Inhibition of histamine accumulation by novel histamine‐degrading species of Staphylococcus sp. isolated from goats and sheep milk

Abstract Histamine is an active amine compound that occurs in various fermented foods that may cause adverse effects on the human health. Certain microorganisms are able to degrade histamine by an oxidative deamination reaction. Therefore, the present study aimed to quantify histamine‐forming and/or ‐degrading activity of the isolates derived from milk of goat and sheep herds, in Iran, by the capillary zone electrophoresis (CZE) method; and we evaluated the molecular characteristics of staphylococcal isolates. Among 243 staphylococcal isolates, 29 histamine‐degrading bacteria were identified. One of these isolates, identified as Staph. epidermidis, No. 605, exhibited the highest activity compared to others, degrading available histamine to 58.33% within 24 h. By polymerase chain reaction (PCR) analysis, the isolate, No. 605 that exhibited remarkable histamine‐degrading activity lacked the genes encoding coagulase and DNase, nor did it harbor any of the five classical enterotoxin genes. This is the first report to show that seven Staphylococcus species, including Staph. chromogenes, Staph. aureus, Staph. haemolyticus, Staph. epidermidis, Staph. pseudintermedius, Staph. agnetis, and Staph. hyicus, were able to degrade histamine, which were hitherto not known to have this capacity. Therefore, histamine‐degrading activity is a definite criterion to introduce fermenting organisms able to decrease histamine content in different food products.


| INTRODUC TI ON
Histamine is well known as the scombroid poisoning agent and it is considered as a natural antinutrition factor which is very important in food hygiene (Gonzaga et al., 2009). It is present in various levels in red wine and beer, fish, milk and cheese, sausage, salami, vegetables, and many other popular foods and beverages (Lorenzo et al., 2007). Histamine in food is mainly formed by microorganisms which are able to decarboxylate histidine. Histidine decarboxylation is catalyzed by l-histidine decarboxylase in the conditions proper for bacterial growth and subsequent decarboxylase activity (Rivas et al., 2008). Food proteolysis during processing or storage can produce free histidine or it can be found naturally in foods.
Therefore, high levels of histamine in food products are usually associated with microbial fermentation. In this sense, we can consider histamine as an index for food hygiene and quality (Maintz & Novak, 2007). In addition, the presence of high amounts of histamine in food is associated with foodborne disease and can be of health concern. Regarding fish consumption, the U.S. Food and Drug Administration (FDA) suggested that histamine content higher than 200 mg/kg can cause histamine (scombroid) fish poisoning (Lehane & Olley, 2000).
Histamine is physiologically inactivated by histamine oxidase.
Recently, the main strategy of using bacteria with histamine degradation activity has appeared as an approach for reduction of food histamine content (Mah & Hwang, 2009;Naila et al., 2010).
Nevertheless, few reports of staphylococcal strains possessing biogenic amine-degrading activity have been described. For instance, Staph. carnosus FS19 was found to possess histamine oxidase capable of degrading histamine up to 29.1% from its initial concentration within 24 h in laboratory experiments (Zaman et al., 2010(Zaman et al., , 2014. Staph. xylosus S81, isolated from artisanal fermented sausages, and Staph. xylosus isolated from the anchovy, also exhibited histamine degradation activity, degraded the histamine content by about 100% and 38% within 48 h, respectively (Lee et al., 2013;Martuscelli et al., 2000).
This research was conducted to isolate and characterize staphylococcal strains possessing histamine-degrading activity from milk of goats and sheep. Histamine-forming and/or -degrading activity of the isolates was quantified and molecular characteristics of the isolates described to classify them regarding food safety.

| Identification of staphylococcal isolates by PCR
The primers used for the identification of staphylococcal strains, Tstag765 and TstaG422, were designed as previously reported (Morot-Bizot et al., 2004) employing a uniplex polymerase chain reaction (PCR) assay. PCR was performed under the following conditions: 3 min at 94°C, then 40 cycles of 1 s at 95°C, 30 s at 55°C, 30 s at 72°C, and a final hold of 3 min at 72°C with a gradient automated thermocycler (Bioer XP Cycler). The PCR mixture was analyzed by 1.4% agarose gel electrophoresis in Tris-acetate-EDTA (TAE) (1×).  (Mah & Hwang, 2009;Martuscelli et al., 2000). After incubation, the culture was centrifuged at 14,000g for 5 min (Eppendorf) at 4°C and the supernatant was filtered with 0.45μm filter paper (Lee et al., 2013).

| Histamine-degrading activity of isolates
One milliliter of the culture broth was taken and frozen at −80°C for quantitation of histamine using capillary zone electrophoresis (CZE) method, as described by Numanoğlu et al. (2008).

| Histamine formation activity of isolates
A similar process was performed for histamine formation activity of isolates. Cells of overnight culture of each isolate (adjusted to 2 × 10 8 CFU/ml) were washed once with phosphate buffer (0.1 M; pH 7.0), pelleted by centrifugation (14,000g for 5 min), and inoculated in 5 ml of sodium phosphate buffer supplemented with histidine (0.5 mM) for 24 h incubation at 37°C. Five ml of histidinesupplemented phosphate buffer (0.5 mM) with no bacterial culture and one incubated with Staph. epidermidis TYH1, histamine-forming strain isolated from fish-miso in Japan (Yokoi et al., 2011), were used as negative and positive controls, respectively. After incubation, the supernatant was removed by centrifugation (at 14,000g for 5 min) at 4°C and filtered through a 0.45μm filter (Lee et al., 2013). The supernatants were preserved at −80°C for CZE analysis.

