Effects of Elaidic Acid, a Predominant Industrial Trans Fatty Acid, on Bacterial Growth and Cell Surface Hydrophobicity of Lactobacilli Qinglong Wu and Nagendra P. Shah
The consumption of trans fatty acids (TFAs) increases the risk of cardiovascular diseases and coronary heart disease in human, and there are no effective ways to remove TFAs after consumption. The aim of this study was to investigate the effects of elaidic acid on bacterial growth, cell surface hydrophobicity of lactobacilli, and metabolism of elaidic acid by lactobacilli. Lactobacilli were inoculated in MRS broth containing 0, 100, 200, and 500 mg/L of elaidic acid. Viable cell counts of lactobacilli were enumerated, concentrations of elaidic acid were determined, and cell surface hydrophobicity of lactobacilli was measured. The results showed that the growth of lactobacilli was significantly inhibited by 500 mg/L of elaidic acid, however, a cell count of 8.50 log10 CFU/mL was still reached for tested lactobacilli after 24-h incubation. In particular, a reduction of elaidic acid was found for tested lactobacilli after 24-h incubation as compared to its initial concentration of 200 mg/L. However, cell surface hydrophobicity showed no correlations with the metabolism of elaidic acid by lactobacilli. Moreover, elaidic acid was able to influence cell surface hydrophobicity, and the decrease in hydrophobicity was more obvious in Lactobacillus paracasei and Lactobacillus casei compared with that in other tested lactobacilli. This study suggests that elaidic acid could change physiochemical surface properties of lactobacilli and the lactobacilli have the potential to reduce TFAs.
Keywords: bacterial metabolism, cell surface hydrophobicity, elaidic acid, lactobacilli
The presence of TFAs in human diets is hard to be avoided due to wide application of industrial trans fats in foods. Elaidic acid, a predominant TFA, showed limited inhibition effect on lactobacilli, and was metabolized and reduced by lactobacilli, particularly the strains from Lactobacillus casei group. This study provides a new insight for using lactobacilli for reduction of elaidic acid.
Practical Application:
Introduction
on modulation of the actions of lactobacilli in the gut by changing physiochemical surface properties of lactobacilli, and PUFAs was found to be assimilated by lactobacilli (Kankaanp¨aa¨ and others 2001; Kankaanp¨aa¨ and others 2004;). Similarly, a recent study reported that the survival of probiotic lactobacilli was affected by oleic acid, linoleic acid, and linolenic acid (Muller and others 2011). However, very few studies have examined the influence of TFAs on LAB cells. TFAs are a family of unsaturated fatty acids with at least 1 double bond in the trans configuration, and are classified into natural and industrial TFAs. Natural TFAs are mainly found in meat products from ruminant animals and are formed due to microbial actions in the rumen. The level of natural TFAs in relevant foods is very low. Industrial TFAs are produced during partial hydrogenation of vegetable oils, in which elaidic acid is the predominant TFAs (Mozaffarian and others 2006; Brouwer and others 2010). Additionally, cooking processes using edible oils including deep frying and heating increase the amount of total TFAs (Bansal and others 2009). Till now, several studies have shown that long-term consumption of TFAs increases the risk of cardiovascular diseases, type 2 diabetes, systemic inflammation, and abdominal obesity (Hu and others 2001; Mozaffarian and others 2004; Mozaffarian and others 2006; Kavanagh and others 2007). Similarly, a quanMS 20140676 Submitted 4/21/2014, Accepted 8/21/2014. Authors Wu and Shah are with Food and Nutritional Science, School of Biological Sciences, The Univ. titative review suggests that all fatty acids with a double bond in of Hong Kong, Pokfulam Road, Hong Kong. Direct inquiries to author Prof. Dr. the trans configuration raise the ratio of plasma LDL- to HDLNagendra P. Shah (E-mail: [email protected]). cholesterol (Brouwer and others 2010). Hence, several countries have enforced the food manufactures to label the level of TFAs in
Lactic acid bacteria (LAB), such as Lactobacillus sp., Lactococcus sp., Bifidobacterium sp., and Streptococcus thermophilus, are widely used for food fermentations such as yogurt, cheese, kimchi. Currently, LAB which confer healthy benefits to human after consumption are encouraged for manufacturing fermented foods (Carminati and others 2010). So far, biofunctionalities of these organisms have been well documented including their antimicrobial activities, cholesterol-lowering properties and immunoregulatory effects (Magnusson and others 2003; Ya and others 2008; Ren and others 2011). Moreover, most LAB cells are resistant to gastric acids and bile salts, which allow them to survive, and colonize in the gastrointestinal tract (Elo and others 1991; Ren and others 2011). Once consumed, LAB cells are exposed to foods that are consumed including polyunsaturated fatty acids (PUFAs) and trans fattys acids (TFAs). High-fat diet is known to reduce the population levels of gut beneficial bacteria such as lactobacilli and bifidobacteria, increase serum lipopolysaccharides content, and induce metabolic syndrome (Cani and others 2008; Zhang and others 2010). Dietary PUFAs have been found to directly impact
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12695 Further reproduction without permission is prohibited
Vol. 79, Nr. 12, 2014 r Journal of Food Science M2485
M: Food Microbiology & Safety
Abstract:
Effects of Elaidic acid on Lactobacilli . . . processed foods. For example, Denmark adopted the mandate in 2004 that all local made and imported foods must contain less than 2% industrial TFAs of total fats or oils. However, consumption of even small amount of TFAs (2% of total energy intake) is consistently associated with an increase in the incidence of coronary heart disease (Micha and Mozaffarian 2009). Moreover, the presence of TFAs in human diets cannot be avoided due to wide use of industrial TFAs, especially in the developing countries (PerezFerrer and others 2010). Orally administrated LAB cells are exposed to TFAs directly in human gastrointestinal tract once consumed. However, there is very little information on effects of TFAs on LAB. Among all the species of LAB, lactobacilli are most widely used as food starter cultures or probiotics. Moreover, lactobacilli after consumption could also be a part of gut microbes to confer their probiotic properties to host. As elaidic acid is predominant industrial TFA, we used it in this study to examine its effects on lactobacilli. In this study, we examined the effects of elaidic acid on the growth of lactobacilli, and whether elaidic acid affect cell surface hydrophobicity of lactobacilli, which is closely associated with the adhesion capability of lactobacilli. Additionally, we aimed to have insights into the potential of removing TFAs by food starters.
M: Food Microbiology & Safety
Materials and Methods Chemicals and reagents ACS grade xylene, methanol, chloroform, and n-hexane were obtained from ACS Chemical Inc. (Point Pleasant, N.J., U.S.A.), while GC grade n-hexane was from Sigma-Aldrich (St. Louis, Mo., U.S.A.). Elaidic acid, pentadecanoic acid, and elaidic acid methyl ester were from Sigma-Aldrich. Boron trifluoride (BF3 ) in methanol (10%, w/w) was purchased from Supelco (Bellefonte, Pa., U.S.A.).
an emulsifier. The elaidic acid stock solutions were filter sterilized through 0.45 μm filter (Corning, New York, N.Y., U.S.A.) and stored in dark at −30 °C before use. Viable cell counts of Lactobacillus sp. were enumerated at the interval of 8 h during the entire incubation by plating serial dilutions of aliquots on MRS agar.
