Az. J. Pharm. Sci. Vol. 41, March, 2010, 67-76.
BIOLOGICAL EVALUATION FOR PROTEIN SOURCE OF WEANING FOOD PREPARATIONS IN RATS Mahmoud, Neven A; Hnaa A Sidky; Aml A AbdElbaky and Fathia K S AboZeid
Special Food And Nutrition Dept. FTRI, ARC, Cairo.
ABSTRACT
The nutritional biochemistry of protein sources for weaning diet was investigated in order to design a more effective program for improved child health. This rat evaluation experiment included the measurement of gained body weight (GBW), organs weight, the histology of liver and kidney, and red and white blood (RBC and WBC) cell count. These were exam end to reach a real biological evaluation employing what so called biological ranking number. It seems that the combination of wheat, date, sweet potatoes, and processed soy protein is the best complimentary food during breast feeding. Although evaluation based on body weight gain (BWG) is always used, it has been recognized; according to the biological facts mentioned here, that BWG of infant is not the most reasonable way of assessment. Surprisingly, G5 of that most equal varied plant ingredients makes the best nutritional combination, even over the control. This has been followed by G4 of certain balance between plant and animal protein sources. Animal protein is important but the untimely and inappropriate introduction of complementary foods should be of not risk for both under- and over-nutrition stunting and micronutrient deficiencies. This food, as in G5, will depend on the science behind current nutrition profiling systems to promote consumption of nutrient-dense foods relative to the current age cycle. The variable combination of plant proteins, from nutrational and medicinal point of view, is more considerable feeding system during this period. It has been concluded that this food preparation in G5 has prevented anemia, immune dysfunction and infant’s liver fro most of: bile duct thickening wall, Kypffer cells activation of cytoplasm, granularity of hepatocytes congestion of central vein, and hepatocyte hydropic degeneration as well as the kidney pathological disorder such as congestion of renal blood vessels, psychosis of the nuclei of epithelial renal tubules.
INTRODUCTION
Yet, principles of nutrition may be assigned very early of man life and nutrition in childhood, as well, influences both the child's present and future health. Weaning, especially, is an important nutritional milestone with implications for present and future health. Health professionals need to educate parents from all socioeconomic groups about good nutrition for their children from birth onwards. For this purposes, Emamghorashi and Heydari (2007) evaluated the growth of 597 infants < 24 months attending health centers in Jahrom according to type of infant feeding. They compared growth curves with those of the National Center for Health Statistics (NCHS). The infants were divided into 3 groups: exclusively breastfed, exclusively bottle-fed and both breast- and bottle-fed and were assessed monthly for the first year and every 2 months in the second year. There were no significant differences between the groups in height and weight in the first few months of birth; later, breastfed infants were significantly heavier and taller. The mean heights and weights of infants were lower that those for NCHS. In order to effectively promote exclusive breastfeeding, it is important to first understand who makes child-care and child-feeding decisions, and why those decisions are made; as in most parts of the world, exclusive breastfeeding until 6 months of age is uncommon in Malawi. To characterize early infant foods in rural northern Malawi, who the decision-makers are, their motivation, and the consequences for child growth, in order to design a more effective program for improving child health and nutrition. Mzuwula and dawale (two herbal infusions), water, and porridge were common early foods (Kerr et al, 2007). Grandmothers introduced mzuwula to protect the children from illness; other foods were usually introduced by mothers or grandmothers in response to perceive hunger. The early introduction of porridge and
dawale, but not mzuwula, was associated with worse anthropometric status. Mzuwula, which is not associated with poor growth, is usually made with boiled water and given in small amounts. Conversely, porridge, which is associated with poor child growth, is potentially contaminated and is served in larger amounts, which would displace breast milk. Promoters of exclusive breastfeeding should target their messages to appropriate decision makers and consider targeting foods that are most harmful to child growth (Kerr et al, 2007). As a food formula, in background, the use of lyophilized meat powder with iron-fortified wheat flour to produce an infant porridge with bioavailability iron, but its acceptability and safety are unknown. In the objective to evaluate the acceptability and safety of porridges containing lyophilized meat powder and iron-fortified wheat flour, Peruvian mothers' input was used to develop porridges without and with meat powder (low or high chicken liver, low or high chicken thigh) (Pachón et al, 2007). To assess the efficacy of a home made energy dense weaning food, containing 110-130 kcal (26-30kJ) per 100 ml on prevention of growth faltering during infancy, methodology used included infants attending four child health welfare clinics in the Medical Officer of Health (Ragama) area and recruited at the age of 4 months (De Silva et al, 2007). The intervention group received a specially designed hand blender, recipe and advice to prepare a weaning food. The control group received weaning foods without any intervention. They were followed up monthly up to the age of 12 months.
