Inhibitory effect of Lactobacillus plantarum metabolites against biofilm formation by Bacillus licheniformis isolated from milk powder products

Food Control 106 (2019) 106721

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Inhibitory effect of Lactobacillus plantarum metabolites against biofilm formation by Bacillus licheniformis isolated from milk powder products

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Ni Wanga, Lei Yuana, Faizan Ahmed Sadiqb, Guoqing Hea,∗ a b

College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacillus licheniformis Milk powder Biofilm Thermophilic spore-forming bacteria Lactobacillus plantarum

Process biofilms of Bacillus licheniformis present worldwide problem to the dairy industry because of their relevance to food spoilage and quality issues. In this study, metabolites of Lactobacillus plantarum were found to effectively inhibit biofilms formed by B. licheniformis. The biofilm formation was delayed by 10 h by the addition of L. plantarum cell free supernatant and controlling pH of the medium. Confocal laser scanning microscope images showed that L. plantarum metabolites decreased the number of B. licheniformis cells adhered to stainless steel and glass surfaces. Gel filtration chromatography results showed that B. licheniformis utilized the peptides in the growth medium (tryptic soy broth) with the molecular weights ranging from 613 to 1486 Da, and produced plentiful small peptides with the molecular weights ranging from 181 to 613 Da. Moreover, 3–10 kDa components of L. plantarum ultra-filtrates were proved to significantly inhibit biofilm formation by B. licheniformis. Thus, the metabolites produced by L. plantarum provide a novel approach for the prevention of B. licheniformis biofilms in the dairy industry.

1. Introduction Spore-forming bacteria are considered as important microbial contaminants in the dairy industry, which can be detected throughout the dairy processing continuum (Sadiq, Flint, & He, 2018; Teh, Flint, Brooks, & Knight, 2015). Thermophilic spore-forming bacteria are thought to originate from raw milk, where they are present in low number (McGuiggan, McCleery, Hannan, & Gilmour, 2002), but they are able to grow in milk or on surfaces during the manufacture of milk powder in different sections of dairy manufacturing plants where temperature elevates up to 55 °C, leading to high counts in the final product (Scott, Brooks, Rakonjac, Walker, & Flint, 2007). Thermophilic spore-forming bacteria potentially decrease the quality and reduce the shelf-life of dairy products by producing acids and spoilage enzymes (Burgess, Lindsay, & Flint, 2010; Yuan, Zhang, & Li, 2018). Bacillus licheniformis is known as one of the most prevalent sporeforming bacterial species which greatly affects the quality of dairy products, particularly powdered milk and products thereof, because of its ability to grow throughout the dairy processing continuum (Sadiq, Li, Liu, Flint, Zhang, & He, 2016; Zou & Liu, 2018). Although non-pathogenic, B. licheniformis is known to produce spoilage enzymes that may decrease the quality of milk powders after reconstitution (Sadiq, Li, Liu, Flint, Zhang, & He, 2016; Sadiq, Li, Liu, Flint, Zhang, Yuan, ∗

et al., 2016). Moreover, B. licheniformis has been proved to be a strong biofilm former at both 37 °C and 55 °C on stainless steel surfaces in the presence of milk (Reginensi et al., 2011; Sadiq et al., 2017; Zain, Bennett, & Flint, 2017). In general, microorganisms embedded in biofilms have been regarded as 100 to 1000 times more tolerant to antibiotics and disinfectants than the corresponding planktonic cells (Gilbert, Allison, & McBain, 2002). Thus, biofilms serve as a persistent source of the contamination of this bacterium in the dairy industry because of the protection this bacterium gains within biofilm matrix against thermal treatments and the cleaning-in-place (CIP) regimes (Bridier, Briandet, Thomas, & Dubois-Brissonnet, 2011; Teh et al., 2014). CIP is widely used to control biofilms in the dairy processing plants. However, the effectiveness of CIP is influenced by many factors including the characteristics of the surface being cleaned, the composition and concentration of the cleaning agents, cleaning temperature and time (Bremer, Fillery, & McQuillan, 2006). Residual microorganisms on equipment surfaces can still be found even after cleaning and sanitizing procedures, and pose threats to the quality and safety of dairy products (Marchand et al., 2012). Biofilms on the other hand are also responsible for process downtime, low system efficiency of processing equipment, particularly heat exchangers as a result of biofouling and corrosion of surfaces. A recent study has reported pitting corrosion on stainless steel

