INNOVATIVE DETOXIFICATION OF MYCOTOXIN CONTAMINATED GRAPE POMACE FOR LIVESTOCK ANIMALS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1030242
• Grant No.: 2023-38821-39581
• Cumulative Award Amt.: $299,998.00
• Proposal No.: 2022-09589
• Project Start Date: Sep 1, 2023
• Project End Date: Aug 31, 2026
• Grant Year: 2023
• Program Code: [EQ]- Research Project

Project Director
Yu, J.

Recipient Organization
NORTH CAROLINA A&T STATE UNIV
1601 EAST MARKET STREET
GREENSBORO,NC 27411

Performing Department
(N/A)

Non Technical Summary
There is an increasing interest of using plant polyphenols as antioxidants and antimicrobial in feed to combat the antibiotic resistance issue. Grape pomace (GP) is a by-product of grapes from wine making and is renewable and sufficient in grape juice and wine producing regions. It is rich in polyphenols and has potential to serve as an antibiotic alternative for livestock animals. However, previous studies show that the GP is often contaminated with toxigenic fungi and high level of OTA which could cause animal sickness and affect animal performance and production, particularly to pigs and poultry. Although studies of OTA detoxification methods have been reported, the research about the feasibility of those methods in real food/feed materials is limited. This project aims to develop an innovative and affordable detoxification approach by combining thermal pressure treatment and probiotic fermentation to make GP a safer feed ingredient, and to evaluate the detoxification efficacy and the impact of feeding detoxified GP on meat quality and the safety using a poultry model. The approach for detoxification will ensure all harmful microbes and spores are killed, and OTA in GP is degraded into non-toxic compounds not just adsorbed. If the detoxified GP is safe for poultry, it will also be safe for other farm animal species because poultry is more sensitive to OTA than most farm animals except swine. The GP detoxified by the approach of this project may serve as reservoir of antibiotic to improve the immunity of livestock animals if regularly consumed at right dose. This will reduce farmers' reliance on antibiotics and increase their profitability. The successful detoxification of OTA in GP will also enhance the value added use of GP which will benefit grape wineries and feed industry.

Animal Health Component

60%

Research Effort Categories

Basic

40%

Applied

60%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
314	1139	1090	40%
711	3220	1150	30%
711	3260	2000	30%

Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals;

Subject Of Investigation
3220 - Meat-type chicken, live animal; 3260 - Poultry meat; 1139 - Grapes, general/other;

Field Of Science
1090 - Immunology; 2000 - Chemistry; 1150 - Toxicology;

Keywords

grape pomace

ochratoxin a

detoxification

antibiotic alternative

chicken health

meat quality

Goals / Objectives
The overall goal of this project is to develop an innovative an affordable detoxification method for grape pomace (GP) by combining thermal pressure treatment and probiotic fermentation to make GP a safer feed ingredient, and to evaluate the detoxification efficacy using a poultry model. The specific objectives of the project are(1) develop GP detoxification method and optimize the detoxification condition,(2) evaluate the effects of feeding detoxified GP on the growth performance and immune-competence of broiler chickens, and (3) evaluate the effects of feeding detoxified GP on the quality chicken meat.

