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1 Effect of solid-state fermentation on proximate composition, anti-nutritional

2 factor, microbiological and functional properties of lupin flour

3
4 Oladapo Oluwaseye Olukomaiyaa*, Oladipupo Qudus Adiamoa, W. Chrishanthi
5 Fernandoa, Ram Mereddyb, Xiuhua Lic, Yasmina Sultanbawaa
6 aCentre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and
7 Food Innovation (QAAFI), The University of Queensland, Coopers Plains, Brisbane,
8 QLD 4108 Australia
9 bQueensland Department of Agriculture and Fisheries, Coopers Plains, Brisbane,
10 QLD 4108, Australia
11 cPoultry
Science Unit, School of Agriculture and Food Sciences, The University of
12 Queensland, Gatton, QLD 4343, Australia
13 *Corresponding author telephone: +61424189693
14
15 *Oladapo O. OLUKOMAIYA; E-mail: o.olukomaiya@uqconnect.edu.au
16 Oladipupo Q. ADIAMO; E-mail: o.adiamo@uq.edu.au
17 W. Chrishanthi FERNANDO; E-mail: chrishanthif2@gmail.com
18 Ram MEREDDY; E-mail: ram.mereddy@daf.qld.gov.au
19 Xiuhua LI; E-mail: x.li1@uq.edu.au
20 Yasmina SULTANBAWA; E-mail: y.sultanbawa@uq.edu.au
21

22 Abstract
23 The effects of solid-state fermentation (SSF) with Aspergillus sojae, Aspergillus

24 ficuum and their co-cultures on proximate composition, anti-nutritional factor,

25 microbiological and functional properties of lupin flour (LF) were investigated. Fibre

26 fractions, in vitro enzyme protein digestion (IVPD), total phenolic content, protein

27 molecular distribution and colour attributes were also evaluated. Samples differed in

28 their proximate composition except ash and fibre contents. The microbial counts of

29 the fermented LFs were generally higher (p<0.05) than that of the unfermented LF.

30 Phytic acid content and IVPD decreased (p<0.05) in the fermented LFs. Also, the

31 fermented LFs showed decreased (p<0.05) water absorption capacity but increased
1

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32 swelling capacity. In addition, fermented LFs demonstrated reduction in colour

33 attributes. Thus, the study indicated that SSF using Aspergillus sojae and Aspergillus

34 ficuum can influence the physical, chemical and functional properties of LF. LF has

35 great potentials in developing new nutritious food products and feed formulations

36 when subjected to SSF.

37 Abbreviations

38 ULF, unfermented lupin flour; ASLF, Aspergillus sojae-fermented lupin flour; AFLF,

39 Aspergillus ficuum-fermented lupin flour; COLF, co-culture fermented lupin flour;

40 SSF, solid-state fermentation

41 Keywords

42 Lupin flour; Aspergillus fungi; solid-state fermentation; protein molecular distribution;

43 functional properties.

44 1. Introduction
45 Lupin (Lupinus spp.) is a legume used as feedstock in food and feed production. Its

46 numerous nutritional and health-promoting benefits has increased its popularity and

47 consumption in recent time (Starkute et al., 2016; Ayyash et al., 2019). Lupin

48 contains substantial quantities of polyphenols, carotenoids, phytosterols,

49 tocopherols, peptides as well as antioxidative, antimicrobial, anticarcinogenic and

50 anti-inflammatory activities (Khan, Karnpanit, Nasar‐Abbas, Huma, & Jayasena,

51 2015). Lupin flour is widely used as a food supplementing in different food products

52 due to its nutritive quality and high functional properties in bakery and pastry

53 products, protein concentrates and other industrial products (Lqari, Vioque,

54 Pedroche, & Millán, 2002; Jiménez‐Martínez, Hernández‐Sánchez, & Dávila‐Ortiz,

55 2003). Functional properties of any protein-rich material are very vital in food
2
56 applications and the functional properties of different lupin species have been

57 previously reported (El-Adawy, Rahma, El-Bedawey, & Gafar, 2001; Muranyi et al.,

58 2016; Ayyash et al., 2019). Lupin flour has been reported to contain high amounts of

59 soluble and insoluble dietary fibre fractions (Hall et al., 2005). The dietary fibre of

60 lupin flour constitutes about 27 - 31% of the kernel weight which is more than that of

61 most other legumes. Other than having high dietary fibre, lupin flour is also high in

62 protein (41 – 44%) making it a food ingredient with high nutritional and commercial

63 value (Evans et al., 1993). Lupin flour has shown possibilities for producing a wide

64 range of edible fibre-enriched baked products with high consumer acceptance (Smith

65 et al., 2006; Jayasena & Nasar-Abbas, 2011). Since lupin flour is a good source of

66 nutrients, it can support microbial growth. Possible nutritional improvement by solid-

67 state fermentation (SSF) will encourage its use in novel food and animal feed

68 production. SSF involves the conversion of complex organic substances into simpler

69 ones by the action of microorganism-derived enzymes as well as modifying the

70 physico-chemical features of the product obtained (Liu, Chen, Shao, Wang, & Zhan,

71 2017; Olukomaiya, Fernando, Mereddy, Li, & Sultanbawa, 2019). Improved nutrient

72 bioavailability and nutrient quality during fermentation are mostly associated to the

73 degradation of anti-nutritional factors (ANFs) and macromolecular proteins into low

