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Combined immobilized cell bioreactor and pulse column technology as a novel approach to food modification
Process Biochemistry 34 (1999) 221 – 229
Combined immobilized cell bioreactor and pulse column technology as a novel approach to food modification Geoffrey H. Bertkau *,1, Sean M. Murphy, Frank J. Sabella Kraft Foods Technical Center, 555 S. Broadway, Tarrytown, NY 10591, USA Received 15 April 1996; received in revised form 6 April 1998; accepted 19 April 1998
Abstract The continuous use of an immobilized cell bioreactor (ICB) and a countercurrent pulse column is a unique approach to removing malic acid from coffee beans prior to roasting. This process recycled a high soluble solids water extract from the green coffee beans through a spiral-wound ICB loaded with food-grade bacteria (Lactobacillus spp. or Leuconostoc spp.) which metabolized malic acid in the green coffee bean extract. The demalated extract was then recirculated to extract the malic acid from the green beans during the countercurrent flow of the ‘malic acid-lean’ extract over the beans. The malic acid level in the beans was reduced by more than 80%. This extract recycle process may have other applications for removing unwanted organics or adding desirable water-soluble components. and is particularly suited to processing beans and grains. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Malic acid; Lactobacillus; Immobilized cell bioreactor; Countercurrent pulse column; Coffee; Malolactic fermentation
1. Introduction For several years Maxwell House Coffee had been interested in identifying and removing malic acid, which was thought to increase the secretion of stomach acid following coffee consumption [1]. Physical, chemical and biological processes were evaluated for the removal of malic acid from green (unroasted) coffee beans and from a water extract prepared from these beans (referred to in the text as green beans and green extract, respectively). Physical and chemical methods were eliminated due to low selectivity, low efficiency and high process costs. An enzymic approach in which green extract was treated with malate dehydrogenase (EC 1·1·1·39) to convert the malic acid to pyruvic acid was also costly and inefficient. Generally Recognized as Safe (GRAS) lactic acid
* Corresponding author. Tel. 1-201-784-7204. 1 Present address: Takasago International, 4 Volvo Drive, Rockleigh, N.J.
bacteria can catabolize malic acid directly to lactic acid and carbon dioxide via the malolactic enzyme (EC 1·1·1·38; Fig. 1). Fermentation parameters and enzyme characteristics have been extensively documented [2–6]. The enzyme obtained from Leuconostoc oenos has a Km of 5·5 mM and a Vmax of 0·71 mM for l-malic acid. The Km and Vmax for this enzyme from Lactobacillus plantarum are 9·5 mM and 1·25 mM, respectively [7]. The primary application of the malolactic fermentation using immobilized lactic acid bacteria is in the wine industry [8–10] where the malic acid content ranges from 0 to 8 g liter − 1. Malic acid levels range from 0·12 to 0·67% dry wt basis (db) in green beans and 0·6 to 4·44 g liter − 1 in green extract, suggesting that these might be suitable substrates for a malolactic fermentation. The objective of this study was to determine the feasibility of combining the ICB process and the green bean extraction process. The overall concept is given in Fig. 2, which illustrates how the malic acid is extracted from green beans and cycled to the ICB for malate removal and then used to extract additional malate from green beans.
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2.2. Cultures and their maintenance
2. Materials and methods
2.1. Preparation of green coffee bean extract (green extract) Coffee beans were moisturized to 40% with steam and loaded in 100 lbs batches into the countercurrent pulse column extractor (extraction column). Hot water was recirculated through the beans for 4 h. The beans were then discarded and another fixed bed of moisturized beans was prepared. The dilute extract used for the first column was recycled through the fresh green bean column at a rate of 5·2 lbs min − 1 for another 4 h. This process was repeated until a 12° brix extract was obtained. For every 100 lbs of moisturized beans, about 76 lbs of extract were produced. The extract was then concentrated to about 26% solids.
Based on microbial screening for malolactic fermentation in green coffee extract, two lactic acid bacteria were selected for this process. Lactobacillus plantarum 8014/1236 was provided by Dr. Thomas Montville at Rutgers University and Leuconostoc oenos 27307 was obtained from the American Type Culture Collection in Maryland. Both bacteria were maintained using the following procedure: the cultures were propagated, centrifuged, resuspended in fresh media and dispensed into cryotube vials containing equal amounts of glycerol. The vials were mixed and stored at − 80°C until needed.
2.3. Description and loading of the immobilized cell bioreactor (ICB) The bioreactor used was a 30× 5 inch spiral-wound
Fig. 1. Biochemical pathway for the malolactic enzyme.
Fig. 2. Schematic of the malic acid removal process for green coffee beans.
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and then through the biosupport from the outside into the unit’s core (opposite direction from loading). The estimated extract residence times in the shroud and biosupport were 5·5 and 6·8 min, respectively. Once through the bioreactor, the malic acid-lean extract passed through a heat exchanger to kill the bacteria. The heated extract was pumped into a collection tank just upstream of the extraction column.
