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Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production
Bioresource Technology 100 (2009) 3618–3624
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Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production Eleftheria Katechaki a, Panayiotis Panas a,*, Yiannis Kourkoutas b, Dionisis Koliopoulos c, Athanasios A. Koutinas a a
Food Biotechnology Group, Section of Analytical Environmental and Applied Chemistry, Department of Chemistry, University of Patras, GR-26500 Patras, Greece Department of Molecular Biology and Genetics, Democritus University of Thrace, Dimitras 19, Alexandroupolis, GR-68100 Alexandroupolis, Greece c AVIGAL S.A., Elliniko, Farres, GR-25008 Achaia, Greece b
a r t i c l e
i n f o
Article history: Received 14 November 2008 Received in revised form 25 February 2009 Accepted 26 February 2009 Available online 28 March 2009 Keywords: Kefir Whey Hard-type cheese Ripening Dried starters
a b s t r a c t In an attempt to seek for suitable dried cultures, thermally-dried kefir was employed as starter in hardtype cheese production and tested in cheeses ripened at 5, 18 and 22 °C. Both free and immobilised on casein kefir cells were used and compared to cheese made without starter culture. Cheese products made with free cells of kefir culture were characterized by longer preservation time, improved aroma, taste, texture characteristics and increased degree of openness. Volatile profiles obtained by GC/MS analysis revealed a 216% increase in total concentration of esters, organic acids, alcohols and carbonyl compounds between cheeses prepared with and without kefir culture. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Recent decades, whey has attracted researchers’ interest due to its nutrient-rich content and its availability in dairy industries as a milk by-product constituting a potential substrate for important biological processes. It corresponds to 85–95% of the total milk volume entering the dairy process and contains about 55% of the nutrients present in the original milk such as proteins, lactose, vitamins and minerals (Siso, 1996). Due to these characteristics whey constitutes a popular waste for the production of beverages, ethanol, single cell protein, polysaccharides, purified lactose, organic acids, flavor enhancers and other useful products (Siso, 1996; Ostojic´ et al., 2005). Kefir is a mixed culture consisting of lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc), yeasts (Kluyveromyces, Candida, Saccharomyces, Pichia) and acetic acid bacteria. This culture is mainly known for its usage in the production of a refreshing fermented beverage called ‘‘kefir” that is popular in Eastern–European countries (Farnworth, 1999; Paraskevopoulou et al., 2003). The main role of such starter cultures with lactic acid bacteria in cheese ripening is the production of lactic acid through lactose
* Corresponding author. Tel.: +30 2610 997123; fax: +30 2610 997105. E-mail addresses: [email protected] (E. Katechaki), [email protected] (P. Panas), [email protected] (D. Koliopoulos), [email protected] (A.A. Koutinas). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.02.061
metabolism and the reduction of pH. Moreover, the potential probiotic and antimicrobial effects of lactic acid bacteria identified in kefir grains have stimulated a major research effort in recent years (Heller, 2001; Kourkoutas et al., 2006; Vuyst and Leroy, 2007). Whey has been extensively used as raw material for kefir fermentation (Rimada and Abraham, 2001; Paraskevopoulou et al., 2003; Koutinas et al., 2005; Assadi et al., 2008; Papapostolou et al., 2008; Papavasiliou et al., 2008). Recently, kefir biomass revealed its suitability as a starter culture in a variety of cheese products, hard-type, feta-type cheese and whey-cheese, mainly due to its effect on quality, preservation time and safety properties of the final product (Kourkoutas et al., 2006; Dimitrellou et al., 2007; Katechaki et al., 2008). In the above studies, kefir culture was used in freeze-dried form and was found to increase the shelf life of the final products and both the variety and concentration of esters, free fatty acids, alcohols and carbonyl compounds. This culture appeared also to be effective in fermentation of a lactose-rich substrate even when dried kefir cells immobilised on casein were used (Dimitrellou et al., 2008). It has been well demonstrated that immobilization technology results in enhanced survival rates and biocatalysts stability during processing and storage (Champagne et al., 1992; Iconomopoulou et al., 2000; Kopsahelis et al., 2007). Drying of cultures, on the other hand, has acquired a significant role in production of commercial cell cultures as it offers long term preservation of micro-organisms, cell stability and convenience in
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transportation, storage and cell dosage of cell cultures (Morgan et al., 2006). Thermal-drying is an alternate approach to the most popular method of freeze-drying that is not suffering from the high investment cost, the use of cryoprotectants and the decreased cell viability. Indeed, the thermal-drying process appeared to be more efficient on cell viability (84%) of kefir culture than that (78%) of freeze-drying (Kopsahelis et al., 2007). In this study, the impact of thermally-dried kefir as a new starter culture in the production of hard-type cheese is evaluated. Results on the effect of thermally-dried kefir culture immobilized on casein on aroma, flavor, textural characteristics, degree of openness and on the microbial associations throughout the ripening process are presented and compared with cheeses made with free cells of kefir. The impact of thermally-dried kefir culture on volatile profiles is presented and discussed.
2. Methods 2.1. Starter strain Kefir culture was isolated from a commercial kefir drink and used as starter in cheese production. Kefir was grown at 30 °C on a sterilized synthetic medium containing (%w/v): 4 lactose, 0.4 yeast extract, 0.1 (NH4)2SO4, 0.1 KH2PO4, and 0.5 MgSO4.7H2O (Merck, Darmstadt, Germany). Pressed wet cells were harvested by centrifugation at 5000 rpm for 10 min. Harvested biomass (4 g) was resuspended directly in 500 mL of whey for aerobic fermentation. Kefir was incubated in whey at 30 °C for further production of starter culture. 2.2. Immobilization process Pressed wet kefir cells were added in commercial pasteurized bovine milk (0% fat) followed by casein isolation as described by Dimitrellou et al. (2008). 2.3. Drying process Thermally-dried kefir cultures were obtained by drying free or immobilized kefir cells in an oven equipped with air circulation (J.P. Selecta, Spain) at 33 °C. The drying process was monitored by weighing the drying material at various time intervals until constant weight. 2.4. Cheese making and sampling Bulk ewes’ milk was obtained from a local dairy factory (AVIGAL S.A., Elliniko, Farres, Achaia). Cheese was produced and ripened using the method of Katechaki et al. (2008). In this method, the starter strain described previously was used instead of freeze-dried kefir culture. Hard-type cheeses were designated as follows: cheese A or rennet cheese (without any starter culture), cheese B with 1.0 g/L added kefir and cheese C with 1.0 g/L added kefir immobilized on casein. Rennet was added in all cheese samples. All experiments were repeated three times. Cheeses were ripened at 5 °C, 18 °C and 22 °C for 90 days. Samples from each cheese were taken at 0, 15, 30, 60 and 90 days. 2.5. Physicochemical analysis Moisture was determined in cheese samples according to AOAC (1995). Samples for lactic acid, ethanol and residual sugar analysis were prepared as follows: 20 g of cheese samples were macerated with warm water (40 °C) up to a total volume of 210 mL followed by filtration.
