Maple sap as a rich medium to grow probiotic lactobacilli and to produce lactic acid

Abstract

Aims:  To demonstrate the feasibility of growing lactobacilli and producing lactic acid using maple sap as a sugar source and to show the importance of oligosaccharides in the processes.

Methods and Results:  Two maple sap samples (Cetta and Pinnacle) and purified sucrose were used as carbon sources in the preparation of three culture media. Compared with the sucrose-based medium, both maple sap-based media produced increased viable counts in two strains out of five by a factor of four to seven. Maple sap-based media also enhanced lactic acid production in three strains. Cetta sap was found to be more efficient than Pinnacle sap in stimulating lactic acid production and, was also found to be richer in various oligosaccharides. The amendment of the Pinnacle-based medium with trisaccharides significantly stimulated Lactobacillus acidophilus AC-10 to grow and produce lactic acid.

Conclusions:  Maple sap, particularly if rich in oligosaccharides, represents a good carbon source for the growth of lactobacilli and the production of lactic acid.

Significance and Impact of the Study:  This study provides a proof-of-concept, using maple sap as a substrate for lactic acid production and for the development of a nondairy probiotic drink.

Introduction

There has been an increased interest in the development of nutraceuticals and functional foods, specifically in probiotics. However, there has been an increase in the demand for nondairy-based probiotic products. In a colloquium of the American Academy for Microbiology held in Baltimore in November 2005 (report available at: http://www.asm.org/Academy/index.asp?bid=2093) and in a recent review (Sleator and Hill 2008), it was reported that the use of probiotics could benefit human and animal health, specifically in the prevention or in the treatment of irritable bowel syndrome, bladder and colon cancer, urogenital infections, Clostridium difficile infections, atopic eczema, asthma and diarrhoea caused by Rotavirus in children. Most probiotics belong to the lactic acid bacteria (LAB) family, with many species of Lactobacillus, including Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum.

In addition to the interest in lactobacilli themselves, lactic acid, which is the primary metabolite of LAB, is widely used in pharmaceutical, chemical, cosmetic, textile and food industries. Lactic acid is also used as an acidulant, a preservative agent, and as precursor for the production of base chemicals or biodegradable polymers such as polylactic acid (PLA; Vickroy 1985; Kharas et al. 1994). The worldwide demand for lactic acid is increasing constantly, and is estimated roughly to range between 130 000 and 150 000 metric tonnes per year (Mirasol 1999). Presently, lactic acid is commercially produced by fermentation of sugar cane (Patil et al. 2006; Timbuntam et al. 2006), corn sugars (Mercier et al. 1992), beet molasses (Kotzamanidis et al. 2002) and whey (Wee et al. 2006). Most of these agricultural feedstocks first require a pretreatment such as extraction or hydrolysis of their sugar content, to allow bacterial fermentation to take place.

Forests in Eastern Canada can be considered as large reservoirs of maple sap, which contains between 10 and 30 g l⁻¹ of sucrose with trace amounts of glucose and fructose. Previous studies revealed that it could be directly used as a raw material without any pretreatment for the production of exopolysaccharides by Enterobacter agglomerans (Morin et al. 1995) or for the production of polyhydroxybutyrate (PHB) by fermentation with Alcaligenes latus (Yezza et al. 2007). The aim of this study was to determine the suitability of maple sap for the production of: (i) a high density of viable probiotic lactobacilli, and (ii) lactic acid, a versatile chemical with potential industrial and biotechnological applications.

Materials and methods

Maple sap

The maple saps were collected during the Spring of 2007, immediately frozen and sterilized by filtration through a 0·22-μm filtration unit (Millipore) before use. Maple sap designated Cetta was provided by Mr Djamel Rabia of the ‘Centre d’Expérimentation et de Transfert Technologique en Acériculture’ (Cetta, Pohénégamook, Quebec, Canada). Pinnacle maple sap was kindly provided by a personal contact in Baldwin Mills (Quebec, Canada). Both sap samples were harvested using tubular conduits collecting system in the middle of March.

