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Ultrasound: Enhance the detachment of exosporium and decrease the hydrophobicity of Bacillus cereus spores
LWT - Food Science and Technology 116 (2019) 108473
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Short communication
Ultrasound: Enhance the detachment of exosporium and decrease the hydrophobicity of Bacillus cereus spores
T
Ruiling Lva, Mingming Zoua, Weijun Chena, Jianwei Zhoua,c, Tian Dinga, Xingqian Yea, Donghong Liua,b,c,∗ a College of Biosystems Engineering and Food Science, National-Local Joint Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang University, Hangzhou, 310058, China b Fuli Institute of Food Science, Zhejiang University, Hangzhou, 310058, China c Ningbo Research Institute, Zhejiang University, Ningbo, 315100, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacillus cereus spores Ultrasound Surface property Exosporium Adhesion
Because of the hydrophobicity and formation of biofilm, Bacillus cereus spores are highly resistant and widespread in food industry. The outermost layer: exosporium of Bacillus cereus spore plays an important role in its surface property. Previous work indicated that ultrasound detached the exosporium of spores, thus the surface properties were further investigated in this study. The detachment of exosporium was observed without damage to inner layers through TEM micrographs. Besides, the size of spores dropped rapidly from 2017.67 to 960.53 nm after 1 min ultrasound treatment. At the same time, the absolute zeta potential value also decreased rapidly. The ratio of hydrophobic spores (RHS) was defined as hydrophobicity, and it dropped-off with the detachment of exosporium. The SDS-PAGE results showed that the intensity of protein bands dropped especially that with higher molecular weight after ultrasound treatment. What's more, a lot of bands disappeared because of the separation of exosporium. In a word, these results indicated that ultrasound could detach exosporium and decrease the hydrophobicity of spores, which had lower adhesion to hydrophobic surface and easier to be cleaned.
1. Introduction
and environment established by the surface layers of spores (exosporium and coat) (Henriques & Moran, 2007). Bacillus cereus spores have a typical balloon-like outermost layer: exosporium, which loosely attaches in the surface occupying a large space (Rönner, Husmark, Henriksson, & livmedelsinstitutet, 1990). The absence of the exosporium would change the size of spores significantly. The exosporium plays important roles in protection, adhesion and virulence of spores (Henriques & Moran, 2007). It is a barrier to larger molecules providing resistance to enzymes and chemicals (Stewart, 2017). The exosporium interacts with environment and host cells of the immune system by virtue of its outermost location (Henriques & Moran, 2007). Furthermore, the presence of exosporium makes great contribution to the remarkable adhesion of Bacillus cereus spores (Faille, Tauveron, GentilLelievre, & Slomianny, 2007). Zhou assessed the effect of germination and sporulation conditions on the adhesion of Bacillus spores with fluid dynamic gauging technique. They found that spores with exosporium (Bacillus cereus) were much more resistant to shear stress than those without exosporium (Bacillus subtilis) (Zhou, Li, Christie, & Wilson, 2017).
Bacillus cereus (B. cereus) is ubiquitous and widespread in food and food industry especially those associated with soil (Tauveron, Slomianny, Henry, & Faille, 2006). It is frequently reported that Bacillus cereus contaminated both raw and processed foods including rice, vegetables, meat or dairy products thus resulted in outbreaks (Hariram & Labbe, 2016). Moreover, as a gram-positive and spore-forming bacterium, B. cereus could survive in hash conditions by forming biofilm and spores. Bacteria and spores in biofilm are highly resistant to cleaning processing which are of great concern to food industry (Zou & Liu, 2018). Previous studies demonstrated that B. cereus strains could produce diarrhoeal toxins and emetic toxins, causing diarrheal or emetic disease (Finlay, Logan, & Sutherland, 2000). Particularly, Bacillus cereus spores are resistant to stresses such as UV radiation, heat, chemicals and can stay dormant for decades. Spores germinate into vegetative cells once the environment outside is suitable for growth. The extraordinary resistance might be related to the multilayer compact structure. Particularly, the interactions between spores
∗ Corresponding author. College of Biosystems Engineering and Food Science, National-Local Joint Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang University, Hangzhou, 310058, China. E-mail address: [email protected] (D. Liu).
https://doi.org/10.1016/j.lwt.2019.108473 Received 4 April 2019; Received in revised form 31 July 2019; Accepted 1 August 2019 Available online 02 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 116 (2019) 108473
R. Lv, et al.
EM UC7) and observed on transmission electron microscope (JEM1230, JEOL, Japan).
Food processing surfaces such as valves, pipes, and pumps (off systems) or conveyors (open systems) in dairy industry, were regularly found to be contaminated by Bacillus spp., Escherichia coli, or Listeria monocytogenes. (Faille et al., 2002). Bacteria could adhere to surfaces, replicate and even form biofilm, causing shorter shelf-life and even safety issues. Compared with vegetative cells, the adhesion of spores was generally much stronger to both hydrophobic and hydrophilic surfaces (Rönner et al., 1990). The main reason was the different structures of spores and vegetative cells. Overall, the surface properties are closely related to the adhesion of Bacillus cereus spores. However, there is no direct evidence showing the relationship between the absence of the exosporium and the surface property. Pervious work evaluated that ultrasound could detach the exosporium of Bacillus cereus spores (Lv et al., 2019). Thus, this study further evaluated the size and surface property changes of Bacillus cereus spores with the detachment of exosporium after ultrasonic treatments.
