Rapid determination of collagen in meat-based foods by microwave hydrolysis of proteins and HPAEC–PAD analysis of 4-hydroxyproline

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MEAT SCIENCE Meat Science 80 (2008) 401–409 www.elsevier.com/locate/meatsci

Rapid determination of collagen in meat-based foods by microwave hydrolysis of proteins and HPAEC–PAD analysis of 4-hydroxyproline M.C. Messia, T. Di Falco, G. Panfili, E. Marconi * DISTAAM, Universita` del Molise, Via De Sanctis, 86100 Campobasso, Italy Received 16 October 2007; received in revised form 10 January 2008; accepted 14 January 2008

Abstract A rapid microwave procedure for protein hydrolysis coupled with High Performance Anion Exchange Chromatography and Pulsed Amperometric Detection (HPAEC–PAD) was developed to quantify the amino acid 4-hydroxyproline in meat and meat-based products. This innovative approach was successfully applied to determine collagen content (4-hydroxyproline  8) as the index quality of meat material employed in the preparation of typical meat sausages (‘‘Mortadella di Bologna PGI” and ‘‘Salamini italiani alla cacciatora PDO”) and fresh filled pastas. Microwave hydrolysis showed a precision and accuracy similar to traditional hydrolysis (RSD% from 0.0 to 6.4; relative error 1.4–10.0%) with a reduction in the hydrolysis time from 24 h to 20 min. HPAEC–PAD allowed detection of 4-hydroxyproline without pre or post-column derivatization and the use of non-toxic eluents. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: 4-Hydroxyproline; Collagen; Microwave hydrolysis; HPAEC–PAD

1. Introduction The protein quality of meat and meat products varies with the amount of extracellular connective tissue proteins present in skeletal, cardiac and smooth muscle tissues (i.e. collagen, elastin, muscle fibre ghosts proteins, proteoglycans, etc.) (Zarkadas et al., 1988). Significant levels of meat of poor quality (both from an economic and nutritional point of view) rich in connective tissue (tendons, skin, etc.) can be shown by the estimation of collagen content. The amino acid sequence of collagen is rich in proline and approximately 50% of proline side chains are hydroxylated post-translationally to form 4hydroxyproline (Delport, Maas, van der Merwe, & Laurens, 2004; Dugan, Thacker, Aalhus, Jeremiah, & Lien, 2000). In contrast with other mammalian proteins, collagen contains a high concentration of the imino acid 4-hydroxy*

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0309-1740/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2008.01.003

proline (Flores, 1980; Vazquez-Ortiz & Gonzalez-Mendez, 1996) through which the collagen content can be estimated (Etherington & Trevor, 1981). 5-Hydroxylysine, another amino acid characteristic of collagen, was also used for the identification and quantification of connective tissue proteins in meat-based products (Zarkadas & Maloney, 1998; Zarkadas et al., 1992; Zarkadas, Karatzas, & Zarkadas, 1996). The amount of collagen relates not only to the economic value of the raw material used (Flores, 1980) but also to nutritional aspects (Flores, 1980; Zarkadas, Yu, Zarkadas, & Minero-Amador, 1995) since the low biological value of protein in connective tissue is due to the deficiencies in lysine, tryptophan and sulphur amino acids (Young & Pellett, 1984). The recommendation of the Food Safety and Inspection Service (FSIS, 1984) of the United States Department of Agriculture (USDA), states that assessment of the quantity of connective tissue/collagen content is a simple and practical method for assessing the protein quality of meat products.

