*2.3.1 Cyclopropane fatty acids presence in food*

*Biochemistry and Health Benefits of Fatty Acids*

will be provided.

*2.2.1 Distribution and structure*

In the following paragraph, information about the distribution of omegacyclohexyl fatty acids, their biosynthesis, and their role on the cellular membrane

Omega-cyclohexyl fatty acids are the principal lipid component of saponifiable

The occurrence of a fully saturated and monosubstituted cyclohexane ring is rare but derivatives of cyclohexyl acid, precursor of omega-cyclohexyl fatty acid biosynthesis, have been isolated from the extract soil and shoots of *Achyranthes aspera* and from several *Streptomyces* antibiotics, including ansatrienin A synthetized by *S. collinus* [8]. However, in this case, omega-cyclohexyl fatty acids do not

In eukaryotes, the identification of 11-cyclohexylundecanoic fatty acid has been

documented as a minor component of butter fat [40], then in sheep fat (0.05% of the total weight of fatty acids) [39] and more recently in cow milk [11]. In this previous work [11], they focused on the identification and characterization of cyclic fatty acids in cow milk to study the effect of diverse types of dairy diet on milk fat composition. 13-cyclohexyl tridecanoic acid methyl ester was well detectable in all milk samples by GC-MS analysis of fatty acid methyl esters (FAME), and its pres-

The presence of omega-cyclic fatty acids in milk could be related to acidic ruminal fermentation patterns. An increase in starch as well as a decrease of less digestible fiber content favors the growth of amylolytic bacteria. This leads to an increased concentration of rumen volatile fatty acids such as butyric and propionic acids, a decrease of rumen odd and branched fatty acids [41] and lactate accumulation, resulting in a pH drop, which favors not only the development of the subacute ruminal acidosis (SARA) [42] but also the growth of thermo-acidophilus bacteria, which produce omega-cyclohexyl fatty acids. In this context, the presence of omega-cyclohexyl fatty acids in milk, especially 11-cyclohexylundecanoic and 13-cyclohexyltridecanoic acids, could be represented as a parameter to detect SARA, as proposed by other authors for odd- and branched-chain fatty acids [41].

However, this hypothesis has never been confirmed by experimental data.

Cyclic fatty acids are generally secondary compounds in fatty acid profiles of food; however, due to the recent discovery, some gaps of knowledge must be fulfilled. In some cases, especially cyclopropane fatty acids, they could reach the g/kg of total fat content in meat and dairy products [12, 43] and their dietary intake

Therefore, it would be interesting to investigate on their metabolism in humans and eventual physiological effects, considering that bacteria produce cyclic fatty acids to enforce their membranes. The aim of these studies is to achieve a first never reported picture of the occurrence of CPFA in humans and their possible health effects.

**2.3 Cyclic fatty acids in human nutrition**

fraction of *Alicyclobacillus acidocaldarius*. They also occur in thermoacidophile strains, such as *A. acidoterrestis* and *A. cycloheptanicus*, and in the mesophile *Curtobacterium pusillum*, where the percentage concentration of these fatty acids in the cellular membrane increases at pH 3–4 as well as at elevated temperatures [8, 37]. In fact, omega-cyclic fatty acids are suggested to have special physiological importance for the cells both at hot temperature and acid pH. Model membranes, consisting of lipids containing omega-cyclohexyl fatty acids, are relatively dense

and closely packed even at the phase transition temperature [8].

seem to play a similar membrane stabilizing role as in *A. acidocaldarius*.

ence was confirmed by mass spectra and the synthetized standard.

**36**

may be not negligible.

Data reported on CPFA, mainly in dairy products, meat, and fish, were obtained in previous publications [10–12]. The content of cyclopropane fatty acids has also been evaluated in other food categories such as probiotic food supplements, vegetable edible oils (e.g., extra virgin olive, corn, soy, and peanuts oils) and cocoa butter, soy-derived products, and mushrooms (data not published). CPFA content in food categories, resulted positive in previous analysis, is shown in **Table 1**.

