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1.
A method of analysis based on hydrolysis with cellulase provides a simple and fast measurement of the improvement in nutritive value achieved by the chemical treatment of cellulosic waste products. The method was adapted for use with alkali-treated straw, and a comparison made with digestibility measured in vitro. The effects of the following were determined: cellulase source, enzyme concentration, pH, incubation time and incubation temperature. There were appreciable differences in the activity of the various cellulase preparations, and activity measurements are necessary if the cellulase source is altered. Variations from 0.5 to 1.5 g enzyme and 24 to 48 h incubation time had no appreciable effects, provided the cellulase was completely soluble. Citrate buffer was preferable to acetate buffer, and with a concentration of 1 molar it was not necessary to adjust the pH of the sample, even with an 8% sodium hydroxide concentration in the straw. The incubation temperature was not critical, though a temperature of 40°C ± 5°C is preferable. The standard deviation of the method was for a single determination ± 0.56 (double determination (s) = ± 0.40). (n = 46). The correlation between enzyme solubility and digestibility in vitro was good (r = 0.91).  相似文献   

2.
A study was made of the accuracy of predicting dry matter digestibility in vivo from the dry matter disappearance of forage samples in pepsin (48 h) followed by Onozuka cellulase (48 h). Forty-five samples representing four tropical pasture species, Cenchrus ciliaris, Chloris gayana, Digitaria spp., Setaria spp. and one temperate grass, Lolium perenne were used.Dry matter digestibility in vivo was significantly correlated with the disappearance in pepsin-cellulase (r = 0.94) and the regression predicting dry matter digestibility in vivo had a residual standard deviation of ± 2.7 digestibility units. Separate regressions for the five grasses had RSD values ranging from ± 1.3 to ± 2.6.It was concluded that the digestibility in vivo of both tropical and temperate grasses in the range 40–76% digestibility could be accurately predicted by the pepsin-cellulase assay, provided samples of known digestibility in vivo similar to those being tested were included as standards in each run.  相似文献   

3.
Low exoglucanase and endoglucanase activities of marine Aspergillus niger cellulase decreased the hydrolyzing ability of cellulase. To increase the activity of halostable cellulase obtained from a marine A. niger, a cellulase with endoglucanase and exoglucanase activity was efficiently expressed by constructing a vector with promoter glaA. Exoglucanase and endoglucanase activities increased from 0.21 and 4.51 U/ml of the original strain to 0.89 U/ml and 15.12 U/ml of the transformant, respectively. Filter paper activity (FPA) increased by 7.1 folds from 0.63 to 4.47 U/ml. The release of glucose by hydrolysis of wheat straw with cellulase from the transformant was 1.37 folds higher than that with cellulase from the original strain under high salinity condition. Cellulase with endoglucanase and exoglucanase activities could be well expressed in marine A. niger. The cellulase from the transformant not only showed higher activity, but also retained halostability. An appreciate proportion of β-glucosidase, exoglucanase, endgolucanasein cellulase was important for hydrolyzing cellulose.  相似文献   

4.
Glucose oxidase (from Aspergillus niger), glucoamylase (from Rhizopus spp.), and cellulase (from Aspergillus niger) of fungal origin are all glycosylated proteins. Dehydration of the three enzymes to a range of water potentials did not affect their activity. However, when more than 10% of the carbohydrate associated with the molecules was removed by periodate oxidation, the enzymes were highly susceptible to dehydration when compared with oxidized controls. Polyvinyl pyrrolidone and Dextran T500 protected the three enzymes in their oxidized state against the effects of dehydration.  相似文献   

5.
W. Schliemann  G. Adam 《Phytochemistry》1982,21(6):1438-1439
The hydrolysis of plumieride to form the aglycone plumieridine was catalysed by a commercial preparation of cellulase from Aspergillus niger.  相似文献   