Corresponding author. College of Biosystems Engineering and Food Science Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, China. E-mail address: [email protected] (G. He).

https://doi.org/10.1016/j.foodcont.2019.106721 Received 19 April 2019; Received in revised form 13 June 2019; Accepted 15 June 2019 Available online 17 June 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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2.4. Biofilm quantification by crystal violet staining

surfaces (grade 304 and 306) caused by biofilms formed by spore forming bacteria (Gupta & Anand, 2018). New biofilm control strategies in combination with CIP, such as the modification of surfaces, the use of quorum quenchers and enzymesbased degradants, are expected to be used at the commercial level in order to reduce the use of chemical agents (Jindal et al., 2016; Laughton, Devillard, Heinrichs, Reid, & McCormick, 2006; Wynendaele, Bronselaer, Nielandt, D'Hondt, Stalmans, Bracke, et al., 2013). Bacteriocin or bacteriocin-producing microorganisms also hold great promises as effective biofilm control agents in the food industry. For example, plantaricin LPL-1 produced by Lactobacillus plantarum LPL-1 downregulated the expression of genes related to biofilm formation by Listeria monocytogenes (Wang, Qin, Zhang, Wu, & Li, 2019). Crude bacteriocin derived from Lactobacillus brevis was proved to have anti-biofilm effects on Escherichia coli and Salmonella typhimurium (Kim, Kim, & Kang, 2019). However, the biofilm control of B. licheniformis by probiotic strains has not been explored yet. The aim of this work was to evaluate the effect of metabolites of L. plantarum on biofilm formation by B. licheniformis and explore the efficient components among the metabolites. This study will help to develop new strategy for biofilm prevention in the dairy industry.

The crystal violet staining assay was employed to assess the biofilm forming ability of B. licheniformis in a 96-well microtiter plate following the method used by Yuan, Burmolle, Sadiq, Wang, and He (2018) with silght modifications. After the cultivation for 24 h, the bacterial suspension was poured off and each well was washed three times with sterile PBS to remove loosely attached cells, followed by drying at room temperature. Firmly attached biofilm cells were fixed by adding 200 μL of methanol (Sinopharm Chemical Reagent Co., Ltd, China) to each well, followed by discarding the liquid and air drying. Then, each well was stained with 200 μL of 0.05% (W/V) crystal violet (Sigma, USA) for 10 min. After that, the crystal violet stain was poured off and the wells were rinsed three times with sterile PBS to remove excess stain followed by air drying. The fixed crystal violet was solubilized in 200 μL of 33% (V/V) glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd, China) added to each well for 10 min. The optical density was measured at 570 nm (OD570) by automatic microplate photometer (Thermo Fisher Multiskan FC, USA).

2.5. Biofilm observation by confocal laser scanning microscope 2. Material and methods

For groups Bl + PBS 6.5 and Bl + Lps + PBS 6.5, sterile coupons of stainless steel AISI 304 with a 2B finish (10 mm × 10 mm) and sterile round glass coverslips (Φ = 10 mm) were used as substrata to grow biofilms by B. licheniformis. These stainless steel or glass coupons were immersed in each well of a 24-well cell culture plate containing 2 mL suspension of mixed bacterial cultures, and the volume of each component was 10 times as described in Table 1. After the incubation at 55 °C for 24 h, the coupons were aseptically removed by sterile forceps followed by rinsing with sterile PBS. The biofilm cells of B. licheniformis on stainless steel and glass were treated with live-stain SYTO 9 and dead-stain propidium iodide (PI) (Flimtracer LIVE/DEAD biofilm viability kit, Thermo Fisher, USA) for 20 min at room temperature, protected from light. After staining, the coupons were rinsed gently with sterile PBS for microscope observation. Biofilms on substrata were examined by confocal laser scanning microscope (CLSM, Zeiss LSM780, Carl Zeiss, Germany), with an EC Plan-Neofluar 40X/1.3 Oil DIC M27 objective. The green and red fluorescence of SYTO 9 and PI were excited using an Ar laser beam at 488 nm (20%) and a He/Ne source at 561 nm (28%), respectively. Two (only x and y: 212.34 × 212.34 μm) or three (x: 212.34 μm, y: 212.34 μm and z: 1 μm) dimensional stacks of horizontal plane images were acquired for each biofilm sample from at least five different areas.

