2.1.4. Steam Network

While most industrial facilities have a major steam source (e.g., a combined heat and power unit (CHP)), the utilization of waste heat from production units in means of steam production is also a common practice [43]. Moreover, the overall steam supply of these local producers can even exceed the major steam source production. Thus, the quality of the supplied steam varies significantly depending not only on the CHP unit's operation but on the whole production network [22,72]. It is therefore vital for process drive sizing to account for steam quality fluctuations.

When analyzing steam networks, as well as any other pipeline systems, momentum and heat transfer must always be considered [73]. Pressure loss caused by friction and/or various installed armatures and potential heat loss to ambient space can a ffect the steam quality significantly. When analyzing the heat transfer, steam–ambient temperature di fference plays the most significant role. The impact of heat loss on the medium quality is thus best observable during extreme ambient conditions, i.e., temperature peaks during winter. Even though most adverse e ffects of ambient temperature changes can be suppressed by e ffective insulation, Hanus et al. [26] showed that the effectivity of outdoor insulation decreases heavily after years of operation and it is in designer's best interest to investigate the true heat conductivity of the used insulation.

Effects of both pressure and heat losses vary greatly depending on pipeline geometry and mostly on the steam network topology and plant infrastructure, i.e., the resulting pipeline length. While the distance (pipe length) of the considered steam drive from the battery limit header can range in terms of tens of meters in some cases, there are units where the pipe length can reach hundreds of meters or even a kilometer. This is mostly the case of mid-twenty century refineries where individual units and their subunits were built far apart for safety reasons [26].

Lastly, it is necessary to assess the impact of steam drive alteration on the steam transport velocity. Naturally, high transport velocities may cause serious erosion of pipeline walls and damage installed armatures. However, low velocities in the pipelines may result in excessive heat loss and decrease in steam quality or even condensate production [26]. Hence, the velocity changes with the steam consumption of the drive, and pipeline suitability assessment is inevitable.

The process described in the previous text draws high-pressure steam from a steam network using a CHP unit as the main steam producer. As illustrated below (Figure 2), steam crossing the fluid catalytic cracking (FCC) unit battery limit is first supplied to the modeled system (propylene recovery unit) and then to other parts of the FCC unit. To account for all above-mentioned aspects, pipeline topology, geometry, and heat transfer properties were incorporated in the Aspen Plus ® process model.

**Figure 2.** Simplified plant steam network. Legend: Red line= HPS pipelines, orange line= middle-pressure steam (MPS) pipelines, dotted red line = turbine exhaust; blue line = condensate.

#### 2.1.5. Combined Heat and Power Unit (CHP) as Marginal Steam Source

Industrial combined heat and power plants (CHP) traditionally employ steam boilers and steam turbines as the cogeneration technology; some of them including gas turbines or combustion engines [40,74]. Their task is to cover the steam network imbalance on all pressure levels (i.e., to serve as a marginal steam source) while the cogenerated electric energy is utilized in the industrial facility or sold to an external grid.  The reason for this specific system feature is explained in more detail in Section 3.2. Techno-economic assessment of a process steam drive sizing inevitably impacts the CHP operation and should be evaluated correctly. Last, but not least, the CHP operation is influenced not only by internal steam demand, but by external factors as well (energy management strategy of the refinery, changeable energy prices and their uncertainty, etc.) [75].

The considered CHP comprised high-pressure steam boilers (9 MPa, 530 ◦C) and a set of backpressure and extraction condensing turbines exporting high-pressure (HPS, 3.5 MPa), medium-pressure (MPS, 1.1 MPa), and low-pressure (LPS, 0.5 MPa) steam to the industrial facility. Constant marginal heat production e fficiency of the CHP and constant marginal backpressure and condensing power production per one ton of steam were assumed. The e ffect of a change in the production unit steam balance on a certain pressure level was transposed to the corresponding change in backpressure electric energy production and fuel consumption in the CHP. A detailed description of the CHP unit operation and its further characteristics can be found elsewhere [26,47].


**2. Literature review strategy and sample selection**

#### **2.1 Review method**

This study uses a systematic literature review to integrate the existing literature on agency theory advancement in the context of international business. The systematic method of the literature review was proposed by Tranfield et al. [9] in medical science literature, to overcome the criticism of the traditional literature review. A traditional literature review involves the subjectivity and bias of the researcher. The main objective of the systematic literature review is to identify research key contributions in a field and descriptively present and discuss them.

There are two important steps in the systematic literature review:

#### *2.1.1 The inclusion criteria*

This study uses the following criteria to include review articles:

*Agency Theory and Internationalization: A Critical Assessment of Literature DOI: http://dx.doi.org/10.5772/intechopen.99192*

#### *2.1.2 Study selection and data source*

This study uses Business Source Ultimate of the EBSCO database to find the relevant articles. The focus of the study was on top ranked international business journals. EBSCO includes all the top ranked journals in the field of international business which includes top international business journals indexed in Web of Science or Scopus databases. The author uses a combination of the keywords: agency and internationalization to find the relevant articles. The search using these keywords in title, abstract, and author keyword in the database yielded a total of 132 articles. Further, the author removes duplicate and below B category journal articles (as per ABDC ranking) and finds 59 articles for further use in the study. The author read the abstract and the introduction of these 59 articles whenever required to find relevant studies. Finally, the author finds 24 relevant articles that meet the objective of the study. **Table 1** lists all the review studies with journal name, the theory used, country or context, method (qualitative/ quantitative), and analytical techniques.

## **3. Findings and discussion**

#### **3.1 Descriptive analysis**

This section discusses various descriptive characteristics of the review articles. There is an increasing trend of publication of studies using agency theory to study MNCs. **Figure 1** shows an increasing trend of using agency theory in the context of firm internationalization after 2010. **Figure 2** shows that review studies are published in various management journals. The highest number of publications from the sample studies are from the International Business Review and Journal of Business Research. Most of the sample studies have integrated agency theory with the resource-based view, resource dependency theory, and institutional theory (see **Table 2**). Firm internationalization is a complex process and requires more than one theory to explain the underlying phenomenon. Most of the articles are based on a single institutional or country context (see **Figure 3**). Most of the articles are empirical and primarily use a diverse set of quantitative techniques (see **Table 3**).

#### **3.2 Corporate governance and agency theory**

As per the agency perspective, the divergence of interest between the principal and agent causes opportunistic behavior by agents to maximize their utility and reduces the shareholders' value [34]. Firms use several governance mechanisms to mitigate agency costs such as monitoring by the board of directors [35], and high ownership by outsiders [36]. There exist internal as well as external mechanisms to control the agency problem [25]. Internal governance mechanism is such as CEO compensation alignment with the shareholders' value maximization [37]. The external mechanism is market control such as the threat of takeover, and competition in the labor and product market [38]. Further, this study divides the literature into various themes.

#### *3.2.1 Board of Directors (BOD)*

BOD has the roles of managing the top management's hiring to firing, providing information and network resources, and assisting top management to decide

#### *Corporate Governance - Recent Advances and Perspectives*


**4.6. Advances for respiratory motions**

To establish a UWB navigator for cardiac MRI in free-breathing mode, a landmark tracking of the heart due to the respiration motion is necessary [66]. The time-dependent UWB radar signal contains mainly respiratory motion of the chest, as in Fig. 32.b, which is not necessarily identical to the mechanical displacement of the heart. Therefore, we compared the UWB detected respiratory motion to simultaneously measured 1D MRI as described in Section 4.5.2. Resulting from that comparison, we extended the UWB configuration to allow for the detection of abdominal respiration, too (Fig. 40.a). The antenna configuration applied in the comparison made in Section 4.5.2 was extended by two additional channels above the chest and a second group with one *Tx* and one *Rx* antenna over the abdominal region.

