TITLE: Bioproduction of ferulic acid and uses thereof

BACKGROUND OF THE INVENTION

Field of the invention

[0001] The present invention relates to novel bioproduction of ferulic acid based on the use of encapsulated feruloyl esterase producing cells and uses thereof.

Description of Prior Art

[0002] Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid), one of the most abundant hydroxycinnamic acids in plants, is a very attractive phenolic compound.

ferulic acid (FA)

[0003] Ferulic acid and its dimers are ubiquitous components of the primary cell walls of plants and commonly found in rice, wheat, barely, oat, sugar beet, maize, roasted coffee, tomatoes, vegetables and citrus fruits. FA is a strong membrane antioxidant and is purported to protect against cancer, cardiovascular diseases, hepatotoxicity, colds, influenza, skin aging, and muscle wasting. The health benefits of FA are now receiving much attention in the research world.

[0004] FA exhibits a number of potential commercial applications such as natural antioxidant, food preservative and anti-inflammatory agent. Other potential uses of ferulic acid are related to pharmaceutical applications as the active ingredient in skin lotions and sunscreen, ergogenic substance in sport foods or as part of the gel matrix of wound dressings. In addition, ferulic acid is reported to have many physiological functions, including antioxidant, antimicrobial, anti-inflammatory, anti-thrombosis, and anti-cancer activities.

[0005] FA can also be transformed by microorganism into "natural" vanillin, the main flavor component of vanilla which is used in the food,

cosmetic and pharmaceutical industries. Ferulic acid is thus a highly versatile phytochemical and its controlled release from plant material, particularly from agro-food by-products, is of economic interest to some industries.

[0006] FA can be produced either by chemical synthesis (but it leads to a mixture of trans- and cis- isomers), by extraction form plant cell walls (usually very expensive since yield is low), from low molecular weight ferulic conjugates (inefficient) or by microbial fermentation (batch process).

Preparation of ferulic acid by chemical synthesis

[0007] It is well-known that ferulic acid can be prepared by the condensation reaction of vanillin with malonic acid catalyzed by piperidine. This method produces ferulic acid as a mixture of trans- and cis-isomers. The yield is high, but it takes as long as three weeks to complete the reaction. An improved method uses benzylamine as the catalytic agent, methylbenzene as the solvent and a reaction temperature of 85-95°C. The improved method increased the yield and reduced the reaction time to 2 h.

Preparation of ferulic acid from natural sources

[0008] There are three pathways to prepare ferulic acid from natural ressources: (1) from low-molecular-weight ferulic conjugates, (2) from plant cell walls, and (3) by tissue culture or microbial fermentation. Several feruloyl esters of triterpene alcohols and sterols were isolated from the methanol extract of rice bran, of which the main component is Y-oryzanol, accounting for about 1.5-2.8% of rice bran oil by weight. Ferulic acid has been prepared in large quantities from rice bran pitch, a blackish brown waste oil discharged in the process of the production of rice bran oil. In this process, the bran oil waste material was hydrolyzed with sodium hydroxide or potassium hydroxide at 90-100 ⁰ C for 8 h under atmospheric pressure, producing crude ferulic acid with purity of 70-90%. The solution containing alkaline salt of ferulic acid was acidified with dilute sulfuric acid to precipitate ferulic acid.

[0009] There are many reports on the use of feruloyl esterases produced by microorganisms to release ferulic acid from plant cell walls.

These enzymes are secreted by fungal, bacterial and yeast microbes, such as Aspergillus niger, Pycnoporus cinnabarinus, Streptomyces avermitilis, Clostridium thermocellum, Bacillus spp, Lactobacilli, Pseudomonas fluorescens and Brettanomyces anomalus. Although feruloyl esterases are not yet commercially used to prepare ferulic acids, extensive research studies have been carried out. Cell suspension cultures of Beta vulgaris, Zea mays and Chenopodium rubrum have been reported to accumulate ferulic acid. In contrast to the plant cultures mentioned above, cell cultures of Ajuga pyramidalis accumulated free ferulic acid also.

[0010] Currently genetically engineered bacterial systems (E.coli and

R.eutropha) are being used and their yield is about 93.3% after 30 hours. Earlier reports illustrate that a molar yield of less than 3% was obtained during fermentation of spent barley grain using fungal feruloyl esterase from A. niger (Bartolome et al.,1997).

[0011] At present, ferulic acid is produced by fermentation and these methods are limited due to batch processes (Bartolome et al.,1997; Faulds et al.,1995).

[0012] None of the fermenting methods known to date can be operated in a continuous mode and thus require a cleaning and reloading of the biorector.

[0013] Therefore, it would be highly desirable to be provided with a novel process for the bioproduction of ferulic acid which would overcome the drawbacks of the prior art.

SUMMARY OF THE INVENTION

[0014] In accordance with the present invention, there is provided a novel process for the bioproduction of ferulic acid which overcome the drawbacks of the prior art.

