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6.1 Introduction

Royal jelly is secreted by the hypopharyngeal gland (sometimes called the brood food gland) of young worker (nurse) bees, to feed young larvae and the adult queen bee. Royal jelly is always fed directly to the queen or the larvae as it is secreted; it is not stored. This is why it has not been a traditional beekeeping product. The only situation in which harvesting becomes feasible is during queen rearing, when the larvae destined to become queen bees are supplied with an over-abundance of royal jelly. The queen larvae cannot consume the food as fast as it is provided and royal jelly accumulates in the queen cells (see Figure 6.1). The exact definition of commercially available royal jelly is therefore related to the method of production: it is the food intended for queen bee larvae that are four to five days old.

The differentiation between queen and worker bees is related to feeding during the larval stages. Indeed, all female eggs can produce a queen bee, but this occurs only when, during the whole development of the larvae and particularly the first four days, they are cared for and fed "like a queen". Queen rearing, regulated by complex mechanisms within the hive, induces in a young larva a series of hormonal and biochemical actions and reactions that make it develop into a queen bee. A queen bee differs from a worker bee in various ways:

in its morphology: the queen develops reproductive organs while the worker bee develops organs related to its work such as pollen baskets, stronger mandibles, brood food glands and wax glands.

in its development period: on average the queen develops in 15.5 days while worker bees require 21 days.

in its life span: the queen lives for several years as compared to a few months for the worker bee,

and its behaviour: the queen lays up to several thousand eggs a day while workers lay eggs only occasionally. Unlike workers, the queen never participates in any common hive activities.

a)	b)
Figure 6.1: a) A 3-day old queen larva floating in royal jelly. The cell is almost ready for harvesting. b) A 5-day old queen larva in a newly sealed cell just before pupation. Not much royal jelly is left.

It is mainly the spectacular fertility and long life-span of the queen, exclusively fed on royal jelly, which have suggestively led people to believe that royal jelly produces similar effects in humans. In the early 1950's, articles began to appear, particularly in the French beekeeping press, in praise of the virtues of royal jelly, referring to research conducted in several hospitals. Chauvin (1968) however, was unable to find the source of such information and therefore considered it unfounded.

The myth of royal jelly started with an amazing biological phenomenon on the one hand and commercial speculation on the other, which, on the basis of initial results obtained by entomologists and physiologists, exploited the suggestibility and imagination of consumers willing to be seduced by the fascination of this rare and unknown product was exploited. In fact, royal jelly was so rare and so little known that it was impossible to verify its actual presence in many products claiming its content.

In the years immediately following its first marketing, royal jelly quickly became widely known and consumed and the increasing demand motivated experts to refine production techniques and led more and more beekeepers to specialize in this activity. At the same time, research on quality control of the commercial product and identification of its biological and clinical properties found growing support.

Consumption of royal jelly has been growing ever since, even without its benefit to human health having ever been scientifically confirmed. The Western medical establishment has always been wary of the effects claimed for this product and in most cases refuses to consider it, largely because of the way royal jelly was initially promoted. In spite of a vast number of publications praising its virtues and the apparently abundant bibliography, there is still a serious lack of scientific data on the clinical effects of royal jelly.

6.2 Physical characteristics of royal jelly

Royal jelly is a homogeneous substance with the consistency of a fairly fluid paste. It is whitish in colour with yellow or beige tinges, has a pungent phenolic odour and a characteristic sour flavour. It has a density of approximately 1.1 g/cm³ (Lercker et al., 1992) and is partially soluble in water. Aqueous solutions clarify during basification with soda.

Viscosity varies according to water content and age - it slowly becomes more viscous when stored at room temperature or in a refrigerator at 5⁰C. The increased viscosity appears to be related to an increase in water insoluble nitrogenous compounds, together with a reduction in soluble nitrogen and free amino acids (Takenaka et al., 1986). These changes are apparently due to continued enzymatic activities and interaction between the lipid and protein fractions. If sucrose is added, royal jelly becomes more fluid (Sasaki et al., 1987). Such changes in viscosity have also been related to the phenomena which regulate caste differentiation in a bee colony (see also 6.4.1).

