Quality of Novel Healthy Processed Cheese Analogue

World Applied Sciences Journal 23 (7): 914-925, 2013 ISSN 1818-4952 © IDOSI Publications, 2013 DOI: 10.5829/idosi.wasj.2013.23.07.13122 Quality of Novel Healthy Processed Cheese Analogue Enhanced with Marine Microalgae Chlorella vulgaris Biomass 1 1 A.G. Mohamed,2B.E. Abo-El-Khair and 3Samah M. Shalaby Dairy Science Department, National Research Science, Dokki, Giza, Egypt 2 Fertilization Technology Department, Algal Biotechnology Unit, National Research Centre, Dokki, Giza, Egypt 3 Food Science Department, Faculty of Agriculture, Ain Shams University, Shoubra EL-Khaima, Cairo, Egypt Submitted: May 26, 2013; Accepted: Jul 6, 2013; Published: Jul 19, 2013 Abstract: Cheese analogue (Ch A) is processed cheese-like product, a nutritious food, can be healthy and attractive when redesigned to be prepared with the addition of a natural ingredient, being Chlorella vulgaris. This microalga is recognized as a rich source of protein, fatty acids, fiber and ash. C. vulgaris also represents a valuable source of essential vitamins and minerals. Chlorella has health benefits, as assisting disorders such as gastric ulcers, wounds, constipation, anemia, hypertension, have immune-modulating and anticancer properties and is a promising ingredient in the food industries. Cheese analogue (Ch A) treatments were enriched with C. vulgaris (1, 2 and 3%) and evaluated for chemical, physical and sensory properties, within three months of cold storage. Chlorella biomass cheeses were richer in the protein, carbohydrates and fiber contents than control samples. On the other hand, addition of Chlorella biomass to the cheese gave products more firmness and gives a stronger network and leads to lower oil separation and meltability values than control. The sensory evaluation indicated that addition of 2% C.vulgaris in Ch A did not have any effect on the overall acceptability of Ch A and in the same time introduced a new healthy alternative Ch A for a healthier lifestyle. Key words: Microalgae Chlorella vulgaris biomass Processed cheese analogue started in the early 1960s (Japan) and nowadays microalgae are mainly marketed as food supplements, commonly sold in the form of tablets, capsules or liquids. Additionally, there is an increasingly growing market for food products with microalgae addition such as pastas, biscuits, bread, snack foods, candy bars or gums, yoghurts, drink mixes, soft drinks, etc., either as nutritious supplement, or as a source of natural food colorant [3]. In Egypt, algae production was achieved as early as 1970s aiming at the massive production of non-traditional protein rich food. Assisting of all inputs was fully reported by El-Sayed [4]. In some countries such as Germany, France, Japan, USA, China or Thailand, food production and distribution companies have already started serious activities to market functional foods with microalgae and cyanobacteria (blue-green algae). The biotechnological exploitation of microalgae resources INTRODUCTION Modern food industry leads to cheaper, healthier and more convenient products, in response to increasingly consumers demand. Microalgae are an enormous biological resource, representing one of the most promising sources for new products and applications [1]. These microscopic organisms can be grown under certain controlled environmental conditions that can stimulate or inhibit the biosynthesis and accumulation of bioactive compounds in large amounts. The possibility of not only harvesting microalgae but also growing them at different conditions enables its use as natural reactors at a large scale [2]. Some microalgae species, such as Chlorella and Spirulina, have been used for many centuries as a nutrient-dense food in Asia, Africa and Mexico. However, commercial large-scale production of microalgae only Corresponding Author: Samah M. Shalaby, Food Science Department, Faculty of Agriculture, Ain Shams University, Shoubra EL-Khaima, Cairo, Egypt. 