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Hygienic Quality of Fermented Pepper Sold in the Markets of Brazzaville

Angélique Espérance Lembella Boumba*, Augustin Aimé Lebonguy, Chancelvy Pahivelle Limingui Polo, Mickaelle Bokamba Moukala, Joseph Goma-Tchimbakala

Ecole Nationale Supérieure d’Agronomie et de Foresterie, BP 69 Brazzaville Congo

Abstract: This study was carried out to determine the hygienic quality of six samples of fermented peppers of different ages sold in the markets of Brazzaville in the Republic of Congo and used for human consumption. To do so, decimal dilution method was performed and only the obtained isolates on Mac conkey agar were identified by sequencing of 16s rRNA gene. Then the phylogenic relationship of the sequences obtained was verified on Phylogeny.fr. The principal component analysis and the similarity dendrogram made it possible to see the resemblance between the different samples. The results showed that the FMAT load varied from 2.33.10⁵ -1.2.10⁷cfu/g with a very significant difference (P <0.001). Only the EB4 sample is of unsatisfactory quality for this parameter. The loads of total and faecal coliforms in EB4 were above the threshold values of Directive 2005/2073/EC relating to the microbiological criteria applicable to foodstuffs. Staphylococcus aureus was absent in all samples except EB4 where the microbial density is 2.6.10² cfu/g. The NCBI blast of the sequences obtained made it possible to identify the following bacterial species with a percentage of similarity equal to or greater than 99%: Enterobacter kobei, E. mori, Klebsiella Pneumoniae and Salmonella Enterica. This work showed that the quality of the peppers fermented depends on fermentation time. Older fermented peppers are of good quality compared to younger ones.

Keywords: hygienic quality, pepper; fermented food, microbial, food safety criteria

Received February 02, 2022; Revised March 04, 2022; Accepted March 11, 2022

Citation: Angélique Espérance Lembella Boumba, Augustin Aimé Lebonguy, Chancelvy Pahivelle Limingui Polo, Mickaelle Bokamba Moukala, Joseph Goma-Tchimbakala, Hygienic Quality of Fermented Pepper Sold in the Markets of Brazzaville. Journal of Food Security, 10 (1). 17-24. January 2022. doi:10.12691/jfs-10-1-3

1. Introduction

The traditional processing of fruits and vegetables is a very widespread practice in the countries of the South, because a significant proportion of these products are lost after harvest, due to the lack of conservation and processing technologies adapted to the local context [1]. Among the processed fruits, chili is one of the most consumed in the world. Its importance stems from the different uses to which it is subject [2]. It occupies an important place in human culture in many countries. Pepper is an important condiment of commercial and medicinal value with antioxidant, anti-cancer and many other properties [3]. Chili fruits are eaten fresh or as a fermented mash. Although there are several species of pepper, the most used for the production of this mash and hot sauce are Capsicum fructescens and Capsicum annum [4]. Their nutrient and pigment composition vary with species, cultivars, environmental growing conditions, and stages of maturity [4]. According to [5] and [6], peppers contain, among other components, fatty acids, volatile compounds, pigments and pungent elements. Regarding pigments, it has been shown that capsaicin, a pigment contained in red pepper, is involved in the elimination of cholesterol [7]. Other important pigments such as β-carotene, violaxanthin and antheraxanthin have also been characterized [8,9,10].

The quality of fermented peppers, like other fermented foods, is strongly related to the fermentation time, the preparation environment and good hygienic practices. In the study by [11], focusing on the diversity of bacterial communities in fermented peppers, it was shown that younger fermented peppers (fermentation time of a few days) contained more pathogen. However, the addition of certain ingredients, such as salt and oil, in the preparation of fermented pepper can make it possible to ensure good preservation of the food and would exert pressure to select certain microorganisms [12]. In addition, it has been pointed out by several authors that lactic acid bacteria (LAB) present in fermented foods produce, among other things, organic acids (which lower the pH), bacteriocin compounds which participate in the elimination of pathogens [13,14]. As fermented pepper is a condiment very appreciated in Congolese households and most often offered in restaurants across the city, its quality must be satisfactory to spare the consuming population from foodborne illnesses. In the study by [15] on the biochemical and microbiological evaluation of fermented pepper sold in local markets in Congo-Brazzaville, a complete absence of pathogens such as Salmonella, Shigella, Staphylococcus aureus and Legionella sp. after 10 days fermentation. However, [11], using an Illumina Miseq high-throughput sequencing method, showed that 10-day-old samples of fermented pepper contained pathogenic bacteria. A food is qualified as good hygienic quality if it meets the criteria defined by a standard. For example, the WHO standard for drinking water (water intended for human consumption) sets the limit values for faecal coliforms and faecal streptococci at 0 cfu / 100 mL of filtered water. On the other hand, the limits set in the food safety criteria (CSA) relating to cut vegetables defined in regulation 2073/2005 / EC are 0 cfu / 25g and 100 CFU / g of food respectively for Salmonella and Listeria monocytogenes. Thus, the objective of this study is to determine the microbial load in the mesophilic aerobic total flora (FAMT), total coliforms (CT), faecal coliforms (CF), Staphylococcus aureus, and to compare them with the standard set by Directive 2005/2073/EC relating to microbiological criteria applicable to foodstuffs in order to check whether the hygienic quality of these fermented peppers is satisfactory.

