Progress 01/15/11 to 01/14/15
Outputs
Target Audience: The target audiences reached include: People who are allergic to peanuts and wheat, and health professionals through news release. Researchers, educators and students who study and work in the area of food allergy through presentations at professional conferences and publications Peanut growers and food industry Members of board of administration, board of trustees and board of visitors of North Carolina A&T State University Members of board of University of North Carolina General Administration (UNC-GA) Faculty and staffs of North Carolina A&T State University Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One graduate student, Davida Harrisonwas trained to conduct her thesis research on enzymatic treatment of roasted peanuts with alcalase and to evaluate the effects of alclase treatment on the proximate composition, functional properties, physiochemical properties and antioxidant activity of defatted peanut flour. Two postdoctoral scholars,Drs. Hao Li and Ying Li, were trained.Dr. Hao Liwas trained to conduct research on peanut allergen reduction by ultrasound assisted enzymatic treatment using sequential treatment of trypsin and chymotrypsin.Dr. Ying Liwas trained to conduct enzyme selection and process optimization for wheat allergen reduction. How have the results been disseminated to communities of interest? The results were disseminated by oral and post presentations at several professional conferences, publications, news releases, andinvited speeches. What do you plan to do during the next reporting period to accomplish the goals? All project goals are completed.However, oral food challenge studies in peanut allergic individuals are needed to evaluate the safety of consuming alcalase-treated peanuts in addition to determining the threshold of alcalase-treated peanut products that would elicit a clinically significant reaction.
Impacts
What was accomplished under these goals?
Major activities completed: During the project period, researchers optimized the treatment condition of roasted peanuts with trypsin, α-chymotrypsin and alcalase. An ultrasound assisted sequential treatment process with trypsin and α-chymotrypsin was developed and optimized with Orthogonal Experimental Design, and a non-digestive protease based process was proposed and optimized with two factor-factorial design. The peanuts from different treatments were evaluated for the protein solubility, residual Ara h 1 and Ara h 2 contents, in vitro IgE-binding potential, and human skin prick tests (SPTs) using peanut allergic individuals. The SPTs were conducted in University of North Carolina, School of Medicine. Two types of enzyme treated peanut sample extracts were tested in comparison with standard peanut extract (SPE) purchased from Greer Laboratories (Lenoir, NC), and untreated roasted peanut extracts. Volunteer Recruitment and Inclusion Criteriawas reviewed and approved by the University of North Carolina Committee on the Rights of Human Subjects (Institutional Review Board). The study was listed on clinicaltrials.gov, NCT01489514. Volunteers were recruited based on advertisements. A combination of a positive clinical history with a positive SPT to SPE was considered to be consistent with a clinically significant peanut allergy. Nine of 22 subjects met the criteria and were selected for SPTs to the treated extracts, a negative saline control, and a positive histamine control. The SPT wheal (hive) and flare (redness) were then recorded 15 minutes after extract application. A 3 mm wheal diameter larger than the negative saline control was considered a positive SPT. The impact of enzyme treatment on the nutritional value,functional properties and physiochemical properties of defatted peanut flour were evaluated. The roasted peanuts were soaked in the phosphate buffer containing 0, 3.03, 6.06 and 9.09 units/100g of alcalase at 37°C for 1, 2, and 3 hours, drained, and dried overnight. The dried peanuts were ground to peanut flour and defatted with hexane. The nutritive values of defatted enzyme treated peanuts were evaluated by the determination of proximate composition and antioxidant activity. The Hunter's L, a, and b values of defatted peanut flour were measured as indicators of color change due to enzymatic treatment. The functional properties evaluated were protein solubility, water/oil binding capacity, emulsification capacity (EC), foaming capacity (FC) and viscosity. Selection of effective proteases for wheat allergen reduction and process optimization: Six proteases were tested at their optimal pH and temperature. Total soluble protein and gliadin contents were determined as indicators to evaluate the effectiveness of enzymes. The hydrolysis conditions of wheat protein by the effective enzymes were optimized using central composite design. The in vitro IgE-bind properties of wheat protein extracts of wheat flour treated with different proteases were compared. The objective 3 was met. Specific objectives met During the whole project period, all specific objectives were met except task 1 (Basophil activation study) of Objective 2. Basophil activation studies (Task 1) were not performed in this cohort of volunteers due to a breakdown of the flow cytometer during study execution. The investigators collectively agreed that continuing with the skin testing (Task 2) would provide more clinically meaningful data for the design of future studies. Significant Results