Abstract: Background and Objective: Spirulina is a rich source of nutrients viz., essential amino acids, essential fatty acids, carotenoids and vitamins. The study was carried out to evaluate of Spirulina maxima addition as source of nutrients, antioxidants and color on processed cheese properties. Materials and Methods: Processed cheese analogue treatments were supplemented with Spirulina maxima powder (1, 2 and 3%). The chemical, physical, color and sensorial properties of processed cheese analogue supplemented with S. maxima were evaluated through 3 months of cold storage (7°C). Also, the antioxidant capacity of S. maxima processed cheese analogue treatments was determined. Results: The spreadable processed cheese analogue with 3% S. maxima powder had higher chemical components except ash compared to control cheese. The results of physical properties showed that the penetrometer reading of the S. maxima processed cheese treatments was higher than those of control allover storage period, while oil separation and melt ability were lower. The S. maxima processed cheeses were more green (a-value) and lower whiter (L-value) than those of control. The highest free radical scavenging activity (57.24%) was recorded for S. maxima processed cheese analogue treatment (3%). From the sensorial results, the S. maxima processed cheese analogue (1 or 2%) treatments was higher acceptable compared to those of 3%. Conclusion: Hence, adding S. maxima powder (1 or 2%) during processed cheese analogue manufacture let the cheese to develop special color (green), high nutritional value, antioxidant activity and sensorial scores.
INTRODUCTION
Algae are a group of microorganisms capable of self-supporting photosynthesis. They get their energy from sunlight1. Spirulina is a primitive organism originating some 3.5 billion years ago2. Spirulina named as tecuitlatl by Aztecs (Mexicans) which means stone’s excrement during 16th century3. Spirulina have classified as Cyanobacterium a Gram-negative bacterium that performs photosynthesis same as that occurs in plants. Spirulina is filamentous, photosynthetic, autotrophic, unbranched and multicellular blue-green algae with symbiotic bacteria that fix atmospheric nitrogen by the process of nitrogen fixation from the air2. Spirulina is mainly found in natural lakes having high pH value i.e. 8-10 all over the earth.
Because of its alimentary value, Spirulina has been consumed from a long past time in many parts of the world as a food supplement by humans as well as animals in various forms like a health drink, tablets and powder, etc. In Europe, Japan and North America cyanobacteria tablet have been sold. Spirulina known to be the richest source of protein and vitamin and can be used to treat children suffering from malnutrition. Spirulina is a rich source of nutrients viz., essential amino acids, essential fatty acids, carotenoids, minerals and vitamins, it has been considered as ‘‘Food of the future’’ and ideal food for astronauts by NASA4,5.
The metabolism of carbohydrate, fats, protein and the reproduction of skin and muscle can be enhanced by the β-carotene, B-group vitamin, vitamin E, iron, potassium and chlorophyll available in the Spirulina. According to the findings of the National Cancer Institute, the United States of America, an intake of 6.0 mg β-carotene daily may be effective in minimizing the risk of cancer. Because Spirulina is well known to have very high iron content, it was tested against a typical iron supplement, iron sulfate. Spirulina-fed rats absorbed 60% more iron than rats fed the iron supplement2.
Spirulina maxima, a filamentous alga, is a cyanobacterium belonging to the family Oscillatoriaceae, which blooms well in alkaline water and contains a unique blend of nutrients that no single source can provide. The alga is characterized by high protein content (60-70%) and a wide spectrum of nutrients that include B-complex vitamins, minerals, γ-linolenic acid and the super antioxidants, β-carotene, vitamin E, trace elements and other uncharacterized bioactive compounds. Because of its apparent ability to stimulate the immune system, Spirulina has several therapeutic functions such as antidiabetic, anti-inflammatory, antiviral and anticancer activities6.
When the algal cells or filaments of Spirulina are transformed into a powder it can provide the basis for a variety of food products, such as soups, sauces, pasta, snack foods, instant drinks and other recipes7. The preparation of fermented foods such as cheese, yogurt and tofu, offered many new possibilities for the use of Spirulina. Diets in which Spirulina provides up to 100% of the protein produced growth rates comparable with those obtained with standard diets in several animal species. As little as 10 g a day of Spirulina brings rapid recovery from malnutrition, especially in infants2.
