Bees’ natural lactic acid bacteria have become bee medicine

Research discovery

In 2005 in Sweden, Dr Tobias Olofsson and his beekeeper grandfather Tage Kimblad and fellow researcher Alejandra Vásquez made a discovery: honey bees carry the largest collection of lactic acid bacteria (LAB) found in their honey stomach (1–3), and they use these LAB in honey production. There are nine different types of lactobacilli and four kinds of bifidobacteria. Lactic acid bacteria are considered to be beneficial and completely harmless.

We started our own research group at Lund University, Lund, Sweden, in 2007 based on this discovery, and Lund University helped us to start our company, ConCellae AB. Today, both Lund University Innovation System AB and SLU (Swedish University of Agricultural Sciences) Holding AB are partners in the company.

Research backgroundOver the years, our research has shown that these bacteria are always present in the honey stomach of honeybees worldwide (4). Nectar from flowers is transported in the honey stomach (Figure 1) for later conversion to honey within the hive. The purpose of the LAB is to protect the nectar from destruction by other microorganisms (bacteria, yeast and mould) while it is being converted into honey. The LAB live in the nectar during this conversion in counts of up to 100 million organisms per gram, but they die as the honey matures. Lactic acid bacteria are also involved in the fermentation and preservation of the beebread made from bee pollen (5).

Figure 1: The sole purpose of the honey stomach (a) is to transport nectar and sometimes water. The bee can swallow water and nectar for consumption, but an organ called the proventriculus (b) prevents the intestinal

contents from entering the honey stomach and destroying the nectar.

Lactic acid bacteria require nutrients in order to grow and to combat other microorganisms that threaten them and their nutrients. The LAB and bees share the same nutritional source, nectar and pollen. Nutrition is also important for both organisms when bees cluster during winter (forthcoming publications). Laboratory testing of 49 different sugars showed us that the various LAB grow either very poorly or not at all on table sugar (Figure 2). However, they all grow very well on fructose and glucose, the dominant sugars contained in honey. They also need other nutrients found naturally in honey and pollen.

Many beekeepers winter their bees using table sugar. We performed a study of four bee colonies, two of which wintered on their own honey and two on table sugar. The purpose was to investigate the LAB count in the honey stomach of bees during winter. One side effect was that the bees that wintered on table sugar barely had any food left when samples were taken in late January; they were aggressive and few in number. Those that wintered on honey were calm, present in larger numbers and had lots of honey left over. The honey crops in bees that wintered on table sugar

contained considerably fewer LAB, 1000 bacteria per honey stomach compared with 100,000 in the honey stomach of bees that wintered on honey (forthcoming publication).

Figure 2: Sugar fermentation test for one of the lactic acid bacteria (LAB) showing that it can only grow on glucose and fructose (yellow), but not on table sugar or any of the other types of sugars, even though other

necessary nutrients are included in the wells with nutrient solution.

Microorganisms in nature are in constant conflict over food and space, succeeding over other organisms through numerical superiority and dangerous substances (Figure 3) that kill their competitors. Lactic acid bacteria too defend themselves when they sense the presence of other microorganisms. Substances on the cell walls of their competitors stimulate growth of antimicrobial substances that the LAB use in defence. A large number of ‘hostile’ microorganisms gain entry into the hive through nectar and pollen, but even though most micro-organisms in stored honey and bee bread are dead, or dormant as spores, such as the bacteria that cause American foulbrood disease, they still stimulate the LAB to constantly produce antimicrobial substances. Consequently bacterial spores remain dormant and invasive microorganisms are countered. If the LAB count is low for any reason, such as poor or inadequate food or treatment of bees with various toxins and antibiotics, the bees’ defences are considerably weakened. Table sugar contains very few or no living or dead micro-organisms compared with honey; consequently, the bees’ defence mechanism is not active during periods of feeding on table sugar, and the bacteria also grow poorly during such times.

