Method for the preparation of skipjack tuna extract having uric acid-lowering effect and the use thereof

Abstract

The present invention discloses a method for the preparation of skipjack tuna extract having hypouricemic effect and the use thereof, which is simple in processing operations, low in production cost, and is free from pollution. The prepared skipjack tuna extract has a potent hypouricemic effect, and has a significant therapeutic effect on hyperuricemia, with no toxic and side effects. The method in an example of the present invention comprises: pretreating skipjack tuna to obtain a skipjack tuna slurry; enzymolysing the skipjack tuna slurry to obtain a crude enzymolysis liquid; removing fishy smell and bitter taste, removing impurities by activated charcoal, and filtering to obtain a refinded enzymolysis liquid; concentrating under vacuum and spray-drying to obtain the skipjack tuna extract. The present invention further discloses use of the skipjack tuna extract in health care products or food products.

Claims

1. A method for the preparation of skipjack tuna (bonito) extract having uric acid lowering effect, comprising: (1) removing the head and entrails of the skipjack tuna before mincing, adding 1 to 2 times of water by weight of the minced skipjack tuna, heating under stirring at 80 to 100 C. for 5 to 30 min, and then lowering the temperature to 50 to 60 C. to obtain a skipjack tuna slurry; (2) adding 0.2% to 3.2% of protease by weight of the minced skipjack tuna to the skipjack tuna slurry, carrying out hydrolyzation for 5 to 9 h while keeping the temperature at 50 to 55 C., then heating to 85 to 95 C. which is kept for 15 to 30 min to deactivate the enzyme, and centrifuging to obtain a supernatant, which is a crude enzymatic hydrolysate; (3) adding 0.5% to 1.0% of activated charcoal by weight of the crude enzymatic hydrolysate to the crude enzymatic hydrolysate, stirring for 0.5 to 1.0 hour at a temperature of 45 to 55 C., and then filtering through a 0.5 m filter paper to obtain a refined enzymatic hydrolysate; (4) concentrating the refined enzymatic hydrolysate under vaccum and spray-drying to obtain the skipjack tuna extract.

2. The method according to claim 1, characterized in that the protease is one or more of NEUTRASE (bacterial protease), FLAVOURZYME (bacterial peptidase), papain, ALCALASE (bacterial protease) and proteolytic enzymes.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the constitutional diagram of the peptide powder of Example 1 after separation by ion-exchange resin.

(2) FIG. 2 shows the analysis of inhibitory activity of each component of the peptide powder of Example 1 on xanthine oxidase, and the antioxidant activity thereof

(3) FIG. 3 is the MALDI-TOF-MS primary mass spectrogram of the peptide powder of Example 1.

DETAILED EMBODIMENTS

(4) The examples of the present invention provide a method for the preparation of skipjack tuna extract having uric acid-lowering effect and use thereof, for reducing the content of uric acid.

(5) The present invention provides a method for the preparation of skipjack tuna extract having uric acid-lowering effect: firstly removing and mincing the head and viscera of the skipjack tuna, adding 1 to 2 times of water by weight of the minced skipjack tuna meat, heating under stirring within 80 to 100 C. for 5 to 30 min, then lowering the temperature within 50 to 55 C. to obtain a skipjack tuna slurry; adding 0.2% to 3.2% of protease by weight of the minced skipjack tuna meat to the skipjack tuna slurry, carrying out hydrolyzation for 5 to 9 h while keeping the temperature within 50 to 55 C., then heating up to 85 to 95 C. which is kept for 15 to 30 min to deactivate the enzyme, the supernatant obtained by centrifugation is a crude enzymatic hydrolysate; removing fishy smell and bitter taste: adding 0.5% to 1.0 wt % of activated charcoal to the crude enzymatic hydrolysate, stirring for 0.5 to 1.0 hour within a temperature of 45 to 55 C., filtering through a 0.5 m filter paper to obtain a refined enzymatic hydrolysate; concentrating the refined enzymatic hydrolysate under vacuum and spray-drying to obtain the skipjack tuna extract; preparing the skipjack tuna extract into a health care product or food product, which can be in the form of oral liquid, capsule, tablet, pill, powder, pulvis, or granule. The skipjack tuna extract can be comprised in an amount of 0.5% to 70% of the health care product or food product by weight.

