# Patent

## Title
Targeted pectin hydrolysis by recombinant E. coli expressing chimeric pectinases to facilitate coffee fermentation

## Abstract

A method for fermenting coffee beans is disclosed. Bacteria secreting chimeric proteins comprising endo polygalacturane A (PGAA) and pectin methyl esterase 1 (PME1), which target the pectin coating for hydrolysis, are described. The net result of this targeted hydrolysis of the inner pectin coating can be the production of coffee beans with unique and unexpected flavor characteristics. Desirable flavors can be created or enhanced without any of the adverse taste qualities associated with artificial flavors. For lower grade coffee beans, this process serves to reduce or eliminate undesirable flavors.

## Background
RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Patent Application No. 61/035,529, filed Mar. 11, 2008, the contents of which are incorporated herein by reference.


SEQUENCE LISTING
This application incorporates-by-reference the material included on a written copy of a sequence listing included with the application, as well as on a computer readable copy of the sequence listing submitted on one compact disc. The disc was created on Jul. 30, 2009, and includes one 20 KB file entitled, “SQL34766 2003 ST25.txt.” The copy of the computer readable form of the sequence listing is identical to the written copy of the sequence listing, and thus, the computer readable copy includes no new matter.
FIELD
The invention relates to the use of pectinases for coffee fermentation.
BACKGROUND OF THE INVENTION
The preparation of coffee beans for consumption is a tightly choreographed process. Coffee cherries are harvested upon ripening and then both mechanical and biological processes are employed to recover a coffee bean from each cherry. The production process begins by the mechanical removal of both the outer covering (exocarp) and the pulp (outer mesocarp) in a process referred to as depulping.
A poorly understood fermentation step that has long been speculated to result in hydrolysis of the inner pectin coating is important for the production of suitable coffee beans. Briefly, this step involves soaking coffee beans in a vat of untreated water for approximately 15 to 20 hours. The completion of the fermentation step is then signaled by coffee beans undergoing textural changes to their pectin coating, which is thought to represent the hydrolysis of this layer. Frequent over fermentation results in spoilage and loss of product. Upon the completion of fermentation, the coffee beans are then washed, dried and are ready for roasting to produce the final product.
The fermentation process has been speculated to target the pectin coating of coffee beans. Pectin itself consists of linear polymers of alpha 1-4 linked D-galacturonic acid1. The carboxyl groups are frequently methylated to yield the corresponding ester. The degree of esterification (DE) refers to the ratio of esterified galactouronic groups to the total number of groups, and is used to classify pectin as either high methoxyl pectin (HM) or low methoxyl pectin (LM) at 50% DE′. The functional significance of the DE is its relationship to pectin solubility, gel forming abilities and availability for hydrolysis. Pectins with a lower degree of esterification can enter solution at a lower temperature and require a lower pH for precipitation due to their relative greater hydrophobicity of ester groups when compared with carboxy groups1.
The hydrolysis of pectin is mediated by four classes of enzymes: pectin methyl esterase (PME), polygalactouronases (PG), pectin lyase (PL) and rhamnogalacturonases (RHG)2. The activity of polygalacturonases may be further classified as having either endo- or exo-polygalacturonase activity. Endo-polygalacturonases catalyze the hydrolysis of 1,4-alpha-D-galacturosiduronic linkages between two non-methylated galacturonic acid residues while exo-polygalacturonases remove terminal residues. While, pectin methyl esterases (PME) function to reduce the esterification of pectin and as a consequence create a substrate that is then available for hydrolysis by polygalacturonases2.