74 molecular weight proteins, peptides or free amino acids (Rayaprolu, Hettiarachchy,

75 Chen, Kannan, & Mauromostakos, 2013). Lupin flour fermented with Bifidobacteria

76 spp and Lactobacterium spp showed health-promoting properties in terms of

77 improved bioactivity (Ayyash et al., 2018; Ayyash et al., 2019). Lupin flour co-

78 fermented with Candida utilis, Saccharomyces cerevisiae, Kluyveromyces lactis

79 increased protein content and decreased phytate, raffinose family oligosaccharides

80 and alkaloid content of lupin flour (Kasprowicz‐Potocka, Zaworska, Gulewicz,

3
 81 Nowak, & Frankiewicz, 2018). In addition, lupin flour fermented with Rhizopus

82 oligosporus was used to reduce ANFs (phytate and tannins), upgrade nutritional

83 value, increase total phenolic contents and antioxidant potential (Jiménez-Martínez

84 et al., 2007; Ortega-David and Rodriguez-Stouvenel, 2014; Khan, Karnpanit,

85 Nasar‐Abbas, Huma, & Jayasena, 2018). Filamentous fungi such as Aspergillus

86 sojae and Aspergillus ficuum are important food-grade fungi which have been used

87 in SSF technologies for many years, especially in Asia for producing traditional

88 fermented foods. These fungi strains have been used in reducing ANFs and

89 increasing nutritional profile of food products and agro-industrial by products (Nair &

90 Duvnjak, 1990; 1991; Ebune, Al-Asheh, & Duvnjak, 1995; Chen, Vadlani, & Madl,

91 2014; Chen, Vadlani, Madl, & Gibbons, 2016). Studies on nutritional improvement of

92 lupin flour by SSF has focused mainly on in vitro investigation of bioactivities,

93 chemical and microbial composition (Ayyash et al., 2018; Ayyash et al., 2019;

94 Kasprowicz‐Potocka, Zaworska, Gulewicz, Nowak, & Frankiewicz, 2018) but effect of

95 SSF on some other aspects such as fibre fractions, protein molecular distribution

96 and functional properties have not been extensively studied, as these parameters

97 are also important in novel food development. Therefore, the aim of this study was to

98 determine the effect of SSF with A. sojae and A. ficuum on proximate composition,

99 anti-nutritional factor, microbiological and functional properties of lupin flour.

100 Moreover, the effect of SSF on fibre fractions, protein molecular distribution and

101 colour attributes were also studied.

102 2. Materials and methods

103 2.1 Materials

4
104 Cracked lupins (Lupinus angustifolius L.) were purchased from a commercial feed

105 mill (Jenco Feeds and Seeds Pty. Ltd., Queensland, Australia). The cracked lupins

106 were ground in a mill (Foss tecator cemotec 1090, Sweden) to pass through a 2 mm

107 sieve to obtain lupin flour (LF).

108 All the reagents and media used in the study were of analytical grade. Folin-

109 Ciocalteu phenol’s reagent, gallic acid, phytic acid (Inositol hexaphosphoric acid)

110 dodecasodium salt and 5-sulfosalicylic acid dihydrate were purchased from Sigma-

111 Aldrich (Saint-Louis, MO, USA).

112 2.2 Preparation of samples

113 2.2.1 Preparation of fungal inocula

114 Lyophilized cultures of fungal strains, Aspergillus sojae (ATCC 9362) and Aspergillus

115 ficuum (ATCC 66876), were purchased from the American Type Culture Collection

116 (ATCC, Manassas, VA, USA). After revival, A. sojae was maintained on PDA (potato

117 dextrose agar; Oxoid Ltd., Basingstoke, UK) slants at 37oC while A. ficuum was

118 maintained on CDA (czapek dox agar; Oxoid Ltd., Basingstoke, UK) slants at 25oC

119 for 7 days. The microbial cultures were later stored at 4oC. Spores were collected by

120 gently washing with 0.1% Tween 80 (Ajax Chemicals, Australia) and counted using a

121 colony counter (Stuart Scientific, UK) to obtain about 107 spores/mL. All

122 microbiological procedures were conducted aseptically.

123 2.2.2 Solid-state fermentation

124 Each SSF set-up was prepared in triplicate in 500-mL Erlenmeyer flasks. Moisture

125 content of the substrates (150 g) enriched with 1 g beef extract, 0.3 g potassium

126 chloride, 0.3 g of iron (II) sulphate and 1 g magnesium sulphate was adjusted by

5
127 45% with reverse osmosis (RO) water. Sterilization was done by autoclaving (Sabac

128 autoclave model T62, Australia) at 121oC for 15 min. The cooled substrates were

129 inoculated with spore suspension and incubated at 30oC for 7 days. Autoclaved but

130 unfermented LF served as the control. The inoculum of A. sojae alone or A. ficuum

131 alone was used for monoculture SSF while a combination of both strains was used

132 for co-culture SSF. All samples (unfermented and fermented) except samples used

133 for microbiological analysis were dried at 65oC for 48 h, cooled, passed through a

134 0.5 mm sieve and stored at -20oC until further analysis.

135 2.3 pH and total titratable acidity (TTA)

136 The pH values were measured using a pH meter (PHM 210, Meterlab, Radiometer

137 Analytical SAS, Copenhagen). TTA was determined using 10 g sample mixed with

138 90 mL of RO water and suspension was titrated with 0.1 mol /L NaOH to obtain a pH

139 value of 8.2. The TTA was expressed as the amount (mL) of NaOH utilized.

