Effect of hydrolysis and microwave treatment on the antibacterial activity of native bovine milk lactoferrin against Cronobacter sakazakii

International Journal of Food Microbiology 319 (2020) 108495

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Effect of hydrolysis and microwave treatment on the antibacterial activity of native bovine milk lactoferrin against Cronobacter sakazakii

T

Saidou Harounaa, Indira Francoa,b, Juan J. Carramiñanaa, Arturo Blázqueza, Inés Abada, ⁎ María D. Péreza, Miguel Calvoa, Lourdes Sáncheza, a

Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA, Zaragoza, Spain b Departamento de Ciencias Naturales, Facultad de Ciencias y Tecnología, Universidad Tecnológica de Panamá, Campus Metropolitano Víctor Levi Sasso, Panamá, Panamá

A R T I C LE I N FO

A B S T R A C T

Keywords: Whey protein Antimicrobial hydrolysates Emergent pathogen Microwaves Infant formula

Bovine lactoferrin (bLF) is an iron-binding glycoprotein used in functional and therapeutic products due to its biological properties, the most important being its antimicrobial activity. In this study, hydrolysates of bovine lactoferrin (bLFH) obtained with pepsin, chymosin and microbial rennet were assayed against Cronobacter sakazakii (104 CFU/mL) in different media: phosphate buffered saline (PBS), bovine skim milk and whey, and reconstituted powdered infant formula (PIFM). The results obtained have shown that hydrolysis of bLF enhances its antibacterial activity against C. sakazakii. The three types of bLFH dissolved in PBS reduced C. sakazakii growth from a concentration of 0.1 mg/mL and inhibited it completely above 0.5 mg/mL, after 4 and 8 h of incubation at 37 °C. The three bLFH (1 and 2 mg/mL) did not show any antibacterial activity in skim milk, whey and reconstituted PIFM after 8 h of incubation at 37 °C. However, C. sakazakii growth was completely inhibited in whey when pepsin and chymosin bLFH (2 mg/mL) were combined with undigested bLF (2 mg/mL), after 8 h of incubation at 37 °C. On the other hand, the combination of any of the three hydrolysates with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM. Furthermore, the effect of temperature after reconstitution (4, 23 and 37 °C), on the antibacterial activity of bLF (2.5 and 5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10–102 CFU/mL) was also investigated. bLF at 5 mg/mL significantly reduced (p < .05) the proliferation of C. sakazakii in reconstituted PIFM at 37 °C until 2 h. C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIFM with or without bLF. The effect of microwave heating (450, 550 and 650 W for 5, 10 and 15 s) on the antibacterial activity and stability of bLF (2.5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10–102 CFU/mL) was also studied. The antibacterial activity of bLF was maintained after treatments at 450 and 550 W for 5 s, which kept 94 and 89% of bLF immunoreactivity, respectively. Moreover, microwave treatments of reconstituted PIFM with or without bLF, at 650 W for 5 s, and at 450, 550 and 650 W for 10 and 15 s, completely inactivated C. sakazakii.

1. Introduction The genus Cronobacter (formerly known as Enterobacter sakazakii), which belongs to the Enterobacteriaceae family, comprises Gram-negative, peritrichous, motile, non-spore-forming and facultative anaerobic rods (Iversen et al., 2008). This genus includes seven species, which are Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter universalis, Cronobacter condimenti, and Cronobacter dublinensis with three subspecies (Hu et al., 2018; Iversen et al., 2008; Joseph et al., 2012). Cronobacter species are opportunistic pathogens that have been associated with sporadic

⁎

infections and outbreaks (FAO/WHO, 2004, 2008). C. sakazakii, C. malonaticus and C. turicensis are the three species most often associated with infant infections (Joseph and Forsythe, 2011). Particularly, C. sakazakii can cause a severe form of neonatal meningitis, bacteraemia, necrotising enterocolitis and necrotising meningoencephalitis (Bowen and Braden, 2006; Van Acker et al., 2001). Although C. sakazakii has caused illness in all age groups of neonates (up to 4–6 weeks of age), preterm, low birth weight or immunocompromised infants are at greatest risk. Such infections have a high fatality rate, of 40–80%, and survivors often suffer from severe neurological disorders (Lai, 2001; Van Acker et al., 2001). The majority of adult cases have occurred in

Corresponding author. E-mail address: [email protected] (L. Sánchez).

https://doi.org/10.1016/j.ijfoodmicro.2019.108495 Received 17 July 2019; Received in revised form 18 December 2019; Accepted 19 December 2019 Available online 28 December 2019 0168-1605/ © 2019 Published by Elsevier B.V.

