**Abstract**

The tourism sector as one of the sectors that has been hit by the Covid-19 pandemic needs a new breakthrough to enter a new normal era. Amid the ongoing paradigm shift and a number of new protocols will be implemented to welcome the new normal conditions in the tourism industry. The tourism sector in Indonesia, which has been absorbing many jobs, has been hit hard by the Covid-19 pandemic. Coupled with the difficulty of predicting when the pandemic will end, it is necessary to take smart steps in maintaining the sustainability of the tourism industry in entering an era of new norms. The new normal era brings new roles, new roads and expectations in the tourism sector. Digitalization that is growing rapidly and rapidly requires adaptation to new conditions and rearranging business strategies and models so that they can survive in the new normal era by adjusting technological developments. The development method used in this research is the Multimedia Development Life Cycle (MDLC). MDCL as a method for designing multimedia tools by emphasizing the 6 stages of multimedia development. The tools used in this application are PT GUI, Eclipse, and Google Maps. The objects of historical and heritage buildings that become the Sate Building, Villa Isola, the Geological Building, the Asian Africa Museum, and the Merdeka Building are made using immersive photography techniques. By representing information in the form of panoramic images, the 3600 makes it easy for users to visually display information from historical and heritage buildings in Bandung. The tourism model is virtually a possible form of tourism in the future.

**Keywords:** new normal, the tourism industry, multimedia development, virtual reality, technological development

#### **1. Introduction**

The tourism sector [1] and the creative economy play an important role [2] in development in Indonesia, this can be seen from the direct and indirect gross domestic product (GDP) and employment [2]. According to data from the Indonesian

Ministry of Tourism and Creative Economy (2012) the tourism sector [2] and creative economy contributed 11.8% to Indonesia's GDP, and 14.66% to total employment [3]. Morison describe tourism as a system into four elements, namely demand, travel,

*Model of Virtual Tourism as an Alternative of the Concept of Architecture Tourism Post…*

hit sectors, there are no more tourists, both domestic and foreign tourists [2].

**2.1 Tourism and the tourism potential of the City of Bandung**

opment and development, especially through the tourism sector [8].

applied in tourism planning based on the logic of activities) [2, 10, 11].

where tourism is profiled and information is disseminated using the internet as a potential business trend. Initially, e-Tourism was intended to fulfill information needs through creative media and the development of innovative information systems [5, 8]. It is believed that e-Tousim's role as a medium for tourism promotion that is low budget and high impact, in which the adoption of appropriate information technology will bridge the high budget for infrastructure and facilitate user understanding.

The four elements are related and influence one another, thus making the system run well. Based on this tourism model, from the demand aspect to the marketing aspect in the context of tourism, it will continue to grow in line with the role of business technology by utilizing increasingly strategic IT in all aspects of the business. Online technology trends such as e-commerce have changed new faces in all lines of human life, including the tourism sector. Especially amid the Covid-19 pandemic, in which all sectors collapsed. One of the economic sectors most affected by Covid-19 is the tourism industry sector. So far, the tourism sector which relies on the potential of tourism objects directly as a force in attracting domestic and foreign tourists is one of the worst-

The use of e-commerce in the tourism sector is known as e-Tourism terminology,

According to Yoety tourism is a trip that is carried out temporarily from one place to another with the reason that it is not for business or earning a living at the place visited but solely for enjoying the trip for sightseeing and recreation or fulfill various desires [9]. Tourism is also several activities, especially those related to economic activities that are directly related to the entry of foreign culture [9]. The wealth of natural resources, cultural diversity, as well as the wealth of historical heritage that Indonesia possesses is a very large base for economic devel-

According to Butowski tourism is the main sector in promoting economic and service growth [2, 10, 11]. So that in tourism planning and development [2, 10–12] it is necessary to consider several approaches [2], including: (a) Continuous Incremental, and flexible approach (in the sense that planning [2] is a process that is takes place continuously based on the needs and the results of existing feddback); (b) System Approach [2] (tourism as a system relationship needs to be planned through system analysis); (c) Comprehensive approach [2] (a tourism development approach that holistically considers institutional elements and the environment as well as socio-economic implications); (d) integrated approach (tourism development approach as an integrated system in plans and forms of development in the area); (e) Environmental and sustainable development approach [2] (a tourism approach starting from the planning process, development and management of natural and cultural resources is maintained/does not experience a decline in quality, and environmental analysis becomes the main point); (f) Community approach [2] (an approach to tourism development by maximizing community involvement starting from planning, making decisions that have an impact on socio-economics); (g) Implementable approach [2] (tourism development that formulates plans and recommendations into reality, as well as application of program techniques and strategies); and (h) Application of systematic planning approach [2] (an approach

destination, and marketing (**Figure 1**) [5–7].

*DOI: http://dx.doi.org/10.5772/intechopen.94015*

**2. Theoretical framework**

**213**

The strategic plan for the development of the tourism industry in collaboration with the ILO and the Australian government as partners of the Indonesian government builds a consensus to build a strong tourism industry. This collaboration involves stakeholders ranging from the government [2], social partners, communities, industry, and the general public [2]. Given the richness of natural and cultural resources that Indonesia has is major potential in the domestic and foreign tourist market [2, 4].

The city of Bandung as one of the tourist destinations in Indonesia relies on tourism potentials such as historical buildings, nature, culture, culinary, fashion, recreation, and entertainment [2]. This potential attraction makes natural resources [2] to come in the framework of (a) heritage tourism; (b) shopping and culinary tours; (c) educational tours; (d) recreational and cultural tourism; and (e) MICE (Meeting, Incentive, Convention, Exhibition) supported by adequate geographical, natural and urban planning, as well as good accessibility [2]. In maintaining the sustainability and existing potential, a programmed, structured, and controlled tourism development model is needed [2]. Tourism activities as the main sector of Bandung City have been started since 1920 [2]. Since the construction of the Cipularang toll road and the construction of a high-speed train linking Bandung City and Jakarta. This tourism sector provides a major contribution to increasing PAD/regional own revenue, almost 70% of Bandung's regional own revenue comes from the tourism sector. Where the tourism sector is a prima donna that continues to need to be improved.

The city of Bandung as one of the cities of past cultural heritage has superior values to be preserved, where the existence of the city of Bandung with its rich architectural styles/styles is its main attraction as a tourist destination and cannot be separated from the history of the development of Bandung City itself [2]. The long history of the city of Bandung has left many historical buildings that make Bandung one of the cities with art deco architectural heritage in the world and was once dubbed the most complete architectural laboratory with various old buildings that inherited the style of architectural beauty in Indonesia [2].

Meanwhile, tourism is a system that develops efforts by emphasizing the value of policies and strategies, as well as promotional patterns that are always synchronized with business development strategies in tourism planning [5, 6]. Mill and

**Figure 1.**

*Tourism elements. Source: Goeldner & Ritchie [6].*

*Model of Virtual Tourism as an Alternative of the Concept of Architecture Tourism Post… DOI: http://dx.doi.org/10.5772/intechopen.94015*

Morison describe tourism as a system into four elements, namely demand, travel, destination, and marketing (**Figure 1**) [5–7].

The four elements are related and influence one another, thus making the system run well. Based on this tourism model, from the demand aspect to the marketing aspect in the context of tourism, it will continue to grow in line with the role of business technology by utilizing increasingly strategic IT in all aspects of the business. Online technology trends such as e-commerce have changed new faces in all lines of human life, including the tourism sector. Especially amid the Covid-19 pandemic, in which all sectors collapsed. One of the economic sectors most affected by Covid-19 is the tourism industry sector. So far, the tourism sector which relies on the potential of tourism objects directly as a force in attracting domestic and foreign tourists is one of the worsthit sectors, there are no more tourists, both domestic and foreign tourists [2].

The use of e-commerce in the tourism sector is known as e-Tourism terminology, where tourism is profiled and information is disseminated using the internet as a potential business trend. Initially, e-Tourism was intended to fulfill information needs through creative media and the development of innovative information systems [5, 8]. It is believed that e-Tousim's role as a medium for tourism promotion that is low budget and high impact, in which the adoption of appropriate information technology will bridge the high budget for infrastructure and facilitate user understanding.

#### **2. Theoretical framework**

Ministry of Tourism and Creative Economy (2012) the tourism sector [2] and creative economy contributed 11.8% to Indonesia's GDP, and 14.66% to total employment [3]. The strategic plan for the development of the tourism industry in collaboration with the ILO and the Australian government as partners of the Indonesian government builds a consensus to build a strong tourism industry. This collaboration involves stakeholders ranging from the government [2], social partners, communities, industry, and the general public [2]. Given the richness of natural and cultural resources that Indonesia has is major potential in the domestic and foreign tourist market [2, 4].

