Degradation of anti-nutritional factors and reduction of immunoreactivity of tempeh by co-fermentation with Rhizopus oligosporus RT-3 and Actinomucor elegans DCY-1

The effects of solid-state fermentation with four filamentous fungi (Rhizopus oligosporus, Actinomucor elegans, Neurospora crassa and Rhizopus oryzae) and their combination on the degradation of protein allergens and anti-nutritional factors (ANFs) of tempeh were evaluated. Glycinin, β-conglycinin, trypsin inhibitor and flatulence-causing oligosaccharides were significantly hydrolysed by all assayed strains but the level of reduction of ANFs depended on the strain. ELISA was conducted to evaluate IgE immunoreactivity using sera from soy sensitive individuals. Results revealed that co-fermentation of R. oligosporus and A. elegans resulted in the highest reduction in IgE immunoreactivity for all sera used and showed greatest degradations on α’, α, β, acidic subunits (100% hydrolysed) and extensive hydrolysis of flatulence-causing oligosaccharides. Tempeh fermented with R. oligosporus and A. elegans displayed high level of soluble protein and peptide (<10 kD) content. Co-fermentation of R. oligosporus and A. elegans was proved to be effective in producing hypoallergenic tempeh.     

酸性条件下毛霉和根霉蛋白酶的催化特性

研究比较了Actinomucor elegans蛋白酶和Rhizopus oligosporus蛋白酶在pH 3.0～6.0的条件下催化大豆分离蛋白(SPI)降解的特性,结果表明:在pH3.0和pH3.5的条件下,SDS-PAGE显示毛霉和根霉蛋白酶都能迅速地催化大豆7S和11S蛋白的降解,但此时水溶性蛋白的得率不高,均在35%以下;在pH5.0、5.5和6.0时,毛霉和根霉蛋白酶催化大豆分离蛋白降解的能力增强,水解度升高,水溶性蛋白的得率增加,但毛霉蛋白酶的水解产物水溶性蛋白得率始终高出约10%,SDS-PAGE显示毛霉蛋白酶催化下的大豆分离蛋白各个亚基降解消失的速度下降,同时伴随有新的条带产生,特别是碱性亚基几乎不能在4h内降解,根霉蛋白酶催化下的大豆分离蛋白各个亚基降解消失的速度在pH5.0和5.5时还比较强,但在pH6.0时明显下降,酸性亚基和碱性亚基均不能在4h内消失,表明在酸性条件下2个霉菌的蛋白酶其催化特性有明显的差异。     

Physicochemical and sensory characteristics of soya protein isolate hydrolysates with added substrate‐like amino acids

Soya protein isolate (SPI) with or without added substrate‐like amino acid was subject to enzymatic hydrolysis catalysed by commercial proteases (Alcalase 2.4 L, flavourzyme and pancreatin). Addition of a small amount of amino acids (amino acid: SPI = 1: 2500, mol g^?1) during hydrolysis would cause a significantly (P < 0.05) reduced protein recovery, increased degree of hydrolysis, and altered amino acid composition and antioxidant activities of SPI hydrolysates. The SPI hydrolysates prepared with added Asp, Arg or Lys exhibited a higher antioxidant activity than the control. The bitterness of SPI hydrolysates was largely reduced upon addition of Met, Asp or Glu during hydrolysis, whilst the umami taste and mouthfeel‐liking were remarkably increased. Therefore, adding amino acid during hydrolysis is a feasible and beneficial approach to improve both the functional and sensory properties of SPI hydrolysate.     

Production of bioactive proteins and peptides from the diatom Nitzschia laevis and comparison of their in vitro antioxidant activities with those from Spirulina platensis and Chlorella vulgaris

This research focuses on green production of bioactive proteins and hydrolysates from Nitzschia. A comparison of antioxidant activities was established between protein extracts and hydrolysates from Nitzschia and two other well‐known microalgae, chlorella and spirulina. Protein hydrolysates from these microalgae were produced using Alcalase^®, Flavourzyme^® and Trypsin. The hydrolysis process enhanced the antioxidant activities in general, especially those obtained using Alcalase^®. Nitzschia showed the highest (P < 0.05) total phenolic content/reducing capacity (2.4 ± 0.02 mg GAE/100 g) after 90 min of hydrolysis with Alcalase^®. The ABTS [2,2′‐Azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid)] radical scavenging activity (66.77 ± 0.00%) was highest (P < 0.05) after 120 min of hydrolysis, but DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl radical) was low (29.59 ± 0.02%). A correlation between ABTS activity and total phenolic contents was the highest (P < 0.05) for protein hydrolysates from all three organisms using Alcalase^®, but superoxide anion radical scavenging activity was intermediate for Nitzschia. Therefore, Nitzschia protein hydrolysates have the potential to be used as antioxidants.     

