Degradation of glucosinolates in rapeseed meal by Lactobacillus delbrueckii and Bacillus subtilis

Zhenyu Zhng*,Min Wen,Yqi Chng

a Animal Nutrition Institute,Sichuan Agricultural University,Chengdu 611130,China

b Institute of Animal Husbandry and Veterinary Medicine,Meishan Vocational Technical College,Meishan 620010,China

Keywords:

Response surface methodology

Glucosinolates

Solid-state fermentation

Lactobacillus delbrueckii

Bacillus subtilis

ABSTRACT

Lactobacillus delbrueckii and Bacillus subtilis were employed as a new combination of strains to treat rapeseed meal by solid-state fermentation,aiming to efficiently degrade the glucosinolates,which are the main toxin in the meal.Single-factor tests and Response surface methodology(RSM)were used to optimize the fermentation parameters.Under the optimum fermentation parameters of 15%total injection volume of the mixture of Lactobacillus delbrueckii and Bacillus subtilis with a ratio of 2:1,bran content of 16%,feed to water ratio of 1:1.5,fermentation temperature of 36°C and fermentation time of 72 h,the content of glucosinolates in rapeseed meal was decreased from 64.558 μmol/g to 3.473 μmol/g,reaching a high degradation rate(94.62%).The high detoxification rate by a consortium of Lactobacillus delbrueckii and Bacillus subtilis provides a bright application prospect in feed utilization of rapeseed meal.

1.Introduction

Rapeseed meal is an abundant by-product of the oil extraction industry. It has high content of protein and balanced amino acid composition [1-3], and is suitable for animal feed use.However,the meal contains glucosinolates[4,5]and other anti-nutrient factors,which can cause animal poisoning symptoms and retard the growth performance [6,7]. Therefore, the use of rapeseed meal in livestock and poultry diets is limited.A number of methods have been developed for rapeseed meal detoxification,e.g., physical [8-10], chemical [11,12] and biological[13,14]detoxification.But physicochemical detoxification has the disadvantages of high cost,severe pollution and significant loss of nutrients,whereas microbial fermentation is an ideal method for detoxification of rapeseed meal because the rapid growth of microorganisms can secrete complex enzymes to decompose the toxic components in the meal.Until now, rapeseed meal fermentation mostly selected single microbial strains,such as Aspergillus niger[15],Rhizopus oligosporus[16-18],etc.Although good fermentation results have been achieved by employing these microbial strains, there are still some points that need to improve:(1)Most of these microbial strains are not listed in the approved strains for feed usage,which indicates safety risks,and cannot be used for feed production;(2)The fermentation process is mostly carried out by aerobic microorganisms, which consume a large number of nutrients in the substrate to produce energy through aerobic breathing.In addition,the aerobic environment is easily to be contaminated by undesired bacteria in the process of operation,which might lead to the failure of fermentation;(3)Using a single microbial strain to ferment the substrate may have defects of longer fermentation time or low detoxification rate.Therefore,it would be better to choose a combination of facultative anaerobic microbes that meet the safety requirements of the feed industry to ferment rapeseed meal.

Both Lactobacillus delbrueckii and Bacillus subtilis belong to the approval list of microorganisms that are Generally Recognized as Safe (GRAS) and can be fed directly to animals[19,20].Lactobacillus delbrueckii can make full use of amino acids in the fermentation substrates to synthesize its own bacteria protein.It also has strong acid resistance and can grow well even when the pH value is as low as 3.0-4.5[21,22].There are barely reports about the application of Lactobacillus delbrueckii in rapeseed meal fermentation yet. As for Bacillus subtilis, it can secrete various enzymes,such as phytase,protease,amylase,cellulase and lipase,which can degrade the macromolecular substances,e.g. protein and cellulose, into small molecular peptides and disaccharides so as to improve the utilization rate of nutrients in fermented substrates [23]. A few studies have proven that Bacillus subtilis is capable of producing iturin A,poly-γ-glutamic acid[24]and functional peptides[25]by fermenting rapeseed meal,but its application on rapeseed meal detoxification is rarely reported.

With the degradation rate of glucosinolates as the dependent variable,this study adopted single-factor design and 33Box-Behnken design based on RSM to explore the optimal parameters of repeseed meal solid-state fermentation using a combination of Lactobacillus delbrueckii and Bacillus subtilis,thereby producing meal safe for animal feed.

