ISSN 1313 - 8820Volume 5, Number 3

September 2013

2013

Scope and policy of the journalAgricultural 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.

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English Editor

Yanka Ivanova (Bulgaria)

2013

ISSN 1313 - 8820 Volume 5, Number 3September 2013

Influence of the amount of milk clotting enzyme with microbial and camel origin on the coagulation time of cow's milk

1 1 1 2P. Panayotov , K. Yoanidu *, P. Boyanova , B. Milenkov

1Department of Milk and Dairy Products, Technological Faculty, University of Food Technologies, 26 Maritza, 4002 Plovdiv, Bulgaria2Department of Computer Systems and Technologies, Technical Faculty, University of Food Technologies, 26 Maritza, 4002 Plovdiv, Bulgaria

Abstract. 3 -2 -3A study of rennet coagulation of cow's milk was conducted using chymosin of microbial and camel origin in the range 10 to 60 cm .10 dm with an equivalent activity to 1:50000. 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

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No 3, pp , 2013339 - 342

Introduction suitable for the production of chymosin in the experiments relating to gene recombination (Rodriguez, 2013).

The application of the different genetic variants of chymosin The development and growth of industry of cheese making and results in changing not only the time of initial coagulation of the milk, reducing the quantity of available sources of milk clotting enzymes but also the duration of the different phases of the coagulation causes demand for new alternative sources of chymosin. Such process. Significant differences were identified at the time of initial alternative is the use of recombinant enzymes (Rodriguez, 2013). coagulation, as well as at the time of complete coagulation of the Although most proteinases may coagulate milk, only a few are used milk.successfully as a substitute of calf chymosin. A prerequisite is: the

Increasing the concentration of milk clotting enzyme to substituent must possess low proteolytic activity, specificity for the accelerate the process of enzymatic coagulation, is likely to lead to a connection Phe105-Met106 of k-casein, and the need to pass in the pronounced bitterness in the taste profile of the finished product. whey (Fox and McSweeney, 1997). As technology for recombining According to Spangler, the optimal concentration of milk clotting DNA, calf chymosin gene was cloned on various microorganisms, enzyme for preparing Gouda type cheese concentrate obtained by which allows the preparation of chymosin by fermentation. Such ultra filtrated milk is 1/3 of the standard; an increase in the rate of types of preparations are widely used in practice (Fox and flocculent added to 2/3 of the standard, leads to significantly bitter McSweeney, 1997).taste profile (Spangler et al., 1991).The enzymatic properties of camel chymosin obtained by

The aim of the study was to establish the influence of the type fermentation were compared with those of calf chymosin; camel and quantity of rennet on the process of initial and total coagulation.chymosin has 70% higher milk clotting activity per unit of cow milk

compared to calf chymosin and only 20% of typical calf enzyme proteolytic activity, respectively camel chymosin has 7 times higher

Material and methodsrate of coagulation (Kappeler et al., 2006). There were no significant differences in the composition and pH of cheeses produced with different coagulants. The extent of proteolysis was significantly In all experiments cow's milk was used collected in the village of lower in cheeses produced with camel chymosin than those Dolnoslav, Asenovgrad area. The milk was accepted in accordance obtained with bovine milk clotting enzyme. There were considerable with the specific requirements and analysis of the main parameters quantitative differences in the peptide profiles of cheeses. However, was conducted. Parameters remained stable during the two days of the levels of the amino acids are similar, with the exception of the execution of experiments, there was a very slight change in the isoleucine, histidine and lysine (Bansal et al., 2009). Camel pH of the milk on the second day of storage. The parameters of cow's chymosin has higher thermal stability than calf chymosin. It was milk (pasteurized at 65°C) were: fat – 3.6% (2.76% after

-3found that camel milk can not coagulate with the use of calf degreasing); SNF – 8.4%; density of milk – 1.027 kg.dm ; protein – chymosin, but with the use of camel chymosin high milk clotting 3.11%, titratable acidity – 16 T; pH = 6.8. Before processing the milk activity was observed (Kappeler et al., 2006). was subjected to partial degreasing to reduce the effect of milk fat on

