*Article* **Impact of Different Epoxidation Approaches of Tall Oil Fatty Acids on Rigid Polyurethane Foam Thermal Insulation**

**Arnis Abolins 1 , Ralfs Pomilovskis 1,2, Edgars Vanags 1 , Inese Mierina 2 , Slawomir Michalowski 3 , Anda Fridrihsone 1 and Mikelis Kirpluks 1,\***

**Abstract:** A second-generation bio-based feedstock—tall oil fatty acids—was epoxidised via two pathways. Oxirane rings were introduced into the fatty acid carbon backbone using a heterogeneous epoxidation catalyst-ion exchange resin Amberlite IR-120 H or enzyme catalyst *Candida antarctica* lipase B under the trade name Novozym® 435. High functionality bio-polyols were synthesised from the obtained epoxidated tall oil fatty acids by oxirane ring-opening and subsequent esterification reactions with different polyfunctional alcohols: trimethylolpropane and triethanolamine. The synthesised epoxidised tall oil fatty acids (ETOFA) were studied by proton nuclear magnetic resonance. The chemical structure of obtained polyols was studied by Fourier-transform infrared spectroscopy and size exclusion chromatography. Average molecular weight and polydispersity of polyols were determined from size exclusion chromatography data. The obtained polyols were used to develop rigid polyurethane (PU) foam thermal insulation material with an approximate density of 40 kg/m3 . Thermal conductivity, apparent density and compression strength of the rigid PU foams were determined. The rigid PU foams obtained from polyols synthesised using Novozym® 435 catalyst had superior properties in comparison to rigid PU foams obtained from polyols synthesised using Amberlite IR-120 H. The developed rigid PU foams had an excellent thermal conductivity of 21.2–25.9 mW/(m·K).

**Keywords:** tall oil fatty acids; ion-exchange resin; lipase enzyme catalyst; high functionality polyols; rigid polyurethane foam


**3. Case studies – results and discussion**

In the preceding items, the general characteristics of the International Amazon were contextualized, with emphasis on the Brazilian Amazonia, in addition to addressing the problems faced in the construction of highways in this region. This Item presents some case studies that sought to circumvent the problems of scarcity of stone material near the construction site and the presence of soil layers with low bearing capacity in the natural foundation (subgrade) of the highways built in the Brazilian Amazonia environment.

**Figure 1** shows the locations of the case studies analyzed here.


*4.3. Sequence Analysis of the SlPKS4 Gene*

The whole genome sequencing data was analyzed by antiSMASH [39]. The phylogenetic analysis was conducted with the MEGA7 software [40], with the amino acid sequences of SlPKS4 and reported PKSs synthesizing isocoumarin derivatives retrieved from National Center for Biotechnology Information (NCBI). The conserved domain of the SlPKS4 protein was scanned by the InterProScan program [41]. Comparative analysis between the gene cluster and other isocoumarin BGCs (Figure S7) was conducted by clinker [42].


**Meet the editor**

Beatriz Peña-Acuña is an associate professor (permanent) accredited by Spanish Quality Agency ANECA (2015). She teaches in the Department of Business Administration and Management to Grades and master's and postgraduate degrees at UCAM (Murcia, Spain) in Multidisciplinary Formation: Hispanic Philology, Diploma in Religious Studies and Journalism. She leads several doctoral theses and

was awarded Extraordinary Doctorate Award (2012). She leads a validated international and multidisciplinary research group called 'Personal Development'. Scientific production is prolific such as *Communication Management and Management Skills* (Dykinson), *Politic Communication* (ACCI), *Research Journalism* (ACCI) and *Specialized Journalism* (two volumes, ACCI). She directs several collections of books in international publishing houses. She has visited several international universities and gives international conferences. She has been awarded the President's Lifetime Achievement Award (2015).

## Contents

#### **Preface XI**


**6. Lessons learned**

plug-in), (ii) expert users regard ERMaster is being a tool that is easy to learn, easy to use and easy to understand and had no trouble remembering how to use it, (iii) ERMaster is designed ergonomically using menus, action bars and easy access icons, but some users requested the addition of options of interest (for example, export DBs to Excel), and (iv) users consider the

template editing, edit tracing, etc.)

83% of users consider it is an easy to use tool, once it is installed. However, they consider that only expert users or those with an advanced level of average knowledge could easily install the tool due to the need of installing first Java and then Eclipse. Certain novel users complain about not having access to this tool due to the difficulty of installing it.

All users consider ERMaster's interface is easy to remember even through

Menus, action bars and icons are easily accessible, but some users asked for the addition of an extra 10% of options of interest (for example,

some time may have passed since they last used it.

The usability techniques have been created for another type of software developments, i.e. they have not been conceived with the specific characteristics of the OSS development process in mind. For this reason, it is necessary to adapt these techniques. These adaptations are based on the adverse conditions these techniques present. Some adverse conditions can be overcome using certain web artefacts (for instance, wikis, forums, blogs, etc.), which are known by the OSS community. As a result, many of these adaptations will be familiar to the members of this community, which favours to a certain extent the application of these usability techniques.

The adaptations of the focus groups technique are mainly two. Firstly, users participate online though a web artefact: a forum. Secondly, the usability expert is replaced by a developer, an experienced user or an HCI student under supervision of a mentor. In our particular case, the

After applying the focus groups technique to the ERMaster project, we were able to confirm that it is very hard to get a representative set of users. We believe that the main reason for this is that users are unmotivated. We had to be persistent and use different communication mechanisms (for example, personal wikis and electronic mails) to get the consent of the principal developers (only one out of five principal developers responded). The biggest problem with applying the focus groups technique was user availability: most users are volunteers and had very little spare time. In fact, the participants did not have the time to enter their

ERMaster work environment to be adequate, as there are Help and Query tools.

In this section we discuss and answer the research question of this study.

**RQ: How to incorporate the focus groups technique in a real OSS project?**

expert was replaced by an HCI student under the supervision of a mentor.

**5. Discussion of results**

**Points to be considered Summary of results**

**Table 4.** Summary of the results obtained from the questionnaire.

13. What problems have you often come across as an ERMaster

14. Do you think that the ERMaster interface is easy to remember?

15. How do you think the ERMaster interface should be changed or

user?

56 Trends in E-learning

added to?

The lessons that we have learned from applying the focus groups technique in the OSS project are as follows:

• OSS projects that want to apply the steps to incorporate techniques and need to find an expert in HCI or an HCI student guided by an expert mentor can do so by publishing advertisements in the webpage, forum, wiki and blogs of the project. Furthermore, the administrator of the OSS project can get in touch with universities to encourage expert usability students to collaborate in the application of techniques to improve the usability of an OSS application.

the working philosophy of the OSS community where anyone who wants to is welcome to participate. In sum, our proposed adaptation does not place any constraints on the number of technique participants. This adaptation is a response to the OSS community working philoso-

Applying a Usability Technique in the Open Source Software Development Process: Experiences…

http://dx.doi.org/10.5772/intechopen.74862

59

The focus groups technique is useful for gathering opinions and suggestions from participant users for the product concept development activity and its results are descriptive. After analysing and applying the focus groups usability technique in requirements engineering activities in OSS developments, we found that there are adverse conditions that are an obstacle to its application like, for example, the shortage of OSS users interested in applying the technique, community geographical and temporal distribution and OSS community motivation.

We believe that, in order to improve the integration of usability techniques in OSS projects, the OSS community has to start attaching importance to and raising awareness about the repercussions that the issues addressed by the HCI field have on software development. Additionally, as HCI techniques need to be adapted to overcome the adverse conditions for adoption in OSS development projects, the OSS community also has to broaden its view of software development in order to consider usability and not focus exclusively on feature development. In the future, we aim to conduct further case studies to adapt and apply other usability techniques in OSS projects. We will analyse other web artefacts that can be adapted to improve communication in OSS communities (e.g., social networks) and gradually raise the awareness of OSS developers about the benefits of applying HCI usability techniques.

This research was funded by the SENESCYT-Ecuador, and Quevedo State Technical University. Also this research was funded by the Spanish Ministry of Education, Culture and Sports FLEXOR (TIN2014-52129-R) and TIN2014-60490-P projects and the eMadrid-CM project (S2013/ICE-2715). Finally, this research received funding from the "DIUDA 22316 Project" of

, Mayra Llerena2

1 Department of Computer Engineering, Autonomous University of Madrid, Madrid, Spain

2 Department of Education Didactics, Autonomous University of Madrid, Madrid, Spain

3 Department of Computer Engineering and Computer Science, University of Atacama,

, John W. Castro3

and Silvia T. Acuña1

**Acknowledgements**

the University of Atacama.

\*, Nancy Rodriguez1

\*Address all correspondence to<EMAIL_ADDRESS>
**Author details**

Lucrecia Llerena1

Copiapó, Chile

phy rather than to an adverse condition posing an obstacle to technique application.


If currency 2 would depreciate, typically no profits (nor losses) occur for central bank 2 as this scenario most likely occurs when central bank 2 has exhausted its foreign reserves. If it had foreign currency reserves, then it would book a profit, or, if it were prudent and conservative, it would book instead revaluation reserves on its liability side. Returning to the case of an appreciation of currency 2: the accounts of country 1 are not affected, but the country 2 central bank books a loss and negative equity. If the Government of country 2 wants to repair this negative equity, then it may issue additional debt (or impose extra taxes on households) and recapitalize the central bank. At the end, the appreciation is at the expense of the wealth of household 2. One could say that the household 2 sold real assets to country 1, but was only paid for half of the value—retroactively because of the devaluation of currency 1 (USD).