In MR-based navigator techniques [67], the position of the diaphragm is monitored because the shift of the diaphragm is the dominant motion component of the heart due to respiration. The displacement of the diaphragm is mainly orientated in head-foot direction. Hence, the pencil-like one dimensional MRI was placed in head-foot direction across the heart. The UWB data of the first antenna group were decomposed by BSS for detection of breast respiration and cardiac cycle and the second group for the abdominal respiration. In the same way, the motion components were decomposed for "pencil-like" MRI.

The UWB detected breast respiration is not suitable to monitor the mechanical heart shift in head-foot direction. In Fig. 41, a delay between the breast respiration and the heart shift is depicted, and especially in Fig. 41.b it becomes obvious that these are different processes. However, the UWB detected abdominal respiration correlates well with heart motion due to respiration. The correlation factor in measurement a) is 0.932 and 0.81 in measurement b).

298 Ultra-Wideband Radio Technologies for Communications, Localization and Sensor Applications

**Figure 39. a)** Selection of the 'pencil' by two crossing slices in antero-posterior direction through the

The trigger events (squares in Fig. 39.b) in the UWB cardiac motion data - representing the point of maximum contraction of the myocardium - were determined by applying the algorithm proposed by us. This did not work with the cardiac components of the MR signal due to the pronounced double peaks in this data set. Comparing the cardiac components simultaneously gained by UWB and MR data, we observe perfectly matching slopes of both signals. However, in contrast to UWB radar the MR signal is affected by the blood velocity in the heart producing the double peaks. Keeping this in mind, we can conclude that both modalities render the same motion. Thus, we can assume the cardiac motion detection by

To establish a UWB navigator for cardiac MRI in free-breathing mode, a landmark tracking of the heart due to the respiration motion is necessary [66]. The time-dependent UWB radar signal contains mainly respiratory motion of the chest, as in Fig. 32.b, which is not necessarily identical to the mechanical displacement of the heart. Therefore, we compared the UWB detected respiratory motion to simultaneously measured 1D MRI as described in Section 4.5.2. Resulting from that comparison, we extended the UWB configuration to allow for the detection of abdominal respiration, too (Fig. 40.a). The antenna configuration applied in the comparison made in Section 4.5.2 was extended by two additional channels above the chest and a second group with one *Tx* and one *Rx* antenna over the abdominal region.

In MR-based navigator techniques [67], the position of the diaphragm is monitored because the shift of the diaphragm is the dominant motion component of the heart due to respiration. The displacement of the diaphragm is mainly orientated in head-foot direction. Hence, the pencil-like one dimensional MRI was placed in head-foot direction across the heart. The UWB data of the first antenna group were decomposed by BSS for detection of breast respiration and cardiac cycle and the second group for the abdominal respiration. In

The UWB detected breast respiration is not suitable to monitor the mechanical heart shift in head-foot direction. In Fig. 41, a delay between the breast respiration and the heart shift is

the same way, the motion components were decomposed for "pencil-like" MRI.

heart; **b)** Detected cardiac motion component by UWB radar and MR "pencil".

UWB radar to be verified.

**4.6. Advances for respiratory motions**

**Figure 40. a)** Extended antenna configuration with second group over the abdominal region; **b)** Placement of the two slices for the 'pencil-like' MRI (Head Foot).

**Figure 41.** Breast and abdominal respiration by UWB radar and mechanical heart shift in head foot direction monitored by MR pencil **a)** with changed breast and abdominal respiration **b)** with fading breast but changed abdominal component.


**1.3.2 The composition analysis of Ti-O film**

Figure 1-3 shows the SEM topography (at different amplifications) of a Ti-O film cathodically-electrodeposited. It can be seen from the figure that under this experimental parameter, the Ti-O film obtained was well-distributedly deposited on TiNi SMA surface. This Ti-O film is closely composed of a lot of tiny particles, the particle's size is in about dozens of nanometers. This close bonding of the particles in nanometer class is conducive to strengthen the TiNi SMA surface properties, and to prevent Ni release from substrate.

Figure 1-4 shows an EDS analysis of the Ti-O film after cathodic-electrodeposition. It can be seen from the figure that this film mainly contains Ti element, as well as a small of amount of Ni element, and the ratio of Ti and Ni reachs 3:1. It illustrates that there is a lot of Ti element in the film. Because EDS might possibly puncture the Ti-O film, thus reflecting the substrate's element, so it could be preliminarily verified that by cathodic-elcetrodeposition, a layer of film mainly including Ti element indeed has been deposited on the surface of TiNi SMA.

Ti-O Film Cathodically-Electrodeposited on

0.0

2.0x103 4.0x103 6.0x103 8.0x103 1.0x104 1.2x104 1.4x104 1.6x104 1.8x104 2.0x104 2.2x104

Intensity

**Ti3s**

Fig. 1-5. XPS survey spectra of sample cathodically-electrodeposited

5.0x103

1.0x104

1.5x104

Intensity

2.0x104

2.5x104

3.0x104

the Surface of TiNi SMA and Its Bioactivity and Blood Compatibility 9

Ti2p

**C1S**

0 200 400<PHONE_NUMBER> 1200

Binding energy (eV)

2p3/2(Ti4+:458.8eV)

2p1/2(Ti4+:464.6eV)

445 450 455 460 465 470 475 480

Binding energy (eV)

Fig. 1-6. Ti2p XPS energy spectrum of sample cathodically-electrodeposited

**Ti(LMM)**

**O O(KLL) 1s**

Figure 1-5 shows an XPS (X-ray photoelectron spectroscope) analysis result of Ti-O film surface after cathodic-electrodeposition of TiNi SMA. It can be seen from the figure that Ti-O film mainly contains the elements of O and Ti, besides element C (an extraneous pollutant), no Ni element exists.

Figure 1-6 shows a high resolution XPS analysis result of Ti2p in Ti-O film. It can be seen from the figure that there are two peaks, at 458.8eV and 464.6eV respectively, both are Ti quadrivalentcations, corresponding with Ti-O bonding energy at the spin state of 2p3/2 and 2p1/2.

Figure 1-7 shows a high resolution XPS analysis result of Ni2p in Ti-O film. It can be seen from the figure that there are no clear peaks emerging, illustrating that no Ni element existing in the Ti-O film.

Figure 1-8 shows a high resolution XPS analysis result of O1s in Ti-O film. It can be seen from the figure that there are two peaks existing, one 530.2eV, this is O2-; another one 530.7eV, and this peak is an integrated OH-. So, the Ti-O film obtained by cathodicelectrodeposition should exist in the form of TiO2 or hydrate of Ti(OH)4.