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In reality there are some commercial collagen-based byproducts used in various meats and meat products, which include skins (rind) and skin trimmings, tendon, beef shank, sinew residues and hand or mechanically separated bones from beef, pork and poultry carcasses (Zarkadas et al., 1995). Non-authentic meat products may involve substitution of meat from a high value species with meat from a lower value species, extension of a meat product with connective tissue or fat (so that these components are present in excess of the amounts naturally associated with the flesh used) and the use of non-meat proteins or other substances (Vallejo-Cordoba, Gonzalez-Cordova, Mazorra-Manzano, & Rodriguez-Ramirez, 2005). For this reason many countries specify the maximum permitted level of collagen, in comminuted meat products such as Protected Designation of Origin (PDO) and Protected Geographic Indication (PGI) products (i.e. Mortadella di Bologna PGI Council regulation (EEC) No. 1549 of 17/07/98, Salamini italiani alla cacciatora PDO Council Regulation (EC) No. 1778 of 07/09/2001, Salchicho´n de Vic PGI Council Regulation (EC) No. 2601 of 28/12/ 2001, Sobrasada de Mallorca PGI Council Regulation (EEC) No. 1107 of 12/06/1996) in order to prevent adulteration, and the partial substitution of high value raw materials with less expensive alternatives. The 4-hydroxyproline content can be measured successfully using different techniques including colorimetric methods (AOAC, 2000), micellar electrokinetic chromatography (Dugan et al., 2000), NIR (Berg & Kolar, 1991; Oh & Grossklaus, 1995), autofluorescence of connective tissue collagens (Egelandsdal, Dingstad, Togersen, Lundby, & Langsrud, 2005; Swatland, 1987; Wold, Lundby, & Egelandsdal, 1999), ultrasonic measurements (Park, Whittaker, Miller, & Hale, 1994) carbon 13-Fourier transform nuclear magnetic resonance spectroscopy (Jozefowicz, Oneill, & Prosser, 1977) and NMR imaging to visualize the spatial distribution of connective tissue (Bonny et al., 2001). Among these, the most commonly employed is the colorimetric method based on hydrochloric acid or sulphuric acid hydrolysis of meat, oxidation of 4-hydroxyproline with chloramine-T and spectrophotometric measurement at 560 nm of the red–purple complex formed. Using this method 4-hydroxyproline is quantitatively determined as a measure of collagenous material in meat and meat products (AOAC, 2000). Although this method is very specific for 4-hydroxyproline, it is difficult to control oxidation, colour formation and the hydrolysis step is very time consuming (16–24 h). One of the most significant and recent developments in the performance of compositional food analysis is the use of microwave radiation energy for protein hydrolysis (Chen, Chiou, Chu, & Wang, 1987; Chiou & Wang, 1989; Fountoulakis & Lahm, 1998). Microwave energy is non-ionizing radiation that produces heat by molecular collision of the dipole rotation of polarized molecules and by ionic conduction without changing the molecular structure (Neas & Collins, 1988). The procedure has been

successfully used for fast protein hydrolysis in determining natural, non-natural single amino acids such as tryptophan (Carisano, 1993), lysine (Marconi et al., 1996), furosine (Marconi, Mastrocola, & Panfili, 1999; Marconi, Panfili, & Acquistucci, 1997) meso-diaminopimelic acid (Marconi, Sorrentino, Mastrocola, & Coppola, 2000) and total amino acids (Chen et al., 1987; Chiou & Wang, 1989; Gilman & Woodward, 1990; Marconi, Panfili, Bruschi, Vivanti, & Pizzoferrato, 1995; Woodward, Gilman, & Engelhart, 1990). Many chromatographic techniques have been developed to resolve amino acids. The original chromatographic approach is ionic-exchange separation using gradient elution followed by post-column reaction to enable detection (Spackman, Stein, & Moore, 1958). The most common post-column derivatizing reagent is ninhydrin, which is used in conjunction with UV absorbance detection. Alternatively, derivatization with ophthalaldehyde can be combined with fluorescence detection. Pre-column techniques can be also used but problems such as the low stability of amino acid derivatives, multiple derivative formation and reagent interference are encountered (Jones, 1986). An alternative to derivatization systems is electrochemical detection (Martens & Frankenberg, 1992; Polta & Johnson, 1983), and in particular integrated amperometry (HPAEC–PAD) (Clarke, Jandik, Rocklin, Liu, & Avdalovic, 1999; Welch, La Course, Mead, & Johnson, 1989) which does not requires amino acid derivatization but the direct detection of amino acids on a platinum or a gold electrode after the separation by anion exchange. In this report the use of microwave hydrolysis coupled to HPAEC–PAD for 4-hydroxyproline analysis in meatbased products was tested. The optimized procedure (microwave hydrolysis and HPAEC–PAD) was used to assess the collagen content (4-hydroxyproline  8) and the quality of commercial meat products. 2. Materials and methods 2.1. Chemicals NaOH 50% (p/v) was purchased from Baker (Mallinckrodt Baker B.V., Deventer, Holland), high-purity laboratory water was produced by means of MilliQ-Plus apparatus (Millipore S.p.A., Milano, Italy); amino acid standard mixture was from Beckman Instruments Inc. (Palo Alto, CA, USA), 4-hydroxyproline and 5-hydroxylysine standards were from Sigma Chemical Co. (St. Louis, MO, USA); all other chemicals and reagents of HPLC grade were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Samples 2.2.1. Sausages Six different brands of cooked non-fermented sausages from pork meat ‘‘Mortadella di Bologna PGI”

