Role of Spirulina platensis as an enhancer of probiotic organisms in whey

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1 Available online at International Journal of Diary Science Research Universal Research Publications. All rights reserved Original Article Role of Spirulina platensis as an enhancer of probiotic organisms in whey Anandi Kavimandan 1 and Sunita sharma* 1 Department of Biotechnology, Madhav Institute of Technology and Science, Gwalior, India ID: sunitasharma75@yahoo.co.in Phone: Received 12 January 2015; accepted 21 January 2015 Abstract Fermented dairy products are rich in beneficial probiotic organisms. A study was carried out to evaluate the effect of dry Spirulina platensis powder in whey inoculated with pure culture of commonly known probiotics, Streptococcus thermophilus NCIM 2412 and Streptococcus cremoris NCIM The sterile whey was inoculated with S. thermophilus and S. cremoris (1% w/v) and supplemented with, and w/v S. platensis powder. The effect was observed by studying ph, titratable acidity and redox potential of whey and viability of both Streptococcus sp. These attributes were studied on 0 th day immediately after inoculation and 1 st, 7 th, 14 th and 21 st day of storage. All results were found significant at P The ph was found between 4-3 at 21 st day. In all outcomes, it was shown that and w/v Spirulina platensis enriched sample gave remarkable increase in studied probiotics upto 7 th day of storage. Therefore S. platensis can be used to enhance probiotics (S. thermophilus and S. cremoris) in whey. The viability of culture at 21 st day of storage was more than7 log cfu/ml; that was satisfactory according to minimum limit of viability of probiotic at time of consumption Universal Research Publications. All rights reserved Keywords: Spirulina platensis, whey, Streptococcus thermophilus, Streptococcus cremoris, Probiotic 1. Introduction Probiotics are the live microbial cells with different beneficiary characteristics. They have been extensively studied and explored commercially in many different products in the world. Their benefits to human and animal health have been proved in hundreds of scientific research. Minimum amount of probiotic bacteria at the time of consumption is cfu/ml in milk [1] but it was also reported that 10 6 to10 8 cfu/ml probiotics culture are sufficient amount at the time of consumption [2]. Spirulina platensis, a cyanobacterium is photoautotrophic microorganism which is widely distributed in nature and has been consumed by human as dietary supplement for decades because of its best known nutritional. The dried biomass nearly contains 3-7% moisture, 55-60% protein, 6-8% lipids, 12-20% carbohydrate, 7-10% ash, 8 to 10% fiber, 1-1.5% chlorophyll a, and a wide range of vitamins [3,4,5].The available proteins in S. platensis have highest economic potential and are water soluble pigments biliproteins (e.g. c-phycocyanin and allophycocyanin). The protein fraction may have a phycocyanin content of up to 20% [4]. Whey is a nutritious byproduct of cheese, chhana and paneer containing valuable nutrients like lactose, proteins, minerals and vitamins etc. which have essential as human food. Whey constitutes 45-50% of total milk solids, 70% of milk sugar (lactose), 20% of milk proteins and 70-90% of minerals and all water soluble vitamins [6]. Considerable work has been done throughout the world to utilize whey for production of whey protein concentrate (WPC), whey powder, lactose, lactic acid, whey paste etc [7].The conversion of whey into beverages through fermentation or without fermentation is one of the most attractive avenues for the utilization of whey for human consumption. In terms of functionality, whey protein enhances protein content of beverage while improving its quality. Apart from the usage of whey in beverage industry, it is also consumed to increase digestive efficiency as it is rich in probiotic organisms. So looking at the nutritional s of whey and essential source of probiotics; it is very useful and cost effective to make a probiotic beverage that has sufficient number of lactic acid bacteria from a waste byproduct of cheese industry e.g. whey. Hence the present study focuses on the effect on the viability of probiotics and other properties of whey. 2. Materials and Methods 2.1 Culture of Spirulina platensis The culture was maintained in Zarrouk s medium [8, 9]. 2.2 Harvesting of biomass and drying After 23 days of incubation, S. platensis culture was taken and biomass was obtained [10]. The collected pellet was then washed with acidified water (ph 4) in order to 1

