Current status and application of lactic acid bacteria in animal production systems with a focus on bacteria from honey bee colonies
Summary
Lactic acid bacteria (LAB) are widely distributed in nature and, due to their beneficial effects on the host, are used as probiotics. This review describes the applications of LAB in animal production systems such as beekeeping, poultry, swine and bovine production, particularly as probiotics used to improve health, enhance growth and reproductive performance. Given the importance of honeybees in nature and the beekeeping industry as a producer of healthy food worldwide, the focus of this review is on the coexistence of LAB with honeybees, their food and environment. The main LAB species isolated from the beehive and their potential technological use are described. Evidence is provided that 43 LAB bacteria species have been isolated from beehives, of which 20 showed inhibition against 28 species of human and animal pathogens, some of which are resistant to antibiotics. Additionally, the presence of LAB in the beehive and their relationship with antibacterial properties of honey and pollen is discussed. Finally, we describe the use of lactic bacteria from bee colonies and their antimicrobial effect against foodborne pathogens and human health. This review broadens knowledge by highlighting the importance of honeybee colonies as suppliers of LAB and functional food.
Introduction
Lactic acid bacteria (LAB) are widely distributed in nature and are part of the microbiota of humans and animals, mainly existing in the digestive tract, oral cavity and the vagina of mammals. The gut microbiota is essential for health because of the metabolic properties and their role in the immune system by providing a barrier against pathogenic micro-organisms (Cariveau et al. 2014; Quinto et al. 2014; Kwong and Moran 2016). Insects are the most abundant clade in the animal kingdom, but few investigations about their relationship with LAB have been carried out. Recent studies have reported that some LAB inhabit the gut of insects; however, our current knowledge of microbial functions in the gut of insects is still limited in contrast to mammalian gut interactions (Engel and Moran 2013).
Lactic acid bacteria belong to a group of Gram-positive, not sporulated, anaerobic or facultative aerobic, catalase negative, cocci or rod bacteria, characterized by producing lactic acid from sugar fermentation (Quinto et al. 2014). Depending on their metabolism, they can be classified into homofermentative, which produce two moles of lactic acid from 1 mol of glucose, or into heterofermentative, which produce CO2, acetic acid, ethanol and lactate besides lactic acid (Hayek and Ibrahim 2013).
Lactic acid bacteria are included into the order Lactobacillales, which encompasses the Aerococcaceae, Arnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae families. The best known genera of LAB are Carnobacterium, Enterococcus, Lactobacillus, Pediococcus, Leuconostoc, Oenococcus, Weissella, Lactococcus and Streptococcus (Whitman 2015).
Fructophilic acid lactic bacteria (FLAB) are a group within LAB that have been described only recently. They can be found in fermented foods, flowers and fruits, and because their optimal substrate is fructose, they are also present in some insects whose diets are fructose-rich. FLAB differ from LAB in that their growth is poor or delayed with glucose as substrate. They produce lactate, CO2 acetate and ethanol. Some FLAB species belong to the Fructobacillus genus (F. fructosus, F. durionis, F. ficulneus, F. pseudoficulneus), and were previously classified as Leuconostoc. Other FLAB have been classified as part of the Lactobacillus genus, such as Lact. kunkeei, Lact. apinorum and Lact. florum (Vasileva et al. 2017; Endo et al. 2018).
Bifidobacteria, even though not considered as LAB, are included in this review because of their similar uses and functions. They were classified into the Bifidobacterium genus, which was included in the Actinomycetaceae family of the Actinomycetales order, because of their rRNA gene sequence (Biavati and Mattarelli 2015).
Some LAB strains are considered probiotic because of their beneficial effects on the host when they are consumed. Consequently, they are added to food products in order to promote human and animal health. Most of the probiotics are bacteria derived from the gut microbiota and correspond to the Lactobacillus and Bifidobacterium genera, although there are some Gram-negative bacteria like Escherichia coli strain Nissle 1917 or fungi, like Saccharomyces, which are considered probiotics (Behnsen et al. 2013; Yahfoufi et al. 2018).
In animal production systems, there are several factors such as diet, medication and stress that can affect the activity of the gut microbiota, and these factors may often be to the detriment of the host. Thus, the gut microbiota is a critical factor in an animal’s overall health, development and productivity. The important functions of the microbiota in increasing productivity have led researchers to explore the use of health-promoting microbes to improve animal health, increase disease resistance, enhance reproductive performance and growth (Yeoman and White 2014). There is evidence of the benefits of using LAB in poultry, swine, bovine and beekeeping production systems. In all animals, including insects, microbial communities are particularly prominent in the digestive tract. In insects the contribution of gut micro-organisms is highly relevant from several perspectives, with links to medicine, agriculture and ecology. The herbivorous insects interact in several ecosystems; for example, they are responsible for pollination of many food crops and also for some agricultural product loss, and through these interactions micro-organisms associated with insects could also have an effect on crops.
Bees are of great importance among insects because of both their role of maintaining the global ecological equilibrium and their beneficial impact on the production of forage and oil crops as well as fruit and vegetable crops. The scale of this pollination service is equal two thirds of the food produced worldwide (Hamdi et al. 2011), since more than 100 crop species depend on either native and wild bees or domesticated bees. Honey bees (Apis mellifera L.) are widespread in the world and they have been domesticated mainly for pollination as well as honey production. In relation to food products, bee products are well known for their nutritional and medicinal use, belonging to the segment of health foods. Some bee products are subjected to natural microbial fermentation and it is evident that there is a connection between the microbiota of honeybees and these products. The consumption of these products in our diet can influence our own gut microbiota.