2.1. Bacterial strains The B. licheniformis strain used in this study was previously isolated from a Chinese milk powder sample (Sadiq, Li, Liu, Flint, Zhang, & He, 2016); similarly, the L. plantarum strain used in this study to inhibit biofilms was isolated from a Chinese traditional sourdough sample (Liu et al., 2016). The bacterial strains were kept in glycerol stock (20%, V/ V) at −80 °C until further use. 2.2. Co-cultivation of B. licheniformis and L. plantarum B. licheniformis was cultured in tryptic soy broth (TSB, Difco, USA) at 55 °C for 8 h on an orbital shaker (150 rpm), then the culture was diluted using fresh TSB to a level where it contained 104 CFU of B. licheniformis per mL. L. plantarum was cultured in De Man, Rogosa and Sharpe (MRS, Merk, Germany) at 37 °C for 24 h without aeration, then the culture was diluted to the concentration of 104 CFU/mL by TSB. A total of 200 μL mixed bacterial suspension with different proportions was cultured in 12 replicate wells of a 96-well cell culture plates (Costar, Corning, USA) at 37 °C for 24 h. For the single-strain groups, the inoculation quantity of B. licheniformis or L. plantarum was 5%. For the two-strain groups, the inoculation ratios of B. licheniformis and L. plantarum were 1:1 (5%:5%), 1:1.5 (5%:7.5%), 1:2 (5%:10%) and 1:2.5 (5%:12.5%) respectively. After the incubation, the final pH of the culture suspension of each group was measured by a pH electrode (Sartorius, Germany). Counts of each species in the culture suspension were determined on tryptic soy agar plates.

2.6. Gel filtration chromatography analysis of peptide components of L. plantarum and B. licheniformis planktonic cultures Fresh TSB and 24-h L. plantarum and B. licheniformis cultures were filtered by 0.22-μm PES membrane (Merk, Germany), and subjected to the ultrafiltration operation with Millipore Amicon Ultra-15 30k Centrifugal Filter Devices (Merk, Germany) to obtain the filtrates containing peptides with molecular weight smaller than 30 kDa. The protein concentrations of filtrates were measured by using modified bicinchoninic acid (BCA) protein assay kit (Shanghai Sangon Biotech Co., Ltd., China). Peptide components of L. plantarum and B. licheniformis cultures were detected by gel filtration chromatography by Superdex peptide10/300 GL gel column (AKTA purifier, GE Healthcare Co., Ltd., USA). The mobile phase was 30% acetonitrile in water (containing 0.1% trifluoroacetic acid, V/V) at a flow rate of 0.5 mL/min with an injection volume of 100 μL, and the detector was UV 215 nm. The peptide standard mixture contained aprotinin (molecular weight of 6511 Da), zinc-bacitracin (1486 Da), L-glutathione oxidized (613 Da) and tyrosine (181 Da).

2.3. B. licheniformis cultivation in the presence of L. plantarum supernatant The 24-h culture of L. plantarum was centrifuged at 3000×g for 10 min and filtered with 0.22-μm polyethersulfone membrane (PES, Merk, Germany), then cell free supernatant (CFS) was mixed with TSB for B. licheniformis cultivation as described in Table 1. Sterile phosphate buffer saline (PBS) was used as a buffering agent to control pH and to minimize growth inhibition due to low pH. Each group had 12 replicate wells, and each well contained 200 μL mixed bacterial solution. After the incubation at 55 °C (or 37 °C, special for group Lp and Bl + Lp) for 24 h, optical density was measured at 600 nm (OD600), and in addition, pH, bacterial counts of cultures, and biofilm forming capacity of B. licheniformis were determined. 2

