Improvement of the Nutritional Value of Cereal Fermented Milk: 1- Soft Kishk Like

Soft Kishk like products were produced from whole wheat, barley and freek burghul with reconstituted skim milk (15% T. S) and addition of different Starter cultures. Physico-chemical, bacteriological and organoleptic properties of soft kishk like samples have been evaluated during 14 days of storage at 5 ± 1°C. The main effect on Soft Kishk like products characteristics was due to the used cereal type more than the started culture. Containing wheat burghul showed the highest pH values, Crude protein content were almost similar between all treatments as a result of the similarity of protein content in the selected seeds. Freek burghul treatment-showed higher total solids and carbohydrates whereas, lower content of ash, crude fiber, fat and crude protein contents compared to the other treatments. The c.f.u on MRS medium (mainly Bifidobacteria) sensitive than other starter microorganisms (mainly lactic acid bacteria) for storage. Cereal fermented dairy products containing Freek; gained the highest scores in the organoleptic properties followed by wheat. While the addition of whole barley burghul had the lowest total score at the end of storage. Therefore, a functional and nutritional Kishk like products have been successfully produced using different cereals and probiotic starter cultures. Improvement of the Nutritional Value of Cereal Fermented Milk: 1Soft Kishk Like Nassar KS1*, Shamsia SM1 and Attia IA2 1Department of Food, Dairy Science and Technology, University of Damanhour, Egypt 2Department of Dairy Science and Technology, Alexandria University, Egypt


Introduction
There is a currently growing interest in certain strains of lactic acid bacteria that have been suggested or shown to provide specific health benefits when consumed as food. The history of those observations had started in the early 1900's, when Élie Metchnikoff noted the longevity of people living in the Balkans, and attributed this to their high consumption of fermented milk products [1].
Following those early observations, scientists reported that yogurt and other fermented milk products contain lactic acid bacteria that are capable of establishing and colonizing the gut, this group of bacteria referred to as probiotic bacteria [2]. Probiotics are live microbial food supplements, which beneficially affect the host animal by improving its intestinal microbial balance [3]. Recently, some foods, as called functional foods that have positive health promoting effects are already on the global market, and especially in the markets of Japan, Europe, and United states. Functional foods are defined broadly as foods that provide more than simple nutrition; they supply additional physiological benefits to the consumer. Yoghurt and other fermented milks containing probiotics may be considered the first functional foods [2][3][4].
Cereals are providing dietary fibers, proteins, energy, minerals and vitamins required for human health. The possible applications of cereal constituents in functional food formulations could be summarized: (a) as dietary fiber promoting several beneficial health or physiological effects; (b) as prebiotics due to their content of specific non digestible carbohydrates; and (c) as encapsulation materials for probiotics in order to enhance their stability [5]. However, the nutritional quality of cereals and the sensorial properties of their products are sometimes poor or inferior in comparison with milk and milk products. Milk proteins have high nutritional value compared to other proteins because of their relatively high content of essential amino acids and good digestibility [6].
During fermentation of dairy products like cheese and yoghurt release bioactive peptides upon enzymatic hydrolysis of milk proteins [7]. Fermentation may be the most simple and economical way of improving nutritional value, sensory properties and functional qualities of the dairy products [8]. Lactic acid fermentation of different cereals has been found effectively to reduce the amount of Phytic acid, tannins and improve protein availability [9]. Increased amounts of riboflavin, thiamine, niacin and lysine due to the action of LAB in fermented blends of cereals where also reported [10]. The traditional foods manufactured from grains usually lack flavor and aroma. Fermentation improves the sensorial value, which is very much dependent on the amounts of lactic acid, acetic acid and several aromatic volatiles such as higher alcohols, aldehydes, ethyl acetate and di-acetyl, produced via the homo-fermentative or hetero-fermentative metabolic pathways [11]. This work aimed to produce probiotic cereals fermented milk supplemented. The cereals were as wheat, barley and Fereek (green wheat), in order to introduce a diet with nutritionally balanced that needed for sensitive or elderly persons.

