Authentication of sibiu salami based on chemometric analysis of spectral data

14 1.2.1 Fatty acid profile analysis of dry-cured sausages by a gas chromatography-mass spectrometric GC-MS technique was used 14 1.2.2 Fatty acid profile analysis dry-cured sausages, s

UNIVERSITY POLITEHNICA OF BUCHAREST THE FACULTY OF APPLIED CHEMISTRY AND MATERIALS SCIENCE

FOOD CHEMISTRY MASTER

AUTHENTICATION OF SIBIU SALAMI BASED ON CHEMOMETRIC

ANALYSIS OF SPECTRAL DATA

Acknowledgements

My sincere appreciation to the scholarship agreement between the government of the socialist republic of Vietnam and the government of Romanian has been extremely beneficial for me

My great thanks go to the Faculty of Applied Chemistry and Materials Science - University Politehnica of Bucharest

I express my deepest gratitude to my supervisor Ms Cristina TODASCA, for her support, motivation and help during my thesis work

The last important word, I would like to remember and thank my family for striving hard to provide a good education for me and always encouraging me to continue my studies

Contents

1 LITERATURE REVIEW 5

1.1 Studies on different sausage products 5

1.1.1 Studies on Sibiu Salami 6 1.1.2 The studies on other dry-cured salami produced abroad 8 1.2 Analytical methods used for composition sausage/salami products measurement 14

1.2.1 Fatty acid profile analysis of dry-cured sausages by a gas chromatography-mass spectrometric (GC-MS) technique was used 14 1.2.2 Fatty acid profile analysis dry-cured sausages, salami by NIR/NIRS/NIT system technique was used 18 1.2.1 Fatty acid profile analysis dry-cured sausages, salami by NMR system technique was used 21 2 ORIGINAL RESULTS AND DISCUSSIONS 29

2.1 Objectives 29

2.2 Collection of the relevant samples of Sibiu Salami and other dry cured salami produced in Romania 29

2.3 Fat extraction from salami samples 31

2.4 GC-MS analysis of the extracted 32

2.5 FT-IR analysis of the extracted fat 35

2.6 NMR analysis of the extracted fat 37

2.7 Chemometric analysis of the spectral data (PCA, AHC) 41

2.7.1 Agglomerative Hierarchical Clustering (AHC) 42 2.7.2 Principal Component Analysis (PCA) 43 3 INSTRUMENTS AND METHODS 45

3.1 Fat extraction – Soxhlet procedure 45

3.2 Transesterification procedure for obtaining FAME 47

3.3 GC-MS Analysis 47

3.4 FT-IR Analysis 48

3.5 NMR Analysis 48

4 CONCLUSION 49

5 REFERENCES 52

LIST OF ABBRFVIATION

1

H-NMR: hydrogen nuclear magnetic resonance spectroscopy

AHC Agglomerative hierarchical clustering

EFA: Essential fatty acids

FA: fatty acid

FAME: fatty acid methyl ester

FT-IR: fourier transform infrared spectroscopy

GC-MS: gas chromatography-mass spectrometry

NIT: near infrared transmittance spectroscopy

NMR: nuclear magnetic resonance spectroscopy

PCA: Principal component analysis

PGI: Protected geographical indication

SFA: Saturated fatty acids

TE: Trolox equivalent

UFA: Unsaturated fatty acids

INTRODUCTION The sausage products are present in many countries throughout the years has become an indispensable dish for many families There are some countries in Europe such as Germany, France, Italy, Romanian, Spain, Denmark and some countries in Asia such us Korea, China with long tradition of salami production

In general, salami can be defined as products made by selecting, chopping, and mixing meat and fat, mixed with seasoning, spices, and authorized additives, that are then ripened and dried (cured) and, sometimes, smoked However, regional traditions, environmental variations, family recipes, and other factors have rise to a wide range of fermented salami, and it can be said that there are almost as many types of salami as there are geographical regions This is also attested in the report were they compare fatty acid composition of salami from different countries and their nutritional implications [19]

In some studies, reveal that, in the processing of sausage they have added additional ingredients such as olive oil, lard, spices This change affects the amount and type of fatty acids found in salami The studies determined the amount of fatty acids in the product enables us to test and refine new methods to improve the quality of products

Today, along with the development of science and technical capabilities, we could use a variety of methods and different analysis system for fatty acid content analysing in salami The main analysis system known as a gas chromatography-mass spectrometry (GC-MS) technique, near infrared spectroscopy technology (NIR), near infrared transmittance (NIT) spectroscopy, nuclear magnetic resonance spectroscopy (1H-NMR)

Itself every method of analysis have its own advantages and disadvantages Some method has dominant time benefits others have lower costs of analysis Hence, researchers continue to drive improvements in analytical methods for fatty acid, to provide accurate process analysis, saving time and chemicals, as well as lower the cost

of analysis for each sample

Fermented salami is products that before consumption passes through a more or less prolonged process of drying and ripening Sibiu Salami is a famous Romanian

fermented dry salami, produced mainly from pork meat and pork back-fat The mix is stuffed into natural casings and then smoked, dried and ripened There is a wide variety

of dry-cured salami produced in Romania, the most appreciated is Sibiu Salami, because of the special taste A similar production procedure is applied for obtaining Dacia, Banatean and Sinaia Salamis They different mainly is the taste and some of them in the production receipt Romania has a tradition of over 100 years in producing

„„Sibiu Salami‟‟, known as „„Salam de Sibiu‟‟ which has been registered as a “protected geographical indication” (PGI) labelled product in the European Union The Sibiu salami‟s producers have already made export to promote this product outside the country

The aim of this thesis was to develop a method for authentication of Sibiu Salami against other meat products of its class, using spectral data combined with chemometrical analysis The GC-MS and NMR spectral data have been used as data base for chemometric analysis

