UTILIZATION OF BYPRODUCTS OF AGRICULTURAL AND FOOD PROCESSING TO ENHANCE FOOD SAFETY AND HUMAN HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1011939
• Project Start Date: Apr 27, 2018
• Project End Date: Feb 28, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
University of Maryland Eastern Shore
11868 College Backborne Road
Princess Anne,MD 21853

Performing Department
Food Science

Non Technical Summary
There is need for continued research on ways to expand the utilization of byproducts of agricultural and aquacultural operations, including use of fish skin and bones, crustacean shells, oilseed meals, fruit peels and pomace, and other such low value biomaterials.Those materials may be suitable for development of biobased films that may be incorporated with antimicrobials to improve food safety and extend shelf life. Hydolysed proteins including peptides with antioxidant functionalities may also be prepared from byproducts of seafood processing. The technical information needed to demonstrate the suitability of those materials for commercial applications in food, feed, nutraceutical, pharmaceutical and other areas is still lacking. This project will investigate ways to develop bio-based films with potential to confer antimicrobial and antioxidant benefits and thereby add value to fishery and other low value materials from agricultural/food processing activities. This can be achieved if films prepared from currently underutilized skins, bones, shells, and oilseed meals are used as vehicles for antimicrobials and/or antioxidants. Fish gelatin films offer several advantages compared to mammalian gelatin films including attractive barrier and color properties. Fish gelatin is recovered from materials that pose no allergenic risk and is completely biodegradable. Another advantage with fish gelatin is that it is kosher or halal, and both attributes are attractive to growing niche market segments. The potential to incorporate antimicrobials into fish skin gelatin films may lead to the development of new products that have commercial potential in diverse platforms. Careful preparation steps may be necessary for these low value-high volume byproducts to be used effectively. Investigating the properties of film-forming solutions, drying to low water activity, characterizing the prepared films, testing on food and packaging will provide insights on their ability to enhance food safety and extend shelf life.

Animal Health Component

75%

Research Effort Categories

Basic

25%

Applied

75%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	3910	2020	80%
502	0819	1000	20%

Knowledge Area
501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
3910 - Cross-commodity research--multiple animal species; 0819 - Natural fisheries, other;

Field Of Science
2020 - Engineering; 1000 - Biochemistry and biophysics;

Keywords

biopolymer films, food safety, value-added processing

packaging, microfluidization, rheology

Goals / Objectives
The project's overal goal is toimprovethe utilization of agricultural resources through valued added processing andto develop novel uses for byproducts of food processing to avoid waste.The overallgoal of the project is to investigate and expand the utilization of regionally important biomass and agricultural by-products, including oilseed (soybean) meal, peels and pomace from fruit processing, aquaculture byproducts including fish skins, bones and crustacean shells, and other related low value byproducts. It is also intended to provide fundamental understanding on the properties of biopolymer films incorporated with antioxidants, antimicrobials, and nanoparticles (e.g. zinc oxide and silver nanoparticles), and to develop antimicrobial packaging films therefrom. Protein (fish gelatin) and other biobased films such as those made from soybean meal (with and without antimicrobials and antioxidants) will be developed and their use on poultry and seafood investigated with long term goal to improve safety and quality of food during normal and refrigerated storage. The offshoot of this work is to provide an opportunityfor minority students to be trained in laboratory skillsthat is necessaryfor workforce development in agriculture and food industry. The research will involve two broad aspects/materials that are important for the Delmarva region, namely (1) fish/poultry-based byproducts (gelatin, skins, and shells) and (2) plant-based byproducts (fruit peels, pomace, and soybean meal). These goals will be achieved though the following objectives (divided into parts A and B):Part A Objectives:Develop fish gelatin- and soybean meal-based antimicrobial films and evaluate their potential to reduce pathogens and microbial spoilage of fish and poultry products.Measure physical properties, solubility in water, and appearance (color) of fish gelatin and soybean meal based films.Evaluate sensory characteristics of chicken breast and fish fillet treated with edible films, including moisture retention, texture, and appearance.Part B ObjectivesDetermine the effect of processing methods (including extraction and drying) on antioxidant and antimicrobial properties of fruit (aronia and grape) pomace extracts.Develop biopolymer films incorporating fruit pomace extracts and investigate their antioxidant and antimicrobial properties on poultry and seafood products.Evaluate the suitability biopolymer films incorporating antimicrobial nanoparticles for food packaging.

