This article was downloaded by: [UQ Library]On: 20 November 2014, At: 08:13Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office:Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Indian Chemical EngineerPublication details, including instructions for authors and subscriptioninformation:http://www.tandfonline.com/loi/tice20

Isolation, Analysis and Identification ofFatty Acids in Probiotic CurdsDebasree Ghosha & Parimal Chattopadhyayb

a Department of Food Technology & Biochemical Engineering, JadavpurUniversity, Kolkata 700032, Indiab Department of Food Technology, Guru Nanak Institute of Technology,Panihati, Kolkata 700114, IndiaPublished online: 09 Apr 2014.

To cite this article: Debasree Ghosh & Parimal Chattopadhyay (2014) Isolation, Analysis andIdentification of Fatty Acids in Probiotic Curds, Indian Chemical Engineer, 56:2, 137-145, DOI:10.1080/00194506.2014.903623

To link to this article: http://dx.doi.org/10.1080/00194506.2014.903623

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis, ouragents, and our licensors make no representations or warranties whatsoever as to theaccuracy, completeness, or suitability for any purpose of the Content. Any opinions and viewsexpressed in this publication are the opinions and views of the authors, and are not the viewsof or endorsed by Taylor & Francis. The accuracy of the Content should not be relied uponand should be independently verified with primary sources of information. Taylor and Francisshall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses,damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly inconnection with, in relation to or arising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any substantialor systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, ordistribution in any form to anyone is expressly forbidden. Terms & Conditions of access anduse can be found at http://www.tandfonline.com/page/terms-and-conditions

Isolation, Analysis and Identification of Fatty Acidsin Probiotic Curds

Debasree Ghosha and Parimal Chattopadhyayb*aDepartment of Food Technology & Biochemical Engineering, Jadavpur University, Kolkata 700032,India; bDepartment of Food Technology, Guru Nanak Institute of Technology, Panihati, Kolkata700114, India

Abstract: Milk and milk products are nutritious food items containing numerous essential nutrients.The saponified fatty acids (SFAs) and conjugated linoleic acid (CLA) in probiotic curd (dahi) producedby Streptococcus thermophilus, Lactobacillus acidophilus and Lactobacillus casei during fermentationand storage at 4°C was determined and compared with the controlled curd. The content of oleic acid,CLA, omega-3 fatty acids, short chain and medium chain fatty acids may promote positive healtheffects. Gas chromatography analysis showed a significant increase in total SFAs during fermentationand storage of curd samples prepared with the addition of different ratios of lactic cultures. Analysis ofSFAs also showed increased concentration of butyric and linoleic acids in the saponified probiotic curdduring the course of fermentation. Lactobacilli appeared to increase the SFAs by lipolysis of milk fatand CLA by using internal linoleic acid.

Keywords: Probiotics, Saponified fatty acids, Cholesterol, Fermentation, Gas chromatography.

IntroductionBovine milk, soy milk and the fermented dairy products have important role in humannutrition. These are the main resources of lipids, proteins, amino acids, vitamins and minerals [1].The lipids in milk are emulsified in globules coated with membranes. Milk contains about33 g total fat/100 g solid materials. Triacylglycerols, which account for about 95% of the lipidfraction, are composed of fatty acids of different length (4–24 C atoms) and saturation [2].Each triacylglycerol molecule is built up with a fatty acid combination turning the molecule

*Author for Correspondence. Email: parimalchattopadhyay@yahoo.com

INDIAN CHEMICAL ENGINEER © 2014 Indian Institute of Chemical EngineersVol. 56 No. 2 June 2014, pp. 137–145Print ISSN: 0019-4506, Online ISSN: 0975-007X, http://dx.doi.org/10.1080/00194506.2014.903623

