APPLICATION OF SOLID-STATE FERMENTATION FOR DEVELOPMENT OF HIGHER VALUE AND SAFETY FOOD PRODUCTS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Erika Mozūrienė

APPLICATION OF SOLID-STATE

FERMENTATION FOR DEVELOPMENT OF

HIGHER VALUE AND SAFETY FOOD

PRODUCTS

Doctoral Dissertation Agricultural Sciences,

Zootechnics (03A)

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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 2012–2016.

Scientific Supervisor

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

Dissertation is defended at the Zootechnical Research Council of the Veterinary Academy of Lithuanian University of Health Sciences.

Chairperson

Prof. Habil. Dr. Romas Gružauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Zootechnics – 03A).

Members

Prof. Dr. Vaidas Oberauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A);

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

Prof. Dr. Pranas Viškelis (Kaunas University of Technology, Physical Sciences, Chemistry – 03P);

Ass. Prof. Dipl.-Ing. Dr. Gerhard Schleining (BOKU – University of Natural Resources and Life Sciences, Vienna, Austria, Technological Sciences, Chemical Engineering – 05T).

Dissertation will be defended at the open session of the Lithuanian University of Health Sciences on the 22nd of December, at 10:00 am in Dr. S. Jankauskas Auditorium of the Veterinary Academy.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA

Erika Mozūrienė

VERTINGESNIŲ IR SAUGESNIŲ MAISTO

PRODUKTŲ KŪRIMAS TAIKANT

AUGALINĖS ŽALIAVOS KIETAFAZĘ

FERMENTACIJĄ

Daktaro disertacija Žemės ūkio mokslai,

zootechnika (03A)

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Disertacija rengta 2012–2016 metais Lietuvos sveikatos mokslų universitete Veterinarijos akademijos Maisto saugos ir kokybės katedroje.

Mokslinė vadovė

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

Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Zootechnikos mokslo krypties taryboje:

Pirmininkas

Prof. habil. dr. Romas Gružauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, zootechnika – 03A).

Nariai:

Prof. dr. Vaidas Oberauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A);

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

Prof. dr. Pranas Viškelis (Kauno technologijos universitetas, fiziniai mokslai, chemija – 03P);

Doc. dr. inž. Gerhard Schleining (BOKU – Gamtos išteklių ir gyvosios gamtos mokslų universitetas, Viena, Austrija, technologijos mokslai, chemijos inžinerija – 05T).

Disertacija bus ginama viešame Zootechnikos mokslo krypties tarybos posėdyje 2016 m. gruodžio 22 d. 10 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos dr. S. Jankausko auditorijoje.

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ABBREVIATIONS

BAs – Biogenic Amines

BIOR – Institute of Food Safety, Animal Health and Environment BLIS – Bacteriocin-Like Inhibitory Substances

GC/MS – Gas Chromatography – Mass Spectrometry GF – Gluten free

GRAS – Generally Recognized As Safe HAA – Heterocyclic Aromatic Amines

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

IF – Intramuscular Fat

KTU – Kaunas University of Technology LAB – Lactic Acid Bacteria

Ls – Lactobacillus sakei

LUHS VA – Lithuanian University of Health Sciences Veterinary Academy MBL – Marinated Beef Loin

MPL – Marinated Pork Loin MRS – de Man Rogosa Sharpe Pa – Pediococcus acidilactici Pp – Pediococcus pentosaceus Rc – Rhaponticum carthamoides

RCMP – Ready-to-Cook Minced Pork meat products Sh – Satureja hortensis L.

Sm – Satureja montana L. SMF – Submerged Fermentation SSF – Solid State Fermentation TPC – Total Phenolic Compounds TTA – Total Titratable Acidity UCC – Unripened Curd Cheese VC – Volatile Compounds

VMU – Vytautas Magnus University VRBA – Violet Red Bile glucose Agar WHC – Water Holding Capacity RSA – Radical Scavenging Activity BaA – Benz[a]anthracene

BbF – Benzo-[b]fluoranthene BaP – Benzo[a]pyrene Chr – Chrysene

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INTRODUCTION

The food industry seeks alternatives to satisfy consumers demands of safe, minimally processed food in which chemical preservatives are replaced by more natural alternatives [19, 141, 246]. Lactic acid bacteria (LAB) have been used in food fermentations all over the world for millennia. LAB are an integral part of food safety, keeping the quality and the nutritional value of many foodstuffs [122].

Traditionally LAB are natural constituents of fermented foods, however, the application of antimicrobial compounds producing protective cultures may provide an additional parameter of bioprotection in several different food matrices and ensure food quality, keeping or enhancing its organoleptic, textural characteristics and nutritional aspects in the final product (e.g., meat or cheese) [19, 41, 115, 122, 277-9, 289]. Many LAB produce a high diversity of different bacteriocins. Bacteriocinogenic LAB are generally recognised as safe (GRAS) and useful to control the frequent development of pathogens and spoilage microorganisms [233]. Bacteriocins can be produced in foods by the activity of bacteriocin-producing LAB strains or when added in foods as food preservatives [346]. The use of bacteriocins has emerged as an important strategy to increase food safety and to minimize the incidence of foodborne diseases due to their minimal impact on the nutritional and sensory properties (low toxicity and stability against proteases and temperature) of food products [83, 119, 309]. They can be used in the production of several foods (meat, chicken, dairy, eggs, seafoods, fruit and vegetables) [117]. The direct addition of bacteriocin-producing cultures into products can be a more practical and economic option for improvings of the safety and quality of the final product [233].

With the advent of biotechnological innovations, mainly in the area of the fermentation technology, many new avenues have been opened [178]. Biotechnology offers significant advantages, such as a high concentration of metabolites, to obtain product stability and the adaptability of microorganisms with a low free water content. Over the last two decades, solid state fermentation (SSF) has gained significant attention for the development of industrial bioprocesses, particularly due to a lower energy requirement associated with higher product yields and a less wastewater production with a lower risk of bacterial contamination. In addition, it is an ecofriendly alternative to produce different fermented values added products like enzymes, organic acids, colours, flavors, pesticides, bio-surfactants, as it mostly utilizes solid plant material, which eventually results in the greener and cleaner environment [123, 234, 376].

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One of the principal challenges of the food industry is to find the best combination among various food processing methods to increase the concentration of bioactive compounds in food to ensure of their nutritional and healthy properties for humans [65, 190, 292, 331]. LAB are used as a flavour, an acidifying agent having biodegradable properties and controlled release and/or increasing the content of specific beneficial compounds [154, 329]. These compounds can be macronutrients, micronutrients (such as vitamins) or non-nutritive compounds [329]. LAB fermentation has been shown to increase the levels of nutrients including folates, soluble dietary fiber, and the total content of phenolic compounds in legumes and dietary fiber-rich pants, thus enhancing their antioxidant activity, improving the protein digestibility [86, 242, 378], increasing lignans and positively influencing the content of alkylresorcinols [308]. Consequently, fermented plants could be an excellent material for enriching the food of animal origin. It is known that savory plants contain compounds with antimicrobial properties [249]. Such a complex (savory plants – bacteriocins producing LAB) could be promising for the preservation of food products. In this way, LAB and plants that combine different functional characteristics could be useful for developing improved or new foods.

