ISSN 1313 - 8820Volume 5, Number 4

December 2013

2013

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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)

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Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2013

ISSN 1313 - 8820 Volume 5, Number 4December 2013

Influence of key factors on the time of initial coagulation of cow's milk using milk-clotting enzyme of camel origin

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

1Department of Milk and Milk 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. The process of rennet coagulation of cow's milk, obtained using the milk-clotting enzyme of camel origin was studied. The rate of the coagulation process conducted by enzymes of various origins is also different. Coagulation process was evaluated using chymosin of camel origin. The influence of the amount of milk-clotting enzyme, the amount of calcium chloride and the active acidity (pH) of the milk on the initial coagulation time were defined by conducting a multifactorial experiment with three factors at two levels. The time of the initial coagulation of milk is determined by the use of heuristic methods. Determination of levels of variation was made on the basis of preliminary conducted experiments and in major industrial values. With the help of mathematical modeling the possibility of studying the process of rennet coagulation of milk and the mutual impact of selected technologically significant factors was established. As a result of analyzing the data obtained, very strong influence of active acidity (pH), strong effect of the amount of milk-clotting enzyme and moderate influence of the amount of CaCl on the time of initial coagulation of cow's milk were established. The values obtained allow for analysis, as well as to 2

predict the course of rennet coagulation of cow's milk carried by chymosin of camel origin.

Keywords: rennet coagulation, chymosin of camel, calcium chloride, acidity

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No 4, pp , 2013463 - 466

Introduction process and maintain certain quality characteristics of the final product.

There are numerous factors, which influence the course of the enzymatic coagulation of milk. During the preparation of cheese, the

Material and methods process of coagulation can be controlled primarily by the addition of varying amounts of calcium dichloride, milk-clotting enzyme and by modification of the active acidity of milk. On this basis, multivariable The process of initial coagulation of milk was studied by a models for initial coagulation in cow and sheep milk were previously multifactorial experiment. The influence of the active acidity of the developed (Castillo, 2006). Different types of milk-clotting enzyme milk and adding amounts of calcium dichloride and milk-clotting are the main factor that varies in already drawn mathematical enzyme was determinated. During the experiment cow's milk is models (Panayotov et al., 2012). As a milk-clotting enzyme originally used, pasteurized at 65°C and cooled to a temperature of the extract of dried stomachs of mammals was only used, coagulation with the following parameters: pH = 6,55; titratable comprising primarily the enzyme chymosin, which coagulates the acidity – 19; fat – 4,40 %; solid non-fat (SNF) – 9,29 %; protein – 3,51

-3milk casein (Knez et al., 1999). Deficiency of this specific enzyme, %; density – 1,030 kg.dm . For each type of enzyme, during the along with the rapid increase in the world production of cheese, multifactorial experiment, 18 samples containing 800 ml of cow's creates a need for new sources of milk-clotting enzymes and, milk were prepared. The milk was heated to a temperature of 38 ÷ 40 therefore, at the present time, the use of various alternative enzymes °C and then cooled to a temperature of 36 ÷ 38 °C, the optimum primarily microbial chymosin. Another alternative for industrial use is temperature for the milk-clotting enzyme activity. The renneted milk, the chymosin of camel origin. It is proved that camel chymosin has in an amount of 300 ml, was then divided in two glass containers. characteristics different from those of the cow. Thus, it has up to 70% The remaining amount of milk was used for “the small sample”, stronger milk-clotting activity than the cow chymosin, and only 20% which serves to heuristically determine initial coagulation. The of the typical enzyme specific protease activity, which leads to 7-fold samples were placed in a water bath at a temperature of 35 ÷ 38 °C higher ratio of coagulation (Kappeler et al., 2006). and were analyzed.

Variations of the active acidity of milk from 6,6 to 5,6, cause a Using mathematical and statistical study (Bethea, 1985; Melas, significant reduction in the time of initial coagulation - about 30 times 2006), the main technological factors allow for the evaluation of the less time to coagulate the milk (MeManhon et al., 1984). Addition of interval in which they actually affect the processes. From the calcium chloride to the milk before coagulation in the preparation of technological factors which affect the time for initial coagulation of cheese, reducing the time to initial coagulation, and the established the milk were selected the dose of calcium dichloride and the milk-quantities of dry calcium dichloride added are about 0,2% clotting enzyme of camel origin and the variation of the active acidity (MeManhon end Brown, 1984). of milk.

