xut touchi m cha c iu kin nghin cu su hn v cc yu t khc ca cng ngh ln men v cc phng php tch, thu hi cht km hm - glucosidaza, cha th nghim trn ng vt v trn ngi v vy cha th ng dng vo thc tin.

II. NI DUNG NGHIN CU Ni dung 1: Phn lp, tuyn chn cc chng nm mc c hot tnh km hm glucosidaza v nghin cu to ch phm ging mc khi ng cho ln men u tng. 1.1. Nghin cu tng quan, xc nh ni dung v phng php nghin cu. 1.2. Phn lp cc chng nm mc ln men u tng to cht km hm glucosidaza. 1.3. Tuyn chn 2-3 chng nm mc c kh nng ln men to cht km hm glucosidaza v nghin cu c im hnh thi ca cc chng nm mc tuyn chn. .

1.4. S dng k thut sinh hc phn t nh tn chng nm mc tuyn chn bng phng php khuych i on ADN cha vng ITS1 - 5,8S- ITS2 (n mc loi). 1.5. Nghin cu xc nh mt s yu t thch hp (thnh phn mi trng, m mi trng, t l ging mc, nhit , thi gian, phng php sy...) cho qu trnh nui cy to ch phm nm mc cho ln men u tng to cht km hm - glucosidaza. Ni dung 2: Nghin cu quy trnh cng ngh ln men u tng to cht km hm - glucosidaza. 2.1. Nghin cu nh hng ca cc loi u tng n kh nng to cht km hm glucosidaza ca chng nm mc tuyn chn. 2.2. Kho st kh nng ln men to cht km hm -glucosidaza ca chng nm mc la chn trn mi trng u tng. 2.3. Nghin cu nh hng ca cc yu t cng ngh ti qu trnh ln men b mt to cht km hm glucosidaza ca nm mc.

- Qa trnh ln men s cp: Nghin cu nh hng ca nhit , m, pH, thi gian nui cy, t l ging, thng kh n s pht trin ca nm mc v kh nng to cht km hm - glucosidaza. - Qu trnh ln men th cp: + Kho st nng mui b sung vo u tng ln men. + Kho st nhit ln men to sn phm c hot tnh km hm -glucosidaza. + Kho st thi gian chn to sn phm c hot tnh km hm -glucosidaza. 2.4. Ti u ha cc yu t nh hng n hot tnh km hm -glucosidaza bng phng php quy hoch thc nghim. Ni dung 3: Nghin cu quy trnh tch chit, thu nhn cht km hm glucosidaza t u tng ln men 3.1. Nghin cu la chn dung mi thch hp cho qu trnh chit xut. 3.2. Nghin cu xc nh cc iu kin thch hp cho qu trnh thu nhn cht km hm -glucosidaza bng phng php ly tm (vn tc, nhit , thi gian ly tm). 3.3. La chn cc phng php thu nhn cht km hm -glucosidaza: phng php sc k, s dng mt s dung mi hu c phn cc... 3.4. Nghin cu la chn phng php sy sn phm v xc nh cc iu kin ti u ca qu trnh sy ch phm to ra duy tr c hot tnh km hm - glucosidaza. 3.5. Xc nh tnh n nh ca cht km hm -glucosidaza: bao b ng gi, nhit , nh sng, thi gian bo qun. Ni dung 4: Sn xut th cht km hm glucosidaza quy m 50 kg nguyn liu/m v ng dng cho sn xut mt s thc phm chc nng.

4.1. Hon thin cc thng s cng ngh ca qui trnh sn xut v sn xut th cht km hm - glucosidase qui m 50 kg nguyn liu/m ng dng cho sn xut thc phm chc nng. 4.2. Nghin cu nh hng ca cht km hm -glucosidaza n mt s enzym tiu ha nh: trypsin, chymotrypsin, pepsin, lipaza. 4.3. Nghin cu ng dng cht km hm - glucosidaza cho sn xut vin thc phm chc nng. 4.3.1. Nghin cu cng thc phi ch to ch phm vin nn cha cht km hm - glucosidaza. 4.3.2. Xc nh tnh n nh ca sn phm: bao b ng gi, nhit , nh sng, thi gian bo qun. 4.3.3. xut quy trnh cng ngh sn xut vin thc phm chc nng cha cht km hm - glucosidaza. 4.4. Nghin cu ng dng cht km hm - glucosidaza cho sn xut tr ho tan. 4.4.1. Nghin cu cng thc phi ch to sn phm tr ha tan cha cht km hm - glucosidaza. 4.4.2. Xc nh tnh n nh ca sn phm: bao b ng gi, nhit , nh sng, thi gian bo qun. 4.4.3. xut quy trnh cng ngh sn xut tr ha tan.

