Patulin-producing mold, toxicological, biosynthesis, and molecular detection of patulin

Mycotoxin is one of the food safety problems that raise concern because of its negative health impact and significant economic losses. Patulin is the most common mycotoxin found in fruits and its products like apple, orange, grape, pear, etc. There are 167 species reported to produce patulin; Penicillium expansum is the major patulin producer among those fungi species. Patulin is not classifiable as to its carcinogenicity to humans, but some studies showed the toxicities of patulin. Patulin has a broad spectrum of toxicity such as acute toxicity, sub-acute toxicity, genotoxicity, embryotoxicity and teratogenicity, carcinogenicity, and immunotoxicity. There are several ways to detect mycotoxins, such as HPLC, TLC, ELISA, and PCR. The popular way to detect mycotoxin molecularly is PCR because of its sensitivity and specificity. To detect patulin molecularly require proper selection of target gene. Therefore, the biosynthesis of patulin, its influencing gene, and its underlying factor needs to be understood.


Introduction
Mycotoxins are secondary metabolites produced by fungi that cause adverse health impacts. Approximately 400 types of mycotoxin are identified, including mycotoxin produced by fungal genera such as Aspergillus, Penicillium, Alternaria, Fusarium, and Claviceps. Mycotoxinproducing fungi can be categorized into two groups (field fungi and storage fungi). Field fungi invade the crops before harvests, such as Cladosporium, Fusarium, and Alternaria spp. In comparison, storage fungi invade the crops after harvest, for instance, Aspergillus and Penicillium spp. Field fungi are categorized into three types: plant pathogens, fungi that grow on senescent or stressed plants, and fungi that colonize at the beginning before harvest and prompt the contamination after harvest (Tola and Kebede, 2016). Mold contaminants that have been found in tropical fruits (apple, avocado, banana, orange, and papaya) are Penicillium citrinum, Collectrotrichum sp., Collectotrichum gleosporioides, Aspergillus niger, Penicillium brevicompactum, Fusarium chlamydosporum, Aspergillus wentii, Mucor sp., and Aspergillus flavus (Rahayu et al., 2019). Several fungal genera produce mycotoxins like aflatoxin, ochratoxin A, fumonisin, zearalenone, trichothecenes, patulin, ergot alkaloids, and alternaria toxins (Marin et al., 2013). According to the type of extrolite, mycotoxin biosynthesis could be categorized as polyketide, terpenoid, and amino acid derivative. Polyketide pathway biosynthesis includes aflatoxin, ochratoxin, fumonisin, and patulin, while deoxynivalenol (DON) is classified as terpenoid pathway biosynthesis (Huffman et al., 2010;Puel et al., 2010).
Mycotoxin can be found in various crops, for instance, grains, nuts, corn, coffee, and fruits (Balendres et al., 2019). Mycotoxin that is mainly found in fruits and fruit-based products is patulin. Patulin (molecular formula C 7 H 6 O 4 ) is colorless, crystalline, and water-soluble (Pal et al., 2017). The chemical structure of patulin can be seen in Figure 1. Patulin contamination has been confirmed in fruits like apple, orange, grape, pear,

Figure 1.
Chemical structure of patulin (Ioi et al., 2017) The maximum level of patulin content for each product is 50 µg/kg (apple products), 25 µg/kg (non-liquid apple based-foods), and 10 µg/kg (apple-based infant foods) (European Commision, 2006). International Agency for Research on Cancer classifies patulin as a group 3 carcinogen, meaning it is not classifiable as to its carcinogenicity to humans (IARC, 1987). However, studies have found that long-term patulin exposure in rats and mice causes carcinogenic health effects (Pal et al., 2017). Furthermore, patulin is resistant to heat; therefore, heat treatment such as pasteurization and distillation is not adequate to remove it (Ioi et al., 2017). Due to its characteristics and adverse effects, the appropriate method is needed to detect patulin. The rapid detection of mycotoxigenic fungi is increasingly popular because classical culture-based methods are time-consuming, has poor reliability, and difficult to standardize (Munaut et al., 2011). Besides, rapid detection is needed for limiting occurrence and consumer exposure to patulin. Rapid detection of mycotoxigenic fungi can be done through conventional PCR, real-time PCR, and PCR-DGGE. This review describes the patulin-producing mold, toxicological properties of patulin, patulin biosynthesis, and molecular tools to detect mycotoxigenic fungi.

