CONJUGATED LINOLEIC ACID-ENRICHED DAIRY PRODUCTS: A REVIEW

Conjugated linoleic acid (CLA) is a family of more than 28 isomers of linoleic acid wherein the isomers cis-9, trans-11 (rumenic acid) and trans-10, cis-12 are the most abundant. It is associated with a number of potential health benefits for human organism. Many foods are a good source of it but it is mosty found in meat and dairy products, derived from ruminants. Dairy products contain CLA in different amounts. The enrichment of these products with CLA is appropriate given the lower CLA content in these products in comparison with the recommended health intake. Modification of CLA concentration can be done by specific animal feeding and diet modification, by direct CLA supplementation in the form of oils or by addition of specific starter culture. The influence of technological treatments on the stability of the final product during storage and maturation is still not completely elucidated. There is a need for further studies on the physiological effects of CLA isomers on humans. The purpose of this review is to summarize the possibilities for increasing CLA content in milk and dairy products and to determine the possible effects of this enrichment on product stability – sensory, chemical, microbiological profile, shelf life and potential health effects of the obtained products.


INTRODUCTION
In recent years, there was an increased discussion about the role of milk fat and its components and their influence on human health. Milk fat is a natural source of high valuable biologically active compounds such as phospholipids, fatsoluble vitamins, short-chain fatty acids and conjugated linoleic acid (CLA) (Bhat and Bhat, 2011). It is believed that the positive effects of these milk fat components are in predominance if compared with the negative influence of saturated fatty acids on human health. Liesbeth et al. (2010) demonstrated that cis-9, trans-11 CLA, presented in significant amounts in the milk of pasture-grazed cows, might offset the adverse effect of the saturated fat content of dairy products. Further research on this dependence can be made in order to determine even more precisely the predominance of the positive health effects of the fatty acid profile of dairy products over the often wrongly alleged unhealthy ones. CLA is naturally formed in the rumen as an intermediate product in the digestion of dietary fat (Song and Kennelly, 2002). According to Meraz-Torres and Hernandez-Sanchez (2012), it is synthetized by two waysthe first is by incomplete biohydrogenation of linoleic and linolenic acids in the rumen by the endogenous bacteria and the second one is the conversion of transvaccenic acid to CLA in the animal tissues which represents 60-95% of the total CLA in the foods produced from animals. CLAs are conjugated dienes of linoleic acid (Fig.1). This name refers to a group of positional and geometric isomers of linoleic acid, characterised by a conjugated system of double bonds, separated by one single bond (National Centre for Biotechnology Information, 2020).

Figure 1 Chemical formulas of CLA isomers
There are 28 potential CLA isomers. The most common isomers are cis-9, trans-11, and trans-10, cis-12 of which rumenic acid (C18:2 cis-9, trans-11) is dominant in milk fat (Virsangbhai et al., 2020). Natural CLA content in milk and dairy products is presented in Table 1.

