INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH AND BIO-SCIENCE

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1 Bhatia A,, 2014; Volume 3(4): INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH AND BIO-SCIENCE SCREENING OF BACTERIAL STRAINS AND OIL SUBSTRATES FOR MICROBIAL PRODUCTION OF 9Z, 11E ISOMER OF CLA BHATIA A, SHARMA A Immunology and Immunotechnology laboratory, Department of Biotechnology Punjabi University, Patiala , Punjab, India. Accepted Date: 03/07/2014; Published Date: 27/08/2014 Abstract: 17 strains belonging Lactic acid bacteria and Bifidobacteria were screened for CLA production using growth medium supplemented with cotton seed oil, sunflower oil and soybean oil as substrates. Bifidobacterium bifidium NCDC 235 was selected as best producer of CLA when nutrient medium was supplemented with 0.1% cotton seed oil. The CLA produced in culture medium was extracted and its isomer was confirmed to be 9Z, 11E CLA through Gas liquid chromatography and ATR spectroscopy. The purity was found to be 90.10% with no other FFA (free fatty acid) peak in GC chromatogram. Keywords: Conjugated linoleic acid, Gas chromatography, vegetable oils, lactic acid bacteria, bifidobacteria Corresponding Author: DR. ARUNA BHATIA Access Online On: PAPER-QR CODE How to Cite This Article: Bhatia A, Sharma A;, 2014; Volume 3(4):

2 Bhatia A,, 2014; Volume 3(4): INTRODUCTION Conjugated linoleic acid (CLA) term is used for geometrical and positional isomers of linoleic acid. Till date 28 isomers have been detected, which includes the 9Z, 11E isomer, the most abundant isomer found in natural dairy and meat products. This fatty acid has drawn particular attention due to its antitumour, antiobese, antiatherogenic, antidiabetic and immunomodulatory activities 1-3. Both Lactic acid bacteria and Bifidobacteria are natural converters for producing CLA. Several studies have been conducted in the past to convert LA present in vegetable oils to CLA 4-7. These alternative substrates are cheap sources of LA for large scale production. The previous researches have revealed that utilization of alternative substrate is not species specific but strain specific This study aims to provide new cost effective alternative substrate and bacterial strain with high potential for CLA production. After evaluating the cultures for probiotic potential they can be used in food industry for production of CLA through in situ fermentation. Chemicals Standard samples of 9Z, 11E conjugated linoleic acid and linoleic acid was procured from Sigma Chemicals (USA) and Himedia (India), respectively. All the other chemicals were of analytical grade and the substrates used were commercially available. Microorganisms and maintenance of culture media Lactic acid bacteria (Lactobacillus casei subsp. casei NCDC 17, Lactobacillus brevis NCDC 403, Lactobacillus delbrueckii NCDC 405, Lactobacillus acidophilus NCDC 14A, Lactobacillus acidophilus NCDC 14B, Lactobacillus acidophilus NCDC 011, Lactobacillus acidophilus NCDC 013, Lactobacillus acidophilus NCDC 14C, Lactobacillus acidophilus NCDC 15A, Lactobacillus acidophilus NCDC 15B, Lactobacillus acidophilus NCDC 15C, Lactobacillus acidophilus NCDC 016, Lactobacillus acidophilus NCDC 0195, Lactobacillus acidophilus NCDC 0291) and bifidobacteria (Bifidobacterium bifidium NCDC 233, Bifidobacterium bifidium NCDC 234, Bifidobacterium bifidium NCDC 235) were procured from National Collection of Dairy Culture, Karnal (India). These strains were maintained and screened on MRS medium supplemented with 0.1% linoleic acid and 1% Tween 80. Lactobacilli and bifidobacteria strains were incubated under aerobic and anaerobic condition respectively at 37 C. Substrates (oil), growth medium and screening conditions Preliminary screening for CLA production was done using 0.1% MRS medium supplemented with pure LA as substrate. Comparative analysis of substrate (oil) utilization efficiency by various probiotic bacterial strains for CLA production was done using commercially available alternative substrates: Cotton seed oil, Soybean oil and Sunflower oil. Each substrate was 190