Results showed that among all the analyzed food categories, the most important CPFA food source is Grana Padano cheese, reaching concentration levels of 1 g/ kg total fat (**Table 1**). CPFA were detected not only in commercial bovine meat (200–400 mg/kg total fat) but also in some species of fish (eels and mullets) with concentrations between 400 and 800 mg/kg total fat [12], probiotics, and in mushrooms (data not published). On the contrary, poultry, pork meat, vegetable oils commonly consumed (e.g., extra virgin olive, corn, soy, and peanuts oils), and cocoa butter were all negative to CPFA (data not shown), indicating that CPFA presence in foodstuffs of animal origin is correlated with the use of silages in the animal feedings, whereas plant organisms generally do not produce CPFA. As a whole, our results demonstrate the bacterial and fungal origin of CPFA in foods [16, 44]. Finally, the estimated daily, weekly and monthly CPFA dietary intake in the total Italian population (all sex and ages) [45] resulted in the milligrams order, so not negligible in view of a possible physiological action by CPFA on humans. Furthermore, food processing, manufacturing, seasoning steps, and fermentation [10] seemed not to affect CPFA content in the analyzed food matrices. Certainly,


*1 Results obtained combining previous analysis [10–12, 43].*

*2 CPFA = cyclopropane fatty acids as the sum of total isomers (dihydrosterculic and lactobacillic acids) as reported by Caligiani et al. [43].*

*SD = standard deviation.*

CPFA can be considered unknown components of the human diet, and additional information about their possible impact on humans is useful to provide a further understanding on the link between diet and human health.

## *2.3.2 CPFA digestibility and potential bioaccessibility*

Triglycerides (TG) are the major components of dietary fats, and once ingested, they are submitted to a hydrolytic process catalyzed by lipases present in gastric and especially in duodenal digestive juices [46]. Nowadays, the evolution of the triglycerides during digestion is a subject of great interest in lipid research, as much as the development of methodologies able to evaluate both qualitatively and quantitatively all the products generated from this process [13].

As reported in the previous paragraphs, no information is present in literature about the fate of CPFA within the human body, and a thorough investigation of how CPFA accumulate and are metabolized in humans is needed. In [13], the rate of CPFA digestibility has been assessed through their lipolysis and resistance to *in vitro* simulated human gastrointestinal (GI) digestion in Grana Padano cheese, one of the most relevant sources of CPFA [43]. Results showed a high percentage of digestibility of the lipid fraction (more than 90% of free fatty acids and 1-monoglycerides were obtained after digestion). Furthermore, CPFA were all released from TG and the cyclopropane ring was not degraded, proving its resistance to GI digestion, mainly due to the acid pH of the gastric environment. Results of CPFA concentration in fat before and after *in vitro* digestion are reported in **Table 2**.

These observations are encouraging, since CPFA seemed to be potentially efficiently absorbable and, ideally, bioavailable. Certainly, additional research is needed to evaluate the diffusion of these unusual fatty acids through the membrane of the small intestine epithelial cells as well as their presence in human plasma. For this purpose, an *in vivo* study needs to be conducted to determine the eventual CPFA presence in human plasma after a CPFA-rich diet.

#### *2.3.3 Potential biological effects of cyclic fatty acids*

Cyclopropane and omega-cyclohexyl fatty acids play a significant role in increasing the chemical and physical stability of bacterial membranes to adverse conditions [8]. To the best of our knowledge, little information is reported in literature about the effect of cyclic fatty acids in higher animals. However, some papers concern about biological activity of cyclopropene fatty acids, mainly sterculic acid, in mammals [9, 47–49]. On the contrary, omega-cyclic fatty acids remain an under investigated lipid class from a nutritional and physiological significance.

Many seed lipids containing cyclopropene fatty acids are extensively consumed by humans, especially in tropical areas [9]. It has been documented that their dietary leads to the accumulation of hard fats and other physiological disorders in animals [9].

Some studies suggested the effect of sterculic acid on lipid metabolism in mammals, especially in dairy sheep [47] and in human Caco2 cells [48], as inhibitor of Δ9-desaturase, which has a key role in the endogenous synthesis of cis-9, trans-11 conjugated linoleic acid (rumenic acid), known to have interesting properties in improving human health.