140 2.4 Proximate analysis

141 Proximate analysis was conducted at the School of Agriculture and Food Sciences,

142 The University of Queensland, Brisbane. Unfermented and fermented samples were

143 analyzed in triplicate for dry matter (method 925.10), crude protein (method 990.03),

144 crude fat by Soxhlet extraction (method 960.39), crude ash (method 923.03), starch

145 (method 996.11) and crude fibre (method 962.09) as described by the methods of

146 AOAC (2000). Soluble carbohydrate (glucose) was measured by an enzymatic

147 method described by Karkalas (1985). Calcium and phosphorus contents were

148 determined using inductively coupled plasma atomic emission spectroscopy (ICP-

149 AES). Results were expressed as a percentage (%) on dry matter basis.

150 2.5 Phytic acid content (PAC)

6
151 The determination of PAC was done according to the modified colorimetric method

152 of Gao et al. (2007). About 1 g of sample was extracted with 20 mL 2.4% HCl with a

153 rotary shaker for 16 h at room temperature. The extracts were centrifuged at 4000 ×

154 g for 10 min at 10oC. The supernatant was mixed with 2 g NaCl for 20 min. After

155 resting for 60 min at 4oC, tubes were centrifuged at 4000 × g for 20 min at 10oC.

156 Exactly 100 µl of the supernatant, 1900 µl reverse osmosis (RO) water, 300 µl

157 Wades reagent were mixed and immediately measured against RO water at 500 nm

158 with a spectrophotometer (Thermo Fisher Scientific GeneSys 20, Australia). Phytic

159 acid (Inositol hexaphosphoric acid) dodecasodium salt was used as standard. PAC

160 was calculated from the standard calibration curve and expressed as mg PA/g

161 sample.

162 2.6 Total phenolic contents (TPC)

163 TPC were determined using the Folin-Ciocalteu reagent as described previously by

164 Singleton and Rossi (1965). Briefly, 0.5 g of sample was extracted twice with 10 mL

165 of 80% methanol for 10 - 15 min. An aliquot of 25 μL extract was mixed with 125 μL

166 of 10% Folin-Ciocalteu reagent, 125 μL of 0.7M sodium carbonate and allowed to

167 incubate in the dark for 30 min at room temperature. Absorbance was measured at

168 750 nm using an automated microplate reader (Tecan infinite F200 96 well plate

169 reader, Austria) using gallic acid as the standard. All analyses were done by triplicate

170 independent assays and TPC results were expressed as milligram gallic acid

171 equivalents (GAE) per gram of dry sample.

172

173

174 2.7 Determination of fibre fractions

7
175 Neutral detergent fibre (NDF) was determined according to the method described by

176 Van Soest (1963 a; b). NDF is defined as the insoluble part of a neutral detergent

177 solution. Acid detergent fibre (ADF) was determined according to the method of Van

178 Soest (1963 a; b). ADF is the fraction of insoluble components in the acid detergent

179 solution. Hemicellulose was calculated by the difference between NDF and ADF

180 contents. Lignin content in ADF was determined according to the method of Van

181 Soest (1963 a; b), which defined it as the insoluble lignin fraction in 72% sulphuric

182 acid.

183 2.8 In vitro enzyme protein digestion (IVPD)

184 IVPD was measured according to the method described by Nalle (2009). One gram

185 of sample was suspended in 5 mL distilled water and incubated at 40oC for 30 min.

186 Then, 0.35 mL 1M HCl was added and pH was adjusted between 2.7 - 2.9. One mL

187 pepsin solution (containing 10 mg pepsin) was added and mixture was incubated at

188 40oC for 1 h. About 0.30 mL of 1M NaOH was later added to the mixture, pH

189 adjusted between 5.9 - 6.10, then 1 mL of 5% pancreatin solution was added

190 followed by incubation at 40oC for 2 h. One mL of 40% 5-sulfosalicylic acid dihydrate

191 was added, vortexing of the mixture was done and allowed to rest for 30 min. At the

192 end of 30 min, the mixture was centrifuged at 2500 × g for 20 min, supernatant

193 decanted and residue was mixed again with 40% 5-sulfosalicylic acid dihydrate.

194 Sample tubes were filled with 10 mL of 95% ethanol, mixture was vortexed,

195 centrifuged twice (at 2500 × g for 20 min) and supernatant decanted. Ten mL of

196 acetone was added, mixed with the residue and centrifuged at 2500 × g for 20 min

197 (repeated twice). The supernatant was finally decanted. The final residue was dried,

198 weighed and analyzed for nitrogen content. The IVPD (%) was calculated as:

8
199

200 where, I = protein content of sample before digestion, F = protein content of sample

201 after digestion.

202 2.9 Microbiological analysis

203 Microbiological analysis using standard plate methods were done as described by

204 Kasprowicz‐Potocka, Zaworska, Gulewicz, Nowak, & Frankiewicz (2018). Total

205 aerobic bacteria in the samples were enumerated on standard plate count agar

206 incubated at 30ºC for 72 h. Total anaerobic bacteria and lactic acid bacteria (LAB)

207 were enumerated on RCA (reinforced clostridial agar; Oxoid Ltd., Basingstoke, UK)

208 and MRS (de Man, Rogosa and Sharpe; Oxoid Ltd., Basingstoke, UK) agar,

209 respectively incubated anaerobically at 30ºC for 72 h. Molds and yeasts were

210 enumerated on PDA (potato dextrose agar; Oxoid Ltd., Basingstoke, UK) incubated

211 at 25ºC for 96 h. Salmonella was determined at Symbio Alliance, Queensland,

212 Australia (a National Association of Testing Authorities (NATA) accredited

213 laboratory) using the horizontal method (procedure M16.1 AS 5013.10).

