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acta facultatis medicae naissensis doi 10 1515 afmnai 2016 0011 udc 613 287 1 5 544 532 135 original article comparative analysis of the physicochemical parameters of breast milk starter ...

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                                                                                          ACTA FACULTATIS 
                                                                                      MEDICAE NAISSENSIS 
                                                                                                                  
            DOI: 10.1515/afmnai-2016-0011                                      UDC: 613.287.1/.5:[544+ 532.135 
                                                                                                                  
                                                        
                                                                            
            Original article 
             
                      Comparative Analysis of the Physicochemical 
              Parameters of Breast Milk, Starter Infant Formulas and 
                                 Commercial Cow Milks in Serbia 
                                                                   
                                     1                   2             3              3                4 
                      Slavica Sunarić , Tatjana Jovanović , Ana Spasić , Marko Denić , Gordana Kocić
                                                                
                            1
                             University of Niš, Faculty of Medicine, Department of Chemistry, Niš, Serbia 
                               2
                                University of Niš, Faculty of Medicine, Department of Physics, Niš, Serbia 
                       3                                 3
                       University of Niš, Faculty of Medicine,  PhD Student at Department of Pharmacy, Niš, Serbia 
                             4
                             University of Niš, Faculty of Medicine, Department of Biochemistry, Niš, Serbia 
             
                                                       SUMMARY 
               
                    Data on the physical properties of cow milk and infant formulas are important since they 
              indicate the differences in physicochemical and rheological characteristics and compatibility with 
              natural breast milk. This fact is important not only for quality control but also for the use of these 
              commercial products as infant diet supplements or as complete breast milk substitutes. This study 
              was undertaken to determine refractive index, surface tension, pH, electrical conductivity, viscosity 
              and titratable acidity of the UHT cow milk, starter infant formulas and breast milk of Serbian 
                                                                                                       he paper 
              mothers in order to compare commercial milk formulations with natural human milk. T
              also presents the measured data of some physical parameters of human milk about which there is 
              little information in the literature. It has been also demonstrated how these parameters were 
              changed by freezing and prolonged storage of breast milk. 
                    In this study, 8 commercial cow milks, 6 starter infant formulas and 15 different samples of 
              colostrum, transition and mature breast milk were included. The titratable acidity, pH, electrical 
              conductivity, refractive index, viscosity and surface tension were measured by using standardized 
                                                                las available on the Serbian market differ in 
              techniques. It has been found that infant formu
              physicochemical parameters compared to breast milk. Regarding these parameters, none of the 
              analyzed formulas fully corresponded to breast milk. It has been also shown that measurement of 
              physical parameters is simple and inexpensive way to monitor the milk shelf-life which is important 
              for human milk banks. 
                       
