Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste

Bacillus sp. nov. SK006 producing four extracellular fibrinolytic enzymes was isolated from fermented shrimp paste, a traditional and popular Asian seasoning. One fibrinolytic enzyme was purified to homogeneity with a molecular mass of 43-46 kDa by SDS-PAGE and gel filtration chromatography. The specific activity was determined to be 11.2 units/mg using plasmin as a standard. The enzyme displayed optimal activity at 30 degrees C and pH 7.2. It was stable below 40 degrees C for 4 h between pH 5.0 and pH 11.0. Zinc ion stimulated the enzyme activity whereas Cu2+, Ca2+, Fe3+, and Hg2+ caused its inhibition. The fibrinolytic activity was strongly inhibited by PMSF and moderately inhibited by EDTA as well as PCMB. The enzyme exhibited a higher affinity toward N-Succ-Ala-Ala-Pro-Phe-pNA and was able to degrade fibrin clots either by forming active plasmin from plasminogen or by direct fibrinolysis. The N-terminal amino acid sequence was found to be AQSVPYEQPHLSQ, which is different from that of other known fibrinolytic enzymes.     

中国传统细菌型豆豉溶栓酶的提取纯化技术研究(简报)

为深入开发利用中国传统发酵细菌型豆豉溶栓酶,对豆豉溶栓酶分离纯化工艺进行了研究探讨,确认了最佳的纯化条件:首先用饱和度75%的硫酸铵沉淀豆豉溶栓酶;然后利用DEAE-Sepharose FF (Diethylaminoethyl Sepharose Fast Flow,二乙基氨基琼脂糖快速凝胶)阴离子交换柱进行层析,优化的层析条件为用10 mmol/L Tris(Trishydroxymethylaminomen,三羟甲基氨基甲烷)-HCI (pH 8.7)作为上样缓冲液,采用线性梯度洗脱,洗脱液为10mmol/L Tris-HCI(pH 8.7)和0.6mol/LNaCl,洗脱流速为1 mL/min;最后经过Sephadex G-75(葡聚糖凝胶G-75)凝胶过滤,得到纯化倍数为11.29倍的豆豉溶栓酶.利用SDS-PAGE (Sodium dodecyl sulphate-polylamide gel electroresis,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳) 显示经纯化后的酶无杂蛋白,初步鉴定其分子量接近31 ku.     

Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.     

传统农家酱产蛋白酶菌株的分离及其在豆浆的应用

豆浆富含植物蛋白,但由于植物蛋白相较动物蛋白不易被人体吸收。利用产蛋白酶菌株发酵豆浆,可将植物蛋白降解成多肽和氨基酸,提高其消化吸收率。传统农家酱是以大豆为主要原料的传统发酵食品,含有大量能够水解大豆蛋白的优良菌株。该研究从传统农家酱中分离到8株产蛋白酶的芽孢杆菌并用于豆浆处理,处理后豆浆的可溶性肽含量显著提高,其中菌株BJ-20处理效果最显著;氨基态氮含量和水解度也有显著变化,其中BJ-6处理后的豆浆最佳,氨基态氮含量提高了10.87%,水解度为23.49%。同时,处理后豆浆的组织状态得到了明显改善。本研究丰富了豆类发酵菌种库,提高了大豆蛋白利用率,丰富了大豆产品种类,为大豆保健产品开发提供参考及理论支持。     

Enzymatic activity and flavor compound production in fermented silver carp fish paste inoculated with douchi starter culture

Silver carp fish pastes inoculated with or without a douchi starter culture containing live Aspergillus oryzae were fermented for 30 days to produce two different fermented products, designated CulF and ConF, respectively. Protein degradation and flavor compound production during the course of fermentation were monitored. Proteolytic activity, generally higher in CulF than in ConF (P < 0.05) and dominated by acidic and serine proteases, declined to an overall minimum after 30 days. Myosin in the CulF and ConF extractives was completely degraded after 1 and 5 days, respectively. The content of free amino acids and low molecular weight (<1.3 kDa) peptides rose rapidly in CulF and progressively in ConF (P < 0.05). Ethanol, silanediol, pyrazine, phenol, and formic acid were prevalent volatile compounds in CulF, whereas butanol, butanoic acid, and acetic acid were abundant in ConF. Therefore, douchi-inoculated fermentation is an attractive process to produce savory fish pastes.     

His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo.