Meat fermentation of is a well-known method to extend the shelf life, transformation and diversification of meat products [108]. Meat and meat products are a concentrated source of proteins, however, they can rapidly spoil and may allow the growth of food-borne pathogenic microorganisms. This is why treatement with fermented savory plant bioproducts become an important preservation technology. LAB and savory plant compounds can prevent the growth of undesirable food pathogens and spoilage bacteria, form texture and taste [199, 210].

Fermented dairy products are the most common fermented foods, because they are convenient, nutritious, stable, natural, and healthy [23]. LAB can show the desired technological and functional potential in milk protein coagulation, production of proteinases [385], lactic acid, acetic acid, ethanol, acetaldehyde, diacetyl, aroma compounds, bacteriocins, exopolysaccharides, and several enzymes of importance to be controlled. These compounds can enhance the shelf life and microbial safety, improve texture, and contribute to the pleasant sensory characteristics of dairy products, but they may also cause spoilage in uncontrolled conditions [134]. Food technology innovation has the potential to deliver many benefits to society from improved food safety and food risk mitigation and improved nutrition to increasing food sustainability and improving food quality [100, 312]. Technological innovation in food production may involve the application of emerging technologies associated with societal disquiet, such

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as biotechnology. However, fermented foods can contain biogenic amines (BAs) whose content should be limited in food [158] and which are often related with BAs poisoning [92]. Also, the main metabolyte of LAB is lactate, which has two otical isomers – L(+) and D(-) lactate. Increased levels of D(-) lactic acid in plasma and urine have been demonstared in cases of intesinal ischaemia, short bowel and appendicitis, and are considered as a marker of dysbiosis and/or increased intestinal permeability [391]. Therefore, the desirable lactic acid isomer in food is L(+).

The aim of the study

To develop new bioproducts by using bacteriocins-producing LAB and plants having a unique chemical composition: the high content of protein, phytoestrogens, inulin, phenolic compounds, and essential oils (EOs), and to adapt them for the production of a higher value and sustainability safer food of animal origin production.

The objectives of the study

1. To select the conditions for plant material fermentation and to evaluate the parameters of fermentation efficiency.

2. To evaluate the content changes of bioactive compounds (alkylresorcinols (ARs), lignans, β-glucans, and volatile compounds (VC)), total phenolic compounds (TPC), and radical scavenging activity (RSA) of plants during biotreatment.

3. To carry out the comparison of SSF and SMF bioprocesses and to select safe, having antimicrobial properties plant bioproducts for the production of higher value and sustainability food.

4. To create a design of technologies for higher value and safer sustainability food products, and to improve the safety of the traditional food technologies by using selected LAB – plant bioproducts.

The scientific novelty and practical usefulness

According to the World Health Organization, access to sufficient amounts of safe and nutritious food is the key to sustaining life and promoting good health. As the world’s population grows, the intensification and industrialization of agriculture and animal production to meet the increasing demand for food creates both opportunities and challenges for food safety. The development of new, higher value and sustainability food technologies and assessing their safety become very important. The exploration of naturally occurring antimicrobials for food preservation receives increasing attention due to consumer awareness of natural food

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products and the growing concern of microbial resistance to conventional preservatives. The use of the LAB-plant complex bioproducts as an alternative to the use of synthetic chemicals to preserve the quality and to increase the functional value of food of animal origin could be a choice. The dissertation is dedicated to solving this particular problem.

The scientific and practical novelty of the thesis is concentrated on developing technologies of the new higher value and safer food of animal origin, based on the treatment with the LAB-plant complex bioproducts or their incorporation in the food formula. For this purpose, in Lithuania cultivated plants (source of biologically active compounds) (I) and SSF with selected LAB (II) (for plant substrate biomodification with the purpose to produce additives for improving the technological and safety parameters of animal origin), will be used. The SSF technology and the unique properties of plants applicated in the food of animal origin production would be novel and perspective to reduce the use of synthetic additives and conservants.

Important practical results will be obtained: (I) LAB-plant complex bioproducts will be created; (II) the new, of higher value and sustainability, safer food of animal origin and its technologies prototypes will be developed, and safe food supplies will support national economies, trade and tourism, contribute to food and nutrition safety, underpin sustainable development.

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LITERATURE REVIEW

1.1. Lactic acid bacteria and their regulation for food uses

LAB are widespread microorganisms which can be found in any environment rich mainly in carbohydrates, such as plants, fermented foods and the mucosal surfaces of humans, terrestrial and marine animals. In human and animal bodies, LAB are part of the normal microbiota or microflora, the ecosystem that naturally inhabits the gastrointestinal and genitourinary tracts, which is comprised by a large number of different bacterial species with a diverse amount of strains [20, 22]. They constitute a Gram-positive heterogeneous group of microorganisms that produce lactic acid as the major metabolite during the fermentation process and initiate rapid and adequate acidification in the raw materials through the production of various organic acids from carbohydrates [12, 27]. The main members of the LAB are Lactococcus, Lactobacillus, Streptococcus, Leuconostoc,

Pediococcus, Carnobacterium, Aerococcus, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus and Weisella. Lactobacillus is the largest

genus of this group, comprising around 80 recognized species [166, 340]. LAB have limited biosynthetic capabilities and thus require continuous supply of purines, pyrimidines, vitamins, and amino acids. These are nonsporing nonmotile organisms and usually categorized as facultative anaerobes. Lactobacillus strains are used in pickle, sauerkraut, beer, wine, juices, cheese, yogurt, and sausage production [22, 306]. Some LAB strains (including Enterococcus faecium, Lactobacillus plantarum, Lactobacillus

acidophilus, Lactobacillus casei subsp. rhamnosus, and several Bifidobacterium and Propionibacterium species) from animal and human

intestinal microflora have been adopted as ”probiotic“ food supplements [374]. However, especially LAB are assessed for their food protective properties. The biopreservative role of LAB is mainly due to the synthesis of a wide range of active metabolites which include: organic acids (lactic, acetic, formic, propionic, and butyric acids), or compounds, such as carbon dioxide, ethanol, hydrogen peroxide, fatty acids, acetoin, diacetyl, antifungal compounds (propioniate, phenyllactate, hydroxyphenyl-lactate, cyclic dipepetides and 3-hydroxy fatty acids), food aromas and flavors (e.g., diacetyl and acetaldehyde) [273, 289], proteinaceous, small heat-stable inhibitory peptides bacteriocins (e.g. nisin, reuterin, reutericyclin, pediocin, lacticin, enterocin), or bacteriocin-like inhibitory substances (BLIS) [12, 263, 293, 317, 352].

