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Pasteurization efficiency of donor human milk processed by microwave heating
LWT - Food Science and Technology 115 (2019) 108466
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Pasteurization efficiency of donor human milk processed by microwave heating
T
Juliana A.S. Leitea,b, Aurea M.A. Migottoa, Mariza Landgrafb,c, Virginia S. Quintald, Jorge A.W. Guta,b, Carmen C. Tadinia,b,* a
University of São Paulo, Escola Politécnica, Department of Chemical Engineering, Main campus, São Paulo, SP, Brazil University of São Paulo, Food Research Center (FoRC/NAPAN), São Paulo, SP, Brazil c University of São Paulo, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, Main campus, São Paulo, São Paulo, SP, Brazil d University of São Paulo, University Hospital, Human Milk Bank, Main campus, São Paulo, SP, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Kinetic parameters Thermal processes Enzymatic inactivation Microbiological quality
The main objective of this work was to verify the efficiency of batch microwave-assisted heating for donor human milk and assess the activity of alkaline phosphatase (ALP) and microbiological quality. Processing was conducted at different temperatures between (50 and 80) °C and times from (5–300) s in order to evaluate the suitability of ALP as a target for the microwave process of donor human milk. The thermal inactivation of ALP followed first-order kinetics with a z-value of 4.4 °C that indicates a strong dependency of ALP inactivation with temperature. The optimal conditions (60 °C for 30 s) were determined, based on ALP inactivation, and the microbiological quality of the processed donor human milk was evaluated. No pathogens (mesophilic bacteria, coliforms at 35 °C, Salmonella spp. and Staphylococcus spp.) were detected after microwave-assisted heating and this suggests that this technology can be applied to ensure adequate safety and quality in Human Milk Banks (HMBs).
1. Introduction Human milk (HM) is universally accepted as the ideal source of nutrition for the first six months of life (Boland, 2005) because it contains vital nutrients such as carbohydrates, lipids, minerals and vitamins, along with proteins with antimicrobial activity including lactoferrin, immunoglobulins and lysozyme (Bjelakovic et al., 2009; Lönnerdal, 2003). In the absence of a mother's own milk, donor human milk (DHM), provided by human milk banks (HMB), may be a lifesaving solution for fragile infants that were born prematurely, have allergies, intolerance to feeding formulas, immunologic deficiencies or infectious diseases (Sherman, Zaghouani, & Niklas, 2014). The HMBs are specialized units located within or close a hospital neonatal unit with the mission to reduce infant mortality by providing the gold standard for feeding vulnerable neonates that cannot have access to their own mother's milk (Brasil, 2008). The main practices consist of donor screening, donor approval by health tests, collection of frozen milk at the donor's home, delivering frozen milk to HMBs, and frozen storage. Prior to pasteurization, the milk is thawed, poured into
flasks and mixed carefully for homogenization. Before and after the pasteurization, a sample of milk is taken for microbial quality control. Then, the milk is quickly cooled in an ice bath and then frozen. Frozen pasteurized DHM is typically stored for no longer than six months (Brasil, 2008; Whitney & Rolfes, 2008). Although DHM is a better alternative than infant formulas to breastfeed term and preterm infants (Escuder-Vieco et al., 2018), microbial contamination may occur in DHM if donors did not follow the hygiene processes established by HMB to extract and handle the milk (Landers & Updegrove, 2010). To reduce the risk of transmission of pathogenic microorganisms to the infant through the DHM, the thermal process known as “Holder” pasteurization or Low-Temperature LongTime, LTLT (heating at 62.5 °C for 30 min), is applied to DHM in most HMBs (Peila et al., 2016). However, heat treatment of DHM may cause reduction of nutrients and some immunological compounds, as immunoglobulins and lactoferrin, which are important to the growth and development of the infant (Arslanoglu et al., 2013; Peila et al., 2016). Some enzymes are considered technological markers for pasteurization in cow milk, such as lactoperoxidase and alkaline phosphatase. Data on lactoperoxidase in human milk are scarce, since its activity is
* Corresponding author. Department of Chemical Engineering, Escola Politécnica, University of São Paulo, Av. Professor Luciano Gualberto, trav. 3, n. 380, São Paulo, SP, 05508-010, Brazil. E-mail address: [email protected] (C.C. Tadini).
https://doi.org/10.1016/j.lwt.2019.108466 Received 23 October 2018; Received in revised form 29 July 2019; Accepted 30 July 2019 Available online 30 July 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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Nomenclature A0 A D DTref teq
Tref T(t) r2 SSE z
initial enzymatic activity [U/mL] enzymatic activity at any time [U/mL] D-value, decimal reduction time of enzymatic activity [s] D-value at reference temperature [s] equivalent time [s]
reference temperature [°C] temperature at any time [°C] coefficient of determination [−] sum of squared errors [−] z-value, temperature increase necessary to obtain a tenfold decrease in D-value [°C]
milk at optimal conditions.
very low; however, alkaline phosphatase (ALP) activity has been found in HM. The ALP activity is under detection limits in human milk after LTLT, as in cow milk after high temperature short time (HTST) pasteurization (Peila et al., 2016). Since ALP is more heat-resistant than bacterial pathogens (Coxiella burnetti and Mycobacterium tuberculosis) (Rankin, Christiansen, Lee, Banavara, & Lopez-Hernadez, 2010), it is considered as a pasteurization target in cow milk. Although ALP function in human milk and its benefits for newborn infants is unknown (Christen, Lai, Hartmann, Hartmann, & Geddes, 2013), the guideline established by NICE (NICE, 2010) recommends bacteriological screening, plus ALP test to assure the quality of pasteurized DHM. A negative result for the presence of ALP in pasteurized DHM is one evidence for an adequate pasteurization method. Besides conventional heating, microwave-assisted pasteurization has also been proven to be effective against a wide range of microorganisms with the advantage of no significant destruction of nutrients and immunological compounds, with minimum sensory changes and greater shelf-life of foods (Escuder-Vieco et al., 2018; Portela et al., 2019). During microwave heating, the heat is volumetrically generated inside the food by conversion of alternating electric field energy to thermal energy, and the rapid raise are responsible for the advantages presented by this treatment (Guo, Liu, Zhu, & Wang, 2011). Portela et al. (2019) studied the inactivation of Salmonella in infant formula during microwave processing and concluded that the treatment at 20 W and processing time greater than 750 s, using 50 mL of samples, was efficient to inactivate the pathogen, with reduction of 9.2 log CFU/ mL. Ben-Shoshan, Mandel, Lubertzky, Dollberg, and Mimouni (2016) reported that microwave irradiation at high-power setting (750 W) and 30 s, using aliquots of 5 mL, led to complete neutralization of cytomegalovirus in human milk. The presence of cytomegalovirus in HM can lead to significant mortality of preterm infants. Clare et al. (2005) compared sensory, microbiological and biochemical parameters of cow milk after two thermal treatments, microwave and UHT, and concluded that microwave processing, operating at 915 MHz in continuous flow, exhibits lower caramelized and stale/fatty flavors compared with UHT method. Both heat treatments presented similar results to microbiological analysis. Although there are many studies about the effect of microwave heating in liquid foods, there is scarce data available for human milk (Clare et al., 2005; Malinowska-Pańczyk et al., 2019; MartysiakZurowska, Puta, & Kielbratowska, 2019; Nemethy & Clore, 1990). Besides that, the few data did not use the inactivation kinetic study of ALP to establish the appropriate heating temperature and time to pasteurize DHM. Microwave-assisted heating can be considered as an alternative to pasteurize DHM in HMBs. Therefore, the equipment needs to be welldimensioned to guarantee the microbiological safety of DHM, avoiding the formation of cold and hot spots, which can degrade vital product sensory and nutritional quality attributes (Atuonwu & Tassou, 2018). The objective of this work was to study the microwave-assisted heating as a new method for donor human milk pasteurization, supplying useful information to design a specific microwave pasteurizer that attends the necessities of the HMBs. The activity of alkaline phosphatase (ALP) was determined as a technological marker of the pasteurization, while the microbiological quality was evaluated to check the efficiency of the microwave-assisted heated donor human
2. Material and methods 2.1. Sample collection Human milk samples from lactating mothers (more than 15 days of lactation) were collected from volunteers from the HMB of the University Hospital of University of São Paulo, São Paulo, Brazil. The collection of the human milk samples was approved by the Research Ethics Committee of University of São Paulo (Process CEP HU/USP: 1461/15, approval: 05/15/15). All samples were collected by health care professionals and delivered frozen to the Food Engineering Laboratory of Escola Politécnica of University of São Paulo and kept at – 30 °C in a plasma freezer (349 FV, FANEM, São Paulo, Brazil) until further thawing, pooling and processing. Prior to processing, milk from 10 different donors was pooled and mixed to provide a sample with an average composition. 2.2. Methods 2.2.1. Microwave-assisted heating Batch microwave processing was performed using a focused microwave reactor (Discover Reflux, CEM, Charlotte, USA) at 2450 MHz with a maximum power of 300 W. To a more reliable temperature monitoring, a fiber optic sensor (Fluoroptic STF-1M, LummaSense Technologies, Santa Clara, USA) was inserted at the center of the liquid. The fiber optic sensor was connected to a data acquisition system (Luxtron 812, LummaSense Technologies, Santa Clara, USA) that allowed temperature registration every 0.5 s to obtain the time-temperature history of the sample. Aliquots of 5 mL of human milk in glass tubes were submitted to different processing conditions (different time-temperature binomials) from (50–80) °C and from (5–300) s. Magnetic stirring in the reactor helped to provide a uniform heating. Once the desired time-temperature binomial was achieved, the glass tube was rapidly removed from the reactor and immersed in an ice-water bath until the temperature achieved approximately 10 °C. The range of temperature (50–80) °C was chosen because it covers the conditions applied in LTLT pasteurization and allows to evaluate the inactivation level of ALP. The processed samples were frozen immediately and maintained at – 30 °C in a plasma freezer (349 FV, FANEM, São Paulo, Brazil) until further analyses. 2.2.2. LTLT pasteurization Aliquots of 100 mL of human milk in glass bottles with a screw cap were submitted to conventional heat treatment in a thermostatic water bath (MA184, Marconi, Piracicaba, Brazil) at 62.5 °C for 30 min (LTLT pasteurization) (Brasil, 2006; NICE, 2010). After that they were cooled immediately in an ice-water bath. The same temperature data acquisition system and probe used in the microwave-assisted heating were used for the LTLT pasteurization. Once removed from the ice-water bath, the flasks were frozen kept until further analyses. The LTLT pasteurization was included in this study to compare its efficiency on microorganism inactivation with microwave-assisted heating at the optimal conditions. 2
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heating.