Bees can forage in several food sources at the same time, and a maximum distance of 10 km can be explored by bees to collect food (Seeley 1995). There are thus good reasons to hypothesize that the beehive is a wide reservoir of LAB from the environment. Furthermore, LAB from bees have important functions and properties that could be applied not only in beekeeping but also in other productions and industries.
In this work, we review and synthesize investigations related to technological applications of LAB in animal production systems. Specifically, we focus on the LAB associated with honey bee colonies and their potential technological use. We argue that the beehive may be an interesting source of biodiversity of LAB with potential beneficial applications for human and animal health, because in this environment several endogenous and exogenous LAB from broad-spectrum sources co-exist. Furthermore, we highlight that some beneficial properties of healthy food such as honey and pollen could be attributable to the presence of some LAB genera.
Methodology
The process of identification, selection and classification of articles for this review was based on the method of integrative literature review (Torraco 2005). The studies were screened with the digital PubMed and Science Direct databases using the following descriptors: [{animal production+probiotics}], [{bees+LAB}], [{bees+probiotic}], [{pollen+LAB}], [{beebread+LAB}], [{honey+LAB}]. The database search provided a total of 91 citations. We reviewed citations from the articles obtained through the search of selected databases. Both recent and old literature sources were included. In order to select the articles, titles and abstracts were analysed and their inclusion was decided by reading the full-text article. Studies which did not meet the inclusion criteria were excluded. A selection of articles from own previous literature review was also made. Thus, a total of 93 articles are cited in this review. The last search was carried out on 11 April 2019.
Applications of LAB in animal production systems
In the animal feed production industry, LAB are mainly used for silage production and as probiotics to improve animal health and their productive capacity. Ensilage, which is the preparation of forage by storing and fermenting crops such as hay and corn, is important for livestock farms because it preserves quality feed for winter consumption. It is particularly important in cool and moist climates where hay drying is a challenge (McGechan 1990). In European countries like the Netherlands, Germany and Denmark, ensilaged forages are highly valued as animal feed. More than 90% of the forages, such as maize, grass, legumes and wheat, are stored as silage (Elfrink et al. 2000).
The interest in the use of biological systems for preserving foods has increased and has been mainly directed at LAB, which are the most common crop silage additive used as inoculants (Muck 2010). For instance, Lactobacillus plantarum, a facultative heterofermentative lactic acid bacterium, is the most used micro-organism to preserve the nutritional quality of ensiled forages by improving lactic acid fermentation and inhibiting deleterious epiphytic microbes (Oliveira et al. 2017), and moreover helps ensure a consistent fermentation in the silage.
Some researchers have also developed biological silages by incorporating reference strains or commercial lactic bacteria to fish residues to be used in aquaculture. Some of those used have been Lact. plantarum and Lactobacillus sp., from waste shrimp fermentation, LAB from yogurt and Lact. plantarum, Pediococcus acidolactici and Lactococcus lactis from a commercial starter (Bello 1997; Ramírez et al. 2013; Fernández Herrero et al. 2015; Libonatti et al. 2017).
Besides being used for technological purposes in the animal feed production industry, some LAB strains are considered probiotics because of their beneficial properties in animals. They are usually added to food to contribute to the treatment or prevention of some diseases and the improvement of health. The definition of probiotics by FAO/WHO (2006) was reinforced in 2013 by an expert panel convened by the International Scientific Association for Probiotics and Prebiotics (Hill et al. 2014). Minor grammatical corrections were considered, and probiotics were finally defined as ‘live micro-organisms that, when administered in adequate amounts, confer a health benefit on the host’.
In some animal production systems in which food is produced for human consumption, many antibiotics are banned, and so in these systems the use of probiotics is an important tool, not only to improve animal health and reduce the incidence of diseases but also to improve productivity and food quality. LAB are part of the animal gut microbiota and have essential functions in their health. In fact, LAB administration to animals has been shown to improve health, increase disease resistance and enhance growth and reproductive performance (Yeoman and White 2014).
The use of LAB in animal production has been widely studied. Many works related to poultry and swine production, whose objective was seeking and selecting LAB from different origins, have focused on different properties that these bacteria provide. Many studies have noted the abilities of LAB to improve growth performance, prevent gastrointestinal infection, regulate host immune systems, enhance gut metabolic capacities and maintain balance in the gut microbiota, capabilities with higher nutrient digestibility, haemato-biochemical and antioxidant status in swine production (Guo et al. 2017; Dowarah et al. 2018). Bacteria used for these purposes are Lactobacillus sp., Pediococcus acidilactici, as Lactobacillus salivarius, Lactobacillus reuteri and Lactobacillus curvatus.
