Non-Traditional Sources for Isolation of Lactic Acid Bacteria
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Ann Microbiol (2016) 66:449–459 DOI 10.1007/s13213-015-1127-9 ORIGINAL ARTICLE Non-traditional sources for isolation of lactic acid bacteria Tsvetanka Teneva-Angelova1 & Dora Beshkova1 Received: 7 April 2015 /Accepted: 26 June 2015 /Published online: 28 July 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract In recent years there has been increasing interest Introduction in lactic acid bacteria isolated from non-dairy products, due to their diverse metabolic profile, unique flavor- Lactic acid bacteria (LAB) are predominantly represented as a forming activities, and potential for use as starters or start- non-taxonomic heterogeneous group of Gram-positive and er adjuncts for the dairy industry. Screening of 400 mi- non-spore-forming facultative anaerobic bacteria. This group crobial isolates obtained from the herbs Geranium includes a wide variety of cell types and physiological and sanguineum L., Hypericum perforatum L., and Panax biochemical characteristics. LAB are designated "generally ginseng C.A. Meyer was performed, and 64 isolates were recognized as safe" (GRAS status), and have long played an selected based on milk coagulation and gas formation important role in food technology—they are responsible for ability and non-specific odour. Using tests involving mul- lactic fermentation in several products, including dairy, meat, tiple transfer and growth in selective and differential me- and silage. LAB can be isolated from traditional sources such dia, 258 single colonies were isolated, of which 98 were as raw milk, dairy products and fermented foods (Wouters affiliated with the lactic acid bacteria group. These bacte- et al. 2002 ; Kimoto et al. 2004; Tamang et al. 2005; ria are homofermentative cocci and rods with a wide pH Nomura et al. 2006; Kostinek et al. 2007; Tanasupawat et al. (5.0–9.6) and temperature range (15–45 °C), high salt 2007;Kivancetal.2011; Venturi et al. 2012;Abegaz2014), tolerance (3.0–10.0 % NaCl), and high acid-producing and from alternative sources including fecal samples, soil and activity (3.50–12.00 g/L). With the use of genotype- plants (Hartnett et al. 2002; Magnusson et al. 2003;Cockand based methods, the plant isolates were identified at the de Stouvenel 2006; Siezen et al. 2008; Trias et al. 2008;Di species level as Enterococcus faecium, Streptococcus Cagno et al. 2009; Cakir 2010;Chenetal.2010; Venugopalan thermophilus and Lactobacillus rhamnosus. et al. 2010; Baradaran et al. 2012; Emerenini et al. 2013; Fhoula et al. 2013; Nguyen et al. 2013; Alemayehu et al. 2014). In recent years, there has been increasing interest in Keywords Lactic acid bacteria . Medicinal plants . Isolation . LAB isolated from non-dairy sources due to their diverse met- 16S rDNA abolic profile and unique flavor-forming activities. Plant-de- rived strains of lactobacteria have demonstrated tolerance to high pH values and salt concentrations, an ability to ferment more types of carbohydrates, and a high level of stress resistance compared to those of dairy origin. Furthermore, studies have noted no significant differences in fermentation characteristics and profiles of enzymes * Dora Beshkova (lipases, peptidases and phosphatases) required for [email protected] obtaining various fermented dairy products with plant- derived and commercial strains of lactobacteria (Nomura 1 The Stephan Angeloff Institute of Microbiology, BAS, 26 Georgi et al. 2006; Michaylova et al. 2007;Siezenetal.2008; Bonchev St., 1113 Sofia, Bulgaria Venugopalan et al. 2010). 450 Ann Microbiol (2016) 66:449–459 The search for new solutions to improve starter systems for such exogenous functional components into their technologi- fermented health foods and for opportunities to realize the cal schemes or through the use of microorganisms as pro- biological potential of LAB has given rise to initiatives for ducers of biogenic substances or microorganisms with probi- exploiting biodiversity in unique natural systems (medicinal otic characteristics (Gobbetti et al. 2010). In this context, plants). These plants are an important ecosystem for isolation Servili et al. (2011) reported the production of a functional of LAB (Siezen et al. 2008;Cakir2010; Venugopalan et al. milk beverage through fermentation of cow's milk that had 2010; Baradaran et al. 2012), and each individual plant species been enriched with phenolic extracts from olive vegetable provides a unique environment in terms of competing micro- water. For this purpose, the authors used starter cultures in- organisms and natural plant antagonists, as well as accessibil- cluding Lactobacillus delbrueckii subsp. bulgaricus ity, type and concentration of the substrate in the various phys- DPPMALDb5 and Streptococcus