Effect of thermoultrasound on aflatoxin M1 levels, physicochemical and microbiological properties of milk during storage

Ultrasonics - Sonochemistry 48 (2018) 396–403

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Ultrasonics - Sonochemistry journal homepage: www.elsevier.com/locate/ultson

Effect of thermoultrasound on aflatoxin M1 levels, physicochemical and microbiological properties of milk during storage

T ⁎

Tania Atzimba Hernández-Falcóna, Araceli Monter-Arciniegaa, Nelly del Socorro Cruz-Cansinoa, , Ernesto Alanís-Garcíaa, Gabriela Mariana Rodríguez-Serranob, Araceli Castañeda-Ovandoc, Mariano García-Garibayb, Esther Ramírez-Morenoa, Judith Jaimez-Ordazc a

Centro de Investigación Interdisciplinario. Área Académica de Nutrición, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Circuito Actopan-Tilcuautla s/n, Ex hacienda La Concepción, San Agustín Tlaxiaca, Hidalgo C.P. 42160. Mexico b Área de Biofisicoquímica, Departamento de Biotecnología, Universidad Autónoma Metropolitana, Av. Michoacán y la Purísima S/N, Col Vicentina, Delegación Iztapalapa, C.P. 09340, Mexico c Área Académica de Química. Instituto de Ciencias Básicas e Ingeniería, Universidad Autónoma del Estado de Hidalgo, Ciudad del Conocimiento, Carretera PachucaTulancingo Km. 4.5, Mineral de la Reforma, Hidalgo, C.P. 42184. Mexico

A R T I C LE I N FO

A B S T R A C T

Keywords: Milk Thermoultrasound Physical stability Color Antioxidant Microbiological Aflatoxin M1

The aim of this research was to determine the physicochemical properties, microbial counts and aflatoxin M1 (AFM1) levels of thermoultrasonicated, pasteurized and untreated milk (control) at days 1, 7 and 14 of storage. Thermoultrasound treatment was performed at a rate of 20 kHz for 10 or 15 min and 95% amplitude on homogenized and non-homogenized milk samples. Results showed that most physicochemical parameters were within the Mexican norms established for milk. Ultrasound treatment for 15 min reduced solids precipitation (p < 0.05) in unhomogenized milk during storage as compared to the pasteurized milk. All samples complied with aerobic mesophilic counts limits set by the Mexican norm except the control and the homogenized milk sample which was thermoultrasonicated for 10 min. Enterobacteriaceae counts of pasteurized and 15 min-thermoultrasound homogenized milks complied with the norm. The lowest levels of AFM1 were found in the 10 minthermoultrasound unhomogenized milk (0.15 ± 0.05 pg AFM1E/mL) one day after storage. Thermoultrasound did not affect the color of samples but homogenized milk treated for 10 min exhibited less total color difference. A high phenolic content was found in thermoultrasound and pasteurized milks on day 1. Thermoultrasound could be an alternative to milk pasteurization that preserves the physicochemical and microbiological quality of milk while reducing AFM1 levels.

1. Introduction In Mexico, annual bovine milk production is over 11.1 billion liters of which more than 20% is used to produce pasteurized and ultrapasteurized milk [1]. Conventional methods such as pasteurization, ultra-high temperature (UHT), low temperature-long time or sterilization are used to achieve microbiological quality and safety of milk [2]. These thermal processes have an impact on the nutritional quality and may not reduce aflatoxins (AF) in milk [3,4]. Milk from dairy cows fed contaminated feedstuffs with aflatoxin B1 (produced by Aspergillus fungi) could contain the AFM1 derivate that may cause liver cancer and other liver damage in humans [5]. Presence of AFM1 in milk is a worldwide problem found in different countries [6–17] and difficult to solve because milk is a staple food. Few studies in Mexico had demonstrated the presence of AFM1 in milk [18,19], but literature is ⁎

Corresponding author. E-mail address: [email protected] (N.d.S. Cruz-Cansino).

https://doi.org/10.1016/j.ultsonch.2018.06.018 Received 30 March 2018; Received in revised form 7 June 2018; Accepted 20 June 2018

Available online 21 June 2018 1350-4177/ © 2018 Elsevier B.V. All rights reserved.

scarce because research has focused primarily on the presence of Aspergillus and mycotoxins quantification in animal feeds and corn [20–22]. Among the technological alternatives that may reduce microbial and mycotoxin contamination of food, ultrasound is one that has been proved to reduce pathogenic microorganisms such as Escherichia coli, Pseudomonas fluorescens and Listeria monocytogenes without altering the food nutritional quality [23]. Despite the bactericidal effect of ultrasound combined with heat (thermoultrasound) [24], the dairy industry does not use this technology for preservation purposes, perhaps because comprehensive studies on the processing of thermoultrasonicated milk are limited [25]. Some thermoultrasound studies have reported the reduction of plasmin activity in skim milk [26], inactivation of mold and yeast in fermented products [27], and spore inactivation in whole milk [28]. There is a lack of studies on the use of ultrasound to reduce or eliminate the presence of AFM1 while

