Buffalo milk has been gaining distinct importance throughout the world. Nowadays, it is the second largest source of global milk production (Khedkar et al., Reference Khedkar, Kalyankar and Deosarkar2016). Besides the absence of β-carotene, buffalo milk differs from bovine milk from a compositional point of view: it contains higher levels of fat, total solids, proteins, caseins, lactose and ash contents (Ahmad et al., Reference Ahmad, Anjum, Huma, Sameen and Zahoor2013). In Brazil, the market for buffalo milk products is growing (Ricci and Domingues, Reference Ricci and Domingues2012). Besides the fact that buffalo milk has a higher yield in the production of derivatives, buffalo mozzarella cheese has already established a market with a promising future in Brazil. This is due to the possibility of adding greater value when compared with cheese made with cow's milk (Sales et al., Reference Sales, Rangel, Urbano, Tonhati, Galvão Júnior, Guilhermino, Aguiar and Bezerra2018). Within this context, two challenges to increasing the potential of buffalo milk production has been inadequate research and a scarcity of information on the subject. It is often falsely presumed that scientific information generated on the characteristics of bovine milk can be extrapolated to buffalo milk (Khedkar et al., Reference Khedkar, Kalyankar and Deosarkar2016).
Milk quality is the most important factor for the success of its industrialization and dairy products. This factor generates a significant increase in the price of milk, and benefits for consumers who acquire better quality products (Figueiredo et al., Reference Figueiredo, Junior and Toro2010). The physicochemical composition (mainly fat, protein, lactose, calcium and total solids) and the microbiological analysis of raw milk samples are important indicators of its quality. Measurement of the freezing point (FP) is used to detect any milk adulteration with water (Pesce et al., Reference Pesce, Salzano, De Felice, Garofalo, Liguori, De Santo, Palermo and Guarino2016). Determining the bulk milk somatic cell count (SCC) is an internationally recognized method to establish milk quality and the udder health status of the cows in the herd (Schukken et al., Reference Schukken, Wilson, Welcome, Garrison-Tikofsky and Gonzalez2003). Microbiological analysis can verify the absence of pathogenic microorganisms (or their presence, within tolerable limits), and show if the product is suitable for human consumption. The analysis of the Standard Plate Count (SPC) determines the concentration of microorganisms present in the milk and is a suitable tool to test the levels of hygiene of the milk production process, from initial management and storage to sampling (Gargouri et al., Reference Gargouri, Hamed and Elfeki2013).
The presence of antimicrobials and other veterinary drugs in milk analysis is another important quality parameter which indicates that the herd has been treated for infection. Therefore, if the withdrawal period is not respected, the milk collected from them is unsuitable for human consumption and processing (Khaniki, Reference Khaniki2007). In Brazil, the National Residue Control Plan (NRCP) defines which residues must be monitored and their maximum limits, aiming mainly to monitor the incidence of residues and prevent potential risks to the population if exposed to those products (Jank et al., Reference Jank, Martins, Arsand, Motta, Hoff, Barreto and Pizzolato2015).
Differences in the composition of buffalo milk in different localities reflect differences in breeds, management, feeding and environmental conditions (Ahmad et al., Reference Ahmad, Anjum, Huma, Sameen and Zahoor2013). These variations would strongly affect the manufacturing conditions, sensory quality and nutritional properties of the dairy products (Khedkar et al., Reference Khedkar, Kalyankar and Deosarkar2016). Despite the high nutritional value and technological importance of buffalo milk, its levels of production and consumption as well as its use in the dairy industry are still very low when compared to bovine milk. The State of Rio Grande do Sul (RS) has not yet planned specific legislation for this type of product. In Brazil, just the Secretariat of Agriculture and Supply (SAA) of the State of São Paulo has published Resolution SAA 24, which is valid only for this state (São Paulo, 1994). At present, the data regarding the microbiological and physicochemical quality of raw buffalo milk, when used as raw material for the production of derivatives in RS, are still limited. As a result, the aim of this study was to characterize samples of raw buffalo milk (whole milk collected from refrigeration tanks) from three farms that provide raw material for the production of buffalo milk derivatives in RS
Materials and methods
Farms and milk samplings
For this study, three farms were chosen for the collection of milk samples. These properties represent 100% of the official buffalo milk market which produces dairy products in RS. The farmers signed a consent form before sample collection. The characteristics of these farms are shown in the online Supplemental File.
