INTRODUCTION:

Lactic acid bacteria (LAB) played a major role in the fermentation of carbohydrate. LAB involved numerous genera of bacteria with the most common genus was arguably the Lactobacilli that could produce various anti-microbial, anti-cancer and immunoregulatory metabolites and so on. However, one of the most notable metabolites reported to possess these biological effects is conjugated linoleic acid (CLA)¹.

Linoleic acid (LA) has formulation of cis-9, cis-11-octadecadienoic acid and included nearly 20 types of isomers with disparity position (7, 9; 8, 10; 9, 11; 10, 12 and 11, 13) and geometric (cis, cis; cis, trans; trans, cis; trans, trans) combination². The majority of biologically active CLA isomers are cis-9, trans-11 (c9, t11) and trans-10, cis-12 (t10, c12)³.

Recently, more researches demonstrated that CLA in dairy product, ruminant meat, vegetable oil contained c9, t11 at 80%. There was undeniable that CLA played vital role in increasing metabolic rate; lowering cholesterol and triglyceride levels; boosting the immune system and enhancing fat burning process. LA represented major unsaturated fatty acid present in practically all oils, but grape seed oil contained a significant percentage of linoleic acid (more than 75%) which was documented as a source for CLA⁴.

Therefore, the study was carried out to seek for an alternative CLA producing source to later contribute to solve the problem of obesity and blood fat. L. fermentum A01 isolated from the gastrointestinal tract as well as other sources was focused to study for this CLA producing ability as a new and essential approach for study further biosynthesis of CLA.

MATERIALS AND METHODS:

Cultivation condition:

Linoleic acid (cis-9, cis-11-octadecadienoic acid) was purchased from Santa Cruz (Dallas, Texas 75220, USA). Grape seed oil was purchased from market in Ho Chi Minh City. Grape seed oil was checked for LA presence prior experiment. All other chemicals were of analytical grade and commercially available. L. fermentum used in this study was isolated from human saliva and cultured in de Man Rogosa and Sharpe (MRS) medium⁵ at 37^oC in 24 - 48h under condition of 5% CO2 and maintained in pH at 6.5. After incubation, the culture was checked for microbial contamination for further use.

Contamination check for GABA-Producing L. fermentum:

L. fermentum was cultured in 10mL medium and incubated from 12h to 48h in MRS broth with or without LA (grape seed oil: O) or purified LA. With treatments involving oil, oil was added to MRS culture in different concentrations: 0.1%, 0.2%, 0.3%, 0.4%, 0.5% (v/v). Incubation time was optimized in 12h, 24h, 36h, 48h. Then, each culture was checked for bacterial growth and the CLA production ability.

Extraction of lipid and fatty acid methylated ester (FAME) preparation:

After incubation time, the culture was centrifuged (13000rpm for 15 min at 4^oC). The culture supernatant was collected and heated to75^oC to harvest the biomass, followed by mixing with chloroform with an equal ratio (v/v) and shaking at room temperature for 2 minutes. The extract was transferred into new tube, evaporated overnight and then lipid was weighed. The esterified fatty acids (EFAs) were transformed into the corresponding FAMEs to be analyzed by GC-MS that were trans-esterified through alkali-catalyzed reaction. The lipid extract was dissolved in 1M potassium meth oxide prepared in methanol solution and the sample was homogenized, then incubated at 55^oC for 1 hour^6-8.

Thin layer chromatography (TLC):

Fatty acid esters were separated by thin layer chromatography⁹. The silica gel plate was soaked in AgNO3 and then dried before samples were loaded on a silica gel plate. The following solvent system was hexane: chloroform (1:3 in v/v).

Gas chromatography- mass spectrum (GC-MS) analysis:

The organic phases containing FAMEs were injected (0.5𝜇L) into a GC apparatus (HP5890 Series II, Agilent, Waldbronn, Germany) equipped with the column (100 mm × 0.25mm, 0.25𝜇m) CP-Select FAME (Varian, Palo Alto, CA, USA). The injector was kept at 270^∘C. To determine the fatty acid (FA) profile, the oven temperature ramped from 135^oC to 250^oC (2.5^oC/min) and was maintained at 250^oC for 23 min. Elution was performed with high-purity helium, with constant column head pressure of 200 kPa. Qualitative analyses of FAMEs were performed with a MS quadrupole detector (HP5972, Agilent). Analysis was identified by comparison with standards (O5632, Sigma Aldrich) and by analysis of their fragmentation patterns (EI, 70eV) with the mass spectrum library NIST 2005 (Gatesburg, USA). Quantitative analyses were performed with a flame ionization detector held at 300^oC⁷.

