Aqueous Two-Phase Micellar System for Nisin Extraction in the Presence of Electrolytes

Liquid–liquid extraction for aqueous two-phase micellar systems (ATPMS) is a promising technique that can either replace or be used as a complementary process to more typical chromatographic operations in order to reduce the costs of downstream processing of many biological products. This method offers attractive conditions when exploited in the extraction/purification of a target protein directly on the culture medium. Nisin, an extracellular antimicrobial peptide, is produced by Lactococcus lactis and is effective at controlling a wide range of Gram-positive bacteria, including multidrug-resistant pathogens. This study evaluates ATPMS composed by a nonionic surfactant, Triton X-114, in the presence or absence of electrolytes, to improve the extraction of nisin. The partitioning behavior of nisin showed that it can be directly extracted from the fermentation media. The partitioning coefficient (K _nis) of the commercial nisin in the presence of electrolytes showed K _nis values of 5.6 and 5.4 for MgSO₄ and (NH₄)₂SO₄, respectively. Similar behavior was observed for the produced nisin where K _nis values were 4.1 and 5.1 for MgSO₄ and (NH₄)₂SO₄. After partition, the commercial nisin activity in the micelle-rich phase, in the absence of electrolytes, was 3.3 logAU/mL, and in the presence of MgSO₄ and (NH₄)₂SO₄, the activity was 5.0 logAU/mL. The produced nisin activity with and without MgSO₄ was around 3.5 logAU/mL; however, in the presence of (NH₄)₂SO₄, nisin activity was 4.5 logAU/mL. The increase in nisin activity after partitioning with salts encourages further researches for the optimization of nisin extraction.     

Chemical and Nutritional Studies on Mesquite Beans (Prosopis juliflora)

Mesquite beans (Prosopis juliflora) consisted of pericarp, hulls and kernels. With the exception of kernels, composition of beans and their fractions were protein, 10–15%; fat, 2–3%; crude fiber, 20–30%; sucrose, 21%; reducing sugars, 2–6%. Kernels contained 38% protein, 3% fat and 9% crude fiber. Bean trypsin inhibitor content was 1.4 TIU/mg. Bean protein isoelectric point was pH 5. Mesquite protein concentrate and isolate were prepared, however, by increasing solubility at the extraction pH (pH 10) with NaCl and decreasing it at the isoelectric point with (NH₄)₂SO₄. First and second limiting amino acids for bean protein were threonine and isoleucine, respectively; the protein was high in total sulfur amino acids and tryptophan. Corrected PER was 1.4.     

Production of feed enzymes from citrus processing waste by solid‐state fermentation with Eupenicillium javanicum

Bingtang sweet orange processing waste was utilised to produce four feed enzymes (Endoglucanase, β‐glucosidase, pectinase and xylanase) by the solid‐state fermentation (SSF) with Eupenicillium javanicum. The factors related with SSF including moisture content, temperature, initial pH, time, carbon source (0.5 g), nitrogen sources (0.05 g), inorganic mineral salts (0.1 g) were investigated separately. The corresponding optimal condition was: moisture content 80% (w/w), temperature 30 °C, natural pH, time 96 h, wheat bran 0.5 g, (NH₄)₂SO₄ 0.05 g or NaNO₃ 0.05 g, CaCl₂ 0.1 g. The L₉(3⁴) orthogonal experiment results showed that the optimal condition for producing above multiple enzymes was: moisture content 80% (w/w), temperature 30 °C, wheat bran 1 g, (NH₄)₂SO₄ 0.05 g, NaNO₃ 0.05 g, CaCl₂ 0.1 g, fermentation time 96 h and natural pH. Under this condition, the average activity of Endoglucanase (CMCase), β‐glucosidase, pectinase and xylanase by E. javanicum could reach 46.80, 49.64, 51.87 and 106.42 U g^?1, respectively, which was significantly higher than those in single factor experiments. Our present results demonstrated that E. javanicum could also be an effective and useful fungus for multienzyme preparation especially for β‐glucosidase and xylanase from citrus processing wastes.     

