Enhanced cholesterol removal ability of lactobacilli via alteration of membrane permeability upon ultraviolet radiation

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ORIGINAL ARTICLE

Enhanced cholesterol removal ability of lactobacilli

via alteration of membrane permeability

upon ultraviolet radiation

Huey-Shi Lye&Abdul-Karim Alias&Gulam Rusul&

Min-Tze Liong

Received: 25 October 2011 / Accepted: 2 February 2012 / Published online: 23 February 2012 # Springer-Verlag and the University of Milan 2012

Abstract This study aimed to evaluate the effects of ultravi-olet radiation (UVA, UVB, and UVC) on the cholesterol removal ability of lactobacilli and altered membrane proper-ties. Viability of lactobacilli decreased (P<0.05) immediately upon UV treatment due to oxidative damage of the cellular membrane, as induced by lipid peroxidation. UVB and UVC showed higher (P<0.05) effects in inducing lipid peroxidation compared to UVA. However, the viability of lactobacilli in-creased significantly (P<0.05) upon fermentation at 37°C for 20 h, which was attributed to the formation of transient pores and membrane permeabilization. These alterations also led to increased (P<0.05) assimilation of cholesterol and incorpora-tion of cholesterol into the cellular membrane upon fermenta-tion. The ratio of membrane cholesterol:phospholipids (C:P) also increased upon UV treatment, accompanied by increased saturation of cholesterol in the polar head, apolar tail and the interface regions of the membrane lipid bilayer.

Keywords Ultraviolet . Lactobacilli . Cholesterol . Membrane . Incorporation

Introduction

Probiotics have been defined as “live microorganisms that

which when administered in adequate amounts confer health

benefits to the host” (FAO/WHO 2001). Past studies have

reported that lactobacilli are the most common probiotic bac-teria that can exhibit several health benefits, including antihy-pertensive effect, alleviate lactose intolerance, activate the

immune system, regulate microbial balance in the gut, and

reduce traveller’s diarrhea (Lye et al.2009; Vesa et al. 1996).

Thus, functional products containing probiotics have gained much attention among the health-conscious population recently. Additionally, lactobacilli have also been reported to reduce serum cholesterol levels in humans and animals that subsequently reduces the risk of cardiovascular disease.

Nguyen et al. (2007) found that the total serum cholesterol

and triglycerides were decreased in hypercholesterolemic mice upon consumption of Lactobacillus plantarum.

Pereira and Gibson (2002) also reported that L. fermentum

was able to remove cholesterol content from culture medium via assimilation of cholesterol. On the other hand, our previ-ous studies found that L. acidophilus BT 1088, L. acidophilus FTCC 0291, L. bulgaricus FTCC 0411, L. bulgaricus FTDC 1311, and L. casei BT 1268 were able to remove cholesterol via assimilation during growth and incorporation of choles-terol into cellular membranes as indicated by increasing satu-ration of cholesterol in regions of polar heads, apolar tails, and upper phospholipids of the membrane bilayer (Lye et al. 2010a, b). Therefore, the ability of lactobacilli to remove cholesterol might relate to membrane properties such as mem-brane permeability.

Ultraviolet radiation (UV) is an electromagnetic radiation that can be categorized into UVA (320-400 nm), UVB

(290–320 nm), and UVC (254 nm) (Qiu et al. 2005). Past

studies have reported that the exposure of UV could affect cell

membrane properties (Smith et al.2009; Howland and Parikh

2010). The membranes of cells become permeabilized to ions

and molecules via formation of pores upon UV treatment at the appropriate dose, and at the same time maintain cell survivability. A similar observation was reported by Bose

and Chatterjee (1995), where the liposomal membrane

becomes more permeabilized due to lipid peroxidation, which subsequently increases transport of substrates in and out of the

H.-S. Lye

:

A.-K. Alias

:

G. Rusul

:

M.-T. Liong (*) School of Industrial Technology, Universiti Sains Malaysia, 11800 USM Penang, Malaysia

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cell upon UV treatment. Hortnag et al. (2010) also reported that growth of Acinetobacter lwoffii was increased upon UV exposure, and that this organism has effective repair mecha-nisms to maintain cell production. Thus, UV treatment could induce permeabilization of cell membranes that not only could increase the removal of cholesterol from medium by lactoba-cilli cells but also maintain cell growth. However, to date, there have been no attempts to utilize such treatments to improve the ability of lactobacilli to remove cholesterol.

The aim of this study was to investigate the effect of UV on the cholesterol removing properties and cell viability of lactobacilli, and membrane properties such as reversible membrane permeability and lipid peroxidation. In addition, changes in the ratio of membrane cholesterol: phospholipids (C:P) and the location of cholesterol enrichment upon treat-ment were also evaluated.

Materials and methods Bacteria cultures

Strains of L. acidophilus BT 1088, L. acidophilus FTCC 0291, L. bulgaricus FTCC 0411, L. bulgaricus FTDC 1311, and L. casei BT 1268 were obtained from the Culture Collection of Bioprocess Division, School of Industrial Technology, Universiti Sains Malaysia (Gelugor, Penang, Malaysia). Each

stock culture was stored at−20°C in 40% (v/v) sterile glycerol.

