Functional food is currently one of the fastest growing sector of food industry because considers a dietary strategy to reduce incidence of human illness (Gatti et al., 2004; Granato et al., 2010; Lobo et al., 2010; Burns et al., 2015). The literature data clearly indicate that functional foods could differently affect human health in relation to the gender. In recent decades, research on the female gender has been neglected, and the results obtained in men have been directly translated to women both in medicine and nutrition fields (Marino et al., 2011). The European Commission's Concerted Action on Functional Food Science in Europe (FuFoSE), coordinated by the International Life Science Institute (ILSI), defines functional foods as “products having beneficial effects on one or more functions of the human organism thus either improving the general and physical conditions or/and decreasing the risk of the evolution of diseases.” Moreover, this Institution states that “the amount and form of functional foods must be compatible with the nutritional parameters of a proper diet for the human organism.” Therefore, probiotic foods have received a lot of attention, because ingestion of probiotic cultures exerts beneficial effects on the gastrointestinal and urogenital tracts (Barrons and Tassone, 2008; Ceapa et al., 2013).

Reproductive-aged women are often subjected to gynecological disorders caused by vaginal microbiota dysbiosis and the possible occurrence of vaginal infections, including vulvovaginal candidiasis, bacterial vaginosis, aerobic vaginitis and sexually transmitted diseases (Workowski and Berman, 2010; Di Vito et al., 2015). Vulvovaginal candidiasis is a pathogenic yeast infections (30–35% caused by Candida albicans) that compromise the quality of life of women (das Neves et al., 2008). Bacterial vaginosis is generally caused by an imbalance in the ecology of the normal vaginal microbiota, characterized by a decrease of Lactobacillus species, which predominate in the vaginal microbiota of healthy women, and an increase in the prevalence of anaerobic pathogenic bacteria (van de Wijgert et al., 2014; Green et al., 2015; Machado and Cerca, 2015). Aerobic vaginitis represents another of the major dismicrobisms of the vaginal microbiota where lactobacilli are replaced with aerobic organisms such as streptococci, enterococci, Escherichia coli and Staphylococcus aureus (Han et al., 2014). Bacterial vaginosis and aerobic vaginitis may negatively interfere with women's reproductive health, increasing the risk of sexually transmitted infections, risk of abortion, preterm delivery, premature rupture of membranes and chorioamnionitis (Geng et al., 2016; Redelinghuys et al., 2016). Conventional therapies used to treat these disorders are generally ineffective and cause frequent relapses. On the contrary, the administration of probiotics has resulted in a very effective strategy to restore the normal vaginal microbiota (Borges et al., 2014). Beyond the vaginal application, probiotic bacteria can be administered orally thanks to their ability to pass from the intestine to the vagina, with a consequent beneficial impact on the vaginal habitat, as demonstrated by several investigations (Reid et al., 2001; Strus et al., 2012; Vitali et al., 2012; Vujic et al., 2013; Heczko et al., 2015).

Recent studies have highlighted the anti-Candida (Parolin et al., 2015) and anti-Chlamydia (Nardini et al., 2016) activities of some Lactobacillus strains isolated from the vagina of healthy women. Considering these important beneficial activities on women's health, the preparation of functional food containing active vaginal lactobacilli can represent a great scientific challenge for the female gender. A wide literature indicates that dairy products, such as fermented milks and cheese, represent excellent carriers of probiotic bacteria, because they are characterized by a high buffer capacity and a protective lipoprotein matrix. These properties allow to better protect the microbial cells during the gastrointestinal transit (Gomes da Cruz et al., 2009; Lollo et al., 2013; Castro et al., 2015). Moreover, dairy products, such as cheese and fermented milks, are perceived positively by consumers because they represent a good source of calcium, phosphorus and vitamins; therefore, the use of a cheese or a fermented milk can be a strategy for the ingestion of probiotic strains capable of positively influencing the healthy status of the human vaginal microbiota, in order to protect the woman from vaginal infections.

In this context, the aim of this work was to evaluate the amensalistic properties and the technological potential of 17 vaginal strains belonging to the species Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus vaginalis, in the perspective to include them in dairy products. In particular, these lactobacilli were evaluated for their antagonistic activity against the pathogenic and spoilage species frequently associated to food products and against the principal etiological agents of bacterial vaginosis, aerobic vaginitis and urinary tract infections. Moreover, the 17 strains were characterized for their bacteriocin production and antibiotic susceptibility. Finally, the fermentation kinetics and viability of these lactobacilli during the refrigerated storage in milk were characterized, as well as the volatile molecule profiles of the obtained fermented milks.