Patulin : Characteristics and Mold-Producing
In the 1940s, patulin was discovered as an antibiotic agent against Gram-positive, Gramnegative bacteria, and fungi. In addition, patulin was suggested to treat common cold because of its antiviral activity. However, clinical studies found that patulin has a negative health impact; therefore, patulin is excluded from antibiotics and categorized as mycotoxin. Because of its toxicity, the regulator limits patulin content in foods (Frisvad, 2018;Moake et al., 2005). Patulin is difficult to remove; several processing steps have an impact on patulin reduction such as clarification/filtration, heat treatment, and fermentation (Ioi et al., 2017). The medium in clarification/filtration techniques are varied, for instance, gelatine, bentonite, and activated carbon/charcoal (Acar et al., 1998;Bissessur et al., 2001;Gökmen et al., 2001). Patulin is resistant to heat; hence the thermal treatment to reduce patulin content is still questioned (Ioi et al., 2017). Pasteurization of 20 min at 90 o C and 100 o C result in 18,81% and 25,99% of patulin reduction (Kadakal and Nas, 2003). Moreover, distillation could reduce patulin by 24% in apple juice (Kubacki, 1986). The fermentation process affects patulin degradation, up to 90% of patulin content is reduced by yeast fermentation (Burroughs, 1977).
The reduction technique of patulin can be divided into 3 ways: biological control agents, chemical additives, and physical treatments. Biological control is a method of detoxifying or adsorption of patulin using microorganisms without changing the characteristics or chemical properties. Examples of biological control agents are lactic acid bacteria, yeasts, and fungi (Ioi et al., 2017). Lactic acid bacteria have been shown to reduce patulin 47 to 80% (Hatab et al., 2012). Chemical additives that have been proven to reduce patulin are ascorbic acid, ammonia, potassium permanganate, sulfur dioxide, ozone, and vitamin B (Ioi et al., 2017). Examples of physical treatments to decrease the patulin content are ultraviolet radiation, pulsed light, and high hydrostatic pressure (Funes et al., 2013;Hao et al., 2016;Tikekar et al., 2014).
Processing steps and reduction techniques were not able to eliminate patulin content. Therefore, the prevention of the presence of patulin-producing mold is better than reduction treatment of patulin. Patulin is secondary metabolism produced by several Penicillium, Aspergillus species, and other fungi genera. The ecological roles of secondary metabolites are virulence determinants, defense compounds, and communication molecules (Macheleidt et al., 2016). For example, patulin acts as an aggressiveness factor during apple infection (Snini et al., 2016). There are 167 species reported to produce patulin; however, not all species were correctly identified in terms of mold strain and mycotoxin produced. As many as 29 species were confirmed to produce patulin consisting of 3 species of Aspergillus ( (Frisvad, 2018). This review will list the patulin-producing mold and the method to confirm its ability to produce patulin. The list of patulin-producing mold can be seen in Table 1.

