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Assessment of The In-Vitro Aflatoxin B1 Adsorption and Probiotic Capacity of Yeasts Isolated From Pacific White Shrimp (Litopenaeus Vannamei)
Assessment of The In-Vitro Aflatoxin B1 Adsorption and Probiotic Capacity of Yeasts Isolated From Pacific White Shrimp (Litopenaeus Vannamei)
Scientific Article
ABSTRACT
This study aimed to isolate and identify yeasts present in the intestinal microbiota of Pacific white shrimp (Litopenaeus
vannamei) cultivated in a tropical estuary and carry out in-vitro assessments regarding their probiotic and aflatoxin
B1 (AFB1) adsorption capacity of isolated Saccharomyces cerevisiae strains. The isolation and identification of
intestinal yeasts from 40 L. vannamei individuals were performed by molecular sequencing. Three S. cerevisiae
strains (C2B, C2D and C9) were chosen for probiotic potential assessments through homologous inhibition, self-
aggregation, co-aggregation, antibacterial activity, gastrointestinal condition viability, and AFB1 adsorption analyses.
The following species were identified: Candida spp., Candida tropicalis, Lodderomyces elongisporus, Rhodotorula spp.,
and S. cerevisiae. All isolated S. cerevisiae strains presented antibacterial activity, with the C9 strain displaying better
performance in the antimicrobial activity, pH viability, and AFB1 adsorption assays. It was, thus, possible to isolate
Candida spp., C. tropicalis, Rhodotorula spp. and S. cerevisiae from L. vannamei shrimp, and our study demonstrated for
the first time that L. elongisporus may be present in the gut of this shrimp species in captive conditions. Furthermore,
the isolated S. cerevisiae strains exhibited in-vitro probiotic and AFB1 adsorption potential.
Keywords: Candida spp.; Lodderomyces elongisporus; Rhodotorula spp., Saccharomyces cerevisiae.
remaining ethanol was dried for 20 minutes at 37 °C. The DNA were resuspended in 4 mL of the same PBS and subjected to
was then resuspended in 10 μL of sterile deionized q.s.p. water, homogenization employing a vortex mixer. The contents were
and the obtained product was dosed using a NanoDrop ND incubated under non-agitation conditions for 2 hours at 37 °C
1,000 spectrophotometer. using a 002CB temperature-controlled culture oven (Fanem
For sequencing, the samples were collected using the Data LTDA®). Subsequently, 2 mL was collected from the upper
Collection 3 program (Applied Biosystems), and the 5.8S ITS portion of the solutions for optical density (OD) determinations
rDNA sequences and sequence comparisons were analyzed using a Biospectro SP-220 spectrophotometer at 600 nm. Initial
using the BLAST software (Basic Local 562 Alignment Serch optical density was standardized to about 0.5. Auto-aggregation
Tool, BLAST 2.0 version 2.215). was expressed by Eq. 1:
by the Slab test method on YPD agar according to Strus YPD broth supplemented with 0.5% bovine bile (Sigma –
(1998). The pathogenic bacterial strains used in this assay were Aldrich®) was adjusted to pH 7 by adding 1 M sodium hydroxide
A. hydrophila INQS 00318 (IOC/FDA 110-36), E. coli INQS solution. At the end of the different incubation times cited
00033 (ATCC 25922), S. aureus INQS 00015 (ATCC 25923), before, 100 μL aliquots were taken for viable cell counting
and S. agalactiae INQS 00128 (ATCC 27853). The bacterial by decimal dilution and seeding by spreading on YPD agar
suspensions were standardized at 600 nm to an optical density surfaces. The plates were then incubated for 24 hours at 37 °C,
of 0.7. The bacteria were then seeded on nutrient agar plates and the assays were performed in duplicate. Controls of the pH
incubated at 37 °C for 48 hours and swabbed onto the surface of tolerance test were performed by submitting each strain to the
150 × 15 mm Petri dishes containing Mueller-Hinton agar. same procedure already described, but with inoculation in YPD
broth at pH 7 and without the addition of bovine bile.
After 48 h, the 14 mm diameter discs were removed from
the YPD agar containing the cultured yeasts under aseptic AFB1 adsorption assay
conditions and distributed over the surface of 150 × 15 mm
The AFB1 adsorption assay was performed according to Bueno
Petri dishes containing Mueller-Hinton agar previously seeded
et al. (2007) and Poloni et al. (2015), with some modifications.
with the test bacteria and incubated at 37 °C for 24 hours. The initial AFB1 solution used in the assay was resuspended in
Each treatment was performed in duplicate. The diameters of the acetonitrile from a dry extract of AFB1 core. Stock solutions of
growth inhibition halos around the agar plates were measured, AFB1 (25 and 50 ng·mL-1) were prepared in PBS (pH 2 and 7).