It is also known that sterculic acid is a potent inhibitor of stearoyl-CoA desaturase (SCD) involved in the biosynthesis of monounsaturated fatty acids (MUFA). SCD catalyzes the NADH- and O2-dependent desaturation of palmitate (16:0) and

**39**

*Cyclic Fatty Acids in Food: An Under-Investigated Class of Fatty Acids*

stearate (18:0) at carbon 9 to produce palmitoleate (cis-9, 16:1) and oleate (cis-9, 18:1), respectively, and has a crucial role in regulation of adipocyte proliferation/ differentiation of adipocytes, mainly in ruminant species [49, 50]. Due to the structural analogies between cyclopropene and cyclopropane fatty acids, it is possible to hypothesize a possible physiological role for CPFA in lipid metabolism as well. Fatty acids (FA) with a cyclopropane in the structure, especially cyclopropaneoctanoic acid 2-hexyl (CPA2H), have also been recently identified in human serum and adipose tissue of obese patients [32, 51] suggesting that they are

*Free (as FFA and MG) and bound (as TG) CPFA in Grana Padano cheese before and after in vitro simulated* 

**Lipid classes CPFA before digestion CPFA after digestion** TG 540 ± 20 mg/kg ≤LOD (60 mg/kg) FFA and MG ≤LOD (60 mg/kg) 520 ± 10 mg/kg *Results are expressed in mg/kg of total extracted fat as mean ± standard deviation. FFA, free fatty acids; MG, monoglycerides; TG, triglycerides. Cyclopropane fatty acids (CPFA) refer to the sum of dihydrosterculic and* 

absorbed as the other fatty acids and can be selected markers of metabolic disorders such as dyslipidemia, inflammation, and increased cardiovascular risk. These studies reported that both obese patients with hypertriglyceridemia and non-obese patients with chronic kidney disease (CKD) presented elevated serum levels of CPA2H, suggesting a positive correlation between high serum levels of CPA2H and high serum TG and cholesterol concentrations rather than to body mass or body mass index (BMI). These results show that CPA2H negatively affect the cellular lipid metabolism; however, the relevance of altered serum concentrations of this

Previously, it has been reported that cyclopropane fatty acids can influence the pathogenicity of *Mycobacterium tuberculosis*, demonstrating they could modulate

TNF is an inflammatory cytokine produced by activated macrophages and plays a key role as a mediator of intestinal inflammation [52]. In [52], they studied select strains of human-derived *Lactobacillus reuteri,* which are involved in human TNF immunomodulatory activity in gut. This work showed that the bacterial enzyme cyclopropane fatty acid synthase is involved in the anti-inflammatory effect of select strains of *L. reuteri*. Indeed, only the strains contained a cyclopropane fatty acid, lactobacillic acid, were able to inhibit TNF in activated macrophages, whereas cyclopropane fatty acid synthase mutants (lacking cyclopropane fatty acid synthase activity) do not suppress the production of the proinflammatory cytokine. However, lactobacillic acid seemed not to be responsible for mediating the repression of human TNF production, indicating that lactobacillic acid indirectly contributed to *L. reuteri* immunomodulatory activity, probably altering the composition and permeability of bacterial membrane, resulting in a decrease of the membrane

Since significative amount of dihydrosterculic acid had been found in foods,

unknown component of human diet. However, no specific works both *in vitro* and *in vivo* about the effect of dihydrosterculic acid are available in the literature, for example on enzyme activity, cellular membranes, and metabolism in mammals.

Future studies should elucidate how and whether this uncommon FA may have a

mainly in dairy products, this fatty acid can be considered a new as well as

biological role and clarify its healthy or unhealthy effects in humans.

*DOI: http://dx.doi.org/10.5772/intechopen.80500*

*lactobacillic acids [13].*

*human GI digestion.*

**Table 2.**

fatty acid remains still unclear.

the host immune response [34, 35].

fluidity or in an altered expression of immunomodulins.

*Cyclic Fatty Acids in Food: An Under-Investigated Class of Fatty Acids DOI: http://dx.doi.org/10.5772/intechopen.80500*


**Table 2.**

*Biochemistry and Health Benefits of Fatty Acids*

understanding on the link between diet and human health.

*2.3.2 CPFA digestibility and potential bioaccessibility*

all the products generated from this process [13].

