Characteristics of Powder Agar Extracted from Different Seaweeds Species and Locations

doi:10.21203/rs.3.rs-2627637/v1

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Characteristics of Powder Agar Extracted from Different Seaweeds Species and Locations

https://doi.org/10.21203/rs.3.rs-2627637/v1

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In Indonesia, there are several production centers of agar-producing seaweeds. Research on agar quality has been frequently conducted everywhere, but agar quality test from several locations has never been done simultaneously. The objective of this experiment is to study the quality and characteristics of powder agar extracted from seaweeds harvested from different species and locations in Indonesia i.e., Gracilaria sp from Serang (Banten Province), Brebes (Central Java), and Ujung Genteng (West Java), as well as Gelidium sp from Ujung Genteng (West Java) and Gunung Kidul (Yogyakarta). Agar quality parameters were analyzed based on sensory (score and hedonic test), chemical (moisture, ash, acid insoluble ash, and sulphate content) and physical (viscosity, gel strength and color) parameters; microbiological (TPC, E.coli, moulds and fungi) and heavy metals (Hg, Pb, Cd, and As) contamination. The environmental condition of seaweed growing habitat was also observed, including Dissolved Oxygen (DO), transparency, pH, temperature, salinity, and nutrient content (nitrate, phosphate, ammonia, nitrite, and sulphite). Sensory analysis of powder agars showed that the highest score was achieved by agar extracted from Gracilaria from Ujung Genteng (8.71), and the highest yield was from Gracilaria from Brebes (10.41%). Viscosity and gel strength of agar extracted from Gelidium were higher than that from Gracilaria. The colour of powder agar was closely related to the salinity and water transparency of the seaweed growing habitat. It was concluded that seaweed species and locations affected agar characteristics and quality.

powder agar

Gracilaria

Gelidium

Indonesia

Seaweed is one of the leading marine biodiversity in Indonesia, accounting for 555 seaweed species in which 25 species have high economic value. From these 25 species, 4 of which being export commodities, ie. Euchema, Gracilaria, Gelidium, dan Sargasum (Yolanda & Agustono 2020). Indonesia is the world's largest agar exporter, followed by South Korea and China (Trade Map 2021). So that powder agar plays a very important role in economic development in Indonesia.

Agar is a hydrocolloid extracted from seaweed belonging to the red algae (Phaeophyceae). Three main seaweed genera producing agar in Indonesia are Gracilaria, Gellidium, and Pterocladia. Agar, having the ability to form a gel, can be used as an emulsifier, stabilizer, gelling agent, or thickener. This product is usually extracted from seaweed using the hot water extraction method in acidic conditions (Asih et al. 2019).

In Indonesia, there are several production centers of agar producing seaweed, among others are Lampung (Hanura et al. 2017; Subaryono & Sinurat 2021), Serang, Bekasi (Rohman et al. 2018), Brebes (Rejeki et al. 2018), Ujung Genteng (Johan et al. 2015), Yogyakarta, Sidoarjo, Bali, NTB, NTT, Luwu, Makasar, Poso, Palopo, and Maluku. Most seaweed used as raw material for agar extraction produced from these regions has met the national Indonesia standard for dry seaweed (BSN 2018), especially seaweeds produced from Serang, Brebes, Ujung Genteng, Palopo, and Makasar. Agar extracted from seaweeds harvested from different production centres could have different qualities and characteristics, which could be affected by species and growth environment, (salinity, nutrition, DO, transparency, pH), besides harvesting age. The objective of this experiment is to study the quality and characteristics of powder agars extracted from seaweed harvested from different species and locations, i.e. Gracilaria sp from Serang (Banten Province), Brebes (Central Java), and Ujung Genteng (West Java), and Gelidium sp from Ujung Genteng (West Java) and Gunung Kidul (Yogyakarta).

2.1. Materials

Dry Gracilaria sp was collected from three different locations i.e. Serang (Banten Province), Brebes (Central Java) and Ujung Genteng (West Java), and Gelidium sp from Ujung Genteng (West Java) and Gunung Kidul (Yogyakarta). The raw materials were packed in a woven plastic bag and transported by car to Pilot plant of Research Center for Marine and Fisheries Product Processing and Biotechnology in Jakarta.

2.2. Methods

2.2.1. Seaweed quality

Parameters used as indicators for seaweed quality are moisture content, clean anhydrous weed, impurities, and heavy metals. Moisture content, clean anhydrous weed, and impurities were analysed using methods suggested by Agency for National Standardisation [15]; heavy metals (Hg, Pb, Cd, and As) using ICP MS.