914 World Appl. Sci. J., 23 (7): 914-925, 2013 for human nutrition purposes is restricted to very few species, due to the strict food safety regulations, commercial factors, market demand and specific preparation [1]. Therefore, microalgae can be used to enhance the nutritional value of food products, due to their well balanced chemical composition as well as a source of highly valuable molecules, such as proteins, carotenoid pigments, vitamins, fatty acids, sterols, polysaccharides, among other biologically active compounds, presenting potential health benefits. However, foods supplemented with microalgae biomass might be sensorial more convenient and varied, thus combining health benefits with attractiveness to consumers [5]. The green alga Chlorella vulgaris is a unicellular green micro-alga that is ubiquitous in freshwater environments. The nutritional value of C. vulgaris was initially determined in 1950s–1960s [6]. It is also known as one of the earliest forms of life, was the first microalga isolated as a pure culture, by Beijernick in 1890s as mentioned by Guiry [7]. It has been used as an alternative medicine in the Far East since ancient times and it is known as a traditional food in the Orient, being considered as a potential source of a wide spectrum of nutrients. In addition to, it widely produced and marketed as a food supplement in Japan, China, U.S and Europe, though it is believed that it does not possess GRAS status and the toxicological test revealed the absence of any toxic effects in C. vulgaris [8]. The nutritive value of outdoor or indoor cultured C. vulgaris is of interest to the food industry, especially in countries where the weather conditions do not allow massive culture of higher plants. Chlorella has health benefits, such as assisting disorders such as gastric ulcers, wounds, constipation, anemia, hypertension, diabetes, infant malnutrition and neurosis. It has also been shown to have immune-modulating and anticancer properties [9-15]. Use of Chlorella vulgaris extract might be considered as an adjunct therapeutic strategy to combat hepatic disorders and other oxidative stress-related diseases [16]. Feeding micro-algae to elderly people or animals has been demonstrated to protect from age-dependent diseases, particularly cardiac hypertension or hiperlipidemia [17-20]. However, Chlorella vulgaris has been named green healthy food by FAO, rich in nutrients, such as proteins, total lipid, total carbohydrate, minerals, dietary fibers, antioxidants and vitamins [21]. C. vulgaris biomass has already been used experimentally in feed [22-25] and food products, such as pasta products, butter cookies and its polyunsaturated fatty acids are added to infant formulas [26, 27]. The incorporation of this biomass can provide a coloring effect and other functional characteristics such as antioxidant activity and providing nutritional supplements (e.g. fiber, fatty acids and oligoelements). Cheese analogue (Ch A) is processed cheese-like product in which milk fat, milk protein or both are partially or wholly replaced by non milk-based components. They are manufactured by blending various edible fats/oils, proteins, other ingredients and water into a smooth homogenous blend with the aid of heat, mechanical shear and emulsifying salts [28]. The market of analogues has grown recently due to the simplicity in producing them and the partial substitution of milk ingredients by cheaper vegetable ones, factors that allow for a reduction in product manufacturing costs [29, 30]. This study’s goal is to develop a new healthy processed cheese analogue (Ch A) enriched with different ratios of Chlorella vulgaris biomass (1, 2 and 3% w/w) in the Ch A formula. Then assessing the changes in the chemical composition, physical properties and sensory acceptance that caused by this addition. MATERIALS AND METHODS Materials: Chlorella vulgaris biomass in the form of freeze-dried was obtained from Algal Biotechnology Unit, National Research Centre, Dokki, Giza, Egypt. Clean growth was performed within 1200 L Zigzag photobioreactor [31] in the presence of the growth media as early described by El-Sayed et al. [32]. For harvesting and cleaning of the obtained biomass a series of precipitation and washing was performed using tap water and cooling centrifuge (HEIDELBERG RUNNE, RSU-20). Ras cheese (one month old), matured Cheddar cheese (8 months old), sodium chlorides and sugar were purchased from local market at Cairo, Egypt. Kasomel emulsifying salt K-2394 was obtained from Rhone-Poulenc Chimie, France. Low heat skim milk powder and butter were from Irish Dairy Board, Grattan House, Lower Mount St., Dublin, Ireland. Chemical composition of the ingredients used in the manufacturing of Ch A is presented in Table 1. Preparation of Acid Casein: Acid casein used in this study was prepared as described by Sawhney et al. [33]. Fresh raw buffalo’s milk was obtained from the herd of the Faculty of Agriculture, Ain Shams University, Cairo, Egypt. The fat was separated from milk to get the skim milk of less than 0.5 g/100 g fat content and the skim milk was pasteurized and cooled to 