Evaluation of Microbial Loads on Dried and Fresh Shiitake Mushrooms (Lentinula edodes) as Obtained from Internet and Local Retail Markets, Respectively

Abstract

Consumer demand for shiitake mushrooms is increasing. However, food safety information regarding the prevalence of microbial pathogens on the products sold via the Internet or at local retail markets is limited. The present study was conducted to assess the microbial load on shiitake mushrooms sold through the Internet and at local (central Virginia) retail markets. A total of 90 shiitake mushroom products, consisting of locally-purchased whole (LW) and sliced (LS) and Internet-procured whole (IW), sliced (IS), and powdered (IP) forms, were tested. High levels of aerobic mesophiles (6.9 ± 1.3 to 7.5 ± 1.1 log CFU/g), yeast and mold (5.8 ± 0.9 to 6.0 ± 0.3 log CFU/g), and coliforms (1.6 ± 1.0 to 1.9 ± 1.1 log MPN/g) were found in locally-acquired mushrooms. One LW sample and 2 of LS contained Listeria spp. Our findings suggest that shiitake mushroom producers and retailers need to be aware of potential microbial hazards associated with handling fresh shiitake mushrooms and consumers should take appropriate precautions when handling fresh shiitake mushrooms to prevent cross-contamination and possible foodborne illness in the home.

1. Introduction

Minimizing adverse impacts from pathogens that affect human health has been a persistent challenge to the food industry. Two important control points for managing foodborne pathogen contamination include the production environment^1–5) and handling both during and after harvest^6–8). Fruits and vegetables are susceptible during growth and harvest to natural contamination from soil, insects, birds, and water, among other sources^9,10). Inadequate and improper handling during harvest, post-harvest processing, packaging, and distribution may further promote the growth or spread of undesirable microorganisms leading to serious cross-contamination, product damage, and/or foodborne illness^11,12). According to the Centers for Disease Control and Prevention (CDC), more than 50 million Americans (about one in six) get a foodborne illness each year, costing the U.S. economy more than $77.7 billion¹³⁾.

Consumer demand for shiitake mushrooms (Lentinula edodes) has made it the second-largest cultivated mushroom crop and the most popular edible mushroom world-wide¹⁴⁾. Sales of shiitake mushroom in the U.S. were 9 million pounds from 2013 to 2014¹⁵⁾. Although shiitake mushroom can be consumed raw and cooked, there was a limited amount consumed raw in the U.S.¹⁶⁾ This demand is due, in part, to their nutritional and health benefits, including antiviral activity, cholesterol reduction, and cardiovascular support^17–19). Other factors that contribute to this growth may include the influx of ethnic groups from areas of the world where shiitake mushroom comprises a common part of the nutritional and medicinal diet, as well as the increased consumption of ethnic foods as consumers of all backgrounds explore and broaden their culinary experiences. Growing demand for shiitake mushrooms in retail, wholesale, foodservice, and consumer markets has attracted the attention of small-scale producers as an alternative crop to increase profitability²⁰⁾ and has led to opportunities for producers interested in a relatively high return on investment²¹⁾.

The Internet has become an important means for producers and distributors within or across countries to market products, including mushroom, directly to consumers. Gold et al²¹⁾ surveyed 104 U.S. shiitake mushroom producers (36% response rate) and reported that 14% of respondents use the Internet to sell their products. These trends suggest that consumer demand for shiitake mushrooms may increase, along with the utilization of the Internet for their marketing, which will create a need for food safety information relating to this market. Currently, there is limited information available concerning the prevalence of microbial pathogens on shiitake mushrooms sold via the Internet or at local retail markets or about foodborne illnesses associated with mushrooms in the U.S.²²⁾ Samadpour et al²³⁾ investigated the microbiological quality of retail food products and found Salmonella and L. monocytogenes in 5% and 1% of mushroom samples tested, respectively. More recently, a recall of mushrooms associated with the potential contamination of L. monocytogenes took place in 2015²⁴⁾. However, neither of these studies examined specifically the extent of contamination on shiitake mushrooms. Consequently, the present study was conducted to assess the microbial load on mostly available shiitake mushroom products sold through Internet and at local (central Virginia) retail markets.

2. Materials and Methods

2-1. Shiitake Mushrooms

A total of 90 shiitake mushroom (Lentinus edodes) products were purchased from Internet and local retail markets (Table 1). Shiitake mushroom products purchased through the Internet included whole (IW), sliced (IS), and powdered (IP) forms. Though a large number of Internet vendors sell shiitake mushroom in dried forms, only a limited number of them sell fresh shiitake mushroom. Therefore, shiitake mushroom samples procured over the Internet were limited to dried forms only. These products represented 22 different brands produced in 4 different countries of origin and marketed in the U.S. by distributors in 12 different states, though two brands were purchased directly from overseas producers. All Internet purchases were made in duplicate between November 2013 and February 2014. The products were packaged in sealed plastic bags or glass containers and were shipped using commercial delivery services.