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Acidity was quantified by titration with 0.1 N NaOH using phenolphthalein as indicator (AOAC, 1995) and expressed as lactic acid content. Lactose, glucose and galactose were determined by high performance liquid chromatography. A Shimadzu chromatograph (Kyoto, Japan) with an SCR-101 N stainless steel column, a LC-9A pump, a CTO-10A oven at 60 °C and a RID-6A refractive index detector was used. Three times distilled water was used as mobile phase at the flow rate of 0.8 mL/min, and 1-butanol was used as an internal standard (IS). Cheese filtrate (0.5 mL) and 1% v/v solution of 1butanol (2.5 mL) were diluted to 50 mL, the final solution was filtered through a 0.2 lm pore size filter, and 40 lL of the filtrate were injected directly into the column. Residual sugar concentrations were calculated using standard curves by correlating the ratio of residual sugar peak areas divided by 1-butanol peak areas to residual sugar concentrations. Ethanol was determined by gas chromatography on a Shimadzu GC-8A system consisting of a Porapac-S packed column, N2 as carrier gas (20 mL/min), and a FID detector. The injection port and detector temperatures were 210 °C and the column temperature was programmed at 120–170 °C, rising by 10 °C/min. 1-Butanol (0.1% v/v) was used as the internal standard and samples of 2 lL were injected directly in the column. Determination of ethanol concentration was carried out using standard curves. Cheese pH was measured using a digital pH meter (CyberScan pH 10 and pH 100, Eutech Instruments, Singapore). 2.6. Solid-phase Microextraction (SPME) gas chromatography/mass spectrometry (GC/MS) analysis Cheese samples ripened at 5 °C for 90 days were assayed for aroma volatiles using SPME GC/MS analysis. Grated cheese samples (7 g each) were placed into a 20-mL headspace vial fitted with a teflon-lined septum sealed with an aluminum crimp seal, through which the SPME syringe needle (Supelco, Bellefonte, PA, USA) was introduced. The internal standard constituted methyl octanoate (Merck, Darmstadt, Germany) at a final concentration of 125.29 lg/kg of cheese. Sample incubation at 80 °C for 30– 35 min was followed by GC/MS analysis of the absorbed volatile compounds. A Shimadzu GC-17A coupled to a GCMS-QP5050A mass spectrometer with a Supelcowax-10 column (60 m– 0.32 mm i.d. 0.25 lm film thickness) and helium as carrier gas (linear velocity of 1.5 mL/min) were used. Oven temperature was set as follows: 35 °C for 3 min; a temperature gradient of 5 °C/ min up to 110 °C; a temperature gradient of 10 °C/min up to 240 °C; and a final extension at 240 °C for 10 min. The injector was operated in splitless mode at 280 °C, detector temperature was set at 250 °C and the mass spectrometer was operated in the electron impact mode with the electron energy set at 70 eV. Identification of compounds was done by comparing the retention times and MS data with those of standard compounds and by MS data obtained from NIST107, NIST21 and SZTERP libraries. The peak areas were measured from the full scan chromatograph using total ion current (TIC). 2.7. Microbiological analysis Inoculum for microbiological analysis was prepared by blending 10 g of cheese from cheese interior of each sample with 90 mL of sterilized 2% tri-sodium citrate solution and subjected to serial dilutions in sterilized Ringer’s solution (1/4 strength). The following microbiological assays were performed: (i) determination of total aerobic counts on plate count agar (Fluka 70188) at 30 °C for 48 h, (ii) enumeration of yeasts and moulds on potato glucose agar (Fluka 70139) at 30 °C for 48 h, (iii) enumeration of Lactococci (gram positive, catalase negative) on M-17 agar (Fluka 63016) at
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37 °C for 48 h, (vii) enumeration of Lactobacilli (gram positive, catalase negative) on acidified MRS agar (Fluka 69964) at 37 °C for 48 h. Microbiological analysis was performed in duplicate using duplicate cheese samples. 2.8. Sensory analysis Sensory assessments were made in cheese samples cut into pieces (5 5 cm) by an internal panel consisting of 10 trained members. Cheese samples were served at room temperature (22 °C). The coded samples were served in a randomized order and were rated for taste, aroma, degree of openness and textural characteristics on a scale of 1–10. Formation of molds on cheese samples was monitored visually and degree of openness was determined on their cross sections similarly by visual inspections. Cross sections were photographed by a digital camera, Canon A430 (Canon Inc., China). Cheese spoilage was determined macroscopically and by sensory tests. The preservation time was defined as the point at which 50% of the panelists rejected the cheese samples. Cheese samples were also compared to commercial hard-type cheeses of Cheddar (US), Emmental (Switzerland), Parmigianoreggiano (Italy), Graviera (Greece) and Kefalograviera (Greece). 2.9. Experimental design and statistical analysis All experiments were carried out in triplicate. In most cases, standard deviation for all values was about ±5%. The effect of the starter culture, ripening temperature and ripening time on final product characteristics was studied 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). 3. Results and discussion 3.1. Effect of thermally-dried kefir starter culture on moisture, preservation time and sensory characteristics of hard-type cheeses Sensory characteristics of cheese products have acquired substantial importance in dairy industry, where their improvement along with the involvement of technologies capable of extending the shelf life of final products constitute major objectives in the development of new products. Free cells of thermally-dried kefir culture as well as immobilised cells on casein were used as starters in hard-type cheese production. Starter culture’s impact on cheese product’s aroma, taste, textural characteristics, degree of openness and preservation time was determined at various ripening temperatures and compared to cheeses made without starter culture
(Table 1). The addition of starter culture and the ripening temperature significantly affected (p < 0.01) taste, flavor, textural characteristics and degree of openness, while a significant interaction (p < 0.05) between the two factors was observed. Similarly, moisture content was significantly affected by the addition of starter culture, ripening temperature and ripening time (p < 0.05) and a significant interaction among the factors was also observed (p < 0.05). Free cells of thermally-dried kefir culture at a concentration of 1 g/L appeared to be the most effective as regards texture, aroma, taste, openness of texture and preservation time especially in cheeses ripened at 5 °C (Table 1). Recent data indicate an increased fermentation activity of immobilised thermally-dried kefir cells on casein compared to free cells (Dimitrellou et al., 2008). In this study, similar results to free cells, but not as high, were also obtained with thermally-dried kefir culture immobilised on casein. Additionally, kefir cheeses were tasted and compared to commercial hard-type cheeses (data not shown). All cheeses scored similar values, whereas rennet cheeses scores were significantly lower (p < 0.05). The preservation time of cheeses, as defined by the test panel, was affected both by the ripening temperature and the addition of starter culture. An extension in preservation time, especially in cheeses ripened at 5 °C and 18 °C, was achieved with the use of 1 g/L kefir culture as starter, followed by cheeses made with immobilized kefir cells on casein. This is possibly a consequence of the lactic acid fermentation due to the action of the starter culture and the relatively rapid pH drop that was observed in cheeses made with starter culture (Table 2). However, resistance to spoilage micro-organisms due to an antimicrobial activity by one or more strains present in this multistrain culture can not be excluded (Santos et al., 2003; Vuyst and Leroy, 2007). On the other hand, the increase in preservation time that was observed in cheeses ripened at room temperature is possibly ought to the very low moisture content of the final products (Table 1). Openness in texture can be formed due to mechanical pressure or the action of micro-organisms that liberate CO2. Depending on the type of cheese, it may be characterized as a quality requirement (Martley and Crow, 1996) and is pursued by the manufacturers. Notably, after 12 days of ripening, increased degree of openness was observed in all cheeses made using thermally-dried kefir (Fig. 1), possibly, due to the activity of heterofermentative lactic acid bacteria, lactose-fermenting and/or non-fermenting yeasts such as Kluyveromyces, Saccharomyces, Candida and Torula included in kefir. 3.2. Effect of thermally-dried kefir starter culture on physicochemical characteristics of hard-type cheeses Hard-type cheeses made with thermally-dried kefir culture, immobilised or free, were monitored during the ripening period
Table 1 Moisture, preservation time, degree of openness, sensory and textural characteristics of hard-type cheeses made without starter culture (rennet cheeses) and cheeses made with the addition of thermally-dried free or immobilized kefir cells, during ripening at various temperatures. Ripening temperature (°C)
Cheese sample
Moisture (%)
Preservation time (days)
Tastea
Aromaa
Textural characteristicsa
Degree of opennessa
5
Rennet cheese 1.0 g/L kefir 1.0 g/L kefir immobilized on casein
50.5 50.1 50.3
38.0 37.8 38.1
20 >90 85
2.86 9.43 8.74
2.83 9.73 8.93
6.29 8.79 8.36
1.61 9.99 6.51
18
Rennet cheese 1.0 g/L kefir 1.0 g/L kefir immobilized on casein
50.4 50.2 50.2
37.2 37.0 37.2
7 32 26
2.69 8.87 9.11
2.91 9.64 9.60
6.24 8.64 8.29
1.64 9.99 6.47
22
Rennet cheese 1.0 g/L kefir 1.0 g/L kefir immobilized on casein
50.4 50.0 50.1
15.8 15.8 16.0
90 >90 >90
3.16 9.54 9.37
3.03 9.76 9.71
1.43 3.27 1.60
1.49 9.81 5.63
a
Taste, aroma, textural characteristics and gas hole formation were assessed on a scale of 1–10.
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Fig. 1. Cross-sections of hard-type cheeses after 12 days of ripening demonstrating the effect of thermally-dried kefir starter culture on texture and degree of openness: (A) rennet cheese; (B) 1.0 g/L kefir; (C) 1.0 g/L kefir immobilized on casein.
