Production of conjugated linoleic acid by probiotic Lactobacillus ...

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Production of conjugated linoleic acid by probiotic Lactobacillus acidophilus La-5 M. Macouzet1, B.H. Lee1,2 and N. Robert1 1 Food Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, QC, Canada 2 Department of Microbiology ⁄ Immunology, McGill University, Montreal, QC, Canada

Keywords CLA, conjugated linoleic acid, Lactobacillus acidophilus La-5, probiotic, production conditions. Correspondence Byong H. Lee, Food Research and Development Centre, Agriculture and Agrifood Canada, 3600 Casavant Blvd. West, Saint-Hyacinthe, QC, Canada J2S 8E3. E-mail: [email protected]

2008 ⁄ 1274: received 23 July 2008, revised and accepted 27 October 2008 doi:10.1111/j.1365-2672.2009.04164.x

Abstract Aims: To study the ability of the probiotic culture Lactobacillus acidophilus La-5 to produce conjugated linoleic acid (CLA), which is a potent anti-carcinogenic agent. Methods and Results: The conversion of linoleic acid to CLA was studied both by fermentation in a synthetic medium and by incubation of washed cells. Accumulation of CLA was monitored by gas chromatography analysis of the biomass and supernatants. While the fermentation conditions applied may not be optimal to observe CLA production in growing La-5 cells, the total CLA surpassed 50% of the original content in the washed cells after 48 h under both aerobic and micro-aerobic conditions. The restriction of oxygen did not increase the yield, but favoured the formation of trans, trans isomers. Conclusions: The capability of L. acidophilus La-5 to produce CLA is not dependant on the presence of milk fat or anaerobic conditions. Regulation of CLA production in this strain needs to be further investigated to exploit the CLA potential in fermented foods. Significance and Impact of the study: Knowledge gained through the conditions on the accumulation of CLA would provide further insight into the fermentation of probiotic dairy products. The capacity of the nongrowing cells to produce CLA is also of great relevance for the emerging nonfermented probiotic foods.

Introduction Conjugated linoleic acid (CLA) is a generic name given to isomers of linoleic acid (LA) in which the carbon pairs involved in the formation of double bonds are adjacent to each other (i.e.: –C=C-C=C–). The most commonly found CLA isomers in nature are the cis-9, trans-11 and the trans-10, cis-12. Both isomers possessing a powerful effect against the spread of colon cancer cells (Soel et al. 2007) have been associated with other health effects, such as body fat modulation (Park and Pariza 2007) and reduction of atherosclerosis lesions (Kritchevsky et al. 2002). Dietary LA is converted to CLA in the rumen by indigenous bacteria (Wallace et al. 2007) and is therefore found naturally in the meat and milk of ruminants (Chin et al. 1992). However, the low concentration of CLA 1886

found in foodstuffs derived from such products does not seem to be enough for any significant therapeutic effect. Other bacterial families, including several species from human gut flora, are able to produce CLA in small amounts (Devillard et al. 2007). Certain bacteria, such as strains of the genera Propionibacterium, Clostridium and Lactobacillus have shown an outstanding capacity to produce CLA and thus have been studied for the possibility of eventual commercial biosynthesis of CLA (Rosson et al. 2004). The most desirable source of CLA, however, would be through the natural enrichment of fermented food products by the action of food grade micro-organisms. Accordingly, food grade lactic acid bacteria as starter cultures for the fermentation of dairy products are of special interest. Although low levels of CLA have been observed in the fermented dairy products, the origin of

