Preface

As a component of food, dietary fibers gained importance when their functional properties came into the limelight. Their role in gut health was identified to be significant, and therefore, they are being incorporated into food products in the market to enhance the health benefits.

This book primarily covers two areas: the fundamentals of dietary fibers and their health benefits. Under the fundamentals of dietary fibers, the chemical, structural, and biochemical aspects of a food component are covered. As for health benefits, emphasis is primarily placed on their benefits imparted to gut microorganisms. The chapters covering these two areas are positioned in such a way that readers who do not come from the relevant academic background can go through the content and realize the importance of dietary fibers for themselves.

As a book that is highly relevant to food chemistry, it is expected that the contents of the chapters will add to the existing knowledge on dietary fibers and also open doors to research that would widen the horizons concerning their importance in health and wellness. From an industrial perspective, it is anticipated that knowledge shared herein would benefit food manufacturers in making scientific judgments about the incorporation and processing of dietary fibers in their food products without causing damage to the functional properties.

I would like to take this opportunity to extend my appreciation to the authors who have contributed all the wonderful and informative chapters to this book. Also, heartfelt appreciation goes to the Publisher IntechOpen with whom I have worked on several publications prior to this one. Last but not least, my appreciation goes to the Publishing Process Managers Ms. Marijana Francetic and Ms. Marina Dusevic who were assigned to this book had rendered their fullest support in putting the materials together and acted on all my requests in a timely manner.

> **Dr. Viduranga Y. Waisundara** Australian College of Business and Technology, Kandy Campus, Kandy, Sri Lanka

### Section 1


**5.1 Introduction**

*Goats (Capra) - From Ancient to Modern*

*<IP_ADDRESS> Mallorquina (15, picture 21)*

*<IP_ADDRESS> Moncaina (16, pictures 22 and 23)*

acceptable milk producer.

*<IP_ADDRESS> Negra Serrana (17, picture 24)*

Closely related to Blanca Celtibérica [14], this breed lives in the mountains near

This breed lives in the higher steep areas of the Balearic Islands, 800 m above sea level in the "Trasmontana Hills." In the past they have even been hunted, although nowadays they are included in conservation programs, living in semi-extensive production systems. The breed resembles the Markhor, with long horns, beards in both sexes, and a red and black coat. Associated with the diet and genotype, these meat aptitude and feral animals have darker, but tenderer, meat than milk lambs [4].

Placed in a mountain area in the Aragón region ("Moncayo"), this breed has genetic connections with the Pirenaica y Guadarrama breeds. It was recognized as a breed in 2002. It has arched horns and a black or chestnut long coat with clearer areas. Meat is its main production, although the breed has been recognized as an

to the Catalonian coast.  Also, rustic-

ity and kid meat are their main productive characteristics.

**36**

Spanish goat systems show a great diversity of farms, although there are several common aspects, such as their familiar character, the lack of technical-economic management, and the lack of a clear structure of the sector [18]. Spanish goat breeds are farmed under different extensive, semi-extensive, semi-intensive, or intensive systems, depending on their productive aptitude and performance.

Thus, in general, those breeds with a high milk production, which can be classified as dairy breeds, are reared under the most intensive systems, in plain geographical areas, with high forage availability to cover their nutritional demands. Sometimes kids are considered as a by-product in these farms. On the other hand, those breeds with a lower milk potential used to be reared in marginal mountainous areas, under extensive or semi-extensive models, following traditional pastoral systems, where kids are the main product (meat breeds). When a particular breed is also milked, it is considered as a dual-purpose goat breed, whose characteristics have been previously defined.

In addition to the genetic base, there is also great heterogeneity in terms of other characteristics of the systems, as are the management of food and reproduction, and the degree of technification, which determines the type of system. Ultimately goat production systems in Spain are mainly determined by the aptitude of the breed such as farm facilities, housing systems, and feeding strategies.


**2. Bacterial hazards associated with fish and fish products**

From the viewpoint of microbiology, fish and related products are a risky foodstuff group. Pathogenic bacteria associated with seafood can be categorized into three general groups [14]: 1) Bacteria (indigenous bacteria) that belong to the natural microflora of fish (*Clostridium botulinum*, pathogenic *Vibrio* spp., *Aeromonas hydrophila*); 2) Enteric bacteria (non-indigenous bacteria) that are present due to faecal contamination (*Salmonella* spp., *Shigella* spp., pathogenic *Escherichia coli*, *Staphylococcus aureus*); and 3) bacterial contamination during processing, storage, or preparation for consumption (*Bacillus cereus*, *Listeria monocytogenes*, *Staphylococcus aureus*, *Clostridium perfringens, Clostridium botulinum, Salmonella* spp.).

*Vibrio parahaemolyticus* has been isolated from sea and estuary waters on all continents with elevated sea water temperatures. *V. parahaemolyticus* is frequently isolated from fish, molluscs, and crustaceans throughout the year in tropical climates and during the summer months in cold or temperate climates [15]. Fish food associated with illnesses due to consumption of *V. parahaemolyticus* includes fish-balls, fried mackerel (*Scomber scombrus*), tuna (*Thunnus thynnus*), and sardines (*Sardina pilchardus*). These products include both raw and undercooked fish products and cooked products that have been substantially recontaminated [9,15]. The most affected by the pathogens are Japan, Taiwan, and other Asian coastal regions, though cases of disease have been described in many countries and on many continents [9,16]. Cases of diseases caused by *V. parahaemolyticus* are occasional in Europe. During 20 years, only two cases of gastroenteritis were recorded in Denmark. The interest in this organism has been widened by the finding that similar organisms, *V. alginolyticus* and group of F *Vibrio* sp. also cause serious disease in humans [17]. *V. cholerae* is often transmitted by water but fish or fish products that have been in contact with contaminated water or faeces from infected persons also frequently serve as a source of infection [1,9,19]. The organism would be killed by cooking and recent cases of cholera in South America have been associated with the uncooked fish marinade seviche (*Cilus gilberti*) [18].

*E. coli* is a classic example of enteric bacteria causing gastroenteritis*. E. coli* including other coliforms and bacteria as *Staphylococcus* spp. and sometimes enterococci are commonly used as indices of hazardous conditions during processing of fish. Such organisms should not be present on fresh-caught fish [9,20,21]. The contamination fish derived food with pathogenic *E. coli* probably occurs during handling of fish and during the production process [20,22]. An outbreak of diarrhoeal illness caused by ingestion of food contaminated with enterotoxigenic *E. coli* was described in Japan [23]. The illness was strongly associated with eating tuna paste. Brazilian authors [24] isolated 18 enterotoxigenic strains of *E. coli* (ETEC) from 3 of 24 samples of fresh fish originating from Brazilian markets; 13 of them produced a thermolabile enterotoxin. Infection with verocytotoxin \_ producing strains of *E. coli* (VTEC) after ingestion of fish was recorded in Belgium [25]. An outbreak caused by salted salmon roe contaminated, probably during the production process, with enterohaemorrhagic *E. coli*  (EHEC) O157 occurred in Japan in 1998 [22]. The roe was stored frozen for 9 months but it appears that O157 could survive freezing and a high concentration of NaCl and retained its pathogenicity for humans [26].

382 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

*Salmonella* spp.).

[18].

are particularly interesting candidates for this technique [1,2,6,7]. Indeed, they are frequently naturally present in food products and are often strong competitors, by producing a wide range of antimicrobial metabolites such as organic acids, diacetyl, acetoin, hydrogen peroxide, reuterin, reutericyclin, antifungal peptides, and bacteriocins [8-10). Hence, the last two decades have seen intensive investigation on LAB and their metabolites

From the viewpoint of microbiology, fish and related products are a risky foodstuff group. Pathogenic bacteria associated with seafood can be categorized into three general groups [14]: 1) Bacteria (indigenous bacteria) that belong to the natural microflora of fish (*Clostridium botulinum*, pathogenic *Vibrio* spp., *Aeromonas hydrophila*); 2) Enteric bacteria (non-indigenous bacteria) that are present due to faecal contamination (*Salmonella* spp., *Shigella* spp., pathogenic *Escherichia coli*, *Staphylococcus aureus*); and 3) bacterial contamination during processing, storage, or preparation for consumption (*Bacillus cereus*, *Listeria monocytogenes*, *Staphylococcus aureus*, *Clostridium perfringens, Clostridium botulinum,*

*Vibrio parahaemolyticus* has been isolated from sea and estuary waters on all continents with elevated sea water temperatures. *V. parahaemolyticus* is frequently isolated from fish, molluscs, and crustaceans throughout the year in tropical climates and during the summer months in cold or temperate climates [15]. Fish food associated with illnesses due to consumption of *V. parahaemolyticus* includes fish-balls, fried mackerel (*Scomber scombrus*), tuna (*Thunnus thynnus*), and sardines (*Sardina pilchardus*). These products include both raw and undercooked fish products and cooked products that have been substantially recontaminated [9,15]. The most affected by the pathogens are Japan, Taiwan, and other Asian coastal regions, though cases of disease have been described in many countries and on many continents [9,16]. Cases of diseases caused by *V. parahaemolyticus* are occasional in Europe. During 20 years, only two cases of gastroenteritis were recorded in Denmark. The interest in this organism has been widened by the finding that similar organisms, *V. alginolyticus* and group of F *Vibrio* sp. also cause serious disease in humans [17]. *V. cholerae* is often transmitted by water but fish or fish products that have been in contact with contaminated water or faeces from infected persons also frequently serve as a source of infection [1,9,19]. The organism would be killed by cooking and recent cases of cholera in South America have been associated with the uncooked fish marinade seviche (*Cilus gilberti*)

*E. coli* is a classic example of enteric bacteria causing gastroenteritis*. E. coli* including other coliforms and bacteria as *Staphylococcus* spp. and sometimes enterococci are commonly used as indices of hazardous conditions during processing of fish. Such organisms should not be present on fresh-caught fish [9,20,21]. The contamination fish derived food with pathogenic *E. coli* probably occurs during handling of fish and during the production process [20,22]. An outbreak of diarrhoeal illness caused by ingestion of food contaminated with

to discover new LAB strains that can be used in food preservation [1,7,11-13].