2.2.3. Determination of activity of alkaline phosphatase (ALP) ALP activity was measured, in triplicate, at 650 nm with a spectrophotometer UV-VIS (700 Plus, FEMTO, São Paulo, Brazil) according to the method 979.13 of Official Methods of Analysis (AOAC, 2005). To assess the residual ALP activity in the DHM samples, a standard curve was obtained using a phenol standard solution. The linear correlation for the ALP standard curve was y = 0.371x + 0.092 (x = concentration of phenol present in milk expressed as μg of phenol/ mL milk; y = absorbance at 650 nm) and the coefficient of determination (r2) was 0.999. The residual activity in the processed DHM samples was expressed as A/A0, wherein A0 is the average initial enzymatic activity and A is the average enzymatic activity after microwave treatment at a specific T of process (Toledo, 2007).
2.2.5. Chemical composition The contents of moisture and ash of the raw human milk samples were determined according to AOAC methods (2005), lipids were determined by the creamatocrit method (Brasil, 2006), protein content was determined from total nitrogen N (g/100 g) × 6.38 based on the Kjeldahl method (AOAC, 2005) and the carbohydrates were calculated by difference. Acidity, expressed as lactic acid, and initial concentration of phenol in human milk were determined according to AOAC methods (2005). All samples were analyzed in triplicate. 2.2.6. Microbial analyses In order to evaluate the efficiency of microwave-assisted heating under optimal conditions (60 °C for 30 s), microbial analyses were conducted using aliquots of 100 mL of human milk. Generally, in HMBs, after pooling, human milk is placed in individual 100 mL bottles before the LTLT pasteurization (Haiden & Ziegler, 2016). Microbial analyses were conducted for aerobic total count (mesophilic) (Morton, 2001), coliforms at 35 °C (Kornachi & Johnson, 2001), Salmonella spp. (Andrews, Flowers, Silliker, & Bailey, 2001) and Staphylococcus spp. (Lancette & Benett, 2001), before and after the microwave treatment and LTLT pasteurization. After both treatments, the milk samples were not frozen and the microbial analyses were conducted immediately, to guarantee that the freezing did not affect the vegetative cells. The microorganisms were selected based on Brazilian standards established in RDC nº 12 (Brasil, 2001) that defined the detection level as absent for coliforms at 35 °C/mL, Staphylococcus coagulase-positive/mL and Salmonella sp/25 mL and up to 102 CFU/mL for mesophilic bacteria in human milk from HMB. Microwave-assisted heating under optimal conditions (60 °C for 30 s) was also tested against some inoculated pathogens. Salmonella spp. (Andrews et al., 2001) and Staphylococcus spp. (Lancette & Benett, 2001), that are considered to be the most resistant to heat within the microorganisms studied, were inoculated in DHM with an initial microbial count of 106 CFU/mL, for each bacteria (Salmonella Typhimurium ATCC14028 and Staphylococcus aureus ATCC 25923), to test the process efficiency. The final critical control point used to consider safe the treatment was at least a 5-log reduction of the each most heatresistant pathogen (Codex, 2004, pp. 1–84).
2.2.4. Kinetic parameters determination The inactivation of microorganisms or enzymes in foods is often described using a first order kinetic model which is characterized by the D-value (decimal reduction time) and the z-value (temperature increase to obtain a tenfold decrease in the D-value). Such model for enzymatic inactivation is presented in Eqs. (1a) and (1b) (Matsui, Gut, Oliveira, & Tadini, 2008; Toledo, 2007).
A t log ⎛ ⎞ = − A D 0 ⎝ ⎠ ⎜
⎟
D = DTref a log
(1a)
(Tref − T ) z
(1b)
For each time-temperature history, that includes heating, holding and cooling periods, it was calculated the equivalent holding time (teq, which is the isothermal holding time at Tref that results in the same “lethal effect”) using an initial estimate parameter (z), based on values from literature. The Tref can be any temperature chosen within the range of study. The integral in Eq. (2) was numerically solved by the trapezoidal method using the recorded time-temperature history from each experiment (Benlloch-Tinoco, Pina-Péres, Martines-Navarrete, & Rodrigo, 2014; Matsui et al., 2008; Tajchakavit, Ramaswamy, & Fustier, 1998): ∞
teq =
∫ a log 0
(T(t ) − Tref ) dt z
(2)
Each experimental run was included in the same worksheet along with the calculation of square errors between the experimental and predicted residual enzymatic activities. The predicted residual activity for each experimental run was calculated from Eq. (3) using an initial guess for DTref. ∞
teq A log ⎛ ⎞ =− = A D 0 T ref ⎝ ⎠model ⎜
⎟
∫ a log 0
(
(Tref − T(t ) )
DT ref
z
)
dt
3. Results and discussions 3.1. Chemical composition and enzymatic activity of raw donor human milk Table 1 presents the chemical composition, acidity and the initial concentration of phenol of the raw DHM at mature stage. The values of moisture, carbohydrates, proteins, lipids, and ash found in this work are in accordance with Shi et al. (2011) and the value of acidity, expressed
(3)
Table 1 Chemical composition, acidity and initial concentration of phenol of raw donor human milk.
To minimize the sum of squared errors (SSE) between experimental and predicted residual enzymatic activities, a non-linear estimation procedure was used, defined in Eq. (4), wherein n is the number of experimental runs and subscript ‘exp’ indicates experimental data (Matsui et al., 2008). n
SSE =
∑ i=1
2
A ⎡⎛ A ⎞ ⎤ −⎛ ⎞ ⎢ A A0 ⎠model ⎥ 0 ⎠exp ⎝ ⎝ ⎣ ⎦ ⎜
⎟
⎜
⎟
(4)
The minimization of SSE was carried out with the solver tool of software Excel 2011 (Microsoft, USA) employing a generalized reduced gradient algorithm (GRG2). Initial estimates of parameters were based on values from the literature and different values were tested to prevent an early convergence to local optima (Matsui et al., 2008). The inactivation kinetic study was conducted only for microwave-assisted
Component [g/100 mL]
This workc
Shi et al. (2011)
Moisture Carbohydratea Proteinb Lipid Ash Acidity, lactic acid Initial concentration of phenol [μg phenol/ mL]
87.73 ± 0.17 7.42 ± 0.61b 1.25 ± 0.05 3.05 ± 0.79 0.21 ± 0.01 0.04 ± 0.01 1.58 ± 0.02
88.51 ± 1.68 6.97 ± 1.22 1.27 ± 0.33 3.04 ± 0.85 0.21 ± 0.06 — —
a b c
3
Calculated by difference. Content of total nitrogen. Mean of three replicates ± standard deviation.
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as lactic acid, is in accordance with Brasil (2006) and Sunaric, Jovanovic, Spasic, Denic, and Kocic (2016). The composition of the human milk protein fraction varies from mother to mother, and changes during the period of breastfeeding. The protein content of term milk is estimated to be approximately (0.9–1.2) g/100 mL (Peila et al., 2016). According to Emmett and Rogers (1997) the lipids are the main source of energy in human milk and appear to be the most variable macronutrient inter- and intra-individuals with maternal nutrition. The cited authors found an average value of human milk lipids content of 4.1 g/ 100 g, superior to that found in this work. The mainly carbohydrate in human milk is lactose (approx. 7 g/100 mL) and its concentration changes only in extreme and unusual feeding conditions (Emmett & Rogers, 1997).