[0015] In accordance with one embodiment of the present invention, there is provided process for producing ferulic acid, which comprises cultivating encapsulated live feruloyl esterase producing cells in a culture

media, wherein said cells are encapsulated in semipermeable polymeric microcapsules.

[0016] In accordance with another embodiment of the present invention, the process further comprises the step of isolating ferulic acid from said culture media.

[0017] The process is preferably a continuous process.

[0018] In accordance with another embodiment of the present invention, the live feruloyl esterase producing cells are feruloyl esterase producing bacteria, feruloyl esterase producing yeast cells and feruloyl esterase producing genetically engineered cells.

[0019] The preferred live feruloyl esterase producing bacteria are feruloyl esterase producing Lactobacillus or Bacillus bacterial cells.

[0020] The preferred feruloyl esterase producing Lactobacillus or

Bacillus bacterial cells are chosen from Lactobacillus fermenum 11976, Lactobacillus leichmanni NCIMB 7854, Lactobacillus farciminis NCIMB 11717, Lactobacillus fermentum NCFB 1751 , Lactobacillus fermentum NCIMB 2797, Lactobacillus reuteri NCIMB 11951 , Bacillus subtilis FMCC 193, Bacillus subtilis FMCC 267, Bacillus subtilis FMCC PL-1, Bacillus subtilis FMCC 511 , Bacillus subtilis NCIMB 11034, Bacillus subtilis NCIMB 3610, Bacillus pumilis ATCC 7661 , Bacillus sphaericus ATCC 14577 and Bacillus licheniformis ATCC 14580.

[0021] The most preferred feruloyl esterase producing Lactobacillus or

Bacillus bacterial cells are chosen from Lactobacillus fermentum 11976 bacterial cells, Lactobacillus fermentum 14932, Lactobacillus reuteri 23272 and Lactobacillus farciminis 29645.

[0022] The preferred live feruloyl esterase producing yeast cells are feruloyl esterase producing Aureobasidium, Pichia, Candida, Rhodotorula or Saccharomyces yeast cells and feruloyl esterase producing genetically engineered cells.

[0023] The microcapsules in accordance with the present invention are made of a material chosen from Alginate-Poly-L-lysine-Alginate [APA], Alginate-Chitosan [AC], Alginate-Chitosan-Polyethylene glycol (PEG)-PoIy-L- lysine (PLL)-Alginate [ACPPA], Alginate -Poly-L-lysine-PEG-Alginate [APPA], Alginate-Chitosan-PEG [ACP], Alginate-Poly-L-lysine - Pectinate-Poly-L- lysine-Alginate [APPPA], Genipin cross-linked alginate-chitosan (GCAC).

[0024] The preferred microcapsules are made of Alginate-Poly-L- lysine-Alginate [APA].

[0025] In accordance with another embodiment of the present invention, there is provided in a process of food preparation, the improvement consisting in the use of encapsulated live feruloyl esterase producing cells, wherein said cells are encapsulated in semipermeable polymeric microcapsules and are producing ferulic acid.

[0026] In accordance with another embodiment of the present invention, there is provided the use of an of encapsulated live feruloyl esterase producing cells in food processes, wherein said cells are encapsulated in semipermeable polymeric microcapsules and are producing ferulic acid, wherein said food processes comprises at least one selected from the group consisting of: a) modification of plant-based food texture; b) baking industry; c) generation of fine chemicals from food waste; d) animal nutrition; and e) wine making.

[0027] In accordance with another embodiment of the present invention, there is provided the use of an of encapsulated live feruloyl esterase producing cells in industrial processes, wherein said cells are encapsulated in semipermeable polymeric microcapsules and are producing

ferulic acid, wherein said industrial processes comprises at least one selected from the group consisting of: a) bleaching high quality paper pulps; b) anti-aging agents; c) antibacterial activity items; d) fragrances; and e) control of germination.

[0028] In accordance with another embodiment of the present invention, there is provided a method of producing the precursor of vanillin which comprises cultivating encapsulated live feruloyl esterase producing cells in a culture medium, wherein said cells are encapsulated in semipermeable polymeric microcapsules.

[0029] The present invention preferably relates to a new continuous ferulic acid production process based on encapsulated feruloyl esterase producing cells with a semi-permeable APA membrane. The encapsulated cells of the present invention is a new and more efficient (yield and cost) way of producing FA. The encapsulated cells of the present invention can be used for continuous fermentation whereas traditional ways of producing FA is by conventional microbial fermentation batches. Also, using encapsulated cells increases cells density and protects them during fermentation, besides which it minimizes the need to clarify or separate the cells from the spent liquor containing the product.

[0030] All references referred herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 illustrates the mode of action for hydrolysis of substrate bound ferulic acid and production of ferulic acid of the microencapsulated lactobacilli of the present invention.

[0032] Figure 2 illustrates feruloyl esterase activity detected by the plate-assay method (A): MRS-EFA agar negative control with no bacteria (0 mm) (B): Lactobacillus fermentum 11976 on MRS-EFA (12.2 + 1mm).

