APPLICATION OF FOOD INDUSTRY BY-PRODUCTS FOR MICROORGANISMS ENCAPSULATION/IMMOBILIZATION AND SUSTAINABLE ANTIMICROBIAL PROPERTIES FEED SUPPLEMENTS PREPARATION

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Paulina Zavistanavičiūtė

APPLICATION OF FOOD INDUSTRY

BY-PRODUCTS FOR MICROORGANISMS

ENCAPSULATION/IMMOBILIZATION

AND SUSTAINABLE ANTIMICROBIAL

PROPERTIES FEED SUPPLEMENTS

PREPARATION

Doctoral Dissertation Agricultural Sciences, Animal Sciences (A 003)

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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 2016–2020.

Scientific Supervisor

Prof. Dr. Elena Bartkienė (Lithuanian University of Health Sciences, Veterinary Academy, Agricultural Sciences, Animal Sciences – A 003). Dissertation is defended at the Animal Sciences Research Council of the Veterinary Academy of Lithuanian University of Health Sciences: Chairperson

Prof. Dr. Vaidas Oberauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Animal Sciences – A 003).

Members:

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

Assoc. Prof. Dr. Darijus Skaudickas (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine (M 001);

Assoc. Prof. Dr. Vilma Kaškonienė (Vytautas Magnus University, Natural Sciences, Chemistry – N 003);

Prof. Habil. Dr. Katarzyna Michałek (West Pomeranian University of Technology, Agricultural Sciences, Animal Sciences – A 003).

Dissertation will be defended at the open session of the Animal Science Research Council of Lithuanian University of Health Sciences on the 18th_of January, 2021, at 10:00 a.m. in Dr. S. Jankauskas Auditorium of the Veteri-nary Academy.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA

Paulina Zavistanavičiūtė

ŠALUTINIŲ GAMYBOS PRODUKTŲ

TAIKYMAS MIKROORGANIZMŲ

ĮKAPSULIAVIMUI / IMOBILIZAVIMUI

BEI TVARIŲ IR ANTIMIKROBINĖMIS

SAVYBĖMIS PASIŽYMINČIŲ

PAŠARŲ PRIEDŲ GAMYBAI

Daktaro disertacija Žemės ūkio mokslai, gyvūnų mokslai (A 003)

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Disertacija rengta 2016–2020 metais Lietuvos sveikatos mokslų universitete Veterinarijos akademijos Maisto saugos ir kokybės katedroje.

Mokslinė vadovė

prof. dr. Elena Bartkienė (Lietuvos sveikatos mokslų universitetas, Veterina-rijos akademija, žemės ūkio mokslai, gyvūnų mokslai – A 003).

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

Pirmininkas

prof. dr. Vaidas Oberauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, gyvūnų mokslai – A 003).

Nariai:

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

doc. dr. Darijus Skaudickas (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

doc. dr. Vilma Kaškonienė (Vytauto Didžiojo universitetas, Gamtos mokslai, chemija – N 003);

prof. habil. dr. Katarzyna Michałek (Ščecino Vakarų Pamario techninių mokslų universitetas, žemės ūkio mokslai, gyvūnų mokslai – A 003).

Disertacija ginama viešame Gyvūnų mokslo krypties tarybos posėdyje 2021 m. sausio 18 d., 10 val. Lietuvos sveikatos mokslų universiteto Veteri-narijos akademijos Dr. S. Jankausko auditorijoje.

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ABBREVENTIONS

AA – acetic acid AFB – aflatoxins AFB1 – aflatoxin B1 Ala – alanine Alb. – albumins AML – amoxicillin

AMP – adenosine monophosphate

ANB – ban of all antibiotics

ARB – arabinogalactan

Asp – asparagine

AST – alanine aminotransferase

B/F – berries/fruits

BA – biogenic amines

BC – bovine colostrum

CFU – colony-forming units

CMP – cytidine monophosphate

DIZ – diameter of inhibition zone

DON – deoxynivalenol

EFAA – essential free amino acids

EOs – essential oils

ERY – erythromycin

FA – fatty acids

FAA – free amino acids

FDA – Food and Drug Administration

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

FUM – fumonisin GEN – gentamycin Gly – glycine Glu – glucose Glut – glutamine GMP – guanosine monophosphate

GRAS – generally recognized as safe

HPLC – high performance liquid chromatography

IgA – immunoglobulin A

IgG – immunoglobulin G

IgM – immunoglobulin M

Ile – isoleucine

IMP – inosine monophosphate

YEX – yeast extract

LA – lactic acid

LAB – lactic acid bacteria

Leu – leucine

LF – lactoferrin

Lys – lysine

LUHS – Lithuanian University of Health Sciences (LSMU)

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Mg – magnesium

MIC – minimum inhibitory concentration

MRS – De Man, Rogosa and Sharpe medium

MRSA – methicillin-resistant Staphylococcus aureus

NFAA – non-essential free amino acids

NMs – nucleotide monophosphates

OTA – ochratoxin A

PA – propionic acid

PBS – phosphate-buffered saline

PLA – phenyllactic acid

POS – pathogenic and opportunistic strains

Pro – proline

PUFA – polyunsaturated fatty acids

QPS – qualified presumption of safety

SACH – saccharose

SCFA – short chain fatty acids

SD – standard deviation Ser – serine T-2, HT-2 – trichothecenes TET – tetracycline Thr – threonine TM – trimethoprim TP – total proteins

UMP – uridine monophosphate

UPLC-MS – ultra performance liquid chromatography – tandem mass spectrometer

UREA – urea

US – ultrasound treatment

Val – valine

v/v – volume per volume

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INTRODUCTION

By 2050, it is estimated that the population of the world is going to increase from 7.6 billion to 9.8 billion people and food production will increa-se with this growing population, not to mention food waste/by-products, which will grow proportionately, especially if nothing is done to solve this problem [1]. Food production and processing create high amounts of waste and by-products resulting in a negative environmental impact as well as significant expenses [2]. However, the industry is showing interest in innova-tions to obtain zero waste, where the waste generated is used as raw materials for new products and applications such as feed or feed supplement products [2]. These actions can have an impact on the Millennium Development Goals, the upcoming Sustainable Development Goals, the Post 2015 Agenda and the Zero Hunger Challenge [2]. The valorisation of food industry by-products can lead to a decrease in malnutrition and hunger in developing countries [2,3]. Whey, for instance, is the major by-product of the dairy industry. Roughly 180 to 190 million tons being produced each year and 47% of this by-product is discharged into drainage systems reaching rivers and soil, causing serious contamination problems [4–6]. A total of 40 million tons/year of whey is pro-duced in the European Union; the annual surplus of whey is 13 million tons, containing about 619,250 tons of lactose [6]. However, the nutritional value of whey is high, as it contains 20% of whole milk proteins (lactalbumin and lactoglobulins) and approximately 4.5% lactose content [7]. The biotechnolo-gical processing of whey generates products of interest to the agro-industrial sector, such as fermented beverages, organic acids, and microbial proteins. The use of cheap carbon sources from renewable resources is now considered an effective approach in the production of lactic acid bacteria (LAB) biomass production [8]. Moreover, microorganisms, which are able to consume the nutrients, mainly the lactose, can be applied to decrease environmental pollu-tion and sustainable products, such as feed supplements, can also be obtained [2].

