ANTIBACTERIAL ACTIVITY AND IDENTIFICATION OF PRODUCED REUTERIN FROM LOCAL Lactobacillus

Millipore S.A.S.- Molsheim, France


INTRODUCTION
In recent years, many biologists have shown a wide interest in studying antibiotics produced by Lactobacilli.This interesting was due to many reasons such as their wide inhibition spectrum for growth of pathogens and food damaging bacteria.In addition, Lactobacilli antibiotics represent a natural inhibitor used in food preservation (Duan et al., 2020), especially acidic and neutral foods.This, antibiotics and bacteriocins of Lactobacilli used commercially as a replacement natural material against chemical preservatives which mostly causes a great harm at the level of food safety and human health (Naimah et al., 2018; Niamah, 2018).One of the Lactobacilli species is Lactobacillus reuteri, which is naturally found in the gastrointestinal tracts of humans, chickens, and other animals.L. reuteri can produce Reuterin, which is an intermediate metabolic compound produced from glycerol fermentation under anaerobic conditions and has a wide inhibition effect against other Gram positive and negative bacteria, fungi, yeasts, and also some protozoa (Greppi et al., 2020).Reuterin is a non-protein compound that is neutral water soluble, and has a low molecular weight (< 200g/mol) and it derivative from glycerol (Lindlbauer et al., 2017).Reuterin is produced from L. reuteri as an intermediate compound during the anaerobic hetero-fermentative metabolic pathway by removal of the water molecule from the glycerol (Talarico et al., 1988).Chemically, reuterin is identified as β-hydroxypropionaldehyde or 3hydroxypropanal (3-HPA) and it is formed during the anaerobic condition growth of L. reuteri by the work of glycerol dehydratase which catalyzes the diversion of glycerol into reuterin compound.This bacterium uses the phoshoketolase pathway for fermentation of carbohydrates to lactate, acetic acid, ethanol, and carbon dioxide.3-hydroxypropanal (3-HPA) is produced as an intermediate step in the conversion of glycerol to 1,3-propanediol, a pathway proposed to regenerate NAD + from NADH and to contribute to improved growth yield (Rodriguez et al., 2003).Many studies were explained to the technique of activity by which the reuterin compound spends its antimicrobial impacts has been so hard to be known.The cause is the aldehyde group of the reuterin, which is highly reactive; thus, the reuterin compound can be converted to different compounds in the hydrous solutions.The reuterin can dimerized to form hydroxypropanal (HPA) dimer (this normally shown in very high concentration of 3-hydroxypropanal); or can be hydrated to form the HPA hydrate; or can also be dehydrated to form the toxic compound 'acrolein'.Depending on the two main hypotheses that have been found to explain its method, The first hypothesis was proposed that aldehyde group of the reuterin is highly reactive with the thiol groups and the primary amines,

Bacteria strains
In the European Nucleotide Archive (ENA), the National Centre for Biotechnology Information (NCBI) and Gene Bank, Lactobacillus reuteri isolate has been checked and registered as a local Iraqi isolate.Under the name "LBIQ1" this isolate was reported (MT259030).The Food Science Department/College of Agriculture/University of Basrah, Iraq supplied E. coli ATCC 25922, Listeria monocytogenes ATCC 15313, Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 25923 and they were used as indicator strains.

