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Thermophilic solid-state fermentation of rapeseed meal and analysis of microbial community diversity
LWT - Food Science and Technology 116 (2019) 108520
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Thermophilic solid-state fermentation of rapeseed meal and analysis of microbial community diversity
T
Xiaoshan Houa,b, Chunhua Daia,b,∗∗, Yingxiu Tanga,b, Zheng Xinga,b, Benjamin Kumah Mintaha,b,c, Mokhtar Dabboura,b,d, Qingzhi Dinga,b, Ronghai Hea,b,∗, Haile Maa,b a
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China Institute of Food Physical Processing, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China c ILSI-UG FSNTC, Department of Nutrition and Food Science, University of Ghana, POB LG 91, Legon, Accra, Ghana d Department of Agricultural and Biosystems Engineering, Faculty of Agriculture, Benha University, P.O. Box 13736, Moshtohor, Qaluobia, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Geobacillus stearothermophilus Rapeseed meal Thermophilic solid-state fermentation Polypeptide Microbial community diversity
To reduce the high cost caused by steam sterilization of fermentation substrates and equipments in mesophilic solid-state fermentation of rapeseed meal (RM), a thermophilic protease-producing strain RM-2 from RM was isolated and employed in thermophilic solid-state fermentation of unsterilized RM for polypeptide preparation. The isolate was identified as Geobacillus stearothermophilus by appearance of colonies and microscopic observation as well as 16S rDNA sequencing. The highest yield of polypeptide (9.67%) was obtained by RM-2 at 50% moisture content, 55 °C and 24 h fermentation time. Analysis of microbial community diversity in fermented RM revealed Geobacillus stearothermophilus as dominant strain inhibiting most mesophilic microorganisms. Thermophilic solid-state fermentation on RM improved the yield of rapeseed polypeptide, as well as the utilization rate of substrate. The application of this technology (thermophilic solid-state fermentation) would be environmentally friendlier and energy-saving to industry.
1. Introduction Rapeseed meal (RM), a by-product after oil extraction from rapeseed seeds, is considered as an important and cheap protein source for food and animal feed (Ebune, Al-Asheh, & Duvnjak, 1995). However, it utilization is limited due to the presence of anti-nutritional factors (ANFs) and high fiber levels (Erdogan & Olmez, 2010). In order to utilize RM effectively, efforts have been channeled in developing techniques to improve its quality, including enzymatic hydrolysis, chemical method and microbial fermentation to prepare rapeseed polypeptide (Drew, Borgeson, & Thiessen, 2007). Of these approaches, enzymatic hydrolysis is the most important. However, the high cost of protease may restrict it application. Other researchers have also used chemical means to prepare rapeseed polypeptide, but in actual production, the degree of hydrolysis is difficult to control, and the nutritional value of protein is damaged greatly, which seriously affects the functional activity of rapeseed polypeptide. In addition, the wastewater from the acid and alkali treatment, upon discharge may lead to environmental pollution (Du & Guo, 2010). Therefore, microbial fermentation is regarded as promising technique applied to detoxify and
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improve the nutrition and bioactivity of RM (Dai et al., 2017a; Lomascolo, Uzan-Boukhris, Sigoillot, & Fine, 2012). For instance, fermentation with Rhizopus oligosporus sp-T3 for 40 h, resulted in the degradation of undesirable factors such as 84% of carbohydrates, 30% of lignin, 47% of total glucosinolates, and other polyphenolic components indigestible by nonruminants when compared with unfermented rapeseed meal (Mejean et al., 2003). Shi et al. (2016) found that fermenting rapeseed meal (FRM) by Aspergillus niger resulted in more crude protein and amino acid (except His) than unfermented RM. Further, the small peptide in FRM increased 2.26 times; and the concentrations of antinutritional substrates including neutral detergent fiber, glucosinolates, isothiocyanate, oxazolidithione, and phytic acid reduced (P < 0.05) by 13.47, 43.07, 55.64, 44.68 and 86.09%, respectively. Moreover, it was reported that the hydrolysates of RM (rapeseed polypeptides) displayed not only the high utilization, fast digestion and absorption rate in the body, but also some physiological activities such as antioxidant, antiinflammatory, anticancer and so on (Zhang et al., 2018; Rodrigues, Carvalho, & Rocha, 2017; Xue, Liu, Wu, Zhuang, & Yu, 2010). Solid-state fermentation (SSF) is defined as a fermentation process executed on non-soluble materials which act as a physical support and a
Corresponding author. School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China. Co-Corresponding author. School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China. E-mail addresses: [email protected] (C. Dai), [email protected] (R. He).
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https://doi.org/10.1016/j.lwt.2019.108520 Received 11 March 2019; Received in revised form 17 July 2019; Accepted 20 August 2019 Available online 20 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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source of nutrients in the absence of free flowing liquid. Nowadays, SSFs are already performed at the commercial scale in the food industry or waste treatment and utilization (Biz et al., 2016; Selvakumar, Ashakumary, & Pandey, 1998). Compared with submerged fermentation, SSF has the characteristic of lower consumption and a mild reaction (Reddy, Wee, Yun, & Ryu, 2008). But in the past, most of the fermentation was performed under mesophilic conditions, along with the necessary process of autoclaving of substrates to kill the bacteria existed in the materials before fermentation in order to avoid contamination of products (Lazim, Mankai, Slama, Barkallah, & Limam, 2009; Prakasham, Rao, & Sarma, 2006). Therefore, mesophilic fermentation requires the use of large amount of steam for sterilization of substrates and equipments. That notwithstanding, rapeseed protein is easily denatured during high temperature and pressure sterilization treatment, which decreases the conversion efficiency of rapeseed protein (Abraham, Gea, & Antoni, 2014; He, He, Chao, & Ju, 2014). Recently, much attention is given to the isolation of thermophilic organisms from compost, hot spring and other extreme environments due to the secreted enzyme in the organism which has application in waste processing (Asad, Asif, & Rasool, 2011; Elazzazy, Abdelmoneim, & Almaghrabi, 2015; Hamilton-Brehm et al., 2010; Li et al., 2019; Zhou, Wu, & Rao, 2016). So far, many researchers have proposed that thermophilic bacteria are better suited for the production of industrial enzymes. Ali et al. (2016) employed open SSF of lignocellulosic biomass to coproduce protease and amylase by thermophilic Bacillus sp. BBXS-2. The concentrations of enzymes reached 12,200 U/g dry matter for protease and 6900 U/g dry matter for amylase under optimal nonsterile fermentation conditions (pH 8.5, 45 °C for 5 d) using wheat straw as substrate. Similarly, SSF involving thermophilic bacteria has also been employed to produce lipase from Pseudomonas sp. S1 (Sahoo, Subudhi, & Kumar, 2014) and xylanase from Promicromonospora sp. MARS (Kumar, Joshi, Kashyap, & Khanna, 2011). At the same time, some researchers thought that thermophilic bacteria were better suited for the production of cellulosic biofuels than mesophilic organisms (Demain, Newcomb, & Wu, 2005; Olson, Sparling, & Lynd, 2015). For example, thermophilic bacteria of Bacillus coagulans, Bacillus sp. NL01, Bacillus sp. 2–6 have been evaluated for producing lactic acid (Ma, Maeda, You, & Shirai, 2014; Ouyang et al., 2013; Wang, Cai, Zhu, Guo, & Yu, 2014; Zhao et al., 2010). There are, however, few information on the application of thermohpilic microorganism in the preparation of bioactive peptides by fermentation of oil seed meal. It is well known, the growth and propagation of most of microorganism are usually restricted when temperature is over 50 °C, even death takes place. Thus, utilization of a nonsterile SSF of RM by thermophiles offers further opportunities for reducing the polypeptide production cost and facilitating the manufacture process due to the elimination of steam-sterilization of the substrate. Generally, the identification of unknown strains is carried out by 16S rDNA sequencing. However, even if a single strain is inoculated as a starter, various microorganisms from raw materials are involved in the actual fermentation process unless they are sterilized before fermentation. Differences in the microbial community of substrate can also lead to differences in metabolites. As a result, identification of the species and characterization of the dominant strains involved in SSF are desirable for stabilizing the fermentation and improving the strains (Fleet, 1999; Gullo & Giudici, 2008; Vegas et al., 2010). Recently, the application of high-throughput next-generation sequencing technologies has provided a better and more comprehensive understanding of dynamics and diversity of microbial community in SSF (Nie, Zheng, Du, Xie, & Wang, 2015). While there are some information on SSF in literature, few data on the application of thermohpilic microorganism in the preparation of bioactive peptides by fermentation of RM exist. This study was aimed to isolate and identify thermophilic protease producing strain for manufacturing bioactive peptides from RM by fermentation at a relatively high temperature. In addition, the diversity
Table 1 Preliminary screening and re-screening of thermo-protease producing strains. Name of strains RM-1 RM-2 RM-3 RM-4
K value 2.03 3.59 1.21 2.54
± ± ± ±
Protease activity (U/mL) c
0.12 0.08a 0.07d 0.08b
24.53 25.50 23.31 24.87
± ± ± ±
0.33b 0.32a 0.58c 0.22ab
Values within a column mean the significant difference among the four strains (P < 0.05).