CPFA can be considered unknown components of the human diet, and additional information about their possible impact on humans is useful to provide a further

Triglycerides (TG) are the major components of dietary fats, and once ingested, they are submitted to a hydrolytic process catalyzed by lipases present in gastric and especially in duodenal digestive juices [46]. Nowadays, the evolution of the triglycerides during digestion is a subject of great interest in lipid research, as much as the development of methodologies able to evaluate both qualitatively and quantitatively

As reported in the previous paragraphs, no information is present in literature about the fate of CPFA within the human body, and a thorough investigation of how CPFA accumulate and are metabolized in humans is needed. In [13], the rate of CPFA digestibility has been assessed through their lipolysis and resistance to *in vitro* simulated human gastrointestinal (GI) digestion in Grana Padano cheese, one of the most relevant sources of CPFA [43]. Results showed a high percentage of digestibility of the lipid fraction (more than 90% of free fatty acids and 1-monoglycerides were obtained after digestion). Furthermore, CPFA were all released from TG and the cyclopropane ring was not degraded, proving its resistance to GI digestion, mainly due to the acid pH of the gastric environment. Results of CPFA concentra-

tion in fat before and after *in vitro* digestion are reported in **Table 2**.

CPFA presence in human plasma after a CPFA-rich diet.

*2.3.3 Potential biological effects of cyclic fatty acids*

These observations are encouraging, since CPFA seemed to be potentially efficiently absorbable and, ideally, bioavailable. Certainly, additional research is needed to evaluate the diffusion of these unusual fatty acids through the membrane of the small intestine epithelial cells as well as their presence in human plasma. For this purpose, an *in vivo* study needs to be conducted to determine the eventual

Cyclopropane and omega-cyclohexyl fatty acids play a significant role in increasing the chemical and physical stability of bacterial membranes to adverse conditions [8]. To the best of our knowledge, little information is reported in literature about the effect of cyclic fatty acids in higher animals. However, some papers concern about biological activity of cyclopropene fatty acids, mainly sterculic acid, in mammals [9, 47–49]. On the contrary, omega-cyclic fatty acids remain an under investigated lipid class from a nutritional and physiological

Many seed lipids containing cyclopropene fatty acids are extensively consumed

Some studies suggested the effect of sterculic acid on lipid metabolism in mammals, especially in dairy sheep [47] and in human Caco2 cells [48], as inhibitor of Δ9-desaturase, which has a key role in the endogenous synthesis of cis-9, trans-11 conjugated linoleic acid (rumenic acid), known to have interesting properties in

It is also known that sterculic acid is a potent inhibitor of stearoyl-CoA desaturase (SCD) involved in the biosynthesis of monounsaturated fatty acids (MUFA). SCD catalyzes the NADH- and O2-dependent desaturation of palmitate (16:0) and

by humans, especially in tropical areas [9]. It has been documented that their dietary leads to the accumulation of hard fats and other physiological disorders in

**38**

significance.

animals [9].

improving human health.

*Free (as FFA and MG) and bound (as TG) CPFA in Grana Padano cheese before and after in vitro simulated human GI digestion.*

stearate (18:0) at carbon 9 to produce palmitoleate (cis-9, 16:1) and oleate (cis-9, 18:1), respectively, and has a crucial role in regulation of adipocyte proliferation/ differentiation of adipocytes, mainly in ruminant species [49, 50]. Due to the structural analogies between cyclopropene and cyclopropane fatty acids, it is possible to hypothesize a possible physiological role for CPFA in lipid metabolism as well.

Fatty acids (FA) with a cyclopropane in the structure, especially cyclopropaneoctanoic acid 2-hexyl (CPA2H), have also been recently identified in human serum and adipose tissue of obese patients [32, 51] suggesting that they are absorbed as the other fatty acids and can be selected markers of metabolic disorders such as dyslipidemia, inflammation, and increased cardiovascular risk. These studies reported that both obese patients with hypertriglyceridemia and non-obese patients with chronic kidney disease (CKD) presented elevated serum levels of CPA2H, suggesting a positive correlation between high serum levels of CPA2H and high serum TG and cholesterol concentrations rather than to body mass or body mass index (BMI). These results show that CPA2H negatively affect the cellular lipid metabolism; however, the relevance of altered serum concentrations of this fatty acid remains still unclear.

Previously, it has been reported that cyclopropane fatty acids can influence the pathogenicity of *Mycobacterium tuberculosis*, demonstrating they could modulate the host immune response [34, 35].

