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Pilot and plant scaled production of ACE inhibitory hydrolysates from Acetes chinensis and its in vivo antihypertensive effect
Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 5956–5959
Short Communication
Pilot and plant scaled production of ACE inhibitory hydrolysates from Acetes chinensis and its in vivo antihypertensive effect Hai-Lun He, Hao Wu, Xiu-Lan Chen, Mei Shi, Xi-Ying Zhang, Cai-Yun Sun, Yu-Zhong Zhang *, Bai-Cheng Zhou State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, China Received 5 June 2007; received in revised form 19 October 2007; accepted 23 October 2007 Available online 3 December 2007
Abstract The angiotensin-I-converting enzyme (ACE) inhibitory oligopeptide-enriched hydrolysates from Acetes chinensis by treatment with the protease from Bacillus sp. SM98011 were produced at pilot scale (100 L) and plant scale (1000 L). The pilot and plant scaled hydrolysate products almost had the same properties as that at laboratory scale. Spray-drying had little effect on the peptide composition and bioactivity of the hydrolysates. The plant scaled hydrolysates were used to study its blood pressure-depressing effect in vivo. It caused reduce of 18.3–38.6 mmHg of the blood pressure of spontaneously hypertensive rats in dose-dependent manner in the range of 100– 1200 mg/kg/day. Histopathologic study showed that the pathologic changes of heart and brain in SHRs got obvious alleviation after treatment of the hydrolysates. 2007 Elsevier Ltd. All rights reserved. Keywords: Hydrolysates; Angiotensin-I-converting enzyme (ACE); ACE inhibitory peptide; Spontaneously hypertensive rats (SHRs); Histopathology
1. Introduction Enzymatic hydrolysis of proteins is a well known method to modify protein physical properties and, more importantly, to improve their nutritional properties. The application of enzyme technology to recover modified seafood protein may produce a broad spectrum of food ingredients or industrial products (Shahidi et al., 1994; Je´roˆme et al., 2002; Tsai et al., 2006). Recent advances in biotechnology have demonstrated the capacity of enzymes to produce novel food products, modified foodstuffs and improved waste management (Afonso and Borquez, 2002). These trends cater to consumer preferences for ‘natural’ products. Among many functional peptides, ones possessing hypotensive activity are thought to be useful as functional food materials for high blood pressure patients.
*
Corresponding author. Fax: +86 531 88564326. E-mail address: [email protected] (Y.-Z. Zhang).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.10.038
This activity is mainly due to the inhibition of the angiotensin-I-converting enzyme (ACE). ACE has been classically associated with the renin–angiotensin system, which regulates peripheral blood pressure via conversion of angiotensin-I to angiotensin-II. Inhibition of ACE may have an antihypertensive effect as a consequence of a decrease in blood pressure (Skeggs et al., 1957). Although synthetic ACE inhibitors, including captopril, enalapril and listinopril, are remarkably effective as antihypertensive drugs, they inevitably cause adverse side effects (Atkinson and Robertson, 1979). Many ACE inhibitory peptides have been reported in hydrolysates from diverse food proteins digested with different proteases (Meisel, 1998). Most of these peptides were identified from hydrolytic products of terrestrial food proteins but less from marine proteins. The composition and primary amino acid sequences of marine proteins are different from those of terrestrial proteins; therefore, marine proteins may become important resources for the exploitation of novel ACE inhibitory peptides by enzymatic hydrolysis.
H.-L. He et al. / Bioresource Technology 99 (2008) 5956–5959
Acetes chinensis growing in Bo Hai Gulf of China is an underutilized shrimp species with a low commercial value, as a consequence of low consumption by humans. Every year, about 300,000 tones of A. chinensis are made into dried small shrimps or shrimp sauce that have low adding value. The advanced utilizing process of this marine resource is a significant topic in marine biologic study. Because of its high content of muscle proteins and chitin, it is a valuable raw material for further processing. Our previous studies had shown that hydrolysates of A. chinensis digested with the crude protease from Bacillus sp. SM98011 possessed ACE inhibitory activity and three novel ACE inhibitory peptides had been found (He et al., 2006a; He et al., 2006b). In the present work, the enzymatic hydrolysis of the shrimp A. chinensis was scaled up to pilot scale and plant scale. The peptide compositions, ACE inhibitory activities, and in vivo anti-hypertensive effect in SHRs of the hydrolysates were also investigated.
