Probiotic butter: Stability, free fatty acid composition and some quality parameters during refrigerated storage

International Dairy Journal 49 (2015) 102e110

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Probiotic butter: Stability, free fatty acid composition and some quality parameters during refrigerated storage
ru c, Bülent Çetin d, Mustafa S¸engül d Tuba Erkaya a, Bayram Ürkek b, *, Ünsal Dog a

Department of Food Processing, Erzurum Vocational High School, Ataturk University, Erzurum, Turkey Siran Mustafa Beyaz Vocational High School, Gumushane University, Gumushane, Turkey c Department of Animal Sciences, College of Agriculture, Ataturk University, Erzurum, Turkey d Department of Food Engineering, College of Agriculture, Ataturk University, Erzurum, Turkey b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 November 2014 Received in revised form 28 April 2015 Accepted 29 April 2015 Available online 19 May 2015

This study was carried out to determine whether butter can be a carrier for probiotics by observing the survivability of selected probiotic strains during cold storage. The effects of using probiotic adjunct cultures (Lactobacillus acidophilus ATCC 4356 and Bifidobacterium bifidum ATCC 29521) in butter on microbiological counts, sensory characteristics, chemical characteristics and free fatty acid (FFA) composition during storage for 60 days were investigated. The butter samples produced with B. bifidum ATCC 29521 maintained the probiotic characteristics, in that the level of viable cells of the probiotic was >106 cfu g
1 until 30 days of storage. The highest scores in sensory assessment were obtained on the first day of storage. FFAs, including C2:0, C6:0, C14:0 and C18:1, were affected significantly by storage period and by the adjunct cultures, however conjugated linoleic acid and C18:2 were not affected by storage period and the use of probiotic adjunct culture. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Probiotic organisms have been generally consumed with fermented foods for thousands of years (Ranadheera, Baines, & Adams, 2010). These consist of viable microorganisms that beneficially affect health of the host by improving microbial balance in the gastrointestinal tract. Probiotic foods are accepted as functional foods (Kaur & Satyanarayana, 2004). Fermented dairy products have been used as functional probiotic foods since ancient times and this situation continues. These foods have therapeutic properties such as prevention of diarrhoea, cancer and childhood infections, inhibition of Helicobacter pylori, prevention of constipation, and improving lactose digestion (S anchez, Reyesn Margolles, & Guemonde, 2009). Probiotic strains of LactoGavila bacillus and Bifidibacterium are used in many foods due to beneficial health effects. Usage of these strains in fermented dairy products has become widespread (Dave & Shah, 1997; Laine, Salminen, Benno, & Quwehand, 2003; Shah & Lankaputhra, 1997). Probiotic bacteria in dairy products have characteristics such as the

* Corresponding author. Tel.: þ90 456 2331032 3606. E-mail address: [email protected] (B. Ürkek). http://dx.doi.org/10.1016/j.idairyj.2015.04.011 0958-6946/© 2015 Elsevier Ltd. All rights reserved.

preservation of dairy products, the production of antimicrobial and flavour compounds, and the raising of the nutritional value of food (Parvez, Malik, Ah Kang, & Kim, 2006). There are many studies reporting on the use of probiotic bacteria in the production of dairy products such as yoghurt, cheese, and ice cream (Gomes et al. 2011; Ranadheera, Evans, Adams, & Baines, 2013; Sagdic, Ozturk, Cankurt, & Tornuk, 2012; Salam et al., 2011; Soukoulis, Lyroni, & Tzia, 2010). Recently, there have been studies on the utilisation of probiotic bacteria in butter production. In these studies, it has been reported that probiotic properties of the bacteria are important. Probiotic bacteria (Lactobacillus maltaramicus AC 3e16 and Lactobacillus casei subsp. casei AB16e65) causes a reduction in the cholesterol content on a fat €
lu & Oner, basis (Alog 2006). However, research about the utilisation of Bifidobacterium bifidum and Lactobacillus acidophilus in butter production has not been encountered in the literature.
dıç, Butter has been produced in Turkey for centuries (Sag €nmez, & Demirci, 2004); however, consumption has decreased Do due to the perceived negative effect on human health of saturated fatty acids (Oeffner et al., 2013). Despite this, milk fat contains essential fatty acids needed in the human diet. Lipid-soluble vitamins such as retinol, carotenoids and tocopherols also function as antioxidants that are important for human health. Physical,

