Hygienic quality, study of enterobacteria isolated from table olives and antimicrobial activity of Olea europaea L from Tetouan, Morocco

. 88 samples of traditional and industrial table olives were collected from various outlets in the city of Tetouan. The physical-chemical controls concerned: pH , ORP (oxydo-reducing potential) and TDE (total dry extract). The microbiological side are: total aerobic mesophilic flora ( TAMB), total coliforms (TC), Staphylococcus aureus (SA) and lactic bacteria (LB) with an identification of the most predominant enterobacteria species and their sensitivity. Also to investigate the antimicrobial activity of Olive (Olea europaea L.) .The obtained results for traditional olives far exceed food hygiene standards, especially when 29.20% of traditional samples are too loaded in TAMB and TC and 16.5% are contaminated with SA. This shows that good hygiene practices are not respected. As for industrial olives, the averages in TAMB and SA are below the thresholds set by the standards, and only 19.8% are contaminated with TC which indicates the compliance of the majority of industrial type. Black table olives are the most contaminated. The identification results of coliforms extracted from traditional olives show the predominance of Citrobacter freundii and klebsiella pneumoniae .The sensitivity test shows that 80% of Enterobacteriaceae tested were sensitive to all antibiotics.The result showed that the ethanolic extrats was active against all bacteria.


Introduction
This tree is characterized by an important medical and food properties within the fruit, leaves or other components.But, during the preparation or storage, olive can be contaminated by pathogens or chemical elements, which threaten the health of consumers, thus several studies have shown this.Like the work of Pereira.A.P (1) on traditional olives marketed in Portugal and which showed the presence of Clostridium sulphite-reducing, coliforms and fecal streptococcus, the same bacteria were detected by Z.Mennane (2)in traditional table olives from Morocco.
Skandamis P.N (3) and Spyropoulou K.E et al(4) who detected Escherichia coli O157:H7 in green olives.So for staphylococcus aureus Asehraou.A (5) reported their presence in olives.The isolation of antibiotic-resistant bacteria from food can aggravate the situation, such as the working case of Z.S El kharoussi (6) and O.O.Bello (7) who were able to isolate Enterobacteriaceae -ESBL in imported or local plant foods.Other work by S. Nabbouti (8) on these plant foods, show the predominance of Klebsiella and Escherichia, and are characterized by a penicillinase-like resistance profile in 30% of the strains tested.All enterobacteria isolated by Nazha Haddia (9) (serratia spp,klebsiella spp and enterobacter spp), have resistance to Amoxicillin, Amoxicillin +clavulonic acid and cefalotin apart from Klebsiella spp Regarding the biological activity of olive leaves, various works have shown their antimicrobial power such as those of H.F. Al-Azzawie (10), A.N. Sudjana (11) and Lahdibi Sahraoui H(12).To protect the health of the consumer, good hygiene practices must be respected during preparation, storage and exposure.
In our study, the hygienic quality of 88 table olive samples were assessed from 4 sites or commercial complex and identification and sensitivity of the most predominant enterobacteria species.Also another objective is to investigate the antimicrobial activity of Aqueous and ethanolic extrats ) of Olive (Olea europaea L.)

Sampling:
This work assesses the hygienic quality of 88 samples of black, green and pitted olives taken from markets in Tetouan region.The samples were taken periodically at nine times (8 traditional sales sites) and 15 industrial samples 1.2 Physical-chemical study:

Total dry extract (TDE):
In a pre-salted porcelain crucible, 10g of olive is added and regulated oven for 24 hours until a constant weight is obtained.

Total Aerobic Mesophilic Flora (TMAF):
The TMAF count was conducted on PCA medium (Plate Count Agar) with incubation at 30 o C for 72 hours [14].

Total Coliforms (CT):
The count was conducted on MacConkey Agar medium and the incubation at 30 o C/24h, the Red Colonies were counted [15].

Staphylococcus aureus (SA):
The count was conducted on Middle Baird Parker and the incubation at 37oC/24h [16]

Lactic bacteria:
Extraction of lactic acid bacteria in MRS medium (Rogosa and Sharpe) .The incubation occurred at 30 o C for mesophilic species for 48 hours.After incubation, round or lenticular colonies were counted.(17)

Identification of coliforms
The identification of extraction was carried out by the search for the biochemical reaction profile using API 20E commercial kit (Bio Mérieux, Marcy l'Etoile, France).

1.4.1Plants collection
Olive leaves were collected in December 2020 from Region Tanger Tetouan .The leaves were washed and dried at room temperature for 2 week, after it were ground to fine powder and stored in a good conditions until extraction.

Preparation of the olive leaves extract 1 1.4.2.1 Aqueous extract
100 g of powder from the leaves were extracted by Soxhlet method for one day in 600 ml of distilled water.Then the decoction is filtered and dehydrated [18].