**2. Bacterial hazards associated with fish and fish products**

*Aeromonas* spp. has been recognized as potential foodborne pathogens for more than 20 years. Aeromonads are ubiquitous in fresh water, fish and shellfish and also in meats and fresh vegetables [27]. The epidemiological results so far are, however, very questionable. The organism is very frequently present in many food products, including raw vegetables, and very rarely has a case been reported. Up to 8.1% of cases of acute enteric diseases in 458 patients in Russia were caused by *Aeromonas* spp. [28]. In this study, *Aeromonas* spp. isolates with the same pathogenicity factors were isolated from river water in the Volga Delta, from fish, raw meat, and from patients with diarrhoea. Most *Aeromonas* spp. isolates are psychrotrophic and can grow at refrigerator temperatures [29]. This could increase the hazard of food contamination, particularly where there is a possibility of crosscontamination with ready-to-eat food products.

*Salmonella* has been isolated from fish and fishery product, though it is not psychrotrophic or indigenous to the aquatic environment [30]. The relationship between fish and *Salmonella*  has been described by several scientists; some believe that fish are possible carriers of *Salmonella* which are harbored in their intestines for relatively short periods of time and some believe that fish get actively infected by *Salmonella* [31]. Most outbreaks of food poisoning associated with fish derive from the consumption of raw or insufficiently heat treated fish and cross-contamination during processing and the U.S. Food and Drug Administration's (FDA) data showed that *Salmonella* was the most common contaminant of fish and fishery products [31]. The highest *Salmonella* incidence in fishery products was determined in Central Pacific and African countries while it was lower in Europe and including Russia, and North America [32]. The most common serovar found in the world was *S.* sub Weltvreden [30, 31]. In seafood the commonest serotype encountered was *S.* sub Worthington followed by *S.* sub Weltevreden.

Enterotoxins produced by *Staphylococcus aureus* are another serious cause of gastroenteritis after consumption of fish and related products. In 3 of 10 samples of fresh fish, higher counts of *Staph. aureus* were detected than permitted by Brazilian legislation [20, 33]. In the southern area of Brazil, *Staph. aureus* was isolated from 20% of 175 examined samples of fresh fish and fish fillets (*Cynoscion leiarchus*). *Staph. aureus* has also been detected during the process of drying and subsequent smoking of eels in Alaska in 1993 [34]. During the process, *S. aureus* populations increased to more than 105 CFU g-1 of the analyzed sample, after 2 to 3 days of processing. Subsequent laboratory studies showed that a pellicle (a dried skin-like surface) formed rapidly on the strips when there was rapid air circulation in the smokehouse and that bacteria embedded in/under the pellicle were able to grow even when heavy smoke deposition occurred.

Lactic Acid Bacteria and Their Bacteriocins: A Promising Approach to Seafood Biopreservation 385

viscera allowed the bacterium to multiply and to produce toxin. *C. perfringens*, an important cause of both food poisoning and non-food-borne diarrhoeas in humans, was found in a number of fish owing to contamination with sewage, which is the main source of this

Seafood products are known to be especially susceptible to both microbiological and biochemical spoilage pathways. The development of effective processing treatments to extend the shelf life of fresh fish products is a must [2]. Additionally, the consumers' demand for high-quality and minimally processed seafood has recently captivated great attention [5, 9]. However, an increase in foodborne illness outbreaks is concomitant with the increase in consumer demand for less processed foods [1]. These trends highlight the importance of studying new microbial stress factors to extend the shelf-life of foods. Until now, approaches to reduce the risk of outbreaks of food poisoning have relied on the search for addition of more efficient chemical preservatives or on the application of more drastic physical treatments such as heating, refrigeration, high hydrostatic pressure (HHP), ionising radiation, pulsed-light, ozone, ultrasound, etc [1,5,50]. In spite of some possible advantage, these types of treatments have many drawbacks and limitation in seafood products: the proven toxicity of many of the commonest chemical preservatives (e.g. nitrites) (3), the alteration of the organoleptic and nutritional properties of seafood by physical treatments due to their delicate nature (e.g. freezing damage, discolouration in case of HHP and ionising radiation) [50,51] and especially recent consumer trends in purchasing and consumption, with demands for healthy seafood products that have been subjected to less extreme treatments (less heat and chill damage), with lower levels of salts, fats, acids, and sugars and/or the complete or the partial removal of chemically synthesized additives [1,2,7]. To harmonize consumer demands with the necessary safety standards, traditional means of controlling microbial spoilage and safety hazards in seafood are being replaced by an alternative solution that is gaining more and more attention: "biopreservation technology" [2,9,13,52,53]. It consists in inoculating food with microorganisms, or their metabolites, selected for their antibacterial properties and may be an efficient way of extending shelf life and food safety through the inhibition of spoilage and pathogenic bacteria without altering the nutritional quality of raw materials and food products [54, 55].

Lactic acid bacteria (LAB) possess a major potential for use in biopreservation because they are safe to consume, and during storage they naturally dominate the microbiota of many foods. Certain LAB species and strains isolated from seafood have been shown to exert strong antagonistic activity against spoilage and pathogenic microorganisms such as *Listeria, Clostridium, Staphylococcus*, and *Bacillus* spp [56-58]. The antagonistic and inhibitory properties of LAB are due to the competition for nutrients and the production of one or more antimicrobially active metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, and antimicrobial peptides (bacteriocins) [10]. Certain LAB are able to grow at refrigeration temperatures and are tolerant to modified-atmosphere packaging, low

organism [21].

**3. Biopreservation**

In ready-to-eat products, cooking, preservation ingredients, and storage atmosphere inhibit the Gram-negative organisms, resulting in a longer shelf life. Such conditions favor the growth of psychotropic pathogens such as *Listeria monocytogenes*, allowing them to grow to dangerous levels [9,35,36]. *L. monocytogenes* is a serious threat to consumer health and safety and has been implicated in several deadly outbreaks around the world [1,2]. This organism is halotolerant (up to 28% w/v for short periods), resistant to freezing temperatures, can grow and multiply during refrigeration, where other competing organisms cannot, and is able to survive at low water activity (aw) [9,14,37]. *L. monocytogenes* is widely distributed in the general environment including fresh water, coastal water and live fish from these areas. Contamination or recontamination of seafood may also take place during processing [37-39]. Moreover, *L. monocytogenes* is a psychrotrophic pathogen with the ability to grow from under 0 to 45°C [40]. This ability to grow at storage temperatures means that this bacterium is the main hazard in this kind of product. The pathogenic bacteria *L. monocytogenes* may grow on fresh seafood. *Listeria* has been found in farmed rainbow trout [41]. The outbreak of listeriosis related to vacuum packed gravad and cold-smoked fish was described in at least eight human cases for 11 months in Sweden [42]. Cold-smoked and gravad rainbow trout (*Oncorhynchus mykiss*) and salmon (*Salmo salar*) have been focused on during recent years as potential sources of infection with *L. monocytogenes* and there are several report on isolation of this food borne pathogen from fish-processing plants environments [14,37,39,43-45]. Seafood treatment is necessary to prevent food-borne illness. However, the pervasive nature of *L. monocytogenes* makes it difficult for processors to fully eliminate the organism from the environment.

Development of new-generation foods, which are mildly processed, contain few or no preservatives, are packaged in vacuum or modified atmospheres to ensure long shelf life and rely primarily on refrigeration for preservation, has raised concerns of potential increases in botulism risk caused by psychrotrophic nonproteolytic group II *Clostridium botulinum* [46]. An average of 450 outbreaks of foodborne botulism with 930 cases have been reported annually worldwide [47]. The main habitat of clostridia is the soil but they are also found in sewage, rivers, lakes, sea water, fresh meat, and fish [48,49]. Most critical are the hygienic conditions for handling the product after smoking. There is a risk of botulism due to the growth of *C. botulinum* type E in smoked fish. The bacterium becomes a hazard when processing practices are insufficient to eliminate botulinal spores from raw fish, particularly improper thermal processing [21]. The growth of *C. botulinum* and toxin production then depends on appropriate conditions in food before eating: the temperature, oxygen level, water activity, pH, the presence of preservatives, and competing microflora [21]. A problem with *C. botulinum* has been encountered with some traditional fermented fish products. These rely on a combination of salt and reduced pH for their safety. If the product has insufficient salt, or fails to achieve a rapid pH drop to below 4.5, *C. botulinum* can grow. There was no evidence that the fish had been mishandled, but a low salt environment in the viscera allowed the bacterium to multiply and to produce toxin. *C. perfringens*, an important cause of both food poisoning and non-food-borne diarrhoeas in humans, was found in a number of fish owing to contamination with sewage, which is the main source of this organism [21].