### *7.3.2 Flexible Exchange Rate Systems*

In Table 7.12, we denominate the accounts of country 1 in currency 1, and those of country 2 in currency 2, and introduce the exchange rate α, i.e. α units of currency 2 are worth one unit of currency 1. Now, **the central bank balance sheet is no longer available for counterbalancing private balance of payment flows**. Instead, the private market participants have to equilibrate the balance of payment on its own. Below, this takes place by letting banks create cross border claims and liabilities between each other, so that eventually the foreign exchange market is in equilibrium. Now call *ca* the capital account imbalance contributed by the household. The total capital account balance will be ca+caB, if we call caB the capital account contribution of the banking system. Necessarily, ca+caB =cu, i.e. the total capital account exactly balances the current account. This obviously implies that caB = cu – ca, i.e. the capital account contribution of the banking system will have to equal the difference between the current account imbalance and the capital account imbalance contributed by the households. If banks are less willing to provide some elasticity be entering crossborder exposures, then the adjustment of the exchange rate to imbalances of payment flows stemming from households will be more violent. In other words, the readiness of the financial system to look through short-term fluctuations of payment flows and to take temporarily cross border exposures is essential in this system to be relatively stable (in conjunction of course with adequate central bank and fiscal policies of the public authorities in both countries). If the private financial sector is only limitedly willing to provide elastic cross-currency liquidity services, this will imply more volatile foreign exchange rates, and possibly require a more activist central bank (through more frequent interest rate adjustments, or even sporadic foreign exchange interventions). This could also be a consequence of tight regulations of banks' risk taking, or a lack of economic capital of banks that makes them unwilling to take risks anyway. Bank 1 has accepted to export capital into country 2 by depositing foreign currency in bank 2, while bank 2 has accepted to import capital by getting indebted towards bank 1.

The net foreign position of the countries has evolved as it would have done under any other international monetary regime—according to the current account imbalance. **The net foreign position is also impacted by the exchange rate**. If the claim is denominated in currency 2, then a devaluation of currency 2 (i.e. an increase in α) implies that the net foreign position of country 1 (in currency 1) declines (while it did not change for country 2), etc. The central bank foreign reserves do not change, i.e. the central bank is neither involved in current account nor capital account flows.

### *7.3.3 The European Monetary Union*

A monetary union like the euro area can be interpreted as a fixed exchange rate system in which the automatic creation of intra-central bank claims and liabilities plays the role of gold/foreign reserves/IMF loans in standard fixed exchange rate systems. The intra-central bank claims and liabilities are in the case of the euro area the so-called TARGET2 balances, which have found some attention starting in 2011 (e.g. Sinn and Wollmershäuser 2012; Bindseil and König 2012; Buiter and Rabhari 2012). The capital flow mechanics in the years up to 2012 are reviewed in more detail in Lane (2013). A recent comprehensive treatment is Hellwig (2019). The system of financial accounts in Table 7.13 assumes that country 1 has a balance of payment surplus and country 2 a balance of payment deficit. We assume that both current account and capital account imbalances originate from the household. The two households contribute to capital flight to the same extent by shifting bank deposits from country 2 to country 1. The payment matching the current account transaction is assumed to be from the account of household 2 with bank 2 to the account of household 1 with bank 1.

The Eurosystem consolidated balance sheet will look as follows is shown in Table 7.14.

Table 7.13 made a number of simplifications: for example, there are no crossborder loans of banks to corporates, and the Eurosystem does not invest into securities. These additional elements could be integrated of course.

**Table 7.12** Two countries' accounts in a flexible exchange rate system

In contrast to foreign reserves, T2 balances are not limited. However, one constraint is the central bank collateral framework and to what extent banks can close the funding gap created by the Balance of Payment deficits through additional central bank credit. This is why Hans-Werner Sinn and other ECB critics have identified the**ECB collateral framework** as one key factors that allowed the Eurosystem to contribute to overcoming the balance of payment crisis associated with the sovereign debt crisis of 2009–2012. Once the cumulated Balance of Payment deficits exceed the initial level of banknotes circulating in country 1, the banking system would be in a liquidity surplus and the Eurosystem consolidated balance sheet would start to lengthen one-to-one with further surpluses of country 1.

**Table 7.13** Two countries' accounts in the European Monetary Union exchange rate system

### *7.3.4 Foreign Reserves*

Although in principle the time of universal fixed exchange rates ended in the 1970s, many central banks continue to manage their exchange rate by letting their foreign reserves fluctuate accordingly. From 2000 to 2013 foreign reserves increased in an unprecedented manner, with China overtaking Japan as the largest holder of official foreign reserves in 2005 and reaching in 2013 close to 4 trillion USD equivalent of foreign reserves. During the same period, the Eurosystem was also surpassed by a number of emerging economies. Switzerland is unique in terms of being a small advanced economy and nevertheless ranking third—reflecting its combat against appreciation in view of safe-haven flows in the context of the euro area sovereign debt crisis. How can one explain this rapid build-up of unprecedented foreign reserves in the years until 2014? IMF (2011, 9) reports the following most frequent answers to the question about the reasons for holding (high) reserves: 80%: "Buffer for liquidity needs"; 60%: "Smoothing of exchange rate volatility"; 30%: "Management of exchange rate level". One might speculate that the frequency of answers also reflect how potentially controversial different explanations are. In reality, the management of the exchange rate level, i.e. preventing appreciation, has likely been the most important reason for the very steep trend of reserve accumulation, which goes beyond needed liquidity buffers.

### **What do foreign reserves consist of?**

As the IMF annual report for 2014 (appendix I, page 1) reveals, foreign reserves at the end of 2014 consisted to a very large extent of **foreign currency** (86%), while **gold** came second (10%) and **IMF related reserves** (including SDRs) were third (4%). The **currency composition of foreign reserves** at the end of 2013 was (according to the IMF annual report for 2014) dominated by USD holdings (66%), followed by the EUR (24%) and the GBP (6%). **In which form were the foreign exchange reserves held?** McCauley and Fung 2003 (see also Borio et al. 2008) report that in the year 2000, 75% were held in the form of securities, and 25% in the form of deposits with banks and money market instruments. The majority of deposits is offshore, i.e. not deposits in USD with US banks, but with banks located in other jurisdictions (mainly London, or other global foreign exchange centres).

Foreign reserves may be built-up in particular in four ways: (i) Accumulated balance of payment surpluses under a fixed exchange rate system (or managed float). (ii) Creation of mutual foreign reserves through a currency swap, possibly including the involvement of an international organisation like the IMF. This neither requires Balance of Payment surpluses, nor will it put pressure on the exchange rate. (iii) Obtaining the foreign claims without counter-flow through e.g. a war indemnity, or a grant. (iv) Acquiring reserves in the market without corresponding balance of payment surpluses, assuming variable exchange rates. Banks will, with some elasticity, finance this, i.e. will accept the corresponding capital account flows. However, the exchange rate of the country accumulating foreign reserves in this way will likely decline to some extent. Eventually balance of payment surpluses may then kick in as a consequence of the devaluation.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **References**

U. Bindseil and A. Fotia, *Introduction to Central Banking*, SpringerBriefs in Quantitative Finance,

https://doi.org/10.1007/978-3-030-70884-9

———. 2013. Central Bank Liquidity Provision, Risk-Taking and Economic Efficiency. ECB Working Paper, No. 1542, May.

Bindseil, Ulrich, Marco Corsi, Benjamin Sahel, and Ad Visser. 2017. The Eurosystem Collateral Framework Explained. Occasional Paper Series, No. 189, May.

———. 2015. The Financial Risk Management of the Eurosystem's Monetary Policy Operations, August, 52.

———. 2020c. Primary and Secondary Lending Programs.

———. 2020d. Speech by Chair Powell on New Economic Challenges and the Fed's Monetary Policy Review. Board of Governors of the Federal Reserve System, 27 August.

———. 2016. Measuring the Natural Rate of Interest Redux. *Business Economics* 51 (2): 57–67.

Lavoie, Marc. 2014. *Post-Keynesian Economics: New Foundations*. Edward Elgar Publishing.

S&P, Standard & Poor's. 2020. 2019 Annual Global Corporate Default and Rating Transition Study.


**2. Textile thermoelectric materials**

with conductors. When temperature difference exists between the two sides of thermoelectric materials, the charge carriers in the materials will be driven to flow from the hot side to the cold side, and thus a thermo-voltage can be formed. As key components in a thermoelectric system, the energy conversion efficiency of a thermoelectric material can be evaluated by a

In this equation, σ is electrical conductivity, S is Seebeck coefficient, *κ* is thermal conductivity, T is absolute temperature, and ZT is a dimensionless value without upper limit. The value of

σ is called power factor, which also can be used to represent the thermoelectric performance of materials where the impact of thermal conductivity is secondary. For example, in polymerbased thermoelectric materials, power factor is widely used, since most polymers have much lower thermal conductivity than inorganic semiconductors and then their thermoelectric per-

Now, two main strategies can be adopted to improve the efficiency of thermoelectric system. One is to increase the ZT value of the thermoelectric materials, and the other is to adjust the structure of TEG for better utilization of heat energy. In past few years, the thermoelectric efficiency of bulk inorganic metal alloys has been improved significantly. A record-breaking high ZT of 2.6 achieved by SnSe single crystals at 923 K was reported in 2014, which gives thermoelectric generators a better application prospect in future [10]. Meanwhile, a new solar heat TEG system has been developed. By combining solar heat collection and thermoelectric

conversion technologies, the new system could possess a peak efficiency of 7.4% [11].

Nowadays, with the rapid development of wearable and flexible electronics, the field of flexible thermoelectric materials and generators were growing dramatically. Various flexible thermoelectric materials were developed in form of films, papers, fabrics, and even fibers. Thermoelectric films are most studied among them, while the research about thermoelectric fabrics and fibers are just beginning very recently. Generally, these flexible thermoelectric materials are mostly made by organic polymers or polymer/inorganic semiconductor composites, so that they are easier to fabricate on large scales. However, the thermoelectric efficiency of these polymer-based flexible materials is much lower than the inorganic semiconductors. Hence, the methods to further improve the thermoelectric efficiency of these flexible materials

Although fabrics, fibers and polymer films are all flexible materials, textile thermoelectric materials have exhibited more competitive characteristics than simple films, especially on the wearable application. Textiles composed of different sets of fibers and yarns have excellent structure design flexibility, which allows them to meet the requirement of flexible, wearable, nontoxic, light weight, and even washable for wearable devices so that we can utilize our human body heat as power source. Until now, many pioneering research works have been conducted on this area and showed gratifying results. Therefore, in this chapter, the development of these textile thermoelectric materials and generators will be thoroughly

*κ T* (1)

thermoelectric figure of merit, also called ZT value [9]. As shown in Eq. (1).

ZT = *σ S*2 \_\_\_

24 Bringing Thermoelectricity into Reality

formance will be mainly decided by the power factor.

will still be a hot research issue in future.

described.

