**14. Antibiotics**

The use of antibiotics is recommeded with preterm premature rupture of membranes (PPROM) based on ORACLE trial and chochrane review which proved that they reduce the time to delivery and the incidence of chorioamnionitis. They also decrease the ioccurance of neonatal sepsis and the need for neonatal surfactant and oxygen therapy. On the other hand; the ORACLE trial did not recommed its use in preterm labour without premature rupture of membranes as there was no difference in the neonatal outcomes [1, 20, 21].

It is also concluded that erythromycin is a better choice compared to coamoxiclav in women with preterm labour associated with premature rupture of membranes due to increased risk of necrotizing enetrocoilitis with the use of co-amoxiclav [20, 21].


**7. References**

44 Management of Organic Waste

Fig. 13. Changes in (a) protease activity, and (b) cellulase activity of grape marc after being processed by the epigeic earthworm species *Eisenia andrei* during the active phase of vermicomposting. Values are means ± SE. Control is the grape marc incubated without

Detritivorous earthworms interact intensively with microorganisms during vermicomposting, thus accelerating the stabilization of organic matter and greatly modifying its physical and biochemical properties. Digestion of the ingested material is the first step in earthworm-microorganism interactions. Passage of organic material through the

earthworms.

**5. Conclusions**

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**3**

**The Sanitation of Animal Waste**

Animal production pose potential hazards of environmental contamination with pathogenic microorganisms. These are particularly related to a subsequent storage processing and utilization of animal organic wastes (manure, fertilizer, wastewater, sludges etc.). A major source of pathogenic microorganisms in the environment are excrements from clinically and

Handling, storage, treatment and use of different forms of animals excrements entails two principle problems: epizootological or epidemiological and hygienical. Solid excrements contain high numbers of common intestinal microflora (*E. coli*, faecal streptococci, lactobacilli etc.), bacteria that are pathogenic also for man (salmonellae, mycobacteria, listeriae etc.), protozoa (*Isospora* spp., *Balantidium coli*) and eggs or larvae of enteronematodes (*Ascaris suum, Oesophagostomum* sp., *Trichuris suis* etc.) (Lauková et al.,

The parasitic propagative stages, mainly endoparasitic protozoa and helminths develop mostly outside their host´s organism. *A. suum* eggs are hygienically the most problematic ones. They are amongst the helminth eggs most resistant to environmental factors and may survive in the nature for many years, therefore, they tend to accumulate in the environment (soil, water) and serve as an infectious entity for both man and animals (Papajová and Juriš, 2009). The cell wall of *A suum* egg is enveloped with an outer layer formed by acid polysaccharides and proteins, central layer consisting of proteins (25%) and lipids (75%, particularly alpha glycosides). Thus this resistant cell wall protects eggs against effects of

Regarding the spread of helminthoses, domestic animals (dogs, cats) are also of great importance because they live in a close contact with man. Infection and way of transmission of the disease depends on the way of breeding and on the breeding environment where the animal occurs. An important factor of the risk of infection transmission is also possibility of animal to move outside its housing (yard, move in nature), or the use of a dog (hunting or social). The most frequent way of transmission of parasitic diseases is through the contact (free-living animals with a domestic ones), or through contaminated environment with

**1. Introduction**

subclinically infected farm animals.

2000; Krupicer et al., 2000).

chemicals and drying (Eckert, 1992).

**Using Anaerobic Stabilization**

Ingrid Papajová and Peter Juriš

*Institute of Parasitology of the Slovak Academy of Sciences*

*Slovak Republic*

Microorganisms and Depends on the Dose of Application of Pig Slurry. *Science of the Total Environment*, vol. 407, No. 20, pp. 5411-5416, ISSN 0048-9697.


**About the Editors**

**Shigemi Kagawa** obtained his BA in Civil Engineering, MA in Information Sciences, and Ph.D. from Tohoku University, Japan, in 2001. Since 2020, he is a distinguished professor at Kyushu University. He is a board member of Ec*onomic Systems Research* (Journal of the International Input-Output Association) and the *Annals of Regional Science*. He was awarded Leontief memorial prize and Sir Richard Stone Prize for his work on environmental input-output analysis from the International Input-Output Association.).

**Hidemichi Fujii** is an associate professor at the Faculty of Economics at Kyushu University. He has been awarded several national research grants on topics such as environmental innovation and energy economics. His work has appeared in academic journals including *Global Environmental Change*, *Energy Economics*, *Business Strategy and the Environment*, *Technological Forecasting & Social Change*, and *Energies*.


3. Conclusions

The primary motivation of this study was to investigate, analyse, characterise and compare laser and mechanical forming processes. The study focussed on the main parameters that influence the bending of plates and their effect on the microstructure and mechanical properties. New theories and discoveries are discussed in the context of contribution to the subject and body of knowledge which is wide and immense in scope. Theories and conclusions are as follows:

### Microstructure and Mechanical Properties of Laser and Mechanically Formed Commercially… DOI: http://dx.doi.org/10.5772/intechopen.81807

The use of thinner gauge material: The study has come up with a new application for the laser formed titanium. Laser formed titanium plates become extremely hard and could be used in the defence industry for bullet-proof body vests and applied on armoured vehicles. Titanium is light in weight and coupled with a hardened surface resulting from laser forming could be a viable solution. Current armoured vehicles are heavy and slow due to the materials used, and venturing into materials like titanium could be a breakthrough to the defence industry. The laser forming process has the ability to customise the mechanical properties of any material, and therefore with this possibility thinner gauge material could be used for the benefit of this industry.

The control of the radius of curvature: Controlling the radius of curvature using the laser forming process is complex and results in uncontrollable bending of the material. Magee et al. saw a great potential for accuracy and controllability on the amount of forming with the laser, but the current study contradicts what he thought possible with the process. Titanium has proved its unpredictability in this study resulting in no proper control of the radius of curvature as envisaged.

The line energy: This is the fraction of the laser power and traverse speed. According to Magee there is a critical energy input below which no plastic straining occurring in each experiment. The study agrees with Magee on the fact that a higher line energy results in more pronounced bending of the material. Maintaining a constant line energy does not result in same bending of plate specimens from the irradiated batch of plates. An increase in laser power increases the line energy and thermal gradient which all determine the extent of bending in titanium. The use of higher line energies compromises fatigue properties of titanium as there is no proper control of temperature, and therefore precise thermo-mechanical control is needed for the success of this process.

The industrial use of laser forming: Contrary to what was envisaged on initiating this study, the laser forming process does not pose a challenge to current popular forming methods. At the moment the best process for forming is mechanical forming due to the ease with which any desired shape can be formed in minimal time. Laser forming is much slower than mechanical forming, and changes brought by the laser forming process could be undesirable to other industrial applications.

The thermal gradient: The low thermal conductivity of titanium means higher thermal gradients are needed for pronounced bending of CP grade 2 titanium. Higher thermal gradients result in higher residual stresses in the material and a complete change in the physical properties of the material. Changes in physical properties could be desirable or less desirable depending on industrial application.

Surface hardening: The laser forming process resulted in surface hardening of CP grade 2 titanium plates. This had a negative effect on the fatigue life of specimens, as there was a reduction in fatigue life. This is contrary to the findings by Konstantino and Altus [11] who reported improvements in fatigue life of Ti-6Al-4 V which behaves in the same manner as CP grade 2 titanium plates. The improvement in fatigue life was achieved by laser heating based on reduced fraction of α (alpha) in the microstructure and a reduction in grain size. In this study there was an increase in grain size as a result of laser heating which completely changed the granular structure of the material. A significant microstructural refinement was observed during this study resulting in the formation of α-martensite. Hardness values are dependent on the line energy generated during the laser forming of titanium. The higher the line energy, the higher will the hardness be for CP grade 2 titanium plates. The laser-irradiated surface hardens as a result of the laser forming process making it difficult to polish and prepare plates for residual stress measurements. In laser formed CP grade 2 titanium plates, the hardness changes with specimen depth as a result of the effects of thermal energy from the laser, which is the heat source.

life as they changed the location of the fracture line. The maximum principal stress at a power of 3000 W is obtained at a depth of 2 mm. The changes in residual stress and strain are closer to the surface of the irradiated plate. The laser forming process increases the hardness of titanium. Residual stresses in this study are a result of interactions between time, temperature and the material. These factors played a major role in the resulting residual stress layout on all laser formed plates. The effect of the thermal gradient is evident when these plates are compared with plates not affected by thermal energy. The highest temperature gradient was obtained at a power of 3500 W. The thermal gradient became a deciding factor in microstructural layout. Even though the line energy was the same from a power of 2500 W up to a power of 3500 W, the effects on the microstructure were not uniform. This led to the conclusion that the thermal gradient is the most influential factor in laser

Titanium Alloys - Novel Aspects of Their Manufacturing and Processing

The maximum stress obtained at 3.5 kW was the second highest at 181.9 MPa (T) and a minimum stress of 1.4 MPa (T). The hardness of plates was equivalent to the parent material, but this is where similarities end. The difference in stress was 185 MPa on the laser-processed plates. This difference in stress is related to changes in hardness of titanium as a result of laser forming process. The differences in temperature between 3000 W and 3500 W played no role in influencing minimum and maximum residual stress. The optimum settings for a line energy of 90 kJ/m

The primary motivation of this study was to investigate, analyse, characterise and compare laser and mechanical forming processes. The study focussed on the main parameters that influence the bending of plates and their effect on the microstructure and mechanical properties. New theories and discoveries are discussed in the context of contribution to the subject and body of knowledge which is wide and

immense in scope. Theories and conclusions are as follows:

forming [8] (Figure 15).

