Probiotic Cheese Production Using Lactobacillus casei Cells Immobilized on Fruit Pieces

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Cell Immobilization
  • Probiotic Cheese Production
  • Reactivation of the Supported Biocatalysts
  • Analyses
  • Solid-Phase Microextraction, Gas Chromatography/ Mass Spectrometry
  • LAB Viable Cell Counts
  • Preliminary Sensory Evaluation
  • Experimental Design and Statistical Analysis
• Results and Discussion
  • SPME and GC-MS Analysis
  • Preliminary Sensory Evaluation
• Conclusions
• Acknowledgements
• Supplementary data
• References
• Copyright

Abstract 

Lactobacillus casei cells were immobilized on fruit (apple and pear) pieces and the immobilized biocatalysts were used separately as adjuncts in probiotic cheese making. In parallel, cheese with free L. casei cells and cheese only from renneted milk were prepared. The produced cheeses were ripened at 4 to 6°C and the effect of salting and ripening time on lactose, lactic acid, ethanol concentration, pH, and lactic acid bacteria viable counts were investigated. Fat, protein, and moisture contents were in the range of usual levels of commercial cheeses. Reactivation in whey of L. casei cells immobilized on fruit pieces after 7 mo of ripening showed a higher rate of pH decrease and lower final pH value compared with reactivation of samples withdrawn from the remaining mass of the cheese without fruit pieces, from cheese with free L. casei, and rennet cheese. Preliminary sensory evaluation revealed the fruity taste of the cheeses containing immobilized L. casei cells on fruit pieces. Commercial Feta cheese was characterized by a more sour taste, whereas no significant differences concerning cheese flavor were reported by the panel between cheese containing free L. casei and rennet cheese. Salted cheeses scored similar values to commercial Feta cheese, whereas unsalted cheese scores were significantly lower, but still acceptable to the sensory panelists.

Key words: probiotic cheese, Lactobacillus casei, cell immobilization, cheese ripening

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Introduction 

An upsurge of interest in developing novel foods containing probiotic microorganisms such as Lactobacillus and Bifidobacterium spp. has taken place in recent years. Such functional foods have great potential for promoting human health by maintaining and improving intestinal microbial balance (Mattila-Sandholm et al., 2002). Probiotic products should be provided with an adequate amount of live bacteria (at least 10⁷ cfu/ g; Oliveira et al., 2002) to survive the acidic conditions of the upper gastrointestinal tract and proliferate in the intestine to obtain the health-promoting benefits. Methods such as cell immobilization, appropriate selection of acid- and bile-resistant strains, use of oxygen-impermeable containers, and stress adaptation have been proposed for improving the viability of probiotic bacteria (Rao et al., 1989; Ishibashi and Shimamura, 1993; Shah, 2000; Champagne et al., 2005).

Fruits and fruit juices have been used extensively as blended additives during production of a variety of foods, especially in dairy industry. Apple (Kourkoutas et al., 2001,2002) and pear pieces (Mallios et al., 2004) have been used successfully as immobilization supports of yeast strains for room temperature and low-temperature wine making. The resulting wines were of improved quality with a distinctive aromatic potential. Likewise, apple pieces have been used in Lactobacillus casei cell immobilization (Kourkoutas et al., 2005) for probiotic additive fermented milk production. In addition, apple pieces proved to be very effective supports for the survival of apple-immobilized yeast (Kourkoutas et al., 2003) and bacteria (Kourkoutas et al., 2005), as the immobilized biocatalysts were able to reactivate after storage of 120 and 129 d, respectively.

Immobilized lactic acid bacteria (LAB) have been used and their effect has been studied during the cheese-making process. Batch and continuous milk prefermentation were performed using entrapped LAB in Ca-alginate beads (Prévost and Diviès, 1987). Dinakar and Mistry (1994) studied the growth and viability of Bifidobacterium bifidum added either as a commercially available powder or immobilized on κ-carageenan freeze-dried preparation in Cheddar cheese. Maximum bifidobacteria counts occurred at 18 and 24 wk for the commercial and immobilized preparations, respectively. Also, alginate-immobilized freeze-dried LAB have been used as starters in cheese making (Champagne et al., 1992) and in cheese ripening (Steenson et al., 1987).

Lactobacillus casei is a homofermentative microorganism (Holzapfel and Schilliger, 2002). It is acid tolerant (Fellows, 1997; Kourkoutas et al., 2005) and could thus survive during cheese ripening.

Feta cheese, one of the most significant and popular dairy products in Greece with worldwide acceptance, is a soft, white cheese, usually ripened in brine. Traditionally, Feta cheese is prepared from ewe's milk using only rennet on small family facilities with simple equipment. Today, most Feta cheese is produced from ewes’ milk or a mixture of ewes’ and goats’ milk in organized, commercial cheese dairies, using yogurt culture for lactic acid production. After a short period, rennet is added for completion of precipitation. A variation of Feta cheese is a traditional Greek soft white cheese known as “Katchochiri” (other local names may also exist), which is produced using ewes’ or goats’ milk, or both, by coagulation using only rennet. Subsequently, treatments such as filtration, salting, and preservation at 4°C are usually carried out. The produced cheese is consumed fresh or after ripening.