In poultry production, LAB have positive effects on growth performance, carcass traits, intestinal microbiota, serum biochemical constituents, immune parameters and caecum microflora, and apparent ileum nutrient digestibility. Since some LABs have an antimicrobial effect against pathogenic bacteria, they could increase the resistance of birds to infections, prevent adverse effects and improve host health. For instance, Pediococcus sp. from rectal samples of chickens has been proved to have antibacterial activity against Salmonella enteritidis and E. coli (Noohi et al. 2016). FloraMax-B11® is a nutritional supplement for poultry production containing Lact. bulgaricus, Lact. casei, Lactobacillus cellobiosus, Lactobacillus fermentum and Lactobacillus helveticus as probiotics (Prado-Rebolledo et al. 2017). These probiotics compete with the pathogenic microflora, generating beneficial effects for the host and consequently, improving productive parameters. Similarly, Lact. salivarius and Lact. reuteri supplements are efficient in avoiding harmful effects of a natural constituent of the grain in wheat-based diets of chickens (Babot et al. 2018).
In beef cattle production, oral administration to calves of LAB isolated from bovine Lact. casei (DSPV 318T), Lact. salivarius (DSPV 315T) and P. acidilactici (DSPV 006T) was evaluated by Frizzo et al. (2010). A significantly higher live weight gain and heart girth values were registered at the end of the experiment, and the starter intake in calves was higher when LAB were supplemented. Studies using Lactobacillus acidophilus showed a reduced incidence of diarrhoea in calves. In addition, Lact. acidophilus was used in combination with Propionibacterium freudenreichii to improve the digestibility of crude protein, neutral detergent fibre and acid detergent fibre in lactating Holstein cows, resulting in increased milk production per day (FAO 2016).
It has recently been found that LAB can be natural biological antagonists against mycotoxigenic moulds and their highly toxic metabolites. Mycotoxins are produced by fungus and have been found in several agricultural products worldwide, especially in maize. The contamination of rations for animal feed by mycotoxins is today a problem worldwide; it is estimated that 25% of crops are contaminated by mycotoxins (Jelinek et al. 1989). A major problem is that mycotoxins cause losses in terms of animal production and health. Aflatoxin B1, a mycotoxin produced by Aspergillus species of fungi, is frequently found in food and feed and may cause adverse health effects to both humans and animals. For instance, this mycotoxin was found in chicken feed and resulted in toxic residues in chicken tissues and products. In this scenario, the use of LAB, such as Lact. acidophilus (ACCC 11073), Lact. plantarum (CICC 21863), and Enterococcus faecium in broiler chicken diets improved growth performance, digestibility and immune function, and reduced the deleterious effect of mycotoxins (Liu et al. 2018). The ability to adsorb several mycotoxins in swine production has also been studied. Lactobacillus sp., isolated from pig rectal swabs, and Lactobacillus rhamnosus can absorb zearalenone, one of the most important toxins causing serious reproductive failures in swine production (Vega et al. 2017). In another study, Lactobacillus delbrueckii subsp. lactis and Pediococcus acidilactici strains have been proved to have a protective effect against antigenotoxicity and precancerous lesions in rats dosed with fumonisin B1, a mycotoxin that constitutes an imminent risk for swine and equines (Khalil et al. 2015).
In apicultural production, whose main product is honey, the improper use of medicines deleteriously affects honey quality and safety because of the presence of drug residues. The use of probiotics in beekeeping could prevent diseases, enhance bee health and consequently, increase honey production (Audisio and Benítez-Ahrendts 2011). Several studies have been conducted in order to evaluate the effect of different micro-organisms as potential bee probiotics using commercial probiotic cultures, and positive effects have been reported. For example, Enterococcus, Lactobacillus and Weissella, isolated from fermented foods, showed in vitro inhibition effects against Paenibacillus larvae, an important bee pathogen (Yoshiyama et al. 2013). Along the same lines, in vivo studies reported that the mortality and number of bee larvae infected by P. larvae decreased with the addition of probiotic bacteria to the honey bee larval food (Forsgren et al. 2009; Al-Ghamdi et al. 2018). Other benefits have also been reported, such as the enhancement of honey bee immunity activation (Evans and Lopez 2004; Yoshiyama et al. 2013) or the development of wax glands (Pătruică and Mot 2012) by using a mix of Lact. acidophilus, Bifidobacterium lactis and Lact. casei. Commercial probiotic preparations such as ‘Biogen-N’, used to stimulate immunity and growth rate of piglets, calves and foals, and ‘Trilac’, a preparation restoring functions of gastrointestinal microflora in humans, were added to pollen supplement and administrated to bees. It was found that these preparations stimulate the growth of the food gland and fat body in bees (Kazimierczak-Baryczko and Szymas 2006).
However, some studies revealed that treatments with inadequate probiotics for honey bees may cause dysbiosis and increase pathogen susceptibility (Ptaszyńska et al. 2016; Schmidt and Engel 2016). According to Ptaszyńska et al. (2016), probiotic bacteria are more beneficial when used as supplements in the diet of the organisms from which they had been isolated. Accordingly, many investigations have been carried out in order to look for promising bacterial strains from honey bees or their environment for its use in beekeeping (Audisio 2017). We will focus on this area of interest in a following section.
Applications of LAB from the honey bee colony environment
Microbial ecology of honey bee colonies
Most insect guts contain relatively few microbial species as compared to mammalian guts. Some insects, however, harbour large gut communities of specialized bacteria that potentially provide many beneficial services to their hosts. In some insects, gut microbiota also have important functions, such as digestion, pathogens defence and insecticide resistance (Cariveau et al. 2014). However, the relationship between the gut microbiota of insects and their immune function remains poorly understood. In social insects, like honey bees, the interactions between individuals of the same colony provide opportunities to transfer gut bacteria, and some of the most distinctive and consistent gut communities with specialized beneficial functions in nutrition and protection have been found in these insects (Engel and Moran 2013).
