Effect of Malting Conditions on Pearl Millet Malt Quality

The effect of malting conditions on pearl millet malt quality in two varieties, SDMV 89004 and SDMV 91018, was investigated. Grain was steeped and germinated at four temperatures, 20°, 25°, 30° and 35°C, over 5 days. Generally, malt quality parameters (percentage of roots and shoots, diastatic power (DP), α‐ and β‐amylase activity, free α‐amino nitrogen (FAN), and malting loss) were significantly affected (P < 0.001) by germination temperature and time, as well as by variety. Malt FAN and malting loss were not affected by variety. A germination temperature of 25–30°C and germination time of 3–5 days were optimal. These conditions resulted in high DP, α‐ and β‐amylase activity, good FAN and moderate malting loss. These malting conditions and the subsequent malt quality of pearl millet are similar to those reported for sorghum. Pearl millet malt can therefore be used for the production of sorghum type beers.     

Changes in enzyme activities and aflatoxin elaboration in sorghum genotypes following Aspergillus parasiticus infestation

Sorghum is a relatively poor substrate for aflatoxin production compared with high‐risk agricultural commodities like maize and groundnut, even though it is susceptible to fungal attack. Fungal infestation of sorghum results in a varied biochemical composition of the deteriorated grain. In this study, six sorghum genotypes (red—AON 486, IS 620; yellow—LPJ, IS 17 779; white—SPV 86, SPV 462) were inoculated with a toxigenic strain of Aspergillus parasiticus (NRRL 2999) in order to evaluate the changes in the activities of various hydrolytic enzymes (α‐ and β‐amylases, protease and lipase) in comparison with those in uninfected grains. Enzyme activities were measured at different times after fungal infestation, and the enzymatic activities were correlated with the aflatoxin production. Alpha‐amylase activity was observed to be greater than β‐amylase activity in all six genotypes under both healthy and infected conditions. The increase in α‐amylase activity during the period of infection was higher in white genotypes than in red sorghum genotypes. Alpha‐amylase activity in all the genotypes increased up to day 6 after fungal infection, but was significantly lower in infected grains than in healthy grains. The variability in the basal enzyme activities among the six sorghum genotypes was quite high compared with the amount of induction of each specific enzyme due to infection and germination. Higher protease activity was observed in the infected grains than in healthy grains. The enzyme activities in high tannin red genotypes were less than those in yellow and white genotypes. The α‐ and β‐amylase activities were positively correlated (r = 0.406 and 0.436; P < 0.05) to aflatoxin production. Inherent lipase activity was highest (on day 0) in AON 486, SPV 462 and SPV 86, as compared with the activity in infected grains. The total aflatoxins produced (quantified by TLC‐fluorodensitometry) were lower in red genotypes than in yellow and white genotypes, suggesting that red genotypes were least susceptible to aflatoxin elaboration among the various genotypes tested. All four aflatoxins, (B₁, B₂, G₁ and G₂) were present in five genotypes (IS 620, LPJ, IS 17 779, SPV 86 and SPV 462) at all the stages of infection, but, aflatoxin could not be detected in the red genotype AON 486 on day 3 after infection. White genotypes SPV 86 and SPV 462) showed maximal aflatoxin (total) production on day 6 after infection. © 2000 Society of Chemical Industry     

Current Developments in Malting and Brewing Trials with Sorghum in Nigeria: A Review

Initially, large‐scale lager beer brewing with sorghum malts proved highly intractable due to a number of biochemical problems including: high malting losses estimated at 10–30% as against 8–10% for barley; high gelatinisation temperatures which limited starch solubilisation/ hydrolysis by the amylolytic enzymes during mashing; low extract yield/low diastatic power (DP) due to inadequate hydrolytic enzyme activities especially β‐amylase; low free α‐amino nitrogen (FAN) due to inadequate proteolysis limiting yeast growth during fermentation; high wort viscosities/beer filtration problems due to low endo‐β‐1,3; 1–4‐glucanase activities on the endosperm cell walls causing the release of some β‐glucans. Strident research efforts using improved Nigerian sorghum malt varieties (SK5912, KSV8 and ICSV400) have reported some encouraging results. The knowledge of the biochemical integrity of the endo‐β‐glucanases of the sorghum malt is helping to elucidate their mode of activity in the depolymerisation of the β‐glucans. This is bound to ensure process efficiency in sorghum beer brewing, reduce beer production costs and ultimately, produce a Pilsner‐type of lager beer with 100% sorghum malt.     

