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    Us 2009004642 A 1

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    Chus Otto Bellosta

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    © All Rights Reserved
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    co») United States c2) Patent Application Publication (o) Pub. No.: US 2009/0004642 Al fey 76) Magaletta et al. IN VITRO METHOD FOR THE DETERMINATION OF GLYCEMIC INDEX OF FOOD PRODUCTS Inventors: Robert L. Magaletta, Branchburg, [NJ (US); Suzanne N. DiCataldo, Florham Park, NJ (US) Correspondence Address: PITCHE 'N TABIN & FLANNERY 120 S. LASALLE STREET, SUITE 1600 cH Appl. No. File: ‘AGO, IL 60603-3406 (US) 12144,086 Jun, 23, 2008, Test Food Sample fe US 2009000866: Al (43) Pub, Date: Jan. 1, 2009 Related US. Application Data (60) Provisional application No, 61/007,534, filed on Jun, 28, 2007 Publication Classification Gl) Inc. cng 128 (2006.01) (2) US.C 4358/4 7) ABSTRACT Te present invention provides an in vitro method for deter sining the glycemic index values for various food products The present invention provides an accurate and inexpensive in vitro method for determining the glycemic index of wide variety ofboth food ingredients and finished food products Coarse Grind Fine Grind Enzyme Digestion Reactions HPLC (determine glucose, fructose, galactose, lactose, sucrose, & mattitol Collect Enzyme Digestion ‘Sample after Fixed Time Period & Stop Enzymatic Determine Protein & Fat Content of Test Food Sample In vitro Gl Predictive equation or predictive technique Multivariate Data Analysis Patent Application Publication —_ Jan. 1, 2009 Sheet 1 of 6 US 2009/0004642 Al Sample & guar gum Figure 1 (Prior Art) Sodium Acetate & Enzyme Mixture Incubate at 375 Aliquot 1 Measure glucose (G,,) Sample at 20 mi Sample at 120 min 30 min at 10 Measure glucose (Bo9) Ethanol; centrifuge Aliquot 2 Measure total glucose (TG) |Add to acetic acid; add Patent Application Publication —_ Jan. 1, 2009 Sheet 2 of 6 US 2009/0004642 Al Figure 2 Test Food Sample Determine Protein Coarse Grind & Fat Content of Test Food Sample In vitro Enzyme Digestion Gl Collect Enzyme Digestion Sample after Fixed Time Period & Stop Enzymatic Reactions HPLC (determine glucose, fructose, galactose, lactose, sucrose, & maltitol Predictive equation or predictive technique Multivariate Data Analysis Patent Application Publication Jan. 1, 2009 Sheet 3 of 6 US 2009/0004642 Al « 3 8 2 2 g 3 2 giz s 3 ois - i Time (minutes) Figure 3 1204 Time (minutes) Patent Application Publication Jan. 1, 2009 Sheet 4 of 6 US 2009/0004642 Al Figure 4 A 120 100 80 60 In vivo Gt 0 20 40 60 80 100 120 In-vitro Gl (Inventive Method) we 0 02 04 0.6 0.8 1 12 RAGI% Available Carbohydrate 0 20 40 60 80 100 120 RAG (g glucose released/100g food) Patent Application Publication Jan. 1, 2009 Sheet 5 of 6 US 2009/0004642 Al 120 Figure 5 10 | A _ 80 3 & eo 3 g 0 20 0 0 20 40 60 80 100, 120 inevitro Gi (Inventive Method) 0 02 4 06 08 1 12 RAG/% Available Carbohydrate 20 40 60 80 100 120 RAG (g glucose released/100g food) Patent Application Publication —_ Jan. 1, 2009 Sheet 6 of 6 US 2009/0004642 Al Figure 6 120 100 804 In-vivo GI 8 0 Sa a 0 20 40 60 80 100 120 In-vitro GI (Inventive Method) US 2009/0004642 AI INVITRO METHOD FOR THE DETERMINATION OF GLYCEMIC INDEX OF FOOD PRODUCTS RELATED APPLICATIONS [0001] This application is based on, and claims benefit of, US. Provisional Application Ser. No, 60/____ with an ‘offective date of Jun. 28, 2007, as well as US. patent appli- «ation Ser.No. 11/770,36, filed on Jun. 28, 2007, which was requested to be converted to Provisional Application Serial [Number 6G/on tun 17,2008, under 35 US.C. $1536) all ‘of which applications are hereby incorporated by reference. FIELD OF THE INVENTION [0002] The present invention relates to an in vitro method for determining the glycemic index (Gl) values for various food products. The present invention