Improvement of bioactivity of soybean meal by solid-state fermentation with Bacillus amyloliquefaciens versus Lactobacillus spp. and Saccharomyces cerevisiae

Accepted Manuscript Improvement of bioactivity of soybean meal by solid-state fermentation with Bacillus amyloliquefaciens versus Lactobacillus spp. and Saccharomyces cerevisiae Chun-Hua Chi, Seong-Jun Cho PII:

S0023-6438(15)30358-3

DOI:

10.1016/j.lwt.2015.12.002

Reference:

YFSTL 5136

To appear in:

LWT - Food Science and Technology

Received Date: 25 May 2015 Revised Date:

26 November 2015

Accepted Date: 1 December 2015

Please cite this article as: Chi, C.-H., Cho, S.-J., Improvement of bioactivity of soybean meal by solidstate fermentation with Bacillus amyloliquefaciens versus Lactobacillus spp. and Saccharomyces cerevisiae, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2015.12.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Improvement of bioactivity of soybean meal by solid-state

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fermentation

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Lactobacillus spp. and Saccharomyces cerevisiae

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.

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Chun-Hua Chi, Seong-Jun Cho*

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Ingredients R&D Center, CJ Cheiljedang, 636 Guro-dong, Guro-gu, Seoul 152051,

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Korea

*Corresponding author

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Ingredients R&D Center

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CJ Cheiljedang, 636 Guro-dong, Guro-gu

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Seoul 152051

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Korea

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Tel: +82-2-2629-5258

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Fax: +82-2-2629-5344

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E-mail [email protected]

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versus

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Seong-Jun Cho

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amyloliquefaciens

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Bacillus

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ABSTRACT

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To evaluate the impact of fermentation with Bacillus amyloliquefaciens U304 on

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nutritional quality and bioactivity of soybean meal (SBM), we analyzed the solid-state

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fermentation process for Bacillus amyloliquefaciens, Lactobacillus spp., and

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Saccharomyces cerevisiae. B. amyloliquefaciens showed significant improvement in

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nutritional quality and bioactivity by removing the protein- and carbohydrate-based

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anti-nutritional factors (ANFs), as well as allergens. The total phenolic content,

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reducing power, free radical scavenging ability, and Ca2+ chelating ability of SBM, as

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indicators of the antioxidant activity, increased to 195.8, 201.7% (at 10 mg/mL), 136.6%

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(at 10 mg/mL), and 122.3%, respectively, after Bacillus fermentation. S. cerevisiae

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decomposed carbohydrate-based but not protein-based ANFs, and fermentation with

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this organism produced similar values of the antioxidant markers of unfermented

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soybean meal, except for the reducing power (160.0% at 10 mg/mL). Lactobacillus spp.

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was only effective for decreasing the activity of trypsin inhibitors, but not other ANFs,

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resulting in lower bioactivity of fermented soybean meal. B. amyloliquefaciens U304

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can substantially improve both the nutritional quality and bioactivity of SBM.

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Keywords: soybean meal, solid-state fermentation, Bacillus amyloliquefaciens, antioxidant activity, Metal chelating activity

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Introduction

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For generations, the soybean has been an important crop in Asia. In addition to

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being a component of such foods as tofu, natto, and tempeh, this crop is used for

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production of soy vegetable oil. Furthermore, the part remaining from processing of

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soybean-based food, defatted soybean meal (SBM), is an important and cheap protein

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source for food and animal feed. SBM is known for its high protein content, balanced

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amino acid composition, and high level of lysine in comparison with other vegetable

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protein sources. However, other components of SBM, namely anti-nutritional factors

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(ANFs) and allergens, cause allergy reactions in children with atopic dermatitis (Kleine-

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Tebbe et al., 2002), and decrease digestibility and absorption based on young animal

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studies (Schneeman & Gallaher, 1986; Gu, Pan, Sun, & Qin, 2010; Goebel & Stein,

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2011), thus limiting its nutrition utilization.

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One of prevalent ANFs is trypsin inhibitor (TI). It is a protein-based ANF, which

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inhibits pancreatic protease, proteolysis, and the absorption of dietary proteins (Liener

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et al., 1988; Perez-Maldonado, Mannion, & Farrell, 2003). Increased secretion of

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trypsin is caused by activity reduction and results in endogenous nitrogen loss,

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especially in sulfur-containing amino acid content (Schneeman & Gallaher, 1986). In

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addition, carbohydrate-based ANFs such as the non-digestible oligosaccharides

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(raffinose and stachyose) can induce increased gas production in humans (Sumarna,

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2008) and diarrhea in poultry due to the absence of endogenous α-(1,6)-galactosidase

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enzyme in these species (Gitzelmann & Auricchio, 1965; Sun, Li, Dong, Qiao, & Ma,

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2008).

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The soybean is one of the “Big 8” food allergens. The allergen proteins account for

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65–80% of total protein content in the soybean and approximately 30% in SBM. The 3

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major allergen proteins are β-conglycinin (α, α’ subunit, β subunit), the 30-kDa allergen

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(Gly m Bd 30), and glycinin. In human subjects, these allergens can induce symptoms

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ranging from skin, gastrointestinal, or respiratory reactions to anaphylaxis (Holzhauser

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et al., 2009). They also cause hypersensitivity in weaned piglets, with the primary

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adverse effect being diarrhea (Hotz & Gibson, 2007).

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Different approaches, including over-heating, chemical treatment, and alcohol

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extraction, were used in order to solve these problems. However, all of these methods

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were able only to denature ANFs and allergens, but not to eliminate them completely. In

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addition to being the most efficient method to remove the ANFs and allergens,

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fermentation provides other benefits. It has been shown that fermentation can help to

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reduce the immunoreactivity and allergic reactions caused by soy products (Frias,

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Young, Martínez-Villaluenga, De Mejia, & Vidal-Valverde, 2008). Fermented soybean

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meal (FSBM) enhanced the bioavailability of nutritious components and decreased the

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incidence of diarrhea in weaned pigs (Egounlety & Aworh, 2003; Teng, Gao, Yang, &

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Liu, 2012), due to the degradation of allergens into peptides. The peptides can be easily

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absorbed by an animal and transported within an organism (Gilbert, Wong, & Webb,

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2008). They also exhibit high specific bioactivities, such as anti-oxidative activity and

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metal-chelating activity. Furthermore, soybean protein hydrolysate has lipid

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peroxidation inhibitory activity attributed mainly to the low-molecular weight (3 kDa)

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peptide (Karki, Maurer, & Jung, 2011; Park, Lee, Baek, & Lee, 2010), and peptide

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fragments with molecular weights of either 14.4 or 8–9 kDa exhibit high Ca2+-binding

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capacity (Bao, Song, Zhang, Chen, & Guo, 2007). Soybean hydrolysate shows high

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peptide content and antioxidant activity (Sefatie, Fatoumata, & Eric, 2013). However,

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the overall benefits of soybean hydrolysate in comparison with FSBM remain unclear.

