Proteolytic and angiotensin‐converting enzyme‐inhibitory activities of selected probiotic bacteria

This study was carried out to examine the proteolytic and angiotensin‐converting enzyme (ACE‐I) activities of probiotic lactic acid bacteria (LAB) as influenced by the type of media, fermentation time, strain type and media supplementation with a proteolytic enzyme (Flavourzyme^®). Lactobacillus casei (Lc210), Bifidobacterium animalis ssp12 (Bb12), Lactobacillus delbrueckii subsp. bulgaricus (Lb11842) and Lactobacillus acidophilus (La2410) were grown in 12% of reconstituted skim milk (RSM) or 4% of whey protein concentrates (WPC‐35) with or without combination (0.14%) of Flavourzyme^® for 12 h at 37 °C. All the strains were able to grow in both media depending on type of strains used and fermentation time. All the strains showed higher proteolytic activity and produced more antihypersensitive peptides when grown in RSM medium at 12 h, when compared to WPC. Combination with Flavourzyme^® also increased LAB growth and proteolytic and ACE‐I activities. Of the four strains used, Bb12 and La2410 outperformed Lc210 and Lb11842. The highest ACE‐I activity and proteolytic activity were found in B. animalis ssp12 combined with Flavourzyme^®. Flavourzyme^® led to an increase in the production of bioactive peptides with ACE‐I activity during 12 h at 37 °C.     

A Systematic Study on Extraction of Lipase Obtained by Solid-State Fermentation of Soybean Meal by a Newly Isolated Strain of <Emphasis Type="Italic">Penicillium</Emphasis> sp

This work aimed to assess the effect of some variables on the lipase extraction from the fermented medium in order to establish the experimental conditions that maximize the yield of the enzyme obtained from solid-state fermentation of soybean meal and a newly isolated strain of Penicillium sp. The experimental design technique was used to investigate the effect of relevant variables on lipase activity. The factors investigated were solvent pH (5.5–8.5), stirring rate (50–150 rpm), temperature (25–49 °C) and solid/liquid ratio (1:20–5:20). The effect of time of contact was evaluated in a kinetic study. Higher lipase activities in the extraction study were obtained using phosphate buffer 100 mM pH 8.5, at 25 °C, 150 rpm, and solid/liquid ratio of 1:20. Extraction studies showed that maximum activity (186 U/g) was obtained in 15 min of extraction.     

Partial Purification and Characterization of Extracellular Fructofuranosidase with Transfructosylating Activity from <Emphasis Type="Italic">Candida</Emphasis> sp.

The present work was carried out with the aim to investigate some properties of an extracellular fructofuranosidase enzyme, with high transfructosylating activity, from Candida sp. LEB-I3 (Laboratory of Bioprocess Engineering, Unicamp, Brazil). The enzyme was produced through fermentation, and after cell separation from the fermented medium, the enzyme was concentrated by ethanol precipitation and than purified by anion exchange chromatography. The enzyme exhibited both fructofuranosidase (FA) and fructosyltransferase (FTA) activities on a low and high sucrose concentration. With sucrose as the substrate, the data fitted the Michaellis–Menten model for FA, showing rather a substrate inhibitory shape for fructosyltransferase activity. The K _m and v _max values were shown to be 13.4 g L⁻¹ and 21.0 μmol mL⁻¹ min⁻¹ and 25.5 g L⁻¹ and 52.5 μmol mL⁻¹ min⁻¹ for FA and FTA activities, respectively. FTA presented an inhibitory factor K _i of 729.8 g L⁻¹. The optimum conditions for FA activity were found to be pH 3.25–3.5 and temperatures around 69 °C, while for FTA, the optimum condition were 65 °C (±2 °C) and pH 4.00 (±0.25). Both activities were very stable at temperatures below 60 °C, while for FA, the best stability occurred at pH 5.0 and for FTA at pH  4.5–5.0. Despite the strong fructofuranosidase activity, causing hydrolysis of the fructooligosaccharides (FOS), the high transfructosilating activity allows a high FOS production from sucrose (44%).     

