Effect of chitosans on in vitro rumen digestion and fermentation of maize silage

The gas in vitro technique was used to study the effects of six types of chitosans, each having different molecular weights and acetylation degrees, on rumen microbial fermentation. In a first trial, a separate concentration of 750 mg/l of culture fluid for each of the six chitosans (CHI1, CHI2, CHI3, CHI4, CHI5, and CHI6) was incubated for 24 h in diluted ruminal fluid with maize silage as the substrate. The ionophore antibiotic monensin (MON) was used as a positive control, and a negative control with no chitosan (CTR) was also included. Each treatment was tested in triplicate for three different periods. At the end of the trial, samples were collected to determine volatile fatty acid (VFA) and ammonia N concentrations, and pH and gas production values were recorded. Methane concentration was estimated stoichiometrically. In vitro true organic matter digestibility (IVOMD) and partitioning factor (PF, mg OM truly degraded/ml gas produced) were also calculated. In a second trial, a separate concentration of 750 mg/l of each of the six chitosans was incubated for 144 h in diluted ruminal fluid with maize silage as the substrate, to study the effects of these compounds on fermentation kinetics.All six chitosans decreased the IVOMD and PF values. Chitosan inclusion did not affect the fermentation of the substrate's soluble fraction, but did reduce the fermentation kinetics of the insoluble but fermentable fraction. However, only CHI5 and CHI6 decreased total VFA concentration. CHI3 and CHI6 decreased the molar proportion of acetate and increased the molar proportion of propionate, thus increasing the propionate-to-acetate ratio. Chitosan inclusion did not affect molar proportions of butyrate. With the exception of CHI2, the molar proportion of branch-chained VFA was lowered by all of the chitosan treatments. Most of the treatments also decreased methane production, also with the exception of CHI2.In conclusion, chitosan extracts may enable the manipulation of rumen microbial fermentation, but further research is required to elucidate the effect of chitosans on ruminal fermentation parameters in commercial diets as well as the adaptability of rumen microflora to these additives.     

Effects of dose and adaptation time of a specific blend of essential oil compounds on rumen fermentation

Essential oils can be used as natural additives in animal feeds. The present study evaluated the effects of three different doses and different adaptation times of a specific blend of essential oils (BEO) on rumen microbial fermentation. Eight dual flow continuous culture fermenters (1320 ml) were used in two periods of 9 days each to study the effects of increasing doses of BEO. Treatments were: CTR (no BEO), D5 (5 mg/l of BEO), D50 (50 mg/l of BEO) and D500 (500 mg/l of BEO). During the last 3 days, samples were taken at 0, 2, 4 and 6 h after the morning feeding and analyzed for large peptide (LPep), small peptides plus amino acid (SPep + AA) and ammonia N concentrations, and at 2 h after feeding for volatile fatty acids (VFA) concentration and profile. The D5 increased total VFA concentration, acetate proportion and acetate to propionate ratio, and decreased propionate and valerate proportion, compared with CTR. The concentration of LPep N tended (P=0.08) to be lower for D5 compared with CTR. In the second experiment, eight sheep were used to study the effects of long-term adaptation of rumen fluid to BEO on ruminal fermentation. Four sheep were assigned at random to the CTR treatment (no BEO) and four sheep were adapted to BEO (110 mg/day of BEO) for 4 weeks (ADBEO). After 4 weeks samples of ruminal fluid were obtained at 0 and 3 h after the morning feeding and in 2 consecutive days using an oro-ruminal probe. Samples were analyzed for LPep, SPep + AA and ammonia N concentrations, total and individual VFA, and pH. Treatment ADBEO tended (P<0.10) to increase acetate proportion and decrease valerate proportion, compared with CTR. Ruminal fluid collected from each of CTR and ADBEO sheep was used to study in vitro fermentation profile of soybean meal, corn meal, alfalfa hay and ryegrass hay. Treatments were: Control fluid (CTR without BEO), CTR fluid plus a single dose of BEO (11 mg/l; CTR + BEO) and ADBEO fluid plus a single dose of BEO (11 mg/l; ADBEO + BEO). Acetate proportion and acetate to propionate ratio was higher, and propionate and isovalerate proportion, and BCVFA and ammonia N concentration were lower in ADBEO + BEO fluid compared with CTR fluid. The addition of essential oils can shift the microbial fermentation in the rumen by increasing the acetate to propionate ratio and inhibiting deamination.     

