novel bioemulsifiers from microorganisms for use in foods

ELSEVIER Journal of Biotechnology 40 (1995) 207-217

,OL’P.NAL 0s

Biotdtnology

Novel bioemulsifiers from microorganisms for use in foods

Rachel Shepherd a, John Rockey b, Ian W. Sutherland ‘, Sibel Roller by* a Leatherhead Food Research Association, Randalls Road, Leatherhead, Surrey KT22 7RY, UK b School of Applied Science, South Bank University, 103 Borough Road, London SE1 OAA, UK

’ University of Edinburgh, Division of Biology, Rutherford Building, Mayfield Road, Edinburgh EH9 3JH, UK

Received 26 January 199.5; accepted 22 March 1995

Abstract

The main objective of this study was to test a range of microorganisms for production of extracellular, high molecular weight emulsifiers for potential use in foods. A standard emulsification assay developed specifically for assessing food emulsifiers was used to examine 24 extracellular microbial products from bacteria, yeasts and algae. Of the 24 products tested, nine had emulsification ability that was as good as and eight had emulsifying properties that were better than those of the commonly used food emulsifiers gum arabic and carboxymethylcellulose. The eight good producer organisms included the yeasts Candida utilis, Candida ualida, Hansenula anomala, Rhodospirid- ium diobovatum and Rhodotorula graminis, the red alga Porphiridium cruentum, and the bacteria Klebsiella spp. and Acinetobacter calcoaceticus. Of these, C. utilis was selected for further study due to the excellent emulsification properties of its extracellular products and the food-grade status of the organism. Crude preparations of the bioemulsifier from C. utilis exhibited low viscosity and had a carbohydrate content of over 80%. Preliminary trials showed that the bioemulsifier from this organism had potential for use in salad cream.

Keywords: Bioemulsifier; Food; Microbial; Extracellular; Candida; Polysaccharide

1. Introduction

Glycerol monostearate (GMS) and carboxymethylcellulose are synthetic emulsifiers widely used in the food industry (Gaonkar, 1991). Although very effective in their intended function, these compounds are gradually losing favour due to increased pressure from consumers to reduce the use of ‘artificial’ or chemically synthe- sized additives in foods. Thus, an increasing con-

* Corresponding author.

sciousness among consumers is driving a steady increase in demand for more natural food ingredients and additives. Some natural, plant-derived, food emulsifiers such as lecithin and gum arabic are already on the market. However, lecithin suffers from limited functionality in many food products subjected to modern food processing conditions. The supply of gum arabic, an import from West Africa, is subject to climatic and polit- ical upheavals (Whistler, 1993). Production of food emulsifiers by microbial cultivation would remove some of the constraints associated with the properties and supply of natural, plant-de-

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208 R. Shepherd et al. /Journal of Biotechnology 40 (1995) 207-217

rived emulsifiers. Experience with xanthan gum, gellan gum and nisin has demonstrated the feasi- bility of producing food-grade additives by large- scale industrial processes (Roller, 1991; Roller and Dea, 1992; Sutherland, 1990).

Biosurfactants from microorganisms have a number of advantages over their chemically syn- thesized counterparts because of their biodegrad- able nature, effectiveness at a wide range of temperatures, pH and salinities, and synthesis under user-friendly conditions, e.g., low temperatures and pressures. Owing to their diverse biosynthetic capabilities, microorganisms are promising candidates for emulsifier production. The majority of microbial emulsifiers have been reported in bacteria (Kosaric, 1993). Generally, bioemulsifiers are microbial metabolites with a hydrophobic moiety that is a fatty acid, and a hydrophilic moiety that is a carbohydrate, amino acid, peptide or phosphate. Some of the most commonly studied microbial emulsifiers have included the glycolipids, e.g., the rhamno lipid R3 from Pseudomonas aeruginosa, the lipopetides, e.g., subtilin from Bacillus subtilis, the polysaccharide-lipid complexes, e.g., emulsan from Acinetobacter calcoaceticus, and the polysaccharide-protein complexes, e.g., liposan from Can- dida lipolytica (Cirigliano and Carman, 1984 and 1985; Fiechter, 1992; Georgiou et al., 1992; Hayes et al., 1986a and Hayes et al., 1986b; Kaplan et al., 1985; Kaplan and Rosenberg, 1982; Kosaric, 1993; Pereilleux, 1979; Zosim et al., 1982). Many of these would not be suitable for use in foods due to the pathogenic nature of the producer organisms. A manno-protein complex with emulsifying properties from the food-grade Saccha- romyces cerevisiae has been reported but has not achieved commercialization (Cameron et al., 1988).

