INHIBITORY EFFECT OF LACTOFERRIN AGAINST CRONOBACTER SAKASAKII ISOLATED FROM INFANT FORMULA MILK POWDER

Assiut University web-site: www.aun.edu.eg

INHIBITORY EFFECT OF LACTOFERRIN AGAINST CRONOBACTER SAKASAKII ISOLATED FROM INFANT FORMULA MILK POWDER

KARIMA G. ABDEL HAMEED

Dept. of Food Hygiene, Fac. of Vet. Med., South Valley University

                Received: 31 December 2016;       Accepted:  19January 2017

INTRODUCTION

Lactoferrin is an 80 kDa iron binding glycoprotein of the transferring family. Lactoferrin is a major component of milk and presents in neutrophil granules or other exocrine secretions such as tears and saliva. It is found in concentration up to 1.5 mg/ml in bovine colostrum (Yekta et al., 2010). Lactoferrin is an important host defence molecule and has diverse physiological functions such as antibacterial, antiviral and anticancer activities.

Many studies have demonstrated the bacteriostatic and bactericidal effect of Lactoferrin, against a wide range of Gram-positive and negative bacteria (Farnaudand Evans, 2003). Lactoferrin inhibits bacterial pathogens by a direct interaction mediated by binding of the lipid A portion of the lipopolysaccharide (LPS) of Gram-negative bacteria (Brandenburg et al., 2001). The expanding demand for adding Lactoferrinto the products due to its nutritional values and physiological benefits has incentivized research workers to find much more simple and economic ways to isolate and purify Lactoferrin.

Corresponding author: Dr. KARIMA G. ABDEL HAMEED

E-mail address: karima_galal2004@yahoo.com

Present address: Dept. of Food Hygiene, Fac. of Vet. Med., South Valley University

C. sakazakii (formerly known as Enterobacter sakazakii) is a Gram negative rod, motile facultative anaerobic bacterium (Iversen et al., 2008).C. sakazakii have associated with severe forms of necrotizing and meningitis especially in neonates with mortality rate varies from 40 – 80% (Healy et al., 2010) and the infective dose is estimated to range from 10³ to ≥ 10⁸ cells (Pagotto et al., 2003).

C. sakazakiihas been isolated from wide range of dairy products (Ye et al., 2014). Moreover, powdered infant formula has been epidemiologically linked to Cronobacter infections in infants (Healy et al., 2010 and Sani et al., 2014). Much research has focused on the presence of C. sakazakii in baby foods. They have been isolated from various infant foods including; powdered infant formula (PIF), herbs and cereals (Sani et al., 2014; Parra et al., 2015 and Li et al., 2016).

Due to immature immune system of infants, researcher have tried to prevent contamination of baby foods with C. sakazakii by irradiation (Osaili et al., 2007), adding probiotic bacteria (Osaili et al., 2008) and control it, if exist, in infant foods by plant essential oil (Al-Nabulsi et al., 2015). Few attempts have been carried out to investigate the inhibition effects of bovine lactoferrin on the growth of this pathogen. Therefore, the objective of the current study was to isolate and purify lactoferrin from bovine colostrum and to investigate its inhibitory effects on the growth of C. sakazakii isolated from IFMP.

MATERIALS AND METHODS

Isolation and Purification of bovine Lactoferrin (bLF)

Bovine colostrum samples were collected within the first day after cow parturition from South Valley University dairy farm. Isolation of bLF was done as described by (Yoshida et al., 2000). Lactoferrin was purified by carboxymethyl Sephadex-C50 chromatography (FPLC, Bio-RAD, USA). The stock solution of bLF was prepared with sterile distilled water to give a final concentration of 10 mg/ml and filter sterilized using 0.22 μm membrane filters. After filtration, the absorbance was monitored by ultraviolet absorption at 280 nm and the concentration of bLF was calculated as recommended by (Yoshida et al., 1999). The final concentration of bLF solutions was adjusted to 1, 2, 5 and 10 mg/ml. The purity of bLF was compared with lactoferrin standard (Sigma-Aldrich, Beijing, China) using (SDS-PAGE) [sodium dodecyl sulphate-polyacrylamide gelelectrophoresis] as described by (Harouna et al., 2015).

Isolation and identification of C. sakasakii

Collection of samples:

A total of 100 powdered infant formula milk (IFMP) (Recommended for infants from birth to 1 year old), were collected from different localities in Qena city then transferred to the laboratory to be examined. The samples were prepared according to FDA (2002) for C. sakazakii detection isolation.

IFMP were pre-enriched by reconstitution in sterilized, distilled water (10g sample/90 ml sterile distalled water) and incubated at 36°C for 24 h, 1 ml of the pre-enrichment culture was inoculated into 9 ml of the Enterobacteriaceae Enrichment Broth (EEB), which was then incubated for 24h at 36°C. 10 μl of the incubated broth was then streaked on the surface of chromogenic media (HiCrome Enterobacter sakazakiiagar) (Oxoid M1577). Suspect blue-green colonies were observed after incubation at 36°C for 24h. Further identification of the isolated C. sakasakii was done according to FDA (2002) and Iversen et al. (2008).

C. sakasakii culture preparation (Harouna et al., 2015)

C. sakasakii isolated from IFMP in this study was kept in (EEB) then incubated for 24h at 36°C. One ml of the inoculated EEB was added to 9 ml of 1% peptone water and decimal dilutions in 1% peptone water were used to yield a suspension of 10⁴ CFU/ml for antibacterial activity assays.

