Development and Characterization of Cultured Buttermilk Fortiﬁed with Spirulina plantensis and Its Physico-Chemical and Functional Characteristics

: In recent years, there has been an unprecedented increase in the demand for fermented dairy products due to medical recommendations and lifestyle preferences. Cultured buttermilk, as an ancient fermented dairy beverage, is an appropriate product choice in this context. This study presents a novel cultured buttermilk formulated by fortiﬁcation with high protein microalgae Spirulina platensis , thus making it valuable and attractive because of its antioxidant properties. The fermentation process, nutraceutical properties, and sensory characteristics of developed cultured buttermilk with various concentrations of Spirulina (0.25, 0.5, and 1%) were compared with the control sample (0% Spirulina buttermilk). Different concentrations of Spirulina in buttermilk result in a signiﬁcant increase in chlorophyll and carotenoid content, boosting its antioxidant properties. The study also evaluated the prebiotic properties of Spirulina , thus, demonstrating its ability to promote a healthy digestive system. It was found that the addition of 0.25% Spirulina was able to ferment the product more quickly and retained the sensory acceptability of the ﬁnished product. The protein content, free radical scavenging activity, chlorophyll, carotenoid, and total phenolic content of 0.25% Spirulina -fortiﬁed buttermilk was 1.83%, 48.19%, 30.9 mg/g, 8.24 mg/g, and 4.21 mg/g GAE, respectively. Based on the results obtained, it was concluded that cultured buttermilk with a high nutritional value and functional health beneﬁts can be developed by fortiﬁcation with 0.25% Spirulina as a natural ingredient.


Introduction
The nutritional and health benefits of traditional fermented dairy products drive growing consumer interest because they promote the intestinal bacterial microbiota thus promising healthy living and longer life expectancy [1,2]. Fermented milk products have been an integral part of Indian diets since ancient times. The products are the result of interactive processes that occur while surplus milk is being stored [3]. However, it was Louis Pasteur who first provided an explanation for these interactive processes through "fermentation theory" [4]. Fermented milk products have enhanced bioavailability, shelf life, and safety contributed by the bioactive metabolites produced by lactic acid bacteria (LAB). Fermented milk products possess desired and predictable sensory, rheological, nutritional, and functional changes, which have increased the economic value of fermented milk products.
When fermented milk or cream is churned into butter, buttermilk is produced as a byproduct, which is classified as a functional food, predominantly due to its emulsifying cream [30][31][32], cheese [33,34], protein bars [35], and others. To develop high-quality foods with a greater nutritional content, microalgae have been commercially exploited and used as functional components [36]. However, there have been no reports to date regarding fortification of buttermilk with Spirulina.
With this background, the current study was planned to produce spiced buttermilk fortified with Spirulina platensis to standardize the optimal concentration of Spirulina as a supplement in spiced buttermilk. Further, the effect of Spirulina on the physicochemical properties and antioxidative activity of the final product as well as its shelf life were assessed.

Materials and Methods
Pasteurized and standardized double-toned milk with 1.5% fat and 9.0% SNF was procured from Amul Milk outlet, Varanasi, India and stored at 4 • C until required. A freezedried direct vat-set (DVS) yoghurt culture (NCDC-167) containing a mixed strain of thermophilic and mesophilic homofermentative bacterial culture was obtained from the National Collection for Dairy Culture, Karnal, India. The culture was stored at −18 • C until used. Spirulina was procured from Heilen Biopharm Pvt. Ltd., India and was stored in a refrigerator until used. The nutritional composition, antioxidant activity, and total phenolic content of the procured Spirulina powder was evaluated adopting a standard protocol.

