Effects of Different Dietary SC/NSC Ratios on in vitro Fermentation Characteristics of Degraded Distilled Grain

YU Qunlian WANG Zhisheng* XUE Bai WU Dan WANG Lizhi

(1.Institute of Animal Nutrition,Sichuan Agricultural University,Ya’an 625014,China；2.Key Laboratory for Animal Disease-resistance Nutrition of China Ministry of Education,Ya’an 625014,China)

The output of distilled grain is more than 21 million tons each year in China[1].It is a nutritional feed stuff resource for beef cattle due to its high content of crude protein and various kinds of bioactive compounds,but it also has a high proportion of lignin which affects palatability and efficiency[1-2].Microbial pretreatment employs lignin degrading microorganisms such as white rot fungi to soften the lignocellulosic structure of plant biomass,making it more amenable for subsequent enzymatic or acid hydrolysis.Some white rot fungi have been reported to selectively degrade lignin leaving the cellulose of the wood virtually untouched[3],and thus making them ideal for microbial pretreatment of lignocellulosic biomass.Phanerochaete chrysosporium is a kind of white rot fungus which produces unique extra-cellular oxidative enzymes(lignin peroxidase,and manganese peroxidase)that can degrade lignin[4].Carbohydrate and protein are the major nutrients of supporting microbial growth,and based on data from both in vitro and in vivo studies,there is a general agreement that the digestion rate of carbohydrate is the major factor of controlling the energy availability for microbial growth[5].Although the protein of degraded distilled grain was improved,the fermentable carbohydrate was consumed during fermentation.Therefore,the objective of this study was to investigate the effects of adding soluble starch on the in vitro fermentation characteristics of degraded distilled grain.

1 MATERIALS AND METHODS

1.1 Experimental materials

The distilled grain was purchased from Ya’an Chunjing Liquor Factory.The main components are corn,rice chaff and rice hulls,which accounted for about 50%.Phanerochaete chrysosporium was provided by Sichuan University.Rumen fluid was from beef cattle(2 years old,450 kg BW).Soluble starch was purchased from Chengdu JinshanChemical Reagent Co.,Ltd.The major composition of soluble starch to provide non-structural carbohydrate(NSC)was corn starch.Double distilled water was used throughout the experiment.

1.2 Equipments

In vitro gas production(GP)was determined in 100 mL glass syringes and the minimum scale was 1 mL.There were soft silicone rubber tubes(about 3.5 cm)onto the front of the glass syringes and clamped end to prevent leakage.Washing and drying the syringes before using,and coating evenly with a small amount of vaseline around the piston.Use an automatic constant temperature water-bath to simulate rumen environment around 39℃.

1.3 Experimental design

Adding soluble starch changed the ratio of structural carbohydrate(SC)/NSC into 1.66,2.06,2.46,2.86,3.26 and 3.66,respectively(Table 1).There was a control group that no sample was added but fermentation fluid.Five replicates were made for each group.

Table 1 The amounts of fermentation substrates and corn starch (DM basis,mg)

1.4 Experimental methods

1.4.1 Degraded lignin of distilled grain

Prepared the liquid culture medium which is full of spore and enzymatic activity of Phanerochaete chrysosporium.The concentration of spore in the liquid culture medium was ensured about 5×105CFU/mL.The lignin of distilled grain was degraded by Phanerochaete chrysosporium at 38℃for 20 days.The moisture of culture was 65%,and the inoculum size was 10%.

1.4.2 In vitro GP

Fermentation substrates were weighed accurately into the culture glass syringes.Incubation medium composition followed the method of Menke et al.[6]and the component proportion of rumen fluid and artificial saliva was 1∶2.The incubation medium was placed in a water-bath to maintain at 39℃with continuous flux of carbon dioxide,and then 30 mL of incubation medium was added into the glass syringes,and the amounts of cumulative GP were recorded at 0,2,4,6,8,12,24,36,48 and 72 h.The data of cumulative GP were corrected through deducting the influence of control group.

1.5 Sampling and measurement

The nutrient composition of untreated and degraded distilled grain was determined according to AOAC methods[7].The dry matter(DM)content was determined by drying at 105℃for at least 24 h.The crude protein(CP)content was determined by Kjeldahl method and the ether extract(EE)content was determined by Soxhlet method.The contents of acid detergent fibre(ADF),acid detergent lignin(ADL)and neutral detergent fibre(NDF)were determined according to van Soest et al.[8].The content of neutral detergent insoluble nitrogen(NDIN)was analyzed according to Licitrd et al.[9].The contents of SC and NSC were calculated by SC(%)=NDFNDIN and NSC(%)=100-(CP+EE+NDF+ASH-NDIN).After 72 h incubation,the fermentation medium was used to analyze ammonia nitrogen(NH3-N),volatile fatty acid[VFA,including total volatile fatty acid(TVFA),acetate,propionate and butyrate]and microbial protein(MCP)concentrations and pH.GP data were analyzed by using curve-fitting programme.NH3-N concentration was determined by the method of Feng et al.[10].Acetate,propionate,butyrate and TVFA concentrations were estimated by gas chromatograph(CP-3800GC,Varian).The pH was determined by pH meter(PHS-3D)and MCP concentration was determined by the method of Makkar et al.[11].

