Microbial keratinase and the bio-economy: a three-decade meta-analysis of research exploit

Microbial keratinase research has been on an upward trajectory due to the robustness and efficiency of the enzyme toward various green technological processes that promote economic development and environmental sustainability. A compendium of research progression and advancement within the domain was achieved through a bibliometric study to understand the trend of research productivity, scientific impacts, authors' involvement, collaboration networks, and the advancement of knowledge gaps for future research endeavours. A three-decade (1990 to 2019) scholarly published articles were retrieved from the web of science database using a combination of terms "keratinas* or keratinolytic proteas* or keratinolytic enzym*", and subsequently analyzed for bibliometric indicators. A collection of 330 peer-reviewed, research, articles were retrieved for the survey period and authored by 1063 researchers with collaboration index of 3.27. Research productivity was most in 2013 with total research output of 28 articles. The top three authors' keywords were keratinase, keratin and protease with a respective frequency of 188, 26 and 22. India, China and Brazil ranked top in terms of keratinase research outputs and total citation with respective article productivity (total citations) of 85 (1533), 57 (826), and 36 (764). This study evaluated the trend of keratinase research outputs, scientific impact, collaboration networks and biotechnology innovations. It has the potentials to influence positively decision making on future research direction, collaborations and development of products for the bio-economy.


Introduction
The global reliance of fossil-based resources lingers on, and the long term sustainability of the non-renewable resources remains a mirage (Abejón 2018). The environmental impacts of the products from fossil-based resource have negatively affected the planet earth. Thus, the imperative for a reliable alternative motivates for the exploration of highly abundant renewable resources for sustainable products. The potentials locked in carbonoclastic materials account for the desire to valorize waste biomass into high-value products, and that ensures sustainability in economic development (Akinsemolu 2018;Nnolim et al. 2020a). The agro and food industry generates lots of solid wastes which may suitably feed into the economy with novel valorization approach. Chitinous, lignocellulosic and keratinous biomass are most Open Access *Correspondence: UNwodo@ufh.ac.za 1 SAMRC Microbial Water Quality Monitoring Centre, University of Fort Hare, Alice 5700, South Africa Full list of author information is available at the end of the article abundant of the wastes in our environment (Hossain et al. 2007).
Keratinous biomass is typically sturdy due to the high cysteine-mediated disulfide cross-linkages that potentiate structural stability of the polymers, and resilience to attack by biotic and abiotic factors (Schweizer et al. 2006;Wang et al. 2016). The slaughterhouses, leather industries and poultry processing farms are some of the sectors that generate keratinous biomass in large quantities. The need to feed the burgeoning global population has led to increased agroindustrial processes with an inevitable generation of high amounts of wastes. The accumulation of the agro-industrial wastes results in varying degree of pollution in the environs (Kalaikumari et al. 2019).
Environmental protection laws significantly promote efficient management of recalcitrant wastes (Akinsemolu 2018), via recycling to essential products (Stiborova et al. 2016). The microbial valorization of keratinous waste biomass may yield high-quality protein hydrolysates (Mazotto et al. 2017), organic fertilizer (Tamreihao et al. 2017), peptone substituted microbial growth media (Taskin and Kurbanoglu 2011), plant growth hormones (Verma et al. 2016), bioenergy feedstocks (Xia et al. 2012), and industrially important enzymes (Cavello et al. 2018). The physicochemical valorization techniques lack these merits. Consequently, the exploration and exploitation of microbial diversity for novel keratinolytic potentials remains topical (Nnolim et al. 2020b). All the identified microbes associated with keratinolysis are, either bacteria or fungi (Brandelli et al. 2010), and the dermatophytes account for most of the fungal species (Brouta et al. 2001). However, associated virulence traits limit the commercial prospects of the dermatophytes. Therefore, bacteria take precedence as the most viable microbe for keratinous waste bioconversion. Autochthonous and/or allochthonous bacterial strains from different ecological niche have been shown to possess potentials for the bioconversion of keratinous biomass into value-added products; some of the earliest studies started with feather degrading Bacillus licheniformis PWD-1 (Williams et al. 1990).