Markiewicz-Kęszycka et al. (2013)
reviewed the fatty acid profile of milk and found that sheep milk was the richest source of conjugated linoleic acid in comparison with cow and goat milk. This fact was confirmed by Prandini et al. (2011). Different factors, such as diet, production system, breed, or stage of lactation could influence on the CLA content in milk and dairy products respectively. Recent review (Ahmad et al., 2019) declared dairy products with natural CLA concentration ranging from 3.4 to 10.7 mg/g fat in milk. Dairy products originating from various producers and from various countries were characterized by similar contents of CLA and diversified contents of trans isomers of C18:1 and C18:2 acids (Paszczyk et al., 2012a) which impose the need of a broader analysis of similar products prepared under different conditions. It can be seen that in some types of dairy products, such as yoghurt, butter, ice cream, fresh cheeses and white pickeled cheeses, large deviations in the reported natural CLA content were found. This imposes on researchers the need to optimize methods of analysis, to seek comparability of results and is an opportunity for future research.The effect of initial fat content on the CLA content of the finished product must be investigated in more depth. The highest CLA content in all analysed cheeses was found in hard cheeses with long ageing time while white pickled cheeses, which were produced without ageing or with short ageing time, contained only low amounts of CLA. From the presented data it is evident that there is a need to study the influence of various factors on the natural content of CLA in dairy products. Collomb et al. (2006) summarized CLA variation and physiological effects on milk fat quality and quantity. According to Kirchnerová et al. (2012) it is not possible to influence some groups of desirable fatty acid, e.g. CLA, without the influence on other fatty acids. In this view, it is recommended that studies dedicated on the current topic provide an overall assessment of the fatty acid profile of the analysed products. Natural CLA concentrations are not sufficient to demonstrate specific health promoting effects. CLA enrichment of foods can cause a number of beneficial health effects on animal and on human organism. In recent scientific research, authors suggested that enrichment of dairy products with CLA may positively influence on human immune system stimulation, may Conjugated linoleic acid (CLA) is a family of more than 28 isomers of linoleic acid wherein the isomers cis-9, trans-11 (rumenic acid) and trans-10, cis-12 are the most abundant. It is associated with a number of potential health benefits for human organism. Many foods are a good source of it but it is mosty found in meat and dairy products, derived from ruminants. Dairy products contain CLA in different amounts. The enrichment of these products with CLA is appropriate given the lower CLA content in these products in comparison with the recommended health intake. Modification of CLA concentration can be done by specific animal feeding and diet modification, by direct CLA supplementation in the form of oils or by addition of specific starter culture. The influence of technological treatments on the stability of the final product during storage and maturation is still not completely elucidated. There is a need for further studies on the physiological effects of CLA isomers on humans. The purpose of this review is to summarize the possibilities for increasing CLA content in milk and dairy products and to determine the possible effects of this enrichment on product stabilitysensory, chemical, microbiological profile, shelf life and potential health effects of the obtained products. present antihypertensive, anticarcinogenic, and antiatherogenic effects and promotes the health benefits by body weight loss (Yang et al., 2015).  Frozen dairy products Fresh goat cheese 6.0 The aim of the present paper was to provide an overview of the CLA-enriched dairy products, to discuss potential health benefits of CLA-enriched dairy products and to identify future directions for research as well as applications. Fig. 2 represents the pathway of CLA from raw milk to dairy products in accordance with the applied process technology. suggested that organic or low-input dairy management was more likely to result in milk higher in beneficial isomers of CLA. This tendency was even more remarkable when producing fermented organic milks with significantly higher amounts of CLA than the same milk before fermentation (Florence et al., 2009). Grana Padano cheese was characterized by a higher CLA content when produced by organic farming system in comparison with the control sample produced by conventional system (Prandini et al., 2009a). The fat from the bio-yoghurts had the highest mean content of CLA (7.62 mg/g) (Paszczyk and Łuczyńska, 2020). The analysis of the considered data showed that the natural content of CLA was influenced by a number of factors. Further investigaions could be done in order to predict more precisely the estimated natural CLA content according to the diet, applied production system, breed type and stage of lactation.

Technological treatments
Heat treatments and homogenization of milk caused oxidation of valuable anticancer CLA through exposure to high temperatures, high pressures, and reduction of fat globule size (Norgauer, 2005). Application of high temperature short time pasteurisation (HTST) resulted in a significant decrease of the cis-9, trans-11 isomer and other minor CLA isomers (Campbell et al., 2003). These results were found in milk that was fortified with CLA at very high doses (30%), which resulted in a significant, but rather moderate loss of CLA (c.a. 10% loss). Refrigerated storage of HTST milk decreased the cis-9, trans-11 isomer concentration after 3 weeks (Campbell et al., 2003). Campbell et al. (2003) found no differences in lipid oxidation between milks with or without added CLA even the fact that fortified HTST milks were supplemented with vitamin E and rosemary extract to prevent lipid oxidation. Ruiz et al. (2016) found that milk processing significantly affected the transferring of CLA from raw milk into dairy products (sweetened condensed milk and powdered milk). Shantha et al.
(1995) found increased CLA concentration in salted butter (1.32 fold) in comparison with unsalted butter (1.27). They stated no changes in CLA content in processed dairy products such as low-fat yogurt, regular yogurt, low-fat and regular ice cream, sour cream or cheeses such as Mozzarella, Gouda and Cheddar. Storage did not affect CLA concentration in any products, suggesting that CLA is a stable component (Shantha et al., 1995). Dave et al. (2002) established no significant influence of milk processing (incorporation of milk powder and heat treatment at 85°C for 30 min, fermentation with yogurt and probiotic bacteria and storage) on CLA-concentrations of probiotic CLAenriched yoghurts. Garcia-Lopez et al. (1994) analysed changes of CLA content in processed cheese during processing and found CLA to increase from 9.5 mg/g of fat in the raw ingredients to 10.7 mg/g of fat in the finished product without apparent changes in the isomer distribution. The culinary utilization and processing did not change the rumenic acid content of Emmental cheese (Chamba et al., 2006). Avilez and Meyer (2014) assessed the effect of processing fresh milk into milk powder and condensed milk. They found 1.35 g CLA/100 g of fatty acids in fresh milk and 1.45 and 0.93 g/100 g fatty acids transferred to the milk powder and condensed respectively. As isomers, the cis-9, trans-11 had higher levels in the powdered milk than fresh milk in 7 months of the 10 months studied with values from 0.50 to 0.84 g/100 g fatty acids. These data were confirmed by Ruiz et al. (2016) who found that sweetened condensed milk presented lower CLA values than raw and powdered milk. No differences were found in the profiles of fatty acids in the fats of milk, sweet cream, and cream, which confirmed their stability during the centrifugation and pasteurisation of milk as well as during the acidification with starter cultures (Bonczar et al., 2016). 7-month cheese aging showed higher CLA content than 4 months-aged cheeses, independently of the type of feeding ( on CLA content in processed cheese food and processed cheese spread caused by mixing (reducing), homogenization and storage period, but heating at 90°C relative to 75°C significantly increased total CLA content in both cheeses. Zengin et al. (2011) studied the influence of milk pasteurization (between 70 and 90°C for 5 min) on its CLA-content in order to produce white pickled cheese and found no effect on CLA concentrations. CLA content in ghee prepared using indigenous method was higher as compared to that of a creamery method ( the factors involved in the cheese making process generally did not affect the CLA content in milk fat because they found no statistically significant differences in CLA concentration when cheeses were produced from the same ruminant species but through different production technologies. Milk processing and cheese aging had a negative effect on CLA concentration in Peccorino cheese (Prandini et al., 2004). The obtained results demonstrated that the applied technological treatment influenced CLA content in milk and dairy products.

