Introduction

Snakehead fish (Channa striata) is the one of abundant tropical freshwater fish in almost region Indonesia  which traditionally known as medicine. It also has been developed in pond cultures due to its commercial value  and health benefit (FAO, 2000)

Scientific studies revealed that the  snakehead fish (SHF) contains albumin which potentially increases the albumin levels in patients with hypoalbuminemia and postoperative surgery and liver function recovery (Santoso, 2009), contains  antioxidant glutathione and its precursors i.e. amino acids glutamine, cysteine and glycine that essential for tissue repair, including pancreas (Sunarno, 2015; Esrefoglu, 2012; Robertson and Harmon, 2007). The previous  pharmacological studies proved that using steamed SHF extract has recovery potential for pancreatic and testicular damaged in diabetic mice as well as lowering their blood sugar toward the normal levels. Furthermore, refer to the malondealdehide  indicated that  SHF extract has role to increase antioxidant capacity in the testis, pancreas and plasma of diabetic mice (Hidayati 2017; Sekartaji and Hidayati, 2016, Abdulgani et al.,  2016)

Steam extraction using distilled water within temperature below 60°C is the one of extraction methods to obtaining the SHF extracts with high yield of valuable soluble protein included albumin (Romadhoni et al. 2016; Nugroho, 2012;  Mustafa,2012; FAO, 1986 ). Albumin content in snakehead fish was reported at value of 6.4%  (Romadhoni et al. 2016) . Those conventional steamed SHF extract (SSHFE) product is recommended to store in freeze temperature (-20°C), not in room temperature. Evaluation to find scientific reason related to the SSHFE storage stability necessary required.

Accordingly, SSHFE is potential to be developed as a nutraceutical,  food /part of a food that safe to eat orally and provides health benefits (Palthur et al, 2010). included for  anti-diabetes.  Therefore, it is necessary to conduct a series of optimization analyzes to obtain SHF nutraceutical products that are feasible to consume before commercial production. The microbiological storage stability evaluation should be investigated for safety purposes,  to provides  important information how to store and   expiring date labeling (Bensley, 2008).

Materials and Methods

The processing of  steam extraction adopted from  Romadhoni et al., (2016). The fins, scale, gill and digestive organs were removed from SHF body.  Then, the remaining tissues primarily consist of flesh were rinsed in running water, cut into smaller pieces and stored in icy container prior to steam extraction. The steaming process was conducted in sterile room using modified steam extractor which consist of the stainless steel steamer- pan with the outlet pipe beneath to collect and directing the evaporated water to the collecting flask. The steamer is digitally connected with temperature and time controller equipment. Steaming extractor was adjusted at 50±10°C for 30 minutes. During steaming process, the droplets of concentrate were directed into the  vessel that connected into the sterilized flask. Then, the flasks were capped and stored in the freeze refrigerator (-20°C) prior to microbiological analysis. In this report, the yielded of steamed SHF extract is called as SSHFE.

The triplicate SSHFE were grouped into four storage stability treatments: 0; 1; 4 and 8 weeks after out from freezer. The culture media without SSHFE were used as control samples. The series of microbiological and the storage stability test were employed to each  sample included total plate count (TPC);   most probable number (MPN) of coliform and Eschericia coli test using Gram staining  and Indol, Methyl Red, Voges Praskauer, Citrate (IMVIC) .

Total Plate Count (TPC) testing was performed in accordance with the procedure of microbial contamination test of  Indonesia National Standard: SNI No.01-2897-1992. The 10 mL SHF extract sample were homogenized in  90 ml of  peptone dilution fluid (PDF), and serial  of 10-fold dilutions (from 10-1 until 10-7) were prepared. The 1 mL aliquot of each dilution was placed into a sterilized petri dish  then added with 15-20 mL of plate count agar (PCA)  then shaken homogeneously and incubated for  48 hours at 37°C. The estimated number of bacteria were computed by multiplying the average number of colonies that appear on each plate with the dilution factor recorded and reported as colony forming units (CFU) (Bambang, 2014; Damongilala, 2009).

Enumeration of Total Coliform Count was conducted using five tubes of most probable number (MPN) method  according to procedure of  analysis colifom and E. coli in fish product that stipulated by Indonesian National Standard SNI 01-2332.1, 2006;  Bartram and  Pedley, 1996).

The presumptive test:   10 ml of SHF extract samples was homogenized with  90 ml of Butterfield’s Phosphate Buffered diluent solution,  then three  consecutive of 10-fold dilutions (10-1; 10-2; 10-3) were prepared. Using sterile pipettes , transfer 1 ml aliquot of each dilution to each of 5 tubes of Lactose Broth (LB) which containing the Durham’s  tube. The tube was incubated for 48 hours ± 2 hours at 35°C ± 1°C and the gas production during 24 hours in Durham’s tube and color change of the media (more turbid) were observed.  The positive tube is characterized by turbidity and gas production in Durham’s tube. Furthermore, the enumeration of Coliform was determined based on the number of positive results from each tube set and compare with MPN standard chart that stipulated by SNI (2006), to give presumptive coliform count per gram sample.