The inner pectin coating has been hypothesized to be degraded by both bacterial and endogenous coffee bean enzymes3,4. The development of an acidic environment (pH 5.3-3.5) during the course of fermentation was thought to reflect the growth of presumed pectinolytic bacteria5. Changes in the texture and feel of the pectin coating after fermentation have been presumed to represent the hydrolysis of the pectin coating and signals to coffee producers the point at which fermentation should be terminated6. An incomplete removal of the inner mesocarp has been postulated to act as substrate for subsequent bacterial fermentation and spoilage of the bean7.
Frequent under- or over-fermentation will result in taste defects in the end product and may require disposal of the product8. Yeast overgrowth from a prolonged fermentation, due to their greater tolerance of an acidic environment is thought to be responsible for the taste defects that arise from over-fermentation. Specifically, ethanol production in addition to other organic acids from yeast is thought to be the principal mediator of the bitter taste associated with over-fermentation9.
More recent studies have suggested that the role of bacterial enzymes in fermentation may be dramatically limited in terms of their significance6. An examination of the fermentation microflora revealed that the proportion of pectinolytic bacteria remained relatively stable during fermentation5. Instead, non-pectinolytic lactic acid bacteria and yeast comprised the majority of cell growth, again presumably due to their greater tolerance of acidic conditions5. Pectinolytic bacteria that were recovered from the fermentation process included only those with limited pectinolytic activity, Erwinia herbicola and Klebsiella pneumoniae5. An innoculum enriched with these organisms failed to accelerate or alter fermentation in any meaningful manner to further refute earlier theories of their significance in coffee bean fermentation10. Yeast capable of pectinolysis have yet to be recovered from a fermentation reaction11.
The pectin coating that covers coffee beans exists in a highly esterified form12. As a consequence of this degree of esterification, the activity of pectin methyl esterases are essential to deesterify the pectin which then allows for subsequent substrate availability and hydrolysis by polygalacturonases. It has been observed that without deesterification the pectin would remain essentially unhydrolyzable by bacterial polygalacturonases2. Bacteria recovered from coffee fermentation reactions have to date been repeatedly shown to lack the activity to depolymerize esterified pectins10. The in vitro pectylase activity that has been recovered from bacteria present in coffee fermentation reactions was relatively inactive at the low pH environment at which the fermentation reaction occurs. Instead in vitro assays showed that the maximum catalytic activity for these enzymes was within the alkaline pH range10. Bacteria with polygalacturonase activity compatible with the acidic conditions of fermentation were present in relatively low levels although as previously mentioned they were unable to hydrolyze highly methylated pectin5. Hence, the current view is that bacterially expressed pectinases have no significant role in coffee fermentation. This notion is further reinforced by examination of coffee beans post fermentation to reveal that only modest depolymerization and limited hydrolysis of the pectin covering occurs6.
In summary, the limited growth of pectinolytic bacteria during the fermentation process, an absence of PME activity and finally the basic pH requirements of the limited pectinolytic activity that was present are all strongly suggestive of the current view that bacteria play a very limited or no role in directly mediating pectin hydrolysis. Avallone et al. have proposed an alternative mechanism that bacterially produced organic acids (lactic and acetic acid) decrease the environmental pH to induce conformational changes in pectin that results in it subsequent depolymerization6. This current view of coffee fermentation is in sharp contrast to previous theories that bacterial enzymes drive fermentation by catalyzing pectin hydrolysis.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the sequences of chimeric proteins comprising pectin methyl esterase (PME1)-Hemolysin A (HlyA): SEQ ID NO: 1, endo-polygalacturonase A (pgaA)-HlyA: SEQ ID NO: 2, hemolysin B (hly B): SEQ ID NO: 3, hemolysin D (hly D): SEQ ID NO: 4.