### **3. Biopreservation**

384 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

heavy smoke deposition occurred.

environment.

surface) formed rapidly on the strips when there was rapid air circulation in the smokehouse and that bacteria embedded in/under the pellicle were able to grow even when

In ready-to-eat products, cooking, preservation ingredients, and storage atmosphere inhibit the Gram-negative organisms, resulting in a longer shelf life. Such conditions favor the growth of psychotropic pathogens such as *Listeria monocytogenes*, allowing them to grow to dangerous levels [9,35,36]. *L. monocytogenes* is a serious threat to consumer health and safety and has been implicated in several deadly outbreaks around the world [1,2]. This organism is halotolerant (up to 28% w/v for short periods), resistant to freezing temperatures, can grow and multiply during refrigeration, where other competing organisms cannot, and is able to survive at low water activity (aw) [9,14,37]. *L. monocytogenes* is widely distributed in the general environment including fresh water, coastal water and live fish from these areas. Contamination or recontamination of seafood may also take place during processing [37-39]. Moreover, *L. monocytogenes* is a psychrotrophic pathogen with the ability to grow from under 0 to 45°C [40]. This ability to grow at storage temperatures means that this bacterium is the main hazard in this kind of product. The pathogenic bacteria *L. monocytogenes* may grow on fresh seafood. *Listeria* has been found in farmed rainbow trout [41]. The outbreak of listeriosis related to vacuum packed gravad and cold-smoked fish was described in at least eight human cases for 11 months in Sweden [42]. Cold-smoked and gravad rainbow trout (*Oncorhynchus mykiss*) and salmon (*Salmo salar*) have been focused on during recent years as potential sources of infection with *L. monocytogenes* and there are several report on isolation of this food borne pathogen from fish-processing plants environments [14,37,39,43-45]. Seafood treatment is necessary to prevent food-borne illness. However, the pervasive nature of *L. monocytogenes* makes it difficult for processors to fully eliminate the organism from the

Development of new-generation foods, which are mildly processed, contain few or no preservatives, are packaged in vacuum or modified atmospheres to ensure long shelf life and rely primarily on refrigeration for preservation, has raised concerns of potential increases in botulism risk caused by psychrotrophic nonproteolytic group II *Clostridium botulinum* [46]. An average of 450 outbreaks of foodborne botulism with 930 cases have been reported annually worldwide [47]. The main habitat of clostridia is the soil but they are also found in sewage, rivers, lakes, sea water, fresh meat, and fish [48,49]. Most critical are the hygienic conditions for handling the product after smoking. There is a risk of botulism due to the growth of *C. botulinum* type E in smoked fish. The bacterium becomes a hazard when processing practices are insufficient to eliminate botulinal spores from raw fish, particularly improper thermal processing [21]. The growth of *C. botulinum* and toxin production then depends on appropriate conditions in food before eating: the temperature, oxygen level, water activity, pH, the presence of preservatives, and competing microflora [21]. A problem with *C. botulinum* has been encountered with some traditional fermented fish products. These rely on a combination of salt and reduced pH for their safety. If the product has insufficient salt, or fails to achieve a rapid pH drop to below 4.5, *C. botulinum* can grow. There was no evidence that the fish had been mishandled, but a low salt environment in the Seafood products are known to be especially susceptible to both microbiological and biochemical spoilage pathways. The development of effective processing treatments to extend the shelf life of fresh fish products is a must [2]. Additionally, the consumers' demand for high-quality and minimally processed seafood has recently captivated great attention [5, 9]. However, an increase in foodborne illness outbreaks is concomitant with the increase in consumer demand for less processed foods [1]. These trends highlight the importance of studying new microbial stress factors to extend the shelf-life of foods. Until now, approaches to reduce the risk of outbreaks of food poisoning have relied on the search for addition of more efficient chemical preservatives or on the application of more drastic physical treatments such as heating, refrigeration, high hydrostatic pressure (HHP), ionising radiation, pulsed-light, ozone, ultrasound, etc [1,5,50]. In spite of some possible advantage, these types of treatments have many drawbacks and limitation in seafood products: the proven toxicity of many of the commonest chemical preservatives (e.g. nitrites) (3), the alteration of the organoleptic and nutritional properties of seafood by physical treatments due to their delicate nature (e.g. freezing damage, discolouration in case of HHP and ionising radiation) [50,51] and especially recent consumer trends in purchasing and consumption, with demands for healthy seafood products that have been subjected to less extreme treatments (less heat and chill damage), with lower levels of salts, fats, acids, and sugars and/or the complete or the partial removal of chemically synthesized additives [1,2,7]. To harmonize consumer demands with the necessary safety standards, traditional means of controlling microbial spoilage and safety hazards in seafood are being replaced by an alternative solution that is gaining more and more attention: "biopreservation technology" [2,9,13,52,53]. It consists in inoculating food with microorganisms, or their metabolites, selected for their antibacterial properties and may be an efficient way of extending shelf life and food safety through the inhibition of spoilage and pathogenic bacteria without altering the nutritional quality of raw materials and food products [54, 55].

Lactic acid bacteria (LAB) possess a major potential for use in biopreservation because they are safe to consume, and during storage they naturally dominate the microbiota of many foods. Certain LAB species and strains isolated from seafood have been shown to exert strong antagonistic activity against spoilage and pathogenic microorganisms such as *Listeria, Clostridium, Staphylococcus*, and *Bacillus* spp [56-58]. The antagonistic and inhibitory properties of LAB are due to the competition for nutrients and the production of one or more antimicrobially active metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, and antimicrobial peptides (bacteriocins) [10]. Certain LAB are able to grow at refrigeration temperatures and are tolerant to modified-atmosphere packaging, low pH, high salt concentrations, and the presence of certain additives such as lactic acid, acetic acid, and ethanol. Because of these benefits, LAB can be used as protective cultures to restrict the growth of undesired organisms such as certain spoilage and pathogenic bacteria, with the subsequent benefits in terms of food safety [9,10,58]. Moreover, these microorganisms may have additional functional properties and, in some circumstances, they can be beneficial for the consumers [6]. LAB represent the microbial group most commonly used as protective cultures, as they are present in all fermented foods and have a long history of safe use [8]. Safety for the consumers is an aspect of great importance, in particular for some seafood products which are not cooked before consumptions, but also for other types of foods.

Lactic Acid Bacteria and Their Bacteriocins: A Promising Approach to Seafood Biopreservation 387

Bacteriocins are ribosomally synthesized peptides, that exert their antimicrobial activity against either strains of the same species as the bacteriocin producer (narrow range), or to more distantly related species (broad range) [1,2,7]. It has been estimated that between 30% and 99% of all bacteria and archaea produce bacteriocins; their production by LAB is very significant from the point of view of their potential applications in food systems and thus, unsurprisingly, these have been most extensively investigated [6,10,12,60,65,66]. It has been noted that the activity of bacteriocins is frequently directed against bacteria that are related to the bacteriocin - producing strain or against bacteria found in similar environments [67]. It has also been noted that some bacteriocins can also play a role in cell signaling. Microorganisms that produce bacteriocins also possess immunity mechanisms to confer self - protection, that is, to protect bacteriocin producers from committing "suicide" [10,68,69]. Besides concern about antibiotic resistance, increasing consumer awareness of potential health risks associated with chemical preservatives has increased interest in bacteriocins. Bacteriocins are naturally produced so they are more easily accepted by consumers [54]. Bacteriocins are usually classified combining various criteria. The main ones being the producer bacterial family, their molecular weight and finally their amino acid sequence homologies and/or gene cluster organization [59,70]. Based on a relatively recent approach [69,71,72] bacteriocins produced by LAB have been categorized into two major classes: the lanthionine - containing bacteriocins or lantibiotics (class I) and the largely unmodified

An important role of meat LAB starter cultures is the rapid production of organic acids; this inhibits the growth of unwanted flora and enhances product safety and shelf life. The types and levels of organic acids produced during the fermentation process depend on the LAB strains present, the culture composition, and the growth conditions [74]. Fermentation of the carbohydrates, glucose, glycogen, glucose-6-phosphate and small amounts of ribose, in meat and meat products, produces organic acids by glycolysis (Embden-Meyerhof Parnas pathway, EMP pathway) or the Hexose Monophosphate, HMP pathway. L (+) lactic acid is more inhibitory than its D (-) counterpart [68]. The antimicrobial effect of organic acids lies in the reduction of pH, and in the action of undissociated acid molecules [75]. It has been proposed that low external pH causes acidification of the cytoplasm. The lipophilic nature of the undissociated acid allows it to diffuse across the cell membrane collapsing the electrochemical proton gradient. Alternatively, cell membrane permeability may be affected, disrupting substrate transport systems [72]. The LAB in particular are able to reduce the pH to levels where putrefactive (e.g. clostridia and pseudomonads), pathogenic (e.g. *Salmonella*  s and *Listeria* spp.) and toxinogenic bacteria (*Staphylococcus aureus*. *Bacillus cereus*, *Clostridium botulinum*) will be either inhibited or killed [7]. Also, the undissociated acid, on account of its fat solubility, will diffuse into the bacterial cell, thereby reducing the intracellular pH and

**4.2. Antimicrobial components from LAB**

linear peptide antimicrobials (class II).

