Haicheng Yin*,Feng Jia,Jin Huang

College of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China

Keywords:

Solid-state fermentation

Bacillus subtilis

Protease

Debitterness Soybean meal

ABSTRACT

The debittering effect of extracellular enzymes from Bacillus subtilis ACCC 01746 was studied using soybean meal as a substrate for solid-state fermentation(SSF).Results showed that B.subtilis produces proteases and carboxypeptidase in the early stage of SSF(0-8 h).Proteases are dominant and can hydrolyze the soybean protein into long-chain peptides with mild bitterness.Carboxypeptidase production is dominant at 8-16 h SSF,at which point soybean protein is further hydrolyzed and bitterness is enhanced.The strain then produces additional carboxypeptidase after 16 h,and bitterness is reduced.We compared the amino acid composition of the hydrolysates from soybean protein isolates to that of the fermented liquid of SSF.In the hydrolysates from soybean protein isolates that exhibit strong bitterness,62.81%of amino acids are hydrophobic and occur in the form of peptides.In the fermented liquid from soybean meal,16.22% of amino acids are hydrophobic and are mainly present in the form of free amino acids.The bitterness of fermented soybean hydrolysate is reduced from 5 to 0 when fermented for 24 h,suggesting that B.subtilis can effectively reduce bitterness,possibly due to the carboxypeptidase.Enzyme analysis shows that B.subtilis excretes carboxypeptidase during growth.The amino acids phenylalanine,alanine,tyrosine,and leucine at the C-terminal of the soybean bitter peptides in hydrolysates are cleaved in the presence of carboxypeptidase,resulting in complete debitterness.

1.Introduction

Soybean meal is a byproduct of soybean oil production and has long been considered a cheap source of plant protein for food and animal feedstuff because of its proteins,rich in essential amino acids.However,its protein utilization has drawbacks,such as poor protein solubility,unpleasant palatability,unappealing dark color,low digestibility,and undesirable anti-nutritional factors,which limit its bioavailability [1].Among the anti-nutritional factors in soybean protein products,the objectionable flavors must be strongly considered because they are important in determining food quality.In addition to being the most efficient method of removing unpleasant flavors and anti-nutritional factors,soybean meal fermentation can also improve palatability,decrease adverse reactions[2]and promote physicochemical functions and nutritional qualities.

Solid-state fermentation(SSF)is a process in which a solid substrate is deposited in a fermenter,cultivated with single or multiple strains,and incubated in a bioreactor for a few days[3].SSF is used successfully in producing microbial products,such as extracellular enzymes,pharmaceutical products,fuel,industrial chemicals,and food [4].Nowadays,SSF has emerged as a new technology for producing new protein or polypeptide feeds[5].During SSF,microorganisms degrade the organic compounds in the substrate into small molecules.As a processing technology,SSF enhances various physiological functions of products and gives them a unique aroma and flavor.Soybean meal SSF with single and multiple microorganisms improves the bioactive properties attributed to the increments of phytochemicals,such as peptides,flavonoids,and other active ingredients[6].Among these ingredients,soybean peptides are the most prevalent;they preserve the nutritional value of the soybean protein and have biological functions.However,the bitter taste after the enzymatic hydrolysis of soybean protein in SSF has limited the application of soybean protein as a food or feedstuff ingredient for a long time[7].Despite controlled research on the bitter taste of soybean protein hydrolysate,the exact mechanism remains unclear;however,low molecular weight peptides composed mainly of hydrophobic amino acids,particularly proline(Pro),leucine(Leu),tyrosine(Tyr),and phenylalanine(Phe),are important factors for health[8,9].

Bacillus subtilis is one of the most commonly used probiotics in the fodder industry because of its safety,high efficacy,low price,and ability to secrete considerable amounts of enzymes,including proteases,lipase,amylase,and carboxypeptidase[10].In particular,the carboxypeptidase generated by B.subtilis during soybean meal SSF is specialized for the hydrophobic amino acids from soybean protein peptides [11].Numerous studies have evaluated the ability of carboxypeptidase to reduce the bitter taste of peptides,but only a few used the enzyme from B.subtilis to investigate debittering[12,13].Thus,the debittering ability of B.subtilis hydrolytic enzyme in soybean meal SSF must be investigated.This work aimed to evaluate the production of extracellular B.subtilis protease via soybean meal SSF and investigate its debittering effect.