2 remove/wash the algae from insoluble salts particles [8]. After centrifugation, algal paste (biomass) was air dried in an oven at 60 C for 6 hrs. After the drying was completed; the pellet was made into very fine and uniform particle powder by using mortar and pestle. 2.3 Maintenance of probiotic strains The Streptococcus thermophilus and Streptococcus cremoris strains were procured from National Collection of Industrial Microorganism, Pune; having strain numbers NCIM 2412, and NCIM 2402 respectively. Both cultures were maintained in M17 media and incubated at C for 48 hrs [11]. 2.4 Preparation of whey Double toned milk was purchased from local Sanchi milk parlor and was warmed to 63 ºC for 30 min [12]. Acidification of the milk was done by adding citric acid 2 % (w/v) solution drop by drop until coagulation occurred. Then it was filtered through a muslin cloth to remove coagulated particles. Whey was collected separately and heated at 60 ºC for 30 min [13]. 2.5 Sample preparation of whey After cooling the whey to 40 C, 50ml of whey was poured in sterile falcon tubes. Then Spirulina platensis powder was added in each tube at various concentrations (0.30% w/v, 0.50% w/v and 0.80% w/v) and control was taken without S. platensis. Each concentration was taken in triplicates. 2.6 Inoculation with Streptococci stains The whey containing various concentrations S. platensis was inoculated with Streptococcus thermophilus and Streptococcus cremoris separately. In each tube, S. thermophilus and S. cremoris was added having cell count to cfu/ml contained in 1ml of culture. The prepared samples were kept for 5 hr. incubation at 40 C for fermentation and after that kept at 4 C for 21 days of storage in refrigerator. 2.7 Enumeration of total viable count Streptococci sp. in different samples was enumerated by pour plate technique [14, 15]. 2.8 ph determination The ph was determined on 0 th, 1 st, 7 th, 14 th and 21 st day by combined glass electrode ph meter (Systronic 361) [16]. 2.9 Titratable acidity measurement Titratable acidity (TA) was measured on 0 th, 1 st, 7 th, 14 th and 21 st day [17] Electrode potential determination The Electrode potential (EP) was determined on 0 th, 1 st, 7 th, 14 th and 21 st day by combined glass electrode ph meter (Systronic 361) [16] Check the purity of whey YGC agar was used for enumeration of yeasts and molds. 100 µl of solution was spreaded on agar plates. The plates were incubated aerobically at 25 C for 5 days [15]. Similarly, presences of coliforms were determined by using Mac conkey agar. The inoculated plates were kept 35 C for 48 hrs aerobically. Presence of E. coli. was determined by using VRB agar. The plates were kept at 37 C for 24 hrs aerobically [18] Statistical analysis All concentrations were studied in triplicates and mean and standard error was calculated for each. The remarkable change in the results was calculated by Student s t test (two tailed test) and two way ANOVA at 5% level of significance. 3. Results 3.1. Effect of Spirulina platensis powder on whey inoculated with Streptococcus thermophilus and Streptococcus cremoris respectively Effect on viable counts of S. thermophilus and S. cremoris The experiment was carried out in sterile condition and whey was inoculated with S. thermophilus and S. cremoris separately. The effect was studied to determine the number of viable counts of S. thermophilus and S. cremoris. The observations showing asterisk sign in Table 1.1 reveals the significant change in viable number of S. thermophilus and S. cremoris. Further, the significant change in observations was also proved by two way ANOVA at 5%. From Table 1.2 the F between column and between row was calculated as and 3.82 respectively, which is more than the F tab (3.25 and 3.49 respectively). At various concentrations of S. platensis, there was increase in number of viable counts of S. thermophilus till 7 days of exposure. However, at 14 and 21 days; it was found that the viability of S. thermophilus decreased. Also, the number of viable cells also showed noticeable increase at with the increase in storage period. Hence, 7 days of storage at S. platensis gave maximum number of viable S. thermophilus. Similar observations for the number of viable cells of S. cremoris were noticed with the increase of storage period at various concentrations. The F for S. cremoris between column and between row was calculated as 1177 and respectively, which is more than the F tab (3.25 and 3.49 respectively) (Table 1.2) Effect on ph of whey inoculated with S. themophilus and S. cremoris separately The variation in ph was studied to understand the change in studied viable counts of probiotic organisms at various concentrations on different days of storage. It was observed that at, ph has reduced from day 1 to 21 days in a significant manner as compared to control (Table 1.3). But on 0 th day, the of S. platensis showed maximum ph. This was due to alkaline nature powder. The significant decrease in ph at various concentrations on different storage period was also proved by calculating student s t test and two way ANOVA. The observations with asterisk depict the significant change in ph of whey. Further, the significant change in observations was also proved by two way ANOVA. The F between column and between row was calculated as and 5.91 for S. thermophilus and and 8.77 for S. cremoris respectively (Table 1.4). This is more than the F tab (3.25 and 3.49 respectively). As the concentration increased there was decreased in ph from day 1 to day 21. But in day wise studies it was clear from the Table 1.3 that there was a noticeable change in ph from day 0 to day 1 but after that the drop in ph was gradual at 21 days of storage of minimum ph was observed in both S. 2