Aqueous two-phase extraction of bromelain from pineapple peels (‘Phu Lae’ cultv.) and its biochemical properties

This study examines the extraction of bromelain from pineapple peels by using an aqueous two-phase system (ATPS). Bromelain from the crude extract predominantly partitioned to the polyethylene glycol rich phase. The best condition for bromelain partitioning was found to be 18% PEG6000-17% MgSO₄, which increased purity 3.44-fold with an activity recovery of 206%. Protein patterns and activity staining showed the molecular weight of bromelain to be about 29 kDa. The bromelain showed the highest relative activity at pH 8.0 and at 60°C. When increasing NaCl concentrations (up to 1.5%, w/v), its activity continuously decreased. The bromelain (0–0.3 units) was applied to hydrolyze collagen. The β, α₁, α₂ components of collagen extensively degraded into small peptides when treated with the bromelain. This study showed that the ATPS can be employed to isolate the bromelain from pineapple peels and the bromelain extract could be used as a meat tenderizing agent.     

Bioprocessing of pineapple waste for sustainable production of bioactive compounds using solid-state fermentation

The bioprocessing of pineapple wastes into value-added bioproducts is a sustainable solution. This study examined a novel alternative for producing bioactive compounds using Aspergillus niger GH1 for solid-state fermentation (SSF) of pineapple peel and core. The results revealed that the chemical composition of pineapple waste was suitable for use in SSF. During the first 32 h of the SSF, free phenols increased by 72.31% and were positively related to antioxidant activity as measured by DPPH (2,2-diphenyl-1-picrylhydrazyl). ß-Glucosidase and cellulase activities were increased by the SSF and were positively associated with free phenolic acids such as 5-caffeoylquinic acid. Scanning electron microscopy (SEM) confirmed mycelial invasion in pineapple waste. Analysis of free and bound phenols by high-performance liquid chromatography-mass spectrometry (HPLC-MS) showed more conjugated phenols in the unfermented than fermented waste. These results provided a broad overview of the chemical compounds with antioxidant capacity that are generated from the growth of A. niger.Industrial relevanceThe market for polyphenols and enzymes is expected to grow exponentially in the coming years. In response to this requirement and to the circular economy that seeks to take advantage of waste by generating new products, solid-state fermentation is applied, which produces in a short time polyphenols with antioxidant capacity and enzymes with possible application in the food industry from the fermentation of organic waste such as pineapple waste.     

Technical note: measurement of ferritin in bovine milk and its clinical significance

A quantitative ELISA was developed for bovine milk ferritin with an assay limit of 0.16 ng/mL of bovine spleen ferritin. Ferritin-binding activity was detected in bovine milk samples, and this binding activity was inhibited by increasing ionic strength with the addition of 0.5 M (NH₄)₂SO₄. Heat treatment (60°C, 20 min) of bovine milk in the presence of 0.5 M (NH₄)₂SO₄ resulted in a 15 to 58% increase in ferritin concentrations compared with untreated samples. Although the recovery of bovine spleen ferritin added to milk was still low (55 to 90%), even in the presence of increased ionic strength with 0.5 M (NH₄)₂SO₄, recovery was improved by heat treatment at 60°C for 20 min (92 to 95%). Milk ferritin concentrations in 30 milk samples from quarters of 25 cows with mastitis (mean ± SE: 134.2 ± 28.7 ng/mL) were significantly higher than those in 17 quarter milk samples from 17 noninfected lactating cows (7.2 ± 1.2 ng/mL), suggesting that bovine milk contains putative ferritin-binding proteins that inhibit immunoassay for milk ferritin and that bovine milk ferritin is an indicator of IMI.     

