Effect of Curcumin Mediated Photodynamic Technology on Salmonella typhimurium Biofilm and Its Bactericidal Mechanism and Application in Milk

Rich in nutrients and welcomed by customers of all ages, milk also provides favorable conditions for foodborne pathogens. It has a high risk of contamination by foodborne pathogens such as Bacillus cereus [1], Cronobacter sakazakii [2] and Salmonella spp. [3] Salmonella is one of the top-four foodborne pathogens leading to high morbidity and mortality [4,5]. Cases of 93.8 million gastroenteritis are caused by Salmonella worldwide each year, resulting in nearly 155,000 deaths [6], and S. typhimurium is one of the most common serotypes [7]. Reports on milk in particular infant formula milk powder contaminated by Salmonella always caused widespread social concern and economic loss [8,9]. Besides bacteria existing in raw milk, bacteria contaminating processing equipment can easily form biofilms then contaminate products. Biofilm has been regarded as an intractable problem in dairy industry, since the formation of biofilm will increase the resistance of bacteria to adverse environment and weaken the effect of disinfection [10]. Therefore, the control of Salmonella biofilm that may contaminate food contact surfaces and planktonic Salmonella in milk is of great importance during dairy product processing.
Traditionally, thermal sterilization is the most commonly employed method to inactivate foodborne pathogens, but it may lead to the loss of heat-sensitive nutrients and the change of organoleptic profiles [11]. Therefore, non-thermal processing technologies such as disinfectant, high-pressure processing and irradiation are considered to be potential for foodborne bacteria disinfection. Recently, however, the shortcomings of these nonthermal techniques were discussed. The problems of toxic residues, high cost, professional requirement for operation and deterioration of nutritional and organoleptic properties of food make it urgent to find a novel alternative non-thermal technique to ensure the quality and safety of milk product [12]. Photodynamic technology (PDT), without above-mentioned disadvantages, is a promising technique to prevent pathogens based on photochemical reactions [13,14].
In this process, the photosensitizer is activated by light at specific wavelength and releases energy to form super-oxide, hydroxyl radical or singlet oxygen which can react with adjacent biological molecules to produce bacterial toxicity, leading to damage or death of pathogens [15-17]. In recent years, increasing PDT studies based on natural edible photosensitizers especially curcumin have showed that PDT can inactivate a wide range of foodborne bacteria with marginal damage to quality of various food such as fruits, vegetables, meat and seafood products [18- 21]. Comparative research studies pointed out that S. typhimurium can be effectively inactivated by PDT although it is more resistant to this treatment as compared to other bacterial species [20,22- 24]. To our knowledge, no investigation has been carried out to test curcumin-PDT effect on bacteria in milk and little literature is available on curcumin-PDT to S. typhimurium biofilm. The aims of this study, therefore, were to investigate curcumin-PDT inactivation effect on S. typhimurium biofilm, explore preliminary inactivation mechanism on planktonic S. typhimurium cells, and apply this technique in milk.

Bacterial culture preparation

S. typhimurium ATCC 14028 was gifted by Guangdong Provincial Center for Disease Prevention and Control (CDC), China. To obtain working culture, a single colony was picked and enriched in 5mL sterile tryptic soy broth (TSB) (Qingdao Hope Bio-Technology Co., Ltd, Qingdao, China), which was agitated at 37 ℃ and 120rpm for 16h. The planktonic cells were harvested by centrifugation at 3532×g for 10min, washed three times and re-suspended in phosphate-buffered saline (PBS) (109CFU/mL).

Photosensitizer and light source

Curcumin powder (>95%, Ci Yuan Biotechnology Co. LTD., Shanxi, China) was dissolved in ethanol (99%) as 20mM stock solution which was then diluted with sterile water to obtain a series of working concentrations. All the solutions were stored in the dark at 4 ℃ before use. The ethanol concentration in working solutions was <1% (v/v). It is worth pointing out that curcumin, as a food additive, can be used in a range of 50-500mg/L or mg/kg in different foods [23].
A blue 450nm laser (LWBL450-10W-F, Beijing Laser wave Optoelectronics Technology Co., LTD, China) was used as the light source and placed at 17cm distance above the sample. Energy density on the surface of sample was (200mW/cm2) measured with a Laser Power Meter (LI-P20W-A, Beijing Laser wave Optoelectronics Technology Co., LTD, China).

