Patent Description:
<CIT> describes the production of compositions containing significant concentrations of inhibitors of MAO A and MAO B from tequilas and from pulque. Reverse osmosis and spinning column technologies are mentioned as techniques for removing alcohol while preserving the activity of these inhibitors, as these inhibitors are volatile and may be formed during fermentation and distillation of tequila and fermentation of pulque. However, details of these processes are not provided. The present invention, as detailed herein below, provides for an improved process for removing ethanol from tequila or other distilled agave fermented extracts, while providing a substantially ethanol-free composition with significant concentrations of MAO A and/or MAO B inhibitors.

<CIT> discloses a device for producing ultra-pure water according to the reverse osmosis principle. <CIT> describes a reverse osmosis seawater desalination system which includes a control device for determining the rate of operation and controlling the pump(s) of the system based on energy consumption price related information provided by an intelligent power network/grid.

As it is known that MAO inhibitors can be used as mood elevators, antidepressants and treatments for various other diseases, including Parkinson's disease, compositions prepared in accordance with the method of the invention are useful in these contexts.

The invention takes advantage of the presence of MAO inhibitors in distilled spirits prepared from fermented agave extracts. The MAO inhibitors are formed during fermentation, or formed during distillation or aging, or combinations of the above. Removing the alcohol from these commercially available spirits by applying a vacuum destroys at least some of the MAO inhibition activity, so the desired compositions must be prepared from these spirits by processes that preserve these volatile components. These processes include reverse osmosis, spinning cone columns, pervaporation and other methods that do not result in the loss of volatile compounds. Particular formats for these processes are shown to have significant advantages.

The present invention provides a method to obtain an improved composition of monoamine oxidase A (MAO A) inhibitors and/or monoamine oxidase B (MAO B) inhibitors from a distillate of fermented agave extract by reverse osmosis, which method comprises:.

Thus, the present invention is directed to improved methods to obtain compositions wherein the activity of inhibitors of monoamine oxidases is preserved but the alcohol content is reduced to less than <NUM>% by volume, or <NUM>% by volume or <NUM>% by volume, often as low as <NUM>% by volume or <NUM>% by volume, and to compositions obtained by this method.

A composition comprising inhibitors of MAO A and/or MAO B obtained by the improved process of the present invention is described herein, which composition comprises inhibitors of MAO A and/or MAO B derived from the distillate of a fermented agave extract and contains less than <NUM>% ABV or <NUM>% ABV or <NUM>% ABV, or even less, as noted above. Described herein are also foodstuffs and drinks that contain these compositions, as well as methods to treat conditions requiring MAO inhibition using any of these. As it has been found that the MAO inhibitors in the compositions are reversible, they are particularly useful for treatment in subjects who are intolerant to irreversible MAO inhibitors.

The agave derived alcoholic beverage may be prepared by heating the stems of agave cactus to hydrolyze complex sugars; shredding and crushing the heated stems to release a syrup; diluting said syrup with water to a <NUM>-<NUM> BRIX level and inoculating with yeast; fermenting the inoculated diluted syrup to obtain a fermentation product; and distilling the fermentation product to obtain a distillate.

Alternatively, the distillate is prepared from a species of agave that does not require processes to break down complex sugars, such as an extract of the maguey plant used to make pulque.

In all cases, a distillate of a fermented extract of an agave plant is treated to remove alcohol in accordance with the method of the present invention, a process that does not also remove the MAO inhibitors, which inhibitors are also volatile compounds.

Reverse osmosis (RO) is a successful method to remove the ethanol from the beverage starting material. A RO membrane with a molecular weight cut-off of approximately <NUM>/mol (<NUM> Daltons), i.e., <NUM>-<NUM>/mol (<NUM>-<NUM> Daltons) separates ethanol from the inhibitors of MAO's formed by microorganisms and/or formed during distillation.

In embodiments of the method of the invention, reverse osmosis (RO) with dilution and subsequent circulation is continued until the product is considered non-alcoholic. Additional pressure adjustments with respect to the flow over the separation membrane may also be required and supplied by use of additional pumps, valves and gauges. The active MAO inhibitors are then contained in the concentrated, non-alcoholic portion and may then be supplemented with vitamins, minerals, amino acids, protein or caffeine, where desired, and may be included in other food or drink preparations as noted above. The alcohol content is reduced to less than <NUM>% by volume or <NUM>% by volume, but may be reduced even further. Illustrative descriptions of this process are provided in the examples below.