provides an accurate _andinexpensve in vitro method for determining the glycemic index ofa wide variety ofboth food ingredients and finished food products BACKGROUND OF THE INVENTION 10003] Imerestin the lyeemie index of food poets as significantly ierease in ect years. The glycemic index is an indicator of the eave glycemic response to dietary ca- boliydatesnagiven food prodet upontuman digestonand allows foods to be ranked base of the rate of release ad shnorption of carbohydrates, In effect, the glycemic index allows identiaton of so- stop the hydrolysis; this sample is used to determine Gy (i, the slucose released after 20 minutes; als refered toas “rapidly available glucose" or RAG) using HPLC. The remainder of the Sample is maintained inthe 37°C. hath for an additional 100 minutes at whic time a second aliquot ofthe sample is removed and add to absolute ethanol with mixing 0 stop the hydrolysis: this samples used to determine Gy Ge the slucose released after 120 minutes)using HPLC. Tremaine ‘ero the sample then eated silted in FIG. by the ‘dition of additional enzymes an then exposure to tempera- tures upto 100° C. to force complet hydrolysis and, thus, ‘determine the total glucose inthe sample. 0010] Comparing foods for which the glycemic index has been measured using the in vivo test method, the Gay or rapidly available glucose values determined sing the Fnglyst method can be eorelated withthe in vivo giveemie index values of known fonds. Using such corelation coefficients, the Bnglyst method can be use to estimate glycemic index ‘aes for test foes 10011] Then vitro Englyst method has, however, met with ‘considerable eriticism, especially from proponents of the in Vivo glycemic index method. For example, Garstti et al, J Am. Coll Nar, 24, 441-487 (2008) wsedthe Enlyst method to.estimate glycemic index values (i. rapidly available pha- ‘ose values) and then compared them with in vivo determined values for food products (ie, cookies) having glycemic indexes in the range of about 35 t0 60. As shown in FIG, 20f Garsett etal (and incorporateby reference herein) scatter plot of in vivo detemined glycemic index values versus rap- ily avilable glucose values (ie, a determined by the Englyst method) yielded a scatter plot with an R® value of 10.25, indicating thatthe in vitro method had litle predictive valve Jan. 1, 2009 [012] Brand-Miller eta, Eur J. li, Nute, 58, 700-701 (2004), determined the in vivo glyeemic index values for commercially available foods (one breakfast cereal and two snack bars) reportedly having low glycemic index values (i.e, 1555) as determined by the Englyst in vitr test method! According to Brand-Mille etal, the three food products aa ‘mean in vivo glycemic index values (2sem) of 7533, 6825, ‘and 6545, respectively, and thus could nat be classifieds law alyeemie index food products. These authors concluded that assumptions that the “laborious tsk of in vivo testing is no Tonger necessary for measuring the Gi values for food” are simply incorrect and “strongly advise agaist the use of any in vitro method for producing GI values for food labeling pur- poses." Finally, the authors “unge food manufactures undertake Gl testing only with experience laboratories using the standardized in vivo method” {0013} Clearly there remains 2 need inthe art for an aceu- rate, precise, and reproducible in vitro method for determin- ing the glycemic index values of food products. Moreover, there remains a noed for such a method which can be applied ‘walarge variety of food products, including solid and liquid food products. The present invention provides such in vitro testing methods SUMMARY OF THE INVENTION {0014} ‘Thepresentinvention provides an in vitro analytical method which allows the improved prediction ofthe slyee- iindex of ood product rom the levels of protein fa and sugary/sugar alcohols generated using a simlated digestion of the food product with enzymes. Curent elycemi