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British Food Journal,Vol. 103 No. 4, 2001, pp. 281-290.

# MCB University Press, 0007-070X

The influence of b-cyclodextrinon goaty flavour

Characterization of synthetic inclusioncomplexes with capric acid and

caprylic acidMarcia M. Meier, Deisy A. Drunkler,

Marilde T. Bordignon Luiz, Roseane Fett and Bruno SzpoganiczFederal University of Santa Catarina, Brazil

Keywords Milk, Taste

Abstract Sensory analysis shows that the addition of 0.4 per cent (w/v) of the macrocyclic b-cyclodextrin (bCD) to goat's milk improves the flavour and reduces its goaty taste, due toencapsulation of molecules responsible of this flavour. In vitro studies of the inclusion of formedcomplexes between bCD and short-chain fatty acids, caprylic (C8 ) and capric acid (C10 ), werecarried out by differential scanning calorimetry (DSC) and proton magnetic resonance ( 1H NMR).Complex formation was confirmed by DSC, through the disappearance of the signs due to meltingof C10 and C8 after the encapsulation process. The 1H NMR technique shows that C10 is includedin the bCD cavity. The results suggest that bCD can encapsulate short-chain fatty acids, makingpossible its use for masking of these compounds in goat's milk and derivatives.

IntroductionGoat's milk is similar to cow's milk in its basic composition. One of the mostsignificant differences from cow's milk is found in the composition andstructure of fat in goat's milk ( Jenness, 1980). A number of flavoursobjectionable in beverage milk are associated with the lipid system. Researchhas shown that the proportion of short-chain (4 to 10 carbon) fatty acids ingoat's milk is approximately double that in cow's milk (Abo-Elnaga et al.,1985).

Flavour is a major attribute that influences the selection and consumption ofbeverages such as milk. With the increased consumption and popularity ofproducts of goat's milk, investigation and resolution of some of the problemshave become economically more important. The powerful attractiveness forgoat's milk comes from medical needs (not just desire) of people, especiallyinfants afflicted with various food intolerances, including sensitivity to cow'smilk proteins. Approximately 50 per cent of all patients sensitive to cow's milkprotein tolerate goat's milk proteins (Nogueira, 1990).

Cyclodextrins are macrocyclic compounds consisting of a variable numberof D-(+)-glucopyranose residues linked through alpha (1?4) bonds (Kollengodeand Hanna, 1987). The geometry of the cyclodextrin gives its the overall shapeof a truncated cone with the wider side formed by the secondary 2- and 3-hydroxyl groups and the narrower side by the primary 6-hydroxyl. The

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number of glucose units determines the dimensions of the cavity. Thecyclodextrins most widely used are alpha (aCD), beta (bCD) and gamma (gCD),containing respectively six, seven and eight glucose units (Linden and Lorient,1996). Cyclodextrins have the remarkable ability to form inclusion compoundswith various components, especially hydrophobic molecules like aliphaticamines, cyclic alcohols (Goldberg et al., 1997), linoleic acid and arachidonic acid(Divakar et al., 1991; Garcia-Carmona et al., 1995) and naproxen (Ganza-Gonzalez et al., 1994). They have been used for the retention of flavourcompounds (Lindner et al., 1981) or the masking of odours in various foods andfood components like enantiomers of limonene and carvone (Chang andReineccius, 1990) and naringin and limonin in grapefruit juice (Shaw andWilson, 1985). Toyoda et al. (1997) and Barrow et al. (1995) reported that bCDwas non-toxic to rats.

Various methods, such as ultraviolet (UV), infrared (IR), X-ray spectroscopyand chromatography are widely used to demonstrate the formation of aninclusion complex. The application of thermal analysis has also been reportedin this field (Fodor et al., 1997; Yilmaz et al., 1995). Nuclear magnetic resonance(NMR) spectroscopy has also been shown to be useful in the study inclusioncomplexes, since the 1H NMR chemical shifts (d) of both the interior protons ofbCD and the guest nuclei can be analysed to provide information about theinclusion mode, stoichiometry and binding affinity of the inclusion complexes(Wilson and Verrall, 1998).