1.6 Statistical analysis

The data of the experiment were analyzed by the method of One-way ANOVA(SPSS 16.0),and the means were compared for significance by LSD at the P＜0.05 or P＜0.01.The linear model(y=b1x+b2)was used to describe the relationship between cumulative GP(y)and SC/NSC ratio(x),and the quadratic model(y=b0+b1x+b2x2)was used to describe the relationship between MCP(y)and SC/NSC ratio(x).

2 RESULTS

2.1 Nutrient composition of untreated and degraded distilled grain

The ADL content of distilled grain was reduced by 16.4%(P＜0.01)and the CP content of distilled grain was increased by 6.04%(P＜0.01)as a result of degradation by Phanerochaete chrysosporium(Table 2).The ADF and NDF contents of distilled grain were also significantly decreased(P＜0.01)by degradation.

Table 2 Nutrient composition of untreated and degraded distilled grain (DM basis,%)

2.2 Effects of different SC/NSC ratios on in vitro GP parameters of degraded distilled grain

As shown in Table 3,the cumulative GP of degraded group was significantly lower than that of untreated group(P＜0.01).The linear correlation between cumulative GP and SC/NSC ratio was formulated as follow:y=-1.262x+18.611(R2=0.940,P＜0.01).With the increase of SC/NSC ratios,the cumulative GP was linearly decreased(P＜0.01).There was no significant difference on the rate of GP between degraded and untreated groups(P＞0.05).The lower the ratio of SC/NSC,the higher the rate of GP(P＜0.05).The lag time of GP was not significantly affected by deg-radation and SC/NSC ratio(P＞0.05).

Table 3 Effects of different SC/NSC ratios on in vitro GP parameters of degraded distilled grain(after 72 h)

2.3 Effects of different SC/NSC ratios on in vitro fermentation parameters of degraded distilled grain

As shown in Table 4,the MCP,NH3-N and TVFA concentrations were significantly affected by degradation and SC/NSC ratio(P＜0.01).Compared with the degraded group,adding soluble starch to change the SC/NSC ratio could significantly increase the MCP concentration(P＜0.01).When the SC/NSC ratio was 2.46,the concentration of MCP was the highest.Through regression analysis to MCP(y),the optimal SC/NSC ratio(x)was 2.39(y=―0.826x2+3.947x+1.189,R2=0.926,P＜0.01).The NH3-N concentration of degraded group was significantly higher than that of untreated and lower SC/NSC ratio(1.66 and 2.06)groups(P＜0.05).When the SC/NSC ratio was 1.66,the concentration of TVFA was the highest,and significantly higher than that of other groups(P＜0.01).Adding soluble starch to change the SC/NSC ratio could significantly increase the propionate/TVFA(P＜0.05),but reduce the acetate/propionate(P＜0.05).No significant differences on pH,acetate/TVFA and butyrate/TVFA were found among all groups(P＞0.05).

Table 4 Effects of different SC/NSC ratios on in vitro fermentation parameters of degraded distilled grain(after 72 h)

3 DISCUSSIONS

3.1 The effects of distilled grain degraded by Phanerochaete chrysosporium

As the results showed that the content of ADL was reduced by fermentation and the content of fibre was also declined by the growth of microorganism,but the content of protein was increased with the MCP concentration increasing.Therefore microbial pretreatment could improve the nutritional value of distilled grain.Huang et al.[12]showed that under the optimal condition,the degradation rate of lignin and crude fibre of rice straw reached 42.97%and 35.39%,respectively.Reducing the lignin content of biomass help to expose the highly-ordered crystalline structure of cellulose and facilitates substrate accessed by hydrolytic enzymes.Liu etal.[13]showed that co-culture two kinds of white rot fungi to degrade corn stalk could degrade 21.3%of crude fiber,and improve 35.6%of CP.Wang et al.[14]showed that Phanerochaete chrysosporium could degrade ADL of distilled grain by 5.93%and improve CP by 13.20%.The above results were consistent with the results of this study.It was concluded that the distilled grain fermented by Phanerochaete chrysosporium could reduce the negative effects of lignin on the digestibility.

