BIOTECHNOLOGICAL SOLUTIONS FOR DAIRY CATTLE FEED PRESERVATION AND THEIR INFLUENCE ON CATTLE HEALTH PARAMETERS AND PRODUCTION QUALITY

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Vita Krunglevičiūtė

BIOTECHNOLOGICAL SOLUTIONS

FOR DAIRY CATTLE FEED

PRESERVATION AND THEIR INFLUENCE

ON CATTLE HEALTH PARAMETERS

AND PRODUCTION QUALITY

Doctoral Dissertation Agricultural Sciences, Zootechnics (03A)

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Dissertation has been prepared at the Department of Food Safety and Quali-ty of Veterinary Academy of Lithuanian UniversiQuali-ty of Health Sciences du-ring the period of 2013–2017.

Scientific Supervisor –

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

Consultant –

Prof. Dr. Rasa Želvytė (Lithuanian University of Health Sciences, Agri-cultural Sciences, Veterinary – 02A).

Dissertation is defended at the Zootechnics Research Council of the Lithuanian University of Health Sciences.

Chairperson –

Prof. Dr. Vida Juozaitienė (Lithuanian University of Health Sciences, Agricultural Sciences, Zootechnics – 03A).

Members:

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

Dr. Violeta Juškienė (Lithuanian University of Health Sciences, Agricultural Sciences, Zootechnics – 03A);

Assoc. Prof. Dr. Antanas Šarkinas (Kaunas University of Technology, Technological Sciences, Chemical Engineering – 05T);

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

Dissertation will be defended at the open session of the Lithuanian University of Health Sciences on the 1st of September, 2017 at 2:00 p.m. in Dr. S. Jankauskas Auditorium of the Veterinary Academy.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA

Vita Krunglevičiūtė

NATŪRALIŲ BIOPRIEMONIŲ

PANAUDOJIMO GALIMYBĖS PIENINIŲ

GALVIJŲ PAŠARŲ KONSERVAVIMUI IR

JŲ ĮTAKA GYVULIŲ SVEIKATINGUMUI

BEI PRODUKCIJOS KOKYBEI

Daktaro disertacija Žemės ūkio mokslai,

zootechnika (03A)

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Disertacija rengta 2013–2017 metais Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Maisto saugos ir kokybės katedroje.

Mokslinė vadovė –

Prof. dr. Elena Bartkienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, zootechnika – 03A).

Konsultantė –

Prof. dr. Rasa Želvytė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A).

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

Pirmininkė –

Prof. dr. Vida Juozaitienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, zootechnika – 03A).

Nariai:

doc. dr. Agila Daukšienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, zootechnika – 03A);

dr. Violeta Juškienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, zootechnika – 03A);

doc. dr. Antanas Šarkinas (Kauno technologijos universitetas, technolo-gijos mokslai, chemijos inžinerija – 05T);

dr. Damian Escribano Tortosa (Barselonos autonominis universitetas, žemės ūkio mokslai, veterinarija – 02A).

Disertacija bus ginama viešajame Zootechnikos mokslo krypties tarybos posėdyje 2017 m. rugsėjo 1 d. 14 val. Lietuvos sveikatos mokslų universi-teto Veterinarijos akademijos Dr. S. Jankausko auditorijoje.

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

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ABBREVIATIONS

ACo – Agriculture Companies

AML – Amoxicillin

AMP – Ampicillin

ANFs – Antinutritional Factors

ARs – Alkylresorcinols

AST – Aspartate Aminotransferase

AU – Activity Units

BAs – Biogenic Amines

BLIS – Bacteriocin-Like Inhibitory Substances

BW – Body Weight

CAD – Cadaverine

CFU – Colony-Forming Units

CHL – Chloramphenicol

CLIN – Clindamycin

CP – Crude Protein

DFM – Direct-Fed Microbials

DM – Dry Matter

DMI – Dry Matter Intake

DON – Deoxynivalenol

EB – Enterobacteria

EFSA – European Food Safety Authority

ERY – Erythromycin

FA – Fatty Acid

FCM – Fat-Corrected Milk

FDA – Food and Drug Administration

FEEDAP – Panel on Additives and Products or Substances used in Animal Feed

GC/MS – Gas Chromatography – Mass Spectrometry

GEN – Gentamicin

GF – Glucose Fermentation

GIT – Gastrointestinal Tract

GRAS – Generally Recognized As Safe

HIS – Histamine

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

KAN – Kanamycin

KTU – Kaunas University of Technology

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Ls – Lactobacillus sakei

MIC – Minimum Inhibitory Concentration

MRS – de Man Rogosa Sharpe

Pa – Pediococcus acidilactici

PHE – Phenylethylamine

Pp – Pediococcus pentosaceus

PUT – Putrescine

RA – Reduction Activity

SARA – Rumen Acidosis

SmF – Submerged Fermentation

SPR – Spermine

SPRD – Spermidine

SSF – Solid State Fermentation

STREP – Streptomycin

TAB – Total Anaerobic Bacteria

TET – Tetracycline

TM – Trimethoprim

TTA – Total Titratable Acidity

TYR – Tyramine

UHPLC – Ultra High Performance Liquid Chromatographs

VFA – Volatile Fatty Acid

WB – Wheat Bran

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INTRODUCTION

Animal feeds play the leading role in the global food industry, enabling the economic production of products of animal origin throughout the world [1]. To help meet the demand for safe and affordable food, feed manufac-turers around the world need to apply a range of processing technologies and engineering in feed manufacture from processes relying on general and skilled labour to fully automated manufacturing systems and make use of a wide range of co-products, by-products and otherwise surplus raw materials from primary agricultural production, food industry and industrial sources [2].

Good animal feeding practices include the practices that help to ensure the proper use of feed on-farm to promote animal health and productivity while minimizing biological, chemical and physical risks to consumers of foods of animal origin and also to reduce the impact on the environment [3]. Feed ingredients should be obtained from safe sources and be subject to a risk analysis where the ingredients are derived from processes or biotech-nologies not hitherto evaluated from the food safety point of view. To increase feed safety and digestibility, biotechnological tools could be recommended (treatment with enzymes or microorganisms). Today, lactic acid bacteria (LAB) play a prominent role in the world feed supply, perfor-ming the main bioconversions in fermented food/feed products [4]. They are generally recognized as safe (GRAS) microorganisms and play an important role in feed fermentation and preservation either as the natural microflora or as starter cultures added under controlled conditions [5]. LAB are part of the gut microbiota and produce ribosomally synthesized antimicrobial peptides or bacteriocins with interest as natural feed preservatives and therapeutic agents [6]. The incorporation of bacteriocins producing LAB as a biopreser-vative ingredient into model feed systems has indeed been studied extensi-vely and has been shown to be effective in the control of pathogenic and spoilage microorganisms; by the way, the direct addition of bacteriocin-producing cultures into products, as compared to bacteriocins, can be a more practical and economic option for improving the safety and quality of the final product [7].