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Although soybean hydrolysate can decompose the protein-based ANFs, the

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carbohydrate-based ANFs do persist. Considering the commercial value improvement,

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solid-state fermentation remains much more economical and beneficial than other

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methods. It has been reported that Bacillus sp. Lactobacillus spp., and Saccharomyces

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cerevisiae could reduce the level of ANFs and improve the nutritional value of soybean

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products (Frias et al., 2008; Murashita et al., 2013; Seo et al., 2011; Hansen, 2012; Zhao,

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Huang, Cai, Hong, & Wang, 2014). However, there are needs of more information on

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the nutritional effects of using solid-state fermentation on SBM.

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In this study, we evaluated the changes in nutritional quality, anti-oxidative activity

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and metal-chelating ability of FSBM depending on the strain chosen for solid

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fermentation and made a comparison.

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2.

Materials and methods

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2.1.

Strains

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Bacillus amyloliquefaciens U304 was isolated from the Korean traditional soybean paste, Cheonggukjang and grown in GYP medium (1% glucose, 0.8% yeast extract, 0.2%

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soy peptone) at 37°C with shaking. Lactobacillus acidophilus and Lactobacillus

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plantarum were isolated from the Korean traditional fermented vegetable, Kimchi and

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grown in MRS medium (Difco) at 37°C anaerobically. Saccharomyces cerevisiae

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CJ1697 was isolated from the Korean rice wine, Makgeolli and cultured in GYP

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medium 30°C with shaking. When the optical density at 660 nm for the growing

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cultures exceeded 5, the strains were used in solid-state fermentation.

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2.2.

Fermentations

SBM with moisture content 45% was steamed at 100°C for 30 min in an autoclave.

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After inoculation with a 10% ratio of B. amyloliquefaciens U304 to L. acidophilus, L.

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plantarum, or S. cerevisiae CJ1697, it was incubated at 37°C for 24 h, 37°C for 36 h

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anaerobically, or 30°C for 48 h, in a constant temperature and humidity test chamber,

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respectively. FSBM was then dried at 60°C for 12 h and ground and then used for

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analysis.

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2.3.

Proximate analysis

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Moisture, protein, ash and fat content of FSBM were determined by the official

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methods and recommended practices of the American Oil Chemists' Society (AOCS,

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2006). Crude protein was measured by the Kjeldahl method using the automatic Kjeltec

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TM8400 system (Foss, Denmark) by AOCS Ba 4d-90. Ash was determined by AOCS 6

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Ba 5a-49 and fat was measured by AOCS Ba 3-38. The nitrogen solubility index (NSI)

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was measured by AOCS Ba 11-65. Shortly, sample was mixed with 40 volumes of

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distilled water and incubated at 30°C for 120 min with agitation, then centrifuged at

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1,500 g for 10 min. The nitrogen content of supernatant and sample was measured by

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Kjeldahl method. The NSI was calculated as follows: nitrogen content of supernatant × 100 total nitogen content of sample

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Methionine and lysine were determined after acid hydrolysis with 6N HCl for 24 h

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at 110°C by high-performance liquid chromatography (Rayner, 1985). Samples were

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assessed in triplicate.

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2.4.

Antinutritional factors

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Trypsin inhibitor (TI) was determined by AOCS Ba 12-75 using BAPNA

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(Benzoyl-DL-arginine-p-nitroanilide) as substrate (AOCS, 2006). The level of raffinose

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and stachyose was analyzed by thin layer chromatography (TLC). The samples were

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extracted in 20 volumes of water and centrifuged at 10,000 g for 5 min following

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filtration using a 0.22-µm filter. The mobile phase consisted of 50% ethyl acetate and 35%

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acetic acid.

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2.5.

Protein molecular weight distribution

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2.5.1.

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

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We used 8 M urea as a solvent to extract proteins with a sonicator (Bandelin, GM

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2070, 70 W power, BR, Germany) at 35% power. After centrifugation at 6,000 g for 10

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min, protein concentration in the supernatant was measured by the BCA assay (Sapan, 7

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Lundblad, & Price, 1999). The samples were denatured with 5× Tris-glycine SDS

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loading buffer, and 30 µg of proteins were loaded and separated using a 10% gel for

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SDS-PAGE (BIO-RAD, Hercules, CA, USA) at 90 V. Protein bands were visualized by

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staining with Coomassie Brilliant Blue R250 (Sigma, St. Louis, MO, USA).

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2.5.2.

Peptide content assay

To calculate the peptide contents, we performed gel permeation chromatography

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(GPC). The protein were extracted as described for SDS-PAGE, and centrifuged at

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10,000 g for 5 min following filtration with a 0.22-µm polyvinylidene fluoride filter.

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The analysis was performed using an Agilent HPLC system with a Superdex 75 10/300

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GL column (GE Healthcare, BKM, UK), and the protein was detected at 214 nm.

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2.6.

Anti-oxidative activity assay

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2.6.1.

Preparation of solvent extracts

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For the anti-oxidative activity assay, SBM and FSBMs were extracted twice by

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shaking at 30°C for 3h with 70% (v/v) ethanol (1:10, w/v) and filtered through

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Whatman No.1 filter paper (Whatman, Maidstone, UK) and freeze-dried.

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2.6.2.

Total phenolic content

The total phenolic content of the samples extracted with ethanol was determined

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by the method of Singleton et al. (1999) using Folin-Ciocalteu reagent with some

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modifications. One milliliter of sample (1mg/mL) mixed with 0.5 mL of 1 N Folin-

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Ciocalteu reagent. After mixing, 2.5 mL of a 20% sodium carbonate solution was added,

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and the mixture was placed in the dark for 40 min and the absorbance was measured at 8

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725 nm.

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mg/g of sample according to the standard curve.

The concentration was calculated and expressed as gallic acid equivalents in

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2.6.3.