and the Pz value (a/b) was calculated, expressed as the ratio The yeasts used in the assay were previously prepared in YPD
between the diameter of the well (a) and the diameter of the broth, and their growth at 107 cells·mL-1 was standardized after
lowest well plus the diameter of the precipitation halo around 48 hours with the aid of a Neubauer chamber.
the proteolysis area (b). Subsequently, 1-mL aliquots of this solution were transferred
Antimicrobial activity was then classified into four categories: to microtubes and subjected to centrifugation for 15 minutes
• Pz 1,000 = no antimicrobial activity; at 5,000 rpm at room temperature. The samples were then
• Pz between 0.999 to 0.7000 = low antimicrobial activity; washed with distilled water, centrifuged again, and mixed with
• Pz between 0.699 to 0.400 = moderate activity; 1 mL of a PBS pH 2 solution containing AFB1, incubated at
• Pz between 0.399 to 0.100 = high antimicrobial activity 30 °C for 30 minutes and shaken manually. Following another
(Ramos et al., 2015). centrifugation step, the pellets were mixed with 1 mL of PBS
at pH 7 containing AFB1 at 25 and 50 ng·mL-1, incubated at
Tolerance to acidic pH and bile salts 30 °C for 60 minutes and subjected to manual agitation every
5 minutes. Cells were then pelleted by centrifugation for
Two assays were performed to determine the viability and
15 minutes at 5,000 rpm at room temperature, and the supernatants
tolerance of the isolated yeast strains, consisting of a simulation
containing unbound mycotoxins were collected and stored for
of intestinal L. vannamei conditions at pH 7 (Alexandre et al.,
high-performance liquid chromatography (HPLC) adsorption
2014) and a test employing variable pH values (2, 7, and 7 with
analyses. The positive (AFB1 only) and negative (PBS only)
the addition of bile salts).
controls were not included for testing. All experiments were
Suspensions of each of the isolated strains (C2B, C2D and
performed in duplicate.
C9) were prepared in peptone water to obtain 107 cells·mL-1,
A high efficiency liquid chromatograph (RF-10AXL-
according to Van der Aa Kuhle et al. (2005), with modifications.
SHIMADZU) was used for AFB1 detection and quantification,
A total of 100 μL of these solutions were mixed with 900 μL with excitation and emission wavelengths of λ 360 and 440 nm,
of YPD broth and adjusted to pH 2 with hydrochloric acid, respectively, equipped with a 20-μL injection volume loop
PA. The solutions containing the inoculums were submitted to (Trucksess et al., 1994) and C18 silica gel reverse phase column
constant agitation at 150 rpm on an SL-180/DT (Solab®) table (SHIM-PACK VP-ODS 150 × 4.6 mm column with 5 μm particle
shaker at room temperature of ± 30 °C. Culture aliquots (100 μL) size). Aliquots (100 μL) of each sample extract were then mixed
were collected at times 0 (control), 4, 8 and 12 h, and viable with 350 μL of a derivatizing solution composed of trifluoroacetic
cell counts were performed by decimal dilution and seeding by acid:glacial acetic acid:water (20:10:70, v/v). The mobile phase
spreading on YPD agar surfaces. The plates were then incubated comprised an isocratic acetonitrile:methanol:water (17:17:66, v/v)
for 24 hours at 37 °C, and the assays were performed in duplicate. system at a 1.5 mL·min-1 flow rate.
An aflatoxin analytical curve was employed measuring Among the total of 40 intestines of L. vannamei analyzed,
the areas, and its interpolation to a curve constructed with 15 yeast strains were obtained, being identified Candida spp.,
different concentrations of an AFB1 standard dissolved Candida tropicalis, Lodderomyces elongisporus, Rhodotorula
in acetonitrile, with the limit of detection established spp. and S. cerevisiae (Table 1).
as 0.4 ng·g-1 and the limit of quantification as 1.2 ng·g-1.
Table 1. Yeast species isolated from Pacific white shrimp
The adsorbed AFB 1 quantifications were established by the
(Litopenaeus vannamei) intestines.
correlation between the two peak areas of the samples and
the standard curve. Adsorption percentages were calculated Yeast species Strain Isolated Frequency (%)
as Eq. 3: Candida spp. C12B 1 6.7
C. tropicalis C2E 1 6.7
Adsorption % = (supernatant area/ C5, C6, C7,
toxin area of the positive control) × 100 (3) C10, C14A,
L. elongisporus 9 60.0
C14B, C15,
C16 e C17
Statistical analyses Rhodotorula sp. C12 A 1 6.7
The treatments were distributed in an entirely randomized C2B, C2D
design as a 3×4 factorial scheme (three yeast strains, four S. cerevisiae 3 20.0
e C9
viability times/four pathogenic bacteria strains) comprising
Total 15 100
two repetitions per treatment for the viability, coaggregation
and antimicrobial activity assays. Regarding the adsorption A statistically significant difference (P<0.05) was
test, a 3×2 factorial scheme was employed (three yeast strains, observed between S. cerevisiae strains regarding self-
two AFB1 concentrations), comprising two repetitions per aggregation capacity, with emphasis on strains C2B and C2D
treatment. Colony counts were transformed into log 10(x+1).