The lactobacilli culture was cultured three times in sterile de Mann Rogosa Sharpe (MRS) broth (Hi-Media, Mumbai,

India) supplemented with 0.15% (w/v)L-cysteine⋅

hydrochlo-ride (Hi-Media) prior to experimental use. Sample preparation and evaluation of viability

Upon activation, cells were collected by centrifugation at 12,000 g for 15 min at 4°C (Braun, Melsungen, Germany). The cell pellet was washed twice with phosphate buffer saline (PBS; 0.01 M; pH 7.4) and then resuspended in the same buffer. Sterile PBS with 10% (v/v) of washed lactobacilli cells was then exposed to different doses of UVA, UVB, and UVC, respectively (Bio-Rad Laboratories, Hercules, CA) and con-stant cooling at 4°C. Past studies have reported that the doses of ultraviolet radiation used proved to be non-lethal and indeed

favored growth (Ogura et al. 1989; Villarino et al. 2003;

Chadsuthi et al.2010). Thus, the doses of UV radiation used

in this study were 30 J m−2, 60 J m−2, and 90 J m−2. Cells in the

stationary growth phase without UV treatment were used as a control. Viability of UV-treated cells immediately upon treat-ment and upon fertreat-mentation at 37°C for 20 h were evaluated using the pour plate method. Briefly, MRS agar supplemented

with 0.15% (w/v)L-cysteine⋅hydrochloride was used for plating

and duplicates plates were incubated at 37°C for 48 h.

Assimilation of cholesterol

UV-treated cells were inoculated into sterile MRS broth

containing 100 μg mL−1 cholesterol (Hi-Media), 0.15%

(w/v) L-cysteine⋅hydrochloride, 0.3% (w/v) oxgall

(Sigma-Aldrich, St. Louis, MO), and 0.1% (w/v) pan-creatin (Sigma-Aldrich). Untreated cells were used as a control. The sample was then adjusted to pH 8.0 in order to mimic the condition of the human small intes-tines, followed by incubation at 37°C for 20 h. Upon incubation, the mixture was centrifuged at 12,000 g at 4°C for 15 min and the supernatant and cells were harvested separately to evaluate the cholesterol

assimi-lation as previously described (Liong and Shah 2005).

Determination of permeabilized cells

Permeabilized cells was determined using fluorescence labeling according to a method modified from that of

Patel and Campbell (1987). Samples were label with

propidium iodide (PI; Sigma-Aldrich). Briefly, 1 mL

sample (OD600 of 0.3) was added to 60 μL PI (1 mg

in 50 mL PBS; pH 7). The mixture was mixed thor-oughly and incubated at 25°C in the dark for 15 min. The mixture was then centrifuged at 3,500 g and 4°C for 10 min and the labeled cell pellet was resuspended in sterile PBS (3 mL) and measured using fluorescence spectrophotometer (Cary Eclipse, Palo Alto, CA). The excitation and emission wavelengths were set at 538 nm and 617 nm, respectively. A positive control with com-pletely permeabilized membrane was prepared by treat-ing the cells with 1% (v/v) sodium dodecyl sulfate for 10 min. The percentage of permeabilized cells was then calculated as following:

%Permeabilized cells¼ A Bð Þ=C 100% ð1Þ

where A is the fluorescence emission of labeled cells immediately after treatment, B is the fluorescence emis-sion of labeled untreated cells and C is fluorescence emission of the positive control.

Lipid peroxidation

Lipid peroxidation of UV-treated cells and untreated cells (control) was examined as described previously

(Giamarellos-Bourboulis et al. 2003). Samples were

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Incorporation of cholesterol into the cellular membrane

Cell suspension (2 mL; OD600 of 0.3) was vortexed for

2 min and placed in an ice bath for 1 min with glass beads (2 g; 0.43–0.60 mm; Sigma-Aldrich). This step was repeated five times. Intracellular content and cellular membrane was collected separately by centrifugation at 12,000 g at 4°C for 15 min. The cellular membrane were then collected and analyzed for incorporation of cholesterol into the cellular membrane via the determination of membrane cholesterol and phospholipid contents, and also the fluidity properties of the membrane by measuring the fluorescence anisotropy (FAn) of fluorescence probes [1,6-diphenyl-1,3,5-hexatriene (DPH; Sigma-Aldrich), 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH; Sigma-Aldrich) and 8-anilino-1-napthalenesulfonic acid (ANS; Sigma-Aldrich)] inserted into the cellular membrane. The extraction of cho-lesterol and phospholipid content from cell membranes was

based on the method of Liong and Shah (2005). The total

cholesterol and phopholipid contents were then examined using commercial enzymatic kits (bioMérieux, Marcy

l’Etoile, France). The FAn of membrane from treated cells

was determined using three fluorescence probes as

previ-ously described (Ooi et al.2010).

Statistical analysis

Data were evaluated with SPSS software (version 15.0) (SPSS, Chicago, IL). Two-way ANOVA was employed to evaluate the significant differences between sample means,

with significance level atα00.05. Mean comparisons were

performed using Tukey’s-test. Unless stated otherwise, all data presented are the mean values of duplicates, obtained from three separate runs.