The city of Bandung as one of the tourist destinations in Indonesia relies on tourism potentials such as historical buildings, nature, culture, culinary, fashion, recreation, and entertainment [2]. This potential attraction makes natural resources [2] to come in the framework of (a) heritage tourism; (b) shopping and culinary tours; (c) educational tours; (d) recreational and cultural tourism; and (e) MICE (Meeting, Incentive, Convention, Exhibition) supported by adequate geographical, natural and urban planning, as well as good accessibility [2]. In maintaining the sustainability and existing potential, a programmed, structured, and controlled tourism development model is needed [2]. Tourism activities as the main sector of Bandung City have been started since 1920 [2]. Since the construction of the Cipularang toll road and the construction of a high-speed train linking Bandung City and Jakarta. This tourism sector provides a major contribution to increasing PAD/regional own revenue, almost 70% of Bandung's regional own revenue comes from the tourism sector. Where the

tourism sector is a prima donna that continues to need to be improved.

inherited the style of architectural beauty in Indonesia [2].

**Figure 1.**

*Tourism*

**212**

*Tourism elements. Source: Goeldner & Ritchie [6].*

The city of Bandung as one of the cities of past cultural heritage has superior values to be preserved, where the existence of the city of Bandung with its rich architectural styles/styles is its main attraction as a tourist destination and cannot be separated from the history of the development of Bandung City itself [2]. The long history of the city of Bandung has left many historical buildings that make Bandung one of the cities with art deco architectural heritage in the world and was once dubbed the most complete architectural laboratory with various old buildings that

Meanwhile, tourism is a system that develops efforts by emphasizing the value of policies and strategies, as well as promotional patterns that are always synchronized with business development strategies in tourism planning [5, 6]. Mill and

#### **2.1 Tourism and the tourism potential of the City of Bandung**

According to Yoety tourism is a trip that is carried out temporarily from one place to another with the reason that it is not for business or earning a living at the place visited but solely for enjoying the trip for sightseeing and recreation or fulfill various desires [9]. Tourism is also several activities, especially those related to economic activities that are directly related to the entry of foreign culture [9].

The wealth of natural resources, cultural diversity, as well as the wealth of historical heritage that Indonesia possesses is a very large base for economic development and development, especially through the tourism sector [8].

According to Butowski tourism is the main sector in promoting economic and service growth [2, 10, 11]. So that in tourism planning and development [2, 10–12] it is necessary to consider several approaches [2], including: (a) Continuous Incremental, and flexible approach (in the sense that planning [2] is a process that is takes place continuously based on the needs and the results of existing feddback); (b) System Approach [2] (tourism as a system relationship needs to be planned through system analysis); (c) Comprehensive approach [2] (a tourism development approach that holistically considers institutional elements and the environment as well as socio-economic implications); (d) integrated approach (tourism development approach as an integrated system in plans and forms of development in the area); (e) Environmental and sustainable development approach [2] (a tourism approach starting from the planning process, development and management of natural and cultural resources is maintained/does not experience a decline in quality, and environmental analysis becomes the main point); (f) Community approach [2] (an approach to tourism development by maximizing community involvement starting from planning, making decisions that have an impact on socio-economics); (g) Implementable approach [2] (tourism development that formulates plans and recommendations into reality, as well as application of program techniques and strategies); and (h) Application of systematic planning approach [2] (an approach applied in tourism planning based on the logic of activities) [2, 10, 11].

The city of Bandung is the prima donna of park tourism in Indonesia apart from Bali and the city of Yogyakarta, where the city of Bandung has uniqueness, beauty, and extraordinary industrial creativity. It is recorded that almost every weekend, both domestic and foreign tourists are always visited. There are 14 (fourteen) potential tourism clusters in Bandung, including: (a) Shopping and health tourism clusters on the Sukajadi-Setrasari-Pasteur road; (b) Cluster tours for traditional arts and culture and handicraft industries along the Padasuka-Suci route; (c) Historical and Heritage tourism clusters on the Braga-Asia Africa-Cikapundung route; (d) The textile industry tourism cluster in Cigondewah; (e) Cluster of traditional cultural arts and MICE tours in East Bandung; (e) Cluster of spiritual tours and shopping tours on the Gegerkalong - Setiabudi route; (g) Clusters of entertainment tours, shopping tours, Geotourism on the Alun-alun - Sudirman - Otista - Gardujadi - Pasirkaliki routes; (h) Cluster of nature recreation tourism, cultural tourism, handicraft industry tourism on the North Dago - Punclut route; (i) Cluster Heritage tourism, education, natural and artificial recreation, convention tours, religion on the Gedung Sate - Gasibu - Sabuga route; (j) Cluster of culinary tours, Heritage tourism, Education, Entertainment and Recreation, Geotourism on the route of Ir. H. Juanda (Dago) - Merdeka - Riau; (k) Heritage tourism clusters, shopping and culinary tours, knitting Industry tours on the Gator Subroto - Binongjati route; (l) Cluster of shopping tourism and handicraft industry tourism in Cibaduyut; (m) Culinary tourism cluster in Burangrang; and (n) Cluster of shopping tourism in Cihampelas [2, 3].

entry of tourists, where logistics and transportation provide access for tourists to a tourist spot [24]. Yu et al. mandate for the government to enforce environmentally friendly practices in logistics and transportation operations, as well as to improve the safety and security of tourists which can reduce the harmful effects on environmental sustainability, which inspire criminal activity, so as to attract foreign tourists [24]. The same research was conducted in Malaysia by Syarif et al. related to the role of tourism, transportation, and globalization that affect the environment

*Model of Virtual Tourism as an Alternative of the Concept of Architecture Tourism Post…*

Syarif's research results show how important logistics and eco-friendly benefits for economic growth in disadvantaged areas, can increase job opportunities, and as a solution for protection and development. The government must encourage the development of low-carbon ecotourism and achieve both tourism and economic

The concept of sustainable tourism development [2] has been discussed since the 1980s. According to Butler [26], sustainable tourism is a constant answer to current tourism problems [2, 13]. Butler divides sustainable tourism into 2 (two) ways, namely: (a) seen from a semantic approach, sustainability as a guarantee of longterm survival following the market, and (b) seen from the concept of sustainable development [2], in the sense of creating sustainable tourism as regional development without violating the principles of sustainable development [2, 14, 15]. Ceballos-Lascurain [29] introduced the concept of ecotourism and since then began to develop [2] with various terms about alternative tourism [16, 17], including green tourism, soft tourism, nature tourism [18, 19], environmentally friendly/ environmentally sensible tourism, responsible tourism, discreet tourism, appropriate tourism, ecoethnotourism [2, 10, 11, 17–25]. With an evaluation approach that juxtaposes new forms of tourism with the old model (mass tourism in nature) [2]. According to Niezgoda sustainable tourism is the relationship between tourism

Based on **Figure 3**, sustainable tourism [2] is aimed at realizing sustainable development and tourism development itself. According to Farrell & Twining-Ward [34] the sustainable tourism approach must be based on an interdisciplinary approach [2], this is because the level of complexity and uncertainty in the behavior of tourism systems and systems that affect it does not guarantee satisfactory results [20, 21]. Furthermore, Farrell & Twining-Ward [34] conveyed a new concept of

using quantitative ARDL approaches [25].

*DOI: http://dx.doi.org/10.5772/intechopen.94015*

[2], the environment, and development [30] (**Figure 2**).

*Conceptions of sustainable tourism. Source: Permana et al. [2].*

development [25].

**Figure 2.**

**215**

**2.3 Sustainable tourism**

The development of this cluster is a consequence of the development and changes of the city as well as market demand, as happened on Jalan Ir. H. Juanda, which was originally a non-commercial area, turned into a commercial area which made it a new destination, which is a very busy shopping tour.

#### **2.2 Globalization and the environment: between economic growth, transport, and environmental degradation**

Globalization training on an integrated market formation process for goods, services, capital, knowledge, and factors of production around the world. Where globalization catalyzes demand and supply scenarios in international markets by means of exchanges of merchandise and services. Globalization is also a growth in the economic growth achieved by developing countries, but if viewed from an ecological aspect it is not sustainable because globalization is not a policy tool. Globalization has a significant effect on the environment and climate change [13–15]. This is in line with the opinion of Jorgenson and Kick regarding the connection between the environment and global economic processes [16]. Globalization has increased the relationship between emissions and economic growth on changing ecological footprint [17, 18]; the influence of globalization on environmental degradation which causes environmental damage [13, 15, 19]. Khan and Ullah applied an autoregressive distribution lag (ARDL) model to examine the relationship between globalization and CO2 emissions in Pakistan [20]; research on two-way causality between globalization and CO2 emissions in Saudi Arabia from 1971 to 2016 [21]; the relationship between financial development, globalization, and CO2 emissions in Asia Pacific Economic Cooperation (APEC) countries from 1990 to 2016 [22, 23].

