ISSN Volume 5, Number 3 September 2013

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1 ISSN Volume 5, Number September 1 1

2 Editor-in-Chief Tsanko Yablanski Faculty of Agriculture Trakia University, Stara Zagora Bulgaria Co-Editor-in- Chief Radoslav Slavov Faculty of Agriculture Trakia University, Stara Zagora Bulgaria Editors and Sections Genetics and Breading Atanas Atanasov (Bulgaria) Ihsan Soysal (Turkey) Max Rothschild (USA) Stoicho Metodiev (Bulgaria) Nutrition and Physiology Nikolai Todorov (Bulgaria) Peter Surai (UK) Zervas Georgios (Greece) Ivan Varlyakov (Bulgaria) Production Systems Dimitar Pavlov (Bulgaria) Dimitar Panaiotov (Bulgaria) Banko Banev (Bulgaria) Georgy Zhelyazkov (Bulgaria) Agriculture and Environment Georgi Petkov (Bulgaria) Ramesh Kanwar (USA) Product Quality and Safety Marin Kabakchiev (Bulgaria) Stefan Denev (Bulgaria) Vasil Atanasov (Bulgaria) English Editor Yanka Ivanova (Bulgaria) Scope and policy of the journal Agricultural Science and Technology /AST/ an International Scientific Journal of Agricultural and Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal and in electronic form. The policy of the journal is to publish original papers, reviews and short communications covering the aspects of agriculture related with life sciences and modern technologies. It will offer opportunities to address the global needs relating to food and environment, health, exploit the technology to provide innovative products and sustainable development. Papers will be considered in aspects of both fundamental and applied science in the areas of Genetics and Breeding, Nutrition and Physiology, Production Systems, Agriculture and Environment and Product Quality and Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings and conference reports will be considered for special issues. Submission of Manuscripts All manuscripts written in English should be submitted as MS-Word file attachments via to ascitech@uni-sz.bg. Manuscripts must be prepared strictly in accordance with the detailed instructions for authors at the website and the instructions on the last page of the journal. For each manuscript the signatures of all authors are needed confirming their consent to publish it and to nominate on author for correspondence. They have to be presented by a submission letter signed by all authors. The form of the submission letter is available upon from request from the Technical Assistance or could be downloaded from the website of the journal. Manuscripts submitted to this journal are considered if they have submitted only to it, they have not been published already, nor are they under consideration for publication in press elsewhere. All manuscripts are subject to editorial review and the editors reserve the right to improve style and return the paper for rewriting to the authors, if necessary. The editorial board reserves rights to reject manuscripts based on priorities and space availability in the journal. The articles appearing in this journal are indexed and abstracted in: EBSCO Publishing, Inc. and AGRIS (FAO). The journal is accepted to be indexed with the support of a project BG51PO Science and business financed by Operational Programme Human Resources Development of EU. The title has been suggested to be included in SCOPUS (Elsevier) and Electronic Journals Submission Form (Thomson Reuters). Internet Access This journal is included in the Trakia University Journals online Service which can be found at Address of Editorial office: Agricultural Science and Technology Faculty of Agriculture, Trakia University Student's campus, Stara Zagora Bulgaria Telephone.: Technical Assistance: Nely Tsvetanova Telephone.: ascitech@uni-sz.bg

