# TheIn Vitro Adsorption Ability of Lactobacillus acidophilus NCFM to Benzo(a)pyrene in PM2.5

Authors: Lili Fu; Yan Ning; Hongfei Zhao; Junfeng Fan; Bolin Zhang
Journal: Journal of Toxicology
 (2021)

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## Abstract

The objective of this work was to explore the ability of lactic acid bacteria strains to bind benzo(a)pyrene (B(a)P) existing in PM2.5. In this study, we examined the ability of Lactobacillus acidophilus NCFM to bind B(a)P in the simulated PM2.5 environment. Among the tested 5 strains, Lactobacillus acidophilus NCFM exhibited the best capacity to bind B(a)P, and its B(a)P binding percentage was 60.00%. Simulations of organic and inorganic systems which represent PM2.5 indicated that B(a)P could be absorbed by strain L. acidophilus NCFM. For the inorganic system of pH 5, L. acidophilus NCFM bound 92.74% B(a)P with a cell concentration of 1 × 1010 cfu/mL at 37°C for 8 hr. Regarding the organic system with pH 6, 73.00% B(a)P was bound by strain L. acidophilus NCFM after this bacterium was incubated at 37°C for 10 min. A quick B(a)P binding by this probiotic bacterium took place in the organic system. The removal of B(a)P from PM2.5 was significantly related to incubation time, cultivation temperature, pH, and cell concentration. Thus, our finding shows that long-term consumption of L. acidophilus NCFM is beneficial for the reduction of B(a)P towards the population who are exposed to PM2.5, although the ability of this bacterium to adsorb B(a)P is partly affected by the differences in the origin of PM2.5.


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## Body

## 1. Introduction

Particulate matter, harmful suspended particles existing in air environment, is referred as small particles less than 10 micrometers. The particulate matter is easily deposited in the upper respiratory tract and causes toxicity to the human body. Such particulate matters which are less than 2.5 micrometers in diameter, i.e., PM2.5, are more dangerous and have a direct damage to lung health due to long-term contact of bronchioles and alveoli [1]. The smaller the particle diameter is, the deeper into the respiratory tract happens. Meanwhile, toxicological data show that particulate matters not only affect the respiratory ability but also are harmful to cardiovascular, nervous, and immune functions [2]. Thus, the World Health Organization (WHO) defines particulate matter as a class of carcinogens and addresses that different degrees of physiological and pathological changes will take place for the particulate matter-carrying population [3].Generally, PM2.5 consists of sulfate, nitrate, ammonium salts, carbonaceous particles, metal particles, minerals, and polycyclic aromatic hydrocarbon (PAH) family [4, 5]. Benzo(a)pyrene (B(a)P), a member of the family PAH, is usually found in its PM2.5. The presence of B(a)P in PM2.5 significantly strongly increases tumor cell migration and invasion, causing mutagenesis and carcinogenicity [6]. B(a)P from the PAH family is regarded as the most abundant and toxic component existing in PM2.5 and has long-term harm to people. Thus, it is practicable to use B(a)P as a marker to elevate the toxicity of PM2.5 [7].In recent years, there has been an increasing interest in PM2.5 which is a significant threat to human health [8]. In addition to activated carbon and modified mesoporous organosilica which are traditionally used as adsorbing agents of PM2.5, microbial community is also becoming an important part of filtration media, and lactic acid bacteria strains could be explored as a bio-binding material for the removal of PM2.5 [9]. Lactobacillus strains, as food-grade microorganisms from lactic acid bacteria, have good intestinal adhesion ability of epidermal cells and thus offer various health-promoting benefits to the human body [10]. The communication between Lactobacillus strains defined as probiotics and intestinal cells can stop the colonization of pathogens from the intestine and enhance the immunity of the body [11]. Lactobacillus strains have been documented to have good adsorption to mutagenic compounds, thus being a biological agent for anticancer or antimutagenic effect [12]. Some Lactobacillus strains are also proved to significantly reduce the mutagenicity of B(a)P via their binding ability [13, 14]. In our cases, L. plantarum CICC 22135 and L. pentosus CICC 23163 exhibited high efficiency in removing B(a)P from the aqueous medium [15]. Moreover, such Lactobacillus strains showed a good B(a)P binding ability under simulated starch conditions, but their B(a)P binding percentages depended on starch concentrations [16]. Studies have shown the importance of bacteria in binding PAHs, and this reduction is significantly affected by initial PAH concentrations, bacterial population, and pH of media; especially, the highest reduction was related to B(a)P [17]. Obviously, the roles of Lactobacillus strains in B(a)P binding are affected by the environments in which they survive. To date, few relevant studies on whether Lactobacilli strains are still able to bind B(a)P in PM2.5 with complex and diverse particulate matter have been done. Therefore, this present study was designed to investigate the possibility of strain L. acidophilus NCFM to remove PM2.5 toxicity in terms of its B(a)P binding ability. Possible factors affecting these tested strains to bind B(a)P were also discussed when they were cultured in the simulated PM2.5 systems. Imaging the cell morphology of L. acidophilus NCFM in the simulated PM2.5 systems via atomic force microscopy (AFM) was presented.


## 2. Materials and Methods

### 2.1. Bacterial Strains

Several LAB strains includingLactobacillus plantarum 121, Leuconostoc mesenteroides DM1-2, Lactobacillus acidophilus NCFM, Lactobacillus paralimentarius 412, and Lactobacillus pentosus ML32 were obtained from the China Center of Industrial Culture Collection (CICC) and tested for this study.


### 2.2. Preparation of Bacterial Suspensions

The lyophilized preparations of five strains transferred into 5 mL de Man–Rogosa–Sharpe (MRS) medium were firstly activated at 37°C, respectively. Then, they were inoculated into the MRS medium at 37°C for 12 hr incubation with 4% inocula. Their cells were collected by centrifugation (4°C, 5000 rpm, and 10 min) and washed twice with sterile water. The cell concentrations of these tested strains were finally adjusted to 5 × 109 cfu/mL prior to use.


### 2.3. Preparation of Two B(a)P Simulation Systems

#### 2.3.1. Inorganic System

The PM2.5-based inorganic compositions simplify the artificial inorganic system. Artificial inorganic system (1.0 mL) consisting of 100 μL of B(a)P working solution (10 μg/mL), 300 μL of sodium sulfate solution (100 μg/mL), 300 μL of ammonium sulfate solution (100 μg/mL), and 100 μL of sterile water was designed according to Dhananjay et al. with some modifications [5].