DESCRIPTION OF THE INVENTION
An embodiment of this invention relates to bacteria secreting chimeric proteins comprising endo polygalacturane A (PGAA) and pectin methyl esterase 1 (PME1), which target the pectin coating for hydrolysis. The net result of this targeted hydrolysis of the inner pectin coating can be the production of coffee beans with unique and unexpected flavour characteristics. Specifically, fruitiness can be enhanced and a new taste note of chocolateness can be created without any of the adverse taste qualities associated with artificial flavours. For lower grade coffee beans, this process serves to reduce or eliminate undesirable flavours such as woody, tobacco, earthy and fermented.
Overview of Recombinant Bacteria
According to an embodiment of this invention, recombinant bacteria have been created that can target the pectin coating present on coffee beans for hydrolysis. The presence of esterified pectin on the surface of the bean can require that a pectin methyl esterase is employed that is able to function under the low pH environment at which coffee fermentation occurs. Once the pectin is demethylated, a second enzyme, a polygalacturonase, can be required to hydrolyze the pectin. Finally, as most recombinant bacterial proteins remain localized within the cell, chimeric forms of these proteins can be required to export these proteins into the extracellular environment
Pectin Methylesterase
Pectin methylesterase (PME) catalyzes the hydrolysis of methyl ester groups linked to galacturonic acid residues that comprise pectin. Partial to complete hydrolysis of these ester groups is a prerequisite for the further hydrolysis of pectin. Qualitatively, enough esters should be hydrolyzed to produce a substantial change in the taste of the coffee. PME has been found in both fruits of several higher plants, bacteria and in filamentous fungi. The PME from higher plants and bacteria typically have optimal activity at a pH range between 7 and 813, whereas fungal PMEs typically operate best at a pH range of 3 to 414,15. Consequently, fungal PMEs may be better suited to the acidic conditions of fermentation.
A total of three fungal PMEs have been reported to date from various Aspergillus species14-16. The PME cloned from Aspergillus oryzae (PMEA) was found to have maximum activity at a pH of 5.0 and temperature of 55° C.14. As the fermentation process would typically not reach this temperature, this isoform of PME may not be well suited to catalyze deesterification during coffee fermentation under typical conditions. The PME cloned from Aspergillus Niger has been reported to have a Tmax of 45° C. (10 to 15 degrees less than A. Oryzae) and an optimal pH of 5.017. It is noted that, under typical conditions, PME from A. niger experiences competitive and non-competitive inhibition from the products of hydrolysis: polygalaturonic acid and methanol17. Finally, the third PME homolog, which can be recovered from Aspergillus Aculeatus15, is the most preferred. The PME recovered from this organism can be ideal for catalysis of coffee fermentation for several reasons15. To begin with, maximum activity occurs at a pH of 4.6 and temperature of 45° C., where 90 percent of the activity is present between 35 to 50 degrees Celsius. Furthermore, the activity of PME recovered from A. Aculeatus was not subject to feedback inhibition from high concentrations of methanol or the presence of polygalacturonic acid (the products of the reaction)15. This is in contrast to PME from A. Niger which was sensitive to feedback inhibition17.
In summary, A. Aculeatus PME1 (Gen Bank Accession Number: 449378) is preferred for deesterification of the coffee bean pectin, under typical conditions, due to its physical functional range and the absence of product feedback inhibition. This protein can be fused at the C-terminus to the hemolysin (HlyA) signal sequence to produce polypeptide SEQ ID NO: 1 to facilitate secretion from recombinant bacteria.
Polygalacturonase
Polygalacturonase catalyzes the hydrolysis of pectin and most isoforms of it hydrolyze deesterified pectin with a much greater efficiency. Plants, fungi and bacteria have all been found to express various PG isoforms, which may be divided into those with endo (can cleave internally) or exo (hydrolyzed the reaction from the ends) activity. Studies of bacterial PG have focused predominantly on PehX, the Erwinia PG18. The low pH at which coffee fermentation occurs would not be well suited to the higher pH requirements of PehX for maximum enzyme activity18.
The source of most commercial sources of PG arises from Aspergillus Niger. Here, seven different PG isoforms exist with different pectin specificites11. Furthermore, this family of enzymes has been shown to have maximum activity under acidic conditions. PG isoforms capable of hydrolyzing pectin with a high degree of esterification included pgaA (PGA) and pgaB (PGB)11. PGB has been shown to have greater activity on methylated pectin, although PGA peak activity occurs at a lower pH (pH=4)19. Ideally, a PG with the pH requirements of PGA and the ability of PGB to hydrolyze highly esterified pectin can be ideal for coffee fermentation. According to an embodiment of this invention, a recombinant bacterial strain expressing both PME and PGA is presented.