2. Material and Methods

2.1. Biological Material

The biological material used consists of six samples of fermented pepper stored in glass bottles purchased in three markets in Brazzaville.

2.2. Methods

2.2.1. Sampling

Five samples of fermented pepper already prepared and packaged in glass bottles (EB1, EB2, EB4, EB5, EB6) were purchased in three markets in Brazzaville, namely Total, Ouénze and Moukondo. Then the bottles were transported to the laboratory. With regard to sample EB3, the fruits were bought at the Moukondo market (Moungali district) then transported to a home located in the same commune where the fermented food was prepared and transferred into a glass bottle before be transported to the laboratory for fermentation at room temperature until use. The names and codes samples, fermentation time and their origins are presented in Table 1.

2.2.2. Microbiological Analysis of Fermented Peppers

The samples of fermented peppers to be analyzed were prepared. It consisted in aseptically introducing 10 g of each sample of chilli pepper into flasks containing 90 mL of sterile physiological water (0.85% w / v). Decimal dilutions ranging from 10–1 through 10–6 were prepared from this initial suspension and then 100 µL of each dilution was seeded into the appropriate agar media poured into petri dishes. After 24 to 48 h incubation, the culture dishes containing between 30 and 300 colonies were selected for manual colony counting. The coliform isolates were subsequently characterized by morphological and metabolic tests and identified after sequencing of 16S rRNA gene.

2.2.2.1. Enumeration of Total Aerobic Mesophilic Flora (FMAT)

The FMAT count was carried out on plate count agar (PCA). The reading was taken after 48 h of incubation at 37°C. The composition of the PCA medium per liter of distilled water is as follows: 5.0 g Tryptone, yeast 2.5 g Extract, 1.0 g Glucose, 15.0 g Agar.

2.2.2.2. Enumeration of Total and Faecal Coliforms

Coliform enumeration was performed on Mac conkey agar medium (Oxoid). Colonies were counted after 24 h incubation at 37°C for total coliforms and at 44°C for fecal coliforms. Mac conkey agar medium contained in g /liter: 17.0 Pancreatic digest of gelatin, 1.5 Tryptone, 1.5 Peptic digest of meat, 10.0 Lactose, 5.0 Sodium chloride, 0.03 Neutral red, 0.001 Crystal violet, 13.5 Agar.

2.2.2.3. Enumeration of Staphylococcus aureus

The enumeration of suspected Staphylococcus aureus was performed on Mannitol Salt Agar. After 48 h of incubation at 37°C, only yellow colored colonies were counted. The composition of Mannitol Salt Agar medium is as follows (g/L): 5.0 Pancreatic digest of casein, 5.0 Peptic digest of animal tissue, 1.0 Beef extract, 10.0 D-Mannitol, 75.0 Sodium chloride, 0.025 Phenol Red, 15.0 Agar.

2.2.3. Molecular Identification of Bacteria

2.2.3.1. Genomic DNA Extraction

Genomic DNA was extracted from the bacterial pellet with Magnetic Bead technology using Omega BioteK Mag-Bind Universal Pathogen Kit according to the manufacturer's instructions.

Table 1. Code, origin and fermentation time of fermented pepper samples

2.2.3.2. PCR Amplification and Sequencing

16S rRNA amplification was done with universal primers 16S-27F and 16S-1492R.The quantities of reagents used for PCR are: 2µl gDNA extracts, 5µl 5X Mix HOTBIOAmp at 12.5 mM MgCl₂, 1.5µl 10X Enhancer, 1µl Primer 16S-27F, 1µl Primer 16S-1492R, 14.5 µl H₂O qsp 30µl. The PCR was carried out under the following conditions: Denaturation at 96°C for 12 minutes, 30 cycles of denaturation at 96°C for 20 seconds, Hybridization at 56°C for 20 seconds, elongation at 72°C for 1min30, a final extension at 72°C for 5 min. The fluorimetric assay of the DNA was carried out with the Quantus device (Quantus and Quantifluor DNA Kit from Promega). A positive control (E.Coli gDNA) as well as a negative control (H2O) was systematically amplified under the same conditions as the bacterial DNAs to be analyzed. The PCR products were separated by enzymatic purification using the ExoSap-It kit. The sequencing was carried out with the BigDyeV3.1 Dye Terminator Kit (Thermo Fisher Scientific) at the BIOfidal laboratory (Lyon, France). The sequences were then purified with the BigDyeXterminator kit (Thermo Fisher Scientific) before being analyzed on the ABI3730XL 96 Capillary Sequencer (Thermo Fisher Scientific).

