Summary Al-Khwarizmi Engineering Journal Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) The Optimum Conditions for Production of Soya Peptone by Acidic Hydrolysis of Soya Proteins Mohammed B. A. G. AL-Bahri * , Safa A. AL-Naimi** and Sundus H. Ahammed *** * Department of Biochemical Engineering/Al-Khwarizmi College of Engineering/University of Baghdad ** Chemical Engineering Department/ University of Technology *** Ministry of Sciences and Technology (Received 6 March 2008; accepted 25 March 2009) Abstract This study was carried out to obtain the optimum conditions necessary for the process of soya protein hydrolysis by using hydrochloric acid (as a chemical catalyst) instead of the papain enzyme (as a biological catalyst), for the production of soya peptone. These conditions are implemented to test the effect of the variables of the process of hydrolysis on the nature and quality of the product. The production of soya peptone was studied for their importance in the process of preparing and producing the culture media used in medical and microbiological laboratories. The process of production of soya peptone includes four main stages:  Preparing the defatted soya flour, firstly.  The soya protein hydrolysis, secondly.  Purifying the product, thirdly , and then,  Drying the product, finally. By following the procedure of the present study, the optimum conditions for the process of soya proteins hydrolysis have been reached in present study are:  Optimum concentration for the hydrochloric acid solution is 1N.  Optimum hydrolysis process temperature ranged between 50-53C.  Optimum period of hydrolysis time ranged between 17.189-19.97 hr. The productivity of soya peptone was 38.071%, by following the procedure and the optimum production conditions of the present study. Keyword: Soya Peptone, Soybean Peptone, Vegetable Peptone. 1. Introduction Soya peptone is a vegetable peptone (1) , It is obtained by the papain hydrolysis (enzymatic hydrolysate) of soya flour (1-3) , prepared under controlled conditions especially for use in microbiological procedures (2) . It is recommended for use in media for the cultivation of a large variety of organisms, including fungi, and is also used in media for microbiological assay (2) . In addition to its nitrogen constituent, this peptone contains a high naturally occurring carbohydrate of the soybean (2) . Typical analysis of soya peptone produced by Oxoid laboratory is shown in Table (1). Peptones are proteins partially digested; they are prepared by the enzymic or acidic hydrolysis of proteinaceous material. These hydrolysates contain secondary protein derivatives such as polypeptides, dipeptides and amino acids. They provide a readily assimilable source of nitrogen which is water soluble, does not coagulate on heating, and is therefore particularly suitable for inclusion in microbiological culture media (1)(4) , hydrolysis proteins yield metaproteins, proteoses, peptones, polypeptides, and finally the amino acids (2) . They are three types of peptones, these types are meat peptones (such as peptone bacteriological, tryptose, etc), vegetable peptone Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 2 (soya peptone), and casein and other milk derived peptones (2, 3) . The hydrolysis of proteins, which breaks them down to their constituent amino acids and peptides, can be achieved by the use of strong acids, strong bases or proteolytic enzymes, there are three main methods of hydrolysis of proteins (3) . Table 1, Average Analysis of Oxoid Soya Peptone (1, 3) . Typical Analysis (w/w%) Oxoid Soya Peptones (1988) Oxoid Soya Peptones (1995) Moisture % 4.1 5.8 Ash% 7.8 13.5 Amino Nitrogen (AN%) 3.1 2.3 Total Nitrogen (TN%) 10.2 9.1 AN/TN% 30 25 pH of 2% solution (after autoclaving) 7 7.2 NaCl% 0.8 0.4 Hydrolysis with strong mineral acid or base (chemical catalysts of biological reactions) is nonspecific, attacking all peptides bonds , degrading proteins and polypeptides to low chain length peptides and amino acids (3) , and also produces a large number of fragments (5) . In this process all peptide bonds are attacked and in theory, complete break down into component parts could be obtained (3) . Acid or base treatment of plant (soybean, corn) or animal (casein) proteins brings about desirable changes in flavor, texture, and solubility. Such treatments also destroy toxins and trypsin inhibitors and are used to prepare protein isolates (6) . One of the