2.2.2. Agar extraction

As much as 2 kg of dry seaweed was soaked in fresh water, washed thoroughly until free from dirt (sand, mud, epiphyte, salt, and other debris). Alkali treatment was conducted using 4% NaOH (with the proportion of 1 seaweed to 20 NaOH solution) at 70–80 ^oC for 2 hrs. The pH of the treated seaweed was neutralized with fresh water by washing it repeatedly until the pH went down to 7–8. Agar extraction was conducted in hot water (with the proportion of 1 seaweed to 15 water) at a temperature of 95–100 ^oC for 2 hrs. The seaweed slurry was then filtered with a 200 mesh size plankton net. The filtrate was put into gelling pans and cooled down at ambient temperature to gelify. The agar gel was then put into cold storage to experience a slow freezing process. The frozen agar gel was thawed, filtered, pressed, dehydrated using isopropyl alcohol and then dried in a mechanical dryer. The dry fibrous agar was milled using a 60 mesh size grinder. The yield was calculated as a percentage of dry agar over the dry seaweed as raw material (Jayasinghe, PS & KKDS 2016).

2.2.3. Agar quality

Agar quality parameters were analysed based on: sensory (score and hedonic test) using 10 expert panelist; chemical test i.e. moisture content, ash content, acid insoluble ash (AOAC 2005); sulphate content(Dodgson & Price 1962); physical analysis: viscosity (1.5% w/v; 60 ^oC) - Brookfield viscometer, gel strength (1.5% w/v; 20 ^oC) - TA. XT Texture Analyzer and color (chromameter). Microbiological analysis (TPC(Tapotubun et al. 2020); E.coli(Jamaluddin et al. 2021), moulds and fungi(Mostafa et al. 2019); heavy metals (Hg, Pb, Cd, and As) using ICP MS (Kalkisim et al. 2019). Some important environmental conditions of seaweed growing habitat were analyzed covering Dissolved Oxygen (DO) using a test kit, pH (pH meter), temperature (digital thermometer), salinity and density (refractometer), nutrient content (ammonia, nitrate, nitrite, phosphate, and sulfite) using colorimetry.

2.3. Statistical analysis

The collected data were analyzed to obtain the mean and standard deviation with a significance level of 95%. An ANOVA (P = 0.05) was used to detect significant parameter differences. Post-hoc analysis (Tukey’s range test) was used to determine the interactive effects of variables. Pearson’s correlation coefficient in IBM SPSS Statistics 25 was used to measure the degree of connection between different variables.

3.1. The characteristic of seaweed as a source of powder agar

Table 1 provides a quality characteristic of seaweed as a source of powder agar, such as moisture content, CAW, impurities, and metals contamination (Hg, Pb, Cd, As).

The moisture content of the seaweed samples ranged from 11.53 to 24.5%. The value of CAW ranged from 44.72 to 84.72%, Impurities of all seaweed samples ranged from 5,8 to 9,33%. The heavy metal analysis results showed that all samples were not detected to contain mercury (Hg); however, the other heavy metals (Pb, Cd and Hg) content was higher than the Indonesian National Standard.

Tabel 1. The quality Characteristic of Seaweed as a source of powder agar

Quality Parameters

Unit

Characteristic of Seaweed

SNI

Chemical

- Moisture content

- Clean Anhydrous Weed (CAW)*

13.16 ± 0.63^b

47.78 ± 2.93^a

11.54 ± 0.11^a

51.39 ± 10.42^ab

24.50 ± 0.19^d

44.72 ± 3.15^a

12.05 ± 0.33^a

65.83 ± 6.61^b

14.65 ± 0.26^c

84.72 ± 0.48^c

Max. 18

Min. 40

Physical contaminant

- Impurities (%)

6.47 ± 1.62^a

5.80 ± 3.08^a

8.80 ± 1.04^a

9.33 ± 1.68^a

7.33 ± 1.70^a

Max. 3

Heavy metals*

- Mercuri (Hg)

- Lead (Pb)

- Cadmium (Cd)

- Arsen (As)

mg/kg

1.31

0.17

17.42

1.31

0.18

8.71

0.26

1.23

11.44

0.35

0.69

5.58

0.84

0.18

0.63

Max. 0.5

Max. 0.3

Max. 0.1

Max. 1.0

Notes:

A: Powder agar from Gracilaria from Serang

B: Powder agar from Gracilaria from Brebes

C: Powder agar from Gracilaria from Ujung Genteng

D: Powder agar from Gelidium from Yogyakarta

E: Powder agar from Gelidium from Ujung Genteng

3.2. The characteristics of powder agar

3.2.1. Yield

The highest yield of powder agar from G. verrucosa seaweed was 10.42%. However, statistically, the yield of G. verrucosa powder agar from Serang and Brebes were not different significantly. Meanwhile, a significant difference was obtained in their yield compared to the yield of powder agar from Ujung Genteng with the same seaweed species.

