Chapter 4: Investigating Acrylamide Mitigation by Potential Probiotics
4.2 Materials and Methods
LAB and Bifidobacterium were isolated from different food products such as dried fish and camel milk and characterized as potential probiotics (Abushelaibi et al., 2017;
Alkalbani et al., 2019; Ayyash et al., 2018). The pure isolated were stored in 50%
(vol/vol) glycerol at -80°C. All cultures were activated in de Man-Rogosa-Sharpe (MRS) broth (LAB-M, Neogen Culture Media, Heywood, UK) by taking a loopful from the isolated bacteria stocks and inoculated in MRS broth followed by incubation at 37°C for 20 h. Prior to any experiments, each culture was sub-cultured twice in MRS broth and incubated for 20 h at 37°C.
4.2.2 Preparation of Stock and Working Solutions of Acrylamide
A 50 mg of acrylamide AA (Sigma Chemical Co., St. Louis, MO) was dissolved in 50 ml deionized-distilled water to obtain a 1 mg/ml (1000 ppm) concentration as a stock
solution. Two working solutions were prepared by diluting the stock solution from 1 mg/ml to 50 µg/ml (50 ppm) in 10 ml of MRS broth in the screening stage and 10 µg/ml in optimization and mechanism of action experiments.
4.2.3 Screening for Acrylamide Mitigation
In 10 ml test tubes of sterilized MRS broth supplemented with 50 µg/ml acrylamide, 1%
of an aliquot of LAB and Bifidobacterium strains were added to the MRS broth,
individually. Acrylamide solution was added after autoclaving MRS broth. The inoculated tubes were incubated at 37°C for 20 h. The bacterial count was enumerated in MRS agar.
For AA analysis, the cultured MRS tubes were centrifuged at 10,000 x g for 10 min at 4°C. The supernatants were collected for AA analysis by LC-MS. The whole experiment
57 was duplicated for each strain (Hernandez-Mendoza et al., 2009b; Serrano‐Niño et al., 2014).
4.2.4 Box-Behnken Design (BBD)
After the screening step, the Box-Behnken design (BBD) was used to optimize the factors that affected the acrylamide removal, including incubation temperature (32°C, 37°C, and 42°C), media pH (4.5, 5.5 and 6.5), incubation time (14, 18 and 22 hours), and the NaCl concentration (0, 1.5 and 3 g/100 g). The Minitab v.21 software was employed to
construct the design and perform statistical analysis and the response surface method.
Twenty-seven experimental runs and three repetitive central points were applied. All 27 trials were carried out using the two selected isolates individually under aerobic and anaerobic conditions. Each experiment was conducted in triplicate, as presented in Table 8. The polynomial equation is described as follows:
Y = β0+ ∑ βiXi+ ∑ βiiXi2+ ∑ βijXiXj
Where Y is the predicted acrylamide removal, and Xi and Xj represent the independent factors, including temperature, pH, NaCl, and incubation time. βₒ is the regression coefficient of the model, and βi is linear, βii is quadratic, and βij is interaction coefficients.
The microbial growth was monitored by the optical density at 600 nm using a microplate spectrophotometer (BioTeck, Santa Clara, CA, USA). At the end of the incubation, the experimental tube was centrifuged at 10,000 x g for 10 min at 4°C, and supernatants were collected to analyze the concentration of the AA remaining using LC-MS.
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Table 8: The BBD with coded variables and the responses of acrylamide removal by percentage (%) for B. breve and Lb. plantarum under anaerobic conditions.
Runs Temperature (ºC) pH Time (h) NaCl (g/100 ml) AA removal (%)
(X1) (X2) (X3) (X4) B. breve Lb. plantarum
1 42 6.5 18 0.0 54.2 18.4
2 42 5.5 18 1.5 47.7 16.3
3 42 5.5 14 1.5 51.5 14.9
4 37 6.5 18 1.5 62.3 5.8
5 37 5.5 22 0.0 64.5 10.7
6 32 5.5 22 1.5 62.9 22.0
7 37 4.5 14 3.0 58.7 12.1
8 37 4.5 18 1.5 56.1 36.2
9 42 5.5 18 1.5 59.4 16.6
10 37 5.5 18 0.0 57.7 20.8
11 37 5.5 14 1.5 55.3 38.0
12 32 6.5 18 3.0 59.6 10.6
13 37 6.5 22 1.5 54.8 24.2
14 37 6.5 14 1.5 52.3 12.1
15 37 5.5 18 0.0 56.9 17.0
16 32 5.5 18 0.0 59.7 22.5
17 37 6.5 18 0.0 49.4 17.5
18 37 5.5 14 3.0 57.2 14.4
19 42 4.5 18 3.0 52.4 11.8
20 32 5.5 14 1.5 60.8 17.0
21 37 4.5 18 1.5 58.3 21.9
22 37 5.5 22 1.5 54.0 20.9
23 32 4.5 18 3.0 60.2 19.3
24 37 5.5 18 3.0 55.4 12.1
25 37 4.5 22 1.5 55.1 25.2
26 32 5.5 18 1.5 61.9 14.7
27 42 5.5 22 1.5 52.5 16.5
AA: acrylamide
4.2.5 In vitro Gastrointestinal Digestion (INFOGEST2.0)
All samples of selected isolates were subjected to in vitro gastrointestinal INFOGEST protocol (Brodkorb et al., 2019). According to the protocol, the simulated salivary fluid (SSF), simulated gastric fluid (SGF), simulated intestinal fluid (SIF), enzymes, and bile concentration were measured prior to experiments. The 2 ml sample containing 50 µg/ml of AA and the 8 logs CFU/mL activated bacteria were subjected to in vitro digestion using the INFOGEST model. Firstly, the 2 ml samples were mixed with salivary salivary
solution (SSF) consisting of 0.19 ml of water, 10 µL of CaCl2 (0.3 M), and 0.2 ml of salivary amylase solution. The mixture was incubated with agitation for 2 min at 37°C and pH of 7.0. In order to mimic the gastric phase in the stomach and reach a pH 3.0 at 37°C, the sample was incubated while mixing for 120 min after adding the following to the sample: 3.2 ml of SGF, 0.2 ml of rabbit gastric extracts RGE, 0.2 ml of pepsin solution, 2 µL of CaCl2 (0.3 M), 0.210 ml of HCl (1 M) and 0.188 ml of water. At the intestinal phase, 1.324 ml of water, 3.4 ml of SIF, 2 ml of pancreatin, 1 ml of bile, 16 µL of CaCl2
(0.3 M), and 0.260 ml of NaOH (1.0 M) were added to the samples prior to incubation for 120 min at pH of 7 and 37°C. During INFOGEST trials, samples were taken to assess the microbial count and AA remaining at oral, gastric, and intestine steps.
