Acid proteinase from Aspergillus niger (activity: 30,000 U/g) was purchased from Donghenghuadao Biotechnology Co., Ltd. (Guangxi, China). Trypsin from porcine pancreas (USP, 1:2,500 U/mg) was purchased from Guangzhou Qiyun Biotechnology Co., Ltd. (Guangdong, China). Whey protein concentrate (WPC with a protein content of 13.45%) was purchased from Guangdong Beric Food Co., Ltd. (Guangdong, China). Commercial synthetic peptides Val-Ala-Gly-Thr-Trp-Tyr, Val-Ala-Gly-Thr-Trp, and Gly-Thr-Trp with 95% purity were purchased from Nanjing Peptide Industry Biotechnology Co., Ltd. (Nanjing, China). β-lactoglobulin with 95% purity was purchased from Sigma Co. (St. Louis, MO, USA). The rest of the reagents and chemicals were purchased from Guangzhou Jingke Glass Instrument Co., Ltd. (Guangzhou, China) and were of the analytical grade or better.

Whey protein was prepared following the alkali-soluble acid precipitation method as described by Zhu et al. (4). The parameters of alkaline extraction were as follows: pH of 11, a ratio of whey protein concentrate (as described previously) to water of 1:10 at 60 ± 1°C for 1 h, centrifugation at 4°C at 8,000 r/min for 20 min, and separation to obtain the supernatant using a centrifuge (GL-21M, Changsha Xiangzhi Centrifuge Instrument Co., Ltd., Changsha, China). The parameters for acid precipitation were as follows: pH of 4.4, centrifugation of 4°C at 8,000 r/min for 20 min, and separation to obtain the precipitate. The precipitate was washed with deionized water at pH 7, vacuum freeze-dried (model: SCIENTZ-18N, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China), and stored at 4°C for further use. After the alkali-soluble acid precipitation method, the total protein content of the prepared whey protein was 78.25%, and the β-lactoglobulin content was 43.46%.

When β-lactoglobulin (two disulfide bonds and a free sulfhydryl group) is heated or enzymatically hydrolyzed under acidic conditions, intermolecular disulfide bonds can maintain macromolecular structure and even cause aggregation of proteins, resulting in low enzymatic hydrolysis efficiency of proteins and difficult release of active peptides (18). However, α-lactalbumin tends to form a “melting ball” state and is easily hydrolyzed by acidic protease under acidic conditions (19). Therefore, this property can be used to selectively hydrolyze α-lactalbumin from whey protein to enriched β-lactoglobulin. β-lactoglobulin was enriched from whey protein using acid proteinase under the following conditions: pH of 4.0, the ratio of whey protein to water of 1:10, and temperature of 50°C. The reaction mixture was then centrifuged at 8,000 r/min and 4°C for 20 min to obtain the precipitation. The precipitation sample was redissolved in Milli-Q water and analyzed using UPLC. The solution was then loaded onto a ZORBOX Eclipse XDB-C₈ UPLC column (specifications: 150 × 4.6 mm, 5 μm; Agilent Technologies, USA). The elution was programmed at a flow rate of 0.25 ml/min and room temperature (25°C) for 20 min, with the mobile phase A (10% acetonitrile and 0.1% trifluoroacetic acid) and mobile phase B (90% acetonitrile and 0.1% trifluoroacetic acid), as 0~1 min 35% B; 1 min~5 min 35%~38% B; 5 min~10 min 38%~42% B; 10 min~ 12 min 42%~46% B; 12 min~15 min 46%~90% B; 15 min~18 min 35%~90% B; and 18 min~20 min 35% B. The elution in gradient mode was monitored at 280 nm with a UV detector (UV201, USA). The sample corresponding to the detected peaks of β-lactoglobulin was collected and freeze-dried for further investigations.

