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1 DIVERSITY OF LACTIC ACID BACTERIA IN INASUA FERMENTATION

3 Ferymon Mahulettea, Nisa Rachmania Mubarikb*, Antonius Suwantoc, Widanarnid

5 1. Ph.D student, Departement of Biology, Bogor Agricultural University,

6 Departement of Biology, Faculty of Mathematics and Natural Sciences,

7 Bogor Agricultural University, Agathis Street, Kampus IPB Dramaga, Bogor, 16680,

8 Indonesia [email protected]

10 2. Department of Biology, Faculty of Mathematics and Natural Sciences,

11 Bogor Agricultural University, Agathis Street. Kampus IPB Dramaga, Bogor, 16680,

12 Indonesia [email protected] ; Phone : +62 858 8028 1352

13

14 3. Professor, Department of Biology, Faculty of Mathematics and Natural Sciences,

15 Bogor Agricultural University, Agathis Street. Kampus IPB Dramaga, Bogor, 16680,

16 Indonesia [email protected]

17

18 4. Professor, Department of Aquaculture, Faculty of Fisheries and Marine Sciences, Bogor

19 Agricultural University, Agathis Street. Kampus IPB Dramaga, Bogor, 16680, Indonesia

20 [email protected]

21

22 * Corresponding author

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27 Abstract

28 Inasua was a traditionally product of wet salt fish fermentation produced by Teon, Nila,

29 and Serua (TNS) communities in Central Moluccas at Indonesia. This product consists of two types,

30 namely inasua with sap and inasua without sap. The objective of research was to study the

31 diversity of lactic acid bacteria during inasua fermentation. The diversity of lactic acid bacteria

32 was studied based on the presence of the 16S rRNA gene. The succession of lactic acid bacteria was

33 strongly influenced by the physicochemical characteristics during fermentation. The dominant

34 bacteria in inasua fermentation were Lactobacillus plantarum. At the end of fermentation, the

35 inasua with sap were dominated by L. plantarum whereas inasua without sap dominated by

36 Leuconostoc mesenteroides. Lactobacillus paracasei was a lactic acid bacteria found only in

37 inasua with sap. The result of Denaturing Gradient Gel Electrophoresis (DGGE) showed that the

38 dominant bacteria in inasua with sap were Lactobacillus, whereas in inasua without sap were

39 Staphylococcus. The bacterial diversity index in inasua with sap was higher whereas the evenness

40 and dominance index was lower than the inasua without sap. The pattern of succession of lactic

41 acid bacteria during fermentation and bacteria composition of inasua with sap was more complex

42 than inasua without sap.

43 Key words : Fermented fish, succession, DGGE, dominance index

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45 Introduction

46 Fish is the main source of protein consumed by the people of Moluccas. The catch of

47 fish is often abundant, so it is necessary to preserve efforts. One of the fish preservation

48 techniques by fermentation is inasua. Inasua is the local wisdom of Teon, Nila, and Serua

49 communities. Beside used as a food reserve during the lean time (17), this fermentation

50 product was used by the communities as a food stock in shipping to sell cloves to other

51 islands in the past. The raw materials used for the processing of inasua are reef fish, salt,

52 and coconut sap. Processing of inasua does not always use coconut sap. In certain

53 conditions fermented fish processing is only using salt as a preservative so known inasua

54 that use coconut sap (designated as inasua-S) and inasua that use salt only (designated as

55 inasua NS). Both of these fermentation products have different sensory characteristics and

56 shelf life.

57 The sensory characteristics and shelf life of a fermentation product are strongly

58 influenced by microbial diversity. Inasua fermentation takes place spontaneously and

59 involves various types of microbes. The main microbe involved in fermentation of fish is

60 lactic acid bacteria (23). The bacteria belong to the category of Generally Recognized as

61 Safe (GRAS) so it is safe to be in a food product (27). The composition of lactic acid

62 bacteria in traditionally fish fermentation is highly determined by the type of carbohydrate

63 and the amount of salt added (29). Lactic acid bacteria that found in inasua fermentation

64 can be developed as a starter in various fermentation products.

65 One method for detecting microbial diversity in a fermentation product is DGGE

66 (19). The results of this analysis can determine the safety aspects of inasua. The objective

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67 of the research was to study the succession of lactic acid bacteria during fermentation and

68 microbial composition in inasua with sap and inasua without sap.

69 Materials and Methods

70 2.1. Inasua Sampling

71 The sample of inasua was taken from traditional producer in Layeni village, Sub

72 district of TNS Waipia, Ceram Island consisting of producer in both inasua with sap and

73 inasua without sap. A total of 5 kg of gurara fish (Lutjanus vitta L) obtained from the sea

74 around the Ceram Island processed into inasua by adding salt only (Inasua-NS).