The administration of probiotics has been shown to be effective in restoring a normal vaginal and gut microbiota, contributing to resolve gynecological disorders (Borges et al., 2014). The rationale behind the present work is to develop a functional food for the female gender capable of delivering probiotic lactobacilli in the vagina in order to promote the gynecological health through nutrition. Taking an active ingredient through a food is a form of administration that best meets the compliance of the consumer and this aspect represents an important added value of a functional food. In particular, the anti-Candida and antibacterial activities of the Lactobacillus strains used in this work can be exploited to restore the healthy status of the human vaginal and gut microbiota, contributing to protect the woman from vaginal dysbiosis and infections. However, the formulation of a functional dairy product should be subordinate to the selection of probiotic strains able to maintain viability and functionality during storage and without negative effects on the hedonistic properties of the carrier food. In fact, to exert positive health effects, the functional microorganisms need to be viable, active, and sufficiently abundant, in concentrations of at least 6 log CFU/g throughout the shelf life, and consequently they have to maintain their viability during the refrigerated storage (Tripathi and Giri, 2014; Burns et al., 2015).

Our data clearly indicated the unsuitability of the studied strains as fermentation starters for the industrial production of dairy products. In fact, the fermentation kinetics in pasteurized whole milk have shown that none of the strains could acidify the milk to pH values below 5 in a considered time of 53 h at the temperature of 37°C. Even the addition of 0.5% tryptone, to provide a source of protein and promote the growth kinetics of the tested strains, did not increase the acidification rate to acceptable levels for the dairy industries. Nevertheless, some Lactobacillus strains maintained a high viability for over a month in pasteurized whole milk at 4°C, indicating their suitability in dairy products as adjunct cultures instead of starters. This is a very important result from an applicative point of view: the use of vaginal lactobacilli as additional cultures in fermented milks and fresh cheeses, which represent the most used carriers for functional microorganisms, allows to overcome the typical sensory defects of the products obtained using probiotics as starters (Castro et al., 2015). Similarly, Patrignani et al. (2006) reported, for the functional strains belonging to the species L. gasseri, L. plantarum, L. rhamnosus isolated from Masaai milks, slow fermentation kinetics, not acceptable for industrial fermentation. In sight of this, the probiotic bacteria belonging to Bifidobacterium spp. and the species L. acidophilus, L. casei, and L. rhamnosus are generally used as additional cultures due to their poor fermentation kinetics and the scarce sensory properties of fermented milk obtained when they are used as starters (Patrignani et al., 2007; Settanni and Moschetti, 2010; Castro et al., 2015).

The data reported in this work showed that some Lactobacillus strains could maintain a high viability for over a month in pasteurized whole milk at 4°C, indicating their suitability as adjunct cultures in dairy products. This is a very important result from an applicative point of view: the use of vaginal lactobacilli as additional cultures in dairy products, such as fermented milks and fresh cheeses, which represent the most used carriers for functional microorganisms, allows to correct the typical sensory defects of the products obtained using probiotics as starters (Castro et al., 2015). On the other hand, to produce a satisfactory fermented probiotic milk product, the viable cell count at the time of consumption should be above 6 log CFU/g in order to comply with the International Dairy Federation standards (IDF 1992) and to ensure the supply of a sufficient “daily dose” of viable bacteria (Patrignani et al., 2006, 2016). Consequently, the most important criterion to select probiotic strains to be employed in fermented milk products as adjuncts, is their ability to maintain high viability during manufacture and refrigerated storage (Kailasapathy and Chin, 2000; Soccol et al., 2010). In fact, several cases of marked loss of viability of the most common probiotics belonging to Bifidobacterium spp. and L. acidophilus species in refrigerated dairy products have been reported in literature (Patrignani et al., 2006).