(6) The method for the preparation of skipjack tuna extract having uric acid-lowering effect according to the present invention is simple in operation, low in production cost, free from pollution, simple and accurate in in vitro screening indexes. Meanwhile, the active polypeptide ingredients of the skipjack tuna were reserved which has a potent uric acid-lowering activity, and can significantly reduce the serum creatinine level, has certain protective effects on renal function, and is free from toxic and side effects. In addition, the raw materials of the present invention come from readily available skipjack tuna. The prepared skipjack tuna extract shows potent uric acid-lowering activity and has significant therapeutic effects against hyperuricemia as verified by animal experiments.

(7) In order to make technicians of this field better understand the technical solutions of the present invention, the present invention is further illustrated in detail through the following specific examples.

EXAMPLE 1

(8) (1) The head and viscera of the skipjack tuna were removed, cleaned completely, and minced by a meat mincer. 500 kg of minced skipjack tuna was weighed, and 500 kg of water was added. The resultant mixture was heated under stirring at 100 C. for 10 min to obtain a skipjack tuna meat slurry. The temperature was then lowered to 50 C.

(9) (2) 5 kg of neutrase and 7.5 kg of flavourzyme were added to the skipjack tuna meat slurry, hydrolyzation was carried out for 5 hours at a temperature kept at 50 C., and then the mixture was heated at 95 C. for 15 min to deactivate the enzyme. Finally, centrifugation was carried out to obtain a supernatant, and a crude enzymatic hydrolysate of skipjack tuna was thus obtained;

(10) (3) 8 kg of activated charcoal was added to the crude enzymatic hydrolysate of skipjack tuna. The resultant mixture was stirred for 1.0 hour at a temperature kept at 50 C., then filtered through a 0.5 m filter paper, and the obtained filtrate was the refined enzymatic hydrolysate of the skipjack tuna.

(11) (4) The refined enzymatic hydrolysate of skipjack tuna was concentrated under vacuum to obtain solid matter with a content of 30%, which was then spray-dried to obtain 50 kg of skipjack tuna extract peptide powder A.

(12) FIG. 1 is the constitutional diagram of the peptide powder A after separation by ion-exchange resin. FIG. 2 shows the analysis of the inhibitory activity of each component of the peptide powder A on xanthine oxidase, and the antioxidant activity thereof. FIG. 3 is the MALDI-TOF-MS primary mass spectrogram of the peptide powder A. As is shown in FIG. 3, the molecular weight of the main effective peptide components of the peptide powder A was determined to be less than 5000 Da by MALDI-TOF-MS mass spectrometry.

EXAMPLE 2

(13) (1) The head and viscera of the skipjack tuna were removed, cleaned completely, and minced by a meat mincer. 500 kg of minced skipjack tuna was weighed, and 1000 kg of water was added. The resultant mixture was heated under stirring at 100 C. for 10 min to obtain a skipjack tuna meat slurry. The temperature was then lowered to 50 C.

(14) (2) 7.5 kg of papain was added to the skipjack tuna meat slurry, hydrolyzation was carried out for 5 hours at a temperature kept at 50 C., and then the mixture was heated at 95 C. for 15 min to deactivate the enzyme. Finally, centrifugation was carried out to obtain a supernatant, and a crude enzymatic hydrolysate of skipjack tuna was thus obtained;

(15) (3) 8 kg of activated charcoal was added to the crude enzymatic hydrolysate of skipjack tuna. The resultant mixture was stirred for 1.0 hour at a temperature kept at 50 C., then filtered through a 0.5 m filter paper, and the obtained filtrate was the refined enzymatic hydrolysate of the skipjack tuna.

(16) (4) The refined enzymatic hydrolysate of skipjack tuna was concentrated under vacuum to obtain solid matter with a content of 30%, which was then spray-dried to obtain 40 kg of skipjack tuna extract peptide powder B.

(17) The molecular weight of the main effective peptide components was determined by MALDI-TOF-MS mass spectrometry to be less than 1000 Da.

EXAMPLE 3

(18) (1) The head and viscera of the skipjack tuna were removed, cleaned completely, and minced by a meat mincer. 500 kg of minced skipjack tuna was weighed, and 500 kg of water was added. The resultant mixture was heated under stirring at 100 C. for 10 min to obtain a skipjack tuna meat slurry. The temperature was then lowered to 50 C.

