The difference of chemical composition between NFM

and FM (Table 12 and 13) was evaluated. As shown in Table 12, FM had higher CP, EE and ash contents but had lower DM, CF, NDF, ADF and lignin compared to NFM. Bacillus licheniformis used in SSF has cellulolytic enzymes indicating the cellulosic materials in the substrate might be degraded by secreted enzymes. That is the reason why FM had lower NDF and ADF contents than those of NFM.

However, CP and EE of FM were higher compared to NFM. This is probably due to bacterial growth. Selected bacteria used fiber

0.00 2.00 4.00 6.00 8.00 10.00

endo β-glu Xylan

Enzyme activity(U/g)

Enzymes

NFM FM

fat contents came from the growth of Bacillus licheniformis sp..

Increased AA (Table 13) can be explained by the same reason. SSF increased total and several AA (asparagine, threonine, glutamine, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, methionine and cysteine).

Table 12. Chemical composition of NFM and FM (DM basis %)

Item NFM FM

DM 92.45 90.65

CP 39.74 43.54

EE 2.09 3.27

CF 7.21 7.02

Ash 6.50 6.87

ADF 11.57 8.76

NDF 49.72 26.29

Hemicellulose 38.15 17.53

Lignin 2.54 1.23

Ca 0.38 0.26

P 0.82 0.84

Table 13. Total amino acid composition of NFM and FM(%)

Since FM had cellulolytic and hemicellulolytic enzymes, in situ degradation study was conducted to if SSF modified degradation characteristics of substrate used for SSF. FM showed significantly higher DM, CP, NDF and ADF disappearance rate at 3, 6 and 24 hrs incubation (Table 14).

Table 14. DM, CP, NDF and ADF disappearance rate of NFM and FM (%)

Incubation time (hr)

Mean

P-value

0 3 6 12 24 Diet Time Diet *

Time DM

NFM 7.76^b 22.96 26.31^b 33.92^b 42.18^b 26.63^b P<0.01 P<0.01 P<0.01 FM 13.01^a 22.78 29.75^a 39.78^a 57.13^a 32.49^a

CP

NFM 8.62 16.99^b 18.27^b 23.32^b 27.55^b 18.95^b P<0.01 P<0.01 P<0.01 FM 10.98 22.58^a 28.58^a 38.61^a 57.17^a 31.58^a

NDF

NFM 8.42^b 13.94 16.47^b 23.67^b 29.36^b 18.37^b P<0.01 P<0.01 P=0.0107 FM 13.69^a 19.58 22.31^a 34.54^a 39.69^a 25.96^a

ADF

NFM 10.38^b 11.15^b 16.71^b 18.32^b 24.44^b 16.20^b P<0.01 P<0.01 P<0.01 FM 15.05^a 16.52^a 19.25^a 25.40^a 34.23^a 22.09^a

a, b indicates significant difference between NFM and FM at the same time points.

Based on this result we calculated degradation kinetics of DM, CP, NDF and ADF of the experimental diets (Table 15). Fraction a and b of DM and CP increased by SSF indicating potentially degradable part was increased by fermentation. Since the value of a+b fraction of FM were higher than those of NFM, effectively degradable value was also higher in case of FM treatment although NFM had bigger value of ‘c’

which means constant rate of degradation of ‘b’ fraction of NFM was higher. Likewise, all degradation kinetics of CP showed same pattern as for DM in two diets. As shown Table 15, only a fraction and ED value of NDF and ADF was significantly different between NFM and FM (p<0.05). The b fraction and value of c were significantly different between the treatments (p<0.05).

Based on nutrient disappearance rate and their kinetics results, it could be postulated that the mixture of WB and SBM was fermented well by selected bacteria and nutrients in the mixture of WB and were converted to more degradable form by SSF. FM had abundant microbial count which makes it excellent candidate for DFM. The fact that FM contains live microbial, enzymes and more degradable nutrients can be good characteristics of a suitable feed additive as a DFM for ruminants.

Table 15. DM, CP and OM degradation kinetics of the feeds in situ study

Items Feeds a¹ b¹ c¹ a+b¹ ED(k=0.05)²

DM

NFM 9.00^b 33.62^b 0.13^a 42.62^b 33.35^b

FM 13.73^a 66.66^a 0.05^b 80.39^a 44.70^a

SEM³ 1.0884 7.8661 0.0189 8.8279 2.5731

CP

NFM 9.38^b 19.00^b 0.12^a 28.38^b 22.76^b

FM 12.28^a 70.45^a 0.04^b 82.73^a 44.28^a

SEM³ 0.7794 12.2319 0.0182 12.7913 4.8453 NDF

NFM 8.48^b 26.50^a 0.07^a 34.99^b 23.61^b

FM 13.08^a 33.45^a 0.08^a 46.53^a 32.66^a

SEM³ 1.1475 2.2847 0.0061 2.9582 2.0364

ADF

NFM 9.97^b 34.92^a 0.04^a 44.89^a 20.73^b

FM 14.01^a 56.00^a 0.02^a 70.02^a 28.95^a

SEM³ 1.0353 9.1386 0.0123 9.9645 1.9082

1 a= readily soluble fraction, b= insoluble but potentially degradable fraction, c= constant rate of degradation of b (%/h).

2 ED = Effective degradability. Rumen passage rate (k) was considered to 0.02, 0.05 and 0.08.

3 SEM means Standard error for means.

a,b Within the same column, means without a common superscript are significantly different (p<0.05).

EXPERIMENT IV

Effects of Bacillus licheniformis Based DFM on Rumen Fermentation and Microbial Population under in vitro Condition

Introduction

There have been numerous attempts to improve production performance of ruminant animals for last decades. The use of antibiotics like monensin, plant extracts, essential oils, rumen protected nutrients, prebiotics and DFM were some of options used to modify rumen microbial ecosystem for the improvement of ruminal digestion animal performances as well as hind-gut health.

Among them, DFM have received much interests from researchers since the use of DFM might give beneficial effects on rumen (modification of fermentation, the improvement of fiber digestion and antimicrobial effects) and hind-gut (pathogen exclusion by competition, stimulation of immune response) (McAllister et al., 2011). Mostly used microbial additives for ruminants were yeast(Nocek & Kautz, 2006;

Nocek et al., 2003) , lactic acid producing bacteria(Nocek et al., 2002;

Raeth-Knight et al., 2007) and lactic acid utilizing bacteria(Stein et al., 2006).

Recently Bacillus sp. like Bacillus subtilis and Bacillus licheniformis have risen as candidates for DFM source because of some beneficial characteristics of these species. Bacillus sp. can develop spore which is helpful not only for survival in harsh conditions (such as low pH in stomach or heat during feed pelleting process) but also for improved

immune response in the intestine without harmful action (Hong et al., 2005; Sanders et al., 2003). Bacillus licheniformis JK7 was isolated from Korea native goat’s rumen, endoglucanase activity was enhanced by genome shuffling and then DFM was prepared by using SSF in the previous study. The objective of this experiment was to investigate the effects of Bacillus sp. -based DFM on rumen fermentation and rumen microbial population under in vitro condition.

Materials & Methods