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5 AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No, pp 9-42, 1 Influence of the amount of milk clotting enzyme with microbial and camel origin on the coagulation time of cow's milk P. Panayotov1, K. Yoanidu1*, P. Boyanova1, B. Milenkov2 1 Department of Milk and Dairy Products, Technological Faculty, University of Food Technologies, 26 Maritza, 2 Plovdiv, Bulgaria Department of Computer Systems and Technologies, Technical Faculty, University of Food Technologies, 26 Maritza, 2 Plovdiv, Bulgaria 2 Abstract. A study of rennet coagulation of cow's milk was conducted using chymosin of microbial and camel origin in the range to cm.-2 dm- with an equivalent activity to 1:. As a control calf chymosin was used. The monitoring includes heuristic, optical and viscosimetric methods. Significant differences in both initial coagulation time, as well as in time of full coagulation of milk were determinated. The data obtained in a research study can be used in laboratory and manufacturing practices using chymosin of camel origin, which can mainly be used in cheese making, using stream dosing of the coagulant and low impact of the enzyme on the cheese ripening process. Keywords: rennet coagulation, chymosin of camel, nonlinear regression, coagulation time Introduction The development and growth of industry of cheese making and reducing the quantity of available sources of milk clotting enzymes causes demand for new alternative sources of chymosin. Such alternative is the use of recombinant enzymes (Rodriguez, 1). Although most proteinases may coagulate milk, only a few are used successfully as a substitute of calf chymosin. A prerequisite is: the substituent must possess low proteolytic activity, specificity for the connection Phe5-Met6 of k-casein, and the need to pass in the whey (Fox and McSweeney, 1997). As technology for recombining DNA, calf chymosin gene was cloned on various microorganisms, which allows the preparation of chymosin by fermentation. Such types of preparations are widely used in practice (Fox and McSweeney, 1997). The enzymatic properties of camel chymosin obtained by fermentation were compared with those of calf chymosin; camel chymosin has 7% higher milk clotting activity per unit of cow milk compared to calf chymosin and only % of typical calf enzyme proteolytic activity, respectively camel chymosin has 7 times higher rate of coagulation (Kappeler et al., 6). There were no significant differences in the composition and ph of cheeses produced with different coagulants. The extent of proteolysis was significantly lower in cheeses produced with camel chymosin than those obtained with bovine milk clotting enzyme. There were considerable quantitative differences in the peptide profiles of cheeses. However, the levels of the amino acids are similar, with the exception of isoleucine, histidine and lysine (Bansal et al., 9). Camel chymosin has higher thermal stability than calf chymosin. It was found that camel milk can not coagulate with the use of calf chymosin, but with the use of camel chymosin high milk clotting activity was observed (Kappeler et al., 6). The most commonly used microorganisms for the preparation of recombinant chymosin by microbial origin are fungi, but this enzyme can be prepared also with the use of bacteria. Three of the most regularly utilized types of microorganisms are Aspergillus niger, Kluyveromices lactis and Escherichia coli, which makes them suitable for the production of chymosin in the experiments relating to gene recombination (Rodriguez, 1). The application of the different genetic variants of chymosin results in changing not only the time of initial coagulation of the milk, but also the duration of the different phases of the coagulation process. Significant differences were identified at the time of initial coagulation, as well as at the time of complete coagulation of the milk. Increasing the concentration of milk clotting enzyme to accelerate the process of enzymatic coagulation, is likely to lead to a pronounced bitterness in the taste profile of the finished product. According to Spangler, the optimal concentration of milk clotting enzyme for preparing Gouda type cheese concentrate obtained by ultra filtrated milk is 1/ of the standard; an increase in the rate of flocculent added to 2/ of the standard, leads to significantly bitter taste profile (Spangler et al., 1991). The aim of the study was to establish the influence of the type and quantity of rennet on the process of initial and total coagulation. Material and methods In all experiments cow's milk was used collected in the village of Dolnoslav, Asenovgrad area. The milk was accepted in accordance with the specific requirements and analysis of the main parameters was conducted. Parameters remained stable during the