LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY. Faculty of Veterinary Medicine. Saba Fatima. Title LT MASTER THESIS.

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1 LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY Faculty of Veterinary Medicine Saba Fatima Title LT Application of Functional Additives for Quality Improvement of Fish Preparations and Heat Treated Products Funkcionaliųjų priedų panaudojimas žuvies pusgaminių ir termiškai apdorotų gaminių kokybės gerinimui MASTER THESIS Food Sciences Supervisor: Prof. Dr. Gintare Zaborskiene KAUNAS, 2020

2 THE WORK WAS COMPLETED IN THE DEPARTMENT OF FOOD SAFETY AND QUALITY CONFIRMATION OF THE AUTHENTICITY OF THE WORK COMPLETED I confirm that the Master Thesis Application of Functional Additives for Quality Improvement of Fish Preparation and Heat Treated Products. 1. Has been completed by me (myself). 2. Was not presented in any other Lithuanian of foreign University. 3. I have not used any other references than indicated in the work and I cite the complete list of used references. I sign by message from my personal mailbox, and the Thesis will be wet signed shortly after the end of quarantine and emergency period due to the COVID-19 pandemic in the Republic of Lithuania. Saba Fatima (date) (author s First and Last name) (signature) CONFIRMATION OF THE PROOF OF ENGLISH LANGUAGE IN THE WORK I confirm the work was reviewed/edited by the English language editing service. I sign by message from my personal mailbox, and the Thesis will be wet signed shortly after the end of quarantine and emergency period due to the COVID-19 pandemic in the Republic of Lithuania. Mohd. Rashid (date) (editor s First and Last name ) ( signature) SUPERVISOR S CONCLUSION REGARDING THE DEFENSE OF THE MASTER THESIS I sign by message from my personal mailbox, and the Thesis will be wet signed shortly after the end of quarantine and emergency period due to the COVID-19 pandemic in the Republic of Lithuania. Gintare Zaborskiene (date) (supervisor s First and Last name) (signature) THE MASTER THESIS HAS BEEN APPROVED IN DEPARTMENT 2

3 I sign by message from my personal mailbox, and the Thesis will be wet signed shortly after the end of quarantine and emergency period due to the COVID-19 pandemic in the Republic of Lithuania. (date of approval) Mindaugas Malakauskas (First and Last name of the Head of Department/institute) (signature) The Reviewers of the Master Thesis I sign by message from my personal mailbox, and the Thesis will be wet signed shortly after the end of quarantine and emergency period due to the COVID-19 pandemic in the Republic of Lithuania. 1) 2) (First and Last name) (signatures) The evaluation of the Defense Commission of Master Thesis: (date) First and Last name of the secretary of the Defense Commission (signature) 3

4 CONTENTS SUMMARY... 6 Santrauka... 7 ABBREVIATIONS... 8 INTRODUCTION LITERATURE REVIEW Nutrition parameters of fish meat Fish products and quality parameters Extracts of plants suitable for fish products Types of fatty acids in a fish METHODOLOGY Study design and research objective Material and methods Material Physical and chemical analysis Sensory analysis Statistical analysis methods RESULT The antioxidant effect of rosemary and pomegranate extracts on fish balls Influence of plant extracts on fatty acids composition of the fish balls Groups of fatty acids Composition of fatty acids Omega-3 and Omega6 fatty acids The influence of plant extracts on texture, viscosity and shear stress of fish ball samples Sensory properties analysis and acceptability of heat treated fish balls DISCUSSION OF RESULTS CONCLUSION ACKNOWLEDGEMENT

5 REFERENCES APPENDIXES

6 SUMMARY Application of Functional Additives for Quality Improvement of Fish Preparation and Heat Treated Products Saba Fatima Master Thesis The aim of this thesis is to applicate functional additives (rosemary and pomegranate) in order to improve the functionality, texture, viscosity and Shear Stress of semi-finished fish balls, analysis of sensory properties and acceptability of heat-treated fish balls and determine the correlation relations between the analyzed physio-chemical quality parameters. The preparation of fish balls were performed in the lab. of Food Safety and Quality Department of Veterinary Academy at the Lithuanian University of Health Sciences (LUHS). In total, Salmon fish were divided into 3 fish samples (control sample, control with rosemary extract, control with pomegranate extract) according to the recipe and made fish balls. Samples were examined by physical chemical and sensory methods: tested on acid and peroxide values, fatty acids content, texture, rheological and sensory properties. Statistical analysis was performed by Excel program. Keeping the fish samples in the freezer for 3 months were tested in fat hydrolysis and oxidation, tests were repeated 3 times, results in the acid value, peroxide and sensory analysis were significantly different. Control minced fish meat samples have adequate results after keeping it in refrigerator for 3 months, rosemary and pomegranate samples showed edible results. Rosemary and pomegranate extracts displayed superior antioxidant activity and slowed down oxidation process of stored fish balls samples. Quality and shelf life of fish balls were enhanced by using additives during processing and storage. Potential effects of rosemary and pomegranate extracts had delayed lipid oxidation, improved textural properties. In addition, additives performed massively in the result of sensory analysis. Adequate performance of rosemary and pomegranate extracts in the result of intensity of total taste and odor, extraneous, residual, sour, cooked fish taste, fatness, chewing, juiciness, hardness in the mouth, intensity of sectional color. Key words: rosemary extract, pomegranate extract, fish balls, sensory analysis. 6