Fig. 1-3. SEM result of Ti-O film

Fig. 1-4. EDS result of Ti and Ni in Ti-O film

Figure 1-5 shows an XPS (X-ray photoelectron spectroscope) analysis result of Ti-O film surface after cathodic-electrodeposition of TiNi SMA. It can be seen from the figure that Ti-O film mainly contains the elements of O and Ti, besides element C (an extraneous

Figure 1-6 shows a high resolution XPS analysis result of Ti2p in Ti-O film. It can be seen from the figure that there are two peaks, at 458.8eV and 464.6eV respectively, both are Ti quadrivalentcations, corresponding with Ti-O bonding energy at the spin state of 2p3/2 and

Figure 1-7 shows a high resolution XPS analysis result of Ni2p in Ti-O film. It can be seen from the figure that there are no clear peaks emerging, illustrating that no Ni element

Figure 1-8 shows a high resolution XPS analysis result of O1s in Ti-O film. It can be seen from the figure that there are two peaks existing, one 530.2eV, this is O2-; another one 530.7eV, and this peak is an integrated OH-. So, the Ti-O film obtained by cathodic-

electrodeposition should exist in the form of TiO2 or hydrate of Ti(OH)4.

pollutant), no Ni element exists.

existing in the Ti-O film.

Fig. 1-3. SEM result of Ti-O film

Fig. 1-4. EDS result of Ti and Ni in Ti-O film

2p1/2.

Fig. 1-5. XPS survey spectra of sample cathodically-electrodeposited

Fig. 1-6. Ti2p XPS energy spectrum of sample cathodically-electrodeposited

Ti-O Film Cathodically-Electrodeposited on

formation of TiO2 is as the reaction formula:

EDT 300 EDT 450

1. 2. 3. 4.

> 1. 2. 3. 4.

Fig. 1-9. XRD result of heat treated Ti-O film

TiNi SMA 300

EDT: electrodeposition TiO2

TiNi SMA 450

Intensity

the Surface of TiNi SMA and Its Bioactivity and Blood Compatibility 11

Figure 1-9 shows an XRD analysis result of TiNi SMA deposited after heat treatments at different temperatures. It can be seen from the figure that in the Ti-O film crystallized at 300C, no TiO2 existing was found besides the main peak reflecting the substrate. But after crystallized at 450C, there is the peak emerging of anatase TiO2 in the figure. Because the substrate also has Ti element existing, to investigate if the anatase TiO2 formed by substrate's thermo oxidation, the same parameters' XRD for the TiNi SMA blank sample before depositing was done. But in the result, no clear TiO2 peak merged after crystallized at 450C, this illustrates that the film obtained by cathodic-electrodeposition is truly an amorphous-state Ti-O film, its composition is mainly in the form of hydrate Ti(OH)4. The

Ti(OH)4 → TiO2 + 2H2O (1.4)

△

△ TiNi (B2)

(Anatase)

● TiO2

15 20 25 30 35 40 45 50 55 60 65 70

2 Theta.

**1.3.3 Corrosion potential test of the materials in simulated physical environment**

Figure 1-10 shows a relation of corrosionpotential-time of the samples before and after depositing in Hank's solution (PH7.45). It can be seen from the figure that the corrosion potential of the deposited sample dipping in solution went down quickly, about 5 minutes after, the corrosion potential did basically not change any more, stabilizing at -0.4V. Furthermore the stable corrosion potential was more positive than the non-depositing sample's, at about -0.2V. This illustrates that the deposited sample has a higher thermodynamic stability than the non-depositing sample's one at PH7.45 in Hank's solution. Figure 1-11 shows a graphofarelation of corrosionpotential-time of the samples before and after depositing in Fusayama solution (PH6.13). It can be seen from the figure that the corrosion potential of the deposited sample dipping in solution went down quickly also, 7 minutes after, basically stabilizing, although somewhat undulating, the potential did not change on the whole, keeping at -0.12V. But the non-depositing sample's potential basically stabilized at -0.15V, which was more negative than the deposited one's. This illustrates that

● ● ●

Fig. 1-7. Ni2p XPS energy spectrum of sample surface cathodically-electrodeposited

Fig. 1-8. O1s XPS energy spectrum of sample cathodically-electrodeposited

835 840 845 850 855 860 865 870 875 880 885 890 895

Binding energy (eV)

1s(O2-:530.2eV) 1s(OH-

:530.7eV)

520 525 530 535 540 545

Binding energy (eV)

Fig. 1-8. O1s XPS energy spectrum of sample cathodically-electrodeposited

Fig. 1-7. Ni2p XPS energy spectrum of sample surface cathodically-electrodeposited

1.7x104 1.7x104 1.7x104 1.7x104 1.7x104 1.8x104 1.8x104 1.8x104 1.8x104 1.8x104

8.0x103 1.0x104 1.2x104 1.4x104 1.6x104 1.8x104 2.0x104 2.2x104 2.4x104 2.6x104 2.8x104 3.0x104

Intensity

Intensity

Figure 1-9 shows an XRD analysis result of TiNi SMA deposited after heat treatments at different temperatures. It can be seen from the figure that in the Ti-O film crystallized at 300C, no TiO2 existing was found besides the main peak reflecting the substrate. But after crystallized at 450C, there is the peak emerging of anatase TiO2 in the figure. Because the substrate also has Ti element existing, to investigate if the anatase TiO2 formed by substrate's thermo oxidation, the same parameters' XRD for the TiNi SMA blank sample before depositing was done. But in the result, no clear TiO2 peak merged after crystallized at 450C, this illustrates that the film obtained by cathodic-electrodeposition is truly an amorphous-state Ti-O film, its composition is mainly in the form of hydrate Ti(OH)4. The formation of TiO2 is as the reaction formula:

$$\text{Ti(OH)}\_{4} \rightarrow \text{TiO}\_{2} + 2\text{H}\_{2}\text{O} \tag{1.4}$$

Fig. 1-9. XRD result of heat treated Ti-O film


**8. Conclusion**

Electrical disturbances are close related to power quality, and service quality, and even safety, can be seriously compromised when two linear systems with different electrical

**Part 3**

**Software Tools**

feeder schemes are deployed one next to other and have even direct electrical contact between them. Electrical systems in DC and AC railway systems are designed separately, especially considering earthing, and this leads to new problems that weren't faced when old DC lines were design, deployed and operated.

Disturbances are a common issue that an engineer has to face while designing a new facility or system. Disturbances can also appear when the system is already in operation. These systems need to be protected from the effects of the disturbances in the most effective ways. These ways are usually the replacement of equipments (or at least part of them) and the shielding against those electrical disturbances.

In the above sections it has been explained the effects that disturbances from high speed lines can cause on conventional DC lines. A measurement methodology has been proposed. This methodology was used in on site measuring campaigns and their results have been used as a validation of the protecting works carried out before the high speed line comes into operation. The determination of the boundaries between what needs to be changed and what does not need to be changed is a complex problem that must be solved in a cost effective way, but without reducing safety issues.

The different ways of disturbance have been presented and explained so a global vision of the problem can be easily achieved.


*Article* **Functional Bakery Snacks for the Post-COVID-19 Market, Fortified with Omega-3 Fatty Acids**

**Haralabos C. Karantonis 1,\* , Constantina Nasopoulou 1 and Dimitris Skalkos 2**

**Abstract:** Flaxseed is a natural ingredient with health benefits because of its rich contents of omega-3 fatty acids and fiber. In this study, whole-meal sliced bread, chocolate cookies, and breadsticks, which were enriched with flaxseed (*Linum usitatissimu*) were produced as a natural enrichment source in order to provide functional baked goods. The three innovative products were tested as sources of omega-3 fatty acids in terms of α-linolenic acid according to EU 1924/2006 as well as for their in vitro antithrombotic/anti-inflammatory effect. The results showed that omega-3 fatty acids had high concentrations (>0.6 g per 100 g of product) in all products even after the heating treatment with constant stability during the time of consumption. All the enriched products exerted higher, but in different grade, in vitro antithrombotic/anti-inflammatory activity compared to the conventional products. The products were evaluated positively by a panel of potential consumers without significant differences compared to conventional corresponding products. Enriched bakery products with omega-3 fatty acids may represent a novel opportunity for the development of functional foods that can be locally consumed, thereby contributing to public health prevention measures that the post-COVID-19 era demands.