Chemical analysis of maple saps

All chemicals were from Sigma-Aldrich. Sucrose, glucose and fructose were analysed using high-performance liquid chromatography (HPLC; Waters Chromatography division, Milford, MA, USA) equipped with an injector model 717, a model 600 pump and a 2414 refractive index detector. The separation was made with an ICsep ICE-ION-300 column (Transgenomic, San Jose, CA, USA) of 300 mm × 7·8 mm i.d. and an ion guard GC-801 column (Transgenomics). The mobile phase consisted of a solution of 0·035 N sulfuric acid (pH 4) flowing at 0·4 ml min⁻¹. Analyses of total organic carbon (EPA Method 415·1 modified 1999), metals (EPA Method 6020, 1994) and total nitrogen (ASTM D5291, 2007) in the sap samples were conducted by Maxxam Analytique Inc. (Montreal, Québec, Canada). Oligosaccharides with various degree of polymerization (d.p.) such as raffinose, stachyose, maltotriose and 1-kestose were analysed by liquid chromatography (LC)-mass spectrometry (MS) using a Bruker micrOTOF-Q mass spectrometer attached to a Hewlett-Packard 1200 series HPLC system (Bruker Daltonics, Milton, Canada). The samples were injected into a 5-μm pore size LC-NH₂ column (4·6 mm i.d. × 250 mm; Supelco, Bellefonte, PA, USA) at 35°C. The solvent system was composed of a mixture of CH₃CN (70% v/v)/H₂O (30% v/v) at a flow rate of 1 ml min⁻¹. For mass analysis, positive electrospray ionization mode was used producing sodium adduct ions [M+Na]⁺. The mass range was scanned from 100 to 800 m/z. In order to better characterize the trisaccharides found in Cetta sap, the LC-MS method was modified by eluting the sap through an LC-NH₂ column using a solvent mixture composed of acetonitrile (82%) and water (18%) at a flow rate 1·5 ml min⁻¹. As described before, trisaccharides were detected as their sodium adduct mass [M+Na]⁺ using electrospray mass spectrometry in the positive ionization mode.

Media

One litre of a sucrose-based medium was prepared by mixing the following autoclaved solutions: 50 ml of a 400 g l⁻¹ stock solution of veggietones pea (Oxoid), 25 ml of a 200 g l⁻¹ stock solution of yeast extract (Difco), 10 ml of a 200 g l⁻¹ stock solution of K₂HPO₄ (Sigma), 10 ml of a 5 g l⁻¹ stock solution of MnSO₄ (Sigma), 10 ml of a 20 g l⁻¹ stock of MgSO₄ (Sigma) and 895 ml of a filtered-sterilized sucrose solution at 22 g l⁻¹ (purified sucrose; EM Science, Gibbston, NJ), providing 20 g l⁻¹ of sucrose in the final mixture. The maple sap-based media were prepared similarly, using 895 ml of either maple sap Cetta or Pinnacle instead of the 22 g l⁻¹ sucrose solution. As determined by HPLC, the final concentration of sucrose in the two maple sap-based media, Cetta and Pinnacle, were 19·00 and 16·47 g l⁻¹, respectively. For some experiments, the Pinnacle-based medium was also amended with trisaccharides such as raffinose and maltotriose, each one added at a final concentration of 1 g l⁻¹.

Micro-organisms and growth conditions

Lactobacillus acidophilus R0240 and Lactobacillus helveticus R0052 were kindly provided by Dr Thomas Tompkins (Institut Rosell-Lallemand Inc., Montreal, Quebec, Canada) and Lactobacillus rhamnosus (designated strain AC-3) was isolated from a fresh, commercial white cheese. Two other lactobacilli, designated as L. casei AC-8 and L. acidophilus AC-10 were isolated from a concentrated nondairy-based commercial probiotic product. The identity of L. rhamnosus AC-3, L. casei AC-8 and L. acidophilus AC-10 was confirmed by comparing their 16S rDNA gene sequence, with the 16S rDNA sequences in the NCBI database (data not shown; Altschul et al. 1997).