214 2.10 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

215 analysis

216 The analysis of protein component degradation by SDS-PAGE was carried out at the

217 Protein Expression Facility of the University of Queensland, Brisbane. Peptide

218 extraction from the crude milled samples was done using a Precellys Evolution

219 homogenizer (Thermo Fisher Scientific, Australia) with either 1.4 mm ceramic beads

220 or 0.5 mm glass beads. Briefly, 0.05 g of sample was added to a 2 mL screw top

221 tubes containing the 1.4 mm ceramic or 0.5 mm glass beads. One mL of lysis buffer

222 (20 mM Tris-HCl, with or without 5 mM Dithiothreitol (DTT), 0.1% SDS and Roche
9
223 complete protease inhibitor) was added to each tube and homogenized at 6500 rpm.

224 Samples were kept on ice throughout the homogenisation process. Samples were

225 centrifuged at 20000 × g for 5 min at 4°C after homogenisation to collect the soluble

226 fraction for SDS-PAGE analysis.

227 2.11 Functional properties

228 2.11.1 Bulk density (BD)

229 The method outlined by Oladele & Aina (2007) was used in determining the bulk

230 density (BD). A 10 mL graduated measuring cylinder was filled with 2 g of sample

231 and its base was gently tapped severally to constant volume. The BD (g/mL) was

232 expressed as weight of flour (g) per flour volume (mL).

233 2.11.2 Water absorption capacity (WAC)

234 Water absorption capacity (WAC) was determined at room temperature using the

235 method of Diniz & Martin (1997). About 1 g of sample was mixed with 10 mL RO

236 water in a centrifuge tube and centrifugation was carried out at 3000 × g for 30 min.

237 The WAC was expressed as percentage increase in the sample weight.

238 2.11.3 Oil absorption capacity (OAC)

239 Oil absorption capacity (OAC) was determined according to the centrifugal method of

240 AOAC (2005). About 0.5 g of sample was added to 3 mL of canola oil in a centrifuge

241 tube and centrifugation was carried out at 3000 × g for 30 min. The OAC was

242 expressed as percentage increase in the sample weight.

243 2.11.4 Swelling index (SI)

10
244 The method of Ukpabi & Ndimele (1990) was used for the determination of swelling

245 index (SI). About 10 g of sample was placed in a measuring cylinder and 50 mL of

246 RO water was added. After standing for 4 h, the SI was determined as the ratio of

247 the swollen volume to the initial volume.

248 2.11.5 Swelling capacity (SC)

249 Swelling capacity (SC) was determined according to the method of Okaka & Potter

250 (1977). A 100 mL graduated cylinder was filled with sample to 10 mL mark and RO

251 water was added to make a total volume of 50 mL. The graduated cylinder was

252 tightly sealed, inverted and shaken. The suspension was inverted after 2 min and left

253 to rest for another 8 min and the final volume occupied by the sample was read after

254 the 8th min.

255 2.11.6 Dispersibility

256 Dispersibility was determined according to the method described by Kulkarni,

257 Kulkarni, & Ingle, (1991). About 1 g of sample was suspended in 20 mL measuring

258 cylinder and RO water was added to reach the 10 mL mark. The set-up was stirred

259 vigorously and allowed to settle for 3 h after which volume of settled particle was

260 measured.

261 2.12 Colour attributes

262 Colour attributes of samples were determined using Minolta colorimeter (Model CR-

263 400, Konica Minolta Co Ltd, Osaka, Japan). Prior to the analysis, the instrument was

264 calibrated with a standard white tile (Y = 88.2, x = 0.3158 and y = 0.3229). The

265 colour of the samples was determined in triplicate in terms of CIE L, a, b values of L

266 (lightness-darkness), a (redness-greenness) and b (yellowness-blueness).

11
267 2.13 Statistical analysis

268 All experiments were conducted in triplicate and all measurements were done in

269 triplicate. The results are expressed on dry matter basis as mean ± standard

270 deviation. Statistical analysis was done using IBM SPSS Statistics V.25 (IBM Corp.

271 NY, USA). The data obtained were analyzed for mean differences with one-way

272 analysis of variance (ANOVA) using Tukey’s procedure with a significance level of

273 p<0.05.