In situ degradability of seven plant protein supplements in heifers fed high concentrate diets with different forage to concentrate ratio

Four Holstein heifers (297.5 ± 27.7 kg BW) fed high concentrate diets were used in a crossover experiment in order to characterize the rumen fermentation pattern, and to estimate by the in situ method rumen degradation kinetics of alfalfa hay and seven plant protein supplements: solvent-extracted soybean meal, solvent-extracted sunflower meal, peas (Pisum sativum L.), lupin seeds (Lupinus sp.), broadbean (Vicia faba L.), horsebean (Vicia faba L. var equina) and vetch (Vicia sativa L.), in high concentrate diets with different forage to concentrate ratio. Heifers were fitted with a ruminal cannula. The experiment was performed in two 30-day periods, 15 days of diet adaptation and 15 days of sampling. At each period, heifers were offered one of two total mixed rations (12:88 versus 30:70 forage to concentrate ratio), two heifers per diet, on ad libitum basis. After the first period, heifers switched treatments. Intake of dry matter (DM), organic matter, crude protein and neutral detergent fibre (NDF), expressed as kg/day, did not differ between treatments, but DM intake, expressed as g/kg metabolic body weight (BW), was higher in the 12:88 diet. Average rumen pH was 6.0 in both diets, and the time pH was below 5.8, which is considered as a critical threshold for fibre degradation, was the same for both treatments (10.4 ± 1.6 h). Average ammonia nitrogen and volatile fatty acid (VFA) concentrations did not differ between treatments and individual VFA proportions were typical of high concentrate diets. Average effective degradability of DM (0.62 ± 0.02) and NDF (0.25 ± 0.03) of alfalfa hay were low and no differences were detected between treatments. The same extent of NDF degradation, together with the same proportions of VFA would indicate that both diets had the same fibrolytic activity. Forage to concentrate ratio did not affect rumen nitrogen degradability of any protein supplements incubated in situ. Corrected effective degradability for small particle losses of sunflower meal (0.78) was higher than legume seeds, which were not statistically different between each other and ranged from 0.63 to 0.66. Soybean meal had the lowest degradability value (0.61). These nitrogen degradation values must be considered more valid for beef cattle formulation of high concentrate diets than data obtained with forage diets.     

Influences of flavomycin,ropadiar, and saponin on nutrient digestibility,rumen fermentation,and methane emission from sheep

This study focused on the effects of three additives given together with a hay/concentrate-based diet on nutrient digestibility, rumen fermentation, and methane emission from sheep. The basal diet consisted of 1.29 kg mixed hay and 0.43 kg concentrate mixture based on dry matter (DM). Treatments consisted of control (no additive), flavomycin⁴⁰ (250 mg/d), ropadiar from an oregano extract (250 mg/d), and saponin in the form of a yucca schidigera extract (170 mg/d). Results indicated that intake and digestibility were unaffected by treatments (P>0.05). The NH₃-N concentration of rumen liquor was lower (P<0.05) for additive treatments versus the control treatment. Higher concentrations of volatile fatty acid (VFA) were observed in the saponin (75.8 mmol/L) and ropadiar (73.1 mmol/L) treatments. The proportion of individual fatty acid of rumen liquor was unchanged, whereas lower ratio of acetate to propionate in the saponin treatment was observed (P<0.05). The average methane production expressed on digested organic matter (OM) and neutral detergent fiber (aNDFom) basis were decreased by approximately 3.3 and 12.0 g/kg, respectively in saponin, and 4.2 and 11.9 g/kg in ropadiar treatment compared to the control. Methane production was positively correlated with the concentrations of NH₃-N, and negatively correlated with total VFA and the proportion of propionate of rumen liquor (P<0.05). The study found that saponin and ropadiar could have the potential to reduce rumen methanogenesis in sheep.     

