Identification of a Novel Peptide from β-Casein That Enhances

Jun 26, 2019 - products prevent cognitive decline and support previously reported observations. KEYWORDS: casein ..... neuronal network is associated ...
0 downloads 0 Views 1MB Size
Download PDF
Article Cite This: J. Agric. Food Chem. 2019, 67, 8160−8167

pubs.acs.org/JAFC

Identification of a Novel Peptide from β‑Casein That Enhances Spatial and Object Recognition Memory in Mice Yasuhisa Ano,*,† Toshiko Kutsukake,† Toshinori Sasaki,† Shinichi Uchida,‡ Koji Yamada,‡ and Keiji Kondo† †

Research Laboratories for Health Science & Food Technologies, Kirin Holdings Co. Ltd., Yokohama, Japan Central Nervous System Research Laboratories, CNS R&D Unit, R&D Division, Kyowa Hakko Kirin Co. Ltd., Shizuoka, Japan

‡

Downloaded via BUFFALO STATE on July 27, 2019 at 06:27:01 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: An increase in the aging population has spurred recent efforts to identify diet and lifestyle changes that help prevent cognitive decline. Several epidemiological investigations and clinical studies have indicated that consuming fermented dairy products prevents cognitive decline. Some peptides from whey including β-lactolin improve memory impairment; the intake of Camembert cheese has been shown to prevent Alzheimer’s in mouse models. To elucidate the molecular mechanisms underlying these preventive effects, we screened peptides from digested casein protein for their ability to improve spatial memory in a scopolamine-induced amnesia mouse model. Administration of KEMPFPKYPVEP peptide from β-casein at 0.5 mg/kg (54.8 ± 2.5) and 2 mg/kg (57.9 ± 3.7) improved memory impairment in the amnesia mice in comparison with control (44.9 ± 3.4; p = 0.031 and p = 0.042, respectively) and increased dopamine (5.9 ± 3.8 [control] and 12.4 ± 6.2 [KEMPFPKYPVEP peptide]) and norepinephrine (7.7 ± 0.8 [control] and 9.9 ± 2.0 [KEMPFPKYPVEP peptide]) levels in the frontal cortex (p = 0.039 and p = 0.031, respectively). Collectively, our findings suggest that peptides in fermented dairy products prevent cognitive decline and support previously reported observations. KEYWORDS: casein, dairy product, dopamine, memory, peptide

■

INTRODUCTION A rapid growth of the aging population has increased the burden of disorders involving dementiaincluding Alzheimer’s diseaseon patients and national healthcare systems. Due to the lack of effective therapeutic approaches for dementia, diet or lifestyle changes that can prevent cognitive decline and dementia are receiving increased attention. Recent epidemiological studies have reported that the consumption of certain dairy products such as yogurt and cheese prevents cognitive decline and dementia in the elderly.1 Study participants who consumed low-fat dairy products once a week were shown to have a higher cognitive ability than those who did not.2 In addition, a study using self-reported health information found that the consumption of low-fat dairy products was associated with the improvement of memory recall and social functioning. This survey investigated more than 1000 Japanese subjects, aged 60−79 years, who were living in the community and showed no signs of dementia. Dietary patterns were analyzed and potential associations with the reduced risk of dementia were reported.3,4 These investigations concluded that certain ingredients in milk and dairy products in their diet prevented cognitive decline and reduced the risk of dementia in the Japanese population. Moreover, a clinical trial reported that eating dairy products improves short-term memory.5 In addition, it was reported that whey protein, which is abundant in α-lactalbumin, improves cognitive function in subjects with stress.6 In our previous study, we demonstrated that the consumption of a dairy product fermented with Penicillium candidum (P. candidum) (i.e., Camembert cheese) prevented © 2019 American Chemical Society

Alzheimer’s disease like pathology in a transgenic mouse model.7 In that demonstration, both oleamide and dehydroergosterol, compounds that are generated by fungi during the fermentation process, were found to suppress microglial inflammation;7,8 however, the specific ingredients responsible for preventing cognitive decline have not been elucidated. During the fermentation processes initiated by fungi, various peptides are produced. Several reports have shown that these bioactive peptides have neuroprotective effects and improve cognitive function. For example, cerebrolysin, a mixture of peptides from pig brain, prevented memory impairment in Alzheimer’s disease patients.9−11 In addition, cerebrolysin reduced inflammation and decreased cognitive impairment in preclinical studies.12−16 Furthermore, a recent study reported that β-lactolin, a peptide from β-lactoglobulin, improved memory impairment in mice.17 These demonstrations encouraged us to search for bioactive peptides from milk proteins that improve memory impairment. To test this, we screened peptidesgenerated from casein proteins (the main protein source in cheese)for their ability to improve cognitive function in a pharmacologically induced amnesia model mouse. Received: Revised: Accepted: Published: 8160

April 21, 2019 June 20, 2019 June 26, 2019 June 26, 2019 DOI: 10.1021/acs.jafc.9b02495 J. Agric. Food Chem. 2019, 67, 8160−8167

