Raman Spectroscopic Study of Structural Changes upon Chilling

Article pubs.acs.org/JAFC

Raman Spectroscopic Study of Structural Changes upon Chilling Storage of Frankfurters Containing Olive Oil Bulking Agents As Fat Replacers A. M. Herrero,*,† C. Ruiz-Capillas,† F. Jiménez-Colmenero,† and P. Carmona‡ †

Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), José Antonio Novais, 10, 28040 Madrid, Spain Institute for the Structure of Matter, Serrano 121, 28006 Madrid, Spain

‡

ABSTRACT: Technological properties and structural characteristics of proteins and lipids, using Raman spectroscopy, of frankfurters formulated with olive oil bulking agents as animal fat replacers were examined during chilling storage. Frankfurters reformulated with oil bulking agents showed lower (P < 0.05) processing loss and higher (P < 0.05) hardness and chewiness. Purge loss during chilling storage was relatively low, demonstrating a good water retention in the products. β-Sheet structures were enhanced by the use of olive oil bulking agents, and this effect was more pronounced in samples containing inulin. Reformulated frankfurters contained the least turns (P < 0.05). A significant decrease of β-sheets and an increase of turns were observed after 85 days of chilled storage. The lowest (P < 0.05) values of IνsCH2/IνasCH2 were recorded in frankfurters reformulated with oil bulking agents, which suggests more lipid acyl chain disorder. Structural characteristics were correlated to processing losses, hardness, and chewiness. KEYWORDS: lipid, protein, olive oil bulking agent, frankfurter, texture, processing loss, structure, Raman spectroscopy

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INTRODUCTION Frankfurters are very popular, frequently consumed meat products. However, they are increasingly being associated with negative aspects relating to health. In order to minimize this negative aspect, frankfurters have been reformulated to improve their lipid composition by reducing fat content and/or replacing the animal fat present in the product with alternative fat whose characteristics are more in line with health recommendations.1 In this context various strategies have been devised to incorporate these fats in meat products, for instance in liquid and solid forms, encapsulated, or as oil-inwater emulsions.1 Our group has developed solid animal fat replacers such as oil bulking agents based on polysaccharide gels, which have been used in the formulation of meat batters.2,3 The potential of a konjac matrix containing healthy oils to improve fat content in meat products has also been evaluated.4−7 To properly develop this type of meat product, it is especially useful to look at reformulation processes that affect technological properties (water and fat binding properties, color, texture, etc.). It has been reported that food processing can potentially alter the structure of food proteins either as the result of intended unit operations or as a consequence of the physicochemical environment a protein encounters during processing.8 In particular, the use of certain reformulation strategies based on replacement of animal fat in the development of healthier lipid meat products could alter both the structural properties of their main components (proteins, lipids, etc.) and technological properties of the final product. An understanding of the interactions that occur between meat proteins and other ingredients is vital for product quality and the development of new processed meat products.9 The interactions of meat proteins with the other ingredients can significantly affect the properties of the end product, since © 2014 American Chemical Society

many of these have a close bearing on the adequate formation of a protein network. Supplementary information relating to the structure/function relationship would clarify the mechanisms that act on them and help determine criteria for adjusting these. It would also aid to find conditions for certified aspects relating to processing and composition and their impact on technological properties of a complex meat system. Raman spectroscopy can provide comprehensive structural information to highlight this structure/function relationship. Raman spectroscopy provides a means to approach the structure,10−16 and particularly the interactions among the different components (protein, lipids, etc.) of these types of healthier-lipid meat products, modified by the addition of animal fat replacers as oil bulking agents. Studies have demonstrated that Raman spectroscopy is a useful tool for directly determining the changes occurring in lipids and proteins of meat batters due to the incorporation of various types of lipids.2,17 These studies identify associations between the protein and lipid structure data obtained using Raman spectroscopy and the technological properties of the product such as texture and water binding.2,17 The primary objective of this work was to determine technological properties (processing loss, purge loss, and texture) and structural characteristics of protein and lipids, using Raman spectroscopy, of frankfurters formulated with olive oil bulking agents, as animal fat replacer. These bulking agents are based on the combination of olive oil, water, a cold set binding agent (alginate), and polysaccharides (inulin or Received: Revised: Accepted: Published: 5963

March 14, 2014 June 4, 2014 June 9, 2014 June 9, 2014 dx.doi.org/10.1021/jf501231k | J. Agric. Food Chem. 2014, 62, 5963−5971

Journal of Agricultural and Food Chemistry

Article

Table 1. Formulation (%) of Frankfurtersa

dextrin). Technological and structural properties were also evaluated as affected by chilling storage (85 days at 2 °C). The secondary objective was to examine the possible relationship between the technological properties and the protein and lipid structures of these frankfurters. A fuller understanding of this relationship could throw more light on the role of oil bulking agents in a meat matrix, which could be useful in optimizing the quality of frankfurters to which they are added.

