Transfructosylating enzyme production from molasses
Molasses is a nutrient-rich sugar source. In this study, molasses was used to produce transfructosylating enzymes by A. pullulans FRR 5284 with addition of only two exogenous nitrogen and phosphorous sources (NaNO3 and Na2HPO4). Figure 1A shows cell production and total sugar consumption with molasses medium and synthetic molasses medium. A. pullulans FRR 5284 grew faster in molasses medium and sugar consumption rate was also higher than that in synthetic molasses medium. Furthermore, Fig. 1B shows the concentration changes of sucrose, glucose and fructose in cultivation. Overall, the consumption of these sugars in molasses medium was faster than that in synthetic molasses medium. Sucrose was almost depleted at 48 h in both media due to the conversion of sucrose to glucose and fructose as well as the synthesis of small amounts of FOS (data not shown). Glucose was consumed more rapidly by A. pullulans FRR 5284 than fructose, consistent with the preference for glucose observed for most microorganisms. The concentration of fructose increased in the middle of the cultivation because of the hydrolysis of sucrose and slow consumption by the strain.
Regarding intracellular transfructosylating enzyme production, as shown in Fig. 2A, the use of synthetic molasses medium led to higher Ut (U/mg cells) based on cell mass from 24 to 120 h with the Ut peaked at 72 h. Figure 2B shows intracellular, extracellular, and total Ut (U/mL) based on cultivation volume. Overall, the use of sugarcane molasses medium led to higher Ut (U/mL) than the use of synthetic medium due to the production of higher concentrations of cell mass using the sugarcane molasses medium. The highest total Ut (123.6 U/mL) was achieved at 72 h, corresponding to the highest intracellular and extracellular Ut of 68.7 U/mL and 54.9 U/mL, respectively. Higher intracellular Ut is preferred as intracellular enzymes can be harvested readily by filtration or centrifugation. The collected cells may be used directly for FOS production. Surprisingly, Uh was not detected in either cells or fermentation broth when sugarcane molasses or synthetic molasses medium was used.
Faster cell growth in sugarcane molasses-based medium than that in synthetic molasses medium was attributed to the abundance and diversity of nutrients in sugarcane molasses (Dorta et al. 2006). The rapid reduction of intracellular Ut (U/mg cells) after 72 h cultivation was likely due to the reduced transfructosylating enzyme production because of the depletion of sucrose (Fig. 1B), which is an inducer for producing inducible transfructosylating enzymes. The activity (U/g cells) reduction may also be attributed to the dilution effect due to the increased cell concentration. The slow reduction of total Ut (U/mL) based on medium volume after 72 h cultivation was mainly due to the increased cell concentrations offsetting the rapid decrease of Ut (U/mg cells). It is worth mentioning that Uh was not detected with the use of molasses-based and synthetic molasses-based media, indicating a relatively low level of Uh. It is expected that the low Uh favors the production of FOS as hydrolysis of produced FOS will be slow.
A previous study showed that with the cultivation of A. pullulans DSM 2404 in a medium containing 50 g/L sucrose, the total Ut (U/flask) of one individual enzyme increased in the first 2 days and then decreased while the activities of all the other four individual enzymes continue to increase during the 3-day cultivation period (Yoshikawa et al. 2006). In another study with the cultivation of A. pullulans KCCM 12017 in a sucrose medium containing 100 g/L sucrose, both intracellular and extracellular total Ut (U/mL) increased in 3-day cultivation (Shin et al. 2004). In the present study, both intracellular and extracellular Ut (both U/mg cells and U/mL) increased first, and then decreased after 72 h cultivation. These different observations indicate that the Ut (U/volume) change during cultivation was affected by cell concentrations and Ut (U/mg cells). Moreover, since the transfructosylating enzymes were likely composed of mixed enzymes (Salinas and Perotti 2009; Shin et al. 2004; Vandáková et al. 2004; Xie et al. 2017; Yoshikawa et al. 2006; Zhang et al. 2016), measured Ut (U/mg cells) was likely the overall activity of total enzymes rather than individual enzymes.
Effect of sucrose/glucose mass ratio on enzyme production
Since transfructosylating enzymes produced by microorganisms likely included both constitutive and inducible enzymes and molasses contained mixture sugars, such as sucrose (an inducer for inducible transfructosylating enzymes) and mono-sugars (glucose and fructose), further study was carried out to understand the effect of different carbon source combinations on the production of transfructosylating enzymes.