principals advantages of acid as compared with base hydrolysis is that the optical activity of the amino acids is not destroyed in the process (7) , on the other hand, acid hydrolysis destroys tryptophan and partially destroys cystine, serine, and threonine. Asparagine and glutamine are converted to their acidic form (3, 7) , and a series of reaction may also take place between carbohydrates and amino acids (Maillard reaction) which gives rise to very dark-brown decomposition products, called “humin” often toxic to the growth of microorganisms (6, 7) . Partial hydrolysis of protein with acids or bases produces mixture of -amino acids, peptides, and polypeptides (8) , as shown in the Figure 1 . CH C H N CH O C N H O CH C O H N CH H2O H + or - OH amino acid units section of a protein chain Mixture of polyeptides, peptides, andamino acids partial hydrolysis R''' R'' R' R Fig.1. Diagram of Partial Hydrolysis of Protein with Acids or Bases Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 3 But complete hydrolysis of proteins with acids or bases, produces mixtures of -amino acids as the principle products (8) , as shown in the Figure 2. CH C H N CH O C N H O CH C O H N CH H2O H + or - OH amino acid units section of a protein chain Mixture of amino acids complete hydrolysis R''' R'' R' R H2N C COOH R (R',R'',etc) H Fig.2. Diagram of Complete Hydrolysis of Protein with Acids or Bases Another method for hydrolysis of proteins by using proteolytic enzymes (biological catalysts) acts on proteins under less severe conditions, they will function at much lower temperatures, and at normal pressure and are usually specific to the peptide bond they will attack (3) . Enzymes commonly used are pepsin, papain, and pancreatin (3) , as shown in the Figure 3. Fig3. Diagram of Enzymatic Action (3) Peptone are proteins partially digested, they are prepared by the enzymic or acidic hydrolysis of proteinaceous material. These hydrolysates contain secondary protein derivatives such as polypeptides, dipeptides and amino acids. They provide a readily assimilable source of nitrogen which is water soluble, does not coagulate on heating, and is therefore particularly suitable for inclusion in microbiological culture media (1) . The hydrolysis of a protein molecule is a gradual process by which the gigantic molecule of protein is converted in to products of successively lower molecular weight (4) , hydrolysis proteins yield metaproteins, proteoses, peptones, Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 4 polypeptides, and finally the chemically simpler amino acids and their analogs (2) . The degree (rate) of hydrolysis of proteins (DH %) is measured by the number of peptide bonds cut, divided by the total number of peptone bonds, multiplied by a hundred and is calculated by the formula of Equation (1) (3) . DH %=[(AN of hydrolysis protein – AN of protein) / TN of protein ] × 100 … (1) Nageli is credited with earliest publications (1880-1882) describing the requirements of microorganisms for a protein component, which he called, as “peptone” (3) . Peptone prepared expressly for bacteriological purposes was first introduced by Difco laboratories in (1914) after many years of preliminary study. This was followed several years later by proteose peptone, and in more recent years other peptones have been added to the group. All peptones are prepared today in the same manner as when first developed (2) . In (1924) Oxoid laboratories were developed the use of culture media. This was also the period when Oxoid laboratories increased investigation into enzymic and acid hydrolysis of meat and vegetable proteins to increase flavor and amino nitrogen content. This work eventually led to the familiar peptones (3) . BDH Laboratory (1985) produces only bacteriological peptone by peptic digest of muscles and animal tissue for general bacteriological purposes (9) . Difco laboratory (1964) produces soya peptone by enzymatic hydrolysate of soya flour (2) , and Sigma laboratory (1988) produces it by enzymatic hydrolysate of soya flour with the typical analysis (total nitrogen (TN) 9.4%, amino nitrogen (AN) 1.8% w/w, AN/TN ratio 20% (10) , and Oxoid laboratory (1988) produces soya peptone also by enzymatic (papain) hydrolysate of soya flour with the typical analysis (TN 10.2% (w/w), AN 3.1% (w/w), AN/TN ratio 30%) (1) , but typical analysis of soya peptone produced by the same company in (1995) is (TN 9.1% (w/w), AN 2.3% (w/w), AN/TN