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Impact of heat treatments and some technological processing on immunoglobulins of Egyptian buffalo's milk
Accepted Manuscript Impact of heat treatments and some technological processing on immunoglobulins of Egyptian buffalo's milk
Mohamed M. El-Loly, Laila K. Hassan, Eman S.A. Farahat PII: DOI: Reference:
S0141-8130(18)32438-3 https://doi.org/10.1016/j.ijbiomac.2018.11.055 BIOMAC 10932
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
20 May 2018 5 November 2018 11 November 2018
Please cite this article as: Mohamed M. El-Loly, Laila K. Hassan, Eman S.A. Farahat , Impact of heat treatments and some technological processing on immunoglobulins of Egyptian buffalo's milk. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.11.055
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ACCEPTED MANUSCRIPT Impact of heat treatments and some technological processing on immunoglobulins of Egyptian buffalo’s milk Mohamed M. El-Loly*, Laila K. Hassan, Eman S. A. Farahat
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Dairy Science Department, National Research Centre, 33 El-Buhouth St. (Former El-Tahrir St.), Dokki, PO 12622, Giza, Egypt * Corresponding author: E-mail: [email protected]
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ABSTRACT
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The effects of heat treatments, ultrafiltration and manufacture of soft cheese on the gross composition and immunoglobulins (Igs) of Egyptian buffalos were investigated. Four Igs (IgG1, IgG2, IgM and IgA) were identified and determined by single radial immunodiffusion (SRID). High concentrations of Igs were found in colostrum which decreased rapidly within the first 72 hrs postpartum parallel to the transition from colostrum to normal milk. IgG (IgG1, IgG2) and IgM were not completely denatured by pasteurization temperature up to 80°C/15 s, while IgA was completely denatured under these conditions. Ultrafiltration of milk resulted in retentate of high values for total IgG (IgG1, IgG2), but low in IgM and IgA content and permeate was free of Igs. Domiati cheese made from UF-milk retentate contained similar levels of Igs to the used retentate.
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1. Introduction
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Keywords: Buffalo colostrum/milk, chemical constituents, immunoglobulins, heat treatments, technological processing.
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Buffalo’s milk is an important source for human nutrition in some parts of the world. It is considered as a richer source of lipids, protein, lactose and minerals that had important for chemical and nutritive properties as well as suitability in the making of traditional and industrial dairy products [1].
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Although the proteins (immunoglobulins or antibodies, lactoferrin, lysozyme and lactoperoxidase) that make up only a minor milk proteins and play an important role as the first line of defense attribute to their specific and non-specific antimicrobial activity, as well as to their other important physiological and health promoting functions [2,3]. The bovine colostrum is a rich source of Igs that confers passive immunity to the newborn during the development of its own immune system [4]. Colostral Igs provide as one of the considerable prospective for consumers health promotion in the future [5]. These are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses [6,7,8,9]. Though Igs represent only 1-2% of the total milk proteins, or about 6% of the total whey proteins [10], while about 70-80% of the total protein in 1
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colostrum [11]. Milk Igs are found the same as in the blood or mucosal secretions, which are proteins molecules, it contains antibody activity, are created by white blood cells called plasma cells. They can be classified into five different classes based on their antigenic properties: IgG, IgM, IgA, IgD and IgE. In normal serum represents about 80, 15, 5 and 0.2% of IgG, IgA, IgM and IgD respectively and a trace of IgE [12]. The major milk Igs are G, A, M types that had higher significantly in colostrum comparing with normal milk, this observation was the same results obtained by [13,14,15,16]. IgG molecule is primary Ig class and dominant in colostrum, milk and blood being about 80-90, 60-70 and 90% of total Igs, respectively [14,17,18]. IgG had various subclasses IgG1 and IgG2 being the major Igs in serum. While IgA was found a major Ig class in mucosal secretions and prevents mucosal infections by agglutination of microbes. But IgM has a low specificity and hence a lower potency in defeating the infection, it appears initially when an organism is exposed to an antigen for the primary infection [19].
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Heat treatment is commonly applied to milk during industrial processing to ensure the microbial safety of dairy products as well as to extend shelf life [20]. Whey proteins denaturation is one of the basic effects of milk heating causing modification the chemical and nutritional properties of milk which leads to partial unfolding the whey proteins with helical structure damage or irreparable [21]. Igs are very important in use for many processing, concentration or isolation when exposing to heat, acid or pressure which may affect the conformation of the protein, and ultimately the immunological activity of it. Thermal treatment is an obligatory step, whereas the temperature and time combination could influence the protein’s structure for both of unfolding and aggregation [22]. IgG activity loss was significantly affected at 65°C/30 m of cow, buffalo and camel milk, while it was the whole activity loss of cow or buffalo milk at 75°C/30 m while the IgG loss being 68.7% in camel [23]. Recently, Bogahawaththa et al., [24] stated that the bovine milk protein structure can be affected directly by the heat treatments that cause changes in associated epitopes, the immunogenic and antigenic potential of milk proteins. The severity of some technological processing impacted to important minor proteins, which may exert immuno-modulatory properties, like a way as to prevent an occurrence of allergies. Also, Daniels et al., [25] illustrated that the effect of temperature using FoneAstra or Flash-heat pasteurization on human milk showed a decrease in the retention of total IgA (78.9 vs. 25.2%) respectively. Furthermore, several investigations related to the pH and temperature stability were reported by many authors, where Varshney et al., [26] indicated that the continuous loss in the secondary structure content in the temperature range of 2095°C. This due to molten globule states for several proteins under various 2
ACCEPTED MANUSCRIPT denaturing conditions. While Rabbani et al., [27, 28, 29] observed that the molecular compactness of the protein increases as the pH of the solution decreases, and the temperature-induced unfolding reveal that the molten globule state is more stable than the other states; as well as the effect of pH on the conformation and thermostability of outer membrane protein (OMP) points towards its heat resistance at neutral pH (7.0 at 69°C). Also, these results suggested that acidinduced unfolded state of OMP at pH 2.0 represented the molten globule state.
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UF technique is used as specific semi-permeable and becomes an essential part in food technology as a tool for concentration and fractionation the liquids and suspensions of the various constituents into two liquids based on its molecular weight, also other factors like the molecular shape and the charge can be affected. The suspended solids and solutes of larger molecules retained at the surface of the membrane are known as retentate or concentrate, while water and smaller molecules solutes will pass freely through the membrane are known as filtrate or permeate [30]. Membrane technology has been used in the dairy industry since the early 1960s and is currently come to the second after the water treatment technology and is a suitable another alternative to traditional dairy processes such as distillation, evaporation or extraction [31].