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(Council Regulation (EEC) No. 1549 of 17/07/98) and three different brands of raw fermented sausages from pork meat ‘‘Salamini italiani alla cacciatora PDO” (Council Regulation (EEC) No. 1778 of 07/09/2001) were purchased from different local supermarkets (Campobasso, Italy).

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2.4. Proximate composition Moisture, protein (Nx6.25), fat, and ash content were determined according AOAC methods 985.14, 992.23, 991.36 and 923.03 (AOAC, 2000). 2.5. 4-Hydroxyproline determination

2.2.2. Fresh filled pastas Eight different samples of fresh filled pastas with meatbased filling were used. The typologies and the ingredients used for the preparation of each fresh filled pastas are reported below: Anolini 1: Soft wheat flour, durum wheat semolina, eggs, bread-crumb, bovine cooked meat, pork cooked meat, sunflower oil, cheese, carrots, wine, onion, flavouring, celery, salt, milk whey powder, concentrated tomatoes. Cappelletti 2: Soft wheat flour, pork and bovine meat, eggs, durum wheat semolina, bread-crumb, cheese, salt, spices. Cappelletti 3: Soft wheat flour, durum wheat flour, eggs, mortadella, pork and bovine meat, bread-crumb, vegetal oil, milk whey powder, salt, flavouring. Tortellini 4: Durum wheat semolina, eggs, water, pork and bovine cooked meat, bread-sticks, potatoes flakes, salt, spices, cheese, flavouring. Tortellini 5: Soft wheat flour, pork and bovine meat, eggs, durum wheat semolina, bread-crumb, mortadella, milk whey powder, soft cheese, flavouring, salt, cheese, spices. Tortellini 6: Soft wheat flour, pork and bovine meat, durum wheat semolina, bread-crumb, mortadella, cheese, flavouring, milk whey powder, salt, spices. Tortelloni 7: Durum wheat semolina, bread-crumb, eggs, pork cooked meat, animal fat, cured ham, salt, flavouring, cheese. Cappelletti 8: Soft wheat flour, durum wheat semolina, pork and bovine meat, eggs, bread-crumb, cheese, salt, spices. All fresh filled pasta samples, from well known Italian manufacturers, packaged with modified atmosphere, were purchased from local supermarkets (Campobasso, Italy). 2.3. Reference material Gristle from pork windpipe was obtained from a local butcher (Campobasso, Italy). Standard collagen from bovine Achilles tendon (C9879) was from Sigma Chemical Co. (St. Louis, MO, USA). Samples of mortadella, salamini and fresh filled pastas were chopped and then homogenized with a steel blade homogenizer, IKA A 10 Labortechnic (Tanke & Kunkel, Gmbh & Co., Staufe, Germany) for no longer than 15– 20 s to minimize any increase in temperature. Samples of fresh filled pasta were analyzed, after grinding and homogenisation, considering either the entire product or the meatbased filling only.