Processed cheese enjoys great popularity all over the world, because it is favorable texture, taste and easy to handle and eat. Processed cheeses are produced by shredding or cutting natural cheeses with different degrees of ripening and mixing them with emulsifying agents under heated conditions, in a partial vacuum or at ambient pressure, until a homogenous mass is obtained8. Per local legislation, other ingredients can be added, such as powdered milk, stabilizers, preservatives, water, meat, fruit and spices, among others. The processed cheese supplemented with Chlorella vulgaris or Spirulina platensis as nutrient source were studied in many studies9-11. Little information is available to add Spirulina maxima as a source of color and antioxidant in the production of processed cheese.
In this study, processed cheese was produced by adding S. maxima powder to increase of the nutritional value and as source of antioxidants and color. Hence, the aim was to investigate the effects of S. maxima powder on the quality characteristics of processed cheese during the storage (3 months at 7°C).
MATERIALS AND METHODS
The study was performed at Department of Dairy Science, National Research Centre, Dokki, Giza, Egypt, from October, 2018 to June, 2019.
Materials:
| • | Spirulina maxima powder was obtained from Algal Biotechnology Unit, National Research Centre, Giza, Egypt |
| • | Ras cheese (one month old) was purchased from Arabic Food Industrial Co. (Domety), 6th October City, Egypt |
| • | Matured cheddar cheese (8 months old) and Kasomel emulsifying salts K-2394 were purchased from International Dairy and Food Co. (milky land), 10th October City, Egypt |
| Table 1: | Chemical composition of ingredients (%) |
| ND: Not detected | |
| Table 2: | Formulation of the various blends for spreadable processed cheese analogue with Spirulina maxima powder |
| • | Low heat skim milk powder and butter were procured from Irish Dairy Board, Grattan House, Lower Mount St., Dublin, Ireland |
| • | Acid curd (Lactic acid casein) was prepared by biological acidification. The skim milk (Dairy Technology Unit, Faculty of Agriculture, Cairo University, Egypt) at pH 6.6 is pasteurized at 72°C for 15 sec. Mesophile, non-gas starter bacteria (Lactococcus lactis, 0.5%) are inoculated at 25°C to pasteurized milk until coagulation. Then the lactic clot is washed and dried under vacuum at 40°C |
Chemical composition of the ingredients used in processed cheese analogue treatments manufacture with Spirulina maxima powder is shown in Table 1.
Methods
Spreadable processed cheese analogue manufacture with Spirulina maxima powder: Spreadable processed cheese analogue treatments (45% total solids and 48% fat-in-dry matter) were manufactured according to the method of Meyer12. Spreadable processed cheese control was produced from Ras and matured cheddar cheeses as a base blend. Spreadable processed cheese analogue treatments were made by replacing of the base cheeses (Ras and cheddar) with acid curd and Spirulina maxima powder (1, 2 and 3%) as presented in Table 2. The control and treatments were manufactured by thoroughly mixing the ingredients in a laboratory style processing kettle of the 10 kg capacity, a pilot machine at National Research Centre. The cheese blends were heated using direct injection of steam at pressure of 1.5 bar to 66°C with controlled agitation for 4 min. The blends were further heated to 80°C in approximately 4 min. The Spirulina maxima powder (1, 2 and 3%) was added to the heated blends at 80°C. The resultant cheeses were filled into sterilized glass jar, covered with aluminum foil and their covers then cooled to 7°C. The resultant cheeses were analyzed when fresh and monthly up to 3 months of cold storage at 7°C.
Methods of analysis
Chemical analysis: The moisture, ash, protein, fat, fiber and total carbohydrate contents of Spirulina maxima powder were determined according to AOAC13. The high performance amino acid analyzer (biochrom 30, Germany) was used for essential amino acids determination of algae according to AOAC13. Total carotenoid content of S. maxima powder was determined. The pigments were extracted with acetone/methanol (4:6)14 and transferred to light-petroleum (40-60°C) in separating funnel and washed thrice thoroughly with distilled water15. The absorbance of the light-petroleum phase was red at 474 nm and the concentration of the carotenoids was determined using the absorption coefficient16 A1%1 cm = 1600. Total carotenoid content was calculated by using the formula17 give below and expressed as μg g–1 of S. maxima powder.