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The present study was focused to evaluate the thermal stability and the technology processing to manufacture soft cheese using UF technique on immunoglobulin fractions content of Egyptian buffalo’s milk within the first 15 th days of lactation. These biophysical properties of buffalo milk Igs are important due to its use as a source for human nutrition and economically helpful and clinically useful.
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2. Materials and methods 2.1. Samples collection
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Individual colostrum and milk samples were collected from three apparently healthy buffaloes of a private farm, Giza Governorate, Egypt, at 0-12, 24, 48, 72 hours; 4, 5, 7 and 15 days postpartum. Immediately samples were collected in clean stoppered bottles in an ice box and transported to the dairy laboratory, National Research Centre. All samples were stored and kept frozen at -20°C until analyzed. The average of duplicate was taken for each sample.
2.2. Precipitation of immunoglobulins using ammonium sulfate Buffalo milk samples were defatted by centrifugation at 4000 rpm for 3 min. Milk whey was prepared from the skim milk by adjusting pH to 4.6 using 1 N Hcl solution and centrifuging at 10000 rpm for 15 min. to remove precipitate casein particles. Total Igs were prepared from whey samples by using saturated 3
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ammonium sulfate (SAS), it prepared by dissolving excess in distilled water until some crystals of ammonium sulfate remained un-dissolved. Working solution of 80% ammonium sulfate solutions was freshly prepared by diluting SAS with a necessary amount of water. Equal volumes of the whey and 40% SAS solution were mixed; the formed precipitate was removed by centrifuging, dissolved in the minimum quantity of distilled water and dialysed until the complete removal of the ammonium salt against distilled water for 24 h at refrigerator with several changes of distilled water during this period. The dialysed extract was kept at -20°C until analyzed [32].
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2.3. Heat treatments
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For evaluate the effect of heat treatments on the Igs content of buffalo milk, the samples were thoroughly mixed, it was defatted and the skim milk of twenty ml of each stopper glass tubes were heat-treated in water bath at 63˚C/30 min., 80˚C/15 s, 100˚C/10 min. and sterilization at 130˚C/15 min., and then followed by rapid cooling to 37˚C for all samples.
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2.4. Preparation of domiati cheese manufactured by ultrafiltration (UF)
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To investigate the effect of UF technique on the Igs content of milk, the milk was heated to 55˚C and ultrafiltrated to CF4 using a carbosep pilot plant (modules 2 S 151 UF system, Orelies, France), equipped with an inorganic tubular membrane. It consists of two ultrafiltration modules of the surface area of 6.8 m2 together with pumps. The samples of milk, retentate, permeate and cheese samples were taken during the UF of milk and cheese processing to determine Igs fractions.
2.5. Determination of some gross chemical constituents
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The total solids (TS), total protein (TP) and ash contents were determined according to the method described by [33].
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2.6. Immunoglobulin quantification by single radial immunodiffusion (SRID) Radial immunodiffusion test in agar gel is a technique that routinely used for measuring the concentrations of various soluble antigens (usually proteins) in the biological fluid. Igs standard and radial immunodiffusion kits (The Binding Site Limited, England) were used for quantitative determination of Igs [IgG (IgG1, IgG2), IgM and IgA] in buffalo milk samples within a first 15th days after birth. It was principally derived from the work of [34,35]. All samples were used without dilution, each sample was placed in a well punched in the layered gel. After sample application, the lid is tightly closed and the plate stored flat with the lid uppermost at 2-8˚C. It is essential that the gel is not allowed to dry out during incubation. To 4
ACCEPTED MANUSCRIPT minimize evaporation, it is suggested that plates should either be resealed in their foil pouches or stored in a moist box (a sealed plastic box containing damp tissue paper) during incubation. The minimum incubation time for complete diffusion is 48 h (72 h for IgM). The diffusion was measured after incubation time, and the concentration of immunoglobulin in each sample can be read directly from the SRID reference table. The calculation was done according to the manufacturer manual.
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3. Results and discussion
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3.1. Gross chemical composition and immunity molecules distribution in buffalo’s milk
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As shown in Table 1, TS values of Egyptian buffalo’s colostrum indicated that the first 12 h had an average of 31.23%, it very rapidly decreased at 48 and 72 hr reaching average values being 16.66 and 15.27% respectively. The reduction was gradual with time progress thereafter until it reached a level 13.70% of normal milk at the end 15th day postpartum. Also, TP content had a mean of 13.53% at 12 hr postpartum, followed by a very rapid decrease to reach the value of 4.07% at 72 hr parturition, then gradually decreased to value 3.01% at the 15 th day of lactation. These results were the same trend with [36,37], but it was higher than that reported by [38] explained that the mean values of TS in buffalo colostrum were gradually decreased with time progress, which ranged of 24.48-13.67% and it was slightly lower for TP being 15.57-3.18% at 1st and 7th day postpartum respectively. As for the ash content was gradually decreased from 0.98 to 0.88% at 12 and 72 hr in order, followed by decreasing to value 0.66% at the end 15 th day parturition. The same thing was observed with [37,38,39,40,41,42,43,44].
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We can be observed that the most changes occur in milk protein content that is reduced more than the one-third at the 3rd day postpartum compared to initial values. Comparing the total Igs (2.41%) to the TP (13.53%) in the 0-12 hr samples indicates that it represent 17.81%. This result explains that the highest levels were in the first few hours of colostrum, and also means that mostly attributed to the Igs sharp decrease. It is completely identical with [40] mentioned that the higher protein values of colostrum were could be due to higher concentration of globulin that serves as the carrier of antibodies for suckling calf against disease producing organism. Moreover, in our investigation the protein content at the 15 th day of lactation was 3.01%, this observation was higher than reported by [45] found that it had ranged of 4.2-4.5% in buffalo milk, while cow milk has 3.6%.