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have also been evaluated.

elderly people with a compromised immune system (Lai, 2001). C. sakazakii is an emerging foodborne pathogen that has been isolated from a wide range of food products of animal and vegetal origin, as well as from other sources, such as water and soil (Friedemann, 2007; Li et al., 2014; Shaker et al., 2007; Yao et al., 2016). Surveillance studies have detected C. sakazakii in a variety of different environments, including households, livestock facilities, and food production operations, particularly in powdered infant formula milk (PIFM) manufacturing facilities (Kandhai et al., 2004; Mullane et al., 2008; Müller et al., 2013). In this sense, the presence of C. sakazakii in PIFM is a cause for most concern (FAO/WHO, 2008). Indeed, the consumption of contaminated PIFM has been epidemiologically or microbiologically linked with several outbreaks and sporadic cases of C. sakazakii infection in infants (FAO/WHO, 2004; Flores et al., 2011; Kalyantanda et al., 2015; Van Acker et al., 2001). PIFM is not a sterile product and, therefore, it may occasionally contain pathogens, among them C. sakazakii. This pathogen is usually inactivated during the pasteurisation process applied through PIFM manufacture. Thus, the presence of C. sakazakii in the final formula may occur by environmental contamination in the post-processing, addition of contaminated ingredients after drying or colonization of poorly cleaned or sanitised equipment and utensils used in the preparation of infant formula (FAO/WHO, 2004). C. sakazakii is a bacterium with high osmotic resistance and tolerance to desiccation that can survive in milk powder for up to 24 months at room temperature (Caubilla-Barron and Forsythe, 2007). This characteristic represents a competitive advantage, which facilitates its prevalence in products with low water content, such as milk powders (Edelson-Mammel et al., 2005; Hu et al., 2018). Lactoferrin is a non-heme iron-binding glycoprotein, belonging to the transferrin family, which is secreted in the milk of the majority of mammals and is also present in the secretions of mucosal surfaces. This multifunctional protein is an essential component of the host defense innate mechanisms (Farnaud and Evans, 2003). Lactoferrin sequesters the iron from the medium, thus avoiding the growth of microorganisms, and it can also interact with the bacterial membrane causing its destabilisation (Sánchez et al., 1992a). In 1991, Tomita et al. discovered that the hydrolysis of bovine lactoferrin (bLF) by porcine pepsin generated a hydrolysate containing a cationic peptide more active against bacteria than the intact protein, which was called lactoferricin (Bellamy et al., 1992a; Bellamy et al., 1992b). This peptide is located at the Nterminal region of lactoferrin, and it seems to be involved in other functions of the protein, such as the binding to the mammal cells. Among the cationic peptides released from bLF, lactoferricin is the most studied and consists of the 17–41 aminoacidic residues that form a loop of 18 aminoacids stabilised by a disulphide bridge (Farnaud and Evans, 2003). Lactoferricin presents a marked amphipathic nature, with the aminoacidic residues at one end and the positively charges at the other end (Gifford et al., 2005). Lactoferricin is situated in a region of the molecule that does not contain any iron-binding site, which reflects that the mechanism of its antibacterial effect is independent of iron-uptaking (Haney et al., 2007; Sinha et al., 2013). Other antimicrobial cationic peptides have been characterised inside the molecule of bLF, such as lactoferrampin and LF1–11, both also located in the N-terminal half of lactoferrin molecule (Haney et al., 2007; Sinha et al., 2013; Wada and Lönnerdal, 2014). It is well known that cationic peptides derived from lactoferrin hydrolysis by pepsin have higher antibacterial activity than the intact protein (Hoek et al., 1997; Tomita et al., 1991); however, there are not many studies on the activity of lactoferrin hydrolysates produced by other proteases. Consequently, the main objective of this study has been to evaluate the effect of proteolytic hydrolysis with different enzymes on the antimicrobial activity of bLF against C. sakazakii in different media including reconstituted PIFM. Moreover, the effect of different temperatures and of microwave heating on the stability and antimicrobial activity of bLF against C. sakazakii in reconstituted PIFM,

2. Materials and methods 2.1. Culture of C. sakazakii The strain of C. sakazakii CECT 858 (equivalent to strain ATCC 29544) was supplied by the Spanish Type Culture Collection (CECT, Valencia, Spain). The freeze-dried culture of bacteria was processed following manufacturer instructions and maintained at −70 °C in sterile cryopreservation vials. The working culture was obtained by transferring a porous bead from the frozen stock culture into 10 mL of Trypticase Soy Broth (TSB, Merck, Darmstadt, Germany) and incubating it at 37 °C for 24 h. The culture was subsequently seeded on Trypticase Soy Agar (TSA, Merck) and after incubation at 37 °C for 24 h, a single colony was transferred to a tube with 10 mL of TSB and incubated at 37 °C for 24 h. This suspension was diluted in 1% sterile peptone water (Difco, Detroit, MI, USA) to be used in the antibacterial assays.