2.4. Operation of the countercurrent pulse extraction column
Fig. 3. Lactobacillus plantarum immobilized on the spiral-wound biosupport (2000 × ).
ribbed NC120 Microporous Plastic Sheet (MPS®) manufactured by FMC Corporation Biosupport Materials (currently produced by Arbor Technologies, Inc.). Its use for several other food-related applications, both microbial and enzymatic, is well documented [11 – 14]. MPS® is a proprietary porous silica particle embedded PVC matrix which is highly suited for the colonization of microbial cells. This unit provided an available surface area of 76·5 ft2 for immobilization and bound an estimated 3 × 1015 CFU (colony forming units). Attachment of bacteria to the biosupport is shown in Fig. 3. The ICB was connected to a 75 liter New Brunswick fermenter and a sterile media reservoir, which were used to ‘load’ the bacteria. L. plantarum was propagated in the 75 liter fermenter (inoculated from a 7 liter fermenter) at 30°C, 200 rpm, 3 psi backpressure and provided with 2·5 LPM sterile air for 18 h in MRS medium. The 50 liters of actively growing bacteria were recycled from the fermenter through the top center of the biosupport. Colonization occurred as the bacteria moved through the biosupport: unbound cells exited from the outer wrap of the spiral unit into the bioreactor shroud and then back to the fermenter. On a routine basis over the 4-day loading period, spent media and cells were removed from the fermenter and replaced with fresh enrichment (MRS) media. Following the loading period, media and free cells were drained from the entire system and the bioreactor was filled with the malic acid-rich green extract that had been clarified by cross-flow microfiltration and sterilized via 0·3 and 0·2 m dead-end filtration. The green extract was pumped into the bottom of the bioreactor shroud at 1·0 – 1·5 lbs min − 1
The extraction column was 33 ft× 4 inches ID. The column, which could be pressurized, was equipped with a feed bin and blowcase on top and a blowcase on the bottom. Moisturized coffee beans were airveyed to this bin at regular intervals. During the run about 6 lbs of ‘demalated’ beans were pulsed out of the column into the blowcase every 30 min. The residence time for beans in the extraction column was 6·5 h (13 pulses). The green extract to bean ratio in the column for this application was about 6·6:1. The extract velocity up the column was 0·22–0·24 ft min − 1, which is equivalent to a flowrate of 1·3 to 1·4 lbs min − 1. Depending on coffee bean type, the column temperature was set between 71° and 83°C. In the blowcase at the bottom of the column, the green extract was blown free of the beans to a holding tank upstream of the microfiltration unit. The ‘demalated’ beans were rinsed free of extract and dried. The rinse water was periodically added to the microfiltration feed tank to prevent extract solids loss. The beans were then roasted at 200–260°C.
2.5. Green extract clarification and sterilization After 6·5 h in the extraction column, the hot extract not only contained malic acid but also coffee bean insolubles and viable microorganisms. The majority of the insolubles were removed via a Romicon microfiltration unit equipped with either 0·8 m or 0·45 m ceramic cross-flow cartridges. The retentate was collected and added back to the extraction column feed tank. The permeate was collected in a holding tank and then pumped through a sterile 0·3 m prefilter and a sterile 0·2 m dead-end filter to sterilize the extract. The sterile, malic acid-rich green extract was cooled from about 71° to 27°C as it passed through a glycol chiller on its way to the ICB.
2.6. Analytical Most of the organic acid analyses were performed with a Dionex HPIC unit using conductivity detection and a Carbopak; PA-1 column (4× 250 mm) and guard
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(4 × 50 mm). The mobile phase was 160 mm sodium carbonate at a flowrate of 1·0 or 1·5 ml min − 1 anti operated at room temperature. Some lactic acid and glucose analyses were performed using a Yellow Springs model 2700 immobilized enzyme system. Sugars were also quantified using a Waters HPLC system equipped with a BioRad Aminex column or more often a Dionex HPIC equipped with pulsed amperometric detection. The column was the same one used for organic acids but the mobile phase was 160 mM NaOH at 1·0 ml min − 1.
3. Results
3.1. Immobilized cell bioreactor During one 72 h run, about 4750 lbs of malic acidrich green extract were pumped through the ICB. Green extract supplied all the required nutrients necessary to support an active lactobacillus population as indicated by the number of cells observed in the exit stream (Fig. 4a). After 15 h, the malic acid-lean exit stream contained an average of approximately 107 CFU ml − 1. The malic acid level in the exit stream decreased rapidly from 2·08 g liter − 1 (initial feed concentration) to 0·2 in the first 12 h and then plateaued to an average of 0·32 g liter − 1. This yielded an 83% reduction in malic acid (Fig. 4b). The malic acid level in the feed also decreased from zero to 50 h and then plateaued at 0·7 g liter − 1. The glucose concentration in the exit stream after 72 h was 56% of the initial feed stream concentration (Fig. 4c). Sugars play a critical role as precursors for flavor development during the roasting process and it was important to balance the catabolism of malic acid with the detrimental microbial utilization of reducing sugars. The immobilized system accommodated this balance, whereas the sugar content of extract after batch fermentation was depleted. Glucose was metabolized to lactic acid by Lactobacillus plantarum as the sole product since this organism is homofermentative (Leuconostoc oenos is a heterofermentative bacterium and will produce ethanol and/or acetate and carbon dioxide and half as much lactic acid on a mole basis). The green extract concentration of lactic acid increased from 2·35 to 5·85 g liter − 1 during the first 50 h and then equilibrated at 6 g liter − 1 (Fig. 4c).