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the D65 illuminant and angle of observation of 10°. The milk sample (50 mL) was tempered at 20 °C before analysis. Color was measured using the CIE–L* a* b* values, where L* indicates lightness (L* = 0 or 100 indicate black and white respectively); a* the axis of chromaticity between green (−) to red (+), and b* the axis between blue (-) to yellow (+). Numerical values of L*, a* and b* were used to obtain chroma (C) and Hue angle (h°) (Eqs. (1) and (2) [33]. Color difference (ΔE) was calculated using the control and pasteurized milk as references, and following Eq. (3).

preserving the physical or chemical characteristics of milk. Therefore, the aim of this study was to evaluate the effect of thermoultrasound on the physicochemical, microbiological properties and AFM1 levels of milk during storage. 2. Materials and methods 2.1. Sample and treatments Milk was donated by a small farm from the Universidad Autonoma del Estado de Hidalgo. Immediately after receiving the milk, several samples were homogenized with a digital homogenizer (IKA-Ultra Turrax, Model T25 basic, IKA Works, Inc, North Carolina, USA) at 11,000 rpm for 5 min. The thermoultrasonication treatment was performed using a 1500 W ultrasonic processor (Sonics & Materials, Inc, VCX 1500 HV., Connecticut, USA) at a constant rate of 20 kHz for 10 and 15 min (according with preliminary studies) and 95% amplitude. Treated milks were the following: homogenized and thermoultrasonicated for 10 min (HU-10), homogenized and thermoultrasonicated for 15 min (HU-15), unhomogenized thermoultrasonicated for 10 min (U-10) and unhomogenized thermoultrasonicated for 15 min (U-15). Milk aliquots of 400 mL were placed in a jacketed vessel through which water was circulated at 45 °C from a water bath (1210610, Cole-Parmer, Vernon Hills, IL, USA) to reduce the heat generated during the ultrasound treatment. The vessel was closed introducing the ultrasound probe of 25 mm. The homogenized pasteurized milk (HP) (85 °C for 15 s) [29], was obtained using the jacketed vessel with circulating water at 87 °C, and the untreated milk (CL) were used for comparison. The inlet and outlet temperatures were recorded (Table 1), and all treated samples were kept at 8 °C and analyzed after 1, 7, and 14 days of storage.

C = [a2 + b2]1/2

(1)

h∘

(2)

=

tg−1 (b/a)

ΔE = [ΔL2 + Δa2 + Δb2]1/2

(3)

2.5. Determination of phenolic content and antioxidant activity 2.5.1. Antioxidant extraction An aliquot of milk (3 mL) and 9 mL of methanol were placed in tubes of 50 mL and the mixture was stirred using an incubator with shaking at room temperature for 15 min at 300 rpm, after it was centrifuged (Allegra 25™, Beckman Coulter., Inc., California, USA) at 10,000 rpm for 10 min at 4 °C. The supernatant was frozen until further analysis [34]. 2.5.2. Analysis of total phenolic content An aliquot of 100 µL of the sample extract was mixed with 500 µL of 1:10 diluted Folin–Ciocalteu reagent. Then, 400 µL of sodium carbonate (7.5%) were added and the mixture was incubated for 30 min at room temperature. The absorbance of the mixture was measured at 765 nm in a microplate reader (Power Wave XS UV-Biotek, software KC Junior, USA). Gallic acid was used as a reference standard and the results were expressed as milligrams of gallic acid equivalent per liter of milk (mg GAE/L) [35].

2.2. Determination of physicochemical properties 2.2.1. Titratable acidity, pH, specific gravity, total solids and solids nonfat Titratable acidity (TA) was determined by acid-base titration and the result was calculated as %TA and pH was measured by direct immersion of the electrode [30]. Specific gravity (SG), total solids (TS) and solids nonfat (SNF) were evaluated in each milk sample after treatment immersing the lactometer and recording the observed reading [31].

2.5.3. Analysis of antiradical capacity by ABTS and DPPH The radical cation 2,2́ azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS%+) was produced by reacting 7 mmol/L of ABTS stock solution with 2.45 mmol/L potassium persulfate in the dark at room temperature for 16 h before being used. The ABTS%+ solution was diluted with deionized water to an absorbance of 0.70 ± 0.10 at 754 nm. An aliquot of 20 µL of extract was added to 980 µL of the diluted ABTS%+ solution, and absorbance measurements were taken after 7 min of incubation at room temperature. The absorbance of the mixture was measured at 754 nm in the microplate reader (Power Wave XS UV-Biotek, software KC Junior, USA), and the results were expressed as micromol Trolox equivalent per liter (µmol TE/L) [36]. Antiradical activity was measured using DPPH% (1,1-diphenyl-2-picrylhydrazyl). An aliquot (500 µL) of ethanolic solution of the stable DPPH% (7.4 mg/L) was prepared and added to 100 µL of extract placed in vials. After the mixture was left to sit at room temperature for 1 h, the absorbance at 520 nm was measured in the microplate reader and was expressed as micromol Trolox equivalent per liter (µmol TE/L) [37].

