BIOTECHNOLOGICAL SOLUTIONS FOR PREPARATION OF SUSTAINABLE AND SAFE PLANT PROTEINS THROUGH THE USE OF LOW-WASTE AND/OR NON-WASTE TECHNOLOGIES

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Vytautė Šakienė

BIOTECHNOLOGICAL SOLUTIONS

FOR PREPARATION OF SUSTAINABLE

AND SAFE PLANT PROTEINS THROUGH

THE USE OF LOW-WASTE AND/OR

NON-WASTE TECHNOLOGIES

Doctoral Dissertation Agricultural Sciences, Zootechnics (03A) Kaunas, 2018 1

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Dissertation has been prepared at the Department of Food Safety and Quality of Veterinary Academy of Lithuanian University of Health Sciences during the period of 2014–2018.

Scientific Supervisor

Prof. Dr. Elena Bartkienė (Lithuanian University of Health Sciences, Veterinary Academy, Agricultural Sciences, Zootechnics – 03A).

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

Assoc. Prof. Dr. Agila Daukšienė (Lithuanian University of Health Sciences, Agricultural Sciences, Zootechnics – 03A).

Members:

Prof. Dr. Asta Racevičiūtė-Stupelienė (Lithuanian University of Health Sciences, Agricultural Sciences, Zootechnics – 03A);

Dr. Violeta Razmaitė (Lithuanian University of Health Sciences, Agri-cultural Sciences, Zootechnics – 03A);

Assoc. Prof. Dr. Vilma Kaškonienė (Vytautas Magnus University, Physical Sciences, Chemistry – 03P);

Dr. Damian Escribano Tortosa (Autonomy University of Barcelona, Agricultural Sciences, Veterinary – 02A).

Dissertation will be defended at the open session of the Zootechnics Research Council of Lithuanian University of Health Sciences on the 7th of September, at 12:00 a.m. in Dr. S. Jankauskas Auditorium of the Veterinary Academy.

Address: Tilžės 18, LT-47181 Kaunas, Lithuania. 2

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA

Vytautė Šakienė

BIOTECHNOLOGINIAI SPRENDIMAI

TVARIŲ IR SAUGIŲ AUGALINIŲ

BALTYMŲ IŠGAVIMUI TAIKANT

BEATLIEKINES IR MAŽAATLIEKINES

GAMYBOS TECHNOLOGIJAS

Daktaro disertacija Žemės ūkio mokslai,

Zootechnika (03A)

Kaunas, 2018 3

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Disertacija rengta 2014–2018 metais Lietuvos sveikatos mokslų universitete Veterinarijos akademijos Maisto saugos ir kokybės katedroje.

Mokslinė vadovė

Prof. Dr. Elena Bartkienė (Lietuvos sveikatos mokslų universitetas, Vete-rinarijos akademija, Žemės ūkio mokslai, Zootechnika – 03A).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterina-rijos akademijos Zootechnikos mokslo krypties taryboje:

Pirmininkė

Doc. Dr. Agila Daukšienė (Lietuvos sveikatos mokslų universitetas, Žemės ūkio mokslai, Zootechnika – 03A).

Nariai:

Prof. Dr. Asta Racevičiūtė-Stupelienė (Lietuvos sveikatos mokslų universitetas, Žemės ūkio mokslai, Zootechnika – 03A);

Dr. Violeta Razmaitė (Lietuvos sveikatos mokslų universitetas, Žemės ūkio mokslai, Zootechnika – 03A);

Doc. Dr. Vilma Kaškonienė (Vytauto Didžiojo universitetas, Fiziniai mokslai, Chemija – 03P);

Dr. Damián Escribano Tortosa (Barselonos autonominis universitetas, Žemės ūkio mokslai, Veterinarija – 02A).

Disertacija ginama viešame Zootechnikos mokslo krypties tarybos posė-dyje 2018 m. rugsėjo 7 d. 12 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Dr. S. Jankausko auditorijoje.

Adresas: Tilžės g. 18, LT-47181 Kaunas, Lietuva.

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Wholemeal and Protein Isolates/Concentrates ... 78 3.5.1. The Total Amino Acids Content in Lupine Wholemeal Samples .... 78 3.5.2. The Free Amino Acids Content in Lupine Wholemeal Samples ... 94 3.5.3. The Amino Acids Profile in Untreated and Biotreated Lupine Protein Isolates/Concentrates ... 111 3.5.4. The Essential and Nonessential Amino Acids – Possibility

to Modulate Their Profile in Lupine Seeds wholemeal and Their

Isolates/Concentrates ... 129 3.6. The Influence of Technological Factors on Formation of Biogenic

Amines in Lupine Products ... 132 3.6.1. The Biogenic Amines Content in Untreated and Biotreated

Lupine Wholemeal ... 132 3.6.2. The Influence of Fermentation on the Biogenic Amines

Content in Lupine Protein Isolates/Concentrates ... 143 3.6.3. The Formation of Biogenic Amines in Plant Based Substrates and Factors Influencing Their Formation ... 152 3.7.The Total Phenolic Compounds Content in Lupine Wholemeal and

Protein Isolates/Concentrates and Their Antioxidant Properties ... 155 3.7.1. The Formation of Antioxidant Properties Showing Compounds and Their Modulation by Using Fermentation ... 160 3.8. Influence of Technological Factors on Isoflavones Content in Lupine Bioproducts ... 162

3.8.1. Isoflavones in Lupine Seeds and Their Possible Changes

During Technological Processes ... 164 3.9. The Trypsin Inhibitors Activity in Lupine Products ... 166

3.9.1. The Perspectives of Reduction of Trypsin Inhibitors in Plant

Based Material ... 168 3.10. The Digestibility In Vitro of Lupine Wholemeal Protein and Protein Isolates/Concentrates ... 168

3.10.1. The Digestibility of Lupine Seeds Protein and Technologies to Improve It ... 171 CONCLUSIONS ... 173 PRACTICAL RECOMMENDATIONS ... 176

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REFERENCES ... 177 PUBLICATIONS ... 203 SUMMARY ... 301 ANNEX ... 331 CURRICULUMVITAE ... 333 ACKNOWLEDGEMENTS ... 334 7

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ABBREVIATIONS

AA – Amino Acids

ANFs – Antinutritional Factors

BAs – Biogenic Amines

BIOR – Institute of Food Safety, Animal Health and Environment

B.w. – Body Weight

CAD – Cadaverine

CFU – Colony-Forming Units

DM – Dry Matter

DW – Dry Water

GC/MS – Gas Chromatography – Mass Spectrometry

HPLC-MS/MS – High-Performance Liquid Chromatography – Mass Spectrometry

HIS – Histamine

GC-FID – Gas Chromatography-Flame Ionization Detector

TRP – Tryptamine PEA – Phenylethylamine VAL – Valine ALA – Alanine GLY – Glycine ILE – Isoleucine LEU – Leucine THR – Threonine HIS – Histamine TYM – Tyramine

KTU – Kaunas University of Technology LAB – Lactic Acid Bacteria

Ls – Lactobacillus sakei

LUHS VA – Lithuanian University of Health Sciences Veterinary Academy

MRS – de Man Rogosa Sharpe

Pa – Pediococcus acidilactici PHE – Phenylethylamine Pp – Pediococcus pentosaceus PUT – Putrescine SMF – Submerged Fermentation SPRM – Spermine SPRMD – Spermidine

SSF – Solid State Fermentation

NF – Nonfermented samples

TIA – Trypsin inhibitors activity TTA – Total Titratable Acidity

SER – Serine PRO – Proline ASP – Asparagine TYR – Tyramine MET – Methionine LYS – Lysine 8

