Speciation of selenium in selenium-enriched seeds,buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> Moench) and quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis> Willdenow)

Buckwheat (Fagopyrum esculentum Moench) and quinoa (Chenopodium quinoa Willdenow) are widely used as food ingredients. The nutritional characteristics of these plants, i.e., high contents of proteins and amino acids suggest that selenium (Se) is preserved as selenoamino acid derivatives, in particular, selenomethionine (SeMet) in proteins, similar to selenized yeast. Therefore, buckwheat and quinoa are expected to be a good nutritional source of Se. Selenized buckwheat and quinoa were cultivated on Se-fortified soil using sparingly soluble Se salts, such as barium selenate and barium selenite. Se concentration in the edible parts of these plants was determined, and Se extraction efficiency with enzyme or alkali was evaluated. In addition, the chemical species of Se in the low molecular weight fraction of these plants were determined by HPLC-ICP-MS. Total Se concentrations in the edible parts of selenized buckwheat and quinoa were 170.4 ± 2.9 μg/g and 102.7 ± 2.4 μg/g wet weight, respectively. Thus, these selenized seeds were found to be Se accumulators. The results indicate that Se in selenized buckwheat exists mainly as SeMet, while Se in selenized quinoa exists not only as SeMet but also as selenate (Se(VI)) and non-protein forms.     

Determination of the daily dietary selenium intake in Croatia

 Data about selenium (Se) concentrations in the environment and its daily dietary intake in Croatia are scarce. The aim of this study was to estimate daily dietary Se intake in a group of female subjects from the Zagreb area using three different approaches: direct measurement of Se in collected daily duplicate portions, daily dietary records and the indirect method of Se analysis in serum. The results were 33.2±8.82, 30.9±7.40 and 37.8±5.45 μg day^–1 (arithmetic mean ±SD) by duplicate portion, daily dietary records and the serum Se method, respectively. The results of dietary Se intake estimation showed that Se intake for the observed group of female subjects from Zagreb area is lower than in the majority of European countries, and lower than the value recommended by the World Health Organisation. Received: 17 May 1999     

Enhanced selenium content in buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> Moench) and pumpkin (<Emphasis Type="Italic">Cucurbita pepo</Emphasis> L.) seeds by foliar fertilisation

The fruit and thin-husked seeds of the pumpkin (Cucurbita pepo L.) and buckwheat grain (Fagopyrum esculentum Moench), both grown in Slovenia, were analysed for selenium (Se) content following foliar application of Se(VI) solution during the period of blooming. Samples were digested by a H₂SO₄-HNO₃-H₂O₂-V₂O₅ mixture and Se determined, based on hydride generation atomic fluorescence spectrometry. The whole procedure from weighing to measuring was carried out in the same Teflon vessel. The detection limit of the method was 0.14 ng g^–1 solution. Buckwheat seeds from untreated plants contained 47 ng g^–1 of Se and 394 ng g^–1 from plants after foliar fertilisation with Se. Pumpkin seeds from untreated plants contained 108 ng g^–1 of Se, and 381 ng g^–1 of Se from Se-treated plants, all per lyophilised sample. Se content in lyophilised pumpkin fruit was 15 ng g^–1 in untreated plants and 20 ng g^–1 in Se-treated pumpkin plants. It is thus feasible to enhance Se content in buckwheat and pumpkin seeds by foliar fertilisation, making them a rich source of dietary Se and useful as a raw material for enriched food products.     

