SpringerLink
Log in

Extraction and Purification of Short-chain Fatty Acids from Fermented Reconstituted Skim Milk Supplemented with Inulin

  • Published:
Food Analytical Methods Aims and scope Submit manuscript

Abstract

Short-chain fatty acids (SCFAs) acetate, propionate, butyrate and lactate were determined in 12 % reconstituted skim milk (RSM) and RSM supplemented with inulin (RSMI). The fermentation was performed with Bifidobacterium animalis subsp. lactis (BB 12) and Lactobacillus rhamnosus LGG ATCC 53013. Fermentation culture activities produced substantial amounts of SCFAs, which were detected and quantitated using a HPLC-UV technique. Using HPLC-UV, we were able to detect low concentrations of lactate and SCFAs from fermented samples; lactate, acetate, propionate and butyrate were detected at 10.10, 12.06, 14.80 and 18.06 μg/mL, respectively. The retention time of all SCFAs and lactic acid were similar to the standard quality control (±0.05), and average recovery ranged between 89.73 and 91.03 %. The experimental conditions and sample preparation were applied to preparative HPLC to isolate and purify SCFAs with concentration range between 0.09 and 2.86 mg/mL. The purity of extracted SCFAs was confirmed using atmospheric pressure chemical ionization/mass spectrometry by determining the molecular masses of target purified compounds. The scaled up validated analytical HPLC-UV method will further enhance and improve the use of this approach to produce purified large-scale SCFAs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Adams MR, Hall CJ (1988) Growth inhibition of food-borne pathogens by lactic and acetic acids and their mixtures. Int J Food Sci Technol 23:287–292. doi:10.1111/j.1365-2621.1988.tb00581.x

    Article  CAS  Google Scholar 

  • Asarat M, Apostolopoulos V, Vasiljevic T, Donkor O (2015a) Short-chain fatty acids produced by synbiotic mixtures in skim milk differentially regulate proliferation and cytokine production in peripheral blood mononuclear cells Int J Food Sci Nutr 66:755–765

  • Asarat M, Vasiljevic T, Apostolopoulos V, Donkor O (2015b) Short-Chain Fatty Acids Regulate Secretion of IL-8 from Human Intestinal Epithelial Cell Lines in vitro Immunological investigations 44:678–693

  • Cherrington CA, Hinton M, Mead GC, Chopra I (1991) Organic acids: chemistry, antibacterial activity and practical applications. In: Rose AH, Tempest DW (eds) Advances in microbial physiology, vol Volume 32. Academic Press, pp 87–108. doi:http://dx.doi.org/10.1016/S0065-2911(08)60006-5

  • Collomb M, Bütikofer U, Sieber R, Jeangros B, Bosset J-O (2002) Composition of fatty acids in cow’s milk fat produced in the lowlands, mountains and highlands of Switzerland using high-resolution gas chromatography. Int Dairy J 12:649–659

    Article  CAS  Google Scholar 

  • Cummings JH, Macfarlane GT (1991) The control and consequences of bacterial fermentation in the human colon. J Appl Microbiol 70:443–459. doi:10.1111/j.1365-2672.1991.tb02739.x

    CAS  Google Scholar 

  • Currie LA (1999) Nomenclature in evaluation of analytical methods including detection and quantification capabilities: (IUPAC Recommendations 1995). Anal Chim Acta 391:105–126, http://dx.doi.org/10.1016/S0003-2670(99)00104-X

    Article  CAS  Google Scholar 

  • De Baere S, Eeckhaut V, Steppe M, De Maesschalck C, De Backer P, Van Immerseel F, Croubels S (2013) Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation. J Pharm Biomed Anal 80:107–115. doi:10.1016/j.jpba.2013.02.032