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INTRODUCTION

Global climate change will present ever increasing challenges in margi-nal, already-stressed agricultural ecosystems; also, the climate change is another factor affecting the goal of feeding the world [1–4]. Globally, 76% of the population derives most of their daily protein obtained from plants [4]. Nevertheless, many people are suffering from a lack or protein malnutrition, especially children [5]. With projected population growth to 9.5 billion by 2050 [6], alongside dietary and demographic changes, future nutritional demands may overwhelm global production of crop [7]. Animal proteins have a competitive advantage over plant-based proteins in terms of their nutritional and functional properties; protein ingredient market is intensively seeking for alternative, underutilized sources of concentrated plant proteins in order to satisfy the demands of consumers with different ethnic, religious, dietary and moral preferences associated with consumption of animal-based products. The demand of the ever-growing world population for protein foods is no longer sustainable through animal products alone. To compensate this deficiency, soya bean has become the prevalent source of plant proteins used for food/feed. Plant proteins are generally of a lower nutritional quality compared to animal proteins due to limited essential amino acids (AA) (lysine in cereals, methionine in legumes) and poor digestibility [8, 9], while animal proteins such as eggs and meat are highly digestible [9]. In recent years, the interest in plant protein sources that could replace soybean meal used for animal nutrition has increased. Soybean meal is the most common plant protein source in animal nutrition. It can be used as the only protein source in diets due to the low level of antinutritional substances and high level of crude protein [10], as well as good amino acid profile, however, soybean is genetically modified [11]. Lupine seeds is a significant alternative to soybean in animal nutrition [12], and the growth of plant protein utilisation has led to the development of new technologies, created to increase bio-availability of plant based protein. There are numerous reasons for increasing global demand of novel, sustainable sources of proteins. Moreover, a high consumption of animal proteins increases gas emission and thus represents an ecological issue [13]. To compare plant protein and animal protein production , the latter is more expensive due to water and energy resources; therefore it is important to find a balance between animal and plant protein in sustainable food/feed systems [14]. The growing demand of plant protein isolates/ concentrates is based on their good functional and technological properties, such as solubility, foaming properties, emulsion stability and viscosity. The

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functional properties of protein can be affected by different protein extraction techniques, protein composition etc. Accordingly, Europe has become heavily dependant on soya bean imports, entailing trade agreements and quality standards that do not fully satisfy the European citizen’s expectations, hence lupine could be established as fundamental crop in various agro-climatic zones and marginal lands of Europe, and their yields and adaptation could be genetically improved to ensure the continuous supply of high-quality grain [15]. Lupine, an autochthonous European legume crop, represents a significant alternative to soya bean due to the following characterisics: it has elevated high-quality protein content (up to 44%); it is well accepted by European consumers, partially as it is not genetically modified; it has high potential in health benefits; and it is well suited for sustainable cultivation. Lupine is a successful protein crop in Australia, where an important industry related to lupine protein has spawned. However, its cultivation in Baltic countries remains insufficient to guarantee a steady supply to the food/feed industry, which in turn must be developed and innovated to produce attractive lupine-based protein-rich food/feed. Yellow lupines were bred in Lithuania until 1995. The narrow-leaved lupine breeding program took off in 1995. The breeding was done in three directions: first, low-alkaloid narrow-leaved lupine varieties were bred for food industry, second – low-alkaloid narrow-leaved lupines were bred for animal feeding, and third – narrow lupines were bred for green manure. The use of low alkaloid narrow-leaved lupine varieties for production of higher value food would be very important for all Baltic region countries. Lupine is a source of seeds that are rich in protein and could be used for animal feeding. However, the high content of alkaloids (from 5 g/kg to 40 g/kg) has been one of the factors limiting the use of white lupine (L. albus) in the past. Alkaloids are compounds that have negative effects on feed intake and nutrient utilisation. Over recent decades, plant breeders have succeeded in developing lupine cultivars with alkaloid content which is close to zero (0.08 g/kg to 0.12 g/kg) [16]. Also, lupine can be considered as an alternative protein-rich crop, capable of promoting socio-economic growth and environmental benefits in Europe [15]. Lupine seeds contain large amount of soluble and insoluble non-starch polysaccharides, oligo-saccharides, phytates and tannins that have anti-nutritional effects including reduced digestion and absorption of amino acids [17, 18]. The antinutri-tional factors (ANFs), such as trypsin inhibitors, phytic acid, saponins, heamagglutinins and tannins are undesirable components in legumes that could hinder utilization of important minerals, particularly calcium, magne-sium, iron and zinc. It interferes with their absorption and utilization and thereby contributes to mineral deficiency [19]. Processing of legumes can

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reduce ANFs. Milling can reduce the levels of iron, zinc and phytate, however, the remaining iron and zinc is more bioavailable; fermentation can degrade phytate and enchance iron and zinc absorption because during the fermentation process low molecular weight organic acids are produced [20– 22]. The isolation and/or concentration of plant proteins reduce ANFs, also the extraction of proteins can improve digestibility, which is similar to animal-based protein [9].

The Aim of the Study

The aim of this work was to evaluate detailed chemical composition of the lupine seed varieties bred newly and locally; and to develop high-value, sustainable and safe plant protein stock/products by applying biotech-nological treatment for whole seed (non-waste technology) and protein isolates/concentrates (low-waste technology).

Objectives of the Study

1. To select samples from the lupine seed varieties that are newly bred in Lithuania and contain the lowest concentration of alkaloids, and to evaluate their detailed chemical composition.

2. To select the most effective conditions of treatment to increase protein digestibility and reduce activity of trypsin inhibitors, by applying sub-merged and solid state fermentation with bacteriocins that produce lactic acid bacteria strains.

3. To evaluate the influence of technological factors on the changes of total phenolic compounds and isoflavones contents, as well as on antioxidant properties of the treated substrate.

4. To perform protein isolation/concentration by analysing their solubility at different pH conditions to obtain the highest protein yield.

5. To evaluate the influence of microbial hydrolysis on the amino acids profile and molecular weight of protein in lupine seeds wholemeal and protein isolates/concentrates.

6. To indicate the influence of technological factors on biogenic amines and formation of D(–) lactic acid isomers in lupine bioproducts.

The Scientific Novelty and Practical Usefulness

The combination of innovative and sustainable processing proposed in this disertation work will allow the researcher to select newly bred lupine varieties and microorganisms used to produce highly proteinaceous products

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with improved properties, as well as potentially novel products with high biofunctionality. Sustainable agriculture that could satisfy the growing European and Global demand for food/feed is becoming a manifest neces-sity in the light of the concerns of growing population, land-use and food/ feed safety. The results of this work will reformulate the prototypes of food/feed stock with high biological value. The newly developed stock with high biological value and their preparation technologies are expected to rapidly reach the populations of defined niche, those who are rejecting the consumption of genetically modified plants and those who prefer convenient and natural food. The main consideration of this scientific novelty and practical benefits of this work are the following: the food/feed stock of higher value can be produced in an environmentally and socio-economically sustainable and beneficial manner, ensuring its safety at global level and its acceptance by the consumer, resulting in impact on the overall sustainability of our society.