Figure 3 shows the effect of the sucrose/glucose mass ratio in synthetic sugar media on Ut and Uh generated by A. pullulans FRR 5284. The highest Ut values (both U/mg cells and U/mL medium) were achieved at 72 h with the use of pure sucrose medium while the inclusion of glucose at the mass ratio from 100:0 to 50:50 in the medium reduced the maximum Ut. With the use of pure glucose medium, the Ut was very low in the beginning of the monitored cultivation period (from 24 to 120 h) and increased gradually with the prolonged cultivation time. The maximum Ut with pure glucose medium was achieved at the end of the cultivation (120 h), which was much lower than that with the media having sucrose/glucose mass ratios of 100:0 – 50:50.
The use of pure sucrose medium also led to the highest Uh values (both U/mg cells and U/mL medium) though the maximum values were observed at the beginning of the monitored cultivation period (from 24 to 120 h). Inclusion of glucose in the medium reduced Uh and the trend was similar to that observed for Ut. Differently from Ut, Uh continued to decrease from 24 to 120 h in the media containing sucrose/glucose mass ratios of 100:0 – 50:50. With the use of pure glucose medium, Uh was not detected in the monitored cultivation period (from 24 to 120 h). It was noted that Uh was not detected in the sucrose-rich molasses and synthetic molasses media though both media contained sucrose, indicating the presence of fructose may also affect the enzyme production.
The above results indicated that transfructosylating enzymes produced by A. pullulans FRR 5284 included both constitutive and inducible enzymes while constitutive enzymes were likely the dominant ones. In the presence of sucrose, both constitutive and inducible enzymes were expressed while in the glucose-based medium, only constitutive enzymes were produced with very low Ut (Fig. 3). The observations in the present study were in line with previous results in the literature which reported the production of constitutive enzymes in the presence of glucose (Nascimento et al. 2019; Yoshikawa et al. 2006, 2008).
FOS production from sugarcane molasses using A. pullulans cells
Following production of transfructosylating enzymes using sugarcane molasses medium, FOS were produced from sugarcane molasses by A. pullulans cells, which contained intracellular transfructosylating enzymes. Although FOS could be produced from high concentrations of sucrose solutions containing 800 g/L sucrose (Zhang et al. 2016), original sugarcane molasses (containing 41 wt% sucrose) was very viscous and had to be diluted to a solution containing 230 g/L sucrose. A. pullulans cells were directly used for FOS production as the cells were easy to collect and recycle. In addition, cells could be freeze-dried for long-term storage.
Figure 4A shows the kinetics of FOS production and the total FOS yields in the production process. For comparison, pure sucrose was included as a control carbon source. As shown in Fig. 4A, FOS production was very rapid and the total FOS yields reached 43–54% within 1 h. The use of molasses led to a lower FOS yield of 43%, which was mainly attributed to the inhibition from existing glucose (Khatun et al. 2020). With sucrose as the substrate, the total FOS yield reached a plateau (56–58%) after 3 h reaction while with molasses, the total FOS yield gradually increased to the highest value of ~ 59% at 9 h. The pure sucrose substrate also led to higher individual FOS concentrations than sugarcane molasses except for the higher GF3 concentration with molasses at 9 h and 12 h. In addition, GF2 was rapidly produced within 1 h and the concentrations of GF2 started to reduce after 1 h while the concentration of GF3 increased in the first 9 h and started to decrease after 9 h reaction. In contrast, the concentrations of GF3 increased steadily in the reaction time. The kinetics of FOS production was in line with previous studies (Sangeetha et al. 2004; Shin et al. 2004; Zhang et al. 2016, 2019). Along with FOS production, significant amounts of glucose and fructose were accumulated and FOS from molasses contained much higher levels of glucose and fructose (Fig. 4B).
FOS content improvement by microbial treatment
Prebiotics such as FOS need to resist the gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption before it can reach intestinal microflora (Gibson et al. 2004). Glucose is the major by-product in FOS solutions and the presence of glucose will reduce the FOS content and delivery efficiency of FOS. In order to improve FOS content in the produced FOS solutions, various approaches have been used to improve FOS content and purity following FOS production, which include expensive ultra-filtration and chromatographic separation technologies (Nobre et al. 2014, 2012; Pinelo et al. 2009). In addition, microbial treatment was also used to improve FOS content (Nobre et al. 2016; Yang et al. 2008). Microbial treatment could not lead to the production of high-purity FOS, but the resultant FOS mixtures may be qualified for feed application. S. cerevisiae (invertase-deficient yeast) is able to convert monosaccharides to ethanol, reducing the contents of digestible sugars and improving the purity of non-digestible FOS.
In the present study, FOS samples (3 and 9 h reaction times) produced from sugarcane molasses and pure sucrose were further treated with an invertase-deficient yeast. Following treatment, glucose in FOS solutions were completely removed and fructose was still present in some samples though the concentrations were reduced while sucrose concentrations remained unchanged (data not shown). Figure 5 shows the changes of FOS contents in total sugars after treatment. FOS contents were improved to 63.0–87.0% after treatment, ~ 20–30% higher than corresponding contents prior to treatment.