1 LITERATURE REVIEW

The issue of product quality is one of the top concern in salami products now There were many countries were different quality problems of dry-cured salami were noticed The studies about the ingredients and the analysis methods were conducted Each study has its advantages and significance of its own, overall analysis of the composition and content of fatty acids in the salami is now improved and very interesting

1.1 Studies on different sausage products

Salami is a type of cured sausage consisting of fermented and air-dried meat, typically beef or pork Historically, salami was popular among southern and central European peasants because it stores at room temperature for up to 40 days once cut, supplementing a potentially meager or inconsistent supply of fresh meat Countries and regions across Europe make their own traditional varieties of salami

1.1.1 Studies on Sibiu Salami

Sibiu Salami, in Romania known as „„Salam de Sibiu‟‟ is a Romanian deli made with pork's meat, pork's fat, salt and condiments Sibiu Salami are considered as a luxury food

Traditional sausages-dried salami is meat products that are part of the traditional daily diet, and also highly valued in modern life with an increasing demand Sausage recipes were developed that took advantage of what people had

The differences between the various types of dry-cured salami are due to the kind of meat they contain the proportion of lean to fat, spices, and the fineness or coarseness of the grind is what distinguishes one regional Salami from another Fermented salami are products that before consumption passes through a more or less

prolonged process of dry and ripening Dry salami is made from different types of meat

which are mainly made from pork In Romania, there are some studies related to salami composition but not much The results showed that GC-MS is a suitable method for quantitative determination of fatty acid in different meat products [1, 2,]

The composition and content of fatty acids in dry salami have been reported in many researches nationwide In a research in Romania, the authors mainly studied in the

composition and content of fatty acids in dry salami The content of different fatty acids

in sausage as such as myristic acid (C14:0), palmitoleic acid (C16:1), palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), stearic acid, arachidonic acid and saturated fatty acids (SFA); unsaturated fatty acids (UFA): essential fatty acids (EFA) The beef meat and the mixture of a sausage meat were compared Research results show the content of different fatty acids in beef meat and sausage Percentage of the fatty acid

of beef meat in respective with sausage meat: myristic acid (C14:1) 0.84- 0.09; palmitoleic acid (C16:1) 1.21 - 0.43; palmitic acid (C16:0) 13.46 - 8.47.; oleic acid (C18:1) 22.7 - 28.54; linoleic acid (C18:2) 0.49 - 6.08; stearic acid 11.97 - 8.88 and saturated fatty acids (SFA) 26.28 - 17.44; unsaturated fatty acids (UFA) 24.30 - 35.05: essential fatty acids (EFA) 1.7 - 7.51 It is interesting to observe that unsaturated fatty acids (UFA) in the sausage mixture is higher than in beef meat and also the total saturated fatty acids (SFA) are higher in beef meat then in the sausage mixture

The changes in fatty acid composition was also mentioned in other studies 19] In another research Dacia sausage (a Romanian fermented dry sausage, produced from raw pork, salt, pepper, garlic, species, additives and starter culture) was analysis The meat mix is stuffed into natural casings and then smoked, dried and ripened During the ripening of fermented sausages, various changes take place, leading to the final characteristics of the sausage Lipids are abundant in fermented meat products and their changes influence the aroma and the flavor of the final product One of many indicators that concern the composition, nutritional and content in fatty acids [3, 4] During the manufacture of Dacia sausage, there was an increase in all of the free fatty acids, with oleic (C18:1) and linoleic (C18:2) being the main fatty acids released The greatest increase in levels of these fatty acids took place during the drying-ripening stage

[1-Adding vegetal oil in smoked and boiled salami resulted in increasing both nutritional value and the protection against oxidation [4] This study has introduced a new meat product, boiled-smoked salami with an addition of sea buckthorn vegetable oil (Hippophặ rhamnosides L.) and walnut oil (Juglans Regia L) in order to improve PUFAs/SFAs and ω-6/ω-3 ratios The vegetable oils have been added for reducing the saturated fatty acids and to increase the poly- and mono - unsaturated fatty acids content Some chemical characteristics of the salami samples were analyzed by NIR spectrophotometry (FoodScan), the antioxidant capacity by photochemical instrument and fatty acids content by GC-MS The fatty acids were determined at 4, 11 and 18 days from processing at refrigeration temperature The lipid profile (g fatty acids/100g fatty acids), the vitamin E content (mg /100g fatty acids) and the antioxidant capacity (µmol

TE /g fat) offer information about the nutritive value of the salami with vegetable oils added, respectively, changing in the lipid content and its profile during the storage The results of this study showed that adding vegetal oil in smoked and boiled salami resulted

in increasing both nutritional value and the protection against oxidation Regarding the nutritional value, the sample prepared with walnut oil had the highest PUFAs content comparing with the sample obtained with sea buckthorn oil that showed higher MUFAs and α-tocopherol values The presence of sea buckthorn oil in salami, in relevant ratio, may cause a 10-day delay of the oxidative processes due to moderate PUFAs and higher antioxidant compounds content

The back-fat sample registered a content of 41.43% of saturated fatty acids, with the palmitic acid as predominant (25.93%) A large percentage of PUFAs was found in walnut oil 73.49g/100g fatty acids, according to the literature values (69.55 ± 0.03 up to 75.54 ± 0.51) (Arimboor & al.) More MUFAs was found in the sea buckthorn oil, 57.25 g/100g fatty acids, higher than the values found in the literature (52.823-54.32%) (Tahira Fatima & al.) Palmitic acid is 36.7% higher in sea buckthorn oil compared to the walnut oil and approximately 50% lower than in meat and back fat The main MUFA acid, the oleic acid, had the highest value in the sea buckthorn oil (70% compared to the walnut oil and 28% compared to the average value of meat and fat)