Certain debris in royal jelly, is a sign of purity as, for example, the ever present fragments of laarval skin (see also 6.8). Wax fragments too, are encountered more or less regularly, but their presence is largely dependent on the collection method. Stored royal jelly often develops small granules due to precipitation of components.

6.3 The composition of royal jelly

Numerous chemical analyses of royal jelly have been published over the years. Only recently though, have highly refined technologies given detailed analyses of the unusual composition and complexity of this somewhat acidic substance (pH 3.6 to 4.2).

The principal constituents of royal jelly are water, protein, sugars, lipids and mineral salts. Although they occur with notable variations (Table 6.1) the composition of royal jelly remains relatively constant when comparing different colonies, bee races and time.

Water makes up about two thirds of fresh royal jelly, but by dry weight, proteins and sugars are by far the largest fractions. Of the nitrogenous substances, proteins average 73.9% and of the six major proteins (Otani et al., 1985) four are glycoproteins (Takenaka, 1987). Free amino acids average 2.3% and peptides 0.16% (Takenaka, 1984) of the nitrogenous substances. All amino acids essential for humans are present and a total of 29 amino acids and derivatives have been identified, the most important being aspartic acid and glutamic acid (Howe et al., 1985). The free amino acids are proline and lysine (Takenaka, 1984 and 1987). A number of enzymes are also present including glucose oxidase (Nye et al., 1973) phosphatase and cholinesterase (Ammon and Zoch, 1957). An insulin-like substance has been identified by Kramer et al. (1977 and 1982).

The sugars consist mostly of fructose and glucose in relatively constant proportions similar to those in honey. Fructose is prevalent. In many cases fructose and glucose together account for 90% of the total sugars. The sucrose content varies considerably from one sample to another. Other sugars present in much lower quantities are maltose, trehalose, melibiose, ribose and erlose (Lercker et al., 1984, 1986 and 1992).

The lipid content is a unique and from many points of view, a very interesting feature of royal jelly. The lipid fraction consists to 80-90% (by dry weight) of free fatty acids with unusual and uncommon structures. They are mostly short chain (8 to 10 carbon atoms) hydroxy fatty acids or dicarboxylic acids, in contrast to the fatty acids with 14 to 20 carbon atoms which are commonly found in animal and plant material. These fatty acids are responsible for most of the recorded biological properties of royal jelly (Schmidt and Buchmann, 1992). The principal acid is 10-hydroxy-2-decanoic acid, followed by its saturated equivalent, lO-hydroxydecanoic acid. In addition to the free fatty acids, the lipid fraction contains some neutral lipids, sterols (including cholesterol) and an unsaponifiable fraction of hydrocarbons similar to beeswax extracts (Lercker et al., 1981, 1982, 1984 and 1992).

The total ash content of royal jelly is about 1 % of fresh weight or 2 to 3 % of dry weight. The major mineral salts are, in descending order: K, Ca, Na, Zn, Fe, Cu and Mn, with a strong prevalence of potassium (Benfenati et al., 1986).

The vitamin content has been the object of numerous studies, from the moment when the first research (Aeppler, 1922) showed that royal jelly is extremely rich in vitamins. Table 6.2 indicates the results obtained by Vecchi et al., (1988) with regard to water-soluble vitamins. Other authors report averages close to the minimum values of Table 6.2 (Schmidt and Buchmann, 1992). Only traces of vitamin C can be found.

As far as the fat-soluble vitamins are concerned, it was initially thought that, given the enormous fertility of the queen bee, royal jelly would contain vitamin E. But tests have shown that it does not. Vitamins A, D and K are also absent (Melampy and Jones, 1939).

During the first studies, much emphasis was placed on the search for sex hormones in royal jelly. The first positive tests were later proven wrong. Melampy and Stanley (1940) showed no gonadotropic effects on female rats and Johansson and Johansson (1958) clearly demonstrated the absence of any human sex hormones. Recently though, with much more sensitive radio-immunological methods, testosterone has been identified in extremely small quantities: 0.012 ~g/g fresh weight (Vittek and Slomiany, 1984). In comparison, a human male produces daily 250,000 to 1 million times the amount present in one gram of fresh royal jelly (Schmidt and Buchmann, 1992). No biological effect has been demonstrated for such small amounts.

