Antibacterial activity of proteins extracted from the pulp of wild edible fruit of Bromelia pinguin L.

Bromelia pinguin L. is a natural source of bioactive compounds. The main purpose of this research was to isolate and characterize bioactive proteins from its fruit. B. pinguin proteins were fractionated by gel filtration chromatography, and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antibacterial activity of the proteins was analyzed against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, and the enzymatic activity was evaluated by protease activity and trypsin inhibitions assays. Protein fraction obtained by gel filtration chromatography exhibited antibacterial activity against E. coli (minimum inhibitory concentration [MIC] 0.3492 mg/mL) and S. aureus (MIC 0.6845 mg/mL). The proteolytic activity of the fraction was 0.985 U_cas/mL. The substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay detected protease inhibitors with molecular weights of 43 and 74 kDa. Antibacterial studies of E.coli and S. aureus were determined by comparing the protein fraction with different antibiotics. The antibacterial activity of proteins extracted from the pulp of the fruit of Bromelia pinguin L. could be related to the presence of enzymes, protease inhibitors and peptides.     

Antiulcerogenic activity of peptide concentrates obtained from hydrolysis of whey proteins by proteases from Cynara cardunculus

Peptide concentrates generated by hydrolysis of whey with aqueous extracts of flowers of Cynara cardunculus were studied for possible protection of the stomach mucosa against ulcerative lesions caused by oral administration of absolute ethanol. Both the whole peptide fraction obtained via hydrolysis of whey protein concentrate (peptide concentrate, PepC) and its fraction below 3 kDa (PepCF) were able to reduce gastric injuries to significant levels (p < 0.05). Single-dose experiments, using 100 mg kg⁻¹ body weight (bw) of either PepCF or PepC, led to 68.5% and 37.4% protection, respectively – which compare well with 93.4% protection by 200 mg kg⁻¹ bw carbenoxolone (a positive control). No dose-response correlation could be demonstrated. Gastric cytoprotection by PepCF appears to depend on sulphydryl-containing moieties, whereas PepC likely protects the gastric mucosa via the prostaglandin cycle and production of nitric oxide.     

Preparation of whey protein hydrolysates with ACE-inhibitory activity using cysteine peptidases from Bromelia hieronymi Mez. (Bromeliaceae)

A proteolytic extract from fruits of Bromelia hieronymi was used to hydrolyse bovine whey proteins. The peptide profile obtained exhibited a gradual fading of the main whey proteins and a decrease in the content of hydrophobic peptides. The 180-min hydrolysate showed a hydrolysis degree of 15.2% and inhibited the angiotensin-converting enzyme (ACE), showing an IC₅₀ of 0.17 mg/mL. By bioinformatics analysis, several theoretical sequences of possible ACE-inhibitory peptides were deduced. These results showed that proteolytic extracts from B. hieronymi can be used to prepare whey hydrolysates that would serve as a potential bioactive ingredient of functional foods.     

Purification and characterization of heat-stable proteases from Bacillus stearothermophilus RM-67

The extracellular proteases of Bacillus stearothermophilus RM-67 were purified by ammonium sulfate fractionation (40 to 70% saturation), gel filtration through Sephadex G-100, and diethylaminoethyl-Sephadex A-50 ion-exchange chromatography. Gel filtration resulted in separation of the enzyme preparation into one minor (protease I) and one major (protease II) peak. The three-step purification scheme resulted in 39.5-fold purification and an overall recovery of 8.1% of protease I and 87.8-fold purification and 59.7% recovery of protease II. Purified proteases had pH and temperature optima of 8.0 and 70 degrees C. Protease I and II, when together, retained 100% activity at 60 degrees C for 30 min. Manganese imported 100% stability to the pooled proteases at 65 degrees C for 30 min. Amino acid analysis of the major peak (protease II) revealed the absence of half cystine and methionine. Protease I and II had molecular weights of 67,610 and 19,950 and Michaelis-Menten constants (casein) of 1.33 and 2.0 mg/ml. Energy of activation was 14,300 cal/mol for protease I and 11,150 cal/mol for protease II. Corresponding heat of activation was l3,620 and 10,470 cal/mol.     