6.
Residual feed intake (RFI) is an alternative measure of feed efficiency (FE) and is calculated as the difference between actual and expected feed intake. The biological mechanisms underlying animal-to-animal variation in FE are not well understood. The aim of this study was to investigate the digestive ability of beef cows selected for RFI divergence as heifers, using two contrasted diets. Fifteen 4-year-old beef cows were selected from a total of 69 heifers based on their RFI following the feedlot test. The selected heifers were ranked into high-RFI (+ 1.02 ± 0.28, n = 8) and low-RFI (−0.73 ± 0.28, n = 7), and a digestibility trial was performed after their first lactation. Both RFI groups were offered two different diets: 100% hay or a fattening diet which consisted of a DM basis of 67% whole-plant maize silage and 33% high starch concentrates over four experimental periods (two per diet). A diet effect was observed on feed intake and apparent digestibility, whereas no diet × RFI interaction was detected (P > 0.05). Intake and apparent digestibility were higher in cows fed the fattening diet than in those fed the hay diet (P < 0.0001). DM intake (DMI) and organic matter apparent digestibility (OMd) were repeatable and positively correlated between the two subsequent periods of measurements. For the hay and fattening diets, the repeatability between periods was r = 0.71 and r = 0.73 for DMI and r = 0.87 and r = 0.48 for OMd, respectively. Moreover, both intake (r = 0.55) and OMd (r = 0.54) were positively correlated (P < 0.05) between the hay and fattening diets. Significant differences between beef cows selected for divergence in RFI as heifers were observed for digestive traits (P < 0.05), DM and organic matter (OM) apparent digestibility being higher for low-RFI cows. Overall, this study showed that apparent digestibility contributes to between-animal variation in FE in beef cows.  相似文献   

7.
Twenty-six winter wheat forages (cultivars Slepjner, Hussar and Cadenza) harvested at three stages of maturity in each of two years, were conserved with or without Maxgrass additive or with urea (20 or 40 g kg−1 dry matter; DM) in 200 l barrels. The forages were analysed for in vivo digestibility in wethers, chemical composition, in vitro rumen fluid-pepsin digestibility, in vitro neutral detergent-cellulase plus gamannase digestibility (NCGD), in vitro fermentation gas production and in situ rumen degradability. Forages were also scanned using near-infrared reflectance spectroscopy (NIRS) and calibration equations developed for predicting in vivo digestibility. In vivo digestible organic matter content (DOMD) was poorly predicted by cell wall content (r2≤0.19), NCGD (r2≤0.41), rumen fluid DOMD (r2≤0.41), rumen degradability (r2≤0.44) and in vitro gas production (r2≤0.26). Although crude protein content was a better predictor (r2≤0.48), the relationship differed (P<0.05) with the year of harvest of the forages. In contrast, NIRS was a more accurate and consistent predictor of DOMD in vivo (r2=0. 87). This study indicates that traditional laboratory-based feed evaluation techniques are unsuitable for predicting the DOMD of WCW, but that NIRS holds promise. However, as only 26 forages were used to derive the calibration equation, further research is required using large (150) data sets to validate the promise shown by NIRS and enable its adoption by the advisory services.  相似文献   