PDT treatment on S. typhimurium biofilm

Every 200μL bacterial suspension and 5mL TSB were transferred to one well of 6-well plate. A microscope cover glass (20mm×20mm, 10212020C, Jiangsu, Citotest Scientific Co., Ltd, China) was put in each well and incubated at 30 ℃ for 48h to form biofilm. Then the TSB was removed, and the cover glass was washed with sterile PBS for three times. The biofilm on cover glass was incubated with a series of curcumin (0, 10, 20, 40, 80μM) in the dark at 37 ℃ for 5min. Then samples were illuminated at room temperature for 0, 1, 10, 20, 30 min, respectively. Experiments were divided into four groups: L-S- group (no light and curcumin), L-S+ group (curcumin alone), L+S- group (light alone) and L+S+ group (both light and curcumin). After illumination, the cover glass was placed in a centrifuge tube containing 5mL saline solution and treated by ultrasound for 10 min. One mL treated suspension was taken for 10-fold serial dilution followed by culturing on TSA plate for counting the viable bacteria. Each experiment was in triplicates.

Preliminary mechanism of bactericidal effect on planktonic S. typhimurium

Agarose gel electrophoresis analysis of S. typhimurium DNA: The planktonic cells were prepared as description in the section of bacterial culture preparation. Each aliquot (1.5mL) of bacterial suspension with 3.5mL curcumin (80μM) or PBS was mixed in one well of 6-well plate then illuminated for 30min. Treated samples were used for extracting bacterial DNA by bacterial total DNA extraction kit (Tian Gen Biochemical Technology Co. LTD., Beijing, China). Harvested DNA products were separated by 1.5% agarose gel electrophoresis, stained by golden view and visualized under UV light.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of S. typhimurium protein: Each aliquot (1.5mL) of bacterial suspension with 3.5mL curcumin (80μM) or PBS was mixed in one well of 6-well plate then illuminated for 30min. Each sample was collected (4mL) by centrifugation at 6149×g for 1min. The pellet was re-suspended in 20μL sterile water first and 20μL 2×SDS-gel loading buffer afterwards. After heating for 5 min at 95 ℃, the samples were centrifuged for another 2min. Ten μL supernatant was loaded and separated by 5% stacking gel and 12% separation gel electrophoresis (PowerPac™ Basic, Bio-Rad, America). Then the gel was stained by Coomassie Brilliant Blue.

Scanning electron microscopy (SEM) observation of S. typhimurium morphology

Each aliquot (1.5mL) of bacterial suspension with 3.5mL curcumin (80μM) or PBS was mixed in one well of 6-well plate and then illuminated for 30min. Each sample was collected (4mL) by centrifugation at 3532×g for 10min and washed with PBS for three times. The pellet was fixed in 1mL glutaraldehyde (2.5%) for 12h at 4 ℃ then dehydrated in ethanol solutions of graded series concentrations (once at 30, 50, 70, 80 and 90%, and twice at 100%) before critical point drying in carbon dioxide. Finally, samples were coated with gold and observed under SEM (Supra55, Zeiss, Germany).

Confocal laser scanning microscope observation of S. typhimurium membrane permeability: Change of cell permeability after PDT treatment was measured by LIVE/DEAD® BacLight™ bacterial viability kit (Invitrogen, Thermo Fisher Scientific, America). Biofilm was formed on the cover glass and exposed to 80μM curcumin combined 30-min light illumination treatment (L+P+) or untreated (L-P-). Subsequently, the glass was picked up and washed for three times by sterile water. Mixture (150μL) of SYTO9 and propidium iodide (PI) was added to cover the sample on glass surface, followed by a 15-min dark incubation at 37 ℃ and washing for three times. Then samples were added a drop of anti-fluorescence quenching solution respectively, prior to their observation by confocal laser scanning microscope (CLSM, LSM880, Zeiss, Germany).

PDT treatment on planktonic S. typhimurium in milk

Table 1: Instruction of milk solution preparation.

