Co-immobilization of selected yeast and bacteria for controlled flavour development in an alcoholic cider beverage

Process Biochemistry Vol. 31, No. 2, pp. I I 1- 117, 1996 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 S 15.1111+ 11.00 ELSEVIER

0032-9592(94)0002

I-6

Co-Immobilization of Selected Yeast and Bacteria for Controlled Flavour Development in an Alcoholic Cider Beverage J. A. Scott* & A. M. O'Reilly School of Chemical Engineering,Universityof Bath, Bath, BA2 7AY,UK (Received 15 February 1995; revisedmanuscriptaccepted 25 March 1995)

A sponge-like material has been used to immobilize both S. cerevisiae and L. plantarum for car~ing out fermentation and partial maturation of ak'oholic cider. 7he ~v)onge ~"open porous network promotes extensive and rapid s,~'ace attachment of nficro-organisms throughout the depth o[the material. The matrix smJhce can be also chemically modified and it was found that basic characteristics enhanced both initial rate o/'uptake and also final loading (in excess of 10~yeast cells/g sponge and I0 m bacteria cells/g sponge). Fermentations curried ottt with immobilized yeast and sequential addition of lactic acid bacteria indicated a route to both enhancing rate of fermentation and controlling positive flavour development.

INTRODUCTION

popular in the industry. Their porous structure can provide natural immobilization for both yeast and bacteria. A mixed microflora can be retained despite vigorous cleaning between batches with ball-head sprayers using a 0-5% sodium carbonate solution at 60°C for 20 min. ~ These organisms can subsequently inoculate a freshly fermented medium, supplementing yeast and bacteria carried over from the fermentation vessel. After prolonged storage under these conditions, the organoleptic character of the final product can reflect, therefore, sequential and complimentary activity between specific yeast and bacteria. In general, maturation, a process of significant commercial impact and value, remains uncontrolled through a reliance on development and activity of a relatively ill-defined and uncoordinated microflora. To produce a consistent product, post-storage blending is often used, while

The production of many fermented fruit based alcoholic beverages, such as wine, perry and cider, can be regarded as two stage processes. Typically, a Saccharomyces cerevisiae fermentation provides an alcoholic base, which after attentuation, is pumped to a storage vat where an indigenous mixed microflora can play a significant secondary role in dictating the finished product's individual character. Prime 'harbours' for these cultures are wood vats, which were traditionally used throughout the process, but for fermentation, have been now mainly replaced by vessels made of food grade materials, in particular stainless steel. For storage maturation of many beverages, including alcoholic ciders, wood vats remain *To whomcorrespondence should be addressed. 111

112

.l. A. Scott, A. M. O'Reilly

the employment of defined co-immobilization of the key organisms could provide a basis for both standardizing and accelerating flavour and aroma development. We are investigating, for example, primary yeast fermentation coupled to secondary bacterial 'maturation' through exploitation of immobilized yeast in conjunction with introduction of a defined bacterial population for malolactic fermentation. For fermentation, the potential benefits of immobilized systems are primarily enhanced rates and increased final alcohol levels. 2-4 These often need to be balanced against limitations associated with current immobilization matrices, 5 in particular those related to maximum support size and adequate internal mass transfer. To address these restrictions, we have successfully used a spongelike material to support Saccharomyces cerevisiae in beer fermentations.~'The support was chosen as it is pliable and can be tailored to suit process requirements regarding net surface charge, shape and size. Unlike many reticulated foam-type matrices, the material used has a very open internal pore network with few 'dead ends' (Fig. 1). This high porosity provides a low pressure drop across the matrix and good mass transfer, which in turn promotes homogeneous conditions, as expressed by internal cell distribution.4

cial cider fermentation and the lactic acid bacterium, Lactobacillus plantarum (University of Bath Culture Collection, 2214). Both yeast and bacteria were cultured separately in 250 ml shake flasks for three days in aerated Tryptone Soya Broth (Unipath, UK) at 25°C.

