Field of Invention:
The present invention relates to a process to prepare cellulase and protease enzyme complex cud liquor from cattle and buffaloes cud for biodegradation of organic materials.
Background of the invention:
For studying biodegradation of Agro Industrial byproducts (organic materials), rumen liquor based in vitro system (Two stage method of Tilley and Terry, 1963) is commonly used through out the world. The prerequisite of rumen liquor collection in this method needs modified "RUMEN FISTULATED" cattle and buffaloes and such surgical operation "RUMEN FISTULATION" is a cruel act under the Prevention of Cruelty to Animal Act, 1960. An alternative method of rumen liquor based biodegradation is in situ method, where Agro Industrial byproducts is placed in the Nylon bag and inserted through the rumen fistula in the rumen fermentation vat (0rskov and MacDonald, 1979). There is a wide variation of end biodegradation results (in terms of In vitro dry matter and crude protein disappearance and degradability) from one series to another or from one laboratory to the other, even after vigorously standardizing the diet of the animals, the sampling conditions and the inoculum preparation (Barnes, 1967,Omedetal. 1989).
Biodegradation of Agro Industrial byproducts, municipal wastes and hospital wastes (organic materials) depends on the reproducibility of end results and such expectation is possible in only enzyme based biodegradation vessel (Jones and Hayward, 1975, MacQueen and VanSoest, 1975, and De Boever et al 1986).
It is a well-established fact that only enzyme based biodegradation vessel could
yield reproducible and fermentation results. Throughout world at present crude
fungal cellulase preparation (Trichoderma viride) manufactured by Sigma Aldrich family, located at USA / European subcontinent, is used as this enzyme has inherent nature of cellulolytic and proteolytic activity but India could not afford such high cost foreign exchange involved commodity for use in biodegradation fermentation vessel.
The object and summary of the invention
The object of this invention is to explore the possibility of using natural source cud from cattle and buffaloes as source of microbial enzyme for use in biodegradation fermentation vessel and thereby economize the cost of biodegradation of organic materials.
To achieve the said objective this invention provides a process of preparing cellulase and protease enzyme complex cud liquor from cattle and buffaloes cud for biodegradation of organic materials comprising:
Step 1 - feeding cattle / buffaloes green leguminous / non-leguminous
fodder ad libitum, Step 2 - withdrawing the feed from said cattle/buffaloes and wait till said
cattle/buffalo is ruminating, Step 3 - inserting loop in the mouth of said cattle / buffaloes, Step 4 - allowing said loop till the cud is embedded on said loop, Step 5 - repeating steps 3 & 4 with different loops and different cattle /
buffaloes, Step 6 - collecting said loops from the mouth of said cattle / buffaloes,
and Step 7 - dipping said loops embedded with cud in at least one litre buffer
nutrient solution to obtain cellulase and protease concentrate
cud liquor.
The feeding of fodder is carried out for 2-3 hours.
The said loop is a fodder loop (having stem and leaves), iron loop or cotton thread loop.
The loop remains in the mouth for at lest 15 -20 minutes for embedding the cud.
The buffer nutrient solution comprises mixing solution no. 1 and solution no. 2 in the ratio of 100:1 wherein: solution no. 1 is:
a) Disodium Hydrogen phosphate - 3. 7g
b) Sodium Bicarbonate 9.80g
Mix both chemicals 'a' and 'b' and make volume - one litre with ammonia free distilled water, and solution no. 2 is
a) Sodium Chloride 4.7g
b) Potassium Chloride 5.7g
c) Calcium Chloride 0.40g
d) Magnesium Chloride 0.60g
Make volume 100 ml with ammonia free distilled water.
At least 4 different loops are inserted in four cattle / buffaloes to get cellulase and protease complex concentrate cud liquor.
The quantity of buffer nutrient solution depends upon the number of cattle/buffaloes used to get cellulase and protease complex concentrate cud liquor.
The instant invention is different from the conventional process in following ways:
(i) In the cud liquor preparation, very minutely minced animal feed particles are present which are evenly distributed while in conventional process (rumen liquor) coarsely Animal feed particles are present therefore concentration of microbial enzymes is more per unit undigested feed particles in cud liquor.
