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. 2020 May 9;10(6):241. doi: 10.1007/s13205-020-02234-0

In vitro prebiotic potential, digestibility and biocompatibility properties of laminari-oligosaccharides produced from curdlan by β-1,3-endoglucanase from Clostridium thermocellum

Krishan Kumar 1, Vikky Rajulapati 1, Arun Goyal 1,✉
PMCID: PMC7211229  PMID: 32405445

Abstract

Curdlan or laminarin, a β-1,3-glucan was hydrolysed by β-1,3-endoglucanase (CtLam81A) from Clostridium thermocellum to produce laminari-oligosaccharides. TLC analysis of hydrolysed curdlan showed the presence of laminari-oligosaccharides of the degree of polymerization, DP2-DP7. This mixture of laminari-oligosaccharides displayed prebiotic properties. Laminari-oligosaccharides showed an increase in the growth of probiotic bacteria such as Lactobacillus plantarum DM5 and Lactobacillus acidophilus, while they did not promote the growth of non-probiotic bacteria (Escherichia coli and Enterobacter aerogenes). Laminari-oligosaccharides showed higher prebiotic activity score of 0.92 ± 0.01 and 0.64 ± 0.08 for L. plantarum DM5 and L. acidophilus NRRL B-4496, respectively, similar to those shown by inulin. Laminari-oligosaccharides showed higher resistance or low digestibility against α-amylase, artificial gastric juice and intestinal fluid than inulin indicating their bioavailability to the probiotic bacteria present in the gastrointestinal tract of human. The probiotic bacteria consumed laminaribiose and laminariotriose more readily than higher laminari-oligosaccharides as carbon source for their growth. The in vitro cytotoxicity assay of laminari-oligosaccharides (1 mg/ml) on human embryonic kidney (HEK 293) cells showed that the cell viability was not affected even after 72 h indicating their biocompatible nature. All the results amply indicated that laminari-oligosaccharides can serve as potential prebiotic additives for functional food products.

Keywords: Curdlan, Laminari-oligosaccharides, Lactic acid bacteria, Human embryonic kidney cells, Human colon cancer cells

Introduction

Prebiotics are compounds which are non-digestible by human or animal and provide beneficial effects to the host by selectively stimulating the growth of beneficial bacteria such as lactobacillus sp. and bifidobacteria in gut (Kerry et al. 2018). A large population in the world face health issues related to digestion such as constipation or irritable bowel movement (Asadi et al. 2016). These problems can be overcome by using the dietary intake that is rich in non-digestible fibres (prebiotics) or probiotic bacteria (such as lactobacillus sp and bifidobacteria) or a mixture of both (Pandey et al. 2015). A prebiotic must tolerate the acidic pH of the stomach and the enzymes present in small intestines so that it can reach the colon. In colon, it can enhance the growth of certain specific bacteria like Lactobacilli and Bifidobacteria (Kerry et al. 2018). Prebiotics also impart several health-promoting benefits such as immune system modulation (Akramiene et al. 2006), antitumor (Khangwal and Shukla 2019) and cholesterol-lowering activities (Kumar et al. 2012). In addition, the fermentation of prebiotics by probiotic bacteria generates the short-chain fatty acids (SCFA) and lactate which help in the suppression of non-desirable pathogenic bacteria in the intestine (Wong et al. 2006; Jacobs et al. 2009).

The most commonly found prebiotics in the natural sources like fruits, vegetables, milk and honey are the fructo-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides and raffinose oligosaccharides (Khangwal and Shukla 2019). The prebiotic, fructo-oligosaccharides are found in the honey, tomato, asparagus, banana, wheat, barley, sugar beet, mushrooms and garlic Khangwal and Shukla 2019). Xylo-oligosaccharides are found in sugarcane bagasse, barley straw bamboo, corncobs, cotton stalk, wheat bran and in a variety of fruits and vegetables (Mano et al. 2018). Bovine and human milk are the sources of the prebiotics, galacto-oligosaccharides (Khangwal and Shukla 2019). The seeds of mustard, peas, legumes, lentils, chickpeas and beans are the natural sources of raffinose oligosaccharides (Khangwal and Shukla 2019). The prebiotic oligosaccharides with different degrees of polymerization (DP) can be generated by chemical or enzymatic treatments. The citrus peel containing the pectin can be hydrolysed with trifluoroacetic acid (TFA) and hydrogen peroxide (H2O2) to produce pectic-oligosaccharides (Zhang et al. 2018). Some of the prebiotics are enzymatically synthesized to get the different DP such as xylo-oligosaccharides (Chapla et al. 2012), manno-oligosaccharides (Ghosh et al. 2015) and fructo-oligosaccharides (Mueller et al. 2016). A number of prebiotics are commercially available, such as inulin, gluco-oligosaccharides, fructo-oligosaccharides and galacto-oligosaccharides (Roberfroid et al. 2010, Khangwal and Shukla 2019). It is therefore of great interest to explore new prebiotic compounds from easily and cheaply available carbohydrate sources.

