THE NUTRITIONAL VALUE, BACTERIAL COUNT AND SENSORY ATTRIBUTES OF LITTLE TUNA (EUTHYNNUS AFFINIS) FLOSS INCORPORATED WITH THE BANANA BLOSSOM

The study aimed to evaluate the addition of banana blossom (12.5, 25, 37.5, and 50% w/w) on nutritional quality, histamine content, bacterial count, and sensory characteristic in the fish floss prepared from little tuna (Euthynnus affinis). The crude protein content, essential amino acids, lipid, and polyunsaturated fatty acids (PUFA) steadily decreased (p <0.05), while the crude fibre, carbohydrate, and ash components of the tuna floss, increased significantly (p <0.05) with increasing levels of banana blossom. The contents of protein, fat, ash, fibre, carbohydrate, and moisture ranged 28.13 – 30.27%, 14.79 – 18.02%, 4.45 – 5.68%, 2.6 – 3.5%, 27.81 – 31.01, and 16.45 – 17.39%, respectively, and most of them met the Indonesian National Standard. For essential and non-essential amino acids, the level varied about 102.82 mg.g-1 to 206.76 mg.g-1 and 79.71 mg.g-1 to 138.76 mg.g-1, respectively in the treated tuna flosses. Moreover, ranging 13.72 – 16.29% of PUFA was found in all treated flosses. The most significant effect was found in the histamine levels of the tuna flosses, especially in the 50% added floss sample. Moreover, bacterial counts and heavy metals content were lower than the maximum limits regulated by the Indonesian National Standard. For sensory evaluation, the banana blossom-added samples significantly increased (p >0.05) the acceptability score for all attributes assessed. Taken together, the tuna floss added with 37.5% of banana blossom may be potentially developed as a low-histamine tuna-based product with high ffibre andEPA+DHA, as well as highly acceptable for consumers.


INTRODUCTION
Banana blossom (Musa acuminata), also commonly called banana flower, is considered as a leftover product after cultivation, which is widely consumed as a vegetable in the Southeast Asian countries, including Indonesia, Malaysia, Thailand, and the Philippines (Liyanage et al.,2016; Wahab, Ismail and Abidin, 2020). Banana blossom contains high nutritional quality, especially dietary fibre. According to Sharma, Shukla and Golani (2019), the banana blossom (100g) contained dietary fibre (5.74g), protein (1.6g), fat (0.6g), carbohydrate (5.7g), calcium (73.3mg), phosphorous (56.4mg) and vitamin E (1.07mg). Dietary fibre plays an essential role in lowering serum cholesterol levels, preventing obesity, normalizing blood glucose and insulin levels (Bhaskar et al., 2012; Elaveniya and Jayamuthunagai, 2014). In addition, banana blossom possesses some bioactive components, such as vitamin C, saponin, tannin, flavonoid, alphatocopherol, and myoinositol phosphates, which could promote health benefits (Somsub et al., 2008;Sheng et al., 2010). Thus, due to being rich in a range of nutrients and biologically active components, the banana blossom has gained considerable attraction as an alternative source of functional food ingredients.
Abon, also known as shredded fish or fish floss, is one of the traditional dried meat products popular among Indonesia and the Asian community. It is recognized by different local names such as serunding in Malaysia, mahu in the Philippines, moo yong in Thailand, thitheotieu in Vietnam, and rousing in China (Hang, 2015). Popular raw materials for producing floss are beef and chicken; however, some fish species are also suitable materials for shredded meat processing. The processing of floss product generally begins with steaming of washed meat until it is tender. The steamed meat is then shredded finely and mixed with some formulated spices, followed by adding coconut milk. After that, the mixture is fried and stirred continuously under heat until the mixture is dry. Finally, excessive oil in the dried meat product is then separated and removed automatically (Huda et al., 2012). Shredded

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Scientific hypothesis
There is a lack of information regarding the use of banana blossom incorporated with tuna floss in increasing fibre content and decreasing histamine levels. Therefore, this study hypothesized that incorporating little tuna floss with banana blossom may give favorable nutritional features, particularly enhancing fibre content and lowering histamine levels. Banana blossom was added up to 500 g.kg -1 in the formulation of little tuna floss. Nutritional aspects such as chemical composition, amino acids, fatty acids, and dietary fibre contents of the formulated little tuna floss were analyzed. Heavy metals content as safely required in the food developments was evaluated. The histamine content and pathogenic bacteria test were also determined. In addition, the sensory properties of the little tuna floss were studied to provide a basis for consumer acceptance and commercial applications. This study may stimulate further study in developing healthy fish floss products for nutritional and functional applications.

