Selected abiotic factors that influence raw cow milk freezing point depression

Freezing point depression (FPD) is an important property of milk that is influenced primarily by milk components connected to osmotic pressure. Under certain conditions it is possible to detect the addition of water to milk. It is necessary to have the right FPD limit in legislation for milk quality control. The aim of this study was to improve the estimation procedure of this limit. Apart from factors related to dairy cow nutrition, cattle breed and milk yield, it is important to take into account CO2 (6%), water steam evaporation and pasteurization under technological conditions. Bulk milk samples (1, 30, 6, 6, 10, 1 according to experiment) from Holstein and Czech Fleckvieh breed (1:1) were used in the experiments and technologically treated. The effects of water addition (water saturated and unsaturated by CO2), carbon dioxide evaporation and pasteurization (80 °C for 22 min) were quantified. Pasteurization aggravation of FPD was -0.00394 ± 0.00171 oC (P < 0.001). Aggravation due to carbon dioxide evaporation could be -0.00383 ± 0.00095 oC (P < 0.001) depending on practice. Increase in FPD is recorded after milking during technological procedures of milk storage, mixing, pumping, transport shaking and warming. During FPD shift, the acuteness of FPD data sets increases. This fact should be considered in the process of deriving standard raw cow milk FPD limits. Similar experimental analysis of milk FPD technological shifts has not been performed in this way until now. Bulk milk, extraneous water addition, pasteurization, vacuum, carbon dioxide saturation Milk freezing point depression (FPD) is an important qualitative milk indicator. It is mostly used for the control of raw or pasteurized milk quality in terms of its incidental milk falsification by adding foreign water (AFW; extraneous water). The main effect on FPD could be caused by the addition of foreign water through defects in milking equipment. Possible effects of automatic milking systems on FPD deterioration have been published (Rasmussen and Bjerring 2005). However, there are other additional reasons for milk FPD variations beside AFW (Wiedemann et al. 1993; Buchberger 1994; Crombrugge 2003; Chládek and Čejna 2005). Average milk FPDs differ significantly among breeds and species of farm mammals, -0.5320 ± 0.0050 and -0.5221 ± 0.0043, -0.5544 ± 0.0293 and -0.6048 ± 0.0691 °C in Holstein and Czech Fleckvieh cattle, White short-haired goat and Tsigai sheep, respectively (Genčurová et al. 2008; Macek et al. 2008). Some European Union countries are using the legislation discrimination limits of FPD for standard cow milk quality ranging from < -0.520 °C to < -0.505 °C (Rohm et al. 1991; Buchberger 1994; Crombrugge 2003). In the Czech Republic, the discrimination limits < -0.515 and < -0.520 °C for raw and pasteurized cow milk have been valid. Council Regulation (EC) 1234/2007 also prescribes the rule that the mean FPD value of drinking milk shall be equal to raw milk FPD mean under local conditions of the collecting area. Lactose content causes 53.8% of the milk FPD. The effect of the milk carbonic acid gas content and its evaporation may be also marked. Significant correlations (P < 0.05) were found by Hanuš et al. (2010) e.g. between FPD and: total milk solids (-0.50), true protein (-0.43), whey protein (-0.47), milk fat (-0.46), lactose (-0.35), somatic cell count (-0.36) and milk citric acid (0.47). Milk citric acid content contributes essentially to FPD ACTA VET. BRNO 2012, 81: 049–055; doi:10.2754/avb201281010049 Address for correspondence: Oto Hanuš Research Institute for Cattle Breeding Rapotín Výzkumníků 267, 788 13 Vikýřovice Czech Republic Phone: +420 583 392 157 Fax: +420 583 392 129 E-mail: oto.hanus@vuchs.cz http://www.vfu.cz/acta-vet/actavet.htm in terms of osmotic pressure support and it is also a suitable indicator of dairy cow energy metabolism (Garnsworthy et al. 2006). However, milk FPD was not found to be influenced dramatically by fat content manipulation (Hanuš et al. 2011). There are questions about possible changes in FPD due to milk storage after milking, its transport and industrial processing caused by business complications. In these cases, milk deliveries are sent from buyer back to seller (between dairy plants) because of unsatisfactory FPD. The aim of this paper was to study, describe and quantify milk FPD changes due to abiotic (technical) factors in various technological experiments. Materials and Methods One bulk raw cow milk sample (Holstein) was split into four subsamples in two rows (n = 1 plus 4 and 4). These were diluted by saturated and original distilled water (without CO2 saturation) from a 0.5 to 2.0% ratio (Fig. 1). In the first case, 800 ml of water were saturated by carbon dioxide (CO2) at 6 oC for 24 h in a pressure bottle, the weight of CO2 was 7.9 g. Set of bulk milk samples (n = 30) from Holstein (Ho) and Czech Fleckvieh (CF) breed (1:1) obtained in the winter season was pasteurized under 80 °C for 22 min (Fig. 2). In three experiments, individual raw cow milk samples (5 ml of one sample in vessel) from Ho and CF cows (1:1) were used (n = 6, 6 and 10). In vacuum, CO2 and partly also water steam were exhausted using a vacuum pump at 20 oC (Figs 3, 4, 5). Milk samples, vacuum and time were variously modified in different experiments as demonstrated in the figures. Six bulk raw cow milk (Ho and CF breed 1:1) and six drinking water samples were saturated by CO2 in pressure bottle for 6 h under refrigerator temperature of 6 oC (Figs 6 and 7). The saturation of liquids (water and milk) was reached using one litre of the working volume and a gas bomb with 7.9 g and 10 ml of the filling. This is equal to 0.1795 mol of CO2 and max. 7.5 MPa pressure (processor data). Calculated pressure according to actual measured paramaters (van der Waals’s equation) was 5.71 MPa. Values of FPD were measured (twice) using the top cryoscopic instrument Cryo-Star automatic Funke-Gerber (Germany) as a reference method. The selected measurement mood was reference Plateau Search. Instrument regularly calibrated by standard NaCl solutions (ISO 5764:2002(E)). The data were processed using regression. Differences were tested by paired t-test. These are shown using box graphs: median is the central short horizontal line; the upper edge of the 1st and 3rd quartiles is the tetragon; variation range, maximum – minimum is the vertical line.