EXPERIMENTAL

Immobilization studies After culturing, cells were then recovered by centrifugation, washed three times with ¼strength Ringer's solution and re-suspended (singularly or together) in 25 ml flasks containing 100 ml of fresh Ringer's solution and either 3 g of sponge (wet weight) or carbon support. The flasks were shaken at 100 rpm and triplicate samples periodically taken to count cells remaining in suspension. For studies of the influence of pH on immobilization, the Ringer's solution was modified by addition of either 1 M NaOH or 1 M H2SO 4.

Micro-organisms and culture media The micro-organisms used were a moderately flocculating S. cerevisiae isolate from a commer-

Fig. l.

Scanning electron micrograph of the immobilization sponge ( x 200 magnification).

Immobilization media A plain sponge (a neutral cross-linked cellulose with no surface treatment) was used along with, PRODUCTIV TM CM (acidic, - C O O - functional group) and PRODUCTIV TM DE (basic, N+(CH2CHfl2H functional group) supplied by BPS Separations, Spennymoor, UK. All sponges were obtained in flat sheets, approximately 0.8 cm thick. For cell uptake measurements and cider fermentations, the material was cut into 0.8 cm sided cubes. After drying sponge samples in an oven at 105°C for 24 h, recorded weight loss was 73+_5% (i.e. l g wet weight gave 0-27 g dry weight). For comparison of immobilization uptake rate, a novel carbon bonded carbon fibre (CBCF) mesh was used, consisting of short, smooth (nonactivated) fibres bonded together by resin to form a random matrix (supplied by B. McEnaney, University of Bath). The CBCF matrix was selected as the internal pores had dimensions similar to those of the sponge.

Scanning electron microscopy (SEM) To investigate cell distribution over internal sponge surfaces, a 0.8 cm sided cube of material was fixed with 2% gluteraldehyde, followed by 1% osmium tetroxide. The sponge was then frozen in liquid nitrogen, fractured in two and dried at -60°C for 18 h (Edwards-Pearse Tissue Dryer EPD3). After drying, sponge sections were mounted and gold coated for 6 rain (Edwards

(bntrolled flavour development in cider

Splutter Coater 515OB) prior to SEM examination (JEOL T330). Fermentation Fermentations were carried out using "standard' strength apple juice based cider (original gravity 1.068_0.001, pH 3.4_+0-1). The medium consisted (per litre) of 85 ml apple juice concentrate, 120 ml glucose syrup, 0.5 g ammonium phosphate, 0.5 g ammonium sulphate, 0.3 g sodium metabisulphite and 795 ml water. 2-0 litres of cider medium was then placed in 2.5 litres fermenters (20°C) which connected via a side-loop to a temperature controlled 1.5 cm i.d. glass column packed with 6 g (wet weight) of sponge pre-ioaded with S. cerevisiae (at 2.2_+ 0.1 x l(F cells/g sponge). The medium was circulated continually upwards through the (nonfluidized) sponge at 60 ml/min for 28 d. At set periods (days 0, 3 or 10) L. plantarum (equivalent to 10 '~ cells/ml) were added to certain fermenters, with others left with just yeast. To act as controls, fermentations were also set up with side-loops that did not contain sponge. At the start of the fermentation, yeast was added to the medium of these fermenters at an initial concentration equivalent to the total amount of cells immobilized on the sponge (i.e. 6.6 x 10 ~' ceils/ml). Sensory evaluation After 28 days, the ciders were clarified by centrifugation and a basic comparative organoleptic evaluation was carried out by a panel of eight people who regularly participated in taste trails.

Fig. 2. Scanning electron micrograph of yeast cells immobilized on DE sponge after 5 h exposure ( x 500 magnification).