Table 2 Adjusted kinetic parameters for microwave-assisted thermal inactivation of alkaline phosphatase in donor human milk. Tref [°C]
50
55
60
65
70
75
80
DTref [s] z [°C]
41217 4.4
2982
215.8
15.61
1.129
0.082
0.009
around the 45° line. Residual activity of ALP at Tref of 70 °C yielded undetectable levels for equivalent times of process above 175 s and temperatures higher than 60 °C (Fig. 2). At 50 °C, the inactivation was minimal. Escuder-Vieco et al. (2018) have proposed high-temperature shorttime pasteurization (HTST) as an alternative for DHM pasteurization and evaluated the microbiological quality and activity of enzyme indicators. They processed 14 DHM batches at three different temperatures: 70 °C, 72 °C and 75 °C at different residence times (5–25) s. In DHM heated at 70 °C there was a small amount of residual ALP activity, ranging 15% after 5 s to 3% after 25 s. According to authors, the ALP was completely inactivated at 72 °C and 75 °C, at the shortest time. After obtaining the kinetic parameters of enzymatic inactivation of ALP in human milk, the optimal conditions for microwave-assisted heating were determined based on the Murthy, Kleyn, Richardson, and Rocco (1992) study, which states that concentrations higher than 1 μg phenol/mL of milk, the product of the enzymatic activity, indicates insufficient pasteurization or contamination by raw milk. Fig. 3 shows the concentrations of phenol in human milk as function of process time. As can be observed the temperature of 55 °C and the retention time of 300 s were not sufficient (1.02 ± 0.03 μg of phenol/ mL of milk) to inactivate the ALP at the desired level, but setting the condition at 60 °C for 30 s, the inactivation of ALP was effective (0.04 ± 0.00 μg of phenol/mL of milk), presenting a concentration of phenol lower than 1 μg of phenol/mL of milk, which is considered the indicator value for adequate pasteurization for cow milk. The conditions of 60 °C for 15 s (0.98 ± 0.04 μg of phenol/mL of milk) was not considered, because the concentration of phenol was very close to the maximum limit.
3.2. Microwave-assisted inactivation kinetics of ALP Fig. 1 shows examples of the time-temperature history acquired during the microwave-assisted heating at 70 °C at different heating times. As can be observed, the temperature achieved was close to set up temperature, for each run, indicating a good temperature control of the system. From the time-temperature history the equivalent holding time (teq) was calculated according to Eq. (2) for a given reference temperature. After the minimization of SSE has been carried out, the corresponding D for each Tref and z-value were summarized in Table 2. The estimated Dvalue ranged from 687 min (41217 s) at 50 °C to 0.01 s at 80 °C with a zvalue of 4.4 °C at the studied temperature interval. Lin and Ramaswamy (2011) studied the thermal inactivation of ALP in cow milk, by conventional batch heating with temperature between (60 and 75) °C and by continuous-flow microwave heating between (65 and 70) °C. They found that the D-value of ALP varied from 183 s at 65 °C to 16.6 s at 70 °C with a z-value of 5.2 °C under conventional batch heating, and it varied from 17.6 s at 65 °C to 1.7 s at 70 °C with a z-value of 5.0 °C under microwave heating, values similar to those obtained in this work for ALP in human milk. The authors concluded that microwave heating was more efficient than conventional treatment to inactivated ALP. Fig. 2 shows the thermal inactivation curves of ALP in human milk obtained from the adjusted kinetic model for different reference temperatures as a function of the equivalent time (teq) of the process; experimental data is also included for comparison. In addition, the parity chart of the calculated residual activity as a function of the experimental residual activity indicates a good fit with spreading of the points
Fig. 1. Time-temperature histories acquired during microwave-assisted heating of donor human milk conducted at 70 °C, for different processing times. 4
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Fig. 2. Thermal inactivation curves of alkaline phosphatase in donor human milk using microwave-assisted heating at different temperatures, and the respective parity chart between predicted and experimental residual activity (A/A0)
while it was not detected after processing at 72 °C for holding time > 5 s. The mentioned authors concluded that processing at 72 °C for, at least, 10 s achieves the microbiological safety objectives currently in HMBs. Christen et al. (2013) studied the effect of ultraviolet irradiation in DHM samples inoculated with five bacteria species. They found exponential reductions of E. coli, S. epidermidis, E. cloacae, S. aureus and B. cereus in treated samples submitted to UV-C irradiation at dosage about 1500 J/L. However, this technique did not cause any reduction of ALP activity. To verify the efficiency of the microwave process at the optimal conditions (60 °C for 30 s) on microorganism inactivation, the human milk was inoculated with Salmonella Typhimurium ATCC14028 and Staphylococcus aureus ATCC 25923, separately, at a concentration of 106 CFU/mL each. After being submitted to microwave-assisted heating, no pathogens were detected. Application of microwave energy at 2450 MHz at 60 °C for 30 s could provide the same reduction of microbial population as LTLT pasteurization, worldwide used method in Human Milk Banks.
Fig. 3. Concentration of phenol (μg phenol/mL of milk) in donor human milk submitted at different process conditions.
4. Conclusions
3.3. Effect of microwave-assisted heating and LTLT pasteurization on microbial quality
The results of the present study indicate that the microwave heating could be a promising alternative to pasteurize human milk because of the obtained reduced residual ALP activity, below the value recommended by healthcare organizations, and achievement of safe
The effect of microwave-assisted heating (MWH) and LTLT on the lethality of mesophilic bacteria, coliforms at 35 °C, Salmonella spp. and Staphylococcus in human milk is detailed in Table 3. Salmonella spp. and Staphylococcus spp. were not detected in raw human milk and after submitted to both methods, whereas mesophilic bacteria, initially with a population of 2.4 × 104 CFU/mL, and coliforms at 35 °C presented low levels of detection (< 10 CFU/mL for mesophilic bacteria and < 0.03 NMP/mL for coliforms at 35 °C). The results show the efficiency of the process. Escuder-Vieco et al. (2018), as already mentioned, evaluated the HTST processing on DHM microbiological quality. They found of about 4 log CFU/mL and 5 log NMP/mL, for enterobacteria and nonfastidious bacteria, yeasts and molds, respectively, in raw DHM. After treatment, they found survivors of B. cereus at the three different temperatures,
Table 3 Survivor populations of indicator microorganisms (Total count and coliforms at 35 °C) and pathogens in donor human milk (DHM) submitted to microwaveassisted heating (MWH) at 60 °C for 30 s and to LTLT pasteurization at 62.5 °C for 30 min, in comparison those obtained from raw human milk.
5
Pathogens
Raw DHM
MWH
HoP
Mesophilic bacteria [CFU/mL] Staphylococcus coagulase positive [CFU/mL] Coliforms at 35 °C [NMP/mL] Salmonella spp. [in 25 mL]
2.4 × 104 Absent < 0.03 Absent
< 10 Absent < 0.03 Absent
< 10 Absent < 0.03 Absent
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microbiological quality. Further studies are required to investigate the effect of microwave heating on proteins such as immunoglobulins and on other bioactive compounds, such as oligosaccharides, to suggest the use of this process in HMBs.