[0033] Figure 3 illustrates photomicrographs of freshly prepared alginate-poly-L-lysine alginate (APA) microcapsules (A) control microcapsules (empty) (B) test microcapsules encapsulating FAE producing Lactobacillus fermentum 11976 cells. Magnification 7Ox, microcapsule size 602+30 μm.

[0034] Figure 4A illustrates overlaid HPLC chromatograms of the samples (10 h) from the experiment in which microencapsulated lactic acid bacteria were used to de-esterify 1.33 mM ethyl ferulate. The peak areas of ferulic acid indicate feruloyl esterase activity of (A) L. farciminis, (B) L. reuteri, (C) L. fermentum 11976, and (D) L. fermentum 14932 microcapsules as compared to sham microcapsule control.

[0035] Figure 4B illustrates overlaid HPLC chromatograms of ferulic acid released over time (0,2,4,6,8, and 10 hours) from ethyl ferulate in vitro. Peak areas of ferulic acid indicate the feruloyl esterase activity of Lactobacillus fermentum 11976 microcapsules. Benzoic acid was used as internal standard for HPLC experiments.

[0036] Figure 5 illustrates feruloyl esterase activity of Lactobacillus fermentum 11976 microcapsules from ethyl ferulate in in vitro conditions. The concentration of ferulic acid is shown over time. Empty microcapsules in simulated intestinal fluid containing ethyl ferulate were used as control.

[0037] Figure 6 illustrates microencapsulated bacterial activity in vitro.

A change in pH of the culture medium was observed due to encapsulated bacterial cell activity when compared to empty microcapsule control after 10 hours of incubation. The experiment was designed to investigate the impact of bacterial metabolic activity on pH, as well as to investigate if change in pH due to the metabolic activity of bacterial cells affected ferulic acid release.

[0038] Figure 7A illustrates free L. fermentum 11976 feruloyl esterase cell viability in low pH conditions.

[0039] Figure 7B illustrates microencapsulated L. fermentum 11976 feruloyl esterase cell viability in low pH conditions.

DETAILED DESCRIPTION OF THE INVENTION

[0040] Biotechnological production of ferulic acid, a precursor of vanillin, is an attractive alternative for various industries due to the high price and demand for natural ferulic acid. Feruloyl esterase has been identified as a key enzyme involved in microbial transformations of ferulic acid to vanillin. Several fungal feruloyl esterases have been purified and characterized for their use in the production of ferulic acid. The present invention relates to the novel use of lactic acid bacteria for the production of ferulic acid. Preferably, Lactobacillus cells and microencapsulation are used to produce ferulic acid continuously using various types of fermentation systems. Bacteria are encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules and the production of ferulic acid by lactobacilli is detected using a real time HPLC based assay. Results show that ferulic acid can be produced using microencapsulated Lactobacillus fermentum (ATCC 11976) with significant levels of biological feruloyl esterase activity.

MATERIALS AND METHOD

1. Bacterial growth media and chemicals:

[0041] Ethyl ferulate (ethyl 4-hydroxy-3-methoxycinnamate), Alginic acid sodium salt (Cat# A2158-250G) and Poly-L-lysine (Cat# P7890-1G) are purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario). De Man- Rogosa-Sharpe (MRS) broth is obtained from Difco (Sparks, Md). The water is purified with an EASYpure Reverse Osmosis System and a NANOpure Diamond Life Science (UV/UF) ultrapure water system from Barnstead/Thermoline (Dubuque, Iowa). Methanol is HPLC-gradient from Fisher Scientific (Fair Lawn, NJ). All other chemicals are purchased from commercial sources and are of analytical or HPLC grade.

2. Bacterial strains and culture conditions:

[0042] The bacterial strains used, Lactobacillus fermentum 11976,

Lactobacillus fermentum 14932, Lactobacillus re uteri 23272 and Lactobacillus farciminis 29644, are known feruloyl esterase producing lactic acid bacteria sourced from ATCC (Manassas, VA). All strains are maintained in Difco's De Man, Rogosa, and Sharpe (MRS) medium containing 30% (v/v) glycerol at -8O ⁰ C and serially propagated three times in fresh MRS medium before experimental use. Lactobacilli are then cultivated in MRS-EFA broth (MRS supplemented with 10% ethyl ferulate w/v in dimethylformamide)). A 1 % inoculum is used, and incubations are performed at 37°C for 2Oh in microaerophilic conditions (5% CO ₂ ).

3. Direct plate assay for confirmation of feruloyl esterase activity:

[0043] Qualitative feruloyl esterase activity of the growing culture is evaluated in a direct agar-plate assay using the method described by Donaghy et al. with some modifications. The assay involves the substitution of the main carbon source (glucose) in MRS agar pH 6.5 with 0.3ml sterile ethyl ferulate (10% w/v in dimethylformamide) at the plate-pouring stage. This supplement is immediately mixed, by swirling, with the agar medium to ensure a homogeneous distribution (a cloudy haze) throughout the plate. Sterile filter disks (Whatman #3) are impregnated in a 2Oh MRS-EFA broth culture of the test strain (L. fermentum 11976) during growth, and placed on MRS-EFA agar plates, and incubated for a maximum of 3 days at 37°C, after which the diameters of the clearance zones are measured. MRS-EFA agar plates in which sterile filter paper disks are placed are used as controls. The formation of a clearing zone around the disks indicates feruloyl esterase production.