113

Half the panel had attended a sensory analysis training course for ciders. As a control, an equal volume of freshly fermented commercial cider was tasted alongside laboratory derived samples and an overall, relative ranking given. This was achieved through the panellists assessing the commercial product before 'blind' tasting the test ciders. All were evaluated for acidity, aroma, fullness, astringency, oxidation, solventness and sulphurness. Each characteristic received a rating of 0-10 (10 corresponding to the commercial product). The scores for each characteristic were added together, averaged over all the tasters and the total adjusted to fall between 0-100% (100% corresponding to the commercial cider).

RESULTS AND DISCUSSION Yeast immobilization Not surprisingly, due to the predominantly negative nature of the yeast outer cell surfaces, when exposed to an acidic sponge (CM), uptake of the weakly flocculating S. cerevisiae cider isolate was virtually zero. Previous work, ~' showed that only a strongly flocculating yeast achieved any level of uptake due to the formation of large flocs in the medium leading to subsequent physical blocking of the pores, whereas, with plain (no surface treatment) and in particular basic (DE) sponges, highly effective rapid internal yeast uptake and immobilization was achieved with weak or nonflocculating strains." SEM analysis indicated that a mono-layer biofilm initially develops over the relatively rough surfaces (Fig. 2), which then acts as a key for other cells to attach to, and/or bud from. In terms of residues from the sponge affecting the fermentation, there were no discernible differences in performance, or resulting taste, after consecutive beer fermentations were carried out with the same sponge and immobilized yeast." The sponge was also useful as it could be autoclaved without any apparent loss in immobilization performance (Fig. 3). However, as was found in yeast immobilization for beer fermentation, (' the medium in which the cells are re-suspended for challenging the matrix does significantly influence uptake. This result was exacerbated by the DE material, with growth medium consistently having the greatest (negative) impact. This suggests coating of surface binding sites by medium constituents interfering with cell attach-

1 14

~

J.A. Scott, A. M. O'Reilly

3.0

l~.l~nger's solution

2.5

Tryptone soya broth

loo [

__

o 80

"~2.0

6o

1.5 o 1.0

i4o

~0.5

20

0.0 0

1

2

3

4

5

Exposure Time (hours) Fig. 3. Effect of autoclavingand cell suspensionmedium on S. cerevisiae (cider isolate) uptake by the DE (basic) sponge matrix. A, o, :z, Autoclaved sponge (121 ° for 15 min), A, o, m, non-autoclaved sponge.

ment. As a consequence, in order to maximize loading, particularly in terms of exposure time, for subsequent immobilization studies, cells were always recovered from their growth medium by centrifugation, washed and re-suspended in strength Ringer's solution prior to exposure to the matrix. Initial uptake by the sponge of the weakly flocculating yeast was clearly restricted to surface attachment. As a gauge of the effectiveness of the material at holding in cells, comparison was made between plain and DE sponge of similar physical characteristics (i.e. pore size distribution and surface area), and a novel type of carbon support. The other medium examined, a carbon bonded carbon fibre (CBCF) mesh, was selected on the basis that it could also be autoclaved and had pore dimensions similar to the sponge, thereby allowing free internal passage of cells. CBCF is made from short fibres randomly stuck together, with the porous structure variable in the range 10 /~m-1 mm by varying fibre length and processing conditions. Results of yeast uptake from Ringer's solution onto 3 g of matrix in shake flasks (100 rpm), again demonstrate the significant advantage the DE sponge has for accumulating S. cerevisiae (Fig. 4). The lower cell loading within the CBCF mesh, as compared to the plain sponge, was attributed, at least in part, to the very smooth surfaces of the chopped fibres. SEM examination indicated that significant cell attachment occurred only at

0

Uptake

Removal

Fig. 4. S. cerevisiae (cider isolate) uptake and removal levels with various immobilization media.

