Progress 10/01/14 to 09/30/18
Outputs
Target Audience:The target audience for this research included (i) Louisiana small and socially disadvantaged farmers with innovative desire to pursue niche market crop production for farm sustainability (ii) cooperative extension service agents, faculty and staff of Southern University Agricultural Land Grant Campus, (iii) undergraduate students of the agricultural sciences program and graduate students of plant and biological sciences, and (iv) local and international specialty mushroom scientific communities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided an opportunity for the PI to visit CePaul Mushroom Nursery which also conducts exploratory research on the growth of medicinal mushrooms such as Ganoderma lucidum, Lignosus rhiroceros and Cordyceps sinensis and to consult with the operator/owner of that farm. Much valuable information on cultivation issues and problems associated with the medicinal mushroom production has been acquired without incurring significant amount of expenses. The visit also resulted in the continuous collaboration and consultation with the owner/operator of CePaul Mushroom Nursery to enhance mushroom research undertakings. The project also provided an opportunity for the PI and associated staff to acquire a hands-on training on laboratory protocol for the phytochemical analysis of the mushroom extracts. How have the results been disseminated to communities of interest?Overview of the project goal and objectives, expected products and outcomes in the form of tour and presentation was presented to scientists, researchers, extension personnel, undergraduate and graduate students at Southern University and a small group of producers. Laboratory tours and a lecture were held for plant science students. What do you plan to do during the next reporting period to accomplish the goals?This project ended.
Impacts
What was accomplished under these goals?
Pure culture of Cordyceps sinensis-SC4 was obtained from commercial grower, Aloha Mushroom of Carson City, Nevada. Pure culture of Cordyceps could be successfully sub-cultured using SPY (sucrose/peptone/yeast: 50g/10g/3g in 1 L purified water) liquid medium, SPY agar (23 g in 1 L. water) medium, and potato dextrose agar medium. Solid culture media were found to be effective in promoting mycelial growth along with liquid medium. Cordyceps under natural environmental conditions in Tibet, grew on caterpillars underground. With this concept in mind, two experiments were conducted to evaluate the unsalted, peeled Louisiana crawfish as a substrate substituting caterpillars for the culture of Cordyceps mushroom. The treatments were 200 ml solid SPY agar, 100 ml SPY solution, brown rice + crawfish (50/50:w/w), 100 g crawfish alone and 100 g brown rice. Brown rice was 65% precooked and crawfish was 100 precooked. Prior to inoculation, all jars were steam-sterilized. The inoculated media were incubated at 160 C. Cordyceps mycelial growth was observed on SPY agar, SPY solution and brown rice alone. No or little growth was observed for either crawfish treatments. The supplements of potassium phosphate Mg sulfate did not enhance mycelial growth in a separate experiment. These observational data indicated that crawfish meat could not be used for Cordyceps cultivation under the 160 C temperature conditions. The effects of temperature on Cordyceps mycelia growth were evaluated using 2x2x3 factorial experiment (two growing substrates: brown nice & millet), (two nutrient solutions: sugar solution consisting of 50g sugar in 1 L water, and SPY) and three temperature treatments (room temperature 230 C, 2-wk at 160 C followed by room temperature and 160 C throughout. When sugar solution was used in conjunction with the substrates, mycelial growth was only observed on both substrates at 160 C throughout treatment. When SPY solution was used in conjunction with the substrates, mycelial growth was observed on all treatments (room temperature, 2-weeks at 160 C followed by room temperature and 160 C throughout). The 160 C throughout treatment showed more significant mycelial growth than those at room temperature and two-weeks in 160 C followed by room temperature. Constant exposure to 160 C of the inoculated media showed significantly higher in mycelial growth that other two treatments. No further observations were carried out due to power failure of the building. In the evaluation of various substrates, seventy ml of purified water was used to soak the substrate medium for 24 hours: The substrate media were brown rice, pearl barley, millet, corn grains, rye, buck wheat for 24 hours and 70 ml. SPY solution on the mycelial growth. The prepared substrates were steam-sterilized at 1210 C for one hour using Steris Century sterilizer. Cordyceps mushroom cultures were inoculated on top of the substrate medium in each six-oz. jar. Forty days after incubation, mycelial growths were observed and recorded. Similar mycelial growth was observed among brown rice, pearl barley, buck wheat substrates with rye substrate and SPY solution having maximum mycelium coverage. From the findings of this study, another experiment was conducted to evaluate rye