Effect of condensed tannins prepared from several forages on the in vitro precipitation of ribulose-1,5-bisphosphate carboxylase (Rubisco) protein and its digestion by trypsin (EC 2.4.21.4) and chymotrypsin (EC 2.4.21.1)

A series of in vitro experiments was undertaken to determine the extent to which Sephadex LH-20 treated extracts from a range of temperate forages precipitated ribulose-1,5-bisphosphate carboxylase (Rubisco) and affected the enzymatic hydrolysis of Rubisco protein by trypsin and chymotrypsin at a range of pH values. Rubisco was chosen because it represents the principal dietary protein for ruminants fed fresh forages. Condensed tannins (CT) or proanthocyanidins (PA) are routinely purified by chromatography using Sephadex LH-20 as a matrix. However, these extracts contained non-CT phenolics together with PA so the term ‘CT extract’ was preferred to ‘PA’ to describe the extracts. The in vitro precipitation of Rubisco provided a means to compare the reactivity of the CT extracts. The amount of CT extract required to precipitate all the Rubisco in 10 μg of total soluble leaf protein from white clover (Trifolium repens) when this protein was incubated with CT extracts of Lotus corniculatus, L pedunculatus and sainfoin (Onobrychis viciifolia) was similar, with between 25 and 50 μg of extract required. The CT extract of sulla (Hedysarum coronarium) also precipitated all the Rubisco, however this only occurred with 50 μg of the extract. The CT extract of dock (Rumex obtusifolius) precipitated all the Rubisco when 5 μg of extract or greater was incubated with total soluble leaf protein. However, the differences between the reactivity of all these CT extracts at a range of pH values appeared to be small. Condensed tannin extracts of L corniculatus and L pedunculatus partially inhibited the hydrolysis of Rubisco by trypsin and chymotrypsin to a similar extent, but the extent of the inhibition was affected by pH. The inhibition was greater at pH 6·0 than 7·0, whilst at pH 8·0, CT extracts had little or no affect on trypsin and chymotrypsin. It was concluded that, although the precipitation of Rubisco provided an ideal method for comparing CT extracts, reactivity alone was unlikely to account for the differences in nutritive value that occur with forages containing CT. © 1998 SCI.     

Controlling the formation of indole and skatole in in vitro rumen fermentations using condensed tannin

Indole and skatole are formed in the rumen from the fermentation of tryptophan and have been correlated to the presence of undesirable pastoral flavours in meat from grazing ruminants. A series of four in vitro rumen fermentation experiments was carried out to determine the effectiveness of condensed tannin (CT) for reducing the formation of indole and skatole. Experiment 1 utilised fresh white clover (WC; Trifolium repens) in ratios with the CT‐containing forage Lotus pedunculatus (LP; 97 g CT kg^?1 dry matter (DM)). Increasing the ratio of LP to WC decreased the formation of indole and skatole. Experiments 2 and 3 used extracts of CT from LP and grape seed added to incubations of fresh non‐CT‐containing WC or perennial ryegrass (PRG; Lolium perenne). Including the CT extracts in incubations at 40 and 80 g kg^?1 DM was more effective at reducing indole and skatole formation than lower concentrations of CT extract (P < 0.05). Including fresh LP in ratios with WC gave a linear decrease in indole and skatole concentration (indicating dilution), while including a CT extract in the incubations gave an exponential decrease in indole and skatole concentration (suggesting binding). Experiment 4 elucidated the mechanisms behind the action of CT by delayed addition of tryptophan and polyethylene glycol (PEG) to incubations of LP. Rumen microbes that were exposed to CT of LP in planta for up to 6 h and then provided with tryptophan were still able to convert tryptophan to indole and skatole. Adding PEG to incubations of LP after 6 h inhibited the activity of plant CT and increased the availability of substrate for indole and skatole formation. These studies have shown that a higher concentration of CT is more efficient for reducing indole and skatole formation and that CT contained within plants acts differently in in vitro rumen fermentations than additions of extracted CT. Under the conditions of these experiments, there was no evidence that CT contained in LP affected the protein present in WC in a mixed fermentation. Copyright © 2007 Society of Chemical Industry     