surface imperfections, indicating the importance of a matrix's physical surface nature, as well as charge. The advantages of the DE material were also highlighted in tests to assess resistance to cell removal. Matrices plus immobilized cells were placed in fresh Ringer's solution and vigorously shaken for 60 min at 250 rpm, after which only 4% of immobilized cells were removed from the DE sponge, compared to around 40% from the CBCF and plain sponge (Fig. 4). Co-immobilization

An aim of this work is co-immobilization of two, or more, selected microbial species for application in aspects of alcoholic beverage production. This includes post-primary fermentation flavour modification, such as yeast for diacetyl reduction in beer and also, mixtures of yeast and bacteria for fruit based beverages. In the production of alcoholic cider and many red wines, a reduction in overall beverage acidity by bacteria dominated malolactic fermentation is generally recognized as an important phase in flavour development. In particular, the vital role of lactic acid bacteria of the genera Lactobacillus, Leuconostoc and Pediococcus, in the conversion of malic to lactic acid has been the subject of extensive review.7-'~ In wine, Leuconostoc is considered predominant in malolactic fermentation,7 but with cider, in our own ~ and other studies, l° Lactobacillus plantarum has been found to be the bacterium present in the largest numbers towards the end of primary fermentation and during storage. L. plantamm was considered, therefore, likely to be

Controlled flavour development in cider

a major contributor to flavour development. Consequently, for co-immobilization studies with eider, the organisms selected were the S. cerevisiae cider isolate, along with L. plantarurn. Further, as part of a programme to determine to what extent microbial sponsored flavour development is generic or variety specific, the bacterial strain used was selected as one not previously isolated from a beverage. Pure cultures of L. plantarum suspended in Ringer's solution exhibited similar degrees of affinity to the yeast to the various sponges, including no recordable uptake by the acidic (CM) material. With these organisms, short-term loading was again superior on the DE, with immobilization restricted to a predominantly monolayer biofilm of cells. As reported for yeast, ~' uptake, in terms of rate and final loading, was found to be independent of the pH in the range 3-9. To determine the extent to which co-immobilization over the same matrix would occur, separately cultured stationary phase S. cerevisiae and L. plantarum were recovered from their growth media, washed, mixed and re-suspended in shake

16

i

lY st

14 12

.~ =

Exp2Yeast

l0

3 ~

Exp3Yeast

8

2 ~

Exp 1 Bacteria

2

~

Exp 3 Bacteria

i

0

i

115

flasks containing 3 g of DE sponge and 100 ml of Ringer's solution. For the three experimental runs depicted in Fig. 5, initial yeast and bacteria concentrations are given in Table 1, along with loading levels after one hour, when the percentage uptake of available yeast cells were similar at 50-60% and attachment was predominately restricted to a mono layer. SEM examination was supported by the data on measured cell loadings, which suggested competition between yeast and bacteria for available binding sites on the DE sponge. In terms of the loadings, the yeast/bacteria ratio between the experiments was of the same order of magnitude. Thus, between experiments 1 and 2, a drop in yeast cell loading of 6.03 x 1 ()~ ceils/g sponge was accompanied by a 1.81 x l0 t" cells/g sponge rise in bacteria numbers. This indicates a ratio of 30 bacteria bound per single yeast cell. The difference between experiments 2 and 3 was a reduction in yeast of 4.50 x 108 cells/g sponge leading to a bacteria increase of 0"82x10 l° cells/g sponge, or 18 bacteria per bound yeast cell. The yeast cells were approximately 4-5 ~ m in diameter and the bacteria had dimensions of 2.5 x 1 ktm, which would suggest, with close packing, around 5-10 bacteria per yeast. However, the yeast cells have been shown to bind initially in a reasonably discrete and spread out manner (Fig. 2). The apparent higher numbers of recorded bacteria per yeast are, therefore, consistent with this and other SEM observations, in as much that the bacteria initially bind in relatively much closer proximity to each other. Hence, it would be expected that a single yeast cell 'occupies" the space required for more than just 5-10 bacteria.

i

1 2 3 4 5 Exposure Time (hours)

Fig. 5. Combined S. cerevisiae and L. plantarum uptake by the DE (basic) sponge matrix.