3.2. Green bean extraction column The extraction column was fed malic acid-lean green extract at an average rate of 78 lbs h − 1 (1·3 lbs min − 1). with an extract to bean ratio of 6·59:1. The feed had a malic acid concentration of 0·32 g liter − 1 at equi-
librium. The malic acid-rich extract draw-off from the column had a malic acid concentration of 0·8 g liter − 1 at equilibrium (pulses 60–130), with a composite concentration of 0·83 g liter − 1 (Fig. 5a). During equilibrium pulses 60–130, the average amount of malic acid extracted from the green beans was equivalent to the average malic acid conversion in the ICB. A material balance performed on the amount of malic acid extracted from beans compared to the amount of malic acid in the extractor draw-off gave good agreement with experimental results—0·78 g liter − 1 versus 0·80 g liter − 1 (experimental) malate concentration. The feed stream was offset by 13 pulses (one bed volume) to account for residence time of the extract in the column. The peak amount of malic acid in the draw-off occurred around pulse 100; this corresponded well with the peak demalation obtained in the product bean from pulses 100 to 120. The beans from the 154 pulses in this trial were dried to yield about 890 lbs of demalated coffee beans. Of these 154 pulses, 60 (70–130) were at equilibrium, yielding about 330 lbs of product beans at an average of 81·6% demalation on a dry basis (Fig. 5b). The pulse size of the demalated beans averaged 11·31 lbs at 59·17% moisture, yielding 2.49 lbs h − 1 when dried to 11·5% moisture. As the beans were pulsed from the column, the surface solids were recovered via a washing step. The wash water, equal to the amount of water the beans would absorb during their residence time in the extractor column, was added back to the extract feed tank every ten pulses. The high solids content in the extractor feed (21·3%) led to lower solids losses across the extraction process (Fig. 5c). On a dry basis, the green bean feed had an average malic acid concentration of 0·38% and the extracted product beans at equilibrium contained 0·077%.
4. Discussion
4.1. Malolactic fermentation By coupling a physicochemical process (countercurrent extraction) with a biological process (malolactic fermentation, MLF) we were able to design the bioreactor component for flexibility, reliability, and reproducibility. The 6·8 min that the green extract spent within the biosupport was sufficient to achieve up to 87% demalation of the extract. This level was high enough to permit malic acid extraction from the green beans to the targeted level. Increased exposure times with the immobilized bacteria would increase the lactic acid content and decrease the glucose level, which was replenished during the extract’s journey up the green bean extraction column.
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4.2. Immobilized cell bioreactor Susceptibility to microbial contamination is always a concern in continuous fermentation processes. The ICB exit stream was routinely monitored for contaminants
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and cell counts. No contamination was observed in any of the ICB pilot plant trials even after operating for a period under non-sterile conditions during one run. It should be fairly difficult for another microbe to gain a foothold on the biosupport, which was totally colo-
Fig. 4. Viable microbial population in the ICB exit stream, (b) malic acid levels in the ICB feed ()and exit ( — ) streams, (c) lactic acid (---) and glucose () in the ICB exit stream.
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Fig. 4. (Continued)
nized with active lactobacilli — approximatel 2·7× 1011 CFU in − 2 —within the 12 – 13 min the extract resides in the bioreactor Benchtop ICB trials operated 6 months without any apparent contamination or loss of MLF efficiency. Besides the long functional lifetime of the MPS® bioreactors, which could be totally cleaned of microflora and reloaded, the bioreactors could be stored refrigerated for up to 3 weeks without significant loss of MLF activity. Two days of recycling fresh MRS media at 30°C restored full metabolic activity to the biosupports (data not shown). One of our major concerns was the possibility of channel plugging, particularly with a 25% total solids substrate. MPS® sheets were manufactured with a variety of rib sizes (width and depth). The standard rib (CF) unit has a channel width of 0·9 mm and a rib height of 0·4 mm (18 channels per one-inch sheet width). If the five-inch diameter spiral-wound unit was unwrapped, it would stretch 37 linear ft (1128 m). Another unit evaluated during this research was the high rib TC190 unit, which has a channel width of 4·8 mm (5 channels per inch), a rib height of 1·0 mm and a linear length unwrapped of 13 ft (3·96 m). Due to periodic precipitation of coffee solids from the extract (e.g. green extract pH dropped below 4·5 while the bioreactor remained idle; this caused solids precipitation), the pressure drop across the standard rib unit increased beyond the 50 – 60 psi maximum recommended operating pressure
after several months of exposure to clarified extract. Without any clarification to remove the insolubles, the rib channels plugged after 48 h of continuous operation. No pressure drop was ever detected across the high rib unit and no plugging was observed. Several of the benchtop and all of the pilot plant trials made use of the intermediate ribbed NC120 unit, which had a channel width of 2·8 mm (9 channels per inch), a rib height of 0·7 mm, and a linear length of 22·4 ft (6·83 m). Over time, the deposition of insoluble solids, including cell mass, could pose a problem for this unit. Periodic high flow-rate flushing may eliminate this potential situation. One of the positive attributes of the bioreactor process was its flexibility. Although only a single unit was required for this application and a single pass of the green extract, the process could be modified for recycling or employment of a series of ICB units manifolded together. A wider rib ‘polishing unit’ (TC190) could follow the intermediate ribbed NC120 biosupport. Also, the length of the biosupport or the linear length of the spiral wrap could be tailored to fit the application requirements. Finally, non-glutaraldehyde treated MPS® is a chemically inert material which can be reused after cleaning with nitric acid and/or hypochlorite solutions. It can be utilized for thermophilic enzymes or micro-organisms up to 90°C. In-place steam sterilization was attempted without success, but was not a major drawback to this process.