The aims of the present study were to investigate 1) the production of a new type of cheese using immobilized L. casei cells on fruit pieces as an adjunct culture, 2) the survival of the probiotic culture during cheese ripening, and 3) the implications of the adjunct culture on physicochemical and sensory characteristics of the final product during ripening.

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Materials and Methods 

Cell Immobilization 

Lactobacillus casei ATCC 393 (DSMZ, Germany) was used. It was grown on MRS broth (Merck, Darmstadt, Germany). Cell immobilization on apple and pear pieces (∼0.5-cm³ cubes) was carried out as described previously (Kourkoutas et al., 2005). In brief, fruit (apples and pears of Starkin and Conference varieties, respectively) pieces (∼500g) were introduced into 1L of L. casei liquid culture (∼10⁹cfu/mL), and allowed to ferment overnight at 37°C without agitation. When immobilization was complete (glucose in the liquid culture was <1g/L; concentration of glucose was determined by HPLC using the same method described for lactose determination below), the fermented liquid was decanted, and the supported biocatalysts were washed twice with pasteurized milk. The biocatalysts were then used in cheese production.

Probiotic Cheese Production 

Ewes’ milk was used for cheese production. It was heated at 65°C for 30min and then cooled at 37°C. Commercial rennet (0.01%) was added and the whole was left undisturbed for 2h for curd formation. Subsequently, the curd was cut in squares (∼1cm diameter), left undisturbed for 10min, and then cloth-filtered. Immobilized L. casei on apple and pear pieces were added separately (50g of immobilized biocatalyst/L of milk used) during cloth filtration, which lasted overnight at room temperature (18 to 22°C) for complete whey removal. Cheese produced from milk containing ∼10⁹cfu/mL of free L. casei, and cheese without L. casei cells (called rennet cheese) were produced for comparison. The effect of salt addition on cheese quality characteristics was studied by rubbing 10g of salt/100g of cheese on the surface. Ripening of the produced cheeses was monitored at 4 to 6°C for 71 d.

Reactivation of the Supported Biocatalysts 

After 7 mo of cheese ripening, apple- and pear-supported biocatalysts (1.4g) were removed from the salted cheese mass, washed twice with 50mL of whey, introduced into 100mL of whey, and tested for their fermentative activity by monitoring pH during lactic acid fermentation. For comparison, the same procedure was followed using equal masses of the remaining salted cheese without fruit pieces, the salted cheese containing free L. casei, and salted rennet cheese.

Analyses 

Twenty grams of cheese sample was macerated with warm water (40°C) to produce a total volume of 210mL, and the whole was then filtered (Kirk and Sawyer, 1991). The filtrate was used for lactic acid, lactose, and ethanol determinations.

Lactic acid was determined by HPLC, using a Shimadzu chromatograph (Shimadzu, Kyoto, Japan) with a Shim-pack IC-A1 stainless steel column, an LC-10A pump, a CTO-10A oven at 40°C, and a CDD-6A conductivity detector. A solution of 2.5mM phthalic acid (Merck) and 2.4mM Tris (hydroxymethyl) aminomethane (pH 4.0; Merck) in triple-distilled water was used as mobile phase with a flow rate of 1.5 mL/min. Samples (0.25mL) were diluted to 25mL, and 60μL was injected directly onto the column. Lactic acid concentrations were calculated using standard curves.

Lactose and ethanol were determined by HPLC, using a Shimadzu chromatograph with an SCR-101N stainless steel column, an LC-9A pump, a CTO-10A oven at 60°C, and a RID-6A refractive index detector. Triple-distilled water was used as mobile phase with a flow rate of 0.8mL/min, and 1-butanol (Merck) was used as an internal standard. Then, 0.5mL of cheese filtrate and 2.5mL of a 1% (vol/vol) solution of 1-butanol were diluted to 50mL, so that the final concentration of 1-butanol was 0.05% (vol/vol). Then, 40μL of the final solution was injected directly onto the column. Lactose and ethanol concentrations were calculated using standard curves prepared with at least 7 standard solutions by correlating the ratio of lactose or ethanol peak areas divided by 1-butanol peak areas to lactose or ethanol concentrations.

Total N expressed as CP on dry weight basis was determined using the Kjeldahl procedure and fat in DM content was determined by the Soxhlet process. The former methods as well as moisture content determination were described previously (Kirk and Sawyer, 1991).