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).     

Improvement of functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle proteins hydrolyzed by Bacillus mojavensis A21 proteases

Functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle protein hydrolysates, with different degrees of hydrolysis (DH from 7.3% to 18.8%), obtained by treatment with Bacillus mojavensis A21 alkaline proteases were investigated. Protein contents for all freeze-dried cuttlefish muscle protein hydrolysates (CMPHs) ranged from 80% to 86%. For the functional properties, hydrolysis by A21 proteases increased (p < 0.05) protein solubility to above 78% over a wide pH range (2.0–11.0). However, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity and foaming stability) decreased with the increase of the DH. All CMPHs exhibited significant metal chelating activity and DPPH free radical-scavenging activity, and inhibited linoleic acid peroxidation. Antioxidant properties of protein hydrolysates increased with protein hydrolysis and the highest activities were obtained at DH of 16%. The IC₅₀ values for DPPH radical-scavenging and metal chelating activities were found to be 0.52 ± 0.01 mg/ml and 0.67 ± 0.13 mg/ml. The obtained results suggested that functional properties and antioxidant activities of cuttlefish muscle protein hydrolysates were influenced by the degree of hydrolysis.The composition of amino acids of undigested and hydrolyzed proteins was determined. CMPHs have a high percentage of essential amino acids such as arginine, lysine, histidine and leucine. They have a high nutritional value and could be used as supplement to poorly balanced dietary proteins.     

Rheological properties and gel strength of Phaseolus lunatus protein/carboxymethylated flamboyant gum systems

The rheological behaviour and gel strength of hydrocolloid mixture systems (HMSs) of carboxymethylated flamboyant gum (CFG) with protein hydrolysates (PHs) of Phaseolus lunatus were examined to evaluate the influence of the protein/polysaccharide ratio (2:1 and 3:1), pH (3 and 9) and concentration of solids, according to a 2³ factorial design. The protein concentrate of P. lunatus was hydrolysed with pepsin–pancreatin enzymes. The flow curve results were fitted to the Ostwald–de Waele model. The flow behaviour index (0.66–0.78) for all conditions studied was indicative of the shear‐thinning behaviour. For the HMS, the consistency index (k) values ranged from 0.4 Pa sⁿ to 1.2 Pa sⁿ. The analyses of variance showed that the ratio of PH/CFG and pH were the main variables that had significant effect on k values (P < 0.05). Only PH system presented a weak gel‐like viscoelastic behaviour. Both functional properties were affected by the protein degree of hydrolysis (DH).     

Comparison of physicochemical properties,antioxidant and metal‐chelating activities of protein hydrolysates from Phaseolus lunatus and hard‐to‐cook Phaseolus vulgaris

Limited and extensive hydrolysates were obtained from Phaseolus lunatus (LHl and EHl) and hard‐to‐cook Phaseolus vulgaris (LHv and EHv) using the enzymes Flavourzyme^®, Alcalase^®, Pancreatin^® and a sequential Pepsin^®–Pancreatin^® system. Degrees of hydrolysis varied from 8.32% to 31.60%. SDS‐PAGE of extensive hydrolysates showed molecular weights smaller than limited hydrolysates. Differential scanning calorimeter (DSC) analysis of LHl and EHl revealed the presence of two endothermic transitions; LHv and EHv had only one. LHv presented a higher content of hydrophobic amino acids whose surface hydrophobicity was 12.17. Functional properties such as nitrogen solubility, foaming capacity and emulsifying activity index in LHv were better than LHl at different pH evaluated. However, the latter showed better foaming stabilities. Amino acids such as His, Tyr, Trp and Arg were observed in greater amounts in both extensive hydrolysates. 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging and metal‐chelating activities in EHv and EHl increased significantly compared to the source material.     