Numerous minor compounds, belonging to diverse chemical categories, have been identified in royal jelly. Among these are two heterocyclic substances, biopterine and neopterine at 25 and 5 ijg/g of fresh weight respectively. These compounds are found in the food of worker bee larvae too, but at about one tenth of these concentration (Rembold, 1965). Other substances identified include several nucleotides as free bases (adenosine, uridine, guanosine, iridin and cytidine) the phosphates AMP, ADP, and ATP (Marko et al., 1964), acetylcholine (1 mglg dry weight, Henschler, 1954) and gluconic acid (0.6% of fresh weight, Nye et al., 1973).

In all popular and scientific literature, there is a fraction of royal jelly described as "other, as yet unknown". This phrase not only emphasizes the incomplete state of analytical knowledge about the product, but also the lack of understanding of the biological activities (proven or presumed) of royal jelly. Up to now, despite many efforts, most of these activities have not been proven definitely, nor have they been attributed to any of the known components.

6.4 The physiological effects of royal jelly

The effect of royal jelly on honeybee larvae, for which it was originally intended as food, is briefly described since in addition to being a fascinating biological phenomenon, it is also the basis of the royal jelly "myth".

In the 1950's, in the wake of new discoveries in the medical field of such wonder drugs as penicillin, hormones and vitamins became "popular" and were seen by many as the simple answers to many biological questions. The elusive "hormonal" effect of royal jelly on honeybee larvae led to the belief that its almost miraculous action on bee larvae could be similar on humans.

By deduction these "hormonal" effects were not only responsible for the caste differentiation between worker and queen bee, but also for the enormous fertility of a queen genetically equal to a worker bee, distinguished apparently only by the food it ate. The same applies to the queen's longevity, unique for an adult insect. Though it is known that royal jelly is a necessary food for the queen's survival and productivity, it is not known which royal jelly fractions are essential, which ones can be replaced and what constitutes minimum or optimum requirements for a queen. Almost all the attention has been focused on the immature stages of development.

Numerous studies were carried out to discover hormones or other substances powerful enough to induce all the necessary changes and give the queen such "superior" qualities. Indeed, the initial studies led to the belief that a "queen determinator" did exist and was an extremely unstable substance (as elusive as eternal life). It appeared to be so unstable that one day after secretion, it was already ineffective. However, the results of other studies did not confirm this hypothesis.

In an attempt to identify the queen determinator, all the components of royal jelly, particularly the more unusual ones or those with known biological activity or present in greater quantity have been tested. In the late 1980's the mystery had still not been solved and a number of contrasting hypotheses had produced equally convincing explanations. Rembold et al. (1974) ware thought to have been close to identifying a specific substance with queen determinator activity which they had isolated; other researchers proposed a differentiation mechanism based on the different proportions of nutrients in the food of worker and queen bee larvae. Weiss (1975) and Asencot and Lensky (1975) believed it was the sugar content of larval food (higher for the young queen bee larvae) that was supposed to cause the differentiation into queens.

More recently, Sasaki et al. (1987) proposed yet another hypothesis incorporating the many contrasting results from other researchers and suggested the "correct" viscosity of royal jelly was a key factor together with higher consumption, but even this theory still has to be substantiated with proof. In other words, it is still not known how royal jelly works nor what is responsible for its amazing effects.

However, if parallels are still being drawn between honeybees and royal jelly, and humans and royal jelly, then they should serve to emphasize the complexity and interdependence of different therapies and factors such as who is taking what, when and how much. Eating royal jelly, or rubbing it into the skin will not make anyone younger or live for a thousand years. On the other hand, using it to supplement and support other diets, activities or medicines may have synergistic effects which cannot be explained by a list of compounds and their individual effects. Tests of such a hypothesis in clinical and scientific trials are needed. There is plenty of circumstantial evidence, reviewed in the following section, that leads us to believe that royal jelly might be highly beneficial to mankind.