Article

Journal of Agricultural and Food Chemistry

■

TOF/TOF mass spectrometer (Bruker Daltonics systems, Billerica, MA, USA). Identification of the peptide from the MALDI-TOF/TOF mass spectra was achieved using MS/MS Ion Search of Mascot (Matrix Science, Boston, MA, USA) against the SWISS-PROT database. Liquid Chromatography (LC)-MS Analysis. To identify the purified peptide with the synthesized peptide, an LC-MS system (Shimadzu LC MS-2010 EV; Shimadzu) with electro spray ionization (ESI) probe and XSelect Peptide CSH C18 XP column (2.1 mm × 100 mm, 130 Å, 2.5 μm) connected to HPLC Model LC20A Prominence (Shimadzu) at 70 °C was used. The elution was started with 5−80% eluent B for 30 min (eluent A = 0.1% formic acid in water; eluent B = 0.1% formic acid in acetonitrile), followed by 5% eluent B for 10 min before re-equilibration to starting conditions. The separation was performed at 0.2 mL/min, and peptides were detected at 210 nm using a photodiode array detector, Model SPD-M20A (Shimadzu). The peptides (KEMPFPKYPVEP, EMPF, PK, and PVEP) were then synthesized and purified to >95% purity (Eurofins, Luxembourg, Brussels), and their effects were evaluated in the scopolamine-induced amnesia model. Spontaneous Alternation Test. To determine which of the peptides improves memory impairment, a spontaneous alternation test in the Y maze using amnesia mice induced by scopolamine was performed in accordance with our previous report.17 Briefly, the Y maze has three arms with equal angles (25 cm long × 5 cm wide × 20 cm high). Mice were initially placed in one arm and explored in the Y maze for 8 min, and the sequence and number of arm entries were monitored. The score of alternation (%) was measured as the ratio of the actual number of alternations to the possible number: % alternation = [(number of alternations)/((total arm entries) − 2)] × 100. The present study used new mice for each experiment. To examine the samples on memory impairment, ICR male mice at 6 weeks old were intragastrically administered samples of casein digest or peptides in distilled water 1 h before evaluation. The control group was administered with distilled water used as vehicle. Memory impairment was induced by an intraperitoneal administration of 0.85 mg/kg−scopolamine hydrobromide trihydrate (Sigma-Aldrich) in saline at 40 min after sample administration. One hour after oral administration, the mice were subjected to the spontaneous alternation test. There were 10 mice per group. Novel Object Recognition Test. To measure object recognition memory, the novel object recognition test was performed during the light period in a polyvinyl chloride box (40 × 40 × 40 cm). In the acquisition step, a pair of triangle poles or pyramids (4.5 × 4.5 × 4.5 cm3) was used for the test. In the retention step, a pair of poles or pyramids and a golf ball (4.5 cm diameter) were used for the test. In each trial, two objects were placed 7.5 cm apart from the box corner. We counterbalanced by exchanging the places of the objects in each trial. In the acquisition step, mice were allowed to explore in the box for 10 min, 1 h after sample administration. Twenty-four h after the acquisition step and 1 h after the sample administration, each mouse was allowed to explore in the box with the familiar and novel object for 5 min. The discrimination index (DI) was calculated by dividing the difference between the time spent exploring the novel object and the time spent exploring the familiar object by the total time spent exploring both objects: ((novel object exploration time) − (familiar object exploration time))/total exploration time. There were 10 mice per group. Monoamine Analysis. To evaluate the levels of dopamine (DA), 5-hydroxytryptamine (5-HT), and its metabolites (3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)) in the brain, removed tissue was immediately frozen in liquid nitrogen and homogenized in 0.2 M perchloric acid (PCA, Wako) containing 100 μM EDTA·2Na (Sigma-Aldrich) using a multibeads shocker (Yasui Kikai, Osaka, Japan). After centrifugation and filtration with a 0.45 μm membrane, the supernatant was analyzed using HPLC with electrochemical detection (ECD). An EICOMPAK SC-5ODS column and a PREPAK column (Eicom, Kyoto, Japan) were used in this analysis. For ECD, the voltage was 750 mV versus an Ag/AgCl reference electrode. The mobile phase was 83% 0.1 M acetic acid in

