Microbial quality and formation of biogenic amines in the meat and edible offal of Camelus dromedaries with a protection trial using gingerol and nisin.

Abstract This study aimed firstly at the investigation of the microbial status of the camel meat and edible offal including liver, kidneys, lungs, rumen, and duodenum distributed at local markets of Egypt. Total plate count, total psychrophilic counts, total Enterobacteriaceae count, the most probable number of coliforms, and total mold counts were monitored at the collected samples. The produced biogenic amines (BA) in the camel meat and offal were further estimated. An experimental trial to investigate the antimicrobial potentials of either nisin, gingerol, or an equal mixture of both using camel muscle as a food matrix was conducted. The achieved results declared a high microbial load in the camel meat and the offal. Duodenum and rumen had the highest microbial counts followed by lungs, kidneys, liver, and muscle, respectively. Similarly, duodenum and rumen had the highest levels of BA, including tyramine, spermine, putrescine, cadaverine, and histamine. Both of nisin and ginger showed significant antimicrobial properties in a concentration‐dependent manner. Thus, efficient hygienic measures during the handling of camel meat are highly recommended. In addition, using nisin, gingerol, or a mixture of both is an efficient strategy for improving the microbiological quality of the camel meat.


| INTRODUC TI ON
Camel (Camelus dromedaries) meat and offal are considered as major sources for animal-derived protein in many parts of the world. These kinds of meat are rich in protein, polyunsaturated fatty acids, and minerals. Camel meat is considered healthier than that of beef and mutton, in terms of less amount of fat, less cholesterol with high protein content (Kadim, Mahgoub, & Purchas, 2008). Camel meat is believed to have many medical values for the treatment of diseases in several Arabian countries, India, and Australia. Camel meat production is increasing worldwide and currently represents about 0.7% of the total world meat production (Suliman, Fadlalmola, Babiker, Arabi, & Ibrahim, 2014).
The high content of protein and moisture are considered as ideal factors that enhance microbial spoilage of the meat and offal.
In addition, poor hygienic measures adopted during slaughtering, evisceration, distribution, and storage of the meat and offal are additional factors for the rapid onset of the microbial spoilage of the meat (Darwish, Eldin, & Eldesoky, 2015). However, there is a clear lack of information about the microbial quality of camel meat and offal in the local markets of Egypt. Indicators of the microbial quality and hygienic status of meat include total mesophilic (plate) count (TPC), total psychrophilic counts (TPsC), total Enterobacteriaceae count (TEC), the most probable number of coliforms (MPN), and total mold counts (TMC) (Vanderzant & Splittstroesser, 2001). One major task of the food control sector is to ensure the safety of marketed meat, and consumer protection. Therefore, the microbial quality of the marketed camel meat and offal in Egypt was investigated in the present work.
Biogenic amines are formed in different food matrices such as meat, fish, milk, and vegetables. Consumption of biogenic amines-contaminated foods may lead to several toxicological implications such as anaphylaxis, nervous, and muscular disorders (Stadnik & Dolatowski, 2010). Concentrations of BA in the food give an indication about the microbial quality of the food and the hygienic status adopted during preparation and processing. However, there is a clear lack of information about the contents of BA in camel meat and offal.
Gingerol is one of the active compounds found in ginger with several biological activities. Of these, its antioxidant and antimicrobial effects as reported before are against periodontal bacteria (Park, Bae, & Lee, 2008). The mechanisms behind gingerol-antibacterial activities are still unclear. In addition, the ameliorative effects of gingerol on the microbial status and formation of BA in the meat had scarcely investigated.
Nisin is an antibacterial peptide that is secreted by several strains of Lactococcus lactis, it has a well-documented bacterial inhibitory effect against a vast array of microorganisms (Abts et al., 2011). The mechanism of the inhibitory effects of nisin is mainly via binding to lipid II, an essential cell wall precursor, thus prevents cell wall synthesis (Hasper, de Kruijff, & Breukink, 2004). However, the preservative effects of nisin on the camel meat had received less attention.
Insight of these factors, the objectives of this study were first to investigate the microbial status of the camel meat and offal (liver, kidney, lungs, rumen, and duodenum) collected from different localities in Egypt via estimation of TPC, TPsC, MPN, TEC, and TMC. Levels of BA in the camel meat and offal were quantitatively estimated using the amino acid analyzer. An experimental trial to investigate the ameliorative effects of either nisin, gingerol, or an equal mixture of both on the microbial quality of the camel meat was conducted.