7 Santrauka Funkcionaliųjų priedų panaudojimas žuvies pusgaminių ir termiškai apdorotų Saba Fatima gaminių kokybės gerinimui Baigiamasis magistro darbas Šio darbo tikslas - pritaikyti funkcinius priedus (rozmarino ir granatų ekstraktus), siekiant pagerinti žuvies pusgaminių ir virtinukų funkcionalumą, fizikinius cheminius rodiklius, juslines savybes ir priimtinumą. Žuvų pusgaminių ir virtinukų paruošimas buvo atliekamas Lietuvos sveikatos mokslų universiteto (LSMU) Veterinarijos akademijoje. Maisto saugos ir kokybės katedros laboratorijoje. Lašišos žuvų smulkinta mėsa pagal receptą buvo padalyta į 3 mėginius (kontrolinį, kontrolinį su rozmarino ekstraktu, kontrolinį su granatų ekstraktu) ir pagamintas faršas bei žuvies virtinukai. Mėginiuose tirta riebalinės fazės rūgščių ir peroksidų skaičius, tekstūra, atliktas reologinių, juslinių savybių testas, atlikta gautų duomenų statistinė analizė. Žuvų faršo mėginiai po 3 mėnesių laikymo šaldiklyje dar kartą buvo tirti dėl riebalų oksidacijos ir hidrolizės, tyrimus kartojome tris kartus. Nustatytas rūgščių ir peroksidų skaičius, juslinės savybės mėginiuose skyrėsi žymiai. Šviežios žuvies faršo mėginiai su rozmarinų ir granatų ekstraktais, buvę 3 mėnesius šaldytuve, buvo priimtini, kitaip negu kontroliniai mėginiai. Rozmarinų ir granatų ekstraktai pasižymėjo antioksidaciniu veikimu ir sulėtino žuvies faršo ir virtinukų oksidacijos procesą. Žuvų virtinukų kokybė ir vartojimo laikas buvo pagerintas gamybos, saugojimo metu, naudojant priedus. Dėl rozmarino ir granatų ekstraktų poveikio sulėtėjo lipidų oksidacija, pagerėjo tekstūra ir reologinės savybės. Be to, mėginių su funkcionaliaisiais priedais atlikti juslinių savybių rezultatai buvo labai geri. Rozmarinų ir granatų ekstraktai turėjo teigiamos įtakos virtinukų bendram skonio ir kvapo intensyvumui, pašaliniam, likutiniam rūgščiam skoniui, virtos žuvies skoniui, riebalingumui, susikramtymui, sultingumui. Raktažodžiai: rozmarino ekstraktas, granatų ekstraktas, žuvies virtinukai, juslinė analizė. 7

8 ABBREVIATIONS EPA- Eicosapentaenoic acid DHA- docosahexaenoic acid SFA-saturated fattyacid MUFA monounsaturated fattyacid PUFA- polyunsaturated fatty acid TVB-N -total volatile base-nitrogen C18:1n-9 - oleic acid AV- acid value POV-Peroxide value FA- fattyacid FAME- FA methyl esters MV-mean value SD-standard deviation TA- texture analysis WHO- World Health Organisation LUHS- Lithuanian University of Health Sciences FIG - figure 8

9 INTRODUCTION Fish is a good source of first class protein and lipid that contains omega-3 fatty acids, especially, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Interestingly, fish is also a good source of various vitamins (A, D, B6, B12, etc.) and minerals such as iron, zinc, iodine, selenium, potassium, sodium, etc [1]. Fish is one of the most valuable commodities among various agricultural products. In developing countries, exported fishes are one of the major sources of foreign revenue. It is not only treated as a food item but also considered as the traditional part while felicitating the guests. Dried fishes are also preferred by many people. Presently fish and fisheries sector contribute 58% of total animal protein intake, 3.78% to GDP, 22.23% to agricultural production and 2.70% of foreign export earning of the nation Bangladesh is the 5th largest inland fisheries producer in the world The present per capita fish consumption is about kg of fish per head per year where as kg of fish is the standard required amount [1]. Fish pond aquaculture has a long tradition in Lithuania. The majority of Lithuanian ponds are used to rear carp which accounts for the largest part of pond aquaculture production and is highly valued among Lithuanian freshwater fish. The main production systems in Lithuania are represented by warmwater and cold water fish ponds. Among the main cultured species, the most important is the common carp (Cyprinus carpio ) which is not an endemic species in Lithuania [2][3]. The rivers of the eastern Baltic region are important for anadromous salmonid reproduction, and they contribute significantly to the wild stocks of sea trout and Atlantic salmon in the Baltic. Many natural, self-reproducing sea trout (stream-dwelling and ana-dromous) and some Baltic salmon populations occur in Lithuanian rivers. The majority of natural salmon populations inhabit the southeastern and eastern coasts of the Baltic Sea in Russia, Estonia, Latvia, and Lithuania (ICES, 2010) [3]. Salmon and sea trout smolts have been well studied and documented in the western part of the Baltic Sea region[4][5]. Salmon is popular as an important aqua cultured freshwater species as well as a food throughout European region [5]. It inhabits fast flowing streams and rivers. Different value added products can contribute to the preservation of fish as well as making a difference in the taste. Hence people can be able to enjoy the same product in different forms and different tastes. Among various value added products, fish ball is the most common and tasty one. Currently consumers particularly urban ones are showing more and more interest in food products which are available in ready-to-eat or ready-to-cook 9