MATERIALS AND METHODS

Chemicals. (−)-Scopolamine hydrobromide trihydrate (SigmaAldrich, St. Louis, MO, USA) was used to induce amnesia. Peptides of KEMPFPKYPVEP, EMPF, PK, and PVEP were synthesized and purified to >95% purity (Eurofins, Tokyo, Japan). For the preparation of casein digestion, casein sodium (Wako, Osaka, Japan) and enzymes (protease M “Amano” 3SD from Aspergillus oryzae [A. oryzae], Thermoase C100 from Bacillus stearothermophilus [B. stearothermophilus], or Papain W-40 [Amano Enzyme Inc., Aichi, Japan]) were used. Animals. Crl:CD1(ICR) male mice, aged 6 weeks (SLC, Inc., Shizuoka, Japan), were maintained at the Kyowa Hakko Kirin Company, Ltd. Experiments were approved by the Animal Experiment Committee of the Kyowa Hakko Kirin Company, Ltd. and were conducted between April 2013 and October 2014 in strict accordance with their guidelines. Mice were maintained at room temperature (23 ± 1 °C) under constant 12 h light and dark cycles. Five mice were housed in one cage, and the cages were exchanged every 3 days. All mice were acclimatized by feeding a standard rodent diet, CE-2 (Clea Japan, Tokyo, Japan), for 7 days. Mice were moved into a soundisolated room 16 h before the test started, and behavioral evaluations were carried out during the light phase. All efforts were made to minimize suffering. After the experiments, the mice were euthanized with carbon dioxide in an appropriate procedure. Preparation of Samples from Casein and Peptide Analysis. To prepare peptide samples for screening, 5% (w/v) casein sodium (Wako, Osaka, Japan) was dissolved in 0.05 M Tris buffer (pH 7.5) and digested with enzymes (0.125% [w/v] of protease M “Amano” 3SD from A. oryzae, Thermoase C100 from B. stearothermophilus, or Papain W-40; Amano Enzyme Inc.) at 50 °C for 4 h. After the enzyme digestion, undigested proteins and enzymes in the sample were filtered using a 10 kDa membrane. The prepared samples were then used for in vivo evaluation. Selected products digested with protease M “Amano” 3SD from A. oryzae were used in the following studies. The casein digestions were fractionated using solid-phase extraction. Each sample dissolved in water was applied to a Sep-Pak C18 column (Nihon Waters, Tokyo, Japan). The bound components were eluted with 0−100% methanol (20% increments). Further fractionation (60−80% methanol elution) was performed using preparative high-performance liquid chromatography (RP-HPLC; LC-Forte/R; YMC Co. Ltd., Kyoto, Japan) using a Mightysil RP18 column (250 × 10 mm, 5 μm size particle; Kanto Chemical Co., Ltd., Tokyo, Japan) with a flow rate of 5.0 mL/min. The gradient employed for this experiment was 40−90% eluent B for 40 min, 90% eluent B for 10 min (eluent A = 5% methanol, eluent B = 100% methanol), followed by 40% eluent B for 10 min before reequilibration to the starting conditions. The elution was monitored at 210 nm. The peptide fractions were collected using an automated fraction collector (0−40 min of fraction siz,e: 2 min/fraction; 40.01− 50 min fraction size, 4 min/fraction) and pooled into five components (Fr 1−5). Purification of Peptide by Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). To purify the active peptide, the active fraction (Fr 2) was subjected to additional chromatography using RP-HPLC and a COSMOSIL Cholester column (250 × 4 mm, 5 μm particle size; Nacalai Tesque, Kyoto, Japan) at 70 °C connected to a HPLC model LC20A Prominence (Shimadzu, Kyoto, Japan). The gradient employed for this experiment was 5−80% eluent B for 30 min (eluent A = 0.1% trifluoroacetic acid (TFA) in water; eluent B = 0.1% TFA in acetonitrile) and 5% B for 10 min before equilibration to starting conditions. The elution was monitored at 210 nm using a photodiode array detector, Model SPDM20A (Shimadzu). Matrix-Assisted Laser Desorption/Ionization-Tandem Timeof-Flight (MALDI-TOF/TOF) Mass Spectrometry (MS) Analysis. To identify the amino acid sequences of the peptide, the purified Fr 2 peptide solution was mixed with α-cyano-4-hydroxycinnamic acid in 90% aqueous acetonitrile/0.5% trifluoroacetic acid. The mixed sample was then spotted onto a MALDI target plate and allowed to crystallize. MALDI-MS was performed using an Ultraflex II MALDI8161

DOI: 10.1021/acs.jafc.9b02495 J. Agric. Food Chem. 2019, 67, 8160−8167

Article

Journal of Agricultural and Food Chemistry

Figure 1. Effects of casein-digested samples on a scopolamine-induced mouse model of amnesia. (A) Effects of 10 mg/kg casein digestion with protease M “Amano” 3SD from Aspergillus oryzae (A. oryzae), Thermoase C100 from Bacillus stearothermophilus (B. stearothermophilus), or Papain W-40 on spontaneous alternations in mice treated with scopolamine. (B) Effects of 2 and 20 mg/kg casein digestion with protease M “Amano” 3SD on spontaneous alternation in mice treated with scopolamine. Data represent the mean, and the error bars indicate the thstandard error of the mean (SEM) of 10 mice per group. p values shown in the graph were calculated by Dunnett’s test. *p < 0.05. citric acid buffer (pH 3.5), 17% methanol (Wako), 190 mg/mL of sodium 1-octanesulfonate sodium (Wako), and 5 mg/mL EDTA·2Na. Quantification of the KEMPFPKYPVEP Peptide in Fermented Products. To quantify the KEMPFPKYPVEP peptide in dairy products, peptides from commercially available yogurts and cheeses (feta, Taleggio, Roquefort, Stilton, Fourme d’Ambert, Camembert, Clarines, chedder, gouda, and mascarpone) were extracted according to a previous study18 with slight modifications. Briefly, cheese (1 g) was homogenized in distilled water (5 mL) and incubated at 40 °C for 60 min. Samples were centrifuged for 30 min at 10000g. The aqueous solution was removed from below the fat layer and filtered through a 10 K membrane (Merck Millipore, Darmstadt, Germany). Yogurts available in the market were diluted with an equal amount of distilled water at 5% (w/w) and the supernatants after centrifugation at 14000g were then filtered through the 10 K membrane. Quantification was performed by LC-MS/MS using a 4000 QTRAP mass spectrometer (ABSciex, Framingham, MA, USA) coupled to a HPLC system (Agilent 1200 series; Agilent Technologies, Santa Clara, CA, USA) according to previous reports.17 Briefly, the samples were separated on a C18 column (TSK GEL ODS-100V, 2.0 × 150 mm, 3 μm; Tosoh, Tokyo, Japan) at 70 °C. The elution proceeded as follows: 0−20% eluent B for 10 min (eluent A = 0.1% formic acid in water; eluent B = 0.1% formic acid in acetonitrile), 20−80% eluent B for 10−30 min, 100% eluent B for 30−40 min, and 0% eluent B for 40−50 min before re-equilibration to the starting conditions with a flow rate of 200 μL/min. The KEMPFPKYPVEP peptide was detected as a doubly charged ion at a mass to charge ratio (m/z) of 731.755. Statistical Analyses. The data represent the mean, and the error bars indicate the SEM. Data were analyzed by one-way ANOVA followed by Dunnett’s test or by Student’s t test as described in the figure legends. All statistical analyses were performed using the Ekuseru-Toukei 2012 software program (Social Survey Research Information, Tokyo, Japan). A value of p < 0.05 was considered as statistically significant.