8.
A set of 180 forages (47 alfalfa hays, 26 grass hays, 52 corn silages, 35 small grain silages and 20 sorghum silages) were randomly collected from different locations of the Po Valley (Northern Italy) from 2009 to 2010. The forages were characterised for chemical composition (11 parameters), NDF digestibility (five parameters) and net energy for lactation (NEL). The latter was calculated according to the two approaches adopted by the 2001 Nutrient Research Council and based on chemical parameters either alone (NEL3x-Lig) or in combination with 48 h NDF degradability in the rumen (NEL3x-48h). Thereafter, a principal component analysis (PCA) was used to define forage populations and limit the number of variables to those useful for obtaining a rapid forage quality evaluation on the basis of the calculated NEL content of forages. The PCA identified three forage populations: corn silage, alfalfa hay and a generic population of so-called ‘grasses’, consisting of grass hays, small grain and sorghum silages. This differentiation was also confirmed by a cluster analysis. The first three principal components (PC) together explained 79.9% of the total variation. PC1 was mainly associated with protein fractions, ether extract and lignin, PC2 with ash, starch, NDF and indigestible NDF (iNDF) and PC3 with NDF digestibility. Moreover, PC2 was highly correlated to both NEL3x-Lig (r = −0.84) and NEL3x-48h (r = −0.94). Subsequently, forage-based scores (FS) were calculated by multiplying the original standardised variables of ash, starch, NDF and iNDF with the scoring factors obtained from PCA (0.112, −0.141, 0.227 and 0.170, respectively). The FS showed a high determination coefficient for both NEL3x-Lig (R2 = 0.86) and NEL3x-48h (R2 = 0.73). These results indicate that PCA enables the distinction of different forage classes and appropriate prediction of the energy value on the basis of a reduced number of parameters. With respect to the rumen in situ parameters, iNDF was found to be more powerful at discriminating forage quality compared with NDF digestibility at different rumen incubation times or rates of NDF digestion.  相似文献   

9.
To test the suitability of Onozuka 3S cellulase as a replacement for the superceded Onozuka SS (P1500) for estimating forage digestibility, a comparison was made using 50 grass and 30 legume samples of known dry matter digestibility in vivo (means of 55.4% and 58.4%, respectively).Mean solubility of dry matter (DMS) following acid pepsin pretreatment and Onozuka 3S or SS (P1500) was 51.3 and 52.8%, respectively for grasses, and 60.2 and 60.7%, respectively, for legumes. The differences in DMS between the two cellulases for both grasses and legumes were not significant (P>0.05).Regressions were calculated relating DMD in vivo to the DMS in pepsin-cellulase with the two cellulase preparations. Errors for the Onozuka 3S and SS (P1500) were ± 2.6 and ± 2.7, respectively, for grasses, and ± 2.6 and ± 2.9, respectively, for legumes.It was concluded that 3S is a satisfactory replacement for SS (P1500) for estimating forage digestibility.  相似文献   

10.
The dried herbage of early and late cut lucerne, red clover, Italian and perennial ryegrasses and timothy was offered in the long form or three processed forms at three levels of feeding (20, 30 and 40 g DM/kg LW) to store lambs in two experiments. Faecal output of organic matter and cell walls was determined.The potential digestibility of the cell walls of these feeds was determined in vitro. The depression in the cell wall digestion from this potential, observed in vivo, was related to water soluble carbohydrate content (WSC) and buffering capacity (BC) of the forages, their fineness of grinding (MF) and the level of feeding (LF).Uniform processing altered the chemical composition of all forages and produced a wide range of MF in the different forages.The digestion of the cell content fraction of the diets was not influenced by herbage species, fineness of grinding or level of feeding.The digestion of organic matter and cell wall was depressed by increasing LF and decreasing MF (P < 0.001) and the interaction between herbage species and processing was significant (P < 0.001).WSC and LF were positively correlated (r = +0.706 and +0.611), and BC and MF were negatively correlated (r = ?0.498 and ?0.498), with the depression observed in cell wall digestion.These four parameters, together with their significant interaction terms (WSC × BC), (WSC × LF) and (BC × MF), accounted for 95.1% of the variance observed in the depression of cell wall digestion and provided a basis for a hypothetical model of factors affecting cell wall digestion.  相似文献   

