Preparation of the rainbow trout bone peptides directed by nutritional properties and flavor analyses

2.1 Materials and chemicals

Rainbow trout bone was supplied by Shandong Yueyi Bio-Technology Co., Ltd (Rizhao, China). The same batch of rainbow trout bone was stored at a temperature of −30°C and thawed at 0–20°C with a flowing water. Neutral protease (35,000 U/g), alkaline protease (35,000 U/g), flavourzyme (35,000 U/g), trypsin (35,000 U/g), and papain (35,000 U/g) were all purchased from Amresco Co. Ltd (Beijing, China).

2.2 Enzyme hydrolysis

Fish bones were cut into a size of blocks of 3–5 cm³ approximately. The chopped fish bones were mixed with water at a ratio of 1:1.2 (w/v) and then transferred in extraction pot with a constant temperature of 121 ± 1°C for 30 min. The enzyme was added after the hot-pressure extraction. The resulting mixture was filtered and centrifuged at 4°C for 20 min at 14,400 g to collect supernatant.

2.3 Determination of the degree of hydrolysis

The percentage of free amino groups separated from protein determines the DH that was calculated as the ratio between α-amino nitrogen and total nitrogen in the samples (Taylor, 1957). The DH was evaluated by the pH-stat method that was based on the consumed volume of standard NaOH solution to maintain the reaction pH constant, using the following equation described by Adler-Nissen (Adlernissen, 1986):

(1)

where h is the content of the -NH₂ group or the –COOH group in the enzymatic hydrolysate, h₀ is the content of the -NH₂ group or the –COOH group in the fish bone, h_tot is the total number of peptidic bonds in the protein substrate.

2.4 Determination of nitrogen recovery

Nitrogen recovery was used as a solubility index of nitrogen to reflect the productivity of the hydrolysis. After the enzymatic hydrolysis, the supernatant was collected by centrifuging at 14,400 g for 20 min. The NR was calculated according to Benkajul and Morrissey (1997):

(2)

2.5 Determination of amino acid composition of hydrolysates

Amino acid identification was performed by high-performance liquid chromatography (HPLC). Briefly, 0.1 g sample power and 1 ml of 0.1% formic acid-0.2% methanol-H₂O were added into an empty tube, which was then added 4 ml of acidic acetone at −30°C for 24 hr. The resulting mixture was filtered and centrifuged at 4°C for 10 min at 14,400 g to collect supernatant. Afterward, the supernatant was purged with nitrogen to eliminate the organic solvent and redissolved with 0.1% formic acid-H₂O.

2.6 Determination of volatile compounds of hydrolysates

The volatile compounds in hydrolysates with different enzymes were analyzed by headspace solid-phase microextraction–gas chromatography mass spectrometry (HS-SPME-GC–MS). Two milliliters of hydrolysates was placed into a 10-ml brown glass vial. In order to make the analyte fully exposed, the SPME devised with polydimethylsiloxane was used for exposing the fiber in the headspace of the vial at 70°C. Take out the fiber, then it was transferred to the gas chromatograph injector port immediately and heated at 260°C for 5 min. The temperature was set as follows: at first, the temperature maintained at 30°C for 3 min, raised to 70°C at a ramp of 2.5°C/min, and to 150°C at 8°C/min. In the end, increased to 260°C at a ramp of 20°C/min and held for 5 min. GC-MS analysis, using HP-5 capillary column (5% phenylmethylsiloxane, 30 mm × 0.25 mm, film thickness 0.25 μm; Agilent, USA), was performed on Agilent 7890A. The instrument detector was operated in electron ionization mode with an ionization voltage of 70 eV. Helium was invoked as the carrier gas at a constant flow rate of 1.5 ml/min. The front inlet was kept at 220°C in the splitless mode. Five microliters of 2,4,6-trimethyl-pyridine (5 × 10⁻⁴ mg/ml) was added to hydrolysate as an internal standard.