75 Processing inasua-S also added coconut sap. Inasua that has been processed subsequently

76 allowed to fermentation at room temperature for 12 weeks in a jar. Analysis of succession

77 of lactic acid bacteria was done in the first week until 12 weeks after fermentation.

78 2.2 Isolation and Characterization of Lactic Acid Bacteria

79 A total of 25 g of sample was mixed with 225 ml of sterile peptone solution and

80 homogenized using a stomacher bags. One ml of the homogenized and diluted samples

81 were poured into Petri dishes, then de Man, Rogosa and Sharp agar (MRSA) media

82 containing 1% CaCO3 with 3%, 5% and 10% NaCl were poured on it and incubated at

83 room temperature for 48 hours. All the isolates obtained were stained with Gram and spore

84 staining, catalase test, and fermentation of carbohydrates (6).

85 2.3 Extraction and Amplification of 16S rRNA Gene Lactic Acid Bacteria

86 DNA extraction was done according to the procedure of Presto TM Mini GDNA Kit

87 (Geneaid). The result of DNA extraction obtained were used to amplify the 16S rRNA

88 gene. Amplification of 16S rRNA gene using PCR machine with primer 63F (5'-CAG GCC

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89 TAA CAC ATG CAA GTC-3 ') and 1387R (5'-GGG CGG WGT GTA CAA GGC-3') (18).

90 The volume of PCR reaction used was 25 μL consisting of 12.5μL of GoTag Green Master

91 Mix 2X (Promega, Madison, W1, USA); 2.5 μL primary 63F and 1387R (concentration 10

92 pmol); 6.5 μL Nuclease Free Water and 1 µL DNA genome as template. The reaction were

93 amplified over 30 cycles. The condition of PCR were pre-denaturation at 95 0C for 5 min,

94 annealing at 550C for 1 min, elongation at 720C for 1,5 min, and extension at 720C for 10

95 min. PCR products were visualized using a electrophoresis machine at 80 volt for 45 min

96 and stained with ethidium bromide.

97 2.4 Construction of Phylogeny Tree

98 The amplified DNA was further sequenced and analyzed using ChromasPro software

99 (Technelysium, AU) for sequencing coupling. The sequence then compared with the

100 GenBank database using the Basic Local Alignment Search Total Nucleotide (BLASTN)

101 software. The homologous sequences then aligned using MEGA 6.0 software (30) with a

102 bootstrap value of 2000 times. Phylogenetic tree construction using Neighbour Joining

103 method.

104 2.5 DNA Extraction and Amplification of Bacterial Genomes from Inasua

105 The DNA isolation of all samples that have been fermented for 3 months based on

106 the Food DNA Isolation Kit protocol (Norgen, Thorold, ON, Canada). Amplification of the

107 16S rRNA gene used a PCR machine to detect bacteria in inasua. Amplification was

108 performed using primers P338F-GC (5'-CGCCCGCCGCGCGCGG-CGGGCGGGG-

109 CGGGGGCCCGGGGGGACTCCGGGAGGCAGCAG-'3) and P518R (5'-ATTA-CCGC-

110 GGCTGCTGG-'3 (24). Reaksi PCR using a mixture consisting of 1 ml of the DNA

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111 template , 2 μl each primer (20 pmol), 1 μl dNTP (100 mM for each dNTP), 5 μl 10 x PCR

112 buffers, 0.25 μl taq polymerase and 40.7 μl H2O. The reaction were amplified over 30

113 cycles. The condition of PCR were initial denaturation at 94 0C for 5 min, denaturation at

114 940C for 30 sec and annealing at 55 0C for 30 sec, then elongation at 72 0C for 30 sec and

115 extension at 720C for 7 min. PCR products were visualized using a electrophoresis machine

116 at 80 volt for 45 min and stained with ethidium bromide.

117 2.6 Denaturing Gradient Gel Electrophoresis (DGGE)

118 The amplified results of 20 μl of DNA mixed with 4 μl of loading dye were migrated

119 to a 6% (b / v) polyacrylamide gel in a TAE 1x buffer (pH 7, 10mM sodium acetate, 0.5

120 mM Na, -EDTA) with a gel prepared from 30 -70% (b / v) acrylamide stock solution

121 (acrylamide-N, N'-methylene bisacrylamide, 37.5: 1) and contains denatures (100%

122 denatures: 7 M urea and 40% (v/v)formamide) (20 ).