Effects of germination time on phenolics,antioxidant capacity,in vitro phenolic bioaccessibility and starch digestibility in sorghum

The effects of germination time on phenolic, flavonoid, anthocyanin, antioxidant capacities, inhibition capacity of α-amylase, bioaccessibility of phenolic and digestibility of starch in sorghum were evaluated in this study. The levels of total phenolic, flavonoid and anthocyanin in germinated sorghum for 48 h increased by 39.74%, 37.28% and 52.21%, respectively. Germination also increased the composition of phenolic, flavonoid and anthocyanin in sorghum, and their antioxidant capacity and inhibitory rates of α-amylase. Additionally, in vitro digestion results showed that phenolic bioaccessibility increased by 10.18%, and digestibility of starch and expected glycaemic index (eGI) decreased by 13.87% and 5.26 in germinated sorghum for 48 h compared with the ungerminated sorghum. These results indicate that germination might be a green method to improve the nutritional quality of sorghum and promote the development of germinated whole-grain food.     

INHIBITORY POTENTIAL OF WINE AND TEA AGAINST α-AMYLASE AND α-GLUCOSIDASE FOR MANAGEMENT OF HYPERGLYCEMIA LINKED TO TYPE 2 DIABETES

ABSTRACT

Natural α‐amylase and α‐glucosidase inhibitors from food‐grade plants offer an attractive strategy to manage postprandial hyperglycemia for type 2 diabetes management via control of starch breakdown and intestinal glucose absorption. In this study, four random sources of red and white wines as well as four types of teas were investigated for α‐amylase and α‐glucosidase inhibitory potential. Water extracts of black tea had the highest α‐glucosidase inhibitory activity, followed by white tea and oolong tea. All the randomly selected red wines had significant α‐glucosidase inhibitory activity compared to white wine. The α‐glucosidase inhibitory activity of the tea and wines correlated to the phenolic content, antioxidant activity and phenolic profile of the extracts. Further, these extracts had less or no α‐amylase inhibitory activity, indicating potential to overcome the side effects of undigested starch. This research has relevance for managing hyperglycemia and related oxidation‐linked dysfunction and concurrently reducing problems of undigested starch.

PRACTICAL APPLICATIONS

In this study anti‐diabetic‐relevant potential of wines and teas were confirmed in four types of red and white wines as well as four types of commonly available teas using in vitro enzyme assays for alpha‐glucosidase and alpha‐amylase inhibitory activities. In vitro inhibitory activities of these enzymes provide a strong biochemical rationale for further in vivo studies and dietary management strategy for type 2 diabetes through the control of glucose absorption. Further this phenolic antioxidant‐enriched dietary strategy using specific beverage combinations can generate a whole food profile that has the potential to reduce hyperglycemia‐induced pathogenesis and also associated complications linked to cellular oxidation stress.     

Quality Assessment of Different Sorghum Varieties for Their Brewing Potential

Four sorghum varieties (SK 5912, KSV 4, KSV 8, ICSV 400) were malted and extracted under similar conditions to assess their quality for brewing. The results showed that, in general, the sorghum varieties had high malting loss which was attributed to the high germination temperature used. The sorghum varieties also developed low levels of amylolytic activity (α‐amylase and β‐amylase), and with similar ratios. When the sorghum malts were mashed at different temperatures with the aid of commercial enzyme preparations, it was observed that mashing temperatures were more important in sugar release than additions of commercial enzymes. This was because at the lower mashing temperature, sorghum starch was not adequately gelatinised. However, when commercial enzyme preparations were added, low levels of enzymes were very effective in reducing wort viscosity and producing free amino nitrogen (FAN). Although, both commercial enzyme preparation and mashing temperature influenced sugar production, the malts produced glucose and maltose at similar ratios. Therefore good quality malts can be produced from sorghum, however mashing will employ commercial enzymes and mashing regimes are not yet optimised.     