of bacterial community in FRM (prepared under different fermentation conditions) was clarified by high-throughput sequencing technology. 2. Materials and methods 2.1. Selection of thermophilic protease producing strains 2.1.1. Screening of protease producers by selective agar plants method Ten grams of RM (stored at room temperature and purchased from Hubei Weipu Biologic Technology Company, Hubei, China) was dissolved in 100 mL of sterile water and stirred (120 rpm, 3 min). After 5 min, 100 mL of LB liquid medium (1% tryptone, 0.5% yeast extract and 1% NaCl) was inoculated with 5 mL of the supernatant and incubated (60 °C, 48 h, 200 rpm) to obtain enriched thermophilic protease releasing strains which can be applied in the production of bioactive peptides by fermentation of RM at a relatively high temperature. The suitable gradient dilution was coated uniformly on the solid selection medium and inoculated at 60 °C for 48 h to form clear colonies. The medium consisted of 0.1% K2HPO4, 0.5% KCl, 0.05% MgSO4.7H2O, 0.01% FeSO4.7H2O, 1% skimmed milk and 1.8% agar, of which skimmed milk was sterilized at 115 °C for 15 min, and the remaining autoclaved at 121 °C for 20 min. Then the single colony with different shapes was streak-inoculated on the LB plate media (containing 1.8% agar) at 60 °C. The ratio of hydrolysis circle to colony diameter (K value) was selected as an index of protease production capacity (Guo et al., 2014), therefore, the strains with larger K value were selected for further strain identification. 2.1.2. Protease activity assay The activity of protease released into fermentation broth by selected strains was measured according to the method of Malathi and Chakraborty (1991) based on the enzymolysis of casein into tyrosine, with a slight modification of incubation temperature 60 °C instead of 42 °C. Fermentation broth was centrifuged at 7000 rpm for 10 min before determining protease activity. After that, 1 mL of casein solution (1%, w/v) and 1 mL of fermentation broth were mixed and incubated at 60 °C for 10 min. Then, 2 mL of 0.4 M trichloroacetic acid was added to stop the reaction. After centrifugation (1000 rpm, 10 min and 4 °C), 1 mL of supernatant was mixed with 5 mL of 0.4 M sodium carbonate and 1 mL of Foline-phenol and incubated at 60 °C for 20 min. The optical density of the soluble constituents was determined at 680 nm by spectrophotometry (UV-1100, Purkinje General Co., Ltd., Beijing, China) and compared with a tyrosine standard (Liu, Zhang, Yang, & Guo, 2014). One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 mg of tyrosine under assay conditions. The activity of protease in fermentation broth was calculated according to the formula:
E=
A1 ·4·n 1 × v 10
where E is the activity of protease (U/mL), A1 is the activity of enzyme in the final diluent of the sample obtained from the standard curve of tyrosine (U/mL), the total volume of reaction reagents was 4 mL, n is the dilution multiple of the sample, v is the volume of the sample (mL), and reaction time was 10 min. 2
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Fig. 1. Phylogenetic tree of RM-2.
et al., 2018). The yield of polypeptide was calculated according to the following formula:
2.1.3. Identification of selected microorganism Among the tested strains, one strain named RM-2 from RM showed promising protease production ability based on the preliminary screening and rescreening. Further identification was on the basis of the 16S rDNA sequence analysis. Genomic DNA was extracted and purified using a genome DNA extraction kit according to the manufacturer's instructions, then it was used as the template for polymerase chain reaction (PCR) (Sangon, Shanghai, China). The 16S rDNA was amplified with the upstream primer 27F 5′-AGA GTT TGA TCC TGG CTC AG-3′ and downstream primer 1541R 5′-AAG GAG GTG ATC CAG CCG CA-3′. PCR amplification was performed under the following conditions: denaturation at 95 °C for 5 min, 29 cycles at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1.5 min, with a final extension at 72 °C for 10 min. DNA sequencing was carried out by Genscript Biotechnology Corporation (Nanjing, Jiangsu, China). The resulting DNA sequence of 1458 bp obtained by 16S rDNA sequencing was analyzed with Blast through the NCBI server. A phylogenetic tree was constructed using MEGA 5.0 (Christensen & Martin, 2016).
P=
C×V × 100 M
where P is the yield of polypeptide (%), C is the concentration of polypeptide in extract (mg/mL), V is the total volume of polypeptide extract (mL), M is the quantity of FRM (mg).
2.4. High-throughput sequencing 2.4.1. Samples treatment The diversity of microbial community structure in different treatment groups was analyzed by fermenting RM with 50% moisture content and ~107 cells/mL inoculum level. In C (control) group, the RM was received no treatment. In NI (no inoculation) group, the unsterilized RM was stirred directly with commensurable distilled water, and then fermented in an incubator at 30 °C for 24 h without inoculating extra microbial strains; while in IM (inoculation of mesophile) group, Bacillus subtilis (CICC 10,160) was inoculated to ferment unsterilized RM at 30 °C for 24 h. IT (inoculation of thermophile) group was prepared by inoculating unsterilized RM with RM-2 and fermenting at 60 °C for 24 h. Fermented samples were stored at −80 °C for analysis of microbial community structure.
2.2. Solid-state fermentation Seed culture of RM-2 was prepared by incubating a single colony into 50 mL of sterile LB medium (~107 cells/mL) contained in a 250 mL flask and incubated for 24 h at 60 °C and 160 rpm, which was used for the SSF of RM. Twenty grams of RM with 42.03% protein content (determined by Kjeldahl method) after grinding to pass 40 mesh were mixed with 20 mL of sterile distilled water in a 250 mL Erlenmeyer flask. After inoculation with a 10% ratio of RM-2 (Jiang, Pan, Xie, He, & Ma, 2015), SSF was conducted at 60 °C, 80% relative humidity, and 24 h fermentation time to obtain FRM. Then, FRM was lyophilized for polypeptide assay and microbial community diversity analysis. The assay of polypeptide production was conducted after SSF at varied time (12, 24, 36, 48, 60 h), temperature (45, 50, 55, 60 °C), and ratio of substrate to liquid (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, g/mL).
