Feasibility of biofilm production capacity by Levilactobacillus brevis isolated from motal cheese and evaluation of biofilm resistance produced in vitro and in yogurt

2.1 Preparation of biofilm in culture medium

One milliliter of L. brevis suspension (1.5 × 10⁸ CFU/mL) inoculated with fresh MRS broth was dispensed in a 24 well microplate and incubated at 30 ℃ for 48 h. After incubation, the medium was poured, and the plates were washed twice with distelited water to remove planktonic cells (Aoudia et al., 2016).

2.2 Preparation of biofilm in milk

This method was developed by modeling the biofilm production in an MRS broth medium (Aoudia et al., 2016). For the preparation of biofilm in real food environments, milk was used as a substrate precursor. The polystyrene straight-sided jar with a volume of 150 mL with polypropylene screw cap was used. The polystyrene straight-sided jar with a volume of 150 mL with polypropylene screw cap was used. 2 mL of strain suspension (1.5 × 10⁸ CFU/mL) inoculated with 18 mL of pasteurized fresh milk (3 % fat) and poured into container and incubated for 48 h at 30 ℃. Then sample was washed and the biofilm was used for yogurt preparation.

2.3 Microstructure

The three-dimensional structure of the biofilm was observed using a LEO1450VP scanning electron microscope (Germany). Biofilm samples were fixed using 2.5 % glutaraldehyde for 24 h at 4 ℃, then washed thrice for 15 min in 10 mM sodium cacodylate buffer by gentle mixing at room temperature and dehydrated in a graded ethanol series 15 min each at 50, 70, 80, 90 and 95, 2 × 15 min at 100 % and 3 × 15 min in t-butyl alcohol. Finally air-dried at room temperature. After sputter coating with gold, the biofilm sample was observed by SEM with resolution 2.5 nm and Maximum Voltage 35kv. Images were taken in different magnifications at a voltage of 20 kV (Kubota et al., 2009).

2.4 Preparation of probiotic yogurt

Sterile and homogenized bovine milk was heated at 92 °C for 12 min and rapidly cooled to 44 °C. The direct starter cultures (Micromilk S.R.I., Italia; 2 kg of batch starter/100 kg milk, consisting of Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus) were added to milk and was transferred to container (150 mL) containing biofilm. The planktonic cells of L. brevis (1.5 × 10⁸ CFU/mL) were inoculated in control group of samples and were incubated at 37 °C. After reached the pH about 4.5, the samples were kept at 4 °C for three weeks to assessment the viability (Li et al., 2017; Yangilar and Yildiz, 2018).

2.5 Enumeration of probiotic bacteria

Sterial dilutions of each sample was prepared.Then were plated on set MRS agar containing 10 mg/L of vancomycin and incubated in a plastic anaerobic jar with C type gas pack sachet (Merck KGaA, Darmstadt, Germany) at 37 °C for 48 h. The total number of viable bacteria was expressed as Log CFU/g (Li et al., 2017).

2.6 Viability in gastrointestinal condition

Potassium chloride (1.12 g/L), sodium chloride (2.0 g/L), calcium chloride (0.11 g/L), and potassium phosphate monobasic (0.4 g/L) were used for preparation Simulated gastric juice (SGJ). After sterilization at 121 °C for 15 min, pepsin (0.26 g/L) was added, and the pH was adjusted (∼2) by adding 1 N HCl. 1 g of the bacterial biofilm and 3 mL of the suspension containing free cells of L. brevis were separately added into the containers 30 mL of the gastric juice and digested on a shaker at a rate of 90 rpm at 37 °C. Bacterial survival was determined by the conventional plate counting method using an MRS agar plate in 0, 30, 60, 90, and 120 min. To assess survival in the gut, bovine bile salt (0.18 g/L) (Sigma-Aldrich, USA) and porcine pancreatin (1.95 g/L) were added to previous containers and adjusted the pH to 7.0 using 1 N NaHCO3. Viable bacteria were cultured in 60, 120, 180, and 240 min (Gebara et al., 2013; Madureira et al., 2011).