Lactic acid bacteria (LAB) play an important role in livestock feed prepa-ration and are in high demand on an industrial scale due to their specifically desirable properties (to inhibit pathogenic bacteria and fungi, to reduce myco-toxins, to degrade antinutritional compounds, to increase bioavailability, etc.) [9]. Alone or in combination with plant or animal based ingredients, LAB compositions can improve sensory properties and increase the nutritional value of feed [9, 10]. However, LAB are sensitive to environmental condi-tions, which can cause difficulties in using them on an industrial scale [8]. Due to LAB sensitivity to environmental conditions, the most important step

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is to protect the viability and stability of LAB during storage [8]. One of the most effective techniques for biomass technological functionalisation is spray-drying. This technique is ancient and the widest encapsulation techni-que is used for the preparation of biologically active material nanocapsules with a size less than 10 µm. LAB can be encapsulated in carrier materials [11]. The encapsulated material is usually referred to as core, fill, or as active, internal or pay load phase, whereas the material used for encapsulation is often called a coating membrane, shell, capsule, carrier material, external phase, or matrix [11]. Spray-drying is exceedingly suitable for industrial applications due to its rapidity, reasonably low cost and high reproducibility, but the high temperature that is not compatible with bacterial viability is the main disadvantage in spray-drying [11, 12]. Usually, LAB strains proliferate in an expensive, commercially available de Man, Rogosa and Sharpe medium, however cheap carbon sources from food industry by-products are now considered to be an effective approach in LAB biomass production [13]. Alternative substrates could include whey/denaturised whey protein or potato juice as membrane material for the encapsulation of technological biomass starters [14, 15]. In addition to this, encapsulated LAB are stable during storage at 4°C and can survive during transit via the gastrointestinal tract, where different factors can reduce biomass viability (including the rumen pH, enzymatic degradation, and the presence of bile salts in the small intestine) [16]. Finally, technological functionalization of biomass is extremely impor-tant, as technological parameters and stability are of the utmost importance in its further application in a variety of different areas. Functional / antimicrobial / technological biomass could be used to increase livestock production in a more sustainable way.

Antimicrobial materials are used in animal production worldwide to improve health and welfare, but also to increase animal growth rates and to raise animal production [17]. More than 90% of antibiotics given to livestock are excreted in urine and faeces, then widely spread through fertilizer, ground-water, and soil, which affects the environmental microbiome [18]. Access to effective and cost-efficient antimicrobials is critical for human and animal health, animal welfare and food security [17]. According to Krajmalnik-Brown et al. [19] the animal intestinal microbiota of livestock is a main organ, which represents a determinant role in the harvesting, storage, and expendi-ture of energy obtained from the feed and these functions can improve the health and weight modification of the animal. FAO [20] reported the possibi-lity of a probiotics application for animal nutrition as gut ecosystem enhan-cers. According to Yirga [21] and Seal et al. [22], LAB-probiotics could be potentially viable as antibiotics replacers due to their multifaceted functions, such as improving growth and reproduction performance, as well as the

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survival rate and health status of animals. Diarrhoea is common in neonatal and young calves, remaining the major cause of productivity and economic losses to cattle producers worldwide. In addition to this, diarrhoea can be fatal, because it causes dehydration and acidosis, which may result in anore-xia and ataanore-xia, and increases the odds of other health complications [23]. Furthermore, treatment often requires excessive use of drugs and antibiotics, increasing public concerns of excessive usage of drugs in dairy farming and the development of antibiotic resistance. To reduce the incidence of diarr-hoea, prophylactic feed additives (usually in milk replacers) should be used to enhance the health of farm animals shortly after birth to stimulate intestinal mucosal immunity and the intestinal microbiome [24]. LAB alone or in combination with prebiotics, essential oils or herbal extracts can be used as feed supplements for livestock as replacements for antibiotics [25]. Pathoge-nic bacteria such Escherichia coli, Salmonella enterica, Clostridium

perfrin-gens, Aeromonas salmonicida, Pseudomonas spp. can cause infection soon

after birth, and LAB, as well as plant-based components, can be used to control them and to improve animal growth [9, 26, 27]. Administration of probiotic strains, both individual and combined, may have a significant effect on the absorption and utilization of feed, the daily increase of body weight and total body weight of various animals, including chickens [28], piglets [29], sheeps [30], cattle [31], and horses [32].

Prebiotics such as arabinogalactan, fruit/berry fibres, are considered preventative agents since they select gastrointestinal microbiota, which not only benefits the host, but can also serve as a barrier to pathogen colonization [33]. Moreover, prebiotics have to adhere to safety regulations required by all nations, such as those that possess Generally Recognized as Safe (GRAS) status, proper dose and side effects evaluations, have no contaminants and impurities, and do not alter intestinal microbiota that would create negative effects on the host [34]. A symbiotic formula of probiotics and prebiotics should have a beneficial effect on the host’s health. A formula may be consi-dered symbiotic if a selective stimulation in the growth of beneficial micro-organisms is confirmed, along with no or limited stimulation in the growth of other microbes. Technological aspects should also be considered [35].

The aim of the study

The aim of this study was to develop a design for the preparation of antimicrobial in sustainable manner feed supplements by using food industry by-products for the stabilisation of technological biomass.

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Objectives of the study

1. To select lactic acid bacteria (LAB) strains according to their charac-teristics (resistance to antibiotics, antimicrobial, antifungal and mycotoxin degrading properties) for the preparation of feed supple-ments.

2. To identify and to select food industry by-products and other (animal and plant-based) ingredients possessing antimicrobial, antifungal, and mycotoxin degrading properties for the preparation of feed supplements.

3. To develop sustainable technology for technological biomass stabili-zation by using spray-drying and/or an immobilistabili-zation technique; 4. To indicate physical and biological methods for the preparation of

sustainable high-value feed supplements at dairy farms.

5. To evaluate the influence of the newly developed feed supplements on the health parameters of newborn calves and endurance horses. The Scientific Novelty and Practical Usefulness

By-products of the food industry are an important environmental issue in many countries, but most agro-industrial by-products contain highly valuable compounds, which are not used efficiently enough. One way to recover these benefits, which has attracted much interest, is the extraction of valuable compounds that can be used as ingredients in the food / feed industry due to their functional and nutritional properties. However, in most cases, solvent extraction is not economically viable and/or involves the use of toxic extrac-tants that hinder their management. Also, extraction involves high tempera-tures, which lead to the degradation of thermolabile compounds. From this point of view, the use of technologies that enable the valorisation of the whole by-product can be a promising way to increase the efficiency and sustainabi-lity of the process. The scientific novelty of this thesis is that food industry by-products were applied to the technological processes without additional treatments for the production of natural antimicrobial compounds or their stabilization. Such an application has a valuable practical usefulness, as the valorisation of these by-products have become economically attractive today. Secondly, the scientific novelty in this thesis is associated with the natural antimicrobial ingredients incorporation in livestock production. The European Union imposed a complete ban on all antibiotics (ANB) as growth promoters in animal feed since January 2006, and according to the regulations by the Food and Drug Administration (FDA), ANB cannot be used for growth promoting purposes across the United States of America (USA) from 2017. For this reason, this study and its aim to develop new natural antimicrobial

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combinations have become very promising. Finally, the combination of innovative suggestions for the valorisation of by-products proposed in this work will allow the industry to develop and design sustainable feed supple-ments. The main consideration of this thesis practical benefits are as follows: (I) the feed supplements can be produced in an environmentally friendly, sustainable manner, reducing the pollution caused by the food industry, while ensuring safety worldwide; (II) combinations developed from valorised food industry by-products, together with animal and plant-based ingredients, can be used as livestock feed supplements to reduce the use of antibiotics; (III) antimicrobial property-possessing feed supplements prepared in a sustainable manner can be used on farms to improve the health parameters of newborn calves and endurance horses.