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4.3. Pulse column extractor It is important to define process equilibrium in terms of the pulse column extractor and the immobilized cell bioreactor. Process equilibrium is defined by operating
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conditions—bean throughput, extract flowrate, extract temperature, ICB extract throughput, etc.—at which the amount of malic acid extracted from green coffee beans per unit time is equal to the amount of malic acid converted to lactic acid in the green coffee extract
Fig. 5. (a) Malic acid levels in the pulse column feed ( ) and draw-off ( ), (b) percent demalation of the product coffee beans exiting the pulse column, (c) solids level in the pulse column feed ( ) and draw-off ().
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Fig. 5. (Continued)
flowing through the ICB per unit time. At these conditions, the product green beans will reach a constant level of demalation:
pulse column was equal to the rate of malic acid conversion in the ICB. Therefore the process can be said to be in equilibrium
d[malic conc. in green beans]/[t]equil =0 These conditions are found between pulses 60 and 130 (Fig. 5b) where the change in concentration of malic acid in the product beans over time was approximately zero. It is at these conditions that a uniform product was produced. A linear regression of percent demalation of the product beans versus time over pulses 60 – 130 gave a slope of 0·2113. At a 95% confidence level, the upper and lower limits were 0·4227 and −4·2 ×10 − 5, respectively. Since the value of zero is encompassed by the limit range, no change was observed. Because the concentration of malic acid in the product from the pulse column is seen to be constant over time, the pulse column process can be said to be operating at ‘steadystate’ conditions. A linear regression of malic acid converted in the ICB versus time from 28 to 52 h gave a slope of − 0 349. At a 95% confidence level. the upper and lower error limits are 0·6378 and − 1·3368, respectively. Again, the value zero is encompassed by the limit range, and hence there is no change in malic acid conversion rate over this time. It can be concluded that at the given time interval pulses 60 – 130 (28 – 52 h of ICB operation) the rate of malic acid extraction in the
4.4. Process summary The immobilized bioreactor was operated in a continuous mode. It had a working volume of 6·35 liter with an average feed residence time of 12–13 min. Up to 87% of the malic acid and 44% of the glucose were removed from the green extract which exited the ICB with 2·1% lactic acid, db per 100 h of operation. 2995 kg of extract was demalated having an average malic acid concentration of 0·32 g liter − 1 at equilibrium. During 100 h of operation 461 kg of green beans were demalated in the pulse column with an average malic acid level of 0·069%, db.
4.5. Applications of pulse column technology coupled with an immobilized bioreactor Alone, each of the unit processes has application. Immobilized cell and enzyme bioreactors have demonstrated commercial success for many years and there is no need to review these applications in this article. The pulse column, however, can provide a unique approach to continuous food processing. An extract can be generated from the material to be treated or alternatively any
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aqueous solution (e.g. brine) or an organic solvent can be employed if solids loss is not an issue. Aqueous extracts can also contain viable micro-organisms and/or enzymes. As discussed above, it is important to precisely determine the extraction reaction, and/or infusion kinetics for the particular food material. Column residence time, temperature, and pressure parameters must be evaluated. When coupling the pulse column extractor with a continuous bioreactor process generation of a high solids extract which will recycle through the bioreactor and pulse column continuously may or may not be desirable. Sterility may not be necessary but microfiltration to remove suspended solids will be required. The immobilized bioreactor can not only microbially or enzymatically treat this extract continuously, but can also be a constant source of microorganisms and extracellular enzymes which can remain functional if the extract is not heat treated downstream of the bioreactor. The rapidly expanding knowledge base on extremophiles and their enzymes might allow the bioreactor to operate under thermophilic or halophilic conditions. Pulse column/bioreactor technology with brine recycling might provide a unique approach to pickle, olive and sauerkraut production.
Acknowledgements We wish to thank Bruce Goldberg for sharing his expertise on the characteristics and use of the biosupport utilized in this process and Professor Thomas Montville at Rutgers University for his valuable expertise in microbial biochemistry and lactobacilli.