LAB can be isolated from different sources: cereal-based products [8], rumen fluid [9], dairy products [10], seafood [11] etc. Cereals are a suitable substrate for the growth of potentially probiotic LAB [12]. In this case, in our study, LAB isolated from spontaneously fermented cereals were tested.

LAB are popular additives for animal nutrition [13]. In cattle, the use of probiotics has the main purpose of preventing and combating digestive

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disorders (especially diarrhea in livestock during lactation), of influencing the ruminal metabolism of nutrients and stimulating the activities of ruminal microorganisms, which helps maintain health and improve productive performance [14]. The rumen is quickly colonized by all types of micro-organisms straight after birth, and the colonization pattern may be influen-ced by several factors such as presence/absence of adult animals, the first solid diet provided, and the inclusion of compounds that prevent/facilitate the establishment of some microorganisms or the direct inoculation of specific strains [15]. The safety issues concerning the use of these organisms have been addressed by various regulatory bodies in different countries [13].

For LAB cultivation, some industrial waste products such as whey and molasses are used [16]. Cereal by-products, also, are perspective for LAB cultivation, but their safety parameters (such as mycotoxins content etc.) should be taken into account. Biotechnological options are available for improving rumen fermentation and enhancing the nutritive value and utilisation of agro-industrial by-products and other forages [17].

To produce feed in environment-friendly conditions, it is not enough to use by-products; the whole technology should be optimized to reduce water consumption or/and other energy sources.

With the advent of biotechnological innovations, mainly in the area of fermentation technology, many new avenues have been opened for the proper valorization of different industrial wastes [18]. Biotechnology is offering unprecedented opportunities for increasing agricultural productivity and for protecting the environment through a reduced use of agro-chemicals. However, the successful application of biotechnology has generally been limited to developed countries [19], and for the conversion of by-products to higher value products sustainable technologies, such as solid state fermentation (SSF), could be adapted.

The last decade has witnessed an unprecedented increase in the interest in SSF for the development of bioprocesses such as bioremediation and biodegradation of hazardous compounds, biological detoxification of agro-industrial residues, biotransformation of crops and crop-residues for nutria-tional enrichment, biopulping, and the production of value-added products such as biologically active secondary metabolites, including antibiotics, alkaloids, plant growth factors, enzymes, organic acids, biopesticides, including mycopesticides and bioherbicides, biosurfactants, biofuel, aroma compounds, etc. SSF systems, which during the previous two decades were termed as “low-technology” systems, appear to be a promising way for the production of value-added ‘low volume-high cost’.

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The aim of the study

The aim of the study is to create a design of feed supplements for cattle feeding by using plant and plant by-products solid state and submerged fermentation with antimicrobial properties showing lactic acid bacteria.

Objectives of the study

1. To evaluate the influence of different fermentation conditions (solid-state and submerged; spontaneous fermentation and fermen-tation with different LAB starters) on changes of the digestibility and the formation of biogenic amines in proteinaceous plants (lupi-ne and soya).

2. To adapt cereal by-products (wheat and barley) and starch produc-tion by-products (potato juice) for selected antimicrobial properties showing LAB cultivation for a safer feedstock with a high content of LAB production.

3. To evaluate the influence of the developed feedstock on cattle health parameters and production quality.

The scientific novelty and practical usefulness

In countries containing large food-processing industries, food-residue disposal is a significant issue. By-product feeds are often described as “waste,” a term that confers an obvious negative message to the consumer. It is estimated that more than half of the industrial waste in the world originates from the food-processing industry, yet livestock can often convert this residue into meat, milk, and wool. It is clear that the use of by-product feeds in combination with the management strategies that improve efficiency will mitigate the environmental and economic impact of animal agriculture. The panel on additives and products or substances used in animal feed (FEEDAP) provides scientific advice on the safety and/or efficacy of additives and products or substances used in animal feed. In ruminants, feed conversion (defined as the amount of dry feed required per unit of weight gain) has been the most common estimate of feed efficiency, because this was the information needed to determine the economic cost of production. Usually, the nutritional and functional value of by-product feeds is low. To convert by-products or/and to increase the nutritional character-ristics and safety parameters of feed of the plant origin, fermentation by using selected microorganisms is recommended. Nowadays, the develop-ment of new biotechnologies of the higher value and sustainability feed becomes very important, because it corresponds with the strategies of reducing the global climate changes.

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The scientific and practical novelty of the thesis is concentrated on the sustainable higher value and safer feed production from proteinaceous plants and/or cereal by-products by using SSF with antimicrobial properties showing LAB for feeding cows and newborn calves. For this purpose, (I) in Lithuania, bred lupine seeds and soya seeds (proteinaceous plants), cereal by-products (wheat and barley) and starch production by-products (potato juice) were used for (II) the cultivation of selected antimicrobial properties showing LAB, and (III) the development of feed stock with a high content of LAB, which was tested for the feeding cows of and newborn calves.

Important practical results will be received: (I) the nutritional and functional value of plants and plant by-products, as well as their safety parameters will be improved and the feed production technology based on SSF will be created; (II) a higher value feed stock for cows and newborn calves will be developed.

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1. LITERATURE REVIEW

1.1. Lactic acid bacteria for feed production 1.1.1. Lactic acid bacteria

Lactic acid bacteria (LAB) constitute a heterogeneous group of gram-positive bacteria, primarily nonsporulating, anaero-aerotolerant and produ-cing lactic acid as the main end metabolite from carbohydrate fermentation. LAB can dominate as the natural microbiota of many fermented substrates of plant and animal origin, where they play the key role in the development of the sensory properties (flavour and texture) and safety [20–22]. From the biochemical perspective, LAB include both homofermenters which mainly produce lactic acid, and heterofermenters which, apart from lactic acid, yield a variety of fermentation products such as acetic acid, ethanol, carbon dioxide and formic acid [23]. The LAB group is restricted to fourteen genera, five of them constituting the core group (Lactobacillus,

Lactoco-ccus, Leuconostoc, Pediococcus and Streptococcus) [22]. Lactobacilli vary

in morphology from long, slender rods to short coccobacilli, which frequently form chains [21]. The bioprotective potential of LAB has been studied since Louis Pasteur (in 1857) first described the lactic acid fermentation and Lister (in 1873) developed the first pure bacterial culture (“Bacterium lactis”, syn.: Lactococcus lactis) [24].

LAB as recombinant microbial cell factories have taken place during the last decades. In this context, it is important to note that the enormous potential of these Generally Recognized As Safe (GRAS) microorganisms by the US Food and Drug Administration (FDA), combined with the development of biotechnological, genomic and proteomic tools experienced during the last years, are expected to convert these microorganisms into emerging platforms for a wide range of applications [25]. Nowadays, it is widely accepted that LAB-derived products from the industrial manufacture of fermented food/feed, as well as lactic acid, antimicrobial peptides and high-value metabolites are by far the most important LAB applications from the economic point of view [26].

Bacteriocinogenic LAB and/or their isolated bacteriocins are considered safe additives (GRAS) useful to control the frequent development of pathogens and spoiling microorganisms in foods/feed production chain [27].