The reduction of surface or interfacial tension has often been used as the primary criterion for screening microorganisms for bioemulsifier production. However, emulsifying and dispersing agents used in food products do not necessarily have the ability to reduce the surface tension of water nor the interfacial tension of the commonly used, but inedible, hydrocarbon hexadecane. Fur- thermore, some microbial emulsifiers such as the

sophorolipids from Torulopsis bombicola have been shown to reduce surface and interfacial tension but not to be good emulsifiers (Cooper and Paddock, 1984). By contrast, liposan has been shown not to reduce the surface tension of water and yet has been used successfully to emulsify commercial edible oils (Cirigliano and Carman, 1985). We have used an emulsification assay developed specifically for assessing food emulsifiers as the primary criterion for screening bioemulsifiers with potential for use in foods (James and Patel, 1988; Pate1 and Fry, 1987). Furthermore, we selected an oil/water ratio of 40:60 to reflect the increasing demand for emulsifiers suitable for use in reduced-fat foods such as mayonnaise-like salad dressings. Finally, we compared all our results to those obtained using the well-known, food-grade emulsifiers gum arabic and carboxymethylcellulose.

In this paper we have concentrated on the high molecular weight surfactants. High molecular weight biopolymers generally exhibit a range of useful properties including high viscosity, ten- sile strength and resistance to shear and are of particular interest to the food industry due to their emulsion stabilizing properties. The hy- drophilicity of polysaccharide-based thickeners has been reported to decrease or the lipophilicity increase when the hydroxyl groups in the monosaccharides have been replaced by less po- lar groups as in aminosugars or the deoxysugars fucose and rhamnose. Increased substitution by acetyl and pyruvate moieties has also been reported to render polysaccharides more lipophilic (Des and Madden, 1986). The properties of the highly lipophilic polysaccharide indican from Bei- jerinckia indica have been attributed to a high content of the deoxysugar rhamnose and a high degree of acetylation (Lawson and Symes, 1981; Symes, 1982).

At this time, only one class of microbial emulsifier, emulsan, is in commercial production and is proposed primarily for secondary oil recovery in the petrochemical industry (Fiechter, 1992; Hayes et al., 1986a,b). The use of emulsan to remove dental plaque in oral hygiene products has been patented but its actual use in such products has not been reported (Eigen and Si-

R. Shepherd et al. /Journal of Biotechnology 40 (1995) 207-217 209

Table 1 Emulsification activity, stability and carbohydrate content of extracellular products from microorganisms

Microorganism Emulsification activity a Emulsification Carbohydrate (OD units) stability a (%) content (%)

Gum arabic (positive control) 0.35 Carboxymethylcellulose (positive control) Kappa-carrageenan (negative control) Low methoxyl pectin (negative control) < Acinetobacter calcoaceticus temulsanl b

Beijerinckia indica ss lacticogenes NCIMB 8846

Can&da kefyr NCYC 188 Candida marina NCYC 784 Candida pseudotropicalis NCYC 744 (sucrose “1

Candida sake NCYC 774 (sucrose ‘1 Candia’a tropicalis NCYC 405 Candida utilis NCYC 769 Candida ualida NCYC 20 (lactose “1 Hansenula anomala NCYC 20 Klebsiella spp. ’ Lipomyces starkeyii NCYC 692 (sucrose ‘1