Protein Export
Recombinant proteins expressed by bacteria typically localize within the cytoplasm or periplasm. Specific signal sequences are required for the export of proteins into the extracellular environment. The hemolysin transporter system can be utilized to export PME and PGA into the extracellular environment. Hemolysin A, a component of the hemolysin transporter system mediates its toxic effect by inducing hemolysis upon its insertion into the plasma membrane of red blood cells20. Acetylation of hlyA by hlyC is required for its toxic activation21. According to an embodiment of the present invention, the hlyC gene is not introduced into the transgenic bacteria and only the C-terminal domain of hlyA, which contains none of the hemolytic properties associated with hemolysin A, can be used. Together, this helps to ensure the partial or complete absence of any pathogenic properties in the resulting recombinant bacteria.
Export mediated by hemolysin proteins requires the C-terminal 50 amino acids of HlyA fused to the target protein22-24, in addition, 2 translocator proteins HlyB (GenBank Accession Number: M81823), HlyD25 and an endogenous outer membrane protein, TolC26 form a trimeric exit pore for release of the hybrid protein. According to an embodiment of the present invention, chimeric forms of PME and PGA can be created by fusing the C-terminal of HlyA to the C-termini of the respective proteins to be target for export. Other molecular export systems as are known in the art can be suitable to export the chimeric proteins. In addition, HlyB and HlyD can be coexpressed to ensure formation of a pore for transport of the pectinases into the extracellular environment.
According to an embodiment of the invention, the DNA encoding the chimeric proteins can comprise a restriction site. The restriction site can be situated between the pectinase and the HlyA sequence and can be useful to allow changes to the DNA construct.
Examples
Materials and Methods
Vector Production
Synthetic gene constructs were purchased of each of the following genes: PME1, PGA, HlyB and HlyD from the GenScript Corporation. The C-terminal 50 amino acid coding sequence of HlyA was fused to the C-terminal of PME1 and PGA to create chimeric forms of these proteins. FIG. 1 provides the amino acid sequence of each protein. The cDNAs were then cloned into the Novagen PetDUET vector which allows for the expression of two recombinant proteins from each vector. The following plasmids were constructed: 1) PME1+PGA and 2) HlyB+HlyD. E. coli strain DH5α was then transformed with both vectors by the GenScript Corporation using methods known in the art.
Fermentation
Ripe coffee cherries were manually depulped prior to fermentation to recover strictly hard bean (shb) Costa Rican coffee beans. Bacteria were grown in Luria-Bertani broth (LB) containing 30 μg/ml kanamycin and 50 μg/ml ampicillin to an optical density (OD595) of 1.0. Bacterial cells were then induced with 4 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 32° C. for 30 minutes. Coffee beans were then added to the inoculum and the reaction incubated for 16 hours at 32° C. Coffee beans were then washed and dried for 2 weeks at room temperature prior to roasting. Coffee beans were roasted only when moisture levels for each bean were less than 10%.
Tasting
Coffee was tasted and scored on a scale of 1-10 for a total of 23 taste sensations: acidity, body, fruity, winey, nutty, chocolatey, floral, smooth, sweet, salty, herbal, woody, astringent, tobacco, musty, earthy, rubbery, phenolic, fermented, chemical, metallic, past crop and baggy. For each trial of tasting, a standard, control (Costa Rican Standard) and experimental sample were prepared in triplicate. This was performed for both intermediate and low-grade coffee beans. Each sample was tasted by 2 independent tasters that were blinded.
Results
The fermentation of coffee beans with recombinant bacteria conferred unique and unexpected taste properties to the end product. Table 1 summarizes the results after treatment of intermediate grade coffee beans. Coffee flavours were scored on a scale of 1 to 10 for each flavour note. Novel flavours were introduced and some desirable flavours were enhanced. New flavours introduced included both chocolate and nutty flavours. Furthermore, the chocolate note was absent of the alcohol note that is characteristically present in artificially flavoured coffees. Flavour notes that were improved included both sweetness and smoothness. For lower grade coffee beans, undesirable flavours were reduced or eliminated. These included woody, tobacco, earthy and fermented flavours. Table 2 summarized these tasting results on a scale of 1 to 10 for each flavour note