thermophilus ical factors. When the plant material is collected and prepared DPPMAST1, and used the probiotic strain Lactobacillus for fermentation, these conditions allow for growth of typical paracasei 15 N and γ-amino butyric acid-producing-strain epiphytic flora from which a population and chain of fermen- Lactobacillus plantarum C48 as adjunct cultures. tation processes is derived. The present study was a preliminary evaluation of LAB Current available information on the use of medicinal isolated from the herbs Geranium sanguineum L., plants as a source for isolation of LAB and their subsequent Hypericum perforatum L. and Panax ginseng C.A. Meyer as potential application as components to form starters for milk potential starters or adjunct starters for fermented dairy prod- fermentation is scarce (Venugopalan et al. 2010). There are no ucts. The main objective of this work was to evaluate the data in the scientific literature regarding the microbial pres- presence of LAB within the context of microbial diversity in ence of different types of bacteria—and LAB in particular— natural biosystems (medicinal plants Geranium sanguineum isolated from herbs of the genera Geranium, Panax or L., Hypericum perforatum L. and Panax ginseng C.A. Meyer) Hypericum. Extracts from various parts of Geranium with desired metabolic activities in order to include them as sanguineum L., known in Bulgaria by the popular name starter or non-starter components during in situ cultivation in "bloody geranium," have significant antiviral, antibacterial, milk, and to identify the isolates at the species level using anti-inflammatory and antioxidant activity (Serkedjieva and genotype-based methods including 16S rDNA and phyloge- Manolova 1992; Hammami et al. 2011). Extracts of netic tree construction. Hypericum perforatum L., commonly known in Bulgaria as Byellow wort^ and as BSt John’swort^ in other countries and on other continents, also possess antibacterial and antiviral Materials and methods properties. St John’s wort has traditionally been used in the form of homeopathic medicines as an alternative treatment for Sample collection various depressive disorders of mild to moderate severity (Barnes et al. 2001). The incorporation of plant extracts in Plant material was collected from following locations: preparations for topical wound treatment has also been suc- Geranium sanguineum L., Eastern Rhodopes (Ivaylovgrad), cessful (Newall et al. 1996). Husain et al. (2011)demonstrated the Sofia region (experimental field of the Institute of significant hypolipidemic activity for the Hypericum Biodiversity and Ecosystem Research [IBER] at the perforatum L. extract in a rat model. In addition, the plant is Bulgarian Academy of Sciences [BAS]) and the Vitosha re- classified by the Council of Europe (2000) as a natural sea- gion (Iskar Dam); Hypericum perforatum L., Western soning for various foods (Barnes et al. 2001). There have been Rhodopes (Persenk Chalet); Panax ginseng C.A. Meyer, reports of high levels of antioxidant activity and pharmaco- Botanical Garden of the Technical University of Dresden, logical effects for extracts of Panax ginseng C.A. Meyer, Germany. All plant samples were collected aseptically in ster- known worldwide as Bginseng,^ including stimulation of ile poly-bags, kept under refrigeration, and transported to the brain function, pain relief, activation of antitumor immunity laboratory for analyses. and improvement in total immunity, regulation of blood pres- sure and blood sugar levels in diabetic patients, tonic effects Lactic acid bacteria isolation procedure on physical fatigue and mental stress, a positive influence on liver function, male potency and side effects of menopause in Individual parts (flower, leaf and stem) of each plant species women, and the inhibition of growth of the AIDS virus (Choi were carefully washed in sterile water, then transferred to test 2008). A new direction in scientific research that has recently tubes with sterile 10 % reconstituted skim milk (RSM; been explored involves obtaining bioactive or biogenic sub- HiMedia Laboratories, Mumbai, India) and incubated at 30 stances extracted from different plants or synthesized during and 37 °C until coagulation of milk occurred. After assess- food fermentation, and the subsequent creation of novel foods ment of milk coagulation, gas formation and non-specific (defined as healthy and functional) by further introduction of odour, samples were selected for subsequent transfer to M17 Ann Microbiol (2016) 66:449–459 451 broth (pH 6.6; Merck Millipore, Darmstadt, Germany) and The presumptive LAB that manifested high acid-producing MRS broth (pH 5.7; Merck Millipore), with the addition of activity were tested for growth at different temperatures (4, 15, 100 μg/mL of cycloheximide (Sigma-Aldrich,