Guo, W., Liu, Y., Zhu, X., & Wang, S. (2011). Temperature-dependent dielectric properties of honey associated with dielectric heating. Journal of Food Engineering, 102, 209–216. https://doi.org/10.1016/j.jfoodeng.2010.08.016. Haiden, N., & Ziegler, E. E. (2016). Human milk banking. Annals of Nutrition & Metabolism, 69, 8–−15. https://doi.org/10.1159/000452821. Kornachi, J. L., & Johnson, J. L. (2001). Enterobacteriaceae, Coliforms, and Escherichia coli as quality and safety indicators. In F. P. Downes, & K. Ito (Eds.). Compedium of methods for the microbiological examination of foods (pp. 69–82). Washington: APHA. Lancette, G. A., & Benett, R. W. (2001). Staphylococcus aureus and staphylococcal enterotoxins. In F. P. Downes, & K. Ito (Eds.). Compedium of methods for the microbiological examination of foods (pp. 387–400). Washington: APHA. Landers, S., & Updegrove, K. (2010). Bacteriological screening of donor human milk before and after LTLT pasteurization. Breastfeeding Medicine, 5, 117–121. https://doi. org/10.1089/bfm.2009.0032. Lin, M., & Ramaswamy, H. S. (2011). Evaluation of phosphatase inactivation kinetics in milk under continuous flow microwave and conventional heating conditions. International Journal of Food Properties, 14, 110–123. https://doi.org/10.1080/ 10942910903147841. Lönnerdal, B. (2003). Nutritional and physiologic significance of human milk proteins. American Journal of Clinical Nutrition, 77, 1537S–1543S. https://doi.org/10.1093/ ajcn/77.6.1537S. Malinowska-Pańczyk, E., Królik, K., Skorupska, K., Puta, M., Martysiak-Żurowska, D., & Kiełbratowska, B. (2019). Microwave heat treatment application to pasteurization of human milk. Innovative Food Science & Emerging Technologies, 52, 42–48. https://doi. org/10.1016/j.ifset.2018.11.005. Martysiak-Zurowska, D., Puta, M., & Kielbratowska, B. (2019). The effect of convective heating and microwave heating on antioxidant enzymes in pooled mature human milk. International Dairy Journal, 91, 41–47. https://doi.org/10.1016/j.idairyj.2018. 12.008. Matsui, K. N., Gut, J. A. W., Oliveira, P. V., & Tadini, C. C. (2008). Inactivation kinetics of polyphenol oxidase and peroxidase in green coconut water by microwave processing. Journal of Food Engineering, 88, 169–176. https://doi.org/10.1016/j.jfoodeng.2008. 02.003. Morton, D. R. (2001). Aerobic plate count. In F. P. Downes, & K. Ito (Eds.). Compedium of methods for the microbiological examination of foods (pp. 63–67). Washington: APHA. Murthy, G. K., Kleyn, D. H., Richardson, T., & Rocco, R. M. (1992). Alkaline phosphatase methods. In R. T. Marshall (Ed.). Standard methods for the examination of dairy products (pp. 412–430). Washington: APHA. Nemethy, M., & Clore, E. R. (1990). Microwave heating of infant formula and breast milk. Journal of Pediatric Health Care, 4, 131–135. https://doi.org/10.1016/0891-5245(90) 90050-G. NICE (2010). Donor breast milk banks: The operation of donor Milk Bank services. Clinical guideline: Vol. 93, (pp. 1–133). London: National Institute for Health and Care Excellence (NICE). Peila, C., Moro, G. E., Bertino, E., Cavallarin, L., Giribaldi, M., Giuliani, F., et al. (2016). The effect of holder pasteurization on nutrients and biologically-active components in donor human milk: A review. Nutrients, 8, E477−495. https://doi.org/10.3390/ nu8080477. Portela, J. B., Coimbra, P. T., Cappato, L. P., Alvarenga, V. O., Oliveira, R. B. A., Pereira, K. S., et al. (2019). Predictive model for inactivation of salmonella in infant formula during microwave heating processing. Food Control, 104, 308–312. https://doi.org/ 10.1016/j.foodcont.2019.05.006. Rankin, S. A., Christiansen, A., Lee, W., Banavara, D. S., & Lopez-Hernadez, A. (2010). Invited review: The application of alkaline phosphatase assays for the validation of milk product pasteurization. Journal of Dairy Science, 93, 5538–5551. https://doi. org/10.3168/jds.2010-3400. Sherman, M. P., Zaghouani, H., & Niklas, V. (2014). Gut microbiota, the immune system, and diet influence the neonatal gut-brain axis. Pediatric Research, 77, 127–135. http://doi.org/10.1038/pr.2014.161. Shi, Y., Sun, G., Zhang, Z., Deng, X., Kang, X., Liu, Z., et al. (2011). The chemical composition of human milk from Inner Mongolia of China. Food Chemistry, 127, 1193–1198. https://doi.org/10.1016/j.foodchem.2011.01.123. Sunaric, S., Jovanovic, T., Spasic, A., Denic, M., & Kocic, G. (2016). Comparative analysis of the physicochemical parameters of breast milk, starter infant formulas and commercial cow milks in Serbia. Acta Facultatis Medicae Naissensis, 33, 101–108. https:// doi.org/10.1515/afmnai-2016-0011. Tajchakavit, S., Ramaswamy, H. S., & Fustier, P. (1998). Enhanced destruction of spoilage microorganisms in apple juice during continuous flow microwave heating. Food Research International, 31, 713–722. https://doi.org/10.1016/S0963-9969(99) 00050-2. Toledo, R. T. (2007). Thermal process calculations. In R. T. Toledo (Ed.). Fundamentals of food process engineering (pp. 301–377). New York: Springer. Whitney, E., & Rolfes, S. R. (2008). Life cycle nutrition: Infancy, childhood, and adolescence. In E. Whitney, & S. R. Rolfes (Eds.). Understanding nutrition (pp. 547–586). Belmont: Thomson Learning.
Acknowledgments The authors acknowledge financial support from the São Paulo Research Foundation (FAPESP) under grants 2014/17534-0 and 2013/ 07914-8, financial support and scholarship from the National Council for Scientific and Technological Development (CNPq) under grants 459177-2014-1 and 306414/2017-1, and the scholarship from the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES) - Finance Code 001. Authors thanks the support from the Food Research Center (FoRC) and from the Human Milk Bank of University of São Paulo (HU/USP). References Andrews, W. H., Flowers, R. S., Silliker, J., & Bailey, J. S. (2001). Salmonella. In F. P. Downes, & K. Ito (Eds.). Compendium of methods for the microbiological examination of foods (pp. 357–380). Washington: American Public Health Association. AOAC. Association of Official Analytical Chemists (2005). Official methods of analysis (18th ed.). Washington DC: AOAC. Arslanoglu, S., Corpeleijn, W., Moro, G. E., Braegger, C., Campoy, C., Colomb, V., et al. (2013). Donor human milk for preterm infants: Current evidence and research directions. Journal of Pediatric Gastroenterology and Nutrition, 57, 535–542. https://doi. org/10.1097/MPG.0b013e3182a3af0a. Atuonwu, J. C., & Tassou, S. A. (2018). Quality assurance in microwave food processing and the enabling potentials of solid-state power generators: A review. Journal of Food Engineering, 234, 1–15. https://doi.org/10.1016/j.jfoodeng.2018.04.009. Ben-Shoshan, M., Mandel, D., Lubertzky, R., Dollberg, S., & Mimouni, F. B. (2016). Eradication of cytomegalovirus from human milk by microwave irradiation: A pilot study. Breastfeeding Medicine, 11, 186–187. https://doi.org/10.1089/bfm.2016.0016. Benlloch-Tinoco, M., Pina-Péres, M. C., Martínez-Navarrete, N., & Rodrigo, D. (2014). Listeria monocytogenes inactivation kinetics under microwave and conventional thermal processing in kiwifruit puree. Innovative Food Science & Emerging Technologies, 22, 131–136. https://doi.org/10.1016/j.ifset.2014.01.005. Bjelakovic, L., Kocic, G., Cvetkovic, T., Stojanovic, D., Najman, S., Pop-Trajkovic, Z., et al. (2009). Alkaline phosphatase activity in human milk during the first month of lactation. Acta Facultatis Medicae Naissensis, 26, 43–47. Boland, M. (2005). Exclusive breastfeeding should continue to six months. Paediatrics and Child Health, 10, 148. Brasil (2001). Ministério da Saúde. RDC nº 12, de 2 de janeiro de 2001. Regulamento técnico sobre padrões microbiológicos para alimentos. Ministry of Health. RDC nº 12, January 2nd 2001. Microbiological standards for foods [original title in Portuguese]. Agência Nacional da Vigilância Sanitária. Brasília: Brasil. Brazil. Brasil (2006). Ministério da Saúde. RDC nº 171, de 04 de setembro de 2006. Regulamento técnico para funcionamento de Bancos de Leite Humano. Ministry of Health. RDC nº 171, September 4th 2006. Technical regulation for the operation of Human Milk Banks [original title in Portuguese]. Brasília: Brasil: Agência Nacional de Vigilância Sanitária. Brasil (2008). Ministério da Saúde. Normas técnicas REDEBLH-BR para Bancos de Leite Humano. Ministry of Health. Technical standards REDEBLH-BR for Human Milk Banks. [original title in Portuguese]. Brasília: Brasil: Agência Nacional de Vigilância Sanitária. Christen, L., Lai, C. T., Hartmann, B., Hartmann, P. E., & Geddes, D. T. (2013). UltravioletC irradiation: A novel pasteurization method for donor human milk. PLoS One, 8, 1–7. https://doi.org/10.1371/journal.pone.0068120. Clare, D. A., Bang, W. S., Cartwright, G., Drake, M. A., Coronel, P., & Simunovic, J. (2005). Comparison of sensory, microbiological, and biochemical parameters of microwave versus indirect UHT fluid skim milk during storage. Journal of Dairy Science, 88, 4172–4182. https://doi.org/10.3168/jds.S0022-0302(05)73103-9. Codex Alimentarius, & Code of hygienic practice for milk and milk products (2004). Joint FAO/WHO food standards programme – Report of thirty-sixth session of the codex Committee on food hygieneWashington: Codex. Emmett, P. M., & Rogers, I. S. (1997). Properties of human milk and their relationship with maternal nutrition. Early Human Development, 49, S7−S28. https://doi.org/10. 1016/S0378-3782(97)00051-0. Escuder-Vieco, D., Espinosa-Martos, I., Rodriguez, J. M., Corzo, N., Montilla, A., Siegfried, P., et al. (2018). High-temperature short-time pasteurization system for donor milk in a Human Milk Bank setting. Frontiers in Microbiology, 9, 1–16. Article 926 https://doi. org/10.3389/fmicb.2018.00926.