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ACCEPTED MANUSCRIPT Table 1 Medians of the total solids (TS), total protein (TP), ash and Igs values in buffalo’s milk during the first 15th days postpartum. Lactation period 48
72
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15.46 3.97 0.85
24.64 10.96 0.93
16.66 4.94 0.91
15.27 4.07 0.88
10.72 44.44 7.24 30.02 5.52 22.89 0.64 2.65 24.12 0.0
10.54 48.11 6.47 29.53 4.38 19.99 0.52 2.37 21.91 -9.16
10.46 50.85 5.74 27.91 3.96 19.25 0.41 1.99 20.57 -14.72
7.24 44.97 4.83 30.00 3.70 22.98 0.33 2.05 16.10 -33.25
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31.22 13.53 0.98
7.20 48.55 4.24 28.59 3.11 20.97 0.28 1.89 14.83 -38.52
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Gross composition (%) TS TP Ash Immunoglobulin fractions (mg/ml) IgG1 % of the total IgG2 % of the total IgM % of the total IgA % of the total Total Igs % of decrease
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15
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14.24 3.92 0.81
13.81 3.87 0.73
13.70 3.01 0.66
6.86 48.21 4.16 29.23 3.02 21.22 0.19 1.34 14.23 -41.00
5.83 46.60 3.63 29.02 2.87 22.94 0.18 1.44 12.51 -48.13
4.05 49.09 2.70 32.73 1.48 17.94 0.02 0.24 8.25 -65.80
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Also, Table 1 explain the immunity molecules distribution of buffalo’s milk, where the obtained median values at the first 12 hours were 10.72, 7.24, 5.52 and 0.64 mg/ml for IgG1, IgG2, IgM and IgA respectively, then dropped to 7.24, 4.83, 3.70 and 0.33 mg/ml in the same order at 72 h postpartum. Moreover, a gradual decrease could be noticed at the following days until the 15 th day being 4.05, 2.70, 1.48 and 0.02 mg/ml in order. Results indicated that a marked reduction in colostrum and milk in all these types with time progression of lactation. It obvious that the trend of changes in total Igs values decreased about 33.25% at the 3rd day and reached 65.8% at 15th day. In addition, the most obvious decrease was detected with both of IgM and IgA types that it decreased slightly levels (22.89 vs 17.94%) and (2.65 vs 0.24%, less than the tenth) at the first hours and the 15 th day of lactation respectively. These data had been established with [17,46]. Confirming to [43] demonstrated in buffalo and cows that the composition of both colostrum approaches those of normal milk within 5 d after parturition, also they found the same trend for IgG and IgM values. Recently, Neama Ashmawy [44] reported that the colostrum IgG content decreases very rapidly with time with average 3260.5, 2654.0, 1900.5 and 1325.25 mg/dl at the zero time, 3, 6 and 24 h after calving in order. 6
ACCEPTED MANUSCRIPT 3.2. Impact of heat treatments on gross chemical composition and immunity molecules distribution of buffalo’s milk
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As thermal processing is a major treatment in the processing of milk and milk products, its influence on whey proteins fractions, whereas the stability of the Igs in milk depends on the severity of the thermal treatment used in the various commercial processes. Milk contains Igs (G, M and A), colostrum being practicality rich in its; the biological function of Igs consists in a passive immunization of the calf via ingested colostrum. From milk processing point of view, the Ig fractions of early lactation milk reduce its heat stability, this is the reason why milk from early stages of lactation is excluded from producing milk. Table 2 shows the effect of thermal treatments of buffalo’s milk, it is clear that the TS content of pasteurized milk samples at 63°C/30 min. and 80°C/15 s were almost the same value as control (11.99, 12.02 and 12.06%) respectively. On the other hand, increasing temperature up to boiling or sterilization led slight losses (14.85 and 17.76%) in TS values, being 10.21 and 9.86% in order. This may attributed to its effect in precipitating some of the milk proteins. The similar trend for TP values 2.19 and 2.15 (with loss of 17.05 and 18.56%) at the same order. While the ash values of different heat-treated samples were almost the same.
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Concerning to Igs distribution (IgG1, IgG2, IgM and IgA) at 63°C/30 min., as result in Table 2, the heat treatment caused losses percentages of 35.06, 33.33, 56.02 and 100% respectively. A similar trend had obtained at 80°C/15 s being 33.33, 31.48, 54.73 and 100% in order. On the contrary, for IgA, it was none retained of treated milk samples that may be due to its low level in raw milk (control), this means that IgA is the most heat sensitive of the Igs. On the other hand, increasing temperature to boiled or sterilized milk did not contain any Igs, which means that these heat treatments had a destructive effect on Igs values. We concluded that the Igs were the least heat stable in the milk whey proteins. Our data suggest that commercial pasteurization process results incomplete denaturation of IgG1, IgG2. Generally, total IgG (IgG1, IgG2) and IgM were not completely denatured at commercial pasteurization up to 80°C/15 s, while IgA was completely denatured at any temperature. Also, this study demonstrates the dependence of buffalo milk Igs stability in milk products on the severity of heat treatment used in various commercial processes. Our results were almost agreed with [47] found that the HTST pasteurization may not destroy IgG activity of bovine milk, being 65-79% retention when comparing to the raw milk, while it was 59-76% by [48]. While IgG1, IgG2 and IgM unfolded at 79.4, 76.7 and 80.3°C respectively [49]. IgG was the most thermo-stable, but IgM was the least one [50]. While [51] illustrated that the Igs are thermo-labile, the bovine IgG molecule can reduce about 40 and 100% at 75°C/5 min. and 95°C/15 s in the same order. Those 7
ACCEPTED MANUSCRIPT changes may be attributed to conformational changes of IgG type causes by heat exposure [52]. IgG and IgM were not completely denatured in buffalo milk between 63-88°C that may be due to the protective effect of the high protein content of buffalo milk, but IgA was completely denatured between 63-88°C [53]. The IgG denaturation ratio at 72°C/15 s and 90°C/5 min. of yak (bovine species) colostral whey reduced by 19.28-37.56 and 44.96-58.97% respectively [54].
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The protein loses a considerable fraction of its changes in the secondary structure during thermal denaturation, also, the presence of intermediate unfolded states in the thermal denaturation of human serum albumin that is structurally similar to bovine serum albumin [55].
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Recently, Klotz et al., [56] illustrated that the mean values of IgA in human milk were 95 and 83% at HTST treatment (62°C/5 s) and standard holder pasteurization (63±0.5°C/30 min.), being 51.7±28.3 and 43.0±22.7 mg/dL in order. Table 2
Control
Heat treatments
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Medians of the total solids (TS), total protein (TP), ash and Igs values in buffalo’s milk as affected by different heat treatments.