2.3. Determination of physical stability An aliquot of milk (10 mL) was weighed and then centrifuged (6500, Hamilton Bell, New Jersey, USA) at 3400 rpm, for 20 min at 20 °C. The supernatant was decanted, and the resulting sediment was weighed. Physical stability was expressed as the percentage (w/w) of solids deposited after centrifugation [32]. 2.4. Determination of color Color was measured using a Hunter Lab colorimeter (Model MiniScan XE™, Hunter associates Laboratory, Inc., Virginia, USA) with Table 1 Temperatures (°C) of milk samples during treatments.

2.6. Microbiological analysis and aflatoxin content

Sample

CL

U-10

U-15

HU-10

HU-15

HP

T1 T2

8 –

31 ± 3.0 48 ± 2.0

31 ± 2.0 55 ± 1.0

30 ± 3.0 50 ± 0.0

29 ± 3.0 55 ± 0.0

8 85

2.6.1. Aerobic mesophilic and enterobacteria The microbial quality of untreated and treated milk samples was determined. Serial dilutions of milk were performed in peptone water solution for microbial count. Standard plate count agar was used to determine total aerobic mesophilic bacteria. Plates were incubated at 37 °C for 24 h [38]. Enterobacteria were determined in violet red bile glucose (VRBG) incubated at 37 °C for 24 h. After the incubation period the number of colonies was counted. Results were expressed as log

T1. Inlet temperature. T2. Outlet temperature. CL: Control. U-10: thermoultrasonicated for 10 min. U-15: thermoultrasonicated for 15 min. HU-10: homogenized and thermoultrasonicated for 10 min. HU-15: homogenized and thermoultrasonicated for 15 min. HP: homogenized and pasteurized. 397

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could be attributed to microbial fermentation [39]. Villafuerte [40] attributed the increase of density to the higher concentrations of protein and lactose during storage of milk which corresponds with the significant increase of SNF. The comparison between treatments at each day of storage showed that U-10 milk exhibited lower values than the control and pasteurized samples. The values obtained for ultrasonicated milks were similar to those described by Bermúdez–Aguirre, et al. [39] who reported lower pH values for thermosonicated milk when compared to the pasteurized and control samples, mainly due to the effect of cavitation on enzymatic action that increases the hydrolysis of esters [41]. Pasteurized milk on day 7 and U-15 sample on day 14 of storage exhibited significantly highest pH values (p < 0.05). The Official Mexican norm [42] suggests a titratable acidity for milk within the range of 0.13–0.17 %, all samples exhibited values of 0.19–0.21% on day 1 and no significant differences were observed between samples, but after 7 days the TA (%) of the control was significantly higher (0.39 ± 0.04) and on day 14 the U-15 milk had the lowest values (0.31 ± 0.02) (p < 0.05). Regarding specific gravity, significant differences were observed on day 7 where the U-10 milk exhibited the highest value (p < 0.05). All milks were within the range recommended by the Mexican norm (1.029 to 1.035 g/mL) [43]. Total soluble solids comprise fat, protein, lactose, vitamins, salts and other components in solution, and should be equal or greater than 12.5% [44]. Milk samples were within those values and significant differences were observed on day 7, where the control was lower than the U-10, U-15 and HU-10 samples. On the other hand, no significant differences were observed for SNF.

colony forming units per milliliter (CFU/mL) of milk [32]. 2.6.2. Assay of aflatoxin M1 The quantitative analysis of AFM1 was performed by competitive enzyme ELISA Kit (5121 AFMF Euro Proxima®, Arnhem, Netherlands). The milk samples were centrifuged at 3500 rpm for 10 min at room temperature. The upper creamy layer was removed with a Pasteur pipette for testing. 100 µl of the AFM1 standard solutions and test samples in duplicate were added to the wells of the micro-titer plate precoated with antibodies for AFM1 and incubated for 30 min at room temperature (20–25 °C) in the dark. Then the liquid was discarded out of the wells and the wells were filled with rinsing buffer and the liquid was discarded out again. This washing procedure was repeated thrice. In the next stage, 100 µL of enzyme conjugate was added to occupy the remaining free binding sites and rinsing buffer were used to remove unbound enzyme conjugates. Then, 50 µL of enzyme substrate and 50 µL of chromogen were added to the wells and incubated for 30 min at room temperature in the dark. The reaction was stopped by adding 100 µL stop solution to each well and absorbance was measured at 450 nm in a microplate reader (Power Wave XS UV-Biotek, KC Junior software, USA). The absorbance of the first standard multiplied by 100 was considered (100%) as zero standard and the absorbance values were expressed as percentage. The results were expressed as picograms aflatoxin M1 equivalents per milliliter (pg AFM1E/mL). 2.7. Statistical analysis All values were obtained from three independent experiments and samples were analyzed in triplicate (n = 9). Results were expressed as means ± standard deviation (SD), and the one-way analysis of variance (ANOVA) test was used to analyze the data. Differences among means were compared with a Duncan test at a level of significance of p < 0.05, using the IBM SPSS Advanced Statistics for Windows, Version 15.0 (SPSS., Inc., Chicago, USA).