*4.2.2. Organic acid production*

*4.2.1. Bacteriocins*


*2.2. Materials, Devices and Spraying Parameters*

The additional material for flame-spraying was obtained by mixing in the ball mill appropriate proportions of aluminum powder (EN AW 1000 series) with carbon nanotubes and aluminum powder in the form of filter dust carburite (Table 1). Carbon nanotubes that were used in the test are commercially available multi-walled carbon nanotubes MWCNTs, produced in the Catalytic Chemical Vapor Deposition (CCVD) process, NANOCYLTM NC7000 (Belgium Nanocyl SA, Sambreville, Belgium) (Table 2).

**Table 2.** Structure and specification of Nanocyl NC 7000 carbon nanotubes and of filter dust carburite. *Materials* **2019**, *12*, x FOR PEER REVIEW 4 of 18

*Materials* **2019**, *12*, x FOR PEER REVIEW 4 of 18

Structure of Nanocyl NC 7000 carbon nanotubes

Surface area m2 /g 250–300 BET Note: 1) pyrolytically deposited carbon on the surface of the NC 7000.

Structure of filter dust carburite

Granulation [mm] % >1 mm up to 10%; <0,06 mm up to 70%

Moisture content % 1 Carburite content % 94 Granulation [mm] % >1 mm up to 10%; <0,06 mm up to 70% The subsonic flame spraying process was carried out cold in accordance with the standard EN 13507:2018 [35] on workstation, equipped with hand-guided modern oxyacetylene system (CastoDyn DS 8000 (Messer Eutectic Castolin, Gliwice, Poland). Final surface preparation was done by shot blasting sheets prior to spraying with sharp-edged cast iron of 0.5–1.5 mm shot grain size in accordance with standard ISO 2063-1:2017 [36]. Final surface roughness of the steel substrate after shot blasting was Ra = 12 μm, Rz = 85 μm. Before the spraying process, steel plates with dimensions of 150 × 150 × 5 mm3 were preheated with a gas burner up to a temperature of 40 °C (the temperature of preheating was measured using pyrometer). The standard modular nozzles regulating the flame outlet SSM 40 (Messer Eutectic Castolin, Gliwice, Poland) and the neutral flame (ratio O2/C2H2 = 1,2) were used. This allowed to obtain the proper spray jet [37,38]. The flame jet burner was guided in a The subsonic flame spraying process was carried out cold in accordance with the standard EN 13507:2018 [35] on workstation, equipped with hand-guided modern oxyacetylene system (CastoDyn DS 8000 (Messer Eutectic Castolin, Gliwice, Poland). Final surface preparation was done by shot blasting sheets prior to spraying with sharp-edged cast iron of 0.5–1.5 mm shot grain size in accordance with standard ISO 2063-1:2017 [36]. Final surface roughness of the steel substrate after shot blasting was Ra = 12 μm, Rz = 85 μm. Before the spraying process, steel plates with dimensions of 150 × 150 × 5 mm3 were preheated with a gas burner up to a temperature of 40 °C (the temperature of preheating was measured using pyrometer). The standard modular nozzles regulating the flame outlet SSM 40 (Messer Eutectic Castolin, Gliwice, Poland) and the neutral flame (ratio O2/C2H2 = 1,2) were used. This allowed to obtain the proper spray jet [37,38]. The flame jet burner was guided in a horizontal position covering the whole surface of the sheet. During the process the spraying direction was changed several times by 90°, until obtained thickness of coating was about 1,0 mm. Distance between the torch and the sprayed surface was 200 mm. The parameters and flame type were The subsonic flame spraying process was carried out cold in accordance with the standard EN 13507:2018 [35] on workstation, equipped with hand-guided modern oxyacetylene system (CastoDyn DS 8000 (Messer Eutectic Castolin, Gliwice, Poland). Final surface preparation was done by shot blasting sheets prior to spraying with sharp-edged cast iron of 0.5–1.5 mm shot grain size in accordance with standard ISO 2063-1:2017 [36]. Final surface roughness of the steel substrate after shot blasting was Ra = 12 µm, Rz = 85 µm. Before the spraying process, steel plates with dimensions of 150 × 150 × 5 mm3 were preheated with a gas burner up to a temperature of 40 ◦C (the temperature of preheating was measured using pyrometer). The standard modular nozzles regulating the flame outlet SSM 40 (Messer Eutectic Castolin, Gliwice, Poland) and the neutral flame (ratio O2/C2H2 = 1,2) were used. This allowed to obtain the proper spray jet [37,38]. The flame jet burner was guided in a horizontal position covering the whole surface of the sheet. During the process the spraying direction was changed several times by 90◦ , until obtained thickness of coating was about 1,0 mm. Distance between the torch and the sprayed surface was 200 mm. The parameters and flame type were constant for each test (Figure 1).

constant for each test (Figure 1).

constant for each test (Figure 1).

horizontal position covering the whole surface of the sheet. During the process the spraying direction was changed several times by 90°, until obtained thickness of coating was about 1,0 mm. Distance between the torch and the sprayed surface was 200 mm. The parameters and flame type were **Sample**

The criterion for visual assessment of the powder coatings' quality, was to make the surface layers characterized by the appropriate thickness, good adhesion to the substrate, low porosity, continuity and uniformity of obtained coatings [39]. Optimal parameters of flame-spraying of aluminum, aluminum with carbon nanotube reinforcement and aluminum with filter dust carburite reinforcement coatings have been determined on the basis of preliminary technological tests (Table 3). The view of representative samples with flame-sprayed coatings on the aluminum matrix are shown in Figure 2. *Materials* **2019**, *12*, x FOR PEER REVIEW 5 of 17 reinforcement coatings have been determined on the basis of preliminary technological tests (Table 3). The view of representative samples with flame-sprayed coatings on the aluminum matrix are shown in Figure 2.

*Materials* **2019**, *12*, x FOR PEER REVIEW 5 of 18

The criterion for visual assessment of the powder coatings' quality, was to make the surface layers characterized by the appropriate thickness, good adhesion to the substrate, low porosity, continuity and uniformity of obtained coatings [39]. Optimal parameters of flame-spraying of aluminum, aluminum with carbon nanotube reinforcement and aluminum with filter dust carburite

**Figure 1.** Flame-spraying process: (**a**) a diagram of handheld flame jet burner; (**b**) a photo from trials of flame-spraying aluminum coatings with CastoDyn DS 8000 burner**. Figure 1.** Flame-spraying process: (**a**) a diagram of handheld flame jet burner; (**b**) a photo from trials of flame-spraying aluminum coatings with CastoDyn DS 8000 burner. materials using CastoDyn DS 8000 torch**. Assist. Gas Number of Mass of**

**Table 3.** Parameters of flame-sprayed aluminum and aluminum coatings reinforced with carbon materials using CastoDyn DS 8000 torch**. Table 3.** Parameters of flame-sprayed aluminum and aluminum coatings reinforced with carbon materials using CastoDyn DS 8000 torch. **Number Type of Powder Oxygen Pressure Acetylene Pressure (Compressed Air) Pressure the Orifice for the Used Powder**

3 Al + 1% CNT 4 0.7 3 2 100.8 57.0 Note: 1) CNT—carbon nanotubes Nanocyl NC 7000 wt.%; 2) C—carburite wt.%. Note: 1) CNT—carbon nanotubes Nanocyl NC 7000 wt.%; 2) C—carburite wt.%.

4 Al + 0.5% C2) 4 0.7 3 2 99.7 59.4 Note: 1) CNT—carbon nanotubes Nanocyl NC 7000 wt.%; 2) C—carburite wt.%.

**Powder Yield [%]**

(a) (b) **Figure 2.** *Cont*.

**Figure 2.** View of test samples with flame-sprayed coatings on the aluminum matrix: (**a**) aluminum powder of the EN AW 1000 series; (**b**) aluminum powder EN AW 1000 series with addition of 0.5 wt.% carbon nanotubes (Nanocyl NC 7000); **(c**) aluminum powder of the EN AW 1000 series with the addition of 1 wt.% carbon nanotubes (Nanocyl NC 7000); (**d**) aluminum powder of the EN AW 1000 series with the addition of 1 wt.% carburite**. Figure 2.** View of test samples with flame-sprayed coatings on the aluminum matrix: (**a**) aluminum powder of the EN AW 1000 series; (**b**) aluminum powder EN AW 1000 series with addition of 0.5 wt.% carbon nanotubes (Nanocyl NC 7000); **(c**) aluminum powder of the EN AW 1000 series with the addition of 1 wt.% carbon nanotubes (Nanocyl NC 7000); (**d**) aluminum powder of the EN AW 1000 series with the addition of 1 wt.% carburite.

#### *2.3. Visual and Metallographic Examination of Coatings 2.3. Visual and Metallographic Examination of Coatings*

In each case, the entire surface of the sample was subjected to visual tests to assess the quality and identify any imperfections in the form of cracks, discontinuities, unevenness, porosity or lack of coating adhesion. Macro and microscopic examinations were performed on Olypmus GX 71 optical microscope (Olympus Corporation, Tokyo, Japan). The observations were made on the cross-section of metallographic samples cut from the centre of element. Samples were polished and etched in Aqua Regia. Selected areas of flame-sprayed coatings (aluminum and aluminum with addition of carbon materials) have been subjected to chemical composition analysis on JEOL 5800LV SEM scanning microscope and also EDX (Jeol Ltd., Tokyo, Japan). In each case, the entire surface of the sample was subjected to visual tests to assess the quality and identify any imperfections in the form of cracks, discontinuities, unevenness, porosity or lack of coating adhesion. Macro and microscopic examinations were performed on Olypmus GX 71 optical microscope (Olympus Corporation, Tokyo, Japan). The observations were made on the cross-section of metallographic samples cut from the centre of element. Samples were polished and etched in Aqua Regia. Selected areas of flame-sprayed coatings (aluminum and aluminum with addition of carbon materials) have been subjected to chemical composition analysis on JEOL 5800LV SEM scanning microscope and also EDX (Jeol Ltd., Tokyo, Japan).