Project Methods
Based on the information obtained from up to date literature, the hypothesis of the project is that the combination of thermal-pressure treatment and fermentation with probiotic bacteria will detoxify OTA-contaminated GP, the poultry diet supplemented with detoxified GP will improve the immune-competence of broiler chickens, the chicken meat and organs of chicken fed with diet containing GP will have significantly lower OTA residue than those from chicken fed with diet containing non-detoxified GP. This hypothesis will be tested by three specific objectives.Objective 1 Develop a detoxification method and optimize the detoxification condition: The fresh GP will be collected from a local winery, which will be treated at high temperature and high pressure for different time to sterilize and to thermal degrade OTA. The LAB and Bifidobacterium strains which can produce OTA degrading enzymes and are resist to high acidity (low pH) will be used to ferment the sterile GP. The efficacy of OTA reduction by the approach will be evaluated by quantifying OTA residual in treated GP in comparison that in untreated GP. The OTA and its degradation product OTα in the treated and untreated GP will be extracted and analyzed by the HPLC-FLD method.Objective 2 Evaluate the effects of feeding detoxified GP on the growth performance and immune-competence of broiler chickens: The detoxification efficiency will be tested by feeding broiler chickens with a diet containing 5% of detoxified GP and monitoring chicken's performance, oxidative status, immune function, liver and kidney functions, and OTA accumulation in selected organs in comparison to those fed undetoxified GP, and controls. Negative control will consist of chicks given a conventional unmedicated corn-soybean meal basal diet, while positive control will consist of chicks given a basal diet with 0.25 g/kg ethoxyquin. Changes induced by GP in cecal microbiome functional composition will also be determined. Experimental birds will be necropsied for the collection of the liver and kidney for the determination of the OTA concentration.Objective 3 Evaluate the effects of diet containing detoxified and undetoxified GPs on the quality and safety of chicken meat: Breast and thigh muscles will also be collected at the end of feeding experiment for the meat quality analysis. The main quality parameters such as appearance, texture, juiciness, proximate composition (moisture, fat, protein and ash), cook loss, and the oxidative stability of muscle meat from chickens fed with detoxified and undetoxified GPs will be measured. The residue OTA in chicken meat samples will be extracted using an enzyme-assisted extraction procedure and quantified by a HPLC-FLD method and compared using post-hoc Tukey test at 5% significance level.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The target audience of this project include poultry farmers, grape vineyard and winery owners, scientists, researchers and students in food science and animal science, people in food and feed industry, food and feed safety regulation agents, and consumers who are interested in usinggrape pomace or other fruit pomace as food ingredient. Changes/Problems: Major problems or delays that may have a significant impact on the rate of expenditure: The project was delayed. In the original proposal, we planned to start objective 2 in second project year. However, we are unable to do so because of 3 major reasons: (1) it took longer than expected time to optimize the thermo-pressure treatment condition for OTA detoxification, (2) the autosampler of UPLC system was suddenly out of work after we completed analyzing acid treated samples andit took us about one month to get an autosampler replacement and to make the UPLC system back to work normally. (3) With the autosampler replacement of the UPLC, the analysis results were significantly different from previously obtained. We had to reanalyze all samples that had been analyzed before the autosampler was out of work. Significant deviations from research schedule or goals; We investigated the impact of pH on the destruction of OTA by pressure-cooking, which is not in the proposal. The reason of doing this that the bacterial strains to be used for fermentation havepH optimal different from the pH of grape pomace. Thereby, we have to test the OTA reduction of thermal-pressure treatment atdifferent pHs in order to find out if there is anyeffects of fermentation on OTA reduction because the fermentation will be conducted after thermal-pressure treatment. unexpected outcomes: The results obtained so far are expected. Because of above issues, both PI and Co-PIs agreed to apply for one year No Cost Extension at the end of second project year. What opportunities for training and professional development has the project provided?One graduate student, Ebenezer Aning-Dei, was supported by this project during this reporting period. He was trained to (1) conduct thermo-pressure treatment of grape pomace at different pH for OTA reduction, (2) OTA and polyphenol extraction and analysis, (3) operate and maintain HPLC system, (4) experiment design and data analysis, and (5) make poster presentation in different conferences and events.He is under training to conduct grape pomace fermentation and towrite thesis and manuscript. How have the results been disseminated to communities of interest?The results were disseminated in three (4) conferences and two outreach events. Conferences 51st NCAFCS Annual Conference, March 1, 2024, Raleigh, NC 21st 1890 ARD Biennial Research Symposiums, April 4-10, 2024, Nashville, TN Poster presentation at 2024 SERMACS (American Chemical Society 2024 Southeastern Regional Meeting). October 23-26, 2024, Atlanta, GA. Poster presentation at 2024 IFT First: Annual Event and Expo, July 14-17, 2024, Chicago, IL Outreach Events 2024 Fall CAES Student Showcase, September 4, 2024, North Carolina A&T State University Farm, Greensboro, NC. 2024 Smalls Farm Field Day, June 20, 2024, North Carolina A&T State University Farm, Greensboro, NC. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, I plan to complete the optimization of fermentation condition to select the best probiotic bacterial strain and fermentation condition (pH and time) for OTA reduction, and then to produce sufficient amount of detoxified grape pomace for animal study (Objective 2).