The prospects of keratinases as suitable enzymes for industrial and biotechnological processes include their robustness in withstanding extreme conditions on like the classical proteases (Verma et al. 2017). Microbial keratinases possess unique properties that bestows a potential suitable for use in a green economy including applications in the formulation of functional feed, detergent formulation, bio-decontamination, bating and tanning processes, personal care product formulation, and nanotechnology (Mitsuiki et al. 2006;Paul et al. 2014b;Gupta et al. 2015;Sanghvi et al. 2016;Kalaikumari et al. 2019;Peng et al. 2020). A continuum in the discovery of keratinases with novel properties and function would, significantly, revolutionize the bio-economy landscape, and the prospects presented by proteolytic enzymes. Hitherto, the narrative on microbial keratinases research highlighting milestones such as isolation of producer microbes, optimization of keratinase production, keratin degradation, enzyme purification, biochemical characterization, and the various keratinases application potentials. Regardless of the frequency for which scientific findings are made, and data accumulated on the valorization of keratinous biomass via microbial enzymatic means, there are no explicit documentations delineating research hotspots, lead authors, relevant sources, regional involvement and collaboration networks on the valorization of keratinous biomass via microbial enzymatic means. To develop a comprehensive report from current bibliographic information in the subject area, meta-analytical approach was adopted for the evaluation of the scientific outputs and bio-innovative developments.
Meta-analysis represents an essential method for handling numerous pieces of literature within a field of research (Belter 2015). It a statistical technique aimed at evaluating the significance and trend of research outputs in a localized research field (Cañas-Guerrero et al. 2013). It can be applied systematically to track the progress in a particular research domain. The bibliometric tools prompt the ease of studying subject evolution, comparing regional performance and scientific collaboration within a research niche through bibliometric indicators (Durieux and Gevenois 2010). Bibliometrics has been applied in assessing the research evolution on the impact of lignin valorization from 2000 to 2016 (Abejón et al. 2018), hemicellulose valorization from 2000 to 2016 (Abejón 2018), enzyme immobilization from 1991 to 2017 (Gonçalves et al. 2019). The gap identified in the absence of meta-analysis on microbial keratinase research evolution motivates for this study. Therefore, the progression of research on microbial keratinase between 1990 and 2019 was systematically articulated. The metaanalysis was, explicitly, implemented through qualitative and quantitative characterization of the bibliographic information available on the subject matter in the web of science (WoS) core collection database.

Data retrieval
The metadata utilized for the meta-analysis were retrieved from the Clarivate Analytics Web of Science core collection (http://webof knowl edge.com/). Web of Science (WoS) is widely used for academic and bibliometric studies as it gives consistent journal coverage of scholarly published articles, high resolution of related records, enhanced metadata for variable information purposes and also, presents more refine options (Li et al. 2018;Birkle et al. 2020). Scientific publications on keratinase studies from 1 January 1990 to 31 December 2019 were retrieved from the WoS on the 10 January 2020 using the key terms "Keratinas* or Keratinolytic proteas* or Keratinolytic enzym*" with title search option. These keywords have been frequently and interchangeably used by authors in the study of microbial keratinases. The incorporation of wildcard (*) and logical operator (or) promoted recovery of relevant published articles with keywords both in singular and plural forms (Capobianco-Uriarte et al. 2019). In addition, title search has been considered most efficient in bibliographic data retrieval against topic search, as it yields more specific results with an infinitesimal loss of sensitivity (Sharma et al. 2018;Olisah et al. 2019). Search results showed a total of 371 publications of different document types. The document types were further refined to exclude review, proceedings, meeting abstract, book chapter, correction, early access and editorial material. After refinement, a collection of 330 articles was retrieved. This document type was chosen as it is generally considered as an original contribution to knowledge (Patience et al. 2017). The documents were further scrutinized for compliance and then, downloaded in the BibTex file format having checked the important fields such as author(s), abstract, addresses, funding information, title, cited references, cited times, language, source, keywords, author identifiers etc. during file exportation. The patented keratinase research discoveries within the three decades of study were retrieved from Google Patents (https ://paten ts.googl e.com/), by using "Keratinase" as a key term.