Results

Viability of lactobacilli cells immediately upon treatment and upon fermentation

The viability of lactobacilli cells decreased significantly

(P < 0.05) immediately upon treatment (Fig. 1) and

decreased with increasing treatment doses. There was a

higher (P<0.05) decrease in viability of cells at 90 J m−2,

compared to lower doses studied; this was most prevalent

for L. bulgaricus FTDC 1311 (Fig.1d), where the viability

was 9.52–11.85% lower than that of the control (P<0.05). Different types of UV also significantly (P<0.05) affected the viability of lactobacilli cells immediately upon treat-ment. Treatment with UVA and UVC showed a lower (P<0.05) viability compared to that of the control and other types of UV treatment studied. This was most marked for

L. bulgaricus FTDC 1311 (Fig. 1d), where reductions of

4.67–10.35% and 10.02–11.52% were observed when cells were treated with UVA and UVC, respectively, compared to that of the control (P<0.05).

Upon fermentation, all UV-treated cells showed growth

values above 8.0 Log10 CFU mL−1 (Fig. 2). A higher

(P<0.05) viability of lactobacilli cells was observed upon

treatment at 30 J m−2compared to higher doses studied and

that of the control. This was most prevalent for L. casei BT

1268 (Fig.2e), where viability was increased by 4.74–13.98%

upon treatment at 30 J m−2, but showed an increase of only

5.24–7.99% in viability for cells treated at 90 J m−2_compared

to that of the control (P<0.05). In addition, treatment with UVB showed a higher (P<0.05) effect in promoting growth upon treatment. This was most noticeable for L. acidophilus

FTCC 0291 (Fig.2b), where an increase of 8.96% in viability

was observed upon treatment at UVB compared to that of the control (P<0.05).

Assimilation of cholesterol

UV radiation significantly (P<0.05) increased the assimila-tion of cholesterol by lactobacilli upon fermentaassimila-tion at 37°C

for 20 h (Fig.3). This effect was most prominent in 60 J m−2

and 90 J m−2-treated L. acidophilus BT 1088 (Fig. 3a),

where cholesterol removal was increased by 75.39–225.58% and 52.82–153.45%, respectively, compared to the control (P<0.05). Additionally, UVB- and UVC-treated cells also showed higher (P<0.05) cholesterol assimilation compared to that of the control and UVA treatment, where an increase of

26.88–225.58% higher than that of the control was observed.

Membrane permeability

UV treatment (P<0.05) contributed to a significantly enhanced membrane permeability of lactobacilli cells upon treatment

(Fig.4). Treatment at dose of 60 J m−2had a higher effect in

increasing the membrane permeability of cells compared to the other doses studied, except in L. acidophilus FTCC 0291

(Fig. 4b) and L. casei BT 1268 (Fig. 4d). In addition, the

membrane permeability of lactobacilli cells also increased significantly (P<0.05) compared to that of the control when cells were treated with UVB and other types of UV treatment. UVB-treated cells showed an increase in membrane perme-ability of more than 66.90% upon treatment compared to that of the control (P<0.05).

Lipid peroxidation

The release of MDA upon treatment was used to measure the degree of membrane lipid peroxidation of lactobacilli

cells (Fig.5). The concentration of MDA released by

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UV-treated cells exhibited a higher production of MDA

upon treatment at 90 J m−2compared to that of the control

and other lower doses studied. This effect was most

preva-lent for L. acidophilus BT 1088 (Fig. 5a), where the

con-centration of MDA released by cells was increased by more than 65.52% compared to that of the control (P < 0.05). Additionally, treatment with UVB and UVC showed a higher (P<0.05) release of MDA by lactobacilli compared to that of the control and UVA treatment, where the concen-tration of MDA released by lactobacilli cells upon UVB and UVC radiation was increased by 27.27–120.69% and

31.82–251.72%, respectively, compared to that of the control.

Meanwhile, cells treated with UVA showed a lower (P<0.05) concentration of MDA released in the medium (1.82–88.24% higher compared to that of the control).

Incorporation of cholesterol into cellular membranes Ratio of cholesterol to phospholipids

A significant increase (P < 0.05) in the ratio of membrane C:P was observed upon UV treatment and upon

fermenta-tion (Fig. 6). The ratio of C:P in the cellular membrane

increased with increasing doses of treatment up to 60 J m−2

0 2 4 6 8 Type of radiation Viability (Log 10 CFU mL -1 ) A _{D: P<0.001; T: 0.028; D x T: 0.003} 0 2 4 6 8 Type of radiation Viability (Log 10 CFU mL -1) 0 2 4 6 8

Control UVA UVB UVC

Control UVA UVB UVC Control UVA UVB UVC

Control UVA UVB UVC

Type of radiation Viability (Log 10 CFU mL -1 ) C

_{D: 0.018; T: 0.005; D x T: 0.265}

without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 0 2 4 6 8 Type of radiation Viability (Log 10 CFU mL -1 ) D: 0.004; T: 0.005; D x T: 0.033 D D: 0.272; T: P<0.001; D x T: P<0.001 B without treatment 30 J m-2 60 J m-2 90 J m-2 0 2 4 6 8 Type of radiation Viability (Log 10 CFU mL -1) D: P<0.001; T: 0.020; D x T: P<0.001 E