2.Materials and methods

2.1.Experimental materials and culture preparations

B.subtilis(ACCC 01746)was used in our experiment as a probiotic producing protease.The strain was acquired from Dr.Guan(College of Bioengineering,Henan University of Technology),maintained in the cryogenic refrigerator at-80°C,and was cultured on peptone beef extract agar plates(peptone 0.5 g,yeast extract 0.25 g,sodium chloride 0.5 g,agar 1.0 g,and water 50 mL)at 30°C for 24 h(for activation and amplification)before use as the SSF strains of the experiment and then stored at 4°C.The strain was inoculated and cultured in a 500 mL conical flask containing approximately 78%distilled water,20%soybean meal,and 2%wheat bran and mixed with a mineral solution (Na2HPO42.0 g/kg,KH2PO41.0 g/kg,CaCl20.01 g/kg,MgSO40.1 g/kg,MnSO40.005 g/kg,CuSO40.002 g/kg,and FeSO40.001 g/kg).The strain was allowed to amplify and adapt to the substrate of the soybean meal via shake-flask liquid culture at 150 r/min and 30°C for 10 h,and the number of bacteria was determined(containing at least 1×108cell/mL).The solution was used as a stock solution for SSF.

Soybean meal and wheat bran were provided by Zhengzhou Jinbaihe Biology Engineering Co.,Ltd.(Zhengzhou,China)and submitted to analyse the lipids(Soxhlet extraction),proteins(Kjeldahl method,N×6.25),moisture(dried,105°C),and ash(incineration,550°C)following the procedures of the AOAC Official Methods of Analysis[14].The samples were grounded in a laboratory mill and sieved to 500-1000 mm particle-size fractions by using a FW100 grinder machine(Tianjin Taisite Instrument Co.,Ltd.,Tianjin,China).The nutritional components of the mixed samples were 51%protein,3%fat,23%carbohydrate,12%moisture,and 11%ash.These substrates were sterilized at 121°C and 103 kPa in an autoclave for 20 min,cooled to 35°C,and stored until further use.The soybean bitter peptides and protein isolated were provided by Kaifeng Bio-Tech Co.,Ltd.(Kaifeng,China).All reagents were of analytical grade and purchased from Nanjing Jiancheng Bioengineering Institute(Nanjing,China).

Each experiment was conducted in triplicate,and results were presented as the mean of three values.

2.2.SSF

Fermentation experiments were conducted in a 500 mL conical flask containing 100 g of solid substrate(with 90.0 g of soybean meal and 10 g of wheat bran)and 10%inoculum(V/W)following the methodology proposed by Rashad et al.[15].The moisture content of the substrate was adjusted to 50%with distilled water.SSF was performed at 30°C for 64 h in a thermostat incubator within a water reservoir to maintain the relative humidity and moisture level of the substrate.The samples were taken every 4 h or 8 h interval for enzyme analysis.

2.3.Comparison of amino acid composition between the enzyme hydrolysate of soybean protein isolated and the SSF soybean meal

In brief,1.0 g of soybean protein isolated and 1.0 g of soybean meal were respectively placed in two 500 mL hydrolysis bottles with 200 mL of(6 mmol/L)hydrochloric acid and hydrolyzed for 24 h at 110°C as accurately as possible.After hydrolysis,the total amino acids for the hydrolyzed substrate were measured using an automatic amino acid analyzer(Hitachi 835-50).The amino acid content after non-catalyzed hydrolysis of soybean protein was isolated and soybean meal was determined using an automatic amino acid analyzer.The amino acid content was calculated using the gap between total amino acids and free amino acids.The amino acid content in the peptides of the soybean protein isolated or soybean meal were obtained from the total amino acids and free amino acids of each sample,respectively.