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Table 1 Different medium compositions for B. licheniformis cultivation. Group name

B. licheniformis culture (μL)

L. plantarum culture (μL)

L. plantarum supernatant (μL)

PBS, pH 6.5 (μL)

PBS, pH 7.2 (μL)

PBS, pH 7.5 (μL)

PBS, pH 8.0 (μL)

TSB (μL)

Bl Lp Bl + Bl + Bl + Bl + Bl + Bl + Bl + Bl + Bl +

12 0 12 12 12 12 12 12 12 12 12

0 12 12 0 0 0 0 0 0 0 0

0 0 0 68 68 68 68 68 68 68 0

0 0 0 0 40 0 0 0 0 0 40

0 0 0 0 0 40 0 0 80 0 0

0 0 0 0 0 0 40 0 0 0 0

0 0 0 0 0 0 0 40 0 80 0

188 188 176 120 80 80 80 80 40 40 148

Lp Lps Lps + PBS 6.5 Lps + PBS 7.2 Lps + PBS 7.5 Lps + PBS 8.0 Lps+2PBS 7.2 Lps+2PBS 7.5 PBS 6.5

Fig. 1. Biofilm formation and planktonic growth of B. licheniformis and L. plantarum in the co-cultivation for 24 h at 37 °C in TSB. (a) biofilm-forming capacity of B. licheniformis tested by crystal violet absorbance values at 570 nm; (b) pH values of mixed-strain culture; (c) cell numbers of the two strains (log CFU/mL). Data represent means ± SD of results obtained from three independent experiments, **P < 0.01, ***P < 0.001 compared with group Bl as control.

Fig. 1. (continued)

2.7. B. licheniformis cultivation in the presence of different L. plantarum ultra-filtrate components After 24 h, planktonic culture of L. plantarum was filtered by 0.22μm PES membrane (Merk, Germany), and ultrafiltered by Millipore Amicon Ultra-15 30k, Ultra-4 10k and Ultra-4 3k Centrifugal Filter Devices (Merk, Germany) in sequence. Four groups of L. plantarum ultra filtrates based on molecular weights (> 30 kDa, 10–30 kDa, 3–10 kDa, < 3 kDa) were collected and diluted with TSB to the concentrations before they were ultra-filtrated according to their enrichment ratios. The diluted L. plantarum ultra-filtrates were used, replacing L. plantarum CFS, during 24-h incubation of B. licheniformis at 55 °C to determine the effect of these ultra-filtrates on its biofilm formation. Other ingredients in the medium were kept the same as group Bl + Lps + PBS 6.5. Biofilm formation of B. licheniformis and pH of the cultures were detected as described above. Fig. 1. (continued)

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Fig. 2. (continued)

Fig. 2. Biofilm formation and planktonic growth of B. licheniformis cultivation with different amounts of L. plantarum CFS at 55 °C for 24 h in TSB. (a) biofilmforming capacity of B. licheniformis tested by crystal violet absorbance values at 570 nm; (b) absorbance values of the B. licheniformis suspension at 600 nm; (c) cell numbers of B. licheniformis culture (log CFU/mL). Data represent means ± SD of results obtained from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 compared with group Bl as control.