T. Erkaya et al. / International Dairy Journal 49 (2015) 102e110

chemical and quality properties of dairy products and human health are affected by milk fatty acid composition. Research has focused on the beneficial effects on human health of fatty acids, such as butyric acid, oleic acid, and conjugated linoleic acid (CLA) €nen, 2006; Ekinci, Okur, (Collomb, Schmid, Sieber, Wechsler, & Ryha Ertekin, & Guzel-Seydim, 2008; Haug, Hostmark, & Harstad, 2007; Ledoux et al., 2005; Yilmaz-Ersan, 2013). Some researchers report that bioactive fatty acids are formed by probiotic strains and can alter fatty acid composition of dairy products (Ekinci et al., 2008; Yadav, Jain, & Sinha, 2007; Yilmaz-Ersan, 2013). In addition, butter produced with probiotic bacteria provide for better sensorial €
lu & Oner, properties (Alog 2006). The goals of this study were: (a) to investigate the survival of probiotic bacteria during storage, and (b) to evaluate the changes in free fatty acid (FFA) composition and CLA content, chemical and microbiological properties in butter samples during 60 d of storage. 2. Materials and methods 2.1. Materials Cream was supplied for butter production from a dairy farm at Atatürk University (Erzurum province, Turkey). Starter culture CHN-22 (Lactococcus lactis subsp. cremoris, Leuconostoc sp., Lactococcus lactis subsp. lactis biovar diacetylactis, Lc. lactis subsp. lactis) used in butter manufacturing was supplied from Chr. HansenPeyma (Istanbul, Turkey). Probiotic cultures, B. bifidum ATCC 29521 and Lb. acidophilus ATCC 4356, were provided by the Food Microbiology Laboratory, Department of Food Engineering, Faculty of Agriculture, Atatürk University. These probiotic strains were chosen because of their high stability in dairy products (Erkaya & Sengul, 2015). 2.2. Methods The butter samples were manufactured at the Atatürk University Dairy Plant. The cream was pasteurised at 85  C for 10 min and allowed to cool to 18e20  C. The cream was divided into three parts for each replicate. All batches were inoculated with direct vat set culture (freeze-dried) at a level of 15 g 500 L
1. Starter culture only was added to one sample of cream (A). Starter culture þ B. bifidum ATCC 29521 were added to a second sample of cream (B). Starter culture þ Lb. acidophilus ATCC 4356 were added to a third sample of cream (C). The inoculation level of B. bifidum ATCC 29521 and Lb. acidophilus ATCC 4356 was 2% (v/v). The ripened cream was churned and washed. The experimental butter samples were packaged (500 g) and stored at 4  C for 60 days until analysis. Production of butter samples was carried out in duplicate. 2.2.1. Microbiological analyses Butter samples (10 g) were dispersed in 90 mL of 0.85% (w/v) sterile NaCl solution and held in a water bath at 45  C until the butter melted. Decimal serial dilutions were prepared. The counts of total aerobic mesophilic bacteria (TAMB) were enumerated on plate count agar (Oxoid, Basingstoke, England) and incubated at 30  C for 48 h (Messer, Behney, & Leudecke, 1985). Presumptive lactobacilli were enumerated on MRS (de Man, Rogosa, Sharpe) agar (Oxoid) by incubating the plates anaerobically at 37  C for 72 h (Speck, 1976). Anaerobic conditions were provided by Anaerocult A sachets (Merck, Darmstadt, Germany). Presumptive lactococci were counted on M17 (modified Rogosa) agar after incubation aerobically at 30  C for 48 h (Cabezas, Sanchez, Poveda, Sesena, & Palop, 2007). B. bifidium counts were determined on MRS-NNLP (nalidixic acid, neomycin sulphate, lithium chloride and paromomycin sulphate) agar and the plates were incubated anaerobically at 37  C

103

for 72 h. The NNLP mix was prepared using neomycin sulphate (100 mg L
1), paramycin sulphate (200 mg L
1), nalidixic acid (50 mg L
1), and lithium chloride (3000 mg L
1) (Dave & Shah, 1997). Lb. acidophilus ATCC 4356 counts were enumerated on MRS-bile agar and incubated anaerobically at 37  C for 72 h (Lima et al., 2009). Coliforms were determined using VRBA (Oxoid) after incubation at 35  C for 48 h (Speck, 1976). Yeasts and moulds were counted on potato dextrose agar acidified with 10% tartaric acid (Oxoid) and incubated at 25  C for 5 days (Koburger & Marth, 1984). 2.2.2. Sensory analysis Sensory analysis was carried out on butter samples by a group of eight panellists from the academic staff working in the Dairy Department (Atatürk University). Panel members assessed the butter samples according to a modified method of Bodyfelt, Tobias, and Trout (1988). Panellists evaluated each butter sample in terms of colour and appearance, structure, flavour, and general acceptability. A five point scale was used for each sensory attributes. Before evaluation, the butter samples were kept at room temperature for 10 min and served with a glass of water and a slice of bread. Sensory analyses were carried out after each storage period for each replicate (5  2 assessments). 2.2.3. Chemical analyses The dry matter (DM), titratable acidity (lactic acid %) and fat contents were determined according to the methods of Metin (2008). For pH measurement, a penetration probe was used and the values were measured using a digital pH meter (Crison model Basic 20, Alella, Spain). The pH of the butter serum was determined according to the method of IDF (1981), and peroxide values (PV) were determined according to the method of Atamer (1993). The thiobarbituric acid (TBA) values were measured according to the method of Egan, Kirk, and Sawyer (1981). For this test, samples (10 g) were weighed and macerated with 50 mL distilled water for 2 min. The mixture was placed in a distillation flask and washed with 47.5 mL distilled water. The pH value of the mixture was reduced to less than 1.5 by adding 2.5 mL 4 M HCl. After boiling the mixture, 50 mL distillate was collected over 10 min. Distillate (5 mL) was transferred into a glass-stoppered tube and added to 5 mL TBA solution (0.2883 g TBA in 100 mL 90% acetic acid). The tube was stoppered and placed in a boiling water bath for 35 min to develop the colour in the dark. The blank was prepared in the same way. The tubes were chilled for 10 min and measurements were carried out against the blank at 538 nm. The TBA values were calculated from a standard curve. The standard curve was prepared with 1,1,3,3-tetramethoxypropane. 2.2.4. Determination of FFA composition For preparation of FFA methyl esters, 50 mg fat samples were weighed and placed into glass tubes. NaOH (2 M, 1.5 mL) was added to the tubes. The tubes were filled with nitrogen gas and heated at 80  C for 1 h. After cooling, 1 mL BF3 in methanol (25%, w/v) was added to the tubes and heated at 80  C for 30 min. At the end of this time, the tubes were cooled again. Hexane (1 mL) was added to the tubes and vortexed. The tubes were re-vortexed after adding ultradistilled water (1 mL). The hexane phase was transferred to a new tube containing anhydrous sodium sulphate by addition of 1 mL of hexane and re-vortexed. All tubes were centrifuged for 10 min at 2879  g and the upper layer was placed into a vial (Metcalfe & Schmitz, 1961). FFA composition was analysed using a GC/MS system (Agilent 6980N series, Agilent Tech. Inc., Santa Clara, CA, USA). Operating conditions for the GC were as follows: flame ionization detector  C; (Agilent Tech. Inc.) at 200 column: DB-23 (60 m  0.25 mm  0.25 mm) held for 15 min at 165  C, then