Curdlan or laminarin is produced as exopolysaccharide by bacteria, such as Alcaligenes faecalis, Agrobaterium rhizogenes, Agrobaterium radiobacter, Pseudomonas aeruginosa, Streptococcus pneumonia etc. (Volman et al. 2008; Xu & Zhang, 2016). The macroalgae, Laminaria digitata, contains laminarin as the storage polysaccharide. Curdlan or laminarin are β-1,3-Glucans, composed of linear β-1,3-linked glucose units. Curdlan is used as a gelling agent for desserts and starch jelly and as thickener or food stabilizer for low calorific value food products (Spicer et al. 1999). The supplementation of β-1,3-glucan stimulated the growth of Bifidobacterium sp. with the production of short-chain fatty acids and lactate in rat (Shimizu et al. 2001; Kuda et al. 2005). The feeding of laminarin to weaning pig reduced the population of Escherichia coli in faecal samples and enhanced their weight (O’Doherty et al. 2010). The ingestion of laminarin by rat modulated the intestinal metabolism by affecting the mucus composition, intestinal pH and SCFA concentration (Devillé et al. 2007). Curdlan is less soluble in water due to its high molecular mass and gelling characteristics (Xiao et al. 2017). This property of curdlan makes it less suitable for food industry applications. Curdlan can be hydrolysed to shorter chains by using β-1,3-glucanase which can make it water soluble (Kumar et al. 2018). The β-1,3-linked glucose oligomers produced by hydrolysis of laminarin (β-1,3-glucan) induced monocytes to release the tumor necrosis factor-alpha (TNF-α) for inhibition of human leukemic U937 cells (Miyanishi et al. 2003) and stimulated phagocytosis of macrophages and granulocytes (Jamois et al. 2005). In tobacco plants, β-1,3-glucan oligosaccharides provided defense against tobacco mosaic virus (TMV) (Klarzynski et al. 2000; Fu et al. 2011). The oligosaccharides produced from the hydrolysis of curdlan showed prebiotic activity by stimulating the lactobacillus growth (Shi et al. 2018).

In this study, the controlled hydrolysis of curdlan was carried out to produce mixed laminari-oligosaccharides (LOS) from curdlan. The LOS mixture was used for studying their effect on the growth of probiotic bacteria. The in vitro analysis of LOS were evaluated for prebiotic properties such as their resistance against artificial gastric juice and intestinal fluid and compared with commercial inulin. The effect of LOS was also analysed by in vitro cytotoxicity assay on human embryonic kidney (HEK-293) cell line.

Material and methods

Chemicals, media and microorganisms

The MRS medium components, TGY medium components and the chemical components for estimation of total carbohydrate and reducing sugar were procured from Hi-Media Pvt. Ltd, India. Curdlan, amylase enzyme, bile salts and pepsin from porcine gastric mucosa were purchased from Sigma-Aldrich, USA. Lactobacillus plantarum DM5 isolated from ethnic fermented beverage Marcha of Sikkim as reported earlier (Das and Goyal 2014) and available with us was used. Lactobacillus acidophilus NRRL B-4495 was procured from Agricultural Research Service Culture Collection (Peoria, USA). The pathogenic bacterial strain Enterobacter aerogenes MTCC 7016 was obtained from Microbial Type Culture Collection (MTCC) and Gene Bank, Institute of Microbial Technology, Chandigarh, India. Thin-layer chromatography plate (TLC Silica gel 60 F254, 20 × 20 cm) was purchased from Merck, India. The Human embryonic kidney (HEK-293) and colon cancer (HT-29) cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India. Dulbecco's Modified Eagle Medium (DMEM) low glucose medium was purchased from Sigma Aldrich, USA.

