Rennet is an aspartyl protease extracted from the Abomasm 4th part of the ruminant Stomach of the suckling calves is secreted in its natural form as an inactive precursor, prochymosin, which is irreversibly converted into active chymosin through limited proteolysis. Rennet is used as the milk clotting agent rennet in cheese manufacture. This enzyme possesses a very high milk clotting activity with a low proteolytic activity and this makes chymosin particularly suitable for the manufacture of cheese. Extraction of milk Rennet were obtained from the Abomasums of the milk fed calf slaughtered between the age of 10-30 days old. These rennet will extracted by the different method i.e 5%, 10%, 15 %20 % Nacl solution, Acidified whey method ,Ultrasonic Extraction the rennet solution obtained from different method and check their activity commercially available rennet
Need of the Project:
Pakistan is the fourth milk producing country in the world with gross production of 46.44 million tons (GOP, 2010-2011) milk. In Pakistan, milk is mainly consumed in liquid form and very small portion is converted into local dairy products. Cheese is the major milk product in Europe and USA, while its production in Pakistan is at a very limited scale. Cheese can be made from cow and buffalo milk after coagulation of milk protein casein by the use of enzyme (Rennet).
Rennet is the most important factor that affects the cheese quality. Nowadays almost all types of cheese are manufactured by clotting the milk with a milk clotting enzyme and then processing the curd in various ways. For this purpose, many enzymes with different sources i.e. from plants, animal tissues and microorganisms are widely used. Cheese prepared from this type of sources has several advantages as compared to those produced by acid clotting methods. Since they are ripened under controlled conditions for a specific time, these type of cheese has a longer shelf life and be more popular with consumers due to their unique characteristics such as texture, color, taste and aroma (1-4).
Chymosin is definitely an aspartyl proteinase secreted within the abomasums from the suckling ruminant. Bovine (cattle) calf chymosin is the milk-clotting enzyme existing mainly within the rennet, that has already been typically employed for tradational cheese making .The purification as well as properties associated with calf rennet have been studied thoroughly (Foltman, 1993).
Rennet originated from the ruminants are extracted from the fourth part of the ruminant stomach (abomasum) of milk fed calves or other young ruminants. The most commonly used milk clotting enzyme , which are used in many cheese varieties worldwide, is calf rennet obtained from 10 to 30 days old milk-fed calves (Yadav et al., 1993). Rennet consists of mainly the rennin (chymosin) and pepsin, rennin being principally responsible for milk clotting activity and the the pepsin has the ability for proteolysis. The rennet extracted from the milk fed calf is rich in rennin (chymosin) 80%, while extracted from the older bovine is rich in pepsin (Akin, 1996).
Garg and Johri (1994) reviewed on rennet extraction. They will explained a throughout the world absence of calf rennet for cheese production have been existed for a number of many years. Bovine pepsin and also to a certain degree porcine pepsin and also plant coagulants are already employed since rennet replacements, yet these kinds of never have recently been commercial productive due to their particular substantial proteolytic nature as well as other inherent drawback. Inspections to produce some other alternates have got introduced the various microbial coagulants which may have identified areas in many nations around the world regardless of specific faults in comparison to standard calf rennet.
Milk clotting nutrients through microbial (Beppu, 1983) as well as grow (Lo Piero, 2002) resources are also utilized in the actual dairy products business for a while. Nevertheless, the caliber of prepared items utilizing these types of nutrients may be discovered to become substandard to that particular produced from livestock leg rennet (Beppu, 1983).
Dairy clotting enzymes coming from microbial (Beppu, 1983) and also plant (Lo Piero, 2002) sources are also used in the dairy industry. However, the quality parameter of these enzyme have been found to dangerous as compared to the calf rennet.(Beppu, 1983). Rennet obtained from the microbial origin usually have low milk clotting and high proteolytic activity leading to break down of milk protein , especially casein, with the releasing of a large number of small peptides, which can produce a bitter taste in the cheese . Such products have limited acceptability (Neelakantan et al. 1999; Li & Walsh, 2000).
Other then the calf , many other animals such as lamb, goat, ,rabbit, pig, bovine and hen contain the enzyme chymosin in their digestive systems (Metin et al., 1998).Other substances used for milk clotting are of vegetable origin and are manufactured from other then the animal sources. Vegetable sources include the cheese-rennet herb, 'Ladies bedstraw' (Galium verum).