TNF is an inflammatory cytokine produced by activated macrophages and plays a key role as a mediator of intestinal inflammation [52]. In [52], they studied select strains of human-derived *Lactobacillus reuteri,* which are involved in human TNF immunomodulatory activity in gut. This work showed that the bacterial enzyme cyclopropane fatty acid synthase is involved in the anti-inflammatory effect of select strains of *L. reuteri*. Indeed, only the strains contained a cyclopropane fatty acid, lactobacillic acid, were able to inhibit TNF in activated macrophages, whereas cyclopropane fatty acid synthase mutants (lacking cyclopropane fatty acid synthase activity) do not suppress the production of the proinflammatory cytokine. However, lactobacillic acid seemed not to be responsible for mediating the repression of human TNF production, indicating that lactobacillic acid indirectly contributed to *L. reuteri* immunomodulatory activity, probably altering the composition and permeability of bacterial membrane, resulting in a decrease of the membrane fluidity or in an altered expression of immunomodulins.

Since significative amount of dihydrosterculic acid had been found in foods, mainly in dairy products, this fatty acid can be considered a new as well as unknown component of human diet. However, no specific works both *in vitro* and *in vivo* about the effect of dihydrosterculic acid are available in the literature, for example on enzyme activity, cellular membranes, and metabolism in mammals.

Future studies should elucidate how and whether this uncommon FA may have a biological role and clarify its healthy or unhealthy effects in humans.

#### **2.4 Cyclic fatty acids in food authentication**

In the last decades, food frauds have been on the rise [53]. For this reason, food authentication represents an important strategic issue for food industry because consumers are becoming increasingly interested in the quality and origin of foods. This is especially true when consumers purchase expensive certified and high added-value products, such as protected denomination of origin (PDO) or protected geographical indication (PGI) products [54].

Assuring food authenticity is not only an economical issue for food industries but it also concerns consumer safety, due to the substitution of food grade materials by cheaper non-food grade materials or to the presence of undeclared ingredients. The broad objective in food authentication is to identify unique or groups of markers to characterize the authenticity of food or their potential adulterants/contaminants and use them to resolve authenticity problem [55, 56].

As previously reported, cyclopropane and ω-cyclohexyl fatty acids, isolated respectively in lactic and rumen bacteria, have been identified in milk and dairy products as well as in meat. The occurrence and the content of cyclopropyl fatty acids in dairy products and meat were mainly correlated with the presence in forage of maize silage, whereas omega-cyclohexyl fatty acids have been proposed as marker of species, especially for ruminant species. In the following paragraphs, we will focus on the role of cyclic fatty acids as quality markers in food authentication mainly in dairy products and meat.

#### *2.4.1 Cyclopropane fatty acids as quality markers in dairy products*

In dairy sector, the most critical issue in authentication is related to PDO cheeses, which are high commercial value products confined according to legislative and proper labeling rules. The higher prices of PDO products encourage more frequent food fraud [57]. Their authenticity is associated with several factors, such as the geographical area of production, materials, and technology used. In fact, cheese production can differ according to the feeding system of the animals providing all the ingredients as milk, the starters used, and the presence or lack of preservatives as well as other parameters (i.e., the heating temperature, the salting, the ripening time). All of these generate defined characteristics, which in turn can be detected by several analytical techniques, and provide a trace of the cheese origin [58, 59].

Among PDO cheeses, Parmigiano-Reggiano is probably the most worldwide appreciated Italian PDO cheese. It represents a fully natural high-quality cheese only made in a very small and specific region of Italy, without the use of additives and from milk of local cows fed with hay and not silage as fodder commonly used worldwide in livestock feeding. It is made according to the Production Specification Rules laid down by Parmigiano-Reggiano Cheese Consortium (CFPR) according to EU regulations (EU 510/2006 and following 1151/12). Due to the high quality of the raw material, the long ripening, and the strict rules of production, it is an expensive cheese, but despite this, a constant growing of the national and international market is registered [60]. Consequently, the problem of fraud is a critical issue. In fact, several grated varieties branded as Parmesan and with "Italian sound" elements on the pack were found to be inauthentic, some of them containing non-declared additives. Therefore, mislabeling is a severe problem concerning unfair competition and deception to the detriment of consumers.