Several research results have been conducted on the impact of globalization, among others: the impact of globalization and; tourism against environmental degradation by Sharif et al. in China in the 1978Q1-2017Q4 period [23]. This is supported by the results of research by Yu et al. in Thailand which show that logistics and operations related to transportation are positively correlated with the

#### *Model of Virtual Tourism as an Alternative of the Concept of Architecture Tourism Post… DOI: http://dx.doi.org/10.5772/intechopen.94015*

entry of tourists, where logistics and transportation provide access for tourists to a tourist spot [24]. Yu et al. mandate for the government to enforce environmentally friendly practices in logistics and transportation operations, as well as to improve the safety and security of tourists which can reduce the harmful effects on environmental sustainability, which inspire criminal activity, so as to attract foreign tourists [24]. The same research was conducted in Malaysia by Syarif et al. related to the role of tourism, transportation, and globalization that affect the environment using quantitative ARDL approaches [25].

Syarif's research results show how important logistics and eco-friendly benefits for economic growth in disadvantaged areas, can increase job opportunities, and as a solution for protection and development. The government must encourage the development of low-carbon ecotourism and achieve both tourism and economic development [25].

#### **2.3 Sustainable tourism**

The city of Bandung is the prima donna of park tourism in Indonesia apart from Bali and the city of Yogyakarta, where the city of Bandung has uniqueness, beauty, and extraordinary industrial creativity. It is recorded that almost every weekend, both domestic and foreign tourists are always visited. There are 14 (fourteen) potential tourism clusters in Bandung, including: (a) Shopping and health tourism clusters on the Sukajadi-Setrasari-Pasteur road; (b) Cluster tours for traditional arts and culture and handicraft industries along the Padasuka-Suci route; (c) Historical and Heritage tourism clusters on the Braga-Asia Africa-Cikapundung route; (d) The textile industry tourism cluster in Cigondewah; (e) Cluster of traditional cultural arts and MICE tours in East Bandung; (e) Cluster of spiritual tours and shopping tours on the Gegerkalong - Setiabudi route; (g) Clusters of entertainment tours, shopping tours, Geotourism on the Alun-alun - Sudirman - Otista - Gardujadi - Pasirkaliki routes; (h) Cluster of nature recreation tourism, cultural tourism, handicraft industry tourism on the North Dago - Punclut route; (i) Cluster Heritage tourism, education, natural and artificial recreation, convention tours, religion on the Gedung Sate - Gasibu - Sabuga route; (j) Cluster of culinary tours, Heritage tourism, Education, Entertainment and Recreation, Geotourism on the route of Ir. H. Juanda (Dago) - Merdeka - Riau; (k) Heritage tourism clusters, shopping and culinary tours, knitting Industry tours on the Gator Subroto - Binongjati route; (l) Cluster of shopping tourism and handicraft industry tourism in Cibaduyut; (m) Culinary tourism cluster in Burangrang; and (n) Cluster of shopping tourism in

The development of this cluster is a consequence of the development and changes of the city as well as market demand, as happened on Jalan Ir. H. Juanda, which was originally a non-commercial area, turned into a commercial area which

**2.2 Globalization and the environment: between economic growth, transport,**

Globalization training on an integrated market formation process for goods, services, capital, knowledge, and factors of production around the world. Where globalization catalyzes demand and supply scenarios in international markets by means of exchanges of merchandise and services. Globalization is also a growth in the economic growth achieved by developing countries, but if viewed from an ecological aspect it is not sustainable because globalization is not a policy tool. Globalization has a significant effect on the environment and climate change [13–15]. This is in line with the opinion of Jorgenson and Kick regarding the connection between the environment and global economic processes [16]. Globalization has increased the relationship between emissions and economic growth on changing ecological footprint [17, 18]; the influence of globalization on environmental degradation which causes environmental damage [13, 15, 19]. Khan and Ullah applied an autoregressive distribution lag (ARDL) model to examine the relationship between globalization and CO2 emissions in Pakistan [20]; research on two-way causality between globalization and CO2 emissions in Saudi Arabia from 1971 to 2016 [21]; the relationship between financial development, globalization, and CO2 emissions in Asia Pacific Economic Cooperation (APEC) countries from

Several research results have been conducted on the impact of globalization, among others: the impact of globalization and; tourism against environmental deg-

radation by Sharif et al. in China in the 1978Q1-2017Q4 period [23]. This is supported by the results of research by Yu et al. in Thailand which show that logistics and operations related to transportation are positively correlated with the

made it a new destination, which is a very busy shopping tour.

**and environmental degradation**

Cihampelas [2, 3].

*Tourism*

1990 to 2016 [22, 23].

**214**

The concept of sustainable tourism development [2] has been discussed since the 1980s. According to Butler [26], sustainable tourism is a constant answer to current tourism problems [2, 13]. Butler divides sustainable tourism into 2 (two) ways, namely: (a) seen from a semantic approach, sustainability as a guarantee of longterm survival following the market, and (b) seen from the concept of sustainable development [2], in the sense of creating sustainable tourism as regional development without violating the principles of sustainable development [2, 14, 15].

Ceballos-Lascurain [29] introduced the concept of ecotourism and since then began to develop [2] with various terms about alternative tourism [16, 17], including green tourism, soft tourism, nature tourism [18, 19], environmentally friendly/ environmentally sensible tourism, responsible tourism, discreet tourism, appropriate tourism, ecoethnotourism [2, 10, 11, 17–25]. With an evaluation approach that juxtaposes new forms of tourism with the old model (mass tourism in nature) [2].

According to Niezgoda sustainable tourism is the relationship between tourism [2], the environment, and development [30] (**Figure 2**).

Based on **Figure 3**, sustainable tourism [2] is aimed at realizing sustainable development and tourism development itself. According to Farrell & Twining-Ward [34] the sustainable tourism approach must be based on an interdisciplinary approach [2], this is because the level of complexity and uncertainty in the behavior of tourism systems and systems that affect it does not guarantee satisfactory results [20, 21]. Furthermore, Farrell & Twining-Ward [34] conveyed a new concept of

**Figure 2.** *Conceptions of sustainable tourism. Source: Permana et al. [2].*

sector. However, in Indonesia, this technology has not been used for matters related to crucial fields such as medicine, industrial/automotive engineering, as well as those related to conservation such as architecture, cities, archeology, and history. Even though the VR application offers a variety of innovative solutions for efforts to

*Model of Virtual Tourism as an Alternative of the Concept of Architecture Tourism Post…*

VR technology [48] is an interface technology between humans and machines that can simulate people as if they are in a natural environment including sight, hearing, movement, and other actions [49]. This condition can not only clearly describe the real environment, but VR also allows users to observe the virtual

VR as one of the innovations that can be used in e-Tourism invites users to interact with an environment that is simulated by a computer. According to LaValle [52], VR displays a pseudo-reality environment [50] by presenting a visual experience displayed on a computer layer [39, 40] or through a stereoscopic [41, 42] viewer where some simulations include additional sensing information such as

VR as technology has made a big difference in the history of human thought and is currently becoming a trend to help improve performance and product quality. VR is a technology created so that users can interact with an environment that is simulated by a computer. A simulated environment can be similar to the real world, a representative virtual reality experience by including a simulation of a combination of sensory results (visual, audio, touch). Computers help simulate a real object by generating a

The main components of this system hardware are the VR engine or computer system, input devices, and output devices. In general, the input device is responsible for interaction, the output device for immersive sense, and the VR engine or computer and its software for accurate control and synchronization of the entire

Based on the image, the input device is a means for users to interact with the virtual world. This device sends signals to the system about the user's actions, thereby providing the appropriate reaction back to the user via the output device in real-time. These devices can be classified for example as tracking devices, pointinput, bio-controllers, and sound devices. VR Engine or computer is a data processor and storage. Real-timing, graphic display, and image processing are some of the important factors and can take up time in the operation of the VR system so it must

The choice of VR Engine depends on the application field, the user itself, input and output devices, the level of immersive and graphic output required, because the VR Engine is responsible for calculating and producing graphic models, object rendering, lighting, mapping, texturing, simulation and so on. to be displayed in real-time. The computer as a VR Engine also handles interactions with users and functions as an interface with input and output devices. The output device is a device that receives feedback from the VR Engine and presents it to the user via a suitable output device to stimulate the senses. Several classifications of output devices based on the senses are graphic (visual), audio (hearing), haptic (touch or style), smell, and taste. The visual display is the most popular output in a VR system

accelerate high technology to maximize performance and products [47].

three-dimensional (3D) atmosphere to make the user physically involved.

be selected according to the character of the application requirements.

environment and feel like they are in that place [23, 32].