2.7 Determination of MIC

According to the active ingredient of each antibiotic, several concentrations (1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 µg mL-1) were prepared (Zhang et al., 2013). 180 µL of culture medium containing 1.5 × 10⁸ CFU mL⁻¹ from L. brevis was poured into each well. 20 µL of each antibiotic concentration was added and incubated for 48 h at 30 °C. After incubation, the culture medium was drained and washed twice with 0.5 mL of 150 mM NaCl solution. The microplate was then stained for 45 min with 1 mL of 0.05 % (v/v) of crystalline violet solution and washed twice. One mL of 96 % ethanol (v/v) was added, and the optical density was determined at 595 nm (Yangilar and Yildiz, 2018).

2.8 Viability of L. Brevis in biofilm

The viability of L. brevis was tested in the storage period (3 days) at 4 °C. 1 mL of biofilm solution was mixed with 9 mL of sterile peptone water (1 g/L). After sequential dilutions, appropriate dilutions were plated on set MRS. Then, they were incubated in an anaerobic jar with C type gas pack sachet (Merck KGaA, Darmstadt, Germany) at 37 °C for 72 h. The total counts of the viable bacteria were reported (Li et al., 2017).

2.9 Antibiotic susceptibility of L. Brevis in biofilm and planktonic forms

Fresh milk with was heated at 92 °C for 10 min and cooled to 42 °C. Then yogurt starter was added to it (3.6 % w/v). The last concentration of each antibiotic that the L. brevis could not grow in the previous step was prepared. It was then added to the milk and mixed thoroughly. 100 mL of antibiotic-contaminated milk was added to each container containing the biofilm. Also, in planktonic samples, 1 mL of the solution with 1.5 × 10⁹ CFU/mL of L. brevis was added to 9 mL of milk (10 mL to 90 mL of antibiotic-contaminated milk). The control sample was yogurt containing biofilm without antibiotic. All samples were incubated at 42 °C till the pH reached 4.6 then stored at 4 °C to be assessed (Li et al., 2017; Yangilar and Yildiz, 2018).

2.10 Antibacterial activity of probiotic biofilm

L. brevis biofilm was formed in in a microplate with 24-well. After washed, 1 mL per well of fresh Brain Heart Infusion broth (BHI; Merck, Germany) was inoculated with 1.5 × 10⁸ CFU/ml of each pathogenic bacteria (L. monocytogenes ATCC7644 and P. aeruginosa PTCC 1074) was dispensed and incubated for 48 h at 30 °C. After the incubation time, the medium was removed from each well, the microplates were washed. The number of L. monocytogenes and P. aeruginosa were counted by the spread plate method in selective media (Oxford-Listeria-Selective-Agar (Base) and Pseudomonas agar base, respectively). Control groups were included biofilm of L. monocytogenes and biofilm of P. aeruginosa Without the presence of biofilm L. brevis (Aoudia et al., 2016).

2.11 Antibacterial activity of planktonic cells

One ml per well of fresh MRS broth culture was inoculated with 1.5 × 10⁸ CFU/ml of L. brevis and was dispensed in a 24-wells microplate. Subsequently, 1 mL of it was added per well of fresh BHI broth inoculated with 1.5 × 10⁸ CFU/ml of each pathogenic bacteria (L. monocytogenes ATCC7644 and P. aeruginosa PTCC 1074) and incubated for 48 h at 30 °C. After washed, evaluation of microorganisms was performed by the spread plate method (Aoudia et al., 2016).

2.12 Statistical analysis

All the tests were performed in triplicate or more replications, and results were presented as mean values and the standard deviation. The one-way analysis of variance (ANOVA) was performed, and significant difference between treatment groups was determined with Duncan’s multiple range test at p < 0.05.using the statistical analysis system (SPSS Inc., Chicago, IL, USA).

