High‐pressure acidified steaming with varied citric acid dosing can successfully detoxify mycotoxins

Abstract Mycotoxins are toxic fungal metabolites that exert various toxicities, including leading to death in lethal doses. This study developed a novel high‐pressure acidified steaming (HPAS) for detoxification of mycotoxins in foods and feed. The raw materials, maize and peanut/groundnut, were used for the study. The samples were separated into raw and processed categories. Processed samples were treated using HPAS at different citric acid concentrations (CCC) adjusted to pH 4.0, 4.5, and 5.0. The enzyme‐linked immunosorbent assay (ELISA) kit method for mycotoxins analysis was used to determine the levels of mycotoxins in the grains, with specific focus on total aflatoxins (AT), aflatoxins B1 (AFB1), aflatoxin G1 (AFG1), ochratoxin A (OTA), and citrinin. The mean values of the AT, AFB1, AFG1, OTA, and citrinin in the raw samples were 10.06 ± 0.02, 8.21 ± 0.01, 6.79 ± 0.00, 8.11 ± 0.02, and 7.39 ± 0.01 μg/kg for maize, respectively (p ≤ .05); and for groundnut (peanut), they were 8.11 ± 0.01, 4.88 ± 0.01, 7.04 ± 0.02, 6.75 ± 0.01, and 4.71 ± 0.00 μg/kg, respectively. At CCC adjusted to pH 5.0, the AT, AFB1, AFG1, OTA, and citrinin in the samples significantly reduced by 30%–51% and 17%–38% for maize and groundnut, respectively, and were reduced to 28%–100% when CCC was adjusted to pH 4.5 and 4.0 (p ≤ .05). The HPAS process either completely detoxified the mycotoxins or at least reduced them to levels below the maximum limits of 4.00–6.00, 2.00, 2.00, 5.00, and 100 μg/kg for AT, AFB1, AFG1, OTA, and citrinin, respectively, set by the European Union, WHO/FAO, and USDA. The study clearly demonstrates that mycotoxins can be completely detoxified using HPAS at CCC adjusted to pH 4.0 or below. This can be widely applied or integrated into many agricultural and production processes in the food, pharmaceutical, medical, chemical, and nutraceutical industries where pressurized steaming can be applied for the successful detoxification of mycotoxins.

Many industrial and domestic processes, including heat treatment (roasting, boiling, and frying), fermentation, and irradiation, among others, have been studied for the removal of mycotoxins in foods (Awuchi, Ondari, Ofoedu, et al., 2021;Ozkan et al., 2023). However, most of these studies reported incomplete or insufficient elimination of these mycotoxins. Mycotoxins are very stable and can withstand the rigors of food processing, thus requiring a technical approach designed for their removal (Bulgaru et al., 2021). Ensuring food and agro-safety/quality is very important to not just food and agricultural industries but also to pharmaceutical and biomedical industries (Awuchi, 2023;Saeed et al., 2023).
The study aimed at addressing the increasing problems of mycotoxins worldwide, especially in developing and underdeveloped regions of the world. A widely applicable, cost-effective, and safe method was considered for the thorough decontamination of mycotoxins from foods, to protect public health and animal safety, and successfully demonstrated that high-pressure acidified steaming (HPAS) can be a reliable method to solve the problem of mycotoxin exposure from dietary and pharmaceutical sources, which are the major sources of human and animal exposure to mycotoxins and their toxic potency. High-pressure acidified steaming is a combination of three techniques (high-pressure [physical], steaming [physical], and acidification [chemical]) with the aim of complete detoxification of the mycotoxins. Citric acid is an edible organic acid used in many products directly consumed by humans, and as such, poses no toxicity when present in normal doses in human foods and animal feed. In this study, we developed a citric acid concentrationdependent high-pressure acidified steaming to successfully detoxify mycotoxins, with specific focus on total aflatoxins (AT), aflatoxins B 1 (AFB 1 ), aflatoxin G 1 (AFG 1 ), ochratoxin A (OTA), and citrinin in foods and feeds. The outcome of this study can be widely applied or integrated into many agricultural and production processes in the food, pharmaceutical, medical, chemical, and nutraceutical industries for the successful detoxification of mycotoxins, such as aflatoxins, citrinin, ochratoxins, deoxynivalenol, and fumonisins.