2.4.2. Microbial diversity analysis DNA samples used in the high-throughput sequencing were amplified using primers targeting the V3–V4 regions of prokaryotic organism 16S rRNA genes (forward primer: 5′-CCT ACG GGN GGC WGC AG-3′; reverse primer 5′-GGA CTA CNV GGG TWT CTA ATC C-3′). Using diluted genomic DNA as template, Vazyme's Taq DNA Polymerase was used for PCR. DNA libraries were multiplexed and loaded onto an Illumina MiSeq instrument according to the manufacturer's instructions (Illumina, San Diego, CA, USA). Sequences were grouped into operational taxonomic units (OTUs) using the clustering program Usearch on the basis of 97% sequence identity (Edgar, 2013; Magoč & Salzberg, 2011). To describe microbial diversity, alpha diversity indices were calculated, including Simpson index (Simpson, 1949) and Shannon index (Shannon, 1948). The bacterial communities of each group were conducted to confirm the dominant species. Principal coordinate analysis (PCoA), as beta diversity analysis, was performed to group microorganisms. Linear discriminant analysis effect size (LEfSe) method (Segata et al., 2011) was applied to identify the communities or species which had significant effect on the division of samples.
2.3. Polypeptide production assay FRM (5 g) was dissolved in distilled water and the solution volume was adjusted to 50 mL. After stirring for 1 h, centrifugation was conducted at 5000 rpm for 15 min. Then the supernatant was mixed with 10% trichloroacetic acid (1:1, v/v) and the mixture was centrifuged (12,000 rpm, 10 min) after 10 min. After that, 2 mL of supernatant was mixed with 8 mL of biuret reagent and incubated in a water bath at 30 °C for 30 min, then centrifugation was conducted (4000 rpm, 10 min). The absorbance of the soluble materials was measured at 540 nm and compared with a Gly-Gly-Tyr-Arg standard curve (Xing 3
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Table 2 Microbial community alpha-diversity characteristics in the fermentation substrate at different inoculum and temperature. Sample ID
Chao1
OTU richness
Shannon
Simpson
Coverage
C NI IM IT
310 87 20 12
301 47 16 10
3.74 0.71 0.19 0.08
0.69 0.45 0.06 0.02
99.93% 99.92% 99.98% 99.98%
2.5. Statistical analysis All experiments were replicated three times unless stated otherwise. The analyses were carried out using one-way ANOVA by Microsoft Office Excel software and SPSS. Probability P < 0.05 indicated statistically significant differences.
3. Results and discussion 3.1. Selection and identification of thermophilic protease producing strains The releasing ability of extracellular proteases of natural isolates and commercial strains from different habitats is usually examined by selective agar medium plates, in which casein is one of the exclusive nutrients. In this study, four strains, named RM-1, RM-2, RM-3, and RM-4 produced distinct hydrolysis rings on the primary screening plate. Five microlitres of LB medium with ~107 cells/mL of these 4 strains were added to the selective agar plate and incubated at 60 °C for 48 h to calculate the K value as described in Section 2.1.1. The bacterial medium was used to measure the protease activity (Table 1). The isolate RM-2 expressed the maximum K value (3.59) and protease activity (25.5 U/mL), demonstrating RM-2 had a strong ability to secrete protease at a relatively high temperature (55–60 °C). Since RM-2 was not commercially available, it identification was essential. The appearance of colonies on solid medium (yellow, flat, round and smooth colonies) and the preliminary microscopic observation (forming spores) suggested that the most likely case was that, RM-2 belonged to a member of sporogenous bacteria. However, reliable identification was further conducted by sequencing 16S rRNA encoding gene. The obtained sequence was compared with the NCBI public database. A 16S rRNA-based phylogenetic tree (Fig. 1) was constructed using MEGA software, showing the position of the selected microorganism in relation to the close strains. Fig. 1 confirmed that RM-2 could be designated as Geobacillus stearothemophilus, the 16S rDNA sequence was deposited at NCBI Genbank with the accession No. NR115284.2. Thermostable protease gene nprT of Bacillus stearothermophilus has been sequenced in 1985, and the DNA sequence was composed of 1644 bases and 548 amino acid residues (Takagi, Imanaka, & Aiba, 1985). Tang, Zhou, Chen, Dai, and Peng (2000) researched thermophilic protease from Bacillus stearothermophilus WF146 and found that more than 600 units of enzyme in 1 mL of fermented culture could be achieved under suitable conditions. The protease, with a molecular weight around 34 kD, exhibited high temperature tolerance and was stable at a wide range of pH. Latiffi, Salleh, Rahman, Oslan, and Basri (2013) investigated secretory expression of thermostable alkaline protease from Bacillus stearothermophilus. It was revealed that, to simplify the purification steps, secretory expression of protease through yeast system were better than inducible system and could improve enzyme's capability with the highest yield of 4.13 U/mL in medium YPTD.
Fig. 2. Effect of fermentation time (1a), temperature (1b) and the ratio of rapeseed meal to water (1c) on polypeptide yield in FRM by RM-2. Vertical bars indicates standard deviations of the mean (n = 3).
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Fig. 3. Beta-diversity in bacterial communities estimated via principal coordinate analysis (PCoA).
that when fermentation temperature is 50 °C or higher, the growth and metabolism of most mesophilic microorganisms are inhibited. Luo, Ma, and Liu (2015) found that the biomass reduced sharply with augmentation of temperature (36–44 °C) in submerged fermentation of soybean by Bacillus subtilis (CICC 10,160). Therefore, it can be inferred that RM2 belong to a kind of thermophilic microorganisms.
3.2. Effect of fermentation conditions on polypeptide yield in fermented rapeseed meal by RM-2 3.2.1. Fermentation time The basic fermentation conditions were inoculum amount ~107 cells/mL, moisture content 50% and fermentation temperature 60 °C. Effects of fermentation time (12, 24, 36, 48, 60 h) on the yields of polypeptide in solid state fermented RM by RM-2 are displayed in Fig. 2a. The results exhibited that polypeptide contents increased significantly (P < 0.05) from 4.19% to 6.86% when fermentation time increased from 0 to 24 h, and then decreased gradually to 4.33% at 60 h. Jiang et al. (2015) reported that rapeseed peptides were about 6.0% under the conditions of solid-state fermentation on rapeseed meal with Bacillus subtilis W1-3 for 24 h at 30 °C. Xing et al. (2018), following solid state fermentation of rapeseed meal using Bacillus subtilis at 36 °C for 48 h, reported polypeptide yield of about 6.0%. Many researchers found that enzyme activity was increased sharply in the earlier stage of SSF, leading to the fermentation substrate decomposition (El-Bakry, Gea, & Sánchez, 2016; Leite, Salgado, Venâncio, Domínguez, & Belo, 2016; Soares, Castilho, Bon, & Freire, 2005). The augmentation of polypeptide may be attributed to the degradation of protein in the RM by protease secreted by RM-2 during fermentation (Dai, Zhang, He, Xiong, & Ma, 2017b). Peptides with relatively low molecular weight were more easily utilized than that with high molecular weight by microorganism (as nutrient for growth and metabolism), resulting in a decrease of polypeptide content with prolongation of fermentation time.
3.2.3. Moisture content The effect of ratio of substrate to water (1:0.5 to 1:2.5, g/mL) on the polypeptide yield in FRM by RM-2 was examined under the fermentation conditions of ~107 cells/mL, 60 °C for 24 h. As shown in Fig. 2c, when the ratio of substrate to water was 1:1 (g/mL), the maximum yield of polypeptide was 10.62%. Wang (2012) revealed that the polypeptide yield was 9.85% in solid-state fermentation of RM by Bacillus subtilis (CICC 10,160) and Actinomucor elegans (CICC 40,252) under 32.1 °C for 111 h, after sterilization at high temperature and pressure. Moisture content is known to be one of the important factors influencing the growth and metabolism of microorganism (Reddy, Jenkins, & VanderGheynst, 2009). Low moisture in substrate would decrease metabolic activity and consequently inhibit microbial propagation. However, excessive water also inhibits microbial activities because of the poor air permeability in the substrate (Rezaei & Vandergheynst, 2010). In the current study, the ratio of substrate to water (1:1, g/mL) gave the maximum yield of polypeptide, and this may be useful information in the generation of polypeptides from RM in industry.
