Final Report for GNE12-046
In 2012, four different log species were inoculated with the WR46 shiitake strain. The log-species used in this investigation include, Sugar Maple (Acer saccharum), Red Oak (Quercus rubra), White Ash (Fraxinus americana) and Ironwood (Carpinus caroliniana). While yields varied among tree species, consumers were unable to detect differences in dried mushroom samples, based on the log species from which they were grown, with greater likelihood than chance. Significant differences were found in the concentrations of phenolics and flavonoids in shiitake mushrooms grown on different tree species, with little correlation between the concentrations of these compounds and the antioxidant capacity as measured by peroxyl-scavenging capacity. This work also explored consumers’ perception of functional foods, in order to identify market niches for forest mushroom growers, in addition to developing a set of adjectives that can be used to describe the flavor of shiitake mushrooms grown on different tree species.
Forest cultivation of shiitake mushrooms can make use of marginal lands to create supplemental income pursuant of small farm viability. With some up-front labor inputs, shiitake cultivation is a low-risk enterprise opportunity. This investigation seeks to contribute to product development and best management practices for this novel forest enterprise by evaluating antioxidant capacity and consumers' perception of shiitake mushrooms, grown using different log-species substrates. Consumer perception will be evaluated using discrimination tests, preference tests, and focus groups to evaluate resulting qualities from specific practitioner decisions. Bio-chemical analysis will describe specific chemical classes that have been associated with human health, specifically, polyphenols, flavonoids, and antioxidant activity. Contrasting biochemical qualities and sensory attributes is novel approach in the assessment of food quality. The results of this work will create greater knowledge capital for mushroom growers to more efficiently market their product, plan production systems more appropriately for their select demographic, and give growers more ammunition in branding and creating a niche for their unique product, not only as a gourmet food item, but also as a functional food. This research will enhance SARE project LNE10-298 Cultivation of shiitake mushrooms as an agroforestry crop for New England.
- Quantify chemical classes that contribute to the antioxidant capacity of forest grown mushrooms
- Evaluate consumer's ability to discriminate between shiitake mushrooms grown on different log species.
- Determine if the consumer can discriminate or have preference for mushrooms produced on different log species substrates.
- Based on preceding sensory results, determine if there are contributing taste factors that influence the ability to discriminate.
- Identify particular keywords or descriptors that differentiate mushrooms grown on different log species.
- Distribute results of work through manual of best management practices, Cultivation of Shiitake as an Agroforestry Crop for New England, stand-alone publication and grower networks/meetings.
In April 2012, ninety-six logs were inoculated with the WR46 strain of shiitake mushrooms. These logs were incubated, outdoors, at the Arnot Forest, in Van Etten, NY for the 1-growing season spawn run. In January 2013, logs were brought inside for a 3-week acclimatization period, at roughly 25°C. Logs consisted of four tree species, Sugar Maple (Acer saccharum), Ironwood (Carpinus caroliniana), White Ash (Fraxinus americana), and Red Oak (Quercus rubrum).
The mushroom logs were split into four blocks for each tree species, containing 5 individuals in each block. Logs were soaked, to induce sporocarp production, on February 21 and March 1 for 12 hours and on February 2 and April 16 for 24 hours. The logs were then allowed to develop sporocarps, in a semi-controlled atmosphere, until the sporocarp had expanded to the point just before the cap unfurls, where upon harvest was initiated. Following harvest, sporocarps were weighed, dried in a Freas mechanical convection oven, for 3 days, at 70°C, to dryness, and weighed again to determine moisture content of sporocarps. Sporocarps were kept, in a desiccation chamber, to maintain dryness, at room temperature, until further analysis.