S2

Textile is an ideal candidate to utilize our human body heat to generate power due to its excellent flexibility, wearability, comfort, and air-permeability properties, especially compared with those rigid bulk electronic materials and impermeable polymer-based flexible films. Thus, it is promising to develop textile material-based thermoelectric materials and generators to utilize our body heat. In the past few years, many research works have been done on both textile thermoelectric materials and fabric generators, which have paved the way and given us inspirations for future wearable thermoelectric systems.

#### **2.1. Fabric-based thermoelectric materials**

Fabric finishing and coating are mature technologies in textile industry that can be easily used for large-scale production. Thus, it is a cost-effective and practical way to added effective thermoelectric materials on fabrics for both organic and inorganic materials. In 2015, Yong Du et al. prepared a textile thermoelectric material by coating commercial polyester fabrics with PEDOT:PSS [12]. Their adopted a simple dip coating process to fabricate textiles with thermoelectric functions. **Figure 1** shows the dip coating process the prepared thermoelectric textile strip by Tong Lin. P-type PEDOT:PSS doped with DMSO was acted as effective thermoelectric materials. Commercial available polyester fabric was used as a flexible substrate. Both advantages of textiles and PEDOT:PSS were utilized. The prepared textile materials exhibit electrical conductivities ranged from 0.5–3 S/cm and Seebeck coefficient of 15.3–16.3 μV/K at 300 K. The highest power factor of 0.045 μWm−1 K−2 was achieved at 390 K. Although the thermoelectric efficiency of this flexible textile thermoelectric material is not quite satisfied, this is

**Figure 1.** (a) A dip coating process and (b) PEDOT:PSS coated polyester fabric by Yong Du et al. [12].

still an initial attempt to applying fabrics into thermoelectric application. The low efficiency may also attribute to the used organic polymer thermoelectric materials. Choosing high efficient inorganic semiconductors especially their nano-sized counterparts as fabric coating materials, the thermoelectric efficiency coated fabrics could be further improved.

Another example is to use nano inorganic materials to produce thermoelectric fabric. In 2015, Chongjian Zhou et al. prepared a thermoelectric fabric made by inorganic nanowires and organic polymer composite [13]. A simple five-step vacuum filtration process was employed. Cu1.75Te nanowires sheet was fabricated by filtration, hot press, and annealing first, and then PVDF solution was drop cast on the nanowire sheet. After a heating process, a flexible composite material was formed. The composite structure and prepared fabric film are shown in **Figure 2**. The prepared flexible fabric film has a high electrical conductivity of 2490 S/cm, and a Seebeck coefficient of 9.6 μV/K at room temperature, which resulted in a power factor of 23 μWm−1 K2 . The performance of prepared fabric could keep steady after 300 cycles of bending tests. The results indicate that this easy to scale-up method is effective to fabricate flexible thermoelectric fabric and can be extended to other effective inorganic materials such as Bi2 Te3 , Ag2 Te, or Ag2 Se nanowires.

In 2016, we proposed a water-processable thermoelectric coating material made of waterborne polyurethane, MWCNT, and PEDOT:PSS composite [15]. The optimal electrical conductivity and Seebeck coefficient could achieve ~13,826 S/m and ~10 μV/K at room temperature respectively, and the resulted power factor is about ~1.41 μWm−1 K−2. Compared with other organic-solventbased thermoelectric polymers, this water-based composite exhibits satisfactory thermoelectric performance and good processability. Then, we coated the prepared water-based composite on polyester and cotton yarns respectively, as shown in **Figure 4**. The results show that the fabricated thermoelectric composite can be successfully coated on textile yarns, and polyester filament is more suitable as coating substrate than staple cotton yarn. Besides, these coated yarns

**Figure 3.** (a) Dip coating process of fibers and (b) comparison of uncoated and coated glass fibers made by Daxin Liang

In 2016, Jae Ah Lee et al. using electro-spinning technology to fabricate thermoelectric nanofibers and then twisted into yarns [16]. Polyacrylonitrile (PAN) nanofiber sheets were elec-

thermoelectric materials, and deposited on two sides of the PAN sheets. After a twisting process, thermoelectric yarns can be formed, as shown in **Figure 5**. The highly porous structure of the electrospun yarns are quite resistant to mechanical damage, which enables the yarns to be knitted and woven into fabrics without the significant changes in their electrical conductivity

**Figure 4.** (a) Yarn coating process and (b) polyester yarn coated with waterborne polyurethane thermoelectric composites

and Sb2

Te3

were selected as n-type and p-type

Thermoelectric Textile Materials

27

http://dx.doi.org/10.5772/intechopen.75474

Te3

can be treated as thermoelectric legs and were further used to fabricate fabric TEG.

trospun on two parallel wire collectors. Bi2

and thermopowers.

made by Jinlian Hu [15].

et al. [14].

The preparation of fabric thermoelectric materials is simple and very easy to fabricate on large scale. The integration of inorganic high performance thermoelectric materials would give fabrics better thermoelectric performance than organic polymers. Although fabrics usually have the same 2D flat structure as polymer films, porous fabric structures would have better airpermeability and wearable comfort than polymer films, which is more appropriate for wearable devices.

#### **2.2. Fiber/yarn-based thermoelectric materials**

In addition to fabrics, fibers and/or yarns can be also fabricated into thermoelectric materials. Since fibers and yarns are the structural basis of fabrics, to offer fibers and yarns thermoelectric properties other than fabric itself will give more design flexibility for flexible textile TEGs. In 2012, Daxin Liang et al. reported a fiber thermoelectric material. PbTe nanocrystals were coated onto glass fibers via a dip coating process [14], as shown in **Figure 3**. The PbTe nanocrystal coated fibers have an electrical conductivity of 104.4 S/m, Seebeck Coefficient 1201.7 μV·K−1, and thermal conductivity of 0.228 W·m−1·K−1 at 300 K. The resulted power factor is 0.15 mW·m−1·K−2 at 300 K. It should be noted that the thin coating layer would lower the electrical conductivity of PbTe nanocrystals compared with PbTe bulk crystals.

**Figure 2.** (a) The composite film structure and (b) the picture of flexible fabric film prepared by Chongjian Zhou et al. [13].

still an initial attempt to applying fabrics into thermoelectric application. The low efficiency may also attribute to the used organic polymer thermoelectric materials. Choosing high efficient inorganic semiconductors especially their nano-sized counterparts as fabric coating

Another example is to use nano inorganic materials to produce thermoelectric fabric. In 2015, Chongjian Zhou et al. prepared a thermoelectric fabric made by inorganic nanowires and organic polymer composite [13]. A simple five-step vacuum filtration process was employed. Cu1.75Te nanowires sheet was fabricated by filtration, hot press, and annealing first, and then PVDF solution was drop cast on the nanowire sheet. After a heating process, a flexible composite material was formed. The composite structure and prepared fabric film are shown in **Figure 2**. The prepared flexible fabric film has a high electrical conductivity of 2490 S/cm, and a Seebeck coefficient of 9.6 μV/K at room temperature, which resulted in a power factor of 23 μWm−1 K2

The performance of prepared fabric could keep steady after 300 cycles of bending tests. The results indicate that this easy to scale-up method is effective to fabricate flexible thermoelectric

The preparation of fabric thermoelectric materials is simple and very easy to fabricate on large scale. The integration of inorganic high performance thermoelectric materials would give fabrics better thermoelectric performance than organic polymers. Although fabrics usually have the same 2D flat structure as polymer films, porous fabric structures would have better airpermeability and wearable comfort than polymer films, which is more appropriate for wearable

In addition to fabrics, fibers and/or yarns can be also fabricated into thermoelectric materials. Since fibers and yarns are the structural basis of fabrics, to offer fibers and yarns thermoelectric properties other than fabric itself will give more design flexibility for flexible textile TEGs. In 2012, Daxin Liang et al. reported a fiber thermoelectric material. PbTe nanocrystals were coated onto glass fibers via a dip coating process [14], as shown in **Figure 3**. The PbTe nanocrystal coated fibers have an electrical conductivity of 104.4 S/m, Seebeck Coefficient 1201.7 μV·K−1, and thermal conductivity of 0.228 W·m−1·K−1 at 300 K. The resulted power factor is 0.15 mW·m−1·K−2 at 300 K. It should be noted that the thin coating layer would lower the

**Figure 2.** (a) The composite film structure and (b) the picture of flexible fabric film prepared by Chongjian Zhou et al. [13].

electrical conductivity of PbTe nanocrystals compared with PbTe bulk crystals.

.

Se

Te, or Ag2

Te3 , Ag2

materials, the thermoelectric efficiency coated fabrics could be further improved.

fabric and can be extended to other effective inorganic materials such as Bi2

**2.2. Fiber/yarn-based thermoelectric materials**

nanowires.

26 Bringing Thermoelectricity into Reality

devices.

**Figure 3.** (a) Dip coating process of fibers and (b) comparison of uncoated and coated glass fibers made by Daxin Liang et al. [14].

In 2016, we proposed a water-processable thermoelectric coating material made of waterborne polyurethane, MWCNT, and PEDOT:PSS composite [15]. The optimal electrical conductivity and Seebeck coefficient could achieve ~13,826 S/m and ~10 μV/K at room temperature respectively, and the resulted power factor is about ~1.41 μWm−1 K−2. Compared with other organic-solventbased thermoelectric polymers, this water-based composite exhibits satisfactory thermoelectric performance and good processability. Then, we coated the prepared water-based composite on polyester and cotton yarns respectively, as shown in **Figure 4**. The results show that the fabricated thermoelectric composite can be successfully coated on textile yarns, and polyester filament is more suitable as coating substrate than staple cotton yarn. Besides, these coated yarns can be treated as thermoelectric legs and were further used to fabricate fabric TEG.

In 2016, Jae Ah Lee et al. using electro-spinning technology to fabricate thermoelectric nanofibers and then twisted into yarns [16]. Polyacrylonitrile (PAN) nanofiber sheets were electrospun on two parallel wire collectors. Bi2 Te3 and Sb2 Te3 were selected as n-type and p-type thermoelectric materials, and deposited on two sides of the PAN sheets. After a twisting process, thermoelectric yarns can be formed, as shown in **Figure 5**. The highly porous structure of the electrospun yarns are quite resistant to mechanical damage, which enables the yarns to be knitted and woven into fabrics without the significant changes in their electrical conductivity and thermopowers.

**Figure 4.** (a) Yarn coating process and (b) polyester yarn coated with waterborne polyurethane thermoelectric composites made by Jinlian Hu [15].

