Microbiological quality of Moroccan labeled Euphorbia resinifera honey

1188 MICROBIOLOGICAL QUALITY OF MOROCCAN LABELED EUPHORBIA RESINIFERA HONEY Abdelkarim Moujanni*1, Anass Terrab2, Rabiaa Eddoha1, Boubker Nasser1, Maryam Benbachir1, Nour Eddine Chaouqy3, Taoufiq Bouzid3 and Abdel Khalid Essamadi1 Address(es): 1Biochemistry and Neurosciences laboratory-Team of Applied Biochemistry and Toxicology. Faculty of Sciences and Technology of Settat. Hassan First University. PB. Box 577 Settat 26000, Morocco. 2Department of Plant Biology and Ecology, University of Sevilla, Ap. 1095-41080. Sevilla, Spain. 3Regional Analysis & Research Laboratory of ONSSA, Agadir (LRARA), BP. 474, Central. Agadir, Morocco. *Corresponding author: [email protected], essama[email protected].ma ABSTRACT Keywords: Morocco, labeled Euphorbia resinifera honey, Bacteriological Quality INTRODUCTION The Euphorbia resinifera is one of the specific and endemic plants of Moroccan Atlas Mountains (Picture 1). Generally, the Euphorbia plants have high adverse effect level (due to the Latex component, which is a powerful alkaloid), so they has been studied for their antifungal and antibacterial properties (Kamba et al., 2010; Benmehdi et al., 2013). In addition, the honeys produced from these plants confirms the antibacterial and antifungal activity (Malika et al.; 2004, Crousilles, 2014; Bouhlali et al., 2016). Likewise, generally the intrinsic properties of honey (osmolality, pH, hydrogen peroxide, phenolic components and flavonoids) affect the growth and survival of microorganisms by bacteriostatic or bactericidal action. (Adock, 1912; White et al., 1962; Iurlina and Fritz, 2005; Kačaniováet al., 2009; Adenekan et al., 2010). Furthermore, the low pH, the low water activity and the high sugar content of undiluted honeys prevent the growth of many species of microorganisms (Snowdon and Cliver, 1996, Snowdon, 1999). In consequence, Euphorbia honey can be expected to contain a small number and limited varieties of microorganisms. It can be noted that vegetative forms of human disease-causing bacteria have not been found in honey and, as bacteria do not replicate in honey, a high count of vegetative bacteria is indicative of a recent contamination from a secondary source (Snowden and Cliver, 1996; McKee et al., 2003; Antúnez et al., 2004). The microorganisms of interest are those that withstand the concentrated sugar, acidity and antimicrobial character of honey. These microorganisms; indicative of sanitary or commercial quality, include yeasts, molds, coliforms, Salmonella, Shigella and some microorganisms such as sporus-forming bacteria, like Bacillus cereus (B. cereus) and Clostridium perfringens (C. perf.), which under certain conditions (e.g. germination and growth in a non-heated-treated product) could cause illnesses in humans (Snowdon and Cliver, 1996; Al-Waili et al., 2012). Otherwise, the Moroccan standards for honey quality (Moroccan Norm 08.05.600, 2012) inspired essentially from Codex Alimentarius Standards (Codex Stan, 2001) and the specifications of the label "GPI" (Moroccan Order, 2012) includes several chemical and physical parameters but do not require microbiological analysis. However, the use of adequate hygienic practices during the product handling is required (Moroccan Law 28-07, 2010; Moroccan Norm 08.0.000, 2008). In addition, various studies have been carried on the palynological and physicochemical parameters of Moroccan Euphorbia honey (Chakir et al., 2011;Aazza et al., 2014, Terrab et al.,2014; Bettar et al., 2015), but a microbiological contamination has not been extensively