3.2.2. Sensory quality of powder agar

Based on the total sensory score among five powder agars extracted from seaweeds from different locations and species (Fig. 2), the best quality agar was achieved by sample C i.e. agar extracted from Gracilaria seaweed from Ujung Genteng, with the total score of 8.71.

The second best were those extracted from Gracilaria seaweed from Brebes (B) and Gelidium from Ujung Genteng (E). These two agars had the same total sensory score of 7.38. The lowest score was agar extracted from Gracilaria seaweed from Serang; however, this score was not significantly different from that of powder agar from Gelidium seaweed from Yogyakarta, which were 6.25 and 6.50, respectively. When the detail of each sensory parameter was further looked at by scoring the product performance, odor, texture and color, as can be seen in Fig. 2, the texture scores were similar among all agar samples, the significant differences were easily detected from their appearance, color and odor. The scores of appearance and color of powder agar extracted from Gracilaria seaweed from Serang were the lowest among all parameters i.e. 5.00 and Gelidium seaweed from Yogyakarta was 5.17. The highest scores of appearance and color, as well as odor, were achieved by powder agar extracted from Gracilaria seaweed from Ujung Genteng (sample C).

3.2.3. The chemical characteristics of powder agar

The moisture content of powder agar extracted from all powder agar obtained from different locations was not different significantly except for G. verrucosa from Brebes, which different significantly from the others (Table 2). The ash content of powder agar from G. verrucosa was different significantly with different locations. However, the ash content between G. verrucosa and Gelidium sp from the same location were not different significantly. Results also showed that the ash content of G. verrucosa and Gelidium sp from Ujung Genteng were not different significantly.

The acid-insoluble ash content of powder agar based on locations and species were different significantly, so as those from different species but the same location. However, acid insoluble ash of cultivated G. verrucosa from Brebes and Serang were not different significantly. The highest sulphate content of powder agar was obtained from G. verrucosa from Brebes, i.e. 2.57%.

Table 2. The Chemical Characteristics of Agar Extracted from Seaweeds from Different Locations

Sample Codes	Moisture content (%)	Ash content (%)	Acid insoluble ash (%)	Sulphate content (%)
A	14.86 ± 2.68^b	2.84 ± 0.02^b	0.08 ± 0.0^a	1.98 ± 0.06^ab
B	9.88 ± 0.20^a	3.57 ± 0.04^d	0.02 ± 0.0^a	2.57 ± 0.04^c
C	15.46 ± 0.04^b	3.42 ± 0.03^c	0.30 ± 0.06^b	2.12 ± 0.07^b
D	15.60 ± 0.12^b	2.63 ± 0.02^a	0.04 ± 0.0^a	2.08 ± 0.03^ab
E	14.02 ± 0.12^b	3.31 ± 0.07^c	0.63 ± 0.03^b	1.87 ± 0.14^ab

Notes:

A: Powder agar from Gracilaria from Serang

B: Powder agar from Gracilaria from Brebes

C: Powder agar from Gracilaria from Ujung Genteng

D: Powder agar from Gelidium from Yogyakarta

E: Powder agar from Gelidium from Ujung Genteng

3.2.4. The Physical quality of agar powder

The viscosity of the extracted powder agar can be seen in Table 3, ranging from 5.03–490.80 cPs.

Table 3. The Physical Quality of Powder agar Extracted from Seaweeds from Different Locations

Sample Codes	Viscosity (cPs)	Gel Strength (g/cm²)	Water Absorption (%)	L*	a*	b*
A	5.03 ± 0.10^a	472.30 ± 11.72^a	1,281 ± 1.41^a	61.84 ± 0.03^a	5.40 ± 0.01^e	21.94 ± 0.03^e
B	5.20 ± 0.05^a	479.50 ± 16.84^a	1,478 ± 1.28^ab	72.42 ± 0.01^c	3.62 ± 0.00^c	19.21 ± 0.02^c
C	13.36 ± 0.47^b	665.25 ± 18.44^b	1,814 ± 0.42^c	77.89 ± 0.04^e	2.86 ± 0.01^a	18.11 ± 0.01^b
D	83.90 ± 2.77^c	888.40 ± 11.46^c	1,804 ± 0.11^c	67.35 ± 0.03^b	3.33 ± 0.01^b	16.26 ± 0.03^a
E	490.80 ± 1.77^d	1403.48 ± 100.47^d	1,610 ± 0.27^bc	72.87 ± 0.01^d	3.96 ± 0.01^d	20.35 ± 0.02^d

*Colour component parameters

Notes:

A: Powder agar from Gracilaria from Serang

B: Powder agar from Gracilaria from Brebes

C: Powder agar from Gracilaria from Ujung Genteng

D: Powder agar from Gelidium from Yogyakarta

E: Powder agar from Gelidium from Ujung Genteng

The gel strength of powder agar ranged from 472.30 to 1,403.48 g/cm². Table 3 shows that the lowest gel strength was agar extracted from Gracilaria from Serang (472.30 g/cm²), and the highest was agar from Gelidium from Ujung Genteng (1403.48 g/cm²). The gel strength of agar extracted from Gracilaria seaweed from Serang was not significantly different from Brebes, but significantly different from gel strength of agar extracted Gracilaria from Ujung Genteng, Gelidium from Yogyakarta, and Gelidium from Ujung Genteng.

