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Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated Bacillus licheniformis BL312

Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated... Purpose This study was conducted to find a microbial milk-clotting enzyme (MCE) with a high and stable milk-clotting activity (MCA) to proteolytic activity (PA) ratio suitable for the cheese industry. Methods Microbial strains were isolated from soil suspensions cultured in solid casein medium. 16S rDNA of representative isolates were sequenced to identify the microbial species. Nutrition and fermentation conditions were systematically examined to optimize MCA of the selected MCE. Protease inhibitors were used to identify the type of MCE. The casein hydrolysis was analyzed through reversed-phase HPLC (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE). Results The Bacillus licheniformis BL312 was identified from 50 bacterial strains. BL312 MCE achieved a maximal MCA (460 ± 15 SU/mL) at 48 h that was 2.7-fold higher than the control, and the MCA/PA ratio (9.0) and pH (6.6) remained stable throughout the fermentation process. Medium containing 30 g/L wheat bran shorts, 5 g/L glucose, and 3 g/L corn steep liquor was sufficient for optimal BL312 MCE production. Fermentation conditions of an inoculum size of 7.0% (v/v), fermentation temperature of 37 °C, agitation speed of 210 rpm, and initial pH 6.6 were required to achieve maximal MCA. BL312 MCE was inhibited by phenylmethanesulfonyl fluoride (PMSF) and high concentrations of ethylenediaminetetraacetic acid (EDTA) (5– 25 mM). The α -casein (α -CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE and calf rennet were different. s s Conclusions BL312 MCE is a serine/alkaline protease that exhibits high MCA and various hydrolysis for caseins in comparison with calf rennet. . . . . . Keywords Isolation Fermentation Bacillus licheniformis Milk-clotting enzyme Serine/alkaline protease Casein hydrolysis Introduction which influences the characteristics of the final product. The MCA/PA ratio is an inherent characteristic of MCE. The ideal Milk coagulation is an important step in cheese production, and MCE is characterized with a high MCA and a low non-specific MCEs play a major role in this process. MCEs facilitate milk PA (Meng et al. 2018). In fact, the high MCA/PA ratio leads to clotting and may influence the flavor and texture of the cheese. better textural and sensorial properties of cheese and a great Calf rennet, the most widely used milk coagulant, is obtained reduction of bitterness. Consequently, the high MCA/PA ratio from the fourth stomach of suckling calves (Liburdi et al. has shown to be crucial in evaluating the applicability of MCEs 2018). However, the use of calf rennet substitutes is becoming (Ben et al. 2017). Plant-derived coagulants can be produced necessary due to the increase in cheese consumption. The se- from seeds, vacuoles, and extracellular spaces (Gagaoua et al. lection of a suitable calf rennet substitute is a critical factor, 2017; Luo et al. 2018), and can be used as calf rennet substi- tutes. However, most plant-derived coagulants, which typically exhibit low MCA/PA ratios, achieve poor cheese yield and * Lianzhong Ai form bitter substances during cheese ripening (Salehi et al. ailianzhong@hotmail.com 2017). In contrast, recombinant chymosins exhibit high MCAs and good thermal stability. However, they are banned Shanghai Engineering Research Center of Food Microbiology, in many countries (Vallejo et al. 2012). School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, 516 Jungong Road, Recently, microbial MCEs have attracted great interest as Shanghai 200093, China promising calf rennet substitutes. Fungal MCEs, especially Beijing Sanyuan Foods Co., Ltd, Beijing 100163, China those from Rhizomucor miehei, R. pusillus,and Endothia 1290 Ann Microbiol (2019) 69:1289–1300 parasitica, are already widely used in commercial cheese pro- (50 mL) at 4 °C. To prepare soil suspensions, 1 g of soil duction (Celebi et al. 2016). Compared with fungal MCEs, sample was suspended in 9 mL of normal saline. Tenfold bacterial MCEs are cheaper to produce, more biochemically serial dilutions of the initial suspension were prepared with −4 diverse, and more easily modified genetically (Hang et al. normal saline until a final dilution of 10 -fold was achieved. 2016). Bacterial MCEs have been reported to exhibit high A120-μL aliquot of each diluted sample was cultured at 37 °C MCA/PA ratios (Luo et al. 2018;Li etal. 2019). Moreover, for 36 h in solid casein medium (pH 6.8) containing 2.5 g/L bacterial submerged fermentation is easier to control and casein peptone, 10 g/L glucose, 1 g/L yeast extract powder, shows a higher material utilization ratio compared with fungi. 25 g/L skim milk powder, and 20 g/L agar. Each strain was However, studies on bacterial MCEs have been less frequently numbered, and the diameters of hydrolysis and precipitation conducted compared with those on fungal MCEs. Few com- rings were examined every 12 h. mercial applications of bacterial MCEs as milk coagulants The isolated strains with high ratios of (i) precipitation ring have been reported. diameter to hydrolysis ring diameter and (ii) precipitation ring Shouguang (Shandong, China) is known as the hometown diameter to colony zone diameter were selected, and their 16S of Chinese vegetables, and its agricultural soil is rich in organ- rDNA were sequenced. For species-level identification, se- ic matter. Importantly, it houses a wide variety of microbial quences were compared with those in the GenBank database species (Zhou et al. 2011), many of which exhibit high prote- using the BLAST program (NCBI). For phylogenetic analy- ase activities. Microorganisms producing proteases (e.g., ser- sis, our data were aligned to a dataset containing 16S rDNA ine and threonine protein kinase) have previously been col- sequences using the BioEdit program. MEGA 7.0 software lected from the agricultural soil in Shouguang (Wang et al. was used to construct the phylogenetic tree (He et al. 2012). 1998; Qiu et al. 2016). In this study, BL312 (a Bacillus The harmful bacteria (e.g., Serratia marcescens, licheniformis strain) was isolated and found to exhibit a high Acinetobacter,and Pneumococcus) were excluded and others and stable MCA/PA ratio during fermentation. Various condi- (e.g., Bacillus licheniformis and Bacillus subtilis)weremain- tions and nutritional parameters were systematically examined tained at − 80 °C in 20% (v/v) glycerin. MCA, PA, and fer- to optimize the condition for MCE production and MCA. mentation pH were measured in subsequent fermentation ex- Large-scale fermentation (using a fermenter) was then used periments using the selected microorganism. to investigate the economic feasibility of BL312 MCE pro- duction. The effects of different protease inhibitors on the MCA of BL312 MCE were examined. The hydrolysis behav- Measurement of MCA and PA ior of BL312 MCE for caseins was analyzed. The fermentation broth was centrifuged at 5000×g for 10 min. The crude MCE was in the supernatant and used for MCA and Materials and methods PA determinations. MCA was determined according to the method of Arima et al. (1968). Skim milk 10% (w/v in deion- Chemicals and reagents ized water) containing 10 mM CaCl was used as the sub- strate. Curd formation was observed by manually rotating Skim milk powder was purchased from Fabrique par Fonterra the test tube from time to time so as to form a thin film on Co., Ltd. (Auckland, New Zealand). Calf rennet was provided its inner surface. The MCA is expressed in Soxhlet unit (SU), from Yuexiang Chemical Co., Ltd. (Shandong, China). PMSF, which is defined as the amount of MCE required to clot 1 mL EDTA, pepstatin A, and DL-dithiothreitol (DTT) were obtain- of milk substrate at 35 °C. The MCA was calculated using the ed from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). following formula: SU = (2400 × V × n)/(t × V ), where V is 1 2 1 The other chemicals used in the study were analytical grade the milk volume (mL), n is the dilution of MCE, t is the milk- from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, clotting time (s), and V is the MCE volume (mL). China). The proteolytic activity was measured following the meth- od of Arima et al. (1968). In brief, 5 mL of 1.2% (w/v) casein Isolation and identification of MCE-producing solution in 0.05 M phosphate buffer (pH 6.5) was added 1 mL bacteria crude MCE. The mixture was then incubated at 35 °C for 10 min. After incubation, 2 mL (0.44 M) of trichloroacetic Soil samples were collected from 10 to 15 cm-deep pits in acid (TCA) was added to quench the reaction. The mixture greenhouses and different agricultural regions in Shouguang was followed