#### 2.3.2. Organic System

Regarding the organic compositions of PM2.5 which belong to the family PHAs, PHA Mix (EPA 610), purchased from Accredited Chemical Testing Lab with a purity ≥98.5%, was used to simulate the organic system of B(a)P. The PHA Mix consists of 16 various concentrations of organic compounds, including benzo[k]fluoranthene of 99.37 ± 1.4 mg/L, chrysene of 99.59 ± 1.41 mg/L, phenanthrene of 98.9 ± 1.31 mg/L, benzo[b]fluoranthene of 202.3 ± 2.69 mg/L, benzo[ghi]perylene of 200.6 ± 2.67 mg/L, fluoranthene of 198.3 ± 2.64 mg/L, fluorine of 200.4 ± 2.66 mg/L, naphthalene of 1000 ± 3.74 mg/L, acenaphthene of 991 ± 6.1 mg/L, acenaphthylene of 2018 ± 12.31 mg/L, anthracene of 101.2 ± 1.34 mg/L, benzo[a]anthracene of 99.98 ± 1.41 mg/L, benzo[a]pyrene of 100.8 ± 1.34 mg/L, dibenz[a,h]anthracene of 202 ± 2.86 mg/L, indeno[1,2,3-cd]pyrene of 98.61 ± 1.39 mg/L, and pyrene of 100.6 ± 1.33 mg/L. The simulated organic system of B(a)P was composed of 100 μL PHAs (100 μg/mL) and 900 μL sterile water.The separation of sixteen kinds of PAHs within 35 min showed good linearity in the range of 0.10–5.00μg/mL, and the detection limit of B(a)P was 0.90 μg/mL [18, 19].


### 2.4. Binding of Tested Strains to B(a)P in Two Simulated Systems

#### 2.4.1. Inorganic System

The artificial inorganic system (100μL) plus 900 μL sterilized water, containing 1.0 μg/mL B(a)P, was added to 1.0 mL bacterial suspension. After incubation at 37°C for 4 hr, the supernatant was collected by centrifugation (3000 r/min, 5 min). Chloroform (500 μL) was added to the supernatant to produce the organic phase for the detection of B(a)P. The control was designed as 1.0 μg/mL aqueous solution of B(a)P without bacterial cell addition. For each sample, B(a)P was detected by HPLC with the following conditions: UV detection of 290 nm wavelength, mobile phase of pure methanol, selection of room temperature as column temperature, flow rate of 1.0 mL/min, and injection volume of 20.0 μL [18].


#### 2.4.2. Organic System

We selected 100μL PHAs plus 900 μL sterilized water which were added to 1.0 mL bacterial suspension. After incubation at 37°C for 4 hr, the level of B(a)P existing in the supernatant collected by centrifugation (3000 r/min, 5 min) was detected by HPLC. The control was 1.0 μg/mL PHAs (1.0 mL) without bacterial cell addition. B(a)P was UV-detected at 290 nm wavelength by HPLC. Acetonitrile and water were used as the mobile phase. Column temperature was room temperature with a flow rate of 1.0 mL/min as well as injection volume of 20.0 μL. For gradient elution analysis, A was acetonitrile and B was water. 60% A plus 40% B was used for isocratic elution for 1 min. After A was elevated from 60% to 100% within 20 min, eluting with 100% A was kept for at least 22 min. Then, elution A was reduced to 60% in 8 min and equilibrated with 60% A for 10 min.


#### 2.4.3. The Binding Percentage of Strains to B(a)P

The binding percentage of the tested bacteria to B(a)P was calculated according to the following equation:(1)Br=Bs−SBs×100%,where Br represents the binding rate of the tested bacterial cells to B(a)P (%); Bs indicates the level of B(a)P from the sample blank (μg/mL); and S is the level of B(a)P for each supernatant harvested from the tested bacterial cultivation (μg/mL).


### 2.5. Factors Affecting the Ability ofL. acidophilus NCFM to Bind B(a)P

#### 2.5.1. Effect of Incubation Time

For either of the simulated inorganic or organic system,L. acidophilus NCFM was grown in both systems for the observation of its B(a)P binding ability under different incubation times at 37°C. After incubated in two simulated systems for 10 min, 60 min, 240 min, 480 min, 1080 min, and 1440 min, respectively, the percentage of strain NCFM to bind B(a)P was detected.


#### 2.5.2. Effect of Temperature

For either of the simulated inorganic or organic system,L. acidophilus NCFM was cultivated in the two simulated systems for the observation of its B(a)P binding ability under different incubation temperatures. The ability of L. acidophilus NCFM to bind B(a)P was evaluated after this strain was incubated at 4°C, 15°C, 23°C, and 37°C for 8 hr, respectively.


#### 2.5.3. Effect of pH

For either of the simulated inorganic or organic system,L. acidophilus NCFM was cultured in the two simulated systems for the observation of its B(a)P binding ability under different pH values at 37°C. The ability of L. acidophilus NCFM to bind B(a)P was evaluated after this strain was incubated at pH 3, 4, 5, 6, 7, 8, and 9 for 8 hr, respectively. The sample was adjusted to a specific pH value using a degassed phosphate buffer solution.


#### 2.5.4. Effect of Cell Concentration

For either of the simulated inorganic or organic system,L. acidophilus NCFM was grown in the two simulated systems for the observation of its B(a)P binding ability under cell concentrations. L. acidophilus NCFM was inoculated into the two simulated systems at cell concentrations of 1 × 108 cfu/mL, 1 × 109 cfu/mL, and 1 × 1010 cfu/mL, respectively. Estimation of cell concentrations during cultures was done by turbidimetry [20]. After incubated at 37°C for 8 hr, the ability of this strain to bind B(a)P was evaluated in terms of its cell concentrations.


### 2.6. The Optimal Parameters forL. acidophilus NCFM to Bind B(a)P

According to Section 2.5, the optimal conditions are obtained from two simulated systems.L. acidophilus NCFM was cultured in the optimal conditions to calculate the best binding percentages of B(a)P.