3 Bhatia A,, 2014; Volume 3(4): added in separate production media (5 ml of the MRS Broth with 1% of tween 80 in each test tubes) at 0.1% and 0.5% (v/v). 5% of the inoculum was inoculated in each test tube and incubated at 37 C for 48 hr. The supernatants were collected after 24 hrs and 48 hrs time period. CLA was extracted through a two-step extraction procedure with isopropanol and hexane by the spectrophotometric method of Barrett et. al., Substrate utilization efficiency was calculated using the following formula: Substrate utilization efficiency = CLA produced from oil x 100 CLA produced from pure LA Lipid analysis Preparation of fatty acid methyl esters Lipid samples were extracted from each production media as per previously described procedure. Fatty acid methyl esters (FAME) were prepared from each sample using 14% boron triflouride (BF 3 ) - methanol complex 9. Briefly, extracted samples were refluxed with NaOH solution containing 0.05N methanol for 10min and were esterified with 14% BF 3 methanol complex for 15 min. Samples were than fractionated with hexane and saturated NaCl. Finally Na 2 SO 4 was added to each reaction mixture and they were sonicated for 5min prior to GC (Capillary gas chromatography) analysis. Gas chromatographic Analysis Amount of 9Z, 11E isomer was analyzed by injecting 1µl of 1mg/ml extract into GC system equipped with 30m DB5 capillary column (with 0.25µm internal thickness) using flame ionization detector (FID). Temperature was programmed to start at 70 C for 1min isotherm (initial temp.) and increased at 10 C/min to 190 C and was held for 2min isotherm and then increased at 5 C/min to 265 C. Injector and detector temperature were kept at 250 C. The carrier gas was helium with a head pressure of 28psi (1.97 kg/cm 2 ). Peaks were then identified through comparison with standard reference. Infrared ATR analysis The FTIR measurements were made directly with a horizontal Attenuated Total Reflectance (ATR). The transmittance spectrum was recorded at 4 cm 1 resolution between 4000 and 400 cm 1 using OPUS software (Bruker optics Inc.). The hexane extract was placed on the ATR crystal and after evaporation (about 3 minutes) the spectrum scan was obtained. Statistical Analysis Results were expressed as Mean ± SEM. 191

4 O.D. at 233nm Research Article CODEN: IJPRNK IMPACT FACTOR : ISSN: Bhatia A,, 2014; Volume 3(4): RESULTS AND DISCUSSION Screening of substrates and bacterium for high CLA production CLA production using pure linoleic acid (0.1% conc. in MRS medium) was estimated through standard graph of CLA (9Z, 11E isomer of CLA) produced by spectrophotometric method (Fig.1, Table 1). Out of the 17 strains tested only 8 were CLA producers viz. Lactobacillus brevis NCDC 403 (LB403), Lactobacillus delbrueckii NCDC 405 (LB405), Lactobacillus casei subsp. casei NCDC 17 (LB 17), Bifidobacterium bifidium NCDC 233 (BIF233), Bifidobacterium bifidium NCDC 234 (BIF 234), Bifidobacterium bifidium NCDC 235 (BIF235), Lactobacillus acidophilus NCDC 14A (LA14a) and Lactobacillus acidophilus NCDC 14B (LB14b). These strains were selected for further optimization of CLA production through alternative substrates. The ability to produce CLA from linoleic acid containing oils was studied for sunflower oil, cotton seed oil and soybean oil. A total no. of 48 sets were prepared using 8 selected strains with combination of 3 vegetable oils as substrates at 0.1% and 0.5 % conc. in growth medium y = 31x Z, 11E CLA (mg/ml) Figure 1. Standard graph of 9Z, 11E CLA. Following data given is the mean of triplicate samples, which did not differ by more than 5%. Table 1. Results of CLA production by various bacterial strains using 0.1% Linoleic acid as substrate after 24hr. The observations given are the mean of triplicate samples, which did not differ by more than 5%. Strains LB403 LB405 LB17 BIF233 BIF234 BIF235 LA14a LA14b CLA Production (mg/ml) Effect of time period and substrate concentration on Substrate utilization efficiency CLA production during cultivation of Lactic acid bacteria and Bifidobacteria in MRS medium supplemented with 0.1% or 0.5% substrates were monitored in two cycles at 24 and 48hr 192