Influence of grain germination on functional properties of sorghum flour

A Sudanese sorghum cultivar (Fetarita) was germinated for five days and protease and amylase activities were measured every 24 h. Results showed that flour obtained from the 3rd germinated sorghum grain had high protease and amylase activities. The functional properties of flours derived from the germinated sorghum seeds were studied and ungerminated seeds were used as a control. Germinated samples had a higher protein solubility compared with the control, and the highest solubility occurred at pH 6. Germination also increased the protein solubility index of sorghum flour. Germinated sorghum flour had a least gelation concentration of 8% compared with 18% for the control. The bulk densities of germinated flours were lower compared to the ungerminated one. Water and oil capacities were increased by germination from 131.34% and 90.56% to 141.64% and 108%, respectively after three days of germination. The emulsifying activities and stabilities of the germinated samples increased significantly. In addition, germination improved the foamability of sorghum flour from unfoam flour to a flour with foam after three days of germination; and the foaming capacity and stability increased significantly with increasing germination time. Thus, the study indicated that germination improved the functional properties of sorghum and it would be possible to design new foods using germinated sorghum.     

The effect of germination and kilning on the cyanogenic potential,amylase and alcohol levels of sorghum malts used for burukutu production

Grain sorghum of the red and white varieties was malted by steeping in water for 18 h, germinated over 5 days and kilned at 50 °C. The malts were analysed for amylase activities and cyanogenic potential and used to produce burukutu, an alcoholic beverage. The alcohol content of the burukutu was recovered by distillation and determined by the refractive index method. α‐Amylase activity peaked on malting day 3 and was higher in the white malts. β‐Amylase activity peaked on day 3 in the red malts and on day 4 in the white malts, but was higher in the red malts. Dhurrinase activity was highest on malting day 4, with a higher activity in the red malts. Kilning at 50 °C reduced the activities of these enzymes. The dhurrin content increased during germination and was consistently higher in the white malts, in which there was evidence of dhurrin mobilisation. In the red malts the dhurrin content increased during germination but decreased progressively after kilning; evidence of dhurrin mobilisation was apparent as from malting day 4. Burukutu produced from the red malts gave higher alcohol contents than that from the white malts. Maximum alcohol yields were obtained on malting day 3 in the red malts and on day 5 in the white malts. © 2000 Society of Chemical Industry     

Time Course for the Development of Enzymes in Barley1

Barley (Hordeum distichon var. Harrington) was steeped, germinated and extracted to observe the order of enzyme development. Different parts of the barley kernel were extracted to observe the order of enzyme development during the malting process. Five enzymes were investigated: carboxypeptidase (EC 3.4.16.1), endo‐β1–3, 1–4‐glucanase (EC 3.2.1.73), endo‐B1‐4‐xylanase (EC 3.2.1.136), arabinofuranosidase (EC 3.2.1.55), and α‐amylase (EC 3.2.1.1). Early development of carboxypepti‐dase, followed by later development of β‐glucanase, then α‐amy‐lase, confirmed earlier reports concerning the sequence of synthesis for these activities. However, xylanase developed during the steeping of barley and early in germination, whereas other authors found this enzyme to develop much later in the malting process. Enzyme activities developed to higher levels in the proximal end of kernels for all enzymes except xylanase, which was evenly distributed throughout the kernel. Enzyme development was tested in sterile barley annuli [embryo‐less cross sections taken through the grain, and thus comprising rings of tissue with husk outermost and starchy endosperm innermost] under four effector conditions. Water controls mirrored the development pattern observed in whole barley kernels. Gibberellic acid (GA₃) promoted higher total enzyme activity and development of all enzymes at the same time. Abscisic acid (ABA) promoted earlier development of late developing enzymes (xylanase, arabinofuranosidase and α‐amylase) and significantly higher levels of xylanase than when treatment was with water alone. Mixtures of GA and ABA showed a non‐exclusive, combined response of higher activity levels and a shifting of the initiation of enzyme development. Treatment with a combination of GA and calcium chloride triggered signifycantly higher carboxy‐peptidase activity and significantly lower xylanase activity as compared to treatment with GA or with GA/ABA mixtures.     