**Keywords:** omega-3 fatty acids; bakery snacks; sensory evaluation; in vitro nutritional functionality; antithrombotic; anti-inflammatory

## **1. Introduction**

In the new post-COVID-19 period, the consumer is searching for quality, healthy foods with natural ingredients in order to protect themself from diseases, protect the environment, and provide sustainability to local economies [1–3].

Bakery snacks are an important part of the human diet and will continue to be one of the main food choices in the new era. Bread is said to be the world's earliest functional food. Functional foods are modified, enhanced, or improved foods that supply important nutrients to the human body when consumed as part of a diverse and balanced diet [4,5].

The addition of probiotics and omega-3 fatty acids to bakery products has become increasingly popular in recent years [6]. The global production of omega-3 products is estimated to be 3.3 million metric tons and worth \$9.1 billion in 2018. Plant omega-3 production values are expected to grow twice as fast as marine production values during the next few years—accounting for 52% of the production value when compared to 48% for marine. In parallel, over the last few years, there have been 255 bread launches containing omega-3, which represent 7.3% of total bread launches [7,8]. Most of these have come from North America (51%), followed by Europe (21%), Asia Pacific (14%), and Latin America (14%).

At the same time linseed is a raw material that is rich in omega-3 fatty acids and fibers, making it an excellent ingredient for fortifying bakery products [9]. A legal list of

**Citation:** Karantonis, H.C.; Nasopoulou, C.; Skalkos, D. Functional Bakery Snacks for the Post-COVID-19 Market, Fortified with Omega-3 Fatty Acids. *Sustainability* **2022**, *14*, 4816. https:// doi.org/10.3390/su14084816

Academic Editor: Dario Donno

Received: 18 March 2022 Accepted: 14 April 2022 Published: 17 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

authorized nutritional claims [10] on food categorization as "source" or "high content" in omega-3 fatty acids has been established and a health claim related to essential fatty acids has been approved and placed in Commission rules [11], stating that "essential fatty acids are needed for the proper development of children".

The majority of research focuses on the experimental production of foodstuffs fortified with omega-3 fatty acids, the study of their sensory quality [12–16], and the bioavailability levels of omega-3 fatty acids in fortified foods [17–19]. Moreover, marine- or plantoriginated omega-3 polyunsaturated fatty acids have been proposed as health promoting constituents for cardiovascular health.

The mechanisms though, through which those fatty acids exert their beneficial activities, are not clear [20–23]. Nevertheless, platelet activating factor (PAF) [24] has been recognized as one of the most potent lipid inflammatory and thrombotic mediators that activates various cells through its specific receptor, such as platelets [25]. Activated platelets are important contributors to thrombosis and inflammation and represent an important linkage between inflammation, thrombosis, and atherogenesis [25,26]. The in vitro inhibition of PAF-induced platelet activation from food components have been used as a research tool to investigate the nutritional functionality of those foods and their possible preventive effect against chronic disease development when consumed as part of a balanced diet [27]. Interestingly, fish oil-derived omega-3 fatty acids have been shown to suppress in vitro a fundamental process in many acute and chronic inflammatory diseases—monocyteendothelium interaction—by inhibiting PAF activity and production [28].

The objective of the present study was to manufacture functional bakery snacks that are enriched with omega-3 fatty acids using linseed as a natural source and investigate their quality in terms of sensory evaluation, omega-3 fatty acids content, and the antithrombotic/anti-inflammatory activity through a nutritive index, as well as forecast the five-year gross sales of the products produced by local companies.

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

*2.1. Manufacture of Bakery Products*

#### 2.1.1. Design of the Products

The first step of the product design was to convene the HACCP food safety team of local enterprises. In this meeting, there was a full description of the products, including the necessary ingredients and raw materials, the manufacturing process of the product, the packaging and distribution, and the final characteristics of the food, as well as the documentation procedure for how to document the allergens in the nutritional details [29]. According to the legislation [30], products are not suitable for consumption for certain sensitive groups, i.e., allergic persons, therefore allergic ingredients are to be referred to on the label of the product.

#### 2.1.2. Design of Flowchart, HACCP Plan—Recipe and Manufacture of the Products

In the design of the flowchart and the confirmation of the HACCP plant in practice, three CCP (Critical Control Points) were confirmed—one for each product in the same stage of thermal processing. The critical limits were: 83 ◦C/1 min for sliced bread, 78 ◦C/40 s for cookies, and 80 ◦C/30 s for breadsticks [29]. During the tests of the recipes, it was decided that 50% of the linseed added to the product should be milled to reduce the feeling of the whole seed and, furthermore, to increase the stability of the fatty acids in the whole grain.

#### 2.1.3. Product Production

The creation of the three functional bakery snacks is depicted in a flow chart in Figure 1, while the ingredients for the recipes are listed in Table 1.

1, while the ingredients for the recipes are listed in Table 1.

**Figure 1.** Flow chart presenting the production of the three functional bakery products. **Figure 1.** Flow chart presenting the production of the three functional bakery products.

**Table 1.** Ingredient recipes for whole-meal sliced bread, chocolate cookies, and breadsticks. **Table 1.** Ingredient recipes for whole-meal sliced bread, chocolate cookies, and breadsticks.

The creation of the three functional bakery snacks is depicted in a flow chart in Figure

Milk (g) 5.00 1 : Functional bakery products enriched with flaxseed as source of omega-3 fatty acids.

#### Honey (g) 3.00 1 : Functional bakery products enriched with flaxseed as source of omega-3 fatty acids. *2.2. Sensory Evaluation*

*2.2. Sensory Evaluation* Omega-3 fatty acid-enriched bakery products, namely whole-meal sliced bread, chocolate cookies, and breadsticks, were evaluated by 16 trained panelists. Each sample was evaluated on six main attributes: appearance, odour, texture, taste, aftertaste, and overall acceptance. Rating of the sensory attributes was carried out using a 9-point hedonic scale where 1 = nonexistent and 9 = too intense (11, 12). The final sensory profile of Omega-3 fatty acid-enriched bakery products, namely whole-meal sliced bread, chocolate cookies, and breadsticks, were evaluated by 16 trained panelists. Each sample was evaluated on six main attributes: appearance, odour, texture, taste, aftertaste, and overall acceptance. Rating of the sensory attributes was carried out using a 9-point hedonic scale where 1 = nonexistent and 9 = too intense [11,12]. The final sensory profile of the products was determined by the average and the acceptable point (5 at the above scale) in duplicate experiments.