5 Percent Substrate Utilization Research Article CODEN: IJPRNK IMPACT FACTOR : ISSN: Bhatia A,, 2014; Volume 3(4): (Table 2). More than 60% substrate was utilized by LB17 (60.46%), BIF234 (64.40%), BIF235 (68.20%) and LB14a (67.40%) when production media was supplemented with 0.1% cotton seed oil as compared to pure linoleic acid (Fig. 2, 3). BIF 235 was found to produce maximum amount of CLA utilizing 0.1% cotton seed oil substrate after 24hr. It was also observed that with BIF235 the production remain stable up to 48hrs. These results are in compliance with the previous observations that CLA production occurs in late log or stationary phase. However increasing the concentration of cotton seed oil to 0.5% resulted in decreased substrate utilization efficiency which might be due to reduction in bacterial growth rate (Fig.4). Table 2. Results of CLA production (mg/ml) by various bacterial strains using 0.1% and 0.5% conc. of oils as substrate after 24hr and 48hr. The observations given are the mean of triplicate samples, which did not differ by more than 5%. Strains LB403 LB405 LB17 BIF233 BIF234 BIF235 LA14a LA14b 24hr 48hr 24hr 48hr 24hr 48hr 24hr 48hr 24hr 48hr 24hr 48hr 24hr 48hr 24hr 48hr Sunflower oil (0.5%) Sunflower oil (0.1%) Cotton seed oil (0.5%) Cotton seed oil (0.1%) Soybean oil (0.5%) Soybean oil (0.1%) LB403 LB405 LB17 Sunflower oil 0.5% BIF233 BIF234 Sunflower oil 0.1% BIF235 LA14a LA14b Cotton seed oil 0.5% Cotton seed oil 0.1% Soybean oil 0.5% Soybean oil 0.1% Figure 2. Bar graph representing percentage substrate utilization pattern of different bacterial strains after 24hr using cotton seed oil, sunflower oil and soybean oil as alternative substrates. The observations are expressed as mean ± SEM (n=3). 193

6 Percent Substrate Utilization O.D. at 600nm Percent Substrate Utilization Research Article CODEN: IJPRNK IMPACT FACTOR : ISSN: Bhatia A,, 2014; Volume 3(4): LB403 LB405 LB17 Sunflower oil 0.5% BIF233 BIF234 Sunflower oil 0.1% BIF235 LA14a LA14b Cotton seed oil 0.5% Cotton seed oil 0.1% Soybean oil 0.5% Soybean oil 0.1% Figure 3. Bar graph representing percentage substrate utilization pattern of different bacterial strains after 48hr using cotton seed oil, sunflower oil and soybean oil as alternative substrates. The observations are expressed as mean ± SEM (n=3) Cotton seed oil Concentration Percent substrate Utilization OD at 600nm Figure 4. Effect of cotton seed oil conc. on substrate utilization efficiency of BIF235 after 24hr. The observations are expressed as mean ± SEM (n=3). Previously several researchers have studied the use of vegetable oils i.e. alfalfa seed oil 10, castor oil 11, safflower oil 12, sesame oil 13, soy oil 14 and sunflower oil 15 for CLA production. Cotton seed oil used in this study contained 24% saturated fatty acid and 76% unsaturated fatty acid (>50% Linoleic acid). However to our best knowledge cotton seed oil has never been used as substrate for CLA production. In certain cases pretreatment with lipase is required before using oils as substrate to produce free LA. Our results indicate that after tween 80 treatment no lipase pretreatment is required. It could partly be due to lipolytic activity of the BIF235 strain 16. The ability to cause isomerization and dehydration of fatty acids is prerequisite criteria for production of CLA from LA. Bacterial biosynthesis of CLA results apparently from two distinct 194

