Kefir Brasil

[Anjali Reyome]

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The t-test analysis showed that both fractions demonstrated a significant effect when administrated at 0.25 and 0.5 mg/mL. Flies treated with these samples showed a lower amyloid content (Fig. 4c) compared to control, neurodegeneration index (Fig. 4d) and antioxidant activity (Fig. 4e) when compared with untreated flies.

The peptide VPPFLQPEV was predicted as the best ligand for BACE. This peptide binds to an important flap that controls enzyme activity by partially covering the substrate-binding cleft localized between the N- and C-terminus lobes (Fig. 5a,b).

The peptide VYPFPGPIPN was also predicted as the best ligand for human AChE. This enzyme has two binding sites: one is a peripheral anionic site (PAS) located at the entrance of the active gorge and the other is a catalytically active site (CAS) located at the base of the active site gorge. The peptide bound to PAS (Fig. 5f,g).

In the present work, we focused on the peptidome of the kefir and in the intact bioactive peptides. The fermentation-generated peptides were identified through a proteomic analysis. Similar results have been described by other research groups30,31, which supports the compositional consistency of kefir despite some variations across samples.

As we used cell-free protein to produce the proteomic profile, the peptidases found using databases from Lactobacillus and Acetobacter could be from cells disrupted (intracellular) by fermentation or by a novel secretory protein degradation system32. Further assays are needed to resolve this question. In previous work, only these two bacteria genera were found in our kefir sample13.

Our in silico analysis indicates that the major part of the peptides generated from our kefir sample during fermentation display several activities, the characteristics of which have been described in other milk-derived peptides33,34. Here, we focused on putative acetylcholinesterase inhibition and antioxidant properties as well BACE inhibition and putative amyloid fibril binding.

These characteristics are especially interesting for AD. Since AD is a multifactorial disease35, researchers have turned their attention to developing multi-target drugs to inhibit the myriad factors involved in AD, including protein misfolding and associated Aβ aggregation, t aggregation, metal dyshomeostasis, oxidative stress and a decrease in AChE levels. Even though the proposed model is based on the amyloidogenic hypothesis, we should expect alterations in other metabolic processes. Previous studies have shown a relationship between amyloid aggregation and both oxidative stress36 and acetylcholinesterase activity37 during AD progression.

Oxidative stress is related to the neuropathological manifestations of AD and implies an increased level of reactive oxygen species (ROS)38,39 and the abnormal homeostasis of bioactive metals40. Otherwise, acetylcholinesterase catalyzes acetylcholine conversion, which is related to the cholinergic cascade and cholinergic neuron loss in AD pathology41.

In an effort to target those processes, antioxidant compounds (e.g. resveratrol) have shown a role in AD prevention42 or as a supportive treatment43. Furthermore, many AD drugs inhibit acetylcholinesterase activity, aiming to increase acetylcholine levels in the brain44. However, these drugs have a limited effect and generate collateral effects45.

Therefore, with a positive prediction from the in silico investigation, we screened the effect of the kefir fractions in vitro. Both antioxidant and anti-acetylcholinesterase properties were confirmed using the FRAP and acetylcholinesterase inhibition assays. To the best of our knowledge, this is the first work to report the anti-acetylcholinesterase activity of a kefir-produced molecule.

In order to verify the in vivo effects of peptides from kefir, we used the D. melanogaster AD-like model. For that, we first provided a confirmation of the model, in which human BACE and APP are overexpressed in fly neurons by the pan-neural driver elav-Gal4.

The BACE1 molecule is formed of three portions: an extracellular N-terminal domain, a transmembrane domain and a C-terminal cytosolic domain. Its cleavage site is between the N- and C-terminal regions, characterized by an aspartate catalytic dyad (Asp32 and Asp228, highlighted in yellow in Fig. 5a,b)51. Near this region, there is a flexible flap (highlighted in green in Fig. 5a,b), perpendicular to the active site. This flexible flap can exist in either an open or closed conformation, and in that way help or hinder the access of a molecule to the enzyme active site52,53. Between the flap and the active site, there is also a space in the BACE1 structure that can be reached by a molecule so that it can access the catalytic dyad54.

