Improving the viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in white-brined cheese by microencapsulation

International Dairy Journal 19 (2009) 22–29

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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj

Improving the viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in white-brined cheese by microencapsulation ¨ zer a, *, Hu¨seyin Avni Kirmaci a, Ebru S¸enel b, Metin Atamer b, Adnan Hayalog˘lu c Barbaros O a

Department of Food Engineering, Faculty of Agriculture, Harran University, Eyyubiye Kampusu, Sanliurfa 63040, Turkey Department of Dairy Technology, Faculty of Agriculture, Ankara University, Ankara 06100, Turkey c Department of Food Engineering, Faculty of Engineering, Inonu University, Malatya, Turkey b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 May 2008 Received in revised form 4 July 2008 Accepted 15 July 2008

The viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 microencapsulated by either an extrusion or an emulsion technique and used in white-brined cheese was monitored. Both microencapsulation techniques were effective in keeping the numbers of probiotic bacteria higher than the level of the therapeutic minimum (>107 cfu g1). While the counts of probiotic bacteria decreased approximately 3 log in the control cheese in which probiotics were used as free cells, the decrease was more limited in the cheeses containing microencapsulated cells (approximately 1 log). Medium- and long-chain free fatty acid contents of the cheeses with immobilized probiotics were much higher than in the control cheese. Similarly, cheeses made with immobilized probiotics contained higher acetaldehyde and diacetyl levels than the control. Experimental cheeses containing microencapsulated probiotics were not different from the control cheese in terms of sensory properties. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Dairy products have a place in delivering probiotic bacteria to the human gut, as these products provide probiotic bacteria with a suitable environment in which their growth and viability are promoted (Ross, Fitzgerald, Collins, & Stanton, 2002). During the past two decades, significant attention has been paid to fermented dairy products containing probiotic bacteria originating from the human intestine (Lee, Nomoto, Salminen, & Gorbach, 1999). The viability of probiotic bacteria in fermented dairy products (Adhikari, Mustapha, Gruen, & Fernando, 2000; Dave & Shah, 1997; ¨ zer, Akın, & O ¨ zer, 2005; Sun & Griffiths, Hussein & Kebary, 1999; O 2000), the physical, chemical and sensory properties of such products (Davidson, Duncan, Hackney, Eigel, & Boling, 2000; Prabha & Shankar, 1997; Rybka & Kailasapathy, 1997) and the probiotic effect (Fooks, Fuller, & Gibson, 1999; Sanders & Klaenhammer, 2001) have been well documented. However, probiotics may show rather low viability in yoghurt and other fermented milk products due to their acidic nature (Gardiner et al., 1999; Vinderola, Prosello, Ghiberto, & Reinheimer, 2000). Because of its higher pH, fat content and more solid consistency, cheese offers certain advantages over fermented milk products in terms of delivering viable probiotics to the human gut and, therefore, has been considered to be an ideal vehicle for probiotic uptake. * Corresponding author. Tel.: þ90 414 247 4195; fax: þ90 414 247 4480. ¨ zer). E-mail address: [email protected] (B. O 0958-6946/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2008.07.001

The protein matrix formed during cheese coagulation affords added protection to the acid-sensitive probiotics both in the cheese and in the gastrointestinal tract (Corbo, Albenzio, de Angelis, Sevi, & Gobbetti, 2001; Vinderola, Mocchiutti, & Reinheimer, 2002). Additionally, the oxygen level of cheese is remarkably reduced by the cheese microorganisms within a few weeks of storage, which provides an almost anaerobic environment favoring the survival of bifidobacteria. It is a well-established fact that although highly strain dependent, the viability of probiotic strains in cheese is restricted by the ¨ zer, & Atasoy, 2004). Gobbetti, presence of salt (Yılmaztekin, O Corsetti, Smacchi, Zocchetti, and de Angelis (1998) claimed that the viability of probiotic strains is hindered considerably when the salt level in cheese exceeds the upper limit of 4 g 100 g1 of cheese. While the viability of probiotics in dry salted hard or semi-hard cheese varieties (e.g., Cheddar) has been more widely studied, fairly limited data are available on the probiotic white-brined cheeses (Ghodussi & Robinson, 1996; Yılmaztekin et al., 2004). Salt is the major limiting factor for the growth of probiotics in white-brined cheeses. Gomes, Malcata, Klaver, and Grande (1995) proposed relatively high inocula of probiotic strains in Gouda cheese to attain a satisfactory level of probiotic bacteria in the final product at the time of consumption. There are a number of alternatives employed in protection of probiotic strains against undesirable environmental conditions. These include cell incubation under sub-lethal conditions; cell propagation in an immobilized biofilm; and microencapsulation.