Preparation of Cultured Buttermilk
Preparation of probiotic buttermilk with Lactobacillus reuteri was optimized by Rodas et al. [37]. The same protocol was used for the development of fortified spiced buttermilk with minor modifications and by incorporation of Spirulina powder.
To produce the Spirulina-milk blend, Spirulina biomass (85-90%) was added to toned milk at a 2% (w/w) concentration at the room temperature (27 • C). The preliminary study indicated that at this rate (2% w/w), Spirulina was completely soluble. Direct addition of Spirulina after the production of buttermilk was found undesirable due to the insoluble particles of Spirulina in the sample. Therefore, a constant concentration of the Spirulinamilk blend was produced. Afterward, to ensure optimal mixing of the Spirulina and milk, the mixture was homogenized using a homogenizer at pressures of 100 kg/cm 2 for the first stage and 200 kg/cm 2 for the second stage. To obtain aliquots of 0%, 0.25%, 0.50%, and 1.00% (w/w) Spirulina biomass, this mixture was diluted using toned milk. Spices (0.4% w/w) were added to the control milk (0%) and the Spirulina-milk blend followed by pasteurization at 90 • C for 10 min in a water bath. The pasteurized samples (100 mL each) were put into polystyrene cups that were pre-sterilized and were allowed to cool to 4 • C. These aliquots received an inoculation of 0.5% (v/w) NCDC-167 stock cultures. All of the samples were carefully wrapped in aluminum foil, stored in low density polyethylene (LDPE) bags, and incubated for seven hours at 42 • C in a temperature-controlled incubator. The samples were observed hourly until they attained a pH of 4.6-4.7 and an acidity of 0.8-0.9%. Curd setting was observed after 7 h of complete fermentation. Then, agitation or breaking of curd was performed using a laboratory blender at a speed of 10,000 rpm for 90 s. As soon as the cultured buttermilk samples were prepared, they were stored under refrigeration at 6-8 • C. The formulated cultured buttermilk samples were referred to as; T 0 (Control Buttermilk), T 1 (0.25% Spirulina Buttermilk), T 2 (0.5% Spirulina Buttermilk), and T 3 (1% Spirulina Buttermilk). Post development, these samples were further analyzed adopting standard scientific methodology after refrigeration for 7, 14, 21, and 28 days.

Physico-Chemical Analysis
After calibrating the pH meter with calibrated pH buffer solutions (4.0, 7.0, and 10.0), the pH of the unfortified and Spirulina-fortified cultured buttermilk samples was determined. Titratable Acidity (TA) was determined and calculated as a percentage of the lactic acid that was produced. The total solids of samples were determined using an oven in which the dry samples were kept at 105 • C overnight till a constant weight was attained. Dry samples were ignited in a muffle furnace at 550 • C to burn all the organic components and measure the amount of ash present. The protein level was determined by the Kjeldhal method using a nitrogen conversion factor of 6.38. The Gerber method was used to determine the sample's fat content [38]. For determination of calcium, the method was adopted from Sehgal [39]. The total carbohydrates and energy (Kcal/100 mL) in the sample were calculated using the procedure of Cunniff [40]. All the analyses were performed in triplicate.

DPPH Inhibition Activity
The method described by Kang and Saltveit [41] was used to measure the free radical scavenging activity of samples using the indicator 1,1-diphenyl-2-picrylhydrazil (DPPH). The DPPH radical scavenging activity was evaluated at 517 nm, and the results were presented as percentage of inhibition. All reactions were carried out in triplicates.

Analysis of Total Phenolic Content
The total phenolic content was determined by the Folin-Ciocalteu method [42] with some modifications. A total of 0.5 mL of the sample extract was taken in a test tube and 2.5 mL of diluted Folin-Ciocalteu (FC) Reagent was added followed by 1 mL of 7.5% Na 2 CO 3 . The test tube was covered with aluminum foil and incubated in the dark at room temperature for 1 h. Absorbance was measured at 760 nm in UV-1800 spectrophotometer. Gallic acid (0-800 mg/L) was used to produce standard calibration curve. The total phenolic content was expressed in mg of Gallic acid equivalents (GAE)/100 g. The standard curve with regression equation (y = 0.0769x + 0.0041, R 2 = 0.998) was obtained using the absorbance against the Gallic acid concentration (mg/100 mg).

Analysis of Pigments
The chlorophyll and carotenoid content in the samples was determined as per Kumar et al. [43] and Lichtenthaler and Wellburn [44].
A total of 1 mL of each sample was centrifuged at 5000 rpm for 10 min. The pellet was dissolved in 1 mL of ethanol and then sonicated at 65 • C for 30 min. After sonication, the solutions were centrifuged at 10,000 rpm for 5 min. The pigment content was estimated by measuring the supernatant absorbance (A) at 666, 653, and 470 nm and calculated using the following equations: Total Chlorophylls Determination of C-Phycocyanin content was executed as per Pan-utai and Iamtham [45]. A total of 1 mL of each sample was centrifuged at 5000 rpm for 10 min. The pellet was dissolved in 1 mL of distilled water and then sonicated for 30 min. The supernatant was collected by centrifugation at 10,000 rpm for 5 min and measured for optical density at 615 and 652 nm. C-Phycocyanin content was calculated using the following equation. For purity, a ratio of A 620 /A 280 was used. C − phycocyanin mg mL = (OD 615 − 0.474 × OD 652 )/5.34