[0044] Feruloyl esterases release aromatic residues, such as ferulic acid, from plant cell wall polymers and a range of esterified substances (Figure 1). Free live cells are tested for feruloyl esterase activity using ethyl ferulate as substrate by a direct plating assay. The feruloyl esterase activity

of the natural isogenic L fermentum 11976 was confirmed by observation of zones of clearance in comparison to the control (Figure 2). After 72 hour incubation, plates containing L. fermentum 11976 feruloyl esterase impregnated discs show a clearance zone of 12.2 + 1 mm, while the control did not show any zone formation.

4. Microencapsulation of bacterial cells and their enumeration:

[0045] Microencapsulation is carried out with 36h old cultures of the bacteria grown in MRS-EFA broth using an lnotech Encapsulator [Inotech Biosystems Intl, Inc., MD, USA]. The cultures are centrifuged at 8,000rpm for 20 mins at 15 ⁰ C. The supernatant is decanted off. The pelleted cell wet weight is re-suspended in saline, pooled and gently added to 60ml of a lightly stirred sterile 1.8% sodium alginate solution. Encapsulation parameters are kept constant: Cell load 4.17g CWW/IOOml alginate, Nozzle diameter 300μm, Vibration frequency 918 Hz, Syringe pump speed 22.0, Voltage > 1.0OkV and Current 2 A. The beads are allowed to gel for 5 min in a gently stirred sterile CaCb solution, followed by a wash in physiological saline (PS). It is consequently coated for 10 min with 0.1% sterile poly-L-lysine (PLL) followed by a wash with PS. A final coat of 0.1% sterile alginate followed for 10 min; trailed by a final wash with PS. Bacteria-free empty capsules are prepared as controls. This procedure is performed in a Microzone Biological Containment Hood (Microzone Corporation ON, Canada) to assure sterility. The microencapsulated lactobacilli are stored in minimal solution (10% MRS and 90% physiologic solution) and kept at 4°C until further use. The bacterial numbers of the Lactobacillus strain in the microcapsules are determined by plate counts on MRS agar. The bacteria are pour-plated at the end of the refrigerated storage time. For each bacterium, storage fluid was aspirated; the microcapsules crushed and re-suspended in 0.9mL of physiological saline (PS). Samples are serially 10-fold diluted in diluent. Triplicate plates are inoculated with 0.5mL samples from the appropriate dilutions and incubated under 5% CO ₂ at 37 ⁰ C for 72 h.

[0046] Artificial cell microcapsules containing feruloyl esterase producing lactobacilli were prepared using the methods described above and were stored at 4°C for use in experiments. Sterile conditions and procedures were strictly adhered to during the process of microencapsulation. APA microcapsules containing live Lactobacillus cells were prepared and the process was optimized using the lnotech Encapsulator. The parameters were optimized for optimal shape and size of the capsules using the following settings: internal nozzle diameter 300 μm; vibrational frequency 918 Hz; syringe pump speed 21.0 and voltage 1.5 kV. The bacterial cells were able to survive the encapsulation process without any significant loss of viability (p>0.05) and grow normally when obtained supernatant was plated after breaking of the microcapsule membrane. The microcapsules contained, on an average, 5 x 10 ¹¹ cfu/mL of bacteria. Figure 3 compares photomicrographs of freshly prepared empty APA microcapsules, which are translucent and lactobacilli containing APA microcapsules, which are opaque on account of bacterial density. Morphological studies by microscopic analysis revealed that the mean capsule diameter was 602 + 30 μm for both empty and bacteria loaded capsules and the encapsulated bacterial cells appeared to grow evenly distributed within the membrane.

5. Investigation of suitability of microcapsule membrane for encapsulation of feruloyl esterase producing bacterial cells:

[0047] The viability and sensitivity of the encapsulated bacteria is evaluated to the APA membrane by storing the encapsulated bacteria in minimal solution at 4 ^° C for two months. The bacterial numbers of the Lactobacillus strain in the microcapsules are determined by plate counts in MRS agar. The bacteria are pour-plated at the end of the refrigerated storage time. For each bacterium, storage fluid is aspirated; the microcapsules crushed and re-suspended in 0.9mL of physiological saline. Samples are serially 10-fold diluted in diluent. Triplicate plates are inoculated with 0.5ml_ samples from the appropriate dilutions and incubated under 5% CO2 at 37°C for 72 h.