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1. LITERATURE REVIEW

1.1. The Perspectives of the Lupine Seeds in Europe

The most important lupine varieties for the food and feed industries are L. angustifolius, L. albus, L. luteus and L. mutabilis [23]. Unlike soybean, lupine seeds can be grown in moderate European climate [15]. The technological properties of lupine seeds are suitable for industrial processing [24]. It is reported in various publications that lupine is high in protein (20– 48%), high in fibre,low in fat and has negligible amount of starch; also, lupine seeds are good sources of vitamins, minerals and bioactive compounds [25–31]. Röder et al. [32] reported that protein content in blue lupine seed is 22–29.8%, 30.1–31.2% in white lupine and 35.2–38.1% in yellow lupine. The high protein content is mainly composed of seed storage proteins, particularly globulins and represent approximately 87% of the total protein content [33]. Seed storage proteins (α-conglutin, β-conglutin, γ-conglutin, and δ-conglutin) account for the main protein fractions in legumes [27, 33, 34]. The globulins are further subdivided into α-conglutin (3–4%), β-conglutin (43–45%), γ-conglutin (5–6%), and δ-conglutin (3– 13%) [27, 33, 34]. However, lupine can cause severe allergic reactions [35– 37], and Jimenez-Lopez et al. [38] reported that β-conglutin has been identified as a major allergen in L. angustifolius seeds.

Lupine, an autochthonous European legume crop, represents a significant alternative to the soya bean for the following benefits: it is rich in high-quality protein content; it is well accepted by European consumers as it is not genetically modified; it has a good potential in health benefits; and it is highly suitable for sustainable cultivation. Lupine is a successful protein crop in Australia, where it has spawned an important industry, related to lupine protein. However, its cultivation in European countries remains insufficient to ensure a steady supply to the food/feed industry, which in turn must be developed and innovated in order to produce attractive lupine-based protein-rich food/feed.

1.2. The Chemical Composition of Lupine Seeds

The main nutrients of lupine seeds. Bähr et al. [39] has reported that lupine seeds can be an alternative to soya, as they contain comparable amounts of protein of a similar AAs profile, but higher fiber content, compared to beans, which is favorable from the dietary point of view for humans, but not for animals. The lupine seeds typically involve 33–47% of

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proteins and 6–13% of oil [40, 16], while the content in dietary fiber ranges from 34–39% [41]. In comparison with other bioactive compounds present in lupine seeds, phenolic compounds are primarily responsible for their antioxidant capacity [42]. The most abundant phenolic compounds detected in lupine seeds belong to the subclasses of phenolic acids, flavones and isoflavones [43]. Lupine is rich in phytochemicals (polyphenols, phytoste-rols, squalene) compared to other legumes [44]. Fiber and protein in lupine seeds are associated with prevention of chronic diseases and improvement of animal health [42]. The digestible carbohydrate content in lupine is lower than in most legumes and comprises oligosaccharides mostly, whereas starch is absent or scarce. The fat content is variable, falling in the interval of 8–12% depending on species, with a high presence of α-linolenic acid (about 8–10% of the oil) [45, 46]. The polysaccharide content in lupine cotyledons mainly consists of galactan and the hull consists of cellulose and/or hemicellulose mostly. The lupine hull comprises 25% of the whole seed and is low in lignin [48]. Lupine kernel is also an excellent source of fibre, containing up to 39% of fibre, composed of 75–80% of soluble fibre, 18–25% of insoluble fibre, and 5–9% of hemicellulose [39].

Apart from its useful nutritional features, it is claimed that lupine is beneficial to management of hyperglycemia [47], prevention of hyperten-sion [49] and cholesterol lowering [50]; what is more, protein seems to be relevant to these beneficial effects [25].

Antinutritional compounds and/or factors (ANF) in lupine seeds. The bioactive phytochemicals in legumes include: enzyme inhibitors, phyto-estrogens, oligosaccharides, phytosterols, saponins, phytates, phenolic acids and flavanoids. The proteinaceous antinutritonal factors (ANF) include lectins, protease (trypsin, chymotrypsin) and amylase inhibitors and lipoxy-genase. Non-proteinaceous compounds include phytic acid, α-galactosides, phenolics, tannins, saponins, cyanogens and toxic AAs [4]. The amount of ANFs, such as alkaloids, saponins, tannins, phytate, trypsin inhibitors (TIs) and lectins is lower in lupine than in other legumes [51]. The polysaccharide content in lupine cotyledons mainly consists of galactan and the hull mainly consists of cellulose or hemicelluloses [52]. Flavonoids and phenolic acids are rich in antioxidant and good physiological and biological properties [53], however, phytic acid is one of the most ANFs in plants. The anti-nutritional activity of phytic acid can be eliminated by adding phytase. Phytic acid or phytate in legume seeds is bound with phosphorus, calcium and magnesium, trace elements, such as iron and zinc, protein compounds and AAs [54].

Saponins are amphiphilic compounds composed of saccharide chains (hexoses and pentoses) soluble in polar water with a non-polar (fat soluble)

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aglycones attached to them [55]. Its glycosylated compounds or glycosides are divided into three main groups, according to the carbon skeleton, of non-polar aglycone region: triterpenoidal glycosides, steroidal glycosides and steroid-alkaloid glycosides [56]. Lupine, beans and peas are the main sour-ces of dietary saponins [57].

Trypsin inhibitors (TIs) are one of the most relevant ANF, because they reduce digestion and absorption of dietary proteins [58]. TIs strongly inhibit the activity of key pancreatic enzymes, particularly trypsin and chymotryp-sin, thereby reducing digestion and absorption of dietary proteins by the formation of complexes that are indigestible even in the presence of high amounts of digestiveenzymes [59].

Other ANFs in lupine seeds are alkaloids. The varieties of L. albus with favorable agronomic characteristics contain toxic quinolizidine alkaloids (1.9–2.7%), which are not suitable for animal consumption [60]. The ANF presence in seeds limits levels of inclusion of legume seeds in diets, espe-cially for young, growing animals [61, 62]. Total alkaloid content in sweet white lupine cultivars has been significantly reduced during the process of domestication and breeding and currently does not exceed 0.02% [63]. Analysis of different qualitative composition of major alkaloids in the seeds of narrow-leafed lupine and was performed, with then following results (% of total alkaloids): lupanine – 46.4%, hydroxylupanine – 35.6%, angusti-foline – 15.5%, and α-isolupanine – 2.5% [64].

Under the environmental condition of high temperature, the accumulation of alkaloids may vary and the alkaloid quantity in sweet lupine seeds may exceed the alkaloid limit for using lupine seeds as a feedstuff; however, pigs are particularly sensitive to alkaloids [65].

Apart from possible reduction of alkaloids during fermentation process, further benefits, such as physiological effects of lactic acid silages of moist lupine grains are the following: the positive effects of lactic acid in terms of physiology of nutrition, for example, the acidification of certain parts of the digestive tract to prevent the proliferation of clostridia and other pathogenic microorganisms; the improvement of the feed quality due to the reduction of other ANF such as oligosaccharides [66, 67]; the elevated contents of essen-tial AA following proteolysis (only when desmolysis can be prevented) [68]; the improvement of feed intake and digestion [69]; the improvement of digestibility of AA (demonstrated for LYS and MET [69]; the possibility to upgrade the quantity of lupine grains in daily ration (broiler, 10%; weaned piglets, 12%) resulting in lower consumption of soy products [70].

Lupine (L. angustifolius) has been identified as a potential source of protein of locally produced plants that could be fed to animals at a positive economic profit and could replace soybean oilcake meals as a raw material

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in ostrich diets. Currently, the price of L. angustifolius is estimated to be 56% of the value of soybean oilcake meal, making it a worthwhile economic alternative. If nutrition expenses could be reduced, this would have a major impact on the profits of the commercial ostrich production enterprise. Concerns have been raised due to the high presence of alkaloids in lupine seeds. Alkaloids are compounds that have a bitter taste which reduces the palatability of the feed. However, there are sweet (low in alkaloids, <0.1%) and bitter (alkaloid-rich, 0.1–4%) varieties within the species [71]. Nonethe-less, lupine can still be included only up to certain levels to be utilized efficiently and to prevent undesirable effects [72]. Lupanine, 13α-hydroxy-lupanine and angustifoline are the main alkaloids of L. angustifolius seeds [73].