The water absorption value of powder agar ranged from 1,281-1,814%. Table 3 showed that the water absorption value of powder agar extracted from Gracilaria seaweed from Serang was not significantly different from Gracilaria from Brebes, but significantly different from Gracilaria from Ujung Genteng, Gelidium from Yogyakarta, and Gelidium from Ujung Genteng.

Color parameters testing resulted in L* (lightness), a* (redness-greenness), and b* (yellowness-blueness) values, as shown in Table 2. Powder agar extracted from different species of seaweed and sampling locations produced L*, a*, and b* different significantly (p < 0.05). This shows that the different types and locations of seaweed origin have a significant effect on the color of the powder agar produced. The lowest L* value was the powder agar extracted from Gracilaria from Serang (61.84), while the highest was from Gracilaria from Ujung Genteng (77.89).

The a* value of the extracted powder agar ranged from 2.86 to 5.40. The lowest was obtained from powder agar extracted from Gracilaria seaweed from Ujung Genteng, while the highest a* value was from Gracilaria from Serang. A negative a* value indicates greenness colour, while a positive a* value indicates the redness colour of the sample.

The lowest b* value was powder agar extracted from Gelidium seaweed from Yogyakarta (16.26), while the highest b* value was powder agar extracted from Gracilaria seaweed from Serang (21.94). A positive b* value indicates a yellow colour.

3.2.5. Microbial contaminants

Results of the micro-contamination analysis can be seen in Table 4. Total microbial content in commercial powder agar (TPC) ranged from 2.2x10² to 1.6x10⁴ cfu/g. There is no E. coli content in all samples. In addition, the total mould content of powder agar ranged from 3.3x10² to 8.4x10³ cfu/g.

Table 4. The Microbial Content of Powder agar Extracted from Seaweeds from Different Locations

Sample Codes	TPC (cfu/g)	E. coli (cfu/g)	Total Mould and Fungi (cfu/g)
A	1.5 x10³	0	3.3 x10²
B	2.0 x10³	0	3.6 x10³
C	1.6 x10⁴	0	4.0 x10³
D	2.2 x10²	0	8.4 x10³
E	1.4 x10⁴	0	2.9 x10³
Inonesian standard (SNI 2021)	1 x 10⁴	1x10²	1 x 10²

Notes:

A: Powder agar from Gracilaria from Serang

B: Powder agar from Gracilaria from Brebes

C: Powder agar from Gracilaria from Ujung Genteng

D: Powder agar from Gelidium from Yogyakarta

E: Powder agar from Gelidium from Ujung Genteng

3.2.6. Heavy metals

Based on Indonesian National Standard for powder agar, analysis of heavy metal content (Hg, Pb, Cd, and As) is required(Kalkisim et al. 2019). The heavy metal content of all powder agar (Table 5) was actually lower than that of the seaweeds as raw materials (Table 1). However, some heavy metal contents of the sampels were still higher than the standard i.e. the content of Pb in sample E,D, and B.

Table 5. The Heavy Metal contents of Powder agar Extracted from Seaweeds from Different Locations

Sample Codes	Hg (mg/kg)	Pb (mg/kg)	Cd (mg/kg)	As (mg/kg)
A	ND	0.25	0.10	0.56
B	ND	0.47	0.02	0.61
C	ND	0.20	0.04	0.74
D	ND	0.38	0.14	0.66
E	ND	0.84	0.18	0.63
Inonesian standard (SNI 2021)	Max 0.5	Max 0.3	Max 1.0	Max 1.0

Notes:

A: Powder agar from Gracilaria from Serang

B: Powder agar from Gracilaria from Brebes

C: Powder agar from Gracilaria from Ujung Genteng

D: Powder agar from Gelidium from Yogyakarta

E: Powder agar from Gelidium from Ujung Genteng

3.3. The environmental condition of Gracilaria and Gelidium seaweed growing habitat based on locations

As can be seen in Table 6 that dissolved oxygen (DO) of waters from Serang, Brebes and Ujung Genteng ranged from 5.10 ± 1.47 to 5.94 ± 1.24. DO values of the three locations were not different significantly. During observation, water transparancy in Serang, Brebes and Ujung Genteng waters ranged from 53.33 ± 5.77 to 200 ± 8.7 cm. Observation on pH values, Serang, Brebes and Ujung Genteng waters raned from 6.57 ± 0.34 to 8.68 ± 0.22. Serang and Brebes waters had pH values not different significantly; only pH value of Ujung Genteng water was lower than Serang and Brebes. The water temperature of the three locations ranged from 30.35 ± 0.68 to 32.41 ± 0.95 ^oC. Serang and Ujung Genteng waters' water temperature was not significantly different, but Brebes's was lower than the other two locations.