(ii) In conventional rumen liquor process there is an immense need to infuse C02 for 20 minutes to maintain strictly anaerobic conditions, while in the instant process CO2 infusion is not required thereby cost of processing is curtailed leading to the huge saving in organic materials biodegradation.
(iii) In testing undigested organic material is to be separated by using sintered glass crucibles (G 1) in conventional rumen liquor based fermentation vessel while in the instant invention we require whatman filter paper No.54 thereby contributing to a huge saving in organic materials biodegradation.
(iv) In the instant invention there is no need of maintaining modified rumen fistulated cattle and buffaloes, which is itself a very costly affair.
Detail description of 51 samples tested is given below:
Batch I: (India)
Groundnut cake, Mustard cake, Soybean meal, Fish meal, Cotton seed, Cotton seed cake, Maize, Barley, Rice polish and Gram churi
Batch II: (India)
Mustard Cake - 10 different locations samples, Cotton Seed cake - 10 different locations samples
Batch III: (Germany)
Mustard cake - 10 different locations samples, Soybean Meal - 8 different locations samples, Maize Gluten Feed-3 different locations samples.
FLOW CHART FOR TESTING BIODEGRADABILITY OF AGRO INDUSTRIAL BYPRODUCTS
(11 Feeds with 51 samples)
In the 1st batch, weigh agro industrial byproducts sample approximately
250 mg. (In triplicate in centrifuge tube).
In 2 batch, repeat same process as mentioned under first batch.
In the 3rd batch, blank centrifuge tube is prepared without any substrate.
• Weigh about 2g sample in the preweighed moisture cup and keep it in the
oven for 18 hours at 80°C for estimating dry matter (AOAC, 1990).
Add 25ml cud liquor in the first and third batch, simultaneously add 25 ml buffer cellulase (Sigma-Aldrich family) solution in the second batch.
Incubate all the three batches centrifuge tubes in the incubator at 39°C for 48 hours.
After the first stage of 48 hours incubation, remove centrifuge tubes of all the three batches from the incubator and add 2 ml of 6N HC1 and 0.2g pepsin (1:10000) powder in each tube. Incubate all these nine tubes for 2nd stage of 48 hours duration.
• Remove all the centrifuge tubes (nine numbers) after second stage of
incubation.
Centrifuge all the tubes, about 2000 gms. for 15 minutes and supernatant is decanted.
Weigh filter paper (whatman No. 54) after keeping for 12 hours at 100°C in the oven and place this filter paper inside the funnel.
• Filter the contents of the tube after washing with warm water 80°C, six
times at least.
Keep the filter paper along with washed material (undigested residue) for drying at 70°C for 48 hours at least.
• Weigh filter paper and dried material (undigested residue) after keeping
in desiccator for 20 minutes.
Preserve undigested residue and filter paper for nitrogen estimation by Kjeldahl method (AOAC, 1990)
CALCULATIONS
(a) In vitro dry matter disappearance per cent (IVDMD%)
Samples dry matter weight - residual dry matter weight - residual dry matter weight of blank Sample dry matter weight
(b) Effective Ruminal dry matter/protein degradability per cent (ED%)
(0rskov and MacDonald, 1979)
(bxc)
P = (a+ )xl00
(c+k)
P= Proportion of dry matter/protein degraded
a= Cold water extractable N/DM as decimal of total N/DM
b= Slowly degradable N/DM as decimal of total N/DM
c= Rate of change constant for "b" fraction or degradation rate k= Ruminal passage rate or ruminal out flow rate
RESULTS
The results related to the biodegradability of dry matter/ protein with respect to Mustard cake (India & Germany), Soyabean meal (India and Germany) and Cotton seed (India) as well as other agro-industrial byproducts were calculated and are given in Tables 1 to 4 for all the 51 samples of 11 different agro-industrial byproducts.
The efficacy of biodegradation fermentation vessel is measured in terms of In vitro dry matter/protein disappearance and degradability and data are presented in Tables 1 to 4. It is quite clear from the perusal of data that cud liquor based fermentation vessel yielded results of In vitro dry matter/protein disappearance and degradability at par with the conventional rumen liquor based. In situ method, as published in an internationally recognised book (AFRC, 1993). In the case of Mustard Cake, effective degradability percent of crude protein (ED%) assessed by In situ method (Sampath, 1990) was 69, while the instant invention based fermentation vessel yielded 74 and 72 ED% using cattle/buffaloes cud liquor respectively. Another testing material Cottonseed Cake also yielded results of ED% on same lines confirming required activity of cellulase and protease in cud liquor of instant invention.