Lipids extraction and methylation All of the lactobacilli were grown aerobically in MRS broth supplemented with elaidic acid at the final concentration of 200 mg/L, and incubated at 37 °C for up to 24 h. MRS without elaidic acid supplementation was used as the control. After incubation, 10 mL of aliquots were collected at 0, 8, 16, and 24 h of incubation. The cultures were then centrifuged at 3724 × g for 15 min at 20 °C. The cell-free supernatants were collected for lipid extraction, and cell pellets at 24 h of incubation were washed twice with PBS buffer (pH 7.4), weighed and stored at −20 °C until analyzed. Before lipid extraction and methylation, pentadecanoic acid (100 mg/L) was added into all of samples as internal standard for calibration and quantification. Lipid was extracted from cell-free supernatants as outlined previously with minor modifications (Jiang and others 1998; Ham and others 2002). Briefly, 1 mL supernatant was mixed thoroughly with 3 mL of chloroform-methanol (2:1, vol/vol) for 5 min before centrifuging at 1000 × g for 10 min at 20 °C. The lower organic layer was then collected using a Pasteur pipette. The upper aqueous phase was extracted again with 2 mL chloroformmethanol, and 2 lower organic fluids were pooled in a 10 mL screw-capped test tube and dried by purging gently with nitrogen gas. Cell pellets suspended in the PBS buffer were sonicated using a sonicator (W-375 Cell Disruptor; Heat Systems UltraSonics Inc., Anaheim, Calif., U.S.A.) for 3 min before the addition of chloroform-methanol. The protocol for lipid extraction procedure of cell pellets was the same as above. The lipids extracts were hydrolyzed to free fatty acids by adding 1 mL of 1 M NaOH in methanol at 50 °C for 30 min (Jiang and others 1998). Then fatty acids methylation was carried out by adding 1 mL of 10% BF3 in methanol at 50 °C for another 30 min in dark. One milliliter of saturated NaCl solution was then added to eliminate excess methoxide. Fatty acids methyl esters (FAMEs) in the mixture were extracted twice with 2 mL of n-hexane and mixed thoroughly for 2 min. Finally, the FAMEs in n-hexane were dried using nitrogen gas and samples stored at −30 °C for further analysis. Lipid extraction was also carried out for MRS broth as a control.
Bacterial strains and cultivation conditions Five dairy starter cultures and 1 wild isolate were used in this study. Four organisms including Lactobacillus rhamnosus ASCC 2607, Lactobacillus paracasei ASCC 279, Lactobacillus zeae ASCC 15820, and Lactobacillus casei ASCC 290 were obtained from Australian Starter Culture Collection Center (ASCC; Werribee, Australia). Lactobacillus acidophilus CSCC 2404 was from CSIRO Starter Culture Collection (CSIRO, Melbourne, Australia) and Lactobacillus plantarum WQ1 was isolated from traditional homemade Chinese sauerkraut (Sichuan, China). Each stock culture was stored in 40% (vol/vol) sterile glycerol at −80 °C. MRS medium is most commonly used for cultivation of lactobacilli as Tween80 in MRS has been shown to be an essential growth factor for lactobacilli (Partanen and others 2001). These organisms were ac- Gas chromatography-mass spectrometry (GC-MS) analysis tivated by growing in DifcoTM lactobacilli MRS broth (BD Co., FAMEs were suspended in n-hexane (GC grade) prior to injectMd., U.S.A.) for 12 h at 37 °C successively for 3 times before ing aliquots to GC-MS instrument. FAMEs were analyzed in an performing experiments. Agilent GC-MS system (6890N-GC with 5973-MS ECD/NPD; Santa Clara, Calif., U.S.A.) equipped with GC/MSD ChemStaViable cell counts tion software (Agilent). FAME analysis was carried out using a The above organisms were grown aerobically in MRS broth silica capillary column (100 m × 0.25 mm × 0.2 μm film thicksupplemented with various concentrations of elaidic acid (100, ness; SP-2560 Supelco, Bellefonte, Pa., U.S.A.), which is a highly 200, and 500 mg/L) for 24 h; MRS broth without elaidic acid polar column. One microliter of aliquot was injected automatsupplementation was used as a control. The pH of MRS with and ically with an inlet temperature of 250 °C and a split ratio of without supplementation with elaidic acid was measured by a pH 50:1. Helium was used as a carrier at a flow rate of 20 cm/s at paper; negligible changes (pH 6.70 to 6.60) were observed (data 175 °C. The oven temperature program was 140 °C and the samnot shown) suggesting that the pH did not affect the bacterial ple was held for 5 min, with 1 ramp to attain 240 °C at a rate of growth. Elaidic acid was added at 15 g/L stock solutions in sterile 4 °C/min, and 240 °C held for 15 min. The detector temperature distilled water containing 2% (vol/vol) of Tween-80 (Sigma) as was 250 °C. M2486 Journal of Food Science r Vol. 79, Nr. 12, 2014
Effects of Elaidic acid on Lactobacilli . . .
Statistical analysis All the presented data are as mean ± standard derivation. The significant difference among groups was determined by one-way analysis of variance (ANOVA) using IBM SPSS Statistics 20.0 version. Differences were considered significant at a P value of < 0.05.
Results and Discussion Effect of elaidic acid on the growth of lactobacilli The effect of elaidic acid on the growth of lactobacilli during the incubation period of 24 h is shown in Table 1. As the concentration of elaidic acid was increased, there was a slight decrease in the viable population of all of lactobacilli after 8 h or 16 h of incubation, except that of L. casei. In some organisms (such as L. paracasei, L. zeae, and L. plantarum), the increased concentration of elaidic acid caused growth inhibition after 24 h of incubation as compared to that after 8 h. In particular, L. casei was not significantly affected by elaidic acid at any concentrations studied during the 24-h incubation. Other 5 dairy lactobacilli showed lower resistance to elaidic acid, but they were still able to survive in the elaidic acid-enriched medium. L. rhamnosus and L. paracasei were significantly (P < 0.05) inhibited by elaidic acid, however, there was no significant bacterial inhibition between 200 and 500 mg/L. L. zeae could grow well at 8 h in 500 mg/L of elaidic acid, but a significant (P < 0.05) inhibition was observed at the 16 and 24 h of incubation. However, there was no significant inhibition of this organism with various elaidic acid supplementations at 16 h, and the lowest count of L. zeae at 500 mg/L of elaidic acid was shown at 24 h. The growth of L. acidophilus was significantly (P < 0.05) affected by 100, 200, and 500 mg/L of elaidic acid as compared to the control, however, it showed almost the same survival in the presence of 100 and 200 mg/L of elaidic acid. L. plantarum showed no inhibition at 8 h and 16 h in the 0 and 100 mg/L of elaidic acid, but was significantly (P < 0.05) inhibited by 200 and 500 mg/L at 8 and 16 h. However, there was no significant difference (P > 0.05) among elaidic acid treated groups at 24 h. For the bacterial growth, 8 h was likely to be the log phase for all lactobacilli, 16 and 24 h as the late stationary phase for L. rhamnosus, L. paracasei, L. casei, and L. acidophilus, and 24 h as a decline phase for L. zeae and L. plantarum. Currently, limited data has shown the inhibitory or stimulatory effect of elaidic acid on the growth of lactobacilli. A previous study revealed that lower concentration of PUFAs showed a significant inhibition of probiotic Lactobacillus strain (Kankaanp¨aa¨ and others
2001). Inhibition of bacterial growth caused by elaidic acid may be associated with the inhibition of fatty acids synthesis such as the reduction of acyl carrier proteins, fatty acids synthetase and acetyl-CoA carboxylase (Weeks and Wakil 1970), or the changes in cellular and membrane lipids in lactobacilli (Kankaanp¨aa¨ and others 2004; Muller and others 2011). In this study, elaidic acid showed slight inhibition of the growth of lactobacilli within the level of 500 mg/L compared with that without supplementation of elaidic acid.