From the above findings, in the current situation, research is focusing on the nutritional composition and quality of dried sausage One of the criteria evaluated are most interested is that the fatty acid composition in dry sausages which are primarily implemented on the system GC-MS analysis This approach is in tune with the trend of research on dry sausage and countries around the world with the advantages of using modern analytical systems

1.1.2 The studies on other dry-cured salami produced abroad

In regard to salami, it is thought that the name is derived from Latin words

“salsiccia” and “salumen” In Europe, the main countries that produce salami are Romania, Germany (Tesco German Salami 125G, Salami pepper Germany), Italy (Cremona salami, Nduja, Calabrese salami), Spain (Iberian Salami, Serrano Ham D.O Teruel) France (Saucisson sec-200g, Garlic cervelat, Chorizo-200g), and Hungary (Bende Teli Hungarian Style Salami) The amount produced is of “several hundred million kg per year.”

Worldwide, there are many different versions of sausages, each with its own cultural and flavour profiles For example, due to immigration to North America, European settlers brought many traditions, including fermented meats such as bologna

or pepperoni Similar types of sausages are found in the Middle East, where various meats such as beef, lamb, and mutton are used, or in China where “Lap Cheong”, translated as “intestines stuffed in the winter” is usually pork Likewise, in Eastern

Europe, Hungarian salami is quite popular This particular type of salami is “intensively smoked and then its surface is inoculated with mould starters or spontaneous mould growth” Additionally, each sausage has its own type of seasonings (garlic, salt, pepper, wine…) and amount of salt, making each flavour and texture unique This wide array of variations of fermented sausages, especially in terms of salami, speaks to its ubiquitous but exclusive nature

In the process of development of traditional sausages as well as processing industry sausage, we are increasingly concerned about the ingredients add new additives that affect the quality of the sausage, and seek to reduce the harmful component to human health In meat products, the content of fatty acids is the important parameter used to establish their quality Additionally, they add flavours and colours to meat that are much appreciated, surpassing meat preservation proposes

The issue of product quality is one of the top concern in sausage products now Ethnic meat products are manufactured mainly by small-scale industries or artisanal producers according to and/or inspired by traditional processes One of the current researches is the quantitative analysis of pork dry-cured sausages to quality [5, 6]

Researchers review the main types of traditional meat sausages produced in the different regions of Portugal When considering the fatty acid profile, on average, 100g

of these products provide about 9g of saturated fatty acids, the minimum and maximum values were found for “presunto” and “chouriço de carne”, providing 11g of monounsaturated fatty acid and 3g of polyunsaturated fatty acids [5]

Quality controls to typical Spanish sausages were performed by proximate analysis (fat, moisture and protein) on the finished product (intact and homogenized) This has been used to provide quality controls at various stages once the finished product was obtained: finished product, storage, distribution, and marketing [6]

Physicochemical properties, fatty acid profile of these different types of sausages

as such as sheep and goat meat and pork was studied The addition of pork back-fat modified the total fatty acid profile, prompting a significant drop in the relative percentages of C14:0, C16:0, C17:0, C17:1, C18:0, together with a marked increase in oleic and linoleic acids [7] The effect of three pork back-fat levels (0% vs 10% vs

30%) on chemical composition, fatty acid profile and sensory properties on sheep and goat meat sausages was studied All physicochemical parameters were affected by the addition of pork back fat in both types of sausages Sausages manufactured with 30% of pork back-fat showed the lowest moisture and protein contents and the highest total fat content The lower aw values in sausages manufactured with higher fat content while in

pH happened the reverse situation Finally, in goat sausages, the fat content significantly affected sensory parameters: taste, texture and overall acceptability (P<0.05) All physicochemical parameters were affected by the addition of pork back fat in both types

of sausages

In this study [7] shows the effect of added pork back fat on the pH, aw and chemical composition of sheep and goat sausages Regarding aw, significant (P < 0.001) differences were observed among batches The G30% showed the lowest values (0.92 ± 0.002) compared to control sausages (0.97 ± 0.002), while G10% presented intermediate values (0.95 ± 0.002) in agreement with Gómez and Lorenzo (2013) who observed lower aw values in sausages manufactured with higher fat content On the other hand, the pH values were affected (P < 0.001) by fat content In their study, both sausages presented higher pH values in samples manufactured with 30% of pork back fat compared to control ones The fat content showed significant differences (P < 0.001) among batches This is an expected result because the batches were manufactured with different fat content The fat content of the goat and sheep sausages was lower than formulated as the batches contained 11.9% and 21.8% and 20.1% and 23.5% fat for goat and sheep sausages, respectively, instead of the 10 and 30% formulated Protein content also showed significant differences (P<0.001) among batches, presenting mean values

of 18.9%, 16.8% and 14.3% for goat sausages manufactured with 0%, 10% and 30% of pork back fat, respectively, and mean values of 18.2%, 15.5% and 14.1% for sheep sausages manufactured with 0%, 10% and 30% of pork back fat, respectively

For the fatty acid composition, their study shows the effect of added pork back fat on the fatty acid profile of sheep and goat sausages The relative percentages of fatty acids differed significantly as a function of the percentage added pork back fat The total amount of SFA in the goat and sheep sausages made with added pork back fat decreased

by approximately 6-9% and 2.5-6.5%, respectively compared to the controls In both

types of sausages, the most abundant fatty acids were, in decreasing order: C18:1n9, C16:0, C18:0 and C18:2n6 fatty acids The relatively high proportion of the latter in sausages manufactured with added pork back-fat (8.85%, on average), which was less than stearic acid, demonstrated the presence of pork back fat as a source of fat in both types of sausages Fresh sheep and goat sausages made with meat from culled animals is

an interesting way to valorise a product with very low market acceptability, satisfying the interest of producers and introducing in the market a new meat product