In brief,10.0 g of soybean protein isolated was placed in a 500 mL hydrolysis bottle with 200 mL of distilled water and 0.8 g of alcalase(enzyme activity 4.29×104U/g)and then hydrolyzed.The mixture was stirred at 60°C and pH 8.0 for 2.5 h.pH value was adjusted using sodium hydroxide,and consumption was recorded until the end of hydrolysis.The enzyme was inactivated at 90°C,and the solution was used for amino acid analysis.Similar to soybean protein methods,amino acid analysis was conducted in the SSF soybean meal at 32 h.

2.4.Evaluation of the bitterness value of soybean meal and bitter peptides during fermentation

In brief,1 g of sample in SSF was dispensed at 4 h interval into a 0.25 L hydrolysis bottle with 100 mL of distilled water,and the bitterness value was tested.

The debittering experiments were conducted in 250 mL flasks containing 1.0 g of soybean bitter peptides,100 mL of distilled water,and 2.0 mL of inoculum(1 ×108CFU/mL B.subtilis).The samples were incubated at 120 r/min for 32 h at 30°C and withdrawn at a 4 h interval.The mixtures were filtered through four layers of gauze,and the obtained fermentation liquid was used to determine the bitterness value.

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2.5.Preparation of carboxypeptidase and debittering experiment

All purification procedures were conducted at 4°C.The culture supernatant of B.subtilis ACCC 01746(1 L)was concentrated to 49 mL via an ultrafiltration membrane 3000 CUT(Millipore Co.,USA).The ultrafiltrate was further concentrated with(NH4)2SO4precipitation,and 80%of the precipitate fraction was collected and dissolved in 0.02 mol/L Tris-HCl buffer(pH= 7.5).The solution was loaded onto the Sephadex G-100 column(Pharmacia Biotech Co.,USA)pre-equilibrated with 10 mmol/L Tris-HCl.The eluted protease fractions (i.e.,the first eluted protein peak) were pooled and concentrated via ultrafiltration.The concentrated fractions were freeze-dried and used as a purified enzyme with a total activity of 42,500 U/g.

In brief,1.0 g of soybean bitter peptides and 20 mg of carboxypeptidase were placed in a 250 mL hydrolysis bottle with 100 mL of distilled water and were hydrolyzed at 30°C.The samples were analyzed for amino acid and bitterness values at a 2 h interval.

2.6.Disruption of B.subtilis cell via ultrasound

B.subtilis cells were extracted from the fermented solution of seed cultures(500 mL)via centrifugation at 10,000 r/min for 30 min at 4°C.The clear supernatant was removed,and the bacterial cells were collected and disrupted using an ultrasound disruptor UP100H(Dr.Hielscher GmbH,Stuttgart,Germany) with a frequency of 30 kHz,100 W power output,and a 1 mm diameter microtip.The supernatant and disrupted cells were utilized for enzyme assays.

2.7.Evaluation of bitterness value

The taste evaluation panel included 10 tasters,5 males and 5 females.The rational judgment of the panel on the bitterness value of polypeptide liquid when tasting and analyzing a drop of each sample was determined in line with the standardized solution.Each test was repeated five times,and the results were presented as the mean of the five textures and flavors.The bitterness value of polypeptide liquid was determined using the method of Ishibashi et al.[16].The grading standard is shown in Table 1.

Table 1 Criterion for bitterness values.

2.8.Proteinase,carboxypeptidase and aminopeptidase assays

Briefly,5 g of SSF sample was mixed with 50 mL of phosphate buffer(pH=7.0),and the mixture was then filtered using four layers of gauze.The filtrates were centrifuged at 8000 r/min,and the supernatant was used as crude enzyme extract.The protease activity in crude enzyme extract was analyzed using Folin method[17].Protease activity was calculated in accordance with the standard curve [18]and expressed as U/g (dwb).One unit of enzyme activity is defined as the amount of 1 mL of enzyme solution required to produce 1 μg of tyrosine in 1 h.