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olive oil bulking agentc

MATERIALS AND METHODS

Materials. Ingredients used for preparation of oil bulking agents as fat replacers included the following: olive oil (13% SFA, 79% MUFA, and 8% PUFA) (Carbonell Virgen Extra, SOS Cuétara SA, Madrid, Spain); sodium alginate (90% carbohydrates) (Tradissimo, TRADES ́ S.A., Barcelona Spain); calcium sulfate (Panreac Quimica, S.A. Madrid, Spain); tetra-sodium pyrophosphate anhydrous (STP) (Manuel Riesgo, S.A. Madrid, Spain); inulin consisting mainly of chicory inulin (>90% inulin) with a molecular weight of 1650 g/mol (TRADES S.A., Barcelona Spain); and white maize dextrin (molecular weight average approximate between 10 and 20 glucose molecule per polymer) (CARGILL S.L.U., CTS Rubi, Barcelona, Spain). Fresh post-rigor pork (mixture of musculus biceps femoris, musculus semimembranosus, musculus semitendinosus, musculus gracilis, and musculus adductor) and pork backfat used for the preparation of frankfurters were obtained from a local meat market. The meat was trimmed of fat and connective tissue, and the pork fat was passed through a grinder with a 0.4 mm plate. Lots of approximately 1 kg were vacuum packed, frozen, and stored at −20 °C until use, which took place within 2 weeks. Other ingredients and additives used were sodium chloride ́ (Panreac Quimica S.A., Barcelona, Spain), sodium nitrite (Fulka Chemie GmbH, Buchs, Germany), and flavoring (Gewürzmüller, GmbH, Münchingen, Germany). Preparation of Olive Oil Bulking Agents. Two different types of olive oil bulking agents were considered:3 a combination of olive oil with sodium alginate, CaSO4, STP, and dextrin, named A/D, or inulin, named A/I. These samples were prepared by mixing sodium alginate (1%), CaSO4 (1%), STP (0.75%), and dextrin (2.25%) or inulin (2.25%) with water (40%) in a homogenizer (Thermomix TM 31, Vorwerk España M.S.L., S.C., Madrid) to prepare A/D or A/I respectively. The mixtures were prepared at 1500 rpm for 20 s. Olive oil (55%) was gradually added to this mixture with the homogenizer running (1500 rpm). Samples of each type were placed in metal molds under pressure to compact them and avoid air bubbles, and stored in a chilling room at 2 °C for 24 h until analysis. Each type of oil bulking agent was prepared in duplicate using two metal molds for each type of sample. These oil bulking agents were used as pork backfat replacers in the formulation of the frankfurters. Preparation of Frankfurters. Frankfurters were prepared following the procedure previously reported.7 Six different products were prepared as reported in Table 1: control normal fat (NFF-PF) and low fat (LFF-PF) frankfurter formulated with meat and pork backfat (PF); four reformulated samples in which the pork backfat was replaced by an equal amount of oil bulking agent A/D or A/I to formulate normal (NFF-A/D, NFF-A/I) and low (LFF-A/D, LFF-A/ I) fat frankfurters. For frankfurter preparation a Stephan Universal Machine UM5 (Stephan u. Söhne GmbH and Co., Hameln, Germany) was used at 2 °C. The meat batter was stuffed into 20 mm diameter Nojax cellulose casings (Viscase S.A., Bagnold Cedex, France), hand-linked, and heat processed in an Eller smokehouse (model Unimatic 1000, Micro 40, Eller, Merano, Italy). After that, the frankfurters were cooled (at room temperature), vacuum-packed in plastic bags (Cryovac BB3050), stored at 2 °C (±1 °C), and analyzed periodically (days 1, 45, and 85). Proximate Analysis. Moisture and ash contents of the frankfurters were determined18 in triplicate. Protein content was measured in triplicate with a LECO FP-2000 Nitrogen Determinator (Leco Corporation, St Joseph, MI, USA). Fat content was evaluated in triplicate.19