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1. LITERATURE REVIEW

1.1. Lactic acid bacteria and their application in the feed industry

1.1.1. Lactic acid bacteria characteristics and their properties Lactic acid bacteria (LAB) are gram-positive bacteria, catalase-negative organisms, which encompass a broad range of natural plant and animal-associated surroundings. LAB are found among the resident microbiota of the gastrointestinal tract of mammals, where they have a positive influence on host health [36, 37]. LAB ferment carbohydrates to gain energy using endo-genous carbon sources as the final electron acceptor instead of oxygen [38]. LAB can be homofermenters, which mainly produce lactic acid (LA), and heterofermenters, which, apart from LA, generate a wide range of fermen-tation products such as acetic acid (AA), ethanol, carbon dioxide, and formic acid (FA), as well as bacteriocins (proteinaceous antimicrobial molecules with a diverse genetic origin) and other materials during fermentation [37, 39]. Due to their health benefits for humans and animals, some LAB are used as probiotics. LAB should not be resistant to antibiotics, tolerate low pH and high concentrations of conjugated and deconjugated bile salts, tolerate lyso-zyme, adhere to the gut epithelial, and should not provoke immune response and antibody production[38, 40]. In addition, LAB are generally recognized as safe (GRAS) and have a Qualified Presumption of Safety (QPS) status [41].

LAB survival in gastric acid (pH 1.5–3.0) for at least 3–4 h is an important characteristic for probiotic strains [40]. It was discovered that the tolerance of the Lactobacillus spp. to pH 2.0, 2.5, and 3.0 for 6 h showed a survival rate on average of 55.8%, 87.5%, and 78.0%, respectively [42]. It was reported that LAB isolates with full tolerance to pH 3.0 for 3 h can be considered high-acid-resistant strains with promising probiotic properties [42, 43]. Also, the resistance to bile salts is a condition for probiotic characterization, which guarantees that the cells could reach the gastrointestinal tract alive. However, LAB resistance in bile salts (at a concentration of 0.15% to 0.5%) is deter-mined at the genus and species level [44]. Bile, secreted in the small intestine, reduces LAB viability by disrupting the cell membrane, by inducing protein misfolding and denaturation, and by damaging DNA [45]. Another characte-ristic suggested for probiotics is adherence to mucus and epithelial cells, but this property is still considered a controversial topic in probiotics research. On the one hand, it is a desirable probiotic trait, as it facilitates colonization of the host and antagonism against pathogens, but on the other hand, it is

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considered a risk factor for translocation [45, 46]. Adhesion to epithelial cells and subsequent colonization of the gastrointestinal tract is a property that confers great advantage to probiotic bacteria and helps them effectively compete and proliferate in the gut [46]. Bacterial adhesion is based on non-specific physicochemical interactions between two surfaces, and therefore, it is usually associated with the characteristics of the cell surface. The adhesion ability to mucin-producing cells is better than to non-mucus-producing ones; thus, the presence of mucus seems to play a major role in adhesion [46, 47]. According to de Souza et al. [48], bacteria with higher hydrophobicity can bind better to epithelial cells and thus influence the adhesion ability to some extent. Hydrophobicity is an important property, which has a positive influen-ce on the first contact between bacteria and host influen-cells [49]. However, overall adhesion is a complex process involving many more different parameters, such as surface exopolysaccharides, S-layer protein, and lipoteichoic acid, which provide potential advantages for microorganisms in colonizing the intestinal tract [49].

LAB showed various degrees of inhibitory activity against pathogenic bacteria [50]. It was reported that LAB possess antimicrobial activities against several pathogenic microorganisms such as Escherichia coli, Pseudomonas

aeruginosa, Staphylococcus aureus, Salmonella enterica, and Listeria mono-cytogenes [50–53]. According to Hu et al. [54], the synergistic effect of diffe-rent compounds produced by LAB during fermentation have higher anti-microbial activity against E. coli and Salmonella, compared to organic acids alone. LAB have great potential as an alternative to chemicals and antibiotics in food/feed technology and some of them displayed antimicrobial activity against opportunistic and pathogenic bacterial strains [55].

However, LAB can be naturally resistant to several antibiotics and may have the potential to acquire resistance to other antimicrobials or to spread the resistance to pathogenic and opportunistic bacteria, which are in the gastrointestinal tract of mammals [56]. The antimicrobial resistance involves several mechanisms of action, which can be associated with the presence of resistant genes that allow the direct inactivation of the active antimicrobial molecules and the loss of susceptibility to the antimicrobials by alteration of the target site and the reduction of antimicrobial assimilation [57]. For example, Shao et al. [58] reported that L. plantarum, which had the aadA and

ant(6) genes, were linked to the resistance to streptomycin, and the excessive

quantity of this antibiotic highly increased the minimum inhibitory concentra-tion (MIC), and also influenced the cross-resistance to other antibiotics of the same class. According to Manini et al. [59], Lactobacillus, Pediococcus and

Leuconostoc species can be resistant to vancomycin (MIC values 128 μg/mL)

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resistance mechanisms are most likely specific to these strains and could not be explained by the acquisition of resistance genes. Inherent or intrinsic (non-transmissible) resistance has a minimum transfer potential between bacterial genus, as resistance genes are sited in the chromosome with a restricted transference to other genera, which indicate a low risk within non-pathogenic bacteria [60]. Moreover, L. plantarum present innate resistance to vancomy-cin, due to the substitution of D-alanine residues of the muramyl pentapeptide cell wall by D-lactate (high-level resistance) or D-serine (low-level resistan-ce) in the chemical structure of the peptidoglycan, thus avoiding the antibiotic interaction [61–63]. Furthermore, enzymatic inactivation in aminoglycosides (neomycin, kanamycin, streptomycin) or quinolones (ciprofloxacin, norflo-xacin, nalidixic acid) prevent the binding of these antibiotics with their specific targets, as observed in Lactobacillus for the 16SrRNA of the 30S ribosomal bacterial subunit and DNA gyrase, respectively, which explains the intrinsic resistance to both groups of antibiotics [62, 64]. In addition to this, if LAB have inherent resistance to antimicrobials, it is considered acceptable for use in food and feed. However, it must be proved that the acquired resistance is in mobile genetic material or was acquired in the process of mutation in the bacterial chromosome (also acceptable for use in foods/feeds) [60]. In contrast to these findings, usually LAB isolated from traditional sour-dough are sensitive to antibiotics such as tetracycline, ceftriaxone, and amoxi-cillin [65–67]. According to Xu et al. [68], L. plantarum isolated from Chine-se fermented cereal food was Chine-sensitive to ampicillin-sulbactam, ceftriaxone, chloramphenicol, tetracycline, and ampicillin and exhibited slight sensitivity to trimethoprim-sulfamethoxazole.

LAB have promising potential as suitable starter cultures for food/feed fermentation and as potential probiotic candidates for further studies.

1.1.2. The influence of lactic acid bacteria on mycotoxins (bio)degradation

Mycotoxins are naturally occurring secondary metabolites of fungi, espe-cially moulds, which have a toxic effect on human and animal health, as well as a negative economic impact [69]. Mycotoxin production is common in species of Aspergillus, Penicillium, Fusarium, Alternaria, and Cladosporium [70]. However, there are more than 400 identified mycotoxins, but aflatoxins (AFB), ochratoxin A (OTA), Fusarium toxins, fumonisin (FUM), zearale-none (ZEA), trichothecenes (T-2, HT-2), and deoxynivalenol (DON), are the most important from the point of view of safety and economic viability [69, 71]. Different methods are used to decontaminate mycotoxin-contaminated material or to reduce the exposure to mycotoxins, but not all methods are

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suitable for food/feed manufacturers [72]. An efficient method for the reduc-tion of mycotoxins should be able to eliminate or inactivate the mycotoxins without producing toxic residues or affecting the technological properties and nutritive value [73]. Many physical (grain cleaning, thermal processing), chemical (alkaline, acids, oxidising reagents, etc.), and biological (feed addi-tives) technologies have been suggested to reduce mycotoxin levels in food/feed [72].