Other group of specific compounds in lupine seeds are phytoestrogens. Phytoestrogens are non-steroidal plant compounds and have structure similar to mammalian estrogen (17-β estradiol). Phytoestrogens and their mammalian metabolites can bind to estrogen receptors found in animal and human cells, and cause a weak estrogenic or anti-estrogenic effect. Intake of phytoestrogens may impair sheep fertility, whereas effects in cattle repro-duction are not consistent. Isoflavones and lignans are metabolised to equol and enterolactone in rumen [74]. Isoflavones exist in plant tissues as the variety of O- or C-glycosylated derivatives, often acylated with malonyl group on sugar moieties. Free aglycones are released in the cells that are under abiotic or biotic stress [75]. Genistein belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants [76]. The Chemical structure of main isofla-vones present in lupine seeds is given in figure 1.2.1. Genistein is a phyto-estrogen, a family of plant-derived compounds that exhibit effects similar to, albeit weaker than, those of mammalian estrogens [77]. Schreihofer et al. [78] has reported that genistein protects neurons from cerebral ischemic injury in rat hippocampus, thus exerting neuroprotective effects in stroke-like injury in vitro. In the brain, genistein can improve spine thickness, as well as their cognitive function, synapse development and regulate the transcription factor of neurotrophic genes in the hippocampal region of adult animals [79]. Also, isoflavones exhibit antifungal activities [80].

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Biochanin-A Daidzein

Genistein Formononetin

Fig. 1.2.1. The chemical structure of main isoflavones present

in lupine seeds

1.3. The Processes of Increasing the Nutritional Value of Lupine Seeds

The nutritional value of legumes depends upon the processing methods, presence or absence of anti-nutritional and/or toxic factors and possible interaction of nutrients with other food/feed components [81]. Roasting improves colour, extends life of shelf, enhances flavor, reduces the ANFs and denatures proteins thereby improving their digestibility [82]. Neverthe-less, treatments such as heat, germination, soaking and fermentation have been reported to reduce the ANFs [83]. It could lead to conclusion that the methods of mechanical, physical and biological processing can help to improve the nutritional value of lupine seeds used in pig nutrition [84, 85], or even affect the nutritional value of complex diets. Other studies have shown that the reduction of the particle size [86], germination [87], hydro-thermal treatment [88] or dehulling [89] can improve nutrient digestibility of lupine seeds in the ileal and total tract in pigs. Despite the above-mentioned information regarding the nutrient digestibility of differentially processed blue lupine meals, there is a lack of information regarding the breakdown of NSP structures and the resulting proximate nutrient, AA and NSP digestibility in complex diets along with gastrointestinal tract of the pig. As indicated by Zijlstra et al. [90], feed processing and enzyme tech-nologies can be valuable tools to enhance digestive utilization of nutrients in NSP-rich feedstuffs. Rutkowski et al. [91] has published that the use of extrusion of yellow lupine seeds had led to relatively minimal changes in nutrient composition, including AAs and ANFs.

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1.4. Lactic Acid Fermentation in Feed Industry – Formation of Desirable and Undesirable Compounds

Lactic acid bacteria (LAB) is a wide variety of bacteria that can produce bacteriocins, and it is mainly Gram-positive [92]. LAB are classified as either homofermentative or heterofermentative based on the metabolism of glucose. Although the terms are typically applied to LAB, many other organisms that generate lactic acid share the features of the same pathways and can be considered to be homofermentative or heterofermentative. As the name implies, a homofermentative lactic acid process can potentially ge-nerate lactate [92]. Fermentation is an easy and economic method to improve nutritional value of feed [93]. Lactobacilli is the most important bacteria for industrial applications related to feed and animal health [94], as well as, fermentation is a major process used in the production of foods from soybeans [95]. Fermentation leads to changes of the physicochemical and sensory properties (colour, flavour) and bioactive compounds of soybean [96]. Fermentation reduces undesirable compounds in legumes and other feed ingredients [97] and lowers the content of flatulence-causing factors in legumes; what is more, it increases protein concentration and improves protein digestibility through the hydrolysis of high-molecular-weight proteins into peptides and AA [98]. According to Amadou et al. [100], fermentation increases trypsin digestibility in vitro and nitrogen solubility under alkaline conditions. A wide variety of microorganisms, mainly yeasts and fungi, are used in the fermentation process. LAB, in-cluding Lactobacilllus, Lactococcus, Streptococcus, Leuconostoc and Pediococcus, are also applied due to their unique characteristic to formate precursors of aromatic compounds and texture forming compounds [99, 100]. During the fermentation of plant based substrates, the growth of LAB enhances conversion of phenolic compounds such as flavonoids to biolo-gically active metabolites via expression of glycosyl hydrolase, esterase, decarboxylase, and phenolic acid reductase [101]. The subsequent reaction of these metabolites with anthocyanidins results in formation of pyrano-anthocyanidins or 3-desoxypyranopyrano-anthocyanidins [102]. Some of these alkyl catechols potently activate Nrf2 (NFE2L2), the main regulator of response to oxidative stress in mammalian cells and thereby induce the expression of antioxidant and detoxifying enzymes protecting against oxidative and chemical damage. Additionally, fermentation can result in the removal of toxic or undesirable constituents such as phytic acid. This plant-associated, anti-nutritional compound chelates divalent metal ions. Fermentation reduces the pH of fermentable substrate, which optimizes the activity of endogenous phytase thus removing the most phytic acid [103,

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104]. AA and its derivatives together with neurotransmitter (e.g. g-ami-nobutyric acid) and immunomodulatory functions are also synthesized during fermentation [105]. Fermentation process can also result in the new compounds with potential health-modulating effects. In addition, fer-mentation can increase the total amount of acids, free AA, antioxidants and other biologically active substances [106]. Fermentation results in the degra-dation of plant cell walls, which can release antioxidants [107, 108]. Modifi-cation of proteins also can be performed by using fermentation processes. For this reason, fermentation is a useful technology for increasing the amount of natural bioactive compounds [110]. The main microbial agents in food and feed technology are LAB, which play an important role in the modification of isoflavone conjugates. It is indicated in the literature sources that fermentation increases the bioavailability of isoflavones [111]. However, some technological processes can also result in significant losses of isoflavones [112].

Lactic acid is the main metabolite in LAB fermentations that is synthesized in amounts often exceeding 1%. Lactic acid was recently evi-denced to reduce pro-inflammatory cytokine secretion of toll-like receptor-activated, bone-marrow-derived macrophages and dendritic cells in the dose-dependent manner [113, 114]. Lactic acid also alters redox status by reducing the burden of reactive oxygen species in intestinal enterocytes [114]. Through LAB metabolic activities (e.g., lipolysis and proteolysis), they also produce important aroma and ﬂavor compounds, while contribut-ing to the texture formation (e.g., by the production of exopolysaccharides) [115, 116]. Lactic acid imparts a sour taste which is an important sensory attribute of LAB fermentation [117]. Supplementation of LAB in neonatal piglets can regulate the formation of the piglet gut microflora, thus benefiting the health of piglets [118]. LAB can inhibit pathogenic bacteria by competing for nutrients in the gut or for binding sites on the intestinal epithelium [119]. However, the metabolic activity of the LAB may also rise the formation of biogenic amines (BAs), and the concentration of AA has an effect on the overall formation of BAs [120]. From this point of view, the fermentation process should be controlled to minimize potential enhan-cement effects on BAs formation; in this case, safe microbial starters should be used [121].