achieved For process optimization: The pretreatment of peanut kernels with ultrasound followed by single digestive protease digestion significantly increased the solubility of peanut protein, completely hydrolyzed Ara h 1but Ara h 2 residual was still detectable. Alpha-Chymotrypsin showed more effectiveness than trypsin in reducing Ara h1 and Ara h2. Trypsin treatment followed by chymotrypsin digestion significantly reduced both Ara h 1 and Ara h 2 concentrations and in vitro IgE binding. Alcalase was the most effective in reduction of Ara h 1 and Ara h 2 in roasted peanuts among three enzymes used. Alcalase was effectively reduced the Ara h 1 and Ara h 2 contents in raw peanuts. Samples treated with ultrasound-alcalase exhibited significantly lowered allergenicity in the SPTs with 9 peanut allergic individuals, while the results of SPTs of samples treated with ultrasound-trypsin/chymotrypsin were not conclusive. All protein extracts treated with alcalase produced smaller average wheal sizes compared to SPE, but extracts without alcalse treatment produced similar wheal size as SPE. Statistically significant reductions in wheal size were observed for extracts from peanuts treated with 4.54-6.05U of alcalase per 100g of peanutsfor 3 hrs (P<0.05). This indicates the reduced allergenicity. The alcalase treatment showed both positive and negative effects on the functional properties of peanut flour under certain conditions. The protein solubility of peanuts increased significantly with enzyme/substrate ratio (E/S) and treatment time (P<0.0001). The enzyme treatment improved the emulsifying properties of peanut flour. The treatment time had a significant impact on the FC (p < 0.0001) of defatted peanut flour, but not E/S (P>0.05). The change of viscosity varied with treatment time. The highest viscosity was observed at the 2 hour treatment time at all E/S ratio tested. However, the enzyme treatment caused a decrease in water binding capacity (P<0.0001) and showed a significant influence on the oil binding capacity (P<0.01). The enzymatic treatment of roasted peanuts under experiment condition used in this study did not cause significant loss of nutrients and did not have a negative effect on the color and flavor of peanuts and defatted peanut flour. Non-digestive had higher ability to reduce allergenic protein content of wheat flour than digestive enzymes. With the alcalase, enzyme to substrate ratio (E/S) and reaction time had significant effects on the gliadin content, and there is a significant interaction between temperature and hydrolysis time. Under the optimal hydrolysis condition for alcalase a 99.96% reduction of gliadin was achieved. This completed the preliminary study on enzyme treatment of wheat for allergen reduction. Key outcomes or other accomplishments realized: This project demonstrates that enzyme-base post-harvest technology has great potential to reduce the allergenicity of peanuts and wheat, thus improving the food safety and adding value to the peanuts and wheat products. The results of this project strongly supported the licensing of our hypoallergenic peanut technology. The results of this research projects is being brought to the marketplace for the benefit of individuals and families whose lives are affected by peanut allergies.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Li, H., Yu, J., Ahmedna, M., & Goktepe, I. (2013). Reduction of major peanut allergen ara h 1 and ara h 2 in roasted peanuts by ultrasound assisted enzymatic treatment. Food Chemistry, 141, 762-768.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Yu, J, Hernandez, M, Li, H., Goktepe, I., Robinette C., Auerbach , A., Peden, D. and Ahmedna, M. (2015). Allergenicity of Roasted Peanuts Treated with a Non-Human Digestive Protease. Food Research International, 69, 341-347.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Ying Li, Jianmei Yu, Ipek Goktepe and Mohamed Ahmedna. Effects of enzymatic treatment of wheat flour on its gliadin content and allergenicity. LWT-Food Science and Technology. (Submitted on March 9, 2015)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Li, H., Yu, J., & Ahmedna, M. (2012). Impact of ultrasonication on the efficiency of enzymatic degradation of Ara h 1 and Ara h 2 in roasted peanut kernels. 2012 IFT Annual Meeting, paper#: 041-08, June 25-28, 2012. Las Vegas Nevada.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Yu, J., Hao Li, Ahmedna, M., Goktepe, I., Hernandez, M., Sanders, M. & Lay, J. (2013). Effects of Alcalase from Bacillus licheniformis on peanut allergenicity. Poster session presented at American Chemical Society 2013 Southeastern Regional Meeting, Atlanta, GA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Davida Harrison and Jianmei Yu. Functional and Nutritional Properties of Roasted Peanuts Flour Treated with Alcalase. The 247th ACS National Meeting, March 16-20, 2014. Dallas, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Ying Li, Jianmei Yu, Mohamed Ahmedna and Ipek Goktepe. Hydrolysis of wheat protein by different proteases to reduce the allergenicity of wheat flour. The 247th ACS National Meeting, March 16-20, 2014. Dallas, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Davida Harrison and Jianmei Yu (2014). Effects of enzyme treatment on the functional and nutritional properties of roasted peanuts. Poster session presented at American Chemical Society 2014 Southeastern Regional Meeting, October 16-19, 2014, Nashville, TN.