by centrifugation (6000×g) for 10 min at 4 °C. (36° 88′ N, 118° 73′ E). The samples were placed into sterile Two milliliters of the clear supernatant was added with 5 mL zipper polythene bags and stored at 4 °C until use. Soil sam- NaOH (0.28 M) solution and 1 mL Folin-Ciocalteu phenol ples taken to the laboratory were passed through a sieve (3– reagent. After incubation at 35 °C for 15 min, the mixture 4 mm mesh) and then were stored in sterile centrifuge tubes was measured for optical density (OD) at 660 nm. One unit 1291 Ann Microbiol (2019) 69:1289–1300 (1 U) of MCE activity is defined as the amount of MCE that The effects of bioprocess parameters on BL312 MCE liberated 1 μgof tyrosine per 1mL in 1 min. production In order to determine bioprocess parameters on MCE produc- Microbial growth and MCE production tion, inoculum size (1, 3, 5, 7, and 9%, v/v), fermentation medium volume (20, 30, 40, 50, and 60 mL), cultivation tem- The strain maintained in laboratory at −80 °C was grown on perature (27, 32, 37, and 42 °C), and agitation speed (120, improved TYC medium, which contained 15 g/L casein pep- 150, 180, 210, and 230 rpm) were studied in 250 mL tone, 5 g/L yeast extract powder, 50 g/L glucose, 0.2 g/L L- Erlenmeyer flasks. The initial pH of fermentation medium cysteine, 1 g/L NaCl, 2 g/L Na HPO ·12H O,and2g/L 2 4 2 was adjustedto4.6,5.6,6.6,7.6,and 8.6 todetermine the NaHCO and was sterilized at 115 °C for 20 min before use. optimal initial pH for MCE production. The one single colony was inoculated on 50 mL of improved TYC medium in a flask of 250 mL and incubated at 37 °C for Time course of BL312 MCE production in a fermenter 16–18 h under shaking (150 rpm) as seed liquid. MCE production was carried out by fermentation with a The MCE production was carried out by fermentation with 5.0% (v/v) inoculum size in three flasks of 250 mL × 20 g/L of optimal parameters in a 7-L fermenter (LiFlus GM, Biotron, wheat bran shorts, which were autoclaved at 115 °C for Korea) containing 4 L fermentation medium, which was 20 min. Wheat bran shorts contained 39.28% (w/w) carbon, autoclaved at 115 °C for 20 min. The initial concentration of 3.12% (w/w) nitrogen, and 6.51% (w/w) moisture. The fer- dissolved oxygen (DO) was regarded as 100% of air mentation conditions were as follows: temperature 37 °C, ro- saturation. tating speed 150 rpm, initial pH 6.6, and liquid volume 50 mL. Determinations on MCA, PA, and pH of fermentation broth The effects of protease inhibitors on BL312 MCE were carried out in a time range of 36–108 h at a 12-h interval. At timed intervals, samples were collected and centrifuged at The supernatant of fermentation broth was brought to ammo- 5000×g for 10 min at 4 °C. The supernatant was used to nium sulfate (60% saturation) at 4 °C for 2 h and harvested by measure MCA, PA, and pH of fermentation broth. centrifugation at 7000×g and 4 °C for 10 min. The precipitate Fermentation conditions were varied by one factor at a time. was dissolved in Tris-HCl buffer (pH 7.0) and centrifuged at 7000×g and 4 °C for 10 min. The crude MCE in the clear The effects of wheat bran concentrations on BL312 supernatant was dialyzed in dialysis bags (8–14 kDa) and MCE production lyophilized to powder for further experiments. The effects of various protease inhibitors including pepstatin A (0.01 and Different concentrations of wheat bran shorts including 20, 0.02 mM), EDTA (1, 5, 10, and 25 mM), PMSF (1 and 30, 40, 50, and 60 g/L were used to determine the optimal concentration for MCE production. MCA was measured in a Table 1 The diameter of hydrolysis and precipitation rings on the solid time range of 36–96 h at a 12-h interval. casein medium CN CD HD PD P/ P/ The effects of carbon and nitrogen sources on BL312 C H MCE production DB215 4.0 ± 1.0 7.0 ± 1.0 11.5 ± 1.5 2.9 1.6 DB218 2.0 ± 0.5 3.0 ± 0.5 10.5 ± 1.5 5.3 3.5 To investigate the optimal carbon source for MCE production, BL312 3.0 ± 0.5 3.5 ± 0.5 18.0 ± 2.0 6.0 5.1 different carbon sources (glucose, sucrose, soluble starch, BL211 2.0 ± 0.5 5.0 ± 1.0 11.0 ± 2.0 5.5 2.2 maltodextrin, and lactose) at 5 g/L were individually added QD111 1.5 ± 0.5 5.5 ± 1.0 11.0 ± 1.5 7.3 2.0 to the basal wheat bran shorts medium. The MCA in the basal QD212 3.0 ± 0.5 4.0 ± 1.0 16.5 ± 1.5 5.5 4.1 wheat bran shorts medium (without other carbon sources) was YN111 4.0 ± 1.0 5.0 ± 1.0 17.0 ± 2.0 4.3 3.4 the control and regarded as 100%. Different organic and inor- YN213 4.0 ± 1.0 6.0 ± 1.0 14.0 ± 2.0 3.3 2.2 ganic nitrogen sources (corn steep liquor, casein peptone, NX112 4.0 ± 0.5 8.0 ± 1.0 13.0 ± 1.0 3.3 1.6 urea, yeast extract powder, ammonium sulfate, and ammoni- um citrate) at 3 g/L were used to determine the optimal nitro- GS113 2.0 ± 0.5 5.0 ± 1.0 10.0 ± 1.5 5.0 2.0 GL111 4.0 ± 1.0 5.0 ± 1.0 9.0 ± 1.0 2.3 1.8 gen source. The MCA of BL312 MCE in wheat bran shorts medium containing 5 g/L glucose (without nitrogen sources) The data are represented as mean ± SD (n = 3). CN, strain number; CD, was the control and taken as 100%. The MCA and MCA of colony zone diameter (mm); HD, hydrolysis ring diameter (mm); PD, following fermentation experiments were measured in a time precipitation ring diameter (mm); P/C, the ratio of PD to CD; P/H, the ratio of PD to HD. The culture time of all strains was 24 h range of 36–84 h at a 12-h interval, unless specified otherwise. 1292 Ann Microbiol (2019) 69:1289–1300 Fig. 1 Phylogenetic position of the isolated BL312 based on 16S rDNA gene sequence analysis. GenBank accession numbers are given in parentheses 2 mM), and DL-dithiothreitol (DTT) (1 mM) were examined Statistical analysis on MCA of the crude MCE according to the method of Salehi et al. (2017) with some modifications. After addition of inhib- Each experiment was performed in triplicate. The results itors, the mixtures were incubated at 25 °C for 30 min. The were expressed as means ± standard deviations. Data ob- samples were evaluated for MCA as mentioned formerly. The tained were analyzed by one-way ANOVA using the soft- control (without inhibitors) was regarded as 100%. ware SPSS 17.0. The level of statistical significance was set at P <0.05. Hydrolysis of caseins by BL312 MCE Results and discussion The α -CN and β-CN hydrolysates generated by BL312 MCE and calf rennet were analyzed by RP-HPLC and SDS-PAGE Separation and identification of Bacillus licheniformis with 12% (w/v) acrylamide according to the method of BL312 Merheb-Dini et al. (2010)and Wasko et al. (2012), respective- ly. The α -CN or β-CN (0.2%, w/v) was mixed with MCEs s Initially, 11 isolated strains were found to form large precipi- (100 SU/mL) in a proportion of α -CN or β-CN:MCEs = s tation rings in the solid casein medium, which indicated high 15:1. For SDS-PAGE analysis, the proportions of α -CN or s MCA (Table 1). Given that a high PA (as indicated by large β-CN:BL312 MCE (10:1, 20:1, and 30:1) were also detected. hydrolysis rings in the solid casein medium) can lead to the The mixtures were incubated at 35 °C for 30 min for reaction. formation of bitter substances in cheese (Merheb-Dini et al. Reactions were quenched by heating the solutions in boiling 2010), strains with a high ratio of precipitation ring to hydro- water for 10 min. lysis ring and precipitation ring to colony zone were selected Fig. 2 The BL312 MCE production (a) and pH of fermentation broth (b) during different fermentation times (36–108 h). Data were presented as means of triplicate measurements; error bars were standard deviations 1293 Ann Microbiol (2019) 69:1289–1300 Table 2 Comparison of PA and Strain Substrate PA MCA/PA ratio References MCA/PA ratio among different derived MCEs B. licheniformis USC13 Casein 43 U/mL 6.7 Ageitos et al. (2007) B. subtilis B1 Casein 174 U/mL 6.5 Ding et al. (2011) B. amyloliquefaciens JNU002 Casein 1109 U/mL 5.9 Ding et al. (2012) A. niger FFB1 Casein 3020 U/mg 0.6 Fazouane et al. (2010) R. miehei (commercial MCE) Casein – 6.6 Thakur et al. (1990) R. miehei (Rennilase L) Casein – 3.9 Thakur et al. (1990) R. pusillus (Noury) Casein – 4.5 Thakur et al. (1990) C. parasitica (Sure curd) Casein – 1.6 Thakur et al. (1990) Calf rennet Casein – 12.8 Thakur et al. (1990) M. mucedo DSM 809 Azocasein 7.9 U/mL 16.5 Yegin et al. (2012) A. rouxii Azocasein 0.7 U/mL – Marcial et al. (2011) Nocardiopsis sp. Azocasein 1.6 U/mL 2.9 Cavalcanti et al. (2005) R. miehei Azocasein 11.2 U/mL 23.9 Yegin et al. (2012) R. miehei (Sigma) Hemoglobin – 2.0 Preetha et al. (1997) A. oryzae MTCC 5341 Hemoglobin 172 U/mg – Vishwanatha et al. (2010) and further examined using 16S rDNA sequencing and fer- good characteristic of BL312 MCE (Ben et al. 2017). In addi- mentation experiments. Accordingly, BL312 was selected tion, for some organisms, the MCA/PA ratio fluctuates during from the 11 