Total carotenoids (μg g–1) = A×V (mL)×104-A1%1 cm×w (g)
Where:| A | = | Absorbance |
| V | = | Total extract volume |
| W | = | Sample weight |
| A1%1 cm | = | Absorption coefficient |
For the spreadable processed cheese analogue treatments, total fat content was measured according to the method of AACC18. Total protein, total solids, fiber and ash contents were analyzed as described by AOAC13. Salt content was determined as mentioned by Bradley et al.19. Carbohydrate content was determined according to the method of Nielsen20. The pH was measured using digital pH meter (HANNA 8417) with combined glass electrode (Electric Instruments Limited).
Physical characterization of the cheese: The penetration of the spreadable processed cheese analogue treatments supplemented with Spirulina maxima powder and control was measured using a penetrometer (Koch Jer Instrument Co. Inc., USA) as described by Gupta and Reuter21. Melting quality was measured as described by Savello et al.22. Oil separation was determined according to the method of Thomas et al.23.
The color of S. maxima processed cheese treatments and control was measured using Hunter colorimeter Model b25 A-2 (Hunter Assoc. Lab. Inc. Va, USA) following the instruction of the manufacturer24. The instrument was first standardized using a white tile (top of the scale) and a black tile (bottom of the scale). A specimen of the cheese (flat layer) was placed at the specimen port; the tri-stimulus values of the color namely, L, a and b were measured, where: L value represents darkness from black (0) to white (100), a value represents color ranging from red (+) to green (-) and b value represents yellow (+) to blue (-).
Antioxidant capacity of cheese samples: Free radical scavenging activity (RSA (%)) assay of S. maxima processed cheese treatments and control was measured using the method of Brand-Williams et al.25 and expressed as percentage inhibition of the DPPH radical and was determined by the following:
Sensorial characterization of the cheese: The sensorial characteristics of S. maxima processed cheese treatments and control were evaluated by the staff members at Department of Dairy Science, National Research Centre. Samples were evaluated according to Bandyopadhyay et al.26.
Statistical analysis: Statistical analysis of the obtained data 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 SAS27 program.
RESULTS AND DISCUSSION
Chemical composition of Spirulina maxima powder: The gross chemical composition of S. maxima powder is shown in Table 3. It could be seen that the protein is the major organic component (70%), followed by carbohydrate (13.48%)+fiber (2.13%). The other components (fat and ash) represent 6.27 and 5.40%, respectively. These results are in agreement with Matos et al.28. The growing world population and consequential efficiency in protein supply for human nutrition lead to increased studies exploring novel and alternative protein sources such as single cell proteins (SCP).
| Table 3: | Gross chemical composition of S. maxima powder |
| *N×4.78 | |
| Table 4: | Essential amino acids of S. maxima powder |
Microorganisms such as algae, bacteria, fungi and yeast/filamentous can be used as a source of SCP, but due to their high level of essential amino acids coupled with low nucleic acid content, algae are preferred over fungi and bacteria as a source of SCP for human consumption29. Special carbohydrates produced by microalgae are important due to their potential therapeutic application. Here, β-1,3-glucan, a natural soluble fiber active as immune stimulator, antioxidant and reducer of blood cholesterol. In addition to the therapeutic use, β-1,3-glucan can be also used in food industry, mainly as fat substitute30. Lipids are constituents of all microalgae cells and the main biological functions include storing energy, signaling and acting as structural components of cell membranes. The lipid content of microalgae varies31 between 1 and 40%. Also, it could be seen from the results that the total carotenoids content of S. maxima powder was 6.4 mg g–1. Carotenoids are organic lipophilic compounds that are produced by plants and algae. They also act as antioxidants that inactivate reactive oxygen species (ROS) formed by exposure to excessive solarradiation32.
The essential amino acid composition of Spirulina maxima powder is shown in Table 4. Among the essential amino acids, leucine was as predominant (5.68%) followed by valline (5.10%). This result is agreement with Al-Dhabi and Arasu33, who reported that the leucine was identified as predominant in all studied samples of Spirulina ( 0.53-7.59%). The lowest essential amino acid percent was recorded for tryptophan (0.98%) followed by methionine (1.88%). Spirulina is very rich in proteins (60-70%) of its dry weight of high biological value containing the essential amino acids needed by both adults and children.
