ANALYSIS OF CHEMICAL COMPOSITION INDICATORS OF EXTRUDED LUPINES (LUPINUS L.), FABA BEANS (VICIA FABA L.) AND PEAS (PISUM SATIVUM L.) AND EFFICIENCY OF THEIR USE IN THE NUTRITION OF DAIRY COWS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Ieva Kudlinskienė

ANALYSIS OF CHEMICAL COMPOSITION

INDICATORS OF EXTRUDED LUPINES

(LUPINUS L.), FABA BEANS (VICIA FABA L.)

AND PEAS (PISUM SATIVUM L.)

AND

EFFICIENCY OF THEIR USE IN THE

NUTRITION OF DAIRY COWS

Doctoral Dissertation Agricultural Sciences, Animal Sciences (A 003)

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Dissertation has been prepared at the Institute of Animal Rearing Technologies of Veterinary Academy of Lithuanian University of Health Sciences during the period of 2014–2020.

Scientific Supervisors:

2018–2020 Prof. Dr. Asta Racevičiūtė-Stupelienė (Lithuanian University of Health Sciences, Agricultural Sciences, Animal Sciences – A 003); 2014–2018 Prof. Habil. Dr. Romas Gružauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Animal Sciences – A 003).

Consultant

Prof. Dr. Meelis Ots (Estonian University of Life Sciences, Agricultural Sciences, Animal Sciences – A 003).

Dissertation is defended at the Animal Sciences Research Council of the Veterinary Academy of Lithuanian University of Health Sciences: Chairperson

Dr. Violeta Juškienė (Lithuanian University of Health Sciences, Agricultural Sciences, Animal Sciences – A 003).

Members:

Assoc. Prof. Dr. Sigita Kerzienė (Lithuanian University of Health Sciences, Agricultural Sciences, Animal Sciences – A 003);

Dr. Ramutė Mišeikienė (Lithuanian University of Health Sciences, Agricultural Sciences, Animal Sciences – A 003);

Assoc. Prof. Dr. Antanas Šarkinas (Kaunas University of Technology, Technological Sciences, Chemical Engineering – T 005);

Prof. Dr. Ayhan Yilmaz (Siirt University (Turkey), Agricultural Sciences, Animal Sciences – A 003);

Dissertation will be defended at the open session of the Animal Sciences Research Council of the Lithuanian University of Health Sciences on the 26th of August, 2020 at 1:00 p.m. in Dr. S. Jankauskas Auditorium of the Veterinary Academy of the Lithuanian University of Health Sciences.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA

Ieva Kudlinskienė

EKSTRUDUOTŲ LUBINŲ (LUPINUS L.),

PUPŲ (VICIA FABA L.) IR ŽIRNIŲ

(PISUM SATIVUM L.) CHEMINĖS

SUDĖTIES RODIKLIŲ ANALIZĖ IR

JŲ PANAUDOJIMO EFEKTYVUMAS

MELŽIAMŲ KARVIŲ MITYBOJE

Daktaro disertacija

Žemės ūkio mokslai, gyvūnų mokslai (A 003)

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Disertacija rengta 2014–2020 metais Lietuvos sveikatos mokslų universiteto Veterinarijos akademijoje, Gyvūnų auginimo technologijų institute.

Moksliniai vadovai:

2018–2020 m. prof. dr. Asta Racevičiūtė-Stupelienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, gyvūnų mokslai – A 003); 2014–2018 m. prof. habil. dr. Romas Gružauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, gyvūnų mokslai – A 003).

Konsultantas

prof. dr. Meelis Ots (Estijos gyvybės mokslų universitetas, žemės ūkio mokslai, gyvūnų mokslai – A 003).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Gyvūnų mokslų krypties taryboje:

Pirmininkė

dr. Violeta Juškienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, gyvūnų mokslai – A 003).

Nariai:

doc. dr. Sigita Kerzienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, gyvūnų mokslai – A 003);

dr. Ramutė Mišeikienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, gyvūnų mokslai – A 003);

doc. dr. Antanas Šarkinas (Kauno technologijos universitetas, techno-logijos mokslai, chemijos inžinerija – T 005);

prof. dr. Ayhan Yilmaz (Siirt universitetas (Turkija), žemės ūkio mokslai, gyvūnų mokslai – A 003).

Disertacija ginama viešajame Gyvūnų mokslų krypties tarybos posėdyje 2020 m. rugpjūčio 26 d. 13 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Dr. S. Jankausko auditorijoje.

Disertacijos gynimo vietos adresas: Tilžės g. 18, LT-47181 Kaunas, Lietuva.

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ABBREVIATIONS

AA ADF ADG ADL AI ANFs B2 B6 BC CF CP DM DMI EU FA FCM GMO HTST KTU LSMU MJ MUFA N2O n-3 n-6 NaOH NDF NEL NFE NH3-N NPN NSP PUFA RDP:RUP SBM SFA TAC TDF – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – Amino acids

Acid detergent fibre Average daily gain Acid detergent lignin Atherogenicity index Antinutritional factors Riboflavin Pyridoxine Before Christ Crude fiber Crude protein Dry matter Dry matter intake European union Fatty acids

Fat corrected milk

Genetically modified organisms High-temperature-short-time Kaunas University of Technology

Lithuanian University of Health Sciences Megajoule

Monounsaturated fatty acids Nitrous oxide

Omega−3 fatty acids Omega−6 fatty acids Sodium hydroxide Neutral detergent fibre Net Energy Lactation Nitrogen-free extracts Ammonia nitrogen Non protein nitrogen Non-starch polysaccharides Polyunsaturated fatty acids

Rumen degradable protein to rumen undegradable protein Soybean meal

Saturated fatty acids

Total available carbohydrates Total dietary fiber

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8 TI TMR UV VFA VOCs – – – – – Thrombogenicity index Total mixed ration Ultraviolet

Volatile fatty acids

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INTRODUCTION

There is a great demand for high-protein materials for livestock feed in Europe agriculture of which 87% is met by imported soybean and soybean meal [1]. The market for plant proteins has three different segments: conventional feed, high-value feed, and food – each with their own economic, social and environmental features. The conventional compound feed market is highly price driven. Livestock farmers focus on ‘value for money’ to meet the nutritional needs of their animals (protein & amino acid content) [2]. Animal supply with high quality feed is a very important factor in the increase of productivity, in rational use of feed and in preservation of animals’ health [3]. Thus, in many animal’s productions systems feed is the biggest single cost and profitability can depend on the relative cost and nutritive value of the feeds available [4].

Soybean or soybean meal is widely used in conventional intensive animal feeding systems because of its known high protein content (38–42%) and good amino acid balance and digestibility [5–9]. However, soybean meal costs and availability are strongly related with the price development of agricultural commodities on the world market [10]. Factors which may influence world market prices include variations in population and economic growth, changes in consumer’s product preferences, also on weather conditions [11, 12]. In pursuit of sustainable and economically-viable farming systems, there is a need for livestock farmers to reduce reliance on imported feedstuffs [4]. Therefore, most researchers have focused on improving the status and utilization of different protein sources in order to reduce costs and maintain optimum performance of animals [13] also improve farm profitability, optimize protein and energy utilization and increase knowledge about environmental pollution [14]. Another important aspect, the worry of the public opinion about the widespread use of genetically modified (GMOs) feedstuffs in animal feeding [15; 16].