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Microencapsulation seems to be the most promising technique for bacterial protection (Krasaekoopt, Bhandari, & Deeth, 2003). Immobilizing cells in a hydrocolloid bead matrix resulted in improved protection of probiotic bacteria against environmental conditions (Picot & Lacroix, 2004). Goderska, Zybals, and Czarnecki (2003) showed that while Lactobacillus rhamnosus colonies microencapsulated in alginate matrix kept their viability up to 48 h at pH 2.0, free cells were inactivated completely under the same conditions. Similarly, increasing the alginate concentration led to an increase in the colony counts of Bifidobacterium longum (Lee & Heo, 2000). Encapsulation by extrusion and emulsion techniques has been used for the protection of probiotic bacteria against adverse environmental conditions (Doleyres & Lacroix, 2005; Jankowski, Zielinska, & Wysakowska, 1997; Kebary, Hussein, & Badawi, 1998). In addition to the increased protection of microbial viability after microencapsulation, the chemical and physical properties of the cheeses containing probiotic bacteria in the encapsulated form may ¨ zer, Uzun, and Kirmaci (2008) demonstrated be affected as well. O that microencapsulation did not affect the basic composition of Kasar cheese; however, the authors noted that development of proteolysis was more pronounced in the cheeses containing probiotic bacteria in the encapsulated form. It was demonstrated that the addition of probiotic cultures, either in the free or encapsulated states, did not seem to significantly affect textural parameters such as springiness and cohesiveness of 7-week-old Feta cheese (Kailasapathy & Masondole, 2005). This study monitored the viability of microencapsulated Lb. acidophilus LA-5 and Ba. bifidum BB-12 in white-brined Turkish cheese. Two different techniques of microencapsulation, namely extrusion and emulsion, were examined. Microbiological, biochemical and sensory properties of the cheeses were monitored throughout 90 days of storage at 4  C.

into sample A at a level high enough to attain 5  109 cfu mL1 in cheese milk. For addition to the samples B and C, Lb. acidophilus LA5 and B. bifidum BB-12 were grown separately at 37  C for 24 h and then microencapsulated (see Section 2.3.). After microencapsulation, the counts of probiotic cells inoculated into the milk were approximately 6  1010 cfu mL1. The inoculated milk was then rested for an hour to allow acidity development and was coagulated by means of calf rennet within 90 min. The coagulated milk was then cut into pieces of 0.5–1.0 cm3 and whey was drained by pressing. Following the removal of whey, fresh cheese was portioned to blocks (7  7  7 cm) and stored in a pasteurized brine solution (12% NaCl, w/v) at 4  C for 90 days. Cheese samples for analyses were taken at days 1, 15, 30, 60 and 90 of storage.

2. Materials and methods

2.3.2. Emulsion technique Probiotic cells were harvested from 80 mL of a 24 h culture (late log phase) by centrifugation at 5000  g. The cells were washed twice with 20 mL of sterile saline solution and resuspended in 10 mL of sterile saline. Afterwards, the cell suspension (20 mL) was added to 2.0 % (w/v) k-carragenan (60 mL, supporting material), containing 0.9% NaCl to improve the dispersability of the k-carragenan, and tempered in a water bath at 47  C. The continuous phase and the emulsifying agent were corn oil (100 mL, Crisco, Interlab A.S.) and Tween-80 (0.1%, v/v) (MERCK, Teknik Kimya A.S., Istanbul, Turkey), respectively. The mixture of corn oil and Tween-80 was stirred using a Calfarmo stirrer at the lowest speed at 40  C for 2– 3 min. The capsule formation was achieved by adding the cell suspension/k-carragenan mixture quickly into the corn oil/Tween80 mixture. To break the emulsion, 1 M CaCl2 (150 mL) was added. The oil phase was removed and the capsules containing bacterial cells were separated from the CaCl2 solution by centrifuging at 350  g for 10 min. The capsules were 0.3–0.4 mm in diameter. The capsules were washed twice with sterile saline solution and stored at 4  C until use.