Reducing activity

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The reducing activity of the samples was measured by the method described in

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Oyaizu (1986). One milliliter of samples with different concentrations was mixed with

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2.5 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 mL of potassium

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ferricyanide and incubated at 50°C for 20 min. Subsequently, 2.5 mL of 10%

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trichloroacetic acid was added to stop the reaction and centrifuged at 3000 ×g for 10

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min. The supernatant (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL

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of 0.1% FeCl3. After 10 min, the absorbance was measured at 700 nm against the

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reagent blank. A higher A700 indicates higher reducing activity.

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2.6.4.

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2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS+) assay

Total radical scavenging capacity was determined by the method described in

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Puchalska et al. (2014)

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with some modifications. ABTS (38.5 mg) and 6.6 mg of potassium persulfate were

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dissolved separately in 5 mL of deionized water and then mixed. The stock solution was

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incubated at room temperature for 12–16 h in the dark. To obtain 0.70 ± 0.02 arbitrary

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units (AU) at 734 nm, the stock solution was diluted with methanol. A 10-µL sample

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was mixed with 990 µL of diluted stock solution and incubated for 10 min. The

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absorbance was measured at 734 nm. A calibration curve was plotted using Trolox, and

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radical scavenging capacity was calculated according to the formula:

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Scavenging capacity% =

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2.7.

 !" # $ %&' ×  !"

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Metal ion-chelating activity Ca2+-chelating activity was measured according to method of Zhao et al. (2014)

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with some modification. One gram of sample was mixed with 50 mL of 2.5 mg/mL

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CaCl2 solution with stirring for 1 h, and centrifuged at 10,000 g for 10 min. One

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hundred microliters of supernatant was mixed with 1 mL of chromogenic agent (0.1

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mM o-CPC, 8 mM 8-hydroxyquinoline, 5 M urea, 25% ethanol, pH 10) at room

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temperature for 20 min. After incubation, the absorbance was measured at 570 nm in the

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spectrophotometer, and calcium content was calculated using the standard curve.

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Fe2+-chelating activity was determined according to Thanigaivel et al. (2014) with

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some modifications. One gram of sample was mixed with 1 mg/mL of FeCl2 solution,

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stirred for 1 h, and then centrifuged at 10,000 g for 10 min. After dilution, 25 µL of

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sample was mixed with 10 µL of deionized water in a microtiter plate. The reaction was

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initiated by the addition of 25 µL 25 mM ferrozine. The mixture was shaken vigorously

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and incubated at room temperature for 10 min. The absorbance was measured at 562 nm

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and Fe content was calculated using the standard curve.

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2.8.

Statistical anaylsis

Analytical values were carried out using three independent determinations.

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Statistical analyses were determined using a statistical software program (SPSS for

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Windows version 17.0). The data were subjected to analysis of variance using the

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general linear model to determine significant differences between samples (p < 0.01).

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3.

Results and discussion

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3.1.

Proximate analysis The protein content in SBM increased after fermentation with B. amyloliquefaciens

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U304, L. acidophilus, L. plantarum, and S. cerevisiae CJ1697 (Table 1) and increased

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by 6.42%, 0.95%, 1.91%, and 5.61%, respectively. The result indicated that B.

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amyloliquefaciens U304 and S. cerevisiae CJ1697 can decompose carbohydrate into

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carbon dioxide gas, reduce the total amount of carbohydrate, and increase the relative

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protein content in FSBM. However, the protein content of FSBMs fermented with L.

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acidophilus and L. plantarum was not changed much, since they convert carbohydrate

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into organic acid in general. The content of soluble protein in FSBM with B.

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amyloliquefaciens U304 significantly increased, suggesting that FSBM with this strain

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can improve the hydrophilic property through the hydrolysis process. However, FSBM

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with L. acidophilus, L. plantarum, and S. cerevisiae CJ1697 decreased the soluble

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protein content to 23.5%, 22.5%, and 55.8% of SBM, respectively. The decrease of NSI

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is caused by the heat damage during steaming. The NSI of FSBM with Lactobacillus

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spp. was lower than that with S. cerevisiae, which may due to the lower pH that can

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cause a change in protein structure and lead to protein precipitation (Wang, Le, Shi, &

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Zeng, 2014).

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The content of methionine in FSBM increased by 22.2% relative to SBM upon

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fermentation with B. amyloliquefaciens U304, 3.5% with L. acidophilus, and 7.4% with

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L. plantarum, but decreased by 11.1% for fermentation with S. cerevisiae CJ1697. The

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lysine content of FSBM fermented with B. amyloliquefaciens U304 remained the same

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as that of SBM, but fermentation with the other species lowered the lysine content

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relative to that of SBM. FSBM produced with B. amyloliquefaciens U304 showed the best improvement in

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the amino acid composition in comparison with that fermented using L. acidophilus, L.

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plantarum, and S. cerevisiae CJ1697 (data not shown).

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3.2.

Antinutritional factors

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After steaming in the autoclave, TI in SBM was denatured by heat and the level of

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TI reduced from 4.77 mg/g to 1.3 mg/g. Furthermore, it is observed that TI could be

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further removed by solid state fermentation with by B. amyloliquefaciens U304, L.

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acidophilus, and L. plantarum, but not with S. cerevisiae CJ1697 (Table 1). We assume

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that B. amyloliquefaciens U304 secretes a strong protease, which is able to decompose

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proteins, including TI (Phengnuam & Suntornsuk, 2013). Alternatively, Lactobacillus

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spp. may cause a structural change, such as precipitation and inactivation of TI, because

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of the lower pH. In contrast, fermentation with S. cerevisiae CJ1697 could not decrease

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TI, because S. cerevisiae, did neither produce protease nor change value of pH.

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We performed a qualitative analysis of raffinose and stachyose using TLC (Fig. 1).

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The levels of both raffinose and stachyose drastically decreased in FSBM with S.

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cerevisiae CJ1697. This effect might have been caused by secretion of α-galactosidase

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and other carbohydrases, which are able to break down raffinose and stachyose (Khattab,

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Arntfield, & Nyachoti, 2009). We also observed that FSBM with B. amyloliquefaciens

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U304 decomposed raffinose and stachyose into an unknown oligosaccharide, which was

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almost completely decomposed. These data suggest that B. amyloliquefaciens U304

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secretes glycolytic enzymes to decompose the carbohydrate-based ANFs into other

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carbohydrates. However, L. acidophilus and L. plantarum had no effect on the

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decomposition of raffinose and stachyose, most probably due to their inability to secrete

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carbohydrases.