2.7 Determination of molecular weight distribution

Molecular weight distribution was determined by gel permeation chromatography on a Superdex Peptide 10/300GL (GE HealthCare, Sweden). The mobile phase composed of 70% (v/v) acetonitrile and 30% (v/v) distilled water, the flow rate is 0.4 ml/min. Peptides that were known molecular weight (MW) were treated as the standard substance to calibrate the column. Cytochrome C (MW = 12,500 Da), aprotinin (MW = 6,512 Da), vitamin B12 (MW = 1,355 Da), glutathione (MW = 307 Da), and glycine (MW = 75 Da) were used as the standards.

2.8 Statistical analysis

All data were expressed as the mean ± SD performed in triplicates. The analysis of variance (ANOVA) was conducted using SPSS 12 software package (SPSS Thailand Co., Ltd., Bangkok, Thailand). The differences between variables were analyzed by Duncan's new multiple range test. Significant differences were evaluated at p < 0.05.

3.1 Chemical composition analysis

The crude protein content of rainbow trout bone is 15.90%, which was higher than that of milk (3.5%) and egg (13.3%). The fat content is 17.70%, and moisture content of rainbow trout bone is 59.60%. Protein extracted from the rainbow trout bone under the condition of 121 ± 1°C for 30 min, while the extraction rate is up to 80%. Besides, the high content of crude protein and fatty acids proves the rainbow trout bone is apposite to add to food to improve nutritional properties or extract the active ingredient that processed into health products.

The yield of crude protein depends on the method of extraction, raw material used, and the type of deboning machine (Kijowski & Niewiarowicz, 1985). It was reported that 47.5% protein was extracted from minced cod head in dilute NaOH (pH 11) and HCl (pH 2–2.6) (Arnesen & Gildberg, 2006). Another work indicated that 50% soluble protein was available from minced cod backbones while no water added (Lanier, 1995). Whereas a study reported the yield of 11%–17% from total protein content in animal bone (Young, 2010), while our study showed that 80% of the crude protein in the rainbow trout bone can be extracted out using hot-pressure extraction method. In a word, the method of hot-pressure process by water can tremendously improve the protein recovery ratio from the bone without using any chemical solvents such as acid or alkali solution and also can avoid the side effects of these solvents.

3.2 Comparison of DH and NR by different enzymes

The DH of rainbow trout bone by five different proteases was shown in Figure 1. With regard to the effects of hydrolysis time, it was showed that DH of rainbow trout bone protein treated with alkaline protease, papain, neutral protease, flavourzyme, or trypsin all increased. The highest DH values (15.03%) were achieved with the reaction time of 180 min and hydrolysate treated with alkaline protease (HA) showed apparently higher DH than that treated with other enzymes. At the same time, hydrolysate treated with trypsin (HT) always had the lowest DH. DH was employed as an indicator of the cleavage of peptide bonds. At first, the DH of hydrolysates demonstrated that part of rainbow trout bone extraction was hydrolyzed during hot-pressure process and decomposed into free amino acids (FAA) and peptides, leading to elevated DH. The curve of DH increased rapidly during the first 2 hr of hydrolysis and then showed a slow increased, which was similar to the results of red hake hydrolysates (Imm & Lee, 1999) and thornback ray gelatin hydrolysates (Lassoued et al., 2015). All peptide bonds susceptible to enzymatic hydrolysis as well as the enzyme inhibition and deactivation could result in the attenuated increasing trend after the starting rise (Ekuwaenyonam, Phillips, & Saalia, 2009).

With the continuation of enzymatic hydrolysis reaction and increase in DH, the NR kept on growing at the same time. NR reflects the yield that can be recovered from the hydrolysis process. Figure 2 showed that NR of rainbow trout bone by five different proteases at the same enzymatic time. The NR value of AH was the highest that reached 83.44%. Changes in DH and NR indicated pretty similar tendency. Similar conclusions were got in tuna waste hydrolysates (Guerard et al., 2002).