investigated. Figure 1 Euphorbia resinifera plant of the “PGI” Tadla-Azilal region of Morocco. ®Photo. Moujanni In the present work, microbiological profile of thirty-seven samples of labeled honey were collected in a Protected Geographical Indication “PGI” area of Tadla-Azilal region, which is an endemic zone of Euphorbia resinifera plant. A profile was assessed using conventional microbial methods, like enumeration, detection and/or germs identification, in accordance with ISO norms. This is the first study in which a honey with Moroccan “PGI” was tested, in order to assess its compliance with bacteriological recommendations. Coliforms (Total and fecal Coliforms), Salmonella spp., Shigella spp., Sporus of Bacillus cereus and Clostridium perfringens were not detected. The numbers of Standard Plate Count “SPC” were less than 102 CFU.g-1 for all samples. The molds and yeasts were found among samples and 32% and 40% of samples were positive, respectively. However, no samples showed a higher value than recommended limit [102 CFU.g-1]. We conclude that samples of labeled euphorbia honey of Tadla-Azilal analyzed present good commercial quality parameters (SPC, molds and yeasts “absence of unwanted fermentations”), a good sanitary quality (absence of coliforms and S. aureus) and are safe (Slam., Shig., Sporus of B. cereus and C. perf.). Standardization (regulation and specifications) and a rationalization of beekeeping techniques throughout Euphorbia “PGI” area studied may further sustainably improve the quality of this unique honey, and ensure it over the years. ARTICLE INFO Received 17. 11. 2016 Revised 1. 2. 2017 Accepted 20. 2. 2017 Published 3. 4. 2017 Regular article doi: 10.15414/jmbfs.2017.6.5.1188-1194 J Microbiol Biotech Food Sci / Moujanni et al. 2017 : 6 (5) 1188-1194 1189 Figure 1 Distribution of samples of labeled monofloral Euphorbia resinifera honeys in “PGI” production area of Tadla-Azilal Euphorbia resinifera honey of Tadla-Azilal region is the first honey labeled “Protected Geographical Indication –PGI-“in Morocco (Moroccan Order, 2012; ADA, 2014). This label was published in the EU Official Journal through the public consultation documents on geographical indications of the Kingdom of Morocco (European Commission, 2013/C). In this context and in concordance, with the importance and good status of this unique monofloral honey, we decided to investigate about its bacteriological profile targeting the major microbiological contaminants (SPC, Total coliform, Fecal coliform, Sporus of Bacillus cereus and Clostridium perfringens, Staphylococcus aureus, Salmonella spp, Shigella spp,) and its fungal profile (Molds and Yeasts) which may cause undesirable fermentation. Their counts being indicative of honey’s commercial and sanitary quality and safety. MATERIAL AND METHODS Sampling Thirty-seven (37) samples of honey “GPI” mono-floral Euphorbia resinifera were supplied directly by the beekeepers of the “PGI” area affiliated to U.C.A.T.AZ cooperative or working individually (Picture 2). Their distribution is indicated in Figure 1. The samples had not been heated or pasteurized. The productions years of all samples were 2013 and 2014 (Table 1). Upon collection, 250g or 500g of each sample are put in clean commercial labeled container and stored at room temperature pending analysis (Table 1). Figure 2 “PGI” Tadla-Azilal production area with aggregate Euphorbia resinifera plant Table1 Information on honey samples studied Sample Locality name Harvest Year P1 FoumOudi 2014 P2 FoumElaancer 2013 P3 AitMhamed 2013 P4 Tanougha 2013 P5 Tabia 2014 P6 Tabaroucht 2013 