two days of the execution of experiments, there was a very slight change in the ph of the milk on the second day of storage. The parameters of cow's milk (pasteurized at 65 C) were: fat.6% (2.76% after degreasing); SNF 8.4%; density of milk 1.27 kg.dm-; protein.11%, titratable acidity 16 T; ph = 6.8. Before processing the milk was subjected to partial degreasing to reduce the effect of milk fat on the obtained results. For each type of enzyme during the single factor experiment, 18 samples containing 8 ml of cow's milk were prepared. The milk was heated to a temperature of 8 C and then cooled to a temperature of 6 8 C, the optimum temperature for the milk * katqioanidu@abv.bg 9

6 Results and discussion Single factor analysis was conducted in order to reveal the influence of the amount of milk clotting enzyme on time of initial and total coagulation of cow's milk. The main focus of the experiment is related to enzymes of camel and microbial origin compared with calf chymosin. The influence of the amount of a microbial enzyme of,,,,, and cm.-2.dm- on the time of initial coagulation of cow's milk (τic, s) was studied. The data from quadruplicate experience are shown in Figure 1, as successive trials are indicated by the numbers 1, 2,, 4, respectively. The average sample was also marked on Figure 1 as microbial av.. Tolerant limits of τic using milk clotting enzyme of 9 microbial 1 microbial 2 microbial microbial 4 microbial average Initial coagulation time, s 8 7 f = (a * b) / (b + x) (1) According to the correlation coefficient R =.998, the quadratic coefficient Rsqr =.9967 was obtained, and the coefficients of significance of the aforementioned values.999, the mathematical model that correctly describes the relation studied has the following form: y = (29 * 4.) / (4. + x) = / (4. + x) (2) The influence of the quantity of chymosin with camel origin in the range of variation from to cm.-2.dm- and step cm. -2.dm- on the time of initial coagulation was studied (τic, s). The determination of τic is identical to the previous experiment. The data obtained from quadruplicate experience are shown in Figure 2. The tolerant limits of τic with the use of the coagulant with camel origin were within a range of variation τic = 59,5 ± 26,2; τic = 28. ± 17.1; τic = 191 ±.5; τic = 1. ± 6.8; τic = 91.5 ± 5.6 and τic = 69 ± 4.1 s. Figure 2 shows that the variation of τic is not linear, but is described similar to a hyperbola. The mathematical processing of data leads to formation of nonlinear regression model, a type of decreasing hyperbola with two coefficients, similar to that in equation 1. According to the coefficient Rsqr =.9967 obtained, mathematical re-processing was needed, also with the use of non-linear regression procedure, but with a decreasing hyperbola with three coefficients of the form: f = y + (a * b) / (b + x) () According to the correlation coefficient R =.9987, Rsqr =.997 was obtained and coefficients of significance of the aforementioned values, respectively, for Ra = ; for Rb = 9 camel 1 camel 2 camel camel 4 camel average Amount of milk clotting enzyme, cm..dm - Figure 1. Variation of time for initial coagulation of cow's milk depending on the amount of milk clotting enzyme of microbial origin microbial origin for the interval of variation are indicated as τic = 69.5 ± 2.4; τic = 7.5 ± 18.8; τic = 27.5 ± 1.6; τic = 216. ± 11.; τic = 187. ± 9.5 and τic = ± 7.8 s, respectively. The above data show that the variation of τic is not linear and is described more like hyperbole. After mathematical processing of the data obtained, nonlinear regression model, a type of decreasing hyperbola with two coefficients was prepared, having the following form: Initial coagulation time, s clotting enzyme activity. The renneted milk, in an amount of ml, was then divided in three glass containers. The remaining amount of milk was used for the small sample, which serves to heuristically determine the initial coagulation. The samples were placed in a water bath at a temperature of 5 8 C and were analyzed by the method of penetrometry by measuring the hardness of the gel in, and min. Detection was carried out on the th and th second, respectively. The quantities of the used milk clotting enzymes are from to cm.