Therefore, various species of home-grown grain legumes, may represent strategically important alternatives to soybean [17]. In Europe, legumes currently account for only 1.5% arable land, while in the world – 14.5% [1]. Grain legumes are an important and low price source of protein, which play a distinct role in agricultural ecosystems with their ability to fix nitrogen (N) symbiotically [18], also represent a great resource in organic agriculture both to satisfy the nutritional requirements of organic livestock feeding and to maintain soil fertility [15]. Growing legumes, a major biological nitrogen source, is also a powerful option to reduce synthetic nitrogen fertilizers use and associated fossil energy consumption [19; 20]. Several grain legumes

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have the potential to replace at least some of the soya currently used in the diets of monogastric animals, ruminants, and fish [1].

The most commonly in dairy cow’s nutrition used legume grains are peas (Pisum sativum L.), faba beans (Vicia faba L.), and lupines (Lupinus L.), it is characterised by high energy density allowed to the high protein, starch and/or fat concentrations, as more than sufficient is their calcium concentration. Within the grain legumes, lupins have higher amount of crude protein (324– 381 g kg–1_{dry matter), compared to faba beans (301 g kg}–1_{dry matter) and} peas (246 g kg–1_{dry matter) [21]. For this reason, legumes may be used in} ruminant diets in order to balance protein content in diet [22]. Legumes are not only a rich source of protein; they also contain fibre, which is essential for normal functioning of the digestive tract [23, 24]. However, choosing protein sources in diets of ruminants, an important criterion is the content of rumen undegradable protein [13]. The diet of high-yielding dairy cows should contain ruminally undegradable protein, which supplies an animal with sufficient amounts of essential amino acids. Thermal treatment of protein feeds is common in feed manufacturing to increase the amount of rumen by-pass protein in order to meet protein needs for milk production [25]. Thus, for production of ruminant livestock, modern nutrition systems suggests an optimum 60 to 40 ratio of rumen degradable protein to rumen undegradable protein (RDP:RUP), of whole protein content [26, 27]. The high protein degradability of legume grains in the rumen has led to investigations into means to protect against fermentation and thus increase the undegraded dietary protein component that escapes the rumen. Consequently, in order to increase the utilization of legumes have aplied a wide range of processing techniques, such as soaking, boiling, autoclaving, radiating, cooking, roasting, dehulling, germinating, fermenting, supplementing with various chemicals and enzymes and utilizing extrusion cooking [28–30].

Extrusion cooking has certain advantages, including versatility, high productivity, low operating costs, energy efficiency, and shorter cooking times. The temperatures reached by the feed during extrusion can be high (up to 200°C), but the residence time at this elevated temperature is short (5 to 10 seconds). This tends to maximize the beneficial effects (preservation of nutrients) of heating feeds, while minimizing the detrimental effects (destruction of nutrients) [31]. Therefore, it has been hypothesized that the extrusion process has a positive effect on nutritional value of legumes (amino and fatty acids composition) and dairy cows’ performance. The effect of extruded peas, lupines and faba beans in dairy cows’ nutrition, has been considered in several studies before, in order to investigate it’s effects on dairy cows’ performance and milk composition [32–34]. However, there hasn’t been done many researches to investigate extruded plants of legume

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family effects on milk sensory and technological properties, amino and fatty acids composition in milk, fermentation processes in the rumen or blood parameters of dairy cows, some of which are important indicators of the animal’s nutritional state and may be useful in identifying metabolic imbalances.

The aim of the study

To analyse the chemical, amino acids and fatty acids composition of untreated and extruded diverse species of legume family; to assess the efficiency of their use in dairy cows’ diets.

Objectives of the study

1. To examine the chemical, amino acid and fatty acids composition of raw and extruded soybeans, lupines, faba beans and peas;

2. To assess the impact of dairy cows` diets supplemented with extruded lupines, faba beans and peas on feed intake and efficiency;

3. To determine the effect of diets supplemented with extruded lupines, faba beans and peas on dairy cows’ performance, milk composition and sensory properties, and the effect of diets with extruded faba beans on milk technological properties, amino acid and fatty acid composition and the formation of volatile flavour compounds;

4. To assess the effect of diets supplemented with extruded lupines, faba beans and peas on fermentation processes in the dairy cows` rumen; 5. To define the effect of diets supplemented with extruded lupines, faba

beans and peas on biochemical blood parameters in dairy cows`. The scientific novelty and practical usefulness

In Lithuania, as in many other European Union countries, industrial by-products such as soybean and rapeseed meal or cake are used mainly to meet the protein demand in cattle diets. However, due to the high price of these raw materials, alternatives are continuously being sought to replace them with local rich in protein crops, it’s of vital relevance in countries where these raw materials are imported. The plants of legume family (Fabaceae) are among the most rich in protein and lowest price raw materials in Lithuania, mainly cultivated lupines (Lupinus L.), faba beans (Vicia faba L.) and peas (Pisum

sativum L.), therefore these seeds can be valuable both in the industrial

production of complete feed and in home-grown feed production. The effect of the extrusion process on the chemical composition of legume family plants

(Fabaceae) and the efficiency of their use in the diets of dairy cows` has been

studied quite extensively in the scientific literature. However, in this dissertation for the first-time were analysed, not only, the chemical and amino

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acid composition, but also the fatty acid composition (before and after the extrusion process) of legume family plants (Fabaceae) included in the national list of plant varieties in Lithuania, and evaluated these materials efficiency in dairy cows` nutrition. Moreover, for the first-time the effect of the extruded faba beans (Vicia faba L.) were evaluated on the milk sensory and technological properties, amino and fatty acids compositions as well as on volatile flavour compounds formation. Furthermore, studies have been carried out to assess the impact of extruded legume family plants (Fabaceae) on fermentation processes in the dairy cows` rumen and on biochemical blood parameters.

Concerning the results of the study carried out, it is possible to establish compound feed recipes and diets for dairy cows supplemented with extruded plants of legume family (Fabaceae) without adversely affecting their per-formance, milk composition and sensory properties.

Scope of the thesis

The thesis consists of the introduction, literature review, description of work methodology, the study results, discussion of results, conclusions, recommendations, thesis summary in the Lithuanian language, use of references (331 sources), list of publications on a thesis topic, participation in conferences, curriculum vitae and acknowledgements. The volume of the thesis is 224 pages, illustrated by 27 tables and 8 figures.

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1. LITERATURE REVIEW

1.1. The extrusion technology, basic features and extruder system Heat treatments are used to improve the nutritional, hygienic, physical and chemical and other animal feed properties. There are many heat treatments, each different in the heat source, construction of the device or process parameters applied, and their efficiency depends on a range of factors. Two unavoidable factors of all heat treatments are temperature and time of their application, although the impacts such as humidity, pressure, shear force and others causing additional effects cannot be neglected either. Combining of these parameters is the starting point for development of all kinds of heat treatments and devices that are used in feed industry. Basically, all the different process techniques increase the temperature of the product. If you are adding moisture in the process, we are talking about hydrothermal treatment. Most of the processes that are used are hydrothermal treatments because even when moisture is not introduced from the outside moisture released from the material to be treated participates in the process. Another effect is mechanical and it can be located in or out of the heat treatment device. No matter where it is carried out, the mechanical treatment causes an additional effect to heat treatments so that they become thermo-mechanical processes. Thus, there are many possible combinations, and types of heat treatments in animal feed processing, and most frequently used are cooking, roasting, popping, steam flaking, toasting, conditioning, pelleting, micro-nisation, expanding and extrusion [35–39].