| Table 5: | Comparison of changes in chemical composition between fresh control and S. maxima processed cheese analogue |
| Means with the same letters in the same row are not significantly different, ND: Not detected | |
Chemical composition of fresh Spirulina maxima spreadable processed cheese analogue treatments: Table 5 show the changes in chemical composition of fresh S. maxima processed cheese analogue treatments compared to control. It could be observed that the total solids, protein and fat in dry matter values of processed cheese with 1 or 2% S. maxima powder were not different from those of control cheese. The spreadable processed cheese analogue with 3 % S. maxima powder had higher chemical components except ash compared to control cheese. Where, the protein, carbohydrates and fiber contents were increased by the high ratio of S. maxima powder (3%) and these may due to high levels of these components in S. maxima composition. These findings are in agreement with Jeon34, who found that the Chlorella addition at levels (0.5 or 1%) did not exhibit any significant differences of chemical composition of processed cheese compared to control. Also, Mohamed et al.9 found a significant differences between the control and only cheese analogue enhanced with 3% Chlorella vulgaris biomass in all the chemical components. Also, it could be noticed that the spreadable processed cheese analogue treatments with different levels of S. maxima powder contained the fiber, which can be attributed to the fiber content in the alga. This finding was in agreement with Tohamy et al.10.
Spreadable processed cheese analogue were made by replacing of the base cheeses (Ras and cheddar) with acid curd and the Cunha et al.35 found the cheese analogue treatments made with acid casein were higher in total solids and protein contents than the control, while the ash content and pH values were lower. The obtained results confirmed this.
Physical characterization of S. maxima processed cheese: The data in Table 6 illustrate the physical properties of S. maxima processed cheese treatments compared to control during storage period. It could be seen that the penetrometer reading of the S. maxima processed cheese treatments was higher than those of control allover storage period and these may be due to the replacement of base cheeses (Ras and cheddar) with lactic casein which gives a softness for cheese. Also, it could be noticed that the penetrometer reading decreased with increasing S. maxima powder level and this is in agreement with Fradique et al.36, who reported that the addition of microalgae resulted in an increase of firmness. The microalgae protein and carbohydrate molecules can also play an important role in the water absorption process, which promotes the increase of cheese firmness, as was reported by Raymundo et al.37. The penetrometer reading of all studied chesses decreased with progressing storage period. Mohamed et al.9 found the penetrometer reading of processed cheese with Chlorella vulgaris biomass decreased with increasing storage period.
The oil separation depends on the state of the fat and protein in the processed cheese emulsion. Also, many factors can affect oil separation, such as type and amount of raw materials in the base formula, degree of creaming action, cooking time and temperature, the type of emulsifying salt and pH value in the final product38. From the results presented in Table 6, the oil separation of S. maxima processed cheese analogue was lower than those of control and this may be due to the replacement of base cheeses (Ras and cheddar) with lactic casein where its casein is intact, strong and able to hold the fat, hence, reduction of oil separation. While, the protein of control was degraded during the storage period resulting in weaker network and hence were less capable of fat hold. Also, the addition of S. maxima powder slightly increased the protein and carbohydrates contents and decreased the fat in dry matter, so the resultant cheeses were lower oil separation compared control.
As shown in Table 6, the meltability of S. maxima processed cheese analogue was lower than those of control and this may be due to the control network is weaker than those of treatments, hence faster melting. The meltability decreased with increasing S. maxima powder level. Cavalier-Salou and Cheftel39 found that melting ability was correlated to soft texture, high degree of casein dissociation and low degree of fat emulsification. Shirashoji et al.40 reported that the high firmness and low melting values in process cheese were correlated with small fat globule sizes. Moreover, Savello et al.41 found that rennet casein cheese melted significantly more than the acid casein cheese.
| Table 6: | Physical characterization of processed cheese with S. maxima powder during storage |
| Means with the same letters in the same row are not significantly different | |
| Table 7: | Color properties of S. maxima processed cheese during storage period at 7°C |
| L: Black (0) to white (100), a: Red (+) to green (-), b: Yellow (+) to blue (-), means with the same letters in the same row are not significantly different | |
| Table 8: | Antioxidant activity of S. maxima processed cheese analogue and control during the storage period at 7°C |
| RSA: Free radical scavenging activity, means with the same letters in the same row are not significantly different | |
Color properties of S. maxima processed cheese: The color parameter changes of S. maxima processed cheese during storage period at 7°C are presented in Table 7. It could be noticed that the L value of control was whiter than those of treatments with S. maxima. The whiteness decreased with increasing storage period or S. maxima levels. The S. maxima processed cheeses were more green (a value) than those control and this may be due to algae chlorophyll content36,42. The green color increased with increasing the level of algae or storage period. Chlorophylls are greenish lipid-soluble pigments, which contain a porphyrin ring and found in all algae. Chlorophyll is registered and approved as a colorant additive and is mostly used in the food pigmentation and dietary supplement industries31. Also, the b value of control was lower than those of with S. maxima allover storage period and increased with increasing algae level.
