Grapes are subject to mold contamination during cultivation, harvest, transport and/or storage. Molded grapes present a safety issue to products derived from grapes because of the presence of mycotoxins. The major mycotoxin in molded grapes is ochratoxin A (OTA) [1
]. The most relevant OTA-producing species are Penicillium verrucosum (P. verrucosum)
, Aspergillus ochraceus (A. ochraceus)
, A. niger
and A. carbonarius
due to their prevalence in foodstuffs (cereals, grapes, coffee, etc.) [3
]. Grape pomace is the residue of grapes after wine making and is a valuable source of phenolic antioxidants, dietary fiber and polyunsaturated lipids. Some of our studies show that GP has great potential to serve as an ingredient in food products such as bread, extruded breakfast and cookies at concentrations up to 5% (dry base) [4
]. There is also increasing interest in using GP as a feed ingredient [7
]. However, previous studies also found the presence of OTA-producing fungi (including Aspergilus niger
, A. carbonarius
, and A. fumigatus
) and a high level of OTA in both wet and dry GP, which makes GP unsafe for human and animal consumption [10
Dietary exposure to OTA represents a serious health issue and has been associated with several human and animal diseases, including poultry ochratoxicosis, porcine nephropathy, human endemic nephropathies and urinary tract tumors in humans [12
]. Livestock consuming OTA-contaminated feed showed pale and grossly enlarged kidney, fatty liver in poultry, altered performance including decreased feed consumption, reduced weight gain, and decreased egg production; the intake of feeds contaminated by OTA was the probable cause of a disease named Denmark nephropathy in pigs. The sensitivities of livestock animals to OTA are in the order of pigs and dogs > poultry > calves > mature cattle [13
]. In humans, various studies have linked OTA exposure with the human diseases Balkan endemic nephropathy (BEN) and chronic interstitial nephropathy (CIN), as well as other renal diseases [15
]. New data available since the last risk assessment conducted by the European Food Safety Authority (EFSA) in 2006 suggest that OTA can be genotoxic by directly damaging DNA, and experts have also confirmed that OTA can be carcinogenic to the kidney [16
Many countries have set limits for OTA, and concentrations need to be reduced to as low as technologically possible in food and feed. For example, in the European Union, a general maximum OTA limit is 5 µg/kg in cereals, 3 µg/kg in processed cereal products, and 10 µg/kg in dry vine fruits [17
]. China’s OTA standard for cereal grains and legumes is 5 µg/kg [18
]. The proposed Canadian OTA regulatory guide is the same as that established in Europe [19
]. Likewise, Israel has applied a 50 µg/kg OTA standard to all cereals and pulses. Switzerland’s OTA standard is 2 µg/kg for all cereal products. Currently, the United States Food and Drug Administration (FDA) has not set regulatory guidelines for OTA in food or feed.
Structurally, OTA consists of a para-chlorophenolic group containing a dihydroisocoumarin moiety that is amide-linked to L-phenylalanine. Its chemical name is L-phenylalanine-N-[(5-chloro-3, 4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyrane-7-yl) carbonyl]-(R)-isocoumarin, and its chemical structure is shown below (Figure 1
Although the most important strategy to control OTA level in food and feed is to prevent fungal growth and OTA production, detoxification becomes necessary if the food and feed materials are contaminated, in order to protect human and animal health, reduce food/feed waste, and even for safe disposal. For cereal grains and legumes, each physical processing step, such as sorting, sieving, floatation, washing, dehulling, milling, and heat treatment (such as cooking and roasting) can remove a certain amount of OTA [22
]. The reported thermal transformation/degradation products of OTA are 2R’-OTA (called 14-(R)-ochratoxin A in the past), 14-decarboxy-ochratoxin A (DC-OTA) and ochratoxin alpha amide [24
]. It was reported that gamma irradiation from 2 to 5 kGy effectively prevented the production of OTA or destroyed it when already produced, and carboxypeptidase at 5 units/50 mL in a liquid medium is very efficient for cleaving the OTA already produced [25
]. Researchers have also discovered a good many microorganisms that could degrade and/or adsorb OTA, including actinobacteria, bacteria, filamentous fungi, and yeast; the degradation of OTA to non-toxic or less toxic OTα via the hydrolysis of the amide bond is the most important OTA biodegradation mechanism [26
]. However, detoxification by microorganisms will cause unavoidable biochemical changes in food and feed stuff due to fermentation.