Limited hydrolysis of β‐casein by cell wall proteinase and its effect on hydrolysates's conformational and structural properties

Optimisation of enzymatic hydrolysis of β‐casein with cell envelope proteinase (CEP) from Lactobacillus acidophilus JQ‐1 to produce the angiotensin‐I‐converting enzyme (ACE) inhibitory peptides using response surface methodology (RSM). Under optimal conditions (enzyme‐to‐substrate ([E]/[S]) ratio (w/w) of 0.132 and pH of 8.00 at 38.8 °C), the ACE inhibitory activity of hydrolysates was 72.06% and the total peptides was 11.75 mg mL^?1. Scanning electron microscopy (SEM) micrographs indicated that the tightness of the β‐casein surface structure was gradually weakened and small holes appeared after enzymatic treatment, while Fourier transform infrared spectroscopy (FTIR) spectra indicated remarkable changes in the chemical composition and macromolecular conformation of β‐casein after enzymatic hydrolysis. Differential scanning calorimetry (DSC) analysis indicated that the corresponding hydrolysates had higher thermal stability. The enzymatic hydrolysis also led to an increase in the free sulfhydryl content of β‐casein hydrolysates compared with raw β‐casein, which led to the increase in the antioxidant activity of β‐casein hydrolysates.     

Biotechnological characterisation of exocellular proteases produced by enological Hanseniaspora isolates

Twenty‐six enological Hanseniaspora isolates were identified by ITS PCR‐RFLP and D1/D2 domain of 26S rDNA sequencing and assayed for exocellular protease production. Based on qualitative data, six isolates, belonging to H. guilliermondii, H. valbyensis and H. occidentalis species, were selected to continue the study. Analytical procedure was optimised, and protease activities were quantified and characterised on the basis of different biotechnological factors. Protease activity was quite glucose, fructose and ethanol content independent, but divalent cation affects activity; these data support that they were aspartic proteases. The effect of 2‐mercaptoethanol suggests the importance of disulphide bonds to maintain the structure of the active centre. Our results show these enzymes could be suitable for using in biotechnological processes at neutral pH, such as cheese, bread and meat industries. This is, to our knowledge, the first work showing the induction process to induce and characterise proteolytic activity in Hanseniaspora isolates.     

Isolation,purification and characterisation of angiotensin I‐converting enzyme–inhibitory peptides derived from catfish (Clarias batrachus) muscle protein thermolysin hydrolysates

The angiotensin I‐converting enzyme (ACE)‐inhibitory activities of catfish (Clarias batrachus) muscle protein hydrolysates were investigated. Thermolytic digests of C. batrachus sarcoplasmic and myofibrillar proteins exhibited inhibitory activity towards ACE and were purified with the aim of ultrafiltration, gel filtration and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The amino acid sequences of hydrolysates with the highest ACE‐inhibitory activities were determined using electrospray quadrupole time‐of‐flight tandem mass spectrometry (ESI‐TOFQ MS/MS). The sequences of GPPP (IC₅₀ = 0.86 μm ) and IEKPP (IC₅₀ = 1.2 μm ) corresponding to the fragments 986–989 and 441–445 of myosin‐I heavy chain were identified for the sarcoplasmic and myofibrillar protein hydrolysates, respectively. Peptide GPPP exhibited a mixed‐type inhibition whereas peptide IEKPP could only bind to the active sites of ACE. The results demonstrate that hydrolysates of C. batrachus muscle proteins obtained by thermolysin may contain bioactive peptides.     

Effects of temperature and relative humidity on growth and enzyme production by Actinomucor elegans and Rhizopus oligosporus during sufu pehtze preparation

Sufu is a Chinese soybean cheese obtained after maturation of solid-state mould-fermented tofu. Ambient temperatures of 30–35 °C during the summer season prohibit the use of the usual starter Actinomucor elegans. We compared the properties of the latter with a potential alternative starter Rhizopus oligosporus that could be used at higher temperatures. The effects of temperature and relative humidity on growth rate of Actinomucor elegans and Rhizopus oligosporus were optimum at 25 °C at RH 95–97%, and 35 °C at RH 95–97%, respectively. Yields of protease (108 U/g pehtze), lipase (172 U/g) and glutaminase (176 U/g) by A. elegans were maximum after 48 h at 25 °C and RH 95–97%, and for α-amylase (279 U/g pehtze) and α-galactosidase (227 U/g) at 30 °C and RH 95–97% after 48 and 60 h of incubation. Highest protease (104 U/g pehtze), and lipase (187 U/g) activities of R. oligosporus were observed after 48 h at 35 °C and RH 95–97%, while maximum α-amylase (288 U/g pehtze) and glutaminase (187 U/g) activities were obtained after 36 h at 35 °C and RH 95–97%. Maximum α-galactosidase activity (226 U/g) by R. oligosporus was found after 36 h at 30 °C and RH 95–97%. It is concluded that R. oligosporus is a potential alternative to A. elegans as sufu pehtze starter during hot seasons.     