Most of LAB bacteriocins are small (<10 kDa) cationic, heat-stable, amphiphilic and membrane-permeabilizing peptides. They can be divided into three major classes; their classification has been constantly revised

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throughout the last decade due to the extensive research [28]. Many of these bacteriocins appear to exhibit a relatively little adsorption specificity [29].

1.1.2. Lactic acid bacteria for feed production and challenges associated with their legislation

LAB are Gram-positive gut commensal bacteria used in the production of silage and animal feed [30]. Silage is the product of anaerobic fermen-tation of water soluble carbohydrates (WSC) to organic acids in forage crops, of which LAB play a dominating role [31]. Animal feed is under threat of fungal decay during storage and feeding. Many studies are concer-ning on in vitro fermentation to explore the effects of LAB inoculants on in

vitro digestibility and improve nutritive value of forage [32, 33]. Also, most

of the LAB show probiotics properties. Apart from the use of probiotics in formulated animal feed, beneficial bacteria used as silage inoculants may also have a probiotic effects in the rumen [34]. Probiotics can (I) increase feed intake without significant improvement in feed conversion ratio (FCR) [35], (II) improve FCR without significant difference in feed intake [36], and (III) increase feed intake along with significant improvement in FCR [37]. However, this response depends on the survival of the inoculants in the silage as the pH drops.

The safety of LAB is discussed in general terms and is not specific to those used in animal feed [38]. Products containing LAB for feeding stuffs are categorized in the EU as feed additives. For these additives, the EU has a completely harmonized and very detailed legislation since 1996. Although the legislation is contained in directives, in practice they are interpreted as the EU regulations, the EU laws directly and literally applicable in all member states. In the EU, a LAB-containing feed additive is subject to a full approval process. Since 2003, the dossier for approval is evaluated by the Standing Committee on Food Chain and Animal Health and by the Scienti-fic Panel on additives and products or substances used in animal feed. It should be noted that a commercial feed additive is a product that contains not only the microorganisms in question but most often also cryoprotectants, excipients, bulking material, and some impurities such as residues from the fermentation broth [39].

The firm wishing to market the product must submit a full dossier that documents the product’s identity, its efficacy as an additive, and its safety. Safety is specifically construed as safety for the animal species consuming the feed, for the worker handling the additive and handling feed containing it (including the farmer), and for the consumer of the animal product. The safety documentation relates both to the microorganisms and to the other

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materials in the product. With few exceptions, all documentation for the microorganism relates to the actual strain in question and not just its species or genus [39, 40].

There is a large discrepancy between the EU regulatory frameworks for LAB in feed. For each regulatory measure introduced, there should be a clearly identified need. The legislation should be flexible and recognize that the primary responsibility for the safety of these cultures rests with the manufacturer. The legislation should be based on scientifically complete risk assessments. Such risk assessments are initiated on the basis of scienti-fically identified hazards such as, for example, the known virulence factors or transfer of antibiotic resistance. Whenever possible, the regulation should use a system of generic approval instead of approving one strain or one use at a time. The EU regulatory frameworks recommend a system that uses the inventories, or lists, of species or subspecies of microorganisms that either have a history of safe use or experimentally have been shown to be safe. The Qualified Presumption of Safety (the EU status) approach, when correctly applied, would serve this end [40–42]. In cases where generic approaches are not applicable, the legislation should allow for the evaluation of microbial strains according to their characteristics and intended use, instead of specifying a fixed list of studies to be done on all microorga-nisms. Bacterial strains used for special purposes may require specific approval or notification procedures [39]. This would result in enormous quantities of safety testing resources being pulled away from genuine safety issues. In addition, the increased demand on laboratory animals would be contradictory to the official EU Commission policy of phasing out all unnecessary animal testing [39, 43].

A food/feed additive in the US is defined extremely broadly to include virtually anything that might come into contact with food/feed, as long as it is not specifically evaluated as GRAS. The responsibility for regulating new strains of microorganisms for foods/feeds as GRAS rests with the Food and Drug Administration (FDA) and its Division of Biotechnology and GRAS notice review. The US has had the concept of GRAS since 1958, and in 1997 the US Government launched a new GRAS programme. The object of the new programme is both to simplify the GRAS concept itself and to simplify the FDA’s administration of substances that due to their relative safety do not merit an extensive review by experts. A GRAS substance is defined in the US Federal Food, Drug, and Cosmetic Act as: generally recognized, among experts qualified by scientific training and experience to evaluate its safety, as having been adequately shown through scientific procedures (or, in the case as a substance used in food before January 1,

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1958, through either scientific procedures or experience based on common use in food) to be safe under the conditions of its intended use [39, 44].

A food/feed company can use a new strain of a bacterium, however, it wishes to do it without any GRAS determination and without ever notifying the FDA. This is fully legal according to the US law. However, in the event of a microbiological food safety incident, the dialogue with the FDA would be much easier and the controversy and legal liability less if a company first has submitted a GRAS determination. In the case when the causes of a particular food safety incident have been unravelled, it could be documented that the FDA and the company have agreed on the quality of the safety evaluation of the new bacterium [39].

In addition, the chosen strain should tolerate the manufacturing, transportation, storage, and application processes, maintaining its viability and desirable characteristics. Also, being non-pathogenic to animals, the microorganisms used as probiotics are selected on the basis of their survival in the gastrointestinal environment and the ability to withstand a low pH and high concentrations of bile acids [38].

LAB have received the GRAS (USA) and the QPS (EU) status, although some species of Enterococcus and Streptococcus are pathogenic in nature [24, 45]. Lactobacillus and Bifidobacterium are probably the safest microorganisms used as probiotics because, first, these microorganisms have been safely used traditionally in various fermented food; second, these microorganisms are naturally present in the gastrointestinal tract (GIT) and other sites in humans [46] and animals in large quantities; and third, infec-tions associated with these microorganisms are extremely rare [47].

Probiotic LAB show attractive therapeutic properties and technological applications such as proteolytic activity, lactose and citrate fermentation, the production of polysaccharides, high resistance to freezing and freeze-drying capacity for adhesion and colonization in the digestive mucosa, production of vitamins and of antimicrobial compounds [48, 49]. Spore-forming bacteria like Bacillus amyloliquefaciens produce extracellular enzymes including α-amylase, cellulase, proteases and metalloproteases [50], which could increase nutrient digestion. Several studies have reported the beneficial effects of LAB inoculants on silage fermentation, and some LAB inoculants have also been reported to improve animal performance by increasing milk yield and feed efficiency [51].

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1.1.3. The influence of lactic acid bacteria on the content of mycotoxins in feed

Mycotoxins are frequent contaminants of cereals and their by-products. LAB, along with their probiotic properties, also reduce mycotoxins in feed. Mycotoxins in feed have a significant negative impact on dairy cattle in terms of various characteristics, thus affecting the economics of dairy cattle husbandry substantially [52].