Milk clotting proteases produces by Fungi are appearing and may easily be isolated from various environments (Tubesha and Al-Delaimy 2003). Three species, includes Rhizomucor miehei, Rhizomucor pusillus and C. parasitica, have been established for large scale. The aspartic protease produced by R. miehei consists of a single polypeptide chain with a high similarity to chymosin in its three dimensional structure (Chitpinityol and Crabbe 1998). As a substitute for native bovine rennet and microbial rennet, recombinant rennet is extensively used for cheese production (Mohanty et al. 1999).
Around 1990 a recombinant technology enzyme with calf chymosin, often denoted as fermentation produced chymosin (FPC), was the first time processing aid for food manufacturing method that was produced with rDNA technology that are registered by the U.S FDA (Flamm 1991).
Genetic engineering may provide almost unlimited amount of appropriate coagulants, but there are, besides some consumer constraints (Grunert et al. 2001; La¨hteenma¨ki et al. 2002), legal regulations in various European countries which prohibit the involvement of genetically modified micro-organisms in the manufacture of Protected Designation of Origin (PDO) and organic cheeses (EC 2007).
Since cheese manufacturing is getting great importance in national dairy industry, so there is need to produce indigenous type of rennet, because of the price of commercially available rennet is too costly that is not affordable for to afford. There is no reliable research work so far has been found in the past in this respect. Thus, the present study merits producing rennet from buffalo calf stomach and evaluating its quality characteristics.
Objective:
To extract rennet from calf stomach
To use Rennet in the production of cheese
To Investigate their Physical and Chemical properties.
REVIEW OF LITERATURE
Oleary and Fox (1975) isolated, ovine ,bovine, caprine pepsin and lamb, pig and kid chymosins (rennins) by basically the same procedure of selective pH precipitation, DEAE-cellulose chromatography in phosphate buffer, pH 6-5 and Sephadex G-100 chromatography. The homogeneity of the preparations was assessed by two steps.(a) re-cycling on Sephadex G-100, (b) chromatography on DEAE-cellulose using piperazine buffer, pH 5-3 and (c) ability to inactivate ribonuclease.
Anifantakis and Margaret (1980) prepared lamb and kid rennets by extraction of dried abomasa with 6 % (w/v) NaCl 2% (w/v) H3BO3 and activation of the proenzymes at pH 2-0. Each gave one zone of precipitation on casein-agar gel diffusion, that is helpful to be differentiated from calf rennet and pig pepsin. After agarose gel electrophoresis, the proteinase activity of lamb rennet occurred in chymosin and pepsin bands only, whereas kid rennet contained an additional proteinase of intermediate mobility. Relative to their milk-clotting activities, lamb and kid rennets contained less pepsin and were less proteolytic on both haemoglobin at pH 1-8. The milk-clotting activities of lamb and kid rennets increased less with decrease in pH and were more stable to storage at both the pH value of maximum stability and lower pH values than that of calf rennet. Neither cathepsin activity nor lipolytic activity on milk fat was detected in any of the 3 rennets, but lamb rennet caused slight hydrolysis of tributyrin. Using abomasums of 2 to 3 week old goat kids as material, the effects of some factors such as ultrasound intensity, extraction time, NaCl concentration in extraction solution and ratio of abomasums mucosa to extraction solution on the extraction yield of goat kid rennet were investigated. On the basis of single factor experiments, the processing parameters for ultrasound extraction were optimized using a fourfactor orthogonal and rotatory combinatorial design. The results showed that the optimum processing parameters for the extraction of goat kid rennet were: ultrasound intensity, 44 W/cm2; extraction time, 25 min; NaCl concentration in extraction solution, 16%; and the ratio of abomasumal mucosa to extraction solution, 1:30. In addition, the ultrasonic extraction, as compared with traditional extraction, proved to be more effective.
Beppu, T. (1983) also extracted bovine chymosin from the abomasum of suckling calves. It is secreted in its natural form as an inactive precursor, prochymosin, which is irreversibly converted into active chymosin through limited proteolysis. Chymosin is used as the milk clotting agent rennet in cheese manufacture. This enzyme possesses a very high milk clotting activity with a low proteolytic activity and this makes chymosin particularly suitable for the manufacture of cheese.
Kim and Zayas (1991) developed an ultrasound method for increased efficiency of chymosin extraction. Ultrasound extraction was carried out with a Tekmar sonic disrupter and control extraction was by shaking. Use of ultrasound resulted in increase in activity and decrease in nitrogen content, thus increasing the specific activity of chymosin extracts. Proteolytic activity of experimental chymosin was higher at 15 and 20°C, but lower at 35 and 40°C than that of controls. Chymosin extracts obtained by ultrasound treatment were darker, less green, less blue, and more turbid than controls. Ultrasound treatment did not affect activation energies for proteolytic activity, total viable cell counts, or storage stability of chymosin extracts.