Production of Single Cell Protein from Cassava Starch in Air-lift Fermenter by Cephalosporium eichhorniae

Batch and continuous cultivations were used to investigate the single cell protein (SCP) production by Cephalosporium eichhorniae 152 in an air-lift reactor. The fungus was grown in an 8L air-lift fermenter. The culture medium consisted of cassava as a sole-carbon source, (NH₄)₂SO₄ as a nitrogen source and KH₂PO₄ as a buffering agent. The pH of the medium was adjusted to 3.8 and was controlled during fermentation by using an automatic pH-controller via 0.5 N NaOH. In batch fermentation, 1% of cassava gave the highest yield of 0.4. The optimum condition for continuous fermentation was 1% cassava in feed medium at a dilution rate of 0.4 h^-1 (396 ml/h where 0.35 was the final yield. The productivity of continuous culture was found to be higher than that of the batch culture (0.135 g/l.h versus 0.04 g/lh).     

A World Survey of Water Activity of Selected Saturated Salt Solutions used as Standards at 25°C

This paper presents and discusses the results of a world survey of water activity (a_w) of selected saturated salt solutions used as standards by different researchers engaged in a_w determinations for food-related applications. Salt slurries cover the range of a_w about 0.57-0.97 at 25°C and are NaBr, NaCl (NH₄)₂SO, KCl, BaC1₂. 2H₂O, KNO₃ and K₂SO₄. The results indicate that there is a good agreement on the exact value to be assigned to NaBr, NaCl, KC1 and BaC1₂.2H₂O, and to a lesser extent, to K₂SO₄. However, a significant discrepancy exists between researchers on the a_w value assigned to (NH₄)₂SO₄ and KNO₃.     

Effect of amino acid,pH and mineral salts on glass transition and flow behaviour of soy protein concentrate

The objective of this study was to assess the effect of proline, mineral salts (NaCl and Na₂SO₄) and pH combined with moisture content on the glass transition temperature (T_g) and flow‐starting temperature (T_f) of soy protein concentrate (SPC). Initial screening of the variables based on fractional factorial design showed insignificant effect of NaCl on T_g and T_f. The design was extended to a face‐centred central composite design (CCD) excluding NaCl and data evaluated by use of response surface methodology. The established model for T_g (R² = 0.824) shows significant negative first‐order effects of moisture, proline and Na₂SO₄, and a positive interaction effect of moisture and Na₂SO₄. The T_f model (R² = 0.937) shows significant negative first‐order effects of moisture and proline, a positive first‐order and negative square effect of pH, and a negative interaction effect of moisture and proline. The main effect on T_g and T_f was 2.2 and 1.3 times higher, respectively, for moisture compared to proline. The study confirms that proline (or other free amino acids) can replace moisture as protein plasticiser in the extrusion process. Minor effects can also be obtained by reduction in pH.     

Nitrate content in lettuce (Lactuca sativa L) heads in relation to plant spacing,nitrogen form and irrigation level

A field study was conducted at two locations (Jordan valley and Al‐Jubeiha) with different rainfall levels, altitudes and temperature ranges. The study was established to evaluate the optimum planting density, nitrogen (N) form and irrigation level to attain the best quality of lettuce crop in terms of minimum nitrate (NO₃) content and to minimise the impact on the environment. Seeds of ‘Amar’ lettuce were sown 1 month before transplanting. Three forms of N fertiliser (Ca(NO₃)₂, (NH₄)₂SO₄ and CO(NH₂)₂) were applied at three times at a total rate of 100 kg N ha^?1. Three in‐row spacings (15, 20 and 25 cm) were assigned. Two irrigation levels were applied: level 1 had twice the amount of irrigation water as level 2, which was achieved by doubling the number of irrigation lines. The results indicated that N form significantly increased both N and NO₃ contents. Ca(NO₃)₂ was the most effective in increasing the N and NO₃ contents in lettuce leaf tissues, followed by CO(NH₂)₂ and then (NH₄)₂SO₄. The outer leaves (green colour) had about five times the NO₃ content of the inner leaves (pale yellow colour). However, the effect of N form on production, total N absorption and N recovery was significant in the following order: (NH₄)₂SO₄ > Ca(NO₃)₂ > CO(NH₂)₂. Increasing the plant spacing resulted in a significant increase in N and NO₃ contents in the lettuce leaves. Copyright © 2004 Society of Chemical Industry     