The most commonly used microorganisms for the preparation the obtained results. of recombinant chymosin by microbial origin are fungi, but this For each type of enzyme during the single factor experiment, 18 enzyme can be prepared also with the use of bacteria. Three of the samples containing 800 ml of cow's milk were prepared. The milk most regularly utilized types of microorganisms are Aspergillus was heated to a temperature of 38 ÷ 40°C and then cooled to a niger, Kluyveromices lactis and Escherichia coli, which makes them temperature of 36 ÷ 38°C, the optimum temperature for the milk

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* e-mail: katqioanidu@abv.bg

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10clotting enzyme activity. The renneted milk, in an amount of 200 ml, microbial origin for the interval of variation are indicated as τic = 20 30 40was then divided in three glass containers. The remaining amount of 693.5 ± 32.4; τic = 407.5 ± 18.8; τic = 273.5 ± 13.6; τic = 216.0 ±

50 60milk was used for “the small sample”, which serves to heuristically 11.3; τic = 187.0 ± 9.5 and τic = 172.8 ± 7.8 s, respectively. The determine the initial coagulation. The samples were placed in a above data show that the variation of τic is not linear and is described water bath at a temperature of 35 ÷ 38°C and were analyzed by the more like hyperbole.method of penetrometry by measuring the hardness of the gel in 20, After mathematical processing of the data obtained, nonlinear

th th40 and 60 min. Detection was carried out on the 30 and 60 second, regression model, a type of decreasing hyperbola with two respectively. coefficients was prepared, having the following form:

The quantities of the used milk clotting enzymes are from 10 to 3 -2 -360 cm .10 dm , respectively, and the results were obtained on the f = (a * b) / (b + x) (1)

basis of a quadruplicate experiment.Camel chymosin of "Christian Hansen" was used as coagulant According to the correlation coefficient R = 0.9983, the

under the name "CHY - MAX" ® M ", with milk clotting activity 1000 quadratic coefficient Rsqr = 0.9967 was obtained, and the IMCU/ml (or strength of the enzyme 1: 70 000), stored refrigerated at coefficients of significance of the aforementioned values 0.9909003, 0 ÷ 8°C. the mathematical model that correctly describes the relation studied

The milk clotting enzyme of microbial origin, produced by "Chr. has the following form:Hansen", was described as activity (1:50 000) and other indicators in the established certificate. For the experiment, the enzyme activity y = (2339 * 4.203) / (4.203 + x) = 9830.817 / (4.203 + x) (2)of the aforementioned coagulants was reduced to 1:50000. Initial coagulation time is determined by the heuristic (Berridge, 1952) and The influence of the quantity of chymosin with camel origin in

3 -2 -3 3viscometric (Boyanova et al., 2012) methods. the range of variation from 10 to 60 cm .10 .dm and step 10 cm . -2 -3Statistical processing of the data obtained in the course of the 10 .dm on the time of initial coagulation was studied (τic, s).

experiment was carried out using specialized mathematical The determination of τic is identical to the previous experiment. software – SigmaPlot 11.0. These mathematical analyses are used The data obtained from quadruplicate experience are shown in to develop mathematical models (Bethea et al., 1985). Figure 2. The tolerant limits of τic with the use of the coagulant with

10 20camel origin were within a range of variation τic = 539,5 ± 26,2; τic 30 40 50= 328.3 ± 17.1; τic = 191 ± 10.5; τic = 130.3 ± 6.8; τic = 91.5 ± 5.6

60and τic = 69 ± 4.1 s. Figure 2 shows that the variation of τic is not Results and discussionlinear, but is described similar to a hyperbola.

The mathematical processing of data leads to formation of Single factor analysis was conducted in order to reveal the nonlinear regression model, a type of decreasing hyperbola with two influence of the amount of milk clotting enzyme on time of initial and coefficients, similar to that in equation 1. According to the coefficient total coagulation of cow's milk. The main focus of the experiment is Rsqr = 0.9967 obtained, mathematical re-processing was needed, related to enzymes of camel and microbial origin compared with calf also with the use of non-linear regression procedure, but with a chymosin. The influence of the amount of a microbial enzyme of 10,

3 -2 -3 decreasing hyperbola with three coefficients of the form:20, 30, 40, 50, and 60 cm .10 .dm on the time of initial coagulation of cow's milk (τic, s) was studied.

