Identification of bioactive peptides from a Brazilian kefir sample, and their anti-Alzheimer potential in Drosophila melanogaster

Abstract

Alzheimer's disease (AD) is the most common form of dementia in the elderly, affecting cognitive, intellectual, and motor functions. Different hypotheses explain AD's mechanism, such as the amyloidogenic hypothesis. Moreover, this disease is multifactorial, and several studies have shown that gut dysbiosis and oxidative stress influence its pathogenesis. Knowing that kefir is a probiotic used in therapies to restore dysbiosis and that the bioactive peptides present in it have antioxidant properties, we explored its biotechnological potential as a source of molecules capable of modulating the amyloidogenic pathway and reducing oxidative stress, contributing to the treatment of AD. For that, we used Drosophila melanogaster model for AD (AD-like flies). Identification of bioactive peptides in the kefir sample was made by proteomic and peptidomic analyses, followed by in vitro evaluation of antioxidant and acetylcholinesterase inhibition potential. Flies were treated and their motor performance, brain morphology, and oxidative stress evaluated. Finally, we performed molecular docking between the peptides found and the main pathology-related proteins in the flies. The results showed that the fraction with the higher peptide concentration was positive for the parameters evaluated. In conclusion, these results revealed these kefir peptide-rich fractions have therapeutic potential for AD.

Figure 1

Proteome and peptidome of the kefir sample. (a) Proteomic analysis of the > 10 kDa fraction digested with trypsin and relationship of the proteins. (b) Proteomic analysis of the < 10 kDa fraction digested with trypsin and relationship of the proteins. (c) Peptidomic analysis of the < 10 kDa non-digested fraction and the quantity of peptides found that are derived from milk. Parts of the figure were drawn by using pictures from Servier Medical Art (https://smart.servier.com/).

Figure 2

In vitro analysis of the effects of kefir fractions. (a) Total antioxidant capacity by the FRAP method of Fe³⁺ reduction analysis. The < 10 kDa fraction shows higher antioxidant activity compared to the others (*p < 0.5), but not as high as ascorbic acid used as the control. (b) Acetylcholinesterase enzyme inhibition capacity. Among the kefir fractions, the < 10 kDa fraction stands out as having the highest inhibition capacity (*indicates p < 0.5).

Figure 3

Validation of the Alzheimer’s disease model. (a) Climbing ability. AD-like flies show reduced motor ability compared to the control genotype at 10–13 days post eclosion (n = 90 in each genotype). (b) Quantification of amyloid by the Thioflavin T method. AD-like flies showed a higher amount of amyloid compared to the control at 10–13 days post eclosion (n = 30 for each genotype). (c) Neurodegeneration index of elav and AD-like flies based on the histopathological analysis, focusing on vacuolar lesions. Indices range from 0 to 5 with 0 indicating no lesions and 5 indicating a neurodegenerative phenotype. Data are presented as mean ± SEM, and significance values are represented as **p < 0.0001, *p < 0.001, **p < 0.01. Representative histopathological images of the elav (d) and AD-like (e) genotypes.

Figure 4

In vivo effects of kefir fractions. (a) The climbing ability of AD-like flies after 5 and 10 days of treatment with the fractions at a concentration 0.25 mg/mL, flies treated with WSF and the < 10 kDa fraction showed an improvement in motor performance (n = 90). (b) The climbing ability of AD-like flies after 5 and 10 days of treatment with the fractions at a concentration 0.5 mg/mL (n = 90). (c) Quantification of amyloid content by the Thioflavin T assay. Flies treated with all fractions at both concentrations showed a reduction in the amyloid content compared to the control (untreated) at 10 days of treatment (n = 30 for each treatment). (d) Index of neurodegeneration based on the histopathological analysis according to vacuolar lesions (n = 10 at 10 days of treatment). The results show a decrease in the index in all treated flies compared to the control. (e) Fe³⁺ reduction capacity by the FRAP method. Flies treated with the fractions show reduced antioxidant activity (n = 30 and 10 days of treatment). (f) Acetylcholinesterase activity. Only flies treated with the < 10 kDa fraction at 0.25 mg/mL demonstrated decreased acetylcholinesterase activity. Data are presented as mean + SEM. Statistically significant differences are indicated by *p < 0.5, **p < 0.1, ***p < 0.01 and ****p < 0.001.

Figure 5

Molecular docking analysis of the VPPFLQPEV and VYPFPGPIPN peptides and their interaction with BACE1, Aβ and acetylcholinesterase. (a) Prediction of the interaction of the VPPFLQPEV peptide with the enzyme BACE1. (b) Zoomed in image of panel (a). (c,d) Prediction of the interaction of the VYPFPGPIPN peptide with β-amyloid plaques between the first and second β-strand regions. (e) Hydrophobic region of interaction of the VYPFPGPIPN peptide with β-amyloid plaque. (f) Prediction of the interaction of the VYPFPGPIPN peptide in the peripheral anionic site of the acetylcholinesterase enzyme. (g) Zoomed in image of panel (f).