Toxicological Properties of Patulin
Patulin toxicological effects can be divided into acute toxicity, sub-acute toxicity, genotoxicity, embryotoxicity and teratogenicity, carcinogenicity, and immunotoxicity (Puel et al., 2010). Acute toxicity of patulin leads to gastrointestinal symptoms such as nausea, vomiting, ulcers, intestinal bleedings, and duodenal lesions (Saleh and Goktepe, 2019). Subacute toxicity of patulin in rats includes weight loss, gastric alteration, intestinal modification, impaired renal function, gastrointestinal disturbances, and inhibition of several enzymes (Puel et al., 2010). The oral LD 50 of patulin for mice and rats ranges from 20 to 100 mg per kg BW depends on characteristics of exposure and the route of ingestion. The oral route is less toxic than intravenous and intraperitoneal routes (Pal et al., 2017). The toxicity of patulin, dose, and its exposure duration can be seen in Table 2.
A study of the patulin genotoxicity mechanism was conducted in V79 cells (Chinese hamster fibroblasts), which showed that patulin treatment causes increased micronuclei and nucleoplasm bridge formation in glutathione depleted cells. Another research has reported that patulin generates irreversible DNA-DNA crosslinks. Patulin has a strong affinity to form a covalent bond with sulfhydryl, amino, thiol, and NH 2 groups. Glutathione serves as a patulin scavenger, the compound against patulin as a cellular defense; up to three glutathione molecules can bind one molecule of patulin. Furthermore, glutathione protects cells against cross-linking substances with the DNA, increased metabolism, and free radical detoxification. The cross-linked sister chromatids did not separate during mitosis forming an anaphase bridge that later converted into a nucleoplasm bridge. The development of micronuclei is the result of mitotic disturbances. The formation of micronuclei and nucleoplasm bridges leads to chromosomal damage, causing cell cycle delays (Glaser and Stopper, 2012;Ioi et al., 2017;Pal et al., 2017). A study on the embryotoxicity of patulin was conducted in NMRI mice; patulin was injected intraperitoneally or given orally on day 12 and 13 of pregnancy. Patulin dose >3,75 mg/kg increased the cleft palates rate (Matthiaschk and Korte, 1986). The teratogenicity effect of patulin was examined in mice, which showed that daily administration of 1,5 mg/kg/day of patulin on days 6-17 pregnancy decreased the average body weight of the fetus (Reddy et al., 1978). Another research showed that patulin also had a teratogenic effect on chicken embryos (Ciegler et al., 1976).
Although patulin is not classifiable as a carcinogen to humans, some studies showed that patulin is highly toxic to kidneys, liver, gastrointestinal tract, organs of the immune system, and endocrine glands (Ramalingam et al., 2019). Patulin causes apoptosis via oxidative stress and induces apoptosis by decreasing superoxide dismutase (SOD), catalase, GSH, and ROS-mediated mitochondrial dysfunction (Wei et al., 2020).
The effect of patulin on renal damage was studied on human embryonic kidney cells (HEK293). The result showed that patulin caused apoptosis on HEK293 cells as well as induced ROS and MDA accumulation. MDA is the primary marker of lipid peroxidation in tissue. Patulin decreases the activities of SOD, GSH, and CAT. SOD, GSH, and CAT are enzymes required in the antioxidant defense system. The toxicology mechanism of patulin is due to the electrophilic activity that binds to sulfhydryl groups in macromolecules. The exposure of patulin on HEK293 cells causes the depletion of glutathione. The glutathione homeostasis disruption is related to oxidative stress. The increase of ROS and MDA along with the loss in activities of SOD, GSH, and CAT will induce oxidative stress. The concentration of patulin increases the number of apoptotic cells .
Immunotoxicity of patulin was shown to increase neutrophil numbers, raise numbers of splenic T lymphocytes, depress delayed hypersensitivity, and decrease serum immunoglobulin concentrations (Bondy and Pestka, 2005). The immunotoxin effect of patulin was also shown in mice and rabbit. The result was reduced concentrations of circulating immunoglobulins in mice and decreased serum immunoglobulins, reduced blast genesis, and reduced chemiluminescence of peritoneal leukocytes in the rabbit. Patulin obstructed DNA synthesis in peripheral lymphocytes, which could be mitigated by cysteine, showing that sulfhydryl binding affects patulin-induced toxicity (Sharma, 1993). Patulin exposure decreased the expression of IL-23, IL-10, and TGF-β in bovine macrophages. Additionally, the exposure of patulin was also reported to reduce the expression of IL-4, IL-3, IFN-gamma, IL-10, and intracellular GSH depletion in human peripheral blood mononuclear cells (Pal et al., 2017).