Antibacterial activity of culture supernatant
The L. reuteri isolate was screened for their antibacterial activity spectra, using the well diffusion agar method (Niamah, 2010) and filter paper disc diffusion method (Mulyani et al., 2019), against E. coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus.First, the cell-free culture supernatants (CFS) were prepared by growing the isolates in tubes of MRS broth (Hi Media, India), adjusting the pH to 6.2±0.2 for 16-18 h at 37 o C, then centrifuging the tubes at 8,000 rpm for 10 min and transforming the cells into new MRS broth (pH 7.2±0.2).After completing the incubation for 24 h at 37 o C, tubes were centrifuged again, and the culture supernatant obtained was sterilised through 0.45 Millipore filters (Millipore S.A.S.-Molsheim, France) to remove all live cells and determine their activity.At the same time, the indicator bacterial cultures were grown for 24 h in nutrient broth (Hi media, India) tubes, and then 0.1 ml (1×10 7 CFU/ml) of each were inoculated by pouring into or spreading on Muller Hinton agar (Hi media, Reuterin was anaerobically manufactured by developing Lactobacillus reuteri LBIQ1 (a local isolate) at 37 °C in the MRS broth medium.Cell biomass was harvested after 18 hours of incubation and used to ferment 278 mMol glycerol for 3 hours in a water-glycerol solution at 37 °C.Cells were then extracted from the suspension solution, which remained at 4 °C until colorimetric, UV, and HPLC assays observed the presence of reuterin in the solution.The reuterin compound has inhibitory activity against Gram-positive and Gram-negative bacteria.The highest inhibition zones measured were 23mm and 21mm against Staphylococcus aureus and Escherichia coli, respectively.The UV chromatogram of produced reuterin showed one peak at 280 nm, while HPLC analysis were appeared three peaks at 6.48, 15.6 min, and 15.49 min for the injection peak, residual glycerol, and produced reuterin, respectively.After incubation for 3 hours, a total yield of 167.382L/ml reuterin was estimated as the productivity of L. reuteri LBIQ1in the glycerol-water solution.The highest inhibition zones of pure reuterin against S. aureus and E. coli were 44 and 39 mm, respectively.Reuterin is an effective compound against microorganisms that can be produced from glycerol media and used in food preservation.
India) plates independently.In the spread method, 6-millimeter wells were formed onto agar plates and then filled with 0.05 ml of the CFS.The plates were kept undisturbed for 2 h and subsequently incubated at 37 o C. for 24 h.After that, the diameters (mm) of the growth inhibition zones were measured (Al-Fekaiki et al., 2017).

Reuterin production and extraction
Reuterin was produced from L. reuteri cultures using the methods described previously by Mohammed et al. 2020 with some modifications.Briefly, the activated cells of the L. reuteri isolate were grown in 20 tubes of 50 ml MRS broth culture, harvested after 18 h of incubation by centrifugation (7000 rpm for 10 min), and washed twice with 50 mMol sodium phosphate buffer (pH 7.2±0.2).The washed cells were then resuspended in 100 ml of 278 mMol water-glycerol solution (pH 7.4±0.2).Then, water-glycerol suspension was distributed on 10 ml screw-capped tubes and incubated under anaerobic conditions at 37°C for 3 h.Reuterin was collected by tube centrifugation (8000 rpm for 15 min) and filtering the supernatant through 0.45 mm Millipore filter paper.The resulting cell-free supernatant was then stored in refrigerator at -4°C before it undergoes the analyses tests for identifying of reuterin containing.

The colour test for detection of reuterin
A colour test, based on the method of Vimont et al. (2019), was used for the detection of 3-HPA content in the glycerol-water solution depending on the 3-HPA dehydration to acrolein, which in turn reacts with the tryptophan reagent (Sigma-Aldrich, Germany) to form a purple complex when compared with standard acrolein.Three millilitres of properly diluted samples and standard acrolein were combined with 6 ml of concentrated HCL and 1.5 ml of tryptophan reagent in a test tube.The mixtures were then incubated for 20 min at 37°C as the purple colour developed, became stable for approximately 15 min, and could be maintained for at least 1 h by cooling the tubes in the refrigerator.

UV chromatogram of sample and standard acrolein
To ensure the detection of 3-HPA in the extract solution of L. reuteri, the UV chromatograms of the sample and standard acrolein were measured to determine the identity between them.The method of Chen et al. (2020) was also used without adding the DL-tryptophan reagent.After Millipore filtration, the 3-HPA sample solution was subjected to dehydration under acidic conditions by the combination of 3 ml of samples, as well as standard acrolein, with 6 ml of concentrated 37% HCL and incubated for 20 min at 37°C then kept in the refrigerator until UV detection was done at the range of wave length between 200 and 800 nm to be compared between peaks obtained as an indicator of 3-HPA detection.