2.1. Materials Raw bovine milk supplied from Harran University Dairy (Sanliurfa, Turkey) was used. Rennet of calf origin was used to coagulate milk in liquid form (coagulating power 1:16,000 IU, Mayasan A.S., Yenibosna, Istanbul, Turkey). Mesophilic cheese starters (a blend of Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris, Code O113) and probiotic strains (B. bifidum BB-12 and Lb. acidophilus LA-5) were obtained from Peyma Chr. Hansen (Gayrettepe, Istanbul, Turkey). All chemicals and the alginate were obtained from Sigma-Aldrich Co. (Interlab A.S., Istanbul, Turkey) and were of analytical grade. 2.2. Cheese-making protocol A batch of raw bovine milk (120 L) was divided into three parts. The first part (Sample A) was inoculated with mesophilic cheese starters and probiotic strains as adjunct culture. Cheese starters plus probiotic strains microencapsulated by extrusion and emulsion techniques were added to the second (Sample B) and third (Sample C) batches, respectively. Cheese making was carried out at pilot plant scale, adapting industrial technology for white-brined Turkish cheese. All batches were converted to cheese at the same time. The milk was heattreated at 72  C for 15 min and cooled to the coagulating temperature (32  C). To restore the ionic calcium balance after heat treatment, calcium chloride was added as a solution (40%, w/v) at a level of 0.02% (v/v). A mixed-strain inoculum (1.0%, v/v) of the cheese starters (Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris) was added to each of the vats. Probiotic strains were incorporated

2.3. Cell microencapsulation Cell microencapsulation was achieved using the two techniques described earlier (Krasaekoopt et al., 2003). 2.3.1. Extrusion technique A probiotic cell suspension was prepared by centrifuging 80 mL of a 24 h culture at 5000  g, as described by Adhikari et al. (2000). Following centrifugation, the cells were washed twice with saline solution (20 mL). Alginate of algal origin was chosen as the supporting material and 2.0% Na-alginate concentration was selected after preliminary studies. The washed probiotic cells were mixed with a Na-alginate solution to form capsules. The mixture of cell suspension and Na-alginate was dripped into a 1 M CaCl2 solution with a sterile syringe. The droplets formed gel spheres instantaneously, entrapping the cells in a three-dimensional lattice of ionically cross-linked alginates. The distance between the syringe and CaCl2 solution was 20 cm, yielding capsules 0.2–0.3 mm in diameter.

2.4. Determination of aroma compounds To determine the aroma compounds of cheese samples, an Agilent Model 6890 Series GC System plus gas chromatograph (Agilent Technologies, Inc., Palo Alto, CA, USA) fitted with an FID detector was used. Volatiles were separated with a capillary column 30 m  320 mm id (HP Innovax Polyethylene glycol, Model Agilent 19091N-13). Nominal film thickness for the column was 0.25 mm. Operating conditions for the gas chromatography (GC) analyses were as follows: column temperature, 50  C; injector temperature, 260  C; FID temperature, 250  C; make-up gas,

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nitrogen; flow rate for make-up gas, 30 mL min1; hydrogen flow rate, 40 mL min1; splitless mode. The temperature of the gas-tight syringe was maintained at 80  C. Vials with a nominal capacity of 10 mL were used for the GC analyses. All glass materials were sterilized before use. Doubly distilled water used for the preparation of standard solutions was boiled for 20 min to remove residual volatiles and subsequently stored in a stoppered glass container. Quantification of the constituents was achieved by means of a computing integrator operated in either external standard or internal standard mode.