Porphiridium cruentum ’ Propionibacterium acidipropionicii NCIMB 1072

Propionibacterium freundenreichii ss shermanii NCIMB 853

Propionibacterium jensenii NCIMB 850

Propionibacterium thoenii NCIMB 854

Rhodospiridium diobouatum NCYC 778 (sucrose ‘1

Rhodotorula glutinis v. glutinis NCYC 59

Rhodotorula glutinis NCIMB 162 Rhodotonda graminis NCYC 502 Rhodotorula rubra NCYC 63 Saccharomyces cerevisiae NCYC 24 (sucrose ‘1

Spirulina spp. ’

0.70 0.11 0.10 1.58

0.42 90 30

0 n/a 78 0.63 42 76 0.63 61 24

0 n/a 70 0 n/a ND 1.14 92 98 0.83 72 68 1.29 94 ND 0.88 95 ND 0.52 (0.4%) 39 (0.4%) ND

0.71 (1%) 0

0

0.69 57 51

0.38 n/a 66

1.4 98 40

0

0.69 51 ND 1.2 64 45 0.54 97 51 0 n/a ND

0.70 47 ND

90 70

n/a n/a

98

ND ND ND ND ND

100 (1%)

n/a

ND 52

n/a 48

n/a 60

* Emulsification activity and stability were determined at a concentration of 0.5% (m/V) unless otherwise indicated in brackets. b Received from Petroferm, USA. Reported protein content: 6.6%. ’ Received from BioEurope, France. Reported protein content: 3.6%. d Received from Photobioreactors, UK. ’ Organisms were grown on media containing glucose as sole source of carbon unless otherwise indicated in brackets following organism name. ND, not determined. n/a, not applicable.

210 R. Shepherd et al. /Journal ofBiotechnology 40 (1995) 207-217

mone, 1986). Few bioemulsifiers have been studied specifically for use in food products.

The primary objective of this work has been to test a range of microorganisms for production of extracellular, high molecular weight emulsifiers for potential use in foods. The secretion of the bioemulsifier out of the cell was considered an important criterion in order to reduce subsequent downstream processing costs.

2. Materials and methods

Chemicals and culture media were supplied by Sigma Chemical Co. and Oxoid (both of UK), respectively, unless otherwise stated. Microbial cultures were obtained from the National Collec- tion of Yeast Cultures (NCYC) and the National Collections of Industrial and Marine Bacteria (NCIMB).

The growth medium used for propionibacteria was that of Reddy et al. (1973). All other microorganisms were cultivated on Modified Cza- pek’s broth containing (per 1): NaNO, 2 g; K,HPO, 0.35 g; KC1 0.5 g; FeSO, .7H,O 0.018 g; MgSO,. 7H,O 0.58 g; agar 10 g and carbon source 30 g, pH 7. The carbon sources were glucose, sucrose, lactose, pyruvate, corn oil or rapeseed oil. Modified Czapek’s Yeast broth was the same as Modified Czapek’s broth except that it also contained 1 g I-’ of yeast extract.

2.1. Growth of microorganisms

Initially, 100 strains of microorganisms were streaked out onto a range of Modified Czapek’s agar plates containing glucose, sucrose, lactose or pyruvate to select those strains and carbon sources which produced the most mucoid colonies, indi- cating polysaccharide production. Organisms were also grown in liquid media containing corn oil or rapeseed oil as sole carbon source and evidence of emulsification was assessed qualitatively following growth. The yeasts were incubated at 25” C and the bacteria at 30°C for up to 3 d.

For the main screening exercise, the yeasts producing the most mucoid colonies on agar plates were grown in 200 ml of culture medium containing the preferred carbon source in l-l

baffled flasks. The flasks were incubated at 25” C for 3 d at 300 rpm. The propionibacteria were grown in a 10-l MBR Mini Bioreactor, sparged with nitrogen, at 30” C for 3 d. Twenty strains of yeasts and bacteria were tested on a small scale (Table 1). Larger amounts of a yeast culture (Candida utilis NCYC 769) were produced using the 10-I reactor mentioned above and a 150-l Model 5100 LH reactor supplied with l-l.5 1 1-l min-’ of air and operated at 25°C and 500 rpm. The pH was monitored but not controlled.