Sensory Evaluation
Sensory evaluation of the control and Spirulina-fortified cultured buttermilk samples was carried out using a 9-point hedonic scale of Like extremely (score-9), Like very much (score-8), Like moderately (score-7), Like slightly (score-6), Neither like nor dislike (Score-5), Dislike slightly (score-4), Dislike moderately (score-3), Dislike very much (score-2), Dislike extremely (Score-1). Twenty trained panelists were selected based on their previous experience and knowledge of sensory evaluation of dairy and milk-based products particularly those who were regular consumers of cultured buttermilk. Color and appearance, body and mouthfeel, flavor, and overall acceptability were the sensory parameters evaluated by the sensory panelists. All cultured buttermilk samples were taken out of the refrigerator and the serving temperature for samples ranged from 10 to 12 • C. Each cultured buttermilk sample was presented in a 100 g plastic cup filled with 50 g cultured buttermilk sample and labeled with a 3-digit code. Order of presentation of samples was randomized. Evaluation was divided into 3 sections: visual characteristics (color and appearance), texture (body and mouthfeel), and flavor evaluations. For visual attributes, the surface of each sample was examined in terms of any variation in color or unevenness. The visual analysis of cultured buttermilk was followed by texture and flavor evaluations.

Enumeration of Microorganisms
The samples comprising 1 mL of each were aseptically removed and serially diluted with 0.9% normal saline. For the enumeration of Streptococcus thermophilus and Lactobacillus bulgaricus M-17 agar and De Man, Rogosa and Sharpe (MRS) agar were used, respectively. The plates were incubated for 48 h at 37 • C [46]. Coliforms, yeast, and mold count were performed using the acidified Potato Dextrose Agar (PDA) and Violet Red Bile Agar (VRB), respectively, in accordance with the Standard Procedures for the Analysis of Milk Products [47]. The data were expressed as log CFU/mL. During the storage period, a microbial study was conducted using the spread plate method on the 1st, 3rd, 6th, 9th, and 12th days.

Statistical Analysis
All analytical determinations were performed at least in triplicate and values were expressed as the mean ± standard deviation (SD). One-way ANOVA and Duncan's multiple comparison tests were used to compare results with significant differences (p < 0.05). IBM SPSS statistics software version 19 (IBM Corp., Armonk, NY, USA) was used to perform all statistical analyses.

Composition of Spirulina Powder
The Spirulina powder used in the study was procured from Heilen Biopharm Pvt. Ltd., Gandhinagar, India. The nutritional composition and antioxidant activities of the Spirulina powder is presented in Table 1. The procured Spirulina powder has 5.12% moisture, 59.27% protein, and 7.02% total ash content. The protein content and ash content of Spirulina as reported by earlier researchers ranged from 53 to 65% and 7.9-8.4%, respectively, when expressed on dry matter basis [48][49][50]. Thus, the protein and total ash contents of the procured Spirulina powder in the present study was within the range of reported values. Further, it was reported that Spirulina extract was able to inhibit 42-50% of free radicals, containing Total Phenolic Content (TPC) 4.51-26.64 mg GAE/g samples on dry matter basis [48,51]. The variable % DPPH inhibition (89.00 ± 1.02%) and total phenolic content (11.26 ± 0.04 mg GAE/100 g) in the procured Spirulina microalga may be due to different stage of maturity and harvest and processing such as grinding and drying conditions.  Figure 1 shows the pH and acidity levels of the different treatments measured over the course of the fermentation (4 hours). During the development of cultured buttermilk, caseinate particles entirely precipitate over a pH range of 4.6-4.7 [52]. Therefore, the duration of fermentation in the current study was determined by the time it took for the pH to reach 4.6-4.7. Due to the strong buffering properties of milk and the rapid bacterial growth during fermentation, increasing rates of titratable acidity and decreasing rates of pH were observed across all treatments [22]. The pH of the Spirulina-fortified buttermilk rapidly drops after 2 h, whereas the T 0 takes a substantially longer period of 3 h. For instance, after 1-2 h of fermentation, the T 3 sample showed a pH drop from 6.42 to 4.29 (a 2.27 reduction), whereas the pH of T 0 declined from 6.42 to 6.2. Spirulina platensis was added to buttermilk samples, and as a result, the time it took to achieve pH 4.5 was considerably shortened (p < 0.05). The samples containing 0.25, 0.5, and 1% Spirulina powder did not significantly differ from one another, though. Additionally, these Spirulina-fortified treatments had a much faster increase in acidity (p < 0.05). It was found that there was a moderate agreement between the results of this study and those of earlier workers [22,25,[53][54][55][56]. Spirulinainoculated milk with S. thermophilus and L. bulgaricus decreased pH levels more rapidly than control samples during fermentation, resulting in a shorter incubation period.