As previously reported, data collected by the authors [10, 11, 43] on hundreds of milk and dairy samples confirm the strict correlation between the use of silages in the feeding and the presence of CPFA in milk fat. Cyclopropane fatty acids (CPFA) were present only in dairy products from cows fed with silages, and their

**41**

**Figure 4.**

*Cyclic Fatty Acids in Food: An Under-Investigated Class of Fatty Acids*

determination has been demonstrated to be molecular markers of quality for PDO cheeses, as Parmigiano-Reggiano, where the use of silage is forbidden [10]. In this context, an example of successful case was the innovative method (UNI11650) that has been developed for Parmigiano-Reggiano cheese authentication based on the presence or absence of CPFA [43]. As results, Grana Padano (GP) samples were always positive to CPFA, reflecting that silages are not forbidden for their production (**Figure 4**). The amount of CPFA found in Grana Padano is variable, and it ranges from 300 to 830 mg/kg of fat, with a global mean value of 540 ± 110 mg/kg [43]. Because grated parmesan is particularly vulnerable to being adulterated with other cheeses, mix of Parmigiano-Reggiano with a cheese produced with milk from ensiled-fed cows have been analyzed to establish the minimum level of adulteration. The GC-MS method [43] was able to detect frauds that included 10–20% of cheaper cheeses. Adulteration behind this value has scarce commercial significance. Furthermore, the optimized GC-MS method was subjected to validation in terms of precision, accuracy, linearity, detection, and quantitation limits following the recommendations of the International Conference on Harmonization [61]. Therefore, due to the innovative and encouraging results, an application for an official standardization of the method (UNI method 11650) has been validated and included in Parmigiano-Reggiano Product Specification Rules among the official controls, enforcing the current analytical methods adopted for the quality controls.

In conclusion, the presence of CPFA in milk and dairy products probably derives from their presence in ensiled feeds, where CPFA can be released by bacteria during fermentation. The environmental conditions developed in silos seem to be essential for the production and release of CPFA from bacteria, whereas shelf-life, manufacturing, seasoning steps, and lactic fermentation did not affect CPFA content [11]. As a whole, CPFA demonstrated to be interesting molecular markers, able to distinguish cheeses obtained from cows fed with or without silages. Moreover, the quantitative

*Enlarged view of CPFA peak elution zone and comparison between a sample of Grana Padano cheese positive* 

*to CPFA and a sample of Parmigiano-Reggiano cheese negative to CPFA [43].*

*DOI: http://dx.doi.org/10.5772/intechopen.80500*

#### *Cyclic Fatty Acids in Food: An Under-Investigated Class of Fatty Acids DOI: http://dx.doi.org/10.5772/intechopen.80500*

*Biochemistry and Health Benefits of Fatty Acids*

mainly in dairy products and meat.

and deception to the detriment of consumers.

**2.4 Cyclic fatty acids in food authentication**

tected geographical indication (PGI) products [54].

nants and use them to resolve authenticity problem [55, 56].

*2.4.1 Cyclopropane fatty acids as quality markers in dairy products*

In the last decades, food frauds have been on the rise [53]. For this reason, food authentication represents an important strategic issue for food industry because consumers are becoming increasingly interested in the quality and origin of foods. This is especially true when consumers purchase expensive certified and high added-value products, such as protected denomination of origin (PDO) or pro-

Assuring food authenticity is not only an economical issue for food industries but it also concerns consumer safety, due to the substitution of food grade materials by cheaper non-food grade materials or to the presence of undeclared ingredients. The broad objective in food authentication is to identify unique or groups of markers to characterize the authenticity of food or their potential adulterants/contami-

As previously reported, cyclopropane and ω-cyclohexyl fatty acids, isolated respectively in lactic and rumen bacteria, have been identified in milk and dairy products as well as in meat. The occurrence and the content of cyclopropyl fatty acids in dairy products and meat were mainly correlated with the presence in forage of maize silage, whereas omega-cyclohexyl fatty acids have been proposed as marker of species, especially for ruminant species. In the following paragraphs, we will focus on the role of cyclic fatty acids as quality markers in food authentication