Today‟s consumers look for foods which provide nutrition and pleasure while safeguarding their health, the result of which is that they increasingly avoid foods containing cholesterol and saturated and trans fatty acids Chemically modified vegetable oils can help tailor meat products to meet this growing need and at the same time fulfil the technological needs of the meat processing industry In the sausage processing industry, some of the pork back-fat was replaced with olive oil The use of olive oil emulsified with alginate modified the fatty acid (FA) composition, increasing monounsaturated fatty acid (MUFA) levels and reducing the saturated fatty acids (FSA) and polyunsaturated fatty acid (PUFA) levels, a composition that remained stable over time and showed higher nutritional value thanks to its higher MUFA content [8, 9].This paper examines [8] variations in the quality of low-salt, inulin enriched Pamplona-style chorizo, in which some of the pork back fat was replaced with olive oil Four different sausage formulations were prepared in which 50% of the pork back fat was replaced with olive oil emulsified with alginate and 58% of the sodium chloride was replaced with 20% potassium chloride and 38% calcium chloride Four lots were prepared, three with 3%, 6% and 10% proportions of added inulin were labeled O–I 3%, O–I 6% and O–I 10%, respectively; while one, lot O, was without inulin These four formulations were compared with a control manufactured according to the traditional formula using pork back-fat The various lots were tested for proximate analysis, pH, processing loss, water activity, lactic acid bacteria, salmonella and listeria monocytogenes, physicochemical composition, instrumental color, texture profile and fatty acid composition during mixing and at days 3, 10, 17, 24 and 31 of the drying process A sensory evaluation was also performed by a seven-member trained panel, to obtain a descriptive analysis of the taste, texture, and appearance of the product The addition of olive oil alginate emulsion and inulin, O–I 6%, resulted in a low-salt, reduced-fat

product (20% less fat than traditional sausage), richer in monounsaturated fatty acids (10%), while retaining sensory notes similar to those of the traditional chorizo used as a control and achieved a good acceptability rating

The fatty acid profiles varied significantly across the different formulations The main fatty acids detected were, in descending order: oleic acid (C18:1n9) (around 40% of the total FA in the control formulation), palmitic acid (C16:0) (22%), linoleic acid (C18:2n6) (15%), stearic acid (C18:0) (10.5%), α-linoleic acid (C18:3n3) (2.3%) and palmitic acid (C16:1n7) (2.05%) This profile coincides with that found in pork fat

by others (Cava et al., 1997; Davenel, Riaublanc, Marchal, & Gandemer, 1999) and in traditional raw cured sausage In conclusion, by adding olive oil emulsified with alginate together with inulin, it is possible to obtain a Pamplona-style chorizo with less salt, less fat, and more unsaturated fatty acids, thus a better nutritional profile than the traditional version

Another study [9]. In this study rapeseed and sunflower oil were used to replace pork back-fat in UK-style sausages by incorporating the oils as pre-formed emulsions Replacing the pork back fat emulsion with rapeseed emulsion at total fat content of about 12%, reduced total saturated fatty acid (SFA) composition from 38% to 14% (4.5 to 1.8g/100 g), increased monounsaturated fatty acid (MUFA) composition from 45% to 59% and increased polyunsaturated fatty acid (PUFA) composition from 15% to 25% Partial replacement of pork back fat with rapeseed at a fat content of about 20% reduced SFA from 38% to 24% (7.2 to 4.8 g/100 g) There were no significant differences in eating quality and overall liking other than slight differences in the attributes „„firmness‟‟ and „„particle size‟‟ Improvement in the fatty acid composition was achieved without adversely affecting color shelf life or lipid oxidation The study suggests that a substantial reduction in SFA can be achieved by incorporating

„„healthy‟‟ oils in UK-style sausages without adversely affecting eating quality or shelf life Six 5 kg batches of sausages were prepared using the formulations Trimmed lean pork and back fat were obtained from the University of Bristol abattoir Treatments 1 and 2 were prepared using a pork back-fat pre-formed emulsion, treatments 3 and 4 with the rapeseed oil emulsion and treatments 5 and 6 with the sunflower oil emulsion Treatments 1, 3 and 5 had a target fat content of about 10% („low fat‟) whilst treatments

2, 4 and 6 had a target fat content of about 20% („„high fat‟‟) the total fatty acid weights per cooked sausage can be calculated as 11,514, 17,812, 12,592 and 20,555 mg/100 g for treatments 1, 2, 3 and 4, respectively i.e very similar to the total fatty acid content in the raw sausages This indicates that oil loss during cooking was minimal so not surprisingly the percentage fatty acid compositions for raw and cooked sausages were very similar Generally, there was no loss of fatty acids on cooking implying that the soya proteins were effective in preventing the fats from escaping

During sausage processing, concerns about the change in the fatty acid profile appear to the authors of the South China University of Technology which did research

on this issue [10] The research was mainly focused on the fatty acid content in different traditional salami and the change of fatty acid profile according to with the various of technical procedures Lipid composition, fatty acid profile, and lipid oxidative stability were evaluated during Cantonese sausage processing Free fatty acids increased with the concomitant decrease of phospholipids The total content of free fatty acids at 72h in muscle and adipose tissue was 7.341 mg/g and 3.067 mg/g, respectively Presents the fatty acid composition of the neutral lipid fractions in muscle and adipose tissues Oleic acid (C18:1) was the most abundant fatty acid followed by palmitic acid (C16:0) Stearic (C18:0) and linoleic acids (C18:2) were also present in relatively large proportions PUFAs only accounted for a small proportion and contained mainly linoleic acid in accordance with the results found in the intramuscular fat of hams Free

fatty acids increased with the concomitant decrease of phospholipids The proportion of