10 form such as fish fillet, finger, cutlet, patties, burger, sausages and fish balls. Present trends of marketing reflect a rapidly growing demand for such processed foods that are more convenient to handle, store and prepare. Factors responsible for such a situation are increasing number of working women, reasonably increase in income, education, awareness and consciousness towards quality, freshness, nutrition, hygiene and health etc. This has led to the development of several fishery products varied in taste, texture and appearance [6]. Different value added products can contribute to the preservation of fish as well as making a difference in the taste. Hence people can be able to enjoy the same product in different forms and different tastes. Among various value added products, fish ball is the most common and tasty one. The purposes of this study were to analyze the proximate composition of raw and dried mrigal fish, raw and dried potato, and to develop fish ball, powder mixture for fish ball and to assess the storage stability and consumer s acceptability of the developed spices mixture product for fish ball [6][7][8]. Omega-3 fatty acids, which are abounding in fish oil, improve the prognosis of several chronic inflammatory diseases although the mechanism for such effects remains unclear. Oxidation is normal process that happens with all fats and oils that contains polyunsaturated fatty acids. In this work using rosemary and pomegranate extracts additives could be reduced oxidation process of omega-3 and controled the consumption time period. AIM OF THE WORK: To applicate functional additives (rosemarry and pomegranate) in order to improve the functionality, physico-chemical quality parameters, sensory properties and acseptability of preparations and heat treated fish balls. OBJECTIVE OF THE WORK: 1. To determine the antioxidant effect of the chosen functional additives - rosemary and pomegranate extracts - on fats quality of semi-finished fish balls during 3 months storage in the freezer; 2. To perform the analysis of texture, viscosity and Shear Stress of semi-finished fish balls; 3. To perform the analysis of sensory properties, acceptability of heat-treated fish balls; 4. To perform the statistical comparative analysis of the obtained results, to determine the correlation relations between the analyzed physio-chemical quality parameters; 10

11 1. LITERATURE REVIEW The global fish supply obtained from both aquaculture and captured fish is increasing day by day. This increase of seafood consumption is likely to lead to improved health. Along with this trend, people want to consume new aquatic species or new seafood products which can be processed in various ways such as fresh, frozen, dried, marinated and coated products (croquettes). Among these processing technologies, the coating technique is one of the leading methods of producing new seafood products. Coating technology can be used to produce food products using non-economic fish or bycatch fish species as well as aquatic species with a high economic value like shrimp and scallop. Fish croquettes are a type of small breaded food produced by mixing minced fish meat and various ingredients and are usually consumed after being deep-fried. The deep-frying is one of the methods of frying most favored by consumers and adds desirable flavor and textural properties to food. Moreover, fishes are rich in amino acids and unsaturated fatty acids especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) [9]. Therefore, the evaluation of sensorial properties of fish croquettes is important EPA and DHA are mostly provided to the humans by the diet, being marine foods (fish, microalgae and some crustaceans) the most important sources. EPA and DHA are mostly provided to the humans by the diet, being marine foods (fish, microalgae and some crustaceans) the most important sources [9]. Table No. 1 describes the sensory appriciation test, quantitative descriptive analysis and their definitions. Table No.1 description and definition of sensory anf quantitative analysis. Descriptior Sensory appreciation test Flavour Odor Texture Appearance General appreciation Quantitative descriptive analysis Saltiness Spiciness Color Hardness Definition general flavor of deep fried fieshballs general odor of deep fried fish balls structure of fish balls is round colour and general look of deep fried fish balls general acceptance considering all parameters density of salt aromatics associated with spices or other ingredients outside color of fried fishballs from light to dark 11

12 Oiliness Frying force required to compress the sample I first bite absorbed oil associated with frying oil frying rate of fishball Fish ball in curry (FBC) was prepared following standard recipe. Prepared FBC was packed in standup pouches and stored at 0 to -2 C. The organoleptic scores for overall acceptability of the FBC were slightly decreased within the storage period (12 days). The ph decreased from 6.38 to 6.07, while peroxide value increased from 2.27 to The total volatile base-nitrogen (TVB-N) increased from 5.04 to The total plate count increased from an initial value of 1.8 x 102 to 2.4 x 104 cfu g-1 during chilled storage. The Staphylcoccus aureus, Escherichia coli and Salmonella sp. were not detected throughout the study period [10]. It was observed that the products stored at 0 to -2 C were acceptable organoleptically, and stable biochemically and microbiologically up to 9 days. Fish and seafood are important sources of protein which provide 40% of the world s protein intake in addition to essential micronutrients which have various health benefits [11][12]. 1.1 Nutrition parameters of fish meat In 2013, global marine fish production reached more than 2.2 million tons, which represents about half the production of diadromous fish and about 6% of freshwater fish production.. Marine fish are mainly produced in Asia (83.1%), followed by Europe (9.2%) and Africa (7.1%). Asia is also the main producer of diadromous fish (39.0%); yet the production is more evenly distributed in the world, with Europe (37.8%) and the Americas (21.9%). For freshwater fish, almost the entire production (93.8%) is realized in Asia [13]. The top ten marine farmed species in 2013 totaled 86.5% of the global production, which was 1,241,149 tons (when excluding groups not identified at the species level). These species are almost exclusively produced in Asia, except for the two leading species, gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax), for which about half of the production is in Europe (Figure 1). 12

13 Fig. No.1. Aquaculture production of the top ten farmed marine species. Gilthead seabream (GS); European seabass (ES); Japanese amberjack (JA); Japanese seabass (JS), subnose pompano (SP); large yellow croaker (LYC); turbot (T); red drum (RD); silver seabream (SS) and cobia (C) Except for few species, such as the common carp (Cyprinus carpio) and the goldfish (Carassius auratus), the bulk of farming has started in the past century [14][15][16]. Most fish species farmed today are not much different from their wild conspecifics. It is estimated that 90% of the global aquaculture industry is based on wild, undomesticated or non-selectively bred stocks [17][18]. Fish represents a valuable source of proteins and other nutrients in the diet of many countries. As with many animal products, fish and fishery products contain water, proteins and other nitrogenous compounds, lipids, carbohydrates, minerals and vitamins. However, the chemical composition of fish varies greatly from one species and one individual fish to another depending on age, sex, environment and season. Proteins and lipids are the major components whereas carbohydrates are detected at very limited levels (less than 0.5 percent) [19]. In human nutrition, fatty acids such as linoleic and linolenic acid are considered essential as they cannot be synthesised by the organism. In marine fish, these fatty acids constitute only around two percent of the total lipids - a small percentage compared with many vegetable oils. However, fish oils contain other "essential" polyunsaturated fatty acids which act in the same way as linoleic and arachidonic acids. As members of the linolenic acid family (first double bond in the third position, n-3 counted from the terminal methyl group), they also have neurological benefits in growing children. One of these fatty acids, eicosapentaenoic (C20:5n-3), has attracted considerable attention since Danish scientists found a significant presence of it in the diet of a group of Greenland Eskimos who proved 13