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3.3.

Protein molecular weight distribution assay

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The protein profile was analyzed by SDS-PAGE (Fig. 2A), a powerful method for

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detection of protein-based ANFs (Gagnon, Poysa, Cober, & Gleddie, 2010). In the SBM

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sample, we observed many protein bands, including β-conglycinin (68-kDa α, 72-kDa α’

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subunit, and 52-kDa β subunit), the 30-kDa allergen (Gly m Bd 30), and glycinin (37-

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kDa acidic subunit and 20-kDa basic subunit). Because of the strong proteolysis that

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occurred during fermentation with B. amyloliquefaciens U304, the protein-based ANFs

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and allergens were almost completely decomposed and hydrolyzed into small

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molecular-weight peptides. The protein larger than 28 kDa was mostly eliminated. At

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the same time, the FSBM with L. acidophilus, L. plantarum, and S. cerevisiae CJ1697

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showed no significant changes. The detailed protein distribution change was analyzed

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by GPC.

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The spectrum results of SBM and the FSBMs (Fig. 2B) showed a change in the

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distribution of proteins. FSBM with B. amyloliquefaciens U304 was different from that

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of the other samples. The large-molecular weight proteins were decreased and the small

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proteins were increased. This result suggests that B. amyloliquefaciens U304 secretes a

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highly active protease that is able to decompose the larger proteins. It is possible that the

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small-molecular weight proteins have anti-oxidative activity and metal ion-chelating

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activities, which can inhibit lipid oxidation and utilization of minerals in the feed

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(Sefatie et al., 2013; Murashita et al., 2013). The change in the protein distribution is

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demonstrated in Table 2. The compositions of proteins under 10 kDa were 17.45%,

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51.43%, 19.37%, 20.03%, and 18.74%; and those under 5 kDa were 12.56%, 33.64%,

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14.32%, 14.46%, and 13.57% in SBM and FSBM with B. amyloliquefaciens U304, L.

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acidophilus, L. plantarum, and S. cerevisiae CJ1697, respectively. It has been reported

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that peptides could be more rapidly utilized than amino acids and proteins (Kodera,

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Hara, Nishimori, & Nio, 2006), which might also contribute to the anti-oxidative

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activity and metal-chelating activity, discussed in detail below in sections 3.5 and 3.6,

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respectively. The increased peptide content can positively affect the bioactivity of SBM,

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including antioxidant activity and metal-chelating activity.

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3.4.

Metal ion-chelating activity

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The result of the metal ion-chelating ability of FSBM is shown in Table 3. FSBM

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with B. amyloliquefaciens U304 showed the highest value for both Ca2+ and Fe2+

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chelating, 122.3% and 107.2% of SBM, respectively. FSBM with L. acidophilus and L.

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plantarum demonstrated lower values, namely 51.5% and 71.9% of SBM for Ca2+

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chelating, and 59.9% and 53.1% of SBM for Fe2+ chelating, respectively. For S.

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cerevisiae CJ1697, the level was 98.7% of SBM for Ca2+ chelating and 102.4% of SBM

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for Fe2+ chelating.

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As shown in Table 2, FSBM produced with B. amyloliquefaciens U304 contained a

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larger amount of small molecular-weight peptides than FSBM produced using the other

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microorganisms, where peptides are known to possess some degree of chelating activity

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(Ghribi et al., 2015; Wattanasiritham, Theerakulkait, Wickramasekara, Maier, & Stevens,

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2015; Zhang, Li, & Zhou, 2010). Therefore, the peptides in FSBM produced with B.

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amyloliquefaciens U304 caused this sample to have the highest metal ion-chelating 14

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ability. The metal chelating ability is related to the antioxidant activity since metal

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chelation helps to inhibit the peroxidation caused by transition metals (Stohs, 1995). In addition to chelating phenolic compounds, metal ions also bind to proteins and

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peptides to transport through the intestinal epithelium (Roullet, Roullet, Martin, &

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McCarron, 1994; J. A. Johnson & Kumar, 1994; Feher & Wasserman, 1979; G. Johnson,

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Jacobs, & Purves, 1983). FSBM with B. amyloliquefaciens U304 showed high potential

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for metal ion transportation.

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3.5.

Anti-oxidative activity assay

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As shown in Fig. 3, the FSBMs exhibited different anti-oxidative activities.

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Soybean contains lots of phenolic compounds, which have anti-oxidative activity and

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can reduce free radicals. The results demonstrate that compared with SBM, not all the

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FSBMs increased the phenolic contents (Fig. 3A). FSBM with B. amyloliquefaciens

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U304 showed a significant increase to 195.8% of SBM and increases of 64.1%, 721.1%

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and 99.4% were observed for FSBM with L. acidophilus, L. plantarum, and S.

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cerevisiae CJ1697, respectively.

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The reducing ability of the ethanol extracts of SBM and FSBM was measured by

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the transformation of Fe3+ to Fe2+. As shown in Fig. 3B, reducing activity was also

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concentration-dependent. FSBM with B. amyloliquefaciens U304 demonstrated the

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highest A700 value followed by S. cerevisiae CJ1697. FSBM with L. acidophilus and L.

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plantarum showed values comparable with that of SBM.

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The ABTS+ assay is an important tool to determine the anti-oxidative activity of

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hydrogen-donating molecules. The percentage of scavenging capacity is dose-dependent.

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In this assay FSBM with B. amyloliquefaciens U304 was the only product surpassing 15

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that of SBM throughout all the concentrations. This result suggests that fermentation

339

with B. amyloliquefaciens U304 gave the best activity with respect to ABTS+ removal

340

and enhanced the scavenging activity. However, FSBM with L. acidophilus and L.

341

plantarum showed lower activity than SBM, and FSBM with S. cerevisiae CJ1697

342

showed activity similar to SBM, which was still lower than FSBM with B.

343

amyloliquefaciens U304.

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It has been reported that the fermentation of soybean with Bacillus can increase the

345

biological activity and physiochemical properties of soybean, such as the polyphenol

346

content, antioxidant activity, reducing activity, and Fe2+ chelating ability (Hu et al., 2010;

347

Juan & Chou, 2010; Moktan, Saha, & Sarkar, 2008; Zhu, Fan, Cheng, & Li, 2008).