f = y0 + (a * b) / (b + x) (3)The data from quadruplicate experience are shown in Figure 1, as successive trials are indicated by the numbers 1, 2, 3, 4,

According to the correlation coefficient R = 0.9987, Rsqr = respectively. The average sample was also marked on Figure 1 as 0.9973 was obtained and coefficients of significance of the “microbial ”. Tolerant limits of τic using milk clotting enzyme of av.

aforementioned values, respectively, for Ra = 0.9885396; for Rb =

Figure 1. Variation of time for initial coagulation ofcow's milk depending on the amount of milk clottingenzyme of microbial origin

900

3 -2 -3Amount of milk clotting enzyme, cm .10 .dm

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microbial 1microbial 2microbial 3microbial 4microbialaverage

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Figure 2. Variation of time for initial coagulation ofcow's milk depending on the amount of milk clottingenzyme of camel origin

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0.9958005 and Ry0 = 0.9803295, the mathematical model that describing the functions of the type decreasing hyperbola with three correctly describes the relation studied has the following form: coefficients were formed. The equation obtained for variations of τtc

using different amounts of a microbial enzyme is:y = -125.5 + 1379 * x / (9.377 + x) (4)

F = 904.3440 + (6938.3959 * 6.7770) / ( 6.7770 + x) (6)mic

An experiment with the use of bovine chymosin as a control was also conducted. Determining τic is analogous to the previous The coefficient of correlation is high (R = 0.9979), and the experiments. The data obtained are shown in Figure 3. squared correlation coefficient Rsqr = 0.9958. All three factors are

The tolerant limits of τic using calf chymosin for the interval of significant, which indicates the correct description of the process of 10 20 30variation are τic = 631.5 ± 31.8; τic = 384.8 ± 18.7; τic = 265.0 ± equation 6.

40 50 6014.2; τic = 189.0 ± 10.3; τic = 147.5 ± 7.2 and τic = 129.3 ± 6.4 s. Variations of τtc depending of the amount of chymosin with camel origin are described by the equation:

F = 607.3132 + (7557.9404 * 4.5226) / (4.5226 + x) (7)cam

The coefficient of correlation R = 0.9999, and quadratic coefficient Rsqr = 0.9997 are significant and equation 7 describes the process properly. Control test on τtc is made using calf chymosin. The equation obtained is:

F = 804.7503 + (6248.2814 * 6.7524) / (6.7524 + x) (8)calf

The coefficient of correlation is R = 0, 9998, and Rsqr = 0, 9996. 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 Figure 3 shows that the variation of τic is not linear and is described

depending on the amount of enzyme is formed and the results as hyperbole. The obtained data were processed statistically, and

obtained are presented in Figure 5. When microbial and calf 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 = 0.997307 obtained shows that the research process is correctly described.

According to the correlation coefficient R = 0.9987 obtained and coefficients of significance of the aforementioned values, respectively, for Ra = 0.9909003 and for Rb = 0.9909003, the mathematical model that correctly describes the dependence investigated is in the form:

y = (2386 * 3.636) / (3.636 + x) = 8675.496 / (3.636 + 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

3 -2 -3 coagulation of the milk, when amounts higher than 30 cm .10 .dmare 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,

Figure 3. Variation of time for initial coagulation ofcow's milk depending on the amount of calf chymosin

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cal f1cal f2cal f3cal f4cal f average

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Figure 4. Variation of τic and τtc of cow's milkdepending of the amount of microbial, camel andcalf chymosin

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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,

3 -2 -3incorporated in amounts of 10 ÷ 60 cm .10 .dm , primarily in cheese making using streaming coagulant dosing. Mathematical models and the resulting relationship between τic and τtc using chymosin of

3 -2 -3microbial, camel and calf origin in amount of 10 ÷ 60 cm .10 .dm can be used in the regulation of syneresis and preparation of technological diagrams to obtain different types of cheeses.

