Analysis of Exopolysaccharides in Lactobacillus casei group Probiotics from Human Breast Milk

Exopolysaccharides get a lot of attention because they can improve the host immune system. Exopolysaccharide is a polysaccharide that is produced and secreted from microbes outside the cell, usually found on the outside of the bacterial structure. The Lactobacillus casei group from human breast milk is thought to have the ability to produce exopolysaccharides. The purpose of the study was to examine the exopolysaccharide of the L. casei group that was isolated from breast milk. The methods used include the gravimetric, the phenol-sulfuric acid and the Fourier Transform Infra-Red (FTIR). The results showed that the L. casei group could produce exopolysaccharides, and had high exopolysaccharide total sugar content. Lactobacillus paracasei had the highest exopolysaccharide and total sugar content of 3660 mg/L and 80.6%, respectively. The FTIR results of the L. casei group exopolysaccharides showed the presence of hydroxyl functional groups O-H (3425.76-3295.98 cm -1 ), methyl C-H (2930.86-2856.70 cm -1 ), carbonyl C=O (1660.11-1647.27 cm -1 ), C-H (1456.16-1373.44 cm -1 ) and C-O-C ether (1071.08-1056.82 cm -1 ) which are specific characters of exopolysaccharides. Since the FTIR profile demonstrates that the L. casei group can produce exopolysaccharides, it has greater potential as a a probiotic.


INTRODUCTION
Changes in lifestyle and poor diet in the community can disrupt the balance of the digestive tract microbiota, resulting in a decrease in the body's immune system (1).
One of the prevention efforts against this is by restoring the balance of the immune system, like improving the health of the (2), the digestive tract is a reflection of the immune system because almost 80% of the components of the immune system are found in the digestive tract.Improving the health of the digestive tract can be done by consuming probiotics.
Lactobacillus is one of the largest genera of lactic acid bacteria (LAB) and is often used as a probiotic.Research related to Lactobacillus probiotics is limited to the requirements that must be met as a probiotic, namely acid and bile salt resistance, can adhere to intestinal cells and survive in the intestinal tract, able to produce antimicrobial compounds, antagonists against pathogens, safe in food and clinically, proven to affect health, and comes from humans (1).The EPS analysis can be done through EPS production and total sugar analysis (5).In addition to these two analyzes, to ensure the compound is an EPS is also needed.One method that can be used is the

Bacterial Cultivation
This study used the L. casei group isolated from human breast milk in the study of Kusmiyati et al. (7).The cultivation of the L. casei group was carried out through culture.One ose of pure culture was taken and grown on 5 mL of MRSA-tilted media at 37 ºC for 24 hours.The rejuvenated species were used for inoculum stock preparation and exopolysaccharide (EPS) production analysis (8).

Exopolysaccharide production analysis
One ose of the isolate was inoculated into 20 mL of MRSB, which 5% sucrose had been added and incubated at 37 ºC for 24 hours.EPS content ( mg L ) = EPS dry weight (mg) media volume (L)

Analysis of total sugar content
The total sugar content of EPS was determined by the phenol sulfuric acid method using glucose as the standard curve.
The standard curve was made by means of 1 mL of a standard glucose solution containing 10, 20, 30, 40, 50 and 60 ppm.Glucose was put into a test tube and 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid were added.The solution was heated in a water bath at 40 ºC for 15 minutes and the absorbance was measured at 490 nm (9).
Exopolysaccharide: as much as 2 mg of crude EPS was dissolved in 50 mL of distilled water.A total of 1 mL of EPS solution was quickly added to 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid and left for 10 minutes.Next, the solution was heated in a water bath at 40 ºC for 15 minutes.The absorbance was measured at 490 nm (9).

Infra-Red (FTIR)
The Fourier Transform Infra-Red provides complete information about the functional groups contained in the structure of a compound.Thus, in this analysis, it can be ascertained that the isolate group can produce exopolysaccharides (10).