2.5.1. Traditional protein hydrolysis Traditional protein hydrolysis was carried out using the procedure reported by Spackman et al. (1958) with some modifications. An aliquot of sample, corresponding to 25 mg of protein, was placed in a pyrex flask (100 mL) with an acid resistant rubber vacuum stopper and 25 mL of 6 N HCl added. In the pyrex flask was created vacuum by means of a water vacuum pump and the flask was put in an oven at 110 °C for 24 h, afterwards cooled and filtered with Whatman paper No. 1. The sample was evaporated to dryness and redissolved in 0.1 N HCl. Before analysis, samples were diluted 1:50–1:100 with ultra-pure water, filtered through 0.20 lm filter (Dionex Corporation, Sunnyvale, CA, USA) and then injected in the chromatographic system. 2.5.2. Microwave protein hydrolysis Microwave hydrolysis was carried out using a Microwave Digestion System, Mod. MDS 2000 (CEM Corporation, Mattews, NC, USA). This microwave oven has a maximum power of 630 ± 50 W and a magnetron frequency of 2435 MHz. The system was equipped with probes to detect and control the pressure and temperature inside the sealed vessel. The sample, corresponding to 25 mg of protein, was placed into a Teflon PFA digestion vessel (capacity volume 50 mL) and 8 mL 6 N HCl was added. The efficiency of microwaves is strictly related to the chemical–physical characteristics of samples, and to allow uniform microwave distribution and absorption, each cycle of hydrolysis (four containers) was carried out on samples of the same typology. The vessel cup was screwed manually; the pressure and fibre optic probes were connected to the vessel with the triple ported cap. After the irradiation cycles the vessels were cooled and then removed, filtered by Whatman paper No. 1, evaporated to dryness and dissolved in 0.1 N HCl. Before analysis, samples were diluted 1:50–1:100 with ultra-pure water, filtered through 0.20 lm filter (Dionex Corporation, Sunnyvale, CA, USA) and then injected in the chromatographic system. The hydrolysis conditions, similar to those previously standardized for total amino acid and lysine determination (Marconi et al., 1995; Marconi et al., 1996), are reported in Table 1. 2.6. Chromatographic determination The 4-hydroxyproline content was determined by High Performance Anion Exchange Chromatography with

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Table 1 Microwave operating conditions for the protein hydrolysis of sausages and meat-based fillings of fresh filled pasta samples

Power (% 630 W) Time (min) Temperature (°C) Pressure max (psi)

1° Cycle

2° Cycle

85 1 100 100

85 5 155 130

Pulsed Amperometric Detection (HPAEC–PAD), using the Dionex system (Dionex Corporation, Sunnyvale, CA, USA) composed of a gradient pump (mod GP50) with an on-line degaser and electrochemical detector (model ED40). Instrument control, data collection and total quantification were managed using Chromeleon chromatography software (Dionex). The flow-through electrochemical cell (Dionex) consisted of a 1 mm diameter gold working electrode, a pH reference electrode, and the titanium body of the cell as the counter electrode. A controlled Rheodyne injector (Cotati, CA, USA) with a 25 lL sample loop was used for sample injection. Separation was performed with an Aminopac PA10 analytical column 250  2 mm, with 8.5 lm particle size (Dionex). Quantitative determination was carried out at a flow rate of 0.25 mL/min using a mobile phase of water, 250 mM sodium hydroxide and 1.0 M of sodium acetate as shown in Table 2 and an optimized time-potential waveform (Table 3) (Caboni et al., 2005). Single amino acid identification and 4-hydroxyproTable 2 Gradient conditions for anion exchange separation of amino acids Time (min)

Water (%)

NaOH (%)

Sodium acetate (%)

