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HEALTHY BEES™:

A UNIQUE SOLUTION TO PROVIDING

BALANCED NUTRITION

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General Background for Healthy Bees™

All animals require a dietary source of essential amino acids (EAAs) which are used for growth, somatic maintenance, and reproduction. EAAs are obtained by consuming the protein found in animals or plants and are in greatest demand during periods of growth early in life (Behmer 2009; Tigreros 2013). In contrast to juvenile animals, adults mainly require amino acids for basic somatic functions (e.g. production of enzymes, peptide or amine signaling, tissue repair, immune function) or reproduction, and their needs for EAAs slowly decline with age (Millward et al. 1997; van de Rest et al. 2013). Reproduction (in the form of allocating resources to eggs or offspring by females (O’Brien et al. 2002)) or the donation of nuptial gifts and the production of sperm by males (Voigt et al. 2008) also places demands on the acquisition of amino acids in adult diets, which is often manifested as a trade-off between lifespan and protein/amino acid ingestion (Fanson et al. 2012; Grandison et al. 2009; Maklakov et al. 2008).

According to Professor de Groot AP. “Protein and amino acid requirements of the honey bee (Apis mellifera L.) Phys Comp Oec. 1953; 3: 197–285”, honey bees need at least all of the ten EAAs listed below:

methionine

tryptophan

arginine

lysine

histidine

phenylalanine

isoleucine

threonine

leucine

valine

Adult eusocial insects such as bees live in colonies of closely-related, largely sterile adults that engage in a division of labor that includes caring for the queen and her brood. In honey bees, behavioral castes correlate with age within the colony. After eclosion, adult worker bees perform within-colony activities such as cleaning, food storage, rearing larvae, and attending to the queen (Winston 1987). These bees continue to perform within-hive behavioral tasks as a function of their exposure to the queen’s mandibular pheromone (QMP); bees near the queen remain more ‘nurse-like’, whereas bees exposed to less of the queen’s pheromones become foragers (Pankiw et al. 1998). Honey bees are unique, even among social

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insects, because nurses provision larvae, other workers, and the queen with glandular secretions as food – royal jelly – which is the main source of protein that larvae receive for the first three instars after hatching (Winston 1987). Because most adult bees are sterile, their nutritional requirements for amino acids should be less than those of queens or drones. However, the production of royal jelly is likely to place metabolic demands on young bees (e.g. nurses) for dietary amino acids (Crailsheim 1990).

Generally-speaking, nutritional requirements for EAAs are greater for young bees, but shift as they age and become foragers.

SOY PROTEIN is an incomplete source of amino acids (i.e. soy’s level of tryptophan is extremely poor: max 0.2%, methionine and isoleucine are both below 2.8%):

SOY – AMINO ACID PROFILE

Many tests have proven that without a complete amino acid profile, proteins are not metabolized properly. Even worse, most (if not all) soy used in bee patties is GMO. GMO soy has been proven to have a negative effect on honey bee gut health. Poor gut health leads to infection, disease, and shorter life spans. SPIRULINA – found in our proprietary and patented Healthy Bees™ formula – is the highest source of vegetal proteins and amino acids (see below details: a total of 18 amino acids) plus a very well-balanced profile of vitamins, particularly B-vitamins (thiamin and riboflavin, 207% and 306% DV, respectively), and dietary minerals including iron (219% DV) and manganese (90% DV). The rate of iron accumulation in honey bees is directly related to the iron levels in their diet, and it can be obtained from pollen or honey (whenever available). In adults, the iron content of the fat body reached a maximum level (2.4 +/- 0.15 micrograms mg-1 tissue), regardless of the amount of iron available for ingestion. Maximal iron levels are reached at the time when honeybee workers commence foraging behavior, suggesting that iron granules may play a role in orientation. Alternatively, accumulation of iron in granules may be a method of maintaining iron homeostasis.

