Ten years of research on bioactive peptides in Brazil: a scientometric analysis

Peptides are biomolecules composed of amino acids linked through peptide bonds present in proteins. These peptides may have biological activities associated with their secondary structure, especially when released from the original protein sequence. The prospect of these biomolecules is the subject of study and research in various sectors, including the food and pharmaceutical industries. This is due to the possibility of using them as substitutes for traditionally marketed drugs, especially those with restrictive legislation on their use, or losing their effectiveness. A scientometric analysis of studies published from 2011 to 2021 by Brazilian researchers demonstrated the growing interest in basic research involving bioactive peptides from numerous protein sources, including food-based and agro-industrial residues. The antioxidant activity, followed by the antimicrobial potential, has been the most studied property of these biomolecules. Several other activities such as anxiolytic, anti-adipogenic, anticoagulant, anticonvulsant, antisclerotic, and cytoregulatory actions have been reported in a limited number of studies, highlighting the potential application of these biomolecules in the development of new products.


Introduction
Bioactive peptides are a group of molecules derived from proteins with 2-50 amino acid residues connected by peptide bonds (Bhandari et al., 2019). These molecules have diverse compositions and arrangements of amino acids along the peptide chain, which define their unimodal or multimodal regulatory actions and lead to health benefits in the organisms consuming them (Akbarian et al., 2022;Liao et al., 2018).
The first study related to the bioactive properties of a peptide was published in 1950 when the receptor-activation ability for umami taste was reported (Chakrabarti et al., 2014). Numerous studies have since been conducted to explore new activities in various bioactive peptides, such as antioxidant, antimicrobial, immunomodulatory, antihypertensive, anticancer, cholesterolemic, and antidiabetic activities (Alberto- Silva et al., 2021;Nascimento et al., 2021b;Santos et al., 2021;Silva et al., 2021).
From a therapeutic point of view, bioactive peptides have shown more benefits than traditionally marketed drugs as they are derived from natural organic macromolecules and tend to have target-tissue-directed activity with little or no toxicity. Moreover, they can exert their biological activity at low concentrations and do not have a cumulative effect on the organisms (Akbarian et al., 2022).
These new revelations and the constant search of consumers for products of natural origin have raised the interest of researchers and industrial sectors to search for bioactive peptides in food sources and agro-industrial residues with high protein content (Acquah et al., 2019;Toldrá & Mora, 2021;Tu et al., 2018a). Therefore, several peptide prospection studies have focused on low-cost proteins, such as milk (Arruda et al., 2012;Silva et al., 2019b), collagen (Hexsel et al., 2017), animal viscera (Aguilar et al., 2020), legumes (Marques et al., 2015), algae , and yams (Nascimento et al., 2021a).
Peptides are inactive when encrypted in the protein chain; however, they are activated after the degradation of the protein chain (Toldrá & Mora, 2021). The cleavage process occurs in vivo through chemical-enzymatic digestion within the gastrointestinal tract, in vitro through food maturation, direct action of enzymes, microbial metabolism, or controlled chemical hydrolysis (Akbarian et al., 2022).
Controlled enzymatic hydrolysis is the most commonly used method in bioprocesses because of the possibility of controlling the physicochemical parameters (Liu et al., 2020). This method allows the optimization of the protein cleavage process and the consequent increase in the final yield of the hydrolyzed material. Moreover, standardization of the steps involving protein cleavage facilitates the reproducibility of the process (Cruz-Casas et al., 2021;Mohanty et al., 2016;Nascimento et al., 2021b).
The scientific literature reports the great possibility of methods and techniques for prospection, purification, recovery, and discovery of numerous activities associated with bioactive peptides (Campos et al., 2022). Therefore, evaluating the quantitative aspects of scientific production and the current state of study trends in the prospection of these substances is crucial, as it will facilitate the identification of relevant fields that require further scientific exploration (Parra et al., 2019). Therefore, we conducted a scientometric analysis to identify trends in the scientific literature on the source and therapeutic purpose of bioactive peptides studied by Brazilian researchers during 2011-2021.

Material and methods
The data were obtained through a bibliographic survey involving research and analysis of the documents from September to December 2021. Two multidisciplinary scientific databases, Web of Science (www.webofknowledge.com) and Scopus (www. scopus.com), were used for this purpose. The keywords used were "Bioactive" and "Peptide".
In this search, documents published from 2011 to 2021 with Brazilian researchers as authors were considered.
Bibliographic review articles were excluded from the search criteria, and only articles of an experimental nature were considered, including full articles, brief communications, and scientific notes. Only the first 200 results found for each argument studied ("bioactive" and "peptide") in each multidisciplinary scientific basis ("Web of Science" and "Scopus") were considered, totaling 800 articles. Duplicate articles or articles that did not match the analysis criteria were discarded as they were characterized as search tools and not evaluation criteria.
The documents suitable for the purpose of this scientometric article were analyzed individually, and the data were systematized through Bardin's content analysis (Bardin, 2006), where data are grouped by the frequency distribution method. In view of this, the following criteria were developed for data systematization: total number and publications related to the search criteria; the number of scientific publications distributed in the evaluated years; Brazilian regions and states; the number of publications in journals according to the factor range of impact, i.e., Journal Citation Reports (JCR); prospect source used; and biological activities tested. The results were exported to an Excel spreadsheet.
Among the evaluated articles, 31 therapeutic purposes were reported; thereafter, the data were grouped into 31 categories according to their functional similarity to facilitate the interpretation of the results (Table 1).