*DOI: http://dx.doi.org/10.5772/intechopen.94015*

sound displayed through headphone speakers [5, 39–43].

virtual environment [24, 44] (**Figure 4**).

*VR system stove. Source: Bahar [48]; Mazuryk and Gervautz [53].*

**Figure 4.**

**217**

**Figure 3.** *Virtual reality. Source: Amazon.com dan VIVE developers.*

sustainable tourism with the term "comprehensive tourism system and complex adaptive tourism systems (CATS)" [2, 21, 22]. Farrell and Twining-Ward [34] divides into 4 (four) areas that affect sustainable tourism, namely: ecosystem ecology, ecological economics, global change science, and complexity theory [2, 21].


2.1.1. *L*1 Penalized Boosting

The core step of PKB is the optimization of the regularized loss function (see step 3 of Table 1). Note that G is the union of the pathway-based learner spaces, thus

$$\begin{aligned} \hat{f}\_{\mathbf{m}} &= \arg\min\_{\mathcal{G}} L\_{\mathcal{R}}(\mathbf{f})\\ &= \arg\min\_{\hat{f}\_{\mathbf{m}}} \{ L\_{\mathcal{R}}(f\_{\mathbf{m}}) : f\_{\mathbf{m}} = \arg\min\_{\hat{f} \in \mathcal{G}\_{\mathbf{m}}} L\_{\mathcal{R}}(\mathbf{f}), m = 1, 2, \dots, M \}. \end{aligned}$$

To solve for ˆ *f* , it is sufficient to obtain the optimal ˆ *fm* in each pathway-based subspace. Due to the way we construct the subspaces, in a given pathway *m*, *f* takes a parametric form as a linear combination of the corresponding kernel functions. This helps us further reduce the optimization problem to

$$\min\_{f \in \mathcal{G}\_m} L\_R(\mathbf{f}) \quad = \min\_{\beta, \mathcal{E}} \frac{1}{N} \sum\_{i=1}^N \frac{q\_{i,t}}{2} (\frac{h\_{i,t}}{q\_{i,t}} + K\_{m,i}^T \beta + \varepsilon)^2 + \Omega(f) \tag{4}$$

$$=\min\_{\boldsymbol{\beta},\boldsymbol{c}} \frac{1}{N} (\boldsymbol{\eta}\_{t} + \boldsymbol{K}\_{\text{m}}\boldsymbol{\beta} + \mathbf{1}\_{N}\boldsymbol{c})^{T} \mathcal{W}\_{t} (\boldsymbol{\eta}\_{t} + \boldsymbol{K}\_{\text{m}}\boldsymbol{\beta} + \mathbf{1}\_{N}\boldsymbol{c}) + \boldsymbol{\Omega}(\boldsymbol{f}),\tag{5}$$

where

$$\begin{array}{rcl} \eta\_{t} &=& (\frac{h\_{1,t}}{q\_{1,t}}, \frac{h\_{2,t}}{q\_{2,t}}, \dots, \frac{h\_{N,t}}{q\_{N,t}})^T, \\\ W\_{t} &=& \text{diag}(\frac{q\_{1,t}}{2}, \frac{q\_{2,t}}{2}, \dots, \frac{q\_{N,t}}{2}), \\\ K\_{\text{m}} &=& \left[K\_{\text{m}}(\mathbf{x}\_{i}^{(\text{m})}, \mathbf{x}\_{j}^{(\text{m})})\right]\_{i,j=1,2,\dots,N}. \end{array}$$

*Km*,*i* is the *i*th column of kernel matrix *Km* and 1*N* is an *N* by 1 vector of 1's. We use the *L*1 norm Ω(*f*) = *λβ*1, as the penalty term, where *λ* is a tuning parameter adjusting the amount of penalty we impose on model complexity. We also prove that after certain transformations, the optimization can be converted to a LASSO problem without intercept

$$\min\_{\beta} \frac{1}{N} \|\tilde{\eta} + \tilde{K}\_{\text{m}} \beta\|\_{2}^{2} + \lambda \|\beta\|\_{1} \tag{6}$$

where

$$\begin{array}{rcl} \eta &=& W\_{\mathfrak{t}}^{\frac{1}{2}} \left[ I\_N - \frac{\mathbf{1}\_N \mathbf{1}\_N^T W\_{\mathfrak{t}}}{tr(\mathcal{W}\_{\mathfrak{t}})} \right] \eta\_{\mathfrak{t}}\\ \mathcal{R}\_{\mathfrak{m}} &=& W\_{\mathfrak{t}}^{\frac{1}{2}} \left[ I\_N - \frac{\mathbf{1}\_N \mathbf{1}\_N^T W\_{\mathfrak{t}}}{tr(\mathcal{W}\_{\mathfrak{t}})} \right] K\_{\mathfrak{m}}. \end{array}$$

Therefore, *β* can be efficiently estimated using existing LASSO solvers. The proof of the equivalence between the two problems is provided in Section 1 of the Supplementary Materials.


**9. Pancreatic metastases from uterine body or cervix cancer**

as part of tertiary cytoreduction. The patient submitted to primary cytoreduction benefitted from a distal pancreatectomy in association with splenectomy which was associated to a total hysterectomy with bilateral adnexectomy, pelvic and para-aortic lymph node dissection and parcelar gastrectomy; the long-term outcome was a favorable one, the patient being diagnosed with relapse at 54 months follow-up. Patients submitted to pancreatic resections as part of secondary cytoreduction experienced a median disease free interval of 32 months and necessitated in all cases a distal pancreatectomy. Postoperatively, two patients developed pancreatic leaks which were treated conservatively in one case and through reoperation in the second case. When it comes to the long-term outcomes, the median overall survival was 36.38 months, all cases being dead of disease at the end of the study. At the time of tertiary cytoreduction, pancreatic resection was performed in a single case, 52 months after the initial diagnostic. Although the early postoperative outcome was favorable, the patient died of disease 10 months later. The authors demonstrated in this way the effectiveness of pancreatic resections as part of cytoreductive surgery in the setting of advanced stage disease as well as

for patients diagnosed with recurrent lesions [45].

necessitating pancreatic resections

necessitating pancreatic resections

necessitating pancreatic resections

necessitating pancreatic resections

necessitating pancreatic resections

**Table 2.** Outcomes after pancreatic resections for pancreatic metastases from ovarian carcinomas.

2001–2004 482 cases, 12 patients necessitating pancreatic resections

2002–2004 6 cases, 2 patients necessitating pancreatic resections

2000–2003 57 cases, 6 patients necessitating pancreatic resections

**No. of patients Type of**

**pancreatic resection**

**Early postoperative outcomes**

DP: 3 cases 0 Not reached at the end of

DP: 6 cases NR Not reached at 36 months

DP: 2 cases 0.6% 106 months—in cases

DP: 9 cases 1% Overall survival after

DP: 2 cases 0 Overall survival after

DP: 17 cases 1.4% Overall survival after

DP: 12 cases Not reported Overall survival after

DP: 13 cases 2% Overall survival after

**Long-term outcomes**

the study (for the entire

follow-up (for the entire

submitted to complete cytoreductive surgery

pancreatic resections: 54

pancreatic resections: not

pancreatic resections: 57

pancreatic resections: 54.6

pancreatic resections: 49

group)

group)

months

reported

months

months

months

**Period of the study**

Chi, 2004 [47] 2001–2002 70 cases, 3 patients

Chi, 2006 [48] 1989–2003 465 cases, 2 patients

Chi, 2009 [49] 2001–2004 210 cases, 9 patients

Chi, 2010 [51] 2001–2006 141 cases, 17 patients

Heitz, 2016 [53] 2005–2010 578 cases, 13 patients

Abbreviations: DP, distal pancreatectomy.