samplesb

meat

pork backfat

NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I

63.0 63.0 63.0 63.0 63.0 63.0

21.0 9.0

A/D

A/I

water

32.5 14.0

13.2 25.2 1.7 20.2 1.7 20.2

32.5 14.0

a

Additives added to all samples: 2.0 g/100 g NaCl; 0.30 g/100 g sodium tripolyphosphate; 0.012 g/100 g sodium nitrite; 0.5 g/100 g flavoring. bFrankfurter: normal fat (NF-) and low fat (LF-) frankfurter (F) formulated with pork backfat (NFF-PF and LFF-PF) or olive oil bulking agent containing dextrin (NFF-A/D and LFF-A/D) or inulin (NFF-A/I and LFF-A/I) as a fat replacer. cOlive oil bulking agent formulated with a combination of olive oil, water, sodium alginate, CaSO4, sodium pyrophosphate, and dextrin (A/D) or inulin (A/I). Energy values were estimated from both protein and carbohydrate content (×4.1 kcal/g) and fat content (×9.1 kcal/g).6 Processing Loss and Purge loss. Processing loss of frankfurters (expressed as % of initial sample weight) occurring after heat processing and chilling overnight at 2 °C was calculated in quadruplicate. Three vacuum packs per formulation were used to determine purge loss during chilling storage for each storage time. After the frankfurters were removed from the package, the surface exudate (tiny drops) was wiped off with paper towels and the frankfurters were weighed again. The purge loss was calculated by weight difference and expressed as a percentage of the initial weight. Texture Profile Analysis. Texture profile analysis (TPA) was performed in a TA.XTplus Texture Analyzer (Texture Technologies Corp., Scarsdale, NY, USA). Cylindrical cores (diameter = 20 mm, height = 20 mm) from each frankfurter formulation were axially compressed to 40% of their original height20 with an aluminum cylinder probe P/25. Six replicates were performed for each frankfurter formulation. Force−time deformation curves were obtained with a 5 kg load cell, applied at a crosshead speed of 1 mm/s. Attributes were calculated as follows: hardness (Hd) = peak force (N) required for first compression; cohesiveness (Ch) = ratio of active work done under the second compression curve to that done under the first compression curve (dimensionless); springiness (Sp) = distance (mm) the sample recovers after the first compression; chewiness (Cw): Hd × Ch × Sp (N × mm). Measurement of samples was carried out at room temperature. FT-Raman Spectroscopic Analysis. Various Raman spectra were measured and used as reference: spectra of heated homogenized raw meat, heated pork backfat, and heated olive oil bulking agent (A/I and A/D). Heating of these samples was performed in a water bath at 70 °C for 30 min (similar conditions to preparation of frankfurters). These Raman spectra were used to appropriately subtract their respective contribution in the spectra of frankfurters analyzed. Spectra of frankfurters formulated according to Table 1 (NFF-PF, LFF-PF, NFF-A/D, LFF-A/D, NFF-A/I, and LFF-A/I) were also measured. Portions (approximately 5 g) of the different samples were measured directly without previous treatment. These portions of samples were transferred to quartz cuvettes (ST-1/Q/10) (Teknokroma, Barcelona, Spain), which were filled to reach 1 cm sample length. For each sample 1500 scans were recorded. This procedure was carried out in triplicate. Measurements were performed on three samples from each formulation. Spectra were excited with the 1064 nm Nd:YAG laser line and recorded on a Bruker RFS 100/S FT-spectrometer. The scattered radiation was collected at 180° to the source, and frequencydependent scattering of the Raman spectra was corrected by multiplying point by point with (νlaser/ν).4 The influence of the 5964

dx.doi.org/10.1021/jf501231k | J. Agric. Food Chem. 2014, 62, 5963−5971


Article

Table 2. Proximate Analysis (%) and Processing Loss (PL, %) of Frankfurtersa samplesb NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I

moisture 60.5 71.7 60.6 72.1 60.9 71.9

± ± ± ± ± ±

0.2 0.1 0.2 0.3 0.2 0.2

fat b a b a b a

20.4 9.9 19.8 9.6 19.6 9.8

± ± ± ± ± ±

protein 0.3 0.3 0.3 0.2 0.4 0.3

a b a b a b

16.2 16.0 15.0 15.5 15.2 15.4

± ± ± ± ± ±

0.5 0.5 0.5 0.6 0.4 0.5

ash a a a a a a

2.90 2.79 4.42 3.49 4.35 3.57

± ± ± ± ± ±

0.06 0.05 0.02 0.01 0.02 0.04

PL (%) c c a b a b

14.7 22.6 13.8 18.9 12.9 17.6

± ± ± ± ± ±

0.7 0.9 0.7 0.9 0.7 0.9

d a e b f c

a Means ± standard deviation. Different letters in the same column indicate significant differences (P < 0.05). bFrankfurter: normal fat (NF-) and low fat (LF-) frankfurter (F) formulated with pork backfat (NFF-PF and LFF-PF) or olive oil bulking agent containing dextrin (NFF-A/D and LFF-A/ D) or inulin (NFF-A/I and LFF-A/I) as a fat replacer.

spectroscopy data. These correlation coefficients range between −1 and +1 and measure the strength of the linear relationship between the variables. In this statistical analysis P-value tests the statistical significance of the estimated correlations. P-values below 0.05 indicate statistically significant nonzero correlations at the 95% confidence level. This statistical analysis was carried out using Statgraphics Plus version 5.0. Inc., Chicago, IL, USA).