Samples
Little tuna (E. affinis) used in this study was obtained from Sedang Biru fish market (Malang, East Java, Indonesia). The size of little tuna samples was approximately 202.8 ±3.3 g and 27.6 ±1.9 cm for weight and length, respectively. Banana blossom (M. acuminata) was purchased from a local market (Malang, East Java, Indonesia), and its weight was around 2.2 ±0.2 kg.

Chemicals
All chemicals and reagents used were of analytical grade. H2SO4, petroleum ether and nitric acid (Merck, Germany) were supplied from a local supplier. Kjeldahl catalyst selenium tablet (Fisher Chemical, USA).

Bacteria and biological material
Bacteria used, including Escherichia coli, Salmonella sp. and Staphylococcus aureus were obtained from the Indonesian Culture Collection (InaCC), Research Center for Biology, Indonesian Institute of Sciences (LIPI). Microbial media used in a recent study were Oxoid (Basingstoke, UK)-based Nutrient agar (NA), MacConkey agar (MCA), Rappaport-Vassiliadis broth (RV), Xylose Lysine Deoxycholate (XLD) agar and Baird Parker agar (BPA).

Description of the experiment Sample preparation
Around 15 kg of little tuna samples were purchased from Sendang Biru fish market (Malang, East Java, Indonesia). Fish samples were kept under cold conditions in an insulated box with ice during transportation (around 2-3 h). Upon arrival, the little tuna samples were washed in running water and were then filleted manually using a sharp knife. The filleted samples, including white and dark muscles, were stored in a freezer until use. For banana blossom, about 5 pieces of samples used were was obtained from a local market (Malang, East Java, Indonesia). Due to fresh banana blossom samples used, the Volume 15 848 2021 detailed preparation was described in the following procedure for addition into little tuna floss. Table 1 presents the distinct formulations for all little tuna floss samples. The banana blossoms added with banana blossom in preparing little tuna floss products were 12.5% to 50% based on the weight of the whole little tuna sample ( Figure 1). The little tuna meat used consists of white and dark muscles with distinct proportions i.e., 80% and 20%, respectively. These treatments were chosen in compliance with the formulated proportion for fish floss recommended by the previous study from Puspita, Kartikaningsih and Dayuti (2019). The control little tuna floss (0%) was used as a control and compared to the treated little tuna floss samples (12.5 -50%) to evaluate the distinct characteristics of the formulation process and its corresponding attributes on the treated little tuna floss produced. During preparation, the thawed little tuna muscles were steamed at 100°C for 15 min (Halco, Indonesia). Separately, the banana flowers were cut into small sizes and then steamed at the same condition of fish treatment. After the steaming process, the steamed tuna was shredded using a fork and all ingredients used were thoroughly mixed into the pan. Afterward, the mixture samples were fried and stirred continuously under heat until the mixtures were dried. The excessive oil in the dried fish floss products was then separated and removed automatically using a spinner machine (Spiner Abon, Indonesia). All experimentations were carried out in triplicate.

Proximate analysis
The proximate analysis of little tuna floss samples was determined according to the method of the Association of Official Analytical Chemists (AOAC, 2000). The Kjeldahl method was used to obtain the crude protein from all dried fish floss samples, while fat content was analyzed using the Soxhlet extractor method. The ash and moisture contents of the samples were examined by the gravimetric method. Carbohydrate content was measured by subtracting total crude protein, fat, ash, and moisture contents from 100%. For energy value (kcal.100g -1 ), the calculation used the following formula provided by AACC (2000): Energy value = (4 × carbohydrate content) + (4 × protein content) + (9 × lipid content) (1)

Dietary fibre analysis
The dietary fibre analysis used in this study referred to the method of AOAC 985.29 (AOAC, 2000). The little tuna floss samples were dried at 100 °C to constant weight using air-oven (M720, Binder, Germany). Around 0.5 g of all samples were added α-amylase enzyme and kept in accordance with its optimum condition (viz. pH 6; at 100 °C, for 30 min). Afterward, the treated samples were added protease with incubating at 60 °C for 30 min at optimum pH (7.5). The protease-treated samples were then added amyl-glucosidase and incubated at pH 6 for 30 min at optimum temperature. Total dietary fibre of all treated samples was measured by precipitating using ethanol, followed by washing and drying. The obtained residues were then weighed manually.