#### *2.4. Hardness Measurements of Coatings 2.4. Hardness Measurements of Coatings*

The coating-hardness measurement was made with the Vickers method using Microhardness Tester 401MVD™ (Wilson Instruments An Instron Company, MA, USA). The examinations were carried out in conformity to ISO 6507-1:2018 standard [41]. The load applied during the hardness measurement was 0.98 N. The hardness measurement was made at the polished cross-section of the samples with flame-sprayed coatings. Ten hardness measuring points were made on the cross-section each sprayed coating. The coating-hardness measurement was made with the Vickers method using Microhardness Tester 401MVD™ (Wilson Instruments An Instron Company, Norwood, MA, USA). The examinations were carried out in conformity to ISO 6507-1:2018 standard [40]. The load applied during the hardness measurement was 0.98 N. The hardness measurement was made at the polished cross-section of the samples with flame-sprayed coatings. Ten hardness measuring points were made on the cross-section each sprayed coating.

### *2.5. Erosive Wear Resistance of Coatings*

*2.5. Erosive Wear Resistance of Coatings* The erosive wear tests of flame-sprayed coatings were carried out in accordance with ASTM G76-95 [42], as shown in Figure 3. Aluminum oxide powder (Al2O3) with particle diameter of 71 µm was used as the erodent material. Particle velocity was set at 70 ± 2 m/s, the erodent expenditure was 2.0 ± 0.5 g/min and the nozzle distance from the sample surface was 10 mm. The tests were carried out at 90° and 30° erodent impact angle. The average weight loss was determined based on three tests. The erosion rate was determined according to the Equation (1), The erosive wear tests of flame-sprayed coatings were carried out in accordance with ASTM G76-95 [41], as shown in Figure 3. Aluminum oxide powder (Al2O3) with particle diameter of 71 µm was used as the erodent material. Particle velocity was set at 70 ± 2 m/s, the erodent expenditure was 2.0 ± 0.5 g/min and the nozzle distance from the sample surface was 10 mm. The tests were carried out at 90◦ and 30◦ erodent impact angle. The average weight loss was determined based on three tests. The erosion rate was determined according to the Equation (1),

$$\text{erosion rate [g/min]} = \text{mass loss of sample [g]} \text{exposure time [min]} \tag{1}$$

However, the erosive wear resistance using Equation (2): However, the erosive wear resistance using Equation (2):

$$\begin{aligned} \text{erosive wear resistance } [0.001 \text{mm}^3/\text{g}] &= \text{volume loss of the sample } [\text{mm}^3] \text{:}\\ \text{total mass of the centroid used in the test } [\text{g}] \end{aligned} \tag{2}$$

*Materials* **2019**, *12*, x FOR PEER REVIEW 7 of 18

**Figure 3.** Erosion resistance testing according to ASTM G 76-95 [42]: (**a**) a schematic view, (**b**) the interior view of the erosion measuring chamber**. Figure 3.** Erosion resistance testing according to ASTM G 76-95: (**a**) a schematic view, (**b**) the interior view of the erosion measuring chamber. *2.6. Abrasive Wear Resistance of Coatings*

#### *2.6. Abrasive Wear Resistance of Coatings 2.6. Abrasive Wear Resistance of Coatings* Metal-mineral wear resistance tests of aluminum matrix coatings were provided in accordance

Metal-mineral wear resistance tests of aluminum matrix coatings were provided in accordance with ASTM G65-00, Procedure E [43]. During the test, the rubber-wheel made 1000 revolutions and the abrasive flow rate of A.F.S. Testing Sand 50–70 was 335 g/min. The force applied pressing the test coupon against the wheel was TL = 130 N (test load-TL). After the abrasive wear resistance test, the test specimen was weighed at weight sensitivity 0,0001 [g]. Converting mass loss to volume loss was as follows: Metal-mineral wear resistance tests of aluminum matrix coatings were provided in accordance with ASTM G65-00, Procedure E [42]. During the test, the rubber-wheel made 1000 revolutions and the abrasive flow rate of A.F.S. Testing Sand 50–70 was 335 g/min. The force applied pressing the test coupon against the wheel was TL = 130 N (test load-TL). After the abrasive wear resistance test, the test specimen was weighed at weight sensitivity 0.0001 [g]. Converting mass loss to volume loss was as follows: with ASTM G65-00, Procedure E [43]. During the test, the rubber-wheel made 1000 revolutions and the abrasive flow rate of A.F.S. Testing Sand 50–70 was 335 g/min. The force applied pressing the test coupon against the wheel was TL = 130 N (test load-TL). After the abrasive wear resistance test, the test specimen was weighed at weight sensitivity 0,0001 [g]. Converting mass loss to volume loss was as follows: volume loss [mm3 ] = mass loss [g]:density [g/cm3 ] x 1000 (3)

] = mass loss [g]:density [g/cm3

$$\text{volume loss [mm}^3\text{]} = \text{mass loss [g]:density [g/cm}^3\text{]} \times 1000\tag{3}$$

] x 1000 (3)

The tests were carried out on abrasion tester shown in Figure 4. The tests were carried out on abrasion tester shown in Figure 4.

volume loss [mm3

The tests were carried out on abrasion tester shown in Figure 4.

[43] (**a**) a schematic view, (**b**) picture of the device used**.**

**3. Results**

**3. Results**

(**a**) (**b**) **Figure 4.** Metal-mineral wear resistance test stand–according to ASTM G65–00, Procedure E standard [43] (**a**) a schematic view, (**b**) picture of the device used**. Figure 4.** Metal-mineral wear resistance test stand–according to ASTM G65–00, Procedure E standard (**a**) a schematic view, (**b**) picture of the device used.

**Figure 4.** Metal-mineral wear resistance test stand–according to ASTM G65–00, Procedure E standard

#### **3. Results** *Materials* **2019**, *12*, x FOR PEER REVIEW 8 of 18

#### *3.1. Metallographic Test Results 3.1. Metallographic Test Results*

The structure of each tested flame-sprayed coating cross-section is presented in Figure 5. The SEM structures of tested aluminum matrix coatings with chemical composition are presented in Figures 6–9. The structure of each tested flame-sprayed coating cross-section is presented in Figure 5. The SEM structures of tested aluminum matrix coatings with chemical composition are presented in Figures 6–9.

**Figure 5.** The macro and microstructure of the flame-sprayed pure aluminum and aluminum matrix with carbon nanotubes and carburite reinforcement coatings; etching: HCl + HNO3*.* **Figure 5.** The macro and microstructure of the flame-sprayed pure aluminum and aluminum matrix with carbon nanotubes and carburite reinforcement coatings; etching: HCl + HNO3.

*Materials* **2019**, *12*, x FOR PEER REVIEW 9 of 18

**Figure 6***.* The structure of EN AW 1000 aluminum powder flame-sprayed coating with marked chemical composition tested areas on SEM*.* **Figure 6.** The structure of EN AW 1000 aluminum powder flame-sprayed coating with marked chemical composition tested areas on SEM. **Figure 6***.* The structure of EN AW 1000 aluminum powder flame-sprayed coating with marked chemical composition tested areas on SEM*.*

flame-sprayed coating with marked chemical composition tested areas on SEM*.* **Figure 7***.* The structure of aluminum powder with 0.5 wt.% Nanocyl NC 7000 carbon nanotubes flame-sprayed coating with marked chemical composition tested areas on SEM*.* **Figure 7.** The structure of aluminum powder with 0.5 wt.% Nanocyl NC 7000 carbon nanotubes flame-sprayed coating with marked chemical composition tested areas on SEM.

*Materials* **2019**, *12*, x FOR PEER REVIEW 10 of 18

*Materials* **2019**, *12*, x FOR PEER REVIEW 10 of 18

**Figure 8***.* The structure of aluminum powder with 1 wt.% Nanocyl NC 7000 carbon nanotubes flamesprayed coating with marked chemical composition tested areas on SEM*.* **Figure 8.** The structure of aluminum powder with 1 wt.% Nanocyl NC 7000 carbon nanotubes flame-sprayed coating with marked chemical composition tested areas on SEM. **Figure** *.* The structure of aluminum with 1 wt.% Nanocyl 7000 nanotubes flamesprayed coating with marked chemical composition tested areas on SEM*.*

**Figure 9***.* The structure of aluminum powder with 0.5 wt.% carburite carbon nanotubes flame-sprayed coating with marked chemical composition tested areas on SEM*.* **Figure 9***.*The structure of aluminum powder with 0.5 wt.% carburite carbon nanotubes flame-sprayed coating with marked chemical composition tested areas on SEM*.* **Figure 9.** The structure of aluminum powder with 0.5 wt.% carburite carbon nanotubes flame-sprayed coating with marked chemical composition tested areas on SEM.