Concentration of indoles and other rumen metabolites in sheep after a meal of fresh white clover,perennial ryegrass or Lotus corniculatus and the appearance of indoles in the blood

The objective of this study was to determine the effect of feeding three fresh forage diets, white clover (WC), perennial ryegrass (PRG) and Lotus corniculatus (LC), on the formation of indole and skatole in the rumen of sheep. The formation of indole and skatole in the rumen and their appearance in the blood were also compared. Peak rumen indole and skatole concentrations per kg crude protein intake (CPI) were significantly higher when feeding WC compared with PRG and LC (P < 0.05) and this was associated with a significantly higher rumen concentration per kg CPI of ammonia, branched chain volatile fatty acids, total nitrogen and soluble nitrogen (P < 0.05). Greater indole and skatole concentrations when feeding WC can be attributed to high solubility and rapid degradation of the forage protein. LC had a similar nutrient composition to WC, but the condensed tannins in LC slowed protein degradation and reduced indole and skatole formation. Indole and skatole concentrations peaked in the plasma 1–2 h after the end of feeding, indicating that skatole and indole are rapidly absorbed from the rumen into the blood. High indole and skatole formation with low intakes of WC indicates that the WC component of traditional New Zealand pastures may be the primary cause of undesirable pastoral flavours that result from the presence of indoles in meat. To ameliorate undesirable flavours, producers reliant on pastoral systems will need to consider using alternative forages such as LC to reduce protein solubility and degradation rate. Copyright © 2007 Society of Chemical Industry     

Effect of different condensed tannin‐containing forages,forage maturity and nitrogen fertiliser application on the formation of indole and skatole in in vitro rumen fermentations

The objectives of this work were to establish the effect of seven different forages with a varied condensed tannin (CT) content, plant maturity and nitrogen fertiliser application on the ruminal formation of skatole and indole using an in vitro method designed to mimic rumen fermentation conditions. After 10 h of incubation, the concentration of indole and skatole was highest when incubating white clover (P < 0.05). Polyethylene glycol addition, to inhibit CT, showed that CT significantly reduced the formation of indole and skatole when incubating Lotus corniculatus, sulla, Dorynium rectum and Lotus pedunculatus (P < 0.01). Mature forage growth resulted in a significantly lower concentration of indole and skatole being formed in vitro compared to the incubation of new spring growth (P < 0.001). A higher application of nitrogen fertiliser to perennial ryegrass‐based pasture resulted in a higher crude protein concentration in the plant and a significantly higher concentration of skatole formed in vitro (P < 0.001). Forages containing CT reduced the conversion of endogenous protein to indole and skatole and plants containing a higher CT concentration tended to be more effective, but compositional differences of CT between forages may also have had an influence. New forage growth or swards that had a high application of nitrogen fertiliser promoted the formation of indole and skatole. Copyright © 2007 Society of Chemical Industry     

Factors affecting in vitro formation of tannin-protein complexes

In interaction of condensed tannins from Desmodium intortum and Lotus pedunculatus and tannic acid (hydrolysable tannin) with salivary mucoproteins (from sheep and goats), plant leaf proteins and bovine serum albumin were evaluated. These studies were carried out over a pH range of 2-0-9-0 and different inorganic ion conditions to simulate conditions in which dietary proteins would interact with tannins in a ruminant digestive tract. Insoluble tannin-protein interactions were found at pH 4–5–5–5 for bovine serum albumin and 3–5–5–5 for plant leaf protein. The present study showed that pH alone was not the sole determinant for tannin-protein complex formation, since tannin-protein complexation was found in the pH range 6-0–6-5 when different inorganic ions were added to the solutions. Insoluble complexes were not formed with salivary proteins, although precipitation by tannic acid was achieved at 5°C. This suggests that tannins may form soluble rather than insoluble complexes with salivary proteins. It was concluded that purified F1 leaf protein (the major protei occurring in leaf tissue) ought to be used as the test protein for evaluating tannin-protein interactions for in vitro assay procedures. Using this method it was calculated that 27–43% and 19–40% of available plant protein may interact with condensed tannins from Desmodium intortum and Lotus pedunculatus, respectively.     