P7 Ait Hamza 2013 P8 Elksibah 2013 P9 Tilougguite 2013 P10 Rfala 2013 P11 Afourer 2013 P12 Ben Driss 2012 P13 BeniMellal 2013 P14 Timoulilt 2013 P15 Bin Elouidane-AitOuarda 2013 P16 AitMazigh 2013 P17 Anergui 2013 P18 Bzou 2013 P19 FoumElaancer 2014 P20 AitOuaarda 2014 P21 AitAamir 2014 P22 Tagzirt 2014 P23 Anergui 2013 P24 Afourer 2013 P25 BeniAayat 2014 P26 FoumJemaa 2014 P27 BeniMellal 2014 P28 Azilal 2014 P29 Tanant 2014 P30 Ouaouizaght - Damnat 2014 P31 Tagzirt 2014 P32 AitAbbass -AitMassad 2014 P33 Assaksi - Tagleft 2014 P34 AitBououlli 2014 P35 FoumElaancer -Tagzirt 2014 P36 Ouaoula-AitMhamed 2014 P37 Elksibah 2014 PGI: Protected Geographical Indication UCATAZ: Union of Beekeepers Cooperative of “PGI” Tadla-Azilal Region, Morocco. Microbiological analysis. Ten grams of each sample were mixed with 90mL of Buffered Peptone Water (Biokar) to prepare the initial dilution. This was used at the mother dilution for further serial dilution. Standard Plate Count (SPC) (ISO Norm 4833-1, 2013): Appropriate serial dilutions (between 10 and 100 colonies per plate) of the samples in the Buffered Peptone Water were placed on standard plate count agar (PCA) (Biokar, France). The plates were incubated at 30°C for 72h. Coliform counts (TC) (ISO Norm 4832, 2006): Were enumerated on Violet Red Bile Lactose Agar (VRBLA). Plates were incubated at 37°C for 24h. Morocco J Microbiol Biotech Food Sci / Moujanni et al. 2017 : 6 (5) 1188-1194 1190 Fecal coliforms (FC) (NF Norm V 08 060, 2009): Were enumerated on Violet Red Bile Lactose Agar (VRBLA). Plates were incubated at 44°C for 24h. Staphylococcus aureus (S. aur.) (ISO Norm 6888-1, 2003): Were enumerated on Baird Parker growth medium (Biolife). Plates were incubated at 37°C for 24h and 48h. Shigella detection (Shig.) (Lampel KA, 2001): 25g of the sample was homogenized in 225mL of selenite broth (Biokar) and the volume was transferred to an Erlenmeyer flask and incubated at 35°C for 20h. After this period, a loopful of this broth was plated onto Petri dishes containing XLD agar (Biolife) and Salmonella–Shigella agar (SS, Biokar). After incubation for 48h at 35°C, five characteristic colonies of Shigella were biochemically tested on TSI agar (Biokar) and API-20E Biomerieux). The colonies were also serologically tested. Clostridium perfringens (C. perf.) (ISO Norm 7937, 2004): Petri dishes are seeded with a specific quantity of the initial suspension. Other dishes were seeded in the same conditions, using decimal dilutions obtained from the mother suspension. The tryptone sulfite cycloserine (Biolife) was added and then a layer of the same medium is added from above. The dishes were incubated anaerobically at 37°C for 20h. The characteristic colonies are counted. Finally, the characteristic colonies are confirmed and the number of C. perfringens per gram of sample is calculated. Bacillus cereus (B. cerus.) (ISO Norm 7932, 2004): Seeding the surface of a solid selective culture medium poured into Petri dishes (MYP Agar) with a specified quantity of the initial suspension. Other dishes were seeded in the same conditions, using decimal dilutions obtained from the mother suspension. The plates are incubated aerobically at 30°C for 18 to 48h. The characteristic colonies are counted and the characteristic colonies are confirmed by hemolysis test and the number of B. cereus per gram of sample is calculated. Salmonella detection (Salm.) (ISO Norm 6579/A1, 2007): For the detection of the presence of Salmonella, 25g of honey sample was homogenized in 225mL of peptone buffered water (Biokar), transferred to an Erlenmeyer flask and incubated