| Study site and sampling area
Samples were randomly drawn from different farm/market outlets in Kampala. The samples were picked using a stainless-steel container and taken to the laboratory for further analyses and processing. The samples were selected based on the fresh grain produce from farms that were aimed at supplying and/or selling to the public or industries.

| Sample collection and preparation
Three representative samples (three each from three different locations) were randomly collected for each of maize and peanuts from three different agro-markets in Kampala. The samples were separated into two categories (raw and processed). Processed samples were treated using high-pressure acidified steaming (HPAS) at different concentrations of citric acid adjusted to pH 4.0, 4.5, and 5.0.
Both processed and raw samples were ground into flour using Art's-Way portable roller mill (PRM30: USA) and labeled accordingly as directed by AOAC (2000aAOAC ( , 2000b. Maximum particle size reduction and thoroughness of mixing of the samples' flour were ensured to achieve effective distribution of contaminated portions.

| High-pressure acidified steaming
The grain flours were subjected to high-pressure acidified steaming (HPAS) using autoclave, made by Thermo Fisher Scientific, Waltham, MA, United States, at the pressure of 15 PSI at steaming temperature, at various concentrations of citric acid adjusted to pH of 4.0, 4.5, and 5.0. Before steam generation, aqueous citric acid was added to pure water to acidify the water before steam generation and the pH was clearly noted at 4.0, 4.5, and 5.0. The method described by Jin et al. (2017), Jessica (2019), and Maya and Rao (1998) was used with slight modification.

| Mycotoxin assay
The ELISA kit method for mycotoxins analysis (AOAC, 2000a(AOAC, , 2000b was employed to determine the concentration of the respective mycotoxins in the samples. Twenty gram of each sample were grinded and added to 100 mL of 70% methanol blended for 3 min. The solutions were filtered through Whatman No. 1 filter and supernatant was collected. Fifty microliter of the filtrate per well was used for the test. Fifty microliter of each of the respective standards (for each mycotoxin assayed) and test samples were added, respectively, to antibody (mycotoxin)-coated microtiter plate wells. The plates were sealed, gently homogenized, and incubated for 30 min at 37°C. Three hundred (300 μL) of wash buffer was added to each well and washed three times, and the plates were inverted on a layer of absorbent towels to remove residual water. One hundred microliter of HRP conjugate was added to each antibody-coated well and incubated at ambient temperature for 30 min. After the incubation period, the plates were washed again with the wash buffer, and the plates were inverted on a layer of absorbent towels to remove residual water.
One hundred microliter of substrate reagent was added to each well and then gently mixed thoroughly. This was then incubated at 37°C for 15 min in dark. Subsequently, 100 μL of stop solution was added to each well and gently mixed and the result read within 5 min after addition of stop solution. The optical density (OD) value of each well was determined at 450 nm (reference wavelength 630 nm) using a microplate reader. The values (corresponding to the concentration of the samples) were extrapolated from a standard curve obtained by plotting the absorbance percentage of each standard on the yaxis against the log concentration on the x-axis.
A: Average absorbance of standard or samples; A 0 : Average absorbance of Standard.

| Statistical analysis
The statistical analysis was done using SPSS. Analysis of variance (ANOVA) was conducted to analyze the data and check for any significant differences. Where p < .05, the differences were considered to be significant. Where there was significant difference in means, the least square difference (LSD) was done to separate the means.
The values are presented as means and standard deviation.

| RE SULTS AND D ISCUSS I ON
The results, their interpretation, and discussion are presented in this chapter in detail. The statistical analysis is also explained.

| Mycotoxins
The results of the mycotoxins are shown in Table 1. The mycotoxins selected for this study include AT, AFB 1 , AFG 1 , OTA, and citrinin.
The removal of these mycotoxins in foods will most likely signify the removal of all the mycotoxins in the foods. The effects of the highpressure acidified steaming on the mycotoxins were analyzed and reported in this section.