In this work, the influence of bCD on removal of the goaty flavour from goat'smilk is described through sensory analysis, and the interactions between bCD andthe short-chain fatty acids, capric (C10) and caprylic acid (C8), are characterized invitro by 1H NMR and differential scanning calorimetry (DSC) techniques.

Materials and methodsMaterialsMilk sample: Pasteurized (658C for 30 mins) goat's milk, Saanen breed, wasobtained from the Dairy Plant of the Department of Food Science andTechnology, Federal University of Santa Catarina, FlorianoÂpolis, SC, Brazil.

ChemicalsbCD was kindly provided by Cereaster, the capric and caprylic acid werepurchased from Fluka and D2O was purchased from Sigma.

Sensory analysisThe trained panelists of both sexes were recruited from the Department of FoodScience and Technology, Federal University of Santa Catarina. The triangletest was used to show whether subjects can detect any difference betweensamples in order to determine the concentration of bCD necessary to minimizethe goaty flavour. Three samples of goat's milk were heated up to 50+/±18C.One sample was designated as the standard (no bCD) and the other two hadbCD incorporated. The results were expressed by the number of correct

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responses (correctly identified) and the number of total responses, in order todetermine whether the number of correct responses for the number tested isequal to or greater than the number indicated in the Table of the CriticalNumber (Minimum) of Correct Answers at 5, 1 and 0.1 per cent levels ofsignificance (Meilgaard et al., 1991).

Chemical compositionTotal solids were determined by drying a 5ml sample at 1058C at constantweight. Total protein (N66.38) was determined by the Kjeldahl method; ash,acidity and lactose content were determined according to standard proceduresoutlined in AOAC (1998). The pH measurement was determined by a MP 220meter (Mettler-Toledo). Fat was determined by the Gerber method (Kirk andSawyer, 1991). All samples were analysed in triplicate.

Statistical analysisResults of chemical composition were analysed using the StatgraphicsProgram (version 7.0 ± STSC, 1985-1991). When the variance analysis, by F test,detected a significant difference between the samples at 5 per cent probability,the Tukey test was applied.

Inclusion complex formation and characterizationSynthesis. bCD, C10 and C8 acids were weight out in a 1 : 1 molar ratio. Themixture was maintained under stirring in aqueous medium (5ml) during 6-7hours at about 408C. The product obtained was filtered and dried undervacuum. The dried sample was kept in a desiccator until analysed.

DSCThe inclusion complex sample (ca. 6mg) was weighed into a tared aluminumcapsule. A Shimadzu DSC-50 Differential Scanning Calorimeter was used forthermal transition analysis. The study was performed in the interval oftemperature of ±10 to 2508C for the complex with the C8 acid, and 20 to 2508Cfor the complex with the C10 acid. The operating circumstances for DSC

analysis were 108C min±1 heating rate and 50ml min±1 N2 flow.

1H NMR spectroscopy analysisThe 1H NMR spectra were obtained for D2O solutions with TMS as reference,and pD=10.4 by addition of KOD. A 200 MHz H1 and C13 Brucker AC-200FNMR spectrometer was used.

Results and discussionSensory analysisThe concentration of bCD necessary to minimize the goaty flavour wasdetermined by sensory analysis. The best results were obtained when theamount of bCD was 0.35 and 0.40 per cent (w/v). The results of triangle testswith these concentrations are given in Table I. A significant difference at the

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level of 0.1 per cent is observed. Additional comments from the panelistsindicated that no further change in colour and appearance of the milk wassensed with these concentrations of bCD. On the other hand the flavour hadundergone significant changes.

Chemical compositionTable II shows data for the chemical composition of the goat's milk sampleswith (treatment control) or without bCD (0.40 per cent w/v) (treatment test).

The results of this study for treatment control are in agreement with thosereported by several researchers (Bonassi et al., 1997; Gajewaska et al., 1996).

Statistical comparison of the control and test treatments demonstratedsignificative differences ( p<0.05) in the acidity, pH and lactose content.