Royal jelly was initially advertised for its rejuvenating effects (De Belfever, 1958). The activities most frequently reported in advertisements and constantly confirmed in the declarations of those who have taken royal jelly are indicated in Table 6.3, citing the contents of one of Europe's most widespread and popular publication on the subject (Donadieu, 1978). Royal jelly, taken orally for 1-2 months by swallowing or letting it melt under the tongue in doses of 200-500 mg a day, is said to act as a tonic and stimulant, with a euphoric effect on healthy humans.

In addition to these indications, users declared that royal jelly had solved most of their health problems. In many cases these were chronic or recurring disorders, for which other treatments did not lead to the desired results, so that the effects obtained by taking royal jelly have been considered "miraculous".

It must be emphasised that these claims are unconfirmed by any scientific studies or documentation. There is no proof that the effects are exclusively or even mostly attributable to royal jelly.

People who have taken royal jelly said that they soon experienced a feeling of general well-being, i.e. an effect on their physical output (resistance to fatigue), intellectual performance (greater learning capacity and better memory) and on their mental condition (greater self-confidence, feeling of well-being and euphoria). In other words, royal jelly appears to act as a general stimulant, improving immune response and general body functions.

Royal jelly is neither toxic when injected into mice and rats at high dosages of up to 3 g per kg body weight per day (Hashimoto et al., 1977) nor mutagenic, as tested on DNA of Salmonella typimurium (Tamura et al., 1985).

Takahashi et al., (1983) reported cases of allergic contact dermatitis in 2 out of 10 patients subjected to patch tests. In the context of allergic reactions it needs to be mentioned that intramuscular or intraperitoneal injections, the most common form of royal jelly administration in early years, have been completely abandoned (even under strict medical supervision) because of the risk of serious allergic reactions (Dillon and Louveaux, 1987) Today, royal jelly is most often administered orally and externally (in cosmetics).

In vitro studies have confirmed that lO-hydroxydecanoic acid in royal jelly has antibiotic activity. The antibiotic effectiveness is thermostable, i.e. is not destroyed by moderate heating, but it decreases with improper or long-term storage. Antibiotic action has been proven against the following microorganisms: Escherichia coli, Salmonella, Proteus, Bacillus subtilis and Staphylococcus aureus (Lavie, 1968; Yatsunami and Echigo, 1985). It shows one quarter of the activity of penicillin against Micrococcus pyrogens and is also fungicidal (Blum et al., 1959). In vitro, antiviral effects have been described (Derivici and Petrescu, 1965) and better resistance to viral infections has been observed in mice.

This same antibiotic action of fatty acids is neutralized by raising the pH above 5.6. Since injection into blood, muscle or the peritoneal cavity will raise the pH to 7.4, and the pH is above 5.6 in the intestines, the therapeutic value of the anti-bacterial activity of fatty acids is likely to be negligible for any internal applications, but will remain effective for topical use.

In studies on the internal effects of royal jelly with live animals or humans the jelly is usually administered either by mouth or by injection. The latter allows better assessment of hormonal activities ascribed to royal jelly but carries a substantial risk of allergic reactions.

Positive effects on reproductivity, though not necessarily due to hormone-like action, have been reported at least for chickens, quails and rabbits. Rabbits reacted to a normal diet supplemented with 100-200 mg of royal jelly per kilogramme of body weight with increased fertility and embryonic development (Khattab et al., 1989). Japanese quail reached sexual maturity sooner and laid more eggs after supplementation of diets with high doses (0.2 g) of lyophilized (freeze-dried) royal jelly (Csuka et al., 1978). Bonomi (1983) increased egg production, fertility and hatching in laying hens by using 5 mg royal jelly per kg of feed, but Giordani (1961) found no histological changes in male or female reproductive organs or weight gain with higher doses of 10 to 40 mg per day.

Growth rates of mice slightly increased with a dosage of 1 g of royal jelly per kg of feed, but decreased with higher dosages (Chauvin, 1968). Bonomi (1983) reported weight increases in chicken, partridges and pheasants with a supplement of S mg royal jelly per kg of feed and Salama et al. (1977) reported weight increases in rats when 10, 20 or 40 mg were injected directly into their stomachs. The administration of 0.02 g of royal jelly to calves less than 7 days old gave a weight gain of 11 - 13 % during the following 6 months in comparison with untreated controls (Radu-Todurache et al., 1978). They also mentioned that the treated calves showed lower mortality and higher resistance to infection.