-2 dm-, respectively, and the results were obtained on the basis of a quadruplicate experiment. Camel chymosin of "Christian Hansen" was used as coagulant under the name "CHY - MAX" M ", with milk clotting activity IMCU/ml (or strength of the enzyme 1: 7 ), stored refrigerated at 8 C. The milk clotting enzyme of microbial origin, produced by "Chr. Hansen", was described as activity (1: ) and other indicators in the established certificate. For the experiment, the enzyme activity of the aforementioned coagulants was reduced to 1:. Initial coagulation time is determined by the heuristic (Berridge, 1952) and viscometric (Boyanova et al., 12) methods. Statistical processing of the data obtained in the course of the experiment was carried out using specialized mathematical software SigmaPlot 11.. These mathematical analyses are used to develop mathematical models (Bethea et al., 1985). -2 Amount of milk clotting enzyme, cm..dm - Figure 2. Variation of time for initial coagulation of cow's milk depending on the amount of milk clotting enzyme of camel origin

7 and Ry =.98295, the mathematical model that correctly describes the relation studied has the following form: y = * x / ( x) describing the functions of the type decreasing hyperbola with three coefficients were formed. The equation obtained for variations of τtc using different amounts of a microbial enzyme is: (4) Fmic = ( * 6.777) / ( x) An experiment with the use of bovine chymosin as a control was also conducted. Determining τic is analogous to the previous experiments. The data obtained are shown in Figure. The tolerant limits of τic using calf chymosin for the interval of variation are τic = 61.5 ± 1.8; τic = 84.8 ± 18.7; τic = 265. ± 14.2; τic = 189. ±.; τic = ± 7.2 and τic = 129. ± 6.4 s. 9 cal f1 cal f2 cal f cal f4 cal f average 7 The coefficient of correlation is high (R =.9979), and the squared correlation coefficient Rsqr = All three factors are significant, which indicates the correct description of the process of equation 6. Variations of τtc depending of the amount of chymosin with camel origin are described by the equation: Fcam = ( * ) / ( x) (7) The coefficient of correlation R =.9999, and quadratic coefficient Rsqr =.9997 are significant and equation 7 describes the process properly. Control test on τtc is made using calf chymosin. The equation obtained is: Fcalf = ( * ) / ( x) -2 Amount of milk clotting enzyme, cm..dm - Figure. Variation of time for initial coagulation of cow's milk depending on the amount of calf chymosin Figure shows that the variation of τic is not linear and is described as hyperbole. The obtained data were processed statistically, and the result is also a non-linear regression model, a type of decreasing hyperbola with two coefficient similar to that in equation 1. The coefficient of correlation Rsqr =.9977 obtained shows that the research process is correctly described. According to the correlation coefficient R =.9987 obtained and coefficients of significance of the aforementioned values, respectively, for Ra =.999 and for Rb =.999, the mathematical model that correctly describes the dependence investigated is in the form: y = (286 *.66) / (.66 + x) = / (.66 + x) (5) The resulting equations show similarity in the course of the coagulation process carried out by the use of microbial and calf chymosin. The equation describing the process of coagulation by chymosin from camel origin also shows similarity considering the fact that the processing of the data from this experiment confirmed the non-linearity of the function, which is also of the type decreasing hyperbola, but unlike the previous two versions there are three coefficients here. The difference is further comprised in that, while the camel chymosin has been actively lowering the times for initial coagulation of the milk, when amounts higher than cm.