### 2.7. Analysis of Atomic Force Microscopy (AFM)

Atomic force microscopy was used to observe the possibility ofL. acidophilus NCFM (109 cfu/mL) to bind B(a)P. Four groups were prepared as follows. The first group was the inorganic system which contains 10 μg/mL B(a)P and strain NCFM; the second was the organic system with 10 μg/mL B(a)P and strain NCFM; the third was 10 μg/mL B(a)P solution plus strain NCFM; and the fourth was only bacterial suspension as the control. These mixtures were incubated at 37°C for 1 hr and then diluted to a final concentration of 10 μg/mL. 5 μL was sampled and dispersed onto a mica carrier (PELCO mica disc 10 mm) for drying at room temperature. AFM images were captured by using a scanning probe microscope (NTEGRA Spectra, NT-MDT Co., Ltd., Moscow, Russia) in the tapping mode.


### 2.8. Statistical Analysis

Three replicates for each experiment were done. Statistical analyses (ANOVA) were performed using SPSS 19.0 statistical software. Results are expressed as means ± standard deviation (SD). Statistical differences are considered to be significant whenP≤0.05 takes place.


## 2.1. Bacterial Strains

Several LAB strains includingLactobacillus plantarum 121, Leuconostoc mesenteroides DM1-2, Lactobacillus acidophilus NCFM, Lactobacillus paralimentarius 412, and Lactobacillus pentosus ML32 were obtained from the China Center of Industrial Culture Collection (CICC) and tested for this study.


## 2.2. Preparation of Bacterial Suspensions

The lyophilized preparations of five strains transferred into 5 mL de Man–Rogosa–Sharpe (MRS) medium were firstly activated at 37°C, respectively. Then, they were inoculated into the MRS medium at 37°C for 12 hr incubation with 4% inocula. Their cells were collected by centrifugation (4°C, 5000 rpm, and 10 min) and washed twice with sterile water. The cell concentrations of these tested strains were finally adjusted to 5 × 109 cfu/mL prior to use.


## 2.3. Preparation of Two B(a)P Simulation Systems

### 2.3.1. Inorganic System

The PM2.5-based inorganic compositions simplify the artificial inorganic system. Artificial inorganic system (1.0 mL) consisting of 100 μL of B(a)P working solution (10 μg/mL), 300 μL of sodium sulfate solution (100 μg/mL), 300 μL of ammonium sulfate solution (100 μg/mL), and 100 μL of sterile water was designed according to Dhananjay et al. with some modifications [5].


### 2.3.2. Organic System

Regarding the organic compositions of PM2.5 which belong to the family PHAs, PHA Mix (EPA 610), purchased from Accredited Chemical Testing Lab with a purity ≥98.5%, was used to simulate the organic system of B(a)P. The PHA Mix consists of 16 various concentrations of organic compounds, including benzo[k]fluoranthene of 99.37 ± 1.4 mg/L, chrysene of 99.59 ± 1.41 mg/L, phenanthrene of 98.9 ± 1.31 mg/L, benzo[b]fluoranthene of 202.3 ± 2.69 mg/L, benzo[ghi]perylene of 200.6 ± 2.67 mg/L, fluoranthene of 198.3 ± 2.64 mg/L, fluorine of 200.4 ± 2.66 mg/L, naphthalene of 1000 ± 3.74 mg/L, acenaphthene of 991 ± 6.1 mg/L, acenaphthylene of 2018 ± 12.31 mg/L, anthracene of 101.2 ± 1.34 mg/L, benzo[a]anthracene of 99.98 ± 1.41 mg/L, benzo[a]pyrene of 100.8 ± 1.34 mg/L, dibenz[a,h]anthracene of 202 ± 2.86 mg/L, indeno[1,2,3-cd]pyrene of 98.61 ± 1.39 mg/L, and pyrene of 100.6 ± 1.33 mg/L. The simulated organic system of B(a)P was composed of 100 μL PHAs (100 μg/mL) and 900 μL sterile water.The separation of sixteen kinds of PAHs within 35 min showed good linearity in the range of 0.10–5.00μg/mL, and the detection limit of B(a)P was 0.90 μg/mL [18, 19].


## 2.3.1. Inorganic System

The PM2.5-based inorganic compositions simplify the artificial inorganic system. Artificial inorganic system (1.0 mL) consisting of 100 μL of B(a)P working solution (10 μg/mL), 300 μL of sodium sulfate solution (100 μg/mL), 300 μL of ammonium sulfate solution (100 μg/mL), and 100 μL of sterile water was designed according to Dhananjay et al. with some modifications [5].


## 2.3.2. Organic System

Regarding the organic compositions of PM2.5 which belong to the family PHAs, PHA Mix (EPA 610), purchased from Accredited Chemical Testing Lab with a purity ≥98.5%, was used to simulate the organic system of B(a)P. The PHA Mix consists of 16 various concentrations of organic compounds, including benzo[k]fluoranthene of 99.37 ± 1.4 mg/L, chrysene of 99.59 ± 1.41 mg/L, phenanthrene of 98.9 ± 1.31 mg/L, benzo[b]fluoranthene of 202.3 ± 2.69 mg/L, benzo[ghi]perylene of 200.6 ± 2.67 mg/L, fluoranthene of 198.3 ± 2.64 mg/L, fluorine of 200.4 ± 2.66 mg/L, naphthalene of 1000 ± 3.74 mg/L, acenaphthene of 991 ± 6.1 mg/L, acenaphthylene of 2018 ± 12.31 mg/L, anthracene of 101.2 ± 1.34 mg/L, benzo[a]anthracene of 99.98 ± 1.41 mg/L, benzo[a]pyrene of 100.8 ± 1.34 mg/L, dibenz[a,h]anthracene of 202 ± 2.86 mg/L, indeno[1,2,3-cd]pyrene of 98.61 ± 1.39 mg/L, and pyrene of 100.6 ± 1.33 mg/L. The simulated organic system of B(a)P was composed of 100 μL PHAs (100 μg/mL) and 900 μL sterile water.The separation of sixteen kinds of PAHs within 35 min showed good linearity in the range of 0.10–5.00μg/mL, and the detection limit of B(a)P was 0.90 μg/mL [18, 19].


