Food Structure Volume 9 Number 1 Article 3 1990 Identification and Characterization of Cocoa Solids and Milk Proteins in Chocolate Using X-ray Microanalysis B. E. Brooker Follow this and additional works at: https://digitalcommons.usu.edu/foodmicrostructure Part of the Food Science Commons Recommended Citation Brooker, B. E. (1990) "Identification and Characterization of Cocoa Solids and Milk Proteins in Chocolate Using X-ray Microanalysis," Food Structure: Vol. 9 : No. 1 , Article 3. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol9/iss1/3 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Food Structure by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. FOOD STRUCTURE, Vol. 9 (1990), pp. 9-21 J046- 705X/90$3 .00+ .00 Scanning Microscopy International, Chicago (AMF 0'!-lare), IL 60666 USA IDENTIFICATION AND CHARACTER IZATI ON OF COCOA SOLIDS AND MILK PROTEINS IN CHOCOL ATE USING X-RAY MICROANALYSIS B.E. Brooker AFRC Institute of Food Research, Reading Laboratory, Shinfield, Reading, Berks RG2 9AT, U.K. Abstract Introduction x-ray microanalysis was used to compare the The sugars , proteins and 1 i pids of mi 1k elemental composition of isolated cocoa solids ingredients used to make milk chocolate take part with milk powder. Whereas mllk powder contained in complex chem ical and physical interactions similar high levels of calcium and potassium, during processing which irtpart important cocoa sol ids were rich in potassium but contained attributes of flavour, colour and texture to the very small amounts of calcium . final product (Kinsella, 1970; Reimerdes & This difference in elemental composition Mehrens, 1988)). The milk proteins in particular allowed the two populations of particles to be possess a range of desirable properties i ncl udi ng distinguished in digital X-ray maps of frozen and surface activity, modiflcation of rheology and a fractured samples of plain , milk and white marked chemical reactivity. It is apparent also c~oco l ates examined by cryo- scanni ng e lectron that the proteins are important determinants of m1croscopy. The size distributions of particles the structural and functional properties of imaged in this way were determined using standard chocolate which, in their turn , influence texture software and were used t o distinguish between two and flavour. Thus, for a given chocolate , changes white chocolates whose particle reducti on only in the nature of the mi 1k added can produce (refining) parameters during processing were s i gifi cant changes in the product (Rei mer des & different. Mehrens, 1988). This technique offers the possibility of Apart from milk sol ids, chocolate contains studying P_article interactions during processing other hydrophilic particles of cocoa solids a~d ~he .1nfluence. of shape, size and spatial (defined here as non-fat cocoa solids) and sugar d1str1but10n of m1lk and cocoa solids on the crysta 1s which, during processing, undergo marked rheology and mechanical properties of chocolate changes and take part in important interact ions without destroying its normal structure . that strongly influence the organoleptic properties of the chocolate (Cook , 1982). Thus, particle size distribution affects the final flavour and mouth feel (Cook, 1982) such that chocolate is perceived as coarse if roore than 20% of the particles are larger than 22 urn (Rostagno , 1969) . However, attempts to study the interactions Initial paper received August 23, 1989 of these particles, their role in the uptake of Manuscript received December 20, 1989 water by chocolate and the way in which their Direct inquiries to B.E. Brooker dimensions are influenced by processing parameters Telephone number: 44 - 734-883103 ext . 2283 have been thwarted by the lack of suitable methods for di scri mi nati ng between, and obtaining quantitative information from, the different populations of particles in bulk samp les of intact chocolate. Whilst it has long been possible to recognise Keywords: Chocolate , X-ray microanalysis , X-ray birefringent crystals of sucrose and cocoa butter mapp1 ng, cryo-scanni ng electr on microscopy, cocoa ln sections of chocolate examined in polarized sol ids, milk protein, plain chocolate, white light (Hanssen, 1970), the ability to specifically chocolate , crumb chocolate , dry mix chocolate . identify cocoa solids and milk protein in a similar way but without the use of solvents and at higher resolution would be a valuable technique. An approach which requires the examination of defatted and stained sections of chocolate by l1ght m1croscopy is limited by resolution and r elies on non-specific staining reactions to visualise mllk protein; there is also the danger B. E. Brooker of altering the spatial relationships of di stribution of elements in intact chocolate particl es by extracti ng the continuous fat phase products, it was