It has been reported that soybean peptide fractions isolated from Korean fermented soybean paste exert angiotensin I converting enzyme (ACE) inhibitory activity in vitro. In this study, further purification and identification of the most active fraction inhibiting ACE activity were performed, and its antihypertensive activity in vivo was confirmed. Subsequently, a novel ACE inhibitory peptide was isolated by preparative HPLC. The amino acid sequence of the isolated peptide was identified as His-His-Leu (HHL) by Edman degradation. The IC(50) value of the HHL for ACE activity was 2.2 microg/mL in vitro. Moreover, the synthetic tripeptide HHL (spHHL) resulted in a significant decrease of ACE activity in the aorta and led to lowered systolic blood pressure (SBP) in spontaneously hypertensive (SH) rats compared to control. Triple injections of spHHL, 5 mg/kg of body weight/injection resulted in a significant decrease of SBP by 61 mmHg (p < 0.01) after the third injection. These results demonstrated that the ACE inhibitory peptide HHL derived from Korean fermented soybean paste exerted antihypertensive activity in vivo.     

Changes in the contents of sugars and organic acids during the ripening and storage of sufu, a traditional Oriental fermented product of soybean cubes

In the present study, sufu, an oriental fermented product of soybeans, was prepared by ripening the tofu cubes in Aspergillus oryzae fermented rice-soybean koji mash for 16 days at 37 °C. The sufu product was further kept at room temperature for another 30 days. Examining the changes in the sugar content revealed that stachyose, raffinose, and sucrose contents of tofu and rice-soybean koji granules decreased while levels of glucose and fructose increased during the ripening period and after storage. Glucose was the most abundant sugar detected. Four organic acids, including oxalic, lactic, acetic, and citric acid, were detected in the sufu product and koji granules after ripening. Generally, the contents of these organic acid increased as the ripening period was extended. Among them, acetic acid was the most highly detected.     

Volatile compounds in Spanish, French, and American oak woods after natural seasoning and toasting

The volatile composition (volatile phenols, phenolic aldehydes, furanic compounds, lactones, phenyl ketones, and other related compounds) of Spanish oak heartwood of Quercus robur, Quercus petraea,Quercus pyrenaica, and Quercus faginea was studied by gas chromatography/mass spectrometry, in relation to the processing in barrels cooperage and in relation to the French oak of Quercus robur (Limousin) and Quercus petraea (Allier) and American oak of Quercus alba (Missouri), which are habitually used in cooperage. The volatile composition of seasoned oak woods varied according to individual trees, species, and origins, and the differences were more significant in Spanish species with respect to American species than with respect to French species. The toasting process influenced the volatile composition of wood. It led to high increases in the concentration of volatile phenols, furanic aldehydes, phenyl ketones, and other related structures, but the effect on w-lactones levels depended on species and origin. The volatile composition in Spanish oak species evolved during toasting like in French and American oak, but quantitative differences were found, especially important in American species with respect to European species.     

Study on furundu, a traditional Sudanese fermented roselle (Hibiscus sabdariffa L.) seed: effect on in vitro protein digestibility, chemical composition, and functional properties of the total proteins

Furundu, a meat substitute, is traditionally prepared by cooking the karkade (Hibiscus sabdariffa L.) seed and then fermenting it for 9 days. Physicochemical and functional properties of raw and cooked seed and of furundu ferments were analyzed. Furundu preparation resulted in significant changes in karkade seed major nutrients. Total polyphenols and phytic acid were also reduced. The increase in total acidity and fat acidity coupled with a decrease in pH indicates microbial hydrolysis of the major nutrients; proteins, carbohydrates, and fats. In vitro digestibility of the seed proteins reached the maximum value (82.7%) at the sixth day of fermentation, but thereafter it significantly decreased. The effect of furundu preparation on N solubility profiles and functional properties, such as emulsification and foaming properties and other related parameters, is investigated in water and in 1 M NaCl extracts from defatted flour samples. The results show that cooking followed by fermentation affects proteins solubility in water and 1 M NaCl. The foaming capacity (FC) from the flour of raw seed decreased as a result of cooking. Fermentation for 9 days significantly increased the FC of the cooked seed, restoring the inherent value. Foam from fermented samples collapsed more rapidly during a period of 120 min as compared to the foam from raw and cooked karkade seeds; stability in 1 M NaCl was lower as compared to those in water. In water, the emulsion stability (ES) from the fermented samples was significantly higher than that of the raw seed flour. Addition of 1 M NaCl significantly decreased the ES of the fermented samples.     