2.Materials and methods

2.1.Materials

Lactobacillus delbrueckii(ATCC9649)and Bacillus subtilis(ATCC 19659)were the standard strains of ATCC collection.

Rapeseed meal and bran were purchased from the local market of Meishan,Sichuan province.The rapeseed meal contained 12.3% water,36.5% crude protein,and the glucosinolates content was 64.558 μmol/g.The bran contained 12.8%water and 13.8% crude protein.These raw substrates were crushed and screened through a 40-mesh sieve for later use.

The MRS medium and beef extract peptone medium were purchased from Beijing Solebao Technology Co.Ltd.,China.

2.2.Activation of the microbial strain

Lactobacillus delbrueckii and Bacillus subtilis colony in the preserved solid culture medium were added to the 50 mL triangular bottle with 15 mL of MRS liquid medium and beef extract peptone liquid medium,respectively.The Lactobacillus delbrueckii was incubated without agitation at 38°C for 48 h and the Bacillus subtilis was cultured in a vibrating incubator at 30°C under vibrating at 180 r/min for 24 h.

2.3.Preparation of the liquid fermention seed

Two percent of activated microbes from the Section 2.2 were inoculated into 50 mL of the liquid medium in a 150 mL triangular bottle (the components of the culture medium were the same as mentioned in Section 2.1),and cultured for 14 to 16 h until they reached the end of logarithmic growth stage.After counting the number of live bacteria on the solid plate, the concentration of bacterial solution was adjusted to 106/mL for subsequent injection.

2.4.Solid-state fermentation process

200 g of rapeseed meal was put into a 10×15 cm anaerobic fermentation bag,and different amounts of bran and water were added into the substrate according to the requirements of the experiment, which were described in Sections 2.5 and 2.6.The substrate was mixed and then sterilized at 115°C for 20 min.After sterilization and cooling,the microbes were inoculated into the substrates in different amounts.The fermentation tests were terminated at different time points and the samples were dried and crushed to determine the content of glucosinolates.

2.5. Determination of the microbial strain combination ratio and total inoculation amount

According to the methods of Section 2.4, Lactobacillus lactate and Bacillus subtilis were added into the fermentation medium in ratios of 1:0,1:1,1:2,2:1,1:3,3:1,0:1,respectively.The total inoculation amount of microbes was 10%,the feed to water ratio was 1:1,and the fermentation was conducted at 30°C for 72 h.The produced meal was dried at 60°C and crushed through a 40-mesh sieve to determine the content of glucosinolates.

After the optimum microbial strain combination ratio was settled,the appropriate total volume to inoculation volume was investigated at a gradient of 10%,15%,20%,25%and 30%(V/V),respectively,with a set feed water ratio of 1:1 and the fermentation temperature of 30°C for 72 h.When the fermentation process was completed, the meal was dried and crushed to determine the content of glucosinolates.

2.6.Single-factor tests to determine the suitable range of each factor

Single-factor tests for the bran addition amount,feed to water ratio, fermentatioin temperature and fermentation time were carried out to explore their appropriate range for the following RSM optimization process.The gradient level set for each factor was as follows:the bran addition amount(5%,10%,15%,20%,25%),feed to water ratio(1:0.8,1:1;1:1.2,1:1.4, 1:1.6), fermentation temperature (30, 32, 34,36,38°C),and fermentation time(24,48,72,96,120 h).

2.7.Optimization of the fermentation parameters by RSM

The optimum value of bran content,feed to water ratio and fermentation temperature was further explored by theRSM design to investigate the response pattern and determine the optimum synergy of variables.The optimization range was bran addition amount (A, 10%-20%), feed to water ratio(B,1:1.2-1:1.6),and fermentation temperature(C,34°C-38°C),respectively.As shown in Table 1,a total of 17 experiments were set up,with 3 replicates at the center point.

Table 1 Box-Behnken design matrix and response.

A quadratic polynomial equation was applied to fit the experimental data,as shown in Eq.(1).

where R is the dependent variable,a0is the fitting response value of the design center point,and a1,a2and a3are the linear coefficients;a12,a13and a23are the cross coefficients;a11, a22, and a33are the quadratic coefficients. Design-Expert software was used to analyze experimental design data and calculate the predicted responses.Then a verification experiment was carried out to confirm the validity of the fitting model.