The mycotoxin problem in public health is long-standing, and all humans and animals are at risk of mycotoxin exposure. Economic losses due to mycotoxins are diverse and can be associated with the reduction of the quality of foods/feeds for humans and animals, of animal production due to feed refusal or diseases, increasing medical costs for toxicosis treatment, increased costs to find alternative foods, to design an adequate management of contaminated supplies, to improve their detection and quantification methods and to develop the strategies that reduce toxin exposure. The animal production industry is most commonly subclinically affected by mycotoxins. Overall, most mycotoxins cause immunosuppression which can make animals more prone to a disease by weakening their immune system or making them less responsive to vaccinations [53].

In terms of effects and average concentration in dairy cattle feed, the most important mycotoxins are deoxynivalenol (DON), T-2 toxin and zearalenone (ZEN) [52]. DON is a Fusarium-produced mycotoxin that is one of the most commonly detected in feeds and is associated with a range of disorders including feed refusals, diarrhea, emesis, reproductive failure, and deaths. The impact of DON on dairy cattle is not well established, but clinical data have shown an association between the DON contamination of diets and poor performance in dairy herds.

The T-2 toxin is a very potent Fusarium-produced mycotoxin that occurs in a low (<10%) proportion of feed samples. It is associated with the reduced feed consumption, loss in yield, gastroenteritis, intestinal hemorrhage, reduced reproductive performance, bloody diarrhea, low feed consumption, decreased milk production, the absence of estrus cycles in cows, and death.

ZEN is a Fusarium-produced mycotoxin, which has the chemical structure similar to that of estrogen. It can produce an estrogenic response in ruminants, including vaginitis, vaginal secretions, poor reproductive perfor-mance and mammary gland enlargement of virgin heifers, and abortions [54]. ZEN has been shown to be immunotoxic and has also been found to be both a suppressor and an inductor of the production of inflammatory cytokines [55, 56].

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It is very difficult to prevent mycotoxin contamination both pre-harvest and during the storage of feeds [57]. Several tools for the neutralization of mycotoxins have been developed to protect animals from the ingestion of contaminated feeds [58]. As recently reviewed, the inclusion of sorbent materials in animal diets or the addition of enzymes or microorganisms capable of detoxifying mycotoxins have been reported to be reliable methods for the prevention of mycotoxicosis in farms. In particular, myco-toxin sequestering agents are compounds able to bind mycomyco-toxins in contaminated feeds without dissociating the toxin-sequestering agent complex; thus, it could pass through the gastrointestinal tract of animals, and the toxin could be eliminated with feces [59], but the natural LAB fermentation of maize meal can substantially reduce the amount of added aflatoxin B1 (AFB1) from the culture medium. The toxicity of the culture

media was also reduced after a 4-day fermentation period with a progressive decrease in the pH of the culture media. This is in agreement with other studies which clearly show that the Lactobacillus strains efficiently remove AFB1 from the culture solution [60].

The activities of bacteria and fungi used in fermentations are responsible for the desired transformations of food/feed components, but hundreds of additional enzymatic activities are present in their cells actively secreted into the food/feed matrix or released from disintegrated cells after autolysis. Some of these activities may transform mycotoxins into non-toxic products, but no microbial strain has been authorized so far as a processing aid targeting mycotoxins [61]. However, fermentation is one of the easiest and cheapest means of food/feed preservation in addition to imparting nutritional and organoleptic benefits to the fermented foods/feeds. Fermen-tation is effected by the natural microbiota of raw materials, microorganisms attached to the fermentation equipments or from externally added starter cultures. Yeasts, especially S. cerevisiae and Candida krusei, and LAB occur as part of the natural microbial population in spontaneous food/feed fermentation and as starter cultures in the food/feed industry [62].

The lower pH of the media could also contribute to the removal of toxins from the media as other studies have shown that the treatment of LAB pellets with hydrochloric acid significantly (p < 0.05) enhanced the binding ability of the bacteria. The lower pH of the media could also have contributed to the removal of toxins from the media as other studies have shown that the treatment of LAB pellets with hydrochloric acid significantly (p < 0.05) enhanced the binding ability of the bacteria.

The lower pH of the media could also have contributed to the removal of toxins from the media as other studies have shown that the treatment of

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LAB pellets with hydrochloric acid significantly (p < 0.05) enhanced the binding ability of the bacteria [63].

To obtain information about the rate and extent of either favorable or adverse fermentations that occur in silages, fermentation end-products are commonly used. To this end, different fermentative quality indexes, such as Flieg–Zimmer’s or Vanbelle–Bertin’s scores, have been proposed to rank between both well- and poorly preserved forages according to the relative amounts of lactic acid, acetic acid, butyric acid or ammonia nitrogen. Recently Gallo et al. [64, 65] have developed an index by using a multi-variate approach (factorial analysis) to evaluate the fermentative quality of MS (whole-crop forage maize). This fermentative quality index was highly correlated with the presence of yeasts and molds in silage, as well as with the concentrations of mycotoxins produced by A. fumigatus, P. roqueforti,

P. paneum and Fusarium spp. Anyway, if a mycotoxin-contaminated forage

is used, it should be recommended to discard the moldy parts or any material contaminated with mycotoxins, to reduce its use in diets by substituting it with other available forages or fibrous by-products and to use the adequate sorbent materials as successively discussed. After economical and management evaluations, feeds proved to be too dangerous for animal health should not be used [65].

According to the FAO, Europe has the most extensive regulations for mycotoxins in feed. Canadian regulations are among the most detailed as they additionally include mycotoxins not regulated in the EU feedstuffs, such as ergot alkaloids and diacetoxyscirpenol (a trichothecene), with China and the Islamic Republic of Iran also having the demanded limits in place. Nevertheless, regulations in the rest of the world undoubtedly focus majorly on AFLs, with only 15 countries in Africa having specific, feed-oriented mycotoxin regulations in place [66].

1.1.4. Lactic acid bacteria and formation of biogenic amines

LAB are generally considered to be non-toxic and non-pathogenic. However, some species of LAB can produce biogenic amines (BAS) in high

concentrations. Establishing the toxic level of BAs is difficult as it depends on the characteristics of different individuals [67].

Biogenic amines are low-molecular-weight organic bases present in all organisms. The most common forms are tyramine, putrescine, histamine, methylamine and tryptamine. At low concentrations, they are essential for the normal growth and differentiation of cells, but in larger quantities (1.4 g per day) they become harmful to humans and livestock [68, 69]. Gram-positive bacteria such as Streptococci and Lactobacilli, as well as

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negative species have the ability to produce BAS from amino acids. The

number of Lactobacillus spp. markedly increased following the addition of fermentable carbohydrate, and the correlation analysis revealed that the number of Lactobacillus spp. was linked to the concentration of ruminal tyramine, putrescine, histamine, methylamine and tryptamine [69].

The pH is an important factor for fermentation and the formation of BAS, because the amino acid decarboxylase activity is higher in an acidic

environment. This may explain why decarboxylase enzymes have the optimum pH of around 5.0. Furthermore, the bacterial growth also increases the amount of BAS by raising the production of the decarboxylase enzyme

[70].