#### *2.3. Chemical Analysis*

The protein content was determined using the AOAC Kjeldahl method [31], the moisture was determined by an air oven [31], the fiber (crude) was determined by digesting the sample in a 1.25% (*v*/*w*) H2SO4 followed by 1.25% (*v*/*w*) NaOH solution [32]. Furthermore, the total fat was determined after acid hydrolysis and diethyl ether/petroleum ether extraction and fatty acids determination was performed with gas chromatography after methylation in methanol with boron trifluoride (BF3) as a catalyst [31]. The fatty acid methyl ester profiles were measured by gas–liquid chromatography on a Shimadzu 2010 chromatograph (Shimadzu Corporation, Tokyo, Japan), which was fitted with an automatic sampler AOC-20 and flame ionization detector. A fused-silica capillary column was used for the FAME analysis; DB-23, 60 m × 0.251 mm i.d., 0.25 µm (J&W, Agilent Technologies, Palo Alto, CA, USA). The oven temperature value sequence was initially 120 ◦C for 5 min, was raised to 180 ◦C at a rate of 10 ◦C per min, then to 220 ◦C at a rate of 20 ◦C per min, and, finally, was isothermal at 220 ◦C for 30 min. The injector and detector temperatures were maintained at 220 and 225 ◦C, respectively. The carrier gas was a high purity helium with a linear flow rate of 1 mL per min and a split ratio of 1:50. The individual FAMEs were identified by comparison with the relative retention time of the FAMEs peaks from the samples with the standard mixtures 37 Component FAME Mix (47885-U Supelco, Bellefonte, PA, USA) and Qualmix Fish S (89-5550 Larodan Fine Chemicals AB, Malmö, Sweden). The carbohydrates and energy were calculated from proximate analysis values.

All products were subjected to the determination of omega-3 fatty acids before and after baking in order to assure their availability after baking.

#### *2.4. Shelf-Life Determination*

The chemical and sensorial shelf life of the enriched bakery products was determined by means of sensory scoring and omega-3 content to meet the legislative limits for nutritional claims for products kept at room temperature for 24 days. The measurements were performed on days 0, 4, 7, 18, and 24. The overall market shelf life of the products was determined as the combination of the chemical and sensory values.

#### *2.5. Anti-Thrombotic and Anti-Inflammatory Activity*

All chemical reagents and solvents were of analytical grade and were supplied by Merck (Darmstadt, Germany). Platelet-activating factor (β-Acetyl-γ-*O*-hexadecyl-*L*-αphosphatidylcholine hydrate) and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA).

#### 2.5.1. Lipid Extraction

An amount of 15.0 mL of methanol was mixed with 3.0 g of flour sample and the mixture was agitated at 200 rpm on a GFL 3017 orbital shaker (GFL, Burgwedel, Germany) for 15 min. After that, 7.5 mL of chloroform was added to the mixture, which was followed by agitation at 200 rpm for 15 min. In the next step, after taking into consideration the moisture content of the samples, 6.0 mL of either distilled water (first version of lipid extraction) or 1 m aqueous sodium chloride solution 0.5% in acetic acid (second version of lipid extraction) was placed in the mixture in order to achieve a ratio for the solvents of methanol/chloroform/water that was equal to 1/2/0.8 (*v*/*v*/*v*), and then an extra agitation was performed at 200 rpm for 15 min. Then, depending on the version of the method, 7.5 mL of methanol were added along with either 7.5 mL of distilled water (first version) or 7.5 mL of 1 m aqueous sodium chloride solution 0.5% in acetic acid (second version), thereby achieving a ratio for the solvents of methanol/chloroform/water that was equal to 1/1/0.9 (*v*/*v*/*v*). After a final agitation at 200 rpm for 15 min, the samples were stored overnight at 4 ◦C. The samples were then centrifuged for 5 min at 2000× *g* in a Hermle Z 383 centrifuge (Hermle Labortechnik, Wehingen, Germany) and the lower phase of the biphasic solvent system was collected into a pre-weighed glass flask along with a 5.0 mL chloroform rinse of the upper phase. The samples were dried using a Lab Tech

EV 311 Rotary evaporator (Lab Tech, Milan, Italy), which was weighed on a KERN ABJ analytical balance (Kern and Sohn GmbH, Balingen, Germany), suspended in 2.0 mL of chloroform/methanol: 1/1 (*v*/*v*), and stored at −40 ◦C until further study.

#### 2.5.2. In Vitro Anti-Thrombotic and Anti-Inflammatory Activity

The in vitro antithrombotic and anti-inflammatory activity of the lipid extracts that were enriched in omega-3 fatty acids or conventional food products were evaluated on a Chrono-Log 500-Ca aggregometer (Chrono-Log Co., Havertown, PA, USA) that was connected to a computer (Aggro/Link software; Chrono-Log, Hawertown, PA, USA) according to their ability to inhibit the thrombotic and inflammatory lipid mediator of PAF towards platelet rich plasma (PRP) [33]. Aliquots of dissolved lipid extracts that were enriched with omega-3 fatty acids or conventional food products and PAF solution were evaporated under a stream of nitrogen and reconstituted in BSA (2.5 mg/mL saline). The platelet response induced by PAF (10−7 M, final concentration) was measured in PRP before (considered as 0% inhibition) and after the addition of various concentrations of the examined sample. Consequently, the plot of the percentage inhibition (ranging from 20 to 80%) versus the different concentrations of the sample is linear. From this curve, the amount of lipids required for a 50% inhibition (inhibitory amount for 50% inhibition; IA50) against PAF was calculated and expressed in µg for the lipid extracts.

#### *2.6. Marketing Plan: Development*

The 5-year marketing plan in this research was investigated in order to target the objectives for the gross sales of the products under study by a local bakery and cheese company that were involved. The plan was based on sales: (a) in the first two years they would be at the local markets, where the company is active and well-known; (b) in the thirdand fourth-year there would be expansion to the national market; and (c) in the fifth year there would be promotion to the European market, targeting two or three specific countries such as Germany, France, and England. Data analysis was based on the last three years of the company's performance gross sales for its conventional products that were successfully promoted in the local and national Greek market. In addition, the overall company's performance and its plans for growth and expansion in new areas of activities—such as healthy food snacks—were considered when formulating the specific marketing plan. Market trends were also considered at the global level by evaluating existing marketing data on omega-3 functional foods in general and on omega-3 bakery products specifically. The formulated plan considered conventional activities such as advertising, exhibitions, events, etc., as well as modern means such as social media, internet usage, etc. for the promotion of the products and their health claims. Based on the proposed activities each year, and combined with the Greek and global market trends, the feasible six-year projected sales were forecasted and presented in the section below.

#### *2.7. Statistics*

A comparison of the results was performed using the statistical package SPSS *v*.21 using the average value comparison assay for independent samples (Student's t-test for independent samples) at a significance level equal to 0.05.

#### **3. Results**

#### *3.1. Manufacture of Bakery Products*

A representative photo of the three products is given in Figure 2.

#### *3.2. Sensory Evaluation of the New Products*

The organoleptic properties and acceptance of the new products are presented in Figure 3. The overall acceptability is above the set limit for acceptance (5.00) and high enough (7.29 and 7.86 for whole-meal sliced bread and chocolate cookies, followed by 6.36 for breadsticks) to indicate that their fate in the market could be successful from a sensorial point of view. A representative photo of the three products is given in Figure 2.

**Figure 2.** Representative photos for the three functional bakery snacks**. Figure 2.** Representative photos for the three functional bakery snacks. breadsticks) to indicate that their fate in the market could be successful from a sensorial point of view.

*Sustainability* **2022**, *14*, x FOR PEER REVIEW 6 of 12

A representative photo of the three products is given in Figure 2.

**Figure 3.** Sensory evaluation of whole-meal sliced bread, chocolate cookies, and breadsticks. Results are expressed as a mean of the responses from 16 trained panelists. **Figure 3.** Sensory evaluation of whole-meal sliced bread, chocolate cookies, and breadsticks. Results are expressed as a mean of the responses from 16 trained panelists.