Figure 15.

Relieved stress (3.5 kW).

are at a power of 2.5 kW [8].

3. Conclusions

42

The residual stress: In all the forming processes analysed, changes in residual stress are greatly influenced by process specifications. In laser forming however these changes are dependent on the process parameters used, as these differ with each laser power. Thermal gradient influences the development of residual stresses. In mechanical forming changes in residual stress are determined by the complexity of the formed shape. It was envisaged at the beginning of the study that residual stress would be enhanced but the laser forming process made undesirable changes to the underlying residual stress distribution. According to Norton [5] good design requires that an engineer try to tailor the residual stresses to a minimum, not create negative effects on the strength and preferably to create positive effects. Fatigue failure is a tensile residual stress phenomenon. The laser forming process resulted in increased tensile residual stress in the specimens due to higher line energies. The use of lower line energies on CP grade 2 titanium results in no bending of the material, and therefore high tensile residual stress remains part of the process if there is forming to be done. The parent material and mechanically formed plates had low residual stress values which was an advantage during fatigue testing as these had a higher fatigue life.

Microstructure: The mechanical forming process has a minimal influence on the microstructure of CP grade 2 titanium plate specimens compared to the effects of laser forming. The laser forming process results in changes in grain size as thermal energy is increased. Changes in the microstructure influenced the mechanical properties of CP grade 2 titanium plates.

Beam interaction time: The beam interaction time is significant in the analysis of resulting mechanical properties as a result of the laser forming process. The time taken to heat up an area influences the mechanical properties and the microstructure of plate samples.

Heat flux: The heat flux is significant in changes observed with titanium plates, and each laser power setting evaluated had a different reading, resulting in varying microstructures on the plates evaluated. The depth of laser penetration depends on the amount of line energy generated.

Laser power: An increase in laser power leads to the oxidation of the passive layer on CP grade 2 titanium plates as a result of the concentrated thermal energy generated by the laser.

Forming parameters: The changing of forming parameters in the laser forming of CP grade 2 titanium succeeded in obtaining optimum operating parameters (in the case of this research, a power of 2.5 kW, a line energy of 90 kJ/m and a scanning velocity of 1.67 m/min) for titanium.

Author details

45

Kadephi Vuyolwethu Mjali1

Port Elizabeth, South Africa

Cape Town, Western Cape, South Africa

provided the original work is properly cited.

\* and Annelize Botes2,3

Microstructure and Mechanical Properties of Laser and Mechanically Formed Commercially…

DOI: http://dx.doi.org/10.5772/intechopen.81807

1 iThemba LABS—Laboratory for Accelerator Based Sciences, Somerset West,

2 Council for Scientific and Industrial Research (CSIR), Pretoria, South Africa

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

3 Department of Mechanical Engineering, Nelson Mandela University,

\*Address all correspondence to<EMAIL_ADDRESS>
Plates: Laser formed titanium plates should not be used in applications requiring prolonged fatigue life. The process is only beneficial in applications where hardness is a priority without a need for high fatigue life, and perhaps the process could be beneficial in military defence applications. Laser formed titanium plates do not bend to the same radius of curvature as proved in this study.

Process control: The process needs precision control of processing parameters as they play a major role in the final microstructural layout and mechanical properties. For good fatigue properties, thermo-mechanical/laser processing of titanium needs to be conducted using lower line energies but then these do not bend the material.

### Acknowledgements

Nelson Mandela University (NMMU) for financial assistance and laboratory facilities. Mr. Victor Ngea-Njoume for technical assistance.

Microstructure and Mechanical Properties of Laser and Mechanically Formed Commercially… DOI: http://dx.doi.org/10.5772/intechopen.81807


**The Microbiota of Edam Cheeses Determined by Cultivation and High-Throughput Sequencing of the 16S rRNA Amplicon**

### **Beata Nalepa 1,\*, Sławomir Ciesielski 2 and Marek Aljewicz 3**

Received: 4 May 2020; Accepted: 10 June 2020; Published: 12 June 2020

**Abstract:** The aim of this study was to evaluate the microbiome of industrially produced ripened Edam cheeses by next-generation sequencing. The samples for analyses were collected in spring and autumn. Spring samples were characterized by significantly higher *Lactococcus* and *Bacillus* counts and lower counts of Enterobacteriaceae, *Enterococcus*, and yeasts than autumn samples. The predominant microorganisms identified by the Illumina high-throughput sequencing technology belonged to four phyla: Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes. The dominant species were starter culture bacteria. *Lactobacillus rhamnosus*, *Lactobacillus kefiri*, *Lactobacillus kefiranofaciens*, *Lactobacillus casei*, *Streptococcus thermophilus*, and *Bifidobacterium* had the highest share of microbial cheese communities. The number of γ-Proteobacteria reads was higher in autumn cheese samples. A high number of reads was also noted in the genus *Clostridium*. The counts of spore-forming bacteria of the genus *Bacillus* were higher in cheeses produced in spring. The study revealed highly similar relationships between the analyzed production periods. The present results contribute to the existing knowledge of cheese microbiota, and they can be used to improve and modify production processes based on the composition of microbial communities, as well as to improve the quality of the final product.

**Keywords:** microbiota; seasonal variations; high-throughput sequencing; NGS

### **1. Introduction**

The quality of fermented foods is determined mostly by microbial genera and species colonizing food products. The above applies to all processing stages, from the raw material to the final product [1–3]. Microorganisms are ubiquitous in the farm environment (soil, water, feed, natural fertilizers, and premises). They are also present on animal skin, in animal digestive tracts, and in animal secretions (such as milk), and they colonize the surface of fodder plants. Microorganisms are found on the surface of processing lines, devices, machines, conveyor belts, pipelines, packaging, and auxiliary raw materials. They are added to products as starter cultures, and they are present on the hands of dairy plant employees [4,5]. Microorganisms spread from the above sources to raw materials and end products, and they influence the attributes of fermented foods. Therefore, cheese microbiota constitute one of the critical factors during cheese ripening and the formation of the sensory properties of cheese. The formation of sensory attributes is a complex biochemical process where environmental and process conditions, such as the heating temperature of the cheese slurry and the

time and temperature of cheese ripening, affect the composition and metabolic activity of microbiota [6]. Microbial communities colonizing fermented foods are highly complex, and they have not been thoroughly investigated to date [1–3]. Many microbial species cannot be cultured in vitro on standard microbiological media, which prevents their identification. Some microorganisms are weakened or sublethally damaged by processing operations, and they enter the viable but not culturable (VBNC) state [7]. Other microorganisms cannot be identified with the use of culture-dependent methods due to their small population size and the overwhelming effect of predominant species, such as starter culture bacteria. A knowledge of the biodiversity of bacterial communities in fermented foods is highly valuable because every microbial species, including low-abundance species, contribute specific enzymes, metabolites, bacteriocins, and genetic material to the metabiome. A better understanding of the interactions between microorganisms, their metabolites, the effects of process parameters, external factors, and disruptions in the production cycle can expand our knowledge of specific microbial communities. This knowledge can be used to develop new tools for designing the most desirable attributes of fermented foods and to precisely control the production process to achieve the desired effects. Considerable advances have been made in molecular analysis since the late 1980s, and they have led to the development of the PCR technique by Kary Mullis in 1983 [8], which supports analyses of the complex ecosystems of fermented foods. Technological progress introduced molecular biology methods to traditional food microbiology. The last decade witnessed the emergence of new methods such as next-generation sequencing (NGS) and new research fields such as metagenomics and metatranscriptomics [9]. Modern NGS techniques support the high-throughput sequencing of genes, metagenomes, and metatranscriptomes, and they deliver new insights into the diversity and metabolic properties of fermented food microbiota, including the microbiota of ripened cheese [10]. The resulting data add to current knowledge about the interactions between microorganisms, the environment, and other microorganisms, and they support the development of strategies for improving the production process and cheese quality. In recent years, metagenomic and/or metatranscriptomic analyses have been conducted on samples of regional cheeses, including water buffalo mozzarella cheese [11], Belgian Herve cheese [12], Italian Plaisentif, Mozzarella, Grana Padano and Parmigiano Reggiano cheese [13,14], Mexican Cotija cheese [15], and French Tomme d'Orchies Livarot-type cheese [16,17], as well as industrially produced Dutch-type [18] and Swiss-type cheese [19]. The microbiological quality of ripened cheese has been widely researched in the literature [20–22]. However, the formerly applied analytical methods have failed to provide comprehensive information about the microbiome of ripened cheese. Recent advances in molecular biology can be deployed to fill this knowledge gap. Therefore, the aim of this study was to determine the microbiota of Edam cheeses produced industrially in Poland with the use of the Illumina technology.

