Solid State Fermentation of Mexican Oregano (Lippia Berlandieri Schauer) Waste

Problem statement: Mexican oregano is recognized for their aromatic c haracteristics and flavor quality. Principal products obtained from th e plant and marketing are the leaves and essential oil; however the extraction of the essential oil ge nerates large amounts of agro industrial wastes; th at can be used as support-substrates in Solid-State Fe rmentations (SSF). Approach: In this study a fungal bioprocess, as solid state fermentation usin g Mexican oregano wastes as support, for the use of these residues to obtain adds value products and/or molecules were developed. The fungal strain was selects by its adaptability to the support. The aqu eous and non polar extracts were obtained kinetical ly until 120 h and then it was partially characterized (hydrolysable tannins, total sugar and proteins contents, antioxidant activity, tymol and carvacrol c ncentration). Results: Solid state fermentation of oregano wastes, with Aspergillus niger PSH, allowed the accumulation of a phenolic compou nd with catechin similar characteristics and could be respo n ible of the biotransformation of small amounts of carvacrol to thymol. Conclusion: These results could give an add value to Mexican o regano wastes and with more investigation the obtained products c an be used in several industries.


INTRODUCTION
Lippia berlandieri Schauer or Lippia graveolens H.B.K. is the most distributed oregano specie in Mexico. Principal products obtained from the plant and marketing are the leaves and the essential oil; however the essential oil extraction generates large amounts of agro industrial wastes. These wastes have been used as animal feed, but the consumed amount is lower than the produced. To be friendlier with the environment, researchers had inverted efforts to reduce the amount of wastes by finding alternative uses (Orzua et al., 2009). One possibility is to use the agro industrial residues as support-substrates in Solid-State Fermentations (SSF).
SSF is defined as the cultivation of microorganisms on moist solid supports, either on inert carriers or on insoluble substrates that can, in addition, be used as carbon and energy source (Holker et al., 2004). The fermentation takes place in the absence or near absence of free water (Pandey et al., 2000); however, the substrate must possess enough moisture to support growth and metabolism of the microorganism (Singhania et al., 2009). This activity has been done by other investigation groups and the molecules that have been released are enzymes, organic acids, polymers, flavors, antibiotics and aroma (Couto, 2008;Swe et al., 2009)) or bioconversions of crops and crop-residues (Holker et al., 2004;Medina-Morales et al., 2011). In this study we report a methodology development to find Mexican oregano wastes applications.

MATERIALS AND METHODS
The Mexican oregano waste was obtained from the rural industry "Oreganeros of central-south of Chihuahua" (in Chihuahua, Mexico). Was placed in black bags and transported to the Microbiology Laboratory of the Food Research Department, were the material was dried at 60°C during 48 h, pulverized in a blender until fine powder, and stored at room temperature in black bags.
Erlenmeyer flasks containing 3 g of oregano waste, 7 m L −1 of Pontecorvo´s culture medium and inoculated with 1×10 7 spores per gram of dry oregano waste powder were incubated at 30°C. Samples were monitored kinetically during 120 h; of each sample was obtained an aqueous and a non polar extract. For aqueous extract the content of one flask was resuspended in 15 m L −1 of distilled deionizated water, filtered and stored in bottles under light protection at -20°C. For the non polar extract the vegetal material, of each flask, were dried and re-suspended in 10 mL of hexane by 8 h at 200 rpm, filtered and stored at 4°C until its analysis. Consumption of total sugars (Dubois et al., 1956), hydrolyzable tannins (Makkar et al., 1993), proteins (Bradford, 1976) and antioxidant activity (Re et al., 1999) were evaluated in all the samples by triplicate.
Thin Layer Chromatography (TLC) was done only with the 120 h extract, following the methodology proposed by Sharma et al. (1998). Mobile phase used was chloroform-ethyl acetate-acetic acid (50:50:1), 2 µL of samples and standards (gallic acid and catequin) were tested and chloride ferric solving in methanol was used to detect the spots.
The non polar extract was filtered by 0.45 µm nylon-membrane and injected into GC-MS (Perkin-Elmer AutoSytmen XL 610N1052906-Perkin-Elmer Turbomax Gold 647N1050901) with ionized electronic detector. Separation was carried out with Perkin Elmer column (30 m length, 0.25 mm diameter, 0.25 µm thickness). Condition works was temperature injector of 220°C, helium as carrier gas and flow rate of 1.0 m L −1 min. The sample and standards (thymol and carvacrol) injection was 1 µL. The oven program was 120°C for 1 min at 1 mL −1 min, 180°C for 7 min at 1 mL −1 min and 180°C for 11 min.

