Adulterants interference on Fourier Transform Infrared analysis of raw milk

Usual major milk composition is of 87.3% water, 4.6% lactose, 3.25% protein, 3.9% fat, 12.7% total solids, and non-fat 8.8% solids. Knowledge of milk components and characteristics is the basis for tests that are designed to investigate modified milk composition. Substantial reduction of solid components could, for example, indicate the fraudulent addition of water to the milk. Altogether, adulteration and variations due to factors such as lactation stage, breed, feeding, environmental temperature, management, milking interval, production and mammary gland health may affect milk composition (Brito et al., 2016). Optimal composition is a preponderant factor for the dairy industry, in processes such as cheese, butter, powdered milk, milk cream, cream cheese and other products (Leite, 2006). 

in the quality evaluation.For example, bronopol is a preservative added to the raw milk sample to avoid quality loss; usually samples may be kept ten days at temperatures up to 10 o C (Leite, 2006).However, other authors have reported decrease in milk fat concentration after seven days, and slight modifications in protein and total solids concentration after storage of six days (Ribas et al., 2004).
In Brazil, new variables were legalized to ensure a minimum standard to be met by all milk producers (Brasil, 2002;2011;2016).Several solid components, IBC, SCC, and drug residues were included in this new regulation for at least monthly monitoring of producers linked to dairies under Federal Inspection.A national net of laboratories was also created to respond to the growing analytical demand (Rede Brasileira de Laboratórios de Análise da Qualidade do Leite; RBQL).These labs are configured with modern electronic equipment for quick and precise analysis, to provide milk quality information in the adequate time (Silveira et al., 2004).
Raw milk must present the original composition, and any component withdrawal, or addition of water, preservatives, contaminants, or any other substance to the raw milk is illegal.The consequence of fraud is consumer misleading and risks including health issues, illegal competition, economical and marketing unbalance.One outstanding example took place in China, where a widespread fraud with melamine addition to raw milk happened in 2008.The outcome was the death of six and more than 300,000 people were sickened.Melamine was added with the intent to artificially increase the protein levels to disguise water illegally added to raw milk (Silva, 2013).

