Screening and selection of Bacillus spp. for fermented corticate soybean meal production



Pairote Wongputtisin, Biotechnology Program, Faculty of Science, Maejo University, Chiang Mai, Thailand. E-mail:



To screen and select the Bacillus spp. from Tua-nao of northern Thailand for fermented corticate soybean meal (FCSBM) production.

Methods and Results

After isolation of Bacillus spp. from Tua-nao was carried out, cellulase, hemicellulases (i.e., β-mannanase and xylanase) and phytase production by isolated Bacillus spp. were determined. B. subtilis isolate MR10 showed the highest β-mannanase, xylanase and phytase production at 280, 41 and 16 U g−1 substrate, respectively, while the highest cellulase production was found in TK8 at 25 U g−1 substrate. FCSBMs produced by single starter and mixed starter of both isolates showed the better properties than those of corticate soybean meal (CSBM), i.e., higher in soluble sugar, protein and phosphate content, smaller sugar molecules and better digestibility and absorbability than those of CSBM. Moreover, FCSBMs had no toxicity effect on mouse fibroblast cell line (3T3) but had an inhibitory effect on lung cancer cell line (CorL23).


B. subtilis isolate MR10 and TK8 were selected for FCSBMs production because of their role as nutritional enhancer for CSBM and their safety.

Significance and Impact of the Study

The results of this study were useful for FCSBM production process that can be applied as feed ingredient for monogastric animals.


Soybean meal (SBM) is a high quality and the most important source of dietary protein for monogastric animals. It is a by-product obtained after oil extraction process of soybean including drying, dehulling, flaking, expanding, extraction and meal drying. SBM contains 47–51% dry matter (DM) of protein, while the other important nutritional compositions are carbohydrates (35% DM), fat (1·2–3·1% DM) and variety of minerals (Wongputtisin et al. 2007). SBM may be classified into two types. The first is SBM without hull or called dehulled SBM, and another type is SBM mixed with hull to adjust protein content. The later is called corticate soybean meal (CSBM) that is cheaper than dehulled SBM. Recently, fermented soybean meal (FSBM) became a candidate of feed additive that has been commercially produced and used in livestock production to replace fishmeal and skim milk in the diets (Wongputtisin 2008). Biotechnological processes have been applied in FSBM production. Microbial fermentation is expected to increase nutritional values and also eliminate the anti-nutritional factors included in SBM such as phytic acid, β-mannan and raffinose family oligosaccharids (RFOs) etc. The benefit of FSBM supplementation for animal health has been reported. Better in feed conversion ratio (FCR) of broiler and piglet, moreover phosphorus and IgM content in broiler serum was improved after fed with FSBM (Feng et al. 2007a,b). The zinc availability in rats fed with FSBM was greater than the control group (Hirabayashi et al. 1998). Mostly, FSBMs were produced by fermentative fungi such as Aspergillus niger (Feng et al. 2007a,b) and A. usamii (Hirabayashi et al. 1998, 1997). In contrast, for fermented soybean (FSB) for human consumption in many regions around the world, Bacillus spp. is the predominant fermentative micro-organism; especially, Bsubtilis.

Tua-nao is a traditional Thai FSB food which is fermented by many Bacillus spp. (Petchkongkaew et al. 2008) and has been consumed for several decades, similar to FSB food of Japan, so-called Natto that is mainly produced by B. subtilis (natto). Many species of Bacilli group; i.e., B. subtilis, B. licheniformis, Bcereus, B. circulans, B. thuringiensis and B. sphaericus are also found in Kinema, a FSB food of India and Nepal, as important fermentative bacteria (Sarkar et al. 2002), while Bsubtilis, B. pumilus and B. licheniformis found in soy-Dawadawa (FSB food of West African countries) was reported (Omafuvbe et al. 2006). Soybean fermentation by Bacillus has been reported on nutritional enhancement of fermented soybean food for human consumption, such as increasing of free amino acids (Sarkar et al. 1997), better digestibility and absorbability (Kiers et al. 2000b), enhancing of flavour, aroma and appetite (Han et al. 2001; Beaumont 2002), and increasing of antioxidant properties (Yang et al. 2000; Wongputtisin et al. 2007). Furthermore, some species such as B. subtilis was reported on its potential used as probiotic for human and swine (Link and Kováč 2006; Sorokulova et al. 2008). Previously, B. subtilis were screened and selected as a starter for soy-Dawadawa production. The criteria used for screening were proteolytic, amylolytic and fat hydrolysis activity, pH of product and growing ability on soybean agar. Furthermore, the acceptance of consumers comparing to spontaneous fermentation was also evaluated (Amoa-Awua et al. 2006; Terlabie et al. 2006).