Stimulation of polygalacturonase production in an immobilized system by <Emphasis Type="Italic">Aspergillus</Emphasis> sp.: effect of pectin and glucose

The synthesis of polygalacturonases (PG) is known to be influenced by Aspergillus growth conditions, namely, environmental factors and pectin content in the cultivation medium containing a mixed carbon source. Optimal conditions were attained at a temperature of 30 °C and an initial pH of 4.5. PG activity (3.29 and 2.48 U/mL) was determined after a two-day culture of Aspergillus sp. HC1 and Aspergillus sp. CC1, respectively, in a basic medium containing 2% citrus pectin as the sole carbon source. The addition of glucose (2% w/v) to the basic medium led to a 2-fold increase in PG production. However, enzyme synthesis was repressed when a higher concentration of glucose was used in the medium containing the mixed carbon source. Spores from the two fungi were immobilized in a 3% Ca–alginate system and the mechanical strength of the gel beads allowed the use of this process system 6-fold longer (288 h) than the free culture. In the Aspergillus sp. CC1 immobilized system, PG production increased nearly 10-fold in the medium with 2% glucose added (5.95 U/mL) in comparison to the medium without sugar (0.55 U/mL). The results demonstrate that a different response in activity was produced by free and entrapped spore systems. PG production remained approximately constant throughout the six 48 h cycles in the medium containing citrus pectin (2% w/v) as the sole carbon source.     

Culture Filtrates from a Fermented Rice Product (Lao-Chao) and Their Effects on Milk Curd Firmness

To develop a new Oriental-style dairy product, the characteristics of culture filtrate from lao-chao with Rhizopus oryzae, used as a milk-clotting agent, and factors (heat-treatments, calcium, sucrose, and curdling temperature) affecting curd firmness were determined. The optimal conditions for proteolytic activity were around 40°C and pH 3. No activity was detected over the range of pH 8 to 11. After high-heat treatment (121°C, 15 min) on skim milk, no clotting was observed. Sucrose resulted in the retardation of milk-clotting. Ca⁺⁺ could be used to increase curd firmness which also increased from 29. 2–2.8g to 80. 3–4.7g when the curdling temperature was increased from 25°C to 45°C.     

Purification and characterisation of β‐mannanase from Lactobacillus plantarum (M24) and its applications in some fruit juices

β‐Mannanase was purified 2619.05‐fold from the Lactobacillus plantarum (M24) bacterium by ammonium sulphate precipitation and ion exchange chromatography (DEAE‐Sephadex). The purified enzyme gave two protein bands at a level of approximately 36.4 and 55.3 kDa in the SDS‐PAGE. The purified mannanase enzyme has shown its maximum activity at 50 °C and pH 8, and it has been also determined that the enzyme was stable at 5–11 pH range and over 50 °C. The V_max and K_m values have been identified as 82 mg mannan mL^?1 and 0.178 mm , respectively. The effects of some metal ions such as Fe²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ on the mannanase enzyme have been also investigated, and it has been determined that all metal ions had significant effects on the activation of the mannanase enzyme. In addition, the effectiveness of the purified mannanase enzyme on the clarification of some fruit juices such as orange, apricot, grape and apple has been investigated. During the clarification processes, the enzyme was more effective than crude extracts on the clarification of the peach juice with a ratio of 223.1% at most.     

Properties of thermostable extracellular FOS-producing fructofuranosidase from <Emphasis Type="Italic">Cryptococcus</Emphasis> sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K _m, K _i and v _max values were shown to be 64 mM, 3 M and 159.6 μmol mL⁻¹ min⁻¹ for FA and 131 mM, 1.6 M and 377.8 μmol mL⁻¹ min⁻¹ for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).     