Statistical analysis:

Statistical Package for the Social Sciences (SPSS) software version 20.0 and Microsoft Excel was employed. Values of P < 0.05 refer to significant differences among experiments.

RESULTS AND DISCUSSION:

Identification of LA in grape seed oil:

In order to use grape seed oil for the study, LA content was detected by TLC and GC-MS. The spot in grape seed oil had similar R_f (0.26) of LA on TLC (Fig. 1). By GC-MS analysis, LA also existed in grape seed oil and occupied for 70% in grape seed oil, equalto 7mg/ml oil.

Fig. 1: The presence of linoleic in A) MRS B) Oil C) LA

Influence of LA on bacterial growth:

Although free LA served as the direct precursor in CLA biosynthesis, it also inhibited bacterial growth, and the tolerance to LA varied among different strains. Therefore, screening bacterial strains with high LA tolerance might be a shortcut to obtain strains that could produce more CLAs. After incubation, the optical density (OD) of cultures ranging between 1.1 and 2.2 illustrated that bacterium was able to grow in the presence of LA at optimal concentrations.

Fig. 2: Optical density of bacteria in MRS-Broth with different concentrations of Lactobacillus fermentum ranging between 0.1% and 0.5% for 12h, 24h, 36h, 48h incubation.

Screening CLA producing ability by thin layer chromatography:

After extraction of lipid and preparation of FAME, the samples were detected on TLC to check for quality before analyzed by GC-MS. Based on the results of TLC, the clear and darker region showed the potential samples in CLA production. As shown in Fig.3, L. fermentum could produce the LA isomers higher in 36-48h when using oil containing 0.5% LA.

Identification of different CLA isomers with GC-MS:

L. fermentum A01could produce 9, 11 di-ene of C18 at the retention time of 30.299 min with 0.67% in case of cultivation in MRS-LA for 36 h. The reason why CLA could not be detected in MRS-O pointed that other fatty acid in oil interfered CLA production in L. fermentum A01although there is most of LA in grape oil. More studies should be carried out to understand mechanism of CLA production in each microorganism.

Fig. 3: The presence of LA after FAMEs of sample that cultured in MRS-LA

L. fermentum A01 could convert LA into CLA in medium containing LA. In the study, L.fermentum A01 produced a few amounts of 9, 11-C18. Normally, free polyunsaturated fatty acids inhibited the growth of anaerobic bacteria, therefore, the saturation reactions were assumed to be detoxification mechanisms.

In L. fermentum A01, 10-octadecenoic acid and 12- octadecenoic acid were produced at high concentration, suggesting that the biological CLA production processes are isomer-selective (Table 1). However, the factors affecting the isomer ratio in CLA of lactic acid bacteria should be determined to further understand the fatty acid transformation reactions, such as isomerization and hydration.

Table 1: Production of individual isomer of LA in the several media

	Main component	% isomer of LA
MRS 36H	· 7-Hexadecenoic acid · 10-Octadecenoic acid · 14-Octadecenoic acid	18.65%
MRS 48H	· 10-Octadecenoic acid · 14-Octadecenoic acid · 11-Octadecenoic acid	17.54%
MRS-O 36H	· 9,12-Octadecadienoic acid (Z,Z) · 9,15-Octadecadienoic acid (Z,Z) · 9,12-Octadecadienoic acid (E,E)	7.59%
MRS-O 48H	· 9,15-Octadecadienoic acid · 9,12-Octadecadienoic acid (Z,Z) · 9,12-Octadecadienoic acid (Z,Z)	12.28%

CONCLUSION:

There are more and more positive effects through a series of disparity mechanism of human gut microbial which are one of the most relevant health promoting, typically lactic acid bacteria. From the obtained results, it is concluded that there were significant differences between the concentration of LA and time for incubation on the CLA production. The effect of LA may be due to the composition of its fatty acids and the biotransformation of linoleic acid into CLA. Depending on the results, we can conclude that there are positive effects for oils on the production CLA of L. fermentum. Moreover, the details of the metabolism, characteristics of the enzymes as isomerase and their gene organization have not been delineated clearly. Therefore, more studies should be done.

ACKNOWLEDGMENT:

Thanks for the fund supported by Hochiminh City International University (HCM IU) for the research signed in the contract (17-BT-2017/HĐSV-QLKH) issued on February 1^st, 2018.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 10.04.2020 Modified on 22.06.2020

Research J. Pharm. and Tech 2021; 14(3):1319-1322.

DOI: 10.5958/0974-360X.2021.00234.1