    Article  Google Scholar 

  • De Sá LRV, De Oliveira MAL, Cammarota MC, Matos A, Ferreira-Leitão VS (2011) Simultaneous analysis of carbohydrates and volatile fatty acids by HPLC for monitoring fermentative biohydrogen production. Int J Hydrog Energy 36:15177–15186. doi:10.1016/j.ijhydene.2011.08.056

    Article  Google Scholar 

  • Di Stefano V et al (2012) Applications of liquid chromatography–mass spectrometry for food analysis. J Chromatogr A 1259:74–85. doi:10.1016/j.chroma.2012.04.023

    Article  Google Scholar 

  • Donkor ON, Nilmini SLI, Stolic P, Vasiljevic T, Shah NP (2007) Survival and activity of selected probiotic organisms in set-type yoghurt during cold storage. Int Dairy J 17:657–665. doi:10.1016/j.idairyj.2006.08.006

    Article  CAS  Google Scholar 

  • Eeckhaut V et al (2011) Butyrate production in phylogenetically diverse Firmicutes isolated from the chicken caecum. Microb Biotechnol 4:503–512. doi:10.1111/j.1751-7915.2010.00244.x

    Article  CAS  Google Scholar 

  • Ewaschuk JB, Zello GA, Naylor JM, Brocks DR (2002) Metabolic acidosis: separation methods and biological relevance of organic acids and lactic acid enantiomers. J Chromatogr B 781:39–56. doi:10.1016/s1570-0232(02)00500-7

    Article  CAS  Google Scholar 

  • FDA U (2001) Guidance for industry: bioanalytical method validation Rockville, MD: CDER

  • Guerrant GO, Lambert MA, Moss CW (1982) Analysis of short-chain acids from anaerobic bacteria by high-performance liquid chromatography. J Clin Microbiol 16:355–360

    CAS  Google Scholar 

  • Helale MI, Tanaka K, Taoda H, Hu W, Hasebe K, Haddad PR (2002) Qualitative analysis of some carboxylic acids by ion-exclusion chromatography with atmospheric pressure chemical ionization mass spectrometric detection. J Chromatogr A 956:201–208

    Article  Google Scholar 

  • Hughes SA, Shewry PR, Gibson GR, McCleary BV, Rastall RA (2008) In vitro fermentation of oat and barley derived beta-glucans by human faecal microbiota. FEMS Microbiol Ecol 64:482–493

    Article  CAS  Google Scholar 

  • Jham GN, Fernandes SA, Garcia CF, da Silva AA (2002) Comparison of GC and HPLC for the quantification of organic acids in coffee. Phytochem Anal 13:99–104

    Article  CAS  Google Scholar 

  • Lima ES, Abdalla DSP (2002) High-performance liquid chromatography of fatty acids in biological samples. Anal Chim Acta 465:81–91. doi:10.1016/s0003-2670(02)00206-4

    Article  CAS  Google Scholar 

  • Moreau NM et al (2004) Rapid measurement of 13C-enrichment of acetic, propionic and butyric acids in plasma with solid phase microextraction coupled to gas chromatography–mass spectrometry. Anal Chim Acta 512:305–310. doi:10.1016/j.aca.2004.02.054

    Article  CAS  Google Scholar 

  • Niven SJ, Beal JD, Brooks PH (2004) The simultaneous determination of short chain fatty acid, monosaccharides and ethanol in fermented liquid pig diets. Anim Feed Sci Technol 117:339–345, http://dx.doi.org/10.1016/j.anifeedsci.2004.09.003

    Article  CAS  Google Scholar 

  • Pan X-d, Chen F-q, Wu T-x, Tang H-g, Zhao Z-y (2009) Prebiotic oligosaccharides change the concentrations of short-chain fatty acids and the microbial population of mouse bowel. J Zhejiang Univ Sci B 10:258–263. doi:10.1631/jzus.B0820261

    Article  CAS  Google Scholar 

  • Roy CC, Kien CL, Bouthillier L, Levy E (2006) Short-chain fatty acids: ready for prime time? Nutr Clin Pract 21:351–366. doi:10.1177/0115426506021004351