In dairy sector, the most critical issue in authentication is related to PDO cheeses, which are high commercial value products confined according to legislative and proper labeling rules. The higher prices of PDO products encourage more frequent food fraud [57]. Their authenticity is associated with several factors, such as the geographical area of production, materials, and technology used. In fact, cheese production can differ according to the feeding system of the animals providing all the ingredients as milk, the starters used, and the presence or lack of preservatives as well as other parameters (i.e., the heating temperature, the salting, the ripening time). All of these generate defined characteristics, which in turn can be detected by several analytical techniques, and provide a trace of the cheese origin [58, 59]. Among PDO cheeses, Parmigiano-Reggiano is probably the most worldwide appreciated Italian PDO cheese. It represents a fully natural high-quality cheese only made in a very small and specific region of Italy, without the use of additives and from milk of local cows fed with hay and not silage as fodder commonly used worldwide in livestock feeding. It is made according to the Production Specification Rules laid down by Parmigiano-Reggiano Cheese Consortium (CFPR) according to EU regulations (EU 510/2006 and following 1151/12). Due to the high quality of the raw material, the long ripening, and the strict rules of production, it is an expensive cheese, but despite this, a constant growing of the national and international market is registered [60]. Consequently, the problem of fraud is a critical issue. In fact, several grated varieties branded as Parmesan and with "Italian sound" elements on the pack were found to be inauthentic, some of them containing non-declared additives. Therefore, mislabeling is a severe problem concerning unfair competition

As previously reported, data collected by the authors [10, 11, 43] on hundreds of milk and dairy samples confirm the strict correlation between the use of silages in the feeding and the presence of CPFA in milk fat. Cyclopropane fatty acids (CPFA) were present only in dairy products from cows fed with silages, and their

**40**

determination has been demonstrated to be molecular markers of quality for PDO cheeses, as Parmigiano-Reggiano, where the use of silage is forbidden [10]. In this context, an example of successful case was the innovative method (UNI11650) that has been developed for Parmigiano-Reggiano cheese authentication based on the presence or absence of CPFA [43]. As results, Grana Padano (GP) samples were always positive to CPFA, reflecting that silages are not forbidden for their production (**Figure 4**). The amount of CPFA found in Grana Padano is variable, and it ranges from 300 to 830 mg/kg of fat, with a global mean value of 540 ± 110 mg/kg [43].

Because grated parmesan is particularly vulnerable to being adulterated with other cheeses, mix of Parmigiano-Reggiano with a cheese produced with milk from ensiled-fed cows have been analyzed to establish the minimum level of adulteration. The GC-MS method [43] was able to detect frauds that included 10–20% of cheaper cheeses. Adulteration behind this value has scarce commercial significance. Furthermore, the optimized GC-MS method was subjected to validation in terms of precision, accuracy, linearity, detection, and quantitation limits following the recommendations of the International Conference on Harmonization [61]. Therefore, due to the innovative and encouraging results, an application for an official standardization of the method (UNI method 11650) has been validated and included in Parmigiano-Reggiano Product Specification Rules among the official controls, enforcing the current analytical methods adopted for the quality controls.

In conclusion, the presence of CPFA in milk and dairy products probably derives from their presence in ensiled feeds, where CPFA can be released by bacteria during fermentation. The environmental conditions developed in silos seem to be essential for the production and release of CPFA from bacteria, whereas shelf-life, manufacturing, seasoning steps, and lactic fermentation did not affect CPFA content [11]. As a whole, CPFA demonstrated to be interesting molecular markers, able to distinguish cheeses obtained from cows fed with or without silages. Moreover, the quantitative

**Figure 4.**

*Enlarged view of CPFA peak elution zone and comparison between a sample of Grana Padano cheese positive to CPFA and a sample of Parmigiano-Reggiano cheese negative to CPFA [43].*

GC-MS method developed is relatively simple, assures a quick sample preparation, and relies on available instrumentation, thus making it suitable for the screening of many samples with a good cost-per-analysis ratio.

#### *2.4.2 Cyclic fatty acids as quality markers in meat*

An increasing critical issue is the substitution of higher commercial valued meats by low-priced ones and the fraudulent labeling of meat species [62]. In this context, there is the need for new, fast, and reliable analytical methodologies and easily quantifiable markers to be used for meat authentication and to protect both consumers and producers from illegal substitutions. Current methods to identify the origin of species present in commercial meat are based on DNA and ELISA, but also UPLC, Raman spectroscopy, low-field NMR, and mass spectrometry have been considered [63].

Fatty acids in food authentication were mainly used for the determination of the cow feeding system, which affect the milk and meat fat composition [64, 65]. Previously, many data have been collected confirming the association between the use of ensiled feeds and the presence of CPFA in the fatty profile of dairy products.