2.6. Hydrophobic measurements The hydrophobic property of spores was measured using a partition method which based on the affinity to hexadecane (Noma et al., 2018; Wiencek, Klapes, & Foegeding, 1990). The initial absorbance at 600 nm of each sample was measured ( A0 ) using a spectrophotometer (1510–02691, Thermo Fisher Scientific Oy Ratastie 2, Fl-01620, Vantaa, Finland). Then, treated and untreated spore suspensions (3 mL) were mixed with 0.6 mL hexadecane and vortexed for 30 s. The absorbance of the aqueous phases was tested at 600 nm after holding for 15 min for phase separation ( Af ), the decrease in absorbance was the value of relative surface hydrophobicity of spores. The hydrophobicity of spores was defined as the ratio of hydrophobic spores (RHS).
RHS (%) = 2. Materials and methods 2.1. Materials
A0 − Af A0
*100%
2.7. Total protein extraction and SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Both nutrient agar and broth were from Hope Bio-Technology Co., Ltd. (Qingdao, China). Hexadecane was obtained from Sigma-Aldrich Co (St. Louis, MO, USA). RIPA lysis buffer, protein loading dye and all the other SDS-PAGE related chemicals were purchased from Sangon Biotech Co., Ltd (Shanghai, China).
Pellets of control and treated samples were obtained by centrifugation at 5, 000 rpm for 10 min at 4 °C. Then, samples were ground for 5 min immersing in liquid nitrogen, and then 1 mL ice-cold RIPA lysis buffer (containing 1 mM PMSF) was added. After agitation at 4 °C for 20 min, samples were centrifuged at 100, 000 g for 5 min. The supernatant containing soluble protein was collected and kept at 4 °C for SDS-PAGE. SDS-PAGE analysis was performed using a mini protean® tetra system (Bio-Rad Laboratories, Hercules, California, USA) (Chen et al., 2019). The stacking and separating gel were 5% and 10% separately, containing 0.1% SDS. Protein solutions (2.5 mg/mL) were diluted 5fold with protein loading dye and boiled for 5 min. The loading volume was 10 μL, electrophoresis was performed in Tris-glycine running buffer (pH 8.3). Gels were stained with Coomassie Brilliant Blue R250 dye and destained with a solution composed of 40% methanol and 10% acetic acid.
2.2. Strains and preparation of spores Spores of Bacillus cereus 14579 (Bio-Technology Co., Ltd., Qingdao, Shandong, China) were used to evaluate the ultrasound-induced detachment of exosporium. The sporulation and enumeration methods were previously described (Lv et al., 2019). 2.3. Ultrasound treatment A Scientz-II D ultrasound device with a 10-mm-diameter titanium probe (Ningbo Scientz Biotechnology Co., LTD., Ningbo, China) was used for ultrasound treatment (Li et al., 2019). The frequency and the maximum power of ultrasound were 20 kHz and 950 W respectively, and the power used in this study was 200 W. Spore suspensions (about 107 CFU/mL) were treated for seven 1 min with 2-min-gaps. The ultrasound treatment was kept at 25 °C in order to avoid thermal-induced inactivation of spores. After ultrasound treatment, the population of spores after ultrasound treatment was also determined.
3. Results and discussions 3.1. Morphological changes by TEM TEM micrographs before and after ultrasound were shown in Fig. 1. The multi-layer structure of untreated spores was clear in Fig. 1A, especially the typical balloon-like outermost layer: exosporium. The exosporium of B. cereus spores was detached as expected without damage to inner layers (Fig. 1B). Besides, the treated spores were smaller than initial, the average length of spores changed from 2.7 to 1.5 μm approximately while width varied not significantly. The population of survival spores after each 1 min ultrasound
2.4. Size distribution and zeta potential To confirm the detachment of exosporium, the size distribution was tested. In addition, as one of important components of the adhesion force between spores and surfaces, zeta potential was also measured (Chung, Yiacoumi, Lee, & Tsouris, 2010). Mean size and zeta potential of treated and untreated spores in aqueous solution (at neutral pH) were determined by Nano-ZS particle size analyzer (Nano ZS 90, Malvern, UK) at 25 °C (Chen et al., 2019). The measurements of the zeta potential were repeated 10 to 20 times and averaged values were obtained by analyzer. The values were measured triply with three independent samples. 2.5. Transmission electron microscopy (TEM) TEM micrographs of Bacillus cereus spores were used to confirm the detachment of exosporium by ultrasound. The pre-process treatments including fixing, dehydration and polymerization were described before (Lv et al., 2019). Samples were sectioned using ultramicrotome (Leica
Fig. 1. TEM micrographs of Bacillus cereus spores: A: untreated; B: seven 1 min ultrasound treated, the exosporium was detached. 2