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T. Erkaya et al. / International Dairy Journal 49 (2015) 102e110

increased to 200  C over 47 min; flow gas: H2 (34 kPa); flow rate of 1 mL min
1; time stable: 200; flow speed: 1/50 (hydrogen/dry air); air flow: 350 mL min
1; H2 flow: 35 mL min
1. The FFA composition analysis was carried out in triplicate. 2.2.5. Statistical analysis The experimental design comprised of an entirely randomized design in a factorial arrangement: three treatments, five storage periods (2, 15, 30, 45 and 60 days) and two replicates. All butters were made from the same cream. Creams were divided into six batches: three replicates that were duplicated. The replicates were not independent. All data were analysed statistically using SPSS statistical software program version 17 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) and Duncan's multiple range test was used to determine significant differences among results. 3. Results and discussion 3.1. Microbiological properties Microbiological characteristics of the butter samples are shown in Table 1. B. bifidum ATCC 29521 and Lb. acidophilus ATCC 4356 counts were less than 6.0 log cfu g
1 at the end of storage and continuously decreased during storage time (Fig. 1). This decrease was higher for Lb. acidophilus ATCC 4356 counts. The counts of B. bifidum ATCC 29521 decreased after 30 days (P < 0.05). Afterwards, along with the storage time, it clearly decreased approximately one logarithmic cycle (Fig. 1). Similarly, Olszewska, Staniewski, and Łaniewska-Trokenheim (2012) reported a reduction in the viable cells of Bifidobacterium lactis in butter during refrigerated storage over 4 weeks, with the number of live cells decreasing by more than one logarithmic cycle in the last week of storage. Pedroso, Dogenski, Thomazini, Heinemann, and FavaroTrindade (2013) examined Bifidobacterium animalis subsp. lactis (BI-01) and Lb. acidophilus (LAC-04) encapsulated in cocoa butter and reported that only 20% of Lb. acidophilus and 72% of B. animalis subsp. lactis cells were viable after 90 days of storage at 7  C. These results were similar to those found in the present work.

Fig. 1. Changes in microbial counts in butter samples produced by the addition of B. bifidum (A) or Lb. acidophilus (-) during storage. Error bars show standard deviation.

The TAMB counts were significantly lower (P < 0.01) in the control sample compared with other samples during storage except for the 2nd and 45th days. Dagdemir, Cakmakci, and Gundogdu (2009) reported that TAMB count varied from 5.52 to 6.92 log cfu g
1, and decreased during storage. In the present study, values were slightly higher than those reported by these authors, and counts increased during storage. The presumptive lactobacilli counts on MRS for butter sample C were higher than those of the control samples at end of the storage. The presumptive lactobacilli counts were higher in samples B and C at the beginning of storage compared with sample A. The presumptive lactobacilli counts did not show a significant difference over time, except for sample A. Results were generally higher than those reported by Dagdemir et al. (2009) for butter produced by the addition of Origanum acuditens and Thymus haussknechtii, and by
diç, Arici, and Sims¸ek (2002) for commercial butters. Sag The counts of presumptive lactococci growth in M17 agar were significantly affected by the addition of probiotic strains and storage time (P < 0.01). All samples showed irregular variations during storage. The presumptive lactococci counts were similar in all samples at 15, 45 and 60 days of storage. As can be seen Table 1, the

Table 1 Effect of adjunct probiotic strains and storage on microbiological characteristics of butters.a Microbial counts (log cfu g
1)

Storage time (days)

Butter samplesa A

TAMB

MRS

M17

Mould-yeast

a

2 15 30 45 60 2 15 30 45 60 2 15 30 45 60 2 15 30 45 60

6.60 6.63 6.63 6.80 6.62 6.03 6.44 6.42 6.42 6.45 6.46 6.69 6.52 6.48 6.51 2.54 3.19 4.98 5.57 5.43

B ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.10 0.05 0.08 0.05 0.34 0.01 0.06 0.21 0.22 0.07 0.08 0.06 0.04 0.23 0.39 0.34 0.06 0.14 0.05 0.45

a,A a,A a,A a,A a,A a,A b,A b,A b,A b,A a,A a,A a,A a,A a,A a,A a,A b,A b,A b,A

6.79 7.13 7.13 7.01 7.05 6.66 6.88 6.60 6.58 6.57 6.75 7.11 7.20 6.89 6.98 2.75 3.16 5.09 5.62 5.77

C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.18 0.16 0.11 0.01 0.08 0.21 0.10 0.05 0.06 0.08 0.25 0.04 0.18 0.07 0.06 0.11 0.01 0.17 0.18

a,AB b,B b,B ab,A ab,B a,B a,A a,A a,A a,AB a,B ab,A b,B ab,A ab,A a,A b,A c,A d,A d,A

6.80 7.08 7.07 7.16 6.92 6.60 6.31 6.29 6.25 6.68 6.67 7.08 7.13 7.09 6.88 2.47 3.45 5.01 5.20 5.83

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.12 0.05 0.23 0.02 0.08 0.43 0.40 0.10 0.08 0.04 0.06 0.04 0.30 0.04 0.61 0.13 0.05 0.42 0.27

a,B ab,B ab,B b,A ab,B a,B a,A a,A a,A a,B a,AB b,A b,B b,A ab,A a,A b,A c,A c,A c,A

Abbreviations are: TAMB, total aerobic mesophilic bacteria; MRS, deMan, Rogosa and Sharpe; M17, Modified Rogosa; A, control butter; B, butter with B. bifidum; C, butter with Lb. acidophilus. Values are means ± SD; values within a row with no common superscript upper case letter differ (P < 0.05), values within a column and data set with no common superscript lower case letter differ (P < 0.05).

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lactococci in only sample A did not exhibit a significant change (P > 0.05) in logarithmic values during storage. In the current study, presumptive lactococci counts on M17 were higher than those re
diç et al. (2002) for commercial butters. ported by Sag The mould-yeast counts of butter samples significantly differed depending upon the storage time (P < 0.01). The mould-yeast count increased over time in all butter samples during storage (Table 1). There were no statistically different results among butter samples during storage period (P > 0.05). These results are in agreement with Dagdemir et al. (2009) who reported that mould-yeast counts of butter samples increased during storage. 3.2. Sensory assessment The sensory evaluation of the butter samples during storage on a scale from 1 (very bad) to 5 (excellent) is presented in radar plots in Fig. 2. The addition of probiotic strains had no effect on flavour, structure and general acceptability scores; however, it was determined that the colour scores were affected by probiotic bacteria at the P < 0.05 level. The significant colour scores from panellists for sample B might be attributed to the halo effect. Mann and Sachdeva (2014) also reported that the colour scores were affected by the inoculum concentration of probiotic bacteria in yoghurt. On the other hand, the flavour, structure and general acceptability scores significantly changed (P < 0.05) throughout the storage period. In general, the highest scores were obtained on the first day of storage, followed by a decrease in scores during storage. The total amount of free fatty acids and the peroxide value may be responsible for the development of flavour defects in butter during storage (S¸enel, € Atamer, & Oztekin, 2011). In particular, the shelf-life of fatty dairy products is affected by these chemical changes through oxidation, and as a result, show an oxidised flavour defect (S¸enel et al., 2011). 3.3. Chemical properties The DM values of butter samples A, B, and C were 85.33 ± 0.30, 86.33 ± 0.23, and 84.63 ± 0.33%, respectively. The DM value of sample B was higher than others and this difference was found to
dıç be significant (P < 0.05). Similar results were reported by Sag et al. (2002, 2004) and Simsek (2011). The fat content of butter samples A, B and C were 85.0 ± 1.1, 85.0 ± 0.5 and 84.0 ± 0.5%, respectively. There was no statistically significant difference between the samples (P > 0.05). The fat values of the butter samples
dıç et al. (2002, 2004) were higher than those determined by Sag and Simsek (2011). Chemical characteristics of the butter samples during storage are presented in Table 2. The addition of probiotic strains and storage significantly affected the pH, acidity and PV of the samples (P < 0.01). The TBA values of the butter samples were significantly affected (P < 0.01) by storage, but were not affected (P > 0.05) by the adjunct probiotic bacteria. The shelf-life of high fatty dairy products is affected by oxidation. Many factors affect the oxidative stability of dairy products, such as concentration of oxygen, antioxidants, metals (Cuþ2, Feþ3), and water activity (O'Connor & O'Brien, 2006). For this reason, it is difficult to comment on PV (S¸enel et al. 2011). PV was affected significantly (P < 0.05) by adjunct Lb. acidophilus ATCC 4356 and B. bifidum ATCC 29521. Some researchers report that some species of lactobacilli have antioxidative activity (Lin & Chang, 2000; Saide & Gilliland, 2005; Wang, Yu, & Chou, 2006). PV showed an increasing and decreasing behaviour during storage (Table 2). Although the PV of all samples significantly decreased at day 30 of storage, the TBA values of all samples significantly increased at day 30 of storage (Table 2). This may result from formation of malondialdehydes as  ndez, Pe rez-Alvarez, result of the degradation of peroxides (Ferna &

Fig. 2. The sensory profiles of butter samples up to 60 d of storage for (A) control butter, (B) butter with B. bifidum, and (C) butter with Lb. acidophilus.