1

#### *3.3. Chemical Analysis of the Products 3.3. Chemical Analysis of the Products*

*3.3. Chemical Analysis of the Products*

**Figure 3.** Sensory evaluation of whole-meal sliced bread, chocolate cookies, and breadsticks. Results are expressed as a mean of the responses from 16 trained panelists. Texture Taste After taste The nutritional composition of the new products is depicted in Table 2. According to Regulation 1924/2006 on nutritional claims [10], whole-meal sliced bread is a "source of fiber" since it contains 5.4 g fibers/100 g of product, while the limit for this claim is 3 g fibers/100 g of product and, furthermore, it is a "source of protein" since 16.7 ± 100% of the energy of the product comes from proteins, while the respective limit for this claim is The nutritional composition of the new products is depicted in Table 2. According to Regulation 1924/2006 on nutritional claims [10], whole-meal sliced bread is a "source of fiber" since it contains 5.4 g fibers/100 g of product, while the limit for this claim is 3 g fibers/100 g of product and, furthermore, it is a "source of protein" since 16.7 ± 100% of the energy of the product comes from proteins, while the respective limit for this claim is 12% according to the formula: % energy from protein s = [(g proteins/100 g × 4 kcal/g)/(total energy/100 g)] × 100. Moreover, chocolate cookies (7.03 ± 0.30 g/100 g product) and breadsticks (7.08 ± 0.12 g/100 g product) are both a "source of fiber".

The nutritional composition of the new products is depicted in Table 2. According to

fibers/100 g of product and, furthermore, it is a "source of protein" since 16.7 ± 100% of the energy of the product comes from proteins, while the respective limit for this claim is

**Table 2.** Nutritional composition of whole-meal sliced bread, chocolate cookies, and breadsticks.

1 : Each result is the mean value of triplicate experiment.

The results of the effect of baking on the content of omega-3 fatty acids are shown in Table 3. According to the results, baking does not influence the omega-3 fatty acids content of the final products.

**Table 3.** Effect of baking on omega-3 fatty acids concentration.

1 : Results from experiments, which were run in triplicate, are expressed as mean value ± standard deviation.

Based on the results and Regulation 1924/2006 on nutritional claims [10], wholemeal sliced bread has a "high content" of omega-3 fatty acids since their concentration is 1.96 ± 0.18 g/100 g of the product, while the respective limit for this nutritional claim is 0.6 g/100 g of the product. Moreover, chocolate cookies and breadsticks also have a "high content" of omega-3 fatty acids (0.95 ± 0.06 and 1.90 ± 0.12 g/100 g product, respectively).

#### *3.4. Shelf-Life Assessment*

The shelf life, both sensorial and chemical, of the new products is presented in Table 4. The shelf life, concerning the minimum concentration of omega-3 fatty acids for the nutritional claim, is 18 days at room temperature for whole-meal sliced bread and more than 24 days for chocolate cookies and breadsticks. The sensorial shelf life is 7 days at room temperature for whole-meal sliced bread and 24 days for chocolate cookies and breadsticks. Given this, the combined shelf life of the new products at room temperature is 7 days for whole-meal sliced bread and 24 days for chocolate cookies and breadsticks, which is acceptable for the food market.

#### *3.5. Lipid Extraction and In Vitro Antithrombotic and Anti-Inflammatory Activity*

In order to optimize lipid extraction, two versions of the Bligh and Dyer method [34] for lipid extraction were evaluated for their capacity in enriched flour samples in triplicate experiments. The results showed that the second version that used 1 m aqueous sodium chloride solution 0.5% in acetic acid instead of distilled water for the first version extracted lipids more efficiently. More specifically, 123.3 ± 8.0 mg of lipids per g of flour were extracted by performing the second version of the extraction versus 106.7 ± 7.2 mg of lipids per g of flour, which were extracted by performing the first version of the extraction (*p* < 0.05). The procedure of the second version was further applied to the prepared

**Attribute Product Day 0 Day 4 Day 7 Day 18 Day 24** Appearance 1 Whole-meal sliced bread 7.7 ± 0.3 8.1 ± 0.3 7.8 ± 0.3 Na 2 NA Chocolate Cookies 7.1 ± 0.4 7.8 ± 0.3 7.0 ± 0.3 6.5 ± 0.3 5.3 ± 0.2 Breadsticks 6.9 ± 0.3 6.9 ± 0.3 6.8 ± 0.3 6.6 ± 0.2 5.8 ± 0.2 Odor 1 Whole-meal sliced bread 7.3 ± 0.4 8.0 ± 0.3 7.7 ± 0.3 NA NA Chocolate Cookies 8.1 ± 0.3 8.0 ± 0.3 7.5 ± 0.3 6.8 ± 0.2 6.0 ± 0.3 Breadsticks 7.1 ± 0.4 7.3 ± 0.4 6.8 ± 0.3 6.1 ± 0.3 5.4 ± 0.3 Texture 1 Whole-meal sliced bread 7.3 ± 0.3 7.3 ± 0.4 7.4 ± 0.4 NA NA Chocolate Cookies 7.4 ± 0.3 7.9 ± 0.4 6.5 ± 0.3 6.0 ± 0.2 5.1 ± 0.2 Breadsticks 6.0 ± 0.3 7.4 ± 0.4 7.3 ± 0.4 6.6 ± 0.3 5.3 ± 0.2 Taste 1 Whole-meal sliced bread 7.1 ± 0.3 6.9 ± 0.3 7.1 ± 0.4 NA NA Chocolate Cookies 8.0 ± 0.4 7.9 ± 0.4 7.5 ± 0.4 6.8 ± 0.3 5.4 ± 0.3 Breadsticks 6.6 ± 0.3 7.6 ± 0.3 7.2 ± 0.3 5.9 ± 0.2 5.2 ± 0.2 After taste 1 Whole-meal sliced bread 7.2 ± 0.2 7.2 ± 0.2 7.3 ± 0.3 NA NA Chocolate Cookies 7.9 ± 0.2 7.8 ± 0.2 7.2 ± 0.3 6.6 ± 0.3 5.2 ± 0.1 Breadsticks 6.2 ± 0.3 7.7 ± 0.2 7.0 ± 0.3 5.8 ± 0.3 5.0 ± 0.3 Overall acceptability 1 Whole-meal sliced bread 7.3 ± 0.4 7.3 ± 0.3 7.3 ± 0.3 NA NA Chocolate Cookies 7.9 ± 0.3 7.9 ± 0.3 7.2 ± 0.3 6.5 ± 0.2 5.2 ± 0.3 Breadsticks 6.4 ± 0.3 7.3 ± 0.3 7.1 ± 0.4 5.9 ± 0.3 5.1 ± 0.3 α-Linolenic acid C18:3n3c (g/100 g product) 3 Whole-meal sliced bread 1.3 ± 0.2 1.0 ± 0.1 1.0 ± 0.1 0.59 ± 0.1 NA Chocolate Cookies 1.3 ± 0.2 0.9 ± 0.1 1.1 ± 0.1 0.9 ± 0.1 1.2 ± 0.1 Breadsticks 2.9 ± 0.1 6.9 ± 0.2 7.2 ± 0.3 6.8 ± 0.4 6.2 ± 0.3

foods to extract their total lipids in order to evaluate their in vitro antithrombotic and anti-inflammatory activities.

1 Results are expressed as mean ± standard deviation of the responses from 16 trained panelists; 2 NA: not analysed because mold has been grown on the product; 3 Results are expressed as mean ± standard deviation from triplicate experiment.