LWT - Food Science and Technology 116 (2019) 108473
R. Lv, et al.
Table 1 Survival of B. cereus spores and hydrophobicity (RHS) after ultrasonic treatments for different durations. Ultrasonic treatment duration (min)
0
1
2
3
4
5
6
7
Surviving population of spores A0 Af RHS (%)
7.42 ± 0.04a
7.37 ± 0.11
7.45 ± 0.03
7.43 ± 0.07
7.40 ± 0.06
7.48 ± 0.08
7.36 ± 0.10
7.38 ± 0.08
1.217 ± 0.007 0.467 ± 0.005 61.616 ± 0.402
1.217 ± 0.005 0.878 ± 0.030 27.881 ± 2.151
1.217 ± 0.005 0.915 ± 0.008 24.793 ± 0.828
1.216 ± 0.004 0.948 ± 0.004 22.065 ± 0.607
1.214 ± 0.004 1.006 ± 0.013 17.132 ± 1.213
1.202 ± 0.008 1.010 ± 0.030 15.990 ± 2.970
1.196 ± 0.017 1.024 ± 0.021 14.396 ± 1.959
1.207 ± 0.004 1.024 ± 0.008 15.190 ± 0.487
a
Data were mean of triplicate measurements ± standard deviation.
for 1 min, RHS dropped rapidly from 61.62% to 27.88%. However, the decrease of RHS was insignificant with the extension of process time (Table 1). Bacillus cereus spores had high hydrophobicity because of the exosporium, which was coincident with previous reference (Rönner et al., 1990). With decreased hydrophobicity, spores were more difficult to adhere to hydrophobic surfaces (Noma et al., 2018). This was of great important to control bacterial spores in food process. Also, the hydrophobic trends were similar with the size and zeta potential, suggesting that the detachment of exosporium had significant influence of the surface property of B. cereus spores.
treatment were tested using plate counting method (Table 1). Previous study indicated ultrasound treatment alone even for 30 min had minor inactivation effect on B. cereus spores (Lv et al., 2019). Unsurprisingly, the current results showed the population of spores had no significant change after ultrasound treatment, indicating this ultrasound treatment had no lethal effect to B. cereus spores. Overall, these results pointed that seven times 1 min ultrasound treatment detached exosporium of B. cereus spores without lethality. 3.2. The size and zeta potential The size and zeta potential of the spores were measured and presented in Fig. 2. The average size of the spores decreased from 2017.67 to 960.53 nm for 1 min treatment after which no significant reduction in size was found. The final size of spores was 633.5 nm after seven 1 min ultrasound treatment. Since the exosporium of spores is a balloon and occupies a large volume, the significant decrease in size also proved the detachment of exosporium (Pizarro-Guajardo, Calderón-Romero, & Paredes-Sabja, 2016). Determining the zeta potential of microorganisms is the fundamental part to clarify the physiology, surface property and mobility (Pizarro-Guajardo et al., 2016). The zeta potential values of B. cereus spores were negative for both untreated and treated samples. Untreated spores had the highest absolute value of the zeta potential, and it decreased with the increasing processing time. Conventionally, the solution with higher absolute zeta potential was considered to be stabler (Chen et al., 2019). The surprising correlation was the decrease of size and absolute zeta potential for the first 1 min ultrasound treatment, indicating that ultrasound detached exosporium of spores in a short time.
3.4. SDS-PAGE analysis SDS-PAGE was conducted to determine the total protein changes of spores after ultrasound treatment. Fig. 3 showed that the protein of untreated spores ranged from 6.5 to 270 KDa, with many clear bands. But clearly, the intensity of all the bands decreased significantly after ultrasound treatment, especially the higher molecular weight of the protein subunits. This was mainly because ultrasound could degrade protein and destroy the structure of protein (Zou, Nguyen, Biers, & Sun, 2019). Moreover, some bands disappeared after ultrasound treatment, indicating that some protein were removed after ultrasound treatment. That might because the detachment of the outer protein shell: exosporium, which consists of over 180 proteins (Stewart, 2017).
3.3. Hydrophobic changes of B. cereus spores The hydrophobic changes of spores after ultrasound treatment were investigated using the ratio of hydrophobic spores (RHS). Once treated
Fig. 2. The size and zeta potential of Bacillus cereus spores after ultrasound treatment.