ratio 25%) (3) . The United Stated Pharmacopoeia (USP 1990) depended only on soya peptone produced by papaic digest of soya flour (11) . But soya peptone prepared expressly for bacteriological purposes was first introduced by Ibn-Al Beitar Center (12) in (1993) by using acidic hydrolysate of soya flour after simple preliminary study. They are prepared soya peptone by simple procedures without any typical analysis of the final product (12) . The aim of present study is the determination of the optimum conditions for preparation of soya peptone, depends on the partial hydrolysis of defatted soya flour by using hydrochloric acid (acid hydrolysis ) instead of papain (enzyme hydrolysis). 2. Manufacture of Peptones The basic steps for preparation of peptone are: 1. Preparation of raw protein materials. 2. Hydrolysis of protein with enzymes or acids. 3. Separation unites to purification of hydrolysis product. 4. Drying the final product to powder form (3) . 3. Materials and Methods 3.1Experimental Trials After clarifying and studying the process of soya protein hydrolysis, by studies the relationship between concentration of acid (HCl) solution, temperature, and time of hydrolysis of soya proteins, and effect of these process variable on the rate of soya proteins hydrolysis , and then, studying the relationship between DH% and TN,AN and AN/TN% ratio of hydrolysized product of soya proteins, as described in a pervious study (13) .The present study which was represented a second step for pervious study by studies the relationship between the rate of hydrolysis of soya proteins DH% and TN, AN, AN/TN% ratio of partially purified hydrolysized product of twenty tests, these texts were carried out previously on each run on the second stage according to Box Wilson experimental design of three variables as described before (13) , to obtain the optimum conditions for production of soya peptone by acid hydrolysis of soya protein. 3.2 Experimental Work Experimental work includes mainly three principal stages, which are: First and second stage: were carried out previously as described before (13) .The following section, clarifying brief for these two steps. First stage: Includes necessary steps for preparation of defatted soya flour from dehulled and defatted soybeans, by using whole mature Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 5 seed of Lee class of soybean provided by IPA Center for Agricultural Researches (Iraq). Second Stage Box-Wilson composite rotatable design is a common type of statistical experiment especially applicable to optimization analysis (14),(15),(16) ,therefore, experiments were designed according to the central composite rotatable design (Box-Wilson composite rotatable design), to can be achieved the purpose of this study . The study was devoted to test the effect of process variables on the rate of hydrolysis of soya protein, the experimental work was designed for the above purpose in the following experimental operating ranges: Variable1: Concentration of HCl solution ranging between 1-7 N. Variable2: Operating temperature of hydrolysis ranging between 35-95 C. Variable3: Duration time of specimen ranging between 0.5-24 hr. Response function was rate of hydrolysis DH%. The center composite rotatable design of three variables was used. According to experimental design of the three variables there are twenty experiments (there are fifteen tests, and five tests are added at the center), were carried out in the sequence as listed in Table (2), where the coded values of +1.732,- 1.732,0 represent the maximum, minimum and average values ,respectively. Table 2, Sequence of Experiments According to Central Composite Design. Exp. No. Coded Variable Real Variable X1 X2 X3 C (N) T (C) t (hr) 1 -1 -1 -1 2.268 47.679 5.466 2 +1 -1 -1 5.732 47.679 5.466 3 -1 +1 -1 2.268 82.321 5.466 4 -1 -1 +1 2.268 47.679 19.034 5 +1 +1 -1 5.732 82.321 5.466 6 +1 -1 +1 5.732 47.679 19.034 7 -1 +1 +1 2.268 82.321 19.034 8 +1 +1 +1 5.732 82.321 19.034 9 -1.732 0 0 1 65 12.25 10 +1.732 0 0 7 65 12.25 11 0 -1.732 0 4 35 12.25 12 0 +1.732 0 4 95 12.25 13 0 0 -1.732 4 65 0.5 14 0 0 +1.732 4 65 24 15 0 0 0 4 65 12.25 16 0 0 0 4 65 12.25 17 0 0 0 4 65 12.25 18 0 0 0 4 65 12.25 19 0 0 0 4 65 12.25 20 0 0 0 4 65 12.25 According to the central composite rotatable design of the experimental work, it explains the relationship between concentration of HCl solution, temperature, and time of hydrolysis of soya proteins, and effect of these variables on the hydrolysis rate of soya proteins, as well as, it studies the relationship between the rate of hydrolysis of soya proteins and TN, AN, AN/TN% ratio of hydrolysized product of soya proteins (13) . Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 6 Third stage: which represents the aim of present study, that which represents the process of partial purifying stage of hydrolysized product, resulting from the second stage by centrifuging it at 8000 rpm at 20 C (12) .At the end, two layers were distinguished within the centrifuged hydrolysized product, upper and lower, each differs in the contents, volume, and colour. The colour of the upper layer was brown while the lower was dark brown. Directly after centrifuging, the upper partially purified hydrolysized product is sucked by a pipette and then separated from the lower layer. The volume of the upper layer was modified to a constant volume for all tests by adding deionized water, and was used to determine TN, AN, and AN/TN% of partially purified hydrolysized product. 3.3 Methods of Analysis  Protein was determined by using the absolute method (17) .  Total protein nitrogen of samples was determined by using 5.71 factor to convert amount of protein to total protein nitrogen TN (18) .  Amino nitrogen was determined by using formaldehyde titration method (3, 19) .  Moisture was determined by using the air oven method (19, 20) .  Oil was determined by using intermitted extraction method (20) .  Ash was determined by using dry ashing method (19, 20) .  Fiber was determined by using the procedure of fertilisers and feeding stuffs regulations 1976 SI No. 840 (20) .  Carbohydrate percentage was determined by subtracting all other components form 100 percent (20) .  Sodium chloride was determined by using the Volhard titration method on the ash residue (20) .  pH value was determined by using pH meter on an autoclaved 2%solution of final product (2,3) . 4. Previewing the Results of Pervious Study First stage: The main purpose of the procedure followed in this stage is to prepare the defatted soya flour to be ready for carrying out the laboratory experiments. After finishing this stage, the defatted soya flour will be ready for the next stage. The chemical analysis of soybeans during the stages of preparing the defatted soya flour can be shown in Table (3). Table 3, Chemical Analysis of Soybeans During the Stages of Preparing the Defatted Soya Flour. Chemical Compositions The Degree Percent of the Chemical Compositions (w/w%) Soybeans Dried Soybeans Dehulled Soybeans Defatted Soybeans First Stage Second Stage Third Stage Moisture 6.856 2.321 2.352 2.887 3.001 3.048 Protein 35.412 37.136 39.932 49.011 50.941 51.733 Oil 20.235 21.22 22.811 5.262 1.531 - Ash 5.634 5.908 5.862 7.195 7.478 7.594 Fiber 5.375 5.637 1.544 1.896 1.97 2.001 Carbohydrate 26.488 27.778 27.499 33.749 35.079 35.624 Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 7 Second stage: The results of the carried out experiments according to the experimental design of Box Wilson are shown in Table(4). Table 4, Results of Experiments Planned According to Composite Rotatable Design. Exp. No. Hydrolysized Product TN gm/100ml AN gm/100ml AN/TN % DH % 1 0.647 0.10613 16.403 8.629 2 0.76967 0.28075 36.477 25.494 3 0.74497 0.2884 38.713 26.233 4 0.70441 0.18455 26.199 16.203 5 0.79686 0.46992 58.971 43.763 6 0.88121 0.51 57.875 47.634 7 0.79826 0.47918 60.028 44.658 8 0.99922 0.81068 81.131 76.673 9 0.61249 0.13901 22.696 11.805 10 0.99257 0.67056 67.558 63.141 11 0.65014 0.17812 27.397 15.582 12 0.87897 0.61963 70.495 58.222 13 0.65997 0.201 30.456 17.792 14 0.85299 0.6078 71.255 57.08 15 0.81341 0.47839 58.813 44.581 16 0.88961 0.47053 52.892 43.822 17 0.88111 0.48393 54.923 45.117 18 0.78502 0.49002 62.421 45.705 19 0.87397 0.47712 54.592 44.459 20 0.79628 0.48432 60.823 45.154 To find out the relation between the DH% and the effect of the following variables on the process of protein hydrolysis: concentration of acid, temperature and the duration of the process of hydrolysis, the conditions of each experiment were fed into a computer program (statistical program). These conditions were entered according to the significance of coded variable and the degree of hydrolysis, which were prepared according to the experimental design shown in Table (4). This process is done to get the coefficients of the polynomial equation of the second order. The resultant equation is as follows: DH% = 44.80638 + 13.33998 X1 + 11.94455 X2+10.64997 X3 + 0.1561251 X1 2 + 3.631374 X2 2 + 2.702626 X3 2 -2.655856 X1X2-2.8462 X1X3- 2.66819X2X3 …(2) Average error = 3.584888% Correlation coefficient = 0.9945404 Standard deviation = 1.892087 Equation (2) has been figured to clarify the effect of each of (C), (T), and (t) on (DH %) by means of Figures from (4), (5), (6), and (7). Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 8 Fig. 5. Degree of Hydrolysis as a Function of Time (hr) at 65C 0.000 6.000 12.000 18.000 24.000 30.000 TIME ( hr ) 0.000 20.000 40.000 60.000 80.000 100.000 D E G R E E O F H Y D R O L Y S IS ( D H % ) 7 N 5.732 N 4 N 2.268 N 1 N Fig. 4. Degree of Hydrolysis as a Function of Temperature (C) at 12.25 hr 0.000 20.000 40.000 60.000 80.000 100.000 TEMPERATURE ( C O ) 0.000 20.000 40.000 60.000 80.000 100.000 D E G R E E O F H Y D R O L Y S IS ( D H % ) 7 N 5.732 N 4 N 2.268 N 1 N Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 9 Fig. 6. Degree of Hydrolysis as a Function of Time (hr) at 4 N 0.000 6.000 12.000 18.000 24.000 30.000 TIME ( hr ) 0.000 20.000 40.000 60.000 80.000 100.000 D E G R E E O F H Y D R O L Y S IS ( D H % ) 95 C O 82.321 C O 65 C O 47.679 C O 35 C O 0.000 2.000 4.000 6.000 8.000 HCL CONCENTRATION ( N ) 0.000 20.000 40.000 60.000 80.000 100.000 D E G R E E O F H Y D R O L Y S IS ( D H % ) 95 C O 82.321 C O 65 C O 47.679 C O 35 C O Fig. 7. Degree of Hydrolysis as a Function of HCL Concentration (N) at 12.25 hr Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 10 5. Results and Discussion of Present Study Third stage involves studying the process of partial purifying of the hydrolysized product resulting from the second stage (mainly proteinous materials), in addition to other materials constituting the hydrolysized product. These materials have somehow long series that cause dark colour at the bottom of the tube after the process of centrifuging. The processes of limiting the value of TN and AN is carried out, and the percentage of TN/AN% is calculated. Results obtained from the processes of analyzing the partially purified hydrolysized product are shown in Table (5). Table 5, Analysis of Partially Purified Hydrolysized Product. Exp. No. Partially Purified Hydrolysized Product TN gm/100ml AN gm/100ml AN/TN% 1 0.24566 0.03203 13.038 2 0.41437 0.10502 25.344 3 0.47088 0.11293 23.983 4 0.36115 0.06436 17.821 5 0.57427 0.21067 36.685 6 .60647 0.23664 39.019 7 0.64339 0.22897 35.588 8 0.77142 0.54485 70.629 9 0.27458 0.0413 15.041 10 0.69386 0.35948 51.809 11 0.33415 0.06255 18.719 12 0.74354 0.35338 47.527 13 0.38424 0.07235 18.829 14 0.65844 0.3386 51.425 15 0.65797 0.22836 34.707 16 0.60226 0.23371 38.805 17 0.65376 0.25737 39.368 18 0.61855 0.25768 41.659 19 0.64137 0.24553 38.282 20 0.59875 0.22596 37.739 This stage represents the process of partial purifying stage and not complete purifying stage of hydrolysized product, resulting from the second stage by centrifuging it. This is due to many reasons: primarily, the process of purifying by the centrifuging is not 100% efficient in separating the long protein series, and also hydrolysis with acid is nonspecific (3),(5),(6),(7) . The effect of DH% on the concentration of TN, AN and the percentage of AN/TN% of the partially purified protein product is shown in Figures (8), (9), and (10).The results predicted from Figures (8),(9), and (10) are concluded in Table (6). Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 11 Fig.9. AN Concentration of Partially Purified Hydrolysized Product as a Function of DH%. 0.000 20.000 40.000 60.000 80.000 100.000 DEGREE OF HYDROLYSIS ( DH % ) 0.00000 0.10000 0.20000 0.30000 0.40000 0.50000 0.60000 A M IN O N IT R O G E N ( A N ) ( g m / 1 0 0 m l ) Fig.8. TN Concentration of Partially Purified Hydrolysized Product as a Function of DH%. 