Identification of Dairy Fungal Contamination and Reduction of Aflatoxin M1 Amount by Three Acid and Bile Resistant Probiotic Bacteria

Introduction

Factors of interest in fungal contamination of dairy products include the ability to grow at low temperatures, fermentation of sucrose and lactose, production of hydrolyzing enzymes for fatty and protein hydrolysis of the product, production of lactic acid and citric acid in the product, and resistance against chemical holders ( Caroli et al., 2011 ). It is notable that probiotics play a paramount role in human health ( Elwood et al., 2007 ). Cow’ milk undergoes a variety of contaminants, which threaten human health ( Khaniki, 2007 ). Although accurate pasteurization, safe milk collection, and storage conditions alleviate the microbial contamination to a high level, they cannot alleviate toxic contaminants, particularly mycotoxins that need more process to be eliminated ( Girma et al., 2014 ).

Mycotoxin small compounds are produced by fungal agents as secondary metabolites that lead to serious disorders and even human death. Aflatoxins (AFs), mainly produced by Aspergillus flavus (A. flavus) and A. parasiticus, as superintendent agents, damage the liver (hepatitis, edema, and hemorrhagic necrosis) or lead to liver, lung, and kidney cancers, as well as immunosuppression ( Lizárraga-Paulín et al., 2011 ).

The four major AFs categories include B₁ (AFB1), B₂, G₁, and G₂. AFB1-containing nutrients by the cow culminate in the formation of the hydroxylated form, known as aflatoxin M1 (AFM₁), released from the cow’s milk within 12 h after the first consumption.

AFM₁ is regarded as a critical carcinogenic compound to humans ( Dhanasekaran et al., 2011 ). Several strategies have been proposed toward the reduction of the levels of this compound in dairy products, including seeding time and density, chemical control, choice of hybrids, extreme lowering of grass moisture and keeping temperature, proper plowing and fertigation, and application of biological routes.

However, dairy products contamination with AFM₁ toxin remains a highly considerable health problem ( Pena-Rodas et al., 2018 ), particularly among developing countries. Noticeably, the high incidence of AFM₁ contamination in cow milk at higher levels, compared to the maximum tolerance limit approved by the European Union is present in Iran ( Ghazani, 2009 ). Therefore, it is of critical importance to determine the AFM₁ levels in various milk products and accurate measures for its reduction.

Probiotics, considered as “live microorganisms”, exert paramount health advantages in favor of existing host when administered in adequate amounts” ( Hill et al., 2014 ). Of particular, dairy strains of Lactobacilli or lactic acid bacteria (LAB) have been introduced to reduce AFB1’s amount in food and dairy products, including yogurt. Accordingly, review studies have revealed the lack of sufficient document data regarding the vast and common application of probiotics as an approved and proper approach toward reducing AFs ( Adebo et al., 2017 ). Owing to the uncertainty in the findings of the studies in this regard, this study aimed to evaluate the milk and cheese contamination level and applicability of three resistance Lactobacilli on the reduction of AFM₁ levels, compared to Natamycin.

Material and Methods

Samples and Fungal Agents. The study protocol was approved by the Research Council of Shiraz University of Medical Sciences, Shiraz, Iran. In total, 85 samples of traditional milk (n=45) and cheese (n=40) were obtained from factories and open markets in Shiraz, Iras, from February to June 2018. All samples were transferred to the laboratory promptly (while shaking) for culture, and the remained were kept in the freezer for two months for further investigations.

Fungal contamination was investigated with phenotypic tests. Moreover, the PCR technique was used for the identification of yeasts and mold for molecular confirmation of strains belonging to one type and genotyping. Obtained sequences were analyzed at the online database of Gene Bank NCBI. In such cases, sequence identification of genes in the areas of S rRNA and ITS was performed by the primers ITS1 and ITS4, as well as NS1 and NS8, respectively. Therefore, molecular identification of yeasts was initiated by sequence detection of gene areas D1/D2 in IBC. Dairy samples were used for the purification and sequencing of isolated yeasts and mold colonies. To ensure, the samples were sent to IBC, and separated colonies were identified and registered in Gene Bank, NCBI.

AFs-levels Assessment. The existence of AFM₁ and natamycin was clarified using High-Performance Liquid Chromatography. Afterward, Aspergillus and Penicillium, as well as Saccharomyces and Yarrowia with ITS and D1/D2 gene sequences, respectively, were isolated following a predefined method. Subsequently, 50 LABs were purchased from Tak-Gene Company (Iran) and coded. Regarding the evaluation of the antifungal traits of the LABs, the samples were cultured in methicillin-resistant Staphylococcus aureus (MRSA) media. AFM₁ vials and natamycin powder were purchased from Farough Company, Iran. In the next stage, the milk and cheese samples were ordered in six groups as follows:

Group 1 (control 1): The traditional milk sample without fungi, AFM₁, or natamycin was kept in Sabouraud dextrose chloramphenicol agar at 22-25˚C for 5 days.

Group 2 (control 2): The traditional milk sample inoculated with fungi and AFM₁ toxin was kept in Sabouraud dextrose chloramphenicol agar at 22-25˚C for 5 days.