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2015
Citation:
Effects of enzyme treatment on the functional, nutritional and physiochemical properties of defatted peanut flour. Davida Harrison,Master of Science. (Defended on March 18, 2015)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Li, Y., Yu, J., Ahmedna, M. & Goktepe, I. (2013). Reducing the allergenicity of wheat flour by proteases. Poster session presented at American Chemical Society 2013 Southeastern Regional Meeting, November 12-16, 2013, Atlanta, GA.
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Progress 01/15/13 to 01/14/14
Outputs
Target Audience: During this reporting period, the research team has been focused on the reduction of wheat allergen and functional properties tests of allergen reduced peanuts. Wheat is the most harvested and consumed crop worldwide. However, many people are allergic to wheat. The rate of sensitization to wheat is 0.4–1.3% in children and 0.2–0.9% among adults. Our previous study indicates that reducing allergenic protein contents in peanuts by ultrasound assisted enzymatic treatment could reduce the risk of peanut allergy. Meanwhile, treatment of wheat flour and peanuts by enzymatic methods results in breakdown of protein chains which may have both positive and negative effects on the functional and sensory properties of products. Therefore, it is important to evaluate such impacts and explore suitable applications for the products with reduced allergenicity. Based on the above description, the target audiences of this project include people who are allergic to wheat, researchers and students who study and work in the area of food allergy, health educators/teachers, health professionals and food manufactures that are interested in develop hypoallergenic products. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One graduate student was trained to perform proximate composition analysis, SDS-PAGE, In vitro IgE-binding assay, and to test the impact of enzyme treatment of peanut on the functional properties of the peanut flour. Her work will be presented in ACS 2014 Spring Meeting. One postdoctoral scholar was trained to explore enzymes and process for effective wheat allergen reduction and optimize the process. How have the results been disseminated to communities of interest? The results were disseminated by oral presentation in the 2013 NIFA/AFRI PD meeting and as poster presentations in 2013 ACS Southeast Regional Meeting in November 2013 in Atlanta, Georgia. What do you plan to do during the next reporting period to accomplish the goals? The principal investor plans to complete 1) sensory evaluation of peanuts treated by both digestive proteases and alcalase, 2) study of functional properties of enzyme treated peanut flour and protein, 3) in vitro IgE-binding test of alcalase and papain treated wheat flour.
Impacts
What was accomplished under these goals?
The effective enzymes for hydrolyzing allergenic wheat proteins in wheat flour were selected. Six proteases including alcalase, papain, flavorzyme, trypsin, α-chymotrypsin and pepsin were tested at their optimal pH and temperature. Total soluble protein and gliadin contents were determined as indicators to evaluate the effectiveness of enzymes. The hydrolysis conditions of wheat protein by the effective enzymes were optimized using central composite design. The impact of enzyme treatment on the nutritional value of peanuts and functional properties of defatted peanut flour were evaluated. The specific objectives met during this reporting periods are Task 2 of objective 1: a preliminary screening of process effectiveness in reducing wheat allergens and Tasks 2 and 3 of objective 3: Evaluate the proximate composition and functional properties of treated peanut and wheat products. Significant results achieved: Effort on wheat allergen reduction showed that proteases of bacterial and plant origin such as alcalase and papain had higher ability to reduce allergenic protein content of wheat flour than digestive enzymes such as pepsin, trypsin and α-chymotrypsin. With the alcalase, enzyme to substrate ratio (E/S) and reaction time had significant effects on the gliadin content, and