3 Table: 1.1. Effect powder on viability of S. thermophilus and S. cremoris in whey S. thermophilus 6.88± ± * 8.94± * 8.6± * 7.82± * S. cremoris 6.87± ± * 8.98± * 8.65± * 7.78± * Mean±S.E. of each sample from day 0-21 is shown. * shows significant at 5% by student s t test (above the of t0.05=2.776 for ν=4) Table: 1.2. ANOVA in viable count of S. thermophilus and S. cremoris at various concentrations and storage period Source of variation Residual Sum of Squares Degree of freedom Viable count (log cfu/ml) of S. thermophilus and S. cremoris Duration of storage Day 0 1 st Day 7 th Day 14 th Day 21 st Day Microorganism Mean sum of squares Sum of Squares Degree of freedom 3 Mean sum of squares Total Table: 1.3. Effect on ph of whey inoculated with S. themophilus and S. cremoris separately S. thermophilus 6.03± ± * 4.25± * 4.09± * 3.86± * S. cremoris 6.04± ± * 4.24± * 4.1± * 3.82± * Mean±S.E. of each sample from day 0-21 is shown. * shows significant at 5% by student s t test (above the of t0.05=2.776 for ν=4) Table: 1.4. ANOVA for ph of whey inoculated with S. thermophilus and S. cremoris at various concentrations of S. platensis and storage period Source of Sum of Degree of Mean sum Sum of Degree of Mean sum variation Squares freedom of squares Squares freedom of squares Residual Total S. thermophilus 6.88± ± ± ± ± S. cremoris 6.87± ± ± ± ± S. thermophilus 6.87± ± ± ± ± S. cremoris 6.88± ± ± ± ± * S. thermophilus 6.88± ± * 8.9± ± * 7.7± S. cremoris 6.88± ± * 8.92± * 8.56± * 7.69± * ph of whey Duration of Storage Day 0 1 st Day 7 th Day 14 th Day 21 st Day Microorganism S. thermophilus 5.94± ± ± ± ± S. cremoris 5.96± ± ± ± ± S. thermophilus 5.97± ± * 4.42± * 4.26± * 4.02± * S. cremoris 6± ± * 4.52± * 4.28± * 4.03± * S. thermophilus 6.± ± * 4.36± * 4.18± * 3.95± * S. cremoris 6.02± ± * 4.36± * 4.2± * 3.93± *

4 Table: 1.5. Effect on Titratable acidity of whey inoculated with S. themophilus and S. cremoris separately TA (%) of whey Duration of storage Day 0 1 st Day 7 th Day 14 th Day 21 st Day Microorganism Mean±S.E. of each sample from day 0-21 is shown. * shows significant at 5% by student s t test (above the of t0.05=2.776 for ν=4) Table: 1.6. ANOVA for TA of whey inoculated with S. thermophilus and S. cremoris at various concentrations of S. platensis and storage period Source of Sum of Degree of Mean sum of Sum of Degree of Mean sum of variation Squares freedom squares Squares freedom squares Residual Total Table: 1.7. Effect on Redox potential of whey inoculated with S. themophilus and S. cremoris separately S. thermophilus 0.16± ± ± ± ± S. cremoris 0.16± ± ± ± ± S. thermophilus 0.15± ± ± * 0.68± ± * S. cremoris 0.14± ± * 0.59± * 0.76± ± * S. thermophilus 0.13± ± * 0.58± * 0.72± * 0.9± * S. cremoris 0.11± ± * 0.63± * 0.8± * 0.94± * S. thermophilus 0.11± ± * 0.62± * 0.78± * 0.92± * S. cremoris 0.09± ± * 0.68± * 0.85± * 0.98± * Redox potential (mv) of whey Duration of storage Day 0 1 st Day 7 th Day 14 th Day 21 st Day Microorganism S. thermophilus ± ± ± ± ± S. cremoris ± ± ± ± ± S. thermophilus ± ± * ± * ± * ± * S. cremoris ± ± * ± * ± * ± * S. thermophilus ± ± * ± * ± * ± * S. cremoris ± ± * ± * ± * ± * S. thermophilus ± ± * ± * ± * ± * S. cremoris ± ± * ± * ± * ± * Mean±S.E. of each sample from day 0-21 is shown. * shows significant at 5% by student s t test (above the of t 0.05=2.776 for ν=4) Table: 1.8. ANOVA for RP of whey inoculated with S. thermophilus and S. cremoris at various concentrations of S. platensis and storage period Source of Sum of Degree of Mean sum Sum of Degree of Mean sum variation Squares freedom of squares Squares freedom of squares Residual Total