When the pH was 7.0 and the temperature increased to 50°C, the β-lactoglobulin dimer dissociated into a monomer, and the structure of β-lactoglobulin became loose (18, 19). Trp was located at the 19th (near the N-terminal) and 61st amino acids of β-lactoglobulin (which is composed of 162 amino acid residue), and the 19th amino acid (Trp) was easy to be exposed to (16). To obtain Trp peptides (near the N-terminal) of the above-prepared β-lactoglobulin, the β-lactoglobulin was hydrolyzed using trypsin under the following optimal conditions: pH of 7.0, a ratio of whey protein to water of 1:10, temperature of 50°C, and incubation time of 4 h. The reaction mixture was then centrifuged at 8,000 rpm and 4°C for 20 min using a centrifuge to obtain the supernatants. It was then vacuum-freeze dried and stored at 4°C. The characterization of the Trp peptide prepared from β-lactoglobulin was conducted using a UPLC-Q-TOF MS/MS (20). An Agilent 1290 series UPLC system (Agilent Technologies CO., Ltd., Guangzhou, China) was equipped with an Agilent ZORBAX RRHD SB-C₁₈ column (2.1 × 50 mm i.d., 1.8 μm, maintained at 30°C). Peptides were analyzed using maXis ImpactTM quadrupole TOF tandem mass spectrometry system (Bruker Daltonics, Beijing, China) fitted with ESI⁺ (Bruker maxis impact) and coupled via MS/MS. All the mass spectrometry data were interpreted by peaks automatic sequencing software and de novo sequencing software (Bruker Compass Data Analysis 4.1 software) for the identification and quantification of peptides (21). The ion peaks of the main individual peptides were extracted, and the relative contents of the main individual peptides were determined by the ion peak area (20, 22).

The adult zebrafish (wild type AB strain, the ratio of ♀ ♂ was 1:1) were bred at the age of 2 months by the Zebrafish Laboratory of South China University of Technology. The temperature was 28.5 ± 0.2°C, pH was 7.5 ± 0.02, tank volume was 3 L, the light-dark cycle was 14/10 h, and conductivity was 450~550 μs/cm. The system water is circulating (automatically updated by 10% every day) and is sterilized by an ultraviolet lamp in real-time. Zebrafish were bred by an independent breeding system and fed two times a day with abundant shrimp eggs. All experiments involving zebrafish were reviewed by the Animal Research Advisory Committee of the South China University of Technology. After a 3-day acclimation period, zebrafish were randomly divided into nine groups (n = 30 per group) as follows: NC group (the normal control group for treatment with normal water); LAWPH and LAWPL (the group for treatment with β-lactoglobulin Trp peptides at a high dose: 500 μg/ml and a low dose: 56 μg/ml); VAGTWYH and VAGTWYL (the group for treatment with Val-Ala-Gly-Thr-Tyr-Trp at a high dose: 500 μg/ml and a low dose: 56 μg/ml); VAGTWH and VAGTWL (the group for treatment with Val-Ala-Gly-Thr-Tyr-Trp at a high dose: 500 μg/ml and a low dose: 56 μg/ml); and GTWH and GTWL (the group for treatment with Gly-Thr-Trp at a high dose: 500 μg/ml and a low dose: 56 μg/ml). The experimental groups (VAGTWY, VAGTW, and GTW) with the zebrafish used the synthetic peptides, and the LAWP experimental group with the zebrafish used the hydrolysates. The maximum tolerated concentration (MTC: 500 μg/ml) of peptides was determined. In the low-dose group and high-dose group, 1/9 MTC and MTC were selected, respectively.

After the fortnight of water-soluble administration, all experimental zebrafish were tested for anxiety-like behavior during the daytime (7:00 am−17:00 pm). To obtain accurate results, the whole behavioral testing process was carried out in a quiet and stable environment.

