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. 2021 Apr 11;11(5):213. doi: 10.1007/s13205-021-02757-0

Production and prebiotic properties of oligofructans from sugarcane juice fermentation by Bacillus subtilis TISTR 001

Boontiwa Ninchan 1,✉, Chanyanuch Noidee 1
PMCID: PMC8039079  PMID: 33928001

Abstract

Oligofructans are potential biological substances that due to their distinctive properties have a positive health-promoting effect. This research aimed to produce oligofructans using sugarcane juice fermentation with Bacillus subtilis TISTR 001 and to study the prebiotic properties in vitro. The results showed that the maximum total oligofructans in the form of free fructose was 2.57% (w/v) at the 84th hour of fermentation with 0.17 g/g reducing sugar and a production yield of 0.031 g/L/h and maximum levansucrase activity of 1.57 × 106 U/mL at that time. The oligofructans contained in the fermented juice had potential functional ingredients that exhibited prebiotic properties that could resist the digestion of enzymes in the gastrointestinal tract under in vitro conditions with digestion of only 6.92%. In addition, the fermented juice promoted the growth of prebiotics, especially Bifidobacterium bifidum TISTR 2129 and inhibited the growth of pathogens using both single culturing and co-culturing with probiotics.

Keywords: Sugarcane juice, Bacillus subtilis, Oligofructans, Prebiotic, Probiotic

Introduction

These days people are particularly interested in taking care of their health, especially when it comes to food. Beyond the considerations of price and taste, the nutritional value of the ingredients to be consumed has become an important factor in selecting and purchasing such products. The potential ingredients contained in the food products must have health benefits or contain nutritional value which can help to reduce the risk of contracting diseases and must promote the overall physical health of a person. Today, there is much research and development on a number of potential ingredients as biological substances to increase the options for consumers and as a response to consumer demand. Consequently, the goal of this research was to attempt the production of a biological substance in form of fructo-oligosaccharides and fructan types (called oligofructans) as potential functional ingredients due to their distinctive properties that promote positive health effects (Sanchez-Martinez et al. 2020; Schorsch et al. 2019; Witczak et al. 2020). Specifically, this research aimed to study the production of oligofructans by Bacillus subtilis fermentation using sugarcane juice as the substrate and to study their prebiotic properties.

B. subtilis is a microorganism which has been certified as safe (Generally Recognized as Safe: GRAS) and is categorized into a probiotic bacterium type that can produce important beneficial substances, such as antibiotics, antigen, enzymes, and various proteins. Furthermore, this microorganism can grow in many types of food, both under anaerobic or aerobic conditions (Olmos and Paniagua-Michel 2014). Past research has shown that B. subtilis can produce levansucrase, which is a fructosyltransferase type of enzyme that is considered the main enzyme used in the production of fructo-oligosaccharides and fructan (Mu et al. 2021; Raga-Carbajal et al. 2016; Santos et al. 2013; Shi et al. 2019; Tanaka et al. 2014). Fructosyltransferase is formed via an enzymatic transformation of fructose sugars connected by beta bonds (β-D-fructose), that enzymes in the primary digestive tract are unable to digest. Therefore, it becomes a food source or prebiotic for living microorganisms in the large intestine. Probiotics are microorganisms that provide health benefits to the body or the host (Jang et al. 2003; Niv et al. 2012; Shah et al. 2020). Prebiotics help to promote the growth of probiotics with the most common probiotic being lactic acid bacteria (LAB), such as Bifidobacterium sp., Lactobacillus sp., Streptococcus sp., Enterococcus sp., and Bacillus sp. (Chlebowska-Smigiel et al. 2017, 2019; Jager et al. 2020; Mu et al. 2021; Raveschot et al. 2020; Roobab et al. 2020). Additionally, they can inhibit the growth of pathogenic microorganisms, such as Clostridium sp., Escherichia coli, and Samonella sp. This is referred to as the bifidogenic effect or prebiotic effect (Gibson 1999; Meyer and Stasse-Wolthuis 2009; Veereman-Wauters et al. 2011). In addition, prebiotics can also aid in lowering the blood levels of triglycerides and cholesterol (hypocholesterolemic effect) (Erejuwa et al. 2014; Raveschot et al. 2020; Santos et al. 2013; Yamamoto et al. 1999) and are beneficial to the immunity system in the human body (immunostimulatory effect) (Niv et al. 2012; Peshev and Van den Ende 2014) in which the benefits are more visible in patients with non-communicable diseases (NCDs) (Rolim 2015). Polymer fructan is also found to have functions in foods by affecting the physical properties of the food products, such as increasing the stability in beverages, such as yogurt, and improving the sensory characteristic of the product (Chlebowska-Smigiel et al. 2017, 2019; Thammarutwasik et al. 2009; Ua-Arak et al. 2017). It has low viscosity, good solubility in water, and has desirable emulsifying properties (Kucukasik et al. 2011; Witczak et al. 2020). Therefore, fructo-oligosaccharides and polymer fructan or oligofructans are beneficial and appealing for use as food additives in functional foods or functional beverages.