Isolation and identification of high pressure‐resistant bacteria naturally contaminating strawberry pulp

The inactivation of bacteria naturally present in strawberry pulp was investigated after high hydrostatic pressure (HHP) treatment at pressure levels up to 600 MPa at 25 °C for 5 ~ 25 min. Five strains of pressure‐resistant bacteria designated as A, B, C, D and E were isolated and identified. The five strains were gram‐positive, spore‐forming, rods or rod in chains. Growth of the strains was observed at 30 ~ 45 °C, and strain B also grew well at 55 °C. They could produce acid from glucose and were catalase‐positive. Analysis of 16S rRNA gene sequences showed that the five strains belonged to the genus Bacillus. Strain A and D exhibited the greatest 16S rRNA gene sequence similarity of 99% with B. licheniformis and B. firmus, respectively. By combination of phenotypic characteristics and 16S rRNA gene sequences, strain C was B. mycoides and E was B. pumilus. On the basis of physiological and biochemical characteristics, gyrB gene sequences analysis and whole‐cell fatty acids analysis, strain B was B. amyloliquefaciens. Further studies showed that strain B (B. amyloliquefaciens) exhibited the highest pressure resistance, and it was reduced by 4.62‐log after treatment at 600 MPa for 25 min at 25 °C as the most effective observed inactivation.     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling     

Purification and characterization of Bacillus subtilis milk-clotting enzyme from Tibet Plateau and its potential use in yak dairy industry

A milk-clotting enzyme named YS-1 was purified from a Bacillus subtilis (B. subtilis) YB-3, which we have isolated from Tibetan Plateau, Gansu, China. The enzyme YS-1 was identified as a metalloproteinase. SDS-PAGE and MALDI-TOF-MS analysis of the purified enzyme gave a molecular weight of 42 kDa. YS-1 was stable over a wide range of temperature from 20 to 60 °C. Purified YS-1 was also active over a wide range of pH from 5.0 to 9.0. It can be activated by Ca²⁺ and Al³⁺ but inhibited by Zn²⁺, Fe²⁺ and Cu²⁺. The milk-clotting enzyme YS-1 exhibited high specificity to substrate β-casein and yak milk casein and led to a 75% more rapid coagulation of yak milk than cow milk, due to high β-casein content in yak milk. Together, our findings confirmed that the enzyme YS-1 has a potential to be used in yak cheese industry.     

Effect of high hydrostatic pressure and high-pressure homogenisation on <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> inactivation kinetics and quality parameters of mandarin juice

The inactivation kinetics of Lactobacillus plantarum in a mandarin juice treated by thermal treatment (45–90 °C), high-pressure homogenisation (HPH) (30–120 MPa at 15 and 30 °C) and high-pressure processing (HPP) (150–450 MPa at 15, 30 and 45 °C) were fitted to different Weibullian equations. A synergic effect between pressure and temperature was observed in HPH and HPP treatments achieving 2.38 log cycles after 120 MPa at 30 °C for 10 s (final T of 45 °C) and 6.12 log cycles after 400 MPa at 45 °C for 1 min (final T of 60 °C), respectively. A combined treatment of 100 MPa at 15 °C for 10 s and 300 MPa at 15–30 °C for 1 min in HPH and HPP, respectively, was needed to the first logarithm microbial population decline. Weibull model accurately predicted microorganism inactivation kinetics after HPH and HPP processing when displaying single shoulder or tail in the survivor curves, whereas when a more complex trend was observed after thermal treatment, the double-Weibull equation was found more appropriate to explain such behaviour. Equivalent treatments that achieved the same degree of microbial inactivation (77 °C–10 s in thermal processing, 120 MPa–10 s at 30 °C in HPH processing and 375 MPa–1 min at 30 °C in HPP) were selected to study the effects on quality parameters. The application of dynamic pressure led to a decrease in sedimentable pulp, transmittance and juice redness, thus stabilising the opaqueness and cloudiness of mandarin juice. Pectin methyl esterase (PME) was found to be highly baroresistant to static and dynamic pressure. Carotenoid content remained unaffected by any treatment. This study shows the potential of high-pressure homogenisation as an alternative for fruit-juice pasteurisation.     