Fig. 1 Viability of control and UV-treated Lactobacillus acidophilus BT 1088 (a), L. acidophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) immedi-ately upon treatment . Control: cells without treatment. Error bars

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(P< 0.05). This effect was most prevalent for L.

acidophi-lus BT 1088 (Fig. 6a), where treatment at 60 J m−2

increased the ratio of membrane C:P up to 154.65% com-pared to that of the control (P < 0.05). Among all types of UV treatment studied, treatment with UVB and UVC showed a higher (P < 0.05) effect in enhancing the ratio of C:P in cellular membrane of lactobacilli cells. Upon treatment with UVB and UVC, the ratio of membrane C:P of L. acido-philus FTCC 0291 was increased by 51.49–115.84% and

78.22–96.04% (Fig. 6b), respectively, compared to that of

the control (P<0.05).

Fluorescence anisotropy

The FAn of ANS (Fig. 7), DPH (Fig. 8), and TMA-DPH

(Fig.9) in cellular membranes increased (P<0.05) upon UV

treatment. Increasing the treatment doses was shown to increase (P<0.05) the FAn of ANS, DPH, and TMA-DPH for lactobacilli cells. This effect was most prevalent for

L. bulgaricus FTDC 1311 (Fig.7d, Fig.8d, Fig.9d), where

a higher (P<0.05) increase of FAn for ANS (47.37–84.21% higher compared to that of the control), DPH (31.25– 90.63% higher compared to that of the control), and

TMA-0 2 4 6 8 10 Type of radiation Viability (Log 10 CFU mL -1 ) A _{D: P<0.001; T: 0.020; D x T: P<0.001} 0 2 4 6 8 10 Type of radiation Viability (Log 10 CFU mL -1 ) D: 0.023; T: 0.023; D x T: P<0.001 D 0 2 4 6 8 10 Type of radiation Viability (Log 10 CFU mL -1 ) D: 0.002; T: 0.001; D x T: P<0.001 B 0 2 4 6 8 10

Control UVA UVB UVC

Type of radiation Viability (Log 10 CFU mL -1) D: P<0.001; T: 0.251; D x T: 0.036 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 C 0 2 4 6 8 10 Type of radiation Viability (Log 10 CFU mL -1 ) D: P<0.001; T: 0.002; D x T: 0.002 E

Fig. 2 Viability of control and UV-treated L. acidophilus BT 1088 (a), L. acidophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) upon fermenta-tion at 37°C for 20 h . Control: cells without treatment. Error bars

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DPH (115.00–250.00% higher compared to that of the

con-trol) was observed upon treatment at 90 J m−2, while a lower

(P<0.05) increase of FAn for ANS (21.05–55.26% higher compared to that of the control), DPH (0.00–40.63% higher

compared to that of the control), and TMA-DPH (80.00–

155.00% higher compared to that of the control) was detected

for lactobacilli cells treated at 30 J m−2. Additionally, UVA,

UVB, and UVC also increased (P <0.05) the FAn of ANS, DPH, and TMA-DPH. Treatment with UVC had shown a stronger (P<0.05) effect in enhancing the FAn of ANS and TMA-DPH, while lactobacilli cells showed a higher (P<0.05)

increase of FAn for DPH when cells were treated with UVB compared to that of the control and other types of UV treatment

studied, except L. acidophilus FTCC 0291 (Fig.7b, Fig.8b,

Fig. 9b) and L. bulgaricus FTCC 0411 (Fig. 7c, Fig. 8c,

Fig.9c).

Discussion

UV radiation has been shown to alter the membrane of microorganisms, and to affect DNA integrity and cell

0 10 20 30 40 50 60 Type of radiation A _{D: P<0.001; T: P<0.001; D x T: P<0.001} 0 10 20 30 40 50 60 Type of radiation D: P<0.001; T: P<0.001; D x T: P<0.001 D 0 10 20 30 40 50 60 Type of radiation B D: P<0.001; T: P<0.001; D x T: P<0.001 0 10 20 30 40 50 60

Control UVA UVB UVC

Type of radiation Cholesterol assimilated (µg mL -1 ) Cholesterol assimilated (µg mL -1 ) Cholesterol assimilated (µg mL -1 ) Cholesterol assimilated (µg mL -1 ) Cholesterol assimilated (µg mL -1 ) D: P<0.001; T: P<0.001; D x T: P<0.001 C 0 10 20 30 40 50 60 Type of radiation D: P<0.001; T: P<0.001; D x T: P<0.001 E without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2

Fig. 3 Removal of cholesterol by control and UV-treated L. acid-ophilus BT 1088 (a), L. acidacid-ophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) upon fermentation at 37°C for 20 h. Control: cells without

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viability. Past studies have reported that, although microbial cells showed a decrease in culturability upon UV radiation

(600–65 kJ m−2_{), cells were able to retain their respiratory}

activity and genomic integrity (Caro et al.1999; Villarino et

al. 2003). In the present study, the viability of lactobacilli

cells decreased immediately upon UV treatment. This may be due to alterations in cellular membranes resulting from the deterioration of membrane lipid causing an injurious

effect on the cells (He et al.2002). Qiu et al. (2005) also

observed that UV radiation was able to inhibit cell division and growth. A similar observation was also reported by Ukuku

and Geveke (2010), where the viability of Escherichia coli

decreased immediately upon UV treatment as more than 90% of the E. coli cells suffered sub-lethal injury immediately upon UV treatment.