Sugarcane juice is a beverage with sucrose as the main component. Sucrose is a double-molecule of sugar composed of single-molecule sugars (glucose and fructose). Sugar juice is suitable as a substrate for fermentation, while, B. subtilis is the producer of levansucrase which then efficiently transforms the sucrose into a chain of fructo-oligosaccharide and polymer fructan. The results of this study are expected to provide important data on oligofructans to aid in the further development production at pilot and then industrial scales and to provide useful information of benefit to human health in the future.

Materials and methods

Materials

The fresh sugarcane juice was purchase from a local market and had a dissolved solids concentration of 15.3°Brix. All chemicals were analytical grade. The standard sugars (sucrose, glucose and fructose) and standard short-chain fructo-oligosaccharides (kestose and nystose) were supplied by Sigma Aldrich (USA). The culture media were purchased from HiMedia Laboratories Pvt. Ltd. (India). Sigma Aldrich Inc. (USA) supplied all the enzymes used in the simulated human gastrointestinal tract in the in vitro experiment: α-amylase from human saliva (A1031), pepsin from porcine gastric mucosa (P7000), α-amylase from porcine pancreas (A6255), and trypsin from porcine pancreas (93615). Sucrase plus β-galactosidase (E-SUCRBG) was supplied by Megazyme Ltd (Ireland). The culture strains (B. subtilis TISTR 001, B. bifidum TISTR 2129, L. casei TISTR 1463, and E. coli TISTR 073) were supplied by the Thailand Institute of Scientific and Technological Research (Thailand) and S. serovar Enteritidis S003 was obtained from the culture collection at the Department of Biotechnology, Kasetsart University, Bangkok, Thailand.

Production of oligofructans from fermenting fresh sugarcane juice with B. subtilis TISTR 001

The first step was to prepare B. subtilis TISTR 001 by culturing in nutrient broth (NB) with added meat extract and peptone at 0.3 and 0.5% (w/v), respectively, incubating at 37 °C with a shaking rate at 150 rpm (model VS-8480SFN, shaking incubator, Vision Scientific Co., Ltd) for 10 h until the turbidity value of the culture medium (OD600) reached 0.7–0.10, so it could be used for inoculum in further fermentation experiments. The fermentation process involved straining the sugarcane juice through muslin cloth to eliminate any dirt contaminating the juice. The strained juice (225 mL) was prepared the fermentation substrate in a 500 mL flask and the following nutrients were added for the fermentation of B. subtilis TISTR 001: yeast extract (0.2% w/v), meat extract (0.3% w/v), peptone (0.5% w/v), (NH4)2SO4 (0.2% w/v), and MgSO4.7H2O (0.06% w/v). Then, the pH was adjusted to 6.8 and sterilized at 121 °C for 15 min. Following this process, the prepared inoculum of B. subtilis TISTR 001 at 10% (v/v) was added to the sterilized sugarcane juice and fermented at 30 °C, with a shaking rate at 150 rpm. The fermentation sample was collected every 6 for 96 h. The B. subtilis cells were separated from the fermented juice prior to conducting the various analyses of: the change in the sugar amounts, the total short-chain fructo-oligosaccharides and total oligofructans in the form of free fructose, as well as analyzing the levansucrase, including the change in total soluble solids that occurred during the fermentation process.

