酶法制备燕麦麸蛋白ACE抑制肽的研究

采用碱溶酸沉法制备了燕麦麸蛋白,并利用7种商业化蛋白酶对其进行酶解以考察生产ACE抑制肽的效果,结果表明Alcalase为最适用酶。优化了Alcalase水解燕麦麸蛋白的工艺条件,在[S]=5.0%,E/S=1.33%,pH7.5,温度60℃条件下酶解90min,酶解产物的ACE抑制活性最强(IC50=0.291mg/mL),此时水解度为11.0%,产物的相对分子质量主要集中在880Da以下。     

VIS/NIR spectroscopy for non-destructive freshness assessment of Atlantic salmon (Salmo salar L.) fillets

Visible/near-infrared spectroscopy has been evaluated for use in freshness prediction and frozen-thawed classification of farmed Atlantic salmon fillets, where fresh samples were stored as whole fish in ice. A handheld interactance probe for performing rapid measurements of single fillets and an imaging spectrometer for online analysis at an industrial speed of one fillet per second, have been used. Freshness as storage days in ice is predicted with an accuracy of 2.4 days for individual fillets, whereas frozen-thawed salmon fillets are completely separated from fresh fillets. The prediction results are comparable to previous results using the Quality Index Method with trained panelists. The region between 605 and 735 nm, which excludes interference by carotenoids and water, is appropriate for both frozen-thawed classification and freshness prediction of salmon fillets. The results indicate that the spectral changes are explained mainly by oxidation of heme proteins during the freeze–thaw cycle and during chilled storage in ice.     

Classification of fresh Atlantic salmon (Salmo salar L.) fillets stored under different atmospheres by hyperspectral imaging

Hyperspectral imaging (HSI) was used to investigate spectroscopic changes in fresh salmon stored under different atmospheres (air, 60% CO₂/40% N₂ and 90% vacuum) and to determine whether HSI can classify fillets by the type of packaging. Hyperspectral images of samples kept at 4 °C were acquired and bacterial growth and lipid oxidation were measured. Principal component analysis was applied to study spectral development of samples during storage and K nearest-neighbour classifier was used for the classification of fillets by packaging type. Partial least squares regression was run to reduce the number of wavelengths included in the classification model. The results demonstrated that spectral variations could be observed in the different packaging atmospheres primarily at the wavelengths 606 and 636 nm. Using HSI, successful classification of fillets according to the packaging used (>88%) was achieved and this was largely dependent on spectral characteristics at the wavelengths 606 and 636 nm, possibly due to the different oxidation states of the haem proteins in the muscles.     

酱油对血管紧张素转换酶抑制作用的研究

考察了若干酱油样品的血管紧张素转换酶(ACE)抑制活性,发现所试样品均表现出ACE抑制活性,其IC50值范围为0.7405～3.0265mg/mL。样品的ACE抑制活性与其蛋白质降解程度、多肽含量及颜色值无明显相关性,应为多种活性物质综合作用的结果。对我国酱油产品中ACE抑制剂的结构表征可为潜在降血压功能性食品的研发提供指导。     

Authentication of Atlantic salmon (Salmo salar) using real-time PCR

A real-time PCR assay based on LNA TaqMan probe technology was developed for the detection and identification of Atlantic salmon (Salmo salar). Among the advantages it is worth highlighting simplicity, rapidity, highest potential for automation and minor risk of contamination of this technique. The TaqMan real-time PCR is the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabelling of this species. The method can be applied to all kind of products, fresh, frozen and processed products, including those undergoing intensive processes of transformation.     

两种来源于牛皮胶原蛋白的新型ACE抑制肽

从牛皮胶原蛋白中鉴定分离出抑制血管紧张素Ⅰ转换酶（ACE）的多肽片段。首先优化牛皮胶原蛋白水解方法,应用胃蛋白酶和碱性蛋白酶获得活性最高的水解产物（抑制ACE的IC50为0.168 mg/m L）。经MW 5000超滤分离后,得到水解液中活性最高的部分（IC50为0.079 mg/m L）。对分离后低于MW 5000的部分经RP-HPLC进一步分离,发现含有大量ACE抑制肽,并应用RP-HPLC-MS/MS鉴定出两种新型ACE抑制肽,氨基酸序列分别为ISVPGPM和LGPVGNPGPA,IC50值分别为0.017 mg/m L（24.3μmol/L）和0.077 mg/m L（87.8μmol/L）。最后,本文模拟了两种抑制肽在体内生理环境下的消化,发现均可被部分降解,且消化产物具有与原始多肽相近的ACE抑制活性。      

