Abstract: Banana by-products viz., stem, pseudostem and leaves were evaluated in three sequential runs measured at 8, 12, 24, 48 and 72 h using Hoheneim gas production test on rumen fermentation pattern. The exponential equation of gas produced by Stem is Y = -6.68+56.71 (1-EXP(-0.0153 t-l)) Lag time: 8.2 h, RSD1.09, pseudostem is Y = -11.14+69.27 (1-EXP(-0.0209 t-l}) Lag time: 8.4 h, RSD1.86 and leaves is Y = -3.79+44.58 (1-EXP{-0.0259 t-l}) Lag time 3.4 h, RSD1.40. The acetate propionate ratio was observed to be relatively higher in leaves than stem and pseudostem. The apparent and true dry matter degradability of the stem was significantly (p<0.05) lower than pseudostem and leaves. Pseudostem and leaves were able to generate microbial production at significantly (p<0.05) higher level than stem at 24 and 36 h of incubation, while stem could support significantly (p<0.05) better than pseudostem and leaves later than 48 h of incubation. Inverse relationship existed between YATP and incubation period. Pseudostem had the lowest partition factor compared to stem and leaves. The values range from 2.24 to 4.99. The half time (T½) was 45.46 h for stem, 33.03 h for pseudostem and 26.8 h for leaves. The microbial biomass production, energetic efficiency of Volatile fatty acids and YATP was significantly (p<0.05) highest in leaves when compared to stem and pseudostem at T½ of c. Hence among by-products evaluated for their fodder potential to cattle, banana leaves rank first with low partition factor, high YATP and high microbial biomass followed by pseudostem and banana stem.
INTRODUCTION
Animal agriculture is one of the most important components of global agriculture. However, a major constraint of livestock rearing in tropical countries is chronic feed shortage. Tapping the available fodder resources is one of the options to bridge the gap. Crop residues are the best resources to be explored in this context; as such attempts do not require additional cultivable land. However, very few of fodder have been screened for their use in animal feed. Many more are yet to be evaluated before being used as animal feed. Banana by-products are one among them.
Banana (Musa paradisiaca) is a traditional plant cultivated widely for its fruits. After harvesting of the fruit, the various other parts of the plant (by products) are not effectively utilised. It has been estimated that a residual biomass (pseudostem and leaves) of 13 to 20 tones dry matter/hectare/year is available. Gupta et al. (2001) suggested that feeding of whole banana plants (stem and leaves) will meet the maintenance requirement of cattle. Hembade and Patel (2004) concluded that banana leaves can be incorporated in the diet of kids. However, at present they are thrown out as waste on roadsides, or allowed to rot away in the fields or some times burnt in the field.
In order to throw light on the efficacy of banana by products to serve as a potential source of roughage to ruminants, a study was undertaken to evaluate the various by-products of banana as a source of feed to ruminants through studying the effect of banana plant by products on rumen fermentation pattern.
MATERIALS AND METHODS
The by-products viz., stem, pseudostem and leaves from various banana growing areas in Tamil Nadu state at India were collected as follows:
| • | Banana stem sample was collected from the portion immediately above the rhizome. Banana pseudostem was collected from the ground level to the (1 feet below) portion of the petiole and banana leaves were collected with mid rib excluding petiole. |
The samples were chopped, dried and ground to pass one mm sieve and stored in airtight container.
Evaluation of Fermentation Characteristics of Banana By-Products
In vitro Gas Production
The banana by-products were evaluated using Hoheneim gas production test
as per the procedure of Menke and Steingass (1988) to evaluate their fermentation
characteristics.
In vitro Gas Production Studies
The rumen liquor was obtained from three Jersey crossbred cattle maintained
on grazing alone to ensure that cellulolysis was optimum. All the laboratory
handling of rumen fluid was carried out under continuous flushing with CO2.
Accurately weighed 500 mg samples and 40 mL rumen buffer volume was taken per 100 mL glass syringe for incubation as described by Blummel and Becker (1997) to minimize the analytical error in gravimetric determinations of apparent and truly degraded substances. The syringe nozzle was fastened with butyl rubber cap designed to fit airtight.
Three blanks containing rumen buffer alone were included for each incubation period. The syringes were rotated at one rotation per minute as described by Menke et al. (1979).