Exopolysaccharide production analysis
Exopolysaccharide production analysis showed that the L. casei group could produce exopolysaccharides (Figure 1).The calculation results show that L. paracasei can produce the highest exopolysaccharide, which is 3660 mg/L.Based on statistical analysis, the results were not different with L.

Exopolysaccharide total sugar analysis
The total sugar content of the exopolysaccharide group of Lactobacillus casei showed different results (Figure 2).The calculation results showed that the total sugar content of the highest exopolysaccharide in L. paracasei was 80.6%.This value was not significantly different in L. casei by 72.6%, and significantly different from L.
rhamnosus.The research of Xu et al. (12) showed that the total sugar content of L. casei exopolysaccharide isolated from sauerkraut, northeastern China, by the phenol-sulfuric acid method was 88%.

Identification of exopolysaccharide functional groups using a Fourier Transform Infra-Red (FTIR) spectrophotometer
The analysis of exopolysaccharide spectra produced by the L. casei group showed different wave numbers (Figure 3).casei group regarding the similarity of the wave numbers obtained for each functional group is presented in Table 1.The L. casei group has eleven to twelve functional groups.
L. casei had more functional groups than L.

Exopolysaccharide production analysis
Genetics and individual phenotypes influence differences in the production of exopolysaccharides in LAB (16).Genetic traits are inherited traits of each bacterial species that are influenced by gene composition, while phenotypic traits tend to be influenced by environmental factors.In addition, the origin and species differences of LAB strains will be able to contribute to the richness and diversity of glucosyltransferase

CONCLUSIONS
The L. casei group was able to produce exopolysaccharides (EPS).L. paracasei was able to produce the highest EPS (3660 mg/L) and total sugar (80.6%).Confirmation by FTIR showed that the L. casei group could produce EPS characterized by hydroxyl, methyl, carbonyl and ether functional groups.
This study shows that the L. casei group has more value than just being a probiotic.

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digestive tract.According to Ahamad et al.
advantage of the Lactobacillus genus is that it has been declared a safe microorganism when added to food because it does not produce toxins, otherwise known as Generally Recognized As Safe (GRAS) microorganisms (3).According to Teame et al. (4), several microorganisms are probiotic which can produce exopolysaccharides.Exopolysaccharides (EPS) are sugar polymers or polysaccharides secreted by microorganisms out of cells.EPS has a health effect because it can improve the immune system, so it is essential to analyze probiotics in producing EPS.According to Ahamad et al. (2), the Lactobacillus casei group derived from human breast milk is eligible as a probiotic.L. casei group consisting of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus can be used as candidates to produce EPS.

Inoculum was added to 20
mL of 10% TCA and homogenized with a 90 rpm shaker incubator for 30 minutes.Then it was centrifuged for 30 minutes at 4 ºC at 4500 rpm.The supernatant was taken and added to 96% cold ethanol twice.The sample was allowed to stand at 4 ºC for 24 hours, then centrifuged at 4 ºC at 4500 rpm for 30 minutes.The pellets were dried in an oven at 60 ºC and the EPS dry weight was weighed.EPS levels were calculated based on the method of Nurhasanah et al. (5) and are presented in Equation 1.
2 mg of dry exopolysaccharide was put into an agate mortar and mixed with 200 mg of potassium bromide (KBr).After that, the material is 32 ground until homogeneous.Next, the mixture is pressed and formed into pellets.The pellet is placed in the cell holder of the FTIR instrument.Dry exopolysaccharides and KBr were suppressed with a hydraulic suppressor with an FTIR spectrum at a wave number of 500-4500 cm -1 (11).qualitative data analysis used the results of the FTIR profile of exopolysaccharide compounds.Product data and total sugar exopolysaccharide were tested for normality using Shapiro-Wilk and homogeneity tested using Levene Test.If the data is normal and homogeneous, then it is analyzed using the Analysis of Variance (ANOVA).If the variance in the data gives a significant difference, then the Duncan Multiple Range Test (DMRT) is carried out with a level of = 5%.
All species have functional groups indicating the presence of an exopolysaccharide in general, namely O-H (hydroxyl), C=O (carbonyl), C-H (methyl), and C-O-C (ether).