Mohanty et al. (1999) described that bovine chymosin is synthesized in vivo as preprochymosin and secreted as prochymosin which is auto catalytically activated to chymosin. Chymosin is bilobular, with Asp 32 and Asp 215 acting as the catalytic residues. Chymosin A and chymosin B have pH optima of 4.2 and 3.8, respectively, and act to initiate milk clotting by cleaving k-casein between Phe 105 and Met 106. The gene encoding chymosin has been cloned and expressed in suitable yeast and bacteria hosts under the control of amino acid, trp-beta, gly A genes, lac, trp and serine hydroxylmethyl-transferase promoters. Protein engineering of chymosin has also been attempted. A number of companies are now producing recombinant chymosin for commercial use in cheese manufacture.
Zayas (1986) extracted rennet by ultrasonic method. He found that improvement of chymosin (rennin) production was limited by a prolonged extraction process. An ultrasonic method of chymosin extraction was developed to increase efficiency. The application of ultrasonic energy significantly reduced time required for the extraction process as a result of abomasum tissue dispersing, destruction of the cells, intensive blending, separation of particles, and increase of the interphase abomasum-extraction medium. The ultrasonic extraction process significantly increased yield of chymosin. Optimal technological parameters for extraction and properties of extraction medium were established: time of ultrasonic treatment (45 min), intensity of ultrasound (3.34 W/cm2), temperature of extraction medium (15°C), NaCl concentration in solution (5%), ratio of abomasum to extracting solution (1:18), NaCl concentration in solution (5%), ratio of abomasum to extracting solution (1:18), pH of solution (4.2 to 4.3), time of chymosin extract activation (7 h), degree of abomasum shredding (strips 5 to 8 mm wide), and time of abomasum swelling (20 min).
Mohanty et al. (1999) also studied that Chymosin is used for curdling of milk and manufacture of cheese. They reported the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molar weight of the purified rennet was 35.6 kDa on SDS-PAGE. Amino acid of N-terminal sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bellshaped stability profile as a function of temperature with maximum activity near 55 oC. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7. The ratio of milk clotting to proteolytic activity was 3. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.
Moschopoulou et al. (2006) worked on the extraction of chymosin (rennet) from the abomasum of the kid. The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3-6 zones at pH 4.6-5.1, while that of kid pepsin was at pH 3.0. Kid pepsin contained 0.37 molecules phosphorous per molecule and was totally inhibited by 5 mM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5.6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin <kid chymosin <kid pepsin.
Zhang and Wang (2007) extracted rennet from kid by using abomasums of 2 to 3 weeks old goat kids as raw material. The effects of some factors such as ultrasound intensity, extraction time, NaCl concentration in extraction solution and ratio of abomasums mucosa to extraction solution on the extraction yield of goat kid rennet were investigated. On the basis of single factor experiments, the processing parameters for ultrasound extraction were optimized using a fourfacto orthogonal and rotatory combinatorial design. The results showed that the optimum processing parameters for the extraction of goat kid rennet were: ultrasound intensity, 44 W/cm2; extraction time, 25 min; NaCl concentration in extraction solution, 16%; and the ratio of abomasumal mucosa to extraction solution, 1:30. In addition, the ultrasonic extraction, as compared with traditional extraction, proved to be more effective.
Materials and Methods
Materials
Calf stomach will be obtained from the Bolan Army Military Farm Okara at the age of 2-3 weeks will be used in the study. Calf abomasums will be obtaine and washed by clean water. Fat and connective tissues on the surface will be removed and then the abomasums will be used for rennet Extraction.
Extraction Method
Acidified Whey:
Rennet will be prepared according to the method described by David B. Fankhauser (2007), in this method; the abomasums from Bolan Military Farm will be used. The abomasums will be dried, Spread the abomasums out on a stainless steel screen and sprinkle salt on the abomasums. Salt will be into the surface of the abomasum. Let it to dry in a cool dry location. The dried, salted abomasum will be laid out, cut the abomasums into strips and soak to the acidified whey pH 4.1 .Put them into the fridge at 4-10 C for 48 hr .Soaked abomasums piece will be removed by pouring through a sieve. Rennet activity will be measured by the method of Arima et al (1967).