6
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Pasteurization efficiency of donor human milk processed by microwave heating
T
Juliana A.S. Leitea,b, Aurea M.A. Migottoa, Mariza Landgrafb,c, Virginia S. Quintald, Jorge A.W. Guta,b, Carmen C. Tadinia,b,* a
University of São Paulo, Escola Politécnica, Department of Chemical Engineering, Main campus, São Paulo, SP, Brazil University of São Paulo, Food Research Center (FoRC/NAPAN), São Paulo, SP, Brazil c University of São Paulo, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, Main campus, São Paulo, São Paulo, SP, Brazil d University of São Paulo, University Hospital, Human Milk Bank, Main campus, São Paulo, SP, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Kinetic parameters Thermal processes Enzymatic inactivation Microbiological quality
The main objective of this work was to verify the efficiency of batch microwave-assisted heating for donor human milk and assess the activity of alkaline phosphatase (ALP) and microbiological quality. Processing was conducted at different temperatures between (50 and 80) °C and times from (5–300) s in order to evaluate the suitability of ALP as a target for the microwave process of donor human milk. The thermal inactivation of ALP followed first-order kinetics with a z-value of 4.4 °C that indicates a strong dependency of ALP inactivation with temperature. The optimal conditions (60 °C for 30 s) were determined, based on ALP inactivation, and the microbiological quality of the processed donor human milk was evaluated. No pathogens (mesophilic bacteria, coliforms at 35 °C, Salmonella spp. and Staphylococcus spp.) were detected after microwave-assisted heating and this suggests that this technology can be applied to ensure adequate safety and quality in Human Milk Banks (HMBs).
1. Introduction Human milk (HM) is universally accepted as the ideal source of nutrition for the first six months of life (Boland, 2005) because it contains vital nutrients such as carbohydrates, lipids, minerals and vitamins, along with proteins with antimicrobial activity including lactoferrin, immunoglobulins and lysozyme (Bjelakovic et al., 2009; Lönnerdal, 2003). In the absence of a mother's own milk, donor human milk (DHM), provided by human milk banks (HMB), may be a lifesaving solution for fragile infants that were born prematurely, have allergies, intolerance to feeding formulas, immunologic deficiencies or infectious diseases (Sherman, Zaghouani, & Niklas, 2014). The HMBs are specialized units located within or close a hospital neonatal unit with the mission to reduce infant mortality by providing the gold standard for feeding vulnerable neonates that cannot have access to their own mother's milk (Brasil, 2008). The main practices consist of donor screening, donor approval by health tests, collection of frozen milk at the donor's home, delivering frozen milk to HMBs, and frozen storage. Prior to pasteurization, the milk is thawed, poured into
flasks and mixed carefully for homogenization. Before and after the pasteurization, a sample of milk is taken for microbial quality control. Then, the milk is quickly cooled in an ice bath and then frozen. Frozen pasteurized DHM is typically stored for no longer than six months (Brasil, 2008; Whitney & Rolfes, 2008). Although DHM is a better alternative than infant formulas to breastfeed term and preterm infants (Escuder-Vieco et al., 2018), microbial contamination may occur in DHM if donors did not follow the hygiene processes established by HMB to extract and handle the milk (Landers & Updegrove, 2010). To reduce the risk of transmission of pathogenic microorganisms to the infant through the DHM, the thermal process known as “Holder” pasteurization or Low-Temperature LongTime, LTLT (heating at 62.5 °C for 30 min), is applied to DHM in most HMBs (Peila et al., 2016). However, heat treatment of DHM may cause reduction of nutrients and some immunological compounds, as immunoglobulins and lactoferrin, which are important to the growth and development of the infant (Arslanoglu et al., 2013; Peila et al., 2016). Some enzymes are considered technological markers for pasteurization in cow milk, such as lactoperoxidase and alkaline phosphatase. Data on lactoperoxidase in human milk are scarce, since its activity is
* Corresponding author. Department of Chemical Engineering, Escola Politécnica, University of São Paulo, Av. Professor Luciano Gualberto, trav. 3, n. 380, São Paulo, SP, 05508-010, Brazil. E-mail address: [email protected] (C.C. Tadini).
https://doi.org/10.1016/j.lwt.2019.108466 Received 23 October 2018; Received in revised form 29 July 2019; Accepted 30 July 2019 Available online 30 July 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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Nomenclature A0 A D DTref teq
Tref T(t) r2 SSE z
initial enzymatic activity [U/mL] enzymatic activity at any time [U/mL] D-value, decimal reduction time of enzymatic activity [s] D-value at reference temperature [s] equivalent time [s]
reference temperature [°C] temperature at any time [°C] coefficient of determination [−] sum of squared errors [−] z-value, temperature increase necessary to obtain a tenfold decrease in D-value [°C]
milk at optimal conditions.
very low; however, alkaline phosphatase (ALP) activity has been found in HM. The ALP activity is under detection limits in human milk after LTLT, as in cow milk after high temperature short time (HTST) pasteurization (Peila et al., 2016). Since ALP is more heat-resistant than bacterial pathogens (Coxiella burnetti and Mycobacterium tuberculosis) (Rankin, Christiansen, Lee, Banavara, & Lopez-Hernadez, 2010), it is considered as a pasteurization target in cow milk. Although ALP function in human milk and its benefits for newborn infants is unknown (Christen, Lai, Hartmann, Hartmann, & Geddes, 2013), the guideline established by NICE (NICE, 2010) recommends bacteriological screening, plus ALP test to assure the quality of pasteurized DHM. A negative result for the presence of ALP in pasteurized DHM is one evidence for an adequate pasteurization method. Besides conventional heating, microwave-assisted pasteurization has also been proven to be effective against a wide range of microorganisms with the advantage of no significant destruction of nutrients and immunological compounds, with minimum sensory changes and greater shelf-life of foods (Escuder-Vieco et al., 2018; Portela et al., 2019). During microwave heating, the heat is volumetrically generated inside the food by conversion of alternating electric field energy to thermal energy, and the rapid raise are responsible for the advantages presented by this treatment (Guo, Liu, Zhu, & Wang, 2011). Portela et al. (2019) studied the inactivation of Salmonella in infant formula during microwave processing and concluded that the treatment at 20 W and processing time greater than 750 s, using 50 mL of samples, was efficient to inactivate the pathogen, with reduction of 9.2 log CFU/ mL. Ben-Shoshan, Mandel, Lubertzky, Dollberg, and Mimouni (2016) reported that microwave irradiation at high-power setting (750 W) and 30 s, using aliquots of 5 mL, led to complete neutralization of cytomegalovirus in human milk. The presence of cytomegalovirus in HM can lead to significant mortality of preterm infants. Clare et al. (2005) compared sensory, microbiological and biochemical parameters of cow milk after two thermal treatments, microwave and UHT, and concluded that microwave processing, operating at 915 MHz in continuous flow, exhibits lower caramelized and stale/fatty flavors compared with UHT method. Both heat treatments presented similar results to microbiological analysis. Although there are many studies about the effect of microwave heating in liquid foods, there is scarce data available for human milk (Clare et al., 2005; Malinowska-Pańczyk et al., 2019; MartysiakZurowska, Puta, & Kielbratowska, 2019; Nemethy & Clore, 1990). Besides that, the few data did not use the inactivation kinetic study of ALP to establish the appropriate heating temperature and time to pasteurize DHM. Microwave-assisted heating can be considered as an alternative to pasteurize DHM in HMBs. Therefore, the equipment needs to be welldimensioned to guarantee the microbiological safety of DHM, avoiding the formation of cold and hot spots, which can degrade vital product sensory and nutritional quality attributes (Atuonwu & Tassou, 2018). The objective of this work was to study the microwave-assisted heating as a new method for donor human milk pasteurization, supplying useful information to design a specific microwave pasteurizer that attends the necessities of the HMBs. The activity of alkaline phosphatase (ALP) was determined as a technological marker of the pasteurization, while the microbiological quality was evaluated to check the efficiency of the microwave-assisted heated donor human
2. Material and methods 2.1. Sample collection Human milk samples from lactating mothers (more than 15 days of lactation) were collected from volunteers from the HMB of the University Hospital of University of São Paulo, São Paulo, Brazil. The collection of the human milk samples was approved by the Research Ethics Committee of University of São Paulo (Process CEP HU/USP: 1461/15, approval: 05/15/15). All samples were collected by health care professionals and delivered frozen to the Food Engineering Laboratory of Escola Politécnica of University of São Paulo and kept at – 30 °C in a plasma freezer (349 FV, FANEM, São Paulo, Brazil) until further thawing, pooling and processing. Prior to processing, milk from 10 different donors was pooled and mixed to provide a sample with an average composition. 2.2. Methods 2.2.1. Microwave-assisted heating Batch microwave processing was performed using a focused microwave reactor (Discover Reflux, CEM, Charlotte, USA) at 2450 MHz with a maximum power of 300 W. To a more reliable temperature monitoring, a fiber optic sensor (Fluoroptic STF-1M, LummaSense Technologies, Santa Clara, USA) was inserted at the center of the liquid. The fiber optic sensor was connected to a data acquisition system (Luxtron 812, LummaSense Technologies, Santa Clara, USA) that allowed temperature registration every 0.5 s to obtain the time-temperature history of the sample. Aliquots of 5 mL of human milk in glass tubes were submitted to different processing conditions (different time-temperature binomials) from (50–80) °C and from (5–300) s. Magnetic stirring in the reactor helped to provide a uniform heating. Once the desired time-temperature binomial was achieved, the glass tube was rapidly removed from the reactor and immersed in an ice-water bath until the temperature achieved approximately 10 °C. The range of temperature (50–80) °C was chosen because it covers the conditions applied in LTLT pasteurization and allows to evaluate the inactivation level of ALP. The processed samples were frozen immediately and maintained at – 30 °C in a plasma freezer (349 FV, FANEM, São Paulo, Brazil) until further analyses. 2.2.2. LTLT pasteurization Aliquots of 100 mL of human milk in glass bottles with a screw cap were submitted to conventional heat treatment in a thermostatic water bath (MA184, Marconi, Piracicaba, Brazil) at 62.5 °C for 30 min (LTLT pasteurization) (Brasil, 2006; NICE, 2010). After that they were cooled immediately in an ice-water bath. The same temperature data acquisition system and probe used in the microwave-assisted heating were used for the LTLT pasteurization. Once removed from the ice-water bath, the flasks were frozen kept until further analyses. The LTLT pasteurization was included in this study to compare its efficiency on microorganism inactivation with microwave-assisted heating at the optimal conditions. 2
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heating.