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1886–1891

M. Macouzet et al.

the LA transformation is generally uncertain and conflicting data have been reported on the capacity of some strains to produce CLA (Sieber et al. 2004). Lactobacillus acidophilus is among the most versatile and appreciated bacterial species that is commonly used as both starter culture and probiotic. Lactobacillus acidophilus La-5 (La-5) is a widely used commercial culture with well-established probiotic properties. This strain has been associated with the production of CLA during the fermentation of milk (Akalin et al. 2007) and cream (Ekinci et al. 2008), but did not show any CLA accumulation in the ripening of cheeses (Bzducha and Obiedzinski 2007). Few other strains of L. acidophilus have been reported to produce CLA, but they are generally not commercially available or the CLA production was marginal and inconsistent (Ogawa et al. 2001; Kim and Liu 2002; Alonso et al. 2003; Lin et al. 2005; Dong and Qi 2006; Van Nieuwenhove et al. 2007). Materials and methods Chemicals and solutions All chemicals were obtained from Sigma-Aldrich (Oakville, ON, Canada). A stock emulsion (14 mmol l)1 LA) was prepared by stirring the linoleic acid in sterile solution of 2% (v ⁄ v) of Tween 80. Milk fat was collected from melted (60C) commercial butter and emulsified in the same way as LA. All solvents, fatty acids and methyl ester standards were GC grade. Heptadecanoic acid was used as an internal standard (10 mg ml)1 stock solution in n-hexane). Methanolic HCl was purchased as a 0Æ5 mol l)1 solution (Supelco, Bellefonte, PA, USA). Culture conditions A commercial frozen starter culture of Lactobacillus acidophilus La-5, supplied by Chr. Hansen (Chicago, IL, USA), was routinely grown in MRS broth (Difco, Sparks, MD, USA) under micro-aerobic conditions at 37C. A pure active culture was prepared by picking a single colony from the MRS agar plate and growing in MRS broth for 24 h. Aliquots of the active culture were used for the scaling up and for the fermentation assays. Fermentation assays The active culture of L. acidophilus (2% v ⁄ v) was inoculated into 20 ml MRS broth supplemented with the following components to generate 4 treatments: (i) control containing the emulsifier used in the other treatments (0Æ2% Tween 80), (ii) 1Æ4 mmol l)1 LA, (iii) 0Æ37% milk fat and (iv) 0Æ37% milk fat + 1Æ4 mmol l)1 LA. All treat-

CLA production by L. acidophilus

ments were prepared in duplicate and incubated for 24 h at 37C. By the end of the incubation time, the samples were centrifuged (3000 g; 15 min) in a Beckman Coulter GS-6R centrifuge (Mississauga, ON, Canada). After the biomass pellets were subjected to two cycles of resuspension (10 ml, 50 mmol l)1 phosphates pH 7) and centrifugation, the washed pellets were stored at )20C for further extraction and methylation of fatty acids. Supernatants were immediately subjected to lipid extraction. Washed biomass assays The active culture of L. acidophilus was inoculated (1% v ⁄ v) into 1Æ5 l of MRS broth and incubated for 24 h at 37C. The biomass was collected and washed as described for the fermentation assays. Washed cells were resuspended in 225 ml phosphate buffer (50 mmol l)1, pH 7) and distributed in aliquots of 10 ml in 15 ml-screw cap tubes. The cell suspensions (100–120 mg dry biomass per sample) were divided into two treatments: (i) control containing the emulsifier used in the other treatment (0Æ2% Tween 80) and (ii) addition of LA to a concentration of 1Æ4 mmol l)1. The samples were further divided in two subtreatments by subjecting them to aerobic or micro-aerobic incubation. For the aerobic incubation, the tubes were capped keeping the air head. For the microaerobic treatment, the tubes were equilibrated for 1 h and hermetically capped in an anaerobic environment (CO2 5%; H2 10%; N2 85%) by means of anaerobic system model 1025 (Forma Scientific, Marietta, OH, USA). The samples were incubated at 22C with gentle oscillatory movement on a compact rocker CR300 (FinePCR, Seoul, Korea) to keep the cells in suspension. Samples of each treatment were collected at 24 h intervals and centrifuged (3000 g; 15 min) to separate biomass and supernatants for further analysis. Extraction and methylation Lipid extraction and formation of fatty acid methyl esters (FAMEs) from bacterial pellets were performed in a single step as described by Dionisi et al. (1999), with minor modifications. Briefly, the frozen biomass samples were lyophilized in a Dura-stop MP stoppering tray dryer (FTS Systems, Stone Ridge, NY, USA). Comminuted freeze dried biomass was mixed with 500 lg internal standard, 1 ml n-hexane and 750 ll of 0Æ5 mol l)1 methanolic HCl. The reaction mixture was vortexed for 20 min, followed by incubation at 60C for further 30 min. Upon cooling the mixture, the reaction was washed with 2 ml of 5 mol l)1 NaCl by gently inverting the tubes several times. The organic phase was transferred to a fresh tube and dried with anhydrous Na2SO4 crystals. Dry extracts


