Fishery products are proposed for consumption as a way to reduce human cardiovascular and other ailments, in addition to meeting the needs of a sizeable section of the Egyptian population. Moreover, they have significantly increased in number over the past few decades in various nations [1
]. Many fish products (including smoked and salted fish) are high in protein, iodine, selenium, amino acid, mineral, lipid, and water-soluble vitamin compositions [2
]. Further, the main nutritional benefit of processed fish products is their composition of extremely healthy fatty acids, which gives them nutritional properties [3
This industry faces a strong neurotoxin produced by Gram-positive, obligately anaerobic, and spore-forming bacteria, such as Clostridium botulinum
]. C. botulinum
is widespread in numerous climatical regions under several environmental conditions, e.g., water bodies, lakes, soils, oceans, seas, and rivers [5
], along with its prevalence in the gastrointestinal tract (GIT) of fish, foods, and animals [6
]. The formation of spores, the main and frequent problem of these bacteria, offers them the opportunity of long-term maintenance in the environment until the beginning of satisfactory situations for growth [7
]. Because C. botulinum
strains may produce highly heat-resistant spores, they are the pathogen of greatest concern in low-acid canned food [8
]. C. botulinum
can produce highly effective neurotoxins, and there are numerous neurotoxin types known (A, B, C, D, E, F, G, HA, and X) that can cause foodborne botulism when they enter the food chain. Botulism has significant fatality rates in both people and animals. Human botulism is brought on by the different toxin types, A, B, E, and F, out of all the toxin types [9
Botulism sickness, which affects a wide range of people, particularly newborns, can result in several issues and symptoms, including drooping eyes, drowsiness, nausea, vomiting, abdominal pains, difficulty swallowing or speaking, and paralysis [4
]. Furthermore, paralysis of the diaphragm, throat, and many upper-airway muscles results in death [10
] due to several factors, including the bacteria’s high potency, neuro-specificity, simplicity of production, and long-time availability. The dependence on molecular techniques has been widely used as they are conducted faster and give more reliable results with high-resolution visions of the structure and diversity of bacterial communities [11
]. This approach has played a vital role in identifying many microorganisms and improving our understanding of the level of microbial complexity. As an effective method, the polymerase chain reaction (PCR) is utilized to identify and detect C. botulinum
. Multiplex PCR is a highly sensitive method and is specific to the target genes in many pathogens, even in low DNA concentrations [12
Nowadays, microorganisms are resistant to many antibiotics, and their tendency to use natural extracts has increased due to their biologically active compounds [13
]. Additives have been approved in food industries due to their antimicrobial and antioxidant properties. These properties can increase the storage period by postponing oxidation and rancidity and preventing off-flavors in fish. The Citrus
genus belongs to the Rutaceae family and includes forty species scattered globally. Indeed, citrus is one of the most vital fruits consumed either fresh and/or as a beverage owing to its pleasant flavor and high nutritive value. The antimicrobial, anticancer, antioxidant, anti-inflammatory, hepato-regenerating, and cardio-protective activities of C. limon
extracts have been investigated [14
leaves have a wide range of therapeutic potentials thanks to their flavonoids and limonoids, which display anticancer anti-inflammation effects [15
]. In another research, Citrus
leaves extracts demonstrated the strongest radical scavenging activity due to their flavonoid contents, including rutin, quercetin, apigenin, kaempferol, and nobiletin. These results suggest the potential utilization of Citrus
leaves as a food supplement for human health [16
]. Moreover, C. limon
and its extracts have been classified as safe products according to the FDA [17
extracts’ utility in food manufacturing is mainly in reducing/inhibiting spoilage, preserving the best quality, increasing safety, and showing strong antimicrobial activity checked against microorganisms, like Pseudomonas aeruginosa
, Salmonella typhimurium
, and Micrococcus aureus
]; hence, it can be safely used as a food preservative.
Therefore, the current study aimed to (I) identify which fishery products might pose a risk of C. botulinum, (II) detect the toxin type that was most prevalent in the samples and tissues of commercial fish from the three investigated Egyptian governorates, and finally, (III) assess which Citrus (C. limon, C. sinensis, or C. unshiu) leaf extract has the most powerful effect on C. botulinum and can be used as a natural preservative.
2. Materials and Methods
2.1. Fishery Samples Collection
A total number of 360 fish samples were collected from three Egyptian governorates, Alexandria, Beheira, and Gharbia; each governorate comprises 120 samples collected from six fish products (i.e., canned tuna, canned sardine, canned mackerel, fesikh, moloha, and Renga), and 20 samples of each product. These samples were randomly collected from local supermarkets and fishery shops in 2022.