Amino acids analysis
The amino acids composition was analyzed using ultrapressure liquid chromatography (UPLC) (Waters, US) according to the method of Nollet and Fidel (2015). About 5 mL of hydrochloric acid 6 N was mixed to 0.1 g of samples. The prepared samples were then hydrolyzed at 110 °C for 24 h (M720, Binder, Germany). The hydrolysed samples were transferred to prepared distilled water. After that, the mixtures were filtered with a 0.45 μm filter paper. The 500 μL filtrates were mixed with 40 μL of 2-Amino-4-boronobutanoic acid (ABBA) and 460 μLof double distilled water. Then, 10 μL of the solution was mixed with 70 μL of AccQ Fluorine Borate and 20 μL of fluorine. The homogenized solutions were incubated at 55 °C for 10 min. Finally, the solutions were injected into the UPLC system to calculate the amino acid composition.

Fatty acids analysis
The fatty acid composition of little tuna floss was analysed by PT. Saraswanti Indo Genetech, Bogor Indonesia. Fatty acid methyl esters (FAMEs) from extracted fat of tuna floss samples were prepared by basic transesterification following the official method (AOAC, 2000), using hexane and hydroxide potassium 2N.

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FAMEs were analyzed by gas-chromatography (GC) (Agilent Technologies, California, US) described by Aquilani et al., (2018). The GC with a flame ionization detector (FID) was used to analyze the amount of fatty acids in the little tuna floss samples. The GC analysis was conducted using capillary column polyethylene glycol, equivalent variants CP-Wax 52 CB (30m × 0.25mm × 0.25µm film thickness). The carrier gas was nitrogen at a flow rate of 1 mL.min -1 . The initial oven temperature was set at 120 °C with an increase of 5 °C per minute until reached 240 °C. The injector and detector temperatures were 260 °C. Individual peaks were measured by comparison of their retention times (RT) with those of standards. Peak areas were calculated, and FAMEs were expressed as the area percentage of total area FAMEs (%).

Heavy metal analysis
Heavy metals analyzed in this study were composed of cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg), and tin (Sn). These microelements were conducted by the method of AOAC (2000) using the Atomic Absorption Spectrophotometer (AAS) (GBC Scientific Equipment, USA).

Histamine determination
The histamine content was carried out by High-Performance Liquid Chromatography (HPLC) (Agilent Technologies, California, US) according to the method from Muscarella et al., (2005). Around 10 g of homogenized little tuna floss was extracted by 20 mL of 5% trichloroacetic acid (TCA). After filtration, 1 mL of the extracted sample was purified with 2 mL of chloroform solution. The mixture was then centrifuged and 20 µL of the supernatant was analyzed by HPLC. The HPLC column used was a Supelcosil LC-ABZ 4.6 × 150 mm, 5 µm film thickness (Supelco, Bellefonte, PA, USA). The decansulphonate phosphate buffer pH 6.9/acetonitrile mixture (85/15) was used as a mobile phase. Isocratic elution was used; the flow rate was 1.2 mL.min -1 . The UVDAD was regulated at the absorption wavelength of 210 nm; the injection loop was 20 µL.

Bacterial count
Bacterial evaluations in the tuna floss consist of total bacteria count, total coliform, Escherichia coli, Salmonella sp., and Staphylococcus aureus with the methods developed by Anang et al. (2018) with some modifications. For total bacteria count, serial dilutions of 10 -1 to 10 -5 were prepared by diluting around 1 g of the little tuna floss samples into 9 mL of distilled water. Approximately 0.1 mL of aliquots from 10 -3 to 10 -5 dilutions were inoculated into Petri dishes containing the Nutrient Agar media. The plates were then incubated at 37 °C for 24 h. After incubation, bacteria colonies formed were counted using the colony counter and recorded as total viable counts. Total coliform was analyzed by dropping 0.1 mL of diluted samples into MacConkey broth with Durham tubes and incubated at 37 °C for 24 h. After incubation, the inoculated tubes were identified as positive total coliform by changing color from purple to yellow, and gas was collected in the Durham tubes. The positive tubes were then transferred into a 5 mL test tube of tryptone solution and incubated at 44 °C for 24 h to test E. coli. Afterward, a drop of Kovacs' reagent was added into the treated tubes. The suspected E. coli in each tube was shown by a red ring color development, indicating the presence of indole. Salmonella sp., the count was evaluated using pre-enrichment of bovine peptone water, followed by enrichment of Rappaport-Vassiliadis (RV) broth. After enrichment, around 0.1 mL of aliquots were inoculated into Xylose Lysine Deoxycholate (XLD) agar. The suspected Salmonella colonies appear colorless with a black center because of H2S production. For S. aureus count, about 1 g of fish floss samples were added into test tubes containing 9 mL of distilled water and the mixtures were serially diluted from 10 -1 to 10 -5. Around 0.1 mL of aliquots (10 -3 to 10 -5) were inoculated into plates prepared with Baird Parker Agar media. Afterward, the inoculated samples were incubated at 36 °C for 48 h. After the incubation period, colonies formed were identified with the suspected colonies were round, convex with a diameter of 2 -3 mm, greyish black color with a clear circle (halo).