TA B L E 1
High-pressure acidified steaming with citric acid detoxified mycotoxins.
6.71 ± 0.01 (17%), 3.03 ± 0.02 (38%), and 5.68 ± 0.02 (19%) μg/kg for maize and groundnut, respectively. At citric acid concentration adjusted to pH 4.5, AFB 1 was not detected in both samples (i.e., 100% detoxification of AFB 1 was achieved), while the AT and AFG 1 in the samples significantly reduced to 6.44 ± 0.05 (36%) and 4.43 ± 0.02 (35%) μg/kg, and 5.82 ± 0.00 (28%) and 4.30 ± 0.14 (39%) μg/kg for maize and groundnut, respectively. Similarly, when the citric acid concentration was increased and adjusted to pH of 4.0, AFB 1 was also not detected in both samples (i.e., same 100% detoxification of AFB 1 was achieved), while AT and AFG 1 significantly reduced to 4.18 ± 0.02 (59%) and 3.91 ± 0.01 (42%) μg/kg, and 4.07 ± 0.02 (50%) and 4.11 ± 0.00 (42%) μg/kg for maize and groundnut, respectively. After HPAS processing at citric acid concentration adjusted to pH 4.0, the mycotoxins either completely detoxified or reduced to safe levels, except for AFG 1 which was relatively higher than the maximum allowable limit. These levels can be further reduced or  Kholif et al. (2021), who made use of size exclusion chromatography followed by HPLC. AFB 1 is the most known toxic of all mycotoxins; its removal in foods is very important to food safety and animal/human health. The mean values of the total aflatoxins reported for maize in this study are slightly higher than the mean value of 3.14 ± 3.01 μg/kg reported by Awuchi et al. (2020), but the mean values for peanut in this study are lower than the mean value of 26.30 ± 11.47 μg/kg reported in the same study. These differences may be due to differences in the locations of the sample collection.
In previous studies, aflatoxins, including AFB 1 and AFG 1 , have been shown to be nephrotoxic, immunotoxic, teratogenic, carcinogenic, mutagenic, hepatotoxic, neurotoxic, and genotoxic, among various toxic potencies. This study provides interesting knowledge into how these toxic effects can be zeroed or significantly reduced by applying HPAS against these mycotoxins in foods, especially Aflatoxins exerted toxic potencies via several mechanisms. Several recent studies have described the various mechanisms involved in aflatoxins toxicities Lai et al., 2022) and their deleterious toxic potency Awuchi, Ondari, et al., 2022). These toxic potencies can be avoided by treating grains with HPAS at the appropriate CCC before consumption. AFB 1 potentiates autophagy mediated by ROS in RAW264.7 and THP-1 cell lines Lai et al., 2022). Navale et al. (2021) Park (2002) reported a 40%-80% reduction in aflatoxins achieved using physical cleaning by removing damaged, mold-infested nuts, seeds, or kernels from the whole grains.
Although the levels of aflatoxins still remained unsafe after this removal. In addition to the physical cleaning, the method developed in this study can be used to either completely remove these mycotoxins or at least reduce them to safe levels. Kaushik (2015) evaluated the effects of food processing operations on my mycotoxin detoxification, including extrusion, roasting, flaking, frying, baking, cleaning, cooking, sorting, and trimming, and reported that processing operations such as thermal, physical, and chemical conditions play important role in detoxifying mycotoxins, with high-temperature processes having more effects. However, they also concluded that all the processes evaluated significantly reduced the concentrations of mycotoxins, but did not completely eliminate them (Kaushik, 2015). This gives the HPAS method used in this study a comparative advantage, as it can completely detoxify most mycotoxins at CCC adjusted to pH 4 or below.

