CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 389 Milk sample Water-baha30°C Figure 10.5 Apparatus for visual determination of the rennet coagulation time of milk of apparatus have been developed. The most popular of these, although with mited use, is the Formograph(Foss Electric, Denmark), a diagram of hich is shown in Figure 10.6a. Samples of milk to be analysed are placed small beakers which are placed in cavities in an electrically heated metal block. Rennet is added and the loop-shaped pendulum of the instrument placed in the milk. The metal block is moved back and forth, creating a dragon the pendulum in the milk. The arm to which the pendulum is attached contains a mirror from which a fashing light is reflected on to photosensitive paper, creating a mark. While the milk is fluid, the viscosity is low and the drag on the pendulum is slight and it scarcely moves from ts normal position; hence a single straight line appears on the paper. As the milk coagulates, the viscosity increases and the pendulum is dragged out of position, resulting in bifurcation of the trace. The rate and extent to which the arms of the trace move apart is an indicator of the strength(firmness of the gel. a typical trace is shown in Figure 10.6b. a low value of r indicates a short rennet coagulation time while high values of a3o and k2o indicate milk with good gel-forming properties A recently developed, and apparently industrially useful, apparatus is the hot wire sensor. a diagram of the original assay cell is shown in Figure 10.7a. A sample of milk is placed in a cylindrical vessel containing a wire uniform dimensions. a current is passed through the wire, generating heat which is dissipated readily while the milk is liquid. As the milk coagul generated heat is no longer readily dissipated and the temperature of the
CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 389 Milk sample Figure 10.5 Apparatus for visual determination of the rennet coagulation time of milk. of apparatus have been developed. The most popular of these, although with limited use, is the Formograph (Foss Electric, Denmark), a diagram of which is shown in Figure 10.6a. Samples of milk to be analysed are placed in small beakers which are placed in cavities in an electrically heated metal block. Rennet is added and the loop-shaped pendulum of the instrument placed in the milk. The metal block is moved back and forth, creating a ‘drag’ on the pendulum in the milk. The arm to which the pendulum is attached contains a mirror from which a flashing light is reflected on to photosensitive paper, creating a mark. While the milk is fluid, the viscosity is low and the drag on the pendulum is slight and it scarcely moves from its normal position; hence a single straight line appears on the paper. As the milk coagulates, the viscosity increases and the pendulum is dragged out of position, resulting in bifurcation of the trace. The rate and extent to which the arms of the trace move apart is an indicator of the strength (firmness) of the gel. A typical trace is shown in Figure 10.6b. A low value of r indicates a short rennet coagulation time while high values of a3, and k,, indicate a milk with good gel-forming properties. A recently developed, and apparently industrially useful, apparatus is the hot wire sensor. A diagram of the original assay cell is shown in Figure 10.7a. A sample of milk is placed in a cylindrical vessel containing a wire of uniform dimensions. A current is passed through the wire, generating heat which is dissipated readily while the milk is liquid. As the milk coagulates, generated heat is no longer readily dissipated and the temperature of the
DAIRY CHEMISTRY AND BIOCHEMISTRY chant papcr Light nash Mirror MILK Ocillating heating hlock 30 min 10.6(a) Schematic representation of the Formograph tus for determining the bifurcate by 20mm, ago is the extent of bifurcation 30 min after rennet addition( the approximate time at which the coagulum is cut in cheesemaking) wire increases, causing an increase in its conductivity, a typical trace is hown in Figure 10.7b. the principle has been commercialized by Stoelting Inc.(Kiel, Wisconsin). The wire probe, in a stainless steel shield, is inserted through the wall of the cheese vat. The output from the wire is fed to a computer which can be used to switch on the gel-cutting knife, permitting
CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 391 Data acquisition (a) reaction coagulation (b) Time after rennet addition Figure 10.7(a) Hot wire sensor for objectively measuring the rennet coagulation of milk.(b) Changes in the temperature of the hot wire during the course of the rennet coagulation of mill automation and cutting of the gel at a consistent strength, which is important for maximizing cheese yield The primary phase of rennet action may be monitored by measuring the formation of either product, i.e. para-K-casein or the GMP. Para-k-casein may be measured by SDs-polyacrylamide gel electrophoresis (PAGE)
CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 391 I 1)ata acquisition Non-enzymatic coagulation c ----------- .. Enzymatic reaction *---- Time after rennet addition Figure 10.7 (a) Hot wire sensor for objectively measuring the rennet coagulation of milk. (b) Changes in the temperature of the hot wire during the course of the rennet coagulation of milk. automation and cutting of the gel at a consistent strength, which is important for maximizing cheese yield. The primary phase of rennet action may be monitored by measuring the formation of either product, i.e. para-lc-casein or the GMP. Para-lc-casein may be measured by SDS-polyacrylamide gel electrophoresis (PAGE)
392 DAIRY CHEMISTRY AND BIOCHEMISTRY Figure 10.8 Schematic representation of hydrolysis and gel formation in renneted milk H=hydrolysis of K-casein: V changes in the viscosity of renneted milk (second stage of coagulation), G= changes in the viscoelastic modulus (gel formation which is slow and cumbersome, or by ion-exchange high performance liquid chromatography(HPLC). The GMP is soluble in TCA(2-12% depending on its carbohydrate content)and can be quantified by the Kjeldahl method or more specifically by determining the concentration of N-acetylneuraminic acid or by reversed phase HPLC(RP-HPLC The activity of rennets can be easily determined using chromogenic peptide substrates, a number of which are available Gel strength(curd tension ) The gel network continues to develop for a considerable period after visible coagulation(Figure 10.8). The strength of the gel formed, which is very important from the viewpoints of syneresis (and hence moisture control) and cheese yield, is affected by several factors the principal ones are summarized in Figure 10.9. The strength of a renneted milk gel can be measured by several types of viscometers and penetrometers. As discussed on p. 389, the Formograph gives a measure of the gel strength but the data can not be readily converted to rheological terms. Penetrometers give valuable information but are single-point determinations. Dynamic rheometers are particularly useful allowing the buildup of the gel network to be studied Syneresis. Renneted milk gels are quite stable if undisturbed but synerese (contract), following first-order kinetics, when cut or broken. By controlling extent of syneresis, the cheesemaker can control the moisture content of neese curd and hence the rate and extent of ripening and the stability of cheese- the higher the moisture content, the faster the cheese will ripen
392 DAIRY CHEMISTRY AND BIOCHEMISTRY 20 Time (min) Figure 10.8 Schematic representation of hydrolysis and gel formation in renneted milk; H = hydrolysis of K-casein; V = changes in the viscosity of renneted milk (second stage of coagulation), G = changes in the viscoelastic modulus (gel formation). which is slow and cumbersome, or by ion-exchange high performance liquid chromatography (HPLC). The GMP is soluble in TCA (2-12% depending on its carbohydrate content) and can be quantified by the Kjeldahl method or more specifically by determining the concentration of N-acetylneuraminic acid or by reversed phase HPLC (RP-HPLC). The activity of rennets can be easily determined using chromogenic peptide substrates, a number of which are available. Gel strength (curd tension). The gel network continues to develop for a considerable period after visible coagulation (Figure 10.8). The strength of the gel formed, which is very important from the viewpoints of syneresis (and hence moisture control) and cheese yield, is affected by several factors - the principal ones are summarized in Figure 10.9. The strength of a renneted milk gel can be measured by several types of viscometers and penetrometers. As discussed on p. 389, the Formograph gives a measure of the gel strength but the data can not be readily converted to rheological terms. Penetrometers give valuable information but are single-point determinations. Dynamic rheometers are particularly useful, allowing the buildup of the gel network to be studied. Syneresis. Renneted milk gels are quite stable if undisturbed but synerese (contract), following first-order kinetics, when cut or broken. By controlling the extent of syneresis, the cheesemaker can control the moisture content of cheese curd and hence the rate and extent of ripening and the stability of the cheese - the higher the moisture content, the faster the cheese will ripen
CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 393 Gel strengt Figure 10.9 Principal factors that affect the strength of renneted milk gels(curd tension); pH O). calcium concentration(O), protein concentration(O), preheat treatment(x) 45 Time after cutting 263 Time after cutting b) Figure 10.10 Effect of temperature(a) and pH(b)on the rate and extent of syneresis in
CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 393 Figure 10.9 Principal factors that affect Gel strength the strength - of renneted milk gels (curd tension); pH (O), calcium concentration (O), protein concentration (O), preheat treatment ( x ). 45’C 40’C 3sc 30’c t Time after cutting pH 6.3 pH 6.4 pH 6.5 pH 6.6 t Time after cutting Figure 10.10 Effect of temperature (a) and pH (b) on the rate and extent of syneresis in cut/broken renneted milk gels