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SPIRULINA – AMINO ACID PROFILE

Let’s take a more in-depth look at the superfood Spirulina found in Healthy Bees™:

Nutritional Value per 100 g (3.5 oz) for Spirulina

Energy 1,213 kJ (290 kcal)

Carbohydrates 23.9 g

Sugars 3.1 g

Dietary fiber 3.6 g

Fat 7.72 g

Saturated 2.65 g

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Monounsaturated 0.675 g

Polyunsaturated 2.08 g

Protein 57.47 g

Tryptophan 0.929 g

Threonine 2.97 g

Isoleucine 3.209 g

Leucine 4.947 g

Lysine 3.025 g

Methionine 1.149 g

Cystine 0.662 g

Phenylalanine 2.777 g

Tyrosine 2.584 g

Valine 3.512 g

Arginine 4.147 g

Histidine 1.085 g

Alanine 4.515 g

Aspartic acid 5.793 g

Glutamic acid 8.386 g

Glycine 3.099 g

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Proline 2.382 g

Serine 2.998 g

Vitamins Quantity% DV†

Vitamin A /equiv. beta-Carotene

lutein zeaxanthin

4%/29 μg

3%342 μg

0 μg

Thiamine (B1) 207%/2.38 mg

Riboflavin (B2) 306%/3.67 mg

Niacin (B3) 85%/12.82 mg

Pantothenic acid (B5) 70%/3.48 mg

Vitamin B6 28%/0.364 mg

Folate (B9) 24%/94 μg

Vitamin B12 0%/0 μg

Choline 13%/66 mg

Vitamin C 12%/10.1 mg

Vitamin D 0%/0 IU

Vitamin E 33%/5 mg

Vitamin K 24%/25.5 μg

Minerals Quantity% DV†

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Calcium 12% /120 mg

Iron 219% /28.5 mg

Magnesium 55% /195 mg

Manganese 90% /1.9 mg

Phosphorus 17% /118 mg

Potassium 29% /1363 mg

Sodium 70% /1048 mg

Zinc 21% /2 mg

Spirulina Summary: Spirulina is a very rich source of protein, and as shown in the charts above and below,

contains all 10 essential amino acids (the amino acids that cannot be synthesized on their own by bees) as

well as 8 unessential amino acids, all of which are necessary for bees to metabolize protein properly.

Amino Acid % Spirulina % Bees (De Groot)

Tryptophan 1.6 1

Methionine 2 1.5

Histidine 1.9 1.5

Phenylanine 4.8 2.5

Lysine 5.2 3

Threonin 5.1 3

Arginine 7.1 3

Isoleucine 5.5 4

Valine 6 4

Leucine 9 4.5

Spirulina contains a myriad of vitamins and minerals such as B1, B2, B3, B5, B6, B9, C, E, K. The B-complex

vitamins are especially important for brood rearing.

Spirulina also contains powerful antioxidants such as phycocyanin, tocopherols, and beta carotene.

Spirulina may also contain sterol 24 methylene cholesterol which helps with hormones and signaling.

Spirulina is a bacteria which means its cell walls break down more easily in the gut compared to animal,

fungal, or plant cells. This means that the nutrients in Spirulina are more bioavailable to the bees.

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Environmentally speaking, Spirulina is a clean food source that has a low risk of contamination. It also

doesn’t promote the spraying of chemicals that kills bees like GMO soy does (GMO soy has been linked to

RoundUp use, as the GMOs found in soy exist to increase the soy’s resistance to RoundUp exposure).

A LOOK AT CARBOHYDRATES: The carbohydrate and lipid metabolism of bees is also affected by QMP (Fischer and Grozinger 2008). QMP maintains the abdominal fat of young bees and increases their resistance to starvation (Fischer and Grozinger 2008). Scientific data indicates that this resistance to starvation may be due to the fact that the presence of QMP maintains their physiological state such that they require far less carbohydrates. Thermal stress is another factor that could influence the within-hive bees’ demand for carbohydrates. In the presence of brood, young bees keep the brood warm using their flight muscles to produce heat when the temperatures outside of the colony drop (Simpson 1961; Fahrenholz et al. 1992; Stabentheiner et al. 2010).