0.0 2.0 12.0 16.0 24.0 40.0 40.1 42.1 42.2 62.0

80 80 80 68 36 36 20 20 80 80

20 20 20 32 24 24 80 80 20 20

0 0 0 0 40 40 0 0 0 0

line quantification were carried out by means of external amino acid standards. 2.7. Calculation of collagen content The collagen content was estimated by multiplying the 4-hydroxyproline content (g/100 g of sample) by eight as provided by AOAC method 990.26 (AOAC, 2000), considering that collagen connective tissue contains 12.5% 4hydroxyproline if the nitrogen-to-protein factor is 6.25. 3. Results and discussion 3.1. Standardization of microwave hydrolysis for 4hydroxyproline analysis A typical pressure/temperature diagram of a meat-based food sample processed by the irradiation program is shown in Fig. 1. The distribution of microwaves was uniform and the temperature/pressure diagram was highly reproducible with good repeatability (temperature RSD max = 4.9% and pressure RSD max = 9.6%). To test the reliability of the microwave program and to check its versatility for different food matrices, the efficiency was tested measuring the 4-hydroxyproline content in the hydrolysates of whole, fresh meat-based filled pasta, meat filling of fresh filled pasta, sausages, gristle from pork windpipe and collagen standard. Results obtained by the combination of the two hydrolysis procedures (traditional and microwaves) with the HPAEC–PAD detection are reported in Table 4. The microwave hydrolysis system had a precision similar to traditional hydrolysis (microwave RSD = 0.0–6.4%; traditional RSD 0.4–9.4%) and a good accuracy (relative error 1.4–10.0%). Analytical performances of the two hydrolysis procedures suggest that microwaves can be used for the rapid protein hydrolysis for the determination of 4hydroxyproline in meat-based foods. Moreover, microwave hydrolysis reduces the hydrolysis time from 24 h to 20 min.

180 160

Table 3 Integrated amperometry waveform used to detect amino acids Potential (V)

0.00 0.04 0.05 0.11 0.12 0.41 0.42 0.56 0.57 0.58 0.59 0.60

+0.13 +0.13 +0.28 +0.28 +0.60 +0.60 +0.28 +0.28 1.67 1.67 +0.93 +0.13

Integration

120

psi/˚C

Time (s)

140 pressure temperature

100 80 60

Begin

40 20

End

0 0

1

2

3

4

5

6

7

8

9

10

Time (min) Fig. 1. A typical temperature/pressure diagram of a Mortadella di Bologna PGI sample during microwave hydrolysis.

M.C. Messia et al. / Meat Science 80 (2008) 401–409 Table 4 Comparison between traditional protein hydrolysis and microwave protein hydrolysis for the determination of 4-hydroxyproline in different meat-based foods Samples

Sausages Mortadella di Bologna 1 Mortadella di Bologna 5 Salamini alla cacciatora 2

4-hydroxyproline (g/100 g protein) Traditional (A)

Microwave (B)

Mean (n = 3)

Mean (n = 3)

RSD%

RSD%

Relative error [(A B)/ A]  100

0.78

2.4

0.74

0.0

5.1

1.84

9.4

1.76

5.7

4.3

0.67

7.2

0.66

6.4

1.5

Whole fresh filled pasta Cappelletti 8 0.20 Tortellini 5 0.74 Tortellini 6 0.53

4.0 3.2 2.9

0.18 0.67 0.48

3.9 3.1 2.9

10.0 9.5 9.4

Meat-based filling of fresh filled Cappelletti 8 0.51 Tortellini 5 2.54 Tortellini 6 1.60

pastas 4.3 4.8 5.9

0.50 2.64 1.56

4.2 4.8 5.9

2.0 3.9 2.5

Standards Pork gristle Collagen from Achilles tendon

2.3 0.4

5.70 10.40

3.4 0.3

1.4 4.0

5.62 10.00

In addition, the accuracy of the innovative analytical system (microwave hydrolysis coupled with HPAEC– PAD analysis) was checked by running a recovery test after the addition of increasing amounts of 4-hydroxyproline (1.5, 3.0, 4.5 mg) to 25 mg of collagen standard from bovine Achilles tendon and to 100 mg of salamini 2 sample. The results of the recovery test were very satisfactory, with values ranging from 97% to 104%. Fig. 2 shows HPAEC–PAD chromatograms of fresh filled pastas hydrolyzed with traditional and microwave procedures. No qualitative or quantitative differences were seen between the two chromatograms further demonstrating the possibility to use the coupled procedure for rapid analysis of 4-hydroxyproline (retention time 9 min). The same HPAEC–PAD chromatographic run could also permit identification and quantification of all other amino acids including 5-hydroxylysine (retention time 2.5 min), another amino acid characteristic of collagen (Zarkadas & Maloney, 1998; Zarkadas et al., 1996) which was previously estimated in its allo-stereoisomer forms with cation exchange chromatography by Zarkadas, Zarkadas, Karatzas, Khalili, and Nguyen (1986). 3.2. Determination of collagen in different transformed meat products 3.2.1. Proximate composition and collagen content in PDO and PGI sausages The addition of low-value meat is generally considered to be the most frequent adulteration of meat-based