Selenium species in buckwheat cultivated with foliar addition of Se(VI) and various levels of UV-B radiation

Common (Fagopyrum esculentum Moench) and tartary (Fagopyrum tataricum Gaertn.) buckwheat was treated by spraying the leaves with a water solution containing 15 mg Se per litre in the form of sodium selenate in the flowering period. The selenium content in all parts of plant was found to be less than 200 ng g⁻¹ in non-treated and in the range 2700–4650 ng g⁻¹ in selenium treated buckwheat. Exposure to UV-B radiation lead to higher Se accumulation in flowers of both Se enriched cultivars. For speciation analysis enzymatic hydrolysis was carried out, separation and detection of selenium species was performed by high performance liquid chromatography–ultraviolet treatment–hydride generation atomic fluorescence spectrometry (HPLC–UV–HG-AFS). In flowers and leaves, on average 11% of the Se content was soluble and in the form of Se(VI), representing between 0.6% (flowers) and 3% (leaves) of the Se content. The remaining soluble non-amino acid organic Se was not detected by HPLC–UV–HG-AFS. In seeds 93% of the selenium content was found in the extracts and the main selenium species was SeMet with 93 ± 5% relative to the selenium content.     

Analysis of phenolic compounds and isoflavones in soybean seeds (Glycine max (L.) Merill) and sprouts grown under different conditions

Soybeans (Glycine max (L.) Merill) are popularly known as a healthy food in many Asian countries and are mostly consumed as soymilk, tofu, and fermented products such as miso, temph, and sufu. The objective of this study was to determine the variation and composition of phenolic compounds and isoflavone contents in soybean seeds [Glycine max (L.) Merill] and sprouts [Kongnamul] grown under dark conditions (producing yellow soybean sprouts) and in green and yellow boxes (producing green soybean sprouts). In seven soybean cultivars, the total phenolic content ranged from 6.67 μg⁻¹ in Pureunkong to 72.33 μg⁻¹ in Poongsannamulkong. The average total phenolic content in the green soybean sprouts (48.33 μg⁻¹) was higher than in the yellow soybean sprouts (29.75 μg⁻¹). The total phenolic content in the yellow soybean sprouts varied from 9.88 μg⁻¹ to 47.71 μg⁻¹, and the total phenolic content in the green soybean sprouts varied from 29.21 μg⁻¹ to 79.70 μg⁻¹. Only four phenolic compounds, p-hydroxybenzoic acid, salicylic acid, p-coumaric acid, and ferulic acid, were detected in all soybean cultivars. Syringic acid was not detected in yellow soybean sprouts, and myricetin was only detected in yellow soybean sprouts (4.65 μg⁻¹) from the Pureunkong cultivar grown under dark conditions. The total isoflavone content in soybean seeds ranged from 2.1 μg⁻¹ in Sowonkong to 33.0 μg⁻¹ in Pureunkong, and the mean total isoflavones was 10.61 μg⁻¹. Green soybean sprouts had higher average total isoflavones (1389.4 μg⁻¹) than yellow soybean sprouts (559.2 μg⁻¹), and the total isoflavone content was highest in the Pureunkong yellow soybean sprouts (756.3 μg⁻¹) and the Sowonkong green soybean sprouts (2791.6 μg⁻¹). In soybean sprouts, the higher the (malonyl)-daidzin or (malonyl)-genistein content, the higher the total isoflavone level. Our study suggests that producing soybean sprouts enriched in isoflavones under coloured-light sources is feasible.     

Physicochemical and Enzymatic Properties of Five Kiwifruit Cultivars during Cold Storage