2.2. Source and preparation of native bovine lactoferrin solutions Native bLF was kindly provided by Tatua Nutritionals Company (Morrinsville, New Zealand). The purity of lactoferrin was checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE), which showed a single band corresponding to 80 KDa molecular mass; therefore, it was used without further purification. The iron saturation of bLF, determined by atomic emission spectroscopy, was below 10%. The stock solution of bLF was prepared in Milli-Q water at a concentration of about 20 mg/mL and sterilised with a low-binding protein 0.22 μm Millipore filter. After filtration, the absorbance of bLF solution was measured at 280 nm to determine the real concentration using a protein absorbance value at 280 nm (A0.1%) of 1.27 mL/cm/mg. The concentration of bLF solutions used in the different assays was adjusted considering the concentration of the stock solution.

2.3. Preparation of bovine lactoferrin hydrolysates Bovine lactoferrin hydrolysates (bLFH) were obtained following the method of Tomita et al. (1991) by using pepsin from porcine gastric mucosa (Sigma-Aldrich, St Louis, MO, USA), microbial rennet from Mucor miehei (Sigma-Aldrich) and recombinant chymosin (Chr. Hansen, Hørsholm, Denmark). A solution of 5% native bLF was prepared in ultrapure water and the pH adjusted to 3.0. Each enzyme was added to achieve a final concentration of 3% (w/w) in bLF solutions. The mixture was incubated at 37 °C for 4 h and afterwards, it was heated at 80 °C for 15 min to stop the hydrolysis. The pH of the solution was adjusted to 7.0 with 0.1 N NaOH and centrifuged at 15,000 xg for 15 min to remove insoluble material. The supernatants obtained, containing the soluble peptides, were lyophilised and stored at −20 °C until required.

2.4. Isolation of cationic peptides from bovine lactoferrin hydrolysates and analysis by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) The bLFH were filtered through a Sartobind S-75 membrane derivatised with sulfonic acid (Sartorius Stedim Biotech, Goettingen, Germany) to isolate the cationic peptides, as described by Ripollés et al. (2015). The identification of the peptides eluted with 2 M ammonium chloride was performed by MALDI-TOF MS, at the Proteomics Platform of Barcelona Scientific Park (Barcelona, Spain), using a 4700 Proteomics Analyser (Applied Biosystems, Foster City, CA, USA). 2

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2.5. Effect of hydrolysis on antibacterial activity of native bovine lactoferrin against C. sakazakii in PBS, skim milk, whey and reconstituted PIFM

2.7. Effect of microwave treatment on antibacterial activity of native bovine lactoferrin against C. sakazakii in reconstituted PIFM

The media used to evaluate the antibacterial activity of native bLF and its hydrolysates were: phosphate buffered saline (PBS) composed by 15 mM monopotassium phosphate, 8 mM dibasic sodium phosphate, 14 mM NaCl and 2 mM KCl, pH 7.4; commercial ultra-high temperature (UHT) bovine skim milk; bovine whey obtained from UHT skim milk by ultrafiltration with 100,000 MWCO hollow fiber, and commercial PIFM. Whey and PBS were filtered through 0.22 μm. PIFM was prepared according to the manufacturer instructions, dissolving 10 g in 90 mL of sterile bottled mineral water. To confirm the absence of C. sakazakii contamination, a 20 μL volume of reconstituted PIFM was inoculated onto Druggan-Forsythe-Iversen (DFI) agar (Oxoid, Basingstoke, England) and incubated at 37 °C for 24 h, not observing growth of C. sakazakii. The DFI agar is selective for C. sakazakii and the typical colonies are visualised in blue-green color (Iversen et al., 2004a). The antimicrobial activity assay was performed in a 96-well microtiter plate. First, C. sakazakii was diluted in 1% peptone water to achieve 104 CFU/mL. A volume of 100 μL of the bacterial suspension was added per well with 100 μL of different media (PBS, skim milk, whey and reconstituted PIFM), containing the bLFH at a final concentration of 0.1, 0.5 and 1 mg/mL in PBS, and of 1 and 2 mg/mL in the other media (skim milk, whey and reconstituted PIFM). Controls consisted of the different media without addition of bLF or bLFH. In the case of PBS, bLF at 1 mg/mL was included as reference. The combination of 2 mg/mL bLFH with 2 mg/mL bLF was also assayed in skim milk, whey and PIFM. The microtiter plates were incubated at 37 °C and the absorbance was measured at 620 nm in an ELISA reader (LabSystems MultiskanRC/ MS/EX Microplate Reader, Pittsburgh, PA, USA), to monitor the bacterial growth at 0, 4 and 8 h of incubation, with shaking for 15 s before reading. To determine the number of viable cells, a volume of 50 μL was taken from each well at 4 and 8 h of incubation in PBS, and at 8 h of incubation in the other media. The 50 μL of suspension with the combination of bLFH with bLF were also taken after 8 h of incubation. After preparing serial dilutions in 1% of peptone water, 20 μl were seeded onto the surface of a TSA plate, except for samples of PIFM, which were seeded onto DFI agar plates, and after incubation at 37 °C for 24 h, the CFU/mL were counted. Each well was seeded by duplicate and at least three independent experiments were performed for each assay.