linoleic acid (C18:2) and arachidonic acid (C20:4) was significantly higher than that in the neutral lipid fraction PUFAs of phospholipids in muscle underwent marked degradation (from 41.05% to 18.97%) during processing, with the proportion of MUFAs increasing from 19.60% to 42.35% PUFA underwent a slight change in the phospholipids from adipose tissue The significant decrease of linoleic (C18:2) and arachidonic (C20:4) acids supported the idea that phospholipids were the main source of polyunsaturated free fatty acids The FFA content rose from 1.108 mg/g to 7.341 mg/g and 0.925 mg/g to 3.067 mg/g in muscle and adipose tissue, respectively In the free fatty acids, MUFAs showed the most significant increase followed by PUFAs The proportion of stearic acid (C18:0) decreased (P <0.05) during processing, suggesting it was poorly liberated Oleic acid (C18:1) and linoleic acid (C18:2) increased most

significantly This is consistent with the data in Table 2 which showed the marked decrease of POL and PSO in lipids from muscle and OOL and SSO in lipid from adipose tissue Additionally, linoleic acid (C18:2) and arachidonic acid (C20:4) were the main free fatty acids liberated from phospholipids These results proved that MUFAs and PUFAs were preferential hydrolyzed from neutral lipid or phospholipids

1.2 Analytical methods used for composition sausage/salami products

measurement

The measurements of the fatty acid derivatives are performed by standard method gas chromatography-mass spectrometry (GC-MS), as well the fat extracted from salami is directly analyzed by fourier transform infrared spectroscopy (FT-IR) and

by high-resolution 1H-NMR spectroscopy analysis

1.2.1 Fatty acid profile analysis of dry-cured sausages by a gas

chromatography-mass spectrometric (GC-MS) technique was used

Determination of free fatty acids of meat sausages can be determined by many different systems as such as GC-MS; NMR; NIS; NIT and NIR In the above analysis systems currently, have and are being used the most to analyze the fatty acid composition in the sausages were GC-MS system [1,11-13]. According to Andreea iordache, Monica culea [1] determination of free fatty acids in sausage meat by a gas chromatography-mass spectrometric (GC-MS) technique was used in Romania Similar studies for fatty acid composition, in recent years, have been carried out in some countries such as Italy [11,13], for a product known as „‟salsiccia salami‟‟ which holds

a PGI quality logo, and Spain in Iberian dry-cured sausages [12]

The studies [1, 11- 13] have been carried out research on the subject of sausage and meat products There are a number of different approaches to the analysis of fatty acid content In some study [5,7-13] the fatty acid is determined by physicochemical properties, sensory characteristics [7] It is also determined simultaneously with amino acids [11], triacylglycerol profiles [12,13]

Table 1.1 The content of different fatty acids in beef meat and various sausages

[1,11,13]

Fatty acid

Beef meat (%)

Sausage meat (mixture: beef, pork, and grease) (%)

Fermented pork sausage (%)

“Salsiccia” Sal20 sausage, pork sausage (%) Myristic acid

With each individual laboratory to apply internal standards and conditions for running own experiments The fatty acids were measured according to with the internal standard quantity and by using the response factors, for detector response correction The response factors were obtained by repetitive injections into GC-MS of the standard mixture containing known quantity of each fatty acid In this study [1] a trace DSQ ThermoFinnigan quadrupole mass spectrometer in the EI mode coupled with a Trace

GC was used The extracts were separated on a Rtx-5MS capillary column, 30m x 0.25mm, 0.25µm film thickness, using a temperature program from 500C, 1 min,

80C/min at 3000C, the flow rate 1ml/min, with helium 5.5 as the carrier gas Undecanoic acid (C11:1) was used as internal standard The qualitative analysis was carried out in

the 50-500 a.m.u mass range The following conditions were ensured: transfer line temperature 2500C, injector temperature 2000C, ion source temperature 2500C, splitter 10:1, electron energy 70eV and emission current 100µA The method was validated by using fatty acid standards The developed method is selective and specific The mass spectra recorded on each chromatographic peak permit the precise identification of the fatty acids, by using NI-ST library of mass spectra Also, the overlapping of the compounds is easily discovered The method was validated by using fatty acid standards Precision gave a lower value than 10% for R.S.D and sensitivity value was lower than 10 ng of fatty acid injected All the samples followed the same extraction and derivatization steps

An innovative procedure for the measurement of free amino acid and fatty acid contents in meat and meat derivatives was developed In the extract obtained from the sausages subjected to analysis 13 amino acid and 6 fatty acid derivatives were unequivocally recognized [11] Advantages of this method allow simultaneous analysis

of 13 amino acids and 6 fatty acids, the results clearly indicated that only 10 min were required for the best performance of the treatment and that more prolonged times are not necessary From all samples, analyzes were recovered with percentage values ranging from 88% to 97% for amino acid and 92% to 98% for free fatty acid

GC-MS identification and quantization of the analyses were performed using a 6890N Network GC System (Agilent Technologies Inc., Palo Alto, CA) equipped with

an HP-5MS (30 m × 0.25 mm i.d., 5% phenyl polysiloxane; film thickness, 0.25 m) capillary column and with a 5973 Network MSD quadrupole mass spectrometer, operated in electron impact ionization mode (70eV) GC-MS analyses were carried out

in splitless mode (1 min purge off), using helium as carrier gas (1 mL/min flow rate) The injection port was heated at 2500C The column was maintained at an initial temperature of 700C for 2 min and then programmed at 100C/min to a final temperature

of 2800C, where it was kept for 10 min All analyses were identified by injecting a real sample spiked with each standard compound and by observing the enhancement of the corresponding peak area The quantitative GC-MS analysis was carried out in splitless mode (1 min purge off), under the same operational conditions used for the identification of the analytes