For about one year (June 2017 to August 2018), milk samples were collected biweekly by the same person, who had been previously trained for the task. They collected the samples from the cooling tank of each farm after homogenizing the contents. For each sampling, three aliquots were obtained: 40 ml in a sterile container with Azidiol (for microbiological analyses), 40 ml in a sterile container with Bronopol (for SCC analyses) and 200 ml in a clean bottle (for physicochemical analyses). The samples were transported under refrigerated conditions to the laboratory. Farmers also provided weekly volume data for buffalo milk production during the study period. A total of 69 samples of bulk milk were collected.
Physicochemical and somatic cell count (SCC) analyses
Fat content (g/100 g), protein (g/100 g), lactose (g/100 g), SNF (g/100 g), and total solids (g/100 g) analyses were performed through mid-infrared spectrometry, according to the ISO 9622 method (2013). SCC analyses were performed using flow cytometry, according to the ISO 13366-2 method (2006). These methods were developed for bovine milk and have been adapted to assess buffalo milk, following standard guidelines. These analyses were carried out by the same accredited laboratory (Milk Quality Laboratory-Univattes, certificate number CRL 0754).
The FP was performed on a digital micro-electronic cryoscope (PZL 7000), in keeping with the manufacturer's instructions. Sample density was verified with a vibrating tube densimeter (DMA 4500, Anton Paar), according to the manufacturer's instructions. Dornic acidity was measured in line with Milk Bank Guidelines (Brazil, 2007). The FP, density and Dornic acidity measurement of each sample was the average of triplicate data. These analyses were performed at the National Agricultural Laboratory at the Ministry of Agriculture, Livestock and Food Supply.
Calcium levels were determined with a Perkin Elmer flame atomic absorption spectrometer (AAnalyst 200 series) at UFRGS’ Chemistry Institute. The samples were digested according to the modified AOAC method 991.25 (2002).
Microbiological analysis
The Standard Plate Count (SPC) was performed according to the standard ISO 4833-1 method (2013). The result was expressed as the mean of the counts multiplied by the dilution factor used in CFU/ml. In addition to SPC, the dairy products were assessed for potential pathogens recognized by Brazilian legislation,. An assessment for coagulase-positive staphylococci was conducted, according to the ISO 6888-1 method (1999). The most probable number (MPN) method was used to quantify the thermotolerant coliforms, in accordance with the ISO 7251 standard (2005). Testing for Salmonella spp. and Listeria spp. in 25 ml samples was carried out using conventional culture-based methods, according to ISO 6579-1 (2017) and ISO 11290-1 (2017), respectively. These analyses were performed at the Institute of Basic Health Sciences at the Universidade Federal do Rio Grande do Sul.
Antimicrobial and antiparasitic residues
A screening method for the analysis of 46 antimicrobial and antiparasitic residues belonging to different classes (Supplementary Material) was performed and analyzed using liquid chromatography–electrospray–tandem mass spectrometry (LC–ESI-MS/MS), as previously described (Rübensam et al., Reference Rübensam, Barreto, Hoff, Kist and Pizzolato2011; Jank et al., Reference Jank, Martins, Arsand, Motta, Hoff, Barreto and Pizzolato2015; Barreto et al., Reference Barreto, Ribeiro, Hoff and Costa2016). These analyses were performed by the Federal Laboratory of Animal and Plant Health and Inspection, at the Ministry of Agriculture, Livestock and Food Supply.
Statistical analysis
The Shapiro–Wilk test was performed to test the normality of data sets. The sizing of the number of samples was calculated considering each farm individually to a maximum error of 1°D for acidity. One-way analysis of variance (ANOVA) was used to compare the means of the parameters that presented all the data sets following normal distributions. In these cases, multiple comparisons were made with the Tukey–Kramer test (Brillinger, Reference Brillinger1984). The Kruskal–Wallis test was used to compare the medians of the parameters that presented at least one data set that did not follow a normal distribution. Multiple comparisons were made with the procedure suggested by Siegel-Castellan (Siegel and Castellan, Reference Siegel and Castellan1988). Pearson correlation coefficients were used to show correlation between parameters.
Results and discussion
The composition of buffalo milk has been studied in several countries and variations have been observed. Besides distinct methods of analysis, these divergences reflect the existence of variability among herds, management, environmental conditions and seasonality.
