3.1.2. Raman Spectra

Raman spectroscopy offers data on protein structure, mostly by analysis of the amide I and III bands, which are associated with secondary structure. This spectroscopic technique also provides bands due to the environment of some side chains of proteins (aliphatic, tyrosine and tryptophan residues) and on the local conformations of disulfide bonds and methionine residues, all associated with a tertiary protein structure [27,29]. Fourier transform (FT) Raman spectrometers with 1064 nm excitation are commonly used to study the structure of these food products [13,27].

The principal Raman bands to establish the secondary structure of meat protein (<sup>α</sup>helix, β-sheet, turn, unordered) are amide I (1650–1658 cm<sup>−</sup>1), which involves mostly C=O stretching, and amide III vibrational modes (1225–1350 cm<sup>−</sup>1) (Figure 4). Amide I, a strong band that involves mostly C=O stretching, is the most commonly used in the study of secondary protein structure. Most studies on the vibrational spectroscopy of proteins highlight the correlation between amide I band frequencies and protein secondary structure [27,29] proteins with grea<sup>t</sup> α-helical content, which display an amide I band centered about 1650–1658 cm–1 (Figure 4), while those with mainly β-sheet structures display the band between 1665 and 1680 cm<sup>−</sup>1, and a grea<sup>t</sup> content of unordered structure is relate to proteins with an amide I band centered at 1660–1665 cm<sup>−</sup>1. The spectral profile of the amide I band is employed in quantifying the secondary structure of proteins in terms of content of α-helix, β-sheet, turn and unordered using different methods mainly centered on frequency determinations at maximum intensity and half-bandwidth of the amide I band [27,29]. However, before this band can be analyzed, the water spectrum must first be correctly subtracted from the spectra. Additionally, other weaker bands can be noticed and analyzed in the Raman spectra. These correspond to the influence of peptide structure on the environment of some side chains such as those of aliphatic (δCHn), tyrosine (Tyr doublet) and tryptophan (Trp) residues (Figure 4), and on the local conformations of disulfide bonds and methionine residues associated to tertiary protein structure [35].

**Figure 4.** Typical Raman spectrum of a cooked sausage (type frankfurter) in the 0–4000 cm<sup>−</sup>1.

There are also several Raman bands allocated to lipids near 1750, 1660, 1470, 1443, 1306, and 1269 cm<sup>−</sup><sup>1</sup> allocated to the C=O stretching modes, C=C stretching modes, CH2 scissoring modes, CH2 twisting modes, and CH in plane deformation modes of lipids [6,7]. The unsaturation level of fat-containing food products can be assessed precisely by analyzing the C=C stretching band (1660 cm<sup>−</sup>1). Nevertheless, one of the most frequently used regions of the Raman spectra in the structural study of lipids in meat and meat products, especially those products with health-enhanced lipid content, is between 2800–3000 cm<sup>−</sup><sup>1</sup> (Figure 4) associated with changes in C-H stretching vibrations (νCH) [6,7]. A CH3 symmetric stretching band near 2897 cm<sup>−</sup>1, a CH2 asymmetric stretching band near 2930 cm<sup>−</sup><sup>1</sup> and a CH2 symmetric stretching motion near 2850 cm<sup>−</sup><sup>1</sup> are found in this region [29]. The symmetric and asymmetric vibrational modes of the CH2 and CH3 groups can offer information about interactions between hydrocarbon chains. The peak height intensity relations I<sup>ν</sup>sCH2/I<sup>ν</sup>asCH2 (I2850/I2890) and I<sup>ν</sup>sCH3/I<sup>ν</sup>asCH3 (I2935/I2890) offer valuable indices to measure lipid packing consequences and establish relative order/disorder of the intermolecular lipid chain [36,37].

Raman spectra also show a broad water band (3100 and 3500 cm<sup>−</sup>1) (Figure 4), associated with OH stretching motions [38,39], and a Raman band in the low-frequency range (below 600 cm<sup>−</sup>1) including the bending (δ) and stretching (ν) vibrations of the O(N)-H..O(N) units, due to interactions of hydrogen bonded to water and protein molecules [38,39].