The results from the in vitro inhibition of PAF-induced platelet activation are presented in Table 5. Flour enriched in omega-3 fatty acids, whole-meal sliced bread, chocolate cookies, and breadsticks showed higher antithrombotic and anti-inflammatory activities compared to the corresponding conventional samples. More specifically, the enriched flour, sliced bread, chocolate cookies, and breadsticks showed ×4.7, ×4.6, ×1.1, and ×1.6 higher activities compared to their respective conventional samples. The results show that the enrichment of omega 3 fatty acids may result in food products with increased nutritional value, but this enrichment may be affected by the processing that is followed to prepare each product.

**Table 5.** In vitro antithrombotic and anti-inflammatory activity.

1 : Inhibitory amount for 50% inhibition of PAF activity toward washed platelets expressed in µg of lipid extracts. Values from experiments in triplicate are expressed as mean ± standard deviation. 2 : Statistically significant difference of enriched versus conventional products at significance level equal to 0.05.

Lipid mediators are a heterogeneous group of molecules that mediate several physiological cellular functions that, when they are dysfunctional, may lead to the development of many chronic diseases. Food-derived bioactive compounds can beneficially influence metabolism, thereby offering an attractive way to prevent the establishment of chronic diseases.

An increased intake of omega-3 fatty acids from foodstuffs is related to a favorable clinical profile of various chronic diseases. Platelet activating factor (PAF) is one of the most potent inflammatory and thrombotic lipid mediators, playing a crucial role in the initiation and propagation of atherosclerosis. Therefore, the omega-3 fatty acid-induced PAF inhibition is very important in terms of their nutritional value.

#### *3.6. Marketing Plan*

The results of the marketing plan developed in terms of forecasted productions and sales over the first 5 years are shown in Table 6. The first two years, when the products will be promoted to the local market, project that the sales will have a 102.7% annual increase, reaching 75,000€. A 120% sales increase is expected in the third year due to the promotion of the products in the overall Greek market, which will be followed by a 45% medium increase the coming year within the same market. Finally in the last year, with the promotion of the products to international markets, an initial 20% increase in sales is expected, which will increase steadily by 20–30% annually in the years to come based on more promotion internationally. A similar annual increase is expected for the production capacity from year to year as well: 2nd from 1st year (103.3%), 3rd from 2nd year (131.7%), 4th from 3rd year (42.1%), and 5th from 4th year (20.9%). The expected differences in production and sales between the three products over the years are based on the company's production and sales for the corresponding conventional products, which are already on the market.

**Table 6.** Expected productions/sales of the new omega-3 bakery products.

1 : Sales at the local market only; 2 : Sales at the Greek market as well; 3 : Sales in targeted European countries as well.

The proposed marketing plan predicts 288,000€ expected annual sales in the 5th year from the three selected products. This amount, for a company of 5,000,000€ total sales with hundreds of products produced, is a satisfactory sales target from three products alone (5.7%), and a good driving force for the company to invest in for the implementation of the five-year proposed marketing plan.

## **4. Discussion**

The relevance and innovation of the present study lies in the incorporation of omega-3 fatty acids as an ingredient for manufacturing healthy bakery snacks that offer new opportunities for the local Greek snack market. The three bakery snacks manufactured are rich in omega-3 fatty acids since their content is higher than 0.6 g of omega-3 fatty acids per 100 g of baked product, and they are also a source of dietary fiber since their content is higher than 3 g of fiber per 100 g of final product. Moreover, whole-meal sliced bread is, in addition, a source of protein since more than 12% of the % energy comes from it.

The produced whole-meal sliced bread, chocolate cookies, and breadsticks have 2.0 g, 1.7 g, and 2.9 g of -linolenic acid, respectively, and daily bread consumption of 160 g [35,36] is higher than that of the other two goods that are taken on a more irregular basis.

Taking into account the frequency and quantity of an adult's average consumption of manufactured products—given that they need 2.22 g of a-linolenic acid per day [37]— whole-meal sliced bread looks to practically meet the daily requirements for a-linolenic acid, being a better source of omega-3 fatty acids than chocolate cookies or breadsticks.

Omega-3 fatty acids incorporation also enhanced the antithrombotic and antiinflammatory bioactivity in terms of the in vitro inhibition of platelet activating factor against platelet rich plasma, thereby indicating health protective properties compared to the conventional formulations.

Omega-3 fatty acid-enriched bakery products were well accepted by panelists for their sensorial attributes.

The findings of this study demonstrated that omega-3 fatty acids might represent a valuable ingredient to improve the nutritional and health-protective properties of bakery snacks. Indeed, linseed (*Linum usitatissimum* L.)—generally known as flaxseed—has been demonstrated to boost immunity against viral infections as well as control cytokine storms and inflammatory mediators [38–43]. Moreover, due to the high number of both soluble and insoluble dietary fibers, flaxseed is beneficial for gut health, thereby boosting the immune system [44,45].

#### **5. Conclusions**

In conclusion, the design and preparation of enriched bakery snacks with flaxseed as a source of omega-3 fatty acids may represent a novel opportunity for the development of functional foods that are sustainable in the food market and could also contribute to the prevention of public health issues when consumed.

**Author Contributions:** Conceptualization, supervision, methodology H.C.K. and D.S.; investigation, writing—original draft preparation and writing—review and editing H.C.K., C.N. and D.S.; software, data curation and resources H.C.K. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**


**4.2 Microorganisms**

Although there are some useful microorganisms as essentially required in important biological processes such as fermentation, many microorganisms have been life-threatening enemies in long history. Especially, food-born infectious microorganisms are very near to our everyday life to often cause epidemics. The recent *E coli* O157-outbreak in Europe carried by fresh vegetables killed more than 50 people and infected about 4400 people. This incidence reminds that we are not well prepared against such outbreaks. Especially the insufficient technical levels in analysis of microorganisms for surveillance and investigation in a global network is of a great concern. While constructing or improving the global infrastructure against such outbreaks, adopting metrological principles as possible will reduce controversy or legal suits associated with the interpretation on the analytical data. In this regard, CCQM of BIPM moves in a swift manner to organize an international symposium and ad hoc Steering Group on Microbial Measurements to ensure food quality and safety. What to do for establishment metrology in microbial measurements are yet to be rummaged among experts in the field and metrologists. Measurements are for detection, identification, and characterization, and enumeration of target microorganisms. Microorganisms are proliferating dependent upon environmental conditions and frequently mutated. Due to these properties, none of quantification, identification, or characterization of a microorganism is straightforward. Characteristic properties and their relevance on infection should be carefully thought and selected for effective measurement. In addition, such measurements had better be in the domain of metrology, which will guarantee the comparability in a global network (Verhoef et al., 2011). Such a grand task is being organized among relevant experts groups and metrological institutes.

#### **4.3 Glycans**

Glycan is a polysaccharide, which is a linear or branched polymer of monosaccharide units linked by glycosidic bonds. Glycans exist in nature as free or glycoconjugates (e.g.