Data analysis
The retrieved data were analyzed for bibliometric indicators using Rstudio software (Rstudio Inc, Boston, USA) with bibliometrix R-package v.3.6.0 (Aria and Cuccurullo 2017). Firstly, the bibliometric analysis was activated in the R environment using the R language "biblioshiny()". The command code opened a biblioshiny web-interface on Google chrome browser which works in synchrony with the R environment. Extracted raw data were imported into the biblioshiny and subsequently converted to a bibliographic data frame. Standard Clarivate Analytics WoS Field Tag codify was used to designate the data frame columns. Descriptive statistics of the dataset implemented include; Author's keyword, keyword plus, annual scientific production, total citation, h_index, most relevant sources, most relevant authors, corresponding author's country, most global cited articles, among others. Bibliographic network matrices were also generated using different bibliographic attributes. Co-occurrence, co-citation, collaboration, among other networks, were automatically computed using bipartite network and visualized by adjusting the network parameters (field, network layout, normalization, node color, clustering algorithm, etc.) and graphical parameters (opacity, number of labels, label size, node shape, edge size etc.) on the biblioshiny web-interface. The percentage frequencies were calculated in Microsoft Excel 2010.

The dynamics of article publication on keratinase research
A total of 330 journal articles on keratinase research were retrieved from the WoS during the defined period of survey (1990-2019), which were published by 142 sources. The metadata contained 1063 authors, from which 1058 authors were in multiple authorships with collaboration index of 3.27 (Table 1). Solo authorship was recorded for 6 articles accounting for about 1.8% of the total documents within the study period. The average citation per article on keratinase research during the three decades of survey was 19.74. The articles' publications were distributed among three languages including English (328 articles; 99.4%), Spanish (1 article; 0.6%) and Portuguese (1 article; 0.6%).
The distribution of articles published on keratinase research between 1990 and 2019 generally showed evolutionary patterns as shown in Fig. 1. Article productivity was relatively low from 1990 to 2000; however, relatively significant number of publications was recorded in 1992

Keywords associated with keratinase study: indicators of research hotspots
A collection of 727 author's keywords (DE) and 635 keywords-plus (ID) were associated with the 330 documents retrieved from the WoS (Table 1). Among the twenty most relevant author's keywords, keratinase which is the core focus of the study showed the highest frequency (n = 188), which indicated that about 56.97% of the 330 retrieved articles included keratinase in the itemized author's keywords (Table 2). Other top five ranked author's keywords included; keratin (n = 26), protease (n = 22), purification (n = 21), and feather degradation (n = 20). The twenty topmost author's keywords could be subdivided into various conceptualizations of which the enzyme's name keratinase, protease, keratinases, and keratinolytic protease form the first clade. The second cluster involves the keratinase inducers (keratin and feather). A common node shoulders the microbial producers (Bacillus, Bacillus licheniformis, Pseudomonas aeruginosa, Bacillus pumilus, and Bacillus cereus). Following similar concept, purification, response surface methodology, characterization, optimization, and solid state fermentation are generally anchored on methodology. Furthermore, feather degradation, dehairing and biodegradation represented the potential application of microbial keratinase. Furthermore, 20 most relevant keywords-plus were also presented in Table 2. The topmost five keywords-plus and their frequencies include purification (n = 147), degradation (n = 87), enzymes (n = 67), strain (n = 64) and protease (n = 53). Author's keywords and keywords-plus presented a couple of similar terms, and they are purification, protease, optimization, feather, keratinase, and B. licheniformis.

The most impactful keratinase research products: relevance and prospects
The contribution of some articles to the development of keratinase research was assessed by considering the topmost impactful twenty research products in a perking order based on the number of citations they have received during the study period (Table 3). The thrust of the studies may be summarized under the following schemes including keratinse production enhancement, keratinase characterization, keratinous-mediated  (Table 3).