Preparation of Cultured Buttermilk
As shown in Figure 2, each product was evaluated for color, flavor, mouthfeel, and overall acceptability. According to the hedonic scale, 82% of the trained sensory panelist indicated a positive response to T 0 and 78% to T 1 . The majority of panelists underrated T 3 with a score of 5 on the hedonic scale. Supplemental Spirulina creates compounds from the oxidation of minerals and lipids that not only function as pro-oxidant molecules but may also produce metallic off-flavors, resulting in an unfavorable flavor [57]. The sample T 1 was not significantly different from T 0 ( Figure 2). When panelists were exposed to buttermilk with a 1% microalgae concentration (T 3 ), it was found to have an unsuitable sensory property (p = 0.001). In addition, insoluble Spirulina particles produced a graininess mostly in T 2 and T 3 , and a significant change in oral texture was observed (p < 0.001). Thus, it showed that the higher concentration of Spirulina decreases the solubility as compared to other samples, therefore higher concentrations were not accepted. The samples of T 1 scored the highest for taste, texture, and mouthfeel control, while treatments containing T 2 and T 3 scored the lowest (p < 0.001). Thus, the highest sensory score and overall acceptability was obtained for T 1 with 0.25% Spirulina-fortified buttermilk. Therefore, the buttermilk supplemented with 0.25% of Spirulina (T 1 ) was selected as the best final product and was evaluated for further analysis.

Physicochemical Analysis
Buttermilk that was enriched with Spirulina (T 1 ) and the control sample (T 0 ) had comparable chemical compositions for most of the attributes. Nonetheless, Spirulina-treated buttermilk was shown to have more total solids (TS), protein, fat, and ash concentrations ( Table 2). Both the control and the Spirulina-fortified buttermilk had total solids of 9.73% and 9.87%, respectively. Between these two samples, there was a significant difference in TS (p < 0.001). The fat content of the control (0.70) and fortified buttermilk sample (0.73) did not differ significantly (p < 0.05). However, the Spirulina-fortified buttermilk had a higher total protein value of 1.83 compared to the control sample (1.71) (p < 0.001). This was most likely caused by the high protein content of Spirulina [58]. The sample treated with Spirulina had the highest calcium concentration (82.05 mg/100 g on dry weight basis) (p < 0.001). This finding is in line with the findings of Ustun-Aytekin et al. [59], who reported that the calcium content of milk appeared to fall noticeably after fermentation, whereas Spirulina-enriched kefir was found to have higher levels of calcium than that of traditional kefir. Due to this high content of calcium, T 1 had the higher ash content. The ash content found in T 1 was 0.76%, which was significantly higher than the ash percentage of T 0 (0.70%) (p < 0.05). When compared to T 0 , the energy value (Kcal/100 mL) reveals a significant difference (p < 0.001), with the highest value for T 1 (40.29). This is due to the presence of the high carbohydrate content present in T 1 (6.59). Changes in these factors, particularly in the amounts of protein, fat, and dietary fiber, may have an impact on other physicochemical characteristics including pH and titratable acidity. As shown in Figure 2, each product was evaluated for color, flavor, mouthfeel, and overall acceptability. According to the hedonic scale, 82% of the trained sensory panelist indicated a positive response to T0 and 78% to T1. The majority of panelists underrated T3 with a score of 5 on the hedonic scale. Supplemental Spirulina creates compounds from the oxidation of minerals and lipids that not only function as pro-oxidant molecules but may also produce metallic off-flavors, resulting in an unfavorable flavor [57]. The sample T1 was not significantly different from T0 ( Figure 2). When panelists were exposed to buttermilk with a 1% microalgae concentration (T3), it was found to have an unsuitable sensory property (p = 0.001). In addition, insoluble Spirulina particles produced a graininess mostly in T2 and T3, and a significant change in oral texture was observed (p < 0.001). Thus, it showed that the higher concentration of Spirulina decreases the solubility as compared to other samples, therefore higher concentrations were not accepted. The samples of T1 scored the highest for taste, texture, and mouthfeel control, while treatments containing The viscosity of a fluid describes its resistance to flow. It contributes directly to the mouthfeel characteristic of cultured buttermilk up to a certain degree. The presence of high viscosity in cultured buttermilk can also cause low phase separation since it reduces the likelihood of whey separation from the network when the viscosity of the buttermilk is high. The binding abilities or stabilizing between the serum and aqueous phase of Spirulina powder may have contributed to the higher viscosity (21.9 cP) values exhibited even in low concentrations of Spirulina powder added into cultured buttermilk. These results are similar with the findings of Mudgil et al. [7].