index determination methods generally requite human subjects and tare bath costly and time consuming. The inventive metho is capable of determining the elycemic index of up to about 1S food samples in one day by one analyst at significantly reduced costs relative tothe tational method using human subjects. Glycemic index values determined usingthe present inventive method show very high correlation with results obiained using the trattional test using human subjects “Moreover this present inventive method can be used with a wie variety of food products, including solid food products aswell a semi-solid fod prodcts(e., yogurt and the ike) and iqud food products (eg, beverages and the lik). [0013] The present invention provides an in vitro method {or detemaining glycemic index fora test food product, said method comprising (0016) (1) determining protein content and fat content of the test food product; [0017] (2) providing the tet food product in an essentially homogeneous and finely divided state to provide atest food sample: {0018} (3) digesting an amount of the tet food sample in the essentially homogeneous and finely divided state su cient to provide a standardized amount of axailalecarbohy- dratewitha mixture of digestive enzymes fora fied period of time to provide a digested sample: {0019} (4)trating the digested sample in is entirety imme diately upon the fixed period of ime to stop enzymatic reac- tions; {0020] (5) determining amountsof glucose andat least two sugars or sugar alcohols selected from the group of fructose, salactose, lactose, sucrose, and malitol in the digestive sample from step (4); and [0021] (6) calculating the glycemic index ofthe test food product from data obtained in steps (1) and (S) far the test US 2009/0004642 AI food product using a predictive equation ora predictive tech- nigue derived from multivariate analysis of calibration data forcalibeation samples wherein the calibration datas of same type and obtained in same manneras the data forthe test food pret determined in steps (1) and (5) and wheeein the ‘calibration samples have known in vivo glycemic index val uss, 10022 ‘The present invention also provides an in vitro method for determining glycemic index for atest food prod- uct, sad method comprising 10023] (1) determining protein content and fat content of the test food prxlct; 0024] (2) providing the test food proiuct in an essentially homogeneous and finely divided state to provide atest food sample wherein the test food product is ground at essentially liquid nitrogen temperatures 10025] (3) digesting an amount of tho test foo sample in the essentially homogeneous and finely divided tate suf ml incre Com GaN OTT OL Pron) Sosy Osis 686 Shoot i) Theta nosis tek Sho Ghesee ps) 364STETHTSsTUE AS NOL Fro) 4820" STIG -e02— ShM0L Laceses) SESE MIT “ost Gaucowe() 7361S sao 001 Nao) nD SASS ‘om US 2009/0004642 AI ‘Ths, the equation wsed to estimate the in vito glycemic index using the MLR method would be: (Gr 630s0014-0.574913 Proteins) — 142 Fa) 436797478 Gent) — 482.5341 Fraoat = 191.38 Lasos)= 497.3615 Glasto) ~ 2582 Matt. ‘The relevant statistical parameters for this model were as {allows Summary of Fit 0161) e anaes Rag) ssest Root Mean Sg Eur fs MeanofRespoase 852981 Osc or San Wet) u Analysis of Variance: [0162] Scone DE SumefSgurer Mea Sguce Fao Moat] mwa aso ais Emr 26 45573 Yad BaF Cal 33 tsemaTs Sino los wee eperinesally decane usage pve ting te mls eerie ol ‘iio values ken om “otemstonal bef yeni iniex and ly fai a ae” Arn). Cin Nie 16595202). Five vlc aken om vey. Nr Res Rv. 162), 16891 C00 "Mao ses te om: enense a, Ez App Pa 8 45- 468 205, A scatter plot ofthis data obtained using the neural net as ‘eure fitisg procedure is shown in FIG. 48. A R® value of {097 isobiaind using this method. The standard deviation for thei vio method (based on duplicate determinations of 4 total samples) was 2108 GI units or 22.33 porent relative. ‘Thus, this