Samples of Abbot, Alison, Bruno, Monty, and Hayward cultivars of kiwifruit (Actinidia deliciosa) were obtained from the Iran Research Center of Citrus (Tonekabon, located in north of Iran) and their physicochemical properties were studied during cold storage (at T = 1 ± 1 °C, RH = 80 ± 5%) at 0-, 9-, and 18-week intervals. The mean chemical composition of the fruits were as follows: ash = 0.66–0.96%, moisture = 75.2–84.7%, starch = 0.3–7.0%, and ascorbic acid = 54.8–261.0; K = 125.0–372.0 mg 100 g⁻¹ fresh weight, Mg = 18.0–32.0 mg 100 g⁻¹ fresh weight, Na = 1.4–3.1 mg 100 g⁻¹ fresh weight, Fe = 0.17–0.52 mg 100 g⁻¹ fresh weight, Cu = 0.04–0.24 mg 100 g⁻¹ fresh weight, Zn = 0.16–0.49 mg 100 g⁻¹ fresh weight, Mn = 0.04–0.10 mg 100 g⁻¹ fresh weight, and P = 25.2–49.3 mg 100 g⁻¹ fresh weight; glucose = 0.7–2.39%, fructose = 1.20–3.13%, and sucrose = 0.0–5.8%. At the same time, the values of the parameters °Brix = 6.5–14.8% and acidity = 1.8–2.5% of the studied cultivars (mutual effects of cultivar and storage time) were investigated. The increase in peroxidase (POX = 0.0–6.65 U ml⁻¹) and the decrease in pectinesterase (PE; poor activity to 0) activities were also determined. The statistical analysis showed that the Bruno cultivar had the highest content of ascorbic acid (115.0–261.0 mg 100 g⁻¹ fresh weight), which is an important compound in fruits during storage, while Hayward had the best overall quality particularly with regards to its resistance to softening. This study confirms that long-term cold storage at 1 ± 1 °C and 80 ± 5% RH is suitable for maintaining the highest quality of Iranian grown cultivars of kiwifruit.     

Analytical procedure based on slurry sampling for determining selenium in organic vegetable samples by graphite furnace atomic absorption spectrometry

This paper presents a simple, fast and sensitive method to determine selenium in vegetable samples by graphite furnace atomic absorption spectrometry through the direct introduction of slurries of the samples into the spectrometer’s graphite tube. The limits of detection and quantification calculated for 20 readings of the blank of the standard slurries (5 mg mL⁻¹ of microcrystalline cellulose) were 0.33 and 1.10 μg L⁻¹. The proposed method was applied to determine selenium in samples of organically grown vegetables and its results proved compatible with those obtained from samples mineralized by acid digestion in a microwave oven.     

富硒大豆低聚肽的制备及其抗疲劳功能的研究

为探究富硒大豆低聚肽对抗运动性疲劳的影响,实验以富硒大豆为原料制备大豆蛋白,并以超滤萃取辅助中性蛋白酶和风味蛋白酶双酶酶解法制取富硒大豆低聚肽,并通过ICP-MS对硒含量进行鉴定,最后通过小鼠运动性疲劳实验综合评估富硒大豆低聚肽的抗疲劳活性.结果显示:富硒大豆低聚肽的总硒含量为(90.03±3.23)μg/kg,主要形...     

Selenium content in selected Portuguese foodstuffs

In the present study, selenium contents for some selected food items commonly consumed in Portugal are presented. Food items, i.e., meat, fish, eggs, dairy products, cereals, fruits and vegetables were sampled from two representative markets of two different regions of Portugal territory. The selenium concentrations in the various food items have been determined by using nuclear activation analysis (NAA) in both versions instrumental (INAA) and replicate sample (RSINAA) through the short-lived nuclide ^77mSe that features a half-life time of 17.5 s. The highest values were found for meat, fish and eggs (87.6–737 μg kg⁻¹); lower values were obtained for vegetables and fruits (1.7–24.9 μg kg⁻¹).     

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%).     

Selenium concentration in St. John’s wort (Hypericum perforatum L.) herb after foliar spraying of young plants under different UV-B radiation levels

St. John’s wort (Hypericum perforatum L.) was grown under different levels of UV-B radiation, with selenium (10 mg l⁻¹ Se applied by foliar spraying in the form of sodium selenate) or without foliar Se application. The different levels of UV-B radiation comprised an enhanced level simulating 17% ozone depletion, ambient level, and a reduced level of UV-B radiation. The concentration of Se in unsprayed plants was from 20 ng g⁻¹ to 120 ng g⁻¹. The concentration of Se in the organs of plants foliarly sprayed with Se ranged from 1000 ng g⁻¹ to 12,000 ng g⁻¹, the highest concentration being detected in plants grown under reduced levels of UV-B radiation. Foliar application of Se fertiliser is feasible and effective in St. John’s wort and results in Se-enriched nutritional supplements.     