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

### *2.1. Cheese Sampling*

The study was performed on Swiss–Dutch-type (Edam) cheeses manufactured by a dairy plant in north-eastern Poland. Raw milk for cheese production was pasteurized (72.5 ◦C for 15 s) and standardized (to 3.0% fat content). Calcium chloride, a coloring agent, and rennet (Chymax, Ch. Hansen, Poland) were added, and milk was inoculated with deep-frozen cheese starters (CSK Food Enrichment, Poland). Milk was inoculated with 0.7% (v/v) of the lactic acid bacterial starter (*Lactococcus lactis* subsp. *lactis*, *Leuconostoc mesenteroides* subsp. *cremoris*) and 0.007% (*v*/*v*) of the propionic acid bacterial starter (*Propionibacterium freudenreichii* subsp. *shermanii*). After brining, cheeses were wrapped in FCC-type (Fesco Pack, Malbork, Poland) heat shrink oxygen barrier bags and cold-stored under controlled conditions. The cheeses were ripened for 10 days at 12 ◦C and a relative humidity of 85%. After 10 days, the ripening temperature was increased to 21 ◦C for 42 days. After 52 days of ripening, cheese samples were randomly collected for analysis. Three random cheese samples were collected each month. The representative cheeses were sampled in compliance with ISO 707:2008 [23]. Before the

experiment, the samples collected over one month (April or October) were grated with a Santos 2 grater (Lyon, France) and combined with the samples collected in the following month (May or November, respectively) of the same season. All cheeses were sampled over a period of 2 years. The resulting averaged sample was used for further analysis. Cheeses produced in April–May were regarded as spring samples, and cheeses produced in October–November were regarded as autumn samples.

### *2.2. Chemical Composition*

Cheese samples were analyzed in triplicate to determine their salt content by the IDF method (IDF 88; ISO 5943:2006) [24], fat content was determined by the Van Gulik method (IDF 222, ISO 3433:2008) [25], and moisture content was determined by oven drying at 102 ◦C (AOAC 926.08) [26].

### *2.3. Determination of the Counts of Selected Bacterial Groups by the Culture-Dependent Method*

The counts of bacteria of the genera *Lactobacillus*, *Lactococcus*, *Leuconostoc*, *Propionibacterium*, Enterobacteriaceae, *Enterococcus*, *Staphylococcus*, *Clostridium*, and *Bacillus*, as well as yeasts, were determined by the standard plate count method on the appropriate culture media. The applied culture media and incubation conditions are presented in Table 1.

### *2.4. DNA Isolation*

Metagenomic DNA was directly isolated from cheese samples with the use of the Genomic Mini AX FOOD kit (A@A Biotechnology, Gda ´nsk, Poland) according to the manufacturer's instructions, and it was stored at −80 ◦C until further analyses.

### *2.5. Amplicon Sequencing and Bioinformatics Analysis*

The microbial communities colonizing the analyzed samples were examined by the sequencing of the V3–V4 region of the 16S rRNA gene. The 16S rRNA gene fragment was amplified with the PCR primers recommended for the Illumina technique. The primers were developed by adding Illumina adapter overhang nucleotide sequences to the PCR primers given by Klindworth et al. [27]. Amplicons were indexed using the Nextera® XT Index Kit (Illumina, Inc. San Diego, CA, United States) according to the manufacturer's instructions. DNA was sequenced in Illumina MiSeq in the 2 × 250 paired-end mode. Demultiplexing and FASTQ file generation were performed using Miseq Reporter v.2.4. (Illumina, Inc. San Diego, CA, United States).

Sequencing results in the form of FASTQ files were uploaded to the MetaGenome Rapid Annotation Subsystems Technology (MG-RAST) server for analysis [28]. Each file underwent quality control (QC), which included quality filtering (removing sequences with ≥5 ambiguous base pairs) and length filtering (removing sequences with a length ≥2 standard deviations from the mean). The UCLUST algorithm was used to cluster the identified rRNA sequences. A representative sequence of each cluster was used for taxonomic identification based on the Greengenes reference database. Illumina metagenomic datasets are available at MG-RAST under accession numbers 4,730,840.3, 4,730,841.3, 4,730,842.3, and 4,734,160.3. Additionally, sequencing data were deposited in the Sequence Read Archive (SRA) NCBI (https://www.ncbi.nlm.nih.gov/) as a Bioproject under the accession code: PRJNA637891.

### *2.6. Statistical Analysis*

The results of chemical composition analysis and bacterial counts were verified for normal distribution and homogeneity of variance. The interactions between production seasons were determined by a one-way ANOVA. The results were statistically analyzed with the Statistica software (Statsoft; 2018, Kraków, Poland) at *p* ≤ 0.05. All data (microbiological and physicochemical parameters) are presented as means ± standard deviation.

### **3. Results and Discussion**

### *3.1. Chemical Composition*

The analyzed cheeses did not significantly differ in water content. The average water, fat, and sodium chloride content of all samples were determined at 42%, 27%, and 1.5%, respectively (Table 2). The production period had no significant effect on the composition of the cheeses. The absence of significant differences between the cheeses could be attributed to adequate standardization of the raw material and the use of more automated production lines.

\* The results are expressed as means ± standard deviation; *n* = 3. The analyses were conducted after 52 days of cheese ripening.

### *3.2. Determination of the Counts of Selected Bacterial Groups by the Culture-Dependent Method*

The abundance of the selected bacterial groups was determined by the traditional culture-dependent method. The applied culture media are presented in Table 3. The microbiological quality of cheese depends on climatic conditions, cow health, cow nutrition, lactation period, and the type and number of starters. Despite the standardization of raw materials and the automation of cheese production, even the same types of cheese sampled from various vats can differ in microbiological quality.

The addition of a starter culture was responsible for the high counts of *Lactococcus*, *Leuconostoc*, and *Propionibacterium*. The statistical analysis revealed that *Lactococcus* counts were significantly influenced by the production season (*p* ≤ 0.05), and the average *Lactococcus* counts in autumn cheeses were determined at log 6.55 cfu/g and were lower than in spring cheeses (log 7.16 cfu/g). Cheeses produced with *Lactococcus* starter cultures were characterized by high *Lactococcus* counts. In turn, high *Lactococcus* counts in mature cheeses were attributed to the availability of suitable carbon and nitrogen energy sources, including bacterial metabolites (low-molecular-weight peptides, free amino acids, citrate, RNA (ribose), and DNA (deoxyribose) produced during starter lactic acid bacteria (SLAB) lysis. Similar results have been reported by other authors [29–31]. Season had no significant effect on *Leuconostoc* and *Propionibacterium* counts, which were determined at 4.43 and 5.75 cfu/g on average, respectively (Table 3). Interestingly, *Lactobacillus* counts were around 11% (*p* > 0.05) higher in the spring cheese than in the autumn cheese samples. Due to seasonal changes in the microbiological quality of the raw material, some dairies add larger amounts of starter cultures to cheese vats to accelerate ripening and obtain cheeses with desirable sensory attributes. The presence of higher *Lactococcus* and *Propionibacterium* counts in the spring cheeses seems to confirm the above observation. During ripening, starter cultures undergo autolysis, which leads to the release of cell wall elements and nucleic acids. These compounds are used by bacteria, including *Lactobacillus* strains, as a source of carbon or nitrogen. Similarly to *Lactobacillus*, *Enterococcus* strains are also classified as non-starter lactic acid bacteria (NSLAB). The high *Enterococcus* counts in mature cheeses could be explained by the fact that *Enterococcus* spp. are highly resistant to pasteurization temperature and that milk fat exerts protective effects [32]. The counts of *Enterococcus* and Enterobacteriaceae were significantly influenced by season. The average counts of *Enterococcus* and Enterobacteriaceae reached 3.63 and 2.50 cfu/g, respectively, and were higher (log 0.50 and log 0.86 cfu/g, respectively) in cheeses produced in spring. *Enterococcus* counts were similar to those reported by other authors in Swiss-type cheese (2–3 cfu/g) [29], but they were significantly lower than in Spanish cheese (6–8 log cfu/g) [33]. The low Enterobacteriaceae counts could probably be attributed to the fermenting activity of Lactic Acid Bacteria (LAB) and the synthesis of antibacterial compounds (bacteriocins, organic acids, ethanol, and H2O2) that reduce microbial counts by increasing the permeability of bacterial cell walls [29,30]. The lower counts of *Enterococcus* and Enterobacteriaceae in autumn cheeses could also indicate that hygiene standards are strictly observed by dairy employees.