Effect of the supplementation of a high-concentrate diet with sunflower and fish oils on ruminal fermentation in sheep

This study was conducted to test the hypothesis that the supplementation of a high-concentrate diet with lipids, reportedly a good strategy for improving the nutritional value of ruminant-derived products, may not necessarily be associated with detrimental effects on ruminal fermentation in sheep. Four ruminally cannulated adult ewes were fed a high-concentrate diet, with no oil (Control diet), for a 14-day adaptation period. Afterwards, they were fed the same basal diet but supplemented with sunflower oil [20 g/kg fresh matter (FM)] and fish oil (10 g/kg FM) (SOFO diet) for a further 11 days, to investigate the impact of the addition of oils on the ruminal fermentation of the diet. On days 0 (Control), 3 and 10 of the experimental period rumen fluid was sampled at 0, 1.5, 3, 6 and 9 h after the morning feeding, for analysis of pH, and ammonia, lactate and total volatile fatty acid (VFA) concentrations. Alfalfa hay was incubated in situ, using the nylon bag technique, for 12 and 24 h to examine the effect of oil supplementation on ruminal disappearance of dry matter (DM), crude protein (CP) and neutral-detergent fibre (NDF). On days 0 and 11, rumen fluid was collected just before the morning feeding and used to incubate alfalfa hay and the Control and SOFO diets by means of the in vitro gas production technique. The mean concentrations of acetate (87.8 mmol/L vs. 73.7 mmol/L) and butyrate (21.2 mmol/L vs. 17.7 mmol/L) were reduced by oil supplementation (P < 0.05) and the total VFA showed a tendency (P = 0.098) to be lower with the SOFO diet (139.0 mmol/L vs. 122.1 mmol/L). However, none of the other in vivo ruminal fermentation parameters were affected by the treatment (P > 0.10). The oil supplementation affected neither in situ rumen disappearance of DM, CP and NDF of alfalfa hay, nor rates of gas production (P > 0.10). On the other hand, a little, but significant reduction in cumulative gas production was observed when the experimental diets were incubated with rumen fluid derived from animals fed the oil-rich diet (P < 0.05).Overall, the results suggest that the supplementation of high-concentrate diets with sunflower oil (20 g/kg FM) plus fish oil (10 g/kg FM) had little effect on ruminal fermentation and therefore its use to improve the nutritional value of ruminant-derived products cannot be precluded.     

Purification and biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

Assessment of the efficacy of varying experimental exogenous fibrolytic enzymes using in vitro fermentation characteristics

This study evaluated a series of recombinant, single activity experimental enzyme products including 13 endoglucanases (END) and 10 xylanases (XY), for their potential to improve in vitro ruminal degradation of alfalfa hay in two experiments. Based on the endoglucanase or xylanase enzymatic activities measured using complex substrates at the optimal conditions (pH 5.4, 37 °C) for the enzymes, a dose level (1 unit/g dry matter [DM]) was chosen for addition of enzymes to substrate. Enzyme products, re-suspended with water, were added to alfalfa hay (0.5 or 1.0 g DM) in culture vials in six replications. Anaerobic buffer medium (20 or 40 ml) adjusted to pH 6.0 and strained ruminal fluid (5 or 10 ml) were sequentially added to the vials and incubated for 18 h. Headspace gas production (GP) was measured throughout the incubation, and degradability of organic matter (OMD) and fibre and volatile fatty acid (VFA) concentrations were determined after 18 h of incubation. The enzyme products had a wide range of added endoglucanase or xylanase activities when determined using pure substrates and physiological conditions typical of the rumen (pH 6.0, 39 °C). In experiment 1, many END, and some XY, products increased GP and OMD. The correlation between added endoglucanase activity determined at ruminal conditions and OMD improvement was high (r = 0.71; P<0.01), whereas added activity of xylanase was not associated with OMD improvement. Two END and two XY products selected from experiment 1 were further assessed because they substantially improved GP and OMD. In experiment 2, all enzyme treatments, alone or in combination, increased total GP and DM and fibre degradabilities (P<0.05). However, the combinations of END and XY did not increase degradation of alfalfa beyond that of the component enzymes. Total VFA production was not affected by enzyme treatments although some products changed the acetate to propionate ratio. Experimental exogenous enzyme products with either endoglucanase or xylanase activity substantially improved in vitro ruminal degradation of alfalfa hay, but further improvement by combining these activities did not occur.     