Kishk like analysis
The samples were analyzed in triplicates for pH, titratable acidity as lactic acid, and Total solids in the fresh products and after 7 and 14 days of storage at 5 ± 1°C, whereas, total protein, carbohydrate, ash, crude fibers and the fat content were determined only in the fresh products.
Total solids content, total protein, fat, ash and crude fibers were determined as described by AOAC [12]. pH values were measured using a pH-meter model HANNA HI9321 microprocessor with a standard, combination glass electrode. Titratable acidity was estimated as percentage of lactic acid according to [14]. The carbohydrate content was calculated in the product according to the following equation: Carbohydrate % = 100 -(Protein % + Ash % + Fat % + Fiber %).
Hansen laboratory, Denmark). Freeze -dried bacterial starters were propagated separately as mother cultures in autoclaved (121°C/20 min) skim milk. The cultures were incubated at 37°C for Bio-yoghurt starter, 32°C for Lactobacillus plantarum and 40°C for yoghurt starter, until curding of milk. Cultures were freshly prepared before using.

Preparation of whole wheat burghul, whole barley burghul and fereek burghul
The polished (wheat, Barley and Fereek) (0.5 kg) was cleaned manually by removing foreign grains and other impurities. Then, it was placed in a stainless steel sieve and washed under a strong stream of tap water with continuous stirring for two minutes followed by rinsing with distilled water. Cleaned and washed polished wheat was cooked in a Stainless-steel pot containing (1.5 L) of water. Cooking time was between 25-30 minutes from the beginning of boiling until the completion of cooking and absorption of the water. The cooked grains were left to cool then broken up a blender.

Preparation of reconstituted skim milk (15 w/w)
150 gm of the dried skim milk were dispersing by stirring in 600 gm distilled water at 40°C, then complete to 1000 gm with distilled water vigorously stirring until completely dissolving

Preparation of cereal fermented dairy products or soft kishk like
Each type of Burghul was mixed with reconstituted skim milk in a ratio of 1:4 (w/w) in addition to, suitable amount of vanillin. The final total solids of mixture were 26%. Mixture was heated to 95°C for 10 seconds, and then rapidly cooled to 45°C, addition 3% of each Starter. The ingredients (reconstituted skim Milk + Cereal + starter culture)  on nutrient agar (NA) according to APHA [16]. The plates were incubated at 32°C for 38-48 hrs.
Spore forming bacterial count: Representative sample was heated at 80°C for 20 min in water bath and then cooled at the room temperature, the same technique as previously mentioned was followed for enumeration the spore formers but using manitol salt agar medium (MSA) Incubation was done at 32°C for 24 hrs [16].

Statistical analysis
Statistical analysis was performed by applying three ways ANOVA and multiple comparisons of means of each treatment (cereals, starter cultures and storage time) using the Least Significant Difference (LSD) test at the confidence level of 95%. Analysis of data was carried out with SAS [19].

Chemical composition
Chemical properties of cereal fermented dairy products are presented in Tables 3 and 4. The results (Table 3) revealed that the effect of cereal type on the chemical composition of the resultant cereal fermented dairy products was more pronounced (P ≤ 0.05) than that of type of starter culture used. There are significant differences (P ≤ 0.05) in pH values and acidity percentages between different cereals fermented dairy products, depending on the type of cereal or starter culture. Data in Table 3 show that the pH values of all samples were decreased gradually till 14 days of the storage, whereas the titratable acidity values were increased at the same period of cold storage. These expected results due to the starter activity similar results were obtained by Hussein [20]. The cereal fermented dairy products containing wheat (W1, W3 and W2, Respectively) were characterized by higher

Sensory evaluation
Organoleptic evaluation was carried out according to the scheme of Clark et al. [15]. The samples were subjected to organoleptic analysis by well-trained members of the Dairy Science and Technology Department (Fac. Agric. Alexandria Univ., Egypt). The sensory attributes evaluated were: The Flavor (1-10 points), Body and Texture (1-5 points) and appearance and Colour (1-5 points).

Microbiological analysis
Preparation of the samples: Eleven grams of each sample were weighted and transferred thoroughly under condition to sterilized flasks, contained 99 ml 2% sodium citrate solution. The necessary serial dilutions using sterilized distilled water were carried out. The samples were mixed by an electric blender for about 2 min. The following microbial were enumerated.

Lactic acid bacterial count (LAB): Using de Man Rogosa Sharpe
Agar medium (MRS) as described by APHA [16]. The plates were incubated at 37°C for 48 hours.
Coliform count: Coliform bacteria were enumerated using Violet Red Bile Agar (VRBA) medium according to Difco [17]. The plates were incubated at 37°C for 24 hours.

