“Masato De Yuca” and “Chicha De Siete Semillas” Two Traditional Vegetable Fermented Beverages from Peru As Source for the Isolation of Potential Probiotic Bacteria
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Probiotics and Antimicrobial Proteins https://doi.org/10.1007/s12602-021-09836-x “Masato de Yuca” and “Chicha de Siete Semillas” Two Traditional Vegetable Fermented Beverages from Peru as Source for the Isolation of Potential Probiotic Bacteria Teresa D. Rebaza‑Cardenas1,2 · Kenneth Silva‑Cajaleón2 · Carlos Sabater1,3 · Susana Delgado1,3 · Nilda D. Montes‑Villanueva2 · Patricia Ruas‑Madiedo1,3 Accepted: 11 August 2021 © The Author(s) 2021 Abstract In this work, two Peruvian beverages “Masato de Yuca,” typical of the Amazonian communities made from cassava (Manihot esculenta), and “Chicha de Siete Semillas,” made from diferent cereal, pseudo-cereal, and legume fours, were explored for the isolation of lactic acid bacteria after obtaining the permission of local authorities following Nagoya protocol. From an initial number of 33 isolates, 16 strains with diferent RAPD- and REP-PCR genetic profles were obtained. In Chicha, all strains were Lactiplantibacillus plantarum (formerly Lactobacillus plantarum), whereas in Masato, in addition to this spe- cies, Limosilactobacillus fermentum (formerly Lactobacillus fermentum), Pediococcus acidilactici, and Weissella confusa were also identifed. Correlation analysis carried out with their carbohydrate fermentation patterns and enzymatic profles allowed a clustering of the lactobacilli separated from the other genera. Finally, the 16 strains were submitted to a static in vitro digestion (INFOGEST model) that simulated the gastrointestinal transit. Besides, their ability to adhere to the human epithelial intestinal cell line HT29 was also determined. Following both procedures, the best probiotic candidate was Lac. plantarum Ch13, a robust strain able to better face the challenging conditions of the gastrointestinal tract and showing higher adhesion ability to the intestinal epithelium in comparison with the commercial probiotic strain 299v. In order to character- ize its benefit for human health, this Ch13 strain will be deeply studied in further works. Keywords Vegetable · Fermented beverage · Food · Probiotic · Lactic acid bacteria · Typing · HT29 Introduction regions [1, 2]. These fermented foods and beverages have an undoubted high nutritional value; also, the microbial Beverages obtained from fermented vegetables have a long transformation of the raw material favors the occurrence of tradition of consumption in many ancestral cultures, such health-promoting components and metabolites [3]. In fact, as those of Latin America, where nowadays some of them it has been proposed that the controlled fermentations of are still widely handcraft-manufactured being part of the ancient protein-rich plants could be a tool to search for alter- sustainable economy of most families living in specifc natives of protein sources [4]. However, we should keep in mind that these approaches must be performed in a sustaina- ble way, ensuring the access of these traditional food sources * Patricia Ruas-Madiedo [email protected] to the local population who have kept the ancestral seeds and their uses. This has been enclosed within the objectives of 1 Department of Microbiology and Biochemistry of Dairy the “Convention on Biological Diversity” dealing to con- Products, Instituto de Productos Lácteos de Asturias – serve the biological diversity and its sustainable use, as well Consejo Superior de Investigaciones Científcas (IPLA- CSIC), Villaviciosa, Asturias, Spain as to ensure the equitable sharing of the benefts that can be obtained for the use of the genetic resources; the last objec- 2 Facultad de Ingeniería Agraria, Universidad Católica Sedes Sapientiae (UCSS), Lima, Peru tive has been implemented through the Nagoya Protocol [5]. These artisanal foods are a natural source for the isolation 3 Group Functionality and Ecology of Benefcial Microbes, Instituto de Investigación Sanitaria del Principado de of novel microorganisms, since they are obtained through Asturias (ISPA), Asturias, Oviedo, Spain spontaneous, non-controlled fermentations, thus keeping Vol.:(0123456789)1 3 Probiotics and Antimicrobial Proteins the biodiversity of the natural microbiota; then, they con- the fermentation can be conducted by the probiotics, or the stitute a valuable source of microbial diversity, ready to be fermented food could contain probiotics, but is always a food explored [6, 7]. However, in spite of the high potential of format [20]. these heritage-fermented foods, studies about their micro- The objective of the present study was to isolate new biota and their genetic diversity are scarce or non-existent LAB from two typical fermented beverages of Peru, “Masato in some cases. One of the microbial groups