Milk freezing point depression (FPD) is an important qualitative milk indicator.It is mostly used for the control of raw or pasteurized milk quality in terms of its incidental milk falsification by adding foreign water (AFW; extraneous water).The main effect on FPD could be caused by the addition of foreign water through defects in milking equipment.Possible effects of automatic milking systems on FPD deterioration have been published (Rasmussen and Bjerring 2005).However, there are other additional reasons for milk FPD variations beside AFW (Wiedemann et al. 1993;Buchberger 1994;Crombrugge 2003;Chládek and Čejna 2005).Average milk FPDs differ significantly among breeds and species of farm mammals, -0.5320 ± 0.0050 and -0.5221 ± 0.0043, -0.5544 ± 0.0293 and -0.6048 ± 0.0691 °C in Holstein and Czech Fleckvieh cattle, White short-haired goat and Tsigai sheep, respectively (Genčurová et al. 2008;Macek et al. 2008).Some European Union countries are using the legislation discrimination limits of FPD for standard cow milk quality ranging from < -0.520 °C to < -0.505 °C (Rohm et al. 1991;Buchberger 1994;Crombrugge 2003).In the Czech Republic, the discrimination limits < -0.515 and < -0.520 °C for raw and pasteurized cow milk have been valid.Council Regulation (EC) 1234/2007 also prescribes the rule that the mean FPD value of drinking milk shall be equal to raw milk FPD mean under local conditions of the collecting area.Lactose content causes 53.8% of the milk FPD.The effect of the milk carbonic acid gas content and its evaporation may be also marked.Significant correlations (P < 0.05) were found by Hanuš et al. (2010) e.g. between FPD and: total milk solids (-0.50), true protein (-0.43), whey protein (-0.47), milk fat (-0.46), lactose (-0.35), somatic cell count (-0.36) and milk citric acid (0.47).Milk citric acid content contributes essentially to FPD in terms of osmotic pressure support and it is also a suitable indicator of dairy cow energy metabolism (Garnsworthy et al. 2006).However, milk FPD was not found to be influenced dramatically by fat content manipulation (Hanuš et al. 2011).
There are questions about possible changes in FPD due to milk storage after milking, its transport and industrial processing caused by business complications.In these cases, milk deliveries are sent from buyer back to seller (between dairy plants) because of unsatisfactory FPD.The aim of this paper was to study, describe and quantify milk FPD changes due to abiotic (technical) factors in various technological experiments.