Evidence of a role for plant proteases in the degradation of herbage proteins in the rumen of grazing cattle

Protein breakdown in the rumen is generally regarded as a two-stage process in which proteases produced by rumen microorganisms cleave plant protein into peptides and amino acids. However, many of the fiber-degrading cellulolytic species in the rumen are not in fact proteolytic, and the proteolytic activity of the entire rumen microbial population is only moderate when compared to the gastric and pancreatic secretions in the abomasum. Moreover, plant cell walls remain largely intact after initial chewing (particularly in cattle), presenting a physical barrier that must be breached prior to their effective colonization. The present study considers the hypothesis that the plant enzymes are at least partly responsible for herbage protein degradation in grazing ruminants. Ryegrass, red clover, white clover, and bird's-foot trefoil were incubated in the presence and absence of rumen microorganisms. The production of volatile fatty acids indicated the level of microbial activity, whereas the relative disappearance of the large subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco LSU) indicated proteolytic activity. In all incubations, the relative abundance of the Rubisco LSU decreased as the incubation progressed. When rumen microorganisms were absent, low molecular weight peptides (below 20 kDa) accumulated as the incubation progressed. This accumulation was not observed in the presence of rumen microorganisms. Therefore we suggest that the intrinsic plant proteases contribute to the initial stages of proteolysis of grazed herbage.     

Comparison of the Precipitation of Alfalfa Leaf Protein and Bovine Serum Albumin by Tannins in the Radial Diffusion Method

The precipitation of protein by condensed and hydrolysable tannins was evaluated with the radial diffusion method of Hagerman (1987) using bovine serum albumin (BSA) and isolated leaf protein from fresh alfalfa (Medicago sativa). Alfalfa leaf protein (AALP) was included at two concentrations, 25 and 156 mg N litre^-1, at pH 6·8 and 39°C to simulate rumen conditions. The condensed tannins were purified from lyophilised samples of Arachis pintoi, Desmodium ovalifolium, Gliricidia sepium, Manihot esculenta and quebracho (Schinopsis balansae). Hydrolysable tannins from tannic acid (TA) were used as well. There was a significant interaction (P<0·001) between tannin and protein source, and protein level on protein precipitation. Most purified condensed tannins (CTs) precipitated more AALP than BSA when protein was included at the same level. Purified CT from quebracho and hydrolysable tannin from TA failed to precipitate AALP at both protein levels. In a second experiment, tannins from crude plant extracts were studied in the radial diffusion method using BSA and two levels of AALP. The crude plant extracts were obtained from lyophilised plant samples of A pintoi, Centrosema macrocarpum, Clitoria ternatea, D ovalifolium, Erythrina berteroana, E poepigiana, G sepium, M esculenta, Pueraria montana and P phaseoloides. The protein precipitated by soluble tannins in the plant samples was correlated to the total phenolic content and to the soluble CT estimated by the acid butanol assay or by the radial diffusion method. Tannins from different plant species precipitated different amounts of BSA and AALP. Therefore, the measures of the biological activity of tannins based on BSA precipitation may not reflect the ability of tannins to precipitate proteins of plant origin such as those commonly found in the diets of herbivores. The present study offers the possibility of using the radial diffusion method with plant proteins at precipitation conditions similar to those in the rumen. © 1997 SCI.     