14 virtually free from arteriosclerosis. Convincing evidence exists now for the significant role fish and fish oils in decreasing the risk of developing cardiovascular diseases and in improving foetal brain development. Proteins of fish comprise structural proteins (actin, myosin, tropomyosin and actomyosin), sarcoplasmic proteins (myoalbumin, globulin and enzymes) and connective tissue proteins (collagen) [19]. Fish proteins contain all the essential amino acids and, like milk, eggs and mammalian meat proteins, have a very high biological value. In addition, fish proteins are an excellent source of lysine, methionine and cysteine, and can significantly raise the value of cereal-based diets, which are poor in these essential amino acids. Also, fish meat is generally a good source of the B vitamins and, in the case of fatty species, of A and D vitamins. Vitamins can be divided into two groups, those that are soluble in fat, such as vitamins A, D, E and K, and those that are soluble in water, such as vitamins B and C [20]. Table no.2 explains the polyunsaturated fatty acids in the flesh of some fish species. Table No.2.Polyunsaturated fatty acids in the flesh of some of fish species found in Hungary (from Csengeri I, 2008) 14

15 1.2 Fish products and quality parameters Ensuring the quality of food, particularly seafood has increasingly become an important issue nowadays. Quality Management Systems empower any organization to identify, measure, control and improve the quality of the products manufactured that will eventually lead to improved business performance. With the advent of new technologies, now intelligent systems are being developed. Quality and shelf life of fish and fish products are often enhanced by using various food additives during handling, processing and storage. Due to potential health hazards, synthetic additives are being widely replaced by their natural counter parts. Extracts containing bioactive compounds isolated from various plant sources viz., spices and herbs, fruit and vegetables, seaweed etc. have shown remarkable in-vitro antioxidant and antimicrobial activities. Based on this, successful application of plant extract treatments have been carried out on chilled, frozen and dried whole fish, fillet as well as fish mince as antioxidant or antibacterial agent. Use of plant extracts also demonstrated to have potential for replacing sulphating agents in crustaceans [21]. 1.3 Extracts of plants suitable for fish products Herbs and spices provide our foods with a wonderful array of flavors, fragrances, and colors. Polyphenolic compounds present in plant extracts demonstrate potential antioxidant properties due to their redox potential; that enable them to act as hydrogen donors, reducing agents, nascent oxygen quenchers, and chelating metal ions in numerous food applications [22]. Rosemary (Rosmarinus officinalis L.), is a popular herb belonging to the Lamiaceae family with high antioxidant activity. Rosemary extracts have known to exhibit a high antioxidant activity and are widely applied in the food industry. The antioxidative properties of rosemary are mainly related to their content of phenolic compounds, which quench free radicals by hydrogen donation [23]. Rosemary (Rosemaria officinalis), belonging to the Lamiaceae family, is well known for its antioxidative properties, and is also used in several pharmaceutical applications. Biologically, rosemary extract improved feed conversion, efficiency of broilers fed diet supplemented with such herb. Cagıltay et al. (2013) reported that parallel to the concentration of rosemary in fed has decreased crude fat and increased amount of crude protein. Also, there have been a few studies on the antioxidative effect of rosemary (Rosmaria officinalis) in fish [24]. Common garden thyme, Thymus vulgaris, is a flowering perennial herb in the Lamiaceae family native throughout the western Mediterranean and into southern 15

16 Italy. Selmi & Sadok (2008) evaluated the combinedeffects of powdered thyme (Thymus vulgaris) sprinkling (0.1% w/w) and vacuum packaging on thequality of tuna (Thunnus thynnus) during storage at0 C and found that thyme treatment minimized auto-oxidation of lipids as evident from lowertbars and higher PUFA values when compared to control [25]. Incorporation of plant extracts into ice was also found useful for inhibiting the microbial and biochemical spoilage of fresh fish compared to conventional icing. Oral et al. (2008) investigated the preservative effects of ice incorporated with wild thyme hydrosol against the spoilage of a freshwater barb and found that this modification in icing system extended its shelf life from 15 to 20 days. In a similar way, Quitral et al. (2009) investigated the effects of ice prepared from rosemary and oregano (Origanum vulgare) extracts on the chemical changes in Chilean jack mackerel during storage and found that the plant extract icing system has delayed the chemical changes compared to traditional ice. In another study, icing with % rosemary extract improved the sensory and chemical quality parameters and extended the shelf life of sardine by 3 days than that stored in traditional ice [26][27]. Shi et al. (2014) demonstrated that clove (Syzygium aromaticum) bud extracts and grape (Vitus vinifera) seed extracts have efficiently inhibited lipid and protein oxidation in silver carp fillets during chilled storage [28][29].Viji et al. (2015) demon-strated that citrus (Citrus aurantium) peel extract hasgood antioxidant and antimicrobial activities andthe shelf life of Indian mackerel by 2 days comparedto control by delaying the spoilage mechanismswhen stored at -2oC. Zaeri et al. (2015) applied acombination of citrus and pomegranate peel extractand chitosan nanoparticles for shelf life extension ofsilver carp fillets. Pomegranate peel extract wassignificantly stronger than orange extract in delay-ing lipid oxidation [30].Yerlikaya & Gokoglu (2010) investigated the positive effects of green tea, grape seed and pomegranate peel extract dip treatment (1% w/v) on the sensory and physical properties of frozen bonito fillets and reported a marked reduction in muscle structure deformation during frozen storage [30]. 1.4 Types of fatty acids in a fish Dietary fat is an essential nutrient providing energy, essential fatty acids and fat-soluble vitamins. Whilst adequate amounts of dietary fat are readily provided by the Western-type diet, the optimal fatty acid composition of the diet is an important factor which can play a role in disease prevention and promote health [31]. The Expert Consultation recognises that grouping of fatty acids into these three broad groups (SFA, MUFA and PUFA) is based on chemical classifications, but it is clear that individual fatty acids within these groups have distinct biological properties. However, most of the epidemiological evidence 16