11.
《New biotechnology》2008,25(6):437-441
Fungal cellulases are well-studied enzymes and are used in various industrial processes. Much of the knowledge of enzymatic depolymerization of cellulosic material has come from Trichoderma cellulase system. Species of Trichoderma can produce substantial amounts of endoglucanase and exoglucanase but very low levels of β-glucosidase. This deficiency necessitates screening of fungi for cellulytic potential. A number of indigenously isolated fungi were screened for cellulytic potential. In the present study, the kinetics of cellulase production from an indigenous strain of Aspergillus niger MS82 is reported. Product formation parameters of endoglucanase and β-glucosidase (Qp + Yp/s) indicate that A. niger MS82 is capable of producing moderate to high levels of both endoglucanase and β-glucosidase when grown on different carbon containing natural substrates, for example, grass, corncob, bagasse along side purified celluloses. Furthermore, it was observed that the production of endoglucanase reaches its maximum during exponential phase of growth, while β-glucosidase during the Stationary phase. Enzyme production by solid-state fermentation was also investigated and found to be promising. Highest production of cellulase was noted at pH 4.0 at 35 °C under submerged conditions. Growth and enzyme production was affected by variations in temperature and pH.  相似文献   

12.
A study was made of the accuracy of predicting dry matter digestibility (DMD) in vivo of 32 legume samples and 50 grass samples from the dry matter disappearance (DMS) of the samples in pepsin (48 h) followed by Onozuka cellulase (48 h). The effects of grinder screen size, incubation temperature and sample size on the accuracy of predicting dry matter digestibility were examined.The DMD in vivo of legumes and grasses was significantly correlated with DMS at 39° C (r = 0.91 and 0.94) and increasing the incubation temperature to 50° C had no effect on the correlations, although DMS was greater. There was no significant difference between the regression equations for grasses and legumes. The regression equation for the combined 82 samples had an RSD of ± 3.0 digestibility units. The corresponding RSD for regressions based on fermentation with rumen fluid in vitro was ± 2.6 digestibility units. Grinding the grass samples through a 0.4 mm screen prior to analysis resulted in regressions with a larger RSD (± 3.3) than when a 1.0 mm screen was used to prepare the samples (P < 0.001).It was concluded that the digestibility in vivo of both legumes and grasses could be predicted by the pepsin-cellulase method by using the same equation, but to eliminate biased results, samples of known digestibility in vivo similar to those being tested should be included as standards in each study. The analytical time could be reduced by 48 h if shorter incubation periods (24 h) were used, but only at the expense of slightly larger errors in predicting DMD in vivo (P < 0.01).  相似文献   

13.
The specificities of acid proteinases from Aspergillus niger, Aspergillus saitoi, Rhizopus chinensis, Mucor miehei, Rhodotorula glutinis, and Cladosporium sp., and that of swine pepsin, were determined and compared with ability of the enzymes to activate trypsinogen. Various oligopeptides containing l-lysine, Z-Lys-X-Ala, Z-Lys-(Ala)m, Z-Lys-Leu-(Ala)2, and Z-(Ala)n-Lys-(Ala)3 (X = various amino acid residues, m = 1–4, n = 1–2) were used as substrates. Of the enzymes which are able to activate trypsinogen, most split these peptides at the peptide bond formed by the carbonyl group of l-lysine. For the peptides to be susceptible to the enzymes it was essential that the chain extended for two or three amino acid residues on the C-terminal side of the catalytic point, and that a bulky or hydrophobic amino acid residue formed the imino-side of the splitting point. The rate of hydrolysis was markedly accelerated by elongation of the peptide chain with l-alanine on the N-terminal side of the catalytic point. Thus, of the substrates used, Z-(Ala)2-Lys-(Ala)3 was the most susceptible to the microbial acid proteinases possessing trypsinogen activating ability. On the other hand, M. miehei enzyme and pepsin, which do not activate trypsinogen, showed very little peptidase activity on the peptides.  相似文献   

