3.2.3.1 pH and microbiological composition of cheese curd during milk
fermentation
Growth of starter and ripening cultures during fermentation of cheese milk for 24 h was monitored by enumeration of viable cell counts and pH measurement (Leclercq-Perlat et al., 2004a). Duplicate samples from each treatment (one from each production) and duplicate samples from each cheese were obtained and analyzed (Note: four samples were obtained).
One mL of milk sample or twenty-five grams of cheese samples was aseptically obtained at various intervals [at initial inoculation (0 h), after milk pre-maturation (3 h), initial draining (6 h), second draining (11 h), and overnight draining (24 h)], and diluted with sterile 0.1% peptone water to make serial dilutions of up to 109 cfu/g or cfu/ml. The diluted samples were plated on appropriate molten agar. Lactic starter bacteria were enumerated on MRS and M17 agar, respectively, while P. camemberti was enumerated on YGC agar (Table 17).
The pH of milk was obtained by direct measurement using a calibrated pH meter (Sartorius, Japan) equipped with a glass electrode (AOAC, 2005b). The pH of the cheese curd was measured using a Sartorious pH meter (PB-20) with cheese electrode (model L8880, Schott). Ten g of cheese curd were mixed with 20 ml of CO2-free distilled water prior to pH measurement (AOAC, 2005b). The pH electrode was rinsed with distilled water after each measurement.
3.2.3.2 Evaluation of cheese during maturation
Sampling
Duplicate cheese blocks from each treatment (one from each production) and duplicate samples from each cheese were obtained (Table 19) and analyzed on days 3, 10, 14 and 21 (Note: four samples were obtained). Using a modified method, 2 mm of the rind (exterior of the cheese), and the inner layer of each cheese including the centre were separated (Graet et al., 1983; Karahadian et al., 1985; Graet and Brule, 1988; Leclercq-Perlat et al., 2004a; Leclercq-Perlat et al., 2004b). The cheese rind was discarded while the body was kept at -80°C overnight, and then grated to produce homogeneous frozen powders (Figure 27). The cheese samples (body, without rind)
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were then analysed for nitrogen fractions [total nitrogen (TN), acid-soluble nitrogen (pH 4.6-SN), non-protein nitrogen (NPN)], free amino acid content (FAA) and pH (Figure 27). Meanwhile, samples (body and rind) were used to determine levels of starter microorganisms and P. camemberti as well as textural firmness of cheese (Figure 27). Aroma compounds in samples were analysed from cheese body (without rind). For respective treatments, one cheese wheel from each of the two independent productions (n=2) was analysed at each sampling interval.
Figure 27. Cheese sampling plan. pH
Ten g cheese powders were mixed with 20 ml of CO2-free distilled water. The pH of the mixture was measured using a calibrated pH meter (PB-20, Sartorious) equipped with cheese electrode (model L8880, Schott) (AOAC, 2005b). The pH electrode was rinsed with distilled water after each measurement.
Analysis of nitrogen content of cheese
Two types of cheese analyses were used to monitor the extent of proteolysis during maturation of Camembert cheese (McSweeney and Fox, 1997; Ardö, 1999). The
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nitrogen content of three fractions of cheese samples was analyzed using the Micro-Kjeldahl method (AOAC, 2005a); these included TN, pH 4.6-SN, and NPN. FAA content of cheese was determined by the Cd-ninhydrin method (Folkertsma and Fox, 1992).
