Optimization of Mixed Solid-state Fermentation of Soybean Meal by Lactobacillus Species and Clostridium butyricum

The dietary protein source of animal feeds directly affects nutrient utilization and subsequent growth performance in weaning animals, particularly in piglets. Soybean meal is the main source of vegetable protein in animal feed due to its low cost and high nutritional value (Cromwell 2012). However, soybean meal contains several anti-nutritional factors, such as trypsin inhibitor, lectin, α-amylase inhibiting factor and soybean antigens (Grant 1989). These anti-nutritional factors attenuate the nutritional value, utilization and digestibility of soybean protein, leading to digestive and metabolic diseases in animals (Li et al. 1990; Herkelman et al. 1992; Zhao et al. 2008).

Probiotics are live microorganisms, which can confer a health benefit for the host when administered in appropriate and regular quantities (Chaucheyras-Durand and Durand 2010). Previous studies have demonstrated that the soybean meal fermented by probiotics can increase nutrient digestibility, reduce diarrhea and improve growth performance in piglets (Kiers et al. 2003; Hong et al. 2004; Mukherjee et al. 2016). Microbial fermentation of soybean meal efficiently eliminates anti-nutritional factors and enhances nutrient value by producing proteolytic enzymes (Hong et al. 2004). Currently, Aspergillus species are widely used to produce fermented soybean meal due to their capacity to produce multiple hydrolysis enzymes (Pinto et al. 2001; Mathivanan et al. 2006; Feng et al. 2007). In addition to fungi-based fermentation, Lactobacillus species, particularly Lactobacillus plantarum, is another facultative anaerobic bacterial strain, which has also been used to ferment soybean meal (Amadou et al. 2010a, 2010b). L. plantarum produces lactic acid and removes trypsin inhibitor contents during fermentation, thereby increasing protein hydrolysis and liberating free amino acids (Amadou et al. 2010a, 2010b).

Other lactic acid-producing Lactobacillus species, including Lactobacillus acidophilus, Lactobacillus delbrueckii and Lactobacillus salivarius also showed beneficial effects on animals (Dumbrepatil et al. 2008; Deng et al. 2012; De Cesare et al. 2017). In addition, Clostridium butyricum is an anaerobic endospore-forming probiotic and has the potential to improve growth performance and immune function in animals (Yang et al. 2012; Zhang et al. 2014). Since different bacteria strains have different enzyme activities, their capacity to degrade anti-nutritional factors and synthesize different metabolites may vary. It has also been reported that the exogenous enzyme supplementation enhances the degradation of soybean protein and inactive anti-nutritional factors in the SSF of soybean meal (Ma and Wang, 2010; Amadou et al. 2011). However, to our knowledge, there have been no reports about the effects of multistrains, such as L. acidophilus, L. delbrueckii, L. salivarius and C. butyricum on SSF of soybean meal. Further, the available data concerning the interaction between exogenous proteases and probiotics in SSF of soybean meal is still scarce.

Thus, the purpose of this study was to investigate the optimal parameters of mixed SSF of soybean meal by Lactobacillus species and C. butyricum and evaluate the nutritional value in fermented soybean meal. The results provide valuable information about the fermentation of soybean meal by multiple strains and are suitable for use on an industrial scale to produce large amounts of functional fermented products.

Optimization of mixed SSF on soybean meal-based medium. Since initial moisture content of fermentation is critical for microorganism growth in SSF, we first investigated the relation between initial moisture content and fermentation duration on soybean fermentation by Lactobacillus species (L. acidophilus, L. delbrueckii and L. salivarius) and C. butyricum. The results showed that 50% initial moisture had the highest bacterial growth compared with other initial moisture contents (Fig. 1A). Similarly, the effect of different initial moisture content on bacterial growth was observed for different fermentation durations (Fig. 1A). The highest water content of fermented soybean meal was also found at the initial moisture of 50% in SSF (Fig. 1B). The lactic acid levels of fermented soybean meal increased linearly with the increased initial percentage of moisture (Fig. 1C). Consistently, the pH of the fermented soybean meal was negatively correlated with the initial percentage of moisture (Fig. 1D). Furthermore, the reducing sugar content in fermented soybean meal was significantly reduced at the initial moisture of 50% in SSF (Fig. 1E). Similar results were also found for different fermentation durations (Fig. 1E).

Fig. 1.