2.2.3.3. Bioinformatics and Statistical Analyzes

The bar diagram used for the enumeration of bacteria was carried out on the software graphprism. Hierarchical classification of samples and principal component analysis were performed with Past3 software. The sequences obtained were aligned with CHROMAS Pro software (Technelysium Ltd) to create the Contig Complete of the 16S gene. The sequences obtained were compared to homologous sequences contained in the sequence database of National Center for Biotechnology Information (NCBI) by using the Basic Local Alignment Search Tools (BLAST) program (http://www.ncbi.gov/Blast.cgi). Identification at the genus or species level was carried out according to the criteria defined by [16]. The phylogenetic tree was built on Phylogénie.fr with the identified strains and three strains obtained from the NCBI database.

3. Results

3.1. Microbial Profiles of Chilli Pepper Samples

Figure 1 shows the load of FMAT, CT, CF and Staphylococcus aureus in the 6 samples of fermented pepper. The total flora density varies from 2.30.10⁵ -1.15.10⁷ cfu /g of feed. The difference in microbial loads between the samples is very significant (P < 0.001). The FMAT load was higher in EB3 (1.15.10⁷ cfu / g) followed by EB4 (7.57.10⁶ cfu / g) then by EB6 (2.33.10⁵ cfu / g). Only the EB1, EB2, EB5 and EB6 samples have FAMT loads below the limit threshold allowed by Directive 2005/2073/EC relating to microbiological criteria applicable to foodstuffs which is10⁶ cfu / g. Concerning total coliforms, they are not present in all the samples with values less than or equal to the limit threshold accepted by Directive 2005/2073/EC relating to microbiological criteria applicable to foodstuffs which is 10³ cfu / g. On the other hand, for faecal coliforms, the bacterial load varies from 0 - 1.6.10² cfu / g and the limit threshold set by Directive 2005/2073/EC relating to microbiological criteria for foodstuffs is 10¹cfu/g. A single EB4 sample has a load of fecal coliforms greater than this threshold. With regard to Staphylococcus aureus, only the EB4 sample is contaminated with a load of 2.6.10² above the threshold which is 10² cfu / g.

Figure 1. Bacterial load of the 6 samples of fermented pepper

3.2. Hierarchical Classification of Samples

Samples were also compared on the basis of the bacterial load present. The similarity dendrogram between samples (Figure 2) shows 2 groups. A 1st group formed by the sub-cluster EB5, EB1 and EB3 with a similarity of 0.85 linked to EB6 with a similarity of about 0.44 and a 2nd cluster, formed by EB3 and EB4 with a similarity of about 0.75.

Principal Component Analysis showed that component 1 expresses 100% of the total deviation results. Samples EB1, EB2, EB5 and EB6 are negatively correlated with axis1 while EB3 and EB4 are positively correlated. Regarding axis 2, only EB4 is positively correlated while the other samples are negatively correlated. These results also indicate that EB1, EB2, EB5 and EB6 form a group and are distant from EB4 and EB3. In addition, EB4 appears to be the most contaminated sample.

Figure 2. Similarity dendrogram between samples

Figure 3. Principal component analysis based on the enumerated genres

3.3. Molecular Identification of Strains

The results of the molecular identification of Enterobacteriaceae isolated from chilli are shown in Table 2. After sequencing by the method of Sanger, the sequences were blasted in comparison with the already identified strains found in the NCBI database. Thus, the strain P1, isolated from EB1 was identified as Klebsiella pneumoniae P1 with a percentage similarity of 100%. Strains P4 and P39, isolated from EB3, were identified as Klebsiella pneumoniae (P4) and Enterobacter kobei (P39), respectively. 4 strains were selected from EB4 and identified as Klebsiella pneumoniae (P38), Enterobacter mori (P13-1), Salmonella enterica (P34), Klebsiella pneumoniae (P8). From sample EB6, 2 strains were selected and identified as Klebsiella pneumoniae (P7), Enterobacter kobei (P21-1). All these strains have been registered on NBCI with the accession numbers present (Table 2).