All bacterial strains used in this study were started from a glycerol stock and streaked on De Man Rogosa Sharpe (MRS) agar (De Man et al. 1960). MRS plates were incubated at 37°C under anaerobic conditions (Gas Pack anaerobic jar system, BBL). One CFU was used to inoculate precultures in 10 ml of MRS broth for 16 h. Two per cent (v/v) of the MRS precultures were used to inoculate 10 ml of a maple sap (Cetta and Pinnacle) or a sucrose-based medium, which was incubated overnight at 37°C in 15 ml conical tubes (Falcon; BD Biosciences, Franklin Lakes, NJ) under static conditions. Subsequently, 20-ml cultures were started in either maple sap or in sucrose-based media by adding 2% (v/v) of the corresponding maple sap or sucrose preculture in 20-ml serum bottles followed by incubation for 16 h at 37°C under static conditions. At T₀ of growth, initial bacterial population was c. 7 log CFU ml⁻¹ for all bacteria, except for L. acidophilus R0240, which was c. 6 log CFU ml⁻¹.

Analysis of culture media

Aliquots of culture medium were collected and analysed at T0 and after 16 h of incubation as follows: (i) 0·1 ml was used to determine the viable cell count (CFU), which was carried out by diluting the samples in 0·1% (wt/v) peptone water, and spread plating in duplicate on soft MRS agar plates, which were incubated at 37°C for 48 h under anaerobic conditions (Gas Pack anaerobic jar system, BBL); (ii) 1 ml was used to determine A_600 nm; (iii) 3 ml was centrifuged at 12 000 g for 15 min, for pH analysis and analysis of lactic acid and residual sugars by HPLC using similar conditions as those described before.

Results

Composition of maple sap samples

Table 1 describes the composition of both Cetta and Pinnacle maple sap samples. As determined by HPLC, both Cetta and Pinnacle sap contained 21·0–18·2 g l⁻¹ of sucrose, respectively, and low concentrations of glucose (0·1–0·15 g l⁻¹) and fructose (0·02–0·08 g l⁻¹). Cetta and Pinnacle saps did not markedly differ in their total organic carbon and total nitrogen concentrations. However, the two sap samples differed in their nitrate, sulfate and sodium content. For example, the concentration of nitrate and sulfate were 5- and 20-fold higher in Cetta sap, but in Pinnacle the concentration of sodium was 50-fold lower (data not shown). Calcium, magnesium and manganese were also present in higher amounts in Cetta sap than in Pinnacle sap; however, the potassium contents were slightly similar in both saps (data not shown).

Table 1.   Composition (in g l⁻¹) of the maple sap samples used

Sap component	Cetta maple sap (Pohenegamook, Quebec, Canada)	Pinnacle maple sap (Balwin Mills, Quebec, Canada)
Fructose	0·080	0·026
Glucose	0·145	0·098
Sucrose	21·001	18·201
Total organic carbon	10·300	9·210
Total nitrogen	<0·100	<0·100

Characterization of oligosaccharides in maple sap

LC-MS analysis of Cetta and Pinnacle saps revealed the presence of oligosaccharides with d.p. ranging from 3 to 5 (Fig. 1). The area of the peaks corresponding to oligosaccharides with a d.p. of 4 and 5 were found to be greater in Cetta than in Pinnacle sap, but those corresponding to trisaccharides (d.p. 3) were found to be c. 11 times greater in Cetta than in Pinnacle sap (5·6 × 10⁷ and 4·8 × 10⁶, respectively) (Fig. 1).

Figure 1.  Liquid-chromatography–mass spectrometry profile of the oligosaccharides with degree of polymerization (d.p.) from 3 to 5 in Cetta (a) and Pinnacle (b) maple saps obtained by ESI-Qq-TOF mass spectrometer using positive ionization mode.