Solid-Phase Microextraction, Gas Chromatography/ Mass Spectrometry 

Salted cheese samples ripened for 30 d at 4 to 6°C were studied for volatile by-product composition using solid-phase microextraction (SPME), gas chromatography/mass spectrometry (GC/MS) analysis. Grated samples of cheeses (∼7g) were placed into a 20-mL heads-pace vial fitted with a Teflon-lined septum sealed with an aluminum crimp seal, through which the SPME syringe needle (bearing a 2-cm fiber coated with 50/30mm divinylbenzene/carboxen on poly-dimethyl-siloxane bonded to a flexible fused silica core; Supelco, Bellefonte, PA) was introduced. The container was then held at a constant temperature of 80°C for 30 to 35min (Bellesia et al., 2003). The absorbed volatile analytes were then analyzed by GC/MS (Shimadzu GC-17A, MS QP5050, capillary column Supelco CO Wax-10; 60m, 0.32mm i.d., 0.25-μm film thickness). Helium was used as carrier gas (linear velocity of 1.5 mL/min). Oven temperature was programmed from 35°C for 3min, 5°C/min to 110°C, then 10°C/min to 240°C, and held at 240°C for 10min. The injector was operated in splitless mode. Injector and detector temperatures were 280 and 250°C, respectively. The mass spectrometer was operated in electron impact mode with the electron energy set at 70eV. Identification was achieved by comparison with standard compounds and data obtained from NIST107, NIST21 (www.nist.gov), and SZTERP (Shimadzu Instruction Manual) libraries.

LAB Viable Cell Counts 

Ten-gram portions of duplicate cheese samples were blended with 90mL of sterilized Ringer solution, and submitted to serial dilutions. Lactic acid bacteria were counted by pour-plating 0.1mL of each dilution on MRS agar (Merck) after 48h at 37°C under anaerobic conditions (Anerocult C, Merck).

Preliminary Sensory Evaluation 

Cheese samples containing probiotic culture were tested for their sensory characteristics and compared with rennet cheese and with commercial Greek Feta cheese. Samples (∼25g) of cheeses ripened for at least 30 d were presented. Sensory evaluation was conducted by 12 laboratory members previously trained using locally approved protocols. The panel was asked to give scores on a 0-to-10 scale (0 = unacceptable, 10 = exceptional) for attributes grouped into 3 categories: aroma, taste, and flavor. Panelists used water to clean their palates between samples and were unaware of the samples they tasted (samples were labeled with codes for identification). Significance was established at P<0.05. Results were analyzed for statistical significance with ANOVA. Duncan's multiple range test was used to determine significant differences among results [coefficients, ANOVA tables and significance (P<0.05) were computed using Statistica v.5.0; StatSoft, Inc., Tulsa, OK].

Experimental Design and Statistical Analysis 

All treatments were carried out in triplicate and the mean values are presented (maximum deviation for all values was±5% in most cases). Duplicate samples from each treatment were collected at various intervals and analyzed for lactic acid, lactose, volatile by-products, and total LAB. After that, cheeses remained at 4 to 6°C for 7 mo to test survival of LAB.

In the experiments conducted, the effect of probiotic L. casei adjunct culture and the effect of salting during cheese ripening were studied. The experiments were designed and analyzed statistically by ANOVA. Duncan's multiple range test was used to determine significant differences among results [coefficients, ANOVA tables and significance (P<0.05) were computed using Statistica v.5.0].

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Results and Discussion 

Ewes’ milk consists of about 66% moisture, 17% total solids, 5.30% fat, and 6.30% total protein, of which 4.60% is casein, 4.60% lactose, and 0.80% ash (Voudouris and Kontominas, 1990). Nonhomogenized ewes’ milk, pasteurized at 65°C for 30min, was used in the present study due to its sensory characteristics.

Milk coagulation was achieved using rennet only, as is common practice in Greece. It is well known that starter cultures may implement an inhibitory effect on probiotic microorganisms (Gomes et al., 1995,1998; Daigle et al., 1999). The strategy adopted was the omission of starter cultures and the use of L. casei culture as an adjunct probiotic culture instead, because L. casei is an acid-resistant microorganism (Fellows, 1997; Kourkoutas et al., 2005) and it survives in the cheese mass during ripening (Broome et al., 1990; Stanton et al., 1998; Antosson et al., 2002).

Immobilized L. casei cells were added to the curd after cloth filtration and the immobilized biocatalyst was distributed uniformly, as much as possible, in the curd mass. Cheese with free L. casei cells and rennet cheese were produced for comparison.

A problem associated with sampling due to uneven distribution of L. casei cells in the cheese mass was noticed in cheeses containing immobilized L. casei on fruit pieces. However, during cheese production, fruit pieces were mixed with the curd and distributed uniformly, as much as possible. All samples withdrawn at various intervals contained cheese and fruit pieces and effort was made to ensure the withdrawn samples contained an equal amount of fruit pieces and were derived from the same depth of the cheese mass. Therefore, average enumeration of LAB is presented in Figure 1, because LAB cell counts in fruit pieces may have been higher. The same problem was observed during determination of physicochemical parameters. Similar problems concerning uneven distribution exist in commercial products that contain fruit pieces and are acceptable by the consumers (e.g., yogurt with fruit).