Fig. 3. SDS-PAGE of total protein extracted from control and ultrasound treated spores. 3
LWT - Food Science and Technology 116 (2019) 108473
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4. Conclusions
Bacillus cereus spores with a damaged exosporium: Consequences on the spore adhesion on surfaces of food processing lines. Journal of Food Protection, 70(10), 2346–2353. https://doi.org/10.4315/0362-028x-70.10.2346. Finlay, W. J. J., Logan, N. A., & Sutherland, A. D. (2000). Bacillus cereus produces most emetic toxin at lower temperatures. Letters in Applied Microbiology, 31(5), 385–389. https://doi.org/10.1046/j.1472-765x.2000.00835.x. Hariram, U., & Labbe, R. G. (2016). Growth and inhibition by spices of growth from spores of enterotoxigenic Bacillus cereus in cooked rice. Food Control, 64, 60–64. https://doi.org/10.1016/j.foodcont.2015.12.024. Henriques, A. O., & Moran, C. P. (2007). Structure, assembly, and function of the spore surface layers. Annual Review of Microbiology, 61, 555–588. Li, J., Cheng, H., Liao, X. Y., Liu, D. H., Xiang, Q. S., Wang, J., et al. (2019). Inactivation of Bacillus subtilis and quality assurance in Chinese bayberry (Myrica rubra) juice with ultrasound and mild heat. Lwt-Food Science and Technology, 108, 113–119. https:// doi.org/10.1016/j.lwt.2019.03.061. Lv, R., Zou, M., Chantapakul, T., Chen, W., Muhammad, A. I., Zhou, J., et al. (2019). Effect of ultrasonication and thermal and pressure treatments, individually and combined, on inactivation of Bacillus cereus spores. Applied Microbiology and Biotechnology, 1. https://doi.org/10.1007/s00253-018-9559-3. Noma, S., Kiyohara, K., Hirokado, R., Yamashita, N., Migita, Y., Tanaka, M., et al. (2018). Increase in hydrophobicity of Bacillus subtilis spores by heat, hydrostatic pressure, and pressurized carbon dioxide treatments. Journal of Bioscience and Bioengineering, 125(3), 327–332. https://doi.org/10.1016/j.jbiosc.2017.09.012. Pizarro-Guajardo, M., Calderón-Romero, P., & Paredes-Sabja, D. (2016). Ultrastructure variability of the exosporium layer of Clostridium difficile spores from sporulating cultures and biofilms. Applied and Environmental Microbiology, 82(19), 5892–5898. https://doi.org/10.1128/AEM.01463-16. Rönner, U., Husmark, U., Henriksson, A., & livmedelsinstitutet, S. I. K. S. (1990). Adhesion of bacillus spores in relation to hydrophobicity. Journal of Applied Bacteriology, 69(4), 550–556. https://doi.org/10.1111/j.1365-2672.1990.tb01547.x. Stewart, G. C. (2017). Assembly of the outermost spore layer: Pieces of the puzzle are coming together. Molecular Microbiology, 104(4), 535–538. https://doi.org/10.1111/ mmi.13651. Tauveron, G., Slomianny, C., Henry, C., & Faille, C. (2006). Variability among Bacillus cereus strains in spore surface properties and influence on their ability to contaminate food surface equipment. International Journal of Food Microbiology, 110(3), 254–262. https://doi.org/10.1016/j.ijfoodmicro.2006.04.027. Wiencek, K. M., Klapes, N. A., & Foegeding, P. M. (1990). Hydrophobicity of Bacillus and Clostridium spores. Applied and Environmental Microbiology, 56(9), 2600–2605. Zhou, K. X., Li, N., Christie, G., & Wilson, D. I. (2017). Assessing the impact of germination and sporulation conditions on the adhesion of Bacillus spores to glass and stainless steel by fluid dynamic gauging. Journal of Food Science, 82(11), 2614–2625. https://doi.org/10.1111/1750-3841.13940. Zou, M. M., & Liu, D. H. (2018). A systematic characterization of the distribution, biofilmforming potential and the resistance of the biofilms to the CIP processes of the bacteria in a milk powder processing factory. Food Research International, 113, 316–326. https://doi.org/10.1016/j.foodres.2018.07.020. Zou, J. H., Nguyen, N., Biers, M., & Sun, G. (2019). Conformational changes of soy proteins under high-intensity ultrasound and high-speed shearing treatments. [Article]. ACS Sustainable Chemistry & Engineering, 7(9), 8117–8125. https://doi.org/ 10.1021/acssuschemeng.8b05713.
This study evaluated that short-time ultrasound treatment could detach exosporium of Bacillus cereus spores without damage to structure of inner layers and survival of spores. Then, spores became much smaller, more hydrophilic with rapidly decrease of absolute zeta potential. Control of the spore hydrophobicity and adhesion is important in food industry. Accordingly, ultrasound treatment could decrease the adhesion to hydrophobic surfaces which might be an alternative choice to clean the facilities in the food processing chain. However, how to achieve industrial application is still an urgent problem which will be our next focus. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Formatting of funding sources This work was supported by the Ministry of Science and Technology of the People's Republic China; National Key Research and Development Program of China (grant number 2016YFD0400301) and the Key Research and Development Program of Zhejiang Province (grant number 2017C02015). References Chen, W., Ma, X., Wang, W., Lv, R., Guo, M., Ding, T., et al. (2019). Preparation of modified whey protein isolate with gum acacia by ultrasound maillard reaction. Food Hydrocolloids, 95, 298–307. https://doi.org/10.1016/j.foodhyd.2018.10.030. Chen, W. J., Zou, M. M., Ma, X. B., Lv, R. L., Ding, T., & Liu, D. H. (2019). Co-encapsulation of EGCG and quercetin in liposomes for optimum antioxidant activity. Journal of Food Science, 84(1), 111–120. https://doi.org/10.1111/1750-3841.14405. Chung, E., Yiacoumi, S., Lee, I., & Tsouris, C. (2010). The role of the electrostatic force in spore adhesion. Environmental Science and Technology, 44(16), 6209–6214. https:// doi.org/10.1021/es101730y. Faille, C., Jullien, C., Fontaine, F., Bellon-Fontaine, M. N., Slomianny, C., & Benezech, T. (2002). Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: Role of surface hydrophobicity. Canadian Journal of Microbiology, 48(8), 728–738. https:// doi.org/10.1139/w02-063. Faille, C., Tauveron, G., Gentil-Lelievre, C. L., & Slomianny, C. (2007). Occurrence of