*3.2. Hardness Measurements*

*3.2. Hardness Measurements*

#### *3.2. Hardness Measurements Materials* **2019**, *12*, x FOR PEER REVIEW 11 of 18

The hardness measurements on flame-sprayed aluminum and aluminum matrix reinforced with carbon material coatings, were carried out on the surface at 5 points along one measuring line (Table 4) and on cross-section of the samples (Figures 10 and 11), according to the scheme showed on Figure 10. The hardness measurements on flame-sprayed aluminum and aluminum matrix reinforced with carbon material coatings, were carried out on the surface at 5 points along one measuring line (Table 4) and on cross-section of the samples (Figures 10 and 11), according to the scheme showed on Figure

**Table 4.** Surface hardness results of the flame-sprayed aluminum and aluminum reinforced with carbon material coatings. 10. **Table 4***.* Surface hardness results of the flame-sprayed aluminum and aluminum reinforced with

**Figure 10.** Hardness measurements scheme and HV 0.1 results of the flame-sprayed aluminum and aluminum reinforced with carbon materials coatings*.* **Figure 10.** Hardness measurements scheme and HV 0.1 results of the flame-sprayed aluminum and aluminum reinforced with carbon materials coatings.

30

*Materials* **2019**, *12*, x FOR PEER REVIEW 12 of 18

**Figure 11.** Comparison of average cross-sectional hardness and standard deviation for each of the investigated coatings*.* **Figure 11.** Comparison of average cross-sectional hardness and standard deviation for each of the investigated coatings.

#### *3.3. Tests Results of the Coatings Erosive Wear Resistance 3.3. Tests Results of the Coatings Erosive Wear Resistance*

Notes: density of aluminum spray coating 2.72 [g/cm3

*3.4. Tests Results of the Coatings' Wear Resistance*

flame-sprayed pure aluminum coating.

The relative erosive wear resistance test results of the flame-sprayed aluminum, aluminum with carbon nanotube reinforcement and aluminum with filter dust carburite reinforcement coatings are presented in Table 5 and Figure 12. The relative erosive wear resistance test results of the flame-sprayed aluminum, aluminum with carbon nanotube reinforcement and aluminum with filter dust carburite reinforcement coatings are presented in Table 5 and Figure 12.

**Table 5.** Summary of results obtained during the erosion wear test ASTM G76−95 [42]. **Table 5.** Summary of results obtained during the erosion wear test ASTM G76−95 [41].

Al + 1% CNT 0.0045 1.654 0.00056 0.10212 Notes: density of aluminum spray coating 2.72 [g/cm3 ], mass of erodent used 16.2 [g], test time 8 [min].

The wear resistance test results of the flame-sprayed aluminum, aluminum with carbon nanotube reinforcement and aluminum with filter dust carburite reinforcement coatings are presented in Table 6 and Figure 13. The metal-mineral type wear resistance of the flame-sprayed aluminum with carbon nanotubes and aluminum with carburite coatings were compared to the

Al + 0.5% C 0.0039 1.434 0.00049 0.08851

], mass of erodent used 16.2 [g], test time 8 [min].

**Figure 12.** The surfaces of flame-sprayed aluminum and aluminum matrix reinforced with carbon material coatings after erosive wear resistance tests; comparison the erosion effect on samples surfaces for each tested angle of erodent particles incidence. **Figure 12.** The surfaces of flame-sprayed aluminum and aluminum matrix reinforced with carbon material coatings after erosive wear resistance tests; comparison the erosion effect on samples surfaces for each tested angle of erodent particles incidence.

#### *3.4. Tests Results of the Coatings' Wear Resistance*

**Table 6.** Summary of results obtained during the abrasive wear test ASTM G65 [43]. **Specimen Designation Number of Specimen Weight Before Test [g] Weight after Test [g] Mass Loss [g] Average Mass Loss [g] Average Volume Loss [mm3] Relative 1) Abrasion Resistance** Al S1-1 43.9675 43.8413 0.1262 0.1418 52.1324 1.00 S1-2 42.3855 42.2281 0.1574 The wear resistance test results of the flame-sprayed aluminum, aluminum with carbon nanotube reinforcement and aluminum with filter dust carburite reinforcement coatings are presented in Table 6 and Figure 13. The metal-mineral type wear resistance of the flame-sprayed aluminum with carbon nanotubes and aluminum with carburite coatings were compared to the flame-sprayed pure aluminum coating.

Al + 0.5% CNT S2-1 56.5170 56.3924 0.1246 0.1286 47.26103 1.10 S2-2 53.8604 53.7279 0.1325

**4. Discussion**

**Table 6.** Summary of results obtained during the abrasive wear test ASTM G65 [42].

Notes: density of aluminum spray coating 2.72 [g/cm3 ]; 1) relative to sprayed coatings of the aluminum without carbon materials. without carbon materials.

#### Specimen designation / Number of specimen

**Figure 13.** The surfaces of flame-sprayed aluminum and aluminum with carbon material coatings after wear resistance metal-mineral tests. **Figure 13.** The surfaces of flame-sprayed aluminum and aluminum with carbon material coatings after wear resistance metal-mineral tests.

slight roughness, lack of porosity and cracks (Figure 2). During the flame-spraying process, carbon material particles added to aluminum powder did not oxidize completely in the oxyacetylene flame.

Visual and metallographic tests of the flame-sprayed aluminum and aluminum with carbon material reinforcement (0.5 wt.% and 1 wt.% of carbon nanotubes Nanocyl NC 7000 and 0.5 wt.% of

#### **4. Discussion**

Visual and metallographic tests of the flame-sprayed aluminum and aluminum with carbon material reinforcement (0.5 wt.% and 1 wt.% of carbon nanotubes Nanocyl NC 7000 and 0.5 wt.% of carburite) have shown that by using proper parameters of the process it is possible to receive coatings with acceptable quality level, characterized by proper adhesion to the substrate, lack of delamination and even thickness over the entire surface. The outer surface of the coatings was characterized by a slight roughness, lack of porosity and cracks (Figure 2). During the flame-spraying process, carbon material particles added to aluminum powder did not oxidize completely in the oxyacetylene flame. Carbon nanotubes (melting point 4526 ◦C [43]) and carburite (melting point 3550 ◦C [44]) in the oxyacetylene flame has formed with aluminum Al-Cx type agglomerates, which due to the large volume and lower heat source temperature than other thermal spraying methods (oxyacetylene flame temperature 3160 ◦C [45]) migrated in large quantities to the coatings. Partially melted and partially only plasticized in a gas flame, Al-Cx agglomerates collided with the substrate at high speed, (Figure 1b) and in this way formed a fine-grained coating structure. Powder flame spraying process (PFS) in comparison with, for example, plasma spraying, increases the probability of stopping carburite and carbon nanotubes (CNT) in flame sprayed composite coating with aluminum matrix. Presence of carbon materials in aluminum powder flame-sprayed coatings is initially confirmed by metallographic microscopic tests, which revealed areas carburite and carbon nanotubes on specimens (Figure 5, Al + 0.5% CNT). Presence of carbon materials can be observed on the entire cross-section of the coating, also at the outer surface. Inside the Al-Cx composite coatings, no cracks were found, only the presence of individual cavities. The tests made using scanning electron microscope have shown presence of some areas consisting small carbon materials inclusions. These were observed in the all-aluminum coatings with carbon material reinforcement. For the coating with 0.5 wt.% CNT, inclusion areas consisted of 33.05 wt.% C; for coating with 1 wt.% CNT, the carbon content was lower in tested area (20.25 wt.% C), while for the coating with 0.5 wt.% of carburite, carbon content was almost two times higher than in the coating with same content of CNTs and amounted to 59.76 wt.% C. In aluminum coating without carbon material addition, carbon and oxygen were found, (Figure 6). A small amount of carbon in the aluminum coating may be caused by the ease of thermal decomposition of acetylene in the gas flame and the physicochemical properties of unsaturated hydrocarbons [46]. Acetylene is dissociated into active carbon atoms (acetylene black, characterized by high purity) and hydrogen molecule. The oxygen content in the aluminum coating is the result of oxidation of the aluminum particles in the gas flame and the atmosphere. The addition of carbon materials to the aluminum powder causes the carbon to bind oxygen as a strong deoxidizer; that is why its presence was not found in composite coatings with aluminum matrix and carbon material (carburite and carbon nanotubes) reinforcement. These results should still be confirmed using more advanced research methods, e.g. XRD X-ray diffraction or Raman spectroscopy. These studies will be completed and presented in another publication.

The hardness measurements of tested coatings were proceeded using standard ISO 6507 [40]. The measurements were done both on the external surface and the cross-section of the sprayed coatings. These tests showed that using addition of 0.5 wt.% and 1 wt.% carbon nanotubes to aluminum coating (34.1 HV 0.1) caused an increase in its hardness of 8.2 HV 0.1 for the 0.5 wt.% of carbon nanotube reinforcement and by 9.5 HV 0.1 for 1 wt.% of carbon nanotube reinforcement. Addition of carburite to aluminum had not significant influence on the coating hardness (Figure 11).

Erosive wear resistance test results have shown that the addition of carbon materials to aluminum powder does not increase the erosive wear resistance of flame-sprayed coatings. During these tests, the aluminum coatings with carbon nanotubes had worn out by erosion with large and small angles of erodent incidence more than aluminum coatings with carburite and much more than aluminum coatings without carbon materials reinforcement. It was observed that for all tested coatings erosive wear resistance was better during using smaller angle of erodent incidence (Table 4).