3.2. Physical stability index The physical stability was evaluated to determine the percentage of settled solids (w/w) after centrifugation. High values indicate low stability due to sedimentation of particles [32]. The values obtained for milk samples are shown in Fig. 1. During the first day of storage most samples exhibited a low percentage of settled solids but at day 14, the values were higher. At each day of storage, when comparing samples, the U-15 milk presented the lower values of precipitated solids (p < 0.05) with percentages of 2.81 ± 0.21, 3.68 ± 0.37 and 3.75 ± 0.33 on day 1, 7 and 14 respectively, which indicates better stability during storage compared to the pasteurized milk that had the

3. Results and discussion 3.1. Physicochemical properties The results obtained among samples throughout storage showed that the pH decreased significantly after 14 days (Table 2) while the values of all other parameters increased. The drop of pH during storage Table 2 Mean values ( ± SD) of physicochemical properties of the samples. Days

CL

U-10

U-15

ax

ay

HU-10 axy

HU-15 axy

HP axy

pH

1 7 14

6.73 ± 0.03 5.90 ± 0.00bz 5.18 ± 0.02cy

6.67 ± 0.06 6.29 ± 0.09by 5.22 ± 0.03cy

6.71 ± 0.06 6.30 ± 0.09by 6.27 ± 0.03bv

6.70 ± 0.03 6.42 ± 0.03bx 5.54 ± 0.04cx

6.70 ± 0.06 6.41 ± 0.06bx 5.69 ± 0.09cw

6.74 ± 0.06ax 6.71 ± 0.01aw 5.05 ± 0.05bz

TA (%)

1 7 14

0.21 ± 0.05cx 0.39 ± 0.04bx 0.68 ± 0.07ax

0.20 ± 0.04bx 0.23 ± 0.03bz 0.66 ± 0.07ax

0.19 ± 0.04cx 0.28 ± 0.02by 0.31 ± 0.02ay

0.20 ± 0.05cx 0.24 ± 0.02bz 0.66 ± 0.03ax

0.20 ± 0.05cx 0.25 ± 0.00bz 0.63 ± 0.05ax

0.20 ± 0.05bx 0.23 ± 0.02bz 0.63 ± 0.07ax

SG (g/mL)

1 7 14

1.032 ± 0.00cx 1.035 ± 0.00by 1.037 ± 0.00ax

1.032 ± 0.00cx 1.037 ± 0.00bx 1.038 ± 0.00ax

1.032 ± 0.00bx 1.036 ± 0.00axy 1.037 ± 0.00ax

1.032 ± 0.00cx 1.036 ± 0.00bxy 1.038 ± 0.00ax

1.032 ± 0.00cx 1.036 ± 0.00bxy 1.038 ± 0.00ax

1.032 ± 0.00cx 1.036 ± 0.00bxy 1.037 ± 0.00ax

TS

1 7 14

11.69 ± 0.34ax 12.51 ± 0.32ay 12.96 ± 0.43ax

11.63 ± 0.43bx 12.79 ± 0.09ax 13.20 ± 0.15abx

11.64 ± 0.47bx 12.73 ± 0.11ax 13.03 ± 0.50abx

11.67 ± 0.54bx 12.75 ± 0.10ax 13.23 ± 0.25abx

11.62 ± 0.40bx 12.66 ± 0.16axy 13.13 ± 0.33abx

11.59 ± 0.40bx 12.66 ± 0.23axy 13.05 ± 0.28abx

SNF

1 7 14

8.69 ± 0.34cx 9.51 ± 0.33bx 9.97 ± 0.64ax

8.64 ± 0.43cx 9.80 ± 0.10bx 10.20 ± 0.15ax

8.64 ± 0.47bx 9.73 ± 0.11ax 10.04 ± 0.50ax

8.67 ± 0.54cx 9.75 ± 0.10bx 10.23 ± 0.25ax

8.63 ± 0.41cx 9.67 ± 0.16bxy 10.14 ± 0.33ax

8.60 ± 0.40cx 9.66 ± 0.23bxy 10.05 ± 0.29ax

a–c Different superscripts indicates significant difference (p < 0.05) between treatments through storage days. w–zDifferent superscripts indicate a significant difference (p < 0.05) between treatments for each day of storage. TA: titratable acidity, SG: specific gravity TS: total solids, SNF: solids non-fat. CL: Control. U-10: thermoultrasonicated – 10 min. U-15: thermoultrasonicated – 15 min. HU-10: homogenized – thermoultrasonicated – 10 min. HU-15: homogenized – thermoultrasonicated – 15 min. HP: homogenized – pasteurized.