Thermal Stabilities of Peroxidases from Fresh Pinto Beans

Heat stabilities of crude and partially purified soluble (SPOX), ionically bound (IPOX) and total peroxidase (TPOX) from fresh pinto beans were investigated at 55–90°C. Heat inactivation of peroxidase (POX) followed first-order reaction kinetics. Each inactivation curve consisted of two linear parts: initial rapid inactivation (heat-labile) followed by slower inactivation (heat-stable). IPOX showed activation during heat treatment with a highly heat-stable isoenzyme (D₉₀=40 min) which was more heat-stable than SPOX. Activation energies for heat-stable parts of crude IPOX and SPOX were, respectively, 12.1 and 36.4 kcalmol-1 with z values 45.4 and 14.1C°. Heat stable SPOX isoenzymes (D₇₀=22.6) were obtained by 65–95% (NH₄)₂SO₄ precipitation from crude SPOX. Two POX fractions (F1 and F2) were separated from TPOX by ion-exchange chromatography.     

Isolation and partial purification of a proteolytic enzyme preparation from Clostridium perfringens

Although two strains of Clostridium perfringens (ATCC 12915 and 13124) exhibited excellent growth on amino acid and peptone media, only one (ATCC 13124) produced measurable proteolytic enzyme activity. Thus, subsequent purification steps concentrated on isolation of a proteolytic enzyme preparation produced by this strain. Purification and concentration were carried out by precipitating the crude enzyme fraction from the culture filtrate with ZnCl₂, extracting with saturated disodium phosphate and reprecipitating by 60% saturation with (NH₄)₂SO₄. The precipitate was then redissolved in borate buffered saline solution and further purified by successively passing it through a Bio-Gel P-100 column, a DEAE-cellulose column and a Bio-Gel P-200 column. The final step resulted in a 159-fold purification with 12% recovery and a final specific activity of 79 azocoll units/milligramme protein. Although each successive purification step eliminated some of the impurities, the final fraction still showed considerable heterogeneity upon disc-gel electrophoresis.     

Distribution of Proteolytic Activity in the Different Protein Fractions of Tropical Shrimp Head Waste

Ammonium sulfate fractionated protein fractions at levels of 25%, 30%, 35%, 40%, 42.5% and 45% ammonium sulfate were recovered from the head waste of tropical marine tiger (MTS), culture tiger (CTS), white (WS) and brown shrimp (BS) and then characterized for protease activity. Distribution of buffer (pH 7.1) extracted protein among ammonium sulfate fractions showed that total protein in 25% fraction of MTS, CTS, WS and BS was 48, 54, 34 and 24 times more than that in the respective 42.5% fraction of the head waste of these shrimps. The highest proteolytic activity was observed in 42.5% (NH₄)₂SO₄ protein fraction of the head waste of MTS, CTS, WS and BS, the values were 19, 1.7, 11.6 and 2 times, respectively, more than that of the corresponding 25% (NH₄)₂SO₄ protein fractions. Highest caseinolytic activity (pH 8.5) and gelatinolytic activity (pH 7.1) was observed in the 42.5% fraction of the head waste of CTS and WS, respectively; but the highest albuminolytic activity (pH 8) was observed in the same fraction of the head waste of both MTS and BS. The optimum pH for highest gelatinolytic and albuminolytic activity of the 42.5% (NH₄)₂SO₄ protein fraction of the head waste of MTS and BS was 4; the same for highest gelatinolytic activity of the same protein fraction of the head waste of WS, and these fractions included acid proteases such as pepsin, the optimum pH for the above activity of the same fraction of CTS was 6–8.5, and the fraction was an alkaline protease such as chymotrypsin. The SDS-PAGE pattern of 42.5% (NH₄)₂SO₄ protein fraction of the head waste of BS, CTS, WS and MTS was almost similar with a dark band close to the marker band 20kDa. Proteases make up to 48% of industrial enzymes and are mostly used in detergents, leather production and food industry.     