(data not shown), a result consistent with our previous report.17 To identify peptides in dairy products that improve the pharmacologically induced memory impairment, casein digestions with enzymes from A. oryzae (fungi), B. stearothermophilus (bacteria), and papain (plants) were orally administered at 10 mg/kg to scopolamine-treated mice and their performance on the Y maze spontaneous alternation test was evaluated 1 h later.(Figure 1A). Of note, no significant increase in spontaneous alternations was detected in mice administered undigested casein protein in comparison with scopolamine-treated mice. In contrast, only the mice administered with casein treated with 10 mg/kg protease M “Amano” 3SD from A. oryzae showed a significant increase in spontaneous alternations compared to scopolamine-treated mice (p = 0.038). A single administration of casein digested with protease M “Amano” 3SD at 20 mg/kg, but not 2 mg/kg, also significantly increased spontaneous alternations (Figure 1B, p = 0.042). These results suggested that casein digested with protease M “Amano” 3SD from A. oryzae contains peptides that improve memory impairment. Screening for Peptides in Protease M “Amano” 3SD Casein Protein Digests That Improve Memory. To identify the specific peptides in casein that improve memory function, peptides from casein protein digested with protease M “Amano” 3SD from A. oryzae were separated by elution using 0−100% methanol (20% increments). Only scopolamine-treated mice receiving a single administration of the 80% methanol elution had significantly more spontaneous alternations in the Y maze test in comparison with scopolamine-treated mice (Figure 2A, p = 0.044). The fraction eluted with 80% methanol was then separated into five fractions using HPLC, as shown in Figure 2B. Among these fractions, administration of scopolamine-treated mice with Fr 2 at 2 mg/kg significantly increased spontaneous alternations (Figure 2C, p = 0.033). To identify the amino acid sequence of Fr 2, Fr 2 was purified using RP-HPLC (Figure 1A in the Supporting Information). The purified peptide was then analyzed using MALDI-TOF/TOF mass spectrometry and LC-MS analysis (Figure 2D) and was identified as the KEMPFPKYPVEP peptide. The MALDI-TOF/TOF mass spectra of m/z 1461 with H ion and m/z 1483 with Na ion (Figure 1B,C, respectively) were also identified as the KEMPFPKYPVEP peptide after analysis of the database. To

■

RESULTS Effect of Digested Casein Peptides on ScopolamineInduced Amnesia Model Mice. Pharmacologically inducing amnesia in mice using scopolamine, a muscarinic antagonist, is a popular method to screen for therapeutic drugs or molecules that improve memory impairment. As a positive control, we first showed that donepezil, an acetylcholinesterase inhibitor, significantly increased spontaneous alternations in the Y maze test in scopolamine-treated mice in comparison to controls 8162

DOI: 10.1021/acs.jafc.9b02495 J. Agric. Food Chem. 2019, 67, 8160−8167

Article

Journal of Agricultural and Food Chemistry

Figure 2. Screening for peptides that improve memory in a mouse model of amnesia. (A) ICR male mice were orally administered 10 mg/kg fractions of casein peptide eluted with methanol and then treated 40 min later with scopolamine. Spontaneous alternations were measured in the Y maze test 1 h after scopolamine injection. Only the 80% methanol elution was found to increase the number of spontaneous alterations. (B) Chromatogram of preparative HPLC of casein digestion. (C) Spontaneous alternation test using 0, 0.5, and 2 mg/kg of the Fr 2 fraction. (D) MALDI-TOF/TOF-MS analysis of the purified Fr 2 peptide. Data represent the mean, and the error bars indicate SEM of 10 mice per group. p values shown in the graph were calculated by Dunnett’s test. *p < 0.05.

Effects of the KEMPFPKYPVEP Peptide on Spatial and Long-Term Memory. To evaluate the effects of the identified peptide on spatial and object recognition memory, scopolamine-treated mice were administered the synthesized KEMPFPKYPVEP peptide and subjected to Y maze and novel object recognition tests. A single administration of the synthesized KEMPFPKYPVEP peptide at 0.5 and 2 mg/kg significantly increased spontaneous alternations in the scopol-

confirm a match between Fr 2 and the synthesized KEMPFPKYPVEP peptide, the Fr 2 peptide and the synthesized KEMPFPKYPVEP peptide were analyzed using LC/MS (Figure 2A,C, respectively). A small peak in Figure 2A corresponded to degradation of Fr 2. We confirmed that the mass spectrum of the Fr 2 peptide matched that of the synthesized peptide (Figure 2B,D, respectively). 8163

DOI: 10.1021/acs.jafc.9b02495 J. Agric. Food Chem. 2019, 67, 8160−8167

Article

Journal of Agricultural and Food Chemistry

Figure 3. Effects of the KEMPFPKYPVEP peptide on memory function. ICR male mice were orally administered 0, 0.1, 0.5, and 2 mg/kg of the KEMPFPKYPVEP peptide and treated with scopolamine 40 min later. Spontaneous alternations (A) and arm entries (B) were measured 1 h later. (C, D) ICR male mice were orally administered 0, 0.1, 0.5, and 2 mg/kg of the KEMPFPKYPVEP peptide and subjected to the novel object recognition test to evaluate long-term memory. The time spent exploring novel and familiar objects during 5 min of re-exploration (C) and the discrimination index = ((time spent with object A) − (time spent with object B))/(total time exploring both objects) (D) were measured. Data represent the mean, and the error bars indicate the SEM of 10 mice per group. p values shown in the graph were calculated by Dunnett’s test. *p < 0.05 and **p < 0.01.