As Tua-nao is a food that contains of Bacillus spp. community and nutritional improvement of soybean can be archived, therefore production of fermented corticate soybean meal (FCSBM) by using a pure culture starter of Bacillus spp. isolated from Tua-nao is very interesting as an alternative fermentative micro-organism. Fermentation by pure culture starter gives many advantages over mixed-natural starter as carried out in a traditional fermentation. The quality consistency of fermented product cannot be obtained when traditional fermentation is conducted. Moreover, the fermented product may be contaminated by harmful micro-organisms from the surrounding environment. This study aims to screen for a suitable fermentative Bacillus spp. from Tua-nao samples for FCSBM production. The criteria used were based on the aspect of monogastric animal feed; i.e., nonstarch polysaccharides (NSPs) degrading enzymes and safety.

Materials and methods

Isolation and identification of Bacillus spp. from Tau-nao

Four fresh Tau-nao samples produced traditionally in Chiang Mai province, Thailand, were collected and screened for Bacillus spp. One gram of Tau-nao was suspended in 9 ml of 0·85% (w/v) NaCl and subsequently incubated at 80°C for 20 min (Chantawannakul et al. 2002) before isolation on nutrient agar. Different colonies were collected and identified for their species according to the biochemical protocols as described by Norris et al. (1981). After that, 16S ribosomal RNA gene partial sequencing was studied in the selected isolates according to the protocol of Niamsup et al. (2003). Primers used were 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 520R (5′-ACCGCGGCKGCTGGCA-3′; Operon, Germany). The reaction consisted of 2 μl of 27F primer, 2 μl of 520R, 20 μl of H2O, 1 μl of 20 ng ml−1 DNA and 25 μl of Qiagen master mix. The amplification was programmed as follows: initial denaturation at 94°C for 5 min, followed by 25 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. The reaction was maintained at 72°C for 5 min after the cycles. PCR products were purified using TaKaRa SUPREC-PCR (Takara, Japan) and sequence analysis was performed by First Base Laboratories Company (Malaysia). All of the sequencing data were analysed using the public database (Blast;

Primary screening for cellulase, hemicellulase and phytase producing Bacillus spp.

Isolated Bacillus spp. were transferred to Luria broth that supplemented with each of 0·1% (w/v) carboxymethyl cellulose (CMC), locust bean gum (LBG) or oat spelt xylan separately, for screening of cellulase, β-mannanase and xylanase activity, respectively. They were incubated on incubator shaker at 150 rev min−1, 37°C for 24 h. The cells were removed from culture broths by centrifugation at 3427 g, and 30 μl supernatant was dropped into the wells (0·5 cm Ø) punched on agar plates supplemented with 0·1% (w/v) of CMC or LBG or xylan. These were incubated at 45°C for 16 h, and subsequently developed by congo red staining and 1 mol l−1 NaCl washing. The formed clear zone represented the enzyme activity.

For phytase screening, each isolate was stabbed onto Luria agar that was supplemented with 0·1% (w/v) phytic acid and then incubated at 37°C for 24 h. After that, clear zone around colony was detected using staining reagents. Briefly, 2% (w/v) CoCl2 solution was poured on agar for 5 min and then followed by the mixture of 6·25% (w/v) ammonium molybdate and 0·42% (w/v) ammonium vanadate for 10 min (Bae et al. 1999).

Secondary screening for cellulase, hemicellulase and phytase producing Bacillus spp.