Table 1. Market source of shiitake mushrooms and their types, procured from 2013 to 2015^a.

Internet market (Dried shiitake mushroom)						Local (Virginia) market (Fresh shiitake mushroom)
Brandb	State where distributed	Country of Origin	Whole (n = 20)	Sliced (n = 28)	Powdered (n = 14)	Brand	State where distributed	Country of Origin	Whole (n = 20)	Sliced (n = 8)
1	California	China	√d	√	√	23	California	USA	√
2	California	China	√	√		24	North Carolina	USA		√
3	California	China	√	√		25	Ohio	USA		√
4	California	China	√	√		26	Pennsylvania	USA	√	√
5	California	China		√		27	Pennsylvania	USA	√	√
6	Florida	China		√	√	28	Pennsylvania	USA	√
7	Florida	China			√	29	Pennsylvania	USA	√
8	Florida	USA	√			30	Pennsylvania	USA	√
9	Illinois	China		√		31	Pennsylvania	USA	√
10	Massachusetts	China		√		32	Pennsylvania	USA	√
11	Nevada	China		√		33	Virginia	USA	√
12	New Jersey	China			√	34	Virginia	USA	√
13	New York	China	√	√	√
14	New York	China		√
15	North Carolina	China	√
16	Ohio	China		√	√
17	Oregon	China			√
18	Utah	China		√
19	Washington	China	√
20	Washington	Japan	√
21		Chinac		√
22		Thailandc	√

^a Samples were purchased from Internet stores between November 2013 and February 2014 and from local stores between April and May 2015, with duplicate samples obtained at each time of purchase.

^b Each brand was assigned an identifying number for the purpose of the study.

^c Samples were directly shipped from the countries.

^d Type of shiitake mushroom procured corresponding to each State.

Shiitake mushrooms purchased from local retail markets included whole (LW) or sliced (LS) products. Most shiitake mushrooms found in local grocery stores were available as fresh product, except in a few stores, which had dried product under the same brand name as those acquired from the Internet. Therefore, shiitake mushroom samples acquired from local retail markets were limited to fresh forms only. All of these retailers were located within a radius of 50 miles from Virginia State University (Petersburg, VA, USA), and all purchases were made during spring (April to May) 2015. Fresh mushrooms purchased were either in perforated plastic containers or as loose bulk held in a plastic bag within a cardboard box at refrigerator temperature (3 ± 1°C) at the point of purchase. Fresh mushrooms were transported to our laboratory in plastic bags on ice inside insulated containers.

Upon arrival at the lab, water activity (Aw) of the samples was determined using a water activity meter (Hygrolab 3, Rotronic Instrument Corp., Huntington, NY, USA). All dehydrated and fresh samples were kept subsequently at ambient room (22 ± 2°C) and refrigerator (4 ± 2°C) temperatures, respectively. Microbial testing was conducted within 2 days of receipt. To open sealed packages for sampling, one corner of each bag was sprayed with 70% ethanol and air-dried before being cut with flame-sterilized scissors.

2-2. Microbial Testing

Microbial testing procedures previously reported by Kim et al²⁵⁾ were used. Unless otherwise stated, all media were Bacto, from Becton Dickinson, Sparks, MD, USA. In brief, each sample portion (25 g, from multiple locations within a sample package) were homogenized in 225 mL of sterile peptone water (0.1%) using a laboratory blender (Masticator Silver, IUL Instruments, Barcelona, Spain) at high speed for 2 minutes. Appropriate dilutions of the homogenate were surface plated using Standard Method Agar (SMA) for aerobic mesophile counts after incubating at 36°C for 48 hours. Yeast and mold counts and presumptive enterotoxigenic Bacillus spp. counts were determined using Acidified Potato Dextrose agar and MYP agar supplemented with egg yolk emulsion and polymyxin B after incubating at 25°C for 5 days and at 30°C for 48 hours, respectively. The detection limits for aerobic mesophiles and for yeast and mold and Bacillus spp. were 100 CFU/g and 10 CFU/g, respectively.

Total coliform and E. coli counts were determined using the three-tube most-probable-number (MPN) evaluation with a detection limit of 3 MPN/g. Samples were incubated at 36°C for 24 to 48 hours, after which a loopful of culture from each positive gassing lauryl sulfate tryptose broth tube was transferred to both brilliant green bile broth (BGBB) and EC broth containing 4-methylumbelliferyl-β-D-glucuronide (EC-mug). After incubating at 36°C for 24 to 48 hours, BGBB tubes with growth and gas production at 36°C confirmed the presence of coliforms at a detection limit of 3 MPN/g. Further evaluation of EC-mug tubes was not conducted due to initial negative results for all tested samples.