Since the main characteristic of bCD is to include hydrophobic molecules inits cavity, the observed change in taste for goaty milk could be due to formationof inclusion complexes with molecules responsible of the flavour, such as theshort-chain fatty acids. This thought induced us to study the in vitro interactionbetween bCD and two short-chain fatty acids: capric and caprylic acid.

bCD/capric acid and bCD/caprylic acid inclusion complexesDSC results. The formation of an inclusion complex can be proved bycomparison of the thermoanalytical curves of the pure bCD, the pure guest

Table I.Triangle test resultsfor the evaluation ofstandard and testsamples with bCDconcentrations of 0.35and 0.40 per cent

bCD concentration(w/v)

Number ofrespondents (n)

Numberof correctresponses

Minimum number of correctresponses required for

significance at 0.1% level*

0.35 21 16 150.40 21 16 15

*Source: Meilgaard, Civille and Carr (1991)

Table II.Chemicalcharacteristics of goat'smilk in the control andtest treatment (0.40 percent bCD)*

Chemical characteristics Control Test

Total solids (% w/v) 11.35 a(1) 11.35 a

Protein (% w/v) 3.19 a(1) 3.12 a

Density at 15ëC 1.0310 a(1) 1.0314 a

Ash (% w/v) 0.67 a(1) 0.69 a

Fat (% w/v) 4.22 a(1) 4.20 a

Acidity** 1.95 a(1) 2.36 b

pH 6.68 a(1) 6.60 b

Lactose content (per cent w/v) 4.15 a(1) 4.35 b

Notes: *Average of three measurements** ml 0.1N NaOH/100ml sample (goat's milk)(1) Different letters in rows denote a significant difference at 5 per cent level of probabilityby Tukey test

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molecule, and the mixture bCD/guest (NovaÂk et al., 1998). When the guestmolecule is complexed, no energy absorption is observed at its normal meltingtemperatures. Since the guest molecule is surrounded by the cyclodextrin andtherefore unable to interact with other guest molecules, there is no crystallineguest structure to absorb energy.

Figure 1 shows the DSC curves for capric acid, bCD and bCD/C10. Theendothermic peak at 33.98C on the DSC curve of pure C10 is due to its melting,up to 1008C the volatilization of C10 takes place. The DSC curve of bCD showsthat during the heating process up to 1308C the sample loses water molecules.The curve of bCD/C10 does not show the melting peak of the guest molecule andthe peak due to water loss occurs at a lower temperature, 1158C, whencompared with pure bCD. During the inclusion process, most of the watermolecules in the bCD cavity are replaced by the capric acid molecule,weakening the bonds between the remaining water molecules and bCD, so thattheir liberation tends to need less energy and to occur at a lower temperature(Yilmaz et al., 1995). This behaviour is characteristic of inclusion complexformation.

Figure 1.DSC curves (a) bCD/C10,

(b) bCD and (c) C10

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Figure 2 shows the DSC curves for the pure C8 acid and bCD/C8 inclusioncomplex. Pure C8 shows an endothermic peak at 13.98C due to its melting, up to100ëC the volatilization of C8 takes place. The bCD/C8 system shows waterrelease in two stages, 110 and 1408C. It indicates that the water molecules arebonded differently in the bCD cavity after C8 is complexed. The melting peak ofpure C8 was not observed for the bCD/C8 system. As with the bCD/C10 system,this is likely to be due to complexation phenomenon. 1H NMR analysisprovided further information concerning the complexation process in aqueoussystems.

1H NMR spectra. The 1H NMR spectra of pure bCD and bCD/C10 inclusioncomplex is shown in Figure 3 and Figure 4, respectively. The bCD/C8 spectradoes not show significant chemical shifts in its protons compared with the purebCD and C8 spectra (not shown).