Physicochemical Analysis
Buttermilk that was enriched with Spirulina (T1) and the control sample (T0) had comparable chemical compositions for most of the attributes. Nonetheless, Spirulina-treated buttermilk was shown to have more total solids (TS), protein, fat, and ash concentrations ( Table 2). Both the control and the Spirulina-fortified buttermilk had total solids of 9.73% and 9.87%, respectively. Between these two samples, there was a significant difference in TS (p < 0.001). The fat content of the control (0.70) and fortified buttermilk sample (0.73) did not differ significantly (p < 0.05). However, the Spirulina-fortified buttermilk had a higher total protein value of 1.83 compared to the control sample (1.71) (p < 0.001). This was most likely caused by the high protein content of Spirulina [58]. The sample treated with Spirulina had the highest calcium concentration (82.05 mg/100 g on dry weight basis) (p < 0.001). This finding is in line with the findings of Ustun-Aytekin et al. [59], who reported that the calcium content of milk appeared to fall noticeably after fermentation, whereas Spirulina-enriched kefir was found to have higher levels of calcium than that of traditional kefir. Due to this high content of calcium, T1 had the higher ash content. The ash content found in T1 was 0.76%, which was significantly higher than the ash percentage of T0 (0.70%) (p < 0.05). When compared to T0, the energy value (Kcal/100 mL) reveals a significant difference (p < 0.001), with the highest value for T1 (40.29). This is due to the presence of the high carbohydrate content present in T1 (6.59). Changes in these factors, particularly in the amounts of protein, fat, and dietary fiber, may have an impact on other physicochemical characteristics including pH and titratable acidity.   Results are expressed as the mean ± standard deviation (n = 3). The values followed by superscript letters ( a and b ) are significantly different between treatment means at a p < 0.05; b p < 0.01. Control (T 0 ) vs. 0.25% Spirulina-fortified buttermilk (T 1 ).
The pH and titratable acidity of T 0 and T 1 samples during the period of 12 days of storage at 4 • C are shown in Figure 2. In the current investigation, the pH of the treated sample dropped (from 4.60 to 4.51) during storage, indicating that lactic acidity (0.22 to 0.35%) developed throughout the storage period. This finding is in agreement with the data of earlier researchers [25,60,61]; they reported that Spirulina-added yoghurt had lower pH values than control samples during the storage period.

Antioxidant Properties
A great deal of recent attention has been paid to the potential role of enriched foods that contain bioactive compounds, generally obtained from microalgae, in the treatment of many human diseases. Microalgae are content with high amounts of free radical scavengers, which explains their high antioxidant activity. Hence, chlorophyll, carotenoid contents, total phenolic content, and DPPH-radical scavenging activity were measured (Table 3) to evaluate its antioxidant capacity. Results are expressed as the mean ± standard deviation (n = 3). Values with superscript letters are significantly different at p < 0.001.