2.2. Fishery Samples Preparation
All fishery samples were refrigerated until they were tested. The surfaces of the cans were cleaned and dried, and the top surfaces were covered with ethanol (96%) and left to stand for 2 min until the ethanol was evaporated. The cans were opened with a sterile can opener and put in large plastic bags to avoid the spread of aerosols. Furthermore, 20 g of each fish was aseptically placed in a sterile mortar with 10 mL of sterile peptone water (0.1%) and then blended for 2 min. Amounts of 2 g of the prepared solid samples were inoculated into two screw-capped bottles; one contained 15 mL of the Trypticase peptone glucose yeast broth (TPGY; purchased from Oxoid Ltd., Basingstoke, Hampshire, UK) while the other contained 15 mL of a cooked meat medium (CMM; obtained from Oxoid Ltd., Basingstoke, Hampshire, UK). The inoculated media were incubated under anaerobic conditions for 7–10 days using an AnaeroGenTM gas-generating kit (bought from Oxoid Ltd., Basingstoke, Hampshire, UK). The Trypticase peptone glucose yeast broth was incubated at 30 °C, overnight (16 h), to obtain subcultures. Then, the bacterial suspension of this overnight culture that was enriched on the TPGY medium was used for C. botulinum isolation and identification using Multiplex PCR (Agilent Technologies, SureCycler 8800 Thermal Cycler, Santa Clara, CA, USA), as well for DNA extraction.
2.3. Isolation and Identification of C. botulinum Using Conventional Methods
2.3.1. Isolation of Pure Culture of C. botulinum
The isolation of C. botulinum in pure culture was enhanced by adding an equal volume of ethanol to 2 mL of enrichment culture. Then, the mixture was incubated at room temperature for 1 h. A loop of the enrichment culture of each sample was streaked on 2 plates of clostridial agar (obtained from HiMedia Laboratories GmbH, Einhausen, Germany). One of the streaked plates was incubated at 37 °C to isolate the proteolytic strains of C. botulinum, while the second plate was incubated at 28 °C to isolate the non-proteolytic strains of C. botulinum. Furthermore, the streaked plates were incubated under anaerobic conditions for 3–5 days by an AnaeroGenTM gas-generating kit (purchased from Oxoid Ltd., Basingstoke, Hampshire, UK). After 3–5 days, all cultured plates were inspected for typical colonies of C. botulinum.
2.3.2. Morphological Identification of C. botulinum Isolates
Morphological identification, microscopic examination, and the determination of catalase, lipase, and proteolytic activities were carried out according to Douillard et al. [18
2.4. Molecular Characterization
2.4.1. Bacterial DNA Extraction
Broth cultures of C. botulinum were centrifuged at 12.500× g/5 min, and the pellet was washed twofold with phosphate-buffered saline (PBS). The DNeasy® blood and tissue kit (Qiagen, Venlo, Netherlands) was used for DNA isolation following the method for Gram-positive bacteria (as described in the instruction manual). DNA was kept at −20 °C until its use in the multiplex PCR.
2.4.2. Multiplex PCR Components and Program
The genotyping of C. botulinum
neurotoxin types (A, B, E, and F) and their detection were carried out using 4 pairs of primers. The sequences was proven in study of De Medici et al. [19
] that used for detection and genotyping of C. botulinum
neurotoxin type (A, B, E, and F) genes. The reaction mixture of the multiplex PCR (50 μL) contained 25 μL of 2× Multiplex PCR master mix (purchased from Qiagen, Spain), 2 μL of 0.3 μM of each primer (obtained from Bioneer, Daejeon, Republic of Korea), and 3 μL of purified DNA template and the volume was completed up to 50 μL using sterile dH2
The amplification was performed in a programmable thermal cycler (Biometra, ND, USA). The reaction conditions were initiated by a hot start at 95 °C/15 min, then they were followed by 35 cycles, each one comprising denaturation at 95 °C/30 s, further annealing at 51 °C/30 s, and extension at 72 °C/90 s, and finally, the reaction ended by a final extension at 72 °C/7 min.
2.4.3. Detection of Multiplex PCR Products by Agarose Gel Electrophoresis
PCR end products were analyzed by 1.5% agarose gel electrophoresis in the presence of a standard DNA ladder (100 bp Nippon genetics) to measure the size of product bands and then visualized under an ultraviolet transilluminator.
2.5. Plant Samples Collection and Extracts Preparation
The leaves of Citrus limon (lemon), Citrus sinensis (orange), and Citrus unshiu (satsuma mandarin) were collected from the Experimental Farm of City of Scientific Research and Technological Applications (SRTA City), New Borg El Arab city, Egypt. The plant leaves were shade-dried for 3 days, successively ground to a fine powder using a blender, extracted in deionized H2O (1:20 w/v), centrifuged (ThermoFisher Scientific Co., Waltham, MA, USA) at 3000× g/15 min, and then filtrated. Aqueous leaf extracts were lyophilized by a vacuum freeze dryer (Lyophilizer, Model FDF 0350, Yangzhong, China) and stored for further analysis.
