Extracellular trypsin-like proteases produced by Cordyceps militaris

A trypsin-like protease, P-1-1, was purified from the culture supernatant of the fungus Cordyceps militaris by (NH(4))(2)SO(4) precipitation, chromatography on DEAE Bio-Gel Agarose, TSKgel CM-5PW, and gel-filtration with HiLoad 26/60 Superdex 75 pg, and its properties were examined. Purified P-1-1 showed a single band by SDS-PAGE and was estimated to have a molecular mass of 23,405 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The optimum pH of the enzyme was between 8.5 and 12.0. It was inhibited strongly by leupeptin and diisopropyl fluorophosphate (DFP), and definitely did by N(alpha)-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. The carbonyl group sides of Arg and Lys were confirmed as the sites of cleavage by the enzyme toward cecropin B. These results indicate that P-1-1 is a trypsin-type serine protease. The N-terminal amino acid sequence of P-1-1 showed a high homology with those of trypsins or chymotrypsin derived from Diptera insects.     

Proteolysis characteristics of Actinomucor elegans and Rhizopus oligosporus extracellular proteases under acidic conditions

Enzymatic hydrolysis of soybean protein isolate by the extracellular proteases from Actinomucor elegans and Rhizopus oligosporus at pH 3.0, 3.5, 5.0, 5.5 and 6.0 was investigated. The activity of the A. elegans protease is lower than that of R. oligosporus, but both proteases exhibit considerable degradation of soybean protein at pH 5.5 and 6.0. The water‐soluble protein content and the degree of hydrolysis of the hydrolysates are increased significantly, and bitterness values are very low. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) reveals that these proteases have different cutting sites on peptide polymers. At pH 5.5, there is a lower content of total free amino acids (39.20 mg per 100 mL; 62.68% hydrophobic amino acids) in the R. oligosporus protease hydrolysate. In conclusion, treatment with R. oligosporus protease at pH 5.5 achieves efficient degradation of soybean protein, suggesting a promising industrial process for making bitterless protein hydrolysates.     

Physicochemical characterisation of eight Spanish mulberry clones: processing and fresh market aptitudes

The objective of this study was to evaluate and compare, for the first time, white and black mulberry species in terms of their main physicochemical characteristics in eight Spanish clones. The results showed significantly different characteristics between the black and white mulberry species. Fruit weight of mulberry species ranged from 2.10 to 4.15 g, and fruit juice yield, from 41% to 62%. Fructose (~61%) and glucose (~39%) were the predominant sugars in all mulberries. The MN1 clone displayed the highest total acidity (>2.6 g L^?1), and malic acid was the most abundant organic acid (6.65 g kg^?1). Cluster analysis has allowed grouping of the clones into three groups (i) MN1 and MN2; (ii) MN3 and MN4; and (iii) MA1, MA2, MA3 and MA4. Experimental results proved that Spanish mulberries have high potential for fresh consumption (attractive dark colour in Morus nigra clones, high sugars content and intense sweetness) and industrialisation (~50% juice yield, attractive juice colour, high content of crude fibre and intense sweetness). This study is also a step towards identification of this fruit as a potential healthy food, which may also be used in food industry and also have pharmaceutical interest.     

Wild Lactobacillus strains: Technological characterisation and design of Coalho cheese lactic culture

To design a specific lactic culture for the controlled manufacture of coalho cheese, 13 Lactobacillus rhamnosus, two Lactobacillus fermentum and one Lactobacillus plantarum strains isolated from artisanal coalho cheeses were identified and characterised. Two Lb. rhamnosus, one Lb. plantarum and one Lb. fermentum were selected and grouped in pairs designing four different culture formulations that demonstrated a good performance in cheesemaking experiments at pilot scale. Further studies to adjust the balance of strains used are necessary to attain adequate sensorial and technological attributes as expected for artisanal cheeses.     

Isolation,characterisation and sulphation of soluble polysaccharides isolated from Cucurbita maxima

Using hot water extraction, a large number of polysaccharides were obtained from Cucurbita maxima. A DEAE‐Sepharose CL‐6B chromatography column was used to isolate the major polysaccharides from C. maxima. Two fractions were obtained (LP2‐1 and LP2‐2). LP2‐1 and LP2‐2 consisted of neutral polysaccharides (MW: 1.02 × 10⁴ and 4.32 × 10⁸ g mol^?1, respectively) comprised mainly of galactose units. Analyses by FT‐IR spectrometry, partial acid hydrolysis, periodate oxidation, Smith degradation and GC‐MS indicated that LP2‐1 consisted of 85.3% (1→4) glycosidic linkages and 1.7% (1→3) or (1→6) glycosidic linkages. The LP2‐1 backbone consisted of (1→4)‐linked galactose units, which occasionally branched at O6 or O3. The branches were composed of (1→4)‐linked galactose and terminated with galactose (13%). Two sulphated derivatives (SLP2‐1 and SLP2‐2) with variable degrees of sulphation (DS) were obtained by the sulphur trioxide–pyridine method, without degradation of the polysaccharide. DS of PL2‐1 and PL2‐2 was 0.19 and 0.20, respectively.     

