Use of scanning electron microscopy and high-performance liquid chromatography to assess the ability of microorganisms to bind aflatoxin M₁ in Minas Frescal cheese

Abstract

The aim of this study was to evaluate the capacity of two strains of lactic acid bacteria (LAB), Lactobacillus rhamnosus and Lactococcus lactis, and a yeast strain, Saccharomyces cerevisiae, inactivated by heat (121 °C, 10 min), from binding to aflatoxin M₁ (AFM₁), as well as the interaction between these microorganisms, aflatoxin M₁ and the Minas Frescal cheese matrix after 2 and 30 days of storage. The ability of LABs and S. cerevisiae to bind AFM₁ to Minas Frescal cheese was evaluated by high performance liquid chromatography (HPLC) composed of a fluorescence detector. The interaction between these microorganisms and AFM₁ was evaluated using a scanning electron microscope composed of a backscattered electron detector with a voltage of 15 kV and magnifications of 1000 ×, 5000 × and 8000 ×. The use of microorganisms as a biological method is efficient in reducing AFM₁ in Minas Frescal cheese and does not affect the microbiological parameters. AFM₁ reduction varied according to the microorganism used in the treatments. S cerevisiae showed greater capacity to bind AFM₁ over time, compared to LABs. Scanning electron microscopy was especially useful, confirming that lactic acid bacteria and S. cerevisae were able to bind AFM₁ particles in Minas Frescal cheese.

Keywords:
S. cerevisiae; lactic acid bacteria; mycotoxins; dairy; aflatoxins

1 Introduction

Aflatoxins are secondary metabolites produced by toxigenic fungal species belonging to the Aspergillus genus, mainly A. flavus, A. parasiticus e A. nomius. Aflatoxin B₁ (AFB₁) is toxic, carcinogenic, teratogenic and/or mutagenic easily found as a contaminant in animal diet. Feeding dairy cows with fodder or any contaminated ingredient of the animal diet with AFB₁ results in the metabolic conversion of AFB₁ to aflatoxin M₁ (AFM₁), which is excreted in milk, in addition, it will be presented in the final product due to heat treatment resistance (Öztürk Yilmaz & Altinci, 2019; Hajmohammadi et al., 2020; Min et al., 2020; Park et al., 2020).

Milk and dairy products are frequently consumed by a portion of the population considered vulnerable, children and the elderly (Park et al., 2019; Ahmadi, 2020). Minas Frescal cheese is a typical Brazilian product, it is one of the most highly consumed lactic products, showing wide national market acceptance. This is a fresh soft white cheese, slightly salted, with a slight lactic acid taste. It is produced by the enzymatic coagulation of pasteurized milk with rennet or other appropriate coagulating enzymes (Prezzi et al., 2020).

The negative impact of aflatoxins on health and economy has led to investigations of strategies to prevent their formation in food, as well as to eliminate, inactivate or reduce the availability of these toxins in contaminated products (Gonçalves et al., 2015a; Bodbodak et al., 2018; Cagri-Mehmetoglu, 2018; Assaf et al., 2019; Martey et al., 2020).

In this context, biological methods of reducing mycotoxins arose, which come from the action of microorganisms, such as bacteria, yeast and others. Biological availability reduction methods have been extensively studied as they are efficient, cost-effective, and in many cases, already used in food production such as lactic acid bacteria and S. cerevisiae (Assaf et al., 2019; Barukčić et al., 2018; Cagri-Mehmetoglu, 2018; Campagnollo et al., 2020; Corassin et al., 2013; Gonçalves et al., 2015b, 2020; Ma et al., 2017).

Scanning electron microscopy is a versatile and useful technique, with a wide variety of commercial, industrial and research applications. It involves a finely collimated electron beam that scans the surface of the sample being analyzed. The beam is focused on a small probe that scans the surface of the sample. The interactions of the beam with the material result in the emission of electrons and photons when electrons penetrate the surface, and emitted particles are collected with the appropriate detector to provide surface information. The end product of the electron beam collision with the sample surface is an image (Lee et al., 2019).

In the present study, scanning electron microscopy was used to characterize the surface of biological additives and aflatoxin M₁ separately and together, depending on the treatment, in order to observe surface changes when aflatoxin is adsorbed. Additionally, the microstructure of cheese samples was evaluated.