Metabolism of elaidic acid by lactobacilli The concentrations of extracellular and intracellular elaidic acid from cell-free supernatants and cell pellets after incubation are shown in Table 2. As shown in the table, the residual elaidic acid in the supernatant after 24 h of incubation was time-dependent and species-specific. The lowest elaidic acid concentration was at 8 h of incubation in all 6 lactobacilli, while there were increases in the concentrations of elaidic acid at 16 and 24 h. The reduction in elaidic acid in the supernatant was observed suggesting the metabolism of elaidic acid by lactobacilli (Table 1). Although oleic acid is the predominant fatty acid in lactobacilli and in MRS broth, Lactobacillus is able to absorb free fatty acids via long-chain fatty acid transport protein (Fujita and others 2007; Eckhardt and others 2013). Thus, free elaidic acid is transported by bacteria, while lipids containing oleic acid needs to be hydrolyzed by bacterial lipase before utilization. Hence, lactobacilli may utilize elaidic acid for fatty acid synthesis at the log phase of 8 h. As compared to the concentrations at 8 h, the concentrations of elaidic acid increased in the supernatant at 16 and 24 h, which may be related with the cell death at 16 and 24 h (late stationary and decline phase). Intracellular elaidic acid was detected by GC-MS and the reduction of elaidic acid was also observed after 24 h of incubation (Table 2). This indicated that lactobacilli was able to absorb and utilize extracellular elaidic acid, however, the metabolism of elaidic acid in lactobacilli was limited which may be related with the specific enzymes involved in the metabolism for oleic acid rather than elaidic acid. Intracellular elaidic acid from cell pellets after 24-h incubation of lactobacilli in elaidic acid-supplemented MRS (200 mg/L) was also determined (Table 2). Intracellular elaidic acid was not detected by GC-MS in lactobacilli cultivated without the supplementation of elaidic acid. The concentration of intracellular elaidic acid among different lactobacilli varied with organism indicating a species-specific metabolism of elaidic acid. The intracellular concentration of elaidic acid in L. casei was the highest while it was lowest for L. paracasei among the 6 lactobacilli. The total amount of intracellular and extracellular elaidic acid decreased after 24 h of incubation compared with the initial concentration of 200 mg/L. L. rhamnosus metabolized the highest level of elaidic acid with the reduction of 71.04 mg/L, while the lowest reduction of 11.85 mg/L was found in L. plantarum. This is possibly due to the incorporation of free fatty acid into bacterial fatty acids as reported in a previous study (Kankaanp¨aa¨ and others 2004). The reduction in elaidic acid concentration was observed indicating the metabolism of elaidic acid by lactobacilli. Also, the reduction in the level of elaidic acid among different lactobacilli was species-specific. It appears that the strains from L. casei group including L. rhamnosus, L. casei, and L. paracasei showed the highest potential to reduce elaidic acid. Previous study indicated a metabolic way for bioconversion or isomerization of elaidic acid into other fatty acids by ruminal microbes (Proell and others 2002). Also, β-oxidation of fatty acids is the general metabolism pathway for unsaturated fatty acids Vol. 79, Nr. 12, 2014 r Journal of Food Science M2487
M: Food Microbiology & Safety
Measurement of cell surface hydrophobicity The ability of bacteria to adhere to hydrocarbons was used to examine the effects of free elaidic acid on the cell surface hydrophobicity of lactobacilli (Lye and others 2010). Briefly, cells were harvested after 24 h of incubation at 4000 × g for 15 min at 4 °C and washed 3 times with pre-cooled sterile saline buffer, and were resuspended in the same buffer. The cell suspension was adjusted to an optical density (OD) of 0.50 to 0.60 at 600 nm with saline buffer. Then, 10 mL of cell suspension was mixed with 2 mL of xylene and was vortexed for 2 min. The 2 phases were allowed to separate naturally for 20 min. The aqueous phase was carefully collected using a Pasteur pipette for the measurement of OD at 600 nm. All the analyses were performed in triplicate. Cell surface hydrophobicity (H%) was calculated according to the equation: H% = ([A0 – A]/A0 ) × 100%, where A0 and A are the absorbances before and after extraction with xylene, respectively.
Effects of Elaidic acid on Lactobacilli . . . Table 1–Effect of various concentrations of elaidic acid on the growth of lactobacilli. Viable cell count after the certain cultivation time (log10 CFU/mL) 8 hd
Organism
Elaidic acid concentration (mg/L)
0h
L. rhamnosus
0 100 200 500 0 100 200 500 0 100 200 500 0 100 200 500 0 100 200 500 0 100 200 500
6.99 ± 0.01
L. paracasei
L. zeae
L. casei
L. acidophilus
L. plantarum
M: Food Microbiology & Safety
9.90 9.24 9.01 9.01 9.31 9.08 9.12 9.15 8.52 8.44 8.54 8.49 8.84 8.92 8.78 8.90 9.78 9.01 9.08 8.87 9.60 9.61 9.43 9.23
7.14 ± 0.14
6.80 ± 0.09
7.62 ± 0.11
6.89 ± 0.08
7.35 ± 0.11
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07a,e 0.06b 0.16c 0.04c 0.03a 0.04b 0.05b 0.09b 0.07a 0.06a 0.06a 0.04a 0.03ab 0.04a 0.04b 0.02a 0.03a 0.08bc 0.02b 0.07c 0.05a 0.04a 0.03b 0.02c
16 h 10.91 10.09 9.54 9.33 10.25 10.19 9.63 9.47 10.35 9.49 9.64 9.60 9.39 9.15 9.49 9.39 9.36 9.60 9.22 9.30 10.56 10.61 10.42 10.26
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
24 h
0.02a 0.01b 0.06c 0.31bc 0.03a 0.06a 0.08b 0.07bc 0.11a 0.10b 0.03b 0.05b 0.21a 0.08a 0.10a 0.09a 0.16ab 0.09a 0.13b 0.03ab 0.08a 0.04a 0.04b 0.03c
10.55 10.19 9.81 9.63 10.10 10.16 9.56 9.48 10.28 9.10 9.23 8.77 9.79 9.91 9.63 9.83 10.82 9.58 9.58 8.96 10.37 9.78 9.72 9.58
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04a 0.11b 0.10c 0.17c 0.07a 0.04a 0.09b 0.18bc 0.05a 0.09b 0.20b 0.07c 0.05ab 0.06a 0.06b 0.09a 0.04a 0.03b 0.09b 0.24c 0.05a 0.09b 0.12b 0.17b
d Samples e
were collected at the time point of 8 h during the entire 24-h incubation. Data were presented as mean ± standard deviation (n = 3). Values with no letters in common within each column are significantly different (P < 0.05).
Table 2–GC-MS analysis of extracellular and intracellular elaidic acid in cell-free supernatant and cell pellets. Extracellular elaidic acid in the cell-free supernatant during the entire cultivation with 200 mg/L of elaidic acid (mg/L)
Organism L. rhamnosus L. paracasei L. zeae L. casei L. acidophilus L. plantarum
0h 191.96 ± 3.77
8 ha 108.31 77.94 103.78 67.78 74.43 88.65
± ± ± ± ± ±
16 h 4.74c 3.63 7.33 0.95 2.52 3.39
126.84 139.16 151.76 130.97 117.96 131.58
± ± ± ± ± ±
Cultivation without elaidic acid
24 h 3.27 2.75 1.21 4.74 5.19 7.59
114.94 138.58 150.06 125.33 141.53 170.03
± ± ± ± ± ±
Intracellular elaidic acid in cell pellets from 10 mL of cultures after 24-h incubation (µg)
6.13 4.88 2.12 4.39 0.96 1.31
N. D.d N. D. N. D. N. D. N. D. N. D.
Cultivation with 200 mg/L of elaidic acid 140.17 42.52 114.54 238.83 124.23 180.42
± ± ± ± ± ±
9.19 2.58 1.54 8.31 4.04 1.63
Reduction in elaidic acid after 24-hb incubation (mg/L)b 71.04 57.17 38.57 50.78 45.90 11.85
± ± ± ± ± ±
5.33 4.81 2.08 3.91 5.44 1.21
a
Samples were collected at the time point of 8 h during the entire 24-h incubation. calculation for reduction in elaidic acid (EA) was based on the initial amount of EA and the residual amount of EA in cell-free supernatants and cell pellets. Data were presented as mean ± standard deviation (n = 3). d N. D.: not detectable concentration by GC-MS. b The c
such as oleic acid in bacteria (Fujita and others 2007). However, the metabolic pathway for oleic acid and elaidic acid in lactobacilli and the effect of conformation change between oleic acid and elaidic acid on lactobacilli are not clear. This merits further investigation.
Effect of elaidic acid on cell surface hydrophobicity of lactobacilli In order to understand the effects of elaidic acid on the surface properties of lactobacilli, we tested the hydrophobicity of 6 lactobacilli during the 24 h of incubation period. The results of hydrophobicity of lactobacilli during the 24-h incubation with and without exposure to 200 mg/L of elaidic acid are shown in Table 3. As shown in the table, the hydrophobicity of lactobacilli was species-specific, and it changed during the incubation period. In general, L. rhamnosus, L. zeae, L. acidophilus, and L. plantarum M2488 Journal of Food Science r Vol. 79, Nr. 12, 2014
exhibited higher hydrophobicity (approximately 80%) toward hydrocarbon and slight changes were observed after exposure to elaidic acid; L. paracasei and L. casei showed lower hydrophobicity (below approximately 60%;) indicating the hydrophilic cell surface and dramatically decreases in hydrophobicity were observed in these 2 species after elaidic acid treatment. Moreover, the hydrophobicity showed no correlations with the decrease in extracellular elaidic acid and increase in intracellular elaidic acid (Table 2 and 3). Thus, cell surface hydrophobicity did not influence the absorption of elaidic acid, and the incorporation of free elaidic acid into the cells was due to the specific long-chain fatty acid binding/transport proteins (Eckhardt and others 2013). Additionally, it appears that the cell surface properties of lactobacilli with lower hydrophobicity such as L. paracasei and L. casei (Table 3) were more easily affected by elaidic acid compared with that in other 4 lactobacilli.