Interrelationships between the concentrations of total condensed tannin,free condensed tannin and lignin in Lotus sp. and their possible consequences in ruminant nutrition

Interrelationships between the concentrations of total condensed tannin (TCT), free condensed tannin (FCT) and lignin were studied to gain knowledge of how to manipulate nutritive value of fresh herbages containing condensed tannins fed to ruminants. FCT was defined as condensed tannin not bound by macerates of fresh plants, with both FCT and TCT being determined with vanillin HCl. Effects of spraying lotus with polyethylene glycol (mol. wt 3350; PEG) upon the relationship between FCT and TCT was also studied. Increasing soil nutrient and climatic stress caused large and similar increases in the concentrations of TCT and of lignin. Over the range 0–90 g kg^?1 DM, 10% of TCT in Lotus sp. was detected as FCT, with increments in TCT above 90 g kg^?1 DM being released almost entirely as FCT. PEG formed much stronger chemical bonds with condensed tannins than did plant proteins, and did not release FCT; consequently PEG application reduced the concentration of condensed tannin that was detectable with vanillin HCl. After disintegration of plant material, it is proposed that most condensed tannin is bound and co-precipitated as an insoluble complex with protein, that FCT is in equilibrium with this complex, and that bound and free tannin are indices of nutritionally beneficial and detrimental effects produced by condensed tannins in fresh forages eaten by ruminants. It was concluded that growing Lotus pedunculatus under conditions of stress leads to depressions in nutritive value through simultaneously increasing concentrations of lignin and FCT, both of which depress rumen carbohydrate digestion and voluntary intake, and that treatment with PEG offers a convenient method of separating effects due to condensed tannins from other factors influencing nutritive value.     

Plant‐mediated lipolysis and proteolysis in red clover with different polyphenol oxidase activities

Polyphenol oxidase (PPO) is an enzyme involved in the browning reaction of red clover leaves, when cut or crushed and exposed to air. PPO starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins. The leaf tissue of two lines of red clover (cv. Milvus, a genotypic mutant with reduced PPO activity (LowPPO) and the wild‐type, NormalPPO) were extracted in phosphate–citrate buffer, and a third treatment was prepared by extracting the LowPPO leaves in phosphate–citrate buffer plus 50 mM ascorbate to inhibit PPO activity (AscPPO). These extracts were compared over a 12 h time course in terms of proteolytic and lipolytic activity. Characterization of the tissues showed PPO activities of 9.11, 1.85 and 0 Δ optical density g^?1 fresh weight min^?1, which were reflected in the extent of phenol (derived from quinones) binding to protein after 12 h incubation 102.3, 83.2 and 45.8 mg bound phenol g^?1 protein (p < 0.001) for NormalPPO, LowPPO and AscPPO, respectively. Proteolysis measured as free amino acids released into the incubation was significantly reduced (p < 0.001) with increasing PPO activity, with values after the 12 h incubation of 0.03, 0.08 and 0.14 g g^?1 protein for NormalPPO, LowPPO and AscPPO, respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (p < 0.001) with increasing PPO activity, with values after the 12 h incubation of 0.12, 0.20 and 0.22 for NormalPPO, LowPPO and AscPPO, respectively. Changes that occurred in the lipid fractions (polar fraction, diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid loses in silo and potentially in the rumen. Copyright © 2004 Society of Chemical Industry     

Potent Antidiabetic Activity and Metabolite Profiling of Melicope Lunu‐ankenda Leaves

Diabetes mellitus is normally characterized by chronic hyperglycemia associated with disturbances in the fat, carbohydrate, and protein metabolism. There is an increasing trend of using natural products instead of synthetic agents as alternative therapy for disorders due to their fewer side effects. In this study, antidiabetic and antioxidant activities of different Melicope lunu‐ankenda (ML) ethanolic extracts were evaluated using inhibition of α‐glucosidase and 2,2‐diphenyl‐l‐picrylhydrazyl (DPPH) radicals scavenging activity, respectively; whereas, proton nuclear magnetic resonance (¹H NMR) and ultra‐high performance liquid chromatography‐tandem mass spectrometric (UHPLC‐MS/MS) techniques were used for metabolite profiling of ML leaf extracts at different concentrations of ethanol and water. Sixty percent of ethanolic ML extract showed highest inhibitory effect against α‐glucosidase enzyme (IC₅₀ of 37 μg/mL) and DPPH scavenging activity (IC₅₀ of 48 μg/mL). Antidiabetic effect of ML extracts was also evaluated in vivo and it was found that the high doses (400 mg/Kg BW) of ML extract exhibited high suppression in fasting blood glucose level by 62.75%. The metabolites responsible for variation among ML samples with variable ethanolic levels have been evaluated successfully using ¹H‐NMR–based metabolomics. The principal component analysis (PCA) and partial least squares(PLS) analysis scores depicted clear and distinct separations into 4 clusters representing the 4 ethanolic concentrations by PC1 and PC2, with an eigenvalue of 69.9%. Various ¹H‐NMR chemical shifts related to the metabolites responsible for sample difference were also ascribed. The main bioactive compounds identified attributing toward the separation included: isorhamnetin, skimmianine, scopoletin, and melicarpinone. Hence, ML may be used as promising medicinal plant for the development of new functional foods, new generation antidiabetic drugs, as a single entity phytomedicine or in combinational therapy.     