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products such as sausages. Suitable markers for identifying the raw material used for sausage preparation are collagen content, water/protein, fat/protein and collagen/protein ratios. The water/protein ratio indicates the amount of meat used in the preparation, while the fat/protein, collagen content and collagen/protein ratios are markers of meat quality. Table 5 shows the proximate analysis of sausage samples. Significant differences between the nutrient content of mortadella and salamini samples were related to the procedure for preparation and to the meat cuts utilized for their preparation. However the chemical composition is very similar to those related to other dry fermented and non-fermented Italian sausages (Coisson, Cerutti, Travaglia, & Arlorio, 2004). All sausage samples fulfilled the amount of protein requested by respective Product Specifications: 13.5% (wet basis) for Mortadella di Bologna PGI and 20.0% (wet basis) for Salamini italiani alla cacciatora PDO. Table 6 shows the 4-hydroxyproline, collagen, protein and the collagen/protein, water/protein and fat/protein ratios of sausages. A wide variability in the collagen content was found in the analyzed samples, with a range between 1.04% and 3.04 % wet basis. The collagen and consequently the collagen/protein ratio increases proportionally with the quantity of connective tissue used in the preparation of meat-based products. Moreover all analyzed samples fulfilled the maximum permitted ratio of collagen/protein fixed by PDO and PGI product specifications ‘‘Salamini italiani alla cacciatora” (collagen/protein = 0.15) and ‘‘Mortadella di Bologna” (collagen/protein = 0.20). Moreover, the collagen/protein variability obtained for salamini samples (RSD% = 16.77) showed a better standardization, than the larger variability (RSD% = 43.1) of Mortadella di Bologna samples, showing the need to revise and establish more restrictive limits for PDO and PGI products. The water/protein and fat/protein ratios also fulfilled the values requested by respective Product Specifications (water/protein max 4.10 for mortadella and max 2.30 for salamini; fat/protein max 2.00 for both sample typologies). However, the fat/protein and collagen/protein found in samples 2 and 3 of mortadella can indicate the use of poor quality meat. 3.2.2. Proximate composition and collagen content in fillings of meat-based fresh filled pasta Fresh meat filled pastas are generally made by using a blend of different ingredients such as cheese, bread-sticks or bread-crumb, which should not contain 4-hydroxyproline. Therefore data relative to the collagen content is not sufficient to characterize the quality of the meat filling and consequently protein/carbohydrates and protein/fat ratios were calculated to provide a better estimate of the amount of meat contained in these foods. The presence of high quantities of carbohydrates in meat-based fillings of fresh filled pastas (Table 7) is evident

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A

Pro(4-OH)

350

His

300

nC

250

Glu

Gly

Arg

200

Cys

Thr

150

Lys

Phe

Ser

Asp

100

Pro

Ala

50

Val

Ile Leu

Tyr

Met

0 0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

35.0

40.0

35.0

40.0

Minutes

B

Pro(4-OH)

350

CI mo (2)2˚

300 Arg Lys(5-OH) 250 Thr

nC

200

Glu

Pro Phe

Lys

150

His

Ser

Asp

Gly

Ala

100

Cys

Val

IleLeu Met

50

Tyr

0 0

5.0

10.0

15.0

20.0

25.0

30.0

Minutes

C

Pro(4-OH)

350 300

CI mo (4)2˚

Arg Lys(5-OH)

nC

250 Thr

200 Lys

150

His

Ser

Glu

Pro Phe

Gly

Asp

Ala

100

Val

50

Ile

Cys

Leu

Tyr

Met

0 0

5.0

10.0

15.0

20.0

25.0

30.0

Minutes Fig. 2. HPAEC–PAD chromatograms of a standard mixture of amino acids (A) and whole fresh filled pasta (tortellini) after traditional (B) and microwave (C) hydrolysis.

