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(11) | EP 0 024 152 B1 |
| (12) | EUROPEAN PATENT SPECIFICATION |
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Continuous method of denitrating tobacco extracts Kontinuierliches Verfahren zur Denitrifikation von Tabakextrakten Procédé en continu pour la dénitrification d'extraits de tabac |
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| Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). |
[0001] This invention relates to a continuous process for reducing the levels of certain nitrogen-containing compounds, notably nitrates, nitrites and ammonium compounds present in tobacco materials using microorganisms.
[0002] It is generally recognised that smoking products having lowered amounts of oxides of nitrogen present in smoke are desirable. Therefore a number of methods have been developed to reduce the delivery of oxides of nitrogen by smoking products. Among these techniques are various methods wherein the nitrate content of the tobacco is altered. For example, methods involving microbial treatment of tobacco to accomplish such nitrate reduction have been proposed.
[0003] Our BE-A-866445 describes a process for anaerobic microbial degradation of nitrates and nitrites in tobacco.
[0004] Our DE-A-2816427 describes a process for microbial degradation of nitrate, nitrite and other nitrogen containing compounds under nitrogen deficiency or oxygen deficiency conditions. The microorganisms may be selected from the genus Aerobacter, Pseudomonas, Micrococcus or Escherichia, with Enterobacter aerogenes being specifically employed in the examples, and obtain their nitrogen or oxygen requirements respectively from nitrate or nitrite degradation. More specifically, in this latter process, an extract of the tobacco is mixed with a culture of the microorganisms and necessary nutrient additives to form a work mixture, and the mixture is maintained under conditions in which the organisms are in their exponential growth phase.
[0005] EP-A-0 005 082 (published October 31, 1979) describes a process for microbial reduction of nitrates in tobacco by dissimilatory denitrification whereby nitrogen gas is the end product. The microorganism specifically suggested for use in the process is Paracoccus denitrificans (or Micrococcus denitrificans). Species of the genera Pseudomonas, Alcaligenes, Bacillus and Propionibacterium can also be employed.
[0006] US-A-3,845,774 to Tso et al. describes a tobacco curing treatment method wherein the tobacco is homogenised and incubated during curing in order to regulate the composition of the final product. Nitrate-nitrogen and total nitrogen are reduced somewhat, but the amount of reduction is not as significant as that of the present process. No specific methods or means for reducing nitrate-nitrogen are suggested.
[0007] Gravely et al., US-A-3,747,608 relates to a method for aerobic microbial digestion of pectin- bound plant material, specifically tobacco materials, using pectolytic enzyme-producing microorganisms.
[0008] Examples 11 and 13 disclose data related to concomitant denitration of tobacco using the microorganism Erwinia carotovora, ATCC 495. This microorganism is unsuitable for use in the present invention since pectolytic enzyme-producing microorganisms, such as Erwinia carotovora, destroy the structural integrity of the tobacco.
[0009] W. O. Atkinson et al. reported a reduction in various tobacco leaf components, including nitrate-nitrogen, by varying homogenization and incubation techniques during curing. (Abstract of Proceedings of the University of Kentucky Tobacco and Health Research Institute, Lexington, Kentucky, Conference Report 4, March 1973, pages 829-33).
[0010] Denitration by means of microorganisms is also known outside the tobacco arts. Representative examples are US-A-3,709,364 to Savage, US-A-3,829,377 to Hashimoto, US-A-4,039,438 to Anderson, and US-A-4,043,936 to Francis et al. which describe denitrification of waste water using anaerobic bacteria to reduce the nitrate to nitrogen gas. Members of the Thiobacillus, Pseudomonas, Chromobacter, Bacillus and Chlostridium genera are among the microorganisms which may be employed. In the Hashimoto patent the use of pressurized systems to increase the amount of methane available to the microorganisms and to facilitate liberation of the nitrogen gas by venting are suggested. The Anderson patent suggests conducting the process at ambient or atmospheric pressure. In the Francis patent the nitrogen gas passes through an exit out of the system. The Savage reference employs pressure to pass the effluent being treated through the filter containing the microorganisms.
[0011] Microorganisms have also been used to modify other tobacco components. For example, US-A-4,037,609 and USA4,O38,993 to Geiss et al. disclose methods for reducing the nicotine content of tobacco by microbial treatment using microorganisms obtained from tobacco, including Pseudomonas putida and Cellulomonas sp. Aerobic fermentation techniques are employed wherein nicotine is degraded via microbial action to 3-succinoylpyridine. The latter microorganism is capable of reducing nitrate to nitrite and actively produces nitrogen gas. Similarly degradation of nicotine to 3- succinoylpyridine by means of the same microorganisms is described in US-4,011,141 to Gravely et al. Lippman et al. US-A-2,000,855 describes microbial denicotinization of tobacco by fermenting moist tobacco while adding acid to overcome the alkaline condition produced by fermentation. Alternatively the patent suggests removal of volatile bases by supplying an air current or employing suction. Fermentation was used to improve aroma and mellowness in US-A-2,644,462 to Frankenburg and in US-A-4,135,521 to Malen et al.