In the report [12] dry-cured sausage production is a major industry in the Spanish economy The most widely consumed types are „chorizo‟ and „salchichón‟ Both products are manufactured from a mixture of chopped meat (pork and beef), lard, sugars, authorized additives (nitrate, nitrite, and antioxidants), starter cultures and spices „„Chorizo‟‟ is characterized by the fact that it includes Spanish paprika and garlic in its formulation while „„salchichón‟‟ includes black pepper Differences in the formulation mixture, an origin of the product, the process of fermentation and ripening determine the quality of the product

Comparable to the studies [1,5,7-11,13] we see in this report [12] the number

of fatty acids are determined up to twelve From C12:0 to C20:1 where 12 peaks were identified In this figure, the peaks of fatty acids eluted according to the atom carbon number and unsaturation number except for C18:3 fatty acids, which eluted after C20:0 fatty acid In fact, the C18:3 and C20:1 fatty acids co-eluted at the end of the chromatogram between 36.2 and 36.9 min

Fatty acid methyl esters (FAMEs) were analyzed following the method proposed by Viera-Alcaide et al and De Pedro et al In a 10 mL screw-top test tube, approximately 0.1 g of the extracted fat (melted 1.5 min in a microwave at 360W) was weighed 8 mL of hexane was added and the mixture was homogenized by shaking 0.4mL of 2N methanolic potassium hydroxide was added, the cap fitted with a PTFE-joint was put on, and the tube was shaken vigorously for 30s After leaving it to stratify until the hexane layer became clear, 1.0 mL of this phase was injected into the gas chromatograph Separation of FAMEs was carried out using a Varian 3800 (Varian Co, Palo Alto, CA USA) gas chromatograph equipped with a flame ionisation detector (FID) and a CP-SIL 88 (highly substituted cyanopropyl phase) silica capillary column

of 50m, 0.25mm ID, coated with a 0.2 μm film thickness of stationary phase (Varian, Palo Alto, USA) The oven temperature was kept at 1700C for 17 min and was then raised to 1900C at a rate of 5.00C min-11 and held isothermally for 9 min The injector and detector temperature was kept at 2500C Helium was used as the carrier (1 mL min -1

column constant flow) and make-up (30 mL min -1) gas Split injection mode was used with a split ratio of 1:80

Variations in the duration of use of the product leading to the change of fatty acid composition this has been mentioned in the report [13] The sausages were sampled and analyzed in triplicate (three different sources) during three different periods of ripening (20 days: samples sal20 and spi20; 40 days: samples sal40, sop40 and spi40;

80 days: samples sal80, sop80 and spi80) In conclusion, GC-MS analysis was used for determining the FFA content and the fatty acid profiles in acylglycerols of three kinds

of dry fermented sausages from Calabria during their ripening The data obtained indicates that lipolysis plays an essential role in developing FFAs during ripening A preferential hydrolysis of linoleic and oleic acid was observed The generation of aldehydic products by the oxidative degradation of unsaturated fatty acid chains was also studied by 1H-NMR The spectroscopic investigation confirmed the data obtained

by Kreis test

Each study has its advantages and significance of its own, overall analysis

of the composition and content of fatty acids in the sausage is now very interesting and complete

1.2.2 Fatty acid profile analysis dry-cured sausages, salami by NIR/NIRS/NIT

system technique was used

As we mentioned above, the determination of fatty acid content is performed

on many different systems and equipments In addition to the GC-MS system, the

NI-RS system is also applied to the analysis of fatty acids in meat and meat products

Some studies focused on assessing the feasibility of near-infrared reflectance spectroscopy (NI-RS) for predicting meat and meat product fatty acid composition [6,14-16] These results show the potential of NI-RS as a rapid, and convenient tool to determine fatty acid in sausage-salami dry

Near-infrared spectroscopy (NI-RS) has proved to be a quick and effective tool

in meat quality assessment The first trials using NI-RS technology on meat were performed over forty years ago, but the majority of research was carried out in the 1990s [6] Near infrared spectroscopy technology (diode array instrument) was used to study the feasibility of applying quality controls to typical Spanish sausages by performing a proximate analysis (fat, moisture, and protein) on the finished product

(intact and homogenized) The research results show us the selected models were calibrated and evaluated by cross and external validation For intact products, the coefficients of determination (R2, r2 and R2EV) for the fat and protein nutritional parameters, both in calibration and validation, were in all selected equations equal to or more than 0.95 The standard errors of prediction (SEP) for external validation in intact products were 1.47%, 0.97% and 1.08% for fat, moisture, and protein, respectively In homogenised products, these values were lower: 0.71%, 0.41% and 0.95%, respectively

In another study, the objectives of their study [15] were to develop calibration models for determination of water activity and the content of fat, dry matter, salt, non-collagen muscle protein and pH in dry cooked sausages

In the report [16] NI-RS accurately predicts several individual fatty acids (FA) (16:0, 18:0, 16:1 n9 cis, 17:1 n9 cis, 18:1 n9 cis, 18:1 n11 cis and 16:1 n9 trans) and several FA groups (total linear saturated FA, total branched saturated FA, total saturated FA, total cis-monounsaturated FA (MUFA), total trans MUFA, total MUFA and total polyunsaturated PUFA) These results show the potential of NI-RS as a rapid and convenient tool to predict the major FA in lamb meat The reflectance spectrum of