Angiotensin I-converting enzyme inhibitory peptides in douchi,a Chinese traditional fermented soybean product

Douchi, a soybean product originating in China, produces angiotensin I-converting enzyme (ACE) inhibitors with the potential to lower blood pressure. The ACE inhibitory activities of douchi qu pure-cultured by Aspergillus Egyptiacus for 48 h, and 72 h were compared with douchi secondary-fermented for 15 d. The results showed that ACE inhibitory activities were improved following the fermentation. ACE inhibitory activities of 48 h-primary-fermented douchi qu did not change dramatically after preincubation with ACE, but increased greatly after preincubation with gastrointestinal proteases. The results suggest they were pro-drug-type or a mixture of pro-drug-type and inhibitor-type inhibitors. The ACE inhibitors in 48 h-fermented douchi qu were fractionated into four major peaks by gel filtration chromatography on Sephadex G-25. Peak 2, which had the highest activity, had only one peptide, composed of phenylalanine, isoleucine and glycine with a ratio of 1:2:5.     

The effects of carbon monoxide on Atlantic salmon (Salmo salar L.)

Atlantic salmon were exposed to carbon monoxide (CO) before the fish were percussively killed and gill cut. The fish were compared against a control group treated identically, without CO. Salmon exposed to CO expressed no adverse reactions and were easily stunned by percussion. CO-treated salmon had an earlier onset of rigor mortis and a faster decrease in muscle pH than the control group. No significant difference in drip loss was found between salmon treated with CO and the control. A significantly deeper red colour of both gills and fillets of CO-treated salmon was observed 10 days post mortem. Significantly higher levels of plasma lactate and potassium were found in CO-treated salmon compared to control, as well as a lower level of pCO₂. Exposure to CO did not increase plasma cortisol, sodium, haematocrit or glucose; however, lactate was high. Exposure of salmon or other fish to CO could improve quality and welfare when slaughtered.     

特异性蛋白酶酶解虾副产物制备ACE抑制肽

ACE抑制肽构效关系(QSAR)的研究认为小肽C末端氨基酸的疏水性和其ACE抑制活性之间呈正相关关系。针对性地选择胰凝乳蛋白酶和脯氨酸蛋白酶两步酶解虾副产物制备ACE抑制肽。在一定温度、pH和加酶量条件下,以蛋白质的水解度和ACE抑制率为指标,确定胰凝乳蛋白酶、脯氨酸蛋白酶的最佳酶解时间均为4h。两步酶解产物ACE抑制的IC50值为1.645mg protein/mL。经透析后,得到0~500u(组分1)和500~1000u(组分2)两个组分,组分1和组分2的IC50值分别降为0.333mg peptide/mL和1.320mg peptide/mL。质谱分析结果表明组分1中含有10个肽,分别由5~14个氨基酸组成;组分2中含有15个肽,分别由7~14个氨基酸组成。25个已知序列多肽中有22个多肽的羧基端是脯氨酸或芳香族氨基酸,与预期相符。      

Angiotensin converting enzyme inhibitory activity of proteolytic digests of peanut (Arachis hypogaea L.) flour

Defatted raw and roasted peanut flour were hydrolyzed with alcalase or sequentially with pepsin and pancreatin, and then the hydrolyzates were fractionated by RP-HPLC and tested for hypotensive potential. This research revealed that proteolytic peanut digests have an inhibitory effect on the activity of angiotensin converting enzyme (ACE). Three fractions from the hydrophobic end of the chromatogram of each hydrolyzate were the most potent for inhibiting ACE activity in comparison to seven other fractions. These potentially potent fractions were then assayed for IC₅₀. Fractions from the alcalase digestion of raw peanut exhibited IC₅₀ values of 8.7-122 μg/ml, and those from roasted flour exhibited values of 12-235 μg/ml. IC₅₀ values of 7.9-65.9 μg/ml, and 11-36 μg/ml for raw and roasted peanut, respectively, from the pepsin-pancreatin system were observed. These values compare to the IC₅₀ value of 0.36 μg/ml of a known commercial ACE inhibitor (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro).     