Measurements
The syringes were incubated in duplicate in three sequential runs. Gas production
was recorded at 8, 12, 24, 48 and 72 h. Piston of the syringes was reset to
10 mL mark by releasing the gas production whenever it reached the level of
60 mL mark. The release of gas was done by withdrawing the gas with the help
of another syringe by piercing through the butyl rubber cap of the 100 mL syringe
with 23G needle. This step prevents the atmospheric air equilibrating the fermented
gas in the process of releasing the excess gas. Net gas volume at each incubation
hour was calculated by subtracting the mean gas volume of blank from mean gas
volume of syringe with sample.
Characteristic of Gas Production
The results of gas volume at various time intervals were fitted to exponential
equation (Blummel and Orskov, 1993) and modified for lag phase as suggested
by Krishnamoorthy et al. (1991).
The Eq. is:
P = a + b (1 – e–ct) |
| Where | ||
| p | = | Gas production |
| t | = | Time |
| a+b | = | Potential gas production |
| c | = | Rate of gas production |
| a, b and c are constant in exponential equation. With lag phase: P = a+b (1 – e-c(t-l)) Where l = initial lag for on set of fermentation. |
Chemical Analysis
At the end of each incubation hours, the whole contents of the syringes
was transferred into 45 mL capped centrifuge tubes. Residues were centrifuged
in an ultracentrifuge (HIMAC, model SCR 20BA, Hitachi) at 20,000 g for 30 min
at 4°C. 2.5 mL of supernatant was collected for VFA and added into glass
tubes, which already contained 0.5 mL metaphosphoric acid (25%).
Volatile Fatty Acids Estimation
Total and individual short chain fatty acids concentrations were measured
by gas chromatograph method as per the procedure of Chase (1990). Netel make
of gas chromatograph model omega QC was used in this study. The internal standard
used in analysis was 2 ethyl butyric acid.
Column Conditions/Requirements
Stainless steel column of 6 inches x 2 mm internal diameter packed with
10 percent SP 1200/1 percent phosphoric acid and on 30/100 chromosorb W(AW),
was used in the present study of separating C2 to C5 volatile
fatty acids in the sample. Column temperature was maintained at 130°C, inlet
temperature and detector temperature were maintained at 170 and 210°C, respectively.
The flow of carrier nitrogen was kept at 40 mL min-1. The flame ionization
detector was used. The concentrations of volatile fatty acids were calibrated
from the standards.
Stoichiometry Derivations
The following stoichiometrical derivations were considered in this study.
| • | Calculation of CO2 and CH4 produced
from VFA (Wolin, 1960): CO2 (moles) = a/2+p/4+1.5 x b CH4 (moles) = a+2b – CO2 Where a = acetate, b = butyrate and p = propionate. In in vitro gas production, additionally 1 mmol of SCFA produces one mmol of CO2 Volumes (v) were calculated as V = mmol x gas constant x temperature k/pressure (p) |
| • | The relationship between SCFA production and ATP production
(Blummel et al., 1997a) 1 mmol acetate = 2 mmol ATP 1 mmol propionate = 3 mmol ATP 1 mmol butyrate = 3 mmol ATP 1 mmol CH4 = 1 mmol ATP |
| • |
| • | Energy efficiency of VFA = 0.622 A+1.092 P+1.56 B/A+P+2B (where A, P and B represent acetic, propionic and butyric acids (Mol percent) (Orskov et al., 1968). |
In vitro Degradability Studies
The degradability studies on banana by-products were studied at 24, 36,
48 and 72 h.
Apparent Digestibility
The apparent digestibility was determined as per Blummel et al. (1997b).
Analysis of in vitro True Degradability
In vitro true degradabilities were determined by refluxing the pellet
contained in the centrifuge tubes to separate the microbial mass from the undegraded
substrate. To achieve this, the pellet was transferred from the tubes into 600
mL spoutless beakers and the centrifuge tubes were thoroughly washed with about
70 mL neutral detergent solution and reflexed for one hour (Van Soest and Robertson,
1985) which was added to the beakers. True degradability was calculated as the
weight of substrate incubated minus the weight of the residue after NDS treatment.
Partition Factor
Partition factor was calculated using the following formula (Blummel
et al., 1994; Blummel et al., 1997a).
Microbial Biomass
The microbial biomass was calculated from the equation quoted by Blummel
et al. (1997a).
Microbial biomass = Substrate truly degraded-Substrate apparently degraded.