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dried and kept at 4 C. Hydrolysates of pilot and plant scale were condensed with ball vacuum concentration tanker (RD500, GuanYi Mechanical Equipment Co., Ltd, China) and were dried with Spray Dryer (BoDa Co., China) at a 10 kg h 1 flow rate with a 170 C inlet temperature and 90 C outlet temperature (Kapel et al., 2006). 2.4. Assay of content of peptides in hydrolysates Content of peptides in the hydrolysates was assayed with the previous method (He et al., 2006a). 2.5. ACE-inhibition activity assay ACE inhibitory activity of the hydrolysates and the IC50 value was assayed with the previous method (He et al., 2007). 2.6. RP-HPLC analysis
2. Methods 2.1. Experimental materials and strains Acetes chinensis was purchased from Chinese Dayudao Group and stored at cold store. Protease-producing bacterium Bacillus sp. SM98011 was preserved in our laboratory. 2.2. Protease preparation and activity assay The strain Bacillus sp. SM98011 was fermented in 200 L fermenter at 28 C for 36 h, and the activity of protease SM98011 in ferment broth reached 4000 U/ml. Then, the ferment broth was centrifuged at 10,000g for 20 min, and the supernatant was collected and used for protein hydrolysis. The activity of protease was determined with the method as before (He et al., 2004). The protease SM98011 was crude serine protease and optimum hydrolysis temperature and pH of protease was 50 C and pH 7.5. 2.3. Acetes chinensis hydrolysates production at pilot scale and plant scale The hydrolysis was carried out at laboratory scale (500 ml Erlenmeyer flask), pilot scale (100 L thermostatically stirred-batch reactor) and plant scale (1000 L thermostatically stirred-batch reactor), respectively. In the batch experiments, minced shrimp was mixed with water at a ratio of 1:1 (w/w) by continuous stirring and the enzyme was added at a ratio of 400,000 U/kg (U/w, enzyme/protein substrate). All reactions were performed at pH 7.5 and 50 C for 5 h with constant agitation (200 rpm). The reactions were stopped by heating the solution to 90 C for 15 min, assuring the complete inactivation of the enzyme. The resulted slurry was centrifuged at 9000g for 20 min and the supernatant was the hydrolysates of A. chinensis. The supernatant of laboratory scale was freeze-
Analysis of peptide composition of the hydrolysates was carried out by RP-HPLC (Waters alliance2695, USA) coupled with Dual Wavelength UV Detector 2487. The separation was performed using a Symmetry C18 column (250 · 4.6 mm). The eluent was 0.1% trifluoroacetic acid (A) and a mixture of eluent A with acetonitrile (2/3, v/v) (B). A linear A–B gradient from 10% to 60% was applied over a period of 30 min followed by a linear A–B gradient from 60% to 10% for 15 min with flow rate 1 ml/min monitored at 214 nm. 2.7. Antihypertensive activity in SHRs Forty male SHRs (12 weeks old, Calco, Italy) and 10 age-matched normotensive Wistar-Kyoto (WKY) rats were kept at an ambient temperature of 23 ± 1 C, with free access to food and water before beginning the experiments. SHRs were divided into five groups (n = 8). A negative control group was orally administered with physiological saline, a positive control group was orally administered with captopril at 10 mg/kg/day and three experimental groups were orally administered with the hydrolysates at doses of 100, 500, 1200 mg/kg/day, respectively. Five untreated WKY rats served as normotensive control group. The other five WKY rats were administered with hydrolysates at a dose of 500 mg/kg/day. Dosages were adjusted every week according to the changes of body weight. The systolic blood pressure (SBP) of the rats was measured by a tail-cuff method (model BP-98, Softron, Tokyo, Japan). During a month of treatment, measurements were taken weekly. Several determinations were made for each animal, and the values were considered valid if five consecutive measurements were within 6 mmHg (Jung et al., 2006). Under anaesthesia with sodium pentobarbital (60 mg/kg i.p.), the animals were sacrificed immediately after the end of treatment. The rats’ hearts, brains were removed and immediately stored in 10% formalin
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H.-L. He et al. / Bioresource Technology 99 (2008) 5956–5959