Keratinase-based products: from laboratory to the market
A few keratinase-based formulations have been made available for commercial uses as presented in Table 4. These products were formulated with keratinases predominantly from B. licheniformis strains. PROTEOS Biotech has four branded formulations in the market which include Keratoclean ® sensitive PB, Keratoclean ® HYDRA PB, Keratoclean ® BP and PURE100 KERATINASE. They are specifically used as topical agents for the treatment of skin problems and related conditions. Valkerase ® and Versazyme ® are products of BioResource International, Inc.; while CIBENZA ® DP100 and FEED-0001 were introduced to the feed market by Novus International, Inc. and Creative Enzymes ® , respectively. These keratinase-based formulations are useful for the improvement of the nutritional values of animal feeds.

Keratinase research innovations and patents
A number of innovative discoveries, pertaining keratinase research, have been patented over the past three decades, and this signified a milestone in this field of research. The bio-innovations may be placed under the following broad categories but not limited to process design, method development and product formulation. The title of the invention, patent number, region, inventor and legal status of the patent are presented in Table 5. A patent grants the inventor an exclusive right of sole ownership of a product or process for a defined period that the invention is protected. Some keratinase invention patents are functionally active. While others have variable legal status, such as expired, abandoned, discontinued, terminated, pending, withdrawn, and invalid ( Table 5). The reasons advanced for the various aforementioned status included patent expiration, post-publication rejection of the patent application, application awaiting examination and approval and intellectual property (IP) right cessation due to unpaid dues. Among the regions that the inventors are domiciled, China recorded the highest number of keratinase patents which invariably indicated the participation of prolific researchers from this region.    The purple coloured collaboration network that involved Paul T, Mondal KC and collaborators constituted authors from India. Shih JCH and Wang J from the USA are pioneers in the keratinase research field. Similar pattern of regional collaboration is applicable to other authors that share similar network connection. The authors without visible collaboration network could be that they are not collaborating with any authors on the top forty or they are independent researchers. Notably, among the lead authors of keratinase research, Brandelli A and Shih JCH recorded the highest number of published articles (18) and total citations (656) respectively (Additional file 1: Table S1). The collaboration networks of top 40 countries are shown in Fig. 3. The collaboration networks among various countries are presented in different colours. Larger nodes demonstrate greater number of collaborations within countries. The thickness of network linkages showcases the strength of collaboration between the two countries. China formed strong collaboration network with India and the USA, while collaborating in varying degrees with Saudi Arabia, Vietnam and Egypt. France formed cluster with Slovenia, Belgium, Switzerland, Bulgaria, Algeria, Tunisia and Brazil. Similarly, Nigeria has South Africa and Malaysia in its network domain. Conversely, Venezuela, Canada, Bangladesh, Iran and Pakistan did not form collaborative clusters with any topmost   Nnolim and Nwodo AMB Expr (2021) 11:12 forty countries that have published scholarly articles on keratinase research during the three decades of study.

Discussion
The evaluation of the macro-state of keratinase research, based on the information available from the WoS database, generally showed a trajectory growth pattern of research products. Notably, the recent past decade recorded the highest number of publications and this may be attributed to the involvement of more active researchers, adequate research funding, availability of state-of-the-art facilities and most importantly, the foreseeable application prospects of microbial keratinase in green technology (Rosenbloom et al. 2015;Hassan et al. 2020).