4
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Short communication
Ultrasound: Enhance the detachment of exosporium and decrease the hydrophobicity of Bacillus cereus spores
T
Ruiling Lva, Mingming Zoua, Weijun Chena, Jianwei Zhoua,c, Tian Dinga, Xingqian Yea, Donghong Liua,b,c,∗ a College of Biosystems Engineering and Food Science, National-Local Joint Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang University, Hangzhou, 310058, China b Fuli Institute of Food Science, Zhejiang University, Hangzhou, 310058, China c Ningbo Research Institute, Zhejiang University, Ningbo, 315100, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacillus cereus spores Ultrasound Surface property Exosporium Adhesion
Because of the hydrophobicity and formation of biofilm, Bacillus cereus spores are highly resistant and widespread in food industry. The outermost layer: exosporium of Bacillus cereus spore plays an important role in its surface property. Previous work indicated that ultrasound detached the exosporium of spores, thus the surface properties were further investigated in this study. The detachment of exosporium was observed without damage to inner layers through TEM micrographs. Besides, the size of spores dropped rapidly from 2017.67 to 960.53 nm after 1 min ultrasound treatment. At the same time, the absolute zeta potential value also decreased rapidly. The ratio of hydrophobic spores (RHS) was defined as hydrophobicity, and it dropped-off with the detachment of exosporium. The SDS-PAGE results showed that the intensity of protein bands dropped especially that with higher molecular weight after ultrasound treatment. What's more, a lot of bands disappeared because of the separation of exosporium. In a word, these results indicated that ultrasound could detach exosporium and decrease the hydrophobicity of spores, which had lower adhesion to hydrophobic surface and easier to be cleaned.
1. Introduction
and environment established by the surface layers of spores (exosporium and coat) (Henriques & Moran, 2007). Bacillus cereus spores have a typical balloon-like outermost layer: exosporium, which loosely attaches in the surface occupying a large space (Rönner, Husmark, Henriksson, & livmedelsinstitutet, 1990). The absence of the exosporium would change the size of spores significantly. The exosporium plays important roles in protection, adhesion and virulence of spores (Henriques & Moran, 2007). It is a barrier to larger molecules providing resistance to enzymes and chemicals (Stewart, 2017). The exosporium interacts with environment and host cells of the immune system by virtue of its outermost location (Henriques & Moran, 2007). Furthermore, the presence of exosporium makes great contribution to the remarkable adhesion of Bacillus cereus spores (Faille, Tauveron, GentilLelievre, & Slomianny, 2007). Zhou assessed the effect of germination and sporulation conditions on the adhesion of Bacillus spores with fluid dynamic gauging technique. They found that spores with exosporium (Bacillus cereus) were much more resistant to shear stress than those without exosporium (Bacillus subtilis) (Zhou, Li, Christie, & Wilson, 2017).
Bacillus cereus (B. cereus) is ubiquitous and widespread in food and food industry especially those associated with soil (Tauveron, Slomianny, Henry, & Faille, 2006). It is frequently reported that Bacillus cereus contaminated both raw and processed foods including rice, vegetables, meat or dairy products thus resulted in outbreaks (Hariram & Labbe, 2016). Moreover, as a gram-positive and spore-forming bacterium, B. cereus could survive in hash conditions by forming biofilm and spores. Bacteria and spores in biofilm are highly resistant to cleaning processing which are of great concern to food industry (Zou & Liu, 2018). Previous studies demonstrated that B. cereus strains could produce diarrhoeal toxins and emetic toxins, causing diarrheal or emetic disease (Finlay, Logan, & Sutherland, 2000). Particularly, Bacillus cereus spores are resistant to stresses such as UV radiation, heat, chemicals and can stay dormant for decades. Spores germinate into vegetative cells once the environment outside is suitable for growth. The extraordinary resistance might be related to the multilayer compact structure. Particularly, the interactions between spores
∗ Corresponding author. College of Biosystems Engineering and Food Science, National-Local Joint Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang University, Hangzhou, 310058, China. E-mail address: [email protected] (D. Liu).
https://doi.org/10.1016/j.lwt.2019.108473 Received 4 April 2019; Received in revised form 31 July 2019; Accepted 1 August 2019 Available online 02 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 116 (2019) 108473
R. Lv, et al.
EM UC7) and observed on transmission electron microscope (JEM1230, JEOL, Japan).
Food processing surfaces such as valves, pipes, and pumps (off systems) or conveyors (open systems) in dairy industry, were regularly found to be contaminated by Bacillus spp., Escherichia coli, or Listeria monocytogenes. (Faille et al., 2002). Bacteria could adhere to surfaces, replicate and even form biofilm, causing shorter shelf-life and even safety issues. Compared with vegetative cells, the adhesion of spores was generally much stronger to both hydrophobic and hydrophilic surfaces (Rönner et al., 1990). The main reason was the different structures of spores and vegetative cells. Overall, the surface properties are closely related to the adhesion of Bacillus cereus spores. However, there is no direct evidence showing the relationship between the absence of the exosporium and the surface property. Pervious work evaluated that ultrasound could detach the exosporium of Bacillus cereus spores (Lv et al., 2019). Thus, this study further evaluated the size and surface property changes of Bacillus cereus spores with the detachment of exosporium after ultrasonic treatments.