The mean fat measurements for all analyzed buffalo milk samples from the period of this study was 5.5 g/100 g, and the fat content ranged from 4.26 g/100 g to 9.57 g/100 g. The overall median for protein was calculated at 4.06 g/100 g with values ranging from 3.42 g/100 g to 4.60 g/100 g. The mean fat and protein contents were lower than those reported in studies conducted in Italy (Pesce et al., Reference Pesce, Salzano, De Felice, Garofalo, Liguori, De Santo, Palermo and Guarino2016; Pasquini et al., Reference Pasquini, Osimani, Tavoletti, Moreno, Clementi and Trobetta2018) and the USA (Han et al., Reference Han, Lee, Zhang and Guo2012), while they were similar to studies performed in Egypt (Elshaghabee et al ., Reference Elshaghabee, Abdel-Hamid and Walte2017) or other parts of Brazil (Filho et al., Reference Filho, Júnior, Rangel, Silva, Novaes, Júnior, Silva and Moreno2014; Bailone et al., Reference Bailone, Borra, Roça, Aguiar and Harris2017; Sales et al., Reference Sales, Rangel, Urbano, Tonhati, Galvão Júnior, Guilhermino, Aguiar and Bezerra2018). Total solids were measured at an average value of 15.5 g/100 g, ranging from 13.56 g/100 g to 19.53 g/100 g. This average was very close to other studies conducted in Brazil (Filho et al., Reference Filho, Júnior, Rangel, Silva, Novaes, Júnior, Silva and Moreno2014; Bailone et al., Reference Bailone, Borra, Roça, Aguiar and Harris2017; Sales et al., Reference Sales, Rangel, Urbano, Tonhati, Galvão Júnior, Guilhermino, Aguiar and Bezerra2018), but lower than those reported by Han et al. (Reference Han, Lee, Zhang and Guo2012). Although some physicochemical characteristics of our samples differ somewhat from those found in international studies, it is known that many of these herds of foreign buffalo are in a more advanced breeding stage, thus increasing total solids. However, the data established in this study are much closer to studies carried out in other parts of the country.
Non-fat solids (SNF) content is the entire residue left after the complete evaporation of water from milk and includes its protein, lactose and mineral content (Bassbasi et al., Reference Bassbasi, Platikanov, Tauler and Oussama2014). In this study, the median for SNF was 9.96 g/100 g with values ranging from 9.44 g/100 g to 10.61 g/100 g. SNF content is an important nutritional parameter of raw milk, and has a significant effect on the quality of milk. As such, it is one of the best factors to analyze for milk quality assurance (Bassbasi et al., Reference Bassbasi, Platikanov, Tauler and Oussama2014).
The overall median for density was 1.034 g/ml and ranged from 1.031 g/ml and 1.037 g/ml. This result is in agreement with what Ahmad et al. (Reference Ahmad, Anjum, Huma, Sameen and Zahoor2013). Sales et al. (Reference Sales, Rangel, Urbano, Tonhati, Galvão Júnior, Guilhermino, Aguiar and Bezerra2018) reported a density value of 1.033 g/ml in research carried out in another region of Brazil. The overall median for acidity was calculated at 16°D, with the values ranging from 13°D to 24°D. The FP of milk is related to its soluble constituents and is usually used to detect water added to milk. It has been reported that the basic FP of buffalo milk is lower than cow's milk (Pesce et al., Reference Pesce, Salzano, De Felice, Garofalo, Liguori, De Santo, Palermo and Guarino2016) and can be affected by seasonality and farm size (El-Salam and El-Shibiny, Reference El-Salam and El-Shibiny2011). The average result for FP was −0.527°C.
The median for lactose during the period of this study was 5.07 g/100 g, and the lactose content ranged from 4.80 g/100 g to 5.30 g/100 g. This result was slightly higher than findings from other studies in Egypt (Elshghabee et al., Reference Elshaghabee, Abdel-Hamid and Walte2017) and Italy (Pesce et al., Reference Pesce, Salzano, De Felice, Garofalo, Liguori, De Santo, Palermo and Guarino2016; Pasquini et al., Reference Pasquini, Osimani, Tavoletti, Moreno, Clementi and Trobetta2018). Compared with other studies within Brazil, our results were higher than those reported by Filho et al. (Reference Filho, Júnior, Rangel, Silva, Novaes, Júnior, Silva and Moreno2014) and Bailone et al. (Reference Bailone, Borra, Roça, Aguiar and Harris2017), but were very similar to those reported by Sales et al. (Reference Sales, Rangel, Urbano, Tonhati, Galvão Júnior, Guilhermino, Aguiar and Bezerra2018). The average calcium content found in the analyzed samples was 0.161 g/100 g, with a maximum value of 0.188 g /100 g. Buffalo milk contains high calcium content (about 1.5-fold more calcium than cow's milk), as was made apparent in several studies (Ariota et al., Reference Ariota, Campanile, Potena, Napolano, Gasparrini, Neglia and Di Palo2007; El-Salam and El-Shibiny, Reference El-Salam and El-Shibiny2011). Breed type, environmental factors, and analytical methods considerably affect the calcium content of buffalo milk (El-Salam and El-Shibiny, Reference El-Salam and El-Shibiny2011).
