WO1980000651A1

WO1980000651A1 - Process for the treatment of tobacco materials

Info

Publication number: WO1980000651A1
Application number: PCT/US1979/000772
Authority: WO
Inventors: D Teng; B Semp
Original assignee: Philip Morris Inc
Priority date: 1978-09-20
Filing date: 1979-09-17
Publication date: 1980-04-17
Also published as: CA1122551A; EP0020472A4; DE2963849D1; EP0020472B1; AU5068879A; AU527386B2; EP0020472A1

Abstract

The use of unexpanded tobacco is not as cost effective as expanded tobacco. This disclosure relates to a process for the treatment of tobacco materials so as to enhance puffing. More particularly, the disclosure relates to a process that comprises contacting tobacco with an aqueous enzyme solution exhibiting cellulase activity, incubating the tobacco-enzyme mixture, and thereafter expanding the tobacco material. Tobacco materials treated in this manner exhibit an enhanced capability for expansion, thereby resulting in a significant increase in filling capacity when compared to untreated expanded tobacco.

Description

PROCESS FOR THE TREATMENT OF TOBACCO MATERIALS

Background of the Invention The use of expanded tobacco materials in smoking products has escalated in recent years for a variety of reasons. For example expanded tobacco comprises an important part of the blend of tobac¬ cos used to produce smoking products and particularly low delivery smoking products. Cost reduction is also realized when expanded tobacco is utilized in that less tobacco is required. A number of tobacco expansion techniques have been described in patents and/or published patent applications in recent years, and these techniques are suitable for use in practicing the present invention.

We have found that enzymatic treatment of tobacco ma¬ terials, especially tobacco leaf, strip, cut filler, or stem, results in a significant increase in the expandability of the tobacco. Tobacco materials that are treated according to the enzymatic process of the present invention may then be subjected to known expansion techniques thereby resulting in a significantly increased filling capacity when compared to untreated tobacco. . Enzymatic modification of tobacco materials has been suggested by others. For example, U.S. Patents 3,256,888 and 3,256,889 disclose the treatment of tobacco with proteolytic or peptidic enzymes whereby the flavor and smoking characteristics are improved. U.S. Patent 3,240,214 -discloses a method for making an improved reconstituted tobacco sheet wherein the tobacco components are treated with catalytic amounts of an enzyme system consisting of cellulase, he icellulase, and pectinase. The resulting sheet product has increased tensile strength and elasticity. U.S. Patent 3,974,838 discloses the treatment of tobacco with an amylolytic enzyme capable of converting the starch contained in the tobacco into sugar whereby the smoking properties are improved. Canadian Patent 970,306 discloses the use of pectolytic enzymes produced by microorganisms for the purpose of effecting substantial fibrilla- tion of plant parts, and particularly tobacco, thereby minimizing the need for mechanical beaters or homogenizers. The thus treated tobacco parts are then fabricated into reconstituted tobacco pro¬ ducts.

U.S. Patent 3,132,651 discloses the treatment of tobacco, preferably uncured, with cellulase to effect a relatively rapid aging or conditioning of the tobacco with a concomitant reduction in nicotine, phenols, and resins. The cellulase used is preferably of the Aspergillus type. The concentration of enzyme used is about 0.001 to 0.1% of the weight of the water, and generally-excessive water is used in the range of 10 to 30 times the weight of the tobacco. After treatment for about 1/2 to 2 hours, the tobacco is drained, repeatedly rinsed with warm water, and dried under moder¬ ate heat. This process suffers several distinct disadvantages. The repeated washing of the tobacco with warm water results in an undesirably high loss of valuable tobacco solubles that contribute desirable flavorants to the tobacco when smoked. Secondly, because of the excessive amounts of water used during treatment, increased energy output is required to dry the tobacco to a moisture content suitable for use in smoking products. U.S. Patent 3,513,857 discloses a process for treating tobacco stems with a solution of polysaccharide-hydrolyzing enzymes exhibiting catalytic pectinase and hemicellulase activity whereby the stems become swollen and softened. The enzyme solution con¬ tains an enzyme to stem weight ratio of between about 1 to 10,000 and 1 to 10, and the concentration of water is about 50 to 1,000%, and preferably about 100 to about 400%, of the weight of the dry stems. Treatment times up to about 48 hours may be employed.

Although many of the enzyme preparations suggested for use contain cellulase as well as pectinase and hemicellulase, the inventor states at column 3, lines 12-15 of the specification that, "Pectinase, however, is the most essential of the enzymes as revealed by chemical analysis of the enzyme treated stems. Cellu-

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WIPO lase is the least essential of the enzymes." Treatment of- stems according to this process results in a product having increased filling power, which may be further improved by freeze-drying. The degree of increased filling power was demonstrated by microscopic examination of the freeze-dried stems as well as by resistance to draw (RTD) measurements.