1.1.5. Lactic acid bacteria resistance to antimicrobials

The development of resistance amongst bacteria to antimicrobials remains a serious concern. For this reason, viable microorganisms used as the active agent(s) in feed additives should not add to the pool of antimicrobial resistance genes already present in the gut bacterial population or otherwise increase the risk of drug resistance transfer [71].

For the assessment of bacteria used as feed additives, strains can be categorised as susceptible or resistant to antimicrobials. Susceptible (S): a bacterial strain is defined as susceptible, when it is inhibited at the con-centration of a specific antimicrobial equal to or lower than the established cut-off value (S ≤ x mg/L). Resistant (R): a bacterial strain is defined as resistant when it is not inhibited at the concentration of a specific antimic-robial higher than the established cut-off value (R > x mg/L). The cut-off values identified should be regarded as a pragmatic response intended to introduce consistency in the separation of strains with the acquired resistan-ce from susresistan-ceptible strains. The cut-off values are not intended for any purpose other than the assessment of microbial products for the possible presence of antimicrobial resistance [71].

When a bacterial strain demonstrates the resistance to a specific antimicrobial higher than the other strains of the same taxonomical unit, the presence of the acquired resistance is indicated and additional information is needed on the genetic basis of antimicrobial resistance [71].

The growing public concern over the widespread use of antibiotics in livestock production and its implication in the emergence of antibiotic-resistant bacteria has stimulated interest in developing alternatives that promote animal performance and health [72]. One potential alternative is the use of direct-fed microbials (DFM) as feed additives [73].

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The emergence of multi-drug-resistant pathogens is now one of the greatest threats to public health around the world [74], although the initial emergence of antibiotic resistance is believed to be the out-safety of probiotics and a potential public health risk. The imprudent use of antibio-tics is believed to be the major cause of widespread antibiotic resistance [38, 75].

The Lactobacillus species, reported to harbour transferable antibiotic resistance genes, are components of some commercial probiotics [38, 76].

All bacterial products intended for use as feed additives must be exami-ned to establish the susceptibility of the component strain(s) to a relevant range of antimicrobials of human and veterinary importance. As the basic requirement, the minimum inhibitory concentration (MIC) of the antimicro bials, expressed as mg/L or µg/mL, should be determined for each of the following substances: ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol and, in specific cases, tylosine, apramycin, nalidixic acid.

Guidance on the assessment of the bacterial antimicrobial susceptibility of sulfonamide and trimethoprim

The FEEDAP Panel considers that any bacterial strain carrying an intrinsic resistance to antimicrobial(s) presents a minimal potential for horizontal spread and thus may be used as a feed additive. Any bacterial strain carrying an acquired resistance to antimicrobial(s) shown to be due to chromosomal mutation(s) presents a low potential for horizontal spread and generally may be used as a feed additive. Any bacterial strain carrying an acquired resistance to antimicrobial(s) that is shown to be due to the acquisition of genetic determinant(s) presents the greatest potential for horizontal spread and should not be used as a feed additive. In the absence of information on the genetic nature of a demonstrated resistance, the strain should not be used as a feed additive [71, 77].

1.1.6. Lactic acid bacteria probiotic properties

Several LAB strains, species belonging to the genera Lactobacillus,

Bifidobacterium and Enterococcus are considered beneficial to the host and

thus have been used as probiotics [78]. Probiotics, defined as a ”live microbial feed supplements” which beneficially affect the host animal by improving its microbial balance, have been proposed for several decades [79, 80]. The potential probiotic strains, characterized as normal inhabitants of the target species, have the ability to adhere to and colonize the epithelial cells of the gut and to survive and grow in the respective ecological units. The strains were genetically stable, able to produce antimicrobial

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ces, antagonistic toward pathogenic or cariogenic bacteria. The strains were able to compete with normal microflora, and resistance to bile and acids can exert one or more clinically documented health benefits. Cell immobiliza-tions, selections of acid and bile-resistant strains, oxygen-impermeable containers have been proposed to improve the viability of probiotic bacteria. In addition, molecular tools based on 16S ribosomal DNA sequences and PCR techniques have been developed for identifying probiotics strains [81, 82]. Probiotics have a number of beneficial health effects in humans and animals, such as reducing lactose intolerance symptoms and enhancing the bioavailability of nutrients. Probiotics help regulate intestinal microflora and immunomodulatory properties [83]. Most of the microorganisms used as probiotics in animals are safe, although some have problems, particularly the enterococci, which may harbour transmissible antibiotic resistance determinants, and the Bacillus cereus group known to produce enterotoxins and an emetic toxin [82, 84]. The most commonly used probiotics in dairy cattle often fall into three categories according to the category of microorganisms: (I) yeast preparation; (II) lactic acid bacteria preparation; (III) Bacillus preparation. These probiotics are primarily targeted for enhancing intestinal health, improving milk production and feed conversion efficiency [33]. Yeast preparations are often fed as live or dead products with or without fermentation extracts as feed additives. Desnoyers et al. [85] reported that yeast supplementation increased dry matter intake (DMI), milk yield and fat-corrected milk; it also increased the rumen pH and volatile fatty acid (VFA) concentrations and decreased the rumen lactic acid concentrations. In in vivo studies, Jiang et al. [86] have found that dietary supplementation of Lactobacillus can significantly increase the milk pro-duction as compared with the control group and reduce the count of somatic cells. Moreover, other researchers found that Bacillus subtilis spores not only increased the antibody level and T cell responses to a co-administered soluble antigen, but also broadened them. These characteristics of Bacillus

subtilis are very important for dairy cattle to enhance their health and milk

safety [33, 87].

Walter [88] has studied the role of lactobacilli in the mammalian intestinal tract and concluded that probiotic strains are particularly efficient for activating the immune system, even though they can hardly persist in the gut. Probiotics, at least in some cases, improve the efficiency of milk production in dairy cows. In the recent years, with the development of molecular methodologies, the advance in molecular methodologies has offered a more precise analytic approach to the analysis of the rumen micro-flora and the intestinal microbial ecosystem, which can help us to better understand their mode of action [33].

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LAB can inhibit pathogenic bacteria by competing for nutrients in the gut or for binding sites on the intestinal epithelium [89]. As most intestinal pathogens must adhere to the intestinal epithelium to colonize the intestine and to produce diseases, some LAB strains have been chosen as probiotics specifically based on their ability to adhere to the intestinal epithelium and thus compete with pathogens for binding sites [90].

Epithelial cells in the gastro-intestinal mucosa create a selectively permeable barrier between the intestinal lumen (which contains harmful substances such as foreign antigens, micro-organisms and toxic materials, as well as beneficial nutrients) and the internal environment of the body [91]. This barrier is the first line of defence against microbes in the GIT [92]. It has a combined defence function incorporating anatomical structures, immunological secretions consisting of the mucus, immunoglobulins, e.g., IgA, antimicrobial peptides, and the epithelial junction adhesion complex. The disease conditions, which cause immunological disturbances, disrupt this barrier, inducing the inflammation of the intestinal wall and intestinal disorders [38, 93]. Experiments in animal models have shown that the improvement in the intestinal barrier function by probiotics is due to the reduction in the permeability of the intestinal epithelium. The translocation of intestinal microbes out of intestinal sites into sites such as the liver, spleen and mesenteric lymph nodes decreased in mice with induced colitis and pre-treated with Lactobacillus probiotics [38, 94].