Group 3 (control 3): The traditional milk sample inoculated with Natamycin was kept in Sabouraud dextrose chloramphenicol agar at 22-25˚C for 5 days.

Group 4 (case 1): Traditional milk samples (n=85) purchased from the marketplaces of Shiraz, Iran, were kept in Sabouraud dextrose chloramphenicol agar at 22-25˚C for 5 days.

Group 5 (case 2): Infected milk samples inoculated with the selected resistant LABs (8×10³ CFU) with turbidity equal to 0.5 McFarland standard value.

Group 6 (case 3): Infected milk samples with natamycin (8×10³ CFU).

To assess the resistance of LABs to bile salts, 1%, 3%, and 5% bile salts were added to the MRSA media. Out of five bile-tolerant LABs containing antifungal features, five LABs were resistant to acid conditions and coded as TD1/2, T21/2, T23/2, TD11, and LAX152. Considering the comparison of the AFs-reducing ability of these three strains, 0.5 ppm of AF vial was inoculated into 1000 mL traditional yogurt and shaken completely by a shaker and left at 37˚C for 120 min. Afterward, each 10 mL was dropped into one tube, and the colonies were cultured in them. After incubation at 30˚C for 72 h, the samples were transported to a laboratory for AFM₁ level evaluation. The test was performed for all five LABs. Furthermore, the milk samples inoculated with 0.5 ppm or 0.2gr natamycin were tested for the level of AFM₁. In the final step, the AFM₁-reducing LABs strains were registered in the NCBI database: https://submit.ncbi.nlm.nih.gov/

Statistical Analysis. The data were analyzed in IBM SPSS software (version 21.0) (IBM Corp. 2012. Armonk, NY: IBM Corp.) The quantitative and qualitative variables were presented as mean±SD and frequency, respectively. Moreover, the groups were compared in terms of the mean level of AFM₁ using ANOVA, and the pairwise comparison was performed by Tukey's test. Categorical variables were also compared using the Chi-square test. A p-value less than 0.05 was considered statistically significant.

Results

This study evaluated 85 samples of dairy products, including cheese (n=40; 47.66%) and traditional milk samples (n=45; 94.52%). Table 1 tabulates the analysis results of the presence of aflatoxin in the samples taken. Of the total samples, 39 (9.45%) and 31 (36.4%) ones were desirable and acceptable, respectively. Moreover, the number of mold colonies and colonies was more than the acceptable limit and considered unacceptable in 15 cases (17.6%). Therefore, 17.6% of the samples had mold and yeast contamination. Furthermore, the results of the test showed no significant difference between the amount of mold and yeast infection (P=0.05).

The dairy product samples were collected from Shiraz (n=85), and among these isolates (i.e., Penicillium strains, Aspergillus niger, Saccharomyces cerevisia, Saccharomyces paradoxus, and Yarrowia lipolytica), the two molds, including Aspergilus Niger and Penicillium spp., had the ability of aflatoxin-bearing.

AFs-reducing traits of LABs

The results revealed that 28 strains could eliminate fungi in the media. Notably, only five out of 28 strains tolerated the bile salts, coded as TD1/2, T21/2, T23/2, TD11, and LAX152. In the final step, considering the assessment of the resistance of the five LABs to acidic conditions, they were kept in MRSA media in acidic pH for 120 min, the results of which revealed three strains with the property of resistance to acidic conditions (i.e., TD1/2, T23/2, and TD11). These three strains were selected as the final sample and referred for the molecular test of PCR with 16srRNA.

The results of analyzing the six groups showed that the group without LABs inoculated with AFM₁, and natamycin indicated no reduction in the level of AFM₁ (0.5 ppm). Among the five groups with five strains of LABs, the mean levels of AFM₁ in the groups inoculated with TD1/2, TD21/2, TD23/2, TD11, and LAX152 were about 0.05, 0.03, 0.03, 0.01, and 0.05, respectively.

The three LABs with resistance to the bile salts and acidic conditions and antifungal property included TD1/2, TD 11, and TD23/2 strains, for which the results of 16s rRNA sequencing are shown in Table 2.

Discussion

In this study, the Penicillium genus was the common dairy-contaminating genotype, followed by Aspergillus niger, Saccharomyces cerevisia, Saccharomyces paradoxus, and Yarrowia lipolytica. Out of 85 dairy product samples collected from Shiraz, the molds were isolated from Aspergillus niger and Penicillium strains. It should be mentioned that most of the isolates had the ability of aflatoxin-bearing.

Out of 50 LABs, only five exhibited antifungal activity and bile salt resistance traits. Inoculation of 0.5 ppm AFM₁ to them was associated with significant amelioration in mean AFM₁ level. These three LABs were sequenced and uncovered as L. fermentum, L. brevis, and E. faecium strains existed.

Several surveys have indicated high levels of AFs in dairy products ( Oveisi et al., 2007 ; Ghazani, 2009 ; Hashemi, 2016 ), necessitating higher attention to the AFs reducing approaches in dairy products. Notably, dairy products are produced and sold in two traditional and industrial forms in the country. Although industrial products are wider, some others prefer traditional dairy products ( Tajkarimi et al., 2008 ; Momtaz et al., 2012 ).