(Table 2).
The obtained data were submitted to an analysis of variance
Table 2. Self-aggregating capacity (%) of Saccharomyces
and a comparison of means by Tukey’s test at 5% significance
(p < 0.05) employing the SAS® University Edition software. cerevisiae strains isolated from the intestinal microbiota of
Pacific white shrimp (Litopenaeus vannamei) intestines*.
presenting high antimicrobial activity for all the tested The presence of pH 7 bile inhibited the multiplication of the
bacteria (Table 4). C2B strain, while the other ones exhibited similar performance
Table 5 summarizes the viability test results of the
S. cerevisiae strains isolated from the intestinal microbiota of to the strains incubated with the controls. All strains remained
L. vannamei shrimp under different pH and bile salt conditions. viable at pH 2 during the cultivation period.
Table 3. Bacterial self-aggregation result and self-aggregation capacity percentages of the Saccharomyces cerevisiae isolated from
the intestinal microbiota of Litopenaeus vannamei and the pathogenic bacteria strains tested herein*.
S cerevisae strains
Bacteria strains tested
C2B C2D C9
Aeromonas hydrophila 24.1 ± 4.5 bA
34.2 ± 2.2 aA
34.0 ± 0.1 Aa
Streptococcus agalactiae 35.8 ± 4.5 abA 40.2 ± 2.3 aA 32.1 ± 0.3 Aa
Staphylococcus aureus 50.7 ± 4.5 aA 40.8 ± 4.7 aA 37.2 ± 0.1 Aa
Escherichia coli 40.0 ± 3.6 abA 23.0 ± 2.8 aA 38.9 ± 0.3 Aa
Score +++ ++ ++
*Means followed by different lowercase letters in the same line and uppercase letters in the same column differ significantly from each other by Tukey’s test at a 1%
probability (P = 0.019). Data are expressed as means ± standard deviations.
Table 4. Antimicrobial activity (Pz Index) of the Saccharomyces cerevisiae strains isolated from the intestinal microbiota of
Litopenaeus vannamei shrimp against pathogenic bacteria strains*.
S. cerevisiae strains
Bacteria strains tested
C2B C2D C9
Aeromonas hydrophila 0.413 a
0.412 a
0.384a
Streptococcus agalactiae 0.368a 0.379a 0.373a
Staphylococcus aureus 0.417 a
0.413 a
0.373a
Escherichia coli 0.384a 0.384a 0.395a
*Means followed by different lowercase letters on the same line differ significantly from each other by Tukey’s test at a 1% probability (P = 0.667); interpretation of the Pz index
= 1,000: no antimicrobial activity; from 0.999 to 0.700: low antimicrobial activity; from 0.699 to 0.400: moderate activity; and from 0.399 to 0.100: high antimicrobial activity.
Table 5. Viability test results for Saccharomyces cerevisiae strains isolated from the intestinal microbiota of Litopenaeus vannamei
shrimp under different pH and bile salt conditions*.
The tested S. cerevisiae strains were able to adsorb both 25 Table 7 summarizes the results of other assessments also
and 50 ng·mL of AFB1 in similar ways, except for the C9 strain,
-1
employing S. cerevisiae strains from various sources in AFB1
which displayed no adsorption at 25 ng·mL (Fig. 2 and Table 6).
-1
adsorption assays at 25 and 50 ng·mL-1.
20,000
15,000
10,000
(a) 5,000
0
-5,000
20,000
15,000
10,000
(b) 5,000
0
-5,000
20,000
15,000
10,000
(c)
5,000
0
-5,000
20,000
15,000
10,000
(d)
5,000
0
-5,000
4,5 5 5,5 6 4 4,5 5 5,5 6
Retenction time Retenction time
Figure 2. Chromatogram presenting AFB1 adsorptions by Saccharomyces cerevisiae strains isolated from the intestinal microbiota of
Litopenaeus vannamei shrimp. (a) Aflatoxin B1 control; (b) C2B strain; (c) C2D strain; (d) C9 strain.
Table 6. AFB1 adsorption by three Saccharomyces cerevisiae strains isolated from the intestinal microbiota of Litopenaeus vannamei shrimp*.
Table 7. Aflatoxin B1 adsorption assays results employing Saccharomyces cerevisiae strains isolated from different environments
reported in other published studies.
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