Evolution of ellagitannins in Spanish, French, and American oak woods during natural seasoning and toasting

The evolution of tannins in Spanish oak heartwood of Quercus robur L., Quercus petraea Liebl.,Quercus pyrenaica Wild., and Quercus faginea Lam. was studied in relation to the processing of wood in barrel cooperage. Their evolution was compared with that of French oak of Q. robur (Limousin, France) and Q. petraea (Allier, France) and American oak of Quercus alba L. (Missouri), which are habitually used in cooperage. Two stages of process were researched: the seasoning of woods during 3 years in natural conditions and toasting. Total phenol and total ellagitannin contents and optical density at 420 nm of wood extracts were determined. The ellagitannins roburins A-E, grandinin, vescalagin, and castalagin were identified and quantified by HPLC, and the molecular weight distribution of ellagitannins was calculated by GPC. During the seasoning process the different ellagitannin concentrations decreased according to the duration of this process and in the same way as those in French and American woods. The toasting process also had an important influence on the ellagitannin composition of wood. Roburins A-E, grandinin, vescalagin, and castalagin decreased during this process in the Spanish wood species, in the same proportion as in the French and American ones. Also, the seasoning and toasting processes lead to qualitative variations in the structure of ellagitannins, especially in the molecular weight distribution, as was evidenced by GPC analysis of their acetylated derivatives.     

Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion

The angiotensin converting enzyme (ACE)-inhibitory activity of several commercial fermented milks was evaluated. Most of these products showed moderate inhibitory activity, but a few exceptions were detected. The high ACE-inhibitory activity found in some cases could be related to the origin of the milk. Two of these products were subjected to an enzymatic hydrolysis process, which simulates physiological digestion, to study the influence of digestion on ACE-inhibitory activity. The activity did not significantly change or increase during simulated gastrointestinal digestion. The peptides generated from one selected product during simulated digestion were sequenced by tandem spectrometry. Most peptides found at the end of the simulated digestion were released after 30 min of incubation with the pancreatic extract. This suggests that physiological digestion promotes the formation of active peptides from the proteins present in these fermented products. The potential ACE-inhibitory activity of the identified peptides is discussed with regard to their amino acid sequences.     

Inhibitory effect of natto,a kind of fermented soybeans,on LDL oxidation in vitro

The oxygen radical scavenging activity of natto (fermented soybeans) and its inhibitory effect on the oxidation of rat plasma low-density lipoprotein (LDL) in vitro were investigated to evaluate the usefulness of the antioxidant properties of natto, which has been shown to have antioxidant activity. Natto was separated into three water-soluble fractions: high-molecular-weight viscous substance (HMWVS; Mw > 100 000), low-molecular-weight viscous substance (LMWVS; Mw < 100 000), and soybean water extract (SWE). LMWVS had the strongest radical scavenging activity for hydroxyl and superoxide anion radicals, as assessed by electron spin resonance. The increase of conjugated dienes in LDL oxidized by copper and an azo pigment was depressed by the addition of LMWVS and SWE. These results demonstrate that natto fractions have inhibitory effects on LDL oxidation as a result of their radical scavenging activity.     

2-chloroethyl fatty acid esters as indicators of 2-chloroethanol in black walnuts, seasoning mixes, and spices

Residues of 2-chloroethyl fatty acid esters (CEEs) and 2-chloroethanol (ECH), by-products of ethylene oxide fumigation, were determined in black walnuts, seasoning mixes, and spices. Extracts containing ECH and CEE were cleaned up by previously described procedures, and residue levels were quantitatively determined using a gas chromatograph equipped with a halogen-selective electrolytic conductivity detector. All food products that contained CEE residues also contained ECH. ECH residues ranged from less than 0.2 to 880 ppm and were less than 0.2-7 times the CEE levels found.     

Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas

To develop safe and cheap thrombolytic agents, a fibrinolytic enzyme productive strain of LSSE-62 was isolated from Chinese soybean paste. This strain was identified as Bacillus amyloliquefaciens by 16S rDNA sequence analysis. Nucleotide and amino acid sequence analysis showed that this fibrinolytic enzyme was identical to subtilisin DJ-4. Chickpeas were used as the substrate for fibrinolytic enzyme production from B. amyloliquefaciens in solid-state fermentation. Under the optimized conditions (34 °C and 50% initial moisture content), the fibrinolytic activity of fermented chickpeas reached 39.28 fibrin degradation units (FU)/g. Additionally, the fermented chickpeas showed anticoagulant activity, and the purified anticoagulant component showed higher anticoagulant activity than heparin sodium. After fermentation, the total phenolic and total flavonoid contents increased by 222 and 71%, respectively, and then the antioxidant activities were improved significantly. This study provided a novel method for the preparation of multifunctional food of chickpeas or raw materials for the preparation of functional food additives and potential drugs.     