In order to intensify and modernize the technological process, Of dry calcium dichloride was prepared a 50% solution that was mathematical models were created and used to describe the used in conducting multifactorial experiments. Camel chymosin with influence of various factors on the enzymatic coagulation process, activity 1:70000 was used as a milk-clotting enzyme. To conduct and through their application it is possible to manage the coagulation experiments that activity is adjusted to 1:10000 and the required

463

* e-mail: katqioanidu@abv.bg

464

amount of coagulant is diluted with water to not less than 1:10.Statistical processing of the data obtained in the course of the

quadruplicate multifactorial experiment was carried out using specialized mathematical software – SigmaPlot 11.0.

Results and discussion

The time of initial coagulation is regarded as y, s and x , x and x 1 2 3

are indicated as factors of experiment. According to a study on a prior information and the preliminary experiments, input factors of the selected model adopted for the design of experiments are the following independent factors: x , x and x , and x – milk-clotting 1 2 3 1

3 -2 -3 -2 -3enzyme, cm .10 dm ; x – calcium chloride , g. 10 dm ; x – pH of 2 3

the milk.The center of the experiment using the developed plan is

3 -2 -3 -2 -3considered as x = 30 cm .10 dm , x = 15 g. 10 dm , x = 6,4 in the 10 20 303 -2 -3 -2 -3range of variation: Δ x = 10 cm .10 dm ; Δ x = 10 g. 10 dm ; Δ x = 1 1 3

0,2. Calf chymosin with activity of 1:10000 is used to determine the limits of the experiment.

Monofactorial experiments conducted on the influence of the active acidity of cow's milk, the amount of milk-clotting enzyme and calcium dichloride within the presumed ranges of variation show linearity in conducting multifactorial experiment, which in turn causes the correct selection of the ranges of variation of the selected factors (Figures 1, 2 and 3).

A matrix of planning was designed for the multifactorial 3experiment – 2 , which is presented in Table 1. Four parallel

experiments were conducted, the results of which, together with the working matrix are shown in Table 2.

As a mathematical model for describing the object of the study were selected polynomial functions of the first degree in the form of regression equations with four and eight coefficients.

ŷ = b + b X + b X + b X (1)0 1 1 2 2 3 3

ŷ = b + b X + b X + b X + b X X + b X X + b X X + b0 1 1 2 2 3 3 12 1 2 13 1 3 23 2 3 123

X X X (2)1 2 3

where ŷ is a function of the response of the explored subject, b , b , 0 1

b , b , b , b , b , b – coefficients of the regression equation.2 3 12 13 23 123

∑ y = 4733 (3)Nj av.2The dispersion of the essay is determined – S v 1÷8

2 2 2S = ∑ (y – y ) / m – 1 = ∑ (y – y ) / 3 (4)v 1÷8 Nj i av. Nj i av.

2 2∑ S = 15468,69 и S = 5774,25Nj v v max Figure 1. Variation of time for initial coagulation (s) of cow's milk depending on the active acidity (рН)

700

Time, scamel chymosin

600

500

400

300

200

1006.8 6.6

Active acidity, рН

6.4 6.2 6

Figure 2. Variation of time for initial coagulation (s) of cow's milk depending on the amount of milk- clottingenzyme of camel origin (cm3.10-2 dm-3).

700

Time, scamel chymosin

600

500

400

300

200

1000 10

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

20 30 40

Figure 3. Variation of time for initial coagulation (s) ofcow's milk depending on the amount of calcium

-2 -3chloride (g.10 dm ).

700

Time, scamel chymosin

600

500

400

300

200

1000 10

3 -2 -3Amount of calcium chloride, cm .10 dm

20 30 40

Table 1. Matrix of planning of the experiment

№

1

2

3

4

5

6

7

8

1

-1

1

-1

1

-1

1

-1

1

1

-1

-1

1

1

-1

-1

1

1

1

1

-1

-1

-1

-1

X1 X2 X3

465

2 2 The critical value of the Fisher criteria is determined according G = S / ∑ S = 0,37 (5)н v max Nj v 2 to the tables in the confidence level α (0,01) with the degrees of The dispersion of the experiment is also determined – S .o

2 2 freedom, N-1 = 8–4 = 4 and N. (m–1) = 24 = 8.3, as F (α, N–1, cr.S = ∑ S / N = 15468,69 / 8 = 1933,58, where (6)o Nj vN.(m–1)) = 4,22, and F > Fn crN = 8 и m – 1 = 4 – 1 = 3.