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Fig. 1. Stability index for milk samples analyzed during storage at 4 °C. a-c Different letters indicate significant differences (p < 0.05) of the same treatment throughout storage days w–zDifferent letters indicate significant difference (p < 0.05) between treatments for each day of storage. CL: Control. U-10: thermoultrasonicated – 10 min. U-15: thermoultrasonicated – 15 min. HU-10: homogenized – thermoultrasonicated – 10 min. HU-15: homogenized – thermoultrasonicated – 15 min. HP: homogenized – pasteurized. % w/w: sediment solids after centrifugation.

the end of storage, the luminosity (L*) of most samples increased significantly (p < 0.05) except for the pasteurized milk. Ultrasound may be partially responsible for the increase of luminosity in treated milks [39] since cavitation decreases fat globules size allowing the refraction of light through the sample [47]. The results for pasteurized milk were similar to those described by Bermudez-Aguirre, et al. [48] who treated samples at 63 °C for 30 min. This decrease in luminosity is probably a consequence of brown pigmented products, such as pyrazines and melanoidins, generated during heat treatment and storage [49]. All samples had a* negative values meaning they were in the yellow space, the lowest a* value corresponded to the pasteurized milk (p < 0.05), while b* values were positive for all samples. Throughout

highest values on days 7 and 14 (3.63 and 10.49% respectively). This lower sedimentation may be attributable to the fragmentation of molecules in smaller particles caused by the high shearing effect that occurs during cavitation [45]. The particles dispersed in the ultrasonicated samples maintaining a stable emulsion until the last day of storage. 3.3. Color Food color is governed by chemical, biochemical, microbial and physical changes that occur during processing and storage [46]. The L*, a* and b* parameters obtained for all samples are shown in Table 3. By Table 3 Mean values ( ± SD) of the difference color parameters during storage. Days

Samples CL

U-10

U-15

HU-10

HU-15

HP

L*

1 7 14

63.02 ± 0.50bz 62.54 ± 0.66bz 70.53 ± 1.25aw

62.53 ± 1.02cz 67.8 ± 0.51by 71.38 ± 0.42av

64.04 ± 0.79cy 69.73 ± 0.24ax 68.04 ± 0.20bx

62.61 ± 1.29cz 71.5 ± 0.16aw 71.38 ± 0.40av

63.34 ± 1.29cyz 69.44 ± 0.26ax 66.98 ± 0.25by

66.61 ± 0.66bx 67.97 ± 0.50ay 62.76 ± 0.27cz

a*

1 7 14

−3.56 ± 0.04bx −4.52 ± 0.23by −2.42 ± 1.91ax

−3.63 ± 0.27axy −4.85 ± 0.03bz −3.67 ± 0.08ay

−3.65 ± 0.27bxy −4.55 ± 0.03cy −3.23 ± 0.02ay

−3.58 ± 0.25bxy −4.61 ± 0.02cyz −3.35 ± 0.06ay

−3.75 ± 0.005by −4.58 ± 0.06cy −2.95 ± 0.03axy

−4.06 ± 0.06az −3.86 ± 0.54ax −4.5 ± 0.03bz

b*

1 7 14

5.46 ± 0.201ay 1.23 ± 0.05cy 4.88 ± 0.15bu

6.12 ± 0.29ay 0.23 ± 0.16cz 2.84 ± 0.05bx

5.41 ± 0.12ay 1.3 ± 0.07cy 3.36 ± 0.05bw

5.72 ± 0.15ax 1.6 ± 0.25cw 2.56 ± 0.08by

4.81 ± 0.32az 1.43 ± 0.09cx 3.89 ± 0.07bv

4.67 ± 0.37az 1.74 ± 0.05bv 1.81 ± 0.12bz

C

1 7 14

6.50 ± 0.19ay 4.69 ± 0.21cx 5.74 ± 0.13bw

7.13 ± 0.13aw 4.85 ± 0.04bx 4.64 ± 0.06cy

6.54 ± 0.14ay 4.73 ± 0.02bx 4.66 ± 0.04 by

6.75 ± 0.21ax 4.89 ± 0.09bx 4.22 ± 0.05cz

6.10 ± 0.28az 4.80 ± 0.04bx 4.89 ± 0.06bx

6.19 ± 0.31az 4.24 ± 0.50cy 4.85 ± 0.05bx

°h

1 7 14

−57.07 ± 0.86by −15.35 ± 1.25aw −58.26 ± 1.53by

−59.28 ± 3.07cz −2.73 ± 1.96av −37.81 ± 0.84bxy

−56.04 ± 2.3cy −15.96 ± 0.89awx −46.05 ± 0.50by

−57.95 ± 1.80cyz −19.06 ± 2.76ay −37.46 ± 1.26bxy

−51.94 ± 1.62bx −17.42 ± 1.28axy −52.79 ± 0.59by

−48.87 ± 1.91cw −24.63 ± 3.18bz −21.87 ± 1.30ax

ΔE

1 7 14

– – –

1.74 ± 0.18cx 5.44 ± 0.21ay 2.37 ± 0.19bz

0.80 ± 0.13cy 7.4 ± 0.36aw 3.62 ± 0.16bx

0.40 ± 0.03cz 8.73 ± 0.24av 3.22 ± 0.26by

0.40 ± 0.16cy 6.77 ± 0.22ax 4.27 ± 0.14bw

3.27 ± 0.08cw 4.39 ± 0.17bz 9.08 ± 0.20av

ΔE

1 7 14

3.71 ± 0.45bx 1.71 ± 0.05cz 8.61 ± 0.14awy

3.22 ± 0.27byz 2.11 ± 0.11cy 5.59 ± 0.19ay

3.03 ± 0.33cz 3.58 ± 0.21bx 8.57 ± 0.31ax

3.61 ± 0.26bx 1.73 ± 0.10cz 5.10 ± 0.14az

3.33 ± 0.13cy 4.96 ± 0.35bw 8.81 ± 0.33aw

– – –

a–c Different superscripts indicate significant differences (p < 0.05) between treatments through storage days. w–zDifferent superscripts indicate a significant difference (p < 0.05) between treatments for each day of storage days. CL: Control. U-10: thermoultrasonicated – 10 min. U-15: thermoultrasonicated – 15 min. HU-10: homogenized – thermoultrasonicated – 10 min. HU-15: homogenized – thermoultrasonicated – 15 min. HP: homogenized – pasteurized.