**Table 3.** Counts of selected microbial groups in Edam cheeses determined by the culture-dependent method.

\* Values are means ± standard deviation for *n* = 6, a,b Means in rows with different superscript letters are significantly different (*p* ≤ 0.05).

Yeast counts were below log 2 cfu/g in spring cheeses, and they were significantly lower in autumn cheeses (log 2.82 cfu/g). The observed yeast counts were below the values given by other authors in Swiss-type cheese (3.48 cfu/g) [29], Gouda-type cheese (3.22–50 cfu/g) [29,31], Chihuahua cheese (5 cfu/g) [34], and Serrano cheese (4–4.5 cfu/g) [30]. Similar results were reported by Sánchez-Gamboa et al. [34]; in their study, yeast counts were 50% higher in cheeses produced in autumn. Different results were reported by de Souza et al., [30]. Higher yeast counts in autumn cheeses probably resulted from the low microbiological quality of brine or cheese contamination during packaging.

Autumn cheeses are made from the milk of pasture-grazed cows, which is why *Clostridium* counts in autumn cheeses should have been lower than in spring cheeses. Despite the above, *Clostridium* counts were approximately 10% (*p* > 0.05) higher in autumn cheeses than in spring cheeses. In a study by Bermúdez et al. [35], *Clostridium* counts were also approximately 9% higher in autumn than in spring cheeses. In recent years, many milk producers have been increasing the proportions of silage in the diets of lactating cows to maximize milk yields and to allow for greater flexibility in managing cattle diets. Such practices contribute to the accumulation of *Clostridium* spores in the bovine digestive tract. Bacterial spores can be transferred to milk during milking, especially if teats are not properly cleaned [35]. Lower *Clostridium* counts in autumn cheeses probably result from higher levels of bacteriocin synthesis by LAB (mainly: *Lactococcus*, *Propionibacterium*, and *Lactobacillus*) [36].

### *3.3. Determination of the Microbiota of Edam Cheeses by High-Throughput Sequencing (HTS)*

The number of 16S rRNA sequence reads ranged from 332,280 to 436,468 in cheese samples after 52 days of ripening. The predominant microbial groups belonged to four phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes (Figure 1).

**Figure 1.** The average relative abundance (%) of the sequences identified at phylum level in samples of Edam cheese.

Eleven predominant genera were identified in the group of 32 genera and taxonomic groups that accounted for at least 0.5% of the bacterial community. They were: *Acetobacter*, *Alcaliphilus*, *Bacillus*, *Bacteroides*, *Bifidobacterium*, *Cellulomonas*, *Clostridium*, *Lactococcus*, *Lactobacillus*, *Leucobacter*, *Propionibacterium*, and one group of γ-Proteobacteria. Similar results were reported in a study of Oscypek, a traditional Polish smoked cheese made of sheep's milk [37]. The authors demonstrated the predominance of the same four phyla and identified nine identical genera and taxonomic groups in 40 operational taxonomic units (OTUs). The proportions of the remaining genera were below 0.5%, and not all genera were identified in all samples. The average bacterial identification levels were similar (75–70%) in the spring and autumn seasons. In autumn and spring samples, 25% and 30% of the sequences could not be identified, respectively. The genera represented by dozens or hundreds of reads, including Archaea and algae (Eukaryota), accounted for a large part of the taxonomic spectrum in cheese samples. A high number of DNA sequences were not classified in the remaining cheese samples (Figure 1). The above could be attributed to the presence of microorganisms that have not yet been classified or microorganisms containing degraded DNA. Fernandes et al. [38] observed that DNA was degraded to very short fragments during corn dough fermentation. The content of bacterial proteases, proteinases, and peptidases increases gradually during cheese ripening and storage. Similarly to bacterial proteases, DNA-degrading enzymes (nucleases) can also be released into the cheese matrix during the autolysis of starter and non-starter lactic acid bacteria [39], or they can be bacteriophage-derived [19].

The highest number of reads was obtained for two out of the three genera of starter bacteria: *Lactococcus* and *Propionibacterium*. In the analyzed group of the SLAB, *Lactococcus* was the predominant

genus in the phylum Firmicutes, and it accounted for 43.78% and 53.35% of the microbial communities in autumn and spring cheese samples, respectively. The second most dominant starter culture belonged to the genus *Propionibacterium*, phylum Actinobacteria. *Propionibacterium* counts were lower, and they were determined at 9.41% and 12.37% in spring and autumn cheese samples, respectively. A comparison of the number of reads for the selected bacterial genera revealed important differences, including in starter bacteria that are added in strictly controlled amounts to every cheese batch. The number of reads for the third genus of starter bacteria, *Leuconostoc*, was very low at a fraction of a percent (Figure 2 and Table S1). The analyzed cheeses were characterized by highly diverse microbiota. Starter bacteria develop in the first stages of the production process and dominate over residual microflora and the microorganisms from secondary infections. During ripening, bacterial starter cells are gradually autolyzed, which promotes the growth of non-starter bacteria that can also influence the attributes of the final product [40]. For this reason, the microbiota of ripened cheese are more diverse than those observed immediately after production or short ripening. Alessandria et al. [41] demonstrated that the microbiota of Grana-like cheese begin to diversify upon brining and undergo significant changes during two months of ripening. In the present study, starter bacteria were predominant in the Edam cheese. *Lactococcus* and *Propionibacterium* from starter cultures accounted for 56–65% of all bacteria, whereas *Leuconostoc* represented only a fraction of percent of the overall microbial community. Similar results were reported by Almeida et al. [42]; in their study, the number of sequence reads for *Leuconostoc* was also low. A predominance of starter bacteria was reported by Ceugniez et al. [16], who found that starter bacteria accounted for 85% of the microbiota in the core samples of Tomme d'Orchies cheese, and by Alessandria et al. [41], who demonstrated that *Lactobacillus* and *Streptococcus* predominated in Grana-like cheese inoculated with natural whey starters (NWS). Duru et al. [19] also found that *Lactococcus*, *Lactobacillus*, and *Propionibacterium* bacteria accounted for 80–82% of the microbiota in industrially produced Swiss cheese. However, in a study by Porcelato et al. [18], starter bacteria (*Lactococcus*, *Leuconostoc*, and *Lactobacillus*) represented more than 99% of the bacterial community in industrially produced Dutch cheese.

**Figure 2.** The average relative abundance (%) of the sequences identified at genus level in samples of Edam cheese.

The results of the SLAB and NSLAB analyses performed with the use of both methods (culture-dependent and NGS) revealed a certain regularity. When plate counts (expressed in log cfu/g) were one log unit higher in one bacterial genus than in another, the corresponding number of reads was approximately 10 times higher (Table 3 and Table S1).

Despite the high proportions of starter bacteria in cheese samples, a high number of reads was noted for the NSLAB. In the phylum Actinobacteria, the abundance of *Cellulomonas*in the microbial community was relatively high, and it ranged from 1% to 3% in all cheese samples. The genera *Bifidobacterium* and *Leucobacter* were less abundant, and their proportions were determined at 0.40%/0.94% and 0.18%/0.33%, respectively. The remaining identified genera of the phylum Actinobacteria accounted for up to 0.2% of the investigated microbial communities (Table S1). Spring cheese samples differed considerably from the remaining samples in the frequency of *Lactobacillus*, which was higher in spring samples (approximately 1.5%) (Table S1). In the genus *Lactobacillus*, the highest number of reads was obtained for *Lactobacillus rhamnosus*, *Lactobacillus kefiri*, *Lactobacillus kefiranofaciens*, and *Lactobacillus casei* (data not shown). The relative frequency of *Bifidobacterium* and *Streptococcus* was 0.6% and 0.3% higher, respectively, only in individual samples relative to the remaining cheese samples (0.08%) (data not shown). Similar results were reported in other studies of industrially produced cheeses. Porcelato et al. [18] detected *Lactobacillus* and *Streptococcus* in Dutch-type cheese, and Duru et al. [19] identified *Enterococcus* and *Streptococcus* in Swiss-type cheese.