Efficiency of Microbial Production
The efficiency of microbial production was calculated using the following
formula:
YATP
YATP was calculated using the following formula:
RESULTS
Evaluation of Degradation Characteristics and Rumen Fermentation Pattern
of Banana By-products by in vitro Studies
The cumulative gas produced (mL) for 500 mg incubation of substrate viz.,
banana stem, banana pseudostem and banana leaves for various hours of incubation
is furnished in Table 1. The description of the gas produced
is furnished through exponential equation as follows:
Stem: |
Y = -6.68+56.71 (1-EXP(-0.0153 t-l)) Lag time: 8.2 h, RSD 1.09, |
Pseudostem: |
Y = -11.14+69.27 (1-EXP(-0.0209 t-l}) Lag time: 8.4 h, RSD 1.86, |
Leaves: |
Y = -3.79+44.58 (1-EXP{-0.0259 t-l}) Lag time: 3.4 h, RSD 1.40, |
| Table 1: | Effect of banana by-products (500 mg) on various in vitro parameters and stoichiometric derivations at various hours of incubation (Mean±SE) |
| Mean of six measurements; Means bearing different superscript within column at concerned incubation hour differ significantly (p<0.05), *Not estimated as it is inappropriate | |
The cumulative gas volumes from incubation of 500 mg of banana by-products ranged from 30.66 to 42.5 mL. The rates © of gas production ranged from 1.5 to 2.6% per hour.
Studies on End Products and Their Stoichiometric Derivations
The mean effect of banana stem, banana pseudostem and banana leaves on in
vitro synthesis of mean short chain fatty acids (mmol L-1), CO2
to CH4 ratio, ATP produced (mmol L-1), percent apparent
and true degradabilities, microbial biomass production, efficiency of microbial
production, YATP production and partition factors (mg/mL) at various
h of incubation are presented in the Table 1.
The molar proportion of VFA across incubation period and across banana by-product studies ranged form 84 to 89% acetate, 1 to 5% propionate and 5 to 14% butyrate. The banana by-products were relatively acetogenic. Among the short chain fatty acids, acetate production in banana leaves was consistently lower than stem and pseudostem (p<0.05) for most of the incubation periods studied. The butyrate production in stem and pseudostem increased steeply from 2.82 and 2.91 mmol L-1 at 24 h to 17.05 and 18.43 mmol L-1 at 72 h, respectively. The corresponding yield (5.23 to 7.84 mmol L-1) differed significantly (p<0.05) from leaves. The propionate production in leaves was significantly (p<0.05) lower than stem and pseudostem from 24 to 72 h of incubation.
The acetate propionate ratio was observed to be relatively higher and consistent at 16.82 to 11.54 for 8 to 72 h in leaves than stem and pseudostem. whereas stem and pseudostem showed wide variations ranging from the ratio of about 20.0 at 8 h of incubation to the ratio of about 6.0 at 72 h of incubation.
The CO2 to CH4 ratio remained similar for all the three substrates. The ATP production (mmol L-1) for the three substrates revealed a spurt at 24 h of incubation for pseudostem and leaves whereas stem recorded gradual elevation from 8 to 36 h.
The apparent as well as the true dry matter degradability of the three substrates at 36, 48 and 72 h of incubation revealed a significantly (p<0.05) lower degradability for stem, compared to pseudostem and leaves. However, at 24 h of incubation, the three substrates had significantly (p<0.05) different true digestibilities of 4.45, 7.81 and 12.71 for stem, pseudostem and leaves respectively. Consequently, the microbial yield also showed similar significant difference at 24 h incubation period. The microbial biomass production at 36 h showed a different pattern with stem being significantly (p<0.05) lower than pseudostem and leaves and no significant difference was observed at 48 h incubation, whereas at 72 h of incubation, stem had maximum microbial production followed by pseudostem and significantly (p<0.05) lowest in leaves.
The YATP calculated based on the efficiency of microbial production
per unit of ATP produced revealed an inverse relationship between YATP
and incubation period. The partition factor derived from the substrate truly
degraded per mL of gas produced indicated pseudostem had the lowest partition
factor when compared to stem and leaves. The values range from 2.24 to 4.99.
| Table 2: | Effect of banana by-products on the ratios of Acetate and Propionate, CO2 to CH4, energetic efficiency, apparent, true digestibility, partition function, ATP production, microbial biomass production, efficacy of microbial production and YATP produced at half time (hours of c during incubation (Mean±SE per 500 mg of each substrate) |
| Numerical before the substrate denote the half time (h) of c during incubation; Mean of six measurements; Means bearing different superscript within row differ significantly (p<0.05) | |
Keeping in view of Blummel and Lebzien (2001) suggestion to evaluate diets at diet specific time (half time) instead of uniform time interval, the generated data in this study was compared according to their half time of c.