(Figure 4D,E, respectively, p = 0.040). The ratio of metabolites to DA of (DOPAC + HVA)/DA was significantly decreased by KEMPFPKYPVEP peptide administration (Figure 4F, p = 0.029), suggesting that the rate of DA metabolism was decreased by KEMPFPKYPVEP peptide treatment. Monoamine analysis was also carried out in the hippocampus, which showed a trend toward increased monoamines (data not shown). The metabolite of NE, 3-methoxy-4-hydroxyphenylglycol (MHPG), was undetectable in the present study. These results indicated that KEMPFPKYPVEP administration increased the total levels of DA and NE in the frontal cortex. Sequences in the KEMPFPKYPVEP Peptide Responsible for Memory Improvement. To evaluate the effects of partial peptide sequences of the KEMPFPKYPVEP peptide on memory improvement, specific peptides, namely EMPF, PK, and PVEP, were spliced by chymotrypsin and evaluated using the spontaneous alternation test. The spontaneous alternation of mice administered with KEMPFPKYPVEP and PVEP was significantly higher than that in control mice (p = 0.039 and p = 0.045, respectively); however, spontaneous alternation in mice administered EMPF or PK was unchanged (Figure 5). These results suggested that the short PVEP peptide at the Cterminus of the KEMPFPKYPVEP peptide could be involved in memory improvement. Quantification of the KEMPFPKYPVEP Peptide in Dairy Products. To evaluate the amount of KEMPFPKYPVEP peptide in commercially available dairy products, we measured its concentration in various yogurts and cheeses using LC-MS/MS. We found that Camembert cheese contained 14.58−39.02 pmol/g of the KEMPFPKYPVEP peptide, while blue cheeses, including Stilton, Roquefort, and

amine-induced amnesia model mice (Figure 3A, p = 0.031 and p = 0.042, respectively), while the total number of arm entries remained unchanged (Figure 3B). These results indicated that the KEMPFPKYPVEP peptide prevented the scopolamineinduced deficit in short-term spatial memory in mice. To evaluate the effects of the KEMPFPKYPVEP peptide on object recognition memory, mice were administered the peptide 1 h before the acquisition step and again 1 h before the retention step and were subjected to behavioral examination using the novel object recognition test. The administration of the KEMPFPKYPVEP peptide at 2 mg/kg increased the time spent with the novel object and reduced the time spent with the familiar object (p = 0.040, Figure 3C) in comparison to control mice, thus increasing the discrimination index (p = 0.034, Figure 3D). These results showed that the KEMPFPKYPVEP peptide enhanced long-term object recognition memory in intact mice. KEMPFPKYPVEP Peptide Increased DA and NE in the Frontal Cortex. DA and its receptors in the brain play crucial roles in the Y maze test and the novel object recognition test,19−21 and norepinephrine (NE) is important for spatial memory in the Y maze test.22 To evaluate the effects of the KEMPFPKYPVEP peptide on the levels of monoamines and their metabolites, we performed HPLC-ECD on tissue homogenates taken from mice 1 h after a single peptide administration. We found that KEMPFPKYPVEP peptide administration significantly increased the level of DA and NE, but not 5-HT, in the frontal cortex (Figure 4A−C, p = 0.039 and p = 0.031, respectively). Levels of the DA metabolite DOPAC did not change, but those of HVA significantly increased after KEMPFPKYPVEP peptide administration 8164

DOI: 10.1021/acs.jafc.9b02495 J. Agric. Food Chem. 2019, 67, 8160−8167

Article

Journal of Agricultural and Food Chemistry

Figure 4. Monoamine and its metabolites in the cortex. Six-week-old ICR male mice were orally given 0 or 2 mg/kg of the KEMPFPKYPVEP peptide. One hour after the administration, the following monoamine levels in the frontal cortex were measured by HPLC: DA (A), NE (B), 5-HT (C), DOPAC (D), and HVA (E). The ratio (DOPAC + HVA)/DA (F) was also determined. Data represent the mean, and the error bars indicate the SEM of 10 mice per group. p values shown in the graph were calculated by Student’s t test. *p < 0.05.

Table 1. KEMPFPKYPVEP Peptide Concentration in Fermented Dairy Productsa

Figure 5. Effects of the KEMPFPKYPVEP peptide and its partial peptides on a scopolamine-induced mouse model of amnesia. ICR male mice were orally given 2 mg/kg of KEMPFPKYPVEP, EMPF, PK, or PVEP peptide and injected intraperitoneally with 0.85 mg/kg of scopolamine 40 min later. At 1 h after the administration, each mouse was allowed to explore the Y maze for 8 min. Spontaneous alternations were then measured. Data represent the mean, and the error bars indicate the SEM of 10 mice per group. p values shown in the graph were calculated by Dunnett’s test. *p < 0.05.

product

concentration (pmol/g)

yogurt A yogurt B feta Taleggio Roquefort Stilton Fourme d’Ambert Camembert A Camembert B Camembert C clarines cheddar gouda mascarpone

N.D. N.D. N.D. 28 11.6 2.49 17.87 39.02 14.58 37.65 8.52 1.52 N.D. N.D.

a

The amount of the KEMPFPKYPVEP peptide in fermented dairy products, including cheese and yogurt, was quantified using LC-MS/ MS.