Preparation of alcalase hydrolysed rice bran protein concentrate and its inhibitory effect on soybean lipoxygenase activity

Rice bran protein concentrate (RBPC) was prepared from defatted rice bran and hydrolysed by alcalase at different hydrolysis times. As the hydrolysis times increased, the degree of hydrolysis (DHs) increased. RBPC hydrolysate obtained at 50 min (RBPCH‐50) had the highest inhibitory efficiency on soybean lipoxygenase (LOX) activity (66%). The inhibition kinetics of the reaction analysed by Lineweaver–Burk plots indicates that RBPCH‐50 is a competitive inhibitor. RBPCH‐50 inhibited soybean LOX with an IC₅₀ of 11.73 μg μL^?1 RBPCH‐50, and the obtained K_I was 4.59 μg μL^?1 RBPCH‐50. LOX inhibitory activity of RBPCH‐50 was significantly higher than that of 50 and 100 ppm butylated hydroxyanisole (BHA) and 50, 100, and 200 ppm butylated hydroxytoluene (BHT) (P ≤ 0.05); however, LOX inhibitory activity of RBPCH‐50 was similar to that of 200 ppm BHA (P > 0.05). Therefore, RBPCH might potentially be used as a natural LOX inhibitor.     

Antioxidant and antibacterial activities of peptide fractions from flaxseed protein hydrolysed by protease from Bacillus altitudinis HK02

Flaxseed protein (FP) hydrolysates by crude protease of strain Bacillus altitudinis HK02 were further separated into five fractions by ultrafiltration membranes with a molecular weight cut‐off of 10, 5, 3 and 1 kDa for the analysis of antioxidant activities and antibacterial ability. The results demonstrated that the fraction of 1‐ to 3‐kDa peptides exhibited higher antioxidant activities on the free radical‐scavenging ability and the reducing power than those of Vit C, Vit E, BHA and other fractions. The fraction with a low molecular weight (<1 kDa) of peptides demonstrated the highest ferrous ion‐chelating ability and a higher inhibition of lipid peroxidation than BHA and other fractions. Moreover, it also exhibited the best growth inhibition of Pseudomonas aeruginosa and Escherichia coli. The results, including the concentration‐dependent effect of peptides fractions, demonstrated that it is feasible to derive functional ingredients of natural antioxidants along with antimicrobial activity from FPs hydrolysed by protease from B. altitudinis HK02.     

Preparation and analysis of hydrolysates from whey protein concentrate using the proteases from Bacillus licheniformis and Aspergillus oryzae

The action of proteases from Bacillus licheniformis and Aspergillus oryzae was studied with the aim of preparing hydrolysates from whey protein concentrate with nutritionally appropriate peptide profile. Various enzyme/substrate ratios were used, and the peptides were fractionated by size‐exclusion HPLC followed by their quantification using the rapid method of correct fraction area (CFA). The protease from B. licheniformis (E:S of 8:100) produced the best peptide with a much lower amount of large peptides (44.61%), greater of di‐ and tripeptides (8.79%) and higher sum of the di‐ and tripeptides with free amino acids (9.99%) than the other hydrolysates. The advantage of using a lower E:S ratio to obtain a nutritionally adequate peptide profile was observed for the protease of A. oryzae when it passed from 3:100 to 2:100.     

Changes in degradation and phosphorylation level of titin in three ovine muscles during postmortem

The objective of this study was to investigate the degradation of titin and its phosphorylation level in three muscles from sheep. The MFI and pH from the longissimus lumborum (LL), semimembranosus (SM) and psoas major (PM) muscles were measured at 30 min, 1, 2, 7, 14, 21 and 28 days postmortem. Myofibrillar proteins were extracted, separated by SDS‐PAGE and quantified by phosphor‐specific staining. Phosphorylation of titin was predicted by Pro‐Q Diamond‐SYPRO Ruby staining. Two days after exsanguination, the pH and MFI of the PM were higher than those of the LL and SM muscles (P < 0.05). The sarcomere length of the PM muscle was also longer than that of the LL and SM muscle (P < 0.05). PM muscles had a highest phosphorylation level (P < 0.05) at 0.5 h postmortem and showed the greatest degree of titin degradation over 28 days. This suggests that phosphorylation of titin might accelerate its degradation.     