Figure 2 .
Figure 2. Total sugar content of the L. casei group of exopolysaccharides

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gtf) genes, which will affect the diversity of enzymes in exopolysaccharide synthesis so that variations in exopolysaccharide production occur.Anindita(16) reproted that four strains of Weisella confusa derived from breast milk produced different exopolysaccharides, namely W. confusa AS3 (1883 mg/L), W. confusa AS14 (1570 mg/L), W. confusa AS18 (1369 mg/L) and W. confusa AS21 (1480 mg/L).These results indicate that the ability to produce exopolysaccharides is influenced by differences in strains.The same species have varying capabilities in the production of exopolysaccharides.Fatih(17) added that the differences in the production of exopolysaccharides were caused by the different enzyme activities of each species.The exopolysaccharide content of L. casei isolated from palm sap was lower (106.33mg/L) (10) compared to the fermented colostrum kefir (1340 mg/L).In another study, L. paracasei exopolysaccharide levels were lower after 48 hours of fermentation (376.4 mg/L) (18) compared to fermented yoghurt (932.0 mg/L) (19).This study showed that L. paracasei produced an exopolysaccharide of 3660 mg/L, which is a fairly high value compared to existing studies.The production of exopolysaccharides in the L. casei group resulted in a high value because the MRSB medium was added with 5% sucrose.According to Malick et al. (20), bacterial culture conditions such as medium composition, pH, temperature, and incubation period have been shown to 36 significantly affect exopolysaccharide production.The exopolysaccharide content of L. rhamnosus with the addition of hydrogen peroxide (H2O2) supplementation after 24 hours of incubation was 567 mg/L (21).The same study showed an increase in exopolysaccharides when given the addition of calcium chloride (CaCl2) and H2O2 after 12 hours, namely 2498 mg/L.Research by Rajoka et al. (22) showed that L. rhamnosus could produce 1808 mg/L exopolysaccharides, while the study of Bertsch et al. (23) L. rhamnosus could produce affects the production of exopolysaccharides is incubation time.In this study, the incubation time used was 24 hours.Incubation time must pay attention to the adequacy of nutrients in the media to ensure that bacteria can still grow by producing exopolysaccharides and do not reach a static or death phase when harvested.According to Angelin & Kavitha (24), exopolysaccharides are synthesized in the logarithmic or final logarithmic, and the stationary phase.However, maximum production occurs only in the late logarithmic phase rather than the stationary phase.According to Fan et al. (25), the production of exopolysaccharides is often associated with the growth phase of bacteria.Some exopolysaccharides are only synthesized in the late logarithmic or stationary growth phase, while others are synthesized during the microbial growth process.The quantity of exopolysaccharide produced varies between strains, media composition, and culture conditions such as pH, temperature and carbon ratio.According to Al-Manhel (26), the concentration of carbon sources is one of the most important factors in achieving high levels of exopolysaccharide production.In this study, the 5% sucrose addition increased the production of exopolysaccharides more than previous studies, which were without the addition of sucrose.According to Bibi et al.Weisella cibaria C43-11 grown in liquid media and enriched with 10% sucrose showed high exopolysaccharide production.The addition of excessive sugar in the culture medium also increased the production of LAB exopolysaccharides.The increase in sucrose concentration in MRS media was suitable for exopolysaccharides production in Lactobacillus confusus TISTR 1498.The exopolysaccharide produced by the L. casei group in this study showed that the L. casei group had the potential to be developed other than as a probiotic.is influenced by the amount of sugar contained in the production medium and the activity of the invertase enzyme, which plays a role in converting the substrate into its constituent sugar monomers (28).According to Imran et al. (29), the total sugar content in the exopolysaccharide L. plantarum NTMI05 was 95.45% and L. plantarum NTMI20 was 