Extrusion is the process in which the material (feedstuff or mixture) is pushed through the barrel by means of screws of different configurations and pressed through the die at the end of barrel. The verb “to extrude” derives from Latin word ex (out) and frudere (to thrust), and means to force, as through a small opening [31]. The simple design of a screw within a barrel chamber has been initially credited to Archimedes of Syracuse, a Greek mathematician and physicist who lived in 287–212 BC. His design of a wooden apparatus devised to move water from a lower level to a higher level with the turn of a screw within a round chamber, amazed the people of his time. This simple design later became the cornerstone of many different industries including material sciences such as metal fabrication, ceramics, concrete, plastic and nonplastic polymers, and, most recently, the food and feed industries [40].

In the last two centuries, extruders have come a long way. Joseph Bramah obtained the first extrusion patent in 1797 for making a lead pipe by having a dummy block placed in a ram type machine and forced out of a die to form a

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continuous profile [40]. Commercial extrusion processing of food and feed products has been practiced for over 60 years. The screw extruder was first used as a continuous cooking device in the late 1930's. The first commercial application of this extrusion cooking process was in the mid 1940's. The end product was direct-expanded corn meal snacks [41]. Extrusion processing, as formerly defined by Smith [42], is thermo-mechanical treatment by which moistened, expansible, starch and/or proteinous materials are plasticized and cooked in a tube by a combination of moisture, pressure, temperature and mechanical shear, and thus pre-define shaped through the die opening at extruder outlet. Extrusion cooking is becoming popular over other common processing methods due to its automated control, high capacity, continuous operation, high productivity, versatility, adaptability, energy efficiency, low cost. Moreover, it also enables design and development of new food products, high product quality, unique product shapes and characteristics, energy savings and no effluent generation [43–45].

Extrusion technology has led to production of a wide variety of cereal-based foods, protein supplements, and sausage products [46]. Today, the extrusion cooker has become the primary continuous cooking apparatus in the commercial production of most dry pet foods and aquatic feed and some applications for livestock feed [41].

The basic concept of extrusion process is a high-temperature-short-time (HTST) physical treatment, whereby the high temperature is a direct result of friction (dry extrusion), or pre-conditioning and steam injection (wet extrusion), or a combination of both. The humidity of treated material in dry extrusion is about 30% while it is up to 80% in wet extrusion [38]. It is mostly carried out at high pressure, temperature and using mechanical shearing, during which flours or starches are subjected to high temperatures and mechanical shearing at relatively low levels of moisture content, however, low-temperature extrusion is also applied in cereal technology [47–50].

Depending on the products being processed, a complete extrusion system generally consists of storage bins, dry mix feeders, liquid pumps and meters, a preconditioner, an extruder assembly, a die, and a cutter (Figure 1) [51]. The system shown is a single-screw extruder, but a twin-screw system consists of the same basic components. To produce an extruded product obviously requires more than extruder alone – all parts of the system are important [52]. Each of the components will be discussed in detail [51].

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Fig. 1.1.1. General view of an extruder system [51]

Raw material preparation prior to extrusion is critical, including:

1) Grinding – particle size can affect texture, degree of cook etc, uniformaly particle size is importanat to process stability;

2) Mixing – uniformity of the feed is critical to product consistency and process stability [52].

A storage bin is used for the storage of dry ingredients. It provides a buffer

of raw material so that an extruder has a continuous and stable supply of feed ingredients. This bin is usually equipped with rotating blades to prevent bridging [51].

Two types of dry feeders are normally used to feed extruders: volumetric feeders and gravimetric feeders. A volumetric feeder provides a constant volume of dry ingredients, but cannot guarantee a constant mass flow rate due to changes in density of feed material. Gravimetric feeders, on the other hand, control feed flow rate based on the mass delivered and, therefore, are more accurate feeding devices [51].

Liquid feeders. Common liquid raw materials used in extrusion include

water, fat, and syrup. Metering of liquid ingredients is critical for successful product manufacturing. Metering of liquids can be achieved either by volume or by mass. Mass flow meters are more accurate than volumetric meters.

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Devices include rotameters, differential pressure meters, fluid displacement meter, velocity flow meters, and mass flow meters [51].

Material from the delivery system is fed into the next section of the extruder, which is called the preconditioner [53]. Preconditioners are mounted between the feeding device and the extruder [51]. A large number of different types of preconditioner are available on market. The type to be used depends upon the application. The capital investment required varies significantly, depending upon the chosen design [52]. Originally, precon-ditioners were of a single-shaft design. The shaft, having mixing elements, rotated at relatively high speeds, resulting in retention times of 30 seconds or less. That was insufficient. Most modern preconditioners have a double-shaft design.. The two shafts have different dimensions, and rotate at different speeds, which result in improved mixing, and retention times of between 2 and 4 minutes [51].

The most important functions of a preconditioner are moisture adjustment and precooking of the raw materials prior to extrusion. During precon-ditioning, raw materials are held in a warm, moist environment where they are mixed for a prescribed time, and then discharged into the extruder. Preconditioning provides the benefits of improved product quality, reduced extruder wear, increased extruder capacity, and reduced power consumption [51]. The preconditioning step initiates the heating process by the addition of steam and water into the dry mash. The preconditioner supplies the extruder with uniformly mixed and hydrated material which improves stability of the extruding system as well as aids the development of certain final product characteristics [54]. In preconditioner the material is heated up to 80–90°C and moistened up to 22–28%. Preconditioning step improves extrusion process in many ways [55]. The main functions of a preconditioner include: 1) mixing of multiple ingredients such as fats, molasses and colors; 2) hydrating the dry mash; 3) precooking, which begins gelatinization of starches and denaturation of proteins; 4) thermal energy addition, generally in the form of steam [54].

After preconditioning, the material is discharged into the extruder barrel where major transformations of raw preconditioned material occur. The types of the processes in the extruder barrel depend on the type of extruder [56]. An extruder is a machine which shapes materials by the process of extrusion [31]. During this process the material is exposed to high temperatures (up to 200°C) for 1–2 minutes or more precisely the material temperature increases progressively within the last 15 to 20 seconds up to the optimum one to achieve the desired effects [38]. Maximal temperatures in extrusion of different feed materials are usually between 100 and 140°C and the pressure, depending of the product formulation, rises to 20 or even 70 bar [57].

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Extruders are available in several designs depending on their application, but are generally classified based on the number of screws. The two general types of extruders are single-screw and twin-screw configurations. Single screw extruders are widely used in the feed industry because of their low initial investment and operating cost compared with twin screw extruders. Single screw extruders operate based on the pressure requirement of the die, slip at the barrel wall and the extent to which the screw is filled, whereas twin screw extruders operate based on the direction the two screws rotate and the extent of intermeshing between the two screws [58].