The data related to In vivo nitrogen degradability per cent estimates for Soyabean meal, Groundnut cake and Fish meal based on In vivo study were compared with the figures obtained by cud liquor (cattle/buffalo) based fermentation vessel of instant invention. It is worth noting that cellulase and protease present in cud liquor is equally effective in biodegrading Agro
Industrial by products as it has been reported on the basis of In vivo study presented in Table 5.
The proteolytic activity of gram negative bacilli enriched cud liquor (cattle/buffalo) vis-a-vis rumen liquor (cattle/buffalo) was tested using casein protein (98.5% CP) and results of crude protein disappearance are presented through pie diagram, as shown in Figure 1 of the accompanying drawings. The overall value of casein crude protein disappearance per cent under In vitro fermentation vessel based on cellulase (Thchoderma Viride), cud liquor (cattle/buffalo), rumen liquor (cattle/buffalo) was 95.51 ± 0.597, 93.60 ± 0.344, 93.44 ± 0.639, 95.1 ± 0.748, 95.39 ± 0.518, respectively. In general, casein protein disappearance was significant (P<0.05) higher with rumen liquor as compared to cud liquor, but the values in both the cases were within the range of published figures (ARC, 1984, Sampath, 1990, Harris and Mitchell, 1941, Hembryetall975).
The concentration of cellulase unit (one unit will liberate 1.0 micro mole of glucose from cellulose in one hour at pH 5.0 at 37° C, 02 hrs incubation time) in cud liquor vis-a-vis rumen liquor are presented in pie diagram, as shown in Figure 2. It was observed that concentration of cellulase in cud liquor and rumen liquor was within the requirement limit as recommended by Jones and Hayward (1973), De Boever etal (1986).
A prediction equation was fitted between dry matter (x) and crude protein (y)
using data obtained by cud liquor (cattle/buffalo) based fermentation vessel,
overall turnover (b) was 0.395 (cattle) and 0.437 (buffalo), yielding prediction
percent (y) value 101 (cattle) and 99.79 (buffalo).
Cattle, Y = 51.434 + 0.395X, r2= 0.608, Sxy% = 2.970
Buffaloes, Y = 50.350 + 0.437X, r2 = 0.643, Sxy% = 3.323
The value of prediction per cent indicate the fitness of cud liquor based fermentation vessel for testing biodegradation of organic materials.
Advantages of the instant invention
1. Cud from cattle and buffaloes is an inexpensive natural source of crude cellulase and protease complex and could replace imported fungal cellulase (Trichoderma Viride) preparation from Sigma - Aldrich family, Sweden.
2. The cud cellulase and protease complex could be used in bioremediation process thereby may be of great assistance to convert and recycle wastes - Municipal and Hospital garbage etc. thereby protecting environmental pollution and adding extra income to the Municipality/Hospitals.
3. Cud liquor may be used as rumen modifiers - optimize rumen function specially under acidosis and anorexia conditions and pharmaceutical industry could make use of this invention and capsule after lypholisation and ultrafiltration could be prepared for treating Anorexia suffering cattle/ buffaloes.
4. This invention is an alternative of rumen fistulation which is banned by Ministry of Social Justice and Empowerment vide notification dated 15 December 1998 under the prevention of cruelty to Animal Act, 1960 (59 of 1960) and provide valuable solution to protect the cattle and buffaloes from the cruel act of rumen fistulation.
5. Cud liquor (cattle/buffalo) having cellulase + protease complex could be used as growth promoter in the Swine ration and present invention provides substitute of imported growth promoter.
6. Cud liquor (cattle/buffalo) could work as a flavouring media for enhancing feed intake of Animal Feed in mash form, pellet form or compact feed block. This is a good substitute of imported costly flavouring agent.
Table : 1 Ruminal Protein degradability, Crude Protein (CP) Rumen degradable protein (RDP) and undegradable
protein (UDP) Content of Indian & Germany Animal feeds