Diet modification
Another possibility to enhance milk CLA content was to modify the animal's diet. Peşmen (2016) summarized the factors affecting CLA content enhancement of milk and dairy products and concluded that fish, sunflower, corn and canola oil had positive effects for this purpose. Oils were added into the diet in the form of protected oils, free oils, processed oilseeds or whole oilseeds (extruded, crushed, roasted or ground) (Virsangbhai et al., 2020). Gonzalez et al. (2003) altered the degree of unsaturation in milk fat by modifying the diets of Holstein cows in order to produce high-oleic and high-linoleic butter and ice cream. Cow and buffalo fed roughage based diet type one berseem (Trifolium alexandrinum) fodder along with wheat straw and type two concentrate mixtures and wheat straw (Tyagi et al., 2015). It was found that total CLA content in milk was higher in berseem fed groups of both the species. lignosulfonate and proved that the use of diets with pelleted sunflower seeds was possible to obtain butter with a desirable nutritional value in dairy products with 23.74mg/g total lipid rumenic acid content. Dairy cows were fed sunflower seeds and pasture in order to increase of conjugated linoleic acid and vaccenic acid in anhydrous milk fat using dry fractionation (Herrera-Meza et al., 2012). The use of linseeds was also applied in order to enrich in CLA ewe's milk (Luna et al.,  2005). The supplementation of elephant grass-based-diets with soybean oil increased CLA content linearly as the level of soybean oil in the diet increased (Lopes et al., 2019). The butter produced from afternoon milk had a lower content of C16:0 and a higher content of cis-9 C18:1 (P < 0.05). Soybean oil and, to a small extent, the selective segregation of milk obtained from afternoon milking sessions are strategies that can be used to improve the fatty acid composition of butterfat. Different approaches can be applied in order to increase CLA in milk and dairy products. New sources can be used to achieve even higher CLA content. prepared milk protein-based conjugated linoleic acid microcapsules and investigated the effect of casein micellar structure on their stability. The authors found adverse effects on the casein micellar structure which suggested a modification of casein micelles in order to improve the efficiency and chemical stability of such microcapsules.

Addition of specific starter culture
The addition of specific starter culture with or without specific substrate addition can be considered as a promising possibility to increase CLA levels in dairy products. The fermentation using microorganisms having linoleate isomerase activity was employed as a promisig technological alternative for the manufacture of fermented milk products with high content of CLA (Gutiérrez, 2016 al., 2012). CLA-content of cheeses ranged from 3.52 to 3.92 mg/g and probiotic differences and storage process had not affected the CLA contents of the samples statistically (Gursoy et al., 2012). A study demonstrated that the type of applied starter culture (Ceska-star Y508 culture) and storage time affected the content of CLA in fermented milk drinks (Paszczyk et al., 2016). The mean content of CLA in fresh drinks reached 3.60 mg/g fat, after 6 days 3.85 g/g fat and after 13 day storage 3.89 mg/g, respectively. Renes et al.