12.02 2.24 0.78
15.15 -
12.06 2.38 0.79
9.85 -
10.21 2.19 0.70
14.85 17.05 -
9.86 2.15 0.75
17.76 18.56 -
2.63 1.80 0.65 0.0
35.06 33.33 56.02 100.00
2.70 1.85 0.67 0.0
33.33 31.48 54.73 100.00
0.0 0.0 0.0 0.0
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0.0 0.0 0.0 0.0
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11.99 2.64 0.75
4.05 2.70 1.48 0.02
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Gross composition (%) TS TP Ash Immunoglobulin fractions (mg/ml) IgG1 IgG2 IgM IgA
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63°C/30 Loss % 80°C/15 Loss % 100°C/10 Loss % 130°C/15 Loss % min. s min. min.
3.3. Gross chemical composition and immunity molecules distribution in Domiati cheese manufacture steps by ultrafiltration technique In traditional cheese making, the milk is first coagulated by addition of starter culture and rennet. The concentration continues when the whey is drained off and curd forms. This whey contains about 25% of protein and 10% of fat contents, whereas some the nutrients are lost in the whey, such as lactose, minerals and soluble vitamins, these losses have a large influence on the economics of the process. UF is an effective means of recovering the by-products that can be used 8
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for further food formulations and also produce cheese milk having substantially the composition of cheese at the end of whey drainage and turn the cheese milk into cheese by the coagulation with adding rennet. Therefore, interesting to use of UF technique to concentrate milk and by-products from milk for subsequent manufacture was in most cheese manufacture. The milk is an ideal liquid for membrane filtration due to its composition, it is concentrated and then followed by starter culture and rennet induced coagulation. Due to the membrane technology was retained larger protein molecules including whey proteins, it means that Igs retained in milk which raised the natural immunity in resultant cheese.
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Renner & Abd EI-Salam, [57] explained that during UF the protein and fat of milk were retained nearly completely in the retentate, whereas lactose, minerals and vitamins are partitioned between the retentate and permeate depending on the degree of concentration. Therefore it is important to follow the Igs content in the method steps of Domiati soft cheese processing, namely milk, retentate, permeate and fresh cheese, which cheese manufacture by UF lends itself to continuous processes and to better control over composition and quality.
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Fig. 1 understood that the TS of milk was 17.90 increased to 33.94% for retentate, while permeate contains only 5.89% including lactose and mineral salts. The protein level of retentate (12.22%) almost equals the protein level of resultant UF Domiati cheese (16.83%) with subtracted the addition amount of salt (4.881.78%). The ash content of milk, retentate and cheese were 0.98, 1.78 and 4.88% respectively. These obtained results are in corresponding to that reported by [58] showed that when buffalo milk ultrafiltered, the resultant retentate had a composition of 24.52, 17.99 and 2.33% for TS, TP and ash contents respectively. Also, it was almost in agreed with Hind Bihari [59] found that the TS, TP and ash constituents of UF buffalo milk were 17.22, 4.15 and 0.76% in the same order.
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While Fig. 2 shows the concerning immunity molecules (IgG1, IgG2, IgM and IgA) that could be increase concentrated using UF technique in both of retentate and fresh UF cheese, being 12.65, 8.23, 2.14 and 0.30 as well as 13.20, 10.0, 2.85 and 0.38 mg/g in the same order. We concluded that as a result using UF technique, both of the resultant retentate and UF soft cheese had higher values of total IgG (IgG1, IgG2), but in a lower level of IgM and IgA, because the buffalo milk already has lower values in both of them compared to the IgG level that it was dominant in colostrum. While the permeate had free Igs because UF membranes retained the larger molecules of milk proteins in the retentate. Finally, Fig. 3 shows the SRID analysis of total IgG (IgG1, IgG2) in buffalo’s milk affected by different heat treatment and technological processing during UF domiatti cheese making steps. 9
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(%)
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Milk
Fresh Cheese
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Permeate
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14 12 10 8 6 4 2 0 IgG2
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Fig. 1. Medians of the total solids (TS), total protein (TP) and ash values in buffalo’s milk during UF Domiati cheese manufacture steps.
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Fig. 2. Medians of the Igs values in buffalo’s milk during UF Domiati cheese manufacture steps.
Fig. 3. Single radial immunodiffusion analysis of total IgG in buffalo’s milk affected by different heat treatment and technological processing during UF Domiati cheese manufacture steps. Wells No: (1&6, 2&7, 3&8, 4&9, 5&10) represent control, 63°C/30 min., 80°C/15 10 s., 100°C/10 min. and 130°C/15 min. respectively, as well as (11,12,13,14) represent milk, retentate, permeate and fresh cheese respectively.