4.2.6 Quantification of Acrylamide by LC-MS-MS
Liquid chromatography (LC) coupled with a mass spectrometer (MS) and equipped with column Hypercarb C18 (2.1 × 100 mm, 5.0 μm) (Thermo Scientific, Waltham, MA, USA) was employed to analyze the remains AA in the supernatants mentioned above. The AA analysis was carried out according to the standard operation procedure (SOP) developed by Dubai Central Laboratory (Dubai, UAE). Briefly, an isocratic mobile phase of 1%
acetic acid in H2O was pumped at a 0.2 ml/min flow rate. An aliquot of 20 μl was
injected, and the column temperature was kept at 35°C. An external standard curve of AA (0, 5, 10, 25, 50, 75, 100, 125, and 150 𝜇g/ml) was prepared to quantify the remaining AA in the samples. The final acrylamide removal (%) was calculated as follow:
AA removal (%) AAstarting time − AAend of incubation
AAstarting time
× 100
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4.2.7 Understanding Mechanism of Acrylamide Removal 4.2.7.1 Preparation of the samples and binding assay
Ten milliliters of sterilized MRS broth containing 10 𝜇g/ml acrylamide were inoculated with 1% activated culture of the selected isolates individually. The inoculated MRS tubes were incubated at 37°C for 18 h. After the incubation, the tubes were centrifuged at 4,000 x g for 10 min at 4°C. The supernatants were decanted, and cell pellets were collected in 0.1 M phosphate buffer (pH 7.0) and stored at -20°C until further analysis.
4.2.7.2 Zeta Potentials
The zeta potential of the selected isolates was measured using Zetasizer Nano ZS-90 (Malvern Instruments Ltd, Worcestershire, UK). The experiment was performed according to Shen et al. (2019a).
4.2.7.3 Fourier Transform Infrared Spectroscopy (FTIR) Analysis
For FTIR, the prepared cell pellets in section 2.7.1 were positioned on a Diamond/ZnSe crystal plate (PerkinElmer, Norwalk, CT, USA). The attenuated total reflectance (ATR)- FTIR spectroscopy (PerkinElmer) was employed to determine the functional groups and assumed binding sites. The FTIR spectral ranged from 400 to 4000 cm-1 (Ge et al., 2017;
Lin et al., 2011; Shen et al., 2019a).
4.2.7.4 Scanning Electron Microscopy Coupled with Energy-dispersive X-ray Spectroscopy (SEM-EDS)
The morphology and elementary composition of the bacterial cell pellets were examined using SEM-EDS. The 2.5% (v/v) glutaraldehyde was used to fix the cell pellets in a 1%
osmium tetroxide mixture. The radius, height, and elemental composition of the cell pellets were assessed by Quanta 250 ESEM according to the detailed method described previously (Ge et al., 2017; Shen et al., 2019a). A small piece of aluminum foil was used to deposit ~ 5 μl of the bacterial pellets. The loaded aluminum foil was attached to the stainless-steel stub with carbon tape and allowed to dry. Finally, the dried sample was loaded on a stub holder and placed in the SEM instrument.
61 4.2.7.5 Transmission Electron Microscopy (TEM) Measurement
The Tecnai G2 transmission electron microscope (TEM) was employed to assess the thickness of the cell pellets. The cell pellets were prepared according to Shen et al.
(2019a). Briefly, the ultramicrotome (UC6, LEICA, Wetzlar, Germany) instrument was employed to cut the cell pellets into 50-60 nm thickness according to the method detailed by (Shen et al., 2019a). Afterward, the prepared cell pellets were imaged using TEM operated at 200 kV.
4.2.8 Statistical Analysis
The response surface method (RSM) was conducted for the optimization stage. The
variance and regression coefficients analyses were performed using Minitab v.21 (Minitab Ltd, Coventry, UK).