For microwave heating, a 2450-MHz household microwave oven with maximum output power of 800 W (Sanyo model EM-S1050, Japan) was used. Commercial formula milk was prepared as above described and 1 mL was contaminated with 101–102 CFU/mL of C. sakazakii and added with 2.5 mg/mL of native bLF. The sample was introduced in sterile plastic vial and was placed at the centre of the microwave oven. The experiment was performed at different power levels (450, 550 and 650 W) for different time intervals (5, 10 and 15 s). The temperature of formula milk samples was measured after microwave treatment with a digital thermometer ( ± 0.1 °C) and the samples were immediately introduced in an ice-water bath. A 20 μl volumen of each treated sample was seeded onto DFI agar plates, by duplicate, counting the colonies after 24 h of incubation at 37 °C. At least, three independent experiments were performed for each treatment condition. 2.8. Effect of microwave treatment on stability of native bovine lactoferrin in reconstituted PIFM The degree of bLF denaturation, after microwave treatment of PIFM samples, was determined by measuring its concentration by radial immunodiffusion using specific antibodies obtained in rabbits, as previously described (Harouna et al., 2015; Sánchez et al., 1992b). The concentrations of bLF standards used were 0.4, 0.2, 0.1 and 0.05 mg/ mL. Milk samples were left to diffuse for 72 h in the gel containing the specific antibodies. Then, the gel was washed with PBS, dryed, stained with Coomassie blue type G (Sigma-Aldrich) and destained. The diameters of radial precipitates corresponding to standards were measured to create a standard curve, in which the values of the diameters of sample precipitates were interpolated to calculate their concentrations. 2.9. Statistical analysis Experiments were performed three times using freshly prepared samples. Mean and standard deviations were calculated from all the data obtained in the experiments performed. Data were statistically evaluated by t-test and ANOVA according to Tukey test using the Graph Pad Prism 8.0 package for Windows. 3. Results 3.1. Analysis of cationic peptides by MALDI-TOF MS

2.6. Effect of temperature on antibacterial activity of native bovine lactoferrin against C. sakazakii in reconstituted PIFM

The analysis of the spectra of hydrolysates obtained with pepsin showed some minor peaks in the range from 1700 to 2592 Da and several peaks around 3000 Da, among them the major peak showing a molecular mass of 3196 Da (Fig. 1a). The pattern of cationic peptides released from bLF after hydrolysis with chymosin (Fig. 1b) and microbial rennet (Fig. 1c) included peptides with molecular masses within a range from 1 kDa to 6.2 kDa.

Rehydrated PIFM prepared as described above was inoculated with a C. sakazakii culture diluted in 1% peptone water to achieve a final concentration of 101–102 CFU/mL. A volume of 1 mL of contaminated milk was added with 100 μl of a native bLF solution to reach final concentrations of 2.5 and 5 mg/mL. The inoculated milk samples were stored at 4 °C (as refrigeration temperature), 23 °C (as room temperature) and 37 °C (as optimum growth temperature of C. sakazakii), including controls without bLF. Formula milk samples at 4 °C were maintained at this temperature up to 6 days, taking samples daily for bacterial counting. From the formula milk samples incubated at 23 °C, aliquots were extracted for bacterial counting every 2 h and from samples at 37 °C every hour, in both cases up to 6 h. The number of viable bacterial cells in formula milk samples was determined by seeding 20 μL onto DFI agar plates, which were incubated at 37 °C for 24 h. Each sample was seeded by duplicate and at least three independent experiments were performed for each assay.

3.2. Effect of hydrolysis on antibacterial activity of native bovine lactoferrin against C. sakazakii in PBS, skim milk, whey and reconstituted PIFM The bLFH obtained with the three enzymes showed a similar slight inhibitory activity against C. sakazakii for the concentration of 0.1 mg/ mL in PBS after 4 and 8 h of incubation at 37 °C, respect to the control (bacterial suspension in PBS) as is shown in Fig. 2 (a, b, c). The inhibitory effect of the three hydrolysates at 0.1 mg/mL was similar to that produced by intact bLF at a concentration of 1 mg/mL, included as reference, after 4 and 8 h. The results showed a total inhibitory effect of bLFH obtained with the three proteolytic enzymes on the growth of C. sakazakii, at concentrations of 0.5 and 1 mg/mL, after 4 and 8 h. The antibacterial activity of bLFH against C. sakazakii was 3