Salmonella and Listeria detections were performed using AOAC-approved or performance-tested methods^26,27). For Salmonella, each sample (25 g) was blended for 2 minutes and pre-enriched in buffered peptone water (225 mL) at 36°C for 20 to 24 hours, which was then followed by enrichment in Rapport-Vassiliadis broth at 42°C for 18 hours and post-enrichment in M broth at 36°C for 6 to 8 hours before enzyme linked immunoassay was performed using a Salmonella Visual Immunoassay test kit (TECRA, Frenchs Forest, Australia). For Listeria, each sample (25 g) was blended in 225 mL of UVM Listeria enrichment broth. After incubation at 30°C for 48 hours, one loopful of the broth was surface-streaked onto Oxford Listeria agar for isolation. Up to three different colonies per isolation were identified to species by API Listeria kits.

2-3. Statistical Analysis

Microbial counts (aerobic mesophiles, Bacillus spp., yeast and mold, and coliform) obtained from duplicate samples of each purchase were log-transformed, averaged, and then analyzed using Analysis of Variance and Duncan’s Multiple Range Test (SAS Institute, Cary, NC, USA) to determine the significance of differences (P ≤ 0.05) between mean values.

3. Results and Discussion

In general, our study suggests that microbial loads tend to be lower on mushrooms (dried forms) purchased via the Internet than on those (fresh forms) from local retail markets. Overall average counts of aerobic mesophiles, yeast and mold, and coliforms were significantly lower (P ≤ 0.05) in mushrooms acquired from Internet markets whereas counts of Bacillus spp. were significantly higher than in those from local retail markets (Table 2). Aerobic mesophile counts from IW (4.2 ± 0.6 log CFU/g) were significantly higher (P ≤ 0.05) than those from IP (3.3 ± 1.6 log CFU/g), whereas levels in IS (3.6 ± 0.3 log CFU/g) were intermediate and did not differ (P > 0.05) from those in either the IW or IP. Counts of Bacillus spp. were significantly higher (P ≤ 0.05) in IW (1.8 ± 0.5 log CFU/g) compared to counts in IS (1.5 ± 0.3 log CFU/g) and IP (1.4 ± 0.7 log CFU/g), which did not differ. In comparison, yeast and mold counts did not differ between IW (2.2 ± 0.6 log CFU/g) and IS (1.9 ± 0.7 log CFU/g), but were significantly lower (P ≤ 0.05) in IP (1.3 ± 0.5 log CFU/g). However, no significant difference (P > 0.05) was found for any tested microbial counts between LW and LS samples.

Table 2. Population of microorganisms recovered from internet and locally procured shiitake mushrooms.

	Microbial population (log CFU/g or log MPN/g)*
	Internet market			Local (Virginia) market
Microorganism	Whole (IW, n = 20)	Sliced (IS, n = 28)	Powdered (IP, n = 14)	Whole (LW, n = 20)	Sliced (LS, n = 8)
Aerobic mesophiles	A 4.2 ± 0.6 b	AB 3.6 ± 0.3 bc	B 3.3 ± 1.6 c	A 6.9 ± 1.3 a	A 7.5 ± 1.1 a
% occurrenceǂ	100.0, 0.0, 0.0	100.0, 0.0, 0.0	85.7, 14.3, 0.0	30.0, 35.0, 35.0	12.5, 25.0, 62.5
Bacillus spp.	A 1.8 ± 0.5 a	B 1.5 ± 0.3 b	B 1.4 ± 0.7 b	A 1.0 ± 0.1 c	A 1.0 ± 0.0 c
Yeast and mold	A 2.2 ± 0.6 b	A 1.9 ± 0.7 b	B 1.3 ± 0.5 c	A 5.8 ± 0.9 a	A 6.0 ± 0.3 a
Coliforms	A 0.5 ± 0.0 b	A 0.5 ± 0.0 b	A 0.5 ± 0.0 b	A 1.6 ± 1.0 a	A 1.9 ± 1.1 a

* Values are mean ± standard error of duplicate samples; means preceded by the same upper-case letters in the same row within each market source are not significantly different (P > 0.05); means followed by the same lower-case letters in the same row are not significantly different (P > 0.05).

^ǂ % occurrence numbers in the row represent aerobic mesophile counts within the range ≤5 × 10⁵ CFU/g, 5 × 10⁵ to 5 × 10⁷ CFU/g, and ≥5 × 10⁷ CFU/g.

The International Commission on Microbiological Specifications (ICMS)²⁸⁾ does not specify the limit of aerobic mesophile counts for raw vegetables and fruits (including mushrooms) for which production and processing history is not known. However, to better understand the microbial loading on mushroom samples acquired for this study, the aerobic mesophile counts associated with the limits established by the ICMS for foods were presented in Table 2. All IW and IS had aerobic mesophile counts ≤5 × 10⁵ CFU/g, while 35% of LW and 62.5% of LS had aerobic mesophile counts ≥5 × 10⁷ CFU/g.