The isolates that showed the satisfactory clear zone diameter were selected for secondary screening. CSBM solid medium was prepared by mixing of 10 g of CSBM and 20 ml of distill water and subsequently sterilized at 121°C for 15 min. After cooling, the 12 h-old starter prepared in nutrient broth was transferred to a sterile CSBM solid medium. The experiment was conducted in triplication. After 3 days fermentation at 37°C, enzymes were extracted using 80 ml of 50 mmol l−1 phosphate buffer pH 7·0. The mixture was stirred well, static stood at 4°C for 1 h and clarified by centrifugation. The β-mannanase, xylanase and cellulase activity was determined using DNS method according to Khanongnuch et al. (1998) and Shah et al. (1999). One unit (U) of enzyme was defined as the amount of enzyme that releases 1 μmol of reducing sugar in 1 min. Phytase activity was determined based on heteropoly blue method described by Kim et al. (1998). One unit (U) of phytase activity was defined as the amount of enzyme that releases 1 μmol of phosphate in 1 min. The strains with the highest enzymes activity were selected for FCSBM production. Enzymes production and growth profile of the selected Bacillus spp. were studied in solid state fermentation using CSBM medium.

FCSBMs preparation

FCSBM was produced using the selected Bacillus spp. strains. Ten gram of CSBM was mixed with 20 ml distill water and sterile at 121°C for 15 min. Inoculum (108 CFU) was transferred to sterilized CSBM and incubated at 37°C for 3 days. In case of mixed starter fermentation, total 108 CFU of inoculum mixed together with the equal ratio was used and fermented under the same conditions. The FCSBMs were freeze dried and ground.

Quality test of FCSBMs

Soluble sugar, soluble phosphate and soluble protein of FCSBMs were analysed. One gram of FCSBM powder was mixed with 9 ml distill water or 0·2 mol l−1 KOH (in case of soluble protein analysis) and mixed well. Supernatant was recovered by centrifugation at 6092 g for 15 min. Soluble reducing sugar, total sugar, available phosphate and protein were analysed according to DNS, phenol-sulfuric acid, heteropoly blue and Lowry's method, respectively (Khanongnuch et al. 1998; Kim et al. 1998).

In vitro digestibility of FCSBMs was studied as follow. One gram of FCSBM powder was mixed with 25 ml of 0·1 mol l−1 sodium phosphate buffer pH 6·0, 10 ml of 0·2 mol l−1 HCl, 1 ml of 1% (w/v) pepsin (Sigma®) and 0·5 ml of 0·5% (w/v) choramphenicol. The mixture was incubated at 39·5°C for 6 h. After that, 10 ml of 0·2 mol l−1 sodium phosphate buffer pH 6·8, 5 ml of 0·6 mol l−1 NaOH and 1 ml of 0·5% (w/v) pancreatin (Sigma®) were added to the mixture and further incubation for 18 h was carried out. Finally, 5 ml of 20% (w/v) sulphosalicylic acid was added and the mixture was allowed to stand for 30 min. The precipitate was recovered by filtration and was subsequently dried. The percent dry matter digestibility could be calculated from the dry matter content remained.

Percent in vitro dry matter absorbability of samples was analysed referring to the method described by Kiers et al. (2000a) based on dialysis method. The retentate was quantitatively collected and its dry matter content was determined. The percent of sample that was able to pass the dialysis membrane (Cellu Sep, cut off 12–14 kDa) was defined as absorbability. Moreover, the ratio of % DM absorbability and digestibility was calculated.

Finally, in vitro cytotoxicity of FCSBMs was studied. Crude extract of CSBM and FCSBMs was prepared by mixing of 10 g of sample powder with 90 ml distil water and stirring under 4°C for 1 h. Crude extract was harvested by centrifugation at 6092 g 15 min and freeze drying. Various concentrations of crude extract were prepared in distill water and sterilized by using sterile cellulose acetate membrane (0·45 μm, Sartorius®). The cytotoxicity effects of crude extract against cancer and normal cell lines were investigated using Sulphorhodamine B (SRB) assay that was originally described by Skehan et al. (1990). Cell survival was calculated as percentage of absorbance compared to control (sterilized water).