Amylase activities in insect (Aelia and Eurygaster)‐damaged wheat

The extent of modification of wheat amylase activities caused by Aelia and Eurygaster attack on wheat grain was determined in different Spanish cultivars subjected to varying degrees of attack. High variation in diastatic activity and α‐amylase and β‐amylase activities was found between cultivars, but no relationship could be established between these activities and bug damage within cultivars. Scanning electron micrographs of the cross‐section of damaged kemels showed an empty cavity under the bite point. The surrounding cell walls and protein matrix were absent, but the starch granules were intact. Since wheat damaged by Aelia and Eurygaster does not have altered amylase activities, it appears that amylolytic enzymes are not involved in the alteration of bug‐damaged wheat. © 2002 Society of Chemical Industry     

The Influences of Steeping Duration and Temperature on the α‐ and β‐Amylase Activities of Six Thai Rice Malt Cultivars (Oryza sativa L. Indica)

A preliminary study of malting conditions for six Thai rice cultivars was conducted. Three non‐glutinous rice cultivars (KDML105, PT60, and WR) and three glutinous rice cultivars (SPT, RD6, and KND) were selected. The steeping durations (24, 48, and 72 h) and temperatures (20, 25 and 30°C) were investigated for their effect on α‐ and β‐amylase, the key enzymes for malt quality evaluation. During steeping, the production of both enzymes was lower than at the germination process. The longer the steeping duration, the lower the maximum β‐amylase activity obtained. The contradictory effect was observed for α‐amylase activity, near the end of the germination time. Additionally, temperature influenced the water absorption content as well as the amylolytic enzyme activity. Particularly at 30°C, the maximum β‐amylase activity (6.7 unit/mg protein) was found in KND malt steeped for 24 h, and maximum α‐amylase activity (20 unit/mg protein) was found in PT60 malt steeped for 72 h. The amount of enzyme production depended on the variety rather than the amylose content in the rice. The optimal condition for malting rice regarding β‐amylase activity and α‐amylase activity was analyzed at 30°C, with steeping for 24 h and germination for 4–5 days.     

Effect of Germination Moisture and Time on Pearl Millet Malt Quality — With Respect to Its Opaque and Lager Beer Brewing Potential

The effect of germination moisture and time on pearl millet malt quality was investigated. Two pearl millet varieties SDMV 89004 and 91018 were germinated at 25°C under three different watering regimes for 5 days. As with sorghum malting, diastatic power, beta‐amylase activity, free α‐amino nitrogen (FAN), hot water extract and malting loss all increased with level of watering. However, pearl millet malt had a much higher level of beta‐amylase and higher FAN than sorghum malt and a similar level of extract. Malting losses were similar or lower than with sorghum. Thus, it appears that pearl millet malt has perhaps even better potential than sorghum malt in lager beer brewing, at least as a barley malt extender, especially in areas where these grains are cultivated and barley cannot be economically cultivated. Also, its increased use in commercial opaque beer brewing, where sorghum malt is currently used, could be beneficial.     

The Impact of Kilning on Enzymatic Activity of Buckwheat Malt

This study investigated the impact of kilning on α‐amylase, β‐amylase (total and soluble), β‐glucanase and protease activities in buckwheat malt. Common buckwheat (Fagopyrum esculentum) was steeped at 10°C for 12 h, germinated at 15°C for 4 days and kilned at 40°C for 48 h. Moisture content and enzymatic activities were determined throughout the kilning period. Results showed moisture content was reduced from 44% to 5% after 48 h of kilning at 40°C. β‐Amylase was found to exist in a soluble and latent form in buckwheat. Maximum activity of (a) α‐amylase, (b) total β‐amylase, (c) soluble β‐amylase, (d) β‐glucanase and (e) protease activity occurred after (a) 8, (b) 7, (c) 30, (d) 0, and (e) 8 h of kilning, respectively. The final malt exhibited very little β‐glucanase and cellulase activity. Proteolytic activity was low in buckwheat malt when compared to the barley malt control. All enzymatic activities were found to decrease during the kilning stage. Results indicated that after prolonged kilning at 40°C, inactivation of hydrolytic enzymes occurred; two‐stage kilning for shorter periods is recommended. Although, amylolytic activity was low in malted buckwheat, buckwheat malt shows potential as an ingredient for the brewing and cereal industry.     