It is important to note that the species of Lactobacillus are the most pre-valent bacterial isolates associated with antifungal activity and also possess bio‐preservation potential [74]. The supernatant of L. acidophilus, L.

amylo-vorus, L. brevis, and L. coryniformis subsp. coryniformis strains can

effecti-vely inhibit the growth of fungus until pH values of 5.0, 5.5 and 6.0 [75, 76].

L. plantarum strains showed high antifungal activity at pH 6.0 and 7.0 and,

therefore, the idea was proposed that antifungal activity was due not only to non-dissociated organic acids, but also to dissociated ones [54]. Organic acids in their protonated or undissociated form are lipophilic and diffuse across the fungal cell membrane and get accumulated in the cytoplasm [74]. LAB, which can produce reuterin during glycerol fermentation, showed higher anti-fungal activity as reuterin is able to inhibit the growth of Aspergillus and

Fusarium [77].

LAB strains can bind AFB to their cell walls or cell wall components, reducing the bioavailability of the mycotoxins, as well as lowering absorption and alleviating the removal of the toxins from the organism [78]. According to Juodeikiene et al. [79], LAB can significantly reduce DON, T-2, HT-2 toxins, and zearalenone (ZEA), but decontamination strongly depends on the LAB strain, which can bind mycotoxins or detoxify mycotoxins with LAB. It is known that a typical cell wall of LAB contains a thick multi-layered pepti-doglycan sacculus, surrounded by a cytoplasmic membrane, which is adorned with polysaccharides, proteins, lipoteichoic acids, and teichoic acids [80]. However, the main LAB components responsible for mycotoxin reduction are polysaccharides and peptidoglycans [72]. Therefore, the differences in myco-toxin reduction strongly depends on LAB cell wall peptidoglycan structures and the number of available binding sites [81]. Nevertheless, small differen-ces in the peptidoglycan structure have influenced the removal capacity of different mycotoxins. It contains linear glycan strands or chains, comprised of N‐acetyl glucosamine and N‐acetyl muramic acid, which are cross‐linked by short peptides, amino acid sequences, which may cause differences in peptidoglycan structures among different LAB strains [81]. It was reported that ZEA and α-zearalenol was trapped in the LAB pellet and no metabolism product of ZEA or α-zearalenol was detected. It was concluded that myco-toxins were removed from media due to binding, not degradation[82]. LAB

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with high antifungal and mycotoxin binding activity would be tremendous in preventing mycotoxin exposure [83].

1.1.3. Influence of microbial feed supplements on animal health, productivity, and production quality

Lactic acid fermentation has been applied to feed preparation for many years due to an increased concentration of functional strains, enzymes, metabolites, and other active compounds [84].

Fermented feeds can improve gut health, as well as growth performance and also decrease serious illness and mortality in pigs [85]. According to Yang et al. [85], feed fermentation with two exopolysaccharide-producing strains of Lactobacillus reuteri reduced the abundance of enterotoxigenic

Escherichia coli in weanling piglets. This can be explained by the ability of

bacteria to produce reuteran or levan, exopolysaccharides that inhibit entero-toxigenic Escherichia coli adhesion to the mucosa [85]. Fermented liquid feed can improve the performance of suckling pigs, weaned pigs, and growing-finishing pigs due to the reduction of pH in the stomach of the pigs. Fermented feed prevents the multiplication of pathogenic bacteria such as coliforms and Salmonella from developing in the gastrointestinal tract [86]. In addition, fermented feed in combination with germinated feed increased nutrient digestibility and improved the intestinal morphology of pigs [86]. Similar tendencies were found by Dowarah et al. [87], who reported that

Pediococcus acidilactici and Lactobacillus acidophilus have a positive

influence on the growth parameters of pigs. Lactic acid bacteria (LAB) increases the faecal beneficial microbial (LAB and bifido bacteria) count and decreases the faecal E. coli and clostridia count, which are responsible for diarrhoeal diseases in pigs [87].

Moreover, LAB has a positive influence on the health parameters of dairy cows and newborn calves. It is known that LAB increases the beneficial microflora population, which decreases the pathogenic microbial establish-ment of ruminants [88]. The inclusion of LAB as a feed suppleestablish-ment improves feed palatability, stimulates cellulolytic bacteria and rumen fermentation, as well as nutrient digestibility, and interferes with rumen pH, due to the decreasing lactic acid production and/or increasing use of lactic acid by some bacteria, thus improving productivity [31, 89]. Feed supplemented with LAB has a positive influence on the supply of glucogenic, aminogenic, and lipogenic substrates, which increase the production of milk protein and fat [31]. According to Krungleviciute et al. [31], a P. acidilactici BaltBio01 and

P. pentosaceus BaltBio02 combination of fermented cereal by-products

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et al. [90], fermented milk with LAB decreases the morbidity and mortality rates of calves and improves nutritional parameters, body condition, and weight gain. A published article also claimed that L. acidophilus CBT and

B. pseudocatenulatum have a positive influence on the health parameters of

calves including a higher growth rate and level of intestinal flora, and decreases in the numbers of faecal enteropathogenic bacteria such as E. coli,

Salmonella spp., and Staphylococcus spp. [91]. Similar tendencies were

found by Kelsey and Colpoys [92], who published that weaned calves receiving a combination of Enterococcus faecium, Lactobacillus acidophilus,

Lactobacillus casei, and Lactobacillus plantarum increased average daily

weight gain compared to the control group, which didn’t receive probiotics. The positive influence of probiotic bacteria can be explained by increasing feed digestibility, decreasing lactate production, and improving ruminal fermentation [93].

Many fermented plant by-products have been used in animal diets to improve meat quality. For instance, fermented apple pomace can replace 11% of the dry matter of alfalfa hay and soybean meal in a sheep’s diet with a positive influence on meat quality [94]. It was published, that sheep loin meat showed less oxidation of lipids after storage at 4°C, and retains good parame-ters of colour, pH, water-retention capacity, drip loss, and tenderness, due to the addition of fermented apple pomace supplement in this diet [94]. More-over, a feed supplement combination of S. faecium, L. acidophilus, L. casei,

L. fermentum, and L. plantarum can reduce E. coli O157:H7 in sheep faeces.

In addition, probiotic bacteria supplements improve sheep meat production performance while reducing E. coli O157:H7 in faeces and these changes lead to higher meat biosafety [95]. Moreover, the positive influence on growth performance and feed efficiency of the sheep fed with the LAB combination might be responsible for changes in the overall microbial balance of the intestinal tract resulting in a reduction of pathogenic microorganisms [96].

1.1.4. Lactic acid bacteria technological functionalisation for convinent use on an industrial scale

There is a growing worldwide interest in using LAB to improve the efficiency of the fermentation process in order to obtain higher quality feed [97]. However, LAB are sensitive to environmental surroundings, which can have a negative impact on their application on an industrial scale [9]. The viability of LAB during storage is one of the most important technological steps [98]. Encapsulation, lyophilisation, immobilisation, and vacuum drying are the most effective methods for improving the viability and stability of LAB [97, 98].