TABLE 1







Mean taste scores from control and 


recombinant bacterial fermentation 


of intermediate-grade coffee









Taste Attribute
Control
Enzyme





Acidity
6
6


Body
5
5


Fruity
2
4


Winey
0
0


Nutty
0
3


Chocolatey
0
8


Floral
0
1


Smooth
2
6


Sweet
0
4


Salty
0
0


Herbal
0
0


Woody
0
0


Astringent
0
0


Tobacco
0
0


Musty
0
0


Earthy
0
0


Rubberry
0
0


Phenolic
0
0


Fermented
0
0


Chemical
0
0


Metallic
0
0


Past Crop
0
0


Baggy
0
0









Coffee production is a multistep process of which a critical step is the fermentation of coffee beans. Current evidence suggests that this step results in only modest depolymerization secondary to acidic changes that occur in the environment and limited hydrolysis of pectin from pectinase activity most likely present within the ripe coffee cherry. The present invention is directed to a novel approach to this process. The process comprises bacteria that secrete chimeric proteins for the purpose of hydrolyzing the pectin coating present on coffee beans. The net result of this novel process is the creation of coffee with unique flavour characteristics. Specifically, a novel chocolate note is present that is absent of the alcohol note that characterizes artificially flavoured coffee. In addition, undesirable flavours of woody, tobacco, earthy and fermented were substantially reduced or eliminated. This novel process results in a superior tasting alternative to existing flavoured or unflavoured coffees.







TABLE 2







Mean taste scores from control and


recombinant bacterial fermentation 


of low-grade coffee beans.









Taste Attribute
Control
Enzyme












Acidity
3.8
5.8


Body
6.8
5.9


Flavour
1.0
5.8


Woody
8.0
0.4


Tobacco
4.5
0


Earthy
7.5
0


Fermented
7.0
0.1









As will be understood by those of ordinary skill in the relevant arts, once they have been made familiar with this disclosure, a variety of different methods can be suitable for use in implementing the present invention. While the invention has been described and illustrated in connection with various embodiments, many variations and modifications, as will be evident to those skilled in the relevant arts, can be made without departing from the spirit and scope of the invention, and the invention is thus not to be limited to the precise details of methodology or construction set forth above as such variations and modifications are intended to be included within the scope of the invention. Except to the extent necessary or inherent in the processes themselves, no particular order to steps or stages of methods or processes described in this disclosure, including the Figures, is implied. In many cases the order of process steps can be varied without changing the purpose, effect, or import of the methods described.
It will be appreciated by those skilled in the relevant arts, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.
All of the references cited herein are hereby incorporated by reference in their entirety.

## Claims

1. A method for fermenting coffee beans through the use of microorganisms, said method comprising the steps of:
combining depulped coffee beans with microorganisms transformed with one or more chimeric proteins comprising pectinases to produce a mixture;
incubating the mixture for a period of time; and
isolating the incubated coffee beans;
wherein the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans to be free from undesirable flavors of woody, tobacco, earthy and fermented, and wherein the woody, tobacco, earthy and fermented flavors are assessed by taste sensation testing,
wherein the pectinases are selected from the group comprising pectin methyl esterases and endo-polygalactouronases, and wherein the pectinases are suitable to use in an acidic environment.