Summary of the Invention The present invention provides an improved process for the enzymatic treatment of tobacco materials with an aqueous solύ- tion of cellulase produced by the microorganism Trichoderma vi ide. Following a suitable incubation period of the tobacco- enzyme mixture, the tobacco is dried and expanded by any of the known expansion techniques. Tobacco treated with cellulase prior to expansion exhibits an increased filling capacity or ability to expand when subjected to known expansion techniques.

Description of Preferred Embodiments The process of the present invention may be adapted for use on green, dried, partially cured, or cured tobacco or homo¬ genized leaf cure tobacco. By tobacco is meant tobacco leaf, strip, stem, midribs, stalk, reconstituted tobacco sheet, or any combination of said components. Use of cellulase produced by microorganisms such as Aspergillus niger, Cellulomonas sp.i Myro- theciu verrucaria, Penicilliu expansum, and various strains of Trichoderma viride such as ATCC 13631, 24449, 26920, and 26921 is possible; however, we have found that using cellulase produced by Trichoderma viride results in highly satisfactory enhanced expand¬ ability of the tobacco. ^' Trichoderma (viride) lonqibrachiatum QM 9414 (ATCC 26291) was purchased from The American Type Culture Collection, Rockville, Maryland 20852. Procedures used for the preparation of cellulase are disclosed generally by Mandels and Sternberg in the Journal of Fermentation Technology, 54(4), 1976, pages 267-286, The Society of Fermentation Technology--Japan. The procedures will be described in detail hereinafter.

Tobacco, preferably in strip or shredded form, is sprayed with a solution of cellulase having an approximate activity of about 0.1 to 0.4 units/ g protein. Only enough solution ι\s utilized to assure even distribution of the enzyme throughout the tobacco thereby minimizing energy output in drying the materials after treatment. Typical methods for applying the cellulase to the tobacco materials include spraying, dipping, or passing the tobacco through a bath containing the enzyme solution. Following applica¬ tion of the enzyme solution, the tobacco typically has a moisture content of about 20 to 50% by weight. The pH of the tobacco-enzyme system is maintained in a range between about 3.5 and 6.5, and optimally at about 4.8. After contacting the tobacco materials with the enzyme solution, the tobacco is placed in a container and incubated at a temperature in the range of about 35 to 70°C, and preferably at about 50°C, for a period of about 4 to 124 hours. Following incubation, the tobacco is dried by .any suit- able means and equilibrated at about 23.9^'°C and 60% relative humi¬ dity (r/h). The tobacco is then expanded using known expansion techniques such as those disclosed^' in Canadian Patent 1,013,640. When tobacco stems are. to be expanded, the expansion techniques as disclosed in U.S. Patent 3,734,104 to Buchanan and Madures produce satisfactory results. Following expansion and reequilibration, the tobacco is ready for use in fabricating cigarettes wherein expanded tobacco comprises a part of the total blend. Tobacco materials treated according to the present invention exhibit enhanced expand¬ ability thereby resulting in an increased filling capacity of about 10 to 30% as compared to expanded tobacco that has not been pre- treated with cellulase.

It will be readily apparent to those skilled in the art that various modifications of the process are possible: For example, for commercial production purposes, shorter reaction times may be required and this may be achieved generally by using either cellulase of increased concentration or cellulase with increased activity; i.e., greater than 0.4 units/mg protein. .The enzyme- containing broths as produced herein may be concentrated by known methods in the art, such as freeze-drying or protein precipitation techniques. In this instance, the concentrated enzyme is resus- pended in a suitable buffered solution to the desired range of

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OMPI activity and thereafter applied to the tobacco materials. -Where time requirements are not of great concern, the more dilute enzyme broth has been found to be adequate to achieve the desired results. Various other modifications may be made and are considered to be within the spirit and scope of the present invention.

Example 1 Cellulase Preparation

A. Induction Broth—The cellulase-containing broth was prepared generally as described by Mandels and Steinberg in the Journal of Fermentation Technology, 54(4), 1976, pages 267-286, The Society of Fermentation Technology— apan and is incorporated herein by reference.

The Trichoderma viride medium for cellulase production was prepared according to the following formulation (grams/liter):

(NHJaSO - 1.4 FeS0_I-7H₂0 - 0.0050

Urea - 0.3 ZnSO HaO - 0.0014

CaCl₂ - 0.3 CoCl₂ - 0.0020 MgSO^H_≥O - 0.3 Cellulose (Avicel PH-105, FMC.Corporation) - 10.0

Proteose peptone (Difco) - 1.0

Tween® 80 - 2.0 pH 5.0 - 6.0

The broth was divided into 250 ml aliquots and placed in 1-liter creased shake flasks. The flasks were capped (metal) and sterilized for 25 minutes at 121°C and 15 psi.