DPPH Radical Scavenging Activity
As shown in Table 3, the experimental samples exhibited high antioxidant properties in terms of DPPH radical scavenging activity. The treatment sample T 1 exhibited significantly higher (48.19% versus 41.99%) (p < 0.001) radical scavenging activity. It has been reported that higher antioxidant activity is observed when a higher ratio of microalga is used [62]. Studies with yogurts fortified with Chlorella vulgaris and Dunaliella sp. have also shown the microalgae-enhanced antioxidant activity of the yogurt. Incorporating Spirulina powders into the diet may increase the levels of chlorophylls [63], carotenoids [64], and phycocyanin [65], resulting in an increase in free radical scavenging.

Chlorophyll Content
Spirulina incorporation significantly improved the chlorophyll content from 0.33 mg/g in T 0 to 30.95 mg/g in T 1 (p < 0.001) besides the enhancement of DPPH inhibition activities (p < 0.05). This result was found to be similar to findings of Barkallah et al. [25]; they investigated the effects of Spirulina-fortified yoghurt and reported that the chlorophyll content of Spirulina-fortified yoghurt was 27.6 mg/g on a dry weight basis as compared to 0.25% in the control yoghurt. According to Ismaiel et al. [63], an increase in free radical scavenging can be attributed to an increase in chlorophyll content. Marzorati et al. described a study of chlorophyll extraction methods using supercritical CO 2 and water [64]. They reported that the total chlorophyll content present in dried Spirulina powder was 9.1 mg/g. In the present investigation, the value obtained was much higher and it is inferred that the yield of chlorophyll content may vary depending on the different extraction processes.

Carotenoid Content
Carotenoids are a class of pigments found in nature. Spirulina contains up to 4000 mg/kg of carotenoids, with β-carotene dominating [65]. Carotenoids contain antioxidant, antiaging, and anti-inflammatory properties, among other things. Carotenoids can quench singlet oxygen and scavenge radicals, resulting in the termination of oxidation chain reactions [66]. Because of their antioxidant characteristics, carotenoids are commonly utilized to treat cardiovascular diseases [67]. Barkallah et al. reported that the carotenoid content in T 1 was 10.86 mg/g on a dry weight basis in their study [25]. Szmejda et al. [68] analyzed antioxidant compounds in ice cream supplemented with Spirulina extract. There was a significant difference (p < 0.001) in the carotenoid level present in the control (T 0 ) and Spirulina extract-supplemented ice cream (T 1 ) which were 0.072 mg/g and 8.24 mg/g, respectively. The study of Szmejda et al. indicated that when compared to non-supplemented flavor variants, the samples enhanced with Spirulina extract have a 3-3.5 times larger magnitude of carotenoids. Marzorati et al. studied the supercritical CO 2 and water extraction methods for carotenoids, and they reported that total carotenoid content present in dried Spirulina powder was equal to 3.5 mg/g [66]. The yield of carotenoid content may also vary depending on the different extraction processes. The carotenoid content present in Spirulina-fortified buttermilk acts as a health-benefiting beverage at considerably low cost. Carotenoid as a color pigment also contributes to the antioxidant compound. However, the pigment quality present in buttermilk decreases upon storage.

C Phycocyanin Content
The C-Phycocyanin level was observed in T 1 with 0.028 mg/g showing a significant difference (p < 0.001). This compound was absent in the control buttermilk. A similar result was observed in the study of Barkallah et al. on the effect of Spirulina-fortified yoghurt. They reported that the C-Phycocyanin content in T 1 was 0.297 mg/g on a dry weight basis [25]. Marzorati et al. reported the yield of C-Phycocyanin using the water extraction method including electrocoagulation, dialysis, and protein salting-out method yielded-250 mg/g of C-Phycocyanin with a very high purity of 2.2 [66]. Khandual et al. described different methods for extracting phycocyanin from Spirulina biomass (dry, wet, and frozen) [69]. They reported a greater phycocyanin content with water as the solvent, 46.65-54.65 mg/g biomass. The phycocyanin concentration of the phosphate buffer at pH 6 was 35.54-37.88 mg/g, while that of the phosphate buffer at pH 7 was 43.13-45.02 mg/g. When we compared different procedures, ultrasonication produced the highest phycocyanin content, 54.65 mg/g with water as the solvent, and the lowest, 35.54 mg/g with phosphate buffer at pH 6. It was observed that ultrasonication retrieved the most phycocyanin, followed by freeze/thaw, and sonication.
Purity of C-Phycocyanin C-Phycocyanin with a purity of 0.7 is regarded as food grade and between 0.7 to 3.9 is called reactive grade, and greater than 3.9 is termed analytical grade [70]. However, the purity of the experimental sample T 1 was 1.32%, which is above 0.7%. This shows that our results were included within the food grade range.