37°C. Hydrochloric acid solution (1.0M) was added to the milk as coagulant and the precipitation was carried out at 37°C and pH 4.7. As soon as the curd was settled, the whey was removed then the curd was washed with fresh water at 41°C and 915 World Appl. Sci. J., 23 (7): 914-925, 2013 pressed. The chemical composition of acid curd is summarized in Table 1. Table 1: Chemical composition (%) of the ingredients used in manufacture of processed cheese analogue Ingredients --------------------------------------------------------------------------------------------------------------------------------------------------------------------Analysis (%) Cheddar cheese Ras cheese Skim milk powder Butter Acid casein Chlorella biomass Total solids 65.80 54.81 96.00 84.00 47.02 94.17 Fat 34.80 24.77 0.97 81.99 ND 12.18 Crude protein 1 1 1 2 43.01 351.45 25.47 22.26 37.13 ND Ash 5.42 5.76 7.89 ND 2.82 9.50 Carbohydrate 0.10 1.64 47.43 ND ND 11.86 ND ND ND ND 9.18 Fiber 1 ND 2 3 Protein% - N × 6.38. Not determined. protein% - N × 4.38 Table 2: Composition (kg/100kg) of different blends used in manufacture of Cheese analogue treatments Ratios of Chlorella vulgaris biomass (%) ---------------------------------------------------------------------------------------------------Ingredient Control 1 2 3 Cheddar cheese 12.80 - - - Ras cheese 38.44 - - - Skim milk powder 5.12 5.75 5.75 5.75 Butter 10.26 25.52 25.52 25.52 Acid curd - 32.89 32.89 32.89 Salt - 1.00 1.00 1.00 Sugar - 1.00 1.00 1.00 Emulsifying salt 2.50 1.85 1.85 1.85 Chlorella biomass - 3.00 4.00 5.00 Water 30.88 28.99 27.99 26.99 Total 100 100 100 100 Manufacture of Cheese Analogue (Ch A): Cheese analogue (Ch A) was manufactured as described by Savello et al. [34]. All experimental Ch A treatments were formulated to yield Ch A with 50-55% moisture and 48-50% fat-in-dry-matter. Control processed cheese was made of young Ras cheese and matured Cheddar cheese as a base blend. Ch A treatments were manufactured by partial replacing of the base cheeses (Ras and Cheddar) with acid casein curd and Chlorella vulgaris biomass in ratios of 1, 2 and 3% as shown in Table 2. Process cheese and Ch A treatments were prepared by blending the dry ingredients with previously warmed (50°C) milk fat into the processing batch type kettle of 10 kg capacity, at the pilot plant unit of the National Research Centre. Cooked was done using direct injection of steam at pressure of 1.5 bar to 66°C with continuous agitation for 4 min. The pH was adjusted to 5.80 using citric acid or sodium hydroxide. The blends were further then heated to a final temperature of 82°C in approximately 4 min. The blends were held at 82°C for 1 min to add the biomass, prior to filling into tin cans then stored at 7°C and analyzed when fresh and monthly up to 3 months of cold storage at 7°C. Analytical Methods: Chemical Analysis of Algae: Chlorella vulgaris biomass nutrient profile was determined by AOAC standard methods [35] in terms of moisture, ash, protein, fat and fiber contents, total carbohydrate contents were calculated by difference. Fatty acids profile was determined using gas chromatography (GC Hewlett Packard 6890) according to the methods described by AOAC [35]. For amino acid profile, the analysis was carried out as described by AOAC [35] using Eppendorf Biotronic LC 3000 amino acid analyzer (Eppendorf-Biotronic, Hamburg, Germany). Chemical Analysis of Ch A: The Ch A samples was tested for fat content (using Gerber method) and protein content (using micro-Kjeldahl method) as described by Ling [36]. Salt content was determined as described by Bradley et al. [37]. 916 World Appl. Sci. J., 23 (7): 914-925, 2013 The moisture and ash contents were determined according to the method by AOAC [35]. The pH values were measured using pH meter model Cole-Armer Instrument Co., USA. species biomass is one of the main reasons to consider them as an unconventional source of protein. In addition, the amino acid pattern of almost all algae compares favorably with that of other food proteins. Table 4 show the presence of nine of essential and seven of Organoleptic Evaluation: Sensory attributes of Ch A samples were evaluated by the staff members at Department of Dairy Science, National Research Center. Samples were evaluated according to the scale mentioned by of Awad et al. [38] as follows: Table 3: Chemical composition of Chlorella vulgaris biomass Firmness: (1 = very soft, 7 = very firm). Stickiness: (1 = very sticky, 5 = not sticky). Crumbliness: (1 = very crumbly, 7 = not crumbly). Sliceability: (1 = poor sliceability, 5 = good sliceability). General acceptability: (1 = very poor acceptability, 7 = very good acceptability). Component (%) Moisture Dry matter Crude protein Carbohydrates* Crude lipid Crude fiber Ash 5.83 94.17 51.45 11.86 12.18 9.18 9.50 *: calculated by differences Table 4: Amino acids composition Chlorella vulgaris biomass Amino acids Concentration (g/100g protein) Statistical Analysis: All data were expressed as mean values. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test with P 0.05 being considered statistically significant using SAS program [39]. Aspartic acid Threonine* Serine Glutamic acid Glycine Alanine Valine* Methionine* Isoleucine* Leucine* Tyrosine Phenylalanine* Histidin* Lysine* Arginine Proline NH4 RESULTS AND DISCUSSION Chemical Composition of Chlorella vulgaris Biomass: The chemical composition of C. vulgaris used in our experiments is shown in Table 3. It can be seen that the main component is the protein which represents more than 50% of the chemical composition of C. vulgaris biomass. The other components such as lipid, carbohydrates, fiber or ash were in the range of 9-12%. These results are in the same trend of Yusof et al. [40], who reported that the protein, ash and fiber contents were quite high in Chlorella vulgaris biomass in all culture conditions, which is comparable with most microalgae, such as Enantiocladia duperrehyi, Amansia multifida and Hypnea musciformi. Also, the results are in agreement with those obtained by Gouveia et al. [8], who mentioned that C. vulgaris biomass has a high protein content (~ 40%), that may have reinforced the dough system of the cookies. On the other hand, the obtained results are different with that of Fradique et al. [41], who found that C. vulgaris (green or orange) were so high in the ash content being more than 34%. The differences in cultivation conditions are the main source of differences in protein and minerals contents of one strain of algae [42]. The high protein content of various microalgae 10.5 5.24 5.08 10.74 5.1 8.44 6.44 1.5 5.01 6.84 5.2 4.2 5.02 5.6 8.2 6.4 6.4 *: Essential amino acids non- essential amino acids in the experimented Chlorella vulgaris biomass. So the Chlorella protein contains all of the essential amino acids for human growth and health [43]. However, information on the nutritive value of the protein and the degree of availability of amino acids of Chlorella vulgaris were reported by Janczyk et al. [42]. Their overall digestibility is high, which is why there is no limitation to use dried whole Chlorella vulgaris microalgae in foods or feeds. In recent years, fatty acids composition in large scale production of microalgae including marine algae has created considerable interest among researchers. This is mainly because of the health benefits of mono and polyunsaturated fatty acids (MUFA and PUFA) that can be found in plants including microalgae. Consumption of 917 World Appl. Sci. J., 23 (7): 914-925, 2013 n-3 PUFAs from both seafood and plant sources may reduce coronary heart disease (CHD) risk as reported by Mozaffarian et al. [44] in a cohort study of 45,722 men. Thus, many health supplement stores now sell preparation of microalgae such as Spirulina and Chlorella packed in capsule or caplets, or even in food acids between different species of Chlorella, as follows 14:0, 16:0, 16:1, 16:2,16:3, 18:0, 18:1, 18:2, 18:3. On the other hand, the obtained results are unlike with that obtained by Yusof et al. [40], who found that respective fatty acids between different species of Chlorella, as follows 15:1, 16:0, 17:0, 18:0, 18:1, 18:2, 18:3 and 20:0. The presence of fatty acids with odd number of carbon atoms 17:1 is a firm proof that the algal culture is contaminated with bacteria as mentioned by Petkov and Garcia [47]. Carbohydrates in Chlorella vulgaris biomass can be found in the form of starch, glucose, sugars and other polysaccharides as mentioned by Hadj-Romdhane et al. [49]. Chlorella vulgaris biomass also represent a valuable source of nearly all essential vitamins (e.g., A, B1, B2, B6, B12, C, E, nicotinate, biotin, folic acid and pantothenic acid) and a good source of Ca, K, Mg and Zn as reported by Yusof et al.