2.8. Statistical analyses The data obtained were analyzed with one-way analysis of variance. Multivariate statistical methods were used to evaluate the effectiveness of the microencapsulation techniques. Principal component analysis (PCA) was performed using the covariance matrix and varimax rotation; hierarchical cluster analysis (HCA) was performed using Euclidean distance and average linkage without standardizing the variables. The SPSSÒ package program (version 9.0, SPSS Inc., Chicago, IL, USA) was used. The entire study including cheese making was repeated three times.

2.5. Determination of free fatty acids 3. Results and discussion

A pour plate method was employed for the determination of microbial groups. A quarter strength Ringer’s solution (Oxoid, Basingstoke, UK) was used in the preparation of serial dilutions. The cheese samples were homogenized by a Stomacher device (Stomacher 400, Seward Medical, West Sussex, UK) to release the entrapped probiotic cells from the microcapsules completely. MRS-D-Sorbitol agar was used in the count of Lb. acidophilus LA-5 (Dave & Shah, 1997). As the heat treatment caused deformation in the structure of D-sorbitol, membrane sterilization was employed. D-sorbitol (10 mL solution of 10%, w/v) was added into 90 mL of sterilized MRS agar. MRS agar containing a membrane sterilized mixture of nalidixic acid (50 mg L1), neomycin sulphate (100 mg L1), lithium chloride (3 g L1) and paramomycin sulphate (200 mg L1), added at a level of 20–80 mL MRS agar (MRS-NNLP; Dave & Shah, 1997), was used for the count of B. bifidum BB-12. Both MRS-D-Sorbitol and MRS-NNLP agars were incubated at 37  C for 72 h. 2.7. Sensory analyses Sensory evaluation of the cheeses was carried out according to the method proposed by Bodyfelt, Tobias, and Trout (1988). The panel group consisted of ten experienced panelists who were familiar with white-brined cheeses. Sensory evaluation and overall acceptability were both based on 10-point hedonic scales (1, dislike extremely; 10, like extremely). Each cheese was scored individually. Samples were presented to the panelists in individual plastic coded containers. Cheeses were randomly presented to the panel group at each session. Panelists were seated in individual booths, and samples and ballots were passed through from an adjoining preparation room. Water was used for mouth rinsing between samples. Three samples were presented to the panel group at each session and each cheese was evaluated in triplicate.

Lb. acidophilus LA-5 and B. bifidum BB-12 were added as adjunct strains during white-brined cheese manufacture in free (sample A) or microencapsulated (samples B and C) form into the vats. Variations in the counts of B. bifidum BB-12 and Lb. acidophilus LA-5 during the ripening period are shown in Fig. 1a and b, respectively. After 1 day of manufacture, the number of B. bifidum BB-12 colonies was present at log10 8.37 cfu g1 in the control cheese (sample A), decreasing continuously during ripening to log10 5.77 cfu g1 at day 90 (a reduction of approximately 31%). This was expected as the relatively low pH and high salt level in white-brined cheese varieties limit the growth of acid and salt sensitive probiotic strains when used as free cells (Ghodussi & Robinson, 1996; Yılmaztekin et al., 2004). In contrast, the counts of B. bifidum BB-12 in samples B and C showed a slight decline during the first 15 days of storage coinciding with the rapid salt penetration from brine to cheese blocks at this period (data not shown). The numbers of B. bifidum BB-12 in these samples remained relatively constant throughout ripening, reaching levels of log10 8.55 and 8.04 cfu g1 at day 90, respectively (reductions of approximately 9% and 15%, respectively).

