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Characterization of refined carrageenan from Kappaphycus alvarezii extracted using marine fungi
To cite this article: S Sulistiawati et al 2020 IOP Conf. Ser.: Earth Environ. Sci. 404 012071
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Characterization of refined carrageenan from Kappaphycus alvarezii extracted using marine fungi
S Sulistiawati1, N H Ain1, Uju1, K Tarman1,2*
1 Department of Aquatic Product Technology, Bogor Agricultural University (IPB University), Bogor, Indonesia
2 Center of Coastal and Marine Resources Studies, Bogor Agricultural University (IPB University), Bogor, Indonesia
*E-mail: [email protected]
Abstract. Carrageenan is a polysaccharide that widely used in food, pharmaceutical, cosmetic, textile and printing industries as a coagulate agent, stabilizer, and gelling agent. The extraction of carrageenan usually is carried out by alkali treatment, but it may have toxic effect for the environment. Therefore, another extraction method is required to reduce the toxic effect from carrageenan. Carrageenan can also be extracted by using a cellulolytic enzyme. A natural cellulolytic enzyme can be produced from endophytic fungi. The objective of this research was to extract carrageenan using endophytic fungal cellulase and to evaluate the effect of fungal cellulase concentration and extraction time on the quality of carrageenan. The materials used in this research were marine endophytic fungus (EN) and dried seaweed Kappaphycus alvarezii.
This research was conducted in three steps. The first step was cultivating of endophytic fungus (EN) in cellulose basal medium. The second step was cellulase extraction from endophytic fungus EN. The last step was extraction of carrageenan by cellulase endophytic fungus. The highest yield was obtained by added 1% of EN cellulase enzyme and extraction period for 3 hours. Characteristics of carrageenan in generally met the standard, but the gel strength and whiteness was still below the standard.
Keywords: algae, cellulose, endophyte, marine fungi, natural
1. Introduction
Carrageenan is a hydrocolloid consisting of the ammonium, calcium, magnesium, potassium, and sodium sulfate ester from galactose and polysaccharide 3.6-anhidrogalaktose (FAO 2007). In the food industry, carrageenan has been widely used because has the functional ability as thickening, gelling, and stabilizing agent, improving cheese texture, controlling the viscosity and texture of pudding, also as a filler and stabilizer in meat processing (Campo et al 2009). Carrageenan is classified into several types such as λ, κ, ι, ε, and μ carrageenan which all contain sulfate 22-35 percent (Necas 2013). Kappa carrageenan (κ-carrageenan) is the most used type of carrageenan in various industrial fields. Kappa
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Cellulolytic enzymes produced by various types of microorganisms, such as fungi. There have been many explorations of cellulolytic enzymes produced by fungi. Aspergillus sp. and Trichoderma sp. are known have ability to produce cellulolytic enzymes with high activity (Sivaramanan 2014, Reddy et al 2014). Carrageenan extraction can be done by utilizing fungi as a cellulolytic enzyme producer. The ability of fungi to produce cellulolytic enzymes is very important in the extraction process, but it is also necessary to use cellulolytic fungi that safe to use. Varadarajan et al (2009) reported that enzymatic carrageenan extraction using Aspergillus niger produced more carrageenan yield than the conventional extraction. Muthezilan et al (2014) has also performed carrageenan extraction using pure enzyme produced from coastal plant endophytic shoots and obtained a higher yield until 52%.
The endophytic fungus strain (EN) isolated from seagrass has been known to produce extracellular cellulase enzyme (Oktavia et al 2014). In previous research, EN fungus produced cellulase with high cellulolytic activity. Irma (2018) has reported that EN fungus could be used in carrageenan extraction from Kappaphycus alvarezii by utilizing EN fungal culture directly. Based on information about EN capability from previous research, further research is needed to know the ability of cellulase enzyme isolated from EN fungus in the carrageenan extraction process. The aims of this research were to obtain carrageenan extract from Kappaphycus alvarezii using EN fungal cellulase and to determine the effect of extraction period, EN cellulase enzyme concentration as well as the interaction between extraction period and concentration of EN cellulase enzyme on carrageenan extract yield.