Confirmed Test of Escherichia coli: A positive presumptive test were inoculated in selective media of eosin methylene blue agar (EMBA) and incubated at 35°C for 24 hours.  The presence of Escherichia coli  colony in the media showed the distinctive (typical) characteristic (the black color  in the middle with or without metallic green).  Presence of typical colonies confirms positive coliform test. Each culture that  positive coliform bacteria was inoculated on Eosin Methylene Blue Agar (EMBA) medium, and then incubated at 37°C for 24 hours. Then, the green colonies with metallic luster and greenish spots on EMBA media were inoculated  on nutrient agar (NA) medium (SNI, 2006). After incubation at 45 °C for 24 hours, medium were tested with gram staining and IMViC assay according to  Harley  and Prescott (2002).

Results

All Result of evaluation the bacterial colony growth using TPC method is presented at  Table 1.

Table 1: Result of TPC:  Bacterial Colony Growth

The control media which not added by  SSHFE at storage stability treatments of 0;1;4,8 weeks were  negatively contaminated  by bacteria ( <10² CFU/ml). It is indicated that the media that used in this research were not contaminated. Media with SSHFE at storage of 0 week after out  from freezer also negative ( <10² CFU/ml).  Whereas on the media that added with SSHFE at storage of 1; 4; 8 weeks after out from freezer, were positively contaminated in level of 1.722×10³ CFU/ml; 1.327×10⁴. CFU/ml and 6.839×10⁴ CFU/ml, respectively. Refer to National Standardization Agency (BSN) these bacterial colony number were still below threshold that stipulated at level of 5×10⁵ CFU/ml.  However, bacterial colonies in SHF extract in the storage of 1; 4; 8 weeks possibly contain coliform bacteria, hence  the coliform test is necessary required.

The MPN test were performed toward the media that containing the bacterial colony , the media that containing  SSHFE at the storage of 1;4;8 weeks. According to the  presumptive test, showed that SSHFE at the storage of 1;4;8 weeks  were positive results  of coliform. These status were indicated by the gas formation and turbidity in Durham’s tube within lactose broth media (LB)  that containing  SSHFE.  Gas and turbidity indicated that  coliform  bacteria  had  used lactose as a carbon source and did the fermentation process  that produced acid and gas (Dad, 2000).

Result of coliform enumeration based on the number of positive results in presumptive test that comparing with MPN standard chart is exhibited at Table 2. The MPN index in media with SSHFE is follow: 24/g (1 week), 350/g (4 weeks), and <1600/g (8 weeks), which all are exceed the Indonesian National standard that stipulated at level of <3/g. Furthermore, the confirmation test of coliform and E coli also shown the positive result. The gram stain and IMVIC test indicated that they were positively contaminated by Escherichia coli.

Table 2. Enumeration result of most probable number (MPN). The MPN index in media with
SSHFE are exceed the Indonesian National standard that stipulated at level of <3/g

Discussion

The media with SSHFE at room temperature with storage of  0 week was negatively contaminated  by bacteria (table 1). Furthermore, during storage  of 1; 4; 8 weeks were observed positively contaminated. The freezing storage prior to storage experiment influence the bacterial behavior.  Several studies reported that freezing technique (-20°C) can decreasing microorganisms in storing foods (Storper et al., 1982; Pankey et al., 1987; Schukken et al., 1989),  decrease in the number of samples positive for Escherichia coli and Arcanobacterium pyogenes (Schukken et al., 1989); lowering the number of   salt tolerant bacteria from 9.8×105 CFU/ml to 7.6×103 CFU/ml and Staphylococcus spp from (3.6x103CFU/ml) to 8.2x101CFU/ml (Alrabadi, 2015). Therefore, the appearance colony at 0 week storage cannot observed yet.

The prolonging of storage treatments is followed by the increasing of bacterial colony growth indicated that media with SSHFE were being suitable substrate for bacteria. Bacterial growth is affecting by several factors included the composition of substrate, initial contamination  and temperature (Koutsoumanis, 2006). SSHFE  consist of high nutrient uch as  proteins with albumin as a major fraction,  fat, glucose and some minerals Zn,Cu, and Fe (Nugroho, 2012;  Mustafa, 2012) hence supporting for bacterial growth. The E. coli initial contamination in SSHFE  also considerable since the SHF lives in river bottom substrate  (Sea Grant, 2008). The temperature influences  to bacterial growth  were shown when SSHFE were placed out from the freezer to room temperature for 1; 4; and 8 weeks. In the other hand, the contamination during the extraction process also considered.  Steam extraction at 60ºC for 30 minutes have risk not sufficient to kill bacteria.  To avoid the presence of E.coli and Salmonella spp. in meat,  it have been promoted to cook at temperatures ranging from 63 to 85°C  (Juneja et al. 1998; USDA, 2011)

Based on this research,  SSHFE is recommended to stored at freeze temperature and immediately consumed after melt in room temperature. Further sterilizing methods for SSHFE processing is suggested.

The research found that snakehead fish extract (SSHFE) that  yielded from distilled water steam extraction using temperature of 60ºC for 30 minutes have risk contaminated by E coli bacteria. SSHFE at the storage of 1;4 and 8 weeks after out  from freeze temperature contain coliform  with MPN range of 24/g -1600/ g which  exceeded Indonesian  National Standard.

Acknowledgements

This research was supported by Indonesia Ministry of Research Technology and Higher Education with contract number: 80/Addendum/ITS/2017. Research was performed in Biology Department Institut Teknologi Sepuluh Nopember (ITS) – Surabaya Indonesia in the Laboratory of Zoology and Animal Engineering; Laboratory of Microbiology. We declare no conflicts of interest.

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