Table 1 shows the FOS profiles after treatment. Treated FOS samples did not contain glucose and had varying GF2/GF3/GF4 ratios. After treatment by the invertase-deficient S. cerevisiae yeast, ethanol and acetic acid were produced. Interestingly, lower concentrations of ethanol and acetic acid were produced from FOS solutions derived from molasses, which had higher glucose concentrations than the corresponding FOS solutions derived from pure sucrose. Under aerobic conditions, the metabolic activities of yeast in the FOS solution derived from molasses were likely very intensive, leading to the generation of large amounts of CO2 and yeast cell mass. In contrast, the FOS solutions derived from pure sucrose solution had little nutrients and the added yeast cells were more like enzyme carriers, which produced ethanol and acetic acid as the main products with the generation of fewer amounts of CO2 and yeast cell mass.
Previously, a two-step fermentation process by using S. cerevisiae and A. pullulans strains improved FOS purity from 51.7 to 81.6%. However, the process needs to be carefully controlled as the S. cerevisiae strain was not invertase-deficient and could consume FOS (Nobre et al. 2016, 2012). In another study, a sequential FOS production and purification was carried with the use of the Aspergillus japonicus strain (FOS producer) and Pichia pastoris (reducing sugar consumer). The Pichia pastoris strain converted glucose to glycerol and improved FOS content from 56.6 to 84.4% (Yang et al. 2008). The removal of glycerol from FOS solution still needs the use of expensive chromatographic technology if the FOS is produced for human consumption. In the present study, glucose was converted to ethanol and acetic acid by the invertase-deficient S. cerevisiae yeast. The ethanol and organic acid-containing FOS may be directly used as feed prebiotics and the presence of ethanol and acetic acid may help to prevent microbial contamination.
Prebiotic activities of treated FOS
For feed application, further removal of impurities in molasses FOS solutions after microbial treatment may not be necessary if the semi-purified FOS solutions have prebiotic activities since molasses is already a commercial feed supplement. Therefore, in vitro prebiotic activity test was carried out to check the prebiotic activities of the semi-purified FOS solutions. A total of nine probiotic strains, including eight Lactobacillus strains and one Bacillus strain were used for in vitro prebiotic activity test. In addition to the FOS samples produced in this study, a commercial FOS (Nutraflora P95) and several non-FOS sugars were also included for comparison. Table 2 shows the compositions of the FOS and non-FOS sugars used for prebiotic activity test at 10 g/L. These FOS and non-FOS sugars used for prebiotic assays had different sugar profiles.
Figure 6 shows the anaerobic growth (as indicated by OD600 value) of two representative probiotic strains in the presence of FOS and non-FOS sugars with the growth of all the nine strains after 12 h and 24 h incubation summarized in Additional file 1: Table S1. Furthermore, the growth improvements of probiotic strains with different carbon sources are summarized in Table 3 in comparison with the commercial FOS (Nutraflora P95). Overall, the use of treated 3-h molasses FOS led to the most significant growth improvements of all the probiotics, followed by the use of treated 9-h molasses FOS. The use of treated 3-h sucrose FOS led to slight growth improvements of most tested probiotics while the use of treated 9-h sucrose FOS resulted in the reduced growth of most tested probiotics compared to the use of Nutraflora P95. Regarding the non-FOS sugars, the use of sucrose resulted in the most significant reductions of most tested probiotics, followed by synthetic molasses and sugarcane molasses. Interestingly, the use of glucose also led to significant growth reductions of most tested probiotics. These results indicated that FOS rather than non-FOS sugars were preferred by most of the tested probiotics, which justified the need to improve FOS content.
In vitro assays are often carried out to assess the prebiotic activities of FOS and other oligosaccharides. In some studies, glucose was included as a reference sugar, which led to either superior, inferior, or comparable growth of tested probiotics to prebiotic oligosaccharides (Huebner et al. 2007; Nobre et al. 2019, 2018). In the present study, the use of glucose led to reduced growth of most tested probiotics. However, it should be noted that the comparison of the growth of probiotics with FOS and non-FOS sugars only indicates the carbon source preference by the probiotics. The better growth with non-FOS sugars, such as glucose does not mean that these non-FOS sugars are prebiotics. In the real animal digestion system, non-FOS sugars, especially glucose, can be readily absorbed and utilized before they reach the intestinal microflora/probiotics. Furthermore, in vivo prebiotic activity assay is also needed in future studies to verify the role and benefits of molasses FOS in improving animal health and promoting animal production.