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For the successful encapsulation of viable LAB cells, it is important to maintain bacterial viability under different processing properties, to use the type of encapsulation material compatible with feed materials, and to ensure viability and stability during processing, storage and gastrointestinal passage [11, 99]. Spray-drying is one of the most popular microencapsulation techni-ques as it permits quick evaporation of water, maintains a low temperature in the particles, and has the added benefit of a lower cost, and a short duration for the process [100, 101]. However, cell membrane and wall destruction, as well as protein and DNA denaturation, are the main problems caused by thermal damage during spray-drying [101]. Optimization of protectants and spray-drying parameters, including inlet temperature and drying matrix, are frequently used as important strategies in the spray-drying process to improve LAB cell survival under thermal stress [102]. The encapsulation effectiveness and loading capacity of bioactive components, such as bacterial cells or lipo-somes, are usually influenced by their core-wall ratio [103]. The protectant volume should regulate the core-wall ratio to achieve the highest encapsu-lation effect and protect LAB cells from the stress of the spray-drying process [101, 103]. Amino acids, proteins, monosaccharides, disaccharides, poly-saccharides, minerals, and their combinations can be used as protective agents during encapsulation by spray-drying [104, 105]. It was reported that treha-lose acts as a critical membrane protecting agent for cells during thermal treatment, dehydration, and environmental stress conditions [106]. Moreover, sodium alginate forms a protective wall around the encapsulated LAB culture, increasing its viability and protecting against environmental stress, such as low pH [107]. The survival of L. rhamnosus E97800 enriched with mono-sodium glutamate after spray-drying was 89.3% [101]. It was reported that a 50/50 mixture of maltodextrin DE5-8 and sucrose improved the survival rate of lactobacilli after the spray-drying process [101, 108]. Furthermore, skim milk powder was selected as a protectant agent for lactobacilli during spray-drying and the LAB survival rate was, on average, 75.5% [109, 110].

For stabilised viability, LAB were successfully vacuum dried and low oxygen levels during drying helped to avoid oxidative stress in sensitive cellular parts, while a low temperature inhibited detrimental thermal reac-tions, due to low pressure [111]. However, vacuum drying is an incredibly long process, as the heat transfer rate from heated shelves to the product is quite low. These changes can also be explained by the low heat conduction in a vacuum [111]. The high storage stability is associated with the compressed LAB structure resulting from shrinkage during vacuum drying [112].

The immobilisation of LAB by entrapment in gel beads can also be a good method to preserve their viability during storage, but the selection of a material and establishment of suitable stability is of the utmost importance,

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as not all substrates are appropriate for LAB immobilisation [113, 114]. A protective coating can prolong survival as it prevents exposure to oxygen during storage and improves resistance to gastric and bile acids [99]. Immobilised LAB in gel and xanthan gum‐based gels, for instance, provide cell viability for 30 min in simulated gastric fluid (pH 2.5) [115]. LAB can also be immobilised in an apple pomace-pectin gel, due to its ability to stabilize bacterial cells, as 84% of the viable LAB count were found after 1 month of storage at 4℃ [114].

Lyophilisation is the most popular process used to preserve LAB, because of its ability to protect from spoilage and its lengthy viability during storage [116, 117]. During freezing, bacterial cells are exposed to mechanical stress due to intra‐ and extracellular ice crystal formation and increased osmotic pressure caused by solutes in the remaining unfrozen fraction. This process can lead to the destruction of bacterial membranes and cause harmful damage [118]. Moreover, during lyophilisation, the removal of water by sublimation additionally increases osmotic pressure and can be harmful for membranes and surface proteins [102].

During lyophilisation, cryoprotectants are usually added to maintain the viability of microorganisms [118]. Substances such as polymers, sugars, milk, honey, polyols and amino acids have been tested for their protective effect during lyophilisation [102, 116, 118]. Disaccharides, such as maltose, sucrose and trehalose, are able to induce shrinkage of the cells by osmosis‐derived dehydration before freezing, thereby reducing intracellular ice formation [118]. Compounds mixing with different protective mechanisms can lead to the increased protection of microorganisms during freezing, as well as drying, compared with single‐component applications, due to the components’ syner-gic protective effects [118]. Skim milk, containing a mixture of lactealbumine and casein, as well as saccharides, are selected as cryoprotectants for many LAB due to their ability to prevent cellular damage by stabilizing the cell membrane and providing a protein-protective coating for the cells [119]. Moreover, different sugars offer high levels of protection for LAB during freeze-drying, due to their ability to replace structural water in membranes after dehydration [120]. It was reported that skim milk powder with lactose or saccharides was the best cryoprotectant for lactobacilli [117]. For instance,

L. lactis CIDCA 8221 lyophilised using milk and sucrose as a cryoprotective

medium, had a high survival rate and ability to recover [121].

Different additives can improve the viability of LAB in different matri-ces, as well as encapsulation, immobilisation or drying processes, increasing the stability of strains, thus alleviating production on an industrial scale.

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1.2. Application of food industry by-products for feed preparation

The food industry produces high levels of waste and by-products, incur-ring significant costs and causing a negative ecological impact [2]. Many by-products are not utilised and they raise serious environmental issues due to microbial expansion, as well as Green House Gas emissions [2, 122]. From an economic point of view, the management of large amounts of different degradable materials poses a challenge, especially since more than 50% of food materials produced are by-products with higher nutritional or functional value compared to the final products [2, 123]. The use of agro-industrial by-products for animal feed lowers the ambient impact of the food industry and improves yields and the valorisation of agricultural wastes, since the valorisation of food industry by-products to animal feed is an effective method to improve poor quality materials into high quality feeds [124]. Furthermore, industrial ecology and circular economy are considered to be key for eco-innovation focusing on a “zero waste” society and an economy where products can be used as raw materials or feed [124]. Food by-products can be classified into the following six categories: (a) crop waste and residues; (b) fruit and vegetable by-products; (c) sugar, starch and confectionary industry by-products; (d) oil industry by-products; and (e) distillery and brewery by-products [125].

Crop by-products. High quantities of by-products are generated by the

cereal industry. For example, global rice production was estimated at 952 million tons in 2016 [126]. It is estimated that it will generate 22.8, 190.4, 76.16, 19.04, and 9.52 million tons of straw, husk, bran, germ, and brewers’ rice, respectively [127]. Rice bran, which has a high amount of vitamin B and E, minerals, essential fatty acids, and dietary fibre, is a waste product of the rice milling industry and constitutes around 10% of the total weight of rough rice [127]. Rice bran has found an application in the food/feed indust-ries for increasing the nutritional quality of processed foods/feeds [128]. Due to its high amount of lysine and methionine, rice bran is regarded as a suitable feed ingredient for livestock [129]. Cereal bran, such as wheat or barley, is considered to be an inescapable by‐product of the milling industry with little commercial value, and is usually used as a supplement for livestock feed preparation [130]. In addition, cereal bran fermentation with LAB can add nutritional value, reduce biogenic amines, and degrade protein, fat, and fibre compounds to a lower molecular weight creating products with better digesti-bility that can be recommended for valorisation as by-products [130]. Accor-ding to Molist et al. [131], wheat bran dietary supplements have a positive influence on the health parameters of weaned pigs, due to the decreased

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number of pathogenic bacteria in their faeces, as well as a reduced incidence of post-weaning diarrhoea. A combination of 56% wheat bran and 44% dried sugar beet pulp can replace 25% of regular grains in a dairy cow diet without any negative impact on milk yield or chemical composition [132]. Similar tendencies were found by Ertl et al. [133], who published that a combination of 25% wheat bran and dried sugar beet pulp in a forage-only diet increased dry matter intake and milk yield in a short time and helped to keep a positive energy balance in mid-lactating cows.