## 2.4. Binding of Tested Strains to B(a)P in Two Simulated Systems

### 2.4.1. Inorganic System

The artificial inorganic system (100μL) plus 900 μL sterilized water, containing 1.0 μg/mL B(a)P, was added to 1.0 mL bacterial suspension. After incubation at 37°C for 4 hr, the supernatant was collected by centrifugation (3000 r/min, 5 min). Chloroform (500 μL) was added to the supernatant to produce the organic phase for the detection of B(a)P. The control was designed as 1.0 μg/mL aqueous solution of B(a)P without bacterial cell addition. For each sample, B(a)P was detected by HPLC with the following conditions: UV detection of 290 nm wavelength, mobile phase of pure methanol, selection of room temperature as column temperature, flow rate of 1.0 mL/min, and injection volume of 20.0 μL [18].


### 2.4.2. Organic System

We selected 100μL PHAs plus 900 μL sterilized water which were added to 1.0 mL bacterial suspension. After incubation at 37°C for 4 hr, the level of B(a)P existing in the supernatant collected by centrifugation (3000 r/min, 5 min) was detected by HPLC. The control was 1.0 μg/mL PHAs (1.0 mL) without bacterial cell addition. B(a)P was UV-detected at 290 nm wavelength by HPLC. Acetonitrile and water were used as the mobile phase. Column temperature was room temperature with a flow rate of 1.0 mL/min as well as injection volume of 20.0 μL. For gradient elution analysis, A was acetonitrile and B was water. 60% A plus 40% B was used for isocratic elution for 1 min. After A was elevated from 60% to 100% within 20 min, eluting with 100% A was kept for at least 22 min. Then, elution A was reduced to 60% in 8 min and equilibrated with 60% A for 10 min.


### 2.4.3. The Binding Percentage of Strains to B(a)P

The binding percentage of the tested bacteria to B(a)P was calculated according to the following equation:(1)Br=Bs−SBs×100%,where Br represents the binding rate of the tested bacterial cells to B(a)P (%); Bs indicates the level of B(a)P from the sample blank (μg/mL); and S is the level of B(a)P for each supernatant harvested from the tested bacterial cultivation (μg/mL).


## 2.4.1. Inorganic System

The artificial inorganic system (100μL) plus 900 μL sterilized water, containing 1.0 μg/mL B(a)P, was added to 1.0 mL bacterial suspension. After incubation at 37°C for 4 hr, the supernatant was collected by centrifugation (3000 r/min, 5 min). Chloroform (500 μL) was added to the supernatant to produce the organic phase for the detection of B(a)P. The control was designed as 1.0 μg/mL aqueous solution of B(a)P without bacterial cell addition. For each sample, B(a)P was detected by HPLC with the following conditions: UV detection of 290 nm wavelength, mobile phase of pure methanol, selection of room temperature as column temperature, flow rate of 1.0 mL/min, and injection volume of 20.0 μL [18].


## 2.4.2. Organic System

We selected 100μL PHAs plus 900 μL sterilized water which were added to 1.0 mL bacterial suspension. After incubation at 37°C for 4 hr, the level of B(a)P existing in the supernatant collected by centrifugation (3000 r/min, 5 min) was detected by HPLC. The control was 1.0 μg/mL PHAs (1.0 mL) without bacterial cell addition. B(a)P was UV-detected at 290 nm wavelength by HPLC. Acetonitrile and water were used as the mobile phase. Column temperature was room temperature with a flow rate of 1.0 mL/min as well as injection volume of 20.0 μL. For gradient elution analysis, A was acetonitrile and B was water. 60% A plus 40% B was used for isocratic elution for 1 min. After A was elevated from 60% to 100% within 20 min, eluting with 100% A was kept for at least 22 min. Then, elution A was reduced to 60% in 8 min and equilibrated with 60% A for 10 min.


## 2.4.3. The Binding Percentage of Strains to B(a)P

The binding percentage of the tested bacteria to B(a)P was calculated according to the following equation:(1)Br=Bs−SBs×100%,where Br represents the binding rate of the tested bacterial cells to B(a)P (%); Bs indicates the level of B(a)P from the sample blank (μg/mL); and S is the level of B(a)P for each supernatant harvested from the tested bacterial cultivation (μg/mL).


## 2.5. Factors Affecting the Ability ofL. acidophilus NCFM to Bind B(a)P

### 2.5.1. Effect of Incubation Time

For either of the simulated inorganic or organic system,L. acidophilus NCFM was grown in both systems for the observation of its B(a)P binding ability under different incubation times at 37°C. After incubated in two simulated systems for 10 min, 60 min, 240 min, 480 min, 1080 min, and 1440 min, respectively, the percentage of strain NCFM to bind B(a)P was detected.


### 2.5.2. Effect of Temperature

For either of the simulated inorganic or organic system,L. acidophilus NCFM was cultivated in the two simulated systems for the observation of its B(a)P binding ability under different incubation temperatures. The ability of L. acidophilus NCFM to bind B(a)P was evaluated after this strain was incubated at 4°C, 15°C, 23°C, and 37°C for 8 hr, respectively.


### 2.5.3. Effect of pH

For either of the simulated inorganic or organic system,L. acidophilus NCFM was cultured in the two simulated systems for the observation of its B(a)P binding ability under different pH values at 37°C. The ability of L. acidophilus NCFM to bind B(a)P was evaluated after this strain was incubated at pH 3, 4, 5, 6, 7, 8, and 9 for 8 hr, respectively. The sample was adjusted to a specific pH value using a degassed phosphate buffer solution.


### 2.5.4. Effect of Cell Concentration

For either of the simulated inorganic or organic system,L. acidophilus NCFM was grown in the two simulated systems for the observation of its B(a)P binding ability under cell concentrations. L. acidophilus NCFM was inoculated into the two simulated systems at cell concentrations of 1 × 108 cfu/mL, 1 × 109 cfu/mL, and 1 × 1010 cfu/mL, respectively. Estimation of cell concentrations during cultures was done by turbidimetry [20]. After incubated at 37°C for 8 hr, the ability of this strain to bind B(a)P was evaluated in terms of its cell concentrations.


## 2.5.1. Effect of Incubation Time

For either of the simulated inorganic or organic system,L. acidophilus NCFM was grown in both systems for the observation of its B(a)P binding ability under different incubation times at 37°C. After incubated in two simulated systems for 10 min, 60 min, 240 min, 480 min, 1080 min, and 1440 min, respectively, the percentage of strain NCFM to bind B(a)P was detected.