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(a)

C. sakazakii (log CFU/mL)

10 ***

8

*

***

6

4

2 nd

nd

0 Control

bLF (1)

bLFH (0.1) bLFH (0.5)

bLFH (1)

Concentration of bLF and bLFH (mg/mL)

(b)

C. sakazakii (log CFU/mL)

10 ***

***

8

**

***

6

4

2 nd

0

Control

bLF (1)

nd

bLFH (0.1) bLFH (0.5)

bLFH (1)

Concentration of bLF and bLFH (mg/mL) Fig. 2. Activity of bLFH obtained with pepsin (a), recombinant chymosin (b) and microbial rennet from Mucor miehei (c) against C. sakazakii (104 CFU/mL) in PBS, after 4 (■) and 8 h ( ) of incubation at 37 °C. bLF in PBS at 1 mg/mL was included as reference. Values are the mean ± standard deviation from nine replicates analysed in three independent experiments. Significant differences for *p < .05, **p < .01 and ***p < .0001 with respect to the control for each time of incubation. nd: not detected.

Table 1 Activity of bLFH at 2 mg/mL, obtained with pepsin, recombinant chymosin and microbial rennet and effect of combination of bLFH at 2 mg/mL with bLF at 2 mg/mL, against C. sakazakii (104 CFU/mL) in whey, skim milk and formula milk, after 8 h of incubation at 37 °C. Values are the mean ± standard deviation from nine replicates analysed in three independent experiments. Significant differences for *p < .05 with respect to the control. and: not detected. Control

Fig. 1. MALDI-TOF mass spectra of the cationic peptides obtained from bovine lactoferrin hydrolysates (bLFH) with pepsin (a), recombinant chymosin (b) and microbial rennet from Mucor miehei (c). The range was from 1000 to 6000 m/z in pepsin and chymosin and 1000–7000 m/z in microbial rennet bLFH.

4

Pepsin

C. sakazakii (log CFU/mL) + Whey 8.70 ± 0.21 Skim milk 8.69 ± 0.21 PIFM 8.63 ± 0.29

bLFH 8.47 8.61 8.54

C. sakazakii (log CFU/mL) + Whey 8.07 ± 0.07 Skim milk 8.47 ± 0.33 PIFM 8.03 ± 0.11

bLF (2 mg/mL) + nda 8.14 ± 0.16* 7.85 ± 0.45

Chymosin

(2 mg/mL) ± 0.20 8.81 ± 0.11 ± 0.19 8.66 ± 0.18 ± 0.29 8.68 ± 0.09

Microbial rennet

8.71 ± 0.20 8.77 ± 0.16 8.53 ± 0.15

bLFH (2 mg/mL) nd 7.86 ± 0.17 8.17 ± 0.09* 8.10 ± 0.09* 8.01 ± 0.26 8.03 ± 0.22

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the control. Nevertheless, the combination of pepsin or chymosin hydrolysates (2 mg/mL) with intact bLF (2 mg/mL) completely inhibited the growth of C. sakazakii in whey, after 8 h of incubation (Table 1). Moreover, it was observed a low significant activity for all hyrolysates with bLF in skim milk, whereas in reconstituted PIFM showed practically no activity.

3.3. Effect of temperature on antibacterial activity of native bovine lactoferrin against C. sakazakii in reconstituted PIFM Reconstituted PIFM inoculated with C. sakazakii was kept at 4 °C for up to 6 days and samples were taken daily for culture. The results showed that C. sakazakii did not grow during storage at 4 °C in PIFM with bLF (2.5 and 5 mg/mL) or without bLF (data not shown). Samples were obtained every hour up to 6 h from inoculated PIFM kept at 37 °C. C. sakazakii growth was only observed in the first hour of incubation in PIFM samples without bLF, while no growth occured in samples with bLF at 2.5 and 5 mg/mL (Fig. 3a). After 2 h of incubation, there was bacterial growth in all samples; however, the growth was significantly lower (p < .05) in PIFM supplemented with bLF at 5 mg/mL, the reduction being about 1 log unit respect to the control. As expected, C. sakazakii counts observed at 23 °C were generally lower than those obtained at 37 °C, and bLF at 5 mg/mL seemed to exert an inhibitory activity until 2 h, although the differences were not significant respect to the control (Fig. 3b).