As a byproduct of grape processing, GP would theoretically have a higher mycotoxin level than the processed products, as in the case of brans of cereal grains [27
]. Although our previous study demonstrated that vacuum drying not only inactivated molds, but also significantly reduced OTA content in GP [11
], research on how to reduce OTA in GP is limited. Therefore, it is important to develop effective methods to destroy molds and transform OTA into less toxic or non-toxic compounds before the GP is added into food formulas to ensure food safety. This study investigated the effectiveness of some common food processing methods, including thermal pressure treatment, acid treatment, baking under slight alkaline conditions and enzymatic treatment on the OTA content of GP.
3. Conclusion and Implication
This study demonstrated that thermal pressure processing, such as autoclave and pressure cooking, could effectively destroy OTA in grape pomace without causing too much damage to polyphenols, but the time of treatment has to be controlled to avoid excess destruction of polyphenols. Treatment using organic acids, such as acetic and citric acid, at concentrations of 0.01 M (pH 2.0) also reduced OTA in GP significantly. Similar to breadmaking, cookie baking could not reduce OTA. Although hydrolytic enzymes such as carboxypeptidase, lipase and protease from Aspegillus niger showed great potential to reduce OTA in the buffer solutions, their efficacies in OTA reduction in GP were very limited, even when the treatment time was 24 h. Therefore, enzyme treatment alone may not be an effective approach for reducing OTA in GP; the combination of thermal pressure treatment and acid/enzyme treatment may reduce OTA further, and may be worth further study. Because most of the degradation products of OTA are reported as being less toxic, it is reasonable to assume that GP treated by thermal processing, acid and enzymes should be safer than untreated GP. However, this assumption needs to be tested by in-vitro and in-vivo toxicity studies using cell cultures and animal model. The limitation of this study is that OTA was quantified by ELISA, which displays high variation and cannot provide information about the degradation products of OTA. More consistent and accurate methods, such as high-performance liquid chromatography (HPLC) or LC-MS/MS, are needed in future studies related to the degradation or transformation of OTA.
4. Materials and Methods
The wet grape pomace samples from seven grape cultivars, including Muscadine Carlos, organic Muscadine Noble, organic Cabernet Franc, Cabernet Sauvignon, Merlot, Sangiovese and Chardonnay were obtained from two North Carolina wineries. They were collected right after press, packed in gallon-size plastic bags separately and stored at −20 °C until use. Purified ochratoxin A (lyophilized powder) from Aspergillus ochraceus, carboxypeptidase A from bovine pancreas, lipase and protease from A. niger, alcalase from Bacillus licheniformis, and pepsin from porcine gastric mucosa were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid, citric acid, lactic acid and hydrochloric acid were purchased from Fisher Scientific (Suwanee, GA, USA).
4.2. Treatments of Grape Pomace and OTA Extraction
4.2.1. Thermal Pressure Treatment
The GP samples were thawed overnight and 100 g of each was weighed into a set of three containers, capped, then autoclaved for 10, 20 and 30 min at 15 psi and 121 °C in a laboratory autoclave. The untreated pomace samples were used as controls. After cooling, 100 mL of DI water was added to each container, and the GP samples were homogenized. OTA was extracted using undiluted methanol. Briefly, 10 g of GP slurry was mixed with 20 mL of methanol in a 50-mL Erlenmeyer flask for 30 min on a magnetic stir, and centrifuged at 3000× g for 20 min. The supernatant was collected for OTA determination. The extraction was conducted in triplicate for each sample.
