Studying the Physiochemical, Sensory and Microbiological Properties of Yoghurt Which are Fortified by Encapsulated Folic Acid

The study aimed to determine the effect of fortification with free and unencapsulated folic acid on the physical, rheological, sensory and microbiological properties of functional yoghurt by fortifying yoghurt with a concentration of 7.5 mg / 100 ml of free folic acid represented by treatment 1 and 7.5 mg / 100 ml encapsulated folic acid as Treatment 2hyd, and the control treatment in which yoghurt was made from milk not fortified with folic acid. In addition to the sensory evaluation immediately after manufacturing and when stored at 5°C for a period of 21 days, the results showed that there were no significant differences in the values of pH and total acidity. Also, Yoghurt fortified with encapsulated folic acid stimulated the growth of starter bacteria, which are beneficial to the human body. The encapsulated folic acid treatment was characterized by an improvement Sensory properties of yoghurt.


Introduction
Vitamins are non-energy producing compound, essential for normal human metabolism that must be supplied in small quantities for diet [1].The importance of vitamins as drugs is primarily in the prevention and treatment of deficiency diseases.Vitamins are traditionally divided into two groups: fat-soluble and water-soluble [2].Water-soluble vitamins (B-complex) are meagrely stored excess is excreted with little chance of toxicity.They act as cofactors for specific enzymes of intermediary metabolism [3].B-vitamins are essential for the functioning of the nervous system, which controls our stress response.Our stress response system determines how we feel in the face of everyday stress [4].Folic acid (vitamin B9) is an essential involved in fundamental functions of nucleic acid biosynthesis and protein metabolism of living organisms [5].Folic acid is composed of p-amino benzoic acid, glutamic acid, and pterin ring.Folic acid, which plays a key in onecarbon metabolism, is essential for biosynthesis of several compounds.The pterin ring changed by reductase enzyme to tetrahydrofolate which receives one carbon fragments from donors ( monocarbonic units)such as serine, glycine, and histidine then transfers them to intermediates in the synthesis of amino acids, purines, thymine, and pyrimidine found in DNA [6].Folic acid represents a focus for nutrition 1259 (2023) 012067 IOP Publishing doi:10.1088/1755-1315/1259/1/012067 2 researches due to its vital roles, including protection, metabolism, and epigenetics [7].Consequently, deficiency of this essential vitamin leads to numerous deleterious health effects including neural tube defects.As an attempt to at least partially eliminate mainly folic acid deficiency, many countries have made fortification of many foods with folic acid mandatory [8].Folic acid fortification of enriched cereal grain products in the USA [9], Folic acid fortification of white wheat flour in Canada [10], and folic acid fortification of bread in Australia are prominent examples.Although mandatory folic acid fortification is not permitted, numerous countries allow voluntary folic acid fortification that has led marketing of a number of folic acid-fortified foods including beverages and yoghurt in those countries [10].The development of accurate packaging techniques and their successful application to protect effective compounds is one of the main things that are resorted to for complete protection when exposed to industrial conditions and storage, which leads to deterioration by temperature, oxygen and humidity, as well as its decomposition in the digestive system when exposed to acidity of the stomach, bile salts and intestinal enzymes [11].ameliorated properties such as enhanced stability, pH dependent release, and improved bioaccessibility or bioavailbility.For example, folic acid has been encapsulated in many food grade matrices such as alginate, aligante-pectin, whey protein , resistant starch, pectin-whey protein concentrate, maltodextrin-whey protein, horse chestnut starch, and ß-cyclodextrin using one or more techniques including ionic gelation, nonospray drying, electrospraying, double emulsions, and spray drying [8].

Materials and Methods
The folic acid FA prepared by the Indian company Qualikems Fine chem.Was used and encapsulated with sodium alginate provided by the American company Sigma alderch according to the method of [8], and the functional yoghurt was fortified with it then, physiological and sensory examination were conducted on it during 21 days of its storage period.The starter was from SAACO-Italy.

Yoghurt Manufacturing
The yoghurt manufactured as the method in [12], by receiving a quantity of row milk, full-fat mixture, from the dairy factory, Department of food science, college of Agriculture Engineering Sciences, University of Baghdad, and the the percentage of solids was adjusted to 12.98% using dry milk.Conducting a heat treatment of milk at 90 °C for 10 min then cooled to temperature suitable for the starter (45 °C) .Then the starter culture was added, and milk was stored in plastic containers with capacity of 120ml.After that 7.5mg/100 ml of encapsulated and unencapsulated folic acid were added then incubated at 42 ° C for 5 h, then stored in refrigerator at 5 °C Table (1) shows the treatments that used in this study.
Table 1.The treatments that used in the study.