3.3 Comparison of total amino acids and FAAs of hydrolysates

Total 18 amino acids were well identified from the rainbow trout bone protein. As showed in Table 1, rainbow trout bone protein had a well-balanced and abundant amino acid composition, the amount of total amino acid was 983 mg/g in rainbow trout bone protein and essential amino acids account for 38.35%. Bitter amino acids account for 43.54% while umami amino acid content was 22.69%. The most abundant amino acid was glutamic acid (132.09 mg/g in rainbow trout bone protein), and the least abundant was tryptophan (3.15 mg/g in rainbow trout bone protein). In general, the enrichment of amino acids will have a positive effect on the diet structure.

3.4 Volatile compounds in different hydrolysates

Volatile compounds in five hydrolysates for 180 min were identified and were listed in Table 2, which mainly belong to different chemical classes such as alkanes, alkene, alcohols, aldehydes, ketones, pyrazines, ester, and others. The main compounds that play a major role in flavor in hydrolysates are alcohols, aldehydes, and pyrazines. Pyrazines played an important role in the hydrolysates because of their low perception threshold and distinctive characteristic odors (Ryan et al., 2004). Results showed that 71 kinds of volatile compounds contained in the hydrolysate of the rainbow trout bone that treated with alkaline protease. Among them, 30 kinds of hydrocarbons, 20 kinds of alcohols, eight kinds of aldehydes, and eight ketones. Besides, 37 kinds of volatile compounds were identified from the hydrolysate of the rainbow trout bone that treated with papain. Among them, 18 kinds of alcohols, seven kinds of aldehydes, and six ketones. The composition and proportion of the volatile compounds changed with the enzymes system.

The hydrolysate that was treated with alkaline protease contained the most volatile compounds. Among them, the content of 2-ethyl-2-hexenal (25.25%), 2-ethyl-5-methyl-pyrazine (13.55%), and 4-methyl-2-propyl-1-n-pentanol (6.35%) were relatively high. Moreover, 2-ethyl-2-hexenal is the most important material that causes the fish oil to produce the smell. It is a significant organic intermediate for the production of octanol and spices. Aldehydes have lower thresholds, which were produced by oxidative degradation of polyunsaturated fatty acids in fish oil under the action of enzymes and microorganisms. A class of substances most commonly found in peanut oil is pyrazine, including 2-ethyl-5-methyl-pyrazine, 2,5-dimethylpyrazine, and 2-ethyl-6-methyl-pyrazine. These compounds played a major role in the formation of rainbow trout oil. Ketones make an important contribution to aroma because of its low odor threshold value. Besides, hydrocarbons that produced by the free radicals of fatty acids have little effect on flavor formation because of its high odor threshold.

3.5 Distribution of peptides molecular weight

The apparent distributions of peptides that are solved by different enzymes were divided into seven ranges and presented in Figure 3. The molecular weight of polypeptides in the five hydrolysates was mainly distributed in the range of 1,000–3,000 Da. The content of small peptides prepared by alkaline protease was the highest, while the content of small peptides hydrolyzed by flavourzyme was the lowest, while the DH of HA was the highest. All these data demonstrated that proteins were degraded into polypeptides and then decomposed into small peptides with the progression of hydrolysis. Similar results were also reported in previous studies (Kristinsson & Rasco, 2000; Ovissipour et al., 2009).

The tendency of the amount of peptides with MW of lower than 500 Da of hydrolysates that digested by different enzymes basically matches the changes in content of FAAs (Table 3). So the components in the fractions with MW lower than 500 Da probably were FAA. Moreover, the highest proportion of the peptides in all of the hydrolysates was the fraction with MW range of 1,000–3,000 Da. Then some peptides were broken down into small peptides, which indicated the potential functional properties and bioactivity of the hydrolysates. Further studies should be investigated to clarify the bioactivity of the peptides.