Carboxypeptidase activity was determined by measuring the release of tyrosine during the enzyme-substrate reaction by using Z-GIu-Tyr (1 mmol/mL)[19].The reaction mixture(1.1 mL)contained 0.1 mL of enzyme extract and 1 mL of Z-GIu-Tyr,and was incubated at 30 °C for 30 min.The reaction was terminated by adding 0.1 mL of ninhydrin solution.The absorbance of the resulting color was measured against the control at 570 nm in a spectrophotometer.The enzyme activity was expressed as above mentioned.

The reaction mixture (1.1 mL) contained 0.1 mL of enzyme extract,1 mL of L-leucine p-nitroaniline hydrochloride,and adequate Tris-HCI buffer solution was incubated at 30°C for 30 min.The absorbance of the resulting color was measured against the control at 405 nm in a spectrophotometer.One unit of aminopeptidase activity was defined as the amount of enzyme releasing 1 μmol paranitroanilinum in 1 min.The enzyme activity was expressed as units(U)per g of dry sample.Standard curve was plotted using 4-nitroaniline following the method of Lei et al.[20].

3.Results

3.1.Protease and carboxypeptidase activity in solid-state fermentation

Fig.1 shows the B.subtilis(ACCC 01746)protease and carboxypeptidase activity in SSF.The enzyme activity increased with prolonged fermentation time(from 0 h to 48 h for protease activity and from 8 h to 48 h for carboxypeptidase).The maximum enzyme activities of protease(469 U/g)and carboxypeptidase(477 U/g)were observed after 48 h of fermentation.At the beginning of fermentation(0-8 h),the strain mainly excreted protease;after 8 h,it then produced carboxypeptidase,which was the dominant enzyme between 30 and 50 h.After 48 h,enzyme activity steadily declined.

3.2.Effects of fermented soybean and soybean meal on bitterness values

Bitterness values were evaluated for the first 32 h of fermentation by using soybean bitter peptides and soybean meal as the substrate.The values of fermented liquid of soybean bitter peptides were reduced from 5 to 0 after 24 h,demonstrating a trend of initially increasing and then decreasing.The bitterness value was initiated at 4 h,peaked at 16 h,and declined to 0(debittered)at 28 h(Fig.2).

Fig.1.Curves of protease and carboxypeptidase activity in solid-state fermentation.

Fig.2.Bitterness values of soybean bitter peptides and fermented soybean meal.

3.3.Effects of the enzyme hydrolysates on the amino acids

Table 2 shows the free amino acids of the enzyme hydrolysate from soybean protein isolated at 2.5 h and fermented liquid following soybean meal SSF at 32 h by using B.subtilis(ACCC 01746).The amount of free amino acids was 18.48%in the hydrolysate and 7.41%in the fermented liquid.The amounts of basic and aromatic amino acids were measured to evaluate the debittering effect in both samples.The three amino acids in the enzyme hydrolysate of soybean protein isolated increased by 12.45%,2.55%,and 3.48%,and those in the fermented liquid increased by 4.54%,1.25%,and 1.62%,respectively.

Table 2 Amino acids in the enzyme hydrolysate of soybean protein isolated and soybean meal SSFa.

Fig.3.Results of the bitterness values and free amino acids in the soybean bitter peptides hydrolyzed using carboxypeptidase.

3.4.Effects of carboxypeptidase on the bitterness value and free amino acids of hydrolyzed soybean bitter peptides

The bitterness value and free amino acid content of the samples were changed because carboxypeptidase hydrolyzed the soybean bitter peptide(Fig.3).The levels of free amino acids in hydrolyzed soybean bitter peptides was gradually increased by carboxypeptidase,with a maximum value(28.5 mg/mL)after 8 h.The bitterness value dropped from 5 to 0 during the first 8 h.Among the free amino acids tested,the content of Phe showed the maximum increase,followed by the content of Leu,Tyr,Ile,and Val(Fig.4).