For rapid determination of microbial growth, optical density of the cultures was measured at 550 nm. Viable counts of Candida utilis were determined using a Spiral Systems spiral plater Model D and Malt Extract Agar (Lab M) plates. Total counts in the 150-I reactor were determined using a Coulter Counter. Dry weight was determined gravimetrically according to Buono and Erickson (1985).

2.2. Screening for emulsification properties

Microbial cultures were recirculated over a 0.45~pm crossflow membrane filter using a Sarto- con Mini (Sartorius) tangential flow filtration unit to remove whole cells from the spent medium. The filtrate was recirculated over three parallel filters with a cut-off of 5000 Da to concentrate extracellular products with a molecular mass over 5000. The concentrated retentate was freeze-dried in a Lyolab II freeze-dryer (Life Science Labora- tories). Cells from the 150-l reactor were removed using a Millipore tangential flow filter (0.45 pm) followed by filtration through an Amicon 10000 Dalton filter prior to freeze-drying.

The freeze-dried extracellular products were dissolved in 0.1 M citrate buffer (pH 5.5) at concentrations ranging from 0.1 to 2% (m/V> and tested for emulsification properties using a standard method developed for food emulsifiers (James and Patek 1988). The aqueous solutions were combined with corn oil in a 60:40 ratio (by weight) in a sealed, air-free, 20-ml screw-capped jar and were homogenized using a Janke and Kunkel Ultraturrax mixer. The mixture of oil and water was homogenized for 1 min at 15000 rpm. The optical density of a 1:250 aqueous dilution of

R. Shepherd et al. /Journal of Biotechnology 40 (I 99s) 207-217 211

the emulsion at 500 nm was defined as emulsification activity. A sample of the emulsion was stored vertically in a syringe for 30 min at room temperature. The optical density of a 1:250 dilution of the lower phase of the stored sample was measured. Emulsification stability was defined as the percentage optical density remaining after 30 min of storage. The errors in the data for emulsification activity were 5 f 0.05 units and f5% for emulsion stability. Gum arabic and carboxymethylcellulose were used as positive controls. Nega- tive controls were kappa-carrageenan and low methoxyl pectin.

Several samples of microbial polysaccharides with potential emulsifying properties were obtained from external sources for inclusion in the screening exercise. Emulsan from Acinetobacter calcoaceticus was obtained from Petroferm, USA; a rhamnose-rich polysaccharide from Kkbsiella spp. was obtained from BioEurope, France; and polysaccharides from the supernatant of Por- phiridium cruentum and Spin&a spp. moist cake were obtained from Photobioreactors Ltd., UK.

2.3. Analytical techniques

Cell-free supematants of selected cultures were analyzed chemically. Ash content was determined gravimetrically following heat treatment at 450- 550°C for 3 h. Kjeldahl nitrogen was determined according to the standard IS0 3593 method (AOAC, 1984). Glucose was determined using Boehringer Mannheim GmbH Diagnostica Test combinations. Protein determinations were carried out using the Pierce Protein Assay Kit (mi- cromethod), described by Bradford (1976). Stan- dard curves were prepared using bovine serum albumin (O-1000 pg ml-‘). Total carbohydrate was determined using the phenol/sulfuric acid assay described by Dubois et al. (1956) with glucose as standard.

The viscosity of a single sample of extracellular product from C. utilis (1% m/V> was determined using a CarriMed 100 Controlled Stress Rheome- ter fitted with a double concentric cylinder (5 cm). The viscosity was measured at 25“ C by ap- plying a gradually increasing tangential stress.

2.4. Determination of type of emulsion formed

Two dyes were used: Methyl orange (water soluble) and Sudan III (oil soluble). Droplets of emulsion (prepared as described in section 2.2) were mixed separately with droplets of each dye on microscope slides. If the emulsion was water- in-oil, Sudan III would colour the emulsion, whereas if the emulsion was oil-in-water, Methyl orange would colour the emulsion.