Production of LOS by hydrolysis of curdlan and analysis by Thin Layer Chromtography

20 reaction mixtures each of 1.0 ml were set, containing 900 µl curdlan (11 mg/ml) dissolved in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 6.5 and added with 100 µl of β-1,3-endoglucanase (CtLam81A, 0.4 mg/ml) produced from Clostridium thermocellum (Kumar et al. 2018). The reaction mixtures were incubated at 75 °C for 5 min. The reaction was stopped by incubating the reaction mixture in boiling water for 5 min. The 1.0 ml reaction mixture was concentrated to 100 µl by evaporating in a hot air oven. The hydrolysed products were analysed by Thin Layer Chromtography (TLC). The 0.6 µl of concentrated reaction mixture and 0.6 µl of the standards, glucose, laminaribiose and laminaritriose each of 2 mg/ml were loaded on TLC (Silica gel 60 F254) plate. The mobile phase was a mixture of ethyl acetate/acetic acid/water: 2/2/1 (Lee et al. 2014). The spots on the TLC plate were visualized by dipping the TLC plate in a solution containing 0.5% (w/v) α-naphthol dissolved in methanol/sulphuric acid: 95/5 solution.

Growth of probiotic bacteria in presence of LOS

The growth of probiotic bacteria (Lactobacillus plantarum DM5 and Lactobacillus acidophilus NRRL B- 4496) was observed in the presence of LOS and commercially available inulin as a standard prebiotic. The freshly grown culture of L. plantarum DM5 (1.6 × 107, CFU (colony forming unit) /ml) or L. acidophilus NRRL B-4495 (2.1 × 106 CFU/ml) were inoculated to 1.0 ml basal MRS medium (pH 6.5) comprising 1% (w/v) glucose (as positive control), inulin or LOS. These cultures were grown at 37 °C for 12 h and 24 h. The growth of nonprobiotic bacteria Escherichia coli and Enterobacter aerogenes MTCC 7016 was also observed in the presence of LOS. E. coli (2.2 × 107 CFU/ml) or Enterobactor aerogenes MTCC 7016 (2.3 × 107 CFU/ml) was inoculated in 1 ml TGY medium comprising 1% (w/v) glucose, inulin or LOS and grown at 37 °C for 12 h and 24 h. The growth of both probiotic and non-probiotic bacteria was observed by spreading 10 µl of grown culture on MRS agar plate (for probiotic bacteria) and on TGY agar plate (for nonprobiotic bacteria) and incubating plates at 30 °C and 37 °C for 18 h. The number of colonies were counted as (CFU)/ml for each culture as reported earlier (Das et al. 2014). The prebiotic activity score of LOS and inulin was calculated by the equation.

Prebiotic activity score=PP24-PP0PG24-PG0-EP24-EP0EG24-EG0,

where PP24 and PP0 are the cell concentrations of probiotics, log CFU/ml grown on prebiotic at 24 h and 0 h, respectively. PG24 and PG0 are the cell concentrations of probiotics, log CFU/ml grown on glucose at 24 h and 0 h, respectively. EP24 and EP0 are the cell concentrations of Enterobacter aerogenes, log CFU/ml grown on prebiotic at 24 h and 0 h, respectively. EG24 and EG0 are the cell concentrations of Enterobacter aerogenes, log CFU/ml on glucose at 24 h and 0 h, respectively.

LOS utilization by probiotic bacteria

The freshly grown culture of L. plantarum DM5 (3.2 × 107 CFU/ml) or L. acidophilus NRRL B-4495 (4.2 × 106 CFU/ml) were inoculated to 1.0 ml MRS medium containing 0.5% (w/v) LOS. These cultures were grown under anaerobic conditions, at 37 °C for 24 h. 50 µl of grown culture was taken out periodically at 6 h, 12 h and 24 h. Then each culture was centrifuged at 13,000g for 10 min. The supernatant of each sample was transferred to fresh microcentrifuge tube. The 1.0 µl of each supernatant sample and 0.6 µl of standards, glucose, laminaribiose and laminaritriose (each of 2 mg/ml) were loaded on TLC plate. The TLC was run and visualized as described above.