Project Methods
Part A Methods:1. Develop fish gelatin- and soybean meal-based antimicrobial films and evaluate their potential to reduce pathogens and microbial spoilage of fish and poultry products.Preparation of experimental films and coatings: Gelatin films will be prepared from deep sea fish and/or catfish skin using procedures developed by Tammineni et al. [26] and Hendrix and Min [6]. Films will be made from fish gelatin extracted from local fish and compared with those made from commercial fish gelatin (Norland Products, Cranbury, NJ). Fish gelatin film solutions at 6.75% and a 1:0.2 ratio of gelatin: glycerol (dry basis) will be used. The protein-based film-forming solutions (FFS) will be prepared and denatured at 90ºC for 30 minutes.Soybean meal will be sourced from a local poultry feed mill in Eastern Shore of Maryland and defatted prior to nanosizing to form antimicrobial-nanocomposite films. A 5% (w/w of water) defatted soybean meal (DSM) solution will be heated at 90 deg. C for 30 min. After cooling, the solution will be mixed with glycerol (5% w/w) as a plasticizer and subjected to high pressure microfluidization at 30,000 psi (LM20 Microfluidizer, Newton, MA) for 2-5 passes. Antimicrobials will be added to the film-forming solution, degassed to remove air before casting on Teflon™ plates. They will be left to dry inside a chamber at room temperature (23±2 deg. C, 50±2% RH) for 24-36 h.Culture-dependent enumerations of natural background microflora in fish and chicken: Bacterial suspension from fish fillet and chicken meat with/without film treatment will be plated on TSA/PCA using the Eddy Jet 2 spiral plater (Neutec Group Inc., Farmingdale, NY, USA) and incubated at the recommended temperature (28°C or 37 °C) for 2-3 days until distinctive colonies appear. Colonies on TSA/PCA will be enumerated as total bacterial populations.Statistical analysis: Student's t-test will be used to evaluate a significant difference of bacterial counts between film treatment and no treatment from the Rti-PCR and culture dependent assays. SPSS Version 22.0 (IBM Corp., Armonk, NY, USA) will be used to explore statistical significance. A confidence interval at the 95% level (P < 0.05) will be considered in all cases.2. Measure physical properties and appearance (color) of fish gelatin and soybean meal based films.Tensile, barrier (WVP), and color properties: The conditioned films will be tested for water vapor permeability (WVP), tensile strength (TS), and percent elongation at break (%E). Elastic modulus (EM), solubility (SM), film thickness, and color of films using CIELAB coordinates L, a, b values will also be determined. The WVP properties will tested based on (ASTM) standard E96-9. A cabinet with a variable speed fan will used at RT (25 ± 1 ºC) to test film WVP. The films will be cut into 50 mmx 8 mm strips and their tensile properties determined.Rheological properties of films: The rheological properties of gelatin and defatted soybean meal based films, as well a flow behavior of film-forming solutions, will be analyzed using a rheometerwith add-on fixtures for measuringviscoelastic properties (loss modulus, E and storage modulus, E'). The E and E' valueswill be measured by heating from minus 10 °C to 60 °C at 5 and 10 °C/min, 1 Hz, and at a strain rate in the linear viscoelastic range [3]. Storage (E'), loss modulus E, and tan δ (E/E') will be recorded.3. Evaluate sensory characteristics of chicken breast and fish fillet treated with edible films, including their moisture retention, texture, and appearance.Texture and overall appearance of both seafood and poultry breast coated with edible films will be evaluated using a 5-point hedonic scale from (1 = highly acceptable) to (5= highly unacceptable). For each sensory attribute, a score above 3.5 will be considered to be