Nacl Solution:
Rennet will be prepared according to the method described by Lambert (1988). After removing the internal contents of the stomach, abomasa will be washed with tape water internally while their veins and fat contents will be removed externally. Then they will be expanding with air just like a balloon and after that it will be dried in wooden- wire mesh cabinet placed in an open air. After drying, each abomasum will be cut into very thin strips separately and soaked in 700 ml solution of the 12 % NaCl and 1% sodium benzoate solution individually. The pH of the solution containing abomasal strips will be reduced to 4.3 with benzoic acid. The solution will be stored at 350C for 72 hrs. The pH of the mixture will be raised to 5.6 with disodium phosphate. The extract will be filtered with filter paper and caramel-like color liquid rennet will be obtained from the individual sample.
Ultrasonic Extraction:
Using thawed abomasums as samples，the ultrasonic extraction device (produced by the Department of Clinical Medicine and Surgery University of Agriculture Faisalabad.) will be used to extract rennet under different ultrasound intensities. NaCl concentrations ratio of abomasums to extracting solution, extracting time, and extracting solution pH． The mixture will be centrifuged at 3,000 rpm for 15 min and unwanted substances will be removed. The supernatant will be adjusted to pH 4.6 by adding 0.1 M HC1 and activated for 24 h at room temperature. The extracting procedure will be shown in the following figure.
Frozen kid abomasums stored at -20C
Cutting
Extraction with NaCl solution
Control group Ultrasonic treatment groups
Both Centrifuged at 3 000 rpm for15min
Activating with pH 4．6 for 12 h
Calf rennet
Assay of rennet activity:
Using the method of Arima et al. rennet activity will be measured. 10 ml of 100 g/l skimmed milk solution will be incubated at 35C for 10 min. 0.5 ml rennet will be added in the solution and mixed immediately; the accurate time needed from adding rennet to milk coagulation will be recorded.
The Soxhlet unit (SU) defined as the amount of enzyme needed to coagulate 1ml 100g/l skimmed milk solution in 40 min. The experimental result is expressed as the Soxhlet unit (SU) of per gram wet abomasums (SU/g).
Soxhlet unit (SU) = 2400/T * 10 /5 * D
In which "T" is coagulating time(s) and "D" is dilution rate.
LITERATURE CITED
Arima K，Shinier I，Gakuzo T． 1967. Milk·clotting enzymes from microorganism ,part
I，screening test and identification of potent fungus．Journal of Agriculture and
Biological Chemistry. 31(5) ：540-545．
Anifantakis, E. and L. Margaret. 1980. Preparation and properties of rennets from lamb's and kid's abomasa. Green Journal of Dairy Research. 47: 221-23.
Beppu T. 1983. The cloning and expression of chymosin (rennin) genes in microorganisms. Trends in Biotechnology. 1:85-90.
Beppu, T. 1983. The cloning and expression of chymosin (rennin) genes in micro-organisms. Trends in Biotechnology. 1: 85-89.
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Foltman, B. 1993 General and molecular aspects of rennets. In Cheese: Chemistry, Physics and Microbiology. (Ed. PF Fox). London: Chapman and Hall. 1: 37-88
Garg, S.K and B.N. Johri . 1994. Rennet: Current trends and future research: Food Reviews International. 3 (10). 313-355.
Kim, S.M and J.F. Zayas. 1991. Effects of ultrasound treatment on the properties of chymosin．Journal of Food Science 56(4) ：926-930.
Kim, S.M and J.F. Zayas. 1991. Comparative quality characteristics of chymosin extracts obtained by ultrasound treatment. Journal of Food Science. 2(52): 406-410.
Kumar, A., J. Sharma, A.K. Mohanty, S. Grover and V.K. Batish. 2006. Purification and characterization of milk clotting enzyme from goat (Capra hircus). Comparative Biochemistry and Physiology, Part B. 145: 108-113.
Mohanty, A.K., U.K. Mukhopadhyay, J.K. Kaushik, S. Grover and K. Virender. 1999. Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis). Batish. Journal of Dairy Research. 70: 37-43.
Mohanty, A.K., U.K. Mukhopadhyay, S. Grover, V.K. Batish. 1999. Bovine chymosin: Production by rDNA technology and application in cheese manufacture. Biotechnology Advances. 17: 205-217.
Moschopoulou1, E.E, I.G. Kandarakis, E. Alichanidis and E.M. Anifantakis. 2006. Purification and characterization of chymosin and pepsin from kid. Journal of Dairy Research. 73: 49-57.
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