                    Key words: breast milk, infant formula, cow milk, physicochemical parameters 
             Corresponding author: 
             Slavica Sunarić  
             e-mail: slavica.sunaric@medfak.ni.ac.rs 
                                   Acta facultatis medicae Naissensis 2016;33(2):101-108                     101 
              Original article 
                     INTRODUCTION                                          starter infant formulas (0-6 months) were purchased 
                                                                           at a local pharmacy and were obtained from 
                     Breastfeeding is universal and the most  representative producers of infant food. Impamil®, 
              appropriate form of nourishing the infants for the first     Nan AR®  and Humana1® were formulas with 
              6 months postpartum (1). When breastfeeding is not           prebiotics, while Hipp Organic® was organic infant 
              possible,  alternate sources of nutrients are required.      milk. Nan 1® contained Bifidobacteriumlactis culture. 
              Human milk is markedly different from cows' milk,                   Human milk samples were collected at the 
              both in terms of macronutrients and micronutrients (2,       Clinical Center Niš, Serbia, from the healthy volunteer 
              3). Cow milk contains high concentrations of proteins        women (n=15) in different period of lactation covering 
              and minerals which impedes digestion. In addition,           colostrum, transition and mature milk. Additionally, 
              cow milk lacks the iron, vitamin C and some fats             the mature milk samples of every woman were 
              important for growing babies. For this reason, cow           categorized into 9 groups depending on the interval 
              milk should not be used as the main drink before 12          of the lactation during ten months.  
              months of age, although small volumes may be added                  All measurements were performed in 
              to complementary foods (4).                                  undiluted samples of commercial and breast milk at 
                     Commercial infant formulas are commonly used          room temperature. Commercial samples were 
              either as baby diet supplements or as complete breast        analyzed immediately after opening. Breast milk 
              milk substitutes. The infant milk substitutes should be      samples obtained in the first 20 days postpartum were 
              properly formulated so that nutritional requirements         analyzed 2 hours after collection, while samples from 
                                                                           the 30th                th 
              for optimal growth are met adequately (5). Most infant                 day to the 5    month were analyzed after 
              formulas are made with cow milk which has been               keeping for 3 months at -20°C. Infant formulas were 
              altered to resemble human milk. The other types of           prepared according to instructions given by 
              formulas are soy-based and protein hydrolysate  producers: amount of 5g of powder milk was 
              formulas. Milk substitute from plant sources does not        weighted in the test tube, dissolved in 50 mL of 
              contain all the nutrients in a healthy balance for infants   deionized water and kept in a warm water bath for 10 
              (6).                                                         min.  
                     The physicochemical characteristics such as                   
              refractive index, surface tension, pH, conductivity,                Apparatus 
              viscosity and titratable acidity are important                       
              parameters in studying quality and nutritional aspects              Milk samples were analyzed for viscosity, 
              of milk and milk products, because the physical and          refractive index, surface tension, conductivity, pH and 
              rheological  properties strongly depend on chemical          titratable acidity. The electrical conductivity was 
              composition (7). The aim of this work was to  measured by HANNA INSTRUMENTS EC 215 
              investigate and compare the physicochemical  conductivity meter  (Hanna Instruments USA, 
              parameters of commercial cow milks, breast milk and          Smithfield, RI). For the measurement of pH 
              starter infant formulas in Serbia. Also, parameters for      CONSORT C830 multi-parameter analyser (Consort 
              the fresh and frozen breast milk were compared and           bvba, Turnhout, Belgium) was used. The refractive 
              discussed.                                                   index was measured by using Abbe’s refractometer 
                                                                           and viscosity by using capillary flow type Ostwald U-
                     MATERIAL AND METHODS                                  tube viscometer. 
                                                                                   
                     Sample collection and preparation                            Methods of measurements 
                                                                                   