ndez-Lo  pez, 1997). Additionally, hydroperoxides are not staFerna ble, as they decompose to other compounds such as flavourful carbonyls (O'Connor & O'Brien, 2006). The PV of all samples was higher at the end of storage (45 and 60 days) compared to the 2nd day of storage (P < 0.05). These results are in agreement with Dagdemir et al. (2009), Ozturk and Cakmaci (2006) and Simsek (2011), who reported that PV values of butter were highest at the end of the storage time; in the present study, values were lower than those reported by these authors (Table 2). TBA values were not affected (P > 0.05) by adjunct Lb. acidophilus ATCC 4356 or B. bifidum ATCC 29521. The TBA contents of all samples were not determined at day 2 of storage. The primary oxidation products are hydroperoxides, which can degrade further into secondary oxidation products. The TBA test measures

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T. Erkaya et al. / International Dairy Journal 49 (2015) 102e110

Table 2 Effect of probiotic bacteria supplementation and storage time on chemical characteristics of butters.a Properties

Storage time (days)

Butter samples A

pH

Acidity (% lactic acid)

Peroxide value (meq O2 kg
1 butter)

TBA (mg malonaldehyde kg
1 butter)

2 15 30 45 60 2 15 30 45 60 2 15 30 45 60 2 15 30 45 60

5.13 5.09 5.09 5.16 5.01 0.36 0.35 0.36 0.39 0.39 0.00 0.32 0.07 0.37 0.37 e 0.09 0.16 0.07 0.04

B ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.06 0.07 0.02 0.06 0.02 0.01 0.01 0.01 0.00 0.00 0.02 0.04 0.01 0.03

ab,AB

± ± ± ±

0.00 0.01 0.03 0.01

b,A

ab,A ab,B b,C a,B ab,A a,A ab,A bc,A c,A a c,B b,A c,AB c,B

c,A ab,A a,A

5.16 5.08 4.85 4.99 4.87 0.36 0.36 0.36 0.39 0.39 0.00 0.25 0.03 0.29 0.31 e 0.06 0.16 0.08 0.02

C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.08 0.06 0.02 0.05 0.16 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.02

c,B

± ± ± ±

0.00 0.01 0.05 0.02

a,A

bc,A a,A ab,B a,AB a,A a,AB a,A a,A a,A a b,A a,A c,A c,AB

b,A a,A a,A

5.05 4.99 4.89 4.87 4.75 0.36 0.39 0.38 0.42 0.42 0.00 0.29 0.01 0.39 0.25 e 0.06 0.16 0.07 0.02

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.08 0.08 0.01 0.06 0.01 0.01 0.03 0.00 0.01 0.00 0.02 0.01 0.04 0.01

c,A

± ± ± ±

0.02 0.00 0.02 0.01

b,A

bc,A ab,AB ab,A a,A a,A abc,B ab,A c,B bc,B a b,AB a,A c,B b,A

c,A b,A a,A

a

Abbreviations are: TBA, thiobarbituric acid; A, control butter; B, butter with B. bifidum; C, butter with Lb. acidophilus. Values are means ± SD; values within a row with no common superscript upper case letter differ (P < 0.05), values within a column and property with no common superscript lower case letter differ (P < 0.05).

secondary products of lipid oxidation (O'Connor & O'Brien, 2006). Since hydroperoxides were not formed in the butters day 2 of storage (PV was determined as 0.00 meq O2 kg
1, see Table 2), the TBA test was not carried out. The level of TBA increased in all butter samples until the 30th day of storage, and thereafter decreased at the end of storage. No significant differences were observed between the butter samples for TBA content during storage (P > 0.05; Table 2). During storage, TBA values exhibited irregular changes, different to the trends for PV (Table 2). Variations of TBA and PV can be explained by the formation of peroxides as, a result of primary oxidation, that quickly change to secondary oxidation products that are unstable. TBA values were lower than those reported by Dagdemir et al. (2009) for butter produced with the addition of Origanum acutidens and T. haussknechtii, and values reported by Simsek (2011) for butters stored at a different temperature, but in these two studies, the TBA values increased in all samples during storage. The pH values of butter samples significantly differed depending upon the adjunct probiotic bacteria and storage time (P < 0.01). The pH values of samples B and C decreased during storage. The pH of fresh and stored butter samples changed from 5.16 ± 0.04 to 4.75 ± 0.06 over time. The pH values will decrease due to continued fermentation by the lactic acid bacteria during storage (Buriti, Rocha, & Saad, 2005; Dave & Shah, 1997; Ranadheera, Evans, Adams, & Baines, 2012). Similar levels were found by Simsek (2011); however S¸enel et al. (2011) reported lower values for Yayık butters samples produced from different animals. The acidity values of the samples containing Lb. acidophilus ATCC 4356 were higher than those containing B. bifidum ATCC 29521 and control samples at 45 and 60 days of storage. There were no significant differences between acidity values at the end of storage between butter containing B. bifidum ATCC 29521 and the control samples. The acidity values of only sample B did not show a significant change during storage. Acidity values were highest in sample C at end of storage time (Table 2). Values of acidity were lower than those reported by S¸enel et al. (2011), but higher than those found by Simsek (2011).