2.5. Preparation of salad cream

The salad cream formulation consisted of sun- flower oil (40%), water (40.3%), spirit vinegar (lo%), whole dried egg (4%), sugar (2%), salt (2%), mustard flour (l%), Mayodan (a mixture of guar and xanthan gums, Grindsted Ltd., 0.2%), Ultratex instant starch (National Starch and Chemical Co. Ltd., 0.5%), and the freeze-dried extracellular products from C. utilis (0.2/0.8%). The dried ingredients were mixed with water in a Silverson homogenizer on medium speed. The vinegar was added when the dried ingredients were dispersed and the oil was gradually incorporated into the mixture. The finished salad creams were stored at 4” C for 1 week prior to informal assessment of appearance.

3. Results and discussion

Prior to the main screening exercise, 100 microorganisms selected from the literature on the basis of their potential for lipophilic polysaccharide production were evaluated for their ability to produce mucoid colonies on agar plates and to emulsify corn and rapeseed oil in liquid media. The criteria used for the selection of the 100 organisms included reported ability to produce mucoid colonies, ability to utilise or degrade hydrocarbons and/or isolation from an oil-rich substrate. The reported ability to produce polysaccharides rich in 6-deoxyhexoses (Crow, 1988; Graber et al., 1988) was also considered a good indication of lipophilic polysaccharide production. Organisms known to cause food poisoning

212 R. Shepherd et al. /Journal of Biotechnology 40 (I 995) 207-217

and those associated with poor hygiene in food processing plants were deliberately excluded from this study. Most of the 100 organisms selected produced mucoid colonies but 20 strains were judged qualitatively, on the basis of colony mu- cosity, to be exceptional polysaccharide producers. Of the four carbon sources tested (glucose, sucrose, lactose and pyruvate), glucose stimulated the production of the most mucoid colonies by all organisms tested except for four yeasts, which produced the most mucoid colonies on sucrose and, in the case of one yeast strain, on lactose (Table 1).

The main screening exercise was carried out using the 20 organisms identified above and four external samples donated by other laboratories, as shown in Table 1. Control samples, prepared using sterile media processed in the same way as the microbial cultures, showed no emulsification activity. An emulsification activity reading of less than 0.1 OD units was obtained when buffer alone with no emulsifiers added was used to prepare the emulsion. Of the total of 24 microbial, extracellular, high molecular weight products tested, seven showed no emulsification activity although the carbohydrate content ranged from 48% for Propionibacterium freundenreichii ss. shermanii to 78% for Candida kefir. The lack of emulsification activity exhibited by the sample from Cundidu tropicalis was in contrast to the emulsifying characteristics reported for this organism by Kappeli and Fiechter (1977). However, these authors studied a polysaccharide-fatty acid complex that was covalently bound to the cell surface of this yeast and was inducible by growth on hydrocarbons. In this study, the yeast was grown on a hydrophilic substrate and the cells were removed from the spent medium prior to testing for emulsifying properties.

Nine organisms produced extracellular emulsifiers that were as good as the positive controls gum arabic and carboxymethylcellulose and their emulsification activity was at or between 0.35 and 0.70 OD units. Amongst these were the products from Beijerinckia indica ss. lacticogenes, a known producer of indican, a polysaccharide with lipophilic properties (Lawson and Symes, 1981; Symes, 1982). Indican owes its lipophilic proper-

ties to a combination of structural features that include a linear, highly acetylated (15-20%) backbone and a high content of the deoxysugar L-rhamnose. These structural features are thought to promote apolar self-association and combine to give viscous, thixotropic solutions at concentrations of 0.5% and above. Indican has been shown to lower surface and interfacial tension and im- part stability to oil-in-water emulsions by the formation of interfacial films (Symes, 1982). In this study, the extracellular products of this organism had an emulsification activity of 0.42 OD units and a stability of 90% but a carbohydrate content of only 30%. The relatively low yield of carbohydrate from the cell-free supernatant of this organism may be explained by the known tendency of indican to bind to the bacterial cap- sule (C. Lawson, personal communication). Therefore, it is possible that some of the indican produced in this study had been removed together with the whole cells during sample processing. Difficulties associated with the separation and extraction of indican from the medium have been considered as one of the principal causes for the lack of further commercial development of indican as an industrial polysaccharide (C. Lawson, personal communication).