Physical Tests
Included water holding capacity, viscosity, whey draining, pH and total acidity.

Total Acidity
Estimated according to [13], method by weighing 9 g of yoghurt in beaker and adding a few drops of phenophthalene reagent to it, then wiping with a base of 0.1 N until the appearance and stability of pink color.

Whey Draining
By adding 50 g of yoghurt in a beaker diagonally for two hours at a refrigerator temperature of 5 0 C the whey was then withdrawn from the surface using a syringe, then the beaker was weighted again, according to [14].

pH Value
The pH of yoghurt was estimated by placing the cathode of a Chinese-made pH directly into the yoghurt samples [15].

Water Holding Capacity
It was estimated, according to the method mentioned in (15) by weighing 10 g of yoghurt, placing it in appropriate test tubes, and exposing it to a centrifuge with a force of 3000 rpm, after which the filtrate was removed, the sediment was weighed, and the water retention capacity was calculated according to the following equation: Water holding capacity: x 100

Viscosity
The viscosity of yoghurt samples was estimated at 10 0 C during the refrigerated storage, using the Fungilab viscometer Alpha Spanish company, according to what [16], mentioned.

Rheological Tests
A sensory rheological properties of yoghurt samples was conducted during the refrigerated storage period of 1, 7, 14 and 21 days f storage by some students in the College of Agriculture Engineering Science.

Microbiological Tests
A count was made for the two type of starter bacteria, Psychrphiles bacteria, yeasts, molds and coliform, which were prepared from NEOGEN-USA.According to [15], method, dilutions are prepared by taking 10g of yoghurt and diluted it with 90 ml of peptone water, then withdrawing 10 ml of the sample and diluting it with 90 ml of peptone water until the dilution reached 10 -, and then the different medias were prepared by dissolving 33 g of Nutrient Agar in 1 L of distilled water with heating to boil, the Macconkey Agar was prepared by dissolving 51.5 g in a liter of distilled water with heating until boiling, potato dextrose medium was prepared by dissolving 40 g of the medium in 1 liter of distilled water with heating until boiling and MRS medium was prepared by dissolving 70 gm in 1 liter of distilled water with heating until boiling, than sterilizing all the media in an autoclave from Optima at a temperature of 120 for 15 minutes.

Statistical Analysis
The Statistical Analysis System-SAS [17], program was used to detect the effect of difference factors in study parameters.Least significant difference -LSD test (Analysis of Variation-ANOVA) was used to significant compare between means in this study.

The Chemical Composition of Milk used in Making Yoghurt
The chemical composition of the milk used in the manufacture of yogurt after adjusting the Total solids (TS) in it using powdered skim milk and as shown in (

Physical Tests
The pH in figure (1) of the pH values of control, T1 and T2, immediately after manufacturing, reached 4.5, 4.5 and 4.5, respectively, which is close to what [15], found.During the cooled storage period, a decrease in the pH is observed, due to the activity of the starter.There was not a significant difference at least between the samples>0.05.found, depending on the activity of the starter.There was not a significant difference at least between the samples <0.05.The whey draining values in figure (3) for the control, T1 and T2 immediately after processing were 0.97 , 0.99 and 0.96 [15], found.There was a significant difference at least between the samples <0.05.The functional properties of food proteins are among the basic factors that must be considered in food processing or in the manufacture of new food products.Proteins give desirable functional properties such as water holding capacity, viscosity, emulsification and foam formation.Large quantities of water in a way that prevents exudation, and protein gel is one of the vital factors in the manufacture of yoghurt because it is linked to the whey draining, which is due to the loss of water during the storage period, and that the whey draining is one of the common quality defects that frequently occur during the manufacture of yoghurt.When the protein bonds show a weak ability to retain water, whey draining occurs at that time.The results show in the figure (4) the ability of holding water capacity for the control treatment, T1 and T2, where the control treatment immediately after manufacturing was 28.1%, which is consistent with what [15], mentioned, and for the treatments T1 and T2 it was 28.3% and 28.5% respectively, and it is clear that there is no significant difference between the samples, but after the storage period of 21 days, an increase in the percentages was observed, and this is consistent with what [20], mentioned, as folic acid led to a change in the structure of the casein, which improves the ability to retain water.There was a significant difference at least between the samples <0.05.Viscosity is one of the important factors in determining the quality indicators of yogurt, which are closely related to each of the stability of the product of fermented milk.The stability of the viscosity of the product is also very important for its quality characteristics.Bacteria play a major role in the production of materials that are considered external cell products called external sugars that interfere with the content of Protein milk, increase its viscosity and improve its quality characteristics [21].