Fractionation of nitrogen content (Sample extraction)
The IDF 337 method (Ardö, 1999) was used for analysing various nitrogen fractions in cheeses. About 10 g grated cheese were dispersed in 50 ml warm 0.5 M tri-sodium citrate (AR, LabServ Pronalys) solution and stirred for 60 min using a magnetic stirrer on a hotplate at 50±2°C. The suspension was cooled to room temperature (about 20°C) and then made up to 200 ml with distilled water. Of the suspension, 10 ml were analyzed by the Macro-Kjeldahl method for TN analysis. SN was fractionated by adjusting 80 ml of the citrate suspension to pH 4.6 using 1.0 M hydrochloric acid (HCl) (AR, LabServ Pronalys). The suspension was shaken vigorously while the pH was being adjusted to 4.35-4.55, and the pH was constantly monitored by a pH meter (PB-20, Sartorious). The volume was then made up to 100 ml by the addition of distilled water. Of the mixture, 25 ml was filtered using quantitative filter paper (model LBS0040.110, LabServ Filtration) and used for pH 4.6-SN analysis. To analyse for NPN, 12% TCA (AR, Normapur) solution was used for extraction. Fifty ml pH 4.6-SN sample were added to 50 ml of 24% TCA. The solution was kept overnight at 4°C and 50 ml filtered samples were then analyzed by Macro-Kjeldahl method.
Nitrogen analysis of cheese samples (Digest of the extract)
The Macro-Kjeldahl method was used to determine the nitrogen content of TN, pH 4.6-SN and NPN using the ‘block digestion/steam distillation system’ analyzer (FOSSTM, Tecator Kjeltec system, Denmark) (Lynch and Barbano, 1999; AOAC, 2005a; AOAC, 2005c). Samples were measured into Kjeldahl tubes (20 tubes per batch). Two Kjeldahl digestion tablets (Kjeltabs, FOSSTM) and 20 ml concentrated sulphuric acid (95-98% pure) (LabServ Pronalys, Reagent Grade) were added into each digestion tube. Digestion was carried out on a block digester (model No. 2020, FOSSTM, Tecator Kjeltec system, Denmark) at 420°C for 4 h until the solution became clear. When the digestion was complete, sample tubes were removed from the digester and allowed to cool overnight. The cooled digest was liquid or liquid with only a few small crystals; a solid cake was not accepted for testing.
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The digestion tubes with samples were placed in an automatic distillation unit (model No. 1026, FOSSTM, Tecator Kjeltec system, Denmark), while 80 ml distilled water and 80 ml of 40% (w/v) sodium hydroxide solution (NaOH) (LabServ, Reagent Grade) were transferred into a water and alkali tank of the distillation unit, respectively. Samples were then distilled against 40 ml of 4% (w/v) boric acid (LabServ, Reagent Grade) solution containing 1% bromocresol green (w/v in 95% ethanol) (LabServ, Reagent Grade) and 0.7% methyl red (w/v in 95% ethanol) (LabServ, Reagent Grade) in a receiving flask (the condenser tip leading into the receiving flask extended below the surface of the boric acid collecting solution) (AOAC, 2005a; AOAC, 2005c). A steam-distillation was set at 5 min (with 100% steam efficiency) and at least 150 ml distillates were collected. During distillation, the presence of nitrogen in sample induced a color change from bright pink/red to green in the boric acid. The nitrogen content of samples was then determined by titrating the samples against standardized 0.1 M HCl solution to a grey-mauve-pink color. The first ‘persistent’ pink color with no hint of grey was taken as the end-point; and the progression of color was from green to grey-green, to grey-mauve color with pink overtones (AOAC, 2005a). The flask was lightly stirred to mix for accurate end-point determination and the volume of HCl used was recorded to the nearest 0.05 ml.
Efficiency of analysis and calculation of proteolysis parameters of cheese
Efficiency of analysis of nitrogen fractions was determined by using 0.12 g ammonium sulfate standard (>99% purity) (LabServ, Reagent Grade) and 0.85 g sucrose (LabServ, Reagent Grade) per tube. The sample was digested and distilled under the same conditions used for the test portion; average recoveries were at least 99%. Digestion efficiency was also evaluated by using 0.18 g tryptophan (LabServ, Reagent Grade) standard with 0.67 g sucrose per tube, with recovery rates above 98%. Analyses of blank samples for TN, SN-pH 4.6 and NPN were conducted for each respective nitrogen fraction analysed.