optics on the spectrometer was eliminated by means of Opus 2.2 software Raman correction command (Bruker, Karlsruhe, Germany). Raman spectra were resolved at 4 cm−1 resolution with a liquid nitrogen cooled Ge detector. The samples were illuminated by laser power at 300 mW, and the frequencies cited are accurate to ±0.1 cm−1. Analysis of the ratios of the intensities of the Stokes and antiStokes Raman bands of the same frequency showed that there was no sample heating by using this laser power. Spectroscopic Data Analysis To Determine Secondary Protein Structure. The secondary protein structure of meat proteins was determined as follows. First, the water spectrum was subtracted from the spectra following the same criteria as described previously.21,22 In order to eliminate any spectral influence of lipids in the spectral region characteristic of proteins (800−1800 cm−1), the corresponding spectrum of the heated oil bulking agent (A/D or A/I) was subtracted from the reformulated frankfurters (NFF-A/D, NFF-A/I, LFF-A/D, or LFF-A/I) by zeroing the bending CH band located close to 1267 cm−1, which is attributed to lipid molecules.2,23 Similarly, the heated pork backfat spectrum was subtracted from the control (all pork backfat) frankfurters (NFF-PF or LFF-PF) following the same criteria. The Phe ν-ring band located near 1003 cm−1 was used as an internal standard to normalize the spectra as it has been reported to be insensitive to the microenvironment.13−15 The visible bands were assigned to vibrational modes of peptide backbone or amino acid side chains by comparing Raman spectra of model polypeptides or monographs of Raman spectra of proteins.13−15 The intensity values of Raman bands from various atomic groups were determined after spectral normalization. Protein secondary structures were determined as percentages of α-helix, β-sheet, turns, and unordered conformations using a parametric method.21 This method permits the determination of the quantitative secondary structure of proteins by using parameters of the Raman amide I band.21 Spectroscopic Data Analysis To Determine Lipid Structure. In order to eliminate any spectral influence of water in the samples in the spectral region 2800−3000 cm−1, the spectral contribution of water was subtracted from the sample spectra.2,21 The water-free spectra of the samples were then appropriately subtracted using the heated raw meat spectrum, based on elimination of the Phe ν-ring band by means of a subtraction factor so that the intensity of this Raman band near 1003 cm−1 is not visible. In this way, spectral influence of protein was eliminated. Raman spectra were processed using Opus 2.2 (Bruker, Karlsruhe, Germany) and Grams/AI version 9.1 (Thermo Electron Corporation, USA) software. For intensity measurements, baselines were considered as straight lines drawn tangentially between the intensity minima occurring on either side of each band. Statistical Analysis. One-way analyses of variance (ANOVA) to evaluate the statistical significance (P < 0.05) of the effect of formulation and two-way ANOVA as a function of formulation and storage time were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Least squares differences were used for comparison of mean values among formulations and Tukey’s HSD test to identify significant differences (P < 0.05) between formulations and storage times. In addition, Pearson product moment correlation (R) was performed to determine the relationships between technological properties (processing loss and texture parameters) and Raman

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RESULTS AND DISCUSSION Proximate Analysis, Processing Loss, and Purge Loss. Proximate analysis of samples (Table 2) was in agreement with the target composition (Table 1). Protein content was similar (P > 0.05) in all frankfurters. Low fat frankfurters showed the highest moisture values according to formulation (Table 2). Ash contents varied between 2.79 and 4.42%. All animal fat samples (NFF-PF and LFF-PF) contained the lowest proportion of ash. These results are consistent with the composition of the components used in the preparation of oil bulking agents, which provide higher (P < 0.05) ash values in reformulated samples. Fat content ranged between 20.38% and 9.59% (Table 2), and the highest (P < 0.05) values were recorded in normal fat frankfurters (NFF-PF, NFF-A/D, and NFF-A/I). The energy content of the oil bulking agent used to replace pork backfat was about 514 kcal/100 g, while the energy content of the pork backfat was around 810 kcal/100 g. This implies that if this oil bulking agent were used as animal fat replacer in the formulation of meat products, their energy content could be reduced by about 36.5%. In addition, frankfurters formulated with this oil bulking agent, which contain 55% olive oil, included around 13.5 g of olive oil per 100 g of product. These observations indicate that given the nature and composition of the lipid components of the olive oil included in the bulking agent, this approach could also be adopted to improve the fatty acid profiles of meat products. Processing loss of frankfurters ranged from 12.9 to 22.6% (Table 2). The values of processing loss were highest (P < 0.05) in LFF-PF, which was also the sample with the most added water (Table 1). Reported values of processing loss in frankfurters normally oscillate between 10 and 20%.7,24,25 Samples reformulated with oil bulking agents, both normal (NFF-A/D, NFF-A/I) and low (LFF-A/D and LFF-A/I) fat, showed significantly less processing loss than their respective counterparts (NFF-PF or LFF-PF) formulated with all pork backfat (Table 2). Processing losses were lowest (P < 0.05) in normal fat frankfurters prepared with A/D and A/I as pork backfat replacers (NFF-A/D and NFF-A/I), significantly more so in NFF-A/I. In these reformulated frankfurters with the added olive oil bulking agent, these agents encapsulated parts of the water and fat in the product, so that less was released during processing (Table 2). In previous assays cooking loss was found 5965



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Table 3. Textural Profile Analysis (TPA) Parameters (Hardness, Springiness, Cohesiveness, Chewiness) of Frankfurtersa during Chilling Storageb days of storage TPA params hardness (N)

cohesiveness (dimensionless)

springiness (mm)

chewiness (N × mm)

samplesa

0

45

85

NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I

27.64 ± 0.40 b1 22.43 ± 0.52 d1 28.46 ± 0.26 a1 24.69 ± 0.52 c1 29.83 ± 0.70 a1 24.67 ± 0.19 c1 0.66 ± 0.03 b3 0.69 ± 0.01 a3 0.69 ± 0.00 a3 0.70 ± 0.01 a2 0.69 ± 0.00 a3 0.69 ± 0.00 a2 6.66 ± 0.06 b2 6.69 ± 0.02 b2 6.95 ± 0.04 a2 6.97 ± 0.05 a2 6.93 ± 0.07 a3 7.01 ± 0.05 a2 121.9 ± 1.3 c1 98.9 ± 3.5 e1 132.5 ± 1.2 a1 117.3 ± 0.8 d1 144.1 ± 1.2 a1 121.3 ± 1.1 c1