Sensory evaluation
Sensory parameters, including appearance, aroma, color, taste, and texture were evaluated using 30 untrained sensory panelists randomly selected among students and this method was adopted from Sęczyk, Świeca and Gawlik-Dziki (2016). The panelists were initially presented with the little tuna floss samples in identical but labeled containers with a three-digit code for their evaluation. Before the sensory session, the tuna floss samples were prepared in triplicate in a randomized permutation. This study used a hedonic test with a 9-point scale to obtain the acceptability score of the little tuna floss products. The likeness scale was arranged in accordance with the above sensory parameters was as follows: 1 = dislike extremely; 2 = dislike very much; 3 = dislike moderately; 4 = dislike slightly; 5 = neither like nor dislike; 6 = like slightly; 7 = like moderately; 8 = like very much; 9 = like extremely.

Statistical analysis
The experimental design applied in this study was a completely randomized design (CRD). All measurements were performed in triplicate. Data were expressed as the mean values ± standard deviation (SD). The differences were determined using a one-way analysis of variance (ANOVA), followed by Duncan's test. The significant difference was established at p <0.05 using SPSS, Version 27, statistical software program (SPSS Inc., Chicago, Ill., USA).

Proximate composition
The proximate composition of little tuna floss (as a control) and floss samples incorporated with banana blossom with different levels (12.5 -50%) is presented in Table 2 and its fibre content was higher than the values determined by SNI and other fish floss products. The higher fibre content is an indication of the better quality of the products with the potential health benefits as above mentioned. The contents of ash, carbohydrate, and moisture increased significantly (p <0.05) with incorporating banana blossom in the little tuna flosses. In terms of ash content, the formulated floss samples agreed with the SNI requirements (>7) for the standardized fish floss product. Moreover, the energy values of treated samples (285.28 -303.69 kcal) were higher than the control (271.66 kcal); but these values were slightly lower than the dietary requirements recommended by the FAO/WHO/UNU (2007) for children and young adult foods. The high energy level and protein content in the tuna floss added with banana blossom indicates its suitability as a supporting diet for children and young adults.
The effect of the addition of banana blossom into a tuna floss formula on histamine content is also depicted in Table 2. Interestingly, the histamine contents of treated tuna floss samples decreased significantly (p <0.05) with increasing the level of banana blossom. Compared to the control sample (319.48 mg.kg -1 ), around 2-fold (144.16 mg.kg -1 ) to 6-fold (52.25 mg.kg -1 ) of histamine content decreased with the increment of at around 12.5% to 50% into tuna floss samples, respectively. The reduction of histamine in the treated tuna floss might be due to the ratio of banana blossom and tuna as main materials in the development of little tuna floss products, in which a higher banana blossom was added, a lower content of histamine found in the tuna floss samples. These results were lower than found in the tuna dumpling (1608 mg.kg -1 ) studied by  Table 3. presents the profile of amino acids found in the control sample and the tuna floss incorporated with different concentrations of banana blossom. Results exhibited that glutamic acid (Glu) was the most dominant amino acid in all little tuna flosses. However, 15-amino acid analyzed in the control sample was a higher amount of total amino acids both essential and non-essential amino acids than found in the treated tuna floss products. This is due to the lower amino acids content observed in banana blossom incorporated with tuna floss samples (Table 3). In terms of essential amino acids, the control and treated samples showed higher values compared to the nonessential amino acids. Furthermore, the content of essential amino acids, including arginine, histidine, isoleucine. leucine, lysine, phenylalanine, threonine, and valine, found in the treated tuna floss samples did not meet the standard recommended by Food and Agriculture Organisation/World Health Organisation/United Nations Nevertheless, fish floss is a non-staple food product like bread, noodle, and rice, which is routinely consumed in large quantities to provide adequate energy, but it is generally combined with other staple foods to enhance nutritional values.