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the days of storage, all samples showed a significant increase in a* on the last day with respect to day 7, except the HP sample which decreased. Contrary, b* values of all samples decreased (p < 0.05) after 14 days of storage. Chroma describes color saturation. All samples at day 1 exhibited the highest saturation (p < 0.05), but during storage the HP milk presented a significantly lower value on day 7 whereas at the end of storage the control had the higher chroma value compared to the other samples. Hue (°h) value is a measure of tonality. All samples during the storage had negative values which increased on day 7 except for HP sample, which showed the highest value on day 14. Samples HU-10 at day 1, HP at day 7 and U-10 at the end of storage showed the lower ΔE values respect to control milk. Total color difference (ΔE) of samples HU-10 at day 1, U-10 at day 7 and HU-15 at days 7 and 14 were the lowest (p < 0.05) compared with the HP milk. According to the standard references of ISO 12647-2 [50], ΔE values < 3 are barely perceptible by the naked eye while values of ΔE > 5 indicate evident color differences. Therefore, one day after treatment all thermoultrasonicated samples were similar in color to the control and pasteurized milk (≤1.74) but during storage most samples exhibited total color differences with the pasteurized milk (ΔE > 3). 3.4. Phenolic content and antioxidant activity 3.4.1. Total phenolic content Phenolic compounds found in bovine milk [51] are derived from pasture, animal metabolism, amino acid catabolism or microbial activity [52]. These compounds contribute to the antioxidant capacity and are found in food of animal and vegetal origin [53]. Total phenolic content in milk samples is shown on Fig. 2a. Highest values were obtained on the first day of storage in the range of 44.26–56.66 mg GAE/L compared with other days, except for HU-15 at day 14. Interactions between proteins and polyphenols may occur via van der Waals interactions, hydrogen bonding and hydrophobic interactions [54]. Sonication could break these interactions [55] which cause the release of phenol and therefore a high content in samples subjected to thermoultrasound. The comparison of samples during storage shows that HU-10 milk on day 7 and HU-15 on day 14 had the highest content (p < 0.05) of phenolic compounds with 29.93 and 55.66 mg GAE/L, respectively. An increase was observed on day 14 in all samples respect to day 7, which could be caused by microorganisms that ferment aromatic aminoacids (tyrosine, phenylalanine and tryptophan), and chemical reactions (deamination, transamination, decarboxylation and dehydrogenation) that could cause the release of phenol groups [56]. 3.4.2. Antiradical capacity by ABTS and DPPH One of the most convenient methods to express antiradical capacity is the measure by ABTS, since ascorbic acid or vitamin C is one of the main active substances responsible for antioxidant capacity [57]. The DPPH method is commonly used to evaluate the free radical scavenging potential of an antioxidant molecule, and it is considered as one of the standard and easy-to-measure colorimetric methods for the evaluation of antioxidant properties of pure compounds [58]. The results of antioxidant capacity measured as ABTS are observed in Fig. 2b, where throughout storage the values of all samples were significantly higher on the first day compared with the last day except for the U-15 milk. Comparing the treatments per day of storage, the pasteurized milk showed the highest content (p < 0.05) among samples with values of 78,762.83 ± 38.21 and 1,213.75 ± 70.40 μmol TE/L for day 1 and 7, respectively. At the end of storage, the control milk had the highest antioxidant activity (1,508.18 ± 102.26 μmol TE/L). Fig. 2c shows the antioxidant activity by DPPH of all milk samples throughout storage. DPPH values for U-10 and HP samples increased

Fig. 2. Phenolic content (a), antioxidant activity by ABTS (b) and DPPH (c) of the different samples during storage. a-c Different letters indicate significant differences (p < 0.05) between days of storage for the same treatment. w–z Different letters indicate significant differences (p < 0.05) between treatments for each day of storage CL: Control. U-10: thermoultrasonicated – 10 min. U-15: thermoultrasonicated – 15 min. HU-10: homogenized – thermoultrasonicated – 10 min. HU-15: homogenized – thermoultrasonicated – 15 min. HP: homogenized – pasteurized. mg GAE/L: milligrams gallic acid equivalent/ liter. µmol TE/L: micromol trolox equivalent/liter.

significantly on day 14 compared to day 1. The HU-15 sample exhibited the highest antioxidant activity among all samples (on day 1) but it decreased with time, while the U-15 and CL milk showed the highest (p < 0.05) values on days 7 and 14, with 24,400 and 12,166 μmol TE/ L respectively.