flour and rye seeds as culture media for Cordyceps mycelial growth. The treatments for rye flour as substrates were purified water, sugar solution (35g sugar in 700 ml water), agar solution (1g/120 g water) and sugar-agar solution (35g sugar+5.38g agar in 700ml water). Rice flour used in each 6 oz. jar was 20 gm with 40 ml of solution, respectively. The treatments for rye seeds as substrates were purified water, sugar solution (35g sugar in 700 ml water), agar solution (1g/120 g water) and sugar-agar solution (35g sugar+5.38g agar in 700ml water). respectively. Rye seeds used in each 6 oz. jar were 40 g with 70 ml of treatment solution, respectively. All the treatment jars were incubated at 160 C in Precision incubator. There was a significant difference in mycelial growth between rye flour and rye seed substrates. The treatment of sugar solution and sugar-agar solution for both rye flour and rye seed were more effective in promoting mycelial growth of Cordyceps than purified water and agar solution. Further observations were conducted by placing all the treatment jars in refrigeration at 400 F with low oxygen and no light. Profuse growth of mycelium was observed at the end of the project, September 30, 2018. However, no fruiting has been observed. Data are still being collected beyond project period. Based on literature, Cordyceps requires 20 weeks of incubation under the stated conditions. It is anticipated fruiting will occur at later date. The UV/VIS method was used to quantify the total phenolic and antioxidant content of Cordyceps mushroom extracts in triplicates. The total polyphenolic content, determined by the Folin Ciocalteu's reagent assay method and expressed as gallic acid equivalent was 7.558 mg/gm. The antioxidant content determined by ABTS reagent assay method, and expressed as Trolox equivalent was 6.583 mg/gm. Results in this study, indicated that Cordyceps mushroom extracts exhibited higher amount of total polyphenol and antioxidant contents than other medicinal mushrooms such as shiitake, Ganoderma lucidum and Pleurotus mushrooms. For the quantification of phenolic acids present in Cordyceps mushroom using UHPLC-MS/MS, 26 phenolic acid commercial standards were used for authentication of the isolated compounds from the extracts. Polyphenolic acids presented in the Cordyceps mushroom extracts were 4-hydroxybenzoic acid, vanillic acid, homovanillic acid, and trace amount of ferulic acid, coumaric acid, and cinnamic acid in addition to Aloha reported 41% polysaccharide, 34% beta-glucan and 0.21 mg/g adenosine-related compound. Nutrient elemental contents such as P, K, Ca, Mg, S, B, Cu, Fe, Zn, and Mn of Cordyceps sinensis C-4 were determined using ARCOS ICP and N was determined by LECO CN 628 Analyzer. Based on triplicated samples, N=3.62%, P=0.58%, K=0.80%, Ca=0.56%, Mg=0.17%, S=0.61%, B=3.84 ppm, Cu=2.17 ppm, Fe=54.05 ppm, Zn=58.64 ppm, Mn=22.40 ppm and Na=2,173.14 ppm. Based on the results obtained from these studies, Cordyceps mushroom requires properly regulated room conditions and temperature for feasible mycelial growth and fruiting. The PIs have concluded that the growing of Cordyceps as a niche market crop is not conducive and economical for small and limited resource farmers production particularly socially disadvantaged farmers because of the requirements of high technical skills and facility investment. The PIs have decided to terminate this research project.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Targeted audience includes a limited number of undergraduate and graduate agricultral science students, a select group of roselle hibiscus home growers and farmers,some Louisiana Cooperative Extension agents, andSouthern University agricultural communities. Changes/Problems:Research approaches will be modified accordingly based on previous results and findings. What opportunities for training and professional development has the project provided?The project provided an opportunity for the project director to consult with the medicinal mushroom expert from CePaul Mushroom Nursery in Malaysia. Mcuh valuable information on cultivation issues and problems had been acquired via this consultancy without incurring significant expenses. No cost continuous collaboration and consultation with CePaul Nursery has enhanced the progress of this research project. How have the results been disseminated to communities of interest?Overview of the project, goals, expected products, and findings of the study were shared with scientists, research and extension personnel, graduate and undergraduate students at Southern University, and also to producers and potential farmers. What do you plan to do during the next reporting period to accomplish the goals?Planned research activities for the coming no-cost extension year include: (i) pure culture of Cordyceps will be re-established (ii) quantitative and qualitative data on Cordyceps extracts by LC/MS, (iii) results on the effects of substrates on the production of mushroom natural products, (iv) in vitro and pin vivo data on anti-human cancer cell growth by mushroom extracts, (v) accomplishment of the project objectives and (vi) presentations on research findings.