4.5. Nghin cu ng dng cht km hm - glucosidaza cho sn xut bt dinh dng chc nng. 4.5.1. Nghin cu t l phi hp cht km hm - glucosidaza trong cng thc sn xut bt chc nng. 4.5.2. Xc nh tnh n nh ca sn phm: bao b ng gi, nhit , nh sng, thi gian bo qun. 4.5.3. xut quy trnh cng ngh sn xut bt chc nng cha cht km hm glucosidaza. Ni dung 5: Nghin cu th nghim cht km hm -glucosidaza. Xy dng tiu chun c s v cng b cht lng sn phm ti Cc An ton V sinh Thc phm - B Y t. 5.1. nh gi cht lng sn phm: cm quan, hot tnh km hm glucosidaza IC50, hm lng protein, lipit, m. 5.2. nh gi tnh an ton ca sn phm: phn tch cc ch tiu l, ha, vi sinh vt, kim loi nng, c cht ca sn phm. 5.3. Xc nh c tnh cp ca cht km hm glucosidaza trn ng vt. 5.4. Xc nh c tnh bn trng din ca cht km hm glucosidaza trn ng vt. 5.5. Th hiu qu gim ng huyt ca cht km hm glucosidaza trn ng vt.

5.6. Nghin cu th nghim lm sng tc dng ca cht km hm glucosidaza trn cc i tng tiu ng: - Sng lc i tng. - Cho i tng dng sn phm v theo di ch n, biu hin sc khe trong sut qu trnh can thip. - nh gi hiu qu gim ng mu trn bnh nhn tiu ng. - nh gi nh hng ca sn phm n cc ch s chc nng gan, thn (GOT, GPT, Ure..) trn bnh nhn tiu ng. - nh gi nh hng ca sn phm n cc ch s chc nng m mu (Cholesterol, triglycerit, HDL- Cholesterol, LDL- Cholesterol). 5.7. Xy dng tiu chun c s cho 03 sn phm thc phm chc nng. 5.8. Cng b cht lng ti Cc An ton V sinh Thc phm - B Y t trn c s cc s liu phn tch v thnh phn dinh dng, cc cht chc nng c hot tnh sinh hc ca sn phm, cc kt qu kho nghim trn ng vt v kt qu nghin cu lm sng trn ngi. III. PHNG PHP NGHIN CU Phng php xc nh hot tnh km hm -glucosidaza s c s dng trong tt c ni dung nghin cu ca ti. La chn ngun -glucosidaza: Cc ngun -glucosidaza chnh do hng Sigma cung cp l: -glucosidaza t Bacillus stearothermophilus, t go, t Saccharomyces

cerevisiae v intestinal acetone powder t chut. Mt s cng trnh nghin cu lm sng t rng hot tnh km hm -glucosidaza khc nhau khi s dng -glucosidaza c ngun gc khc nhau. Vi mc tiu ca ti l to c cht km hm -glucosidaza v ng dng cho bnh nhn tiu ng nn chng ti la chn -glucosidaza t chut cho cc th nghim nh tnh v nh lng hot tnh km hm -glucosidaza. C hai phng php nh lng thng c s dng xc nh hot tnh km hm -glucosidaza: Phng php 1: tin hnh theo phng php ca Yamaki v Mori (2006). Nguyn l: 4-nitrophenyl -D-glucopyranoside -glucosidaza -D-glucose + 4-nitrophenol Hot tnh km hm -glucosidaza c xc nh bng o s hnh thnh 4-nitrophenol bi -glucosidaza sau khi phn ng vi 4-nitrophenyl -D-glucopyranoside (4-PNP) Phng php 2: tin hnh theo phng php ca Miwa v cng s. Nguyn l Sucrose -glucosidaza glucose + fructose Hot tnh km hm -glucosidaza c xc nh bng lng glucose gii phng bng kit thng mi (glucose test wako) s dng ngun c cht sucrose. ti la chn phng php 1 do ha cht dng trong phng php ny d t mua hn. 1. Phng php phn lp, tuyn chn cc chng nm mc c hot tnh km hm -glucosidaza v nghin cu to ch phm ging mc khi ng cho ln men u tng. - ti s dng cc phng php vi sinh truyn thng phn lp cc chng nm mc. - Tuyn chn mt s chng nm mc c hot tnh km hm -glucosidaza theo phng php ca Yamaki and Mori. - S dng k thut sinh hc phn t nh tn chng nm mc tuyn chn bng phng php khuyn i on ADN cha vng ITS1 - 5,8S- ITS2. - S dng phng php vi sinh xc nh thnh phn, m mi trng, t l ging mc, nhit , thi gian thch hp cho to ch phm ging mc khi ng duy tr c