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014

into a liquid form at the body temperature. Other milk lipids are diacylglycerol (about 2%),cholesterol (<0.5%), phospholipids (about 1%) and free fatty acids (FFAs) accounting to<0.5% of total milk lipids [3]. Increased levels of free fatty acids in fermented dairy productsmight result in off-flavours and the free volatile short chain fatty acids contribute to thecharacteristic flavour of the ripened cheese [4]. More than half of the milk fatty acid residuesare saturated. The concentration of fatty acids was found to be increased in the fermenteddairy product curd. The health effects of esterified individual fatty acids were extensivelystudied. The saturated fatty acids, namely, myristic acid (14:0) and palmitic acid (16:0) havelow-density lipoprotein (LDL) and high-density lipoprotein (HDL), respectively, withincreased cholesterol properties [5]. Increased intake of these acids raises blood cholesterollevels and our diets rich in saturated fatty acids cause heart diseases, weight gain and obesity[6]. Several intervention studies have shown that the diet containing low-fat dairy productsare associated with favourable changes in serum cholesterol. However, fat consumptionthrough probiotic curd has been shown to have less adverse effects on serum lipids than whichcould be expected from fat of normal milk [7]. Probiotic lactic acid bacteria are able toincrease the production of FFAs by lipolysis of milk fat [8]. It was observed that theconjugated linoleic acids produced by lactic acid bacteria provide nutritional and therapeuticbenefit to human health after consumption of curds [2].

Fatty acids play a vital role in health benefits [9]. Currently there are many products inthe market that claim to contain healthy omega 3 fatty acids. They only contain α linoleicacid (ALA), not eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). DHA andEPA are produced by microalgae that live in seawater. People with blood circulatoryproblems benefit from supplements containing EPA and DHA, which stimulate bloodcirculation [10], increase the breakdown of fibrin, a compound involved in clot and scarformation, and additionally shown to reduce blood pressure [11]. On the other hand, fattyacids play important role in cancer prevention [12] and HDL is also necessary for ourbody [9].

In this present study, we have studied the changes in saponifiable fatty acids afterfermentation of milk during the production of fermented dairy products (cow milk and soymilk curd), which will be very useful for further study. We know that soymilk contains almostall the nutrients except iron and calcium and this can be the reason for poor growth ofLactobacillus acidophilus in soymilk. Moreover, vigorous stimulators of growth of L.acidophilus increase the amount of thiol groups, whereas peptone and trypsin stimulate itsacid production (Vuyst 2000). Soy milk is low in amino-acids containing sulphur and this canbe a reason for poor growth of L. acidophilus in the growth medium. In this study, we haveused natural iron containing natural source like beet pulp for iron fortification of soy milkduring curd formation by lactic acid bacteria. This can be recommended as safe functionalfood for better health [13].

Materials and MethodsBacterial CulturesLactic acid bacteria, namely, Streptococcus thermophilus DG-1, isolated from home-madecurd and Lactobacillus plantarum (MTCC no. 1325), and Leuconostoc mesenteroides (MTCC

138 GHOSH and CHATTOPADHYAY

INDIAN CHEMICAL ENGINEER Vol. 56 No. 2 June 2014

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014

no. 107) collected from Institute of Microbial Technology, Chandigarh, India were used indifferent concentration to form milk curd and soy curd.

Preparation of InoculumsFifty millilitre of soy milk and 50 ml cow milk were boiled for 10 minutes and cooled to 37°Cand were inoculated with a loopful of lactic cultures from MRS agar stab culture, andincubated at 37°C for 24 hours. These inoculums were used in the preparation of soy curdand cow milk curd for the subsequent investigation.

Preparation of SoymilkThe raw material, i.e., soybean, was purchased from the local market. Beans were carefullyselected and the infected ones were discarded. The beans were soaked in water for 8 hours,dehulled and again soaked in water overnight to absorb sufficient water (water/bean ratio6:1). The weight of the soybean was increased two to three times. The soaked beans wereground in a blender for 15 minutes. It was boiled and cooled to destroy trypsin inhibitor andto improve beany flavour, filtered through fine cloth and was heated to boiling for 10 minutesand then cooled to 37°C for subsequent studies [14].

Preparation of Soy Milk CurdSoy milk was inoculated with 2% inoculum (v/v) from each of single strain and mixed culturein 1:1 (v/v) ratio of L. plantarum and S. thermophilus with addition of 20% (w/v) skimmedmilk (AMUL, Gujarat) and 2% v/v beet pulp and 1:1:1 (v/v) ratio of L. plantarum,L. mesenteroides and S. thermophilus with addition of 20% (w/v) skim milk (AMUL, Gujarat)and 2% v/v beet pulp, respectively, and were incubated at 37°C for 16 h to form soy milk curd[15]. Skimmed milk was added to supply lactose in the soy milk.

Preparation of Cow Milk CurdCow milk was boiled for 10–12 minutes and cooled to 37°C and 2% inoculum (v/v) from eachsingle strain and mixed culture in 1:1 (v/v) ratio of L. plantarum and S. thermophilus and 1:1:1(v/v) ratio of L. plantarum, L. mesenteroides and S. thermophilus, respectively, were addedaseptically and incubated at 37°C for 16 hours to form cow milk curd [15].