Fermentation characteristics,aerobic stability and ruminal degradation of ensiled pea/wheat bi‐crop forages treated with two microbial inoculants,formic acid or quebracho tannins

This study evaluated the effects of two commonly used microbial inoculants (Lactobacillus buchneri (LB) and Lactobacillus plantarum (LP)), formic acid (FA) and quebracho tannins (QT) on the fermentation quality, aerobic stability and in situ rumen degradation of pea/wheat bi‐crop forages. Precision‐chopped spring pea (Pisum sativum, var Magnus) and wheat (Triticum aestivum, var Axona) bi‐crops (3:1 pea/wheat ratio) harvested at a combined dry matter (DM) content of 301 g kg^?1 were used for the study. The bi‐crops were conserved without (Control) or with inoculants based on lactic acid bacteria (LB (10⁵ CFU g^?1 fresh weight (FW)) or LP (10⁶ CFU g^?1 FW)), QT (16 g kg^?1 FW) or FA (2.5 g kg^?1 FW) in laboratory silos of 1.5 kg capacity, with each treatment being replicated six times. The pH, chemical composition, aerobic stability and in situ rumen degradation of DM, nitrogen (N) and neutral detergent fibre (NDF) after 112 days of ensilage were measured. The average pH at silo opening was 4.0, suggesting that the silages were well fermented. There were no significant effects of additive treatment on water‐soluble carbohydrate, total N, soluble N, ammonia N and NDF. Lactic acid and acetic acid were the main fermentation products. High concentrations of acetic acid were found in all the treatments, indicating a heterofermentative pathway. Although FA treatment gave the most aerobically stable silage, the Control and QT‐treated silages did not heat up by more than 1 °C until after 6 days of exposure to air. There were no effects of additives on DM degradation characteristics. However, the inoculants increased the rate of N and NDF degradation in the rumen, and both FA and QT reduced the effective and potential degradation of N. © 2001 Society of Chemical Industry     

Potential protein degradation balance and total metabolizable protein supply to dairy cows from heat‐treated faba beans

The effects of pressure toasting (100, 118 and 136 °C for 3, 7, 15 and 30 min) on potential protein nutritional value of faba beans were evaluated with the NRC 2001 dairy model, by determining undegraded (RUP) and degraded rumen protein (RDP), undegraded (RUST) and degraded rumen starch (RDST), truly absorbed undegraded protein (ARUP), microbial protein (MCP_RDP) synthesized in the rumen from rumen‐available protein, truly absorbed rumen synthesized microbial protein (AMCP), truly absorbed rumen endogenous protein (AECP), total metabolizable protein (MP) in the small intestine, and the protein degradation balance (PDB). The treatments increased RUP, RUST, ARUP and MP (p < 0.001), and decreased RDP, RDST, MCP_RDP and PDB (p < 0.001), the effects increasing with increasing temperature and time. The treatments increased (p < 0.001) ARUP without affecting AECP and AMCP, so that the net absorbable total MP in the small intestine was increased. The PDB was reduced (p < 0.001) but never became negative. These results indicated that potential microbial protein synthesis would not be impaired due to sufficient nitrogen in the rumen, but the high positive PDB values with most treatments, except 136 °C for 15 min (PDB 2.0 g kg^?1 DM) indicated that there were large potential losses of nitrogen in the rumen, particularly for the control with a value of 88.9 g kg^?1 dry matter. It is concluded that predicted potential protein degradation balance and total metabolizable protein supply from faba beans were improved by the treatments. Copyright © 2005 Society of Chemical Industry     