2.8. Statistic analysis The results are expressed as mean ± standard deviation (SD) with each experiment containing three replicates. Statistical significance was determined by Independent-Samples T Test (SPSS, version 20, SPSS Inc., Chicago, USA). CLSM pictures were processed with Image J (version d 1.47, National Institutes of Health, USA). 3. Results 3.1. Planktonic growth and biofilm formation in co-cultivation of B. licheniformis and L. plantarum For mono-species biofilms, the OD570 values of B. licheniformis were 3.614 and 0.001, respectively. However, significant reductions in OD570 values of B. licheniformis were observed when co-cultured with different proportions of L. plantarum (Fig. 1a). The pH values of single-strain culture suspensions of L. plantarum and B. licheniformis were 4.94 and 8.11, respectively. When B. licheniformis was co-cultured with L. plantarum, the pH of the growth medium decreased (P < 0.001) as compared to the pH of medium in which only B. licheniformis was grown. For groups Bl + Lp (1:1), Bl + Lp (1:1.5), Bl + Lp (1:2) and Bl + Lp (1:2.5), the pH values were recorded as 5.77, 5.47, 5.01 and 4.95, respectively (Fig. 1b). Cell numbers of B. licheniformis also decreased in the co-cultivation growth condition. As shown in Fig. 1c, cell numbers of L. plantarum remained around 8.31 log CFU/mL in each group, showing no significant difference (P > 0.05). Log CFU/mL of B. licheniformis was 7.88 for its single-species culture, 7.62 for group Bl + Lp (1:1) (P < 0.01), 7.31 for group Bl + Lp (1:1.5) (P < 0.001), and 5.70 for group Bl + Lp (1:2) (P < 0.001), showing an apparent downtrend. For group Bl + Lp (1:2.5), B. licheniformis did not grow.

Fig. 2. (continued)

3.2. Effect of L. plantarum supernatant on planktonic growth and biofilm formation by B. licheniformis As shown in Fig. 2a, CFS of L. plantarum also inhibited the biofilm 4

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Fig. 3. Planktonic growth curves and biofilm development of B. licheniformis cultivation with L. plantarum CFS after controlling pH, at 55 °C for every 2 h within 24 h in TSB. (a) absorbance values of the B. licheniformis suspension at 600 nm; (b) biofilm forming capacity of B. licheniformis tested by crystal violet absorbance values at 570 nm. Data represent means ± SD of results obtained from three independent experiments.

groups Bl + Lps + PBS 6.5, Bl + Lps + PBS 7.2, Bl + Lps + PBS 7.5 and Bl + Lps + PBS 8.0, planktonic cell growth of B. licheniformis did not decrease, but was increased at 24 h. The greatest growth activity was observed in group Bl + Lps + PBS 6.5, with the OD600 of 0.984 and cell number of 8.88 log CFU/mL (P < 0.01). Superfluous PBS (in group Bl + Lps+2PBS 7.2 and group Bl + Lps+2PBS 7.5) diluted the medium, resulting in a reduction in cell number and biofilms formation by B. licheniformis (P < 0.001). In addition, in the absence of L.

formation by B. licheniformis. Compared to the untreated group of Bl, the OD570 value of group Bl + Lps was decreased to 0.039, showing a significant reduction in biofilm forming tested by the crystal violet staining assay (P < 0.001). As for pH-controlled groups Bl + Lps + PBS 6.5, Bl + Lps + PBS 7.2, Bl + Lps + PBS 7.5 and Bl + Lps + PBS 8.0, the OD570 values were 0.297, 0.301, 0.285 and 0.329, respectively. The OD600 values and cell numbers of the cultures were also determined and are shown in Fig. 2b and c. For pH-controlled

Fig. 3. (continued) 5

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Fig. 4. CLSM analysis of B. licheniformis biofilm adhered on surfaces of stainless steel and glass. (a) B. licheniformis cultivation on stainless steel coupons; (b) B. licheniformis cultivation with L. plantarum CFS on stainless steel coupons; (c) B. licheniformis cultivation on glass coupons; (d) B. licheniformis cultivation with L. plantarum CFS on glass coupons; (e) numbers of live and dead cells adhered on the coupons in the view of CLSM. The images were acquired for each biofilm sample from at least five different areas.