Table 2 Residual sugar, ethanol, pH and acidity of hard-type cheeses made without starter culture (rennet cheeses) and cheeses made with the addition of thermally-dried free or immobilized kefir cells, during ripening at various temperatures. Cheese sample
Ripening temperature (°C)
Rennet cheese 5
18
22
1.0 g/L kefir 5
18
22
1.0 g/L kefir immobilized on casein
5
18
22
Ripening time (days)
Lactose (g/ 100 g cheese)
Glucose (g/ 100 g cheese)
Galactose (g/ 100 g cheese)
Ethanol (g/ 100 g cheese)
pH
Acidity as lactic acid (g/ 100 g cheese)
0 15 30 60 90 15 30 60 90 15 30 60 90
1.84 1.00 0.81 0.82 0.81 0.98 0.45 0.41 0.39 1.09 1.04 0.82 0.69
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.02 0.00 0.01 0.01 0.00 0.03 0.01 0.00 0.00 0.00 0.02 0.01 0.00
6.58 5.75 5.24 5.49 5.48 5.70 5.26 5.45 5.46 5.72 5.22 5.49 5.47
0.11 0.41 0.56 0.20 0.23 0.41 0.52 0.29 0.25 0.41 0.59 0.20 0.25
0 15 30 60 90 15 30 60 90 15 30 60 90
0.70 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.86 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.65 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.01 0.02 0.03 0.02 0.03 0.01 0.01 0.00 0.02 0.02 0.01 0.01
6.53 5.10 5.06 5.04 5.12 5.04 5.15 5.27 5.30 5.02 4.98 4.98 5.01
0.10 0.68 0.82 0.84 0.67 0.76 0.66 0.49 0.48 0.85 0.90 0.89 0.86
0 15 30 60 90 15 30 60 90 15 30 60 90
1.18 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.31 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.01 0.00 0.01 0.03 0.03 0.03 0.01 0.01 0.00 0.02 0.01 0.00 0.01
6.52 5.30 5.26 5.19 5.23 5.17 5.20 5.24 5.33 5.08 5.02 5.08 5.21
0.10 0.47 0.49 0.62 0.60 0.65 0.62 0.59 0.46 0.78 0.82 0.79 0.60
by determining lactose, galactose, glucose, ethanol, pH and lactic acid and compared to cheese made without starter culture (rennet cheese) (Table 2). The addition of starter culture, ripening temperature and ripening time significantly affected concentration of lactose (p < 0.05) and acidity (p < 0.05). Content of glucose and galactose were affected by the starter culture and the ripening time (p < 0.05), but not by ripening temperature (p > 0.05). No significant differences were observed in ethanol content and pH (p > 0.05). The acidity of rennet cheese (as g of lactic acid per 100 g of cheese) was significantly lower (p < 0.05) compared to the other types of cheeses ripened at the same temperature. At the end of the ripening process at 5 °C and 22 °C, a 2.9- and 3.4-fold
increase in lactic acid concentration, compared to rennet cheese, were, respectively, observed in cheeses made with free cells of kefir culture. The fermentation of lactose by lactic acid bacteria present in kefir culture is possibly responsible for the rapid increase in lactic acid during the ripening period and the subsequent drop in pH. The increase of lactobacilli counts (Table 3) may also contribute to lactic acid production through the hydrolysis of sugars released from the glycomacropeptide of casein as well as the glycoproteins associated with the fat globule membrane (Rynne et al., 2007). The slight increase of pH values that was observed in all cheeses after the 2nd month of ripening may be caused by proteolysis, lipolysis or lactic acid metabolism of yeasts (Azarnia
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Table 3 Microbial counts of hard-type cheeses made without starter culture (rennet cheeses) and cheeses made with the addition of thermally-dried free or immobilized kefir cells, during ripening at various temperatures. Ripening temperature (°C)
Ripening time (days)
Total aerobic counts (log CFU/g)
Yeasts and molds (log CFU/g)
Lactococci (log CFU/g)
Lactobacilli (log CFU/g)
Rennet
1.0 g kefir/L milk
1.0 g immobilized kefir/L milk
Rennet
1.0 g kefir/L milk
1.0 g immobilized kefir/L milk
Rennet
1.0 g kefir/L milk
1.0 g immobilized kefir/L milk
Rennet
1.0 g kefir/L milk
1.0 g immobilized kefir/L milk
0 15 30 60 90
3.78 6.53 6.17 6.00 5.99
7.19 9.06 8.32 8.21 7.67
7.30 9.33 9.02 8.72 8.19
3.84 6.38 6.17 5.47 5.37
7.36 8.99 8.07 7.81 7.32
7.50 9.06 8.63 8.15 7.86
3.48 3.69 3.00 2.89 2.77
7.10 7.01 6.69 5.72 5.00
7.35 7.18 6.90 6.04 5.63
3.00 6.18 6.79 6.94 6.90
7.02 7.33 7.56 7.88 8.01
7.38 7.57 7.83 8.00 8.16
18
15 30 60 90
6.84 6.83 6.37 6.12
9.33 8.50 8.37 7.82
7.82 9.51 8.89 8.35
5.79 5.69 5.47 5.28
9.05 8.63 8.31 7.81
7.76 9.22 8.80 8.04
3.78 3.07 3.00 2.90
7.25 6.80 5.98 5.18
7.49 7.12 6.36 6.02
5.83 6.57 6.80 6.87
7.41 7.90 8.05 8.22
7.78 7.96 8.13 8.29
22
15 30 60 90
6.61 6.52 6.29 5.89
9.22 8.56 8.04 7.16
9.40 8.78 8.31 7.42
5.95 5.80 5.07 5.00
9.11 8.23 8.05 7.52
9.18 8.67 8.29 7.60
3.69 3.11 3.02 2.60
7.20 6.72 5.63 4.60
7.50 7.20 6.02 5.87
6.05 6.61 6.89 5.63
7.57 7.99 8.23 7.51
7.92 7.98 8.04 7.65
5
et al., 2006; Pisano et al., 2006). Lactose content in kefir cheeses was significantly lower even at the start of the ripening period possibly due to the action of lactic acid bacteria during curd formation. In these cheeses the remaining lactose was metabolised in the first 15 days of ripening. This rapid decrease in lactose as well as the increase in glucose and galactose associated with the addition of kefir culture at the start of the ripening process (Table 2) agree with previous studies where kefir was used as starter culture in fetatype and whey-cheese production (Kourkoutas et al., 2006; Dimitrellou et al., 2007). Ethanol content ranged in low or undetectable levels in both kefir and rennet cheese. 3.3. Effect of thermally-dried kefir starter culture on microbial associations of hard-type cheeses Microbial counts of total aerobic counts, yeasts and moulds, lactococci and lactobacilli, were also determined during ripening of cheeses at all temperatures (Table 3). The addition of starter culture, ripening temperature and ripening time significantly affected (p < 0.05) all microbial counts. The initial concentration of all microbial counts was greater than 7.02 log CFU/g in cheeses made using kefir culture and less than 3.84 log CFU/g in rennet cheese. All microbial taxa were generally at maximum levels on the 15th day of ripening period. Exceptions were observed in total aerobic counts as well as in yeasts and moulds in cheeses made with immobilized kefir culture, as maximum levels were observed on the 30th day of ripening. On the other hand, lactobacilli counts continued to increase in all cheese samples reaching maximum levels in those made at 18 °C with free and immobilized cells of kefir culture (up to 8.22 log CFU/g and 8.29 log CFU/g, respectively). However, in cheeses ripened at 22 °C a slight drop of lactobacilli was detected on the last day of the ripening process possibly due to the low moisture content of those cheeses (Table 1). Our results indicate a significant increase in lactobacilli at the end of the ripening process associated with the use of kefir culture. This may constitute an important tool in the development of novel probiotic foods given the general concern with regard to viability of such cultures at the time of consuming in order to be effective. The decrease of most microbial counts after the 15th day of ripening can be attributed to the inhibitory effect of the low pH, the consumption of sugars as well as to the moisture loss (Swearingen et al., 2001; Pisano et al., 2006), or even to an antimicrobial activity originating from members of the lactobacilli taxon (Santos et al., 2003; Vuyst and Leroy, 2007). The latter would also agree with the simultaneous increase of lactobacilli counts. Generally, the addition of
kefir culture in cheese samples significantly increased all microbial counts. 3.4. Effect of thermally-dried kefir starter culture on flavor and aroma of hard-type cheeses Proteolysis, lipolyisis and carbohydrate degradation are the most important biochemical processes that occur during cheese ripening due to their contribution in volatile compounds, key components of cheese flavor (Engels et al., 1997). Lactic acid bacteria starter cultures, such as kefir, produce a plethora of enzymes that contribute to the formation of volatiles via proteolysis, lipolysis and carbohydrate degradation during ripening. Such enzymes are peptidases which are involved in the transformation of casein into free amino acids which are further degraded to volatile aroma compounds. Other enzymes are esterases and lipases that hydrolyze triglycerides of milk fat in free fatty acids and transform alcohols and glycerides to esters (Skeie et al., 2008). GC/MS analysis was employed to determine volatile compounds in cheese made with thermally-dried free cells of kefir culture and ripened for 90 days at 5 °C given that this type of cheese had the highest overall score as regards preservation time, textural characteristics, taste and aroma (Table 1). Control cheese made only with rennet was also assayed (Table 4). In total, 20 volatile compounds were detected in rennet cheese while 33 compounds in cheese made with 1 g/L kefir culture contributing to total concentrations of 49.37 lg/ kg and 155.82 lg/kg, respectively. Increase in volatile composition of different types of cheese by kefir starter cultures has also been observed in previous studies (Beshkova et al., 2003; Kourkoutas et al., 2006; Dimitrellou et al., 2007). The main contribution of kefir in concentration of volatiles was observed in organic acids (315% increase) and carbonyl compounds (1401% increase) in which major differences (83%) between rennet and kefir cheese as regards diversity were also observed. Organic acids are important components of cheese flavor and may originate either from milk fat lipolysis or from breakdown of amino acids (Urbach, 1993), while carbonyl compounds and particularly methyl ketones such as 2heptanone and 2-undecanone are formed by enzymic oxidative decarboxylation of fatty acids (Welsh et al., 1989) and may give a mould-ripened character (Engels et al., 1997). Kefir culture was also associated with an important increase in ester concentration (191%). Esters are mainly derived from fatty acids (Engels et al., 1997). Most of the esters that were detected have a fruity aroma attribute. Butanoic, hexanoic, heptanoic, octanoic and nonanoic acids provide a cheesy aroma character, while acetic and decanoic
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Table 4 SPME-GC/MS analysis of aroma-related compounds (lg/kg cheese) isolated from cheeses ripened for 90 days at 5 °C and made either without starter culture (rennet cheese) or using thermally-dried kefir (1.0 g/L) as starter culture. Compound
Identification method
Rennet cheese
Kefir cheese
Esters Ethyl butyrate Ethyl hexanoate Ethyl octanoate Ethyl nonanoate Ethyl decanoate Ethyl-9-decenoate Ethyl-9-hexadecenoate Ethyl dodecanoate Ethyl tetradecanoate Ethyl pentadecanoate Phenyl ethyl acetate Total esters
RTa, KIb, MSc RT, KI, MS RT, KI, MS RT, MS RT, MS MS MS KI, MS KI, MS MS MS
0.13 1.10 3.47 0.00 5.26 0.00 0.00 0.00 0.00 1.35 1.21 12.52
2.25 1.45 5.74 3.93 12.47 3.14 0.10 3.12 0.05 0.00 4.27 36.52
Organic acids Acetic acid Butanoic acid Hexanoic acid Heptanoic acid Octanoic acid Octadecanoic acid Nonanoic acid Decanoic acid Dodecanoic acid Tetradecanoic acid Total organic acids
KI, MS KI, MS KI, MS KI, MS RT, KI, MS KI, MS RT, KI, MS RT, KI, MS KI, MS KI, MS
0.00 0.00 0.02 0.00 5.65 0.07 0.00 2.37 1.06 10.1 19.27
4.41 10.07 9.63 6.25 8.71 0.00 7.06 2.56 1.93 29.53 80.15
Alcohols Ethanol 2-Hexanol 3-Methyl-1-butanol 3-Methyl-2-buten-1-ol 2-Undecanol 2-Nonanol 2,3-Butandiol 1,3-Butandiol 1-Tridecanol Phenyl ethanol Total alcohols
RT, KI, MS MS RT, MS MS MS MS KI, MS KI, MS KI, MS RT, KI, MS
10.08 0.00 0.12 0.00 0.25 0.00 1.60 1.05 0.00 3.75 16.85
18.84 0.12 0.26 2.13 3.18 0.50 1.82 0.00 0.62 5.72 33.19
Carbonyl compounds Acetoin 2-Heptanone 2-Undecanone Nonanal Benzaldehyde 2-Butenal Total carbonyl compounds
MS RT, MS KI, MS KI, MS KI, MS KI, MS
0.67 0.00 0.00 0.06 0.00 0.00 0.73
6.14 0.24 0.51 0.00 3.90 0.17 10.96
a b c
RT: positive identification by retention times that agree with authentic compounds and by mass spectra of authentic compounds generated in the laboratory. KI: tentative identification by Kovats’ retention index. MS: positive identification by mass spectra obtained from NIST107, NIST21 and SZTERP libraries.