[40]. This microalga is also rich in pigments like chlorophyll (0.5% to 1% of dry weight), carotenoids (0.1% to 0.2% of dry weight (on average) and up to 14% of dry weight for â-carotene of Dunaliella) and phycobiliproteins. Gouveia et al. [23] used the dry biomass obtained from stressed cells of Chlorella vulgaris (rich in carotenoids pigments) in animal feed, instead of the commercial synthetic pigment and strongly suggested that yolk pigmentation was comparable to that obtained using commercial pigments. Therefore, Chlorella vulgaris biomass is able to enhance the nutritional content of conventional food preparations and hence, to positively affect the health of humans, this is due to their original chemical composition. Table 5: Fatty acid composition of oil extracted from Chlorella vulgaris biomass (% of TFA) Fatty acid Name Area (%) C14:0 C16:0 C17:1 C18:0 C18:1 C18:2 C18:3 TSFA TUSFA Myristic Palmitic Heptadecanoic Stearic Oleic Linoleic Linolenic - 1.85 52.56 15.88 8.83 5.42 6.56 8.87 63.24 36.73 TSFA : total saturated fatty acids. TUSFA: total unsaturated fatty acids. and beverages known to have therapeutic values in treating hypercholesterolemia, hyperlipidaemia and atherosclerosis [45, 46]. Fatty acids composition of oil extracted from Chlorella vulgaris biomass are presented in Table 5. As expected the studied oil had elevated amount of total saturated fatty acids (63.24%). This oil had superior content in palmitic acid; its percentage was 52.56%, followed by stearic acid (8.83%). It could be noticed that the most predominant unsaturated fatty acids was hepatadecanoic (C17:1), its percentage was 15.88%. While, the omega 3 fatty acids linolenic in the second order having a percentage of 8.87%, followed by linoleic (6.56%) the omega 6 fatty acid, then the mono-unsaturated fatty acid oleic (5.42%). These finding in accordance with that found by Petkov and Garcia [47], which the palmitic acid was the major fatty acid in their Chlorella vulgaris biomass. It can be noticed that neither fatty acid containing four double bonds, nor any with 20 carbon atoms were observed. Present results show that Chlorella has quite a simple qualitative fatty acid composition compared to almost all green algae. However, saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) displayed a significant interactive effect of growth modes. The results of fatty acids composition of experimented Chlorella vulgaris biomass presented in Table 5 are in agreement with those reported by Petkov and Garcia [47] and Liu et al. [48],who found that fatty Chemical Composition of Ch A: Table 6 shows the chemical composition of Ch A treatments. It is interesting to note that both ingredients i.e., milk protein bases (acid casein) and Chlorella vulgaris biomass exhibited significant albeit small effects on the chemical composition of cheese analogues. There were significant differences between the control and only cheese analogue enhanced with 3% Chlorella vulgaris biomass in all the chemical components (moisture, fat, carbohydrate and salt content) except the ash content, while it were not significant between the cheese analogues themselves. These differences between Ch A treatments reflected differences between formulations. The main effects on chemical composition values were associated with the different milk protein base type; products made from acid curd had a higher protein and lower salt content and pH than that made from normal cheeses. Ch As are in agreement with those reported by Muir et al.[50], who found that the total solids and protein 918 World Appl. Sci. J., 23 (7): 914-925, 2013 contents were higher in cheese analogues than the control, while the fat and ash contents and pH values were lower in cheese prepared with acid casein than the control. The same trend was also mentioned by Cunha et al. [30], who reported that the analogue cheeses were higher in protein and lower in salt content and pH values than control. On the other hand, addition of the algal Table 6: Changes in gross chemical composition (%) of processed cheese analogues enhanced with Chlorella vulgaris biomass. Chemical composition (%) Ratios of Chlorella vulgaris biomass (%) -----------------------------------------------------------------------------------------------------------------------------------------------Control 1 2 3 Total solids Fat/ dry matter Protein1 Carbohydrates Ash Salt in moisture Fiber pH 44.86b 50.16a 13.56b 2.90 b 4.36 a 3.75 a ND2 5.80 a 1 45.25ab 49.72a 14.38ab 3.95 a 3.60 a 2.35 ab 1.18 c 5.42 b 45.66ab 48.53ab 14.49 ab 3.91 a 3.74 a 2.20 b 2.42 b 5.40 b 45.91a 47.92b 14.70 a 4.85 a 3.76 a 2.18 b 3.66 a 5.39 b Protein% - N × 6.38. 