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[29] G. Rabbani, J. Kaur, E. Ahmad, R.H. Khan, S.K. Jain, Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi. Appl. Microbiol. Biotechnol. 98 (2014) 2533-2543. [30] W.S. Winston Ho, K.K. Sirkar, In: Membrane Handbook (Ed. W.S. Winston Ho and K.K. Sirkar), Van Nostrand Reinhold (1992) 3-16. [31] A. Saxena, B.P. Tripathi, M. Kumar, V.K. Shahi, Membrane based techniques for the separation and purification of proteins: An overview, Adv. Colloid Interface Sci. 145 (2009) 1-22. [32] M.A. Nawar, The optimal ammonium sulphate concentration for high recovery of immunoglobulins prepared from buffaloes’ blood serum and colostrums, Alex. J. Agric. Res. 44 (1999) 151-159. [33] E.R. Ling, A Text Book of Dairy Chemistry, Vol. II. 3rd ed, (1963) Practical Chapman and Hell, L.T.D. London. [34] T.L. Fahey, E.M. Mckelvey, Quantitative determination of serum immunoglobulins in antibody-agar plate, J. Immunol. 94 (1965) 84-90. [35] G. Mancini, A.O. Carbonara, J.F. Heremans, Immunochemical quantitation of antigens by single radial immunodiffusion, Immunochemistry 2 (1965) 235254. [36] J. Malhi, Evaluation of physico-chemical quality of fore milk, mid milk and stripping of Kundhi buffalo, M.Sc. Thesis, (2000) Sindh Agriculture Univ., Tandojam. [37] H.H. Arain, M. Khaskheli, M.A. Arain, A.H. Soomro, A.H. Nizamani, Heat stability and quality characteristics of postpartum buffalo milk, Pakist. J. Nutr. 7 (2008) 303-307. [38] S.H. Sadek, M.A. Fahmy, H. Nour El-Houda Mahmoud, A.M. Hassanein, Chemical studies on the colostrum, Assiut J. Agric. Sci. 43 (2012) 19-26. [39] A.M. Kholif, Effect of supplementing rations with buffers on the productive performance of dairy buffalo, Ph.D. Thesis, (1989) Fac. of Agric., Ain Shams Univ., Cairo, Egypt. [40] S.C. Nickerson, Milk Production: Factor Affecting Milk Composition, Blackie Academic and Professionals (1995) 22-23. [41] J. Prasad, Animal Husbandry and Dairy Science. Kalyani publishers, New Delhi, (1997) p. 316. [42] M.A. Nawar, Antimicrobial factors in buffalo milk: changes during the first month of lactation, Egypt. J. Dairy Sci. 34 (2006) 133-138. [43] A.M. Abd El-Fattah, F.H.R. Abd Rabo, S.M. El-Dieb, H.A. El-Kashef, Changes in composition of colostrum of Egyptian buffaloes and Holstein cows, BMC Veterinary Res 8 (2012) 1-7. 13
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[58] R.S. Patel, H. Reuter, D. Prokopek, S. Sachdeva, Manufacture of low lactose powder using ultrafiltration technology, Food Sci. Technol. 24 (1991) 338340. [59] Hind Bihari, Production of milk with enhanced protein content, M.Sc. Thesis, (2012) Dairy Technology Division, National Dairy Res. Instit. (ICAR), Deemed Univ., Karnal-132001, Haryana, India.
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Mohamed M. El-Loly, Laila K. Hassan, Eman S.A. Farahat PII: DOI: Reference:
S0141-8130(18)32438-3 https://doi.org/10.1016/j.ijbiomac.2018.11.055 BIOMAC 10932
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
20 May 2018 5 November 2018 11 November 2018
Please cite this article as: Mohamed M. El-Loly, Laila K. Hassan, Eman S.A. Farahat , Impact of heat treatments and some technological processing on immunoglobulins of Egyptian buffalo's milk. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.11.055
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ACCEPTED MANUSCRIPT Impact of heat treatments and some technological processing on immunoglobulins of Egyptian buffalo’s milk Mohamed M. El-Loly*, Laila K. Hassan, Eman S. A. Farahat
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Dairy Science Department, National Research Centre, 33 El-Buhouth St. (Former El-Tahrir St.), Dokki, PO 12622, Giza, Egypt * Corresponding author: E-mail: [email protected]
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ABSTRACT
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The effects of heat treatments, ultrafiltration and manufacture of soft cheese on the gross composition and immunoglobulins (Igs) of Egyptian buffalos were investigated. Four Igs (IgG1, IgG2, IgM and IgA) were identified and determined by single radial immunodiffusion (SRID). High concentrations of Igs were found in colostrum which decreased rapidly within the first 72 hrs postpartum parallel to the transition from colostrum to normal milk. IgG (IgG1, IgG2) and IgM were not completely denatured by pasteurization temperature up to 80°C/15 s, while IgA was completely denatured under these conditions. Ultrafiltration of milk resulted in retentate of high values for total IgG (IgG1, IgG2), but low in IgM and IgA content and permeate was free of Igs. Domiati cheese made from UF-milk retentate contained similar levels of Igs to the used retentate.
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1. Introduction
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Keywords: Buffalo colostrum/milk, chemical constituents, immunoglobulins, heat treatments, technological processing.
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Buffalo’s milk is an important source for human nutrition in some parts of the world. It is considered as a richer source of lipids, protein, lactose and minerals that had important for chemical and nutritive properties as well as suitability in the making of traditional and industrial dairy products [1].
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Although the proteins (immunoglobulins or antibodies, lactoferrin, lysozyme and lactoperoxidase) that make up only a minor milk proteins and play an important role as the first line of defense attribute to their specific and non-specific antimicrobial activity, as well as to their other important physiological and health promoting functions [2,3]. The bovine colostrum is a rich source of Igs that confers passive immunity to the newborn during the development of its own immune system [4]. Colostral Igs provide as one of the considerable prospective for consumers health promotion in the future [5]. These are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses [6,7,8,9]. Though Igs represent only 1-2% of the total milk proteins, or about 6% of the total whey proteins [10], while about 70-80% of the total protein in 1
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colostrum [11]. Milk Igs are found the same as in the blood or mucosal secretions, which are proteins molecules, it contains antibody activity, are created by white blood cells called plasma cells. They can be classified into five different classes based on their antigenic properties: IgG, IgM, IgA, IgD and IgE. In normal serum represents about 80, 15, 5 and 0.2% of IgG, IgA, IgM and IgD respectively and a trace of IgE [12]. The major milk Igs are G, A, M types that had higher significantly in colostrum comparing with normal milk, this observation was the same results obtained by [13,14,15,16]. IgG molecule is primary Ig class and dominant in colostrum, milk and blood being about 80-90, 60-70 and 90% of total Igs, respectively [14,17,18]. IgG had various subclasses IgG1 and IgG2 being the major Igs in serum. While IgA was found a major Ig class in mucosal secretions and prevents mucosal infections by agglutination of microbes. But IgM has a low specificity and hence a lower potency in defeating the infection, it appears initially when an organism is exposed to an antigen for the primary infection [19].