3.4. Effect of microwave treatment on antibacterial activity of native bovine lactoferrin against C. sakazakii in reconstituted PIFM The C. sakazakii initial population was not reduced in the PIFM samples without bLF after microwave treatments at 450 and 550 W for 5 s, which reached temperatures of 42 °C and 50 °C, respectively (Table 2). However, the microwave treatment at 650 W for 5 s that reached a similar temperature (53 °C), showed a total inactivation of C. sakazakii. Power levels of 450, 550 and 650 W for 10 and 15 s, produced temperatures above 70 °C (71 to 85 °C) and, therefore, the C. sakazakii initial population was reduced to undetectable levels. For reconstituted PIFM samples supplemented with bLF (2.5 mg/ mL), treatments at 450 and 550 W for 5 s produced similar increase of temperature (42 °C and 50 °C, respectively), but showed lower counts of C. sakazakii. This implied an approximate reduction of about 0.3 and 0.8 log cycles, respectively, with respect to the same microwave treatments of PIFM samples without bLF. This reduction was significant (p < .05) for microwave treatment at 550 W for 5 s. As it was observed in PIFM without bLF, treatment of 650 W for 5 s (53 °C), showed a total inhibitory effect on C. sakazakii growth. Treatments of 450, 550 and 650 W for 10 and 15 s also produced temperatures above 70 °C (71 to 88 °C) and, consequently, total inactivation of C. sakazakii.

Fig. 3. Effect of incubation temperatures, 37 °C (a) and 23 °C (b), on the antibacterial activity of bLF at 2.5 ( ) and 5 ( ) mg/mL against C. sakazakii (101–102 CFU/mL) in formula milk. A control without bLF (■) was included. Growth of C. sakazakii was determined every hour for 37 °C (until 6 h) and every 2 h for 23 °C (until 6 h). The values represent the mean ± standard deviation of nine replicates analysed in three independent experiments. Significant differences for *p < .05 with respect to the control. nd: not detected.

drastically reduced when the activity of the three hydrolysates was assayed in skim milk, whey or reconstituted PIFM, compared to that obtained in PBS (Table 1). The hydrolysates at concentrations of 1 mg/ mL (data not shown) and 2 mg/mL did not show any antibacterial activity on C. sakazakii in those media after 8 h of incubation, respect to

Table 2 Effect of microwave heating (at 450, 550 and 650 W for 5, 10 and 15 s), on the inhibitory activity of bLF at 2.5 mg/mL, against C. sakazakii (101–102 CFU/mL) in formula milk. Values are the mean ± standard deviation from nine replicates analysed in three independent experiments. Significant differences for *p < .05 respect to formula milk without bLF subjected to the same treatment. and: not detected. Power (W)

Temperature (°C) 5 s

Log (CFU/mL)

Temperature (°C) 10 s

Log (CFU/mL)

Temperature (°C) 15 s

Log (CFU/mL)

Without bLF 450 550 650

42 ± 6.7 50 ± 7.9 53 ± 3.0

2.2 ± 0.1 2.1 ± 0.2 nd

71 ± 1.7 72 ± 1.9 76 ± 5.4

nda nd nd

73 ± 2.0 81 ± 1.8 85 ± 1.1

nd nd nd

1.9 ± 0.2 1.3 ± 1.0* nd

71 ± 1.5 82 ± 6.1 83 ± 4.1

nd nd nd

77 ± 3.1 85 ± 4.1 88 ± 0.9

nd nd nd

With bLF (2.5 mg/mL) 450 42 ± 2.2 550 50 ± 2.4 650 53 ± 10.1

5

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Concentration of bLF (%)

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100 90 80 70 60 50 40 30 20 10 0