The guts of worker honey bees are dominated by a core of 7–12 regularly occurring phylotypes or bacterial species clusters that constitute between 95 and 99·9% of the bacteria found in the gut of a bee (Anderson et al. 2013; Kwong and Moran 2016). Gram-negative bacteria constitute 70% of Apis mellifera microbiota, with Snodgrassella alvi, Gilliamella apicola and members of the Proteobacteria phylum being the predominant species. Among Gram-positive bacteria that constitute 30% of the microbiota, the most abundant have been termed the Lactobacillus Firm-4 and Lactobacillus Firm-5 clades; these are species clusters in the Firmicutes phylum and mainly founded in the rectum. Bifidobacterium asteroids, which belongs to the Actinobacteria phylum, is also another species cluster from the rectum of bees. Less numerous, and also less prevalent, are the Proteobacteria species, Frischella perrara, Bartonella apis, Parasaccharibacter apium and a Gluconobacter (Kwong and Moran 2016).
With regard to LAB, at least 45 bacteria species have been isolated and identified from bees, bee products and flowers (Table 1). Lactobacillus was the most frequent genus found in the beehive, constituting 90·9% of the bacteria present in honey, 74·6% in pollen, 83·9% in beebread, 93·3% in royal jelly and 30·3% in whole gut (Asama et al. 2015). For example, Lact. kunkeei, a FLAB, has been found to be one of the dominant bacterial species in bees, being also the most frequent species in bee products (Vásquez et al. 2012; Anderson et al. 2013; Endo and Salminen 2013; Kieliszek et al. 2018).
| Bacterium | Isolated from | Effect against bee pathogens | |
|---|---|---|---|
| 1 | Bifidobacterium sp. | Stomach or crop (Anderson et al. 2013), royal jelly beebread (Asama et al. 2015) | |
| 2 | B. catenulatum | Honey (Olofsson et al. 2016) | |
| 3 | B. longum | Honey (Olofsson et al. 2016), gut (Disayathanoowat et al. 2012) | |
| 4 | B. asteroides | Stomach or crop (Olofsson and Vásquez 2008), gut (Moran 2015; Baffoni et al. 2016; Kwong and Moran 2016), honey (Moran 2015; Kwong and Moran 2016; Olofsson et al. 2016), beebread (Janashia and Alaux 2016) | A mixture of 2, 3, 5, and 6 sowed in vitro and in vivo effect against P. larvae (Forsgren et al. 2009) |
| 5 | B. coryneform | Stomach or crop (Olofsson and Vásquez 2008) | A mixture of 2, 3, 4, 6, 7, 8 sowed in vivo effect against N. ceranae (Baffoni et al. 2016) |
| 6 | B. indicum | Gut (Scardovi and Trovatelli 1969; Disayathanoowat et al. 2012) | |
| 7 | Lactobacillus sp. | ||
| 8 | L. kunkeei | Stomach (Olofsson and Vásquez 2008; Vásquez et al. 2012; Anderson et al. 2013; Uğraş, 2017; Vasileva et al. 2017), gut (Disayathanoowat et al. 2012; Asama et al. 2015; Baffoni et al. 2016), honey (Olofsson and Vásquez 2008; Endo and Salminen 2013; Asama et al. 2015), pollen and royal jelly (Asama et al. 2015), beebread (Asama et al. 2015; Janashia and Alaux 2016), flowers (Neveling et al. 2012; Anderson et al. 2013) | In vivo against P. larvae and N. ceranae (Arredondo et al. 2018), in vitro and in vivo against M. plutonius (Endo and Salminen 2013; Vásquez et al. 2012) |
| 9 | L. johnsonii | Gut (Audisio et al. 2011; Baffoni et al. 2016) | In vitro against P. larvae (Audisio 2011) and in vivo against N. ceranae (Maggi et al. 2013; Audisio and Sabaté 2015; Audisio 2017) |
| 10 | L. plantarum | Gut (Mudroňová et al. 2011, Baffoni et al. 2016), beebread (Asama et al. 2015) | In vitro against P. larvae (Mudroňová et al. 2011) |
| 11 | L. apinorum | Stomach or crop (Olofsson et al. 2014; Endo et al. 2018) | |
| 12 | L. apis | Stomach or crop (Killer et al. 2014) | In vitro against P. larvae and M. plutonius (Killer et al. 2014) |
| 13 | L. helsingborgensis | Stomach (Olofsson et al. 2014), gut (Baffoni et al. 2016) | |
| 14 | L. kimbladii | Stomach (Olofsson et al. 2014), gut (Baffoni et al. 2016) | |
| 15 | L. kullabergensis | Stomach (Olofsson et al. 2014), gut (Baffoni et al. 2016) | |
| 16 | L. mellifer | Stomach (Olofsson et al. 2014) | A mixture of 2, 3, 6, 9, 10, 11, 12, 13, 14, 15, 16 sowed in vitro and in vivo effect against M. plutonius (Vásquez et al. 2012) and P. larvae (Lamei et al. 2019) |
| 17 | L. mellis | Stomach (Olofsson et al. 2014) | |
| 18 | L. melliventris | Stomach (Olofsson et al. 2014) | |
| 19 | L. acidophilus | Stomach or crop (Olofsson and Vásquez 2008) honey (Aween et al. 2012) | |
| 20 | L. alvei | Gut and pollen (Asama et al. 2015) | |
| 21 | L. brevis | Gut (Disayathanoowat et al. 2012) | |
| 22 | L. buchneri | Stomach or crop (Olofsson and Vásquez 2008) | |