The meta-analysis of papers published on the effects of yeast probiotics in all ruminant species reared for milk or meat [85] found much variability in response, with an overall average increase in DMI of 0.44 g/kg body weight and the total tract organic matter digestibility by 0.8%, the effects being too small to warrant probiotic addition [38].

The quality control of the probiotic strain production and the subse-quent shelf viability is a critical component of trials assessing the effect they have when fed, and often in nutrition trials this is inadequately dealt with and could be a reason for the variability in animal response among the trials [38].

Enforced restrictions on the use of antibiotics as growth promoters (AGPs) in animal production have prompted investigations into alternative feed additives in the recent decades, and the probiotics are currently the main feed additive used in livestock [95].

Potentiated probiotics are bio-preparations containing production strains of microorganisms and the synergistically acting components of natural origin that potentiate the probiotic effect on both the small intestine and the colon. Potentiated probiotics are more effective than their components

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separately, and their potentiated protective and simulative effects were expressed in all parts of the digestive tract [82, 96].

1.2. The effects of lactic acid bacteria on the health parameters and productivity of dairy cattle and calves

Gut microbial balance is one of the most important factors to provide for a good health status in young animals, particularly calves, because the immature immune system is prone to debilitating diarrhea and respiratory diseases [97]. LAB increased the lactic acid content of silage and had the potential to increase dry matter (DM) digestibility and to decrease ruminal methane production [98]. The use of LAB has been identified as a tool to maintain the intestinal microbial balance and to prevent the establishment of opportunistic pathogenic bacterial populations. LAB can be used as growth promoters in calves instead of antibiotics to counteract the negative effects of their widespread use [99]. Nutrition has an important impact on the reproductive performance of dairy and beef cattle. The impact of nutrition on the fertility of dairy herds may be caused by both direct and indirect effects of nutrients on the reproductive traits [100]. In clinical trials, LAB have been reported to enhance the growth of many domestic animals including cows, neonatal calves, piglets, and broilers. Therefore, the studies on the efficacy of LAB strains must be performed in target species/animal categories. The claims for microbial products are: improved performance and feed conversion for the target species; reduced morbidity or mortality; benefits for the consumer through the improved product quality [82, 101]. However, feeding with diets high in cereals is often associated with multiple metabolic disorders like rumen acidosis, laminitis, fatty liver [102], displaced abomasums, and bloat. Among these diseases, subacute rumen acidosis (SARA) represents one of the most important metabolic disorders in intensive dairy farms, which affects rumen fermentations, animal welfare, productivity and farm profitability and is characterized by reduced pH values (5.8–5.0) in the rumen content, occurring either for longer periods or repeatedly [69,103]. Supplementation with the live LAB culture may reduce the incidence of acute ruminal acidosis. Although the mode of action of the LAB for reducing the incidence of rumen acidosis is not completely clear, the presence of lactate-producing bacteria is thought to modify the rumen microbial population and help the rumen microbes adapt to the presence of lactic acid [103]. In addition, Baah et al. [72] have suggested that supplementation with 12 × 107 CFU/kgdiet DM of a mixed culture of LAB

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to barley grain and barley silage-based feedlot cattle diets could improve ruminal fermentation.

Neonatal ruminants are unique in that, at birth, they are physically and functionally two different types of animals with respect to their GI system. The intestine of a newly born calf is sterile, and the colonization of the GI tract begins immediately after birth. Thereafter, a complex and dynamic microbial ecosystem with high densities of living bacteria is established in the large intestine as animals grow to maturity [78]. Stress in young calves frequently leads to scours or diarrhea and weight loss [38]. The early expe-rience of ingesting feeds increases the preference and later the consumption of these feeds by animals. Early dietary experiences have a greater and more lasting effect than those occurring later in life. Different processes (neurological, morphological, and physiological) may be involved early in life and can be altered so that animals can better manage in the environment in which they are reared from birth [15]. Other studies using LAB also showed a reduced incidence of diarrhea in calves [38, 104].

In young pre-ruminants, the Lactobacillus species generally targets the lower intestine and represents an interesting means to stabilize the gut microbiota and decrease the risk of pathogen colonization. LAB are a well-known supplement for young calves, regarded as applicable in regular feeding practices. Previous findings support the beneficial effects of these products in balancing the GI tract microbiota as well as in animal nutrition and health [78].

Also, LAB have been used to control the effects of pathogens such as

Salmonella spp. and Escherichia coli. These two pathogens are the most

frequent bacterial etiologic agents in calf scours during the first week of life [99, 105].

Dairy and beef cattle fed direct-fed microbials (DFM) showed impro-ved growth performance, milk and meat production, and feed efficiency in many experiments [106, 107]. Also, LAB for adult ruminants have mainly been selected to improve fiber digestion by rumen microorganisms. Such LAB have positive effects on various digestive processes, especially cellulo-lysis and the synthesis of microbial proteins [78].

Limiting of rumen acidosis. Regarding bacterial additives for adult ruminants, lactate producing bacteria (Enterococcus, Lactobacillus) which sustain lactic acids are a more constant level than Streptococcus bovis and may represent a possible means of limiting acidosis in high-concentrate-fed animals, especially feedlot cattle [78, 108].

The condition is referred to as subacute ruminal acidosis (SARA) when the pH drops below 5.6 and remains between 5.2 and 5.6 for at least 3 hours per day. This condition is economically very important as milk production

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by the suffering animal is reduced due to loss of appetite, diarrhea, dehydra-tion, debilitadehydra-tion, impaired rumen motility, and impaired fibre digestibility [109]. Lactic acidosis is the more severe form of ruminal acidosis where the pH drops below 5.2 due to lactate accumulation. Bacterial additives are effective in preventing or treating ruminal acidosis [110].

The use of rumen manipulators is an option to enhance animal produc-tivity. Rumen manipulation can be done by the use of many growth stimu-lants including hormones and antibiotics. However, it has potential risks of two prevailing public health problems such as the development of antibiotic resistance genes and milk and meat antibiotic residues [111]. The potential alternative is feeding of microbials as probiotics also called DFM [112].

Improving the animal growth rate. In animal nutrition, microor-ganisms used as probiotics were linked with a proven efficacy on the gut microflora. Administration of probiotic strains separately and in combi-nation significantly improved feed intake, feed conversion rate, daily weight gain and total body weight in chicken, pig, sheep, goat, cattle, and equine [82, 113]. Selected microorganisms can increase the weight gain of rumi-nants. For example, additives containing a mixture of microorganisms (L. reuteri DDL 19, L. alimentarius DDL 48, E. faecium DDE 39, and

B. bifidium DDBA) isolated from a healthy ruminant, when fed to ruminants

for eight weeks commencing at 75 days of age, resulted in the improvement of the average body weight by 9% [38, 114].