We then examined whether the different initial percentage of moisture and fermentation duration have an impact on the degradation of soybean oligosaccharides in SSF. The results showed that soybean oligosaccharides, including raffinose and stachyose were more efficiently degraded at the initial moisture of 50% compared with other initial moisture contents (Fig. 2A). In addition, two days of SSF was sufficient to degrade soybean oligosaccharides compared with four and six days of SSF (Fig. 2A). Furthermore, we investigated whether the different initial percentage of moisture and fermentation duration could affect the degradation of soybean crude protein in SSF. Consistently, two days of fermentation duration in combination with 50% of initial moisture content in SSF was sufficient to degrade soybean crude proteins compared with other conditions (Fig. 2B, indicated by an arrow). After six days of fermentation, degradation of soybean proteins was achieved at almost all initial moisture conditions (Fig. 2B, indicated by an arrow). Taken together, these results demonstrate that the ideal initial percentage of moisture and fermentation duration in mixed SSF of soybean meal by Lactobacillus species and C. butyricum are initial moisture of 50% and two days of fermentation. Therefore, 50% moisture level was selected as the optimum parameter for the proliferation of multiple strains of probiotics and used for the next optimization process.

Fig. 2.

Effect of mixed probiotics in combination with protease supplementation on SSF of soybean meal. We further tested whether proteases and probiotics could further improve the nutritional value of soybean meal at the initial moisture of 50% and two days of SSF. The results showed that bacterial growth was further increased in the presence of both proteases and probiotics in SSF of soybean meal from 12 hours to 48 hours of fermentation compared with probiotics supplementation alone (Fig. 3A). The spore production was further elevated in the presence of both proteases and probiotics from 36 to 48 hours of fermentation compared with probiotics alone (Fig. 3B). The additive effect of proteases and probiotics on the water content of fermented soybean was only observed at 48 hours of SSF (Fig. 3C). Probiotics and protease supplementation further increased the lactic acid levels of fermented soybean meal compared with probiotics supplementation alone (Fig. 3D). The pH of fermented soybean meal was significantly elevated by protease supplementation compared with probiotics supplementation alone, while the pH of fermented soybean meal was further decreased in the presence of both probiotics and protease supplementation (Fig. 3E). Similar to previous results, probiotics supplementation significantly reduced the reducing sugar content in fermented soybean in SSF (Fig. 3F). However, the reducing sugar content was further remarkably decreased in the presence of both probiotics and protease supplementation (Fig. 3F).

Fig. 3.

We then examined whether proteases and probiotics could affect the degradation of soybean oligosaccharides in SSF. As expected, probiotics supplementation significantly promoted oligosaccharide degradation in fermented soybean meal from 12 to 48 hours of SSF (Fig. 4A). However, protease supplementation did not affect the degradation of soybean oligosaccharide in fermented soybean meal in SSF (Fig. 4A). In addition, supplementation of both proteases and probiotics in SSF did not further increase the degradation of soybean oligosaccharide in fermented soybean meal in SSF (Fig. 4A). Furthermore, we investigated whether supplementation of both proteases and probiotics in SSF could affect the degradation of soybean crude protein in SSF. Similar to previous findings, probiotics supplementation gradually degraded soybean proteins during the fermentation (Fig. 4B, indicated by an arrow), whereas protease supplementation alone did not have a significant effect on the degradation of soybean crude proteins. Furthermore, the degradation of soybean protein was rapidly achieved in the presence of both proteases and probiotics at the early stage of SSF (Fig. 4B, indicated by an arrow). In addition, we also demonstrated that fermented soybean meal harvested from two days of SSF with supplementation of proteases and probiotics showed antimicrobial activity against S. aureus (Fig. 5A) and E. coli (Fig. 5B) compared with ampicillin. These results suggest that the supplementation of both proteases and probiotics in SSF is able to improve the nutritional value of fermented soybean meal.

Fig. 4.

Fig. 5.

In this study, we demonstrated that the optimal initial percentage of moisture and fermentation duration for L. acidophilus, L. delbrueckii, L. salivarius, and C. butyricum was 50% and two days in SSF of soybean meal. Supplementation of exogenous protease in combination with probiotics further improved the SSF of soybean meal. Fermented soybean meal was able to inhibit the growth of S. aureus and E. coli in vitro.