Intravenous injections cause slight vasodilation (temporary enlarging of blood vessels) and have a hypotensive effect (lowering blood pressure); both due to acetylcholine in royal jelly (Jacoli, 1956; Shinoda et al., 1978).

Injections of royal jelly solutions induced higher blood sugar levels than oral applications (Chauvin, 1968). No hypoglycemic (insulin-like) reaction could be shown in rats (Fujii et al., 1990). Afifi et al. (1989) reported weight increases in guinea pigs after injection of 100-300 ing royal jelly per kilogramme of body weight. Small doses injected into cats raised haemoglobin and erythrocyte counts and repeated doses of up to 10 mg/kg of body weight stimulated motor activity and weight gains in mice. Repeated higher doses of 100 mg/kg in mice, however, caused weight loss and impaired cerebrocortical (brain cortex) cellular metabolism (Lupachev, 1963).

In other studies human diseases were simulated in animals in order to identify the mechanisms of royal jelly action. Thus it is known that royal jelly can reduce blood plasma levels of cholesterol and triglycerides (Cho, 1977) and cholesterol and arterial cholesterol deposits in rabbits when these disorders were induced experimentally (Carli et al. 1975). Nakajin et al., (1982) stated that although royal jelly has no effect on lipid levels in blood plasma in normal rabbits, it can reduce the cholesterol content in the blood of animals fed on a diet which induced high levels of blood cholesterol.

Vittek and Halmos (1968) found that royal jelly promoted bone healing in rabbits. The healing of skin lesions was accelerated and anti-inflammatory action was shown for rats by Fujii et al. (1990).

Other researchers tested royal jelly and some of its compounds on tumour cell cultures, showing the inhibitory action of lO-hydroxydecanoic acid (Townsend et al., 1960) and certain dicarboxylic acids. However, they also showed that the same acids could induce tumours in mice when royal jelly is mixed with the culture medium (several mg/ml at less than pH 5) prior to injection into the test animals (Morgan et al., 1960). Wagner et al., (1970) found no significant effects of prolonged survival in mice irradiated against experimentally induced tumours and treated with royal jelly (20 mg/kg of body weight) as compared to control mice which did not receive any royal jelly. More recently, Tamura et al., (1987) have shown tumour growth inhibition in mice with prophylactic and therapeutic oral administration of royal jelly. Inhibition of rapid-growth cancers (leukaemia) was insignificant but it was noticeable on slow-growing, solid tumours (Ehrlich and Sarcoma strains).

Studies of the effects of royal jelly on humans are extremely numerous, particularly in Eastern Europe. A few early studies were presented in Russian by Braines (1959, 1960 and 1962). Most studies however, arc difficult to evaluate for the scientific value of the reported information. Although many are presented as scientific publications, they often lack details on test methods, use parameters difficult to quantify (well-being, euphoria and rejuvenation) do not entirely exclude effects from other concurrent treatments, or use subject numbers too small to exclude accidental effects or natural variation. Of all the works consulted and selected for this chapter, of which a few are summarized in Table 6.4, not one is totally without criticism. The information presented therefore must be considered only as an indication of possible effects requiring further clinical testing.

The mechanisms of royal jelly's activity is not known and none of the numerous hypotheses have been confirmed. An early explanation (Johansson and Johansson, 1958) claiming high vitamin content as a contributory factor can be refuted on the grounds that the same effects should then be achievable with vitamin supplements or a glass of milk, which contains amounts of vitamins similar to the usual dose of royal jelly. Beneficial effects on intestinal flora through selected anti-microbial action can mostly be excluded due to pH. The action of some compounds on endocrine glands, or becoming part of enzyme systems or directly affecting intermediate metabolism has been suggested by Bonomi (1983).