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Heat treatment is commonly applied to milk during industrial processing to ensure the microbial safety of dairy products as well as to extend shelf life [20]. Whey proteins denaturation is one of the basic effects of milk heating causing modification the chemical and nutritional properties of milk which leads to partial unfolding the whey proteins with helical structure damage or irreparable [21]. Igs are very important in use for many processing, concentration or isolation when exposing to heat, acid or pressure which may affect the conformation of the protein, and ultimately the immunological activity of it. Thermal treatment is an obligatory step, whereas the temperature and time combination could influence the protein’s structure for both of unfolding and aggregation [22]. IgG activity loss was significantly affected at 65°C/30 m of cow, buffalo and camel milk, while it was the whole activity loss of cow or buffalo milk at 75°C/30 m while the IgG loss being 68.7% in camel [23]. Recently, Bogahawaththa et al., [24] stated that the bovine milk protein structure can be affected directly by the heat treatments that cause changes in associated epitopes, the immunogenic and antigenic potential of milk proteins. The severity of some technological processing impacted to important minor proteins, which may exert immuno-modulatory properties, like a way as to prevent an occurrence of allergies. Also, Daniels et al., [25] illustrated that the effect of temperature using FoneAstra or Flash-heat pasteurization on human milk showed a decrease in the retention of total IgA (78.9 vs. 25.2%) respectively. Furthermore, several investigations related to the pH and temperature stability were reported by many authors, where Varshney et al., [26] indicated that the continuous loss in the secondary structure content in the temperature range of 2095°C. This due to molten globule states for several proteins under various 2
ACCEPTED MANUSCRIPT denaturing conditions. While Rabbani et al., [27, 28, 29] observed that the molecular compactness of the protein increases as the pH of the solution decreases, and the temperature-induced unfolding reveal that the molten globule state is more stable than the other states; as well as the effect of pH on the conformation and thermostability of outer membrane protein (OMP) points towards its heat resistance at neutral pH (7.0 at 69°C). Also, these results suggested that acidinduced unfolded state of OMP at pH 2.0 represented the molten globule state.
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UF technique is used as specific semi-permeable and becomes an essential part in food technology as a tool for concentration and fractionation the liquids and suspensions of the various constituents into two liquids based on its molecular weight, also other factors like the molecular shape and the charge can be affected. The suspended solids and solutes of larger molecules retained at the surface of the membrane are known as retentate or concentrate, while water and smaller molecules solutes will pass freely through the membrane are known as filtrate or permeate [30]. Membrane technology has been used in the dairy industry since the early 1960s and is currently come to the second after the water treatment technology and is a suitable another alternative to traditional dairy processes such as distillation, evaporation or extraction [31].
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The present study was focused to evaluate the thermal stability and the technology processing to manufacture soft cheese using UF technique on immunoglobulin fractions content of Egyptian buffalo’s milk within the first 15 th days of lactation. These biophysical properties of buffalo milk Igs are important due to its use as a source for human nutrition and economically helpful and clinically useful.
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2. Materials and methods 2.1. Samples collection
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Individual colostrum and milk samples were collected from three apparently healthy buffaloes of a private farm, Giza Governorate, Egypt, at 0-12, 24, 48, 72 hours; 4, 5, 7 and 15 days postpartum. Immediately samples were collected in clean stoppered bottles in an ice box and transported to the dairy laboratory, National Research Centre. All samples were stored and kept frozen at -20°C until analyzed. The average of duplicate was taken for each sample.
2.2. Precipitation of immunoglobulins using ammonium sulfate Buffalo milk samples were defatted by centrifugation at 4000 rpm for 3 min. Milk whey was prepared from the skim milk by adjusting pH to 4.6 using 1 N Hcl solution and centrifuging at 10000 rpm for 15 min. to remove precipitate casein particles. Total Igs were prepared from whey samples by using saturated 3
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ammonium sulfate (SAS), it prepared by dissolving excess in distilled water until some crystals of ammonium sulfate remained un-dissolved. Working solution of 80% ammonium sulfate solutions was freshly prepared by diluting SAS with a necessary amount of water. Equal volumes of the whey and 40% SAS solution were mixed; the formed precipitate was removed by centrifuging, dissolved in the minimum quantity of distilled water and dialysed until the complete removal of the ammonium salt against distilled water for 24 h at refrigerator with several changes of distilled water during this period. The dialysed extract was kept at -20°C until analyzed [32].
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2.3. Heat treatments
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For evaluate the effect of heat treatments on the Igs content of buffalo milk, the samples were thoroughly mixed, it was defatted and the skim milk of twenty ml of each stopper glass tubes were heat-treated in water bath at 63˚C/30 min., 80˚C/15 s, 100˚C/10 min. and sterilization at 130˚C/15 min., and then followed by rapid cooling to 37˚C for all samples.
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2.4. Preparation of domiati cheese manufactured by ultrafiltration (UF)
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To investigate the effect of UF technique on the Igs content of milk, the milk was heated to 55˚C and ultrafiltrated to CF4 using a carbosep pilot plant (modules 2 S 151 UF system, Orelies, France), equipped with an inorganic tubular membrane. It consists of two ultrafiltration modules of the surface area of 6.8 m2 together with pumps. The samples of milk, retentate, permeate and cheese samples were taken during the UF of milk and cheese processing to determine Igs fractions.
2.5. Determination of some gross chemical constituents
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The total solids (TS), total protein (TP) and ash contents were determined according to the method described by [33].
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2.6. Immunoglobulin quantification by single radial immunodiffusion (SRID) Radial immunodiffusion test in agar gel is a technique that routinely used for measuring the concentrations of various soluble antigens (usually proteins) in the biological fluid. Igs standard and radial immunodiffusion kits (The Binding Site Limited, England) were used for quantitative determination of Igs [IgG (IgG1, IgG2), IgM and IgA] in buffalo milk samples within a first 15th days after birth. It was principally derived from the work of [34,35]. All samples were used without dilution, each sample was placed in a well punched in the layered gel. After sample application, the lid is tightly closed and the plate stored flat with the lid uppermost at 2-8˚C. It is essential that the gel is not allowed to dry out during incubation. To 4
ACCEPTED MANUSCRIPT minimize evaporation, it is suggested that plates should either be resealed in their foil pouches or stored in a moist box (a sealed plastic box containing damp tissue paper) during incubation. The minimum incubation time for complete diffusion is 48 h (72 h for IgM). The diffusion was measured after incubation time, and the concentration of immunoglobulin in each sample can be read directly from the SRID reference table. The calculation was done according to the manufacturer manual.
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3. Results and discussion
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3.1. Gross chemical composition and immunity molecules distribution in buffalo’s milk
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As shown in Table 1, TS values of Egyptian buffalo’s colostrum indicated that the first 12 h had an average of 31.23%, it very rapidly decreased at 48 and 72 hr reaching average values being 16.66 and 15.27% respectively. The reduction was gradual with time progress thereafter until it reached a level 13.70% of normal milk at the end 15th day postpartum. Also, TP content had a mean of 13.53% at 12 hr postpartum, followed by a very rapid decrease to reach the value of 4.07% at 72 hr parturition, then gradually decreased to value 3.01% at the 15 th day of lactation. These results were the same trend with [36,37], but it was higher than that reported by [38] explained that the mean values of TS in buffalo colostrum were gradually decreased with time progress, which ranged of 24.48-13.67% and it was slightly lower for TP being 15.57-3.18% at 1st and 7th day postpartum respectively. As for the ash content was gradually decreased from 0.98 to 0.88% at 12 and 72 hr in order, followed by decreasing to value 0.66% at the end 15 th day parturition. The same thing was observed with [37,38,39,40,41,42,43,44].