A Log cfu g-1 cheese

2.6. Microbiological studies

3.1. Cheese microbiology

10 8 6 4 2 0

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Total and individual free fatty acids (FFAs) were determined by the method of Deeth, Fitzgerald, and Fron (1983). Cheese (5 g) was ground with 2.5 g of Na2SO4 and then, 5 mL of an internal standard (C7) and 300 mL H2SO4 were added. The mixture was mixed thoroughly for 1 min and hexane (5 mL) was added. Samples were rested for 1 h before the liquid phase was removed and mixed with 2 mL of 6% solution of formic acid/ether mixture. This mixture was centrifuged at 2000  g for 10 min. The clear part was transferred into the vials and the vials were stored at 18  C until use. The volume of the cheese samples analyzed was 5 mL. The chromatography system used consisted of an Agilent Model 6890 instrument fitted with an FID detector. The column used was an Agilent-FFAP capillary 300  250 mm  0.25 mm. The conditions of the determination were as follows: injection temperature, 250  C; split, 1/10; flow rate of the sample, 2 mL min1; flow rate of H2, air and make-up gas, 33 mL min1, 30 mL min1 and 30 mL min1, respectively.

12 10 8 6 4 2 0

1

15

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Ripening time (days) Fig. 1. Variation in the counts of (A) Bifidobacterium bifidum BB-12 and (B) Lactobacillus acidophilus LA-5 in the cheeses throughout 90 days of storage (n ¼ 3): B, sample A (control, containing probiotic bacteria in free state); ,, sample B (containing probiotic bacteria microencapsulated by extrusion technique); 6, sample C (containing probiotic bacteria microencapsulated by emulsion technique). Values represent means of (SD) of duplicate analyses of three cheeses from trials 1–3.

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by calcium alginate polymers containing starch were susceptible to disintegration in Feta cheese ripened in brine. On the other hand, it should be borne in mind that apart from slow disintegration of microcapsules during ripening, the salt might penetrate into the beads and affect the viability of probiotic bacteria.

Similar to the case with B. bifidum colonies, the number of Lb. acidophilus LA-5 colonies decreased in all samples, more remarkably in sample A. At day 1, the numbers of Lb. acidophilus LA-5 in the samples A, B and C were log10 8.97, 10.41 and 10.26 cfu g1, respectively. These figures reached log10 6.69, 9.33 and 8.74 cfu g1 after 90 days of storage, respectively (reductions of approximately 25%, 16% and 14%, respectively). In general, B. bifidum and B. longum are among the strains shown to demonstrate good suitability through the processing and storage of cheese (Bolyston, Vinderola, Ghoddusi, & Reinheimer, 2004). Both microencapsulation techniques were efficient enough to keep the number of probiotic bacteria above the threshold level for therapeutic minimum (107 cfu g1 of cheese). These findings were in good agreement with previous studies carried out on various probiotic-containing foods including yoghurt (Adhikari et al., 2000; Khalida et al., 2000), frozen dairy desserts (Kebary et al., 1998) and mayonnaise (Khalil & Mansour, 1998). However, in contrast, Godward & Kailasapathy (2003) claimed that microencapsulation of probiotic cells in Feta cheese caused higher cell loss, either by preventing encapsulated bacteria from interacting with the environment for survival or inhibiting disposal of cell metabolites that may be accumulating inside the encapsulated capsules causing death. Similar conclusions were drawn by Godward (2000) for Cheddar cheese, the dense matrix of which was claimed not to be conducive for free exchange of metabolites and nutrients to and from the entrapped capsules. Here, the cheese type and microencapsulation technique were factors for viability of the bacterial cells. In the present study, the experimental cheese matrix is known to be more open than Cheddar and, to some extent, Feta cheese. One possible reason for the limited decline in the number of microencapsulated bacteria in samples B and C may be the slow disintegration of the microcapsules during ripening as a result of the effect of the salt in the brine. It may be possible that exchanging sodium ions with calcium ions binding alginate capsules together led to disintegration of the capsules, releasing probiotic bacteria into the medium. The difference between the counts of microencapsulated and non-encapsulated probiotic bacteria in the experimental cheeses may indicate that during time-dependent release of probiotic bacteria, a slight salt adaptation might have developed in the probiotic cells and the death of cells remained relatively limited. The role of sodium chloride in the disintegration mechanism of k-carragenan capsules is not clear and needs further investigation. Similar results were reported by Kailasapathy and Masondole (2005) who demonstrated that microcapsules formed 120