s lng bo t v hot lc cao, n nh. 2. Phng php nghin cu cng ngh ln men u tng to cht km hm glucosidaza. - S dng phng php x l nguyn liu v ln men b mt chng nm mc theo phng php ca Jing Chen v cng s. - X l s liu theo phng php ton hc thng k bng phn mm ANOVA. - S dng phng php quy hoch thc nghim ti u ha cc yu t nh hng n qu trnh sinh tng hp cht km hm -glucosidaza. 3. Phng php nghin cu tch chit, thu nhn cht km hm -glucosidaza t u tng ln men - Phng php tch chit s dng cc dung mi phn cc thng dng. - Phng php thu nhn s dng cc phng php hin i nh: phng php sc k, s dng mt s dung mi hu c phn cc. - Thm d mt s phng php sy cho to cht km hm -glucosidaza dng bt. - nh gi cht km hm -glucosidaza sau bo qun. 4. Phng php nghin cu cng ngh sn xut th cht km hm glucosidaza quy m 50 kg nguyn liu/m v ng dng cho sn xut mt s thc phm chc nng. - Sn xut th cht km hm - glucosidaza qui m 50 kg nguyn liu/m ng dng cho sn xut thc phm chc nng da trn cc kt qu nghin cu ni dung 2 v 3. - Sn xut 20 kg cht km hm glucosidaza da trn quy trnh cng ngh t c.

- nh gi nh hng ca cht km hm -glucosidaza n mt s enzym tiu ha nh: trypsin, chymotrypsin, pepsin, lipaza bng phng php th km hm enzym. - La chn cc ph gia thch hp c s dng trong thc phm chc nng to cc sn phm cha cht km hm - glucosidaza. - nh gi tnh n nh ca sn phm sau bo qun. 5. Phng php th hot tnh ca cht km hm -glucosidaza. Xy dng tiu chun c s v cng b cht lng sn phm ti Cc An ton V sinh Thc phm - B Y t. - nh gi cht lng v tnh an ton ca sn phm: s dng cc phng php vi sinh, ha sinh nh gi sn phm, c th l: o Xc nh m ton phn bng phng php Kjelhdan. o Xc nh hm lng cht bo bng phng php Sochlex. o Xc nh hm lng hydratcacbon theo phng php Betrand. o Phng php xc nh gi tr IC50 theo Rammohan S., 2008. o Phng php nh gi cm quan: theo tiu chun Vit Nam 3215-79. o Xc nh tng s vi sinh vt hiu kh theo TCVN 4884:2005, tng s nm men, nm mc theo TCVN 5166-90, E. Coli theo TCVN 6846-2001, Coliforms theo TCVN 4882:2001, B. cereus theo TCVN 4992:2005, C. perfringens theo TCVN 4991-2005. o Xc nh c t aflatoxin theo TCVN 6685 : 2000 (ISO 14501 : 1998) hoc AOAC. o Xc nh c tnh cp ca sn phm theo phng php ca Trung m (theo quyt nh 371 BYT/Q ca B Y t): nh gi ch s LD trn ng vt. o Xc nh c tnh bn trng din ca sn phm theo hng dn kho st c tnh ca thuc (quyt nh 371 BYT/Q ca B Y t).

o Th hiu qu gim ng huyt ca sn phm trn ng vt gy i tho ng bng alloxan monohydrat. o Phng php th nghim lm sng: th nghim trn 30- 40 bnh nhn tiu ng. Cho i tng dng sn phm v theo di ch n, biu hin ca sc khe ca i tng trong sut qu trnh can thip. nh gi tnh trng trc v sau khi dng sn phm. nh gi ch s ng huyt, cc ch s chc nng gan, thn (GOT, GPT, Ure..), cc ch s chc nng m mu (Cholesterol, triglycerit, HDL- Cholesterol, LDL- Cholesterol) trc v sau khi dng sn phm. - Xy dng tiu chun c s cho sn phm thc phm chc nng h tr lm gim lng ng trong mu. - Cng b cht lng ti Cc An ton V sinh Thc phm - B Y t.

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