燕麦发芽过程中淀粉及其相关酶活性的动态变化

研究了裸燕麦发芽过程中淀粉及其相关酶活性的动态变化。结果表明,发芽过程中,燕麦还原糖和可溶性糖含量及α-淀粉酶、β-淀粉酶和总淀粉酶活力明显地先增加后降低;直链淀粉、支链淀粉和总淀粉的含量均随着发芽的进行呈下降趋势,发芽72 h分别降低了25.86%、11.08%和17.31%。相关性分析表明,燕麦发芽期间还原糖、可溶性糖含量分别与α-淀粉酶、β-淀粉酶及总淀粉酶活力呈显著正相关,而直链淀粉、支链淀粉及总淀粉含量均与淀粉酶活力呈显著负相关。     

Effect of preincubation of sorghum flour with enzymes on the digestibility of sorghum gruel

A low-tannin sorghum cultivar M-35-1 was used in this study. Sorghum was germinated for 6 days and protease and amylase activities were measured every 24 h. Results showed that the 5th day germinated sorghum had a higher protease activity and a lower amylase activity. Sorghum flour was incubated for 30 min with the extract from germinated sorghum or with 0.01, 0.05 or 0.1 mg ml⁻¹ papain or trypsin prior to cooking in water. Results showed increase in in vitro protein digestibility (IVPD) with the 5th day germination extract. Pretreatment of sorghum flour with small amounts of papain or trypsin (0.01 mg ml⁻¹) improved the IVPD without affecting the paste viscosity, whereas the germinated sorghum extract led to very low paste viscosity. ©     

Extraction,optimisation and characterisation of phenolics from Thymus vulgaris L.: phenolic content and profiles in relation to antioxidant,antidiabetic and antihypertensive properties

The objectives of this study were to examine varying extraction conditions of Thymus vulgaris L. as related to phenolic content and profiles of the extracts and their antioxidant, antihypertensive and antidiabetic properties. Phenolics were extracted under various conditions pertaining to free and bound phenolics, solvent type and combination of extraction time and temperature, and these extracts were evaluated in terms of their antioxidant activities and inhibitory activities of angiotensin‐converting enzyme (ACE), α‐glucosidase and α‐amylase. The acetone–water solvent mixture (1:1; v/v) produced the extract with the greatest phenolic content, antioxidant activity and inhibitory activities of ACE and α‐glucosidase. The optimal extraction temperature for maximum phenolic content and antioxidant activity associated with methanol extraction was 60 °C, whereas a lower temperature at 40 °C was required to maximise inhibitory activities for ACE, α‐glucosidase and α‐amylase. An inverse relationship was seen between antioxidant and glucosidase inhibitory activities vs. the ACE and α‐amylase inhibitory activities, which suggests the need for extractions to be directed to specific bioactivities of thyme extracts. Generally, the results indicate major differences in phenolic profiles among the tested extraction conditions with thymol as the predominant phenolic seen in most extractions, while gallic acid, rosmarinic acid or diosmin also predominated in other extracts. Extracts with the same predominant phenolic compound and similar phenolic content showed major disparities in their ACE, glucosidase and α‐amylase inhibitory activities, indicating that the major phenolic profiles of thyme extracts may not be necessarily related to the degree of inhibition of ACE, glucosidase and α‐amylase enzymes.     

Screening grape seeds recovered from winemaking by‐products as sources of reducing agents and mammalian α‐glucosidase and α‐amylase inhibitors

Grape seeds collected from vinification of various grape varieties were extracted by supercritical CO₂ for oil recovery. The defatted residues thus obtained were considered as a re‐utilisable co‐product and assessed for phenolic content, reducing capacity and inhibitory activities against mammalian α‐amylase and α‐glucosidase enzymes. Supercritical CO₂ treatment led to higher recovery of anthocyanins. Reducing capacity of phenolic extracts reached up to ~2200 mmolFe(II) kg^?1, much higher than that of various natural phenolic sources. The anthocyanin‐rich extracts showed the highest inhibitory effectiveness towards α‐glucosidase (I₅₀ value equal to ~40 μg gallic acid equivalents (GAE)/mL ~ half than acarbose). Inhibitory effectiveness towards α‐amylase activity was similar among grape varieties, with I₅₀ values comparable to that of acarbose and correlated with proanthocyanidin contents. These results could pave the way for an efficient processing of grapes, including cascade processes, namely: winemaking, oil extraction from recovered grape seeds and phenolic extraction from defatted grape seeds as potential cost‐effective nutraceuticals.     