Preparation and identification of angiotensin I-converting enzyme inhibitory peptides from sweet potato protein by enzymatic hydrolysis under high hydrostatic pressure

Sweet potato protein hydrolysates (SPPH) with angiotensin I-converting enzyme (ACE) inhibitory activity were prepared by papain, pepsin and alcalase under high hydrostatic pressure (HHP, 100–300 MPa). HHP significantly increased degree of hydrolysis (DH), nitrogen recovery (NR) and molecular weight (MW) <3 kDa fractions contents of SPPH by all three enzymes (P < 0.05). MW < 3 kDa peptide fractions from SPPH by alcalase under 100 MPa showed the highest ACE inhibitory activity (IC₅₀ value 32.24 µg mL⁻¹), and was subjected to purification and identification by semi-preparative RP-HPLC and LC-MS/MS. Fifty-four peptides ranged from 501.28 to 1958.88 Da with 5–18 amino acids were identified and matched sporamin A and B sequences. Five identified peptides with sequences of VSAIW, AIWGA, FVIKP, VVMPSTF and FHDPMLR displayed good ACE inhibitory activity with the contribution of Val, Trp, Phe and Arg. Thus, SPPH by enzymatic hydrolysis under HHP can be potentially used in functional food.     

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase^®, chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2 ± 1.5% at 2 mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Biological functions of all fractions were assayed, and P₄ was found to display a high ACE inhibitory activity. The IC₅₀ values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P₄ were 1.2 ± 0.09 and 0.81 ± 0.013 mg/ml, respectively. Further, P₄ showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P₄ was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.     

鱼鳞血管紧张素转化酶(ACE)抑制肽的制备工艺研究

采用酶法酶解鱼鳞制备血管紧张素转化酶（angiotensin-I converting enzyme,ACE）抑制肽。以鱼鳞水解度、相对分子质量分布、游离氨基酸含量以及ACE抑制活性为指标,从五种蛋白酶中选取来源于枯草芽孢杆菌的中性蛋白酶作为水解鱼鳞制备ACE抑制肽的最适酶种。经Box-Behnken响应面分析法得到其最佳酶解工艺条件为:pH6.4,温度50℃,加酶量（[E]/[S]）5%,底物浓度（[S]）5%（w/v）,水解3h。在此条件下得到的水解度为19.26%。酶解产物富含脯氨酸和甘氨酸,其次是丙氨酸,缺乏色氨酸。疏水性氨基酸约占总氨基酸的28%,相对分子质量主要分布在80⁸00之间,约为2⁶个氨基酸的寡肽。      

The impact of fermentation and in vitro digestion on formation angiotensin converting enzyme (ACE) inhibitory peptides from pea proteins

Pea seeds were fermented by Lactobacillus plantarum 299v in monoculture under different time and temperature conditions and the fermented products were digested in vitro under gastrointestinal conditions. After fermentation and digestion ACE inhibitory activity was determined. In all samples after fermentation no ACE inhibitory activity was noted. Potentially antihypertensive peptides were released during in vitro digestion. The highest DH (68.62%) were noted for control sample, although the lowest IC₅₀ value (0.19 mg/ml) was determined for product after 7 days fermentation at 22 °C. The hydrolysate characterised by the highest ACE inhibitory activity was separated on Sephadex G10 and two peptides fractions were obtained. The highest ACE inhibitory activity (IC₅₀ = 64.04 μg/ml) for the first fraction was noted. This fraction was separated by HPLC and identified by LC–MS/MS and the sequence of peptide derived from pea proteins was determined as KEDDEEEEQGEEE.     