[0012] Further, US-A-2,149,179 relates to an accelerated aging method for tobacco wherein the aging is effected by means of fermentation with exclusion of oxygen employing microorganisms capable of growing in the absence of oxygen. The microorganisms may be those which are bred on noble tobaccos or anaerobic yeasts. By means of the process, fermentation times of only days, rather than months are required. The purpose of the claimed fermentation process is to improve the bouquet of the tobacco. Nicotine content in the tobacco is also reduced. According to the patent, a prior process of Suchsland, which used microorganisms to decompose complex organic substances in tobacco into simpler compounds, did not prove practical since the oxidation effected by oxygen during the fermentation was ignored.
[0013] We have now unexpectedly discovered that by employing carefully controlled conditions, it is possible to effect denitration via an aerobic assimilatory metabolic pathway on a continuous basis. Specifically, it has been discovered that by controlling the denitration conditions, it is possible to coordinate the microorganisms' growth rate with the tobacco extract treatment rate, whereby a denitration process is provided which is easily adapted to other continuous tobacco treatment processes, can be employed on a continuous basis for extended periods with relatively little or no supervision and permits treatment of greater amounts of tobacco extract and results in a higher production rate relative to batch processes. That is, the present process provides a method whereby nitrates, nitrites and ammonium compounds can be efficiently eliminated from tobacco via an assimilatory metabolic process on a large, technical scale under economical conditions, with a minimal requirement of manpower or energy and minimal addition to or transformations of the tobacco extract components, other than such denitration.
[0014] Accordingly, the invention is characterised in that sterile tobacco extract and additives are continuously added to the work mixture at a dilution rate which does not exceed the growth rate of the microorganisms, while the pH, temperature and aeration are maintained at levels which promote aerobic assimilation, the extract, phosphate and a carbon source being added in amounts such that the overall addition thereof is 0.1-7.5 g nitrate ions per liter added, 1.0 to 10 g P04 ions per liter added and sufficient carbon source to provide at least 16.5 assimilative carbon atoms/N03 ion added, and that work mixture is continuously withdrawn at such a rate as to keep the total volume of work mixture constant. The microrganisms may subsequently be removed from the withdrawn mixture. Preferred microorganisms for use in the present process are Candida yeasts. Denitration with such yeasts may be effected continuously as above described employing a dilution and withdrawal rate of 0.1 to 0.35 liter of additives and extract per liter of work mixture per hour while maintaining a pH of 3.5 to 7.2, a temperature of 25° to 37°C and an aeration rate of 0.8 to 2.5 liters per liter work mixture per minute. Enterobacter aerogenes may also be used in the present process. Denitration with such microorganisms may be effected on a continuous basis employing a dilution and withdrawal rate of 0.1 to 0.25 liter of additives and extract per liter of work mixture per hour while maintaining a pH of 5.5-8, a temperature of 30°-40°C and an aeration rate of 1.0 to 3 liters air per liter work mixture per minute.
[0015] In the drawings, Figure 1 is a flow diagram of a tobacco denitration system for the extraction of tobacco through the denitration steps of the present invention up to the reapplication of the denitrated extract to the extracted tobacco. It is possible to run a system employing the denitration method of the invention for extended periods with little or no supervision.
[0016] Continuous assimilatory denitration is effected according to the present invention by introducing material to be treated into a fermentation vessel while withdrawing treated extract from the fermentor at the same rate, such that the overall volume of material in the fementor, that is, the work mixture, remains constant. Moreover, the rate of extract introduction and withdrawal and the process conditions are such that there is no need to repeatedly inoculate the work mixture; rather after a single inoculation a system employing the present denitration process can be run for extended periods without reinoculation.
[0017] The metabolic pathway employed in assimilatory denitration can be represented as follows:
Such assimilatory denitration thus involves the use of nitrate as a nitrogen source to build up cell material.
[0018] In the practice of the present invention, microorganisms capable of assimilatory denitration must be employed. Various yeasts, particularly Candida yeasts, are capable of nitrate assimilation. Among the Candida yeasts, the Candida utilis NCYC 707, 321 and 359 strains, the Candida utilis DSM 70167 strain, which is the same as the NCYC 359 strain, and the Candida berthetii CBS 5452 strain have been found particularly effective in the practice of the present invention. Microorganisms, such as Enterobacter aerogenes, particularly Enterobacter aerogenes ATCC 13048, which is the same as the DSM 30053 strain, may also be employed in the practice of the present invention.
[0019] These cultures are available at the culture banks indicated by the abbreviations. The meaning of the abbreviations is as follows:
NCYC Nation Collection of Yeast Cultures Brewing Industry Researth Foundation
CBS Central Bureau of Mold Cultures
ATCC American Type Culture Collection
DSM German Collection of Microorganisms
Tables I, II, III are descriptions of the cultures.
[0021] The work mixture comprises suitable microorganisms in tobacco extract under conditions which promote aerobic assimilation of nitrogen-containing compounds. Generally, to start assimilation 30-100 g of starter culture mass of microorganisms are inoculated per liter of tobacco extract under conditions favorable to aerobic assimilation.