2. Materials and methods
2.1. Cultivation of endophytic fungus producer of cellulolytic enzyme
The cultivation was carried out by inoculation 10% of EN fungus on Basal Medium without carbon source (NaNO3 2.0 g/L; KH2PO4 1.0 g/L; MgSO4.7H2O 0.5 g/L; FeSO4 10.0 mg/L). EN endophytic fungus was cultivated on 500 mL Erlenmeyer flask with 200 mL of media and added with carbon source of dried Kappaphycus alvarezii seaweed (1%). The culture was incubated for seven days at room temperature and shaked with 120 rpm. Furthermore, for cellulase enzyme production, inoculation of 10% EN culture in 500 mL of the same production media (basal medium and seaweed) was done. The culture was incubated at room temperature and shaked with 120 rpm for 9 days (Oktavia et al 2014).
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2.2. Cellulase enzyme extraction from en fungus
The fungus culture was filtered using filter paper. The filtrate was centrifuged at a rate of 3,000 rpm for 20 min. Centrifugation has done to obtained crude cellulase extract. The crude cellulase extract was purified by precipitation using ammonium sulfate (Muthezilan et al 2014).
2.3. Carrageenan extraction from Kappaphycus alvarezii using cellulase enzyme
The carrageenan extraction process was carried out by suspending 12.5 g of flour Kappaphycus alvarezii with 250 mL of distilled water (Varadarajan et al 2009) and added with three different concentrations (0 %, 1 % and 2%) of cellulase enzymes from EN endophytic fungus. Sample suspension was heated on shaking incubator with two different extraction times (2 h and 3 h). Centrifugation of suspension with speed at 7,000 rpm and 4°C for 15 min (Varadarajan et al 2009). The supernatant was mixed with a 2-propanol solution with a ratio of 1:1. To remove the liquid part, the sample was centrifuged with 7,000 rpm at a temperature of 4°C for 20 min. The sample was then dried using an oven.
2.4. Analysis of carrageenan extract
2.4.1. Yield. The yield of carrageenan extraction was calculated based on the ratio between carrageenan weight produced and weight of dried seaweed that was processed respectively.
2.4.2 Sulfate content. The carrageenan was weighed 1 g and put into the Erlenmeyer flask 50 mL of HCl 0.2 N then refluxed for 6 h until the solvent became clear. This solvent was transferred into a cup glass and heated to boiling. Furthermore, 10 mL of BaCl2 solution was added over the water bath for 2 h. The precipitate formed was filtered with a non-gray filter paper and washed with boiling water to chloride free. The filter paper was dried into the dryer ovenat 1,000°C until white ash was obtained. Ash was cooled in a desiccator and then weighed (FMC Corp 1977).
2.4.3 Viscosity. Viscosity measurement was done by dissolving 1.5% carrageenan water then heated in a boiling water bath until the solvent temperature reached 75°C. Viscosity was measured using viscometer. The hot solvent was adjusted to a precise, viscometer was turned on and the temperature of the solvent was measured. When the solvent temperature reached 75°C and the viscosity value was measured on a scale of 1 to 100. The measurement was done after one minute of a full rotation for spindle number three (FMC Corp 1977).
2.4.4 Gel strength. The 1.6% carrageenan solution and 0.16% KCl were heated in a bath of boiling water with regular stirring to 80°C. The volume of the solution was 50 mL. The hot solution was then put into the 4 cm diameter mould and kept at 10°C for two hours. The gel in the mould was inserted into the measuring instrument (Rheoner RE-3305). The plunger that will be in contact with the gel was in the middle. The plunger was activated and the samples were measured. Evaluation of measurement results was done by reading the resulting graph. The maximum force (gel strength) can be read on the recorder (FMC Corp 1977).
2.4.5 Acid-insoluble ash content (FMC Corp. 1977). The carrageenan was boiled with 25 mL of 10%
HCl for 5 minutes. The unsolved ingredients were filtered using a non-gray filter paper, then cooled in a desiccator for further weight.