Therefore, with a confidence level α (0,01) the mathematical X , X and X are encrypted (to be used in a natural state a 1 2 3model does not describe the studied process, it is therefore

recalculation is needed).necessary for it to be completed. 2The dispersion of the experiment (S ) is defined considering o ŷ = b0 + b X + b X + b X + b X X + b X X + b X X + 2 1 1 2 2 3 3 12 1 2 13 1 3 23 2 3the dispersions of parallel experiments (S , v = 1 ÷ 8). The v b X X X (15)123 1 2 32calculated values are shown in Table 2. A comparison of all S is v Complementary factors and observable values received using

made, corresponding to the Cochrane criteria for equality of all Student's criteria are:b = 22,900 t = 1,48 b = 12,100 t = 0,77 12 н12 123 н123dispersions and proof if the following condition is correct: b = -120,650 t = 7,76 b = -25,075 t = 1,7113 н13 23 н23G < G (7)n cr. Therefore, b and b are significant coefficients, and the 13 23 When G = 0,37, Gcr. is determined using mathematical tables n complementary mathematical model type is: according to confidence level α (0,05), and two degrees of freedom ŷ = b0 + b X + b X + b X + b X X + b X X = 591,625 – 1 1 2 2 3 3 13 1 3 23 2 3m–1 (4–1 = 3) and N (8), meaning Gcr. (α, m-1, N) = 0,4377. The 176,850X – 55,03X + 206,275X – 120,650X X – 25,075X X (16)1 2 3 1 3 2 3inequality is correct and therefore all variances are equal.

The dispersion of the complementary model is equal to: b = (∑ y )/N (8) 2 20 Nj iav. S = (m/N-к)).∑ (y – y ) = (4/(8-6)). 5407 = 10814 (17)ad Nj iav. imodl.b = (∑ y X )/N (9) 2 2 j Nj iav i F = S / S = 5,59 (18)n av. о

The coefficients are identified: b = 591,625; b = -176,850; b = -0 1 2 The critical value of the Fisher criteria is determined according 55,025; b = 206,275.3 to the tables in the confidence level α (0,01) with the degrees of

2The dispersions of the experiment S (1933,58) and the o freedom N-к = 8 – 6 = 2 and N.(m–1) = 8.3 = 24, and F (α, N–к, cr. 2coefficients S , are obtained as:bj N.(m–1)) = 5,61 и F < F Therefore, with a confidence level α (0,01) n cr.

2 2S = S / N = 241,7 (10)bj o the model is adequate and properly describes the studied process.2 -2S = (S ) = 15,55 (11) The resulting correlations 12 and 16 clearly show that the most bj bj

significant effect on the timing of initial coagulation of cow's milk, s Using the Studant criteria, an assessment of the significance of has active acidity (pH). The requirement of completing model 12 the coefficients was made. The observable values of criteria were shows that the combined effect involving the active acidity had some defined as: significant impact on the adequacy of the model. Analysis of the t = abs(b) / S , where j = 1÷4.nj j bj

results determined that the combined effect of the amount of milk-The critical value of the Student's criteria is determined clotting enzyme and calcium dichloride and that of all three factors is according to the tables in the confidence level α (0,05) with a degree insignificant.of freedom N. (m–1) = 8.3=24, as t = 1,711, and the cr. (α, N.(m-1))

Using the same algorithm and part of the obtained results are observable values in the first equation are: t = 11,37; t = 3,54; t = n1 n2 n3 2composite plans for multifactorial experiment as well – 2 where x = 313,25.constant, respectively, for pH = 6,6 and pH = 6,2.The condition for the significance of a precise coefficient is t > nj

The active acidity is constant, i.e. x = constant = 6,6 in cow's 3t , therefore, all coefficients are significant and the equation cr. (α, N.(m-1))

milk, free of developed secondary acidity. Experimental data from has the following form:the analysis are presented in Table 3.y = b0 + b X + b X + b X = 591,625 - 176,850X - 55,03X + 1 1 2 2 3 3 1 2

b = 797,900; b = –297,500; b = 35,000; b = –80,1000 1 12 2206,275X (12)3

Therefore, the significant coefficients are b b и b , and the 1, 2 12Verification of the adequacy of the obtained mathematical model type is:model is made using Fisher criteria:

2 2 ŷ = 797,900 – 297,500X - 80,100X + 35,000 X X (19)1 2 1 2S = (m/N-k)).∑ (y – y ) (13)ad. Nj iav. imodl. 2 2F = S / S = 0 / 2926,15 = 0n ad. оwhere: m – number of parallel experiments; N – number of F is defined according to the tables in the confidence level α cr.experiments; к – number of significant coefficients (к ≤ n-1); n –