A single-screw extruder assembly consists of a screw and a barrel. The

conventional practice is to rotate the screw within a stationary barrel, but the converse is available. Different screw configurations provide different shearing conditions and pressure profiles, which are uniquely suited for the production of different products. According to Harper [59] the extruder barrel for a single-screw extruder can be divided into three sections in terms of functionality: feeding, compression, and final metering zones. In this design, ground feed or ingredients enter the hopper, and the rotating action of the screw conveys the feed material to the transition section where the screw channel becomes more narrow and compacted. The mechanical energy causing compaction generates heat, which is dissipated to increase the temperature of the material, resulting in gelatinization of starch and cohesion. As the feed material continues to be transported by the metering section, it is pushed through the die opening. Twin screw extruders differ from single screw extruders and have several advantages including: no pre-conditioning, self-cleaning, greater range in length to diameter ratios; good mixing; shorter residence time and good heat transfer; and capable of handling a wide range in moisture content and types of feed ingredients [60]. In the extruder barrel very high temperatures can be achieved, but the residence time of the feed at such elevated temperatures is very short (5-10 sec). This high temperature short time process maximizes the benefits of heating feed ingredients (improved digestibility, inactivation of antinutritional factors and pasteu-rization) while minimizing nutrient degradation [56].

The screw forces the material through the die, where the material is formed and expanded at the outlet of the die [61]. The die plate functions as a restriction and forming device mounted at the end of the barrel. Die have two major functions. It provides restriction to product flow causing the extruder develop the required pressure and shear. In addition, it shapes extrudate as the product exits the extruder. By adjusting the die opening, the pressure, retention time, and the dimensions and shape of the final product can be controlled. The amount of expansion can be controlled by formula mani-pulation and open area in the die. The relative speed of the knives and the

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linear speed of the extrudate results in the desired product length [62]. For small scale extruders, the die assembly has only one opening. Multiple openings are available for large or commercial scale extruders [51].

The product is cut with rotating knife into the desired length [52]. A cutter consists of a group of rotating knives mounted in front of the die plate. The rotational speed of the knives is adjustable. The length of extrudate is determined by the number of knives and the rotational speed of the knives [51].

After extrusion must correctly implemented, and my include: 1) Drying – important for preservation in some products, and also has a major effect on texture, and affects product destiny; 2) Coating – to add flavor of fat [52]. After treatment, the product is often quite different, from nutritional point of view, compared to the raw material from which it is composed [63].

1.2. Chemical composition of untreated and extruded plants of the legume family (Fabaceae) and their use in ruminants nutrition 1.2.1. The plants of legume family (Fabaceae)

The legume family (Fabaceae, syn. Leguminosae) is the third largest family of angiosperms, comprising over 750 genera and 19.000 species ranging from small herbs to large trees [64]. The botanical family of grain legumes is known as Fabaceae, also referred to as Leguminosae. Grain legumes are cultivated primarily for their seeds which are harvested at maturity, and which are rich in protein and energy. The mature dry seeds of grain legumes are used either as animal feed ingredient or for human consumption [15]. Grain legumes such as faba bean (Vicia faba L.), pea (Pisum sativum L.) and lupines (Lupinus L.) are old crops cultivated in all arable continents [65]. In Europe, amongst others, the major grain legumes cultivated are peas (Pisum sativum), faba beans (Vicia faba L.) and lupines (Lupinus L.), whereas in Argentina, Brazil, China, India, and the United States soybeans dominate [66].

The unique capacity of leguminous plants in conjunction with rhizobium symbionts to biologically fix and utilize atmospheric N enables that inorganic N-fertilisers with rising prices and high requirement of energy in manufacturing are not required. Indeed, the emissions of a potent greenhouse gas N2O from legume cultivation are generally lower than those from N-fertilised crops [1]. The seed yield potential of grain legumes under optimal conditions is similar or exceeding that of conventional protein crops. These

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advantages make legumes increasingly attractive in the intensive farming in addition to current wide spread use in the low-input and organic farming [65].

Lupines belong to the Genisteae family, Fabaceae or Leguminosae [67]

and more than 400 species are known, from which only four are of agronomic interest [68] Lupinus albus (white lupin), Lupinus angustifolius (narrow leaf or blue lupin), Lupinus luteus (yellow lupin) and Lupinus mutabilis (Andean lupin). Cultivated species of lupins (Lupinus L.) used as feed ingredient for pigs, ruminants and poultry mainly include Lupinus albus, Lupinus

angus-tifolius and Lupinus luteus, and they all originate from the Mediterranean area

[69]. The European white lupin (L. albus) shows white or violet-blue flowers, and typically seed weights are of about 35 g/100 seeds, although some European cultivars may have weights of about 50 to 60 g/100 seeds [70]. On the other hand, flowers of the narrow-leafed lupin or Australian sweet lupin (L. angustifolius) are normally blue, thus sometimes being referred to as blue lupin [71, 72] with a typical seed weight of about 15 g/100 seeds [70]. In contrast, the yellow lupin (L. luteus) has golden yellow flowers and a typical seed weight of about 12 g/100 seeds [70].

Faba beans (Vicia faba L.), also known as field beans, horse beans, broad

beans or tick beans, represent an annual legume that is well adapted to cool climates, and thus is preferably cultivated in regions with mild winters and adequate summer rainfall [73]. Beans are one of most consumed legume worldwide [74]. According to their seed size, Vicia faba can be classified in three subspecies: Vicia faba minor (small seeded), Vicia faba major (large seeded) and Vicia faba equina (intermediate seed size) [75]. The faba bean (Vicia faba L.) is widely used for animal feed. [76]. Faba beans represent a well established ingredient in diets for horses and ruminants Recently, they have been receiving growing attention as protein supplement in diets for pigs, particularly in Europe, due to the low production of protein feed ingredients within the European Union [73].

Peas are a genus of the family Fabaceae. It contains one to five species,

depending on taxonomic interpretation. Pisum sativum (the field or garden pea), is domesticated and is a major human food crop [77]. Cultivation of peas (Pisum sativum L.) provides a good cool-season alternative for regions not suited for growing soybeans due to their climate conditions, as peas are less frost sensitive and thus may tolerate low temperatures for germination and growth [78]. Two subspecies of peas are grown in Europe, namely Pisum

sativum hortense and Pisum sativum arvense. Pisum sativum hortense is

characterised by white flowers, whereas Pisum sativum arvense shows dark-coloured flowers [79]. Peas are primarily grown for human consumption, however, over the last years, they have been increasingly used in pig nutrition as well, especially in Canada, the Northern United States and Australia [73].

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In the short-term, grain legumes are presumably the most promising alternatives to soybean (Glycine max. L. Merr) and rapeseed in the temperate areas because their cultivation practices are already available and implemented. However, grain legume seeds are edible by humans as well. Therefore, the utilisation of human-inedible feeds for ruminants and/or feeds the production of which require less or not at all arable land should be encouraged to improve further the sustainability of food production system in the longer term [65].

1.2.2. Chemical composition of the legume family plants (Fabaceae) Legumes are good sources of protein, essential amino acids (particularly rich in lysine, leucine, isoleucine, phenylalanine and valine), unsaturated fatty acids (linolenic acid and linoleic acids), dietary fiber, resistant starch [80], and vitamins (Vitamin C) [81].