**3.1. Two-dimensional textile TEGs**

with an internal resistance of 1270 Ω.

output Pmax of ~12 nW.

maximum output power Pmax could achieve 12.29 nW.

A 2D flat TEG structure is also adopted in the first generation of textile TEGs. In 2012, C. A. Hewitt et al. developed a PVDF/CNT composite-based fabric TEG. Several n-type and p-type CNT composite films were alternatively arranged between the insulated PVDF films. The PVDF films are shorter than the CNT composite films. Thus, n-type and p-type CNT composites could form p-n junctions to connect the generator legs by hot press the stacked films, and resemble a felt fabric TEG [17]. The schematic structure of they prepared fabric TEG is shown in **Figure 6**. The prepared TEG composed of 72 layers fabric could generate power about 137 nW

Thermoelectric Textile Materials

29

http://dx.doi.org/10.5772/intechopen.75474

In 2015, Yong Du et al. prepared a 2D fabric TEG by using PEDOT:PSS coated polyester fabric strips [12]. To fabricate the TEG, a whole PEDOT:PSS coated polyester fabric was cut into several strips first. Then, these strips were further adhered on a polyester fabric substrate by silver paint and connected in series with silver wires. Thus, a fabric TEG only composed of p-type materials can be prepared. The TEG arrangement is shown in **Figure 7**. Temperature difference will generate along the fabric length direction. The fabric TEG prototype consisting of five fabric strips could generate 4.3 mV when temperature difference ΔT is 75.2 K. The

The same arrangement can also be achieved by thermoelectric yarns. In 2017, Ryan et al. reported a highly durable thermoelectric silk yarn made by dyeing with PEDOT:PSS, which could be produced with a length of up to 40 m and keep steady after repeated machine washing and drying [18]. Then, they embroidered these yarns into a felted wool fabric substrate and connected them end-to-end with silver wires to form a fabric TEG. The structure is illustrated in **Figure 8**. In an in-plane fabric TEG prototype composed of 26 yarn legs, the internal resistance is about 8.7 KΩ, and the output voltage of Vout/ΔT is about 313 μV K−1 when temperature difference ΔT is about 120°C. An output current of 1.25 μA can be observed when ΔT is 66°C, and resulted a maximum power

**Figure 6.** (a) Alternative arranged multilayer fabric TEG structure and (b) fabric film TEG prepared by Hewitt et al. [17].

**Figure 5.** Schematic illustration of the conversion of thermoelectric sheet into a yarn.

The studies of yarn and fiber thermoelectric materials are just beginning. The same coating process of fabrics can be further used to prepare fiber and yarn thermoelectric materials. Additionally, more complicated fabrication technique such as electrospinning and twisting process can also be adopted to prepare fiber and yarn materials with better performance. It can be imagined that there will be more ingenious methods in future to fabricate high performance thermoelectric fiber or yarn materials, so that a fully textile-based thermoelectric generator can be achieved.


**1. Introduction**

Since ancient times, medicinal plants have been used for human well-being as they are a source of healthy metabolic compounds. They still are widely used for the production of food, medicines and fuel. The improvement of the biological properties of plants, vegetables and herbs can be achieved through fermentation technology, which can transform complex substrates and change the quantity of some bioactive compounds [1]. Additionally, the fermentation process allows the preservation of food and beverages while improving the nutritional and organoleptic quality of the products [2]. Selected microorganisms are widely applied to obtain fermented foods and beverages. Among them, yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) are the main organisms with beneficial technological applications and for which a wide knowledge of use is available [3,4]. In some cases, mixed cultures are preferred over single cultures since they can offer improved processes and higher flavor and bioactive compound content [5], as well as health benefits [6]. Applying selected microbial cultures to suitable raw materials, the fermentation process can be modulated, also enhancing the content of bioactive metabolites and other compounds with antioxidant activity [7–10]. Since the production of specific metabolites is a strain-specific trait, the choice of microorganisms for the intended use is fundamental to obtain fermented products with enhanced properties [11–13].

**Citation:** Gadhoumi, H.; Gullo, M.; De Vero, L.; Martinez-Rojas, E.; Saidani Tounsi, M.; Hayouni, E.A. Design of a New Fermented Beverage from Medicinal Plants and Organic Sugarcane Molasses via Lactic Fermentation. *Appl. Sci.* **2021**, *11*, 6089. https://doi.org/10.3390/ app11136089

Academic Editor: Maria Kanellaki

Received: 26 May 2021 Accepted: 28 June 2021 Published: 30 June 2021

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

**Copyright:** © 2021 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/).

A comprehensive inventory of microorganisms and their status as GRAS (generally recognized as safe) and/or QPS (qualified presumption of safety) organisms for the intended use is provided by Bourdichon and Casaregola [14].

In particular, LAB strains can produce key volatile compounds, active amino acids, peptides and sugars that contribute to the final flavor of fermented food products [15–17].

Moreover, due to the probiotic activity of some LAB, they can confer further attributes to fermented foods and beverages [18,19].

Plants are also suitable substrates for the growth of LAB [20–22]. Aromatic and medicinal plants are particularly valuable for selective bioprocesses as they already contain many bioactive compounds, including phenolic compounds, carotenoids, anthocyanins and tocopherols [23,24]. Moreover, the content of these compounds can be increased by the metabolic activity of the microorganisms involved in the fermentation process [25–27]. The metabolism of phenolic compounds correlated with increased antioxidant activity was observed during lactic acid fermentation [23,28].

Several authors have successfully obtained fermented products with improved antioxidant activity from vegetables, fruits, sugarcane molasses or plants [29,30]. Functional beverages that contain significant amounts of bioactive compounds can offer several health benefits. For instance, they can prevent some damaging physiological activities including metabolic and cardiovascular diseases [21–33].

Several LAB strains have been proposed as candidate probiotics due to their ability to provide functional attributes to foods and beverages.

Within the *Bifidobacterium* genus, some species are reported as potential probiotics for the treatment of enteric disorders in newborns such as infantile colics or as preventive agents for infantile diarrhea of bacterial origin. This function was also associated with strong antimicrobial activity against coliforms and other pathogenic bacteria [18]. Moreover, *Lactobacillus* species show several probiotic traits and are widely used in dairy and no dairy foods and beverages production. Among the functional attributes, those contributing to lower cholesterol levels are reported to be related to the action of strains belonging to the *Lactobacillus acidophilus* species [19]. The selection of appropriate organisms is the first step in designing and implementing bioprocesses for obtaining functional foods and beverages, as well as the availability of strain cultures in culture collections with known and validated functions [34,35].

This study aims to explore the effect of single and mixed culture of LAB strains, previously indicated as candidate probiotics, for the production of functional beverages starting with plants and organic sugarcane molasses as the raw material. The main fermentation parameters, the biochemical composition (carbohydrate, polyphenols, proteins), the volatile content and the antioxidant activity of the obtained beverages is assessed.

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

#### *2.1. Plant Material and Fermentation*

The plant material used for the formulation of three fermented plant beverages is shown in Table 1. Some plants were collected from Orbata National Park Mountain (Gafsa, Tunisia) with coordinates: N 34◦22 49.8" and E 9◦3 23.4" and others were purchased from the commercial central market of Tunisia. A voucher specimen was deposited in the Laboratory of Extremophile plants, Center of Biotechnology at the Ecopark of Borj-Cédria, and was identified by Prof. Abderrazak Smaoui. The selected plant materials were harvested and mixed with 30 g of organic sugarcane molasses solubilized in 1 L of distilled water. After the adjustment of the pH at 7.8, the solution was sterilized by pasteurization (80 ◦C in 15 min). Three equivalent batches were prepared and fermented using the selected strains *Lactobacillus acidophilus* ATCC 43121 and *Bifidobacterium breve* B632, provided by the Microbiology laboratory at the Centre of Biotechnology of Borj–Cédria.

**Table 1.** Plant material used for the fermentation process.

Specifically, a single culture of each strain, *L. acidophilus* ATCC 43121 and *B. breve* B632, was used to ferment batches named FB-lc and FB-sp, respectively. In addition, a mixed culture from both the strains was prepared and used to ferment the batch named FB-lcsp. The LAB cultures were previously grown on de Man Rogosa Sharpe (MRS broth, Oxoid, UK) at 37 ◦C for 24 h. Then, 10 mL of each culture inoculum with a concentration of 6 log cells/mL was added to each batch. After 30 days of fermentation, the three beverages were sterilized by filtration (0.2-μm filters) and preserved at +4 ◦C until analysis.

#### *2.2. Fermentative Parameters*

Titratable acidity (TA) was measured by titration using 0.1 mol/L NaOH solution and phenolphthalein as an indicator. The pH was detected by a digital potentiometer. The evaluation of reducing sugars was assessed using the dinitrosalicylic acid (DNS) method described by Warwick et al. [36]. Total proteins were measured using Bradford methods and the free amino acid according to the method described by Hermosín et al. [37]. Viable cell counts were made by inoculating 0.1 mL of samples, subjected to serial dilution in phosphate buffered saline (PBS), on MRS agar plates. Bacteria colonies were counted after 2 days of incubation at 30 ◦C.

#### *2.3. Total Phenolic Content*

According to the Folin–Ciocalteu method described by Tlili et al. [38], the total phenolic content was detected. A volume of 0.5 mL of Folin–Ciocalteu reagent and 1.25 mL of Na2CO3 (7% *w*/*v*) were added to 0.125 mL of each fermented beverage. The absorbance of each sample was measured at 765 nm after incubation of the tubes for 90 min in the dark. The total polyphenols content was expressed as μg gallic acid equivalents per mL of fermented beverage (μg GAE/mL).

### *2.4. Total Flavonoid Content*

The total flavonoid content was assessed according to the method reported by Dewanto et al. [39]. Total flavonoids, expressed as μg of quercetin equivalent per mL of fermented beverage (μg QE/mL), were estimated concerning the quercetin standard curve (concentration range: 100–750 μg/mL).

#### *2.5. Condensed Tannins Contents*

The condensed tannins were assessed according to the method reported by Sun et al. [40]. The number of condensed tannins, expressed as μg catechin equivalent per mL of fermented beverage (μg CE/mL), was measured using a standard calibration curve.