The mean SCC of all samples in this study was 95 × 103 cells/ml, ranging from 24 × 103 cells/ml to 216 × 103 cells/ml. This result was lower than what had been published in previous research (Filho et al., Reference Filho, Júnior, Rangel, Silva, Novaes, Júnior, Silva and Moreno2014; Pasquini et al., Reference Pasquini, Osimani, Tavoletti, Moreno, Clementi and Trobetta2018), albeit close to findings that Bailone et al. reported (Reference Bailone, Borra, Roça, Aguiar and Harris2017). To ensure the quality of manufacturing cheese, especially coagulation, Tripaldi et al. (Reference Tripaldi, Palocci, Miarelli, Catta, Orlandini, Amatiste, Bernardini and Castillo2010) suggest that the buffalo milk should not contain an SCC above 200 × 103 cell/ml. Considering the results found regarding the criterion of SCC, we can affirm that the quality of the milk collected for our study is excellent.
The mean SPC of the analyzed samples was of 9.0 × 104 CFU/ml, with results varying between 1.1 × 103 CFU/ml and 9.4 × 105 CFU/ml. This parameter is extremely important in the manufacture of derivatives. A high SPC alters the coagulation of the mass and the texture of the cheese, generating a negative result in the product yield. Durability and sensory characteristics are also affected (Teixeira et al., Reference Teixeira, Bastianetto and Oliveira2005). Our results corroborate those of many previous studies (Chye et al., Reference Chye, Abdullah and Ayob2004; Filho et al., Reference Filho, Júnior, Rangel, Silva, Novaes, Júnior, Silva and Moreno2014; Bailone et al., Reference Bailone, Borra, Roça, Aguiar and Harris2017; Sales et al., Reference Sales, Rangel, Urbano, Tonhati, Galvão Júnior, Guilhermino, Aguiar and Bezerra2018), and are only greater than the findings of some studies (Figueiredo et al., Reference Figueiredo, Junior and Toro2010; Pasquini et al., Reference Pasquini, Osimani, Tavoletti, Moreno, Clementi and Trobetta2018).
In relation to the microbiological analyses of pathogenic microorganisms, neither Salmonella spp. nor Listeria monocytogenes was detected in any of the samples. Regarding coagulase-positive staphylococci, 36 (52%) of the 69 analyzed samples were positive. Samples with coagulase-positive staphylococci were linked to all three farms: 12 from farm A, 13 from farm B and 11 from farm C. For the determination of thermotolerant coliforms, the average was 1.6 × 102 MPN/ml, with a maximum value equal to 1.1 × 103 MPN/ml. The occurrence of the coliform group and E. coli in milk indicates poor hygiene or fecal contamination. Although this milk is subsequently processed, the dairy industry must show concern for the safety of dairy products because the entry of pathogens into processing plants may lead to the persistence of these pathogens in biofilms and the subsequent contamination of processed dairy products. Furthermore, pasteurization may fail to destroy all foodborne pathogens in milk. Meanwhile, information on health hazards associated with contaminated raw milk should be made available to the public, to help prevent the consumption of untreated raw milk.
As expected, a correlation was established between acidity values, total solids, protein and fat values. Interestingly, there was also a significant correlation between calcium values, protein and total solid values. According to Ahmad et al. (Reference Ahmad, Anjum, Huma, Sameen and Zahoor2013), most of the calcium is found in insoluble form, mainly because of the high casein contents of buffalo milk, and represents 67.6–82.6% of the total calcium. For this reason, milk that has a higher protein content also has higher associated levels of calcium.
We compared our results with the parameters established in the SSA 224 (São Paulo, 1994), the only current legislation in Brazil for buffalo milk. The SSA 24 rules that the minimum fat content should be 4.5 g/100 g. While taking into account uncertainties about the method of measurement, we believe that the majority of our results satisfy this requirement. Regarding the SNF parameter, the SSA 24 rules that the minimum should be 8.57 g/100 g – a score all analyzed samples obtained. The ideal SSA density range for buffalo milk is between 1028 and 1034, the same as stated in bovine milk legislation. Some samples in this study presented values above this range, with the average result bordering the upper limit. Nevertheless, this parameter proposed by the SSA 24 could be easily questioned because, as shown in this study, buffalo milk has its own characteristics that make it denser than bovine milk. With respect to the acidity of buffalo milk, the SSA 24 describes a range between 14°D and 23°D. Once more, while the uncertainty of the method of measurement has been taken into account, all values were within this range. Regarding the freezing point (FP), the average result of this study falls within the allowed range which, according to the SSA 24, varies between −570°C and −520°C.