3.4. Free fatty acid composition The FFA composition, including conjugated linolenic acid (CLA), is shown in Fig. 3. Butyric acid showed one of the lowest levels amongst FFAs. S¸enel et al. (2011) also reported that the butyric acid content of butter samples produced from cows', goats' and ewes' milk yoghurt was found at a trace level. The level of butyric acid was 0.65% at the beginning of storage, and 0.25% at the end of storage in sample B. The level of caproic acid changed between 0.45% and 2.53% during storage (Table 3). The caprylic acid content of sample B (1.22%) was higher than samples A (0.99%) and C (1.00%) at the beginning of storage. Compared with the control sample (2.58%), samples B (%3.37%) and C (3.08%) had higher level of capric acid at the 2nd day of storage (P < 0.05) (Fig. 3). SametBali, Ayadi, and Attia (2009) commented that changes in the fatty acid composition could be associated with the degradation of fat by heating and oxidation. The content of lauric acid ranged from 3.40% to 3.97% during storage, but it was not affected by storage time and adjunct probiotic strains (P > 0.05). The changes for acetic, butyric, caproic, caprylic, myristic and palmitic acids were statistically significant (P < 0.01) during storage. The amount of saturated fatty acids was higher than unsaturated. The major fatty acids of butter samples were myristic (12.33e13.97%), palmitic (36.98e40.27%), stearic (8.67e9.09%) and oleic (20.53e22.88%) acids (Table 3). Similar results were reported by Samet-Bali et al. (2009) who studied physicochemical and microbiological characteristics, fatty acid composition, and thermal stability of traditional Tunisian butter. CLA is mainly present in milk and dairy products, and the most abundant source is milk fat (Ledoux et al., 2005). Ledoux et al. (2005) reported that the CLA content of butter varied, depending upon the season. CLA levels ranged between 0.77 and 0.99% during storage. This change was not significant during storage among butter samples (P > 0.05). Significant differences were only obtained for CLA levels during storage in sample A (P < 0.05; Fig. 3). Addition of probiotic strains did not cause a significant difference in CLA levels of butter samples. This result is in agreement with other studies carried out by Dave, Ramaswamy, and Baer (2002), Ekinci

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Table 3 Effect of probiotic bacteria supplementation and storage on fatty acid composition of butters.a Properties

Butter samples

Storage time (days) 2

C2:0

C4:0

C6:0

C8:0

C10:0

C12:0

C14:0

C14:1

C15:0

C15:1

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

CLA

SFA

MUFA

PUFA

A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C

0.31 0.65 0.70 0.40 0.65 0.42 1.10 1.23 2.5 0.99 1.22 1.00 2.58 3.37 3.08 3.40 3.89 3.93 12.97 12.33 13.50 1.29 1.23 1.34 1.53 1.53 1.61 0.43 0.43 0.45 37.7 36.98 38.7 1.90 1.89 1.92 9.02 8.89 8.69 22.9 21.13 20.53 0.65 0.63 0.74 2.04 2.16 2.01 0.99 0.86 0.84 69.99 70.7 74.10 26.5 24.67 24.23 3.67 3.65 3.58

15 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a,A

0.04 0.15 b,A 0.06 b,A 0.02 a,A 0.14 a,B 0.04 a,A 0.01 a,B 0.28 a,B 0.4 b,B 0.01 a,A 0.02 b,B 0.01 a,A 0.02 a,A 0.23 b,A 0.08 b,A 0.21 a,A 0.27 a,AB 0.02 a,A 0.26 b,A 0.04 a,A 0.05 c,A 0.01 b,A 0.01 a,A 0.00 c,A 0.01 a,A 0.01 a,A 0.01 b,A 0.00 a,A 0.00 a,A 0.01 b,A 0.5 ab,A 0.02 a,A 0.5 b,A 0.00 a,A 0.01 a,A 0.02 a,A 0.25 a,A 0.04 a,AB 0.07 a,A 0.6 b,B 0.23 a,AB 0.04 a,A 0.02 a,A 0.07 a,A 0.06 a,A 0.01 a,AB 0.04 a,B 0.10 a,BC 0.04 a,B 0.03 a,A 0.08 a,A 0.09 a,AB 1.0 a,A 0.24 b,B 0.6 b,B 0.23 a,A 0.01 a,A 0.04 a,B 0.06 a,C 0.11 a,B

0.23 0.37 0.25 0.27 0.38 0.24 1.00 0.91 1.3 0.56 0.82 0.58 2.2 2.8 2.2 3.53 3.97 3.6 13.51 13.75 13.9 1.36 1.36 1.40 1.63 1.62 1.65 0.47 0.45 0.47 39.4 38.73 39.2 2.01 1.98 2.00 8.97 8.67 8.8 21.28 20.75 21.0 0.64 0.56 0.57 2.12 2.10 2.06 0.86 0.79 0.81 71.29 72.01 71.7 25.11 24.54 24.9 3.61 3.45 3.44