necessary to use cryo-SEM i n with sol vent (Heathcock, 1985). Procedures for conjunction with X-ray mi croanalysis. The the fixation, embedding and sectioning of application of a low tempe"ature technique, using chocolat e for examination by transmission e lectron an EMscope SP2000 cryo-preparation unit with microscopy (TEM) are successful in i dent i fyi ng transfer device and microscope cold stage, ensured particles of milk powder and cocoa sol ids, but the the stabi 1 i ty of the specimens and the specimens used are of necessity very small and the iiTITlobilization of their components during fixation time is very extended (Cruickshank, 1976; examination in the electron beam. A modified Heathcock, 1985). Although some work has been specimen holder was used (Fig. 1) which, unlike reported in which metal replicas of frozen and the standard EMscope holder, was not fitted with fractured chocolate were ex ami ned by TEM to obtain the shroud that was ori ginally designed to prevent fine details of the structural relationships the formation of ice on the specimen during between these components ( Buchhei m & Knoop, 1971 ; transfer betwee n the preparation chamber and the Heathcock, 1985), again, only small samples can be SE M. However , under the conditions of specimen used with this technique and an overall handling used in this study, experience has shown evaluation of particle size , distribution and t:-hat ice does not form on the surface of specimens relative position i s difficult to obtain . during transfer 1f sufficient care is taken. Co ld stage scanning electron microscopy The vacuum system of the EMscope SP2000 cryo­ (cryo-SEM) has the great advantage that it allows preparation unit was specially modified to allow the examination of frozen and fractured bulk the more efficient and more controlled evaporation samples of food materia l s which are quite stable of carbon duri ng specimen coating. By f itting a in t he electr on beam and in which details of the Leybold 50 1 turbomolecular pump between the work internal structure can often be seen (Schmidt & chamber and the rotary pump (Fig. 2), carbon van Hooydonk, 1980; Brooker et al., 1986, Brooker, 1988a, 1988b) , but chocolate is an exception . ~~:~ora~b<?.% ~~~r per:ao;hmeerd t~~n a afr~~~urpere~~~;e~ Thus, whilst Heathcock (1985) and Sargent (1988) ach~eved with a cryo-assisted rotary pump ( 10-2_ were ab l e to recognise occasional particles of 10- Torr). Used in this way, several specimen dried milk and sugar in bulk samples of chocolate coatings could be ob tained from a single length of examined in this way, the identification of all carbon cord. such components, i ncl udi ng cocoa solids , exposed Some preliminary studies reported by at the surface of the fracture cannot be done Heathcock ( 1 985) suggested that freezing chocolate reliably using mor phological criteria only. In the frorn amb ient temperature produced fractures which present study , an improved technique is described passed on ly through the continuous fat phase. It in which X-ray spectrometry is used in conjunction was suggested that chocolate should be heated to with cryo-SEM and in which the identification of 40° C to melt the fat phase before freezing and particles is based on their elemental composition . fracturing the specimen to reveal the internal particulate components . Under these circumstances, Materials and Meth ods the plane of fracture wa s believed to pass around the cant i nuous cocoa butter phase rather than Choco late solids and milk powder through it, thus permitting the popu l ations of The d1stnbut1on of d1 fferent elements in particles to be seen more easily and, by particles of spray dried skillliled milk powder and inference, to produce a better x-ray s i gnal. i n the cocoa sol ids of cocoa mass was studied Preliminary observations did not support these ~!~~;).energy dispersive X- ray spectrometry (see resu l ts but in some experiments the X-ray spectra and maps from chocolate prepared at 40°C in this Cocoa mass was obtained from a commercial way were compared with those obtained using the source and defatted using several changes of a standard method described below. 60:40 mixture of chloroform and methanol . Between Generally , samples were placed in an oven at washings with this solvent, insoluble cocoa solids 28°C for at least lh before use to allow intact were al lowed to sediment and after the final wa sh, sl ices of chocolate, 2 mm thick, to be cut with a excess sol vent was evaporated. The layer of cocoa razor blade without crumbl ing . Sli ces were then solids produced in this way wa s dispersed by placed in the groove of a c ircular carbon insert crush1ng gently wlth a spatula and small portions on a pre-warmed (28°C) specimen holder and secur ed of the resulting powder were then app l ied as a in place using carbon cement (F i g.