Impacts
What was accomplished under these goals?
Research data of all the experiments conducted thus far had indicated the use of crawfish meat was not the conducive substrate medium for Cordyceps which grew symbiotically with caterpillars under natural environmental conditions in the mountainous plateau of Tibet. Sucrose, peptone and yeast were essential medium components for Cordyceps mycelial growth but not sucrose alone imbedded with substrate media. Results indicated Cordyceps extracts exhibited higher amount of total polyphenol and antioxidant contents than other medicinal mushrooms such as shiitake, Ganoderma lucidum, and Pleurotus species.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Target audience include undergraduate and graduate agricultural sciences students, a select group of limited resource farmers, Louisiana cooperative extension agents and Southern University scientific community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The projectprovided an opportunity for the project director to visit CePaul Mushroom Nursery, Kajang, Malaysia and discuss some of the issue, problems and culture of Cordyceps mushroom. How have the results been disseminated to communities of interest?Overview of the project,goals and expected products, outcomes, etc.of the study were presentedto scientists, research and extension personnel, graduate and undergraduate students at Southern University, and also to agroup of producers. What do you plan to do during the next reporting period to accomplish the goals?Review the shortcomings and pitfalls of 2015/16 research data and plan and design new experiments that can be implemented with minimum pitfalls.
Impacts
What was accomplished under these goals?
Five substrate media in 16 oz. Mason jars were preliminarily evaluated in three replications for the Cordyceps mycelium growth under two temperature treatments (160C and room temperature at 230C). The growing substrate of 100 g each were: SPY (50 g sucrose, 11 g peptone, 3 g yeast/liter solution), SPY agar (SPY in 23 g of agar), solution, brown rice + crawfish (50g/50g) , crawfish and brown rice alone. Brown rice and crawfish were precooked at 65% and 100%, RESPECTIVELY. A standard-size of 0.25 sq.cm of Cordyceps mycelium was used to inoculate the medium.The growth of mycelium as the percent of substrate surface covered was recorded 9 days after inoculation. The percent of surface area covered by Cordyceps mycelium was recorded.The percent of surface covered were 45%, 25%, 25%, 5% and 20% for SPY agar, SPY solution, brown rice + crawfish, crawfish and brown rice, respectively under room temperature treatment.The percent of surface covered were 80%, 25%, 25%, 0% and 35% for SPY agar, SPY solution, brown rice + crawfish, crawfish and brown rice, respectively under 160C treatment.A preliminary experiment was conducted to evaluate the effect ofthe addition of potassiumphosphate + Mg sulfate at 0.5% by weight to the 65% precooked millet substrate. SPY solution was used in the precooking of millet. The addition of both potassium phosphate and Mg sulfate did not enhance mycelium growth under SPY cooking solution.In the comparison between brown rice and millet substrates precooked in distilled water, millet was found to be a better substrate for Cordyceps mycelium growth than brown rice. In a 2x2x3 factorial experiment to evaluate the effect of two substrates (brown rice and millet) prepared with sugar solution and SPY solution, respectively and three temperature treatments (room temperature at 230C, 2-week at 160C followed by room temperature culture, and 160C throughout, respectively). In the brown rice and millet substrates precooked with sugar solution, mycelium growth was only observed in 160C treatment for both brown rice and millet. In the brown rice and millet substrates precooked with SPY solution, mycelium growth was observed in both substrates under three separate temperature treatments. Millet was found to be more conducive substrate for Cordyceps mycelium growth than brown rice.
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