Analysis of fermentative parameters

Total soluble solids

The samples of fermented sugarcane juice, from which the cells had been separated, were measured using a refractometer (OPTi Digital Handheld Refractometer, Bellingham Stanley).

Reducing sugar amount determined using the Somogyi–Nelson method

The samples of fermented juice (1 mL) that had been water-diluted to the appropriate concentration, were added with alkaline copper reagent (1 mL), shaken well together and boiled underwater for 15 min (sealed in a glass bead) and subsequently cooled immediately in an ice bath. Nelson Reagent solution (1 mL) was then added to the samples, shaken and left at room temperature in a darkened room for 30 min. Thereafter, 5 mL of distilled water was added and mixed well. Then immediately, the samples were measured at 520 nm using a spectrophotometer (Genesys 30, Thermo Scientific Co., Ltd., USA) and the standard glucose solution was used to calculate the amount of reducing sugar produced during the fermentation process.

Amounts of sucrose, glucose, fructose, and short-chain fructo-oligosaccharides determined using high-performance liquid chromatography

The fermented sugarcane juice, in which the cells had been separated, was diluted to the appropriate level of concentration and filtered using cellulose acetate (0.45 μm diameter) and then the amounts of various sugars was analyzed using high-performance liquid chromatography (HPLC; Shimadzu Corporation, Japan). The substances were measured using a refractive index detector (RID-10A, Shimadzu Corporation, Japan) with VertiSep™ Sugar CMP column (7.8 × 300 mm, 8 μm; Vertical Chromatography Co., Ltd., Thailand) at 80 °C. The mobile phase used was deionized distilled water at a flow rate of 0.4 mL per minute using the standard solutions for sucrose, glucose, and fructose to study the changes in the various types of sugar during the fermentation process. For the analysis of short-chain fructo-oligosaccharides, a Shodex Asahipak NH2P-50 4E column (4.6 mm I.D. × 250 mm, Showa Denko America, Inc., Japan) was applied at 40 °C. The mobile phase used was acetone nitrite/water at a ratio of 70:30 with a flow rate of 1 mL per minute. Standard fructo-oligosaccharide kestose and nystose were used to calculate the amount of short-chain fructo-oligosaccharides.

Analyzing amount of total oligofructans content in form of free fructose

1 mL of the fermented sugarcane juice, in which the cells had been separated, was added with 2 mL absolute ethanol to precipitate the fructo-oligosaccharides and fructan, prior to the sample contents being centrifuged at 10,000 rpm for 10 min (Hettich Rotina 35R refrigerated tabletop centrifuge, UK). Then, the supernatant was discarded and the precipitated portion was hydrolyzed with 2 mL of 0.1 N hydrochloric acid, then boiled for 30 min to break the bonds of the oligosaccharides and polymer fructan into monosaccharides. The amount of hydrolyzed monosaccharides as reducing sugar was then analyzed using the Somogyi–Nelson method with the standard solution as fructose to calculate the total free fructose content used in forming the oligofructans chain.

Analyzing levansucrase activity

The fermented juice obtained by separating the B. subtilis cells was considered as the crude enzyme of levansucrase. The enzyme activity was analyzed by preparing a 10% (w/v) sucrose solution (substrate) in 1 mL of pH 6.8 phosphate buffer solution and incubating at 30 °C for 5 min. Then, 1 mL of crude enzyme levansucrase was added and incubated for 30 min; the enzyme reaction was stopped by boiling in water. The reducing sugar produced were analyzed using the Somogyi–Nelson method. Standard curves of glucose were used to analyze the free glucose produced before and after the enzyme addition.