By analyzing the 3D structure of BACE1 and the peptide VPPFLQPEV (Table 1 docking of smaller global energy value, Fig. 5a,b), it was possible to verify that it did not interact with the BACE1 active site. Despite this, the peptide interacted with ALCA, and was able to alter its conformation, strategically located between the large N- and C-terminal portions of BACE1. This peptide position could block the access of other molecules (substrates) to the enzyme active site, thereby inhibiting its action. As a consequence, BACE1 could lose its capacity to cleave APP, consequently reducing Aβ peptide production and amyloid plaque accumulation. In this way, it could be possible to decrease the progression of neurodegeneration through the amyloidogenic pathway.

The amyloidogenic pathway results in the production of Aβ peptides of distinct sizes, depending on the γ-secretase cleavage region. Amongst those, the 42 amino acid peptide (Aβ42) is the most neurotoxic55,56. Based on that, we also evaluated molecular docking between the bioactive peptide VYPFPGPIPN and amyloid plaques generated by the Aβ42 peptide.

The present study extends the existing literature by providing evidence that peptides derived from cow milk kefir can modulate the AD phenotype in AD-like flies by decreasing the relative β-amyloid level in the brain. Consequently, this intervention decreases neuronal tissue damage, improves motor ability and decreases acetylcholinesterase activity. In summary, our study was able to identify bioactive peptides present in kefir (

Kefir grains were obtained as a donation from the local population in Uberlndia, Brazil, inoculated (4% w/v) in UHT (ultra-high temperature) whole cow milk for fermentation process and left for 24 h at room temperature in a glass container. A genetic fingerprint was previously published by our group13. The fermented product was collected by removing the grains by filtration, followed by centrifugation at 4 C, 4900g for 10 min. The resulting supernatant went through a series of filtering processes, where we obtained the three fractions that were further tested.

ToxinPred ( ) was used to calculate the physicochemical characteristics of each characterized peptide and the expected toxicity level, while the Peptide Property Calculator from Innovagen ( -tools) was used to analyze solubility. For the bioactivity prediction, PeptideRanker ( ) was used. Finally, the Milk Bioactive Peptide Database ( ) was used to determine the origin of peptides from milk protein and their putative biological functions. The peptides with a rank above 0.5 were considered as having potential bioactivity.

Total antioxidant capacity was evaluated by FRAP (ferric reducing antioxidant power) assay20. This method consists of evaluating the capacity if a compound reduction Fe3+ to Fe2+ Kefir fractions (WSF and both Amicon samples) were solubilized in distilled water at 500 g/mL. Ascorbic acid was used at the same concentration as the positive control and sodium acetate buffer was used as the blank.

The solutions were prepared in the following way: the enzyme was diluted to 0.2 U/mL in buffer I, DTNB 0.1% was diluted in buffer III, acetylcholine iodide solution (substrate) 0.4% v/v was diluted in Milli-Q water, and the inhibitor Galantamine (positive control) diluted in Milli-Q water.

For the assay, 25 L of each sample were added to 125 L of DTNB solution, 50 L of buffer II, 25 L of acetylcholine iodide solution and 25 L of acetylcholinesterase solution. Using a spectrophotometer, the absorbance was measured at 405 nm for 20 min at 30 C. Acetylcholinesterase inhibition was calculated using the following equation:

To evaluate the total antioxidant activity for each treatment, the modified FRAP method was used. Ten fly heads (in triplicate) after 10 days of treatment were homogenized with PBS and centrifuged (2 min, 1000g at 4 C). For the assay, 10 L of each supernatant was mixed with 50 L of FRAP reagent 1:1 in distilled water. Antioxidant activity was evaluated as previously described for the in vitro analysis.

Putative bioactive components were used for docking analysis. The 3D peptide structures were created using PEP-FOLD 2.0. The Protein Data Bank (PDB) files for BACE (3TPJ), acetylcholinesterase (AChE-3LII) and 42-Residue Beta-Amyloid Fibril (2MXU), were retrieved from PDB. The docking between the peptides and these enzymes was performed using PathDocking. The best model was chosen based on both global energy and atomic contact energy contribution to the global binding energy.

The authors are thankful to the Institute of Cell Biology for providing access to the Light Microscope and Software LAS EZ and to the Pathology Laboratory of the Faculty of Odontology of the Federal University of Uberlndia for the microscope sections. We are grateful for the technical and material support of Professor Dr. Luiz Ricardo Goulart, from the Biotechnology Institute of the University of Uberlndia, in memoriam.

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