92.35%.This indicates the presence of total sugar in the exopolysaccharide produced by each different strain.In the same study, the total sugar content of L. helveticus MB2-1 was obtained at 95.45%.In another study, the total sugar content of L. pentosus 14FE was 81.38%, L. plantarum 47FE was 83,28%, L. pentosus 68F was obtained 85.19%, indicating that all exopolysaccharides consist of carbohydrates (30).The total sugar content of Bacillus subtilis exopolysaccharide in the research of Razack et al. (31) with media without supplementation showed a yield of 53%, while media supplemented with 2% sucrose showed a yield of 76%.This indicates that the medium has an affect on the total sugar content of the exopolysaccharide.Exopolysaccharide production and exopolysaccharide sugar content obtained from the three bacteria in the L. casei group showed varying results due to different species or strains.According to Nurhasanah et al. (5) differences in yield and total sugar content of exopolysaccharides due to differences in strains between species.Differences in bacterial strains cause the number of metabolites produced to also be different.Fatih (17) added that the number of exopolysaccharides produced by different LAB was caused by inherited traits or genetic traits.The analysis of the total sugar exopolysaccharide of the L. casei group in this study showed a relationship between the total sugar content of the exopolysaccharide and the crude production of exopolysaccharides.This analysis supports the presence of polysaccharides produced by the L. casei group and improves the quality of the probiotics of the L. casei group.that the L. casei group is capable of producing exopolysaccharides.Exopolysaccharides can be used as a form of self-defence by the L. casei group.In addition, exopolysaccharides in lactic acid bacteria can also be used to improve the host immune system.According to Jurášková et al. (32), exopolysaccharides produced by lactic acid bacteria can increase macrophage activity and cytokine production and stimulate the formation of immunoglobulin-A (IgA).According to Mundiri et al. (33), exopolysaccharides are known to have the potential to act as immunomodulators that play a role in the innate immune system in 38 digestion.Exopolysaccharides of lactic acid bacteria can enhance the innate immune system through the role of gut-associated lymphoid tissue (GALT).The use of exopolysaccharides as immunomodulators has been widely carried out and has been proven in-vitro and in-vivo to increase macrophage activity, cytokine production and ability to stimulate IgA formation.from lactic acid bacteria, Bifidobacterium longum can increase the production of IFN-γ , IL-1 β , and IL-6.According to Domingos-Lopes et al. (35), exopolysaccharides from the lactic acid bacteria Leuconostoc citreum L3C1E7 can suppress the synthesis of allergen-specific IgE and exopolysaccharides from lactic acid bacteria have been shown to have immunomodulatory activity with non-toxic effects.According to Rajoka et al. (36), the exopolysaccharides of Lactobacillus contain various functional groups (such as hydroxyl groups, phosphate groups, and carbonyl groups), which help to exert their immunomodulatory, antimicrobial, antioxidant, and anticancer activities.Exopolysaccharides from Lactobacillus can be used as nutritional and therapeutic agents to regulate the immune system, which can help fight various diseases such as cancer, diabetes, and hypertension.Therefore, the addition of Lactobacillus as exopolysaccharide agents immunomodulatory in functional foods has potential in the future.

Nur
Kusmiyati: substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data, drafting the article or revising it critically for important intellectual content, final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Yuni Puspitasari: rafting the article or revising it critically for important intellectual content.Ulfah Utami and Anggeria Oktavisa Denta: agreement to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Table 1 .
FTIR spectra of exopolysaccharides from the L. casei group Exopolysaccharide FTIR results in L. casei have similarities to previous studies.Differences in previous studies often occur in shifting wavenumbers but still show the similarity of functional groups.The L. casei