The best metal-mineral type wear resistance had the aluminum coating with carburite. The wear resistance of this coating was 19% higher than pure aluminum coating. The aluminum coatings with addition of 0.5 wt.% and 1 wt.% of carbon nanotubes in comparison to pure aluminum coating had better relative wear resistance by 10% and 11% (Table 5). The cause of decreasing wear using aluminum coatings with carbon material addition was increased glide of ceramic abrasive particles on metal.

Based on the conducted study and the obtained results, it can be concluded that it is possible to introduce carbon particles in the form of carbon nanotubes (CNT) and also carburite into the aluminum matrix by means of flame spraying method. The flame spraying is an effective and cheaper alternative to the technology of laser surface treatment of metals. Properly selected parameters of the flame spraying process allow to preserve the properties of particles of carbon materials, their even distribution in the coating, proper bonding with the matrix and prevent the effects of their thermal degradation. The produced composite is characterized by a low friction coefficient. The tribological characteristics of produced test coating of aluminum reinforced by carbon particles in the form of carbon nanotubes and carburite show that the coatings can be classified as sliding materials. Additionally, the coatings are characterized by high wear resistance. The obtained result should be considered as a preliminary information on a new group of materials, which can find application in the automotive industry. They are the basis for the design and optimization of friction materials operated at elevated temperatures (e.g. pistons, engine blocks), systems subject to intensive wear (e.g. brake discs, cylinders), as well as in propulsion systems (e.g. bearings), providing low friction coefficient and also high ability for absorption of vibration. Further research should be focused on the investigation of the effect of doping the aluminum matrix with carbon nanotubes (CNT) on the wear mechanisms, change of microstructure of the counter-specimen and also tests which will allow to determine the tribological characteristics of the materials at elevated temperatures.

### **5. Conclusions**

The analysis carried out comparing properties of flame-sprayed EN AW 1000 aluminum coatings and aluminum matrix coatings with carbon materials reinforcement (0.5 wt.% and 1 wt.% of Nanocyl NC 7000 carbon nanotubes and 0.5 wt.% of carburite) resulted in the following conclusions:

**Funding:** This research was financed from the own resources of the Silesian University of Technology. **Conflicts of Interest:** The author declare no conflict of interest.


**1. Introduction**

The COVID-19 pandemic appeared in China in the fall of 2019 and has since spread worldwide [1]. In February 2021, when the third wave occurred, the virus had infected hundreds of millions of people and millions had died [2,3]. Moreover, numerous severe cases led to hospitalizations, putting the healthcare system and professionals under heavy pressure over a prolonged period of time [4].

Nurses have been among the most sought-after healthcare workers during the pandemic. During the different waves of COVID-19, they faced death, uncertainty, and work overload every day [4]. Such circumstances are likely to elicit stress, depressive symptoms, insomnia, and anxiety [5,6]. In turn, stress can lead to cognitive overload that can result in errors at work [7,8] and feelings of a lower quality of life (QoL), which is a strong correlate of nurses' engagement in dire contexts [9–11]. QoL is defined by the World Health Organization as 'an individual's perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards, and concerns [12]. This definition highlights the multi-dimensionality of the QoL concept that articulates four broad domains: physical health, psychological wellbeing,

**Citation:** Jubin, J.; Delmas, P.; Gilles, I.; Oulevey Bachmann, A.; Ortoleva Bucher, C. Protective Factors and Coping Styles Associated with Quality of Life during the COVID-19 Pandemic: A Comparison of Hospital or Care Institution and Private Practice Nurses. *IJERPH* **2022**, *19*, 7112. https://doi.org/10.3390/ ijerph19127112

Academic Editors: V. K. Kumar, Jasmin Tahmaseb-McConatha andPaulB.Tchounwou

Received: 13 April 2022 Accepted: 8 June 2022 Published: 10 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

social relationships, and environment. During the COVID-19 crisis, QoL was found to be associated with the use of adaptive strategies, particularly, resilience and coping [13,14]. QoL is thus a key indicator of an individual's well-being or life experiences during crises and it allows us to understand how nurses adapt to crises [15].

Nurses' QoL has been extensively explored during the COVID-19 pandemic [16]. However, most researchers have adopted a 'pathogenic' perspective [17], focusing on risk factors that impair health [18,19]. Such a pathogenic approach is limited because it tends to categorize individuals as sick or healthy and considers that disease prevention consists solely in avoiding or minimizing risk factors [20]. Hence, this approach does not necessarily provide nurses with tools to anticipate or cope with crises such as a pandemic. Thus, identifying protective factors seems to be a more efficient strategy to allow crisis anticipation and provide nurses with actionable resources, particularly when the sources of stress cannot be avoided. Thus, in the present study, we adopted a salutogenic approach, which aims at identifying factors that protect one's QoL and health [17]. In the context of the COVID-19 pandemic and its duration, investigating which protective factors might reinforce nurses' ability to deal with stress is of paramount importance. Salutogenicoriented studies could help us understand how to maintain or improve nurses' QoL and health. Protective factors are relevant to guiding the development of interventions aimed at educating nurses to help them cope with prolonged a crisis [17,21,22].

Nevertheless, investigators have taken little interest in identifying protective factors. We identified three key resources to help nurses cope with stress during the pandemic. First is resilience, which reflects the ability of an individual to face the difficulties of life and bounce back after a traumatic event [23,24]. Resilience has been found to mediate the association between sources of stress and psychological distress during COVID-19, and several studies recommend training nurses to become resilient to prepare them for crises [5,25,26]. The second is social support, which measures the psychological and material support provided by an individual's entourage [27,28]. Lastly, the third, is coping strategies that individuals use to react and adapt to stressful situations [29]. Maladaptive coping during the pandemic has been shown to be associated with poor outcomes [6]. On the other hand, several studies point to the conjoint effect of resilience, social support, and coping strategies [26]. For example, resilience combined with other adaptive coping strategies has been shown to help manage stress and sustain personal growth [30]. To our knowledge, no study has explored the role of these factors in maintaining nurses' QoL. Therefore, the main objective of this study was to identify which protective factors were most helpful in maintaining the nurses' QoL during the pandemic in France.

Studying the protective role of resilience, social support, and coping strategies is important in France because nurses can work as employees at hospitals or care institutions (NHCIs) (e.g., retirement homes, care homes) or have a private independent practice (NPPs) where they provide care prescribed by a physician at patients' homes [31]. Both categories of nurses had to adapt to multiple sources of stress during the pandemic. A survey of French NPPs in 2020 found that around a third of the respondents did not have sufficient medical supplies to do their work, and half of them reported having to reorganize their work schedules and feeling stressed by their work during the pandemic [32]. However, to our knowledge, no study has investigated the similarities and differences in how these two groups reacted to the pandemic. Such knowledge would allow us to provide nurses with the necessary support to help them adapt to the specific sources of stress in their practice during crisis times. Thus, the second objective of the current study was to investigate the role of different protective factors in determining QoL specific to the two types of practice (NHCI and NPP) and across both groups.

#### **2. Materials and Methods**

#### *2.1. Design and Population*

This cross-sectional survey was conducted in France between February and March 2021 during the third COVID-19 wave. An invitation to participate in the study accompanied by a link to the online self-administered questionnaire was sent to all registered nurses through the Ordre National des Infirmiers (French National Order of Nurses), followed by three weekly reminders. About 400,000 nurses are registered at the Ordre National des Infirmiers. Participation was anonymous and voluntary. The questionnaire was developed with Sphinx iQ2 v<IP_ADDRESS>. The data used in this study were part of a larger study focused on nurses' QoL and health-protecting factors in European countries [33].


**4.3 Electrospinning of composite nanofibers**

The prepared polymer spinning solution or sol–gel solution was immediately loaded in plastic syringe of 5 ml capacity and a blunt ended 20-gauge stainless steel needle fitted in it. The nozzle equipped syringe was attached to the syringe pump. The positive electrode of high voltage power supply capable of generating DC voltages up to 40 kV attached with the needle whereas negative electrode was connected to the aluminum foil covered collecting plate. The ground electrode of the power supply was attached to a piece of flat aluminum foil used as the collector plate [57, 58]. The needle tip to collector distance was 10 cm to collect the nanofibers. The ES process was conducted in ambient air at room temperature (25 ± 2°C) having relative humidity of 65 ± 5%. A solution feeding rate was set to 1 ml/h with the help of a syringe pump. The steady deposition of nanofibers on the collector plate was ensured by frequent cleaning of nozzle clogging intermittently. The experiment was conducted by applying positive high voltage of 18 kV across the needle and the collector plate. At such

#### **Figure 3.**

*Process flowchart for the preparation of nanofibers by electrospinning method.*

environmental condition fluid jet ejected continuously from the nozzle and accelerated jet moved towards the collector plate. During this movement of jet the solvent get evaporated leaving ultra thin fibers on the collector plate. The ES process continued until all the solution in the syringe was exhausted. The complete sequence of spinning solution preparation and nanofiber synthesis, its calcination and final morphological feature are schematically represented in **Figure 3**.