Total Phenolic Content (TPC)
A significant difference was observed between T 0 and T 1 for TPC (p < 0.001). As a result of the addition of Spirulina platensis to the cultured buttermilk, the total phenolic content was greatly increased in T 1 . A similar response was observed when the microalgae was added to gluten-free pasta samples [64]. In addition, Niccolai et al. identified an increase in phenol content as a result of fermentation of Spirulina biomass [71]. Previously, Liu et al. reported that Spirulina platensis is high in phenolic content (19.47 mg/g GAE) and fermentation enhances its effects [72].

Enumeration of Microorganism
The changes observed in the cultures of L. bulgaricus and S. thermophilus throughout the period of 15 days of storage at 4 • C are shown in Figure 3. After the fermentation process, the L. bulgaricus and S. thermophilus counts of the buttermilk samples were analyzed, and the initial counts of the microorganisms were found to be 9.12 log CFU/g and 9.13 log CFU/g, respectively. The results showed that Spirulina platensis had a significant impact on the lactobacilli and streptococci counts of treated samples compared to control sample milk throughout 12 days of cold storage (p < 0.05). Comparable results were reported by Güldas and Irkin [73] and Sudheendra [74]. Similarly, Beheshtipour et al. [22] found that lactic acid bacteria were more likely to survive in the presence of Spirulina platensis. According to Alizadeh Khaledabad et al. [60], probiotic populations of yoghurt samples were boosted by an increase in Spirulina platensis during its shelf-life. Furthermore, Spirulina platensis is known to contain further growth-inducing components, such as adenine, free amino acids, and linoleic acid [75][76][77]. This indicates that Spirulina has some prebiotic efficiencies that could help the growth of the culture. Varga and co-workers [55] were the first to identify this symbiotic relationship between Spirulina platensis and lactobacilli. Additionally, Gupta et al. [77] investigated how blue green algae affected probiotic bacteria. Due to the prebiotic properties of Spirulina, the product in our study may be regarded as a symbiotic product, although further evaluations and studies need to be conducted to determine Spirulina's prebiotic efficiency. None of the samples had any evidence of mold, yeast, or coliform bacteria during storage. Even after 12 days of storage at 4 °C, the lack of these bacteria demonstrated that the buttermilk were secure and uncontaminated. These findings imply that buttermilk processing took place in clean and hygienic conditions.

Conclusions
As an innovative and attractive ingredient, Spirulina powder can be successfully incorporated into spiced buttermilk as a means of enhancing the nutritional and physicochemical properties without significantly altering its sensory acceptability. The study indicated that fortifying buttermilk with 0.25% Spirulina enhanced nutritional and functional properties by enhancing its antioxidant activity. The microbial evidence of this investigation suggest that Spirulina platensis may have the ability to encourage the growth of lactic acid bacteria and can be the effective prebiotic algal source. Thus, it can be used as a high-quality dietary supplement to enhance the nutritive and bioactive properties of food and dairy products. Further research is required to overcome its unpleasant flavor by modern encapsulating techniques and the efficient extraction of bioactive components such carotenoids and C-Phycocyanin using improved extraction techniques.  None of the samples had any evidence of mold, yeast, or coliform bacteria during storage. Even after 12 days of storage at 4 • C, the lack of these bacteria demonstrated that the buttermilk were secure and uncontaminated. These findings imply that buttermilk processing took place in clean and hygienic conditions.

Conclusions
As an innovative and attractive ingredient, Spirulina powder can be successfully incorporated into spiced buttermilk as a means of enhancing the nutritional and physicochemical properties without significantly altering its sensory acceptability. The study indicated that fortifying buttermilk with 0.25% Spirulina enhanced nutritional and functional properties by enhancing its antioxidant activity. The microbial evidence of this investigation suggest that Spirulina platensis may have the ability to encourage the growth of lactic acid bacteria and can be the effective prebiotic algal source. Thus, it can be used as a high-quality dietary supplement to enhance the nutritive and bioactive properties of food and dairy products. Further research is required to overcome its unpleasant flavor by modern encapsulating techniques and the efficient extraction of bioactive components such carotenoids and C-Phycocyanin using improved extraction techniques.