Results were calculated as follows in Equation 7:
ࡺ࢚࢘ࢍࢋΨ ൌ Ǥૠൈሺ܄ܛି܄܊ሻൈۻ܅ Equation 7
Where Vs and Vb = volumes of HCl (titre) used for test portion and blank (mL), respectively; M = molarity of HCl solution (g/L); and W = weight of test portion (g)
Degree of proteolysis in cheese samples was determined by the ‘ripening index’ as pH 4.6-SN and NPN against TN (Leclercq-Perlat et al., 2004a; Sullivan et al., 2005;
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Guizani et al., 2007) through calculation using Equations 8 and 9:
ሺΨሻ ൌ୮ୌସǤିୗ ൈ ͳͲͲ Equation 8
ሺΨሻ ൌ ൈ ͳͲͲ Equation 9
In addition to the calculated ‘ripening index’, cheese proteolysis was also evaluated based on the estimated values of protein nitrogen, casein nitrogen and peptide nitrogen content (Delgado et al., 2010; Delgado et al., 2011) through calculation using Equations 10, 11 and 12:
Protein Nitrogen = TN - NPN Equation 10 Casein Nitrogen = TN – pH 4.6-SN Equation 11 Peptide Nitrogen = pH 4.6-SN - NPN Equation 12
Determination of total FAA in cheese samples
Total FAA level was determined in pH 4.6-SN fractions of the cheeses using the Cd-ninhydrin method (Folkertsma and Fox, 1992; Hayaloglu, 2007; Hayaloglu et al., 2008; Hayaloglu, 2009). A 100-μl sample was diluted to 1 ml with distilled water and 2 ml Cd-ninhydrin reagent was added [0.8 g ninhydrin (AR, LabServ, Pronalys) dissolved in a mixture of 80 ml of 99.5% ethanol (LabServ, Reagent Grade) and 10 ml acetic acid (LabServ, Reagent Grade), followed by the addition of 1 g Cadmium chloride (CdCl2) (AR, LabServ, Pronalys) dissolved in 1 ml of distilled water]. The mixture was heated at 84f2°C for 5 min, cooled and the absorbance was measured in a spectrophotometer (UV-VIS model No. 1800, SHIMADZU) at 507 nm. A calibration curve of L-leucine (AR, LabServ, Pronalys) for concentrations of 0.001-0.1 mg/ml was used. The results were expressed as mg Leucine per 100 g cheese.
Key aromatic compounds analysis SPME
Since obtaining complete aroma profiles of cheese samples is difficult to accomplish (Mariaca and Bosset, 1997; Reineccius, 2006; Taylor and Linforth, 2010), key volatile compounds responsible for characteristic sensory properties of cheese during ripening were analysed by the SPME/GC-MS. The key volatile compounds are good indicators of the main metabolic reactions responsible for flavour synthesis
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(Delahunty and Drake, 2004; Quere, 2004). The methods of Kourkoutas et al. (2006), Katechaki et al. (2008), Rehn et al. (2011), Vitova et al. (2006; 2007), Kaminarides et al. (2007), Hayaloglu et al. (2008), and Leclercq-Perlat et al. (2004b) were adapted, with some minor modifications. For each shredded cheese sample (approximately 4 g) and 0.4 ml internal standard (IS) solution (0.008μg/ml) were transferred into a 20 ml headspace vial (fitted with a Teflon-lined septum sealed with an aluminum crimp seal) (SHIMADZU). The SPME programme used in this study is shown in Appendix 3.5. Volatiles were equilibrated by keeping the sample vial at 60°C (thermostatically controlled) for 30 min, with agitation at 500 rpm. In this experiment, sampling was done by using an auto-sampler (AOC-5000, AUTO-INJECTOR, SHIMADZU); volatile compounds extraction was achieved by inserting a 2-cm SPME fiber coated with 85 μm CAR/PDMS fused silica film (divinylbenzene-carboxen on polydimethylsiloxane, pH 2-11, working temperature 250-310oC, maximum heating time 2 h) (SUPELCO, Bellefonte, PA) into the vial through the septum, exposing it to the headspace for 20 min at 60°C. The fiber was positioned at 22.0 cm in each run. Both PA and PDMS fibers have been widely used for sampling volatiles from dairy samples (Pinho et al., 2001). In this experiment, PDMS was preferred due to its greater extraction efficiency (Chin et al., 1996; Guillen et al., 2004).