Effects of Elaidic acid on Lactobacilli . . . Table 3–Hydrophobicity of lactobacilli cultivated in MRS broth without and with the addition of 200 mg/L of elaidic acid during 24-h incubation. Hydrophobicity (%) 8ha Organism L. rhamnosus L. paracasei L. zeae L. casei L. acidophilus L. plantarum
− Elaidic 94.75 4.35 91.35 44.75 97.17 80.84
± ± ± ± ± ±
acidb 1.11d 1.65 2.55 3.99 2.63 2.17
16 h + Elaidic 93.13 8.43 94.22 29.42 99.53 76.19
± ± ± ± ± ±
acidc 2.33 1.33 2.57 7.42 0.73 2.78
− Elaidic acid 80.12 23.60 89.82 52.83 94.59 87.47
± ± ± ± ± ±
6.04 3.35 3.67 5.76 4.76 0.54
24/0 h + Elaidic acid 88.66 11.57 85.22 13.88 87.12 84.02
± ± ± ± ± ±
2.48 3.40 2.42 2.77 1.64 4.57
− Elaidic acid 87.44 32.19 83.88 55.16 92.36 84.67
± ± ± ± ± ±
2.11 3.80 1.75 2.46 2.12 2.21
+ Elaidic acid 93.50 2.16 94.96 55.25 84.46 88.53
± ± ± ± ± ±
6.64 0.76 1.01 5.04 1.19 2.46
a
Samples were collected at the time point of 8 h during the entire 24-h incubation. acid (200 mg/L) was not added into the MRS broth before incubation. Elaidic acid (200 mg/L) was added into the MRS broth before incubation. d Data were presented as mean ± standard deviation (n = 3).
Lactobacillus sp. is the gram-positive bacteria and does not have References outer membrane and has simple composition in cell surface Bansal G, Zhou WB, Tan TW, Neo FL, Lo HL. 2009. Analysis of trans fatty acids in deep frying oils by three different approaches. Food Chem 116(2):535–41. compared with that in gram-negative bacteria. It has been well Brouwer IA, Wanders AJ, Katan MB. 2010. Effect of animal and industrial trans fatty acids on documented that cell surface hydrophobicity is closely associated HDL and LDL cholesterol levels in humans—a quantitative review. PLOS One 5(3). Cani PD, Bibiloni R, Knauf C, Neyrinck AM, Neyrinck AM, Delzenne NM, Burcelin R. with the adhesion property (Vadillo-Rodriguez and others 2005; 2008. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in Pan and others 2006; Wang and others 2010), and is highly conhigh-fat diet-induced obesity and diabetes in mice. Diabetes 57(6):1470–81. D, Giraffa G, Quiberoni A, Binetti A, Su´arez V, Reinheimer J. 2010. Biotechnology veyed by cell surface proteins known as S-layer proteins in lacto- Carminati of lactic acid bacteria: novel applications. In: Mozzi F, Raya RR, Vignolo GM, editors. bacilli (van der Mei and others 2003). Hence, it appears that elaidic Advances and trends in starter cultures for dairy fermentations. New York: John Wiley & Sons, Ltd. p. 177–92. acid was able to induce specific changes to the cell surface proteins Eckhardt TH, Skotnicka D, Kok J, Kuipers OP. 2013. Transcriptional regulation of fatty acid in lactobacilli, probably in lactobacilli with lower hydrophobicbiosynthesis in Lactococcus lactis. J Bacteriol 195(5):1081–9. ity, leading to the decrease in surface hydrophobicity which may Elo S, Saxelin M, Salminen S. 1991. Attachment of Lactobacillus casei strain GG to human colon carcinoma cell line Caco-2: comparison with other dairy strains. Lett Appl Microbiol in turn affect the adherence to the mucosal cells to exhibit the 13(3):154–6. Fujita Y, Matsuoka H, Hirooka K. 2007. Regulation of fatty acid metabolism in bacteria. Mol probiotic properties to the host.
Conclusions Elaidic acid showed low growth inhibition to selected lactobacilli up to the level of 500 mg/L and was metabolized by lactobacilli. Incorporation of free elaidic acid into the lactobacilli was observed by GC-MS. Moreover, the metabolism of elaidic acid was species-specific. Bacterial cell surface hydrophobicity showed no correlation with the absorption and metabolism of elaidic acid in lactobacilli. However, elaidic acid was able to influence the cell surface hydrophobicity, and the decrease in hydrophobicity was more obvious in lactobacilli with lower hydrophobicity (L. casei and L. paracasei) compared with that in lactobacilli with higher hydrophobicity when incubated in MRS containing 200 mg/L of elaidic acid. A changed surface hydrophobicity may reflect the particular changes in surface S-layer proteins which affect the adhesion property of lactobacilli. Results suggest that lactobacilli were able to metabolize elaidic acid providing a new way for reducing dietary TFAs. However, further works are necessary to understand the metabolic pathway of oleic acid and elaidic acid in lactobacilli.
Acknowledgments The authors would like to thank Ms. Jessie H.Y. Lai in HKU School of Biological Sciences for providing assistance in GC-MS analysis of fatty acids.
Author Contributions N.P. Shah & Q. Wu designed the study; Q. Wu conducted the experiments and drafted the manuscript; N.P. Shah revised and edited the manuscript.
Microbiol 66(4):829–39. Ham JS, In YM, Jeong SG, Kim JG, Lee EH, Kim HS, Yoon SK, Lee BH. 2002. Screening of conjugated linoleic acid producing lactic acid bacteria from fecal samples of healthy babies. Asian Austral J Anim 15(7):1031–5. Hu FB, Manson JE, Stampfer MJ, Colditz G, Liu S, Solomon CG, Willett WC. 2001. Diet, lifestyle, and the risk of type 2 diabetes mellitus in women. New Engl J Med 345(11):790–7. Jiang J, Bjorck L, Fonden R. 1998. Production of conjugated linoleic acid by dairy starter cultures. J Appl Microbiol 85(1):95–102. Kankaanp¨aa¨ PE, Salminen SJ, Isolauri E, Lee YK. 2001. The influence of polyunsaturated fatty acids on probiotic growth and adhesion. FEMS Microbiol Lett 194(2):149–53. Kankaanp¨aa¨ P, Yang B, Kallio H, Isolauri E, Salminen S. 2004. Effects of polyunsaturated fatty acids in growth medium on lipid composition and on physicochemical surface properties of lactobacilli. Appl Environ Microb 70(1):129–36. Kavanagh K, Jones KL, Sawyer J, Kelley K, Carr JJ, Wagner JD, Rudel LL. 2007. Trans fat diet induces abdominal obesity and changes in insulin sensitivity in monkeys. Obesity 15(7):1675– 84. Lye HS, Rusul G, Liong MT. 2010. Removal of cholesterol by lactobacilli via incorporation and conversion to coprostanol. J Dairy Sci 93(4):1383–92. Magnusson J, Strom K, Roos S, Sjogren J, Schnurer J. 2003. Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiol Lett 219(1):129– 35. Micha R, Mozaffarian D. 2009. Trans fatty acids: effects on metabolic syndrome, heart disease and diabetes. Nat Rev Endocrinol 5(6):335–44. Mozaffarian D, Katan MB, Ascherio A, Stampfer MJ, Willett WC. 2006. Medical progress— trans fatty acids and cardiovascular disease. New Engl J Med 354(15):1601–13. Mozaffarian D, Pischon T, Hankinson SE, Rifai N, Joshipura K, Willett WC, Rimm EB. 2004. Dietary intake of trans fatty acids and systemic inflammation in women. Am J Clin Nutr 79(4):606–12. Muller JA, Ross RP, Sybesma WFH, Fitzgerald GF, Stanton C. 2011. Modification of the Technical Properties of Lactobacillus johnsonii NCC 533 by supplementing the growth medium with unsaturated fatty acids. Appl Environ Microb 77(19):6889–98. Pan WH, Li PL, Liu ZY. 2006. The correlation between surface hydrophobicity and adherence of Bifidobacterium strains from centenarians’ faeces. Anaerobe 12(3):148–52. Partanen L, Marttinen N, Alatossava T. 2001. Fats and fatty acids as growth factors for Lactobacillus delbrueckii. Syst Appl Microbiol 24(4):500–6. Perez-Ferrer C, Lock K, Rivera JA. 2010. Learning from international policies on trans fatty acids to reduce cardiovascular disease in low- and middle-income countries, using Mexico as a case study. Health Policy Plann 25(1):39–49. Proell JM, Mosley EE, Powell GL, Jenkins TC. 2002. Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes. J Lipid Res 43(12):2072–6. Ren J, Sun KJ, Wu ZJ, Yao J, Guo BH. 2011. All 4 bile salt hydrolase proteins are responsible for the hydrolysis activity in Lactobacillus plantarum ST-III. J Food Sci 76(9):M622-M8. Vadillo-Rodriguez V, Busscher HJ, van der Mei HC, de Vries J, Norde W. 2005. Role of Lactobacillus cell surface hydrophobicity as probed by AFM in adhesion to surfaces at low and high ionic strength. Colloid Surface B 41(1):33–41.