HPLC chromatogram of sample and standard reuterin
After centrifugation and Millipore filtration of glycerol-water solution, the extracted reuterin product was sent to characterized by HPLC technique.Samples from both extracted and standard reuterin were sent to Gahan Shemi Kuster Company laboratories /Tehran, Iran, where the analysis of the HPLC test was done by using a Shimadzu binary pump with an automatic injector and the acetone used as eluent (Mauro and Garcia, 2019).

Quantification of reuterin by standard curve of acrolein
Depending on the method of Burge et al. (2015), a standard calibration curve of acrolein, as an analytical standard, was performed for different concentrations (0.1, 0.5,0.5 ,1, 1.5, 2, 2.5, and 3 mg) prepared in water mixed with 0.75 ml of DLtryptophan reagent and 3 ml of HCL 37% in a test tube.At the same time, a sample of 3-HPA extract was filtered through 0.22-mm Millipore filter paper, and then 1 ml of the sample was mixed with 0.75 ml of DL-tryptophan reagent and 3 ml of HCL 37%.Tube acrolein samples and standards were then incubated for 20 min at 37°C, and the optical density was measured immediately at 560 nm.If necessary, samples were diluted with distilled water to ensure an OD560 within the range of the OD560 of standard acrolein..

Pure reuterin activity against bacteria
The bioactivity of the pure reuterin were assessed by using the wells diffusion method.Both of the pure reuterin were produced in a test tube of glycerol-water solution with the pH neutralised to 7.4 for the prevention of organic acid production.Tubes were then subjected to centrifugation at 8000 rpm for 10 minutes and filtered through 0.45-micron Millipore filter papers.Fifty microliters of pure reuterin were transferred onto the wells of Muller-Hinton agar that had been previously inoculated with E. coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus and left for 60 min at room temperature until they diffused, then incubated at 37°C for 24 h.After incubation, the zone of growth inhibition was measured in millimetres (mm).

Statistical analysis
The obtained data has been treated with statistical analysis by mean ± standard deviation.One-way ANOVA table was used to analyze the using chi square to count of the significant values at (P<0.05) level using the statistical analysis program SPSS version 16.

Antibacterial spectrum of L. reuteri culture supernatants
Table 1 shows the inhibition effect of the culture supernatant of the L. reuteri isolate against four indicator strains (E.coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus).The inhibition zones were measured at (23, 22) mm against Gram-positive S. aureus and L. monocytogenes, while they were 21 mm and 16 mm for Gram-negative E. coli and P. aeruginosa, respectively.This result gives a sign that the isolates under study have the ability to produce an antibacterial substance against pathogenic bacteria.We then discovered that the antimicrobial bioactive compounds are found in the culture supernatant rather than within the bacterial cells.Our results were in agreement with many past studies that indicated the antibacterial activity of reuterin produced by L. reuteri.

The colour test for detection of reuterin
After a short period of incubation at 37°C, the colour of both sample and standard acrolein solutions began to develop to red, then converted to red-purple and finally to deep purple which was left at the end of the incubation period until the purple color became stable of both tubes (Figure 1).The test gave a colour indicator for the chemical conversion of 3-HPA to acrolein as it subjected it to the dehydration process under the effect of concentrated HCL.This result was in agreement with Sun et al. (2018), who mentioned that this method is specific for acrolein detection as a replacement for 3-HPA, which in turn reacts with tryptophan to form a purple complex..

Figure 1
The color test of standard acrolein and acrolein yielded from 3-HPA dehydration.