274 3. Results and discussion

275 3.1 Proximate composition

276 The proximate composition of unfermented and fermented lupin flours is shown in

277 Table 1. Samples differed significantly (p<0.05) in their proximate composition

278 except ash and fibre contents. The highest crude protein content was recorded in

279 COLF (36.40%) followed by ASLF (36.00%), ULF (35.77%) and AFLF (35.27%).

280 Increase in crude protein during lupin fermentation with Candida utilis (Kasprowicz-

281 Potocka et al., 2015) and dried baker’s yeast (Saccharomyces cerevisiae) combined

282 with a commercial polybacterial preparation (Zaworska, Frankiewicz, & Kasprowicz-

283 Potocka, 2017) have been reported. The increase in protein content could be

284 attributed to the microorganisms using the substrate as carbon and energy sources

285 during SSF to produce fungal protein. However, the slight reduction in crude protein

286 content of AFLF may be associated to the observed increase in soluble carbohydrate

287 and starch. The fat content observed in COLF (6.69%) ASLF (6.42%) and AFLF

288 (6.26%) were higher than that of ULF (6.05%). Increase in crude fat content of lupin

289 fermented with various yeast strains was previously reported by Kasprowicz-Potocka

290 et al. (2016). This may partially be due to the reduction in carbohydrate content
12
291 during SSF. Soluble carbohydrate (glucose) ranged from 4.71% for AFLF, 4.46% for

292 ULF, 4.15% for COLF and 3.71% for ASLF. The decrease in carbohydrate content of

293 fermented flours (ASLF and COLF) compared to ULF may be attributed to the use of

294 carbohydrate (glucose) as a source of energy by microorganism. In the study of

295 Kaczmarska, Chandra-Hioe, Zabaras, Frank, & Arcot (2017), fermentation increased

296 the glucose content of lupin and soy flours. Fermentation might have increased the

297 activity of α-amylase causing the hydrolysis of polysaccharides into glucose and also

298 the fermentative ability of the fungi might have activated the release of bound sugar

299 during SSF, thus, the observed increase in the glucose content of AFLF. The starch

300 content of ASLF (1.40%), AFLF (1.04%) and COLF (0.84%) were significantly higher

301 than that of ULF (0.13%). The observed result agrees with a previous report on

302 increased starch content after fermentation of sweet lupins (Kaczmarska, Chandra-

303 Hioe, Zabaras, Frank, & Arcot, 2017). In contrast, fermented flours usually have

304 lower starch and higher sugar contents (Granito et al., 2002; Kasprowicz‐Potocka,

305 Zaworska, Gulewicz., Nowak, & Frankiewicz, 2018), therefore, more studies are

306 required to investigate the differences in results. Calcium content increased from

307 0.07% (ULF) to 0.08% (ASLF), 0.08% (AFLF) and 0.09% (COLF). Similarly,

308 phosphorus content increased from 0.31% (ULF) to 0.34% (ASLF), 0.35% (AFLF)

309 and 0.34% (COLF). The increase in calcium and phosphorus contents after SSF

310 may be attributed to the efficiency of fermentation in increasing mineral bioavailability

311 (Kiplamai, Tuitoek, & Ethangatta, 2010). It is likely that the degradation of phytic acid

312 that complex with minerals might have resulted to the increase in concentration of

313 calcium and phosphorus.

314 3.2 pH and total titratable acidity (TTA)

13
315 The result of the pH revealed that there was a reduction (p<0.05) in pH from 6.01

316 (ULF) to 5.96, 5.91 and 5.73 in COLF, ASLF and AFLF, respectively (Table 2).

317 Kasprowicz-Potocka et al. (2016) had previously reported a reduction in pH of LF

318 fermented with different yeast strains. A drop in pH could be attributed to increase in

319 organic acids produced by the microorganisms during SSF (Canibe, Miettinen, &

320 Jensen, 2008). Moreover, microbial activities during SSF can degrade carbohydrates

321 into organic acids (Wakil & Onilude, 2009). TTA gradually increased (p<0.05) from

322 the initial value of 0.73 (ULF) to 1.05, 1.08, 1.34 in AFLF, ASLF and COLF,

323 respectively but the level of increase did not differ (p>0.05) among the fermented

324 flours. An increase in TTA is important to inhibit the proliferation of undesirable

325 microorganisms that may cause poor fermentation (Mbata, Ikenebomeh, & Alaneme,

326 2009).

327 3.3 Fibre fractions

328 The fibre fractions of the lupin flours are presented in Table 2. There was a variation

329 in the cellulose content among the lupin flours. Compared to cellulose content of

330 17.48% in ULF, cellulose content reduced to 16.47% in ASLF but increased to

331 19.96% in COLF, although, the cellulose content of ULF did not differ (p>0.05) from

332 that of AFLF. Martín-Cabrejas et al. (2004) previously reported a decrease in

333 cellulose content of fermented common bean flour. The decrease in cellulose

334 content suggests the degradation of the polysaccharides carried out by the

335 enzymatic activities of the microorganisms during SSF process. In contrast, increase

336 in cellulose content was found in sugarcane bagasse fermented with either

337 Aspergillus oryzae, Aspergillus sojae or Aspergillus awamori (Ramli, Imura,

338 Takayama, & Nakanishi, 2005). This increase may be explained as possible buildup

14
339 of acid, alkaline or neutral detergent insoluble substances during SSF, thus, making

340 the fibre values to be overestimated. Cellulose has a defensive role on colon by

341 increasing the number of apoptotic epithelial cells in the large intestine (Mastutik,

342 Putra, & Martoprawiro, 2004). In addition, it makes stool massive helping to

343 eradicate possible carcinogens and reduce bowel transit time (Nakaji et al., 2004).

344 3.4 In vitro enzyme protein digestion (IVPD)

345 There was a reduction (p<0.05) in the IVPD of LF after SSF (Table 2). Proso millet

346 flour was also observed to have shown a reduced IVPD after fermentation (Gulati,

347 Sabillón, & Rose, 2018). In contrast, LF fermented with Lactobacillus sakei,

348 Pediococcus acidilactici and Pediococcus pentosaceus showed higher IVPD

349 (Bartkiene, Krungleviciute, Juodeikiene, Vidmantiene, & Maknickiene, 2015). The

350 unexpected reduction in IVPD of the fermented flours may be due to protein being

351 locked within the fibre matrix, thus, reducing the hydrolytic action of the enzymes. In

352 addition, partial protein denaturation during drying might have also lowered protein

353 dispersibility and solubility, thus, resulting in a reduced IVPD.