| MATERIAL S AND ME THODS
The experimental work in the present study is divided into two experiments. Experiment 1 aimed at the investigation of the microbial status and BA content in the camel meat and edible offal.
Experiment 2 aimed at investigating the antimicrobial potentials of either nisin, gingerol or an equal mixture of both using camel muscle as a food matrix.

| Collection of samples
One hundred and twenty samples including 20 each of camel meat (round), liver, kidneys, lungs, rumen, and duodenum were collected randomly from different butchery shops in Egypt. Samples were collected from October 2017 to March 2018. The collected samples were transferred cooled directly without delay to the laboratory for the microbiological examination.

| Microbiological examinations
Samples were prepared according to recommendations of the American Public Health Association (APHA) (Vanderzant & Splittstroesser, 2001). In brief, 25 g of each sample weighed and homogenized with 225 ml of 0.1% of sterile buffered peptone water (LAB104, LAB M) for 1-2 min at 450 g using a sterile homogenizer (type M-p3-302, mechanic, precyzina). Such homogenate represents the dilution of 10 −1 and then followed by the preparation of tenfold decimal serial dilutions.

| Total plate count (TPC)
Total plate count was estimated using the method of APHA (Vanderzant & Splittstroesser, 2001). In brief, one ml from each dilution was pipetted to a clean and sterile Petri dish. About 12-15 ml of plate count agar (Difco Laboratories) cooled to 45 ± 1°C were added to each Petri dish and left to solidify at room temperature and then incubated at 35 ± 2°C for 48 hr. Colonies including pinpoint size were counted as TPC in plates with 25-250 colonies per dish.

| Determination of total psychrophilic count (TPsC)
The same method used for TPC was performed. Petri dishes were incubated at 7°C for 10 days. Results were calculated and recorded in the same way as TPC.

| Determination of the most probable number (MPN) of Coliforms
Three tubes' most probable number (MPN) method was used (Vanderzant & Splittstroesser, 2001). In brief, one ml of each dilution was used to inoculate separately into three test tubes containing MacConkey broth with inverted Durham's tubes. The inoculated tubes were incubated at 37°C for 24-48 hr. Positive tubes showing acid (yellow color) and gas production in inverted Durham's tubes were recorded. The most probable number of coliforms was calculated according to the recommended tables.

| Determination of total Enterobacteriaceae count (TEC)
From the original and the subsequently prepared dilutions, one ml was poured onto an empty presterilized Petri dish, and then 12-15 ml of violet red bile glucose agar (Difco Laboratories) cooled to 45 ± 1°C was poured to each Petri dish. The plates were incubated at 37°C for 24 hr. All large pink to red colonies were counted (Vanderzant & Splittstroesser, 2001). Results were calculated and recorded in the same way as TPC.

| Determination of the total mold count (TMC)
Total mold counts were determined by culturing duplicate plates on Sabouraud's dextrose agar media (Oxoid) supplemented with chloramphenicol 100 mg/L followed by incubation in dark at 25°C for 5-7 days. During the incubation time, the plates were examined daily for fungal growth. Estimation of total mold was obtained by direct counting of the cultured agar plates (Vanderzant & Splittstroesser, 2001).

| Content of biogenic amines (BA)
The extraction of BA was conducted by homogenizing 10 g of each sample with 100 ml of 10% trichloroacetic acid. The homogenate had been extracted for 1 hr followed by centrifugation at 2,800 g, 4°C for 20 min. The supernatants were filtered through a Whatman filter No. 1. The filtrates were stored at 4°C until analysis. The analysis of biogenic amines was performed using an amino acid analyzer (L-8900, HITACHI), as described in a recent study (Kononiuk & Karwowska, 2019). Content of the BA (histamine, tyramine, putrescine, cadaverine, spermidine, agmatine, and spermine) was determined with a reference to the amine standards (Merck KGaA).
Detection limits for the tested BA ranged from 0.004 to 0.01 mg/g of meat. The biogenic amine concentrations were reported as mg/g meat.

| Improvement of the microbiological status of camel meat using gingerol and nisin
Reduction trials for the microbial load of camel meat were done using food grades of 6-gingerol (Biopurify Phytochemicals) and nisin (Food grade, SIDLEY chemical, Linyi city, China) under 0.5, 1.5, and 2.5% separate or in a combination. A block of camel meat (5 kg) was purchased from the local market. This block was divided into 10 groups (each group includes five meat pieces, 100 g/piece) and used in the experimental trial. The 1st group was soaked in corn oil for 30 min at room temperature and used as a control (nontreated) in this study. The 2nd, 3rd, and 4th groups were soaked for 30 min at room temperature in nisin 0.5%, 1.5%, and 2.5% in corn oil, respectively. The 5th, 6th, and 7th groups were soaked in 6-gingerol 0.5%, 1.5%, and 2.5% in corn oil, respectively at the same conditions. The synergistic effects of equal volumes of nisin and 6-gingerol were additionally tested. The 8th, 9th, and 10th groups were soaked for 30 min at room temperature in nisin/gingerol (1:1) 0.5%, 1.5%, and 2.5% in corn oil, respectively. TPC, TPsC, MPN, TEC, TMC, and changes in the content of the BA were evaluated as mentioned before.