method appears to have very high precision and high preticive value EXAMPLES 10168] fer fing te original aplication, work was con- tinned andadtional data points have been ded and certain ageurate and precise in vitro method. As noted in Example 2 above the pH doring the enzyme digestion step in Example 2 ‘was too low. The proper pl value for the enzyme digestion step should be about $; other than that modification, the samples were treated inthe same maner a in Example 2 ‘Thus, 31 of the samples used in Example 2 were rerun to ‘obiain data atthe more optimal enzyme digestion pH. Adi- tionally, 34 new samples have been added to thererun sample from Example 2 to bring the total number of samples to 65) This in vitro method (current as ofthe date of this continua- tion-in-part application) isbased onsamplesenzyme digested atthe more optimal pH value and is preseated heen. OF course, as one skilled in the art realizes, the accuracy and precision of this method is also ikely wo be increased even fther 2s additional in vivo data fr calibration samples and! brstandards hocomes available ands incorporated into the in vitro model during the calibration procedure, [0169] The following model parameters were obtained using the MLR method: Tem Vie Str tRio_Pob> one Darivate2uNewe aor Protan 0) Shoat aaias “koe Shoot Ft) Chustis “oon 1522 Shoot Ghevee es) LON} snomas3 3039 Sao tre) SLsinis axaI6Ns Sivot (Ghose (6) =TR16OK2-—S48H6I 3.33 OMS bustisyertin (4-826) “Thus, the equation used to estimate the in vitro glycemic index using the MLR method and the expanded data set would be: = 2676860967251 Pin) — 34715 Fa) +611.40013 Gon) + SO19SUL4 Laos) + 169.05988 Maoh) — 15 10614) ~0.05718 Protas) ~ 267, The relevant statistical parameters for this model were as follows ‘Summary of Fit [0170] = Rag) sn oot Meas Sgure oe ‘55889 US 2009/0004642 Al Jan. 1, 2009 12 -contined continued Meat pane wa = vite tsa Si We) s * = s Fao oan Shc -tisrast Fate 00) Das Analysis of Variance: oo) tiga Gancoe(%) Dues 0171) HI-Matit si9s90 Gr menen oe Sows DF _souctSyuies esse F8sie As noted in Example 2, the neural net method! doesnot pro- Nos aaalsase a9 sisi __videa simple prediction equation. Rather, his method pro- Eerste 3659 FoF vides a series of parameter weights anda network topology oe Which ean then be used to obiin rected results using a eee <0 suitable software program (inthis ease the JMP Statistica Softwar) The fit obained using this method is illstated {0172} The folowing model parameters were obtsined graphically in FIG. 4A. The relevant parameters fr this using the PLS mato with ive latent variables model were as fellows: Te Tae Sei roe “ast ened H fe vet ey fot im Neer Pe = foe) 2003 Mitton 0 tance Bhonn Come Cee 9 ae) “eiaton Results: Ths, the equation sed to estimate the in vite glyeenie index using te PLS metiod woul be: 10176) = 1620409- L818 Prin) — spenTe 028185 Fa) 60.784 Ghee) Se S299 Faces) 2817 Lene) ~ 5 Be 61.2107 Ga ~H6888 Mab, 2» Conepes Bot ° ‘come! Wane Ta Bet » Stokes Fla The relevant statistical parameters for this model were as o Resale follows y Stas SEO Sommary of it G_aeans SIRO _ aor On 0173) = coo {0177] The resulting model parameters were then sed t0 Rad tsaoxn Caleulate tein vit glycemic index values sing each ofthe Root Mat gue Er Sis data fiting methods asin Example 2. The results from the Mea of epee sia following produets, about 33 more than in Example 2, were Oecatins o Sun Wet) 8 [0174] The following model parameter weights were ‘obiained using the neural net method: used to calculate correlation parameters fr each model. The resulting in vitro glycemic index values determined using eachof the data fiting models or methods with the expanded dataset are compared tothe in vivo glycemic index values: Neural Net Table I (0175) asin Gh Paste Weight vie sere InyneGlt_PLS_MUR_NN sinew ‘anisiares “all Mix Bled om om US 2009/0004642 Al Jan. 1, 2009 2B -contnned -continaed iinet