Fermentation In commercial cider and most wine operations, the presence and introduction of lactic acid bacteria is generally uncontrolled, such that the

Table 1. Defined co-immobilization of L. plantarum and S. cerevisiae from Ringer's solution by DE sponge

Experiment

1 2 3

Initial cell concentrations

Loading after 1 h

L. plantarum

S. cerevisiae

L. plantarum

S. cerevisae

(cells/ml)

(cells/ml)

(cells/g sponge)

(cells/g sponge)

0'34 x 10 m 1"53 × 10 l° 1'38 x 10 m

7"75 X 1 0 7 4"85 × 107 2"45 x 107

0"61 x 10 "~ 2"42 x 10 l° 3"24 x 10 m

1"47 × 10 ~ 8"67 x 108 4'17 x 10 ~

116

J. A. Scott, A. M. O'Reilly

timing and degree of any microbial sponsored transformations are unregulated and ill-defined. As a typical cider fermentation may be 14-21 days, followed by one to eight weeks' maturation, this represents a considerable production period over which little or no systematic control of microbial interactions is exerted. A possible solution is to identify the optimum time at which to introduce these bacteria, such that each batch fermentation is exposed to the same level of microbial activity. Following the cell uptake studies co-immobilization of S. cerevisiae and L. plantarum was investigated. A series of fermenters were set up, some with attached columns containing S. cerevisiae (cider isolate) pre-immobilized onto 6 g of DE sponge (2.2 × 109 cells/g sponge). At specific times during the fermentation, L. plantarum was added (i.e. at the beginning, after three days when aeration was stopped, or after 10 days). The bacteria were added in free suspension at a concentration of 1.0 × 109 cells/ml, a level much higher than encountered in maturing cider fermentation systems' (i.e. 104-107 cells/ml), to ensure a bacterial impact on the fermentation. Control fermentations were also prepared with no sponge (all yeast remained in free suspension), but with circulation to ensure equivalent levels of mixing. The fermentations attenuated between 18 and 21 days (Fig. 6), but were allowed to remain undisturbed for a further week to provide a short

[]

1.06

Immob.

post-fermentation maturation period. In Fig. 7, the SEM of a sponge sample extracted after 21 days fermentation (L. plantarum added at day 10), illustrates that both types of cell were successfully accommodated over the matrix, even though the yeast had prior exposure. In terms of ethanol production, the presence of the bacterium did not affect levels, but a consistent feature of yeast immobilization was an increased rate of production and a slightly higher final concentration (Fig. 8). We have speculated from single culture work, 4 that higher ethanol yields reflect build up of greater yeast numbers due to the sponge providing an improved micro-environment,

Fig. 7. Scanning electron micrograph of S. cerevisiae and L. plantarum immobilized on DE sponge after a 21 day cider (OG 1.068, 20°C) fermentation ( x 2000 magnification).

•--•'8.5•

[]

Immob. (0)*

[]

~1.04

Immob. (3)* [] Immob. (10)* []

i,.o

Non-Immob.

~1.02

* day laotio aoid

baoteria added 1.00

i

2

t

i

,

5 7 11 18 22 Fermentation Time (days)

Fig. 6. Decline is specific gravity in various cider (OG 1'068, 20°C) fermentations using S. cerevisiae either preimmobilized or in free suspension, with and without added L. plantarum.

7.5

None Day 0 Day 3 Day 10 Day 0 Day 10

~

Lactic Acid Bacteria Addition [ ] Ethanol [ ] Flavour Fig. 8. Final flavour assessments and ethanol levels in various cider (OG 1.068, 20°C) fermentations using S. cerevisiae either pre-immobilized or in free suspension, with and without added L. planmrum.