there is a significant interaction between temperature and hydrolysis time. Under the optimal hydrolysis condition for alcalase a 99.96% reduction of gliadin was achieved. Results from functionality tests indicated that enzymatic treatment of roasted peanuts with alcalase increased protein solubility and foaming capacity of defatted peanut flour, but weakened the emulsifying and the thickening power of peanut flour compared with the untreated peanut flour due to the break of polypeptide chain by enzymes. Non-digestive food grade proteases such as alcalase and papain have great potential to reduce the allergen levels of peanuts and wheat flour, thus reduce the risks to consumers. However, the change in the functional properties of needs to be considered when enzymatic treated peanuts and wheat flours are used in food product formulation, because some properties are enhanced and some are weakened.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Li, H., Yu, J., Ahmedna, M., & Goktepe, I. (2013). Reduction of Major Peanut Allergen Ara h 1 and Ara h 2 in Roasted Peanuts by Ultrasound Assisted Enzymatic Treatment. Food Chemistry, 141, 762-768.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Yu, J., Hao Li, Ahmedna, M., Goktepe, I., Hernandez, M., Sanders, M. & Lay, J. Effects of Alcalase from Bacillus licheniformis on peanut allergenicity. Poster presented at American Chemical Society 2013 Southeastern Regional Meeting, November 12-16, 2013, Atlanta, GA.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Li, Y., Yu, J., Ahmedna, M. & Goktepe, I. Reducing the allergenicity of wheat flour by proteases. Poster session presented at American Chemical Society 2013 Southeastern Regional Meeting, Poster presented at American Chemical Society 2013 Southeastern Regional Meeting, November 12-16, 2013, Atlanta, GA Atlanta, GA.
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Progress 01/15/12 to 01/14/13
Outputs
OUTPUTS: A previous study showed that treatment of peanut kernels using digestive enzymes such as trypsin and chymotrypsin significantly reduced contents of the major peanut allergens Ara h 1 and Ara h 2. During the reporting period, we applied many approaches, including ultrasound assisted enzymatic hydrolysis of peanut allergenic protein, use of the food grade reducing agent sodium ascorbate. In addition, the use of a food grade protease alcalase produced by the bacterium Bacillus licheniformis (Novozyme, Corp) has been tested to optimize the enzymatic process for allergen reduction of peanut. In addition, skin prick tests using patients with confirmed peanut allergies have been conducted in our subcontractor's facility, University of North Carolina at Chapel Hill (UNC Chapel Hill). The effects of ultrasound, enzyme concentration and enzyme treatment time on the concentrations of soluble protein and major allergenic proteins (Ara h 1 and Ara h 2) of roasted peanut kernels were investigated. A 3-factor (time of ultrasonication, enzyme concentration, and enzyme digesting time), five-level orthogonal experimental design was implemented. Porcine trypsin and chymotrypsin, alcalase were used separately or in combination for enzymatic treatment of peanuts. The pretreatment of peanuts with reducing was conducted by soaking peanuts in sodium acerbate solutions (1, 10, 100 mM, pH 7.27), then digesting with 0.25% trypsin or chymotrypsin at 37 Degree Cesius for 1 hour. Treated peanuts were dried in a vacuum oven and ground to paste. Proteins were extracted using phosphate buffer (pH7.5). SDS-PAGE and Sandwich ELISA techniques were used to evaluate the effectiveness of the treatments on the reduction of Ara h 1/2. The in vitro IgE-binding of treated samples was tested by a competitive inhibition ELISA using human plasma and compared to that of untreated samples. Protein extracts of samples with lowest Ara h 1 and Ara h 2 contents and lower in vitro IgE-binding capacity were used to perform skin prick tests in comparison with untreated peanut extract, standard peanut extract and saline. All sample treatment, Ara h 1 and Ara h 2 quantification and in vitro IgE binding tests were conducted in North Carolina A&T State University, while skin prick