2.6. Phytochemical Screening of Citrus Leaf Extracts
Antioxidant compounds were determined in each leaf extract, as prescribed by Sobhy et al. [20
], to assess their biological effect. Secondary metabolites, e.g., saponin and tannins, were estimated quantitatively [21
]. In addition to secondary metabolites, antioxidant compounds, such as flavonoids, phenolics, and ascorbate, were assayed following the methods of Chang et al. [23
]. Total antioxidant capacity, by DPPH and phosphomolybdate assay (PMA), was determined according to the methods of El Sohaimy et al. [24
]. Moreover, Ferric reducing antioxidant power was evaluated following the method of Saeed et al. [25
2.7. Determination of Antibacterial Activity of Citrus Leaves Extracts and Their Minimum Inhibitory Concentration (MIC)
The wild microbial strains were grown in a nutrient broth at 37 °C/24 h, and the C. botulinum
suspension of grown cultures was prepared and adjusted to a density of 106
colony-forming units (CFU)/mL, and then spread on MHM plates. After dryness, three Citrus leaf extracts (C. limon
, C. sinensis
, and C. unshiu
) were loaded onto each separate disk (20 µL were taken from 100 mg/mL from each leaf extract concentration), and the plates were maintained at 4 °C/30 min and then incubated at 37 °C/24 h. The clear inhibitory zones obtained were recorded in mm, considering the anti-C. botulinum
activity of these Citrus leaf extracts [26
]. Moreover, a set of 7 concentrations of reconstituted aqueous leaf extracts, i.e., 1.56, 3.1, 6.25, 12.5, 25, 50, and 100 mg/mL, were examined to determine the minimum inhibitory concentration (MIC) of C. limon
, C. sinensis
, and C. unshiu
leaf extracts against pathogenic strains (C. botulinum
2.8. Storage Study and Shelf Life of Tuna Supplemented with of C. limon Leaf Extracts
The fishes were prepared as fillets weighing approximately 100–150 g. After that, the fillets were assigned into five groups: (1) the control group (untreated with an extract), (2) control tuna meat, which was dipped in chilled distilled water for 20 min, and groups 3, 4, and 5, which were infected with a C. botulinum
CFU/mL) and treated by being dipped in C. limon
leaf extract at concentrations of 10%, 20%, and 30%, respectively, for 20 min. After dipping, the chunks were drained at ambient temperature for 3 min. The fillets were placed in sterile polythene bags and stored at 4 °C. The samples were randomly removed from each treatment to assess the preservative effect of C. limon
leaf extracts on the shelf-life of tuna fillets under several storage periods, i.e., 0, 2, 4, 6, 8, 10, and 12 days. The samples representing all regions of the chunks of the respective lots (in correct quantities) were weighed and transferred for microbiological analysis at every 2-day interval [27
]. For the purpose of microbial analyses, the cooled samples were homogenized for 1 min and then incubated at 37 °C/24 h in a CO2
incubator. An amount of 1 mL was added to 9 mL of the peptone broth and incubated at 37 °C/24 h in a CO2
incubator. The total anaerobic plate counts were taken on TPGY agar under anaerobic conditions.
2.9. Sensory Evaluation
The tuna samples were maintained at room temperature, 25 °C/10 min, prior to assessment. The panelists evaluated the tuna sensorial attributes for both the control and samples treated with different concentrations (10%, 20%, and 30%) of C. limon
leaf extract based on the following criteria: odor, taste, color, texture, and overall acceptability (10 points each item), with a scale ranging from 1 to 9, where 9 = excellent, 8 = very good, 7 = very good, 6 = good, 5 = medium, 4 = fair, 3 = poor, 2 = very poor, and 1 = very, very poor, as described by Hamad et al. [28
2.10. Statistical Analysis
The obtained results were statistically analyzed based on the SPSS software (version 23, IBM SPSS Statistics for Windows, IBM Corp., New York, NY, USA) using a one-way analysis of variance (ANOVA) to determine the degree of significance for the obtained variations of the used treatments. The expressed data were the mean of three the replicates ± the standard deviation, and the significant level was estimated at p < 0.05.
This study reported the high incidence of C. botulinum in all tested fishery products from three different Egyptian governorates, which is considered one of the main biological threats that cause foodborne pathogenesis. This study highlights the control of C. botulinum (a) in contaminated fish products using the leaf extracts of Citrus limon, Citrus sinensis, and Citrus unshiu as control agents. The current findings demonstrate the antioxidant activities of Citrus leaf extracts. In addition to their broad potential to suppress the growth of C. botulinum, these bioactive components can be used to treat bacterial contamination and as preservatives.
In addition, all Citrus extracts inhibited C. botulinum growth by increasing the inhibition zone, with C. limon being the most potent extract, followed by C. sinensis and C. unshiu. Overall, the high antioxidant and anti-Clostridium powers of C. limon leaf extract, which indicate its preservative activity in fishery products during storage, can be concluded. C. limon leaf extract has the potential to prevent C. botulinum growth and serve as a promising natural preservative agent for keeping fishery products fresh and safe. Further investigations should be carried out into the potential toxicity of Citrus leaf extracts at high concentrations in both in vitro and in vivo studies.