After the behavioral tests, the collected brain tissues were frozen in liquid nitrogen for further studies. The concentrations of Trp, 5-HT level, and kynurenine (KYN) were determined by Elisa assay kits following the instructions of the manufacturer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). Briefly, 100 mg of brain tissue was homogenized with 9 volumes of phosphate-buffered saline (PBS, pH 7.2) at 6°C using a homogenizer (Xianou-24, Nanjing Xianou Instrument Co., Ltd., Nanjing, China) and centrifuged for 20 min (3,000 r/min, 4°C) to collect the supernatants. The concentrations of Trp, 5-HT level, and KYN of the obtained supernatants from brain tissues were determined using the abovementioned ELISA method. The activity of TPH was determined by Elisa and colorimetric assay kits (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) as described by Zhu et al. (10) with some modifications.

All experimental results were expressed as “mean ± standard deviation” for reporting. The statistical software, Origin 6.1 (Origin Lab Corporation, USA), was used by the primary contributor to the present study. Analysis of variance and significant difference tests were performed using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). ANOVA was performed to evaluate the difference, followed by the Duncan post-hoc test. Statistical significance was defined as a p-value of < 0.05.

The content of β-lactoglobulin in whey protein (prepared above) was 43.46 ± 1.02%, as shown in Figures 1A, B. In addition, the content of β-lactoglobulin was increased with the hydrolysis time and the content was the highest (71.17 ± 1.45%) when the hydrolysis time was 120 min (Figure 1A). However, as the hydrolysis time continued increasing, the content of β-lactoglobulin decreased. As shown in Figure 1C, most of α-lactalbumin in whey protein was hydrolyzed and β-lactoglobulin was hardly hydrolyzed when the hydrolysis time was 120 min. Then, β-lactoglobulin began to hydrolyze and the degree of hydrolysis gradually increased with the increase in time (Figures 1A, D). Under acidic conditions, α-lactalbumin tends to form a “melting ball” state and was easily hydrolyzed by acidic protease (19). This property can be used to selectively hydrolyze α-lactalbumin from whey protein to enriched β-lactoglobulin. Our results showed that α-lactalbumin in whey protein was preferentially hydrolyzed by acidic protease at pH 4.0, which was consistent with previous studies. In summary, the optimal hydrolysis parameters of enriched β-lactoglobulin from whey protein by selective hydrolyzed technology were as follows: whey protein concentration of 10%, pH of 4.0, and hydrolysis temperature of 50°C for 120 min.

After 4 h of hydrolysis, trypsin produced a large number of peptides from the β-lactoglobulin (enriched above) hydrolysate. Then, three major and novel Trp peptides, Gly-Thr-Trp, Val-Ala-Gly-Thr-Trp, and Val-Ala-Gly-Thr-Trp-Tyr, were identified from the β-lactoglobulin hydrolysate (Figure 2A). Trypsin only cleaves K and R (not A, W, and Y) based on the cleavage patterns predicted by ExPASy PeptideCutter (23). Theoretically, GTW and VAGTWY cannot be released from QTMKGLDIQKVAGTWYSLAM; however, these two peptides were identified in our present study, which may be due to miscleavage resulting from weak peptide–protease interaction (23, 24). The total yield of Trp peptides was 23.46% (w/w), and the relative contents of Gly-Thr-Trp, Val-Ala-Gly-Thr-Trp, and Val-Ala-Gly-Thr-Trp-Tyr were 6.03, 15.57, and 1.86% (w/w), respectively. The strong signals detected in positive-ion mode at 363.1659, 533.2725, and 696.3362 m/z corresponded to the ionized peptides Gly-Thr-Trp (Figure 2B), Val-Ala-Gly-Thr-Trp (Figure 2C), and Val-Ala-Gly-Thr-Trp-Tyr (Figure 2D). Counting from the N-Term, the sequences of Trp peptides prepared from β-lactoglobulin were all located at the position between the 15th and 20th amino acids (Figure 2E). In addition, the three peptides identified were all made up of Trp peptides (the 19th amino acid of β-lactoglobulin). Based on these obtained results, trypsin could hydrolyze β-lactoglobulin to produce Trp peptides consisting of 19th tryptophan.