14.
Samples of 1 kg of wheat straw, oat straw and paspalum hay were separated manually into botanical fractions, and the three largest fractions of each forage were analysed chemically for cell-wall constituents, silica and nitrogen. Proportions of digested dry matter, cellulose and hemicellulose, and potential digestibility for each of these major botanical fractions were determined when chaffed samples were placed in nylon bags and incubated for 12, 24, 48, 72 and 96 h in the rumen of sheep fed on lucerno. Cross-sections of botanical fractions were stained with safranin and fast green, and proportions of lignified tissue determined by light-microscopy and planimetry.Large differences in dry-matter digestibility between wheat straw and oat straw were attributed to the different proportions of botanical fractions. Within forages, stem was the largest fraction, the most lignified and had the lowest potential digestibility. Proportions of digested dry matter from botanical fractions at 12 h were poorly correlated with lignin content of dry matter (r = 0.25) but at 72 h were negatively correlated with lignin content of dry matter (r = ?0.84, P<0.01) and with proportions of lignified tissue (r = ?0.67, P<0.05) in the respective botanical fractions. Proportions of cellulose and hemicellulose digested at 72 h were strongly correlated with lignin content of cell walls (r = ?0.90, P<0.01; r = ?0.85, P<0.01, respectively). Proportions of lignified tissues were less closely correlated with all measurements of digestibility than were proportions of lignin in cell walls determined chemically. Development of a technique for measuring intensity of lignification might enhance the value of light-microscopy measurements.  相似文献   

15.
Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, Km (15.6260 g/L) and the rate constant of deactivation, Kde (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, Vmax (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.  相似文献   

16.
This study investigated the effects of, and interactions between, dietary grain source and marginal changes in alfalfa hay (AH) particle size (PS) on digestive processes of dairy cows. A total of eight Holstein dairy cows (175 days in milk) were allocated in a replicated 4 × 4 Latin square design with four 21-day periods. The experiment was a 2 × 2 factorial arrangement with two levels of theoretical PS of AH (fine = 15 mm or long = 30 mm) each combined with two different sources of cereal grains (barley grain alone or barley plus corn grain in a 50 : 50 ratio). Results showed that cows consuming diets supplemented with corn had greater dry matter and nutrient intakes (P < 0.01), independent of forage PS. In addition, the apparent digestibility of fiber fractions was greater for diets supplemented with corn (P = 0.01). The feeding of barley grain-based diets was associated with greater apparent digestibility of non-fiber carbohydrates, and this variable was even greater when long AH was fed (P = 0.04). Moreover, the feeding of long AH resulted in longer time spent eating (P = 0.03) and higher pH (P < 0.01), as well as a tendency for higher acetate-to-propionate ratio in the rumen fluid (P = 0.06) at 3 h post feeding. In conclusion, the results indicated that the marginal increase of PS of AH may prolong eating time and improve rumen fermentation, particularly in diets based on barley grain. Partial substitution of barley grain by corn can improve feed intake and fiber digestibility in mid-lactation dairy cows.  相似文献   