Extraction of Lipids from Curd SamplesTwo grams each of curd samples were taken in test tubes and to these 5 ml each of 2:1 (v/v),1:1 (v/v) and 1:2 (v/v) methanol-chloroform solution were added and the mixture wereblended in a Lourdes homogeniser for 2 minutes at 37°C. Then the homogenates werecentrifuged and supernatants were carefully decanted and the residues were re-extracted threetimes with 38 ml of methanol–chloroform–water in the ratio of 2:1:0.8 (v/v), 1:1:0.8 (v/v) and1:2:0.8 (v/v). After homogenisation for 2 minutes and then centrifugation, the combinedsupernatants were diluted with 20 ml each of chloroform and water and the phases wereseparated by centrifugation. The lower chloroform layer was withdrawn and concentrated ina rotary evaporator at 30–35°C (benzene was added to aid in removal of traces of water), andthe residue was dissolved in 10 ml of chloroform. The whole experiment of lipid extraction

Isolation, Analysis and Identification of Fatty Acids 139

INDIAN CHEMICAL ENGINEER Vol. 56 No. 2 June 2014

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014

from both the cow milk curd and soymilk curd was repeated thrice with different chloroformto methanol ratios of 2:1 (v/v), 1:1 (v/v) and 1:2 (v/v), respectively.

Determination of Unsaponifiable Matter in Extracted Lipid Layer of Curd SampleHexane method was used for the estimation of unsaponifiable matter as per proceduredeveloped by Paqust et al. [16].

Identification and Determination of Sterols by Gas Chromatography (GC)Ten milligrams of lipid sample was refluxed with 2 ml of 1M solution of potassium hydroxidein 95% ethanol for 1 hour. The solution was cooled and 5 ml of water was added. Themixture was extracted thoroughly with hexane–diethyl ether (1:1 v/v). The solvent extract wasthen washed with water for six times, dried over anhydrous sodium sulphate and theunsaponifiable materials were recovered after removal of the solvent in a rotary evaporator ata temperature of 40°C. The water washings were added to the aqueous layer, which wasacidified with 6 M hydrochloric acid and extracted with diethyl ether–hexane (1:1 v/v). Thefree fatty acids were recovered after washing the extract with water, drying it over anhydroussodium sulphate and removing the solvent by evaporation. Precautions were taken all alongto prevent autoxidation of lipids by adding mercaptoethanol because polyunsaturated fattyacids autoxidise very rapidly in air and it may not be possible to obtain an accurate analysisby chromatographic means. Once it has been initiated, the reaction preceded autocatalyti-cally. The experiment was carried out according to the method described by William [17].

After saponification, the unsaponifiable matter of the curd sample was separated. Thesterols were isolated from the unsaponifiable matter by thin-layer chromatography (TLC) toensure the presence of sterols in the unsaponifiable matter and to qualitatively determine theclass of lipid fraction present in the sample. The raised sterol layer from TLC was collected byscooping from the TLC plate and subjected to further analysis by gas chromatography (GC).The chromatogram of the sterol was analysed. The gas chromatograph (model 6890N;Agilent Technologies Wilmington, DE, USA) was equipped with a ‘DB-Wax’ capillarycolumn (part no. 122–7032), with a length of 30 m, an internal diameter of 0.25 mm and afilm thickness of 0.25µm. The method of lipid analysis was developed using methyl stearate(18:0) as the locking compound. In this method, the retention time of methyl stearate waslocked to 14 minutes and the peaks of saponified fatty acids generated were identified by thestandard fatty acids scale attached with the GC machine [18]. Chromatograms of raw cowand soy milk yoghurt have already been reported by Souza et al. [19].