**1. Introduction**

*Candida albicans* is the most important opportunistic commensal yeast that asymptomatically colonizes the skin, oral cavity, gastrointestinal and genitourinary tracts in healthy people. However, it can cause superficial and invasive infections, especially in immunocompromised individuals [1–3]. Actually, invasive infections due to *Candida* species are considered among the main causes of morbidity and mortality in hospitalized patients. Although there are at least 15 *Candida* species related to human disease, more than 90% of the invasive diseases are related to *C. albicans*, *Candida glabrata*, *Candida tropicalis*, *Candida parapsilosis*, and *Candida krusei* [4–6]. *C. albicans* infections is considered the fourth most common

#### **Figure 1.**

*Mechanisms of action of main antifungals families in the fungal cell. (a) Azoles disrupt the ergosterol synthesis by inhibiting the enzyme 14-α-lanosterol demethylase (ERG11) involved in the transformation of lanosterol into ergosterol in the endoplasmic reticulum. (b) Polyenes disrupt the cell membrane by binding to ergosterol resulting in pore formation. (c) Echinocandins inhibit 1,3-*β*-d-glucan synthase (FKS ½) which causes disruption of the cell wall.*

opportunistic infection in hospitals. Invasive candidiasis (IC) is fatal in about 42% of the reported cases, despite the use of antifungal therapies [7, 8].

Nowadays, the most widely used antifungal drugs for IC include: A) azoles, for instance fluconazole (FLZ), itraconazole (ITC), voriconazole, posaconazole, isavuconazole; B) polyenes such as amphotericin B (AMB); C) echinocandins like caspofungin, micafungin, and anidulafungin [9–11].

These antifungal compounds act on different parts of the fungal cell (**Figure 1**). Azoles interrupt the ergosterol biosynthesis, the main component of the fungal membranes [10, 12, 13]. Polyenes such as AMB interact with ergosterol making pores in the cell membrane [10, 12–14]; while echinocandins act blocking the synthesis of β-d-glucan located in the fungal cell wall [13, 15]. The gradual risk increment for Candida infection and the greater use of antifungal agents has increased resistance towards *Candida* spp. Pharmacological failures in *Candida* spp. treatments have drawn attention to the problem of resistance to antifungals and their molecular mechanisms. *C. albicans* inherently is susceptible to azoles, polyenes, and echinocandins. Mono-resistance to azoles or echinocandins has been reported, as well as combined resistance to azoles and amphotericin, but resistance to multiple compounds that covers all three drug classes is a rare phenomenon and few cases have been reported in *C. albicans* [10, 12, 16].

The following chapter offers an overview of the main genetic mechanisms contributing to the antifungal resistance in *C. albicans*, besides giving an approach for alternative-compounds proposed against their infection.

### **2. Molecular mechanisms of antifungal resistance**

#### **2.1 Azoles**

Fungi cell membrane is mainly integrated by ergosterol, a sterol contributing to several cellular functions, besides modulating membrane fluidity and the structure and function of membrane proteins. The azoles mechanism of action is to inhibit

#### *Molecular Mechanisms of Resistance to Antifungals in* Candida albicans *DOI: http://dx.doi.org/10.5772/intechopen.96346*

14α-lanosterol demethylase, encoded by the *ERG11* gene, which converts lanosterol to ergosterol in the cell membrane (**Figure 1**). This enzyme contains an iron protoporphyrin unit in its active site. Azoles bind to iron causing the blockage of the ergosterol biosynthetic pathway [17–19]. The interruption of ergosterol synthesis allows the accumulation of 14α-methyl sterols, which alters the membrane's stability, permeability, and the action of the enzymes bound to it [20].

The evolution of antimicrobial agent's resistance is common, as there are many microbes able to develop strategies against drugs action. The incremented azoles resistance is mainly a result of their fungistatic rather than fungicidal nature [17–19]. The mechanisms of resistance to azole antifungal agents have been elucidated in *Candida* spp. species and can be classified mainly as: 1) changes in cell wall or in plasma membrane, leading to poor drug absorption; 2) alterations in the affinity of the target drug (i.e. *ERG11* gene), due to a site mutation or its overexpression; 3) drug efflux mediated by membrane transporter proteins belonging to the transporters of the ATP-binding cassette (ABC), namely CDR1 and CDR2 or the transporter of the major facilitator superfamily (MFS), CaMDR1; 4) biofilm formation [18–21]. Although the resistance described in *C. albicans* strains is usually a combination of the mechanisms mentioned above (**Figure 2**) [16].

## *2.1.1 Mutations of the ERG11 target enzyme*

Mutations in the *C. albicans ERG11* gene reduce the affinity for fluconazole and have a moderate effect on posaconazole [17–19]. Several point mutations have been identified in the *ERG11* gene. In resistant strains, there are more than 140 substitutions reported, most of them have a functional additive effect. Two of the most common alterations in *C. albicans* (R467K and G464S), are located near the hemebinding site [20]. Other substitutions related to resistance are A114S, Y132H, Y132F, K143R, Y257H, and K143Q, which contribute to a significant increased resistance (more than four times) to fluconazole and voriconazole [22].

Some clinical isolates share common mutations with environmental azoleresistant strains, suggesting that some azole-resistant clinical isolates could have their origin in the environment [23]. This resistance appears to be driven by the

#### **Figure 2.**

*Schematic overview of the main mechanisms of drug resistance against azoles, AMB, and echinocandins adopted by* Candida albicans*. (a) Alteration of the enzyme target (azoles and echinocandins), (b) overexpression of drug efflux proteins (azoles), (c) Reduction of sterols in the plasma membrane (AMB), (d) increased stress tolerance and altered the fungal cell wall (echinocandins and AMB).*

agricultural use of azoles. In patients without azoles treatment, resistance has been identified derived from the environment. These cases involved a Cyp51A substitution at position 98 (from leucine to histidine), and a 34 base tandem repeat (TR) in the cyp51A promoter, leading to overexpression. Both changes are necessary to confer resistance. In particular, these resistant isolates can be crossed with susceptible strains, suggesting that resistance could be transferred through the sexual cycle. Strains with these alterations have emerged throughout Europe and beyond. Additionally, a new environmentally selected resistance mutation (TR46, Y121F, T289A) was reported among patients in the Netherlands [20].

#### *2.1.2 Dysregulation of the target enzyme ERG11*

One way to decrease the drug effective concentration is the overexpression of *ERG11* [17]. This overexpression is common among azole-resistant *C. albicans* clinical isolates. This contributes directly to resistance, since an increase in the target requires more drug for inhibition, reducing susceptibility [19]. *ERG11* overexpression arises either from genetic dosing through gene duplication or from positive regulation of the gene by trans-acting factors [23]. Multiple mechanisms explain the constitutive overexpression of *ERG11* in azole-resistant clinical strains. First, amplification of the *ERG11* gene can occur by the formation of an isochromosome with two copies of the left arm of chromosome 5 [i (5 L)], in which *ERG11* resides, or by duplication of the entire chromosome. Second, the activation of mutations in the gene encoding the transcription factor Upc2 positively regulates most of the ergosterol biosynthesis genes [18, 20].

### *2.1.3 Alteration of the ergosterol biosynthesis pathway (point mutations in ERG genes)*

Brief exposures of two to three hours to azoles cause transient upregulation of the *ERG* gene family in *C. albicans.* These data suggest a common regulation of ergosterol biosynthetic pathway in the presence of inhibitors. Longer *in vitro* exposures to azoles (minimum 24 h) leads to constitutive up-regulation of the *ERG* genes decreasing drug susceptibility [23].

Modification of the metabolic pathway can be effective at different points, as example, alteration of the last steps of biosynthesis through the inactivation of the *ERG3* gene results in no toxic methylated sterols production, leading to azole crossresistance. Furthermore, mutations in non-essential genes of this pathway (*ERG3, ERG6, ERG24,* and *ERG2*) also lead to a decrease, or even a total absence, of ergosterol in the plasma membrane [17]. Lanosterol demethylase inactivity or defectiveness due to azoles induce ergosterol depletion and toxic 14α-methyl-3,6-diol sterols accumulation. The presence of 14α-methyl sterols can modify the function and fluidity of the plasma membrane [21]. The additive mutation in the *ERG3* gene prevents the formation of this toxic product from 14α-methylfecosterol and leads to the accumulation of non-toxic sterols (Mishra, 2007; Shukla, 2016). Although this mechanism is not the most frequent one, it has been identified in several clinical isolates of *C. albicans* [23]. Mutations in *ERG3* are sufficient to induce azole resistance in *Candida* spp., but they are rarely associated with high resistance [20].

Four clinically isolated *C. albicans* erg3 mutants (CA12, CA488, CA490, and CA108) were reported as resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under CLSI test conditions. Importantly, CA12 and CA108 retained an azole-resistant phenotype even when tested in the presence of FK506, a multi-drug flux inhibitor. In contrast, CA488, CA490, along with three isolates (CA6, CA14, and CA177, in which ergosterol comprised more than 80% of the total sterol

#### *Molecular Mechanisms of Resistance to Antifungals in* Candida albicans *DOI: http://dx.doi.org/10.5772/intechopen.96346*

fraction and ergosta 7,22-dienol was undetectable) exhibited azole sensitive phenotypes in the inhibitor FK50 presence. CA108 mutant strain contains multiple amino acid substitutions in ERG3, but only a single conserved polymorphism (E266D) in sterol 14α-demethylase (ERG11). CA12 contains a substitution (W332R) in ERG3 and no residue changes in ERG11. Furthermore, CA488 and CA490 were found to harbour multiple residue changes in both ERG3 and ERG11 [24]. Furthermore, the residue 193 in ERG3 was found to play an important role in azole resistance [25].