The infants in the intervention group gained significantly more weight than the control group. Both groups showed a drop in the Z score for mean weight for age during the study period but this was less marked in the intervention group (De Silva et al, 2007).
Current development in infant formulae, e.g. nucleotides; long chain polyunsaturated fatty acids, prebiotics, etc, should be a priority and can help to avoid several nutritional problems later on, such as excessively faddy eating, faltering growth, constipation, iron deficiency anemia and obesity (Harrod-Wild 2007). Contaminated water and food are major causes of malnutrition and mortality in the developing world, particularly among children. Infants are most vulnerable to diarrheas illnesses when introduced to fluids and foods as they are weaned from breastfeeding to a mixed diet. Dietetic professionals have the training to empower individuals and communities with skills to create a safe water and food environment (Marino, 2007). Simply, it is small, frequent meals of easily digestible, smooth, semisolid nutrient- and energy-dense complementary foods. This should initially be offered while gradually increasing variety in both the type and texture of food. The quantity and quality of infant's diet, as seen before, are essential. Timely introduction of appropriate complementary foods is vital for the immediate and long-term health of the infant and caregivers should be accordingly advised on feeding at this age (Van der Merwe, et al 2007).
Protein and carbohydrates intake should be increased with the infant's age while preference should be given to foods rich in micronutrients. Iron, in particularly, differs from other minerals because iron balance in the human body is regulated by absorption only and there isno physiological mechanism for excretion. On the basis of intakedata and isotope studies, iron bioavailability has been estimatedto be in the range of 14–18%
for mixed diets and 5–12%for vegetarian diets in subjects with no iron stores, and thesevalues have been used to generate dietary reference values forall population groups (Harrell and Egli 2010). Moreover, weaning food must contain several other constituents. Vitamin A is an example. Ross and Li, (2007) inferred that the lungs depend, at least in part, on the uptake of dietary vitamin A, probably from chylomicrons, to develop RE stores in the post weaning growth period. Moreover, a complementary food is essential. Vitamin A supplements, again, are administered to infants in developing countries at immunization contacts; doses of 50000 IU vitamin A are recommended. Doses of 100000 IU are given to children aged 0.5-1 y. The efficacy of these doses has not been adequately determined. Therefore, Surles et al, 2007 aimed to quantify liver vitamin A after the administration of vitamin A doses to piglets, the good model for infants because of their similar size, gastrointestinal anatomy, and vitamin A requirements. Energy with efficient range of micronutrients is key ward. Most recently, the science behind current nutrition profiling systems to promote consumer intake of nutrient-dense foods was facilitated (Casgrain, et al, 2010; Kapica, 2010; Drewnowski, 2010; Kramer, 2010 and Lupton, et al 2010; Schack-Nielsen, et al, and 2010; Philip, et al 2010), meanwhile, the most recent biochemical category in biological sciences is what so called bioactive proteins.
Here, it focuses on the effects of milk peptides on immune function and attempts to provide an overview of the knowledge available in this field. Bioactive peptides encrypted within the native milk proteins can be released by enzymatic proteolysis, food processing, or gastrointestinal digestion. These peptides possess a wide range of properties, including immunmodulators properties (Politis and Chronopoulou 2008). The first months of life represent a critical period for the maturation of the immune system because a tolerance for nutrient
molecules should be developed while that for pathogen-derived antigens is avoided. Evidence has accumulated to suggest that milk peptides may regulate gastrointestinal immunity, guiding the local immune system until it develops its full functionality (Politis and Chronopoulou 2008). Milk contains a variety of components that provide immunological protection and facilitate the development of neonatal immune competence. Two main categories of milk compounds are thought to be associated with immunological activity. The first category includes cytokines, of which neonates are not produce efficiently. Cytokines present in milk are thought to be protected against intestinal proteolysis and could alleviate immunological deficits, aiding immune system maturation (Politis and Chronopoulou 2008). The second category of milk compounds includes milk protein peptides. Milk peptides may affect mucosal immunity possibly by guiding local immunity until it develops its full functionality (Politis and Chronopoulou 2008).