Juice extraction from mango pulp using an enzymatic complex of Trichoderma sp. produced by solid-state fermentation

In this study, 2 Trichoderma strains (T-I and T-II) were evaluated for the production, by submerged (SmF) and solid state fermentation (SSF) of an enzymatic complex with the capacity to degrade the cell components of mango peels, using these as support (in SSF) and source of nutrients. Highest enzymatic activity (7,552.5 units of endo-glucanase/L) was found in T-II by SSF. Efficiency of this crude enzymatic extract in the extraction of mango juice was evaluated, improving the yield up to 79%, representing an alternative to give an added value of mango peels improving the yields of production of mango juice.     

Effect of Germination Environment on Nutrient Composition of Alfalfa Sprouts

Alfalfa seeds were germinated for 120 hr utilizing solutions of 0.05M (NH₄SO₄, 0.05M urea, 0.05M KNO₃, 0.1M KNO₃ or distilled water for rinsing. One of the water rinsed samples was flushed with CO₂ after each rinse. Proximate analysis, NPN, amino acids and reduced ascorbic acid contents were measured to determine the effect of germination environment on the nutrient composition. The sprouts had significantly increased crude protein values but reduced fat and carbohydrate concentrations as compared to the seeds. True protein content increased significantly in the sprouts rinsed with 0.05M (NH₄SO₄ or 0.1M KNO₃. The essential amino acid patterns of these were not significantly affected. Although the treatments used did not appear to affect adversely the composition of the sprouts, visually the sprouts did suffer somewhat.     

Partition Behaviors of Different Polar Anthocyanins in Aqueous Two-Phase Systems and Extraction of Anthocyanins from <Emphasis Type="Italic">Nitraria tangutorun</Emphasis> Bobr. and <Emphasis Type="Italic">Lycium ruthenicum</Emphasis> Murr.

This study aimed to investigate the partition behaviors of various polar anthocyanins in NaH₂PO₄/(NH₄)₂SO₄-ethanol aqueous two-phase systems (ATPS) and to extract anthocyanins from Nitraria tangutorun Bobr. and Lycium ruthenicum Murr. Anthocyanins in Hibiscus sabdariffa L., Morus atropurpurea Roxb., N. tangutorun, and L. ruthenicum were profiled using HPLC-ESI-MS/MS and HPLC-DAD, and the partition behaviors of total anthocyanins and main anthocyanins were studied. The partition coefficient of anthocyanins increased with increased hydrophobicity, and low-polarity anthocyanins exhibited a higher preference for the top phase in NaH₂PO₄/(NH₄)₂SO₄-ethanol ATPS. Additionally, the NaH₂PO₄-ethanol ATPS gave higher selectivity and total anthocyanin yield than the (NH₄)₂SO₄-ethanol system. Extraction at 65 °C for 45 min and at 45.5 °C for 45 min using 28% NaH₂PO₄ and 26% ethanol (w/w) led to the recovery of 98.91 ± 0.03% of N. tangutorun anthocyanins (3.62 ± 0.05 mg/g) and 99.84 ± 0.01% of L. ruthenicum anthocyanins (13.16 ± 0.29 mg/g) from raw material; more than 70% of total sugars were removed in a single step. NaH₂PO₄-ethanol aqueous two-phase extraction is a promising method for extracting anthocyanins from N. tangutorun and L. ruthenicum.     

Formulation of Culture Medium with Agroindustrial Waste for β-Galactosidase Production from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> ATCC 16045