Keywords associated with keratinase research spotlight the hotspots and dimensions of researches over the study period. Author keywords are useful in the identification of scientific concepts advanced by an article. Their frequency therefore indicates the authors' involvement, a N/A not available and also expresses the trend of research in that particular field (Tripathi et al. 2018). Keywords-plus which the WoS extracts automatically from the metadata of a particular research field as indexing terms also assist in the determination of a knowledge structure, although they may be less comprehensive in divulging the intrinsic aim of a research (Zhang et al. 2016). It may be inferred from the keywords frequency that bacterial keratinases have been predominantly explored, of which the lead producers are Bacillus spp. (Nnolim et al. 2020b). Animal hide dehairing potentials of some candidate keratinases from various bacterial strains have been adequately evaluated which may serve as green depilatory strategy in leather processing (Fang et al. 2017a). However, there are yet to be commercially marketed keratinase based products for the processing of hides and skins in leather production. The cost-efficient and eco-friendly nature of these biobased processes have become an attraction for biotechnologists, hence the continuum in the development of the field. Moreso, a few empirical experimentations have lately suggested other promising applications of keratinase which include nanotechnology, bio-bleaching, and bio-energy (Gupta et al. 2015;Patinvoh et al. 2016;Zhang et al. 2019). Keratinases from wild microorganisms have been predominantly characterized and evaluated for various biotechnological and industrial advancements (Srivastava et al. 2020). However, a couple of limiting factors including prolonged fermentation period and low enzyme yield may thwart the commercial prospects of these keratinases. Therefore, cloning and overexpression vis-à-vis molecular optimization of the keratinase expression in industrial suitable hosts portend the tendency to ameliorate the productivity hitch fostered by the physiology of the wild microbial producers (Fang et al. 2019). The most cited articles were published during the first two decades of the survey period and the significant number of citations accrued by these papers may suggest their relevance and scientific contributions toward the development of keratinase research field (Agarwal et al. 2016). Notably, with an exception of three studies, the topmost cited papers were basically self-funded research. This might have contributed to the fluctuations in the number of research outputs and sluggish development of keratinase research during the first decades of the survey period. Research funding has been identified as a catalyst that promotes and encourages scientific development and innovation (Rosenbloom et al. 2015). Therefore, it is imperative that funding agencies and other stake holders Table 6 The top twenty productive countries on keratinase research based on corresponding authors affiliation CP BCA country productivity based on corresponding author, SCP single country production, MCP multiple countries production, TC total citations, AAC average article citations, TC PC total citation per country a Malaysia and Turkey also ranked twelfth position adequately support this research niche that intrinsically drives bio-based industry for sustainable bioeconomy. The study by Lin et al. was an advancement of the work previously reported by Williams et al. (1990) on feather degradation potential of B. licheniformis PWD-1 isolated from a laboratory poultry waste digestor. The discovery of this bacterial isolate landscaped new vistas of exploiting microbial keratinases as important candidates in green technology. Hence, the exponential growth and development of keratinase research in the recent past were based on the fundamentals of strain PWD-1characterization and its offshoots including PWD-1 keratinase characterization (Cheng et al. 1995), PWD-1 keratinase gene (kerA) cloning (Lin et al. 1995), PWD-1 keratinase gene expression in B. subtilis (Lin et al. 1997), comparative keratinase activity of B. licheniformis PWD-1 and recombinant B. subtilis FDB-29 (Wang and Shih 1999), PWD-1 keratinase immobilization (Wang et al. 2003).
The robust keratinolytic system of B. licheniformis therefore prompted the development of a few keratinase based formulations at commercial scale. Therefore, BioResource International, Inc. formulations; Versazyme ® and Valkerase ® preparations were efficient and sustainable means of utilizing avian feather wastes as low cost and good quality protein sources for animal husbandry (Potera 2013). Evaluation of Versazyme on the growth performance of birds showed that it significantly improved the feed conversion, body weight, and breast yield of poultry birds (Wang et al. 2006). The keratinase inclusion in animal feeds promotes the bioavailability of essential proteins which ultimately improves nutrient utilization and less excretion of nitrogen by fed animals (Potera 2013). Furthermore, the products of PROTEOS Biotech were formulated by microencapsulation of keratinase from B. licheniformis. These gentle natural formulations serve as alternatives to the synthetic alpha hydroxyl acids (AHAs), urea and thioglycolates predominantly utilized in cosmetics as cell renewing, moisturizing, and anti-hair growth agents. The commercialization of keratinase-based formulations presents the prospects India performance in terms of keratinase research suggests the involvement of many active researchers from this region. The significant number of articles recorded by a few countries may be attributed to the availability of requisite facilities, adequate research funding, and good collaboration network (Lee and Bozeman 2005;Rosenbloom et al. 2015). Generally, the countries on the list were more in intra-national publication than multinational production and this single country production considerably promoted scientific productivity (Scarazzati and Wang 2019).