123 2.7 Analysis of Microbial Diversity

124 Analysis of diversity index based on interpretation of CLIQS ID software. Analysis

125 of relative abundance and OTU dominance values of DGGE results using past3 software

126 (7). Microbial diversity was analyzed using the Shannon-Wienner diversity index obtained

127 based on the OTU richness and the proportion of abundance of each OTU. The formula of

128 Shannon-Wienner index was H '= -Σ (pi log pi). H = = diversity index, pi = the proportion

129 of the number of individuals of a OTU to the total number of individual samples in the plot

130 (n/N) (11)

131 Results

132 1. Diversity of Culturable Lactic Acid Bacteria

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133 A total of 53 isolates of lactic acid bacteria were isolated during inasua fermentation

134 consist of 25 isolates from inasua-NS and 28 isolates from inasua-S. Sequences analysis of

135 gene encoding 16S rRNA with data on GenBank using the BLAST-N program showed that

136 all isolates were lactic acid bacteria that were closely related to the Lactobacillus and

137 Leuconostoc groups, except 3 isolates found at the end of the fermentation of the inasua

138 without sap that were closely related to the Staphylococcus group. The percentage of

139 sequence similarity with target of 16S rRNA gene in genBank database were 96-99%

140 (Table 1).

141 These bacteria undergo a succession during fermentation. In fermentation of

142 inasua-S, Lactobacillus plantarum and L. rhamnosus were lactic acid bacteria found at the

143 beginning of fermentation. After fermentation for 4 weeks found L. paracasei and

144 Leuconostoc mesenteroides at the end of fermentation. The dominant of lactic acid bacteria

145 in fermentation inasua-S was L. plantarum. Lactobacillus plantarum and L. rhamnosus

146 were also found at the beginning of fermentation inasua-NS. After fermentation for 8 week

147 found L. mesenteroides. The dominant of lactic acid bacteria at the end of fermentation

148 inasua-NS was L. mesenteroides (Figure 1).

149 Analysis of the phylogenetic tree showed isolates of lactic acid bacteria found in the

150 fermentation were association with Lactobacillus and Leuconostoc, except for 3 isolates

151 found at the end of inasua without sap fermentation closely related to Staphylococcus

152 (Figure 2).

153 2. Metagenomic Diversity

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154 The result of the separation of PCR products using DGGE showed that bacterial

155 community pattern based on 16S rRNA gene varied in both samples. The distribution

156 pattern of bacterial community in the inasua-NS (4 bands) was less varied than inasua-S

157 (8 band) in polyacrylamide gel (Figure 3). The comparision with database in GenBank

158 obtained 4 bands in inasua-NS were identified as Lactobacillus curiae, Staphylococcus

159 pasteuri and two other bands were S. epidermidis, while 8 bands in inasua-S were

160 identified as L. apinorum, Escherichia fergusonii, L. nagelii, L. paracasei, L. curiae and

161 two other bands were L. hilgardii. The percentage of similarity sequences of DGGE results

162 with the target genes in GenBank data were 90-99% (Table 2)

163 The phylogenetic analysis based on the 16S rRNA encoding gene constructed using a

164 neighbour joining model with bootstrap 2000x showed the bands found in two type of

165 inasua classified in the Lactobacillus, Staphylococcus and Escherichia groups (Figure 4).

166 3. Relative Abundance and Diversity of Microbial

167 The rank abundance curve showed the relationship between richness and evenness of OTU

168 in each inasua community (Figure 5). The index of Shanon-Winner diversity (H ') indicates

169 that bacterial community diversity of inasua-S was 1.42 (medium category), whereas

170 inasua without-NS was 0.90 (low category). The evenness and dominance index of these

171 two types of inasua was different. The evenness index of inasua-NS was 0.68 (high

172 category), whereas inasua nira was 0.52 (medium category). The dominance index of

173 inasua without sap was 0,52, whereas inasua with sap was 0,37 (Figure 6).

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176 Discussion

177 The succession of Lactobacillus, Leuconostoc and Staphylococcus during

178 fermentation was strongly influenced by the characteristics of inasua. The factor that

179 greatly affects the diversity of lactic acid bacteria in inasua-NS was salt content. In contrast

180 in inasua-S, the limiting factors that affect the microbial diversity were acid and alcohol

181 contents. Lactic acid bacteria have varying tolerance to salinity, acid and alcohol. One of

182 the lactic acid bacteria that tolerant of high salt, acid and alcohol and low oxygen

183 conditions was Lactobacillus (2). Lactobacillus usually grows optimum at 5-6% salt

184 content (28), tolerant to pH below 4.5 (3), and 4% alcohol content (8).