**Name, year of the study**

142 Advances in Pancreatic Cancer

Eisenhauer, 2000

Hoffman, 2007

Rodriguez, 2013

[50]

[52]

[43]

Pancreatic metastases from uterine primaries are other rare eventualities, only few cases being described so far [1, 54–56]. The main pattern of spread responsible for the apparition of pancreatic metastases with endometrial origin consists of hematogenous disseminations and it is usually responsible for the apparition of other distant lesions such as hepatic, pulmonary or splenic metastases [57]. Due to this aspect, pancreatic metastases from uterine carcinomas can be rarely treated with curative intent. The first authors who reported performing a surgical procedure for a pancreatic metastasis originating from an endometrial carcinoma came from the USA, in 1998; it was the case of a patient known with previous history of endometrial cancer who presented for upper digestive stenosis 3 years later. At this time, a 4 cm tumor located at the level of the uncinate process of the pancreas was found, so the patient was successfully submitted to surgery; unfortunately the authors did not report the performed surgical procedure or the outcome of this patient [2]. The first successful pancreatic resection for pancreatic metastases from endometrial cancer came from Dan Blazer, at *M.D. Cancer Center, Houston, Texas, United States of America,* in 2008. It was the case of a 56-year-old patient who had been previously submitted to surgery for endometrial cancer, at that moment a total hysterectomy with bilateral adnexectomy, pelvic and para-aortic lymph node dissection being performed; postoperatively, the patient was submitted to adjuvant radiotherapy. However, 31 months later, she was diagnosed with a pancreatic lesion measuring 3 × 3 cm in the pancreatic tail. The fine needle biopsy confirmed the metastatic origin, so the patient was resubmitted to surgery, a distal pancreatectomy being performed. The histopathological studies confirmed the metastatic origin of the lesion; however, 6 months later, the patient remained free of recurrent disease [55].

Pancreatic oligometastases with uterine cervix origin is another rare situation, a successful resection of such a lesion being reported for the first time by Wastell et al. in Westminster, London. The authors reported the case of a patient who had been initially treated by radiotherapy with curative intent for a squamous cell carcinoma; however, 5 years later, the patient was diagnosed with a pancreatic head tumor. At that moment a pancreatoduodenectomy was performed, the histopathological studies confirming the metastatic origin of this lesion; unfortunately the postoperative course was complicated by the apparition of a bronchopneumonia, the patient being dead 16 days later [58].

**11. Pancreatic cancer from sarcomas**

ity of resection with curative intent [7].

6 years after pancreatic resection [63].

still alive at the time of publishing the case [64].

curative resection is performed [65, 66].

**12. Conclusion**

Metastatic lesions with sarcomatous origin are usually associated with an extremely poor outcome due to the biological aggressiveness of such primaries. When encountered, pancreatic metastases with sarcomatous origin are reported as part of the systemic disease so most often surgery is no longer a valid therapeutic option. In cases presenting oligometastatic disease, surgery might be proposed whenever the biological status of the patient will permit it. A particular problem in such cases is related to the multifocality of such lesions and to the feasibil-

Pancreatic Resections for Metastatic Disease http://dx.doi.org/10.5772/intechopen.75571 145

Successful resection of pancreatic metastases from soft tissue sarcomas has been reported by the Japanese authors in two cases. The first patient had been initially diagnosed with a mesenchymal chondrosarcoma of the left thigh in 1986; 3 years later, the patient was diagnosed with isolated pancreatic lesions, the patient being submitted to surgery with curative intent; the patient remained alive for the next 10 years. The second case was initially diagnosed with a synovial sarcoma followed by pulmonary resection for metastatic disease; the case was further diagnosed with a solitary pancreatic lesion for which she was submitted to pylorus preserving pancreatoduodenectomy with good results, the patient remaining alive for more than

Another extremely interesting situation was reported by another Japanese team in 2016. The authors reported the case of a 44-year-old woman who had been previously submitted to surgery for a right fibular head osteosarcoma; 3 years later, the patient was diagnosed with a metastasis in the distal pancreas, so a laparoscopic distal pancreatectomy with spleen preservation was successfully performed; the histopathological studies confirmed the metastatic origin from the initial osteosarcoma. Although the patient also reported the apparition of lung metastases, 1 year later, she was resubmitted to surgery with curative intent, the patient being

A particular situation is represented by patients diagnosed with pancreatic metastases from uterine sarcomas, in cases presenting oligometastatic disease, surgery being considered as a valid option. Most often these lesions occur in patients who had been previously diagnosed with uterine leiomiosarcomas and might experience good long-term outcomes whenever a

Isolated pancreatic metastases suitable for resection are rare eventualities; the renal cell carcinoma origin being the most frequently reported situations. When diagnosed as metachronous isolated lesions, such metastases can be submitted to surgery with curative intent, long-term survival rates being reported. Another primary with good outcomes after pancreatic resections for metastatic disease is represented by ovarian cancer, debulking surgery to no residual disease

Another interesting case was reported by the Japanese authors in 2013 [59]. The authors reported the case of a 44-year-old patient who had been initially diagnosed with a stage IB uterine cervix cancer, the histopathological studies reporting a mixed adeno-neuroendocrine carcinoma; 8 years later, the patient was diagnosed with an isolated pancreatic tumor which was biopsied, a metastatic neuroendocrine tumor being revealed. At that moment a central pancreatectomy was performed, the histopathological studies confirming the presence of a metastatic lesion; however, only the neuroendocrine component seems to be responsible for the apparition of the recurrent disease. The long-term outcomes were favorable, the patient being free of disease at a 7 month follow-up [59].


**Secondary Metabolites and Antioxidant Activity of the Solid-State Fermentation in Apple (***Pirus malus* **L.) and Agave Mezcalero (***Agave angustifolia* **H.) Bagasse**

**Diego Ibarra-Cantún 1 , María Elena Ramos-Cassellis 2,\* , Marco Antonio Marín-Castro 3 and Rosalía del Carmen Castelán-Vega 3**

Received: 30 June 2020; Accepted: 3 August 2020; Published: 18 August 2020

**Abstract:** Solid-state fermentation (SSF) is used in enzyme and antibiotic production, bioethanol and biodiesel as an alternative energy source, biosurfactants with environmental goals, and the production of organic acids and bioactive compounds. The present project determined the quantity of secondary metabolites and the antioxidant activity of the extracts obtained by the solid-state fermentation of apple and agave mezcalero bagasse over 28 days, inoculated with the *Pleurotus ostreatus* strain. The extraction was carried out with three solvents: acetone and water (80:20 *v*/*v*), 100% methanol and 100% water. The results showed a higher presence of phenolic compounds, flavonoids, total triterpenes and antioxidant activity in the apple bagasse from the SSF on day 21 in the extract of acetone and water (80:20 *v*/*v*), 100% methanol and aqueous; while the agave bagasse showed a significant presence of phenolic compounds and flavonoids only in the aqueous extract. In conclusion, the presence of secondary metabolites exhibiting antioxidant activities from the solid-state fermentation in the residues of the cider and mezcal industry is an alternative use for wasted raw material, plus, it reduces the pollution generated from the agroindustrial residues.

**Keywords:** agave mezcalero bagasse; apple bagasse; solid-state fermentation; secondary metabolites; *Pleurotus ostreatus*

#### **1. Introduction**

The residues generated by the agroindustry are, in most cases, not processed, causing environmental pollution and adverse effects on health [1]. In Mexico, this situation is mainly reflected in the production of the cider industry in Puebla and the production of mezcal in the state of Oaxaca. The former reported an annual production of 150 thousand bottles [2], where the processed fruit residues fluctuate between 15% and 30% of said production, depending on the state of maturation and the used technology [3]. The latter generated 122,696 tons of bagasse in 2012, which is usually dumped in rivers or streams, endangering the region's environment [4].

Most of these wastes degrade slowly due to their solid and organic nature, which corresponds to their lignocellulosic biomass, rich in cellulose, hemicellulose and lignin. In addition, they are not subjected to an adequate process of exploitation, giving as a result an utterly deficient disposal of the waste [5,6]. Therefore, the aim of the present investigation was to determine the content of the total phenolic compounds,

total flavonoids and total triterpenes, and the antioxidant activity, by 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2′ -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS•+) test methods resulting from the solid-state fermentation of apple bagasse and agave mezcalero bagasse substrates inoculated with the *Pleurotus ostreatus* strain, in order to optimize the utilization of residues of the cider and mezcalero industry in the attaining of secondary metabolites.

*Pleurotus ostreatus* synthesizes bioactive compounds, such as: polysaccharides (alpha and beta glucan) and polyphenols [7,8]. The macromycetes, such as *Pleurotus* and *Agaricus*, contain tetracyclic triterpenoids, with the sterols being the most abundant ones. At the same time, in *Pleurotus mutilis* and *Clitopilus passeckerianus* (called *Pleurotus passeckerianus* in the past), pleuromutilin and diterpene were identified as having antibiotic action against microplasmatic infections in animals [9,10].

Singhania et al. [11] and Thomas et al. [12] describe solid-state fermentation (SSF) as a process that takes place in a solid matrix that is within an inert support or substrate, or near free water, with just enough to promote the growth and metabolic activity of microorganisms. The SSF reduces the production costs due to the lower energy consumption and production area (smaller reactors), proving that it is an economic, interesting and environmentally friendly process for the production of secondary metabolites from agroindustrial and forest residues [13]. The SSF has generated great interest in recent years due to its variety of uses, such as bioremediation, biofuels (biodiesel, bioethanol, biobutanol, and biohydrogen among others) and the production or extraction of bioactive compounds [14].