[0022] For optimum results and to avoid lag phase, a starter culture which is in exponential growth phase, and preferably late exponential growth phase, and which has been pregrown on tobacco extract is employed. Such a starter culture of Candida yeast can be prepared for example by inoculating tobacco extract with 2 loops of yeast, incubating the inoculated yeast for 9 hours, employing 10 ml of the resultant solution as an inoculum for 200 ml of fresh extract substrate and thereafter incubating for 15 hours. Typically the amount of such a starter culture used to inoculate the work mixture is sufficient to produce a final concentration of culture in mixture of at least O.5-1 %.
[0023] Conditions which promote effective aerobic assimilation' are maintained in the work mixture during the practice of the process. Generally suitable conditions for Candida yeasts are an aeration rate of 0.8 to 2.5 liters air/liters work mixture/minute, a pH value within the range of 3.5 to 7.2 and a temperature at a point between 25° and 37°C which is favorable for a large proportion of nitrate elimination relative to carbon added plus adequate agitation.
[0024] In the continuous operation of the process, a sterile additive mixture made up of tobacco extract and additives is added to the work mixture at a dilution rate which does not exceed the growth rate of the microorganisms employed. This dilution rate is measured as liters of additive solution per liters work mixture per hour. Generally, dilution rates of 0.1 to 0.35 I/l/hr are acceptable. Sterilization of the additive mixture can be accomplished by heating.
[0025] The additive mixture may be added as a single solution containing the tobacco extract and additives or the individual materials may be separately introduced into the work mixture. Overall the total additions to the work mixture comprise 0.1 to 7.5 grams nitrate/liter of total additive mixture depending on the microorganism employed and 1.0 to 10 grams phosphate/liter of total additive mixture, as well as a carbon source at a concentration sufficient to provide at least 16.5 assimilative carbon atoms per molecule of nitrate added.
[0026] During the practice of the process, a portion of the work mixture is continuously removed at a rate such as to keep the volume of the work mixture constant. The withdrawn work mixture may be further treated to remove the biomass therefrom, that is, the microorganisms are removed, whereby denitrated extract is obtained which is of substantially the same composition as the original extract except for the removal of the nitrate.
[0027] It is preferable to allow the starter culture to reach the exponential growth phase in the work mixture prior to commencing continuous operation of the process. However, aside from the one-time starting phase, whose products can be discarded, the process can be operated on a continuous basis with maintainance and regulation of conditions for the effect desired with little or no supervision. In contrast, various treatment phases are required in a noncontinuing, charged, so called batch process and if mistakes are made with a batch process, new conditions for assimilation have to be achieved, which could take hours. Production by batch techniques is, thus, costlier and requires more personnel than with the process described in the invention.
[0028] The aqueous tobacco extract employed in the process of the invention may be obtained in a conventional manner. One method comprises contacting tobacco with water in a 1:10 ratio, commonly at elevated temperature. The insoluble tobacco residue is thereupon separated from the aqueous extract by suitable solid/liquid separation techniques, such as centrifugation, pressing or the like. The insoluble residue may then be dried or subjected to reconstitution. If necessary, the concentration of nitrate is adjusted for addition to the work mixture by evaporation or dilution of the tobacco extract.
[0029] In contrast to batch methods, wherein the phosphate present in stem pool extracts is sufficient, phosphate must be added in the practice of the present process. Thus the additive mixture must contain phosphates, as well as a carbon source, in amounts sufficient for cell growth and total nitrate absorption. Typically 1.0 to 10 grams of phosphate/liter of additive mixture and enough carbon source to provide at least 16.5 assimilative carbon atoms/molecule of nitrate added are adequate for additive mixtures containing 3-7.5 grams nitrate. Preferably the concentrations of carbon source and phosphate are such that they are consumed during assimilation of the nitrate and thus do not reach the final denitrated extract. However, higher concentrations of these materials can be tolerated in the practice of the present invention.
[0030] The carbon source may be any material that will provide the necessary carbons in an organic form usable by the microorganism to assimilate nitrates, nitrites and the like. Various carbon sources have been found suitable in the practice of the invention. For example, glucose, dextrose monohydrate and beet molasses have proven satisfactory. The carbon may also be derived from the acid employed to adjust the pH of the work mixture, for example, from lactic acid. With the Candida yeasts, glucose, sucrose, maltose, cellobiose, ethanol, glycerin or citrate are all suitable carbon sources. With Enterobacter aerogenes, lactose may additionally be employed.
[0031] Generally 16.5 assimilative carbons are required as an absolute minimum for assimilation of a molecule of nitrate. On the other hand, the amount of carbon source is preferably kept as close as possible to the threshold, since any excess will remain in the final denitrated extract. The threshold is generally about 20±5 carbons/nitrate molecule. With good aeration, a maximum of about 6.2 g/I N03 can be assimilated with a 4% glucose solution. In general, with nitrate levels of 3-7.5 g/I added to the work mixture, a concentration of 2.4-6% glucose is required when employing Candida yeasts.