Figure 2.DSC curves for (a) bCD/C8 and (b) C8

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Table III shows a small variation in chemical shift (Dd) for the methyleneprotons of C8. The apolar cavity protons, H3 and H5, on the bCD cavity and thealkyl chain protons of C10 displayed the largest Dd value. This indicates theformation of an inclusion complex, the interactions occuring primarily betweenthe hydrophobic regions of the bCD cavity and the hydrocarbon chain of C10

(Wilson and Verrall, 1998).The chemical shifts observed for the protons on the alkyl chain of the acids

are due to the change in the medium polarity. The deprotonated free acid isdissolved in a polar medium (D2O). However, when inclusion occurs, themedium inside the cavity of cyclodextrins is less polar than D2O. The lowervalues for Dd in the bCD/C8 system are due to the higher contribution ofdissociated species.

The results suggest guest inclusion through the hydrocarbon chain ofC10 or C8 in the bCD cavity, the carboxylic group remaining outside thecavity.

Figure 3.1H NMR spectrum of

pure bCD in D2O,pD=10.4

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Conclusionsb-Cyclodextrin improves the quality of goat's milk flavour in theconcentrations of 0.35 and 0.40 per cent (w/v), minimizing the goaty flavour.The values from physical chemical analysis of total solid, protein, density, fat

Figure 4.1H NMR spectrum ofbCD/C10 in D2O,pD=10.4

Table III.Chemical shiftschanges (Dd=dpure±dcomplex) of bCDprotons (H1-H6) andacid protons (the lettersafter the signal arerepresented in scheme2), solvent: D2O,pD=10.4

SignalDd (parts per million)

bCD/C10 bCD/C8

H1 0.00 0.04H2 0.01 ±0.05H3 0.06 0.06H4 0 0.05H5 0.11 0.06H6 0.01 0.02CH3 (A) ±0.11 0(CH2)n (B) ±0.09 0CH2 (C) ±0.15 0CH2 (D) ±0.09 0.01

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and ash did not show significant differences for the standard and test milksamples.

The DSC results for the synthesised complex show characteristics ofinclusion complex formation, due to the disappearance of the melting signal ofthe pure acid in the presence of bCD. The chemical shifts observed in the1H NMR spectrum indicate that the alkyl chain of the acids is in the cavity ofbCD, due to its hydrophobic characteristics, and the carboxylic groups remainoutside of the cavity. The studies in vitro indicate that goat's milk bCD canencapsulate short-chain fatty acids, such as C8 and C10, providing an improve-ment in the flavour and smell of the milk.

References

Abo-Elnaga, I.G., el-Dahan, A.S. and Ridha, S.H. (1985), `̀ The fatty acid composition of Iraqiewe's, goat's and cow's milk fat'', Nahrung, Vol. 29 No. 3, pp. 241-45.

Association of Official Analytical Chemist (AOAC) (1998), `̀ Official methods of analysis'', 16thed., AOAC International Method, Nutley, NJ, USA.

Barrow, P.C., Olivier, P. and Marzin, D. (1995), `̀ The reproductive and developmental toxicityprofile of beta-cyclodextrin in rodents'', Reprod Toxicol, Vol. 9 No. 4, pp. 389-98.

Bonassi, I.A., Martins, D. and RocËa, R.O. (1997), `̀ ComposicËaÄo quõÂmica e propriedades fõÂsico-quõÂmicas do leite de cabra'', CieÃncia e Tecnologia de Alimentos, Vol. 17 No. 1, pp. 57-63.

Chang, Y.-I. and Reineccius, G.A. (1990), `̀ Interaction of b-cyclodextrin with enantiomers oflimonene and carvone'', Journal of Food Science, Vol. 55 No. 6, pp. 1686-95.

Divakar, S., Jyothirmayi, N. and Ramadoss, C.S. (1991), `̀ Nuclear magnetic resonance studies ofcyclodextrin complexes of linoleic acid and arachidonic acid'', Agric Food Chem, Vol. 39No. 12, pp. 2123-7.

Fodor, M., NovaÂk, Cs., RaÂkosa, R., Tomor, K., Pokol, G. and GaÂl, S. (1997), `̀ Solid and liquid stateinvestigations of mandelic acid cyclodextrin complexes'', Journal of Thermal Analysis,Vol. 48, pp. 515-25.