nasal = owes pis win ww SE yw ms MR . Xr ws ww Taine = ee Mat ees Tali ot hog on 2 See Tha i eds 3 X22 Trvaonbaecwegainanly docmind ana pace ho aera BB BERAGSIMECIS fan imei able oflyemicindr al hy Sei et e sR ‘semic oad values,” Am. J, Clin, Nutr, 76, 556 (2002), - Ses wee 66 BERSE Si at in ample Friis Coo 7 77) ahaa aes ee mrt Fri Fill Cale 2 Gg os BARISEROM OSS ae dein or Pt tear SSMS cececaemn andres wh art lc Sie Cc e® de FACS un an naa sic of gcc nan eyes Bae Cer tetera I ln Nat $36 3 ten eed, isi okie S50 $957 Betray tm nein Ee Baar Chip Sa a ¢ RSeisckret gunn Wk We Cnc ne ot thwinvoa rut Fees ge of ot ie eeaeaecean Sf A tamales tee nc camo? Beaks Coral Ge SK 557s ben om Liven, Ne Res Rew 1(2), 163-9 (20), Briss Cereal 2 a yee ‘Tn vivo values taken frocn: Jenjens & al, Eur J. App. Physiol. 88, 459-465. Bia Col om a 8 a oO as SET 2 tom sutra detain wig ‘Muli-irai nstaut Outre! 7 maT curve fitting procedure is shown in FIG. $A. A R? value of toi il SG a 0bisobtained tsingthismetiod The standard deviation for Cage Chee in Ps 2 5S 1 theinvitomethod (ose on duplicate determinations of 6 fou Fk 16% 18 tal samples) was 4 GT units or 46 percent eave, Thus, Baie Pos Si iG this method ppears to have very high prvision and high feng Bi 22 predictive vue Bens Bo? 3% 8 4 [0179) Theinvitometiadcanthenbeusedtocleuitethe ere Bs 393 init Gl ale fora enknown fond prods or food ing Energy bard 3 3 ient by simply carrying out the method described herein for Coeecutet SiS St hesampleprepartion and analysand using the data andthe Wasle Milk 2 ee desired predictive equation determined from the calibration aie 4 aul Ofcoune, as demonstrated by comparing Examples 2 rma ee? S93 theclibeatonreslisand preditive equation data il Cary aa i eelpiy spearpser sam teats mn ot Wet et an Gate stand unknown sample ae analyzed (specially a meetin 7 7h repanto the enzyme digestion sep). However, a long asthe Saeead TT same procedre is viet forall samples, versions in the Smee Tao alysis procedure donot appear to signify eft the Pre 5% So prodetve ability ofthe present method even wen the pre- Foy Moe Bate Fe) Th 73 Gitive equations ifr widely This, we believe, demon- asi aa Min 535 52. staesherobusines of his ia vito method. Asthesize ofthe Rice, Brown, Long Gniin 2 2 calibration data set is increased, either in number of samples ie We Lo Gin St & andor agcuracy ofthe in vivo G values sed the accra feeinicun sick 80m 52 andprecision othe method is expeted wo inrease ae © © 2 —_|MIs0) _Asnotedabove, Garseti etal provideaseaterplot Gres 42 48 ith an R? value of 0.25 forthe in vitro Englyst method, thus Oran ssw indicating that this method had little predictive value. For Peaees, Came 45 45 45° further comparison purposes, the rapidly available glucose a 2 © @AGesonpsal ogame poe lta in20 ita Ame 2 3 2 jong) a obtained sing he calulionechigues wed at Cae Yow She Ree) Say jintheEnglystmetiod(., only based on he glucose lease as SSS Sater 20 minutes digestion) on the data fom Examples 2 and oon 2S. Swe separately coreltd to in vivo GI voles using the ee © 22 caloulations and parameters desert in then vitro Enalst ar gE G_ethod Ge, singonly glucose relesed afer 20 minutes) as Gtewe 529998 eta in Enlyst et al, Brit. Noe, 75, 327-337 (1996) Sane Sg se F1G-4B)and Englyst eal, Am. 1. Clin. Nu, 69, 48-454 Fricoee 2118 26 (1999) (FIG. 4C). Such seatter plots are shown in PIGS. 4B. to ff 4 ond 4C using the Example 2 dat and in FIGS. SB and SC using the Example 4 data. In FIGS, 4B and $B, the in vivo Gl US 2009/0004642 AI is ploted versus RAG/(% Available Carbohydrates) for the Example 2and Example 3 data, respectively: in FIGS. 4C and C; the in vivo Gl is pote versus RAG for the Example 2 ‘and Example 3 data, respectively. When such calculations ‘were carried out using Example 2 data, the R° values of the Engst et al. method inereased to about 041 and about 0.68 in FIGS. 4B and 4C, respectively; using the Example 3 data, the