■

DISCUSSION Previous epidemiological studies have reported that the consumption of fermented dairy products prevents cognitive decline or dementia, particularly in the elderly.3,23 Our previous study also demonstrated that the consumption of Camembert cheese prevents Alzheimer’s disease like pathology in 5×FAD model mice.7 Collectively, these reports strongly suggest that the consumption of fermented dairy products is

Fourme d’Ambert, contained 2.49−17.87 pmol/g. Yogurt and cheese without fungi contained small or undetectable amounts of the KEMPFPKYPVEP peptide (Table 1). These results showed that only yogurt and cheese fermented with fungi contained the KEMPFPKYPVEP peptide. 8165

DOI: 10.1021/acs.jafc.9b02495 J. Agric. Food Chem. 2019, 67, 8160−8167

Article

Journal of Agricultural and Food Chemistry

improvements might vary based on age, microbiome, and other factors. To ingest enough KEMPFPKYPVEP peptide to benefit, development of supplements or nutraceuticals rich in the peptide might be helpful. In addition, other ingredients in dairy products may also improve cognitive function.17 To resolve these limitations, further study will be required. In summary, the present study identified the KEMPFPKYPVEP peptide as an ingredient contained in fermented dairy products that improves memory impairment in mice. Future studies using Alzheimer’s model mice and clinical studies will help clarify its beneficial effects on cognitive function.

beneficial for the prevention of age-related cognitive decline and dementia; however, the exact ingredients in dairy products that are beneficial have not been identified. In the present study, we used a pharmacologically induced mouse model of amnesia to screen for peptides in the enzymatic digestion of casein protein that prevent memory impairment. Our study identified the 12 amino acid peptide KEMPFPKYPVEP from β-casein as a component that improved memory impairment in these mice. Interestingly, the KEMPFPKYPVEP peptide not only improves spatial memory in amnesic mice but also enhances long-term object recognition in healthy mice. DA and NE are crucial for working memory and memory retention.24−27 We found that the KEMPFPKYPVEP peptide increased the total levels of DA and NE in the frontal cortex and reduced the ratio of DA metabolites. Previous deonstrations have shown that DA and NE are involved in frontal cortex dependent memory functions such as spatial working memory and object recognition memory.28−32 Indeed, the DA neuronal network is associated with cognitive performance, including working memory and episodic memory, and the DA precursor levodopa has been shown to improve task-based learning rates and task performances in elderly people.33 NE is also important for synaptic transmission in the frontal cortex circuitry.34 These reports suggest that the KEMPFPKYPVEP peptide in dairy products may have a beneficial effect on memory function by increasing the levels of DA and NE in the brain. To test whether increased DA and NE levels contribute to the memory improvement seen with the KEMPFPKYPVEP peptide, DA and NE release should be measured using microdialysis, and the effects of DA or NE antagonist treatment on memory improvement by the KEMPFPKYPVEP peptide should be studied. We previously reported that some peptides, including the GTWY peptide of β-lactolin, inhibit monoamine oxidase B (MAO-B) and increase dopamine levels in the brain, resulting in improved memory impairment.17,35 Monoamine oxidase B is attracting increased attention as a preventive target in Alzheimer’s disease.36,37 KEMPFPKYPVEP peptide may also be associated with the inhibition of MAO-B activity, but the underlying mechanisms still need to be investigated. The KEMPFPKYPVEP peptide is normally degraded by digestive enzymes to be absorbed into the body when ingested orally. The KEMPFPKYPVEP peptide is possibly degraded into K, EMPF, PK, Y, and PVEP. Our results showed that the PVEP peptide increased spontaneous alternations; thus, the sequence at the C-terminal side of the KEMPFPKYPVEP peptide appears to have an important effect on memory improvement. Further study is needed to elucidate the pharmacokinetics of the KEMPFPKYPVEP peptide. Finally, the concentration of the KEMPFPKYPVEP peptide in common fermented dairy products was measured. The KEMPFPKYPVEP peptide was found in various commercially available cheeses, particularly in fungal fermented cheeses such as Camembert and Roquefort. These demonstrations support recent epidemiological surveys that the consumption of fermented dairy products prevents cognitive decline and dementia.3,4 However, intake of more than 10 kg of Camembert cheese would be required for the KEMPFPKYPVEP peptide to have an effect in humans, and cheese contains large amounts of saturated fatty acids that are associated with cardiovascular disease. In the present study, we evaluated the effects of the KEMPFPKYPVEP peptide using young mice, but the epidemiological study surveyed the elderly. Thus, the effective amount needed to produce memory

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02495.

■

MALDI-TOF/TOF-MS analysis of the Fr 2 peptide and LC-MS analysis of the purified Fr 2 peptide and the synthesized KEMPFPKYPVEP peptide (PDF)

AUTHOR INFORMATION

Corresponding Author

*Y.A.: e-mail, [email protected]; tel, +81-45-3309007; fax, +81-45-782-4042. ORCID

Yasuhisa Ano: 0000-0001-5957-8710 Author Contributions

Y.A. conducted most of the experiments, analyzed the results, and drafted the paper. T.K. prepared the peptide sample. T.S. conducted the MALDI-TOF/TOF-MS analysis. K.K. discussed the results and drafted the manuscript. S.U. and K.Y. supervised the experimental techniques. All authors revised and approved the submitted manuscript. Notes

The authors declare the following competing financial interest(s): Yamada, K and Uchida, S. are employed by Kyowa Hakko Kirin Co. Ltd. and Ano, Y., Kutsukake, T., Sasaki, T., and Kondo, K. are employed by Kirin Holdings Co. Ltd. All other authors declare no competing interests.