L lumborum muscle in the visible and near infrared range was measured on ground samples and on intact non-ground samples The non-ground muscle sample was a slice

of intact muscle tissue Ground and non-ground tissue samples were thawed at room temperature for approximately 2 h Portions weighing about 3 g were then scanned in the reflectance mode (400-2500 nm) in a NI-RS 6500 scanning monochromator (NIR Systems, Silver Spring, MD, USA) using ISI software, version 3.01, from Infrasoft International (Infrasoft International, South Atherton St State College, PA 16801, USA) equipped with a spinning module Muscle samples were scanned in a circular cup (diameter 50 mm, depth 10 mm) (Part number IH-0307, NIR Systems, Infrasoft International, South Atherton St State College, PA 16801, USA), compressed and sealed with a disposable paperbacked wrap Reflectance data were recorded at 2 nm intervals and stored as log (1/reflectance) Special, they used the reference method to fatty acid analysis by gas-liquid chromatography (GLC), using the Peri 2100 chromatograph model (Perichrom, Saulz-les-Chartreux, France) equipped with a CP-Sil

88 glass capillary column (Varian, USA) (length 100 m, ID 0.25 mm, H2 as carrier gas)

Temperatures of the split/splitless injector and of the flame ionisation detector were 200 and 2300C respectively, and the oven temperature was programmed as follows: 700C for

4 min, 70-1750C at 200C/min, 1750C for 25 min, 175-2150C at 100C/min and a final plateau at 2150C for 42 min

In view of the present findings, it may be concluded that the NIR technique could provide an exceptional opportunity for the quality control of dry-cured sausages The feasibility of using near infrared reflectance spectroscopy to evaluate the accurate information through a rapid determination of the fatty acid profiles in pork sausages was analyzed Individual fatty acids contents together with the saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA) groups were determined

Consumption of traditional dry-cured pork sausages is widespread in Mediterranean countries In Spain, the most popular dry-cured sausages are known as

„„chorizo” and „„salchichón” These foodstuffs are made following a traditional process

of mixing ingredients, casing, curing and ripening, which is regulated by the Spanish quality standard for crude and dry-cured sausages [14] Conventional procedures used for the determination of fatty acids involve the extraction of fat with diethyl ether followed by conversion of the fatty acids to their methyl esters and analysis by capillary gas chromatography This analysis is expensive, time-consuming and generates hazardous waste Near-infrared spectroscopy (NI-RS) has proven to be a rapid and effective tool in meat quality analysis for a wide variety of products and parameters There is evidence showing that NI-RS technology can quantitative fatty acid contents in meat or sausage [14, 16]

They used NI-RS system (Foss-NIR Systems 6500 SY-II monochromator, Foss NIR Systems, Silver Spring, MD, from 400 to 2498 nm, every 2 nm spectral bandpass 10 nm ± 1 nm) determine fatty acid content then used reference method on a gas chromatograph Agilent 6890N Network GS System, (Agilent, Santa Clara, CA, equipped with a flame ionisation detector and fitted with an HP-88 capillary column ,100m, 0.25mm i.d., 0.2 lm film thickness) to compare [14] The fatty acid profiles of the samples of sausages determined The results show that distribution of laboratory values for some of the analytical parameters (C12:0, C14:0, C16:1, C17:0, C18:2,

C20:0, MUFA and PUFA) was not uniform, particularly for higher and lower values, where it could be found samples whose extreme laboratory values were relatively distant from those shown by the next group of samples

Research results published twelve kinds of fatty acids and saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) Once again showed differences in the content of these fatty acids are identified [1,11,13, 14]

Determination of fatty acid using the near-infrared transmittance (NIT) spectroscopy system is very new, especially those studying in meat and meat products near-infrared transmittance (NIT, 850 to 1048 nm) spectroscopy was used to predict groups of fatty acids (FA), namely saturated FA (SFA), monounsaturated FA (MUFA) and polyunsaturated FA (PUFA)

In some studies, meat and a mixture of different ingredients such as bread, cheese, spices and additives were first analyzed by near infrared transmittance (NIT) instrument for spectral information and reference FA values were obtained by gas chromatographic analysis [17] Moreover, FA groups were expressed on the total fresh meat Fat content, protein content, collagen content and moisture were determined using the FoodScan (FOSS Electric A/S, Hillerød, Denmark) Finally, the present study underlined the ability of NIT spectroscopy to predict FA groups in commercial ground meat products Better predictions were obtained for SFA, MUFA and PUFA expressed

on fresh meat than on the total FA This study confirmed the positive effect of mincing the samples on the development of robust prediction models The calibration models developed in this study can be used by the meat industry to address the requirements of

EC Regulation 1169/2011

1.2.1 Fatty acid profile analysis dry-cured sausages, salami by NMR system

technique was used

High-resolution 1H-NMR spectroscopy was proposed for the determination of the fatty acid chain profile of lipids in pork meat products during ripening Quantitative NMR analysis provided the fatty acid chain profiles of total lipid extracts The

transesterification of total lipid extracts furnished the fatty acid methyl ester (FAME) mixtures that enabled quantitation of fatty acid acyl chains in the acylglycerol and free fatty acids (FFA) portions In all cases, alkyl chains were predominant, and high amounts of polyunsaturated fatty acid chains were observed The proposed spectroscopic method allowed also the estimation of the most important nutritional parameters of dry fermented meat products, high resolution 1H-NMR spectroscopy (NMR) was used in this study [18]

Figure 1.1 High-resolution 1 H-NMR spectra of TLE (total lipid extract), FAME (fatty acid methy ester), and FFA (free fatty acid) recorded for the analysis of ‘soppressata dolce’

Samples were prepared after analyzing the 1H-NMR system For 1H-NMR analysis, aliquots of 100 mg of the lipid extracts were dissolved in 0.7 ml of CDCl3 in a

5 mm NMR tube All spectra were recorded at 25 ± 0.10C under temperature control, using a Bruker Avance 300 Ultrashield spectrometer equipped with a 5-mm probe with Z-axis gradient coils Spectra were obtained at the 1H frequence of 300.132 MHz applying a standard zg90 pulse sequence, with the lock on the deuterium resonance of the solvent Acquisition parameters were as follows: sweep width (SW) of 4194.631Hz,