3.2.2. Fermentation temperature As shown in Fig. 2b, it could be noted that fermentation temperature played an important role in the production of polypeptide during RM fermentation by RM-2, under the conditions: 50% moisture content, ~107 cells/mL inoculum level and 24 h fermentation time. Polypeptide contents were increased with the fermentation temperature (45–55 °C), and the maximum value (9.67%) was obtained at 55 °C, after which the polypeptide content decreased with increasing temperature (60 °C). Thus, it can be concluded that the capacities of growth and metabolism of RM-2 was the strongest at 55 °C, leading to production of more protease and consequently higher polypeptide yield in FRM. It is known
3.3. Microbial alpha and beta diversity in the fermentation substrate The microbial alpha diversity indices, including the OTU richness, the Shannon-Wiener index, the Simpson index and Chao 1 index were estimated in the fermentation medium under four fermentative conditions (Table 2). The results demonstrated that all samples had a high coverage (greater than 0.99) of bacteria sequences and the difference of microbial alpha diversity was obvious. Compared with NI, IM and IT group, the C group showed higher OTU richness, Shannon-Wiener and Simpson index for bacterial communities, indicating SSF could inhibit the growth of some microorganisms. Moreover, the alpha diversity 5
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Fig. 4. Profiling histogram of species in different classification levels of samples. a, family level; b, genus level.
3.4. Analysis of bacterial community diversity in fermentation substrate
indices were lowest in the IT group, demonstrating that bacterial diversity was the lowest in the substrate under thermophilic SSF, which strongly confirmed that fermentation at high temperature had good inhibition on most mosephilic microorganisms. Principal coordinate analysis (PCoA), as the beta diversity, displayed different results among the bacterial communities in FRM with four different fermentative conditions. As shown in Fig. 3, both NI and IM groups clustered together away from the C and IT groups, while the C group was also far from IT group. It was demonstrated that the difference between NI group and IM group was little, which indicated that SSF with or without inoculation of mosephilic bacteria had little effect on the microbial community in the medium, and the evolution process of the species contained in these two groups was similar. However, fermentation by thermophile was different from the other groups as regards microbial community diversity.
The bacterial communities of the four treatments at family levels are provided in Fig. 4a. In the raw materials, Chloroplast (60.32%) was the dominant family, followed by Sphingomonadaceae (4.69%), Pseudomonadaceae (4.36%), and Flavobacteriaceae (3.44%). However, in NI, IM and IT groups, Bacillaceae was the dominant species, and the relative abundance was about 96.48%, 99.81% and 99.97% respectively, while 1.47% was observed in the C group. The relative abundance of Bacillaceae increased sharply, after SSF for 24 h, irrespective of inoculation as well as fermentation temperature, revealing Bacillaceae could make full use of the nutrients in RM for propagation. It was reported that the spores formed by the bacteria of this family had very low water content and strong resistance to stress. At the same time, they could withstand high temperature, ultraviolet radiation, and ionizing radiation (Coleri Cihan, Karaca, Ozel, & Kilic, 2017). 6
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Fig. 5. Linear discriminant analysis effect size (LEfSe) analysis of the four samples (a) and the contribution of each species (b).
3.5. Evaluation of species in fermentation substrate with linear discriminant analysis effect size (LEfSe) method
Additionally, in order to analyze the diversity of bacterial community in fermentation medium under different fermentation conditions, especially the differences between IM and IT groups, analysis on the genus levels was conducted and displayed in Fig. 4b. The relative abundance of Geobacillus in IT group was 72.83%, and Geobacillus was the dominant bacteria in the process of SSF. Geobacillus is an aerobic or facultative anaerobic thermophilic bacillus with optimum growth temperature of 55–65 °C. It metabolites which have been reported include protease by Geobacillus toebii LBT 77, acetic acid kinase by Bacillus stearothermophilus, alpha-amylase by Geobacillus sp. LH8 and so on (Mollania, Khajeh, Hosseinkhani, & Dabirmanesh, 2010; Nakajima, Suzuki, & Imahori, 2017; Thebti, Riahi, & Belhadj, 2016). But in NI group, the relative abundance of species all decreased (except Bacillus) compared with the family level. The reason might be that some reads (sequence of DNA) failed in finding the corresponding genera.
LEfSe analysis emphasizes statistical significance and biological relevance (Segata et al., 2011). Effect of each component on the significant differences in C, NI, IM and IT groups was identified by linear discriminant analysis (LDA) coupled with effect size (LEfSe). Subsequently, it was to identify the communities or species which had significant effect on the division of samples (Fig. 5a). As shown in Fig. 5b, the main species in IT and IM groups in genus level was Geobacillus and Bacillus, respectively, which showed significant difference among four groups. In this study, the threshold value was set to 2 so that about 157 species in C group were not shown in Fig. 5b because they had few contributions to the division of samples.
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4. Conclusion
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In this work, thermophilic microorganism (TM) was isolated from RM in order to prepared polypeptide from RM without sterilization process. TM was identified as Geobacillus stearothermophilus by appearance of colonies and microscopic observation as well as 16S rDNA sequencing. The highest yield (9.67%) of polypeptide was achieved with Geobacillus stearothermophilus at 50% moisture content, 55 °C and 24 h fermentation time in solid-state fermentation of RM. The result of bacterial community diversity indicated that thermophilic fermentation decreased the number of species in the medium. Conflicts of interest statement We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements This study was funded by National Primary Research & Developement Plan (2016YFD0401401), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJA550002), North Jiangsu Science and Technology Project (SZYC2018011), sponsored by Qing Lan Project and a project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Abraham, J., Gea, T., & Antoni, S. (2014). Substitution of chemical dehairing by proteases from solid-state fermentation of hair wastes. Journal of Cleaner Production, 74, 191–198. Ali, C. H., Chisti, Y., Ahmad, A., Majeed, H., Qureshi, A. S., & Khushk, I. (2016). Coproduction of protease and amylase by thermophilic Bacillus sp. BBXS-2 using open solid-state fermentation of lignocellulosic biomass. Biocatal. Agric. Biotechnol. 8, 146–151. Asad, W., Asif, M., & Rasool, S. A. (2011). Extracellular enzyme production by indigenous thermophilic bacteria: Partial purification and characterization of α-amylase by Bacillus sp. wa21. Pakistan J. Bot., 43, 1045–1052. Biz, A., Sugai-Guérios, M. H., Kuivanen, J., Maaheimo, H., Krieger, N., Mitchell, D. A., et al. (2016). The introduction of the fungal d-galacturonate pathway enables the consumption of d-galacturonic acid by Saccharomyces cerevisiae. Microbial Cell Factories, 15, 1–11. Christensen, A., & Martin, G. D. A. (2016). Identification and bioactive potential of marine microorganisms from selected Florida coastal areas. Microbiology Open, 6 e448. Coleri Cihan, A., Karaca, B., Ozel, B. P., & Kilic, T. (2017). Determination of the biofilm production capacities and characteristics of members belonging to Bacillaceae family. World Journal of Microbiology and Biotechnology, 33, 1–13. Dai, C. H., Ma, H. L., He, R. H., Huang, L. R., Zhu, S. Y., Ding, Q. Z., et al. (2017a). Improvement of nutritional value and bioactivity of soybean meal by solid-state fermentation with Bacillus subtilis. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 86, 1–7. Dai, C. H., Zhang, W. W., He, R. H., Xiong, F., & Ma, H. L. (2017b). Protein breakdown and release of antioxidant peptides during simulated gastrointestinal digestion and the absorption by everted intestinal sac of rapeseed proteins. LebensmittelWissenschaft und -Technologie- Food Science and Technology, 86, 424–429. Demain, A., Newcomb, M., & Wu, D. (2005). Cellulase, clostridia, and ethanol. Microbiology and Molecular Biology Reviews, 69, 124–154. Drew, M. D., Borgeson, T. L., & Thiessen, D. L. (2007). A review of processing of feed ingredients to enhance diet digestibility in finfish. Animal Feed Science and Technology, 138, 118–136. Du, D. M., & Guo, H. (2010). Research progress in preparation and application of rapeseed polypeptide. Food and Nutrition in China, 11, 37–39. Ebune, A., Al-Asheh, S., & Duvnjak, Z. (1995). Production of phytase during solid state fermentation using Aspergillus ficuum NRRL 3135 in canola meal. Bioresource Technology, 53, 7–12. Edgar, R. C. (2013). UPARSE: Highly accurate OTU sequences from microbial ampliconreads. Nature Methods, 10, 996. El-Bakry, M., Gea, T., & Sánchez, A. (2016). Inoculation effect of thermophilic microorganisms on protease production through solid-state fermentation under non-sterile conditions at lab and bench scale (SSF). Bioprocess and Biosystems Engineering, 39, 585–592. Elazzazy, A. M., Abdelmoneim, T. S., & Almaghrabi, O. A. (2015). Isolation and characterization of biosurfactant production under extreme environmental conditions by
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thermostable, haloalkaline, solvent stable, and detergent compatible serine protease from Geobacillus toebii strain LBT 77. BioMed Research International 2016. Vegas, C., Cerezo, A. B., Mateo, E., Callejón, R. M., Poblet, M., Torija, M. J., et al. (2010). Effect of barrel design and the inoculation of Acetobacter pasteurianus in wine vinegar production. International Journal of Food Microbiology, 141, 56–62. Wang, L. M., Cai, Y. M., Zhu, L. F., Guo, H. L., & Yu, B. (2014). Major role of NADdependent lactate dehydrogenases in the production of L-lactic acid with high optical purity by the thermophile Bacillus coagulans. Applied and Environmental Microbiology, 80, 7134–7141. Wang, X. F., Ju, X. R., Wang, L. F., Yuan, J., & He, R. (2012). Study on the optimum medium conditions for rapeseed peptide production by solid-state fermentation with mixed bacteria. Food Science and Technology, 23, 147–153. Xing, Z., Hou, X. S., Tang, Y. X., He, R. H., Mintah, B. K., Dabbour, M., et al. (2018). Monitoring of polypeptide content in the solid-state fermentation process of rapeseed meal using NIRS and chemometrics. Journal of Food Process Engineering, 41, e12853. Xue, Z. H., Liu, Z. W., Wu, M. C., Zhuang, S. W., & Yu, W. C. (2010). Effect of rapeseed peptide on DNA damage and apoptosis in Hela cells. Experimental & Toxicologic Pathology, 62, 519–523. Zhang, M. Y., Yan, Z. B., An, C. M., Wang, D., Liu, X., Zhang, J. F., et al. (2018). Rapeseed protein-derived antioxidant peptide RAP alleviates renal fibrosis through MAPK/NFκB signaling pathways in diabetic nephropathy. Drug Design, Development and Therapy, 12, 1255–1268. Zhao, B., Wang, L. M., Ma, C. Q., Yang, C. Y., Xu, P., & Ma, Y. H. (2010). Repeated open fermentative production of optically pure L-lactic acid using a thermophilic Bacillus sp. strain. Bioresource Technology, 101, 6494–6498. Zhou, J. W., Wu, K., & Rao, C. V. (2016). Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production. Biotechnology and Bioengineering, 113, 2156–2167.
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Thermophilic solid-state fermentation of rapeseed meal and analysis of microbial community diversity
T
Xiaoshan Houa,b, Chunhua Daia,b,∗∗, Yingxiu Tanga,b, Zheng Xinga,b, Benjamin Kumah Mintaha,b,c, Mokhtar Dabboura,b,d, Qingzhi Dinga,b, Ronghai Hea,b,∗, Haile Maa,b a
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China Institute of Food Physical Processing, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China c ILSI-UG FSNTC, Department of Nutrition and Food Science, University of Ghana, POB LG 91, Legon, Accra, Ghana d Department of Agricultural and Biosystems Engineering, Faculty of Agriculture, Benha University, P.O. Box 13736, Moshtohor, Qaluobia, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Geobacillus stearothermophilus Rapeseed meal Thermophilic solid-state fermentation Polypeptide Microbial community diversity
To reduce the high cost caused by steam sterilization of fermentation substrates and equipments in mesophilic solid-state fermentation of rapeseed meal (RM), a thermophilic protease-producing strain RM-2 from RM was isolated and employed in thermophilic solid-state fermentation of unsterilized RM for polypeptide preparation. The isolate was identified as Geobacillus stearothermophilus by appearance of colonies and microscopic observation as well as 16S rDNA sequencing. The highest yield of polypeptide (9.67%) was obtained by RM-2 at 50% moisture content, 55 °C and 24 h fermentation time. Analysis of microbial community diversity in fermented RM revealed Geobacillus stearothermophilus as dominant strain inhibiting most mesophilic microorganisms. Thermophilic solid-state fermentation on RM improved the yield of rapeseed polypeptide, as well as the utilization rate of substrate. The application of this technology (thermophilic solid-state fermentation) would be environmentally friendlier and energy-saving to industry.
1. Introduction Rapeseed meal (RM), a by-product after oil extraction from rapeseed seeds, is considered as an important and cheap protein source for food and animal feed (Ebune, Al-Asheh, & Duvnjak, 1995). However, it utilization is limited due to the presence of anti-nutritional factors (ANFs) and high fiber levels (Erdogan & Olmez, 2010). In order to utilize RM effectively, efforts have been channeled in developing techniques to improve its quality, including enzymatic hydrolysis, chemical method and microbial fermentation to prepare rapeseed polypeptide (Drew, Borgeson, & Thiessen, 2007). Of these approaches, enzymatic hydrolysis is the most important. However, the high cost of protease may restrict it application. Other researchers have also used chemical means to prepare rapeseed polypeptide, but in actual production, the degree of hydrolysis is difficult to control, and the nutritional value of protein is damaged greatly, which seriously affects the functional activity of rapeseed polypeptide. In addition, the wastewater from the acid and alkali treatment, upon discharge may lead to environmental pollution (Du & Guo, 2010). Therefore, microbial fermentation is regarded as promising technique applied to detoxify and
∗
improve the nutrition and bioactivity of RM (Dai et al., 2017a; Lomascolo, Uzan-Boukhris, Sigoillot, & Fine, 2012). For instance, fermentation with Rhizopus oligosporus sp-T3 for 40 h, resulted in the degradation of undesirable factors such as 84% of carbohydrates, 30% of lignin, 47% of total glucosinolates, and other polyphenolic components indigestible by nonruminants when compared with unfermented rapeseed meal (Mejean et al., 2003). Shi et al. (2016) found that fermenting rapeseed meal (FRM) by Aspergillus niger resulted in more crude protein and amino acid (except His) than unfermented RM. Further, the small peptide in FRM increased 2.26 times; and the concentrations of antinutritional substrates including neutral detergent fiber, glucosinolates, isothiocyanate, oxazolidithione, and phytic acid reduced (P < 0.05) by 13.47, 43.07, 55.64, 44.68 and 86.09%, respectively. Moreover, it was reported that the hydrolysates of RM (rapeseed polypeptides) displayed not only the high utilization, fast digestion and absorption rate in the body, but also some physiological activities such as antioxidant, antiinflammatory, anticancer and so on (Zhang et al., 2018; Rodrigues, Carvalho, & Rocha, 2017; Xue, Liu, Wu, Zhuang, & Yu, 2010). Solid-state fermentation (SSF) is defined as a fermentation process executed on non-soluble materials which act as a physical support and a
Corresponding author. School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China. Co-Corresponding author. School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China. E-mail addresses: [email protected] (C. Dai), [email protected] (R. He).