2 Materials and methods

2.1 Material

Whole milk, probiotic culture composed of Lactobacillus rhamnosus (LRB, SACCO, Cadorago, Italy) and Lactococcus lactis (MWO 040, SACCO, Cadorago, Italy), a Saccharomyces cerevisiae strain (Fermentis K-97, SafAle, Belgium), liquid rennet (chymosin and bovine pepsin, Ha-La, Christian-Hansen®, Brazil) and Aflatoxin M₁ standard (Sigma Aldrich®), were used in this study.

2.2 Manufacture of Minas Frescal cheese

The Minas Frescal cheese were manufactured according to Fernandes et al. (2012). The cheese was produced at the Food Microbiology and Mycotoxicology Laboratory of the Faculty of Animal Science and Food Engineering at the University of São Paulo, Pirassununga campus, from milk produced on the same campus.

The cheese samples (16) were produced in duplicate for each of the following treatments: T1) negative control - cheese only; T2) positive control, cheese with addition of AFM₁ (0.5 µg/kg); T3) cheese with the addition of L. rhamnosus and L. lactis (10¹⁰ cells/g); T4) cheese with the addition of L. rhamnosus, L. lactis and AFM₁; T5) cheese with the addition of S. cerevisiae (concentration 10¹⁰ cells/g); T6) cheese with the addition of S. cerevisiae and AFM₁; T7) cheese with the addition of L. rhamnosus, L. lactis and S. cerevisiae; T8) cheese with the addition of L. rhamnosus, L. lactis, S. cerevisiae and AFM₁. In all treatments, the lactic acid bacteria, S. cerevisiae and AFM₁ were in the same concentration.

The LAB and S. cerevisiae ability to bind AFM₁, as well as the interaction between these microorganisms and aflatoxin M₁ in the Minas Frescal cheese were evaluated on days 2 and 30 after manufacture.

2.3 Sample preparation and determination of aflatoxin M₁

AFM₁ determination in cheese samples was performed as described by Jager et al. (2013). Two grams of NaCl, 22 mL of methanol and 13 mL of ultrapure water were added to a tube containing 8 g of the sample. The mixture was centrifuged at 2,078 x g for 15 min., and the obtained supernatant was filtered. 20 mL of the filtrate was collected in another flask, and 40 mL of ultrapure water was added. Purification was performed by passing the total volume (60 mL) through a manifold-coupled immunoaffinity column (Aflatest WB, Vicam®) at 2-3 drops/sec flow. After this procedure, the column was washed by passing 20 mL of ultrapure water. Subsequently, the toxin was eluted by passing 1 mL of HPLC grade methanol. Then, the eluent was evaporated to dryness under a stream of nitrogen at 40 °C and resuspended with 1 mL solution of methanol and ultrapure water (50:50 v/v).

Final extracts of cheese samples were injected (20 µL) into a Shimadzu 10VP liquid chromatograph (Kyoto, Japan), equipped with a 10 AXL fluorescence detector (excitation at 360 nm and emission above 440 nm). A Kinetex C₁₈ column (Phenomenex, Torrance, CA, USA) 4.6 × 150 mm, 2.6 μm particle size and an in-line filter of 0.5 μm were used. The isocratic mobile phase consisted of methanol/water/acetonitrile (61.4:28.1:10.5, v/v/v) with a flow rate of 0.50 mL/min. The equipment used in this study included vortex mixer (Fanem, São Paulo, Brazil); centrifuge (SOLAB SL-700, Piracicaba, Brazil); column manifold (Supelco, Bellefonte, Pennsylvania, USA) and vacuum pump (Model 131, type 2V, Primatec, Itu, Brazil).

Calibration curves to detect AFM₁ from cheese samples were prepared using standard solution of AFM₁ (Sigma®), diluted in acetonitrile at concentrations of 0.625, 1.25, 5, 10, 20 μg/L. Integrated peak areas were linearly correlated with the concentrations. The limits of detection (LOD) and quantification (LOQ) were calculated for each method of analysis based on signal:noise ratio of 3:1 and 10:1, respectively. Linearity was evaluated by verifying the coefficient of determination (r²) and visual inspection of residual plots of analytical curves. The performance of methods used for determination of AFM₁ in cheese samples in the laboratory has been presented elsewhere (Jager et al., 2013), describing limits of detection (LOD) and quantification (LOQ) values of 0.017 and 0.055 µg/kg, in cheese samples respectively.