acid are known for their acetic and sour aroma profile (Moio et al., 2000; Qian and Reineccius, 2002). Dodecanoic acid provides a soapy odour and tetradecanoic acid a waxy and oily odour. The lowest increase in volatile compounds associated with the use of kefir culture was observed in alcohols (97%). Alcohols can be derived from amino acids transamination and oxidative deamination followed by decarboxylation (Moio et al., 2000; Qian and Reineccius, 2002) and are common in hard-type cheeses such as Emmental, Parmezan, Gouda, Cheddar and Comté (Engels et al., 1997; Mariaca et al., 2001). Ethanol had a higher concentration in kefir cheese samples (18.84 lg/kg) rather than in rennet cheese (10.08 lg/kg) possibly due to mesophilic starters present in kefir culture (Massouras et al., 2006). Among the alcohols related to the use of kefir as starter culture, only 2-hexanol has also been detected in other cheeses such as Cheddar, Emmental, Comté, Camembert, Provolone and Parmezan (Engels et al., 1997; Mariaca et al., 2001). Our results indicate a significant contribution of kefir culture in volatile compounds as the composition of esters, organic acids, alcohols and carbonyl compounds was radically modified.
3.5. Effect of the drying process on cheese characteristics Data presented in this study as regards the effect of thermallydried kefir culture on moisture, preservation time, degree of openness, sensory and textural characteristics of hard-type cheese products agree with a recent study from our laboratory involving the use of freeze-dried kefir cells in hard-type cheese production (Katechaki et al., 2008). However, the drying process appears to affect the volatile profile of these products (Table 5). Both number and concentration of total volatiles were greater in cheese products made with thermally-dried kefir. Significant differences were observed not only in alcohol and carbonyl compound concentration but also in the variety of volatiles belonging to these groups (33% different). Given the detrimental effect of freeze-drying on cultures viability (Morgan et al., 2006) especially when compared to thermal-drying (Kopsahelis et al., 2007), the effect of the drying process on microbial associations of the starter culture is more likely the main reason for such alterations in the volatile profile. It is clear from our results that important quality characteristics of
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Table 5 Effect of freeze- and thermally-dried kefir culture on aroma-related compounds of hard-type cheese products compared with cheese made without kefir inoculants. Kefir culture
Increase in total number of volatile compounds (%)
Freeze-dried Thermally-dried
48 57
Increase in concentration (%) Total compounds
Esters
Organic acids
Alcohols
Carbonyl compounds
179 216
164 192
323 316
20 97
1683 18167
cheese products can not only be obtained by the use of kefir culture as starter in hard-type cheese production but also by the method of drying this culture. 4. Conclusion Our results indicate the suitability of thermally-dried kefir culture as starter in hard-type cheese production. Both free and immobilised cells of kefir culture led to the production of improved cheese products as regards preservation time, sensory and textural characteristics. Kefir culture’s cheese products were characterized by high lactobacilli counts at the end of the ripening process, thus constituting a promising probiotic vehicle given the general concern with regard to viability of such cultures at the time of consuming in order to be effective. Finally, thermal drying appeared to contribute to the volatile composition of the final product giving a different character in cheeses made with thermally-dried kefir culture even when compared to cheeses made with the alternate method of freeze-drying. The thermal drying process used in this study was simple, and of low cost, lower than that of other energy consuming methods such as freeze drying and therefore may constitute a potential process of drying cultures in cheese industry. Acknowledgments This work has been performed within the framework of the Regional Operational Programme (ROP) of Western Greece and was co-funded by the European Regional Development Fund and the Region of Western Greece with final beneficiary the Greek General Secretariat for Research and Technology. References AOAC, 1995. Moisture in Cheese, 33.7.04 Method 948.12, Official Methods of Analysis, 16th ed., Association of Official Analytical Chemists, Arlington, VA. Assadi, M.M., Abdolmaleki, F., Mokarrame, R.R., 2008. Application of whey in fermented beverage production using kefir starter culture. Nutr. Food Sci. 38, 121–127. Azarnia, S., Robert, N., Lee, B., 2006. Biotechnological methods to accelerate Cheddar cheese ripening. Critic. Rev. Biotechnol. 26, 121–143. Beshkova, D.M., Simova, E.D., Frengova, G.I., Simov, Z.I., Dimitrov, Z.P., 2003. Production of volatile aroma compounds by kefir starter cultures. Int. Dairy J. 13, 529–535. Champagne, C.P., Morin, N., Couture, R., Gagnon, C., Jelen, P., Lacroix, C., 1992. The potential of immobilized cell technology to produce freeze-dried, phageprotected cultures of Lactococcus lactis. Food Res. Int. 25, 419–427. Dimitrellou, D., Kourkoutas, Y., Banat, I.M., Marchant, R., Koutinas, A.A., 2007. Whey cheese production using freeze-dried kefir culture as a starter. J. Appl. Microbiol. 103, 1170–1183. Dimitrellou, D., Tsaousi, K., Kourkoutas, Y., Panas, P., Kanellaki, M., Koutinas, A.A., 2008. Fermentation efficiency of thermally-dried immobilized kefir on casein as starter culture. Process Biochem. 43, 1323–1329. Engels, W.J.M., Dekker, R., de Jong, C., Neeter, R., Visser, S., 1997. A comparative study of volatile compounds in the water-soluble fraction of various types of ripened cheese. Int. Dairy J. 7, 255–263. Farnworth, E.R., 1999. Kefir: from folklore to regulatory approval. J. Nutraceut. Funct. Med. Foods 1, 57–68. Heller, K.J., 2001. Probiotic bacteria in fermented foods: product characteristics and starter organisms. Am. J. Clin. Nutr. 73, 374–379.