Increasing milk yield. Supplementing animal feed with selected LAB has a beneficial effect on the subsequent milk yields, fat and protein content [115]. The blood and milk parameters were significantly improved as shown by higher concentrations of serum cholesterol and total lipids and a higher milk fat and protein content [82, 116]. Selected LAB can improve the milk yield in dairy animals. Increased feed intake together with improved microbial digestion of feed could be the possible mode of action for improved animal performance. In contrast, Dutta et al. [117] found no diffe-rences in DM intake, body weight gain, milk yield, and milk composition when yeast was added in the diet for lactating cattle [118].

Researchers have tested the L. plantarum strain and found that increa-sing the microbial biomass yield (MBY) in ruminal in vitro fermentation has well-documented effects of increasing milk production. They have also included the fatty acid (FA) treatment as a positive control, because it is known to improve silage protein preservation [119, 120].

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1.3. Lupine and soya chemical composition and their use for cattle feeding

1.3.1. Lupine and soya chemical composition

About 400 species of lupine (genus Lupinus) have been found in nature. Lupine, a crop with high protein and fibre contents, is a rich source of phytochemicals, importantly bioactive peptides, alkaloids, polyphenols, phytosterols, tocopherols, etc. [121]. In the dry matter of seeds of lupine varieties (Lupinus albus, L. angustifolius, L. luteus), individual constituents are in a relatively wide range depending on the variety and climatic conditions: (g/kg) proteins 317.1–458.9, fats 52.2–125.8, fibre 101.2–154.2, nitrogen-free extract 285.9–436.5, starch 41.3–102.6, organic matter 951.8– 966.2, ash 33.8–48.2, calcium 2.3–5.1, phosphorus 4.6–8.0, magnesium 1.4–2.5, and acid-detergent fibre 133.1–209.3 [122]. Lupine is increasingly used as a protein source in European countries as a replacement for poten-tially genetically modified soya products. Lupines are successful protein crops in Australia too, where an important industry has developed to use lupine protein and other fractions, yet lupine production in Europe is insufficient to guarantee the stable and sufficient supply required for its use by the food/feed industry.

Lupine is grown in several European countries, and although its grain yield is the world’s highest in some parts of Europe, its cropping area remains modest and yields are highly variable. White lupine (Lupinus albus

L.), yellow lupine (L. luteus L.), and narrow-leafed lupine (L. angustifolius L.), are native European legumes that represent a significant alternative to

soya bean. Their seed protein content is high (up to 44%) and its quality is good, they offer potential health benefits and contribute to the sustainability of cropping systems [123]. Yellow lupines were bred in Lithuania until 1995. The narrow-leaved lupine breeding program was started in 1995. The breeding work is done in three directions: first, low-alkaloid narrow-leaved lupine varieties bred for food industry, second – low-alkaloid narrow-leaved lupines bred for animal feeding, and third – narrow lupines bred for green manure [124].

The nutritional value of lupine proteins is higher than that of beans or peas, mainly because of the high concentrations of the essential amino acids lysine, leucine, and threonine, which are higher only in soya beans [125]. Compared to soya protein, the amino acid composition is characterized by a low content of methionine, cysteine, lysine, threonine, and tryptophane, while the arginine content is significantly higher [126]. A high arginine content is characteristic of lupine protein [122].

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In Europe, grain legumes are grown on only 5,726 thousand ha in 2013 (2.8% of the global area), of which soybean by 3,176 thousand ha (2.8% of global) and produced 5,943 thousand tons (2.8% of global production) [127]. Soybean is currently one of the most important agricultural crops in the world, holding over 100 million ha sown annually, the 4th place after wheat, rice, and corn. The extent which soybean cultivation has reached in the recent decades is due to the chemical composition of the crop, rich in biochemical constituents with a high biological value (approximately 20% of lipids and around 40% of proteins), which form a significant proportion of essential fatty acids and amino acids in the human body; very varied possibilities of processing and use of the crop (edible fats, animal feed concentrates, protein preparation for food, biofuels, other uses as a raw material in very different industries); the importance as a leguminous plant for land fertility improvement in crop rotations [127]. Soya bean has become the prevalent source of plant proteins for food/feed, and Europe de-pends on soya bean imports for 70% of its plant protein requirements [123]. It is important that lupine seeds are characterised by a high content of pro-teins: in yellow varieties it largely exceeds the protein content in soybeans. Compared to soybeans, lupine seeds grown in Europe have a significantly lower content of fat, ranging from 52.2 to 125.8 g/kg [128] in the original matter of the monitored varieties. As regards white lupine of the Amiga variety, some authors [129, 130] found its fat content to be 107.7 g/kg [122].

Lupine has a high content of essential amino acids, protein, fibre and a low fat content if compared with soya bean [131, 132]. These features provide a huge industrial potential for this legume. Lupine is considered as a cheap alternative stock to other legume crops such as soya beans as it contains a comparable quantity of proteins with a similar profile of amino acids [133]. Also, in Mediterranean environments, lupine seeds are a poten-tial alternative to soybean meal in organic breeding systems [134], more-over, they are legumes suitable for rotation with winter wheat [135].

The reduction of protein digestibility in lupine and soya seeds has been associated with the presence of protease inhibitors as well as antinutritionals such as fibre and oligosaccharides [135, 136]. Sweet lupine seeds (Lupinus

albus and L. angustifolius) have a much lower amount of inhibitors

(< 0.1 mg/g) than soya beans (26.2 mg/g) [137]. In the last few years, fermentation has been performed to increase the content of bioactive pheno-lic compounds in legumes, thus enhancing their antioxidant activity [138]. The digestibility of lupine protein could be improved also by lactic acid fermentation [139].

Soybeans and soybean meals were fermented by Aspergillus oryzae GB-107 in a bed-packed solid fermentor for 48 hours. After fermentation,

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their nutrient contents as well as the trypsin inhibitor were measured and compared with those of raw soybeans and soybean meals. Proteins were extracted from fermented and non-fermented soybeans and soybean meals, and the peptide characteristics were evaluated after electrophoresis. Fermen-tation increased the protein content, eliminated trypsin inhibitors, and reduced the peptide proportion in soybeans and soybean meals. These effects of fermentation might make soy foods more useful in human diets as a functional food and benefit the livestock as a novel feed ingredient [140]. Diverse anaerobic microbes in adult ruminant animals possess a unique ability to employ microbes to degrade protein sources to ammonia, volatile fatty acids and gases [141].

1.3.2. The use of lupine and soya for cattle feeding, their anti-nutrient factors and the possibilities to reduce them

Legumes offer important opportunities for sustainable grassland-based animal production, because they can contribute to important key challenges by 1) increasing forage yield, 2) substituting inorganic N-fertilizer inputs with symbiotic N2 fixation, 3) mitigating and facilitating adaptation to

climate change, as elevated atmospheric CO2, warmer temperatures and

drought-stress periods increase, and 4) increasing the nutritive value of herbage and raising the efficiency of herbage conversion to animal protein [142].