2.4 Scanning electron microscopy assay

The cheese samples were prepared for scanning electron microscopy according to proposed by Lobato-Calleros et al. (2002) and Fritzen-Freire et al. (2010) with adaptations. Cylindrical samples of 0.5 cm diameter by 0.5 cm height were fixed in 2% buffer glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for 6 h. The samples were dehydrated in increasing concentrations of aqueous ethanol solutions (50%, 60%, 70%, 80%, 90% and 100%, 15 min for 3 times in each, totalizing 45 min) and placed in acetone for 1 h. After that, each sample was fractured perpendicular to its long axis and mounted on stubs with fractured face upwards. A scanning electron microscope (Hitachi, TM 3000, Japan) with a high sensibility semiconductor backscattered electron detector was used at 15 kV to observe each sample at a magnification of 1000×, 5000× and 8000×.

Image analysis

The analysis of the scanning electron microscopy images was performed without the use of a specific software, besides the SEM - Hitachi 3000 device itself, which was used only for image capture. The images were evaluated one by one, being selected according to the following criteria: (1) image sharpness and (2) magnification used, the magnification that presented the sharpest images in all treatments was sought. After choosing the best magnification, which was 5000×, the clearest images and the best images (day 2 and day 30) were screened for each treatment through a new screening.

2.5 Microbiological assay

Determination of Salmonella sp., Listeria monocytogenes, thermotolerant coliforms and coagulase positive staphylococci in Minas Frescal cheese were performed on days 2 and 30 after manufacture following the procedures described by American Public Health Association (2004).

3 Results and discussion

Table 1 shows the results of the microbiological analyzes performed on Minas Frescal cheese after 2 and 30 days of storage. Thermotolerant coliforms counts in the samples is in conformity with Brazilian legislation, and all confirmatory tests for Salmonella sp. and Listeria monocytogenes were negative, as were the coagulase tests to verify the positive coagulase staphylococci count (Brasil, 2001). Therefore, it can be said that the use of BAL and S. cerevisiae as a biological method to reduce AFM₁ in Minas Frescal cheese had no negative effect on the shelf-life of this cheese, which is generally 21 days. The shelf-life of Minas Frescal cheese is also related to the raw material used, the production process and the initial quantity of microorganism present (Prezzi et al., 2020).

Table 1
Microbiological analysis of Minas Frescal cheese produced with or without the addition of inactivated cells of lactic acid bacteria and yeast and aflatoxin M₁ after 2 and 30 days of storage.

Treatment	Probiotic culturea (cells/kg)	Yeastb (cells/kg)	AFM1 in curd (µg/kg)	Microbiological parameters during storagec
				Thermotolerant coliforms (NMP/g)		Salmonella sp.		Listeria monocytogenes		Coagulase positive staphylococci (CFU/g)
				Day 2	Day 30	Day 2	Day 30	Day 2	Day 30	Day 2	Day 30
T1	0	0	0	26	273	ND1	ND1	ND1	ND1	<102	<102
T3	2 x 1010	0	0	28	250	ND1	ND1	ND1	ND1	<102	<102
T5	0	1010	0	26	99	ND1	ND1	ND1	ND1	<102	<102
T7	2 x 1010	1010	0	33	270	ND1	ND1	ND1	ND1	<102	<102
T2	0	0	0.5	1	270	ND1	ND1	ND1	ND1	<102	<102
T4	2 x 1010	0	0.5	16	19	ND1	ND1	ND1	ND1	<102	<102
T6	0	1010	0.5	42	89	ND1	ND1	ND1	ND1	<102	<102
T8	2 x 1010	1010	0.5	0	160	ND1	ND1	ND1	ND1	<102	<102

• ^a
  Commercially available lyophilized product (SACCO Brazil) containing L. rhamnosus at 1.0 x 10¹⁰ cells/g + L. lactis at 1.0 x 10¹⁰ cells/g;
• ^b
  Commercially available brewer's biological dry yeast (Fermentis K-97, SafAle, Bruggeman, Belgium) containing 1.0 x 10¹⁰ yeast cells/g;
• ^c
  Values expressed as mean of samples analyzed in triplicate;
• ¹
  ND - Not detected.
• NMP – most likely number. CFU - colony forming unit.

The concentration of AFM₁ in cheese from treatments which AFM₁ was not included was below the detection limit of the analytical method (Table 2), while the concentration of AFM₁ in treatments that included AFM₁ ranged from 0.29 µg of AFM₁/kg until undetected.

Table 2
Concentrations and percentage reductions of aflatoxin M₁ in Minas Frescal cheeses manufactured with or without the addition of AFM₁ and heat-killed cells of lactic acid bacteria and yeast during 30 days of storage.