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b Elaidic c
Effects of Elaidic acid on Lactobacilli . . . van der Mei HC, van de Belt-Gritter B, Pouwels PH, Martinez B, Busscher HJ. 2003. Cell surface hydrophobicity is conveyed by S-layer proteins—a study in recombinant lactobacilli. Colloid Surface B 28(2–3):127–34. Wang LQ, Meng XC, Zhang BR, Wang Y, Shang YL. 2010. Influence of cell surface properties on adhesion ability of bifidobacteria. World J Microb Biot 26(11):1999–2007. Weeks G, Wakil SJ. 1970. Studies on the control of fatty acid metabolism. II. The inhibition of fatty acid synthesis in Lactobacillus plantarum by exogenous fatty acid. J Biol Chem 245(8):1913– 21.
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Ya T, Zhang QJ, Chu FL, Merritt J, Bilige MH, Sun TS, Du RT, Zhang HP. 2008. Immunological evaluation of Lactobacillus casei Zhang: a newly isolated strain from koumiss in Inner Mongolia, China. BMC Immunol 9:68. doi:10.1186/1471–2172–9–68 Zhang CH, Zhang MH, Wang SY, Han RJ, Cao YF, Hua WY, Mao YJ, Zhang XJ, Pang XY, Wei CC, Zhao GP, Chen Y, Zhao LP. 2010. Interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice. ISME J 4(2):312–3.
The consumption of trans fatty acids (TFAs) increases the risk of cardiovascular diseases and coronary heart disease in human, and there are no effective ways to remove TFAs after consumption. The aim of this study was to investigate the effects of elaidic acid on bacterial growth, cell surface hydrophobicity of lactobacilli, and metabolism of elaidic acid by lactobacilli. Lactobacilli were inoculated in MRS broth containing 0, 100, 200, and 500 mg/L of elaidic acid. Viable cell counts of lactobacilli were enumerated, concentrations of elaidic acid were determined, and cell surface hydrophobicity of lactobacilli was measured. The results showed that the growth of lactobacilli was significantly inhibited by 500 mg/L of elaidic acid, however, a cell count of 8.50 log10 CFU/mL was still reached for tested lactobacilli after 24-h incubation. In particular, a reduction of elaidic acid was found for tested lactobacilli after 24-h incubation as compared to its initial concentration of 200 mg/L. However, cell surface hydrophobicity showed no correlations with the metabolism of elaidic acid by lactobacilli. Moreover, elaidic acid was able to influence cell surface hydrophobicity, and the decrease in hydrophobicity was more obvious in Lactobacillus paracasei and Lactobacillus casei compared with that in other tested lactobacilli. This study suggests that elaidic acid could change physiochemical surface properties of lactobacilli and the lactobacilli have the potential to reduce TFAs.
Keywords: bacterial metabolism, cell surface hydrophobicity, elaidic acid, lactobacilli
The presence of TFAs in human diets is hard to be avoided due to wide application of industrial trans fats in foods. Elaidic acid, a predominant TFA, showed limited inhibition effect on lactobacilli, and was metabolized and reduced by lactobacilli, particularly the strains from Lactobacillus casei group. This study provides a new insight for using lactobacilli for reduction of elaidic acid.
Practical Application:
Introduction
on modulation of the actions of lactobacilli in the gut by changing physiochemical surface properties of lactobacilli, and PUFAs was found to be assimilated by lactobacilli (Kankaanp¨aa¨ and others 2001; Kankaanp¨aa¨ and others 2004;). Similarly, a recent study reported that the survival of probiotic lactobacilli was affected by oleic acid, linoleic acid, and linolenic acid (Muller and others 2011). However, very few studies have examined the influence of TFAs on LAB cells. TFAs are a family of unsaturated fatty acids with at least 1 double bond in the trans configuration, and are classified into natural and industrial TFAs. Natural TFAs are mainly found in meat products from ruminant animals and are formed due to microbial actions in the rumen. The level of natural TFAs in relevant foods is very low. Industrial TFAs are produced during partial hydrogenation of vegetable oils, in which elaidic acid is the predominant TFAs (Mozaffarian and others 2006; Brouwer and others 2010). Additionally, cooking processes using edible oils including deep frying and heating increase the amount of total TFAs (Bansal and others 2009). Till now, several studies have shown that long-term consumption of TFAs increases the risk of cardiovascular diseases, type 2 diabetes, systemic inflammation, and abdominal obesity (Hu and others 2001; Mozaffarian and others 2004; Mozaffarian and others 2006; Kavanagh and others 2007). Similarly, a quanMS 20140676 Submitted 4/21/2014, Accepted 8/21/2014. Authors Wu and Shah are with Food and Nutritional Science, School of Biological Sciences, The Univ. titative review suggests that all fatty acids with a double bond in of Hong Kong, Pokfulam Road, Hong Kong. Direct inquiries to author Prof. Dr. the trans configuration raise the ratio of plasma LDL- to HDLNagendra P. Shah (E-mail: [email protected]). cholesterol (Brouwer and others 2010). Hence, several countries have enforced the food manufactures to label the level of TFAs in
Lactic acid bacteria (LAB), such as Lactobacillus sp., Lactococcus sp., Bifidobacterium sp., and Streptococcus thermophilus, are widely used for food fermentations such as yogurt, cheese, kimchi. Currently, LAB which confer healthy benefits to human after consumption are encouraged for manufacturing fermented foods (Carminati and others 2010). So far, biofunctionalities of these organisms have been well documented including their antimicrobial activities, cholesterol-lowering properties and immunoregulatory effects (Magnusson and others 2003; Ya and others 2008; Ren and others 2011). Moreover, most LAB cells are resistant to gastric acids and bile salts, which allow them to survive, and colonize in the gastrointestinal tract (Elo and others 1991; Ren and others 2011). Once consumed, LAB cells are exposed to foods that are consumed including polyunsaturated fatty acids (PUFAs) and trans fattys acids (TFAs). High-fat diet is known to reduce the population levels of gut beneficial bacteria such as lactobacilli and bifidobacteria, increase serum lipopolysaccharides content, and induce metabolic syndrome (Cani and others 2008; Zhang and others 2010). Dietary PUFAs have been found to directly impact
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12695 Further reproduction without permission is prohibited
Vol. 79, Nr. 12, 2014 r Journal of Food Science M2485
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Abstract:
Effects of Elaidic acid on Lactobacilli . . . processed foods. For example, Denmark adopted the mandate in 2004 that all local made and imported foods must contain less than 2% industrial TFAs of total fats or oils. However, consumption of even small amount of TFAs (2% of total energy intake) is consistently associated with an increase in the incidence of coronary heart disease (Micha and Mozaffarian 2009). Moreover, the presence of TFAs in human diets cannot be avoided due to wide use of industrial TFAs, especially in the developing countries (PerezFerrer and others 2010). Orally administrated LAB cells are exposed to TFAs directly in human gastrointestinal tract once consumed. However, there is very little information on effects of TFAs on LAB. Among all the species of LAB, lactobacilli are most widely used as food starter cultures or probiotics. Moreover, lactobacilli after consumption could also be a part of gut microbes to confer their probiotic properties to host. As elaidic acid is predominant industrial TFA, we used it in this study to examine its effects on lactobacilli. In this study, we examined the effects of elaidic acid on the growth of lactobacilli, and whether elaidic acid affect cell surface hydrophobicity of lactobacilli, which is closely associated with the adhesion capability of lactobacilli. Additionally, we aimed to have insights into the potential of removing TFAs by food starters.