#### **4. Application of Vibrational Spectroscopy in Meat Products with Healthier Lipid Content**

Once this basic knowledge about vibrational spectroscopic techniques has been established, their application on the development of EGs and OBAs and healthier meat products in which these structured lipids are incorporated will be described separately.

#### *4.1. Infrared and Raman Spectroscopy to Study Lipid Material Based on Structured Lipids*

Many researchers have studied the possibilities of vibrational spectroscopy to establish fat content and the fatty acid composition of animal fat (adipose tissue from beef, lamb, pork and chicken, etc.) [40–43] Raman spectroscopy has been used to predict PUFA, MUFA and SFA content, and the degree of unsaturation (IV) in melted fat and adipose tissue of pork [41]. Raman spectral regions between 775 and 1800 cm<sup>−</sup>1, and between 2600 and 3100 cm<sup>−</sup>1, were selected for regression models since these contain bands related to lipids. Results showed that Raman spectroscopy is an interesting technique to measure the fatty acid composition of pork adipose tissue with the benefit that this technique is non-invasive and measurements can be completed online [41]. Vibrational spectroscopy has also been used extensively to achieve structural information about oils [31,32,43–45], which are the basis of lipid materials. Based on the useful results obtained from the analysis of animal fat and oils, many studies were later conducted on lipid materials, especially structured lipids.

FT-IR has been employed to study conformational modifications of oils emulsified with proteins ( α-lactalbumin and β-lactoglobulin) where these proteins are adsorbed in the emulsion formation process [46,47]. This process induces changes in their secondary structure [46,47]. The results showed the creation of an intermolecular, antiparallel β-sheet upon adsorption due to protein self-aggregation. Studies to obtain details on protein secondary structures of olive oil-in-water emulsions stabilized with various protein systems based on caseinate or soy protein have been conducted using FT-IR [48,49]. The relationship between emulsion structure and its physical properties was also evaluated. Protein secondary structures changed to more orderly protein backbones, mainly involving the α-helical structure upon creation of the olive oil-in-water emulsion. These structural properties could be associated with the firmer textural characteristics found in soy and caseinate emulsions. A better interpretation of the potential relationship between the structural and textural characteristics of olive oil-in-water emulsions could help in selecting the best emulsion components to obtain specific textural characteristics. In this connection, FT-IR and FT-Raman play an important role. Structured lipids such as emulsion gels (EGs) are an interesting possibility to structuring edible oils. These are described as emulsions with a gel-like network structure and solid-like mechanical characteristics [2,3], making them a good alternative to animal fat in meat products with a healthy lipid profile. It is relevant to note the function of structure and lipid phase interactions in the stability of EGs and their technological properties [4,5]. IR, especially attenuated total reflectance (ATR)-FTIR spectroscopy, has proven valuable insofar as it is a fast, non-destructive analytical method capable of offering analytical and structural information on various EGs. Molecular structures of polyphenol–soy EGs have been studied using this spectroscopic technique owing to their potential as release systems for bioactive compounds (polyphenols, MUFAs, PUFAs). Studies on polyphenol–soy EGs have shown that phenolic compounds seem to become confined in the emulsion matrix network, due to its textural properties, exhibiting lower gel strength than EGs without polyphenols [50]. ATR-FTIR spectroscopy has also been used to examine the structural characteristics of chia EGs. Analyses of the 3000–2500 cm<sup>−</sup><sup>1</sup> region indicate that the order/disorder of the oil lipid chain, associated to lipid interactions and droplet size in the emulsion gels, could be important in controlling their textural properties [4]. Raman spectroscopy has also been used to examine the lipid structure of different chia EGs and spectroscopic results, mainly significant differences were found in the area 2800–3000 and I2854/I2900, and indicated differences depending on these EG compositions in the lipid structure and interactions in terms of lipid acyl chain mobility (order/disorder). These lipid structural properties of chia oil EGs linked significantly with a particular textural behavior [9].