Iconomopoulou, M., Psarianos, K., Kanellaki, M., Koutinas, A.A., 2000. Delignified cellulosic material supported biocatalyst as freeze dried product in alcoholic fermentation. J. Agric. Food Chem. 48, 958–961. Katechaki, E., Panas, P., Kandilogiannakis, L., Koutinas, A., 2008. Production of hardtype cheese using free or immobilised freeze-dried kefir cells as starter culture. J. Agric. Food Chem. 56, 5316–5323. Kopsahelis, N., Panas, P., Kourkoutas, Y., Koutinas, A.A., 2007. Evaluation of thermally-dried immobilized cell of Lactobacillus delbrueckii subsp. Bulgaricus on apple pieces as a potent starter culture. J. Agric. Food Chem. 55, 9829–9836. Kourkoutas, Y., Kandylis, P., Panas, P., Dooley, J.S.G., Nigam, P., Koutinas, A.A., 2006. Evaluation of freeze-dried kefir coculture as starter in feta-type cheese production. Appl. Environ. Microbiol. 72, 6124–6135. Koutinas, A.A., Athanasiadis, I., Bekatorou, A., Iconomopoulou, M., Blekas, G., 2005. Kefir yeast technology: scale-up in SCP production using milk whey. Biotechnol. Bioeng. 89, 787–796. Mariaca, R.G., Imhof, M.I., Bosset, J.O., 2001. Occurrence of volatile chiral compounds in dairy products, especially cheese – a review. Eur. Food Res. Technol. 212, 253–261. Martley, F.G., Crow, V.L., 1996. Open texture in cheese: the contribution of gas production of microorganisms and cheese manufacturing practices. J. Dairy Res. 63, 489–507. Massouras, T., Pappa, E.C., Mallatou, H., 2006. Headspace analysis of volatile flavour compounds of teleme cheese made from sheep and goat milk. Int. J. Dairy Technol. 59, 250–256. Moio, L., Piombino, P., Addeo, F., 2000. Odour-impact compounds of Gorgonzola cheese. J. Dairy Res. 67, 273–285. Morgan, C.A., Herman, N., White, P.A., Vesey, G., 2006. Preservation of microorganisms by drying, a review. J. Microbiol. Meth. 66, 183–193. Ostojic´, S., Pavlovic´, M., Zˇivic´, M., Filipovic´, Z., Gorjanovic´, S., Hranisavljevic´, S., Dojc´inovic´, M., 2005. Processing of whey from dairy industry waste. Environ. Chem. Lett. 3, 29–32. Paraskevopoulou, A., Athanasiadis, I., Kanellaki, M., Bekatorou, A., Blekas, G., Kiosseoglou, V., 2003. Functional properties of single cell protein produced by kefir microflora. Food Res. Int. 36, 431–438. Papapostolou, H., Bosnea, L.A., Koutinas, A.A., Kanellaki, M., 2008. Fermentation efficiency of thermally dried kefir. Biores. Technol. 99, 6949–6956. Papavasiliou, G., Kourkoutas, Y., Rapti, A., Sipsas, V., Soupioni, M., Koutinas, A.A., 2008. Production of freeze-dried kefir culture using whey. Int. Dairy J. 18, 247– 254. Pisano, M.B., Fadda, M.E., Deplano, M., Corda, A., Cosentino, S., 2006. Microbiological and chemical characterization of Fiore Sardo, a traditional Sardinian cheese made from ewe’s milk. Int. J. Dairy Technol. 59, 171–179. Qian, M., Reineccius, G., 2002. Identification of aroma compounds in parmigianoreggiano cheese by gas chromatography/olfactometry. J. Dairy Sci. 85, 1362– 1369. Rimada, P.S., Abraham, A.G., 2001. Polysaccharide production by kefir grains during whey fermentation. J. Dairy Res. 68, 653–661. Rynne, N.M., Beresford, T.P., Kelly, A.L., Guinee, T.P., 2007. Effect of milk pasteurisation temperature on age-related changes in lactose metabolism, pH and the growth of non-starter lactic acid bacteria in half-fat cheddar cheese. Food Chem. 100, 375–382. Santos, A., San Mauro, M., Sanchez, A., Torres, J.M., Marquina, D., 2003. The antimicrobial properties of different strains of Lactobacillus spp. Isolated from kefir. Syst. Appl. Microbiol. 26, 434–437. Siso, M.I.G., 1996. The biotechnological utilization of cheese whey: a review. Biores. Technol. 57, 1–11. Skeie, S., Kieronczyk, A., Naess, R.-M., Østlie, H., 2008. Lactobacillus adjuncts in cheese: their influence on the degradation of citrate and serine during ripening of a washed curd cheese. Int. Dairy J. 18, 158–168. Swearingen, P.A., O’Sullivan, D.J., Warthesen, J.J., 2001. Isolation, characterization, and influence of native, nonstarer lactic acid bacteria on Cheddar cheese quality. J. Dairy Sci. 84, 50–59. Urbach, G., 1993. Relations between cheese flavor and chemical composition. Int. Dairy J. 3, 389–422. Vuyst, L., Leroy, F., 2007. Bacteriocins from lactic acid bacteria: production, purification, and food applications. J. Mol. Microbiol. Biotechnol. 13, 194–199. Welsh, F.W., Murray, W.D., Williams, R.E., 1989. Microbiological and enzymatic production of flavor and fragrance chemicals. CRC Critic. Rev. Biotechnol. 9, 105–169.
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Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production Eleftheria Katechaki a, Panayiotis Panas a,*, Yiannis Kourkoutas b, Dionisis Koliopoulos c, Athanasios A. Koutinas a a
Food Biotechnology Group, Section of Analytical Environmental and Applied Chemistry, Department of Chemistry, University of Patras, GR-26500 Patras, Greece Department of Molecular Biology and Genetics, Democritus University of Thrace, Dimitras 19, Alexandroupolis, GR-68100 Alexandroupolis, Greece c AVIGAL S.A., Elliniko, Farres, GR-25008 Achaia, Greece b
a r t i c l e
i n f o
Article history: Received 14 November 2008 Received in revised form 25 February 2009 Accepted 26 February 2009 Available online 28 March 2009 Keywords: Kefir Whey Hard-type cheese Ripening Dried starters
a b s t r a c t In an attempt to seek for suitable dried cultures, thermally-dried kefir was employed as starter in hardtype cheese production and tested in cheeses ripened at 5, 18 and 22 °C. Both free and immobilised on casein kefir cells were used and compared to cheese made without starter culture. Cheese products made with free cells of kefir culture were characterized by longer preservation time, improved aroma, taste, texture characteristics and increased degree of openness. Volatile profiles obtained by GC/MS analysis revealed a 216% increase in total concentration of esters, organic acids, alcohols and carbonyl compounds between cheeses prepared with and without kefir culture. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Recent decades, whey has attracted researchers’ interest due to its nutrient-rich content and its availability in dairy industries as a milk by-product constituting a potential substrate for important biological processes. It corresponds to 85–95% of the total milk volume entering the dairy process and contains about 55% of the nutrients present in the original milk such as proteins, lactose, vitamins and minerals (Siso, 1996). Due to these characteristics whey constitutes a popular waste for the production of beverages, ethanol, single cell protein, polysaccharides, purified lactose, organic acids, flavor enhancers and other useful products (Siso, 1996; Ostojic´ et al., 2005). Kefir is a mixed culture consisting of lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc), yeasts (Kluyveromyces, Candida, Saccharomyces, Pichia) and acetic acid bacteria. This culture is mainly known for its usage in the production of a refreshing fermented beverage called ‘‘kefir” that is popular in Eastern–European countries (Farnworth, 1999; Paraskevopoulou et al., 2003). The main role of such starter cultures with lactic acid bacteria in cheese ripening is the production of lactic acid through lactose
* Corresponding author. Tel.: +30 2610 997123; fax: +30 2610 997105. E-mail addresses: [email protected] (E. Katechaki), [email protected] (P. Panas), [email protected] (D. Koliopoulos), [email protected] (A.A. Koutinas). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.02.061
metabolism and the reduction of pH. Moreover, the potential probiotic and antimicrobial effects of lactic acid bacteria identified in kefir grains have stimulated a major research effort in recent years (Heller, 2001; Kourkoutas et al., 2006; Vuyst and Leroy, 2007). Whey has been extensively used as raw material for kefir fermentation (Rimada and Abraham, 2001; Paraskevopoulou et al., 2003; Koutinas et al., 2005; Assadi et al., 2008; Papapostolou et al., 2008; Papavasiliou et al., 2008). Recently, kefir biomass revealed its suitability as a starter culture in a variety of cheese products, hard-type, feta-type cheese and whey-cheese, mainly due to its effect on quality, preservation time and safety properties of the final product (Kourkoutas et al., 2006; Dimitrellou et al., 2007; Katechaki et al., 2008). In the above studies, kefir culture was used in freeze-dried form and was found to increase the shelf life of the final products and both the variety and concentration of esters, free fatty acids, alcohols and carbonyl compounds. This culture appeared also to be effective in fermentation of a lactose-rich substrate even when dried kefir cells immobilised on casein were used (Dimitrellou et al., 2008). It has been well demonstrated that immobilization technology results in enhanced survival rates and biocatalysts stability during processing and storage (Champagne et al., 1992; Iconomopoulou et al., 2000; Kopsahelis et al., 2007). Drying of cultures, on the other hand, has acquired a significant role in production of commercial cell cultures as it offers long term preservation of micro-organisms, cell stability and convenience in
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transportation, storage and cell dosage of cell cultures (Morgan et al., 2006). Thermal-drying is an alternate approach to the most popular method of freeze-drying that is not suffering from the high investment cost, the use of cryoprotectants and the decreased cell viability. Indeed, the thermal-drying process appeared to be more efficient on cell viability (84%) of kefir culture than that (78%) of freeze-drying (Kopsahelis et al., 2007). In this study, the impact of thermally-dried kefir as a new starter culture in the production of hard-type cheese is evaluated. Results on the effect of thermally-dried kefir culture immobilized on casein on aroma, flavor, textural characteristics, degree of openness and on the microbial associations throughout the ripening process are presented and compared with cheeses made with free cells of kefir. The impact of thermally-dried kefir culture on volatile profiles is presented and discussed.
2. Methods 2.1. Starter strain Kefir culture was isolated from a commercial kefir drink and used as starter in cheese production. Kefir was grown at 30 °C on a sterilized synthetic medium containing (%w/v): 4 lactose, 0.4 yeast extract, 0.1 (NH4)2SO4, 0.1 KH2PO4, and 0.5 MgSO4.7H2O (Merck, Darmstadt, Germany). Pressed wet cells were harvested by centrifugation at 5000 rpm for 10 min. Harvested biomass (4 g) was resuspended directly in 500 mL of whey for aerobic fermentation. Kefir was incubated in whey at 30 °C for further production of starter culture. 2.2. Immobilization process Pressed wet kefir cells were added in commercial pasteurized bovine milk (0% fat) followed by casein isolation as described by Dimitrellou et al. (2008). 2.3. Drying process Thermally-dried kefir cultures were obtained by drying free or immobilized kefir cells in an oven equipped with air circulation (J.P. Selecta, Spain) at 33 °C. The drying process was monitored by weighing the drying material at various time intervals until constant weight. 2.4. Cheese making and sampling Bulk ewes’ milk was obtained from a local dairy factory (AVIGAL S.A., Elliniko, Farres, Achaia). Cheese was produced and ripened using the method of Katechaki et al. (2008). In this method, the starter strain described previously was used instead of freeze-dried kefir culture. Hard-type cheeses were designated as follows: cheese A or rennet cheese (without any starter culture), cheese B with 1.0 g/L added kefir and cheese C with 1.0 g/L added kefir immobilized on casein. Rennet was added in all cheese samples. All experiments were repeated three times. Cheeses were ripened at 5 °C, 18 °C and 22 °C for 90 days. Samples from each cheese were taken at 0, 15, 30, 60 and 90 days. 2.5. Physicochemical analysis Moisture was determined in cheese samples according to AOAC (1995). Samples for lactic acid, ethanol and residual sugar analysis were prepared as follows: 20 g of cheese samples were macerated with warm water (40 °C) up to a total volume of 210 mL followed by filtration.