SUGAR contains none of the micronutrients bees need to thrive nor the amino EAAs they require. Iron, B-

vitamins, antioxidants, and all the key building blocks of a healthy diet are nowhere to be found in sugar.

The scientific evidence available is overwhelming and clearly shows that poor nutrition has a direct effect

on honey bee health. This lack of nutrition forces beekeepers to give their bees antibiotics, which ultimately

further weakens honey bee gut health.

Healthy Bees™, on the other hand, contains DEXTROSE – a far more efficient carbohydrate that translates

right into ATP (energy). Other sugars like sucrose and fructose need to go through extra steps in order to

be metabolized, while the latter is also known to form HMF (a toxic chemical) under acidic/hot conditions.

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Healthy Bees™ also contains 10 carefully selected essential oils that contribute to the nutritional diet of

honey bees with a balanced set of active principles:

Essential oil Main active principles/components and their specific activities of the

selected compounds

Essential oil #1

It contains mucopolysaccharide acemannan (immunostimulant), cinnamic

acid (germicide, fungicide, drainage, analgesic), crisofanic acid (antifungal),

emodin (bactericidal), aloe-emodin (antivirus), salicylic acid (anti-

inflammatory), isobabaloina (analgesic), socaloine, capaloine, barbaloin

(strong antibacterials)

Essential oil #2 It contains geraniol, borneol, citronellol, linalool, termineol, limonene,

phellandrene, pinene, methyl-eugenol (all highly active antioxidants)

Essential oil #3 It contains thymol (very active against varroa), carvacrol, borneol, cineol,

metnone and pinene, geraniol, linlol thujanol-4 or alpha-terpineol.

Essential oil #4 It contains thymol (very active against varroa), carvacrol, borneol, cineol,

metnone and pinene, geraniol, linlol thujanol-4 or alpha-terpineol.

Essential oil #5 It contains carvacrol, thymol, origanol, pinene, sabinene, termineolo,

terpinene, terpineol, camphene. It contains also tannins, flavonoids

(apigenol, borneol), rosmarinic acid, caffeic acid- very important to contrast

neonicotenoids side effects.

Essential oil #6 It contains borneol, camphene, cymol, dipentene, eugenol, geraniol, linalol,

pinene, sapol, terpineol, myristicin, safrol. It is rich in triterpens (canfene,

pinene), monoterpenic alcohols (terpinol, borneol) and safrol, eugenol,

miristicine. High antioxidant activity was found in monoterpenoid extracts

including terpinene-4-ol (3), alpha-terpineol (4), and 4-allyl-2,6-

dimethoxyphenol (12) with a particularly high antioxidant activity and

contrasting effect of the neonicotinoids formulated pesticides. Three

antifungal lignans were isolated and identified as erythro-austrobailignan-6

(EA6), meso-dihydroguaiaretic acid (MDA), and nectandrin-B (NB).

Essential oil #7 It contains crocin (color), picrocrocin (taste), and safranal (fragrance):

fundamental for increasing the strength of bee immune defense systems

Essential oil #8 It contains Origanol, carvacrol, and thymol to combat varroa.