Statistical analysis

Experiments were performed in triplicate and data were analysed using statistix software (Analytical Software, Tallahassee, FL, USA).


Isolation and biochemical identification of Bacillus spp. from Tua-nao

After heat treatment of Tua-nao samples, total 34 isolated spore forming bacteria were collected. Biochemical identification revealed that 16 isolates (47%) were identified as Bacillus subtilis, while the others were aerobic and spore forming bacteria that were close to B. megaterium, B. macerans, B. circulans, B. firmus and B. pumilus. Moreover, we found the other five isolates that were heat tolerant but nonspore forming bacteria. The isolates that were identified as B. subtilis were isolate TM4, TM6, TM7, TM10, TM11, TK5, TK8, MR1, MR2, MR3, MR9, MR10, MR12, MT2, MT4 and MT5. They hydrolysed starch, casein and showed positive result on catalase test and VP test. Among these B. subtilis, we found some differences in their character and morphology. For example, the colony surface of isolate TK8 was dry and flat while one of isolate MR10 was thick and slimy. Data from our unpublished work revealed that MR10 produced slimy and sticky substance that called poly-gamma-glutamic acid (γ-PGA) and could produce γ-PGA at 77 mg g−1 SBM after growing on solid medium composing of soybean meal as a sole substrate and water in a ratio of 1 g per 3 ml.

Screening of cellulase, hemicellulase and phytase producing Bacillus spp. for FCSBM production

To reduce the number of further tested isolates, gel diffusion method was used to primarily screen for cellulase, hemicellulases and phytase producing isolates. It was found that each isolate showed different activity level. The wider clear zone might be assumed as higher activity production (Downie et al. 1994). Therefore, there were 14 isolates of Bacillus spp. that showed satisfactory clear zone diameter, i.e. T4/4, T4/10, TK2, TK5, TK8, TM4, TM5, TM6, TM8, TM11, MR1, MR6, MR10 and BS. These isolates were subsequently cultured on solid state fermentation using CSBM as a sole substrate. The results were shown in Table 1. Isolate MR10 showed the highest β-mannanase, xylanase and phytase activity at 280, 41 and 16 U g−1 CSBM; respectively, while TK8 exhibited the highest cellulase activity at 25 U g−1 CSBM. Therefore, isolate MR10 and TK8 were then selected for the FCSBM production in this study. Moreover, production of FCSBM by the mixed starter of isolate MR10 and TK8 was studied parallel with single starter fermentation. Both isolates were subsequently confirmed for their species using the molecular technique based on partial sequencing of 16S rRNA gene. The results confirmed that isolate MR10 was close to B. subtilis strain AX20 with 99% identity, and isolate TK8 was close to B. subtilis strain CICC10079 with 98% identity.

Table 1. Size of the clear zone formed by the action of enzymes and enzyme activity produced by some Bacillus spp. isolates
CZa (cm)Activity (U g−1)CZa (cm)Activity (U g−1)CZa (cm)Activity (U g−1)CZa (cm)Activity (U g−1)
  1. Values of enzyme activity were shown in mean with standard deviation of triplication experiment.

  2. a

    ‘CZ’ represents clear zone diameter.

  3. b

    Reported CZ diameters caused by phytase activity were average diameter because some CZ(s) were irregular shape.

T4/43·090 ± 51·815 ± 3·32·010 ± 2·50·611 ± 3
T4/103·595 ± 61·99 ± 0·42·29 ± 41·013 ± 4
TK22·454 ± 41·225 ± 3·01·57 ± 10·55 ± 3
TK53·5155 ± 342·06 ± 1·01·83 ± 0·51·417 ± 5
TK83·041 ± 32·015 ± 1·22·225 ± 3·50·83 ± 0·2
TM4086 ± 132·018 ± 5·11·49 ± 30·56 ± 1
TM52·614 ± 30·81 ± 0·52·44 ± 1·61·85 ± 2
TM63·5165 ± 172·011 ± 4·01·613 ± 3·20·816 ± 4
TM83·540 ± 181·840 ± 182·15 ± 0·20·50
TM112·8191 ± 11·813 ± 32·015 ± 31·61 ± 0·2
MR13·3240 ± 182·06 ± 31·515 ± 2·50·74 ± 0·6
MR63·5211 ± 121·77 ± 11·93 ± 11·02 ± 1
MR104·0280 ± 162·241 ± 51·93 ± 0·21·816 ± 1·3
BS3·3133 ± 172·014 ± 31·76 ± 1·61·715 ± 2·9