Seven strains of the genus Kluyveromyces were screened for β-galactosidase activity and Kluyveromyces marxianus ATCC 16045 was selected as the best enzyme producer for culture medium optimization. The production of β-galactosidase by submerged cultivation was evaluated using a factorial design and response surface methodology. The culture medium containing whey and parboiled rice effluent was formulated to maximize the production of β-galactosidase. The effects of the initial pH and the concentrations of whey lactose, peptone, (NH₄)₂SO₄, yeast extract, and parboiled rice effluent on enzyme production were studied using a 2 _IV ^6-2 fractional design. A CCRD (24 trials plus axial and central points) was used for the four variables selected from the fractional design (lactose, peptone, (NH₄)₂SO₄ and yeast extract), with β-galactosidase activity as the response. The optimum conditions established for production were a whey (lactose) concentration of 120 g/L, a yeast extract concentration of 5 g/L, a peptone concentration of 15 g/L, a (NH₄)₂SO₄ concentration of 15 g/L, a parboiled rice effluent concentration of 30 g/L, and a pH value of 4.0. Under these conditions, the highest enzymatic activity of 10.4 U/mL was measured, being 9.5–9.7 as the values predicted by the proposed model, showing an enzymatic activity increase of 30% using alternative sources of lactose and nitrogen for β-galactosidase production.     

Cytotoxicity of the white ginseng extract and red ginseng extract treated with partially purified β-glucosidase from Aspergillus usamii KCTC 6954

Red ginseng extract (RGE) and white ginseng extract (WGE) were treated with partially purified β-glucosidase to increase a production of minor ginsenosides. The enzyme produced from Aspergillus usamii KCTC 6954 was precipitated with (NH₄)₂SO₄. Ginseng extracts were treated with a crude extract possessing β-glucosidase activity (1,089.2 μM/mL·min) at 60°C for 72 h. The results of HPLC showed that enzyme-treated RGE and enzymetreated WGE have increased amounts of minor ginsenosides compared to each controls implying that the ginsenoside Rb₁ in WGE and RGE is converted enzymatically to Rd, F₂, Rg₃, and compound K. In cytotoxicity study, 2.5 mg/mL of RGE, 1.25 mg/mL of ERGE, and 5 mg/mL of WGE and EWGE were effective against the HepG2, AGS, and DLD-1, but HeLa and SK-MES-1 were not affected at any concentration. The results suggested that cytotoxicity of ginseng extracts treated with β-glucosidase were greater than that of each control against cancer cells.     

Development of a quantitative method for headspace analysis of methylsulfanyl-volatiles from kiwifruit tissue

Minor fruit volatiles are likely to be missed using sampling techniques optimized for the extraction of major compounds. This can be a disadvantage if these minor compounds contribute to characteristic fruit flavors. In this comparative study, headspace solid-phase microextraction (HS-SPME) and dynamic headspace sampling (DHS) parameters were systematically optimized to ensure highest extraction yields of methylsulfanyl-volatiles from kiwifruit tissue samples. A significant “salting-out” effect from the fruit matrix was observed using both sampling techniques after (NH₄)₂SO₄ saturation. HS-SPME at optimized conditions (polydimethylsiloxane-divinylbenzene-coated fiber, (NH₄)₂SO₄ saturation, 5 min equilibration and 20 min sampling at 40 °C) was faster and more convenient to use than DHS for qualitative purposes. Despite this, the qualitative and quantitative methylsulfanyl-volatile profile was improved using optimized DHS ((NH₄)₂SO₄ saturation; sampling time 20 h; flow rate 30 mL min^− 1) compared with HS-SPME, making this the more sensitive and preferred method for quantitative studies. The optimization strategies for increasing headspace extraction yields of trace compounds presented in this study can easily be applied to tissue samples from other fruit.     

Purification and characteristics of an endogenous alpha-amylase and trypsin inhibitor from wheat seeds

The bifunctional endogenous alpha-amylase and trypsin inhibitor was purified 151-fold from a crude extract of wheat grain by (NH₄)₂SO₄ fractionation, ion exchange chromatography and gel filtration. The inhibitor coded A/T-WI was an albumin protein, relatively heat stable with a molar mass of 23233 g/mol and optimum activity towards native alpha-amylase and trypsin at pH 5.1 and 7.1 respectively. Amino acid analysis indicated the presence of about 15 half-cystine residues per mole. The protein was homogeneous as assessed by polyacrylamide gel electrophoresis. In the presence of detergent the inhibitor was dissociated into two subunits. A ?double head”? in nature the inhibitor was almost inactive in the presence of detergent with reducing agent. At neutral pH over 80% of inhibitor was adhered to wheat starch granules.