Seaweed has been used in the field of aquaculture as a source of antioxidant, anti-pathogen and immunostimulant. The carrageenan content of seaweed is a potential immunostimulant that can act against bacterial attacks, such as Vibrio alginolyticus on white shrimp culture. This study aims to examine nutrition profile changes in red seaweed Kappaphycusalva-reziiafterfermentation using Saccharomyces cerevisiae.Fermentation process was done under aerobic conditions at 26-28 oC with an agitation of 125 rpm using. The treatments were varied based on inoculum size and comprised of 5% (v/v), 10% (v/v) and 15% (v/v) inoculum, where each treatment was analyzed in triplicates.Crude protein and fat increased in all treatments. The amino acid and fatty acid content rose to 20% and 48%, respectively as a result of biosynthesis by Saccharomyces cerevisiae. FermentedKappaphycusalvareziiisnutritionally more beneficial compared to the unfermented type, supporting its potential as a feed supplement for white shrimp.
Keywords: Kappaphycusalvarezii, fermentation, Saccharomyces cerevisiae, feed supplement.
Seaweed is commonly used in the industry as a source of phycocolloids in the form of alginates, carrageenan and agars which are obtained through an extraction process (Gressleret al., 2010). The extracts can be incorporated as ingredients for food, cosmetics and fertilizers or be further developed into thickening agents and feed additives. Seaweed can also be used as feed supplements for fish and shrimp. Eclonia maxima, Laminaria japonica, Ulvarigida, Carpoblepharisflaccida, Glacilariagracilis and Ulvalactura are examples of species that can potentially be used as a nutritional source for fish and shrimp.
Seaweed contains a complete source of nutrition in varying amounts. The content of seaweed can vary due to differences in species, location and seasonal temperature, condition of harvest, and age of harvest (Machado et al., 2003; Dawczynskiet al., 2007). The fat content can vary from 1 – 6%, while the fiber and protein content range from 33 – 50% and 5.6 – 24%, respectively (Dawczynski, 2007; Gressleret al., 2010). The essential amino acid content of seaweed is considered high(45 – 49%) compared to the total amino acid content (Dawczynskiet al., 2007). In the field of aquaculture, seaweed is known to have antioxidant, anti-pathogen and immunostimulant properties (Thanigaivelet al., 2016). The carrageenan content of seaweed is a potential immunostimulant that can act against bacterial attacks, such as Vibrio alginolyticus on white shrimp (Yehand Chen, 2008).
A commonly cultivated red algae (Rodhophyta) that serves as a source of carrageenan is Kappaphycusalvarezii. Indonesia is a large producer of red algae Kappaphycusalvarezii, previously known as Eucheumacottonii, where an estimated of 11 million tons was produced in 2016. Kappaphycusalvarezii is recognized to exhibit fast vegetative and generative growth, being able to double its biomass within 15-30 days after culturing.
The fermentation of seaweed using yeast can enhance its nutritional value by enriching the protein, vitamin, mineral, essential amino acid and fatty acid content, as well as improve its digestibility value (Uchida, 2003; Uchida and Murata, 2004; Felix and Pradeepa, 2011; Felix and Brindo, 2014). The employment of Saccharomyces cerevisiae is expected to to increase the nutritional value of the seaweed and to act as a potential immunostimulant from the presence of -glucans in the cell walls of the yeast. Thus, the objective of this study is to explore the potential of fermented Kappaphycusalvareziias a feed supplement for white shrimp (Litopenaeusvannamei) based on its nutritional profile.
MATERIALS AND METHODS
Pretreatment of Seaweed
The seaweedKappaphycusalvarezii(Eucheumacottonii) originated from Bali in dried conditions and without pretreatment. The washing step was done to remove salt and dirt that remained on the surface of the seaweed. A sorting process was also done during the washing step. The drying step was done using a convection system oven at a temperature below 90 °C to prevent protein denaturation. The milling step was performed using a disc mill with a filter size of 5 mm, followed by a sieving step using a 250 µm metal sieve. The yield of the overall process was 25% of the total initial amount of dried seaweed used.