As noted above, the MAO inhibitors produced in fermented agave sources are volatile compounds that typically follow ethanol through evaporative separation techniques. The compounds are selective inhibitors of MAO A or MAO B and the ratio of MAO A:MAO B inhibitory activity vary considerably from brand-to-brand of distillates of fermented agave extract, but are consistent within each brand. While most have inhibitory activity to both MAO A and MAO B, samples of tequila from certain specific manufacturers inhibited only MAO B activity. Certain other agave derived products were found to inhibit only MAO A activity.

An important aspect of the agave derived MAO inhibitors for their use in food or drink products is that they are reversible rather than irreversible inhibitors, as demonstrated in Example <NUM> below. Irreversible MAO inhibitors are pharmaceutical products now in limited use due to this mechanism of action that permanently neutralizes the target protein. Tequila consumption is not known to be associated with the characteristic side effects of irreversible inhibitors, such as serotonin syndrome (excess MAO B inhibition) or "the cheese effect" (excess MAO A inhibition). The uncontrolled (by medical supervision) amounts of composition consumed when included in food or drink mandates the property of reversibility.

The compositions prepared by the method of the present invention may be used as a food supplement and/or included in foods or medicines, or used to treat depression, Parkinson's disease or general malaise, and may also be used as an adjunct or substitute for coffee or in decaffeinated products. The compositions may be mixed with one or more carriers, excipients and/or diluents that are pharmaceutically or nutritionally acceptable. Thus, in general, the compositions may be included in juices or other soft drinks such as colas or fruit flavored sodas, or can be consumed directly. The compositions may also be included in foodstuffs such as uncooked liquids, for example salad dressings, and consumed along with the solid components of the salad. The amounts of compositions to be consumed or administered to subjects is highly dependent on the nature of the condition to be treated, as well as the concentrations of the MAO inhibitors determined to be in the composition itself. Levels of MAO inhibitors useful for various medical indications are known in the art and these guidelines can be followed. For use as food supplements, this is a matter of the judgment of the nutritionist or other practitioner.

The subjects to be treated are generally human, but other mammals such as livestock and companion animals may also benefit. Thus, the compositions obtained by the method of the present invention may be used in pharmaceutical and veterinary compositions. Laboratory models, such as murine models, may also be used as subjects.

In the method of the present invention, wherein RO is used as described above, commercial water purification systems, such as (Osmonics E2/EZ2 Series, GE water) can be modified to separate MAO inhibitors from ethanol in fermented agave plant sources and is illustrated below. Brass components may be replaced with stainless steel, and the flow path altered, and electronic flow control components removed. The modified RO system separates the agave derived MAO inhibitors from ethanol while operating at low pressure, which has the advantage of requiring less energy than conventional alcohol removal RO systems. The osmotic pressure of a solution is directly related to the concentration of solute and, in order for a reverse osmosis system to function, the pressure across the membrane must be higher than the osmotic pressure of the solution.

The unmodified GE Water system shown in <FIG> is capable of processing brackish water at an operating range of <NUM> - <NUM> MPa (<NUM> - <NUM> psi) across the membrane, which is considered low pressure compared to systems running at <NUM> - <NUM> MPa (<NUM> - <NUM> psi) used to process sea water. Low pressure water purification systems such as the GE Water unit require a feed supply (<NUM>) delivered to the RO pump (<NUM>) at a minimum pressure equivalent to that found in a municipal water supply [<NUM> - <NUM> MPa (<NUM> - <NUM> psi)]. The RO pump boosts the pressure of the feed to supply the membrane (<NUM>), water migrating across the membrane (permeate) (<NUM>) is normally captured for drinking, while solution not passing through the membrane (concentrate) (<NUM>) is discarded.

In the method underlying the present invention, the flow path of the conventional system was altered to redirect the concentrate from the membrane to the feed supply.

In the Examples below, specific illustrations of the improvements in the present method to obtain compositions substantially free of alcohol that nevertheless contain the volatile inhibitors of monoamine oxidase are shown. These are not limiting, but illustrative. In some cases, an extra pump is added to adjust suitable pressure in addition to the single pump generally supplied with a reverse osmosis apparatus that is commercially available. A gauge and/or valves may also be included. In addition, these systems are redesigned so as to recycle the concentrate from each pass through the membrane into a reservoir so that the concentrate remaining in the reservoir is lower in alcohol percentage but contains the desired monoamine oxidase inhibitors. In some instances, the level of liquid contained in the reservoir is maintained at a desired level, but such approach is not according to the method of the present invention. In accordance with the method of the present invention, the additional water for dilution of the recycled concentrates is supplied immediately upstream of the membrane. The distilled agave ferment is diluted in the feed reservoir of the reverse osmosis system prior to the reverse osmosis process.