Antioxidant capacity of S. maxima cheese: Antioxidants are compounds that help combat cell and DNA damage that leads to cancer, heart disease and other chronic diseases. The antioxidant activity of S. maxima processed cheese analogue treatments and control during the storage period (3 month) are shown in Table 8.
| Table 9: | Sensorial properties of S. maxima processed cheese analogue and control during the storage period at 7°C |
Spreadability: (1: Poor spreadability, 7: Good spreadability), Softness: (1: Very firm, 7: Very soft), Crumbliness: (1: Very crumbly, 7: Not crumbly), Stickiness: (1: Very sticky, 7: Not sticky), Acceptability: (1: Very poor acceptability, 7: Very good acceptability), means with the same letters in the same row are not significantly different |
|
It could be observed that, the free radical scavenging activity (RSA (%)) of S. maxima processed cheese analogue treatments was higher than those of control and this may be due to antioxidant compounds of S. maxima. Spirulina happens to contain several compounds shown to have antioxidant properties, including phenolic compounds, phycocyanins, tocopherols and beta-carotene43. Also, the RSA (%) increased with increasing S. maxima powder level and decreased with progressing of storage period. This result is in agreement with Mohamed and Shalaby44, who reported that the percentage of RSA gradually decreased during cold storage (three months) for all cheese treatments. The highest RSA (57.24%) was recorded for S. maxima processed cheese analogue treatment (3%).
Sensorial characterization of the S. maxima processed cheese: Sensorial properties are important indicator for consumer preferences, so the sensorial properties of S. maxima processed cheese treatments compared to control is shown in Table 9. It could be noticed from the obtained results that the all studied treatments were acceptable all over storage period and gained high scores. The spreadability and softness scores of S. maxima processed cheese treatments were higher than those of control during storage period and these scores decreased with increasing S. maxima levels. This may be due to the replacement of base cheeses (Ras and cheddar) with lactic casein which gives softness for cheese. The spreadability and softness scores of processed cheese analogue with 3% S. maxima powder were lower than those with 1 or 2% Spirulina maxima and this result is in agreement with those obtained by Pereira et al.45, who reported that, the cheeses with lower moisture and higher algae contents (Table 5) were firmer than cheeses with higher moisture content. Crumbliness is the strength of the internal bonds of product body. The crumbliness of processed cheese analogue with 1 or 2% S. maxima powder was higher than those with 3% S. maxima throughout the storage period and this could be due to pH of S. maxima processed cheese analogue (3%) was lower than other treatments. Also, the crumbliness slightly decreased with progressing the storage period. This is mainly due to the decrease in pH values that occurred throughout the storage period which makes the protein more crumbly9. Stickiness is the resist separation of cheese from a material it contacts46. No stickiness was detected for all studied treatments during storage period. The S. maxima processed cheese analogue (3%) had the highest stickiness score throughout the studied storage period. This result is in agreement with those observed by Mohamed et al.9, who mentioned that the cheese analogues with 2 and 3% Chlorella biomass showed very slight stickiness and this may be due to effect of biomass on the cheese protein solubility. From the aforementioned sensorial results, the S. maxima processed cheese analogue (3%) treatment was lower acceptable compared to other treatments.
From the aforementioned results, the Spirulina maxima powder can be added at level 2% during production of spreadable processed cheese analogue because of its sensorial properties and showed acceptable green color and high free radical scavenging.
CONCLUSION
Microalgae are promising organisms for sustainable products for use as feed stocks for food, feed and biofuels. It could be concluded from the results that the addition of S. maxima powder during processed cheese analogue manufacture leads to cheese with high chemical components, free radical scavenging activity, good physical properties and special color (green) compared to control cheese. The processed cheese analogue with S. maxima (1 or 2%) was more acceptable than those of 3%.
SIGNIFICANCE STATEMENT
This study confirmed that the alga of Spirulina maxima can be used during processed cheese analogue production as a source of natural antioxidants, special color and nutrients. The study contributes to produce of Spirulina maxima processed cheese analogue with green color, high nutritional value, antioxidant activity and sensorial scores.