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Use of central composite design to optimize working conditions of Streptomyces griseus enzymatic method in estimating in vitro rumen undegraded crude protein of feedstuffs
- A. Gallo, P. Fortunati, S. Bruschi, G. Giuberti, F. Masoero
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- Journal:
- The Journal of Agricultural Science / Volume 156 / Issue 1 / January 2018
- Published online by Cambridge University Press:
- 24 January 2018, pp. 100-109
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The aim was to identify optimized combinations of Streptomyces griseus protease concentration (CONC), incubation length (TIME), or amount of crude protein (CP) incubated in buffered enzymatic solution (CPW) to predict the in vitro rumen-undegraded feed CP (RUP) of 26 different feeds (soybean, rapeseed or sunflower meals, wheat bran, distillers dried grains with solubles, maize co-products and alfalfa hay). Different levels of CONC (0.08, 0.19, 0.44, 0.69 and 0.80 enzymatic units [U] of S. griseus protease/ml), TIME (6, 10, 18, 26 and 30 h) and CPW (69, 118, 235, 353 and 401 mg CP) were tested in agreement with a central composite design (CCD) with four replications of the central point to calculate second-order polynomial equations of main tested effects. The RUP was estimated by incubating samples in a buffered rumen fluid for 16 h or by adopting different enzymatic approaches as planned a priori in CCD. Differences between rumen and enzymatic RUP (ΔRUP) were estimated and regression terms of second-order polynomial equations for estimating ΔRUP were calculated between and within feeds. These equations were optimized using the non-linear generalized reduced gradient method with the objective set at ΔRUP equal to 0. The adoption of CCD permitted identification of optimized enzymatic combinations of CONC (0.12 U of S. griseus protease/ml), TIME (18 h) and CPW (from 233 to 458 mg CP for distillers dried grains with solubles and soft white wheat bran, respectively) to predict RUP accurately in all feed categories except for soybean meal, where optimized combinations were 0.47 U of S. griseus protease/ml, 18 h and 435 mg CP.
Study of the effects of PR toxin, mycophenolic acid and roquefortine C on in vitro gas production parameters and their stability in the rumen environment
- A. GALLO, G. GIUBERTI, T. BERTUZZI, M. MOSCHINI, F. MASOERO
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- Journal:
- The Journal of Agricultural Science / Volume 153 / Issue 1 / January 2015
- Published online by Cambridge University Press:
- 01 May 2014, pp. 163-176
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Moulds belonging to Penicillium section roqueforti are common contaminants of feedstuffs and produce several mycotoxins that can cause health hazards when ingested by farm animals. Among these, PR toxin (PR), mycophenolic acid (MY) and roquefortine C (RC) have been frequently detected in forages, particularly silages. The aims of the current trials were to study the effects of the presence of pure mycotoxins on in vitro rumen fermentation parameters and to assess their stability in the rumen environment. Two successive in vitro gas production experiments were carried out: a central composite design with four replications of central point (CCD) and a completely randomized design with a fully factorial arrangement of treatments (FFD). In CCD, the effects of PR, MY and RC concentrations in diluted rumen fluid (i.e. 0·01, 0·30, 1·01, 1·71 and 2·00 μg of each mycotoxin/ml) were tested. Gas volume produced after 48 h of incubation (Vf) decreased linearly as concentrations of RC and MY in diluted rumen fluid increased, with marginal effects similar for two mycotoxins, being respectively −14·6 and −13·4 ml/g organic matter (OM) for each 1·0 μg/ml of increment in mycotoxin concentration. Similarly, total volatile fatty acid (VFA) production decreased quadratically as concentrations of RC and MY increased, with marginal effects about two times higher for MY than RC, being −4·22 and −2·62 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration. With respect to maximum Vf (i.e. 410·6 ml/g OM) and VFA (98·06 mmol/l) values estimated by the model, decreases of 13·6 and 15·2% were obtained when incubating the highest RC and MY concentrations, respectively. The PR did not interfere with rumen fermentation pattern and it was not recovered after 48 h of incubation, whereas the stabilities of MY and RC in rumen fluid were similar and on average equal to about 50%. On the basis of CCD results, a second experiment (FFD) was carried out in which only effects of MY and RC concentrations (i.e. 0, 0·67, 1·33 and 2·00 μg of each mycotoxin/ml of diluted rumen fluid) were tested. Data from FFD showed Vf decreased linearly when concentrations of MY and RC increased, with marginal effect two-folds higher for MY than for RC (−11·1 ml/g OM and −6·7 ml/g OM, respectively). Similar marginal effects of MY and RC in decreasing VFA production were recorded: −2·38 and −2·86 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration, respectively. At the highest RC and MY tested concentrations, Vf and VFA decreased by 8·7 and 10·7%, respectively, over maximum estimated values. In FFD, the average amounts of MY and RC recovered in rumen fluid after 48 h of incubation were 79·0 and 40·6%, respectively. In conclusion, the MY and RC from standards interfered with rumen microorganisms at relatively low levels and were partially stable in the rumen environment after 48 h of incubation. These findings suggested that MY and RC could interfere with digestive processes and might represent a potential risk for ruminants fed diets containing feeds contaminated by mycotoxins produced by P. roqueforti.