Fatty acids composition
The effect of the formulation of tuna floss with banana blossom on the fatty acid composition is presented in Table 4. The content of fatty acids in the tuna floss samples, both control and treated samples with different levels of banana blossom, varied considerably (p <0.05). The most abundant fatty acids in all treated floss samples were oleic acid (C18:1) (34.03 -40.63%), followed by palmitic acid (C16:0) (27.19 -31.84%) and linoleic acid (C18:2) (11.20 -13.30%), but these fatty acids were slightly lower compared to the control sample's fatty acids. It might be due to a low-fatty acids content observed in the banana blossom (0.39 -1.28%). In addition, all tuna floss incorporated with banana blossom had lower content of saturated (33.84 -41.44%), monounsaturated (34.37 -41.00%), and polyunsaturated (13.72 -16.29%) fatty acids than those contained in the control. However, among the identified fatty acids, omega-3 (n-3) and omega-6 (n-6) fatty acids seem to be the most important, due to their multiple biological roles, such as reducing oxidative stress, influencing the inflammatory cascade, presenting neuroprotection, and cardiovascular protection. The total n-6 and n-3 in the treated samples ranged at around 11.28 -13.40% and 2.44 -3.23% respectively, with an n-6/n-3 ratio of about 3.89 -4.62%. These values within the range (1 -5) of omega-6 and omega-3 ratio per day recommended by some food experts that should be consumed to prevent undesirable diseases related to the lack of essential fatty acids intake (EFSA, 2010). Moreover, amongst n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are required essentially by the human body and approximately 0.2-2.0 g/day recommended by most health organizations (Desai et al., 2018), and all formulated tuna floss samples met the requirements.

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Compared to the control treatment, all identified elements were relatively the same in the contents except amount of Sn, which the content of Sn increased significantly (p <0.05). However, all heavy metals contained in the treated samples agreed with the Indonesian National Standard for fish floss specification (SNI 01-3707, 1995). It indicates that the tuna floss incorporated with a banana blossom is potentially safe for human beings.
Bacterial test Table 6. shows the bacterial counts for all tuna floss samples evaluated with different tests, including total count, Salmonella sp., E. coli, S. aureus, and total coliform. For total bacterial count, a non-selective media was used, and the results presented that no significant difference (p >0.05) was exhibited in the total bacterial count and slightly higher compared to scad fish (Decapterus sp.) floss (Kasmiati et al., 2020). However, all treated samples did not exceed the maximum permissible limits of microbial contamination in food products regulated by the Indonesian National Standard (SNI 7988, 2009). The identification of Salmonella sp., E. coli, S. aureus in the tuna floss samples using specific media showed no growth (negative) after incubation and these results agreed with the requirement for microbial contamination in the dried fish product (SNI 7988, 2009). Furthermore, all treated samples, both augmented and unaugmented with banana blossom, showed a similar count (<3 MPN.g -1 ) to the total coliform. These results revealed that the total coliform in all tested samples were within the acceptable microbial quality of the Indonesia National Standard. From those microbial evaluations, the tuna floss product used in the present study agrees with the SNI 7988: 2009.

Figure 2
Physical appearance and sensory attributes of control tuna floss and tuna floss incorporated with different levels (12.5 -50%) of banana blossom. The same letters denote the lack of statistically significant differences between the results at p <0.05 (n = 30).

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Assessment of sensory attributes (appearance, aroma, color, taste, texture, and overall quality) of tested tuna floss by the panelist is depicted in Figure 2. Generally, the addition of banana blossom at the level from 12.5% to 50% to tuna flosses had a significant influence (p <0.05) on its sensory characteristics and consequently on consumer acceptance. Amongst these formulations, the 37.5% added floss showed the highest scores in almost assessed attributes compared to other treatments and control samples.
In addition, the mean score of overall acceptability in the tested floss samples was approximately 7.11, which implies that the treatment of tuna flosses is preferred moderately among the consumers. This acceptability value agreed with the requirement recommended by the Indonesian National Standard (5) (SNI 01-3707, 1995), and even much higher than the score accepted by the SNI. Also, these results are in accordance with the studies from Candra and Tunoq (2018)

CONCLUSION
Taken together, the addition of tuna floss with different levels (12.5% -50%) of banana blossom reduced histamine level and increased the dietary fibre content in the little tuna floss samples. In addition, microbial and toxic elements were permissible limits regulated by the Indonesian National Standard (SNI) in all the treated tuna flosses. Our results suggested that 37.5% of banana blossom incorporated into the tuna flosses was selected as an appropriate formula due to its attributes, such as being more acceptable for overall sensory evaluation, high contents of EPA+DHA, and dietary fibre, as well as a low histamine content. However, our further research concerns the nutritional value of the selected tuna floss sample, especially on the enrichment of essential amino acids composition through fortification strategy. Also, functional properties like antioxidant and antimicrobial activities will be further studied.