Results and DiscussionThe total amount of extracted lipid, unsaponifiable matter and the cholesterol percentage inprobiotic curd samples were analysed and reported in Table 1. From the table value, it can beseen that cow milk curd contains maximum amount of cholesterol compared to soymilk curddue to its bovine origin. We know that soybean of plant origin consists of lesser amount oflipid compared to the animal source like cow milk and so it produces lower amount ofcholesterol [17]. Soy milk samples were fortified with the beet pulp (2% v/v) to supplementiron, which is not present in required amount in soy milk as the lactic acid bacteria were notable to grow without requisite iron in soy milk. Therefore, addition of beet pulp was effective

140 GHOSH and CHATTOPADHYAY

INDIAN CHEMICAL ENGINEER Vol. 56 No. 2 June 2014

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014

in iron fortification of soy milk curd. On the other hand, beet pulp had significant effect onthe changes of fatty acid concentration in soy milk curd. From a study, we have found thataddition of beet pulp to the soy milk before fermentation yielded a product that showedsynergistic role in the reduction of free fatty acid (Table 2). It was effective for qualityimprovement in iron-fortified soy milk product. In our previous study on iron fortification ofsoy milk curd (communicated), we discussed its effect on growth of lactic acid bacteria in soymilk curd before and after addition of beet pulp. It was also compared with the cow milk curdand found that the growth of lactic acid bacteria was greater than 106 CFU/ml in soy milkcurd samples after addition of beet pulp.

From the gas liquid chromatogram (Figs. 1–4) it was clear that the fatty acids, whichwere present in maximal amounts in saponified matter, included palmitic acid (16:0), stearicacid (18:0) and oleic acid (18:1). These values depended on the area percentage of peak shownin the chromatogram. The position of the peak at different retention times was also shown inFigs. 1–4. The concentration of saponified fatty acids present in the curd samples are given inTable 2. It showed that oleic acid was present in maximum amount in cow milk curd whereasin soymilk curds (inoculated with different ratios of lactic cultures), palmitic acid, stearic acidand linoleic acids were present in higher amount. From these results, it can be concluded thatsoy curd inoculated with 2% (v/v) inoculum of S. thermophilus contained linoleic acid,whereas due to addition of different ratios of lactic cultures (1:1 v/v of L. plantarum andL. mesenteroides and 1:1:1 v/v of S. thermophilus, L. plantarum, L. mesenteroides) maximumproduction of oleic acid, stearic acid was obtained (Table 2). This difference in fatty acidconcentration was due to the fermentation by different ratios of mixed lactic cultures. It hasbeen shown that maximum production of oleic acid was observed from the chromatogram(Fig. 1, Table 2) when cow milk was inoculated with 2% (v/v) of S. thermophilus whereasother saponifiable fatty acids, namely, myristic acid, palmitic acid, etc. were present ininsignificant amount and was difficult to determine in the curd sample due to its volatilenature (Table 2). On the other hand, in case of soy milk curd, the addition of one or more

Table 1. Amount of extracted lipid, unsaponifiable matter and cholesterol in curd samples

Samples

Amount ofextracted lipid

(g/100 g of curd)

Unsaponifiablematter

(g/100 g of curd)

Cholesterol(g/100 g of extracted

lipid)

Cow milk curd 5.2 ± 0.18a 4.1 ± 0.12a 0.25 ± 0.03b

Soy milk curd 3.4 ± 0.19a 1.03 ± 0.06b

2% (v/v) inoculum of S. thermophilus 1.6 ± 0.17a 1.9 ± 0.11a 0.06 ± 0.01b

1:1 (v/v) L. plantarum andS. thermophilus with 2% (v/v) beetpulp 1:1:1 (v/v) L. plantarum, L.mesenteroides and S. thermophiluswith 2% (v/v) beet pulp

1.4 ± 0.16a 2.5 ± 0.11a

Note: Means ± SD with different supercripts in the same row were significantly different (p <0.001; n = 4).

Isolation, Analysis and Identification of Fatty Acids 141

INDIAN CHEMICAL ENGINEER Vol. 56 No. 2 June 2014

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014

lactic cultures in different ratios showed quite different pattern of fatty acids due to the effectsof lactic fermentation of soy milk containing lipids [8]. We know that fermentation of milk byL. acidophilus, Lactobacillus bulgaricus and S. thermophilus resulted in significant increase inthe levels of saturated fatty acids and oleic acid with a concomitant decrease in the levels of

Fig. 1. Gas liquid chromatography (GLC) chromatogram of fatty acid profile of cow milk curdinoculated with 2% (v/v) S. thermophilus.