Treatments Cereals Dairy base Vanillin Whole Wheat Burghul (W) Whole Barley Burghul (B)
Freek Burghul (F) Re-constituted Skim milk (15%) 1: (3% Yoghurt starter) 2: (2% Yoghurt starter + 1% Bio-yoghurt starter) 3: (2% Yoghurt starter + 1% Lactobacillus plantarum) fermented dairy products that fermented with only Yoghurt starter culture (1) were characterized with higher pH than that fermented with mixed cultures contain Yoghurt and Bio-Yoghurt starter (2) and mixed cultures of Yoghurt starter culture and Lactobacillus plantarum (3), respectively. During values of the storage at 5 ± 1°C for 14 days, significant differences (P ≤ 0.05) were recorded in pH of different cereal fermented dairy products. Moreover, a gradual decrease in pH could be observed in all samples of cereal fermented dairy products, with extending the cold storage period. The decrease in pH could be attributed to a limited growth of different bacterial starter cultures and the slow fermentation of residual lactose [20,21].
The fermentation activity can be observed by acidity and/or pH measurements [22]. During fermentation of cereal fermented dairy products, the amount of organic acids other than lactic acid production were small and neglected in acidity calculations [23]. Unlike pH, it is normal expected that the higher acidity rates would lead to lower pH values. The rates of acidity were higher in Barley samples than other samples of Wheat and Fereek [24][25][26][27]. The higher acidity of cereal fermented dairy products made with mix of Yoghurt starter culture and Lactobacillus plantarum culture can be attributed to the high activity of yogurt starter for splitting lactose into glucose and galactose as the first step of fermentation [28]. Table 4 illustrates the percentages of total solids, carbohydrates, ash, crude fiber, fat and crude protein of cereal fermented dairy products. There were no significant differences (P < 0.05) in the crude protein for all samples, but significant (P ≤ 0.05) in the carbohydrates, ash, crude fiber and fat contents between samples depending on type of cereals usage. The cereal fermented dairy products containing Freek were characterized with higher total solids and carbohydrates whereas, lower content of ash, crude fiber, fat and crude protein contents, compared with the other treatments. Also, depending on the type of starter culture, the cereal fermented dairy products fermented with only Yogurt starter showed very slight increase in the crude protein and carbohydrate contents, but decrease in moisture content than that fermented with mixed starter cultures except moisture content at (S. B. 1) treatment. As expected the crude fibers were higher in barley products than other samples because of its higher percent of crude fiber which reached 2.51%. Also, it ranged from 0.702% to 0.709% in whole barley burghul fermented with reconstituted skim milk, but unlike contain whole wheat burghul and Freek products that ranged between   0.381-0.388 and from 0.282% to 0.285%, respectively. These differences of the chemical properties of cereal fermented dairy products, due to its chemical composition of cereal burghul and blends. It was reported that the type of starter culture used in the fermentation did not affect in the Total solids, fat and crude protein of yogurt, bio-yogurt [20][21][22][23][24][25][26][27][28][29]. Also, Salama [30] noticed that the stirred fermented milk made by ABT-4 culture (Lb. acidophilus, Str. thermophilus and B. bifidum) observed slight decrease in crude protein than made by used YO-FLEX yogurt culture (Lb. delbruckii spp. lactis, Lb. delbruckii spp. bulgaricus and, Str. thermophilus); this may be due to the limited proteolysis of milk protein by lactic acid bacteria. Table 5 illustrates the changes in the viable cell counts appeared on MRS medium for fresh and 14 days stored of cereal fermented dairy products at 5 ± 1 ○ C. Slight decrease were observed after 14 days of storage. Generally, the decreasing values were about 0.45 (c.f.u × 10 3 g -1) . On the other hand, it can be noticed that the decline on MRS+L-Cysteine medium were less than that obtained in the case of MRS medium. The decreasing in values was 0.32, 0.43 and 0.44 (c.f.u × 10 3 g -1) for S.W. 2, S.B. 2 and S.F. 2 treatments, respectively. All the samples did not contain any growth in 0.1 gm on SDA, VRBA, NA and MSA media in either fresh or stored products through the storage period. These results are revealed the good hygiene sanitation during manufacture different products. From the above results, it could be concluded that the c.f.u on MRS medium (mainly Bifidobacteria) sensitive than other starter microorganisms (mainly lactic acid bacteria) for storage.