0.000 20.000 40.000 60.000 80.000 100.000 DEGREE OF HYDROLYSIS ( DH % ) 0.20000 0.30000 0.40000 0.50000 0.60000 0.70000 0.80000 T O T A L N IT R O G E N ( T N ) ( g m / 1 0 0 m l ) Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 12 Table 6, Equations Concluded from Figures (1), (2), and (3). No. of Fig. Equation Average Error % Kind of Relationship Extent of Application 8 TN(gm/100ml)=0.1438015+0.0137331(DH%)- 6.946058E-5 (DH%) 2 ……(3) 4.485 Non-linear Relationship 76.673DH%8.629  9 AN(gm/100ml)=5.451805E-4+2.988322E- 3(DH%)+5.036919E-5(DH%) 2 ……(4) 4.497 Non-linear Relationship 76.673DH%8.629  10 AN/TN%=10.34685+0.4300127 (DH%)+4.221502E-3 (DH) 2 ……(5) 4.369 Non-linear Relationship 76.673DH%8.629  From the relationship connecting TN of the partially purified hydrilysized product with DH% which shown in Equation (3) and Figure (8), it could be noticed that whenever the protein hydrolysis degree increases, the TN of the partially purified hydrolysized product increases. At the same time concentration of the protein materials removed from the hydrolysized product by means of centrifuging is reduced. Contrary to this case, whenever the protein hydrolysis degree comes closer to zero. Regarding to the relationship between AN of the partially purified hydrolysized product and DH% which shown in Equation (4), and Figure(9), whenever the protein hydrolysis degree increases, AN value of the partially purified hydrolysized product came closer to the TN value of the same product, due to the increase in the number of broken peptide bonds with the increase of the hydrolysis degree. The opposite behavior happened when the hydrolysis degree is low. To find out and clarify the indirect effect of the system variables studied before (13) (concentration of the acid, temperature, and time of the hydrolysis) on the concentration of TN and AN of the partially purified hydrolysized product, are shown in Figures (11) to (16). Fig.10. AN/TN% Concentration of Partially Purified Hydrolysized Product as a Function of DH%. 0.000 20.000 40.000 60.000 80.000 100.000 DEGREE OF HYDROLYSIS ( DH % ) 10.000 20.000 30.000 40.000 50.000 60.000 70.000 80.000 ( A N / T N ) % Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 13 Fig.11. TN Concentration of Partially Purified Hydrolysized Product as a Function of Hydrolysis Temperature (C) at 12.25 hr. 0.000 20.000 40.000 60.000 80.000 100.000 TEMPERATURE ( C O ) 0.20000 0.30000 0.40000 0.50000 0.60000 0.70000 0.80000 T O T A L N IT R O G E N ( T N ) ( g m / 1 0 0 m l ) 7 N 5.732 N 4 N 2.268 N 1 N 0.000 6.000 12.000 18.000 24.000 30.000 TIME ( hr ) 0.20000 0.30000 0.40000 0.50000 0.60000 0.70000 0.80000 T O T A L N IT R O G E N ( T N ) ( g m / 1 0 0 m l ) 7 N 5.732 N 4 N 2.268 N 1 N Fig.12. TN Concentration of Partially Purified Hydrolysized Product as a Function of Hydrolysis time (hr) at 65 C. Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 14 0.000 6.000 12.000 18.000 24.000 30.000 TIME ( hr ) 0.20000 0.30000 0.40000 0.50000 0.60000 0.70000 0.80000 T O T A L N IT R O G E N ( T N ) ( g m / 1 0 0 m l ) 95 C O 82.321 C O 65 C O 47.679 C O 35 C O Fig.13. TN Concentration of Partially Purified Hydrolysized Product as a Function of Hydrolysis time (hr) at 4N. 0.000 20.000 40.000 60.000 80.000 100.000 TEMPERATURE ( C O ) 0.00000 0.10000 0.20000 0.30000 0.40000 0.50000 0.60000 A M IN O N IT R O G E N ( A N ) ( g m / 1 0 0 m l ) 7 N 5.732 N 4 N 2.268 N 1 N Fig.14. AN Concentration of Partially Purified Hydrolysized Product as a Function of Hydrolysis Temperature (C) at 12.25 hr. Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 15 The following Table (7) clarifies the effect of concentration of each TN, AN and AN/TN% of the hydrolysized product before and after the process of partial purifying which has been carried out with the protein hydrolysis degree. 0.000 6.000 12.000 18.000 24.000 30.000 TIME ( hr ) 0.00000 0.20000 0.40000 0.60000 A M IN O N IT R O G E N ( T N ) ( g m / 1 0 0 m l ) 95 C O 82.321 C O 65 C O 47.679 C O 35 C O Fig.16. AN Concentration of Partially Purified Hydrolysized Product as a Function of Hydrolysis Time (hr) at 4N. 0.000 6.000 12.000 18.000 24.000 30.000 TIME ( hr ) 0.00000 0.10000 0.20000 0.30000 0.40000 0.50000 0.60000 A M IN O N IT R O G E N ( A N ) ( g m / 1 0 0 m l ) 7 N 5.732 N 4 N 2.268 N 1 N Fig.15. AN Concentration of Partially Purified Hydrolysized Product as a Function of Hydrolysis Time (hr) at 65 C. Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 16 Table 7, Comparison Between TN, AN and AN/TN% of the Hydrolysized Product Before and After the Process of Partial Purifying. Hydrolysis Degree of Soya Protein The Hydrolysized Product Before the Process of Partial Purifying The Hydrolysized Product After the Process of Partial Purifying TN gm/100ml AN gm/100ml AN/TN% TN gm/100ml AN gm/100ml AN/TN% 0 0.57732 0.01678 - - - - 8.629 0.62776 0.10613 17.584 0.25713 0.03008 14.372 76.673 1.02546 0.81068 78.557 0.78842 0.52578 68.134 100 1.1618 1.05221 - - - - 5.1 Limiting the Appropriate Degree of Soya Protein Hydrolysis The compositions of soya peptone product by Oxoid company have been limited with regard to TN, AN and AN/TN%. Accordingly, the characteristic of the soya peptone produced by this company at the years 1988 and 1995 (as shown in Table (8)) were depended as maximum and minimum ranges of the soya peptone produced by acidic hydrolysis of soya protein in the present study. Table 8, A Comparison between the Characteristic of Soya Peptone Analysis Produced by Oxoid Company for 1995 and 1988. Analysis Soya Peptone Analysis of 1995 (w/w%) Soya Peptone Analysis of 1988 (w/w%) TN 9.1 10.2 AN 2.3 3.1 AN/TN% 25 30 Now, the soya peptone analysis in Table (8) converted, from weight percentage formula (w/w%) to (gm/100ml) formula, that has been adopted in the laboratory for this study, Table (9) clarifies this operation. Table 9, Characteristics of the Soya Peptone Produced by Oxoid Company for 1995 and 1988 in gm/100ml. Analysis Soya Peptone for 1995 Soya Peptone for 1988 (w/w%) gm/100ml (w/w%) gm/100ml TN 9.1 0.47177 10.2 0.52879 AN 2.3 0.11924 3.1 0.16071 AN/TN% 25 25 30 30 The values in Table (9) were used as a characteristics range of the soya peptone produced by the acidic hydrolysis of the soya protein which studied in the present study. The extent range of the characteristics of the soya peptone with regard to TN, AN, AN/TN% which shown in Table (9) are plotted in Figures (8),(9) and (10), the extent within which the degree of protein hydrolysis DH% ranges is limited, is shown in Table (10). Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 17 Table 10, Soya Protein Hydrolysis Degree Limited by Using the Acid. Analysis Hydrolysis Degree 1995 1988 TN (gm/100ml) 27.787 33.818 AN (gm/100ml) 27.226 34.053 AN/TN % 27.365 34.757 Hence, it can be concluded that the best extent within which the soya protein hydrolysis degree values (provided that the product is a soya peptone that have characteristics ranging between the characteristics of the soya peptone produced by Oxoid company for the year 1988 and 1995) ranges between 27% and 34%. 5.2 Optimum Conditions for Soya Peptone Production By using a computer program at a BASIC language , has been formulated, to find out the optimum conditions for producing the soya peptone This program gives the optimum extent for the time of soya protein hydrolysis at degree ranges between 27% and 34% by using Equation (2) (concluded from the pervious study (13) ), as shown below, which shows the effect of the variables of the process (the concentration of the acid (N), temperature (C) and the time of hydrolysis (hr)) on the soya protein hydrolysis degree . DH% = 44.80638 + 13.33998 X1 + 11.94455 X2 + 10.64997 X3 + 0.1561251 X1 2 +3.631374X2 2 + 2.702626 X3 2 - 2.655856 X1X2 - 2.8462 X1X3 - 2.66819 X2X3 …(2) Average error = 3.584888% Correlation coefficient = 0.9945404 Standard deviation = 1.892087 To determine the optimum conditions for the process of hydrolysis, 1N of HCl has been chosen in this process for the following reasons: 1. Decreasing the intensity of the occurrence of side-reactions. 2. Decreasing the amount of salt NaCl. 3. Decreasing the opportunities of destruction of protein and then its loss of the biological characteristics. With regard to temperature, the best extent within which the optimum temperature of the process of the soya protein hydrolysis range is from 50 to 53 C has been chosen for the following reasons: 1. Decreasing the intensity of the occurrence of side-reactions. 2. Decreasing the opportunity increasing the effectiveness of the acid. 3. Preventing changes in the nature of the protein itself at low temperatures. After performing this computer program, the optimum time necessary for soya protein hydrolysis was obtained : from 17.189 to 19.97hr when dealing with the following conditions of hydrolysis: 1N of HCl at 50 to 53 C, to reach a hydrolysis degree ranges from 27% to 34% for the soya protein. 6. Conclusions  The process of hydrolysizing the soya protein by using the papain enzyme is not economic when produced locally. For this, enzyme is extracted from a special fruit found only in equatorial places.  Whenever the protein hydrolysis degree increase, TN and AN of the hydrolysized product of centrifuging come closer to TN of this product before centrifuging and to TN of the soya protein before the acidic treatment. This is due to the low concentrations of the separated proteinous materials during the processes of filtering and centrifuging.  