354 3.5 Phytic acid content (PAC)

355 The PAC (anti-nutritional factor) of the lupin flours is presented in Table 3. There was

356 a significant difference (p<0.05) in the PAC reducing from 14.38 mg/g (in ULF) to

357 6.71 mg/g, 3.86 mg/g and 5.00 mg/g in ASLF, AFLF and COLF, respectively,

358 although, the PAC in AFLF did not differ (p>0.05) from COLF. Reduction in PAC of

359 LF by fermenting with Lactobacillus spp (Van Vo, Bui, Nguyen, & Fotedar, 2015) and

360 Saccharomyces cerevisiae (Ilham, Hapsari, & Fotedar, 2018) have been previously

361 reported. The decrease in PAC may be due to SSF increasing the activity of inherent

362 phytase which can break non-soluble organic complexes with minerals or a possible
15
363 phytate-degrading ability of the Aspergillus strains. It has been shown that phytic

364 acid degradation is pH dependent (Zhao, Guo, & Zhu, 2017) and the optimal pH for

365 most phytases ranges between 4.0 - 6.0 (Lei & Porres, 2003; Vats & Banerjee,

366 2004). Possibly, a reduction in the pH of the fermenting flours could have activated

367 phytase from the fungi to reduce the levels of the PAC. Likewise, Zamudio et al.

368 (2001) also stated that phosphatase from the microorganisms can also hydrolyze

369 phytic acid.

370 3.6 Total phenolic contents (TPC)

371 The TPC content of 2.54 mg GAE/g, 2.65 mg GAE/g and 2.34 mg GAE/g in ASLF,

372 AFLF and COLF were significantly higher (p<0.05) compared to the ULF (1.93 mg

373 GAE/g). Increase in TPC after fermentation is consistent with the report of Zhao,

374 Guo, & Zhu (2017). Phenolic compounds are secondary metabolites that possess

375 health-promoting benefits such as antioxidant and anticancer activities

376 (Balasundram, Sundram, & Samman, 2006). Thus, SSF may support the conversion

377 of bound to free phenolics, thus, improving their bioavailability (Dey, Chakraborty,

378 Jain, Sharma, & Kuhad, 2016). The increase in TPC may be explained by the

379 proteolytic activities of fungi leading to the release of bound to free TPC.

380 3.7 Microbiological composition

381 The SSF process significantly affected the microbiological status of lupin flour (Table

382 4). After sterilization, all the samples had no microbial counts (data not shown). After

383 7 days of fermentation, the content of all the estimated microorganisms in the

384 fermented flours was generally higher (p<0.05) than in the unfermented flour except

385 for Salmonella that was not detected in any of the samples. Total aerobic bacteria in

386 ULF (2.56 log CFU/g) was significantly (p<0.05) lower than in ASLF (4.22 log

16
387 CFU/g), AFLF (4.15 log CFU/g) and COLF (4.48 log CFU/g). Total anaerobic

388 bacteria in ULF (1.05 log CFU/g) was significantly (p<0.05) lower than in ASLF (2.65

389 log CFU/g), AFLF (2.95 log CFU/g) and COLF (2.57 log CFU/g). The increase in

390 bacterial count may be attributed to varying reactions and changes in pH as well as

391 other biochemical parameters within the fermenting material (Achi, 2005). ASLF and

392 AFLF had higher lactic acid bacteria (LAB) counts of 3.61 and 3.70 log CFU/g,

393 respectively compared to COLF (3.00 log CFU/g) and ULF (1.95 log CFU/g).

394 Kasprowicz-Potocka et al. (2015) previously reported increase in LAB counts during

395 lupin fermentation with Candida utilis. The increase in LAB counts may be attributed

396 to a reduction in pH as acidic conditions can encourage the growth of LAB. Mold in

397 ULF (1.22 log CFU/g) was significantly (p<0.05) lower than in ASLF (3.30 log

398 CFU/g), AFLF (3.66 log CFU/g) and COLF (3.00 log CFU/g). It is possible that

399 increase in mold count may also be due to the duration and conditions of SSF

400 encouraging the growth of mold. Yeast in ULF (1.00 log CFU/g) was significantly

401 (p<0.05) lower than in ASLF (2.56 log CFU/g), AFLF (2.39 log CFU/g) and COLF

402 (2.10 log CFU/g). Increase in yeast count may be associated to reduction in pH as

403 yeast are low pH tolerant (Kasprowicz-Potocka et al., 2015).

404 3.8 SDS-PAGE analysis

405 The changes in the electrophoretic patterns was examined by SDS-PAGE (Figure 1).

406 The difference in protein molecular weights was quantified by a densitometric

407 analysis using Image Lab version 5.0 (Bio-Rad Laboratories, Hercules, CA, USA).