Fig. 4. (continued)

Fig. 4. (continued)

3.3. Effect of L. plantarum supernatant on planktonic growth and biofilm formation by B. licheniformis under controlled pH Planktonic growth curves and biofilm development in two groups (Bl + PBS 6.5 and Bl + Lps + PBS 6.5) were determined after every 2 h within 24 h in order to monitor the dynamic process of biofilm formation by B. licheniformis. A similar growth trend was observed for these two groups, however, an increase in B. licheniformis cell number in group Bl + Lps + PBS 6.5 was slower as compared to the increase in cell number observed in group Bl + PBS 6.5. Despite the fact that they both entered logarithmic phase after 4 h (Fig. 3a), biofilm formation by B. licheniformis was delayed for about 10 h in the presence of L. plantarum CFS (Fig. 3b). Group Bl + PBS 6.5 started forming biofilm after 8 h, which developed rapidly after 18 h. At the 24th h, OD570 of group Bl + Lps + PBS 6.5 was noted as 1.751, while group Bl + PBS 6.5 was

Fig. 4. (continued)

plantarum CFS, the pH of B. licheniformis culture increased from 7.07 to 8.07 after the incubation period of 24 h. Compared with the control group, group Bl + Lps + PBS 6.5 showed stable pH values ranging from 6.69 to 6.78 within 24 h.

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Fig. 4. (continued)

3.4. CLSM analysis of biofilms formed by B. licheniformis

2.992, presenting that L. plantarum metabolites had a significant inhibitory effect on biofilm formation by B. licheniformis. It is worth mentioning that the biofilm formed by group Bl + Lps + PBS 6.5 was easily removed during rinsing.

CLSM was used to observe live and dead B. licheniformis cells adhered to surfaces of stainless steel and glass. Results showed that cell numbers of B. licheniformis grown on the surface of stainless steel and glass coupons decreased with the addition of L. plantarum CFS (Fig. 4). In group Bl + PBS 6.5, there were 432 cells (about 9.6 × 105 cells/cm2

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Fig. 5. Gel filtration chromatography analysis of peptide components of L. plantarum and B. licheniformis planktonic cultures. (a) protein concentrations of B. licheniformis and L. plantarum ultra-filtrates; (b) gel filtration chromatography analysis of peptide components of L. plantarum and B. licheniformis ultra-filtrates. Data represent means ± SD of results obtained from three independent experiments.

3.5. Effect of different components from L. plantarum ultra-filtrates on biofilm formation by B. licheniformis

in total) on the surface of stainless steel under the microscopic view; 63.2% of which were live, while 46.5% in group Bl + Lps + PBS 6.5 were live. On glass coupons, there were 423 cells under the microscopic view of group Bl + PBS 6.5 (about 9.4 × 105 cells/cm2), 52.0% of which were live, but only 19.1% in group Bl + Lps + PBS 6.5 were live. With L. plantarum metabolites, total cell and live cell numbers on glass surface significantly decreased. Furthermore, 3D mode results (data not shown) revealed that B. licheniformis forms monolayer biofilms on glass and steel surfaces rather than multilayer biofilms.

The total protein concentrations of B. licheniformis CFS, L. plantarum CFS and fresh TSB were 9.86 × 103, 1.02 × 104 and 1.18 × 104 μg/mL, respectively (Fig. 5a). Results of gel filtration chromatography showed that B. licheniformis utilized peptides with molecular weights ranging from 613 to 1486 Da, and produced plentiful peptides with molecular weights ranging from 181 to 613 Da (Fig. 5b). The 3–10 kDa components of L. plantarum ultra-filtrates significantly inhibited the biofilm formation by B. licheniformis (P < 0.001), as the OD570 value was

Fig. 5. (continued) 8

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Fig. 6. Effect of different components from L. plantarum ultra-filtrates on biofilm formation by B. licheniformis. (a) biofilm formation abilities of B. licheniformis tested by crystal violet absorbance values at 570 nm; (b) pH values of B. licheniformis planktonic cultures with different components. Data represent means ± SD of results obtained from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 compared with group TSB as control.