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Fig. 3. Content of (a) aerobic mesophilic bacteria, (b) Enterobacteriaceae count and (c) aflatoxin M1. pg AFM1E/mL: picograms aflatoxin M1 equivalent/mL. CL: Control. U-10: thermoultrasonicated – 10 min. U-15: thermoultrasonicated – 15 min. HU-10: homogenized – thermoultrasonicated – 10 min. HU-15: homogenized – thermoultrasonicated – 15 min. HP: homogenized – pasteurized.

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3.5. Microbiological and AFM1 content.

Acknowledgements

The microbial quality was determined through the aerobic mesophilic bacteria and Enterobacteriaceae counts, and AFM1 levels and shown in Fig. 3. Throughout storage and for the three microbiological parameters, the control (unpasteurized milk) exhibited the highest values. For day 1, 7 and 14 days of storage the lower values (p < 0.05) of aerobic mesophilic bacteria counts were for HP, HU-15 and U-15 (2.50 ± 0.00, 2.45 ± 0.08 and 4.8 ± 0.02 Log CFU/mL, respectively) (Fig. 3a). The Mexican norm [59] establishes a value of ≤5 Log CFU/mL for raw milk, control and HU-10 milk samples exceeded these established values. According to the specifications of the NOM-243-SSA1-2010 [59], the Enterobacteriaceae count in milk should be < 1 Log CFU/mL; HP and HU-15 milks were the only ones that complied with the standard (Fig. 3b). One day after treatment, the HP sample did not have detectable microbial load but had low counts at the end of storage (3.41 ± 0.06 Log CFU/mL). Only the U-15 milk, on day 7 showed the lowest Enterobacteriaceae count. The decrease of microbial load (aerobic mesophilic and enterobacteria) in ultrasonicated samples occurred due to cavitation and the changes in pressure that weaken the cell membranes generating lysis and death of microorganisms [60]. According to the USDA [61] ultrasound time and amplitude also affect microbial inactivation, i.e. long times of exposure and high amplitudes improves microbial reduction as was observed in milks treated for 15 min (U-15 and HU-15). On the other hand, bacterial increase at the end of storage could have benefited from the availability of cellular content [62,63] after membrane rupture caused by ultrasonic waves [64]. Recovery of respiration and biosynthesis of macromolecules would allow regeneration of the cell membrane and the physiology and structural integrity of the bacteria [65]. Regulations for aflatoxin M1 levels in milk and dairy products differ between countries. According to the Mexican norm the maximum allowed AFM1 level in milk is of 500 pg/mL [43], same as Brasil [66], China and USA [9,67], European Union have stablished levels 10-folds lower (50 pg/mL) [68] while Nigeria had more permissible values (1000 pg/mL) [69]. The values in the whole milk studied were within the levels established by the Mexican norms (around 12 pg/mL). Levels of AFM1 (Fig. 3c) increased in all the samples by the end of storage, except for HU-15 milk in which the aflatoxin decreased from 9.41 ± 0.13 to 4.51 ± 0.17 pg AFM1E/mL. These results may be interpreted carefully since the kit used to measure the aflatoxin may not detect the AFM1 linked to other compounds (i.e. proteins), which may have been degraded during storage by the microorganisms present in the samples [70]. AFM1 values for the control remained similar during storage (12.04 ± 0.20 to 12.84 ± 0.05 pg AFM1E/mL), while in pasteurized milk increased from 1.98 ± 0.03 to 8.02 ± 0.13 pg AFM1E/ mL. Differences between treatments showed that on day 1, U-10 milk had significantly the lowest aflatoxin content (0.15 ± 0.05 pg AFM1E/ mL) while on day 14 the U-15 was the sample with lower levels (2.61 ± 0.05 pg AFM1E/mL). Studies on the detoxification of AFM1 in milk by thermoultrasound are lacking, but these results may be partially explained by the findings of Mohammad [71] who observed reduction of aflatoxin in standard solutions subjected to ultrasound, due to changes in the molecular structure of the micotoxin as hydrolysis of the lactone ring or rupture of the furan double ring.