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Materials and Methods Chemicals and reagents ACS grade xylene, methanol, chloroform, and n-hexane were obtained from ACS Chemical Inc. (Point Pleasant, N.J., U.S.A.), while GC grade n-hexane was from Sigma-Aldrich (St. Louis, Mo., U.S.A.). Elaidic acid, pentadecanoic acid, and elaidic acid methyl ester were from Sigma-Aldrich. Boron trifluoride (BF3 ) in methanol (10%, w/w) was purchased from Supelco (Bellefonte, Pa., U.S.A.).
an emulsifier. The elaidic acid stock solutions were filter sterilized through 0.45 μm filter (Corning, New York, N.Y., U.S.A.) and stored in dark at −30 °C before use. Viable cell counts of Lactobacillus sp. were enumerated at the interval of 8 h during the entire incubation by plating serial dilutions of aliquots on MRS agar.
Lipids extraction and methylation All of the lactobacilli were grown aerobically in MRS broth supplemented with elaidic acid at the final concentration of 200 mg/L, and incubated at 37 °C for up to 24 h. MRS without elaidic acid supplementation was used as the control. After incubation, 10 mL of aliquots were collected at 0, 8, 16, and 24 h of incubation. The cultures were then centrifuged at 3724 × g for 15 min at 20 °C. The cell-free supernatants were collected for lipid extraction, and cell pellets at 24 h of incubation were washed twice with PBS buffer (pH 7.4), weighed and stored at −20 °C until analyzed. Before lipid extraction and methylation, pentadecanoic acid (100 mg/L) was added into all of samples as internal standard for calibration and quantification. Lipid was extracted from cell-free supernatants as outlined previously with minor modifications (Jiang and others 1998; Ham and others 2002). Briefly, 1 mL supernatant was mixed thoroughly with 3 mL of chloroform-methanol (2:1, vol/vol) for 5 min before centrifuging at 1000 × g for 10 min at 20 °C. The lower organic layer was then collected using a Pasteur pipette. The upper aqueous phase was extracted again with 2 mL chloroformmethanol, and 2 lower organic fluids were pooled in a 10 mL screw-capped test tube and dried by purging gently with nitrogen gas. Cell pellets suspended in the PBS buffer were sonicated using a sonicator (W-375 Cell Disruptor; Heat Systems UltraSonics Inc., Anaheim, Calif., U.S.A.) for 3 min before the addition of chloroform-methanol. The protocol for lipid extraction procedure of cell pellets was the same as above. The lipids extracts were hydrolyzed to free fatty acids by adding 1 mL of 1 M NaOH in methanol at 50 °C for 30 min (Jiang and others 1998). Then fatty acids methylation was carried out by adding 1 mL of 10% BF3 in methanol at 50 °C for another 30 min in dark. One milliliter of saturated NaCl solution was then added to eliminate excess methoxide. Fatty acids methyl esters (FAMEs) in the mixture were extracted twice with 2 mL of n-hexane and mixed thoroughly for 2 min. Finally, the FAMEs in n-hexane were dried using nitrogen gas and samples stored at −30 °C for further analysis. Lipid extraction was also carried out for MRS broth as a control.
Bacterial strains and cultivation conditions Five dairy starter cultures and 1 wild isolate were used in this study. Four organisms including Lactobacillus rhamnosus ASCC 2607, Lactobacillus paracasei ASCC 279, Lactobacillus zeae ASCC 15820, and Lactobacillus casei ASCC 290 were obtained from Australian Starter Culture Collection Center (ASCC; Werribee, Australia). Lactobacillus acidophilus CSCC 2404 was from CSIRO Starter Culture Collection (CSIRO, Melbourne, Australia) and Lactobacillus plantarum WQ1 was isolated from traditional homemade Chinese sauerkraut (Sichuan, China). Each stock culture was stored in 40% (vol/vol) sterile glycerol at −80 °C. MRS medium is most commonly used for cultivation of lactobacilli as Tween80 in MRS has been shown to be an essential growth factor for lactobacilli (Partanen and others 2001). These organisms were ac- Gas chromatography-mass spectrometry (GC-MS) analysis tivated by growing in DifcoTM lactobacilli MRS broth (BD Co., FAMEs were suspended in n-hexane (GC grade) prior to injectMd., U.S.A.) for 12 h at 37 °C successively for 3 times before ing aliquots to GC-MS instrument. FAMEs were analyzed in an performing experiments. Agilent GC-MS system (6890N-GC with 5973-MS ECD/NPD; Santa Clara, Calif., U.S.A.) equipped with GC/MSD ChemStaViable cell counts tion software (Agilent). FAME analysis was carried out using a The above organisms were grown aerobically in MRS broth silica capillary column (100 m × 0.25 mm × 0.2 μm film thicksupplemented with various concentrations of elaidic acid (100, ness; SP-2560 Supelco, Bellefonte, Pa., U.S.A.), which is a highly 200, and 500 mg/L) for 24 h; MRS broth without elaidic acid polar column. One microliter of aliquot was injected automatsupplementation was used as a control. The pH of MRS with and ically with an inlet temperature of 250 °C and a split ratio of without supplementation with elaidic acid was measured by a pH 50:1. Helium was used as a carrier at a flow rate of 20 cm/s at paper; negligible changes (pH 6.70 to 6.60) were observed (data 175 °C. The oven temperature program was 140 °C and the samnot shown) suggesting that the pH did not affect the bacterial ple was held for 5 min, with 1 ramp to attain 240 °C at a rate of growth. Elaidic acid was added at 15 g/L stock solutions in sterile 4 °C/min, and 240 °C held for 15 min. The detector temperature distilled water containing 2% (vol/vol) of Tween-80 (Sigma) as was 250 °C. M2486 Journal of Food Science r Vol. 79, Nr. 12, 2014
Effects of Elaidic acid on Lactobacilli . . .
Statistical analysis All the presented data are as mean ± standard derivation. The significant difference among groups was determined by one-way analysis of variance (ANOVA) using IBM SPSS Statistics 20.0 version. Differences were considered significant at a P value of < 0.05.
Results and Discussion Effect of elaidic acid on the growth of lactobacilli The effect of elaidic acid on the growth of lactobacilli during the incubation period of 24 h is shown in Table 1. As the concentration of elaidic acid was increased, there was a slight decrease in the viable population of all of lactobacilli after 8 h or 16 h of incubation, except that of L. casei. In some organisms (such as L. paracasei, L. zeae, and L. plantarum), the increased concentration of elaidic acid caused growth inhibition after 24 h of incubation as compared to that after 8 h. In particular, L. casei was not significantly affected by elaidic acid at any concentrations studied during the 24-h incubation. Other 5 dairy lactobacilli showed lower resistance to elaidic acid, but they were still able to survive in the elaidic acid-enriched medium. L. rhamnosus and L. paracasei were significantly (P < 0.05) inhibited by elaidic acid, however, there was no significant bacterial inhibition between 200 and 500 mg/L. L. zeae could grow well at 8 h in 500 mg/L of elaidic acid, but a significant (P < 0.05) inhibition was observed at the 16 and 24 h of incubation. However, there was no significant inhibition of this organism with various elaidic acid supplementations at 16 h, and the lowest count of L. zeae at 500 mg/L of elaidic acid was shown at 24 h. The growth of L. acidophilus was significantly (P < 0.05) affected by 100, 200, and 500 mg/L of elaidic acid as compared to the control, however, it showed almost the same survival in the presence of 100 and 200 mg/L of elaidic acid. L. plantarum showed no inhibition at 8 h and 16 h in the 0 and 100 mg/L of elaidic acid, but was significantly (P < 0.05) inhibited by 200 and 500 mg/L at 8 and 16 h. However, there was no significant difference (P > 0.05) among elaidic acid treated groups at 24 h. For the bacterial growth, 8 h was likely to be the log phase for all lactobacilli, 16 and 24 h as the late stationary phase for L. rhamnosus, L. paracasei, L. casei, and L. acidophilus, and 24 h as a decline phase for L. zeae and L. plantarum. Currently, limited data has shown the inhibitory or stimulatory effect of elaidic acid on the growth of lactobacilli. A previous study revealed that lower concentration of PUFAs showed a significant inhibition of probiotic Lactobacillus strain (Kankaanp¨aa¨ and others
2001). Inhibition of bacterial growth caused by elaidic acid may be associated with the inhibition of fatty acids synthesis such as the reduction of acyl carrier proteins, fatty acids synthetase and acetyl-CoA carboxylase (Weeks and Wakil 1970), or the changes in cellular and membrane lipids in lactobacilli (Kankaanp¨aa¨ and others 2004; Muller and others 2011). In this study, elaidic acid showed slight inhibition of the growth of lactobacilli within the level of 500 mg/L compared with that without supplementation of elaidic acid.