acceptable [22]. Treated samples will be evaluated at each sampling time (0, 5, 10, 20, and 30 days) over the course of the microbiological study by three panelists for color, texture, aroma, and overall appearance only. Uncoated and uninoculated fish fillets and poultry breast will serve as a control. Moisture retention for the treated fish will be determined during the storage period gravimetrically. The same protocol will be repeated in the case of soybean meal-based films.Part B Methods:4. Determine the effect of processing methods on antioxidant and antimicrobial properties of fruit pomace extracts.Aronia berry pomace will be processed and incorporated into films or healthy fruit leathers. The pomace contains residual polyphenols that may deteriorate if not extracted and utilized appropriately. It is possible that drying and extraction of phytochemicals in pomace may cause changes or reduce the expected value [16]. Following drying, antioxidant activity and texture of films will be measured. The pomace will be ground and stored at -20 ºC to prevent degradation. Phytochemical contents, including total phenolic, flavonoid, anthocyanin contents, and antioxidant capacities, including DPPH radical scavenging capacity (DPPH) will be determined using spectrophotometric and other methods [10, 11, 24].5. Develop biopolymer films incorporating fruit pomace extracts and investigate their antioxidant and antimicrobial properties on poultry and seafood products.Fruitpomace, sourced from local farms and vineyards, will be freeze-dried, ground, nitrogen-flushed in plastic packaging, and stored at -20 ºC for later analyses. Dried pomace will be extracted using organic solvents [27] thenused to determine amounts of antioxidant compounds as in Part B, Objective 1. Grape pomace extracts will be processed in like manner [29] and used to prepare to films for testing. Texture analyzer will be used to measure tensile strength (TS) and elongation at break (EB) [7]. The prepared polysaccharide and/or protein films incorporated with the various pomace extracts will be applied to compatible meat/poultry products. Half of the packaged raw samples will be stored at 4 ºC for 10 days and another half will be cooked an oven, packaged, and stored at 4 ºC for 7 days. Lipid oxidation, measured by 2-thiobarbituric acid reactive substance (TBARS) method; and color, measured using a colorimeter with CIE L*a*b* color system, will be determined at 0, 5, and 10 days of storage for raw samples and at 0, 3, and 7 days of storage for cooked meat [12].6. Evaluate the suitability biopolymer films incorporating antimicrobial nanoparticles for food packagingPolysaccharide and gelatin based films with and without antimicrobial nanoparticles (ZnO and AgNP) will be prepared by solution casting [2, 18, 19]. The film forming solutions will be degassed and denatured (for protein-based materials) before casting onto custom-made Teflon plates followed by drying in a controlled humidity chamber [8]. Dried films will be peeled off and inoculated with different concentrations of common food-borne pathogens such as Salmonella and E. coli using standard methods [4, 5, 14] to test antimicrobial activity in our food microbiology lab. Test will be done as described Part B, Objective 2. Prepared films with and without antimicrobials will be inoculated following the method of Tammineni et al [25] then treated with UV-C light [4]. The treatments and controls will be observed and analyzed to assess microbial inhibition. Polysaccharide and protein based films incorporating facile nanoparticles [2, 17, 21] and pomace extract will be applied to poultry products to test their antimicrobial activity and oxidative stability. Samples will be taken off the poultry surface and tested on day zero, three (mid), and day 7 (end of shelf life for meats).