To better characterize the d.p. 3 oligosaccharides shown in Fig. 1, a modified LC-MS method was employed to improve separation of overlapping signals to enhance their detection. Figure 2a represents a typical LC-MS chromatogram of trisaccharides in Cetta maple sap. The peak at 25·2 min exhibits a sodium adduct mass [M+Na]⁺ at 527 Da with a retention time (r.t.) similar to that of 1-kestose (β-d-Fruf-(2→1)β-d-Fruf(2→1)α-d-Glup). The minor LC-MS peak was observed with a r.t. at 30·1 min and a [M+Na]⁺ at 527 Da and matched the two trisaccharides, raffinose (O-α-galactopyranosyl(1→6)α-d-glucopyranosyl-β-d-fructofuranoside) and maltotriose (O-α-dd-glucopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→4)-d-glucose), with each showing the same r.t. at 30·1 min and [M+Na]⁺ at 527 Da. The identities of these sugars and the major compound eluting at 21·5 min will be discussed latter.

Figure 2.  Extracted ion chromatograms ([M+Na]⁺ at m/z 527) of the trisaccharides from Cetta maple sap (a), 1-kestose spiked (b), raffinose spiked (c) and maltotriose spiked (d) Cetta maple sap.

Growing lactobacilli in sucrose- and maple sap-based media

The growth of L. acidophilus R0240, L. helveticus R0052 and L. acidophilus AC-10 resulted in a drop in pH (from 7·2 to below 4·5, not shown), and significant sucrose consumption (1·3–6·0 g l⁻¹; Table 2). Whichever medium was used, with the exception of L. acidophilus R0240 that had low final bacterial counts, the viable counts of all other strains reached c. 9 log CFU ml⁻¹ after 16 h of fermentation at 37°C (Fig. 3a). Extending the fermentation time from 16 to 24 h did not result in an increased viable count (data not shown). While L. rhamnosus AC-3, L. acidophilus R0240 and L. casei AC-8 grew similarly in the sucrose- and maple-based culture media, L. helveticus R0052 and L. acidophilus AC-10 grew four to seven times more in the maple sap-based media (Fig. 3a) and also produced more lactic acid (1·89–4·94 g l⁻¹) than the other strains (Fig. 3b). No other volatile organic acids such as acetic acid were detected in the cultures. For all cultures, except for L. casei AC-8, Cetta maple sap led to higher lactic acid production than the Pinnacle maple sap. It is interesting to note that L. helveticus R0052 and L. acidophilus AC-10 both consumed increased amounts of sucrose when grown in maple sap-based media (Table 2). On the other hand, Table 2 shows that the three other strains (AC-3, R0240 and AC-8) generally consumed comparable amounts of sucrose in sucrose-based and in Pinnacle-based media, but much less in Cetta-based medium. To understand what factors in maple sap could contribute to increased growth, sucrose consumption and lactic acid production, complementary experiments of bacterial growth in maple sap-based media were performed.

Table 2.   Sucrose consumption (in g l⁻¹) by various lactobacilli after 16 h of fermentation in purified sucrose-based medium or in maple sap-based media*

Strains	Sucrose-based medium	Cetta-based medium	Pinnacle-based medium
*The initial concentrations of sucrose in sucrose-, Cetta- and Pinnacle-based media were respectively, 20·0, 19·0 and 16·5 g l^−1. Glucose and fructose were totally consumed by all cultures.
Lactobacillus rhamnosus AC-3	1·05 ± 0·13	0·00 ± 0·04	1·01 ± 0·16
Lactobacillus acidophilus R0240	2·41 ± 0·29	1·26 ± 0·09	2·47 ± 0·13
Lactobacillus helveticus R0052	3·42 ± 0·04	5·64 ± 0·11	4·41 ± 0·08
Lactobacillus casei AC-8	0·92 ± 0·11	0·48 ± 0·09	0·95 ± 0·24
Lactobacillus acidophilus AC-10	3·20 ± 0·10	5·95 ± 0·03	4·57 ± 0·12

Figure 3.  Bacterial viable count (a) and lactic acid produced (b) by lactobacilli in sucrose-based (black), Cetta (white) or Pinnacle (grey) sap-based media after 16 h of fermentation at 37°C. CFU, colony-forming units; L., Lactobacillus. Values indicate averages from two distinct cultures and bars represent the SE.