| 23 | L. casei | Gut (Disayathanoowat et al. 2012; Janashia et al. 2018) and beebread (Janashia et al. 2016) | |
| 24 | L. crispatus ST1 | Honey (Olofsson et al. 2016) | |
| 25 | L. helveticus | Gut (Disayathanoowat et al. 2012) | |
| 26 | L. Firm-4 | Gut (Moran 2015; Alberoni et al. 2016; Kwong and Moran 2016) | |
| 27 | L. Firm-5 | Gut (Anderson et al. 2013; Moran 2015; Alberoni et al. 2016; Kwong and Moran 2016) | |
| 28 | L. floricola | Pollen (Asama et al. 2015) | |
| 29 | L.florum | Flowers (Endo et al. 2018) | |
| 30 | L. flumenti | Pollen (Asama et al. 2015) | |
| 31 | L. insectis | Stomach or crop, gut, pollen and royal jelly (Asama et al. 2015) | |
| 32 | L. intestinalis | Pollen (Asama et al. 2015) | |
| 33 | L. kefiranofaciens | Gut and honey (Asama et al. 2015) | |
| 34 | L. mucosae | Beebread (Asama et al. 2015) | |
| 35 | L. reuteri | Pollen (Asama et al. 2015) | |
| 36 | L. rossiae | Honey (Olofsson et al., 2016) | |
| 37 | L. ozensis | Pollen (Asama et al. 2015) | |
| 38 | L. versmoldensis | Honey (Olofsson et al. 2016) | |
| 39 | Lactococcus lactis | Pollen (Belhadj et al. 2010) | |
| 40 | Enterococcus durans | Gut and beebread (Janashia et al. 2016) | |
| 41 | E. faecium | Gut (Audisio et al. 2011), honey (Ibarguren et al. 2010) | In vitro against P. larvae (Audisio 2011) |
| 42 | E. faecalis | Stomach or crop (Anderson et al. 2013) | |
| 43 | E. raffinosus | Honey (Olofsson et al. 2016) | |
| 44 | Fructobacillus sp. | Gut, beebread and flowers (Anderson et al. 2013) | |
| 45 | F. fructosus | Stomach or crop (Salman and Saleh 2018), gut (Endo and Salminen 2013; Janashia and Alaux 2016), honey (Syed Yaacob et al. 2018), flowers (Endo and Salminen 2013; Syed Yaacob et al. 2018) | |
| 46 | F. pseudoficulneus | Gut (Janashia and Alaux 2016) | |
| 47 | F. tropaeoli | Gut (Janashia and Alaux 2016) | |
| 48 | Oenococcus sp. | Gut and beebread (Mattila et al. 2012) | |
| 49 | Weissella sp. | Beebread and flowers (Anderson et al. 2013) | |
| 50 | W. paramesenteroides | Beebread (Libonatti et al. 2018) |
The microbiota of bees is acquired through vertical transmission and from environmental sources such as flowers. According to Vásquez et al. (2009), LAB communities and their abundance were found to vary with bees foraging activity. It is therefore plausible that some LAB associated with honey bees (A. mellifera) may be environmentally transmitted, while others are maternally inherited, that is vertically transmitted (McFrederick et al. 2012). Some FLAB species, like Lact. kunkeei and Fructobacillus fructosus, are microbial components in the digestive tract of honey bees that are also found in flowers, and therefore flowers provide bees with both fructose-rich diets and microbes (Endo and Salminen 2013). For instance, Lact. kunkeei is an important bacterium found in the crop of bees, usually called the honey stomach, and it is also present in high proportions in the beebread (Anderson et al. 2014).
Bees collect nectar and pollen from flowers to produce their own food, honey and beebread, both being stored in different cells within the colony. Beebread is a mixture of pollen, nectar and bee salivary secretions stored in the cells like an ensilage. After being stored in the cells, lactic acid fermentation is produced by micro-organism derived from both pollen and bees. Oenococcus, Bifidobacterium and Lactobacillus have been found in the beebread, suggesting that they can be responsible of pollen fermentation (Olofsson and Vásquez 2008; Mattila et al. 2012; Anderson et al. 2013; Kieliszek et al. 2018). This food, preserved by fermentation, constitutes the main protein source for larvae, newly emerged bees, and contains LAB, which, as mentioned earlier, may play a role against honeybee diseases (Anderson et al. 2014). These bacteria could equally come from both flowers (exogenous) and bees (endogenous) because bees add endogenous bacteria to beebread. In fact, the same LAB found in honeybee colonies were also isolated from nectar and pollen (Anderson et al. 2013; Asama et al. 2015).
As mentioned above, honeybees produce honey from nectar by adding their own substances and promoting the evaporation of water from the nectar. Honey is a viscous substance with a high concentration of sugar that represents the main source of carbohydrates and energy for bees. The intrinsic characteristics of honey make it an unfavourable environment for microbial growth. Nevertheless, some LAB have been found in honey such as Lactobacillus acidophilus and it has been hypothesized that some beneficial properties of honey are due to these LAB present in the honey (Aween et al. 2012).