Eight of the 24 organisms screened produced extracellular, high molecular weight materials with emulsification activities greater than 0.70 OD units at a concentration of 0.5% (m/V) and were therefore judged to be better emulsifiers than the food emulsifiers gum arabic and carboxymethylcellulose. The highest emulsification activity and stability were recorded for emulsan, the bioemulsifier from Acinetobacter calcoaceticus. Emulsan has been characterized as a het- eropolysaccharide rich in N-acetylgalactosamine and uranic acids, containing lo-15% of its dry weight as fatty acids, covalently linked to the polysaccharide via ester linkages (Zuckerberg et al., 1979; Shabtai and Gutnick, 1985).

Emulsification activities superior to those of the positive controls were also obtained for the extracellular products of the yeasts Rhodospirid- ium diobovatum, Rhodotorula graminis, Hansen&a anomala, Candida valida and Candida utilis, the red alga Porphiridium cruentum and the


bacterium Klebsiefla spp. The polysaccharide from Klebsiella spp. was, at the time of this study, under development by the company BioEurope, France, as a source of rhamnose for the manufac- ture of furaneol, a flavour precursor. Many algae, including P. cruentum, are well-known producers of polysaccharides and some of these are used widely as thickeners and viscosifiers in food products (Yalcin et al., 1994). It was therefore not surprising to find that the sample from P. cruen- turn tested in this study showed excellent emulsion stabilizing properties. All of the microbial emulsifiers showing superior emulsifying activity, except R. graminis and C. valida, also showed excellent stabilising properties (> 90% at 0.5% m/V>. The carbohydrate content of the bioemulsifier from C. utilis was highest at 98%. For this reason, and due to its long history of safe human consumption, C. utilis was chosen for further study (Scrimshaw, 1975; Solomons, 1983).

Larger quantities of the bioemulsifier from C. utilis were prepared using 10-l and 150-l reactors to allow for more detailed functional and chemical analyses to be carried out. Figs. 1 and 2 show the emulsififcation activity and stability, respectively, of the extracellular products from C. utilis over a range of concentrations and grown under different conditions (shake-flask, 10-l and 150-l reactors). The results show that both emulsification activity and stability of the C. utifis bioemulsifier were better than those of the food emulsifiers gum arabic and carboxymethylcellulose, irre- spective of growth conditions. The improvement in performance was particularly evident at the lower concentrations of emulsifiers tested (0.1, 0.2 and 0.5%). However, the results of chemical analyses showed that the composition of the preparations from the three types of culture was somewhat variable as shown in Table 2, suggest- ing that stricter control of some of the growth parameters may be required to achieve good re- producibility. Thus, carbohydrate content varied from 79 to 98%. Residual glucose was absent from all samples. Protein content determined by the Pierce method failed to confirm calculated protein values based on Kjeldahl nitrogen, sug- gesting that much of the nitrogen may have been present in the form of amino sugars rather than

Actlvlty (00 units)

2

1.75

1.5

1.25

1

0.75

0.5

0.25

.__

SQ-

60 -

70 -

60-

50-

40 -

30 ’ I,,,I,,,/,#, ,,I

0 0.2 0.4 0.6 0.6 1 1.2 1.4 1.6 1.6 2 Concmtmtion(X,w)

Fig. 1. Emulsification activity of extracellular products from C. utilis. Cells were grown in a shake-flask (+), a 10-l reactor (0) and a 150-l reactor (*I. Curves for the positive standards gum arabic (0) and carboxymethylcellulose t 0) and the negative standards kappa-carrageenan (01 and low methoxyl pectin ( * ) are also shown.