Controlling microbial growth on surfaces and removing biofilms are a mandatory part of an effective cleaning and sanitation program. CIP is a conventional and indispensable process of cleaning the interior surface of tanks, pasteurizers, pipelines and other equipment in the dairy industry (Kumari & Sarkar, 2016; Thomas & Sathian, 2014). However, bacteria may still remain on equipment surfaces and accumulate in areas especially in dead ends, crevices, gaskets and valves, which are difficult to be completely eradicates using CIP. For instance, a previous study provided evidence of the survival of pathogenic and spoilage microorganisms on food contact surfaces following cleaning and CIP, as 45 hugely diverse bacterial isolates, including both spoilage and pathogenic bacteria, were recovered from eleven different food contact surfaces in milk powder processing lines after CIP processes (Zou & Liu, 2018). New strategies for biofilm control are required and should be used in combination with CIP in the dairy industry to control biofilms. Based on its inhibition activity of microorganisms, L. plantarum has presented prospects of its practical use in controlling biofilms

decreased from 1.208 to 0.156. Also, a reduction in biofilm by B. licheniformis by 65.3% was observed under the influence of CFS components with size < 3 kDa (Fig. 6a). After cultivation, the pH value of control group was 7.45. Among the other four treatment groups, pH values of the cultures varied significantly, and showed both an increase and or decrease in pH. For group of > 30 kDa components and 10–30 kDa components, pH values increased to 7.63 and 7.54, respectively. For group of 3–10 kDa components and < 3 kDa components, pH values decreased to 7.24 and 7.07, respectively (Fig. 6b).

4. Discussion Biofilms formed by B. licheniformis in the milk powder processing environment presents serious consequences for dairy product manufacturers. Controlling biofilms in the milk processing environment can not only improve the quality of dairy products, but can also help making the overall processing cost effectiveness (Bremer et al., 2006). 9

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containing CFS of L. plantarum was modified to keep the pH value around 6.5, in order to exclude the effect of low pH on biofilm formation. The biofilm formation by B. licheniformis was delayed by 10 h by the addition of L. plantarum metabolites. The absence of biofilms by B. licheniformis in double-volume PBS groups resulted from low numbers of bacterial cells due to TSB dilution. It is indicated that L. plantarum metabolites affected biofilm forming by B. licheniformis, regardless of causing any influence on cell count of planktonic fractions, which showed that biofilm inhibition occurred due to negative effects of metabolites on bacterial systems particularly involved in biofilms. CLSM images showed that L. plantarum metabolites impaired the adhering ability of B. licheniformis cells to the surfaces of stainless steel and glass. When used alone, the SYTO 9 stain labeled all B. licheniformis cells on the coupons, while PI penetrated only into bacteria with damaged membranes. With an appropriate mixture of the SYTO 9 and PI stains, live cells with intact cell membranes stained fluorescent green, whereas dead cells with damaged membranes stained fluorescent red. Difference in the proportion of the total cell number and live cells on stainless steel and glass surfaces reflected the relationship between material and biofilm formation by B. licheniformis. The addition of L. plantarum CFS had no effect on TSB nutrients, thus the growth of B. licheniformis was not compromised, however, the CFS had anti-biofilm metabolites which directly affected the biofilm forming ability of B. licheniformis. In this study, we further found, based on results of gel filtration chromatography, that B. licheniformis utilized and decomposed large peptides with molecular weight 613–1486 Da into peptides of smaller size (181–613 Da), which may have a role in the regulation of biofilm formation. However, L. plantarum metabolites, especially of the size 3–10 kDa, are possibly involved in affecting the biofilm formation cascade of B. licheniformis through a mechanism which we did not investigate in this study – merit further studies. During the cultivation of B. licheniformis with L. plantarum ultra-filtrates, it was indicated that 3–10 kDa components have a role in inhibiting B. licheniformis biofilm formation. Wasfi, Abd El-Rahman, Zafer, and Ashour (2018) also found that biofilm cells of Streptococcus mutants, once treated with CFS of a Lactobacillus sp., showed reduced expression of genes related to exopolysaccharide production, acid tolerance and quorum sensing, mainly because of bacteriocin or bacteriocin-like polypeptides that had a small molecular weight of < 10 kDa. Vahedi Shahandashti, Kasra Kermanshahi, and Ghadam (2016), reported a purified bacteriocin from L. plantarum ATCC 8014 and a partially purified bacteriocin from Lactobacillus acidophilus ATCC 4356 which displayed noticeable inhibitory activity against the growth and biofilms of Serratia marcescens strains. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) analysis revealed that the molecular weight of bacteriocin from L. plantarum and Lactobacillus acidophilus was 63 kDa, and 68 or 48 kDa, respectively. Aslim, Yuksekdag, Sarikaya, and Beyatli (2005) found that bacteriocin-like substances with inhibitory activity on autoinducer type 2 quorum sensing were identified as 29 kDa peptide by SDS-PAGE. A recent study revealed that a specific Bacillus substance, fengycins - a specific class of lipopeptides which are partly peptide and partly lipid, are able to inhibit Staphylococcus aureus through inhibiting its quorum sensing system (Piewngam, Zheng, Nguyen, Dickey, Joo, Villaruz, et al., 2018). The wild strain Bacillus subtilis ATCC 21332 produced two cyclic lipopeptides: surfactin and fengycin, as secondary metabolites (Chtioui et al., 2014; Chtioui, Dimitrov, Gancel, & Nikov, 2010; Gancel, Montastruc, Liu, Zhao, & Nikov, 2009), providing more evidences on interspecific interaction in response to population density. Although the finding of this research has great implications for the control of B. licheniformis biofilms, however, the mechanism of this inhibition still remain unknown and will be explored in the future.