5 thermophilus and S. cremoris inoculated whey Effect on Titratable acidity of whey inoculated with S. themophilus and S. cremoris separately Titratable acidity (TA) showed the trends of sharp increase with the increase in concentration and with storage period (Table 1.5). Student s t test and ANOVA has proved the results to be significant at 5%. The asterisk sign showed significant results. The F between column and between row was calculated and for S. thermophilus fermented whey and and 7.38 respectively for S. cremoris fermented whey which is more than F tab (Table 1.6) Effect on Redox potential of whey inoculated with S. thermophilus and S. cremoris separately The Redox potential (RP) showed increased trend on 0 and 7 days of storage with the increase in concentration. However, the pattern of change in RP was varied on 14 th and 21 st days of exposure. The redox potential decreased after 7days of exposure (Table 1.7). The F between column and between row was calculated as and for S. thermophilus fermented whey and and for S. cremoris fermented whey (Table 1.8). 4. Discussion Whey is important product of dairy industry and consumed by human to improve the digestive efficiency due to presence of sufficient amount of proiotics availability. Therefore, the need of increasing the storage period of whey is essential. The enrichment of whey with S. platensis was done and the effect on two microbes acting as probiotics i.e. S. thermophilus and S. cremoris was studied at various concentrations on different days of storage. The observations of S. thermophilus and S. cremoris inoculated whey supplemented with S. platensis showed very little variation at different concentrations and storage period. A study also supports our observations. They studied the influence powder on yoghurt and acidophilus milk stored at 4ºC for 30 days period. 1% S. platensis enriched samples showed low ph compared to control and S. platensis enriched samples at 30 days of storage. There were also large difference in ph between 0 th day and 1 st day. Some sample showed decrease in number of lactic acid bacteria and some gradually decrease from 5 th day. 30 th day findings showed least viable cells [19]. When determined the impact of Arthospira platensis on probiotics of yoghurt. Three concentrations of A. platensis were taken (0.25%, 0.50% & 1%) and a control sample was taken. They evaluate the effect on different parameters e.g. ph, titratable acidity and redox potential and viability of bacteria during fermentation and 28 days of storage at 5 C. The supplementation of A. platensis significantly increases the bacterial counts of L. acidophilus and bifidobacteria at the end of fermentation and during storage period. The A. platensis treated samples showed slower ph decline, faster acidity increase, longer incubation time and greater final acidity than control. The samples 1% enrichment of algal biomass showed better in both probiotic microorganism inoculated samples. It was found that enriched sample were also gave more than 10 7 cfu/ml of bacterial count until the end of storage [20]. Similarly some studied changes in acid production of mesophilic lactic acid bacteria grown in milk. Milk samples enriched with Spirulina at different concentration (0%,, & ) were inoculated at the rate of 1% with the mesophilic LAB strains to be tested. Result of the study showed that Spirulina concentration were effective to increase the acidity of lactococci (Lc. lactis ssp. lactis Ha-2 and Lc. lactis ssp. cremoris W-24) containing milk. The cyanobacterial biomass used to was significantly increase (P<0.05) the acidity development by lactococci between 6 th and 12 th hr. of the fermentation process [21]. Microbes in whey supplemented with S. platensis were grown drastically till 7 days of storage and later less support was provided by S. platensis in the growth of probiotics (S. thermophilus and S. cremoris). The viability of the microbes decreased after 7 th day due to substantial decrease in ph and increase in acidity. The ph below 4.5 did not support the growth of S. thermophilus and S. cremoris both. Therefore no further increase in viable number of cell was noticed. But S. platensis enriched samples showed more viability than control. Infact w/v suplementend samples showed higher number of bacteria. The low ph or high acidity were also inhibit the growth of spoilage other undesirable microorganism. The decrement of viability may be due to the loss of nutrient or oxygen in sample containers. Lack of oxygen is one of the reasons of lower redox potential after 7 th day. But more viable count in S. platensis supplemented samples proved that this dried microalgae work as nutrient for S. thermophilus and S. cremoris. The continuous decrease in ph of whey is due to production of lactic acid by EMP pathway. 5. Conclusion S. platensis can maximally manage the growth of studied probiotics present in whey upto one week at. The growth of S. thermophilus and S. cremoris was start reduced after 1 st week. But the study support the fact that supplementation would be beneficial for probiotic microorganism. With this the intake of such kind of food product would be more advantageous for human as they will get probiotics plus Spirulina platensis dry powder; which nutritive is known by all. Therefore storage life of whey can be prolonged for one week at refrigerated storage by supplementing it with of S. platensis in whey and make it healthy dietary supplement. Acknowledgement This research was done in completion of degree of M. 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