The enzyme production profiles of B. subtilis MR10 and TK8 when cultured in CSBM solid medium were determined and the results were shown in Fig. 1. It was investigated that the β-mannanase activity of both strains was detected after incubation for 1 day. The highest β-mannanase activity of MR10 at 220 U g−1 CSBM was found at day 1, while the one of TK8 reached the highest activity at 55·8 U g−1 CSBM after 2 days of fermentation. After reaching the highest point, β-mannanase of MR10 rapidly decreased while the one of TK8 only slightly decreased. The profile of xylanase production was consistent to β-mannanase. On the other hand, the cellulase activity of MR10 was lower than that of TK8. Both MR10 and TK8 cellulase were detected since the first day. Isolate MR10 produced phytase along with fermentation time until it reached the highest activity at day 4, while TK8 produced very low phytase activity that was consistent to the results in Table 1. Growths of MR10 and TK8 in CSBM solid medium were investigated and the results were shown in Fig. 2. The highest growths were found in Day 1 that associated to β-mannanase production and after that growth declined slightly.

Figure 1.

The activity of β-mannanase (a), cellulase (b), xylanase (c) and phytase (d) produced by MR10 (■) and TK8 (□) culturing in corticate soybean meal (CSBM) medium at various fermentation times.

Figure 2.

The growth of Bacillus subtilis MR10 and TK8 when cultured in corticate soybean meal (CSBM) solid medium. □, TK8; ■, MR10.

Quality tests of FCSBMs

We found that the fermentation of CSBM by B. subtilis MR10, TK8 and mixed starter could increase the content of soluble reducing sugar, protein and phosphate significantly (< 0·05) as shown in Table 2. These contents were approximately 8, 4 and 2–3 folds higher than those of CSBM, respectively. We also found that the size of soluble sugar as presented in term of ‘degree of polymerization’ or DP decreased approximately 10 folds.

Table 2. The soluble sugar, protein and phosphate content of fermented corticate soybean meals (FCSBMs)
SamplesSoluble sugar (mg g−1 sample)Soluble protein(mg g−1 sample)Soluble PO4(mg g−1 sample)
Reducing sugarTotal sugarDP a
  1. Values were shown in mean with standard deviation of triplication experiment. Values with different letters in each column are significantly different at < 0·05 by LSD test.

  2. a

    DP is degree of polymerization that is calculated by ‘DP = amount of total sugar/amount of reducing sugar’.

  3. b

    FCSBM (MR10) is FCSBM that produced by isolate MR10.

  4. c

    FCSBM (TK8) is FCSBM that produced by isolate TK8.

  5. d

    FCSBM (mixed Bacillus) is FCSBM that produced by mixed starter of isolate MR10 and TK8.

CSBM10·9 ± 1·0b188·0 ± 15·2a17·256·7 ± 6·4b2·2 ± 0·5d
FCSBM(MR10)b85·6 ± 7·6a149·2 ± 9·0b1·74196·1 ± 13·0a7·0 ± 1·1a
FCSBM (TK8)c74·4 ± 8·0a145·4 ± 11·0b1·95190·7 ± 10·5a3·7 ± 0·6c
FCSBM (mixed Bacillus)d81·4 ± 4·6a117·4 ± 5·5b1·44192·2 ± 9·0a5·0 ± 0·7b

The % DM digestibility of CSBM slightly increased but not significantly at < 0·05 from 68·6% up to 69·5–70·1% after fermentation (Table 3). However, we found that the % DM absorbability of FCSBMs was obviously higher significantly (< 0·05) than that of CSBM as also shown in Table 3. The ratio between % absorbability and digestibility was also increased from 0·27 to 0·348–0·364 after fermentation. This ratio was used to estimate a part of digestible matter that was absorbable.