Antioxidant properties of polar and non‐polar extracts of some tropical green leafy vegetables

BACKGROUND: The higher consumption of vegetables and fruits could be a practical approach to the management of oxidative stress. The present study sought to compare the antioxidant properties of polar and non‐polar constituents of some tropical green leafy vegetables (Struchium sparganophora, Amaranthus cruentus, Telfairia occidentalis, Ocimum gratissimum, Talinium triangulare, Cnidoscolous aconitifolius and Vernonia amygdalina). RESULTS: The polar antioxidant constituents (total phenol (3330–17 572 mg kg^?1), total flavonoid (1668–4306 mg kg^?1) and vitamin C (224–642 mg kg^?1)) were higher than the non‐polar antioxidant constituents (total phenol (703–3115 mg kg^?1), total flavonoid (130–1303 mg kg^?1) and carotenoids (132–1303 mg kg^?1)). Furthermore, the polar extracts had a significantly higher (P < 0.05) 1,1‐diphenyl‐2‐picrylhydrazyl radical scavenging ability (except T. triangulare), total antioxidant capacity, reducing power (except T. triangulare and A. cruentus) and Fe(II) chelating ability (except C. aconitifolius and S. sparganophora). However, the polar and non‐polar extract of O. gratissimum had the highest antioxidant properties while that of T. triangulare had the least antioxidant properties. CONCLUSION: The polar extract of most of the vegetables had higher antioxidant properties than the non‐polar extract, with O. gratissimum extracts having the highest antioxidant properties. Copyright © 2008 Society of Chemical Industry     

Antigenotoxicity of extracts from Pleurotus citrinopileatus

While Pleurotus citrinopileatus is a widely used edible mushroom, little is known about its physiological effects. Extracts, including aqueous extract, water‐soluble polysaccharide (WSP), crude protein solution (CPS) and residue from chloroform–ethyl acetate–methanol elution (CEM), were obtained first from fruiting bodies, through a solid‐state culture, and then from the mycelium, through a submerged culture. This study explored the antigenotoxicity effects of these extracts from Pleurotus citrinopileatus via the Ames test and a spore rec‐Assay. The results showed that, regardless of where the extract came from, the fruiting body or the mycelium, the antigenotoxicity effect was highest for CEM, followed by CPS, aqueous extract and WSP. The results of the Ames test indicated that, among several mutagens, CEM had the highest inhibition rate against AFB_l in TA98 and TA100 and the lowest inhibition rate against NQNO. The concentrations of the various extracts were as follows: water extracts were 1 mg ml^?1 and 5 mg ml^?1 WSP, while CPS and CEM were 0.4 mg ml^?1 and 2 mg ml^?1, respectively; the higher the concentration of the extract, the higher the antimutagenicity effect. The results of the rec‐Assay indicated that CEM had the highest anti‐DNA‐damaging activity with or without the S9 mixture; the higher the concentration, the more significant the effect (p < 0.05). The anti‐DNA‐damaging activities were lower in the water extract concentrations, at 30 µg disc^?1 dry weight^?1, while the WSP, CPS and CEM at 12, 150 and 60 µg disc^?1, respectively, were high. Copyright © 2004 Society of Chemical Industry     

Feeding value,in vitro digestibility and in vitro gas production of different by‐products for ruminant nutrition