solution of 4 C. Then, the fixed tissue was dehydrated in graded ethanol solutions, and embedded in paraffin. Sections were cut on a MICRON hm-310 microtome at a thickness of 5 lm and were stained with Hematoxylin and eosin (HE). Images were captured by a high-resolution video camera (Sony ccd-iris). 3. Results and discussion 3.1. Characterization of the hydrolysates from A. chinensis produced at pilot and plant scale The content of peptides in shrimp hydrolysates was 48.7 ± 0.51% at pilot scale product and 47.9 ± 1.8% at plant scale product, which had a little reduce compared with that of laboratory scale product (51.3 ± 0.2%). The ACE inhibitory IC50 value of the product was 0.96 ± 0.03 mg/ml at pilot scale and 0.97 ± 0.05 mg/ml at plant scale. Both of them were almost the same as that at laboratory scale (0.98 ± 0.02 mg/ml). The results showed that, after scaled up to pilot and plant scale, the hydrolysates almost had the same ACE inhibitory activity. It is satisfactory that the total peptide contents, and the ACE inhibitory activity of the hydrolysates produced at pilot and plant scales are almost the same as that of product at laboratory scale. 3.2. Comparison of the peptide chromatogram in the hydrolysates produced at laboratory, pilot and plant scales The HPLC chromatographic analysis of the products at laboratory, pilot and plant scales showed that there were no significant differences of peptide composition among the products at different scales. Therefore, the high correspondence of the products shows that the hydrolysis conditions at the laboratory scale can be used at pilot and plant scales. This standardization of the peptide chromatogram can be used for the quality control and quality detection of the industrial production of the hydrolysates. There have been many reports on the ACE inhibitory activity of peptides derived from food proteins by enzymatic hydrolysis. However, there is relatively less information on the ACE inhibitory activity and hypertensive activities of marine proteins. High conversion and ACE inhibitory activity of the hydrolysates from the marine shrimp are promising. It can be produced at plant scale to be utilized in functional food as bioactive peptides.
air caused little change on the peptide composition of the hydrolysates, especially for the ACE inhibitory peptides. Therefore, the oligopeptide-enriching ACE inhibitory hydrolysates from shrimp A. chinensis are thermostable. 3.4. Blood pressure-depressing activity of the hydrolysates in SHRs The hydrolysates from A. chinensis have good ACE inhibitory activity (He et al., 2006a). Five inhibitory peptides had been isolated from the hydrolysates, and three of them were novel ACE inhibitory peptides, giving further evidences for the ACE inhibitory activity of the hydrolysates in vitro (He et al., 2006b). In this article, the in vivo experiments had been done. At the beginning of the study, SBPs of SHRs were already higher than that of the WKY rats and these differences persisted as the time progressed. As shown in Fig. 1, the average SBP of the ACE inhibitor group was 163 ± 1.5 mmHg just before the administration. After oral administration of the spray-dried hydrolysates each day, the SBPs of SHRs started to decrease in dosedependent manner. After a month administration in the range of 100–1200 mg/kg/day, the decrease of the blood pressure in SHRs was 18.3–38.6 mmHg. The blood pressure-depressing effect of the highest dose of the hydrolysates was similar to that of commercial antihypertensive drug Captopril at a dose of 10 mg/kg/day. Otherwise, no obvious decrease of SBP was observed in control SHRs group administrated with physiological saline. On the other hand, administration of the hydrolysates at a dose of 500 mg/kg/day did not show any significant hypotensive effect on WKY rats. These results showed that the marine shrimp hydrolysates possess a significant and safe antihypertensive effect on SHRs. Because the hydrolysates used
3.3. Effect of spray-drying on the peptide composition in the hydrolysates at plant scale The peptide chromatogram of the hydrolysates before and after spray-drying was analyzed. There was little difference of the peptide spectra between them. The spray-dried hydrolysates still kept its high ACE inhibitory activity at an IC50 value of 0.97 mg/ml, which is almost the same as that of the hydrolysates at laboratory scale. These results showed that exposure of hydrolysates to high temperature
Fig. 1. Blood pressure-depressing activity of the hydrolysates from Acetes chinensis: (a) SHR negative control (with physiological saline); (b) low dose group (with hydrolysates 100 mg/kg/day); (c) medium dose group (with hydrolysates 500 mg/kg/day); (d) high dose group (with hydrolysates 1200 mg/kg/day); (e) positive control (with captopril 10 mg/kg/day); and (f) WKY normotensive control (with hydrolysates 500 mg/kg/day).