Indeed, the two sap samples, Cetta and Pinnacle, varied in their chemical compositions (Table 1) and we attributed the variation in lactic acid production (no significant difference was observed on bacterial growth) between the two samples as due to cumulative effect of maple sap components. To demonstrate the effect of oligosaccharides in promoting lactobacilli growth and in increasing their lactic acid production, we monitored the disappearance of the oligosaccharides initially present in the Cetta maple-based medium. After 16 h of incubation, LC-MS analysis showed that L. helveticus R0052 and L. acidophilus AC-10 consumed c. 70% of the d.p. 4 oligosaccharide appearing at 19·2 min and 50% of the major d.p. 3 oligosaccharides shown in Fig. 2a (data not shown). Since Pinnacle sap contained less oligosaccharides, we thus amended the Pinnacle sap-based medium with a mixture of two d.p. 3 oligosaccharides, raffinose (1 g l⁻¹) and maltotriose (1 g l⁻¹) and used it to grow L. acidophilus AC-10. Because A_600 nm was previously found to perfectly correlate with CFU (data not shown), this parameter was used to measure bacterial growth. The lactobacilli grew approximately three times more in the Pinnacle medium amended with raffinose and maltotriose, and produced double the concentration of lactic acid than when grown in the nonamended Pinnacle medium (Table 3). It should be noted that the average lactic acid concentration obtained in the nonamended Pinnacle sap-based medium presented in Table 3 is different from the average lactic acid concentration previously shown Fig. 3b (2·95 and 1·89 g l⁻¹, respectively). Despite the fact that the 20-ml, 16-h culture was done similarly, the incubation times of the precultures varied between the two experiments explaining the variation observed.

Table 3.   Growth of Lactobacillus acidophilus AC-10 as measured by the A_600 nm and production of lactic acid in Pinnacle-based medium and Pinnacle-based medium amended with raffinose (1 g l⁻¹) and maltotriose (1 g l⁻¹) after 16 h of incubation at 37°C

Medium	A600 nm	Lactic acid (g l^−1)
Values indicate averages from two distinct cultures.
Pinnacle sap	0·58 ± 0·058	2·95 ± 0·102
Amended Pinnacle sap	1·60 ± 0·066	5·95 ± 0·260

Discussion

In this study, we have investigated the use of maple sap as a new feedstock for the production of probiotic lactobacilli and lactic acid. The LAB strains were selected based on their ability to yield high concentrations of viable cells when grown on maple sap-based media (Fig. 3a). Our results clearly demonstrate that (i) maple sap constitutes a good carbon source; (ii) supported the growth of L. rhamnosus AC-3, L. helveticus R0052, L. casei AC-8 and L. acidophilus AC-10 at c. 9 log CFU ml⁻¹ and (iii) allowed L. helveticus R0052 and L. acidophilus AC-10 (the best lactic acid producers of the study) to produce up to 5 g l⁻¹ of lactic acid after 16 h of fermentation at 37°C in Cetta sap-based medium (Fig. 3b). Figure 3 also shows that L. rhamnosus AC-3 and L. casei AC-8 are good candidates for production of probiotics because lactic acid if produced would be in trace amounts. Viable bacterial counts and lactic acid production were correlated with significant sucrose consumption. These results show that maple sap was favourable compared with other sucrose-based feedstocks, such as beet juice and soymilk containing 58 and 28 g l⁻¹ of sucrose respectively, each of which were shown to support the growth of lactobacilli (8–9 log CFU ml⁻¹) and the production of lactic acid (2·3–5·3 g l⁻¹) under similar culture conditions, i.e. batch cultures conducted without pH control or lactic acid removal (Garro et al. 1998; Yoon et al. 2005).