Antibacterial activity assay (CLSI, 2011)

Antibacterial activity of bLF was done by disc diffusion method using Mueller–Hinton agar (Oxoid) according to the recommendation of the Clinical Laboratory Standards Institute (CLSI, 2011). bLF discs were prepared according to Barry (1976) in which empty sterilized discs (What man no. 6 mm diameter) were impregnated with 50 μL per disc with different bLF concentrations (1, 2, 5 and 10 mg/ml). The discs were placed on and swabbed over the surface of the plates that inoculated with 50 μl of the previously prepared inoculum (10⁴CFU/ml) then, were incubated for 24 h at 36°C. The susceptibility of C. sakasakii was determined by measuring the zone of growth inhibition around the discs. Inhibition of bacterial growth in the plates containing tested bLF was judged by comparison with growth in blank control plates without bLF discs (Harouna et al., 2015).

DISCUSSION

Lactoferrin from bovine colostrum has become increasingly important because of its diverse range of biological activities, such as anti-infective activities toward a broad spectrum of species. Therefore it was important to isolate and purify lactoferrin from bovine colostrum. The purity of bLF in this study was checked by SDS-PAGE, which showed a single band corresponding to a protein of about 80 KDa and the purification efficiency was about 90% (photo 1). Moradian (2014) and Harouna et al. (2015) obtained the same purity. A higher purity of 91.3 % was recorded by Yafei et al. (2011) using SPEC 70 SLS cation exchange resin. Lower bLF purity of 87% by SP Sepharose Big Bead ion exchange column was obtained by Kong et al., (2012). However the same authors recorded a purity estimated to be >95% using SDS-PAGE. From the above mentioned results it could be concluded that the stated method result in isolation of highly pure bLF indicating simple, low-cost and efficient method on preparation of bLF without loss its bioactivity, as compared with other previous methods of purification of lactoferrin.

Regarding the Incidence of C. sakazakii in IFMP, it was found that 21% of the total (100) IFMP samples examined were contaminated with C. sakazakii (Table 1) higher results of 24% and 23% were detected by El-Gamal et al. (2013) and Li et al. (2016), respectively as they use the same isolation media (HiCrome Enterobacter sakazakii agar) and using the FDA enrichment procedure.

The current results were higher than the previous studies by Iversen and Forsythe (2004), they isolated C. sakazakii from 2.4% out of 82 analyzed samples of IFMP. Oonaka et al. (2010); Shetty et al. (2011); Fu et al. (2011) could detect C. sakazakii in 6.6% (9/149), 5.4% (11/202) and 3.9% (3/77) IFMP samples, respectively. However, Sani and Yi (2011) and Putthana et al. (2012) did not detect any positive samples in 390, 30 and 7 IFMP evaluated, respectively. The other C. species was not detected in the examined IFMP samples.

According to the current and previous studies, there was a direct relationship between IFMP and C. sakazakii, despite the fact that formulas are exposed to heat treatment during processing. That means post-pasteurization contamination of IFMP with C. species may occur via the addition of dry ingredients (as vitamins and minerals) or during packaging. However, the prevalence of the organism following the drying and survival in powdered foods for a long time may be partially due to the organism’s ability to resist desiccation and osmotic stress (Arku, 2008). Therefore, hygienic measures and practices must be applied during the manufacture of formula to minimize entry of contaminants into the process.

Antibacterial activity is a biological function attributed to Lf (Farnaud and Evans, 2003). The mechanisms that account for the antibacterial properties have been reported to be iron dependent and iron independent (Orsi, 2004); the latter implies direct interaction of Lf with the bacterial cell surface (Brandenburg et al., 2001).

Regarding the antibacterial effect of bLf at different concentrations (1, 2, 5 and 10 mg/ml) on C. sakazakii growth, the results indicated that bLF at concentration of 1mg/ml had the least inhibitory effect whereas maximum inhibitory effect was recorded for 10 mg/ml against C. sakazakii (Table 2). These findings are in parallel to those reported by Wakabayashi et al. (2008), who found that Apo-LF at 0.5 mg/ml weakly suppressed the growth of C. sakazakii and Apo-LF at 2 to 8 mg/ml completely inhibited its growth. As well Maria et al. (2014) stated that bLF at a concentration of 10 mg/ml, inhibit adherence of C. sakazakii to intestinal epithelium.

It was noted that bLF at concentration of 2mg/ml was difficult to draw clear-cut conclusions about inhibition versus growth of the tested C. sakazakii (Table 2). This was contrary to the results of Moradian et al. (2014), they concluded that bLF above 1 mg/ml inhibited the bacterial growth especially for gram negative bacteria.

Bovine LF has been used as a supplement to some infant formulas (Wakabayashi et al., 2006). Bovine LF heated at 80°C showed similar anti-Enterobacter activity to non heated bLF at above 1 mg/ml. This observation suggests that bLF in the powdered infant formula may retain its antibacterial activity to some extent after reconstitution with hot water. In conclusion, bovine LF may have potential usefulness for the prevention of infection by C. sakazakii in foods such as infant formula.

RESULTS

Table 1: Incidence of C. sakazakii in powder infant formula milk (IFMP).

Table 2: The inhibitory effect of different bLF concentrations on growth of C. sakasakii.

growth (+)                  no growth (−)

Photo 1: Bovine Lactoferrin (bLF) by SDS-PAGE

Lan LS: Lactoferrin Standard,

Lan L: Laddar (kDa) Molecular weight markers, transferrin (76 kDa),

Lan 1-3: bLF positive samples,

Lan 4-6: bLF negative samples.

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