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It is well known that the production of acid based compounds play an important role in the preservative effect of LAB and can inhibit growth of the food spoilage microorganims [263, 314, 317]. The antimicrobial effect of acids is due to the fact that undissociated acids can pass through the microbial lipid membranes and disrupting the host cell proton motive force [263, 317]. These bacteria not only produce lactic acid but also preserve nutrients, vitamins and are used as starter cultures to convert sugars into lactic acid and other end products which give the typical flavour to fermented products [275]. The production of LAB antimicrobial compounds is dependent on the selected strain, growth conditions, and the interactions between metabolites [213].

Second group of antimicrobial compounds produced by LAB – bacteriocins, are generally defined as ribosomally synthesized peptides that have bacteriostatic or bactericidal activity against other related and unrelated microorganisms [128]. These peptides are considered natural biopreservatives and their potential application in the food industry has received great interest [68]. Their main advantage over chemical preservatives is their ability to preserve without affecting the sensory qualities of the food while adhering to the demand for natural preservatives. The ideal bacteriocin should be potent at low concentrations, active against a range of spoilage and pathogenic organisms, innocuous to the host and economical to produce [74; 309]. Bacteriocins in combination with other antimicrobial factors may be useful tools for the implementation of methods intended to significantly reduce the load of food spoilage and foodborne pathogenic bacteria [155]. In our experiment used L. sakei KTU05-6, P.

pentosaceus KTU05-10 and P. acidilactici KTU05-7 produced BLIS

(sakacin 05-6, pediocin Ac05-7, pediocin 05-8, pediocin 05-9 and pediocin 05-10, respectively) show wide-ranging antimicrobial activities against gram positive and gram negative strains [59]. L. sakei KTU05-6 produced BLIS inhibited both B. subtilis substrains: B. subtilis subsp. subtilis and B.

subtilis subsp. spizizenii. Among gram negative bacteria 7 from 13 Pseudomonas spp. strains were inhibited by L. sakei KTU05-6 produced

BLIS, whereas other tested pathogenic bacteria strains were not affected by this BLIS. P. acidilactici and P. pentosaceus strains produce BLIS were active against gram positive B. subtilis substrains only, whereas P.

pentosaceus KTU05-10 produced BLIS additionally inhibited the P. fluorescens biovar [59].

Currently, the European Union (EU) focuses very intensely on food safety, and especially on both chemical and microbiological hazards. Microbiological hazards are not only pathogenic microorganisms that coincidently find their way into the food chain, but also can be microbial

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cultures deliberately added during food production. Of all 25 EU member states, only Denmark and France have legislation that explicitly regulates the addition of microbial cultures to food, and the EU itself only regulates them in infant formulae and only as to the configuration of the lactic acid molecule [96]. Vice versa, by Food and Drug Administration (FDA, USA) LAB have a GRAS status [12, 53, 98, 109, 138, 275, 294, 296, 405], and lactic acid fermentation has been associated with the reduction of certain naturally occurring or otherwise formed toxins in foods of plant origin. On the other hand, microbially produced lactic acid is usually a mixture of the L(+)- and D(-)- isomers (Figure 1.1.1). As the latter can not be metabolized by humans, excessive intake can result in acidosis, which is a disturbance in the acid-alkali balance in the blood. The potential toxicity of D(-)-lactic acid is of particular concern for malnourished and sick people. Since elevated levels of D(-)-lactic acid is harmful to humans, L(+) lactic acid is the preferred isomer in food industrie as humans have only L-lactate dehydrogenase that metabolizes L(+) lactic acid. The fermentation processes to obtain lactic acid can be classiﬁed according to the type of bacteria used. In the heterofermentative process, equimolar amounts of lactic acid, acetic acid, ethanol, and carbon dioxide are formed from hexose, whereas in the homofermentative process only lactic acid is produced from hexose metabolism. Unlike the higher animals and plants which produce exclusively the L(+) isomer, species of LAB produce either D(-)- or L(+)-lactate or even both isomers. The FAO/WHO-experts [161] suggested limiting the daily intake of D(-)-lactate to 100 mg/kg body weight and attempts to favour the L(+) isomer content in fermented food are in progress.

Figure 1.1.1. Chemical structures of L(+) and D(-)-lactic acid isomers.

A regulation for reasons of safety should be proportional to perceived risks, risk being a function not only of severity but also of probability of the adverse effect taking place [402]. The European Food Safety Authority (EFSA) has stated that several LAB strains can be considered to have “Qualified Presumption of Safety” QPS-status [196].

a) L – lactic acid b) D – lactic acid

OH HO H _CH 3 O OH HO O H₃C _H

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The main aim of lactic acid fermentation is the conversion of carbohydrates to lactic acid. Therefore the action of LAB is desirable for the production of fermented food products. However, some of the bacteria involved in fermentation can produce BAs (Figure 1.1.2). Member States informed the EFSA that findings of certain levels of toxic BAs in fermented food could be of concern and reported a recent increase of BA content in some fermented foods. Formation of BAs in all foods of animal origin having high protein contents, as well in foods of plant origin, has been reported. It can occur as a result of activities of spoilage microflora and/or intentionally added microorganisms. The consumption of food containing higher amounts of toxic BAs may cause food intoxication with symptoms including flushing, headaches, nausea, cardiac palpitations, and increased or decreased blood pressure; in extreme cases the intoxication may have fatal outcome and indicates the need for a better hygiene process and other controls [92].

Figure 1.1.2. Chemical structures of main BAs.

The most frequent food-borne intoxication caused by BAs involves histamine (HIS) and tyramine (TYR) [92, 379]. Histamine causes dilation of peripheral blood vessels, capillaries and arteries, thus resulting in hypotension, flushing, and headache. The toxicological effect depends on histamine intake concentration, presence of other different amines, amino-oxidase activity and the intestinal physiology of the individual [150, 307].

Putrescine Cadaverine Spermidine _Spermine Histamine Tyramine β-Phenyltethylamine Tryptamine H2N NH2 H₂N NH₂ H₂N N NH2 H H2N N N NH₂ H H H2N N NH H2N OH H₂N H2N N H

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For cured products, the FDA consider it a danger to health if the histamine level is equal to 500 mg/kg [206]. Harmful effects resulting from the consumption of foods rich in BAs can be expected only when these amines gain access to the bloodstream [150]. Dietary TYR and trace amines cause vasodilatation of the mesenteric vascular bed, increasing blood flow at the gastrointestinal level and thus facilitating their absorption [43]. Direct effects associated with specific receptors have also been reported at the cardiovascular level, causing an increase in heart rate [110]. The vasoconstriction effect of TYR, phenylethylamine (PHE) and tryptamine (TRP) cause hypertension, but other symptoms such as headache, perspiration, vomiting, pupil dilatation, migraine cases caused by the consumption of food potentially rich in TYR [92]. In this respect, the control of BAs accumulation in food products is one of the present challenges of the food industry [392].