Structural characterisation and immunomodulatory effects of polysaccharides isolated from Dendrobium aphyllum

A crude polysaccharide extract of Dendrobium aphyllum (cDAP, yield 38.15 ± 0.20%) was generated. The D. aphyllum polysaccharide (DAP, M_w 471.586 kDa), purified by DEAE‐Sepharose and Sephadex‐G200 Fast Flow, was composed of mannose (71.3%) and glucose (28.7%), according to GC–MS analysis. Its backbone was composed of β‐d ‐mannopyranose and β‐d ‐glucopyranose residues, as revealed by infrared spectroscopic analysis. Its glycosidic bond was mainly 1, 4‐linked, and the O‐acetyl groups were mainly linked to mannose residues, according to periodate oxidation and Smith degradation analysis. The DAP units polymerised into a filiform‐shaped spatial pattern, as characterised by atomic force microscopy and scanning electron microscopy. DAP treatment enhanced cytokine secretion (nitric oxide, interleukin‐6 and tumour necrosis factor‐α) and pinocytic and phagocytic capacities of RAW 264.7 mouse macrophages. The complement receptor 3 and mannose receptor were identified to be the receptors of DAP on RAW 264.7 cells, indicating that the Akt/mTOR/MAPK and IKK/nuclear factor‐?B pathways could be involved in DAP‐activated immunomodulation.     

In vitro characterisation of probiotic properties of Enterococcus faecium and Enterococcus durans strains isolated from raw milk and traditional dairy products

In this study, some probiotic characteristics such as antimicrobial activity, vancomycin resistance, growth ability at pH, resistance to bile salts, bile salt deconjugation and hydrophobicity of 30 Enterococcus faecium and Enterococcus durans strains isolated and identified from raw milk and various dairy products were investigated. According to the study results, antimicrobial activity profiling, pH and bile salt resistance and bile salt deconjugation ability of Enterococcus strains varied depending on the species and strains and all the strains showed resistance to the tested bile salt concentrations. It was concluded that the E. faecium and E. durans strains tested showed probiotic characteristics and have the potential to be used in food production.     

Milk‐clotting enzymes produced by Aspergillus flavo furcatis strains on Amazonic fruit waste

Six strains of Aspergillus flavo furcatis were screened to investigate milk‐clotting enzyme production by fermentation in natural liquid medium. The growth media comprised extracts of cupuaçu exocarp+rice bran [10% or 20% (v/v) CE+RB] and açai waste+rice bran [10% or 20% (v/v) AW+RB] with or without supplementation of 0.1% (w/v) yeast extract and 0.5% (w/v) gelatin. Significant values of milk‐clotting activity were determined by A. flavo furcatis DPUA 1461 and DPUA 1608, in the standard and natural media, respectively. According to criteria of clot and whey formations, 8.3% of the samples tested were classified as strong coagulation, 41.70% showed weak coagulation and in 50% was not observed milk coagulation. The enzyme optimal action of DPUA 1608, the selected strain, was at 40 °C and pH 7.0. Milk‐clotting proteases were inhibited by pepstatin (94.72%) and moderately inhibited by the others metal ions tested.     

Production,mode of action and sequencing of the corresponding gene of a bacteriocin produced by Lactococcus lactis 19.3

Lactococcus lactis 19.3 produces a bacteriocin with a wide inhibitory spectrum, including the food pathogen Listeria monocytogenes. The producing strain was able to grow and produce similar amounts of bacteriocin in MRS medium, cow's milk and soya milk, respectively. The mode of action of this bacteriocin was further investigated using two indicator strains, Lactobacillus delbrueckii subsp. bulgaricus LMG 6901^T and Listeria monocytogenes ATCC 1911‐1. The bacteriocin displayed a bactericidal effect, causing a rapid decrease of the cell viability. Bacteriocin treatment of the sensitive strains resulted in major morphological changes, including pore formation, as shown by scanning electron microscopy. Finally, the bacteriocin‐encoding gene was identified by sequencing. The presence of nisin gene was confirmed. Based on all our data gathered so far, L. lactis 19.3 is a good candidate for a starter or protective culture in the manufacturing of both fermented dairy and vegetarian food products.     