[0032] In the practice of the invention, temperature affects the amount of carbon required. At temperatures of about 28-30°C, minimal amounts, i.e., about 16.5 carbons, may be used. Outside this temperature range, the amount of carbon must be increased. Further temperature and growth rate of the microorganisms are directly related. Thus higher temperatures favor increased growth of microorganisms. However, increases in temperature also increase the fermentation rate, with resultant alcohol formation rather than growth. The rate of fermentation may be checked by measuring ethanol formation during the process. Temperatures which minimize fermentation while maximizing growth are thus preferred. In the case of Candida utilis 30°C is the preferred temperature.
[0033] To regulate and maintain pH, acids and/or bases are employed in the work mixture. Orthophosphoric acid and/or potassium hydroxide are preferred for this purpose. The agent employed to adjust pH may also be the phosphate or carbon source, for example phosphoric, lactic or citric acid or mixtures thereof. Thus, where phosphoric acid is employed to regulate pH, no other addition of phosphate in the additive mixture is required.
[0034] Aeration of the work mixture is generally at a rate which is sufficient to avoid fermentation while favoring assimilation. Generally, a rate of 0.8 liters air per liter work mixture per minute is the threshold aeration rate required to avoid fermentation where maximum carbon levels are employed. Overall, aeration rates of between 0.5 and 2.5 are suitable in the practice of the process, with rates of 1.0 to 2.0 being particularly effective.
[0035] In order to overcome the effects of air injection, it may be necessary to employ a mechanical foam breaker or antifoam agent. Paracum 05/12A and 24/sw have both been found satisfactory. Addition of 225 ppm to the work mixture is adequate but levels of 250 ppm are preferred to ensure trouble-free operation.
[0036] The precise conditions employed in the practice of the present invention will depend upon the precise organism employed. In general, when two of the three conditions for aerobic assimilation are optimized, the third variable can be changed empirically. Further, it should be noted that since the nitrate extracts being treated are solutions of naturally occurring products whose components vary, optimum conditions are not always the same, but will vary within the ranges indicated. For example, in the case of Candida utilis NCYC 707, optimum conditions for the practice of the invention in the treatment of some extracts are an aeration rate of 1.5 I/I/min., a temperature of 30°C and a pH of 5.5. Thus, very good results are obtained with an aeration of 1.5 liters/liters/minute, a pH value of 5.5 and a temperature between 26° and 37°C; with an aeration of 1.5 liters/liters/minute, a temperature of 30°C and a pH between 3.9 and 5.5; or with a temperature of 30°C, a pH of 5.5 and aeration between 0.5-1.0 liters/liters/minute.
[0037] A denitration system employing the process of the invention is depicted in the flow diagram of Figure 1. The reference numbers refer to the denitration stages as follows:
1. Tobacco supply
2. Water tank
3. Supply for additives
4. Washer with partition
5. Mixer
6. Sterilization section
7. Sterilization section
8. Mixer
9. Dosage pump
10. Work container (possibly fermentor)
12. Pasteurization
13. Centrifuge
14. Treatment section
15. Device for readding of materials
17. pH regulator
18. Temperature regulator
19. Aerator
20. Stirrer
21. Device for adding antifoaming agent
22. Section for reconstitution
23. Drier
[0038] The material to be treated, for example, tobacco stems, is added from tobacco supply 1 and mixed with water from water tank 2 in washer 4. The soluble components are separated from the insoluble tobacco residue. The insoluble residue is passed to drier 23 or reconstitution stage 22. The extracted soluble components are conveyed to 6 where sterilization by heating and thereafter cooling takes place. Specifically, the treatment in sterilization sections 6 and 7 may consist of preheating to 100°C, sterilization of 110°C for over 40 minutes and cooling to 30°C.
[0039] The necessary additives, mostly phosphate and glucose, travel from supply 3 with water to mixer 5 and as solution to section 7, where they are sterilized by heating and thereafter cooled. The solutions from the two treatment sections 6 and 7 are mixed in mixer 8 and are by way of dosage pump 9 transferred into work vessel 10. To start, fermentor 10 may contain a work mixture, comprising the product solution with the necessary additives and an inoculum of the desired microorganism, from which all the nitrates, nitrites and ammonium compounds will generally be eliminated after about 8-20 hours, whereupon the continuing process can be started by the dosage pump 9 at the rate desired for dilution and regulated in such a way as to keep the volume of the work mixture in fermentor 10 constant. According to the products and microorganisms used, the working conditions are regulated in such a way as to totally eliminate nitrates, nitrites and ammonium compounds contained in the product solution and to completely use all additives from mixer 5 during this assimilation. The treated work mixture in fermentor 10 is removed. The biomass A is removed from the treated work mixture in centrifuge 13 and may be saved for further usage. If necessary, the treated work mixture may be pasteurized in section 12 as shown in Figure 1 or pasteurization of the liquid portion resulting from biomass removal in section 13 may be effected. The remaining treated liquid is conveyed to treatment section 14 as final solution for concentration, as by evaporation. This final solution, containing most of the components of the product solution, except the nitrates, nitrites and ammonium compounds may now be used in any way. The solution may for example, be sprayed onto the dried or reconstituted tobacco residue with device 15 for readding materials. The reconstituted product B resembles tobacco sheets.