(19) (2) 7.5 kg of alcalase was added to the skipjack tuna meat slurry, hydrolyzation was carried out for 5 hours at a temperature kept at 50 C., and then the mixture was heated at 95 C. for 15 min to deactivate the enzyme. Finally, centrifugation was carried out to obtain a supernatant, and a crude enzymatic hydrolysate of skipjack tuna was thus obtained;

(20) (3) 8 kg of activated charcoal was added to the crude enzymatic hydrolysate of skipjack tuna. The resultant mixture was stirred for 1.0 hour at a temperature kept at 50 C., then filtered through a 0.5 m filter paper, and the obtained filtrate was the refined enzymatic hydrolysate of skipjack tuna.

(21) (4) The refined enzymatic hydrolysate of skipjack tuna was concentrated under vacuum to obtain solid matter with a content of 30%, which was then spray-dried to obtain 60 kg of skipjack tuna extract peptide powder C.

(22) The molecular weight of the main effective peptide components was determined by MALDI-TOF-MS mass spectrometry to be less than 1000 Da.

EXAMPLE 4

(23) (1) The head and viscera of the skipjack tuna were removed, cleaned completely, and minced by a meat mincer. 500 kg of minced skipjack tuna was weighed, and 500 kg of water was added. The resultant mixture was heated under stirring at 100 C. for 10 min to obtain a skipjack tuna meat slurry. The temperature was then lowered to 50 C.

(24) (2) 2.5 kg of Alcalase and 5 kg of hydrolysis protease were added to the skipjack tuna meat slurry, hydrolyzation was carried out for 5 hours at a temperature of 50 C., and then the mixture was heated at 95 C. for 15 min to deactivate the enzyme. Finally, centrifugation was carried out to obtain a supernatant, and a crude enzymatic hydrolysate of skipjack tuna was thus obtained;

(25) (3) 8 kg of activated charcoal was added to the crude enzymatic hydrolysate of skipjack tuna. The resultant mixture was stirred for 1.0 hour at a temperature kept at 50 C., then filtered through a 0.5 m filter paper, and the obtained filtrate was the refined enzymatic hydrolysate of the skipjack tuna.

(26) (4) The refined enzymatic hydrolysate of the skipjack tuna was concentrated under vacuum to obtain solid matter with a content of 30%, which was then spray-dried to obtain 60 kg of skipjack tuna extract peptide powder D.

(27) The molecular weight of the main effective peptide components was determined by MALDI-TOF-MS mass spectrometry to be less than 5000 Da.

EXAMPLE 5

(28) (1) The head and viscera of the skipjack tuna were removed, cleaned completely, and minced by a meat mincer. 500 kg of minced skipjack tuna was weighed, and 700 kg of water was added. The resultant mixture was heated under stirring at 80 C. for 30 min to obtain a skipjack tuna meat slurry. The temperature was then lowered to 60 C.

(29) (2) 1.0 kg of Alcalase were added to the skipjack tuna meat slurry, hydrolyzation was carried out for 9 hours at a temperature of 55 C., and then the mixture was heated at 85 C. for 30 min to deactivate the enzyme. Finally, centrifugation was carried out to obtain a supernatant, and a crude enzymatic hydrolysate of skipjack tuna was thus obtained;

(30) (3) 6 kg of activated charcoal was added to the crude enzymatic hydrolysate of skipjack tuna. The resultant mixture was stirred for 1.0 hour at a temperature kept at 45 C., then filtered through a 0.5 m filter paper, and the obtained filtrate was the refined enzymatic hydrolysate of the skipjack tuna.

(31) (4) The refined enzymatic hydrolysate of the skipjack tuna was concentrated under vacuum to obtain solid matter with a content of 30%, which was then spray-dried to obtain 55 kg of skipjack tuna extract peptide powder E.

EXAMPLE 6

(32) (1) The head and viscera of the skipjack tuna were removed, cleaned completely, and minced by a meat mincer. 500 kg of minced skipjack tuna was weighed, and 1000 kg of water was added. The resultant mixture was heated under stirring at 90 C. for 15 min to obtain a skipjack tuna meat slurry. The temperature was then lowered to 55 C.