at 35°C for 24h. After the incubation period, 1mL was added to a tube containing 10mL of tetra-thionate broth (Biolife). The Rappaport broth (Biokar) received 0.1mL from pre-enrichment and the tubes were incubated at 37°C and 41.5°C for 24h, respectively. After this period, a loopful of each selective broth was plated into Petri dishes containing xylose lysine desoxycholate agar (XLD- Biolife) and CHRO-Magar (Rambach). After incubation for 24h at 37°C, five typical colonies from each agar plate were biochemically tested on TSI agar (Biokar) and API-20E (Biomerieux). The colonies were also serologically tested with polyvalent somatic and flagellar antisera (Probac).  Mold and yeast counts (ISO Norm 21527-1, 2008): Petri dishes prepared using a defined selective culture medium (Glucose Chloramphenicol Agar- Biolife) are seeded. In the number of colonies expected, a specific amount of the initial suspension or decimal dilutions sample / suspension are used. Additional Petri dishes can be seeded in the same conditions; using dilutions decimal obtained from the initial suspension. Plates are incubated aerobically at 25°C for five days. Then, if necessary, the agar plates are allowed to stand in daylight for one to two days. Colonies/propagules are then counted, and if necessary (to distinguish yeast colonies of bacteria colonies), the identity of suspicious colonies is confirmed by examination under the binocular or microscope. The number of yeasts and molds per gram is calculated from the number of colonies/ propagules/germs obtained on Petri dishes selected to dilution ratios to obtain colonies that can be counted. Molds and yeasts are counted separately. Statistical analysis All determinations were made in triplicate and the data was processed using XLSTAT, 2015 software. RESULTS AND DISCUSSION Results of the microbiological analyzes are given in Table 2. The standard plate count (SPC) also referred to as the aerobic plate count or the total viable count, is one of the most common tests applied to indicate the microbiological quality of food. The Moroccan legislation (Moroccan Order, 2004) does not set values for SPC in honey but establishes only that you follow good hygiene practices in handling and processing of this product because entire microbial load in honey can indicate the possible presence of pathogens (Moroccan Norm 08.5.600, 2010). The SPC were isolated from all samples of honey. Their number varied between 10 and 340CFU.g-1 with a mean value equal to 76.76±82.93CFU.g-1. This result was inferior to those obtained, for the same type of Moroccan honey, by Malika et al., (2005). Compared to other foreign honeys, our results are below Argentinean and French honeys which had main SPC values 244CFU.g-1 and 227 CFU.g-1 respectively (Iurlina and Fritz, 2005; Tysset et al., 1981), while Portuguese commercial honey had better SPC levels [2.101CFU.g-1] (Gomes et al., 2010). This variation of SPC values could be related to the type of sample, the age and the honey harvest time. In addition, these vegetative forms can be made by secondary contamination, which would also explain the high counts sometimes found in honey (Snowdon and Cliver, 1996). Coliforms (TC and FC) are indicators of fecal contamination and poor hygienic processing conditions. In this study, TC and FC were not detected (level of quantification is 10CFU.g-1) and suggest a respect of good practices for extraction and processing of honey were followed. Our results corroborate with data found by Rall et al., (2003), Gomes et al. 2010, Iglesias et al., (2012), Rios et al., (2014) and Kunovà et