although pervasively widespread in the world , iron is the nutrient with a most pronounced deficiency in humans 1 , 2 many remedial actions have been undertaken ; nevertheless , the problem persists for a number of reasons : unspecific diet supplements , poor dietary food combinations and low bioavailability in the iron - bearing products consumed 3 - 5 dietary iron occurs in two forms : 1 ) non - heme iron ; derived mainly from vegetable products , and 2 ) heme iron ; only found in products of animal origin 5 , 6 .
heme iron is more easily absorbed in the diet than inorganic iron ; because the absorption process is different for each 7 .
there are experiences on the use of bovine and pig blood to exploit their high nutritional value and coincidentally eliminate a highly polluting effluent 8 .
these experiences show that a base product can be obtained with a high heme iron concentration and good bioavailability for direct consumption or food supplements 5 , 9 - 11 .
for example , in 1993 walter et al 12 , achieved this with biscuits ; pallares et al 13 added bovine blood to a cereal with milk ; aznar and gonzlez 14 , developed a protein - mineral food supplement ( trofin ) , using dried whole blood with hydrolyzed proteins as a base .
many techniques are available for studying iron bioavailability , which can be classified as : in vitro studies and live studies depending on the model used the latter in either animals or humans 6 .
the in vitro studies are cheaper and permit a greater control over the experimental variables .
the main limitation of these methods is that they can not simulate physiological states or some physical and chemical properties and adaptive responses that influence the bioavailability of iron 15 .
although recent methods have been developed attempt to correct these limitations and approach the absorption conditions in the small intestine 16 .
absorption studies in humans continue to be the gold standard in estimating the bioavailability of iron ; nevertheless , animals tests are often more practical for the prediction of nutrient bioavailability 6,17 .
the pig is considered a model that shares more human structure and function similarities than other animals .
more variables can be measured , such as those related to the diet or growth rate . for these reasons , was chosen as the model for this study 18 .
the objective of this work was to evaluate the bioavailability of a heme iron concentrate product incorporated into a chocolate flavor biscuit filling .
all analyses were performed by two replicates . a previous study ( data not shown ) used a factor - by - factor procedure 19 , to evaluate several ingredients and formulas ( hydrocolloids among them ) ; from this the definitive one was selected , consisting of 9 ingredients ( table 1 ) . by means of the researchers ' direct observation and using an arbitrary scale ( from 0 to 5 ) , the spreadability , flow , colour , smell , flavor , adhesion , gumminess , firmness , cutting effort and radiance characteristics were measured .
microbiological checks were applied during the process and to the end product , making measurements of total counts , enterobacteria , moulds and yeasts , salmonella spp . and
the heme iron used for the fortification is a dark colour dust , obtained from animal blood from healthy pigs that have passed veterinary inspections satisfactorily in registered slaughter houses and hygienically collected after the addition of an appropriate anticoagulant .
after the separation of the corpuscular fraction by centrifuging , a pressure pump is used to liberate the hemoglobin contained in the erythrocytes .
the hemoglobin is later enzymatically hydrolyzed by a proteolytic enzyme ( alcalase ; novo - nordisk .
denmark ) working within its specific conditions ( ph 8,5 and 50c ) . afterwards , globin peptones were separated from the heme group enriched concentrate by ultrafiltration .
protein , fat , humidity and ash were analyzed following the methodology proposed in the aoac 20 ; iron and zinc were measured by flame atomic absorption photospectrometer in a perkin - elmer photospectrometer , 2100 model 21 . the sample size calculation and the study itself
was made with 20 common pigs ( sus scrofa domesticus ) , belgian pietrain ; females , 4 weeks old , with an average weight of 7 kg , that only received 100 mg of dextrane iron administration at birth with the intention of producing a slight iron deficiency at weaning . by means of random selection two groups of 10 piglets each
this group was fed a low iron diet ( 452.7 mg / kg ) and sandwich biscuits with a chocolate flavor filling with added heme iron .
this group was fed normal food , which contained ferrous sulphate ( 537.1 mg / kg ) .
the iron supplement was given during 20 weeks , making daily inspections , taking their weight and blood samples every fortnight , applying antiparasitic treatment as required and a veterinary check - up monthly .
the blood samples were extracted from the superior cave vein after the pigs were tranquilized with azaperone ( stresnil ) .