Other procedures illustrated herein for reducing the alcohol content in distilled spirits derived from agave include harvesting the monoamine oxidase inhibitors under reduced pressure as demonstrated below. A further example below demonstrates that the method of the present invention may be applicable to distillates obtained from fermented pulque, since it is demonstrated that fermented agave species that result in pulque also contain the desired inhibitors. Distillates of pulque are not commercially available for reasons not related to the applicability of the invention.

The following materials and procedures were used in the examples below:.

The ethanol assay was from <NPL>). A master mix containing per <NUM> assays:.

An optical density plate reader and compatible <NUM>-well clear plates are used for this assay. To each well, <NUM>µl of master mix is first added, then <NUM>µl of each standard and samples at <NUM>- and <NUM>-fold dilutions. The standard curve is constructed by diluting <NUM>% ethanol <NUM>-fold and serially diluting fs, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>. The plate is incubated at room temperature for <NUM>-<NUM> minutes and read in the plate reader at OD340 nm.

The MAO assay was from<NPL>. A master mix containing per <NUM> assays:.

Recombinant MAO A and MAO B are from Corning. The proteins are thawed and aliquoted <NUM>µl per tube and refrozen at -<NUM>. For the assays, a tube of each protein is removed from the freezer and <NUM>µl of buffer is added, then <NUM>µl is added to each assay.

A black fluorescence compatible plate is used and <NUM>µ! of sample is added to each well, then <NUM>µ! of master mix is added, followed by <NUM>µ! of MAO recombinant protein. The plate is sealed and placed in an incubator at <NUM> for <NUM> minutes. After incubation, the sealer is removed and <NUM>µl of 2N NaOH is added. The plate is read at excitation <NUM>/<NUM> emission in a fluorescence spectrophotometer.

Tequila samples were assayed for MAO inhibitory activity with or without pre-incubation at <NUM> for <NUM> minutes with recombinant MAO A or MAO B. Kynuramine substrate was then added to all assays before <NUM> minute incubation at <NUM> according to the assay protocol.

Reversible inhibitors show no change in inhibitory activity with pre-incubation with the proteins as seen in Table <NUM>. Irreversible inhibitors show increased inhibition with pre-incubation due to chemical interaction between the compound and protein.

A modified RO system is shown in <FIG>. In this system, <NUM> of Kirkland anejo tequila was diluted with distilled water to a volume of <NUM> in a reservoir (<NUM>) connected to a centrifugal pump (<NUM>) (HP75SS-<NUM>-<NUM>-<NUM>-<NUM>-1T6, Price Pumps). The centrifugal pump supplied the RO pump (<NUM>) with the diluted tequila feed at <NUM> MPa (<NUM> psi) and the RO pump boosted the feed supply pressure to the membrane (<NUM>). After exiting the membrane housing, the concentrate (<NUM>) was channeled back to the reservoir to be continuously recycled while the permeate flow (<NUM>) (ethanol and water) was discarded after analysis. Pressure across the membrane was adjusted by a valve restricting the concentrate flow and the pressure was monitored by a gauge installed between the membrane and the valve. The volume of concentrate decreased with continuous recycling at a pressure of <NUM> MPa (<NUM> psi) across the membrane, while the concentration of MAO inhibitors and ethanol increased relative to the <NUM>-fold dilution. The machine was run until the volume of concentrate equaled the original <NUM> volume.

The strategy behind this RO hardware configuration and operational protocol was successful in separating MAO inhibitor compounds from ethanol, but required an initial <NUM>-fold dilution of tequila and produced <NUM>% ABV concentrate, much higher than the desired <NUM>% ABV. Presumably, the limit to ethanol reduction is related to osmotic pressure of the concentrate and low operating pressure of the system. During the <NUM>-to-<NUM> volume reduction run of the machine, fifteen containers at <NUM> each of permeate were collected and ethanol assays were run on containers <NUM>, <NUM>, <NUM> and <NUM>. The results are shown in Table <NUM>.