al., (2015). The absence of these microorganisms in analyzed honey was expected since bacteria growth needs water activity more than 0.91 (Ribeiro and Seravalli, 2004). Snowdon and Cliver, (1996) already reported that the population of FC in honey varied from 10 to 102CFU.g-1. In contrast, in 70 samples of honey analyzed in Nigeria Coliforms and E. coli were isolated at rate of 95.7% (Kokubo et al., 1984). Also, Dûmen et al.,(2013) and Sherwani et al.,(2013).reported respectively that 16% of 80 honey samples were contaminated by coliforms in Turkey and two Pakistanian honey samples over six presents coliforms (0.2×101 and 0.4×101 CFU.g-1) S. aureus is the causative agent of the numerous outbreaks of foodborne disease worldwide. Poisonings generally occur after an intake of enterotoxins through the alimentary track. The absence of this bacterium in this study constitutes another sanitary index in favor to the quality of this product. In a similar study done on Turkish honeys, 13.4% of the 67 samples analyzed contained S. aureus (Dûmen et al., 2013). B. cereus and C. perfringens, as producers of spores, are considered as health indicator including uncontrolled land-based, environmental or human contamination. High levels of B. cereus in honey constitute a risk to the consumer, as ingestion of 105 spores can result in food-borne illness (Stenfors et al., 2008). The results of this study demonstrate a negative result regarding detection of sporus of B. cereus and C. perfringens. However, Pucciarelli, (2014) found the incidence of Clostridium and Bacillus (42.85 and 39% respectively) in yateí honey from Argentina. In addition, Ragazani et al., (2008) studying honey marketed in several Brazilian states found 11% were Clostridium genus and 28% of the genus Bacillus. Erkan et al.,(2015) and Sherwani et al.,(2013) reported respectively 5.5×101±6.3×101 B. cereus mean count in Turkish honey and presence of B. cereus in all (six) samples of Pakistanis (Karachi) honey tested. In respect to safety, none of the 37 samples contained Salmonella and Shigella. The absence of these pathogens was expected since in addition to its antibacterial properties, honey has low water activity and a pH, which are not in favor to the development of such bacteria (Snowdon and Cliver, 1996; Alves et al., 2015; Matuella and Torres, 2000). In the same way, study of the microbiologic quality of honey samples produced in the surroundings of a large garbage dump in Brazil showed the absence of Salmonella. These results confirm the conclusion of Anses that the Salmonella survival duration in honey does not exceed one month (Anses, 2012). Table 2 Distribution of microorganisms detected in “GPI” Moroccan Euphorbia resinifera honeys Nº Microorganisms count (CFU.g-1) Per 25g Sporus SPC TC FC S.aur Yeasts Molds Salm. Shig. C.perf B.cereus P1 50 <10 <10 <102 <10 <10 Abs Abs <10 <10 P2 340 <10 <10 <102 10 <10 Abs Abs <10 <10 P3 320 <10 <10 <102 70 90 Abs Abs <10 <10 P4 270 <10 <10 <102 20 <10 Abs Abs <10 <10 P5 250 <10 <10 <102 93 60 Abs Abs <10 <10 P6 60 <10 <10 <102 10 <10 Abs Abs <10 <10 P7 90 <10 <10 <102 <10 <10 Abs Abs <10 <10 P8 30 <10 <10 <102 <10 <10 Abs Abs <10 <10 P9 50 <10 <10 <102 30 10 Abs Abs <10 <10 P10 70 <10 <10 <102 30 10 Abs Abs <10 <10 J Microbiol Biotech Food Sci / Moujanni et al. 2017 : 6 (5) 1188-1194 1191 P11 40 <10 <10 <102 <10 <10 Abs Abs <10 <10 P12 30 <10 <10 <102 <10 <10 Abs Abs <10 <10 P13 40 <10 <10 <102 <10 <10 Abs Abs <10 <10 P14 10 <10 <10 <102 <10 <10 Abs Abs <10 <10 P15 110 <10 <10 <102 20 20 Abs Abs <10 <10 P16 30 <10 <10 <102 10 <10 Abs Abs <10 <10 P17 80 <10 <10 <102 <10 <10 Abs Abs <10 <10 P18 20 <10 <10 <102 <10 <10 Abs Abs <10 <10 P19 30 <10 <10 <102 <10 <10 Abs Abs <10 <10 P20 70 <10 <10 <102 50 10 Abs Abs <10 <10 P21 60 <10 <10 <102 20 10 Abs Abs <10 <10 P22 40 <10 <10 <102 <10 <10 Abs Abs <10 <10 P23 120 <10 <10 <102 80 20 Abs Abs <10 <10 P24 10 <10 <10 <102 <10 <10 Abs Abs <10 <10 P25 70 <10 <10 <102 <10 <10 Abs Abs <10 <10 P26 10 <10 <10 <102 <10 <10 Abs Abs <10 <10 P27 50 <10 <10 <102 50 <10 Abs Abs <10 <10 P28 50 <10 <10 <102 20 <10 Abs Abs <10 <10 P29 10 <10 <10 <102 <10 10 Abs Abs <10 <10 P30 20 <10 <10 <102 <10 <10 Abs Abs <10 <10 P31 50 <10 <10 <102 <10 <10 Abs Abs <10 <10 P32 50 <10 <10 <102 <10 10 Abs Abs <10 <10 P33 50 <10 <10 <102 <10 10 Abs Abs <10 <10 P34 10 <10 <10 <102 <10 <10 Abs Abs <10 <10 P35 60 <10 <10 <102 <10 <10 Abs Abs <10 <10 P36 110 <10 <10 <102 60 20 Abs Abs <10 <10 P37 80 <10 <10 <102 <10 <10 Abs Abs <10 <10 Mean 76.76 <10 <10 <102 38.20 23.33 n.a. n.a. <10 <10 SD 82.93 n.a. n.a. n.a. 27.20 25.35 n.a. n.a. n.a. n.a. Min 10 n.a. n.a. n.a. 10 10 n.a. n.a. n.a. n.a. Max 340 n.a. n.a. n.a. 93 90 n.a. n.a. n.a. n.a. n.a.: not applicable- Abs: Absence Level of quantification = 102 CFU.g-1 for S. aureus Level of quantification = 10 CFU.g-1 for all microorganisms tested Level of quantification = 10 sporus for C.perf. and B.cereus Table 3 Comparing the results of molds and yeasts counts in honey from different countries References Nb. samples Molds and yeast Count CFU.g-1 Country Incidence Positives/Tot al analyzed Limit recommen ded CFU.g- 1 [a] [55] Min Max Mean Iurlina and Fritz (2005) 23 0 4.7×102 1.64×102 Argentina 59% 1.0×102 Finola et al., (2007) 23 <1.0×101 <1.0×101 - Argentina - 1.0×102 Rios et al, (2014) 58 - - - Argentina 17% (7%) 1.0×102 Pucciarelli et al., (2014) 28 1.2* 4.7* 3.02* Argentina - 1.0×102 Rall et al., (2003) 100 <1.0×102 1.5×105 - Brazil 64% 1.0×102 Sereia et al., (2010)[b] 11 1.9×102 1.1×103 5.3×102 Brazil - 1.0×102 Sereia et al., (2010)[c] 6 1.8×101 2.5×102 1.0×102 Brazil - 1.0×102 Pontara et al, (2012) 12 <1.0×101 <1.0×101 - Brazil - 1.0×102 Ananis et al., (2013) 35 <1.0×101 5.0×102 - Brazil 45.71% 1.0×102 Alves et al., (2015) 15 2.2×107 3.4×107 - Brazil 20% 1.0×102 Giraldo et al., (2013) 7 0 0 0 Colombia 0 1.0×102 [d] Mahmoudi et al., (2016) [e] 34 - - - Iran 5.8%-32.3% - Ayansola, (2012) [f] 108 1.0×101 2.0×103 - Nigeria - - Ummulkhair, (2014) 15 1.0 ×104 1.2 ×105 - Nigeria 26.66% - Malika et al, (2005) 10 <1.0×101 3.0×101 - Morocco 30% - Present study (yeasts) 37 1.0×101 9.3×101 3.82×101 Morocco 32% - Present study (molds) 37 1.0×101 9.0×101 2.33×101 Morocco 40% - Sherwani, (2013) 6 0 <1.0×101 Pakistan 20% - Różańska and Osek (2012) 245 <5.0×101 8.0×104 Poland - 1.0×102 Gomes et al. (2010) 5 1.1×101 2.1×101 Portugal 60% 1.0×102 Feás et al., (2010) 45 1.0×101 8.0×101 2.2×101 Portugal 100% 1.0×102 Duman Aydin et al., (2008) 20 1.0×102 1.0×103 Turkey 40% 1.0×102 J Microbiol Biotech Food Sci / Moujanni et al. 2017 : 6 (5) 1188-1194 1192 Dûmen et al., (2013) 500 - - - Turkey 16%-32% - Erkan et al., (2015) [g] 50 1.0 ×102 1.2×103 3.5×102 Turkey 26% - Erkan et al., (2015) [h] 50 7.4 ×103 1.4×105 5.4×104 Turkey 46% - *Count in log CFU.g-1 a: The value recommended by MERCOSUR (Agreement on the Southern Common Market) and CNEVA. b: Organic honey c: Inorganic honey d: The value specified by Colombian Resolución N°1057 e: Count of fungi (Aspergillus, Penicillium, candida and other yeasts) f: Count of total heterotrophic fungi g: Count for Mold vegetative form h: Count for Yeast vegetative form The results obtained for standard counting of molds and yeasts showed that 32% and 40% of