30 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a,A

0.02 0.11 a,A 0.00 a,A 0.12 a,A 0.15 a,AB 0.07 a,A 0.21 a,AB 0.28 a,AB 0.4 a,A 0.20 a,A 0.05 a,A 0.22 a,A 0.5 a,A 0.06 a,A 0.6 a,A 0.26 a,A 0.01 a,B 0.6 a,A 0.04 a,AB 0.16 a,B 0.7 a,A 0.02 a,AB 0.01 a,A 0.09 a,A 0.01 a,B 0.02 a,A 0.01 a,A 0.01 a,A 0.01 a,B 0.01 a,AB 0.5 a,B 0.21 a,B 0.8 a,A 0.04 a,A 0.03 a,A 0.01 a,AB 0.04 a,A 0.11 a,A 0.3 a,A 0.18 a,A 0.21 a,A 0.6 a,A 0.01 a,A 0.09 a,A 0.03 a,A 0.02 a,B 0.04 a,B 0.06 a,C 0.01 a,AB 0.06 a,A 0.04 a,A 0.27 a,B 0.07 a,A 0.7 a,A 0.25 a,A 0.16 a,A 0.5 a,A 0.02 a,B 0.11 a,BC 0.12 a,AB

0.29 0.42 1.2 0.27 0.39 0.35 1.0 0.71 1.0 0.68 0.90 0.83 2.22 2.4 2.7 3.44 3.93 3.84 13.24 14.1 13.63 1.31 1.39 1.36 1.62 1.3 1.63 0.45 0.47 0.48 39.4 40.3 39.0 1.99 1.96 1.98 9.09 9.10 9.12 21.6 21.49 21.5 0.55 0.50 0.63 2.08 1.87 1.81 0.82 0.71 0.86 71.2 71.43 71.9 25.3 24.6 24.8 3.4 3.04 3.28

45 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a,A

0.03 0.26 ab,A 0.4 b,B 0.05 a,A 0.17 a,AB 0.06 a,A 0.4 a,B 0.25 a,AB 0.4 a,A 0.08 a,A 0.24 a,AB 0.16 a,A 0.22 a,A 1.0 a,A 0.4 a,A 0.21 a,A 0.11 a,AB 0.24 a,A 0.28 a,A 0.4 b,B 0.02 ab,A 0.03 a,A 0.20 a,A 0.01 a,A 0.01 a,B 0.6 a,A 0.02 a,A 0.01 a,A 0.01 ab,D 0.00 b,B 0.4 a,B 0.8 a,C 1.1 a,A 0.02 a,A 0.18 a,A 0.01 a,AB 0.21 a,A 0.08 a,BC 0.23 a,A 0.4 a,A 0.28 a,BC 0.6 a,A 0.13 a,A 0.13 a,A 0.18 a,A 0.12 a,B 0.06 a,A 0.11 a,A 0.09 a,A 0.16 a,A 0.13 a,A 0.7 a,B 0.06 a,A 0.4 a,A 0.4 a,AB 1.0 a,A 0.6 a,A 0.3 a,AB 024 a,A 0.19 a,B

0.22 0.30 0.22 0.28 0.25 0.26 0.45 0.51 0.62 0.62 0.53 0.68 2.36 2.08 2.44 3.73 3.42 3.72 14.12 13.68 13.95 1.42 1.35 1.38 1.68 1.68 1.67 0.48 0.46 0.48 40.57 41.06 40.34 1.7 2.08 2.03 9.10 9.21 9.14 21.65 22.1 21.65 0.53 0.54 0.61 1.86 1.92 1.85 0.78 0.79 0.83 70.42 71.3 71.67 24.3 25.5 25.06 3.12 3.22 3.25

60 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a,A

0.11 0.18 a,A 0.07 a,A 0.04 a,A 0.03 a,A 0.06 a,A 0.04 a,A 0.16 a,A 0.01 a,A 0.01 a,A 0.11 a,A 0.06 a,A 0.15 a,A 0.23 a,A 0.18 a,A 0.13 a,A 0.17 a,A 0.11 a,A 0.08 a,B 0.21 a,B 0.10 a,A 0.01 b,B 0.03 a,A 0.01 ab,A 0.00 a,B 0.01 a,A 0.00 a,A 0.01 a,A 0.00 a,CD 0.01 a,B 0.28 a,B 0.28 a,C 0.22 a,A 0.6 a,A 0.01 a,A 0.00 a,AB 0.09 a,A 0.18 a,C 0.13 a,A 0.22 a,A ± 0.4 a,C 0.25 a,A 0.09 a,A 0.04 a,A 0.03 a,A 0.05 a,A 0.11 a,A 0.02 a,AB 0.05 a,A 0.04 a,A 0.02 a,A 0.17 a,AB 0.6 ab,A 0.24 b,A 0.8 a,A 0.4 a,A 0.23 a,A 0.08 a,A 0.18 a,AB 0.01 a,B

0.37 0.27 0.23 0.36 0.25 0.36 0.98 0.71 0.7 0.81 0.57 0.7 2.6 2.4 2.6 3.70 3.85 3.9 13.4 14.02 14.23 1.34 1.40 1.45 1.61 1.67 1.68 0.40 0.45 0.48 39.5 40.2 39.9 2.00 2.05 2.05 8.1 9.06 8.95 22.0 21.70 21.3 0.65 0.57 0.73 1.94 1.87 1.85 0.88 0.80 0.88 68.7 71.69 71.8 24.78 25.11 24.8 3.43 3.21 3.42