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We can be observed that the most changes occur in milk protein content that is reduced more than the one-third at the 3rd day postpartum compared to initial values. Comparing the total Igs (2.41%) to the TP (13.53%) in the 0-12 hr samples indicates that it represent 17.81%. This result explains that the highest levels were in the first few hours of colostrum, and also means that mostly attributed to the Igs sharp decrease. It is completely identical with [40] mentioned that the higher protein values of colostrum were could be due to higher concentration of globulin that serves as the carrier of antibodies for suckling calf against disease producing organism. Moreover, in our investigation the protein content at the 15 th day of lactation was 3.01%, this observation was higher than reported by [45] found that it had ranged of 4.2-4.5% in buffalo milk, while cow milk has 3.6%.
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ACCEPTED MANUSCRIPT Table 1 Medians of the total solids (TS), total protein (TP), ash and Igs values in buffalo’s milk during the first 15th days postpartum. Lactation period 48
72
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15.46 3.97 0.85
24.64 10.96 0.93
16.66 4.94 0.91
15.27 4.07 0.88
10.72 44.44 7.24 30.02 5.52 22.89 0.64 2.65 24.12 0.0
10.54 48.11 6.47 29.53 4.38 19.99 0.52 2.37 21.91 -9.16
10.46 50.85 5.74 27.91 3.96 19.25 0.41 1.99 20.57 -14.72
7.24 44.97 4.83 30.00 3.70 22.98 0.33 2.05 16.10 -33.25
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31.22 13.53 0.98
7.20 48.55 4.24 28.59 3.11 20.97 0.28 1.89 14.83 -38.52
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Gross composition (%) TS TP Ash Immunoglobulin fractions (mg/ml) IgG1 % of the total IgG2 % of the total IgM % of the total IgA % of the total Total Igs % of decrease
Days 5
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15
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14.24 3.92 0.81
13.81 3.87 0.73
13.70 3.01 0.66
6.86 48.21 4.16 29.23 3.02 21.22 0.19 1.34 14.23 -41.00
5.83 46.60 3.63 29.02 2.87 22.94 0.18 1.44 12.51 -48.13
4.05 49.09 2.70 32.73 1.48 17.94 0.02 0.24 8.25 -65.80
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Also, Table 1 explain the immunity molecules distribution of buffalo’s milk, where the obtained median values at the first 12 hours were 10.72, 7.24, 5.52 and 0.64 mg/ml for IgG1, IgG2, IgM and IgA respectively, then dropped to 7.24, 4.83, 3.70 and 0.33 mg/ml in the same order at 72 h postpartum. Moreover, a gradual decrease could be noticed at the following days until the 15 th day being 4.05, 2.70, 1.48 and 0.02 mg/ml in order. Results indicated that a marked reduction in colostrum and milk in all these types with time progression of lactation. It obvious that the trend of changes in total Igs values decreased about 33.25% at the 3rd day and reached 65.8% at 15th day. In addition, the most obvious decrease was detected with both of IgM and IgA types that it decreased slightly levels (22.89 vs 17.94%) and (2.65 vs 0.24%, less than the tenth) at the first hours and the 15 th day of lactation respectively. These data had been established with [17,46]. Confirming to [43] demonstrated in buffalo and cows that the composition of both colostrum approaches those of normal milk within 5 d after parturition, also they found the same trend for IgG and IgM values. Recently, Neama Ashmawy [44] reported that the colostrum IgG content decreases very rapidly with time with average 3260.5, 2654.0, 1900.5 and 1325.25 mg/dl at the zero time, 3, 6 and 24 h after calving in order. 6
ACCEPTED MANUSCRIPT 3.2. Impact of heat treatments on gross chemical composition and immunity molecules distribution of buffalo’s milk
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As thermal processing is a major treatment in the processing of milk and milk products, its influence on whey proteins fractions, whereas the stability of the Igs in milk depends on the severity of the thermal treatment used in the various commercial processes. Milk contains Igs (G, M and A), colostrum being practicality rich in its; the biological function of Igs consists in a passive immunization of the calf via ingested colostrum. From milk processing point of view, the Ig fractions of early lactation milk reduce its heat stability, this is the reason why milk from early stages of lactation is excluded from producing milk. Table 2 shows the effect of thermal treatments of buffalo’s milk, it is clear that the TS content of pasteurized milk samples at 63°C/30 min. and 80°C/15 s were almost the same value as control (11.99, 12.02 and 12.06%) respectively. On the other hand, increasing temperature up to boiling or sterilization led slight losses (14.85 and 17.76%) in TS values, being 10.21 and 9.86% in order. This may attributed to its effect in precipitating some of the milk proteins. The similar trend for TP values 2.19 and 2.15 (with loss of 17.05 and 18.56%) at the same order. While the ash values of different heat-treated samples were almost the same.
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Concerning to Igs distribution (IgG1, IgG2, IgM and IgA) at 63°C/30 min., as result in Table 2, the heat treatment caused losses percentages of 35.06, 33.33, 56.02 and 100% respectively. A similar trend had obtained at 80°C/15 s being 33.33, 31.48, 54.73 and 100% in order. On the contrary, for IgA, it was none retained of treated milk samples that may be due to its low level in raw milk (control), this means that IgA is the most heat sensitive of the Igs. On the other hand, increasing temperature to boiled or sterilized milk did not contain any Igs, which means that these heat treatments had a destructive effect on Igs values. We concluded that the Igs were the least heat stable in the milk whey proteins. Our data suggest that commercial pasteurization process results incomplete denaturation of IgG1, IgG2. Generally, total IgG (IgG1, IgG2) and IgM were not completely denatured at commercial pasteurization up to 80°C/15 s, while IgA was completely denatured at any temperature. Also, this study demonstrates the dependence of buffalo milk Igs stability in milk products on the severity of heat treatment used in various commercial processes. Our results were almost agreed with [47] found that the HTST pasteurization may not destroy IgG activity of bovine milk, being 65-79% retention when comparing to the raw milk, while it was 59-76% by [48]. While IgG1, IgG2 and IgM unfolded at 79.4, 76.7 and 80.3°C respectively [49]. IgG was the most thermo-stable, but IgM was the least one [50]. While [51] illustrated that the Igs are thermo-labile, the bovine IgG molecule can reduce about 40 and 100% at 75°C/5 min. and 95°C/15 s in the same order. Those 7
ACCEPTED MANUSCRIPT changes may be attributed to conformational changes of IgG type causes by heat exposure [52]. IgG and IgM were not completely denatured in buffalo milk between 63-88°C that may be due to the protective effect of the high protein content of buffalo milk, but IgA was completely denatured between 63-88°C [53]. The IgG denaturation ratio at 72°C/15 s and 90°C/5 min. of yak (bovine species) colostral whey reduced by 19.28-37.56 and 44.96-58.97% respectively [54].
