Isolation and Identification of Dextran-Producing Weissella cibaria from Pickled Sauerkraut

The presence of certain bacteria in food products are particularly desirable. Probiotic strains of lactic acid bacteria (LAB), which have a number of favorable characteristics, including antimicrobial properties that support the development of beneficial microflora, are among them. In addition, because of their biochemical activity, they have an impact on the dietary, nutritional, and organoleptic qualities of food products. Before being released into the market, each strain must undergo independent testing to ascertain its probiotic qualities and potency. The purpose of this study was to separate and identify the LAB strain (Weissella cibaria bacteria) at the molecular level from a sample of pickled Sauerkraut that might be employed in the dairy sector and maybe is regarded as a probiotic in the future. Directly from the can, the shredded picked sauerkraut was brined in 2.25% salt (NaCl) for 48 hours before being inoculated aseptically into deMan, Rogosa, and Sharpe agar (MRS) and incubated for two days at 37 °C. Gram’s reaction, catalase production, and sugar fermentation tests employing glucose, sucrose, maltose, lactose, and fructose were among the traditional assays performed on isolation. The sequence was acquired and checked against those in the GenBank database using the BLAST tool at the NCBI-GenBank database after the isolate was extracted using the AccuPrep Genomic DNA Extraction kit, amplified, and agarose gel electrophoresis. The isolated LAB strains were validated using SEM and a genetic PCR assay, and the matching rate of the two isolated strains was 99%. The obtained strains showed their ability to produce dextran by observing the viscous growth characteristic when they were grown in MRS medium supplemented with 15% sucrose. To identify the bacterium, many culture and biochemical tests were carried out. The overall study concluded that the strains of Weissella cibaria bacteria can be easily isolated from pickled Sauerkraut using the developed procedure with the possibility of using the dextran produced by the bacteria as a prebiotic in the food sector.


Introduction
Exopolysaccharides (EPS) studies have recently accelerated due to the industrial demand for natural polymers.EPS are found in all living organisms, including bacteria, animals, plants, and algae.However, due to their ease of extraction and high purity, microbial polysaccharides have received a great deal of attention [1].Since lactic acid bacteria (LAB) are generally recognized as safe (GRAS) [2], their EPS can be directly incorporated into food production and fermentation processes to enhance the texture, flavor and stability of dairy products, a source of particular interest for researchers is LAB 1259 (2023) 012069 IOP Publishing doi:10.1088/1755-1315/1259/1/012069 2 EPS [3][4][5].EPS is a byproduct of several LAB produced from various sources, including milk and dairy products.Weissella species, Leu.citreum, Lactobacillus plantarum and Leuconostoc mesenterica are examples of often investigated EPS-producing LAB [6,7].According to the kind of monosaccharide they include, LAB EPS can be classified into two categories: homologous polysaccharides, which contain only one type of monosaccharide, and heterogeneous polysaccharides, which contain two or more types of monosaccharides.Due to their commercial significance, Dextrans, a family of homologous polysaccharides made of glucose, have been thoroughly studied: According to Patel, Majumder, and Goyal [8], α-D-glucan has been employed in the fields of food science, material science, and medicine, among other things, as a plasma expander, β-Glucan is also often found in food and skincare [9].The structure and chemical characteristics of dextran's differ, despite the fact that they are always constructed entirely of glucose molecules.According to reports, the bulk of dextran from Weissella species include primarily branched α-1,6 glycoside linkages with 2.4-7% and α-1,3 glycosidic bonds [10,11].Dextran from strains of Leu.citreum, however, shows substantially greater branching [12][13][14].

Isolation of Weissella Cibaria from Pickled Sauerkraut
The Weissella Cibaria strains were isolated from locally supplied, pickled sauerkraut.In Iraq, Pickled foods are highly respected for their ability to promote health and serve as an excellent source of natural probiotics.After being thoroughly cleaned with water, the white leaves of Brassica oleracea Var.capitata were cut into strips.They were then put in a sterilized glass beaker, and 2.25% NaCl salt was added by evenly sprinkling it over the chopped leaves.The mixture was then compressed by applying pressure.Following this, the beaker head was tightly covered, and it was put in the incubator for two days at a temperature of 37 ºC.During this time, samples were taken by withdrawing 1.0 mL of the juice and planting it in the medium.To isolate and characterize Weissella Cibaria strains from Sauerkraut, the procedures in Figure 1 was followed.