Study of a microwave digestion method for total arsenic determination in marine mussels by electrothermal atomic absorption spectrometry: application to samples from the Ria de Arousa

Arsenic determination in mussel tissue was performed by electrothermal atomic absorption spectrometry (ETAAS) with Zeeman background correction and using iridium as a chemical modifier. Samples were digested by microwave heating using a mixture of nitric and sulphuric acids. This mixture makes possible the destruction of organoarsenic compounds, specifically arsenobetaine, prior to the graphite furnace determination. Optimum pyrolysis and atomization temperatures were 1,100 and 1,800 °C, respectively. The method was precise (with RSD% < 10), accurate (study of a certified reference material: 18.4 ± 1.4 μg As g⁻¹ vs. a certified content: 18.0 ± 1.1 μg As g⁻¹; recoveries between 90 and 104%) and sensitive (LOD 0.21 μg g⁻¹ on a dry weight basis). The method was applied to the determination of arsenic in aquaculture mussels collected in four sampling campaigns from the productive Ría de Arousa (estuary sited in Galicia, NW of Spain).     

Supercritical CO<Subscript>2</Subscript> extraction of <Emphasis Type="Italic">Alkanna</Emphasis> species and investigating functional characteristics of alkannin-enriched yoghurt during storage

Alkannin is a potent pharmaceutical substance with a wide spectrum of biological activities. In the scope of this study, supercritical CO₂ extraction and sonication with hexane were applied to various Alkanna species, which were then subjected to hydrolysis. Total alkannins were quantified by HPLC/DAD and incorporated into yoghurt. Viscosities, pH values and microbial analyses were reported at 7 days of intervals for 21 days of storage. A. tinctoria possessed the highest amounts of alkannins and total phenols (686.3 mg GAE/g extract). The results revealed no significant changes in pH values (4.1–4.0), viable counts of Streptococcus salivarius ssp. thermophilus (80–150 × 10⁶ cfu g⁻¹) and slightly lower viscosities of enriched yoghurts (8,250–6,750 cPs) compared with the control (4.15–4.0; 110–105 × 10⁶ cfu g⁻¹; 12,600–11,310 cPs) during storage. However, viable counts of Lactobacillus delbrueckii ssp. bulgaricus of enriched yoghurts (87 × 10³ cfu g⁻¹) were much better than the control (191 × 10³ cfu g⁻¹), indicating a significant decrease in post acidification and generation of bitter peptides. Among the species investigated, A. tinctoria is the most promising source, obtained at higher yields via supercritical fluid extraction technology as a green alternative to solvent extraction and thus can be utilized at industrial scale in order to develop yoghurt products with improved health benefits.     

Selenium Content of Raw and Cooked Marine Species Consumed in Portugal

The present study evaluates the effects of different cooking methods (grilling, frying and boiling) on selenium contents of six marine species commonly consumed in Portugal. Forty-two composite samples of sardine, horse mackerel, gilthead seabream, silver scabbardfish, hake and octopus were digested in a microwave system and analysed by electrothermal atomic absorption spectrometry. The described method is adequate for the analysis of selenium in marine species and meets the requirements of validation and quality control. Mean selenium contents in raw species ranged from 0.35 mg kg⁻¹ to 1.24 mg kg⁻¹. Cooked samples presented mean selenium contents from 0.38 mg kg⁻¹ to 1.85 mg kg⁻¹ in grilled fish, from 1.22 mg kg⁻¹ to 1.28 mg kg⁻¹ in fried fish and from 0.84 mg kg⁻¹ to 0.87 mg kg⁻¹ in boiled fish. No statistically significant differences were determined for selenium levels in raw and cooked samples and in different marine species. Estimated selenium intake agrees well with recommendations and is far below the Upper Tolerable Nutrient Level. This is the first study concerning the evaluation of the effects of cooking methods on selenium contents of marine species consumed in Portugal.     