Food Chemical Analysis
Diets rich in polyphenols are epidemiologically associated with lower risk of developing age-related diseases in humans (Halliwell, 2008). Quantification of phenolics was performed using a modified method described by Singleton and Rossi (1965). Samples were thawed in a 37?C water bath and diluted to 10% in a small, centrifuge tube so that the resulting volume was 1mL. Samples were centrifuged at 12000g for 15minutes. 100µL of sample was mixed with 400µL of deionized water in a glass culture tube. 100µL of Folin-Ciocalteu Reagent was added to each tube and let to stand for 5-8minutes. 1mL of 7% sodium carbonate and 0.8mL of deionized water were added, mixed, and let to stand for 90 minutes at room temperature. 200µL aliquots were added to a 96-well plate and absorbance was read using an MRX II Plate Reader at 760nm. Gallic Acid was used as the standard and treated like samples in analysis. Phenolic content was expressed, based on the standard, in gallic acid equivalents (GAE) per 100g of sample and are reported as mean ± SD. All analyses were performed in triplicate.
Quantification of flavonoids was performed using an modified method described by He, et al (2008). Tested extracts were added to test tubes and dried to dryness in < 2 h using nitrogen gas and reconstituted in 1 mL of Tetrahydrofuran (THF)/EtOH (1:1, v/v). Catechin standards were prepared fresh before use in 1 mL of THF/EtOH (1:1, v/v) into test tubes. 0.5 mL 50.0 mM NaBH4 solution and 0.5 mL 74.6 mM AlCl3 solution were added to each test tube. The tubes were shaken on the Lab-Line orbital shaker at room temperature for 30 min on setting 8. An additional 0.5 mL 50.0 mM NaBH4 solution was added into each test tube with continuing shaking for another 30 min at room temperature. Following shaking, 2.0 mL cold (4 ºC) 0.8 M acetic acid solution was added to each test tube. The solutions were protected from the light and shaken on the orbital shaker room temperature for 15 min after a thorough mix. Then, 1 mL 20.0 mM chloranil was added to each tube. The tubes were placed in water in the dry bath incubator set at 95 ºC, on the Red Rotor orbital shaker with shaking at setting 4 for 60 min. The dry bath incubator wells were topped up with hot water as needed. The reaction solutions were cooled using tap water. The solutions were transferred to glass culture tubes. The test tube was flushed at least twice with methanol and brought to 4.0 mL volume with methanol. 1 mL 16% vanillin methanol solution was added to each tube and mixed. Then 2 mL 12 M HCl was added to each tube, mixed, and kept in dark at room temperature (20-25 ºC) for 15 min.The solutions in the glass culture tubes were centrifuged at 2500 rpm for 3 min. 200 μL of each solution was added into a 96-well plate in duplicate and absorbance were measured at 490 nm using an MRX Microplate Reader with revelation work station. Flavonoids content was expressed as mg of catechin equivalent (CE) per 100g of sample and are reported as mean ± SD. All analyses were performed in triplicate.
Peroxyl Scavenging (Antioxidant) Capacity (PSC)
Antioxidant data in the literature primarily describes the water soluble portions of food extracts (Adom and Liu 2005). The additional capacity to measure lipophilic antioxidants creates a clean picture on food’s ability to prevent degenerative diseases. An assay, developed by Adom and Liu (2005), measures the ability of both water and fat soluble vitamins to scavenge peroxyl radicals. This assay utilizes oxidation of dichlorofluorescin diacetate by peroxyl radicals to yield, in a dose dependent manner, fluorescence at 485nm and 583nm using a fluorescent spectrophotometer. The reaction is carried out at 35? C for 36 min in the case of hydrophilic and 40 min in the case of lipophilic. Antioxidant capacity is expressed as an EC50 using AUC measurements and the SigmaPlot Software.