Controlled flavour development in cider

including acting as a nucleation site for CO2, thereby leading to enhanced gas removal from solution. Although good immobilization and fermentation rates of alcoholic beverages can be achieved using the sponge, the key factors of colour, aroma, texture and above all, taste remain of paramount importance. Consequently, as part of this programme, carried out regularly with the aid of a 'blind' tasting panel, evaluations of end products are made. The comparative assessment results for various fermentations, whilst subjective, were nevertheless consistent. They were carried out on the basis of a ranking in relation to a product fermented and matured with a natural mixed microflora (100% rating). The results, also given in Fig. 8, clearly indicated that the bacterium, even though not a cider isolate, did significantly enhance flavour development. Furthermore, the time of addition was important and from the data obtained, should be delayed until towards the end of fermentation. Considering that only two organisms were involved and these were "competing' against an established mixed microflora, and that lactic acid bacteria numbers were high, the results are encouraging.

CONCLUSIONS A pliable sponge material proved an excellent immobilizer of both yeast and bacteria important in the production of fruit based alcoholic beverages. A surface treatment that provided basic characteristics gave the highest cell loadings and subsequent resistance to removal. Rate of yeast uptake and final loading was, however, influenced by the medium in which the cells are suspended, with a rich organic medium having greatest (negative) impact. Commercial cider fermentations typically take two to three weeks, and post-fermentation maturation one to eight weeks, so there is significant potential for the concept of controlled

117

and defined co-immobilization to accelerate and closely regulate full development of the beverage. This is likely to be in the form of a two stage system, with yeast for the first few days, followed by sequential addition of bacteria that subsequently co-immobilize with the yeast. The results from alcoholic cider fermentation confirmed that introduction of lactic acid bacteria does have a significant, positive impact on final flavour. Timing is important since although the time when bacteria were added did not affect fermentation rate, it clearly did influence the organoleptic character of the final beverage. REFERENCES 1. Swaffield, C. H. & Scott, J. A., Existence and development of natural microbial populations in wooden storage vats used for alcoholic cider maturation. J. Am. Soc. Brew. Chem. (In press). 2. Hayes, S. A., Power, J. & Ryder, D. S., Immobilized cell technology for brewing: a progress report. Part two: physiology of immobilized cells and the application to brewing. Brewers Dig., 66 ( 1991 ) 28-33. 3. Masschelein, C. A., Ryder, D. S. & Simon, J.-P., Immobilized cell technology in beer production. Critic. Rev. Biotectmol., 14 (1994) 155-77. 4. O'Reilly, A. M. & Scott, J. A., Use of an ion-exchange sponge to immobilisc yeast in high gravity apple based (cider) alcoholic fermentations. Bioteclmol. Techn., 15 (1993) 1061-6. 5. Masschelein, C. A., State-of-the-art and future developments in fermentation. J. Am. Soc. Bren'. Chem., 52 (1994) 28. 6. Scott, J. A. & O'Reilly, A. M., Use of a flexible sponge matrix to immobilize yeast for beer fermentation. J. Am. Soc. Brew. Chem., 53 (1995)67-71. 7. Davis, C. R., Wibowo, D., Eschcnbruch, R., Lee, T. H. & Fleet, G. ll., Practical implications of malolactic fermentation: a review. Am. J. Viticulture, 36 (1985) 290-9. 8. Davis, C. R., Wibowo, D., Lee, T. H. & Fleet, G. H., Growth and metabolism of lactic acid bacteria during fermentation and storage of some Australian wines. Food Technol. Australia, 38 (1986) 35-40. 9. Kunkee, R. E., Some roles of malic acid in the malolactic fermentation in wine making. Ft:MJ; Microbiol. Rev., 88 (1991) 55-72. 10. Carr, J. G. & Davies, R A., The ecok)gy and classification of strains of Lactobacillus: a bacterium commonly fotmd in fermenting ciders. ,/. Appl. BacterioL, 35 (1972) 463 71.