This work was financially supported by the PRODEP (Programa para el Desarrollo Profesional Docente), project for the integration of Thematic Networks of Academic Collaboration (grant No. DSA/103.5/ 15/14172). The first and second authors participated in this research and received their Bachelor’s degree in Nutrition (act numbers 216945/ 2017 and 4256/2017, respectively) from the Universidad Autónoma del Estado de Hidalgo. Conflict of interest The authors declare that there are no conflicts of interest. References [1] SAGARPA. México. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación, Panorama de la Lechería en México, 2015. Available from: < http:// www.siap.gob.mx/wp-content/uploads/boletinleche/b_lecheenemar2015. pdf > (accessed 08.07.15). [2] V.L. Barraquio, Which milk is fresh, Int. J. Dairy Sci. Process 1 (2014) 1–6. [3] M.H. Iha, C.B. Barbosa, I.A. Okada, M.W. Trucksess, Aflatoxin M1 in milk and distribution and stability of aflatoxin M1 during production and storage of yoghurt and cheese, Food Control 29 (2013) 1–6. [4] M. Tabari, K. Tabari, O. Tabari, Aflatoxin M1 determination in yoghurt produced in Guilan province of Iran using immunoaffinity column and high-performance liquid chromatography, Toxicol. Ind. Health 29 (2013) 72–76. [5] L.E. Chase, D.L. Brown, G.C, Bergstrom, S.C. Murphy, Aflatoxin M1 in Milk, Dai. Nut. Fact Sheet, 2013, pp. 70. [6] WHO. World Health Organization, Food Additives Series: 47, Aflatoxin M1 safety evaluation of certain mycotoxins in food. 2001. Available from: < http://www. inchem.org/documents/jecfa/jecmono/v47je01.htm > (accessed 11.01.17). [7] S.A.A. Sefidgar, M. Mirzae, M. Assmar, S.R. Naddaf, Aflatoxin M1 in pasteurized milk in Babol city, Mazandaran Province, Iran, Iran J. Public Health 40 (2011) 115. [8] V. Siddappa, D.K. Nanjegowda, P. Viswanath, Occurrence of Aflatoxin M1 in some samples of UHT, raw and pasteurized milk from Indian states of Karnataka and Tamilnadu, Food Chem. Toxicol. 50 (2012) 4158–4162. [9] N. Zheng, P. Sun, J.Q. Wang, Y.P. Zhen, R.W. Han, X.M. Xu, Occurrence of Aflatoxin M1 in UHT milk and pasteurized milk in China market, Food Control 29 (2013) 198–201. [10] J. Kos, J. Lević, O. Đuragić, B. Kokić, I. Miladinović, Occurrence and estimation of Aflatoxin M1 exposure in milk in Serbia, Food Control 38 (2014) 41–46. [11] N. Bilandžić, Đ. Božić, M. Đokić, M. Sedak, B.S. Kolanović, I. Varenina, Ž. Cvetnić, Seasonal effect on Aflatoxin M1 contamination in raw and UHT milk from Croatia, Food Control 40 (2014) 260–264. [12] O. Golge, A survey on the occurrence of Aflatoxin M1 in raw milk produced in Adana province of Turkey, Food Control 45 (2014) 150–155. [13] D.Y. Sarica, O. Has, S. Tasdelen, Ü. Ezer, Occurrence of Aflatoxin M1 in milk, white cheese and yoghurt from Ankara, Turkey markets, Biol. Chem. Res. (2015) 36–49. [14] A. Ismail, S. Akhtar, R.E. Levin, T. Ismail, M. Riaz, M. Amir, Aflatoxin M1: prevalence and decontamination strategies in milk and milk products, Crit. Rev. Microbiol. 42 (2016) 418–427. [15] S. Armorini, A. Altafini, A. Zaghini, P. Roncada, Occurrence of Aflatoxin M1 in conventional and organic milk offered for sale in Italy, Mycotoxin Res. 32 (2016) 237–246. [16] L. Goncalves, A. Dalla-Rosa, S.L. Gonzales, M.M.C. Feltes, E. Badiale-Furlong, G.C. Dors, Incidence of Aflatoxin M1 in fresh milk from small farms, Food Sci. Technol. 37 (2017) 11–15. [17] M.H. Movassaghghazani, M. Ghorbiani, Incidence of Aflatoxin M1 in Human and Cow Milk in Kashan, Iran, J. Food Qual. Hazards Control 4 (2017) 99–102. [18] J. Pérez, R. Gutiérrez, S. Vega, G. Díaz, G. Urbán, M. Coronado, A. Escobar, Ocurrencia de Aflatoxina M1 en leches cruda, ultrapasteurizada y orgánica producidas y comercializadas en el Altiplano Mexicano, Rev. Salud Anim. 3 (2008) 103–109. [19] W. Reyes-Velázquez, S. Patricio-Martínez, V.H. Isaías-Espinosa, M.A. Nathal-Vera, E.D. Lucas-Palacios, F. Rojo, Aflatoxinas totales en raciones de bovinos y AFM1 en leche cruda obtenida en establos del estado de Jalisco, México, Téc. Pecu. Méx. 47 (2009). [20] S. Hernández-Delgado, M.Á. Reyes-López, J.G. García-Olivares, N. Mayek-Pérez, C.A. Reyes-Méndez, Incidencia de hongos potencialmente toxígenos en maíz (Zea mays L.) almacenado y cultivado en el norte de Tamaulipas, México, Rev. Mex. Fitopatol. 25 (2007) 127–133. [21] G.N. Montes, M.C.A. Reyes, R.N. Montes, A.M.A. Cantu, Incidence of potentially toxigenic fungi in maize (Zea mays L.) grain used as food and animal feed, CYTA-J Food 7 (2009) 119–125. [22] C.M.F. Ortiz, L.B.H. Portilla, J.V. Medrano, Contaminación con micotoxinas en alimento balanceado y granos de uso pecuario en México en el año 2003, Rev. Mex. Cienc. Pecu. 44 (2012) 247–256. [23] A.A. Gabriel, Microbial inactivation in cloudy apple juice by multi-frequency Dynashock power ultrasound, Ultrason. Sonochem. 19 (2012) 346–351. [24] M.L. Garcia, J. Burgos, B. Sanz, J.A. Ordonez, Effect of heat and ultrasonic waves on

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