B. Inocυla Preparation—A potato dextrose agar (Difco) slant was inoculated with 0.05 ml of a sterile water solution containing spores of Trichoderma (viride) longibrachiatum QM 9414 (ATCC 26921). This material was spread with a sterile loop, the tube sealed (screw cap), and incubated for 96 hours at 24+1°C. At this point, the material may be stored at 0 to 5°C until further usage is desired. Transfer of cultures of Trichoderma viride every 21 days gives satisfactory results.

If immediate usage is desired, the slants are removed from the incubator and three milliiiters of sterile water is introduced. .The slant is then shaken (Vortex Genie Mixer, Scienti¬ fic Products) for 1 minute.

The suspension from the slant is asceptically transferred to a 500 ml creased flask containing 100 ml of potato dextrose broth (Difco), capped^", and placed in New Brunswick Scientific gyro- tory® water bath shaker set at 24+1°C. This material is shaken at 80 RPM, for 96 hours at which time it is removed and placed in a refrigerator at 0 to 5°C for at least 12 but no longer than 48 hours prior to usage. This material will be referred to herein- after as the seed broth.

C. Induction—Four milliiiters of seed broth were trans¬ ferred to the flasks containing 250 ml of cellulase induction broth as prepared in Step (A). The flasks were then placed in a New B Brruunnsswwiicckk SScciieennttiiffiicc ggyyrroottoor water bath .shaker and incubated at 60 RPM, 24+1°C for 14 days.

D. Fungal Removal—The Trichoderma viride-containing broth was removed from the shaker and quickly chilled in an ice bath. The chilled broth was centrifuged at 12,000 g for 30 minutes in a refrigerated centrifuge (Beckman Model J-21C). The superna- tant broth was then passed through a 0.2 μ milipore filter, poured into sterile flasks packed in ice, and stored in a refrigerator at 0 to 5°C. This cellulase-containing culture broth was then ready for enzyme assays.

E. Enzyme Assays—In order to determine the specific enzyme activity of the materials prepared in Steps (C) and (D), assays as disclosed by Mandels et al . in Biotechnology and Bio- engineering, Volume XVI, pages 1471-73, 1974 were used.

O...PI . VWvI-.FrOU Assay 1: Cx Activity—One half of a ml of the enzyme solution obtained in Step (D) is added to 0.5 ml of 1% CMC-0.5 DS in 0.1 M citrate buffer, pH 4.8. The mixture is incubated at 50°C for 30 minutes. Three ml of DNS is added and the mixture is boiled for 5- minutes. Eight ml of water is added to the mixture and the optical density is read at 550 mμ on a spectrophotometer (Hitachi Model 124). Units of activity = mg glucose produced/ml.

Assay 2: C_x Activity—To 5 ml of the enzyme solution obtained in Step (D) is added 250 mg Avicel PH 105 and the mixture is adjusted to pH 4.8 using.0.1 M citrate buffer, pH 4.8. The mixture is incubated at 50°C for 24 hours and then filtered. To the filtrate is added 1 ml of.0.1 M citrate buffer, pH 4.8 and 3 ml DNS. The mixture is boiled for 5 minutes, diluted with 8 ml of water and the optical density is read at 550 mμ on a spectropho- tometer.

Assay 3: Protein Measurement—Lowry's Folin phenol reagent method is used to determine the protein content in the enzyme broth. (Journal of Biological Chemistry, Volume 193, pages 265-275, 1951.) Units of enzyme activity = mg glucose produced/ml. Tv cellulase activity = C_x + C_x activities.

Example 2 One kg of bright tobacco cut filler was sprayed with 300 ml of 0.5 M citric-sodium citrate buffer at pH 4.8 containing 750 units of total Tv cellulase activity (Ci + Cx = 0.86 units/mg protein) as prepared in Example 1(D). The tobacco was placed in a container and incubated at 50°C for 72 hours. The treated tobacco material was then air-dried and equilibrated at about 23.9°C and 60% r/h. This material was then expanded using the process as disclosed in Canadian Patent 1,013,640. The expanded material was equilibrated at 23.9°C and 60% r/h, and the filling capacity was determined by the cylinder volume determination of Wakeham et al. as disclosed in Tobacco Science, Volume XX (1976), pages 157-160. One kg of bright tobacco cut filler, which was sprayed with 300 ml of 0.5 M citric-sodium citrate buffer at pH 4.8 and treated as described above, was used as a control. The average results, comprised of three separate- runs, (8 replicates) are listed below.

Control Cellulase-Treated

Cylinder Volume 83.4 99.4 (cc/10 g)

The increased filling capacity of the cellulase-treated tobacco samples ranged from about 12% to a high of about 29% with an average of about 19.1% when compared to expanded tobacco samples that had not been treated with cellulase. Enhanced expandability of cellulase-treated tobacco is evident from the above results.