GC-MS
GC-MS programmes used in this study were set up as shown in Appendix 3.5. Thermal desorption of the extracted volatiles was performed in the GC injection port at 250°C for 5 min in splitless mode, with helium as the carrier gas at a flow rate of 1.0 mL min-1. Gas chromatography (GC-2010, SHIMADZU) was performed with a mass spectrometer detector (GC-MS-QP 2010 PLUS, SHIMADZU). The injection port was equipped with a narrow-bore (0.75 mm. i.d.) glass liner (SUPELCO, Bellefonte, PA) to minimize peak broadening. The split valve was opened 5 min after injection. The mass spectrometer was programmed as follows: the ion source temperature set at 220°C, the interface temperature set at 200°C, detector voltage set at 0.8 kV, mass scan range from 50 m/z to 300 m/z and scan speed of 526 ms. For volatile compound analysis of milk and dairy products, a polar column is desirable for separation (Mariaca and Bosset, 1997). Other workers have recommended use of DB-Wax and Co-Wax columns (Pinho et al., 2001; Kourkoutas et al., 2006; Vitova et al., 2007; Katechaki et al., 2008) but these were not available in our study. Therefore, a low polarity column was used in this study; temperature-program of column was optimized to obtain higher resolution and eliminate peak co-elution and broadening. Volatile compounds were separated on a fused silica RESTEK X-5 column (low
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polarity phase, crossbound, diphenyl dimethyl polysiloxane (60 m, 0.25 mm i.d., 0.25
μm thickness, and temperature range of 20°C-230°C). The column temperature was held at 30°C for 3 min (during desorption), then increased at 0.25°C/ min to 46°C, after which it ramped at 5°C/min to 115°C. The temperature was then raised at 20°C/min to 230°C. A final extension was applied at 230°C for 20 min for column clean up, giving a total run time of 121.55 min.
Compound identification and quantification
In this study, dimethyl sulfide, 1-octen-3-one, 1-octen-3-ol, 2-heptanone, 2-nonanone, 3-methyl-butanol, butyric acid (or butanoic acid), 3-methylbutanal (or isovaleraldehyde) and butane-2,3-dione (or diacetyl) were used as external aromatic standards and methyl octanoate was used as internal standard (Leclercq-Perlat et al., 2004b; Kourkoutas et al., 2006; Katechaki et al., 2008; Vitova et al., 2007). For each standard (internal and external), single stock solution was prepared separately using ethanol. Mixed standard solutions (2 ml) were made at various concentrations (0.0008-0.32 μg/ml), using prepared external standard stock solutions, IS solution and Millipore water [Table 34, Appendix 3.5]. In each mixed standard solution, the same amount (0.4 ml) of IS solution (0.008μg/ml) was used. The prepared mixed standard solutions were transferred into 15 ml headspace vials and sealed for analysis. All the above standard compounds were pure GC grade or reagent grade (>95%) and purchased from Fluka and Sigma-Aldrich Pty Ltd (NZ). Millipore water was used for carrying standard solutions instead of organic solvents, although all standards were only slightly soluble in the water. Millipore water was used because most of the organic solvents have low boiling points. When organic solvents are used for headspace analysis, high concentration of solvent would appear in vapour phase which could easily cause the detector being over saturated making mass scan difficult.