Results and discussion
According to the search criteria used, only 174 out of 800 publications were analyzed. A total of 594 studies were repeated, and 32 did not fit the research objective; therefore, they were disregarded from the analysis. Figure 1 shows the distribution of scientific publications on bioactive peptides studied by researchers from the Brazilian States during 2011-2021. The general trend of promoting research on the prospection of bioactive peptides can be justified by a change in the lifestyle of consumers, who prefer to include new natural-origin products in their diet (Toldrá et al., 2018). Therefore, research institutions and industrial sectors have intensified their studies to obtain novel potential bioactive compounds with beneficial biological activities in vivo and in vitro (Khedri et al., 2021;Mohanty et al., 2016;Toldrá & Mora, 2021).
However, the results indicated a decline in the total number of publications related to the topic in 2021, with only 22 articles (a reduction of 40.55%) compared to the year 2020 with 37 articles; this was in stark contrast to the upward trend observed during previous years. In 2020, the SARS-CoV-2 pandemic began, which affected the world population in terms of public health and negatively impacted the industrial, scientific, and technology-based sectors, especially in developing countries such as Brazil and in underdeveloped countries (Candido et al., 2021).
While analyzing the data from each suitable publication, Brazilian researchers from five regions were verified to have contributed to the research related to the prospection and evaluation of the biological activities of peptides during 2011-2021.
The data demonstrate that the Southeast region led the publication ranking, followed by the South, Northeast, and Midwest regions, with 98, 35, 21, 19, and 1 scientific publications about bioactive peptides, respectively. In fact, studies about this theme have gained significant prominence in the number of publications over time. This is primarily due to increased investments in basic research and partnerships and collaborations with good scientific institutions, such as those located in the Southeast region (Sidone et al., 2016).
The southeast region of Brazil accounted for approximately 54% of the total bioactive peptide studies published by Brazilian researchers. Additionally, this region also ranks the highest for the most diverse scientific and technological topics owing to the high level of financial investment (Oliveira, 2016). The interaction between universities, research institutes, and the private sector occurs in a punctual and concentrated manner in some regions of the country. This helps meet the demand for interests aligned with specific regional economic activity segments (Camargo et al., 2021).
Although the distribution map elucidated increased research on bioactive peptides in Brazil, the focus on specific regions shows a significant gap in the investments and interests of Brazilian researchers in terms of scientific studies aimed at prospecting peptides across different states and countries. Notably, only one work was reported from the North region, corresponding to 0.57% of the total scientific publications. The primary factor contributing to the slow evolution of scientific and technological development in this region is the delayed implementation of the industrialization process (Spíndola et al., 2015).
While analyzing the data from each suitable publication, Brazilian researchers from 17 states and the Federal District were verified to have contributed to the research related to the prospection and evaluation of the biological activities of peptides during 2011-2021, according to the data shown in Figure 2.