initial isolates for MCE production. fermentation (Hashem 1999). However, the MCA/PA ratio of The 16S rDNA sequences of BL312 were compared to all BL312 MCE was found to be stable, indicating that BL312 sequences in the GenBank for species identification. Our ob- MCE production and activity can be more easily controlled tained nucleotide sequence showed the best match (99% ho- during fermentation compared with other MCEs. mology) to those of Bacillus licheniformis strain DSM 13 (NR118996.1), Bacillus licheniformis strain BCRC 11702 (NR116023.1), and Bacillus licheniformis strain NBRC The effects of different carbon and nitrogen sources 12200 (NR113588.1). A phylogenetic tree was constructed on BL312 MCE production using the neighbor-joining method (Fig. 1), and BL312 was identified as Bacillus licheniformis using phylogenetic analy- Wheat bran shorts contains several sources of carbon and sis. Bacillus licheniformis BL312 was deposited in the China nitrogen. It is a low-cost raw material that supports the growth of many microorganisms. Importantly, wheat bran is an General Microbiological Culture Collection Center (CGMCC No. 15009). The MCA and PA of BL312 fermentation are shown in Fig. 2a. BL312 MCE achieved the maximal MCA (169 ± 6 SU/mL)andPA(19±2 U/mL)at84h.The MCA/PA ratio was found to be 9.0 throughout the fermentation. As indicated in Fig. 2b, the pH of the fermentation broth was 6.6 and remained unchanged throughout. These results suggested that Bacillus licheniformis BL312 did not produce acid during fer- mentation, indicating that curd formation was caused by MCE. It is likely that BL312 is applicable for MCE production. Table 2 shows that the MCA/PA ratio of BL312 MCE was generally similar to that of commercial MCEs (e.g., calf rennet and R. miehei MCE). However, BL312 MCE exhibited a higher MCA/PA ratio in comparison with B. amyloliquefaciens JNU002, A. niger FFB1, and C. parasitica MCE. The MCA/ Fig. 3 The effects of different concentrations of wheat bran shorts on PA ratio plays an important role in the selection of MCEs. The BL312 MCE production during fermentation (36–96 h). Data were MCEs with high MCA/PA ratio achieve great cheese yield and presented as means of triplicate measurements; error bars were standard decreased bitter substances in cheese production, indicating a deviations 1294 Ann Microbiol (2019) 69:1289–1300 effective nutrient source for MCE production during fermen- starch, maltodextrin, and sucrose (Fig. 4a, b). Given our re- tation (Chwen-Jen et al. 2009). Our results indicated that sults, 5 g/L glucose was used to supplement the basal fermen- MCA of BL312 MCE increased with increasing concen- tation medium in subsequent experiments. Next, the effects trations of wheat bran shorts (up to 30 g/L), but de- of four organic nitrogen sources (corn steep liquor, casein creased at higher concentrations (30–60 g/L) (Fig. 3). peptone, urea, and yeast extract powder) and two inorgan- The maximal MCA (260 ± 9 SU/mL) was achieved after ic sources (ammonium sulfate and ammonium citrate) on 48 h of fermentation using 30 g/L wheat bran shorts. MCE production were examined at different fermentation Accordingly, 30 g/L of triturated wheat bran shorts was time points (Fig. 4c, d). Our data indicated that BL312 used as the basal fermentation medium in the following MCE achieved the maximal MCA in the presence of 3 g/ optimization experiments. L corn steep liquor (302 ± 10 SU/mL). Compared with the BL312 was cultured in conditions supplemented with dif- control, MCA increased by 10% and 5% in the presence ferent carbon sources, and MCE production was measured at of corn steep liquor and urea, respectively. However, different time points to select the most suitable source of car- MCA was reduced in the presence of casein peptone, bon supplementation (Fig. 4a, b). The carbon sources included yeast extract powder, ammonium sulfate, and ammonium glucose, sucrose, soluble starch, maltodextrin, and lactose. citrate (Fig. 4c, d). Our results indicated that BL312 MCE achieved the highest Taken together, these results indicated that glucose (5 g/L) MCA in the presence of glucose (275 ± 9 SU/mL, 6% higher and corn steep liquor (3 g/L) supplementation in wheat bran than the control). While the MCA was not altered in the pres- shorts fermentation medium produced the best culture condi- ence of lactose, it was reduced in the presence of soluble tion for BL312 MCE production. MCE synthesis can be Fig. 4 a, b The effects of different carbon sources on BL312 MCE YP, yeast extract powder; AM, ammonium sulfate; and AC, ammonium a–g production during fermentation (36–84 h). Glu, glucose; Suc, sucrose; citrate. Values in the same row with different superscripts are SS, soluble starch; MD, maltodextrin; and Lac, lactose. c, d The effects significantly different (P<0.05).Datawerepresented as meansof of different nitrogen sources on BL312 MCE production during triplicate measurements; error bars were standard deviations fermentation (36–84 h). CSL, corn steep liquor; PC, casein peptone; 1295 Ann Microbiol (2019) 69:1289–1300 induced or suppressed by various materials and nutrient The effects of bioprocess parameters on BL312 MCE sources (Shata 2005). As BL312 growth and MCE production production could be achieved using the low-cost wheat bran shorts with modest nutrient supplementation (Fig. 4), our results sug- As presented in Fig. 5, the bioprocess parameters were further gested that large-scale production of BL312 MCE would be optimized to achieve a maximal MCA (442 ± 12 SU/mL) in economically feasible. Over-supplementation of carbon and BL312 MCE. Our results indicated that the optimal inoculum nitrogen sources inhibits the growth of microorganisms and size was 7% (v/v) and medium volume for BL312 MCE pro- MCE production (Sen et al. 2009). According to our results, duction was 40 mL (Fig. 5a, b). The maximal MCA was ob- organic nitrogen sources (corn steep liquor and urea) produced served at a fermentation temperature of 37 °C and an agitation better BL312 MCE yield than inorganic nitrogen sources (am- speed of 210 rpm (Fig. 5c, d). Deviations from the optimal monium sulfate and ammonium citrate) (Fig. 4c, d). It could inoculum size and fermentation temperature both decreased be possible that organic nitrogen contains kinds of amino MCE production. acids, which can be absorbed directly by BL312. In contrast, It is known that both the inoculum size and fermentation BL312 first synthesized inorganic nitrogen into amino acids, temperature profoundly affect the production of microbial reducing the growth of microorganisms. The MCE production MCEs. The biomass production was determined by inoculum is thus limited by the reduced biomass (Cai et al. 2004). These size during fermentation (Sen et al. 2009). When the inoculum results were different from previous reports. Glucose and or- size was increased to 9% (v/v), MCA decreased significantly. ganic nitrogen sources are not considered good nutrient It could be faster growth of BL312 and larger biomass, which sources for MCE production in Penicillium oxalicum and caused the shortage in nutrients. To achieve the maximal MCE Nocardiopsis sp. (Hashem 1999; Cavalcanti et al. 2005). production, it is necessary to maintain the balance of biomass However, glucose is considered a suitable carbon source for production and nutrient availability (Patel et al. 2005). The Mucor miehei (Sun et al. 2014). optimal temperature for MCE production in BL312 (37 °C) Fig. 5 The effects of inoculum size (a), volume (b), cultivation composition was wheat bran shorts (30 g/L), glucose (5 g/L), and corn temperature (c), and agitation speed (d) on BL312 MCE production steep liquor (3 g/L). Data were presented as means of triplicate measure- during different fermentation times (36–84 h). Dissolved oxygen (DO) ments; error bars were standard deviations and medium volume were the negative correlation. The medium 1296 Ann Microbiol (2019) 69:1289–1300 F34 (Wu et al. 2008), Fusarium subglutinans (Ghareib et al. 2001), and Amylomyces rouxii (Yu and Chou 2005), respectively. BL312 MCE production using a 7-L fermenter A large-scale production of BL312 MCE was next conducted in a 7-L fermenter using the optimized condition (Fig. 7). The BL312 MCE production was initiated using 4 L of the optimal fermentation medium (wheat bran shorts with glucose and corn steep liquor supplementation). The fermentation condi- tion was as follows: inoculum size 7.0% (v/v), temperature 37 °C, agitation speed 210 rpm, and ventilation 1.7 vvm. Compared with using shake flasks, BL312 MCE achieved a Fig. 6 The effects of initial pH of fermentation medium on BL312 MCE higher maximal MCA (460 ± 15 SU/mL) in the fermenter. production during different fermentation times (36–84 