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Similar incidents have come to light in the last years in Brazil, with several cases of systematic adulteration being detected in inspected dairy industries.Common aspects of these cases included water addition to the raw milk together with substances to counterbalance properties modification of the milk due to the extraneous water.For example, to restore density and freezing point, salt, sugar, and other substances are used.Impairment of the milk nutritional value is a direct consequence of these frauds.Additionally, preservative is illegally added to decrease microbial growth in low quality raw milk (Rosa-Campos et al., 2011;Santos et al., 2013;Silva, 2013;Abrantes et al., 2014).
Although several analytical methods have been used for a long time to investigate raw milk adulteration in the dairy industry, usually they are time consuming and expensive.However, equipments with relatively low analytical cost have been developed in recent decades.Mid infrared equipment, for example, has expanded the analytical capacity for compositional analysis, while flow cytometers have been used for somatic cell and bacterial counting in raw milk (Oliveira et al., 2012).
The infrared equipment is based in differential IR absorption in specific wavelengths of chemical groups present in fat, protein, and lactose in milk (Biggs et al., 1987).For milk, the mid infrared spectrum (MIR) represents great advantages when compared to previous techniques, as the simultaneous reading of several milk components, without any previous sample treatment, with high output and possibility of process automation (Rodriguez-Otero et al., 1997).The IR technique matched the reference methods (Silveira et al., 2004), with major milk components being simultaneously analyzed.
The automated routine analysis of raw milk with electronic equipment usually includes the main milk components total solids, fat, protein, casein, ureic nitrogen, and lactose among others through IR analysis, and somatic cells and bacterial counting by flow cytometry.Nowadays, old IR equipment based on filters are being replaced by FTIR based equipment, a new and promising technology (Oliveira et al., 2012).
Although the infrared methods have been extensively evaluated for milk quality analysis, not much has advanced in the study of extraneous substances used as adulterant in milk.One of the concerns is related to the potential interference of these substances in the analysis of milk components because of superimposed absorptive wavelengths.Moreover, since the current methods used to detect adulterants in raw milk are relatively expensive, highly dependent on labor and time-consuming, innovative screening methods to simultaneously detect extraneous substances added to milk would represent an expressive advance in the dairy sector.
The objective of the current work was to evaluate the potential interference of extraneous substances, added to milk as adulterants, in the component analysis based on Fourier Transform Infrared technique.
Six batches of bulk tank raw milk were obtained from August to October 2016, from healthy cows, under veterinary supervision.To each batch sucrose was added (0.1g/100mL; 0.5g/100mL and 1g/100mL) or starch (0.1g/100mL; 0.5g/100mL and 1g/100mL) and aliquoted in 50mL vials.Samples for compositional analysis and somatic cell counts were added with bronopol, and for bacterial counting with azidiol.
Compositional analyses were done by FTIR 400® (LactoScope FTIR, Delta Instruments, Drachten, Netherlands) and some spectra are shown in Figure 1.Somatic cell count by flow cytometry in FTIR 400® (SomaScope, Delta Instruments, Drachten, Netherlands) and total bacterial counting by flow cytometry in Bactoscan FC® (Foss, Hillerod, Denmark).Multiple Linear Regression was used for statistical analysis with a confidence interval of 95%, using the program Stata version 12.0 (Stata..., 2011).
Milk analyzed for SCC and TBC were monitored to ensure IR readings deviations for components were not due to quality issues.However, starch and sucrose additon affected both values.Starch addition resulted in increasing SCC and TBC, while sucrose addition resulted in reducing SCC readings (Table 1).
Storage time was an influential factor, with SCC reduction after 24h, but this effect was more pronounced with storage at 25±2°C and starch addition.Previous report by Leite (2006), in raw milk without adulterants, has found SCC reduction storage at 30ºC during five days, while TBC increased.In the current work, TBC increased was noticed in the seventh day of storage at 25ºC.Every experimental repetition presented extremely low p-values for the all dependent variables, except for TBC, which was significant only in the last repetition.This indicates the strict control of the milk used in the experiment, once the variables that could adversely affect the compositional results outcome, such as lactation stage, calving order, milking management and hygiene, feeding, among others were controlled in the experimental repetitions (Brito et al., 2016).Similarly, high Adjusted R 2 value was found for all dependent variables, except for TBC in the milk added with sucrose treatment.This indicates that the multiple linear regression was well adjusted to the sample data (Triola, 2008).
Composition results for milk added with starch deviated from the control with increase of parameters fat, protein, lactose, total solids, solids non fat and reduction of casein and MUN using FTIR (P< 0.05).However, the addition of sucrose to milk modified FTIR results for lactose, TS, SNF and MUN with increasing these values and decreasing protein and casein (Table 2).Lactose IR readings were slightly higher in milk added with starch, however, after sucrose addition, lactose content readings reached values as high as 5.19% (Table 2).
Freezing point of milk decreased with increasing sucrose concentration, and this is due to the increased amount of soluble substance.As a colligative property, the freezing point is correlated to the vapour pressure at the solution according to Raoult's Law.On the other hand, since starch molecules are longer and have higher molecular weight than sucrose, it is expected that starch addition to the milk will have lesser effect on freezing point.
The use of sucrose and starch as milk adulterants is a very common fraud.For example, Rosa-Campos et al. (2011) analyzed 72 pasteurized milk samples and in some cases found sucrose addition in 100% of some brands samples.
MUN values obtained by IR were lower (P< 0.05) after starch addition, even during storage at 7ºC and 25ºC.On the other hand, MUN values slightly increased in the control group during storage.Casein results were lower in milk added with starch or sucrose than in control milk.
In conclusion, infrared analysis of raw milk added with extraneous substances used as adulterants presented deviating results for component analysis due to superimposed absorptive wavelengths.
Starch addition changed expected compositional results of raw milk in a different direction and intensity when compared with sucrose, with interference in the major components, fat and protein.Simultaneously, increasing individual bacterial count by flow cytometry were obtained after starch addition.Keywords: raw milk, FTIR, startch, sucrose, composition analysis ACKNOWLEDGEMENTS Fapemig -APQ1179-14; CNPq PQ309801/2014-1

Figure 1 .
Figure 1.FTIR spectra of milk control and added starch and sucrose, in three different concentrations.

Table 1 .
Mean and standard deviation (sd)of the SCC and TBC in raw milk added with starch and sucrose and analyzed by flow cytometry