19.39 ± 0.64 b2 14.54 ± 0.71 d2 18.85 ± 0.94 b2 16.41 ± 0.47 c2 21.42 ± 0.88 a2 16.61 ± 1.05 c2 0.73 ± 0.01 d2 0.75 ± 0.01 c2 0.73 ± 0.01 d2 0.75 ± 0.01 b1 0.74 ± 0.01 d2 0.76 ± 0.01 a1 7.03 ± 0.04 b1 7.04 ± 0.06 b1 7.24 ± 0.10 a1 7.31 ± 0.16 a1 7.23 ± 0.07 a3 7.24 ± 0.17 a2 96.2 ± 1.8 2 75.6 ± 1.5 e3 99.5 ± 1.4 b2 90.6 ± 1.0 d3 114.6 ± 2.4 a2 88.5 ± 2.7 d2

18.98 ± 0.71 a2 16.37 ± 0.80 c3 19.91 ± 0.85 a2 17.79 ± 1.05 b2 19.47 ± 1.06 a2 16.27 ± 0.85 c2 0.73 ± 0.00 c1 0.77 ± 0.01 a1 0.77 ± 0.01 a1 0.75 ± 0.01 b1 0.77 ± 0.02 a1 0.77 ± 0.01 a1 7.00 ± 0.12 b1 7.08 ± 0.11 b1 7.18 ± 0.15 a1 7.20 ± 0.11 a1 7.30 ± 0.18 a1 7.40 ± 0.15 a1 97.8 ± 1.3 b2 80.1 ± 1.9 e2 103.2 ± 1.8 a2 95.5 ± 1.3 c2 104.3 ± 2.0 a3 88.4 ± 1.9 d2

a

Frankfurter: normal fat (NF-) and low fat (LF-) frankfurter (F) formulated with pork backfat (NFF-PF and LFF-PF) or olive oil bulking agent containing dextrin (NFF-A/D and LFF-A/D) or inulin (NFF-A/I and LFF-A/I) as a fat replacer. bMeans ± standard deviation. Different letters in the same column indicate significant differences (P < 0.05), and different numbers in the same row indicate significant differences (P < 0.05).

matrix,27,28 a structure associated with harder products in normal fat samples (Table 3). The incorporation of fat replacers also seems to have affected the texture. Reformulated frankfurters containing a fat replacer, irrespective of fat content, produced stronger structures with higher (P < 0.05) hardness and chewiness values (Table 3). Given similar compositions (and the same fat level), the differences in hardness and chewiness were presumably attributable to the bulking agent, which should facilitate the formation of a gel/emulsion matrix in the samples, so that samples should be harder/firmer. Previous results indicated that meat products reformulated using fat replacers based on a mixed konjac/gellan gum gel,29 oil in a konjac glucomannan matrix,7 or pre-emulsified oils were more firm than those formulated with pork backfat.17,24,25,30,31 In general, the TPA parameters were affected (P < 0.05) by chilling storage (Table 3). However, no clear relationship was identified between these changes in texture during storage and the type of sample (Table 3). Results showed a significant decrease of hardness and chewiness and an increase (P < 0.05) of cohesiveness and springiness in all samples during chilling storage (Table 3). Previous studies have detected similar texture behavior in frankfurters formulated with emulsified oils as fat replacers.25 FT-Raman Spectroscopic Analysis. Protein Structure. Figure 1 shows representative Raman spectra in the 950−1800 cm−1 region from normal fat frankfurters (NFF-PF, NFF-A/D, and NFF-A/I), following elimination of the spectral influence of water and lipids (see Materials and Methods). Amide III Band. This Raman band includes C−N stretching and N−H in-plane bending vibrations of the peptide bond.

to be lower in meat batters where fat was replaced by polysaccharide matrices containing olive oil than in samples made with all pork backfat.2 Similar behavior has been found in other products such as low fat pork batters formulated with animal fat replacers based on polysaccharides such as konjac gel.26 Purge loss is associated with unwanted purge accumulation in the packaged product during storage. This accumulation is undesirable for both commercial and microbiological reasons. Results showed that purge loss levels over storage were relatively low (ranging from 0.5 to 1.5%) and similar to those reported by other authors.7,24,25 This fact was associated with appropriate stability during storage in terms of fat and water binding properties of the new meat derivate. The good stability found was independent of the formulation. Texture Profile Analysis (TPA). Both types of formulation and chilling storage significantly influenced textural properties. Samples with normal fat content (NFF-PF, NFF-A/D, and NFF-A/I) presented the highest (P < 0.05) hardness and chewiness (Table 3). All frankfurters reformulated with oil bulking agents showed the highest (P < 0.05) springiness, while there was no significant difference in cohesiveness between samples. This difference in textural properties would appear to be due to two factors, the differences in fat and moisture content, and incorporation of an oil bulking agent as animal fat replacer. In normal fat frankfurters an increase in fat content and a decrease in water (Table 2) will raise the “effective” concentration of the protein that acts to form the gel/emulsion matrix. An increase in protein concentration generally entails the formation of a denser and more compact protein 5966