The researchers, in their respective collaboration networks, have contributed significantly to the development of keratinase research and innovation. Paul and collaborators have sufficiently advanced the use of microbial keratinase for different benefits including valorization of keratinous wastes into high value functional peptides and essential amino acids (Paul et al. 2014a, b), cleaning properties of keratinase with an admixture of detergent (Paul et al. 2014a, b), plant growth promotion by keratinasederived organic hydrolysates (Paul et al. 2013) and many other innovative studies.
Fang and coworkers have shown their expertise by the discovery of novel keratinases from Stenotrophomas maltophilia (Fang et al. 2013a), keratinase production process optimum construction (Fang et al. 2013b), the biotechnological development of the bacterial strain for the overproduction of keratinase (Fang et al. 2014). They have also employed various protein engineering approaches to improve keratinase biotechnological and industrial values which were detailed in studies including truncation of keratinase PPC domain for catalytic efficiency improvement (Fang et al. 2016a), keratinase domain exchange for an improved catalytic efficiency (Fang et al. 2016b), keratinase substrate specificity alteration (Fang et al. 2015), thermostabiliy improvement through rational protein engineering approaches (Fang et al. 2017b), and cloning and overexpression of keratinase in an heterologous industrial host (Fang et al. 2019). These rigorous investigations therefore underpin the industrial and biotechnological potentials of S. maltophilia keratinolytic protease.
PWD-1 keratinase was a product of Shih, Wang and collaborators' innovative researches (Wang and Shih 1999;Wang et al. 2004). Its dexterity in degradation of recalcitrant keratinous wastes and augmentation of their nutritional value has become an attraction for researchers, nutritionists and feed producers. Consequently, the formulation of two patented products Versazyme ® (launched 2005) and Valkerase ® (launched 2006) by Shih and coworkers at BioResource International was on the basis of this thermostable keratinase (Potera 2013). The Shih founded BioResource International further extended the market of keratinase-based products outside the US in 2008 by partnering with a China based animal nutritional company Novus International. This partnership promoted the distribution of these keratinase-based formulations in the Asian market which attracted significant revenues (Potera 2013). Therefore, scientific collaboration of researchers in a particular research domain ensures expansion of knowledge base and promotes sustainable growth and development of that field (Fang et al. 2020). Funding bodies especially government agencies strictly facilitate collaboration as one of the formal contractual agreements during grant awards (Lee and Bozeman 2005), as it brings about crosspollination of ideas that would enhance productivity and innovation. International collaboration of scientific community fosters high quality scientific production and also promotes mobility of researchers with the ultimate goal of enhancing their scientific capacity (Chinchilla- Rodríguez et al. 2018). Moreso, networking of researchers at both domestic and international levels positively influences the growth of budding researchers by provision of new insights and career boost (Scarazzati and Wang 2019). However, international collaborations could suffer variable hiccups but not limited to language barrier, project delay, travel issues and political instability which may affect research productivity and innovation.
In conclusion, this study provided insight on the development of keratinase research domain vis-à-vis research outputs, scientific impacts, authors' involvement, collaboration pathways and bioinnovations over the past three decades. This study would imperatively subsidize the time investment by researchers to understand the trends of researches, fundamental hotspots, as well as assisting in the identification of knowledge gaps and prioritization of future research endeavours for innovative developments. The commercially available keratinase-based products currently in use are either for bio-decontamination, upgrading of animal feed nutritional value or skin care. Therefore, there is a need to carefully develop commercial keratinase-based products that lack collagenase activity for sustainable production of high quality leather materials. Moreso, other bacterial keratinases that have shown promising properties may be exploited for the development of novel products at commercial scale since all the existing keratinase-based products were based on B. licheniformis keratinases.