Suitability of heat treatments, good hygiene and microbiological analysis of characteristics of different blends
Medina and Jordano [31] studied the survival of constitutive microflora in one batch of fermented milk containing Bifidobacteria during storage at 7°C. Levels of Streptococcus thermophilus, Lactobacillus bulgaricus and Bifidobacterium spp. in initial population were 2.6 × 10 8 , 5.1 × 10 7 and 7.4 × 10 6 c.f.u/ml respectively. Streptococcus thermophilus slightly increased after 10 days and then decreased. Numbers of Bifidobacterium and Lactobacillus bulgaricus decreased faster during storage. Also Shah et al. [32] studied the survival of Lactobacillus acidophilus and Bifidobacterium bifidum in commercial yoghurt during five weeks period under refrigerated storage. Viable cells of Lactobacillus acidophilus were 10 7 to 10 8 /g in three of the five products, whereas the other two products contained less than 10 5 /g. Initial count of Bifidobacterium bifidum was 10 6 to 10 7 /g in two of five products, whereas the viable numbers were less than 10 3 /g in other three products. All the products showed a similar decline in the viable count of Lactobacillus acidophilus and Bifidobacterium bifidum during storage. Krasackoopt et al. [33] investigated the survival of the microcapsulated probiotics, Lactobacillus acidophilus 547, Bifidobacteria bifidum ATCC1994 and Lactobacillus casei 01 in stirred yoghurt from UHT and conventionally treated milk during low temperature storage at 4°C for 4 weeks. They found that the survival of encapsulated probiotic bacteria was higher than free cells bacteria were mentioned above. Also, they pointed that the viability of probiotic bacteria in yoghurt from both treatments was not significantly different.
The cereals can be used as fermentable substrates for the growth of probiotic microorganisms. Also, Lactobacillus acidophilus exhibited the poorest growth in malt, barley and wheat media, probably due to substrate deficiency in specific nutrients [25][26][27][28][29][30][31][32][33][34]. The optimum final    pH and the concentration of lactic and acetic acid in fermented cereal product in relation to the properties of each specific probiotics strain have to be investigated in order to maximise the viability during storage for practical application; a pH value of the final product must be maintained above 4.6 to prevent the decline Bifidobacteria population [35].

The sensory evaluation
The sensory evaluation is the main factor affecting the consumption of the products and its acceptance. It was realized that the sensory evaluation could contribute pertinent, valuable information related to marketing consequences and simultaneously provide direct actionable information [15]. The scores for organoleptic properties of cereal fermented dairy products prepared using whole wheat burghul or whole barley burghul or freek burghul with different types of starter cultures, during storage at 5 ± 1°C for 14 days were presented in Table  6 and Figure 2. It was clear that in fresh products (first day of storage), the addition of wheat or Freek burghul didn't affect the general acceptability of them, whereas the addition of barley burghul had lowering the total score. General acceptability of the cereal fermented dairy products containing Freek; especially when fermented with yogurt culture (S.F.1) treatment; was gained the highest scores in the organoleptic properties followed by their containing of wheat (S.W.2) product. The cereal fermented dairy products containing of Freek were characterized with perfect flavor, body and texture as well as whiteness appearance and colour. On the other hand, the pronounced malt flavor and light brown color or the decline of color to un-natural, were noticed in cereal fermented dairy products containing of barely; especially at (S.B.2) treatment, these results are in agreement with [20]. During storage at 5 ± 1°C for 14 days, Sensory evaluation was decreased for all products due to the acidity increased in all samples gradually and effect on organoleptic properties and decreased its total score. Vijayalakshmi et al. [36] found that during storage of cereal based low fat fruit yogurt, acidic or malt flavor, firm or ropy body and texture, shrunken or free whey appearance, as well as light brown color were increased in different cereal fermented dairy products at the end of storage.
At the end of storage time, whole wheat burghul treatments had the highest total scores and then, the Freek burghul samples, while the addition of Whole barley burghul had the lowest total score because of high viscosity and unnatural color and still not acceptable at the end of storage. Blanddino et al. [8] mentioned that during cereal fermentations several volatile compounds are formed, which contribute to a complex blend of flavors in the products [37]. Moreover, the presence of aromas representative of Diacetyl acetic acid and butyric acid make fermented cereal-based products more appetizing. Also, Salmeron et al. [38] found that incubation with the probiotic LAB caused a significant change in the aroma profile of the four cereal broths. In barely, considerable amounts of new volatiles were generated after the fermentation. In general, the volatile production depends more on the substrate than on the microorganisms.

Conclusion
From the above results it could be concluded that either Whole  Wheat Burghul, Whole Barley Burghul and Whole Freek Burghul can be used to produce an acceptable product as functional foods suitable for elderly persons or infants weaning foods. This formula has a high nutritional value and fiber content beside the presence probiotic bacteria with a lot of health benefits.