Protein hydrolysate derived from blue-backed fish

and Methods

[0259]1.1. Cell Cultures

[0260]BV2:

[0261]BV2 cells are derived from a neonatal murine microglial cell line immortalized by the raf / mycet system, provided by Dr. Watterson (NorthWestern University, USA). They were grown in complete medium containing RPMI-Glutamax with 2 mM glutamine (Invitrogen, Life Technologies, France) supplemented with 10% inactivated fetal calf serum and 1% penicillin (100 U / mL)-streptomycin (100 μg / mL; Sigma-Aldrich, France) under a humid atmosphere with 5% CO2 at 37° C. as described by De Smedt-Peyrusse et al. (8).

[0262]When the cells had reached 80% to 90% confluence, they were treated for 24 hours with:[0263]DHA at the concentration of 16μM (Sigma-Aldrich, France). This concentration of DHA has been validated in the laboratory as an effective dose to demonstrate an anti-inflammatory effect.[0264]the hydrolysate according to the invention providing 16μM of DHA.

[0265]The cells were then treated for 2 h, 6 h or 24 h with 1 μg / mL of LPS (lipopolys

Effect of the Hydrolysate

[0355]2.1. Effect of H1 Hydrolysate on Neuroinflammation of BV2 Microglial Cells

[0356]Microglial cells are the immunocompetent cells of the brain, responsible for the production of cytokines. Inflammatory stress was induced in these cells (BV2 cells) by liposaccharide (LPS) using the protocol described in Example 1.1. The H1 protein hydrolysate was then tested for its ability to decrease the expression of the pro-inflammatory cytokines IL-6 (interleukin 6), IL-1β (interleukin 1beta) and TNF-α (tumor necrosis factor alpha), in these cells, according to the protocol described in Example 1.4.5.

[0357]The results show that in the control condition, LPS induces inflammatory stress with high expression of IL-6, IL-1β and TNF-α. DHA, which has anti-inflammatory capabilities, reduces LPS-induced IL-6 and IL-1β expression. Surprisingly, treatment with H1 hydrolysate decreases LPS-induced IL-6 and IL-1 expression with a greater effect than DHA (FIGS. 1a and 1b). The H1 hy

ffect of the Hydrolysate

[0368]For these in vivo tests, the animals were reared, fed and divided into different groups according to the protocol described in Example 1.2.

[0369]3.1. Effect of H2 Hydrolysate on Anxiety-Like Behaviour

[0370]The stress response involves the hypothalamic-pituitary-adrenal axis (HPA axis). In the presence of stress, the brain secretes cortisol in humans and corticosterone in mice, stress hormones that influence the immune system and allow a return to homeostasis. Once this return to homeostasis has been achieved, cortisol acts by negative feedback on the brain to reduce its own secretion.

[0371]In people suffering from anxiety, which accounts for 10% of the elderly, this axis is disrupted, leading to damage to the brain, particularly the hippocampus.

[0372]The effects of protein hydrolysate supplementation according to the invention on anxiety-like behaviour were studied by means of the so-called open-field (OF) test. The protocol of this test is described in Ex