References

Bansal N, Drake MA, Piraino P, Broe ML, Harboe M, Fox PF and McSweeney PLH, 2009. Suitability of recombinant camel (Camelus dromedarius) chymosin as a coagulant for Cheddar cheese. International Dairy Journal 19, 510-517.Bethea RM, Duran BS and Boullion TL, 1985. Statistical Methods for Engineers and Scientists. New York: Marcel Dekker, Inc 1985, ISBN 0-8247-7227-X.Berridge NJ, 1952. Some observations on the determination of the activity of rennet. The Analyst, 77, 57-62.Boyanova P, Panayotov P, Milenkov B and Dinkov H, 2012. Monitoring of milk acid coagulation by rotational viscometer. Agricultural Science and Technology, 4, 4, 456 - 458, ISSN: 1314-412X.Fox PF and McSweeney PLH, 1997. 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, 1-49.Kappeler S, van den Brink H, Nielsen H, Farah Z, Puhan Z, Hansen E and Johansen E, 2006. Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk. Biochemical and Biophysical Research Communications 342, 647-654.Montgomery D, Peck E and Vining G, 2006, Introduction to Linear Regression Analysis, Wiley-Interscience; 4th edition.Rodriguez Е, 2013. 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, 1991. Ultrafiltered Gouda Cheese: Effects of Preacidification,

chymosin are used, the obtained ratios are similar, as the differences Diafiltration, Rennet and Starter Concentration, and Time to Cut. ranged from 1.8 to 27.8%. Ratios using camel chymosin vary from Journal of Dairy Science, 74, 9, 2809- 2819.6.20 to 27.38. These ratios show a rapid rate of coagulation during

Figure 5. Variation of the ratio τic and τtc of cow's milkdepending on the amount of microbial, camel and calfchymosin

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25

20

15

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calf microbialcamel

Figure 6. Variation of τic using microbial and calf chymosinin relation with τic of camel chymosin and variation of τtcobtained by microbial and calf chymosin in relation with τtc of camel chymosin

3 -2 -3Enzyme amount, cm .10 dm

ic - mic/camel

ic - mic/camelic - calf/camel

ic - calf/camel

4

3

2

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Figure 7. Variation of τic using microbial and camelchymosin in relation with τic of calf chymosin andvariation of τtc obtained by microbial and camelchymosin in relation with τtc of calf chymosin

ic - mic/camel

ic - mic/camelic - camel/calf

ic - camel/calf

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Review

Genetics and Breeding

Nutrition and Physiology

Fibromelanosis in domestic chickens H. Lukanov, A. Genchev

Rumi and IPK Nelina – new cotton varietiesA. Stoilova, Hr. Meluca

Drying of seeds from common wheat (Triticum aestivum L.) by using Silica gel forex situ storageP. Chamurlyisky, N. Tsenov, S. Stoyanova

Breeding evaluation of newly stabilized lines of maizeV. Valkova

Apricot breeding for resistance to SharkaV. Bozhkova, S. Milusheva

Dry matter accumulation in the varieties of wheat (Triticum aestivum L.)according to previous cropA. Ivanova, N. Tsenov

®Reproductive performance of weaning saws after treatment with FertipigS. Dimitrov, G. Bonev

Reproductive performance of Polish Large White and Polish Landrace sowsB. Szostak, V. Katsarov

Effect of the feeding of products stimulating the development of bee coloniesR. Shumkova, I. Zhelyazkova

Investigations on kidney function in mulard ducklings with experimental aflatoxicosisI. Valchev, N. Grozeva, L. Lazarov, D. Kanakov, Ts. Hristov, R. Binev, Y. Nikolov

Rumen fermentation in yearling rams fed different rationsV. Radev

Effect of different lipid and protein dietary levels on rumen ciliate fauna and cellulolytic activity in yearling ramsV. Radev, I. Varlyakov, R. Mihaylov

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Production Systems

Agriculture and Environment

Product Quality and Safety

Efficacy and selectivity of antibroadleaved herbicides at durum wheat against volunteers of coriander, Clearfield canola, Clearfield sunflower and ExpressSun sunflowerG. Delchev

Investigations on friction coefficients of cow hooves with different dairy farm floor typesT. Penev, Z. Manolov, I. Borissov, V. Dimova, Tch. Miteva, Y. Mitev, V. Kirov

Productivity of irrigation cotton cultivated under different inter-row spacesI. Saldzhiev, A. Muhova

Stability evaluation of mixtures among preparations with different biological effect on the basis of grain yield in spring vetchG. Delchev, N. Georgieva, I. Nikolova

Biological activity of plant protection products against Tuta absoluta (Meyrick) in tomato grown in greenhousesN. Valchev, V. Yankova, D. Markova