Metabolism of elaidic acid by lactobacilli The concentrations of extracellular and intracellular elaidic acid from cell-free supernatants and cell pellets after incubation are shown in Table 2. As shown in the table, the residual elaidic acid in the supernatant after 24 h of incubation was time-dependent and species-specific. The lowest elaidic acid concentration was at 8 h of incubation in all 6 lactobacilli, while there were increases in the concentrations of elaidic acid at 16 and 24 h. The reduction in elaidic acid in the supernatant was observed suggesting the metabolism of elaidic acid by lactobacilli (Table 1). Although oleic acid is the predominant fatty acid in lactobacilli and in MRS broth, Lactobacillus is able to absorb free fatty acids via long-chain fatty acid transport protein (Fujita and others 2007; Eckhardt and others 2013). Thus, free elaidic acid is transported by bacteria, while lipids containing oleic acid needs to be hydrolyzed by bacterial lipase before utilization. Hence, lactobacilli may utilize elaidic acid for fatty acid synthesis at the log phase of 8 h. As compared to the concentrations at 8 h, the concentrations of elaidic acid increased in the supernatant at 16 and 24 h, which may be related with the cell death at 16 and 24 h (late stationary and decline phase). Intracellular elaidic acid was detected by GC-MS and the reduction of elaidic acid was also observed after 24 h of incubation (Table 2). This indicated that lactobacilli was able to absorb and utilize extracellular elaidic acid, however, the metabolism of elaidic acid in lactobacilli was limited which may be related with the specific enzymes involved in the metabolism for oleic acid rather than elaidic acid. Intracellular elaidic acid from cell pellets after 24-h incubation of lactobacilli in elaidic acid-supplemented MRS (200 mg/L) was also determined (Table 2). Intracellular elaidic acid was not detected by GC-MS in lactobacilli cultivated without the supplementation of elaidic acid. The concentration of intracellular elaidic acid among different lactobacilli varied with organism indicating a species-specific metabolism of elaidic acid. The intracellular concentration of elaidic acid in L. casei was the highest while it was lowest for L. paracasei among the 6 lactobacilli. The total amount of intracellular and extracellular elaidic acid decreased after 24 h of incubation compared with the initial concentration of 200 mg/L. L. rhamnosus metabolized the highest level of elaidic acid with the reduction of 71.04 mg/L, while the lowest reduction of 11.85 mg/L was found in L. plantarum. This is possibly due to the incorporation of free fatty acid into bacterial fatty acids as reported in a previous study (Kankaanp¨aa¨ and others 2004). The reduction in elaidic acid concentration was observed indicating the metabolism of elaidic acid by lactobacilli. Also, the reduction in the level of elaidic acid among different lactobacilli was species-specific. It appears that the strains from L. casei group including L. rhamnosus, L. casei, and L. paracasei showed the highest potential to reduce elaidic acid. Previous study indicated a metabolic way for bioconversion or isomerization of elaidic acid into other fatty acids by ruminal microbes (Proell and others 2002). Also, β-oxidation of fatty acids is the general metabolism pathway for unsaturated fatty acids Vol. 79, Nr. 12, 2014 r Journal of Food Science M2487
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Measurement of cell surface hydrophobicity The ability of bacteria to adhere to hydrocarbons was used to examine the effects of free elaidic acid on the cell surface hydrophobicity of lactobacilli (Lye and others 2010). Briefly, cells were harvested after 24 h of incubation at 4000 × g for 15 min at 4 °C and washed 3 times with pre-cooled sterile saline buffer, and were resuspended in the same buffer. The cell suspension was adjusted to an optical density (OD) of 0.50 to 0.60 at 600 nm with saline buffer. Then, 10 mL of cell suspension was mixed with 2 mL of xylene and was vortexed for 2 min. The 2 phases were allowed to separate naturally for 20 min. The aqueous phase was carefully collected using a Pasteur pipette for the measurement of OD at 600 nm. All the analyses were performed in triplicate. Cell surface hydrophobicity (H%) was calculated according to the equation: H% = ([A0 – A]/A0 ) × 100%, where A0 and A are the absorbances before and after extraction with xylene, respectively.
Effects of Elaidic acid on Lactobacilli . . . Table 1–Effect of various concentrations of elaidic acid on the growth of lactobacilli. Viable cell count after the certain cultivation time (log10 CFU/mL) 8 hd
Organism
Elaidic acid concentration (mg/L)
0h
L. rhamnosus
0 100 200 500 0 100 200 500 0 100 200 500 0 100 200 500 0 100 200 500 0 100 200 500
6.99 ± 0.01
L. paracasei
L. zeae
L. casei
L. acidophilus
L. plantarum
M: Food Microbiology & Safety
9.90 9.24 9.01 9.01 9.31 9.08 9.12 9.15 8.52 8.44 8.54 8.49 8.84 8.92 8.78 8.90 9.78 9.01 9.08 8.87 9.60 9.61 9.43 9.23
7.14 ± 0.14
6.80 ± 0.09
7.62 ± 0.11
6.89 ± 0.08
7.35 ± 0.11
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07a,e 0.06b 0.16c 0.04c 0.03a 0.04b 0.05b 0.09b 0.07a 0.06a 0.06a 0.04a 0.03ab 0.04a 0.04b 0.02a 0.03a 0.08bc 0.02b 0.07c 0.05a 0.04a 0.03b 0.02c
16 h 10.91 10.09 9.54 9.33 10.25 10.19 9.63 9.47 10.35 9.49 9.64 9.60 9.39 9.15 9.49 9.39 9.36 9.60 9.22 9.30 10.56 10.61 10.42 10.26
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
24 h
0.02a 0.01b 0.06c 0.31bc 0.03a 0.06a 0.08b 0.07bc 0.11a 0.10b 0.03b 0.05b 0.21a 0.08a 0.10a 0.09a 0.16ab 0.09a 0.13b 0.03ab 0.08a 0.04a 0.04b 0.03c
10.55 10.19 9.81 9.63 10.10 10.16 9.56 9.48 10.28 9.10 9.23 8.77 9.79 9.91 9.63 9.83 10.82 9.58 9.58 8.96 10.37 9.78 9.72 9.58
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04a 0.11b 0.10c 0.17c 0.07a 0.04a 0.09b 0.18bc 0.05a 0.09b 0.20b 0.07c 0.05ab 0.06a 0.06b 0.09a 0.04a 0.03b 0.09b 0.24c 0.05a 0.09b 0.12b 0.17b
d Samples e
were collected at the time point of 8 h during the entire 24-h incubation. Data were presented as mean ± standard deviation (n = 3). Values with no letters in common within each column are significantly different (P < 0.05).