Organic extracts
Extraction by the Soxhlet method of 100 g of leaves, for one day in 600 ml of ethanol and subsequently evaporated by a rotary evaporator, [19]

Physical-chemical study:
For physical-chemical analyses ( table 1)of traditional samples, the black-type pH is close to neutrality, the (ORP) is low and the (TDE) is high which is explained by the presence of the nucleus.Regarding the green type, the pH is very low, the (ORP) and the (TDE) are very high (nucleus presence).Then the pitted type is characterized by a high pH and ORP.
Comparing between the three traditional sites, there is no significant difference between the pH of the different types of the same category and, that the (ORP) and (TDE) values are reconciled.Regarding industrial samples of black olives (mark1) and candied black olives , the results are different especially at the level of ORP that is negative for the first and that is for medium confit.Also the TDE is high for mark 1.For the samples of the cooperative, the pH is low, then the ORP is the highest when comparing with the other brands, and the TDE is high (nucleus presence) For industrial pitted olives there is no difference between the brands and their Values of pH, POR and TDE are close .pH is correlated with the amount of free fatty and organic acids produced by microorganisms (20)

Microbiological study:
The results of microbiological characterization( table 2and 3) show that the hygienic quality of traditional samples is insufficient, so that of industrial olives is 80% satisfactory.
The coliform contamination rate of traditional black olives ranges from 40% at the Mandari and Bab Noadar sites to 67% at the Lamhanech site and for SA it varies between 0 at the Bab Noadar site to 37.5% at the Mandari site.
So, for the green variety coliform contamination ranges from 25% at the Mandari center to about 30% at the lamhanach and Bab Noadar sites and for SA varies between 10% at the bab noadar site to 29% at the lamhanach site

Table 1: Physical-Chemical Composition of Traditional and Industrial Olives
For pitted olives samples from the Mandari and Bab Noadar sites were loaded with coliforms with a rate of 29 and 25% successively, so 15 and 12.5% of these foodstuffs were contaminated with SA in the same order.So those at the Lamhanach site were free of contamination.

Other studies have shown the contamination of olives by coliforms such as those of Pereira.A.P (1) .For Portuguese olives, Spyropoulou K.E et al(4) for green olives, Skandamis P.N (3) ,Moumene (23) in the Marrakesh and A region. MAOUNI (25) for olives from the three Moroccan cities (Fez, Marrakech and Tetouan).
For staphylococcus aureus asehraou detected these germs in 55% of the analyzed black olives.Industrial candied black and pitted type samples are 100% compliant.Probably the sterilization of candied olives, and the use of preservatives for the pitted type are the causes.Monica Österblad (30) showed that in vegetable samples, the Enterobacter spp were most frequent, Escherichia coli was rare.No resistance was found to cefotaxime, aztreonam, imipenem, gentamicin, nalidixic acid or ciprofloxacin Rabia Razzaq (31) found E. coli, Enterobacter spp.and Salmonella spp in fresh vegetables.Also all the strains were sensitive to ampicillin, erythromycin, kanamycin and nalidixic acid.

Antimicrobial activity of some extracts of Olive (Olea europaea L.)
Ethanolic extracts from olive leaves have shown inhibitory activity towards all bacteria.Especially on Chrysobacterium indoligenes and Chrysobacterium spp with an inhibition diameter of 40mm followed by methicillin-resistant staphylococcus and in third position comes klebsiella pneumoniae, Serratia marcescens and CA with a diameter of 24mm; thereafter comes Citrobacter freundii and Klvyra spp.But the aqueous extract is only effective on Klvyra spp with a diameter of 15mm.(Table 5) These results encourage the use of these ethanolic extracts of olive leaves for the preservation of table olives.Also Sahraoui (12) found that methanolic extracts of the leaves are active on enterobacteria like K.pneumoniaa, Citrobacter freundii, enterobacter spp and staphylococcus.But some aqueous extracts of sahrawi are active on all bacteria tested Other works have also shown their antimicrobial power such as those of H.F. Al-Azzawie (10)
Microbiological tests show the contamination of about 30% of traditional samples by fecal flora and that is a good indicator of deplorable hygiene conditions.In terms of pathogenic bacteria, we mention staphylococcus aureus with a rate of 16% which poses a threat to the consumer.the results of identification of enterobacteria isolated from olives show the predominance of Citrobacter freundii and klebsiella pneumoniae.This requires informing the preparers of this situation, to raise their awareness and train them to respect the conditions of hygiene (thermal treatment, cleaning in accordance with the standards and the proper packaging and storage of their finished products).The sensitivity test shows that the majority were sensitive to all antibiotics and that ethanolic extracts were active against all bacteria