However, inactivation or sub-lethal injury of cells upon treatment due to the alteration of cellular membrane is reversible and the bacteria may still be viable. It has been reported that the pores formed on the cellular membrane are

transient (Coronado et al.2005). Our results show that the

viability of lactobacilli cells increased upon fermentation. This may be due to the resealing of pores, which enables the cells to return to their normal state and regain their vital activities. It has also been reported that microorganisms

0 20 40 60 80 Type of radiation A D: P<0.001; T: P<0.001; D x T: P<0.001 0 20 40 60 80 Type of radiation B _{D: P<0.001; T: P<0.001; D x T: P<0.001} D 0 20 40 60 80

Control UVA UVB UVC

Type of radiation Membrane permeability (%) Membrane permeability (%) Membrane permeability (%) Membrane permeability (%) Membrane permeability (%) D: P<0.001; T: P<0.001; D x T: P<0.001 C 0 20 40 60 80 Type of radiation Type of radiation E _{D: P<0.001; T: P<0.001; D x T: 0.021} 0 20 40 60 80 D: P<0.001; T: P<0.001; D x T: P<0.001 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2

Fig. 4 Membrane permeability of control and UV-treated L. acid-ophilus BT 1088 (a), L. acidacid-ophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) immediately upon treatment. Control: cells without treatment. Error

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possess various repair mechanisms against UV damage that operate during exposure to UV to maintain cell viability

(Beggs2002). Therefore, UV-treated cells may be recovered

via cellular repair systems that allow injured cells to repair their biological damage and maintain cell viability.

In addition, the temporary pores formed on the cellular membrane could increase membrane permeability, which will allow free access of molecules into cells (Coronado

et al.2005). Thus, this effect might increase the uptake of

cholesterol from the medium and enhance the assimilation of cholesterol by lactobacilli. In the present study, the

removal of cholesterol by UV-treated cells increased upon

fermentation at 37°C for 20 h. Runyan et al. (2006) also

found that an increase in permeability of the membrane in Pseudomonas aeruginosa resulted in increased diffusion of large-sized proteins from the microbial cell. This phenome-non paralleled the increased membrane permeabilization upon UV treatment.

Our results also showed that UVB and UVC had a higher effect in promoting cell membrane permeability compared to UVA. This was likely due to UVA radiation destabilizing only the outer membrane of the microorganism, while UVB

0 0.5 1 1.5 2 2.5 Type of radiation D: 0.001; T: P<0.001; D x T: 0.225 A 0 0.5 1 1.5 2 2.5 Type of radiation D _{D: 0.001; T: 0.045; D x T: 0.016} 0 0.5 1 1.5 2 2.5 Type of radiation D: P<0.001; T: P<0.001; D x T: 0.848 E 0 0.5 1 1.5 2 2.5 Type of radiation B D: 0.003; T: 0.014; D x T: 0.724 0 0.5 1 1.5 2 2.5

Control UVA UVB UVC

Type of radiation Concentration of malondialdehyde (nmole mL -1) Concentration of malondialdehyde (nmole mL -1) Concentration of malondialdehyde (nmole mL -1) Concentration of malondialdehyde (nmole mL -1) Concentration of malondialdehyde (nmole mL -1) C _{D: 0.004; T: 0.001; D x T: 0.440} without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2

Fig. 5 Membrane lipid peroxidation of control and UV-treated L. acidophilus BT 1088 (a), L. acidophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) immediately upon treatment. Control: cells without treatment. Error

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and UVC could have a higher impact on the cell membrane

bilayer via lipid peroxidation (Berney et al.2007a). Berney

et al. (2007a) also reported that the degree of

permeabiliza-tion was related closely to lipid peroxidapermeabiliza-tion and other membrane damage caused by UV radiation. Considering that lipids are one of the components that form the cellular membrane of microorganisms, oxidative damage to the cel-lular membrane upon exposure to UV radiation due to lipid peroxidation may be observed. However, this may cause the formation of pores on the cellular membrane that subsequently

increase membrane permeability. Thus, the change in mem-brane permeability was due to alteration of the cell envelope. When lipid peroxidation occurs, it could cause an alter-ation in membrane structure via formalter-ation of pores that could increase membrane permeability (Kantar et al.