Microbial biofilm activity and physicochemical characterization of biodegradable and edible cups obtained from abdominal exoskeleton of an insect

A contemporary focus in food industry is the use of edible bio-based products with properties such as antimicrobial and biodegradable to replace the synthetic harmful petroleum-based polymers. Among the natural polysaccharides, chitin has generated considerable research interest thanks to its biocompatibility and abundance. This study investigated the production of chitin bio-cups from abdominal exoskeleton of an insect as an alternative to synthetic materials in food processing industry. The physicochemical properties of the obtained chitin and chitosan cups were studied by FT-IR, TGA, XRD and SEM analyses. The purity of the extracted chitin was examined by chitinase digestive test. The microbial biofilm formation on the cups was tested and no growth was recorded for the common food pathogen bacteria (Listeria monocytogenes) and yeast (Candida albicans). Considering the antimicrobial, antioxidant, nontoxic and edible nature of chitin and chitosan, these cups can be suggested as an alternative bioplastic for food protection.Industrial relevanceIn recent years much research has focused on the use of nontoxic and edible biopolymers as film and coating material in food industry to eliminate the use of carcinogenic and harmful petroleum products. Among the biopolymers, chitin and its deacetylated form, chitosan, are attracting widespread interest thanks to their nontoxic, biodegradable and edible properties. Here in this study, we investigated the production of chitin bio-cups from abdominal exoskeleton of an insect as an alternative to synthetic materials in food processing industry.     

Composition and characterization of cordyxanthins from Cordyceps militaris fruit bodies

Fruit bodies of Cordyceps militaris are widely used as functional food in China, Southeast Asia and North America. Cordyceps carotenoids are the important active components in the fruit bodies. However, chemical composition and property of the cordyceps carotenoids are still unknown. In this study, the novel carotenoids from C. militaris fruit bodies were separated and identified as xanthophylls and named as cordyxanthin-I (2, 3, 2′, 3′-tetradehydro-18, 16′, 17′, 18′-tetranor-ε, ε-carotene-5, 5′, 1′ -triol), cordyxanthin-II (2, 3, 2′, 3′-tetradehydro-18, 1′, 16′, 17′, 18′-pentanor-ε, ε-carotene-5, 5′, -diol), cordyxanthin-III (2, 3, 2′, 3′-tetradehydro-18, 17′, 18′-trinor-ε, ε-carotene-5, 5′, -diol) and cordyxanthin-IV (2, 3, 2′, 3′-tetradehydro-18, 18′-dinor-ε, ε-carotene-5, 5′, -diol). The four cordyxanthins proved to be highly water-soluble and could be directly quantified at 447 nm with β-carotene as a standard. Compared with traditional cultivation, the contents of the four cordyxanthins could be significantly increased by treatment of pink light (2/3 of 620–630 nm + 1/3 of 450–460 nm).     

草鱼消化道粗蛋白酶的部分性质

用丙酮脱脂然后用磷酸盐缓冲溶液从草鱼肠道中提取一种主要含有酸性蛋白酶和碱性蛋白酶的粗蛋白酶。该粗酶水解血红蛋白的最适pH=3～4，水解酪蛋白的最适pH值有2个，分别为pH4．4和PH10．3。水解牛血红蛋白的最适作用温度为37℃，水解酪蛋白的最适作用温度为45℃。酸性蛋白酶在50℃保温30min后只有大约20％的最初活性被保留，而要使碱性蛋白酶丧失80％的最初活力需要在60℃保温30min。     

Bioactivity of hydrolysates obtained from bovine casein using artichoke (Cynara scolymus L.) proteases