■

ABBREVIATIONS USED DA, dopamine; DI, discrimination index; DOPAC, 3,4dihydroxyphenylacetic acid; ECD, electrochemical detection; EDTA, ethylenediaminetetraacetate; 5-HT, 5-hydroxytryptamine; HVA, homovanillic acid; LC-MS, liquid chromatography−mass spectrometry; MALDI, matrix assisted laser desorption/ionization; MHPG, 3-methoxy-4-hydroxyphenylglycol; MS, mass spectrometry; NE, norepinephrine; PCA, perchloric acid; RP-HPLC, reversed-phase high performance liquid chromatography; TFA, trifluoroacetic acid

■

REFERENCES

(1) Ano, Y.; Nakayama, H. Preventive Effects of Dairy Products on Dementia and the Underlying Mechanisms. Int. J. Mol. Sci. 2018, 19 (7), 1927. (2) Crichton, G. E.; Bryan, J.; Murphy, K. J.; Buckley, J. Review of dairy consumption and cognitive performance in adults: findings and methodological issues. Dementia Geriatr. Cognit. Disord. 2010, 30 (4), 352−61. (3) Ozawa, M.; Ninomiya, T.; Ohara, T.; Doi, Y.; Uchida, K.; Shirota, T.; Yonemoto, K.; Kitazono, T.; Kiyohara, Y. Dietary patterns 8166

DOI: 10.1021/acs.jafc.9b02495 J. Agric. Food Chem. 2019, 67, 8160−8167

Article

Journal of Agricultural and Food Chemistry and risk of dementia in an elderly Japanese population: the Hisayama Study. Am. J. Clin. Nutr. 2013, 97 (5), 1076−82. (4) Ozawa, M.; Ohara, T.; Ninomiya, T.; Hata, J.; Yoshida, D.; Mukai, N.; Nagata, M.; Uchida, K.; Shirota, T.; Kitazono, T.; Kiyohara, Y. Milk and dairy consumption and risk of dementia in an elderly Japanese population: the Hisayama Study. J. Am. Geriatr. Soc. 2014, 62 (7), 1224−30. (5) Ogata, S.; Tanaka, H.; Omura, K.; Honda, C.; Hayakawa, K. Association between intake of dairy products and short-term memory with and without adjustment for genetic and family environmental factors: A twin study. Clin. Nutr. 2016, 35 (2), 507−513. (6) Markus, C. R.; Olivier, B.; de Haan, E. H. Whey protein rich in alpha-lactalbumin increases the ratio of plasma tryptophan to the sum of the other large neutral amino acids and improves cognitive performance in stress-vulnerable subjects. Am. J. Clin. Nutr. 2002, 75 (6), 1051−6. (7) Ano, Y.; Ozawa, M.; Kutsukake, T.; Sugiyama, S.; Uchida, K.; Yoshida, A.; Nakayama, H. Preventive effects of a fermented dairy product against Alzheimer’s disease and identification of a novel oleamide with enhanced microglial phagocytosis and anti-inflammatory activity. PLoS One 2015, 10 (3), No. e0118512. (8) Ano, Y.; Kutsukake, T.; Hoshi, A.; Yoshida, A.; Nakayama, H. Identification of a novel dehydroergosterol enhancing microglial antiinflammatory activity in a dairy product fermented with Penicillium candidum. PLoS One 2015, 10 (3), No. e0116598. (9) Rainer, M.; Brunnbauer, M.; Dunky, A.; Ender, F.; Goldsteiner, H.; Holl, O.; Kotlan, P.; Paulitsch, G.; Reiner, C.; Stossl, J.; Zachhuber, C.; Mossler, H. Therapeutic results with Cerebrolysin in the treatment of dementia. Wiener medizinische Wochenschrift (1946) 1997, 147 (18), 426−31. (10) Alvarez, X. A.; Cacabelos, R.; Sampedro, C.; Aleixandre, M.; Linares, C.; Granizo, E.; Doppler, E.; Moessler, H. Efficacy and safety of Cerebrolysin in moderate to moderately severe Alzheimer’s disease: results of a randomized, double-blind, controlled trial investigating three dosages of Cerebrolysin. European journal of neurology 2011, 18 (1), 59−68. (11) Plosker, G. L.; Gauthier, S. Cerebrolysin: a review of its use in dementia. Drugs Aging 2009, 26 (11), 893−915. (12) Rockenstein, E.; Mante, M.; Adame, A.; Crews, L.; Moessler, H.; Masliah, E. Effects of Cerebrolysin on neurogenesis in an APP transgenic model of Alzheimer’s disease. Acta Neuropathol. 2007, 113 (3), 265−75. (13) Rockenstein, E.; Desplats, P.; Ubhi, K.; Mante, M.; Florio, J.; Adame, A.; Winter, S.; Brandstaetter, H.; Meier, D.; Masliah, E. Neuro-peptide treatment with Cerebrolysin improves the survival of neural stem cell grafts in an APP transgenic model of Alzheimer disease. Stem Cell Res. 2015, 15 (1), 54−67. (14) Alvarez, X. A.; Lombardi, V. R.; Fernandez-Novoa, L.; Garcia, M.; Sampedro, C.; Cagiao, A.; Cacabelos, R.; Windisch, M. Cerebrolysin reduces microglial activation in vivo and in vitro: a potential mechanism of neuroprotection. Journal of neural transmission. Supplementum 2000, 59, 281−92. (15) Zhang, Y.; Chopp, M.; Meng, Y.; Zhang, Z. G.; Doppler, E.; Winter, S.; Schallert, T.; Mahmood, A.; Xiong, Y. Cerebrolysin improves cognitive performance in rats after mild traumatic brain injury. J. Neurosurg. 2015, 122 (4), 843−55. (16) Xing, S.; Zhang, J.; Dang, C.; Liu, G.; Zhang, Y.; Li, J.; Fan, Y.; Pei, Z.; Zeng, J. Cerebrolysin reduces amyloid-beta deposits, apoptosis and autophagy in the thalamus and improves functional recovery after cortical infarction. J. Neurol. Sci. 2014, 337 (1−2), 104−11. (17) Ano, Y.; Ayabe, T.; Kutsukake, T.; Ohya, R.; Takaichi, Y.; Uchida, S.; Yamada, K.; Uchida, K.; Takashima, A.; Nakayama, H. Novel lactopeptides in fermented dairy products improve memory function and cognitive decline. Neurobiol. Aging 2018, 72, 23−31. (18) Bütikofer, U.; Meyer, J.; Sieber, R.; Wechsler, D. Quantification of the angiotensin-converting enzyme-inhibiting tripeptides Val-ProPro and Ile-Pro-Pro in hard, semi-hard and soft cheeses. Int. Dairy J. 2007, 17 (8), 968−975.