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• Figure 1. 

  Lactic acid bacteria counts in probiotic white cheese produced by immobilized Lactobacillus casei cells on fruit pieces and free L. casei cells during ripening for 71 d at 4 to 6°C.

Physicochemical parameters for unsalted and surface-salted cheeses during ripening are presented in Table 1. Addition of adjunct culture, salting, and ripening had a significant effect on pH and concentrations of lactose and ethanol (P<0.01), and there was a strong interaction among them (P<0.01). In contrast, lactic acid concentration was only affected by adjunct culture and ripening (P<0.01), but not by salt addition (P>0.05). However, a strong interaction between salting and adjunct culture (P<0.01), between adjunct culture and ripening (P<0.01), and among adjunct culture, salting, and ripening (P<0.01) affecting lactic acid concentration significantly was observed. During ripening, lactose content was significantly decreased and lactic acid was generally increased. Ethanol content was generally at a very low level (0.03 to 0.6 g/100g of cheese in most cases), whereas pH decreased during ripening in salted cheeses. However, pH was in the range of levels usually observed in commercial products (Table 1 The opposite effect was observed for unsalted cheeses, probably due to invisible spoilage after ripening for more than 30 d (Spencer and Spencer, 1997). The smear microorganisms that develop on the surface of the cheese play a role in the ripening process, both through the action of proteolytic and lipolytic enzymes, and the formation of many alkaline products, mainly due to the assimilation of lactate that penetrated the body of the cheese. (Reps, 1993; Smacchi et al., 1999; Addis et al., 2001; Corsetti et al., 2001).

Table 1. Quality characteristics of probiotic white cheese produced by immobilized Lactobacillus casei cells on fruit pieces and free L. casei cells during ripening for 71 d at 4 to 6°C¹

Analysis	Day of ripening	Cheese type
		L. casei on apple pieces		L. casei on pear pieces		L. casei free cells		Rennet cheese
		No salt	Salt	No salt	Salt	No salt	Salt	No salt	Salt
Lactose (g/100g of cheese)	1	1.80±0.1	1.15±0.01	1.90±0.1	2.09±0.1	1.19±0.07	1.57±0.05	2.03±0.1	1.74±0.1
	4	1.38±0.06	1.04±0.001	1.79±0.08	1.27±0.05	1.25±0.05	1.26±0.03	1.73±0.1	1.40±0.05
	15	1.29±0.05	0.92±0.04	0.62±0.03	1.13±0.04	0.59±0.01	0.71±0.02	1.70±0.1	0.89±0.02
	30	0.85±0.02	0.77±0.02	0.47±0.01	0.79±0.01	0.30±0.01	0.70±0.02	0.78±0.03	0.84±0.02
	71	0.71±0.01	0.48±0.01	0.14±0.01	0.21±0.01	Tr	0.34±0.01	0.33±0.01	0.56±0.01
Lactic acid (g/100g of cheese)	1	0.42±0.01	0.52±0.02	0.48±0.02	0.50±0.05	0.55±0.02	0.39±0.02	0.15±0.002	0.10±0.002
	4	0.62±0.02	0.60±0.03	0.49±0.02	0.60±0.08	0.61±0.03	0.43±0.02	0.22±0.01	0.15±0.002
	15	0.80±0.07	0.63±0.03	0.45±0.02	0.60±0.08	0.64±0.03	0.46±0.02	0.13±0.002	0.13±0.002
	30	0.53±0.02	0.68±0.04	0.45±0.02	0.69±0.03	0.68±0.03	0.48±0.02	0.12±0.002	0.12±0.002
	71	0.54±0.02	0.68±0.04	0.68±0.04	0.94±0.04	0.86±0.04	0.64±0.03	0.86±0.04	0.50±0.01
Ethanol (g/100g of cheese)	1	0.21±0.01	0.09±0.002	0.22±0.01	0.12±0.005	0.22±0.01	0.09±0.001	0.28±0.01	0.11±0.001
	4	0.29±0.01	0.05±0.001	0.09±0.001	0.20±0.005	0.09±0.001	0.15±0.01	0.28±0.01	0.16±0.01
	15	0.50±0.01	0.05±0.001	0.21±0.01	0.31±0.01	0.14±0.01	0.56±0.01	0.27±0.01	0.12±0.001
	30	0.66±0.02	0.03±0.001	0.18±0.01	0.11±0.005	0.14±0.01	0.51±0.01	0.19±0.01	0.08±0.001
	71	0.09±0.001	1.19±0.01	0.05±0.001	2.12±0.1	0.27±0.01	0.12±0.001	0.34±0.01	0.90±0.05
pH	1	5.3±0.1	5.1±0.1	5.2±0.1	5.1±0.1	5.3±0.1	5.1±0.1	5.9±0.1	5.9±0.1
	4	5.3±0.1	4.9±0.1	5.1±0.1	4.8±0.1	5.2±0.1	5.0±0.1	5.7±0.1	5.4±0.1
	15	5.2±0.1	4.6±0.1	5.0±0.1	4.7±0.1	5.0±0.1	4.9±0.1	5.8±0.1	5.4±0.1
	30	5.4±0.1	4.4±0.1	5.7±0.1	4.3±0.1	5.5±0.1	4.9±0.1	7.0±0.1	5.0±0.1
	71	5.8±0.1	4.6±0.1	6.4±0.2	4.5±0.1	5.9±0.2	4.9±0.1	7.3±0.2	4.8±0.1
Infection	1	−	−	−	−	−	−	−	−
	4	−	−	−	−	−	−	−	−
	15	−	−	−	−	−	−	−	−
	30	−	−	−	−	−	−	+	−
	71	+	−	+	−	+	−	+	−