2.6. Hydrophobic measurements The hydrophobic property of spores was measured using a partition method which based on the affinity to hexadecane (Noma et al., 2018; Wiencek, Klapes, & Foegeding, 1990). The initial absorbance at 600 nm of each sample was measured ( A0 ) using a spectrophotometer (1510–02691, Thermo Fisher Scientific Oy Ratastie 2, Fl-01620, Vantaa, Finland). Then, treated and untreated spore suspensions (3 mL) were mixed with 0.6 mL hexadecane and vortexed for 30 s. The absorbance of the aqueous phases was tested at 600 nm after holding for 15 min for phase separation ( Af ), the decrease in absorbance was the value of relative surface hydrophobicity of spores. The hydrophobicity of spores was defined as the ratio of hydrophobic spores (RHS).
RHS (%) = 2. Materials and methods 2.1. Materials
A0 − Af A0
*100%
2.7. Total protein extraction and SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Both nutrient agar and broth were from Hope Bio-Technology Co., Ltd. (Qingdao, China). Hexadecane was obtained from Sigma-Aldrich Co (St. Louis, MO, USA). RIPA lysis buffer, protein loading dye and all the other SDS-PAGE related chemicals were purchased from Sangon Biotech Co., Ltd (Shanghai, China).
Pellets of control and treated samples were obtained by centrifugation at 5, 000 rpm for 10 min at 4 °C. Then, samples were ground for 5 min immersing in liquid nitrogen, and then 1 mL ice-cold RIPA lysis buffer (containing 1 mM PMSF) was added. After agitation at 4 °C for 20 min, samples were centrifuged at 100, 000 g for 5 min. The supernatant containing soluble protein was collected and kept at 4 °C for SDS-PAGE. SDS-PAGE analysis was performed using a mini protean® tetra system (Bio-Rad Laboratories, Hercules, California, USA) (Chen et al., 2019). The stacking and separating gel were 5% and 10% separately, containing 0.1% SDS. Protein solutions (2.5 mg/mL) were diluted 5fold with protein loading dye and boiled for 5 min. The loading volume was 10 μL, electrophoresis was performed in Tris-glycine running buffer (pH 8.3). Gels were stained with Coomassie Brilliant Blue R250 dye and destained with a solution composed of 40% methanol and 10% acetic acid.
2.2. Strains and preparation of spores Spores of Bacillus cereus 14579 (Bio-Technology Co., Ltd., Qingdao, Shandong, China) were used to evaluate the ultrasound-induced detachment of exosporium. The sporulation and enumeration methods were previously described (Lv et al., 2019). 2.3. Ultrasound treatment A Scientz-II D ultrasound device with a 10-mm-diameter titanium probe (Ningbo Scientz Biotechnology Co., LTD., Ningbo, China) was used for ultrasound treatment (Li et al., 2019). The frequency and the maximum power of ultrasound were 20 kHz and 950 W respectively, and the power used in this study was 200 W. Spore suspensions (about 107 CFU/mL) were treated for seven 1 min with 2-min-gaps. The ultrasound treatment was kept at 25 °C in order to avoid thermal-induced inactivation of spores. After ultrasound treatment, the population of spores after ultrasound treatment was also determined.
3. Results and discussions 3.1. Morphological changes by TEM TEM micrographs before and after ultrasound were shown in Fig. 1. The multi-layer structure of untreated spores was clear in Fig. 1A, especially the typical balloon-like outermost layer: exosporium. The exosporium of B. cereus spores was detached as expected without damage to inner layers (Fig. 1B). Besides, the treated spores were smaller than initial, the average length of spores changed from 2.7 to 1.5 μm approximately while width varied not significantly. The population of survival spores after each 1 min ultrasound
2.4. Size distribution and zeta potential To confirm the detachment of exosporium, the size distribution was tested. In addition, as one of important components of the adhesion force between spores and surfaces, zeta potential was also measured (Chung, Yiacoumi, Lee, & Tsouris, 2010). Mean size and zeta potential of treated and untreated spores in aqueous solution (at neutral pH) were determined by Nano-ZS particle size analyzer (Nano ZS 90, Malvern, UK) at 25 °C (Chen et al., 2019). The measurements of the zeta potential were repeated 10 to 20 times and averaged values were obtained by analyzer. The values were measured triply with three independent samples. 2.5. Transmission electron microscopy (TEM) TEM micrographs of Bacillus cereus spores were used to confirm the detachment of exosporium by ultrasound. The pre-process treatments including fixing, dehydration and polymerization were described before (Lv et al., 2019). Samples were sectioned using ultramicrotome (Leica
Fig. 1. TEM micrographs of Bacillus cereus spores: A: untreated; B: seven 1 min ultrasound treated, the exosporium was detached. 2
LWT - Food Science and Technology 116 (2019) 108473
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Table 1 Survival of B. cereus spores and hydrophobicity (RHS) after ultrasonic treatments for different durations. Ultrasonic treatment duration (min)
0
1
2
3
4
5
6
7
Surviving population of spores A0 Af RHS (%)
7.42 ± 0.04a
7.37 ± 0.11
7.45 ± 0.03
7.43 ± 0.07
7.40 ± 0.06
7.48 ± 0.08
7.36 ± 0.10
7.38 ± 0.08
1.217 ± 0.007 0.467 ± 0.005 61.616 ± 0.402
1.217 ± 0.005 0.878 ± 0.030 27.881 ± 2.151
1.217 ± 0.005 0.915 ± 0.008 24.793 ± 0.828
1.216 ± 0.004 0.948 ± 0.004 22.065 ± 0.607
1.214 ± 0.004 1.006 ± 0.013 17.132 ± 1.213
1.202 ± 0.008 1.010 ± 0.030 15.990 ± 2.970
1.196 ± 0.017 1.024 ± 0.021 14.396 ± 1.959
1.207 ± 0.004 1.024 ± 0.008 15.190 ± 0.487
a
Data were mean of triplicate measurements ± standard deviation.