According to the characteristic of soya peptone produced by Oxoid company 1988 and 1995,the appropriate hydrolysis degree is 27-34%.  The optimum conditions necessary to get this degree are:  Concentration of HCl: 1 N Mohammed B.A.G.AL-Bahri Al-Khwarizmi Engineering Journal, Vol. 5, No. 1, PP 1-19 (2009) 18  Temperature: 50-53 C  Time: 17.189-19.97 hr  The productivity of soya peptone which prepared at the optimum conditions limited in the present work was 38.071%. Abbreviations DH% degree of protein hydrolysis TN concentration of total protein nitrogen AN concentration of amino nitrogen of protein, peptides, and amino acids C concentration of hydrochloric acid solution in normality (N) T operation temperature in C t duration time of specimen in hr X1 concentration coded variable X2 temperature coded variable X3 time coded variable Acknowledgments The authors thank the Department of Chemical Engineering / University of Technology, and the Ministry of Industry and Minerals for their help in providing facilities, during the period of achieving this work… 7. References [1] “The Oxoid Manual”, United Kingdom, (1988). [2] “Difco Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures”, 9th ed., Detroit 1, Michigan, U.S.A., (1964). [3] Bridson, E.Y., “The Oxoid Manual”, 7th ed., United Kingdom,(1995). [4] Brewster, R. Q., and W. E. Mcewen, “Organic Chemistry”, 3rd ed., (1958). [5] Weininger, S. J., Stermitz, “Organic Chemistry”, International Edition, (1984). [6] Friedman, M., and R. Liardon, J. Agric. Food Chem., 33, 666, (1985). [7] Haurowitz, F., “Biochemistry”, An Introductory Textbook, John Wiley & Sons, Inc., New York, (1955). [8] Hendrickson, J. B., D. J. Cram, and G. S. Hammond, “Organic Chemistry”, 3rd ed., International Student Edition, McGraw-Hill Kogakusha, Ltd., (1970). [9] “BDH Chemicals Ltd”, laboratory chemicals & biochemicals, Manual, England, (1984/1985). [10] “Sigma Chemical Company”, Manual, U.S.A, (1988). [11] “The United States Pharmacopoeia (USP XX II)”, January (1990). [12] Hossain, N, Report, Ibn Al-Beitar Center (Ministry of Industrial and Minerals-Iraq), (1993). [13] AL-Bahri, M. B. A. G, “Preparation of Soya Peptone and Potato Extract for Use in Production of Some Culture Media for Medical Laboratories”, M.Sc. Thesis, University of Technology , (2001). [14] Cochran, W. G., and G. M. Cox, “Experimental Design”, John Wiley and Sons, Inc., New York, (1957). [15] Montgomery, D. C., “Design and Analysis of Experiments”, John Wily and Sons, New York, (1976). [16] Perry’s, R. H., and C. H. Chilton, “Chemical Engineers Handbook”, McGraw-Hill, 5th ed., (1973). [17] Whitaler, J. R., and P. E. Granum, Anal. Biochem., 109, 156, (1980). [18] Apostolatos, G., J. Fd. Technol., 19, 639, (1984). [19] “AOAC-Official Methods of Analysis”, 12th ed., Association of Official Agricultural Chemists, Washington D. C. (1975). [20] Lees, R., “Food Analysis: Analytical and Quality Control Methods for the Food Manufacturer, and Buyer”, 3 rd ed.,Leonard Hill Book, (1975). 200 )19-1، صفحة 1، العذد 5مجلة الخىارزمً الهىذسٍة المجلذ محمذ باسل علً غالب 9) 19 الظروف المثلى إلوتاج ببتىن الصىٌا بىاسطة التمٍئ ألحامضً لبروتٍه الصىٌا محمذ باسل علً غالب البحري * ، صفاء الذٌه عبذ هللا الىعٍمً ** ، سىذس حمٍذ احمذ *** خايؼت بغذاد/كهٍت ُْذست انخٕاسصيً/ قسى انُٓذست انكًٍٍائٍت االحٍائٍت* اندايؼت انخكُٕنٕخٍت / قسى انُٓذست انكًٍٍأٌت** انخكُٕنٕخٍا ٔٔصاسة انؼهٕو *** الخالصة حى اَداص ْزِ انذساست ألخم إٌداد انظشٔف انًثهى انضشٔسٌت انالصيت نؼًهٍت حًٍئ بشٔحٍٍ انصٌٕا باسخخذاو حايض انٍٓذسٔكهٕسٌك ْزِ انظشٔف قذ اخخٍشث باخخباس حأثٍش يخغٍشاث . إلَخاج ببخٌٕ انصٌٕا (كؼايم يساػذ حٍٕي)بذال يٍ اإلَضٌى انباباٌٍ (كؼايم يساػذ كًٍٍائً) . ػًهٍت انخًٍئ ػهى طبٍؼت ٔخٕدة انًادة انًُخدت حى دساست إَخاج ببخٌٕ انصٌٕا ألًٍْخّ فً ػًهٍت ححضٍش ٔإَخاج األٔساط انضسػٍت انًسخخذيت فً يخخبشاث انخحهٍم انطبً ٔ . انًاٌكشٔبإٌنٕخً :إٌ ػًهٍت إَخاج ببخٌٕ انصٌٕا حخضًٍ أسبغ يشاحم سئٍسٍت  ،ٍْأٔال إػذاد طحٍٍ انصٌٕا يضال انذ .  ػًهٍت حًٍئ بشٔحٍٍ انصٌٕا،ثاٍَا.  حُقٍت انُاحح، ثانثا.  ًٔأخٍشا حدفٍف انُاحح انُٓائ. بأحباع خطٕاث انؼًم انًٕضحت فً انذساست انحانٍت،أيكٍ انخٕصم إنى انظشٔف انًثهى انالصيت نؼًهٍت حًٍئ بشٔحٍٍ انصٌٕا، ٔ أنًبٍُّ فً : أدَاِ  ْٕ ع1أفضم حشكٍض نًحهٕل حايض انٍٓذسٔكهٕسٌك أنالصو نؼًهٍت حًٍئ بشٔحٍٍ انصٌٕا .  ٍٍو °53 –50أفضم دسخت حشاسة نؼًهٍت حًٍى بشٔحٍٍ انصٌٕا حخشأذ ب .  ٍٍساػت19.97 – 17.189أفضم فخشة صيٍُت نخًٍئ بشٔحٍٍ انصٌٕا حخشأذ ب . %.38.071 بهغج اإلَخاخٍت نببخٌٕ انصٌٕا بإحباع خطٕاث انؼًم ٔ انظشٔف انًثهى نإلَخاج انًٕضحت فً انذساست انحانٍت إر