As it is of that great metabolic role, this animal protein source was examined here using some biological parameters to measure the most protein healthful diet that support the healthy growth of rat infant.
MATERIA AND METHOD
Rationales: The food materials: soy protein extrudates (SP) is a product of the FTRI, meanwhile, semisolid date (D), powdered sweet potatoes (SWP) and skimmed milk (SM) as well as honey, apple, and spices used in preparation of special additives (AD) were obtained from local market in Cairo. Six different formulations have been prepared as seen in Table 1 at different ratios of these food ingredients to test the appropriate level role of animal portion, keeping protein level at 16 -/+ 1% diet.
Animals: A 9 day age of average weight 55 +/- 2 g male albino rats of western strains were housed in the Ophthalmology Research Institute, Giza, Egypt. These were the offspring of pregnant females’ rats, which have been accomplished in proper time according to UFAW (2000). Animal groups (5x6 = 30 animals) were either partially fed on basal diet according to Bowman et al, (1990), meanwhile salt and vitamins mixtures have followed that of AOAC (1984) plus infant commercial cereals at half and half level as a control (P1), or grouped from 2 to 5 as outlined in Table (1). The food and water were daily given ad labtium. Each mother was kept with its offspring of equal numbers in a separate cage.
To test the biological effect of these food formulations, rats’ blood samples were taken and animals were anesthetized after two weeks. The organ were separated and weighed when the trial has been terminated.
For cell count, Excel 80 blood cell counter (Danam, USA) was used to count the red (RBC) and weight blood cells (WBC).
Histopathological examination: The liver and kidney were collected and post-mortal examination was done as soon as possible. Fixation was carried out in 10% of natural formalin, dehydrated, cleared, and ended paraffin then sectioned at (4-6 mm), and stained with Harris hematoxylin, and casein for histopathological examination (Frankel and Reithman, 1963). A value of average of 6 samples was calculated to be recorded.
Table (1) Diet formula plus 1% AD as weaning foods Groups Skim
Milk
Soy protein
Date Sweet
Potatoes
Wheat Flour
Vit.
Mix
Fat Starch Ad G1 50% a common commercial weaning food in basal diet.
G2 20 10 20 10 25 5 9 0 1
G3 0 20 20 10 25 5 9 10 1
G4 20 10 10 20 25 5 9 0 1
G5 0 20 10 20 25 5 9 10 1
SM, sckimmilk; SP, extrudate soy protein; SWP, dried sweet potato; W, Wheat; MIX, salt and vitamin mixtures; AD special additives.
RESULTS AND DISCUSSION
The formula of weaning food with or without 20% Sckimmilk (SM), the most common animal protein source used in these cases, has been arranged as seen in Table (1). Vegetables ingredients, e.g., both SP and D portions were ranged between 10 to 20% in counterpartation of dried sweet potatoes (SWP). All other ingredients, e.g., wheat flour (W), salt, fat and vitamin mixtures (MIX) were fixed at
their levels. Occasionally, SM and SP have been varied to keep equal dietary rates of proteins at 16- /+1% using 0 or 10% corn starch. This special weaning practice of priority was taken to avoid several nutritional problems like excessively faddy eating, faltering growth, constipation, iron deficiency anemia and obesity (Harrod-Wild 2007). Unfortunately, some of these clinical characteristics have been noticed in the control within three weeks. A great body weight (BW) variation was occurred between the control and both plants’ or SM’ diets. Table 2 shows also that group 3, an animal protein free diet, was the most similar group to the control among all others. However, high energy density home made complementary food may improve the BWG of infants during the weaning period (De Silva et al, 2007), which should scientifically be examined experimentally.