In vitro production of short-chain fatty acids from resistant starch by pig faecal inoculum
- G. Giuberti, A. Gallo, M. Moschini, F. Masoero
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The need to improve the knowledge of fermentation processes within the digestive tract in pigs is growing, particularly for ingredients that may act as potential prebiotic sources, such as resistant starch (RS). A study (based on enzymatic digestion followed by in vitro fermentation) was conducted to investigate whether various sources of RS, obtained from eight native starches characterized by inherent heterogeneous starch chemistry and structure, can influence short-chain fatty acid (SCFA) concentrations and relative production kinetics. Total and individual SCFA productions were evaluated over time and up to 72 h of incubation. The in vitro hydrolysis of native starches allowed a classification from very high [⩾650 g/kg dry matter (DM)] to low (<50 g/kg DM) RS amount. The total SCFA production was similar between ingredients, whereas acetate and butyrate molar ratios in the SCFA profile differed (from 0.48 to 0.56 and from 0.17 to 0.25, respectively; P < 0.05). Differences in fermentation kinetic parameters for total and individual SCFA productions were observed (P < 0.05). Considering the total SCFA production after 72 h of incubation, the time at which half of the maximum production has been reached (T1/2), the maximum rate of production (Rmax) and its time of occurrence (Tmax) differed between ingredients (P < 0.05), with values ranging from 6.1 to 11.9 h, from 0.459 to 1.300 mmol/g DM incubated per hour and from 5.1 to 9.8 h, respectively. Overall, a similar trend was observed considering individual SCFA productions. In particular, T1/2 ranged from 6.4 to 12.5 h, from 5.5 to 12.5 h and from 6.7 to 11.3 h for acetate, propionate and butyrate, respectively (P < 0.05). For Rmax, differences were obtained for propionate and butyrate productions (P < 0.05), whereas no difference was recorded for acetate. In summary, our findings indicated that both quantitative and qualitative production of SCFA and related kinetics were influenced by fermentation of RS obtained from native starches characterized by heterogeneous starch characteristics. Current findings are based on an in vitro approach, and thus require further in vivo validations.
Plasma glucose response and glycemic indices in pigs fed diets differing in in vitro hydrolysis indices
- G. Giuberti, A. Gallo, F. Masoero
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Different dietary starch sources can have a great impact in determining starch digestion potential, thus influencing the postprandial blood glucose response. Our objectives were to define: (i) the incremental plasma glucose response in pigs fed diets containing various sources of starch differing in in vitro digestion patterns, (ii) the in vivo glycemic index (GI) values for the same diets, (iii) the possible relationship between in vitro and in vivo data. Diets, formulated with 70% of starch from five heterogeneous sources, were characterized in depth by using two distinct in vitro evaluations. The first one was based on the Englyst-assay for nutritional classification of starch fractions, whereas the second one was based on a time-course multi-enzymatic assay up to 180 min from which the hydrolysis indices (HIs) were calculated and used as a link between the physicochemical properties of starch from diets and the in vivo responses. For the in vivo study, five jugular-catheterized pigs (35.3 ± 1.1 kg body weight) were fed one of the five diets for 6-day periods in a 5 × 5 Latin square design. On day 5, blood was collected for 8 h postprandially for evaluating glucose appearance. On day 6, blood was collected for 3 h postprandially for the estimation of the GI. Starchy diets differed for rapidly digestible starch (from 8.6% to 79.8% of total starch (TS)) and resistant starch contents (from 72.5% to 4.5% of TS). Wide between-diets variations were recorded for all the kinetic parameters and for the HI calculated from the in vitro digestion curves (P < 0.05). On the basis of the obtained HI, diets contained starch with a very low to a very high in vitro digestion potential (ranging from 26.7% to 100.0%; P < 0.05). The glucose response differed among diets (P < 0.05), with marked differences between 15 and 120 min postprandial. Overall, the ranking of incremental glucose appearance among diets agreed with their in vitro HI classification: high HI diets increased plasma glucose response more (P < 0.05) than low HI diets. Lastly, different in vivo GIs were measured (ranging from 30.9% to 100.0%; P < 0.05). The relationship between HI and GI showed a high coefficient of determination (R2 = 0.95; root mean square error (RMSE) = 15.8; P < 0.05). In conclusion, diets formulated with starches with a wide range in HI potential can strongly affect the postprandial glucose response in pigs.