17 reviewed by the experts uses broad groupings, which makes it difficult to distinguish and disentangle the effects of individual fatty acids [33]. SFA refers to the major SFA in our diet, namely C14, C16, C18, except in the case of milk and coconut oil where SFA range from C4 to C18. MUFA refers to the major monounsaturated fatty acid in Western diets, which is oleic acid (C18:1n-9). It should be recognized that in some populations, a major monounsaturated fatty acid is erucic acid (C22:1n-9), as for example, found in culinary oils derived from some Brassica spp. such as rapeseed and mustard seed. PUFA refers to the major PUFA in our diet, which includes mainly linoleic acid (C18:2n-6), a lower proportion of alpha-linolenic acid (C18:3n-3), and depending on seafood intake a variable but relatively low proportion of long chain PUFA such as AA, EPA, DPA and DHA [33]. For the purposes of food labelling, the terms EFA, PUFA, long chain PUFA, n-6 and n-3 lack precision and should not be used without fully specifying the actual fatty acids and their amounts [34]. Omega 3 fatty acids. Fish oils can be obtained from eating fish or by taking supplements. Fish that are especially rich in the beneficial oils known as omega-3 fatty acids include mackerel, tuna, salmon, sturgeon, mullet, bluefish, anchovy, sardines, herring, trout, and menhaden. Fish oil supplements are usually made from mackerel, herring, tuna, halibut, salmon, cod liver, whale blubber, or seal blubber. Fish oil supplements often contain small amounts of vitamin E to prevent spoilage. They might also be combined with calcium, iron, or vitamins A, B1, B2, B3, C, ord. Fish oil is used for a wide range of conditions. There are two families of essential fatty acids, the omega- 6 and omega-3 polyunsaturated fatty acids (PUFA) [34]. Intake of Omega-3 Fatty Acids. A number of countries (Canada, Sweden, United Kingdom, Australia, Japan) as well as the World Health Organization and North Atlantic Treaty Organisation have made formal population-based dietary recommendations for omega-3 fatty acids. Typical recommendations are 0.3 to 0.5 g/d of EPA+DHA and 0.8 to 1.1 g/d of - linolenic acid. Recently, the Food and Nutrition Board, Institute of Medicine, and The National Academies, in collaboration with Health Canada, released the Dietary Reference Intakes for Energy and Macronutrients. The Acceptable Macronutrient Distribution Range (AMDR) for -linolenic acid is estimated to be 0.6% to 1.2% of energy, or 1.3 to 2.7 g/d on the basis of a 2000-calorie diet. This is 10 times the current intake of EPA+DHA [35]. Omega -3 is effective for: Lowering fats called triglycerides. High triglycerides are associated with heart disease and untreated diabetes. To reduce the risk of heart disease, doctors believe it is important to keep triglycerides below a certain level. Now researchers believe that fish oil, though not as effective as gemfibrozil, can 17

18 reduce triglyceride levels by 20% to 50%. One particular fish oil supplement called Lovaza has been approved by the FDA to lower triglycerides. Lovaza contains 465 milligrams of EP and 375 milligrams of DHA in 1-gram capsules; Preventing heart disease and heart attacks. Fish oil may be effective in keeping people with healthy hearts free of heart disease. People who already have heart disease may be able to lower their risk of dying from heart disease by taking fish oil. Omega-6 Fatty acids. The important ω-6 fatty acids and their source: Gamma-linolenic acid, 18:3 (Δ 6, 9, 12)- It is present in evening primrose oil (7 10 %) (Hoy et al. 1983), black current (15 20 g/100 g), borage (18 26 g/100 g) (Lawson and Hughes 1988). It is also present in small amounts in organ meats and in human milk (Horrobin 1990); Di homo-gamma-linolenic acid, C 20:3 (Δ 8, 11, 14)- It is synthesized from GLA; Arachidonic acid, C 20:4 (Δ 5, 8, 11, 14)- It is found in meat, eggs and dairy products [35][36][37]. 18