Table 2–GC-MS analysis of extracellular and intracellular elaidic acid in cell-free supernatant and cell pellets. Extracellular elaidic acid in the cell-free supernatant during the entire cultivation with 200 mg/L of elaidic acid (mg/L)
Organism L. rhamnosus L. paracasei L. zeae L. casei L. acidophilus L. plantarum
0h 191.96 ± 3.77
8 ha 108.31 77.94 103.78 67.78 74.43 88.65
± ± ± ± ± ±
16 h 4.74c 3.63 7.33 0.95 2.52 3.39
126.84 139.16 151.76 130.97 117.96 131.58
± ± ± ± ± ±
Cultivation without elaidic acid
24 h 3.27 2.75 1.21 4.74 5.19 7.59
114.94 138.58 150.06 125.33 141.53 170.03
± ± ± ± ± ±
Intracellular elaidic acid in cell pellets from 10 mL of cultures after 24-h incubation (µg)
6.13 4.88 2.12 4.39 0.96 1.31
N. D.d N. D. N. D. N. D. N. D. N. D.
Cultivation with 200 mg/L of elaidic acid 140.17 42.52 114.54 238.83 124.23 180.42
± ± ± ± ± ±
9.19 2.58 1.54 8.31 4.04 1.63
Reduction in elaidic acid after 24-hb incubation (mg/L)b 71.04 57.17 38.57 50.78 45.90 11.85
± ± ± ± ± ±
5.33 4.81 2.08 3.91 5.44 1.21
a
Samples were collected at the time point of 8 h during the entire 24-h incubation. calculation for reduction in elaidic acid (EA) was based on the initial amount of EA and the residual amount of EA in cell-free supernatants and cell pellets. Data were presented as mean ± standard deviation (n = 3). d N. D.: not detectable concentration by GC-MS. b The c
such as oleic acid in bacteria (Fujita and others 2007). However, the metabolic pathway for oleic acid and elaidic acid in lactobacilli and the effect of conformation change between oleic acid and elaidic acid on lactobacilli are not clear. This merits further investigation.
Effect of elaidic acid on cell surface hydrophobicity of lactobacilli In order to understand the effects of elaidic acid on the surface properties of lactobacilli, we tested the hydrophobicity of 6 lactobacilli during the 24 h of incubation period. The results of hydrophobicity of lactobacilli during the 24-h incubation with and without exposure to 200 mg/L of elaidic acid are shown in Table 3. As shown in the table, the hydrophobicity of lactobacilli was species-specific, and it changed during the incubation period. In general, L. rhamnosus, L. zeae, L. acidophilus, and L. plantarum M2488 Journal of Food Science r Vol. 79, Nr. 12, 2014
exhibited higher hydrophobicity (approximately 80%) toward hydrocarbon and slight changes were observed after exposure to elaidic acid; L. paracasei and L. casei showed lower hydrophobicity (below approximately 60%;) indicating the hydrophilic cell surface and dramatically decreases in hydrophobicity were observed in these 2 species after elaidic acid treatment. Moreover, the hydrophobicity showed no correlations with the decrease in extracellular elaidic acid and increase in intracellular elaidic acid (Table 2 and 3). Thus, cell surface hydrophobicity did not influence the absorption of elaidic acid, and the incorporation of free elaidic acid into the cells was due to the specific long-chain fatty acid binding/transport proteins (Eckhardt and others 2013). Additionally, it appears that the cell surface properties of lactobacilli with lower hydrophobicity such as L. paracasei and L. casei (Table 3) were more easily affected by elaidic acid compared with that in other 4 lactobacilli.
Effects of Elaidic acid on Lactobacilli . . . Table 3–Hydrophobicity of lactobacilli cultivated in MRS broth without and with the addition of 200 mg/L of elaidic acid during 24-h incubation. Hydrophobicity (%) 8ha Organism L. rhamnosus L. paracasei L. zeae L. casei L. acidophilus L. plantarum
− Elaidic 94.75 4.35 91.35 44.75 97.17 80.84
± ± ± ± ± ±
acidb 1.11d 1.65 2.55 3.99 2.63 2.17
16 h + Elaidic 93.13 8.43 94.22 29.42 99.53 76.19
± ± ± ± ± ±
acidc 2.33 1.33 2.57 7.42 0.73 2.78
− Elaidic acid 80.12 23.60 89.82 52.83 94.59 87.47
± ± ± ± ± ±
6.04 3.35 3.67 5.76 4.76 0.54
24/0 h + Elaidic acid 88.66 11.57 85.22 13.88 87.12 84.02
± ± ± ± ± ±
2.48 3.40 2.42 2.77 1.64 4.57
− Elaidic acid 87.44 32.19 83.88 55.16 92.36 84.67
± ± ± ± ± ±
2.11 3.80 1.75 2.46 2.12 2.21
+ Elaidic acid 93.50 2.16 94.96 55.25 84.46 88.53
± ± ± ± ± ±
6.64 0.76 1.01 5.04 1.19 2.46
a
Samples were collected at the time point of 8 h during the entire 24-h incubation. acid (200 mg/L) was not added into the MRS broth before incubation. Elaidic acid (200 mg/L) was added into the MRS broth before incubation. d Data were presented as mean ± standard deviation (n = 3).
Lactobacillus sp. is the gram-positive bacteria and does not have References outer membrane and has simple composition in cell surface Bansal G, Zhou WB, Tan TW, Neo FL, Lo HL. 2009. Analysis of trans fatty acids in deep frying oils by three different approaches. Food Chem 116(2):535–41. compared with that in gram-negative bacteria. It has been well Brouwer IA, Wanders AJ, Katan MB. 2010. Effect of animal and industrial trans fatty acids on documented that cell surface hydrophobicity is closely associated HDL and LDL cholesterol levels in humans—a quantitative review. PLOS One 5(3). Cani PD, Bibiloni R, Knauf C, Neyrinck AM, Neyrinck AM, Delzenne NM, Burcelin R. with the adhesion property (Vadillo-Rodriguez and others 2005; 2008. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in Pan and others 2006; Wang and others 2010), and is highly conhigh-fat diet-induced obesity and diabetes in mice. Diabetes 57(6):1470–81. D, Giraffa G, Quiberoni A, Binetti A, Su´arez V, Reinheimer J. 2010. Biotechnology veyed by cell surface proteins known as S-layer proteins in lacto- Carminati of lactic acid bacteria: novel applications. In: Mozzi F, Raya RR, Vignolo GM, editors. bacilli (van der Mei and others 2003). Hence, it appears that elaidic Advances and trends in starter cultures for dairy fermentations. New York: John Wiley & Sons, Ltd. p. 177–92. acid was able to induce specific changes to the cell surface proteins Eckhardt TH, Skotnicka D, Kok J, Kuipers OP. 2013. Transcriptional regulation of fatty acid in lactobacilli, probably in lactobacilli with lower hydrophobicbiosynthesis in Lactococcus lactis. J Bacteriol 195(5):1081–9. ity, leading to the decrease in surface hydrophobicity which may Elo S, Saxelin M, Salminen S. 1991. Attachment of Lactobacillus casei strain GG to human colon carcinoma cell line Caco-2: comparison with other dairy strains. Lett Appl Microbiol in turn affect the adherence to the mucosal cells to exhibit the 13(3):154–6. Fujita Y, Matsuoka H, Hirooka K. 2007. Regulation of fatty acid metabolism in bacteria. Mol probiotic properties to the host.
Conclusions Elaidic acid showed low growth inhibition to selected lactobacilli up to the level of 500 mg/L and was metabolized by lactobacilli. Incorporation of free elaidic acid into the lactobacilli was observed by GC-MS. Moreover, the metabolism of elaidic acid was species-specific. Bacterial cell surface hydrophobicity showed no correlation with the absorption and metabolism of elaidic acid in lactobacilli. However, elaidic acid was able to influence the cell surface hydrophobicity, and the decrease in hydrophobicity was more obvious in lactobacilli with lower hydrophobicity (L. casei and L. paracasei) compared with that in lactobacilli with higher hydrophobicity when incubated in MRS containing 200 mg/L of elaidic acid. A changed surface hydrophobicity may reflect the particular changes in surface S-layer proteins which affect the adhesion property of lactobacilli. Results suggest that lactobacilli were able to metabolize elaidic acid providing a new way for reducing dietary TFAs. However, further works are necessary to understand the metabolic pathway of oleic acid and elaidic acid in lactobacilli.
Acknowledgments The authors would like to thank Ms. Jessie H.Y. Lai in HKU School of Biological Sciences for providing assistance in GC-MS analysis of fatty acids.
Author Contributions N.P. Shah & Q. Wu designed the study; Q. Wu conducted the experiments and drafted the manuscript; N.P. Shah revised and edited the manuscript.
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