One unit of enzyme was defined as the amount of enzyme that produced 1 μmol/mL of free glucose in 1 min under the conditions used for enzyme activity analysis.

Study of prebiotic properties of sugarcane juice fermented containing oligofructans under in vitro conditions

Sugarcane juice was fermented for 84 h and then the fermented cane juice was pasteurized at 70 °C for 30 min and cooled immediately by soaking in cold water. Testing for prebiotic properties in a simulated human gastrointestinal tract was divided into two stages. The first step tested for the resistance to digestion in the upper digestive tract (mouth, stomach, and small intestine). The second step took the sample of sugarcane juice that had been digested in the upper digestive system and studied its efficacy for the growth of microorganisms, both probiotics and pathogens, under the simulated conditions in the colon.

Stage 1: This consisted of an enzyme digestion test in the upper digestive tract (mouth, stomach, and small intestine) (Ayimbila and Keawsompong 2018). The pasteurized sugarcane juice was mixed with artificial saliva adjusted to pH 6.8 and then incubated at 37 °C, simulating the digestion conditions in the mouth and adding the amylase enzyme (human salivary α-amylase) to the pasteurized fermented cane juice. The final enzyme concentration was 0.33 U/mL, incubated for 10 min. Samples were collected at 0, 5, and 10 min, after which the enzyme reaction was stopped immediately by adjusting the pH to 2 (simulating gastric conditions) and incubating at 37 °C as previously. Pepsin enzyme was then added to achieve the final enzyme concentration of 20 U/mL, which was then incubated for 4 h. Sugarcane juice samples were collected at 0, 0.5, 1, 2, and 4 h. The enzyme reaction was stopped by adjusting the pH to 7.5 immediately (simulating the conditions in the small intestine) and incubating at 37 °C. The enzyme mixture of α-amylase (pancreatic α-amylase), trypsin, and sucrase plus β-galactosidase was added to the final concentration of each enzyme (0.75 U/mL) and incubated for 6 h. Samples were collected at 0, 0.5, 1, 2, 4, and 6 h. All enzyme reactions were stopped by boiling in water for 10 min. All collected samples were analyzed for reducing sugar, which was the product after digestion of each enzyme, and the percentage of hydrolysis was calculated using the formula:

%Hydrolysis=Reducing sugar at timefinal-Reducing sugar at timeinitial×100Reducing sugar at timeinitial

Stage 2: This involved studying the efficacy of micro-organisms growth in cultures with the addition of fermented cane juice in conditions that simulated the upper digestive tract. This study was conducted using a single culture of each species and co-cultures of probiotics and pathogens.

For the single culture growth of microorganisms to study the growth of each microorganism, both probiotic bacteria (B. bifidum TISTR 2129 and L. casei TISTR 1463) and pathogens (E. coli TISTR 073 and S. serovar Enteritidis S003) were first prepared for each type of inoculum in a specific medium, namely liquid DeMan, Rogosa and Sharpe broth (MRS broth) + 0.05% (w/v) L-cysteine, MRS broth, Luria–Bertani (LB) medium, and NB, respectively. Each sample was cultured at 37 °C for 24 h in aerobic conditions, except for the B. bifidum TISTR 2129 that was cultured in anaerobic conditions with the turbidity value of the culture medium (OD600) equal to 0.5–0.6, after which each type of culture was re-cultured in a specific medium. Then, 10% (v/v) of fermented sugarcane juice was added to the specific medium for each bacterial species. All cultures were cultivated at 37 °C in aerobic condition, except for the B. bifidum TISTR 2129. Samples were collected every 3 for 48 h. The pH of the culture medium and the growth of the individual strains were analyzed and compared with and without the addition of digested fermented sugarcane juice that had passed through the simulated upper digestive system. The results in this study were described using counts of the microbial colonies (CFU/mL) in the different media, namely MRS agar + 0.05% (w/v) L-cysteine, MRS agar, nutrient agar (NA), and MacConkey agar, which were the selective media for B. bifidum TISTR 2129, L. casei TISTR 1463, E. coli TISTR 073, and S. serovar Enteritidis S003, respectively.