Gajewaska, R., Nabrzyski, M., Ganowiak, Z. and Kucharska, D. (1996), `̀ Evalution of the healthaspects of goat's milk'', Rocz-Panstn-Zakl-Hig, Vol. 47 No. 3, pp. 295-301.

Ganza-Gonzalez, A., Villa-Jato, J.L., Anguiano-Iega, S., Otero-Espinar, F.J. and Blanco Mendez, J.(1994), `̀ A proton nuclear magnetic resonance of the inclusion complex of naproxen withbeta-cyclodextrin'', International Journal of Pharmaceutics, Vol. 106, pp. 179-85.

Garcia-Carmona, F., SaÂnchez-Ferrer, A. and LoÂpez-NicolaÂs, J.M. (1995), `̀ Use of soluble lipids forbiochemical processes: linoleic acid-cyclodextrin inclusion complexes in aqueoussolutions'', Biochemistry Journal, Vol. 308, pp. 151-4.

Goldberg, R.N., Mayhew, M.P., Rekharsky, M.V., Ross, P. D., Inoue, Y. and Yamashoji, Y. (1997),`̀ Thermodynamic and nuclear magnetic resonance study of the reactions of a- andb-cyclodextrins with acids, aliphatic amines, and cyclic alcohols'', J Phys Chem B, Vol. 101,pp. 87-100.

Jenness, R. (1980), `̀ Compositions and characteristics of goat's milk: a review 1968-1979'', Journalof Dairy Science, Vol. 63 No. 10, pp. 1605-30.

Kirk, R.S. and Sawyer, R. (1991), Pearson's Composition and Analysis of Foods, 9th ed.,Longmans, London.

Kollengode, A.N.R. and Hanna, M.A. (1987), `̀ Cyclodextrin complexed flavurs retention inextruded starches'', Journal of Food Science, Vol. 62 No. 5, pp. 1057-60.

BFJ103,4

290

Linden, G. and Lorient, D. (1996), BioquõÂmica agroindustrial. RevalorizacioÂn alimentaria de laproducËaÄo agrõÂcola, Acribia, Spain, pp. 175-185.

Lindner, K, Szente, L., Szejtli, J. (1981), `̀ Food flavouring with b-cyclodextrin-complex flavoursubstances'', Acta Alimentaria, Vol. 10 No. 3, pp. 175-85.

Meilgaard, M., Civille, G.V. and Carr, B.T. (1991), `̀ Sensory evaluation techniques'', CRC Press,Inc., Boca Raton, FL.

Nogueira, M.H.L. (1990), `̀ CaracterizacËaÄo e utilizacËaÄo do leite de cabra no desenvolvimento dealimentos hipoalergeÃncios'', DissertacËaÄo (Mestrado em CieÃncia e Tecnologia de Alimentos),Universidade Federal de VicËosa, Brasil, 1990.

NovaÂk, Cs., Fodor, M. Pokol, G., Izvekov, V., Sztatisz, J. Arias, J.M. and GineÂs, J.M. (1998),`̀ Investigation of cyclodextrin complexes of mandelic acid'', Journal of Thermal Analysis,Vol. 51, pp. 1039-48.

Shaw, P.E. and Wilson, C.W. (1985), `̀ Reduction of bitterness in grapefruit juice withb-cyclodextrin polymer in a continous flow process'', Journal of Food Science, Vol. 50,pp. 1205-7.

Toyoda, K., Shoda, T., Uneyama, C., Takada, K. and Takahashi, M. (1997), `̀ Carcinogenicitystudy of beta-cyclodextrin in F344 rats'', Food Chem Toxicol, Vol. 35 No. 3-4, pp. 331-6.

Wilson L.D. and Verrall, R.E. (1998), `̀ A 1H NMR study of cyclodextrin hydrocarbon surfactantinclusion complexes in aqueous solutions'', Canadian Journal of Chemistry, Vol. 76, pp. 25-34.

Yilmaz, V.T., Karadag, A. and IcËbudak, H. (1995), `̀ Thermal decomposition of b-cyclodextrininclusion complexes of ferrocene and their derivatives'', Thermochimica Acta, Vol. 261,pp. 107-18.