∗∗
https://doi.org/10.1016/j.lwt.2019.108520 Received 11 March 2019; Received in revised form 17 July 2019; Accepted 20 August 2019 Available online 20 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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source of nutrients in the absence of free flowing liquid. Nowadays, SSFs are already performed at the commercial scale in the food industry or waste treatment and utilization (Biz et al., 2016; Selvakumar, Ashakumary, & Pandey, 1998). Compared with submerged fermentation, SSF has the characteristic of lower consumption and a mild reaction (Reddy, Wee, Yun, & Ryu, 2008). But in the past, most of the fermentation was performed under mesophilic conditions, along with the necessary process of autoclaving of substrates to kill the bacteria existed in the materials before fermentation in order to avoid contamination of products (Lazim, Mankai, Slama, Barkallah, & Limam, 2009; Prakasham, Rao, & Sarma, 2006). Therefore, mesophilic fermentation requires the use of large amount of steam for sterilization of substrates and equipments. That notwithstanding, rapeseed protein is easily denatured during high temperature and pressure sterilization treatment, which decreases the conversion efficiency of rapeseed protein (Abraham, Gea, & Antoni, 2014; He, He, Chao, & Ju, 2014). Recently, much attention is given to the isolation of thermophilic organisms from compost, hot spring and other extreme environments due to the secreted enzyme in the organism which has application in waste processing (Asad, Asif, & Rasool, 2011; Elazzazy, Abdelmoneim, & Almaghrabi, 2015; Hamilton-Brehm et al., 2010; Li et al., 2019; Zhou, Wu, & Rao, 2016). So far, many researchers have proposed that thermophilic bacteria are better suited for the production of industrial enzymes. Ali et al. (2016) employed open SSF of lignocellulosic biomass to coproduce protease and amylase by thermophilic Bacillus sp. BBXS-2. The concentrations of enzymes reached 12,200 U/g dry matter for protease and 6900 U/g dry matter for amylase under optimal nonsterile fermentation conditions (pH 8.5, 45 °C for 5 d) using wheat straw as substrate. Similarly, SSF involving thermophilic bacteria has also been employed to produce lipase from Pseudomonas sp. S1 (Sahoo, Subudhi, & Kumar, 2014) and xylanase from Promicromonospora sp. MARS (Kumar, Joshi, Kashyap, & Khanna, 2011). At the same time, some researchers thought that thermophilic bacteria were better suited for the production of cellulosic biofuels than mesophilic organisms (Demain, Newcomb, & Wu, 2005; Olson, Sparling, & Lynd, 2015). For example, thermophilic bacteria of Bacillus coagulans, Bacillus sp. NL01, Bacillus sp. 2–6 have been evaluated for producing lactic acid (Ma, Maeda, You, & Shirai, 2014; Ouyang et al., 2013; Wang, Cai, Zhu, Guo, & Yu, 2014; Zhao et al., 2010). There are, however, few information on the application of thermohpilic microorganism in the preparation of bioactive peptides by fermentation of oil seed meal. It is well known, the growth and propagation of most of microorganism are usually restricted when temperature is over 50 °C, even death takes place. Thus, utilization of a nonsterile SSF of RM by thermophiles offers further opportunities for reducing the polypeptide production cost and facilitating the manufacture process due to the elimination of steam-sterilization of the substrate. Generally, the identification of unknown strains is carried out by 16S rDNA sequencing. However, even if a single strain is inoculated as a starter, various microorganisms from raw materials are involved in the actual fermentation process unless they are sterilized before fermentation. Differences in the microbial community of substrate can also lead to differences in metabolites. As a result, identification of the species and characterization of the dominant strains involved in SSF are desirable for stabilizing the fermentation and improving the strains (Fleet, 1999; Gullo & Giudici, 2008; Vegas et al., 2010). Recently, the application of high-throughput next-generation sequencing technologies has provided a better and more comprehensive understanding of dynamics and diversity of microbial community in SSF (Nie, Zheng, Du, Xie, & Wang, 2015). While there are some information on SSF in literature, few data on the application of thermohpilic microorganism in the preparation of bioactive peptides by fermentation of RM exist. This study was aimed to isolate and identify thermophilic protease producing strain for manufacturing bioactive peptides from RM by fermentation at a relatively high temperature. In addition, the diversity
Table 1 Preliminary screening and re-screening of thermo-protease producing strains. Name of strains RM-1 RM-2 RM-3 RM-4
K value 2.03 3.59 1.21 2.54
± ± ± ±
Protease activity (U/mL) c
0.12 0.08a 0.07d 0.08b
24.53 25.50 23.31 24.87
± ± ± ±
0.33b 0.32a 0.58c 0.22ab
Values within a column mean the significant difference among the four strains (P < 0.05).
of bacterial community in FRM (prepared under different fermentation conditions) was clarified by high-throughput sequencing technology. 2. Materials and methods 2.1. Selection of thermophilic protease producing strains 2.1.1. Screening of protease producers by selective agar plants method Ten grams of RM (stored at room temperature and purchased from Hubei Weipu Biologic Technology Company, Hubei, China) was dissolved in 100 mL of sterile water and stirred (120 rpm, 3 min). After 5 min, 100 mL of LB liquid medium (1% tryptone, 0.5% yeast extract and 1% NaCl) was inoculated with 5 mL of the supernatant and incubated (60 °C, 48 h, 200 rpm) to obtain enriched thermophilic protease releasing strains which can be applied in the production of bioactive peptides by fermentation of RM at a relatively high temperature. The suitable gradient dilution was coated uniformly on the solid selection medium and inoculated at 60 °C for 48 h to form clear colonies. The medium consisted of 0.1% K2HPO4, 0.5% KCl, 0.05% MgSO4.7H2O, 0.01% FeSO4.7H2O, 1% skimmed milk and 1.8% agar, of which skimmed milk was sterilized at 115 °C for 15 min, and the remaining autoclaved at 121 °C for 20 min. Then the single colony with different shapes was streak-inoculated on the LB plate media (containing 1.8% agar) at 60 °C. The ratio of hydrolysis circle to colony diameter (K value) was selected as an index of protease production capacity (Guo et al., 2014), therefore, the strains with larger K value were selected for further strain identification. 2.1.2. Protease activity assay The activity of protease released into fermentation broth by selected strains was measured according to the method of Malathi and Chakraborty (1991) based on the enzymolysis of casein into tyrosine, with a slight modification of incubation temperature 60 °C instead of 42 °C. Fermentation broth was centrifuged at 7000 rpm for 10 min before determining protease activity. After that, 1 mL of casein solution (1%, w/v) and 1 mL of fermentation broth were mixed and incubated at 60 °C for 10 min. Then, 2 mL of 0.4 M trichloroacetic acid was added to stop the reaction. After centrifugation (1000 rpm, 10 min and 4 °C), 1 mL of supernatant was mixed with 5 mL of 0.4 M sodium carbonate and 1 mL of Foline-phenol and incubated at 60 °C for 20 min. The optical density of the soluble constituents was determined at 680 nm by spectrophotometry (UV-1100, Purkinje General Co., Ltd., Beijing, China) and compared with a tyrosine standard (Liu, Zhang, Yang, & Guo, 2014). One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 mg of tyrosine under assay conditions. The activity of protease in fermentation broth was calculated according to the formula:
E=
A1 ·4·n 1 × v 10
where E is the activity of protease (U/mL), A1 is the activity of enzyme in the final diluent of the sample obtained from the standard curve of tyrosine (U/mL), the total volume of reaction reagents was 4 mL, n is the dilution multiple of the sample, v is the volume of the sample (mL), and reaction time was 10 min. 2
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Fig. 1. Phylogenetic tree of RM-2.
et al., 2018). The yield of polypeptide was calculated according to the following formula:
2.1.3. Identification of selected microorganism Among the tested strains, one strain named RM-2 from RM showed promising protease production ability based on the preliminary screening and rescreening. Further identification was on the basis of the 16S rDNA sequence analysis. Genomic DNA was extracted and purified using a genome DNA extraction kit according to the manufacturer's instructions, then it was used as the template for polymerase chain reaction (PCR) (Sangon, Shanghai, China). The 16S rDNA was amplified with the upstream primer 27F 5′-AGA GTT TGA TCC TGG CTC AG-3′ and downstream primer 1541R 5′-AAG GAG GTG ATC CAG CCG CA-3′. PCR amplification was performed under the following conditions: denaturation at 95 °C for 5 min, 29 cycles at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1.5 min, with a final extension at 72 °C for 10 min. DNA sequencing was carried out by Genscript Biotechnology Corporation (Nanjing, Jiangsu, China). The resulting DNA sequence of 1458 bp obtained by 16S rDNA sequencing was analyzed with Blast through the NCBI server. A phylogenetic tree was constructed using MEGA 5.0 (Christensen & Martin, 2016).