| Assay for histamine content (CZE analysis of histamine)
The histamine content was determined by the CZE analysis described by Numanoğlu et al. (2008) with some modification. A CZE apparatus (Prince Autosampler, Model 1-Lift, 450 Series) was applied for histamine analysis. This system was supplied with a thermometer and a UV-visible (UV-vis) detector (set at 25°C and 210 nm, respectively). CZE data were then analyzed by Data Acquisition and Analysis Software, DAx. The separation was accomplished with phosphate buffer (50 mM, pH 2.5) and the injection of samples (hydrodynamically at 50 mbar for 3 s) was performed under constant voltage conditions of 20 KV and normal polarity. The capillary utilized had a proper length of 52 cm and 75 μm of internal diameter (Prince Autosampler). Peak area was used for the determination of histamine in samples.

| Molecular characterization of histaminedegrading staphylococcal isolates
Molecular characteristics of the isolates obtained in the present study were performed by identification of genus and detecting coagulase (coa), thermostable nuclease (nuc), and staphylococcal enterotoxin (SE) (sea, seb, sec, sed, and see) genes.

| DNA extraction
Genomic DNA was isolated from staphylococcal strains with histamine-degrading activity, using a DNeasy Blood and Tissue kit (Qiagen GmbH) with modification, based on manufacturer's instructions.

| Identification of Staphylococcus genus
The identities of histamine-degrading isolates were further confirmed by amplifying and sequencing a single 899 bp band of rpoB gene, encoding the beta subunit of RNA polymerase, using primers described by Mellmann et al. (2006). Amplifications were carried out as follows: initial denaturation 94°C for 5 min followed by 35 cycles of denaturation (94°C for 45 s), annealing (52°C for 1 min), elongation (72°C for 90 s), and then a final elongation at 72°C for 10 min (Mellmann et al., 2006). The amplified genes were finally extracted TA B L E 1 Nucleotide sequences and predicted size of polymerase chain reaction (PCR) products for the staphylococcal-specific oligonucleotide primers (SEA, SEB, SED), were used as enterotoxin producers (Rahmdel et al., 2018). The products of PCR were detected by electrophoresis in a 1.4% agarose gel in TAE (1×).

| Statistical analysis
All the experiments were carried out in triplicate and the results were expressed as mean values and standard deviations. Data analyses were performed using SPSS software Version 16.0 for Windows.
The mean comparison was performed using the Duncan's Multiple Range Test (DMRT) at p < .05 significant difference following analysis of variance (ANOVA).

| RE SULTS AND D ISCUSS I ON
Based on biochemical and morphological characteristics, we differ-  in 5 ml of sodium phosphate buffer containing 0.5 mM histidine.
Isolate No. 53 was the only one with histamine-forming activity, forming histamine to about 0.052 ± 0.0002 mM after incubation of 24 h at 37°C, and was identified as Staph. haemolyticus, also possessing histidine decarboxylase activity. This isolate also degraded 29.16% of initiate histamine content in our assay. All amine oxidase-positive strains showed simultaneous amino acid decarboxylase activity (Voigt & Eitenmiller, 1978). Out of other isolates from our collection tested, 29 isolates, which exhibited histaminedegrading ability, did not show histamine-forming activity. The positive control, Staph. epidermidis TYH1, and negative control exhibited 0.41 ± 0.0003 and 0.0 mM histamine content, respectively.
According to the histamine-forming ability of Staphylococcus strains isolated from various foods, Simonova et al. (2006) reported that Staph. carnosus SO2/F/2/5, isolated from Slovak traditional meat products, was the only one strain which exhibited production of the biogenic amine. Several authors (Bover-Cid et al., 2001;Casaburi et al., 2005;Karovicova & Kohajdova, 2005) reported similar results of biogenic amine production by Staphylococcus strains.
The results of molecular identification of histamine-degrading staphylococcal isolates are shown in Table 3 Other isolates were PCR-negative for the coa gene ( Figure 1a; Table 3). In our study, the coa gene appeared as one of the two different-sized amplicons, viz., 603 and 720 bp. All isolates were observed to produce only one type of amplicon, either 603 or 720 bp.
Amplification with nuc primers revealed that out of 29 histaminedegrading isolates, 21 were positive (72.4%) for the amplification of a 270-300 bp specific band (Figure 1b) (Podkowik et al., 2013). In the pres-   (Rall et al., 2010). Collery et al. (2008) showed that of the classical enterotoxin genes, SEA, SEB, and SEC were detected commonly among Staph. aureus with SEB the most common, which was likewise observed in our study. Hence, the finding of this research indicated that, within the staphylococcal strains isolated from milk of goats and sheep, 29 possessed the potential to degrade histamine from 6.25% to 58.33%.

| CON CLUS IONS
However, bacterial histamine oxidase activity in a simple medium of phosphate buffer will be different from their behavior in complex substances, as well as in different incubation times. We observed the histamine degradation amount, two times more than that reported by Zaman et al. (2010), after an incubation time of 24 h.
Therefore, the effects of the complexity of culture media and different incubation times on the bacterial histamine degradation should be explored in further researches.

ACK N OWLED G M ENTS
The authors would like to thank Dr. M. Chahardahcherik and Mr.
H.A. Shamsaei for their technical assistance and Mr. K. Yokoi for providing Staph. epidermidis TYH1 used in this study.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The additional data will be available upon requesting the corresponding author.