As Stanton et al. (1998) reported, during the early period of cheese ripening, a population of nonpathogenic organisms referred to as nonstarter lactic acid bacteria (NSLAB) mainly composed of lactobacilli (Lb. plantarum, casei, and brevis) and pediococci (Pediococcus pentosaceus) survive pasteurization in an attenuated state and proliferate as the cheese is ripened. Number of nonstarter lactic acid bacteria increase rapidly, reaching maximum levels of 10⁷ to 10⁸ cfu/g in ripened Cheddar cheese within a few days (Stanton et al., 1998). Salting of cheese contributes positively to cheese flavor, and has an impact on microorganisms’ growth as salt diffuses in the cheese mass, so that differences concerning salt content in the cheese center and periphery decrease with ripening (Mocquot, 1979; De Leon-Gonzalez et al., 2000). Viability of bifidobacteria and other bacteria is inversely related to the salt concentration (Gomes et al., 1998; Vinderola et al., 2002). In our study, LAB counts were generally increased up to 30 d of cheese ripening, reaching maximum levels of about 10⁹ to 10¹² cfu/g (Figure 1). The increase was higher in unsalted cheeses than in salted cheeses. After 30 d, LAB cell counts were decreased except in the case of salted samples with immobilized L. casei cells on fruit pieces, in which some deviations were recorded (Figure 1). The reduction was probably due to high salt in moisture, low pH, and low ripening temperature as recently reported (Stanton et al., 1998). The fact that almost equal initial LAB counts were observed in cheeses containing immobilized L. casei and in rennet cheese could be attributed to similar concentration of L. casei on the fruit pieces after immobilization to levels of NSLAB in rennet cheese (∼10⁷ to 10⁸ cfu/g). Therefore, increased levels of lactic acid bacteria should not be expected in the cheeses that contained immobilized L. casei. Heat treatment at 65°C was applied during raw milk pasteurization in the present study to standardize cheese quality, as is traditionally recommended in Greece, and because it has been claimed that cheeses made from raw, unpasteurized milk possess a better flavor (Adams and Moss, 1997). Consequently, thermophilic LAB might have survived; and thus, levels of 10⁷cfu/g of NSLAB in the rennet cheese at the first day of cheese ripening are justified (Adams and Moss, 1997). Similar results have been reported in previous studies describing production of a similar type of cheese using raw milk (Hatzikamari et al., 1999; Nikolaou et al., 2002).

Salting resulted in slightly higher protein content in cheese containing free L. casei cells and in rennet cheese compared with cheeses containing immobilized L. casei on fruit pieces (Table 2), probably because the presence of salt may have an inhibitory effect on proteolytic enzymes. In contrast, cell immobilization may have a protective effect on proteolytic activity. Salting resulted in slightly higher fat content in cheeses containing immobilized L. casei on fruit pieces, whereas the opposite effect was observed in cheeses containing free L. casei cells and in rennet cheeses. The above results could be because the majority of L. casei cells were immobilized on fruit pieces and therefore did not contribute to fat lipolysis. Moisture was relatively lower in cheeses produced in the laboratory compared with commercial Feta cheese, as the latter was ripened and stored in brine solution.