UV chromatogram of sample and standard acrolein
The UV chromatogram result (Figure 2) showed one peak identity between the sample and standard acrolein solutions at 280 nm, indicating that acrolein was yielded by dehydration of 3-HPA content in the extraction solution under acidic conditions.The asymmetric length of the sample and standard peaks is probably due to the differences in purity and concentration between sample and standard, despite many attempts we made to obtain further identity in the peaks of UV detection.This method was derived from the work of Burge et al. (2015), who used both acrolein (an analytical standard) in the colorimetric method and the UV chromatogram of citric acid (an internal analytical standard) in the separation and analysis of synthetic 3-HPA by HPLC assay.Therefore, this analytical method is appropriate for the detection of 3-HPA production capability efficiently and effectively based on the use of a UV chromatogram for acrolein as a diagnostic standard.
Figure 2 UV chromatogram detection at 280nm for (A) acrolein standard solution (B) acrolein sample solution.

HPLC analysis of extracted and standard acrolein
Results showed the high purity of reuterin, which was represented by the sharp reuterin peak in the purified, extracted sample in comparison with the standard (Figure 3).The first small peaks in both (A) and (B) represent the injection peak, which appeared at 6.71 and 6.48 of standard and sample reuterin, respectively, while the reuterin peaks appeared at 15.6 min and 15.49 min, respectively.The third peak that shown in figure (B) the sample was represented the residual glycerol in the production solution.The incorporeal treatments of the regression equation for the prediction of the reuterin (3-HPA) concentrations that are produced in the supernatant of L. reuteri suspension were significant (P < 0.05) depending on optical density (OD560) for any sample.So, by compensating for the absorption value of the sample supernatant in the equation, we can predict its concentration.For obtaining the actual concentration of sample supernatant, the obtained value from the equation has been multiplied by the sample dilution factor.According to this result, the predicted concentrations of the produced 3-HPA were calculated in the glycerolwater supernatant of the L. reuteri suspension.As clarified in table (2), the regression equation above was applied in the calculation and prediction of 3-HPA concentrations (column Y) using the optical density OD560 (column X) obtained for L. reuteri supernatant.The OD560 data has been recorded after each 30 minutes for the production period (total period = 3 hours), which started beyond 18 hours of growth in the MRS broth and the transformation of cells into a glycerol-water solution.Figure 4 shows the slope and the straight increasing in concentration gradually with incubation time, depending on the absorption indicator of the supernatant.From the monitoring of the production process by this curve, we observed that 148.173μL/ml began to yield after 30 minutes of incubation and, when the incubation time continued to 3 hours, a total yield of 167.382μL/ml reuterin was estimated as the productivity of L. reuteri LBIQ1, under study, in the glycerol- Liang and Sung (2001) reported that the fermentation optimum conditions for reuterin production were 100mg/ml in 300mM glycerol-water suspension of L. reuteri incubated anaerobically at 37°C for 1 h.A notable variance in reuterin concentrations and susceptibility among bacteria strains has been reported.4000 activity units (AU)/ml of reuterin were obtained from using L. reuteri strain 12002 in a 2-step fermentation process for the production of the greatest amount of bacterial biomass, which was then collected and re-suspended in a glycerol-water solution and incubated anaerobically to produce reuterin.The produced reuterin was then water-diluted and used in food decontamination, where MIC values against E. coli and Listeria monocytogenes were 4 AU/ml and 8 AU/ml, respectively (EI-Ziney et al., 1999).The reason of this reuterin activity was clarified by Fujiwara et al. (2017) who's referred to HPA system which contains three compounds, were used together in food preservation, there were HPAhydrate and HPA-dimmer which beat equilibrium state.