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Acidity was quantified by titration with 0.1 N NaOH using phenolphthalein as indicator (AOAC, 1995) and expressed as lactic acid content. Lactose, glucose and galactose were determined by high performance liquid chromatography. A Shimadzu chromatograph (Kyoto, Japan) with an SCR-101 N stainless steel column, a LC-9A pump, a CTO-10A oven at 60 °C and a RID-6A refractive index detector was used. Three times distilled water was used as mobile phase at the flow rate of 0.8 mL/min, and 1-butanol was used as an internal standard (IS). Cheese filtrate (0.5 mL) and 1% v/v solution of 1butanol (2.5 mL) were diluted to 50 mL, the final solution was filtered through a 0.2 lm pore size filter, and 40 lL of the filtrate were injected directly into the column. Residual sugar concentrations were calculated using standard curves by correlating the ratio of residual sugar peak areas divided by 1-butanol peak areas to residual sugar concentrations. Ethanol was determined by gas chromatography on a Shimadzu GC-8A system consisting of a Porapac-S packed column, N2 as carrier gas (20 mL/min), and a FID detector. The injection port and detector temperatures were 210 °C and the column temperature was programmed at 120–170 °C, rising by 10 °C/min. 1-Butanol (0.1% v/v) was used as the internal standard and samples of 2 lL were injected directly in the column. Determination of ethanol concentration was carried out using standard curves. Cheese pH was measured using a digital pH meter (CyberScan pH 10 and pH 100, Eutech Instruments, Singapore). 2.6. Solid-phase Microextraction (SPME) gas chromatography/mass spectrometry (GC/MS) analysis Cheese samples ripened at 5 °C for 90 days were assayed for aroma volatiles using SPME GC/MS analysis. Grated cheese samples (7 g each) were placed into a 20-mL headspace vial fitted with a teflon-lined septum sealed with an aluminum crimp seal, through which the SPME syringe needle (Supelco, Bellefonte, PA, USA) was introduced. The internal standard constituted methyl octanoate (Merck, Darmstadt, Germany) at a final concentration of 125.29 lg/kg of cheese. Sample incubation at 80 °C for 30– 35 min was followed by GC/MS analysis of the absorbed volatile compounds. A Shimadzu GC-17A coupled to a GCMS-QP5050A mass spectrometer with a Supelcowax-10 column (60 m– 0.32 mm i.d. 0.25 lm film thickness) and helium as carrier gas (linear velocity of 1.5 mL/min) were used. Oven temperature was set as follows: 35 °C for 3 min; a temperature gradient of 5 °C/ min up to 110 °C; a temperature gradient of 10 °C/min up to 240 °C; and a final extension at 240 °C for 10 min. The injector was operated in splitless mode at 280 °C, detector temperature was set at 250 °C and the mass spectrometer was operated in the electron impact mode with the electron energy set at 70 eV. Identification of compounds was done by comparing the retention times and MS data with those of standard compounds and by MS data obtained from NIST107, NIST21 and SZTERP libraries. The peak areas were measured from the full scan chromatograph using total ion current (TIC). 2.7. Microbiological analysis Inoculum for microbiological analysis was prepared by blending 10 g of cheese from cheese interior of each sample with 90 mL of sterilized 2% tri-sodium citrate solution and subjected to serial dilutions in sterilized Ringer’s solution (1/4 strength). The following microbiological assays were performed: (i) determination of total aerobic counts on plate count agar (Fluka 70188) at 30 °C for 48 h, (ii) enumeration of yeasts and moulds on potato glucose agar (Fluka 70139) at 30 °C for 48 h, (iii) enumeration of Lactococci (gram positive, catalase negative) on M-17 agar (Fluka 63016) at
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37 °C for 48 h, (vii) enumeration of Lactobacilli (gram positive, catalase negative) on acidified MRS agar (Fluka 69964) at 37 °C for 48 h. Microbiological analysis was performed in duplicate using duplicate cheese samples. 2.8. Sensory analysis Sensory assessments were made in cheese samples cut into pieces (5 5 cm) by an internal panel consisting of 10 trained members. Cheese samples were served at room temperature (22 °C). The coded samples were served in a randomized order and were rated for taste, aroma, degree of openness and textural characteristics on a scale of 1–10. Formation of molds on cheese samples was monitored visually and degree of openness was determined on their cross sections similarly by visual inspections. Cross sections were photographed by a digital camera, Canon A430 (Canon Inc., China). Cheese spoilage was determined macroscopically and by sensory tests. The preservation time was defined as the point at which 50% of the panelists rejected the cheese samples. Cheese samples were also compared to commercial hard-type cheeses of Cheddar (US), Emmental (Switzerland), Parmigianoreggiano (Italy), Graviera (Greece) and Kefalograviera (Greece). 2.9. Experimental design and statistical analysis All experiments were carried out in triplicate. In most cases, standard deviation for all values was about ±5%. The effect of the starter culture, ripening temperature and ripening time on final product characteristics was studied 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). 3. Results and discussion 3.1. Effect of thermally-dried kefir starter culture on moisture, preservation time and sensory characteristics of hard-type cheeses Sensory characteristics of cheese products have acquired substantial importance in dairy industry, where their improvement along with the involvement of technologies capable of extending the shelf life of final products constitute major objectives in the development of new products. Free cells of thermally-dried kefir culture as well as immobilised cells on casein were used as starters in hard-type cheese production. Starter culture’s impact on cheese product’s aroma, taste, textural characteristics, degree of openness and preservation time was determined at various ripening temperatures and compared to cheeses made without starter culture
(Table 1). The addition of starter culture and the ripening temperature significantly affected (p < 0.01) taste, flavor, textural characteristics and degree of openness, while a significant interaction (p < 0.05) between the two factors was observed. Similarly, moisture content was significantly affected by the addition of starter culture, ripening temperature and ripening time (p < 0.05) and a significant interaction among the factors was also observed (p < 0.05). Free cells of thermally-dried kefir culture at a concentration of 1 g/L appeared to be the most effective as regards texture, aroma, taste, openness of texture and preservation time especially in cheeses ripened at 5 °C (Table 1). Recent data indicate an increased fermentation activity of immobilised thermally-dried kefir cells on casein compared to free cells (Dimitrellou et al., 2008). In this study, similar results to free cells, but not as high, were also obtained with thermally-dried kefir culture immobilised on casein. Additionally, kefir cheeses were tasted and compared to commercial hard-type cheeses (data not shown). All cheeses scored similar values, whereas rennet cheeses scores were significantly lower (p < 0.05). The preservation time of cheeses, as defined by the test panel, was affected both by the ripening temperature and the addition of starter culture. An extension in preservation time, especially in cheeses ripened at 5 °C and 18 °C, was achieved with the use of 1 g/L kefir culture as starter, followed by cheeses made with immobilized kefir cells on casein. This is possibly a consequence of the lactic acid fermentation due to the action of the starter culture and the relatively rapid pH drop that was observed in cheeses made with starter culture (Table 2). However, resistance to spoilage micro-organisms due to an antimicrobial activity by one or more strains present in this multistrain culture can not be excluded (Santos et al., 2003; Vuyst and Leroy, 2007). On the other hand, the increase in preservation time that was observed in cheeses ripened at room temperature is possibly ought to the very low moisture content of the final products (Table 1). Openness in texture can be formed due to mechanical pressure or the action of micro-organisms that liberate CO2. Depending on the type of cheese, it may be characterized as a quality requirement (Martley and Crow, 1996) and is pursued by the manufacturers. Notably, after 12 days of ripening, increased degree of openness was observed in all cheeses made using thermally-dried kefir (Fig. 1), possibly, due to the activity of heterofermentative lactic acid bacteria, lactose-fermenting and/or non-fermenting yeasts such as Kluyveromyces, Saccharomyces, Candida and Torula included in kefir. 3.2. Effect of thermally-dried kefir starter culture on physicochemical characteristics of hard-type cheeses Hard-type cheeses made with thermally-dried kefir culture, immobilised or free, were monitored during the ripening period
Table 1 Moisture, preservation time, degree of openness, sensory and textural characteristics of hard-type cheeses made without starter culture (rennet cheeses) and cheeses made with the addition of thermally-dried free or immobilized kefir cells, during ripening at various temperatures. Ripening temperature (°C)
Cheese sample
Moisture (%)
Preservation time (days)
Tastea
Aromaa
Textural characteristicsa
Degree of opennessa
5
Rennet cheese 1.0 g/L kefir 1.0 g/L kefir immobilized on casein
50.5 50.1 50.3
38.0 37.8 38.1
20 >90 85
2.86 9.43 8.74
2.83 9.73 8.93
6.29 8.79 8.36
1.61 9.99 6.51
18
Rennet cheese 1.0 g/L kefir 1.0 g/L kefir immobilized on casein
50.4 50.2 50.2
37.2 37.0 37.2
7 32 26
2.69 8.87 9.11
2.91 9.64 9.60
6.24 8.64 8.29
1.64 9.99 6.47
22
Rennet cheese 1.0 g/L kefir 1.0 g/L kefir immobilized on casein
50.4 50.0 50.1
15.8 15.8 16.0
90 >90 >90
3.16 9.54 9.37
3.03 9.76 9.71
1.43 3.27 1.60
1.49 9.81 5.63
a
Taste, aroma, textural characteristics and gas hole formation were assessed on a scale of 1–10.
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Fig. 1. Cross-sections of hard-type cheeses after 12 days of ripening demonstrating the effect of thermally-dried kefir starter culture on texture and degree of openness: (A) rennet cheese; (B) 1.0 g/L kefir; (C) 1.0 g/L kefir immobilized on casein.