2Not determined. biomass had insignificant effects on protein, carbohydrates and fiber contents in Ch A treatments. It could be due to the small proportions of biomass added to the Ch A and these increases are a logical consequence of the chemical composition of Chlorella vulgaris biomass (Table 3). The pH, moisture, fat and salt contents were decreased in the Ch As enhanced with biomass by increasing of the ratio of Chlorella vulgaris biomass to 3%. On the contrary, protein, carbohydrates and fiber contents were increased by the high ratios of added biomass (2 and 3%) according to the high content of these components in the Chlorella vulgaris biomass (Table 6). These obtained results are in the same line with the results obtained by Fradique et al. [41], who found that pasta prepared with Chlorella vulgaris and Spirulina maxima presented a chemical composition richer than the control pasta, namely in protein, total fat and ash contents. resulted in analogues that were clearly dissimilar in some textural attributes, especially firmness (Fig. 1). Treatments with high moisture content and pH around 5.70 (control) were in general softer, while those with lower moisture and pH values, around 5.40 were firmer and easier to handle (Fig. 1). The decrease in firmness caused by the increase in the moisture content of the treatments was expected. It occurs due to the greater hydration and consequent weakening of the casein network [51]. Similar behavior was reported by Lawrence et al. [52] and Tunick et al. [53] for natural cheeses and by Fox et al. [54] for processed cheese products. On the other hand, Solowiej [55] observed that hardness of the processed cheese analogues obtained only on the base of acid casein was very high at pH 4.5–5.0; however it decreased significantly with an increase of pH. Addition of microalga resulted in a significant (P<0.05) increase in the Ch As firmness in comparison with the control sample (Fig.1). Furthermore, raising biomass concentration (1–3.0%) results in general tendency for an increase in firmness. These results evidence the positive effect of the alga in the cheese structure protection. This can be related to the fact that Chlorella vulgaris biomass has a high protein Physical Properties of Ch As Firmness Values: The compositional differences in the experimental Ch As (Table 6) were in line with the expected differences from the formulations used and 919 World Appl. Sci. J., 23 (7): 914-925, 2013 Fig. 1: Penterometer reading (mm) of processed cheese analogues enhanced with Chlorella vulgaris biomass Fig. 2: Oil separation index (%) of processed cheese analogs enhanced with Chlorella vulgaris biomass Fig. 3: Melting index (%) of processed cheese analogues enhanced with Chlorella vulgaris biomass content (>50%) [8]. The microalgae protein and carbohydrate molecules can also play an important role on the water absorption process, which promotes the increase of cheese firmness, as was observed by Raymundo et al. [27]. However, the obtained experimental results are in agreement with the results of Fradique et al. [41], who reported that the addition of Chlorella vulgaris biomass (0.5 -2%) resulted in a significant increase in the raw pasta firmness. By the same way, Gouveia et al. [8] found that the firmness of the biscuits increased linearly and significantly with Chlorella vulgaris biomass content (0.5 -3%). protein in the resultant processed cheese emulsion. Also, many factors can affect oil separation, such as type and amount of used raw materials in the base formula, degree of creaming action, cooking time and temperature, the type of emulsifying salt used and pH value in the final product etc [38]. As shown in Fig. 2, a marked significant difference (p<0.05) was observed in the oil separation indexes between control and Ch A treatments. Batches made with acid curd enhanced with algal biomass showed lower oil separation indexes than the control one. This may be attributed to that the control samples had higher fat content and lower protein content than other formulas. Also, when the levels of the added biomass increased, the oil separation indexes of Ch As were more decreased. As mentioned before, the addition of Chlorella vulgaris biomass increased the protein and carbohydrates contents and decreased the fat in dry matter, so the resultant emulsion for Ch As were more stable than the control. These results are in agreement Oil Separation Index: Oil separation indexes of Ch As made by replacing the hard cheeses in the base blend with acid casein curd enhanced with Chlorella vulgaris biomass at ratios of 1, 2 and 3% when fresh & after 1, 2 and 3 months of cold storage at 7°C are presented in Fig. 2. The oil index depends on the state of the fat and 920 World Appl. Sci. J., 23 (7): 914-925, 2013 with the results of Awad and