## 2.5.2. Effect of Temperature

For either of the simulated inorganic or organic system,L. acidophilus NCFM was cultivated in the two simulated systems for the observation of its B(a)P binding ability under different incubation temperatures. The ability of L. acidophilus NCFM to bind B(a)P was evaluated after this strain was incubated at 4°C, 15°C, 23°C, and 37°C for 8 hr, respectively.


## 2.5.3. Effect of pH

For either of the simulated inorganic or organic system,L. acidophilus NCFM was cultured in the two simulated systems for the observation of its B(a)P binding ability under different pH values at 37°C. The ability of L. acidophilus NCFM to bind B(a)P was evaluated after this strain was incubated at pH 3, 4, 5, 6, 7, 8, and 9 for 8 hr, respectively. The sample was adjusted to a specific pH value using a degassed phosphate buffer solution.


## 2.5.4. Effect of Cell Concentration

For either of the simulated inorganic or organic system,L. acidophilus NCFM was grown in the two simulated systems for the observation of its B(a)P binding ability under cell concentrations. L. acidophilus NCFM was inoculated into the two simulated systems at cell concentrations of 1 × 108 cfu/mL, 1 × 109 cfu/mL, and 1 × 1010 cfu/mL, respectively. Estimation of cell concentrations during cultures was done by turbidimetry [20]. After incubated at 37°C for 8 hr, the ability of this strain to bind B(a)P was evaluated in terms of its cell concentrations.


## 2.6. The Optimal Parameters forL. acidophilus NCFM to Bind B(a)P

According to Section 2.5, the optimal conditions are obtained from two simulated systems.L. acidophilus NCFM was cultured in the optimal conditions to calculate the best binding percentages of B(a)P.


## 2.7. Analysis of Atomic Force Microscopy (AFM)

Atomic force microscopy was used to observe the possibility ofL. acidophilus NCFM (109 cfu/mL) to bind B(a)P. Four groups were prepared as follows. The first group was the inorganic system which contains 10 μg/mL B(a)P and strain NCFM; the second was the organic system with 10 μg/mL B(a)P and strain NCFM; the third was 10 μg/mL B(a)P solution plus strain NCFM; and the fourth was only bacterial suspension as the control. These mixtures were incubated at 37°C for 1 hr and then diluted to a final concentration of 10 μg/mL. 5 μL was sampled and dispersed onto a mica carrier (PELCO mica disc 10 mm) for drying at room temperature. AFM images were captured by using a scanning probe microscope (NTEGRA Spectra, NT-MDT Co., Ltd., Moscow, Russia) in the tapping mode.


## 2.8. Statistical Analysis

Three replicates for each experiment were done. Statistical analyses (ANOVA) were performed using SPSS 19.0 statistical software. Results are expressed as means ± standard deviation (SD). Statistical differences are considered to be significant whenP≤0.05 takes place.


## 3. Results

### 3.1. The Ability of the Tested Strains to Bind B(a)P

As shown in Figure1, the five tested bacteria had different abilities to bind B(a)P. L. acidophilus NCFM showed the best capacity for the adsorption of B(a)P, followed by strain 121, strain ML32, strain DM1-2, and strain 412. 60.00% B(a)P was bound by L. acidophilus NCFM in the present study. It was seen that the ability of lactic acid bacteria to bind B(a)P was strain-dependent. To better understand the roles of various factors in determining the efficiency of B(a)P binding, only L. acidophilus strain NCFM was chosen for further study.Figure 1
B(a)P binding percentage of lactic acid bacteria strains. Note:∗P<0.05 and ∗∗P<0.01 compared with strain NCFM.


### 3.2. Factors Affecting the Ability ofL. acidophilus NCFM to Bind B(a)P

Several factors including incubation time, incubation temperature, pH, and cell concentrations have been reported to directly affect the possibility of potential probiotic bacteria to bind B(a)P if they were presented to different media [21]. To show the possible potential of L. acidophilus NCFM to bind B(a)P in PM2.5, we designed two simulated systems, i.e., inorganic and organic systems, as imaginary PM2.5 to observe the B(a)P adsorbing ability of this strain was subjected to various variables.


#### 3.2.1. Incubation Time

As shown in Figure2, the highest percentage of strain NCFM to bind B(a)P in the simulated inorganic system was 96.34% when this bacterium was incubated for 8 hr. As the simulated inorganic system was concerned, no clear correlation between the B(a)P binding percentage of strain NCFM and its incubation time was observed (Figure 2(a)). For the organic system, however, the binding of L. acidophilus NCFM to B(a)P was a quite quick process. It only took 10 min for this bacterium to bind most B(a)P. 25.66% B(a)P was absorbed by this strain after 10 min incubation. It was seen that the binding percentage of strain NCFM to B(a)P in the inorganic system was higher than that in the organic system at the same incubation time. Thus, the component’s complex from PM2.5 might significantly affect the adsorption of strain NCFM to B(a)P.Figure 2
Factors affecting the binding ability ofL. acidophilus NCFM to B(a)P existing in two simulated PM2.5, i.e., inorganic system and organic system: (a) effect of incubation time on B(a)P binding by strain NCFM, (b) effect of temperatures on B(a)P binding by strain NCFM, (c) effect of pH on B(a)P binding by strain NCFM, and (d) effect of cell concentrations of strain NCFM on its B(a)P binding. Note: ∗P<0.05 and ∗∗P<0.01 compared with the control group.
(a)(b)(c)(d)


#### 3.2.2. Temperature

The temperature ranges from 4°C to 37°C were chosen to investigate the role of incubation temperature in affecting the ability ofL. acidophilus NCFM to bind B(a)P in PM2.5 (Figure 2(b)). 85.64% B(a)P was bound by strain NCFM in the simulated inorganic system, but only 29.60% B(a)P was bound in the simulated organic system at 37°C. In both simulated systems, however, it was noted that an elevating incubation temperature was helpful for L. acidophilus NCFM to bind more B(a)P.