#### *2.6. Polysaccharides Analyses*

#### 2.6.1. Extraction

The polysaccharides dissolved in the filtrate obtained from the fermented beverages were precipitated by adding absolute ethanol (four times the volume of the filtrate) and were incubated at 4 ◦C for 24 h. The solution was then centrifuged at 4500× *g* rpm for 10 min. The precipitate obtained was collected and again dissolved in 20 mL of distilled water deproteinized with 2 mL of Sevag reagent (chloroform/butanol 4: 1, *v*/*v*) according to the method described by Navarini et al. [41]. Finally, the solution obtained was dialyzed using a dialysis membrane (2 μm) against bi-distilled water.

#### 2.6.2. Infrared Spectrum Analysis

The organic functional groups were investigated by Fourier transform infrared (FTIR) analysis, which was performed using an FTIR spectrophotometer (Bruker, Ettlingen, Germany) with a spectral range of 4000–400 cm−1 [42]. Briefly, 2 mg of each sample of polysaccharides was diluted with 200 mg spectroscopic grade potassium bromide (KBr) powder, ground and pressed into 1 mm pellets.

#### *2.7. Headspace Solid-Phase Microextraction of Volatile Compounds*

The volatile compounds were analyzed by Headspace Solid-Phase Microextraction (HS-SPME) coupled with Gas Chromatography–Mass Spectrometry (GC–MS) as previously described by Tian et al. [43]. The GC–MS headspace analysis was performed with an iron sources temperature of 240 ◦C and an ionization voltage of 70 eV. The mass spectrometer was operated in scan mode from *m*/*z* 50 to 350. Peak areas were determined for each compound by integrating a selected ion unique to that compound. The Kovats retention index (RI) was calculated with a homologous series of n-alkanes (C6-C28) under the same conditions applied for the sample analyses. The volatile compounds (OAV > 1) were considered to contribute to the aroma of fermented beverages [44,45].

#### *2.8. Antioxidant Activity Determination*

The antioxidant activities of different fermented beverages were determined by the following tests. The α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method was performed according to Tlili et al. [38]. The reducing power was determined using the method described by Oyaizu [46]. The appearance of the blue green color was measured at 700 nm, vitamin C was used as the control. The total antioxidant capacity of each extract was estimated using the method described by Prieto et al. [47]; the absorbance was detected at 695 nm. Moreover, a 2,2-azino-bis diammonium (ABTS•+) radical scavenging assay was assessed using the method described by Hayouni et al. [48]. A total of 25 μL of the fermented beverages or the standard trolox was added to 2 mL of diluted ABTS•+ solution and the absorbance was measured at 734 nm. The scavenging ability was expressed as IC50 (μg/mL), which is defined as the concentration inhibitive at 50% of free radical scavenging.

#### *2.9. Statistical Analyses*

All the experiments were performed in triplicate. The results were reported as mean values of three replicates ± the standard deviation (sd). The differences between the average variables were considered significant at *p* < 0.05. XLSTAT statistical software version 2016 (Addinsoft, New York, USA) was used for data processing.

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

#### *3.1. Fermentation Trials*

The colony counts of the selected LAB cultures, starting from the value of 6.5 log cfu/mL, increased during the 30 days of the fermentation trials. The highest value of 12 log cfu/mL was reached in the samples fermented with the mixed culture (FB-lcsp), after 20 days of fermentation. Later, the LAB population decreased, and the final values detected were 11, 10 and 8 log cfu/mL for FB-lcsp, FB-lc and FB-sp, respectively (Figure 1a). A substantial decrease of the pH and a corresponding increase of the titratable acidity was observed during the fermentation process (Figure 1b). The lowest pH value (3.7) and the highest TA value (3.4 mL NaOH 0.1N) were assessed for the FB-lcsp samples.

**Figure 1.** LAB cells count, expressed as log of colony forming unit/mL (**a**), pH and titratable acidity (**b**) of the three different products during the lactic acid fermentation process. Results are reported as mean values of 3 replicates.

A decrease of total and reducing sugars was observed, probably due to the acid production by the LAB. In addition, a degradation of the total proteins and a decrease of the free amino acid content occurred (Table 2). These results demonstrate the effectiveness of the fermentation, confirming the degradation of sugars and proteins by the LAB and the production of organic acids and new metabolites. It is interesting to note that the variation of all the considered parameters was higher for the trial obtained from FB-Lcsp, due to the combined effect of *B. breve* B632 and *L. acidophilus* ATCC 43121 cultures. During the fermentation process, the soluble components of proteins, carbohydrates and free sugars were readily available, and the consumption of these nutrients by LAB led to a drop in pH levels [49]. Moreover, the beverage was obtained by selecting hetero-fermentative LAB results that had a sparkling attribute, due to CO2 produced, which is desired by consumers [49].

**Table 2.** Total sugars and proteins, total phenolic compounds, flavonoids and tannins contents of obtained beverages.

FB-sp: obtained beverage fermented by *Bifidobacterium breve B632*. FB-lcsp: the obtained beverage fermented by a mix of both strains. FB-lc: the obtained beverage fermented by *L. acidophilus* ATCC 43121. \* μg of gallic acid equivalents per mL of beverages. \*\* μg of rutin equivalent per mL of beverages. \*\*\* μg of catechin equivalent per mL of beverages. Results are expressed as the mean of 3 replicates.

#### *3.2. Polyphenol, Flavanoids and Condensed Tannins Content*

The polyphenols represent the major group of antioxidants of plant materials [50,51]. They include a wide variety of bioactive metabolites that contain at least one aromatichydroxyl group in addition to other substituents with large spectra of biological activities [45]. They are divided into different groups such as flavonoids, tannins and phenolic acids [45]. In this study, the amount of total phenolics, flavonoids and condensed tannins varied in the three beverages (Table 2). A high level of polyphenols, with values of 315.6; 289.7 and 182.5 μg GAE/mL, were observed for FB-lcsp, FB-sp and FB-lc, respectively. The highest content of flavonoids (152.1 μg RE/mL) and tannins (93.6 μg CE/mL) was found in the FB-lcsp trial. It is well known that the secondary metabolites possess a wide range of biochemical and pharmacological properties [48]. These results suggest that the mixed culture improves the extraction of secondary metabolites such as flavonoids, tannins and phenolic acids. During fermentation, many changes of composition occur, leading to a modified ratio of nutrients and anti-nutrients and, therefore, the properties of the product, such as bioactivity and digestibility, are modified [52]. Lactic fermentation of plants has been shown to increase the concentration of several phenolic compounds [53]. So, the richness of our fermented plant beverages with phenolic compounds, especially FB-lcsp, could be an important starting point to explore their biological activities.

### *3.3. Infrared Spectrum*

The infrared spectrum was used to identify the polysaccharides by detecting the stretching vibration of related groups, which allows identification of a variety of functional groups, the type of sugar and glycosides. Figure 2 shows the FTIR spectra with the characteristic absorptions of the beverages before and after fermentation.

**Figure 2.** Infrared spectrum of polysaccharides, recorded for wavelengths 500–4000 cm−1. D refers to the polysaccharides detected in not fermented beverage (**a**). A, B, and C refer to the polysaccharides detected in the beverages fermented with the selected cultures FB-sp, FB-lcsp, and FB-lc, respectively (**b**).

Substantial differences between the spectra were, indeed, observed, and the intensity of the peaks was higher before the fermentation (Figure 2a) and decreased in the fermented beverages (Figure 2b), indicating an almost total degradation of sugars.

In particular, the infrared spectrum analysis of the polysaccharides, recorded for wavelengths 500–4000 cm−1, showed an intense peak between (3415–3395 cm−1), representative of the vibrations of the hydroxyl groups (-OH), followed by a weak vibration of the C-H band at 2930 cm−1 [54]. Peaks with vibration bands between (3415–2930 cm−1) are unidentified organic groups (Figure 2a). The peak at 1612 cm−1 is attributed to the vibration of the C = O group in its protonated carboxylic form. The peak at 1415 cm−1 is attributed to the vibration of the COO- group in its deprotonated carboxylic form polysaccharide extract [55]. Moreover, the peaks detected at 1200–1000 cm−1 indicate that the fractions are pyranose forms of carbohydrate [56]. The peaks with vibration bands at 992 cm−1 and 942 cm−1 (Figure 2b) derive from the antisymmetric stretching of tetrahydropyran, suggesting the presence of an arrangement in the carbohydrate units [57]. The peak at 605 cm−1 is related to D-glucopyranose. Additionally, the vibration at 766 cm−1 is related to rhamnoside present in the polysaccharide. The functional groups, such as –OH, –COOH

and C=O, largely found in carbohydrate in addition to anionic and cationic groups, have a good ability to counteract oxidative stress, which is expressed by excessive production of reactive oxygen species [58].

#### *3.4. Volatile Compounds*

Twenty-four volatile compounds were identified with remarkable differences between the three beverages (Table 3). Among the volatile compounds detected, some are typically produced by plants and other compounds are generated by the LAB metabolism [59]. The data showed high variations in aroma compounds. Some compounds were present only in FB-sp (1-pentanol 2-methyl, and 2-butyl acetate), and others were detected only in FB-lcsp (isoamyl alcohol, propanoic acid- 1-methylpropyl ester, α-pinene, camphene, l-phellandrene, and α-terpinene). Lactic acid, acetic acid, propyl ester, propanoic acid, propyl ester; 1,8-cineole and fenchone were also found in the three beverages. During fermentation, many changes of composition occur, leading to a modified ratio of nutrients and anti-nutrients and, therefore, the properties of the product, such as bioactivity and digestibility, are modified [60]. Among volatile compounds, alcohols are considered the principal compounds in the aromatic profile of fermented plant beverages and are a common result of the metabolism of carbohydrate and amino acids [61]. Alcohols can represent essential preservative and aromatic components in fermented plant beverages. Moreover, they can also be used as a solvent for other volatile compounds and, thus, can contribute more to the global flavor [62]. The presence of ester compounds in the fermented beverages due to the esterification of free acids with alcohol was also observed. Several volatile components in fermented plant beverages can contribute to sweet aromatic notes [63]. Our results suggest that the mixed culture of LAB could provide a suitable aroma to the fermented plants beverages.

**Table 3.** Aromatic compounds identified from fermented beverage extracts of by GC–MS head space.

FB-sp: beverage fermented by *Bifidobacterium breve B632*. FB-lcsp: beverage fermented by a mix of both strains. FB-lc: beverage fermented by *Lactobacillus acidophilus* ATCC 43121. NF: not found. BV: baseline and valley points (start at a baseline point, end at a dropline from a valley point). VB: Valley dropline and baseline points. PV: penetration point and valley point. BB: start at a baseline point, end at a dropline baseline point. VV: start and dropline at a valley point.