Characterizations Tests of Weissella Cibaria
The collected bacteria were recognized as Weissella cibaria by examining their morphological chrematistics and performing biochemical tests in accordance with the taxonomic keys stated in the specialist scientific sources.

Morphological Chrematistics
In terms of colony shape, size, color, edge, height, and capacity to manufacture dextran through mucoid colony formation, the phenotypic features of colonies developed on MRS media were evaluated.

Microscopic Examination
Following Gram staining, isolated colonies of bacterial cells were examined under a microscope to determine the form and kind of the bacteria present, in other words, how they interacted with the Gram stain.

Biochemical Tests
The following tests were carried out to identify the sex and type of bacteria in accordance with the taxonomic and diagnostic keys found in Bergey's Manual, 1986, on bacterial colonies that could make dextran and had characteristics resembling those of Weissella cibaria: Catalase enzyme test, temperature influence on bacterial growth, 55 ºC growth at 3% and 6.5% NaCl concentrations for 15 minutes, followed by 30 minutes at 60 ºC, and the formation of dextran.

Bacteria Culture Test
Colonies developing on MRS media enriched with sucrose underwent a confirmatory assessment of their morphological characters (shape, color), and their capacity to make dextran was investigated by examining the viscous growth characteristic when grown in the aforementioned medium.

DNA Extraction Weissella Cibaria
The wizard genomic DNA purification kit (Promega, USA) was used to extract the DNA of bacterial isolates in accordance with the manufacturer's instructions, as shown in

Bacterial DNA Extraction
The procedure for extracting DNA from the bacterial suspension was carried out in accordance with the manufacturer's methodology.Before centrifuging for 2 minutes at a speed of 13,000 rpm, the bacteria were first activated on the sterile liquid Broth Nutrient medium prepared at a temperature of 37 ºC for 24 hours.The active bacteria were then placed in a centrifuge tube and centrifuged for two minutes at a speed of 13,000-16,000 rpm.The precipitate (bacterial cells) was produced after the filtrate was removed.The bicardial cells were then incubated for 30 minutes at 56 ºC and cooled to room temperature.After obtaining the bacterial cells, the following steps were followed:  Add (200 µL) of Protein Precipitation Solution to remove protein and mix with a Vortex mixer for 10 seconds. Put the tube in ice for 5 minutes. The centrifuge tube was placed in the centrifuge at 13,000-16,000 rpm for 2 minutes. Transfer the filtrate to a clean tube containing 600 µL of isopropanol and mix at room temperature. The tube was placed in a centrifuge and the filtrate was disposed of and the bottom sediment was kept, which contains "Pellet Cells" in the form of granules which is the DNA of bacteria. 600 μL of 70% ethanol was added to purify DNA, at room temperature with mixing  The tube was placed in the centrifuge for 2 minutes at a speed of 13,000 -16,000 rpm. Withdraw the ethanol and air dry the granules for 10-15 minutes. Rehydration of DNA in (100 μL) DNA Rehydration Solution for 1 hour at 65 ºC. After obtaining the DNA, the DNA was preserved at a temperature of (-20 ºC) until the time of replication by PCR technique.

Measuring the Concentration of Extracted DNA
The concentration of extracted DNA was measured using QuantiFluor chromatography indicator.A 199 μL of Quantus chromatids was added to 1.0 μL of bacterial DNA sample, left for 5 minutes, and then the purified DNA concentration was measured using a Quantus™ Fluorometer.