1992). Bose and Chatterjee (1995) also showed that lipid

peroxidation has a direct positive correlation with mem-brane permeability. In the present study, lipid peroxidation occurred upon UV treatment. UVB- and UVC-treated cells showed a higher production of MDA in medium compared

0 0.5 1 1.5 2 2.5 Type of radiation A _{D: P<0.001; T: P<0.001; D x T: P<0.001} 0 0.5 1 1.5 2 2.5 Type of radiation D: P<0.001; T: P<0.001; D x T: P<0.001 B 0 0.5 1 1.5 2 2.5 Type of radiation D _{D: P<0.001; T: P<0.001; D x T: P<0.001} 0 0.5 1 1.5 2 2.5

Control UVA UVB UVC

Type of radiation Ratio of Cholesterol: Phospholipids Ratio of Cholesterol: Phospholipids Ratio of Cholesterol: Phospholipids Ratio of Cholesterol: Phospholipids Ratio of Cholesterol: Phospholipids D: P<0.001; T: 0.786; D x T: 0.002 C without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 0 0.5 1 1.5 2 2.5 Type of radiation D: 0.007; T: P<0.001; D x T: 0.001 E

Fig. 6 Ratio of cholesterol and phospholipids (C:P) of control and UV-treated L. acidophilus BT 1088 (a), L. acidophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) upon fermentation at 37°C for 20 h. Control: cells

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to cells that treated with UVA. It has been reported that UVB and UVC could produce more reactive oxygen species (ROS) that attack outer and inner layer of membrane of cells, while in the presence of UVA the ROS impact only the outer

mem-brane (Berney et al.2007b). Therefore, more phospholipids

and unsaturated lipids were oxidized by ROS when exposed

to UVB and UVC. He et al. (2002) reported that, upon

treatment, UVB radiation caused a higher increased in lipid peroxidation and/or oxidative stress by producing a higher MDA concentration due to the formation of ROS compared to UVA. This may subsequently result in a decrease in the “survivability” of cells (or the cell survival); however, this damage was reversible and the cells recovered upon treatment

(He et al.2002). This result is in agreement with data showing

decreased viability of lactobacilli cells immediately upon treatment but an increase upon fermentation.

UV radiation has also been found to depolarize the mem-brane potential and reduce memmem-brane resistance, which subse-quently increases membrane permeability, allowing the uptake of molecules from the medium into the cell membrane. These effects were reversible and the cell membrane returns to steady

state upon treatment (Doughty and Hope 1973). Our results

showed that the C:P ratio increased upon UV treatment, as indicated by a deterioration in the phospholipid composition

via lipid peroxidation. Bose and Chatterjee (1995) also

reported an increased ratio of C:P upon treatment, as lipid peroxidation leads to oxidization of membrane phospholipids. Nevertheless, cholesterol has been shown to mediate a protec-tive effect on UV stress by enhanced packing of lipid

mole-cules in the membrane lipid bilayer (Pandey and Mishra1999).

Therefore, cholesterol from the medium may replace that oxidized through UV radiation, thus repairing the damage.

0 0.5 1 Type of radiation ANS (FAn) A _{D: 0.043; T: P<0.001; D x T: 0.005} 0 0.5 1 Type of radiation ANS (FAn) B D: P<0.001; T: P<0.001; D x T: P<0.001 0 0.5 1 Type of radiation ANS (FAn) D _{D: P<0.001; T: P<0.001; D x T: 0.045} 0 0.5 1 Type of radiation ANS (FAn) E D: P<0.001; T: P<0.001; D x T: P<0.001 0 0.5 1

Control UVA UVB UVC

Type of radiation ANS (FAn) D: P<0.001; T: P<0.001; D x T: P<0.001 C without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2

Fig. 7 Fluorescence anisotropy (FAn) of 8-anilino-1-napthalenesul-fonic acid (ANS) of control and UV-treated L. acidophilus BT 1088 (a), L. acidophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) upon

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We have also previously reported that cholesterol was removed from the medium via incorporation of cholesterol

into the cellular membrane (Lye et al.2010b). The increase

in the incorporation of cholesterol into the cellular mem-brane may subsequently lead to an increased ratio of C:P in the membrane.

Therefore, three fluorescent probes—ANS, DPH, and TMA-DPH—were used to determine the location of choles-terol enrichment. ANS is an anionic probe that binds to the interface of apolar and polar regions of phospholipids. DPH is a neutral hydrophobic probe that is incorporated into the inner hydrophobic core of bilayer phospholipids, while TMA-DPH is a cationic derivative of DPH where the charged TMA group is anchored in the polar head of phos-pholipids and the hydrophobic DPH group is incorporated

into the apolar tail of phospholipids (Liong et al.2007). In

the present study, UV treatment increased the FAn of ANS, TMA-DPH, and DPH of the cellular membrane upon fer-mentation. This suggests that the cholesterol was incorpo-rated into different regions of the cellular membrane, including the interface of hydrophobic and hydrophilic regions of phospholipids, hydrocarbon regions of the mem-brane, upper regions of lipid acyl chain, and hydrophilic regions of the phospholipids.