Table 2. Concentration of saponifiable fatty acids in cow milk curd and soy milk curd

Concentration of saponified fatty acids (g/100 g of curd)

Soy milk curd

Name of the fattyacids

Carbonnumber

Cow milk curd 2% v/vinoculum

2% v/vinoculum

1:1 (v/v)inoculum

1:1:1 (v/v)inoculum

Capric acid 10:0 ND ND ND 1.0Lauric acid 12:0 ND ND ND 0.4Myristic acid 14:0 ND ND 2.0 3.3Palmitic acid 16:0 29.9 11.3 14.0 34.4Palmitoleic acid 16:1 2.2 ND 2.0 4.2Stearic acid 18:0 17.4 67.2 64.0 50.5Oleic acid 18:1 33.2 13.7 27.0 NDLinoleic acid 18:2 2.2 2.8 6.8 NDά linoleicacid (ALA)

18:3 0.74 ND 0.92 ND

γ linoleicacid (GLA)

18:3 ND ND ND 6.2

Eicosapentaenoicacid (EPA)

20:5 ND ND 5.0 ND

Behenic acid 22:0 ND ND 6.9 NDErucic acid 22:1 ND ND 12.5 ND

Note: The experiment was carried out in triplicate (n = 3).ND = not detectable.

142 GHOSH and CHATTOPADHYAY

INDIAN CHEMICAL ENGINEER Vol. 56 No. 2 June 2014

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014

linoleic and linolenic acids in the glyceride fraction [8]. There were significant increase in thelevels of stearic and oleic acids in soy milk curd whereas addition of beet pulp to the soy milkreduced the production of free fatty acids. As a result, soy milk inoculated with 1:1:1 v/v of L.plantarum, L. mesenteroides, and S. thermophilus had very little effect on changes in theconcentration of saponifiable fatty acids, namely, oleic acid, linoleic acid, ALA etc. (Table 2,Fig. 4). The insignificant concentration of SFAs was due to the degradation of fatty acids in

Fig. 4. GLC chromatogram of fatty acid profile of soymilk curd inoculated with 1:1:1 v/v ofL. plantarum, L. mesenteroides and S. thermophilus.

Fig. 2. GLC chromatogram of fatty acid profile of soymilk curd inoculated with 2% (v/v)S. thermophilus.

Fig. 3. GLC chromatogram of fatty acid profile of soymilk curd inoculated with 1:1 v/v ofL. plantarum and S. thermophilus.

Isolation, Analysis and Identification of Fatty Acids 143

INDIAN CHEMICAL ENGINEER Vol. 56 No. 2 June 2014

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014

very little amount by the lactic acid bacteria in the curd samples. The peaks obtained fromGC were not measurable for the concentration of fatty acid or it may be due to thevolatilisation of the short chain fatty acids (especially 14:0, 16:0) present in the milk samplesduring fermentation by the lactic acid bacteria [8, 20].

Comparison of the saponifiable fatty acids between cow milk curd and soy milk curdproduced by using different ratios of lactic acid bacteria is shown in Table 2, which indicatedthat changes of fatty acids might be due to the fermentation by the different ratios of mixedlactic cultures.

ConclusionThe present study indicated that saponified milk fat is rich in oleic acid and it has a very highratio of oleic acid/polyunsaturated fatty acids whereas the saponified soymilk is rich inpalmitic acid and stearic acid. We know that oleic acid is more stable to oxidation than theomega-3 and omega-6 fatty acids and it can partly replace these fatty acids in bothtriacylglycerols and membrane lipids. A high ratio between oleic acid and polyunsaturatedfatty acids will protect lipids, i.e., LDL from the attack of oxidative stressors such as cigarettesmoke, ozone and other oxidants. A diet rich in milk fat, i.e., milk curd may help to increasethis ratio in total dietary saponified fatty acids. From our studies it has also been observedthat soy milk curd contains lower amount of cholesterol as well as total lipid compared tocow milk curd; therefore, soy milk curd is more beneficial for human health.

AcknowledgementsThe authors gratefully acknowledge the financial support of University Grants Commission (UGC),India to carry out the research work. The authors are thankful to Dr (Mrs) Mahuya Ghosh of theDepartment of Applied Chemistry (Oil Technology), University of Calcutta for gas chromatographyanalysis.

References

[1] Insel, P., Turner, R.E. and Ross, D., Nutrition, 2nd edn, American dietetic association, Jones andBartlett, USA (2004).

[2] Haug, A., Hostmark, A.T. and Harstad, O.M., “Bovine Milk in Human Nutrition–A Review”,Lipids Health Dis., 6, pp. 1–34 (2007).

[3] Jensen, R.G. and Newburg, D.S., “Bovine Milk Lipids”, Handbook of Milk Composition, JensenR.G., ed., Academic Press, London (1995).