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References [1] Cohen S, Booth. GH Jr. Gastric acid secretion and lower esophageal sphincter pressure in response to coffee and caffeine. N Engl J Med 1975;18:897 – 9. [2] Kunkee RE. Malo-lactic fermentation. Adv Appl Microbiol 1967;9:235 – 79. [3] Kunkee RE. A second enzymatic activity for decomposition of malic acid by malo-lactic bacteria. In: Carr JG, editor. Lactic Acid Bacteria in Beverages and Foods. New York: Academic, 1773:29 – 42. [4] Garvie EI. Leuconostoc oenos sp. Nov. J Gen Microbiol 1967;48:431 – 46. [5] Caspritz G, Radler F. Malolactic enzyme of Lactobacillus plantarum. J Biol Chem 1983;259:4907– 10. [6] Chagnaud P, et al. Purification and properties of a malolactic enzyme from a Lactobacillus sp. able to perform the malolactic fermentation of wines. Biotechnol Appl Biochem 1989;11:445– 58. [7] Henick-Kling T. Growth and metabolism of Leuconostoc oenos and Lactobocillus plantarum wine. PhD thesis. University of Adalaide, 1986, pp. 88 – 94. [8] Spettoli P. Technological improvement of malolactic fermentation in wine by immobilized microbial cells in a continuous flow reactor. Ann NY Acad Sci 1988;523:386 – 9. [9] McCord JE, Ryu DD. Development of malolactic fermentation process using immobilized microbial cells in a continuous flow reactor. Am J Enol Vittic 1985;36:214 – 8. [10] Gestrelius SH, Kjaer JH. Reducing acidity of wine by malolactic fermentation using cells of Leuconostoc oenos encapsulated in beads of alginate gel. NOVO Industries. AS US Patent 4380559, 1983. [11] Goldberg BS. Method of immobilizing proteinaceous substances. Amerace Corporation, US Patent 4169014, 1979. [12] Goldberg BS. A novel immobilized enzyme reactor system. 24th Annual Spring Symposium, on Non-conventional Reactor Systems AICHE, 1984. [13] Bakken AP, Hill CG, Amundson. CH. Hydrolysis of lactose in skim milk by immobilized b-galactosidase in a spiral flow reactor. Biotechnol Bioeng 1989;33:1249 – 57. [14] Lakhwala F, et al. A polymeric membrane reactor for biodegradation of phenol in wastewater. Bioact Compat Polym 1990;5:439 – 59.
Combined immobilized cell bioreactor and pulse column technology as a novel approach to food modification Geoffrey H. Bertkau *,1, Sean M. Murphy, Frank J. Sabella Kraft Foods Technical Center, 555 S. Broadway, Tarrytown, NY 10591, USA Received 15 April 1996; received in revised form 6 April 1998; accepted 19 April 1998
Abstract The continuous use of an immobilized cell bioreactor (ICB) and a countercurrent pulse column is a unique approach to removing malic acid from coffee beans prior to roasting. This process recycled a high soluble solids water extract from the green coffee beans through a spiral-wound ICB loaded with food-grade bacteria (Lactobacillus spp. or Leuconostoc spp.) which metabolized malic acid in the green coffee bean extract. The demalated extract was then recirculated to extract the malic acid from the green beans during the countercurrent flow of the ‘malic acid-lean’ extract over the beans. The malic acid level in the beans was reduced by more than 80%. This extract recycle process may have other applications for removing unwanted organics or adding desirable water-soluble components. and is particularly suited to processing beans and grains. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Malic acid; Lactobacillus; Immobilized cell bioreactor; Countercurrent pulse column; Coffee; Malolactic fermentation
1. Introduction For several years Maxwell House Coffee had been interested in identifying and removing malic acid, which was thought to increase the secretion of stomach acid following coffee consumption [1]. Physical, chemical and biological processes were evaluated for the removal of malic acid from green (unroasted) coffee beans and from a water extract prepared from these beans (referred to in the text as green beans and green extract, respectively). Physical and chemical methods were eliminated due to low selectivity, low efficiency and high process costs. An enzymic approach in which green extract was treated with malate dehydrogenase (EC 1·1·1·39) to convert the malic acid to pyruvic acid was also costly and inefficient. Generally Recognized as Safe (GRAS) lactic acid
* Corresponding author. Tel. 1-201-784-7204. 1 Present address: Takasago International, 4 Volvo Drive, Rockleigh, N.J.
bacteria can catabolize malic acid directly to lactic acid and carbon dioxide via the malolactic enzyme (EC 1·1·1·38; Fig. 1). Fermentation parameters and enzyme characteristics have been extensively documented [2–6]. The enzyme obtained from Leuconostoc oenos has a Km of 5·5 mM and a Vmax of 0·71 mM for l-malic acid. The Km and Vmax for this enzyme from Lactobacillus plantarum are 9·5 mM and 1·25 mM, respectively [7]. The primary application of the malolactic fermentation using immobilized lactic acid bacteria is in the wine industry [8–10] where the malic acid content ranges from 0 to 8 g liter − 1. Malic acid levels range from 0·12 to 0·67% dry wt basis (db) in green beans and 0·6 to 4·44 g liter − 1 in green extract, suggesting that these might be suitable substrates for a malolactic fermentation. The objective of this study was to determine the feasibility of combining the ICB process and the green bean extraction process. The overall concept is given in Fig. 2, which illustrates how the malic acid is extracted from green beans and cycled to the ICB for malate removal and then used to extract additional malate from green beans.
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 8 ) 0 0 0 6 5 - X
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G.H. Bertkau et al. / Process Biochemistry 34 (1999) 221–229
2.2. Cultures and their maintenance
2. Materials and methods
2.1. Preparation of green coffee bean extract (green extract) Coffee beans were moisturized to 40% with steam and loaded in 100 lbs batches into the countercurrent pulse column extractor (extraction column). Hot water was recirculated through the beans for 4 h. The beans were then discarded and another fixed bed of moisturized beans was prepared. The dilute extract used for the first column was recycled through the fresh green bean column at a rate of 5·2 lbs min − 1 for another 4 h. This process was repeated until a 12° brix extract was obtained. For every 100 lbs of moisturized beans, about 76 lbs of extract were produced. The extract was then concentrated to about 26% solids.
Based on microbial screening for malolactic fermentation in green coffee extract, two lactic acid bacteria were selected for this process. Lactobacillus plantarum 8014/1236 was provided by Dr. Thomas Montville at Rutgers University and Leuconostoc oenos 27307 was obtained from the American Type Culture Collection in Maryland. Both bacteria were maintained using the following procedure: the cultures were propagated, centrifuged, resuspended in fresh media and dispensed into cryotube vials containing equal amounts of glycerol. The vials were mixed and stored at − 80°C until needed.