128K data points (TD), a recycle delay (D1) of 5.0s, for an acquisition time (AQ) of 15.624s Two prior dummy scans (DS) were applied for each spectrum and, in order to attain the best S/N ratio, 64 scans (NS) were recorded Under these instrumental conditions, the total time of analysis were 22.46 min After acquisition, FIDs were zero-filled up to 512K data points A line broadening (LB) of 0.1Hz was applied to all FIDs, prior the Fourier transformation, to minimize noise and enhance resolution The zero and first order phase-correction was effectuated manually Chemical shift values (б) were reported in ppm by referencing them to TMS used as the internal standard The results were compared with a gas-chromatographic system (Agilent Technologies Inc., Palo Alto, CA) Separation was carried out with an HP-5MS PhMesiloxane capillary column (30m long, 0.25mm i.d., 1 lm film thickness) Injector and detector temperatures were 2500C and 2800C, respectively The mass detector was operated in the electron impact ionization mode (EIMS) with electron energy of 70eV The temperature ramp program was as follows: the initial column temperature was 1500C, which was kept for 2 min, and then the temperature was raised to 2800C at 20C/min, where it was maintained for 10 min The split/splitless injector was used with a split/splitless ratio of 1/50 He was used as carrier gas at 1 ml/min flow The total time

of analysis were 77 min

The analysis of the acyl chain composition of the lipid extracts obtained from two typical Calabrian salami was carried out by high-resolution 1H-NMR The common unsaturated (oleyl, linoleyl, and linolenyl) fatty acid chains present in the lipid fractions

of the two meat products case of study, either as FFA or acylglycerol constituents, can easily be quantitated by high-resolution 1H-NMR spectroscopy Furthermore, this method allowed the determination of the total amounts of saturated (myristyl, palmityl, and stearyl) fatty acid chains Finally, it is possible to estimate the S/U and P/S ratios, two important quality parameters indicating the main nutritional parameters of meat products As for as the two typical dry-fermented salami are concerned, the high values

of oleic acid and PUFA, and the characteristic values of the S/U and P/U ratios provided

by the NMR method could be indicative of healthy products, which can be consumed in

a balanced diet From this investigation, it is possible to conclude that the routine application of high-resolution 1H-NMR techniques in the screening of the quantitative composition of fatty acid chains is a useful addition to the methods for the analysis of

the lipid fractions of meat products The NMR method is accurate, does not imply transformations of matrices, and minimizes sample handling The short time required for sample preparation and spectra recording can allow the analysis of a larger number

of matrices per day Furthermore, the high reliability and consistency between the data obtained by the NMR and GC-MS methods for the Calabrian „„salsiccia piccante‟‟ and

„„soppressata dolce‟‟ [18], demonstrated that high-resolution 1

H-NMR spectroscopy could be used as a powerful tool in alternative, to the classical chromatographic methods for the determination of fatty acid chain compositions in meat products

A comparative study of the fatty acid composition of salami from different countries and their nutritional implications were published „„Milano-type‟‟ salami from

13 European and American countries were analysed to establish their nutritional value

in relation to fat and fatty acid composition [19]

The fat content, fatty acid profile, ratio of n-6/n-3 polyunsaturated fatty acids, ratio of hypocholesterolemic/hypercholesterolemic fatty acids (h/H) and the contribution to the daily intake recommended for the population were studied Differences (P<0.05) in the dry matter, pH, water activity, fat content, and percentages

of saturated, monounsaturated and polyunsaturated fatty acids were found Principal component analysis permitted samples to be separated into three different groups: (1) salamis from Chile, Mexico, and Brazil, with high n-6/n-3 ratios (14 -16), medium iodine index (73 -76) and high h/H ratios (2.6-2.7); (2) European salamis and salamis from Costa Rica and USA, with medium and high n-6/n-3 ratios (8-15), low iodine index (62-72) and low h/H ratios (2.1-2.6); and (3) products from Peru, with the lowest n-6/n-3 ratio (7.2), the highest iodine index (about 80) and medium h/H ratio (2.5)

Results of the study indicated that the fatty acid composition (% of total FA) of the different Milano type salamis manufactured in different European countries Significant differences (P < 0.05) were found in almost all fatty acids detected, except for C10:0, C12:0, C14:0, C16:0, C18:1n-7 and C18:2n-7 Dcis 9, trans 11 The most abundant fatty acid was C18:1n-9 found in a proportion from about 38% (Spain) to 44% (Hungary) Also, significant differences (P < 0.05) were found among the saturated fatty acids (SFA) with values ranging from 37% (Germany and Hungary) to about 41% (Denmark, Holland, Spain and Italian prosciutto salami) The percentages of MUFA

varied from 46% (Denmark, Holland, Spain and Italian prosciutto salami) to 51.5% (Hungary) The PUFA content was between 11.5% (Hungary) and 14.6% (Spain)

Table 1.2 Fatty acid composition (of total fatty acids) of the Milano-type salamis

manufactured in different European countries [19]

The results obtained in this study indicate that „Milano-type‟ salamis show different fat contents and fatty acid compositions depending on the country of manufacture According to the results reported above, an average salami covered about 33%, 71%, 28%, 20%, 37%, 9.5%, 8% and 12% of the daily intake recommended for fat, SFA, MUFA, total PUFA, C18:2n-6, C18:3n-3, and total n-3, respectively

Through the process of studying about the fatty acids in sausage - salami dry, through research situation at local and abroad, we find that the study of fatty acid composition in the sausages as an important indicator to determine the quality of products Fatty acid content in the sausage in each country and region is different; the cause of this phenomenon is mainly composed of sausage ingredients It is the starting material or traditional formula has created its own flavor and characteristics of each type of sausage The studies aim to analyze the exact content of substances in the