A flip-flop process has been suggested to account for the translocation of compounds through the membrane lipid

bilayer (Kotova et al. 2011). Kotova et al. (2011) reported

that hydrophobic pores were formed upon treatment and that these pores were more permeable to amphiphilic com-pounds such as cholesterol. Thus, cholesterol could be trans-ported from the outer layer of the membrane into the inner layer of membrane due to increased cholesterol binding to

0 0.5 1 Type of radiation D: P<0.001; T: P<0.001; D x T: 0.162 A 0 0.5 1 Type of radiation D: P<0.001; T: P<0.001; D x T: 0.038 B 0 0.5 1 Type of radiation D D: P<0.001; T: P<0.001; D x T: 0.440 0 0.5 1 Type of radiation E _{D: P<0.001; T: P<0.001; D x T: P<0.001} 0 0.5 1

Control UVA UVB UVC

Type of radiation

TMA-DPH (FAn)

TMA-DPH (FAn) TMA-DPH (FAn)

TMA-DPH (FAn) C D: P<0.001; T: P<0.001; D x T: 0.002 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2

Fig. 8 FAn of 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) of control and UV-treated L. acidophilus BT 1088 (a), L. acidophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) upon

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hydrophobic regions of the phospholipids. It has also been reported that the replacement of lipids may take place to repair cellular membrane damage and to rebuild the cellular

membrane against further damage (He et al.2002). Therefore,

the UV-treated cells may take up cholesterol, and incorporate this cholesterol into the cellular membrane to repair the cellu-lar membrane via hydrophobic pores; thus, there was an increase in the cholesterol incorporated in the hydrophobic regions of phospholipids upon UV treatment.

In addition, the replacement of damaged lipids also facil-itated the recovery of growth from inhibition due to UV

radiation (He et al. 2002), as indicated by the increased

viability of lactobacilli upon fermentation. Nevertheless,

Smith et al. (2009) also suggested that not only hydrophobic

pores but also hydrophilic pores/channels were formed on the cellular membrane upon UV exposure, which may lead to increased free access of molecules. Moreover, exchange of phospholipids between the two monolayers is favored upon treatment due to lipid peroxidation (Kotova et al.

2011). This may also enable the cholesterol to move around

the membrane lipid bilayer and thus be incorporated into apolar and/or polar regions of phospholipids, and also inter-face between the apolar and polar regions of phospholipids. In conclusion, UV radiation caused alterations in the cell membrane that increased membrane permeability due to increased lipid peroxidation. This caused a decrease in the viability of lactobacilli cells immediately upon treatment. However, membrane permeability due to pore formation

0 0.5 1 Type of radiation DPH (FAn) A _{D: 0.068; T: 0.001; D x T: P<0.001} 0 0.5 1 Type of radiation DPH (FAn) E _{D: 0.002; T: 0.001; D x T: P<0.001} 0 0.5 1 Type of radiation DPH (FAn) D: P<0.001; T: P<0.001; D x T: 0.016 D 0 0.5 1 Type of radiation DPH (FAn) B _{D: P<0.001; T: P<0.001; D x T: P<0.001} 0 0.5 1

Control UVA UVB UVC

Type of radiation DPH (FAn) C _{D: 0.001; T: P<0.001; D x T: 0.596} without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2 without treatment 30 J m-2 60 J m-2 90 J m-2

Fig. 9 FAn of 6-diphenyl-1,3,5-hexatriene (DPH) of control and UV-treated L. acidophilus BT 1088 (a), L. acidophilus FTCC 0291 (b), L. bulgaricus FTCC 0411 (c), L. bulgaricus FTDC 1311 (d), and L. casei BT 1268 (e) upon fermentation at 37°C for 20 h. Control: cells without

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was transient, and such treatment effects on cells were revers-ible. Upon fermentation, the viability of lactobacilli then increased. The increase in membrane permeability led to increased assimilation and incorporation of cholesterol into cellular membrane. These occurrences also led to an increase in the ratio of C:P in cellular membrane of lactobacilli, accom-panied by increased saturation of cholesterol in apolar regions of phospholipids, polar regions of membrane, and interface between apolar and polar regions of the membrane bilayer.

Acknowledgments This work was supported by the RU grant (1001/ PTEKIND/811089), IPS-Research Fund Grant, and the USM Fellowship (1001/441/CIPS/AUPE001) provided by Universiti Sains Malaysia.

References

Beggs CB (2002) A quantitative method for evaluating the photoreac-tivation of ultraviolet damaged microorganisms. Photochem Photobiol Sci 1:431–437

Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007a) Assessment and interpretation of bacterial viability by using the LIVE/DEAD Baclight kit in combination with flow cytometry. Appl Environ Microbiol 73:3283–3290

Berney M, Weilenmann HU, Egli T (2007b) Adaptation to UVA radiation of Escherichia coli growing in continuous culture. J Photochem Photobiol B 86:149–159

Bose B, Chatterjee SN (1995) Correlation between UVA-induced changes in microviscosity, permeability, and malondialdehyde formation in liposomal membrane. J Photochem Photobiol B 28:149–153

Caro A, Got P, Lesne J, Binard S, Baleux B (1999) Viability and virulence of experimentally stressed nonculturable Salmonella typhimurium. Appl Environ Microbiol 65:3229–3232

Chadsuthi S, Wong-ekkabut J, Triampo W, Doungchawee G, Triampo D (2010) Comparison of the effects of UVA radiation on Leptospira interrogan serovar Bataviae Canicola and Pomona. Afr J Biotechnol 9:3196–3206

Coronado JM, Soria J, Conesa JC, Bellod R, Adan C, Yamaoka H (2005) Photocatalytic inactivation of Legionella pneumophila and an aerobic bacteria consortium in water over TiO2/SiO2fibers in a continuous reactor. Top Catal 35:279–286