4. **Stability** — confirms the integrity of nuclear genomes using cytogenetics and SNP

5. **Pluripotency** — confirms the ability of hESCs to generate various differentiated cell types using teratoma formation and characterization, as well as embryoid bodies formation. Appropriateness of such tests is to be determined by experts in the field, and the quantities here are more likely method-dependent. As discussed in the later section, metrology for such quantities needs to be established to support 'metrological execution' of the given measurement protocols, where those protocols is not fully established yet. In this regard, the advancement of cell testing protocols should be closely tracked for prompt implementation

Counting of well specified cells is rather straight forward in terms of quantification. CCQM BAWG has executed or is preparing for two international comparisons: 1) flow cytometric determination (CCQM-P102) and 2) microscopic determination (CCQM-P123). It is noteworthy that physical parameters such as motility and morphology could be important in characterization of cells. Among NMIs, NIST scientists are in particular active in such

Although there are some useful microorganisms as essentially required in important biological processes such as fermentation, many microorganisms have been life-threatening enemies in long history. Especially, food-born infectious microorganisms are very near to our everyday life to often cause epidemics. The recent *E coli* O157-outbreak in Europe carried by fresh vegetables killed more than 50 people and infected about 4400 people. This incidence reminds that we are not well prepared against such outbreaks. Especially the insufficient technical levels in analysis of microorganisms for surveillance and investigation in a global network is of a great concern. While constructing or improving the global infrastructure against such outbreaks, adopting metrological principles as possible will reduce controversy or legal suits associated with the interpretation on the analytical data. In this regard, CCQM of BIPM moves in a swift manner to organize an international symposium and ad hoc Steering Group on Microbial Measurements to ensure food quality and safety. What to do for establishment metrology in microbial measurements are yet to be rummaged among experts in the field and metrologists. Measurements are for detection, identification, and characterization, and enumeration of target microorganisms. Microorganisms are proliferating dependent upon environmental conditions and frequently mutated. Due to these properties, none of quantification, identification, or characterization of a microorganism is straightforward. Characteristic properties and their relevance on infection should be carefully thought and selected for effective measurement. In addition, such measurements had better be in the domain of metrology, which will guarantee the comparability in a global network (Verhoef et al., 2011). Such a grand task is being

organized among relevant experts groups and metrological institutes.

Glycan is a polysaccharide, which is a linear or branched polymer of monosaccharide units linked by glycosidic bonds. Glycans exist in nature as free or glycoconjugates (e.g.

profile

of metrology in such testing protocols.

investigations (Ni and Chiang, 2007).

**4.2 Microorganisms**

**4.3 Glycans**

glycoproteins, proteoglycans, and glycolipids) with cell or tissue. Their function in living organisms is important. Especially the conjugated glycans in proteins and lipids affect physicochemical properties, such as solubility, stability as well as biological properties. Particularly, glycosylated biopharmaceuticals contain various glycoforms, and the heterogeneity of carbohydrate moiety is altered according to the manufacturing process. For this reason, glycan analysis is an essential part of protein characterization in development and quality control of biopharmaceuticals as well (Harazono et al., 2011). Therefore, the study of protein with post-translational modifications has increased explosively in recent proteomic research, and this phenomenon represents importance of glycans in this area (Wei and Li, 2009). Though the advanced mass spectrometry plays a versatile role in current glycan analysis, due to the complexity of structure and the difficulty of isolation of glycans, there is no single method which can clearly and efficiently analyze all of them (Zaia, 2010). The glycan analysis is processed by a combination of several methods, such as monosaccharide composition analysis after the hydrolysis (Harazono et al., 2011), profiling of liberated glycans (Pabst and Altmann, 2011), and structural assignment (Zaia, 2010). A number of methods have been developed for glycomics using either chromatographic, electromigratory or mass spectrometric method as qualitative and quantitative methods. Glycomics has been recognized as an emerging technology in this post-genome generation in worldwide. The term 'glycomics' includes the systematic study of all glycan structures as well as the comprehensive study in genetic, physiologic and pathologic areas. The dynamic complexity and variety, and the essential roles in physiology, drive the need of generally accepted methodology for glycoconjugates. However, the challenging task to implement metrological rigorousness in such complex analysis is yet to be launched. As new glycomics and glycoproteomics problems will require unique solutions, a wide range of methodological and instrumental techniques will be needed to address them.


*Edited by Heimo Mikkola*

Owls are one of the most fascinating bird families. They have inspired much folklore and superstition worldwide. This book discusses this nocturnal species and their unique physiology and anatomy. It includes owl studies from countries including Bulgaria, Ecuador, Iraq, Slovakia, Syria, and Africa.

Published in London, UK © 2023 IntechOpen © Alexander Pytskiy / iStock

Owls - Clever Survivors

Owls

Clever Survivors

*Edited by Heimo Mikkola*


5.2.1 Introduction

For a copper capsule, the difference of ε between dry a soil and moderately wet (e.g., less than 15% water) is negligible (less than 1%); therefore, it is not necessary in this case to know exactly the soil humidity to perform the correction, which is

Surrounding media 40K Uranium Thorium

Use of Gamma Radiation Techniques in Peaceful Applications

C soil 0.96 0.95 0.94 0.89 0.95 0.91 C soil + 15% water 0.97 0.95 0.96 0.90 0.96 0.91 S soil 1.03 0.97 1.10 0.95 1.08 0.96 S soil + 15% water 1.05 0.98 1.13 0.96 1.11 0.97 S soil + 30% water 1.06 0.98 1.16 0.97 1.14 0.98 S soil + 50% water 1.07 0.96 1.20 0.95 1.17 0.96 Pure water 1.35 1.01 1.82 1.03 1.70 1.03

Encapsulating Encapsulating Encapsulating Polyethylene Copper Polyethylene Copper Polyethylene Copper

In practice, the limestone soil and the siliceous soil are two extreme cases, which make only a difference of 5% for copper on the value of ε, so only a coarse knowledge of the cashing medium is necessary for the calculation of e with an high accuracy. The variations of ε for 40K with the absorber or medium are less important than for uranium or thorium. This is due to a smaller contribution of low energies to the

An important consequence of the foregoing is that, for the experimenter, copper (or a close material) is well used when the composition (especially moisture) of the soil is not well known. The error made a priori on the value of ε will thus be

On the other hand, in most cases, the dispersion on ε related to the relative intensity of the three gamma radiation sources is insignificant. Indeed, the potassium-40, uranium and thorium series generally intervene for approximately 1/3

In general, it is difficult to determine the respective contribution of the potassium-40, uranium and thorium series to the total dose rate. However, in most cases, the contribution of these three radioelements does not exceed 60% and approxi-

The results obtained with our program under the same conditions regarding the

dosimeter, the surrounding environment, the capsules and the sources of the gamma rays are slightly superior to the theoretical results of G. Valladas [7]. This small difference (in the order of 3%) can be explained by the fact that the nominal values for capsule thickness have been used instead of the mean values which take into account geometry, also to a lesser extent by the fact that self-absorption has not been taken into account in our calculation. The effect of this last correction was

each, and significant deviations from this proportion are rare.

Values of ε for infinite uniform medium with 1 g m2 capsule wall thickness [5].

• ε = 0.97 0.01 for S soil + 15% water

• ε = 0.92 0.01 for dry C soil.

estimated to be less than 1%.

122

mate values on the whole with acceptable dispersion may be proposed.

an advantage.

Table 2.

reduced.

For example,

40K spectrum (no low energy lines).

The gamma radiation self-absorption coefficient is of great interest in activation analysis. Since it is difficult to measure this coefficient, various calculation methods have been developed.

Measuring the self-absorption coefficient is not a simple thing. The physicists who have faced this problem, for a long time, have always used methods of statistical or non-statistical computation: Parallel beam methods, Monte-Carlo method and many other methods. For our part, we have developed an original technique calculating the self-absorption coefficient of multienergetic γ-radiations [13].

In this chapter, we are presenting a method that we have developed, this which allows us control and calibrate the activation analysis experiments [13]. This method consists of simulating the interaction processes of gamma rays induced by neutron activation of various samples by using the Monte Carlo method adapted to experimental conditions.