The phylum Firmicutes also includes bacteria that cause technological defects in cheeses. In the group of contaminating bacteria, the highest number of reads was obtained for spore-forming bacteria of the genus *Clostridium*. The relative proportions of *Clostridium* were 0.97% higher in spring samples than in autumn samples. The genus *Clostridium* was represented mainly by three species: *Clostridium butyricum*, *Clostridium longisporum*, and *Clostridium tyrobutyricum*. In spring samples with the highest number of reads for the genus *Clostridium*, the predominant species was *C. tyrobutyricum*, whose abundance was around 10-fold higher in comparison with *C. butyricum* and *C. longisporum* in the remaining samples (data not shown). In the current study, next-generation sequencing supported the detection of *C. tyrobutyricum*, which could be responsible for late blowing, a cheese defect resulting from butyric acid fermentation [43,44]. This is a very valuable observation that can be used to introduce the required adjustments during cheese production, ripening, and storage. Spore-forming bacteria of the genus *Bacillus* also had a relatively high share of microbial communities, particularly in spring samples where the average frequency of *Bacillus* was 0.81% higher than in autumn samples. The proportions of spore-forming bacteria of the genus *Bacillus* were higher in spring cheeses than in autumn cheeses. The observed variations can be probably attributed to dietary factors because cows graze naturally on grass in summer and autumn. Henderson et al. [45] demonstrated that the abundance of bacterial genera in the bovine rumen changed with the cows' diet. The presence of bacteria in milk can also differ because raw milk microbiota are much more diverse and contain more environmental bacteria and microorganisms from the bovine digestive tract in summer and autumn. In winter and spring, cows are fed with hay and silage, which increases the counts of spore-forming bacteria of the genus *Bacillus* in the environment, milk, and, consequently, cheese. The autumn samples were characterized by the greatest differences in the frequency of *Enterococcus* spp. (0.06%), and they differed (*p* > 0.05) from spring samples (Table S1). The analyzed cheeses also contained trace amounts of DNA of the microorganisms colonizing the natural environment and cows. The remaining identified bacterial genera of the phylum Firmicutes accounted for up to 0.2% of the analyzed microbial communities. On rare occasions, selected genera, including *Veillonella*, *Laceyella*, *Streptococcus*, *Eubacterium*, and *Ruminococcus*, were more abundant (Table S1). Bacteria of the phylum Proteobacteria accounted for 3.26% (spring samples) and 8.69% (autumns samples) of microbial communities. They were mostly represented by members of the class γ-Proteobacteria whose DNA sequences could not be identified to the genus level. The proportion of γ-Proteobacteria ranged from 1.11% in spring samples to 7.11% in autumn samples (Figure 2 and Table S1). Among the identified genera, the predominant class was α-Proteobacteria of the genus *Acetobacter*, which accounted for 0.39–1.01% of microbial communities in

spring and autumn samples, respectively. The class γ-Proteobacteria of the genus *Acinetobacter* was also abundantly represented in spring samples at 1.56% (Table S1). The phylum Bacteroidetes accounted for 0.52–0.84% of microbial communities in the analyzed cheese samples, and it was represented mostly by the genus *Bacteroides*, whose abundance ranged from 0.15% in spring samples to 0.30% in autumn cheese samples. Other genera were sporadically noted, including *Prevotella* and *Chryseobacterium* (Table S1). Cheeses were also colonized by bacteria of the genera *Acinetobacter*, *Chryseobacterium*, and *Alkaliphylus* which are ubiquitous in soil and water, as well as *Cellulomonas*, *Eubacterium*, *Rothia*, and *Ruminococcus*, which are found in the bovine digestive tract [11,13,41]. Interestingly, the unexpected bacterial genera *Alloscardovia*, *Prevotella* and *Veillonella* from animal respiratory and digestive systems [45,46]—as well as *Laceyella*, which are found in bottom sediments, soil, and patients with farmer's lung disease [47]—were also identified at relatively high concentrations in individual samples. The DNA of *Erysipelothrix rhusiopathiae*, a bacterium responsible for swine erysipelas disease [48], was detected in a single sample in our study. The proportion of this bacterium in the sample was surprisingly high at 0.22%, which can probably be attributed to the accidental contamination of raw milk by veterinarians or farm personnel. Bokulich and Mills [49] also identified various bacterial and fungal genera that were not added as starter cultures. The presence of the DNA of unexpected bacterial genera and species provides detailed information about the quality of raw milk, and it can be used to prevent contamination of milk and the final product.

In the present study, the variations in bacterial genera colonizing the evaluated samples were very high, despite the fact that all samples were obtained from the same dairy plant, and the raw milk was produced in the same Polish region. Alessandria et al. [41] also found differences in the composition of microbial populations colonizing cheeses from the same dairy plant. They observed considerable microbial diversity of cheeses produced in spring at weekly intervals. The above could imply that minor fluctuations in the production process significantly influence the composition of cheese microbiota. Differences in microbiological quality can also result from various stages of cheese ripening, variations in the microbial composition of raw milk, environmental factors, other disruptive factors, and minor deviations from production procedures. In this study, certain seasonal changes in cheese microbiota were also noted, though only in communities of contaminating microorganisms.

### **4. Conclusions**

High levels of milk production in dairies and the combination of raw materials from different milk producers (different regions of the country) contribute to the high microbial biodiversity of cheese and the absence of significant differences in the OTU reads across production seasons. This study demonstrated that despite the application of different testing techniques, the relationships between the analyzed production periods were highly similar. Both next-generation sequencing and culture-dependent approaches are sources of bias, but they provide complementary information about the microbial ecology of cheese. The evaluated Edam cheeses were characterized by a predominance of starter bacteria and the presence of non-starter lactic acid bacteria from the microflora of the dairy plant and the cattle environment. Trace amounts of bacteria that are pathogenic for cattle were also identified. High-throughput sequencing also supported the identification of low-abundance bacteria, as well as bacteria that had not been previously detected in ripened cheeses. Advanced analytical tools developed by "omics" sciences can provide new insights into the metagenomes of fermented foods, including ripened cheeses. Metagenomic profiling has created unprecedented opportunities for analyzing cheese ecology. The identification of the genes and genomes of microbial communities in ripened cheeses supports the detection of the most desirable strains, the description of their development and transfer, and the identification of microbial contaminants and pathogens.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3417/10/12/4063/s1, Table S1: Percentages of the most abundant taxonomic groups in Edam cheese samples after 52 days of ripening.

**Author Contributions:** B.N. developed the research concept and administered the research project. B.N., S.C., and M.A. procured the materials, developed the methodology, analyzed and curated the data, performed formal

analyses, and wrote the manuscript. B.N. and M.A. acquired research funds, visualized data, and reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The project was financially supported by the Minister of Science and Higher Education under the program entitled "Regional Initiative of Excellence" for the years 2019–2022, Project No. 010/RID/2018/19, amount of funding PLN 12,000,000 and by University of Warmia and Mazury in Olsztyn (statutory research, Projects No. 17.610.015-300 and 17.610.003-110).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).


*Edited by Kaan Kirali*

Heart valves are the basic functional structure of the heart to obtain, sustain, and maintain blood circulations in the body without energy loss, allowing blood regurgitation or obstructing blood flow. There are two basic designs of heart valves: actively protected via subvalvular apparatus (atrioventricular valves) and passively strengthened (ventriculoarterial valves). The embryologic and morphologic development of heart valves is the key stage to continue normofunctional structure and physiological behavior. Fetal anomalies, embryologic defects, or non-development pathologies may damage the structure of heart valves and cause functional deterioration at different grades. In this book, we have focused on specific pathologies, which require special knowledge and treatment modalities.

Published in London, UK © 2018 IntechOpen © PublicDomainArchive / pixabay

Structural Insufficiency Anomalies in Cardiac Valves

Structural Insufficiency

Anomalies in Cardiac Valves

*Edited by Kaan Kirali*


4.2.2. WiFi

WiFi is a wireless technology used for WLAN implementation within the IEEE 802.11 standard. It provides a frequency spectrum with a data rate of 2-600 Mbps and covers up to 100 meters distance. High penetration of WiFi as the Internet infrastructure makes it a good candidate for HAN and FAN applications.


**5. Conclusions**

This MedDiet intervention induces only modest changes in adherence to the MedDiet and consequently in metabolic biomarkers related to lipid metabolism. Further research should confirm these results based on larger study samples in workplace interventions with powerful study designs.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/12/3610/s1, Figure S1: Flowchart of participants at time point 1 and 2 of the Feeding America's Bravest trial, Figure S2: Associations of unit changes in mMDS score by SD changes in plasma biomarkers between month 6 (time point 2) and baseline (time point 1) in all participants grouped together, Figure S3: Association between biomarkers and the PREDIMED score adherence (cross-sectional analysis), Table S1: The 6 months effect of the Mediterranean Diet intervention on metabolic biomarkers (linear mixed model analysis with *p*-values corrected for multiple testing).

**Author Contributions:** All authors have participated su fficiently and meaningfully to the research study and the preparation of this manuscript. M.S.-P. formulated the study question and design, interpreted the results, and drafted the manuscript. M.S.-P. and M.R.-C. were involved in statistical modeling. M.S.-P., S.N.K., Y.S. and S.M. contributed to the conception and design of the study and acquisition of the data. All authors contributed to the interpretation of data and critical revision of the manuscript and approved the final version. M.S.P., M.R.-C. and S.N.K. share primary responsibility for the final content. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by EMW-2014-FP-00612, US Department of Homeland Security, a Ohio University OURC grant and a CHSP Research Innovation grant, and the 2018 Southeast Center for Integrated Metabolomics Pilot & Feasibility Project. M.S.-P. holds a Ramón y Cajal contract (RYC-2018-025069-I) from the Ministry of Science, Innovation and Universities and FEDER/FSE and FIS grant PI20/00896. The funding agencies had no role in study design, data collection and analysis, interpretation of results, manuscript preparation or in the decision to submit this manuscript for publication

**Acknowledgments:** We appreciate the participation of the Indianapolis Fire Departments as well as the firefighters and their spouses. We also thank Noora Kanerva and Heli Julkunen for their help with data analysis, as well as the Indiana Clinical and Translational Science Institute for helping with sample processing, the Kroger Company (coupons and customer loyalty discounts), Barilla America (Barilla Plus Products), Arianna Trading Company, Innoliva and Molino de Zafra, Spain (extra virgin olive oil samples and discounts) and the Almond Board of California (free samples of roasted unsalted almonds) for study support. The sponsors had no involvement in the overall study design; collection, analysis and interpretation of data; writing of the report; or the decision to submit the report for publication.