19 2. METHODOLOGY 2.1 Study design and research objective The research of the final work was performed at the Lithuanian University of Health Sciences, Veterinary Academy, Faculty of Veterinary Medicine, Department of Food Safety and Quality in the period from 10/06/2019 to 12/06/ Material and methods Material The object of the research. 1 kg of freezed salmon meat purchased from supermarket X - the meat was minced in an electric meat grinder Meissner SR 130/2 (Germany, 380 V), divided into 330 g and placed in 3 separate bowls. On 3 different fish minced recipes were studied with physicochemical parameters. The recipes were created by adding a functional additive to the minced fish pomegranate and rosmarry extracts. The control mince was without functional additives. Minced fish was divided into 3 groups of samples: Group 1 control - 330g minced fish + 20ml water+chrushed carrot 11.11g+fine chopped onion 35.45g+ garlic powder 0.397g, blackpaper 0.72g., ginger powder 0.20g., salt 1.19g.+egg g only white part; Group 2-330g minced fish + 20ml water+rosemarry extract 3.380g +chrushed carrot 11.11g+ fine chopped onion 35.45g+ garlic powder 0.397g+blackpaper 0.72g+ginger powder 0.20g+salt 1.19g.+egg g only white part Group 3-330g minced fish + 20ml water+pomegranate extract 2.37g +chrushed carrot 11.11g+ fine chopped onion 35.45g+ garlic powder 0.397g +blackpaper 0.72g+ginger powder 0.20g+salt 1.19g.+egg g only white part. All ingredients were mixed by hand (about ± 3 min.) Formed fish balls samples were divided in to 2 parts: 1 st part was analyzed for acid and peroxide values, texture, rheological parameters, fatty acid composition. Boiled fish balls were analyzed for sensory properties, acseptability. 2 nd part was transferred to freezer -18 degree Centigrate for storage 3 months. After storage fish balls were analysed only for acid value, peroxide value. 19

20 2.2.2 Physical and chemical analysis Acid value (AV) of the tested samples was used to determine the degree of hydrolytic rancidity of fish ball lipids. AV of the extracted lipids was determined according to EN ISO 660: procedure. One gram of extracted lipids were dissolved in 50 ml neutral solvent solution (diethyl ether (50 ml), ethyl alcohol (50 ml), and 1 ml 1% phenolphthalein solution). The acid number is determined from the volume of 0,1 N KOH solution used to titrate the free fatty acids in the molten and in the extracted fats of ethyl alcohol / ether mixtures, calculated by the coefficient (5,6I). The titration was carried out at constant shaking until the formed pink color persisted for 15 s. Peroxide value (POV) of the studied lipids is determined by standard (EN ISO 3960:2010) iodometric method and was presented as meqv O2/kg lipids. About 3.0 g fat was mixed with 50 ml of the solvent mixture (glacial acetic acid:chloroform 3:2), 1 ml freshly prepared saturated potassium iodide solution, and 100 ml water. The titratation was carried out with 0.01 mol/l sodium thiosulfate solution, using 1 ml starch solution and 0.1 g of Thyodene indicator until the blue color disappear. Fatty acids (FA) content, the amount of FA was determined by the method of gas chromatography using flame ionization detector. For the analysis of FA, the samples were prepared according to the standard LST EN ISO :2017. FA were methylated using anhydrous KOH methanol solution. Chromatographic analysis of FA methyl esters was performed using gas chromatograph (GC_MS) PerkinElmer Clarus 680, PerkinElmer Clarus SQ8T column, following the methodology established in EN ISO 15304:2003/AC: Analysis conditions: Chromatography column temperature - 60 C for 1 min, 12 C / min to 180 C for 10 min.; Spectrometer temperature mode - 5 C / min. to 300 C for 2 min.; Evaporator temperature C; Gas carriers - nitrogen; The FA methyl esters (FAME) were identified by comparison of each retention time with Supelco 37 Component FAME mix (catalog No U). Texture determination of fish preparations was performed using a Brookfield CT3 texture analyzer (Brookfield AMETEK, Inc., USA). The test samples were pressed with a cylinder (TA4 / 1000, 38.1 mm D, 20 mm L) with a diameter of 20 mm. The average value of the texture parameter was determined for each sample (average value from 3 measurements). 20

21 The rheological properties (stress and viscosity) of fish preparations were measured with a Rheolab QC viscometer (Germany). Conditions: The sample is placed in an external fixed container, the measurements are recorded every second, the points are plotted and a curve is drawn according to the hysteresis equation (Pa / s). The stability of the analyzed system is understood as follows: the lower the energy used to rotate the plunger, the less stable the analyzed structure.(see the Appendix) Sensory analysis Sensory analysis was performed after 15 min. after heat treatment. Intensity of taste and aroma properties were evaluated by analyzing samples of fish balls. The evaluation was performed by a group of 10 people who were introduced to the evaluation of balls before the study. To avoid changes in olfactory and taste receptors, participants should not eat or drink (other than water) or smoke prior to performing sensory evaluation. Cooked products from different batches were provided to each assessor. Assessors are not aware of the recipes.when analyzing the samples of boiled fish balls, the following was evaluated: intensity of total, extraneous, residual, sour, cooked fish taste, fatness, chewing, juiciness, fiberiness, hardness in the mouth, intensity of sectional color. Also the intensity of the common, sour, foreign and boiled fish smell. The intensity of sensory properties is assessed on a 10-point scale. The acceptability of the tested semi-finished products and products is assessed by an emotional test, according to an analogous 10-point digital scale (10 excellent,5- acceptable, 1 - bad / unacceptable). From the obtained data, using the methods of mathematical statistics, a profile of sensory properties is formed for each sample, showing the intensity of each property by marking it on the given scales. Based on it, we compared the samples according to their individual properties and their intensities Statistical analysis methods Obtained data was analyzed with Microsoft Office Excel 2010 and the data were reported as a mean of standard deviation. Mean comparison and separation were done using t-test. Means are significantly different, than p