Table (2) Infant body weight growth with 55.0+/-3.5g SBW response to vegetarian diet formula.
Groups Group 1st wk % SBW 2nd wk % SBW 3rd wk % SBW RN1
G1 P1 69.7 123 119.5 211 123.0 217 1
G2 P2 57.9 102 73.2 129 78.3 138 5
G3 P3 62.4 110 87.0 153 90.7 160 2
G4 P4 55.3 96 76.5 135 81.0 143 4
G5 P5 61.4 113 75.3 133 82.4 145 3
RN, ranking number; SBW, starting body weight
Organs weight gave a fluctuated data since each of which had its own size response to the effect of used diets. Table 3 showed that G5 and G2 are the best and the worst of all for liver, spleen and lung, respectively, meanwhile, kidney goes another way and heart seems to be unaffected.
Table (3) Organs in gram as response to vegetarian diet formula.
Group L S K H Lu
G1in grams 5.50 0.45 0.80 0.50 0.80
G2* 33 56 81 70 63
G3* 99 56 69 60 56
G4* 99 78 63 70 81
G5* 100 78 63 70 69
L liver, S spleen, K kidney, H heart, and Lu lung. * %P1
In more details, excluding G1, with which all groups of organs were generally smaller than, it is possible to say that G4 may consider the most acceptable one. It is also true that the G1 group is heavier than all other groups for all organs examined. Since catch up growth is not as important as hyperplasia one, other biological parameters should be encountered. Van der Merwe, et al (2007) stated that untimely and inappropriate introduction of complementary foods have been shown to be risk factors for both under- and over-nutrition with resultant under- or overweight, stunting and micronutrient deficiencies. Optimal timing for the introduction of complementary foods will depend on the infant's physiological and developmental status. Applying another organ biological parameter such as tissues histopathology clearly discovered that groups 2 and 4 may suppress that of the control. Tables 4 and 5 listed the liver and kidney histology reveals that these groups are the most acceptable feeding systems.
Table (4): Effect of feeding infant rat on vegetarian formulations on liver histopathology.
Groups G1 G2 G3 G4 G5
Bile duct thickening wall.
Kypffer cells activation of cytoplasm + Slight granularity of hepatocytes. +
Congestion of central vein. +
Hepatocyte hydropic degeneration. +
RN2 1 1 4 1 2
Table (5): Effect of feeding rat on vegetarian formulations on kidney histopathology.
Groups G1 G2 G3 G4 G5
Congetion of renal blood vessels. +
Pylcnosis of the nuclei of epithelial renal tubules. +/-
RN3 1 1 2 1 1
RN, ranking number
Moreover, he blood cell counts in the present study were significantly affected by diet formula. Table 6 illustrated the highest counting for both WBC and RBC for G5 followed by G4. Prevention of anemia and immundysfunction at this early age cycle is a central dogma of nutrition. Optimal feeding keeps blood constituents at normal ranges. In an assessment of fortified complementary foods addition, alpha-amylase treatment increases hemoglobin. These 9-mo-old Zambian infants feeding experiment may be used to improve the nutritional status of infants in developing countries (Owino et al, 2007).
Table (6) Blood cell counts as affected by vegetarian diet formula
Group WBC %G1 RBC %G1 RN4 WBC RN5 RBC
G1 9.53 100 6.88 100 3 3
G1 12.43 130 6.78 99 2 4
G1 9.01 95 6.86 100 4 3
G1 12.68 133 6.94 101 2 2
G1 16.13 170 7.63 111 1 1
Table 7 mathematically uses the ranking numbers observed for the above mentioned biological tests in a collective assessment. This includes some more parameters such as anemia and development of immune system as appropriate selective medical tool to be considered in this case besides the BWG. So far, it is to recognize that BW evaluation is not reasonable way for biological and nutritional assessment hence the net figures in Table 7 gave G5, but not G1, the superior biological range.
Table (7) Calculated biological net ranking number for the effect of dietary system used.
Group RN1 RN2 Liver Histology
RN3 kidney histology
RN4 WBC
RN5 RBC
Total Ranking*
Net Ranking
G1 1 1 1 3 3 9 2
G1 5 1 1 2 4 13 4
G1 2 4 2 4 3 15 5
G1 4 1 1 2 2 10 3
G1 3 2 1 1 1 8 1
The total of each row.