6. Experimental method of ferulic acid production by encapsulated live bacterial cells:

[0048] An HPLC method is used to investigate the real time feruloyl esterase activity of the microencapsulated feruloyl esterase producing lactobacilli. For this, five grams of microencapsulated Lactobacillus reuteri, Lactobacillus farciminis, Lactobacillus fermentum 11976 and Lactobacillus fermentum 14932. In all experiments empty capsules with no bacterial cells are used as control. For each experiment fresh 15ml MRS broth, supplemented with 1.33mM ethyl ferulate, adjusted to pH 6.6 prior to autoclaving is used.

[0049] Samples are taken after 10-hours of incubation and the ferulic acid concentration is assayed in the reaction vessels using the method discussed below.

7. Real time assay of the Feruloyl esterase activity of microencapsulated bacterial cells: a. HPLC standard curve preparation:

[0050] Ferulic acid is dissolved in minimal volume of ethanol prior to mixing with water and standards are constructed with 0, 100, 300, 500, 700 μM ferulic acid in 1 mL total volume of MRS. The standards are acidified with 0.1 ml of 0.35M H ₂ SO ₄ , vortexed and supplemented with 0.3 mL of 1mM benzoic acid as an internal standard. 0.1mL of 0.7 M NaOH is added and the standards are then filtered with 0.45μm Millipore filter. b. Feruloyl esterase assay method:

[0051] The assay is modified from Mastihuba et al. and is based on the measurement of ferulic acid released from ethyl ferulate (Mastihuba et al.,2002). Both the microcapsules and ethyl-ferulate-MRS broth are preheated to 37°C before mixing. Microencapsulated lactic acid bacteria and controls are incubated in the reaction broth at 37 ^° C with 5% CO ₂ and sampled after 10 hours. At the time points, a 0.3mL sample of the supernatant is transferred to a sterile centrifuge tube, promptly acidified with 0.1 ml of 0.35M H ₂ SO ₄ and

vortexed. Then 0.3 mL of 1mM benzoic acid and 0.1 ml_ of 0.7 M NaOH are added and the entire mixture frozen in an eppendorf tube at -20°C. c. HPLC based sample analysis:

[0052] Samples are thawed at room temperature, and the solution is mixed by vortexing, passed through a 0.45μm syringe filter, and analyzed by HPLC. Analysis is performed on a reverse-phase C-18 column: LiChrosorb RP-18, 5 μm, 250 x 4.6mm (Richard Scientific, USA). The HPLC system comprised of two ProStar 210 solvent delivery modules, a ProStar 335 Diode Array Detector (DAD), a ProStar 410 autosampler, and Star LC Workstation Version 6.41 software. The acidic components of the samples are eluted with a mixture of water: acetic acid: 1-butanol (350:1 :7, vol/vol/vol) with a flow rate of 1 ml/min. A linear gradient of methanol (ramped up to 100% methanol in 5 min) is used to wash off the unreacted ethyl ferulate.

8. Investigation of effect of low pH conditions on survival of microencapsulated L. fermentum 11976 cells:

[0053] Low pH survival of microencapsulated L. fermentum 11976 feruloyl esterase cells is examined by incubating the microcapsules in MRS medium adjusted to pH 1.5, 2.0 and 3.0 with 1.0M HCI. Incubation is performed at 37 ⁰ C and 150 rpm agitation for 2 hours. The bacterial numbers of the bacterial strain are determined by plate counts in MRS agar. The bacteria are spread-plated at intervals of 0, 20, 40, 60, 90 and 120 mins. At each time-point, media is aspirated; the microcapsules crushed and re- suspended in 0.9mL of PS. Samples are serially 10-fold diluted using PS. Duplicate plates are inoculated with 0.5mL samples from the appropriate dilutions and incubated under 5% CO ₂ at 37 ⁰ C for 72 h.

9. Feruloyl esterase activity of microencapsulated L. fermentum 11976 cells:

[0054] Figure 4A shows superimposed HPLC chromatograms of ferulic acid in reaction media at 10 hours. The peaks of ferulic acid and benzoic acid

were identified by comparison with the pure phenolic acids in MRS based on the retention time. The increased peak area of ferulic acid indicates feruloyl esterase activity of lactobacilli in the microcapsules as compared to control.

[0055] Figure 4B shows the de-esterification of ethyl ferulate and consequent release of ferulic acid over time by L fermentum 11976 feruloyl esterase producing microcapsules. The activity of L fermentum 11976 feruloyl esterase producing microcapsules was compared with control sham (empty) microcapsules. L fermentum 11976 containing microcapsules de- esterified ethyl ferulate (P < 0.001) at a significantly greater rate than the sham empty microcapsules. As seen in Figure 5, the average amount of ferulic acid liberated from ethyl ferulate as a function of time was 28.0 + 0.8 micrograms ferulic acid /g microcapsule/h for microcapsules containing L. fermentum 11976 and 0.1 + 0.02 micrograms ferulic acid /g microcapsule/h for empty microcapsules. Maximal activity was reached within three hours with 28.0 + 0.8 micrograms ferulic acid released/g microcapsule/h. After the fourth hour, there was a gradual decrease in the feruloyl esterase activity of the microencapsulated cells. At the tenth hour, feruloyl esterase activity had somewhat stabilized at 24.2 + 0.6 micrograms ferulic acid released/g microcapsule.