Figure 4 shows the phylogenetic tree built on the Phylogeny.fr server with the strains identified in this study and the 3 related strains obtained on NCBI.

All the strains identified as Klebsiella formed the same group associated with Salmonella enterica. However, the Enterobacter kobei (P39) and Enterobacter mori (P13-1) strains identified form a sub-cluster with the Enterobacter mori strain taken on NCBI, forming another cluster with the Klebsiella michiganensis strain taken on NCBI. Finally, the Pseudomonas aeroinosa MDR2 MH078568.1 strain forms an external group.

Table 2. Identification of Enterobacteriaceae isolated from chilli by the 16s rRNA technique

Figure 4. Phylogenetic tree of identified strains

4. Discussion

The World Health Organization and the United Nations Food Organization emphasize compliance with hygiene and safety rules, the sole guarantor of consumer health. Therefore,

The control of a food product intended for human consumption cannot be done without evaluating its quality. It is about the nutritional, organoleptic and hygienic quality. This latter is the subject of the study of germs of hygienic interest which are, among others: FMAT, total and feacal coliforms and Staphylococcus aureus [17]. Indeed, pathogenic microorganisms (the most frequently encountered in food) belong to the groups of fecal coliforms, total coliforms, Staphylococcus aureus, Shigella and Samnolla and are sometimes responsible for health disorders [18,19]. In this context, standards have been established by certain organizations such as AFNOR, ISO, US-EPA and WHO to establish the quality of the food consumed. The fermented pepper consumed in the Congo, which is also a food, must meet these hygienic quality requirements to preserve the health of consumers. The objective of this work was to assess the bacteriological quality of 6 samples of fermented pepper purchased and prepared in Brazzaville. Bacteriological analysis of these samples by the decimal dilution technique on PCA medium showed that for FAMT, the microbial load varied from 2.3310⁵ -1.20107 cfu/g. The difference in microbial loads between the samples is very significant (P <0.001). The EB3 and EB4 samples have a FAMT load greater than the limit thresholds accepted by the European Codex, which is 10⁶ cfu/g. they are therefore of unsatisfactory quality. On the other hand, samples EB1, EB2, EB4 and EB6 have loads in FAMT below the limit threshold (10⁶ cfu/g), they are of satisfactory quality. In the study of [15], although the FAMT load was not compared to a standard, nevertheless 15.55% of the samples were non-compliant with respect to the threshold set by directive 2005/2073/EC relating to the criteria microbiological applicable to food. The 84.44% of the samples were of satisfactory quality. Other authors have enumerated FAMT in other fermented foods and spices. [20], for example, showed that African mustard produced from fermented seeds of Parkia biglobosa and Glycine max had a FAMT load of less than 10⁶, therefore of satisfactory quality. [21] also obtained in Guedji, a condiment made from fermented and dried fish, FAMT concentration lower than the standard. The high load of FAMT in the EB3 and EB4 samples dating back a few days could be explained by the unsanitary environment in which the product was manufactured, the raw material and the utensils used [11,22]. The presence of this flora allows to assess the overall bacterial load present in a food. It is an indicator for the application of good manufacturing and hygiene practices. Also, the FAMT load allows to learn about the state of freshness or decomposition of the food [1].

This study also revealed that all samples had a total coliform load of less than or equal to 10³ cfu/g of food. They therefore comply with the standards set by Directive 2005/2073/EC relating to microbiological criteria applicable to food. No study to our knowledge has enumerated total coliforms in fermented peppers in Congo. Nevertheless [15] enumerated Enterobacteriaceae, a group in which coliforms are found. In the study by these authors, the number of bacteria belonging to the Enterobacteriaceae family varied between 10⁵-10⁶ cfu/g. Thirteen samples did not have Enterobacteriaceae and the rest had values ​​between 10⁵ and the study by [23] on the evaluation of the microbiological quality of 3 spices including chilli in Bangladesh, total coliforms were counted. Their results showed that the unpackaged peppers had a load of 7.2010¹ in TC and the packaged ones did not. As for the study of [24] on fermented milk, the total coliforms load in 7 samples out of 18 varied from 10³-104 CFU/mL. According to [25], the presence of total coliforms is not necessarily a direct indication of faecal contamination. The presence of Coliforms in some samples of chili peppers is evidence of poor processing hygiene that may result from the process, the equipment in contact and/or the immediate environment of the food. These bacteria are generally not dangerous from a health point of view except in cases of extremely abundant proliferation or particular sensitivity of the consumer [25]. These microorganisms could degrade the products by altering the taste, the smell, the appearance, in short, the merchantability [26]. However, [23] point out that coliform bacteria appear sporadically and usually in small populations in spices and are associated with feacal contamination.