Essential oil #9 It contains thymol (44.599%), 1,8-cineole (23.613%), α-phellandrene

(4.815%) and β-cymene (4.019%) and other monoterpenes and

sesquiterpene – all very important to attract bees to eat the solution

Essential oil #10 It contains betalaine e betaine that attract bees and provides antibacterial

agents

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Scientific Tests for Healthy Bees™

Healthy Bees, LLC conducted several (many 6+ months) independent researches in Italy, Turkey, Argentina, Brazil, USA, and New Zealand. All the tests were performed against a control group; both the groups were fed with water plus sugar (or candy) diluted in water with the addition of our product, Healthy Bees™. In some of the studies, Healthy Bees™ was tested against competing products. Sugar, candy, and their respective quantities were similar to quantities normally used by beekeepers in their daily routine. The bees fed with Healthy Bees™ (in the tests performed during the last 5 years) clearly showed higher vitality, higher activity, stronger health, and better overall aspects. Our USA tests at the USDA about antioxidant and anti-virus/herbicide efficacy along with the gene expressions tests at University of Maryland provided irrefutable evidence to the unique value of Healthy Bees™. The research performed at the University of Maryland was conducted to evaluate the differences in gene expression patterns on fat bodies of honey bees fed with different nutritional (Healthy Bees™) conditions. The primary purpose of this research was to study fat metabolism and evaluate gene expression differences in honey bees fed with a natural food source (honey) and two ‘artificial’ food sources, namely high fructose corn syrup (HFCS) and Healthy Bees™. Results showed that several genes were differently regulated once fed Healthy Bees™, suggesting that Healthy Bees™ may increase a bee’s ability to detoxify toxins as well as fight infections. The experimental group developed higher concentrations of fat bodies when compared to the control group. This suggests an increase in the efficiency of fat body development under the supplementary diet. The cDNA analyses (obtained from the RNA extracted from both groups) indicated which genes were expressed along with the gene expression variability in the two compared groups. A total of 67 differentially expressed genes (DEGs) were found in the data set. The wide variety of genes observed indicates that diet has a global effect on honey bee metabolism. 20 DEGs showed an up-regulation in the presence of Healthy Bees™ (including two important immune genes: one in the humoral defense pathway, and another in the cellular immune defense - see picture below). The up-regulation of these immune defense genes is a clear sign of increased immune system strength. 47 DEGs showed down-regulation in presence of Healthy Bees™ (including two P450 detoxification genes), which is also a clear indication of improved health. It is clear that Healthy Bees™ significantly improved honey bee metabolism on many different levels.

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Table 1. Differentially expressed genes (DEGs) on honey bees under Healthy Bees™ diet. Values expressed in Fragments Per

Kilobase of transcript per Million mapped reads (FPKM).

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In the USDA studies, for the first time, a nutritional system (Healthy Bees™) was able to limit the detrimental

effects of a pesticide/herbicide – a huge advance in improving honey bee health in the field. After

demonstrating that the immune genes showed significant improvement under the effects of Healthy

Bees™, it was also demonstrated that it enhanced the behavior of the detoxification system. Bees activate

their detoxification systems to fight exogenous components such as pesticides. The side effect is that this

activation generates molecules (Reactive Oxygen Species (ROX)) that can potentially damage honey bee

tissues. Under the Healthy Bees™ effect, bees expressed fewer of the many genes responsible for the

detoxification system, a strong indication that Healthy Bees™ was helping to detoxify the bees’ systems.

Data on the chart below shows that Healthy Bees™ contains not only 17 - 38 times more antioxidant power

than other products for bees on the market, but it also shows that Healthy Bees™ is stronger than vitamins

B6, B12 and beta-carotene combined. No other product on the market has shown such standards for

antioxidative capacity as Healthy Bees™. Dr. Steven Cook at the United States Department of Agriculture

(USDA) has confirmed the unprecedented antioxidant power of Healthy Bees™. Antioxidants are the key

factor in controlling ROX and stopping the damage caused by oxidative stress.

CHART: Antioxidant capacity (mean ± se mM uric acid equivalents) of lipophilic (dark bars) and hydrophilic (light bars) test substances. Substances are B12 = Vitamin B12; B6 = Vitamin B6; BC = Beta Carotene, BHT = Butylated Hydroxytoluene; BVP = Healthy Bees™; BVPc = Healthy Bees™ in 2M sucrose; CHO = 2M sucrose; HBHc = Honey Bee Healthy® in 2M sucrose; HBHw = Honey Bee Healthy® in molecular grade water; VC = Sodium Ascorbate; VE = Vitamin E (mixed tocopherols).