Water salinity of Serang, Brebes and Ujung Genteng waters ranged from 33.22 ± 1.85 to 38.31 ± 7.19 ppt. Nitrate content in Serang, Brebes, and Ujung Genteng waters ranged from 3.04 ± 0.96 to 7.13 ± 0.65 ppm, and phosphate content from 0.08 ± 0.01 to 0.25 ± 0.05 ppm. Ammonia content in Serang, Brebes and Ujung Genteng waters ranged from 0.08 ± 0.01 to 0.27 ± 002 ppm. Nitrite content of Serang, Brebes and Ujung genteng waters ranged from 0.003 ± 0.00 to 0.05 ± 0.01 ppm. The maximum sulfite content was 0.016 ± 0.01 ppm.

Table 6. The Environmental Conditions of Seaweed Growing Habitat (brackish water ponds) of Gracilaria

Parameters	Locations
Parameters	Serang	Brebes	Ujung Genteng
DO	5.10 ± 1.47^a	5.83 ± 0.88^a	5.94 ± 1.24^a
Transparency (cm)	53.33 ± 5.00^a	145.00 ± 5.48^b	200 ± 26.35^c
pH	7.00 ± 0.79^a	6.58 ± 0.34^a	8.69 ± 0.22^b
Temperature (^oC)	32.18 ± 1.00^b	30.35 ± 0.68^a	32.41 ± 0.95^b
Salinity (ppt)	38.31 ± 7.19^a	33.22 ± 1.85^a	34.11 ± 0.93^a
Nitrate (ppm)	5.26 ± 1.33^b	7.13 ± 0.65^c	3.04 ± 0.96^a
Phosphate (ppm)	0.26 ± 0.05^b	0.15 ± 0.09^a	0.08 ± 0.01^a
Amonia (ppm)	0.21 ± 0.07^b	0.08 ± 0.01^a	0.27 ± 002^c
Nitrite (ppm)	0.004 ± 0.00^a	0.009 ± 0.00^a	0.05 ± 0.01^b
Sulphite (ppm)	0.016 ± 0.01^b	0.03 ± 0.00^c	0.00 ± 0.00^a

The moisture content of seaweed is significant as a critical quality parameter since it will affect the shelf life of the dry seaweed right from the production site, during storage in the warehouse, trading up to the processing plants. Moisture content will affect the price as well during trading. The lower the moisture content of the seaweed, the slower is the quality degradation process resulted from chemical process as well as enzymic and microbial activities (Nur & Sunarharum 2019). Based on Indonesian Standard (SNI), the maximum moisture content of dry Gracilaria seaweed is 18% (BSN 2018). Moisture content of seaweed from various production centers mostly have met the Indonesian Standard; only seaweed from Ujung Genteng had higher moisture content i.e., 24.5%. The reference moisture content of the seaweed sample (Gracilaria verrucosa) from Karawang, which was studied by (Waluyo; et al. 2019), had a moisture content of 11,9%. The moisture content of the commercial Gracilaria verrucosa sample from Sinjai (South Sulawesi) collected by Utomo & Satriyana (2006) was 18.8%.

The other important parameter of seaweed quality is Clean Anhydrous Weed (CAW). This parameter indicates the cleanliness or purity of the seaweed (Subaryono & Sinurat 2021). It can be seen from Table 1 that the CAW depends on the location where the seaweed was collected. Based on the Indonesian National Standard that the minimum CAW value is 40% (BSN 2018), dry seaweed samples from all representing locations had CAW values are well within the standard. The CAW of commercial Gracilaria verucosa from Sinjai (South Sulawesi) was 64.1% (Utomo & Satriyana 2006). The impurities were mainly dirt. The proportion of clean anhydrous weed was high since no other type of seaweed was found in the sample, and only a little sand was included.

Impurities of all seaweed samples did not meet the National standard of dry seaweed with maximum impurities of 3% (BSN 2018). This showed that the post-harvest handling of the seaweed was not good, this high impurities eventually affected the agar yield, with the highest yield was only 10.42% (Fig. 1). As a reference, Gracilaria seaweed harvested from brackish water pond in Kerawang (Waluyo; et al. 2019) showed that the impurities of dry seaweed were 8.44%.

Results of the heavy metal analysis, all samples were not detected to contain mercury (Hg), so that all samples were well within the National standard of 0.5 mg/Kg (BSN 2018). Some samples did not meet the standard for the other heavy metals (Pb, Cd and Hg) since the heavy metal content was higher than the standard. However, the agar extracted from these seaweeds had heavy metal content lower than the agar standard(CODEX STAN 192–1995: General standard for food additives 1995).