348

Soymilk fermented with Lactobacillus exhibited about 171.2% antioxidant activity

349

compared to the parent material, and the reducing power of soybean fermented with

350

Sacchromyces was quantified as 191.2% (Marazza, Nazareno, de Giori, & Garro, 2012;

351

Romero, Doval, Sturla, & Judis, 2004). We assume that the difference in the results

352

obtained with fermentation by Lactobacillus and Sacchromyces might be derived from

353

the different fermentation processes. Liquid-phase or higher moisture fermentation

354

could enhance the activity of the microorganism. Furthermore, the strength of the

355

protease secreted by the microorganism can affect the bioactivity and physiochemical

356

properties of FSBM. Also, FSBM produced with certain microorganisms may have

357

enhanced total phenolic content and radical scavenging activity relative to SBM

358

(Rashad, Mahmoud, Abdou, & Nooman, 2011; Samruan, Oonsivilai, & Oonsivilai, 2012;

359

Wang et al., 2014)

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360

In this study, FSBM produced with B. amyloliquefaciens U304 exhibited

361

significant improvement of the phenolic content, reducing ability, and free radical 16

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scavenging ability relative to parent SBM; these values were also higher than those

363

achieved with other microorganisms. Furthermore, FSBM produced with L. acidophilus

364

and L. plantarum exhibited significantly low antioxidant activity. This may be caused

365

by the lower pH of FSBM produced with L. acidophilus and L. plantarum. A lower pH

366

in complicated materials will reduce the susceptibility towards oxidation by the proton

367

shield. The improved antioxidant activity of FSBM produced with B. amyloliquefaciens

368

U304 is mainly attributed to the increased phenolic content and the formation of

369

bioactive peptides. Other constituents such as vitamins and inorganic ions also have

370

antioxidant activity, but were not significantly changed by fermentation (data not

371

shown).

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It was reported that the phenolic compounds act as reducing agents, metal

373

chelators, and single-oxygen quenchers (Moktan et al., 2008). In addition to the

374

phenolic compounds, the bioactive peptides can also improve the antioxidant activity.

375

Protease treatment during microorganism solid-state fermentation shows higher degree

376

of hydrolysis and higher antioxidant activity than just fermented products (Moktan et al.,

377

2008).

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4. Conclusions Fermentation is an effective way to improve the nutritional quality and bioactivity

380

of SBM. The level of improvement depends on the microorganism used for

381

fermentation. Some microorganisms positively impact the physicochemical properties

382

only, whereas others improve the biological properties only, and others can improve

383

both sets of properties. Herein, we compared the properties of FSBM produced with

384

several microorganisms together to evaluate the improvement in the properties. In

385

contrast with FSBM produced with L. acidophilus, L. plantarum, and S. cerevisiae,

386

FSBM produced with B. amyloliquefaciens U304 exhibited obvious improvements,

387

including a decrease in the level of the carbohydrate-based and protein-based ANFs and

388

increased antioxidant activity and metal-chelating ability, derived mainly from

389

increased levels of phenolic compounds and bioactive peptides. To confirm the

390

bioactivity of FSBM produced with B. amyloliquefaciens U304, we intend to continue

391

our research with an animal model.

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References

393

AOCS. (2006). Official methods and recommended practices (5th ed.). Urbana, IL:

394

Association of Oil Chemists Society. Bao, X. L., Song, M., Zhang, J., Chen, Y., & Guo, S. T. (2007). Calcium-binding ability

396

of soy protein hydrolysates. Chinese Chemical Letters, 18(9), 1115–1118.

397

doi:10.1016/j.cclet.2007.07.032

RI PT

395

Egounlety, M., & Aworh, O. C. (2003). Effect of soaking, dehulling, cooking and

399

fermentation with Rhizopus oligosporus on the oligosaccharides, trypsin inhibitor,

400

phytic acid and tannins of soybean (Glycine max Merr.), cowpea (Vigna

401

unguiculata L. Walp) and groundbean (Macrotyloma geocarpa Ha. Journal of Food

402

Engineering., 56(2-3), 249–254. doi:10.1016/s0260-8774(02)00262-5

M AN U

SC

398

Feher, J. J., & Wasserman, R. H. (1979). Intestinal calcium-binding protein and calcium

404

absorption in cortisol-treated chicks: effects of vitamin D3 and 1,25-

405

dihydroxyvitamin D3. Endocrinology, 104(2), 547–51. doi:10.1210/endo-104-2-

406

547

TE D

403

Frias, J., Young, S. S., Martínez-Villaluenga, C., De Mejia, E. G., & Vidal-Valverde, C.

408

(2008). Immunoreactivity and amino acid content of fermented soybean products.

410

AC C

409

EP

407

Journal

of

Agricultural

and

Food

Chemistry,

56(1),

99–105.

doi:10.1021/jf072177j

411

Gagnon, C., Poysa, V., Cober, E., & Gleddie, S. (2010). Soybean Allergens Affecting

412

North American Patients Identified by 2D Gels and Mass Spectrometry. Food

413

Analytical Methods, 3(4), 363–374. doi:10.1007/s12161-009-9090-3

414

Ghribi, A. M., Sila, A., Przybylski, R., Nedjar-Arroume, N., Makhlouf, I., Blecker, C.,

415

Attia, H., Dhulster, P.,Bougatef, A., & Besbes, S. (2015). Purification and 19

ACCEPTED MANUSCRIPT

416

identification of novel antioxidant peptides from enzymatic hydrolysate of

417

chickpea (Cicer arietinum L.) protein concentrate. Journal of Functional Foods, 12,

418

516–525. doi:10.1016/j.jff.2014.12.011 Gilbert, E. R., Wong, E. a., & Webb, K. E. (2008). Board-invited review: Peptide

420

absorption and utilization: Implications for animal nutrition and health. Journal of

421

Animal Science, 86, 2135–2155. doi:10.2527/jas.2007-0826

423

Gitzelmann, R., & Auricchio, S. (1965). the Handling of Soya Alpha-Galactosides By a

SC

422

RI PT

419

Normal and a Galactosemic Child. Pediatrics, 36(2), 231–235.

Goebel, K. P., & Stein, H. H. (2011). Ileal digestibility of amino acids in conventional

425

and low-Kunitz soybean products fed to weanling pigs. Asian-Australas. J. Anim.

426

Sci, 24, 88–95.

M AN U

424

Gu, C., Pan, H., Sun, Z., & Qin, G. (2010). Effect of soybean variety on anti-nutritional

428

factors content, and growth performance and nutrients metabolism in rat.

429

International

430

doi:10.3390/ijms11031048

TE D

427

Journal

of

Molecular

Sciences,

11(3),

1048–56.