The relationship between the volatile fatty acids supply and the nitrogen retention in growing sheep nourished by total intragastric infusions

Twelve four-month old Suffolk × Small-tail-Han male sheep (live weight 21–26 kg), fitted with rumen and abomasum fistulas and nourished by total intragastric infusions, were used to study the relationship between the volatile fatty acids (VFA) supply and the nitrogen (N) retention in sheep. The animals were randomly divided into four groups and four levels of mixed VFA energy (the molar proportion of acetic acid, propionic acid and butyric acid was 65:25:10), i.e. 333, 378, 423 and 468 kJ kg^?1 W^0.75 d^?1, were infused into the rumen, as treatments I, II, III and IV, respectively. The results showed that the N retention was significantly increased (P < 0.05) with the VFA infusion level. Significant regression relationship was found between the VFA supply (x, g d^?1) and the N retention (y, mg d^?1): y = 2.75x ? 403, r² = 0.86, n = 12, P < 0.01. It was concluded that to get efficient utilization of dietary N and high N retention in sheep, it is necessary to supply enough dietary energy.     

Effect of polyethylene glycol,urea and sunflower meal on olive (Olea europaea var. europaea) leaf fermentation in continuous fermentors

A continuous culture system, inoculated with rumen liquor from goats or sheep, was used to study fermentation characteristics of olive leaves (OL). The effects of adding polyethylene glycol (PEG 4000 MW; 0, 2 or 20 g/100 g OL) and/or supplementing with urea (U) or sunflower meal (SM) (1.0 g N/100 g OM) were also studied. Olive leaf fermentation promoted low VFA production (35.2 mmol/d), predominantly of acetic acid, and low efficiency of VFA production (4.91 mol/kg digestible carbohydrates, DCHO). Both values increased with N supplementation, but effects of PEG were variable. No differences ascribed to the rumen inoculum origin were observed. The ammonia N concentration was increased only by supplementation with U. Total and amino acid N output was low and increased with N addition, but it was not affected by PEG treatment. No differences ascribed to the inoculum origin were observed concerning microbial N production rate or efficiency (g N/kg DCHO). There was no clear difference between sources of supplementary N regarding bacterial protein synthesis. On the basis of PEG results, the effect of tannins on OL fermentation was not important.     

Interpretation of rumen degradability of concentrate feeds with a Gompertz model

An investigation of the kinetics and synchronicity of rumen crude protein and starch degradability was performed for maize, flaked maize, ensiled maize cob, barley, flaked barley, wheat, oat, sorghum and triticale grain, using the in situ polyester bag technique. Kinetics of rumen degradability were corrected for particle losses from the bag before degradation. Washing losses were measured by shaking feed samples in polyester bags in tap water at 20°C for 1 h and recovering the particle losses in fibre glass filter. Mean washing losses of dry matter were 442 g/kg DM (soluble fraction 17%); mean washing losses of nitrogen were 446 g/kg nitrogen, with 52% being water soluble. Starch escaped to a higher extent from the bags in the washing machine (average washing losses were 581 g/kg starch, with a soluble fraction of 7%). Degradability data for dry matter, nitrogen and starch measured at each time of incubation were corrected for the respective particle losses and fitted with both first order and Gompertz (sigmoidal) models. The difference between the estimated parameters obtained with the two models was negligible, although differences occurred for the immediately soluble fraction (a) of dry matter and starch and for total degradable fractions of dry matter, nitrogen and starch. No differences were observed between effective degradabilities, independent of rumen outflow rate (0.04 and 0.08/h), apart for the mean effective degradability of nitrogen at 0.08/h, which was higher for the first order model (577 versus 564 g/kg, P < 0.001). The advantage of using the Gompertz model to interpret the kinetics of rumen degradability of cereals was due to the possibility of studying synchronicity of nitrogen and starch released into the rumen and ranking feeds according to their degradability pattern, which required the calculation of the first derivatives of the Gompertz model for nitrogen and starch and weighted for their respective amounts in the feeds. The difference of these ratios from the optimum value of 30 mg N per 1 g starch gives the instantaneous synchronicity of the cereals. Maize, flaked maize, maize cob and sorghum had negative and almost constant values during the first 8 h of incubation. Barley, flaked barley, wheat, triticale and oat changed from an initially negative value to a positive value from about 4 h onwards; oats had a similar pattern, but a positive value was observed only after 6 h.     