One of the main applications of the solid fermentation of agroindustrial wastes is the extraction of secondary metabolites, also called phytochemicals, which include carotenes, terpenes, sterols, tocopherols and polyphenols. These can be used in the treatment and prevention of various diseases [15], since they have antitumor, anti-inflammatory, antithrombotic, antimicrobial, immune system modulating, and hypoglycemic properties; also, they reduce the concentration of lipids in the blood and display antioxidant activities [16,17]. Waste from agribusiness, such as the peel of fruits and vegetables, has been employed recently as a potential source of production of bioactive compounds that are commonly known as phenolic compounds [18]. Therefore, the secondary metabolites obtained from the solid fermentation of residues can be used as natural bioactive triggers for the formulation of functional foods or serve as additives in food products to extend their shelf life [19].

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

#### *2.1. Obtaining Plant Material*

The apple bagasse was collected from Huejotzingo's cider industry, a town located in the state of Puebla, Mexico, and the agave mezcalero bagasse was collected from San Pablo Villa de Mitla's mezcal industry, a town located in the state of Oaxaca, Mexico.

These bagasses were transported in airtight containers at 20 ◦C in less than 4 h. They were subsequently bleached at 70 ◦C for 5 min and dried in a forced air dryer (HTP 72 Lumistell Celaya, Gto. México) for 8 h at 60 ◦C until the moisture content was less than 10%. Once dried, the bagasse was crushed in a mill to obtain a particle of 0.841 mm to be used during solid fermentation.

Physicochemical tests were performed on the crushed bagasse in order to discover its attributes and adjust its physicochemical properties to make them suitable for the growth of the *Pleurotus* fungus. The bagasse reconditioning was carried out according to Staments and Chilton [20] where it is mentioned that *Pleurotus ostreatus* culture must have the following conditions: a humidity percentage of 80–90%, temperature from 23 to 26 ◦C, and pH from 6.5 to 7.5. The temperature and relative humidity were controlled during the process. The physicochemical tests performed were: total soluble solids (◦Brix) [21], pH [21], moisture percentage [21], water activity (A*w*) [21], and titratable acidity percentage [21]; the meq value of malic acid were 0.067, the meq value of acetic acid were 0.060 and direct reducing sugars were also used [21].

#### *2.2. Solid-State Fermentation*

This process was accomplished with the contribution of the Mycology Laboratory of the Departamento de Investigación de Ciencias Agrícolas (Agricultural Sciences Research Department) of the Benemérita Universidad Autónoma de Puebla (Benemeritus Autonomous University of Puebla), which provided the *Pleurotus ostreatus* strains.

The solid-state fermentation was carried out in 9.7 × 2.6 cm glass tubes with a Bakelite cork, and each contained 10 g of each residue adjusted to a humidity percentage of 80% and a pH of 6.5, which was then sterilized in an autoclave ((CV300 AESA Tecámac, Edo. México, México)) at 120 ◦C and 15 psi for 45 min.

Then, a 1 cm diameter mycelium-agar circle of the *Pleurotus ostreatus* strain, corresponding to 3 mg of dry weight biomass, was incorporated into each tube and incubated in an oven (E51 Riossa Monterrey, N.L., México) at 25 ◦C. Samples were taken at 0, 7, 14, 21 and 28 days.

#### *2.3. Solid Waste Extraction*

After the fermentation days, 12 mL of each solvent was added to each glass tube for extraction. The used solvents were acetone and water (80:20 *v*/*v*), 100% methanol and distilled water. The samples were placed in a shaker (KJ-201BD Orbital Shaker Westtune Hangzhou, Zhejiang, China) at 80 rpm for 1 h, filtered and placed in a centrifuge (Z200A Hermle Wehingen, Germany) at 47 g, and the ensuing supernatant was stored in Eppendorf tubes at −20 ◦C for further analysis.

#### *2.4. Quantification of Total Phenolic Compounds (TPC)*

The analysis was performed using the method described by Singleton and Rossi [22], with some modifications. A total of 250 µL of 50% Folin–Ciocalteu reagent was added to 50 µL of the extract; then it was stirred and allowed to stand in the dark for 8 min. Subsequently, 1.25 mL of 5% CaCO3 (*w*/*v*) was added and allowed to stand again for 30 min in the dark at room temperature. An absorbance at 725 nm was read afterwards on a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). A calibration curve for gallic acid (Sigma Aldrich, CAS 149-91-7, Steinheim, Germany) was used in the range of 0 to 0.3 g/L. The results were expressed in mg gallic acid equivalent per g substrate on dry weight (mg GAE/g substrate dw). These analyses were performed in triplicate.

#### *2.5. Total Flavonoid Quantification (TF)*

The methodology proposed by Chang et al. [23] was used, with some modifications. A total of 500 µL of the extract, 1.5 mL of 80% ethanol (*v*/*v*), 100 µL of 10% hexahydrate aluminum chloride solution, 100 µL of 1 M potassium acetate, and 2.8 mL of distilled water were placed, stirred and incubated for 30 min at room temperature.

Subsequently, an absorbance at 415 nm was read on a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). A quercetin calibration curve (Sigma Aldrich, CAS 117-39-5, Germany) was used at a concentration of 0 to 0.1 g/L. The results were expressed in mg quercetin equivalents per g substrate on dry weight (mg QE/g substrate dw). These analyzes were performed in triplicate.

#### *2.6. Total Triterpenes Quantification (TT)*

The vanillin-acetic acid method was used, with some modifications [24]. A total of 120 µL of each extract, 100 µL of vanillin (Sigma Aldrich, CAS 121-33-5, Germany) 5% (*w*/*v*), and 400 µL of perchloric acid in 0.1 N glacial acetic acid were placed together in a test tube at 60 ◦C for 15 min.

Then, the mixture was cooled down to room temperature and 2.5 mL of glacial acetic acid were added. The absorbance was measured at 550 nm in a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). The results were expressed as mg ursolic acid per g substrate on dry weight (mg UA/g substrate dw). A calibration curve for uric acid (Sigma Aldrich, CAS 77-52-1, Germany) was used with a concentration from 0 to 120 g/L. These analyses were performed in triplicate.

#### *2.7. Antioxidant Activity by 1,1-Diphenyl-2-Picril Hydracil (DPPH*•*)*

The DPPH• (2,2-Diphenyl-1-picrilhydrazyl) radical in methanol solution was developed by Brand-Williams et al. [25], and was used with some alterations. A total of 990 µL of DPPH (Sigma Aldrich, CAS 1898-66-4, Germany) 0.188 mM was added to 10 µL of the extract. The mixture was then homogenized and kept in the dark for 30 min at room temperature.

The absorbance at 517 nm was measured in both the control (methanol (A0)) and the test samples (A1) on a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). In order to calculate the percentage of inhibition, Equation (1) was used. These analyses were performed in triplicate.

Subsequently, the results were expressed as the inhibitory concentration (IC50) and the percentage of inhibition of four different concentrations was calculated to obtain a linear regression curve. These analyses were performed in triplicate.

$$\text{Percent inhibition} = \left[\frac{\text{A0} - \text{A1}}{\text{A0}}\right] \ast 100 \tag{1}$$

A0 = Control absorbance A1 = Sample absorbance.

#### *2.8. Antioxidant Activity by 2,2*′ *-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid Radical Cation (ABTS*•+*)*

The method proposed by Re et al. [26] was used. An ABTS•+ radical cation was formed from 0.0033 g of potassium persulfate and 0.0194 g of ABTS reagent (2,2'-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid (Sigma Aldrich, CAS 30931-67-0, St. Louise, MO, USA) mixed in 5 mL of distilled water. The mixture was stirred and kept in the dark for 16 h at room temperature. After this, a mixture was made with absolute ethanol and the ABTS•+ radical cation until an absorbance of 0.70 ± 0.02 at 754 nm was obtained.

A total of 3920 µL of the ABTS radical solution were then added and the initial absorbance (Ai) was recorded; 80 µL of extract was afterwards added and mixed. After 7 min, the final absorbance (Af) was recorded with the use of a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). The inhibition percentage was calculated according to Equation (2) to obtain the medium inhibitory concentration (IC50) by a curve of lineal regression using the inhibitory percentage of the four different concentrations. These analyses were performed in triplicate.

$$\text{Percent inhibition } \text{(\%)} = \left[ \frac{\text{Ai} - \text{Af}}{\text{Ai}} \right] \* 100 \tag{2}$$

#### *2.9. Statistic Analysis*

The data analysis was performed through a completely randomized design. The experiment consisted of the inoculation of *Pleurotus ostreatus* in two bagasses (two treatments with five replicas) at 25 ◦C for 28 days. Samples of the substrates were taken at 0, 7, 14, 21 and 28 days of fermentation (5 periods) to obtain the extracts with three organic solvents: acetone and water (80:20 *v*/*v*), 100% methanol, and water at 100% (3 extracts); the secondary metabolite content and antioxidant activity were determined as well. Samples were analyzed in quadruplicate (*n* = 4).