Fruit and vegetable by-products. Fruit and vegetable processing

by-pro-ducts have a high amount of bioactive compounds, such as carotenoids, phe-nolics, flavonoids, antioxidants, antimicrobials, vitamins, or dietary fibres, which contain appropriate functional, technological, and nutritional proper-ties [134]. Apple by-products, for instance, have a high amount of phytoche-micals in the form of simple sugars, pectin, dietary fibres, and natural antioxidants [135]. However, several million tons of apple by-products are produced every year and due to their high moisture content (70–75%), their use is limited [136]. High levels of unused apple by-products violate pollution control standards and industrial safety issues, but also pose a risk to public health [135, 136]. In addition to this, fruit and vegetable processing by-products have been applied in animal diets as main feed compounds due to their positive effect on animal health parameters [137]. It was reported that dried fruit (apples, strawberries, blackcurrants) and vegetable (carrots and tomatoes) by-products in pig fattening feed combinations have a positive influence on some meat quality parameters and the performance of pigs [137, 138]. This can be explained by the fact that fruit by-products have organic acids, such as malic and citric acids, which improve the flavour of feed and promote the secretion of gastric juices, thus increasing feed intake [137]. Citrus and winery by-products can improve dry matter intake, nutrient digestibility, rumen fermentation efficiency, as well as growth performance and the overall health and welfare of ruminant animals [139]. Dried lettuce, green cabbage, red cabbage and cauliflower residues have a high amount of nutrients and can be used as feed for livestock [140]. According to Wadhwa et al. [141], dried cauliflower by-products combined with minerals and common salt, have a positive influence on the body weight of goats and their health parameters. Moreover, 30% of apple pomace can be combined in the diet of lactating Holstein cows, without any negative impact on milk yield or composition [72]. The replacement of ground maize with 50–75% of dried apple pomace in the concentrate supplement fed to Jersey cows similarly did not have a negative impact on milk fat and milk protein contents. However, a 4% decrease in the fat-corrected milk yield and fat yield were observed [142].

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Sugar, starch and confectionary industry by-products. One of the major

by-products of the sugar industry is sugar beet pulp, which contains highly digestible fibre and a relatively low content of sugar [143, 144]. Sugar beet pulp is a by-product representing a world production of over one hundred million tons each year [1]. Sugar beet pulp can be used for ruminants as a feed supplement as high amounts of digestible fibre stimulate acetate production in rumen [143]. Therefore, sugar beet pulp can be used as feed fresh, dried, or fermented [143, 145]. Dried sugar beet pulp contains 89.52% of dry matter, 10.71% of crude protein, 21.54% of crude fibre, 3.25% of ash, 63.86% of nitrogen-free extract, 2.83% of lignin, etc. [143]. Ruminants such as cattle or sheep can consume dried sugar beet pulp, which can replace up to 50% of the energy source rations for growing and fattening beef cattle, as well as in finishing rations for sheep resulting in high animal performance and high feed efficiency [146, 147]. Moreover, sugar beet pulp can be used as a replacement for oats in equine diets, showing the same positive effects on their performan-ce and metabolism [148].

Another by-product of the sugar industry is cane molasses. 100 tons of sugar cane will yield 10–11 tons of sugar and 3–4 tons of molasses [1, 149]. Molasses is an excellent source of minerals (sodium, potassium, magnesium and sulphur) and can be used as a feed supplement due to its ability to increase feed intake and improve palatability [149]. Microbes easily ferment molasses in the rumen, thus providing energy release and are therefore, very useful in feed supplements [150]. In addition, molasses improves the digestion of the feed and increases milk yield [149]. Similar tendencies were found by Ly et al. [151], who observed that molasses had positive effects on daily weight gain when used as a feed additive for pigs.

Oil industry by-products. Oilseed by-products are the residues left after

the removal of the oil from oilseeds. Oilseed by-products can be produced from soybeans, groundnuts, cottonseeds, rapeseed, sunflower seeds, coco-nuts, palm kernels, linseed and sesame seeds [152]. The defatted cake left after oil production represents 35% of the initial oil seed weight in the case of soybeans, 45% in the case of cottonseeds and 50% in the case of peanuts [153]. Tons of rapeseed by-products are used as animal feed, because they are unsuitable for human consumption due to antinutritional compounds, such as glucosinolates, phytates or erucic acid [154].

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However, cold-pressed rapeseed cake has a high energy content (due to fat) and is a good protein source, especially for organic diets. Cold-pressed rapeseed cake has been proven to be suitable for dairy cows as it improves its milk fatty acid profile (decreasing saturated fat, palmitic acid (C16:0) and increasing the content of oleic acid (C18:1), as well as the amount of other desirable nutrients) [155, 156]. In addition to this, rapeseed press cake has a positive impact on dairy cow diets including lower methane emissions and a better milk profile, making rapeseed press cake a promising feed component [155]. Cold-pressed sunflower cake is an oil industry by-product rich in crude fat and linoleic acid, and can be used as a feed supplement to moderate rumen fatty acid profiles [157]. Furthermore, cold-pressed sunflower cake supple-ments in dairy cow feed did not have a negative impact on milk production, digestibility, feed intake, or milk composition, and even increases flavour and the overall acceptability of milk [158]. In addition, feed with cold-pressed sunflower cake improved the fatty acid composition of milk to a higher polyunsaturated to saturated fatty acids ratio and increased the content of polyunsaturated fatty acids creating a positive effect on human health [158]. Hempseed cake can be included in the diets of livestock as a beneficial source of crude protein and essential fat without a major impact on production traits, yet still retaining a positive influence on the fatty acids in the products of the animals [159]. Hempseed cake supplement improved the fatty acid

compo-sition of raw sheep’s milk and also prevented lipid oxidation [160].

Distillery and brewery by-products. The brewing industry produces

mil-lions of tons of by-products, which can cause ecological and economic prob-lems [161]. It is estimated that 200 tons of wet, spent brewers’ grain is produ-ced worldwide every year [161].

Brewer’s grain is a by-product of the brewing industry and can be used as feed material for livestock due to its nutritional value [162]. Furthermore, brewer’s grain can be used for dairy and beef cattle due to its high quality protein and fibre, as well as its positive effect on milk yields and the fat and dry matter content of milk [162, 163]. In addition, 10% of brewer’s grain in sheep feed had a positive influence on body weight growth compared with the control group [163]. According to Radzik-Rant et al. [162], a 35% mixture of wet brewer’s grain improved the body weight of lambs and shortened their fattening period. It also decreased meat fattiness and increased the polyunsa-turated fatty acids and conjugated linoleic cis9 and trans-11 acid content of the meat. Distillery by-products such as spent barley grains and yeast cells recovered after alcoholic fermentation are suitable for ruminant diets, but their use is restricted to poultry and pig feed due to their low energy density and high fibre content [164]. However, the use of barley by-products for animal feed decreases the cost of feeding [164].

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The second major by-product of the brewing industry is spent brewer’s yeast [165]. This is a primary fermentation product containing inactive yeast cells and metabolites and about 0.7–1.1 kg compressed yeast per hectolitre of finished beer remains after the brewing process [165, 166]. It is a good source of protein, non-starch polysaccharides, B-complex vitamins, minerals, and several other unidentified growth factors, but utilisation of spent brewer’s yeast is a problem for the breweries [165, 166]. However, several trials have been published which observe the use of spent brewer’s yeast as feed additives for ruminants, horses, poultry and swine [93, 167–169]. Spent brewer’s yeast has a beneficial impact on ruminant animal health and produc-tivity, the stabilisation of the rumen environment, the inhibition of pathogenic bacteria and proliferation in the gastrointestinal tract and the modulation of the immune response. It increases fibre degradation and fermentation, nutrient availability and utilisation, animal growth performance and milk production [93, 170]. Spent yeast improved dry matter intake, growth, and feed efficiency and reduced diarrhoea in calves [93]. Spent brewer’s yeast hydro-lysate, when added to the diet of growing pigs, had a positive influence on feed efficiency and increased the digestibility of nutrients, as well as body weight. In addition to this, the content of blood urea nitrogen was also improved [171]. 3% distillery yeast additive in a diet significantly improved weight gain in quails, due to higher feed intake and the biological value of the protein present in the feed additive [172]. Spent brewer’s yeast can replace soybeans providing protein without adverse effects on growth performance, carcass characteristics or the internal organs of broiler chickens [173].