4.2.2. Acid Treatment
The reason to select acid treatment is that most of polyphenols (anthocyanins, catechins and flavonoids) in GP are stable under acidic conditions even at high temperatures [34
]. The GP samples were treated with acetic acid (AA), citric acid (CA), lactic acid (LA) and hydrochloric acid (HCl) at pH 2.0, respectively, for 24 h at 37 °C. Briefly, 50 g of wet GP was suspended in 0.01 M of acid solution in a glass container at a GP-to-acid ratio of 1:1 (w/v) and adjusted to pH 2 using 2 M HCl and 2 M NaOH, then incubated at 50 °C for 3, 6, 9, 18 and 24 h. GP samples without added acid were used as the control. After homogenization, OTA was extracted using 100% methanol as described in Section 4.2.1
According to our previous studies, cookies are one of the food products suitable for GP application [4
]; thus, cookie making was used as the baking model in this study. The vacuum-dried GP was ground and sieved, and the portion passed through a 40-mesh screen was used for baking. The OTA content of GP powder and flour were determined before cookie making. Cookie dough was formulated with all-purpose flour, sugar, butter, egg, baking soda, vanilla extract, and 5% GP with known OTA content. The dough was then spiked with 5 ppb of OTA and mixed thoroughly with a kitchen aid mixer; the dough was then divided into 6 equal balls, 3 were baked at 350 °F (178 °C) for 20 min in a lab oven, and 3 were used to determine total OTA before baking. The dough and cookie samples spiked with OTA but without GP were used as controls. For OTA extraction, the dough was homogenized with 80% methanol, while cookie was broken into small pieces, ground into powder, and then extracted with 80% methanol. The moisture of dough and cookie were determined by drying spread samples in a vacuum oven for 24 h at 80 °C.
4.2.4. Enzymatic Treatment
Alcalase, flavourzyme, lipase, pepsin, and carboxypeptidase A were used for enzymatic treatment. For lipase, alcalase and flavourzyme, the treatments were conducted in pH 7.5 phosphate buffer (PB), the pepsin treatment was conducted in simulated gastric fluid at pH 1.5, papain treatment was conducted in PB at pH 6, and carboxypeptidase treatment was conducted in Tris buffer at pH 8.0. Purified OTA solution was diluted to 30 ng/mL in a set of test tubes with different buffers containing different enzymes. The enzyme concentration was 10 mg per 1µg OTA as described by Abrunhosa and colleagues [43
]. The treatment was conducted at 37 °C in a water bath shaker for 0–24 h at the optimal pH of that enzyme, and samples were taken at 0, 3, 6, 9 and 24 h. After inactivation of the enzyme in a boiling water bath, the OTA concentration was determined by using an ELISA kit. The enzymes that resulted in obvious OTA reduction were selected to treat GP. Before enzyme treatment, the GP was sterilized by autoclaving to inactivate microorganisms, followed by homogenization to ensure the even distribution of spiked OTA and enzyme. The OTA content of the GP slurry was determined, and slurry was then spiked with 10 ng/g of OTA. After thorough mixing, the selected enzyme was added and GP slurry samples were incubated in the water bath shaker for 24 h at 37 °C followed by enzyme inactivation. OTA-spiked GP slurry without added enzyme but incubated under same conditions was used as a control. The OTA in treated GP samples was extracted in triplicate as described in Section 4.2.1
4.3. OTA Determination
The OTA contents of extracts were determined in triplicate by a rapid immune assay using the AgraQuant® Ochratoxin Assay Kit (RomerLabs, Newark, DE, USA) according to the manufacturer’s instructions. All OTA extracts were adjusted to pH 7.0 ± 0.10 using 2 M HCl or 2 M NaOH, centrifuged again to remove any particles, and quantitatively diluted with 80% methanol before ELISA assay. The OTA content was expressed as ng/kg sample according to sample weight, extract volume and dilution factor. The OTA contents of dough and cookies were based on dry sample weight.
4.4. Polyphenol Extraction and Analysis
The polyphenols in treated and control samples were extracted in the same way as for OTA extraction. Total polyphenol (TP) was determined by the Folin–Ciocalteu method [44
] modified for microplate assay. Total flavonoids (TF) was determined by the aluminum chloride (AlCl3
) colorimetric method [45
4.5. Data Analysis
Data were analyzed by post-ANOVA Duncan test using SAS version 9.4 (SAS Institute, Cary, NC, USA). The percentages of OTA reduction under specific treatments were calculated for each sample.