The co-culture growth of probiotics and pathogens investigated a co-culture of B. bifidum TISTR 2129 with S. serovar Enteritidis S003 and a co-culture of L. casei TISTR 1463 with E. coli TISTR 073. Each inoculum was prepared as in the previous experiment for the single culture study. The liquid NB was prepared with 10% (w/v) digested fermented sugarcane juice from the simulated upper gastrointestinal tract and then 5% (w/v) of each inoculum strain was added, followed by culturing at 37 °C in anaerobic conditions for the co-culture of B. bifidum TISTR 2129 with S. serovar Enteritidis S003 and in aerobic conditions for L. casei TISTR 1463 and E. coli TISTR 073. Samples were collected every 3 for 48 h and the pH of the culture medium was analyzed. The growth rates of each strain in the selective media were analyzed using the same parameters as for the single culture experiment, and the specific growth rate (μ) was calculated using the formula:

μh-1=lnNt-lnN0/t

where μ = specific growth rate (h−1), Nt = the log CFU/mL of bacteria at time interval (t), and N0 = the initial number of bacteria.

Statistical analysis

Three replicates of all experiments and parameters were determined. The Statgraphics XVII-X64 software program was used for analysis based on Duncan's multiple range test and significance was tested at the P < 0.05 level.

Results and discussion

Oligofructans production from fermenting fresh sugarcane juice with B. subtilis TISTR 001

Oligofructans (fructo-oligosaccharides and fructan), which occurred during the fermentation of the fresh sugarcane juice, contain sucrose as the main component; the results of the transformation occurring in the various sugars were investigated. In theory, B. subtilis produces levansucrase to hydrolyze or digest the bonds of sucrose, a double-molecule sugar, to become a single-molecule sugar (glucose and fructose), also known as reducing sugar. Levansucrase would trigger the occurrence of free fructose sugar to form a chain or a long-chained bond of fructo-oligosaccharides or long-chained polymer fructan. From the experimental results of changes in sucrose, glucose, and fructose including the amounts of dissolved solids in the juice and reducing sugar produced during fermentation (Fig. 1), it was found that the sucrose content decreased continuously while the contents of glucose and fructose increased gradually at the same rate until the 6th hour of fermentation, after which the two single sugars underwent different changes. Glucose tended to increase continuously until the 72nd hour of fermentation, after which it tended to decrease. On the other hand, fructose tended to decrease slightly and then decreased significantly after the 54th hour. The reduction in the free fructose content was the result of the fructose molecules being linked together to form a long chain of fructo-oligosaccharides or possibly polymer fructose by the levansucrase enzyme that was produced (Fig. 2a). Therefore, the amount of free fructose was less than the amount of glucose (Fig. 1a). The results of the change in each type of sugar were supported by the reduction of the total soluble solids and the increase in the reducing sugars, as shown in Fig. 1b. The fermentation still contained the remaining free glucose after sucrose hydrolysis, so the reducing sugar tended to increase (Fig. 1b).

Fig. 1.

Fig. 1

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Changes in sugar concentrations (sucrose, glucose, and fructose) (a); reducing sugar and total soluble solids (b); during sugar cane juice fermentation for oligofructans production by B. subtilis TISTR 001

Fig. 2.

Fig. 2

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Concentration of total oligofructans in form of fructose and levansucrase activity (a); and short-chain fructooligosaccharides (kestose and nystose) (b); during fermentation of sugar juice by B. subtilis TISTR 001

This was consistent with the production of oligofructans (Fig. 2). The total oligofructans content was analyzed by hydrolyzing with hydrochloric acid at high temperatures, resulting in the oligosaccharides and polymer fructose being digested and then analysis was undertaken of the free fructose after digestion, as shown in Fig. 2a. The total oligofructans content increased continuously and was correlated positively with levansucrase activity. It tended to decrease significantly after the 84th hour of fermentation when the free fructose had been used up (Fig. 1a). At the 84th hour of fermentation, the total amount of oligofructans in the form of free fructose sugar had its highest content of 2.57% (w/v), resulting in 0.17 g/g of reducing sugar with a production rate of 0.031 g/L/h and the maximum levansucrase activity was 1.57 × 106 U/mL (Fig. 2a). During the bond formation of the oligofructans chain, short-chain oligosaccharides (kestose and nystose) were also being formed continuously, as shown in Fig. 2b.