(19) Lee, K. N.; Chirwa, S. Blocking Dopaminergic Signaling Soon after Learning Impairs Memory Consolidation in Guinea Pigs. PLoS One 2015, 10 (8), No. e0135578. (20) Yang, K.; Broussard, J. I.; Levine, A. T.; Jenson, D.; Arenkiel, B. R.; Dani, J. A. Dopamine receptor activity participates in hippocampal synaptic plasticity associated with novel object recognition. Eur. J. Neurosci 2017, 45 (1), 138−146. (21) da Silva, W. C.; Kohler, C. C.; Radiske, A.; Cammarota, M. D1/ D5 dopamine receptors modulate spatial memory formation. Neurobiol. Learn. Mem. 2012, 97 (2), 271−275. (22) Ayabe, T.; Ohya, R.; Taniguchi, Y.; Shindo, K.; Kondo, K.; Ano, Y. Matured Hop-Derived Bitter Components in Beer Improve Hippocampus-Dependent Memory Through Activation of the Vagus Nerve. Sci. Rep. 2018, 8 (1), 15372. (23) Camfield, D. A.; Owen, L.; Scholey, A. B.; Pipingas, A.; Stough, C. Dairy constituents and neurocognitive health in ageing. Br. J. Nutr. 2011, 106 (2), 159−74. (24) Lara, A. H.; Wallis, J. D. The Role of Prefrontal Cortex in Working Memory: A Mini Review. Front. Syst. Neurosci. 2015, 9, 173. (25) Funahashi, S. Working Memory in the Prefrontal Cortex. Brain Sci. 2017, 7 (5), 49. (26) Arnsten, A. F. Through the looking glass: differential noradenergic modulation of prefrontal cortical function. Neural Plast. 2000, 7 (1−2), 133−46. (27) Arnsten, A. F. Catecholamine influences on dorsolateral prefrontal cortical networks. Biol. Psychiatry 2011, 69 (12), No. e89. (28) Puig, M. V.; Rose, J.; Schmidt, R.; Freund, N. Dopamine modulation of learning and memory in the prefrontal cortex: insights from studies in primates, rodents, and birds. Front. Neural Circuits 2014, 8, 93. (29) Surmeier, D. J. Dopamine and working memory mechanisms in prefrontal cortex. J. Physiol. 2007, 581, 885. (30) Berridge, C. W.; Spencer, R. C. Differential cognitive actions of norepinephrine a2 and a1 receptor signaling in the prefrontal cortex. Brain Res. 2016, 1641, 189−96. (31) Courtney, S. M.; Petit, L.; Maisog, J. M.; Ungerleider, L. G.; Haxby, J. V. An area specialized for spatial working memory in human frontal cortex. Science (Washington, DC, U. S.) 1998, 279 (5355), 1347−51. (32) Ghazizadeh, A.; Hong, S.; Hikosaka, O. Prefrontal Cortex Represents Long-Term Memory of Object Values for Months. Curr. Biol. 2018, 28 (14), 2206−2217. (33) Chowdhury, R.; Guitart-Masip, M.; Lambert, C.; Dayan, P.; Huys, Q.; Duzel, E.; Dolan, R. J. Dopamine restores reward prediction errors in old age. Nat. Neurosci. 2013, 16 (5), 648−53. (34) Xing, B.; Li, Y. C.; Gao, W. J. Norepinephrine versus dopamine and their interaction in modulating synaptic function in the prefrontal cortex. Brain Res. 2016, 1641, 217−233. (35) Ano, Y.; Ayabe, T.; Ohya, R.; Kondo, K.; Kitaoka, S.; Furuyashiki, T. Tryptophan-Tyrosine Dipeptide, the Core Sequence of beta-Lactolin, Improves Memory by Modulating the Dopamine System. Nutrients 2019, 11 (2), 348. (36) Wang, Y.; Sun, Y.; Guo, Y.; Wang, Z.; Huang, L.; Li, X. Dual functional cholinesterase and MAO inhibitors for the treatment of Alzheimer’s disease: synthesis, pharmacological analysis and molecular modeling of homoisoflavonoid derivatives. J. Enzyme Inhib. Med. Chem. 2016, 31 (3), 389−397. (37) Borroni, E.; Bohrmann, B.; Grueninger, F.; Prinssen, E.; Nave, S.; Loetscher, H.; Chinta, S. J.; Rajagopalan, S.; Rane, A.; Siddiqui, A.; Ellenbroek, B.; Messer, J.; Pahler, A.; Andersen, J. K.; Wyler, R.; Cesura, A. M. Sembragiline: A Novel, Selective Monoamine Oxidase Type B Inhibitor for the Treatment of Alzheimer’s Disease. J. Pharmacol. Exp. Ther. 2017, 362 (3), 413−423.

8167

DOI: 10.1021/acs.jafc.9b02495 J. Agric. Food Chem. 2019, 67, 8160−8167