h). The medium Throughout the fermentation process, the MCA/PA ratio and composition was wheat bran shorts (30 g/L), glucose (5 g/L), and corn pH remained at 9.0 and 6.6, respectively. During the growth steep liquor (3 g/L). Data were presented as means of triplicate measurements; error bars were standard deviations phase of BL312, DO concentration drastically decreased by 78% compared to the initial concentration, but increased after was different from that for Paenibacillus sp. BD3526 (30 °C) 36 h of fermentation. The maximal MCA of BL312 MCE (Hang et al. 2016)and Bacillus methanolicus LB-1 (30 °C) (Li observed here was higher than that reported for et al. 2019). The agitation speed and liquid volume also played B. licheniformis USC13 (290 SU/mL) (Ageitos et al. 2007), an important part in BL312 MCE production as they affected B. subtilis YB-3 (200 SU/mL) (Li et al. 2012), and M. mucedo the amount of DO during fermentation. DSM 809 (130 SU/mL) (Yegin et al. 2012). The BL312 MCE exhibited lower PA compared with B. subtilis B1 and B. amyloliquefaciens JNU002 MCE (Table 2). Besides, the The effects of initial pH on BL312 MCE production stable MCA/PA ratio and pH suggested that the submerged fermentation of BL312 was easy to control, implying that it is BL312 MCE production reached the maximum when initial valuable in potential application. pH of the fermentation medium was adjusted to 6.6 (Fig. 6). Deviations from this initial pH decreased MCA. The initial pH is an important factor for many enzymatic and membrane The effects of protease inhibitors on BL312 MCE transport processes (Moon and Parulekar 2010). The optimal initial pH for MCE production is dependent on the fermenta- The MCA values of crude BL312 MCE treated with different tion medium and the species of microorganism. The maximal protease inhibitors are shown in Table 3. Our results indicated MCA was achieved at the original medium pH (6.6). It was in that BL312 MCE was not affected by pepstatin A (0.01– favor of the production and industrialization of BL312 MCE. 0.02 mM), DTT (1 mM), and low concentrations of EDTA The optimal initial pH values for the maximal MCA were (1 mM). However, it was significantly inhibited by PMSF (1 reported to be 4.5, 6.0, and 7.0 for Chinese distiller’syeast and 2 mM) and high concentrations of EDTA (5–25 mM) Fig. 7 Changes in the MCA, PA, DO, and pH in a 7-L fermenter with respect to fermentation times. Data were presented as means of triplicate measurements; error bars were standard deviations 1297 Ann Microbiol (2019) 69:1289–1300 Table 3 The effects of different Inhibitor Concentration (mM) Relative MCA (%) Inhibition rate (%) protease inhibitors on BL312 MCE None – 100 0 Pepstatin A 0.01 95.7 ± 1.5 4.3 0.02 96.9 ± 1.0 3.1 EDTA 1 97.1 ± 2.1 2.9 5 83.0 ± 2.3 17.0 10 70.6 ± 3.2 29.4 25 59.6 ± 2.1 40.4 PMSF 1 9.7 ± 1.7 90.3 2 7.1 ± 2.2 92.9 DTT 1 98.6 ± 1.4 1.4 The data are represented as mean ± SD (n =3) (Table 3). Protease inhibitors can be used to reveal the type addition, the BL312 MCE identified in this study is different and active site of enzymes. It is known that PMSF, pepstatin from the MCEs of Enterococcus faecalis TUA2495L (Sato A, and DTT were serine, aspartate, and cysteine protease in- et al. 2007), B. subtilis YB-3 (Li et al. 2012), and hibitor, respectively (Uda et al. 2017). The alkaline proteases Paenibacillus sp. BD3526 (Hang et al. 2016). were inhibited by high concentrations of EDTA (Tang et al. 2004). Our results showed that BL312 MCE exhibited the Hydrolysis behavior of BL312 MCE properties of serine and alkaline protease, indicating that it was a serine/alkaline protease. Unlike BL312 MCE, the The RP-HPLC chromatograms of peptides were shown in MCA of Bacillus licheniformis 5A5 MCE is enhanced in the Fig. 8. The BL312 MCE was more hydrolytic on α -CN and presence of PMSF and EDTA (Ahmed and Helmy 2012). In β-CN than calf rennet. As shown in Fig. 8c–f,the α -CN and Fig. 8 RP-HPLC chromatograms of α -CN (a)and β-CN (b); RP- HPLC chromatograms of α -CN (c)and β-CN (d)hydrolysates generated by calf rennet; RP- HPLC chromatograms of α -CN (e)and β-CN (f) hydrolysates generated by BL312 MCE 1298 Ann Microbiol (2019) 69:1289–1300 Fig. 9 SDS-PAGE patterns of α -CN (a) and β-CN (b) hydrolysates 2–5: BL312 MCE. The α -CN:MCEs or β-CN:MCEs ratios were 15:1 in s s generated by BL312 MCE and calf rennet. Lane M: molecular weight lanes 1 and 3, 10:1 in lane 2, 20:1 in lane 4, and 30:1 in lane 5 marker proteins; lane A: α -CN; lane B: β-CN; lane 1: calf rennet; lanes β-CN hydrolysates generated by BL312 MCE and calf rennet hydrolyze α -CN, indicating it was potential to be used in mak- were significantly different at retention times of 10–50 min. ing soft or semi-hard cheese. However, the hydrolysates were generally similar at retention times of 50–80 min, where few peaks could be observed in chromatograms. The β-CN hydrolysates at retention times of Conclusions 60–80 min represent the bitter peptides, which are important for the flavor during cheese ripening (Lee and Warthesen In this study, a Bacillus licheniformis BL312 strain was iden- 2010). Although lack of the sensory analysis, the likely pep- tified and exhibited high MCA. A remarkable MCA (460 ± 15 tide compositions were indicated through the comparison of SU/mL) was achieved after 48 h of fermentation in culture two chromatograms of hydrolysates generated by BL312 medium containing low-cost wheat bran shorts (30 g/L), glu- MCE and calf rennet (Fig. 8d, f). It is concluded that the β- cose (5 g/L), and corn steep liquor (3 g/L). An initial pH of CN hydrolysates generated by BL312 MCE were few bitter 6.6, fermentation temperature of 37 °C, agitation speed of peptides. 210 rpm, and ventilation of 1.7 vvm were required for the For SDS-PAGE analysis, Fig. 9 shows that the hydrolytic optimal MCA. The BL312 MCE exhibited a stable MCA/ abilities of BL312 MCE were getting weaker with the increase PA ratio of 9.0 and pH of 6.6 throughout the fermentation of α -CN:MCE or β-CN:MCE ratios. The α -CN was mainly s s process, suggesting that it is suitable for prolonged fermenta- hydrolyzed into apparent fragments (12–18 kDa) by BL312 tion. The MCA of BL312 MCE was significantly inhibited by MCE, which were much smaller than those by calf rennet PMSF and high concentrations of EDTA, implying that it was (Fig. 9a). As presentedinFig. 9b,the β-CN hydrolysates gen- a serine/alkaline protease. Importantly, our results revealed erated by BL312 MCE were about molecular masses of 12– that BL312 MCE was characterized with various hydrolysis 14 kDa and 17–18 kDa. Different casein hydrolysates generat- for caseins. Further characterization and application of BL312 ed by BL312 MCE and calf rennet indicated different cleavage MCE should be conducted in the future. sites in caseins and might lead to various cheese flavors (Majumder et al. 2015). In addition, the BL312 MCE hydro- Acknowledgments The authors gratefully acknowledge the National Key R&D Program of China (grant no. 2018YFD0502306) and lyzed α -CN to a greater level than β-CN, which was in line Shanghai Engineering Research Center of Food Microbiology (grant with RP-HPLC analysis result. It is known that the hydropho- no. 19DZ2281100) for the granted fellowships. bic peptides generated by β-CN hydrolysis were responsible for the bitterness in cheeses (An et al. 2014). Weak hydrolysis Funding information This work was supported by the National Key level of β-CN for BL312 MCE led to a decreased amount of R&D Program of China (grant no. 2018YFD0502306) and Shanghai Engineering Research Center of Food Microbiology (grant no. bitter peptides in cheese production. Besides, various MCEs 19DZ2281100). exhibited different preferences for α -CN and β-CN hydroly- sis. As reported with researchers, the hydrolysis of β-CN was Compliance with ethical standards stronger than that of α -CN for Paenibacillus spp. BD3526 MCE (Hang et al. 2016). Importantly, the meltability and hard- Conflict of interest The authors declare that they have no conflict of ness of cheese were closely related to the α -CN and β-CN interest. hydrolysis. The cheese meltability increased with α -CN deg- radation. In contrast, the cheese was hard at a high β-CN hy- Informed consent This manuscript is approved by all authors for drolysis level (Hayaloglu et al. 2014). BL312 MCE preferred to publication. 1299 Ann Microbiol (2019) 69:1289–1300 Lee KD, Warthesen JJ (2010) Mobile phases in reverse-phase HPLC for References the determination of bitter peptides in cheese. 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Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated Bacillus licheniformis BL312

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Springer Journals