Fig. 2. Emulsification stability of extracellular products from C. utilis. Cells were grown in a shake-flask (+I, a 10-I reactor (0) and a 150-l reactor (*). Curves for the positive standards gum arabic (0) and carboxymethylcellulose (0) are also shown.

protein. The presence of proteinaceous material not covalently bound to a polysaccharide-based bioemulsifier would lead to erroneous conclusions about the emulsification properties of that polysaccharide as proteins are known for their surfactancy (Pate1 and Fry, 1987). The concentration of bioemulsifier varied from 0.26 to 0.93 g


1-l. These results indicated that optimization of the growth conditions used for the organism was required to ensure consistent activity, stability, composition and concentration of the bioemulsifier. Studies on the optimization of process conditions for C. utilis will be reported separately.

The surface tension (in water) and interfacial tension (against hexadecane) of a single sample of the C. utilis bioemulsifier (0.1%) were 51.1 and 17.3 mN m-l, respectively (courtesy of D. Byrom, Zeneca). This compares favorably with the reduction in the surface tension of water (51.6 mN m-i) reported in the presence of the same concentration of Tween 80 (Martin0 et al., 1991). The sophorolipids from Torulopsis bombicola have been shown to reduce surface and interfacial tension; however, unlike the bioemulsifier from C. utilis, the sophorolipids had been shown to be poor emulsifiers (Cooper and Pad- dock, 1984). By contrast, the extracellular bioemulsifier liposan from C. lipolytica, consisting of 83% carbohydrate and 17% protein, failed to reduce the surface tension of water (72.8 mN m-l) although it emulsified and stabilized oil-in- water emulsions effectively (Cirigliano and Car- man, 1985).

Water-soluble Methyl orange rather than the oil-soluble Sudan III dye was incorporated into an emulsion formed with 1% (m/V) of the bioemulsifier from C. utilis, showing that this product promoted the formation of oil-in-water emulsions.

The viscosity of a 1% (m/V) solution of the bioemulsifier from C. utilis was low (0.125 N me2

Table 2

Composition and properties of extracellular products from C. uti1i.s

Property/composition l-l flask

Emulsification activity (0.5% m/V) 1.14

Emulsification stability (0.5% m/V) 91

Carbohydrate (%) 98

Kjeldahl nitrogen (%I 3.8 Calculated protein content (N X 6.25) 23.8 Protein content by Pierce method (%) ND Moisture (%I ND

Ash (%) ND

Concentration of product (g I ‘) 0.73

s) and comparable to that of gum arabic (0.132 N mm2 s at 1.22% m/V> as reported by Taft and Malm (1931). Like gum arabic, the new bioemulsifier formed solutions that were Newtonian in character. Gum arabic is a unique plant polysaccharide in that it has excellent emulsifying properties and despite its relatively high molecular weight (approx. 400000) gives solutions of very low viscosity. The component responsible for the gum’s emulsifying ability has recently been shown to consist of arabinogalactan blocks of molecular weight 200 000 linked together by a main polypeptide chain. The low viscosity has been ascribed to the highly branched, globular nature of the arabinogalactan/protein complex (Wil- liams et al., 1990). It can be speculated that the low viscosity of the bioemulsifier from C. utilis may also have been due to a highly branched, globular structure.

Ogawa et al. (1978, 1988, 1990) have identified and structurally characterized a glucomannan as the principal component of the cell walls in C. utilis. The glucomannan consisted of an a-(1,6)- linked o-mannosyl backbone partially substituted with side chains of one, two, three or four o-man- nosy1 units connected by a-(1,2) linkages; each repeating unit had an additional side chain in which o-glucose residues were linked through a-(1,6) linkage to the nonreducing ends of each of four o-mannosyl units. Although it is possible that the extracellular bioemulsifier from C. utilis identified in our laboratory was a component of the cell wall secreted by the organism, it is un- likely that a pure glucomannan would have

10-I reactor 150~litre reactor

1.10 0.91 89 64

83 79

3.9 2.3 24.4 14.4

4.9 2.2 5.8 8.1 9.7 5.4 0.93 0.26

ND, not determined.

R Shepherd et al. /Journal of Biotechnology 40 (1995) 207-217 215

demonstrated such strong emulsification properties. The substantial nitrogenous component of the crude bioemulsifier may indicate that the emulsifying properties were due to the presence of aminosugars.