1887


M. Macouzet et al.

Table 1 LA and CLA analysis of Lactobacillus acidophilus La-5 biomass after 24 h growth in MRS based broth (20 ml) Biomass yield* MF

X X

X

LA in cells

Range

(mg g)1)

Range

CLA in cells

CLA in medium

0Æ08 0Æ00 0Æ01 0Æ01

ND 3Æ18 ND 2Æ39

– 0Æ36 – 0Æ14

ND ND ND ND

ND ND ND ND

MF, milk fat supplementation at 0Æ37%; LA, linoleic acid at 1Æ4 mmol l)1; CLA, total conjugated linoleic acid; X, treatment applied; ND, concentration below 0Æ06 mg g)1. *Yield of freeze dried biomass. Concentration in dry biomass.

(a) pA 15

11

10t, 12c*

12

t, t mixture

CLA isomers

LA

13

9c, 11t *

Detector response

14

10

Detector response

(b) pA

21

22

23

25

24 CLA isomers

LA

20 18 16 14

11c, 13t 10t, 12c

20

t, t mixture

Results

(g l ) 2Æ02 1Æ50 2Æ02 1Æ94

X

GC analysis FAMEs were analysed in an Agilent 6890 GC System (Agilent Technologies Canada, Kirkland, QC, Canada). Injection (1 ll) was performed automatically with an inlet temperature of 240C and a split ratio of 100 : 1. Hydrogen was used as a carrier through a DB-23 capillary column (60 m · 320 lm). The oven worked at an initial temperature of 140C for 3 min, with four ramps to attain 176, 200, 205 and 250C at a rate of 9, 4Æ5, 0Æ25 and 10C min)1 respectively. Detection was carried out by flame ionization at 260C. The signal correction factor (CFx) to compute CLA concentration was calculated by using the ratio of the internal standard response over that obtained by the same weight of LA. As LA and CLA have identical active carbon numbers and unsaturations, they produce similar detector responses and share the same CFx. Thus, the experimental CFx for LA and CLA was 0Æ923.

LA

)1

9c, 11t & 9t, 11c

were transferred by decantation to 2 ml fresh vials that were subjected to complete evaporation at room temperature in a Zymrak Turbo Vap evaporator (Caliper Life Sciences, Hopkinton, MA, USA). Residual FAMEs were dissolved in 600 ll of 2, 2, 4-trimethylpentane. Supernatants were extracted as described by Jung et al. (2006) with some modifications. Samples were extracted with 2x 500 ll n-hexane after addition of 1 g NaCl crystals to facilitate diffusion. Extraction proceeded by gentle inversions until salt was completely dissolved. The recovered organic phase was dried with anhydrous Na2SO4 and mixed with the internal standard (500 lg) and methanolic HCl (5 mol l)1, 1 ml). After the reaction mixture was incubated at 60C for 30 min, the reaction products were treated as described for the biomass samples.

12 10

From the fermentation studies using the active growing cells, no discernable accumulation of CLA was detected in the medium or in the biomass after the growth of 24 h. Similar results were observed with or without milk fat in the culture medium (Table 1). When the study was performed with the washed cells, LA was readily incorporated into the biomass, reaching up to 14% of lipids in 72 h. In the same period, the concentration of CLA found in the cells represented 27% to 30% of that of LA (Fig. 2). The accumulation of CLA was evident in the biomass after 24 h incubation (Fig. 1a). It accumulated faster during the first 48 h under aerobic conditions (Fig. 2a), as compared with the micro-aeobic incubation (Fig. 2b). After the growth of 72 h, however, total CLA accumulation was similar in both cases reach1888

20

21

22 23 Retention time (min)

24

25

Figure 1 Partial chromatograms of Lactobacillus acidophilus La-5 samples at the CLA region. (a) Extract from washed cells, 24 h in 50 mmol l)1 phosphates pH 7, 1Æ4 mmol l)1 LA, 22C; (b) Methyl ester standards of LA and CLA. (LA) linoleic acid; (CLA) conjugated linoleic acid; (t) trans.; (c) cis and (*) presumptive isomer.

ing an average concentration of 388 lg g)1 (CLA ⁄ dry biomass) or 4Æ18 mg l)1 (CLA ⁄ cell suspension). While aerobic incubation seemed to slightly favour the formation of the cis-9, trans-11 isomer, restriction of oxygen resulted in a preferred accumulation of trans, trans species (Fig. 3a,b).