-2.dm- are used, the other two enzymes show a tendency to reduce the differences between successive τic. The time of total coagulation of cow's milk using microbial, camel and calf chymosin (τtc, s) is also established during the experiment. Mathematical analysis were performed by using the data obtained (Figure 4) and non-linear regression equations, (8) The coefficient of correlation is R =, 9998, and Rsqr =, These results obtained by analysis show the significance of the coefficients, respectively equation 8 describes the process correctly. The data obtained for variations of the time of initial and total coagulation of cow's milk depending on the amount of microbial, camel and calf chymosin are shown in Figure 4. From the figure it is apparent that the times for total coagulation of milk are shorter when camel chymosin is used in comparison to bovine and microbial chymosin. The ratio time for initial coagulation to total coagulation time depending on the amount of enzyme is formed and the results obtained are presented in Figure 5. When microbial and calf Time, s Initial coagulation time, s 8 (6) Amount of enzyme, cm.-2.dm- Figure 4. Variation of τic and τtc of cow's milk depending of the amount of microbial, camel and calf chymosin 41

8 Enzyme amount, cm.-2 dm- camel microbial calf Figure 5. Variation of the ratio τic and τtc of cow's milk depending on the amount of microbial, camel and calf chymosin 4 the first two phases and the relatively slow pace in the next. Differences related to ratios using microbial enzyme increased from 12.7 to 152.1%. The data on the relationship between τic and τtc using microbial and calf enzyme to the time for initial and total coagulation of the enzyme of camel origin are shown in Figure 6. The Figure shows that differences in the relationship between τic involving microbial chymosin were lower than those in the initial coagulation time of calf chymosin, while the relationship in τtc is close and there is a relatively small change in the range of varying the amount of enzyme. Dependencies on the times of coagulation, obtained using calf chymosin are presented in Figure 7. The data shows small variations depending on the amount of the enzymes used. Conclusion The mathematical models obtained can be used in laboratory and manufacturing practice using chymosin of camel origin, incorporated in amounts of cm.-2.dm-, primarily in cheese making using streaming coagulant dosing. Mathematical models and the resulting relationship between τic and τtc using chymosin of microbial, camel and calf origin in amount of cm.-2.dm- can be used in the regulation of syneresis and preparation of technological diagrams to obtain different types of cheeses. 2 1 References -2 Enzyme amount, cm. dm- ic - calf/camel ic - calf/camel ic - mic/camel ic - mic/camel Figure 6. Variation of τic using microbial and calf chymosin in relation with τic of camel chymosin and variation of τtc obtained by microbial and calf chymosin in relation with τtc of camel chymosin Enzyme amount, cm. dm ic - camel/calf ic - camel/calf - ic - mic/camel ic - mic/camel Figure 7. Variation of τic using microbial and camel chymosin in relation with τic of calf chymosin and variation of τtc obtained by microbial and camel chymosin in relation with τtc of calf chymosin chymosin are used, the obtained ratios are similar, as the differences ranged from 1.8 to 27.8%. Ratios using camel chymosin vary from 6. to These ratios show a rapid rate of coagulation during 42 Bansal N, Drake MA, Piraino P, Broe ML, Harboe M, Fox PF and McSweeney PLH, 9. Suitability of recombinant camel (Camelus dromedarius) chymosin as a coagulant for Cheddar cheese. International Dairy Journal 19, Bethea RM, Duran BS and Boullion TL, Statistical Methods for Engineers and Scientists. New York: Marcel Dekker, Inc 1985, ISBN X. Berridge NJ, Some observations on the determination of the activity of rennet. The Analyst, 77, Boyanova P, Panayotov P, Milenkov B and Dinkov H, 12. Monitoring of milk acid coagulation by rotational viscometer. Agricultural Science and Technology, 4, 4, , ISSN: X. Fox PF and McSweeney PLH, Rennets: their role in milk coagulation and cheese ripening. In: Law, B.A. (Ed.), Microbiology and Biochemistry of Cheese and Fermented Milk, second ed. Blackie Academic and Professional, London, UK, Kappeler S, van den Brink H, Nielsen H, Farah Z, Puhan Z, Hansen E and Johansen E, 6. Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk. Biochemical and Biophysical Research Communications 42, Montgomery D, Peck E and Vining G, 6, Introduction to Linear Regression Analysis, Wiley-Interscience; 4th edition. Rodriguez Е, 1. Microbial production of recombinant chymosin. The Keck Science Department of the Claremont Colleges. Spangler PL, Jensen LA, Amundson CH, Olson NF and Hill Jr CG, Ultrafiltered Gouda Cheese: Effects of Preacidification, Diafiltration, Rennet and Starter Concentration, and Time to Cut. Journal of Dairy Science, 74, 9,