The results were expressed as a mean and standard deviation, and were examined through the Statistical Analysis System (SAS) version 9.0 statistical package [27]. Additionally, an analysis of variance (ANOVA) means test (Tukey α = 0.05) and Pearson correlation were performed.

#### **3. Results**

#### *3.1. Physicochemical Characterization*

Table 1 shows the physicochemical characterization of the residues before fermentation. The values in percent humidity and A*w* are similar since both bagasses were subjected to the same drying process at 60 ◦C for 12 h and then they were crushed. ≤ ≤ ≤ ≤

**Table 1.** Physicochemical characteristics of apple bagasse and agave mezcalero bagasse.

Values are the mean ± standard deviation. Means with the same letters within each column do not differ statistically by Tukey's test (*p* ≤ 0.05). 1 Direct reducing sugars before washing apple bagasse; 2 direct reducing sugars after washing apple bagasse.

#### *3.2. Secondary Metabolites*

#### 3.2.1. Quantification of Total Phenolic Compounds (TPC)

In Figure 1, it is shown the total phenolic compounds resulting from the solid-state fermentation of agroindustrial wastes. The TPC had its highest concentration at the beginning of the experiment (day 0) in both residues, and after fermentation it decreased, increasing again after day 21.

**Figure 1.** Total phenolic compounds (mg gallic acid equivalent per g substrate on dry weight (mg GAE/g substrate dw)) of the solid-state fermentation of agroindustrial residues: (**a**) Apple bagasse; (**b**) Agave mezcalero bagasse. Acetone and water (80:20 *v*/*v* ( ( )); methanol extract ( ol extract ( ); aqueous extract ( aqueous extract ( ). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.

Apple bagasse extract (Figure 1a) increased its concentration 21 days after fermentation, with intervals of 0.385 and 0.587 mg gallic acid equivalent/g substrate dw. The highest concentration was present in the aqueous extract with a value of 0.587 ± 0.013 mg gallic acid equivalent/g substrate dw. The TPC content of agave mezcalero bagasse from the acetone and water (80:20 *v*/*v*) and methanol extracts tended to decrease during fermentation, while the aqueous extract increased its concentration after day 21, until it reached 0.976 ± 0.126 mg gallic acid equivalent/g substrate dw at 28 days.

#### 3.2.2. Total Flavonoid Quantification (TF)

In Figure 2, the total flavonoids in apple bagasse and mezcalero agave are shown. First, the apple increased its TF content from day 21, giving final values (on day 28) of 0.015 ± 0.018, 0.013 ± 0.02, and 0.023 ± 0.012 mg QE/g substrate dw in extracts of acetone and water (80:20 *v*/*v*), methanol and the

≤

≤

aqueous extract, respectively; the latter was the extract with the highest amount of flavonoids. Then, the agave mezcalero bagasse registered a similar tendency to the content of TPC. The total flavonoid concentrations in extracts of acetone and water (80:20 *v*/*v*) and methanol decreased during fermentation, while in the aqueous extract there was an increase in the TF content at the end of the fermentation time, with a final concentration of 0.012 mg QE/g substrate dw.

≤

≤

**Figure 2.** Total flavonoids (mg QE/g substrate dw) of the solid-state fermentation of agroindustrial residues: (**a**) apple bagasse; (**b**) agave mezcalero bagasse. Acetone and water (80:20 *v*/*v* ( ( )); methanol extract ( ol extract ( ); aqueous extract ( aqueous extract ( ). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.

#### 3.2.3. Total Triterpenes Quantification (TT)

In Figure 3, the total triterpenes content is shown. Apple bagasse presented TT contents which increased from day 21 in extracts of acetone and water (80:20 *v*/*v*) and methanol, with final values (on day 28) of 8.043 ± 0.696, and 9.411 ± 2.512 mg ursolic acid/g substrate dw, respectively. The aqueous extract showed steady growth from the beginning of the solid-state fermentation of triterpenes, and reached a concentration of 26.440 ± 0.949 mg ursolic acid/g substrate dw at 28 days. Despite the fact that the TT in agave mezcalero bagasse had the highest value at the beginning of the fermentation, its concentration decreased in all extracts during this process.

**Figure 3.** Total triterpenes (mg UA/g substrate dw) of the solid-state fermentation of agroindustrial residues: (**a**) apple bagasse; (**b**) agave mezcalero bagasse. Acetone and water (80:20 *v*/*v* ( ( )); methanol extract ( ol extract ( ); aqueous extract ( aqueous extract ( ). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.

≤

*J. Fungi* **2020**, *6*, 137

≤

≤

### *3.3. Antioxidant Activites*

The antioxidant activity of the extracts obtained by the solid-state fermentation of *Pleurotus* from apple and agave mezcalero bagasses was expressed as the 50% inhibitory capacity of the radical in question (IC50), which refers to the concentration of antioxidant required to reduce the initial amount of the DPPH and/or ABTS radical by 50%. This is the parameter used to measure the antioxidant properties of a substance [28], that is, the lower the IC50 value, the greater its antioxidant activity.

#### 3.3.1. Antioxidant Activity by 1,1-Diphenyl-2-Picril Hydracil (DPPH•)

In apple bagasse, the IC50 of the DPPH assay ranged from 14.67 to 385.01 g/L residue. On the other hand, the agave mezcalero bagasse presented IC50 values from 61.80 to 377.75 g/L residue (Figure 4a,b). A tendency for IC50 to increase was observed from day 0 to day 21, however, a decrease was perceived from day 28 in different extracts.

**Figure 4.** 50% inhibitory concentration in 2,2-diphenyl-1-picrylhydrazyl (DPPH (IC50/DPPH)) of the solid-state fermentation of agroindustrial residues: (**a**) apple bagasse; (**b**) agave mezcalero bagasse. Acetone and water (80:20 *v*/*v* ( ( )); methanol extract ( ol extract ( ); aqueous extract ( aqueous extract ( ). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.

3.3.2. Antioxidant Activity by 2,2′ -Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid Radical Cation (ABTS•+)

In the ABTS test, the apple bagasse presented a range of residue values from 60.20 to 423.09 g/L; meanwhile, the agave mezcalero bagasse ranged from 74.65 to 390.86 g/L (Figure 5a,b). The obtained results in the apple bagasse showed that the initial values (on day 0) were higher than the agave mezcalero bagasse with intervals of 61.80 to 91.42 g/L of residue.

●

≤

≤

≤

′

′ **Figure 5.** A 50% inhibitory concentration in 2, 2′ -azino-bis (3-ethylbenzothiazoline)-6 ammonium sulfonate (ABTS (IC50/ABTS)) of the solid-state fermentation of agroindustrial residues: (**a**) apple bagasse; (**b**) agave mezcalero bagasse. Acetone and water (80:20 *v*/*v* ( ( )); methanol extract ( ol extract ( ); aqueous extract ( aqueous extract ( ). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.

The acetone and water (80:20 *v*/*v*) and aqueous extracts increased in IC50 during the days of fermentation. It should be noted that the methanol extract displayed a decrease in IC50 at day 28, which means that the secondary metabolites produced at the same time of solid fermentation denoted the antioxidant property of this residue.

#### **4. Discussion**

In both residues, the pH showed values lower than 5.0, lower than the value recommended for the adequate growth of *Pleurotus* (5.5–6.5) [29]. The pH was adjusted for proper fungus growth and fermentation development. The highest amounts of total soluble solids (◦Brix), acidity percentage and reducing sugars were found in the apple bagasse because the residue had a significant amount of polysaccharides and organic acids [30]. This limited the growth of *Pleurotus*, thus, bagasse was subjected to a wash with tridestilated water. After washing the apple bagasse released sugars, it was observed that its content of reducing sugars decreased (Table 1), which was resulted in an adequate growth of the *Pleurotus* strain in the residue.

Apple bagasse presented a significant difference in the aqueous extract of the total triterpenes test, having the highest value; the other extracts showed no significant difference in the other tests performed. In the agave mezcalero bagasse, the significant difference between extracts was manifested in the tests of total flavonoids and total triterpenes; the aqueous extract had the highest value, while the others did not show significant differences in the rest of the performed tests.