Prebiotic properties of fermented cane juice containing oligofructans under in vitro conditions

The prebiotic properties were studied in two stages. First, the resistance was tested of digestive enzymes in the upper digestive tract (mouth, stomach, and small intestines). Prebiotics are rarely digested by enzymes in the upper digestive tract. This means there were little or indigestible amounts of: (1) enzymes in the mouth, such as alpha-amylase (human salivary α-amylase); (2) gastric enzyme, such as pepsin; and (3) small intestinal enzymes, such as pancreatic α-amylase, trypsin, and sucrase plus β-galactosidase. The fermented sugarcane juice that passed through the digestive tract would become a type of food, known as a prebiotic acting as good microbes or beneficial microorganisms living in the large intestines, or a probiotic, which would stimulate the growth of probiotics and additionally help to inhibit the growth of pathogens, which was investigated in the second phase of the experiment.

The bioavailability of fermented sugarcane juice in the upper digestive tract was studied based on the digestion efficiency of the enzymes from the reducing sugar content, the product obtained from digestion. If the reducing sugar content increased through enzymatic digestion, it could indicate that oligosaccharides or fructose polymer were digested. The content of reducing sugars was calculated as a hydrolysis percentage. From Fig. 3, at the beginning of hydrolysis, the initial content of reducing sugar prior to the enzymatic digestion test of fermented cane juice was 0.75% (w/v). When fermented juice was digested in the mouth (mouth condition) using human salivary α-amylase for 10 min, the result was that there was very little or almost no change in the amount of reducing sugar (Fig. 3: M0-M10), which implied that the amylase enzyme digested very little if any of the fermented sugar cane juice at all. Reducing sugar was increased to 0.77% (w/v), for which the hydrolysis was 2.95% after oral digestion was completed. The reaction was stopped by adjusting to pH 2, similar to the gastric condition. This early acidic condition may have caused the fermented sugar cane juice to be digested, thus the reducing sugar content increased during the phase transition from the mouth to the stomach (Fig. 3: M10-G0). During 4 h (240 min) of digestion with an enzyme simulating gastric digestion using pepsin, there was little decrease in reducing sugar (Fig. 3: G0-G240). The digestion percentage (% hydrolysis) at the completion of gastric digestion was 5.50%. Similarly, at the end of the gastric digestion period, the enzyme reaction was stopped by adjusting to pH 7.5, to simulate the intestinal conditions. The change was likewise increased (Fig. 3: G240-I0). In the small intestine digestion model, three enzymes were used: alpha-amylase (pancreatic α-amylase), trypsin, and sucrase plus β-galactosidase. The experimental digestion was 6 h (360 min). From the results, it was found that the reducing sugar content increased slightly as well and hydrolysis was 6.92%. All the results of enzyme digestion in the upper digestive tract showed slight increases in reducing sugar that positively related to a slight increase in the hydrolysis rate, anyway, the percentage of hydrolysis was not significant in each interval condition (mouth condition, gastric condition, and intestinal condition) but the results were significant during phase transition (M-G and G-I phases) due to the pH change (Fig. 3) as previously explained. It was found that at the end of the digestive simulation in the upper digestive tract, the amount of fermented sugar cane juice digested was approximately 7%.

Fig. 3.