2012a, 2012b; Murdock and Matthews, 2002; Venkitanarayanan et al., 1999). This reduced effect could be attributed to possible interference of salts, divalent cations or proteic, lipidic or glucidic components (AlNabulsi et al., 2009; Bellamy et al., 1993; Branen and Davidson, 2000; Murdock and Matthews, 2002). However, the growth of C. sakazakii was completely inhibited in whey when pepsin and chymosin bLFH were combined with whole bLF. The hydrolysates could cause disruption of the cell membrane, contributing to the destabilisation of bacterial membrane and the iron chelating-mediated antimicrobial activity produced by intact bLF (Farnaud and Evans, 2003; Jenssen and Hancock, 2009). Therefore, the results obtained in our study showed that it would be particularly interesting to investigate some procedures to improve the antibacterial activity of bLFH combined with bLF in milk products, by modifying or eliminating some interfering components. On the other hand, the combination of any of the three types of bLFH with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM, under the same conditions. In this type of media, the higher concentration of divalent cations can increase bacterial membrane stability by interacting with the negative charges of the core oligosaccharide chain of lipopolysaccharide (LPS) molecules (AlNabulsi et al., 2009; Conghlin et al., 1983; Ellison et al., 1988). This lack of bLF activity in skim milk and PIFM could also be due to its interaction with casein micelles, as this interaction has been previously reported by Anema and de Kruif (2011) and Croguennec et al. (2012), although its biological significance is not known. The molecular mass pattern of peptides obtained by hydrolysis of bLF was very different for the three enzymes, though all showed similar antibacterial activity in PBS. In the case of pepsin bLFH, the antimicrobial activity was probably due to lactoferricin, as it has been identified a peak of 3196 Da in the analysis by MS-MALDI TOF, which is close to its reported molecular mass (Bellamy et al., 1992b; Conesa et al., 2010; Plate et al., 2006; Shin et al., 1998). A fragment derived from lactoferricin, with a molecular mass of 1718 Da, has been reported with a weak antibacterial activity against various microorganisms such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Pseudomonas fluorescens and Escherichia coli (Hoek et al., 1997). This fragment could coincide with the peak at 1719 Da observed in the mass spectral analysis of pepsin bLFH. Moreover, in this spectrum, two peaks with molecular masses of 2433 and 2592 Da, respectively, could be associated with peptides that have been described in previous studies as having antibacterial activity against different microorganisms (Dionysius and Milne, 1997; Recio and Visser, 1999). On the other hand, the mass spectra recorded for bLFH obtained by recombinant chymosin and microbial rennet, showed many peptides within a wide molecular mass range. In the case of the recombinant chymosin, peptides with molecular masses of 3123; 3194; 3308 and 5850 Da with antibacterial activity against E. coli L361 have been described. Some of those peptides share high structure homology with lactoferricin B (from the bovine protein) (Hoek et al., 1997). However, the molecular masses of the peptides present in the spectrum obtained in our work do not coincide with those described in the work of Hoek et al. (1997), which seems to indicate that other different peptides have been generated, also showing antibacterial activity. The hydrolysis of bLF with microbial rennet has been reported to generate peptides with molecular masses of 990, 1545; 2338 and 2506 Da, which present antibacterial activity against E. coli and B. subtilis (Elbarbary et al., 2010). Some of cationic peptides obtained in our study, could be associated with those reported by Elbarbary et al. (2010), because of its close molecular mass. Considering that some infant formulas are supplemented with bLF, it is essential to study the effect of temperature and time after reconstitution and microwave heating, on the stability and antibacterial activity of bLF. Reconstituted formula is an excellent medium for grow of C. sakazakii (Gurtler and Beuchat, 2007; Iversen et al., 2004b). This pathogen can grow over a wide temperature range (6–47 °C), the optimum ranging from 37 °C to 43 °C (Iversen et al., 2004a, 2004b).

Power level (W)/holding time (s) Fig. 4. Effect of microwave treatment (450, 550 and 650 W for 5, 10 and 15 s), on the concentration of bLF determined by radial immunodiffusion using polyclonal specific antibodies. Immunoreactivity is expressed as the relative concentration respect to the non-treated bLF.

3.5. Effect of microwave treatment on stability of native bovine lactoferrin in reconstituted PIFM The estimated concentrations of bLF in microwave treated PIFM containing this protein, determined by its immunoreactivity, are shown in Fig. 4. In general, the concentrations of bLF decreased as the microwave power level and treatment time increased. At 450 W the loss of immunoreactivity with respect to the non-treated bLF was of 6, 17, and 67% at 5, 10 and 15 s, respectively. When the power level was increased to 550 W, the reduction of immunoreactivity also increased with treatment time, being of 11, 28, and 78% at 5, 10, and 15 s, respectively, with respect to the control. At 650 W, the greatest reduction in bLF concentrations was observed, being of 17, 44, and 89% at 5, 10 and 15 s, respectively.

4. Discussion Lactoferrin has been proposed in food industry as a natural alternative, alone or combined with thermal or non-thermal treatments, for controlling foodborne pathogens (Al-Nabulsi and Holley, 2007; Murdock and Matthews, 2002). Specifically, bLF is used as a supplement in several foods, including infant milk products, to reinforce the infant natural defenses and also to prevent the contamination by pathogen microorganisms. Although bLF is normally added to products as the whole protein, lactoferrin hydrolysates have been extensively investigated due to their enhanced antibacterial activity (Hoek et al., 1997; Tomita et al., 1991). Bioactive peptides derived from lactoferrin are usually obtained with porcine pepsin, trying to mimic the physiological situation in the gastrointestinal tract (Branen and Davidson, 2000). However, other proteases normally used in cheese manufacture could be applied, such as chymosin or microbial rennet. This approach could be interesting to obtain lactoferrin hydrolysates with other biological properties, and also to be added in products directed to communities for which the use of porcine derivatives is not allowed (Elbarbary et al., 2010). In this study, the antibacterial activity of bLFH obtained with pepsin, chymosin and microbial rennet has been found to be higher than that observed for intact bLF in PBS. The potent inhibitory activity exerted by the three types of bLFH against C. sakazakii in a simple medium, such as buffered phosphate, was almost completely lost when assayed in skim milk, whey and reconstituted PIFM. Our results are in agreement with previous reports which have shown a reduced bactericidal effect of bLFH in food systems (Branen and Davidson, 2000; Chantaysakorn and Richter, 2000; Conesa et al., 2010; Del Olmo et al., 6

