Evaluating Contamination Level of Raw and Roasted Nuts Distributed in Commercial Markets in Mazandaran Province, Iran

Nuts are generally consumed in many parts of the world directly as a snack with high nu- tritive value and desirable taste, accepted by almost everyone. Also, nuts are used in var- ious parts of the food industry such as infant food, complementary food for athletes and kids, cooking, and extraction of oils is com- mon for home and industrial uses. Types of nuts, hazelnut, pistachio, almonds, cashew nut, and peanuts, are rich in high-quality proteins, unsaturated good fats, minerals,  and vitamins, and they have low water con- tent; therefore, nuts are highly vulnerable to microbial spoilage especially fungal attack. Microorganisms may enter into the nuts ' shell while still on the trees and this usually occurs when the pods or hard shells of the nuts are split open and the seeds are attacked by insects or pests which make space for the microbial spores to access the developing seeds. Other possible ways of contamination of nuts by microorganisms include harvest- ing process, sorting practices, and washing of the nuts before storage. If the nuts are not properly treated during these stages, it could lead to bacterial and mould growth especial- ly when nut seeds are not properly dried to the safe moisture content before storage or distribution (Adetunji et al., 2014). As men- tioned above, both fungi and bacteria are re- sponsible for microbial biodeterioration of food crops resulting in food and economic losses, hence reducing the consumer accep- tance value of the food products. The fungal contamination occurs without necessarily showing any form of moldiness, moulds pro- duce mycotoxins as secondary metabolites, which are consumed by humans and lead to different forms of diseases such as cancers, spontaneous abortion, cirrhosis, and other liver diseases, and also immune suppression

interference with micronutrient metabolism and therefore stunted growth (Abbas et al., 2005; Luttfullah and Hussain, 2011; Ritter et al., 2011).

This research has focused on evaluating the microbiological quality and safety of dif- ferent types of nuts and the effect of packag- ing type through analyzing the total colony counts, total coliform bacteria, fecal coliform bacteria, Staphylococcus aureus and fungal (yeasts and moulds) counts.  The  objective of the present study was to investigate the microbial quality of raw and roasted (salted and unsalted) nuts, distributed in commercial markets and consumed in Mazandaran prov- ince, Iran.

Materials and Methods

Sampling

The analyses of the bulk nut samples were conducted in four different sampling times (interval between successive samples, also called the sampling interval), for a total pe- riod of four months (April-July 2018). The sampling procedure was conducted accord- ing to sampling guidelines of the European Commission for nuts (in a shell and shelled or otherwise prepared). So, the random se- lection was done from different  parts  of  the consignment, at  least  five  convention- al points (one at the top, three at different depths in the middle, and one at the bottom). The recommended minimum quantity for each final sample was 100-500 gr. In this study, the sample size was equivalent to 200 gr. For sample collection, five different types of bulk nuts (raw, roasted and salted, roasted and unsalted) including shelled hazelnut, un- shelled pistachio, shelled almonds, cashew nut and shelled peanuts were obtained from local markets of different cities in Mazanda-

ran province (Iran). Samples were collected from four different markets in each city. The mixed samples were blended aseptically us- ing a mixer grinder and 100 g portions kept in zip lock envelopes. Samples that were not used immediately were kept at −20 °C be- fore analysis.

Chemicals

All were purchased from Sigma Chemical Co. (St. Louis, USA).

Moisture content

Moisture content was determined accord- ing to Iran national standard (2015) for dry fruits. Briefly, 5 gr of samples were ov- en-dried in a pre-weighed dish at 105 °C for 4 h and cooled in a desiccator until a constant weight was obtained. The moisture content was calculated from the reduction in weight and expressed as a percentage of the original weight.

Total viable count

Ten grams of each nut sample  (shelled) were weighted in a sterilized stomacher bag and homogenized using a sterile physiolog- ical normal saline solution (0.85% NaCl), then serial dilutions were prepared. Pour plate method was applied for aerobic me- sophilic bacteria count, using PCA (Plate Count Agar). PCA plates were incubated at 30 ºC for 48 h (Iran national standard, 2015). Total coliform bacteria and fecal coli-

form bacteria

For total coliforms counts, the method de- scribed by Iran national standard (2007) was used. Growth of coliforms was detected us- ing VRBA (Violet Red Bile Agar) incubated at 37 ºC for 48 h, and confirmation step was performed through subculturing of suspected colonies into BGLB (Brilliant Green Lactose Bile Broth) and evaluating gas production at 37 ºC for 48 h, and at 44 ºC for 24 h for fecal coliforms.

Staphylococcus aureus

To detect the presence of S.aureus, 0.2 ml of dilution 1 (first dilution) was spread onto an of Baird Parker Agar plate complement- ed with 5% of Egg Yolk Tellurite Emulsion. The plates were incubated at 37 ºC for 48 h, and then they were evaluated for the typical morphology of S. aureus colonies (Iran na- tional standard, 2005).

Fungal contamination

One gram of each blended and well-mixed nut was added to 9 ml of sterile peptone water in a sterile test tube and the solution was dilut- ed decimally. 20 ml of sterilized molten Pota- to Dextrose Agar (PDA) plates supplemented with 0.01% chloramphenicol were cooled to 45 °C and poured into plates (in triplicate) and

0.1 ml aliquots of each sample were added. The plates were gently swirled and allowed to solidify. The PDA plates were incubated at 25

°C for 5 days for the determination of fungal counts (Spangenbergi and Ingham, 2000).

Statistical analysis

Means and standard divisions of the mi- crobial counts and moisture content were analyzed using SPSS 22.0 by the One-Sam- ple t-test. Means were compared by the least significant difference (LSD) test when the P-value was <0.05.