Optimization of process conditions for the production of a prolylendopeptidase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 11414 in solid state fermentation

The effect of 8 factors [(with/without) daily mixing and moisture control, incubation time (t), temperature, ratio between dry substrate mass and bed’s cross section area (MA), inoculum size (spores/g), wheat germ content (WG), initial pH, and moisture content (M)] in the production of a prolyl endopeptidase (PEP) by Aspergillus niger ATCC 11414 in solid state fermentation (SSF) was tested. Contribution of all the factors was significant (p<0.05); main effects were those of MA, t, and M. The 4 interactions that presented high interaction severity indexes involved the WG. Under optimized conditions PEP and protease activity were 9.76±0.06 and 3.6×10⁶±1.5×10⁵ U/kg, respectively. The enzyme was partially purified (ammonium sulfate precipitation, dialysis, DEAE-Sepharose ionexchange); it has a molecular weight of 66 kDa (SDS-PAGE), and maximum activity was exhibited at pH 4 and 50°C. The enzyme is stable in a wide pH range (2.2–10) and at temperatures lower than 70°C.     

Novel anticoagulant compound from fermented red alga <Emphasis Type="Italic">Pachymeniopsis elliptica</Emphasis>

Natural fermentation was tested as a method of releasing active compounds during screening for potential anticoagulant activity in three types of algae (Pachymeniopsis elliptica, Sargassum horneri, and Ulva pertusa). Freeze dried algae samples (2.5 g) were fermented by adding 75 g of sugar and 500 mL of water and thereafter kept at room temperature (25 °C) for 3 months. Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were measured every 2 weeks for 3 months to determine the optimum time for the highest activity. Fermented P. elliptica, (which had the highest activity) was subjected to anion exchange chromatography (DEAE-cellulose) and sepharose 4B gel permeation chromatography. The purified sample was analyzed by agarose-gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) to confirm the purification and to determine the molecular mass, respectively. The 360 μg/mL of purified compound (Mwt > 500,000 Da) had both APTT and PT activities (>1,000 s). However, at the concentrations of 180 μg/mL, purified compound and heparin showed 540 and >1,000 s APTT activity, respectively. Though, the purified compound of P. elliptica considered as a weaker anticoagulant than heparin, this purified anticoagulant polysaccharide could be considered as a good alternative source as an anticoagulant. Moreover, the technique of fermentation is an inexpensive and feasible, this purified anticoagulant polysaccharide compound could be used in pharmaceutical and biomedical industry. Further investigations need to be performed to determine the mechanism of this novel anticoagulant compound. The authors Prashani Mudika Ekanayake and Chamilani Nikapitiya contributed equally to this work.     

Synthesis of raffinose by fungal α-galacotosidase from Absidia corymbifera

In order to investigate the optimal conditions for raffinose synthesis, α-galactosidase was purified from Absidia corymbifera IFO8084 with a recovery yield of approximately 8.1% (8.36 mg). The molecular weight of the wild-type α-galactosidase was about 83 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native molecular mass of the enzyme was approximately 330 kDa by gel filtration chromatography, indicating that α-galactosidase from A. corymbifera IFO8084 is a homotetrameric enzyme. The purified enzyme displayed optimal enzyme activity at pH 4.5 and 60°C. When the purified α-galactosidase was incubated in a substrate solution of sucrose and D-galactose for 48 hr at 37°C, raffinose was synthesized and was confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and ¹³C-nuclear magnetic resonance (¹³C-NMR) spectrometer analysis. Maximum rates of conversion were observed with 1.67 M galactose, 2.04 M sucrose, and 100 U α-galactosidase at pH 6.0 and 70°C. Under the optimized conditions, the overall conversion ratio was 10%(w/v), representing 2.5 times the synthesis yield that would be possible without the optimized conditions.     

Purification and properties of a heat-stable enzyme of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 °C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn²⁺ has positive effect on activity and can increased the heat resistance capability, while Ca²⁺ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL⁻¹. The enzyme showed typical heat-stable behavior. After treatment of 160 °C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 °C in buffer with Mn²⁺ was approximately 12 s. Among several main milk proteins, Ht13 can cleave α_s-casein, β-casein and κ-casein. The sequence of 1st–16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.     