2.5 Functional group test by FTIR
Potassium bromide (KBr) powder was finely grounded by 0.1 g, then weighed carrageenan samples of 0.01 g were added and crushed together until smooth and homogeneous. The water content was removed by heating in an oven 60°C for 48 h. Further FTIR spectrophotometric analysis was performed to determine the functional group (Smith 2011).
3. Results and discussion
3.1. Cellulase enzyme production
The cellulase enzyme in this study was obtained through the cultivation of EN fungus in the basal medium. Cultivation was done by growing EN fungus in the basal medium with dried Kappaphycus alvarezii as its substrate source. The cellulase enzyme activity of the crude extract was measured after obtaining enzyme on fungal culture broth after nine days cultivation. The crude extracts enzyme was obtained by the filtration process and the centrifugation of culture broth of EN fungus. The supernatant from the centrifugation was the crude extract enzyme that has been produced. The value of enzyme activity of crude extract obtained was 0.0015 U/mL.
The semi-pure enzyme was obtained by precipitation using 80% of ammonium sulfate. The value of semi-pure cellulase enzyme activity was 0.014 U/mL. This semi-pure cellulase enzyme was used in the carrageenan extraction. This result was still low if compared to the cellulolytic activity of the best known producer of cellulases Trichoderma reesei. Zhao et al (2018) reported the cellulase production of T.
reesei cultured in different media and pretreatment. The cellulase production by T. reesei cultured in steam-treated willow was 0.7 U/mL and increased to 1.6 U/mL when the culture medium was induced with acetic acid.
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3.2. Yield of carrageenan
Yield is the ratio of the amount of produced carrageenan by the amount of seaweed extracted. The seaweeds used as the raw material and carrageenan were weighed in dried form. The results of carrageenan extraction can be seen in figure 1.
Figure 1. Effect of cellulase enzyme concentration and time of extraction on carrageenan yield.
Based on the results of yield analysis, it is known that the enzyme concentration factor, extraction time and interaction between the two factors show the significant effect with p-value <0.01. The highest yield produced in this study was carrageenan obtained from 1% enzyme concentration and 3 h of extraction time. The highest yield value was 30.96%.
The yield of this treatment had a value of 6.44% higher than the treatment without enzyme addition.
However, when compared to some previous studies on enzymatic extraction of carrageenan, the yield value in this study was still lower than other studies. Varadarajan et al (2009) yielded 45% of carrageenan, while Muthezilan et al (2014) obtained carrageenan yield of 52% by extraction using purified enzyme with dialysis. This is allegedly due to differences in the value of cellulase enzyme activity used in carrageenan extraction. The research of Varadarajan et al (2009) used a cellulase enzyme with an activity value of ≥700 U/g in the carrageenan extraction process.
The value of yield with 1% enzyme concentration was the highest accounting for 28.42%. There was 4.51% difference if compared to the treatment with no enzyme addition. This means that the addition of 1% cellulase enzyme from EN fungus increased the productivity of carrageenan extraction by 4.51%.
The highest value of yield based on the extraction period was obtained after 3 hours extraction with 27.89%. This result shows that the extension of extraction period increased the carrageenan by 2.72%.
3.3. Sulfate content
Sulfate content is a parameter used for various types of polysaccharides contained in red algae (Winarno 1996). The sulfate content affects the gel strength of carrageenan. The value of carrageenan sulfate content in this study can be seen in figure 2.
a b c
d
e f
0 5 10 15 20 25 30 35
2 h 3 h 2 h 3 h 2 h 3 h
0% 1% 2%
Yield (%)
Treatment
Figure 2. Effect of cellulase enzyme concentration and time of extraction on carrageenan sulfate content.
Based on the result of sulfate content analysis using ANOVA, it is known that the interaction between enzyme concentration and time of extraction did not have a significant effect on the sulfate content with p-value>0.01. The sulfate content of carrageenan in this study ranged from 16.05% to 16.95%, the value still meets the quality standard of carrageenan according to FAO (2007) with sulfate content ranged from 15-50%. Although the sulfate content in this study still meets the standards, this value remains very low when compared to the sulfate content of the carrageenan produced using alkali compounds.
Wenno et al (2012) in his research obtained the value of sulfate content in carrageenan was 27.43%.