#### *3.5. Antioxidant Activity*

The metabolization and depolymerization of phenolic compounds, correlated with increased antioxidant activity, have been observed during lactic acid fermentation [64]. In this study, the results of total antioxidant capacity (TAC) showed a similar motif to secondary metabolite contents. The TAC differed significantly among the strains used (Table 4). The three beverages showed high antioxidant activity, in particular, FB-lcsp (82.69 μg GAE/mL). Lower values were detected in samples FB-sp and FB-lc with 73.2 and 64.83 μg GAE/mL, respectively.

**Table 4.** Total antioxidant capacity and the free radical scavenging activity of DPPH, ABTS and the reducing power essay of the three fermented beverages.

The scavenging ability was expressed as IC50 (μg/mL). The reducing power activity (FRPA) was expressed as EC50 (μg/mL). Results are expressed as mean of 3 replicates ± standard deviation.

> The DPPH scavenging activity of the fermented beverages also varied among the fermentation trials (Table 4). Samples of FB-lcsp showed the highest antioxidant activity, compared to FB-sp and FB-lc. The free radical scavenging activity ABTS•+ of each sample was also different (Table 4). This activity was higher in samples FB-lcsp (IC50; 17.4 ± 0.96 μg/mL) compared to those detected in FB-sp and FB-lc samples (IC50; 19.3 ± 1.1 and 24.6 ± 1.6 μg/mL, respectively).

> Regarding the reducing power activity (FRPA) of fermented beverages, expressed as EC50 (μg/mL), all the beverages were able to lower Fe3+ to Fe2+, but the results differed among the three samples. Specifically, the highest FRPA was detected in FB-lcsp samples (Table 4). A significantly lower activity was found in FB-sp and FB-lc samples, indicating that the double culture was able to enhance and improve the antioxidant activity in the fermented beverages more than the monoculture. Indeed, it is well known that the total antioxidant capacity is essentially due to the presence of phenolic and bioactive compounds. Several studies reported that the polyphenols present in fermented beverages possess high scavenging activity against oxidative stress [65–67]. Recently, fungal fermentation has been used to increase the antioxidant and anti-cholesterolemic activities of food and medical compounds as well as for food preservation [68]. Moreover, many bioactive compounds, formed during seaweed fermentation, have been reported to scavenge free radicals and reactive oxygen species [69].

#### **4. Conclusions**

In this study, medicinal plants and organic sugarcane molasses were tested as raw materials to produce functional beverages via lactic fermentation. *L. acidophilus* ATCC 43121 and *B. breve* B632 were selected and used in single and multiple cultures. In particular, the strain *B. breve* B632 is widely described in the literature as a potential probiotic that is useful for the treatment of enteric disorders in newborns. Strains belonging to the *L. acidophilus* species are also valuable for functional attributes among which is the ability to lower cholesterol level.

In our work, the three fermented beverages revealed an enhanced level of polyphenols, flavonoids and tannins, especially those obtained by using the mixed culture FB-lcsp. In addition, the detection of aromatic compounds in the fermented beverages showed twenty-four interesting volatile compounds, which could give pleasant-positive aroma attributes to the flavor of the beverages. The highest antioxidant activity was observed in the beverage obtained by the mix culture strains. This innovative approach, based on

lactic fermentation performed by a mixed LAB culture, can generally be used to ameliorate secondary metabolites production and extraction from plant materials.

Accordingly, the assessment of selected strains that are able to efficiently ferment medicinal plants and organic sugarcane molasses is a crucial step for designing new functional beverages with well-being effects on human health.

**Author Contributions:** H.G.: conceptualization, methodology, original draft; E.A.H., E.M.-R., and M.S.T. designed the study and performed the experiments. H.G., M.G., and L.D.V., contributed to drafting the manuscript, validation, data curation, visualization, writing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by University of Tunis EL Manar; University of Applied Sciences, Neubrandenburg, Germany; University of Modena and Reggio Emilia, Italy; Institute of Pasteur of Tunis for the infrastructure and facilities, and Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology (LR15CBBC06) at the Ecopark of Borj-cédria. BP-901, 2050 Hammam-Lif. Tunisia.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are grateful, and provide sincere thanks, to the honorable Vice Chancellor of the University of Applied Sciences. Neubrandenburg, Germany, and the University of Modena and Reggio Emilia, Italy for the infrastructure and facilities. This study was supported by a grant from the Ministry of Tunisia, Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology (LR15CBBC06) at the Ecopark of Borj-Cédria. BP-901, 2050 Hammam-Lif. Tunisia.

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

### **References**


**1. Introduction**

Pandemics and epidemics of infectious origin are large-scale outbreaks that can greatly increase morbidity and mortality globally or over a wide geographic area, respectively [1]. Pandemics have occurred throughout history and appear to be increasing in frequency in the last centuries. Noteworthy examples include the Black Death at the end of the Middle Ages, Spanish flu in 1918, the 2014 West Africa Ebola epidemic or the current COVID-19 pandemic. The direct impact of pandemics on health can be dramatic. These large outbreaks can disproportionally affect younger or active workers, but vulnerable populations such as the elderly are at a particular high-risk. Pandemics can cause acute, short-term as well as longer-term damage to economic growth due to public health efforts, health system expenditures, and aid to affected sectors. Evidence suggests that epidemics and pandemics can have significant social and political consequences too, by debilitating institutions, amplifying political tensions, stigmatizing minority populations, or encouraging sharp differences between social classes [2].

Outbreaks by respiratory ribonucleic acid (RNA) viruses such as influenza or coronaviruses entail the principal threat due to their ease of spreading among humans, their potential severity and recurrence. However, other RNA viruses such as flaviviruses (Zika) or filoviruses (Ebola) must be taken into consideration due to a great overall burden of morbidity and mortality [3]. Antiviral drugs can help mitigate a viral outbreak by reducing the disease in infected patients or their infectiousness. While these drugs can be very successful against some viruses (*e.g*. hepatitis C virus [HCV]) [4], they are not universally effective as exemplified in the current SARS-CoV-2 pandemic [5]. Nowadays, having effective vaccines may be the only tool to reduce susceptibility to infection and thus, prevent the rate of virus spread [2].

Vaccination has dramatically decreased the burden of infectious diseases. Vaccines have saved hundreds of millions of lives over the years [6]. It has been estimated that approximately 103 million cases of childhood diseases were prevented in the United States through vaccination between 1924 and 2010 [7]. The eradication of smallpox in 1980 through vaccination is considered one of the crown accomplishments of medicine. Despite these achievements, effective vaccines have been developed against just over 30 pathogens among bacteria and viruses. There are many pathogens, including viruses such as human immunodeficiency virus (HIV) or respiratory syncytial virus (RSV), for which all efforts for vaccine development have failed so far. In addition, current available vaccines for worldwide important viral diseases like influenza are suboptimal, especially in the elderly, resulting in vulnerability among billions of at-risk populations [6]. On the other hand, having a new effective and safe vaccine in time to control highly contagious emerging viruses that cause epidemic or pandemic threats is an almost impossible task considering the timeframes for vaccine development. This includes preclinical and clinical research, its approval by the regulatory authorities, as well as its production and distribution [3].

Altogether, it has been postulated that one possibility of filling the gap between the appearance of a viral outbreak by an emerging pathogen and the availability of a specific vaccine is to take advantage of the heterologous protection of some existing vaccines, in order to increase the non-specific resistance of the host through trained immunity [8, 9].

### **2. Specific anti-infectious vaccines**

Conventional (specific) anti-infectious vaccines are biological preparations containing live-attenuated or dead microorganisms, their antigens or nucleic acids encoding for them, designed for specific pathogens. The purpose of vaccination is to induce a long lasting adaptive immune response against key antigens able to confer host resistance for future encounters with the corresponding pathogen. Either the production of antibodies, generation of T helper/effector cells, or both, may play a critical role in such a resistance, which greatly depends on the type of pathogen, the route of entrance and the host-pathogen relationship (*e.g.,* extracellular and/or intracellular) [10]. Successful vaccines are highly effective not only in inducing long-lasting immunity against disease-causing pathogens, but also in providing herd immunity to the community that substantially restricts the spread of infection [6].

Most of the vaccines available today have been developed empirically and used successfully long before their mechanism of action on the immune system was understood. Early protection is associated to induction of antigen-specific antibodies, being their quality (avidity, specificity, or neutralizing capacity) key factors for

#### *Trained Immunity-Based Vaccines: A Ready-to-Act Strategy to Tackle Viral Outbreaks DOI: http://dx.doi.org/10.5772/intechopen.95765*

their efficacy. Long-term protection relies on the persistence of vaccine antibodies and availability of immune memory cells capable of rapid and effective reactivation with subsequent microbial exposure. On the other hand, T cells have a critical role in the induction of high affinity antibodies and immune memory. Furthermore, T cells have a direct role in protection conferred by some vaccines, including the tuberculosis Bacille Calmette-Guérin (BCG) vaccine [11].

Vaccines using whole pathogens have been classically classified as either live attenuated or inactivated (killed). Subunit vaccines contain just selected antigens (*e.g.,* proteins, polysaccharides). Recently, due to a growing availability of bioinformatics and sequencing tools, there has been an increase interest on so-called "rational" vaccine design approaches for subunit vaccines, such as the reverse vaccinology [12]. In this regard, modern vaccines include recombinant proteins or nucleic acids [13]. Rather than administering the antigen itself, DNA and mRNA vaccines targeting dendritic cells (DCs) encode the antigen of interest that will be produced by the vaccinated host, representing a new era in vaccinology [14]. In fact, the first RNA vaccine licensed for humans in Western countries has been recently developed for SARS-CoV-2.