Detection and quantification of Escherichia coli and Pseudomonas aeruginosa in cow milk by near‐infrared spectroscopy

This work investigated the potential of NIR technology to be applied in the dairy industry for the detection of micro‐organisms. To this end, two types of cow milk samples were studied, one in which only bacterial biomass was considered and the other in which bacteria were cultured and grown in milk for 24 h. The study was carried out using two micro‐organisms Escherichia coli and Pseudomonas aeruginosa. Both types of samples with different counts of both micro‐organisms were analysed by a NIR analyser in the range 10 000–4000 cm^?1 based on transmittance measurements. Multivariate models indicated that a better discrimination between micro‐organisms was attained in those milk samples in which micro‐organisms have been grown.     

Tannase from Aspergillus melleus improves the antioxidant activity of green tea: purification and biochemical characterisation

Tannase is an inducible enzyme used extensively in food, feed, pharmaceutical and chemical industries. In this study, tannase production and its biochemical properties were evaluated. From 42 Aspergillus strains analysed for potential tannase selection, Aspergillus melleus yielded the best results. Production was analysed using a complete factorial planning of 2³. Maximum activity (452.55 U mL^?1) was obtained in the optimal conditions of substrate (5.0 g), initial moisture (60%), tannic acid (2%) and 48 h of fermentation. The molecular weight of the purified enzyme was estimated as 69.52 kDa; its optimum temperature and pH were 40 °C and 5.5, respectively. Regarding the chemical effectors used, tannase was inhibited by ZnCl₂, ZnSO₄, Triton X‐100 and SDS. The addition of tannase to green tea improved its antioxidant potential by approximately 85% when compared to the control. The present results suggest that tannase may be used as an adjuvant to increase the antioxidant potential of green tea.     

Diversity of traditional Caciocavallo cheeses produced in Italy

This paper presents the results of a survey carried out in 68 dairies in southern Italy on the manufacturing processes of traditional Italian Caciocavallo cheese varieties. Following a study of the relevant literature, the various cheesemaking processes were analysed and the implications of different cheesemaking procedures were explored. The manufacturing variations able to influence the organoleptic characteristics of Caciocavallo cheese were milk and rennet types, procedures for curd acidification and stretching, salting and ripening conditions, and smoking treatment. This survey is designed to guide producers and consumers alike with respect to the perceivable effects of manufacturing variants on cheese quality.     

发酵豆酱中蛋白酶高产细菌的筛选及所产蛋白酶的酶学特性研究

从发酵豆酱中分离出四种产蛋白酶细菌,采用Folin-phenol法测定其蛋白酶活力,筛选出产酶活力最强的菌株,并进行酶学特性的研究。结果表明:该酶的最适反应温度为55℃;最适作用pH为8.5;该酶具有较好的稳定性;在pH6.0～8.5条件下稳定;NH+4对该蛋白酶有明显的激活作用,Pb2+、Fe3+则对其表现出强烈的抑制作用;在55℃、pH8.0条件下,该酶解反应的表观米氏常数为0.056%。     

Biotechnological Applications of Proteases in Food Technology

This review presents some of the hottest topics in biotechnological applications: proteases in biocatalysis. Obviously, one of the most relevant areas of application is in the hydrolysis of proteins in food technology, and that has led to a massive use on proteomics. The aim is to identify via peptide maps the different proteins obtained after a specific protease hydrolysis. However, concepts like degradomics are also taking on a more relevant importance in the use and study of proteases and will also be discussed. Other protease applications, as seem in cleaning (detergent development), the pharmaceutical industry, and in fine chemistry, will be analyzed. This review progresses from basic areas such as protease classification to a discussion of the preparation of protease‐immobilized biocatalysts, considering the different problems raised by the use of immobilized proteases due to the peculiar features of the substrates, usually large macromolecules. Production of bioactive peptides via limited hydrolysis of proteins will occupy an important place in this review.     

Streptococcus thermophilus strains of plant origin as dairy starters: Isolation and characterisation

Streptococcus thermophilus strains have been isolated mainly from dairy environments. To prospect for new strains of S. thermophilus, isolation was made from different plant sources. In this study, 74 plant isolates were characterised as S. thermophilus by a polyphasic approach and by 16S rRNA sequencing. The isolates were further evaluated for their physiological and biochemical properties. Plant isolates exhibited good acid production and varying proteolytic activity. All the isolates showed acetaldehyde and capsular exopolysaccharide (EPS) production and few revealed diacetyl production. In contrast to industrial strains, six isolates were able to ferment galactose and 25 were found to have no urease activity. Both the plant isolates and reference dairy cultures were found to possess similar physiological and biochemical properties and can be considered for developing new starters.     