                     Cow milk samples were obtained from the                      Titratable acidity (TA) was determined by 
              market and included the most representative  using titrimetric method on this way: 9.0 mL of milk 
              producers in Serbia. These milk samples were  or dissolved infant formula sample is titrated with 
              different in terms of milk fat and vitamin content. One      0.1N sodium hydroxide (NaOH) in the presence of 
              of them was fortified with vitamins A and D. Also,           phenolphthalein as indicator. The obtained total 
              one of the samples was obtained by organic  acidity was expressed as % of lactic acid and was 
              production and one was without lactose. Samples of           calculated according to the equation:                
              102                        Acta facultatis medicae Naissensis 2016;33(2):101-108 
                                                                                       Slavica Sunarić, Tatjana Jovanović, Ana Spasić et al. 
                     % lactic acid=(Vg×100× 0.009)/Vm (1)                   ions. Lactic acid and its salts are also important 
              where Vg is the added volume of NaOH solution                 conductors. On the other hand, the non-electrolytes 
              (mL); Vm is the milk volume used for titration (mL)           (lactose, urea, proteins, peptides, sugars and fats) do not 
              and 0.009 is equivalent of lactic acid normality (8).         directly contribute to the electrical conductivity, but they 
                     Viscosity was determined by the flow time of           affect it through their influence on the viscosity and the 
              the fluid and by gravity through the Ostwald’s                ions migration. Thus, in a heterogeneous system such as 
              viscometer at 25°C. The time in seconds was  milk, fats obstruct the ions migration and decrease the 
              converted into dynamic viscosity by using the  conductivity (9-11). 
              diameter of the capillary and the formula:                           The conductivity range of all the examined 
                                           ρ ⋅t                             commercial milk samples was from 3.84 mS/cm to 4.06 
                                 η =ητ x x (2)                              mS/cm, as shown in Table 1. The organic produced 
                                   x    0 ρ ⋅t                              commercial cow milk Bello organic® with 2.8% of milk 
                                             0  0
                                                                            fat showed the lowest value of the conductivity. The 
              where, ρx and ρ0 are densities of examined samples            electrical conductivity of the fresh breast milk was in the 
              and water and ητ  is the coefficient of viscosity of          range of 1.46-1.89 mS/cmand decreased with the period 
                                  0                                         of lactation. Frozen mature breast milks had slightly 
              water at 25°C.                                                lower values of conductivity than fresh mature milk. The 
                     The coefficient of surface tension was  values of the conductivity of infant formulas were lower 
              determined by using stalagmometer by the drop  than those of commercial cow milk. Among the infant 
              number method. The mean values of the number of               formula samples, the lowest conductivity was found for 
              drops for the sample and water (nxand no) were used           Humana 1® and the highest was for the Impamil®. 
              to calculate the surface tension of the sample fluid          These conductivities are more adjusted to those for 
              according to equation:                                        breast milk, especially to colostrums, than conductivities 
                                           σ n                              of commercial cow milks. Only Humana 1® had 
                                 T =T x 0(3) 
                                   x     0 σ n                              electrical conductivity similar to those of transition and 
                                             0 x                            mature breast milk. 
              where, T0 is the surface tension coefficient of distilled             
              water, σ                                                             Refractive index (RI) 
                       x and σ0 are the specific weights of the sample 
              and distilled water.                                                  
                                                                                   Optical properties of milk determine the 
                     RESULTS AND DISCUSSION                                 appearance of milk and milk products. The refraction of 
                                                                            light by solution depends on the individual molecular 
                                                                            species present and their concentrations (9). The 
                     The physicochemical parameters of the  refractive index of milk is the sum of the refractive index 
              commercial cow milks, infant formulas and human milk          of the solvent and solutes, therefore, this parameter is 
              are given in Tables 1, 2 and 3, respectively.  The            useful for estimation of total solids in milk or the content 
              measurements were repeated at least in triplicate and         of water which was added to milk. The refractive index 
              the results are presented as mean value ± standard            of milk is difficult to estimate due to light scattering by 
              deviation.                                                    casein micelles and fat globules; however, by using 
                                                                            Abbe’s refractometer it is possible to make satisfactory 
                     Electrical conductivity                                measurements (12).  
                                                                                   The refractive index of the fresh breast milk 
                     The conductivity test was primarily standardized       samples were found to be 1.3494 to 1.3510, that is lower 
              for the rapid quality control of cow’s milk and it is         than RI of commercial milks and most of the infant 
              important to detect abnormal milk (9). The electrical         formulas. On the other hand, most of the infant formulas 
              conductivity of milk is mainly due to the presence of         had similar RI values to commercial cow milks, although 
              various electrolytes (soluble salt fraction), non-            slight variations were found for this parameter in all of 
              electrolytes and fat content. The main conductors in milk     examined samples. Thus, Nan AR® had RI similar to 
              and milk products are sodium, potassium and chlorides         transitional milk, while Impamil ® had RI close to 
                                                                            colostrum.
                                          Acta facultatis medicae Naissensis 2016;33(2):101-108                                   103 
               Original article 
                        Titratable acidity (TA) and pH                            occur by the activity of bacteria even at -20°C (12). The 