Changes of some agro-chemical parameters of Pellic Vertisol (FAO) soil type in growing cereal crops under organic system of agricultureV. Koteva

Carcass composition and meat quality in lambs reared indoors and on pasture T. Popova, P. Marinova

Fatty acids and lipid indices of buffalo milk yogurtN. Naydenova, T. Iliev, G. Mihaylova

Effect of supplementary honey and artificial sugar feeding of bees on the composition of royal jellyR. Balkanska, I. Zhelyazkova, M. Ignatova, B. Kashamov

Influence of the amount of milk clotting enzyme with microbial and camel origin on the coagulation time of cow's milkP. Panayotov, K. Yoanidu, P. Boyanova, B. Milenkov

Determining chlorophyll and carrotenoid content in Bombyx mori L. excreta by Near Infrared SpectroscopyS. Atanasova, M. Panayotov, D. Pavlov, M. Duleva

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tables and figures, accompanied by the statistical parameters needed for the evaluation. Data from tables and figures should not be repeated in the text.Tables should be as simple and as few as possible. Each table should have its own explanatory title and to be typed on a separate page. They should be outside the main body of the text and an indication should be given where it should be inserted.Figures should be sharp with good contrast and rendition. Graphic materials should be preferred. Photographs to be appropriate for printing. Illustrations are supplied in colour as an exception after special agreement with the editorial board and possible payment of extra costs. The figures are to be each in a single file and their location should be given within the text. Discussion: The objective of this section is to indicate the scientific significance of the study. By comparing the results and conclusions of other scientists the contribution of the study for expanding or modifying existing knowledge is pointed out clearly and convincingly to the reader.Conclusion: The most important conse- quences for the science and practice resulting from the conducted research should be summarized in a few sentences. The conclusions shouldn't be numbered and no new paragraphs be used. Contributions are the core of conclusions. References:In the text, references should be cited as follows: single author: Sandberg (2002); two authors: Andersson and Georges (2004); more than two authors: Andersson et al.(2003). When several references are cited simultaneously, they should be ranked by chronological order e.g.: (Sandberg, 2002; Andersson et al., 2003; Andersson and Georges, 2004).References are arranged alphabetically by the name of the first author. If an author is cited more than once, first his individual publications are given ranked by year, then come publications with one co-author, two co-authors, etc. The names of authors, article and journal titles in the Cyrillic or alphabet different from Latin, should be transliterated into Latin and article titles should be translated into English. The original language of articles and books translated into English is indicated in parenthesis after the bibliographic reference (Bulgarian = Bg, Russian = Ru, Serbian = Sr, if in the Cyrillic, Mongolian =

Мо, Greek = Gr, Georgian = Geor., Japanese = Jа, Chinese = Ch, Arabic = Аr, etc.)The following order in the reference list is recommended:Journal articles: Author(s) surname and initials, year. Title. Full title of the journal, volume, pages. Example:Simm G, Lewis RM, Grundy B and Dingwall WS, 2002. Responses to selection for lean growth in sheep. Animal Science, 74, 39-50Books: Author(s) surname and initials, year. Title. Edition, name of publisher, place of publication. Example: Oldenbroek JK, 1999. Genebanks and the conservation of farm animal genetic resources, Second edition. DLO Institute for Animal Science and Heal th, Netherlands.Book chapter or conference proceedings: Author(s) surname and initials, year. Title. In: Title of the book or of the proceedings followed by the editor(s), volume, pages. Name of publisher, place of publication. Example: Mauff G, Pulverer G, Operkuch W, Hummel K and Hidden C, 1995. C3-variants and diverse phenotypes of unconverted and converted C3. In: Provides of the Biological Fluids (ed. H. Peters), vol. 22, 143-165, Pergamon Press. Oxford, UK.Todorov N and Mitev J, 1995. Effect of level of feeding during dry period, and body condition score on reproductive perfor-

thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, Sept.11 – 14, Berlin, Germany, p. 302 (Abstr.).Thesis:Penkov D, 2008. 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, 314 pp.

The Editorial Board of the Journal is not responsible for incorrect quotes of reference sources and the relevant violations of copyrights.EthicsStudies 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”.

Volume 5, Number 3September 2013