The initial moisture content of dry substrates is a critical factor in the proliferation of bacteria (Zhao et al. 2008). It has been reported that initial moisture content of 50% in SFF increases the growth of L. plantarum (Patel et al. 2004). The maximum viable counts of L. reuteri was observed at an initial moisture content of 50% in SSF (Zhang et al. 2014). Similarly, we herein found that the maximum viable counts of probiotics were achieved at the initial moisture content of 50% in SSF of soybean meal by Lactobacillus species and C. butyricum. Moreover, prolonged fermentation duration did not significantly improve bacterial growth in SSF of soybean meal. This result was in accord with the previous studies (Patel et al. 2004; Ying et al. 2009; Zhang et al. 2014). The correlation between initial moisture content, water content and viable counts of bacteria in fermented soybean meal in SSF are relatively less studied. Here, we found that initial moisture content was strictly associated with water content in fermented soybean meal. However, the water content in fermented soybean meal was not positively correlated with viable counts of probiotics, particularly after six days of fermentation. It seems that an optimal range of water content for bacteria growth may occur in SSF of soybean meal. It has been reported that the pH of the fermented product shows a gradual drop in the initial stage of fermentation and then remains steady (Ying et al. 2009; Zhang et al. 2014). In the present study, we used Lactobacillus species and C. butyricum to ferment the soybean meal and demonstrated that the lactic acid production was linearly increased at the same initial moisture content during the fermentation. The pH value of fermented soybean meal was also consistently decreased at the same initial moisture content after six days of fermentation. These results are similar to the previous findings that lactic acid content increased with increasing initial moisture content during SSF, while the pH value decreased (Dumbrepatil et al. 2008; Ying et al. 2009). Since C. butyricum also could reduce pH value in the fermented products by producing butyric acid during fermentation (Zhang et al. 2009), the concentration of butyric acid during SSF of soybean meal in the present study remains to be further examined. It has been shown that residual reducing sugar content is negatively associated with viable counts of bacteria in SSF (Zhang et al. 2014). Our findings were generally in line with an earlier report that lower reducing sugar content indicates that the bacteria proliferated more rapidly (Zhang et al. 2014).

Several reports have demonstrated that microbes could produce a variety of enzymes during fermentation and these enzymes could degrade the soybean oligosaccharides and crude proteins (Hong et al. 2004; Adeyemo and Onilude 2014). We also found that soybean oligosaccharides and crude protein content decreased with increasing initial moisture content in SSF, implying that some hydrolytic enzymes were produced by Lactobacillus species or C. butyricum. It has been demonstrated that protease supplementation significantly improved the nutritional value of soybean meal in SSF by degradation of the large molecular weight of soybean protein (Wang et al. 2014). The proteases are classified into acidic, neutral and alkaline proteinase according to its optimum pH value. Since Lactobacillus species are lactic acid-producing probiotics, we then used acidic protease in the present study. Similar to a previous study (Wang et al. 2014), we found that the viable counts of bacteria were significantly increased in the presence of both protease and probiotics. It is possible that amino acids hydrolyzed from soybean proteins by exogenous protease were efficiently utilized by bacteria for growth. Our results including lactic acid production, pH value, residual reducing sugar content and degradation of soybean crude protein supported this hypothesis. Herein, we also found that the spore production of C. butyricum was further increased in the presence of both proteases and probiotics in SSF. These results may contribute to the Lactobacillus species becoming the dominant strains at the final stage of fermentation and then create a more acidic environment by lactic acid production. Thus, the increased spore production of C. butyricum was achieved to resist the harsh environment at the later stages of fermentation. In addition, the more acidic environment caused by Lactobacillus species during fermentation also further enhanced the enzyme activity of exogenous acid proteinase. Therefore, the degradation of soybean protein was greatly increased in the presence of both protease and probiotics in SSF compared with supplementation of protease or probiotics alone.

A previous study reported that the pH value was elevated linearly with increased levels of protease supplementation in SSF (Wang et al. 2014). In the present study, we also observed that protease supplementation alone in SSF also consistently increased the pH value.

It has been reported that increased antimicrobial activity of fermented product is mainly due to bacterial metabolites, including acidic substances and bacteriocins (Fuller 1989). Lactic acid from Lactobacillus species is able to inhibit the synthesis of bacterial proteins (Wang et al. 2015). C. butyricum also can reduce pH value to inhibit the growth of pathogens by producing butyric acid (Zhang et al. 2009). In the current study, the lactic acid production was linearly increased during fermentation and the fermented soybean meal also possessed antimicrobial activity. Thus, it is possible that the antimicrobial activity in the fermented soybean meal might contribute to lactic acid or butyric acid, although the butyric acid production was not measured in our study.

The multistrain probiotics for fermentation have been reported to be more effective than monostrain probiotics, but related research or products are limited (Timmerman et al. 2004; Choi et al. 2011). The previous research reported that the viable counts of Lactobacillus species were significantly increased when Bacillus subtilis was added to the solid substrate (Zhang et al. 2014). B. subtilis followed by Enterococcus faecium supplementation effectively improved the quality of corn-soybean meal mixed feed (Shi et al. 2017). Further, fermented soybean meal using multistrain probiotics in combination with protease supplementation improved the nutritional value of soybean meal (Wang et al. 2014). Here, we also demonstrated that SSF by multistrain probiotics also has beneficial effects. Whether the anti-nutritional factors, such as trypsin inhibitor are reduced in SSF and the effects of fermented soybean meal on animals remain to be further investigated.

In conclusion, our results showed that the optimal initial moisture content and fermentation duration for mixed SSF of soybean meal is 50% of initial moisture and two days fermentation. The exogenous protease supplementation in mixed SSF of soybean meal was able to improve the nutritional value of fermented soybean meal.