#### 3.2.3. pH

As the pH ranges from 3 to 9 were concerned, most B(a)P was bound byL. acidophilus NCFM when pH of both simulated systems was 5 (Figure 2(c)). Strain NCFM bound 94.11% B(a)P for the simulated inorganic system at pH 5 and 54.93% B(a)P for the simulated organic system at pH 6. An acidic to near-neutral medium surrounding seemed to be useful for L. acidophilus NCFM to bind more B(a)P from PM2.5, although this bacterium bound B(a)P more in the simulated inorganic system than in the simulated organic system.


#### 3.2.4. Strain Concentration

It is apparent that the higher the L. acidophilus NCFM cell concentrations are in both simulated systems, the more B(a)P will be bound (Figure2(d)). About 53.75% B(a)P was absorbed by strain NCFM when its cell concentration was 1 × 1010 cfu/mL in the simulated inorganic system, while 41.85% B(a)P was bound in the same cell concentration.


### 3.3. The Optimal Conditions forL. acidophilus NCFM to Bind B(a)P

Summary of the aforementioned available data indicated that strain NCFM was able to bind B(a)P either in the inorganic or organic system. When this bacterium was incubated with a cell concentration of 1 × 1010 cfu/mL at 37°C for 8 hr in the inorganic system of pH 5, strain NCFM bound the highest B(a)P (92.74%). The optimal parameters for strain NCFM to bind B(a)P were 1 × 1010 cfu/mL cell concentration and 37°C for 10 min of incubation in the organic system of pH 6. About 73.00% B(a)P was absorbed by this strain under the optimal conditions. It was seen from our present data that at least 60.00% B(a)P could be removed in the presence of L. acidophilus NCFM. Obviously, L. acidophilus NCFM strain might have an ability to remove B(a)P from PM2.5. Thus, consumption of this probiotic bacterium should be beneficial for humans to reduce the damage of PM2.5.


### 3.4. AFM Analysis

The morphology of the cells was measured by the AFM contact mode, and the single-cell topography of the control group and the three experimental groups was obtained (Figure3). The morphology of strain NCFM binding B(a)P in the simulated inorganic system or organic system was shrinkage and edge-damaged, whilst the cells of the control group were smooth and intact, showed as normal Lactobacilli cells (Figures 3(a)–3(d)). Moreover, a decrease in the surface roughness of binding cells was also observed for the organic or inorganic system (Figure 3(e)).Figure 3
Factors affecting the binding ability ofL. acidophilus NCFM to B(a)P existing in PM2.5, i.e., inorganic system and organic system: (a) AFM images of strain NCFM, (b) AFM images of B(a)P binding by strain NCFM, (c) AFM images of the inorganic system, (d) AFM images of the organic system, and (e) surface roughness analysis using AFM images (a, b, c, and d). Note: ∗P<0.05 and ∗∗P<0.01 compared with strain NCFM.
(a)(b)(c)(d)(e)


## 3.1. The Ability of the Tested Strains to Bind B(a)P

As shown in Figure1, the five tested bacteria had different abilities to bind B(a)P. L. acidophilus NCFM showed the best capacity for the adsorption of B(a)P, followed by strain 121, strain ML32, strain DM1-2, and strain 412. 60.00% B(a)P was bound by L. acidophilus NCFM in the present study. It was seen that the ability of lactic acid bacteria to bind B(a)P was strain-dependent. To better understand the roles of various factors in determining the efficiency of B(a)P binding, only L. acidophilus strain NCFM was chosen for further study.Figure 1
B(a)P binding percentage of lactic acid bacteria strains. Note:∗P<0.05 and ∗∗P<0.01 compared with strain NCFM.


## 3.2. Factors Affecting the Ability ofL. acidophilus NCFM to Bind B(a)P

Several factors including incubation time, incubation temperature, pH, and cell concentrations have been reported to directly affect the possibility of potential probiotic bacteria to bind B(a)P if they were presented to different media [21]. To show the possible potential of L. acidophilus NCFM to bind B(a)P in PM2.5, we designed two simulated systems, i.e., inorganic and organic systems, as imaginary PM2.5 to observe the B(a)P adsorbing ability of this strain was subjected to various variables.


### 3.2.1. Incubation Time

As shown in Figure2, the highest percentage of strain NCFM to bind B(a)P in the simulated inorganic system was 96.34% when this bacterium was incubated for 8 hr. As the simulated inorganic system was concerned, no clear correlation between the B(a)P binding percentage of strain NCFM and its incubation time was observed (Figure 2(a)). For the organic system, however, the binding of L. acidophilus NCFM to B(a)P was a quite quick process. It only took 10 min for this bacterium to bind most B(a)P. 25.66% B(a)P was absorbed by this strain after 10 min incubation. It was seen that the binding percentage of strain NCFM to B(a)P in the inorganic system was higher than that in the organic system at the same incubation time. Thus, the component’s complex from PM2.5 might significantly affect the adsorption of strain NCFM to B(a)P.Figure 2
Factors affecting the binding ability ofL. acidophilus NCFM to B(a)P existing in two simulated PM2.5, i.e., inorganic system and organic system: (a) effect of incubation time on B(a)P binding by strain NCFM, (b) effect of temperatures on B(a)P binding by strain NCFM, (c) effect of pH on B(a)P binding by strain NCFM, and (d) effect of cell concentrations of strain NCFM on its B(a)P binding. Note: ∗P<0.05 and ∗∗P<0.01 compared with the control group.
(a)(b)(c)(d)


### 3.2.2. Temperature

The temperature ranges from 4°C to 37°C were chosen to investigate the role of incubation temperature in affecting the ability ofL. acidophilus NCFM to bind B(a)P in PM2.5 (Figure 2(b)). 85.64% B(a)P was bound by strain NCFM in the simulated inorganic system, but only 29.60% B(a)P was bound in the simulated organic system at 37°C. In both simulated systems, however, it was noted that an elevating incubation temperature was helpful for L. acidophilus NCFM to bind more B(a)P.


### 3.2.3. pH

As the pH ranges from 3 to 9 were concerned, most B(a)P was bound byL. acidophilus NCFM when pH of both simulated systems was 5 (Figure 2(c)). Strain NCFM bound 94.11% B(a)P for the simulated inorganic system at pH 5 and 54.93% B(a)P for the simulated organic system at pH 6. An acidic to near-neutral medium surrounding seemed to be useful for L. acidophilus NCFM to bind more B(a)P from PM2.5, although this bacterium bound B(a)P more in the simulated inorganic system than in the simulated organic system.