Regarding fecal coliforms, only the EB4 sample has a bacterial load greater than 10 CFU/g of food (limit threshold). It is therefore of unsatisfactory quality. Although few studies have enumerated feacal coliforms in fermented peppers, several authors have nevertheless performed the enumeration of these in fermented foods. In the study by [20], the enumeration of feacal coliforms did not show any colony of bacteria from this group. The work of [27] showed that in most crushed peppers sold in the markets, enteric bacteria and feacal coliforms were absent, except in samples produced under unsanitary conditions. The presence of feacal coliforms in a food indicates feacal contamination. This group contains pathogenic bacteria such as Klebsiella sp., Enterobacter sp. E. coli, and Serratia sp. Several authors who have used high throughput sequencing methods have confirmed the presence of these pathogenic bacteria in fermented peppers and other fermented foods [11,28,29,30]. The absence of total coliforms in the EB2 and EB5 samples is probably due to the observance of good preparation practices by the producers. In addition, it is possible that the metabolic processes during fermentation led to the decrease in pH, the production of organic acids, and the production of inhibitory substances making the environment hostile to the growth of pathogens [31,32,33,34]

About Staphylococcus aureus, only 1 of the 6 samples had a bacterial concentration of 2.6.10² cfu / g. This value is greater than the maximum acceptable value (100 cfu/g) set by directive 2005/2073 / EC. Thus, the quality of the EB4 sample is therefore unsatisfactory. In the study by [23] no strain of S. aureus was found in the 2 types of peppers (fresh and fermented) analyzed. The presence of these pathogenic bacteria in this sample may be due to non-compliance with good preparation practices, in particular non-cleaning of the hands. Staphylococcus aureus is considered the third most important cause of foodborne illness in the world [35,36]. Indeed, this bacterium secretes thermostable enterotoxins released in the food during its growth. This enterotoxin causes gastroenteritis [37].

To group the samples according to their hygienic quality based on the count, a hierarchical classification was performed. Thus, the samples EB5, EB2 and EB1 and EB6 form a cluster. The samples EB4 and EB3 form a second cluster. This variability is due to the duration or time of fermentation. The older samples form one group and the younger ones form another group. The study of [11,38] had shown that fermentation time influences the microbial diversity of fermented foods. The older the sample, the less diverse it is, so the fewer pathogenic bacteria there are. On the other hand, the younger the sample, the more bacteria there are and the more pathogens can be found. The principal component analysis confirms the grouping obtained from the hierarchical classification. However, the EB4 sample contains Staphylococci which differentiates it from EB3.

Sequences analysis by BLAST on NCBI showed that the identified strains are the following species: Klebsiella pneumoniae, Enterobacter mori, Enterobacter Kobei and Salmonella Enterica. The study of [39] identified the genus Enterobacter, Klebsiella, Escherichia, Proteus vulgaris and Shigella from fermented milk. The study of [11,40] showed by high throughput sequencing methods the presence of the genera Klebsella and Enterobacter in fermented Brazzaville pepper and the genus Enterobacter in fermented apple. Some of these genera are classic human contaminants and can grow on organic material. These bacteria are most likely present in the air where they can contaminate raw materials and used utensils. The review by [19] and the work of [28] reveal that cases of microbial pathogens have been reported in association with several fermented foods, such as cheese, sausages, fermented fish and cereals such as Bacillus cereus, Escherichia coli, Salmonella sp., Escherichia coli O157: H7, Staphylococcus aureus, Vibrio cholera, Listeria monocytogenes, Aeromonas, Klebsiella, Campylobacter and Shigella sp. The presence of its pathogenic bacteria alters the hygienic quality of these foods.

5. Conclusion

This study allowed to assess the hygienic quality of fermented pepper sold in 3 markets in Brazzaville. It showed the variation of the bacterial groups counted from one sample to another, from one market to another, but also the influence of the fermentation time on the quality of the fermented pepper. All samples are of satisfactory quality compared to the 1995 Codex Alimentarius with the exception of sample EB4. Then, nine isolates belonging to the feacal coliform group were selected and identified by sequencing the 16S rRNA gene. These isolates belong to 4 species. These are Salmonella enterica, Enterobacter kobei, Enterobacter mori and Klebsiella pneumoniae. These strains were registered on NBCI. These results highlight a need to expand this study in several departments of the Congo in order to have a better appreciation of the hygienic quality of fermented pepper. Too to see the influence of controlled fermentation after more than 6 months on the enterobacteriaceae species colonizing the raw material.

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