[0056] After the 10h incubation, all the strains of microencapsulated bacteria were shown to have achieved considerably similar decreases in pH over time when in the culture media, indicating substrate utilization and consequently organic acid production on account of cell metabolism/growth. It was observed that the pH of the test medium for each of the encapsulated bacterium strains decreased from pH 6.6 to 3.2 over the 10 hour incubation period.

10. Survival of microencapsulated feruloyl esterase producing bacterial cells:

[0057] Viable counts of the microencapsulated L. fermentum 11976 feruloyl esterase cells showed an increase from pre-treatment values of 10.9 log cfu/ml to 12.3 log cfu/ml when exposed to assay medium MRS over a 10

hour period. The APA microcapsules were found to remain intact at the end of the incubation period and the microencapsulated L. fermentum 11976 feruloyl esterase cells lowered batch pH of MRS media after the 10 hour incubation in comparison to control (Figure 6).

[0058] Microcapsules containing L. fermentum 11976 feruloyl esterase cells were investigated for survival at pH 1.5, 2.0 and 3.0 at 37 ⁰ C and 150 rpm agitation. In all experiments, pH variation in the medium due to the incubation of microencapsulated live cells was less than 10% during the 2 hour period.

[0059] Figure 7A shows that the cells were less stable and viable at pH 3. The viability decreased by 3.37 log cfu/mL over 60 minutes but there was only a 0.93 log cfu/mL reduction in viability over the next 60 minutes. With increased acidic conditions of pH 2, the corresponding loss of cell viability over an hour's interval was shown to be 5.04 log cfu/mL. The loss in viability was not so drastic during the next 60 minutes (1.66 log cfu/mL). The total number of retained cells was found to be 8.1 , 7.3, 6.9, 6.2 and 5.2 log cfu/ml after 20, 40, 60, 90 and 120 minutes respectively. In pH 1.5 conditions, microencapsulated cells were found to lose all viability within 40 minutes of exposure to acidic conditions. Cells were not enumerated beyond this period for this set.

[0060] Figure 7B shows that at pH 3 there is virtually no loss of cell viability. Cell viability was retained over the first 40 minutes and then decreased slightly, resulting in a total cell loss of approximately 1.3 log cfu/ml after two hours. In contrast to pH 3.0, at pH 2.0, microencapsulated L. fermentum 11976 cells showed a gradual loss of viability during the first 90 minutes. This was followed by a leveling off effect resulting in a total cell loss of approximately 3.9 log cfu/ml after two hours. The total number of retained cells was found to be 11.2, 10.1 , 9.3, 8.0 and 8.0 log cfu/ml after 20, 40, 60, 90 and 120 minutes respectively. In pH 1.5 conditions, microencapsulated cells were found to lose all viability and were thus not enumerated after 60 minute of exposure to acidic conditions.

[0061] Feruloyl esterase activity of other microencapsulated lactobacilli was determined to evaluate the potential for continuous production of ferulic acid. Known quantities of ethyl-ferulate were added to MRS broth and 0.3 ml_ sample aliquots were analyzed for ferulic acid concentration using the modified HPLC feruloyl esterase assay. Using 1.OmM benzoic acid internal standard, correlation of determinant factors (R ² ) of 0.9826 was obtained and used in all experiments. The de-esterification of ethyl-ferulate after 10 hours by feruloyl esterase producing Lactobacillus bacteria containing microcapsules was determined. The activity of feruloyl esterase producing Lactobacillus containing microcapsules was compared with control empty microcapsules. The feruloyl esterase activity of each of the microencapsulated lactobacilli towards ethyl-ferulate was also analyzed (Table 1).

[0062] HPLC analysis of feruloyl esterase activity of microencapsulated lactobacilli. Ethyl-ferulate was used as a substrate and the experimentation in- vitro was carried out at 37°C with 5% CO ₂ . De-esterification activity of feruloyl esterase was determined after 10 hours of incubation.

Table 1

Bacterial strain Feruloyl esterase activity

(μg ferulic acid released/g CWW*/h)

Microencapsulated L. fαrciminis 12.24

Microencapsulated L. reuteri 6.88

Microencapsulated L. fermentum 11976 35.32

Microencapsulated L. fermentum 14932 28.67

"CWW- capsule wet weight

[0063] The feruloyl esterase activity of 5 g capsule wet weight of microencapsulated lactic acid bacteria was quantified as the amount of product formed in a 10-hour period from the substrate. All the Lactobacillus

microcapsules de-esterified ethyl-ferulate at a significantly greater rate (P < 0.05) than the sham microcapsules. As seen in Table 1 , the average amount of ferulic acid liberated from ethyl-ferulate in 10 hours was 35.32 μg/g microcapsule/h for L fermentum 11976 microcapsules, 28.67 μg/g microcapsule/h for L. fermentum 14932 microcapsules, 6.88 μg/g microcapsule/h for L. reuteri microcapsules, 12.24 μg/g microcapsule/h for L. farciminis microcapsules and 0.10 μg/g microcapsule/h for empty microcapsules.