185 Lactobacillus plantarum was a dominant lactic acid bacteria found in fish (21). The

186 ability of L. plantarum utilizes several types of amino acids as substrates that cause these

187 bacteria to survive in fish with relatively low carbohydrate content (13). The optimum

188 growth at 4-6% salt and pH 4-9 causes these bacteria to be found in various ecological

189 niches (31). Other lactic acid bacteria found in fish are Lactobacillus rhamnosus (21).

190 Some of these bacterial strains have potential as probiotics (33). The dominant of lactic

191 acid bacteria at the end of fermentation inasua-NS was L. mesenteroides. These bacteria are

192 obligate heterofermentative bacteria (4). The increase of pH at the end of the fermentation

193 inasua supports the growth of this bacterium. Salt tolerant bacteria also found in inasua-NS

194 fermentation were Staphylococcus epidermidis, S. galinarum, and S. arlettae. High

195 tolerance of salinity causes these bacteria to be found in inasua-NS fermentation.

196 After 4 weeks fermentation found L. paracasei in inasua-S fermentation. The

197 presence of this bacterium was caused alcohol content that have decreased at the end of

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198 fermentation. Lactobacillus paracasei has a lower tolerance to alcohol than L. plantarum

199 (4). These bacteria play a role in the fermentation of coconut sap and as probiotics (22).

200 The dominant of lactic acid bacteria at the end of fermentation inasua-S still dominated by

201 L. plantarum. Besides being as dominant of lactic acid bacteria in fish, L. plantarum was

202 also the dominant bacteria in fermented sap (32).

203 The results of DGGE showed that most of the bacteria found in inasua-NS was

204 Staphylococcus. The addition of salt above 9% in fish fermentation can suppress the growth

205 of Lactobacillus which plays a role in fish fermentation and supports the growth of salt

206 tolerant pathogen bacteria, such as Staphylococcus (25). The high salt contents can inhibit

207 the growth of spoilage bacterial, but also cause the rate of fermentation to be slow (26).

208 Staphylococcus epidermidis and S. pasteuri naturally are not found in fish. The presence of

209 these two bacteria in fermented fish products because contact with humans during the

210 preparation and processing of less hygienic (14). The low water activity and high salt

211 content strongly support the growth of halophilic and halotoleran bacteria, including

212 Staphylococcus in fish fermentation (10)

213 Other bacteria that found in inasua-NS was L. curieae. This bacterium was found in

214 the two type of inasua. Lactobacillus curiae was lactic acid bacteria that lives in various

215 environments. The presence of L. curiae in some fermented products with salt addition

216 indicates that the bacteria was tolerant to salt during the fermentation.

217 The dominant of bacteria found in inasua-S was lactic acid bacteria. These bacteria

218 come from fish, sap and processing of inasua. Beside from the sources, the diversity of

219 lactic acid bacteria was also influenced by salinity, acid and alcohol contents during

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220 fermentation. The salt content of less than 7% can increase the growth of lactic acid

221 bacteria that play an important role in fish fermentation (25). Decreasing pH and high

222 ethanol contents at the beginning of fermentation was a limiting factor for the growth of

223 pathogenic and spoilage bacteria in inasua-S.

224 Lactobacillus apinorum, L. hilgardii, L. paracasei, L. nagelii and L. sucicola were

225 lactic acid bacteria found in fermented sap. The dominance of the bacteria from coconut

226 sap in inasua due to the fact that coconut sap contains simple carbohydrates easily utilized

227 by bacteria rather than the complex carbohydrates contained in fish. Lactobacillus

228 apinorum is commonly found in flowers and fruits with high sugar content. The bacteria

229 originally isolated from the honeybee stomach was a fructophilic lactic acid bacteria that

230 tends to utilize fructose rather than glucose as a substrate for its growth (16). Coconut sap

231 was a source of nutrients for microbial growth because it contains high fructose (1).

232 Lactobacillus hilgardii was also found in inasua-S fermentation. This bacteria was

233 often found in sap fermentation because it was resistant to high alcohol contents.

234 Lactobacillus hilgardii produce lactic acid and grow at an optimum pH below 4.5 (5). This

235 bacterium was a heterofermentative lactic acid bacteria. The ability of L. hilgardii to

236 produce alcohol and a variety of organic compounds might add to the sensory quality of

237 inasua-S.

238 The other lactic acid bacteria found in inasua-S was L. sucicola. Lactobacillus

239 sucicola (sucus = sap) was capable to produce lactic acid through homofermentative

240 pathways. These bacteria are commonly found in fermented sap to produce traditionally

241 alcoholic beverages (12). Tolerance to high alcohol and acid contents causes L. nagelii to

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242 grow in fermented sap (9). The last lactic acid bacteria found in inasua-S was L. paracasei.

