**1. Introduction**

Main causes of deterioration of dried fruits and seeds are fungi (yeasts and molds); thanks to their low water activity preventing bacterial growth for the most part. The edible part of the seeds is also usually protected from spoilage by thick, durable shells [1]. However, dried fruits are often sold to consumers with the shell removed, which exposes the edible portion to microbial contamination and spoilage. Some species of molds are capable of producing toxic substances, such as aflatoxins, ochratoxin, and patulin.

In Mexico, purchasing fresh produce loose or in bulk at traditional markets is a common practice, even by small (i.e., non-corporate) consumers, such as families and individuals. These points of sale tend to be loosely regulated, which translates also to a lack of knowledge of the sanitary conditions of the product being sold and of potential risks to the health of consumers. More knowledge about the mycobiota of these food products can help make better, more informed policy decisions, as well as help in the development of solutions to potential problems caused by fungi.

Peanuts are an important food crop all around the world, valued for their edible seed as well as a source of edible oils. As a crop that grows underground, peanuts are highly susceptible to microbial contamination. Mycobiota of peanuts mostly consists of *Aspergillus* and *Penicillium* species, including *A. flavus* and *A. parasiticus*, both known to produce highly toxic aflatoxins [2, 3]. Due to their low water activity, spoilage of peanuts is often caused by molds; however, it is only likely to happen when storage conditions are inadequate and water activity of the kernels is allowed to rise above the threshold for mold growth.

Tree nuts are similar to peanuts in composition and spoilage characteristics; they have a high lipid content and low water activity. Contamination of tree nuts with molds occurs mostly when they are handled improperly after being dehulled since their thick shells are usually able to protect them from microbial contamination. After being dehulled, tree nuts are susceptible to spoilage by molds when their water activity is allowed to increase above the threshold at which molds are capable of growth. Tree nuts are important food crops all around the world, and their nutritional and flavor properties, as well as their relatively high market price, make them of economic importance to the countries in which they are produced.

The seeds from different species of squash or gourds are consumed either on their own or as an ingredient on several dishes all around the world. The species *Cucurbita pepo* (winter squash) and *Cucurbita argyrosperma* (silver-seed gourd) are of particular importance for their production of edible seeds [4]. Amongst edible seeds, squash seeds are unique in that they have a relatively high moisture content, which makes them more susceptible to microbial spoilage [5]. Despite this, little information exists concerning the mycobiota and spoilage of edible squash seeds.

In recent years, the identification of molds is more commonly carried out via molecular identification techniques. However, morphological analysis remains an accessible and effective, if labor-intensive, technique for the identification of fungal isolates. Although often dismissed by some specialists as difficult and inconsistent, it has certain advantages over molecular techniques. In particular, many mold species produce polymerase-inhibiting substances, which hinder identification via techniques based on the polymerase chain reaction or PCR [6]. Lack of knowledge about microbiota of foods is a common problem in developing nations, more accessible methods for isolation and identification of microorganisms can help researchers in these areas to investigate such matters more effectively. For these purposes, further study and development of morphological techniques for mold identification are not only useful but also essential.

### **2. Methodology**

Peanut (*Arachis hypogaea*), pecan (*Carya illinoinensis*), walnut (*Juglans regia*), and squash seed (*C. argyrosperma*) samples were purchased in municipal produce markets of cities and towns across different regions of Mexico, where dried fruits can be purchased in bulk. Samples consisted of at least 250 g of the selected dried fruit dehulled, unsalted, and unroasted. A total of 30 samples were obtained and transferred to the Laboratory of Food Microbiology of the Universidad de las Americas Puebla for further analysis. None of the samples showed visible signs of fungal growth/deterioration.

Total yeast and mold content were determined on dried fruit samples. 10 g of each dried fruit were weighed and placed in sterile sampling bags. Samples were diluted with 90 mL of 0.1% peptone (Becton-Dickinson, Mexico) solution, and homogenized using a Stomacher ® laboratory blender (Seward, United Kingdom.).