Salama [56], who mentioned that adding boiled egg to the formula of processed cheese would give a stronger network and leads to lower oil separation. Furthermore, cold storage of the samples increased the free oil index; this may be due to the changes in pH value and soluble nitrogen (SN) content during storage. The lower pH values and higher SN content (more protein decomposition) would lead to lower degree of product emulsification and higher fat leakage [38]. Organoleptic Evaluation of Ch A Treatments: Sensory analysis of cheeses is an important contribution to their possible future commercialization, since it gives a perspective of the potential consumer’s acceptance. Organoleptic properties scores of resultant Ch As when fresh and during cold storage (7°C) are summarized in Table 7. Firmness appreciation refers to the product resistance to attain a given deformation. The results in Table 7 revealed that the addition of Chlorella vulgaris biomass to the analogue cheese made by acid casein produced the firmest cheese (Fig.1), while the control was the softest. The difference was insignificant (P<0.05) among the data, this could be attributed to the small ratios of microalgae biomass in the mixture. The panelists preferred the microalgae cheeses, particularly those with the microalgae content (2%), in comparison with the other microalgae samples (1 and 3% microalgae). In the same time, all Ch A treatments became more firm when stored up to 3 months which is in accordance with the results found by Awad et al. [38]. Stickiness is the work necessary to overcome the attractive forces between the surface of the food and the other materials with which the food comes in contact, it’s also meaning the resist separation of cheese from a material it contacts [61]. All Ch A treatments were nonsticky and enhanced cheese analogues with 2 and 3% biomass showed very slight stickiness. This could be due the effect Chlorella biomass on the cheese protein solubility. This effect may be due to the hydrolysis of emulsifying salts during storage causing a decrease in pH value, which hardened the cheese samples. Meltability Index: Figure 3 shows the relationship between meltability (mm) of Ch A treatments as affected by algal biomass when fresh & after 1 and 3 months of storage at 7°C. Melting index of Ch As made by replacing the hard cheeses in the base blend with acid casein enhanced with 1, 2 and 3% of Chlorella vulgaris biomass had significantly the lowest meltability values than that control (p<0.05). On the other hand, the results indicated that meltability of the Ch As insignificantly decreased with increasing algal biomass amount (Fig. 3). The cheese meltability showed a tendency to decrease after storage at 7°C for 3 months. This could be due to the changes in pH and SN content of processed cheese, as mentioned by Awad et al. [38]. The obtained results of meltability of Ch As are in the same trend with those of firmness (Fig. 1) and chemical composition (Table6). The progressive increase in hardness and decrease in flow ability values (Fig. 3), at increased biomass ratio may be related to the observed decrease in fat globule sizes. Similarly, Cavalier-Salou and Cheftel [57] found that melting ability was correlated to high pH, soft texture, high degree of casein dissociation and low degree of fat emulsification. Shirashoji et al. [58] also reported that high firmness and low melting values in process cheese were associated with small fat globule sizes. By the same way, Gupta and Reuter [59] reported that the softer body of processed cheese foods with higher moisture content might help in providing higher meltability as f1owability improves. Moreover, Savello et al. [34] found that rennet casein cheese melted significantly more than the acid casein cheese. Otherwise, Mounsey and O’Riordan [60] noted that imitation cheese with poor melting characteristics is often desirable when used in products that are deep-fat-fried. Table 7: Sensory evaluation of processed cheese analogues enhanced with Chlorella vulgaris biomass during storage at 7°C for 3 months Character Assessed Storage period (month) Control Ratios of Chlorella vulgaris biomass (%) -------------------------------------------------------------------------------1 2 3 Firmness (1-7) Fresh 6.80a 6.82a 921 6.90a 6.90a World Appl. Sci. J., 23 (7): 914-925, 2013 1 3 6.82a 6.90a 6.87 a 6.91 a 6.95 a 6.98 a 6.92b 6.95 a Stickiness (1-5) Fresh 1 3 5.00a 4.93a 4.93a 4.91a 4.86ab 4.79ab 4.95a 4.90ab 4.81ab 4.86 a 4.52b 4.43b Crumbliness (1-7) Fresh 1 3 7.00a 7.00a 