Leguminous grains have a high protein content, a considerable

concen-tration of energy and calcium. Their protein are highly degradable in the rumen. By comparing some of them, it can be concluded that there is a higher content of proteins in lupine (324–381 g kg–1_{of dry matter), compared to} beans (301 g kg–1_{dry matter) and peas (246 g kg}–1_{dry matter) [21]. Acording} White et al. [82] grain legume seeds differ in the chemical composition, the CP content ranging from 240 (peas) to 400 g kg–1_{DM (soybeans). The protein} in grain legume seeds, faba beans and lupin seeds in particular, is low in methionine , which is often the limiting AA for the lactation performance of dairy cows [83].

The protein of grain legumes consists mainly of globulins, with this fraction being higher in lupins and soybeans than in faba beans or peas. The globulins themselves are composed of two major proteins characterised by their sedimentation coefficients, namely the 7S and the 11S globulins [84]. In faba beans and peas, these globulins are called vicillin (7S) and legumin (11S). The ratio between these two globulins differs from one species to another, e.g. in soybeans and lupins the 7S-like protein is found in a higher proportion than the 11S-like protein. Contrary, 11S-like protein (legumin) dominates in peas and faba beans [85].

The protein of faba beans and peas contains similar or even higher proportions of lysine (70 and 80 g kg–1_{CP, respectively), when compared to} protein from SBM (69 g kg–1_{CP) or lupins (51 to 54 g kg}–1_{CP). The} proportion of threonine in grain legume protein (38 to 42 g kg–1_{CP) is similar} to that in SBM (45 g kg–1_{CP) [21]. The protein content of the peas is about} 25–26% of dry matter. In terms of the amino acid profile, high lysine and low tryptophan content as compared to soybean meal [79]. The protein fraction is

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easily degradable in the rumen while the starch content is outstanding (more than 40% DM), soluble and easily degradable [86].

The crude fat content (ether extract) in peas and faba beans is generally rather low compared to lupins. For example, crude fat contents of faba beans and peas range from 15 to 20 g kg–1_{DM, thus being in a similar range as} values for SBM (15–28 g kg–1_{DM) [87, 88]. In lupins, the crude fat content} varies between species, with values of about 57 g kg–1_{DM (L. luteus, L.}

angustifolius) to 88 g kg–1_{DM (L. albus) [87]. However, there is general} agreement that the ether extract or crude fat content in grain legumes represents an inadequate measure for the lipid contents in these feed ingredients, as these compounds comprise significant amounts of non-nutritive lipids (e.g. waxes, cutin) while lipids of high nutritional value (triacylglycerol, including fatty acids) are often incompletely extracted [89]. Alternatively, by means of gas chromatography, detailed information on fatty acid content and composition can be obtained [89, 90]. Using this method, linoleic acid (480 mg g-1_{of total lipids) and oleic acid (260 mg g}–1_{of total} lipids) were identified as predominant fatty acids in peas, whereas the total lipid content (sum of fatty acids) amounted to 18 g kg–1_{DM [91]. In faba} beans, palmitic and oleic acid were found to be the major fatty acids (170 and 150 mg g–1_{of total lipids, respectively), while the total lipid content (sum of} fatty acids) was about 39 g kg–1_{(air-dry basis) [92]. In contrast, Duc et al.} [93], observed a lower total lipid content in faba beans (18 g kg–1_{DM; sum} of fatty acids), with linoleic and oleic acid being the predominant fatty acids (52 and 28 mg g–1_{of total lipids, respectively). The predominant fatty acids} of lupins are oleic acid (210 to 530 mg g–1_{of total lipids) and linoleic acid} (172 to 473 mg g–1_{of total lipids), but the ratio of these fatty acids may vary} between different lupin species [70].

The carbohydrate fraction includes the low molecular-weight sugars, starch and various NSP [94]. The NSP and lignin are the principal components of cell walls and are commonly referred to as dietary fibre [94– 96]. Generally, faba beans and peas are rich in starch (422–451 and 478–534 g kg–1_{DM, respectively) [87, 97, 98], whereas lupins have comparatively low} levels of starch (42–101 g kg–1_{DM) [10; 87]. Legume starch contains a} higher proportion of amylose (30–35% amylose) when compared with cereal starches (20–30% amylose). High amylose starch is more susceptible to retrogradation than low amylose starch, and retrograded starch becomes more resistant to enzymatic degradation, thereby increasing the amount of starch entering the large intestine [97]. Lignin content is low and ranges from 1 to 7 and 6–9 g kg–1_{of dry matter, respectively [10]. However, it needs to be} emphasised that the determination of starch in grain legumes may be confounded by the analytical method used [99].

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Faba beans and peas contain rather low amounts of fibre fractions in comparison to lupins [94, 98] and with regard to lignin content, faba beans and L. angustifolius have similar amounts of lignin (1 to 7 and 6 to 9 g kg–1 DM, respectively), whereas the lignin content in peas is of minor importance (0.4–3 g kg–1_{DM) [71, 98]. The NSP fraction of faba beans consists mainly} of cellulose (89–115 g kg–1_{DM), with lower levels of hemicellulose (21–57} g kg–1_{DM) [71, 98, 100]. Hemicellulose contents in peas range from 23 to 95} g kg–1_{DM and cellulose contents range from 52 to 77 g kg}–1_{DM [71, 98,} 100]. Lupins contain high levels of NSP, with contents of cellulose generally being higher than hemicellulose (131 to 199 vs. 40 to 66 g kg–1_{DM) [71, 94,} 98, 100], and they also have considerable amounts of oligosaccharides [71, 94).

Grain legumes contain a number of secondary plant metabolites, also referred to as bioactive substances, which may exert a wide range of different effects on the animals that consume them [101]. These effects have been described as positive, negative or both [101, 102]. Antinutritional factors (ANFs) in legumes can be divided into several groups based on their chemical and physical properties such as non-protein amino acids, quinolizidine al-kaloids, cyanogenic glycosides, pyrimidine glycosides, isoflavones, tannins, oligosaccharides, saponins, phytates, lectins or protease inhibitors [103]. Faba beans contain antinutritional factors such as vicine and convicine [104], lupins quinolizidine alkaloids [105] and peas lectins and tannins [106]. Since many of the ANFs are toxic, unpalatable or indigestible, their elimination can be achieved by selection of plant genotypes or through post-harvest pro-cessing (germination, boiling, leaching, fermentation, extraction). The main antinutritional substances found in lupin seeds are various alkaloids of the quinolizidine group [72, 107, 108], but the levels of others undesirable constituents, such as phytic acid, oligosaccharides, trypsin inhibitors, and lectins and saponins are lower in comparison with other legumes [109]. Faba beans and peas are devoid of alkaloids [110]. These secondary plant metabolites may be divided into two major categories: a heat-labile group, such as protease inhibitors and lectins, which is sensitive to temperatures eventually occurring during feed processing, and a heat-stable group including condensed tannins, alkaloids, pyrimidine glycosides and saponins which is stable under processing temperatures [111].