### 3.2.4. Strain Concentration

It is apparent that the higher the L. acidophilus NCFM cell concentrations are in both simulated systems, the more B(a)P will be bound (Figure2(d)). About 53.75% B(a)P was absorbed by strain NCFM when its cell concentration was 1 × 1010 cfu/mL in the simulated inorganic system, while 41.85% B(a)P was bound in the same cell concentration.


## 3.2.1. Incubation Time

As shown in Figure2, the highest percentage of strain NCFM to bind B(a)P in the simulated inorganic system was 96.34% when this bacterium was incubated for 8 hr. As the simulated inorganic system was concerned, no clear correlation between the B(a)P binding percentage of strain NCFM and its incubation time was observed (Figure 2(a)). For the organic system, however, the binding of L. acidophilus NCFM to B(a)P was a quite quick process. It only took 10 min for this bacterium to bind most B(a)P. 25.66% B(a)P was absorbed by this strain after 10 min incubation. It was seen that the binding percentage of strain NCFM to B(a)P in the inorganic system was higher than that in the organic system at the same incubation time. Thus, the component’s complex from PM2.5 might significantly affect the adsorption of strain NCFM to B(a)P.Figure 2
Factors affecting the binding ability ofL. acidophilus NCFM to B(a)P existing in two simulated PM2.5, i.e., inorganic system and organic system: (a) effect of incubation time on B(a)P binding by strain NCFM, (b) effect of temperatures on B(a)P binding by strain NCFM, (c) effect of pH on B(a)P binding by strain NCFM, and (d) effect of cell concentrations of strain NCFM on its B(a)P binding. Note: ∗P<0.05 and ∗∗P<0.01 compared with the control group.
(a)(b)(c)(d)


## 3.2.2. Temperature

The temperature ranges from 4°C to 37°C were chosen to investigate the role of incubation temperature in affecting the ability ofL. acidophilus NCFM to bind B(a)P in PM2.5 (Figure 2(b)). 85.64% B(a)P was bound by strain NCFM in the simulated inorganic system, but only 29.60% B(a)P was bound in the simulated organic system at 37°C. In both simulated systems, however, it was noted that an elevating incubation temperature was helpful for L. acidophilus NCFM to bind more B(a)P.


## 3.2.3. pH

As the pH ranges from 3 to 9 were concerned, most B(a)P was bound byL. acidophilus NCFM when pH of both simulated systems was 5 (Figure 2(c)). Strain NCFM bound 94.11% B(a)P for the simulated inorganic system at pH 5 and 54.93% B(a)P for the simulated organic system at pH 6. An acidic to near-neutral medium surrounding seemed to be useful for L. acidophilus NCFM to bind more B(a)P from PM2.5, although this bacterium bound B(a)P more in the simulated inorganic system than in the simulated organic system.


## 3.2.4. Strain Concentration

It is apparent that the higher the L. acidophilus NCFM cell concentrations are in both simulated systems, the more B(a)P will be bound (Figure2(d)). About 53.75% B(a)P was absorbed by strain NCFM when its cell concentration was 1 × 1010 cfu/mL in the simulated inorganic system, while 41.85% B(a)P was bound in the same cell concentration.


## 3.3. The Optimal Conditions forL. acidophilus NCFM to Bind B(a)P

Summary of the aforementioned available data indicated that strain NCFM was able to bind B(a)P either in the inorganic or organic system. When this bacterium was incubated with a cell concentration of 1 × 1010 cfu/mL at 37°C for 8 hr in the inorganic system of pH 5, strain NCFM bound the highest B(a)P (92.74%). The optimal parameters for strain NCFM to bind B(a)P were 1 × 1010 cfu/mL cell concentration and 37°C for 10 min of incubation in the organic system of pH 6. About 73.00% B(a)P was absorbed by this strain under the optimal conditions. It was seen from our present data that at least 60.00% B(a)P could be removed in the presence of L. acidophilus NCFM. Obviously, L. acidophilus NCFM strain might have an ability to remove B(a)P from PM2.5. Thus, consumption of this probiotic bacterium should be beneficial for humans to reduce the damage of PM2.5.


## 3.4. AFM Analysis

The morphology of the cells was measured by the AFM contact mode, and the single-cell topography of the control group and the three experimental groups was obtained (Figure3). The morphology of strain NCFM binding B(a)P in the simulated inorganic system or organic system was shrinkage and edge-damaged, whilst the cells of the control group were smooth and intact, showed as normal Lactobacilli cells (Figures 3(a)–3(d)). Moreover, a decrease in the surface roughness of binding cells was also observed for the organic or inorganic system (Figure 3(e)).Figure 3
Factors affecting the binding ability ofL. acidophilus NCFM to B(a)P existing in PM2.5, i.e., inorganic system and organic system: (a) AFM images of strain NCFM, (b) AFM images of B(a)P binding by strain NCFM, (c) AFM images of the inorganic system, (d) AFM images of the organic system, and (e) surface roughness analysis using AFM images (a, b, c, and d). Note: ∗P<0.05 and ∗∗P<0.01 compared with strain NCFM.
(a)(b)(c)(d)(e)