2.8.Determination of glucosinolates and the formula to calculate the degradation rate

The content of glucosinolates was determined by spectrophotometry according to the method of[26].The degradation rate of glucosinolates was calculated as Eq.(2):

where Y stands for the degradation rate of glucosinolates,A and B represent the content of glucosinolates in unfermented and fermented rapeseed meal,respectively.

2.9.Data analysis

All experiments were carried out in triplicate.Graphpad 8.0 was used for the single-factor experiment data analysis and graph plotting. Design expert 7.0 was used for RSM Design and statistical analysis.P<0.05 indicates a significant difference,while P>0.05 indicates the difference was not significant.

3.Results

3.1.Determination of the microbial strain ratio and total inoculation amount

The content of glucosinolates in the meal fermented with Lactobacillus delbrueckii,Bacillus subtilis and their 1:1 combination was shown in Fig.1a.Inoculation of either Lactobacillus delbrueckii or Bacillus subtilis alone could degrade glucosinolates substantially, while their combination had the highest degradation efficiency,indicating a synergistic effect between them.As to their various combination ratios,the 2:1 ratio of Lactobacillus delbrueckii to Bacillus subtilis has the best degradation efficiency on glucosinolates (Fig. 1b).When the total inoculation quantity was 15%,the content of glucosinolates in the fermented meal was the lowest(Fig.1c).Therefore,the 2:1 of Lactobacillus delbrueckii to Bacillus subtilis and a total inoculation quantity of 15%were adopted to conduct the following single-factor and RSM tests.

3.2.Single-factor fermentation test

3.2.1. Effect of bran content on the degradation rate of glucosinolates

Fig.1.Determination of the microbial strain ratio and total inoculation amount.

Bran can not only provide a necessary carbon source for microbial growth but also change the dense structure of the fermentation matrix,making it loose and porous,allowing the enzymes to become fully mixed with the substrate and perform their function.Therefore,the bran content is one of the critical factors for solid-state fermentation.As shown in Fig.2a,when the bran content was 15%,the degradation rate of glucosinolates was the highest.However,with a further increase of bran content,the degradation rate of glucosinolates decreased, which may be accountable to the anaerobic nature of Lactobacillus delbrueckii.Thus,15%of bran was appropriate for the growth of both facultative anaerobic Lactobacillus delbrueckii and aerobic Bacillus subtilis to achieve the best degradation rate of glucosinolates.

3.2.2.Effect of feed to water ratio on the degradation rate of glucosinolates

The amount of water in the substrate is critical for the porosity of the substrate and the content of oxygen in it.Meanwhile,the water tension also affects the growth of microorganisms.As shown in Fig.2b,when the feed to water ratio was 1:1.4, the glucosinolates in the substrate meal had the highest level of degradation,reaching 87.33%.

3.2.3.Effect of fermentation temperature on the degradation rate of glucosinolates

Each kind of microbes and the biological enzyme has its own optimal temperature to perform the biological functions.In order to give full play to each microbe and achieve a synergistic effect between them,the proper temperature must be maintained in the fermentation process.As shown in Fig.2c,the degradation rate of glucosinolates increased to the highest value from 30 °C to 36 °C, then decreased when futher rising the temperature.The decrease may be accredited to the high fermentation temperature,which is not conducive to the retention of water in the culture medium and inhibits the growth of microbes.

Fig.2.Single-factor test of solid-state fermentation.

3.2.4. Effect of fermentation time on the degradation rate of glucosinolates

The degradation rate of glucosinolates showed a rapid enhancement trend from 24 h to 72 h,and the maximum degradation rate of 83.67%(at a content of 10.451 μmol/g) was reached after 72 h fermentation (Fig. 2d). After that, the degradation rate of glucosinolates increased slightly but not significantly with the increase of fermation time.Thus,a 72 h fermentation time would be most suitable for actual production to save time and reduce material loss.

3.3.Optimization by RSM

3.3.1.RSM design and analysis

The bran content,feed to water ratio and fermentation temperature were chosen according to the single -factor test results to carry out a 33RSM analysis.The test factor levels and results are shown in Table 1.

The experimental data were analyzed and a quadratic multinomial regression equation was obtained as in Eq.(3):

where R is the content of glucosinolates;A,B and C represent for the bran content,feed to water ratio,and fermentation temperature,respectively.