Progress 04/27/18 to 02/28/21

Outputs
Target Audience:Students were reached through instruction on the application of films for food packaging. These students learned about the importance of applying natural coatings or films to improve food safety and extend shelf life. One student working on a different but related project (high pressure processing) investigated the use of polymer films for packaging of food under HPP for improved food safety and to extend shelf life. Films with oxygen transmission rate (OTR) of more than 10K cm3/cm2/24 hr can be used in seafood and meat packaging. The targeted students also learned about sustainable food packaging technologies thatminimize the negative impacts on the environment. The studentsinstructed onfood processing methods (FDST 805) and individual studies in food and nutrition (NUTD 680)received knowledge on methods and instrumentation for testing food packaging films, including understanding of water and oxygenbarrier properties which are important for ensuring food safety, quality,and shelf life extension. The food industry, especiallyseafood and poultry, are the other target users that stand to benefit from these investigations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided ample opportunities to train students on edible films and the potential uses of byproducts of agricultural processing. In the previous reporting period, one student was able to present his work virtually at a scientific conference (ASABE Annual International Meeting). Collaboration with the food industry to test a commercial electrostatic spray to inactivate Salmonella on chicken thigh meat provide another opportunities for training of students and profesional development. One MS student investigatedthe integrity of 16 oz plastic containers (with film seals that allowed more than10K cm3/cm2/24 hr oxygen transmission rate)to extend the shelf life of crab meat. One other studenthas been recruited and will continue investigations and be trained on the development of films incorporating chitosan nanoparticles to extend shelf life of strawberries. These are opportunities that the project has provided for students to learn about food packaging and novel processing techniques. How have the results been disseminated to communities of interest?The results were disseminated through oral (virtual) presentations to researchers and other students at seminars and scientific conferences. The PD had the opportunity to participate as Co-PD on "value-added production of antioxidant peptides from crab processing waste generated in the Delmarva area (the Delmarva Cooperative Land Grant Summit in 2020, Arlington, VA). As previously indicated, a manuscript titled "Effect of measurement method on poultry digesta viscosity" was presented virtuallly at ASABE International Meeting giving professional development opportunities to students. One related productwas published in a peer-reviewed journal. One abstract was prepared and accepted forpresentationat the 2022 ARD Meeting to report the results of the containerintegrity testing of 16 oz plastic cups (with and without 10K films) and their ability to extend the shelf life of cooked crab meat. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The activities were divided into A and B to develop gelatin films and utilize byproducts of agricultural processing. We reported on the utilization of crabmeat wasteby extracting peptides with potential antioxidant properties. One student was graduated who worked onrheology of poultry digesta. This student has completed writing a manuscript that is ready for publication. One related product (on antimicrobial coatings) was published during this reporting period. We plan to extend the work started on the various byproducts and include instrumental evaluation of sensory characteristics of chicken breast and fish products. Most of the byproducts could not be sourced due to the pandemic situation. However, despite thatsitution the project has increased the collaboration with the food industry to explore the use of and conduct research on elctrostatic spray cabinets to inactivate salmonella on chicken meat; and secondly to begin the investigation of high pressure processing of seafood and poultry meat. The latter approach will allow to better understand the incorporation or coating of these natural antimicrobialson poultry meatunder high presure. This project revealed the potential to develop and apply edible coations on perishable fruits such as strawberries. A student has been recruited and will be trained on the development of compatible films incorporatingnano-chitosanto extend the shelf life of strawberries. Flexible packaging, including films with different barrier properties will be tested on those products. Preliminary testingof 10K films on plastic cups wasconducted for packaging of crab meat.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Punchihewage-Don, A.J., Parveen, S., Schwarz, J., Hamill, L., Nindo, C., Hall, P., Vimini, B. 2021. Efficacy and quality attributes of antimicrobial agent application via a commercial electrostatic spray cabinet to inactivate Salmonella on chicken thigh meat. Journal of Food Protection, 84 (12), 22212228. https://doi.org/10.4315/JFP-21-206