P=
C×V × 100 M
where P is the yield of polypeptide (%), C is the concentration of polypeptide in extract (mg/mL), V is the total volume of polypeptide extract (mL), M is the quantity of FRM (mg).
2.4. High-throughput sequencing 2.4.1. Samples treatment The diversity of microbial community structure in different treatment groups was analyzed by fermenting RM with 50% moisture content and ~107 cells/mL inoculum level. In C (control) group, the RM was received no treatment. In NI (no inoculation) group, the unsterilized RM was stirred directly with commensurable distilled water, and then fermented in an incubator at 30 °C for 24 h without inoculating extra microbial strains; while in IM (inoculation of mesophile) group, Bacillus subtilis (CICC 10,160) was inoculated to ferment unsterilized RM at 30 °C for 24 h. IT (inoculation of thermophile) group was prepared by inoculating unsterilized RM with RM-2 and fermenting at 60 °C for 24 h. Fermented samples were stored at −80 °C for analysis of microbial community structure.
2.2. Solid-state fermentation Seed culture of RM-2 was prepared by incubating a single colony into 50 mL of sterile LB medium (~107 cells/mL) contained in a 250 mL flask and incubated for 24 h at 60 °C and 160 rpm, which was used for the SSF of RM. Twenty grams of RM with 42.03% protein content (determined by Kjeldahl method) after grinding to pass 40 mesh were mixed with 20 mL of sterile distilled water in a 250 mL Erlenmeyer flask. After inoculation with a 10% ratio of RM-2 (Jiang, Pan, Xie, He, & Ma, 2015), SSF was conducted at 60 °C, 80% relative humidity, and 24 h fermentation time to obtain FRM. Then, FRM was lyophilized for polypeptide assay and microbial community diversity analysis. The assay of polypeptide production was conducted after SSF at varied time (12, 24, 36, 48, 60 h), temperature (45, 50, 55, 60 °C), and ratio of substrate to liquid (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, g/mL).
2.4.2. Microbial diversity analysis DNA samples used in the high-throughput sequencing were amplified using primers targeting the V3–V4 regions of prokaryotic organism 16S rRNA genes (forward primer: 5′-CCT ACG GGN GGC WGC AG-3′; reverse primer 5′-GGA CTA CNV GGG TWT CTA ATC C-3′). Using diluted genomic DNA as template, Vazyme's Taq DNA Polymerase was used for PCR. DNA libraries were multiplexed and loaded onto an Illumina MiSeq instrument according to the manufacturer's instructions (Illumina, San Diego, CA, USA). Sequences were grouped into operational taxonomic units (OTUs) using the clustering program Usearch on the basis of 97% sequence identity (Edgar, 2013; Magoč & Salzberg, 2011). To describe microbial diversity, alpha diversity indices were calculated, including Simpson index (Simpson, 1949) and Shannon index (Shannon, 1948). The bacterial communities of each group were conducted to confirm the dominant species. Principal coordinate analysis (PCoA), as beta diversity analysis, was performed to group microorganisms. Linear discriminant analysis effect size (LEfSe) method (Segata et al., 2011) was applied to identify the communities or species which had significant effect on the division of samples.
2.3. Polypeptide production assay FRM (5 g) was dissolved in distilled water and the solution volume was adjusted to 50 mL. After stirring for 1 h, centrifugation was conducted at 5000 rpm for 15 min. Then the supernatant was mixed with 10% trichloroacetic acid (1:1, v/v) and the mixture was centrifuged (12,000 rpm, 10 min) after 10 min. After that, 2 mL of supernatant was mixed with 8 mL of biuret reagent and incubated in a water bath at 30 °C for 30 min, then centrifugation was conducted (4000 rpm, 10 min). The absorbance of the soluble materials was measured at 540 nm and compared with a Gly-Gly-Tyr-Arg standard curve (Xing 3
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Table 2 Microbial community alpha-diversity characteristics in the fermentation substrate at different inoculum and temperature. Sample ID
Chao1
OTU richness
Shannon
Simpson
Coverage
C NI IM IT
310 87 20 12
301 47 16 10
3.74 0.71 0.19 0.08
0.69 0.45 0.06 0.02
99.93% 99.92% 99.98% 99.98%
2.5. Statistical analysis All experiments were replicated three times unless stated otherwise. The analyses were carried out using one-way ANOVA by Microsoft Office Excel software and SPSS. Probability P < 0.05 indicated statistically significant differences.
3. Results and discussion 3.1. Selection and identification of thermophilic protease producing strains The releasing ability of extracellular proteases of natural isolates and commercial strains from different habitats is usually examined by selective agar medium plates, in which casein is one of the exclusive nutrients. In this study, four strains, named RM-1, RM-2, RM-3, and RM-4 produced distinct hydrolysis rings on the primary screening plate. Five microlitres of LB medium with ~107 cells/mL of these 4 strains were added to the selective agar plate and incubated at 60 °C for 48 h to calculate the K value as described in Section 2.1.1. The bacterial medium was used to measure the protease activity (Table 1). The isolate RM-2 expressed the maximum K value (3.59) and protease activity (25.5 U/mL), demonstrating RM-2 had a strong ability to secrete protease at a relatively high temperature (55–60 °C). Since RM-2 was not commercially available, it identification was essential. The appearance of colonies on solid medium (yellow, flat, round and smooth colonies) and the preliminary microscopic observation (forming spores) suggested that the most likely case was that, RM-2 belonged to a member of sporogenous bacteria. However, reliable identification was further conducted by sequencing 16S rRNA encoding gene. The obtained sequence was compared with the NCBI public database. A 16S rRNA-based phylogenetic tree (Fig. 1) was constructed using MEGA software, showing the position of the selected microorganism in relation to the close strains. Fig. 1 confirmed that RM-2 could be designated as Geobacillus stearothemophilus, the 16S rDNA sequence was deposited at NCBI Genbank with the accession No. NR115284.2. Thermostable protease gene nprT of Bacillus stearothermophilus has been sequenced in 1985, and the DNA sequence was composed of 1644 bases and 548 amino acid residues (Takagi, Imanaka, & Aiba, 1985). Tang, Zhou, Chen, Dai, and Peng (2000) researched thermophilic protease from Bacillus stearothermophilus WF146 and found that more than 600 units of enzyme in 1 mL of fermented culture could be achieved under suitable conditions. The protease, with a molecular weight around 34 kD, exhibited high temperature tolerance and was stable at a wide range of pH. Latiffi, Salleh, Rahman, Oslan, and Basri (2013) investigated secretory expression of thermostable alkaline protease from Bacillus stearothermophilus. It was revealed that, to simplify the purification steps, secretory expression of protease through yeast system were better than inducible system and could improve enzyme's capability with the highest yield of 4.13 U/mL in medium YPTD.
Fig. 2. Effect of fermentation time (1a), temperature (1b) and the ratio of rapeseed meal to water (1c) on polypeptide yield in FRM by RM-2. Vertical bars indicates standard deviations of the mean (n = 3).