Table 1 presents the means of parameters of buffalo milk from each farm that follow normal distributions and the medians of parameters where at least one measured group does not follow a normal distribution. When comparing the physicochemical results of the analyzed samples of the three sites, farm B presented a significantly higher average of fat, protein, total solids and SNF than farms A and C. Samples collected from farm B also presented higher acidity. This result matches parameters reported elsewhere, since it is known that acidity correlates to total solids and fat (Ahmad et al., Reference Ahmad, Anjum, Huma, Sameen and Zahoor2013). Density and FP did not present a significant difference among samples from the three farms. This similarity also occurred in the comparison of the microbiological parameter of thermotolerant coliforms. In the comparison of sites, samples from farm A demonstrated a significantly higher lactose content than those from farm B. Samples from farm B showed a higher average calcium content, followed by farm C and then farm A. Regarding the microbiological analyses, farm B showed the highest microbiological quality with the lowest significant SPC results. This parameter may be related to the SCC result, which was also lower for farm B.
Table 1. Normal distribution means of buffalo milk parameters and medians of buffalo milk parameters that do not follow normal distributions from each farm
TS, total solids; SNF, not-fat solids; FP, freezing point; SCC, somatic cell counts; SPC, standard plate count; CFU, colony-forming unit; TC, thermotolerant coliforms; MPN, most probable number.
Means and medians with the same superscript letter do not present a significant difference. Means and medians with different superscript letters present a significant difference (ρ < 0.05).
Table 2 presents the mean measures of quality parameters of all analyzed buffalo milk samples which presented significant differences between seasons. When the results of all samples were assessed according to seasons, we verified statistically significant differences for the parameters of protein, SNF, lactose and density. These results were analyzed together with the average volume of milk produced in each season. SNF and protein levels were higher (P < 0.05) in summer and spring and lower in autumn and winter. These results corroborate those previously found by Filho et al. (Reference Filho, Júnior, Rangel, Silva, Novaes, Júnior, Silva and Moreno2014). It is reasonable to think that higher values of SNF (g/100 g) in hotter seasons relate to lower milk production, increasing the concentration of this component in the milk (Araújo et al., Reference Araújo, Rangel, Soares, Lima, Lima Júnior and Novaes2011). For the same reason, we could explain the significantly higher density in summer when compared to autumn. Unlike other studies that did not report a significant difference in lactose levels in raw buffalo milk (Bailone et al., Reference Bailone, Borra, Roça, Aguiar and Harris2017; Pasquini et al., Reference Pasquini, Osimani, Tavoletti, Moreno, Clementi and Trobetta2018), we observed that the summer and spring results of our samples were higher when compared to those collected in autumn. It needs to be taken into account that we were unable to account for possible stage-of-lactation effects when examining effects of season. No antibiotic or antiparasitic residues were detected in any of the samples analyzed during the research period. This suggests that the farms have followed legal recommendations.
Table 2. Mean measures of quality parameters of all analyzed buffalo milk samples that presented significant differences between seasons
SNF. not-fat solids. Means with the same superscript letter do not present a significant difference. Means with different superscript letters present a significant difference (ρ < 0.05).
*average volume of milk produced weekly by the three farms.
In conclusion, buffalo milk used as raw material for dairy products in southern Brazil showed satisfactory physicochemical and microbiological characteristics and presented data in accordance with the revised literature. This first study took place over one year and was subject to the range of parameters characteristic of these farms. Understanding the buffalo milk composition of this region allows the dairy industry to have greater control over efficiency and a better standardization of the physical, chemical and organoleptic characteristics of mozzarella cheese, thus ensuring stability in quality for the consumer. Moreover, such data may support the design of specific technical regulation that is not yet available in RS.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S002202992000093X
Acknowledgements
The authors sincerely thank the farms for access to the milk samples, the Laticínio Kronhardt and Cooperativa Sulriograndense de Bubalinocultores for their assistance with sample collection, and the Laboratório Nacional Agropecuário for allowing us to carry out tests at their laboratories.