DAIRY PRODUCTS ENRICHED IN CLA
Bell and Kennelly (2011) summarized the potential of "designer milk" for the production of dairy products. According to Meraz-Torres and Hernandez-Sanchez (2012) all dairy products were a source of CLA and can be considered as functional foods. CLA-enriched fresh, ripened and other dairy products were developed in order to enhance their nutritional and biological values. The need, the interest and the possibilities to produce dairy products from CLA-enriched milk are reviewed below.

CLA-enriched fresh dairy products
The effects of added conjugated linoleic acid (CLA) (1 and 2% in the form of triglyceride oil) on the sensory, chemical, and physical characteristics of 2% total fat fluid milk were studied by Campbell et al. (2003). They established lower consumer acceptability for CLA-fortified milks compared to control milks, but the addition of chocolate flavour increased acceptability. Similar results were obtained by Jones et al. (2005) for CLA-enriched ultra-high temperature (UHT) treated milk, which sensory profile differed slightly from those of the control sample. Although the sensory profiles of the CLA-enriched milk and fresh cheese differed from those of the control product with respect to some attributes, the overall impression and flavour did not differ. Rodríguez-Alcalá and Fontecha (2007) evaluated the changes of CLA isomers composition in fresh dairy products (milk, fermented milk, yoghurt, fresh cheese, milk-juice blend) after processing and storage. They detected a significant decrease of total CLA in fresh cheese samples after 10 weeks of refrigerated storage, but not in CLA isomers C18:2 cis-9, trans-11 and C18:2 trans-10, cis-12. In the same research, the disappearance of some of the minor CLA isomers and a significant increase of C18:2 trans, trans isomers from cis, trans; trans, cis; and cis, cis in fermented milk, yogurt, and milk-juice blend were established. Gutiérrez (2016) revised the effects of technological treatments on CLA content variations in milk and fermented milks. Some substrates (maltodextrine and fructooligosaccharides) in combination with probiotic starter cultures showed to be an effective method for enhancing the concentration of CLA in fermented milks (Akalın et al., 2007). In contrary, it has been reported that the addition of sucrose, fructose and lactose (60 g/L) led to an inhibition of the CLA production in yogurts elaborated using Lactobacillus acidophilus and Lactobacillus delbrueckii ssp. bulgaricus (Lin, 2000).

Other CLA-enriched dairy products
A recent study evaluated the effects of three widely practiced cows feeding systems in the United States, Europe, and Southern Hemisphere regions on the characteristics, quality, and consumer perception of sweet cream butter (O'Callaghan et al., 2016a). It was found a significantly higher concentration of conjugated linoleic acid (cis-9, trans-11) in butter produced from pasture-derived milks than TMR butter. They observed alterations in the fatty acid composition of butter contributed to significant differences in textural and thermal properties of the butter. Few researches about ice cream CLA-fortification were published (Gonzalez et al., 2003). They found that control ice cream mixture had higher viscosity compared with high-oleic and high-linoleic, but the firmness of all ice creams was similar when measured between -17 and -13°C. Enriched anhydrous milk fat (AMF) and fractions (at 25, 20, 15, 10, 5, 0 and -5°C) showed CLA increase by 31% in enriched AMF and 59% after fractionation (liquid fraction at 0°C) when compared to control samples (Herrera-Meza et al., 2012). According to the authors this new technology, called "dry fractionation", was an inexpensive chemical-free process that may be used to produce CLA-rich products such as butter, cream, cookies and bread, in a way that would not be possible with conventional dairy processing equipment. have found that feeding butter with elevated content of trans-10, cis-12 conjugated linoleic acid to lean rats did not impair glucose tolerance or muscle insulin response.

CONCLUSION
The most recent data concerning CLA-enriched dairy products were presented in this review. While there are a lot of data for the possibilities to enrich dairy products with CLA, this area needs further investigations on some issues. Further studies are needed to determine the most appropriate group of dairy products for CLA enrichment. Additional studies should be performed to determine new antioxidants, their respective concentrations and methods for prevention of CLA oxidation. CLA content variations of different dairy products enriched with CLA remain still not completely clear. It is still unknown why some strains of microorganisms produce greater amounts of CLA than others. The influence of the substrate in CLA formation must be further clarified. There is a need of more recent findings in the physiological effects of nutritionally desirable CLA and its isomers, presented in dairy enriched products, on human organism. There is a need of improved knowledge about the improvement of milk fat composition through various factors as feeding regime, production system, breed, or stage of lactation. All of these concerns are in correspondence with the requirements of the growing market of functional foods.