Hydrolysis of LOS by α-amylase

The α-amylase enzyme (100 U/ml) was added to 1 × PBS (phosphate buffer saline) buffer and its pH was adjusted to 5, 6, 7 or 8 as reported earlier (Al-Sheraji et al. 2012). 0.5% (w/v) LOS or inulin mixed with 2 ml of α-amylase enzyme (200 U/ml) in 1 × PBS buffer of each pH was incubated at 37 °C for 30 min to 5 h. 20 µl of sample was taken out periodically from each pH sample containing LOS or inulin. The reducing sugar content was measured by Nelson and Somoygi method (Nelson 1944; Somogyi 1945) and total carbohydrate content present in the sample by phenol–sulphuric acid method (Dubois et al, 1956) using glucose as standard. The percentage hydrolysis of LOS and inulin was estimated by using the formula:

Hydrolysis%=Reducing sugar released×100Total sugar content-Initial sugar content

LOS digestibility by artificial gastric juice

Artificial human gastric juice was prepared by using phosphate buffer saline (PBS) containing (in g/L): NaCl, 8; Na2HPO4·2H2O, 8.25; KCl, 0.2; NaHPO4, 14.35; MgCl2.6H2O, 0.18 and CaCl2·2H2O, 0.1. The pH of artificial gastric juice was adjusted to 1, 2, 3 and 4 and supplemented with 1000 U/ml of pepsin (Korakli et al. 2002). 0.5% (w/v) LOS or inulin mixed with 2 ml of artificial gastric juice of each pH was incubated at 37 °C for 30 min to 5 h. 20 µl of sample was taken out periodically from each pH of gastric juice sample containing LOS or inulin. The percentage hydrolysis of LOS and inulin was estimated by the method as described above.

Digestibility of LOS by the intestinal fluid

The artificial intestinal fluid contained 0.5% (w/v) bile salt and 1,000 U/ml of trypsin dissolved in 1 × PBS and the pH were adjusted to 8.0 as reported earlier (Fernandez et al. 2003). 0.5% (w/v) LOS or inulin was mixed with 2 ml of artificial intestinal fluid and incubated at 37 °C for 30 min to 5 h. 20 µl of the sample was taken out periodically from an intestinal fluid containing LOS or inulin. The percentage hydrolysis of LOS and inulin was estimated by the method described above.

Production of lactic acid and SCFA by probiotic bacteria in the presence of LOS

The freshly grown culture of L. plantarum DM5 (1.6 × 107 CFU/ml) or L. acidophilus NRRL B-4495 (2.1 × 106 CFU/ml) were inoculated to 1.0 ml basal MRS medium (pH 6.5) supplemented with 1% (w/v) LOS. These cultures were grown at 37 °C for 12 h and 24 h and then centrifuged at 13,000g for 10 min. The supernatant was filtered through 0.2 μm membrane and analysed for lactic acid and short-chain fatty acids (SCFA) released in the medium by using Ultra-Fast Liquid Chromatography (UFLC, Prominence, Shimadzu, Japan) equipped with Aminex HPX-87H column (Bio-Rad Laboratories, USA). A mobile phase of 5 mM H2SO4 was passed through the column at a flow rate of 0.6 ml/min and keeping the oven temperature at 50 °C. Lactic acid and SCFA (Acetic acid, propionic acid and butyric acid) were detected by refractive index (RI) detector.

In vitro biocompatibility assay of LOS on mammalian cells

The effect of LOS on the viability of normal Human Embryo Kidney (HEK-293) and colon cancer (HT-29) cells was studied by cell viability assay using colorimetric method involving 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) (Mosmann 1983). HEK-293 (1 × 104 cells) or HT-29 (1 × 104 cells) were seeded in 200 µl of complete Dulbecco's Modified Eagle Medium (DMEM) low glucose medium and Roswell Park Memorial Institute (RPMI) 1640 medium, respectively and incubated in 5% (v/v) CO2 atmosphere at 37 °C for 12 h. After the incubation, the medium was completely removed and washed with 1 × PBS (pH 7.2). 200 µl of complete medium containing varying concentrations of LOS (100–1000 µg/ml) was added and incubated in 5% (v/v) CO2 atmosphere at 37 °C for 48 and 72 h. After the incubation, the medium containing LOS from each well was removed gently and 100 µl of MTT solution (0.5 mg/ml in PBS) was added to each well and incubated at 37 °C for 4 h. After 4 h, the MTT solution of each well was replaced with an equal volume of dimethyl sulfoxide (DMSO). The plate was then vortexed gently to dissolve the precipitate completely and the absorbance at 570 nm was detected with a reference wavelength of 690 nm using a microplate reader (Tecan, Infinite 200 Pro). The percentage of cell viability was calculated by the following equation.