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Researchers, students, and small producers. These audiences have been reached through symposia (ASABE Annual International Meeting and Delmarva Cooperative Land-Grant Summit) where poster and oral presentations were made, respecively. Students were instructed on the development of films in graduate Food Processing (FDST 805) and Individual Studies in Food and Nutrition (NUTD 680) classes, and provided opportunities for undergraduates on how to use analytical instruments to learn about food material science. Changes/Problems:The initial objectives were much broader (divided into parts A and B), but the unforeseen shutdown of lab research activities for most of 2020 due the Covid-19 pandemic is leading us to rethink the approach (materials and methods) and/or the potential byproducts of food and agricultural to be investigated. The main objective remains the same but we will focus more on value-added processing, food safety implications, and the broad impact of byproducts utilization. What opportunities for training and professional development has the project provided?Undergraduate and graduate students are trained on the utilization of byproducts of food processing, including development and characterization of bio-based films. Students enrolled in the FDST 805 Food Processing and NUTD 680 Individual Studies in Food and Nutrition courses are learning about the importance of value-added processing and the opportunities to increase the utilization of byproducts of food and agro-processing. One undergraduate student (graduating in spring 2021) was provided a hands-on training opportunity. The project provided opportunity for one graduate student to present his work at a virtual scientific conference (ASABE 2020) and network with other researchers. The PD is using the lessons learned to improve instruction and to generate data needed to apply for extramural funding. How have the results been disseminated to communities of interest?The PD worked in a team of researchers from other public institutions. A presentation titled "Value-added production of antioxidant peptides from crab processing waste generated inDelmarvaarea (Huang et al., 2020) was made to the Delmarva Cooperative Land Grant Summit, held at Virginia Tech National Capital Regional Research Center - Arlington, Arlington, VA, March 5, 2020. An oral presentation titled "Review of measurement methods for poultry digesta viscosity" (Barnas, M & Nindo, C.) was made at ASABE 2020 Annual International Virtual and On-Demand Meeting, July 13-15, 2020. What do you plan to do during the next reporting period to accomplish the goals?We will continue to investigate the incorporation of crab waste protein hydrolysates and related seafood byproducts into films and test their mechanical and barrier properties (continuation of activities in Part A Objectives). One student has been recruited and will study value-added processing and explore the development of new products from powder, film preparation, and application of high pressure microfluidization in liquid media to modify the properties of film-forming solutions.

Impacts
What was accomplished under these goals? Project was initiated in the last half of 2018 and one graduate student was recruited in 2019 but had to defer his work because of health reasons. In 2021, one student has been recruited to work on value-added processing activities. An undergraduate student was given opportunity to learn about the preparation and testing of films. One student graduated with PhD. Part of his work on rheology of poultry digesta was presented during ASABE 2020 Virtual Meeting to communicate the importance of utilizing byproducts of agricultural/food processing. Students were trained and graduated to meet workforce needs in agriculture.

Publications

• Type: Other Status: Published Year Published: 2020 Citation: Barnas, M. and Nindo, C. Review of measurement methods for poultry digesta viscosity. ASABE 2020 Annual International Virtual and On-Demand Meeting, July 13-15.
• Type: Other Status: Other Year Published: 2020 Citation: Huang, H; Nindo, C. Liu, C.; Yin, Y. Value-added production of antioxidant peptides from crab processing waste generated in Delmarva area. Delmarva Cooperative Land Grant Summit, Virginia Tech National Capital Region Research Center - Arlington, VA, March 5, 2020.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Researchers and students. The audiences have been reached through symposia (2019 ARD Research Symposium) where a poster was presented. Students were instructed on development of films (food processing class) and how to use of analytical instruments. Changes/Problems:1) Timeline of project will require adjustment by another year. A graduate student who was recruited in 2018 could not continue due to health reasons. 2) A high pressure equipment (HPP) that was donated in 2019 will support the project. The PD deemed this important and related to project goals. To adequately incorporate it, additional time is necessary for installation works and training of operators by the vendor. What opportunities for training and professional development has the project provided?Graduate students are being trained on value-addition and utilization of byproducts of food processing, including development and characterization of bio-based films. Students enrolled in the PD's class (FDST 805 food processing) are learning the importance of value-added processing, use of novel technologies, and ways to avoid waste (preparation of edible films). One undergraduate student has been identified to be provided the opportunity to gain research experience working on the project. A corollary study involves the extraction of antixodants from hibiscus and potential use of pomace. This project provided the PD and one graduate student the opportunity to attend a scientific conference and to network with other researchers. Lessons learned from those events have helped the PD to improve classroom instruction and to gather preliminary data needed to apply for extramural funding. How have the results been disseminated to communities of interest?In 2019 a poster was presented at the 1890 ARD Research Symposium, Jacksonville, FL. What do you plan to do during the next reporting period to accomplish the goals?The development and testing of value-added products (films and related materials) will be continued. We are creating linkages with local vineyards to source grape pomace and data will be collected using aronia berries that were planted at the UMES farm in 2018. This crop is just getting established. We obtained an initial harvest of aronia berries (fall 2019) that will be processed into juice and the residual pomace used as natural antioxidants. Coatings or films will be prepared following freeze-drying, micronization and high pressure microfluidization (HPM) in liquid media for various food uses. Physical and functional properties of the films will be determined. We will continue investigating the rheological properties to understand the effect of micronization, HPM and potential usage effects in poultry.