3.1 Microstructure of biofilm

The microstructure of the biofilm showed (Fig. 1) a coherent and three-dimensional network that, similar to a strong skeleton, was able to maintain this unique biological network. Probiotic bacteria in this coherent and natural structure have gained more resistance to environmental stresses such as changes in temperature, pH and the presence of antibiotic residues compared to their counterparts in the plankton state, so their survival rate has increased (Jones and Versalovic, 2009; Kubota et al., 2008; Rueda-Robles et al., 2022).

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Fig. 1

Scanning electron microscopy images of biofilm of L. brevis in (a, b) MRS agar and (c, d) milk.

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3.2 Viability during storage time

The viability of probiotic bacteria in biofilm and planktonic form is compared in Fig. 2. The bacterial population in the biofilm showed an increase about 0.25 log during storage time, while in the planktonic form there was a 3.5 log decrease in the probiotic population.The results show the protective effect of biofilm on probiotics. There is no significant difference between the whole and pulverized biofilm, and this result can make the application of biofilm easier and more convenient on an industrial scale. Biofilm is unique structure that enable bacteria to survive against environmental challenges such as osmotic changes, metal toxicity, dehydration, radiation, host immunity, antimicrobial agents, and disinfectants (Z Rezaei et al., 2021). This significant and protective effect is due to the network structure consisting of hydrophilic and hydrophobic exopolysaccharides that exist in the biofilm (Zeinab Rezaei et al., 2021a). Survival of probiotic bacteria until consumption and in the digestive tract is of significant importance (Kalantarmahdavi et al., 2021). In this study, the viability of bacteria increased with the probiotic biofilm production method (González-Ferrero et al., 2018; Holkem et al., 2017).

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Fig. 2

Viability of L. brevis during storage for 21 days at 4 °C.

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3.2.1

3.2.1 Syneresis and pH of yogurt

The results of syneresis and pH are shown in Fig. 3. Yogurt samples containing pulverized biofilm and whole biofilm have a lower pH than yogurt samples containing plankton form of probiotics, which is probably due to the protective effect of biofilm on probiotics. Biofilm has been able to maintain the bacterial population because of its protective effect. The pH of yogurt is affected by the activity of yogurt bacteria becuse more lactic acid will be produced (Koohestani et al., 2018).

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Fig. 3

Physicochemical properties of probiotic yogurt with L. brevis.

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The amount of syneresis of yogurt can have a significant effect on the appearance quality and overall acceptance of the product. In the dairy industry, compounds such as pectin and gum are used to reduce or prevent syneresis (Lee and Lucey, 2010). The study of the biofilm structure shows the presence of exopolysaccharide compounds in it (Terraf et al., 2016). Therefore, when probiotic bacteria are used in the form of biofilm in dairy products, in addition to the health benefits they can also technically effective in the product and reduce the consumption of stabilizers.

3.3 Sensory evaluation

The color of yogurt containing biofilm was significantly better than the control samples. It also smelled better.(Fig. 4, A). It can be concluded that the acidity of yogurt can affect its pleasant smell. On the other hand, the presence of polysaccharide and protein compounds in the biofilm causes an increase in yogurt solids, and as a result, more light scattering occurs and the color of yogurt becomes whiter (Mousavi et al., 2019). Yogurt texture is also an important parameter. The biofilm showed a more appropriate texture in yogurt and provided more acceptable feeling than control samples. This property was due to the extracellular polysaccharides in biofilm.

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Fig. 4

Sensory evaluation of probiotic yogurt made by planktonic and biofilm forms of probiotic.

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(p < 0.05).Fig. 4, B). The present results showed that the taste of yogurt samples prepared with biofilm was significantly better than the control samples and this property was maintained during the storage time (Fig. 4, C). Biofilm contains about 6–7 % protein and polysaccharides, these compounds may affect other tissue properties (Mousavi et al., 2019; Terraf et al., 2016). In addition, the number of bacteria in yogurt has increased in the form of biofilm, which increases the production of lactic acid and improves the acidity and taste of the product. The new yogurt showed industrial potential and a higher overall acceptance score in sensory evaluation. (Fig. 4, D). Comparing the results of this study with other techniques such as nanoencapsulation and microencapsulation(Iravani et al., 2015; Yao et al., 2020), showed that biofilms do not cause negative sensory changes and can be used to improve the organoleptic properties of yogurt and similar products.