Cell viability%=Nt/Nc×100.

where Nt is the absorbance of treated cells and Nc is the absorbance of untreated cells.

Result and discussion

Production of LOS

The LOS generated by CtLam81A after the hydrolysis of curdlan were run on TLC (Fig. 1). The chromatogram showed the presence of laminaribiose, laminaritriose and higher laminari-oligosaccharides. These LOS were used for investigating their prebiotic properties and compared with the commercially available prebiotic, inulin. Several studies have been reported on the production of prebiotic oligosaccharides by enzymes. Some of the oligosaccharides produced are xylooligosaccharides from corncob xylan (Chapla et al. 2012), manno-oligosaccharides from copra meal galactomannan (Ghosh et al. 2015), fructooligosaccharides from fructan (Mueller et al. 2016) and pectic-oligosaccharides from Citrus limetta (Chakraborty et al. 2018).

Fig. 1.

Fig. 1

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TLC analysis of hydrolysed products of curdlan by β-1,3-glucanase (CtLam81A). Lane 1 (G1, G2 and G3 are glucose, laminaribiose and laminaritriose, respectively, as standards) and lane 2 (hydrolysed curdlan)

Growth of probiotic and pathogenic bacteria in the presence of LOS

A prebiotic compound can tolerate acidic pH of stomach and enzymes of the small intestine and increases the growth of selective bacteria, such as Lactobacilli and Bifidobacteria. The growth of probiotic bacteria (L. plantarum DM5 and L. acidophilus B-4496) in the presence of glucose, inulin or LOS was studied at 37 °C for 24 h. The growth of probiotic bacteria in the presence of LOS was similar to the growth of bacteria in the presence of glucose or inulin (Table 1). LOS stimulated the growth of L. plantarum DM5 and L. acidophilus NRRL B-4496, by increasing the number of cells from 7.2 ± 0.02 to 10 ± 0.02 and 6.3 ± 0.02 to 8.0 ± 0.2 Log10 CFU/ml, respectively, in 24 h. The growth of probiotic bacteria may vary with respect to the probiotic strain and prebiotic carbohydrate. The growth of Lactobacillus paracasei 1195 was significantly higher with inulin than galacto-oligosaccharides, while the growth of L. acidophilus 33,200 was higher with galacto-oligosaccharides than inulin (Huebner et al. 2007). This may be due to the presence of carbohydrate degrading enzyme present in bacteria. The growth of non-probiotic bacteria (E. coli and E. aerogenes MTCC 7016) was not supported by the LOS (Table 1). These results showed that LOS supports the growth of probiotic bacteria and do not support the growth of non-probiotic bacteria or pathogenic bacteria indicating the attributes of a prebiotic. The probiotic bacteria grow more in the presence of prebiotic because probiotic bacteria have necessary enzymes to hydrolyze the prebiotic oligosaccharides (Swennen et al., 2006). As the enteric bacteria such as E. coli and Enterococcus species lack the enzymes to hydrolyze the prebiotic oligosaccharides, so they are unable to utilize prebiotic for their growth. The prebiotic activity score defines the ability of a given compound to support the growth of probiotic strains relative to non-probiotic strains and non-prebiotic substrate glucose. LOS showed the prebiotic activity score of 0.92 ± 0.01 and 0.64 ± 0.08 for L. plantarum DM5 and L. acidophilus NRRL B-4496, respectively. However, Inulin displayed the prebiotic activity score of 0.86 ± 0.02 and 0.79 ± 0.06 for L. plantarum DM5 and L. acidophilus NRRL B-4496, respectively. The prebiotic activity score of LOS was higher than dextran RBA12 from Weissella cibaria RBA12 (0.26 for L. plantarum DM5 and 0.3 for L. acidophilus NRRL B-4495) (Baruah et al. 2017), while, the prebiotic activity score of inulin was comparable to dextran RBA12 (0.85 for L. plantarum DM5 and 0.66 for L. acidophilus NRRL B-4495) (Baruah et al. 2017). These results showed the comparable prebiotic activity score of LOS to commercial inulin, thereby indicating their use as potential prebiotics.

Table 1.