## 4. Discussion

Currently, public sectors take regulation actions to reduce emissions and prevent inhalation in order to cope with the PM2.5 problem [22], but there is still no good solution for mitigating the injuries of toxins on the human body. To date, various physical and chemical ways have been tried to reduce the damage of PM2.5 to the human body. As a new type of biological filter medium, microorganisms have been used in indoor and automobile air purifiers [9]. Lactobacillus has been evaluated for its ability to remove various toxins, but very little was found in the literature on the studies of Lactobacillus as the main biological filter medium [23]. Our present study is designed to use lactic acid bacteria strains to remove the toxicity from PM2.5 in terms of their B(a)P binding ability.Our previous studies confirmed thatLactobacillus plantarum 121, Leuconostoc mesenteroides DM1-2, Lactobacillus acidophilus NCFM, Lactobacillus paralimentarius 412, and Lactobacillus pentosus ML32 had good adsorption effects on B(a)P [21]. In our present study, it is interesting to note that five tested bacteria exhibited some ability to bind B(a)P, and at least 36.70% B(a)P was absorbed by these lactic acid bacteria strains. Additionally, their B(a)P absorbing capacity is observed to be species-specific. Among the tested strains, L. acidophilus NCFM has the best ability to bind B(a)P (60.0%). As potential decontaminating agents of aflatoxin B1 (AFB1) and B(a)P, the removal ability of lactic acid bacteria to the mutagenic compounds differs from strain to strain in various medium surroundings. Our results are in accordance with what have been reported so far [14, 24].Regarding the composition of PM2.5, it includes sulfate, nitrate, ammonium salts, carbonaceous particles, metal particles, minerals, and the polycyclic aromatic hydrocarbon (PAH) family. The current studies have implicated that B(a)P is one of the causative agents in colon cancer [25]. Epidemiological studies provide evidence for dietary intake of PAHs which is associated with colorectal cancer risk [26]. On the question of the mechanism of colon tumorigenesis as a result of B(a)P ingestion, B(a)P could lead to a differential induction of cytochrome P450 both in the liver and colon, and its ability of malignant transformation of colon epithelial cells has also been documented [27, 28]. Lactobacillus mostly colonized rats’ colon and ileum and has shown promise in preventing colon carcinomas in rats [29]. In addition to bind B(a)P, Lactobacillus kefir strains were reported to interact with metal ions via their binding ability [30]. Similar studies showed that Lactobacilli strains had good ability to bind various chemicals in various simulated environments [16, 31]. Interestingly, these studies have confirmed that the B(a)P binding efficiency is species-specific and depends on Lactobacillus cell structures. Several reports have shown that the adsorption of mycotoxins to the cell wall of Lactobacillus was attributable to their surface properties and mainly to their hydrophobicity [32, 33]. In addition, literatures have revealed that the adsorption of toxins by microorganisms mainly depends on the specific chemical composition of the cell wall such as the peptidoglycan, teichoic acids, and teichuronic acids of Gram-positive bacteria [34–36]. Therefore, it seems that adsorption of Lactobacillus is dependent on the hydrophobicity and cell wall structure. A note of caution is due to the limited binding site on the cell wall since binding efficiency changes with different environments. Most components in PM2.5 could be absorbed by Lactobacillus cells, but previous studies have not reported how the organic and inorganic systems which resemble PM2.5 affect Lactobacillus strains to bind with B(a)P. Dutton et al. directly simplified the most complex component environments into two types, i.e., organic and inorganic systems [37]. The simplified systems easily allow us to evaluate the toxicity of PM2.5 supplemented with B(a)P. In our case, we used the organic and inorganic systems which were supplemented with B(a)P in vitro to simulate PM2.5. Then, the ability of the tested L. acidophilus NCFM to bind B(a)P was evaluated in PM2.5. It was seen that the tested L. acidophilus NCFM bound B(a)P, and its B(a)P binding percentages were high in the simulated organic and inorganic systems. Thus, it is presumed from our in vitro test that use of selected lactic acid bacterial strains as a biofilter agent should have potential in removing the toxicity of PM2.5.Numerous studies state that the roles of lactic acid bacteria in absorbing various chemicals such as ochratoxin A and aflatoxins depend largely on parameters such as incubation time, cultivation temperature, pH values, and viable cell counts [31]. The stability of the aflatoxin B1-bacteria complex appears to be species-specific [31]. This study confirms that the binding percentage of the mycotoxin by Lactobacillus is associated with incubation time [38]. Piotrowska found the ability of three lactic acid bacteria species in removing ochratoxin A, and heating made higher toxin-binding ability [39]. Aflatoxin B1 bound by lactic acid bacteria was pH-dependent, and a similar finding was also reported by Serrano et al. [40, 41]. Bacterial concentration is the other factor that strongly affects the removal of PAHs [17]. Studies showed that removal of aflatoxin M1 was significantly affected by microbial concentration [42]. Thus, the maximum adsorption of lactic acid bacteria cells to benzopyrene should be optimized. In our case, the optimized conditions are as follows: incubation temperature of 37°C, incubation time of 8 hr, pH of 5, and 1 × 1010 cfu/mL for the inorganic system. In this case, 92.74% B(a)P is removed by L. acidophilus NCFM. For the organic system with pH of 6, L. acidophilus NCFM binds 73.00% B(a)P only within 10 min when this strain is cultured at 37°C with a cell concentration of 1 × 1010 cfu/mL. In vivo, pH of the colon is stable at around 6.8, and the imbalance of pH in tissues is directly linked to diseases such as cancer [43]. These results suggest that Lactobacillus acidophilus NCFMTM may potentially prevent colon cancer development, and Lactobacillus acidophilus NCFMTM significantly suppressed AOM-induced colon carcinogenesis in a dose-dependent manner [44]. This corresponds with our earlier observations, which showed that the optimized conditions were suitable for B(a)P binding in the colon.The presence of PM2.5 caused the B(a)P-binding cells to be changed in their morphology, as observed in our study (Figure 3). A similar report indicated that PM2.5 treatment destroyed the integrity of bacterial cells which shrank or suffered defects [15]. Importantly, the cell wall-prone status decreased because the main component of the cellular wall is composed of peptidoglycans [15]. This also accords with our earlier observations, which showed that more compounds existing in the complex PM2.5 were bound by NCFM strain cells compared to B(a)P environment only. Thus, analysis of AFM further proved that our selected NCFM strain was possible to be used as a precautionary agent for the reduction of PM2.5 pollutants due to its B(a)P binding ability.


## 5. Conclusions

Five tested lactic acid bacteria strains showed ability to bind B(a)P to some certain extent, butL. acidophilus NCFM exhibited the best capacity to bind B(a)P. At least 60% B(a)P was bound by this probiotic bacterium. This process of L. acidophilus NCFM to bind B(a)P was affected by incubation time, cultivation temperature, pH, and cell concentration. The simplification of PM2.5 into inorganic and organic systems provided a model for us to evaluate the roles of lactic acid bacteria strains in reducing the damage of the polluted environments to human beings. There were significant differences in the binding percentage of L. acidophilus NCFM to B(a)P between the two simulated systems, but our in vitro data indicated that strain NCFM exhibited good possibility to remove B(a)P existing in simulated PM2.5. Currently, environmental pollution situation becomes more and more serious, and thus, using probiotic bacteria to relieve environmental hazards will be a new attempt.



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*Source: 6290524-2021-01-07.xml*