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Dr. Steven Cook at the USDA: Bee Research Laboratory performed a cage study exposing bees fed with

Healthy Bees™ to Paraquat (a strong herbicide) and, after a week, measured the levels of reactive oxygen

species (ROS). ROS are the molecules that can potentially cause tissue damage to the bees, making them

weak to the point of death. Healthy Bees™ could impair the amount of ROS formed by the herbicide,

demonstrating for the first time that a nutritional system can lower the detrimental effects of pesticides.

Tests revealed a significant reduction in lipid peroxide levels (inversely represented in the figure below) in honey bees from both the CHO and CHO+P treatments with the addition of Healthy Bees™. Reduced damage from oxidative stress in honey bees from CHO Treatment with the addition of Healthy Bees™ was shown, despite somewhat elevated ROS levels in tissues of the same bees (Figure 2A), suggesting that the metabolism of Healthy Bees™ may induce some oxidative stress in honey bees, but effectively lowers overall cellular damage from ROS produced. The bolstering effect of Healthy Bees™ to total antioxidant capacity of honey bee extracts in the presence of Paraquat is responsible for the reduced damage in tissues of honey bees from the CHO + P + Healthy Bees™ treatment. No effect on lipid peroxide levels was observed in honey bees from CHO + IM Treatment with the addition of Healthy Bees™, again possibly due to differences in mode of action of Imidacloprid and/or its metabolism in exposed honey bees.

CHART: Mean (± standard error) levels of reactive oxygen species (ROS) (A) and lipid peroxides (B) in tissue homogenates of honey bee samples that had fed for one week on 2M sucrose solution (CHO), CHO with the addition of two exogenous sources of chemical stress, Imidacloprid (CHO+IM) or Paraquat (CHO+P), and the above treatments with the addition of BVP (BVP = Healthy Bees™): (CHO+BVP, CHO+IM+BVP, CHO+P+BVP). Horizontal lines with asterisks indicate significant differences from student’s post hoc tests (* + P < 0.05, ** P <0.01).

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To further confirm the results, a commercial fluorometric kit (Cell BioLabs) was used to measure the level of reactive oxygen species in honey bee tissues after one week in cages provisioned with a pollen patty (BeePro®), and provided 1—2 molar sucrose solution; 2—2M sucrose containing 20 ug/ml Paraquat; and 3—2M sucrose containing 20ug/mL Paraquat and 35ug/mL Healthy Bees™.

2M Sucrose (CHO) CHO + Paraquat CHO + Paraquat

(@ 20 ug/mL) + Healthy Bees™ (@ 35 ug/mL)

CHART: Mean ± s.e.m. levels of reactive oxygen species (ROS) in micromolecular hydrogen peroxide equivalents,

measured using commercial fluorometric assay kit (Cell BioLabs). The addition of Healthy Bees™ reduced the level of ROS in honey bees exposed to the toxin Paraquat.

A FINAL NOTE: You can safely use Healthy Bees™ year-round (depending on location/seasons) since NO

residuals or contaminants have been found whatsoever in the honey, wax, or royal jelly. Low forage is

unpredictable – ensure your bees have access to balance nutrition all year long in order to maintain their

health.

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Thank you for taking the time to read some of our studies!

Should you have any questions, feel free to reach out to us at any time.

PHONE: (786) 524-9200

EMAIL: info@healthybeesllc.com

WEBSITE: www.healthybeesllc.com

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• Schmitzova J, Klaudiny J, Albert S, Schroder W, Schreckengost W, Hanes J, Judova J, Simuth J. A family of major royal jelly proteins of the honeybee Apis mellifera L. Cell Mol Life Sci. 1998;54(9):1020–1030. doi: 10.1007/s000180050229. [PubMed] [CrossRef]

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