The highest phenolic content was at the beginning, since the phenolic compounds are shown in the subtracts (bagasses). In addition, it contains lignin, which is a phenolic polymer made up of three principal monomerics: paracoumaryl alcohol, synapilic alcohol and conyferyl alcohol [31,32]. This acts as a carbon source for the *Pleurotus* fungi for its growth and development (trophophase) after the mycelial growth. However, the nutrient exhaustion produces the biosynthesis of secondary metabolites in the idiophase. In this period (T21) there is a metabolite increase according to Ferrer-Romero et al. [33] and Vamanu [34] which supports the presence of *Pleurotus* and its survival in the residue [35]. Therefore, to observe a higher antioxidant activity, idiophase time must be increased in order.

The TPC values generated from *Pleurotous* in other residues, such as the mixture of pineapple and rice straw residues [36], reported values of 0.179–0.650 mg GAE/g substrate dw. Other authors reported greater values than the ones obtained in this research: Hamdipour et al. [37] showed values of 5.53 to 11.6 mg GAE/g dry weight in the fermentation of apple residues with *Rhizopus oligosporus*, and Ajila et al. [38] obtained values of 4.6 to 16.12 mg GAE/g dry weight during the solid-state

fermentation of apple residues with *Phanerocheate chrysosporium*. Meanwhile, Ferrer-Romero et al. [33] obtained 0.1 g of total phenols/g of glucose by submerged fermentation from the mycelial biomass of *Pleurotus ostreatus*.

The *Pleurotus ostreatus* strain of this study led to the degradation of lignin to obtain the necessary nutrients for its development, given that growth was observed in both lignocellulosic substrates: apple bagasse and mezcalero agave bagasse [39]. In addition, the genus *Pleurotus* has the capacity to produce laccase enzymes, which have a fundamental role both in the degradation of lignin and in the biosynthesis of phenolic compounds in white-rot fungi [40].

Boonsong et al. [41] conducted an investigation with the purpose of evidencing the high nutritional value of edible fungi. This investigation reported intervals from 2.29 to 2.61 mg QE/g in aqueous extracts of *Pleurotus* strains, which are higher values compared to the ones obtained in this project.

Furthermore, Braga et al. [42] reported significant concentrations of total flavonoids (1.76 mg QE/g) in grape marc residue, and 1.70 mg quercetin equivalent/g in mango residue; Dulf et al. [43] found slightly lower values of 0.29 and 0.36 mg QE/g, resulting from the solid-state fermentation of apricot residue with *Aspergillus niger* and *Rhizopus oligosporus* for 14 days; while Lessa et al. [44] found concentrations of TF with values of 31.5 mg QE/100 g in fermented cocoa flour by means of *Penicillium roqueforti*. Therefore, the low concentration of this type of metabolites maybe be determined, once again, by the used strain, that degrades lignin during solid-state fermentation.

Yang et al. [45] found a higher concentration of triterpenes, 47.10 mg/g, 30 days after the solid-state fermentation of citrus residues with the fungus *Antrodia cinnamomea*. In this investigation, the total triterpenes values have great importance, especially those found during solid fermentation in the apple bagasse because this type of secondary metabolite has antibacterial properties against plant pathogens; in addition, it has demonstrated effectiveness against oxidative damage through the elimination of free radicals and the modulation of enzymatic activity [46]. The authors mention solvents with low polarity for the extraction of triterpenes, such as ethyl acetate [47,48]. Nevertheless, aqueous extracts have been used to identify secondary metabolites, like triterpenes, in other macromycete fungi, *Phellinus* and *Ganoderma* [49]. As a consequence, it was decided to use this type of solvent, due to polyphenols, sterols, triterpenes and anthracenes being moderately abundant [50].

The production of secondary metabolites can be considered similar to the tendency of antioxidant activity, due to the fact that since day 21 there was an increase in antioxidant activity in the apple bagasse, which can be attributed to a higher concentration of polyphenols present in acetone and water (80:20 *v*/*v*) and aqueous extracts. As mentioned by Lu and Foo [51], the antioxidant activity is caused by polyphenols in apple residues. Meanwhile, Ajila et al. [38] report IC50 values of 12.24 ± 5.20 to 14.27 ± 2.50 µg in acetone and water (80:20 *v*/*v*) extracts and 40.16 ± 2.40 to 50.18 ± 2.00 µg in aqueous extracts in fermented apple residue.

While there is little information on antioxidant activity in agave mezcalero through solid-state fermentation, some authors have found this propertyin certain agave species. For example, Carmona et al. [52] mention the antioxidant activities of *Agave lechuguilla*, and Araldi et al. [53] mention it regarding *Agave sisalana*. Both species are used as a source of raw material in the production of biofuels from solid fermentation.

Although the values obtained can be considered low, the obtained methanol extract from the solid-state fermentation of the agave mezcalero can be used as an alternative for the production of compounds with antioxidant properties.

Overall, the IC50 values of apple bagasse are lower; they displayed greater inhibitory activity against the DPPH radicals and ABTS radicals in comparison to those of the agave mezcalero bagasse. On the other hand, Ignat et al. [54] express that the different results in the metabolite content may be due to the extraction conditions, type of solvent, and its susceptibility to degradation. This way, it is possible to indicate the variability of secondary metabolites and antioxidant activity in agroindustrial waste.

### **5. Conclusions**

The growth of *Pleurotus ostreatus* was observed in both the apple bagasse and agave mezcalero bagasse residues due to their ability to secrete extracellular, non-specific ligninolytic enzymes during secondary metabolism. Spectrophotometric studies have demonstrated that, because of solid-state fermentation, there was presence of secondary metabolites—phenolic compounds, flavonoids and triterpenes.

The highest concentration of compounds of biological interest occurred in apple bagasse after 21 days, with the aqueous extract presenting the highest extraction of the different phytochemical compounds. This behavior was reflected in the antioxidant activity of the different extracts during the same fermentation time. Minor amounts of phenolic and flavonoid compounds were found in the aqueous extract of the agave bagasse from day 21 of fermentation.

The biosynthesis of secondary metabolites displaying antioxidant activities in the solid-state fermentation of *Pleurotus ostreatus* in the apple residues of the cider industry and in the agave residues of the mezcalera industry can contribute to the use of these wastes, as they can be used in functional food formulation or, due to their medical properties, in the pharmaceutical industry.

**Author Contributions:** Conceptualization, D.I.-C., M.A.M.-C., R.d.C.C.-V. and M.E.R.-C.; methodology, D.I.-C., M.A.M.-C., and M.E.R.-C.; validation, D.I.-C., M.A.M.-C., and M.E.R.-C.; formal analysis, D.I.-C., M.A.M.-C., R.d.C.C.-V. and M.E.R.-C.; investigation, D.I.-C.; resources, M.A.M.-C. and M.E.R.-C.; data curation, D.I.-C., M.A.M.-C. and M.E.R.-C.; writing—original draft preparation, D.I.-C.; writing—review and editing, D.I.-C., M.A.M.-C., R.d.C.C.-V. and M.E.R.-C.; visualization, D.I.-C., M.A.M.-C. and M.E.R.-C.; supervision, M.A.M.-C. and M.E.R.-C.; project administration, M.A.M.-C., R.d.C.C.-V. and M.E.R.-C.; funding acquisition, M.A.M.-C., R.d.C.C.-V. and M.E.R.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Programa de Desarrollo Profesional Docente para el Tipo Superior (PRODEP) Folio PTC-BUAP.PTC-560.

**Acknowledgments:** The authors thank the Posgrado de Ciencias Ambientales—BUAP and CONACyT for the post-doctoral of Diego Ibarra-Cantún.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*


**ANTIPHOSPHOLIPID SYNDROME**

Edited by **Alena Bulikova**

http://dx.doi.org/10.5772/1060 Edited by Alena Bulikova

#### **Contributors**

Silvia Sanchez-Ramon, Áurea García-Segovia, Margarita Rodríguez Mahou, Jakub Swadzba, Jolanta Kolodziejczyk, Perumal Thiagarajan, Hideto Yamada, Kenji Tanimura, Yashuhiko Ebina, Joost Meijers, Cetin Agar, Philip De Groot, Lilia Cedillo, Elizabeth Herrera-Saldivar, Antonio Yáñez, David Bañuelos, Constantino Gil, Rocco Manganelli, Serena Manganelli, Salvatore Iannaccone, Mario Iannaccone, Josep Ordi-Ros, Miquel Vilardell-Tarrés, Jesus Castro-Marrero, Ph.D., Eva Balada, Katarzyna Fischer, Jacek Fliciński, Marek Brzosko, Kjell Haram, Silvia Pierangeli, Maria Corina Serban, Alexandru Caraba, Alena Bulikova

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