Effect of rare earth elements on in vitro rumen microbial fermentation and feed digestion

The objectives of this study were to investigate the effects of rare earth elements (REEs) on in vitro rumen fermentation, gas production, microbial protein synthesis and nutrient digestion using in vitro batch culture and continuous culture technique. A mixture of REE containing (g/kg) 380 g of LaCI₃·6H₂O, 521 g of CeCI₃·6H₂O, 30 g of PrCI₃·6H₂O and 69 g chlorides of other light REEs. The experimental diet consisted of 885 g/kg barley grain, 84 g/kg barley silage and 31 g/kg supplement (dry matter (DM) basis). Diet supplemented with different dosages of REE (control, no additional REE; low, 400 mg/kg REE; and high, 800 mg/kg REE, DM basis) were incubated for 4, 8, 14 and 24 h in diluted rumen fluid. At the end of 24 h of incubation, gas production and concentration of volatile fatty acid (VFA) linearly increased with increasing REE supplementation; whereas, influence of REE supplementation on VFA profile was marginal. Dry matter disappearance was not affected (P>0.10). Six dual-flow continuous culture fermenters were used in a replicated 3 × 3 Latin square with same treatments and same diet used in the batch culture. Mean ruminal pH (5.71) and total VFA (93.6 mM) concentration were not affected by supplementation of REE. The molar proportion (mol/100 mol) of acetate (39.1) and propionate (50.5) was similar among the treatments. However, the proportion (mol/100 mol) of butyrate was higher with the high REE (6.6) than with low REE (5.3) or the control (5.8). Ruminal true digestibilities of organic matter (OM) (0.785, 0.811 and 0.828), acid detergent fibre (0.360, 0.431 and 0.432) and crude protein (0.496, 0.590 and 0.589) for control, low and high REE, respectively, linearly increased with increasing REE supplementation, whereas, the increase in ruminal digestibility from low to high dosage of REE was minimal. Microbial nitrogen (N) production (g/day) and microbial efficiency (g N/kg of truly fermented OM) were not affected by treatments. Improvement of ruminal digestibility of OM due to REE supplementation was attributed to the increase in digestibility of fibre and degradability of protein. The results suggest that REE supplementation improved ruminal fibrolytic and proteolytic activities.     