Polysaccharides, a structured lipid, can be employed either individually or in mixture to form a diversity of gel structures which may be appropriate for immobilizing oil droplets working as oil-bulking agents. Raman spectroscopy has also been employed to determine lipid and polysaccharide interactions in different polysaccharide gels elaborated for use as oil-bulking agents [5]. Raman spectroscopic results show that these structured lipids were stabilized by hydrogen bonding between oil carbonyl groups and water and/or carbohydrate molecules. This structural behavior may clarify the differences in textural characteristics. Structural studies of lipid materials (EGs and OBA) contribute to a better interpretation of the correlation between their technological properties and the structural characteristics of their ingredients. Understanding these correlations ultimately helps in selecting the most suitable materials.

This information is of particular interest given that these lipid materials can help to improve or maintain the quality of the food to which they are added. Particularly important in this regard is their use as animal fat replacers, for instance in the development of healthenhanced meat products without compromising the properties of the final product.

#### *4.2. Infrared and Raman Spectroscopy to Examine Meat Products with Healthier Lipid Content*

Vibrational spectroscopic techniques, particularly FT-Raman and FT-IR, provide information about the secondary structure of proteins and the structure of lipids in meat and meat products [6,13,27,28]. These spectroscopic techniques have been used to build models that can predict sensory and technological characteristics, and also to evaluate structural modifications appearing in proteins during processing [13,28,29]. Studies evaluating the feasibility of FT-IR spectroscopy in determining structural properties have focused on health-enhanced meat products reformulated with lipid material such as olive oil-in-water emulsions stabilized with casein or soy protein to replace animal fat [51]. The results of these studies sugges<sup>t</sup> that this spectroscopic technique offers relevant information about changes in protein secondary structure caused by changes in the amide I band profile. Moreover, changes in the 3000–2500 cm<sup>−</sup>1, particularly the half-bandwidth in the 2922 cm<sup>−</sup><sup>1</sup> band, has suggested modifications in lipid chain order or lipid–protein interactions. These results underscore the relevance of the stabilizing system employed in the preparation of oil-in-water emulsions as it impacts both the structural and technological characteristics of the reformulated meat products.

Lipid materials based on structured lipids such as chia EGs have been applied as animal fat substitutes to develop frankfurters with a health-enhanced lipid profile. In this case, ATR-FTIR spectroscopy was used to study how replacing animal fat with chia EGs affects the structural characteristics of lipids [52]. Results from the 3000–2500 cm<sup>−</sup><sup>1</sup> region showed that frankfurter reformulated with chia emulsion gels implicate more lipid–protein interactions which correlated significantly with processing loss (mainly loss of water and fat) and the textural behavior of these samples [52]. ATR-FTIR spectroscopy was also used to study health-enhanced frankfurters reformulated with structured lipids based on polyphenol-EGs as animal fat replacers (Figure 5). The spectroscopic result of the acyl chain region (between 2950 and 2830 cm<sup>−</sup>1) of the ATR-FTIR spectrum presented greater (*p* < 0.05) inter- and intramolecular lipid disorder irrespective of the presence of polyphenol in the EG [53].

FT-Raman spectroscopy was also employed to study the structural properties of healthenhanced frankfurters reformulated with structured lipids such as oil-bulking agents to replace fat [6]. Analysis of the amide I band furnished valuable information on secondary protein structures regarding enrichment of β-sheet structures in these frankfurters reformulated with oil-bulking agents [6]. The high content of β-sheet structures in these healthenhanced frankfurters indicate the creation of a denser network, resulting in increased hardness. Regarding lipid structure, analysis of the 2800–300 cm<sup>−</sup><sup>1</sup> regions and intensity ratios I<sup>ν</sup>sCH2/I<sup>ν</sup>asCH2 (I2850/I2890) and I<sup>ν</sup>sCH3/I<sup>ν</sup>asCH3 (I2935/I2890) revealed that the addition of oil-bulking agents as animal fat replacers in these health-enhanced frankfurters increased lipid acyl chain disorder and implicated more lipid −protein interactions [6].

**Figure 5.** Analysis of frankfurters reformulated with structured lipids based on polyphenol-EGs as animal fat replacers using FT-IR with an ATR device.