As some LAB associated with honey bees may be also environmentally transmitted, in this review we refer to ‘LAB from the honey bee colony environment’, as those bacteria from flowers, bees and bee products found in bee hives.
Applications of LAB in beekeeping
The application of endogenous LAB in beekeeping has mainly focused on colony health status, pathogen defence, nutrition and modulation of immune response. Budge et al. (2016) analysed the microbiota of different beehives and concluded that Lactobacillus and Leuconostoc were the genera most frequently associated with healthy hives and their strength was measured as the number of bee combs (Budge et al. 2016). On the other hand, it has been reported that the more active the Bifidobacterium community was in bee guts, the lower the activity of pathogen bacteria. This suggests that Bifidobacterium may modulate the immune response or exclude pathogens, providing protection for honey bees against infection (Mattila et al. 2012).
Some endogenous bacteria of bees can have an immune-boosting role. For instance, Bifidobacterium asteroides and Fructobacillus pseudoficulneus isolated from worker honeybee gut significantly upregulated the expression of Apidaecin1, an antimicrobial peptide that plays an important role in humoral immunity of bees. In addition, they showed the downregulation of hexamerin 70b, a larvae and pupae storage protein with a key role in the development towards adult stage (Janashia and Alaux 2016).
Even though there is a wide range of LAB in the hive, most research has focused on 15 species that showed inhibitory effects on bee pathogens (Table 1). In particular, Lact. kunkeei and Lactobacillus johnsonii have been widely studied in this regard. A mixture of Lact. kunkeei strains isolated from the bee midgut has been reported to reduce the number of infected larvae by Paenibacillus larvae, a bacterium that causes American foulbrood, a disease of bee larvae. This mixture of Lact. kunkeei strains likewise had antimicrobial effects against Melissococcus plutonius, a causative pathogen of European foulbrood that also affects bee larvae (Endo and Salminen 2013). Similar results have been reported by Vásquez et al. (2012), who showed that Lact. kunkeei, used with a mixture of 12 LAB, inhibits M. plutonius in vitro and in vivo. In vitro inhibition of both P. larvae and M. plutonius produced by Lactobacillus apis, isolated from the stomach of bees, was also demonstrated (Killer et al. 2014). In vivo studies, showed that the addition of LAB to the food of young honeybee larvae exposed to M. plutonius decreases the number of larvae infected (Vásquez et al. 2012).
On the other hand, it has been reported that Lact. johnsonii CRL1647 and E. faecium SM21 isolated from bee gut inhibit in vitro P. larvae (Audisio et al. 2011). The effect of Lact. johnsonii on Nosemosis, a disease of adult bees caused by the enteric parasite N. ceranae or N. apis, has been also documented (Audisio and Sabaté 2015; Audisio 2017). Moreover, the administration of Lact. johnsonii CRL1647 to bees stimulated the egg laying by the queen, which therefore produced an increase in bee number and a higher honey yield in bee colonies. Apparently, the increase in population levels and the survival of bees during winter are due to the metabolites produced by this bacterial strain (Audisio and Benítez-Ahrendts 2011). The metabolites reported are organic acids such as lactic acid, phenyl-lactic acid and acetic acid and when these metabolites were orally administrated with syrup to honeybees, the intensity of the N. ceranae pathogen was reduced (Maggi et al. 2013).
Different mixtures of Lactobacilli and Bifidobacteria isolated from honeybees have been studied (Table 1). These LAB mixtures reduced the levels of important bee diseases, such as American foulbrood and nosemosis. In bioassays, the number of infected larvae with P. larvae was significantly reduced when LAB were added to the larval food (Forsgren et al. 2009). A mixture of LAB administered with sugar syrup for 13 days was also effective in reducing the infection level of N. ceranae in adult bees (Baffoni et al. 2016).
Potential use of LAB in the food industry
In the food industry, LAB isolated from the bee colony environment become relevant because, as mentioned above, they are part of bee products, namely honey, pollen and royal jelly, that are consumed as food by humans. In many cases, these products are considered functional foods or nutraceuticals because of their numerous beneficial properties, some of which are given by the LAB. Among these, the antimicrobial activity of bee products against human and animal pathogens and foodborne pathogens has been extensively studied.
Antibacterial properties of honey against pathogenic micro-organisms have been demonstrated in many studies (Libonatti et al. 2014; Ramos et al. 2018). However, the antimicrobial effect of different honeys varies and there is controversy about which components account for this effect. Differences between the antimicrobial capacity of several honeys could be explained by the presence of the different LAB strains in honey. Recent studies have reported that LAB from honey produces bioactive compounds, such as proteins and free fatty acids, that contribute to several of the antibacterial and therapeutic properties of honey. For instance, some Lact. acidophilus strains isolated from honey produce antimicrobial compounds that are stable to heat, treatment with proteolytic enzymes and pH adjustments (Aween et al. 2012). In addition, some free fatty acids produced by LAB were detected in freshly stored honey, suggesting that these substances do not disappear with time (Olofsson et al. 2016). These antimicrobial compounds produced by LAB that remain in honey for a long time may enhance the medicinal benefit and value of honey.