3.4. Viscosity
Viscosity is the power of molecular flow in the solution system. Viscosity test was done to know the degree of viscosity of carrageenan solution at a certain concentration and temperature. In this study, viscosity measurements were performed using 1.5% carrageenan solution at 75°C and measured by a Brookfield viscometer. The result of viscosity analysis can be seen in figure 3.
Figure 3. Effect of cellulase enzyme concentration and time of extraction on carrageenan viscosity.
Based on the results of viscosity analysis using ANOVA, it was found that enzyme concentration factor, extraction time and interaction between two factors showed a significant effect with a p-value<0.01.
Viscosity of carrageenan extracted for 2 h was higher than that of 3 h extraction with a difference of 2.67 cP. This result shows that the extension of extraction time decreased the viscosity value by 2.67 cP.
a a a a a a
0 2 4 6 8 10 12 14 16 18
2 h 3 h 2 h 3 h 2 h 3 h
0% 1% 2%
Sulfate content (%)
Treatment
a a
b
c d d
0 5 10 15 20 25 30 35
2 h 3 h 2 h 3 h 2 h 3 h
0% 1% 2%
Viscosity (cP)
Treatment
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The enzyme concentration had a significant effect on the viscosity value, with the highest value at the enzyme concentration level of one percent and the lowest for the treatment with no enzyme addition.
The results of this analysis indicate that the addition of enzyme concentration increased carrageenan viscosity with a concentration limit of 1%. The highest viscosity value 29.5 cP was found in the interaction of treatment level of enzyme concentration of 1% and 2 h of extraction time. Overall, the carrageenan viscosity value still follows the quality standard of carrageenan according to FAO that is above 5 cP.
3.5. Gel strength
Gel strength is one of the characteristics to determine the use of carrageenan. The gel strength of carrageenan is in correlation with its sulfate content. The gel strength value of carrageenan gel produced in this study can be seen in figure 4.
Figure 4. Effect of cellulase enzyme concentration and time of extraction on carrageenan gel strength.
The gel strength values generated in this study ranged from 14.6 gF to 24.75 gF, with the highest value in the SB carrageenan with treatment 3 h of extraction time and 1% of enzyme concentration. SB carrageenan has the lowest sulfate content compared with other treatments. This result is similar to that of Wenno et al (2012) that the smaller of sulfate content increases the consistency of carrageenan gel strength.
3.6. Acid-insoluble ash content
The acid-insoluble ash content is an indicator of the hygiene of carrageenan. In this study it was known that the acid-insoluble ash value was in the range of 0.02 to 0.21% of each treatment, this value is still under the maximum limit according to FAO (2007) that is 1%. This low acid-insoluble ash content shows that the carrageenan was produced in this study have a high level of hygiene.
3.7. Functional group test by FTIR
The identification using FTIR aims to show the functional groups present in the carrageenan samples.
The FTIR data of carrageenan from this study were compared to commercial carrageenan. FTIR analysis showed that the results of this research have a number of waves that are similar to the commercial carrageenan, hence it can be stated that the carrageenan produced in this research was confirmed. The FTIR spectrum of commercial carrageenan and the results of this study can be seen in figure 5.
20.2
24.75
20.04 21.75
14.6
22.2
0 5 10 15 20 25 30
2 h 3 h 2 h 3 h 2 h 3 h
0% 1% 2%
Gel strength (gF)
Treatment
Figure 5. Functional groups of each hydrolysis product with different enzyme concentration, FTIR spectrum (a) commercial carrageenan, (b) carrageenan with 0% enzyme, (c) carrageenan with enzyme
1% (d) carrageenan with 2% enzyme.
4. Conclusion
The enzyme of marine fungus EN can be used to extract carrageenan. Enzyme concentration, extraction period and interaction of extraction time and enzyme concentration are affect the yield value, viscosity and gel strength of the carrageenan.
Acknowledgements
Acknowledgements are given to Ministry of Research, Technology and Higher Education of the Republic of Indonesia who has funded this research through Competency-Based Research Scheme to Dr. Kustiariyah Tarman, SPi, MSi with contract number 1442 / IT3.11 / PN / 2017 and 1608 / IT3.11 / PN / 2018.
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