As commented before, a vaccine response is linked to the induction of T and B cell specific responses to the antigens contained in the vaccine. This requires lymphocyte activation, proliferation and differentiation on specialized lymphoid tissues (*e.g* lymph nodes), where antigen presenting cells, like DCs for T cells or follicular dendritic cells (FDCs) for B cells, are present. Mature DCs are recruited into the T cell areas of lymph nodes from the periphery, *e.g.,* at the site of injection of the vaccine. DCs express pattern recognition receptors (PRR) that recognize evolutionary conserved pathogen-associated molecular patterns (PAMPs) that are not contained in self-antigens and are identified as "danger signals" [15]. When immature DCs are exposed to the vaccine-derived antigens at the site of vaccination, they uptake them and become activated [16]. This activation will lead to their maturation with the expression of homing receptors at their surface, triggering DC migration to the draining lymph node through afferent lymphatic vessels, where the activation of T and B lymphocytes will occur. Mature DCs process the up-taken antigens and present them to naïve T cells associated to molecules of the major histocompatibility complex (MHC) within the T cell areas of lymph nodes. On the other hand, unprocessed native antigens, either free or complexed with antibodies or complement, access the B cell areas of lymph nodes (lymphoid follicles) where they are captured by FDCs and displayed from their cell surface to the B cells. Antigen-specific B cells will rapidly proliferate forming a germinal center and differentiate into plasma cells producing low-affinity immunoglobulin (Ig) M antibodies. The B cells will then receive additional signals from activated T cells, undergoing isotype antibody switch from IgM to IgG or IgA and affinity maturation of the antibodies produced.

For a vaccine to be immunogenic enough, DC activation, that can be achieved by adjuvants, is essential. Live attenuated and inactivated whole-cell vaccines are considered "self-adjuvanted" as they naturally present sufficient PAMPs to activate innate immune cells, including DCs; thus, promoting a robust antigen-specific immune response. In contrast, subunit vaccines generally require different types of adjuvants to enhance and/or drive the immune response in the desired direction [15, 17].

#### **2.1 Difficulties for novel specific vaccines in a viral outbreak situation**

Viral outbreaks appear when there is a sufficient number of susceptible individuals within a nearby population. Although susceptibility is a balance between host factors (high/low resistance) and pathogens (high/low virulence), in many cases it reflects a

lack of prior contact with a given pathogen. In general, this is related to the emergence of new viruses or the lack of effective vaccines against known viruses. As pointed above, the development of effective vaccines is not an easy task against certain viruses. We are still lacking vaccines for some of the most lethal viral infections, including HIV and MERS-CoV, among others. These pathogens are difficult to tackle, as we do not fully understand their mechanisms to evade the immune system or how to elicit protective immunity against them [13]. However, encouraging progress is being made against these pathogens and there are currently several "pipeline vaccines" in development, such as RSV, universal influenza vaccine, and SARS-CoV-2 [18–20]. Apart of SARS-CoV-2 for obvious reasons in the current pandemic, there is an urgency to have a universal influenza vaccine that provides a broad and durable protection from influenza virus infection. Yet, the high level of antigenic diversity and variability, and antigenic drift in the surface antigens, enable these viruses to escape antibody-mediating neutralization [21]. On the other hand, there is a number of vaccines currently licensed, including the influenza A virus vaccine, that provide incomplete protection, especially in high-risk groups [22]. Mumps outbreaks observed in Ireland, United Kingdom and United States in vaccinated subjects with Measles Mumps Rubella (MMR) vaccine is another example [23]. Different factors have been postulated to contribute to mumps outbreak, including waning immunity and primary and secondary vaccine failure. Yet, their actual contribution is not fully understood [23].

Vaccine efficacy must consider different target populations as well. Adaptive immune response to vaccines may be limited in newborn and the elderly. Early in life, immune responses are dampened compared to adults [24, 25]. Neonates have underdeveloped germinal centers in lymph nodes and the spleen, and low expression of B cell receptors which in turn results in low levels of primary IgG responses to infections and vaccines [26]. As we age, our immune system undergoes agerelated changes that lead to progressive deterioration of the innate and the adaptive immune responses, this is termed immunosenescence. The most common features of immunosenescence are short-lived memory responses, impaired response to new antigens, increased predisposition to autoimmune diseases and low-grade systemic inflammation (*inflammaging*) [27, 28]. Immunosenesence results in increased susceptibility to infections and deficient response to vaccination causing high hospitalization and mortality rates. For example, influenza vaccine efficiency has been reported to be 17–53% in the elderly, compared with the 70–90% efficacy in young adults [29]; and vaccination with Varicella zoster virus (VZV), also an important pathogen in elderly people, only partially prevents reactivation of herpes zoster [27].

If the difficulties listed above are outlined for existing or developing vaccines, quickly obtaining an effective vaccine to urgently control a new virus outbreak is almost an impossible task in the short-term as pointed above. This is well exemplified by the SARS-CoV-2 vaccine race pushed by the devastating COVID-19, with more than 100 vaccine candidates in the running. It is considered that no less than 1 year will last the time until the first licensed vaccine can provide protection in the best scenario [30]. This, in spite of greatly shortening the usual clinical development time and regulatory obstacles for a new vaccine and, therefore, without knowing its true performance and/ or safety in the medium term compared to other authorized vaccines [31].

## **3. Trained immunity and infections**

It has become evident from epidemiological, clinical and experimental data that some conventional whole-cell vaccines, like BCG and others, also provide resistance to infectious diseases not related with the specific pathogen targeted by the vaccine [32–34]. Much of these non-specific "heterologous" effects appear to depend on

#### *Trained Immunity-Based Vaccines: A Ready-to-Act Strategy to Tackle Viral Outbreaks DOI: http://dx.doi.org/10.5772/intechopen.95765*

the activation of innate immune cells by the PAMPs contained naturally in these vaccines [10], although other mechanisms such as cross-reactive epitopes between different pathogens could also account for this protection in some cases [35].

Immunological memory, understood as the ability to "remember" past encounters with pathogens, has been classically attributed to the adaptive branch of the immune system exclusively, by virtue of the antigen-driven clonal expansion of T and B lymphocytes and exemplified by the mechanism of conventional specific vaccines pointed above. However, the notion that innate immunity was unable to induce immunological memory has been challenged in recent years, particularly from studies in organisms that lack adaptive immunity, such as plants or invertebrates, as well as early studies in mice lacking the adaptive immune system [8, 36]. Altogether, the term 'trained immunity' was coined to define an innate immune memory that lead the innate immune system to an enhanced response to secondary challenges [37]. Importantly, trained immunity seems to be underlying the heterologous effects of an increasing number of vaccines [38–40].

## **3.1 The concept**

*What is trained immunity? -* Trained immunity is defined as the memory of the innate immune system, where an encounter with a first stimulus (*e.g.* a microbial insult) results in a subsequent long-term adaptation and enhanced non-specific response by innate immune cells against a secondary challenge (the same or unrelated), thus providing non-specific, broad-spectrum, long-term protection in case of infection [8, 9, 37, 41].

*Which cells can be trained? -* Trained immunity properties have been defined for distinct cell subsets of the innate immune system [9, 42], including natural killer (NK) cells and innate lymphoid cells [43]. Of note, training of myeloid cells [42], particularly monocytes and macrophages [44, 45], and more recently DCs [46, 47] and hematopoietic stem cells [48], have been extensively studied. Finally, the acquisition of this immunological memory has also been demonstrated to a lesser extent for non-immune cells [49].

*How to get trained? -* A wide variety of stimuli can train innate immune cells, particularly when considering monocytes and macrophages [9, 50]. Among infectious agents, live microorganisms such as the tuberculosis vaccine BCG [51], *Candida spp* [52] or viruses [53, 54]; bacterial components, such as flagellin, lipopolysaccharide, muramyl dipeptide [55], fungal components as β-glucan [52] or even helminth products [56]. In general, microbial ligands engaging some PRR, like C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain-like receptors (NLRs) are well established training inducers, whereas those engaging toll-like receptors (TLRs) may have opposite effects depending on the TLR type and concentration [55, 57]. Intriguingly, not only infectious agents but also endogenous inducers and metabolites such as oxidized low-density lipoprotein or mevalonate can induce trained immunity [50].

*What hallmarks define trained immunity?* - In contrast to adaptive immune responses, epigenetic reprogramming of transcriptional pathways — rather than gene recombination — mediates trained immunity. This training phenomenon comprises three key hallmarks that occur at the intracellular level: increased cytokine production upon rechallenge, changes in the metabolism and epigenetic reprogramming [9, 58, 59], which eventually support increased protection upon infection.

Among those cytokines whose production is augmented after re-exposure in trained cells, proinflammatory molecules such as tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1β and interferon γ (IFN-γ) are fairly constant [45, 52, 55, 60, 61]. Modulation of IL-10 varies between studies [45, 52, 56, 62, 63]. A noted

shift from oxidative phosphorylation to aerobic glycolysis (Warburg effect) is the main change in cellular metabolism during the induction trained immunity [64]. Moreover, glutaminolysis, cholesterol synthesis and the tricarboxylic acid cycle are non-redundant pathways required for trained immunity to take place [64, 65]. Epigenetic reprogramming, mainly mediated by histone modifications, is one of the bases for the long-lasting effect of trained immunity [8, 66–68]. Immune pathway activation and changes in metabolism serve as basis for epigenetic rewiring [65]. As a result, epigenetic modifications have been found at the level of important promoters for the training process, which makes chromatin more accessible and conditions gene expression patterns of trained cells upon stimulation with a secondary challenge [69].

As a result of the whole process, enhanced, broad-spectrum, non-specific protection mediated by innate immune cells is found upon infection. This crossprotection has been observed for a wide range of human pathogens including fungi [51, 52], parasites [70, 71] and different bacterial infections [72–75]. Importantly, induction of trained immunity has been proved to be effective against viral infections including yellow fever [76], influenza A virus [77] and others [78, 79]. In this line, the induction of this phenomenon has been also proposed as a tool for reducing susceptibility to emergent SARS-CoV-2 infection, as will be described at the end of the chapter [78, 80].

*How long does trained immunity last?* – Trained immunity phenotypes have been observed for months and up to one year after the training insult. This was initially controversial, as trained immunity properties had been attributed to short-lived myeloid cells such as monocytes or DCs [38]. In this regard, several studies have shown that modulation of bone marrow progenitors is also an integral component of trained immunity, supporting the long-lasting effect of this phenomenon [9, 81]. In this way, trained immunity inducers [82–85] would be able to reprogram and induce expansion of hematopoietic progenitors with a particular bias to the myeloid lineage. Thus, bone marrow-derived mature cells would be also trained [86], showing improved clearance of infection [83].