R? values of the Engst et l- method increase to about 10.56 and about 0.58 in FIGS. $B and $C, respectively. Although these represent improvements as compared 10 the ‘Garsett et al scatter pl, the preitve value ofthe method issill poor, [0181] We believe, as demonstrated by the R? valve of about 0.94 to about 0.97 (depending on the specific curve fitting method used andthe conditions der which the data is generated) forthe present inventive method, that forthe fist time, ani vitro method has been provided whieh canbe used to accurately and precisely determine glycemic index valves for a wide variety of food products andlor food ingredients ‘This methodean be used to easily, quickly and inexpensively ‘obtain accurate glycemic index values which compare favor ably in tems of accuracy tothe much more time consuming ‘and expensive human subjet in vivo testing curently rexom- mended. Moreover, the accuracy and precision ofthis method islikely tobe increased even further a additonal in vivo d for calibration samples andor standands becomes available and is incorporated int thei vitro model during the ealibra- tion procedure. This advance in the art should increase the usefulness of glycemic index and related values (e,wlyee- mic load values determined by multiplying the alyeemic index by the amount of carbohydrate in grams provided by @ food and dividing the total by 100) in human nutiton EXAMPLE 4 0182] ‘The samples used in Example 2 (using a less than ‘optimal pH value in the enzymatic reaction step) were rerun in Example 3 using 2 more optimal pH value, The data from Example for only the rerun Example 2 data set (i. exelud- ing the addtional samples added in Example 3) was used to generate a new set of the various curve fitting parameters ‘This examplewhich is based on 31 of the 34 samples of Example 2—effectvely illustrates the in vitro analysis of Example 2 withthe samples run at @ more optimal pH value ‘This allows for a more direct comparison ofthe corelation, ‘ceefficients or parameters from Example 2 and Example 3 ‘where only the numberof samples in each dataset was varied [0183] The following model parameters for this modified data set were obtained using the MLR method: im Vie Sinoe ¢ Ratio Prob ince game aaeST 9m) nor Protein) Sa7s169 UIs 578 Sime r09) Tasis6es —_nweses “S42 SimoL ‘Glico ST3sS S123 L180 Maid (5) Lanes S190835 28.00 (Guess KS 210 nso Prosi 100829) Jan. 1, 2009 ‘Thus, the equation used to estimate the in vit glycemic index using the MLR method: (= 3051296075769 Pras) — S69 Fag 575.0928 Gs) + 29961246 Lasua) + 147.0386 Mit) = 20.3994 Ghove4) D009 Prin) ~100825) ‘The relevant statistical parameters for this model were as follows: ‘Summary of Fit lo184) © oes Root Mea Squire Ener ‘303218 Mano Response sss ‘Obearcas or Stn Wat) 3 Analysis of Variance: (or8s} Souse DF SimofSquree Mea Se Ratio a Ener 931 obo F [0186] The following model parameters were obtained tsing the PLS method with five latent variables: “ken Vite nee aio Prot) Siow fee) Siusais Fructose (5) 268.4388 ieee) Saisie alco) ma Nati 05) 22713 ‘Thus, the equation used to estimate the in vit glycemic index using the PLS method would be: (= 48 43999-00376 Prin 8) — D915 Fa) 448.0721 Goa) 2654988 rates) 3878816 Lactose) — STHI6T Galas) - 42.3773 Mion, US 2009/0004642 AI 1s “The relevant statistical parameters for this model were as {allows Sommary of Fit 0187] © ome Rad) ans 0216 n Root Mean Sq Bor Mamet Repasse Oectins o Sun Ws) [0188] The following model parameter weights were ‘obtained using the neural net method: Neural Net Table 0189) Pare Wee vie is inenepe ora HisGucose) ass Hk Fics) paren Hu Laos) -honaieste Hi Guster 6) 0 260808955 Fils Mahtol 8) onss203198 Gites Sauztenses AAsmotedin Examples 2 and3, the neural net method! does not provide a simple prediction equation. Rather, this method provides a series of parameter weights and a network topol- ‘ogy which can then be used to obtain predicted