(Table Removed)
N.B., 1. Detail of 11 feed samples is given on page 5 of this specification
2. DM, Dry matter
3. AFRC, Agricultural Food Research Council, U.K., 1993
Table : 2 Rumlnal Dry matter and protein degradation of some feed ingredients (Figures obtained by C.L. based in vitro method vis-a-vis values of other methods)

1. (Table Removed)
Table 25 Ruminal Dry matter and protein degradation of some feed ingradients (Figures obtained by C.L. based in Xfitro method vis-a-vis values of other methods.

(Table Removed)
Table *^ Ruminal Dry matter and protein degradation of some feed ingradients (Figures obtained by C.L. based in vitro method vis-a-vis values of other methods)

(Table Removed)

Table5Nitrogen degradability (%) estimates for soybean meal groundnut cake and fish meal
(Comparison of In-viuo* vis-a-vis In-uitro C.L. based method)

(Table Removed)
SIDDONS, R.C., PARADINE, J., GALE, D.L. and EVANS, R.T. (1985) Estimation of the degradability of dietary protein in the sheep rumen by in uiuo and in uilro procedures. British Journal of Nutrition 54: 545-561.
MARY, P.F., KLOPFENSTEIN, T. and BRITTON, R.A. (1985) Evaluation of laboratory Techniques for predicting Ruminal Protein degradation. J. Dairy Sci., 68: 829-39.

claim:
Recess of preparing cellulase and protease enzyme complex cud liquor from cattle and buffaloes cud for biodegradation of organic materials comprising: Step 1 - feeding cattle / buffaloes green leguminous / non-leguminous
fodder ad libitum, Step 2 - withdrawing the feed from said cattle/buffaloes and wait till said
cattle/bu is ruminating, Step 3 - inserting loop in the mouth of said cattle / buffaloes, Step 4 - allowing said loop till the cud is embedded on said loop, Step 5 - repeating steps 3 & 4 with different loops and different cattle/
buffaloes, Step 6 - collecting said loops from the mouth of said cattle / buffaloes,
and Step 7 - dipping said loops embedded with cud in at least one litre buffer
nutrient solution to obtain cellulase and protease concentrate.
A process as claimed in claim 1 wherein the feeding of fodder is carried out for 2-3 hours.
A process as claimed in claim 1 wherein said loop is a fodder loop (having stem and leaves), iron loop or cotton thread loop.
A process as claimed in claim 1 wherein the loop remains in the mouth for at lest 15 -20 minutes for embedding the cud.
5. A process as claimed in claim 1 wherein the buffer nutrient solution
comprising mixing solution no. 1 and solution no. 2 in the ratio of 100:1
wherein:
solution no. 1 is:
a) Disodium Hydrogen phosphate - 3. 7g
b) Sodium Bicarbonate 9.80g
Mix both chemicals 'a' and 'b' and make volume - one litre with ammonia free distilled water, and solution no. 2 is
a) Sodium Chloride 4.7g
b) Potassium Chloride 5.7g
c) Calcium Chloride 0.40g
d) Magnesium Chloride 0.60g
Make volume 100 ml with ammonia free distilled water.
6. A process as claimed in claim 1 wherein at least four different loops are inserted in four cattle/buffaloes to get cellulase, protease concentrate.
7. A process as claimed in claim 1 wherein the quantity of buffer nutrient solution depends upon the number of cattle/buffaloes used to get cellulase, protease concentrate cud liquor.
8. Cellulase and protease enzyme complex cud liquor whenever prepared by the process as claimed in the preceding claims.
9. A process of preparing cellulase and protease enzyme complex cud liquor from cattle and buffaloes cud for biodegradation of organic materials
substantially as herein described with reference to the accompanying drawings.
10. Cellulase and protease enzyme complex cud liquor substantially as herein described with reference to the accompanying drawings.