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Copyright © 2019 by Università degli studi di Milano
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
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1869-2044
DOI
10.1007/s13213-019-01513-3
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Abstract

Purpose This study was conducted to find a microbial milk-clotting enzyme (MCE) with a high and stable milk-clotting activity (MCA) to proteolytic activity (PA) ratio suitable for the cheese industry. Methods Microbial strains were isolated from soil suspensions cultured in solid casein medium. 16S rDNA of representative isolates were sequenced to identify the microbial species. Nutrition and fermentation conditions were systematically examined to optimize MCA of the selected MCE. Protease inhibitors were used to identify the type of MCE. The casein hydrolysis was analyzed through reversed-phase HPLC (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE). Results The Bacillus licheniformis BL312 was identified from 50 bacterial strains. BL312 MCE achieved a maximal MCA (460 ± 15 SU/mL) at 48 h that was 2.7-fold higher than the control, and the MCA/PA ratio (9.0) and pH (6.6) remained stable throughout the fermentation process. Medium containing 30 g/L wheat bran shorts, 5 g/L glucose, and 3 g/L corn steep liquor was sufficient for optimal BL312 MCE production. Fermentation conditions of an inoculum size of 7.0% (v/v), fermentation temperature of 37 °C, agitation speed of 210 rpm, and initial pH 6.6 were required to achieve maximal MCA. BL312 MCE was inhibited by phenylmethanesulfonyl fluoride (PMSF) and high concentrations of ethylenediaminetetraacetic acid (EDTA) (5– 25 mM). The α -casein (α -CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE and calf rennet were different. s s Conclusions BL312 MCE is a serine/alkaline protease that exhibits high MCA and various hydrolysis for caseins in comparison with calf rennet. . . . . . Keywords Isolation Fermentation Bacillus licheniformis Milk-clotting enzyme Serine/alkaline protease Casein hydrolysis Introduction which influences the characteristics of the final product. The MCA/PA ratio is an inherent characteristic of MCE. The ideal Milk coagulation is an important step in cheese production, and MCE is characterized with a high MCA and a low non-specific MCEs play a major role in this process. MCEs facilitate milk PA (Meng et al. 2018). In fact, the high MCA/PA ratio leads to clotting and may influence the flavor and texture of the cheese. better textural and sensorial properties of cheese and a great Calf rennet, the most widely used milk coagulant, is obtained reduction of bitterness. Consequently, the high MCA/PA ratio from the fourth stomach of suckling calves (Liburdi et al. has shown to be crucial in evaluating the applicability of MCEs 2018). However, the use of calf rennet substitutes is becoming (Ben et al. 2017). Plant-derived coagulants can be produced necessary due to the increase in cheese consumption. The se- from seeds, vacuoles, and extracellular spaces (Gagaoua et al. lection of a suitable calf rennet substitute is a critical factor, 2017; Luo et al. 2018), and can be used as calf rennet substi- tutes. However, most plant-derived coagulants, which typically exhibit low MCA/PA ratios, achieve poor cheese yield and * Lianzhong Ai form bitter substances during cheese ripening (Salehi et al. ailianzhong@hotmail.com 2017). In contrast, recombinant chymosins exhibit high MCAs and good thermal stability. However, they are banned Shanghai Engineering Research Center of Food Microbiology, in many countries (Vallejo et al. 2012). School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, 516 Jungong Road, Recently, microbial MCEs have attracted great interest as Shanghai 200093, China promising calf rennet substitutes. Fungal MCEs, especially Beijing Sanyuan Foods Co., Ltd, Beijing 100163, China those from Rhizomucor miehei, R. pusillus,and Endothia 1290 Ann Microbiol (2019) 69:1289–1300 parasitica, are already widely used in commercial cheese pro- (50 mL) at 4 °C. To prepare soil suspensions, 1 g of soil duction (Celebi et al. 2016). Compared with fungal MCEs, sample was suspended in 9 mL of normal saline. Tenfold bacterial MCEs are cheaper to produce, more biochemically serial dilutions of the initial suspension were prepared with −4 diverse, and more easily modified genetically (Hang et al. normal saline until a final dilution of 10 -fold was achieved. 2016). Bacterial MCEs have been reported to exhibit high A120-μL aliquot of each diluted sample was cultured at 37 °C MCA/PA ratios (Luo et al. 2018;Li etal. 2019). Moreover, for 36 h in solid casein medium (pH 6.8) containing 2.5 g/L bacterial submerged fermentation is easier to control and casein peptone, 10 g/L glucose, 1 g/L yeast extract powder, shows a higher material utilization ratio compared with fungi. 25 g/L skim milk powder, and 20 g/L agar. Each strain was However, studies on bacterial MCEs have been less frequently numbered, and the diameters of hydrolysis and precipitation conducted compared with those on fungal MCEs. Few com- rings were examined every 12 h. mercial applications of bacterial MCEs as milk coagulants The isolated strains with high ratios of (i) precipitation ring have been reported. diameter to hydrolysis ring diameter and (ii) precipitation ring Shouguang (Shandong, China) is known as the hometown diameter to colony zone diameter were selected, and their 16S of Chinese vegetables, and its agricultural soil is rich in organ- rDNA were sequenced. For species-level identification, se- ic matter. Importantly, it houses a wide variety of microbial quences were compared with those in the GenBank database species (Zhou et al. 2011), many of which exhibit high prote- using the BLAST program (NCBI). For phylogenetic analy- ase activities. Microorganisms producing proteases (e.g., ser- sis, our data were aligned to a dataset containing 16S rDNA ine and threonine protein kinase) have previously been col- sequences using the BioEdit program. MEGA 7.0 software lected from the agricultural soil in Shouguang (Wang et al. was used to construct the phylogenetic tree (He et al. 2012). 1998; Qiu et al. 2016). In this study, BL312 (a Bacillus The harmful bacteria (e.g., Serratia marcescens, licheniformis strain) was isolated and found to exhibit a high Acinetobacter,and Pneumococcus) were excluded and others and stable MCA/PA ratio during fermentation. Various condi- (e.g., Bacillus licheniformis and Bacillus subtilis)weremain- tions and nutritional parameters were systematically examined tained at − 80 °C in 20% (v/v) glycerin. MCA, PA, and fer- to optimize the condition for MCE production and MCA. mentation pH were measured in subsequent fermentation ex- Large-scale fermentation (using a fermenter) was then used periments using the selected microorganism. to investigate the economic feasibility of BL312 MCE pro- duction. The effects of different protease inhibitors on the MCA of BL312 MCE were examined. The hydrolysis behav- Measurement of MCA and PA ior of BL312 MCE for caseins was analyzed. The fermentation broth was centrifuged at 5000×g for 10 min. The crude MCE was in the supernatant and used for MCA and Materials and methods PA determinations. MCA was determined according to the method of Arima et al. (1968). Skim milk 10% (w/v in deion- Chemicals and reagents ized water) containing 10 mM CaCl was used as the sub- strate. Curd formation was observed by manually rotating Skim milk powder was purchased from Fabrique par Fonterra the test tube from time to time so as to form a thin film on Co., Ltd. (Auckland, New Zealand). Calf rennet was provided its inner surface. The MCA is expressed in Soxhlet unit (SU), from Yuexiang Chemical Co., Ltd. (Shandong, China). PMSF, which is defined as the amount of MCE required to clot 1 mL EDTA, pepstatin A, and DL-dithiothreitol (DTT) were obtain- of milk substrate at 35 °C. The MCA was calculated using the ed from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). following formula: SU = (2400 × V × n)/(t × V ), where V is 1 2 1 The other chemicals used in the study were analytical grade the milk volume (mL), n is the dilution of MCE, t is the milk- from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, clotting time (s), and V is the MCE volume (mL). China). The proteolytic activity was measured following the meth- od of Arima et al. (1968). In brief, 5 mL of 1.2% (w/v) casein Isolation and identification of MCE-producing solution in 0.05 M phosphate buffer (pH 6.5) was added 1 mL bacteria crude MCE. The mixture was then incubated at 35 °C for 10 min. After incubation, 2 mL (0.44 M) of trichloroacetic Soil samples were collected from 10 to 15 cm-deep pits in acid (TCA) was added to quench the reaction. The