Nine by‐products used in animal nutrition were examined for their nutritive value by determining the chemical composition (crude protein, crude fat (ether extract), neutral detergent fibre, acid detergent fibre, lignin), in vitro organic matter digestibility and rumen fermentation kinetics (from gas production curves measured in vitro). The by‐products studied were giant pumpkin, red pepper, stem broccoli, brewer's grain, fresh artichoke, scalded artichoke, lemon peel, orange peel and melon. The nutritive value was very variable, depending on the by‐product and on the process applied to the material during industrial processing. In vitro gas production was measured for 500 mg dry matter in quadruplicate at 39 °C after 6, 12, 24, 36, 48 and 72 h of incubation and fitted to single‐pool exponential equations. The fermentation kinetics indicated that brewer's grain was the only by‐product which fitted well with the p = a + b(1 ? e^?ct) equation model; all other by‐products had a very fast degradation rate and their gas production fitted the equation p = b(1 ? e^?c(t?L)), because in the other exponential model a was negative. © 2002 Society of Chemical Industry     

The condensed tannin content of vegetative Lotus pedunculatus,its regulation by fertiliser application,and effect upon protein solubility

The condensed tannin concentration in Lotus pedunculatus (cv. Grasslands ‘Maku’) was 8–11% DM when grown in acid soils without fertiliser application and 2–3% DM when grown in high fertility soils. Application of P & S fertiliser to the acid soils increased DM yield and reduced condensed tannin content to 4–5% DM, with over 88% of the variation in condensed tannin content being explained by variation in DM yield; it also increased plant total N concentration and halved the molar ratio of condensed tannin: protein (MR). Condensed tannins quantitatively precipitated soluble protein in lotus and also effectively precipitated protein in mixtures of lotus with white clover. The minimum concentration of condensed tannin necessary to precipitate protein was 2–4% DM, corresponding to MR values of 6–13. However, protein from such mixtures could be deaminated by fermentations with rumen fluid in vitro, but potentially soluble protein in pure lotus (MR 29) was protected from deamination by condensed tannins. A portion of lotus tannin could not be bound by plant constituents in finely ground fresh herbage and was designated ‘free’ tannin. This fraction was linearly related to total plant tannin content, was predicted to be zero at 1.8% DM total tannin, and increased at 0.15 units per unit increase in total tannin content above this figure. Maximum ‘free’ tannin comprised 17% of total condensed tannin. Effects of total and ‘free’ tannin content upon the intake and digestion of lotus by sheep are discussed.     

Influence of degree of hydrolysis on functional properties and angiotensin I‐converting enzyme‐inhibitory activity of protein hydrolysates from cuttlefish (Sepia officinalis) by‐products

BACKGROUND: In Tunisia the cuttlefish‐processing industry generates large amounts of solid wastes. These wastes, which may represent 35% of the original material and constitute an important source of proteins, are discarded without any attempt at recovery. This paper describes some functional properties and the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of protein hydrolysates prepared by hydrolysis of cuttlefish (Sepia officinalis) by‐products with crude enzyme extract from Bacillus licheniformis NH1. RESULTS: Cuttlefish by‐product protein hydrolysates (CPHs) with different degrees of hydrolysis (DH 5, 10 and 13.5%) were prepared. All CPHs contained 750–790 g kg^?1 proteins. Solubility, emulsifying capacity and water‐holding capacity increased while fat absorption and foaming capacity decreased with increasing DH. All hydrolysates showed greater fat absorption than the water‐soluble fraction from undigested cuttlefish by‐product proteins and casein. CPHs were also analysed for their ACE‐inhibitory activity. CPH3 (DH 13.5%) displayed the highest ACE inhibition (79%), with an IC₅₀ value of 1 mg mL^?1. CONCLUSION: Hydrolysis of cuttlefish by‐product proteins with alkaline proteases from B. licheniformis resulted in a product with excellent solubility over a wide pH range and high ACE‐inhibitory activity. This study suggests that CPHs could be utilised to develop functional foods for prevention of hypertension. Copyright © 2010 Society of Chemical Industry     

Electrophoresis to determine the molecular weight distribution in soluble proteins from various foods and their rumen‐resistant residues in cattle