3.4 Gastrointestinal tolerance

In gastric conditions, the free cell viability of L. brevis was decreased to 3.2 Log CFU/mL after 30 min and decreased to undetectable levels after 90 min (Fig. 5a). Survival continued in the biofilm form in the intestine and by the fourth hour of digestion there were 10⁶ viable bacteria in the intestine. (Fig. 5b). In comparison, the viability of L. brevis was greatly enhanced by biofilm in the high acid condition (pH 2.0) to the extent that only 0.25 Log CFU/mL reduction were observed in 120 min. The comparison of the results of this study with studies conducted on commercial strains shows the greater survival power of indigenous strains, which can be attributed to the intra-strain difference of probiotics. In recent years,studies were focused on the isolation and identification of probiotic bacteria from traditional dairy sources. On the other hand, more studies should be done on the annual economy and losses caused by starter imports (Zeinab Rezaei et al., 2021a). Even compared to different methods of increasing survival, including nanoencapsulation and encapsulation, the biofilm technique showed a higher power for the survival of native probiotic (Huq et al., 2017; Liao et al., 2019; Sohail et al., 2011).

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Fig. 5

Viability of lactic acid bacteria biofilm during the exposure to (a) the simulated gastric condition and (b) the simulated intestine condition.

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3.5 Antibiotic resistance

3.5.1

3.5.1 MIC

Enrofloxacin showed the most growth inhibitory effect on the probiotic strain. In the presence of other antibiotics, there was growth in different concentrations of antibiotics, the results of which are shown in the (Fig. 6a). Comparison of the results of this study with the study conducted by Rezaei et al. on commercial strains showed that the native strain of Lactobacillus brevis has significant resistance to the presence of tetracycline antibiotic compared to commercial strains (Zeinab Rezaei et al., 2021b).One reason for the existence of this greater inherent resistance can be considered intra-species differences and the type of source of bacterial isolation (Aoudia et al., 2016).

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Fig. 6

The minimum inhibitory concentration of antibiotic (MIC) in vitro (a), Biofilm and planktonic antibiotic susceptibility (b).

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3.6 Antagonistic activity

In this study, the antagonistic power of the indigenous probiotic strain against two important food pathogens was investigated. The results are given in the Fig. 7. L. brevis probiotic strain was able to reduce the bacterial population of Listeria monocytogenes and Pseudomonas aeruginosa by 1.4 and 1.2 log, respectively, in planktonic form. In contrast, in the biofilm form of. L. brevis, the bacterial population of the pathogens Listeria monocytogenes and Pseudomonas aeruginosa showed a 4.9 and 3.2 log decrease in growth, respectively. Another useful approach of probiotics is to inhibit the growth of pathogens. Probiotics have been shown to reduce their infections and symptoms (Tung et al., 2009). Numerous studies have shown that indigenous strains separated from indigenous dairy products are capable of inhibiting some gastrointestinal diseases compared to commercial strains and have significant antibacterial properties (Z Rezaei et al., 2021). Organic acids such as lactic acids, short chain fatty acids, proteins, active peptides, bacteriocins and enzymes create many well -known roles in antagonistic activity (Chen et al., 2020). Comparison of the results of the antagonistic activity of commercial probiotic bacteria on food pathogens with the present study showed that the indigenous probiotic strain had more antagonistic activity either in the planktonic form or in the biofilm form. Considering that the biofilm mechanism showed more antagonistic power than the planktonic form of bacteria, it can be concluded that the biofilm structure has been able to maintain the microbial population due to its protective role and more antimicrobial compounds are produced by bacteria in the biofilm form (Zeinab Rezaei et al., 2021a).

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Fig. 7

Antagonistic activity of L. brevis in biofilm and planktonic form on the growth of food pathogen.

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