Materials and Methods
One bulk raw cow milk sample (Holstein) was split into four subsamples in two rows (n = 1 plus 4 and 4).These were diluted by saturated and original distilled water (without CO 2 saturation) from a 0.5 to 2.0% ratio (Fig. 1).In the first case, 800 ml of water were saturated by carbon dioxide (CO 2 ) at 6 ºC for 24 h in a pressure bottle, the weight of CO 2 was 7.9 g.
In three experiments, individual raw cow milk samples (5 ml of one sample in vessel) from Ho and CF cows (1:1) were used (n = 6, 6 and 10).
In vacuum, CO 2 and partly also water steam were exhausted using a vacuum pump at 20 ºC (Figs 3,4,5).Milk samples, vacuum and time were variously modified in different experiments as demonstrated in the figures.Six bulk raw cow milk (Ho and CF breed 1:1) and six drinking water samples were saturated by CO 2 in pressure bottle for 6 h under refrigerator temperature of 6 ºC (Figs 6 and 7).The saturation of liquids (water and milk) was reached using one litre of the working volume and a gas bomb with 7.9 g and 10 ml of the filling.This is equal to 0.1795 mol of CO 2 and max.7.5 MPa pressure (processor data).Calculated pressure according to actual measured paramaters (van der Waals's equation) was 5.71 MPa.Values of FPD were measured (twice) using the top cryoscopic instrument Cryo-Star automatic Funke-Gerber (Germany) as a reference method.The selected measurement mood was reference Plateau Search.Instrument regularly calibrated by standard NaCl solutions (ISO 5764:2002(E)).The data were processed using regression.Differences were tested by paired t-test.These are shown using box graphs: median is the central short horizontal line; the upper edge of the 1 st and 3 rd quartiles is the tetragon; variation range, maximum -minimum is the vertical line.

Results
Experimental results are shown in graphs (Figs 1-7).Significant effect (P < 0.001) of AFW represented a milk FPD increase by 0.006 ºC (Fig. 1).A similar effect with water saturated by CO 2 was 0.004 ºC.Significant (P < 0.001) increase in FPD by milk pasteurization was Fig. 1.Regression rendering of effect of weak dilution of raw cow milk by saturated (with carbon dioxide, CO 2 ) and unsaturated distilled water on its freezing point depression.
R 2 -determination coefficient, r -correlation coefficient, P -probability of significance 0.00394 ºC (Fig. 2).We also found a significant milk FPD increase due to CO 2 exhausting and consequent FPD decrease due to water steam evaporation (Figs 3,4 and 5).In this context, the raw cow milk FPD increase by CO 2 evaporation (normal content in milk is 6%) was estimated as a practical effect with a value 0.00383 ºC (Fig. 4).In the experiments shown in Figs 6 and 7 we demonstrated a significant effect of milk and water saturation by CO 2 on FPD decrease under the mentioned circumstances.Milk FPD was decreased by 0.0896 ºC (Fig. 7).51 Fig. 2. Rendering of pasteurization effect on raw cow milk freezing point depression.n -number of cases, t -criterion of pair t-test, P -probability of significance, A -raw cow milk, B -pasteurized milk.Pasteurization was carried out at 80 °C for 22 minutes.Thirty bulk milk samples from Holstein and Czech Fleckvieh dairy cow herds in winter feeding season (from February to April); one sample is equal to 300 ml.
H -original milk, it means 6 individual raw cow milk samples from two breeds (Holstein and Czech Fleckvieh), I -CO 2 evaporation, J -water steam evaporation.There were 5 ml of milk in one sample on Petri dish.CO 2 exhausting was performed at 40 and 80 kPa (I and J) during 60 and 120 second of vacuum time.