1.2. Design of the fermentation processes – solid state and submerged fermentation

The demand for faster, more efficient, controllable and largescale fermentation has resulted in a careful selection of starter microorganisms to guarantee the reproducibility of fermentation at industrial scale and to obtain a product with specific properties [48]. The choice of starter culture and fermentation conditions has critical impact on the final quality of fermented foods. Fermentation with well-characterized cultures, yeast or LAB, could be a potential tool to improve the palatability, processability and the nutritional value of fermented products or high-fiber ingredients [335]. The main criteria used to select microbial starters are desirable technological, sensory and nutritional aspects. The main technological factors of interest for fermentation are cells growth and acidification rate [62], synthesis of antimicrobial compounds [169] antifungal activity [63], exopolysaccharide (e.g. glucan and fructan) [55, 116], and sweeteners (e.g., mannitol) production [148].

Technologically interesting potential starter strains are usually selected from the food matrix they are going to be used for [62]. The composition of a fermentation medium influences the supply of nutrients and metabolism of cells in a bioreactor and, therefore, the productivity of a fermentation process also depends on the culture medium used. Of the major culture nutrients, carbon and nitrogen sources generally play a dominant role in fermentation productivity because these nutrients are directly linked with the formation of biomass and metabolites [49]. The understanding and modeling of microbial growth kinetics and transport phenomena play

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important roles in fermentation. The parameters like pH, temperature, agitation and aeration also need to be controlled. Moreover, such understanding is very much required in the design, scale up and process control in fermentation [238].

Fermentation processes may be divided into two systems: submerged fermentation (SMF), which is based on the microorganisms cultivation in a liquid medium containing nutrients, and solid state fermentation (SSF), which consists of the microbial growth and product formation on solid particles in the absence (or near absence) of water, which is similar to the fermentation reaction occurring in nature; however, substrate contains the sufficient moisture to allow the microorganism growth and metabolism [118, 287]. In recent years, SSF has received more interest from researchers since several studies have demonstrated that this process may lead to higher yields and productivities, better product characteristics, less investment, and low energy, water volume and sterility demand than SMF [77]. SSF has become a very attractive alternative to SMF for specific applications [287]. SMF requires the consumption of large amounts of water, energy, and space. SSF, has not gained significant use because of engineering and standardization issues, especially concerning scaling-up the SSF process to an industrial scale process [270]. These biotechnological processes, especially SSF, can be applied to reduce costs and enable the use of enzymes for human and animal consumption [270]. Many microorganisms are capable of growing on solid substrates [118]. In SSF, the water content is quite low and the microorganisms are almost in contact with gaseous oxygen and substrate, unlike in the case of SMF. The water activity levels are very low, the risk of contaminating bacterial or fungal growth is greatly reduced, thereby reducing the high energy cost of strict aseptic and sterile conditions. SSF produces products that are more heat and pH stable at a reduced risk of enzyme inhibition and protease degradation [198].

1.3. Fermented plant products for the food of animal origin nutritional, technological, and safety parameters improving

LAB improve technological characteristics and the nutritional value of foods during fermentation by increasing the protein content and its digestibility, reducing saccharides content and antinutritional factors (phytates, tannins, and polyphenols), improving the bioavailability of minerals [238] increasing availability of functional compounds (e.g. soluble fiber, soluble arabinoxylans, free phenolic acids, bioactive peptides) [185] and increasing the energy density by hydrolyzing starch into simpler compounds such as glucose and fructose [360]. Incorporation of animal

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origin with plant additives can be a simple way to improve their nutritional value and biofunctionality [142]. Health enhancing ingredients such as plant proteins, dietary fibers, herbs and spices are rapidly increasing worldwide [399]. Plantbased products provide the majority of the carbohydrates, some proteins, oils, dietary fiber (arabinoxylans, β-glucans, cellulose, lignin and lignans), sterols, tocopherols, tocotrienols, ARs, phenolic acids, vitamins and microelements, and have positive health benefits: significantly reduce the risk of some chronic health conditions such as type 2 diabetes, cardiovascular disease, and cancer [14, 54, 209, 300, 344, 349, 382]. The technological processes such as mechanical, thermal, chemical, and biological are used to reduce antinutritional factors content and to improve the bioavailability of nutrients. Unlike thermal, chemical, and mechanical processes which can deteriorate quality of food, fermentation is one of the processes that decreases the level of antinutrients in plant food and increases the starch and protein digestibility, and nutritive value [133, 320]. During fermentation, the plant substrate constituents are modified by the action of both endogenous and bacterial enzymes, including amylases, proteases, hemicellulases and phytases [305], esterases, xylanases, phenoloxidases, thereby affecting their structure, bioactivity, and bioavailability.

Changes of the plants bioactive compounds – ARs, lignans, and VC – during lactic acid fermentation. Fermentation with LAB positively influences nutritional and functional value of plants: increase the levels of free phenolic acids, TPC, soluble dietary fiber, lignans [168, 229, 300, 308], improve the protein digestibility [16, 93]. Also, LAB are essential for the transformation of natural compounds, e.g., lignans and ARs, to perform their bioactivities. The intake of ARs is beneficial because they reduce the absorption of cholesterol, regulate metabolism of triacylglycerols and affect levels of lipid-soluble vitamins [197; 200]. ARs (Figure 1.3.1) contain phenolic ring and belongs to antioxidants. However, in vitro investigations showed that their antioxidant activity was very low compared to some other bioactive compounds found in cereal grains such as tocols [322]. ARs display also antibacterial and antifungal activities [200; 323]. Because ARs contain hydrophobic alkyl chains, they easily react with proteins including enzymes and thereby inhibit their catalytic activity. Even at low concentrations (e.g. 900 mM), ARs considerably decrease the activities of human digestive enzymes (proteases, aldose reductases and a-glucosidases) and this way they reduce the absorption of some nutrients [323].

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Figure 1.3.1. The five major cereal ARs homologues have saturated odd-numbered alkyl chains with 17-25 carbon atoms

(C17:0-25:0, top to bottom).

Second group of biological active compounds in plants, which are increasing during fermentation, is lignans (Figure 1.3.2). Lignans are present in a wide range of foods, such as flaxseed, cereals, vegetable, fruit, and beverages. They afford protection against cardiovascular diseases, hyperlipidemia, breast cancer, colon cancer, prostate cancer, osteoporosis and menopausal syndrome, dependent on the bioactivation of these compounds to enterolactone and enterodiol [113, 211]. They have anticarcinogenic, antioxidant, estrogenic and activities [5, 255].