22 3. RESULT 3.1 The antioxidant effect of rosemary and pomegranate extracts on fish balls Fig. no.2 represent the number of acids in the samples by chemical analysis. In sample1(control) number of fatty acids is 1.10±0.55 which is less than 2.25 ml/koh. It means in control sample used fresh fish meat but Sample no.2 (rosemary) is better than sample no. 1, it has acid value 0.7±0.5 ml/koh which means the antioxidant effect of rosemary extract performed. Sample no.3 (pomegranate) has more good quality of acid value than other two samples, 0.5±0.4 which is better quality than the control sample. After 3 months of keeping it in the freezer, the same test was repeated again on the samples, results are average. In sample no.1(control) the value of fatty acids is 3.04 which is more than 2.25ml/KOH, are not suitable for eating. But sample no.2 and sample no.3 has 1.6 ml/koh and 1.7 ml/koh, which is not bad. Mean value of samples no.1 (control)1.65 ml/koh, sample no.2 (rosemary)1.2 ml/koh and 3 (pomegranate) 0.9 ml/koh and deviation of sample no. 1 (control) , sample no.2 (rosemary) , sample no.3 (pomegranate ) sample1 sample2 sample3 acid value after 3 months acid value Fig. No.2. determination the number of acids value, ml/koh, in the samples In the fig. no. 3, peroxide value has good result in sample no.3 than other two samples. sample no. 3 has 0.03 ±.005 meq/g value in the graph which means sample no.3 is fresh. But sample no. 2 and 3 has 0.05±.005 meq/g as well as 0.04±.005meq values which mean sample 2 and 3 is fresh but unfit for storage. 22

23 After 3 months keeping it in the refrigrator, performed the same test again on the samples. Sample no.1 peroxide value 0.07 meq/g shows suspicious freshness (need to be thawed). But sample no. 2 and 3 still has good results 0.04 ±.005 meq/g and 0.05±.005 meq/g repectively, which means fresh but unfit for storage. Mean value of the samples no.1 (control) 0.06 meq/g, sample no.2(rosemary)is meq/g, and sample no.3(pomegrante) is meq/g and deviation of sample no meq/g, sample no.2 is meq/g and sample no.3 is meq/g sample1 sample2 sample3 peroxide value after 3 months peroxide value Fig. No.3 determination the of peroxide value, meq/g, in the samples by chemical analysis. 3.2 Influence of plant extracts on fatty acids composition of the fish balls Groups of fatty acids The influence of plant extract on the group of fatty acids are totally different from each other. In the fig. no.4, comparison between the group of saturated fatty acids, mono unsaturated and poly unsaturated fatty acids for sample no. 1 to sample no. 2 not showing huge variation. Difference between the percentage of saturated fatty acids in sample no.1 and sample no.2 are only 1.04 percent but in sample no.3 it is showing a huge difference. The percentage of saturated fatty acids sample no.3 is percent. Comparison of mono unsaturated fatty acids between the sample no.1, sample no. 2, sample no.3, are relatively 42.31percent, percent,45.03percent. Percentage of saturated fatty acids and monounsaturated fatty acids in sample no.3 is higher than other samples. But the percentage of 23

24 polyunsaturated fatty acids in sample no. 3 is less than sample no.1and sample no.2. Sample no. 3 polyunsaturated fatty acids percentage is only 29.92±0,79 percent from total fatty acid Saturated Monounsaturated Polyunsaturated Fig. No.4 Fatty acids groups, percentage from total fatty acids amount Composition of fatty acids In the fig.no. 5 identifies fatty acids composition, names and their percentage in the samples. Oleic acid is more than 40 percent of total fatty acids content in all samples but sample no. 3 has higher in percentage than other two samples. After that linoleic acid has good percentage. Eicosapentaenoic acid has low percentage in comparision to other fatty acids. Docosahexaenoic acid is little bit high in percentage than eicosapentaenoic acid, linolenic acid and γ-linolenic acid is about similar in percentage. After linoleic stearic acid is more than 10 percent sample 1 sample 2 sample 3 Fig.No. 5 Fatty acids composition, names and percentage amount from total fatty acid 24

25 3.2.3 Omega-3 and Omega6 fatty acids Fig. no.6 shows the percentage of omega-3 fatty acids in sample no.1 is percent from total faty acids content, sample no.2 is and in sample no.3 is The value of omega-3 fatty acids in sample no. 1 and sample no. 2 is almost equal but the value percentage of omega-3 in sample no. 3 is very less in comparison to sample no. 1 and sample no.2. The percentage value of omega-6 fatty acids in sample no. 1,2,3 are relatively 6.79, 6.30, The ratio omega6/omega3 fatty acids in sample no.1 is The ratio omega6/omega3 fatty acids in sample no.2 is 0.43 and the sample no.3 is sample 1 sample 2 sample 3 omega-3 FA omega-6 FA Ratio omega6/ omega3 Fig.No.6 ratio of omega3 fatty acid and omega 6 fatty acid Note- Correlation coefficient between omega3 fatty acids and polyunsaturated fatty acids is medium correlation. The R value of correlation between omega3 and poly unsaturated fatty acid is 0.98, which is less than 1. Also there is Medium correlation between omega6 and polyunsaturated fatty acids, R value is The influence of plant extracts on texture, viscosity and shear stress of fish ball samples In the fig. no.7 is about the texture of the fish ball samples. Texture are the same of all 3 samples showing in the fig.7. Sample no. 1,2 and 3 has 0.3 mj texture value. Values are the same because the texture results are the same 0.3mJ. which means all the samples have good texture. Sample upload control 65g, rosemarry,61g, pomegranate 60g, but the result was same for all three samples 0.3mJ. 25

26 texture(mj) sample1 sample 2 sample3 Fig.No.7 texture analysis of fish ball sample no.1, sample no.2 and sample no. 3. The viscosity and shear stress average values are varying from each other. The graphical values and fig.8 and 9 is different. The average value of viscosity is showing high percentage than shear stress. 35 Shear Stress [Pa] sample1 sample2 sample3 Fig.No. 8 comparison between the percentage of shear stress in fish ball samples 26

