**4. Micropropagation of Plants for Production of Large Therapeutic Molecules (LTM) Including Fructooligosaccharides Classified as Prebiotic**

Li et al. [18] has called on LTMs crop plants to be cultivated for production of large molecules such as proteins and polysaccharides and engineered crops (GM) with the ultimate goal of producing drugs or vaccines at low cost. The LTM's crops are sources of

proteolytic enzymes such as papain isolated from *Carica papaya* L., bromelain from fruits and stems of pineapple, and the bioactive momordica anti-HIV from *Momordica charantia* L. We included in the LTM's species those that supply prebiotic dietary fibers that are carbon sources for fermentation pathways in the colon to support digestive health. In this section, we focus on these prebiotic fibers to highlight how micropropagation may be used to create a stable supply of crops that produce LTMs.

Fructooligosaccharides/inulin also known as FOS are universally agreed-upon prebiotics [77], and species that are rich sources of dietary fibers have tremendous effect on gut microbiome. Humans cannot digest FOS. Instead, the gut microbiome ferments these non-digested carbohydrates and produces short chain fatty acids with health benefits such as reducing the risk of cancer and increasing the absorption of both calcium and magnesium. Research on the gut microbiome has increased exponentially, revealing that the intestines greatly affect human health, especially in relation to the immune system and behavior [78,79].

FOS are present in fruits, bulbs, rhizomes, and roots of banana, onion, garlic, and species belonging to the Agavaceae and Asteraceae, which are the richest sources of FOS including chicory (*Chicorium intybus* L.), globe artichoke (*Cynara cardunculus* var. *scolymus* L. Fiori, Jerusalem artichoke (*Helianthus tuberosus* L), elecampane *(Inula helenium* L.), bear's foot (*Smallanthus uvedalia* (L.) Mack. ex Mack and yacon (*Smallanthus sonchifolius* (Poepp.) H. Rob.). According to Roberfroid [78] chicory roots provide the commercial source of FOS for industrial applications, also known as inulin, which are extracted and then processed into short-chain fructans, such as the oligofructose with 2–10 degree of polymerization by partial enzymatic hydrolysis. López and Urías-Silvas [79] reviewed the use of Agave/FOS as prebiotics called agavins whose molecular structure is composed of a complex mixture of fructans. The agavins stimulated the growth of *Bifidobacterium breve* and *Lactobacillus casei* more efficiently than most commercial inulin [80]. Melilli et al. [81] evaluated (*Cyanara cardunculus* var. *scolymus* L.) germplasm for inulin with a high degree of polymerization in the Mediterranean environment, to reduce breeding time and offer growers uniform, healthy globe artichoke plants. Ozsan and Onus [82] compared in vitro micropropagation response of open-pollinated cultivars with F1 hybrids in maturity and height. They concluded that open-pollinated cultivars are cheaper than F1 and could be used for in vitro mass propagation.

The food industry considers FOS/inulin a natural ingredient that improves sensory characteristics such as taste and texture, the stability of foams, emulsions and mouthfeel in a large range of food applications like dairy products and baked goods reducing sugar and fat content while improving health [83]. Padalino et al. [84] added inulin with different degree polymerization with whole meal flour to improve quality of functional wheat spaghetti as example of processed food. The Global Market Insights reported that inulin's (FOS) market size in 2015 was 250 kilo tons and it is expected gains of 8.5% for 2023, likely to be worth more than US\$ 2.5 billion [85]. The consumers of FOS are Europe, China, Japan and North America, with Japan being the world's largest market. The COVID-19 pandemic reinforced the major role of microbiota on the immune response and well-being. We expect that more consumers will pay more attention to prebiotics that modulate the gut microbiome.

Recent studies on the traditional food yacon (*Smallanthus sonchifolius* (Poeppig & Endlicher) H. Robinson), an Andean species, demonstrated that its roots are also a rich source of FOS with a smaller degree of polymerization than chicory. It has great potential as a prebiotic and sugar substitute due to its sweet taste that is related to degree of polymerization [86,87]. The role of yacon as FOS supplementation favors a healthy microbiota while reducing pathogenic population in the gut. Furthermore, short chain fatty acids produced by the beneficial bacteria improve glucose homeostasis and lipid metabolism. Clinical studies confirm that consumption of yacon as flour or syrup prevented and treated chronic diseases [88,89]. The beneficial compounds present in storage roots of yacon classify the spices as functional food (Figure 2).

**Figure 2.** Yacon (*Smallanthus sonchifolius*) functional constituents.

Brazil is one of the largest agricultural producers in the world, but does not produce inulin/FOS from either source (chicory or artichoke) for applications in the food industry. However, in the vicinity of Sao Paulo City, yacon is produced for fresh consumption for its health benefits. Thus, to initiate any production system for supplying FOS as an ingredient with applications in the food and pharmaceutical industries, rhizospheres [90], along with storage roots may be better utilized to extract FOS. Micropropagation of yacon can still be done using axillary buds as explants of healthy plantlets for cultivation. Table 3 shows the published in vitro protocols of FOS producing plants for development of the business models of fructans as prebiotic.

Given the predicted increase in FOS/inulin consumption, supply of these LTM's crop plants will be necessary in a way such as the one suggested by McChesney et al. [20] a sustainable system to meet the demand. Yacon micropropagation is an example to stabilize the supply of crop plants as source of LTMs, thus ensuring that stability of production.


**Table 3.** Micropropagation protocols of FOS producing species.

#### **5. Functional Food Crops**

Metabolic syndrome is a global economic and social burden, understanding the origins, relevant factors contributing to high rates of obesity and its physiological impacts may reveal potential therapeutic targets.

Maintaining a healthy gut microbiome is one of the therapeutic goals that improve human health [83]. Dietary fibers promote wellbeing, and thus they are classified as functional food. Wildman [96] refers as functional food, the food, either natural or formulated, which will enhance physiological performance or prevent or treat disease and disorders.

Royston and Tolesfbol [97] refer to term epigenetic diet class of bioactive dietary compounds such as resveratrol in grapes, genistein in soybean, apigenin in celery, allicin in garlic, phenolic compounds in berries and omega 3 in Portulaca oleracea L. also known as purslanen [98] and other consumed foods, which have been shown to defend against the development of many different types of tumors. Compounds that act as epigenetic modulators prevent initiation and the progression of oncogenesis [97]. Micropropagation is an important tool for the propagation of selected lines in various breeding programs, as well as the recovery of pathogen-free material, or even for slow growth storage and the cryopreservation of valuable germplasm of fruit and vegetable crops.

#### **6. Conclusions**

Humans have long used plants to address various problems, the solutions to which often brought unintended consequences, such as overharvesting and environmental degradation. These negative consequences teach us the solution to our problems ought to be sustainable. Through a literature review, this paper argues that micropropagation can be a part of a strategy to reinforce the supply and quality of crops used for medicinal purposes: (1) small therapeutic molecules, (2) standard therapeutic extracts, (3) large therapeutic molecules, and (4) functional foods.

**Author Contributions:** Conceptualization, R.M.M. writing original draft R.M.M., A.L.C., and M.V.L. All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** The authors thank Marcos Cerdeira for helping with editing.

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