Anxiety-like behaviors in the novel fish tank test after the fortnight of treatment with Trp peptides are summarized in Table 1. The result shows that anxiety-like behaviors were all decreased for the Trp peptide treatment groups compared with the NC zebrafish (Table 1). Notably, the number of times of shuttle up/down (14.24 ± 4.78) in the NC group was significantly (p < 0.05) lower than that in all the Trp peptide treatment groups. The number of times of shuttle up/down in the VAGTWH group (26.17 ± 4.66) was significantly higher (p < 0.05) compared to the LAWP (H and L), GTW (H and L), and VAGTWY (H and L) groups. The number of times of shuttle up/down was decreased in the following order: VAGTWH > VAGTWL > GTWL > GTWH > LAWPH > VAGTWYH > LAWPL > VAGTWYL > NC. The percentage of retention time in the top half of the tank increased significantly (p < 0.05) in the LAWPH, GTW (H and L), and VAGTW (H and L) groups compared to the NC group (22.52 ± 1.43%). However, there was no significant difference (p > 0.05) in the LAWPL and VAGTWY (H and L) groups in the percentage of retention time in the top half of the tank compared to the NC group. Similarly, the percentage of retention time in the top half of the tank in the VAGTWH group was also significantly higher (p < 0.05) than all the other groups. Hawkey et al. (25) showed that the novel fish tank test was a direct, repeatable method for measuring zebrafish anxiety-like behaviors. When placed in a novel tank, zebrafish often dive to the bottom of the tank (avoidance). Zebrafish will also be stimulated by a novel area (top of a fish tank) and develop an urge to explore; thus, zebrafish form an anxious state of avoidance/exploration. According to previous studies, our results indicated that β-lactoglobulin Trp peptides could alleviate anxiety-like behaviors of zebrafish in the novel fish tank test after the fortnight treatment (effectiveness: VAGTW > GTW > VAGTWY).

The light-dark test, a kind of anxiety stress model, is a classical method for screening anti-anxiety drugs and establishing and evaluating the zebrafish anxiety model, which has been widely used in basic research in stress response, psychopharmacology, or neuropharmacology (26, 27). Anxiety-like behaviors in the light/dark test after the fortnight treatment with Trp peptides are summarized in Table 2. After the fortnight treatment of this study, the number of times of shuttling light/dark (30.33 ± 2.08) in the NC zebrafish was significantly lower (p < 0.05) than that in the Trp peptide treatment groups (Table 2). However, the number of occasions of shuttling light/dark in the VAGTWH (43.08 ± 3.97) group was significantly higher (p < 0.05) than all the other groups. Notably, the difference in the number of times of shuttling light/dark among the high or low dose of the LAWP, GTW, and VAGTWY groups was insignificant (p > 0.05); however, Trp peptide supplementation increased the number of times of shuttle light/dark all in a dose-dependent manner. Except for the LAWPL and VAGTWYL groups, the percentage of time spent in the light area was significantly (p < 0.05) lower in the NC group compared to all the other groups (22.52 ± 1.43%). The percentage of time spent in the light area was slightly higher in the VAGTWH group, but there was no significant difference (p > 0.05) among the LAWPH, GTW (H and L), VAGTW (H and L), and VAGTWYH groups. Trp peptide supplementation increased the percentage of time spent in the light area, all in a dose-dependent manner. Studies that employed the light-dark task found that animals exhibit a strong preference for the dark compared to the light compartment, and higher levels of anxiety-like behaviors are associated with more time spent in the dark compartment (26, 27). Accordingly, our results indicated that β-lactoglobulin Trp peptide ingestion could reduce the anxiety-like behaviors of zebrafish in the light-dark test in a dose-dependent manner. In terms of anti-anxiety behaviors, VAGTW seemed more effective (p < 0.05) than GTW and VAGTWY.