Article

control and in reformulated samples (NFF-PF, NFF-A/D, and NFF-A/I) (Figure 2). Some frequency upshifting from 1667.8 to 1668.3 cm−1 was detected, particularly in normal fat samples containing the oil bulking agent with inulin (Figure 2). This result may be related to increased β-sheet structure content in the meat protein in NFF-A/I. When the amide I bands of LFFPF and reformulated frankfurters (LFF-A/D and LFF-AI) were compared, shifts of the frequency maximum from 1665.7 to 1666.1 cm−1 in LFF-A/D and from 1665.7 to 1667.2 cm−1 in LFF-A/I (Figure 2) were observed. These frequency upshifts are related to increased β-sheet structure content in the meat protein due to the use of a pork backfat replacer in the low fat frankfurters. As in the case of normal fat frankfurters, this effect was more pronounced in samples where the oil bulking agent contained inulin. Quantitative Percentages of Secondary Structure. In depth examination of amide I spectra profiles offered quantitative information about the percentages of secondary structure in the different frankfurters (Table 4). In general, values of α-helix content were lowest (P < 0.05) in normal fat frankfurters. Quantitative results of protein secondary structure confirm that β-sheet content was higher (P < 0.05) in normal than in low fat frankfurters, either control or reformulated. Among the normal fat frankfurters, β-sheet content was higher (P < 0.05) in samples reformulated with oil bulking agents as fat replacers (NFF-A/D and NFF-A/I) and highest (P < 0.05) of all in the sample with inulin in the oil bulking agent (NFFA/I) (Table 4). Similar results were found when only low fat frankfurters were compared. β-Sheet content was significantly higher in LFF-A/D and LFF-A/I, with the highest values (P < 0.05) in the samples containing inulin in the oil bulking agent. These structural results were consistent with previous studies indicating that the use of animal fat replacers like olive oil bulking agents in meat batters, or oil-in-water emulsions in frankfurters, produces an increase in β-sheet structure.2,32,33 The presence of aggregated intermolecular β-sheets in frankfurters has been associated with the formation of a compact network imparting more firmness.2,17,32,33 These findings are consistent with our results (Tables 3 and 4). The formation of intermolecular β-sheets may result from the selfaggregation of the proteins. Studies of FT-Raman indicated the effect of modified celluloses, pectins, and NaCl on the secondary structure of gluten proteins. In general, a decrease in α-helix conformation and an increase in less ordered structures were observed with the addition of hydrocolloids.34 It has been reported that certain systems based on mixes of milk proteins such as whey protein concentrate and sodium alginate were distinguished by a tendency to protein aggregation.35 Interactions among muscle proteins and hydrocolloids, reflected in an enhancement of hydrogen bonding, have been suggested.36 The structural results show that, regardless of fat content, turn content was lowest (P < 0.05) in reformulated frankfurter, while differences among samples in term of unordered structure were nonsignificant. Table 4 shows a decrease (P < 0.05) in β-sheet structure, in general accompanied by an increase in turn content at 85 days of chilled storage, while the rest of the protein secondary structure parameters (α-helix and unordered) remained constant throughout storage. Relationship between Protein Structure and Technological Properties. Results showed that reformulation-induced changes in the secondary structure of meat protein were

Figure 1. Raman spectra in the 950−1800 cm1 region from the normal fat frankfurters with pork backfat (NFF-PF) or olive oil bulking agent containing alginate (A) and dextrin (NFF-A/D) or inulin (NFF-A/I) as a fat replacer.

Contributions from Cα−C stretching and CO in-plane bending13−15 were also detected (Figure 1). The amide III band provides information about the secondary structure of proteins. The amide III band in the spectra comprises a complex pattern of bands in the 1225−1350 cm−1 range (Figure 1) and for that reason is difficult to interpret. Amide I Band. Figure 1 shows the amide I band, which offers valuable information about protein secondary structure.13−15 The amide I band comprises mainly CO stretching and, to a lesser extent, N−H in-plane bending of peptide groups.13−15 In all spectra the amide I band was analyzed to elucidate the structural changes occurring in the meat protein due to the use of an olive oil bulking agent as pork backfat replacer. Figure 2 shows the amide I region (1620−1720 cm−1) from the different frankfurters in detail. The alterations of frequency and intensity in this band revealed mainly changes in the secondary structure of meat proteins and variations in local environments. The highest frequency values were recorded in normal fat frankfurter when compared with the low fat one, both in

Figure 2. Raman spectra in the 1620−1720 cm−1 region from frankfurters formulated as normal fat (NFF-) and low fat (LFF-) formulated with pork backfat (NFF-PF and LFF-PF) or olive oil bulking agent containing alginate (A) and dextrin (NFF-A/D and LFF-A/D) or inulin (NFF-A/I and LFF-A/I) as a fat replacer. 5967



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Table 4. Percentages of Protein Secondary Structure (α-Helix, β-Sheet, Turns, and Unordered) of Frankfurters during Chilling Storagea days of storage protein secondary structure params (%) α-helix

β-sheet

turn

unordered

samplesb NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I

0 26.7 29.0 26.5 30.2 27.9 27.7 40.5 36.7 43.1 38.5 45.6 41.9 20.1 21.8 18.3 18.8 16.2 18.0 12.7 12.5 12.1 12.5 10.3 12.4

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

0.6 0.5 0.5 0.6 0.7 0.9 0.8 0.7 0.6 0.5 0.6 0.7 1.6 1.2 1.4 1.5 1.9 1.5 1.1 0.9 1.2 0.8 2.2 1.1