Fig. 6. (continued)

(Goncalves de Almeida Junior et al., 2015; Kachouri, Ksontini, & Hamdi, 2014; Ramos et al., 2015). In this study, L. plantarum CFS is reported to contain effective anti-biofilm peptides which can effectively inhibit biofilm formation by B. licheniformis. Low pH in co-culture growth conditions, involving Lactobacillus ssp., is another important factor responsible for growth inhibition of B. licheniformis. Inhibition of growth at low pH may be related to bacteriocin production as it is known that low pH is important for Lactobacillius spp. to produce bacteriocin. For example, Taheri, Samadi, Ehsani, Khoshayand, and Jamalifar (2012) found that neutralization of CFS of Lactococcus lactis to pH 6.5 significantly reduced the anti-microbial activity of its bacteriocin. L. plantarum has been reported to limit the growth of microorganisms by decreasing the pH of the medium, which eventually influences the trans-membrane pH gradient and decreases the amount of available energy for cells to grow, leading to their death (Wee, Kim, & Ryu, 2006). A variety of anti-microbial metabolites produced by Lactobacillius spp., including organic acids, ethanol, fatty acids, acetoin, hydrogen peroxide, diacetyl, bacteriocins (nisin, reuterin, reutericyclin, pediocin, lacticin, enterocin and others) and bacteriocin-like inhibitory substances, are also reported to be effective against the growth of several pathogens (Chtioui, Dimitrov, Gancel, Dhulster, & Nikov, 2014; Reis, Paula, Casarotti, & Penna, 2012; Sobrino-Lopez & Martin-Belloso, 2008; LeBlanc, Laino, Juarez, del Valle, Vannini, van Sinderen, Taranto, et al., 2011; Oliveira, Oliveira, & Gloria, 2008). Some acids, such as acetic acid, are critical to the metabolism of lactobacilli but inhibitory to members of the genus Bacillus (Reis et al., 2012). The pH is also a key factor that affects the biofilm formation by B. licheniformis (Almasoud et al., 2016; Bai, Zhong, Wu, Elena, & Gao, 2019). In this study, the results showed that an alkaline environment was suitable for the planktonic growth of B. licheniformis, while a neutral environment proved to be ideal for biofilm formation. TSB

5. Conclusions The inhibitory effect of L. plantarum metabolites against biofilm 10

Food Control 106 (2019) 106721

N. Wang, et al.

formation by B. licheniformis was explored in this study. L. plantarum was found to inhibit planktonic cell growth as well as the biofilms of B. licheniformis in co-cultivation under pH controlled conditions. A 3–10 kDa peptide component in the CFS of L. plantarum was found responsible for the inhibition of biofilms formation by B. licheniformis. Effective prevention of biofilm formation by B. licheniformis was also demonstrated on stainless steel and glass surfaces. These findings provide new and safe strategies for the removal of biofilms, which is a paramount requisite to increase dairy process efficiency and product quality.

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