(33) (2) 6 kg of Alcalase and 10 kg of hydrolysis protease were added to the skipjack tuna meat slurry, hydrolyzation was carried out for 6 hours at a temperature of 50 C., and then the mixture was heated at 90 C. for 20 min to deactivate the enzyme. Finally, centrifugation was carried out to obtain a supernatant, and a crude enzymatic hydrolysate of skipjack tuna was thus obtained;

(34) (3) 15 kg of activated charcoal was added to the crude enzymatic hydrolysate of skipjack tuna. The resultant mixture was stirred for 0.5 hour at a temperature kept at 55 C., then filtered through a 0.5 m filter paper, and the obtained filtrate was the refined enzymatic hydrolysate of the skipjack tuna.

(35) (4) The refined enzymatic hydrolysate of the skipjack tuna was concentrated under vacuum to obtain solid matter with a content of 30%, which was then spray-dried to obtain 62 kg of skipjack tuna extract peptide powder F.

(36) The molecular weight of the main effective peptide components was determined by MALDI-TOF-MS mass spectrometry to be less than 5000 Da.

EXAMPLE 7

(37) Proportion of parts by weight: Smilacis Glabrae Rhizoma, 18; Cichorii Herba, 13; Plantaginis Herba, 10; Coicis Semen, 17; Pueraria Lobata Radix, 7; Alismatis Rhizoma, 3; the skipjack tuna extract D as described in Example 4, 0.3; with which a health care food product having the uric acid-lowering function was prepared.

(38) Animal tests were conducted using the skipjack tuna extracts A, B, C and D as prepared in the above-described Examples.

(39) Since rats share 90% genes with human beings, rats are globally recognized experimental model in the physiological and disease studies on human bodies. During the experiments for verification of uric acid-lowering effects, due to the presence of uricase in rat body, the uric acid will degrade during the metabolic process. Therefore, in the present animal experiment, rats were intragastricly administered with potassium oxonate everyday to block the effects of uricase in the rats, thus the serum uric acid level of the rat increases, and rats having a uric acid content of greater than 110 umol.Math.L.sup.1 were determined to be successful models and were further used for the experiments by intragastric administration.

(40) 72 SD rats (SPF grade, male, 20020 (g)) were provided by Laboratory Animals Centre, Guangzhou University of Chinese Medicine (License Number: SCXK(Yue)2013-0020). Reagents: allopurinol tablets (Guangdong P.D. Pharmaceutical Co., Ltd., Approval Number: National Drug Approval No. H44021368); potassium oxonate (Shandong Zhongke Taidou Chemical Co., Ltd., Batch No. 120901); sodium carboxymethyl cellulose (Shanghai Celluloid Factory, Product Standard No. GB2760); uric acid, urea nitrogen (BUN) assay kit (NanJing Jiancheng Bioengineering Institute, Production batch No. 20140306).

(41) Animal grouping and modeling: the rats in modeling group were intragastricly administered with potassium oxonate for one week (once daily), anaesthetized with 3% pentobarbitol sodium (i.p., 30 mg.Math.kg.sup.1), and blood (0.5 ml) was collected from retro-orbital vein plexus and centrifuged at 4 C., 3000 rpm for 15 min. The supernatant sera were taken for determining the content of uric acid. Rats in normal control group were intragastricly administered with equal volume of solvent. Those rats having a uric acid content of greater than 110 mol.Math.L.sup.1 were determined to be successful models. The successfully modeled rats were randomly divided into 6 groups (12 rats/group) according to uric acid contents, including one normal group, one model group (equal volume of distilled water), and four skipjack tuna peptide groups. The skipjack tuna peptide groups were intragastricly administered with skipjack tuna extracts A, B, C and D, respectively, with a volume of 10 ml/kg. The model group was administered with equal volume of distilled water. On 7th and 14th day after treatment with the above-described samples, 50 min after the last administration and anesthetization with 3% pentobarbitol sodium (i.p., 30 mg.Math.kg.sup.1), blood (0.5 ml) was collected from retro-orbital vein plexus and the serum uric acid content was determined. On 21st day after treatment, the rats were anaesthetized with 3% pentobarbitol sodium, 5 ml of blood was collected from abdominal aorta, and the serum uric acid content was determined. Serum uric acid assay was carried out according to tungstic acid method, which was performed and determined strictly according to the instructions of the kit. Statistical processing: all the data were expressed in (xs) and processed with spss 16.0 statistical software.