27 sample1 sample2 sample3 Viscosity [mpa s] Fig.No.9 variation of viscosity between the sample no.1,2and Sensory properties analysis and acceptability of heat treated fish balls In the sensory analysis and acceptability test on sample 1, sample 2 and sample 3 shows different results in the fig. no.10. Total odor intensity of sample no.1 shows 60 percent in the graph, but sample no.2 and 3 shows higher odor intensity in comparison to sample no.1. Greasiness percent is very high in sample no. 2, is about 90 percent. Graph shows very low greasiness in sample no. 1. Sample no.1 in graph shows ticking about 60percent, sample no.2 and 3 shows the same percentage. It means sample no.1 is less in ticking property than other two sample. Juiciness property makes a sample very smooth and soft in texture. under this property all three sample are the same in percentage, showing by the fig. It shows 70 percent which is good for product. After having the taste of all three samples. Sample no.1 more soft than other two sample. Sample no.1 90percent soft in mouth and sample no. 2 is a little bit hard in mouth because of rosemary and pomegranate extract. Marvellous color intensity in sample no.2 and 3 because of extract. Plant extract gives slightly different color in samples.obviously odor intensity is good in sample no.2 and 3(90percent). Odor intensity is low in sample 1(only 50 percent). Boiled fish balls smell intensity intensity but the sample no.2 and 3 shows good result in fig.10. Smell intensity after boiling is different than raw. After boiling, sample no.1 is average in smell. 27

28 sample1 sample2 sample3 Total Odor Intensity 10 Boiled fish smell Intensity 8 6 Greasiness 4 The intensity of sour odor 2 Ticking 0 Extranal Odor Intensity Juiciness Sectional color Intensity Hardness in the mouth Fig. No.10 sensory properties of fish balls sample no.1,2and 3. Total taste intensity in all three samples different. The chart structure showing their taste intensity level. Sample no.1 showing high in residual taste intensity in fig.no.11. Sample 1 Boiled fish Flavor Intensity The total taste intensity Residual taste intensity Extranal Taste intensity Fig. No.11 showing total taste intensity of fish balls sample no.1 28

29 Sample 2 The total taste intensity Boiled fish Flavor Intensity Residual taste intensity Extranal Taste intensity Fig. No. 12 shows total taste intensity of fish balls sample no.2. Sample no.2 shows higher in external taste intensity in fig. no.12. But sample no.3 shows high external taste intensity, boiled fish flavor intensity as well as total taste intensity. Overall, sample no.3, fig. no.13 showing better total taste intensity than sample no.1 and 2. Sample 3 Boiled fish Flavor Intensity The total taste intensity Residual taste intensity Extranal Taste intensity Fig No.13 showing total taste intensity of fish balls sample no.3. 29

30 Acceptability percentage of all three samples showing in the fig.no.14. Sample no. 3 shows 90 percentage acceptability, which means sample 3 is better than other two sample. sample no.2 shows more than average percentage. But sample no.1 showing low percentage than other samples in fig.no acceptability sample1 sample2 sample3 Fig. No.14 showing the acceptability of fish balls sample no.1,2 and 3. 30

31 4. DISCUSSION OF RESULTS Extracts containing bioactive compounds isolated from numerous plant sources. Plant extracts have shown remarkable antioxidant and antimicrobial activity. Natural antioxidant and antimicrobial activity of plant extracts have been recognized for many years and applied in several food systems as antioxidant or antibacterial agents[38)[39]. The investigated influence of plant extract on fatty acids composition is tremendous. In the result of chemical analysis of fish balls, plant additives rosemary and pomegranate extract works as an antioxidant to slowed down the lipid oxidation as well as antimicrobial activity to inhibit the microbial growth [40]. Which makes it more healthy and favorable fish balls. After adding the rosemary and pomegranate extract, quality of fish balls improved. Rosemary and pomegranate extracts gave satisfactory results in comparison to control samples. In fish experiment found quality difference between control, rosemary and pomegranate extract samples. In sensory analysis found the major quality difference in texture, odor, greasiness, boiling fish ball odor intensity, ticking, juiciness, softness in mouth, sectional color intensity, external odor intensity, the intensity of sour odor, total odor intensity in all three samples. Plant additives sample gave valuable results because of their properties. Plant additive (rosemary and pomegranate) gave marvelous color to fish balls and aroma intensity. Pomegranate sample and rosemary samples higher in acceptability, which means additives makes it more acceptable product. In all the result samples, pomegranate sample has remarkable figure except shear stress and viscosity. In all figures rosemary has high value, which means in the texture and shear stress rosemary extract is good for fish meat products. In the picture of total taste intensity, pomegranate and rosemary has high result in total taste intensity, external taste intensity, boiled fish flavor intensity, averge in residual taste intensity [27][31]. Keeping fish in the refrigerator, after three months performed the same test and found different results. When performed this experiment 3 times then I found accurate result. Plants additives rosemary and pomegranate helps to improve texture quality and increase antioxidant property of fish balls. It gives a good odor and flavor intensity. The AV of the tested samples was used to determine the degree of hydrolytic rancidity of fish ball lipids. AV of the extracted lipids was determined according to EN ISO 660: procedure. POV of the studied lipids is determined by standard (EN ISO 3960:2010) iodometric method and was presented as meqv O2/kg lipids. Acid value and peroxide value result are good in fresh sample after keeping the samples in to the refrigerator the control sample performing bad in result. In the texture analysis of fish ball samples control, rosemary and pomegranate have same result 0.3mJ because the texture of fish balls was the same. Result of shear stress are 26.4 of pomegranate,