Salad creams prepared using 0.2% and 0.8% m/V of the bioemulsifier from C. utilis were shown to separate to a lesser extent than controls prepared with reduced amounts of egg and no stabiliser (guar and xanthan gums) when stored at 4“ C for 1 week (Table 3). Further optimization of bioemulsifier concentrations and other compo- nents of the formulation would be required to achieve a satisfactory product.

Few food-grade microorganisms have been reported to produce bioemulsifiers. Recently, Buss- cher et al. (1994) have reported biosurfactant production by several strains of Streptococcus thermorphilus isolated from biofilms formed on heat exchanger plates in a milk pasteuriser. Ex- cellent emulsification and whipping properties have been claimed for extracts prepared from whole cells of C. utilis; however, all these preparations have been reported to contain at least 50% or more protein (Anheuser-Busch, 1976; Schachtel, 1981a,b; Schnell et al., 1976). There- fore, this report of the production of an extracellular, carbohydrate-based bioemulsifier by the edible C. utilis is novel. For many industrial applications, such as in foods, a pure polysaccharide preparation is not necessary provided the organism is inactivated (by pasteurisation, for example).

Table 3 Assessment of salad creams containing C. utilk bioemulsifier

Several microbial products now used in foods such as xanthan gum and nisin are not purified to any great extent. Therefore, a crude bioemulsifier from a culture of C. utilis may provide a proms- ing new focus for further investigation as a novel, functional food ingredient.

4. Conclusions

In this study, several microorganisms have been identified as good producers of extracellular bioemulsifiers with potential for exploitation by the food industry. Of the 24 extracellular, polymeric bioemulsifiers tested, eight had demonstrated emulsification activity that was better than that of the known food emulsifiers gum arabic and carboxymethylcellulose. The good producer organisms included the yeasts C. utilis, C. valida, H. anomala, R diobovatum, R. graminis, the red alga P. cruentum, and the bacteria Klebsiella spp.

and A. calcoaceticus. The emulsification properties of the extracellu-

lar, polymeric compounds from C. utilis have been shown to be sufficiently promising to war- rant further study. The novel bioemulsifier may allow direct replacement of some chemically syn- thesized emulsifiers currently used in the food industry as well as providing opportunities for creating novel texture modifications currently not achievable with conventional emulsifiers. Fur- thermore, since C. utilis has a history of human

Sample

Positive control: (4% m/V egg, 0.2% m/V Mayodan “1

Negative control: (1% m/V egg, no Mayodan “1

Experimental 1: 0.2% m/V C. utihk bioemulsifier (1% m/V egg, no Mayodan “)

Experimental 2: 0.8% m/V C. utilk bioemulsifier (1% m/V egg, no Mayodan “)

Appearance of salad creams after 1 week at 4” C

Creamy yellow in colour. Thick and smooth with no separation into layers. Formed soft peaks, which quickly disappeared. Barely spoonable. Yellow in colour. Very thin and slightly lumpy. About 30% separated out as an aqueous, bottom layer. Some creaming. Creamy-yellow in &our. About 20% separated out as an aqueous, bottom layer. Top layer was thick and spoonable rather than pourable. Creamy-yellow in colour. About 10% separated out as an aqueous, bottom layer. Top layer was thicker than the positive control or Experimental 1.

a Mayodan = commercial stabilizer consisting of guar and xanthan gum.


consumption without reported adverse effects (Scrimshaw, 1975; Solomons, 19831, legislative ap- proval for use of a product from it as a food additive may be expedited. Chemical characteri- sation of the novel bioemulsifer, determination of functional properties at a range of temperatures and ionic concentrations, evaluation in a range of food products and optimization of yields would need to be undertaken before a final conclusion about commercial potential could be drawn.

Acknowledgements

We are indebted to K. Aitken and A. Jarmuz for their technical assistance. We would also like to thank the Ministry of Agriculture, Fisheries and Food (UK), CPC International, Heinz, Kraft General Foods, Tate and Lyle, Unilever and Zeneca for sponsoring this work.

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