Table 2. Fat, protein, and moisture content of probiotic white cheese produced by immobilized Lactobacillus casei cells on fruit pieces and free L. casei cells after ripening for 71 d at 4 to 6°C

Analysis	Cheese type								Commercial Feta
	L. casei on apple pieces		L. casei on pear pieces		L. casei free cells		Rennet cheese
	No salt	Salt	No salt	Salt	No salt	Salt	No salt	Salt
Fat, % in DM	50.0±1.0	64.4±1.4	51.8±0.9	66.7±1.7	64.1±1.4	52.3±1.3	61.0±2.2	55.9±0.9	53.0±1.8
Protein, % in DM	37.9±1.6	33.1±1.9	44.9±1.6	30.1±2.5	31.8±2.4	39.9±1.4	32.1±2.8	37.9±2.4	44.3±1.6
Moisture, %	52.3±1.8	50.1±2.0	54.6±2.1	52.3±2.7	54.5±1.7	42.3±1.9	58.8±1.9	49.6±2.1	57.1±2.2

Survival of L. casei cells was investigated by reactivating separately 1) the immobilized biocatalysts, 2) the remaining part of the cheeses that contained the immobilized biocatalysts, 3) a sample of the cheese that contained free L. casei, and 4) a sample of the rennet cheese after ripening for 7 mo. In a final LAB enumeration carried out after 7 mo in salted cheeses, viable cell counts were 10.15, 11.15, 7.69, 7.56, 7.66, and 7.65 cfu/ g in apple pieces only, in pear pieces only, in the remaining cheese mass without apple and pear pieces, in the cheese sample containing free L. casei, and in rennet cheese, respectively. The above results showed increased LAB cell counts in fruit pieces compared with the remaining cheese mass, which could be attributed to survival of the immobilized L. casei cells. This was in agreement with results obtained during reactivation of the immobilized L. casei cells, as shown in Figure 2. Lactobacillus casei cells immobilized on fruit pieces and especially on apple pieces showed a higher rate of pH decrease and lower final pH value (4.15) compared with other types of cheeses after 7 mo of ripening. The higher pH (4.45) obtained during lactic acid fermentation using pieces of rennet cheese (Figure 1) showed that fruit pieces are an effective support for the acid-tolerant L. casei cells during cheese ripening.

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• Figure 2. 

  Kinetics of pH during lactic acid fermentation of whey by: a) Lactobacillus casei cells immobilized on apple pieces only, withdrawn from salted cheese (■), b) L. casei cells immobilized on pear pieces only, withdrawn from salted cheese (●), c) remaining mass of salted cheese produced by L. casei cells immobilized on apple pieces (▴), d) remaining mass of salted cheese produced by L. casei cells immobilized on pear pieces (▾), e) salted cheese containing free L. casei cells (♦), and f) salted rennet cheese (+), after ripening for 7 mo.

SPME and GC-MS Analysis 

For evaluation of the aromatic profile, probiotic white cheeses produced by immobilized L. casei on apple, pear pieces, and free cells were analyzed with SPME GC/ MS and compared with rennet cheese and commercial Feta cheese. Only salted cheeses were analyzed, because an increase in the pH during 30 d of maturation in the unsalted cheeses was observed, accompanied by surface cheese infection and a bad odor. This analysis allowed a qualitative comparison of the aroma-related compounds produced during cheese ripening. The results are summarized in Table 3.

Table 3. Peak areas (×10⁵) of volatile by-products isolated in probiotic salted white cheeses produced by immobilized Lactobacillus casei cells on fruit pieces and free L. casei cells after ripening for 30 d at 4 to 6°C using solid-phase microextraction gas chromatography-mass spectrometry¹

In general, the most important compounds that are usually identified by SPME GC/MS technique in cheeses are esters, organic acids, alcohols, carbonyl compounds, sulfur, and miscellaneous compounds (Barbieri et al., 1994; Engels et al., 1997; Izco and Torre, 2000; Qian and Reineccius, 2002). Nevertheless, this does not imply that some components are less important than others. The flavor of the cheeses seems to depend not on particular key components, but rather on a “critical balance” or a “weighted concentration ratio” of all components present (Izco and Torre, 2000). In regard to the present study, more compounds were detected in cheeses containing immobilized L. casei cells on apple and pear pieces than in the other types of cheeses produced. These compounds were probably derived from the fruit pieces introduced in the cheeses. In total, 61 compounds were detected in cheese containing L. casei cells immobilized on apple pieces, 71 compounds in cheese containing L. casei cells immobilized on pear pieces, 39 compounds in cheese with free L. casei cells, 51 compounds in rennet cheese, and 35 compounds in commercial Feta cheese. Presence of L. casei cells in cheese with free L. casei cells (Bachmann et al., 1997; Bosset et al., 1997; Champagne et al., 2005) and presence of salt (Tzanetakis and Litopoulou-Tzanetaki, 1992; Gerasi et al., 2003) in commercial Feta cheese probably inhibited development and action of some Lactobacillus species that produce aroma-related compounds in cheeses. Adjuncts of lactobacilli may suppress the growth of undesirable bacteria in cheese (Martley and Crow, 1993). Vinderola et al. (2002) observed inhibition in growth of LAB in the presence of probiotic bacteria. Furthermore, high salt in moisture may result in a dramatic decline in starter numbers during the early weeks of ripening (Boylston et al., 2004). A comparison between cheese with free L. casei cells and rennet cheese showed that the rennet cheese contained more alcohols and aldehydes, which constitute mainly products of proteolysis, and esters than the cheese with free L. casei cells.