tests were conducted in UNC Chapel Hill. PARTICIPANTS: Dr. Jianmei Yu, an Asian female, North Carolina A&T State Univeristy Dr. Mohamed Ahmedna, African male North Carolina A&T State University Dr. Ipek Goktepe, a Caucasian female, North Carolina A&T State University Dr. Michelle L Hernandez: University of North Carolina at Chapel Hill. Dr. David Peden, Professor of Pediatrics, University of North Carolina at Chapel Hill Dr. John Lay, University of North Carolina at Chapel Hill Dr. Hao Li: Postdoctoral scholar, North Carolina A&T State University Amy R. Auerbach, University of North Carolina at Chapel Hill TARGET AUDIENCES: The target audiences of this project include: - People who are allergic to peanuts and wheat - Researchers and Students who study and work in the area of food allergy - Educators/teachers - Health professionals - Food Industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Peanut and wheat are two economically and nutritionally important crops that can also cause severe allergies. Due to the large consumption and wide use of peanuts in processed foods and food service settings, there is a need to reduce or eliminate allergens in peanuts and wheat before they are mixed with other food ingredients. The results from the research activities during this reporting period show that all tested enzymes significantly increase the solubility of peanut protein. Trypsin digestion decreased concentration of Ara h 1 in both soluble and insoluble portions but not Ara h 2 in the insoluble portion of peanuts. Chymotrypsin showed more effectiveness than trypsin in reducing Ara h 1 and Ara h 2. Trypsin digestion followed by chymotrypsin digestion significantly reduced both Ara h 1 and Ara h 2 concentrations. Ultrasound pretreatment enhanced the solubilization of peanut protein and increased the Ara h 2 reduction by trypsin digestion. Pretreatment with ultrasound followed by sequential enzyme treatment with trypsin-chymotrypsin reduced Ara h 1 and Ara h 2 in both soluble and insoluble portions of peanuts to the undetectable level, and significantly lowered the in vitro IgE-binding of the peanuts. Alcalase was the most effective in reducing Ara h 1 and Ara h 2 among three enzymes used. Peanut kernels treated alcalase alone significantly increased the solubility of peanut protein and decreased concentrations of Ara h 1 and Ara h2. The maximum reductions of Ara h 1 and Ara h 2 were obtained under alcalase concentration of 0.15 unit/100g peanut and treatment time of 3 hours. Samples obtained under this condition also showed lowest in vitro IgE-binding with pooled plasma from 10 individuals allergic to peanuts and lowest in vivo allergenicity in the skin prick tests with 8 peanut allergic individuals. Pretreatment with reducing agent sodium ascorbate did not enhance the reduction of Ara h 1 and Ara h 2. The results achieved during this project period show that post-harvest technology has great potential to reduce the allergenicity of peanuts and reduce the incidence of peanut reactions due to the accidental consumption of foods containing peanut ingredients, thus enhancing food safety. The process developed during this project period is simple and safe. Therefore, the process may be used to produce hypoallergenic peanuts, thus adding value to the peanut products and enhancing the profitability and sustainability of peanut industry and peanut farmer. The product produced by this process may also be a good choice of peanut samples for immunotherapy. The research protocols developed in this project may also be applied to other allergenic foods such as wheat, dairy products, tree nuts, soybeans and other legumes.
Publications
- Li, H., Yu, J., & Ahmedna, M. (2012). Impact of ultrasonication on the efficiency of enzymatic degradation of Ara h 1 and Ara h 2 in roasted peanut kernels. 2012 IFT Annual Meeting, paper#: 041-08, June 25-28, 2012. Las Vegas Nevada.
- Li, H., Yu, J., Ahmedna, M., & Goktepe, I. (2012). Reduction of Major Peanut Allergen Ara h 1 and Ara h 2 in Roasted Peanuts by Ultrasound Assisted Enzymatic Treatment. Food Chemistry (submitted).
- Ahmedna M, Yu J and Goktepe I. Safer peanuts closer at hand. Re: Search, Vol. 8 (2011), 26-27.