mixture greenhouses and different agricultural regions in Shouguang was followed by centrifugation (6000×g) for 10 min at 4 °C. (36° 88′ N, 118° 73′ E). The samples were placed into sterile Two milliliters of the clear supernatant was added with 5 mL zipper polythene bags and stored at 4 °C until use. Soil sam- NaOH (0.28 M) solution and 1 mL Folin-Ciocalteu phenol ples taken to the laboratory were passed through a sieve (3– reagent. After incubation at 35 °C for 15 min, the mixture 4 mm mesh) and then were stored in sterile centrifuge tubes was measured for optical density (OD) at 660 nm. One unit 1291 Ann Microbiol (2019) 69:1289–1300 (1 U) of MCE activity is defined as the amount of MCE that The effects of bioprocess parameters on BL312 MCE liberated 1 μgof tyrosine per 1mL in 1 min. production In order to determine bioprocess parameters on MCE produc- Microbial growth and MCE production tion, inoculum size (1, 3, 5, 7, and 9%, v/v), fermentation medium volume (20, 30, 40, 50, and 60 mL), cultivation tem- The strain maintained in laboratory at −80 °C was grown on perature (27, 32, 37, and 42 °C), and agitation speed (120, improved TYC medium, which contained 15 g/L casein pep- 150, 180, 210, and 230 rpm) were studied in 250 mL tone, 5 g/L yeast extract powder, 50 g/L glucose, 0.2 g/L L- Erlenmeyer flasks. The initial pH of fermentation medium cysteine, 1 g/L NaCl, 2 g/L Na HPO ·12H O,and2g/L 2 4 2 was adjustedto4.6,5.6,6.6,7.6,and 8.6 todetermine the NaHCO and was sterilized at 115 °C for 20 min before use. optimal initial pH for MCE production. The one single colony was inoculated on 50 mL of improved TYC medium in a flask of 250 mL and incubated at 37 °C for Time course of BL312 MCE production in a fermenter 16–18 h under shaking (150 rpm) as seed liquid. MCE production was carried out by fermentation with a The MCE production was carried out by fermentation with 5.0% (v/v) inoculum size in three flasks of 250 mL × 20 g/L of optimal parameters in a 7-L fermenter (LiFlus GM, Biotron, wheat bran shorts, which were autoclaved at 115 °C for Korea) containing 4 L fermentation medium, which was 20 min. Wheat bran shorts contained 39.28% (w/w) carbon, autoclaved at 115 °C for 20 min. The initial concentration of 3.12% (w/w) nitrogen, and 6.51% (w/w) moisture. The fer- dissolved oxygen (DO) was regarded as 100% of air mentation conditions were as follows: temperature 37 °C, ro- saturation. tating speed 150 rpm, initial pH 6.6, and liquid volume 50 mL. Determinations on MCA, PA, and pH of fermentation broth The effects of protease inhibitors on BL312 MCE were carried out in a time range of 36–108 h at a 12-h interval. At timed intervals, samples were collected and centrifuged at The supernatant of fermentation broth was brought to ammo- 5000×g for 10 min at 4 °C. The supernatant was used to nium sulfate (60% saturation) at 4 °C for 2 h and harvested by measure MCA, PA, and pH of fermentation broth. centrifugation at 7000×g and 4 °C for 10 min. The precipitate Fermentation conditions were varied by one factor at a time. was dissolved in Tris-HCl buffer (pH 7.0) and centrifuged at 7000×g and 4 °C for 10 min. The crude MCE in the clear The effects of wheat bran concentrations on BL312 supernatant was dialyzed in dialysis bags (8–14 kDa) and MCE production lyophilized to powder for further experiments. The effects of various protease inhibitors including pepstatin A (0.01 and Different concentrations of wheat bran shorts including 20, 0.02 mM), EDTA (1, 5, 10, and 25 mM), PMSF (1 and 30, 40, 50, and 60 g/L were used to determine the optimal concentration for MCE production. MCA was measured in a Table 1 The diameter of hydrolysis and precipitation rings on the solid time range of 36–96 h at a 12-h interval. casein medium CN CD HD PD P/ P/ The effects of carbon and nitrogen sources on BL312 C H MCE production DB215 4.0 ± 1.0 7.0 ± 1.0 11.5 ± 1.5 2.9 1.6 DB218 2.0 ± 0.5 3.0 ± 0.5 10.5 ± 1.5 5.3 3.5 To investigate the optimal carbon source for MCE production, BL312 3.0 ± 0.5 3.5 ± 0.5 18.0 ± 2.0 6.0 5.1 different carbon sources (glucose, sucrose, soluble starch, BL211 2.0 ± 0.5 5.0 ± 1.0 11.0 ± 2.0 5.5 2.2 maltodextrin, and lactose) at 5 g/L were individually added QD111 1.5 ± 0.5 5.5 ± 1.0 11.0 ± 1.5 7.3 2.0 to the basal wheat bran shorts medium. The MCA in the basal QD212 3.0 ± 0.5 4.0 ± 1.0 16.5 ± 1.5 5.5 4.1 wheat bran shorts medium (without other carbon sources) was YN111 4.0 ± 1.0 5.0 ± 1.0 17.0 ± 2.0 4.3 3.4 the control and regarded as 100%. Different organic and inor- YN213 4.0 ± 1.0 6.0 ± 1.0 14.0 ± 2.0 3.3 2.2 ganic nitrogen sources (corn steep liquor, casein peptone, NX112 4.0 ± 0.5 8.0 ± 1.0 13.0 ± 1.0 3.3 1.6 urea, yeast extract powder, ammonium sulfate, and ammoni- um citrate) at 3 g/L were used to determine the optimal nitro- GS113 2.0 ± 0.5 5.0 ± 1.0 10.0 ± 1.5 5.0 2.0 GL111 4.0 ± 1.0 5.0 ± 1.0 9.0 ± 1.0 2.3 1.8 gen source. The MCA of BL312 MCE in wheat bran shorts medium containing 5 g/L glucose (without nitrogen sources) The data are represented as mean ± SD (n = 3). CN, strain number; CD, was the control and taken as 100%. The MCA and MCA of colony zone diameter (mm); HD, hydrolysis ring diameter (mm); PD, following fermentation experiments were measured in a time precipitation ring diameter (mm); P/C, the ratio of PD to CD; P/H, the ratio of PD to HD. The culture time of all strains was 24 h range of 36–84 h at a 12-h interval, unless specified otherwise. 1292 Ann Microbiol (2019) 69:1289–1300 Fig. 1 Phylogenetic position of the isolated BL312 based on 16S rDNA gene sequence analysis. GenBank accession numbers are given in parentheses 2 mM), and DL-dithiothreitol (DTT) (1 mM) were examined Statistical analysis on MCA of the crude MCE according to the method of Salehi et al. (2017) with some modifications. After addition of inhib- Each experiment was performed in triplicate. The results itors, the mixtures were incubated at 25 °C for 30 min. The were expressed as means ± standard deviations. Data ob- samples were evaluated for MCA as mentioned formerly. The tained were analyzed by one-way ANOVA using the soft- control (without inhibitors) was regarded as 100%. ware SPSS 17.0. The level of statistical significance was set at P <0.05. Hydrolysis of caseins by BL312 MCE Results and discussion The α -CN and β-CN hydrolysates generated by BL312 MCE and calf rennet were analyzed by RP-HPLC and SDS-PAGE Separation and identification of Bacillus licheniformis with 12% (w/v) acrylamide according to the method of BL312 Merheb-Dini et al. (2010)and Wasko et al. (2012), respective- ly. The α -CN or β-CN (0.2%, w/v) was mixed with MCEs s Initially, 11 isolated strains were found to form large precipi- (100 SU/mL) in a proportion of α -CN or β-CN:MCEs = s tation rings in the solid casein medium, which indicated high 15:1. For SDS-PAGE analysis, the proportions of α -CN or s MCA (Table 1). Given that a high PA (as indicated by large β-CN:BL312 MCE (10:1, 20:1, and 30:1) were also detected. hydrolysis rings in the solid casein medium) can lead to the The mixtures were incubated at 35 °C for 30 min for reaction. formation of bitter substances in cheese (Merheb-Dini et al. Reactions were quenched by heating the solutions in boiling 2010), strains with a high ratio of precipitation ring to hydro- water for 10 min. lysis ring and precipitation ring to colony zone were selected Fig. 2 The BL312 MCE production (a) and pH of fermentation broth (b) during different fermentation times (36–108 h). Data were presented as means of triplicate measurements; error bars were standard deviations 1293 Ann Microbiol (2019) 69:1289–1300 Table 2 Comparison of PA and Strain Substrate PA MCA/PA ratio References MCA/PA ratio among different derived MCEs B. licheniformis USC13 Casein 43 U/mL 6.7 Ageitos et al. (2007) B. subtilis B1 Casein 174 U/mL 6.5 Ding et al. (2011) B. amyloliquefaciens JNU002 Casein 1109 U/mL 5.9 Ding et al. (2012) A. niger FFB1 Casein 3020 U/mg 0.6 Fazouane et al. (2010) R. miehei (commercial MCE) Casein – 6.6 Thakur et al. (1990) R. miehei (Rennilase L) Casein – 3.9 Thakur et al. (1990) R. pusillus (Noury) Casein – 4.5 Thakur et al. (1990) C. parasitica (Sure curd) Casein – 1.6 Thakur et al. (1990) Calf rennet Casein – 12.8 Thakur et al. (1990) M. mucedo DSM 809 Azocasein 7.9 U/mL 16.5 Yegin et al. (2012) A. rouxii Azocasein 0.7 U/mL – Marcial et al. (2011) Nocardiopsis sp. Azocasein 1.6 U/mL 2.9 Cavalcanti et al. (2005) R. miehei Azocasein 11.2 U/mL 23.9 Yegin et al. (2012) R. miehei (Sigma) Hemoglobin – 2.0 Preetha et al. (1997) A. oryzae MTCC 5341 Hemoglobin 172 U/mg – Vishwanatha et al. (2010) and further examined using 16S rDNA sequencing and fer- good characteristic of BL312 MCE (Ben et al. 2017). In addi- mentation experiments. Accordingly, BL312 was selected