(0,01) with the degrees of freedom N-1 = 4 - 3 = 1 и N.(m-1) = 4.3 = number of coefficients.2 2 12, and F (α, N-1, N.(m-1)) = 4,22. The inequality is satisfied F < Fcr. n cr.F = S / S = 126637,44 / 1933,58 = 65,51 (14)n ad. о

3Table 2. Experimental data from the multifactorial experiment – 2 using milk- clotting enzyme of camel origin

№

1

2

3

4

5

6

7

8

х1

40

20

40

20

40

20

40

20

х2

25

25

5

5

25

25

5

5

х3

6.6

6.6

6.6

6.6

6.2

6.2

6.2

6.2

y1

465

985

547

1216

313

402

350

484

y2

492

1028

586

1289

333

431

374

521

y3

469

997

561

1232

319

410

358

496

y4

395

911

488

1105

275

360

311

428

yav1÷4

455.3

980.3

545.5

1210.5

310.0

400.8

348.3

482.3

2Sv 1÷8

1754.92

2459.59

1715.83

5774.25

614.67

887.59

716.25

1545.59

Ymod1÷4

566.5

920.2

676.55

1030.25

153.0

506.7

263.05

616.75

Ymod1÷6

420.6

1015.3

580.5

1175.5

299.2

411.6

359.1

471.5

466

The resulting model 19 is adequate with confidence level α influence of time on initial coagulation of cow's milk by chymosin of (0,01) and correctly describes the studied process in milk without a camel origin, the active acidity of milk has the most significant effect, developed secondary acidity under 6,6. followed by the amount of milk-clotting enzyme and quantities of

The time dependence of the initial coagulation of the milk and calcium dichloride.the amount of milk-clotting enzyme dominates. The mutual influence The full factorial experiment with two factors of two levels and of the amount of milk-clotting enzyme and the amount of calcium the resulting regression equation 19 show that the dominant factor in dichloride is significant. the timing for initial coagulation of cow's milk with pH=6,6 is the

For milks, in the initial phase of development of secondary amount of chymosin of camel origin.active acidity, the active acidity can be considered constant, i.e. x = Analysis also demonstrated the relevance of the influence of 3

2 the amount of milk-clotting enzyme and that of calcium dichloride. In constant = 6,2. Experimental data from multifactorial experiment – 2 equation 20 low acidity (pH = 6,2) leads to ignore the relevance of the and pH= 6,2 are presented in Table 4.combined effects of the quantity of the enzyme and calcium b = 385,35; b = -56,20; b = -29,95; b = 10,800 1 2 12

dichloride.The analysis identified as significant factors b and b , and the 1 2

model has the following form:ŷ = b0 + b X + b X + b X X = 385,350 – 56,200X – 1 1 2 2 12 1 2 1

References29,950X (20)2

Supplementing the model by the coefficient b does not 12

Castillo M, 2006. The effect of temperature and inoculums significantly improve the results.2 2 concentration on rheological and light scatter properties of milk F = S / S = 121,0 / 941,03 = 0,13n ad. о

coagulated by a combination of bacterial fermentation and F is defined tabulated according to the confidence level α cr. chymosin. Cottage cheese- type gels/ M. Castillo, J.Lucey, F.Payn. (0,01) with the degrees of freedom, N -1 = 4 – 3 = 1 and N.(m–1) = 4.3

International Dairy Journal,16,131-146.= 12, and F (α, N–1, N.(m–1)) = 4,22. The inequality Fn < Fcr. is cr. Bethea R.M., B.S. Duran and T.L. Boullion. 1985. Statistical satisfied. Therefore, with a confidence level α (0,01) model 20 is Methods for Engineers and Scientists. New York: Marcel Dekker, Inc adequate and properly describes the studied process in milk with a 1985, ISBN 0-8247-7227-X.developed secondary acidity within 6,2. MeManhon DJ and Brown RJ, 1984. Enzymic Coagulation of

The analysis of cow's milk with a developed secondary acidity Casein Micelles: A Review. Department of Nutrition and Food (pH – 6,2), results in an adequate mathematical model in which the Sciences Utah State University Logan 84322. Journal of Dairy major factors determining the time of initial coagulation are the Science, 67, 5, 919-929.amount of milk-clotting enzyme and calcium dichloride. It has been Knez V and Sedlackova H, 1999. Cheese and Preparation of found that the complementation of the model does not improve the Cheese Curd. SNTL, Prague.results obtained and in that case, the mutual influence of the Melas VB, 2006. Functional Approach to Optimal Experimental aforementioned two factors is not important for the progress of the

Design. Lecture Notes in Statistics 184. Springer-Verlag. ISBN 0-research process of initial coagulation of cow's milk.