It is not only the fact of balanced and dense diet concept, special proteins play special roles. G1 may convey certain bioactive protein. Using weaning piglet as the model has suggested that several milk peptides down regulate various immune properties at a time (one to two weeks after weaning) that coincides with immaturity of the immune system. The protein kinase A system and/or the exchange protein directly activated by cyclic AMP (Epac-1) are implicated in the mechanism through which milk peptides can affect immune function in the early post weaning period. Despite the fact that the research in this field is in its infancy, the evidence available suggests that milk protein peptides may promote development of neonatal immune competence (Politis and Chronopoulou 2008). G5 then G1 here may represent a special fragment as an example of a particular feeding pattern. They help avoiding several nutritional problems such as excessively faddy eating, faltering growth, constipation, iron deficiency anemia and obesity (Harrod-Wild 2007). Non-purified plant food sources might not be considered as the best for iron. Dietary factors that influence iron absorption, such as phytate, polyphenols, calcium, ascorbic acid, and muscle tissue, have repeatedly been shown to influence iron absorption in single-meal isotope studies, whereas in multimeal studies with a varied diet and multiple inhibitors and enhancers the effect of single components was more modest.The importance of fortification with iron and food additives suchas
erythorbic acid on iron bioavailability from a mixed diet needs clarification. Bioactive proteins in groups 6 and 1 may regulate these metabolic functions. The iron status of theindividual and other host factors, such as obesity, play a keyrole in iron bioavailability, and iron status generally hasa greater effect than the diet composition (Hurrell and Egli 2010). It is, therefore, a sort of multiple inhibitors and enhancers that affect a single components or nutrients to biologically conceive.
Likewise, the influence of vitamin A, carotenoids,and no digestible carbohydrates on iron absorption and the nature of the "meat factor" remain unresolved. Again, for simple biological evaluation received data that converted to ranking numbers proposed for BWG, WBC, RBC, and both Liver and kidney histology, were collectively calculated to get the net figures of a roughly biological assessment. In Table (7) G5 formula then G1, was the best over all, meanwhile, group 3 is the worst.
In summary, a particular plant protein combination might be considered sufficient; meanwhile, animal portion despite its importance, it should be carefully administrated. However, animal protein acceptability was determined by maternal hedonic scoring, 9-day infant intake, and videotape analysis of how well infants liked porridge. Dry and cooked porridges and meat ingredients were tested for microorganisms; meats were tested for pesticides. As a result, mothers gave higher acceptability scores to the non-meat porridge, followed in order by low and high quantities of meat powder. Microbiologic safety was acceptable except for marginally acceptable molds and yeasts in dry ingredients. No pesticide residues were detected (Pachón et al, 2007). Moreover,protein and carbohydrate intake, in general, should increase with the infant's age while preference should be given to foods rich in micronutrients. It should be observed that certain foods, such as fresh cow's milk and egg white, because of their allergenic properties, as well as fat-free and high-fiber foods, excessive fruit juice and low nutrient value drinks such as tea are not recommended (Van der Merwe,et al 2007).
In conclusion, the untimely and inappropriate introduction of complementary foods have been shown to be risk factors for both under- and over-nutrition with resultant under- or overweight, stunting and micronutrient deficiencies. Optimal timing for the introduction of complementary foods will depend on the infant's physiological and developmental status (Van der Merwe, et al 2007). In this concern, it is to be mentioned that G5 may convey the right food internal structure between the macronutrients, particularly proteins, and the micronutrients that facilitates the production of special bioactive proteins helps to generate the right hormonal balance of this physiological status. The bioactive protein is one of the most important branches of biological role in nutritional biochemistry. Some plant proteins are recognized nowadays to play a great role in preventing some pathological diseases (Ahmed et al, 2005). More evidence is recently published regarding WBC data in this study, evidence has been accumulated to suggest that milk peptides may regulate gastrointestinal immunity, guiding the local immune system until it develops its full functionality (Politis and Chronopoulou 2008).
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