7 ml of blood was extracted , of which 3 ml was put in a tube with edta to make the red series analyses ; the rest of the blood ( 4 ml ) was emptied into a tube with separating serum gel to make the biochemical analyses .
the samples were transported from the farm to the laboratory refrigerated and they were immediately processed .
the hematological measurements were made with an advia ( tm ) 120 ( bayer diagnostics europe sl , 22 , and the biochemical measurements with an cobas mira 89 ( roche ) chemical analyzing system , using the ferrozine iron a11a00091 abx commercial kit .
the measurement of iron in the liver was made by atomic absorption spectrophotometer with perkin - elmer equipment , model 2100 , using a sample taken immediately after slaughtering and deep frozen to -20 c until the analysis 15 days later .
the end - point criteria for the experiment was either the existence of incapacitating or incurable disease or the completion of study ( 20 weeks ) ; in both cases , slaughtering was done by administering sodium pentobarbital on the same pig farm 23,24 . the statistical analysis was done with the spss 13.0 software .
first , descriptive analysis was performed , using duplicate determination of initial and final values , and comparing independent averages of the results of both groups .
a previous study ( data not shown ) used a factor - by - factor procedure 19 , to evaluate several ingredients and formulas ( hydrocolloids among them ) ; from this the definitive one was selected , consisting of 9 ingredients ( table 1 ) . by means of the researchers ' direct observation and using an arbitrary scale ( from 0 to 5 ) ,
the spreadability , flow , colour , smell , flavor , adhesion , gumminess , firmness , cutting effort and radiance characteristics were measured .
microbiological checks were applied during the process and to the end product , making measurements of total counts , enterobacteria , moulds and yeasts , salmonella spp . and escherichia coli .
the heme iron used for the fortification is a dark colour dust , obtained from animal blood from healthy pigs that have passed veterinary inspections satisfactorily in registered slaughter houses and hygienically collected after the addition of an appropriate anticoagulant .
after the separation of the corpuscular fraction by centrifuging , a pressure pump is used to liberate the hemoglobin contained in the erythrocytes .
the hemoglobin is later enzymatically hydrolyzed by a proteolytic enzyme ( alcalase ; novo - nordisk .
denmark ) working within its specific conditions ( ph 8,5 and 50c ) . afterwards , globin peptones were separated from the heme group enriched concentrate by ultrafiltration .
protein , fat , humidity and ash were analyzed following the methodology proposed in the aoac 20 ; iron and zinc were measured by flame atomic absorption photospectrometer in a perkin - elmer photospectrometer , 2100 model 21 .
the sample size calculation and the study itself was made with 20 common pigs ( sus scrofa domesticus ) , belgian pietrain ; females , 4 weeks old , with an average weight of 7 kg , that only received 100 mg of dextrane iron administration at birth with the intention of producing a slight iron deficiency at weaning . by means of random selection two groups of 10 piglets each
this group was fed a low iron diet ( 452.7 mg / kg ) and sandwich biscuits with a chocolate flavor filling with added heme iron .
this group was fed normal food , which contained ferrous sulphate ( 537.1 mg / kg ) .
the iron supplement was given during 20 weeks , making daily inspections , taking their weight and blood samples every fortnight , applying antiparasitic treatment as required and a veterinary check - up monthly .
the blood samples were extracted from the superior cave vein after the pigs were tranquilized with azaperone ( stresnil ) .
7 ml of blood was extracted , of which 3 ml was put in a tube with edta to make the red series analyses ; the rest of the blood ( 4 ml ) was emptied into a tube with separating serum gel to make the biochemical analyses .
the samples were transported from the farm to the laboratory refrigerated and they were immediately processed .
the hematological measurements were made with an advia ( tm ) 120 ( bayer diagnostics europe sl , 22 , and the biochemical measurements with an cobas mira 89 ( roche ) chemical analyzing system , using the ferrozine iron a11a00091 abx commercial kit .
the measurement of iron in the liver was made by atomic absorption spectrophotometer with perkin - elmer equipment , model 2100 , using a sample taken immediately after slaughtering and deep frozen to -20 c until the analysis 15 days later .
the end - point criteria for the experiment was either the existence of incapacitating or incurable disease or the completion of study ( 20 weeks ) ; in both cases , slaughtering was done by administering sodium pentobarbital on the same pig farm 23,24 .
first , descriptive analysis was performed , using duplicate determination of initial and final values , and comparing independent averages of the results of both groups .
table 2 shows the composition of the filling formulated ; it may be observed that heme contribution of iron is 2,6 mg for each gram of filling , so a single biscuit containing 5 grams of product will contribute up to 13 mg of iron , covering 100% of the recommended iron intake for humans 25 .
the 14.1% protein content , being of hemoglobin and milk serum , is of high biological quality 26 .
the filling obtained was directly observed to have a creamy appearance , chocolate smell and flavor and appropriate spreadability .