The main metabolite of LAB is the lactic acid, which can be obtained as two optical isomers, particularly L(+) and D(-) and/or their mixture. Increased levels of D(-) isomer in plasma and urine have been demonstrated in cases of intestinal ischaemia, short bowel and appendicitis, and are con-sidered as the indicator of dysbiosis and/or increased intestinal permeability [122]. Therefore, the desirable lactic acid isomer in fermentation of food

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and feed is L(+) [123]. LAB also produce organic acids with several com-pounds, hydrogen peroxide, diacetyl, acetaldehyde, D(-) isomer of AA, reuterin and bacteriocins [124]. D(-) isomer can be produced during fermen-tation by using Lactobacillus spp., hence glucose and glycerol are the main sources of carbon used for fermentation of D(-) isomer [125]. The silage used for ruminant nutrition may contain considerable amounts of D(-) isomer [2]. However, the main fraction is metabolised or converted to the L(+) in rumen which seldom leads to D(-) acidosis and neurotoxity. D(-) isomers in the mammalian body are mainly of microbiological origin and they are often located in the digestive tract. Consequently, the control of D(-) in feed is very important. Several analytical procedures for D(-D(-) isomer have been introduced, but it is absolutely mandatory to distinguish this metabolite from the much more abundant and naturally occurring L(+) stereoisomer. When enzymatic analytical methods are used, it is conse-quently essential to eliminate the response from L(+) and the dehydrogenase of ubiquitous enzyme L(+), which will interfere with the D(–) isomer determination heavily [3]. The fermentation is very important in the process of ensiling because it affects the nutritional quality of the silage and the animal performance. If the fermentation is not effective, it will result in a completely spoiled feed that has potential risk for animal health. Well-fermented silage can be used in diets for ruminant animals without any risk for their health and without compromising the productive performance [126]. The silage metabolites, such as organic acids (lactic acid, acetic acid etc.) formation is important for the fermentation quality of ensiled forage [127]. The main genera of LAB commonly associated with silage fermen-tation are Lactobacillus, Pediococcus, Leuconostoc and Lactococcus [126, 128]. The lactic acid production and the rate of pH decrease are responsible for the disappearance of enterobacterial and clostridial secondary fermentations. Others studies have shown that spontaneous fermentation results in higher concentrations of both acetic acid and BAs which adversely affect the palatability of fermented feed [129, 130]. However, it is not necessary to use spontaneous fermentation, as the quality of spontaneously fermented feed can be improved by adding copper to the fermentation medium which speeds up the production of lactic acid [131]. BA are non-volatile low-molecular-weight nitrogenous organic bases, derived through decarboxylation of corresponding AAs. They can be both formed and degraded as a result of normal metabolic activities in animals, plants and microorganisms. Putrescine and cadaverine are known to potentiate these effects. Moreover, these amines are thermo-stable [132]. BAs can produce a wide range of toxicological effects [133], histamine and tyramine being the main BA, regarding their toxic effect. BAs in the gastrointestinal tract are

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important metabolites of dietary protein and AAs due to their support to digestive enzymes and microbes in gut, which play a crucial role in the regulation of intestinal functions, including digestion, absorption and local immunity. However, high concentrations of BAs can induce adverse reactions and are harmful to animal health [134]. Özyurt et al. [135] re-ported that due to the activity of various endogenous and bacterial decar-boxylase enzymes, fish silage contains considerable levels of FAA that constitute the precursors for BAs. In fish silages, the concentration of spermidine, tryptamine, phenylethylamine and spermine was lower than the concentration of putrescine, cadaverine, histamine, tyramine and agmatine. Formation of BAs can be reduced by restricting fermentation in the silage or by achieving rapid acidification during the first phase of ensiling [136]. Bacterial proteolysis, the presence of BAs is associated with a decrease in the protein content and nutritional value of the silage. Negative effects of BAs on animal health have been reported; the BAs have been implicated to be causative factors in ketonemia, and absorption of ruminal histamine should be considered a major cause of systemic histaminosis in acidotic ruminants [138, 139]. Although hyperketonemia may result from con-sumption of silage with high content of butyric acid, high concentrations of putrescine or other amines in the feed may contribute to the development of ketonemia both in early and further period of lactation. BAs notably decrease palatability of silage and reduce dry matter intake and cattle performance [140]. However, Van Os et al. [137] and Mao et al. [141] have shown that in sheep adapted to silage with high levels of BAs, amine accumulation in the rumen is prevented by the increase in the amine de-grading capacity of rumen microbes [142]. Formation of BAs can be affected by several factors such as temperature, rapidity of pH decrease during the initial stage of fermentation, and oxygen availability. Effects of BAs on cow health also differ depending on the composition of the mixture; TRP, PUT and CAD form a group of highly intercorrelated substances with similar effects [143]. Exogenous dietary putrescine can increase the growth rates of neonatal animals under nutritional stress. Dietary putrescine prevents damage to the intestinal mucosa and has beneficial effects on the height and width of villus in tissues [144]. Addition of putrescine could stimulate the intestinal epithelial DNA, RNA, and protein synthesis [145] and accelerate the development of small intestinal of ducks during the posthatch period [146]. Spermine modulates depression, shortens the immobility time of animals in a dosage-dependent manner, presumably by activating N-methyl D-aspartate receptors [147]. Supplementing newly hatched chicks with putrescine has shown no positive or even adverse effects on development of small intestine [148]. The proper concentration of

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dietary protein and AA is very important to ensure optimal nutrition of livestock [134].

1.5. Technologies for Preparing Protein Isolates/Concentrates of Lupine Seeds

More than 90% of protein is considered to be isolate and lower than 90% of protein is considered to be concentrate [149], however, other opinion exists stating that lupine protein isolates contain >85% of protein and concentrates contain >60% of protein content. The main protein extraction processes can be classified into dry and wet methods. In general, wet extraction methods can be applied for preparing both: protein concentrates and isolates at levels of 70% and 90% protein and higher, respectively. However, it should be noted that currently there is no universal classi-fication scheme which separates concentrate from an isolate for all the legumes. The various wet extraction processes include acid/alkaline extrac-tion – isoelectric precipitaextrac-tion, ultrafiltraextrac-tion and salt extracextrac-tion. Legume flour dispersed in aqueous solutions typically shows high solubility when subjected to alkaline or acidic extraction conditions at pH 8-10 and below pH 4, respectively [150, 151]. Important conditions for the development of plant-based alternatives are the latter: the availability of highly functional plant protein concentrates and isolates produced in a sustainable manner. Current protein extraction processes are inefficient due to the use of organic solvents, acids, bases and large amounts of water, resulting in little environ-mental gain which is even lower than theoretically possible [149]. Conven-tional plant protein production methods involve the use of solvents, con-centrated acids and alkali that can result in denaturation of protein and loss of solubility, thereby reducing the quality and functionality of protein in-gredients [152, 153]. There are two techniques usually applied in isolation of protein: the alkaline extraction with subsequent isoelectric precipitation and the salt-induced extraction followed by dilutive precipitation. The alkaline extraction and the salt-induced extraction techniques provide protein isolates with different properties [154]. The consistency of lupine protein precipitate obtained by applying isoelectric technique is rough, compact and curdy and its microstructure is unfolded and has large protein agglomerates, while protein precipitate obtained by applying dilutive precipitation is smooth, pasty, mellifluent at room temperature and features a fat-like texture [154, 155]. Muranyi et al. [156] compares alkaline extraction and isoelectric precipitation with salt-induced extraction followed by dilutive precipitation. It was shown that alkaline extraction has had a higher protein denaturation, and summarized that depending on the desired