The results of the analysis of the yield of powder agar from the extraction of G. verrucosa and Gelidium sp from different locations are shown in Fig. 1. Based on the analysis of the yield of powder agar, the location of the growth obtained affects the yield of powder agar. The same result was also obtained from the research of Chapman & Chapman (1980) in (Murdinah et al. 2008) that the yield of Gracilaria was higher than that of Gelidium sp. still, the gel strength of Gelidium agar was higher than that of Gracilaria. The high and low yield of agar is influenced by the type of seaweed, the extraction process, environmental conditions, geographical distribution, and harvest season (Santika et al. 2014; Ibrahim et al. 2015). The low agar yield of Gelidium from Ujung Genteng might be caused by high impurities and the harvesting age of the seaweed since it was harvested from the wild crop. However, the high agar yield of Gracilaria from Brebes could result from the high CAW value and the low moisture content of the dry seaweed (Table 1). Agar yield is very much affected by impurities, clean anhydrous weed, and moisture content of the extracted seaweed (Utomo & Satriyana 2006).

From sensory evaluation, it can be concluded that the best quality agar among all evaluated samples was achieved by powder agar from Gracilaria seaweed from Ujung Genteng with an overall sensory score of 8.71. Based on the score of each parameter (appearance, odor, texture and color), odor had the lowest score among all sensory parameters i.e. 8.00 out of 9.00 for the maximum score. The high sensory score of powder agar extracted from Gelidium from Ujung Genteng might be affected by the high CAW value of the raw material and the good environmental condition such as the high water transparency and suitable pH value (Tabel 5).

The higher the moisture content, the higher the powder agar weight and lower agar moisture content to lower the powder agar weight. However, based on the requirements of the EU commission, the moisture content of powder agar obtained still meets the requirements, namely a maximum of 16% of the total weight of powder agar. While according to the export quality standard for agar, according to Food and Agriculture Organization (FAO), the quality standard of agar moisture content is 15–21% (Yolanda & Agustono 2020). The moisture content affects the shelf life of the agar product and the stability and quality index. Powder agar with low moisture content is not easily damaged compared to powder agar with high moisture content (Insan & Dwi Sunu Widyartini 2012).

The ash content of seaweed raw materials that grow in the sea is higher than in ponds because of its salinity (Andiska et al. 2019). In addition to salinity levels, seaweed harvesting age also affects the mineral content of agar it contains. The longer the harvest age of the seaweed, the higher the ash content will be (Waluyo; et al. 2019). The high ash content was closely related to the high mineral content of the agar (Wenno et al. 2012; Andiska et al. 2019).

Acid insoluble ash is an indication of the minerals contained in the agar. This analysis shows that the mineral content of seaweed that grows in the sea is higher than that of seaweed in ponds even though the species are the same (Wenno et al. 2012; Andiska et al. 2019). Another factor that causes high ash content is imperfections in washing seaweed raw materials before extraction and incomplete filtration; impurities are not filtered and included (Santika et al. 2014).

The sulfate content affects the gel strength of the agar. The higher the sulfate ester content in the agar, the lower the strength of the gel formed (Chapman & Chapman, 1980 in Dina et al.,(Wenno et al. 2012). Base pretreatment can catalyze the 6-sulfate group of the galactopyranose unit also that the sulfate content of agar is lower(Darmawan et al. 2020). The other information is that indications of high sulfate levels indicate high levels of impurities in the agar (Waluyo; et al. 2019). Refers to Suryaningrum (1988) in Dina et al., (Wenno et al. 2012), sulfate levels can be influenced by seaweed species, seaweed origin, extraction method, and harvest age. Increasing the age of harvest can respond to a decrease in sulfate content. Based on the type of seaweed, the agarose content of Gracilaria sp is higher than that of Gelidium sp Chapman & Chapman, (1980) in Fransiska & Murdinah (2007).

Based on this research, Gelidium produces powder agar with a higher viscosity than Gracilaria. The highest viscosity obtained in powder agar extracted from Gelidium from Ujung Genteng, significantly different from the whole powder agar analyzed. This viscosity is much higher than powder agar from Gelidium from Pameungpeuk, with a range of 26.2 to 47.5 cPs(Darmawan et al. 2020). As well as flour extracted from Gelidium sesquipedale, which only has a viscosity value of 11.11 cP (Nil et al. 2016). Meanwhile, the lowest viscosity was obtained from agar flour extracted from Gracilaria from Serang, 5.03 cPs. This value was not significantly different from that of powder agar from Gracilaria from Brebes but significantly different from Gracilaria Ujung Genteng (C). Meanwhile, powder agar from Serang (A) and Brebes (B) has a viscosity that is not significantly different (p > 0.05). Rebello, Ohno, Ukeda, & Sawamura (1996) reported that a high viscosity (240–370 cP) was obtained from agar extracted from Gracilariopsis from Japan, while a low viscosity was obtained from the seaweed G. edulis from Indonesia (8.0 cP) after 10% NaOH treatment. These results indicate that the powder agar extracted from the Gracilaria spp. with different geographical origins has different viscosities.