Biosynthesis of Patulin
Mycotoxin biosynthesis is influenced by a pathway-specific transcription factor and a global transcriptional factor. Pathway-specific transcription factor that regulates genes within the cluster in patulin biosynthesis is the C6 transcription factor encoded by PatL. The global transcriptional factor in patulin biosynthesis is the velvet complex involved in responding to environmental signals such as pH, nutrition, light, temperature, and stresses (Li et al., 2019;Alkhayyat and Yu, 2014).
The scheme of patulin biosynthesis can be seen in Figure 2. The first stage is the condensation of one acetyl-CoA and three malonyl-CoA to form 6-methylsalicylic acid (6MSAS) by PatK. 6MSAS is converted to mcresol by 6MSA decarboxylase. The methyl group of m-cresol forms an aldehyde group by oxidation. The next step is the formation of gentisaldehyde by hydroxylation reaction. The intermediate stage was not clearly identified; the conversion of gentisaldehyde into patulin needs the opening of a ring by a mechanism mediated by monooxygenase or dioxygenase. The precursors of patulin were identified as isoepoxydon, phyllostine, neopatulin, and ascladiol. The patulin biosynthesis scheme renewed, as shown in Figure 3, is divided into ten steps involving 15 genes (PatA-PatO). The 15 genes include one putative transcription factor, three transporters, and 11 biosynthetic enzymes. The first step of patulin biosynthesis is the formation of 6MSAS from the condensation of one unit of acetyl-CoA and three units of malonyl-CoA carried out by PatK. The second step is the decarboxylation of 6MSAS to m-cresol by PatG. The next step is converting m-cresol to m-hydroxybenzyl alcohol and the formation of gentisyl alcohol from m-hydroxybenzyl alcohol catalyzed by PatH and PatI. This step is followed by the hydroxylation reaction, which causes the formation of gentisaldehyde. The conversion from gentisaldehyde to patulin requires opening the ring due to a mechanism mediated by monooxygenase or dioxygenase. The enzyme involved in this step remains unknown. The sixth step is the conversion of gentisaldehyde to isoepoxydone by both PatJ and PatO, followed by the conversion of isoepoxydone to phyllostine by PatN. The eighth stage is the conversion of phyllostine to neopatulin by PatF. Neopatulin converted into ascladiol through reduction reaction by PatD. The last step is ascladiol converted to patulin by PatE.
Patulin biosynthetic gene cluster consists of 15 genes (PatA -PatO). Besides Penicillium expansum, Aspergillus clavatus is also known as patulin producer. However, there are differences in the order of gene clusters. In Penicillium expansum, the gene cluster is started with PatH and ended with PatK. While in Aspergillus clavatus, the first gene is PatA and the last gene is PatO (Tannous et al., 2014). There are eight genes (PatE-PatL) that are strictly necessary in patulin biosynthesis. The deletion of those genes will eliminate patulin production ability (Li et al., 2019). The description of genes is shown in Table  3  .
Scheme of patulin biosynthesis in the cell can be seen in Figure 4. Most of the proteins (PatB, PatD, PatF, PatG, PatK, and PatN) are located in the cytosol, which is a place to produce enzymes, substrates, and cofactors (Keller, 2015). PatH and PatI are catalyzed in two successive stages, the third and fourth stages located in the endoplasmic reticulum. PatE plays a role in the final stage of biosynthesis, the conversion of ascladiol to patulin, which is identified as a secreted protein.
The cytotoxicity of ascladiol is lower than patulin (Maidana et al., 2016). Patulin biosynthesis at the cell wall effectively prevents mold cell damage due to toxic secondary metabolites (Li et al., 2019).   (Brakhage, 2013) Secondary metabolism is correlated with fungal development due to biotic and abiotic factors (El Hajj Assaf et al., 2018). The velvet family proteins regulate the secondary metabolism and the differentiation process in filamentous fungi, including VeA, VelB, VelC, and VosA. The deletion of VeA and VelB in Penicillium expansum was reported to obstruct the production of patulin completely. Additionally, the deletion of VelC in Penicillium expansum results in a decrease of <50% patulin production. However, the deletion of VosA did not affect the production of patulin. Therefore, it can be concluded that velvet family proteins (VeA, VelB, and VelC) except VosA regulate patulin biosynthesis (Li et al., 2019).

Factors Affecting Patulin Biosynthesis
The environment also affects the biosynthesis of patulin through transcription factor action. Environmental factors such as carbon source, nitrogen source, temperature, light, pH, and a w are essential for the biosynthesis of secondary metabolites. The correlation of environmental factors and regulatory protein can be seen in Figure  5. The effect of each factor on patulin biosynthesis described below: Carbon Patulin production is influenced by C source. P. expansum showed better mycelial growth and sporulation in media that contains C sources of maltose, glucose, fructose, mannose, sucrose, and starch than lactose, A-pectin, C-pection, cellulose, and maleic acid. Patulin production is increased in media containing C source from maltose, sucrose, and glucose. Contrary, there was no patulin detected in media with malic acid or cellulose . The regulatory protein responsible for processing the physiological signal for carbon in filamentous fungi is catabolite-responsive element A (CreA). Additionally, CreA influences the virulence in some fungi and its expression induced by sucrose in P. expansum (Alkhayyat and Yu, 2014;Li et al., 2020;). Nitrogen N source has a more substantial impact on patulin production and sporulation than the mycelial expansion of P. expansum. Beef extract and peptone stimulate colony expansion of P. expansum better than L-Asparagine, glycine, arginine, glutamic acid, urea, and yeast extract. Among inorganic N sources, media with sodium nitrite and calcium nitrate have higher mycelial growth, sporulation, and patulin production than ammonium sulphate. Peptone, glutamic acid, and yeast extract are the best patulin production enhancer. Generally, media with organic N sources is more favorable than inorganic N sources for patulin production . The regulatory protein responsible for processing physiological signals for nitrogen in filamentous fungi is AreA (Alkhayyat and Yu, 2014).