As suggested by Lolli et al. [12], cyclopropane fatty acids (mainly dihydrosterculic acid) have also been detected in animal fat, especially in bovine meat fat. As results, CPFA were detected in the fatty profiles of commercial bovine meat samples but they were absent in the samples of certified meat from cows not fed with fermented forages, reflecting the same correlation observed in dairy products [10, 11, 43]. In the case of meat of other animal species (pork, pork cured meat, and chicken), results did not show the presence of cyclopropane fatty acids as shown in **Table 3**. These preliminary results suggested that CPFA might be proposed as markers of silage feedings and for the authentication of high quality costly meat whose producers declare the absence of silages in the feeding as in dairy products. Certainly, it will require the construction of a robust database of certificated meat for the feeding system. Moreover, CPFA (mainly lactobacillic acid) were recently found [12] in farmed fish. Therefore, this approach could also be extended to fish to eventually distinguish farmed from wild fish.

Regarding omega-cyclohexyl fatty acids, they have been detected [11] (mainly 11-cyclohexyl undecanoic and 13-cyclohexyl tridecanoic fatty acids) in the GC-MS fatty profile of cow milk, suggesting their presence in milk fat could represent a reliable method to evidence rumen acidosis in cows. However, as mentioned above, this hypothesis has never been confirmed by experimental data.

Recently, they have been also identified in animal fat, mainly in meat of ruminants, especially bovine and ovine meat [14]. Preliminary data (not published) showed that omega-cyclohexyl fatty acids, combined with other fatty acids as branched chain fatty acids [41], permitted to discriminate beef from pork meat.


**43**

**Figure 5.**

*Cyclic Fatty Acids in Food: An Under-Investigated Class of Fatty Acids*

As suggested by Marseglia et al. [11], 11-cyclohexyl undecanoic fatty acid methyl ester from milk and meat fat eluted in the chromatographic region of isomers of the oleic acid, so it was detected by the characteristic molecular ion 282 m/z in the mass

13-cyclohexyl tridecanoic fatty acid methyl ester was detectable in the GC-MS profile as it elutes just before the eicosanoic acid (arachidic acid) and after eicosenoic acid. However, due to the presence of interfering signals, the identification of 13-cyclohexyl tridecanoic was confirmed by the mass spectrum with the characteristic molecular ion 310 m/z and the previous biosynthesized compound [11]. The characteristic mass spectra of 13-cyclohexyl tridecanoic fatty acid detected by

The results from the GC-MS analysis showed that omega-cyclohexyl fatty acids, both 11-cyclohexylundecanoic acid and 13-cyclohexyltridecanoic acid, were present only in bovine and ovine meat samples with values between 90–230 and 20–200 mg/kg of the total meat fat, respectively [14]. On the contrary, they were absent in pork, horse, chicken, and rabbit, reflecting the ruminal origin and a possible application for the detection of bovine/pork ratio in commercial

As mentioned above, current analytical methods in meat authentication are mainly based on protein or DNA measurement, which are not directly comparable to labeled meat expressed as percentage (w/w) [66]. Furthermore, analytical procedures based on protein analysis are sensitive to heat treatment. Therefore, they could not be applied to cooked products for the quantitative analysis. In this context, a quantitative GC-MS method is going to be developed on mixtures of beef and pork meat, both raw and cooked (ragout), based on the method previously applied to determine CPFA [43] and combining other fatty acids, as iso-branched

Preliminary results [14] showed that omega-cyclohexyl and iso-branched chain fatty acids content decreased in minced meat, both raw and cooked, as function of

Therefore, the analysis of omega-cyclohexyl fatty acids combined with that of specific iso-branched chain fatty acids was able to detect until 20% of pork meat in beef, representing potential markers for ruminant meat, also detectable in complex

*DOI: http://dx.doi.org/10.5772/intechopen.80500*

GC-MS analysis is shown in **Figure 5**.

chain fatty acids, of ruminal origins [41].

bovine meat percentage in the sample, as shown in **Table 4**.

matrix and after thermal treatment in ragout samples.

*Mass spectra of 13-cyclohexyl tridecanoic fatty acid methyl ester [11].*

spectrum.

minced meat [14].