Supplementary feeding of horses with processed sorghum grains and oats

Two feeding experiments and in vitro hind gut fermentation tests were carried out to study the effect of processing sorghum grain on digestion of starch and on the gastrointestinal (GI) tract environment of the horse. In experiment 1, 12 yearling Australian stock horses were blocked on the basis of sex then randomly divided into four equal groups, each containing one castrated male and two females of approximately the same age and weight. Horses were offered at 0800 and 1500 h, 3 kg medium quality liverseed grass (Urochloa panicoides) hay and 2 kg of either oats (O), dry rolled sorghum (DRS), steam-flaked sorghum (SFS) or expanded sorghum (ES). Lanthanum was used as external solid marker for the measurements of apparent total tract digestibility. Fresh water was available ad libitum. Horses were allowed 18 days to adapt to the diets followed by a 3-day faecal collection period. Digestibility of dry matter (DM), and acid detergent fibre (ADF) were higher (P<0.01) for dry rolled and expanded sorghum diets. Digestibility of starch was similar across treatments, averaging 0.98 ± 0.009. Faecal pH values were lower (P<0.05) for the sorghum treatments but remained slightly below neutral (≥6.42) for the steam-flaked sorghum. Sub-samples of fresh faeces were supplemented with glucose and used for in vitro measurement of potential lactate production and final pH. The 20 h in vitro pH was also lower (P<0.05) for the steam-flaked sorghum group. All horses maintained good health and completed the experiment successfully.Results of this experiment indicate that digestibility of starch of processed sorghum is comparable to that of oats (0.97 versus 0.98), but processing sorghum grains may produce conditions favourable for lactate production and accumulation.In experiment 2, eight Australian stock horses were used to study changes in pH and accumulation of VFA and lactate in the different parts of the gastrointestinal tract. In this experiment SFS was compared with DRS. Horses were divided into two groups on the basis of sex and body weight. Each group contained two females and two castrated males. The nutritional management of the horses was the same as for the DRS and SFS groups in experiment 1. At the end of the adaptation period horses were slaughtered at intervals of 1.5, 3.0, 4.5, and 6.0 h after morning feeding, one horse per interval per treatment. Samples were collected from the different anatomical regions, and analysed for pH, VFA, and l- and d-lactate. Fermentation in the pre-glandular stomach produced mainly lactic acid and reduced pH to 4.0–4.3 while VFA was the main product in the hind gut. Total and d-lactate were higher (P<0.01) for SFS and a similar trend was observed for total and individual VFA and l-lactate but differences lacked significance (P>0.05). It was concluded that steam flaking of sorghum exacerbates starch fermentation, which may lead to acidosis-related damage to the epithelium of the pre-glandular stomach.     

Effects of dietary whole cottonseed and crude protein level on rumen protozoal population and fermentation parameters

In this investigation in vitro and in vivo trials were performed to determine the efficacy of a cottonseed to limit protozoal population and fermentation parameters. The composition of diets given to the different treatments were as follow: (1) control (without whole cottonseed), 16% crude protein (CP), 3.2% ether extract (E.E.); (2) 20% whole cottonseed, 16% CP, 6.5% E.E.; (3) 20% whole cottonseed, 13% CP, 6.4% E.E. and (4) 20% crushed whole cottonseed, 13% CP, 6.4% E.E. DM disappearance (DMD) and fermentation characteristics of the treatments were determined by in vitro incubation studies. In the in vivo trial, ruminal fluid was taken by rumenocentesis (3 h after feeding) on days 1, 3, 5, 7, 9, 11, 14, 21 and 28 from four sheep fed about treatment diets. The pH and protozoal counts were determined in each sample, while ammonia nitrogen and volatile fatty acid (VFA) were determined in samples taken on days 7, 14, 21 and 28. The in vitro DMD after 24 h incubation decreased (p < 0.01) with the addition of cottonseed in diets 3 and 4 and DMD after 72 h incubation was highest (p < 0.01) for the control diet. The fractional rate of gas production (c) for the control and diet 2 was higher (p < 0.05) than for the diets 3 and 4. Feeding crushed whole cottonseed decreased molar proportion of propionate (p < 0.05) and increased molar proportion of butyrate (p < 0.01). Low crude protein level increased the molar proportion of propionate (p < 0.05) and decreased molar proportion of butyrate (p < 0.05) and cellolytic protozoa population (p < 0.05). Feeding cottonseed decreased (p < 0.05) the total protozoa population from approximately 500,000 to 250,000 ml⁻¹ and Holotrich and cellulolytic protozoa disappeared from the rumen of sheep and only Entodinium sp., remained. This was associated with lower concentration of ammonia nitrogen in rumen fluid of sheep fed diets 4 (p < 0.05) and 2 (p < 0.01). It was concluded that cottonseed reduced rumen fauna and ammonia nitrogen, but had no effect on ruminal VFA while the crushed whole cottonseed decreased molar proportion of isovalerate only. In vivo molar proportion of propionate and butyrate and valerate were increase and decrease, respectively, by decreasing CP percentage in treatment diets.     

Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation

Sheep rumen contents were used as inoculum for an in vitro semi-continuous incubation system to study whether preservation method affects microbial fermentation pattern. Rumen fluid was filtered and either used immediately as inoculum (CTL) or dispensed into 110 mm × 16 mm tubes, that were stored refrigerated at 6 °C for 4 h (REF) or frozen at ?20 °C (FRZ), frozen in liquid N (FLN) or added with 0.04 glycerol and frozen in liquid N (FGL) for 48 h. Frozen inocula were thawed at 39 °C for 2 min before use (16 ml per bottle). Two 24 h incubations with four bottles per treatment were completed. The microbial utilisation of added glycerol after thawing in FGL increased total gas production (P<0.05) and 24 h volatile fatty acid (VFA) production (P<0.05), and also increased propionate and butyrate proportions at the expense of acetate. The other freezing inocula (i.e., FLN and FRZ) reduced the rate of gas production (as ml/g dry matter per hour), compared with CTL in the first 2 and 4 h of incubation (P<0.05), but this was compensated by increased fermentation at 8 and 12 h, respectively. Differences in gas production did not manifest a different VFA pattern at either 6 or 24 h incubation. Bacterial diversity was slightly affected by the preservation process, and the similarity index between untreated inocula and the 24 h incubated CTL samples was 0.690–0.724. Similarity between bacterial communities in FRZ and FLN with that in CTL after incubation was 0.678. The freezing preservation method of rumen inocula for subsequent in vitro gas production studies does not affect microbial fermentation pattern or bacterial biodiversity, provided that processing is rapid enough by using a high surface to volume ratio. Freezing in liquid N is more appropriate than at ?20 °C.     

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.     

α-Amylase from germinating soybean (Glycine max) seeds – Purification,characterization and sequential similarity of conserved and catalytic amino acid residues

Starch hydrolyzing amylase from germinated soybeans seeds (Glycine max) has been purified 400-fold to electrophoretic homogeneity with a final specific activity of 384 units/mg. SDS–PAGE of the final preparation revealed a single protein band of 100 kDa, whereas molecular mass was determined to be 84 kDa by MALDI–TOF and gel filtration on Superdex-200 (FPLC). The enzyme exhibited maximum activity at pH 5.5 and a pI value of 4.85. The energy of activation was determined to be 6.09 kcal/mol in the temperature range 25–85 °C. Apparent Michaelis constant (K_m(app)) for starch was 0.71 mg/mL and turnover number (k_cat) was 280 s^?1 in 50 mM sodium acetate buffer, pH 5.5. Thermal inactivation studies at 85 °C showed first-order kinetics with rate constant (k) equal to 0.0063 min^?1. Soybean α-amylase showed high specificity for its primary substrate starch. High similarity of soybean α-amylase with known amylases suggests that this α-amylase belongs to glycosyl hydrolase family 13. Cereal α-amylases have gained importance due to their compatibility for biotechnological applications. Wide availability and easy purification protocol make soybean as an attractive alternative for plant α-amylase. Soybean can be used as commercially viable source of α-amylase for various industrial applications.     

Rumen enzyme profile and fermentation characteristics in sheep as affected by treatment with sodium lauryl sulfate as defaunating agent and presence of ciliate protozoa

The efficiency of sodium lauryl sulfate as a defaunating agent and effect of rumen protozoa on nutrient utilization, fermentation characteristics and enzyme profile were evaluated in adult sheep maintained on a mixed ration containing 65:35% Pala (Ziziphus numularia) leaf: concentrate. Twenty-one adult Malpura sheep divided into three equal groups (DF, RF and F) were either defaunated by oral administration of sodium lauryl sulfate at the rate of 8 g/100 kg body weight (DF), or defaunated and again refaunated (RF), or maintained faunated (F). Daily dry matter intake was similar in defaunated, refaunated and faunated sheep. However, digestibility of cell wall and cell wall contents (NDF, ADF and cellulose) were lower (P < 0.01) in defaunated than refaunated and faunated sheep. Irrespective of the presence or absence of rumen protozoa, daily intake of DCP and DE were similar in the three experimental groups. Even with similar DM, DCP and DE intake, N-retention, blood glucose level, ruminal concentration of total VFA and total-N were higher (P < 0.01), while rumen pH and NH₃-N concentration were lower (P < 0.01) in defaunated sheep. Ruminal activity of amylase, xylanase, protease and urease enzymes were not influenced by presence or absence of ciliate protozoa. However, carboxymethyl cellulase enzyme activity was lower (P < 0.01) in the rumen of defaunated sheep. The total and differential counts of rumen protozoa were similar in refaunated and faunated sheep indicating lack of residual toxic effect of sodium lauryl sulfate. It is concluded that absence of ciliate protozoa increased ruminal TVFA, total-N with lower NH₃-N concentration and fibre digestibility in sheep. Moreover, sodium lauryl sulfate was fully effective for complete removal of rumen ciliate protozoa and successfully defaunated the sheep.     