408 Intense protein bands ranging between 5.31 – 224.51 kDa were observed in ULF

409 (lane 2). Overall, the results suggest that soluble proteins from ASLF, AFLF and

410 COCM were mainly polypeptides with less than 77.6 kDa molecular weights. The

411 molecular weight of the sample proteins in the present study fall within the range (20
17
412 - 100 kDa) found in lupin proteins as reported by Burgos-Díaz et al. (2019). In

413 comparison to ULF, the proteins from ASLF (lane 3), AFLF (lane 4) and COLF (lane

414 5) showed faint protein bands. The results indicate that SSF had caused the soluble

415 proteins in LF to be degraded into fragments with low molecular weight smaller than

416 20 kDa. It is possible that proteolytic enzymes produced by the fungi strains during

417 SSF hydrolyzed the proteins to some extent, thereby reducing the molecular weight

418 of the sample proteins.

419 3.9 Functional properties and colour attributes

420 The effect of SSF on functional properties and colour attributes of LF samples is

421 presented in Table 5. The WAC of 220% (ULF) significantly reduced (p<0.05) to

422 210% (ASLF), 185% (AFLF) and 195% (COLF). This result is consistent with a

423 previous report on decreased WAC after fermentation of pigeon pea seed flour

424 (Adebowale & Maliki, 2011). The decrease in WAC may be due to reduced

425 hydrophilic groups which bind water molecules during the fermentation (Afoakwa,

426 Sefa-Dedeh, Budu, Sakyi-Dawson, & Asomaning, 2007) suggesting their potential

427 use for producing thin gruels in food formulations. However, swelling capacity (SC)

428 of 1.54% (ULF) significantly increased (p<0.05) to 3.28% (ASLF), 2.09% (AFLF) and

429 2.24% (COLF) which agrees with increased SC after the fermentation of pearl millet

430 flour (Adebiyi, Obadina, Mulaba-Bafubiandi, Adebo, & Kayitesi, 2016). The increase

431 in SC suggests high affinity for water indicating improved flour functionality, which

432 would ultimately produce a good product. Also, the dispersibility of ASLF (94.21%)

433 was the lowest among all the treatments. The values obtained were higher than the

434 range (51.50 to 56.75%) reported for LF (Tizazu & Emire, 2010). High dispersibility

435 would probably enhance better reconstitution of starch in water to give fine and

436 constituent paste (Eke-Ejiofor, 2015). SSF significantly (p<0.05) reduced the colour
18
437 attributes (L* and b*) as compared to the unfermented LF with the least a* value

438 recorded in the co-culture fermented LF. Similarly, Bilgiçli & İbanoğlu (2007) reported

439 that fermentation reduced L*, a* and b* values of tarhana (a wheat flour-yoghurt

440 mixture). A reduction in colour values could be due to thermal degradation of

441 pigments responsible for colour or change in pH during SSF causing discolouration

442 of the samples.

443 Conclusion

444 SSF using the filamentous fungi strains of Aspergillus sojae, Aspergillus ficuum and

445 their co-cultures influenced the proximate composition, fibre fractions, protein band

446 intensity and functional properties of lupin flour. SSF improved the nutritional quality

447 especially the crude protein content and reduced the phytic acid content in lupin

448 flour. In addition, SSF increased the total phenolic contents, enhanced the functional

449 properties (swelling capacity and dispersibility) and reduced the colour attributes of

450 lupin flour. In summary, the nutritional features and functional properties of the solid-

451 state fermented lupin flours showed that they could be suitable ingredients for animal

452 feed and human food formulations.

453 Acknowledgments

454 The authors are sincerely grateful for the support of the University of Queensland.

455 The scholarship support through the Research Training Program Scholarship

456 provided to Oladapo O. Olukomaiya during his PhD study at the University of

457 Queensland, Australia and technical support from the Queensland Department of

458 Agriculture and Fisheries are gratefully appreciated.

459 Conflict of interest

19
460 The authors declare that they have no conflict of interest.

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690
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698
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704
705 Figure legends:
706 Figure 1. SDS-PAGE patterns of unfermented and solid-state fermented lupin
707 proteins.
708

25
Figure 1. SDS-PAGE patterns of unfermented and solid-state fermented lupin
proteins. Lane 1: molecular weight marker; lane 2: unfermented lupin flour (ULF);
lane 3: Aspergillus sojae fermented lupin flour (ASLF); lane 4: Aspergillus ficuum
fermented lupin flour (AFLF); lane 5: co-culture fermented lupin flour (COLF).
 Table 1. Proximate composition of unfermented and solid-state fermented lupin
flours (DM basis)
Parameters ULF ASLF AFLF COLF
Crude protein (%) c
35.77±0.06 36.00±0.1 b 35.27±0.06 36.40±0.00a
d