Pollen collected by bees from flowers is considered a functional dietary food supplement with bioactive properties (Kroyer and Hegedus 2001). In our laboratory, we evaluated the antimicrobial capacity of pollen extracts against Listeria monocytogenes, Staphylococcus aureus, E. coli and Bacillus cereus (Libonatti et al. 2014). The results showed high inhibition against S. aureus and B. cereus. Isolation of LAB from pollen extracts that had the highest inhibition power was subsequently made and evaluation of in vitro inhibition against the foodborne pathogens E. coli enterophatogenic, S. aureus and Pseudomona aeruginosa was carried out. A suitable inhibition power was observed by Lactobacillus sp., Bifidobacterium sp. and Weisella sp. isolated from pollen and beebread (Table 2) (Libonatti et al. 2015; Vega et al. 2018). We conclude that the antimicrobial activity of pollen could be attributed mainly to these bacteria, which are a component of pollen. The antimicrobial activity of LAB isolated from pollen against foodborne pathogens was also demonstrated by Belhadj et al. (2010) (Table 2). In addition, some LAB isolated from beebread may inhibit fungal strains such as Candida sp., one of the most common causes of human fungal infections worldwide, and Aspergillus niger that produces carcinogenic mycotoxins (Janashia et al. 2018; Kieliszek et al. 2018). LAB species that have shown inhibition are Lact. kunkeei, F. fructosus and F. tropaeoli isolated from breebead (Table 2). Additionally, Lact. kunkeei also inhibited the frequent food-spoiling agent Zygosaccharomyces rouxii (Janashia et al. 2018). The antimicrobial activity of LAB against foodborne pathogens suggests that some LAB species associated with pollen and beebread could be used as a preservative in the food industry. It should be noted that pollen is consumed as a dietary food supplement; consequently, some LAB present in pollen may also influence human health (Selhub et al. 2014).
| Lab strain | Pathogen inhibited |
|---|---|
| Fructobacillus fructosus | Aspergillus niger and Candida sp. (Janashia et al. 2018); Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae (Syed Yaacob et al. 2018) |
| Fructobacillus tropaeoli | A. niger and Candida sp. (Janashia et al. 2018) |
| Enterococcus faecium | Listeria sp. (Ibarguren et al. 2010; Audisio et al. 2011); S. aureus (Belhadj et al. 2010) |
| Enterococcus faecalis | E. coli, Salmonella typhi, S. aureus, Bacillus cereus (Belhadj et al. 2010) |
| Lactobacillus plantarum | |
| Lactococcus lactis | |
| Leuconostoc mesenteroides | |
| Lactobacillus acidophilus | S. aureus, Staphylococcus epidermis, B. subtilis (Aween et al. 2012) |
| Lactobacillus johnsonii | S. aureus, B. cereus, E. coli O157:H7, L. monocytogenes (Audisio et al. 2011) |
| Lactobacillus kunkeei | A. niger and Candida sp. (Janashia et al. 2018); Candida albicans, Citrobacter freundii, Klebsiella aerogenes, Serratia marcescens, Acinetobacter, E. coli, Enterobacter cloacae, S. aureus (Olofsson et al. 2016); E. coli, L. monocytogenes, Yersinia pseudotuberculosis (Uğraş 2017); P. aeruginosa (Olofsson et al. 2016; Berríos et al. 2017; Uğraş, 2017) |
| Weisella sp. | E. coli, S. aureus, P. aeruginosa (Libonatti et al. 2015; Vega et al. 2018) |
| A mixture of Lactobacillus kunkeei, Lactobacillus apinorum, Lactobacillus mellis, Lactobacillus mellifer, Lactobacillus kullabergensis, Lactobacillus kimbladii, Lactobacillus helsingborgensis, Lactobacillus melliventris, Lactobacillus apis, Bifidobacterium coryneforme, Bifidobacterium asteroids and Bifidobacterium sp. | S. aureus, Enterococcus faecalis, E. coli, Enterobacter cloacae, Proteus mirabilis, P. aeruginosa, Fascioloides magna, Staphylococcus pettenkoferi, Streptococcus dysgalactiae, Morganella morganii, Bacteroides fragilis, Klebsiella oxytoca (Butler et al. 2016) |
| Acinetobacter, Enterococcus faecalis, C. albicans, E. coli, Klebsiella aerogenes, K. oxytoca, S. aureus, Citrobacter freundii, Serratia narcescens, Enterobacter cloacae, P. aeruginosa (Olofsson et al. 2016) | |
| S. aureus, Staphylococcus hyicus, Streptococcus dysgalactiae, Staphylococcus chromogenes, Staphylococcus fleurettii, Streptococcus uberis, Streptococcus dysgalactiae, E. coli, K. pneumoniae (Piccart et al. 2016) |
In the animal feed production industry, as mentioned earlier, LAB are also used as inoculants for ensilage of animal food, mainly because LAB reduce the silage pH more rapidly and attain a lower final pH. This rapid drop to a low pH would reduce the activity of enterobacteria, clostridia and bacilli, which can affect silage quality. The use of LAB from honeybee hives is poorly documented in this regard. Recently, the use of Weisella paramesenteroides isolated from beebread has been tested with potential use in fish biological silage with promising results (Libonatti et al. 2018). The addition of W. paramesenteroides to fish waste avoids the growth of spoilage and pathogenic micro-organism, thus being a reliable alternative to ferment fish residues, which are used in animal feed.
Application of LAB in human and animal health
The relationship between LAB from the beehive environment and human and animal health has again been reported. It has been shown that LAB isolated from the bee guts produce bioactive metabolites with an antibacterial effect against pathogens. Specifically, 20 LAB species isolated from honey bee colony environment were studied and showed antibacterial activity against pathogens (Table 2).