A loop of Saccharomyces cerevisiaeculture was streaked on to PDA (Potato Dextrose Agar) medium and incubated at room temperature 28 – 30 °C for 24 – 48 h. Strain activation was later done by subculturing 1-2 loops of culture into 100 ml of activation medium, followed by incubation at room temperature 28 – 30 °C for 24 h with an agitation of 125 rpm. The activation step was then repeated using 10% (v/v) of inoculum from the previous culture. Following activation, the cells of Saccharomyces cerevisiae were counted until it reached a density of 106 cell/ml.
Subsequently, activated cultures of Saccharomyces cerevisiae were adapted onto the substrate that will be used during fermentation. The adaptation procedure was done three times using the following medium composition; (1) 75 % microbial growth medium + 25 % fermentation medium, (2) 50 % microbial growth medium + 50 % fermentation medium, and (3) 25 % microbial growth medium + 75 % fermentation medium.
The fermentation process was done by incorporating the different inoculums into a medium containing 20 g of seaweed flour, 1.5 g of maize flour, 1 g of glucose and 0.4 g of urea into an Erlenmeyer flask of 500 ml containing 200 ml of deionized water. The different treatment conditions comprised of 5 % (v/v) inoculum denoted as treatment I, 10 % (v/v) inoculum denoted as treatment II and 15 % (v/v) inoculum denoted as treatment III. The incubation was done at room temperature 28 – 30 °C with an agitation of 125 rpm for 72 hours. Parameters tested during fermentation are pH and microbial count (TPC and direct counting) which was done every six hours.
A drying step using an electrical conventional oven at 60 – 75 °C for 24 – 48 was performed after the fermentation process had finished. The temperature was important since too high of a temperature can affect the nutritional content and denaturate the proteins. Once the seaweed has dried (water content ≤ 5%), it was milled through a disc mill using a filter size of 5 mm. Milled seaweed was then further sieved using sieve No. 60 (size 250 µm).
The nutritional content of the seaweed was analyzed using the following methods: proximate analysis consisting of dry matter content (oven method), crude protein content (Kjhedal titration), crude fat content (Soxhlet method) (Machadoet al., 2004), energy (Bomb calorimetry), extract matter without nitrogen (calculated), fatty acid analysis (gas chromatography), crude fiber content as well as amino acid analysis (HPLC) (Ortizet al., 2006).
RESULT AND DISCUSSION
Proximate Analysis of Seaweed Without Fermentation
Proximate analysis was done to examine nutrition value of pretreated seaweed Kappaphycusalvarezii. The result shows similar values asother studies byIstianiet al.(1986) and Liem (2013) (Table 1). Ash, fat, protein and crude fiber content of the seaweed was observed to be lower compared to other red seaweed species reported by Gressler et al. (2010), which are Gracilariadomingensis, Gracilariabirdiae, Laurenciafiliformisand Laurencia intricate. This condition may occur as a result of differences in species, geographical condition and season (Kaehlerand Kennish, 1996). However, these values are still in accordance with the acceptable ranges published by Dawczynskiet al. (2007) and Gressler et al. (2008).
Table 1. Proximate Analysis of the red algae Kappaphycusalvarezii
|Measured||Reported by Istianiet al.(1986)||Reported by Liem (2013)|
|Total Energy (kal)||2883.8||–||–|
|Dry matter (%)||91.1||86.1||90.05|
|Water content (%)||8.9||13.9||9.95|
|Crude fat (%)||0.22||0.37||0.53|
|Crude protein (%)||3.26||2.69||3.82|
|Non protein nitrogen (%)||0.56||–||–|
|Extract matter without N (%)||64.34||65||73.81|
|Crude Fiber (%)||4.6||0.95||4.15|
Amino Acid& Fatty Acid Analysisof Seaweed Without Fermentation
The amino acid content of the measured seaweed was found to be comprehensive, although it still has lower values compared to other species (Table 2). Based on the analysis, the most abundant amino acid was found to be aspartate and glutamate at 0.29% and 0.35%, respectively. The two amino acids were also reported to be highest in the comparing species.
Table 2. The amino acid content of red algae
|Amino acid (%)||Kappaphycusalvarezii||Species reported by Gressler et al. (2010)|
The amino acid content is linearly dependent on the protein content of the seaweed where a low protein content would translate to a low amino acid content. Thus, similar to the factors affecting protein, the amino acid content is also affected by the difference in species, environment and age of harvest.