Researchers affiliated with the institutions in São Paulo
State had the most publications, with 72 publications during the study period. In general, the growth trend of Brazilian scientific outcomes is closely correlated with public sector investments, which in turn are directed toward the education of human capital and the improvement of the infrastructure of universities and research institutes (Helene & Ribeiro, 2011;Souza et al., 2020).
The Universidade Estadual de Campinas and the Universidade de São Paulo, located in the State of São Paulo, had the highest number of publications nationwide, with 23 (13.21%) and 19 (10.91%), respectively. The leading institutions are located in the Southeast and South regions, followed by the Northeast region (Universidade Federal de Pernambuco) and the Center-West region (Universidade de Goiás) of Brazil.
Regarding the periodicals used for publications, a total of 95 journals were used for collecting data on bioactive peptides studied by Brazilian researchers during 2011-2021. The lowest value of the JCR impact factor was 0.579 for the "Brazilian Archives of Biology and Technology". The highest was 9.642 for "Bioresource Technology", each journal having only one published article. Figure 3 shows an overview of the 174 scientific publications on bioactive peptides published by Brazilian researchers according to the JCR impact factor intervals for the journals. A total of 52 and 46 articles on the topic studied were published in journals with a JCR value between 3.001-4.000 and 4.001-5.000, respectively. These results suggest that the studies conducted by Brazilian researchers on bioactive peptides over the last 10 years have been gaining strength and recognition in the scientific environment. Among the 95 journals included in the present study, seven with the highest number of publications were selected (Figure 4), along with their respective JCR scientific impact factors.
Our results demonstrated that the "Food Research International" journal published 14 articles with the highest publication rate and currently has an impact factor of 4.972. The impact factor is a bibliometric indicator commonly used to assess the relative quality of a journal within its field of study and measure the frequency with which a journal article was cited in each period (Sharma et al., 2014).
The prospecting sources studied in the 174 scientific articles evaluated in the present study are shown in Figure 5.
Our observations based on 108 (62.06%) publications suggested that Brazilian researchers used animal products as the primary source of bioactive peptides. In contrast, protein matrices of plant, microbial, and synthetic origin were discussed in 35 (20.11%), 18 (10.35%), and 13 (7.48%) publications, respectively.
Bioactive peptides exhibit bioactivity (unimodal or multimodal) based on the length and arrangement of amino acids in their chain (Bezerra et al., 2020;Cruz et al., 2020;Silva et al., 2021). Figure 6 shows the number of published studies according to the underlying therapeutic purpose.
The data analysis revealed that some scientific articles reported more than one activity for the prospected peptide, thereby characterizing the multimodal activity of these peptides. The analysis of specific activities revealed that antimicrobial and antioxidant activities were most frequently reported, with 55 (35.05%) publications reporting them.
Several studies related to testing the antioxidant properties of peptides could be justified by the existence of specific legislation that limits the use of synthetic substances, particularly in the preservation of foods and medicines. Therefore, the development of potentially safe natural-origin products, such as bioactive peptides with antioxidant activity, is always desirable (Marques et al., 2015).
Since conventional antimicrobial drugs are losing their effectiveness owing to indiscriminate usage and increasing multidrug resistance in the microbial population, interest in exploring peptides with antimicrobial activity has grown over the years (Jia et al., 2019). Peptides with antimicrobial action effectively control pathogens owing to their valuable characteristics. These substances have broad-spectrum microbicidal activity associated with high specificity for microbial cells. Moreover, the rare induction of microbial resistance and the ability to act synergistically with commonly marketed antibiotics make them a promising choice for antimicrobials (Kobbi et al., 2018;Osman et al., 2021).
Studies related to other observed biological activities are expanding, demonstrating a high potential for their application in future therapeutic studies. In view of the data analysis, only five of the 174 articles included cytotoxicity studies. Despite a lower rate of peptide-associated toxicity, biomolecules can possibly generate toxic substances, such as lysinoalanine, D-amino acids, and biogenic amines, capable of causing deleterious effects in organisms. Therefore, extensive toxicity studies must be performed (Liu et al., 2020).
The in vitro cytotoxicity, hemolytic activity, and genotoxicity tests of bioactive peptides must be assessed before their development for human consumption. Additional in vivo, animal, acute oral toxicity, chronic, and repeated-dose toxicity assessments must be performed (Shivanna & Nataraj, 2020). Major limitations include the complexity and cost of assays to determine toxicity and/or allergenicity in vitro and in vivo, particularly with several peptides under evaluation (Lin et al., 2018).
However, in silico methods are considered a practical alternative to predict toxicity, allergenicity, and peptide bioactivity (Lin et al., 2018;Tu et al., 2018b). Although such analysis is unsuitable for reliable comparison between in vitro and preclinical toxicity testing, bioinformatics tools can help researchers screen and select peptides with potential pharmacological applications (Tu et al., 2018a).
Given these data, the increasing development of bioactive substances, especially bioactive peptides, is evident. The prospection of novel natural-origin products can generate health benefits; therefore, they can serve as alternatives to increase the nutritional scope since these substances are primarily dietary constituents (Romani et al., 2020).

Conclusion
The results of the analyzed publications based on the prospection of bioactive peptides and evaluation of their biological activities over the last 10 years (2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)(2020)(2021) in Brazil demonstrate the increasing number of publications and their respective impact on contributing to the scientific and technological development of the country. The Brazilian region that has the largest number of researchers who carry out studies related to the prospection of bioactive peptides is the Southeast region; however, it has been possible to observe the rise of scientific development over the last few years in the South, Northeast, and Midwest regions. Among the numerous sources of prospecting for bioactive peptides, food proteins of animal origin are the most used, and studies published by Brazilian researchers demonstrate the potential for multimodal application of bioactive peptides, as several biological activities are reported. Antioxidant, broad-spectrum antimicrobial, and antihypertensive activities were the most researched in the evaluated period; however, it was possible to identify other activities that demonstrate the potential for the development of new bioactive formulations based on peptides. Câmara, G. A., Nishiyama, M. Y., Kitano, E. S., Oliveira, U. C., Silva Jr., P. I., Junqueira-de-Azevedo, I. L., & Tashima, A. K. (2020). A Multiomics approach unravels new toxins with possible in silico antimicrobial, antiviral, and antitumoral activities in the venom of Acanthoscurria rondoniae. Frontiers in Pharmacology, 11, e1075. https://doi.org/10.3389/fphar.2020.01075 Camargo, R. B., Batisti, D. L. S., Souza, V. K. G., Neumann, M., & Moritz, C. M. F. (2021. Reflexões sobre a importância do aporte financeiro para a pesquisa científica brasileira com produtos naturais. Research, Society and Development, 10 (5)