*BS is base saturation

Infant formula (Friso, Friesland Campina (Hong Kong) Limited, China), instant milk powder (Devondale, Murray Goulburn Co- Operative Co. Limited, Australia) and instant skim milk powder (Devondale, Murray Goulburn Co-Operative Co. Limited, Australia) were purchased from local supermarket in Guangzhou. Different types of milk solutions were prepared according to instructions given by manufacturers (Table 1). Curcumin stock solution (0.10mL, 20mM) compounded with 24.90mL and 16.57mL milk solutions respectively to gain 80μM and 120μM turmeric milk.
To test the effect of curcumin concentration on bactericidal efficacy, 1mL bacterial aliquot was mixed with the equal volume of PBS, 80μM and 120μM turmeric milk solutions respectively in a 6-well plate. One mL or 2mL bacterial aliquot was mixed with the equal volume of PBS and 120μM turmeric milk solutions respectively to test the effect of liquid level (0.2cm or 0.4cm) on bactericidal efficacy. Then the turmeric milk solutions were diluted to gain double dilution milk, and 1mL bacterial aliquot was mixed with the equal volume of PBS, 120μM raw turmeric milk and 120μM double diluted turmeric milk solutions respectively to test the effect of milk dilution on bactericidal efficacy. All plates after incubation in the dark at 37 ℃ for 5 min, were placed under the blue light to illuminate for 30min. One mL treated sample was then taken for 10-fold serial dilution, and 100μL of each dilution was spread on TSA plate and incubated at 37 ℃ for 16h for counting viable cells. The infant formula milk solution was diluted to gain twofold and tenfold dilution milk for testing light transmittance. The light transmittance of milk samples was measured by UV spectrophotometer. Each experiment was in triplicates.

Statistical analysis

All data are expressed as mean ± standard deviation (SD) from three separate experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) using Origin 9.0 software.

PDT inactivation of S. typhimurium biofilm

Effect of exposure time: The effect of illumination time on PDT inactivation of S. typhimurium biofilm was shown in Figure 1. Illumination alone did not affect the S. typhimurium biofilm viability obviously, even the illumination time was prolonged to 30min. Curcumin (20μM) combined with blue light had the inactivation effect on S. typhimurium biofilm, and 1.9lg/(CFU/mL) viability decrease was obtained after 30 min PDT treatment.

Figure 1: Effect of illumination time on PDT inactivation of S. typhimurium biofilm. Different upper letters and lowercase letters represent significant difference in S- group (without curcumin) and S+ (with curcumin, 20μM) group respectively.

Effect of curcumin concentration: Data in Figure 2 demonstrated that curcumin had a mild bactericidal effect by itself. Eighty μM curcumin could reduce approximate 0.6lg/(CFU/mL) biofilm viability. When combined with blue light, curcumin showed more significant inactivation effect on S. typhimurium biofilm. The inactivation effect was strengthened when curcumin concentration increased, and 80μM curcumin reduced the most biofilm viability (more than 2lg/(CFU/mL)) after 20-min illumination. A previous study reported that curcumin treatment alone caused cell membrane damage and cell death because the insertion of curcumin into the liposome bilayer enhanced cell membrane permeability [25,26], but it took longer time to achieve the same bactericidal effect as PDT. Previous studies pointed out that grampositive bacteria were more susceptible than gram-negative bacteria [23,24] and bacterial biofilm inactivation by PDT was more difficult than planktonic bacteria [27,28]. Present viability results suggested that curcumin-PDT could penetrate extracellular polymeric substance (EPS) and injured bacteria inside. Penha et al. [23] examined the bactericidal effect of 75μM curcumin and 30 min blue LED (λmax 470nm) illumination, showing a 2.82log CFU/mL reduction of S. typhimurium. This treatment conditions and results were quite similar to that in present study, indicating that curcumin-PDT can also be a potential strategy for controlling both planktonic S. typhimurium and its biofilm. Bonin et al. [24] exposed S. typhimurium to the combined treatment of 10μM eosin Y and 15-min green LED (λmax 490-570nm) illumination, causing a 1.7 log CFU/mL reduction. Santos et al. [22] compared PDT effects mediated by eosin and rose bengal respectively on S. typhimurium, and 100μM eosin combined 15-min illumination (530±40nm) could only lead to about 2 log reduction. In contrast, 50μM rose bengal with 15-min illumination (530±40nm) led to the total inactivation. Although, some photosensitizers in PDT treatment could result in very desirable inactivation effects, they are synthesized dyes and not suitable for food application.

Figure 2: Effect of curcumin concentration on PDT inactivation of S. typhimurium biofilm. Different upper letters and lowercase letters represent significant difference in L- (without light) group and L+ (with light, 20min) group respectively.