H.-L. He et al. / Bioresource Technology 99 (2008) 5956–5959
in this experiment were the crude hydrolysates without any purification, the dose of the hydrolysates used was relatively high. Our previous results showed that most of the ACE inhibitory peptides in the hydrolysates from A. chinensis are short peptides with molecular mass lower than 3000 Da and the ACE inhibitory IC50 value of the hydrolysates decreased to 0.22 mg/ml from 0.98 mg/ml after an ultra filtration with an Amicon PM-3 membrane (He et al., 2006b). Therefore, if the hydrolysates were purified by such an ultrafiltration and the dried filtrate was used, it could be reasonably conjectured that the dose used would be dramatically reduced. 3.5. Effects of long-term administration of the hydrolysates on the organs of SHRs After administrated with the hydrolysates for thirty days, the rats were sacrificed to study its effects on the organs of SHRs. In the negative control group SHRs, the thickness of arteriolar wall in cardiac muscle increased significantly. After administration of the hydrolysates at a dose of 1200 mg/kg/day, significant reduction in the development of cardiac and vascular hypertrophy in treated SHRs was observed. Moreover, no side effects were observed in WKY rats with similar treatment of hydrolysates. Effect of long-term administration of the hydrolysates on arterioles of frontal cortex was also investigated. In the negative control group SHRs, the walls of intracerebral arterioles revealed magnification hypertrophy and the lumina of these arterioles became narrow and even occlusion. No signs of apparent nervous tissue damage were found in cerebral areas investigated. After administered with hydrolysates, there were much less pathologic changes in intracerebral arterioles in treated SHRs than in untreated ones. In addition, there was no hypertrophy in the wall of arterioles in WKY group. Histopathologic study showed that these pathologic changes got obvious alleviation after administration of the hydrolysates. Accordingly, the inhibitory ACE activity might be one of the major mechanisms for the organ protection produced by long-term treatment with hydrolysates in SHRs (Shan et al., 1999; Pu and Schiffrin, 2001). Acknowledgments This work was supported by Grants from the National High Technology Research and Development Program of China (Nos. 2001AA625040 and 2003AA625040), and Program for New Century Excellent Talents in University (NCET-06-0578).
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References Afonso, M.D., Borquez, R., 2002. Review of the treatment of seafood processing wastewaters and recovery of proteins therein by membrane separation processes-prospects of the ultrafiltration of wastewaters from the fish meal industry. Desalination 142, 29– 54. Atkinson, A.B., Robertson, J.I.S., 1979. Captopril in the treatment of clinical hypertension and cardiac failure. Lancet 2, 836–839. He, H.L., Chen, X.L., Li, J.W., Zhang, Y.Z., Gao, P.J., 2004. Taste improvement of refrigerated meat treated with cold-adapted protease. Food Chemistry 84, 307–311. He, H.L., Chen, X.L., Sun, C.Y., Zhang, Y.Z., Zhou, B.C., 2006a. Preparation and functional evaluation of oligopeptide-enriched hydrolysates from shrimp (Acetes chinensis) treated with crude protease from Bacillus sp. SM98011. Bioresource Technology 97, 385–390. He, H.L., Chen, X.L., Sun, C.Y., Zhang, Y.Z., Zhou, B.C., 2006b. Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis. Journal of Peptide Science 12, 726–733. He, H.L., Chen, X.L., Wu, H., Sun, C.Y., Zhang, Y.Z., Zhou, B.C., 2007. High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis. Bioresource Technology 98, 3499– 3505. Je´roˆme, T., Laurent, M., Jean-Luc, G., 2002. Angiotensin-I-converting enzyme inhibitory peptides from tryptic hydrolysate of bovine aS2casein. FEBS Letters 531, 369–374. Jung, W.K., Mendis, E., Je, J.Y., Park, P.J., WhaSon, B., Kim, H.C., Choi, Y.K., Kim, S.K., 2006. Angiotensin I-converting enzyme inhibitory peptide from yellow sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chemistry 94, 26–32. Kapel, R., Chabeau, A., Lesage, J., Riviere, G., Ravallec-Ple, R., Lecouturier, D., Wartelle, M., Guillochon, D., Dhulster, P., 2006. Production in continuous enzymatic membrane reactor of an antihypertensive hydrolysate from an industrial alfalfa white protein concentrate exhibiting ACE inhibitory and opioid activities. Food Chemistry 98, 120–126. Meisel, H., 1998. Overview on milk protein-derived peptides. International Dairy Journal 8, 363–373. Pu, Q., Schiffrin, E.L., 2001. Effect of ACE/NEP inhibition on cardiac and vascular collagen in stroke-prone spontaneously hypertensive rats. The American Journal of Hypertension 14, 1067–1072. Shahidi, F., Synowiecki, J., Balejko, J., 1994. Proteolytic hydrolysis of muscle proteins from Harp seal (Phoca groenladica). Journal of Agricultural and Food Chemistry 42, 2634–2638. Shan, Z.Z., Dai, S.M., Su, D.F., 1999. Relationship between baroreceptor reflex function and end organ damage in spontaneously hypertensive rats. American Journal of Physiology 277, 1200–1206. Skeggs, L.T., Kahn, J.E., Shumway, N.P., 1957. The preparation and function of the angiotensin-converting enzyme. Journal of Experimental Medicine 13, 295–299. Tsai, J.S., Lin, T.C., Chen, J.L., Pan, B.S., 2006. The inhibitory effects of freshwater clam (Corbicula fluminea, Muller) muscle protein hydrolysates on angiotensin I converting enzyme. Process Biochemistry 41, 2276–2281.
Bioresource Technology 99 (2008) 5956–5959
Short Communication
Pilot and plant scaled production of ACE inhibitory hydrolysates from Acetes chinensis and its in vivo antihypertensive effect Hai-Lun He, Hao Wu, Xiu-Lan Chen, Mei Shi, Xi-Ying Zhang, Cai-Yun Sun, Yu-Zhong Zhang *, Bai-Cheng Zhou State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, China Received 5 June 2007; received in revised form 19 October 2007; accepted 23 October 2007 Available online 3 December 2007
Abstract The angiotensin-I-converting enzyme (ACE) inhibitory oligopeptide-enriched hydrolysates from Acetes chinensis by treatment with the protease from Bacillus sp. SM98011 were produced at pilot scale (100 L) and plant scale (1000 L). The pilot and plant scaled hydrolysate products almost had the same properties as that at laboratory scale. Spray-drying had little effect on the peptide composition and bioactivity of the hydrolysates. The plant scaled hydrolysates were used to study its blood pressure-depressing effect in vivo. It caused reduce of 18.3–38.6 mmHg of the blood pressure of spontaneously hypertensive rats in dose-dependent manner in the range of 100– 1200 mg/kg/day. Histopathologic study showed that the pathologic changes of heart and brain in SHRs got obvious alleviation after treatment of the hydrolysates. 2007 Elsevier Ltd. All rights reserved. Keywords: Hydrolysates; Angiotensin-I-converting enzyme (ACE); ACE inhibitory peptide; Spontaneously hypertensive rats (SHRs); Histopathology
1. Introduction Enzymatic hydrolysis of proteins is a well known method to modify protein physical properties and, more importantly, to improve their nutritional properties. The application of enzyme technology to recover modified seafood protein may produce a broad spectrum of food ingredients or industrial products (Shahidi et al., 1994; Je´roˆme et al., 2002; Tsai et al., 2006). Recent advances in biotechnology have demonstrated the capacity of enzymes to produce novel food products, modified foodstuffs and improved waste management (Afonso and Borquez, 2002). These trends cater to consumer preferences for ‘natural’ products. Among many functional peptides, ones possessing hypotensive activity are thought to be useful as functional food materials for high blood pressure patients.