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The protein loses a considerable fraction of its changes in the secondary structure during thermal denaturation, also, the presence of intermediate unfolded states in the thermal denaturation of human serum albumin that is structurally similar to bovine serum albumin [55].
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Recently, Klotz et al., [56] illustrated that the mean values of IgA in human milk were 95 and 83% at HTST treatment (62°C/5 s) and standard holder pasteurization (63±0.5°C/30 min.), being 51.7±28.3 and 43.0±22.7 mg/dL in order. Table 2
Control
Heat treatments
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Medians of the total solids (TS), total protein (TP), ash and Igs values in buffalo’s milk as affected by different heat treatments.
12.02 2.24 0.78
15.15 -
12.06 2.38 0.79
9.85 -
10.21 2.19 0.70
14.85 17.05 -
9.86 2.15 0.75
17.76 18.56 -
2.63 1.80 0.65 0.0
35.06 33.33 56.02 100.00
2.70 1.85 0.67 0.0
33.33 31.48 54.73 100.00
0.0 0.0 0.0 0.0
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0.0 0.0 0.0 0.0
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11.99 2.64 0.75
4.05 2.70 1.48 0.02
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Gross composition (%) TS TP Ash Immunoglobulin fractions (mg/ml) IgG1 IgG2 IgM IgA
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63°C/30 Loss % 80°C/15 Loss % 100°C/10 Loss % 130°C/15 Loss % min. s min. min.
3.3. Gross chemical composition and immunity molecules distribution in Domiati cheese manufacture steps by ultrafiltration technique In traditional cheese making, the milk is first coagulated by addition of starter culture and rennet. The concentration continues when the whey is drained off and curd forms. This whey contains about 25% of protein and 10% of fat contents, whereas some the nutrients are lost in the whey, such as lactose, minerals and soluble vitamins, these losses have a large influence on the economics of the process. UF is an effective means of recovering the by-products that can be used 8
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for further food formulations and also produce cheese milk having substantially the composition of cheese at the end of whey drainage and turn the cheese milk into cheese by the coagulation with adding rennet. Therefore, interesting to use of UF technique to concentrate milk and by-products from milk for subsequent manufacture was in most cheese manufacture. The milk is an ideal liquid for membrane filtration due to its composition, it is concentrated and then followed by starter culture and rennet induced coagulation. Due to the membrane technology was retained larger protein molecules including whey proteins, it means that Igs retained in milk which raised the natural immunity in resultant cheese.
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Renner & Abd EI-Salam, [57] explained that during UF the protein and fat of milk were retained nearly completely in the retentate, whereas lactose, minerals and vitamins are partitioned between the retentate and permeate depending on the degree of concentration. Therefore it is important to follow the Igs content in the method steps of Domiati soft cheese processing, namely milk, retentate, permeate and fresh cheese, which cheese manufacture by UF lends itself to continuous processes and to better control over composition and quality.
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Fig. 1 understood that the TS of milk was 17.90 increased to 33.94% for retentate, while permeate contains only 5.89% including lactose and mineral salts. The protein level of retentate (12.22%) almost equals the protein level of resultant UF Domiati cheese (16.83%) with subtracted the addition amount of salt (4.881.78%). The ash content of milk, retentate and cheese were 0.98, 1.78 and 4.88% respectively. These obtained results are in corresponding to that reported by [58] showed that when buffalo milk ultrafiltered, the resultant retentate had a composition of 24.52, 17.99 and 2.33% for TS, TP and ash contents respectively. Also, it was almost in agreed with Hind Bihari [59] found that the TS, TP and ash constituents of UF buffalo milk were 17.22, 4.15 and 0.76% in the same order.
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While Fig. 2 shows the concerning immunity molecules (IgG1, IgG2, IgM and IgA) that could be increase concentrated using UF technique in both of retentate and fresh UF cheese, being 12.65, 8.23, 2.14 and 0.30 as well as 13.20, 10.0, 2.85 and 0.38 mg/g in the same order. We concluded that as a result using UF technique, both of the resultant retentate and UF soft cheese had higher values of total IgG (IgG1, IgG2), but in a lower level of IgM and IgA, because the buffalo milk already has lower values in both of them compared to the IgG level that it was dominant in colostrum. While the permeate had free Igs because UF membranes retained the larger molecules of milk proteins in the retentate. Finally, Fig. 3 shows the SRID analysis of total IgG (IgG1, IgG2) in buffalo’s milk affected by different heat treatment and technological processing during UF domiatti cheese making steps. 9
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(%)
TP
Retentate
Milk
Fresh Cheese
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Ash
Permeate
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TS
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14 12 10 8 6 4 2 0 IgG2
IgM
Fresh Cheese Permeate Retentate Milk
IgA
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IgG1
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(mg/ml)
Fig. 1. Medians of the total solids (TS), total protein (TP) and ash values in buffalo’s milk during UF Domiati cheese manufacture steps.
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Fig. 2. Medians of the Igs values in buffalo’s milk during UF Domiati cheese manufacture steps.
Fig. 3. Single radial immunodiffusion analysis of total IgG in buffalo’s milk affected by different heat treatment and technological processing during UF Domiati cheese manufacture steps. Wells No: (1&6, 2&7, 3&8, 4&9, 5&10) represent control, 63°C/30 min., 80°C/15 10 s., 100°C/10 min. and 130°C/15 min. respectively, as well as (11,12,13,14) represent milk, retentate, permeate and fresh cheese respectively.
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