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1.2.3. Effect of extrusion on chemical composition of the legume family plants (Fabaceae)

During the extrusion process the temperature, moisture, pressure and shear forces act on the product during relatively short residence time and cause changes in product components [112]. The chemical reactions that take place during extrusion, especially the breakdown of polymeric compounds, depend on many parameters concerning the raw materials, additives, technical equipment, and processing conditions. Rheological and physicochemical properties of the dough, and physical properties of the extruded product, are influenced by different variables such as the type of extruder, feed rate, moisture content, residence time, temperature profile in the different heating zones, screw configuration and its geometry, screw speed etc. [44]. The effect of heat treatment is not always positive. Depending on the nature of raw materials, as well as the conditions applied in heat treatment conducting, it is possible to find both positive and negative effects on product quality. Knowledge on both positive and negative impacts is important for animal feed manufacturers in order to better set up and handle the technological process, as well as for consumers to know what quality of products is available to them [35, 39, 113–115].

Extruding has the following beneficial effects on the feed mixture:1) increased digestibility of components (starch modification, protein denaturation, fineness and solubility of fiber); 2) structuring and forming of individual components and formulations such as fish feed or texturing of high-protein components; 3) high water absorption ability; 4) different shapes of the product; 5) abrasion free pellets; 6) flavor enhancement; 7) destruction of antinutritional and toxic components (trypsin inhibitors, lecitins, gluco-sinolate); 8) inactivation of undesirable enzymes (urease, peroxidase, lipoxi-genase); 9) destruction of microorganisms (bacteria, salmonella, yeasts), etc. [112].

However, changes in the extruder can also have some negative effects: 1) destruction of temperature sensitive vitamins and supplements (vitamins A, C, B1, pigments, etc.); 2) inactivation of enzymes (amylase, phytase); 3) destruction of amino acids (lysine); 4) undesirable substances (Maillard, starch-lipid), etc [112].

Proteins are a group of highly complex organic compounds that are made

up of a sequence of amino acids. Protein nutritional value is dependent on the quantity, digestibility and bioavailability of essential amino acids [15; 116]. Most protein undergo structural unfolding and/or aggregation when subjected to moist heat or shear. This often leads to insolubilization and to inactivation (when the nature molecules posses a biological activity) [31]. Extrusion

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promotes cross-linking and polymerization among proteins and starches to form expanded matrices due to shear, heat pressure and oxygen and thus alters protein structure, solubility and digestibility [117]. Vasanthan et al. [45] reported the interactions between proteins and fiber during extrusion of barley flour, which was observed by analyzing the nitrogen content of the dietary fiber fractions of the extrudates.

Protein concentration, moisture content, and the physical and mechanical parameters of the extruder significantly affect the physical and sensory qualities of extrudates [118]. Therefore, functional properties like expansion index, water absorption and solubility indices of the product determine texture and sensory properties of products [119].

Soybeans and oilseeds or legumes provide a good example of improved protein digestibility and bioavailability of sulphur amino acids through thermal unfolding of the major globulins, and thermal inactivation of trypsin inhibitors and other growth-retarding factors such as lectins [31]. However, extensive lysine loss can take place when legume or cereal legume blends are extruded bunder severe conditions of temperature or shear forces (> 100 rpm) at low moisture (<15%), especially in the presence of reducing sugars (23% glucose, fructose, maltose, lactose) [120]. This damage depends on the maillard condensation between &-NH2 groups of lysine residues and C=O groups of reducing sugars. [31].

Several changes occur during extrusion of which denaturation is undoubtedly the most important. Extrusion may improve protein digestibility by denaturating proteins and exposing enzyme-accessible sites [121–123]. Enzymes and enzyme inhibitors generally lose activity due to denaturation. Protein digestibility value is higher for non-extruded products. The possible cause might be the denaturation of proteins and inactivation of ANFs that impair digestion. The extensive studies have been done and reported on the effects of extrusion on protein nutrition especially for animal feeds and for human weaning foods [124] The extrusion operations have very little effect on the protein denaturation [125]. Maillard reactions occur during extrusion particularly at high barrel temperature, low moisture, and high shear. All processing variables have different effects on protein digestibility [126]. High shear extrusion conditions in particular promote denaturation [127], although mass temperature and moisture are also important factors. In a model system of wheat starch, glucose and lysine, low pH favours Millard reactions, as measured by increased colour [128].

Cooking extruders for processing high-protein materials into palatable foods is very common today. Many new applications have been developed for protein extrusion during the past decade. Improvements in functional characteristics of proteins may be achieved through modification of

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temperature, screw speed, moisture content, and other extrusion parameters [126]

Lipids. The nutritional value of lipids could be affected during extrusion

as a result of oxidation, hydrogenation, isomerization or polymerization [50]. Heat treatments affect the quality of fats in two ways. Large friction and high pressure lead to destruction of cell walls and release of oil from spherosome, which increases oil digestibility. At the same time, complexes of fats and carbohydrates are created and stability is enhanced, i.e. the oxidation processes is prevented, through inactivation of lipolytic enzymes [129, 130].

Oxidation rate is affected by many factors such as fat type, fat content, moisture content and expansion degree where the unsaturation in fats increases the preservation challenge. In addition, trace minerals, iron, in particular, and the use of biological antioxidants may play a significant role in oxidation post-extrusion [131, 132].

Under specific extrusion conditions, complexes of protein or lipid-starch can be formed. For example, high moisture and high temperature conditions can increase the hydrolysis of lipids which increases potential interactions with the side chains of amino acids in proteins. Free fatty acids and polar lipids are especially reactive in these situations. If formation of amylose-lipid complexes does not occur to a large extent, it will not impair the utilization of the fat [133].

Extrusion-inactivation of lipase and lipoxidase helps protect against oxidation during storage. Higher temperatures reduce the lipase activity and moisture level, thereby decreasing favoring free fatty acids development. However, the expanded porous nature of the extrudate causes the feed to be susceptible to the development of oxidation during storage, even though deterioration due to extrusion may not be immediately apparent [31].

Carbohydrates. Berrios et al. [134] determined carbohydrate composition

of raw and extruded pulse flours. Dry pea showed the highest concentration of total available carbohydrates (TAC), followed by chickpea and lentil. Formulated pulse flours demonstrated a beneficial increase in dietary fiber. In addition, carbohydrate quality may be modified by thermo-mechanical treatments through the gelatinization of starch [135] or a shift to the development of resistant starch [136]. Extrusion also increases the soluble fiber content of fibrous materials such as plant cell-wall rich materials, brans and hulls of various cereals and legumes [137–143]

Extrusion processing may change dietary fibre content and composition [144]. The morphology (i.e. size, shape, aspect ratio) of fiber is also modified during extrusion. Redgwell et al. [145] using light microscopy observed that under extrusion, the particle size of citrus fiber was reduced. The decrease in sectional expansion when increasing insoluble dietary fiber content often

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leads to an increase in longitudinal expansion [146, 147]. Recently, it was shown that extrusion technology increases the level of dietary fiber in non-gluten-free ready-to-eat expanded snacks made from cereal and vegetable co-products [148, 149]. An increase in the total dietary fiber (TDF) also was observed in barley and wheat flour upon extrusion [150]. Stojceska et al. [151] reported the advantage of using extrusion processing for increasing dietary fiber level in gluten-free products. The formation of gluten-free expanded products with high dietary fiber levels can be achieved by controlling extrusion conditions, such as temperatures, solid feed rate, and screw speed combinations and the selection of appropriate raw ingredients. Zhang et al. [152] confirmed that extrusion process improves the functionality of soluble dietary fiber in oat bran. The yield, composition, thermal properties, rheological behavior, and functionality of soluble dietary fiber in extruded oat bran were compared with those of soluble dietary fiber in untreated oat bran. During extrusion processing, the extent of soluble dietary fiber increment largely depended on the temperature and pressure in the extruder barrel. The higher the temperature and pressure, the higher the success of breakdown of polysaccharides glucosidic bonds was. This led to the release of oligosaccharides and eventually to the increase in soluble dietary fiber [153].

