The main purpose of this research was to monitor the effect of dry Spirulina platensis powder in whey inoculated with pure culture of Streptococcus lactis NCIM 2080 and Streptococcus faecalis NCIM 2093, respectively during their 21 days storage at 4°C. The whey samples were supplemented with 0.3, 0.5 and 0.8% w/v S. platensis powder and without S. platensis powder (control). All samples were inoculated with 1% v/v single strain of streptococci. All samples were inoculated with same viable counts. Whey samples were filled in 50 mL tightly capped sterilized falcon tubes. Five hours of incubation were given to all samples each in three replicates at 40°C. The effect was studied on following parameters: pH, titratable acidity and redox potential of whey and viability of streptococci in current sample. These attributes were checked on 0th day (before incubation), 1st, 7th, 14th and 21st day of storage. The most significant results were obtained at 0.8% S. platensis supplemented samples. At the day 7 both streptococci gave the highest viable counts throughout the 21 days of storage. The lowest pH and highest titratable acidity was observed in S. lactis at 21st day of storage.
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The dried biomass of S. platensis contains 3-7% moisture, 55-60% protein, 6-8% lipids, 12-20% carbohydrate, 7-10% ash, 8-10% fiber, 1-1.5% chlorophyll a and a wide range of vitamins (Belay, 1997; Cohen, 1997; Vonshak, 1997). Spirulina have not cellulose in its cell wall, a trait that makes it a suitable and vital foodstuff for patients who have poor intestinal amalgamation and for old patients (Richmond, 1984).
In India, it is predictable that about 100 million kg of whey is annually derived as a byproduct which may cause substantial loss of about 70,000 t of nutritious whey solids (Parekh, 2006), containing valuable nutrients like lactose, proteins, minerals and vitamins etc., which have essential value as human food. Whey constitutes 45-50% of total milk solids, 70% of milk sugar (lactose), 20% of milk proteins and 70-90% of milk minerals and most important all the water soluble vitamins are present in milk (Horton, 1995). 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. (Panesar et al., 2007).
A probiotic is commonly defined as a live microbial supplement that has an effect on the host by developing its intestinal microbial balance (Fuller, 1989). There are many health benefit for regular intake of viable probiotics which includes lactose tolerance (Kim and Gilliland, 1983), antimicrobial (Yildirim and Johnson, 1998), anticarcinogenic (El-Gawada et al., 2004), hypercholesterolemic (Kikuchi-Hayakawa et al., 2000; El-Gawada et al., 2005) and antimutagenic (Hsieh and Chou, 2006) and various others profitable effects.
Probiotic has a good and healthy source of diet for human from centuries. On the other side, algae are emerging as dietary supplements. So, looking at the nutritional values of whey, S. platensis powder and probiotics; it is very useful and cost effective to make a probiotic beverage that has sufficient number of lactic acid bacteria from a waste by product e.g., whey.
Schleifer and Kilpper-Balz (1987) applying classification and serological techniques demonstrated three separate, independent and different genera: Streptococcus, Lactococcus and Enterococcus from old streptococci genus (Schleifer and Kilpper-Balz, 1987) lactococci (formerly group N streptococci) are the major mesophilic microorganisms used for acid production in dairy fermentations. Although five species are recognized, only one, L. lactis, is of significance in dairy fermentations (Law and Haandrikman, 1997).
The genus Enterococcus includes the Lancefield group D (fecal) streptococci. Now Streptococcus faecalis and S. faecium are known as E. faecalis and E. faecium (Stiles and Holzapfel, 1997; Klein et al., 1998). Enterococci are used as food safety indicators and have a possible involvement in foodborne illness. Enterococci are also used as starter cultures in some southern European cheeses. In addition, they are commercially available as probiotics for prevention and treatment of intestinal disorders. Among enterococci only E. faecalis and E. faecium are important as probiotics. They are readily differentiated by fermentation of arabinose and sorbitol and by their growth temperatures (Klein et al., 1998).
Enterococci are applied in fermentation process of fermented food production because their contribution to ripening and aroma development, probiotic properties and the production of antimicrobial substances (Giraffa, 2002; Klein, 2003; Moreno et al., 2006). Many E. facalis and E. faecium strains isolated from dairy products were shown to be good producer of acetaldehyde, ethanol, diacetyl and acetone when grown in milk, thus further contributing in the development of aroma and flavour of cheese (Andrighetto et al., 2001; Sarantinopoulos et al., 2001).