Evaluation of chemical indicators for monitoring freshness of food and determination of volatile amines in fish by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A headspace solid-phase microextraction and gas chromatography–mass spectrometric (HS-SPME-GC-MS) method was developed to monitor methylamine (MA), dimethylamine (DMA) and trimethylamine (TMA) in fish. The alkylamines were extracted using carboxen/divinylbenzene/polydimethylsiloxane (CAR/DVB/ PDMS) fibres at 25 °C and exposure time of 5 min. Calibration curves showed good linearity in the concentration range of 0.5–10 μg ml⁻¹ and the corresponding coefficients of correlation were all above 0.99. Detection limits were estimated to be 0.10 μg ml⁻¹ for MA and DMA; and 0.15 μg ml⁻¹ for TMA. Intra-day and inter-day variation of spike samples at 2.0 μg g⁻¹ were 4.3–6.2% and 5.4–10%, respectively. Recovery of the method for TMA was found to be comparable to that of a standard titration method with the mean deviation of the two methods being 6.7%. The validated method was applied to study the decay profiles of the concerned alkylamines in two fish species that were stored at 0 and −20 °C; and the results obtained were compared with that obtained by using the classical parameter of total volatile basic nitrogen (TVBN).     

Selenium species in leaves of chicory,dandelion, lamb’s lettuce and parsley

Lamb’s lettuce, dandelion, parsley and four cultivars of chicory were cultivated aeroponically for 41 days with nutrient solution containing 7  mg Se/L in the form of Na₂SeO₄. Se compounds were determined by high performance liquid chromatography–ultraviolet treatment–hydride generation atomic fluorescence spectrometry (HPLC–UV–HG-AFS) in the green parts of the selected plants. Se species were extracted by water and by enzymatic hydrolysis with Protease XIV. Separation of Se^IV, Se^VI, SeMet, SeMeSeCys and SeCys₂ was made by a combination of anion and cation exchange chromatography in which the columns were connected on-line to a UV–HG-AFS detection system. Se accumulated efficiently in plant leaves up to 480 μg/g dry mass, mostly as Se^VI, i.e. the form of Se in the nutrient solution. Beside inorganic Se, selenomethionine (6–21%), selenomethylselenocysteine (0.5–4.4%) and selenocistine (<DL-0.8%) were determined in the extracts after enzymatic hydrolysis. Some unidentified peaks were also observed in the chromatograms.     

Effect of year‐to‐year variation and genotype on trypsin inhibitor level in common bean (Phaseolus vulgaris L) seeds

Changes in trypsin inhibitor (TI) level in common bean (Phaseolus vulgaris L) seeds were investigated in relation to protein content, year‐to‐year variation and genotype. Twenty‐one local populations from the Basilicata region (southern Italy) were tested over 3 years (1995–97). The populations were cultivated in the same environment in which they were traditionally cultivated and are currently grown. A wide variation of TI content, expressed as units of inhibitor per milligram of dry matter (TIU mg^?1 DM), was found within the populations of the collection (x? ± 2σ: 27.67 ± 2.72 in 1995, 25.31 ± 2.82 in 1996, and 23.39 ± 2.14 in 1997). It was found that 16 populations showed a decrease of TI levels from 1995 to 1997; one population showed the opposite trend, in two populations the TI level remained unchanged, and in another two it reached its maximum in the intermediate year. A t‐test showed that only the 1995–97 variation is highly significant (p < 0.02). The increase of TI expression might possibly be related to the drought stress suffered by plants during the vegetative growth stage: rainfall received in 1995 during the growing season was lower (171 mm) than in 1996 (477 mm) and 1997 (388 mm); however, other abiotic or biotic factors cannot be excluded. It is suggested that the extent of TI variation contains a genetic component. Copyright © 2003 Society of Chemical Industry     