Table 2 Residual sugar, ethanol, pH and acidity of hard-type cheeses made without starter culture (rennet cheeses) and cheeses made with the addition of thermally-dried free or immobilized kefir cells, during ripening at various temperatures. Cheese sample
Ripening temperature (°C)
Rennet cheese 5
18
22
1.0 g/L kefir 5
18
22
1.0 g/L kefir immobilized on casein
5
18
22
Ripening time (days)
Lactose (g/ 100 g cheese)
Glucose (g/ 100 g cheese)
Galactose (g/ 100 g cheese)
Ethanol (g/ 100 g cheese)
pH
Acidity as lactic acid (g/ 100 g cheese)
0 15 30 60 90 15 30 60 90 15 30 60 90
1.84 1.00 0.81 0.82 0.81 0.98 0.45 0.41 0.39 1.09 1.04 0.82 0.69
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.02 0.00 0.01 0.01 0.00 0.03 0.01 0.00 0.00 0.00 0.02 0.01 0.00
6.58 5.75 5.24 5.49 5.48 5.70 5.26 5.45 5.46 5.72 5.22 5.49 5.47
0.11 0.41 0.56 0.20 0.23 0.41 0.52 0.29 0.25 0.41 0.59 0.20 0.25
0 15 30 60 90 15 30 60 90 15 30 60 90
0.70 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.86 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.65 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.01 0.02 0.03 0.02 0.03 0.01 0.01 0.00 0.02 0.02 0.01 0.01
6.53 5.10 5.06 5.04 5.12 5.04 5.15 5.27 5.30 5.02 4.98 4.98 5.01
0.10 0.68 0.82 0.84 0.67 0.76 0.66 0.49 0.48 0.85 0.90 0.89 0.86
0 15 30 60 90 15 30 60 90 15 30 60 90
1.18 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.31 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.01 0.00 0.01 0.03 0.03 0.03 0.01 0.01 0.00 0.02 0.01 0.00 0.01
6.52 5.30 5.26 5.19 5.23 5.17 5.20 5.24 5.33 5.08 5.02 5.08 5.21
0.10 0.47 0.49 0.62 0.60 0.65 0.62 0.59 0.46 0.78 0.82 0.79 0.60
by determining lactose, galactose, glucose, ethanol, pH and lactic acid and compared to cheese made without starter culture (rennet cheese) (Table 2). The addition of starter culture, ripening temperature and ripening time significantly affected concentration of lactose (p < 0.05) and acidity (p < 0.05). Content of glucose and galactose were affected by the starter culture and the ripening time (p < 0.05), but not by ripening temperature (p > 0.05). No significant differences were observed in ethanol content and pH (p > 0.05). The acidity of rennet cheese (as g of lactic acid per 100 g of cheese) was significantly lower (p < 0.05) compared to the other types of cheeses ripened at the same temperature. At the end of the ripening process at 5 °C and 22 °C, a 2.9- and 3.4-fold
increase in lactic acid concentration, compared to rennet cheese, were, respectively, observed in cheeses made with free cells of kefir culture. The fermentation of lactose by lactic acid bacteria present in kefir culture is possibly responsible for the rapid increase in lactic acid during the ripening period and the subsequent drop in pH. The increase of lactobacilli counts (Table 3) may also contribute to lactic acid production through the hydrolysis of sugars released from the glycomacropeptide of casein as well as the glycoproteins associated with the fat globule membrane (Rynne et al., 2007). The slight increase of pH values that was observed in all cheeses after the 2nd month of ripening may be caused by proteolysis, lipolysis or lactic acid metabolism of yeasts (Azarnia
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Table 3 Microbial counts of hard-type cheeses made without starter culture (rennet cheeses) and cheeses made with the addition of thermally-dried free or immobilized kefir cells, during ripening at various temperatures. Ripening temperature (°C)
Ripening time (days)
Total aerobic counts (log CFU/g)
Yeasts and molds (log CFU/g)
Lactococci (log CFU/g)
Lactobacilli (log CFU/g)
Rennet
1.0 g kefir/L milk
1.0 g immobilized kefir/L milk
Rennet
1.0 g kefir/L milk
1.0 g immobilized kefir/L milk
Rennet
1.0 g kefir/L milk
1.0 g immobilized kefir/L milk
Rennet
1.0 g kefir/L milk
1.0 g immobilized kefir/L milk
0 15 30 60 90
3.78 6.53 6.17 6.00 5.99
7.19 9.06 8.32 8.21 7.67
7.30 9.33 9.02 8.72 8.19
3.84 6.38 6.17 5.47 5.37
7.36 8.99 8.07 7.81 7.32
7.50 9.06 8.63 8.15 7.86
3.48 3.69 3.00 2.89 2.77
7.10 7.01 6.69 5.72 5.00
7.35 7.18 6.90 6.04 5.63
3.00 6.18 6.79 6.94 6.90
7.02 7.33 7.56 7.88 8.01
7.38 7.57 7.83 8.00 8.16
18
15 30 60 90
6.84 6.83 6.37 6.12
9.33 8.50 8.37 7.82
7.82 9.51 8.89 8.35
5.79 5.69 5.47 5.28
9.05 8.63 8.31 7.81
7.76 9.22 8.80 8.04
3.78 3.07 3.00 2.90
7.25 6.80 5.98 5.18
7.49 7.12 6.36 6.02
5.83 6.57 6.80 6.87
7.41 7.90 8.05 8.22
7.78 7.96 8.13 8.29
22
15 30 60 90
6.61 6.52 6.29 5.89
9.22 8.56 8.04 7.16
9.40 8.78 8.31 7.42
5.95 5.80 5.07 5.00
9.11 8.23 8.05 7.52
9.18 8.67 8.29 7.60
3.69 3.11 3.02 2.60
7.20 6.72 5.63 4.60
7.50 7.20 6.02 5.87
6.05 6.61 6.89 5.63
7.57 7.99 8.23 7.51
7.92 7.98 8.04 7.65
5
et al., 2006; Pisano et al., 2006). Lactose content in kefir cheeses was significantly lower even at the start of the ripening period possibly due to the action of lactic acid bacteria during curd formation. In these cheeses the remaining lactose was metabolised in the first 15 days of ripening. This rapid decrease in lactose as well as the increase in glucose and galactose associated with the addition of kefir culture at the start of the ripening process (Table 2) agree with previous studies where kefir was used as starter culture in fetatype and whey-cheese production (Kourkoutas et al., 2006; Dimitrellou et al., 2007). Ethanol content ranged in low or undetectable levels in both kefir and rennet cheese. 3.3. Effect of thermally-dried kefir starter culture on microbial associations of hard-type cheeses Microbial counts of total aerobic counts, yeasts and moulds, lactococci and lactobacilli, were also determined during ripening of cheeses at all temperatures (Table 3). The addition of starter culture, ripening temperature and ripening time significantly affected (p < 0.05) all microbial counts. The initial concentration of all microbial counts was greater than 7.02 log CFU/g in cheeses made using kefir culture and less than 3.84 log CFU/g in rennet cheese. All microbial taxa were generally at maximum levels on the 15th day of ripening period. Exceptions were observed in total aerobic counts as well as in yeasts and moulds in cheeses made with immobilized kefir culture, as maximum levels were observed on the 30th day of ripening. On the other hand, lactobacilli counts continued to increase in all cheese samples reaching maximum levels in those made at 18 °C with free and immobilized cells of kefir culture (up to 8.22 log CFU/g and 8.29 log CFU/g, respectively). However, in cheeses ripened at 22 °C a slight drop of lactobacilli was detected on the last day of the ripening process possibly due to the low moisture content of those cheeses (Table 1). Our results indicate a significant increase in lactobacilli at the end of the ripening process associated with the use of kefir culture. This may constitute an important tool in the development of novel probiotic foods given the general concern with regard to viability of such cultures at the time of consuming in order to be effective. The decrease of most microbial counts after the 15th day of ripening can be attributed to the inhibitory effect of the low pH, the consumption of sugars as well as to the moisture loss (Swearingen et al., 2001; Pisano et al., 2006), or even to an antimicrobial activity originating from members of the lactobacilli taxon (Santos et al., 2003; Vuyst and Leroy, 2007). The latter would also agree with the simultaneous increase of lactobacilli counts. Generally, the addition of
kefir culture in cheese samples significantly increased all microbial counts. 3.4. Effect of thermally-dried kefir starter culture on flavor and aroma of hard-type cheeses Proteolysis, lipolyisis and carbohydrate degradation are the most important biochemical processes that occur during cheese ripening due to their contribution in volatile compounds, key components of cheese flavor (Engels et al., 1997). Lactic acid bacteria starter cultures, such as kefir, produce a plethora of enzymes that contribute to the formation of volatiles via proteolysis, lipolysis and carbohydrate degradation during ripening. Such enzymes are peptidases which are involved in the transformation of casein into free amino acids which are further degraded to volatile aroma compounds. Other enzymes are esterases and lipases that hydrolyze triglycerides of milk fat in free fatty acids and transform alcohols and glycerides to esters (Skeie et al., 2008). GC/MS analysis was employed to determine volatile compounds in cheese made with thermally-dried free cells of kefir culture and ripened for 90 days at 5 °C given that this type of cheese had the highest overall score as regards preservation time, textural characteristics, taste and aroma (Table 1). Control cheese made only with rennet was also assayed (Table 4). In total, 20 volatile compounds were detected in rennet cheese while 33 compounds in cheese made with 1 g/L kefir culture contributing to total concentrations of 49.37 lg/ kg and 155.82 lg/kg, respectively. Increase in volatile composition of different types of cheese by kefir starter cultures has also been observed in previous studies (Beshkova et al., 2003; Kourkoutas et al., 2006; Dimitrellou et al., 2007). The main contribution of kefir in concentration of volatiles was observed in organic acids (315% increase) and carbonyl compounds (1401% increase) in which major differences (83%) between rennet and kefir cheese as regards diversity were also observed. Organic acids are important components of cheese flavor and may originate either from milk fat lipolysis or from breakdown of amino acids (Urbach, 1993), while carbonyl compounds and particularly methyl ketones such as 2heptanone and 2-undecanone are formed by enzymic oxidative decarboxylation of fatty acids (Welsh et al., 1989) and may give a mould-ripened character (Engels et al., 1997). Kefir culture was also associated with an important increase in ester concentration (191%). Esters are mainly derived from fatty acids (Engels et al., 1997). Most of the esters that were detected have a fruity aroma attribute. Butanoic, hexanoic, heptanoic, octanoic and nonanoic acids provide a cheesy aroma character, while acetic and decanoic