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 a,A 0.13 a,A 0.08 a,A 0.12 a,A 0.02 a,A 0.19 a,A 0.07 a,AB 0.03 a,AB 0.3 a,A 0.28 a,A 0.19 a,A 0.3 a,A 0.5 a,A 0.4 a,A 0.8 a,A 0.07 a,A 0.29 a,AB 0.6 a,A 0.6 a,AB 0.08 a,B 0.09 a,A 0.06 a,AB 0.01 a,A 0.01 a,A 0.07 a,B 0.02 a,A 0.05 a,A 0.08 a,A 0.00 a,BC 0.02 a,B 0.9 a,B 0.5 a,C 1.5 a,A 0.06 a,A 0.05 a,A 0.09 a,B 2.0 a,A 0.00 a,BC 0.16 a,A 0.4 a,AB 0.14 a,BC 0.5 a,A 0.04 ab,A 0.03 a,A 0.01 b,A 0.08 a,AB 0.00 a,A 0.00 a,AB 0.04 b, AB 0.02 a,A 0.01 b,A 1.4 a,A 0.23 b,A 0.6 b,A 0.12 a,A 0.18 a,A 0.6 a,A 0.16 a,AB 0.05 a,AB 0.00 a,AB

a Abbreviations are: A, control butter; B, butter with B. bifidum, and C, butter with Lb. acidophilus; CLA, conjugated linoleic acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly-unsaturated fatty acids. Values are means ± SD; values within a row with no common superscript upper case letter differ (P < 0.05), values within a column and data set with no common superscript lower case letter differ (P < 0.05).

et al. (2008), Xu, Boylston, and Glatz (2005, 2006), and YilmazErsan (2013). Amongst the long-chain unsaturated fatty acids, oleic acid content was the highest in all samples. During storage, the amount of oleic acid in the control sample was measured at between 22.88 and 22.04%. In contrast, the content in probiotic butter samples was

lower than in the control sample (Fig. 3). Yilmaz-Ersan (2013) found that oleic acid values were higher in cream fermented with B. lactis than in cream fermented with Lb. acidophilus and Lb. rhamnosus. The linoleic acid level ranged from 0.50% to 0.74% during storage. However, the linoleic acid level in sample B (0.57%) was lower than in sample C (0.73%) at end of storage (P < 0.05).

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Fig. 3. Percentage of free fatty acids (FFAs) after ( ) 2, ( ) 15, ( ) 30, (-) 45, and ( ) 60 d of storage for (A) control butter, (B) butter with B. bifidum, and (C) butter with Lb. acidophilus. CLA, conjugated linoleic acids; SFA, saturated fatty acids; MUFA, mono-unsaturated fatty acids; PUFA, poly-unsaturated fatty acids. Error bars show standard deviation.

Linolenic acid content in butter samples was between 2.01 and 2.16% at the beginning of storage, whereas it was between 1.85 and 1.97% at the end of storage (Table 3). These results are not in agreement with those of Glew, Okolo, Chuang, Huang, and VanderJagt (1999), Idoui, Benhamada, and Leghouchi (2010),
dıç et al. (2004). Idoui, Rechak, and Zabayou (2013), or Sag In this study, it was found that the saturated fatty acid (SFA) content of the control sample was lower than for probiotic butters at all times measured during storage. The total content of SFA ranged from 68.71% to 74.10%. Statistically significant differences (P < 0.01) were determined for the SFA contents of the butters, whereas they were not affected by the storage period. Yadav et al. (2007) also reported a similar result. Total polyunsaturated fatty acid (PUFA) content was significantly affected by storage period

(P < 0.01) and values ranged from 3.67% to 3.04%during storage. The PUFA value was found to between 3.65% and 3.21% in sample B during storage. The monounsaturated fatty acid (MUFA) content was between 3.65% and 3.21% at day 2 and 24.78% at day 60 in sample A. The storage period and probiotic bacteria adjunct did not affect (P > 0.05) the MUFA content. 4. Conclusions The results of this study show that storage period had significant effects on chemical properties such as pH, acidity, PV, TBA and microbiological characteristics such as TAMB, M17, mould-yeast counts (P < 0.05). Live cell counts of the probiotic strains decreased approximately one logarithmic cycle at the end of

T. Erkaya et al. / International Dairy Journal 49 (2015) 102e110

storage. Based on the results obtained, fresh butter can be recommended as a probiotic source for consumers in terms of viable counts of B. bifidum ATCC 29521 and Lb. acidophilus ATCC 4356. The use of B. bifidum ATCC 29521 is recommended for up to 30 days of storage to obtain probiotic butter. Commercialised probiotic products with health claims must meet a minimum criterion of one million viable probiotic cells per mL or gram of product (Bunselmeyer & Buddendick, 2010). According to our result, 25 g of butter per day provides sufficient probiotic product. Moreover, this product will contribute to an increased diversity of probiotic products on the market. Chemical changes in the butter samples resulted in a negative impact on the sensory scores during storage. Unsaturated FFAs were not affected by the addition of probiotic strains, except for oleic acid. Saturated FFAs were not affected by storage (P > 0.05), but they were affected by adjunct probiotic strains (P < 0.01). Adjunct probiotic strains did not cause a significant difference in CLA levels of butter samples. The B. bifidum ATCC 29521 counts decreased most rapidly after the 30th day of storage (P < 0.05). Acknowledgements The authors wish to thank the Atatürk University Research Centre for the financial support of this project. Project No: 2012⁄ 254. References €
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