Discussion
An increase in milk FPD by 0.005 ºC is equal to 1% AFW (extraneous water; Wiedemann et al. 1993;Buchberger 1994).In Fig. 1, the effect (P < 0.001) of water addition to milk is shown.In the case of water addition, the FPD increase is approximately 52 Fig. 4. Graphical rendering of carbon dioxide evaporation and consequently also water steam evaporation on raw cow milk freezing point depression by vacuum and time of exposition.OM -original milk, it means 6 bulk (herd) raw cow milk samples from two breeds (Holstein and Czech Fleckvieh cattle breed), from J to N -CO 2 evaporation, O -water steam evaporation.There were 5 ml of milk in one sample.CO 2 exhausting was performed at 10, 20, 40, 60, 80 and 100 kPa (from J to O) with time duration of vacuum approach and its consequent decrease, up to reaching of demanded vacuum value.
OM -original milk, it means 10 bulk (herd) raw cow milk samples from two breeds (Holstein and Czech Fleckvieh cattle breed), P -CO 2 evaporation, Q and R -water steam evaporation.5 ml of milk were in one sample.There was performed addition of foreign water into one sample (10%).CO 2 exhausting was carried out at 60 kPa (P; increase 20 second and decrease to 0 kPa 167 second), 100 kPa (Q; increase 87 second, tenacity 60 second and decrease) and at 100 kPa (R; increase 87 second, tenacity 120 second and decrease).
in accordance (0.006 ºC) with this figure.However, in the case of water saturated by CO 2 the corresponding FPD increase is markedly lower (by 1/3, about 0.004 ºC) compared to pure water addition.Carbon dioxide is a natural raw milk component after milking and its volume is decreased from this time point.It can have important effects on milk FPD changes.
As it is well known, an important technological step in processing in the milk food chain is pasteurization.Fig. 2 shows the effect of pasteurization on milk FPD.There was a significant (P < 0.001) aggravation of FPD following pasteurization.Under the mentioned circumstances, the difference was -0.00394 ± 0.00171 ºC.This could  be caused by milk protein complex heat shock, although it is possible that water steam evaporation in opened pasteurization systems (experimental or practical) could mitigate the described effect on FPD due to milk concentration.However, this effect may be very limited.Rohm et al. (1991) and Roubal et al. (2004) described higher (worse) mean FPD in pasteurized cow milk than raw bulk milk under practical conditions.Similarly, Janštová et al. (2007) determined a higher FPD for pasteurized goat milk compared to raw milk (-0.5488 ± 0.0046 > -0.5513 ± 0.0046 °C).These factors could explain why heat treated milk FPDs are usually worse than raw milk in practice.
Evaporation of natural CO 2 from milk was simulated using vacuum generation.Using experiments with evaporation simulation, it is shown in Figs. 3, 4 and 5 that vacuum CO 2 evaporation leads to FPD aggravation (increase) according to vacuum level and time.Consequently there is a technological and quality improvement (decrease) in milk FPD which is determined probably due to water steam evaporation and milk concentration.This effect was also observed in Fig. 5.One milk sample with AFW (10%) was ranked.This fact is evident as greater FPD variation range and mean value skew in terms of frequency distribution.Experimental raw cow milk FPD aggravation (increase) by CO 2 evaporation could be estimated to be about -0.00383 ± 0.00095 ºC regarding practice under natural conditions (Fig. 4).This could be compared to 1% of AFW in milk (Wiedemann et al. 1993;Buchberger 1994;Crombrugge 2003;Rasmussen and Bjerring 2005;Hanuš et al. 2010).The effect of milk CO 2 concentration on FPD is demonstrated similarly as supporting proof but in reverse (Fig. 6) by model milk CO 2 saturation in a pressure vessel.Under these conditions, FPD was decreased ("improved") dramatically by -0.0896 ± 0.00562 ºC.This result was compared to the similar method of drinking water CO 2 saturation (Fig. 7).From the results, in accord with Rohm et al. (1991) and as shown in Fig. 8, the FPD shifts markedly in the period after milking during technological procedures of milk storage, mixing, pumping, transport shaking and warming without AFW.During technological FPD shifts the peak of data sets for milk quality increases and variance tapers.This fact should be considered in the process of 54 deriving standard raw cow milk FPD limits for legislative materials apart from specific country, breed and milk yield conditions.
In conclusion, the results can be considered in derivation procedures for objective standard raw cow FPD limits.These selected abiotic FPD factors must be considered in statistics aside from such variables as country conditions (climate, soil, feeding type), cattle breed and actual milk yield in FPD discrimination limits.This is necessary for maximal probability of objective separation of standard milk deliveries without AFW from milk with AFW.It is important in procedures for milk quality payment and control of the quality of the milk food chain.Similar solution and experimental analyses of milk FPD technological shifts have not been performed in this way until now.

Fig. 3 .
Fig. 3. Graphical rendering of carbon dioxide evaporation and consequently also water steam evaporation on raw cow milk freezing point depression by vacuum and time.

Fig. 5 .
Fig. 5. Rendering of impact of carbon dioxide evaporation and consequently water steam evaporation on raw cow milk freezing point depression by vacuum and time.

Fig. 6 .
Fig. 6.Rendering of raw cow milk saturation by carbon dioxide on milk freezing point depression value.

Fig. 7 .
Fig. 7. Rendering of impact of drinking water saturation by carbon dioxide on its freezing point depression measured by milk cryoscope.

Fig. 8 .
Fig. 8. Hypothetical model graph of raw cow milk FPD shift (increase) after milking.Change of freezing point depression during milk storage, transport, mixing and technological treatment in dairy plant, which can be caused by different abiotic factors, based on presumption of normal frequency distribution.