3.5.Comparison of carboxypeptidase and aminopeptidase in intracellular and extracellular environments

Table 3 shows the carboxypeptidase and aminopeptidase activities in the intracellular and extracellular enzyme systems of B.subtilis (ACCC 01746)during 64 h of incubation.Carboxypeptidase activity was extremely high only in the extracellular enzyme system,and aminopeptidase was not detected in either enzyme systems.

4.Discussion

Microbial proteases are enzymes that can hydrolyze peptide bonds in target proteins,and are utilized in food production,and pharmaceutical applications [18].Among microbes,B.subtilis is an important proteaseproducer due to its safety and the usefulness of its extracellular proteins in hydrolyzing soybean proteins[16,21].Maximum enzyme secretion by the Bacillus species usually occurs during the exponential phase of late growth and the stationary phases of early growth[22].Different types of proteases,such as extracellular carboxypeptidase,are produced at various times during fermentation[23].Our conclusions on the production of carboxypeptidase are consistent with previous results on different strains,such as Aspergillus oryzae[19].As such,a difference in fermented products exists,thus affecting the flavors produced during fermentation.Fig.1 and 2 show that B.subtilis(ACCC 01746)yields extracellular proteases and carboxypeptidase at the beginning of fermentation(0-8 h),and these enzymes can hydrolyze the proteins of soybean meal into long-chain peptides with slight bitterness.At 8-16 h of fermentation,the long chain peptides are further hydrolyzed into middle and short chain peptides,thereby increasing bitterness.Carboxypeptidase significantly increases after 16 h,and amino acids are removed from the C-terminal of peptides,thereafter decreasing bitterness.

Table 3 Analysis of enzyme activities produced by B.subtilis(ACCC 01746)in seed cultures.

The standard levels of bitterness for soybean proteins are associated with the amount of hydrophobic amino acids in protein [24,25].This amount allows us to infer the level of bitterness and speculate that peptides with a high number of hydrophobic amino acids have bitter hydrolysates.Certain hydrophobic amino acids,including Lys,Val,Leu,Pro,Phe,Tyr,and Trp [26],have a positive correlation with bitterness.This study shows that the percentage of nonhydrolyzed hydrophobic amino acids in the soybean meal peptide bonds is 29.05%of the total amino acid content,which is less than that in the soybean protein isolated(62.81%).The high levels of hydrophobic amino acids in soybean protein isolates are the basis of the strong bitterness of hydrolysates.This phenomenon explains the reduced bitterness after soybean meal is fermented and is agreement with previous observations by Kukman et al.[27].

The bitterness values of fermented soybean solution were reduced from 5 to 0(Fig.2)upon fermentation with B.subtilis for 32 h.The debittering ability of the strain is possibly caused by carboxypeptidase.Enzyme analysis also shows that the strain can excrete carboxypeptidase during growth(Table 3),thereby increasing hydrophobic amino acids (Fig.4) and decreasing the bitterness(Fig.3).These results are consistent with the findings of Liu et al.[28]and Li et al.[29].

Fig.4.Results of the amino acids of soybean bitter peptides hydrolyzed using carboxypeptidase.

5.Conclusion

Others have tried to improve the bitterness of food hydrolysates by using activated carbon,isoelectric precipitation,and extraction with alcohol.At present,there are few researches on the debittering ability of B.subtilis are few,and the effect of extracellular proteinase on the bitterness of soybean protein peptides remains unclear.The present study isolated a hydrolytic enzyme from B.subtilis(ACCC 01746)and revealed that the enzyme has a debittering activity by cleaving the adjacent hydrophobic amino acid residues(i.e.,Phe,Ala,Tyr,and Leu)at the C-terminal of soybean peptide hydrolysates.The carboxypeptidases from B.subtilis(ACCC 01746)efficiently decrease the bitterness of peptides by liberating Phe,Ala,Tyr,and Leu.Further research on the free amino acids should be conducted to gain insights into soybean peptide structure and its correlation with bitterness.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by Grain&Corn Engineering Technology Research Center,State Administration of Grain(GA2017004),Science and Technology Research Project of Henan(172102110205);Henan University of Technology:Integration of Science and Education(30).