that are mostly de Yuca” (from Manihot esculenta) and “Chicha de Siete always present in fermented products are the lactic acid Semillas,” a poli-cereal mixture combined with legumes bacteria (LAB), which are the main drivers of lactic fer- and aromatic plants. Furthermore, the selection of the most mentation. In recent years, special attention has been payed robust strains, able to survive to the gastrointestinal transit to the isolation and characterization of LAB from diferent and to transitionally adhere to the colon, will be achieved. plant-based foods in the Latin American geography, such Following this “roadmap,” the best candidates will be as “Tocosh” and “Tunta” from potato in Peru [8, 9], Chi- selected to undertake future studies about their potential cha from maize in Argentina [10], or fermented “Chia” also probiotic capabilities. This work was conceived in the frame- in Argentina [11], to cite some of them. A wide variety of work of the ProInfant-CYTED project, a multidisciplinary LAB genera have been described, although members Lac- and collaborative work of several Latin American, Spanish, tobacillus spp. are often found; this genus has been recently and Italian research groups, in which the fnal long-term reclassifed and some species renamed [12]. The reason aim is to develop high nutritional value, vegetable-based behind the interest in the LAB is the multiple applications fermented foods, containing well-adapted probiotic strains of this bacterial group, not only as a way to standardize able to reduce malnutrition-related diseases, such as diarrhea and improve the hygienic-sanitary quality of the fermented and respiratory infections. products, but also as drivers of the “identity signature” of foods with geographical indications (GIs). In this regard, it has been proposed that the microbial resource informa- tion could be integrated into the context of the fermented Material and Methods food GIs [13]. LAB are able to modify components of the food matrix, which might improve their bioaccessibility and Food Sample Collection digestibility, thus increasing the nutritional values [14]; but, also the metabolites resulting from the bacterial activity are Two types of traditional Peruvian fermented foods were used responsible for the aroma, favor, viscosity, and texture of to search for LAB (Table 1). The “Chicha de Siete Semil- the fermented product [15]. Besides, some specifc strains las” (hereafter referred to as Chicha) was collected from are able to produce other molecules along the fermentations, 5 producers located in the province of Huanta (Ayacucho such as vitamins, neuroactives, bioactive peptides, bacteri- Department) which sell their products on specialized cel- ocins, among others, that might have an infuence on our lars (“bodegas”), whereas the “Masato de Yuca” (hereafter health [16]. In this regard, some of the LAB having positive referred to as Masato) was obtained from 6 producers that efects on our well-being are known as probiotics, which ofer their products in local markets or street selling in the have been defned by FAO-WHO in 2001 as “live micro- provinces of Chanchamayo and Satipo, both belonging to organisms that, when administered in adequate amounts, the Peruvian Amazonia. The collection of the food sam- confer a health beneft on the host” [17]. The probiotics ples was performed following the Nagoya protocol, with the intended for food (and feed) applications must be safe for corresponding permission of the local Peruvian authorities human (or animal) consumption; therefore, only certain spe- (Contract No. 001 2018-MINAGRI-SERFOR/DGGSPFFS- cies are Generally Recognized as Safe (GRAS, according to DGSPF). For LAB isolation, serial dilutions were made FDA administration), or are included in the Qualifed Pre- in 0.1% (w/v) of peptone water (HiMedia, India) which sumption of Safety (QPS) list [18]. Among them, the most were spread in the surface of agar-MRS (pH 6.4, Conda, common belong to genera Bifdobacterium and Lactobacil- Spain) supplemented with 50 μg/mL cycloheximide to pre- lus and there are multiple mechanisms by which probiotic vent fungi overgrowth. Plates were incubated at 32 °C for strains might exert the benefcial action. The positive efect 48–72 h. Afterwards, colonies of diferent morphology were on health must have strong scientifc evidence behind, before picked up to perform a Gram staining; those Gram-positives being used for clinical applications [19] or being included were screened for the absence of catalase activity and spore in the formulation of foods with health claims. In relation to formation, using standard procedures. The fnal isolates were this, it has been recently reported the diferences among the stored at − 20 °C in MRS broth containing 30% glycerol. concept probiotic, fermented food, and probiotic fermented Samples were sent to IPLA-CSIC directly