RESULTS
Tested fungal strains did not growth in the Mexican oregano plants. However all the fungal strains growth in Mexican oregano wastes, but the lag phase duration and the radial growth rate was different for each (Fig. 1).
A. niger PSH grew faster, 16 h earlier, than the others strains and had the greatest invasion capacity, average of 16 mm. A. niger GH1 was the second in importance but its lag phase (32 h) is significant different to A. niger PSH. The rest of the strains had little growth, from 11-2.4 mm. Based on the adaptability to support-substrate (phase lag duration) and the invasion capacity of the wastes, selected strain for the fermentation process was A. niger PSH.
In the chemical partial-characterization of extracts the results showed that proteins, hydrolysable tannins and total sugar amount had similar behavior. The maximum value is at 20 h of fermentation time (1.05, 4.5 and 169.54 mg g −1 dry matter respectably). Minimum value for proteins and total sugar was at 60 h of fermentation time (0.73 and 72.04 mg g −1 dry matter respectably), while hydrolysable tannins was at 40 h of fermentation time (2.65 mg g −1 dry matter). However, after this, the values rise again. On the other hand, the antioxidant activity was higher at the beginning of the kinetics (73.2%) and it totally decreased at 80 h of fermentation time. In HPLC profiles is possible to see a release compound since time 60-120 h, it has a singular peak with retention time of 14.5 min (Fig. 2). This compound agrees on time where proteins, total sugar and hydrolysable tannins had its lower values; however the retention time of the compound did not match with any standard (13.3 min for catechin, 7.15 min for gallic acid and 18.34 min for ellagic acid). As result in TLC, only one compound could be observed and its chemical properties seem to catechin; the Rf of the standards were 0.12 for gallic acid and 0.75 for catechin, while in the samples were 0.76. In the no polar extract we found that main terpenes (thymol and carvacol) not were extracted totally in essential oil. Chromatograms showed that the carvacrol signal was lower with increasing fermentation time, while the thymol signal was higher (Fig. 3).

DISCUSION
Oregano plants are not possible using as supportsubstrate in SSF because the main antimicrobial compounds (tymol and carvacrol) (Portillo-Ruiz et al., 2005) may not allow fungal growth and by consequence the bioprocess; instead, in the Mexican oregano wastes all the fungal strains grew because the thymol and carvacrol concentrations was minimal and culture media provide the necessary nutrients to the fungal metabolism.
Fungal strains, especially filamentous organisms, with a potential enzymatic machine to degrade chemical complex compounds are one of the most useful microorganisms in solid state fermentation (Banerjee et al., 2005, Medina et al., 2010. A. niger PSH had been used to produce tannases and release gallic and elagic acids from tar bush, creoseote bush (Ventura et al., 2008) and pomegranate residues (Robledo et al., 2008) as others uses of agroindustrial wastes to obtain compounds with industrial applications; these reports support the strain selection to the bioprocess in Mexican oregano wastes.
Proteins, total sugar and hydrolyzed tannins consumption is major at the beginning of the fermentation because the microorganism needs them to start its reproduction phase. After several days, when the principal nutrients were consumed, the microorganism releases enzymes to degraded complex molecules, obtaining by this way sugars and other nutriments. If the metabolism of the microorganism is able, can be accumulated other molecules with industrial applications and it should be purified at downstream process. Antioxidant activity in the samples can be attributed to hydrolysable tannins present in earlier fermentation kinetics times.
With the results of HPLC and TLC is able to think that the main compound present in the aqueous extract of fermented oregano waste is a phenolic compound with catechin in its molecule or a catechin derivate. This can be supported because the major amount groups of molecules that can be found in oregano plants and essential oil are monoterpenes and phenolics acids (Silva-Vazquez et al., 2008) but flavonoids (Arcila-Lozano et al., 2004) are present too. Bhat et al. (1998) mentioned that the distribution of tannins, based on their structure is hydrolysable and condensed; condensed tannins are composed by flavonoid units, however there is an intermediate position group formed by catechin tannins. We can propose that as consequence of fungal invasion, condensed or catechin tannins are formed with the components of the oregano wastes and this is the reason of the observed HPLC chromatogram profile.
On the other hand, thymol and carvacrol molecules are isomers, so we made the hypothesis that A. niger PSH makes a bioconversion (Bicas et al., 2009;Leutou et al., 2009;Ramachandran et al., 2008;Shimoda et al., 2006) during fermentation process and for these reason the thymol content was higher in later fermentation times than earlier. The isomerization from carvacrol to thymol can be a new method to purify this compound and then it can be used in pharmacy, food or perfumes industries (Carrera et al., 2005;Lambert et al., 2001;Tsimogiannis et al., 2006).

CONCLUSION
With the results above described, we propose that the compound released or/and accumulated at 120 h of SSF did not had antioxidant activity because it has tannins condensed structure, more similar to catechin than other molecules, as flavonoids. Also we have the hypothesis that A. niger PSH makes possible monoterpene isomerization of carvacrol to thymol. This possibilities gives an add value to Mexican oregano wastes, but is necessary to do more investigation about that.

ACKNOWLEDGMENT
This project is part of a collaborative agreement between DIA-UAdeC and FCQ-UACH and it was financed by the Mexican funding program CONACYT-CONAFOR (C04-13) Author Melendez-Renteria thanks the financial support of CONACYT fellowship.