Because of their occurrence in gastrointestinal tract, enterococci are often used as indicator of faecal contamination through food production chain (Giraffa, 2002; Moreno et al., 2006). However, enterococci are now also considered as normal parts of the food microflora and not only as indicator for poor hygiene (Klein, 2003). The differentiation of apparently safe and non-safe enterococci strains is not simple, especially because virulence genes can be easily exchanged between strains (Messi et al., 2006; McGowan et al., 2006).
For applications of enterococci in foods, antibiotic resistance is of special concern because genetic determinants of resistance in these bacteria are generally located in conjugative plasmids or transposons (Hasman et al., 2005; Zanella et al., 2006). Antibiotic multi-resistance has been more commonly reported for E. faecalis due to its notorious ability to acquire and transfer antibiotic resistance genes (Citak et al., 2004; McBride et al., 2007). So it is a dualistic in nature.
MATERIALS AND METHODS
Spirulina platensis biomass: The culture was obtained from Madhav Institute of Technology and Science and grown in Zarrouks medium and maintained (Zarrouk, 1966; Vonshak, 2002).
Harvesting of biomass and drying: Twenty three days old S. platensis culture was taken and biomass was obtained (Arun et al., 2012). The collected pellet then washed with acidified water (pH 4) in order to remove insoluble salts particles. Algal pastes (biomass) were air dried in an oven at 60°C for 6 h for making dried powder (Vonshak, 2002).
Procurement and maintenance of streptococci strains: The Streptococcus lactis and Streptococcus faecalis strain were procured from National Collection of Industrial Microorganism, Pune; having strain numbers NCIM 2080 and NCIM 2093, respectively. Both cultures were maintained in M17 media incubated at 37-40°C for 48 h (Terzaghi and Sandine, 1975).
Preparation of whey: Double toned milk was warmed to 63°C for 30 min (USPHA., 1995). 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°C for 30 min (Shafiee et al., 2010).
Sample preparation: After the cooling of whey to 40°C; sterile falcon tubes were taken and whey was poured in it. Each tube was containing 50 mL of whey sample. Then S. platensis powder was added in each tube according to 0.30% w/v, 0.50% w/v and 0.80% w/v and control without S. platensis. Each concentration has been taken in three replicates.
Inoculation with streptococci stains: Streptococci culture were taken; having cell count 1.2x108 to 1.8x108 CFU mL-1 and all whey samples were inoculated with 1% v/v. Each streptococci strain was taken at a time.
Incubation and storage of samples: After preparing the samples kept it for 5 h incubation at 40°C for fermentation after that kept these at 4°C for 21 days of storage in a refrigerator. All analysis of samples was done at 0th day (before fermentation), 1st, 7th, 14th and 21st day of storage.
pH determination: The pH was determined by combined glass electrode pH meter (Systronic 361) (Shafiee et al., 2010).
Electrode potential determination: The Electrode Potential (EP) was determined by combined glass electrode pH meter (Systronic 361) (Shafiee et al., 2010).
Titratable acidity measurement: Titratable Acidity (TA) was measured by the method described by Dave and Shah (1997).
Enumeration of total viable count: Streptococci present in samples have been enumerated by pour plate technique (Harrigan, 1998; Varga et al., 2002).
Detection of contamination: The chance of any kind of contamination was checked in 1st week of storage. Presence of yeast and mold was check by spread plate technique using YGC agar (Varga et al., 2002). Presence of coliforms and E. coli were checked by using MacConkey and VRB agar, respectively (APHA., 1992).
Statistical analysis: All data were interpreted in Mean±SE. The significant change in the results was calculated by Students t test (two tailed test) and two way ANOVA at 5% level of significance.
RESULTS AND DISCUSSION
Effect of Spirulina platensis powder on whey inoculated with Streptococcus lactis and Streptococcus faecalis, respectively: The pure culture of S. lactis and S. faecalis were inoculated in to whey separately at 8.89 log CFU mL-1 in 1% v/v ratio. There was no yeast, mold, E. coli and coliform contamination found in both S. lactis and S. faecalis fermented whey samples.