Impacts
What was accomplished under these goals? Major activities completed: During thereporting period of September 1, 2023 - August 30, 2024, we completed thermal pressure treatment of GP with and without added acid oralkaline using a pressure cooker. Without addition of acid/alkaline, the treatment was done at 115 decree Celsiusand 12 psifor 15-60 min. For acid treatment, acetic acid and citric acid were added to GP to the concentrations of 10, 100 and 1000 mM followed by pressure cooking for 45 minutes. For the Alkaline treatment, sodium bicarbonate solution was added to the GP and the pH was adjusted to 5, 6, and 7. The GP samples were then pressure cooked for 15-60 minutes. Followed the thermal treatment, OTA and polyphenols were extracted and their contents were evaluated. Specific objectives met: Task 1 and Task 2 in Objective 1 are completed. We have submitted IBC protocol and are waiting for the approval to start Task 3. Significant results achieved, including major findings, developments, or conclusions (both positive and negative): The grape pomace was acidic with pH 4.3. The pressure-cooking without pH adjustment reducedOTA and total anthocyanin contents in the GPwith increasing cooking time powerly. A 45 min of pressure cooking decreased the OTA by 35%, but no further OTA reduction was observed with continue increase of cooking time. The total flavonoid and total polyphenol content went down first, and then went up gradually. The results indicate that pressure-cooking under home cooking condition can moderately reduce OTA content in GP in a reasonable duration. With the addition of either acetic acid or citric acid, the efficacy of thermal-pressure treatment for OTA reduction was significantly enhanced with the highest OTA reduction being 55% at acid concentration of 1000 mM. At same acid concentration, the OTA reduction in the presence of aceticacid was slightly but significantly higher than the presence of citric acid.Although the total anthocyanin content of GP decreased with acid concentration, the reduction of anthocyanin was significantly smaller than that without acid addition. The extractable total flavonoids and total polyphenol increased with acid concentration near linearly. This suggests that GP polyphenols are more stable under acidic condition, and the addition of organic acid in the GP could protect polyphenols from heat destruction, meanwhile enhance the efficacy of OTA reduction. In addition, extractability of polyphenols might be increased under acidic condition and high temperature. Thermal treatment (pressure-cooking) after adjusting pH to 7 by adding sodium bicarbonate solution resulted in a significant increase of OTA reduction. The reductions of OTA was 18-36% at pH 4.3 and 57 - 63% at pH 7 depending on the treatment time. Although the total anthocyanin concentration decreased obviously with increasing pH and treatment time (P<0.0001), the total flavonoids increased with pH and treatment time (P<0.05), while total polyphenol concentrations decreased with pH but increased with treatment time. The results indicate that higher pH could enhance the detoxification efficacy of thermal-pressure treatment but increased the loss of polyphenols, particularly, anthocyanins. Key outcomes or other accomplishments realized This study found that the efficiency of thermal-pressure treatment on OTA reduction of grape pomace is affected by pH. At very low or neutral pH, the OTA reduction is significantly higher. More than 60% of OTA can be destroyed by pressure-cooking for 45-60 minutes at neutral or slightly alkaline pH. This finding has not been previously reported and will contribute to the body knowledge of OTA reduction by thermal-pressure treatment. This method can be easily applied to apple pomace, grains and legumes in food industry and home to enhance food safety, and reduce the health risk from consuming OTA contaminated food and feed, particularly in the regionswith food shortage. Although most of the anthocyanin in the grape pomace was lost, the total available flavonoids and polyphenol increased due to pressure-cooking, the health benefit of grape pomace may not be significantly affected.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Aning-Dei, E., Rehrah, D., and Yu, J. Effects of Pressure Cooking on Ochratoxin A and Polyphenol Contents of Grape Pomace. Poster presentation at 2024 ARD Biennial Research Symposiums, April 4-10, 2024, Nashville, TN.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Aning-Dei, E., Rehrah, D., and Yu, J. Destruction of Ochratoxin A (OTA) in Grape Pomace by Pressure Cooking. Poster presentation at 2024 IFT First: Annual Event and Expo, July 14-17, 2024, Chicago, IL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Aning-Dei, E., and Yu, J. Destruction of Ochratoxin A (OTA) in Grape Pomace by Pressure-Cooking at Different pH. Poster presentation at 2024 SERMACS (American Chemical Society 2024 Southeastern Regional Meeting). October 23-26, 2024, Atlanta, GA.