A variance analysis was conducted for the above regression model.F-value and P-value were used to test the significance of the influence of each variable on the response value. According to the ANOVA results in Table 2, the model's F-value was 63.51,and the P-value was<0.0001,while the P-value for the misfit term was 0.4704,indicating that the model was significant.The value of R2and Adj R2was 0.9879 and 0.9723,respectively,indicating the model was close fit to reality with high reliability. Thus, this model can be used to analyze and predict the glucosinolates content in the fermented meal to get an optimal result.

Table 2 also indicated that the three factors of bran content,feed to water ratio,and fermentation temperature all had a significant impact on the glucosinolates content(P < 0.05). Their influence on the content of glucosinolates in order of significance was as follows:fermentation temperature>bran content>feed to water ratio.The interaction between feed to water ratio and fermentation temperature also had a significant impact on the glucosinolates content(P<0.05),whereas the interaction between bran content and feed to water ratio and between bran content and fermentation temperature had no significantimpact on glucosinolates content(P>0.05).The quadratic terms of the model were all significant.

Table 2 Analysis of variance for the fitted model and lack of fit for glucosinolates content.

3.3.2.Response surface analysis

Response surface diagrams can reflect the importance of each factor and their interaction in a more direct way.The contour diagram of the effect of the interaction between bran content and feed to water ratio on glucosinolates content was shown in Fig.3.When the feed to water ratio was set,the glucosinolates content showed a trend of decreasing and then increasing with the rising bran content.Whereas when the bran content remained unchanged,the glucosinolates content showed a falling trend as the feed to water ratio increased.When the bran content was 16%and the feed to water ratio was about 1:1.45,the glucosinolates content reached the minimum.

Fig.4 showed the influence of the interaction between bran content and fermentation temperature on the glucosinates content.With the set bran content,the glucosinolates content first decreased and then increased with the rising fermentation temperature.The minimum glucosinolates content was reached at the bran content of 16.5%and the fermentation temperature of 35.5°C.

Fig.5 indicated a significant influence of the interaction between feed to water ratio and fermentation temperature on the glucosinates content. With the feed to water ratio set at a fixed value,the glucosinolates content first decreased and then increased with the rising fermentation temperature. The glucosinolates content reached the minimum when the feed to water ratio was 1:1.48 and the fermentation temperature was 35.5°C.

3.3.3.Verification of the model

The minimum level of glucosinolates was predicted to be 3.547 μmol/g at bran content of 16.05%,feed to water ratio of 1:1.47,and fermentation temperature of 35.56°C.

Fig.3.The response plot of bran content vs.feed to water ratio on the glucosinolates content while keeping fermentation temperature at a constant of 36°C.

To verify the validity of the model, the above optimized parameters were rounded up to the nearest whole number, i.e., bran content of 16%, feed to water ratio of 1:1.5, and fermentation temperature of 36 °C, and the rounded parameters were adopted to test the glucosinolates content in fermented rapeseed meal. The validation test was carried out 3 times. The mean value of glucosinolates content in fermented rapeseed meal was 3.473 μmol/g, the relative error being 2.09% compared to the theoretical prediction value. It can be seen that this model can predict the degradation of glucosinolates in the fermented meal well.

4.Conclusion

To degrade the glucosinolates in rapeseed meal,two facultative anaerobic microbes, i.e., Lactobacillus delbrueckii and Bacillus subtilis, which are generally regard as safe,were employed to ferment the meal.With a total injection volume of 15%(Lactobacillus delbrueckii:Bacillus subtilis=2:1),bran content of 16%,feed to water ratio of 1:1.5 and fermentation temperature of 36°C,Lactobacillus delbrueckii and Bacillus subtilis degraded the glucosinolates in rapeseed meal by 94.62%.The feeding effect of the detoxified rapeseed meal needs further animal experiments to confirm.

Fig.4.The response plot of bran content vs.fermentation temperature on the glucosinolates content while keeping feed to water ratio at a constant of 1:1.4.

Fig.5.The response plot of feed to water ratio vs.fermentation temperature on the glucosinolates content while keeping bran content at a constant of 15%.

Conflicts of interest

Authors declare that there are no confilcts of insterest.

Acknowledgments

This work was financially supported by the Education Department of Sichuan Province(18ZB0289).Authors greatly acknowledge Dr.Dan Alderson for providing language help.