45 d12 b1 d1 a12 c1 c1 d1 f1 b1 e1 a1 c1 b1 b1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1

25.5 29.5 27.4 29.0 27.6 28.6 39.4 37.0 43.4 39.1 43.4 37.3 22.5 21.2 19.3 19.5 16.9 20.2 12.6 12.3 11.9 12.1 12.1 13.9

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

0.8 0.7 0.9 0.7 0.8 0.5 0.6 0.4 0.7 0.6 0.5 0.7 1.2 1.6 1.0 1.2 1.7 1.2 1.0 1.1 1.4 1.0 0.9 1.7

85 c2 a1 b1 a2 b1 ab1 12 d1 a1 1 b2 d2 b12 b1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1

26.9 30.0 27.2 30.5 27.4 27.9 38.6 35.3 40.3 37.5 41.2 37.5 22.4 21.8 19.9 19.8 18.6 20.8 12.1 12.9 12.6 12.5 12.8 13.8

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

0.79 b1 0.8 a1 0.8 b1 0.5 a1 0.6 b1 0.8 b1 0.5 b2 0.8 c2 0.6 a2 0.4 b2 0.5 a3 0.7 b2 1.4 b2 1.1 b1 1.5 a12 1.1 a12 1.8 a2 1.5 a2 1.3 a1 0.9 a1 0.8 a1 1.3 a1 1.0 a1 0.9 a1

Means ± standard deviation. Different letters in the same column indicate significant differences (P < 0.05). bFrankfurter: normal fat (NF-) and low fat (LF-) frankfurter (F) formulated with pork backfat (NFF-PF and LFF-PF) or olive oil bulking agent containing dextrin (NFF-A/D and LFF-A/ D) or inulin (NFF-A/I and LFF-A/I) as a fat replacer. a

Materials and Methods), the 2800−3025 cm−1 region was studied to elucidate the lipid structure (Figure 3). This region contained bands that are characteristic of symmetric and asymmetric C−H stretching vibrations of methyl and methylene groups in aliphatic molecules.23,39 There were prominent bands: a CH2 asymmetric stretching band at 2897 cm−1, a CH3 symmetric stretching band at 2931 cm−1, and a

accompanied by changes in processing loss and texture. Thus, a correlation was found between processing loss and β-sheet (R = −0.81, P < 0.0005) and turn (R = 0.74, P < 0.0005) content. These correlations seem to indicate that in these types of meat products processing loss could be due to a specific meat protein structure in terms of β-sheet and turn content. In addition, the results showed a positive correlation between β-sheet structure and hardness (R = 0.77, P < 0.05) and chewiness (R = 0.92, P < 0.005). There was also a negative correlation between turn and hardness (R = −0.86, P < 0.005) and chewiness (R = −0.92, P < 0.005). These findings indicate that reformulation-induced modifications in protein secondary structure could affect textural properties of the final meat product. Previous studies have reported a similar correlation between β-sheet content and hardness in frankfurters prepared with an olive oil-in-water emulsion.32,33 Similarly, a positive correlation has been reported between β-sheet structures and textural behavior in meat products prepared with different lipids (pork fat, soybean oil, and dairy butter).17 In particular, meat batters reformulated with soybean presented greater hardness, springiness, cohesiveness, chewiness, and resilience than meat batters made with pork fat or butter. These specific textural properties were accompanied by an increase of β-sheet structures.17 This is consistent with the observation of increases of hardness associated with increases in β-sheet and turn structure in meat systems to which cold-set binding systems such as transglutaminase or fibrinogen/thrombin from porcine blood plasma have been added.37,38 Lipid Structure. In the Raman spectra obtained after removing the water and protein spectral contribution (see

Figure 3. Raman spectra in the 2800−3025 cm−1 region from the low fat frankfurters with pork backfat (LFF-PF) (solid line) or olive oil bulking agent containing alginate (A) and dextrin (LFF-A/D) (dashed line) or inulin (LFF-A/I) (dotted line) as a fat replacer. 5968



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Table 5. Relative Intensity of IνsCH2/IνasCH2 and IνsCH3/IνasCH3 of Frankfurters during Chilling Storagea days of storage samplesb IνsCH2/IνasCH2

IνasCH2/IνsCH3

NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I NFF-PF LFF-PF NFF-A/D LFF-A/D NFF-A/I LFF-A/I

0 0.94 0.99 0.91 0.90 0.89 0.91 1.12 1.08 1.14 1.13 1.12 1.11

± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.05 0.02 0.03 0.02 0.01 0.03 0.04 0.02 0.01 0.03 0.04

45 a1 b1 c1 c1 c1 c1 a1 a1 a1 a12 a1 a1

0.93 1.05 0.90 0.93 0.85 0.92 1.13 1.11 1.11 1.15 1.13 1.12

± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.01 0.04 0.05 0.03 0.02 0.02 0.03 0.01 0.02 0.02 0.03

85 ab1 a1 ab1 ab1 b2 ab1 a1 a1 a1 a1 a1 a1

0.93 1.02 0.92 0.89 0.88 0.83 1.15 1.14 1.15 1.13 1.14 1.11

± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.03 0.01 0.02 0.03 0.04 0.02 0.05 0.03 0.02 0.03 0.01

b1 a1 bc1 bc1 c2 d1 a1 a1 a1 a1 a1 a1

a Means ± standard deviation. Different letters in the same column indicate significant differences (P < 0.05). bFrankfurter: normal fat (NF-) and low fat (LF-) frankfurter (F) formulated with pork backfat (NFF-PF and LFF-PF) or olive oil bulking agent containing dextrin (NFF-A/D and LFF-A/ D) or inulin (NFF-A/I and LFF-A/I) as a fat replacer.