Crude fat (%) 6.05±0.02 d 6.42±0.03 b 6.26±0.02c 6.69±0.02a

Crude fibre (%) 13.60±0.80 14.60±0.26 14.90±0.10 12.40±2.10
Crude ash (%) 3.57±0.18 3.66±0.25 3.70±0.41 3.83±0.16
Soluble carbohydrate (glucose, %) 4.46±0.03 b 3.71±0.04 d 4.71±0.07 a 4.15±0.10c
Starch (%) 0.13±0.03 d 1.40±0.03 a 1.04±0.05 b 0.84±0.03c
Calcium (%) 0.07±0.01b 0.08±0.01ab 0.08±0.01ab 0.09±0.01a
Phosphorus (%) 0.31±0.01 b 0.34±0.01a 0.35±0.01a 0.34±0.01a
Values are given as mean ± standard deviation (n = 3). Values in the same row
having different superscripts differ significantly (p<0.05). ULF: unfermented lupin
flour; ASLF: Aspergillus sojae fermented lupin flour; AFLF: Aspergillus ficuum
fermented lupin flour; COLF: co-culture fermented lupin flour.
Table 2. pH, total titratable acidity, fibre fractions (DM basis) and in vitro enzyme
protein digestion of unfermented and solid-state fermented lupin flours
Parameters ULF ASLF AFLF COLF
pH 6.01±0.01 a 5.91±0.03 b 5.73±0.01 c 5.96±0.03ab
Total titratable acidity 0.73±0.04 b 1.08±0.05 a 1.05±0.20 a 1.34±0.14a
Neutral detergent fibre, % 28.87±5.09a 32.15±2.78a 37.38±4.46a 39.04±7.28a
Acid detergent fibre, % 20.82±1.51 a 20.88±1.41 a 22.99±1.40 a 24.00±1.27a
Hemicellulose, % 8.05±3.58a 11.27±3.54a 14.39±4.09a 15.04±6.94a
Cellulose, % 17.48±0.81 ab 16.47±1.13 b 17.80±1.74 ab 19.96±1.04a
Lignin, % 3.33±2.24a 4.42±1.45a 5.19±2.87a 4.04±2.31a
IVPD, % 44.74±0.32 a 34.25±0.26 c 37.58±0.34 b 30.22±0.53d
Values are given as mean ± standard deviation (n = 3). Values in the same row
having different superscripts differ significantly (p<0.05). ULF: unfermented lupin
flour; ASLF: Aspergillus sojae fermented lupin flour; AFLF: Aspergillus ficuum
fermented lupin flour; COLF: co-culture fermented lupin flour; IVPD: In vitro enzyme
protein digestion.

Table 3. Effect of solid-state fermentation on phytic acid (anti-nutritional factor) and

total phenolic contents of unfermented and solid-state fermented lupin flours
Parameters Phytic acid, mg PA/g DM % reduction TPC, mg GAE/ g DM % increase
ULF 14.38±0.58a - 1.93±0.01b -
ASLF 6.71±0.40 b 53.27 2.54±0.23a 31.6
AFLF 3.86±0.38 c 73.16 2.65±0.21 a 37.3
COLF 5.00±0.40c 65.23 2.34±0.04a 21.2
Values are given as mean ± standard deviation (n = 3). Values in the same column
having different superscripts differ significantly (p<0.05). ULF: unfermented lupin
flour; ASLF: Aspergillus sojae fermented lupin flour; AFLF: Aspergillus ficuum
fermented lupin flour; COLF: co-culture fermented lupin flour; TPC: Total phenolic
contents
 Table 4. Microbiological composition of unfermented and solid-state fermented lupin
flours
Parameters ULF ASLF AFLF COLF
Total aerobic bacteria, log CFU/g b
2.56±0.11 4.22±0.20 a 4.15±0.25 4.48±0.21a
a

Total anaerobic bacteria, log CFU/g 1.05±0.50b 2.65±0.26a 2.95±0.34a 2.57±0.10a

Lactic acid bacteria, log CFU/g c
1.25±0.15 3.61±0.10 a 3.70±0.26a 3.00±0.30b
Salmonella, /25 g ND ND ND ND
Molds, log CFU/g c
1.22±0.28 3.30±0.30 a 3.66±0.33 3.00±0.55a
a

Yeasts, log CFU/g b

1.00±0.26 2.56±0.26 a 2.39±0.15a 2.10±0.30a
Values are given as mean ± standard deviation (n = 3). Values in the same row
having different superscripts differ significantly (p<0.05). ULF: unfermented lupin
flour; ASLF: Aspergillus sojae fermented lupin flour; AFLF: Aspergillus ficuum
fermented lupin flour; COLF: co-culture fermented lupin flour; ND: not detected; CFU
- colony forming units.

Table 5. Functional properties and colour attributes (L, a and b values) of

unfermented and solid-state fermented lupin flours
Parameters ULF ASLF AFLF COLF
Bulk density, g/mL 0.64±0.01 a 0.66±0.01 a 0.66±0.01 a 0.66±0.01a
WAC, % 220±0.00 a 210±10.00 ab 185±5.00 c 195±5.00bc
OAC, % 66.70±0.00a 75.00±8.30a 66.70±0.00a 63.35±3.35a
Swelling index 2.06±0.07 a 1.80±0.07a 1.86±0.00a 1.80±0.20a
Swelling capacity 1.54±0.04c 3.28±0.08a 2.09±0.09b 2.24±0.04b
Dispersibility, % 95.74±0.13 a 94.21±0.20 95.48±0.00 95.54±0.20a
b a

Colour
L 80.14±0.10a 72.10±0.82b 69.68±1.29c 69.62±0.89c
a 33.73±0.11a 35.14±0.18a 34.02±1.15a 30.89±0.24b
b 94.85±0.05 a 87.41±0.46b 83.32±0.99c 83.63±0.58c
Values are given as mean ± standard deviation (n = 3). Values in the same row
having different superscripts differ significantly (p<0.05). ULF: unfermented lupin
flour; ASLF: Aspergillus sojae fermented lupin flour; AFLF: Aspergillus ficuum
fermented lupin flour; COLF: co-culture fermented lupin flour; WAC: Water
absorption capacity; OAC: Oil absorption capacity; L: lightness-darkness; a: redness-
greenness; b: yellowness-blueness.