[0040] In a preferred mode the present invention comprises extracting tobacco with water employing a 10:1 water to tobacco ratio at 90°C for 60 minutes. The extract thus formed is separated from the insoluble tobacco residue. If necessary, the nitrate concentration in the extract is adjusted to the desired level by conventional means such as dilution or evaporation. The extract at a dilution of 3 to 7.5 g N03/liters, preferably 4.5-5.5 g/I and most preferably 5 g/I, is thereupon combined with sufficient K2HP04 to give a phosphate concentration of 1.1-1.5, preferably 1.25, and glucose is added to a concentration of 4%, along with 250 ppm antifoam, such as Paracum 24/sw. The pH is adjusted to 5.5 employing KOH. The mixture may then be sterilized at 110°C for forty minutes. Alternatively, the extract and additives may be separately sterilized prior to mixing.
[0041] The sterilized extract solution containing the additives is thereupon introduced into a fermentation vessel containing a work mixture at a rate of 0.18-0.22 I/I/hr, preferably 0.2 I/I/hr. The work mixture contains a suitable microorganism and is preferably a starter culture of Candida utilis NCYC 707 yeast in exponential, most preferably late exponential, growth phase which has been built up as above described. The pH of the work mixture is maintained at about 5.5±0.3 preferably by addition of a mixture of 9 parts lactic acid to 1 part o-phosphoric acid and/or KOH. The temperature of the mixture is maintained at 30±3°C. The vessel containing the work mixture is aerated at a rate of 1.4-1.6 and preferably 1.5 I/I/min. and the mixture is agitated. In smaller vessels it may be desirable to shut off the air for one minute every two hours, whereby the pressure is reduced and the condenser on the outgoing air is purged. This can be accomplished by means of an electromagnetic valve coupled with a time on the incoming air. Such purging avoids wetting of the sterile filter. Such purging is generally unecessary when working in larger fermentors, as for example, when a 500 I working volume is employed in a 750 I fermentor.
[0042] Simultaneously with and at a rate equal to the introduction of the sterilized solution, a portion of the work mixture, i.e., treated extract is withdrawn from the fermentation vessel so that the volume of work mixture remains constant. The treated extract is thereupon pasteurized, separated from the biomass and concentrated. The thus denitrated, concentrated extract may then be applied to the dried and/or reconstituted insoluble tobacco residue. Employing the above procedure, the process of the invention has been practiced continuously for five weeks with production of 2400 liters denitrated extract per day which is equal to one-fifth of the volume of the fermentor employed per hour, i.e., 100 liters fermented denitrated extract per hour.
[0043] Where Enterobacter aerogenes ATCC 13048, or other bacterium, is employed the conditions of the work mixture are adjusted to a pH of 5.5-8.0, preferably 7.0, and a temperature of 30°-40°C, preferably 37°C, and the process is operated at an aeration rate of 1.0-3, preferbaly 2 I/I/min., a dilution rate of 0.1-0.25, preferably 0.2 I/I/hr., with the addition mixture containing 0.1-7.5 g nitrate/I, preferably 5 g/I.
[0044] The invention is preferably used in treatment of tobacco extracts, but is not limited to that usage. Elimination of nitrates, nitrates and ammonium compounds from foods and other consumer items may also be desirable. Where these materials are in liquid form, they may be used as the nitrate solution for treatment in the practice of the invention. Otherwise an aqueous solution can be obtained by washing, which solution, following denitration, may be recombined with the insoluble fraction of the material to form the final denitrated product.
[0045] In the case of tobacco the work conditions can be gauged by the nitrate concentration of the product solution. To determine working conditions for foods and other consumer items, the concentration of the sum of all compounds to be eliminated, i.e., nitrates and nitrites and ammonium compounds should be considered. This total concentration of these materials in the overall additive mixture should be between 3 and 7.5 g/liter. The remaining parameters may be the same as in treatment of tobacco. Thus, although the invention has been described in terms of its application to tobacco, it may-apart from the limitations described before-just as well be applied in the treatment of foods and other consumer goods.
Examples 1-10
[0047] Tobacco stems were extracted with water and the resultant extracts were treated with Candida utilis NCYC 707 according to the process of the invention using the conditions specified in Table IV. The results are set forth in Table IV. "0" indicates an amount, which is not detectable using normal analysis conditions; it is smaller than 10 ppm in the case of carbon and phosphate and is less than 1 ppm for nitrates, nitrites and ammonium compounds.
Example 11
[0048] Seven tobacco extracts were prepared by separately washing tobacco stems and by-products with water at a 1:10 tobacco to water ratio and combining a stem extract with a by-product extract. With appropriate installations, it would be possible to effect the extraction, as well as the denitration, on a continuous basis. The nitrate levels of each tobacco extract are set forth in Table V. To each tobacco extract were added glucose, KH2po4 and Paracum 24/sw antifoam as indicated in Table V. the pH was adjusted to 5.5 with KOH. The extract and additives were sterilized at 110°C for 40 min.