Properties and applications of β‐glycosidase from Bacteroides thetaiotaomicron that specifically hydrolyses isoflavone glycosides

To modify the glycan part of glycosides, the gene encoding β‐glycosidase was cloned from Bacteroides thetaiotaomicron VPI‐5482. The cloned gene, bt_1780, was expressed in Escherichia coli MC1061 and the expressed enzyme was purified using Ni‐NTA affinity chromatography. The purified enzyme, BTBG, showed optimal activity at 50 °C and pH 5.5. Interestingly, this enzyme did not have any hydrolysing activity on ordinary β‐linkage–containing substrates such as xylobiose, lactose and cello‐oligosaccharide, but specifically hydrolysed isoflavone glycosides such as daidzin, genistin and glycitin. Compared to a commercial beta glucosidase, BTBG selectively hydrolysed isoflavone glycosides in soybean extract mixture solution. These results suggest that BTBG may be a specialized enzyme for the hydrolysis of glycosides and that the substrate specificity of BTBG is applicable for the bioconversion of isoflavone glycosides in the food industry.     

Identification of active peptides from backbones of Nemipterus japonicus and Exocoetus volitans by electrospray ionisation–mass spectrometry

In our present investigation, Nemipterus japonicus and Exocoetus volitans backbone protein were hydrolysed by proteases like trypsin and pepsin, respectively. The protein hydrolysates were purified by different chromatographic methods, and the resulted purified peptides were analysed for their amino acid sequences by electrospray ionisation–MS/MS. The analysis of peptides showed sequences as Gly‐His‐Met‐Ser (451.8 Da) and Leu‐Glu‐Val‐Lys‐Pro (596.9 Da) for N. japonicus and E. volitans muscle, respectively. The presence of hydrophobic amino acids contributes more to the antioxidant activities of peptides than other amino acids. Moreover, sequence of amino acids in peptides plays an important role in their antioxidant activities.     

Developing a potential prebiotic of yogurt: growth of Bifidobacterium and yogurt cultures with addition of glycomacropeptide hydrolysate

This study is aimed at investigating the feasibility of casein glycomacropeptide (GMP) hydrolysates as potential prebiotics in yogurt. The growth performance of Bifidobacterium animalis subsp. lactis (Bb12) was determined in the presence of GMP hydrolysates produced by the action of proteolytic enzymes (trypsin and papain) with various degrees of hydrolysis. The growth‐promoting effect of GHP on Lactobacillus bulgaricus and Streptococcu thermophilus were also evaluated. Results showed that the optimum hydrolysis time of GMP with trypsin or papain for Bb12 growth promotion was 2 and 0.5 h, respectively. Compared with GMP and its trypsin hydrolysate, the GMP hydrolysate produced with papain (GHP) was the best for Bb12 growth, which obtained the highest viable count (9.3 log cfu mL^?1) and the lowest pH of 4.69. The obtained GHP significantly improved the growth of S. thermophilus (P < 0.05), while it had little effect on the growth of L. bulgaricus (P > 0.05)., The viable count of Bb12 of the yogurt obtained with the addition of 1.5% GHP was about four times higher than that of the control without GHP addition. The growth‐promoting effect of GHP might be related to its high content of Glu, Leu and Ala while had no direct relationship with sialic acid content.     

Bioactive action of β‐glucosidase enzyme of Bifidobacterium longum upon isoflavone glucosides present in soymilk

Bioconversion of isoflavone glucosides and antioxidant activity by probiotic strain (Bifidobacterium longum) during soymilk fermentation was investigated, as well as partial characterisation of the produced enzyme β‐glucosidase. The enzyme has higher affinity for genistin than for other substrates assayed. Maximum activity occurred at 42 °C and at pH 6.0; keeping 70–80% of activity for 60 days stored at low temperatures. Bifidobacterium longum grew well in soymilk (8.26 log CFU mL^?1 and pH of 3.9 at 24 h) and were produced in good quantities of organic acids. High hydrolysis degree of isoflavone glucosides (81.2%) was observed at 24 h. Enhancements in bioactivity were assessed in fermented soymilk by monitoring the radical‐scavenging activity, antioxidant activity and DNA protective action. The use of probiotic Bifidobacterium strain as β‐glucosidase producer increased bioactive isoflavone content and demonstrated that this enzyme plays a key role in the bioavailability of soymilk isoflavones, reducing the bioconversion time compared to other studies.