(42) The results are shown in Table 1 and Table 2. As is shown in Table 1, there is no significant difference in body weight amongst those groups of rats before and after treatment of the model animal with antigout peptides. Before administration, the body weights of the rats are comparable, and thus the results are comparable.

(43) TABLE-US-00001 TABLE 1 Effects of antigout peptides on body weight of rats having hyperuricemia induced by potassium oxonate (X s) Before Groups Animal number administration Treatment 7 d Treatment 14 d Treatment 21 d Normal group 12 222.2 16.7 262.3 20.5 291.3 27.6 319.2 30.1 Model group 12 230.8 11.7 274.3 27.6 306.3 23.9 324.1 24.5 Group A 12 220.7 14.7 270.3 22.2 284.0 27.8 302.1 44.0 Group B 12 230.8 18.4 266.2 16.4 299.5 25.5 314.3 28.1 Group C 12 221.4 8.3 268.9 9.5 291.8 14.0 301.7 12.2 Group D 12 217.9 14.4 276.4 24.5 290.2 28.6 301.3 31.2

(44) TABLE-US-00002 TABLE 2 Effects of different treating duration of antigout peptides on serum uric acid content of rats having hyperuricemia induced by potassium oxonate (X s) Before administration Treatment 7 d Treatment 14 d Treatment 21 d Uric acid value Uric acid value Uric acid value Uric acid value Groups n (mol/L) n (mol/L) n (mol/L) n (mol/L) Normal 12 74.9 28.7 12 75.2 23.2 12 81.4 21.2 12 72.7 26.0 Group Model 12 225.8 41.7 12 216.6 32.1a 12 249.8 40.6a 12 212.0 30.0a Group Group A 12 211.4 31.0 12 214.5 47.0f 12 205.4 47.5c 12 205.4 47.5c Group B 12 258.8 49.4 12 213.3 70.0f 204.5 62.1c 12 210.7 27.1f Group C 12 235.2 27.0 12 166.2 59.8c 12 187.9 34.8e 12 189.8 23.9c Group D 12 201.1 54.1 12 280.2 40.8e 12 277.6 56.0f 12 157.8 36.7e Notes: as compared to normal control group: ap < 0.01; as compared to model group: ep < 0.01, cp < 0.05, fp < 0.05.

(45) As is shown in Table 2, on 7th, 14th and 21th days after treatment with potassium oxonate, the blood uric acid contents in rats of the model group are significantly higher than those in normal control group (p<0.01), which indicates a successful animal modeling.

(46) The hyperuricemic mice were treated with the skipjack tuna extracts. The skipjack tuna extract groups A, B, C and D showed an uric acid-lowering effect (p<0.05, p<0.01). The uric acid content in group C was reduced from 235.2 mol/L to 189.8 mol/L (reduced by 19.3%) and remained stable, indicating the skipjack tuna extract in group C has a stable effects of reducing the uric acid content and inhibiting the increase of uric acid on hyperuricemic rats that induced by potassium oxonate, while group A had certain uric acid-lowering effect only on 14th day. Group D was found to be most effective, as the uric acid content was significantly reduced on 21st day from 201.1 mol/L to 157.8 mol/L (the uric acid content was reduced by 21.5%), while the uric acid content in the model group was reduced from 225.8 mol/L to 212.0 mol/L (reduced by 6.1%). The data above indicates that the extract of the present invention has a significant uric acid-lowering effect.

(47) The above-described Examples are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above-described Examples. Any other alteration, modification, substitution, combination and simplification without departing from the spirit and principle of the present invention are all equivalent alternatives, and are all included within the protection scope of the present invention.

(48) The peptide powder prepared according to the present invention is capable of reducing the serum uric acid levels, and the effect is the best especially when Alcalase and proteolytic enzyme are used for enzymolysis during the preparation process.

(49) The results show that the skipjack tuna extract prepared according to the method of the present invention has a potent uric acid-lowering effect, a significant therapeutic effect on hyperuricemia, has a certain protective effect on renal function; and is free from toxic and side effects as the extract of the present invention is obtained from an edible raw material, i.e., skipjack tuna.