17.
Two experiments were undertaken using the in vitro gas production technique of Theodorou et al. [Anim. Feed Sci. Technol. 48 (1994) 185] to compare rumen liquor (RL) and faeces (FA) as inocula for fermenting gramminaceous forages over 96 h periods. Experiment 1 used 12 forages of differing in vivo digestibility (ammonia treated wheat straw, field-cured hay (Lolium perenne) and 10 artificially dried grasses (L. perenne) harvested at different maturities). Experiment 2 used seven maize-silage based forages (whole plant, stover, leaf, lower stem, middle stem, upper stem and husk). In both experiments, rumen liquor and faeces were obtained from two cows in early lactation, each fed daily with 9.4 kg DM of grass silage and 9.0 kg DM of concentrate. Rumen contents were sampled through the fistula, before morning feeding; faeces were sampled from the rectum, immediately afterwards. Rumen liquor (250 ml) was prepared by straining contents through two layers of muslin, adding the solids after blending with 250 ml of buffer and re-straining. Faeces were prepared by mixing (300 ml) with 150 ml of buffer and straining through two layers of muslin and adding a homogenate of the solids and 150 ml of buffer after straining. Data were fitted to the model of France et al. [J. Theor. Biol. 163 (1993) 99]. All model parameters showed FA to have a poorer fermentation capacity than RL. In both experiments, potential gas production volumes (A) were lower (on average 52.9 ml (18.5%)) and lag times longer (on average 2.9 h) for FA compared to RL. Fractional rate of fermentation at half asymptote (T/2) was generally greater for RL than FA (overall means, 0.042, 0.028) and the time required to T/2 being less (overall means, 21.9, 35.4 h). However, potential gas production (A) was highly correlated between RL and FA: Experiment 1 (r2=0.94, 11 forages, excluding ammonia treated straw) and Experiment 2 (r2=0.83, six forages, excluding middle stem). In Experiment 1, organic matter digestibility in vivo (OMDIV) was also highly correlated with both OMDFA (r2=0.77, 11 forages) and OMDRL (r2=0.89, 11 forages); OMDRL and OMDFA were also highly correlated (r2=0.81). Similar correlations occurred in Experiment 2. It is concluded that faeces have potential as an alternative inoculum to rumen liquor for in vitro gas production techniques, but methods of overcoming the longer lag phase with faeces require further research.  相似文献   

18.
Coffee pulp was subjected to a solid-state fermentation process, using Aspergillus niger. The initial moisture content of the pulp, as well as the fermentation time and temperature, had a significant effect on the increase in total amino acid content of the material. The increase in total amino acids showed a significant correlation with the dry matter recovered (r = −0.98) and the increase in pH during the process (r = 0.98). With a moisture content of 80%, a pH of 3.5, a temperature of 35°C, and an aeration of 8 liters/min per kg as fermentation conditions, it was found that the maximum concentration of total amino acids was attained after 43 h. The fermented product had a higher total amino acid content and a lower cell wall constituent value (primarily cellulose and hemicellulose) than the original pulp. A growing chicken's ration containing 10% of the fermented product had a feed efficiency (2.14) similar to that of the standard ration (2.19) and was significantly better than that of the diet containing 10% of the original pulp (2.53). The difference observed in feed intake and weight gain between the standard diet and that with 10% of the fermented product is considered to be due to palatability factors which should be studied further.  相似文献   

19.
The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.  相似文献   

20.
The impact of fibre level and fibre source on digestibility, gastrointestinal tract (GIT) development, total tract mean retention time (MRT) and growth performance was studied in indigenous Mong Cai (MC) and exotic Landrace × Yorkshire (LY) pigs. The diets were based on maize, rice bran, soyabean meal, fish meal and soyabean oil, and cassava residue (CR) or brewer's grain (BG) as fibrous ingredient sources in the high-fibre diets (HF) and were fed ad libitum. A low-fibre diet (LF), containing around 200 g NDF/kg dry matter (DM), was formulated without CR and BG as feed ingredients. The HF diets (HF-CR and HF-BG) were formulated to contain around 270 g NDF/kg DM. The experiment was arranged as a 2 × 3 factorial completely randomized design with six replications, and lasted 27 days. Increased dietary fibre level resulted in a reduction (P < 0.05) in average daily gain, digestibility of organic matter (OM), CP and gross energy (GE) at the ileum and in the total tract, and in MRT, and an increase (P < 0.05) in the feed conversion ratio and in the weight of the GIT (except for small intestine and caecum). The coefficients of total tract digestibility of fibre fractions were higher in HF diets than in the LF diet, with highest values for diet HF-CR, which had a high proportion of soluble non-starch polysaccharides. MC pigs had longer MRT of digesta than LY pigs (P < 0.05), resulting in higher digestibility at the ileum and in the total tract. Across diets and breeds, the total tract apparent digestibility of OM, CP and GE was positively related (R2 = 0.80 to 0.84) to the MRT of solids, whereas the MRT was negatively related to the DM intake (R2 = 0.60).  相似文献   

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