2.2.3. Determination of activity of alkaline phosphatase (ALP) ALP activity was measured, in triplicate, at 650 nm with a spectrophotometer UV-VIS (700 Plus, FEMTO, São Paulo, Brazil) according to the method 979.13 of Official Methods of Analysis (AOAC, 2005). To assess the residual ALP activity in the DHM samples, a standard curve was obtained using a phenol standard solution. The linear correlation for the ALP standard curve was y = 0.371x + 0.092 (x = concentration of phenol present in milk expressed as μg of phenol/ mL milk; y = absorbance at 650 nm) and the coefficient of determination (r2) was 0.999. The residual activity in the processed DHM samples was expressed as A/A0, wherein A0 is the average initial enzymatic activity and A is the average enzymatic activity after microwave treatment at a specific T of process (Toledo, 2007).
2.2.5. Chemical composition The contents of moisture and ash of the raw human milk samples were determined according to AOAC methods (2005), lipids were determined by the creamatocrit method (Brasil, 2006), protein content was determined from total nitrogen N (g/100 g) × 6.38 based on the Kjeldahl method (AOAC, 2005) and the carbohydrates were calculated by difference. Acidity, expressed as lactic acid, and initial concentration of phenol in human milk were determined according to AOAC methods (2005). All samples were analyzed in triplicate. 2.2.6. Microbial analyses In order to evaluate the efficiency of microwave-assisted heating under optimal conditions (60 °C for 30 s), microbial analyses were conducted using aliquots of 100 mL of human milk. Generally, in HMBs, after pooling, human milk is placed in individual 100 mL bottles before the LTLT pasteurization (Haiden & Ziegler, 2016). Microbial analyses were conducted for aerobic total count (mesophilic) (Morton, 2001), coliforms at 35 °C (Kornachi & Johnson, 2001), Salmonella spp. (Andrews, Flowers, Silliker, & Bailey, 2001) and Staphylococcus spp. (Lancette & Benett, 2001), before and after the microwave treatment and LTLT pasteurization. After both treatments, the milk samples were not frozen and the microbial analyses were conducted immediately, to guarantee that the freezing did not affect the vegetative cells. The microorganisms were selected based on Brazilian standards established in RDC nº 12 (Brasil, 2001) that defined the detection level as absent for coliforms at 35 °C/mL, Staphylococcus coagulase-positive/mL and Salmonella sp/25 mL and up to 102 CFU/mL for mesophilic bacteria in human milk from HMB. Microwave-assisted heating under optimal conditions (60 °C for 30 s) was also tested against some inoculated pathogens. Salmonella spp. (Andrews et al., 2001) and Staphylococcus spp. (Lancette & Benett, 2001), that are considered to be the most resistant to heat within the microorganisms studied, were inoculated in DHM with an initial microbial count of 106 CFU/mL, for each bacteria (Salmonella Typhimurium ATCC14028 and Staphylococcus aureus ATCC 25923), to test the process efficiency. The final critical control point used to consider safe the treatment was at least a 5-log reduction of the each most heatresistant pathogen (Codex, 2004, pp. 1–84).
2.2.4. Kinetic parameters determination The inactivation of microorganisms or enzymes in foods is often described using a first order kinetic model which is characterized by the D-value (decimal reduction time) and the z-value (temperature increase to obtain a tenfold decrease in the D-value). Such model for enzymatic inactivation is presented in Eqs. (1a) and (1b) (Matsui, Gut, Oliveira, & Tadini, 2008; Toledo, 2007).
A t log ⎛ ⎞ = − A D 0 ⎝ ⎠ ⎜
⎟
D = DTref a log
(1a)
(Tref − T ) z
(1b)
For each time-temperature history, that includes heating, holding and cooling periods, it was calculated the equivalent holding time (teq, which is the isothermal holding time at Tref that results in the same “lethal effect”) using an initial estimate parameter (z), based on values from literature. The Tref can be any temperature chosen within the range of study. The integral in Eq. (2) was numerically solved by the trapezoidal method using the recorded time-temperature history from each experiment (Benlloch-Tinoco, Pina-Péres, Martines-Navarrete, & Rodrigo, 2014; Matsui et al., 2008; Tajchakavit, Ramaswamy, & Fustier, 1998): ∞
teq =
∫ a log 0
(T(t ) − Tref ) dt z
(2)
Each experimental run was included in the same worksheet along with the calculation of square errors between the experimental and predicted residual enzymatic activities. The predicted residual activity for each experimental run was calculated from Eq. (3) using an initial guess for DTref. ∞
teq A log ⎛ ⎞ =− = A D 0 T ref ⎝ ⎠model ⎜
⎟
∫ a log 0
(
(Tref − T(t ) )
DT ref
z
)
dt
3. Results and discussions 3.1. Chemical composition and enzymatic activity of raw donor human milk Table 1 presents the chemical composition, acidity and the initial concentration of phenol of the raw DHM at mature stage. The values of moisture, carbohydrates, proteins, lipids, and ash found in this work are in accordance with Shi et al. (2011) and the value of acidity, expressed
(3)
Table 1 Chemical composition, acidity and initial concentration of phenol of raw donor human milk.
To minimize the sum of squared errors (SSE) between experimental and predicted residual enzymatic activities, a non-linear estimation procedure was used, defined in Eq. (4), wherein n is the number of experimental runs and subscript ‘exp’ indicates experimental data (Matsui et al., 2008). n
SSE =
∑ i=1
2
A ⎡⎛ A ⎞ ⎤ −⎛ ⎞ ⎢ A A0 ⎠model ⎥ 0 ⎠exp ⎝ ⎝ ⎣ ⎦ ⎜
⎟
⎜
⎟
(4)
The minimization of SSE was carried out with the solver tool of software Excel 2011 (Microsoft, USA) employing a generalized reduced gradient algorithm (GRG2). Initial estimates of parameters were based on values from the literature and different values were tested to prevent an early convergence to local optima (Matsui et al., 2008). The inactivation kinetic study was conducted only for microwave-assisted
Component [g/100 mL]
This workc
Shi et al. (2011)
Moisture Carbohydratea Proteinb Lipid Ash Acidity, lactic acid Initial concentration of phenol [μg phenol/ mL]
87.73 ± 0.17 7.42 ± 0.61b 1.25 ± 0.05 3.05 ± 0.79 0.21 ± 0.01 0.04 ± 0.01 1.58 ± 0.02
88.51 ± 1.68 6.97 ± 1.22 1.27 ± 0.33 3.04 ± 0.85 0.21 ± 0.06 — —
a b c
3
Calculated by difference. Content of total nitrogen. Mean of three replicates ± standard deviation.
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as lactic acid, is in accordance with Brasil (2006) and Sunaric, Jovanovic, Spasic, Denic, and Kocic (2016). The composition of the human milk protein fraction varies from mother to mother, and changes during the period of breastfeeding. The protein content of term milk is estimated to be approximately (0.9–1.2) g/100 mL (Peila et al., 2016). According to Emmett and Rogers (1997) the lipids are the main source of energy in human milk and appear to be the most variable macronutrient inter- and intra-individuals with maternal nutrition. The cited authors found an average value of human milk lipids content of 4.1 g/ 100 g, superior to that found in this work. The mainly carbohydrate in human milk is lactose (approx. 7 g/100 mL) and its concentration changes only in extreme and unusual feeding conditions (Emmett & Rogers, 1997).