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antibacterial activity of pepsin and chymosin bLFH was enhanced in whey when used in combination with intact bLF. These results are promising for the future use of bLFH as natural preservatives in foods or as functional ingredients. However, further research should be done to improve the antibacterial activity of those compounds by modifying or eliminating some interfering components in foods. According to the results obtained in this study, the use of bLF may be a promising approach for the efficient control of C. sakazakii in reconstituted PIMF held at abusive temperatures (23 to 37 °C) for short periods. Moreover, low intensity microwave treatments of reconstituted PIFM would maintain the nutritional properties of product and the antibacterial activity of bLF practically intact with an immunoreactivity of about 90%.

Moreover, the relatively short generation time and lag time may allow C. sakazakii to grow rapidly to a dangerously high level, if the contaminated rehydrated PIFM remains at room temperature for too long during preparation and holding time (FAO/WHO, 2004). The use of natural antimicrobials in formula milks could help to avoid the growth of pathogens following reconstitution and reduce the risk of infection. In this sense, the results obtained in the present study showed that bLF reduced the proliferation of C. sakazakii in reconstituted PIFM for short periods of time (until 2 h). Therefore, these results suggest that addition of bLF may be useful to minimise the growth of C. sakazakii in PIFM held at non-refrigeration temperatures. In this work, as it could be expected, C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIMF, with or without bLF. Our results are in agreement with those of other authors (Beuchat et al., 2009; Magalhäes et al., 2012), who reported that the growth of C. sakazakii was prevented in PIMF when stored at 4 °C. In any case, PIFM prepared for later administration should be refrigerated immediately following reconstitution and used within 24 h (FAO/WHO, 2004). To reduce the risk of Cronobacter species presence in infant formula, FAO/WHO (2004) recommends the reconstitution with water at temperatures ≥70 °C. Nowadays, practices at home and childcare settings involve microwave heating of reconstituted PIFM and possible reheating after an incomplete feed (Pina-Pérez et al., 2014). Respect to microwave heating, our results showed that the inhibitory capacity of bLF on C. sakazakii in reconstituted PIFM was maintained high after 450 W for 5 s (42 °C) and 550 w for 5 s (50 °C), since bLF immunoreactivity was of 94 and 89%, respectively. The effect of bLF slightly increased the effect of microwave heating. This implied an approximate reduction of about 0.3 and 0.8 log cycles, respectively, with respect to counts obtained after the same treatments in reconstituted PIFM without bLF. It is noteworthy that reconstituted PIFM samples with or without bLF treated at 650 W for 5 s (53 °C), showed total inhibitory effect on the growth of C. sakazakii. This result indicates that microwaves themselves would affect the bacteria, since the temperature reached was below the inactivation temperature of C. sakazakii (70 °C). This is in agreement with some studies, which attributed nonthermal electromagnetic radiation effects on microorganisms processed by microwaves, permitting higher reduction levels to be reached with lower temperatures and shorter treatment times than by conventional heat processing (Heddleson and Doores, 1994; Najdovski et al., 1991; Tang et al., 2008). On the other hand, no C. sakazakii was recovered after treatments at 450, 550 and 650 W for 10 and 15 s (71 to 88 °C) in reconstituted PIFM with or without bLF. With regard to the few studies that have previously evaluated the effect of microwave processing on C. sakazakii in reconstituted PIFM, other researchers have obtained higher reductions of C. sakazakii than those obtained in our study. However, they applied higher intensity treatments, used larger quantity of inoculum and samples were treated in different containers and volumes. Kindle et al. (1996) reported that microwaving infant formula at 600 W in baby bottles for 85–100 s to a temperature of 82–93 °C, could result in a > 4 log cycles destruction of C. sakazakii. In the study of Pina-Pérez et al. (2014), power levels of 800 and 900 W for 120 s, reduced the C. sakazakii initial population to undetectable levels (≥8 log cycles) in reconstituted PIFM, reaching maximum temperatures of 78 °C and 88 °C, respectively. According to some authors, there are factors that can influence the effectiveness of microwave heating of milk using household equipment (2450 MHz frequency). Among them, product quantity, vessel geometry, inoculated microorganism and food composition (Barbosa-Cánovas et al., 2014; Sieber et al., 1996). In conclusion, the results derived from this study have indicated that hydrolysis of native bLF with different proteolytic enzymes generates peptides that showed higher antibacterial activity against C. sakazakii in PBS than the intact bLF. We also observed that antibacterial activity of bLFH against C. sakazakii was low when it was evaluated in bovine skim milk, whey and reconstituted PIFM. Nevertheless, the

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