Total Mercury, Inorganic Mercury and Methyl Mercury Determination in Red Wine

This study deals with the development of a method for total Hg, inorganic Hg and methylmercury (MeHg) determination in red wine by using flow injection-cold vapour generation–inductively coupled plasma mass spectrometry (FI-CVG-ICP-MS) and gas chromatography-ICP-MS (GC-ICP-MS). For Hg speciation analysis, a derivatization step was carried out using a 1% (m/v) sodium tetraphenylborate (NaBPh₄) solution, followed by extraction of Hg species and their quantification by GC-ICP-MS. The main parameters evaluated were the make-up gas flow rate, volume of the NaBPh₄ solution, time for derivatization reaction/analyte extraction and solvent used for Hg species extraction. Accuracy was evaluated by analyte recovery, whereas recoveries ranged from 99% to 104% for Hg(II) and MeHg. The limits of detection (LODs) for Hg(II) and MeHg were 0.77 and 0.80 μg L⁻¹, respectively. Wine from Argentina, Brazil, Chile and Uruguay were analysed. The wine samples were also acid digested for total Hg determination by FI-CVG-ICP-MS. The LOD of the method used for total Hg determination was 0.01 μg L⁻¹. The concentrations of Hg species in red wine measured by GC-ICP-MS were lower than the respective LODs. Only total Hg was detected in the analysed samples, where the highest concentration of Hg found was 0.55 ± 0.02 μg L⁻¹.     

Bioactive properties of Snake fruit (Salacca edulis Reinw) and Mangosteen (Garcinia mangostana) and their influence on plasma lipid profile and antioxidant activity in rats fed cholesterol

Two exotic fruits (Snake fruit and Mangosteen) were characterized by polyphenols, proteins and antioxidant potentials and by their influence on plasma lipids and antioxidant activity in rats fed cholesterol. The content of polyphenols (14.9±1.5 and 9.2±0.8 mg GAE g⁻¹) and antioxidant potential (46.7±4.7 and 72.9±7.4 μmol TE g⁻¹) in Snake fruit was significantly higher than in Mangosteen (P<0.05). Twenty male Wistar rats were divided into four dietary groups: Control, Chol, Chol/Snake and Chol/Mangosteen. After 4 weeks of the experiment diets supplemented with Snake fruit and to a lesser degree with Mangosteen significantly hindered the rise in plasma lipids and hindered a decrease of antioxidant activity. Changes were found in fibrinogen fraction, such as solubility and mobility by the number of protein bands detected in SDS-electrophoresis: Chol/Snake differed from Chol/Mangosteen. In conclusion, Snake fruit and Mangosteen contain high quantity of bioactive compounds, therefore positively affect plasma lipid profile and antioxidant activity in rats fed cholesterol-containing diets. Such positive influence is higher in rats fed diet with added Snake fruit.     

Evaluation of Protease-producing Ability of Fish Gut Isolate Bacillus cereus for Aqua Feed

Extracellular protease production by Bacillus cereus isolated from the intestine of fish Mugil cephalus has been investigated in shake-flask experiment using different preparations of tuna-processing waste such as raw fish meat, defatted fish meat, alkali hydrolysate, and acid hydrolysate as nitrogen source. Among the tuna preparations tested, defatted fish meat supported the maximum protease production (134.57 ± 0.47 U ml⁻¹), and 3% concentration of the same was found to be optimum for maximizing the protease production (178.50 ± 0.28 U ml⁻¹). Effect of carbon sources on protease production in the optimized concentration of defatted tuna fish meat revealed that galactose aided the higher protease production (259.83 ± 0.04 U ml^–1) than the other tested carbon sources and a concentration of 1.5% galactose registered as optimum to enhance the protease production (289.40 ± 0.16 U ml⁻¹). The halotolerancy of B. cereus for protease production indicated that 3% of sodium chloride was optimum to yield maximum protease (301.63 ± 0.20 U ml⁻¹). Among the surfactants tested, protease production was high in Triton X 100-added medium (298.63 ± 0.12 U ml⁻¹) when compared to other surfactants, and its optimum concentration recorded was 0.8% (320.57 ± 0.17 U ml⁻¹) for more protease production. Partial characterization of crude enzyme revealed that pH 7.0 (278.90 ± 0.08 U ml⁻¹) and 60°C temperature (332.37 ± 0.18 U ml⁻¹) were optimum for better protease activity by B. cereus.