[4] Ontsouka, C.E., Bruckmaier, R.M. and Blum, J.W., “Fractionized Milk Composition duringRemoval of Colostrum and Mature Milk”, J. Dairy Sci., 86(20), pp. 5–11 (2003).

[5] Grundy, S.M., “Influence of Stearic Acid on Cholesterol Metabolism Relative to Other Long-chain Fatty Acids”, Am. J. Clin. Nutr., 60, pp. 986S–990S (1994).

[6] Eichholzer, M. and Stahelin, H., “Is There a Hypocholesterolemic Factor in Milk and MilkProducts?” Int. J. Vitam. Nutr. Res., 63, pp. 158–167 (1993).

[7] Bosaeus, I., “Milk and Cholesterol”, Vår. Föda., 43, pp. 98–101 (1991).[8] Rao, D.R. and Reddy, J.C., “Effects of Lactic Fermentation of Milk on Milk Lipids”, J. Food Sci.,

49(7), pp. 48–750 (1984).

144 GHOSH and CHATTOPADHYAY

INDIAN CHEMICAL ENGINEER Vol. 56 No. 2 June 2014

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014

[9] Mensink, R.P., Zock, P.L., Kester, A.D.M. and Katan, M.B., “Effects of Dietary Fatty Acids andCarbohydrates on the Ratio of Serum Total to HDL Cholesterol and on Serum Lipids andApolipoproteins: A Meta-analysis of 60 Controlled Trials”, Am. J. Clin. Nutr., 77(11), pp. 46–55 (2003).

[10] Sandstrom, B., Marckmann, P. and Bindslev, N., “An Eight-month Controlled Study of a Low-fatHigh-fibre Diet: Effects on Blood Lipids and Blood Pressure in Healthy Young Subjects”, Eur. J.Clin. Nutr., 46, pp. 95–109 (1992).

[11] Seidel, C., Deufel, T. and Jahreis, G., “Effects of Fat-modified Dairy Products on Blood Lipids inHumans in Comparison with Other Fats”, Ann. Nutr. Metab., 49(4), pp. 2–8 (2005).

[12] German, J.B., “Butyric Acid: A Role in Cancer Prevention”, Nutr. Bull., 24(29), pp. 3–9 (1999).[13] Bandyopadhyay, M., Chakraborty, R. and Raychaudhuri, U., “Effect of Beet and Honey on

Quality Improvement and Carotene Retention in a Carrot Fortified Milk Product”, Inn. Food Sci.Emerg. Technol., 9, pp. 9–17 (2008).

[14] Anon., “Making Soy Milk (Milking the Soy Bean, Part 1)”, (2006).[15] Ghosh, D., Chattoraj, D.K. and Chattopadhyay, P., “Studies on Changes in Microstructure and

Proteolysis in Cow and Soy Milk Curd during Fermentation Using Lactic Cultures for ImprovingProtein Bioavailability”, J. Food Sci. Technol., 50(5), pp. 979–985 (2013).

[16] Paqust, C. and Hautfenne, A., “Standard Methods for the Analysis of Oils, Fats and Derivatives”,7th revised and enlarged edn, Blackwell Scientific Publication Ltd, Oxford, London (1987).

[17] William, W.C., Gas Chromatography and Lipids, Published by Barnes, P.J. & Associates (The OilyPress Ltd), Bridgwater (1989).

[18] Soyeurt, H., Dardenne, P., Dehareng, F., Lognay, G., Veselko, D., Marlier, M., Bertozzi, C.,Mayeres, P. and Gengler, N., “Estimating Fatty Acid Content in Cow Milk Using Mid-InfraredSpectrometry”, J. Dairy Sci., 89, pp. 3690–3695 (2006).

[19] Souza, N.V.D., Coró, F.A.G., Paula, L.N., Dias, L.F., Pedrão, M.R. and Júnior, O.O.S., “FattyAcid Content of Bovine Milkfat From Raw Milk to Yoghurt”, Am. J. Appl. Sci., 9(8), pp. 1300–1306 (2012).

[20] Vuyst, D.E., “Technology Aspects Related to the Application of Functional Starter Cultures”,Food Technol. Biotechnol., 38(1), pp. 5–12 (2000).

Isolation, Analysis and Identification of Fatty Acids 145

INDIAN CHEMICAL ENGINEER Vol. 56 No. 2 June 2014

Dow

nloa

ded

by [

UQ

Lib

rary

] at

08:

14 2

0 N

ovem

ber

2014