    Article  Google Scholar 

  • Rutkowska J, Adamska A, Bialek M (2012) Fatty acid profile of the milk of cows reared in the mountain region of Poland. J Dairy Res 79:469–476

    Article  CAS  Google Scholar 

  • Shin HS, Lee JH, Pestka JJ, Ustunol Z (2000) Growth and viability of commercial Bifidobacterium spp in skim milk containing oligosaccharides and inulin. J Food Sci 65:884–887. doi:10.1111/j.1365-2621.2000.tb13605.x

    Article  CAS  Google Scholar 

  • Stein J, Kulemeier J, Lembcke B, Caspary WF (1992) Simple and rapid method for determination of short-chain fatty acids in biological materials by high-performance liquid chromatography with ultraviolet detection. J Chromatogr B Biomed Sci Appl 576:53–61, http://dx.doi.org/10.1016/0378-4347(92)80174-O

    Article  CAS  Google Scholar 

  • Tan L, Ju H, Li J (2006) Extraction and determination of short-chain fatty acids in biological samples. Se Pu 24:81–87

    CAS  Google Scholar 

  • Thomson BA (1998) Atmospheric pressure ionization and liquid chromatography/mass spectrometry—together at last. J Am Soc Mass Spectrom 9:187–193. doi:10.1016/s1044-0305(97)00285-7

    Article  CAS  Google Scholar 

  • Top** DL, Clifton PM (2001) Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol Rev 81:1031–1064

    CAS  Google Scholar 

  • Torii T, Kanemitsu K, Wada T, Itoh S, Kinugawa K, Hagiwara A (2010) Measurement of short-chain fatty acids in human faeces using high-performance liquid chromatography: Specimen stability. Ann Clin Biochem 47:447–452

    Article  CAS  Google Scholar 

  • Trufelli H, Palma P, Famiglini G, Cappiello A (2011) An overview of matrix effects in liquid chromatography–mass spectrometry. Mass Spectrom Rev 30:491–509. doi:10.1002/mas.20298

    Article  CAS  Google Scholar 

  • Wong JMW, de Souza R, Kendall CWC, Emam A, Jenkins DJA (2006) Colonic health: fermentation and short chain fatty acids. J Clin Gastroenterol 40:235–243

    Article  CAS  Google Scholar 

  • Zhao J, Cheung PCK (2011) Fermentation of β-glucans derived from different sources by Bifidobacteria: evaluation of their bifidogenic effect. J Agric Food Chem 59:5986–5992. doi:10.1021/jf200621y

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge Victoria University, Australia, for their financial and technical support.

Author information

Authors and Affiliations

  1. Advanced Food Systems Research Unit, College of Health and Biomedicine, Victoria University, Werribee Campus, P.O. Box 14428, Melbourne, VIC, 3030, Australia

    M. Asarat, T. Vasiljevic, M. Ravikumar & O. Donkor

  2. Faculty of Veterinary Medicine, Tripoli University, P.O. Box 13662, Tripoli, Libya

    M. Asarat

  3. Centre for Chronic Disease, Victoria University, St Albans, VIC, 3021, Australia

    V. Apostolopoulos

Authors
  1. M. Asarat
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T. Vasiljevic
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Ravikumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. V. Apostolopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. O. Donkor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Asarat.

Ethics declarations

Human and Animal Rights and Informed Consent

This article does not contain any studies with animal or human subjects.

Conflict of Interest

Authors declare that they have no conflict of interest.

Informed Consent

Not applicable.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Asarat, M., Vasiljevic, T., Ravikumar, M. et al. Extraction and Purification of Short-chain Fatty Acids from Fermented Reconstituted Skim Milk Supplemented with Inulin. Food Anal. Methods 9, 3069–3079 (2016). https://doi.org/10.1007/s12161-016-0471-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12161-016-0471-0

Keywords

Search

Navigation