These phytochemicals are not typical to animal origin and could be an opportunity to industry develop novel food products with enhanced nutritional and health benefits, improved shelf-life, quality and usefull for animal origin functional food production [180, 203].

During the fermentation process VC (acids, alcohols, aldehydes, ketones, and esters) are produced, and they are associated with the sensory characteristics of the fermented products [203]. The SSF showed positive changes of the phenolic content with the development of special flavour compounds, and the influence of SSF on the content of biologically active compounds depended on the type of microorganisms (LAB or yeast) and the used LAB strain [176]. Volatile organic compounds comprise a chemically diverse group of organic compounds, generally with a molecular weight in the range of 50-200 Da, which exhibit appreciable vapor pressure under ambient conditions. For humans, volatiles are important as scents and contribute to the flavor of foods (flavor volatiles). As food aromas, volatiles contribute to palatability and to our appreciation of foods, and along with

OH HO OH HO OH HO OH HO OH HO OH HO

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sugars, organic acids, salts, and other components affecting taste receptors, are responsible for the flavor of food [203].

Figure 1.3.2. Chemical structures of some dietary lignan precursors.

Plants and plant products for the food of animal origin quality improving. Pea fiber. Pea dietary fiber (DF) gives the opportunitie to make innovative, healthy products. DF is the edible part of plants or analogous carbohydrates; it consists of polysaccharides that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine [221]. The physiological effects of DF are dependent on its physicochemical properties, which are mainly influenced by particle size, cell wall structure, solubility, degree of polymerisation and substitution, distribution of side chains and degree of cross-linking of the polymers. Recent results demonstrate the efficacy of fermentation to increase the bioavailability of DF related compounds such as free ferulic acid, lignans and phenolic acids together with other phytochemicals [186, 265]. The important technological characteristics of dietary fiber that determine the possibilities for their application are water holding capacity (WHC), capacity of fat binding, viscosity, gel forming ability, chelating capacity, and the influence on food texture. The WHC is associated with the length and density of the fibers. Also, the pH of the environment affects the water retention capacity. Capacity of fat binding is more dependent on the porosity of the fibers, than the molecular affinity. The ability to form a gel is the most important feature in using fibers as a fat replacer. This ability is

Secoisolariciresinol Matairesinol Lariciresinol Pinoresinol OH OH H3CO HO OCH3 OH H3CO HO OCH₃ OH O O O OH OCH3 OH H3CO HO OCH3 OH O O H₃CO HO

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provided by cross-linking of polymeric units and by retention of water or other solvents in the gel structure. This characteristic depends on a number of factors, such as concentration, temperature, the presence of certain ions, and the pH of the environment [85]. This is why DF and high-fiber food products have attracted great attention because of their significant health benefits to consumers [14, 57].

Soya. Soybean is one of the main agricultural commodities cultivated

worldwide. It is an excellent source for plant protein, oligosaccharides, VB,

VE and mineral substance [72]. The health benefits of soybean intake are

attributed to various phytochemicals: phenolic acids (mainly vanillic acid, caffeic acid, ferulic acid, protocatechuic acid, and coumaric acid), flavonoids (mainly quercetin and the glycosylated isoforms of isoflavones genistin, daidzin, and glycitin), carotenoids, and tocopherols [90, 205]. They are well known for their antioxidant, antiinflammator, anticarcinogenic activities and they can reduce the risk of cardiovascular diseases [13, 90]. Different from phenolic acids and flavonoids, which are hydrophilic constituents, carotenoids and tocopherols are lipophilic and are found in the oil fraction. Carotenoids possess antioxidant activity due to their provitamin A and RSA, and they present the capability to prevent carcinogenesis, coronary, and neurodegenerative diseases [204, 261]. SSF and SMF are current processing techniques traditionally used to preserve and to enhance the nutritional quality and health promotion properties of legumes [174, 378]. SSF of the soybean is a more economical and simple fermentation technology in order to produce probiotics carrier food [410]. The type of microorganism plays a key role in the fermentation process [174, 378]. Fermentation of soya brings several advantages: decreases the non-nutritional factors (phytates, tannins, trypsin inhibitors and oligosaccharides), improves nutrient digestibility, reduces their allergenicity [111, 318, 364], microbial enzymes bring about the bioconversion of polyphenols into more biologically active compounds [220], reduces the toxins and release many small peptides by the hydrolysis of soybean proteins by microbial proteases [241, 338, 378].

[395] have found that lactic fermentation reduced stachyose and raffinose content and transformed b-glucoside-, acetyl- and malonyl-glucosides isoflavones in soymilk into aglycone, the bioactive form of isoflavones. Furthermore, it has also been found that fermentation enhanced the antioxidant and antimutagenic activity. This is why fermentation with LAB is very usefull on purpose to improve the protein content, supply essential amino acids, reduced the tannin, phytate, trypsin inhibitor and protease inhibitor [398].

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Jerusalem artichockes (Helianthus tuberosus L.) Jerusalem artichoke

(JA) is a perennial sunflower species with origins in central North America and has been grown in Europe since the 17th century [370]. Fresh tuber of JA contains 75-80% water, 15-20% carbohydrates (main sources of inulin (70-90%)) and 2% protein [164]. Inulin from JA is a non-starch carbohydrate known as a fructan which is considered as a functional food ingredient with similar characteristics to DF and important technological benefits [78, 326, 395]. Because of its desirable textural and nutritional properties inulin from JA has been used as a prebiotic [326], a source of low glycemic index food [310], foam/emulsion stabilizer and a fat/sugar replacer and texturizer [81] while it promotes further health benefits [240].

JA is a good source of minerals (calcium, iron, selenium, potassium, phosphorus) and vitamins (vitamin B complex, vitamin C and β – carotene) [177], and rich in biologically active substances, including the naturally occurring isomers of caffeoylquinic acid [179], coumarins, unsaturated fatty acids, polyacetylenic derivatives and sesquiterpenes [285], and polyphenols (antioxidants) [334, 343]. The higher antioxidant activity of JA tubers prevent from oxidative stress [299]. Phenolics are secondary plant metabolites found in the majority of herbs, vegetables with well pronounced RSA [121, 247]. The phenolic content exhibits various medicinal properties, such as antioxidant, anticancer, antiallergenic, anti-inflammatory and antiviral [232].

Flaxseed. Flaxseed (Linum usitatissimum L.) accumulates many

biologically active compounds and elements including linolenic acid, linoleic acid, phenolic compounds such as lignans, phenolic acids (p-coumaric, ferulic, p-hydroxybenzoic, caffeic, and sinapic acids), and their glucosides, as well as flavonoids (herbacetin and campherol diglucoside), cyclic peptides, polysaccharides, alkaloids, cyanogenic glycosides, and cadmium. Defatted flaxseed contains high levels of dietary fibers and phytochemicals such as lignans [182, 183, 351, 394], are resistant to oxidation [7], and could be incorporated in many types of food as defattening agent [152]. Among the phenolic compounds, flaxseed lignans are in focus because of their estrogenic/antiestrogenic and antioxidant activity [358]. Therefore, flaxseed and their products (whole seed, ground seed and partially defatted flaxseed, which contains the highest content of dietary fiber) are used as a component of functional food [282, 351]. Besides, flaxseed is a good source of soluble and insoluble fibers and has been used as a traditional medicine for centuries to treat constipation.