M. Macouzet et al.


(a) 500

1600

450

1200 400

1000

350

800 600

300 250

400 0

20

40

60

80

120

200

Incubation time (h)

Relative increment (%) in 72 h

1400

LA concentration (µg g–1)

CLA concentration (µg g–1)

(a)

100 80 60 40 20 0

1600 1400

450

1200 400

1000

350

800 600

300

400

250

200 0

20

40 60 Incubation time (h)

80

Figure 2 Time course accumulation of linoleic and conjugated linoleic acids in the biomass of Lactobacillus acidophilus La-5. Washed cells incubated in 50 mmol l)1 phosphates pH 7 supplemented with 1Æ4 mmol l)1 linoleic acid at 22C. Error bars represent the range (n = 2). (a) Aerobic incubation, (b) micro-aerobic incubation. ( ) linoleic acid and ( ) conjugated linoleic acid.

Discussion Evidence has been published on the formation of CLA by including La-5 in the fermentation of dairy products (Akalin et al. 2007; Ekinci et al. 2008). Conversely, when we incubated this strain in MRS + LA broth for 24 h, no detectable accumulation of CLA was observed. This observation is in agreement with those of other L. acidophilus strains that produced very little CLA in MRS, if at all, but appeared to produce higher amounts when cultured in a milk-based medium (Kim and Liu 2002). To determine any relationship on milk fat in the production of CLA, we supplemented the culture medium with milk fat, but no difference was observed on the CLA content at the end of the fermentation. While time course monitoring of the CLA production was not performed, the 24 h fermentation period was expected to generate detectable amounts of CLA based on reports indicating that other L. acidophilus strains produced measurable amounts between 12 and 24 h of growth (Kim and Liu 2002; Alonso et al. 2003) with a

9c, 11t

10t,12c

t, t mix

(b) 100 Proportion of CLA isomers (%)

500

LA concentration (µg g–1)

CLA concentration (µg g–1)

(b)

90

20

23

80 70 60

39

39

41

38

Aerobic

Anaerobic

50 40 30 20 10 0

Figure 3 Production of conjugated linoleic acid (CLA) isomers in Lactobacillus acidophilus La-5 cells. Washed cells were incubated in 50 mmol l)1 phosphates pH 7 supplemented with 1Æ4 mmol l)1 linoleic acid at 22C for 72 h. (a) Relative increment of each CLA isomer. Blanc bars correspond to the aerobic treatment while those filled with a dotted pattern represent the micro-aerobic incubation. Height of bars represents the average of 2 analyses performed on independent incubations. Error bars symbolize the range. (b) Proportion of CLA isomers. The trans, trans mixture; trans-10, cis-12 and cis-9, trans-11 isomers are represented by black, white and grey bars respectively.

maximum at 21 h (Dong and Qi 2006). The fact that CLA production could not be confirmed for growing cells of La-5 under the narrow conditions tested, does not disqualify the micro-organism as a potential producer under different circumstances. The analysis of potential factors conducting to the production of CLA highlighted two critical variables to be tested. First, it was considered that the growth stage might be a determinant factor. This hypothesis was based on reports showing that L. acidophilus produced CLA only after having reached the stationary growth phase (Alonso et al. 2003). A second potential factor affecting the CLA production was considered to be oxygen availability. When L. acidophilus AKU 1137 was incubated in the presence of LA under aerobic conditions, no