for 1 min, RHS dropped rapidly from 61.62% to 27.88%. However, the decrease of RHS was insignificant with the extension of process time (Table 1). Bacillus cereus spores had high hydrophobicity because of the exosporium, which was coincident with previous reference (Rönner et al., 1990). With decreased hydrophobicity, spores were more difficult to adhere to hydrophobic surfaces (Noma et al., 2018). This was of great important to control bacterial spores in food process. Also, the hydrophobic trends were similar with the size and zeta potential, suggesting that the detachment of exosporium had significant influence of the surface property of B. cereus spores.
treatment were tested using plate counting method (Table 1). Previous study indicated ultrasound treatment alone even for 30 min had minor inactivation effect on B. cereus spores (Lv et al., 2019). Unsurprisingly, the current results showed the population of spores had no significant change after ultrasound treatment, indicating this ultrasound treatment had no lethal effect to B. cereus spores. Overall, these results pointed that seven times 1 min ultrasound treatment detached exosporium of B. cereus spores without lethality. 3.2. The size and zeta potential The size and zeta potential of the spores were measured and presented in Fig. 2. The average size of the spores decreased from 2017.67 to 960.53 nm for 1 min treatment after which no significant reduction in size was found. The final size of spores was 633.5 nm after seven 1 min ultrasound treatment. Since the exosporium of spores is a balloon and occupies a large volume, the significant decrease in size also proved the detachment of exosporium (Pizarro-Guajardo, Calderón-Romero, & Paredes-Sabja, 2016). Determining the zeta potential of microorganisms is the fundamental part to clarify the physiology, surface property and mobility (Pizarro-Guajardo et al., 2016). The zeta potential values of B. cereus spores were negative for both untreated and treated samples. Untreated spores had the highest absolute value of the zeta potential, and it decreased with the increasing processing time. Conventionally, the solution with higher absolute zeta potential was considered to be stabler (Chen et al., 2019). The surprising correlation was the decrease of size and absolute zeta potential for the first 1 min ultrasound treatment, indicating that ultrasound detached exosporium of spores in a short time.
3.4. SDS-PAGE analysis SDS-PAGE was conducted to determine the total protein changes of spores after ultrasound treatment. Fig. 3 showed that the protein of untreated spores ranged from 6.5 to 270 KDa, with many clear bands. But clearly, the intensity of all the bands decreased significantly after ultrasound treatment, especially the higher molecular weight of the protein subunits. This was mainly because ultrasound could degrade protein and destroy the structure of protein (Zou, Nguyen, Biers, & Sun, 2019). Moreover, some bands disappeared after ultrasound treatment, indicating that some protein were removed after ultrasound treatment. That might because the detachment of the outer protein shell: exosporium, which consists of over 180 proteins (Stewart, 2017).
3.3. Hydrophobic changes of B. cereus spores The hydrophobic changes of spores after ultrasound treatment were investigated using the ratio of hydrophobic spores (RHS). Once treated
Fig. 2. The size and zeta potential of Bacillus cereus spores after ultrasound treatment.
