A Mechanistic Study of the Antiaging Effect of Raw-Milk Cheese Extracts

Abstract

Many studies have highlighted the relationship between food and health status, with the aim of improving both disease prevention and life expectancy. Among the different food groups, fermented foods a have huge microbial biodiversity, making them an interesting source of metabolites that could exhibit health benefits. Our previous study highlighted the capacity of raw goat milk cheese, and some of the extracts recovered by the means of chemical fractionation, to increase the longevity of the nematode Caenorhabditis elegans. In this article, we pursued the investigation with a view toward understanding the biological mechanisms involved in this phenomenon. Using mutant nematode strains, we evaluated the implication of the insulin-like DAF-2/DAF-16 and the p38 MAPK pathways in the phenomenon of increased longevity and oxidative-stress resistance mechanisms. Our results demonstrated that freeze-dried raw goat milk cheese, and its extracts, induced the activation of the DAF-2/DAF-16 pathway, increasing longevity. Concerning oxidative-stress resistance, all the extracts increased the survival of the worms, but no evidence of the implication of both of the pathways was highlighted, except for the cheese-lipid extract that did seem to require both pathways to improve the survival rate. Simultaneously, the cheese-lipid extract and the dried extract W70, obtained with water, were able to reduce the reactive oxygen species (ROS) production in human leukocytes. This result is in good correlation with the results obtained with the nematode.

Keywords: Caenorhabditis elegans; DAF-16; longevity; oxidative stress; p38 MAPK; raw-milk cheese.

Figure 1

Preparation of the milk extracts. Ovals designate the milk extracts used for our biological studies (FDM (freeze-dried milk), milk lipid extract, LFM (lipid-free milk), and extracts MA (obtained with dichloromethane), MB (obtained with ethyl acetate), MC (obtained with absolute ethanol) and RSM (residual solid milk)).

Figure 2

Influence of the FDC (freeze-dried cheese) (A) and the cheese-lipid extract (B) on the lifespan of C. elegans GR1307 strain. The worms were incubated on the medium supplemented with the dried extracts at day 0 and regularly fed with HK E. coli OP50. The conditions were considered significantly different when the p-value was lower than 0.05 (*) or 0.01 (**) (log-rank test). The asterisks next to the curves represent the differences with the control condition CC2. The asterisks next to the legend represent the differences between the extracts.

Figure 3

The influence of the aqueous extracts WF (A), W40 (B) and W70 (C) on the C. elegans GR1307 strain lifespan. The worms were incubated on the medium supplemented with the dried extracts at day 0 and regularly fed with HK E. coli OP50. The conditions were considered significantly different when the p-value was lower than 0.001 (***) or 0.0001 (****) (log-rank test). The asterisks next to the curves represent the differences with the control condition CC1. The asterisks next to the legend represent the differences between the extracts.

Figure 4

Comparison of the effect of the aqueous extracts WF, W40 and W70 at 0.5% (A) and 1% (B) concentration on the C. elegans GR1307 strain lifespan. The worms were incubated on the medium supplemented with the dried extracts obtained with water at day 0 and regularly fed with HK E. coli OP50. The conditions were considered significantly different when the p-value was lower than 0.001 (***) or 0.0001 (****) (log-rank test). The asterisks next to the curves represent the differences with the control condition CC1. The asterisks next to the legend represent the differences between the extracts.

Figure 5

The effects of the FDC (freeze-dried cheese) and the extracts on DAF-16 cellular localization in C. elegans transgenic strain TJ356 expressing DAF-16::GFP, after 2 h and 4 h of incubation on the supplemented medium. The arrows indicate the accumulation of the transcription factor in the nuclei. Scale bar: 100 µm.

Figure 6

Relative survival rates of the wild-type C. elegans N2 strain on an oxidative medium after 5 days of incubation on a medium supplemented with FDC (freeze-dried cheese) and cheese-lipid extract (A) or aqueous extracts WF, W40 and W70 (B). The conditions were considered significantly different from the control when the p-value was lower than 0.05 (*), 0.01 (**), 0.001 (***) (Kruskal–Wallis test).

Figure 7

The relative survival rates of the C. elegans GR1307 strain (DAF-16 loss-of-function) (A,B) and IG10 strain (TOL-1 loss-of-function) (C,D) on an oxidative medium after 5 days of incubation on a medium supplemented with the cheese extracts (FDC (freeze-dried cheese), cheese-lipid extract and aqueous extracts WF, W40 and W70). The conditions were considered significantly different from the control when the p-value was lower than 0.05 (*), 0.01 (**), 0.001 (***) or 0.0001 (****) (Kruskal–Wallis test).

Figure 8

The representation of the insulin-like pathway and the p38 MAPK pathway studied in this article. The following genes were tested: daf-16 (studied with a mutant and a transcriptomic analysis), tol-1 (studied with a mutant), and pmk-1 and sek-1 (studied with a transcriptomic analysis).

Figure 9

The effect of the cheese-lipid extract, WF, W40 and W70 on the ROS production in the leukocytes (A) and the viability of the leukocytes (B). The cells were treated with the indicated concentrations of the extract for 2 h, and measurements were made every 30 min. Data were expressed as relative production or viability in comparison with the control which was fixed at 100%. The conditions were considered significantly different from the control when the p-value was lower than 0.05 (*), 0.01 (**) (Kruskal–Wallis test).

Figure 10

The influence of FDM (freeze-dried milk) (A), milk lipid extract (B), extracts MA (obtained with dichloromethane) (C), MB (obtained with ethyl acetate) (D), MC (obtained with ethanol) (E) and RSM (residual solid milk) (F) on the lifespan of wild-type C. elegans N2 strain. The worms were incubated on the medium supplemented with the dried extracts at day 0 and regularly fed with HK E. coli OP50. The conditions were considered significantly different when the p-value was lower than 0.05 (*) or 0.0001 (****). The asterisks next to the curves represent the differences with the control condition CC1. The asterisks next to the legend represent the differences between the extracts (log-rank test).

Figure 11

The influence of the LFM (lipid-free milk) on the lifespan of the wild-type C. elegans N2 strain. The worms were incubated on the medium supplemented with the dried extract at day 0 and regularly fed with HK E. coli OP50. The conditions were considered significantly different from the control conditions CC2 when the p-value was lower than 0.0001 (****) (log-rank test).

Figure 12

The relative survival rates of the wild-type C. elegans N2 strain on an oxidative medium after 5 days of incubation on a medium supplemented with FDM (freeze-dried milk), milk lipid extract, extract MA (obtained with dichloromethane), MC (obtained with ethanol) and RSM (residual solid milk) (A) or LFM (lipid-free milk) (B). The conditions were considered significantly different from the control when the p-value was lower than 0.05 (*) or 0.001 (***) (Kruskal–Wallis test).