tion, for some organisms, the MCA/PA ratio fluctuates during from the 11 initial isolates for MCE production. fermentation (Hashem 1999). However, the MCA/PA ratio of The 16S rDNA sequences of BL312 were compared to all BL312 MCE was found to be stable, indicating that BL312 sequences in the GenBank for species identification. Our ob- MCE production and activity can be more easily controlled tained nucleotide sequence showed the best match (99% ho- during fermentation compared with other MCEs. mology) to those of Bacillus licheniformis strain DSM 13 (NR118996.1), Bacillus licheniformis strain BCRC 11702 (NR116023.1), and Bacillus licheniformis strain NBRC The effects of different carbon and nitrogen sources 12200 (NR113588.1). A phylogenetic tree was constructed on BL312 MCE production using the neighbor-joining method (Fig. 1), and BL312 was identified as Bacillus licheniformis using phylogenetic analy- Wheat bran shorts contains several sources of carbon and sis. Bacillus licheniformis BL312 was deposited in the China nitrogen. It is a low-cost raw material that supports the growth of many microorganisms. Importantly, wheat bran is an General Microbiological Culture Collection Center (CGMCC No. 15009). The MCA and PA of BL312 fermentation are shown in Fig. 2a. BL312 MCE achieved the maximal MCA (169 ± 6 SU/mL)andPA(19±2 U/mL)at84h.The MCA/PA ratio was found to be 9.0 throughout the fermentation. As indicated in Fig. 2b, the pH of the fermentation broth was 6.6 and remained unchanged throughout. These results suggested that Bacillus licheniformis BL312 did not produce acid during fer- mentation, indicating that curd formation was caused by MCE. It is likely that BL312 is applicable for MCE production. Table 2 shows that the MCA/PA ratio of BL312 MCE was generally similar to that of commercial MCEs (e.g., calf rennet and R. miehei MCE). However, BL312 MCE exhibited a higher MCA/PA ratio in comparison with B. amyloliquefaciens JNU002, A. niger FFB1, and C. parasitica MCE. The MCA/ Fig. 3 The effects of different concentrations of wheat bran shorts on PA ratio plays an important role in the selection of MCEs. The BL312 MCE production during fermentation (36–96 h). Data were MCEs with high MCA/PA ratio achieve great cheese yield and presented as means of triplicate measurements; error bars were standard decreased bitter substances in cheese production, indicating a deviations 1294 Ann Microbiol (2019) 69:1289–1300 effective nutrient source for MCE production during fermen- starch, maltodextrin, and sucrose (Fig. 4a, b). Given our re- tation (Chwen-Jen et al. 2009). Our results indicated that sults, 5 g/L glucose was used to supplement the basal fermen- MCA of BL312 MCE increased with increasing concen- tation medium in subsequent experiments. Next, the effects trations of wheat bran shorts (up to 30 g/L), but de- of four organic nitrogen sources (corn steep liquor, casein creased at higher concentrations (30–60 g/L) (Fig. 3). peptone, urea, and yeast extract powder) and two inorgan- The maximal MCA (260 ± 9 SU/mL) was achieved after ic sources (ammonium sulfate and ammonium citrate) on 48 h of fermentation using 30 g/L wheat bran shorts. MCE production were examined at different fermentation Accordingly, 30 g/L of triturated wheat bran shorts was time points (Fig. 4c, d). Our data indicated that BL312 used as the basal fermentation medium in the following MCE achieved the maximal MCA in the presence of 3 g/ optimization experiments. L corn steep liquor (302 ± 10 SU/mL). Compared with the BL312 was cultured in conditions supplemented with dif- control, MCA increased by 10% and 5% in the presence ferent carbon sources, and MCE production was measured at of corn steep liquor and urea, respectively. However, different time points to select the most suitable source of car- MCA was reduced in the presence of casein peptone, bon supplementation (Fig. 4a, b). The carbon sources included yeast extract powder, ammonium sulfate, and ammonium glucose, sucrose, soluble starch, maltodextrin, and lactose. citrate (Fig. 4c, d). Our results indicated that BL312 MCE achieved the highest Taken together, these results indicated that glucose (5 g/L) MCA in the presence of glucose (275 ± 9 SU/mL, 6% higher and corn steep liquor (3 g/L) supplementation in wheat bran than the control). While the MCA was not altered in the pres- shorts fermentation medium produced the best culture condi- ence of lactose, it was reduced in the presence of soluble tion for BL312 MCE production. MCE synthesis can be Fig. 4 a, b The effects of different carbon sources on BL312 MCE YP, yeast extract powder; AM, ammonium sulfate; and AC, ammonium a–g production during fermentation (36–84 h). Glu, glucose; Suc, sucrose; citrate. Values in the same row with different superscripts are SS, soluble starch; MD, maltodextrin; and Lac, lactose. c, d The effects significantly different (P<0.05).Datawerepresented as meansof of different nitrogen sources on BL312 MCE production during triplicate measurements; error bars were standard deviations fermentation (36–84 h). CSL, corn steep liquor; PC, casein peptone; 1295 Ann Microbiol (2019) 69:1289–1300 induced or suppressed by various materials and nutrient The effects of bioprocess parameters on BL312 MCE sources (Shata 2005). As BL312 growth and MCE production production could be achieved using the low-cost wheat bran shorts with modest nutrient supplementation (Fig. 4), our results sug- As presented in Fig. 5, the bioprocess parameters were further gested that large-scale production of BL312 MCE would be optimized to achieve a maximal MCA (442 ± 12 SU/mL) in economically feasible. Over-supplementation of carbon and BL312 MCE. Our results indicated that the optimal inoculum nitrogen sources inhibits the growth of microorganisms and size was 7% (v/v) and medium volume for BL312 MCE pro- MCE production (Sen et al. 2009). According to our results, duction was 40 mL (Fig. 5a, b). The maximal MCA was ob- organic nitrogen sources (corn steep liquor and urea) produced served at a fermentation temperature of 37 °C and an agitation better BL312 MCE yield than inorganic nitrogen sources (am- speed of 210 rpm (Fig. 5c, d). Deviations from the optimal monium sulfate and ammonium citrate) (Fig. 4c, d). It could inoculum size and fermentation temperature both decreased be possible that organic nitrogen contains kinds of amino MCE production. acids, which can be absorbed directly by BL312. In contrast, It is known that both the inoculum size and fermentation BL312 first synthesized inorganic nitrogen into amino acids, temperature profoundly affect the production of microbial reducing the growth of microorganisms. The MCE production MCEs. The biomass production was determined by inoculum is thus limited by the reduced biomass (Cai et al. 2004). These size during fermentation (Sen et al. 2009). When the inoculum results were different from previous reports. Glucose and or- size was increased to 9% (v/v), MCA decreased significantly. ganic nitrogen sources are not considered good nutrient It could be faster growth of BL312 and larger biomass, which sources for MCE production in Penicillium oxalicum and caused the shortage in nutrients. To achieve the maximal MCE Nocardiopsis sp. (Hashem 1999; Cavalcanti et al. 2005). production, it is necessary to maintain the balance of biomass However, glucose is considered a suitable carbon source for production and nutrient availability (Patel et al. 2005). The Mucor miehei (Sun et al. 2014). optimal temperature for MCE production in BL312 (37 °C) Fig. 5 The effects of inoculum size (a), volume (b), cultivation composition was wheat bran shorts (30 g/L), glucose (5 g/L), and corn temperature (c), and agitation speed (d) on BL312 MCE production steep liquor (3 g/L). Data were presented as means of triplicate measure- during different fermentation times (36–84 h). Dissolved oxygen (DO) ments; error bars were standard deviations and medium volume were the negative correlation. The medium 1296 Ann Microbiol (2019) 69:1289–1300 F34 (Wu et al. 2008), Fusarium subglutinans (Ghareib et al. 2001), and Amylomyces rouxii (Yu and Chou 2005), respectively. BL312 MCE production using a 7-L fermenter A large-scale production of BL312 MCE was next conducted in a 7-L fermenter using the optimized condition (Fig. 7). The BL312 MCE production was initiated using 4 L of the optimal fermentation medium (wheat bran shorts with glucose and corn steep liquor supplementation). The fermentation condi- tion was as follows: inoculum size 7.0% (v/v), temperature 37 °C, agitation speed 210 rpm, and ventilation 1.7 vvm. Compared with using shake flasks, BL312 MCE achieved a Fig. 6 The effects of initial pH of fermentation medium on BL312 MCE higher maximal MCA (460 ± 15 SU/mL) in