Preliminary mechanism of PDT inactivation of S. typhimurium biofilm

Damage of bacterial DNA: In Figure 3, the intensity of DNA band in L-S+ group showed a marginal decrease compared with bands in L+P+ and L+P- groups, indicating that light alone could not damage bacterial DNA but 80μM curcumin treatment alone might lead to a slight DNA damage. The DNA band in L+S+ group was the weakest, showing the most significant damage compared with other groups, but still not disappeared completely. It is consistent with the DNA damage of Vibrio parahaemolyticus by MB-PDT reported by Deng et al. [29]. Other studies have shown that PDT enhanced the control of pathogens by attenuating quorum sensing (QS) dependent factors, such as production of exopolysaccharide and alginate, swimming ability and virulence factors [30-32]. However, this study only showed the PDT effect on total bacterial DNA by SDS-PAGE for a preliminary conclusion. The quantification of gene damage and analyzing specific gene damage caused by PDT can be further carried out by real-time polymerase.

Figure 3: Agarose gel electrophoresis analysis of S. typhimurium DNA. M: Marker, DL 2000. L-S-: no light and curcumin. L+S-: light alone (30min). L-S+: curcumin alone (80μM). L+S+: both light (30min) and curcumin (80μM).

Damage of bacterial protein: In Figure 4, protein degradation of S. typhimurium was analyzed by SDS-PAGE. Intensity of protein bands in L-P- (control), L-P+ (only curcumin), L+P- (only light) groups did not change obviously, showing that compared with control group, neither individual curcumin nor only light treatment could affect S. typhimurium protein. In contrast, slight decreasing intensity of protein band was observed in L+P+ group, suggesting that bacterial protein degradation might be caused by 80μM curcumin in combination with 30 min illumination treatment. Results obtained in this study proved curcumin-PDT caused damage of S. typhimurium total protein. Similarly, Li et al. [21] observed a slight degradation of total Salmonella spp. protein by riboflavinmediated PDT. Wu et al. [33] showed that PDT damaged bacterial outer membrane protein. But hardly any report has further studied other PDT-targeted proteins of bacteria. Actually, the oxidative burst of reactive oxygen species (ROS) produced by PDT will break the balance of microorganism homeostasis and attack those substances playing important physiological roles in cells such as RNA and lipid, besides DNA and protein [34].

Figure 4: SDS-PAGE analysis of S. typhimurium protein. M: Marker (5-245kDa). L-S-: no light and curcumin. L+S-: light alone (30min). L-S+: curcumin alone (80μM). L+S+: both light (30min) and curcumin (80μM).

Change of bacterial morphology: Morphological change of S. typhimurium after PDT treatment was shown in Figure 5. The bacteria of L-S- group were oval and plump, while bacterial cells in L+S+ group were wrinkled and irregular, even some cracks occurred on the surface (see arrows in Figure 5B). This result visually showed that curcumin-PDT distorted morphology of S. typhimurium but did not cause collapse of cells. Chai et al. [35] observed no significant change of L. monocytogenes morphology after curcumin-PDT by SEM, but further transmission electron microscopy (TEM) observation found partial cytoplasm cavitation. Therefore, the main target of PDT might be the disruption of intracellular biological damage.

Figure 5: SEM images of S. typhimurium. A: L-S-, no light and curcumin. B: L+S+, both light (30min) and curcumin (80μM).

Change of membrane permeability: Since membrane integrity is indispensable for bacterial survival, based on the different transmembrane ability of SYTO 9 and PI, live and dead cells can be distinguished by double stains SYTO 9 and PI. SYTO 9 can cross cell membrane and label all bacteria with green fluorescence when used alone while PI can only penetrate cells when membrane injured or disrupted and present red fluorescence [36]. CLSM was used to observe the permeability of bacteria in biofilms after PDT treatment of 80μM curcumin in combination with 30-min illumination. As shown in Figure 6, cells in L-S- group were green, showing bacterial membrane remaining intact and blocked PI stain outside the cells. In contrast, most cells in L+S+ group presented red, indicating PDT treatment caused membrane permeability damage and PI can went inside. Therefore, the result suggested that curcumin-PDT could penetrate or disrupted bacterial EPS and induced S. typhimurium permeability damage, leading to lethal injuries of inside bacteria. However, how PDT interacted and affected protective bacterial EPS could not be explained in present study. Further study on PDT effect on EPS in detail needs to be carried out.

Figure 6: Live/dead fluorescent staining images of S. typhimurium. A: L-S-, no light and curcumin. B: L+S+, both light (30 min) and curcumin (80μM).