The gel strength in this study was more significant than the results of research by Praiboon et al., (2006), which reported that the gel strength of agar extracted from Gracilaria edulis was 334.50 g/cm². Similarly, powder agar extracted from G. tenuistipitata has the highest gel strength of 482 g/cm² with 5% NaOH treated (Yarnpakdee et al. 2015).The gel strength of agar is related to its sulfate content. The presence of charged groups such as sulfate groups can disrupt intermolecular hydrogen bonds to form a double helix, affecting the value of gel strength (Lahaye & Rochas 1991). The high viscosity and gel strength are related to the length of the agar polymer, where the longer the agar polymer is positively correlated with the viscosity and gel strength values (Santika et al. 2014).

The water absorption value of the powder agar in all treatments met the requirements of SNI 01-2802-1995, which requires that the flour can absorb water at least five times or equivalent to 500%. The sulfate content in agar and some other factors are also considered to relate to the water-absorbing behaviour of agar (Dumitriu 1998). The determining factor for the level of water absorption in agar is determined by the relaxation of the polymer chains so that it affects the rate of water penetration into the agar gel (Witono et al. 2014).

The high lightness of powder agar follows sensory results, which show the highest value for the color of powder agar from Ujung Genteng (Fig. 3). Powder agar from all samples was categorized as having high lightness. The lightness value of powder agar in this study was similar to that of Yarnpakdee et al. (2015) of 58.02–84.80. The pigment content influences the lightness of the color in the seaweed, which may be leached during extraction (Yarnpakdee et al. 2015).

Based on the analysis results, powder agar showed positive results indicating a red color. The red color comes from the phycoerythrin content in Gracilaria sp. (Sudhakar 2014). By Christaki, Bonos, Giannenasa, & Florou-Paneria (2013) states that the presence of carotene pigments affects the color of a product. The carotene content in red seaweed Gracilaria causes flour to produce an increased intensity of yellow color. This is also by the statement of Hidayati, Yudiati, Pringgenies, Oktaviyanti, & Kusuma (2020) that Gracilaria contains carotene (0.528 ± 0.009 mol/g).

The results of the analysis of microbial contamination observed were by the requirements of (BPOM 2019), including TPC, E. coli, moulds, and fungi. A total of 5 codes meet the requirements of Indonesian Standard (BSN 2021), but 2 codes did not meet the requirements, namely codes C and E that are greater than 1 x 10⁴ cfu/g. For the parameter E. coli the value is 0 for all treatments, so it meets the requirements of Indonesian Standard (BSN, 2021). All samples did not meet the requirements of the Indonesian Standard (BSN, 2021). The high content of total bacteria (TPC), mould and Fungi could be related to the high moisture content of the powder agar bacteria, mould, and fungi grow well in a suitable moisture content (Nur & Sunarharum 2019).

Seaweed is a marine organism that can absorb and accumulate heavy metals found in aquatic environments. Heavy metal testing is used as ne way to determine contamination metal biota that are worried can affect the nutritional content and cause poisoning (Nufus et al. 2017). The heavy metal value analysis results can be seen in Table 5. All sample codes meet the requirements of Hg, Cd and As in (BSN 2021). Powder agar from Gracilaria from Serang and Ujung Genteng meet the requirements of Pb, but powder agar from Gracilaria from Brebes and powder agar from Gelidium from Yogyakarta and Ujung Genteng did not meet the requirements, which is more to the standard limit of 0.3 mg/kg. This is following the study results of Purwaningsih & Deskawati (2021), which stated that Gracilaria seaweed originating from Banten was below the threshold for heavy metal levels required by BSN (2021). This shows that some heavy metals of the agar extract could be reduced during the extraction and filtering process (Table 5). An experiment conducted by Erniati et al. (2018) showed that agar extraction using hot water could reduce the heavy metal content.