Concentration (mg kg-1)

100

25

3.2. Variations in FFAs The concentrations of the even-numbered FFAs in 90-day-old experimental cheeses are illustrated in Fig. 2. The concentrations of myristic (C14:0), palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids were the highest in the experimental cheeses. Throughout the ripening period, the concentrations of FFAs increased in all samples. The concentrations of short- and medium-chain FFAs (C4:0–C12:0) did not differ significantly between the cheeses containing microencapsulated cells (samples B and C) at each sampling age (P > 0.05), and were significantly higher than the cheese containing non-encapsulated cells (sample A). The levels of myristic (C14:0), palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids were significantly higher in samples B and C compared with sample A. Although a slight increase in the concentration of palmitic acid (C16:0) was noted in sample A during ripening, this FFA fraction increased considerably at day 90 in samples B and C. Similar results were recorded for stearic (C18:0) and oleic (C18:1) acids. Linoleic acid (C18:1) was identified only in samples B and C; the level of this FFA in sample A was at a trace level. Total levels of FFAs of samples A, B and C at day 1 were 39, 103 and 123 mg kg1, respectively. These figures increased to 55,289 and 270 mg kg1 at day 90, respectively. It could be assumed that FFAs contributed to the sensory properties of the cheeses in the present study. The higher total levels of FFAs in the cheeses containing microencapsulated probiotic cells indicated that the microcapsules containing probiotic cells disintegrated partially or wholly and the probiotic bacteria, released into the medium, showed lipolytic activity. Although both B. bifidum and Lb. acidophilus are known to have weak to medium lipolytic properties, the increase in the levels of FFAs in samples B and C could only be explained by the lipolytic and esterolytic actions of these bacteria. In contrast to our results, Gobbetti et al. (1998) found that B. bifidum showed a limited activity on long-chain FFAs in Crescenza cheese. Other researchers (Corbo et al., 2001) also demonstrated that incorporation of Bifidobacterium spp. into the hard or semihard Italian cheeses resulted in increases in the concentrations of butyric (C4:0), caproic (C6:0), capric (C10:0) and oleic (C18:1) acids.

Sample A Sample B Sample C

80 60 40 20 0 C4

C6

C8

C10

C12

C14

C16

C18:0

C18:1

C18:2

Free fatty acids (FFA) Fig. 2. Individual free fatty acids contents of 90-day-old cheeses: sample A, control, containing probiotic bacteria in the free state; sample B, containing probiotic bacteria microencapsulated by extrusion; sample C, containing probiotic bacteria microencapsulated by emulsion. Values represent means (SD) of duplicate analyses of three cheeses from trials 1–3.

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The FFAs results were compared applying PCA and HCA (Fig. 3). Both PCA and HCA showed that all the samples were divided into three groups on principal component 2 (PC2) and samples B and C were clearly differentiated from sample A. Sample A positively correlated with PC2 (cumulative variation of 16.5%). Samples B and C showed a separate group at day 90 (positive correlation with principal component 1 (PC1), cumulative variation 87.2%). PCA results were further justified by HCA which yielded three clusters on the basis of microencapsulation and storage (Fig. 3). With the exception of day 90, samples B and C grouped in the same cluster, and sample A (with all ages) grouped in one cluster. The third cluster was formed by the 90-day-old samples B and C. 3.3. Variations in the volatile compounds Some selected volatile compounds were analyzed in the cheeses at different sampling ages by GC. Quantitative differences in

acetaldehyde, acetone, diacetyl and ethanol (EtOH) contents were evident in the experimental cheeses (Fig. 4a–d). Acetaldehyde accumulation was fairly low in all cheeses during the first 2 weeks of ripening (Fig. 4a). Afterwards, the level of acetaldehyde in the samples A and C increased up to 2.0 mg kg1 and 2.5 mg kg1, respectively. At later stages of ripening, the accumulation of acetaldehyde in sample B was accelerated and reached the highest level at day 90 (4.1 mg kg1). As is known, both mesophilic and thermophilic lactic acid bacteria can produce acetaldehyde (Fox, Guinee, Cogan, & McSweeney, 2000). Glycine is a growth-promoter for mesophilic cheese starters (especially Lactococcus spp.) and is produced from threonine metabolism, yielding acetaldehyde as a by-product. Since the same mesophilic strains were used as cheese starters in the current study, the quantitative differences in acetaldehyde levels of the experimental cheeses could be due to the metabolic activities of probiotic bacteria incorporated into the manufacture of