Temperature
The optimal temperature for P. expansum growth is 25 o C because it has the shortest lag phase and larger biomass compared to 4 o C, 8 o C, 16 o C, and 30 o C. However, the highest patulin concentration was found at 16 o C. The increase in temperature to 25 o C and 30 o C decreased patulin production. These results showed that suboptimal temperature is favourable to patulin production .

Light
The presence of light results in P. expansum unable to penetrate the apples' wall or emerge from the fruit to complete its cycle. The light disturbed the formation of coremia and synnemata. Velvet complex (VelB, VeA, and LaeA) is responsible for the light response. In the presence of light, the expression of VeA is low and located in the cytoplasm. But, VeA will be transported to the nucleus in the absence of light by KapA. VeA interacts with VelB and forms a bridge with LaeA in the nucleus. LaeA is the master regulator of secondary metabolism. This phenomenon described the connection of light signals with secondary metabolite biosynthesis (El Hajj Assaf et al., 2018;Alkhayyat and Yu, 2014).
VeA contributes to the pathogenicity and secondary metabolism production of P. expansum. VeA plays a role in several cellular processes such as morphogenesis, oxidative stress response, lightdependent control of sexual development, asexual development, and secondary metabolism. The growth of the null mutant VeA strain was decreased by as much as 12%. It also has some abnormalities macroscopically and microscopically, such as the absence of coremia, biverticillated conidiophores with inflated metulae and larger conidia. The null mutant VeA strain has a rot volume of infected apples 50% smaller than the wild type and complemented strain. Moreover, no patulin was detected in the apples infected with the null mutant VeA strain (El Hajj Assaf et al., 2018). pH The optimal pH value for patulin production depends on the mold strain ranging from 3-5. If the pH value is higher than 7, the accumulation of patulin in the medium will be reduced .
This result is also in line with Tannous et al., 2016, reporting no significant differences in the growth of P. expansum on three different pH values (2.5, 4, and 7). However, the highest patulin concentration was found at pH 4. The lowest patulin was detected at pH 2.5 and the patulin content was also decreased when the pH of the medium increased from 4 to 7. The initial pH 7 in the medium decreased during the 7-day incubation to pH 6 because of organic acid production. This ability to change the ambient pH is to develop a favourable environment for fungi development. PacC is the regulatory protein responsible for processing the physiological signal for pH in filamentous fungi. It is responsible for pH adaptation and is required for mycelial growth, conidiation, and virulence of P. expansum. The alkali condition resulted in proteolysis of PacC (Alkhayyat and Yu, 2014;Li et al., 2020). a w P. expansum showed optimum growth at a w of 0.99 because it has the shortest lag phase and highest growth rate. Lowering the a w from 0. 99 to 0.85 results in a decrease in P. expansum growth rate. Patulin production is also influenced by water activity in the medium. The a w of 0.99 significantly increases patulin production. On the other hand, there is no patulin detected at a w of 0,85 .

Molecular Tools Used for Detection of Mycotoxigenic Mold
Polymerase chain reaction (PCR) is the most popular molecular method to detect mycotoxigenic fungi because of its high sensitivity, specificity, and rapidity. PCR process is the repetitive cycles (25-40) of denaturation, annealing, and extension that generate billions of DNA copies. PCR assays require four materials such as template DNA, primers, nucleotide, and DNA polymerase. The strategy to develop a PCR assay to detect mycotoxigenic could be divided into several processes: primer design, primer alignment, first PCR program, PCR optimization, primer specificity, the applicability of the PCR, DNA extraction, and PCR application (Konietzny and Greiner, 2003).
Several approaches to detect mycotoxigenic fungi have been reported previously, such as universal primer based, mycotoxin biosynthetic primer based, and species-specific primer based (El Sheikha, 2019). The explanation of each approach is described below:

Universal primer based
Universal primer is based on the conserved DNA region on the microorganism. On the fungal, the conserved regions are small subunit / SSU (18S), internal transcribed spacer / ITS (5,8S), and large subunit / LSU (28S) ribosomal DNA. ITS region is the most common target gene to identified fungal species. The ITS region is divided into 2 parts: ITS1, located between 18S and 5,8S, and ITS2, located between 5,8S and 28S. The location of SSU, ITS, and LSU is shown in Figure 6 (Raja et al., 2017). The ribosomal gene cluster is repeated several times in individual strains, with the repeated modules linked by an intergenic spacer (IGS). Since their high variability, ITS1, ITS2, and IGS act as markers for discrimination between taxa (Richard et al., 2008). However, ITS and IGS regions are not directly responsible for mycotoxin production, so they could not be used as a differentiation tool between toxic and nontoxic producers (Konietzny and Greiner, 2003).