Table 3. The in vitro digestibility and absorbability of corticate soybean meal (CSBM) and fermented corticate soybean meals (FCSBMs)
Samples% DM digestibility% DM absorbabilityAbsorbability/digestibility
  1. Values were shown in mean with standard deviation of triplication experiment. Values with different superscript letters in each column are significantly different at < 0·05 by LSD test.

CSBM68·6 ± 6·6a18·6 ± 0·9b0·270
FCSBM (MR10)70·1 ± 2·0a25·2 ± 1·0a0·359
FCSBM (TK8)69·5 ± 3·5a24·1 ± 1·1a0·348
FCSBM (mixed Bacillus)69·8 ± 3·0a25·4 ± 1·5a0·364

According to the in vitro cytotoxicity test of crude extract of CSBM and FCSBMs to animal cell lines, the results were shown in Table 4. The toxicity of crude extract of CSBM and FCSBMs to 3T3 (mouse fibroblast) was not found, although cells were tested at the highest concentration of 200 μg ml−1. On the other hand, growth of CorL23, the lung cancer cells, was suppressed after exposed to the extract for 48 h.

Table 4. The % survival of CorL23 and 3T3 after exposed to corticate soybean meal (CSBM) and fermented corticate soybean meals (FCSBMs) extract
Crude extracts% Survival of cell line at various extract concentration
3T3 (μg ml−1)CorL23 (μg ml−1)
  1. ‘day1 and day2’ represents ‘incubation cell and extract for 1 day and 2 days’ respectively.

Mixed Bacillus


In this study, Bacillus spp. from Tua-nao was proposed to be used as FCSBM producer. These bacteria are predominant microbes found in FSB foods, and they are also able to enhance the nutritive values of soybean as reviewed previously. Almost of them have been guaranteed as generally recognized as safe; especially B. subtilis (Wongputtisin et al. 2007; Hong et al. 2008). We found B. subtilis population with a high proportion in Tua-nao (43%) comparing to all heat tolerant-spore forming bacteria. However, this proportion was lower than that found in Kinema. Sarkar et al. (2002) reported that Bacillus spp. diversity in soybean Kinema was diverse. They found B. subtilis (88%), B. licheniformis (3·6%), B. cereus (2·4%), B. circulans (2·4%), B. thuringiensis (2·4%) and B. sphaericus (1·2%).

Ability of NSPs degrading enzymes and also phytase production of selected Bacillus spp. was mentioned, because these enzymes are not normally produced in monogastric animals, the target of FCSBM use (Nortey et al. 2007; Selle and Ravindran 2008). Therefore the FCSBM producing bacteria should be able to produce β-mannanase, xylanase, cellulase and phytase to hydrolyse β-mannan, xylan, cellulose and phytic acid containing in CSBM. However, isolated Bacillus with the highest activity of every considered enzyme above was not found. Isolate MR10 was an interesting strain with the highest β-mannanase, xylanase and phytase production but very low at cellulase activity that is necessary for hydrolysing of cellulose included in hull portion. The mixed starter fermentation using MR10 and TK8 that produced the highest cellulase was then an interesting process.

High soluble sugar, protein and phosphate of FCSBMs might be attributed to bacterial cellulase, hemicellulases, proteases and phytase activity. Smaller DP value of soluble sugar than that of CSBM is a good point of FCSBMs indicating that able to be easier utilized than bigger molecules. Furthermore, we expected that β-mannan, an important anti-nutritional factor containing in CSBM, was hydrolysed by the action of β-mannanase. Galactomannan is the main β-mannan found in soybean grain (Mullin and Xu 2001). This polysaccharide is virtually undigestible in the digestive tract of animals due to the absence of appropriate enzymes (Ward and Fodge 1996); furthermore, water dissolved-galactomannan has high ability of water holding and results the viscous mixture. That makes galactomannan have a severe anti-nutritive effect depressing performance, nutrient digestibility and also interferes normal nutrient digestion and absorption process; especially on monogastric animals (Jackson et al. 1999). Therefore, β-mannanase treatment of feed diets can reduce those anti-nutritive effects in monogastric animals (Jackson et al. 1999; Isshiki et al. 2000). Cellulose also cannot be digested and utilized in digestive tract of monogastric animals; therefore hydrolysed cellulose in feed diets may increase nutrient availability and metabolic energy for animals. The significant increasing of soluble protein was due to the activity of Bacillus proteases that hydrolysed soy protein molecules to be smaller that be easily utilized than larger one and could enhance bioavailability for animal feed. Therefore, the solubility of protein can be considered as a good quality index for feed (Parsons et al. 1991).