Growth of probiotic and non-probiotic bacteria in the presence of prebiotics. (number of cells expressed as log10 CFU/ml)

Bacterium 0 h Glucose Inulin Laminari-oligosaccharides
12 h 24 h 12 h 24 h 12 h 24 h
L. plantarum DM5 7.2 ± 0.02 9.7 ± 0.15 10.1 ± 0.03 9.0 ± 0.07 9.5 ± 0.05 9.6 ± 0.05 10.0 ± 0.02
L. acidophilus 4495 6.3 ± 0.02 8.1 ± 0.17 8.8 ± 0.19 7.8 ± 0.08 8.1 ± 0.17 7.3 ± 0.10 8.0 ± 0.20
E. coli DH5α 7.2 ± 0.04 9.6 ± 0.05 9.9 ± 0.11 7.2 ± 0.24 7.0 ± 0.0 7.1 ± 0.17 7.2 ± 0.24
E. aerogenes 3030 7.3 ± 0.04 9.7 ± 0.06 10 ± 0.06 7.2 ± 0.17 7.1 ± 0.21 7.5 ± 0.11 7.4 ± 0.15
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LOS utilization by probiotic bacteria

The fermentation of LOS by L. plantarum DM5 and L. acidophilus NRRL B-4495 was carried out at 37 °C for different time intervals. The utilization pattern of LOS by L. plantarum DM5 and L. acidophilus NRRL B-4495 was analysed by running the supernatant (unutilized LOS) on TLC (Fig. 2a). The TLC analysis of L. plantarum DM5 fermented LOS showed the consumption of laminaribiose and laminaritriose within 6 h and laminaritetraose in 24 h (Fig. 2a). However, L. acidophilus NRRL B-4495 utilized LOS at a slower rate than L. plantarum DM5 and consumed only laminaribiose and laminaritriose in 24 h and did not utilize laminaritetraose within 24 h (Fig. 2b). LOS of DP2 and DP3 were consumed readily by both L. plantarum DM5 and L. acidophilus NRRL B-4495 within 12 h. This indicated that probiotic bacteria prefers to utilize LOS of DP2 and DP3 more favourably than higher DPs as a carbon source. Therefore, LOS of DP2 and DP3 can be used as an additive for functional foods. Similar results were reported for fructo-oligosaccharides, where the growth of probiotic bacteria was higher in the presence of lower DP fructo-oligosaccharides and lower in the presence of higher DP fructo-oligosaccharides (Mueller et al. 2016).

Fig. 2.

Fig. 2

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TLC analysis of the utilization pattern of LOS by probiotic bacteria. aLactobacillus plantarum DM5 and bLactobacillus acidophilus NRRL B-4495. The samples containing oligosaccharides from both fermenting cultures were withdrawn at 0 h, 6 h, 12 h and 24 h. G1, G2 and G3 are glucose, laminaribiose and laminaritriose respectively, the standards

Effect of α-amylase on LOS

The hydrolysis of LOS and inulin by α-amylase increased with the increase in the time period of incubation and also with the increase in pH. LOS showed only 2.3, 2.6, 2.9 and 3.1% hydrolysis at pH 5, 6, 7 and 8, respectively, after 5 h of incubation (Fig. 3a). However, inulin showed higher, 8.9, 10.4, 12.6, and 18.6% of hydrolysis at pH 5, 6, 7 and 8, respectively, after 5 h (Fig. 3b). Therefore, higher resistance (over 97%) of LOS than inulin against α-amylase indicated its suitability as a prebiotic compound. The presence of β-1,3-linked glucose in LOS makes it resistant to cleavage by α-amylase, which can only hydrolyse α-1,4-glucan linkages. The LOS digestibility in the presence of α-amylase was comparable to other reported prebiotics like glucan-DM5 from Lactobacillus plantarum DM5 (Das et al. 2014), dextran from Weissella cibaria (Tingirikari et al. 2014) and manno-oligosaccharides (Ghosh et al. 2015). However, LOS showed lower percentage of digestibility than gentio-oligosaccharides from Leuconostoc mesenteroides (Kothari and Goyal 2015), oligosaccharides from Pitaya (Wichienchot et al. 2010), gluco-oligosaccharide from Gluconobacter oxydans (Wichienchot et al. 2006) and exopolysaccharides from Mangifera pajang (Al-Sheraji et al. 2012).

Fig. 3.