Results

Different samples of nuts, including ha- zelnut, pistachio, almond, cashew nut, and peanut,  collected  from  the   local   markets of Mazandaran province (Iran) during four months were analyzed. The samples included raw and roasted-salted and roasted-unsalted nuts. The moisture content of all the samples was measured and presented in Table 1. The moisture content of raw and roasted hazelnut, pistachio, almond, and cashew nut samples

was above the standard limits (5%, 5 and 3%,

5 and 4%, 4%, respectively) according to the Iran national standards. The moisture contents of the raw peanuts were within the permitted

limits (max 9%) but that of roasted peanut samples was above the permissible recom- mended moisture limit of 5% (Iran national standard, 2015).

Table 1. Moisture content in different nut products

Moisture content (%)

* Time of sampling

** Data are presented as mean ± standard deviation

a Mean values with the same superscript along the same column are not significantly different (P< 0.05).

The whole samples were evaluated mi- crobiologically to detect the levels of a total viable count, coliforms, fecal coliforms, S. aureus, yeasts, and moulds (Tables 2 and 3). For yeasts and moulds (Table 2), except all almond samples and 56% of cashew nut samples, all kinds of pistachio products

ended up being loaded  with  unsatisfacto- ry limits (>2.69logcfu/gr) (Iran national standard, 2014). There is still no microbi- ological permissible limit for hazelnut and peanut in Iran national standards but it is obvious that yeast and mould count in pea- nut samples is high.

Table 2. Fungal contamination of different nut products

* Time of sampling

** Data are presented as mean ± standard deviation

a Mean values with the same superscript along the same column are not significantly different (P< 0.05).

The total viable count was within the stan- dard limits in hazelnut, almond, and cashew nut samples (less than 4log cfu/gr). Satis- factory limits of coliforms (lower than 1log cfu/gr) were observed in all almond sam- ples, 91.6% of hazelnut and16.7% of pista-

chio samples (Table 3). No fecal coliforms were detected in samples (results are not shown). S.aureus contamination was found in 6.5% of hazelnut, 8% of almond, 25% of cashew nut, and 33% of peanut samples.

  Table 3. Results of bacteriological contamination of different nut products

Total viable count (log cfu/g)                                                                                     Total coliform (log cfu/g)

  * Time of sampling ** Data are presented as mean ± standard deviation a Mean values with the same superscript along the same column are not significantly different (P< 0.05).

Discussion

The moisture contents of all the raw and roasted nuts, except for raw peanut, were above the permissible recommended mois- ture limit. In comparison to our study, lower moisture content, 5.10-7.2%, and 6.48-7.05% were reported for peanuts from different states of Nigeria by Adetunji et al. (2018) and Oyedele et al. (2017), respectively. Also, the moisture content of our raw and roasted cashew nuts was higher than the results of Adebajo and Diyaolu (2003) who reported a range of moisture content of 4.1–6.8% and higher than the findings of Oluwafemi et al. (2009) who reported a low moisture content range of 3.7–4.3 and 3.2–5.4% for cashew nuts during the dry and raining seasons. Sim- ilar results to our work were found by Ade- tunji et al. (2018) who obtained 5.2-8.6% of moisture content for cashew nuts. There is no report on the moisture content of hazelnut, pistachio or almond. As nuts are hygroscop- ic materials, they absorb moisture from the surrounding atmosphere. The higher mois- ture content of the raw samples could be as  a result of their raw nature as they have not yet been treated by any form of the drying process. The high moisture contents of the samples may also be as a result of inappro- priate packaging, use of improper packaging compositions, and harvesting methods in the farms (Oladapo et al., 2014).

Nuts are vulnerable to fungal attacks at different stages of cultivation, harvesting, sorting, processing, and storage. If the nuts are not properly handled at these stages, it could result in mould development especial- ly when they are not dried enough to be con- sidered as the safe moisture level and stored under conditions suitable for mould growth such as high humidity and temperature (Ab-

dulla, 2013; Adetunjiet et al., 2014). Howev- er, the total bacteria, total coliforms, and fun- gi load of the almond samples were less than the limits recommended in nuts by the Iran National Standard Commission on Speci- fication for almond as 104cfu/gr, 101cfu/gr and102cfu/gr, respectively. As reported by Adetunji et al. (2018), total viable count of groundnut  and  cashew  nut  in  Nigeria was

4.85 and 4.47 log cfu/gr, respectively, which is almost the same as our findings of peanut samples and a little higher than our results from cashew nut microbiological analysis. The lower counts of viable organisms in the almond, hazelnut and cashew nut samples possibly is due to the presence of amygda- lin in almond and phenolic  compounds  in all these three nuts which exert inhibitory effects against bacteria, yeast, and fungi. Although the cashew nut is known to be a rich substrate for microbial growth, an out- break of intoxication and infections through its consumption had rarely been reported. Also, Torquato et al. (2004) observed  that the cashew nut contains the anacardic acid which is an inhibitor for microbial growth. Anacardic acid has lateral chains that are ef- fective against the growth of S. aureus. Con- taminated food handlers, surfaces, and types of equipment may result in staphylococcal infections that is one of the most important pathogens as a cause of foodborne intoxica- tion. The results show S.aureus contamina- tion was observed in all groups of nuts sam- ples except the pistachio. It seems that the shell of pistachio decreases the probability of staphylococcal cross-contamination through handlers or surfaces. Adetunji et al. (2018) reported the presence of S.aureus in ground- nuts and roasted cashew nuts in Nigeria (2 and 2.1 log cfu/gr). Also, Oluwafemi et al. (2009)  announced  high  contamination of S.