Treatment	Probiotic culturee (cells/kg)	Yeastf (cells/kg)	AFM1 in curd (µg/kg)	Aflatoxin M1 in cheese during storage
				Day 2		Day 30
				Concentration (µg/kg)g	% Reductionh	Concentration (µg/kg)g	% Reductionh
T1	0	0	0	<LOD	-	<LOD	-
T3	2x1010	0	0	<LOD	-	<LOD	-
T5	0	1010	0	<LOD	-	<LOD	-
T7	2x1010	1010	0	<LOD	-	<LOD	-
T2	0	0	0.5	0.29 ± 0.01	42aA	0.23 ± 0.03	54aB
T4	2x1010	0	0.5	0.09 ± 0.02	82bA	0.03 ± 0.01	94bB
T6	0	1010	0.5	0.13 ± 0.02	74cA	<LOD	100cC
T8	2x1010	1010	0.5	0.13 ± 0.01	74cA	<LOD	100cB

• ^a–dIn the same column, means followed by different letters differ significantly (P < 0.05); ^A–CIn the same line, means followed by different letters differ significantly (P < 0.05);
• ^e
  Commercially available lyophilized product (SACCO Brazil) containing L. rhamnosus at 1.0 x 10¹⁰ cells/g + L. lactis at 1.0 x 10¹⁰ cells/g;
• ^f
  Commercially available brewer's biological dry yeast (Fermentis K-97, SafAle, Bruggeman, Belgium) containing 1.0 x 10¹⁰ yeast cells/g;
• ^g
  Values expressed as mean ± standard deviation of samples analyzed in triplicate;
• ^h
  Cumulative percentages of reductions related to the concentration of aflatoxin M₁ added in cheese curd during processing. LOD: Limit of detection (0.017 μg/kg).

In the present study, the scanning electron microscopy assay was useful to support the results obtained through the analysis by HPLC, showing that there is a connection or interaction between the surfaces of LAB, yeast and aflatoxin M₁, separately and in associated treatment. In addition, it was possible to observe few changes in (1) LAB and S. cerevisiae when aflatoxin was attached and (2) in the microstructure of Minas Frescal cheese with aflatoxin and LAB and/or S. cerevisiae added versus cheese containing only these microorganisms.

Figures 1 to 3 show the micrographs of the different treatments applied to Minas Frescal cheese evaluated in this work, after days 2 and 30 of storage. Differences were observed in the microstructure of the cheese and in the morphology of the microorganisms used to reduce AFM₁.

Figure 1
Scanning electron microscopy images of treatments 3 and 4 on days 2 and 30 after manufactured.

The cheese microstructure is a spatial arrangement of casein micelles that join in groups and chains to form a viscoelastic protein network through which moisture, fat globules, miners and bacteria are dispersed (Aldalur et al., 2019).

Differences were observed in the microstructure of the cheese and in the structure of the lactic acid bacteria (Figure 1). Comparing the images of treatments 3 and 4 on days 2 and 30 (T3 and T3.1 and T4 and T4.1, respectively) it is possible to observe that the cheese matrix it has a more compact structure on day 2 of both treatments, and it is not possible to even visualize the lactic acid bacteria. After 30 days of production, the cheese network is less compacted, and it is possible to observe lactic acid bacteria in treatment 3 (T3.1) and in treatment 4 (T4.1), it is possible to observe AFM₁ (white dots) linked to lactic acid bacteria.

Fritzen-Freire et al. (2010) and Madadlou et al. (2007) reported that during cheese storage, structural changes may occur due to moisture loss and biochemical changes, as well as due to changes in cheese properties. In addition, another factor that can contribute to the formation or changes in the microstructure of the cheese is the use of lactic acid bacteria, due to their ability to improve the body and texture of the cheese (Centeno et al., 2002; Merrill et al., 1996). Also, Abdelmotilib et al. (2018) observed that strains of Lactobacillus plantarum, Lactobacillus acidophilus and Bifidobacterium bifidum when in contact with AFM_1, had spots on the cell wall after adsorption of the toxin.

The effects of the microorganisms used to reduce AFM₁ can be observed in the LAB treatment (T4), supported by the SEM images. Analyzes by HPLC showed that LAB has a high capacity to reduce AFM_1. On day 2, a reduction of 82% was observed (Table 2), followed by an increase in the percentage of reduction on day 30, reaching 94%.