[0049] The work mixture comprised Candida Utilis 707, lactic acid, KOH and Paracum-24/sw and had a pH of 5.5. The 14 I fermentor, which was employed, was equipped with an electromagnetic valve on the incoming air coupled with a timer. The incoming air was shut off for 1 min. every two hours to thus purge the condenser on the outgoing air.
[0050] About 1700 I of extract were denitrated on a continuous basis except for the first two weekends where the system was cooled down with agitation reduced to 300 rpm and air flow reduced to 30%. After the first week there was no supervision over the weekends or during the nights. Except for a problem with the weight control system, which resulted in the fermentor's being empty one morning, the operation ran smoothly.
[0051] During denitration 200 ml of lactic acid were consumed per 3 kg of extract. This is probably explained by the fact that the acid was being used by the microorganisms as a carbon source.
[0052] The biomass was removed from the denitrated extract by centrifugation. The resulting 1478 g of extract containing 3% tobacco solubles was thereupon concentrated to give an average concentration of 39.06 tobacco solubles and reapplied to dried tobacco stems.
[0053] The average values of the composition of several samples were measured after extraction, after sterilization of the combined extract and additives and following concentration of the denitrated extract. These values are set forth in Table VI.
Example 12
[0054] A continuous one week pilot plant trial was carried out in a 750 I fermentor (working volume 500 I) with tobacco stem extract. Operating conditions are given in Table VII.
[0055] The stems were continuously washed in a screw extractor at a stem to water ratio of 1:10. The extraction was carried out at 90°C and the tobacco extract (out of the extractor) to water (into the extractor) ratio was 0.74. The tobacco extract was then sterilized by pumping it through 3 heat exchangers: the first to pre-heat it to 110°C, the second to hold it at that temperature for 40 minutes and the third to cool it down to room temperature. Analytical values are given in Table VIII.
[0056] A dextrose solution was prepared batchwise, but then continuously pumped through a second line of 3 heat exchangers for sterilization using the above conditions. The two flows, i.e., sugar solution and tobacco extract, were then regulated to the desired sugar concentration in the tobacco extract and then pumped into the fermentor. Nitrate and sugar values are given in Table VIII.
[0057] Before the start of continuous operation, the fermentor was filled with 480 kg of tobacco extract, 20.2 kg of dextrose, 2.4 kg of KH2po4 and 120 ml of an antifoaming agent, and then sterilized at 120°C for 40 minutes. After the fermentor had been cooled down, it was inoculated with 13 I of a starter culture of Candida Utilis 707 grown in tobacco extract. After 12 hours there was no more sugar or nitrate in the batch and the yeasts were in the exponential phase. At this point continuous operation was started. The operating conditions are given in Table VII. The pH regulation was done with phosphoric acid at 25%. The fermentor was equipped with a mechanical foam separator, a turbine aeration/agitation system, and a weight control system.
1. A method for denitrating an aqueous tobacco extract by means of microorganisms
which are capable of aerobic metabolic assimilation of nitrogen-containing compounds,
in which the extract is mixed with a culture of the microorganisms and necessary nutrient
additives to form a work mixture, and the mixture is maintained under conditions in
which the organisms are in their exponential growth phase, characterised in that sterile
tobacco extract and additives are continuously added to the work mixture at a dilution
rate which does not exceed the growth rate of the microorganisms, while the pH, temperature
and aeration are maintained at levels which promote aerobic assimilation, the extract,
phosphate and a carbon source being added in amount such that the overall addition
thereof is 0.1-7.5 g nitrate ions per liter added, 1.0 to 10 g P04 ions per liter added and sufficient carbon source to provide at least 16.5 assimilative
carbon atoms/N03 ion added, and that work mixture is continuously withdrawn at such a rate as to keep
the total volume of work mixture constant.
2. A method according to claim 1 characterised in that the microorganisms are removed
from the withdrawn mixture.
3. A method according to claim 1 characterised in that the microorganism is Candida
utilis NCYC 707, 321 or 359, or Candida Berthetii CBS 5452 yeast.
4. A method according to claim 3 wherein the pH is maintained between 3.5 and 7.2.
5. A method according to claim 3 or 4 characterised in that the temperature is maintained
between 25°C and 37°C.
6. A method according to claim 3, 4 or 5 characterised in that the dilution rate is
between 0.1 and 0.35 5 I/I/hr r.
7. A method according to any of claims 3 to 6 characterised in that the aeration rate
is between 0.5 and 2.5 I/I/min, preferably 1.0-2.0 1/1/min.
8. A method according to any of claims 3 to 7 characterised in that the overall nitrate
addition is 3-7.5 g nitrate per liter added, preferably 4.5 to 5.5 g per liter added.
9. A method according to claim 1 or 2 characterised in that the microorganism is Enterobacter
aerogenes, preferably Enterobacter aerogenes ATCC 13048.
10. A method according to claim 9 characterised in that the pH is maintained between
5.5-8.0, preferably 7.0.
11. A method according to claim 9 or 10 characterised in that the temperature is maintained
between 30° and 40°C, preferably at 37°C.
12. A method according to claim 9, 10 or 11 characterised in that the dilution rate
is between 0.1 and 0.25 I/I/hr., preferably 0.2 I/I/hr.