Hansen, O. K. (2012). Fermented protein product. Washington, DC: U.S.

432

Holzhauser, T., Wackermann, O., Ballmer-Weber, B. K., Bindslev-Jensen, C., Scibilia,

433

J., Perono-Garoffo, L., Utsumi, S., Poulsen, L., & Vieths, S. (2009). Soybean

435

AC C

434

EP

431

(Glycine max) allergy in Europe: Gly m 5 (beta-conglycinin) and Gly m 6 (glycinin) are potential diagnostic markers for severe allergic reactions to soy. The

436

Journal

437

doi:10.1016/j.jaci.2008.09.034

of

Allergy

and

Clinical

Immunology,

123(2),

452–8.

438

Hotz, C., & Gibson, R. S. (2007). Traditional Food-Processing and Preparation

439

Practices to Enhance the Bioavailability of Micronutrients in Plant-Based Diets. J. 20

ACCEPTED MANUSCRIPT

440

Nutr., 137(4), 1097–1100. Hu, Y., Ge, C., Yuan, W., Zhu, R., Zhang, W., Du, L., & Xue, J. (2010).

442

Characterization of fermented black soybean natto inoculated with Bacillus natto

443

during fermentation. Journal of the Science of Food and Agriculture, 90(7), 1194–

444

1202. doi:10.1002/jsfa.3947

446

Johnson, G., Jacobs, P., & Purves, L. R. (1983). Iron binding proteins of iron-absorbing rat intestinal mucosa. Journal of Clinical Investigation, 71(5), 1467.

SC

445

RI PT

441

Johnson, J. A., & Kumar, R. (1994). Renal and intestinal calcium transport: roles of

448

vitamin D and vitamin D-dependent calcium binding proteins. In Seminars in

449

nephrology (Vol. 14, pp. 119–128).

M AN U

447

450

Juan, M.-Y., & Chou, C.-C. (2010). Enhancement of antioxidant activity, total phenolic

451

and flavonoid content of black soybeans by solid state fermentation with Bacillus

452

subtilis

453

doi:10.1016/j.fm.2009.11.002

14715.

Food

Microbiology,

27(5),

586–91.

TE D

BCRC

Karki, B., Maurer, D., & Jung, S. (2011). Efficiency of pretreatments for optimal

455

enzymatic saccharification of soybean fiber. Bioresource Technology, 102(11),

456

6522–8. doi:10.1016/j.biortech.2011.03.014

EP

454

Khattab, R. Y., Arntfield, S. D., & Nyachoti, C. M. (2009). Nutritional quality of

458

legume seeds as affected by some physical treatments, Part 1: Protein quality

459 460

AC C

457

evaluation.

LWT

-

Food

Science and

Technology,

42(6),

1107–1112.

doi:10.1016/j.lwt.2009.02.008

461

Kleine-Tebbe, J., Wangorsch, A., Vogel, L., Crowell, D. N., Haustein, U.-F., & Vieths,

462

S. (2002). Severe oral allergy syndrome and anaphylactic reactions caused by a Bet

463

v 1– related PR-10 protein in soybean, SAM22. Journal of Allergy and Clinical 21

ACCEPTED MANUSCRIPT

464

Immunology, 110(5), 797–804. doi:10.1067/mai.2002.128946 Kodera, T., Hara, H., Nishimori, Y., & Nio, N. (2006). Amino acid absorption in portal

466

blood after duodenal infusions of a soy protein hydrolysate prepared by a novel

467

soybean protease D3. Journal of Food Science, 71(7), S517–S525.

RI PT

465

Liener, I. E., Goodale, R. L., Deshmukh, A., Satterberg, T. L., Ward, G., DiPietro, C.

469

M., Bankey, P. E. & Borner, J. W. (1988). Effect of a trypsin inhibitor from

470

soybeans (Bowman-Birk) on the secretory activity of the human pancreas.

471

Gastroenterology, 94(2), 419–27.

SC

468

Marazza, J. A., Nazareno, M. A., de Giori, G. S., & Garro, M. S. (2012). Enhancement

473

of the antioxidant capacity of soymilk by fermentation with Lactobacillus

474

rhamnosus.

475

doi:10.1016/j.jff.2012.03.005

Journal

of

M AN U

472

Functional

Foods,

4(3),

594–601.

Moktan, B., Saha, J., & Sarkar, P. K. (2008). Antioxidant activities of soybean as

477

affected by Bacillus-fermentation to kinema. Food Research International, 41(6),

478

586–593. doi:10.1016/j.foodres.2008.04.003

TE D

476

Murashita, K., Akimoto, A., Iwashita, Y., Amano, S., Suzuki, N., Matsunari, H., …

480

Yamamoto, T. (2013). Effects of biotechnologically processed soybean meals in a

481

nonfishmeal diet on growth performance, bile acid status, and morphological

483

AC C

482

EP

479

condition of the distal intestine and liver of rainbow trout Oncorhynchus mykiss. Fisheries Science, 79(3), 447–457. doi:10.1007/s12562-013-0617-6

484

Oyaizu, M. (1986). Studies on products of browning reaction: Antioxidative activities

485

of products of browning reaction prepared from glucosamine. The Japanese

486

Journal of Nutrition and Dietetics, 44, 307–315.

487

Park, S. Y., Lee, J.-S., Baek, H.-H., & Lee, H. G. (2010). Purification and 22

ACCEPTED MANUSCRIPT

488

characterization of antioxidant peptides from soy protein hydrolysate. Journal of

489

Food Biochemistry, 34, 120–132. Retrieved from http://dx.doi.org/10.1111/j.1745-

490

4514.2009.00313.x Perez-Maldonado, R. A., Mannion, P. F., & Farrell, D. J. (2003). Effects of heat

492

treatment on the nutritional value of raw soybean selected for low trypsin inhibitor

493

activity.

494

doi:10.1080/0007166031000085463

Poultry

Science,

44(2),

299–308.

SC

British

RI PT

491

Phengnuam, T., & Suntornsuk, W. (2013). Detoxification and anti-nutrients reduction

496

of Jatropha curcas seed cake by Bacillus fermentation. Journal of Bioscience and

497

Bioengineering, 115(2), 168–72. doi:10.1016/j.jbiosc.2012.08.017

M AN U

495

Puchalska, P., Marina, M. L., & García, M. C. (2014). Isolation and identification of

499

antioxidant peptides from commercial soybean-based infant formulas. Food

500

Chemistry, 148, 147–54. doi:10.1016/j.foodchem.2013.10.030

TE D

498

Rashad, M. M., Mahmoud, A. E., Abdou, H. M., & Nooman, M. U. (2011).

