Development of an innovative methodology combining chemical fractionation and in vivo analysis to investigate the biological properties of cheese

Abstract

With the ever-increasing human lifespan, age-related affections have become a public health issue. The health sector is looking for new bioactive compounds to respond to this demand. The unexplored microbial biodiversity and its metabolites represent a major source of innovative bioactive molecules with health potential. Fermented foods, such as raw-milk cheese, have already been investigated for their rich microbial environment, especially for their organoleptic qualities. But studies remain limited regarding their effects on health and few metabolites of microbial origin have been identified. An efficient methodology was developed in this study to investigate the biological effect of raw-milk cheese, combining a chemical fractionation, to isolate the most metabolites from the cheese matrix, and an in vivo biological test using Caenorhabditis elegans. C. elegans was brought into contact with cheese extracts, obtained by means of chemical fractionation, and with freeze-dried whole cheese by supplementing the nematode growth medium. A longevity assay was performed to evaluate the effects of the extracts on the worms. Our results demonstrate the feasibility of the method developed to bring the worms into contact of the cheese extracts. The evaluation of the effects of the extracts on the longevity was possible. Some extracts showed a beneficial effect as extract W70 for example, obtained with water, which increases the mean lifespan by 16% and extends the longevity by 73% (p < 0.0001).

Fig 1. Preparation of cheese fractions.

Rings indicate the cheese extracts used for biological tests (FDC, lipid fraction, fractions A, B, C, WF, W40, W70 and RSC).

Fig 2. Influence of amphotericin B on C. elegans wild-type N2 strain lifespan.

Comparison between CC1 and CC2. Worms were incubated on the medium (with or without antifungal) at day 0 and fed every three days with HK OP50. The asterisks indicate the p-values (log-rank test) (“*”: p <0.05; “**”: p <0.01; “***”: p <0.001; “****”: p <0.0001).

Fig 3. Influence of FDC and lipid fractions on C. elegans wild-type N2 strain lifespan.

Worms were incubated on fractions at day 0 and fed every three days with HK OP50. The asterisks indicate the p-values (log-rank test) (“*”: p <0.05; “**”: p <0.01; “***”: p <0.001; “****”: p <0.0001). The asterisks around the curves are the comparisons with the control conditions. The asterisks around the legend are the comparisons between the fractions.

Fig 4. Influence of fractions E, WF, W40, W70 and RSC on C. elegans wild-type N2 strain lifespan.

Worms were incubated on fractions at day 0 and fed every three days with OP50 HK. The asterisks indicate the p-values (log-rank test) (“*”: p <0.05; “**”: p <0.01; “***”: p <0.001; “****”: p <0.0001). The asterisks around the curves are the comparisons with the control conditions. The asterisks around the legend are the comparisons between the fractions.

Fig 5. Comparison of the effect of aqueous fractions WF, W40 and W70 on C. elegans wild-type N2 strain lifespan.

Worms were incubated on aqueous fractions at day 0 and fed every three days with OP50 HK. The asterisks indicate the p-values (log-rank test) (“*”: p <0.05; “**”: p <0.01; “***”: p <0.001; “****”: p <0.0001). The asterisks around the curves are the comparisons with the control conditions. The asterisks around the legend are the comparisons between the fractions.