7.00a 6.96a 6.90a 6.80a 6.90a 6.92a 6.85a 6.86a 6.82a 6.80a Sliceability (1-5) Fresh 1 3 4.85a 4.62a 4.38a 4.57ab 4.45ab 3.78b 4.60ab 4.48ab 4.25a 4.32b 4.00b 3.56b Acceptability (1-7) Fresh 1 3 7.00a 6.94a 6.92a 6.80a 6.75a 6.60ab 6.95a 6.92a 6.90a 6.77a 6.63a 6.40b a, b, c Means not followed by the same lower case letter in each row are significantly different (p 0.05). These obtained results are in the same trend of the obtained firmness results and also are in agreement with those obtained by Pereira et al. [62], who mentioned that cheeses with lower moisture content were, in general, firmer, curdier and less sticky than cheeses with higher moisture content. Products with intermediate moisture content resulted, in general, intermediate scores for these attributes. The scores of stickiness decreased throughout the entire storage period, this suggesting that the cheese matrix became sticky during storage as a resulted of increased mobility during storage as observed by Awad et al. [38]. Crumbliness refers to the strength of the internal bonds making up the body of the product. There was no crumbliness detected in fresh cheese samples except the cheese enhanced with 3% Chlorella biomass that was insignificant slightly crumbly. This could be due to the low pH value (Table 6) in the sample, which makes the protein more crumbly. The same observation was also noticed after 1 month of storage. After 3 months, slight crumbliness was detected in the rest of all treatments. This change is mainly due to the decrease in pH values that occurred during the storage period [38]. Sliceability is the ability of the product to cut into slice. The Ch A treatments showed very good sliceability being significantly good in control and analogue cheese enhanced with 2% Chlorella vulgaris biomass. Analogue enhanced with 3% algae biomass showed a slightly breakable texture and did not give good cheese slice. The sliceability was related to the cheese firmness and decreased during storage. Ch A enhanced with 3% chlorella biomass showed significantly decrease in sliceability. General acceptability of cheese is concerned with texture, odor and flavor attributes. The flavor was found to be the most important attribute that affect the acceptability of a cheese analogue containing chlorella vulgaris biomass in different ratios. However, all analogues treatments scored positively (>6) revealing a good acceptance by the panelists. There were no significant differences detected by the panelists between analogous with microalgae biomass and the control. Although some individuals detected a strange flavor in the batches prepared with 2.0 and 3% of Chlorella vulgaris biomass. Storage of cheese samples lowered the acceptability and the cheese enhanced with 3% chlorella biomass showed significantly lower acceptability after 3 months of cold storage. From the sensory evaluation results, it could be concluded that the addition approximately 2% Chlorella vulgaris biomass in the Ch A did not have significant effect on the overall acceptability score of it and in the same time introduced a new healthy alternative processed cheese analogue. CONCLUSION Significant attention has recently been drawn to the use of microalgae for developing functional food, as microalgae produce a great variety of nutrients that are essential for human health. Chlorella vulgaris has been named green healthy food by FAO, rich in nutrients, such as proteins, total lipid, total carbohydrate, minerals, dietary fibers, antioxidants and vitamins. Chlorella vulgaris biomass provides a functional ingredients for feed additive, pharmaceutical and nutraceutical purposes. A novel processed cheese analogue was successfully produced by adding microalgae biomass to cheese blend. Ch A treatments prepared with Chlorella vulgaris presented a chemical composition richer than the 922 World Appl. Sci. J., 23 (7): 914-925, 2013 control, namely in protein, carbohydrates, fiber and ash. The physical quality of analogues was improved by including microalgae, the textural characteristics of the resultants, namely firmness, were positively affected by the inclusion of microalgae, when compared to the control samples. The increase of analogue firmness may be related to the addition of components rich in protein that, probably have a significant influence in the structure of the cheese network. In the same time, the resultant emulsion Ch As was more stable than the control, by giving a stronger network and leads to lower oil separation. 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