Polymerase Chain Reaction (PCR) Test
Utilize the lyophilized version of the initiator that purchased from the Macrogen Company (Korea), the current test was performed.To generate the final solution, the lyophilized initiator is dissolved in free nucleic acid to achieve a concentration of 100 pmol/L.Next, 10 mL is withheld and added to 90 mL of free nucleic acid to produce a solution with a concentration of 10 µL.Then a 100 µL of the total volume are for use at work.

PCR Master Mix Calculation
Master Mix5 is a ready-made solution that contains all reagents required for PCR (except template, initiator, and water).The (Ready to Load) format also includes a compound necessary for loading onto the chromium gel and the guide dyes Bromo phenol blue are used to monitor progress during the electrophoresis process.It is a ready-to-use solution for DNA amplification by PCR.This set was obtained from Solis Bio Dyne / Europe.The PCR polymerase mix used to detect bacterial DNA includes:  4.0 μL from Master Mix 5X  2.0 μL of template DNA  2.0 μL from the initiator  9.0 µL of nucleic acid free ions to complete the amplification mixture to 20 µL.Table 1 illustrates the primaries used during the PCR experiment.

Process of Preparing a Polymerase Chain Reaction (PCR) Mixture
The total volume of the polymerase chain reaction PCR is 20 μL, according to the components of the mixture as in the aforementioned Tables (3-6) from the manufacturer, prepare the reaction volume in the PCR tube, the mixture is rotated down and then the PCR tube is placed in the heat rotor, as the amplification reactions for the PCR reaction start according to the thermal conditions.In Table (3)(4)(5)(6)(7), it consists of an initial denaturation at 95 ºC for 3 minutes, followed by 30 cycles with denaturation at 95 ºC for 45 seconds, and the primer bonding at 61 ºC for 1 minute and 45 seconds, first inflation at 72 ºC for 50 sec, and final inflation at 72 ºC for 10 min.The agarose gel prepared in paragraphs (3-3-7) was mixed with 5 μL of red dye.A bandwidth of the DNA was imaged by electrophoresis and was captured with a reliable gel banding system.

Gel Electrophoresis Test
The electrophoresis procedure was carried out as instructed by (Maniatiset et al., 1982).On 2% agarose, all PCR components were combined as follows:  Make the gel according to the step (3-3-7). Pour 5 mL of Red Safe TM into this gel mixture. Put adhesive tape on both sides of a plastic plate and secure it so that the gel won't leak when you pour it on the plate in order to prepare it to retain the gel while the model cools.After that, place the comb one centimeter from one end, wait for the gel to set, and then slide the comb off. After removing the adhesive, place the gel in the electrophoresis apparatus containing the ready-made TBE buffer solution described in paragraphs (3-3-6).Each hole on the gel should have 10 µL of the PCR mix containing bacterial DNA added to it, followed by 5 µL of the labeler in the first hole.As a molecular indicator, use it to calculate the PCR mixture's volume.
 Pour µL of the labeler into the first hole on the gel before adding 10 µL of the PCR mixture containing bacterial DNA to each one.As a molecular indicator, use it to calculate the PCR mixture's volume. Before putting it into the device, around 3 µL of Bromo phenol blue dye, or in a ratio of 1: 3 was added to DNA that had been isolated. Using an electrophoresis device set to 100 volts and 70 amps for an hour long enough to observe the bacterial DNA bundles when the electrophoresis is complete. Lifting the gel plate to expose it to UV light for the purpose of identifying and photographing bacterial DNA strands.

DNA Sequencing
Before being transmitted to Macrogen, the PCR result was verified on an agarose gel to make sure it included a sample of bacterial DNA in the appropriate volume.The automated DNA sequencing method AB13730XL was used for gene sequencing.BLAST, Geneoius, and sequencing results analyses were used to examine the outcome and identify the transmitted isolates.

SEM Test
This examination was carried out on the bacteria within 24 hours of their production of dextran by culturing the bacteria on a plate containing solid MRS medium and leaving it in the incubator for 24 hours at a temperature of 37 °C and observing the formation of dextran and then this examination is carried out by the device where a very small amount is taken coated from the sample with a gold layer.The parts of the device are painted over and the morphology is observed.The surface microscopic shape of the EPS sample was observed using SEM with an acceleration voltage of 10.0 kV under magnifications of 400×, 1000× and 2000.