13. A method according to any of claims 9 to 12 characterised in that the overall
nitrate addition is 5.0 g per liter added.
14. A method according to any of claims 9 to 13 characterised in that the aeration
rate is between 1.0 and 3.0 I/I/min, preferably 2.0 I/I/min.
15. A method according to any of claims 1 to 14 characterised in that the carbon source
is one or more of glucose, dextrose, sucrose, maltose, cellobiose, lactose, ethanol,
glycerol and citrate.
16. A method according to claim 15 characterised in that the carbon source is glucose
added at a concentration of 2.4-6%, preferably 4%.
17. A method according to any of claims 1 to 16 characterised in that the overall
phosphate addition is 1.1-1.5 g per liter added, preferably 1.25 g per liter added.
18. A method according to any of claims 1 to 17 characterised in that antifoam is
added to the work mixture, preferably at a level of at least 250 ppm.
1. Kontinuierliches Verfahren zur Denitrifikation von Tabakextrakten mittels Mikroorganismen,
die zur aeroben metabolischen Assimilation von stickstoffhaltigen Verbindungen befähigt
sind, wobei der Extrakt zur Bildung eines Arbeitsgemischs mit einer Kultur der Mikrooganismen
und nötigen Nährstoffzusätzen gemischt und das Gemisch unter Bedingungen gehalten
wird, in denen die Organismen in deren exponentieller Wachstumsphase vorliegen, dadurch
gekennzeichnet, dass dem Arbeitsgemisch steriler Tabakextrakt und Zusätze kontinuierlich
mit einer Verdünnungsrate zugesetzt werden, die die Wachstumsrate der Mikroorganismen
nicht übersteigt, während pH-Wert, Temperatur und Belüftung solcherart eingestellt
werden, dass aerobe Assimilation angeregt wird, der Extrakt, Phosphat und eine Kohlenstoffquelle
in solchen Mengenanteilen zugesetzt werden. dass die Gesamtzugabe davon 0,1―7,5 g
Nitrationen pro zugesetztem Liter, 1,0―10 g P04-lonen pro zugesetzten Liter und genügend Kohlenstoffquelle zur Schaffung von mindestens
16,5 assimilativen C-Atomen pro zugesetzten N03-lonen ausmachen, und dass das Arbeitsgemisch kontinuierlich in solchem Ausmass abgezogen
wird, dass das Gesamtvolumen des Arbeitsgemischs konstant bleibt.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass die Mikroorganismen aus
dem abgezogenen Gemisch entfernt werden.
3. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass man als Mikroorganismus
Candida utilis NCYC 707,321 oder 359, oder Candida Berthetii CBS 5452 Hefe verwendet.
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, dass der pH-Wert zwischen 3,5
und 7,2 gehalten wird.
5. Verfahren nach Anspruch 3 oder 4, dadurch gekennzeichnet, dass die Temperatur zwischen
25 und 37°C gehalten wird.
6. Verfahren nach Anspruch 3, 4 oder 5, dadurch gekennzeichnet, dass die Verdünnungsrate
zwischen 0,1 und 0,35 I/I/h beträgt.
7. Verfahren nach einem der Ansprüche 3-6, dadurch gekennzeichnet, dass die Belüftungsrate
zwischen 0,5 und 2,5 I/I/min, vorzugsweise 1,0-2,0 I/I/min, beträgt.
8. Verfahren nach einem der Ansprüche 3-7, dadurch gekennzeichnet, dass die Nitratzugabe
insgesamt 3-7,5 g Nitrat pro zugesetzten Liter, vorzugsweise 4,5-5,5 g pro zugesetzten
Liter, beträgt.
9. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass als Mikroorganismus
Enterobacter aerogenes, vorzugsweise Enterobacter aerogenes ATCC 13048, verwendet
wird.
10. Verfahren nach Anspruch 9, dadurch gekennzeichnet, dass der pH-Wert zwischen 5,5
und 8,0, vorzugsweise bei 7,0, gehalten wird.
11. Verfahren nach Anspruch 9 oder 10, dadurch gekennzeichnet, dass die Temperatur
zwischen 30 und 40°C, vorzugsweise bei 37°C, gehalten wird.
12. Verfahren nach Anspruch 9, 10 oder 11, dadurch gekennzeichnet, dass die Verdünnungsrate
zwischen 0,1 und 0,25 I/I/hr, vorzugsweise 0,2 I/I/h, beträgt.
13. Verfahren nach einem der Ansprüche 9-12, dadurch gekennzeichnet, dass die Nitratzugabe
insgesamt 5,0 g pro zugesetzten Liter beträgt.
14. Verfahren nach einem der Ansprüche 9-13, dadurch gekennzeichnet, dass die Belüftungsrate
zwischen 1,0 und 3,0 I/I/min, vorzugsweise 2,0 I/I/min, beträgt.
15. Verfahren nach einem der Ansprüche 1-14, dadurch gekennzeichnet, dass als Kohlenstoffquelle,
Glukose, Dextrose, Sucrose, Maltose, Cellobiose, Lactose, Ethanol, Glycerin oder Citrat
oder ein Gemisch solcher Substanzen verwendet wird.