Mycotoxin biosynthetic gene primer based
The detection of patulin-producing mold is widely known using isoepoxydon dehydrogenase (idh) gene. The first idh gene was sequenced from Penicillium urticae, which was renamed Penicillium griseofulvum (Fedeshko, 1992). Nevertheless, some of the non-patulin-producing mold also create the same sized PCR product. Therefore, other alternatives to the biosynthetic gene to detect patulin such as 6-methyl-salicylic acid synthase (6MSAS) and PatF. 6MSAS is the gene involved in the first step of patulin biosynthesis, while PatF plays a role in the eighth step to convert phyllostine to neopatulin. PCR assay with PatF gene showed that this gene is a specific patulin biosynthetic gene of P. expansum (Mostafa et al., 2012;Tannous et al., 2015).

Species-specific primer based
The species-specific primer was designed to detect mycotoxigenic fungi and ensure that the PCR product is restricted to those species. It was created using a gene that responsible for mycotoxin biosynthesis. In this case, the responsible gene for patulin biosynthesis is idh gene. Several species of Penicillium are the most common patulin-producing fungi. The development of species-specific primer is to identify the presence of specific Penicillium species that can produce patulin. The list of primers is shown in Table 4. The species-specific primers approach can be used to detect the presence of Penicillium species and their ability to produce patulin in apples or their product rapidly and specifically (Dombrink-Kurtzman and McGovern, 2016).  (Rodríguez et al., 2012) The method to detect mycotoxigenic fungi is divided into conventional PCR, real-time PCR, and PCR DGGE. The list of tools and primers to detect patulin can be seen in Table 5. The most critical factor in PCR-based detection is primer design which should meet the criteria like GC content (40-60%), Tm values (50-80ºC), 5'-end stability, 3'-end specificity, and minimum interaction of primer-primer and hairpins. Detection of PCR amplification on conventional PCR uses agarose gels at the final stage of the PCR reaction (Ur Rahman et al., 2020). Real-time PCR allows the detection of PCR amplification during the early stage of the reaction and detects and quantifies DNA of toxigenic mold in food product. Real-time PCR is used not only for quantification of fungal contamination but also for estimation of mycotoxin level. Some researchers have done the detections of patulin and its producing mold with real-time PCR. Their method showed that it could quantify patulin producers from idh gene and also found a positive correlation between P. expansum DNA content and patulin concentration Tannous et al., 2015).
Based on the amount of primer being used in PCR is classified into simplex, duplex, and multiplex PCR. Simplex PCR uses a single primer to detect the microbial target, while duplex PCR applies two primers. Multiplex PCR uses multiple primers in PCR mixture to generate amplicons of varying sizes for different DNA sequences. The application of multiplex PCR to detect mycotoxigenic fungi combines several biosynthesis genes from some mycotoxin. For instance, mycotoxins such as aflatoxin, ochratoxin, and patulin, have been found in various foodstuffs like nuts, fruits, spices, and dry-cured meat products (Rodríguez et al., 2012).
PCR-DGGE is an efficient molecular tool that allows the identification of microbes in environmental samples. The steps of PCR-DGGE are a collection of environmental samples, extraction of total DNAs, specific amplification of microbial DNA (16, 26, 28S rDNA) by using universal primers, and separation of microbial DNA by acrylamide gel. The examples of PCR-DGGGE application to detect toxigenic mold in food have been conducted in grapes, shea tree fruits, coffee, cheese, and fermented tea (El Sheikha, 2019).

Conclusion
Patulin is a mycotoxin mainly found in fruits produced by molds diverse from Aspergillus, Byssochlamys, Emericella, Paecilomyces, and Penicillium genus. The toxicological effects of patulin differ from acute to chronic effects. Patulin is difficult to remove; therefore, the reduction technique is only to minimize it. The best way is to prevent the growth of patulinproducing mold. Patulin biosynthesis is influenced by a pathway-specific transcription factor and global transcriptional factor. The favourable conditions for patulin production are acidic, glucose-containing sugar complex N sources, dark, suboptimal temperature, and a high value of a w . There are some approaches and methods to detect mycotoxigenic fungi. Several target genes are reported to detect patulin-producing mold, but the most widely used is idh gene. However, the development of another target gene is needed because idh gene is also detected in non-patulinproducing molds.