Fig. 3. Classification of the experimental cheeses by (a) principal component analysis (PCA) and (b) hierarchical cluster analysis (HCA) on the basis of free fatty acids data obtained at different stages of ripening: B, sample A (control, containing probiotic bacteria in the free state); 6, sample B (containing probiotic bacteria microencapsulated by extrusion); ,, sample C (containing probiotic bacteria microencapsulated by emulsion).

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B

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Fig. 4. Variation in the carbonyl compounds (B, acetaldehyde; B, acetone; C, diacetyl; D, ethanol) of cheeses throughout ripening: A, sample A (containing probiotic bacteria in the free state); -, sample B (containing probiotic bacteria microencapsulated by extrusion technique); :, sample C (containing probiotic bacteria microencapsulated by emulsion technique). Values represent means (SD) of duplicate analyses of three cheeses from trials 1–3.

probiotic cheeses. It is a fact that acetaldehyde production capabilities of the probiotic bacteria are limited compared with thermophilic yogurt starters. Among the Bifidobacterium spp., B. bifidum strains are able to produce acetaldehyde at higher levels (Yuguchi, Hiramatsu, Doi, & Idachi, 1989). The microencapsulation of probiotic bacteria affected the acetaldehyde contents of the cheeses significantly (P < 0.05). It is thought that aldehyde dehydrogenase synthesised by B. bifidum BB-12 especially was released from the matrix during ripening and contributed to the acetaldehyde production in cheese. During the first 2 weeks of ripening, the acetone contents of the cheese samples did not differ significantly (Fig. 4b). In this period, sample B had a higher level of acetone than samples A and C (P < 0.05). The levels of acetone increased in all cheeses during ripening, more remarkably in sample A. At day 90, samples A, B and C had acetone contents of 8.5, 5.3 and 4.1 mg kg1, respectively. Acetone accumulation was found to be independent from microencapsulation of probiotic bacteria. Similarly, Sarantinopoulos, Kalantzopoulos, and Tsakalidou (2002) demonstrated that the acetone level of probiotic Feta cheese made by using Enterococcus faecalis increased slightly at the early stages of ripening and then remained almost unchanged during the rest of the storage period. Diacetyl was not detected in any of the samples at day 1 (Fig. 4c). After 2 weeks of manufacture, while samples B and C had diacetyl levels of 3.4 and 3.3 mg kg1, respectively, no diacetyl accumulation was detected in the sample A. At later stages of ripening, the levels of diacetyl increased in all the experimental cheeses. The accumulation of diacetyl increased significantly in samples B and C after 60 days of ripening. The diacetyl values of samples A, B and C at day 90 were 7.2, 19.9 and 27.8 mg kg1, respectively. Diacetyl, which gives a creamy flavour, is considered as an important contributor to the cheese flavour (Fox et al., 2000). However, excessive diacetyl accumulation in cheese may cause development of off-flavour. Ethanol (EtOH) was the most abundant of the volatile compounds in the cheeses at each sampling age (Fig. 4d). Sample A