results using ‘suitable software program (inthis case the JMP Statistical Software), The relevant parameters for this model were as follows Sosy Tia Noes v Overt Pens ot Mss teat By Comer Citron oot Results 0190) SE ey Tal N a0 Comers ABest Ccomered We Tha Best Jan. 1, 2009 continued 7 ‘Sukon Fir » Faldo inprove ° Resch Ma her y SE Sisal ROSE GRMN ALT come ‘The resulting model parameters were then used to caleulate the in vitro glycemic index values using each of the data fiting methods, The results from the following commercially availble products were used to calculate the correlation parameters. The resulting in vitro glycemic index values determined using each ofthe data fitting methods are com- pared to the in vivo glycemic index values: vio or Near Sample ms MIR Ne “Tal Mixed ‘Trl Mix led? Tr Mix Bled} Tr Mix lend ‘rl Mix led ‘ra Mic led eae Fr ill Cookie Fr Fld Croke 2 Pon rte Bar Sine Crater Bute Caster Chocolate Cone Babel Cake Chip ‘Whole Wit Cree Checolae Cg Cone ow Fabs nen Bur ney Bar? Ene Bu3 xen Bard Clarice App uce Ghose Stone Prato ito Galtone 6 @ » ie » B o » o a o o > 8 » is 6 we @ » » ‘Sethe eal ed in apes? and wee die dey wee a sto daa. ‘Stamos we tke fom Example 3 whe tiry wee derives pln tei, [0191] A scatter plot ofthis data obtained using the neural net curve fitting procedure is shown in FIG. 6. R? value of (0.96 is obtained using this method. Thus, again this method appears to have very high precision andhigh predictive valve ‘Theresuts of Examples 3 and 4, based on dierent umber of samples butall run under similar conditions, generally agree. [0192] As demonstrated in these Examples (especially in Examples 3 and 4) as the number of samples inereases, the values ofthe various coefficients ad other parameters used in the various curve fitting procedures are expocteto he refined tw ochieve the best fit for the data set, Additional samples US 2009/0004642 AI ‘aed to the data stare expected to increase the diversity of food products and better represent larger populations of food products ‘What is claimed is: 1.An in vitro method for determining glycemic index fora test food produet, said method comprising (1) determining protein content and fat content ofthe test Tod produ (2) providing the test food product in an essentially homo- eneous and finely divided state to provide a test food sample (@) digesting sufficient amount of the test food sample in the essentially homogenecus and finely divided state to provide a standardized amount of available carbohy- rate with a mixture of digestive eazymes for a fixed period of ime to provide a digested sample; (4) eating the digested sample init eatirely immediately ‘upon the fixed period of time to stop enzymatic eae ‘ions; (6) determining amounts of glucose and at last wo sugars ‘orsugar alcohols selected rom the group consisting of fuctose, galactose, lactose, suerose, and malttl inthe digestive sample from step (4); and (6 calculating the glycemic index ofthe test food product rom data obtained in steps (1) and (5) forthe test food product using a predictive equation or a predictive ‘method derived from multivariate analysis of calibration data fr calibration samples wherein the calibration data, js of sume ype and oblained in same manner asthe data forthe tes food product determined in steps (1) and (5) ‘and wherein the calibration samples have known in vivo alycemic index values 2. The in vitro method of claim 1, wherein the amounts of al ised sugars and sugars alcohols instep (4) are determined and used to caleulated the glycemic index in step (6). 3. The in vitro method of claim 1, wherein the test food praduct is obtained ia the essentially homogeneous and finely divided state by grinding the test food product at a tempera ture bel about 40°C. 4. The in vitro method of claim 2, wherein the test food pprxluct is obtained inthe essentially homogeneous and finely

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