Proteins capable of inhibiting the proteolytic activity of digestive enzy-mes are common constituents of leguenzy-mes. These protease inhibitors are generally believed to be largely responsible for the poor digestibility of the protein of legumes. The phytohemagglutinins also play an important role contributing to the poor nutritive value of some legumes, particularly those belonging to the genus Phaseolus [143].

The use of legumes as a source of protein is somewhat limited by the low digestibility of most plant proteins [144]. Previous digestibility studies of protein obtained from legumes have shown interactions between antinutritional compounds, such as trypsin inhibitors and tannins, and the decreased proteolytic susceptibility of protein complexes, thereby decree-sing the food/feed value of plant proteins [145].

The possibilities of using lupine in the feed rations of milk cows and in the fattening of bullocks are summarized by Homolka et al. [146]. Inclusion of lupine seed meal in the feed rations for high-utility milk cows assumes the crushing and flaking of seeds and their inclusion into 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 advantage is that, unlike soybeans,

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they do not need to be heat-treated. Depending on the lupine variety, the degradability of lupine seed protein in rumen ranged from 71 to 79%. The lipidic component of lupine has a favourable effect on the milky efficiency and milk fat, and changes in the concentration of fatty acids with long strings in milk are positive from the viewpoint of human nutrition. Cattle can use even whole fodder plants as fresh fodder or ensilage [122].

Lupine seed is a legume with a high crude protein (CP) content (35– 40%), which has a high degradability (665 g/kg of CP) and contains both the low starch content and the high content of non-starch polysaccharides. The seeds of traditional varieties contain substantial amounts of alkaloids (5–20 g/kg DM) [147] which can reduce intakes, but newer varieties of sweet lupine have a low alkaloid content (<0.05% DM), especially sweet white lupine (Lupinus albus L.). They also appear to be free of other antinutritional factors such as lecithin, antitrypsin and hemagglutinin. Ho-wever, the high ruminal degradability of protein makes difficult their use as replacements for soybean meals in the ruminant rations, but this issue is controversial [135, 148].

Marley et al. [149] investigated the effects of feeding concentrates containing either yellow lupines or soya beans to mature dairy cows, and it was indicated that lupines could play a role in replacing imported soya bean in the diet of dairy cows in many countries, but that further research is necessary to determine the effects of other legume grains on the fertility of cows. Therefore, this study was planned to provide alternative feeding strategies for dairy farming comparing the diets based on different protein sources (soya bean, pea, and faba bean) to assess their effect on and metabolic and reproductive responses of early-lactating dairy cows [100].

One constraint to the use of legume-based cow systems is the presence of phytoestrogens in the forage, which reduces cow fertility. Phytoestrogens are a group of naturally occurring plant-derived non-steroidal compounds which have the ability to cause estrogenic and/or anti-estrogenic effects in livestock species [150]. On the other hand, different plant species contain different types and also different concentrations of these estrogen-mimi-cking compounds, which may have led to a contrary anecdotal evidence as to the effects of legumes per se on ruminant fertility. So, there is a need to develop clear messages on the effects of these forage legumes on ruminant fertility for the farming industry, messages which consider the availability of new varieties, current understanding and future research requirements to overcome the practical constraints for their use in dairy farms [100].

Lupines are a major toxic problem in range sheep. However, lupines are toxic when ingested at 1% or less of ruminant body weight [151]. Quinolizi-dine alkaloids are found in various plants including those belonging to the

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genus Lupinus, although the nature and level of these alkaloids is highly variable among species. While they are not the only alkaloids found in lupines, they are the major concern in relation to human and animal health. The levels of alkaloids in seeds or meal can be reduced through the de-bittering process involving soaking or washing with water. The level of alkaloids in lupines after the de-bittering process is reported to be approxi-mately 500 mg/kg. Data indicate that the mean alkaloid content of market-able sweet lupine seeds is on average 130–150 mg/kg [151, 152].

Many feedstuffs commonly used in preparing diets for animals contain antinutritional factors (ANFs). These factors, which cause depressions in growth and feed efficiency and/or affect animal health, can be defined as antinutritional factors (ANFs). In plants and seeds, these ANFs primarily act as biopesticides, protecting them against moulds, bacteria, and birds. According to the above definition, dietary fibre (nonstarch polysaccharides, NSPs) can also be classified as ANFs [28]. As mentioned by Green et al. [153], lupine (Lupinus spp.) contains teratogenic piperidine alkaloids. Piperidine alkaloids are acutely toxic to adult livestock species and produce musculoskeletal deformities in neonatal animals. These teratogenic effects include multiple congenital deformities in cattle, pigs, sheep, and goats [122, 153].

The nutritive value of soybean is limited mainly by trypsin and chymo-trypsin inhibitors, pectins and the protein as regards immunology activity. In animals, they cause lowered nitrogen retention, decreased performance results and an increased metabolic nitrogen excretion [154]. The trypsin inhibitor activity is very low in lupines, ranging from 0.1 to 0.2 mg/g in

L. albus [155]. The reaction of animals to antinutritional substances in

soybean depends on animal species and age. Adult ruminants are not sensitive to these substances, whereas a decreased growth of chicken, pigs, calves and rats was observed when raw soybean had been given to them. Soybeans contain several antigenic proteins, which can stimulate the immune system sensitivity of calves, pigs and humans [156]. These proteins are not sensitive to temperature. Lectins (hemagglutinins) are proteins that bind to carbohydrates. Raw soybean can decrease the growth and increase mortality rate in animals [154]. The soybean requires the processing to eliminate anti-nutrient factors, particularly in the feeding of non-ruminants. The anti-nutrient substances such as protease inhibitors, lectins, pectins, urease, lipooxygenases, and anti-vitamin substances are decomposed at a high temperature or in fermentation processes [157].

Different technological methods are used to improve the nutritional value of legumes: thermal treatment (cooking, extrusion), germination, fermentation, etc. [158]. Various studies have demonstrated a reduction in

BIOTECHNOLOGICAL SOLUTIONS FOR DAIRY CATTLE FEED PRESERVATION AND THEIR INFLUENCE ON CATTLE HEALTH PARAMETERS AND PRODUCTION QUALITY

Vita Krunglevičiūtė

BIOTECHNOLOGICAL SOLUTIONS

FOR DAIRY CATTLE FEED

PRESERVATION AND THEIR INFLUENCE

ON CATTLE HEALTH PARAMETERS

AND PRODUCTION QUALITY

Vita Krunglevičiūtė

NATŪRALIŲ BIOPRIEMONIŲ

PANAUDOJIMO GALIMYBĖS PIENINIŲ

GALVIJŲ PAŠARŲ KONSERVAVIMUI IR

JŲ ĮTAKA GYVULIŲ SVEIKATINGUMUI

BEI PRODUKCIJOS KOKYBEI

TABLE OF CONTENTS

ABBREVIATIONS

INTRODUCTION

1. LITERATURE REVIEW