Effect of by-product gypsum application rate to grass pasture,grass hay,and corn silage on fermentation by rumen microorganisms in continuous culture

A dual-flow continuous culture fermenter system was used to investigate ruminal fermentation in response to increased by-product gypsum application rate of three forages. The treatments included 0, 22, 45, and 90 tonnes/ha by-product gypsum applied to grass plots and 0, 22, and 45 tonnes/ha by-product gypsum applied to corn plots. Forage was harvested to represent grass pasture (GP), grass hay (GH), and corn silage (CS), dried, ground, and fed to fermenters at a rate of 60 g dry matter (DM)/day. Organic matter (OM) and neutral detergent fiber (aNDF) digestibilities, rumen pH, total volatile fatty acid (VFA) production, and N metabolism were not affected by gypsum application rate for all forage types. The GH had greater sulfur content than recommended as the maximum tolerable level by the National Research Council (NRC). The results of this study indicate that ruminal fermentation was not compromised when by-product gypsum was applied to GP, GH, or CS at rates up to 90 tonnes/ha. By-product gypsum application to pastures and crops shows promise as an economical soil amendment to reduce dissolved phosphorus loss in runoff, although potential animal health issues should be further evaluated.     

Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme

Under the optimal conditions, 10 U/ml of glucoamylase was produced by the marine yeast Aureobasidium pullulans N13d. It was noticed that the crude glucoamylase actively hydrolyzed potato starch granules, but poorly digested raw corn starch and sweet potato starch, resulting in conversion of 68.5, 19 and 22% of them into glucose within 6 h of incubation in the presence of 40 g/l of potato starch granules and 20 U/ml of the crude enzyme. When potato starch granules concentration was increased from 10 to 80 g/l, hydrolysis extent was decreased from 85.6 to 60%, while potato starch granules concentration was increased from 80 to 360 g/l, hydrolysis extent was decreased from 60 to 56%. Ratio of hydrolysis extent of potato starch granules to hydrolysis extent of gelatinized potato starch was 86.0% and the hydrolysis extent of potato starch granules by action of the crude glucoamylase (1.0 U/ml) was 18.5% within 30 min at 60 °C. Only glucose was detected during the hydrolysis, indicating that the crude enzyme could hydrolyze both α-1,4 and α-1,6 linkages of starch molecule in the potato starch.     

Debranching enzyme concentration effected on physicochemical properties and α-amylase hydrolysis rate of resistant starch type III from amylose rice starch

The effect of debranching enzyme concentration on physicochemical properties and α-amylase hydrolysis rate of resistant starch type III from high amylose rice starch were studied. The pullulanase enzyme (8, 10, 12, 14 and 16 U/g starch) was introduced to modify amylopectin molecules of 15% (w/w) gelatinized rice starches at 55 °C for 16 h. The debranched starches with different degrees of hydrolysis (0.14–5.27%), and having 66.60–98.82% β-amylolysis limit were then induced at 4 °C for 16 h, afterward a one cycle of freeze–thaw process (?10/30 °C) was applied. The results showed that a pullulanase hydrolysis improved the degree of syneresis (51.64–54.85% from 8 to 16 U/g starch). Resistant starch content increased sharply as the amount of the enzyme increased, reaching the highest (19.81%) for a 12 U/g starch and decreased to 13.16% by 16 U/g starch. α-Amylase hydrolysis rate showed that incompletely-debranched had a lower estimated glycemic index than completely debranched rice starches. Microstructure of the selected RS III samples using X-ray diffraction and scanning electron microscopy revealed a crystal pattern change from A- to V-type pattern and formed a coarse honeycomb-like and a filamentous network structure.