While a chemistry-based approach to food quality is important, the truth lies with the consumer. Formal sensory analysis procedures have been developed in order to eliminate the incredible amount of variance that is associated with sensory perception (Lawless and Heymann, 2010). Roughly 63 patrons at the Trumansburg Farmer’s Market in Trumansburg, NY were surveyed on two dates (31 on June 12 and 32 on June 26) in the summer of 2013. Participants were surveyed to determine their ability to distinguish between mushrooms grown on different tree species, Quercus rubra, Carpinus caroliniana,and Acer saccharum. Fraxinus americana was not included in this portion of the survey because this species did not yield sufficient quantities.
This analysis examines consumer’s (panelist) ability to discriminate between the sample and the standard. Forty-five grams of dried mushroom, from previous sections, were ground into individually sealed packages and mixed with 0.17g of coarse ground kosher salt. On the day of analysis, packages were mixed with 1L of water, brought to a boil, and then allowed to cool to a palatable temperature. The panelist was presented with three samples, of 1 oz. each, that were labeled with a random three-digit code. Two of the samples were of one treatment, while the remaining sample is an alternative treatment. The panelists were forced to choose the sample that they feel is the most different. Statistical significance is compared with the entire panel’s ability to accurately discriminate samples and is compared to chance (33%) to generate a one-tailed z-score.
Focus groups are a qualitative, non-statistical method that utilizes small groups of people to generate ideas and probe issues. In this study, culinary professionals were recruited to evaluate fresh mushroom samples and identify key adjectives that could be used to describe the flavor of mushrooms grown on different tree species. Panelists were permitted to cook fresh mushroom samples, in a way that was commensurate with their training. Following preparation, groups of culinary professionals were asked to taste the mushrooms and discuss certain flavor characteristics that represent the different treatment groups.
Participants were presented with a short survey to gauge their preferences and purchasing behavior for not just shiitake mushrooms, but mushrooms in general. In addition to gauging consumer behavior, participants were asked to rate their perceptions of functional foods. The survey was multiple choice with the option for free-listing for consumer preferences and purchase behavior. The perceptions of functional foods were gauged using a 5-point likert scale.
Food Chemistry Analysis
Shiitake mushrooms were found to have moderate concentrations of phenolic compounds, when compared to fruits, roughly equivalent to pineapples, bananas, and peaches. However, the phenolic concentrations[i] were greater than many vegetables; surpassing the concentration of broccoli, when compared to measurements from previous study (Chu, et al 2002). The flavonoid concentration in shiitake mushrooms[ii] was comparable to the peel of apples (Wolfe, et al 2003). The peel of the apple is the site of the greatest concentration of health promoting compounds. While the wives tale suggests that “an apple a day may keep the doctor away”, it may be more appropriate to say that “a mushroom a day would more appropriately keep the doctor away”. The antioxidant capacity of shiitake mushrooms[iii] was quite low compared to other foods (Sun, et al 2002). While significant differences do exist between shiitake mushrooms grown on different tree species for concentrations of both phenolics and flavonoids (no significant differences were found in antioxidant capacity), it must also be noted that chemical measures of food quality may not be the most appropriate metric and that other compounds, not included as a focus of this work, may also be responsible for the health benefit of shiitake mushrooms.
In general, patrons were not able to distinguish between shiitake mushrooms grown on different tree species with greater likelihood that pure chance. This does not mean that there are not differences in the flavor of shiitakes grown on different tree species; it simply means that the differences, if there were any, were too subtle to be detected by the average consumer. Culinary professionals, who may have a greater ability to distinguish tastes, were presented with cooked shiitake mushrooms that were labelled with the tree species from which they came. These culinary professionals, in a focus group setting, generated a list of descriptive adjectives that differentiate the subtleties of flavor between shiitakes grown on different tree species[iv]. Preference tests were not conducted during this study because sample population was not able to discriminate between shiitakes grown on different tree species.
This survey also took the time to explore the perceptions and behaviors of consumers who purchase mushrooms. Consumers were more likely to purchase fresh mushrooms[v] and indicated a preference for seasonality that does not correspond to the traditional production season[vi]. This conclusion suggests the need for greater consumer education efforts, or an exploration into season extension strategies for shiitake mushroom cultivation (although likely a combination of the two).