In this work, we demonstrated that several vaginal lactobacilli, mainly belonging to L. crispatus species, exhibited a remarkable antagonistic activity against spoilage and pathogenic microorganisms of food interest, as well as against urogenital pathogens. In addition, previous literature data had clearly indicated that the vaginal lactobacilli considered in this study, especially L. crispatus strains, were characterized by significant anti-Candida (Parolin et al., 2015) and anti-Chlamydia (Nardini et al., 2016) activities. The results obtained in this research showed the absence of bacteriocin production by all the tested strains. The highest antagonistic activities of L. crispatus BC1, BC4, BC5, and BC6 can be attributed to the highest production of organic acids as demonstrated by the pH values of the cell free supernatants, and the volatile molecule profiles of the inoculated milks registering the highest abundances of acetic acid and short chain fatty acids (hexanoic and butanoic acid). The high levels of acetic acid detected in the volatile molecule profiles of the L. crispatus strains can explain their high antimicrobial activities against the target microorganisms. In fact, it is well reported that short-chain organic acids, such as acetic acid and lactic acid, can exert a strong antimicrobial activity against both gram-positive and gram-negative microorganisms, due to the diffusion of the acids through the cell membrane of microorganisms (Varalakshmi et al., 2014; Poppi et al., 2015). Also Annuk et al. (2003) attributed the strong antimicrobial activity of intestinal lactobacilli against both Gram-negative and Gram-positive bacteria to the high production of organic acids, such as lactic acid, succinic acid and acetic acid. Moreover, Breshears et al. (2015) showed that high concentrations of acetic acid could inhibit the growth of Neisseria gonorrhoeae on porcine vaginal mucosal model at pH 5.5. In addition, all the tested lactobacilli showed the production of diacetyl, acetoin, and also acetaldehyde, which are important for the sensory characterization of dairy products as well as for their antimicrobial activity against both gram-positive and gram-negative pathogenic species (Ott et al., 1997; Lanciotti et al., 2003; Patrignani et al., 2007, 2016). Such molecules are considered key factor for the characterization of dairy products (Escamilla-Hurtado et al., 2000; Lanciotti et al., 2003, 2007). Furthermore, the capacity to generate diacetyl and acetoin is considered a general trait relevant for the selection of starter lactic acid bacteria (Cruciata et al., 2014). Moreover, acetaldehyde is considered a fundamental compound for fermented milks quality as it gives a characteristic flavor in yogurt and fermented milks (Smit et al., 2005; Patrignani et al., 2007). These results are particularly important since the ability to impart good sensory properties is an important selection criterion for starter cultures or probiotic adjuncts (Patrignani et al., 2006; Cheng, 2010; Routray and Mishra, 2011; Widyastuti and Febrisiantosa, 2014).

The determination of the antibiogram is considered a prerequisite in protocols for the selection of starter, co-starter or functional microorganisms by EFSA (Wedajo, 2015).

The antibiogram results are in agreement with literature data (Ammor et al., 2007; Nueno-Palop and Narbad, 2011; Fguiri et al., 2015). In fact, the lactobacilli showed a variable spectrum of susceptibility in relation to the species and the strain considered. The majority of the tested strains resulted very sensitive to Amoxicillin, Ampicillin, Penicillin G and Erythromycin. Some lactobacilli were found to be less susceptible to different antibiotics. In these case, further studies are needed to better characterize the resistance mechanism, before including these strains in food products. Other authors have observed a high susceptibility of different lactic acid bacteria strains to these antibiotics (Danielsen and Wind, 2003; Temmerman et al., 2003; Nueno-Palop and Narbad, 2011). In general, the spectrum of susceptibility to antibiotics results variable in function of the lactic acid bacteria strain considered. In fact, several studies have shown the natural resistance of lactic acid bacteria to a wide range of antibiotics (Botes et al., 2008; Fguiri et al., 2015).

Although strains belonging to L. casei (L. paracasei), L. rhamnosus, and L. plantarum are widely used in probiotic dairy products (Kaushik et al., 2009; Zheng et al., 2013), only few reports are focused simultaneously on their functional and technological properties (Olasupo et al., 2001; Patrignani et al., 2006). Some aspects, i.e., viability in milk during refrigerated storage, antagonistic activity against pathogenic and spoilage species, volatile molecule profiles and production of key sensory compounds such as dyacetyl, are particularly important for the selection of adjunct cultures to produce dairy functional foods since they are the main choice criteria for the consumers and the dairy industries. L. crispatus BC1, BC2, BC3, BC4, and BC5, L. gasseri BC9 and BC12, and L. vaginalis BC15 maintained a high vitality during refrigerated storage and at the same time they showed a good antimicrobial activity against both spoilage and pathogenic microorganisms. In addition, they showed interesting volatile molecule profiles, without negative aroma compounds for dairy products. These strains, with proven anti-Candida and antibacterial activities, appear to be the most promising candidates to be used as adjunct cultures for the production of a functional food capable of improving the health of the female gender.

The results obtained represent a first towel for the selection of adjunct cultures to produce new functional dairy products, directed to the women well-being, contributing also to innovate the dairy sector. In fact, in recent years there has been a significant increasing demand for tailor made microbial strains able to contribute to food innovation in terms of functionality, sensory, nutritional and rheological characteristics. The present study has identified a pool of strains having technological and functional properties promising for the production of a dairy functional food specifically directed to female gender.

All the authors contributed to the experimental design of the work; as well as to the acquisition, analysis and interpretation of the obtained results; Moreover, all the authors contributed to the writing and the critical revision of the manuscript.