The extrusion of starchy foods results in gelatinization, partial, or complete destruction of the crystalline structure and molecular fragmentation of starch polymers, as well as protein denaturation, and formation of complexes between starch and lipids and between protein and lipids [154]. Robin et al. [155] investigated starch transformation in bran-enriched extruded wheat flour. Cooking extrusion of wheat flour enriched with wheat bran significantly modified the physicochemical properties of the starch. In the tested extrusion conditions, starch crystallites were fully dissociated. However, a remaining amorphous starch structure that could hydrate, swell, and burst under shear in hot water was observed. The estimated starch solubility was only increased at the highest bran concentration. It showed that higher bran levels led to a higher amount of free water and a decrease in starch glass and melt temperatures. The extrusion cooking reduced the starch and non-starch polysaccharides (NSP) contents in pea and kidney bean seed meals [124], whereas the rumen starch degradability was increased in extruded peas with no effect on milk yield [32].

Vitamins and minerals. Athar et al. [156] studied the effect of extrusion

processing conditions on the stability of vitamins. They observed that extrudates obtained from short barrel (90 mm) extruders had a higher retention rate of B vitamin group (44–62%) compared to 20% for long barrel extruders. Anuonye et al. [157] studied the stability of vitamins during

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extrusion and observed a 6% decrease in Riboflavin (B2), a 86.36% decrease in pyridoxine (B6), and no significant change in ascorbic acid content. Athar et al. [156] observed that the retention of vitamins during the extrusion process is not related to initial levels of the vitamins and varies with the cereal type. High temperature, short-time extrusion cooking is also reported to influence the stability of fat soluble vitamins such as vitamin A and E [158]. For example Zielinski et al. [159] observed a significant decrease (about 63%) in vitamin E content of buckwheat during extrusion cooking. Sensitivity of various forms of vitamin E varies with extrusion process variables for example a significant decrease in a-tocopherol was reported with an increase in extrusion temperature and a significant decrease in g-tocopherol with increase in moisture content during extrusion of grass peas was reported by Grela et al. [160].

Effect of extrusion cooking on mineral bioavailability in pea and kidney bean seed meals was studied by Alonso et al. [124]. Moisture content decreased and iron increased in extruded compared with non-treated seed meals. Starch and NSP were reduced in both pea and kidney bean seed meals. Raffinose, stachyose, and verbascose also dropped in kidney bean meal after extrusion, but only stachyose was reduced by thermal treatment in pea flours. The apparent absorption of Fe, Ca, and P from unsupplemented pea-based diets significantly increased in extruded compared with raw seed meals [124].

Antinutritional factors. Rathod and Annapure [161] observed that

extrusion was the best method to abolish trypsin inhibitors (99.54%), phytic acid (99.30%) and tannin (98.83%) without altering the protein content. Furthermore, it was also found that the associated thermal treatment was most effective in improving protein and starch digestibility (up to 89% and 96%, respectively) when it was compared with traditional thermal processes. Karla et al. [162] observed that the extrusion significantly decreased antinutrients and water solubility, water absorption index, and in vitro protein and starch digestibility were improved by the extrusion process. Moreover, the changes in mycotoxins during extrusion process of cereal-based extruded products has also been reported [163]. Therefore, technological processing methods such as extrusion with high pressure, heat and/or steam is used to minimize the negative effects of anti-nutritional factors in legumes. For example, the extrusion of faba beans decreased the concentrations of trypsin inhibitor, phytic P and resistant starch [164].

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1.2.4. Use of the legume family plants (Fabaceae) in the diets of ruminants

In animal nutrition, grain legumes are mainly used as protein supplements, but also as a valuable energy source, due to their partly high contents of starch (faba beans, peas) and lipids (lupines) [71, 79, 94]. A further search for alternative sources of protein has led to increased interest in the use of legumes in ruminant nutrition [13]. The feasibility of the use of alternative grain legumes in ruminant diets is determined not only by their chemical composition, but also by the rate and extent of degradation of nutrients in the rumen. Dietary proteins that reach the small intestine of ruminants consist of two protein fractions: microbial and protein undegradable at the rumen level. Microbial protein is produced by the action of the rumen flora, which breaks down the dietary protein to peptides, amino acids and ammonia, after which these materials are used for the synthesis of own proteins [165]. In the course of the decomposition and synthesis some losses occur (typically about 20%, but sometimes higher). Thus, reduced amount of amino acids reaches the location where digestion and adoption of proteins occur, which means that the needs of high-yielding meat breeds cannot be satisfied by the microbial protein synthesis from the usual sources of protein and energy [166, 167]. Therefore, in order to ensure optimal pool of amino acids for a particular production, it is necessary to provide protein fraction which avoids degradation of the protein in the rumen (undegradable protein) [168, 169].

The degradability of faba bean, pea and lupin protein in the rumen is often over 80% [1] that is significantly higher than those of soya bean or rapeseed expellers. In addition, the heat treatment of faba beans, peas or lupin seeds to lower ruminal degradability has seldom improved animal performance [1; 82]. It is plausible that the high-protein degradability in the rumen together with suboptimal AA profile in the undegraded protein of alternative grain legume seeds limit their production responses in high-yielding ruminants. Replacing protein in soya bean meal partially or completely with faba beans, blue lupin, white lupin or peas has resulted in rather similar bovine lactation performances [1]. Furthermore, the milk fat concentration of medium chain saturates has been lower and those of cis-9 18:1 and 18:2 n-6 higher in cows fed white lupins seeds relative to soya bean meal [82].

In contrast, the milk production responses of alternative grain legumes are often inferior compared to the rapeseed meal in dairy cow nutrition [1]. Substitution of rapeseed meal with faba beans has typically decreased milk protein yield and increased milk urea concentration and the proportion of N excreted in urine suggesting less efficient use of protein in faba beans than in rapeseed [170], thus leading to increased N emissions from animals. Partial

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or total replacement of soya bean or rapeseed protein by faba beans, lupin seeds or peas has not significantly altered ADG or meat chemical composition in growing sheep or cattle. Besides replacing protein in ruminant diets, starchy faba beans and peas and lupins with higher metabolisable energy content than cereals [1] have potential in replacing cereals as well. Indeed, the substitution of cereal grains by grain legumes in dairy cow diets generally increases milk production [1; 82]. Furthermore, starch in peas and faba beans has lower degradability in the rumen than cereal starch [1] that lowers the risk for acidosis.

Di Francia et al. [171] have assessed the effect of partial replacement of soybean cake with extruded peas in the diet of cows during the first 100 days of lactation, and found that peas construe an attractive source of protein (GMO formulations free) in diets for cows whose production is based on organic principles.