[0064] L. fermentum 11976 microcapsules liberated the greatest amount of ferulic acid (μg ferulic acid released/g microcapsule/h) from the ethyl-ferulate (Table 1). In a 10-h reaction, of the original 1.33mM of ethyl- ferulate, 0.606mM was converted to ferulic acid, corresponding to a yield of 45.59%.

[0065] Bacterial density of approximately 4 g of cell isolate (wet weight) per 100 ml of alginate at the time of microencapsulation, as mentioned above, resulted in a concentration of 5 x 10 ¹¹ cfu/ml of bacteria. When the microcapsules were incubated in ethyl-ferulate supplemented MRS for 10 hours, it was seen that the bacteria retained their viability and even vigorously propagated in the media resulting in a higher viable count per ml of capsules. The capsules were stored at 4°C and as Table 2 shows, no significant loss in viability of the bacteria occurred even after 2 months of refrigerated storage. Further, microcapsule integrity was not found to be significantly compromised during storage or in the course of the experiment.

Table 2

Viability of microencapsulated lactobacilli after refrigerated storage for 2 months

[0066] At refrigerated storage, the viable count of the microencapsulated L. fermentum 11976 cells was found to be 3.24 x 10 ¹² cfu/ml. When these microencapsulated cells were exposed to low pH conditions at pH 2.0 for 60 minutes, it resulted in a viability decrease of 1.6 log cfu/ml. The microcapsules retained viability at approximately 10 ¹⁰ cfu/ml. The alginate core shrank significantly in acidic conditions from 602 + 30 μm to 531 + 20 μm. However, its mechanical stability remained intact at pH 2.0 and there was no significant release of bacteria into the gastric incubation medium.

[0067] Using microencapsulated L. fermentum 11976 a molar yield of

45.59% was obtained over 10 hours in this experiment which compares favorably to earlier reports wherein a molar yield of less than 3% was obtained during fermentation of spent barley grain using fungal feruloyl esterase from A. niger (Bartolome et al.,1997).

11. Other potential uses of the present invention:

[0068] Feruloyl esterase producing bacteria also have considerable potential as biocatalysts for the food and allied industries such as:

[0069] the modification of plant-based food texture;

[0070] the baking industry to improve flavors and textures;

[0071] the generation of fine chemicals from food waste;

[0072] the potential use in animal nutrition;

[0073] the improvement of sensory properties of wine;

[0074] the bleaching high quality paper pulps;

[0075] anti-aging agents;

[0076] antibacterial activity items;

[0077] fragrances;

[0078] control of germination;

[0079] bioconversion of agricultural waste into industrially valuable products; and

[0080] part of the gel matrix of wound dressings.

[0081] In addition, due to interest in the use of ferulic acid in the biotechnological and pharmaceutical industry, these microencapsulated feruloyl esterase producing lactobacilli may well be useful in oral therapeutic formulations for cancer, cardiovascular diseases, hepatotoxicity, colds, influenza, skin aging, and muscle wasting.

Beer production:

[0082] Beer is produced using yeast fermentation. The encapsulated bacteria of the present invention could be present during the yeast fermentation to add flavor to the beer.

Baking Industry:

[0083] Vanillin is quantitatively one of the most important flavor additives in the world. Vanillin is an important aromatic flavor compound used in foods, beverages, perfume and pharmaceuticals. It is produced on a large scale in the industry through chemical synthesis. However, the vanillin obtained by chemical synthesis cannot be considered as natural. Since the demand for natural vanillin cannot be accommodated from vanilla pod extraction, biotechnological processes based on microorganisms become more and more important. Thus, there are many attempts to produce vanillin based on bioconversion of natural sources such as lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid or aromatic amino acid. Current commercial processes for biotechnological vanillin production suffer from the relatively high price of the natural substrate ferulic acid, which is due to its limited accessibility from lignin by biological means. Ferulic acid can be used to produce vanillin through biotransformation in microorganisms by three major pathways. These biotransformations have been discovered in many bacteria, fungi and yeasts. Use of enzymes secreted by these microorganisms, rather than direct use of microorganisms, is the practical pathway to produce vanillin from ferulic acid, as vanillin is toxic to most microorganisms and the formed vanillin will be further oxidized to the less toxic vanillic acid.

[0084] In accordance with the present invention, a novel way to isolate ferulic acid as a fine chemical from a synthetic substrate using microencapsulated lactobacilli is disclosed. Ferulic acid is found in numerous plants and in significant quantities in agro-industrial derived by-products. The efficient enzymatic removal of ferulic acid from sugar beet pulp and other substrates could thus represent a cheap substrate for the industrial production of natural vanillin. It can also be used in the bioconversion of waste from industries such as agriculture, food, etc. as a waste treatment method. It can also be used to produce value added products from such waste materials.