Savory plants. A long time ago, herbs and spices have been added to

different types of food to improve the flavour and organoleptic properties [51]. Consumers increasingly demand natural antimicrobials as alternative

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preservatives in foods because the safety of additives has been questioned in the last few years. Alternative preservation techniques with such naturally derived ingredients are under investigation for their application in food products. Due to negative consumer perceptions of chemical preservatives, attention is shifting toward alternatives that consumers perceive as natural, especially plant extracts, including the EOs and essences of plant extracts [129]. In this context, they have attracted increasing interest because of their relatively safe status (many of them are considered GRAS by the FDA); they are easily decomposed, environmentally friendly and non phytotoxic [71, 129]. EOs are volatile, natural, complex compounds characterized by a strong odor and are formed by aromatic plants as secondary metabolites [8]. Many spices and herbs exert antimicrobial activity due to their EOs fractions can be used in food system like antifungal, antibacterial and antioxidant agents by inhibiting the growth of pathogenic microorganisms, ensuring the microbiological safety of food products [ 246, 345]. Their inherent antimicrobial activity is commonly related to the chemical structure of their components, the concentration in which they are present, and their interactions, which can affect their bioactive properties. They also may contain various antioxidant compounds such as polyphenols, phenols, flavonoids, etc. which have been thought to be the basis of their antimicrobial properties [71]. Antimicrobials are used in food for two main reasons: (1) to control natural spoilage processes (food preservation), and (2) to prevent/control growth of microorganisms, including pathogenic microorganisms (food safety). There is considerable potential for utilization of natural antimicrobials in food, especially in fresh fruits and vegetables. However, mechanisms of action, as well as the toxicological and sensory effects of natural antimicrobials, are not completely understood [371].

Satureja montana L. and Satureja hortensis L. S. montana L.,

commonly known as winter savory or mountain savory, belongs to the Lamiaceae family, Nepetoideae subfamily and Mentheae tribe and is a perennial semi-shrub (20–30 cm) that inhabits arid, sunny and rocky regions. S. montana L. is native to the Mediterranean and is found throughout Europe, Russia and Turkey. This is a intensive aromatic herb and has been used for centuries as a spice for food and teas; is used in Mediterranean cooking, mainly as a seasoning for meats and fish and in flavoring agents for soups, sausages, canned meats and spicy sauces [280].

S. montana L. has biological properties that are related to the presence of its

major EOs chemical compounds, in general, carvacrol, p-cymen and thymol are main phenolic compounds of savory species oil [167]. S. montana L. have been demonstrated antibacterial, antifungal, antioxidant, anti-diabetes, anti-HIV, anti-hyperlipidemic, reproduction stimulatory, expectorant and

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vasodilatory activities. Carvacrol and thymol play the fundamental roles in antimicrobial activity of this genus [257, 280, 345]. S. hortensis L. (Lamiaceae), summer savory, is an annual, herbaceous aromatic and medicinal plant native to southern Europe and naturalized in parts of North America. This herb is popular in most regions of the world and are widely used in foods as a flavor component, in herbal teas and possess at a broad spectrum of potent antibacterial and antifungal activities. In folk and traditional medicines this herb is used to treat ailments such as nausea, cramp, indigestion, muscle pain, diarrhea and infectious diseases. Evidence shows that this plant contains phenolic compounds such as thymol and carvacrol with a relatively wide spectrum of antimicrobial activity [311].

Rhaponticum carthamoides CD. R. carthamoides CD. (Willd.) Iljin, a

member of the Asteraceae family, is a perennial, herbaceous species naturally growing in the mountains of South Siberia, Middle Asia, and Mongolia [194]. This species often occurs in scientific literature equally under synonyms Leuzea carthamoides (Willd.) DC., preferred primarily in Eastern Europe and post-Soviet countries, or less common Stemmacantha

carthamoides (Willd.) [409]. R. carthamoides CD. possess a wide range of

biological activities: adaptogenic or anabolic dietary supplement, cardioprotective, immunomodulatory, antihyperglycemic, antioxidant (radical scavenging) and antimicrobial effects. It is valued as a rich natural source of ecdysteroids that are present in all parts of the plant [194]. R.

carthamoides CD. are considered to be highly promising in developing new

classes of biologically active food additives and ecologically safe products against pests [239] and functional food preparation [44]. Therefore the use of certain aromatic plants as innovative food additives may help to prevent the external growth of fungal spoilers and thus avoid consumer exposure to mycotoxins. In addition, recent research on spices and aromatic herbs suggests that they may be more effective in improving flavour and preserving food than artificial flavourings [225].

1.4. Changes of the meat during lactic acid fermentation process and the influence of the lactic acid bacteria on smoked meat products safety

parameters

Fermentation of meat causes number of physical, biochemical and microbial changes, which eventually result in functional characteristics of the products. Those changes include acidification (carbohydrate catabolism), solubilization and gelation of myofibrilla and sarcoplasmic proteins, degradation of proteins and lipids, reduction of nitrate into nitrite, formation of nitrosomyoglobin and dehydration [137]. Decrease in pH

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caused by lactic acid affects the firmness, colour, aroma, texture and flavour development and preservation effect of meat. The hardness of meat is a measure of degree of maturation, resulting from the denaturation and gelation of meat proteins and the loss of water [132]. Fast pH decrease can influence myofibrillar protein functionality, thereby altering meat tenderness, colour, WHC, and meat protein binding ability [175]. Weight loss in meat products is mainly associated with loss in water and WHC of meat. Increasing amounts of released and expressible water are possibly responsible for an increase in weight loss caused by proteolysis and denaturation of proteins during fermentation. Denaturation of sarcoplasmic proteins contributes to the decreased WHC of pork myofibrils [403] and that may cause the increased drip loss [258]. The taste of fermented meat products is mainly due to lactic acids and production of low molecular weights flavor compounds such as peptides and free amino acids, aldehydes, organic acids and amines resulted from proteolysis of meat [264]. It is well known that Lactobacillus species are weakly lipolytic [259]. Lipid oxidation products, free fatty acids, and VC produced from the process of fermentation are responsible for the aroma of a meat product [60, 284]. In most food fermentations, lactic and acetic acids produced by LAB and the resulting decrease in pH are responsible for the preservation effect. In meats, the main organic acid formed is lactic acid, and only low concentrations of acetic acid are acceptable from a sensory point of view [231]. Influence of microorganisms on the change in colour can be associated with acid production, protein denaturation. The production of organic acids is undoubtedly the determining factor on which the shelf life and the safety of the final product depends. The inhibition of pathogenic and spoilage flora is also dependent on a rapid and adequate formation of these organic acids [286]. Meat is an excellent source of protein in human diet and it is highly susceptible to microbial contaminations, which can cause spoilage and food borne infections in human, resulting in economic and health loses [195]. Many factors influencing meat shelf life can promote spoilage, bacterial growth and oxidative processes during storage. These in turn provoke deterioration in the flavour, texture and colour of meat [79]. Biopreservation has gained increasing attention as means of naturally controlling the shelf-life and safety of meat products, where antagonistic microorganisms or their antimicrobial metabolites can prevent the growth of pathogenic bacteria and fungus in food [389]. Consumer demand for greater stringency in relation to hygiene and safety of fresh and processed meat products with natural flavor and taste, free from chemical additives and preservatives [1]. Some microorganisms commonly associated with meat have proved to be antagonistic towards pathogenic and spoilage bacteria