*
Corresponding author. Fax: +86 531 88564326. E-mail address: [email protected] (Y.-Z. Zhang).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.10.038
This activity is mainly due to the inhibition of the angiotensin-I-converting enzyme (ACE). ACE has been classically associated with the renin–angiotensin system, which regulates peripheral blood pressure via conversion of angiotensin-I to angiotensin-II. Inhibition of ACE may have an antihypertensive effect as a consequence of a decrease in blood pressure (Skeggs et al., 1957). Although synthetic ACE inhibitors, including captopril, enalapril and listinopril, are remarkably effective as antihypertensive drugs, they inevitably cause adverse side effects (Atkinson and Robertson, 1979). Many ACE inhibitory peptides have been reported in hydrolysates from diverse food proteins digested with different proteases (Meisel, 1998). Most of these peptides were identified from hydrolytic products of terrestrial food proteins but less from marine proteins. The composition and primary amino acid sequences of marine proteins are different from those of terrestrial proteins; therefore, marine proteins may become important resources for the exploitation of novel ACE inhibitory peptides by enzymatic hydrolysis.
H.-L. He et al. / Bioresource Technology 99 (2008) 5956–5959
Acetes chinensis growing in Bo Hai Gulf of China is an underutilized shrimp species with a low commercial value, as a consequence of low consumption by humans. Every year, about 300,000 tones of A. chinensis are made into dried small shrimps or shrimp sauce that have low adding value. The advanced utilizing process of this marine resource is a significant topic in marine biologic study. Because of its high content of muscle proteins and chitin, it is a valuable raw material for further processing. Our previous studies had shown that hydrolysates of A. chinensis digested with the crude protease from Bacillus sp. SM98011 possessed ACE inhibitory activity and three novel ACE inhibitory peptides had been found (He et al., 2006a; He et al., 2006b). In the present work, the enzymatic hydrolysis of the shrimp A. chinensis was scaled up to pilot scale and plant scale. The peptide compositions, ACE inhibitory activities, and in vivo anti-hypertensive effect in SHRs of the hydrolysates were also investigated.
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dried and kept at 4 C. Hydrolysates of pilot and plant scale were condensed with ball vacuum concentration tanker (RD500, GuanYi Mechanical Equipment Co., Ltd, China) and were dried with Spray Dryer (BoDa Co., China) at a 10 kg h 1 flow rate with a 170 C inlet temperature and 90 C outlet temperature (Kapel et al., 2006). 2.4. Assay of content of peptides in hydrolysates Content of peptides in the hydrolysates was assayed with the previous method (He et al., 2006a). 2.5. ACE-inhibition activity assay ACE inhibitory activity of the hydrolysates and the IC50 value was assayed with the previous method (He et al., 2007). 2.6. RP-HPLC analysis
2. Methods 2.1. Experimental materials and strains Acetes chinensis was purchased from Chinese Dayudao Group and stored at cold store. Protease-producing bacterium Bacillus sp. SM98011 was preserved in our laboratory. 2.2. Protease preparation and activity assay The strain Bacillus sp. SM98011 was fermented in 200 L fermenter at 28 C for 36 h, and the activity of protease SM98011 in ferment broth reached 4000 U/ml. Then, the ferment broth was centrifuged at 10,000g for 20 min, and the supernatant was collected and used for protein hydrolysis. The activity of protease was determined with the method as before (He et al., 2004). The protease SM98011 was crude serine protease and optimum hydrolysis temperature and pH of protease was 50 C and pH 7.5. 2.3. Acetes chinensis hydrolysates production at pilot scale and plant scale The hydrolysis was carried out at laboratory scale (500 ml Erlenmeyer flask), pilot scale (100 L thermostatically stirred-batch reactor) and plant scale (1000 L thermostatically stirred-batch reactor), respectively. In the batch experiments, minced shrimp was mixed with water at a ratio of 1:1 (w/w) by continuous stirring and the enzyme was added at a ratio of 400,000 U/kg (U/w, enzyme/protein substrate). All reactions were performed at pH 7.5 and 50 C for 5 h with constant agitation (200 rpm). The reactions were stopped by heating the solution to 90 C for 15 min, assuring the complete inactivation of the enzyme. The resulted slurry was centrifuged at 9000g for 20 min and the supernatant was the hydrolysates of A. chinensis. The supernatant of laboratory scale was freeze-