Table 5 Proximate composition of sausages (Mean ± SD; n = 3) Samples

Moisture (%)

Protein (% dry weight)

Fat (% dry weight)

Ash (% dry weight)

Mortadella di Bologna PGI Mortadella 1 57.6 ± 0.05 Mortadella 2 51.2 ± 0.01 Mortadella 3 52.6 ± 0.06 Mortadella 4 58.1 ± 0.04 Mortadella 5 58.1 ± 0.02 Mortadella 6 52.3 ± 0.00

41.4 ± 0.84 31.3 ± 0.23 34.3 ± 0.31 38.3 ± 0.11 40.6 ± 0.00 34.8 ± 0.02

53.1 ± 0.24 57.4 ± 0.32 52.8 ± 0.17 54.0 ± 0.21 51.9 ± 0.14 52.4 ± 0.12

6.91 ± 0.007 8.14 ± 0.005 7.17 ± 0.009 8.18 ± 0.012 7.46 ± 0.041 7.94 ± 0.008

Salamini italiani alla cacciatora PDO Salamini 1 26.8 ± 0.01 Salamini 2 25.1 ± 0.02 Salamini 3 24.9 ± 0.01

38.4 ± 0.11 45.5 ± 0.11 41.2 ± 0.05

46.8 ± 0.21 40.8 ± 0.14 43.8 ± 0.41

10.07 ± 0.012 8.80 ± 0.130 9.50 ± 0.078

M.C. Messia et al. / Meat Science 80 (2008) 401–409

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Table 6 4-Hydroxyproline, collagen and ratios collagen/protein, water/protein, fat/protein in sausages Collagen (% wet basis)

Protein (% wet basis)a

Collagen/ proteinb

Mortadella di Bologna PGI Mortadella 1 0.13 ± 0.00 Mortadella 2 0.38 ± 0.01 Mortadella 3 0.36 ± 0.01 Mortadella 4 0.16 ± 0.05 Mortadella 5 0.30 ± 0.04 Mortadella 6 0.22 ± 0.02 Mean 0.26 RSD% 40.4

1.04 3.04 2.88 1.28 2.40 1.76 2.07 40.4

17.6 15.3 16.3 16.1 17.0 16.6 16.5 4.8

0.06 0.20 0.18 0.08 0.14 0.11 0.13 43.1

3.28 3.35 3.23 3.62 3.41 3.15 3.34 4.9

1.28 1.83 1.54 1.41 1.28 1.51 1.48 14.0

Salamini italiani alla cacciatora PDO Salamini 1 0.24 ± 0.01 Salamini 2 0.23 ± 0.01 Salamini 3 0.23 ± 0.00 Mean 0.23 RSD% 2.5

1.92 1.84 1.84 1.87 2.5

28.1 34.1 30.9 31.0 9.7

0.07 0.05 0.06 0.06 16.7

0.95 0.74 0.81 0.83 12.8

1.22 0.90 1.06 1.06 15.1

Samples

4-Hydroxyproline (% wet basis) (Mean ± SD; n = 3)

Water/ protein

Fat/protein

a Minimum level for Mortadella di Bologna fixed by Council Regulation (EEC) No. 1549, 17/07/1998 = 13.5% (wet basis). Minimum level for Salamini italiani alla cacciatora fixed by Council Regulation (EEC) No. 1778, 07/09/2001 = 20.0% (wet basis). b Maximum level for Mortadella di Bologna fixed by Council Regulation (EEC) No. 1549, 17/07/1998 = 0.20. Maximum level for Salamini italiani alla cacciatora fixed by Council Regulation (EEC) No. 1778, 07/09/2001 = 0.15.