Microbiological Characteristics
The bacteria present in the yoghurt are considered to be beneficial to Human health, although the number of starter bacteria differs in the different types of yoghurt and fermented milk products.But it is required that there numbers be not less then 10 7 U include Lactobacillus bulgaricus and Streptococcus thermophilus .Lactobacilli are characterized by their ability to inhibit the growth of bacteria throughout the production of antimicrobial materials such as bacteriocins and biosurfactants [22].The advantage of lactic acid bacteria is that they are important in food and therapeutic processing and have multiple characteristics that they grow in aerobic and anaerobic conditions, their rapid growth, non-producing toxins, most of them are non-pathogenic, and they resist low pH, and their production of flavoring materials in food as well as their ability to preserve food because of their products.The metabolites resulting from fermentation, such as bacteriocins, have been used as probiotics, which are single or combined cultures of microorganisms that have beneficial effects on health, and are important in treating many diseases that affect humans and animals [23].The method of pouring dishes mentioned by [24] using the solid nutrient media(Nutrient agar) in estimating the total number of microorganisms (total plate count) , using the solid MacConkey agar in estimating the total number of coliform bacteria , using the solid Potato Dextrose Agar in estimating the total number of yeasts and molds and using the solid MRS agar in estimating the numbers of the starter bacteria Table (3) showed the results of estimating the number of microorganisms from starter bacteria that included both types, and examining coliform bacteria, Psychrphiles bacteria, yeasts and molds for the control, T1 and T2.Where the numbers of total starter bacteria of the control, T1 and T2 , Immediately after processing, where 90 x 10 6 , 92 x 10 6 and 91 x 10 6 U which are numbers close to what [15], found.There was a significant difference at least between the samples <0.05.Decrease was observed in the numbers due to the increase in acidity, which negatively effected the grows of the starter bacteria, and this is consistent with what [25] mentioned.[15], found Immediately after manufacturing and during the refrigerated storage period (21 days), The reason for this is due to the previous heat treatment of milk before making yoghurt, which eliminated coliform bacteria and contaminated microbes.There was not a significant difference at least between the samples (>0.05).

The Sensory Tests
The sensory evaluation of the yogurt was conducted according to what was mentioned by [26], Table (5) showed the sensory evaluation of control, T1 and T2 during the storage period of 21 days, where T2 was the best treatment in terms of evaluation Immediately after manufacturing, reaching 99.5 0 100 0 , which is similar to the degree of sensory evaluation of the control treatment of 99.5 0 0 during the storage period, there was no significant change in the degree of the sensory evaluation of this treatment, as it reached 80.2 0 0 on the 21-day of cooled storage.There was a significant difference at least between the samples <0.05.
Table 5.Effect of treatment and period in Sensory parameters.

Conclusion
The fortification of yoghurt with encapsulated folic acid (T2) did not lead to significant changes in the physical properties and the results showed that the T2 treatment was the best in preserving the sensory characteristics of the fortified yoghurt, compared to the unencapsulated folic acid fortified yoghurt.Therefore it is qualified to use folic acid as one of the nutritional fortification methods.

Figure 1 .
Figure 1.pH values in yoghurt.The total acidity percentages in figure(2) of control, T1 and T2, immediately after manufacturing.Were 0.62 , 0,63 and 0.62, respectively, and they differed with what (18) found and close to what(19) found, depending on the activity of the starter.There was not a significant difference at least between the samples <0.05.

Figure 2 .
Figure 2. The total acidity of yoghurt.Whey draining is the protein matrix of yoghurt leaving and floating on the surface.It occurs automatically without any external influence.This characteristic is undesirable, which occurs as a result of several reasons, including a low pH below 4.4 and insufficient heating during ,manufacturing.

Figure 4 .
Figure 4. Shows the water capacity of Yoghurt.

6
The results in Figure(5) show the viscosity values of the control treatment and for treatment 1 and 2, where it was for the control treatment 300 and for treatment 1 and 2 it reached 250 and 330 respectively for the first day after manufacturing directly.A decrease in viscosity was observed during the refrigerated storage period of 21 days, due to the high acidity, as the two are inversely proportional This is consistent with what[14], found.

Figure 5 .
Figure 5.The viscosity in the yoghurt.

Table 2
[15]r each of the percentages of moisture, fat, protein, total solids and non-fat solids are 87.19,2.81,4.51 and 11.32 and 8.51, respectively, and these percentages are within the normal limits for milk and close to what[15]found.

Table 2 .
The chemical composition of the milk used in the manufacture of yoghurt.

Table 3 .
Effect of treatment and period in Starter bacteria.

Table 4 .
Shows the numbers of contamination microorganisms in yoghurt.

Period storage days Yeasts and molds Coliform Psychrphiles
NS( NO significant difference).Each number mentioned in the table is an average of two repeats.