CH2 symmetric stretching motion near 2853 cm−1 (Figure 3). Another Raman band at 3007 cm−1 attributable to cis-olefinic group C−H stretching vibration is clearly visible. Intensity Ratios IνsCH2/IνasCH2 and IνasCH2/IνsCH3. It has been reported that the peak height intensity ratios IνsCH2/ IνasCH2 (I2850/I2880) and IνasCH2/IνsCH3 (I2880/I2935) are valuable references for gauging lipid packing effects and determining relative order/disorder of the intermolecular lipid chain.40 In particular, the IνsCH2/IνasCH2 index reflects primarily interchain interactions whereas the IνasCH2/IνsCH3 intensity ratio evaluates effects resulting from modifications in intrachain trans/gauche isomerization superimposed on the chain−chain interactions. Intensity ratio IνasCH2/IνsCH3 was similar (P < 0.05) for all samples (Table 5). However, the intensity ratio values IνsCH2/IνasCH2 were lowest (P < 0.05) in frankfurter reformulated with olive oil bulking agent as pork backfat replacer (Table 4). These significant differences indicate that there was more lipid acyl chain disorder in frankfurters containing oil bulking agents than in samples made with animal fat, regardless of the fat content. There are a number of mechanisms that may explain fat stabilization in gel/emulsion meat products like frankfurters.41 An “emulsion theory” has been proposed to the effect that a thin layer of myofibrillar proteins forms around the fat globules. The hypothesis assumes that this is accompanied by physical entrapment so that fat globules are stabilized within a gelled protein matrix.41 On that assumption, the higher lipid chain disorder observed in frankfurters formulated with an oil bulking agent could be due to the fact that in reformulated samples more meat protein chains can be inserted between the acyl chains of the olive oil bulking agent than in control normal and low fat frankfurters containing pork backfat. This would imply more lipid−protein interactions in the reformulated products, which in turn implies more lipid acyl chain disorder (Table 4). Similar behavior has been found in meat batters in which oil bulking agents containing polysaccharide gels were used as fat replacers.2 In general, IνsCH2/IνasCH2 and IνasCH2/IνsCH3 were not significantly affected by chilling storage (Table 4). Relationship between Lipid Structure and Technological Properties. The possible relationship between lipid structure and processing loss and texture was also evaluated. Results showed that the observed changes in lipid structure due to the use of an olive oil bulking agent as pork backfat replacer were

accompanied by changes in processing loss and textural properties of frankfurters. Thus, a positive significant correlation (R = 0.80; P < 0.05) was found between processing loss and intensity ratios IνsCH2/IνasCH2 related to lipid acyl chain order/disorder. This correlation suggests that disordering in oil acyl chains or lipid−protein interaction as a result of reformulation could affect the water and fat binding properties of the final meat product. In addition, order/disorder of lipid acyl chain correlated negatively or inversely with hardness (R = −0.73, P < 0.005) and chewiness (R = −0.81, P < 0.05). The reformulation strategy employed could be used to reduce the energy content and improve the fat content of frankfurters. The implications of this study are that technological and structural characteristics of proteins and lipids were affected by the incorporation in frankfurters of olive oil bulking agent based on polysaccharides and cold-set binding agent like alginate as animal fat replacers. This reformulation process reduced processing loss while increasing hardness and chewiness of the frankfurters. The behavior of samples during chilling storage in terms of the technological properties considered was similar in all products regardless of the formulation. Raman spectroscopy provided useful information on secondary protein structures in terms of enrichment of βsheet structure, which was more pronounced in frankfurters containing inulin in the oil bulking agent. The high presence of aggregated intermolecular β-sheets in frankfurters suggests the formation of a denser network producing greater hardness. In addition a reduction in turn content was observed in response to reformulation. In relation to how lipid structure was affected by this reformulation process, we should stress that it caused increased lipid acyl chain disorder, which involved more lipid−protein interactions when an oil bulking agent was used as a pork backfat replacer. This study presented further advances in terms of how these reformulation processes influence the structures of the principal component proteins and lipids in these meat products, and how this affects the technological properties of the final meat product. This information can be helpful in improving these conditions and hence could be valuable for the development and formulation of healthier-lipid meat products. In addition, these correlations could be decisive for understand the 5969



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implications of both protein and lipid interactions in specific functional or technological properties such as texture or processing loss, which are very important for the acceptance of consumer.

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AUTHOR INFORMATION

Corresponding Author

*Phone:+34 915616800. Fax: +34 91 5645557. E-mail: ana. [email protected]. Funding

This research was supported by projects AGL2010-19515 and AGL2011-29644-C02-01. Notes

The authors declare no competing financial interest.

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Raman Spectroscopic Study of Structural Changes upon Chilling

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