Volpelli et al. [172] investigated that the substitution of a long-used feed such as SBM with flaked pea and faba beans in diets for Reggiana dairy cows did not induce negative effects on concentrate intake, milk yield and composition, and milk aptitude for cheesemaking. Faecal and blood para-meters were also unaffected, and the slight increase in blood urea observed in treated group fed flaxed pea and faba beans, was irregular and within the normal range.

Froidmont and Bartiaux-Thill [173] has examined, that coarsely ground lupin seeds appeared suitable to replace 75% of soybean meal on a DM basis in high-producing dairy cow feed, whereas the protein content of the pea was too low. Lupin protein was used as efficiently as soybean meal protein. Total soybean meal substitution by lupin seeds on a N basis is therefore possible without any loss of milk production. However, lupin seed incorporation in dairy cow feeding should be limited to 6 kg d–1_{in order to avoid an excess of} some dietary fatty acids with practical feeding.

Masoero et al. [33] evaluated the nutritive value of raw, extruded or expanded peas relative to soybean meal in lactating dairy cows feeding. The results of this study shown, that the inclusion of peas, either processed or unprocessed, in diets for lactating dairy cows did not produce negative effects on milk yield, composition and cheese-making properties. The increased milk yield when feeding extruded pea might justify the cost effort of the technological treatment put into the processed feed.

Morbidini et al. [174] has examined the influence of soybean meal and beans on the performance of Italian Merino lambs. It was found that the use of beans in the diet resulted in a depressive effect on the performance of lambs. The explanation lies in the increasing content of NPN compounds and anti-nutritional factors. Hence, the recommendations for the period upon the

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early weaning of lambs is: the use of the rumen undegradable protein in the diet, given that in this period, the rumen is not a fully functional protein synthesis and is less efficient. In addition, for the same reason, in this period, the animals are not able to neutralize the possible anti-nutritive factors. The negative side of legume grains is that many of them contain anti-nutritional factors (lecithin, trypsin inhibitors, tannins, saponins, phytase). However, ruminants are not susceptible to most of them because of microbial meta-bolism and degradation in the rumen [1].

Lanza et al. [175] have conducted a comparative study in order to compare the effects of soybean meal and peas (39 and 18% in the mixture), on fattening performance and meat quality of Barbaresa lambs. Protein source showed no significant impact on average daily gain (0.218, 0.29 and 0.250, respectively), feed conversion ratio (4.7, 4.8 and 4.1 kg, respectively), dressing percentage (50.1, 50.8 and 51.2%, respectively), as well as the physical and chemical properties of M. longissimus dorsi.

Similar studies were conducted by Cutrignelli et al. [176] on two groups of bulls, average age of 129 days, fed diets based on bean and soybean meal, weighing up to 620 kg. Source of protein has not significantly affected other parameters, except body weight (173 and 186 kg, in treatment with beans and soybean meal, respectively). The chemical composition of the Muscullus

longissimus thoracis, amino acid composition, cholesterol content and

sensory profile were not affected by the tested treatment. These results show that the beans can be used as an alternative source of protein, with no adverse effects on the biological effectiveness of growth, feed conversion index and meat quality of ruminants. Nunes [177] characterized the common bean as a product of low acceptability and digestibility, with recommendations for inclusion of up to 15% in concentrates for fattening cattle.

Magalhães et al. [178] evaluated the inclusion of common beans (0%, 13%, 26% and 39%) in concentrates for lactating cows and found that the inclusion of beans resulted in the reduction of milk production of the animals.

De Tonissi e Buschinelli de Goes et al. [179] indicated the inclusion of beans in the diet for cattle in feedlot did not affect animal performance, interfering in fermentation patterns and nutrient digestibility.

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2. MATERIALS AND METHODS

2.1. Investigation venue, time, objects and the experimental phase The scientific work was carried out in the Animal and Aquaculture Productivity and Product Quality Assessment Laboratory of Institute of Animal Rearing Technologies of Veterinary Academy of Lithuanian University of Health Sciences (LSMU), The Research Centre of Digestive Physiology and Pathology of the Department of Anatomy and Physiology, Practical Training and Trial Centre, Institute of Animal Science, Food institute of Kaunas University of Technology (KTU), State Enterprise “Pieno tyrimai”, AB “Kauno grūdai” and dairy cattle farm of JSC “AUGA Grūduva” in the period 2014–2020.

The directions of activity in which the objectives of the work were imple-mented are depicted in the principle scheme of this work (Fig. 2.1.1.). As it can be observed from Fig. 2.1.1 outline, the thesis study was carried out in two stages. In the first stage, the chemical, fatty acids and amino acids composition of diverse species of legumes were analysed before and after the extrusion process. A feeding trial was subsequently carried out on dairy cows using diverse species of extruded legumes in their diet, and the effects on cow performance, milk composition, sensory properties, fermentation processes in the cows’ rumen and blood parameters were assessed. The first stage of the study aimed at finding out which species of extruded legumes are most suitable for use in dairy cows’ diets, replacing extruded soybeans without compromising the performanse, milk composition and physiological processes. After evaluation of the results, it was found that extruded faba beans are the most appropriate in the diet of dairy cows, but it is not clear whether the results are dependent on the extruded process used to treat the faba beans before the trial, thus, it was decided to carry out further studies.

In the second stage, the chemical, fatty and amino acid composition of faba beans were analysed before and after the extrusion process. A feeding trial was subsequently carried out on dairy cows using untreated and extruded faba beans in their diet, assessing their effects on cow performance, milk composition, sensory and technological properties, as well as the composition of milk fatty and amino acids and the effects on the formation of volatile flavour compounds in milk.

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Fig. 2.1.1. The basic outline of the thesis

STAGE I

Chemical, amino acids and fatty acids analysis of untreated and extruded soybeans ‘Amadine’, lupine ‘Vilniai’, faba beans ‘Bioro’ and peas ‘Casablanca’

Feeding trial with dairy cows, in the diets partially replacing extruded soybeans with extruded diverse plants of the legume family

effect of partial replacement of extruded soybeans with extruded lupines, faba beans and peas on the productivity of dairy cows

effect of partial replacement of extruded soybeans with extruded lupines, faba beans and peas on milk composition and sensory properties of dairy

cows

effects of partial replacement of extruded soybeans with extruded lupines, faba beans and peas on fermentation processes in the rumen and

health ofcdairy cows STAGE II

Chemical, amino acids and fatty acids analysis of extruded and raw faba beans of cultivar ‘Fuego’ Feeding trial with dairy cows, when diets supplemented with untreated and extruded faba beans

effect of extruded faba beans on the productivity of dairy cows effect of extruded faba beans on composition, sensory and technological

properties of dairy cow milk

effects of extruded faba beans on the milk amino acids and fatty acids composition and the formation of volatile flavour compounds

ANALYSIS OF CHEMICAL COMPOSITION INDICATORS OF EXTRUDED LUPINES (LUPINUS L.), FABA BEANS (VICIA FABA L.) AND PEAS (PISUM SATIVUM L.) AND EFFICIENCY OF THEIR USE IN THE NUTRITION OF DAIRY COWS

Ieva Kudlinskienė