Available phosphate of FCSBMs that was determined by heteropoly blue method was also increased significantly comparing to that of CSBM. It could be explained that increasing of phosphate might be mainly due to phytase activity from fermentative Bacillus strains used, as the major part of phosphorus in cereals and oilseeds is in the form of phytic acid (Blaabjerg et al. 2007). However, among two isolates of B. subtilis MR10 and TK8, different phytase activity was found leading to different available phosphate content in FCSBMs. FCSBM produced by MR10 contains available phosphate higher than one produced by TK8; it might because MR10 produces phytase activity higher than strain TK8 or strain TK8 utilized phosphate for its metabolisms greater than MR10. Supplementation of exogenous phytase to animal feeds has been conducted generally in livestock production to reduce the effect of phytic acid, also one of anti-nutritional factor in CSBM, leading to enhance animal performances and also reduce environmental problems (Wodzinski and Ullah 1996; Hirabayashi et al. 1997, 1998; Kim et al. 1998; Guggenbuhl et al. 2007). In this study, Bacillus phytase could eliminate phytate molecule instead of exogenous phytase supplementation.

The high content of fibre results in lower digestibility of sample. Digestibility of FCSBMs was only slightly higher than CSBM, similar to the results obtained during Tempe fermentation of soybean seed (Kiers et al. 2000a). It might be explained that even fibre degrading enzymes of Bacillus spp. degraded CSBM fibres, but polymers composing in cell wall of Bacillus spp. might affect the digestibility of FCSBMs. Kiers et al. (2000a) recommended that to obtain maximum levels of total digestibility, soybean samples should be defatted prior analysis. Raw material used in our study was CSBM; a by-product obtained after soybean oil extraction process, thus digestibility values of samples might not be affected by oil remaining in CSBM. Moreover, the study of fermented soybean seed by Kiers et al. (2000b) found that Bacillus fermentation showed the better digestibility and absorbability value than that fermented by mold. Although this was only an in vitro investigation, it could be assumed that predigestion of CSBM by Bacillus fermentation leads to obtain a feed additive product with high nutrient bioavailability for monogastric animals.

As the inhibitory effect of FCSBMs on cancer cell line was found, it might be due to isoflavones and their glycosides containing in samples. The later substances could be generated by glycosidase activity of fermentative Bacillus spp. (Wongputtisin et al. 2007). There are many reports that reported on the effect of isoflavones and their glycosides on cancer cells suppression (Fukutake et al. 1996; Barnes 1997; Ren et al. 2001; Yamamoto et al. 2003; Jung et al. 2006). However, no inhibitory effect of FCSBMs on normal cell line was found. That might guarantee the safety of selected B. subtilis before using it as FCSBM producer and applying to the animals.

It could be concluded that B. subtilis MR10 and TK8 were suitable fermentative bacteria for FCSBM production according to their ability of cellulase, hemicellulases and phytase production. The FCSBMs produced by both isolates showed many satisfactory properties, i.e. higher in soluble sugar, protein and phosphate, smaller sugar molecules and better digestibility and absorbability than those of CSBM. These bacterial enzymes predigested soybean polysaccharides, protein and the other nutrients; moreover these enzymes were expected to digest biopolymers remaining in feed. It is very useful for monogastric animals because they lack of endogenous cellulase, hemicellulases and phytase. As each selected isolates could not produce all considered enzymes, therefore mixed starter fermentation seemed to be an interesting process. Finally, cytotoxicity test revealed that FCSBMs produced by B. subtilis isolate MR10 and TK8 were safe for animal use.


This work was granted by The Office of Higher Education Commission, Thailand.