1889


M. Macouzet et al.

detectable amounts of CLA were produced; but it accumulated significant amounts of CLA under micro-aerobic conditions (Ogawa et al. 2001). Both potential factors were tested simultaneously by incubating the fully grown cells under aerobic and micro-aerobic conditions. As expected, the washed cells in the ‘stationary’ physiological state permitted the accumulation of CLA (Figs 1 and 2). Therefore, the fact that no detectable accumulation of CLA was observed in the early phase of growth in MRS + LA might be explained by the relative freshness of the culture. However, caution should be taken in the comparison of both approaches as the incubation temperature was different and the amount of biomass available for the CLA analysis was three to four times higher in the case of the washed cell assay. Thus, the possibility that growing cells had produced small amounts of CLA, which was not detected because of the size of the sample, should also be taken into consideration. The oxygen availability turned out not to be a decisive factor for the production of CLA, but it affected the relative composition of the isomers. Oxygen restriction did not improve CLA accumulation, but caused an undesirable enrichment in trans, trans isomers (Fig. 3). The amount of LA incorporated by the La-5 cells was of the same order as that reported for other Lactobacilli species (Jenkins and Courtney 2003). The proportion of CLA formed was clearly higher than that of other lactic bacteria (Jenkins and Courtney 2003), with the exception of L. reuteri ATCC 55739, which accumulated about four to five times the concentration found in La-5. These results confirm the capacity of La-5 to produce CLA, but refute any dependency on milk fat or on oxygen for the CLA production. This is desirable in the sense that it allows great versatility in using the La-5 cells. Regarding the reported negative CLA accumulation in the ripening of cheeses (Bzducha and Obiedzinski 2007), we noticed that the authors mistakenly assumed that the produced CLA would be esterified in the form of triacylglycerides. Based on this assumption, they chose the wrong methylation method for lipid analysis by GC that does not work on free fatty acids (Christie et al. 2001). They were induced into error by the fact that other L. acidophilus strains were able to produce CLA from esterified LA (Alonso et al. 2003; Xu et al. 2004); however, they did not consider that the conversion happens after lipolysis and that the bulk of CLA produced is thus found in free form (Ogawa et al. 2001). As reported for other L. acidophilus strains, the CLA produced by La-5 tends to remain in the cells or associated with the cells (Ogawa et al. 2001). This might be an advantage in that probiotic supplements could be naturally enriched in CLA (Wall et al. 2008); however, consid1890

ering that probiotics are not digestible by human or monogastric animals, the availability of the accumulated CLA remains uncertain and will depend on the capacity of the bacteria to release the CLA in the intestines. Also, it is important to note that the capacity to produce and accumulate CLA is not common to every strain; for instance, the homologous popular probiotic L. acidophilus NCFM has shown to be rather insensitive to LA and did not incorporate any CLA into its membrane as shown by the known CLA producers (Jenkins and Courtney 2003). The capacity of the nongrowing cells to produce CLA is of great relevance for the emerging nonfermented probiotic food industry. Probiotic supplementation of food products is frequently superior to 107 CFU g)1 (Macouzet and Champagne 2007); such a concentration of La-5 cells might result in a significant accumulation of CLA during storage and commercialization. Therefore, it would be interesting to test this premise and conduct optimization studies in diverse food systems under different storage temperatures. In conclusion, the capability of La-5 to produce CLA is not dependant on the presence of milk fat or anaerobic conditions. The CLA accumulation capacity of La-5 is of interest for the development of probiotic supplements or health food products, but further studies should be performed in particular food systems to maximize the enrichment in CLA and enhance the health functionality of the product. To obtain a better understanding on the production of CLA by L. acidophilus, we are presently conducting a study at both functional and genetic levels including several selected strains. Acknowledgements The authors thank Dr Claude Champagne for his valuable input during the planning and progress of the study. The authors also thank Yves Raymond and Catherine Avezard for their technical advice on gas chromatography. References Akalin, A.S., Tokusoglu, O., Gonc, S. and Aycan, S. (2007) Occurrence of conjugated linoleic acid in probiotic yoghurts supplemented with fructooligosaccharide. Int Dairy J 17, 1089–1095. Alonso, L., Cuesta, E.P. and Gilliland, S.E. (2003) Production of free conjugated linoleic acid by Lactobacillus acidophilus and Lactobacillus casei of human intestinal origin. J Dairy Sci 86, 1941–1946. Bzducha, A. and Obiedzinski, M.W. (2007) Influence of two probiotic Lactobacillus strains on CLA content in model ripening cheeses. Pol J Food Nutr Sci 57, 65–69.