Shelf life and quality of probiotic yogurt produced with Lactobacillus acidophilus and Gobdin

This study evaluated the effect of dry white mulberry and walnut paste (Gobdin, a traditional Turkish food) in probiotic yogurt on the survival of Lactobacillus acidophilus and yogurt properties. Six different yogurts were produced with 0%, 5% and 10% Gobdin using Lactobacillus bulgaricus + Streptococcus thermophilus and with 0%, 5% and 10% Gobdin using L. bulgaricus + S. thermophilus + L. acidophilus. The physical, chemical, microbiological and sensorial properties of the yogurts were evaluated based on storage at 4 ± 1 °C. Probiotic shelf life and the most suitable combinations were determined. The highest L. acidophilus count (8.65 log cfu g^?1) was found in the 5% Gobdin‐supplemented yogurt on the 7th day of storage, while the lowest count (8.11 log cfu g^?1) was found in the probiotic control yogurt on the 21st day. Although the L. acidophilus counts in the probiotic yogurts declined during storage, all values found throughout the 21‐day storage period were >8 log cfu g^?1. This is above the level necessary to provide the desired therapeutic effect in probiotic products (10⁶–10⁷ cfu g^?1). The highest overall acceptability score was obtained on the first day from the yogurt with 5% Gobdin. However, all yogurt samples had general acceptability scores between 7 and 8 points from a 9‐point maximum. Thus, this study determined that a new functional yogurt can be produced using L. acidophilus with 5% Gobdin.     

Detection of Salmonella spp., Yersinia enterocolitica,Listeria monocytogenes and Campylobacter spp. by real‐time multiplex PCR using amplicon DNA melting analysis and probe‐based assay

Syto9 and probe‐based multiplex real‐time PCR assays for simultaneous detection of a group of foodborne pathogens (named SYLC group), targeting Salmonella spp. (invA gene), Yersinia enterocolitica (ystA gene), Listeria monocytogenes (hly gene) and Campylobacter spp. (rrna gene), have been developed. The Syto9 assay generates amplicon DNA melting curve with four peaks of 86.5 ± 0.5, 84 ± 0.5, 81.5 ± 0.5 and 90.5 ± 0.5 °C corresponding Salmonella spp., Y. enterocolitica, L. monocytogenes and Campylobacter spp. targets, respectively. The sensitivities of the Syto9 and TaqMan assays in artificially inoculated chicken wing rinses were in a range of 3.2 × 10² to 3.1 × 10⁴ and 9.8 × 10² to 1.9 × 10⁴ colony‐forming units per millilitre, respectively, depending on the pathogen. All tested target strains (n = 100) were correctly detected by the both assays, whereas nontarget strains (n = 100) demonstrated no cross‐reactivity representing 100% specificity. The assays are suitable for application in qualitative and quantitative detection of SYLC group pathogens in food matrices.     

Partial purification and characterisation of endoglucanase from an edible mushroom, Lepista flaccida

A crude extract was prepared from the fruiting body of Lepista flaccida, an edible mushroom and endoglucanase activity of the extract was increased 14-fold with ammonium sulphate precipitation. Maximum enzyme activity was seen at pH 4.0 and 50 °C when carboxymethylcellulose was used as a substrate. K_0.5 and V_max values of the partially purified endoglucanase were 7.7 mg/ml and 25 ± 0.9 U/mg protein, respectively. The enzyme was quite stable over a broad range of pH (2.0–9.0) at 4 °C. When it was incubated at temperatures between 20 °C and 60 °C for 12 h, it conserved much of its original activity (over 40%). The activity of the enzyme increased by 234 ± 3.6% in the presence of 1 mM Mn²⁺. The endoglucanase was inhibited by EDTA, PMSF, β-ME and DDT. In conclusion, pH and thermal stability of the L. flaccida endoglucanase could make it useful for industrial purposes.     

两种蛋白酶对米渣蛋白的酶促降解作用

碱性蛋白酶和木瓜蛋白酶均可水解从米渣中提取的蛋白质,前者酶解米渣蛋白的效率明显优于后者。通过分析底物浓度、酶用量、溶液酸碱度、酶解温度和时间对酶促降解米渣蛋白效率的影响,得出碱性蛋白酶和木瓜蛋白酶各自水解米渣蛋白的最适条件,即碱性蛋白酶作用底物的浓度、酶的用量、溶液pH、酶解温度和时间分别为0.6%、50U/ml、8.5、45℃和4h;木瓜蛋白酶则依次为0.9%、300U/ml、6.0、70℃和4h。