PDT inactivation of S. typhimurium in milk

Results in Figure 7 showed that when PDT was applied in milk, type of milk, curcumin concentration, illuminated liquid level and liquid transmittance all affected inactivation efficacy. In Figure 7A, PDT inactivation effect on S. typhimurium in full cream milk was the weakest in terms of three types of milk. Increasing curcumin concentration to 120μM could decrease milk-borne S. typhimurium in full cream milk by around 0.5lg/(CFU/mL) while about 0.8lg/ (CFU/mL) decline was observed in other two kinds of milk. The difference might be attributed to the lipid content in milk powder. Since the external structural integrity of gram-negative bacteria is closely related to lipopolysaccharide (LPS), the higher level of lipid content in milk may be more conducive to maintaining the lipid soluble structure of bacteria. Undesirable bactericidal effect in milk was also observed by Wang et al. [37] Staphylococcus aureus in milk was just reduced by 0.42 log after Na-chlorophyllin- PDT treatment, while 4.5 log bacteria could be reduced in saline solution. Such a significant difference was presumed to be caused by large solid particles in milk which sheltered and returned light to block PDT reaction. Galstyan [38] considered that reduction of bactericidal effect in milk was due to aggregation effect of casein and whey protein on photosensitizer, which changed the maximum absorption spectrum of photosensitizer. The existence of cysteine could also quench the singlet oxygen produced during the photodynamic process. In addition, calcium and magnesium ions in the emulsion could stabilize the negative charge in oligosaccharide chains and might seriously affect the binding of photosensitizer to gram-negative bacteria.
Figure 7B showed that the PDT effect did not change rapidly when the liquid level of full cream and skim milk changed, while the bactericidal effect on infant formula milk was totally inhibited when the illuminated liquid level changed from 0.2cm to 0.4cm. It might be that the infant formula milk contained more trace elements and minerals (iron, zinc, vitamins), so blue light could not penetrate the emulsion effectively, and the light intensity reaching the surface and bottom of the sample was unequal, which greatly reduced the bactericidal effect.
Besides, the bactericidal effect was significantly enhanced with the increase of milk dilution ratio (Figure 7C). Especially for skim milk, S. typhimurium reduction in double diluted milk was the most, reaching almost 1.4lg/(CFU/mL). Figure 7D revealed that transmittance of milk increased with the increase of dilution ratio, indicating the positive correlation between PDT efficacy and solution transmittance. Wang et al. [37] also proved it by comparing the PDT inactivation effect on S. aureus in clear and cloudy litchi juice. Therefore, application of PDT in cloudy liquid food will be more difficult.

Figure 7: Factors involved in PDT effect on S. typhimurium in milk.
A. Curcumin concentration (80μM+0.2 cm; 120μM+0.2cm).
B. Liquid level (120μM+0.2cm; 120μM+0.4cm).
C. Milk dilution ratio (raw milk; double diluted milk).
D. Light transmittance of milk with different dilution ratio.

This study showed that curcumin in combination with blue light could inactivate S. typhimurium biofilm by more than 2lg. For planktonic S. typhimurium, curcumin-PDT could cause genomic DNA damage and protein degradation, and distorted S. typhimurium morphology, induced membrane damage and permeability change, leading to S. typhimurium cell death. When curcumin-PDT was adopted to control S. typhimurium in milk, type of milk, curcumin concentration, illuminated liquid level and liquid transmittance all affected PDT efficacy. Curcumin (120μM) in combination with illumination (30min) obtained the best bactericidal effect in double diluted skim milk with a liquid level of 0.2cm.
Even though inactivation effects of curcumin-PDT were observed on both S. typhimurium biofilm in vitro and planktic S. typhimurium cells in milk, further investigations are necessary to be implemented. Firstly, the curcumin-PDT inactivation of S. typhimurium biofilm in food or food contact surfaces should be carried out. Secondly, it is important to figure out the interactions between milk compositions (such as protein and fat) and curcumin and light, and the attributes change of milk before and after treatment. Thirdly, since its limited penetrability in cloudy solution, the possibility of combination with other bactericidal technologies also deserves future investigation. Furthermore, comparative studies of curcumin-PDT in various products and foodborne pathogens can be valuable and helpful to identify its accurate application.

The authors are grateful to the National Key Research and Development Project (2018YFG1602504), China-New Zealand Joint Research Center for Food Safety & Nutrition (20200502) and Fujian Provincial Collaboration Project (2020I0010) for their financial supports.

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