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Fig. 3. Beta-diversity in bacterial communities estimated via principal coordinate analysis (PCoA).
that when fermentation temperature is 50 °C or higher, the growth and metabolism of most mesophilic microorganisms are inhibited. Luo, Ma, and Liu (2015) found that the biomass reduced sharply with augmentation of temperature (36–44 °C) in submerged fermentation of soybean by Bacillus subtilis (CICC 10,160). Therefore, it can be inferred that RM2 belong to a kind of thermophilic microorganisms.
3.2. Effect of fermentation conditions on polypeptide yield in fermented rapeseed meal by RM-2 3.2.1. Fermentation time The basic fermentation conditions were inoculum amount ~107 cells/mL, moisture content 50% and fermentation temperature 60 °C. Effects of fermentation time (12, 24, 36, 48, 60 h) on the yields of polypeptide in solid state fermented RM by RM-2 are displayed in Fig. 2a. The results exhibited that polypeptide contents increased significantly (P < 0.05) from 4.19% to 6.86% when fermentation time increased from 0 to 24 h, and then decreased gradually to 4.33% at 60 h. Jiang et al. (2015) reported that rapeseed peptides were about 6.0% under the conditions of solid-state fermentation on rapeseed meal with Bacillus subtilis W1-3 for 24 h at 30 °C. Xing et al. (2018), following solid state fermentation of rapeseed meal using Bacillus subtilis at 36 °C for 48 h, reported polypeptide yield of about 6.0%. Many researchers found that enzyme activity was increased sharply in the earlier stage of SSF, leading to the fermentation substrate decomposition (El-Bakry, Gea, & Sánchez, 2016; Leite, Salgado, Venâncio, Domínguez, & Belo, 2016; Soares, Castilho, Bon, & Freire, 2005). The augmentation of polypeptide may be attributed to the degradation of protein in the RM by protease secreted by RM-2 during fermentation (Dai, Zhang, He, Xiong, & Ma, 2017b). Peptides with relatively low molecular weight were more easily utilized than that with high molecular weight by microorganism (as nutrient for growth and metabolism), resulting in a decrease of polypeptide content with prolongation of fermentation time.
3.2.3. Moisture content The effect of ratio of substrate to water (1:0.5 to 1:2.5, g/mL) on the polypeptide yield in FRM by RM-2 was examined under the fermentation conditions of ~107 cells/mL, 60 °C for 24 h. As shown in Fig. 2c, when the ratio of substrate to water was 1:1 (g/mL), the maximum yield of polypeptide was 10.62%. Wang (2012) revealed that the polypeptide yield was 9.85% in solid-state fermentation of RM by Bacillus subtilis (CICC 10,160) and Actinomucor elegans (CICC 40,252) under 32.1 °C for 111 h, after sterilization at high temperature and pressure. Moisture content is known to be one of the important factors influencing the growth and metabolism of microorganism (Reddy, Jenkins, & VanderGheynst, 2009). Low moisture in substrate would decrease metabolic activity and consequently inhibit microbial propagation. However, excessive water also inhibits microbial activities because of the poor air permeability in the substrate (Rezaei & Vandergheynst, 2010). In the current study, the ratio of substrate to water (1:1, g/mL) gave the maximum yield of polypeptide, and this may be useful information in the generation of polypeptides from RM in industry.
3.2.2. Fermentation temperature As shown in Fig. 2b, it could be noted that fermentation temperature played an important role in the production of polypeptide during RM fermentation by RM-2, under the conditions: 50% moisture content, ~107 cells/mL inoculum level and 24 h fermentation time. Polypeptide contents were increased with the fermentation temperature (45–55 °C), and the maximum value (9.67%) was obtained at 55 °C, after which the polypeptide content decreased with increasing temperature (60 °C). Thus, it can be concluded that the capacities of growth and metabolism of RM-2 was the strongest at 55 °C, leading to production of more protease and consequently higher polypeptide yield in FRM. It is known
3.3. Microbial alpha and beta diversity in the fermentation substrate The microbial alpha diversity indices, including the OTU richness, the Shannon-Wiener index, the Simpson index and Chao 1 index were estimated in the fermentation medium under four fermentative conditions (Table 2). The results demonstrated that all samples had a high coverage (greater than 0.99) of bacteria sequences and the difference of microbial alpha diversity was obvious. Compared with NI, IM and IT group, the C group showed higher OTU richness, Shannon-Wiener and Simpson index for bacterial communities, indicating SSF could inhibit the growth of some microorganisms. Moreover, the alpha diversity 5
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Fig. 4. Profiling histogram of species in different classification levels of samples. a, family level; b, genus level.
3.4. Analysis of bacterial community diversity in fermentation substrate
indices were lowest in the IT group, demonstrating that bacterial diversity was the lowest in the substrate under thermophilic SSF, which strongly confirmed that fermentation at high temperature had good inhibition on most mosephilic microorganisms. Principal coordinate analysis (PCoA), as the beta diversity, displayed different results among the bacterial communities in FRM with four different fermentative conditions. As shown in Fig. 3, both NI and IM groups clustered together away from the C and IT groups, while the C group was also far from IT group. It was demonstrated that the difference between NI group and IM group was little, which indicated that SSF with or without inoculation of mosephilic bacteria had little effect on the microbial community in the medium, and the evolution process of the species contained in these two groups was similar. However, fermentation by thermophile was different from the other groups as regards microbial community diversity.
The bacterial communities of the four treatments at family levels are provided in Fig. 4a. In the raw materials, Chloroplast (60.32%) was the dominant family, followed by Sphingomonadaceae (4.69%), Pseudomonadaceae (4.36%), and Flavobacteriaceae (3.44%). However, in NI, IM and IT groups, Bacillaceae was the dominant species, and the relative abundance was about 96.48%, 99.81% and 99.97% respectively, while 1.47% was observed in the C group. The relative abundance of Bacillaceae increased sharply, after SSF for 24 h, irrespective of inoculation as well as fermentation temperature, revealing Bacillaceae could make full use of the nutrients in RM for propagation. It was reported that the spores formed by the bacteria of this family had very low water content and strong resistance to stress. At the same time, they could withstand high temperature, ultraviolet radiation, and ionizing radiation (Coleri Cihan, Karaca, Ozel, & Kilic, 2017). 6
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Fig. 5. Linear discriminant analysis effect size (LEfSe) analysis of the four samples (a) and the contribution of each species (b).
3.5. Evaluation of species in fermentation substrate with linear discriminant analysis effect size (LEfSe) method
Additionally, in order to analyze the diversity of bacterial community in fermentation medium under different fermentation conditions, especially the differences between IM and IT groups, analysis on the genus levels was conducted and displayed in Fig. 4b. The relative abundance of Geobacillus in IT group was 72.83%, and Geobacillus was the dominant bacteria in the process of SSF. Geobacillus is an aerobic or facultative anaerobic thermophilic bacillus with optimum growth temperature of 55–65 °C. It metabolites which have been reported include protease by Geobacillus toebii LBT 77, acetic acid kinase by Bacillus stearothermophilus, alpha-amylase by Geobacillus sp. LH8 and so on (Mollania, Khajeh, Hosseinkhani, & Dabirmanesh, 2010; Nakajima, Suzuki, & Imahori, 2017; Thebti, Riahi, & Belhadj, 2016). But in NI group, the relative abundance of species all decreased (except Bacillus) compared with the family level. The reason might be that some reads (sequence of DNA) failed in finding the corresponding genera.
LEfSe analysis emphasizes statistical significance and biological relevance (Segata et al., 2011). Effect of each component on the significant differences in C, NI, IM and IT groups was identified by linear discriminant analysis (LDA) coupled with effect size (LEfSe). Subsequently, it was to identify the communities or species which had significant effect on the division of samples (Fig. 5a). As shown in Fig. 5b, the main species in IT and IM groups in genus level was Geobacillus and Bacillus, respectively, which showed significant difference among four groups. In this study, the threshold value was set to 2 so that about 157 species in C group were not shown in Fig. 5b because they had few contributions to the division of samples.
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4. Conclusion
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