**Conflicts of Interest:** The authors declare no conflict of interest.


**4. Other autoantibodies**

especially in the context of SLE, Sjögren's syndrome, subacute cutaneous lupus and neonatal lupus congenital heart block [31, 32]. However, the presence of anti-Ro52 alone, without anti-Ro60, was reported in inflammatory myopathy and in systemic sclerosis. It was also observed, that anti-Ro52 antibodies are, to larger extent than anti-Ro60, associated with primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) with co-expression of anti SLA antibodies

Anti-SS-B/La antibodies are less common and usually coexist with anti-SS-A antibodies, and their presence in other systemic diseases, especially in SLE (25% of patients with TRU), is associated with skin lesions (erythema, alopecia), inflammation of the serous membranes, leukopenia, symptoms of dryness and they often coexist with the presence of anti-SS-A antibodies (secondary SS) [28]. Rao et al. have noted that anti-SSB antibodies are important for the SLE diagnosis. They are associated with cheek erythema, alopecia, serositis, secondary Sjögren's syndrome, and hematological changes such as leukocytopenia, thrombocytopenia, and immunoglobulins elevation. They are often accompanied by the presence of anti-/Ro or anti-SSA/Ro52 antibodies [34]. These autoantibodies may also be found before the SLE-

Rheumatoid factor (RF) is an autoantibody directed against the CH2 and CH3 domains of an Fc region of a class G immunoglobulin (IgG). RF is produced by plasmatic cells (RF-PCs) that are formed from B cells activated both dependently and independently of T lymphocytes. Thus, RF producing B cells (RF-PC) become cells with ability of antigen presentation (APC) and of binding IgG. As this cascade of events constitutes a method of immune response against the infectious antigens, the RF production during infections protects the host organism. This phenomenon explains the occurrence of RF in the course of many viral (e.g., HCV, Herpes virus, and HIV), bacterial (e.g., subacute bacterial endocarditis, *Chlamydia pneumoniae*, *Klebsiella pneumoniae*, tuberculosis, and syphilis) and even parasitic (malaria, onchocerciasis, and toxoplasmosis) infections. In those autoimmune diseases, in which B-cell hyperactivity occurs, rheumatoid factors, particularly clinically relevant RF-IgM, also appears [36]. It should be remembered that RF appears in 4% of a healthy population and its incidence increases with age; after 75 years of life, RF can be observed even in 10–25% of individuals [37, 38]. The frequency (%) of RF in various CTD is presented in **Table 3**. The primary Sjögren's syndrome is one of the autoimmune diseases in which the majority of patients have a rheumatoid factor (some authors report up from 60 to 90% of patients)—specifically its most common IgM class isotype. The presence of RF IgM is associated with the occurrence of leukopenia, increased erythrocyte sedimentation rate (ESR), higher concentration of gamma globulins and lower C4 complement component concentration. Observations of a positive correlation of the rheumatoid factor with symptoms of dryness, hypergammaglobulinemia, presence of higher ANA antibody titers, presence of

(soluble liver antibodies) [33].

124 Autoantibodies and Cytokines

*2.3.2. Anti-SS-B/La antibodies*

specific antibodies can be detected [35].

**3. Rheumatoid factor**

#### **4.1. Anti-centromere autoantibodies**

Anti-centromere antibodies (ACA) are directed to six antigens associated with centromere (composed of a complex of kinetochore proteins). Currently identified anti-centromere antibodies (CENP) have been assigned designations with letters from A to F. The most common are CENP-A, B and C. CENP-B is also the most frequently occurring anti-centromere antibody in patients with pSS. The incidence of ACA antibody described in the literature ranges from 3.7 to 4% [40, 41]. This antibody, with a mass of 80 kDa, is a DNA-binding protein involved in the heterochromatin folding. Anti-centromere antibodies (A, B, C) occur mainly in limited systemic sclerosis (LSSc) and are associated with the prevalence of telangiectasia, higher severity of Raynaud's symptom, lung involvement such as interstitial lung disease [ILD] and fibrosis [42]. Their relationship between the presence of ACA antibodies and the involvement of endocrine glands has been demonstrated; in the ACA+ group, anti-SS-A and anti-SS-B autoantibodies were less frequent [42].

The association of ACA antibodies with non-Hodgkin's lymphomas (CENP-F) including MALT lymphomas was also described [43], as well as the case reports of CENP-B presence in small-cell lung cancer [44].

Interestingly, it has also been demonstrated that the presence of anti-CENP-B antibodies is associated with prolonged survival in a breast cancer [45].


**5. Discussion**

The discussion of results centers on revealing the interpretive flexibility and closure of meaning that characterizes smart home devices. When technology is interpretively flexible, it means that the "interaction of technology and organizations is a function of the different actors and socio-historical contexts implicated in its development and use" [43].

In terms of awareness, business respondents tended to provide definitions of IoT in terms of its structural properties, that is, connectedness. NGO respondents, instead, defined the IoT more in terms of its function and the problems the IoT can solve. In this view, the IoT's identity is intrinsically connected to its pragmatic aspect, that is, its role in a context or "situatedness." This might explain why the wider UK population awareness is greater for the expression "smart home" (90% of people are aware of "smart home") than for the expression IoT (47% of people are aware of "IoT") [3], since "smart home" indicates a recognizable context for use of these devices.

Business respondents are uncertain about the public awareness of IoT. This finding was also reflected in [3]. A deeper awareness of IoT examples and functions may be crucial. Zeng and Roesner [55] point out in fact some of the limitations of current smart home devices design, for example, in regard with the management of multiple users and sometimes lacking basic access control. Hence, promoting awareness of functionalities of this kind may also stimulate adoption in the home, and different players in the industry may need to act in concert to stimulate this functional awareness.

The lack of awareness is also related to the need to have specific technical knowledge and skillsets to be able to grasp both the connectedness and functionality of IoT. This requirement for a technical mindset and expertise could place adopting the IoT beyond the reach of the layperson, particularly those who are less well-educated since usually, it is the "more highly educated individuals who tend to adopt innovations sooner" [56]. Also, [3] survey showed how those with high and medium levels of education were early adopters of smart home devices, though those with less education were catching up.

Business and NGO respondents feel privacy and security issues are not sufficiently part of IoT awareness for the wider public, which is consistent with the finding that 59% of the wider population are not aware of media reports of security incidents involving smart home devices [57].

Previous research [58] showed that the smart home industry is insufficiently emphasizing measures to build consumer confidence in data security and privacy. The industry respondents we recruited, felt they possessed the skillset to judge the security-preserving capacity of smart home devices, but were unsure about the public possessing adequate skillsets. This suggests there is a perceived need to educate the population in regard to security issues pertaining to IoT. This is consistent with the survey finding that consumers' security concerns are likely to impact negatively on IoT adoption. In regard to privacy, both business and NGO respondents raised privacy issues as an industry-wide IoT concern. Hence, privacy as an obstacle to the adoption of the smart home emerges as a stable and established meaning of the smart home. The specific issues respondents raised concern data collection being always on, the uncertainty of data use, illegal malicious data use, and legal but harmful data use. Particular emphasis was placed on the importance of trusting smart home systems' integrity—the belief that the entity is honest and will fulfill its promise to the client [59]—for successful smart home adoption; this view reflects the finding that public trust in companies *not* using data produced by smart home devices without consumers' explicit consent, was fairly low [3]. Significantly, the issue of the influence of friends and experts may have on Privacy Decision Making (e.g., allowing or denying data collection) was not mentioned by any of the participants but this was shown to be an important factor for IoT adoption [60].

One respondent outlined what was perceived to be the neutral position of the public in regard to the likelihood of privacy breach, which was also reflected in our survey [3]. However, in our survey, the public's neutrality changed when the emphasis was placed on understanding the impact or consequences of a privacy breach. Again, this emerging feeling was consistent with our survey finding that the UK public tends to agree that the impact of privacy-related incidents is high [3].