Analysis of peptide composition of the hydrolysates was carried out by RP-HPLC (Waters alliance2695, USA) coupled with Dual Wavelength UV Detector 2487. The separation was performed using a Symmetry C18 column (250 · 4.6 mm). The eluent was 0.1% trifluoroacetic acid (A) and a mixture of eluent A with acetonitrile (2/3, v/v) (B). A linear A–B gradient from 10% to 60% was applied over a period of 30 min followed by a linear A–B gradient from 60% to 10% for 15 min with flow rate 1 ml/min monitored at 214 nm. 2.7. Antihypertensive activity in SHRs Forty male SHRs (12 weeks old, Calco, Italy) and 10 age-matched normotensive Wistar-Kyoto (WKY) rats were kept at an ambient temperature of 23 ± 1 C, with free access to food and water before beginning the experiments. SHRs were divided into five groups (n = 8). A negative control group was orally administered with physiological saline, a positive control group was orally administered with captopril at 10 mg/kg/day and three experimental groups were orally administered with the hydrolysates at doses of 100, 500, 1200 mg/kg/day, respectively. Five untreated WKY rats served as normotensive control group. The other five WKY rats were administered with hydrolysates at a dose of 500 mg/kg/day. Dosages were adjusted every week according to the changes of body weight. The systolic blood pressure (SBP) of the rats was measured by a tail-cuff method (model BP-98, Softron, Tokyo, Japan). During a month of treatment, measurements were taken weekly. Several determinations were made for each animal, and the values were considered valid if five consecutive measurements were within 6 mmHg (Jung et al., 2006). Under anaesthesia with sodium pentobarbital (60 mg/kg i.p.), the animals were sacrificed immediately after the end of treatment. The rats’ hearts, brains were removed and immediately stored in 10% formalin
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H.-L. He et al. / Bioresource Technology 99 (2008) 5956–5959
solution of 4 C. Then, the fixed tissue was dehydrated in graded ethanol solutions, and embedded in paraffin. Sections were cut on a MICRON hm-310 microtome at a thickness of 5 lm and were stained with Hematoxylin and eosin (HE). Images were captured by a high-resolution video camera (Sony ccd-iris). 3. Results and discussion 3.1. Characterization of the hydrolysates from A. chinensis produced at pilot and plant scale The content of peptides in shrimp hydrolysates was 48.7 ± 0.51% at pilot scale product and 47.9 ± 1.8% at plant scale product, which had a little reduce compared with that of laboratory scale product (51.3 ± 0.2%). The ACE inhibitory IC50 value of the product was 0.96 ± 0.03 mg/ml at pilot scale and 0.97 ± 0.05 mg/ml at plant scale. Both of them were almost the same as that at laboratory scale (0.98 ± 0.02 mg/ml). The results showed that, after scaled up to pilot and plant scale, the hydrolysates almost had the same ACE inhibitory activity. It is satisfactory that the total peptide contents, and the ACE inhibitory activity of the hydrolysates produced at pilot and plant scales are almost the same as that of product at laboratory scale. 3.2. Comparison of the peptide chromatogram in the hydrolysates produced at laboratory, pilot and plant scales The HPLC chromatographic analysis of the products at laboratory, pilot and plant scales showed that there were no significant differences of peptide composition among the products at different scales. Therefore, the high correspondence of the products shows that the hydrolysis conditions at the laboratory scale can be used at pilot and plant scales. This standardization of the peptide chromatogram can be used for the quality control and quality detection of the industrial production of the hydrolysates. There have been many reports on the ACE inhibitory activity of peptides derived from food proteins by enzymatic hydrolysis. However, there is relatively less information on the ACE inhibitory activity and hypertensive activities of marine proteins. High conversion and ACE inhibitory activity of the hydrolysates from the marine shrimp are promising. It can be produced at plant scale to be utilized in functional food as bioactive peptides.