Most esters encountered in analyzed cheeses are described as having fruity, floral notes. Ethyl acetate was identified in cheese containing immobilized L. casei cells on apple and pear pieces and in rennet cheese, whereas ethyl butanoate and propyl butanoate were present only in commercial Feta cheese. Likewise, ethyl octanoate was identified in cheeses containing pear-immobilized biocatalyst and in rennet cheese. The above esters are known for their fruity aroma contribution (Pérès et al., 2001; Qian and Reineccius, 2002) and may derive from milk (Urbach, 1995).

Free fatty acids are important components in cheese flavor and may originate from milk fat lipolysis or from breakdown of amino acids (Urbach, 1993). Acetic and hexanoic acids (identified only in commercial Feta cheese) provide a pungent flavor (Pérès et al., 2001). Octanoic acid (present in all cheeses produced in the laboratory) and decanoic acid (present in all samples tested except in cheese containing free L. casei cells; Table 3) are known for their goaty, rancid flavor (Kim Ha and Lindsay, 1992; Molimard and Spinnler, 1996). A number of long-chain fatty acids were also detected such as undecylenic, dodecanoic, tetradecanoic, pentadecanoic, hexadecanoic, octadecenoic, and 9-octadecenoic acids. However, fatty acids with >12 carbon atoms play a minor role in cheese flavor, because they have a high perception threshold (Molimard and Spinnler, 1996).

Alcohols identified included alcohols of the aliphatic series, fusel alcohols, and phenols. Ethanol (identified in all cheese samples) provides an alcohol, mild flavor note, and occurs in fresh milk (Urbach, 1995) and may derive from lactose metabolism, whereas 1-octen-3-ol (detected only in cheese containing immobilized L. casei on pear pieces) is well known for its raw mushroom odor (Molimard and Spinnler, 1996). Aliphatic primary alcohols such as 1-pentanol (identified in all cheese samples produced in the laboratory) and 1-hexanol (found in cheese samples containing immobilized L. casei cells on apple and pear pieces and in rennet cheese; Table 3) may impart a fruity, nutty note to the flavor of cheese (Engels et al., 1997). 4-Methyl-phenol (p-cresol) was detected in cheeses containing immobilized and free L. casei, whereas 2-methyl-phenol (o-cresol) was detected only in cheese containing immobilized L. casei on pear pieces. In general, cresols and phenols may play an important role in cheese produced by sheep milk providing a distinctive sheep-like or sheepyard-like flavor (Kim Ha and Lindsay, 1992).

Carbonyl compounds identified included mainly aldehydes, ketones, and lactones. Straight-chain aldehydes such as acetaldehyde, propanal, 2-butenal, pentanal, hexanal, 2-hexenal, heptanal, 2-heptenal, octanal, 2-octenal, nonanal, decanal, and 2-decenal were detected. The majority of these aldehydes are natural constituents of fresh milk (Urbach, 1995), formed during β-oxidation of unsaturated fatty acids (Collin et al., 1993; Moio et al., 1993; Lee et al., 1996). Hexanal and 2-hexenal provide a green note of immature fruit, whereas octanal, nonanal, and decanal result in an aromatic note resembling orange (Molimard and Spinnler, 1996). Benzaldehyde (identified in cheese produced by immobilized L. casei on apple pieces and free cells and in commercial Feta cheese; Table 3) is described as having an aromatic note of bitter almond (Molimard and Spinnler, 1996), and may originate from α-oxidation of phenylacetaldehyde or from β-oxidation of cinnamic acid (Casey and Dobb, 1992).

Methyl ketones are formed in a metabolic pathway connected to the β-oxidation pathway. 2-Butanone (detected only in commercial Feta cheese) provides an acetone note, whereas 2-undecanone (detected in cheeses containing L. casei immobilized on apple and pear pieces and in rennet cheese) provides a floral, herbaceous aroma (Molimard and Spinnler, 1996). Likewise, 2-tridecanone with a fruity, green flavor note (Molimard and Spinnler, 1996) was found only in cheese containing pear-supported biocatalyst and free L. casei cells. 3,5-Octadien-2-one was detected in cheese containing immobilized L. casei on pear pieces and in rennet cheese, whereas 6,10,14-trimethyl-2-pentadecanone was detected only in cheese containing immobilized L. casei on pear pieces. 3-Hydroxy-2-butanone (acetoin) was detected only in cheese containing free L. casei cells; it may derive from diaketyl reduction, due to the reducing cheese environment.