387-98741-X.Panayotov P, Boyanova P and Milenkov B, 2012. Study on the coagulation process of cow's milk by conducting a full factorial experiment. Paper of MGPU, 2 ,13, 81-84.Conclusions Kappeler SR, van den Brink H, Rahbek-Nielsen H, Farah Z,

The conducted multifactorial experiment with three factors at Puhan Z, Hansen E and Johansen E, 2006. Characterization of two levels and the resulting regression equation 16 shows that the

recombinant camel chymosin reveals superior properties for the obtained mathematical model correctly reproduces the studied

coagulation of bovine and camel milk. Biochemical and Biophysical process of the time of initial coagulation of cow's milk by chymosin of Research Communications 342, 647-654.camel origin. It has been found that, within the indicated interval of

2Table 3. Experimental data from multifactorial experiment – 2 using milk- clotting enzyme of camel origin at рН 6,6.

2Table 4. Experimental data from multifactorial experiment – 2 using milk- clotting enzyme of camel origin at рН 6,2

№

1

2

3

4

№

1

2

3

4

х1

1

-1

1

-1

х1

1

-1

1

-1

х2

1

1

-1

-1

х2

1

1

-1

-1

х1

40

20

40

20

х1

40

20

40

20

х2

25

25

5

5

х2

25

25

5

5

y1

465

985

547

1216

y1

313

402

350

484

y2

492

1028

586

1289

y2

333

431

374

521

y3

469

997

561

1232

y3

319

410

358

496

yav1÷4

455.3

980.3

545.5

1210.5

yav1÷4

310.0

400.8

348.3

482.3

2Sv 1÷8

1754.92

2459.59

1715.83

5774.25

2Sv 1÷8

614.67

887.59

716.25

1545.59

Ymod1÷4

455.3

980.3

545.5

1210.5

Ymod1÷4

299.2

411.6

359.1

471.5

y4

395

911

488

1105

y4

275

360

311

428

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Study of bee honey by spectral analysis in the near infrared spectrumI. Zhelyazkova, S. Atanasova , K. Elencheva – Karaneycheva

Comparative GC/MS analysis of lavender (Lavandula angustifolia Mill.) inflorescence and essential oil volatilesT. Zagorcheva, S. Stanev, K. Rusanov, I. Atanassov

Influence of key factors on the time of initial coagulation of cow's milk using milk-clotting enzyme of camel originP. Panayotov, K. Yoanidu, P. Boyanova, B. Milenkov

Production Systems

Agriculture and Environment

Product Quality and Safety

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Instruction for authors

Preparation of papersPapers shall be submitted at the editorial office typed on standard typing pages (A4, 30 lines per page, 62 characters per line). The editors recommend up to 15 pages for full research paper ( including abstract references, tables, figures and other appendices)The manuscript should be structured as follows: Title, Names of authors and affiliation address, Abstract, List of keywords, Introduction, Material and methods,Results, Discussion, Conclusion, Acknowledgements (if any), References, Tables, Figures.The title needs to be as concise and informative about the nature of research. It should be written with small letter /bold, 14/ without any abbreviations. Names and affiliation of authorsThe names of the authors should be presented from the initials of first names followed by the family names. The complete address and name of the institution should be stated next. The affiliation of authors are designated by different signs. For the author who is going to be corresponding by the editorial board and readers, an E-mail address and telephone number should be presented as footnote on the first page. Corresponding author is indicated with *.Abstract should be not more than 350 words. It should be clearly stated what new findings have been made in the course of research. Abbreviations and references to authors are inadmissible in the summary. It should be understandable without having read the paper and should be in one paragraph. Keywords: Up to maximum of 5 keywords should be selected not repeating the title but giving the essence of study. The introduction must answer the following questions: What is known and what is new on the studied issue? What necessitated the research problem, described in the paper? What is your hypothesis and goal ?Material and methods: The objects of research, organization of experiments, chemical analyses, statistical and other methods and conditions applied for the experiments should be described in detail. A criterion of sufficient information is to be possible for others to repeat the experi-ment in order to verify results.Results are presented in understandable

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 4December 2013