16. Verfahren nach Anspruch 15, dadurch gekennzeichnet, dass als Kohlenstoffquelle
Glukose in einer Konzentration von 2,4―6% vorzugsweise 4%, zugesetzt wird.
17. Verfahren nach einem der Ansprüche 1-16, dadurch gekennzeichnet, dass die Phosphatzugabe
1,1-1,5 g pro zugesetzten Liter, vorzugsweise 1,25 g pro zugesetzten Liter, beträgt.
18. Verfahren nach einem der Ansprüche 1-17, dadurch gekennzeichnet, dass dem Arbeitsgemisch
Antischaummittel, vorzugsweise in einer Konzentration von mindestens 250 ppm, zugesetzt
wird.
1. Procédé de dénitrification d'un extrait de tabax aqueux au moyen de microorganismes
qui sont capables d'assimilation en métabolisme aéobie, de liaisons contentant de
l'azote, selon lequel l'extrait est mélangé à une culture des microorganismes et à
des additifs nutritifs nécessaires de façon à former un mélange de travail et ce mélange
est maintenu dans des conditions dans lesquelles les dits organismes se trouvent dans
leur phase de croissance exponentielle, caractérisé en ce que l'extrait de tabac stérile
et les additifs sont ajoutés en continu au mélange de travail sous un taux de dilution
qui n'excède pas le taux de croissance des microorganismes, tandis que le pH, la température
et l'aération sont maintenus à des niveaux tels que l'assimilation aérobie est promue,
l'extrait, un phosphate et une source de carbone étant ajoutés en quantités telles
que leur somme totale atteint 0,1-7,5 g d'ions nitrate par litre ajouté, 1,0-10 g
d'ions P04 par litre ajouté et une quantité suffisante de source de carbone, pour produire au
moins 16,5 atomes assimilateurs de carbone par ion N03 ajouté, et en ce que le mélange de travail est retiré en continu à un taux tel que
son volume total est maintenu constant.
2. Procédé selon la revendication 1, caractérisé en ce que les microorganismes sont
enlevés du mélange retiré.
3. Procédé selon la revendication 1, caractérisé en ce que le microorganisme est une
levure de Candida utilis NCYC 707, 321 ou 359, ou Candida Berthetii CBS 5452.
4. Procédé selon la revendication 3, caractérisé en ce que le pH est maintenu entre
3,5 et 7,2.
5. Procédé selon les revendications 3 ou 4, caractérisé en ce que la température est
maintenue entre 25° et 37°C.
6. Procédé selon les revendications 3, 4 ou 5, caractérisé en ce que le taux de dilution
est compris entre 0,1 et 0,25 I/I/h.
7. Procédé selon l'une quelconque des revendications 3 à 6, caractérisé en ce que
le taux d'aération est compris entre 0,5 et 2,5 I/I/min, de préférence entre 1,0 et
2,0 I/I/min.
8. Procédé selon l'une quelconque des revendications 3 à 7, caractérisé en ce que
l'ajonction totale de nitrate est de 3 à 7,5 g nitrate par litre ajouté, de préférence
4,5 à 5,5 g par litre ajouté.
9. Procédé selon la revendication 1 ou la revendication 2, caractérisé en ce que le
microorganisme est Enterobacter aerogenes et de préférence Enterobacter aerogenes
ATCC 13048.
10. Procédé selon la revendication 9, caractérisé en ce que le pH est maintenu entre
5,5 et 8,0, de préférence à 7,0.
11. Procédé selon la revendication 9 ou la revendication 10, caractérisé en ce que
la température est maintenue entre 30°C et 40°C, de préférence à 37°C.
12. Procédé selon les revendications 9, 10 ou 11, caractérisé en ce qu le taux de
dilution est compris entre 0,1 et 0,25 I/I/h et a de préférence la valeur 0,2 I/I/h.
13. Procédé selon l'une quelconque des revendications 9 à 12, caractérisé en ce que
l'adjonction totale de nitrate est de 5,0 g par litre ajouté.
14. Procédé selon l'une quelconque des revendications 9 à 13, caractérisé en ce que
le taux d'aération est compris entre 1,0 et 3,0 I/I/min, et a de préférence la valeur
2,0 I/I/min.
15. Procédé selon l'une quelconque des revendications 1 à 14, caractérisé en ce que,
comme source de carbone, on utilise une ou plusieurs des substances glucose, dextrose,
sucrose, maltose, cellobiose, lactose, ethanol, glycérine et citrate.
16. Procédé selon la revendication 15, caractérisé en ce que la source de carbone
est le glucose à une concentration de 2,4 à 6%, de préférence 4%.
17. Procédé selon l'une quelconque des revendications 1 à 16, caractérisé en ce que
l'adjonction totale de phosphate est de 1,1 à 1,5 g par litre ajouté, de préférence
1,25 g par litre ajouté.
18. Procédé selon l'une quelconque des revendications 1 à 17, caractérisé en ce qu'un
produit de prévention de la mousse est ajouté au mélange de travail, de préférence
à une concentration d'au moins 250 ppm.