Butyrolactone, which has a pungent, buttery, fetid impact (Molimard and Spinnler, 1996) was only detected in commercial Feta cheese, whereas delta-undecalactone was identified in cheese containing immobilized L. casei (Table 3). Generally, hydroxy fatty acids, which are lactone precursors, may be present in milk.

A number of miscellaneous compounds were identified, some groups of which are known to contribute to the complexity of cheese aroma, such as aromatic and nonaromatic hydrocarbons and furanic derivatives. Cheese with free L. casei contained more hydrocarbons than did rennet cheese. The presence of free L. casei cells probably inhibited the action of enzymes or microorganisms that could convert these compounds to other by-products. Similar results were observed after addition of L. casei cells in raw milk during production of Swiss Emmental cheese, in which inhibition of propionic acid fermentation and enterococci was reported (Bachmann et al., 1997). Toluene (identified in all cheese samples) and limonene (identified only in cheese containing free L. casei cells) may occur in fresh milk and milk butter, respectively. β-Myrcene (detected only in cheese containing free L. casei cells) is a monoterpene found in essential oils. A number of nonaromatic hydrocarbons such as n-decane, 2,4,6-trimethyloctane, 3-methyldecane, 4,5-dimethylnonane, undecane, 1-decyne, dodecane, tridecane, 2,3,5,8-tetramethyl-1,5,9-decatriene, 2,4-dimethyldodecane, hexadecane, octadecane, and 3,7,11,15-tetramethyl-2-hexadecene was detected. n-Alkanes such as decane, undecane, and dodecane were also reported as volatile flavor compounds of buffalo Mozzarella cheese in a previous study (Moio et al., 1993). However, the hydrocarbons belong to a family of secondary products of lipid antioxidants (Barbieri et al., 1994) that do not have a major contribution to aroma in cheese, although these compounds may serve as precursors for the formation of other aromatic compounds (Arora et al., 1995). Furans such as 2-pentyl-furan, furfural, 2-furanmethanol, and 5-hydroxymethyl-2-furancarboxaldehyde were likely formed by the Maillard reaction (Nursten, 1981). 2-Furanmethanol (present in all cheese samples produced in the laboratory) provides a strong baked aroma (Qian and Reineccius, 2002), whereas 2-pentyl-furan (present in cheese produced with immobilized L. casei cells), which may also occur as autoxidation product of linoleic and linolenic acids, has a liquorice aroma (Belitz and Grosh, 1987). Furfural (detected in cheese containing immobilized L. casei and in rennet cheese) is known for its cooked, heated milk odor; it has been also reported as volatile constituent of cheeses (Moio et al., 1993).

Regarding peak areas of the identified compounds, the highest maximum peak areas were observed for fatty acids, followed by peak areas of ethanol, aldehydes, ketones, esters, and lastly, hydrocarbons. Peak areas of ethanol were much higher compared with other alcohols in all samples. However, to understand the respective contribution of each aroma-bearing compound to the overall cheese aroma, quantitative studies are necessary to relate the concentration of an individual component with respect to its threshold level, and to observe any synergistic effect at the odor port.

Preliminary Sensory Evaluation 

Feta is a very popular cheese in Greece and therefore, the sensory characteristics of the produced cheeses were compared with commercial Feta cheese. Commercial Feta cheese was characterized by a more sour taste. More acids were detected in commercial Feta cheese during GC/MS analysis (Table 3). A fruity taste was predominant in cheeses containing L. casei immobilized on fruit pieces, whereas no significant differences concerning cheese flavor were reported by the panel between cheese containing free L. casei and rennet cheese. Salted cheeses scored similar values to commercial Feta cheese (P>0.05; data not shown), whereas unsalted cheeses scores were significantly lower (P<0.01) than those of commercial Feta. However, the unsalted cheeses were accepted by the panel, which is an important consideration for people with high blood pressure.

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Conclusions 

As a final consideration, it is concluded that 1) no spoilage was observed in salted cheeses after ripening for 7 mo, 2) fruit pieces proved to be a very effective support for survival of the L. casei cells during cheese ripening, and 3) the cheeses produced had a distinctive taste and acceptable sensory characteristics when compared with the very popular Feta cheese.

Cheeses produced using immobilized L. casei on fruit pieces could be manufactured with some additions to the traditional cheese-making practice. The production of probiotic cheese in which the probiotic culture would survive and develop during manufacture and throughout its shelf life could lead to a major economic advantage. The fact that immobilized L. casei on fruit pieces were reactivated after storage for 7 mo is very promising with regard to survival of probiotic bacteria in an acidic environment such as cheese mass. Probiotic bacteria immobilized on fruit pieces would reach the colon, because the fruit pieces contain cellulose, which is not digested. It is strongly believed that future clinical tests will ensure the beneficial effects of fruit based probiotics.

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Acknowledgements 

The authors would like to thank Ms Taboukou for supplying the ewes’ milk.

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Supplementary data 

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