Some LAB from the bee crop had antibacterial activity against pathogens isolated from human wounds such as methicillin-resistant S. aureus, P. aeruginosa, vancomycin-resistant Enterococcus and E. coli (Butler et al. 2016; Olofsson et al. 2016). In addition, the antimicrobial spectrum against human foodborne pathogenic bacteria has also been documented. For instance, E. coli O157:H7, S. aureus, B. cereus and L. monocytogenes were all inhibited by the lactic acid produced by Lact. johnsonii CRL1647, isolated from the intestinal tract of honeybee. Additionally, L. monocytogenes was inhibited by Lact. kunkeei isolated from the stomach of bees (Uğraş 2017) and a cell-free supernatant of E. faecium SM21 (Audisio et al. 2011).
Among foodborne pathogens, those that are able to produce biofilms are very important for public health because of their participation in several infectious diseases and their greater resistance to antibiotic therapies when they are residing in the biofilm. In particular, P. aeruginosa is an opportunistic pathogen responsible for several infections in humans which is also able to infect plants and farm animals. It is interesting to note that Lact. kunkeei, which is frequently found in honey stomach, guts, honey, beebread and pollen, can form biofilms in vitro that can inhibit the formation of P. aeruginosa biofilms (Berríos et al. 2017).
In addition to antimicrobial properties, LAB may have probiotic effects due to their contribution to the protection of the host, since they modulate the intestinal microbiota, inhibit the adherence of pathogens to the intestinal mucosa, prevent the translocation and infection of other organs and stimulate the host immune system. For example, Lact. kunkeei has been studied in relation to this trait. The concentration and secretion of IgA, an antibody that plays a crucial role in the immune function of mucous membranes, was increased in human saliva when a heat-killed form of Lact. kunkeei YB38 isolated from honeybee products was administered to humans (1000 mg day−1 for 4 weeks) (Asama et al. 2015). In addition, the same strain decreased the influenza virus infection in BALB/c mice, induced IgA secretion at the infection site and prevented the infiltration of inflammatory immune cells or production of excessive IL-6, resulting in less damage to lung tissues (Asama et al. 2017).
The effect of Lact. kunkeei YB38 on human intestinal microbiota has been also reported. Bacteroides fragilis is part of the normal microbiota of the human colon, but its increase is a risk factor for inflammatory bowel disease and colon cancer. When a heat-killed form of Lact. kunkeei YB38, isolated from honeybee pollen, was administered to patients at a dose of 10 mg day−1, the high levels of Bact. fragilis decreased. As a result, Lact. kunkeei intake may be effective to modulate the intestinal microbiota, improving the intestinal defence and thereby protecting the host against infections. It has been also demonstrated that Lact. kunkeei YB38 increased bowel movement in healthy women with a tendency for constipation. A possible explanation of this fact is that Lact. kunkeei raises the levels of acetic acid, which stimulates the motility of the large bowel (Asama et al. 2016).
The effects of LAB in relation to animal health have been poorly studied, and most of the limited research has focused on the use of LAB mixture with honey to heal animal wounds. Olofsson et al. (2016) showed that heather honey formulation made with viable LAB isolated from honey stomach promoted horse wound healing, showing a reduction in the wound size and an effective healing. They concluded that the LAB and honey together have additional benefits for wound healing than just honey on its own. The fact that these LAB have showed in vitro antimicrobial effects against wound pathogens may explain the greater effectiveness of the mixture in healing wounds. In other studies, the same mixture of lactobacilli and bifidobacteria from a honey crop has been shown to inhibit mastitis pathogens (Table 2) (Piccart et al. 2016). The honey stomach microbiota could therefore be a future promising alternative for the treatment and prevention of bovine mastitis.
Concluding remarks
Modern research highlights the potential value of using LAB as probiotics in animal production systems in which food is obtained for human consumption. Use of LAB is an important tool, not only to improve animal health and reduce the incidence of diseases but also to ensure food quality. The new challenge of the food industry focuses on producing antibiotic-free foods, and the evidences suggest that the use of LAB in production systems could represent one of the most important tools in pursuit of this achievement.
The beehive is a wide reservoir of LAB, as at least 43 species have been identified. The microbiota of bee colonies includes LAB from a broad range of environmental sources and this microbiota, essential for the proper functioning of the bee colony, could be a source of bacteria to be used with different purposes. Although there is evidence that LAB enhance bee health and could be used in beekeeping, their use in the food production system is not widespread as commercial products have not yet been developed.
Evidence of the antimicrobial effect of LAB from honey bee colony against foodborne pathogens suggests a potential use of these LAB as bio-preservatives in the food industry. We supply documentary evidence that 20 LAB strains isolated from beehives inhibited at least 28 species of human and animal pathogens, some of which are resistant to antibiotics. Nevertheless, more research is necessary to evaluate the effect of LAB against these pathogens in vivo.
As described in this review, some bee products that include LAB are consumed as foods, and therefore there is ample justification for hypothesizing a relationship between the potentially beneficial foods derived from the hive and their effect on human health.
Acknowledgements
This study was supported by the Science, Art and Technology Office (SECAT) of the National University of Buenos Aires Province Centre. The authors thank PhD Peter Purslow for his help with English translation.
Conflict of Interest
The authors have no conflict of interests.