Dairy industry by-products. Whey is a by-product of manufacturing hard

and semihard cheeses made from milk. Whey can be acidic or sweet depending on casein precipitation [174]. It was published that 4 kg of raw milk produces 3 kg of whey [175]. It was estimated that whey production worldwide is around 160 million tons per year and only 30–50% of this is used [176]. However, whey has a negative environmental impact on soil and water due to its high mineral and lactose concentration and is considered to be a potential environmental pollutant [174, 177].

Bovine milk whey is a valuable dietary energy source, as well as a palatable product that can replace a part of the maize used in a diet, thus decreasing costs [177, 178]. Whey can be fed to animals in a variety of forms, such as liquid, condensed or dried [175]. It was reported that 12–20 L of sweet whey given daily to lactating cows increased milk yield, calcium and magne-sium concentration in milk, and improved the technological properties of the milk [175]. According to Salem and Fraj [179], whey can be an economical alternative to the half substitution of the concentrate in a dairy diet without any negative impact on milk production or composition. Skim milk powder

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and whey products can also be used as a milk replacement for newborn calves. It was concluded, that skim milk powders can be completely replaced by whey products due to the positive daily gain of the calves until the age of 6 months [180]. Moreover, liquid whey permeate can partly replace barley grain in lamb diets without a negative impact on dry matter intake, average daily gain, and digestibility [181]. A number of studies have been conducted regarding the fundamental principle of the dietary influence of whey protein on animals, including growth performance, nutrient digestibility, and its metabolic process [182–184]. It was reported that 0.1% of a whey powder feed supplement has a positive influence on the texture and sensory properties of aged pork [183]. Moreover, liquid whey feeding changed the faecal microbiota of mostly mature pigs as lactobacilli and bifido bacteria were in higher abundances in whey-fed pigs, while lower counts were observed in opportunistic species. Whey can be considered a useful feed supplement due to its positive effect on animal health and nutrition [185]. The feeding of whey to ruminants and monogastric animals offers the possibility of using large quantities of by-product nutrients for the economical production of meat, while alleviating disposal and environmental problems [175, 185].

Animal nutrition as a science demands a constant scrutiny of new by-products to use in feeds. In addition to this, it is important to discover the appropriate ways of combining by-products with other feedstuffs to obtain a better balance of nutrients and to improve animal health parameters. How-ever, studies about the valorisation of acid whey remain scarce.

1.3. The application of essential oils in livestock production

It is known that essential oils (EOs) have been used on both humans and animals since ancient times due to their antibacterial, antiviral, fungicidal, insecticidal, and acaricidal activities [186, 187]. EOs can contain more than 100 individual volatile and non-volatile compounds, which predominate in the determination of a chemotype [186, 188]. Moreover, EO components can have antimicrobial or other biological effects, but synergistic and additive effect functions of the various molecules of the EOs and their monoterpenoid components have also been indicated [188, 189].

EOs have a positive influence on pig and poultry growth performance due to higher nutrient absorption and improved feed digestibility [190, 191]. This can be explained by the EOs ability to improve the digestibility of feed and speed of passage through the digestive tract. This can impact bile synthe-sis, increasing the secretion of saliva, bile, and mucus while enhancing enzy-me activity [186]. Therefore, EOs as additives in livestock feed have roles as

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hypolipidemic and immune-modulating agents, as well as heat stress allevia-tors [192]. It was reported that EO additives in feed improve weight gain, feed intake, and feed conversion by 2.0%, 0.9%, and 3.0% for piglets and 0.5%, 1.6%, and 2.6% for poultry, respectively [190, 193]. Furthermore, oregano EO and sweet chestnut wood extract (both were used as feed additives) induced higher levels of glutathione peroxidase and glutathione reductase and prevented lipid oxidation in pork, but did not affect cooking loss, drip loss, or the chemical composition of the pork [193].

Ruminal microbial activity is important for the synthesis of high-quality protein for ruminants, but microbial fermentation could have a negative impact on energy and protein losses due to the production of methane and ammonia [194]. EOs could be used to manipulate ruminal metabolism and to selectively inhibit rumen methanogenesis, due to their antimicrobial proper-ties, while reducing CH4 emissions [194, 195]. However, Origanum vulgare,

Thymus vulgaris, and Cinnamomum zeylanicum EOs and their main

consti-tuents do not have effects on the growth, carcass composition, or meat quality of lambs or beef cattle [196, 197], or on milk yield or the milk compositions of dairy cows [198]. Simitzis et al. [194] found that the oral administration of 0.06 mL of rosemary EO improved the organoleptic properties of lamb’s meat without changing its chemical and physical properties, and a higher content of polyunsaturated fatty acids (PUFA), in particular n = 3 FA, were also established. According to Dudko et al. [199], the inclusion of Origanum

vulgare and Citrus spp. EOs in sheep diets have a positive effect on the

intensity and prevalence of coccidian infection in sheep flocks and also improved the growth of the sheep. Moreover, it was published that dietary supplements containing a combination of O. vulgare and Citrus spp. EOs increased lamb muscularity and significantly decreased fattiness [199]. The addition of EOs in calf starters also increased feed intake and body weight gain, as well as beneficial bacteria in the gut flora [200, 201]. Feeding EOs to young calves can be beneficial due to their ability to regulate ruminal bacterial species belonging to separate phylogenetic lineages [202]. Finally, EOs can effectively modulate the ruminal environment and microbiome of young bovine animals resulting in improved nutrition, performance, and health during the productive stages of their lives [202].

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1.4. Sustainable plants in the feed industry

Sustainable agriculture that can satisfy the ever-rising global demand for food and feed is becoming a necessity for both a growing population and farms as people become more concerned about increasing land use for food and feed [203]. Organic farming requires crop rotation, so legumes can be used for nitrogen formation and weed control, while high amounts of the grass grown is suitable for ruminant feeding [204]. Sustainable crops play an important role in organic farming, hence the increased interest in hemp and lupines as protein sources for animal nutrition in recent years [203].

Cannabis sativa L. has a long history of cultivation and can be used as

feed materials in the European Union and the countries of the European Free Trade Association [205]. Different types of feed materials may be derived from the hemp plant, such as hemp seed (full-fat), hemp seed meal/cake (after lipid removal, mainly cake from mechanical pressing), hemp seed oil and the whole hemp plant (which may include hemp hurds, fresh or dried), as well as hemp flour (ground dried hemp leaves) and hemp protein isolate (from seeds) [205]. It was reported that 20% of hemp seed/press cakes can be used for laying hens without a negative impact on laying performance or egg sensory characteristics, whereas linoleic acid and alpha-linolenic-acid increased in egg yolks [206]. In addition to this, the insertion of hemp seed can be effective in maintaining the oxidative stability of egg lipids compared to hempseed cake or sunflower oil in the diets of laying hens. This can be explained by the level and type of polyunsaturated fatty acids, the level of α-tocopherol and the duration of egg storage, which significantly affected the oxidative stability of eggs [207]. 20% of hemp meal could be used in growing sheep diets without negative effects on nutrient utilisation [208]. Diets containing 14% hemp seed could be fed to yearling steers for 166 days without negative effects on gain, gain to feed ratio and carcass traits [209]. Data indicate that a 14% additive of hemp seed cake or daily amounts of 0.5 to 1.5 kg whole hemp plant dry matter can be used in a total mixed ration for dairy cows and improve rumen function [205, 210].