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properties of the products,it is an appropriate isolation technique. Muranyi et al. [156] has reported that lupine protein isolates extracted in alkaline solution have resulted in higher protein content (26.4–31.7%), compared to salt-induced extraction (19.8–30%) and combined alkaline salt-induced extraction (23.3-25.6%). Other Can Karaca’s et al. studies [157] have proven that isolates prepared from faba bean, chickpea, lentil, pea and soybean by applying an alkaline extraction using isoelectric technique have had higher overall protein content (85.6%), compared to those prepared applying a salt extraction method (78.4%). Moreover, it was reported that both legume source and protein extraction method along with their interaction have had significant effects on protein levels of the isolates, and also on physicochemical and emulsifying properties. The overall surface charge, solubility, hydrophobicity and creaming stability of isolates produced via isoelectric technique was higher, compared to isolates produced applying salt extraction. Acid extraction involves the preliminary extraction of proteins under acidic conditions. This technique could result in high solubilization of proteins prior to protein recovery (isoelectric technique, ultrafiltration), as proteins tend to be more soluble under acidic conditions below pH 4.0 [82]. Sussmann et al. [158] reported that protein isolation method based on salt-induced extraction followed by a dilutive precipitation has shown relevant processing parameters; different raw materials have been investigated systematically to obtain high yields of protein preparations with characteristic of fatlike textural properties. In the case of full-fat lupine ﬂakes, a protein yield of 38% was achieved. The sensory proﬁle of the lupine protein isolate revealed unique creamy, smooth and fatlike characteristics due to the formation of micellar aggregate [154, 159]. The membrane separation methods have shown protein isolates with higher functionality and were effective in reducing levels of anti-nutritional compounds, which include protease and amylase inhibitors, lectins and polyphenols [160, 161]. Ultrafiltration and microfiltration are membrane-based methods of fractionation using pressure as the driving force for separation [162]. For preparation of protein by using ultrafiltration, alkaline or acidic extraction is followed by the processing of supernatant using either ultrafiltration or diafiltration together to isolate the protein material, also ultrafiltration is often combined with diafiltration to improve protein recovery [163]. To increase the life of shelf of the protein isolate, ultrafiltration may be followed by spray drying. If desired, the oil, which ends up for 0.5–0.6 g in the fibre-rich fraction, can be recovered by an additional oil extraction step. Overall, it seems that scope exists to lower the environmental impact on the extraction of water- and dilute salt-soluble proteins from legume materials. This is required to facilitate the transition

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from animal-based protein towards plant-based protein in a sustainable manner [164]. The aqueous fractionation without appliance of an organic solvent but with defatting using and organic solvent can be used for isolation of lupine protein; what is more, the aqueous fractionation is a sustainable alternative, because the oil extraction step is omitted and thereby the organic solvent is not used [165] for defatting using an organic solvents, such as hexane. The defatted flour is then solubilized in water or a buffer at alkaline pH; later on, insoluble parts are separated from the protein-rich supernatant. However, since oil is not removed prior to purification of protein, the oil might become oxidized during processing and have a negative effect on the quality of the obtained protein isolate. The protein is separated from other soluble solids, such as sugars, by isoelectric precipitation of the protein. Wet fractionation of lupine seeds without oil extraction results in lupine protein isolates containing a few percents of oil together with functional properties similar to those of wet fractionated lupine protein isolates, which generally do not contain oil [166]. The lupine protein isolates obtained by applying wet fractionation techniques have a low capacity of gelling and high heat stability [167]. The fractionation is followed by drying process to stabilise the protein isolate. This might not be necessary if the final application contains or requires water. Therefore, it was explored whether the process of drying could be omitted [166]. Dry and wet fractionation processes differ in their separation principle, the use of resources, the unit operations needed, and the yield and composition of obtained fractions. The efficiency of these fractionation processes is evaluated through calculating and visualizing mass, energy and exergy flows. Dry fractionation by fine milling is based on the physical disen-tanglement of protein bodies from fibres and other cellular components, which allows their subsequent separation by air classification [82, 168]. Wet fractionation is based on the differences in solubility of different compo-nents in organic solvents, water and saline solutions. Dry fractionation of lupine seeds leads to protein-enriched flours (>50 g protein/100 g). Wet fractionation of lupine seeds can yield protein concentrates (>70 g tein/100 g) and further fractionation leads to protein isolates (>90 g pro-tein/100 g). Papalamprou et al. [169] have reported that milder processing techniques, rather than the composition of the protein isolate, improved the functional properties of chickpea protein isolates in terms of increased protein solubility, reduced minimum protein concentration needed for gel formation, and improved gel elasticity. The effect of the drying method on protein functionality depends on the drying method and on the type of protein. Freeze-drying influences the morphology and size of the protein particles and the surface hydrophobicity of proteins by partial denaturation,

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due to such stresses as low temperature, freezing stress (e.g. phase se-paration, pH change and formation of ice crystals) and drying stress [170, 171]. Spray drying has reduced the solubility of a lentil protein isolate less than vacuum drying, however, it has thermally damaged lupine protein isolates [172]. Since freeze-drying is generally perceived as the mildest form of drying, this drying method was chosen for comparison with ultrafiltration [167, 173].

1.6. Lupine Seeds as the Material of Animal Feed

Legumes belong to the family of Leguminosae or Fabacae and are considered the second most important crop worldwide, cereals being the first. They are grown on about 180 million hectares, equivalent to 12-15% of Earth’s arable land [174]. In contrast to many crops, the lupine is reasonably yielding even on sandy soils with a low pH value, also it im-proves the soil structure by mobilizing soil-bound phosphorus and fixing atmospheric nitrogen, thus providing nutrients to the succeeding crop. Despite its value in crop rotations, lupine so far is an underutilized crop due to its low grain yield stability. To improve yield stability and lupine yield potential, intense breeding efforts are required [175]. Global legume pro-duction is currently growing due to the increasing nutritional and economic significance of legume seeds [176]. In animal nutrition, especially in intensive livestock systems, soya bean is the most utilized protein source, mainly administered as extract of meal solvent, a by-product of the oil industry, where soya bean seeds are treated with high temperature and organic solvents. However, recently some obstacles have limited the use of soya bean: the ban in organic livestock [177] due to the chemical treatment, its cost and availability strongly related to the price developments of agricultural commodities on the world market. Ensiling lupine seeds may be an economical and ecologically advantageous alternative to produce a high-protein feed of local origin that can be used in both conventional and organic farming [178]. Also, lupine seeds are important for both animal feed and human foodstuff due to the production of lupine flour and isolate proteins, whereas yellow lupine is mostly used in the livestock chain [179, 180]. Nevertheless, they are still considered to be rich in nutrients and phytochemicals, which makes them an important, inexpensive seed in many developing countries not just a for feed uses, but also for supplementing human diets based on legumes, cereals and roots; consequently, it is suggested to be one of the best solutions to malnutrition in these countries [181, 182].

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Lupine Seeds for Poultry Feeding. The growing interest in cultivation

and the introduction of legume seeds into poultry diets have been observed in recent years. It was discovered that biologically active legume proteins, rich in lysine and owing antioxidant potential can be a good source for production of animal feed [183–189].

Legume seeds have been used for broiler chickens [190, 191], turkeys [192, 193] and laying hens [194, 195] feeding. The inclusion of lupine seed meal in feed mixtures for broilers did not have a negative effect on the chemical composition of breast and femoral musculature. Unlike the control group, the experimental group of chickens taking lupine in the proportion of n-3 and n-6 fatty acids, had the outcome of breast and femoral musculature narrowing for both pullets and cockerels, which is a proof of an increased dietetic value of musculature. On the other hand, replacement of soybean meal with yellow lupine seeds meal in turkey nutrition (0%, 8%, 16%, and 24%) did not have a positive effect on feed intake and body weight gain. Increase in the concentration of polyunsaturated fatty acids (PUFA) was noted in turkey meat of those fed with lupine-based feed; this did not change the n-6/n-3 PUFA ratio, but improved the value of the atherogenic and thrombogenic indices [196]. In the previous studies during which yellow lupine seeds were used (raw and extruded) in broiler diets in a share of 5– 30%, it was found that the extrusion improved digestibility of fats from seeds as well as nitrogen retention in chickens, and enhanced the apparent metabolizable energy correcting it to zero value of N balance in seeds [197]. However, the inclusion of 25% or 30% of either raw or extruded yellow lupine seeds into diets significantly decreased the performance index of broiler chickens. Using 10% or 20% of extruded seeds positively affected the ratio of growth and feed conversion of birds in comparison to those fed with raw seeds [197].