The Variation of β‐amylase Activity and Protein Fractions in Barley Grains as Affected by Genotypes and Post‐anthesis Temperatures

The variation of β‐amylase activity and protein fractions in barley grains was evaluated using 148 barley genotypes grown in the field and two cultivars under in vitro culture with two temperature treatments during grain development. The results showed that there was significant genotypic variation in β‐amylase activity and protein fraction content. Regression analysis indicated that β‐amylase activity was positively correlated with total protein and the level of each of the protein fractions, with the correlation coefficient between β‐amylase activity and hordein content being the highest. Furthermore, higher post‐anthesis temperatures (32/26°C, day/night) significantly enhanced β‐amylase activity and protein fraction content, presumably as a result of reduced starch content. Albumin and glutelin were the least and most affected, respectively, in comparison with the plants under lower temperature (22/16°C). Temperature post‐anthesis also influenced the morphology of the starch A granule and the number of B granules, suggesting the altered starch structure may also be a reason for deteriorated malting quality under high temperatures.     

ANTI-AMYLASE, ANTI-GLUCOSIDASE AND ANTI-ANGIOTENSIN I-CONVERTING ENZYME POTENTIAL OF SELECTED FOODS

α‐Amylase and α‐glucosidase have been targeted as potential avenues for modulation of postprandial hyperglycemia through mild inhibition of the enzymatic breakdown of complex carbohydrates to decrease meal‐derived glucose absorption. Water‐soluble extracts with optimized phenolic content of selected American and Asian foods were investigated for inhibitory activity against α‐amylase and α‐glucosidase, as well as angiotensin I‐converting enzyme (ACE), which has been linked to hyperglycemia‐associated hypertension. Porcine pancreatic α‐amylase (PPA) was allowed to react with each phenolic‐optimized food extract, and the derivatized enzyme–phytochemical mixtures obtained were characterized for residual amylase activity. The α‐glucosidase and ACE activities were determined in the presence of each phenolic‐optimized food extract. The amylase activity was inhibited more than the glucosidase activity in the presence of these phytochemical extracts, and more so by Asian foods than by American foods. The Asian spice ginger was found to possess strong ACE inhibitory activity in addition to significant anti‐amylase activity. The α‐amylase enzyme inhibition was positively associated with extract antioxidant activity and negatively with extract protein content. The significance of food‐grade, plant‐based amylase inhibitors for modulation of carbohydrate breakdown and control of glycemic index of foods in the context of preventing hyperglycemia and diabetes mellitus complications in the long term and ACE inhibitors for modulation of associated hypertension is hypothesized and discussed.     

Modelling and Optimizing of Mashing Enzymes — Effect on Yield of Filtrate of Unmalted Sorghum by Use of Response Surface Methodology

The effect of commercial enzymes on liquefaction of starch from unmalted sorghum was studied. The effects which these enzymes had on rates of filtration were evaluated. Models were developed, validated and optimized to establish the actions of enzymes, either alone or in combination. Preliminary studies on the sorghum cultivars Safrari, Madjeru and S.35 showed that α‐amylase was the backbone enzyme for starch liquefaction among the enzymes used (α‐amylase, Filtrase, protease and β‐amylase). Models confirmed this observation as α‐amylase individually in its first order (X₁) contributed 25, 11 and 17%, and in its sum of first and second orders (X₁+X₁²) contributed a 29, 31 and 36% yield of filtrate for Safrari, Madjeru and S.35 respectively. The ease of starch liquefaction, assessed by summing the first and second orders of individual intervention of all enzymes, was found to be in the order of Madjeru, S.35 and Safrari (79, 70 and 56% of yield of filtrate respectively). The importance of the enzyme combination in starch liquefaction in Safrari, S.35 and Madjeru was shown to be 44, 30 and 21% respectively. Enzyme combinations giving maximal starch liquefaction, as identified from a Doehlert experimental matrix, displayed a similar yield of filtrate (Safrari: 85 mL, Madjeru: 84 mL and S.35: 81 mL) after filtration of a 130 mL mash during 1 h. Validation of the models revealed the model developed for Madjeru was the most reliable (R² = 0.994), while those developed for Safrari (R² = 0.987) and S.35 (R² = 0.976) were slightly less reliable. Model optimization gave theoretical enzyme (Brewers Amyliq TS, Filtrase NLC, Brewers Protease and β‐amylase) combinations of 25 mg, 5.68 mg, 100 mg and 67.4 U for Safrari, 15.06 mg, 0.51 mg, 24.32 mg and 53.8U for Madjeru and 19.01 mg, 6.36 mg, 58.76 mg and 43.48 U for S.35, with a resulting yield of filtrate of 94, 87.7 and 83.8 mL respectively.