Fig. 3

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Effect of α-amylase on hydrolysis of a laminari-oligosaccharides and b standard prebiotic inulin (B) at pH 5, 6, 7 and 8 for 5 h. 0.5% (w/v) LOS or inulin mixed with 2 ml of α-amylase (200 U/ml) in 1 × PBS buffer of each pH was incubated at 37 °C for 30 min to 5 h

Effect of artificial gastric juice on LOS digestibility

The percentage of hydrolysis of LOS or inulin by gastric juice at pH 1, 2, 3 and 4 was estimated at different time intervals (30 min-5 h) (Fig. 4a, b). LOS was hydrolysed only by 4.0, 2.5, 1.9 and 1.3% at pH 1, 2, 3 and 4, respectively, after 5 h of incubation (Fig. 4a). However, inulin was significantly hydrolysed, by 42.3, 19.6, 6.3, and 1.5% at pH 1, 2, 3, and 4, respectively, after 5 h of incubation (Fig. 4b). LOS showed 11-, 8-, 3- and 1.2-fold higher resistance at pH 1, 2, 3 and 4, respectively to hydrolysis against gastric juice than inulin. This results indicated that LOS will be available in the small intestine of gastrointestinal tract in human as it showed greater resistance to hydrolysis at low pH of gastric juice from the stomach. The resistance of LOS to acidic environment makes it useful for acidic foods such as yoghurt and dairy products as also reported earlier (Huebner et al. 2007). The results of resistance of LOS to hydrolysis are comparable to other reported prebiotics like glucan-DM5 from Lactobacillus plantarum DM5 (Das et al. 2014), dextran from Weissella cibaria (Tingirikari et al. 2014) and manno-oligosaccharides (Ghosh et al. 2015).

Fig. 4.

Fig. 4

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Effect of artificial gastric juice on hydrolysis of a mixed laminari-oligosaccharides and b standard prebiotic inulin at pH 1, 2, 3, and 4 at 37 °C for 5 h. 0.5%, (w/v) LOS or inulin mixed with 2 ml of artificial gastric juice of each pH was incubated at 37 °C for 30 min to 5 h

Effect of artificial intestinal fluid on LOS

A prebiotic must be able to tolerate the bile juice and enzymes of the intestinal fluid so that it can reach in intact form to the probiotic bacteria present in the colon. The percentage of hydrolysis of LOS and inulin increased with time in the presence of intestinal fluid. The maximum hydrolysis of LOS was 0.8% in intestinal fluid after 5 h of incubation at 37 °C (Fig. 5). However, the standard prebiotic inulin showed hydrolysis of 8.9% after 5 h of incubation at 37 °C (Fig. 5). Thus, LOS showed 11-fold higher resistance than inulin. This result indicated that LOS possesses high resistance to the intestinal fluid of small intestine and over 99% of it will be available in intact form as a carbon source for probiotic bacteria.

Fig. 5.

Fig. 5

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Effect of artificial intestinal juice on hydrolysis of mixed laminari-oligosaccharides and standard prebiotic inulin at pH 8 for 5 h. 0.5% (w/v) LOS or inulin was mixed with 2 ml of artificial intestinal fluid and incubated at 37 °C for 30 min to 5 h

Production of Lactic acid and short-chain fatty acid by probiotic bacteria

The production of lactic acid and SCFA (acetic acid, propionic acid and butyric acid) by L. plantarum DM5 or L. acidophilus NRRL B-4495 in the culture medium were analysed at 0 h, 12 h and 24 h. Both probiotic bacteria, L. plantarum DM5 and L. acidophilus NRRL B-4495 produced lactic acid, acetic acid and propionic acid as the metabolites in the presence of LOS, but butyric acid was not formed (Table 2). Initially, at 0 h, the concentration of lactic acid, acetic acid and propioninc acid in the medium were 0, 28.9 ± 0.4 and 2.6 ± 0.1 mM, respectively, which increased to 5.4 ± 1.0, 32.5 ± 0.7 and 3.3 ± 0.6 mM for L. plantarum DM5 (Table 2). Similarly, for L. acidophilus NRRL B-4495, lactic acid increased from 0 to 10.2 ± 0.3 mM and acetic acid from 28.9 to 31.1 ± 0.8 mM and propionic acid from 2.6 to 5.1 ± 0.7 mM after 24 h. The lactic acid and SCFA released from microbial fermentation of dietary fibers showed various metabolic and regulatory functions like lactic acid helps in immune modulation and inhibit histone deacetylases (Koh et al. 2016). The lactic acid and acetic acid produced by the probiotic bacteria can be further utilized by cross-feeding butyrate-producing bacteria in the colon for producing butyric acid (Belenguer et al. 2006). Acetic acid was found to stimulate long-chain fatty acid and cholesterol in the liver (Delzenne et al. 2011). Propionic acid reduced cholesterol synthesis and found to be involved in the activation of G-protein-coupled receptors (GPR-41, and GPR-43) to release satiety hormones (Kimura et al. 2011).