2.3. Description and loading of the immobilized cell bioreactor (ICB) The bioreactor used was a 30× 5 inch spiral-wound
Fig. 1. Biochemical pathway for the malolactic enzyme.
Fig. 2. Schematic of the malic acid removal process for green coffee beans.
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and then through the biosupport from the outside into the unit’s core (opposite direction from loading). The estimated extract residence times in the shroud and biosupport were 5·5 and 6·8 min, respectively. Once through the bioreactor, the malic acid-lean extract passed through a heat exchanger to kill the bacteria. The heated extract was pumped into a collection tank just upstream of the extraction column.
2.4. Operation of the countercurrent pulse extraction column
Fig. 3. Lactobacillus plantarum immobilized on the spiral-wound biosupport (2000 × ).
ribbed NC120 Microporous Plastic Sheet (MPS®) manufactured by FMC Corporation Biosupport Materials (currently produced by Arbor Technologies, Inc.). Its use for several other food-related applications, both microbial and enzymatic, is well documented [11 – 14]. MPS® is a proprietary porous silica particle embedded PVC matrix which is highly suited for the colonization of microbial cells. This unit provided an available surface area of 76·5 ft2 for immobilization and bound an estimated 3 × 1015 CFU (colony forming units). Attachment of bacteria to the biosupport is shown in Fig. 3. The ICB was connected to a 75 liter New Brunswick fermenter and a sterile media reservoir, which were used to ‘load’ the bacteria. L. plantarum was propagated in the 75 liter fermenter (inoculated from a 7 liter fermenter) at 30°C, 200 rpm, 3 psi backpressure and provided with 2·5 LPM sterile air for 18 h in MRS medium. The 50 liters of actively growing bacteria were recycled from the fermenter through the top center of the biosupport. Colonization occurred as the bacteria moved through the biosupport: unbound cells exited from the outer wrap of the spiral unit into the bioreactor shroud and then back to the fermenter. On a routine basis over the 4-day loading period, spent media and cells were removed from the fermenter and replaced with fresh enrichment (MRS) media. Following the loading period, media and free cells were drained from the entire system and the bioreactor was filled with the malic acid-rich green extract that had been clarified by cross-flow microfiltration and sterilized via 0·3 and 0·2 m dead-end filtration. The green extract was pumped into the bottom of the bioreactor shroud at 1·0 – 1·5 lbs min − 1
The extraction column was 33 ft× 4 inches ID. The column, which could be pressurized, was equipped with a feed bin and blowcase on top and a blowcase on the bottom. Moisturized coffee beans were airveyed to this bin at regular intervals. During the run about 6 lbs of ‘demalated’ beans were pulsed out of the column into the blowcase every 30 min. The residence time for beans in the extraction column was 6·5 h (13 pulses). The green extract to bean ratio in the column for this application was about 6·6:1. The extract velocity up the column was 0·22–0·24 ft min − 1, which is equivalent to a flowrate of 1·3 to 1·4 lbs min − 1. Depending on coffee bean type, the column temperature was set between 71° and 83°C. In the blowcase at the bottom of the column, the green extract was blown free of the beans to a holding tank upstream of the microfiltration unit. The ‘demalated’ beans were rinsed free of extract and dried. The rinse water was periodically added to the microfiltration feed tank to prevent extract solids loss. The beans were then roasted at 200–260°C.
2.5. Green extract clarification and sterilization After 6·5 h in the extraction column, the hot extract not only contained malic acid but also coffee bean insolubles and viable microorganisms. The majority of the insolubles were removed via a Romicon microfiltration unit equipped with either 0·8 m or 0·45 m ceramic cross-flow cartridges. The retentate was collected and added back to the extraction column feed tank. The permeate was collected in a holding tank and then pumped through a sterile 0·3 m prefilter and a sterile 0·2 m dead-end filter to sterilize the extract. The sterile, malic acid-rich green extract was cooled from about 71° to 27°C as it passed through a glycol chiller on its way to the ICB.
2.6. Analytical Most of the organic acid analyses were performed with a Dionex HPIC unit using conductivity detection and a Carbopak; PA-1 column (4× 250 mm) and guard
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(4 × 50 mm). The mobile phase was 160 mm sodium carbonate at a flowrate of 1·0 or 1·5 ml min − 1 anti operated at room temperature. Some lactic acid and glucose analyses were performed using a Yellow Springs model 2700 immobilized enzyme system. Sugars were also quantified using a Waters HPLC system equipped with a BioRad Aminex column or more often a Dionex HPIC equipped with pulsed amperometric detection. The column was the same one used for organic acids but the mobile phase was 160 mM NaOH at 1·0 ml min − 1.