Leguminous plants are important for sustainable agricultural production, as well as livestock feeding [211]. Lupine species could be a realistic sustainable alternative source of protein for animal feeding. Lupine seeds could be applied to monogastric and ruminant feed and replace soybeans without loss in quantity [211, 212]. The use of lupines as livestock feed for ruminants has many advantages as they can be used as concentrate (whole seeds, ground seeds or other processed seeds) or as forage (whole-crop, silage or hay) [212, 213]. The replacement of cereal grains with an equivalent amount of lupines in dairy cow diets increased the yields of milk, fat, and protein, and

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a higher fat concentration was also observed [214]. This can be explained by the fact that lupines have a higher metabolisable energy content compared to cereal grains [214]. Similar tendencies were found by Buryakov and Aleshin [215], who observed that 24% of lupine supplements in regular feed can increase daily yields of milk, as well as fat and protein contents. According to Lestingi et al. [216], lupines combined with fava beans as feed improved lamb growth performances and blood parameters. According to Pieper et al. [217], polished and hydrothermally treated blue sweet lupines can be used as an alternative to soybean meal for the diets of growing pigs, due to higher nutritive value.

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2. MATERIALS AND METHODS

2.1. Investigation venue

The experiments were conducted between 2016 and 2020 at the Lithuanian University of Health Sciences (LSMU) Department of Food Safety and Quality, Microbiology and Virology Institute, Research Center of Digestive Physiology and Pathology, Department of Pharmaceutical Technology and Social Pharmacy (Kaunas, Lithuania); at the Kaunas University of Technology Institute of Materials Science, Department of Food Science and Technology (Kaunas, Lithuania); Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry (Babtai, Lithuania); University of Naples “Federico II” hereinafter “UNINA”, Department of Pharmacy (Napoli, Italy); Institute of Food Safety, Animal Health and Environment BIOR (Riga, Latvia).

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2.2. Materials

Microorganisms used in experiments. Leuconostoc mesenteroides

LUHS225, Lactobacillus plantarum LUHS122, Enteroccocus pseudoavium LUHS242, Lactobacillus casei LUHS210, Lactobacillus curvatus LUHS51,

Lactobacillus farraginis LUHS206, Pediococcus pentosaceus LUHS183, Pe-diococcus acidilactici LUHS29, Lactobacillus paracasei LUHS244, bacillus plantarum LUHS135, Lactobacillus coryniformis LUHS71, Lacto-bacillus brevis LUHS173, LactoLacto-bacillus uvarum LUHS245 were acquired

from the Lithuanian University of Health Sciences collection (Kaunas, Li-thuania). These strains were used for antimicrobial compositions develop-ment, and Lactobacillus plantarum LUHS135, Lactobacillus paracasei LUHS244, and Lactobacillus uvarum LUHS245 strains were used in experi-ments with animals.

Strains before the experiment were stored at –80°C (PRO-LAB Diagnos-tics, Bromborough, United Kingdom), supplemented with 20% glycerol. Before the experiment, LAB were propagated in MRS broth (CM 0359, Oxoid Ltd, Hampshire, United Kingdom) at 30°C for 48 hours.

Klebsiella pneumoniae, Salmonella enterica 24SPn06, Pseudomonas aeruginosa 17-331, Acinetobacter baumanni 17-380, Proteus mirabilis, MRSA M87fox, Enterococcus faecalis 86, Enterococcus faecium 103, Bacillus ce-reus 18 01, Streptococcus mutans, Enterobacter cloacae, Citrobacter freun-dii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pasteurella multocida were obtained from the Lithuanian University of Health Sciences

(Kaunas, Lithuania) collection. These strains were used for antimicrobial activity of the tested compounds determination as the indicator strains.

Aspergillus fischeri, Aspergillus nidulans, Penicillium oxalicum, Penicil-lium funiculosum, Fusarium poae, Alternaria alternata, Fusarium grami-nearum C, Fusarium gramigrami-nearum B were obtained from the collection of

the Vytautas Didysis University, Department of Biology (Kaunas, Lithuania). These strains were used for antifungal activity of the tested compounds determination as the indicator strains.

Other materials used for technological functionalization and antimic-robial compositions development. The agar powder (Gelidium sesquipedale

algae) was purch from the Rotmanka (Gdansk, Poland). The agar powder was used for Lactobacillus plantarum LUHS135, Lactobacillus paracasei

LUHS244, Lactobacillus brevis LUHS140, Pediococcus acidilactici LUHS29, and Pediococcus pentosaceus LUHS100 immobilisation.

The wheat starch (Amilina, Panevezys, Lithuania) and Vaccinium

oxyco-ccus (obtained from the local market, Kaunas, Lithuania) were used for

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O. basilicum (obtained from a local market, Kaunas, Lithuania) was used

for antimicrobial basil-LAB bioproducts immobilized in agar preparation. The essential oils (EOs) of clove (Eugenia caryophyllata), fennel

(Foeni-culum vulgare), eucalypt (Eucalypti globuli), thyme (Thymus vulgaris),

grapefruit (Citrus paradis L.), lavender (Lavandula angustifolia), mandarin (Citrus reticulata L.), mint (Mentha piperita L.), sage (Salvia sclarea), lemon (Citrus limon L.), rosemary (Rosmarinus officinalis), ginger (Zingiber

officinale), Cinnamonn (Cinnamomum verum), aniseed (Pimpinella anisum),

muskat (Myristica fragrans), oregano (Origanum compactum) were purcha-sed from the Sigma-Aldrich (Saint-Louis, MO, USA). EOs were upurcha-sed for antimicrobial combinations development.

Raspberries (Rubus idaeus, variety “Poliana”), blackcurrants (Ribes

nigrum, variety “Ben Alder”), apples (variety “Auksis”), and rowanberries

(wild Sorbus aucuparia) by-products were obtained from the Institute of Hor-ticulture, Lithuanian Research Centre for Agriculture and Forestry (Babtai, Kaunas distr., Lithuania) in 2018. Berries/Fruits (B/F) by-products were used for antimicrobial compositions development.

Arabinogalactan (ARB) (D-galactose and L-arabinose in a 7.5:1 ratio, extracted from Larix spp. wood, Siberia, Russia, purchased from SME “Ro-kiskio pragiedruliai”, Rokiskis, Lithuania) in combination with selected LAB was used for antimicrobial compositions development and in experiments with new born calves.

Whey (lactose 4.0%, protein 0.8%, lactic acid 0.5%, minerals 0.6%, total solids 6.5%) was purchased from JSC “Pieno žvaigždės” (Kaunas, Lithuania). Whey was used for selected LAB strains technological functionalization (as a coating for nanocapsules preparation, and as criogenic agent to increase LAB viability during lyophilization).

Yeast extract, gucose and sucrose were purchased from the Biolife (Milano, Italy). Above mentioned additives were used for selected techno-logical, antimicrobial and functional LAB biomass preparation.

Bovine colostrum (BC) was obtained within the 2 hours of calf delivery from the agricultural company “Linas Agro” (Luksiai, Lithuania). BC was used for multifunctional antimicrobial compositions development.

APPLICATION OF FOOD INDUSTRY BY-PRODUCTS FOR MICROORGANISMS ENCAPSULATION/IMMOBILIZATION AND SUSTAINABLE ANTIMICROBIAL PROPERTIES FEED SUPPLEMENTS PREPARATION

Paulina Zavistanavičiūtė