502

Improvement of nutritional quality and antioxidant activities of yeast fermented

503

soybean curd residue, 10(28), 5504–5513. doi:10.5897/AJB10.1658

EP

501

Rayner, C. J. (1985). Protein hydrolysis of animal feeds for amino acid content. Journal

505

of Agricultural and Food Chemistry, 33(4), 722–725. doi:10.1021/jf00064a039

AC C

504

506

Romero, A. M., Doval, M. M., Sturla, M. A., & Judis, M. A. (2004). Antioxidant

507

properties of polyphenol-containing extract from soybean fermented with

508

Saccharomyces cerevisiae. European Journal of Lipid Science and Technology,

509

106(7), 424–431. doi:10.1002/ejlt.200400953

510

Roullet, C. M., Roullet, J.-B., Martin, A.-S., & McCarron, D. A. (1994). In vivo effect

511

of calcitriol on calcium transport and calcium binding proteins in the 23

ACCEPTED MANUSCRIPT

512

spontaneously hypertensive rat. Hypertension, 24(2), 176–182. Samruan, W., Oonsivilai, A., & Oonsivilai, R. (2012). Soybean and Fermented Soybean

514

Extract Antioxidant Activities. World Academy of Science, Engineering and

515

Technology, 6(12), 1223–1226.

516

RI PT

513

Sapan, C. V, Lundblad, R. L., & Price, N. C. (1999). Colorimetric protein assay

517

techniques.

518

doi:10.1111/j.1470-8744.1999.tb00538.x

and

Applied

Biochemistry,

29(2),

99–108.

SC

Biotechnology

Schneeman, B. O., & Gallaher, D. (1986). Nutritional and Toxicological Significance of

520

Enzyme Inhibitors in Foods. (M. Friedman, Ed.) (Vol. 199). Boston, MA: Springer

521

US. doi:10.1007/978-1-4757-0022-0

M AN U

519

Sefatie, R. S., Fatoumata, T., & Eric, K. (2013). In Vitro Antioxidant Activities of

523

Protein Hydrolysate from Germinated Black Soybean (Glycine max L.), 5(4), 453–

524

459.

TE D

522

Seo, S. H., Cho, S.-J., Hong, Y.-H., Ryu, J.-H., Kang, J. H., Chi, H., Park, S.-W., Pyun,

526

Y. R., Cho, S. C., Kook, M. C., & Park, H. H. (2011). Method for preparing a

527

fermented soybean meal using bacillus strains. Geneva,

EP

525

Singleton, V. L., Orthofer, R., & Lamuela-Raventos, R. M. (1999). Analysis of total

529

phenols and other oxidation substrates and antioxidants by mean of Folin-

530 531 532

AC C

528

Ciocalteu reagent. Methods Enzymol, 299, 152–178.

Stohs, S. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radical Biology and Medicine, 18(2), 321–336. doi:10.1016/0891-5849(94)00159-H

533

Sumarna. (2008). Changes of raffinose and stachyose in soy milk fermentation by lactic

534

acid bacteria from local fermented foods of Indonesian. Malaysian Journal of

535

Microbiology, 4(2), 26–34. 24

ACCEPTED MANUSCRIPT

536

Sun, P., Li, D., Dong, B., Qiao, S., & Ma, X. (2008). Effects of soybean glycinin on

537

performance and immune function in early weaned pigs. Archives of Animal

538

Nutrition, 62(4), 313–321. doi:10.1080/17450390802066419 Teng, D., Gao, M., Yang, Y., Liu, B., Tian, Z., & Wang, J. (2012). Bio-modification of

540

soybean meal with Bacillus subtilis or Aspergillus oryzae. Biocatalysis and

541

Agricultural Biotechnology, 1(1), 32–38. doi:10.1016/j.bcab.2011.08.005

RI PT

539

Thanigaivel, S., Vijayakumar, S., Mukherjee, A., Chandrasekaran, N., & Thomas, J.

543

(2014). Antioxidant and antibacterial activity of Chaetomorpha antennina against

544

shrimp

545

doi:10.1016/j.aquaculture.2014.07.003

Vibrio

parahaemolyticus.

Aquaculture,

M AN U

pathogen

SC

542

433,

467–475.

Wang, N., Le, G., Shi, Y., & Zeng, Y. (2014). Production of Bioactive Peptides from

547

Soybean Meal by Solid State Fermentation with Lactic Acid Bacteria and Protease.

548

Advance Journal of Food Science and Technology, 6(9), 1080–1085.

TE D

546

549

Wattanasiritham, L., Theerakulkait, C., Wickramasekara, S., Maier, C. S., & Stevens, J.

550

F. (2015). Isolation and identification of antioxidant peptides from enzymatically

551

hydrolyzed

552

doi:10.1016/j.foodchem.2015.06.057

bran

EP

rice

protein.

Food

Chemistry,

192,

156–162.

Zhang, L., Li, J., & Zhou, K. (2010). Chelating and radical scavenging activities of soy

554

protein hydrolysates prepared from microbial proteases and their effect on meat

555 556

AC C

553

lipid

peroxidation.

Bioresource

Technology,

101(7),

2084–9.

doi:10.1016/j.biortech.2009.11.078

557

Zhao, L., Huang, S., Cai, X., Hong, J., & Wang, S. (2014). A specific peptide with

558

calcium chelating capacity isolated from whey protein hydrolysate. Journal of

559

Functional Foods, 10, 46–53. doi:10.1016/j.jff.2014.05.013 25

ACCEPTED MANUSCRIPT

560

Zhu, Y. P., Fan, J. F., Cheng, Y. Q., & Li, L. T. (2008). Improvement of the antioxidant

561

activity of Chinese traditional fermented okara (Meitauza) using Bacillus subtilis

562

B2. Food Control, 19(7), 654–661. doi:10.1016/j.foodcont.2007.07.009

AC C

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1

Table 1 - Proximate and trypsin inhibitor (TI) analysis of soybean meal (SBM) and fermented soybean

2

meal (FSBM). Constituent (dry basis) Protein (%)