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[98]. LAB mainly the species Lactobacillus sakei, Lactobacillus curvatus,

Staphylococcus carnosus, Staphylococcus xylosus and Staphylococcus saprophyticus are therefore often used as starter and bioprotective cultures

in industrial meat fermentation [131]. LAB are essential agents during meat fermentation improving hygienic and sensory quality of the final product. Its fermentative metabolism prevents the development of spoilage and pathogenic microflora by acidification of the product, also contributing to its colour stabilization and texture improvement [98]. pH is one of the most important environmental parameters affecting food fermentation. pH is closely related to microbial growth and the structural changes in phytochemicals during fermentation [367]. Reducing the pH of meat and meat products to 5.0 causes a reduction in lipoxygenase activity. An increase in the enzyme’s oxidative activity was noted for the pH ranging between 6.0–9.0 by [171]. The main cause of the quality defect is denaturation of sarcoplasmic proteins and myosin, leading to a decrease in water-binding capacity of the protein, as a result, a decrease in the WHC of the meat [26]. Low pH and high temperature conditions caused protein denaturation, including denaturation of µ-calpain, and as a consequence, limited post-mortem proteolysis and pale colour of the meat [191]. It is important to be able to predict a high WHC of meat because it is responsible for weight loss in raw, cooked and processed meats. WHC is also responsible for poor colour development in cured meat products, such as ham, and can influence meat palatability traits [328]. During meat processing, one common problem is water loss, which is frequently expressed as drip loss, expressible water, cook loss, and cooling loss depending upon the stage during processing in which it was measured [56]. As a consequence of these processes, the muscle protein coagulates, resulting in the slice ability, firmness and cohesiveness found in the final product. The development of curing colour occurs also in acidic conditions when nitric oxide is produced from nitrite and can then react with myoglobin [210]. The safety of meat and meat products is the most attention problem for consumers. BAs are related to quality and freshness of meat and meat products [281, 354] and has been used as a quality index of unwanted microbial activity [379]. Due to increases in the global demand for foods of animal origin, suppliers are obliged to implement specific controls to guarantee food safety and high quality [379]. BAs are basic nitrogenous compounds present in food and produced by different mechanisms, such as decarboxylation of amino acids or by the normal cellular metabolism of tissues [76, 345].

Biodegradation of toxic compounds by LAB is one of the most important mechanisms for the breakdown of organic compounds and the

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microorganisms are the most important agents for such degradation. It is known that LAB are able to adsorb some toxic substances (p-cresol, heterocyclic aromatic amines (HAA), ochratoxin A) to their cell wall [2, 271, 272, 302, 377] and remove several toxic compounds [136, 145].

Smoking of meat and meat products is one of the oldest processing methods applied in food preservation. Smoke not only gives special taste, colour and aroma to food but also enhances preservation due to the dehydrating, bactericidal and antioxidant properties of smoke [369]. However, it is known that polycyclic aromatic hydrocarbons (PAHs) can be found in smoked meats and they are a large group of organic compounds, belonging to the food and environmental contaminants [390] (Figure 1.4.1).

Figure 1.4.1. Chemical structures of some policyclic aromatic hydrocarbons.

These contaminants generate considerable interest, because some of them are highly carcinogenic in laboratory animals and have been implicated in breast, lung, and colon cancers in humans. Dietary intake of PAHs constitutes a major source of exposure in humans [153]. Food can be contaminated by PAHs that are present in air, soil or water, by industrial food processing methods (e.g. heating, drying and smoking processes) and during domestic food preparation (e.g. grilling and roasting processes) [159, 304].

1.5. Lactic acid fermentation in unripened cheese production

LAB are an important group of industrial starter cultures applied in the production of fermented dairy products [260, 380, 381]. The application of antimicrobial compounds producing LAB in the manufacture of dairy

Benz[a]anthracene

Benzo-[b]fluoranthene

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products, which can be incorporated into fermented or nonfermented dairy products, implies a processing additional advantage to improve the safety and increase the quality of dairy products, reduce the likelihood of food-borne diseases [19].

Fresh curd cheeses (acid curd cheeses, tvarogs, cottage cheeses) count among traditional dairy products. The technology of their production varies in different regions and different dairy traditions all over the world. For this reason, white fresh cheeses have strongly varied quality characteristics, especially those concerning their chemical composition and sensory characteristics [36]. They are regarded high quality products in our diet, that are rich in protein, macro elements, organic acids and vitamins [368]. Curd cheese are a large and diverse group of fermented dairy products. A common feature of all curd cheese is their processing, which is the coagulation of milk protein (mainly casein) by lactic acid fermentation or an acid-rennet combination (a coagulant enzyme simultaneously in conjunction with the LAB). Curd cheeses have mild, clean, slightly acidic taste and smell. Their structure and texture are uniform, compact, without lumps, and slightly loose, and it may be slightly granular. The colour of curd cheeses should be white to light cream and be uniform throughout the whole cheese [412].The manufacture of dairy foods is not a sterile process, and BA producers are likely to enter the food chain as non-starter LAB that are indigenous to the raw material. The presence of BAs in nonfermented foods generally indicates inadequate or prolonged storage; on the other hand, their presence in fermented foods could be unavoidable due to the diffusion of decarboxylases LAB [362]. BA formation is only possible if there is availability of the free substrate amino acids and the environment conditions are favorable to the decarboxylation activity [330]. Formation of BA in cheese depends on various factors; such as ripening time, ripening temperature, pH, the presence of microorganisms having BA-producing capability through their proteolytic and decarboxylase “activities” [226] and the bacterial density and synergistic effect between microorganisms are the most important [363].