Complementary to progenitor reprogramming, peripheral trained immunity induction would take place in tissue-resident cells [9]. This is especially relevant at the mucosal level, where cells encounter most of the infectious training inducers. Alveolar macrophage (AM) memory was demonstrated following viral infection [87, 88]. Training of these long-living cells led to increase antimicrobial properties, independently of systemic immunity [87, 89]. This local training of AM was further reproduced following respiratory mucosal administration of tuberculosis vaccine, being crucial for *Mycobacterium tuberculosis* clearance [90]. On the other hand, training of NK cells lead to long-lived, self-renewing, stable expanded cells with memory-like properties, both in an antigen-dependent or independent manner [91–93]. Finally, it was also reported that self-renewing long-living skin epithelial stem cells exhibited local trained immunity, providing faster wound healing in primed mice than in naïve mice [94, 95].

#### **3.2 Trained immunity on ongoing immune responses**

### *3.2.1 Effect on adaptive immunity*

Non-specific effects of vaccines have been extensively studied and reported over the last decades. Although trained innate cells could partially account for these effects, involvement of adaptive immunity has also been suggested [96]. An adaptive immune mechanism of non-specific effects could be heterologous immunity; vaccine antigens can give rise to T cell cross-reactivity against other antigens that may confer some protection against unrelated pathogens [96, 97].

#### *Trained Immunity-Based Vaccines: A Ready-to-Act Strategy to Tackle Viral Outbreaks DOI: http://dx.doi.org/10.5772/intechopen.95765*

However, innate immune cells constitute the bridge between the intrusion of microbial threats and the activation of adaptive immunity. As said before, following sensing of pathogens by PRRs, activated innate immune cells secrete different factors and act as antigen-presenting cells (APCs) to initiate activation of adaptive immunity [98]. Thus, it would not be unexpected that trained innate immune cells, within their acquired enhanced properties, would be able to induce stronger adaptive immune responses [39]. In this regard, BCG vaccine, a well-known trained immunity inducer, has shown to enhance the antibody titer and alter heterologous T cell responses against a wide range of vaccines and unrelated infections [99–101]. In different experimental models, BCG-mediated protection against viral and *Plasmodium* infections was abrogated in the absence of T cells. In these models, BCG vaccination has been mainly associated with modulation of CD4+ T helper (Th) 1 responses. Similar observations have been found in different clinical studies [99]. Of note, BCG vaccinated human volunteers displayed a long-lasting heterologous Th1 and Th17 response upon stimulation with unrelated pathogens and TLR-ligands [38]. To some extent, similar observations have been found in other vaccines such as diphtheria-tetanus-pertussis (DTP) or measles vaccine [99].

As said before, trained immunity properties have been recently described also for DCs. As being the most professional APCs, they emerge as crucial bridge for potentiating adaptive immune responses. In this sense, DCs with high immunostimulatory properties that enhance adaptive immune responses via IL-1β release had been described [102]. More recently, programmed memory DCs have shown to increase Th1/Th17 immunity and confer protection during cryptococcosis [46]. Finally, different polybacterial preparations of whole-cell inactivated bacteria, have shown to prime DCs and induce enhanced Th1, Th17 and IL-10 T cell responses against related and unrelated stimuli [103, 104]. This capability of modulating heterologous T cell responses by APCs have been also described to suppress pathogenic T cell immunity in experimental models of autoimmune encephalomyelitis [56].


**2.3 Anatomical studies**

The free hand sections of the collected specimens were taken and observed under the light microscope. Measurements of anatomical characteristic features were made using standard methods.

## **2.4 Molecular studies**

Total genomic DNA was extracted from 7-day old seedlings of wild rice species; *O.rufipogon*, *O. nivara*, *O. eichingeri* and *O. rhizomatis* respectively using Promega Plant DNA extraction kit. A total of twelve SSR primer pairs were used (**Table 3**) for molecular study. SSR markers were obtained from Gramene (http://www.grame ne.org/). All SSR PCR amplification reactions were carried out in a total volume 30 μl of which consist 1 x PCR buffer, 1 mM dNTPs, 2 μM SSR primers, 2 mM

**Table 3.** *SSR markers used for the molecular studies.* MgCl2, 50 ng of genomic DNA and 0.5 Units of *Taq* DNA polymerase. SSR alleles were resolved on Poly Acrylamide Gel. The SSR banding patterns were identified using Poly Acrylamide Gel Electrophoresis (PAGE).

#### **2.5 Analysis of Data**

Gathered data were analyzed with univariate, bivariate and multivariate statistical procedures. Suitable statistical software was employed in the analysis of data. In addition, data mining analysis were also attempted for the data gathered from the study to reduce the noise in the data set.

Molecular data were analyzed using Genemapper 4.1 software and SSR profiles were analyzed using PowerMarker 3.25.

### **3. Results**

#### **3.1 Morphological studies**

The mean values of the parametric morphological measurements of wild rice species are given in **Table 4**. According to the table, the species *O. rufipogon* indicated highest mean for the plant height (153.23 cm) and the culm length (94.11 cm) and minimum plant height was observed in *O. eichingeri* (99.25 cm). Similarly, the highest leaf length and breadth were found in the samples of *O. nivara* and narrow leaves were occurred in samples of *O. rufipogon*. The variation of ligule length indicated that *O. nivara* possessed a higher ligule length with respect to other species included in the study. The summary of the ANOVA carried out on the parametric lamina morphological characters are shown in **Table 5**, except ligule length, panicle length, the rest of the characters are significantly varying across the wild rice species.

The association of the non-parametric characters with wild rice species included in the study is shown in **Table 6**. The characters such as leaf blade pubescent, awn after full heading and intermodal color after full heading are not significantly differ across the species (p > 0.05). However, the rest of the characters are significantly associated with the wild rice species and are of potential characters in separating wild rice species.

#### **Table 4.**

*Summary of the parametric morphological characters of the four wild rice species (Mean value and standard deviation below mean value).*

#### *Characterization of Wild Rice -* Oryza *Species Complexes in Sri Lanka DOI: http://dx.doi.org/10.5772/intechopen.97244*

#### **Table 5.**

*Summary of the ANOVA performed on the parametric morphological characters of the four wild rice species.*

#### **Table 6.**

*Result of χ2 test performed on the non-parametric morphological characters of the wild rice species included in the study.*

A total of three clusters were resulted from the cluster analysis of morphological characters (**Figure 5**) and species were grouped under each cluster with respect to their similarities. The samples of *O. nivara* and *O. rufipogon* were intermingled and separated into two groups. Meanwhile the samples of *O. eichingeri* and *O. rhizomatis* well-separated from 80% similarity level and from rest of the clusters representing two populations. However, one sample of *O. eichingeri* was grouped with O*. rhizomatis.* The phylogenetic tree (**Figure 6**) constructed by morphological characters clearly showed a well separated cluster of *O. rhizamatis*. The samples of *O. nivara* and *O. rufipogon* were intermingled and separated into four groups. Findings of the study led to conclude that wild rice species in Sri Lanka are "ecological swarms" and represents allopatric or sympatric populations.

**Figure 5.**

*Dendrogram produced by cluster analysis of 22 morphological characters of wild rice species*, O. nivara*,* O. rufipogan*,* O. rhizomatis *and* O. eichingeri*.*

#### **Figure 6.**

*Phylogenetic Tree constructed by 22 morphological characters of wild rice species,* O. nivara*,* O. rufipogan*,* O. rhizomatis*.*

#### **3.2 Anatomical studies**

The variation of anatomical characters, especially the laminar anatomical features across the wild rice species are given in **Table 7**. Comparatively, the magnitude of mean values of bundle sheath cell width indicated a considerable variation between the wild rice species *O. eichingeri* (11.77 μm) and *O. rufipogon* (10.74 μm). The summary of the ANOVA (**Table 8**), indicated that the all the anatomical

*Characterization of Wild Rice -* Oryza *Species Complexes in Sri Lanka DOI: http://dx.doi.org/10.5772/intechopen.97244*

#### **Table 7.**

*Summary of the parametric lamina anatomical characters of the wild rice species (Mean value and standard deviation below mean value).*

#### **Table 8.**

*Summary of the ANOVA performed on the laminar anatomical characters of the four wild rice species.*

characters except mesophyll height and bundle sheath height. The anatomy of the culm and leaf sheath of wild rice species indicated that the characteristic features of the structures reflect the habitat conditions (**Figures 7** and **8**).

The result of the cluster analysis of anatomical characters of wild rice species is shown in **Figure 9**. Comparatively, the Dendrogram resulted from the anatomical

**Figure 7.** *Laminar anatomical characters of 4 wild rice species collected during the study.*

**Figure 8.**

*(a) The transverse section of culm of* O. rhizomatis *(b) the section through a portion of* O. rufipogon *culm encircled by leaf sheath (c) the culm section of* O. nivara*.*

#### **Figure 9.**

*Dendrogram produced by cluster analysis of anatomical characters of wild rice species,* O. nivara*,* O. rufipogan*,* O. rhizomatis *and* O. eichingeri*.*

features indicated that anatomical characters well-separate the samples of each wild rice species. The samples of *O. rhizomatis* formed a unique group at similarity level of 80%. The pattern of the sample grouping was similar to the results obtained from the cluster analysis of morphological characters. However, samples were homogenized representing each wild rice species by pure tree branch.

The dendrogram resulted from the morphological and anatomical characters are shown in **Figure 10**. The grouping pattern of wild rice samples obtained from the analysis of morphological characters and anatomical characters reflect the same pattern observed in previously (**Figures 5** and **9**).

#### **3.3 Molecular studies**

A total of three clusters were resulted from the cluster analysis of molecular data (**Figure 11**) and species were grouped under each cluster with respect to

### *Characterization of Wild Rice -* Oryza *Species Complexes in Sri Lanka DOI: http://dx.doi.org/10.5772/intechopen.97244*

**Figure 10.**

*Dendrogram produced by cluster analysis of morphological and anatomical characters of Wild rice cultivars,* O. nivara*,* O. rufipogan*,* O. rhizomatis *and* O. eichingeri*.*

#### **Figure 11.**

*Dendrogram produced by cluster analysis of molecular data of wild rice species,* O. nivara*,* O. rufipogan *and* O. rhizomatis*.*

their genetic similarities. The samples of *O. nivara*, *O. rufipogon* and *O. rhizomatis* were very well separated from 40% similarity level confirming their distant relationship with each other and of independent evolution within Sri Lanka.