9 AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No, 1 CONTENTS 1/2 Review Fibromelanosis in domestic chickens H. Lukanov, A. Genchev 29 Genetics and Breeding Rumi and IPK Nelina new cotton varieties A. Stoilova, Hr. Meluca 247 Drying of seeds from common wheat (Triticum aestivum L.) by using Silica gel for ex situ storage P. Chamurlyisky, N. Tsenov, S. Stoyanova 252 Breeding evaluation of newly stabilized lines of maize V. Valkova 257 Apricot breeding for resistance to Sharka V. Bozhkova, S. Milusheva 261 Dry matter accumulation in the varieties of wheat (Triticum aestivum L.) according to previous crop A. Ivanova, N. Tsenov 264 Reproductive performance of weaning saws after treatment with Fertipig S. Dimitrov, G. Bonev 269 Reproductive performance of Polish Large White and Polish Landrace sows B. Szostak, V. Katsarov 272 Nutrition and Physiology Effect of the feeding of products stimulating the development of bee colonies R. Shumkova, I. Zhelyazkova 276 Investigations on kidney function in mulard ducklings with experimental aflatoxicosis I. Valchev, N. Grozeva, L. Lazarov, D. Kanakov, Ts. Hristov, R. Binev, Y. Nikolov 282 Rumen fermentation in yearling rams fed different rations V. Radev 29 Effect of different lipid and protein dietary levels on rumen ciliate fauna and cellulolytic activity in yearling rams V. Radev, I. Varlyakov, R. Mihaylov 294

10 AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No, 1 CONTENTS 2/2 Production Systems Efficacy and selectivity of antibroadleaved herbicides at durum wheat against volunteers of coriander, Clearfield canola, Clearfield sunflower and ExpressSun sunflower G. Delchev 299 Investigations on friction coefficients of cow hooves with different dairy farm floor types T. Penev, Z. Manolov, I. Borissov, V. Dimova, Tch. Miteva, Y. Mitev, V. Kirov 5 Productivity of irrigation cotton cultivated under different inter-row spaces I. Saldzhiev, A. Muhova 9 Stability evaluation of mixtures among preparations with different biological effect on the basis of grain yield in spring vetch G. Delchev, N. Georgieva, I. Nikolova 1 Biological activity of plant protection products against Tuta absoluta (Meyrick) in tomato grown in greenhouses N. Valchev, V. Yankova, D. Markova 18 Agriculture and Environment Changes of some agro-chemical parameters of Pellic Vertisol (FAO) soil type in growing cereal crops under organic system of agriculture V. Koteva 22 Product Quality and Safety Carcass composition and meat quality in lambs reared indoors and on pasture T. Popova, P. Marinova 25 Fatty acids and lipid indices of buffalo milk yogurt N. Naydenova, T. Iliev, G. Mihaylova 1 Effect of supplementary honey and artificial sugar feeding of bees on the composition of royal jelly R. Balkanska, I. Zhelyazkova, M. Ignatova, B. Kashamov 5 Influence of the amount of milk clotting enzyme with microbial and camel origin on the coagulation time of cow's milk P. Panayotov, K. Yoanidu, P. Boyanova, B. Milenkov 9 Determining chlorophyll and carrotenoid content in Bombyx mori L. excreta by Near Infrared Spectroscopy S. Atanasova, M. Panayotov, D. Pavlov, M. Duleva 4

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Todorov N and Mitev J, Effect of level of feeding during dry period, and body condition score on reproductive performance in dairy cows,ixth International Conference on Production Diseases in Farm Animals, Sept.11 14, Berlin, Germany, p. 2 (Abstr.). Thesis: Penkov D, 8. Estimation of metabolic energy and true digestibility of amino acids of some feeds in experiments with muscus duck (Carina moshata, L). Thesis for DSc. Agrarian University, Plovdiv, 14 pp. The Editorial Board of the Journal is not responsible for incorrect quotes of reference sources and the relevant violations of copyrights. Ethics Studies performed on experimental animals should be carried out according to internationally recognized guidelines for animal welfare. That should be clearly described in the respective section Material and methods.

12 Volume 5, Number September 1