According to Diana, Nirmala, & Soelistyowati (2014), the DO parameter was correlated with seaweed quality. DO values of the research locations were not suitable for seaweed growth. Research results (Widiastuti 2011) revealed suitable DO values for planting Gracilaria verrucosa ranged from 6.0 to 7.0 ppm. Water transparency is a variable that is correlated with light penetration into the water (Amir 2019). Light penetration represents how far light can penetrate the water to support the photosynthesis process or seaweed growth (Amir 2019). The ideal water transparency is when the sunlight can reach the seaweed (Amir 2019).pH were still suitable for seaweed growth based on the observation conducted (Syam et al. 2020) that most seaweed can grow well at pH 7–8.This condition was still good for seaweed growth (Rohman et al. 2018), ranging from 30.70 to 33.40°C. According to Diana et al. (2014) water quality parameters correlated to seaweed quality were temperature, DO, PO₄-, P, and NH₃-N. Based on the study conducted by Rukmi, Sunaryo, & Djunaedi (2012), optimum water salinity for seaweed growth ranged from 15 to 50 ppt so that saweed can grow well at the three locations. Asni (2015) mentioned that water transparency becomes a significant contribution to seaweed production during the rainy season while nitrate, salinity, and transparency become significant contributions during the dry season.

Nitrate and phosphate are two macronutrients that determine water fertility (Daud et al. 2014). Nitrate and phosphate content become water quality parameters for seaweed growth (Daud et al. 2014). Nitrate and phosphate are the nutritional elements in the form of ions, functioning as support to increase plant and algae for their metabolism process. The optimum nitrate content for plant growth is between 0,9–3,5 ppm, and phosphate 0,10–0,20 ppm (Daud et al. 2014).

Nitrite, ammonia, and sulfite act were supporting parameters in water and become limiting factors for the growth of aquatic plants. According to Daud, Mulyaningrum, & Tjaronge (2014), the presence of nitrite in the water does not significantly affect seaweed growth. Nitrite content from 0.0033 to 0.0406 did not significantly affect the growth of Gracilaria seaweed at Bone, Pangkep, and Takalar (Daud et al. 2014). Nitrite and nitrate content in water is usually used as an indicator of water pollution level (Juliasih et al. 2017). In natural waters, nitrite (NO₂) is usually found as a trace element, less than nitrate, since it is not stable in the presence of oxygen, nitrite acts as an intermediate form of ammonia and becomes nitrate in the nitrification process, and from nitrate becomes nitrogen in denitrification (Juliasih et al. 2017). Based on the study conducted by Daud et al. (2014), that value still can be tolerated and even become a supporting parameter for the seaweed growth. Ammonia is the main nitrogen product in the water originated from the aquatic organism. Ammonia is a toxic substance that can kill aquatic organisms in a high concentration. The ability of Gracilaria in absorbing nitrogen in water polluted with organic materials, is 0,4 gram N/m² per day (Daud et al. 2014). Sulfite is one of the water pollution parameters and is hazardous to aquatic organisms. Sulfite (SO₃²⁻) will be formed if sulfur reacts with oxygen in the water (Madjid et al. 2018). This is still safe for the aquatic organism. The maximum limit of sulfite content is 0.05 ppm (Madjid et al. 2018). Sulfur in SO42 becomes an available form for the aquatic organism and becomes an important nutrient for plant growth (Aisyah et al. 2015). In the form of sulfate, sulfur acts as an essential fertilizer to elevate the plant's metabolism, such as MgSO₄ fertilizer (Ayunda et al. 2021).

This research shows that the quality and characteristics of powder agar are very much affected by seaweed species and the locations. Viscosity and gel strength of powder agars extracted from Gelidium are higher than that of agar from Gracilaria. Sensory analysis of the agars shows that powder agar extracted from seaweed from Ujung Genteng has a higher sensory quality, especially the colour parameter, than Serang and Brebes. It could be affected by water salinity and transparency where the seaweed as raw material was harvested.

Funding Statement

This research was supported by the Ministry of Marine Affairs and Fisheries, Indonesia

Data availability

All data generated or analysed during this study are included in this published article.

Conflicts of interest/Competing interests

The authors have no competing interests.

Authors' contributions

ES: Conceptualization, Methodology, Validation, Investigation, Data Curation, Writing—Original Draft, Visualization. DF: Conceptualization, Methodology, Resources, Writing— Review & Editing, Project administration. BSBU: Formal analysis, Writing—Review & Editing. SB and NH: Resources, Writing—Review & Editing, Data Curation, Project administration

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Characteristics of Powder Agar Extracted from Different Seaweeds Species and Locations

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials

2.2. Methods

2.2.1. Seaweed quality

2.2.2. Agar extraction

2.2.3. Agar quality

2.3. Statistical analysis

3. Results

3.1. The characteristic of seaweed as a source of powder agar

3.2. The characteristics of powder agar

3.2.1. Yield

3.2.2. Sensory quality of powder agar

3.2.3. The chemical characteristics of powder agar

3.2.4. The Physical quality of agar powder

3.2.5. Microbial contaminants

3.2.6. Heavy metals

3.3. The environmental condition of Gracilaria and Gelidium seaweed growing habitat based on locations

4. Discussion

CONCLUSION

Declarations

References

Additional Declarations

Status:

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