3.2 Effects of different SC/NSC ratios on in vitro GP parameters of degraded distilled grain

GP techniques were based on the principle of anaerobic microbial digestion of carbohydrate to gas(CO2and CH4,primarily)and VFA.Menke et al.[15]showed that high correlation was found between in vitro GP and in vivo apparent digestibility.GP is a comprehensive reflection of the degree of fermentation and rumen microbe activity.Distilled grain fermented by Phanerochaete chrysosporium reduced the amount of fermentable carbohydrate that affected the GP parameters.Adding soluble starch to change SC/NSC ratio increased the content of fermentable organic matter,the activity of rumen microbe,and the amount of GP.Wang et al.[16]showed that higher amount of GP was in the treatments of high starch,and the treatments of high cellulose had lower amount of GP.Results presented by Chai et al.[17]indicated that the majority of starch degradation normally occurred between 6 and 24 h,and concluded the fermentation of starch and other non-soluble components occurred simultaneously.The 1ag time of GP was positively related to the hemicellulose content.And if the content of fiber in the substrate was higher,the in vitro fermentation would be lagged.Whereas the content of NSC in the substrate was higher,the in vitro fermentation would be quickly start-up[18].But the lag time of this experiment had no significant influence.In this experiment,the in vitro fermentation may also be affected by other factors,such as microbial types and different contents of rumen fluid.

3.3 Effects of different SC/NSC ratios on in vitro fermentation parameters of degraded distilled grain

The results showed that a high proportion of NSC may lead to ruminal microbe fermentation producing more VFA.With the soluble starch inclusion increasing,the proportion of acetate was decreased,and the proportion of propionate was increased.These were similar to the reports of other studies[19-20].Ruminal fermentation resulted in redistribution of feed organic matter into microbial mass,VFA and gas,which were in variable proportions.Chen et al.[21]showed that there was a linear positive correlation between acetate production and the amount of cellulose,and a linear positive correlation between propionate production and the amount of soluble carbohydrate.Sutton et al.[22]reported that increasing the content of starch in concentrate resulted in higher rumen propionate concentration.McCarthy et al.[23]indicated that increasing the content of ruminal fermentable starch enhanced the TVFA concentration.

NH3-N was regarded as the most important nitrogen source for microbial protein synthesis in rumen.Suitable concentration of ruminal NH3-N could stabilize rumen ecology and improve microbial protein synthesis.The content of crude protein in degraded distilled grain was higher than that in untreated distilled grain which led to higher NH3-N concentration of in vitro fermentation.Moreover,adding suitable amount of soluble starch not only increased the utilization of NH3-N,but also balanced the carbon and nitrogen proportion of substrate.Satter et al.[24]showed that the concentration of NH3-N in media of in vitro fermentation ranging from 20 to 50 mg/dL could ensure the fastest growth of microorganisms.Adding suitable amount of soluble starch increased the content of VFA,and accelerated the synthesis of microbial protein.

MCP was a response of the level of fermentation.With the proportion of NSC increasing,the supply of ferment able substrate for ruminal fermentation was increased.Ruminalmicrobial growth and protein synthesis are dependent on adequate supplies of degradable protein and available carbohydrate[5].Stokes et al.[25]showed that in continuous culture studies,increasing the proportion of nonstructural carbohydrate(NSC)and reducing the NDF of diets could result in higher yield of microbial protein and digestibility of carbohydrate.Feng et al.[26]reported that using excessive NSC,or a combination of high NSC and rapidly degraded NDF could depress ruminal microbial yield.

The pH of rumen fluid is a comprehensive reflection of fermentation process for it affects the amount and activity of ruminal microbe.In this experiment,the rumen pH of all groups was within the range from 6.0 to 7.0,which had been reported as an optimal range for microbial digestion of fiber and protein.It also showed that adding an appropriate amount of soluble starch would not cause acidosis.

Tan[27]reported that the ruminal fermentation could achieve the best efficiency when the SC/NSC was about 2.64.Starch degradation rate was an important factor to determine the microbial ecology of the rumen.The exact responses in the partitioning of organic matter in the rumen depend on class and digestibility of carbohydrate as well as dynamic properties,such as degradation rate and the interaction between ferment able carbohydrate and nitrogen compounds[28].Zhou[29]showed that in the condition of equivalent nitrogen,the efficiency of microbial protein synthesis was affected by SC/NSC ratio.Only to ensure the appropriate ratio of carbohydrate,the synthesis of microbial protein can have a higher efficiency.

4 CONCLUSIONS

①The microbial pretreatment using Phanerochaete chrysosporium could reduce the lignin content of distilled grain,and improve the nutritional values and digestibility of distilled grain.

②In vitro cumulative GP of degraded distilled grain was poorer than that of the untreated group for the consumption of ferment able organic matter in the process of microbial pretreatment.

③Adding a suitable amount of soluble starch could improve the fermentation characteristics of degraded distilled grain,and the optimal SC/NSC ratio for in vitro fermentation was 2.39.

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