After the administration process, the activity of THP was significantly lower (p < 0.05) in the normal control (447.07 U/L) group than in all Trp peptide treatment groups (Figure 3). The activity of TPH in the VAGTWH group was significantly (p < 0.05) higher than all the other groups. The activity of TPH in the GTWH and VAGTWL groups was significantly (p < 0.05) higher than the LAWP (H and L), GTWL, and VAGTWY (H and L) groups, while they were significantly (p < 0.05) lower than the VAGTWH group. Nevertheless, there was no significant difference (p > 0.05) among the LAWP (H and L), GTWL, and VAGTWY (H and L) groups. The level of 5-HT was significantly lower (p < 0.05) in the NC (352.10 ng/ml) group than in all Trp peptide treatment groups, except in the VAGTWYH group. The level of 5-HT was slightly higher in the VAGTWH group, but there was no significant difference (p > 0.05) among the LAWP (H and L), GTW (H and L), and VAGTW (H and L) groups. Notably, the level of 5-HT in the VAGTWH group was significantly higher (p > 0.05) than that in the VAGTWY (H and L) group. As the key rate-limiting enzyme, the activity of TPH in brain tissue directly affects the level of 5-HT in the central nervous system (28). The abnormal level of 5-HT in the brain is closely related to anxiety disorders (28, 29). Previous studies showed that most anti-anxiety drugs act by influencing the neurotransmitter, including the increase in the levels of 5-HT in the brain (10). According to the results of this study, Trp peptides had stimulatory effects on the level of 5-HT, and such effects likely occurred via boosting the activation of TPH.

After the fortnightly treatment of this study, the NC group had a normal Trp concentration, and 5-HT/Trp (1,562.88) and KYN/Trp (1,421.52) ratios in brain tissues for the group have been indicated in Table 3. The concentrations of Trp in the LAWP (H and L), GTWH, VAGTW (H and L), and VAGTWYH groups were significantly higher (p < 0.05) than those in the NC, GTWL, and VAGTWYL groups. The concentrations of Trp in the GTWL and VAGTWYL groups were slightly higher, but there was no significant difference (p > 0.05) compared with the NC group. Excepting for the VAGTWYL group, the ratio of 5-HT/Trp in the NC group was significantly lower (p < 0.05) than that in all other Trp peptide treatment groups. The ratio of 5-HT/Trp decreased in the following order: GTWL > LAWPL > VAGTWH > LAWPH > GTWH > VAGTWL > VAGTWYH > VAGTWYL > NC. However, the ratio of KYN/Trp in the NC group was significantly higher (p < 0.05) than in all Trp peptide treatment groups, except the GTWL and VAGTWYL groups. Additionally, the ratio of KYN/Trp increased in the following order: LAWPH < VAGTWYH < GTWH < VAGTWH < VAGTWL < LAWPL < GTWL < VAGTWYL < NC. These results suggest that Trp peptides significantly increased the level of 5-HT in the zebrafish, possibly by influencing Trp concentration, 5-HT/Trp ratio, and KYN/Trp ratio of brain tissue. A large number of studies showed that Trp and its metabolite were critical for monitoring anxiety disorders (30, 31), and a positive relationship was found between dietary Trp and the level of 5-HT in the brain (7). Our results were consistent with those of previous studies that showed that ingestion of Trp peptide diets did, in fact, increased the concentration of Trp in brain tissues, with VAGTWH being more effective. Previous research showed that the ratio of 5-HT/Trp was reduced and the ratio of KYN/Trp was increased in nervous and mental diseases (such as anxiety or depression) (32). Our result was consistent with the previous studies that showed that ingestion of Trp peptide diets, in fact, enhanced the ratio of 5-HT/Trp, with VAGTWH being more effective. Thus, Trp peptide might act more effectively in anti-anxiety via enhancing the concentration of Trp and the ratio of 5-HT/Trp and reducing the ratio of KYN/Trp.