2. The method according to claim 1, wherein the chimeric proteins comprise a hemolysin polypeptide.

3. The method according to claim 1, wherein the microorganisms are also transformed with hemolysin B and hemolysin D.

4. The method of claim 1, wherein the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans to have a chocolate flavor, and wherein the chocolate flavor is assessed by taste sensation testing.

5. The method of claim 1, wherein the microorganisms are bacteria.

6. The method of claim 1, wherein the depulped coffee beans are combined, in any order, with water and the microorganisms transformed with one or more chimeric proteins comprising pectinases to produce the mixture.

7. The method of claim 1, wherein the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans by decreasing taste scores for undesirable flavors of woody, tobacco, earthy and fermented by 2 or more units on a scale of 0 to 10 when assessed by taste sensation testing.

8. The method of claim 1, wherein the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans by decreasing taste scores for undesirable flavors of woody, tobacco, earthy and fermented by 4 or more units on a scale of 0 to 10 when assessed by taste sensation testing.

9. The method of claim 1, wherein: the chimeric proteins comprise a hemolysin polypeptide; the microorganisms are also transformed with hemolysin B and hemolysin D; the depulped coffee beans are combined, in any order, with water and the microorganisms transformed with one or more chimeric proteins comprising pectinases to produce the mixture;
and the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans to have a chocolate flavor, and wherein the chocolate flavor is assessed by taste sensation testing.

10. The method of claim 1, wherein: the chimeric proteins comprise a hemolysin polypeptide; the microorganisms are also transformed with hemolysin B and hemolysin D; the depulped coffee beans are combined, in any order, with water and the microorganisms transformed with one or more chimeric proteins comprising pectinases to produce the mixture; the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans by decreasing taste scores for undesirable flavors of woody, tobacco, earthy and fermented by 2 or more units on a scale of 0 to 10 when assessed by taste sensation testing; and the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans to have a chocolate flavor, and wherein the chocolate flavor is assessed by taste sensation testing.

11. The method of claim 2, wherein the microorganisms are also transformed with hemolysin B and hemolysin D.

12. The method of claim 2, wherein the microorganisms are also transformed with hemolysin B and hemolysin D, and wherein the depulped coffee beans are combined, in any order, with water and the microorganisms transformed with one or more chimeric proteins comprising pectinases to produce the mixture.

13. The method of claim 6, wherein the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans to have a chocolate flavor, and wherein the chocolate flavor is assessed by taste sensation testing.

14. The method of claim 2, wherein the microorganisms are also transformed with hemolysin B and hemolysin D, wherein the depulped coffee beans are combined, in any order, with water and the microorganisms transformed with one or more chimeric proteins comprising pectinases to produce the mixture, and wherein the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans by decreasing taste scores for undesirable flavors of woody, tobacco, earthy and fermented by 2 or more units on a scale of 0 to 10 when assessed by taste sensation testing.

15. The method of claim 2, wherein the microorganisms are also transformed with hemolysin B and hemolysin D, wherein the depulped coffee beans are combined, in any order, with water and the microorganisms transformed with one or more chimeric proteins comprising pectinases to produce the mixture, and wherein the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans by decreasing taste scores for undesirable flavors of woody, tobacco, earthy and fermented by 4 or more units on a scale of 0 to 10 when assessed by taste sensation testing.

16. The method of claim 15, wherein the activity of the pectinases during the fermentation process alters the flavor of coffee produced from the isolated coffee beans to have a chocolate flavor, and wherein the chocolate flavor is assessed by taste sensation testing.

17. The method of claim 1, wherein the pectin methyl esterase comprises an amino acid sequence as shown in SEQ ID NO: 1 and the endo-polygalactouronase comprises an amino acid sequence as shown in SEQ ID NO: 2.

18. The method of claim 1, wherein the chimeric proteins comprise the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

19. The method of claim 6, wherein the chimeric proteins comprise the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

20. The method of claim 13, wherein the chimeric proteins comprise the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.