containing non-encapsulated probiotic strains had higher levels of EtOH than the samples containing encapsulated probiotics. While the EtOH level of sample A increased continuously during ripening, the EtOH contents of samples B and C remained almost unchanged during the first 4 weeks of ripening. Microencapsulation techniques employed had no significant effect on the EtOH concentrations of the samples, except for 90-day-old cheeses. A major source of EtOH in cheese is from citrate metabolism by Lactococcus spp. In general, acid-producing mesophilic cheese starters have limited capability of producing EtOH (Baron, Roy, & Vuillemard, 2000). EtOH can also be produced through acetaldehyde degradation by alcohol dehydrogenase (Tamime & Robinson, 1999). Higher EtOH levels and lower acetaldehyde concentrations in sample A may indicate higher alcohol dehydrogenase activities in this sample. It has been reported that using Cit(þ) Lactococcus strains in combination with B. bifidum in cheese production caused increases in EtOH levels (Starrenburg & Hugenholtz, 1991). EtOH is of importance for the quality of cheese (especially for scalded varieties) (Gyosheva, Stefanova, & Bankova, 1986); however, to the best of our knowledge, no upper limits for EtOH have been established for commercial cheeses. Results of PCA of carbonyl compounds showed that the samples were divided into two distinct groups (not shown). Sample A was clearly differentiated from samples B and C. Sample A positively correlated with PC1 (cumulative variation 66.7%). In contrast, samples B and C negatively correlated with PC2 (cumulative variation 21.2%). This shows that overall levels of carbonyl compounds in samples B and C were significantly higher than sample A, indicating higher citrate metabolism in the microencapsulated probiotic cells. 3.4. Sensory evaluation Sensory attributes evaluated were appearance and colour, aroma and flavour, texture and overall acceptability. The results for 60- and 90-day-old cheeses are given in Fig. 5a and b, respectively.

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A

Appearance & colour 10 8 6 4 2

Overall acceptability

0

Aroma & flavour

probiotic cells (1.3–1.7 log for Lb. acidophilus and 0.8–1.4 log for B. bifidum BB-12 in samples B and C, respectively). Microencapsulation induced the formation of acetaldehyde and diacetyl, and increased the concentration of long-chain FFAs. According to the sensory evaluations, the microencapsulation did not adversely affect the appearance and colour, texture and overall acceptability of the experimental cheeses. The model cheeses were found not to be different from the control cheese, except for the aroma and flavour attributes. Considering the total counts of probiotic bacteria in the final product, the cheeses containing microencapsulated probiotic cells can be regarded as probiotic. Acknowledgement

Texture

B

The authors thank the Turkish Scientific and Research Council (TUBITAK) for financial support for this research (project no.: TOVAG 3227)

Appearance & colour 10 8 6

References

4 2

Overall acceptability

0

Aroma & flavour

Texture Fig. 5. Sensory scores of (A) 60-day-old cheeses and (B) 90-day-old cheeses: >, sample A (control, containing probiotic bacteria in the free state); ,, sample B (containing probiotic bacteria microencapsulated by extrusion); C, sample C (containing probiotic bacteria microencapsulated by emulsion). Values represent means (SD) of duplicate analyses of three cheeses from trials 1–3.

No significant differences were noted between samples A and C in terms of external and internal appearances of the cheeses. The aroma and flavour scores of the samples revealed insignificant differences between samples A and C. Sample B received lower scores with regard to this attribute (P < 0.05). The major aroma and flavour sources in cheese are proteolysis and lipolysis. The data for proteolysis revealed no difference between the cheeses containing encapsulated probiotic strains (samples B and C) (data not shown). Similarly, no difference was noted between samples B and C in terms of short- and medium-chain FFAs. The relatively higher CaCl2 concentration used in the preparation of extrusion capsules in sample B was thought to result in a salty taste in sample B. None of the panelists found the experimental cheeses unacceptable in terms of aroma and flavour attributes. The cheeses received similar scores for texture (P > 0.05). The presence of the capsules was not regarded as a limiting factor for the acceptability of the cheeses by the panel group. The panelists found clear differences between the 60- and 90-day-old cheeses in terms of textural properties. Overall, no significant sensory differences were noted between the experimental cheeses. The effect of ripening time on the overall acceptability of the cheeses was found to be significant (P < 0.05). 4. Conclusion Both microencapsulation techniques (extrusion and emulsion) were successful in keeping counts of Lb. acidiophilus LA-5 and B. bifidum BB-12 in the cheese high enough for the therapeutic minimum. While the counts of probiotic cells declined approximately 3 log in the control cheese (sample A) during ripening, this was far more limited in the cheeses containing microencapsulated

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