The increased lactic acid production and sucrose consumption by L. helveticus R0052 and L. acidophilus AC-10 in Cetta maple sap compared with Pinnacle maple sap led us to search for variations in their chemical compositions. The maple sap samples came from two different regions of Quebec and literature indicates that the location and several other factors that include, the age of maple trees, period and method of maple harvest, will largely influence sap composition in terms of sugars, minerals, phenolic compounds, vitamins and organic acids (Morselli 1975; Kuentz et al. 1976; Stuckel and Low 1996). Despite the fact that some differences between the two saps were noted in the concentrations of nitrate, sulfate, sodium, magnesium and manganese, the amendment of sap with culture medium components such as phosphate, yeast extract, veggietones pea, manganese and magnesium likely rendered these differences insignificant. Also because sucrose was always partially consumed by the lactobacilli cultures, the difference in sucrose concentration does not explain the difference in growth and lactic acid production observed in the two sap samples, Cetta and Pinnacle.

However, the most significant difference observed between the two maple sap samples was in their content of oligosaccharides. Both maple saps revealed the presence of oligosaccharides with a d.p. ranging from 3 to 5. We found that Cetta sap that contained a higher content of oligosaccharides, particularly trisaccharides, showed a slightly higher bacterial growth and higher yield of lactic acid than Pinnacle. For example, Cetta sap was found to contain 1-kestose as one of the two major trisaccharides as confirmed by comparison with a reference standard (Fig. 2a,b). The second major trisaccharide was tentatively identified as neokestose (Fig. 2a) based on previous assignment made by Haq and Adams (1961) who also reported neokestose as a major trisaccharide in maple sap. Whereas the minor LC-MS peak with a r.t. at 30·1 min was tentatively identified as raffinose as confirmed by Porter et al. (1954) who reported the presence of this sugar based on detailed structural analysis of the trisaccharide in maple sap. However, more recently Bazinet et al. (2007) suggested the trisaccharide to be a maltotriose based on HPLC/refractive index analysis alone. Further confirmation to our assignments of the three trisaccharides was obtained by proper spiking of each analyte separately during HPLC analysis (Fig. 2b–d).

Literature reports indicated that raffinose-like oligosaccharides could enhance the acidification rate and the population levels of strains of L. acidophilus and Bifidobacterium lactis (Martínez-Villaluenga et al. 2005). More recently, it was shown that also fructo-oligosaccharides can enhance the production of various bacteriocins by LAB (Chen et al. 2007) and the soybean fructo-oligosaccharides, inulin and raffinose, are able to enhance the growth of various probiotic bacteria (Su et al. 2007). In the present study, when the Pinnacle-based medium was amended with the two trisaccharides, raffinose (1·0 g l⁻¹) and maltotriose (1·0 g l⁻¹), L. acidophilus AC-10 grew approximately three times faster and the lactic acid yield increased from 2·95 g l⁻¹ to 5·95 g l⁻¹.

In conclusion, our results revealed that maple sap can be considered as a remarkable renewable feedstock for developing a nondairy drink with probiotic lactobacilli. Among the strains tested, L. rhamnosus AC-3 and L. casei AC-8 represented the best choices for the probiotic drink owing to their high viable cells count and low lactic acid production. Finally, maple sap-based media may serve as a convenient substrate for significant lactic acid production by L. helveticus R0052 and L. acidophilus AC-10 without pretreatments of maple sap.

Acknowledgements

The authors wish to thank Stéphane Deschamps, Chantale Beaulieu, Louise Paquet, Karine Trudel and Alain Corriveau for their excellent technical assistance. They also thank Punita Mehta for the revision of the manuscript.