The lack of information on the inclusion of lupine in ostrich feeding and the ways in which dietary inclusion might affect the production and quality of the meat, leather and feathers of slaughter birds. However, the use of locally produced feed sources would benefit the local legume industry and the ostrich industry. In an attempt to decrease the feeding costs of ostriches, animal nutritionists apply low cost diet formulations and the use of lupine as a raw material could contribute to these formulations [198]. Zdunczyk et al. [199] have analysed performance of growing, gastrointestinal function and meat quality in growing-finishing turkeys that were on diets with different levels (6%, 12% and 18%) of L. luteus seed meal. During the first phase of feeding, yellow lupine meal has decreased feed intake and body weight gain linearly due to significant deterioration in the feed conversion ratio. An opposite trend was noted in the second phase of feeding. Body weight gain

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and feed conversion ratio improved significantly. The effects of dietary replacement of soybean meal with blue lupine meal on the gastrointestinal tract function, growth performance and meat quality in growing-finishing turkeys were studied by Mikulski et al. [200].

Lupine Seeds for Pig Feeding. The use of lupine in weaner diets has

been limited from 50 to 100 g/kg on the basis of idea that pigs would have limited ability to deal with the high fibre content in lupine wholemeal. Other studies have shown that yellow lupine seeds could be included at up to 150 g/kg in weaner diets without compromising performance of pigs [201]. In this regard, it is possible that a similar or greater amount of lupine seeds could be used in a weaner diet. Moreover, there is a general perception that removal of the hull, which is indigestible, from lupines may offer the opportunity for even higher levels of inclusion (i.e. >150 g/kg) as increased amounts of insoluble fibre may physically limit the quantity of lupine stock that can be incorporated in a diet for weaner pigs. However, despite a relatively low concentration of anti-nutritional factors such as alkaloids in lupine varieties, the use of blue lupine meal over soybean meal in swine diets may have some limitations due to the profile of imbalanced AAs, which leads to lower protein digestibility, lower proximate nutrient and putative negative interaction between the relatively high concentrations of non starch polisaccharides (NSP) and the digestion process of other nutrients [202]. Use of sweet lupine wholemeal up to 240 g/kg in diets for weaner pigs did not affect performance and indices of intestinal health most likely because of the insoluble and non-soluble polysacharides in the hull, that in turn could have possibly altered physico-chemical properties of the digesta. However, feeding animals with a diet containing greater than 180 g/kg of dehulled lupine seeds significantly compromised feed intake and hence growth of the pigs, and stimulated the proliferation of β-haemolytic strains of E. coli in the gastrointestinal tract [203]. However, the data about lupine feed stock influence on the efficiency of production of pigs are not homogenous. Reduced feed intake and growth of pigs fed a diet containing 150–430 g/kg of L. albus seeds have been reported by Zettl et al. [204]. Applying the inclusion of 30% of white lupine in the feed mixture, they have noticed a reduced feed intake, lower conversion of nutrients and growth of depression; however, they have not observed a positive effect of dehulling and supplementation with AAs. Conversely, in pigs fed a diet with L. angustifolius, compared to a barley- and soy-based diet, Gdala et al. [205] have not observed growth of depression. Positive results of feeding with yellow lupine seeds of Juno variety were achieved by Flis et al. [206]. The nutritional value of diets with various contents of cultural lupines (L. angustifolius and L. albus) in fattened pigs have been studied by Zralý et al.

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[207], who proved that the animal protein or soybean could be completely replaced by lupine in the diet for fattened pigs. Also, it was reported that limitation of AA was balanced and the nutritional value was increased by supplementation of fat and lupine seed dehulling. No antinutritional effect was observed in the applied representation of lupine seeds in experimental diets. Effects of feeding finisher pigs with chicory or lupine seeds for one or two weeks before slaughter with respect to levels of Bifidobacteria and Campylobacter have been studied by Jensen et al. [208]. This study has shown that even a short-term strategy of alternative feeding with probiotics added in the diet of pre-slaughter pigs elicited changes in the composition of the intestinal microbiota, where lupine has increased the level of bifidobacteria in the caecum and reduced the level of Campylobacter spp. excretion after one week.

Lupine Seeds for Sheep and Lamb Feeding. Yilkal et al. [209] have

re-ported positive responses of washera sheep on a hay based supplementation of different forms of processed lupine seeds. However, Lestingi‘s et al. [210] experiment has shown that the lupine protein is highly degradable in the rumen and this may partly explain the poor performance of lambs observed. It is possible that combined use of the lupine seeds and faba beans in lamb feeding could achieve a better balance and thus improve the animal performances. The use of lupine and faba bean seeds as the main protein sources provides lamb growth performance and slaughtering data compa-rable to those obtained when faba beans are used alone. However, when faba seeds were used as the sole protein source in the diet, the half-carcasses presented a higher percentage of loin than the half-carcasses of lambs fed with the two protein supplements combined. The use of lupine and faba bean protein sources together improved lamb growth performances and decreased the percentage of leg bone compared to lupine used alone. Blood parameters were little affected by dietary treatments. Both lupine and faba protein sources in lamb feed have a positive effect on lamb growth and slaughtering data [211].

Lupine Seeds for Ruminants Feeding. The lupine could be an excellent

protein and energy source for ruminants, and it can be fed as wholemeal, ground seeds, whole plant – silage. However, its bitter taste due to a high alkaloid content remains to be a big challenge [212]. Homolka et al. [213] have reported that lupine can be used in the feed rations of milk cows and in fattening of bullocks. Inclusion of lupine seed meal in the feed rations for high-utility milk cows requires crushing and flaking of seeds and their inclusion in the feed mixture for bullocks up to 30% (0.5 kg/100 kg of live weight), for milk cows up to 20% (0.4 kg/100 kg live weight). Their advan-tage is that unlike soybeans, they do not need heat treating. Depending on

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the lupine variety, the degradability of lupine seed protein in rumen ranges from 71 to 79%. Cattle can even use whole fodder plants as a fresh fodder or ensilage. In sheep, Somchit-Assavacheep et al. [214] have monitored the effect of short-term nutritional supplementation with lupine seeds (L. luteus) on folliculogenesis, concentrations of hormones and glucose in plasma and follicular fluid. The numbers of follicles have increased in the group fed by lupine, glucose and insulin levels were also higher.

Lupine Seeds for Fish Feeding. Ranjan and Bavitha (2015) have

re-ported that lupine kernels is a suitable meal for fish. The lupine kernels is a cheap source of protein, which shows high protein digestibility, high phosphorus retention, does not cause enteritis is salmon unlike soya, and protein in it is not damaged by heating processes [215]. By replacing 60% of fish meal with Lactobacillus spp. fermented lupine wholemeal, the performance of barramundi was improved [216]. Phytic acid is ANFs in plant based feedstuffs. Most of the fish do not have endogenous enzymes to break down the phytate and release nutrients, and the feedstuff is not completely digestable. However, the advantages of phytase are the follow-ing: phytase reduces the release of nutrients into the environment by making more bound phosphorus available to fish for growing; phytase added to diets improves protein and amino acid digestibility in fishes; it can improve the metabolic energy of feeds by breaking down the phytate-lipid complex; plant protein sources can be substituted with animal protein sources (e.g., fishmeal), reducing feed cost [217]. Hoerterer et al. [217] have reported that lupine wholemeal and lupine kernel meal have great potential as a sustai-nable, locally produced replacement for fishmeal in diets for the carnivorous European sea bass with no negative effects on their growth.