Effect of S. platensis on viable counts of S. lactis and S. faecalis: When the effect of S. platensis powder was studied on viability of S. lactis and S. faecalis then it was clear that all samples were inoculated with same bacterial counts. On the 0th day viability in all samples were approximately 6.89 log CFU mL-1 shown. On the 1st day observation it was clear that the viable counts were increased so rapidly. Control was having least viability and other treatments were increases in viable numbers as the concentration increases. From the Table 1 and 2 the highest viability was observed on 7th day observations then viability start to decreases and after 14th day there was major downfall occurred. The decrement in viability was due to the lack of nutrient in whey samples or may be lack of oxygen in tubes or deposition of toxic waste from bacteria. Low pH may be a cause of less viability because lowest pH was observed on 21st day of storage among all four streptococci inoculated samples (Kavimandan and Sharma, 2015). Streptococcus thermophilus was lowest in viability at 7th day observation among all streptococci and highest in viable number at 21st day of storage with equal to S. faecalis. Most significant results were obtained in 0.8% w/v enriched samples.
Effect on pH of whey inoculated with S. lactis and S. faecalis, respectively: As shown in Table 3 and 4 at the time of inoculation (before incubation) S. platensis powder supplemented samples were shown high pH value as compare to control.
|Table 1:||Effect of S. platensis powder on viable counts of Streptococcus lactis in whey|
|Values are in Mean±SE where, n = 3 significant at p≤0.05 by two way ANOVA. The calculated F1 (between column) = 876.27 and F2 (between row) = 13.82 (F1tab = 3.25 and F2tab = 3.49). *Significant results at 5% level by students t test (for t0.05 = 2.776 for v = 4)|
|Table 2:||Effect of Spirulina platensis powder on viable counts of Streptococcus faecalis in whey|
|Values are in Mean±SE where, n = 3 significant at p≤0.05 by two way ANOVA. The calculated F1 (between column) = 860.16 and F2 (between row) = 13.63 (F1tab = 3.25 and F2tab = 3.49). *Significant results at 5% level by students t test (for t0.05 = 2.776 for v = 4)|
|Table 3:||Effect of S. platensis powder on pH of whey inoculated with S. lactis|
|Values are in Mean±SE where, n = 3 significant at p≤0.05 by two way ANOVA. The calculated F1 (between column) = 247.88 and F2 (between row) = 8.96 (F1tab = 3.25 and F2tab = 3.49). *Significant results at 5% level by students t test (for t0.05 = 2.776 for v = 4)|
|Table 4:||Effect of S. platensis powder on pH of whey inoculated with S. faecalis|
|Values are in Mean±SE where, n = 3 significant at p≤0.05 by two way ANOVA. The calculated F1 (between column) = 272.36 and F2 (between row) = 8.67 (F1tab = 3.25 and F2tab = 3.49). *Significant results at 5% level by students t test (for t0.05 = 2.776 for v = 4)|
These findings indicate that the S. platensis powder is alkaline in nature. From both the tables it was concluded that as the concentration of S. platensis powder increases in the whey; samples shows higher value of pH. After the 5 h of incubation the pH falls down drastically as the observation were taken on 1st day. When the observation were taken on 7th, 14th and 21st day of storage period all results shown that the pH value was stable compared to their previous observations. The falling in the pH was very slow but there was a significant difference between the value of control and other S. platensis added samples (at 5% level of significance). From the Table 3 and 4 it was clear as the concentration increased the pH value decreased and control was having highest pH value. Most significant change was occurred at 0.8% w/v enriched whey samples. When the data of pH was compared between S. lactis and S. faecalis then no major difference were found in them. But S. lactis show high acidic nature. When the results were compared with previously published results of study by Kavimandan and Sharma (2015), then no large difference was found in pH among S. thermophilus, S. cremoris, S. lactis and S. faecalis inoculated whey samples.