| Ochratoxin A
The values of OTA in the raw samples were 8.11 ± 0.02 and 6.75 ± 0.01 μg/kg for maize and groundnut (peanut), respectively (see Table 1). These values are far higher than the maximum limits of 5 μg/kg for OTA in grains and grain products set by the European Union, WHO/FAO, and USDA (Agriopoulou et al., 2020;EFSA, 2020;Giovati et al., 2015). After HPAS processing at citric acid concentra-  Qing et al. (2022). OTA and aflatoxins often cooccur in foods and feeds, often along with other mycotoxins (Agriopoulou et al., 2020;Awuchi et al., 2020). Their removal is very important for food safety and public health. This study has developed a reliable method and processing regimen for completely detoxifying mycotoxins in foods and feeds or at least reducing them to safe levels (i.e., levels at which the body can easily detoxify them without them exerting any form of toxicity). Awuchi et al. (2020) and Awuchi, Owuamanam, and Ogueke (2021) reported the effects of ochratoxins on the nutritional and functional properties of foods. Gan et al. (2017) reported that in vitro OTA nephrotoxic effects in primary porcine splenocytes and PK15 cells showed that 0.5-4.0 and 2.0-8.0 mg/mL per day, respectively, induced apoptosis and cytotoxicity by phosphorylation and signaling of ERK and p38. These toxic effects can be avoided by employing the HPAS method to completely detoxify OTA or reduce its levels to safe levels.
OTA exerts many toxic effects in humans and animals, including nephrotoxic, hepatotoxic, carcinogenic, immunotoxic, neurotoxic, genotoxic, and teratogenic effects, among other toxic effects. causes time-and dose-dependent (6-72 h) reduction in differential and proliferative viability; differentiated neurons have less vulnerability to toxins compared to proliferating NSCs (Bhat et al., 2016;Gill & Kumara, 2019). These toxicities can simply be avoided by either eliminating or at least reducing the levels of OTA using high-pressure acidified steaming methods as described in this study.
Many methods have also been explored in other studies in an attempt to reduce the toxic effects of ochratoxin A and other mycotoxins. Leitão and Enguita (2021) conducted research on filamentous fungal proteomes' enzymes that can degrade ochratoxins in a systematic structure-based manner. They concluded that filamentous fungi are rich in hydrolases that can potentially degrade ochratoxins, and may detoxify many food commodities. However, the successful application of these enzymes in real-time is yet to be ascertained.
The method developed in this study can easily be applied in real time and large-scale food and agricultural production, as many industries already make use of steam generation and application in many processes.  (Gayathri et al., 2015;Jaus et al., 2022). Many in vitro studies showed that citrinin toxicity involves decreased cytokine production nitride oxide gene expression's inhibition, increase in ROS, inhibition of DNA and RNA synthesis, oxidative stress induction, and apoptotic cell death activation through the caspase cascade system and signal transduction pathways (European Food Safety Authority, 2012).

| Citrinin
Citrinin and its metabolite (dihydrocitrinon) have been detected in urine by Ali et al. (2015) in 82% and 84% of urine samples, respectively. Citrinin mostly targets the kidney (Jaus et al., 2022). These toxic effects of citrinin and other mycotoxins can be prevented by treating foods and feeds using HPAS in citric acid concentration adjusted to pH 4.5 or 4.0 or below prior to further processing and/ or consumption.

| CON CLUS ION
Mycotoxins are very toxic and persistent in foods and feeds. They escape the rigors of many processes used in food, pharmaceutical, and nutraceutical industries. We developed widely applicable, costeffective, and safe method for mycotoxins decontamination, and successfully demonstrated that high-pressure acidified steaming (HPAS) can be a reliable method to solve the problem of mycotoxin exposure from dietary and pharmaceutical sources. The results of this study showed that the HPAS can completely detoxify mycotoxins, such as AT, AFB 1 , AFG 1 , OTA, and citrinin, or at least reduce them to safe levels. The study strongly recommends and encourages the application of the HPAS method at industrial scale in the food, pharmaceutical, nutraceutical, medical, and chemical industries where citric acid-containing pressurized steaming can be applied for the detoxification of mycotoxins.

ACK N OWLED G M ENTS
The authors are thankful to Kampala International University, Bushenyi, Uganda, and all the labs in both Uganda and Nigeria that provided/assisted with the research facilities used for this study.
The authors are also thankful to all the academic staff at Kampala International University who contributed to the success of this study, in one way or the other.

CO N FLI C T O F I NTER E S T S TATEM ENT
All authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Additional data for this study can be made available from the corresponding author upon request.

E TH I C A L A PPROVA L
The study does not involve any human or animal testing.