3. Results
3.1. Immobilized cell bioreactor During one 72 h run, about 4750 lbs of malic acidrich green extract were pumped through the ICB. Green extract supplied all the required nutrients necessary to support an active lactobacillus population as indicated by the number of cells observed in the exit stream (Fig. 4a). After 15 h, the malic acid-lean exit stream contained an average of approximately 107 CFU ml − 1. The malic acid level in the exit stream decreased rapidly from 2·08 g liter − 1 (initial feed concentration) to 0·2 in the first 12 h and then plateaued to an average of 0·32 g liter − 1. This yielded an 83% reduction in malic acid (Fig. 4b). The malic acid level in the feed also decreased from zero to 50 h and then plateaued at 0·7 g liter − 1. The glucose concentration in the exit stream after 72 h was 56% of the initial feed stream concentration (Fig. 4c). Sugars play a critical role as precursors for flavor development during the roasting process and it was important to balance the catabolism of malic acid with the detrimental microbial utilization of reducing sugars. The immobilized system accommodated this balance, whereas the sugar content of extract after batch fermentation was depleted. Glucose was metabolized to lactic acid by Lactobacillus plantarum as the sole product since this organism is homofermentative (Leuconostoc oenos is a heterofermentative bacterium and will produce ethanol and/or acetate and carbon dioxide and half as much lactic acid on a mole basis). The green extract concentration of lactic acid increased from 2·35 to 5·85 g liter − 1 during the first 50 h and then equilibrated at 6 g liter − 1 (Fig. 4c).
3.2. Green bean extraction column The extraction column was fed malic acid-lean green extract at an average rate of 78 lbs h − 1 (1·3 lbs min − 1). with an extract to bean ratio of 6·59:1. The feed had a malic acid concentration of 0·32 g liter − 1 at equi-
librium. The malic acid-rich extract draw-off from the column had a malic acid concentration of 0·8 g liter − 1 at equilibrium (pulses 60–130), with a composite concentration of 0·83 g liter − 1 (Fig. 5a). During equilibrium pulses 60–130, the average amount of malic acid extracted from the green beans was equivalent to the average malic acid conversion in the ICB. A material balance performed on the amount of malic acid extracted from beans compared to the amount of malic acid in the extractor draw-off gave good agreement with experimental results—0·78 g liter − 1 versus 0·80 g liter − 1 (experimental) malate concentration. The feed stream was offset by 13 pulses (one bed volume) to account for residence time of the extract in the column. The peak amount of malic acid in the draw-off occurred around pulse 100; this corresponded well with the peak demalation obtained in the product bean from pulses 100 to 120. The beans from the 154 pulses in this trial were dried to yield about 890 lbs of demalated coffee beans. Of these 154 pulses, 60 (70–130) were at equilibrium, yielding about 330 lbs of product beans at an average of 81·6% demalation on a dry basis (Fig. 5b). The pulse size of the demalated beans averaged 11·31 lbs at 59·17% moisture, yielding 2.49 lbs h − 1 when dried to 11·5% moisture. As the beans were pulsed from the column, the surface solids were recovered via a washing step. The wash water, equal to the amount of water the beans would absorb during their residence time in the extractor column, was added back to the extract feed tank every ten pulses. The high solids content in the extractor feed (21·3%) led to lower solids losses across the extraction process (Fig. 5c). On a dry basis, the green bean feed had an average malic acid concentration of 0·38% and the extracted product beans at equilibrium contained 0·077%.
4. Discussion
4.1. Malolactic fermentation By coupling a physicochemical process (countercurrent extraction) with a biological process (malolactic fermentation, MLF) we were able to design the bioreactor component for flexibility, reliability, and reproducibility. The 6·8 min that the green extract spent within the biosupport was sufficient to achieve up to 87% demalation of the extract. This level was high enough to permit malic acid extraction from the green beans to the targeted level. Increased exposure times with the immobilized bacteria would increase the lactic acid content and decrease the glucose level, which was replenished during the extract’s journey up the green bean extraction column.
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4.2. Immobilized cell bioreactor Susceptibility to microbial contamination is always a concern in continuous fermentation processes. The ICB exit stream was routinely monitored for contaminants
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and cell counts. No contamination was observed in any of the ICB pilot plant trials even after operating for a period under non-sterile conditions during one run. It should be fairly difficult for another microbe to gain a foothold on the biosupport, which was totally colo-
Fig. 4. Viable microbial population in the ICB exit stream, (b) malic acid levels in the ICB feed ()and exit ( — ) streams, (c) lactic acid (---) and glucose () in the ICB exit stream.