Functional Foods are foods that naturally contain or are supplemented with compounds that contribute to health effects beyond standard nutrition. Mushroom consumers generally agree that foods help to prevent disease, however when specific foods were probed, consumers indicated less certainty in their ability to prevent disease. Still, the same consumers indicated they that would be more likely to purchase and pay more for a food with greater functional qualities[vii]. In terms of health claims associated with functional foods, consumers indicated they had the most confidence in a claim made in the scientific literature, followed by a medical professional[viii]. This means that if a farmer in a farmer’s market is advertising the health benefits of their products, then they should be sure to include the source of their claims for improved authority and trustworthiness.
[i] Total phenolic contents of shiitake sporocarps cultivated on different tree species
Total phenolic contents of 3 mushroom (mean ± SD, n = 3). Bars with no letters in common are significantly different (P < 0.05).
*Values with different letters in each line are significantly different (P< 0.05).
[ii] Total flavonoid contents of shiitake sporocarps cultivated on different tree species
Total flavonoid contents of 3 mushroom (mean±SD, n=3). Bars with no letters in common are significantly different (P< 0.05).
*Values with different letters in each line are significantly different (P< 0.05).
[iii] Peroxyl Scavening Capacity (PSC) values of shiitake sporocarps cultivated on different tree species
Peroxyl Scavening Capacity (PSC) values of Log-grown Shiitake (Lentinula edodes) grown on 4 different log-tree species (mean±SD, n=3). No significant differences were observed (P < 0.05).
*PSC value is the EC50 expressed as micromoles of ascorbic acid equivalents per 100 g fresh weight. Values with different letters in each line are significantly different (P< 0.05).
[iv] Focus Group Generated List of Adjectives Used to Describe Fresh Shiitake Mushrooms Grown on Different Tree SpeciesFocus Group Generated List of Adjectives Used to Describe Fresh Mushrooms Grown on Different Tree Species
Umami, smooth, mild, clean
roasted, sweet, caramel flavor, banana, buckwheat pancakes
Meaty, heavy, hearty
[v] Frequency of Purchase by Consumers of Fresh and Dried Mushroom
Frequency of Purchase by Consumers of Fresh and Dried Mushroom (n=96; Respondents were not forced to answer)
[vi] Seasonality of Mushroom Purchase
Seasonality of Mushroom Purchase as indicated by survey respondents. 96 respondents were presented with the question. Multiple answers were allowed. No forced choice. Number inside of pie-chart indicates number of affirmative responses.
[vii] Consumer’s Perceptions of Functional Foods
Consumer’s Perceptions of Functional FoodsResponses based on consumer’s agreement with statement on a 5 point Likert Scale, where 1 (Strong Disagreement) and 5 (Strong Agreement)
[viii] Level of Trust Consumers Communicated in Health Claims
Level of Trust Consumers Communicated in Health Claims Made by the Respective Entities on a 5 point Likert Scale, Where 1 (No Trust) and 5 (Strong Trust)
The impacts of this work will help to provide information for the development of log-grown shiitake enterprise.
Education & Outreach Activities and Participation Summary
- Results of this work were included in a manual of best management practices, Best Management Practices for Log-Based Shiitake Cultivation in the Northeaster United States.
- A presentation was given at the New York Organic Farmers' Association (NOFA) Winter 2013 Meeting based on preliminary results. The primary scope of this work was to educate farmers on what they can and cannot say about health claims of their products, with a focus on mushrooms. This presentation was part of a series of Cornell/UVM grower meetings.
- Poster presentation at the Northeast SARE summer meeting
Farmers expressed interest in the work being conducted; however, as adoption rates were not formally evaluated conclusions would only be circumstantial.