### *Isolation and Identification of Molds in Selected Dried Fruits and Seeds Sold in Bulk in México DOI: http://dx.doi.org/10.5772/intechopen.99973*

Serial dilutions of the sample were prepared using the same 0.1% peptone solution and plated in potato dextrose agar (PDA, Becton-Dickinson, Mexico) plates acidified with 1.6 mL/100 mL of a 10% tartaric acid solution to a final pH of 3.5. Colonies in inoculated plates were counted after being incubated at 25°C over a period of 5 days. For samples in which *Rhizopus* spp. colonies were readily apparent, Dicloran Chloramphenicol Rose Bengal agar (DRBC, Becton-Dickinson, USA) was used instead of PDA to limit colony growth and allow for more accurate quantification of yeasts and molds. Assays were carried out by triplicate; results of these trials showed generally low counts and are not presented in this article.

Individual mold colonies were taken from counting plates and inoculated in PDA plates, without tartaric acid, for isolation. Plates were incubated at 25°C for 7 days, or 3 days when a *Rhizopus* spp. the colony was apparent and inspected afterward for contamination. Resampling and inoculation of the mold colonies were repeatedly carried out until only a single mold species was apparent in the agar plate. Afterwards, molds were point-inoculated in malt extract agar (MEA, Becton-Dickinson, Mexico), and Czapek Yeast Extract agar (CYA, Becton-Dickinson, Mexico) or Czapek Dox agar (CZD, Becton-Dickinson, Mexico). For identification, molds were incubated at 25°C for 7 days, or 3 days when a *Rhizopus* spp. the colony was readily apparent. Colony diameter, front and back colony color, colony texture and shape, presence and color of exudates, as well as presence and color of soluble pigments in the different media were the main criteria used for mold identification.

After incubation of the fungal isolates, colony macro and microscopic morphology was observed and identification was carried out following dichotomous keys as outlined by Pitt and Hocking [1], and Samson *et al*. [7]. For microscopic observation and measurement, a small (2 mm2 , approx.) and shallow (<1 mm deep) sample were cut from the outer edges of a colony using a sterile dissection needle, including a small portion of the culture media along with the mold sample. The colony sample was then placed on a glass microscope slide and a drop of aniline blue solution (0.1% aniline blue, Química Meyer, Mexico) in 85% lactic acid (Química Meyer, Mexico) was added, as mounting fluid. Afterwards, a drop of 70% ethanol was added to help disperse conidia and aid in the visualization of fruiting structures. A slide cover was then placed on top of the sample, and the sample was carefully heated with a Meker-Fisher burner to melt the culture medium. The sample was then observed under 100-x magnification in an optical microscope (American Optical, USA) equipped with an Axiocam ERc 5S camera (Zeiss Microscopy, Germany) and associated software for microphotography and measurements. Shape, size, texture, and color of conidia and fruiting structures were the main characteristics used for discrimination. Data is only presented for media in which the sampling for microscopic observation yielded measurable, observable structures and images of satisfactory quality.

For identification of *Penicillium* subgenus *Penicillium* species, an additional culture was prepared using creatine sucrose neutral agar (CSN), as described by Pitt and Hocking [1]. Briefly, 10 g of sucrose, 5 g of creatine, 1 g of monopotassium phosphate, 0.05 g of bromocresol purple, 10 mL of a solution containing potassium chloride, magnesium sulfate heptahydrate, ferrous sulfate, zinc sulfate, and copper (II) pentahydrate in trace amounts, and 15 g of bacteriological agar were dissolved in 1000 mL distilled water and heated to a boil before being autoclaved at 121°C for 15 min. The medium adds an additional criterion for discrimination of the otherwise similar species of *Penicillium* subgenus *Penicillium* by producing an acid (yellow), alkaline (violet), or neutral (gray) reaction based on the capacity and extent to which the species metabolizes sucrose and/or creatine. CSN reaction, along with culture morphology, is part of the criteria used in the dichotomous keys presented by Pitt and Hocking [1].