The fatty acid content of the measured seaweed was found to be lower compared to other species. The highest amount of fatty acid observed was palmitate (C16:0) at 0.15%. However, this value was considerably lower than other species reported by Gressleret al.(2010). Factors affecting fatty acid content can include type of seaweed, temperature of environment, characteristic of seaweed, intensity of light, mineral content, nitrogen content and period of life cycle the seaweed is in (Takagiet al., 1985; Dawczynskiet al., 2007).
Growth Curve of Saccharomyces cerevisiae
Growth curve of Saccharomyces cerevisiae on PDB (Potato Dextrose Broth) can be divided into two phases, a logarithmic phase between 0 – 28 hours and a stationary phase between 28 – 48 hours (Figure 1). The absence of a lag phase indicate that the culture was in an active state and had been well-adapted to the PDB medium.
The growth of Saccharomyces cerevisiae used in this study was relatively fast. A significant change in cell density was observed between t0 andt4 where it rose from 2.1 x 106 cells/ml to 1.07 x 107 cells/ml. The significant increase of cell density indicate that the growth is at the logarithmic phase where the yeast is actively undergoing cell division. The highest growth rate on PDB medium was observed at t2 where µ was equivalent to 0.39 h-1.
The pH value of the medium changed during the growth of S. cerevisiae where it dropped from an initial value of 4.52 to 3.82 (Figure 1). The decrease of pH suggests that there is a production of acid from the metabolism of carbohydrate in the seaweed sample. The decrease in pH corresponded well to the increase of cell mass. This decrease of pH was observed until t12, while at t14the pH increased signifying the end of growth. During cell lysis, ammonia can be released into the medium causing the pH to rise (Boulton and Quain, 2001).
Fig. 1. Growth curve of Saccharomyces cerevisiae on PDB medium at 26 – 28 °C and agitation of 125 rpm
The growth curve of Saccharomyces cerevisiae on fermentation medium (Figure 2) has similar trend with its growth curve on PDB medium, as it displayed a logarithmic phase followed by a stationary phase. The highest growth rate observed for each treatments;10% (v/v), 15% (v/v) and 5% (v/v); were 0.38 ± 0.05h-1, 0.37 ± 0.06 h-1 and 0.23 ± 0.03 h-1, respectively.
All the fermentation medium contained the same nutritional content but a different initial cell density. Therefore, the ratio of nutrition compared to the initial cell density ofS. cerevisiaeis different. The treatment with 5% (v/v) inoculum has the highest availability ratio of nutrient over cells, as compared to 10% (v/v) and 15% (v/v) inoculum, respectively.
Fig. 2. Growth curve of Saccharomyces cerevisiae on fermentation medium at 26 – 28 oC and agitation of 125 rpm
With time, the available nutrition will become limited where the cells will then lose energy and the ability to grow, resulting in autolysis. Environmental factors can also affect growth, for example the increasing concentration of alcohol that can inhibit enzymes and cause cell death (Boulton and Quain, 2001). The autolysis that occurred during cell death is due to the work of hydrolytic enzymessuch as proteinases and glucanases that are present in the intracellular matrix.
The fermentation medium consists of several different carbohydrate sources; carrageenan and other polysaccharides from Kappaphycusalvarezii, glucose which was added to the medium, and starch from maize flour. During the cultivation of S. cerevisiaein the fermentation medium, glucose will be the first resources to be utilized as it is the simplest form of carbon source to assimilate. Saccharomyces cerevisiae will also prefer glucose since it will suppress the use of the other available carbon sources (Boulton and Quain, 2001). The yeast can utilize dextrose, galactose, sucrose, maltose, raffinose, trehalose but not lactose as their carbon source.
The pH of all the treatments during fermentation decreased from an average of 6.48 to 4.6. The pH of the medium will greatly affect the growth of the yeast, where the optimum range is 4 – 5. A pH value of 3 or lower will inhibit the growth of yeast and the fermentation will run at a much slower pace (Adamet al., 1985). Saccharomyces cerevisiae produces organic acids from non-acidic compounds such as carbohydrates. These acids can take the form of pyruvic acid, citric acid and succinic acid that are formed by the metabolism of sugars in the medium. These organic acids can accumulate and decrease the pH of the medium. A drop of pH may also caused by the use of nitrogen where the yeast will use a cation resulting in a free nitrate ion that will react with water to form nitrate acid. As the growth enters the death phase, the pH of the medium will become more basic from the release of ammonia during autolysis.
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