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Progress 01/15/11 to 01/14/12
Outputs
OUTPUTS: Peanut and wheat are two economically and nutritionally important crops that can cause severe allergies. While avoidance is the best way to guard against the manifestation of food allergies, the ubiquitous use of peanut and wheat products by the food industry makes it very hard for allergic individuals to avoid accidental ingestion. Given the common use of peanuts and wheat as ingredients in a variety of processed food products and food service settings, there is a need to reduce or eliminate allergens before they are mixed with other foods to protect consumers from allergic reactions. Our previous study demonstrates that the treatment of peanuts with certain proteases under specific experimental conditions apparently reduces major peanut allergens. However, the enzymatic process developed can be optimized to further reduce or eliminate the allergenic proteins in different types of foods. In addition, in vivo validation is needed to confirm the absence of known peanut allergens, and any new process-related allergens (degradation products) that may appear in the treated peanuts, in order to demonstrate that the treated peanut products are safe for human consumption. Such a step is critical for potential commercial use of hypoallergenic peanut and wheat products and approval of claims or label content by regulatory agencies. During this project period, our research team has completed the following: The initiation of the project; postdoctoral hiring, and the allocation of funds between North Carolina A&T State University, the lead institution, and A&T's collaborating institution, the University of North Carolina at Chapel Hill. We also started the execution of project activities including the pretreatment of roasted peanuts by ultrasonication and sodium ascorbate to enhance the effectiveness of the enzymatic degradation of peanut allergens, as well as human skin tests of the products produced, according the approaches laid out in the proposal. These activities are essential for achieving the project objectives 1) to evaluate the effectiveness of post-harvest processing of peanut and wheat with endopeptidases and physicochemical treatment in reducing the concentrations of allergens, and (2) to confirm the reduction of allergic potential in patients through clinical testing. The monetary rescore was used to purchase supplies needed, and to support one postdoctoral scholar to conduct research activities, which is essential to ensure the execution and completion of project objectives and goals. Our research activities were disseminated through local television broadcasts, the School of Agriculture and Environmental Sciences homepage web video at http://www.ag.ncat.edu, and in Re:search, the magazine of the Agricultural Research Program at North Carolina Agricultural and Technical State University. This magazine is distributed among stakeholders and farmers in the land grant university system. PARTICIPANTS: Dr. Jianmei Yu: Co-PI of the project, acted as project manager due to the absence of PI, Dr. Mohamed Ahmedna. She Initiated the project, communicated with Co-PIs in University of North Carolina at Chapel Hill, recruited a postdoctoral scholar and managed the supplies needed for the research activities. Dr. Hao Li: Postdoctoral visiting scholar. She was recruited from China to conduct specific project activities. Dr. Michelle L Hernandez: Co-PI of the project in University of North Carolina at Chapel Hill. She was responsible for recruiting patients who are allergic to peanuts and conduct skin prick tests. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Overall, the research protocols developed and results achieved during the project period show that post-harvest technology has great potential to reduce the allergenicity of some food products such as peanuts, thus enhancing food safety, adding value to the peanut products and enhancing the profitability and sustainability of peanut industry. New knowledge developed under this project include the finding that the treatment of roasted peanut kernels using endopeptidase such as trypsin and chymotrysin, significantly reduced the major peanut allergens Ara h 1 and Ara h 2, and that blanching showed some positive effect in terms allergen reduction. However, we also noticed that blanching caused loss of soluble components including those contributing to the specific flavor of roasted peanuts. Therefore, we replaced blanching with ultrasound pretreatment and tested the influence of ultrasound on the efficiency of enzyme diffusion and peanut allergen degradation. Researchers investigated the effects of ultrasound, enzyme concentration and enzyme treatment time on the concentrations of soluble protein and major allergenic proteins (Ara h 1 and Ara h 2) of roasted peanut kernels. A 3-factor (time of ultrasonication, trypsin/chymotrypsin concentration, and trypsin digesting time), five-level orthogonal experimental design was implemented with 0, 0.5, 1.0, 1.5 and 2.0 hrs for time of ultrasonication, 0.1 0.15, 0.2, 0.25 and 0.3 g/100mL for trypsin/chymotrypsin, and 0, 1, 3, 5, 7 and 9 hrs for enzyme treatment time. SDS-PAGE and Sandwich ELISA techniques were used to evaluate the effectiveness of the treatments. The results of this experiment indicates that the pretreatment of peanut kernels with ultrasound, followed by protease digestion, significantly increases the solubility of peanut protein. Results indicate that trypsin digestion decreased concentration of Ara h 1 but not Ara h2. Trypsin digestion followed by chymotrypsin digestion significantly reduced both Ara h 1 and Ara h 2 concentrations. The highest soluble protein and lowest Ara h1 and Ara h2 was achieved under conditions of 1 hour ultrasound, 1 hour trypsin hydrolysis at concentration of 0.25 g/ml and 1 hour chymotrypsin hydrolysis at concentration of 0.2g/ml. Samples with lowest Ara h 1 and Ara h 2 contents, and untreated sample were used to perform skin prick tests. Skin prick test was conducted with 3 patients. These results were not conclusive. Other new knowledge emerging from the project indicates that ultrasonication increased the solubility of peanut proteins which made proteins more available for proteolytic enzymes. The results also show that less specific enzymes, or the enzyme preparation containing multiple proteases, may be needed. Therefore, further process optimization and more skin tests will be conducted in the project period 01/2012-12/2013.
Publications
- No publications reported this period
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