FSBM with

SBM 54.37 ± 0.12

e

B. amyloliquefaciens 60.79 ± 0.20a

L. acidophilus 55.32 ± 0.15d

L. plantarum 56.28 ± 0.18c

S. cerevisiae 59.98 ± 0.07b

6.71 ± 0.12b

7.41 ± 0.10a

6.66 ± 0.13b

6.64 ± 0.07b

7.29 ± 0.10a

Fat (%)

1.21 ± 0.02b

0.91 ± 0.01c

1.16 ± 0.02c

0.99 ± 0.01d

1.29 ± 0.01a

NSI (%)

17.69 ± 0.59b

70.37 ± 0.44a

4.15 ± 0.53d

3.98 ± 0.22d

9.87 ± 0.09c

c

a

bc

b

0.48 ± 0.01d

Methionine (%)

0.54 ± 0.01

0.56 ± 0.01

Lysine (%)

3.44 ± 0.02a

3.44 ± 0.02a

3.12 ± 0.01c

3.30 ± 0.01b

3.33 ± 0.02b

TI (mg/g)

4.77 ± 0.21a

0.67 ± 0.08cd

0.83 ±0.19c

0.53 ± 0.09d

1.25 ± 0.06b

pH

6.80 ± 0.01b

7.56 ± 0.02a

4.52 ± 0.02e

4.72 ± 0.03d

6.43 ± 0.01c

M AN U

Each value represents the mean of three replications ± standard deviation. Means in a column with different letter were significantly different (p<0.01). NSI : nitrogen solubility index, TI : trypsin inhibitor

3

AC C

EP

TE D

4

1

0.58 ± 0.02

SC

0.66 ± 0.02

RI PT

Ash (%)

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Table 2 – Protein molecular weight distribution of soybean meal (SBM) and fermented soybean meal

6

(FSBM)

B. amyloliquefaciens L. acidophilus L. plantarum

>75 30–75

a

50.58 ± 1.50 21.47 ± 0.80a

c

a

12.24 ± 0.12 12.72 ± 0.35c

50.25 ± 0.40 20.00 ± 0.64b

10–30

10.50 ± 0.34c

23.61 ± 0.46a

10.38 ± 0.15c

5–10

4.89 ± 0.29c

17.80 ± 0.38a

5.05 ± 0.09c

S. cerevisiae

b

48.66 ± 0.26 51.39 ± 0.51a 20.10 ± 0.42b 19.42 ± 0.25b 11.21 ± 0.17b 10.45 ± 0.16c 5.57 ± 0.23b

5.18 ± 0.25bc

<5 12.56 ± 0.06e 33.63 ± 0.01a 14.32 ± 0.04c 14.46 ± 0.04b 13.56 ± 0.04d Each value represents the mean of three replications ± standard deviation. Means in a column with different letter were significantly different (p<0.01).

SC

7 8

FSBM with SBM

RI PT

Molecular weight (kDa)

9

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Table 3. Metal-chelating ability of soybean meal (SBM) and fermented soybean meal (FSBM). Chelating activity (mg/g) Ca

33.12 ± 0.02

B. amyloliquefaciens b

40.49 ± 2.21

a

FSBM with L. acidophilus L. plantarum 17.07 ± 0.24

c

S. cerevisiae

c

32.70 ± 1.67b

22.58 ± 0.07d

43.49 ± 1.21b

19.84 ± 0.09

EP

TE D

M AN U

SC

RI PT

Fe2+ 42.49 ± 0.42b 45.54 ± 0.13a 30.53 ± 0.87c Each value represents the mean of three replications ± standard deviation. Means in a column with different letter were significantly different (p<0.01).

AC C

12 13

2+

SBM

3

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Fig. 1

M AN U

SC

RI PT

1

2

AC C

EP

TE D

3

1

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Fig. 2.

5

A

SC

RI PT

4

6

B

10

AC C

9

EP

TE D

8

M AN U

7

2

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A

13 14 15 16 17 18 19

600 500 400

b

b

300

2.5

SBM U304 LA LP CJ1697

2.0

Absorbance at 700 nm

25 26 27

b

1.0

a 0.5

a

0.0

a

d

b c cc

cd c d

1

5 10 15 concentration (mg/mL)

SBM U304 LA LP CJ1697

80

b

a

c

b a

60

b

b

d

b 40

c c

a b cc

20

aba

20

a

AC C

100

d

c c

EP

b bb

Scavenging capacity (%)

c c

b

29

34

b

a

a

C

CJ1697 a

1.5

28

33

LP

TE D

B

U304

M AN U

SBM

24

32

LA

0

23

31

c

100

22

30

c 200

20 21

a

RI PT

12

SC

Fig. 3.

Total phenolic content (mg/100g)

11

b b

e

cc

bbb

0

1

5 10 15 concentration (mg/mL)3

20

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Figure Legends

36

Fig. 1. Qualitative analysis of raffinose and stachyose in soybean meal (SBM) and Fermented soybean

37

meal (FSBM) by thin layer chromatography.

38

M: marker, 1: SBM, 2: FSBM with B. amyloliquefaciens U304, 3: FSBM with L. acidophilus

39

4: FSBM with L. plantarum, 5: FSBM with S. cerevisiae CJ1697

RI PT

35

40

Fig. 2. The protein profiles of soybean meal (SBM) and fermented soybean meal (FSBM) by sodium

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dodecyl sulfate-polyacrylamide gel electrophoresis (A). The spectrum results of soybean meal and

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fermented soybean meal by gel permeation chromatography (B).

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M : marker, 1: SBM, 2: FSBM with B. amyloliquefaciens U304, 3: FSBM with L. acidophilus

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4: FSBM with L. plantarum, 5: FSBM with S. cerevisiae CJ1697

M AN U

SC

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Fig. 3. Analyses of total phenolic contents (A), reducing power (B), and ABTS+ radical scavenging

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capacity (C) of soybean meal (SBM) and Fermented soybean meal (FSBM). Each value represents the

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mean of three replications ± standard deviation. Values with different letters are significantly different

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(P<0.01)

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SBM: soybean meal, U304: FSBM with B. amyloliquefaciens U304

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LA: FSBM with L. acidophilus, LP: FSBM with L. plantarum, CJ1697 : FSBM with

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CJ1697

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EP

AC C

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TE D

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4

S. cerevisiae

ACCEPTED MANUSCRIPT Highlights
Bacillus fermentation is optimal for improving soybean meal nutritional value.
Antioxidative and chelating activity can be improved by solid-state fermentation

AC C

EP

TE D

M AN U

SC

RI PT


Bioactive peptide is important for improving fermented soybean meal bioactivity.