M. Macouzet et al.

Chin, S.F., Liu, W., Storkson, J., Ha, I.L. and Pariza, M.W. (1992) Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J Food Compos Anal 5, 185–197. Christie, W.W., Sebedio, J.L. and Juaneda, P. (2001) A practical guide to the analysis of conjugated linoleic acid (CLA). Inform 12, 147–152. Devillard, E., McIntosh, F.M., Duncan, S.H. and Wallace, R.J. (2007) Metabolism of linoleic acid by human gut bacteria: different routes for biosynthesis of conjugated linoleic acid. J Bacteriol 189, 2566–2570. Dionisi, D., Golay, P.A., Elli, M. and Fay, L.B. (1999) Stability of cyclopropane and conjugated linoleic acids during fatty acid quantification in lactic acid bacteria. Lipids 34, 1107–1115. Dong, M. and Qi, S. (2006) Conjugated linoleic acid production by fermentation. Int J Food Eng 2, 1–12. Ekinci, F.Y., Okur, O.D., Ertekin, B. and Guzel-Seydim, Z. (2008) Effects of probiotic bacteria and oils on fatty acid profiles of cultured cream. Eur J Lipid Sci Technol 110, 216–224. Jenkins, J.K. and Courtney, P.D. (2003) Lactobacillus growth and membrane composition in the presence of linoleic acid or conjugated linoleic acid. Can J Microbiol 49, 51–57. Jung, M.Y., Kim, G.-B., Jang, E.S., Jung, Y.K., Park, S.Y. and Lee, B.H. (2006) Improved extraction method with hexane for gas chromatographic analysis of conjugated linoleic acids. J Dairy Sci 89, 90–94. Kim, Y.J. and Liu, R.H. (2002) Increase of conjugated linoleic acid content in milk by fermentation with lactic acid bacteria. J Food Sci 67, 1731–1737. Kritchevsky, D., Tepper, S.A., Wright, S. and Czarnecki, S.K. (2002) Influence of graded levels of conjugated linoleic acid (CLA) on experimental atherosclerosis in rabbits. Nutr Res 22, 1275–1279. Lin, T.Y., Hung, T.H. and Cheng, T.-SJ (2005) Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus. Food Chem 92, 23–28.


Macouzet, M. and Champagne, C.P. (2007) Les bacte´ries probiotiques : innovations et tendances de de´veloppement technologique. Centre que´be´cois de valorisation des biotechnologies. Bioveille 6, 1–24. Ogawa, J., Matsumura, K., Kishino, S., Omura, Y. and Shimizu, S (2001) Conjugated linoleic acid accumulation via 10-hydroxy-12-octadecanoic acid during microaerobic transformation of linoleic acid by Lactobacillus acidophilus. Appl Environ Microbiol 67, 1246–1252. Park, Y. and Pariza, M.W. (2007) Mechanisms of body fat modulation by conjugated linoleic acid. Food Res Int 40, 311–323. Rosson, R.A., Grund, A.D., Deng, M.-D. and Sanchez-Rivera, F. (2004) Linoleate isomerase. Patent US 6,743,609 B1. Sieber, R., Collomb, M., Aeschlimann, A., Jelen, P. and Eyer, H. (2004) Impact of microbial cultures on conjugated linoleic acid in dairy products – a review. Int Dairy J 14, 1–15. Soel, S.M., Choi, O.S., Bang, M.H., Park, J.H.Y. and Kim, W.K. (2007) Influence of conjugated linoleic acid isomers on the metastasis of colon cancer cells in vitro and in vivo. J Nutr Biochem 18, 650–657. Van Nieuwenhove, C.P., Oliszewski, S.N., Gonzalez, S.N. and Perez Chaia, A.B. (2007) Conjugated linoleic acid conversion by dairy bacteria cultured in MRS broth and buffalo milk. Lett Appl Microbiol 44, 467–474. Wall, R., Ross, R.P., Fitzgerald, G.F. and Stanton, C. (2008) Microbial conjugated linoleic acid production – a novel probiotic trait? Food Sci Technol Bull: Functional Foods 4, 87–97. Wallace, R.J., McKain, N., Shingfield, K.J. and Devillard, E. (2007) Isomers of conjugated linoleic acids are synthesized via different mechanisms in ruminal digesta and bacteria. J Lipid Res 48, 2247–2254. Xu, S., Boylston, T.D. and Glatz, B.A. (2004) Effect of lipid source on probiotic bacteria and conjugated linoleic acid formation in milk model systems. J Am Oil Chem Soc 81, 589–595.


1891