Results and Discussion
On MRS culture medium, a bacterial strain of gram-positive Bacillus or Staphylococcus that can produce EPS of 15% sucrose as a substrate was isolated from pickled Sauerkraut.It was determined by a genetic diagnostic test that the germs are Weissella.Due to their capacity to generate significant amounts of dextran during the past 10 years, these strains have drawn a lot of interest.These bacteria are non-motile, short-lived, coccoid Gram-positive bacilli that range in length from 1.0 to 0.4.(Fig. 2).This study showed that bacteria grow in the presence of 3% -6.5% sodium chloride and high or low temperatures through tests of the medium of NaCl salt tolerance and growth at high temperatures ranging between 55-60 ºC and also growth at low temperatures ranging between 4-10 ºC through the formation of turbidity and the change in the color of the medium is an indication of its growth and conducting a co-enzyme test by adding 3% hydrogen peroxide by adding several drops and the absence of gas bubbles, which gave a negative result.The obtained isolates showed their ability to produce dextran by observing the viscous growth characteristic when they were grown in MRS medium supplemented with 15% sucrose (Fig. 3) and this result was identical.The results of the SEM test provide a clear picture of the characteristics of polymers and the surface micromorphology of biomolecules, which helps to understand their physical and functional characteristics.The polymeric matrix of the EPS was smooth and uniform at a 1000x magnification.This demonstrates the structural prerequisite for dextran production and qualifies the structure's porosity for the synthesis of such a matrix.Dextran has high solubility, water retention ability, and thermal stability in a consistent polymer; these results support its use as a food additive and environmental protection as shown in Figure 4.The laboratory strain was identified as Weissella cibaria during the genetic analysis conducted by the Korean company's analysis of the DNA sequencing.The laboratory strain was then kept and subjected to the remaining tests.The primers for the aforementioned gene were used in polymerase chain reaction to amplify the 16S rRNA gene.The existence of distinct bands for the amplified genetic material suggests that primers have bound to the intended gene without the other components of the isolated DNA being removed (Fig. 5).

Conclusion
As dextran-producing bacteria, Weissella cibaria strains were isolated from pickled Sauerkraut and analyzed under the microscope and during culture.The isolation strains were then validated using SEM and a genetic PCR assay, and the matching rate of the two isolated strains was 99%.

Figure 1 .
Figure 1.Scheme shows the isolation steps of Weissella Cibaria strains from the picked Sauerkraut.

Figure 2 .
Figure 2. The optical macroscope image of the Gram staining of the cell morphology of strains for Weissella cibaria.

Figure 3 .
Figure 3.The Colony morphology of the examined strains.

8 Figure 5 .
Figure 5.The amplification of 16s RNA gene of Unknown bacterial species were fractionated on 1.5% agarose gel electrophoresis stained with Eth.Br.M: 100bp ladder marker.Lanes 1-4 resemble 1500bp PCR products.PCR products were supplied by Macrogen Corporation, Korea, for Sanger sequencing utilizing ABI3730XL automated DNA sequences.After analyzing the results with the BLAST program at the National Center for Biotechnology Information (NCBI) and comparing them with the available information, it was discovered that there is a match between the two isolated strains of Weissella bacteria with those registered in NCBI and also these results matched with many modern finds discovered by many researchers in molecular diagnostics (Yu et al., 2018; Bounaix et al., 2010; Li et al., 2022; Gao Y et al., 2021).

Table 1 .
The Wizard genomic 1259 (2023) 012069 DNA purification kit components contain of the following: Cell Lysis Solution, Protein Precipitation Solution, DNA Rehydration Solution and RNase A Solution.

Table 1 .
The primers used during the PCR experiments.

Table 2 .
The components of the mixture that required for the PCR experiments.

Table 3 .
Lists the guidelines for using the PCR equipment to amplify genetic material.