7 Bhatia A,, 2014; Volume 3(4): pathways which are strain dependent: direct isomerisation of LA or via 10-hydroxyoctadecenoic acid in some Lactobacillus, Propionibacterium, Bifidobacterium and Clostridiumlike bacteria of the human gut The CLA production using BIF235 can be attributed to 10- hydroxy-octadecenoic acid pathway. Qualitative and quantitative analysis of 9Z, 11E isomer through Gas chromatography The bioconverted CLA (BCLA) produced using 0.1% cotton seed oil as substrate through BIF235 was estimated in supernatant of production media. Methyl esters of BCLA showed retention time of min as compared to min of the 9Z, 11E standard (CLA isomer). Only single sharp peak was observed and isomer was found to be 91.10% pure as compared to the standard. The above results were calculated after applying base line correction to GC graphs (Fig.5, Table 3). The detection of single prominent peak could be the result of degradation of other (C16:0, C16:1) fatty acid by BIF235. Previous researches have also showed that 9Z, 11E (C 18:2) CLA isomer is the most abundant and naturally occurring isomer of CLA 19. Figure 5. GC Chromatogram of Standard [A] 9Z, 11E CLA methyl ester (Sigma) and [B] BCLA methyl ester. Table 3. Qualitative and quantitative parameters of Gas chromatographic analysis. Sample Peak No. Retention Time (min) Area (1*uV*sec) Area (%) 9Z, 11E CLA methyl ester BCLA methyl ester Previously, Oh et. al., 2003 and Chung et. al., 2008 found that most efficient bacterial species viz. B. breve KCTC 10462, B. pseudocatenulatum KCTC 10208, B. breve LMC 017and B. sp. LMC 052 were only able to produce 9Z, 11E isomers of CLA from pure linoleic acid as substrate These species were able to convert more than 90 % of free LA acid to 9Z, 11E CLA. However in our study high purity was achieved using cheap substrate which makes the production process cost effective. 195

8 Bhatia A,, 2014; Volume 3(4): Confirmation of 9Z, 11E isomer of CLA through FTIR-ATR analysis ATR spectrum of 9Z, 11E CLA methyl ester (Sigma) and BCLA methyl ester were obtained using sample conc. of 0.5mg/ml in hexane. In both the samples peaks were observed at: 2956, 2922, 2861, 1461, 1378, 724, 521, 509, 477 and 465 cm -1 (Fig. 6). The method used in this study is fast and only requires the evaporation of hexane prior to sample scanning which provides no hexane (hydrocarbon) interference in C-H absorption pattern. After analysis of the spectrum it was confirmed that BCLA sample contained 9Z, 11E isomer of CLA. Figure 6. ATR spectrum of [A] 9Z, 11E CLA methyl ester (Sigma) and [B] BCLA methyl ester. In the present study the method of screening and detection of an essential fatty acid has been developed. This method produces conjugated linoleic acid through bacterial biotransformation of vegetable oils. The optimized method developed here was very reproducible and could be scaled up to accommodate larger sample volumes. This is necessary for more systematic bioactivity screening of this compound. Finally, the methods described can be further optimized for production and detection of other isomers of CLA with similar properties. CONCLUSION The various strains of Lactic acid bacteria and Bifidobacteria have variable capacity to biotransform CLA from LA. Vegetable oils containing high LA content can be used as cheaper substrate than LA. Combination of oil and bacteria is required to get the best CLA production. Further potential for in vivo CLA production by using Bifidobacterium bifidium NCDC 235 as probiotic along with cotton seed oil as food and feed supplement can be exploited. REFERENCES 1. Wahle KWJ, Heys SD and Rotondo D: Conjugated linoleic acids: are they beneficial or detrimental to health? Progress in Lipid Research. 2004; 43: Bhattacharya A, Banu J, Rahman M, Causey J and Fernandes G: Biological effects of conjugated linoleic acids in health and disease. Journal of Nutritional Biochemistry. 2006; 17:

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