Changes in low molecular weight phenolic compounds in Spanish, French, and American oak woods during natural seasoning and toasting

The evolution of low molecular weight polyphenols in Spanish oak heartwood of Quercus robur,Quercus petraea, Quercus pyrenaica, and Quercus faginea was studied by HPLC, in relation to the processing of wood in barrel cooperage. The polyphenolic composition of Spanish woods subjected to natural seasoning for 3 years and to the toasting process was studied in relation to those of French oak of Q. robur (Limousin) and Q. petraea (Allier) and American oak of Q. alba (Missouri), which are habitually used in cooperage. The concentrations of benzoic and cinnamic acids and aldehydes of Spanish woods increased during seasoning depending on the duration of this process and in the same way as those of French and American woods. The process having the main influence on the phenolic composition of wood was the toasting. It led to high increases in the concentration of phenolic aldehydes and acids, especially cinnamic aldehydes (sinapic and coniferylic aldehydes), followed by benzoic aldehydes (syringaldehyde and vanillin) and benzoic acids (syringic and vanillic acids). This polyphenolic composition in Spanish oak species evolved during toasting as in French and American oak, but quantitative differences were found, which were especially important in American species with respect to the others.     

New monascus metabolites with a pyridine structure in red fermented rice

Monascus purpureus produces several hitherto unknown compounds in addition to pigments and the mycotoxin citrinin. In red fermented rice (angkak, red koji) obtained as cultures of M. purpureus DSM1379 and DSM1603, we detected two compounds with identical UV absorption spectra and maxima at 306-307 nm. They were isolated by HPLC, and their structures were elucidated by intensive MS and NMR studies. Monascopyridine A (3) contains a gamma-lactone, propenyl group, hexanoyl side chain, and a pyridine ring, whereas the more lipophilic compound, monascopyridine B, is a higher homologue of monascopyridine A with the more lipophilic octanoyl instead of the hexanoyl side chain. This is the first report of Monascus metabolites with a pyridine ring.     

Extractives content in cooperage oak wood during natural seasoning and toasting; influence of tree species,geographic location,and single-tree effects

The chemical composition of cooperage oak wood is highly variable, depending upon the tree species (Quercus robur L. versus Quercus petraea Liebl.), its geographic location, and the single-tree effect. In the process of cask-making, natural seasoning and toasting contribute strongly to the modification of the oak wood chemical composition and therefore influence wine cooperaging. HPLC and GC quantification of ellagitannins and volatile compounds such as whiskey-lactones, eugenol, and vanillin over a sample set of 61 pedunculate oaks and 72 sessile oaks originating from six different forests showed that natural drying leads to a decrease of the ellagitannins and total extractives content level and a quasi constant level of the volatile compounds. Toasting (medium type) drastically enhanced the loss of ellagitannins and the gain in volatile compounds. Statistical treatment showed that the species effect remained significant throughout the process of drying and toasting, but not the provenance. The poor correlation with ring width of extractives levels measured on fresh timber remained unchanged as did the single-tree effect, with high variability found for all chemical parameters. These results provide further evidence that cooperage oak selection should not be based solely on the wood grain or the provenance but rather on a species-provenance combination.     

Key odorants of Oscypek, a traditional Polish ewe's milk cheese

The unique flavor of Oscypek, a Polish ewe's milk smoked cheese, is described as slightly sour, piquant, salted, and smoked. In this paper with the application of gas chromatography-olfactometry (GC-O) and combination of aroma extract dilution analysis (AEDA) 20 potent odorants of this cheese have been identified within the flavor dilution factor (FD) range of 4-2048. Among them, 2-methoxyphenol, 2-methoxy-4-methylphenol, 4-methylphenol, and butanoic acid showed the highest FD factors. Quantification results based on labeled standard addition followed by calculation of odor activity values (OAV) of 13 compounds with the highest FD factors revealed that 11 compounds were present at concentrations above their odor threshold values and therefore mostly contribute to the overall aroma of smoked ewe's milk cheese. Six of those compounds were represented by phenolic derivatives, with the highest OAV for 2-methoxyphenol (1280). Analysis of key odorants of an unsmoked cheese sample showed that the smoking process had a fundamental influence on Oscypek aroma formation.