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2. MATERIALS AND METHODS

2.1. Investigation venue

The experiments were conducted between 2012 and 2016 at the Lithuanian University of Health Sciences Veterinary Academy (LUHS VA) Department of Food Safety and Quality; Institute of Animal Rearing Technologies Laboratory of Meat Characteristics and Quality Assessment (LUHS VA), Kaunas University of Technology (KTU) Department of Food Science and Technology; Vytautas Magnus University (VMU) Department of Biology / Environmental Research Centre; Kaunas Botanical Garden (VMU); University of Latvia (LU) Centre of Food Chemistry (Riga, Latvia); Institute of Food Safety, Animal Health and Environment – “BIOR” (Riga, Latvia); at the enterprises “Nematekas” (Dovainonys, Lithuania), and “Judex” (Kaunas, Lithuania).

2.2. Materials

Microorganisms used in experiments. Lactobacillus sakei KTU05-6,

Pediococcus acidilactici KTU05-7, Pediococcus pentosaceus KTU05-8 and Pediococcus pentosaceus KTU05-9 previously isolated from spontaneous

rye sourdough and selected due to their preliminary inhibiting properties [82] were obtained from the culture collection of the Kaunas University of Technology, Department of Food Science and Technology, cereal and cereal products research group collection (Kaunas, Lithuania).

Bacillus cereus ATCC 10876, Bacillus subtilis, Escherichia coli 1.10, Escherichia coli ATCC25922, Escherichia coli, Listeria monocytogenes

1.1, Pseudomonas fluorescens biovar. V and Pseudomonas fluorescens biovar. III were obtained from the Institute of Botany of the Nature Research Centre (Vilnius, Lithuania).

Pseudomonas aeruginosa NCTC 6570, Pseudomonas aeruginosa

VUL-13, Staphylococcus aureus ATCC 9144, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 8739, Listeria monocytogenes ATCC 35152,

Salmonella enterica serovar typhimurium ATCC 13311, Bacillus cereus

ATCC 11778, and Yersinia enterolitica DSM 13030 and Yersinia

pseudotuberculosis III HH 146-36/84, previously isolated from the pork

production chain, were obtained from the LUHS VA Department of Food Safety and Quality collection (Kaunas, Lithuania).

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Plant material. In experiment used plant material is presented in Table 2.2.1.

Defatted soy flour (Maxima, Kaunas, Lithuania) and pea fiber (M plant

Ltd., Hamburg, Germany) were purchased at a local pharmacy (Kaunas,

Lithuania).

Winter savory (Sm), Summer savory (Sh) and Maral root or

Rhaponticum carthamoides (Rc) were grown and collected in an

experimental field located in the Kaunas Botanical Garden of VMU in 2012. Savory plants (Sm ans Sh) were used for RCMP production and for the beef and pork meat treatment.

Table 2.2.1. In experiment used plant material. The main characteristic of selected

plant

Plant

With the high content of proteins Defatted soy flour With the high content of phenolic

compounds

Winter savory (Satureja montana L.) (Sm), Summer savory (Satureja hortensis L.) (Sh),

Maral root or Rhaponticum (Rhaponticum carthamoides CD.) (Rc)

With the high content of inulin Jerusalem artichokes (Helianthus tuberosus L.) With the high content of lignans Defatted flaxseed (Linum usitatissimum L.) With the high content of dietary

fiber

Pea fiber

Rc products were used for unripened curd cheese (UCC) production. Defatted flaxseed (Institut Wlokien Naturalnych, Poznan, Poland) were purchased at a local super-market (Kaunas, Lithuania). Defatted flaxseeds were used for the production of functional additives with a high content of lignans production.

Jerusalem artichoke (Helianthus tuberosus L.) (harvest of 2011) was received from the Lithuanian Institute of Horticulture Experimental Farm (Babtai, Lithuania). Jerusalem artichoke tubers were used for ready-to-cook minced pork meat products (RCMP) enrichement [365].

Pea fiber was used for gluten-free RCMP production.

Material of animal origin. Fresh pork and beef meat from five different muscles including neck, shoulder, ham, M. longissimus dorsi and loin were obtained from the local market (Kaunas, Lithuania) (less than 4 days after slaughter). Pork and beef muscles were specifically chosen to span as large range of the concentrations of protein, moisture and fat as possible. All meat samples were cut into chops with a thickness of 2.5 cm, placed into plastic containers and stored under refrigeration (+4 ± 1 °C) for 12 hours and then

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were used for marination with selected LAB-based marinades, and Sm and Sh bioproducts were used for fresh pork and beef loin treatment.

Fresh, raw, boneless chicken breast, drumsticks and thigh muscles were obtained from the local market (Kaunas, Lithuania) and from an ecological farm (Kaunas, Lithuania).

Fresh pork loin (used for minced meat production) was obtained from the local market (Kaunas, Lithuania) (less than 4 days after slaughter). Loin was cut into chops with a thickness of 2.5 cm, placed into plastic containers and stored under refrigeration (+4 ± 1 °C) for 12 hours. Fresh pork loin minced meat was used for RCMP production.

Fresh pork and frozen back fat (used for sausage production) was obtained from the local market (Kaunas, Lithuania), minced and used for sausage production.

Raw cow milk was collected from a local farm (Mazeikiai, Lithuania). Before cheese production, the raw cow milk was stored in a refrigerator at +4 ± 1 °C no longer than 12 hours. Raw cow milk was used for UCC production.

2.3. Lactic acid bacteria cultivation and plant material fermentation

LAB cultivation. LAB (L. sakei; P. acidilactici; P. pentosaceus 8,

P. pentosaceus 9) before the experiment had been stored at -80 °C (PRO-LAB Diagnostics, United Kingdom) supplemented with 20% of glycerol.

Before the experiment, LAB had been defrosted and propagated in de Man Rogosa Sharpe (MRS) broth (CM 0359, Oxoid Ltd, Hampshire, United Kingdom): L. sakei at 30 °C, P. acidilactici at 32 °C, P. pentosaceus 8,

P. pentosaceus 9 at 35 °C temperature by keeping for 48 hours in a

thermostat (Binder, Germany). Before the use, 40 mM of fructose and 20 mM of maltose had been added.

Plant bioproducts production. The fermentation of plant products was performed with pure P. acidilactici, L. sakei, and P. pentosaceus strains (2%, m/m). The substrate water content was calculated with reference to the moisture content of the raw materials, water absorption capacity and the required humidity of the end product for SSF was ≤ 50%, and for SMF was ≥ 70%. The fermentation was carried out for 48 hours at optimal temperatures for P. acidilactici, L. sakei and P. pentosaceus (30 °C; 32 °C and 35 °C, respectively).

Fermentation of defatted soy flour and pea fiber was carried out for 48 hours at optimal temperatures for LAB cultivation, under SSF and SMF conditions.

APPLICATION OF SOLID-STATE FERMENTATION FOR DEVELOPMENT OF HIGHER VALUE AND SAFETY FOOD PRODUCTS

Erika Mozūrienė