The half time (T½) was found to be 45.46 h for stem, 33.03 h for pseudostem and 26.8 h for leaves. Therefore, the data generated at 48 h for stem, 36 h for pseudostem and 24 h for leaves were compiled for relative values viz., ratio/efficiencies and presented in Table 2. The relative values were given more weightage for the purpose of comparison. Stem had significantly (p<0.05) highest acetate to propionate ratio, lowest energetic efficiency of VFA than pseudostem and leaves. The energetic efficiency was significantly (p<0.05) highest in leaves and CO2:CH4 ratio was lowest in leaves. Similarly the microbial biomass production, energetic efficiency of VFA and YATP was significantly (p<0.05) highest in leaves when compared to stem and pseudostem at T½ of c.
DISCUSSION
Evaluation of Degradation Characteristics and Rumen Fermentation Pattern
of Banana By-Products by in vitro Studies
The initiation of gas production varied with substrate, banana leaves at
3.4 h banana stem at 8.2 h and pseudostem at 8.4 h. Even though, leaves produced
gas at 3.4 h itself, subsequent measurement of gas at 48 and 72 h revealed lower
cumulative gas production than banana pseudostem but higher than banana stem.
However, the cumulative gas production in leaves was significantly (p<0.05)
lower than pseudostem and significantly (p<0.05) higher than banana stem
at 72 h of incubation.
The observed cumulative gas production measurements were in agreement with Brenda Keir et al. (1997) and Ly et al. (1997) for banana leaves. The description of gas production for banana leaves through Y = A+B (1 – e-ct-l) also in close agreement with those described by Ly et al. (1997). However, literature reviewed did not provide information on the gas production values for banana stem and banana pseudostem.
The proximate principles and fibre fractions of the substrates viz., banana stem, banana pseudostem and banana leaves (Amarnath, 2002) do not explain the different course of the gas production by the three substrates, except for banana stem having lower organic matter resulting in delayed onset as well as lower gas production. However, mathematical modelling predicts higher potential gas production in stem than leaves.
Spurt in ATP production (mmol L-1) at 24 h of incubation for pseudostem and leaves may probably indicate the optimal incubation duration required for the concerned substrate to express its nutrient delivery to the host.
The in vitro apparent dry matter digestibility values for stem (20.02%) at 48 h incubation obtained in this study found to be lower than the IVDMD value observed by various authors (Geoffroy et al., 1978; Pieltain et al., 1999). Similarly the in vitro apparent dry matter degradability values (24.02%) of pseudostem at 48 h also found to be lower than the values reported by various authors (Viswanathan et al., 1989; Pieltain et al., 1999).
The dry matter apparent digestibility value (24.16%) of leaves at 48 h of incubation period in this study found to be lower than the values 61.7% as reported by Shem et al. (1995). The in vitro apparent digestibility results obtained in this study were lower than the in vivo results of degradability of pseudostem (Viswanathan et al., 1989) and leaves (Shem et al., 1995). The true dry matter digestibility for banana stem observed in the study was in dis-agreement with Goeffroy et al. (1978) who reported IVDMD for banana stem as 78-82% and with Pieltain et al. (1999) with the value of 68.4% IVOMD. Similarly, the IVDMD and IVOMD values reported by various authors (Viswanathan et al., 1989; Pieltain et al., 1999) were higher than the values obtained in this study for pseudostem as well as for leaves reported by Pezo and Fanola (1980).
The only agreement with the reported values was Pieltain et al. (1999) who reported that IVOMD for banana leaves were as 35.2%. The in vivo digestibility figures reported for stem (Pieltain et al., 1999), for pseudostem (Ffoulkes and Preston, 1978) and leaves (Reddy and Reddy, 1991; Shem et al., 1995) were all higher than the corresponding values obtained in this study. Variety difference could only be the possible reason between reported and observed values.
The efficiency of microbial production based on the microbial production for truly degradable substrate indicated the pseudostem and leaves were able to generate microbial production at significantly (p<0.05) higher level than stem at 24 and 36 h of incubation. The stem was able to support microbial production significantly (p<0.05) better than pseudostem and leaves later than 48 h of incubation.
Banana leaves had significantly (p<0.05) highest microbial biomass production, energetic efficiency of VFA and YATP compared to stem and pseudostem at T½ of c. Since T½ of c is the right period to judge the relative merit of the various resources, among the three banana by-products evaluated for their fodder potential to cattle, banana leaves rank first with low partition factor, high YATP and high microbial biomass followed by pseudostem and banana stem in that order.
ACKNOWLEDGMENT
The authors gratefully acknowledge the Tamil Nadu Veterinary and Animal Sciences University authorities for the facilities rendered in conducting this study as a part of M.V.Sc Thesis.