The objective of this work was to obtain casein hydrolysates with aspartic proteinases present in extracts from the artichoke flower (Cynara scolymus L.) and evaluate their antioxidant, antimicrobial, and angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. The casein hydrolysates produced by the action of C. scolymus had elevated antihypertensive and antioxidant activity due to their high hydrophobic peptide content (93.84, 96.58, and 90.54% at 2, 4, and 16 h of hydrolysis, respectively). Hydrolysis time and molecular weight (<3 kDa) had a significant influence on the hypertensive and antioxidant activity of the hydrolysates, which were greater at hydrolysis times of 4 and 16 h and corresponding to the <3 kDa fractions. The <3 kDa fraction of the 16 h hydrolysate had an ACE inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) of 71.77 µg peptides per mL; DPPH and ABTS^•+ radical scavenging activities of 6.27 µM and 6.21 mM Trolox equivalents per mg of peptides, respectively; and iron (II) chelation activity with an IC₅₀ of 221.49 µg of peptides per mL. Antimicrobial activity against Enterococcus faecalis was also observed in the hydrolysates. From the peptide sequences identified in the hydrolysates, we detected 22 peptides (from the BIOPEP database) that were already in their bioactive form (AMKPWIQPK, AMKPWIQPKTKVIPYVRYL, ARHPHPHLSFM, DAQSAPLRVY, FFVAPFPEVFGK, GPVRGPFPII, KVLPVPQK, LLYQEPVLGPVRGPFPIIV, MAIPPKKNQDK, NLHLPLPLL, PAAVRSPAQILQ, RELEELNVPGEIVESLSSSEESITR, RPKHPIKHQ, RPKHPIKHQGLPQEVLNENLLRF, SDIPNPIGSENSEK, TPVVVPPFLQP, VENLHLPLPLL, VKEAMAPK, VLNENLLR, VYPFPGPIH, VYQHQKAMKPWIQPKTKVIPYVRY, VYQHQKAMKPWIQPKTKVIPYVRYL) and are reported to display antioxidant, antimicrobial, and ACE inhibitory activity. We also identified 12,116, 14,513, and 25,169 peptide sequences in the hydrolysates at 2, 4, and 16 h, respectively, that were contained in the primary sequence, and these are reported to display ACE inhibitory, antioxidant, dipeptidyl peptidase IV inhibition, antithrombotic, opioid, immunomodulation, antiamnesic, anticancer, chelating, and hemolytic bioactivity.     

Total phenolic content and antioxidant capacity of extracts obtained from six important fruit residues

The extracts from kinnow peel, kinnow seeds, litchi pericarp, litchi seeds, grape seeds, and banana peel were screened for total phenolic content (TPC), trolox equivalent antioxidant capacity (TEAC), 1,1 diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, as well as reducing power. Kinnow peel extract exhibited the highest reducing power, TEAC, and DPPH free radical scavenging activity, whereas, the phenolic content of 37.4 mg GAE/g-dw was highest for grape seed extract. Banana peel extract with a low TPC showed the lowest reducing power, TEAC as well as DPPH free radical scavenging activity among the fruit residue extracts examined in the present study. Correlation analysis between the reducing power and DPPH radical scavenging ability; reducing power and ABTS radical scavenging activity; and ABTS and DPPH radical scavenging abilities showed a high degree of correlation (r² = 0.85-0.91). However, r² of 0.36, 0.66, and 0.49 between TPC and DPPH radical scavenging activity; TPC and reducing power; and TPC and ABTS radical scavenging ability, respectively, indicated that some non-phenolic compounds also contributed to the total antioxidant activity in fruit residue extracts examined in this study. To the best of our knowledge, this is the first paper presenting comprehensive data on TPC, reducing power, and antioxidant activity for the six fruit residues. This study demonstrated that kinnow peel, litchi pericarp, litchi seeds, and grape seeds, can serve as potential sources of antioxidants for use in food and pharmaceutical industry.     

Partial characterization of dextran-degrading enzyme obtained from blue cheese

Degradation of dextran beads was observed when the water-soluble fraction of a blue cheese extract was applied to the top of a Sephadex G-150 or G-200 column. This phenomenon suggests the presence of a specific enzyme that can hydrolyze dextran. After removal of casein components from the blue cheese fraction, ammonium sulfate treatment and gel filtration chromatography were performed to isolate the enzyme fraction. The enzymatic products were analyzed by thin-layer chromatography and gel filtration chromatography and identified as isomaltooligosaccharides. The isoelectric point of this enzyme fraction was approximately 4.9, as determined by isoelectric focusing using Rotofor, and the molecular weight of the fraction was 65 kDa, as estimated by sodium dodecyl sulfate (SDS)-PAGE. Optimum pH for enzymatic activity was 5.0 to 5.3. A partial N-terminal amino acid sequence of 20 residues was determined to be ATPDEWRSRSIYFMLTDRGA from an enzyme fraction further purified by ion-exchange chromatography and native PAGE. This sequence showed a maximum homology of 80% with alpha-amylase or Taka amylase that originated from various microorganisms.     