Impacts
What was accomplished under these goals? Project was initiated in the last half of 2018 and one graduate student was recruited in 2019 but had to defer his work because of health reasons. More emphasis is on recovery of materials and their functionalities. Efforts are being made to acquire research equipment. A high pressure processing (HPP) donated to the program by a local business will be used to train students and farmers/processors on food safety. Instruments needed to test moisture barrier properties (Permatran-W Model 1/50 G+ for WVP), water activity (Aqualab 4TE) were purchased and an existing rheometer will be relied on to investigate film properties. One graduate student is currently using the rheometer to study properties of poultry digesta in an attempt to understand performance of birds fed different diets that might include byproducts of food processing (corn/soy). Elements of this work was presented at the 2019 ARD Research Symposium held in Jacksonville, FL.

Publications

• Type: Other Status: Published Year Published: 2019 Citation: Barnas, M. Modeling the Rheological Properties of Poultry Digesta. Presented at 1890 ARD Research Symposium. March 30-April 2, 2019, Jacksonville, FL.

Progress 04/27/18 to 09/30/18

Outputs
Target Audience:Researchers, students, and commodity groups. These audiences have been reached through symposia (2nd 1890 ARD-USDA Food safety Symposium) where a paper was presented, lectures and hands-on experience was provided to students taking a food processing class (FDST 805), and another presentation was made to Maryland Soybean Board members. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students are being trained on the utilization of byproducts of food processing, including development and characterization of biobased films. The students enrolled in the project director's class (FDST 805 Food processing) are learning the importance of adding value to food processing byproducts to avoid waste and gaining valuable skills on how to use film preparation and analysis instruments. The project provided opportunity for the PD to attend a food safety symposia and network with other researchers. How have the results been disseminated to communities of interest?A presentation was made to Maryland Soybean Board (Denton, MD) in reponse to a call for proposals for projects with goals that add value soybean. A paper was also presented to students and researchers at the 2nd 1890 - USDA ARS Food Safety Symposium, Beltsville MD. What do you plan to do during the next reporting period to accomplish the goals?We will continue with the development and testing of films as stated in Part A (Objectives 1 and 2). Variously prepared film-forming solutions will be subjected to high pressure microfluidization to modify the sizes of antimicrobial particles incorporated in the films. Inclusion or preparation of nanoparticles will focus on natural products, especially those that are deemed important to the Delmarva region. Measurement of other film properties such as glass transition and mechanical (tensile) strength will be conducted. Later on, testing of seafood and poultry deli meat in packaging coated with natural antimicrobials will be conducted (Part B, Objective 1).

Impacts
What was accomplished under these goals? Project was initiated in the last half of 2018 and one graduate student has been recruited to develop biopolymer packaging films that would be suitable for use with compatible foods to enhance safety and extend shelf life. The PD has started assembling relevant instruments needed to test moisture barrier properties (water vapor permeability tester - Permatran-W Model 1/50 G+), water activity (Aqualab 4TE), and a rheometer to investigate properties of films and film-forming solutions to address Part A, Objectives 1 and 2. Commercial fish gelatin powder was purchased and will be used to conduct the initial studies. The importance of this work and the potential of using food processing byproducts and/or co-products of seafood processing to improve food safety and extend shelf life was presented at professional meetings during the reporting period.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Nindo, C.I. Development of biopolymer films for food safety and shelf life extension. Presented at 2nd 1890 ARD & USDA-ARS Food Safety Symposium, USDA ARS, Beltsville Agricultural Research Center (BARC), Beltsville, MD. April 23-25, 2018.