Table 7 Proximate composition of meat-based filling of fresh filled pastas (Mean ± SD; n = 3) Samples Meat-based filling Anolini 1 Cappelletti 2 Cappelletti 3 Tortellini 4 Tortellini 5 Tortellini 6 Tortelloni 7 *

Moisture (%)

Protein (% dry weight)

of fresh filled pastas 38.9 ± 0.11 26.5 ± 0.93 40.7 ± 0.04 27.6 ± 0.56 36.8 ± 0.05 19.3 ± 0.29 32.0 ± 0.02 23.2 ± 0.08 40.7 ± 0.09 26.1 ± 0.50 41.6 ± 0.34 27.7 ± 0.07 32.3 ± 0.26 18.2 ± 0.05

Fat (% dry weight)

Ash (% dry weight)

Carbohydrates* (% dry weight)

20.0 ± 0.79 25.0 ± 0.37 22.5 ± 0.36 20.8 ± 0.51 26.8 ± 0.17 25.3 ± 1.13 29.6 ± 0.25

5.55 ± 0.092 4.15 ± 0.410 3.39 ± 0.042 4.30 ± 0.141 5.04 ± 0.177 5.56 ± 0.424 5.56 ± 0.191

47.9 43.2 54.9 51.7 42.1 41.4 46.7

Calculated by difference.

in all samples and demonstrates the use of carbohydraterich ingredients such as bread-crumb, bread-sticks and potato flakes, substituting for higher value ingredients such as meat which should be the bulk of the filling. The fat content found in anolini 1 and tortellini 4 was lower than in the other meat fillings because of the use of vegetables in the

filling (as reported on the sample’s labels), while the higher percentages of fat found in tortellini 5 and tortelloni 7 is probably due to the use of cheese and sausages or meat cuts rich in fat. As for the collagen content and collagen/protein ratios (Table 8), a wide variability was found, with a range

Table 8 4-Hydroxyproline, collagen, ratios collagen/protein, protein/carbohydrates, protein/fat in meat-based filling of fresh filled pastas Samples

4-Hydroxyproline (% wet basis) (Mean ± SD; n = 3)

Meat-based filling of fresh filled pastas Anolini 1 0.16 ± 0.04 Cappelletti 2 0.14 ± 0.05 Cappelletti 3 0.42 ± 0.04 Tortellini 4 0.58 ± 0.13 Tortellini 5 0.41 ± 0.04 Tortellini 6 0.21 ± 0.01 Tortelloni 7 0.29 ± 0.05 Mean 0.32 RSD% 51.2

Collagen (% wet basis)

Protein (% wet basis)

Collagen/ protein

Protein/ carbohydrates

Protein/fat

1.28 1.12 3.36 4.64 3.28 1.68 2.32 2.52 51.2

16.2 16.4 12.2 15.8 15.5 16.2 12.3 14.9 12.5

0.08 0.07 0.27 0.30 0.21 0.10 0.19 0.17 53.3

0.55 0.64 0.35 0.45 0.62 0.67 0.39 0.52 24.4

1.32 1.10 0.86 1.11 0.97 1.09 0.61 1.01 22.3

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between 1.12 and 4.64% wet basis and between 0.07 and 0.30% wet basis, respectively. In particular, the collagen and collagen/protein values obtained for cappelletti 3, tortellini 4 and 5 and tortelloni 7, suggest that the meat used for their preparation was rich in gristle; the use of low quality ingredients in the preparation of cappelletti 3 and tortelloni 7 was also confirmed by the protein/carbohydrate (0.35 and 0.39, respectively) and protein/fat ratios (0.86 and 0.61, respectively), which denote the use of breadsticks, bread-crumb and ingredients with high fat contents. The collagen content coupled to collagen/protein and protein/carbohydrates ratios (Table 8) found in anolini 1 and cappelletti 2 and tortellini 6 attested to both the high quantity and quality of meat used in their preparation. However, it is important to remember that the lower collagen/ protein ratios could have been due to the use of non-meat protein in the preparation of the products, diluting the lean meat. 4. Conclusions Low-value meat replacing more expensive meat is a frequent way of adulterating meat-based foods. The combined use of the microwave procedure for protein hydrolysis and HPAEC–PAD for the determination of 4hydroxyproline supplies a rapid tool to assay the protein quality of different meat-based foods (sausages and meatbased filling of fresh filled pastas) and provides: (i) A reduction of sample hydrolysis time from 24 h (traditional hydrolysis) to 20 min (microwave hydrolysis). (ii) Direct 4-hydroxyproline detection with no need either for pre or post-column derivatization or for toxic eluents use. (iii) The possibility to evaluate in the same chromatographic run other amino acids.

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