air caused little change on the peptide composition of the hydrolysates, especially for the ACE inhibitory peptides. Therefore, the oligopeptide-enriching ACE inhibitory hydrolysates from shrimp A. chinensis are thermostable. 3.4. Blood pressure-depressing activity of the hydrolysates in SHRs The hydrolysates from A. chinensis have good ACE inhibitory activity (He et al., 2006a). Five inhibitory peptides had been isolated from the hydrolysates, and three of them were novel ACE inhibitory peptides, giving further evidences for the ACE inhibitory activity of the hydrolysates in vitro (He et al., 2006b). In this article, the in vivo experiments had been done. At the beginning of the study, SBPs of SHRs were already higher than that of the WKY rats and these differences persisted as the time progressed. As shown in Fig. 1, the average SBP of the ACE inhibitor group was 163 ± 1.5 mmHg just before the administration. After oral administration of the spray-dried hydrolysates each day, the SBPs of SHRs started to decrease in dosedependent manner. After a month administration in the range of 100–1200 mg/kg/day, the decrease of the blood pressure in SHRs was 18.3–38.6 mmHg. The blood pressure-depressing effect of the highest dose of the hydrolysates was similar to that of commercial antihypertensive drug Captopril at a dose of 10 mg/kg/day. Otherwise, no obvious decrease of SBP was observed in control SHRs group administrated with physiological saline. On the other hand, administration of the hydrolysates at a dose of 500 mg/kg/day did not show any significant hypotensive effect on WKY rats. These results showed that the marine shrimp hydrolysates possess a significant and safe antihypertensive effect on SHRs. Because the hydrolysates used
3.3. Effect of spray-drying on the peptide composition in the hydrolysates at plant scale The peptide chromatogram of the hydrolysates before and after spray-drying was analyzed. There was little difference of the peptide spectra between them. The spray-dried hydrolysates still kept its high ACE inhibitory activity at an IC50 value of 0.97 mg/ml, which is almost the same as that of the hydrolysates at laboratory scale. These results showed that exposure of hydrolysates to high temperature
Fig. 1. Blood pressure-depressing activity of the hydrolysates from Acetes chinensis: (a) SHR negative control (with physiological saline); (b) low dose group (with hydrolysates 100 mg/kg/day); (c) medium dose group (with hydrolysates 500 mg/kg/day); (d) high dose group (with hydrolysates 1200 mg/kg/day); (e) positive control (with captopril 10 mg/kg/day); and (f) WKY normotensive control (with hydrolysates 500 mg/kg/day).
H.-L. He et al. / Bioresource Technology 99 (2008) 5956–5959
in this experiment were the crude hydrolysates without any purification, the dose of the hydrolysates used was relatively high. Our previous results showed that most of the ACE inhibitory peptides in the hydrolysates from A. chinensis are short peptides with molecular mass lower than 3000 Da and the ACE inhibitory IC50 value of the hydrolysates decreased to 0.22 mg/ml from 0.98 mg/ml after an ultra filtration with an Amicon PM-3 membrane (He et al., 2006b). Therefore, if the hydrolysates were purified by such an ultrafiltration and the dried filtrate was used, it could be reasonably conjectured that the dose used would be dramatically reduced. 3.5. Effects of long-term administration of the hydrolysates on the organs of SHRs After administrated with the hydrolysates for thirty days, the rats were sacrificed to study its effects on the organs of SHRs. In the negative control group SHRs, the thickness of arteriolar wall in cardiac muscle increased significantly. After administration of the hydrolysates at a dose of 1200 mg/kg/day, significant reduction in the development of cardiac and vascular hypertrophy in treated SHRs was observed. Moreover, no side effects were observed in WKY rats with similar treatment of hydrolysates. Effect of long-term administration of the hydrolysates on arterioles of frontal cortex was also investigated. In the negative control group SHRs, the walls of intracerebral arterioles revealed magnification hypertrophy and the lumina of these arterioles became narrow and even occlusion. No signs of apparent nervous tissue damage were found in cerebral areas investigated. After administered with hydrolysates, there were much less pathologic changes in intracerebral arterioles in treated SHRs than in untreated ones. In addition, there was no hypertrophy in the wall of arterioles in WKY group. Histopathologic study showed that these pathologic changes got obvious alleviation after administration of the hydrolysates. Accordingly, the inhibitory ACE activity might be one of the major mechanisms for the organ protection produced by long-term treatment with hydrolysates in SHRs (Shan et al., 1999; Pu and Schiffrin, 2001). Acknowledgments This work was supported by Grants from the National High Technology Research and Development Program of China (Nos. 2001AA625040 and 2003AA625040), and Program for New Century Excellent Talents in University (NCET-06-0578).
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