Our results suggest that fresh mushrooms have a higher potential for coliform contamination (Table 3); specifically, coliforms were detected in 70% to 88% of local samples, whereas no coliforms were found in any Internet samples. Findings from fresh mushrooms in this study also indicate that the amount of aerobic mesophile loads on mushrooms does not necessarily point to the presence of coliforms. While E. coli, Salmonella, and L. monocytogenes were not detected in any of the locally-acquired samples, about 11% of the 28 local mushroom samples were positive for Listeria spp., and L. weihenstephanensis and L. grayi were isolated from 1 and 2 samples, respectively. Listeria spp. was more frequently detected in LS (25%) than in LW (5%), which may be due to the further processing and handling used for sliced mushrooms.

Table 3. Occurrence of bacterial contamination in shiitake mushrooms procured for this study.

Organism	Number of samples positive for each microorganism
	Internet market			Local (Virginia) market
	Whole (IW, n = 20)	Sliced (IS, n = 28)	Powdered (IP, n = 14)	Whole (LW, n = 20)	Sliced (LS, n = 8)
Coliforms	0	0	0	14	7
E. coli	0	0	0	0	0
Listeria spp.	0	0	0	1	2
L. monocytogenes	0	0	0	0	0
Salmonella	0	0	0	0	0

Although no L. monocytogenes was detected in the samples acquired for this study, the presence of any Listeria spp. in food may be indicative of unsanitary conditions²⁹⁾ that may also be favorable for L. monocytogenes^30,31). A high prevalence of Listeria spp. on locally-acquired mushrooms therefore may suggest the potential for L. monocytogenes contamination, given that it is an opportunistic pathogen. Murray et al³²⁾ also indicated that the discovery of Listeria on mushrooms may not be unexpected, due to the fact that the propagation of mushrooms occurs on organic substrate under relatively warm (10–20°C) and humid (relative humidity 85%) conditions. For example, Samadpour et al²³⁾ reported the detection of L. monocytogenes in 1% of mushroom samples obtained from various retail stores in the city of Seattle, WA.

Fresh, locally-acquired shiitake mushroom products were characterized by high water activity (Aw 0.99) whereas Internet products had low Aw in the range of 0.34 to 0.40. Since Aw is a measure of the amount of unbound water available in a food product to support the growth of microorganisms, the higher microbial levels found in locally-acquired mushrooms may have been attributed to the high Aw. Beelman et al³³⁾ studied the postharvest deterioration of fresh mushroom and found that moisture in packages can prevent mushroom dehydration but can also encourage microbial growth. Studies of retail mushrooms in Spain have also reported total microbial counts in the range of 10^4.4 to 10^9.4 CFU/g³⁴⁾.

The Internet has become a venue-of-choice for some producers to market and sell directly to consumers. While the Internet offers a great deal of convenience, there may be some risks associated with purchasing food products online. Although the present study has shown low occurrence of microbial contamination on products purchased via the Internet, previous studies in our lab found that there is actually a great chance of getting pathogen-contaminated products from online sources^12,25,35,36). For example, sprout seeds purchased via the Internet had presumptive enterotoxigenic Bacillus spp. counts averaging 1.1 log CFU/g³⁵⁾, and meat samples purchased via the internet had a high occurrence of L. monocytogenes (27.2%) and E. coli (25%)²⁵⁾.

Additionally, in our current study, due to the presumed premium quality of products and appealing packaging materials (ie, glass), the price range (dried weight basis per 100 g) for mushrooms acquired from Internet distributors varied from $3.5–$25.0 for whole, $4.3–$25.9 for sliced, and $4.6–$41.5 for powdered forms, while locally-procured products (fresh weight basis per 100 g) ranged from $3.0–$3.8 for whole and $2.4–$2.8 for sliced forms.

In conclusion, this study identified differences in the microbial load on shiitake mushrooms sold through the Internet and at local retail markets. Given the high level of microbial counts and the occurrence of Listeria spp., consumers can prevent possible foodborne illness by using careful post-purchase handling of fresh shiitake mushrooms. Findings from this study will provide shiitake mushroom producers with baseline knowledge and research-based guidance concerning microbial load on the product.

Abbreviations:Aw, water activity; BGBB, brilliant green bile broth; CFU, colony forming unit; EC-mug, EC broth containing 4-methylumbelliferyl-β-D-glucuronide; ICMS, International Commission on Microbiological Specifications; IP, Internet-procured powdered; IS, Internet-procured sliced; IW, Internet-procured whole; LS, locally-purcahsed sliced; LW, locally-purchased whole; MPN, most-probable-number