Figure 3: A gel with nutrients seeded with Paenibacillus larvae (yellow), the bacteria that cause American foulbrood disease. A transparent zone containing bactericidal substances can be seen surrounding the lactic acid

bacteria (LAB) (white) in the middle as a manifestation that the bacteria outside are being counteracted.

Development of bee medicineAided by our technical equipment, as well as earlier research and knowledge, we have now developed an excellent product for bees and their LAB. We strive to mimic nectar insofar as possible to attract the bees, while maintaining optimal nutrient composition for the bacteria. We use a mixture composed of one-third honey and two-thirds table sugar, as well as bee pollen. The mixture is sterilized to avoid spreading bee diseases. The bee medicine ‘SymBeeotic’ (Figure 4) should be given shortly before and after the bees cluster for winter. The purpose of the active LAB is to prevent disease among bees and their larvae, or hopefully cure them, while providing a small nutritional supplement.

Figure 4: SymBeeotic.

Each bag contains 50 g honey, 5 g bee pollen, and LAB. The bag is equivalent to one course of treatment, sufficient to feed one colony once daily for 3 days. The beekeeper mixes the contents of the bag with water in a container such as a honey jar and allows the jar to stand with its lid on for 2 days at room temperature, or preferably warmer (maximum 40� C). This allows the bacteria to flourish and to become active for 5 days. On day 3, just before giving the bees the liquid for the first time, 100 g of table sugar is added and blended into the mixture. Next, 60 ml of the liquid is

administered with a syringe directly onto the bees, evenly distributed between all the upper frames in the hive (Figure 5). The latter process is repeated on days 4 and 5. In Sweden, this process is performed during the late autumn (in October) and again in February.

Figure 5: SymBeeotic is being administered by syringe onto the bees between the frames in the top box of the hive.

Previous results and ongoing studiesOur tests on diseases affecting bees and their larvae have to date demonstrated in the laboratory that LAB are effective against both American and European foulbrood disease (4, 6).

In ongoing international collaborations we have tested SymBeeotic on colonies that were heavily infected with Nosema apis and N. ceranae. The colonies were given either SymBeeotic or placebo and the results were highly encouraging. The spore counts among almost all colonies that received SymBeeotic were sharply reduced after 2–3 months (forthcoming publication).

These findings are as yet preliminary. We are continuing to test SymBeeotic in the laboratory and in field studies against Nosema, American foulbrood, the parasitic mite Varroa destructor and deformed wing virus (DWV) virus, which are spread to bees by the mites.

SymBeeotic is at its launch in conjunction with Apimondia Kiev, Ukraine, mainly available online through our subsidiary www.apicellae.se. In the future it is planned to make the product available in beekeeping stores and through distributors.

References

1. Olofsson TC, Vásquez A (2008) Detection and Identification of a Novel Lactic Acid Bacterial Flora Within the Honey Stomach of the Honeybee Apis mellifera. Current Microbiology 57: 356–363.

2. Olofsson TC, Vásquez A, Sammataro D, Macharia J (2011) A scientific note on the lactic acid bacterial flora within the honeybee subspecies; Apis mellifera (Buckfast), A. m. scutellata, A. m. mellifera, and A. m. monticola. Apidologie. pp 1–4.

3. Vásquez A, Olofsson TC, Sammataro D (2009) A scientific note on the lactic acid bacterial flora in honeybees in the USA – a comparison with bees from Sweden. Apidologie 40: 26–28.

4. Vasquez A, Forsgren E, Fries I, Paxton RJ, Flaberg E, Olofsson TC (2012) Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honey Bees. PLoS ONE 2012, 7.

5. Vásquez A, Olofsson TC (2009) The lactic acid bacteria involved in the production of bee pollen and bee bread. Journal of Apicultural Research 48: 189–195.

6. Forsgren E, Olofsson TC, Vásquez A, Fries I (2010) Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae. Apidologie 41: 99–108.

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