Pure reuterin activity against bacteria test
The bioactivity of the pure reuterin against E. coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus test bacteria revealed a significant difference (p<0.05) between their frequencies.The diameter of the inhibition zones for the pure reuterin was 39, 38, 30, and 44 mm, respectively (Table 3 and Figure 5).This result clarifies that the purity of reuterin is important for its mode of action.As mentioned previously, the aldehyde group of reuterin is highly reactive, which leads to the conversion of reuterin in a glycerol-water solution to various compounds, such as HPA dimer or HPA hydrate, termed the "reuterin system," or to the toxic acrolein compound.L. reuteri use glycerol as the raw material for secreting high levels of reuterin when grown or incubated in the presence of excess amounts of glycerol.When the glycerol amount decreased, the amount of reuterin decreased too.Doi (2019) reported that glycerol is metabolised in two pathways: the oxidation by a dehydrogenase to dihydroxyacetone (DHA), which is phosphorylated by DHA kinase and enters the glycolysis pathway; or the reductive pathway, where glycerol is converted to 1,3-propanediol via 3HPA as an intermediate compound.

CONCLUSION
The results obtained in this research explained that reuterin compound is a strong natural disinfecting agent able to inhibit many species of Gram positive and negative bacteria.This opens up one of the alternate ways to tackle the antibioticresistance menace that is rampant around the world in this century.In addition, it uses a preservative as an alternative to chemical additives in food.In the reuterin pathway, glycerol is converted to 1,3-propanediol via 3HPA as an intermediate compound that has inhibitory activity against bacteria.The purification method of the reuterin compound increases the inhibitory activity against the bacterial species tested.
therefore, reuterin compound could inactivate or inhibited the proteins and the small molecules that containing such groups (Katryniok et al., 2013).Reuterin is effective against a wide range of microorganisms, including Candida species (C.albicans, C. dubliniensis, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis) (Jørgensen et al., 2017), Bacillus subtilis, E. coli, Pseudomonas aeruginosa and Staphylococcus aureus (Mohammed et al., Campylobacter jejuni and Campylobacter coli (Asare et al., 2020).The present study was aimed at the production and extraction of reuterin from a local isolate of Lactobacillus reuteri, followed by a study of the antibacterial activity of extracellular metabolites offered in cell-free supernatant and the extraction and characterization of the reuterin product.

Figure 3
Figure 3 HPLC chromatogram of reuterin compound.(A)standard solution, (B)sample solution.Quantification of reuterin by the standard curve of acroleinDepending on Burge et al. (2015), who referred to 1 mol of 3-HPA dehydrating to 1 mol of acrolein, the quantification of 3-HPA content in the extracted samples was assessed by the analytical calibration curve of the standard acrolein in a range of concentrations tested.The linear equation was obtained between the prepared concentrations of standard acrolein and its optical density (OD) at 560 nm (Figure4).

Figure 4
Figure 4 Standard curve of standard acrolein concentrations at optical density (OD) at wavelength 560 nm.
The yield of reuterin produced at different glycerol concentrations in glucose and other sugars as carbon dioxide sources depends on the action of the catabolite repression element sequence existent in the DNA of L. reuteri bacteria, where this result was distinguished in comparison with other previous studies.

Figure 5
Figure5The activity of pure and crude reuterin (CFS) against staphylococcus aureus test bacteria.

Langa et al. (2018) reported
that L. reuteri reacts to the invasion of pathogens with the production of the antimicrobial substance reuterin.Mohammed et al. (2020) mentioned that the reuterin compound is produced in anaerobic conditions with the presence of glycerol and that it causes oxidative stress in pathogenic bacterial cells while lactobacilli, streptococci, and other commensals are more resistant to reuterin.Numerous experiments have tested the antimicrobial effects of biologically derived agents against these bacteria to date, with a new review disclosing the antimicrobial activities of L. reuteri supernatant against Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mutans (

Yang et al., 2021). Table 1
Antibacterial activity spectrum of bacteria L. reuteri culture supernatants * The mean values with standard deviation (SD), n = 3 for samples.

Table 2
Concentrations of produced reuterin (3-HPA) per each 30min of incubation period, predicted according to acrolein OD560 absorption.

Table 3
The diameters inhibition zones (mm) of pure reuterin activity against bacteria test * Mean values with standard deviation (SD), n = 3 for samples.