Example 3 In a similar manner to Example 2^', one kg of tobacco filler was sprayed_.with 300 ml of 0.5 M citric-sodium citrate buffer at pH 4.8, which contained 375 units of total cellulase activity (Ci + C_x = 0.86 units/mg protein). The tobacco was placed in a container and incubated at 50°C for 72 hours. The treated tobacco material was then air-dried and equilibrated at 23.9°C and 60% r/h. The equilibrated tobacco filler was expanded as in Example 2 and equilibrated as before. The cylinder volume was determined using the method of Wakeham et al. One kg of tobacco filler, which was sprayed with 300 ml of 0.5 M citric-sodium citrate buffer at pH 4.8 and treated as described above, was used as a control. The results are tabulated below.

Control Cellulase-Treated

Cyl inder Volume 75.9 85.9 (cc/10 g)

The results show that the enzyme-treated filler expanded to a greater degree; i.e., 13.2% greater than the untreated control tobacco.

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/ Example 4 In a similar manner to Example 2, 1 kg of tobacco stems was sprayed with 300 ml of 0.5 M citrate-sodium citrate buffer at pH 4.8. The solution contained 750 units of total cellulase acti- vity (Ci + Cx = 0.86 units/mg protein). The enzyme-stem mixture was incubated for 12 hours at 50°C. ControT stems were sprayed with the same buffer solution containing no cellulase and treated in a similar manner. The enzyme-treated stems and control stems were expanded according to the methods disclosed in U.S. Patent 3,734,104 to Buchanan and Madures. Following equilibration at 23.9°C and 60 r/h, the filling capacity of the expanded stems was determined using the cylinder volume method. The results are tabulated below.

Control . Cellulase-Treated Cylinder Volume 39.5 43.0 cc/10 gm

The results indicate that the cellulase-treated stems expanded to a greater degree, specifically 8.9% greater, than the untreated expanded control.

Example 5

To demonstrate that the expansion enhancement is pre¬ dominantly a function of the cellulase enzyme, tobacco filler was treated with pectinase, hemicellulase, and pectinesterase purchased from Sigma Chemical Company. One kg of tobacco filler was treated with 300 ml of appropriate buffer, which contained 1 g of the designated enzyme. The tobacco-enzyme mixture was incubated at the specified optimum temperature for the particular enzyme for 24 hours. Following prolonged incubation at the designated tempera¬ ture range and moisture content, some degree of spoilage was noted. The enzyme-treated tobacco and control tobacco treated in a similar manner were dried, equilibrated, and expanded as in Example 2. The experimental conditions and results are tabulated below.

The results indicate that there is no significant change in the cylinder volume when tobacco is treated with the above-cited enzymes and then expanded using the method disclosed in Canadian Patent 1,013,640. .

Example 6 To demonstrate the use of the present invention in a typical tobacco production run, the cellulase enzyme produced by Trichoderma viride was incorporated into the casing solution and applied to the tobacco. The casing solution, which is comprised of a mixture of hygoscopic agents and volatile or nonvolatile flavor¬ ing agents is generally sprayed on the tobacco to condition it for further processing.

One kg of tobacco filler was sprayed with a buffered solution containing 750 units of total cellulase activity and tobacco casing additives. Control tobacco was sprayed with a similar buffered casing solution containing no cellulase. The treated tobacco and control were incubated for 72 hours at 50°C and then expanded and equilibrated as in Example 2. The cylinder volume of the treated and control tobacco were determined and the results are tabulated below.

Control Cel l ulase-Treated

Cyl inder Vol ume 77.6 . 101 .8 (cc/10 g)

The cel l ul ase- treated tobacco increased 31 .2% more in vol ume than the untreated control tobacco.

Claims

WHAT IS CLAIMED IS:

CLAIM 1. A process for treating tobacco materials to achieve increased filling power that comprises the steps of: a. contacting tobacco material with a buffered solution at a-pH in the range of about 3.5 to 6.5 and containing cellulase in an amount sufficient to effect enhanced expandability; b. incubating the tobacco-cellulase mixture for a period of time between 4 and 124 hours and at a temperature within the range of 35 to 70°C; and c. drying and expanding the cellulase-treated tobacco materials.

CLAIM 2. A process.of Claim 1 wherein the buffered solution of cellulase contains from 0.1 to 0.4 units/mg of protein.

CLAIM 3. A process of Claim 1 wherein the tobacco material is selected from stem, strip, midribs, cut filler, or reconstituted tobacco.

CLAIM 4. A process of Claim 1 wherein the cellulase used is produced by the microorganism Trichoderma viride.

CLAIM 5. A process of Claim 1 wherein the cellulase- treated tobacco of Step (b) has a moisture content in the range of about 20 to about 50% by weight.