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2. MATERIALS AND METHODS

2.1. Investigation Venue

The experiments were conducted between 2014 and 2018 at the Lithuanian University of Health Sciences Veterinary Academy (LSMU VA) Department of Food Safety and Quality (Kaunas, Lithuania); Institute of Food Safety, Animal Health and Environment – “BIOR“ (Ryga, Latvia); University of Leipzig Institute of Food Hygiene (LU IFH) (Leipzig, Germany).

2.2. Materials

2.2.1. Plant Material. The seeds of narrow-leafed lupine varieties Vilciai

and Vilniai, as well as hybrid lines No.1700, No.1701, No.1702, No.1072, No.1734 and No.1800 (Lupinus angustifolius L.) seeds were obtained from the Lithuanian Institute of Agriculture (Voke, Lithuania) in 2014.

2.2.2. Microorganisms Used for Experiments. Lactobacillus sakei

KTU05-6, Pediococcus acidilactici KTU05-7, Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 were selected due to their good technological, functional and antimicrobial properties, above mentioned microorganisms were obtained from the Kaunas University of Technology, Department of Food Science and Technology, Cereal and Cereal Products research group collection [218].

2.3. The Lupine Wholemeal Biotreatment and Protein Isolation 2.3.1. The Lupine Wholemeal Fermentation

Lupine seeds were crushed (particle size 3 mm) using a household mill (Braun, Germany). The water content was calculated with reference to the moisture content of raw materials and the required 45% moisture content of the solid state fermentation (SSF) end product and 65% moisture content of the submerged fermentation (SMF) end product. Fermentation was carried out for 48 h at the optimal temperature for the cultivation of Lactobacillus sakei (30 °C), Pediococcus acidilactici (30 °C), Pediococcus pentosaceus strains KTU05-8, KTU05-9, and KTU05-10 (35 °C). Ten different samples from each seeds variety were prepared by using different LAB strains and different fermentation technologies (SSF or SMF). Nonfermented lupine seeds were used as a control.

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2.3.2. The Lupine Protein Isolation

Lupine protein was isolated according to procedure described by Muranyi et al. [156]. For preparation of isoelectric lupine protein isolates full-fat lupine seeds where crushed using a Grindomix GM 200 (Retsch GmbH, Haan, Germany) and suspended in DI water at a ration 1:8 (w/v). For alkaline protein extraction the pH value was adjusted to pH 10 with 0.5 mol/L NaOH and this suspension stirred for 1 h at room temperature to maximise the extend of solubilized proteins. After separation through a sieve (mesh size: 1.5 mm) removing the fibre fraction, the suspension was centrifuged at 3300 g for 10 min., and the supernatant was removed and acidfied to pH 4.5 with 0.5 mol/L HCl. For an exhaustive protein preci-pitation, the crude protein suspension was left for 18 hours at 1 °C in refrigerator. The precipitate was recovered by centrifugation and the supernatant was discarded. The protein precipitate was washed 3 times with DI water (ration 1:10 (w/v)) to eliminate effectively surplus salts. After a last centrifugation step was the precipitated concentrate/isolate stored at -20 °C. For futher analysis the protein isolate was directly frozen at -80 °C and lyophilized in a freeze-drier (Beta 18, Christ GmbH, Osterode, Germany). Protein isolates/concentrates were prepared from nonfermented and fermented seeds.

2.4. The Methods used for Analysis of Lupine Seeds and Their Bioproducts

2.4.1. The Evaluation of Lupine Seeds Proximate Composition

Chemical composition of lupine seeds was investigated according to the ICC standard methods. Moisture content was determined by drying the samples at 105 ± 2°C to constant weight (ICC 109/01:1976. Determination of the moisture content of cereals and cereal products). Ash content was determined by calcinations at 900°C (ICC 104/1:1990. Determination of ash in cereals and cereal products). Nitrogen content was determined using Kjeldahl method with a factor of 5.7 to determine protein content (ICC 105/2:2001. Determination of crude protein in cereals and cereal products for food and for feed). The total lipid content was determined by extraction in the Soxhlet apparatus (“Boeco”, Germany) with hexane technical grade (Fisher Scientific, USA) (ICC 136:1984. Cereals and cereal products – Determination of total fat content). Carbohydrates content in lupine seeds was calculated by the following formula: 100 − (weight in grams [protein + fat + water + ash] in 100 g of seeds). Energy value was calculated by

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multiplying the content of protein, fat and carbohydrates by the appropriate factor – 4, 4 and 9 for protein, carbohydrates and fat, respectively.

2.4.2. The Analysis of Fatty Acids Composition in Lupine Seeds

Fatty acid (FA) composition of lupine seed oil was determined using a gas chromatography-flame ionization detector (GC-FID), gas chromato-graph Agilent 6890N (Agilent Technologies, USA). Methyl esters of FAs were dissolved in anhydrous 99.5% (Sigma-Aldrich, Germany) cyclohexane (100 mg in 4 mL) and were prepared by transmethylation using 8 mL 1.5% sulphuric acid (≥95%, Sigma-Aldrich, Germany) in the pure (99.9%) methanol (Sigma-Aldrich, Germany), and kept at 60°C for 12 h in the dark. Samples were cooled, shaken for 30 s and centrifuged for 10 min, at 3000 relative centrifugal force at 17°C and injected (100 µL of the upper part of supernatant, diluted before in cyclohexane 1:9, respectively) into a capillary BPX90 column (60 m × 0.32 mm, ID × 0.25 µm film thickness) (SGE, USA). The following conditions were used: flame ionization detector – 280°C, H2 flow – 40 ml/min, air flow – 450 ml/min, helium (carrier gas) flow – 1 ml/min , injector – 250°C (split 1:10), oven temperature – 50°C (2 min), 4°C ml/min to 245°C and 245°C for 15 min. The identification of FA was carried out by retention times and expressed as percentage of the total peak area of all the fatty acids in the oil sample.

2.4.3. The Analysis of Macro- and Microelements in Lupine Seeds

Determination was carried out using inductively coupled plasma mass spectrometry (ICP-MS). The seeds were milled and homogenised (final particle size ≤150 µm). For the analysis the following chemicals were used: nitric acid (concentration ≥69.0%), for-trace element analysis (Sigma-Aldrich, France), hydrogen peroxide, 30% w/w (weight/weight), extra pure (Scharlau, Spain), multielement standard solution V for ICP-MS calibration (Sigma-Aldrich, France). Agilent 7700x ICP-MS (Agilent Technologies, Japan), software Mass Hunter Work Station for ICP-MS, version B.01.01 (Agilent Technologies, Japan) were used for analysis. For sample prepa-ration for ICP-MS analysis, 0.3 g of milled lupine seeds was accurately weighed in a microwave vessel. 2 mL of de-ionized water, 8 mL of con-centrated nitric acid and 2 mL of concon-centrated hydrogen peroxide were added and waited for 2–8 h for reaction stabilization until the formation of bubbles had finished. The vessel was sealed and heated in the microwave system. The following thermal conditions were applied: 150 °C temperature was reached in approx. 20 min and remained such for 30 min, and then

BIOTECHNOLOGICAL SOLUTIONS FOR PREPARATION OF SUSTAINABLE AND SAFE PLANT PROTEINS THROUGH THE USE OF LOW-WASTE AND/OR NON-WASTE TECHNOLOGIES

Vytautė Šakienė