Electrophoresis was used to visually identify and determine the molecular weight (MW) distribution of rumen‐degradable and rumen‐resistant or escape peptides in soluble proteins from 12 agricultural and distillers' food raw materials (RM) and their residues (RU) following 18 h of in sacco rumen incubation (dg₁₈) in cattle. Soluble proteins were extracted by using water, salt, acid and alkali in succession to represent albumins, globulins, glutalin 1 and glutalin 2 respectively. RM and RU differed substantially in the MW range, number and intensity of bands for various soluble proteins. The bands were mostly below the MW range of 66 kDa. Low‐MW (<25 kDa) peptides were greater in number than high‐MW (>25 kDa) peptides in almost all soluble proteins from RM. Individual peptides behaved differently during rumen incubation. Their resistance to or escape from rumen degradation varied with the class of food, type of soluble protein and their MW range. On average, low‐MW albumins in agricultural foods were more resistant to rumen degradation (0.41 RM vs 0.12 RU; 29%) than their high‐MW counterparts (0.12 RM vs 0.02 RU; 21%). In contrast, high‐MW glutalin 1 was more resistant (0.03 RM vs 0.22 RU) than low‐MW glutalin 1 (0.09 RM vs 0.26 RU) in most agricultural foods. Globulins contained the least and glutalins the most resistant peptides in distillers' foods. While this study reveals an association between dg₁₈ and protein type, structure and size, we do not recommend the immediate use of electrophoresis for routine food evaluation unless more studies are undertaken. It may, however, be suitable for further characterisation of the degradation of specific, selected peptides by specific micro‐organisms. © 2001 Society of Chemical Industry     

Pilot‐scale brewing using self‐cloning bottom‐fermenting yeast with high SSU1 expression

A pilot‐scale fermentation was performed using SSU1‐overexpressing bottom‐fermenting yeast strains constructed by ‘self‐cloning’. In these strains, the gene SSU1, encoding a plasma membrane protein that excretes sulphite, was highly expressed. The rate of fermentation of the two SSU1‐overexpressing strains tested showed some reduction during the mid‐fermentation phase as compared with the parental strain. These differences, however, did not affect overall fermentation and the final apparent extracts had decreased to a level normally obtained during brewing. The concentration of hydrogen sulphide in the wort remained low during fermentation in the case of the two self‐cloning strains compared with the parent. The concentration of 2‐mercapto‐3‐methyl‐1‐butanol, a sulphur compound that causes an ‘onion‐like’ off‐flavour, was also reduced in the case of the self‐cloning strains, a result confirmed by sensory evaluation of the beer immediately after bottling. Furthermore, with these strains the anti‐oxidation potential of bottled beer, as measured by electron spin resonance, was improved and the concentration of trans‐2‐nonenal in bottled beer after 7 days of accelerated aging at 37°C was decreased. These observations, together with the lower stale flavour score determined by sensory evaluation of bottled beer after a month of aging at 25°C, indicated that the flavour stability of the beer had been successfully improved. Copyright © 2013 The Institute of Brewing & Distilling     

Antioxidant activity of two yeasts and their attenuation effect on 4‐nitroquinoline 1‐oxide induced in vitro lipid peroxidation

To obtain functional yeast with antioxidant ability for food industry, the antioxidant activity of intact cell and intracellular cell‐free extract of Pichia fermentans BY5 and Issatchenkia orientalis BY10 was investigated. Both intact cell and extract of them demonstrated antioxidant activity ranged from 49% to 68%. The ability to scavenge 1, 1‐diphenyl‐2‐picrylhydrazyl free radicals were 12–41%. Furthermore, the reducing activity, Fe²⁺‐chelating ability, scavenging of reactive oxygen species of extracts illuminated these two isolates had excellent antioxidant ability. And then, the attenuated effect of cell‐free extracts from these two strains was evaluated using 4‐nitroquiunoline 1‐oxide (4‐NQO) as an inducing reagent. The results indicated that the addition of extraction inhibit the lipid peroxidation induced by 4‐NQO, which mainly caused by the protective intracellular protein rather than the polysaccharides. Therefore, these two yeast strains have potential to be utilised for production of functional foods.