the expected values column 27 in table 3 gives some hematological reference parameters for piglets of similar ages and weights and , despite a relatively high variation coefficient , can be used as a guide to verify that the results obtained for most of the parameters studied were within the norm .
we observed a high weight variation ( variation coefficient 0.23 ) , indicating a quite heterogeneous study group .
given the heterogeneousness of the group , care was exercised to ensure randomness in the separation into groups .
some differences can be noted ; nevertheless in all cases these were not statistically significant , therefore we can assume that the groups were not different and that the splitting process was satisfactory . of the total number of pigs when the study began , 6 died during the trial , 5 from control group ( 50% ) and 1 from heme group ( 10% ) .
there was a risk that the pigs that died shared a common differentiating characteristic compared to the survivors , thereby slanting the results .
however , comparing the values of variables of both groups , we observed that although there were slight differences in some variables they had no statistical significance , leading us to consider that there was no factor which influenced mortality differentially in the study groups , since both were subjected to the same treatment and therefore the same stress , probably the cause of the high mortality 28 - 30 .
figure 1 shows the behavior of both study groups ' weights ; there was a slight not statistically significant difference between them from week 8 until week 13 ( p>0,05 ) ; from which point the heme supplement group increasingly took off until the end of the study in week 20 .
both groups started with an average weight of 6.9 ( 1.6 ) kg ; but the increase was much greater in the heme group , their final weight being 56.7 kg , an increase of 49.8 kg .
in contrast , the control group , whose final weight average reached 43.9 kg , an increase of 37 kg , giving a statistically significant ( p<0.05 ) difference of 12.8 kg between the groups , and agreeing with the results obtained by perestrelo and perestrelo 31 , who used amino - quelated iron in their supplement and concluded that it reduces mortality and improved growth . on average ,
the heme group pigs consumed 10.5 kg of sandwich biscuits ( 498 pieces ) , a total of 46,737 kcal , which , when converted to animal weight at the rate of 9,200 kcal per kg 32 , gives a total of 5.1 kg of weight attributable to biscuit consumption .
therefore , the actual difference exclusively due to the heme iron source were 7.7 kg ( 17.2% ) in favor of the heme iron supplement group .
table 4 shows the iron measured in tissues and subsequently calculated for each tissue , as well as the sum of each and the percentage relative to the total iron consumed by the pigs throughout the study , according to the supplement group they belonged to .
we can observe significant differences between the groups with respect to iron in the blood , but not in the other body parts , although the heme group showed values higher than the ferrous sulphate supplement group as regards both the liver and hemoglobin .
the difference in absorption percentage between the groups was significant ( p<0.05 ) ; the absorption of heme iron was shown to be 23% superior to ferrous sulphate . in conclusion ,
the adverse characteristics of heme iron ( dark colour , blood smell and flavor ) can be transformed and exploited by its incorporation into a suitable base , which in this case was a chocolate flavored biscuit filling .
mortality was less in the heme iron supplement group of pigs ; the weight also was greater in this group and there was a clear difference in favor of the heme iron group in the total measured iron averages ; although the heme group pigs consumed less iron ; therefore , we can conclude that the measured bioavailability of heme iron was greater than that of ferrous sulphate .
 the objective of this work was to evaluate the bioavailability of heme iron added to biscuit filling . 
 it comprised two stages : first , the development of the heme iron enriched biscuit filling ; second , the evaluation of the bioavailability of the mineral in fattening piglets . 
 two groups were selected randomly and fed : a ) low iron feed and biscuits with heme iron supplemented filling ; b ) normal feed ( with ferrous sulphate ) . 
 weight and blood parameters were measured every fifteen days . 
 averages were compared after duplicate analyses . 
 the filling had a creamy appearance , chocolate taste and smell , appropriate spreadability , heme iron content of 2.6 mg per gram and a shelf - life of a month . 
 the heme iron supplemented pigs registered a greater ( p<0.05 ) weight gain ( 27.8% more than the control group ) . 
 mortality in the heme iron group was 10% , compared to 50% in the control group . 
 the amount of iron measured in the different compartment was greater in the heme group ( 3315 mg ) than in the control group ( 2792 mg ) . 
 however , the amount of iron consumed in the latter was greater . we show that an acceptable product with high heme iron content can be formulated , suitable for use as biscuit filling . 
 the heme iron supplement produced better weight increase and lesser mortality in fattening pigs . 
 the bioavailability of heme iron was 23% greater ( p<0.05 ) compared to ferrous sulphate . 
INTRODUCTION
MATERIAL AND METHODS
Development of biscuit filling
Microbiological analyses
Working ingredient
Bioavailability evaluation
Statistical analysis
RESULTS AND DISCUSSION