Table 2 Adjusted kinetic parameters for microwave-assisted thermal inactivation of alkaline phosphatase in donor human milk. Tref [°C]
50
55
60
65
70
75
80
DTref [s] z [°C]
41217 4.4
2982
215.8
15.61
1.129
0.082
0.009
around the 45° line. Residual activity of ALP at Tref of 70 °C yielded undetectable levels for equivalent times of process above 175 s and temperatures higher than 60 °C (Fig. 2). At 50 °C, the inactivation was minimal. Escuder-Vieco et al. (2018) have proposed high-temperature shorttime pasteurization (HTST) as an alternative for DHM pasteurization and evaluated the microbiological quality and activity of enzyme indicators. They processed 14 DHM batches at three different temperatures: 70 °C, 72 °C and 75 °C at different residence times (5–25) s. In DHM heated at 70 °C there was a small amount of residual ALP activity, ranging 15% after 5 s to 3% after 25 s. According to authors, the ALP was completely inactivated at 72 °C and 75 °C, at the shortest time. After obtaining the kinetic parameters of enzymatic inactivation of ALP in human milk, the optimal conditions for microwave-assisted heating were determined based on the Murthy, Kleyn, Richardson, and Rocco (1992) study, which states that concentrations higher than 1 μg phenol/mL of milk, the product of the enzymatic activity, indicates insufficient pasteurization or contamination by raw milk. Fig. 3 shows the concentrations of phenol in human milk as function of process time. As can be observed the temperature of 55 °C and the retention time of 300 s were not sufficient (1.02 ± 0.03 μg of phenol/ mL of milk) to inactivate the ALP at the desired level, but setting the condition at 60 °C for 30 s, the inactivation of ALP was effective (0.04 ± 0.00 μg of phenol/mL of milk), presenting a concentration of phenol lower than 1 μg of phenol/mL of milk, which is considered the indicator value for adequate pasteurization for cow milk. The conditions of 60 °C for 15 s (0.98 ± 0.04 μg of phenol/mL of milk) was not considered, because the concentration of phenol was very close to the maximum limit.
3.2. Microwave-assisted inactivation kinetics of ALP Fig. 1 shows examples of the time-temperature history acquired during the microwave-assisted heating at 70 °C at different heating times. As can be observed, the temperature achieved was close to set up temperature, for each run, indicating a good temperature control of the system. From the time-temperature history the equivalent holding time (teq) was calculated according to Eq. (2) for a given reference temperature. After the minimization of SSE has been carried out, the corresponding D for each Tref and z-value were summarized in Table 2. The estimated Dvalue ranged from 687 min (41217 s) at 50 °C to 0.01 s at 80 °C with a zvalue of 4.4 °C at the studied temperature interval. Lin and Ramaswamy (2011) studied the thermal inactivation of ALP in cow milk, by conventional batch heating with temperature between (60 and 75) °C and by continuous-flow microwave heating between (65 and 70) °C. They found that the D-value of ALP varied from 183 s at 65 °C to 16.6 s at 70 °C with a z-value of 5.2 °C under conventional batch heating, and it varied from 17.6 s at 65 °C to 1.7 s at 70 °C with a z-value of 5.0 °C under microwave heating, values similar to those obtained in this work for ALP in human milk. The authors concluded that microwave heating was more efficient than conventional treatment to inactivated ALP. Fig. 2 shows the thermal inactivation curves of ALP in human milk obtained from the adjusted kinetic model for different reference temperatures as a function of the equivalent time (teq) of the process; experimental data is also included for comparison. In addition, the parity chart of the calculated residual activity as a function of the experimental residual activity indicates a good fit with spreading of the points
Fig. 1. Time-temperature histories acquired during microwave-assisted heating of donor human milk conducted at 70 °C, for different processing times. 4
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Fig. 2. Thermal inactivation curves of alkaline phosphatase in donor human milk using microwave-assisted heating at different temperatures, and the respective parity chart between predicted and experimental residual activity (A/A0)
while it was not detected after processing at 72 °C for holding time > 5 s. The mentioned authors concluded that processing at 72 °C for, at least, 10 s achieves the microbiological safety objectives currently in HMBs. Christen et al. (2013) studied the effect of ultraviolet irradiation in DHM samples inoculated with five bacteria species. They found exponential reductions of E. coli, S. epidermidis, E. cloacae, S. aureus and B. cereus in treated samples submitted to UV-C irradiation at dosage about 1500 J/L. However, this technique did not cause any reduction of ALP activity. To verify the efficiency of the microwave process at the optimal conditions (60 °C for 30 s) on microorganism inactivation, the human milk was inoculated with Salmonella Typhimurium ATCC14028 and Staphylococcus aureus ATCC 25923, separately, at a concentration of 106 CFU/mL each. After being submitted to microwave-assisted heating, no pathogens were detected. Application of microwave energy at 2450 MHz at 60 °C for 30 s could provide the same reduction of microbial population as LTLT pasteurization, worldwide used method in Human Milk Banks.
Fig. 3. Concentration of phenol (μg phenol/mL of milk) in donor human milk submitted at different process conditions.
4. Conclusions
3.3. Effect of microwave-assisted heating and LTLT pasteurization on microbial quality
The results of the present study indicate that the microwave heating could be a promising alternative to pasteurize human milk because of the obtained reduced residual ALP activity, below the value recommended by healthcare organizations, and achievement of safe
The effect of microwave-assisted heating (MWH) and LTLT on the lethality of mesophilic bacteria, coliforms at 35 °C, Salmonella spp. and Staphylococcus in human milk is detailed in Table 3. Salmonella spp. and Staphylococcus spp. were not detected in raw human milk and after submitted to both methods, whereas mesophilic bacteria, initially with a population of 2.4 × 104 CFU/mL, and coliforms at 35 °C presented low levels of detection (< 10 CFU/mL for mesophilic bacteria and < 0.03 NMP/mL for coliforms at 35 °C). The results show the efficiency of the process. Escuder-Vieco et al. (2018), as already mentioned, evaluated the HTST processing on DHM microbiological quality. They found of about 4 log CFU/mL and 5 log NMP/mL, for enterobacteria and nonfastidious bacteria, yeasts and molds, respectively, in raw DHM. After treatment, they found survivors of B. cereus at the three different temperatures,
Table 3 Survivor populations of indicator microorganisms (Total count and coliforms at 35 °C) and pathogens in donor human milk (DHM) submitted to microwaveassisted heating (MWH) at 60 °C for 30 s and to LTLT pasteurization at 62.5 °C for 30 min, in comparison those obtained from raw human milk.
5
Pathogens
Raw DHM
MWH
HoP
Mesophilic bacteria [CFU/mL] Staphylococcus coagulase positive [CFU/mL] Coliforms at 35 °C [NMP/mL] Salmonella spp. [in 25 mL]
2.4 × 104 Absent < 0.03 Absent
< 10 Absent < 0.03 Absent
< 10 Absent < 0.03 Absent
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microbiological quality. Further studies are required to investigate the effect of microwave heating on proteins such as immunoglobulins and on other bioactive compounds, such as oligosaccharides, to suggest the use of this process in HMBs.