Actions in the form of responses to privacy challenges revolved mainly around taking responsibility for mitigating privacy-related risks. This is key because it has been shown that even when users do indeed trust device manufacturers to protect their privacy, they do not verify that these protections are in place [61]. For business respondents taking responsibility to address privacy-related risks involved taking direct action and experimenting with the technology in order to find new ways to protect privacy. Business respondents felt that a big part of the responsibility toward guaranteeing data integrity was with big service providers. On the other hand, NGO respondents responded to privacy challenges by emphasizing standards, applying pressure to improve industry practices toward data use, and persuading consumers that their data is properly curated and looked after. They also called for collaboration with external, noncommercial, and nongovernmental players, such as academic institutions and researchers. Synergy among industry or industry-relevant stakeholders emerges in this view as the key mechanism toward responding to the privacy challenges of the smart home. When it came to security, both NGO and business respondents associated security issues with the public's lack of awareness of security and uncertainty over making security judgments about a device, which is consistent with the survey finding that people seem to be more concerned about the likelihood of a security incident rather than its impact [3] (unlike for privacy, where it is the other way round), suggesting that there is an education gap in regard to the practical consequences of security breaches.

NGO and business respondents alike thought that security risks were exacerbated by problems at the level of regulation. Specifically, NGO respondents felt that the issue is with a fragmented security-regulation effort, with security being too thinly spread as an issue across government, which is therefore unable to provide a solid answer to this challenge. Steps have been made toward providing a unified approach, with the UK government producing the Code of Practice for Consumer Internet

### *How Is the Internet of Things Industry Responding to the Cybersecurity Challenges of the Smart… DOI: http://dx.doi.org/10.5772/intechopen.106012*

of Things Security [9]. However, this effort may not be sufficient to unify security improvement practice in the sector. Brass et al. [62] point to the proliferation of non-governmental *de facto* standards for smart home cybersecurity produced by businesses, trade associations, and interest groups, as well as NGOs themselves. For businesses, the issue with regulation is felt through a lack of enforcement.

Addressing a specific security concern, one NGO respondent felt that liability may be exacerbated through the public-wide lack of awareness of security issues of smart home devices. Businesses felt that a key security issue is the lack of a marketing incentive for smart home cybersecurity, a feeling that reflects a wider trend with cybersecurity in the private sector in general. Gordon et al. [63] underline how, in general, firms invest in cybersecurity activities at a level below what would be optimal. The issue is particularly significant in regard to small to medium enterprises (SMEs), which are deemed to be potentially the ones most at risk [64], as they often neglect cybercrime prevention [65] and do not possess adequate knowledge in cyber security [66].

In terms of actions, we found NGOs to be leading with the range of responses to the security challenges posed by smart home devices, as they primarily aim to make security a default positioning of devices. They stressed the key role of government in changing the *discourse* around smart home security. The choice of the socio-philosophical term "discourse" refers to the fact that it is both ideas and actions [67] around security that should be promoted and performed, a task for which the government is held to be both capable and responsible for. This perception underlines how it is important that the consumer does not feel he or she is solely responsible for smart home security. However, this feeling contrasts with the attitudes of the public, who ranked the service provider (e.g., Google, Amazon, and Apple) as the main actor responsible for the security of smart home devices, followed by the consumer and the manufacturer, with the government ranking fifth only [57]. This misalignment of perception across NGO experts and consumers may represent an opportunity for intervention for a number of players in the smart home ecosystem. Finally, the global marketplace for smart home devices reminds us that responsibility toward ensuring the security of smart home devices requires an international effort.


*3.2. PV-PRC Implementation Analysis*

Equation (3) shows that the premise of introducing active reserve is that the current maximum output power value *P*mpp is a known amount. Therefore, the maximum power point estimation (MPPE) link is necessary for the PV-PRC operation, and the active power can be introduced when the MPPE link is found to be *P*mpp. For the sake of simplicity, the MPPE method in the reference [17] is used, as shown by the red dashed box in Figure 4, taking two sets of PV modules or arrays of the same model and quantity as an example. When operating under the same operating conditions, PV1 operation in MPPT mode, its output power can be used as the available power value of PV2 (*P*mpp1 = *P*mpp2). However, in [17], the PV output power is controlled by controlling the output voltage. The calculation of the voltage command in the process leads to a cumbersome control process. In view of this, this paper improves it by using direct power control to control the output power. In addition, in this kind of control mode, through the lowest voltage limit, the PRC runs with the *V*2 side, and the control block diagram is shown in Figure 4.

**Figure 4.** PV-PRC block diagram.

#### **4. PV-PRC-VSG Control Strategy and Reserve Ratio Analysis**

In PRC operation mode, a part of the power up-regulation capability is maintained in the PV system, so that the output of the PV system can be adjusted within a certain range, and the regulation of the reserve power has the same effect as the charging and discharging of the energy storage system, and the active output can be adjusted. With the VSG technology, the inertial response and the participating power system primary frequency response can be realized. In this mode, the output power of the virtual prime mover is as shown in Equation (5).

$$\begin{cases} J\omega \frac{d\omega}{dt} = P\_{\rm m} - P\_{\rm e} \\ P\_{\rm m} = P\_{\rm deload} + D\_{\rm P}(\omega\_{\rm n} - \omega) \end{cases} \tag{5}$$

Equation (5) can be reduced to:

$$P\_\mathbf{e} = P\_\text{deload} - J\omega \frac{\mathbf{d}\omega}{\mathbf{d}t} + D\_\mathbf{p} (\omega\_\mathbf{n} - \omega) \tag{6}$$

where from left to right are PV output power, inertial response process demand power and primary frequency modulation demand power.

Define *Pf* = −*J*ωdωd*t*+ *D*p(ωn − ω), where *Pf* is the required power for frequency response.

Assuming that the PV system operates in PRC mode initially and maintains a certain active reserve Δ*P*, when the frequency change of the grid is detected to exceed the dead zone (±0.03 Hz), the required power *Pf* for the PV system to participate in frequency regulation is calculated through the VSG control link. Then, by release or increasing the reserve power, it can participate in the frequency regulation of the power grid to provide power support for the power grid. The control block diagram of the whole system is shown in Figure 5.

**Figure 5.** Principle of PRC-VSG implementation.

The PRC operation mode of the PV system essentially abandons part of the PV resources, which is in contradiction with the efficient use of energy and relevant national network codes. However, considering that the realization of PV-PRC-VSG technology can improve the stability of the power system, enhance the acceptance of the power grid to PV energy, and save the investment of ESSs, it is necessary to keep the power reduction within a certain range, and the proportion of the power reserve in the actual operation process is mainly constrained by the following factors:

To sum up, considering the limitation of abandoning PV energies and the requirement of the VSG for primary frequency modulation, it is more appropriate to reduce power by 10%*P*mpp. During the operation of the power system, when the system frequency increases, the active output of the PV

system further reduces the frequency change of the response system by increasing the value of Δ*P*. When the system frequency decreases, the value of Δ*P* of the PV system needs to be reduced to release the reserve active power. It is noteworthy that when *Pf* > Δ*P*, there is no active power up-regulation ability in the PV system. In addition, Δ*P* is still limited by the upper limit. When Δ*P* increases to 30%*P*mpp, it cannot be increased any more. As shown in Equation (7), because of the limitation of active reserve capacity, the ability of PV-PRC-VSG to participate in primary frequency regulation is limited.

$$
\Delta P' = \begin{cases}
30\%P\_{\rm mpp} & , P\_f < -20\%P\_{\rm mpp} \\
\Delta P - P\_f & , -20\%P\_{\rm mpp} \le P\_f < \Delta P \\
0 & , P\_f \ge \Delta P
\end{cases}
\tag{7}
$$

where Δ*P* is a reference value of reserve power during primary frequency response.

## **5. Simulation of Proposed Method**

To verify the effectiveness of the proposed control strategy, the corresponding simulation model was built in MATLAB/Simulink. The model topology adopts the grid-connected structure of the PV system in Figure 2. The corresponding experimental verification was made for a DC/DC inverter working in PRC mode and DC/DC and DC/AC inverter coordinated control participating in power system frequency response.

## *5.1. PV-PRC Simulation and Analysis*

Firstly, the PRC operation control strategy of the DC/DC side proposed in the second section is simulated and analyzed. Taking the parameters of a single 240-W PV module (JLS60P240W) as an example, the parameters of PV modules are shown in Table 2. The maximum power value of the PV module is calculated in the MPPE process, and then the active reserve is introduced. For the convenience of calculation, the reserve ratio (*R*) is introduced. The value of *R* can be calculated by Equation (8). In the operation process control, *R* is the direct control object.

$$R = \Delta P / P\_{\rm mpg} \times 100\% \tag{8}$$

where: *V*oc(*V*)-Open circuit voltage; *I*sc(A)-short-circuit current; *V*mpp(V)-Maximum Power Voltage; *I*mpp(A)-Maximum Power Current; β*V*oc(%/◦C)-temperature coefficient of open circuit voltage; α*I*sc(%/◦C)-temperature coefficient of short circuit current.