An additional improved RO system is shown in <FIG>. In this system, <NUM> of Kirkland anejo tequila was diluted to <NUM> in a <NUM> reservoir (<NUM>) fitted with a float switch (<NUM>) (Omega Engineering, LVK-<NUM> x2; solid state relay SSRL2) connected to a peristaltic pump (<NUM>) (Cole Parmer). The float switch was adjusted to maintain a volume of <NUM> in the reservoir by energizing the peristaltic pump to deliver distilled water (<NUM>) when the level in the reservoir fell below <NUM>. MAO inhibitors were separated from ethanol with this method and the ethanol concentration was significantly lower in the final concentrate compared to Example <NUM>. Fifteen containers at <NUM> each of permeate were collected and ethanol assays were run on all containers. Results for containers <NUM>, <NUM>, <NUM> and <NUM> are shown in Table <NUM>.

This strategy was successful in separating MAO inhibitor compounds from ethanol at low system pressure using a <NUM>-fold initial dilution of tequila producing <NUM>% ABV concentrate after processing <NUM> of distilled water. This method was a significant improvement over Example <NUM>, since the ethanol concentration was even lower than the <NUM>:<NUM> dilution of tequila.

A method of production in accordance with the present invention is shown in <FIG>. The system was configured as in Example <NUM>, except that distilled water was introduced immediately before the membrane by the peristaltic pump at arrow (<NUM>) instead of at the reservoir. MAO inhibitors were also successfully separated from ethanol with this method and the ethanol concentration was significantly lower in the final concentrate compared to Example <NUM>, but the ethanol concentration was not as low as Example <NUM>. In this method, <NUM> of water was used rather than <NUM> as in Example <NUM> during the processing. Each container of permeate was collected and ethanol assays were run on all containers. Results for containers <NUM>, <NUM>, <NUM> and <NUM> are shown in Table <NUM>.

This strategy was successful in separating MAO inhibitor compounds from ethanol at low system pressure using a <NUM>-fold initial dilution of tequila producing <NUM>% ABV concentrate after processing <NUM> of distilled water.

The inhibitors are volatile compounds that typically co-evaporate with ethanol, but may be separated from ethanol under conditions of reduced pressure according to the following procedure:.

A <NUM> polypropylene microfuge tube containing <NUM> of anejo tequila (<NUM>% ABV), was placed in a centrifugal vacuum concentrator (Savant Speed Vac) under a vacuum of <NUM> Torr at <NUM> for <NUM> minutes, which conditions resulted in a final volume of <NUM>µl. Using an enzymatic assay, the ethanol concentration was measured at <NUM>% ABV and dilutions of the concentrated sample and tequila were assayed for MAO A and MAO B inhibitors. The <NUM>-fold diluted tequila sample (<NUM>% ABV) shows <NUM>% inhibition of MAO A activity and <NUM>°/ inhibition of MAO B activity and the <NUM>-fold concentrated sample (<NUM>% ABV) shows <NUM>% inhibition of MAO A and <NUM>% inhibition of MAO B. This demonstrates that the MAO inhibitors can be separated from ethanol under reduced pressure.

This method of separation of MAO inhibitors from ethanol in fermented agave sources illustrates proof of concept for the successful commercial production using spinning cone column (SCC) or pervaporation techniques of ethanol removal. Conditions would be adjusted to maximize the desired results, as in ratio of MAO inhibitors, for the final product because the inhibitors are volatile compounds that may be selectively isolated from ethanol depending upon vacuum/temperature.

Agave derived MAO inhibitors are also found in the fermentation products of aguamiel (honey water) produced from the maguey plant or Americana agave. The fermented beverage from aguamiel is known as pulque and may be produced from agave other than Blue Weber agave, which is restricted for the production of tequila. For pulque production, the maguey plant sap is harvested and fermented immediately since the sap contains simple fermentable sugars. Reports of microorganisms include thermo bacteria (which produce ethanol) in addition to yeasts.

Claim 1:
A method to obtain an improved composition of monoamine oxidase A (MAO A) inhibitors and/or monoamine oxidase B (MAO B) inhibitors from a distillate of fermented agave extract by reverse osmosis, which method comprises:
diluting the distillate in a feed reservoir of a reverse osmosis system;
recycling said diluted distillate across a separation membrane to obtain a permeate and a concentrate that is returned to the feed reservoir at each cycle, wherein the method further includes diluting the recycled concentrates with water immediately prior to recycling said concentrates past the membrane, wherein the additional water is supplied immediately upstream of the membrane; and
continuing to recycle each successive concentrate to obtain a final concentrate that contains less than <NUM>% alcohol by volume (ABV), thus obtaining an improved composition of said inhibitors.