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Fig. 4. (Continued)
nized with active lactobacilli — approximatel 2·7× 1011 CFU in − 2 —within the 12 – 13 min the extract resides in the bioreactor Benchtop ICB trials operated 6 months without any apparent contamination or loss of MLF efficiency. Besides the long functional lifetime of the MPS® bioreactors, which could be totally cleaned of microflora and reloaded, the bioreactors could be stored refrigerated for up to 3 weeks without significant loss of MLF activity. Two days of recycling fresh MRS media at 30°C restored full metabolic activity to the biosupports (data not shown). One of our major concerns was the possibility of channel plugging, particularly with a 25% total solids substrate. MPS® sheets were manufactured with a variety of rib sizes (width and depth). The standard rib (CF) unit has a channel width of 0·9 mm and a rib height of 0·4 mm (18 channels per one-inch sheet width). If the five-inch diameter spiral-wound unit was unwrapped, it would stretch 37 linear ft (1128 m). Another unit evaluated during this research was the high rib TC190 unit, which has a channel width of 4·8 mm (5 channels per inch), a rib height of 1·0 mm and a linear length unwrapped of 13 ft (3·96 m). Due to periodic precipitation of coffee solids from the extract (e.g. green extract pH dropped below 4·5 while the bioreactor remained idle; this caused solids precipitation), the pressure drop across the standard rib unit increased beyond the 50 – 60 psi maximum recommended operating pressure
after several months of exposure to clarified extract. Without any clarification to remove the insolubles, the rib channels plugged after 48 h of continuous operation. No pressure drop was ever detected across the high rib unit and no plugging was observed. Several of the benchtop and all of the pilot plant trials made use of the intermediate ribbed NC120 unit, which had a channel width of 2·8 mm (9 channels per inch), a rib height of 0·7 mm, and a linear length of 22·4 ft (6·83 m). Over time, the deposition of insoluble solids, including cell mass, could pose a problem for this unit. Periodic high flow-rate flushing may eliminate this potential situation. One of the positive attributes of the bioreactor process was its flexibility. Although only a single unit was required for this application and a single pass of the green extract, the process could be modified for recycling or employment of a series of ICB units manifolded together. A wider rib ‘polishing unit’ (TC190) could follow the intermediate ribbed NC120 biosupport. Also, the length of the biosupport or the linear length of the spiral wrap could be tailored to fit the application requirements. Finally, non-glutaraldehyde treated MPS® is a chemically inert material which can be reused after cleaning with nitric acid and/or hypochlorite solutions. It can be utilized for thermophilic enzymes or micro-organisms up to 90°C. In-place steam sterilization was attempted without success, but was not a major drawback to this process.
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4.3. Pulse column extractor It is important to define process equilibrium in terms of the pulse column extractor and the immobilized cell bioreactor. Process equilibrium is defined by operating
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conditions—bean throughput, extract flowrate, extract temperature, ICB extract throughput, etc.—at which the amount of malic acid extracted from green coffee beans per unit time is equal to the amount of malic acid converted to lactic acid in the green coffee extract
Fig. 5. (a) Malic acid levels in the pulse column feed ( ) and draw-off ( ), (b) percent demalation of the product coffee beans exiting the pulse column, (c) solids level in the pulse column feed ( ) and draw-off ().
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Fig. 5. (Continued)
flowing through the ICB per unit time. At these conditions, the product green beans will reach a constant level of demalation:
pulse column was equal to the rate of malic acid conversion in the ICB. Therefore the process can be said to be in equilibrium
d[malic conc. in green beans]/[t]equil =0 These conditions are found between pulses 60 and 130 (Fig. 5b) where the change in concentration of malic acid in the product beans over time was approximately zero. It is at these conditions that a uniform product was produced. A linear regression of percent demalation of the product beans versus time over pulses 60 – 130 gave a slope of 0·2113. At a 95% confidence level, the upper and lower limits were 0·4227 and −4·2 ×10 − 5, respectively. Since the value of zero is encompassed by the limit range, no change was observed. Because the concentration of malic acid in the product from the pulse column is seen to be constant over time, the pulse column process can be said to be operating at ‘steadystate’ conditions. A linear regression of malic acid converted in the ICB versus time from 28 to 52 h gave a slope of − 0 349. At a 95% confidence level. the upper and lower error limits are 0·6378 and − 1·3368, respectively. Again, the value zero is encompassed by the limit range, and hence there is no change in malic acid conversion rate over this time. It can be concluded that at the given time interval pulses 60 – 130 (28 – 52 h of ICB operation) the rate of malic acid extraction in the
4.4. Process summary The immobilized bioreactor was operated in a continuous mode. It had a working volume of 6·35 liter with an average feed residence time of 12–13 min. Up to 87% of the malic acid and 44% of the glucose were removed from the green extract which exited the ICB with 2·1% lactic acid, db per 100 h of operation. 2995 kg of extract was demalated having an average malic acid concentration of 0·32 g liter − 1 at equilibrium. During 100 h of operation 461 kg of green beans were demalated in the pulse column with an average malic acid level of 0·069%, db.
4.5. Applications of pulse column technology coupled with an immobilized bioreactor Alone, each of the unit processes has application. Immobilized cell and enzyme bioreactors have demonstrated commercial success for many years and there is no need to review these applications in this article. The pulse column, however, can provide a unique approach to continuous food processing. An extract can be generated from the material to be treated or alternatively any
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aqueous solution (e.g. brine) or an organic solvent can be employed if solids loss is not an issue. Aqueous extracts can also contain viable micro-organisms and/or enzymes. As discussed above, it is important to precisely determine the extraction reaction, and/or infusion kinetics for the particular food material. Column residence time, temperature, and pressure parameters must be evaluated. When coupling the pulse column extractor with a continuous bioreactor process generation of a high solids extract which will recycle through the bioreactor and pulse column continuously may or may not be desirable. Sterility may not be necessary but microfiltration to remove suspended solids will be required. The immobilized bioreactor can not only microbially or enzymatically treat this extract continuously, but can also be a constant source of microorganisms and extracellular enzymes which can remain functional if the extract is not heat treated downstream of the bioreactor. The rapidly expanding knowledge base on extremophiles and their enzymes might allow the bioreactor to operate under thermophilic or halophilic conditions. Pulse column/bioreactor technology with brine recycling might provide a unique approach to pickle, olive and sauerkraut production.
Acknowledgements We wish to thank Bruce Goldberg for sharing his expertise on the characteristics and use of the biosupport utilized in this process and Professor Thomas Montville at Rutgers University for his valuable expertise in microbial biochemistry and lactobacilli.
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