samples were positive respectively for molds and yeasts. However, the detection of yeasts and molds remains at low levels ([1.0×101- 9.3×101CFU.g-1] for yeasts with mean=3.82×101±2.72×101CFU.g-1 and [1.0×101- 9.0×101CFU.g-1] for molds with average 2.33×101±2.53×101CFU.g-1). Withal, note that no result exceeds the recommended threshold for yeast (102 CFU.g-1), nor the fermentation honey line (5.0×102 CFU.g-1) (Fléché et al., 1997). Furthermore, total most and yeast counts can vary greatly, typically between 0 and 105 CFU.g-1, although high counts are not palatable because of the increased rate of fermentation and the honey is unlikely to pass quality control (changing the taste and the flavor of honey) (White, 1975). From that point a view, a few hundred CFU.g-1 of yeast are more likely to be found in honey samples. Table 3 below gives a comparison of the values found in several studies, conducted in the world relating to molds and yeasts in honey. It appears from reading the table that our results are close to those of Malika et al., (2005), Gomes et al., (2010) and Feás et al., (2010). Finola et al., (2007) and Giraldo et al., (2013) reported no or low values (<1.0×101CFU.g-1) of molds and yeasts for respectively Moroccan, Portuguese, Argentinian and Colombian honeys. However, Różańska and Osek, (2012) from Poland, Mahmoudi et al., (2016) from Iran, Rall et al., (2003) from Brazil, Erkan et al., (2015) from Turkey, Ummulkhair, (2014) from Nigeria and other authors reported a higher counts of molds and yeasts (Table 3). CONCLUSION At the end of this study, it was observed that Euphorbia resinifera honey has an acceptable microbiological profile. In fact, none of the analyzed sample contained any microorganisms that have an impact on human health. Additionally, the low levels of microbial contamination associated to a very low rate of mold and yeast indicate that this honey does not undergo any significant degradation and, therefore, this product always keeps its commercial quality. However, it is has to be emphasized the importance of continuous monitoring throughout the honey processing, to ensure the marketing of a reliable food. It is also recommended for governments and producers, to ensure a continuous control and to set up specification conditions during storage (moderate temperatures, increased humidity, granulation of the honey and elevated yeast counts). Finally, standardization, by national and/or “PGI” specifications, of microbial contamination limits is very important to further improve the quality of honey, and ensure it sustainability over the years. Acknowledgments: The authors extend their appreciation to the president of the Union of beekeeping cooperative in the Tadla-Azilal region “UCATAZ” and all producers of “PGI” Euphorbia resinifera honey who have helped us to conduct this research. We also thank M. Hatym Ramhy, from geological department of FST of Settat, for his help in designing the map of the study. Conflict of interest: The authors declare that there no conflicts of interests. REFERENCES AAZZA, S., LYOUSSI, B., ANTUNES, D., MIGUEL, M.G. (2014). Physicochemical characterization and antioxidant activity of 17 commercial Moroccan honeys, Int. J. Food Sci. Nutr. 65 (4), 449-457. http://dx.doi.org/10.3109/09637486.2013.873888 ADENEKAN, M.O., AMUSA, N.A., LAWAL, A.O., OKPEZE, V.E. (2010). Physicochemical and microbiological properties of honey samples obtained from Ibadan. J. Microbiol. Antimicrob. 2(8), 100-104.[Online] Available in http://www.academicjournals.org/JMA ADOCK, D. (2016). 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