243 This bacterium was also found in inasua-S fermentation with culture method since 4 week

244 until the end of fermentation.

245 The uncontrolled processing of inasua allow the presence of microbial contaminants

246 involved in fermentation. The presence of E. fergusonii in fermentation inasua with sap

247 probably comes from the process of sap tapping which was less aseptic or the process of

248 inasua less hygienic. The presence of these bacteria probably comes from the equipment

249 used in sap tapping (1). Escherichia fergusonii can be found in low salt fish processing

250 (15).

251 Conclusion
252 The succession of lactic acid bacteria during inasua fermentation is

253 strongly influenced by physicochemical characteristics of inasua. The

254 dominant lactic acid bacteria is Lactobacillus plantarum during

255 fermentation. At the end of inasua-S fermentation is dominated by L.

256 plantarum, whereas inasua-NS is dominated by Leuconostoc

257 mesenteroides. Lactobacillus paracasei is a lactic acid bacteria found

258 only in the inasua-S fermentation. The result of Denaturing Gradient Gel

259 Electrophoresis show that the dominant bacteria in inasua-NS is

260 Staphylococcus, whereas inasua-S is Lactobacillus. The bacterial

261 diversity index inasua-S higher than inasua-NS.

262 Acknowledgement

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263 The study was funded by the Ministry of Research, Technology and Higher Education

264 through Excelent Strategic Research Scheme on 2017 to second author and Domestic

265 Scholarship for Graduate Education 2014 to first author.

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275 LIST OF TABLE

276 Table 1 Bacterial isolates obtained from two type of inasua fermentation

Sample Isolate Description Identity (%)

Inasua- ITN-05 Lactobacillus rhamnosus NBRC 3425 99%

NS ITN-06 Lactobacillus plantarum CIP 103151 99%

ITN-12 Lactobacillus plantarum CIP 103151 99%

ITN-13 Lactobacillus rhamnosus NBRC 3425 99%

ITN-17 Leuconostoc mesenteroides ATCC 8293 99%

ITN-23 Staphylococcus epidermidis NBRC100911 99%

ITN-24 Staphylococcus galinarum VIII1 94%

ITN-25 Staphylococcus arlettae ATCC 43957 96%

Inasua-S IN-01 Lactobacillus rhamnosus NBRC 3425 99%

IN-05 Lactobacillus plantarum JCM 1149 99%

IN-13 Lactobacillus rhamnosus NBRC 3425 99%

IN-15 Lactobacillus plantarum CIP 103151 99%

IN-17 Lactobacillus paracasei NRBC 15906 98%

IN-19 Lactobacillus plantarum CIP 103151 96%

IN-27 Leuconostoc mesenteroides ATCC 8293 99%

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280 Table 2 Bands obtained from two type of inasua

Sample No. Band Description Identity (%)

Inasua-NS 1 Staphylococcus epidermidis NBRC 100911 98%

2 Staphylococcus epidermidis NBRC 100911 99%

3 Staphylococcus pasteuri ATCC 51129 95%

4 Lactobacillus curieae S1L19 95%

Inasua-S 4 Lactobacillus curieae S1L19 95%

5 Lactobacillus sucicola NRIC 0736 90%

6 Escherichia fergusonii ATCC 35469 98%

7 Lactobacillus apinorum Fhon13N 94%

8 Lactobacillus hilgardii NBRC 15886 94%

9 Lactobacillus hilgardii NBRC 15886 94%

10 Lactobacillus nagelii JCM 12492 86%

11 Lactobacillus paracasei NBRC 15889 94%

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287 LIST OF FIGURE

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295 (A) (B)

296 Figure 1 Sucession of lactic acid bacteria in two type of inasua.

297 Inasua-NS (A), inasua-S (B)

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321 Figure 2 The phylogenetic tree of bacterial isolates that obtained from two type of

322 inasua. Methanococcus vannielii as an outgroup

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334 (A) (B)

335 Figure 3 DGGE profiles of 16S rRNA in two type of inasua (A).

336 Ilustrations of DGGE banding patterns employing phoretix 1D software (B)

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368 Figure 4 The phylogenetic tree of 11 sequences of 16S bacterial origin rRNA

369 obtained from the DGGE analysis. Methanococcus vannielii as an

370 outgroup

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Abundance
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Abundance
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377 Rank of bands Rank of bands

378 (A) (B)

379 Figure 5 Rank abundance curve based 16S rRNA of OTU in two type of inasua.

380 Inasua-NS (A), Inasua-S (B)

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401 Figure 6 Index of diversity, evenness and dominance in two type of inasua

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