Table 2.

Production of lactic acid and SCFAs released by lactic acid bacteria

Bacterium Lactic acid (mM) Acetic acid (mM) Propionic acid (mM)
0 h 12 h 24 h 0 h 12 h 24 h 0 h 12 h 24 h
L. plantarum DM5 0 6.4 ± 0.2 5.4 ± 1.0 28.9 ± 0.3 32.3 ± 0.2 32.5 ± 0.7 2.6 ± 0.1 3.3 ± 0.6 3.3 ± 0.6
L. acidophilus NRRL B-4495 0 9.3 ± 0.4 10.2 ± 0.3 28.9 ± 0.3 30.6 ± 0.6 31.1 ± 0.8 2.6 ± 0.1 5.1 ± 0.7 5.3 ± 0.7
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Effect of LOS on mammalian cells by in vitro assay

The effect of LOS (with varying concentrations from 100 µg/ml to 1000 µg/ml) on viability of mammalian cells (HEK-293) and cancer cells (HT-29) was analysed for 48 h and 72 h. The viability of both HEK-293 and HT-29 cells was studied by the MTT assay. The viability of both HEK-293 and HT-29 cells was not significantly affected even at higher concentrations of LOS (1000 µg/ml) after 72 h (Fig. 6a, b). The nontoxic effect of LOS on human embryonic kidney cells (HEK-293) displayed its biocompatible nature. Therefore, LOS can be considered as biocompatible and safe for consumption as a functional food. LOS did not show any direct inhibitory effect on the proliferation of cancer HT-29 cells (Fig. 6b). However, it has been reported that, when monocytes are treated with laminari-oligosaccharides, leads to induction of TNFα production displaying its anticancer activity (Miyanishi et al. 2003).

Fig. 6.

Fig. 6

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The cell viability test of a HEK-293 cells and b HT-29 cells by treatment with varying concentrations of LOS (100–1000 µg/ml) for 48 h and 72 h by MTT assay

Conclusion

The hydrolysis of curdlan by β-1,3-endoglucanase (CtLam81A) from Clostridium thermocellum showed the production of laminari-oligosaccharides (LOS), viz. laminaribiose, laminaritriose and higher laminari-oligosaccharides on TLC. LOS stimulated the growth of probiotic bacteria (Lactobacillus acidophilus and Lactobacillus plantarum DM5), while it did not support the growth of non-probiotic bacteria (E. coil and E. aerogenes) indicating the presence of β-1,3-glucanase in the probiotic bacteria. The prebiotic activity score of LOS was comparable to commercial inulin. The probiotic bacteria more rapidly utilized LOS of DP2 and DP3 than higher DPs as a carbon source. Higher resistance (over 95%) of LOS against digestibility by α-amylase, gastric juice and intestinal fluid than commercial inulin confirmed that it can act as a potential prebiotic that can reach the colon and can stimulate the growth of probiotic bacteria. Stability of LOS in the lower pH environment of gastric juice makes it useful for acidic food like yogurt and other dairy products. The probiotic bacteria in presence of LOS releases lactic acid, acetic acid and propionic acid which impart several beneficial properties to the host like immune modulation, histone deacetylase inhibition and the activation of G-protein-coupled receptors. LOS showed nontoxicity to the human embryonic kidney (HEK-293) cells displaying the biocompatible nature. The overall results of this investigation confirmed that the LOS can be used as potential prebiotic additives for functional foods.

Acknowledgments

The authors are grateful to Departmental Central Instrument Facility, Biosciences and Bioengineering, IIT Guwahati for providing the cell culture facility.

Author contribution

AG conceived the idea and designed the objectives. KK performed the prebiotic evaluation of laminarioligosaccharides and VR performed in vitro biocompatibility study. AG, KK and VR wrote the paper.

Compliance with ethical standards

Conflict of interest

There are no conflicts of interest to declare.

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