Isolation and partial characterization of CD36 from skim milk

CD36, a common milk fat globule membrane glycoprotein, was isolated from skim milk by methods similar to those previously utilized for the isolation of sulfhydryl oxidase. Two separate methods that were employed, gave similar purity as observed by electrophoresis. The first was based on differential centrifugation and size-exclusion chromatography, whereas the second combined ultrafiltration and affinity chromatography. After significant purification, the protein was identified by Western blotting and sequence analysis. Deglycosylation decreased the apparent molecular mass from approximately 85 to 57 kDa. These results suggested tissue-specific glycosylation. The purified fractions also exhibited low levels of sulfhydryl oxidase activity, the significance of which will require further study.     

蛋壳膜中透明质酸的提取及部分特性研究

分别采用胰蛋白酶和胃蛋白酶降解蛋壳膜,从中提取透明质酸.通过正交实验考察酶解温度、pH、酶用量、料液比和酶解时间对透明质酸提取量的影响,确定采用胰蛋白酶酶解的最佳条件为:温度为50℃,时间为7h,料液比为1:40.酶用量为8000U/g,pH为8.5,提取率为13.294mg/g;采用胃蛋白酶酶解的最佳条件为:温度为37℃,时间为5h,料液比为1:40,酶用量为11000U/g,pH为3.0,提取率为24.494mg/g.结果表明,胃蛋白酶提取透明质酸的效果好于胰蛋白酶,且壳膜中含有重要数量的透明质酸.通过红外光谱分析和测定葡萄糖醛酸、氨基葡萄糖的含量,对提取的透明质酸进行分析鉴定.     

Hydrolysis degree,peptide profile and phenylalanine removal from whey protein concentrate hydrolysates obtained by various proteases

The action of various proteases was tested for preparing whey protein concentrate (WPC) hydrolysates with high degree of hydrolysis (DH), appropriate peptide profiles and reduced phenylalanine (Phe) content. The peptide profile analysis included the fractionation of hydrolysates by size‐exclusion HPLC. The rapid correct fraction area method was used to quantify the components of the chromatographic fractions. Activated carbon (AC) was used to remove Phe, and its efficiency was evaluated by measuring the amount of Phe by second‐derivative spectrophotometry. The results showed that the DH of WPC hydrolysates increased and that the protease from Aspergillus oryzae yielded the highest DH value. This protease also produced the best peptide profile, that is, the highest di‐ and tripeptide content (16.14%), the highest amounts of free amino acids (18.43%) and the lowest amount of large peptides (18.76%). The proteases from both A. oryzae and Bacillus subtilis produced the highest Phe removals (79.0 and 77.8%, respectively).     

Crude lipoxygenase from pig muscle: partial characterization and interactions of temperature, NaCl and pH on its activity

Crude lipoxygenase (LOX) was extracted from fresh pig bacon belly and studied of partial characteristics. The interactions of temperature, sodium chloride (NaCl) and pH on LOX activity were investigated by response surface methodology (RSM). Kinetic studies indicated that the Michaelis constant (K(m)) and maximum velocity (V(max)) for LOX activity using linoleic acid as substrate were 68 μM and 0.26 U/min at 20°C, respectively. The optimal conditions for this reaction were: substrate concentration 3.47 mM, reaction temperature 39°C and pH ≧9.0. The NaCl critical value for LOX activity was 3.0% (w/w) at 20°C, above which the LOX activity began to decrease. Temperature had significant interactions (p<0.05) with NaCl and pH. The temperature critical value decreased with NaCl content increasing, while increased with pH increasing. These indicated that LOX activity and enzyme-catalyzed lipid oxidation in dry-cured meat products could be regulated by controlling process factors during the processing.