**Table 2.** PV module parameter data sheet.

The primary task of PRC operation is to calculate the value of *P*mpp at each moment; that is, the MPPE process. MPPE plays a vital role in the whole PRC operation process, which directly affects the accuracy of the power reserve. In Figure 6a, the red dotted line is the maximum output power (actual *P*mpp value) of the PV system in MPPT mode, and the black dotted line is the *P*mpp value calculated by MPPE, which deviates little from the actual value, thus laying a foundation for subsequent experiments.

(**a**) Output power contrast diagram (**b**) Reserve ratio contrast diagram

(**c**) Output power contrast diagram

**Figure 6.** PV-PRC mode simulation waveform.

As shown in Figure 6b, there is no active reserve in the PV system before 0.4 s, and the PV system works in MPPT mode; between 0.4–2 s, the PV system maintains the active reserve in PRC mode. In Figure 5b, the solid line is the given value of the reserve rate, and the dotted line is the actual reserve rate value in the simulation operation. It has good tracking accuracy in the whole operation process. The output power is shown by the blue solid line in Figure 6a. It is noteworthy that the direct power reduction method used in this paper can work in MPPT and PRC modes in a time-sharing manner according to the actual needs, and there is no need to switch control modes, which provides greater convenience for engineering implementation.

Figure 6c is a comparison of output voltage of MPPT mode and PRC mode. It was found that the output voltage in PRC mode is always greater than that in MPPT mode. It is proved that in PRC mode, the output voltage always works on the right side of the P–V curve, which is the same as the expected result in Section 3. It is interesting to find that the DC voltage ripple in PRC mode is much smaller than that in MPPT mode.


*4.5. Political Dangers in Practice*

Hazony is a binary thinker who presents the reader and political actor with monolithic ideological blocks. The construction of ideological enemies both on a religious and political level especially serves the goal to bring out one's own polarizing binary Weltanschauung. However, neither religious nor political traditions are monolithic buildings without any historical and social context. There are different schools and practices of liberalisms. These also necessitate critique, but there is no monolithic violent liberalism as such 16. One highly dangerous result of rejecting liberalism as such is the nearly complete neglect of the individual and their rights within and against the collective. Hazony's National Conservativism presents us a divinely instituted, hierarchical and gendered structure of the political community. The individual only exists within the framework of the collective, identified with the majority. This is particularly dangerous for women's rights and minority rights, which have been in the center of the struggle for liberal democracies throughout decades.

#### **5. Reflecting the Theo-Political Consequences**

Let us finally reflect some theo-political consequences of National Conservativism as presented by Yoram Hazony. This school of thought and its related international movement are characterized by an entanglement of political and religious actors, which intend to delegitimize liberal democracy, human rights, international cooperation and pluralism in the name of a religiously based political doctrine. It reads the Hebrew Bible as a nationalist vademecum and history as a conflict between empires and national states, the former identified with any Weltanschauung showing universalist tendencies, and a monolithic liberalism in particular.

National Conservativism and Hazony in particular is a prominent example for the significance of debates around sex, gender and family in contemporary theo-political conflicts. Scott (2018) underlines the focus of political conflicts between the secular and the religious on this range of topics from the early 19th century onwards. Already during that era, the conflict line was not in between the secular and the religious, but in between progressive and traditionalist actors within the respective communities and Weltanschauungen. Similarly, this is true for contemporary theo-political conflicts. National Conservativism is not the single representative of a religiously motivated political ideology, but unites traditionalist Jewish and Christian, sometimes even secular, intellectuals against those who are identified as violent imperialists. The focus on the traditional, patriarchal family serves as an essential focal point for the movement.

Within National Conservativism, religion is not an open, inclusive concept, which could serve as an integrating factor for a pluralist society, as suggested by Bellah's civil religion, but the exclusivist marker of the homogenous community called "nation". Calling for the end of institutional separation between politics and religion, the central pillar of a secular-liberal democracy, we observe the return of an institutionalized state religion.

However, who serves whom in this doctrine? Do religious actors use the idea of National Conservativism in order to implement their religious worldview on a political level or do political actors use religious motives and invoke "Judaeo-Christianity" as a bulwark for protecting their power, without any sincere interest in faith?

For the Edmund Burke Foundation, whose founder and president is Hazony, National Conservativism is not a mere academic enterprise. It aims at bringing together political theory and political activism, particularly supporting figures such as Victor Orbán, who famously coined the term "illiberal democracy" (for a first impression see Vormann and Weinman 2020; the most comprehensive and systematic overview is currently given by Sajó et al. 2021) 17. "Illiberal democracy" is the practical implementation of National Conservativism, and National Conservativism is the intellectualized version of "illiberal democracy". As Orbán (2018) points out in a speech at Bálványos Summer Open University, Christian democracy and liberal democracy are contradictions; Christian democracy can only be "illiberal democracy" based on a prioritization of "Christian culture", the rejection of immigration and the focus on the "Christian family model" of father, mother, child.

Applebaum (2020, part. pp. 15–17) critically discusses the role of intellectuals in Europe, supporting the rise of contemporary authoritarian politics, and calls them "clercs", a term coined by Julien Benda in the 1920s. Hazony and his co-fighters within the Edmund Burke Foundation can be summarized in this category. It is a group of Jewish, Christian and a few secular intellectuals who intend to offer a theologically and philosophically justified version of what they call "conservativism", but what is actually in many aspects an anti-democratic, authoritarian enterprise, which silently delegitimizes both liberal democracy as such and religion as a productive, peace-building political force within democratic processes.

National Conservativism does not overcome the definitely existing aspects of violence in some versions of liberalism, but religiously legitimates political authoritarianism at the expense of minority rights, individual freedom and particularly women's rights; women who are widely reduced to reproductive units beyond political power. Nevertheless, National Conservativism and his key thinker Hazony call for a reflection on the relation between nation, state and religion. Are there any inclusive, non-violent forms of nationalism, overcoming religious exclusivism? Palaver (2021), following Henri Bergson, has already suggested the helpful distinction between static and dynamic religions, and supports an open patriotism. This is an important task that both theology and political theory need to commence in an age of rising populism and religiously inspired political extremism. Interestingly, Palaver points to Hans Kohn's distinction between civic nationalism and ethnic nationalism and his warnings of abusing essential Jewish concepts such as "chosen people" for a fierce nationalism, which is not tempered anymore by ethics and universalism. The same Buber-student Hans Kohn is criticized by Hazony (2000, pp. 212, 249) as representative of Jewish anti-Zionism.

National Conservativism is one voice within the rising front of theo-political networks fostering authoritarianism. They are attractive, because they touch hot issues within our current crises, i.a., the growth of pluralism, rising migration, the decline of traditional institutional religions, the shattering of established gender roles and family structures, and the loss of trust in liberal democracy. Theologians and political theorists are called to pay attention to the abyss that is re-opened again by National Conservativism and its fore-thinker Hazony. On a theoretical level, academia needs to reflect sincerely what a "good nationalism" in accordance with democracy could look like, especially with regard to the ever-present plurality in political communities and the quest for human flourishing in equality and freedom. Special attention has to be given to the role of religion and religious actors within this process. What is the persistent political contribution of religious actors in a fragmented world, resisting the seduction of identifying with one political doctrine? With exclusivist, ethnocentric schools showing anti-democratic, authoritarian sympathies?

Hazony's National Conservativism displays many serious problems of the contemporary entanglements of politics and religion, but can hardly serve as a solution. In contrast, as pointed out, in addition to its highly problematic (mis-)readings of religion, history and philosophy, it will lead to deeper struggles, theo-politically fostering new waves of exclusion and violence. Especially with regard to minority rights, women's rights and the role given to equality as an essential principle of democracy, National Conservativism raises serious questions whether it is still compatible with the basic outlines of a democratic political system.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The author declares no conflict of interest.

## **Notes**

increasingly spreading his networks in Europe too. Dreher and the Danube Institute, which hosted him in Budapest, are closely affiliated with the Edmund Burke Foundation and National Conservativism. The Danube Institute was one of the sponsors of the 2020 National Conservativism Conference in Rome. Dreher himself is a regularly invited speaker at National Conservativism Conferences (as is Orbán), e.g., in Florida 2021, where he spoke on "What Conservatives Must Learn from Orbán's Hungary" (Dreher 2021).

has contextualized the social teaching of John Paul II within the historical context of his pontificate. Admirand (2012) summarizes his legacy in intra- and interreligious dialogue.

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*Participant 2:*

*"There are significant challenges because Botswana does not monetize. So, I had to ask my friend from Zimbabwe to create the account on my behalf, using Zimbabwean YouTube. And another challenge is that even the money is not being credited into our bank accounts in Botswana because I do not qualify for the trading. Therefore, it has to be credited to someone in South Africa or Zimbabwe, then they send the money to me here. Those are the challenges we face."*

From the last research question, as shown in **Figure 3**, the participants' responses were distributed as follows:

**Figure 3.** *Distribution of themes from participants' responses to research question 3.*