the fermenter. production during different fermentation times (36–84 h). The medium Throughout the fermentation process, the MCA/PA ratio and composition was wheat bran shorts (30 g/L), glucose (5 g/L), and corn pH remained at 9.0 and 6.6, respectively. During the growth steep liquor (3 g/L). Data were presented as means of triplicate measurements; error bars were standard deviations phase of BL312, DO concentration drastically decreased by 78% compared to the initial concentration, but increased after was different from that for Paenibacillus sp. BD3526 (30 °C) 36 h of fermentation. The maximal MCA of BL312 MCE (Hang et al. 2016)and Bacillus methanolicus LB-1 (30 °C) (Li observed here was higher than that reported for et al. 2019). The agitation speed and liquid volume also played B. licheniformis USC13 (290 SU/mL) (Ageitos et al. 2007), an important part in BL312 MCE production as they affected B. subtilis YB-3 (200 SU/mL) (Li et al. 2012), and M. mucedo the amount of DO during fermentation. DSM 809 (130 SU/mL) (Yegin et al. 2012). The BL312 MCE exhibited lower PA compared with B. subtilis B1 and B. amyloliquefaciens JNU002 MCE (Table 2). Besides, the The effects of initial pH on BL312 MCE production stable MCA/PA ratio and pH suggested that the submerged fermentation of BL312 was easy to control, implying that it is BL312 MCE production reached the maximum when initial valuable in potential application. pH of the fermentation medium was adjusted to 6.6 (Fig. 6). Deviations from this initial pH decreased MCA. The initial pH is an important factor for many enzymatic and membrane The effects of protease inhibitors on BL312 MCE transport processes (Moon and Parulekar 2010). The optimal initial pH for MCE production is dependent on the fermenta- The MCA values of crude BL312 MCE treated with different tion medium and the species of microorganism. The maximal protease inhibitors are shown in Table 3. Our results indicated MCA was achieved at the original medium pH (6.6). It was in that BL312 MCE was not affected by pepstatin A (0.01– favor of the production and industrialization of BL312 MCE. 0.02 mM), DTT (1 mM), and low concentrations of EDTA The optimal initial pH values for the maximal MCA were (1 mM). However, it was significantly inhibited by PMSF (1 reported to be 4.5, 6.0, and 7.0 for Chinese distiller’syeast and 2 mM) and high concentrations of EDTA (5–25 mM) Fig. 7 Changes in the MCA, PA, DO, and pH in a 7-L fermenter with respect to fermentation times. Data were presented as means of triplicate measurements; error bars were standard deviations 1297 Ann Microbiol (2019) 69:1289–1300 Table 3 The effects of different Inhibitor Concentration (mM) Relative MCA (%) Inhibition rate (%) protease inhibitors on BL312 MCE None – 100 0 Pepstatin A 0.01 95.7 ± 1.5 4.3 0.02 96.9 ± 1.0 3.1 EDTA 1 97.1 ± 2.1 2.9 5 83.0 ± 2.3 17.0 10 70.6 ± 3.2 29.4 25 59.6 ± 2.1 40.4 PMSF 1 9.7 ± 1.7 90.3 2 7.1 ± 2.2 92.9 DTT 1 98.6 ± 1.4 1.4 The data are represented as mean ± SD (n =3) (Table 3). Protease inhibitors can be used to reveal the type addition, the BL312 MCE identified in this study is different and active site of enzymes. It is known that PMSF, pepstatin from the MCEs of Enterococcus faecalis TUA2495L (Sato A, and DTT were serine, aspartate, and cysteine protease in- et al. 2007), B. subtilis YB-3 (Li et al. 2012), and hibitor, respectively (Uda et al. 2017). The alkaline proteases Paenibacillus sp. BD3526 (Hang et al. 2016). were inhibited by high concentrations of EDTA (Tang et al. 2004). Our results showed that BL312 MCE exhibited the Hydrolysis behavior of BL312 MCE properties of serine and alkaline protease, indicating that it was a serine/alkaline protease. Unlike BL312 MCE, the The RP-HPLC chromatograms of peptides were shown in MCA of Bacillus licheniformis 5A5 MCE is enhanced in the Fig. 8. The BL312 MCE was more hydrolytic on α -CN and presence of PMSF and EDTA (Ahmed and Helmy 2012). In β-CN than calf rennet. As shown in Fig. 8c–f,the α -CN and Fig. 8 RP-HPLC chromatograms of α -CN (a)and β-CN (b); RP- HPLC chromatograms of α -CN (c)and β-CN (d)hydrolysates generated by calf rennet; RP- HPLC chromatograms of α -CN (e)and β-CN (f) hydrolysates generated by BL312 MCE 1298 Ann Microbiol (2019) 69:1289–1300 Fig. 9 SDS-PAGE patterns of α -CN (a) and β-CN (b) hydrolysates 2–5: BL312 MCE. The α -CN:MCEs or β-CN:MCEs ratios were 15:1 in s s generated by BL312 MCE and calf rennet. Lane M: molecular weight lanes 1 and 3, 10:1 in lane 2, 20:1 in lane 4, and 30:1 in lane 5 marker proteins; lane A: α -CN; lane B: β-CN; lane 1: calf rennet; lanes β-CN hydrolysates generated by BL312 MCE and calf rennet hydrolyze α -CN, indicating it was potential to be used in mak- were significantly different at retention times of 10–50 min. ing soft or semi-hard cheese. However, the hydrolysates were generally similar at retention times of 50–80 min, where few peaks could be observed in chromatograms. The β-CN hydrolysates at retention times of Conclusions 60–80 min represent the bitter peptides, which are important for the flavor during cheese ripening (Lee and Warthesen In this study, a Bacillus licheniformis BL312 strain was iden- 2010). Although lack of the sensory analysis, the likely pep- tified and exhibited high MCA. A remarkable MCA (460 ± 15 tide compositions were indicated through the comparison of SU/mL) was achieved after 48 h of fermentation in culture two chromatograms of hydrolysates generated by BL312 medium containing low-cost wheat bran shorts (30 g/L), glu- MCE and calf rennet (Fig. 8d, f). It is concluded that the β- cose (5 g/L), and corn steep liquor (3 g/L). An initial pH of CN hydrolysates generated by BL312 MCE were few bitter 6.6, fermentation temperature of 37 °C, agitation speed of peptides. 210 rpm, and ventilation of 1.7 vvm were required for the For SDS-PAGE analysis, Fig. 9 shows that the hydrolytic optimal MCA. The BL312 MCE exhibited a stable MCA/ abilities of BL312 MCE were getting weaker with the increase PA ratio of 9.0 and pH of 6.6 throughout the fermentation of α -CN:MCE or β-CN:MCE ratios. The α -CN was mainly s s process, suggesting that it is suitable for prolonged fermenta- hydrolyzed into apparent fragments (12–18 kDa) by BL312 tion. The MCA of BL312 MCE was significantly inhibited by MCE, which were much smaller than those by calf rennet PMSF and high concentrations of EDTA, implying that it was (Fig. 9a). As presentedinFig. 9b,the β-CN hydrolysates gen- a serine/alkaline protease. Importantly, our results revealed erated by BL312 MCE were about molecular masses of 12– that BL312 MCE was characterized with various hydrolysis 14 kDa and 17–18 kDa. Different casein hydrolysates generat- for caseins. Further characterization and application of BL312 ed by BL312 MCE and calf rennet indicated different cleavage MCE should be conducted in the future. sites in caseins and might lead to various cheese flavors (Majumder et al. 2015). In addition, the BL312 MCE hydro- Acknowledgments The authors gratefully acknowledge the National Key R&D Program of China (grant no. 2018YFD0502306) and lyzed α -CN to a greater level than β-CN, which was in line Shanghai Engineering Research Center of Food Microbiology (grant with RP-HPLC analysis result. It is known that the hydropho- no. 19DZ2281100) for the granted fellowships. bic peptides generated by β-CN hydrolysis were responsible for the bitterness in cheeses (An et al. 2014). Weak hydrolysis Funding information This work was supported by the National Key level of β-CN for BL312 MCE led to a decreased amount of R&D Program of China (grant no. 2018YFD0502306) and Shanghai Engineering Research Center of Food Microbiology (grant no. bitter peptides in cheese production. Besides, various MCEs 19DZ2281100). exhibited different preferences for α -CN and β-CN hydroly- sis. As reported with researchers, the hydrolysis of β-CN was Compliance with ethical standards stronger than that of α -CN for Paenibacillus spp. BD3526 MCE (Hang et al. 2016). Importantly, the meltability and hard- Conflict of interest The authors declare that they have no conflict of ness of cheese were closely related to the α -CN and β-CN interest. hydrolysis. The cheese meltability increased with α -CN deg- radation. In contrast, the cheese was hard at a high β-CN hy- Informed consent This manuscript is approved by all authors for drolysis level (Hayaloglu et al. 2014). BL312 MCE preferred to publication. 1299 Ann Microbiol (2019) 69:1289–1300 Lee KD, Warthesen JJ (2010) Mobile phases in reverse-phase HPLC for References the determination of bitter peptides in cheese. 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Annals of Microbiology – Springer Journals

Published: Sep 10, 2019

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