Effect on titratable acidity of whey fermented with S. lactis and S. faecalis, respectively: From the Table 5 and 6 it was depicted that at 0th day observation the TA of control was having highest percentage showing higher acidity in comparison to other three S. platensis treated samples for both S. lactis and S. faecalis fermented samples, respectively. On the next day (1st day), observations control was having less TA with compare to other three concentration samples in both streptococci inoculated whey. On other days observation e.g., 7th, 14th and 21st day it was clear that control was having least TA as compared to other three treatments. As the concentration increased the TA showed higher percentage and on 21st day observation it showed highest acidity. The most significant value was observed at 0.8% w/v enriched samples. In comparison to both streptococci the S. lactis show high acidic nature.
|Table 5:||Effect of S. platensis powder on titratable acidity of whey inoculated with S. lactis|
|Values are in Mean±SE where, n = 3 significant at p≤0.05 by two way ANOVA. The calculated F1 (between column) = 186.40 and F2 (between row) = 5.23 (F1tab = 3.25 and F2tab = 3.49). *Significant results at 5% level by students t test (for t0.05 = 2.776 for v = 4)|
|Table 6:||Effect of S. platensis powder on titratable acidity of whey inoculated with S. faecalis|
|Values are in Mean±SE where n = 3 significant at p≤0.05 by two way ANOVA. The calculated F1 (between column) = 196.46 and F2 (between row) = 5.37 (F1tab = 3.25 and F2tab = 3.49). *Significant results at 5% level by students t test (for t0.05 = 2.776 for v = 4)|
|Table 7:||Effect of S. platensis powder on redox potential of whey inoculated with S. lactis|
|Values are in Mean±SE where, n = 3 significant at p≤0.05 by two way ANOVA. The calculated F1 (between column) = 1384.27 and F2 (between row) = 137.73 (F1tab = 3.25 and F2tab = 3.49). *Significant results at 5% level by students t test (for t0.05 = 2.776 for v = 4)|
When the data compared with the previously performed research of kavimandan and Sharma similar of results occurred in all studied streptococci (Kavimandan and Sharma, 2015). High acidic value shows there was very less chance of contamination or presence of spoilage microorganism in whey.
Effect on redox potential in S. lactis and S. faecalis fermented whey samples: As shown in the Table 7 and 8 all samples were showed high RP value on 0th day observation. It was clear that as the concentration increases the sample shown high RP value compared to control. It was happened due to the support of S. platensis to oxidation capacity of S. lactis. As the viability of S. lactis increased the RP value of samples also increases. Higher the concentration of S. platensis higher the RP value due to the oxidant present in S. platensis powder. It was depicted from tables that the RP value decreases on 14th and 21st day of observations. This happened due to the lack of oxygen in sample containers. The highest RP values were observed on the 7th day observation. The highest viability was also found in 7th day observation and as the viability increases (in increment of concentration) RP value also rose. The RP value started to decreases after 7th day.
|Table 8:||Effect of Spirulina platensis powder on redox potential of whey S. faecalis|
|Values are in Mean±SE where n = 3 significant at p≤0.05 by two way ANOVA. The calculated F1 (between column) = 2447.80 and F2 (between row) = 3.25 (F1tab = 3.25 and F2tab = 3.49). *Significant results at 5% level by students t test (for t0.05 = 2.776 for v = 4)|
Streptococcus faecalis was having very low RP compared to S. lactis may be because of S. faecalis is a facultative anaerobe. It was also concluded that S. faecalis was having reducing capacity and S. platensis powder support the same due to the presence of some reducing agents in it. When the data was compared from the previously published study by Kavimandan and Sharma (2015) then it was clear that S. thermophilus is very oxidative in nature and S. faecalis is reducing in nature. At the day of inoculation there was a large difference among the RP of all the streptococci enriched whey samples.
This is concluded that S. platensis enhances the growth of streptococci species. As the concentration increases; difference between the results of control and concentration increases. There was no large difference between the pH and TA of all species observed after 1st day. The acidity of sample increase slowly. With that effect there were not any spoilage microorganism found. So, the role of S. platensis powder as prebiotics was justified. Most significant results were found at 0.8% w/v enriched samples. The study also supports the role of S. platensis powder as enhancer of storage life of whey for prolong period (more than seven days). High acidity was one of the factors to deplete the growth of probiotic microorganism in whey after 1st week of storage. The 21st day viability also proved that there was sufficient number of microorganisms present in whey samples which are up to the minimum limit of probiotic at time of consumption. When compared to current study with previously performed study the same results were concluded (Kavimandan and Sharma, 2015). With that present study also shows some more opportunities for beverages and other products made from combination of probiotic microorganism and microalgae.
This study was done in completion of degree of M. Tech. by author from Madhav Institute of Technology and Science, Gwalior, India. The author is grateful toward Institute for the fulfillment of facilities provided during research and the author was also pay her gratitude to National collection of Industrial Microorganism (NCIM), Pune for providing Streptococcus cultures.
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