| Statistical analysis
All values were expressed as means ± SE, and all measurements were carried out in duplicates. Bacterial counts were converted into base logarithms of colony-forming units per g (log CFU/g). Statistical significance was evaluated using the Tukey-Kramer HSD test. In all analyses, p < .05 was taken to indicate statistical significance using JMP statistical package; SAS Institute Inc.

| Microbial status of camel meat and edible offal
The microbial status of the examined camel meat and edible offal samples was investigated using TPC, TEC, MPN, TPsC, and TMC.
recorded high mold counts in the refrigerated camel meat collected F I G U R E 2 Biogenic amine content in the camel meat and offal. The content of (a) cadaverine, (b) histamine, (c) putrescine, (d) spermine, and (e) tyramine, and total biogenic amines in the examined camel meat and edible offal including duodenum, rumen, lungs, kidneys, and liver. Values represent means ± SE (mg/g) of twenty samples from each. Columns carrying different superscript letters differ significantly among examined samples at p < .05 from different markets in Iran (Fallah et al., 2008). Fungal contamination of meat in this study indicates inadequate sanitary measures performed starting from slaughtering, evisceration, and storage. The conditions of the environment in the refrigerators, cutting boards and workers' hands, and clothes are very suitable for the development of mold spores (Mižáková, Pipová, & Turek, 2002;Reij, Den Aantrekker, & ILSI-Europe Risk Analysis in Microbiology Task Force., 2004). Fungal contamination of meat may lead to their spoilage and production of mycotoxins with potential health hazards to humans due to their carcinogenic effects, liver diseases, and organ damage (Darwish, Ikenaka, Nakayama, & Ishizuka, 2014).
The overall obtained results in the current investigation revealed the lack of proper hygienic measures adopted during the handling and marketing of the examined camel meat and edible offal, leading to poor microbial quality of such products. Edible offal had significantly higher microbial load compared with the meat. This could be explained as offal usually receives less attention during washing or preparation compared with the muscle and may be kept in the wastewater until the time of marketing (Darwish et al., 2018).

Production of BA gives an indication about the shelf life and
the hygienic status of the meat and meat products (Ruiz-Capillas & Jimenez-Colmenero, 2005). The recorded levels of BA in the current study revealed that duodenum followed by rumen had the highest BA concentrations. The recorded total BA levels were 2.89 ± 0.19, 2.63 ± 0.17, 1.61 ± 0.14, 1.50 ± 0.09, 1.44 ± 0.71, and 1.03 ± 0.11 mg/g in the examined duodenum, rumen, kidneys, liver, lungs, and muscle,  reduced the formed BA either individually or in total in a concentration-dependent manner (Table 2). In agreement with the obtained results, Avery and Buncic recorded the anti-Listerial activity of nisin especially in an in vitro approach (Avery & Buncic, 1997 (Table 1). Furthermore, the used nisin-gingerol combination significantly reduced the formed BA in a concentration-dependent fashion (Table 2). Similarly, Cao et al. had successfully used ginger at a combination with onion and garlic to improve the shelf life of stewed-pork during refrigerated storage (Cao et al., 2013). Furthermore, Gharsallaoui et al. mentioned that nisin has strong antimicrobial abilities and can be used alone or in combination to improve the microbial quality of the meat and meat products (Gharsallaoui, Oulahal, Joly, & Degraeve, 2016).
Therefore, a combination of nisin-gingerol 2.5% is highly recommended to achieve the highest improvement in the microbial quality of the camel meat.

| CON CLUS IONS
In conclusion, the results of this study indicated unsatisfactory hygienic measures adopted during the handling of camel meat and edible offal in the slaughterhouses and local markets in Egypt, leading to poor meat quality of the retailed products. Therefore, strict hygienic practices should be followed during the handling of camel meat and offal. Furthermore, using nisin, gingerol, or preferably a combination of both is a promising tool to reduce the microbial load of the camel meat and edible offal. TA B L E 2 Ameliorative effects of nisin, gingerol, and a mixture of both on the production of biogenic amines in the camel meat

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M ED CO N S ENT
Written informed consent was obtained from all study participants.