Doughty CJ, Hope AB (1973) Effects of ultraviolet radiation on the membranes of Chara coralline. J Membr Biol 13:185–198 FAO/WHO (2001) Health and nutritional properties of probiotics in

food including powder milk with live lactic acid bacteria—Joint Food and Agricultural Organization of the United Nations and World Health Organization Expert Consultation Report, Córdoba, Argentina. Available from: http://www.who.int/foodsafety/ publications/fs_management/probiotics/en/index.html. [Accessed 7 August 2010]

Giamarellos-Bourboulis EJ, Skiathitis S, Dionyssiou-Asteriou A, Hatziantoniou S, Demetzos K, Dontas I, Papaioannou GT, Karatzaz G, Helen G (2003) Lipid peroxidation by Pseudomonas aeruginosa in the pathogenesis of nosocomial sepsis. J Postgrad Med 49:11–16

He YY, Klisch M, Hader DP (2002) Adaptation of Cyanobacteria to UVB stress correlated with oxidative stress and oxidative damage. Photochem Photobiol 76:188–196

Hortnag P, Perez MT, Sommaruga R (2010) Contrasting effects of ultraviolet radiation on the growth efficiency of freshwater bacteria. Aquat Ecol 45:125–136

Howland MC, Parikh AN (2010) Model studies of membrane disrup-tion by photogenerated oxidative assault. J Phys Chem 114:6377– 6385

Kantar A, Giorgi PL, Curatola G, Fiorini R (1992) Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study. Biol Cell 75:135–138

Kotova EA, Kuzevanov EV, Pashkovskaya AA, Antonenko YN (2011) Selective permeabilization of lipid membranes by photodynamic action via formation of hydrophobic defects or pre-pores. BBA Biomembr 1808:2252–2257

Liong MT, Shah NP (2005) Acid and bile tolerance and the cholesterol removal ability of bifidobacteria strains. Biosci Microflora 24:1–10 Liong MT, Dunshea FR, Shah NP (2007) Effects of a symbiotic containing Lactobacillus acidophilus ATCC 4962 on plasma lipid profiles and morphology of erythrocytes in hypercholesterolemic pigs on high- and low-fat diets. Br J Nutr 98:736–744

Lye HS, Kuan CY, Ewe JA, Fung WY, Liong MT (2009) The improve-ment of hypertension by probiotics: effects on cholesterol, diabetes, rennin, and phytoestrogens. Int J Mol Sci 10:3755–3775

Lye HS, Rusul G, Liong MT (2010a) Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract. Int Dairy J 20:169–175

Lye HS, Rusul G, Liong MT (2010b) Removal of cholesterol by lactobacilli via incorporation and conversion to coprostanol. J Dairy Sci 93:1383–1392

Nguyen TDT, Kang JH, Lee MS (2007) Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol lowering effects. Int J Food Microbiol 113:358–361

Ogura R, Sugiyama M, Sakanashi T, Suematsu T, Hidaka T, Morikawa F, Kon Y (1989) Membrane responses of B-16 melanoma cells to single exposure to ultraviolet light. Arch Dermatol Res 280:481–486 Ooi LG, Bhat R, Rosma A, Yuen KH, Liong MT (2010) A synbiotic

containing Lactobacillus gasseri CHO-220 and inulin improves irregularity of red blood cells. J Dairy Sci 93:4535–4544 Pandey BN, Mishra KP (1999) Radiation induced oxidative damage

modification by cholesterol in liposomal membrane. Radiat Phys Chem 54:481–489

Patel AK, Campbell AK (1987) The membrane attack complex of com-plement induces permeability changes via thresholds in individual cells. Immunol 60:135–140

Pereira DIA, Gibson GR (2002) Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut. Appl Environ Microbiol 68:4689–4693

Qiu XY, Sundin GW, Wu LY, Zhou JZ, Tiedje JM (2005) Comparative analysis of differentially expressed genes in Shewanella oneidensis MR-1 following exposure to UVC, UVB, and UVA radiation. J Bacteriol 187:3556–3564

Runyan CM, Carmen JC, Beckstead BL, Nelson JL, Robison RA, Pitt WG (2006) Low frequency ultrasound increases outer membrane permeability of Pseudomonas aeruginosa. J Gen Appl Microbiol 52:295–301

Smith HL, Howland MC, Szmodis AW, Li Q, Daemen LL, Parikh AN, Majewski J (2009) Early stages of oxidative stress induced membrane permeabilization: a neutron reflectometry study. J Am Chem Soc 131:3631–3638

Ukuku DO, Geveke DJ (2010) A combined treatment of UV light and radio frequency electric field for the inactivation of Escherichia coli K-12 in apple juice. Int J Food Microbiol 138:50–55 Vesa TH, Marteau P, Zidi S, Briet F, Pochart P, Rambaud JC (1996)

Digestion and tolerance of lactose from yoghurt and different semi-solid fermented dairy products containing Lactobacillus acidophilus and bifidobacteria in lactose maldigesters—is bacterial lactase important? Eur J Clin Nutr 50:730–733 Villarino A, Rager MN, Grimont PAD, Bouvet MM (2003) Are