Fig. 3

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Product of hydrolysis in form of reducing sugar and rate of hydrolysis of fermented sugar cane juice passing through simulated human gastrointestinal tract under in vitro conditions (M = Mouth condition; G = Gastric condition; I = Intestinal condition). Different letters represent significant (P < 0.05) differences in rate of hydrolysis through the human gastrointestinal (M-G-I) tract

Efficacy of microorganism growth in culture medium with addition of fermented sugarcane juice through upper digestive tract

Growth of microorganisms in single culture of probiotics and pathogens

The results of adding digested fermented sugarcane juice into each culture medium compared with the control which was a non-fermented sugarcane culture medium on the growth of each probiotic (B. bifidum TISTR 2129 and L. casei TISTR 1463) and pathogen (E. coli TISTR 073 and S. serovar Enteritidis S003), are shown in Fig. 4. The fermented juice containing oligofructans promoted the growth of microorganisms, especially B. bifidum TISTR 2129. It was evident that when fermented sugar cane juice was added to the culture medium, the Bifidobacteria content was higher than in the control medium (Fig. 4a). Additionally, the effect of pH of the culture medium during growth provided supporting information in terms of the growth of probiotics. This was consistent with Chlebowska-Smigiel et al. (2017), Meyer & Stasse-Wolthuis (2009), and Shen et al. (2013) who reported that the growth of beneficial bacteria in the intestine, such as Bifidobacteria and Lactobacilli, caused the production of lactic acid or short-chain organic acids, which increased the acidity of the colon and the pH tended to decline. As a result of this experiment, it could be seen that under probiotic culture conditions, the pH decreased rapidly during the first 12 h of culture, followed by a steady trend. This was consistent with the period when B. bifidum TISTR 2129 and L. casei TISTR 1463 had very high growth rates and after the 12th hour of fermentation B. bifidum TISTR 2129 continued to grow until the 30th hour and then it started to decrease, while L. casei TISTR 1463 grew very rapidly in the first 12 h and thereafter, it tended to drop more than the control; however, the pH tended to decrease, corresponding to the growth of both probiotic strains (Fig. 4a, b). On the other hand, the growth of pathogens (E. coli TISTR 073 and S. serovar Enteritidis S003) was different from that of probiotics (B. bifidum TISTR 2129 and L. casei TISTR 1463). In the first 6 h of that culture, it was found that both E. coli TISTR 073 and S. serovar Enteritidis S003 showed no difference in growth in both the cultured medium with and without fermented cane juice. However, after the 6th hour of culturing, it was found that the growth tendency in both strains of pathogens reduced. This was evident growth of E. coli TISTR 073 in the medium containing fermented cane juice which was lower than the control after 27 h (Fig. 4c). In addition, the pH of the pathogen medium tended to increase (Fig. 4c, d), which was significantly different from the pH of the probiotic medium (B. bifidum TISTR 2129 and L. casei TISTR 1463) (Fig. 4a, b). From the results of a single culture experiment, it was found that fermented sugar cane juice containing oligofructans had a relatively clear effect on stimulation of the growth of B. bifidum TISTR 2129 and reduction of E. coli TISTR 073.

Fig. 4.

Fig. 4

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Growth of single culture probiotics (B. bifidum TISTR 2129 (a), and L. casei TISTR 1463 (b)), and pathogens [E. coli TISTR 073 (c), and S. serovar Enteritidis S003 (d)] in medium without (control) and with fermented hydrolysate