Guo, W., Liu, Y., Zhu, X., & Wang, S. (2011). Temperature-dependent dielectric properties of honey associated with dielectric heating. Journal of Food Engineering, 102, 209–216. https://doi.org/10.1016/j.jfoodeng.2010.08.016. Haiden, N., & Ziegler, E. E. (2016). Human milk banking. Annals of Nutrition & Metabolism, 69, 8–−15. https://doi.org/10.1159/000452821. Kornachi, J. L., & Johnson, J. L. (2001). Enterobacteriaceae, Coliforms, and Escherichia coli as quality and safety indicators. In F. P. Downes, & K. Ito (Eds.). Compedium of methods for the microbiological examination of foods (pp. 69–82). Washington: APHA. Lancette, G. A., & Benett, R. W. (2001). Staphylococcus aureus and staphylococcal enterotoxins. In F. P. Downes, & K. Ito (Eds.). Compedium of methods for the microbiological examination of foods (pp. 387–400). Washington: APHA. Landers, S., & Updegrove, K. (2010). Bacteriological screening of donor human milk before and after LTLT pasteurization. Breastfeeding Medicine, 5, 117–121. https://doi. org/10.1089/bfm.2009.0032. Lin, M., & Ramaswamy, H. S. (2011). Evaluation of phosphatase inactivation kinetics in milk under continuous flow microwave and conventional heating conditions. International Journal of Food Properties, 14, 110–123. https://doi.org/10.1080/ 10942910903147841. Lönnerdal, B. (2003). Nutritional and physiologic significance of human milk proteins. American Journal of Clinical Nutrition, 77, 1537S–1543S. https://doi.org/10.1093/ ajcn/77.6.1537S. Malinowska-Pańczyk, E., Królik, K., Skorupska, K., Puta, M., Martysiak-Żurowska, D., & Kiełbratowska, B. (2019). Microwave heat treatment application to pasteurization of human milk. Innovative Food Science & Emerging Technologies, 52, 42–48. https://doi. org/10.1016/j.ifset.2018.11.005. Martysiak-Zurowska, D., Puta, M., & Kielbratowska, B. (2019). The effect of convective heating and microwave heating on antioxidant enzymes in pooled mature human milk. International Dairy Journal, 91, 41–47. https://doi.org/10.1016/j.idairyj.2018. 12.008. Matsui, K. N., Gut, J. A. W., Oliveira, P. V., & Tadini, C. C. (2008). Inactivation kinetics of polyphenol oxidase and peroxidase in green coconut water by microwave processing. Journal of Food Engineering, 88, 169–176. https://doi.org/10.1016/j.jfoodeng.2008. 02.003. Morton, D. R. (2001). Aerobic plate count. In F. P. Downes, & K. Ito (Eds.). Compedium of methods for the microbiological examination of foods (pp. 63–67). Washington: APHA. Murthy, G. K., Kleyn, D. H., Richardson, T., & Rocco, R. M. (1992). Alkaline phosphatase methods. In R. T. Marshall (Ed.). Standard methods for the examination of dairy products (pp. 412–430). Washington: APHA. Nemethy, M., & Clore, E. R. (1990). Microwave heating of infant formula and breast milk. Journal of Pediatric Health Care, 4, 131–135. https://doi.org/10.1016/0891-5245(90) 90050-G. NICE (2010). Donor breast milk banks: The operation of donor Milk Bank services. Clinical guideline: Vol. 93, (pp. 1–133). London: National Institute for Health and Care Excellence (NICE). Peila, C., Moro, G. E., Bertino, E., Cavallarin, L., Giribaldi, M., Giuliani, F., et al. (2016). The effect of holder pasteurization on nutrients and biologically-active components in donor human milk: A review. Nutrients, 8, E477−495. https://doi.org/10.3390/ nu8080477. Portela, J. B., Coimbra, P. T., Cappato, L. P., Alvarenga, V. O., Oliveira, R. B. A., Pereira, K. S., et al. (2019). Predictive model for inactivation of salmonella in infant formula during microwave heating processing. Food Control, 104, 308–312. https://doi.org/ 10.1016/j.foodcont.2019.05.006. Rankin, S. A., Christiansen, A., Lee, W., Banavara, D. S., & Lopez-Hernadez, A. (2010). Invited review: The application of alkaline phosphatase assays for the validation of milk product pasteurization. Journal of Dairy Science, 93, 5538–5551. https://doi. org/10.3168/jds.2010-3400. Sherman, M. P., Zaghouani, H., & Niklas, V. (2014). Gut microbiota, the immune system, and diet influence the neonatal gut-brain axis. Pediatric Research, 77, 127–135. http://doi.org/10.1038/pr.2014.161. Shi, Y., Sun, G., Zhang, Z., Deng, X., Kang, X., Liu, Z., et al. (2011). The chemical composition of human milk from Inner Mongolia of China. Food Chemistry, 127, 1193–1198. https://doi.org/10.1016/j.foodchem.2011.01.123. Sunaric, S., Jovanovic, T., Spasic, A., Denic, M., & Kocic, G. (2016). Comparative analysis of the physicochemical parameters of breast milk, starter infant formulas and commercial cow milks in Serbia. Acta Facultatis Medicae Naissensis, 33, 101–108. https:// doi.org/10.1515/afmnai-2016-0011. Tajchakavit, S., Ramaswamy, H. S., & Fustier, P. (1998). Enhanced destruction of spoilage microorganisms in apple juice during continuous flow microwave heating. Food Research International, 31, 713–722. https://doi.org/10.1016/S0963-9969(99) 00050-2. Toledo, R. T. (2007). Thermal process calculations. In R. T. Toledo (Ed.). Fundamentals of food process engineering (pp. 301–377). New York: Springer. Whitney, E., & Rolfes, S. R. (2008). Life cycle nutrition: Infancy, childhood, and adolescence. In E. Whitney, & S. R. Rolfes (Eds.). Understanding nutrition (pp. 547–586). Belmont: Thomson Learning.
Acknowledgments The authors acknowledge financial support from the São Paulo Research Foundation (FAPESP) under grants 2014/17534-0 and 2013/ 07914-8, financial support and scholarship from the National Council for Scientific and Technological Development (CNPq) under grants 459177-2014-1 and 306414/2017-1, and the scholarship from the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES) - Finance Code 001. Authors thanks the support from the Food Research Center (FoRC) and from the Human Milk Bank of University of São Paulo (HU/USP). References Andrews, W. H., Flowers, R. S., Silliker, J., & Bailey, J. S. (2001). Salmonella. In F. P. Downes, & K. Ito (Eds.). Compendium of methods for the microbiological examination of foods (pp. 357–380). Washington: American Public Health Association. AOAC. Association of Official Analytical Chemists (2005). Official methods of analysis (18th ed.). Washington DC: AOAC. Arslanoglu, S., Corpeleijn, W., Moro, G. E., Braegger, C., Campoy, C., Colomb, V., et al. (2013). Donor human milk for preterm infants: Current evidence and research directions. Journal of Pediatric Gastroenterology and Nutrition, 57, 535–542. https://doi. org/10.1097/MPG.0b013e3182a3af0a. Atuonwu, J. C., & Tassou, S. A. (2018). Quality assurance in microwave food processing and the enabling potentials of solid-state power generators: A review. Journal of Food Engineering, 234, 1–15. https://doi.org/10.1016/j.jfoodeng.2018.04.009. Ben-Shoshan, M., Mandel, D., Lubertzky, R., Dollberg, S., & Mimouni, F. B. (2016). Eradication of cytomegalovirus from human milk by microwave irradiation: A pilot study. Breastfeeding Medicine, 11, 186–187. https://doi.org/10.1089/bfm.2016.0016. Benlloch-Tinoco, M., Pina-Péres, M. C., Martínez-Navarrete, N., & Rodrigo, D. (2014). Listeria monocytogenes inactivation kinetics under microwave and conventional thermal processing in kiwifruit puree. Innovative Food Science & Emerging Technologies, 22, 131–136. https://doi.org/10.1016/j.ifset.2014.01.005. Bjelakovic, L., Kocic, G., Cvetkovic, T., Stojanovic, D., Najman, S., Pop-Trajkovic, Z., et al. (2009). Alkaline phosphatase activity in human milk during the first month of lactation. Acta Facultatis Medicae Naissensis, 26, 43–47. Boland, M. (2005). Exclusive breastfeeding should continue to six months. Paediatrics and Child Health, 10, 148. Brasil (2001). Ministério da Saúde. RDC nº 12, de 2 de janeiro de 2001. Regulamento técnico sobre padrões microbiológicos para alimentos. Ministry of Health. RDC nº 12, January 2nd 2001. Microbiological standards for foods [original title in Portuguese]. Agência Nacional da Vigilância Sanitária. Brasília: Brasil. Brazil. Brasil (2006). Ministério da Saúde. RDC nº 171, de 04 de setembro de 2006. Regulamento técnico para funcionamento de Bancos de Leite Humano. Ministry of Health. RDC nº 171, September 4th 2006. Technical regulation for the operation of Human Milk Banks [original title in Portuguese]. Brasília: Brasil: Agência Nacional de Vigilância Sanitária. Brasil (2008). Ministério da Saúde. Normas técnicas REDEBLH-BR para Bancos de Leite Humano. Ministry of Health. Technical standards REDEBLH-BR for Human Milk Banks. [original title in Portuguese]. Brasília: Brasil: Agência Nacional de Vigilância Sanitária. Christen, L., Lai, C. T., Hartmann, B., Hartmann, P. E., & Geddes, D. T. (2013). UltravioletC irradiation: A novel pasteurization method for donor human milk. PLoS One, 8, 1–7. https://doi.org/10.1371/journal.pone.0068120. Clare, D. A., Bang, W. S., Cartwright, G., Drake, M. A., Coronel, P., & Simunovic, J. (2005). Comparison of sensory, microbiological, and biochemical parameters of microwave versus indirect UHT fluid skim milk during storage. Journal of Dairy Science, 88, 4172–4182. https://doi.org/10.3168/jds.S0022-0302(05)73103-9. Codex Alimentarius, & Code of hygienic practice for milk and milk products (2004). Joint FAO/WHO food standards programme – Report of thirty-sixth session of the codex Committee on food hygieneWashington: Codex. Emmett, P. M., & Rogers, I. S. (1997). Properties of human milk and their relationship with maternal nutrition. Early Human Development, 49, S7−S28. https://doi.org/10. 1016/S0378-3782(97)00051-0. Escuder-Vieco, D., Espinosa-Martos, I., Rodriguez, J. M., Corzo, N., Montilla, A., Siegfried, P., et al. (2018). High-temperature short-time pasteurization system for donor milk in a Human Milk Bank setting. Frontiers in Microbiology, 9, 1–16. Article 926 https://doi. org/10.3389/fmicb.2018.00926.
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