In Figure 2 its possible comparing the images of treatments 5 and 6 (T5, T5.1, T6 and T6.1 on days 2 and 30, respectively). Differences were observed in the cheese matrix between days 2 and 30, both between T5, T5.1 and between T6 and T6.1, in both cases there was an increase in the pores of the network. Both in T6 and T6.1 it is possible to observe AFM₁ binding to the wall of S. cerevisiae, which, when the toxin is bound, changes its shape, becoming elongated or oval.

In agreement with our study, Hamad et al. (2017) reported that SEM analysis showed that the microorganism's surface is able to adsorb the toxin particles that appear as a small dot on the microorganism's cell walls. Abdelmotilib et al. (2018) reported in their study that non-viable strains of the yeasts Kluyveromyces lactis and S. cerevisiae showed changes in the cell wall after adsorption of AFM₁.

Supporting SEM images, HPLC analyzes showed S. cerevisiae, as a microorganism choice to reduce AFM₁, once it presented a lower percentage of reduction than LAB initially, 74%, however, after 30 days it was more efficient, reaching a percentage reduction of AFM₁ of 100%.

Treatments 7 and 8 (T7, T7.1, T8 and T8.1 on days 2 and 30, respectively) are in Figure 3. Differences in the cheese matrix between treatments without AFM₁ (T7 and T7.1) and with AFM₁ (T8 and T8.1) and in the same treatment were observed between days, day 2 (T7 and T8) and day 30 (T7.1 and T8.1). When comparing T7 and T7.1 to T8 and T8.1, it is possible to observe that the first presents a cheese matrix with a more compact structure than the other. According to Fox et al. (2017) a less compacted microstructure may be the result of demineralization (loss of calcium and phosphate in the casein micelle) that occurs due to the decrease in pH. A fact that may have occurred with Minas Frescal cheese over the course of thirty days, there may have been a decrease in pH due to the increase in lactic acid. Fritzen-Freire et al. (2010) reported that after 28 days, the Minas Frescal cheese with a higher lactic acid content showed a less compacted structure and less deformations.

Figure 2
Scanning electron microscopy images of treatments 5 and 6 on days 2 and 30 after manufactured.

Regarding AFM₁ binding to the yeast, when comparing T7 and T7.1 with T8 and T8.1 in Figure 3, it is possible to observe the stains on the cell wall of S. cerevisiae in T8 and T8.1. These spots indicate that AFM₁ is adsorbed to the yeast cell wall. In agreement with our study, Abdelmotilib et al. (2018) and Hamad et al. (2017) reported that the surface of the microorganism is able to adsorb AFM₁ particles that appear as small dots or spots on the cell wall of microorganisms. Supporting SEM images, the result obtained through HPLC showed that on day 2, the treatment that received LAB and S. cerevisiae combined showed the same percentages of reduction as the treatment that received only S. cerevisiae. On day 2, a reduction of AFM₁ of 74% was observed and on day 30, 100%.

Figure 3
Scanning electron microscopy images of treatments 7 and 8 on days 2 and 30 after manufactured.

4 Conclusions

The use of microorganisms as a biological method to reduce AFM₁ is efficient in Minas Frescal cheese and does not affect the microbiological parameters of the product. The reduction in AFM₁ varied according to the microorganism used in the treatments. Saccharomyces cerevisiae showed greater capacity to bind AFM₁ over time, compared to LABs. Scanning electron microscopy was especially useful, confirming that both lactic acid bacteria and S. cerevisiae were able to bind AFM₁ particles in Minas Frescal cheese. In addition, it allowed visualizing the changes that occurred in the cheese protein matrix over time and according to the different treatments.

Acknowledgements

The authors would like to thanks: São Paulo Research of Foundation, FAPESP [Grants #2017/20081-6 and #2017/19683-1], Brazilian National Council for Scientific and Technological Development, CNPq [Grant #302195/2018-1], and this study was financed in part by the Coordenação de Aperfeiçoamento Pessoal de Nível Superior - Brasil (CAPES) – Finance Code 001.

• Practical Application: One of the main objectives of the food industry is to avoid contamination by mycotoxins in processes and products. Methods that can verify the effectiveness of decontamination of aflatoxin M₁ by microbial action are extremely important.

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Publication Dates

• Publication in this collection
  01 Feb 2021
• Date of issue
  2022

History

• Received
  07 Sept 2020
• Accepted
  15 Sept 2020