Areas needing additional study
Food Chemical Analysis
When evaluating the biological efficacy of novel chemical compounds, more biologically based models such as cell-culture lines or animal models may be more representative of in vivo conditions. Therefore, results from chemical antioxidant assays should be interpreted loosely. Similarly, the relevance of in vitro chemical antioxidant assays to in vivo mechanisms is poorly understood, despite the popularity of antioxidant claims in food and food products (Huang, et al 2005). Future work should concentrate on cell, animal, or even clinically based trials, in order to understand the effects dietary bioactive compounds may have on the human population.
Agroforestry systems, such as log-grown shiitake production systems have the ability to generate positive environmental feedbacks. This agriculture system does not carry the brand or certification status of an organic cropping system. However, these practices may be described as being closer to ecological foundations than other cropping systems because, especially in the case of forest farming, the ecosystem diversity rivals the equivalent, native lands. The inter-species interactions in a diverse ecosystem create both positive and negative feedbacks, acting as pest, pathogen, and positive synergist, while a diversity of organisms interact in the forest landscape. Through this work, a cropping system, such as forest farming, that has been shown to have a positive impact on the environment and society may also provide the benefit of producing food products that have a greater ability to prevent disease. Additional study should explore differences within as well as between production systems to provide sufficient evidence for claims that may be included on a product label. Similarly, the impact of labelling shiitake mushrooms as ‘log-grown’, ‘agroforestry product’, or other similar labelling scheme, should be evaluated to determine a scheme with the greatest impact on stimulating sales.
The commoditization of a product, as defined by regulations or custom, refers to the conformity of a product to consumer standards (Peri 2006). Industrially produced products seek to maintain a familiar consistency to build a loyal customer following. However, not all products can be judged based on their consistency. Wines, for example, are valued for their seasonal variation as well as variation between brands, so they must be judged in a stylistic manner. The stylistic judging of wines provided a basis and guideline for this study. However, our study was limited in the variables that were selected for evaluation. Future work should explore the differences of additional tree species, other production variables (i.e. temperature, moisture, sunlight exposure, etc), or even go as far to evaluate variation in the different cropping systems that are used to produce shiitake mushrooms.
The focus of this study is limited in the participants surveyed. To generate a more robust data set, we recommended surveying a greater diversity of participants. In addition, qualitative methods would help to increase the depth of understanding. Similarly, purchasing behavior depended on recall data, which may generate inaccurate results. However, ethics of consumer tracking and information collection may be reasonably questioned, leaving recall data the best alternative.
Adom, K. K., & Liu, R. H. (2005). Rapid peroxyl radical scavenging capacity (PSC) assay for assessing both hydrophilic and lipophilic antioxidants. Journal of agricultural and food chemistry, 53(17), 6572–80.
Chu, Y.-F., Sun, J., Wu, X., & Liu, R. H. (2002). Antioxidant and antiproliferative activities of common vegetables. Journal of agricultural and food chemistry, 50(23), 6910–6.
Halliwell, B. (2008). Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Archives of biochemistry and biophysics, 476(2), 107–12.
He, X., Liu, D., & Liu, R. H. (2008). Sodium borohydride/chloranil-based assay for quantifying total flavonoids. Journal of agricultural and food chemistry, 56(20), 9337–44. doi:10.1021/jf070954+
Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of agricultural and food chemistry, 53(6), 1841–56.
Peri, C. (2006). The universe of food quality. Food Quality and Preference, 17(1-2), 3–8.
Singleton, V. L., & Rossi, J. A. J. (1965). Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. American Journal of Enology and Viticulture, 16, 144–158.
Sun, J., Chu, Y.-F., Wu, X., & Liu, R. H. (2002). Antioxidant and antiproliferative activities of common fruits. Journal of agricultural and food chemistry, 50(25), 7449–54.
Wolfe, K., Wu, X., & Liu, R. H. (2003). Antioxidant activity of apple peels. Journal of agricultural and food chemistry, 51(3), 609–14.