Fig. 3. SDS-PAGE of total protein extracted from control and ultrasound treated spores. 3
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4. Conclusions
Bacillus cereus spores with a damaged exosporium: Consequences on the spore adhesion on surfaces of food processing lines. Journal of Food Protection, 70(10), 2346–2353. https://doi.org/10.4315/0362-028x-70.10.2346. Finlay, W. J. J., Logan, N. A., & Sutherland, A. D. (2000). Bacillus cereus produces most emetic toxin at lower temperatures. Letters in Applied Microbiology, 31(5), 385–389. https://doi.org/10.1046/j.1472-765x.2000.00835.x. Hariram, U., & Labbe, R. G. (2016). Growth and inhibition by spices of growth from spores of enterotoxigenic Bacillus cereus in cooked rice. Food Control, 64, 60–64. https://doi.org/10.1016/j.foodcont.2015.12.024. Henriques, A. O., & Moran, C. P. (2007). Structure, assembly, and function of the spore surface layers. Annual Review of Microbiology, 61, 555–588. Li, J., Cheng, H., Liao, X. Y., Liu, D. H., Xiang, Q. S., Wang, J., et al. (2019). Inactivation of Bacillus subtilis and quality assurance in Chinese bayberry (Myrica rubra) juice with ultrasound and mild heat. Lwt-Food Science and Technology, 108, 113–119. https:// doi.org/10.1016/j.lwt.2019.03.061. Lv, R., Zou, M., Chantapakul, T., Chen, W., Muhammad, A. I., Zhou, J., et al. (2019). Effect of ultrasonication and thermal and pressure treatments, individually and combined, on inactivation of Bacillus cereus spores. Applied Microbiology and Biotechnology, 1. https://doi.org/10.1007/s00253-018-9559-3. Noma, S., Kiyohara, K., Hirokado, R., Yamashita, N., Migita, Y., Tanaka, M., et al. (2018). Increase in hydrophobicity of Bacillus subtilis spores by heat, hydrostatic pressure, and pressurized carbon dioxide treatments. Journal of Bioscience and Bioengineering, 125(3), 327–332. https://doi.org/10.1016/j.jbiosc.2017.09.012. Pizarro-Guajardo, M., Calderón-Romero, P., & Paredes-Sabja, D. (2016). Ultrastructure variability of the exosporium layer of Clostridium difficile spores from sporulating cultures and biofilms. Applied and Environmental Microbiology, 82(19), 5892–5898. https://doi.org/10.1128/AEM.01463-16. Rönner, U., Husmark, U., Henriksson, A., & livmedelsinstitutet, S. I. K. S. (1990). Adhesion of bacillus spores in relation to hydrophobicity. Journal of Applied Bacteriology, 69(4), 550–556. https://doi.org/10.1111/j.1365-2672.1990.tb01547.x. Stewart, G. C. (2017). Assembly of the outermost spore layer: Pieces of the puzzle are coming together. Molecular Microbiology, 104(4), 535–538. https://doi.org/10.1111/ mmi.13651. Tauveron, G., Slomianny, C., Henry, C., & Faille, C. (2006). Variability among Bacillus cereus strains in spore surface properties and influence on their ability to contaminate food surface equipment. International Journal of Food Microbiology, 110(3), 254–262. https://doi.org/10.1016/j.ijfoodmicro.2006.04.027. Wiencek, K. M., Klapes, N. A., & Foegeding, P. M. (1990). Hydrophobicity of Bacillus and Clostridium spores. Applied and Environmental Microbiology, 56(9), 2600–2605. Zhou, K. X., Li, N., Christie, G., & Wilson, D. I. (2017). Assessing the impact of germination and sporulation conditions on the adhesion of Bacillus spores to glass and stainless steel by fluid dynamic gauging. Journal of Food Science, 82(11), 2614–2625. https://doi.org/10.1111/1750-3841.13940. Zou, M. M., & Liu, D. H. (2018). A systematic characterization of the distribution, biofilmforming potential and the resistance of the biofilms to the CIP processes of the bacteria in a milk powder processing factory. Food Research International, 113, 316–326. https://doi.org/10.1016/j.foodres.2018.07.020. Zou, J. H., Nguyen, N., Biers, M., & Sun, G. (2019). Conformational changes of soy proteins under high-intensity ultrasound and high-speed shearing treatments. [Article]. ACS Sustainable Chemistry & Engineering, 7(9), 8117–8125. https://doi.org/ 10.1021/acssuschemeng.8b05713.
This study evaluated that short-time ultrasound treatment could detach exosporium of Bacillus cereus spores without damage to structure of inner layers and survival of spores. Then, spores became much smaller, more hydrophilic with rapidly decrease of absolute zeta potential. Control of the spore hydrophobicity and adhesion is important in food industry. Accordingly, ultrasound treatment could decrease the adhesion to hydrophobic surfaces which might be an alternative choice to clean the facilities in the food processing chain. However, how to achieve industrial application is still an urgent problem which will be our next focus. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Formatting of funding sources This work was supported by the Ministry of Science and Technology of the People's Republic China; National Key Research and Development Program of China (grant number 2016YFD0400301) and the Key Research and Development Program of Zhejiang Province (grant number 2017C02015). References Chen, W., Ma, X., Wang, W., Lv, R., Guo, M., Ding, T., et al. (2019). Preparation of modified whey protein isolate with gum acacia by ultrasound maillard reaction. Food Hydrocolloids, 95, 298–307. https://doi.org/10.1016/j.foodhyd.2018.10.030. Chen, W. J., Zou, M. M., Ma, X. B., Lv, R. L., Ding, T., & Liu, D. H. (2019). Co-encapsulation of EGCG and quercetin in liposomes for optimum antioxidant activity. Journal of Food Science, 84(1), 111–120. https://doi.org/10.1111/1750-3841.14405. Chung, E., Yiacoumi, S., Lee, I., & Tsouris, C. (2010). The role of the electrostatic force in spore adhesion. Environmental Science and Technology, 44(16), 6209–6214. https:// doi.org/10.1021/es101730y. Faille, C., Jullien, C., Fontaine, F., Bellon-Fontaine, M. N., Slomianny, C., & Benezech, T. (2002). Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: Role of surface hydrophobicity. Canadian Journal of Microbiology, 48(8), 728–738. https:// doi.org/10.1139/w02-063. Faille, C., Tauveron, G., Gentil-Lelievre, C. L., & Slomianny, C. (2007). Occurrence of
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