According to the significance of fungal infection and AFs produced for human health ( Prandini et al., 2009 ; Dhanasekaran et al., 2011 ), various studies have evaluated the presence of LABs in dairy products for reducing their fungal contamination. It was observed that 28 out of 50 LABs exerted antifungal activity and three of them were highly potent for reducing AFM₁. In a previously conducted study, 16/30 of LABs isolated from the curd tolerated bile salts and acids ( Prabhurajeshwar and Chandrakanth, 2017 ), highlighting our findings that not all LABs exert antifungal traits requiring prior evaluation. The LABs from a variety of dairy products have shown the ability to reduce AFs ( Abdelmotilib et al. ; Hernandez-Mendoza et al., 2010 ; Elsanhoty et al., 2014 ).

Sadeghi et al. (2016) isolated L. acidophilus and L. brevis from traditional sourdough and examined inhibitory growth on A. flavus and reduction of AFB1. The results showed a significant reduction in the AFB1 level; accordingly, these LABs (especially non-viable cells) were supposed as proper bioremediation for dairy products. In another study, L. brevis was the most sensitive agent to reduce the AFM1 level in milk (Verma et al., 2013).

The findings of these studies (Verma et al., 2013; Sadeghi et al., 2016) highlight our results regarding the effect of L. brevis on AF levels. Fazeli et al. (2009) isolated various LABs from sourdough and found a significant reduction of AFB1 by L. casei, L. plantarum, and L. fermentum. The results of this study highlighted the inhibitory effect of L. fermentum on AFs levels. Moreover, Nazhand et al. (2017) studied 20 LABs from which two strains of E. faecium had the highest ability to eliminate Coumarin (similar to AFs). These findings are in line with the results of the present study on the satisfactory effects of E. faecium. Although all the above-mentioned studies highlighted our observations on the AFs reducing effect of these three LABs, the investigated LAB strains and AF types varied among studies, and the three strains in the present study have been introduced here for the first time.

Various agents determine the efficiency of LAB in AFs reduction and fungal development, such as the bacterial strain. Various bacteria employ different mechanisms for the removal of AFs, such as binding to the fungal membrane (for which the cell wall peptidoglycans and polysaccharides of the bacteria are important) and preventing the absorption of amino acids leading to dissimilar antifungal potencies for various LABs (Perczak et al., 2018).

The strains were evaluated in this study with antifungal property and resistance to bile salts and acids for their applicability in milk and cheese, and only three of the 50 LABs had all three properties. Furthermore, these three LABs had different potencies for reducing AFM1 levels; moreover, L. brevis and L. fermentum had the highest reduction of AFM1 levels.

However, the best incubation period and the temperature were supposed about 48 h at 25-30˚C (Dalié et al., 2010). A variety of incubation periods and temperatures have been used in this regard. Fazeli et al. (2009) incubated LABs in the existence of AFB1 at 37˚C for 72 h and observed a difference between the 72- and 24-h cultures in terms of AFB1 removal by the strains.

Noticeably, our samples were incubated at 22-25˚C for five days, and sufficient results were observed in this regard. Furthermore, different inoculum amount of treatment has been reported as sufficient bacterial population for the elimination of AFs in different studies (Abdelmotilib et al.; Hernandez-Mendoza et al., 2010). In a study conducted by Sadeghi et al. (2016), the required bacterial population for L. brevis was 2×10³ CFU. Fazeli et al. (2009) also reported 2×10³ CFU as a sufficient amount for the removal of AFs in L. casei, L. plantarum, and L. fermentum.

In the present study, regarding the 8×103 CFU, the mean values of 3×10³ and 5×10³ CFU were obtained for L. fermentum and E. faecium, respectively. Moreover, the results showed a sufficient inoculum dose of treatment for these strains. This survey could successfully isolate the LABs with antifungal traits and resistance to bile salts and acidic conditions, compared to natamycin.

Conclusion

It was observed that out of 50 LABs, 28 ones contained antifungal properties. Regarding their applicability in human body conditions, their resistance to bile salts and acids was tested, and the results outlined that only three LABs had all the beneficial characteristics. The ability to reduce AFM₁, compared to natamycin, revealed that the LABs sequenced as L. brevis, L. fermentum, and E. faecium strains had the highest ability to reduce AFM₁. The details of the three strains were registered in the NCBI database. Accordingly, it is suggested to employ these three strains to the traditional studied milk and cheese samples, which had a high level of fungal contamination.

Authors' Contribution

Study concept and design: F. F. Sh.

Acquisition of data: F. F. Sh.

Analysis and interpretation of data: M. T. E. and M. B.

Drafting of the manuscript: F. F. Sh. and V. R.

Critical revision of the manuscript for important intellectual content: M. T. E. and M. B.

Statistical analysis: F. F. Sh. and J. H.

Administrative, technical, and material support: F. F. Sh. and M. T. E.

Ethics

All samples were collected using aseptic methods and in conformity with the ethical guidelines of the 1975 declaration of Helsinki.

Grant Support

This was self-funded study.

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