Cosmetics:

[0085] Ultraviolet light is an exogenous factor that may cause skin damage, resulting in both precancerous and cancerous skin lesions and acceleration of skin aging. Reactive oxygen species are believed to be largely responsible for some of the deleterious effects of UV light upon skin. Particularly, prolonged skin exposure to UV light results in a severe decrease of its antioxidant content. Overproduction of nitric oxide from keratinocytes seems to play a major role in the integrated response leading to erythema production and inflammation processes following UV radiation exposure. Owing to the high degree of conjugated unsaturation (as a strong UV absorber) and its permeability to the skin, ferulic acid constitutes an active ingredient in many skin lotions, sunscreens and hair creams designed for photoprotection and for skin tumor inhibition.

Food additives:

[0086] Ferulic acid can be used to preserve foods because of its antioxidant and antimicrobial activities. Ferulic acid was first used in Japan in 1975 to preserve oranges and to inhibit the autooxidation of linseed oil, lard and soybean oil. Ferulic acid is an effective antioxidant in some food-related system, such as lecithin-liposomes and aqueous emulsions.

[0087] Compared with other phenolic substances, ferulic acid has two advantages. First, ferulic acid has strong antioxidant activity. Another advantage is that ferulic acid is much less affected by pH changes than other phenolic compounds, such as chlorogenic acid, caffeic acid and gallic acid. This is very important for foods subjected to alkali processing, which is commonly used to recover proteins from cereals and legumes, to induce the formation of fiber-forming meat analogue vegetable protein, and to prepare peeled fruits and vegetables. However, ferulic acid also has shortcomings. It has been reported that ferulic acid formed 4-vinylguaiacol by decarboxylation during processing, contributing off-flavors in cooked foods, beer and orange juice.

[0088] Another preservative function of ferulic acid comes from is its antimicrobial activity. Ferulic acid can inhibit the growth of bacteria, fungi and yeasts. Ferulic acid is an active ingredient of extracts of some plants showing antimicrobial activity. It was reported that feruloyl oligosaccharide ester showed inhibitory effects against many kinds of Gram-positive and -negative bacteria. Ferulic acid, at a concentration of 500 mg I ^"1 , can also appreciably inhibit growth of yeasts such as Pichia anomala, Debaryomyces hansenii and Saccharomyces cerevisiae.

Ergogenic substance in sport foods:

[0089] As ferulic acid is a potent antioxidant and is believed to stimulate hormone secretion in humans, it is widely used as an ergogenic substance in sport foods.

Cross-linking agent for the preparation of food gels and edible films:

[0090] As ferulic acid is known to cross-link with polysaccharides, it is used to increase the viscosity and form gels from some polysaccharides such as pectin and arabinoxylans. Gel formation can also be effected from feruloylated arabinoxylans and proteins using cross-linking agents such as ammonium sulfate, hydrogen peroxide and peroxidase, and laccase. This property is very important, as ferulic acid can make it possible to use polysaccharides of lower molecular weight, which have low viscosity and poor gel formation capacity, to make new gels in food processing.

[0091] Ferulic acid and its oxide, quinoid ferulic acid, can react with some amino acids in proteins such as tyrosine, lysine, cysteine and cross-link protein molecules. Thus, it can be used as a cross-linking agent to improve the properties of protein-based edible films. Incorporation of ferulic acid into the film-forming solution made from soy protein isolate (SPI) increased the tensile strength, the percentage of elongation at break, and decreased the water vapor permeability and gas permeability of the SPI film. Ferulic acid at 5.5 mmol I ^"1 markedly enhanced the heat stability of milk at 140 ⁰ C. The addition of caffeic acid resulted in a reduction of the reactive lysine and

sulphydryl content and inhibited the dissociation of κ-casein-rich protein from the casein micelles in milk on heating. These results suggest that caffeic acid and its related phenolic compounds, such as ferulic acid, may find their prospective applications in the processing of milk products.

[0092] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

REFERENCES

Bartolome, B., Faulds, C. B., and Williamson, G. (1997) Enzymic release of ferulic acid from barley spent grain Journal of Cereal Science 25 (3), 285-288

Donaghy, J., Kelly, P. F., and Mckay, A. M. (1998) Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli Appl. Microbiol. Biotechnol. 50 (2), 257-260

Faulds, C. B., Kroon, P. A., Saulnier, L., Thibault, J. F., and Williamson, G. (1995) Release of Ferulic Acid from Maize Bran and Derived Oligosaccharides by Aspergillus-Niger Esterases Carbohydrate Polymers 27 (3), 1 87- 190

Krings, U. and Berger, R. G. (1998) Biotechnological production of flavors and fragrances Applied Microbiology and Biotechnology 49 (1 ), 1-8

Mastihuba, V., Kremnicky, L., Mastihubova, M., Willett, J. L., and Cote, G. L. (10-1-2002) A spectrophotometric assay for feruloyl esterases Anal. Biochem. 309 (1 ), 96-1 01

Priefert, H., Rabenhorst, J., and Steinbuchel, A. (2001 ) Biotechnological production of vanillin Applied Microbiology and Biotechnology 56 (3-4), 296-3 14