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Table 4 SPME-GC/MS analysis of aroma-related compounds (lg/kg cheese) isolated from cheeses ripened for 90 days at 5 °C and made either without starter culture (rennet cheese) or using thermally-dried kefir (1.0 g/L) as starter culture. Compound
Identification method
Rennet cheese
Kefir cheese
Esters Ethyl butyrate Ethyl hexanoate Ethyl octanoate Ethyl nonanoate Ethyl decanoate Ethyl-9-decenoate Ethyl-9-hexadecenoate Ethyl dodecanoate Ethyl tetradecanoate Ethyl pentadecanoate Phenyl ethyl acetate Total esters
RTa, KIb, MSc RT, KI, MS RT, KI, MS RT, MS RT, MS MS MS KI, MS KI, MS MS MS
0.13 1.10 3.47 0.00 5.26 0.00 0.00 0.00 0.00 1.35 1.21 12.52
2.25 1.45 5.74 3.93 12.47 3.14 0.10 3.12 0.05 0.00 4.27 36.52
Organic acids Acetic acid Butanoic acid Hexanoic acid Heptanoic acid Octanoic acid Octadecanoic acid Nonanoic acid Decanoic acid Dodecanoic acid Tetradecanoic acid Total organic acids
KI, MS KI, MS KI, MS KI, MS RT, KI, MS KI, MS RT, KI, MS RT, KI, MS KI, MS KI, MS
0.00 0.00 0.02 0.00 5.65 0.07 0.00 2.37 1.06 10.1 19.27
4.41 10.07 9.63 6.25 8.71 0.00 7.06 2.56 1.93 29.53 80.15
Alcohols Ethanol 2-Hexanol 3-Methyl-1-butanol 3-Methyl-2-buten-1-ol 2-Undecanol 2-Nonanol 2,3-Butandiol 1,3-Butandiol 1-Tridecanol Phenyl ethanol Total alcohols
RT, KI, MS MS RT, MS MS MS MS KI, MS KI, MS KI, MS RT, KI, MS
10.08 0.00 0.12 0.00 0.25 0.00 1.60 1.05 0.00 3.75 16.85
18.84 0.12 0.26 2.13 3.18 0.50 1.82 0.00 0.62 5.72 33.19
Carbonyl compounds Acetoin 2-Heptanone 2-Undecanone Nonanal Benzaldehyde 2-Butenal Total carbonyl compounds
MS RT, MS KI, MS KI, MS KI, MS KI, MS
0.67 0.00 0.00 0.06 0.00 0.00 0.73
6.14 0.24 0.51 0.00 3.90 0.17 10.96
a b c
RT: positive identification by retention times that agree with authentic compounds and by mass spectra of authentic compounds generated in the laboratory. KI: tentative identification by Kovats’ retention index. MS: positive identification by mass spectra obtained from NIST107, NIST21 and SZTERP libraries.
acid are known for their acetic and sour aroma profile (Moio et al., 2000; Qian and Reineccius, 2002). Dodecanoic acid provides a soapy odour and tetradecanoic acid a waxy and oily odour. The lowest increase in volatile compounds associated with the use of kefir culture was observed in alcohols (97%). Alcohols can be derived from amino acids transamination and oxidative deamination followed by decarboxylation (Moio et al., 2000; Qian and Reineccius, 2002) and are common in hard-type cheeses such as Emmental, Parmezan, Gouda, Cheddar and Comté (Engels et al., 1997; Mariaca et al., 2001). Ethanol had a higher concentration in kefir cheese samples (18.84 lg/kg) rather than in rennet cheese (10.08 lg/kg) possibly due to mesophilic starters present in kefir culture (Massouras et al., 2006). Among the alcohols related to the use of kefir as starter culture, only 2-hexanol has also been detected in other cheeses such as Cheddar, Emmental, Comté, Camembert, Provolone and Parmezan (Engels et al., 1997; Mariaca et al., 2001). Our results indicate a significant contribution of kefir culture in volatile compounds as the composition of esters, organic acids, alcohols and carbonyl compounds was radically modified.
3.5. Effect of the drying process on cheese characteristics Data presented in this study as regards the effect of thermallydried kefir culture on moisture, preservation time, degree of openness, sensory and textural characteristics of hard-type cheese products agree with a recent study from our laboratory involving the use of freeze-dried kefir cells in hard-type cheese production (Katechaki et al., 2008). However, the drying process appears to affect the volatile profile of these products (Table 5). Both number and concentration of total volatiles were greater in cheese products made with thermally-dried kefir. Significant differences were observed not only in alcohol and carbonyl compound concentration but also in the variety of volatiles belonging to these groups (33% different). Given the detrimental effect of freeze-drying on cultures viability (Morgan et al., 2006) especially when compared to thermal-drying (Kopsahelis et al., 2007), the effect of the drying process on microbial associations of the starter culture is more likely the main reason for such alterations in the volatile profile. It is clear from our results that important quality characteristics of
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Table 5 Effect of freeze- and thermally-dried kefir culture on aroma-related compounds of hard-type cheese products compared with cheese made without kefir inoculants. Kefir culture
Increase in total number of volatile compounds (%)
Freeze-dried Thermally-dried
48 57
Increase in concentration (%) Total compounds
Esters
Organic acids
Alcohols
Carbonyl compounds
179 216
164 192
323 316
20 97
1683 18167
cheese products can not only be obtained by the use of kefir culture as starter in hard-type cheese production but also by the method of drying this culture. 4. Conclusion Our results indicate the suitability of thermally-dried kefir culture as starter in hard-type cheese production. Both free and immobilised cells of kefir culture led to the production of improved cheese products as regards preservation time, sensory and textural characteristics. Kefir culture’s cheese products were characterized by high lactobacilli counts at the end of the ripening process, thus constituting a promising probiotic vehicle given the general concern with regard to viability of such cultures at the time of consuming in order to be effective. Finally, thermal drying appeared to contribute to the volatile composition of the final product giving a different character in cheeses made with thermally-dried kefir culture even when compared to cheeses made with the alternate method of freeze-drying. The thermal drying process used in this study was simple, and of low cost, lower than that of other energy consuming methods such as freeze drying and therefore may constitute a potential process of drying cultures in cheese industry. Acknowledgments This work has been performed within the framework of the Regional Operational Programme (ROP) of Western Greece and was co-funded by the European Regional Development Fund and the Region of Western Greece with final beneficiary the Greek General Secretariat for Research and Technology. References AOAC, 1995. Moisture in Cheese, 33.7.04 Method 948.12, Official Methods of Analysis, 16th ed., Association of Official Analytical Chemists, Arlington, VA. Assadi, M.M., Abdolmaleki, F., Mokarrame, R.R., 2008. Application of whey in fermented beverage production using kefir starter culture. Nutr. Food Sci. 38, 121–127. Azarnia, S., Robert, N., Lee, B., 2006. Biotechnological methods to accelerate Cheddar cheese ripening. Critic. Rev. Biotechnol. 26, 121–143. Beshkova, D.M., Simova, E.D., Frengova, G.I., Simov, Z.I., Dimitrov, Z.P., 2003. Production of volatile aroma compounds by kefir starter cultures. Int. Dairy J. 13, 529–535. Champagne, C.P., Morin, N., Couture, R., Gagnon, C., Jelen, P., Lacroix, C., 1992. The potential of immobilized cell technology to produce freeze-dried, phageprotected cultures of Lactococcus lactis. Food Res. Int. 25, 419–427. Dimitrellou, D., Kourkoutas, Y., Banat, I.M., Marchant, R., Koutinas, A.A., 2007. Whey cheese production using freeze-dried kefir culture as a starter. J. Appl. Microbiol. 103, 1170–1183. Dimitrellou, D., Tsaousi, K., Kourkoutas, Y., Panas, P., Kanellaki, M., Koutinas, A.A., 2008. Fermentation efficiency of thermally-dried immobilized kefir on casein as starter culture. Process Biochem. 43, 1323–1329. Engels, W.J.M., Dekker, R., de Jong, C., Neeter, R., Visser, S., 1997. A comparative study of volatile compounds in the water-soluble fraction of various types of ripened cheese. Int. Dairy J. 7, 255–263. Farnworth, E.R., 1999. Kefir: from folklore to regulatory approval. J. Nutraceut. Funct. Med. Foods 1, 57–68. Heller, K.J., 2001. Probiotic bacteria in fermented foods: product characteristics and starter organisms. Am. J. Clin. Nutr. 73, 374–379.
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