Relevance of calpain and calpastatin activity for texture in super-chilled and ice-stored Atlantic salmon (Salmo salar L.) fillets

The aim of the present experiment was to measure the protease activities in ice-stored and super-chilled Atlantic salmon (Salmo salar) fillets, and the effect on texture. Pre-rigour fillets of Atlantic salmon were either super-chilled to a core temperature of −1.5 °C or directly chilled on ice prior to 144 h of ice storage. A significantly higher calpain activity was detected in the super-chilled fillets at 6 h post-treatment compared to the ice-stored fillets and followed by a significant decrease below its initial level, while the calpastatin activity was significantly lower for the super-chilled fillets at all time points. The cathepsin B + L and B activities increased significantly with time post-treatment; however, no significant differences were observed at any time points between the two treatments. For the ice stored fillets, the cathepsin L activity decreased significantly from 6 to 24 h post-treatment and thereafter increased significantly to 144 h post-treatment. There was also a significantly lower cathepsin L activity in the super-chilled fillets at 0 h post-treatment. No significant difference in breaking force was detected; however, a significant difference in maximum compression (F_max) was detected at 24 h post-treatment with lower F_max in the super-chilled fillets. This experiment showed that super-chilling had a significant effect on the protease activities and the ATP degradation in salmon fillets. The observed difference in F_max may be a result of these observed differences, and may indicate a softening of the super-chilled salmon muscle at 24 h post-treatment.     

Detection of frozen-thawed salmon (Salmo salar) by a rapid low-cost method

The aim of this study was to evaluate a rapid low-cost system based on impedance spectroscopy as an alternative method to differentiate between fresh and frozen-thawed salmon. Samples of fresh salmon and others submitted to freezing at −18 °C or to 2 freezing cycles, kept in frozen storage for different times, were analysed. In general, no significant differences in moisture, total volatile basic nitrogen, pH, texture parameters, K₁ value, or microbial counts between the different samples were observed. This revealed that the freezing process, storage time or number of freezing cycles did not affect the physico-chemical parameters of fish samples, except for water holding capacity, which was significantly lower in all frozen samples compared with fresh salmon. The results showed that impedance spectroscopy was unable to differentiate between different storage times under frozen conditions; however, this technique could be a useful tool to detect fish submitted to freezing process.     

Angiotensin I-converting enzyme inhibitory and hypocholesterolemic activities: Effects of protein hydrolysates prepared from Achatina fulica snail foot muscle

The angiotensin I-converting enzyme (ACE) inhibitory activity and hypocholesterolemic effect of Achatina fulica snail foot muscle protein hydrolysates (SFMPH) and its hydrolysates were studied. The SFMPHs were prepared at a temperature of 121°C for 60 min. To obtain the enzymatic hydrolysates, the SFMPHs were further hydrolysed with three proteases (papain, trypsin, or alcalase). Among all the hydrolysates, alcalase hydrolysate showed the highest degree of hydrolysis and was dominated by a small molecular size fraction (189–686 Da). The SFMPH treated by alcalase was effective in disintegrating intact cholesterol micelles. Furthermore, alcalase hydrolysate with a hydrolysis time of 60 min showed a strong ACE inhibitory activity in vitro with an IC₅₀ of 0.024 mg/mL. Therefore, alcalase hydrolysate may be a promising ingredient for the use in functional foods.     

Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats

In order to utilize yellowfin sole (Limanda aspera) frame protein, which is normally discarded as industrial waste in the process of fish manufacture, yellowfin sole frame protein was hydrolysed by α-chymotrypsin. Yellowfin sole frame protein hydrolysates (YFPHs) were fractionated into three ranges of molecular weight (YFPH-I, 30–10 kDa; YFPH-II, 10–5 kDa; YFPH-III, below 5 kDa) using an ultrafiltration (UF) membrane bioreactor system. Angiotensin I-converting enzyme (ACE) inhibitory activity was detected on YFPH-III, and the ACE inhibitory peptide (YFP) was purified from YFPH-III using consecutive chromatographic techniques. The YFP with a molecular mass of 1.3 kDa consisted of 11 amino acids, Met-Ile-Phe-Pro-Gly-Ala-Gly-Gly-Pro-Glu-Leu, and its IC₅₀ value was 28.7 μg/ml. Lineweaver–Burk plots suggest that YFP acts as a non-competitive inhibitor to inhibit ACE. Antihypertensive effects of YFP on spontaneously hypertensive rats (SHR) following oral administration was determined as the blood pressure significantly decreased after peptide ingestion.     

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC₅₀ = 132 μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC₅₀ = 703 μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.