Identification of unknown compounds was achieved by comparing retention time of standard compounds and data from database library (Real Time Analysis Database Library, SHIMADZU). However, the use of retention time alone for compound identification is considered weak (Quere, 2004). Therefore, mass spectra of the compounds provided by MS were also used to provide additional data supporting identification beyond GC data. For quantification of volatile compounds, peak areas of external and internal standard (IS) at various concentrations were used to generate calibration curves. Calibration curves were constructed for each standard by plotting concentration of volatile compound against the peak area ratio of analytes and
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internal standard determined (Equation 13). Calculation of the volatile compounds in test samples was determined using Equation 14.
୮ୣୟ୩ୟ୰ୣୟୗୈ
୮ୣୟ୩ୟ୰ୣୟ୍ୗୗୈൌ ൈ Ǥ כ Equation 13 Where STD=external standard, ISSTD=internal standard, con.STD=concentration of external standard (μl/ml), ‘a’ is a factor, ‘b’ is a constant. *since concentration of internal standard (0.008 μl/ml) was applied constantly in both standard solutions and samples, standard curve was not determined using concentration ratio of STD/ISSTD (IOFI, 2011; Biedermann et al., 2007). In each standard, a linear relationship between response and concentration was obtained over the desired range for the analysis of cheese samples used in this study.
ൌቀ
౦ౡ౨ౙౣ౦౫ౚ ౦ౡ౨ీ ିୠቁ
ൈୡ୦ୣୣୱୣ୵ୣ୧୦୲ ൈ106 Equation 14 Where the concentration of volatile compounds in cheese samples is calculated in Ϥg/kg,
cheese weight (g).
Microbiological analysis
Samples (body and rind) were analyzed on days 3, 10, 14 and 21 to evaluate microbial changes of cheese during maturation. Duplicate cheese from each treatment (one from each production) and duplicate samples from each cheese were used, giving four samples. Description of microbial analyse of cheese fermentation profiles can be found in Section 3.2.3.1. Twenty-five grams of each of cheese sample were aseptically withdrawn at various intervals for analysis. Lactic starter bacteria were enumerated on MRS and M17 agar (IDF, 2003; Thage et al., 2005; IDF, 2010), while P. camemberti was enumerated on YGC agar (IDF, 2004).
Instrumental texture analysis
Textural profile of cheese samples (with rind) during ripening was assessed on days 3, 10, 14 and 21; duplicate cheese from each treatment (one from each production) and duplicate samples from each cheese were used, giving four samples.
Texture analysis of Camembert cheese samples were performed using a TA-TX2 Texture Analyzer (SMS Stable Micro System Ltd, United Kingdom), operating in the compression mode (Guamisa et al., 1997; Antoniou et al., 2000; Saldo et al., 2000). Cubic cheese samples with rind (20 mm diameter × 20 mm high) were obtained using a knife lubricated with food grade Canola™ oil (vegetable oil) to prevent fracture
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during cutting. Prepared samples were kept at room temperature (20°C) for 2 h prior to analysis. Texture analysis of samples was done in a temperature-controlled room set at 20°C. During texture analysis, test pieces of cheeses were compressed between 50 mm diameter flat upper plate probe and a square stainless steel bottom plate to 50% of the original height. A pre-test speed of 1.0 mm/sec, test speed of 2.0 mm/sec, post-test speed of 10.0 mm/sec, and an automatic trigger force of 5.0 g were used [Appendix 3.4]. Both sample surfaces in contact with the plates were lubricated with Canola™ oil to prevent friction.
The TX.TA2 equipment was connected to a computer with data transfer rate for force, displacement and time data. The test measurements were analyzed using the Texture Analyzer TE32 software. Data were collected in the form of force/time/displacement curves, using the software Texture Expert (Stable Micro System). Hardness of the cheese samples was determined as the end force required to compress samples (point of maximum deflection of the curve during the first pressing cycle).