                                                                                  elevated TA values of frozen breast milk are not 
                       The natural acidity is an important characteristic         acceptable and those milk samples are unsuitable for 
               of fresh milk and is mainly due to the presence of                 infant nutrition.   
               proteins, phosphates, carbon dioxide and citrates.                         
               Another important characteristic of milk is its buffering                 Viscosity 
               capacity. The principal buffering compounds in milk are                    
               its salts (soluble calcium phosphate, citrate and                         Viscosity is a parameter that defines flow 
               bicarbonate) and acidic and basic amino acid side-chains           properties of milk or milk products. Milk and milk 
               on proteins (particularly the casein) (9). Titratable acidity      products are fluids with Newtonian rheological 
               (TA) is a parameter suitable for expressing the natural            properties, meaning that they exhibit a  direct 
               acidity and buffering capacity, because it is a measure of         proportionality between shearing stress and rate of 
               the total acid content (dissociated and undissociated).            shear. The viscosity of milk and milk products is mainly 
                                                                       +
               On the other hand, pH is a measure of the free H  ions             influenced by composition, concentration of 
               concentration obtained from dissociation of acids  components, pH, temperature and thermal history. 
               present in milk. Therefore, there is no direct relationship        Casein and fats are the principal contributors  to the 
               between pH and TA. It is possible to obtain high pH and            viscosity of milk (7). Viscosity is an important 
               high TA values, which suggests high concentrations of              rheological property of breast milk because it determines 
               proteins and other buffering compounds.                            the ease of swallowing by infants. There are few recent 
                       Titratable acidity of our fresh human milk  data about the viscosity of human milk (15, 16). 
               samples varies from 0.02% to 0.07% of lactic acid. Higher                 The examined fresh mature breast milk samples 
               TA value for colostrum can be explained due to its high            had the lowest viscosity, followed by the infant formulas 
               protein content. In the infant formula samples, the                and commercial cow milks. Frozen mature breast milks 
               average TA was 0.0608±0.014% of lactic acid, which is              had lower viscosity than fresh mature milk (average 
               more similar to that of colostrum and transition milk              1.36±0.04  Pa·s 10-3
               than that of mature breast milk. Based on the standard                                  ), which is probably related with 
               deviation, it can be concluded that the differences in             reactions explained above. Among the products for 
               titratable acidity are relatively high among infant  infant feeding, the lowest value for viscosity was found 
               formulas. The values of TA for commercial cow milk                 for Nan AR® and the highest value was for Humana1®. 
               (average 0.166 ± 0.007 % of lactic acid) were significantly        The samples of Humana1® were very viscous, so the 
               higher than those for infant formulas or breast milk,              measurement could not be done with satisfactory 
               because cow milk has higher protein content (3.4%                  accuracy and precision. Further, Aptamil® and Hipp 
               versus 1.0% for human milk) and particularly high                  Organic® had viscosity similar to colostrum, while 
               casein content (9).                                                viscosity of Nan AR® was mostly like those for 
                       Commercial milks and some of the infant  transition and mature breast milk. In the liquid-packed 
               formulas have pH values in the range of 6.5-6.65. Only             commercial cow milk samples, the UHT milk fortified 
                                                                                  with the vitamins A and D, Imlek A+D
               two of the investigated infant formulas had pH round                                                               3®, had the 
               6.90 that is very similar to pH of the fresh human                 lowest viscosity and the Zdravo® with 3.5% of milk fat 
               colostrums. All of the infant formulas had pH lower                had the highest viscosity.  
               than fresh transitional and mature breast milk. This                       
               result suggests that commercial products for infant                       Surface tension 
               feeding from our market are not fully adjusted to the                      
               composition of the breast milk.                                           The surface tension is defined as the work 
                       The examined mature breast milk samples which              required to increase the surface area of a solution. The 
               were frozen for three months at -20°C had significantly            main surfactants in milk are proteins, phospholipids, 
               lower values of pH and higher values of TA than fresh              mono- and diglycerides and salts of free fatty acids (7). A 
               breast milk (Table 3). This is due to the lipolysis as well        higher content of non-polar molecules results in lower 
               as bacterial growth (7). During cold or frozen storage of          values of the surface tension of the liquid. Thus, the 
               human milk, lipoprotein lipase causes hydrolysis of milk           increase of fat content results in surface tension decrease. 
               triglycerides which yields to release of the free fatty acids             In the examined fresh breast milk samples, the 
               (13). Also, the conversion of lactose to lactic acid may           average value of surface tension was (46.99 ± 1.21) × 10-3 
               104                           Acta facultatis medicae Naissensis 2016;33(2):101-108 
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...Acta facultatis medicae naissensis doi afmnai udc original article comparative analysis of the physicochemical parameters breast milk starter infant formulas and commercial cow milks in serbia slavica sunari tatjana jovanovi ana spasi marko deni gordana koci university nis faculty medicine department chemistry physics phd student at pharmacy biochemistry summary data on physical properties are important since they indicate differences rheological characteristics compatibility with natural this fact is not only for quality control but also use these products as diet supplements or complete substitutes study was undertaken to determine refractive index surface tension ph electrical conductivity viscosity titratable acidity uht serbian he paper mothers order compare formulations human t presents measured some about which there little information literature it has been demonstrated how were changed by freezing prolonged storage different samples colostrum transition mature included using s...

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