Purification and Properties of a Thermostable Inulinase (β-D-Fructan Fructohydrolase) from Bacillus stearothermophilus KP1289

A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41 °C to 69 °C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight (M_r) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively. The enzyme was active between 30 and 75 °C and at pH 4.5—8.6 with an optimum at 60 °C and pH 6.1. At 69 °C and pH 7.0 the half-life of the enzyme was 10 min. The enzyme released fructose exo-wise from the non-reducing end of inulin (M_r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60 °C and pH 5.0 were 80 mM (360 mg/mL), 460 s^—1, and 5.8 s^—1 mM^—1, respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0.50:0.16. The enzyme was classified as a thermophilic thermostable β-D -fructan fructohydrolase (EC 3.2.1.80).     

Novel milk-clotting enzyme produced by Coprinus lagopides basidial mushroom

Submerged cultured higher basidiomycetes were screened for milk-clotting activity. The native liquid of Coprinus lagopides demonstrated high milk-clotting activity, while having relatively low general proteolytic activity. Optimization of growth media composition and ultrafiltration of the native liquid allows increasing the ratio of milk-clotting and proteolytic activities. The Enzyme was purified and characterized.     

Changes in activity during storage and characteristics of superoxide dismutase from hen eggs (<Emphasis Type="Italic">Gallus gallus domesticus</Emphasis>)

Hen eggs are one of the most popular food stuffs. Moreover, they can be the source of not only nutrients but also factors of biological origin, which may be used for food preservation and food additives. The aim of the study was to determine and describe the activity of superoxide dismutase isoforms (SOD; EC 1.15.1.1) in hen eggs (Gallus gallus domesticus). Our electrophoretic studies confirmed the presence of SOD isoenzyme bands with molecular weight of 14–15 and 50–70 kDa in egg yolk. By contrast, in egg white, we confirmed the presence of a protein with molecular weight of 13–14 and 50–55 kDa. Zymografic pattern confirmed the activity of SOD isoforms of the enzyme present in the egg yolk; however, it did not confirm enzyme activity in egg white (at the level of error of the used method). Study has also shown SOD activity during storage at 4 °C for 9 days in egg yolk and egg white. In start time, SOD activity in egg yolk is clearly different from a small activity in the protein (respectively, 90.5 ± 22.2 and 7.9 ± 3.9 U g⁻¹). It did not change during 6 days storage but between 6th and 9th day, it decreased significantly in egg yolk while remained low but unchanged in egg white. Present study confirmed the presence of SOD and its activity in hen egg yolk.     

Characterisation of a milk-clotting extract from Balanites aegyptiaca fruit pulp

Milk-clotting proteases were extracted and characterised from Balanites aegyptiaca fruit pulp traditionally used in sub-Saharan countries to thicken gruel. The aim was to improve the extraction conditions and milk-clotting in the context of future cheese making. B. aegyptiaca fruits were disinfected, husked and soaked in different buffers and saline solutions. The extracts obtained were diafiltered, concentrated and decoloured with active charcoal. The effects of extract concentration, milk pH and temperature on clotting-time were studied. The use of specific inhibitors showed that the B. aegyptiaca extract contains two types of proteases: an aspartic protease and a serine protease, with an optimum activity at pH 5.0 and pH 8.0, respectively, and an optimal temperature at 50 °C. The proteolytic activity is more thermostable than bovine chymosin at pH 5.0. Partial purification by anion exchange chromatography and sodium docecylsulphate polyacrylamide gel electrophoresis allowed separation of the two milk-clotting proteases.     

Microbiological quality of Brazilian UHT milk: Identification and spoilage potential of spore‐forming bacteria

Counts of aerobic mesophilic micro‐organisms and aerobic mesophilic spore‐forming bacteria were determined in 91 ultra‐high‐temperature (UHT) commercial Brazilian samples. Forty‐six spore‐forming bacteria were identified and characterised in terms of their spoilage potential. Among the 20 brands evaluated, 45% had counts of aerobic mesophilic micro‐organisms higher than 1.0 × 102 cfu/mL. The sporulated bacteria were identified as Bacillus subtilis/amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus megaterium, and 31% and 33% showed proteolytic and lipolytic activity, respectively. Our findings indicate that there is a potential risk of UHT milk samples becoming spoiled during their commercial shelf life.