Growth of microorganisms in co-culture of probiotics and pathogens

Figure 5 shows the results of the co-culture experiment between probiotic and pathogenic microorganisms to study the efficacy of co-growth of microorganisms in the medium with and without the addition of digested fermented sugarcane juice and to study the efficacy of probiotic bacteria in inhibiting the growth of pathogens in the co-culture. The pH changes of the culture medium during the co-culture of B. bifidum TISTR 2129 with S. serovar Enteritidis S003 and L. casei TISTR 1463 with E. coli TISTR 073 are shown in Fig. 5a. It was found that the pH of the culture medium during the culture of B. bifidum TISTR 2129 together with S. serovar Enteritidis S003 was lower than for the culture of L. casei TISTR 1463 with E. coli TISTR 073. The co-culture showed a significant decrease in pH until the 12th hour, which was confirmed with the growth of probiotics, as shown in Fig. 5b, c. Thereafter, it was relatively stable. As with the previous results of probiotic growth, the pH tended to decrease (Fig. 4a, b), while the pathogen cultures tended to have a higher pH (Fig. 4c, d). It was possible that the effect of the pH of the co-culture was influenced by probiotic growth, or that the probiotics outgrew the pathogens. Confirmation of this is shown with Fig. 5b, c. The growth rates of B. bifidum TISTR 2129 and L. casei TISTR 1463 were significantly higher than those of S. serovar Enteritidis S003 and E. coli TISTR 073 in co-culture. These results confirmed that the probiotics were clearly effective in inhibiting the pathogens. The specific growth rate of each strain in co-culture (Fig. 6) showed that in the co-culture between B. bifidum TISTR 2129 and S. serovar Enteritidis S003 during the first 12 h, B. bifidum TISTR 2129 had a significantly higher specific growth rate than S. serovar Enteritidis S003 (Fig. 6a). When comparing, the specific growth rate of B. bifidum TISTR 2129 under fermented hydrolysate was significantly higher than for the control during the first 12 h. S. serovar Enteritidis S003 had a significantly lower specific growth rate (in fact very slow growth) during the first 12 h of co-culturing. After the 12th hour of co-culturing, the specific growth rates of both strains tended to decline. The co-culture of L. casei TISTR 1463 with E. coli TISTR 073 (Fig. 6b) produced similar results. Therefore, all results supported that the fermented cane juice containing oligofructans had prebiotic properties which promoted probiotic growth and inhibited the growth of pathogens, as shown in Figs. 5 and 6.

Fig. 5.

Fig. 5

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Change in pH value (a); during cultivation of co-culture: B. bifidum TISTR 2129 + S. serovar Enteritidis S003 (b); and L. casei TISTR 1463 + E. coli TISTR 073 (c); in medium without (control) and with fermented hydrolysate

Fig. 6.

Fig. 6

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Specific growth rate of the co-culture: B. bifidum TISTR 2129 + S. serovar Enteritidis S003 (a); and L. casei TISTR 1463 + E. coli TISTR 073 (b); in medium without (control) and with fermented hydrolysate. Different letters represent significant (P < 0.05) differences for each co-culture

Conclusion

Oligofructans were produced by fermenting fresh sugarcane juice (15.3°Brix) using B. subtilis TISTR 001 that produced the enzyme levansucrase to hydrolyze sucrose and to make long-chain fructo-oligosaccharides and polymer fructose as the target products. The maximum total oligofructans content was achieved at the 84th hour of fermentation in the form of free fructose of 2.57% (w/v) with a production yield of 0.17 g/g reducing sugar at a production rate of 0.031 g/L/h, while the maximum levansucrase activity was 1.57 × 106 U/mL at that time. Additionally, the oligofructans contained in the fermented cane juice acted as a potential biological ingredient exhibiting prebiotic properties because it was able to resist digestion of enzymes in the gastrointestinal tract under in vitro conditions with digestion of 6.92%. Furthermore, the oligofructans promoted the growth of prebiotics, especially B. bifidum TISTR 2129, and inhibited the growth of pathogens in both single culturing and co-culturing with probiotics that resulted in lower growth rates than for probiotic growth, for both E. coli TISTR 073 and S. serovar Enteritidis S003. Consequently, all results provide important data to help guide further development to pilot scale and industrial production and to support research on future benefits to human health. In addition, the attempt to produce a functional ingredient substance in form of oligofructans based on sugarcane juice as the substrate for fermentation and the testing of their prebiotic properties, suggested that the fermented sugarcane juice containing oligofructans could be preferably developed into an alternative functional drink as a beverage options with health benefits for consumers in the future.

Acknowledgements

This research received research grants in the 2019 academic year from the Kasetsart University Research and Development Institute (KURDI), Bangkok, Thailand.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

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