6.5 Uses and marketing of royal jelly

Royal jelly can be sold in its fresh state, unprocessed except for being frozen or cooled, mixed with other products, or freeze-dried for further use in other preparations. The fresh production and sale can be handled by enterprises of all sizes since no special technology is required. In its unprocessed form it can also be included directly in many food and dietary supplements as well as medicine-like products or cosmetics. For larger industrial scale use, royal jelly is preferred in its freeze-dried form, because of easier handling and storing. Freeze-dried royal jelly can be included in the same products as the fresh form. The production of freeze-dried royal jelly requires an investment of at least US$ 10,000 for a freeze-dryer, sufficient production volume and an accessible market for the raw material or its value added products. The discussion below describes some of the value added products in which royal jelly has been included in the past.

Since the assumed benefits of royal jelly have not been sufficiently proven, statements in advertisements and on package labels should be very careful to avoid suggestions which are not well-founded. Any kind of fraudulent or exaggerated statements and claims are in the long run more damaging than any short-term benefit that may be derived from, for example, an increase in the price of a product. Products containing royal jelly should be specially marked or packaged in order to distinguish them from similar products without it.

Royal jelly belongs to a group of products generically described as "dietary supplements" These are products which are consumed not for their caloric content nor for pleasure, but to supplement the normal diet with substances in which it might be lacking. In reality, however, the use of royal jelly is not so much linked to its high content in "noble" substances, but to its assumed stimulant and therapeutic value. However, it cannot be defined as a medicine because the data required for classification in this category are lacking. If it were declared a medicine, its use would become dependant on medical prescriptions and the production and marketing of royal jelly-based products would become the exclusive domain of the pharmaceutical industry.

A large amount of royal jelly is sold and consumed as it is harvested. In its unprocessed, natural state, it is preferred by most producers, because it does not require any special technology, and by consumers because of its unaltered "naturalness". The fact that its taste is not very pleasant, instead of deterring consumers appears to enhance its image as a "medicine". For those who do not appreciate this particular medicinal aspect, royal jelly can be mixed with a little honey, sugar syrup or water, or it may be encapsulated.

Unprocessed royal jelly is usually packaged in small, dark glass bottles of sizes that correspond to the duration of a "treatment" e.g. 10, 15 or 20 g. A tiny plastic spatula is usually included for the "correct" dosage of 250 - 500 mg (see Figure 6.2) Special isothermal packaging (usually a moulded polystyrene box) is sometimes used to make the product look even more precious and protect it perhaps from brief temperature fluctuations. In Italy, in the past, it was also sold in special glass syringes, allowing more precise dosages and giving greater protection against oxidation.

Producers also sell pure royal jelly in its original queen cells after having removed the larvae and sealed the cells. The cells may be sealed with another wax queen cell cup, with liquid wax or by squeezing the ends of the cell together. The queen cells thus prepared can be packaged in small plastic boxes or glass jars together with a small spatula. The main disadvantage of this type of packaging is that the royal jelly does not keep well (two weeks in a refrigerator or a few months when frozen immediately) and only sells well directly from the producer to the consumer. On the other hand such sales can be extremely profitable and are also attractive to consumers who can be sure that the product is untreated and fresh. Given the normal variation in content of queen cells the net weight must be given for the smallest possible quantity (e.g. minimum content 250 mg/cell).

Royal jelly sold in any of the above forms must always be kept at or below 5~ C during storage, during transportation and in the retail store. Empty packages can be displayed while full containers are stored in a refrigerator.

A mixture of royal jelly in honey (1-3 % royal jelly) is probably the most common way in which royal jelly is used as a food ingredient. Among the advantages of this product are that no special technology is required and the honey masks any visible changes in the royal jelly. The final product is pleasant-tasting and it provides the beneficial effects of both products. One teaspoon of the mixture typically contains 100 - 300 mg of royal jelly, about the dosage of royal jelly that is most commonly recommended. Nothing is known however about the preservation of royal jelly in such a mixture. It should, therefore, be kept refrigerated.

Another food frequently enriched with royal jelly in some European countries is yogurt, which has an acidity similar to royal jelly and also requires refrigeration. Yoghurt is already a popular food for health-conscious consumers who often appreciate its further enrichment with royal jelly. The higher price that is usually charged reflects what the market will bear rather than the extra production costs, i.e. the market value added to such a product by the royal jelly is higher than the cost of the jelly and extra production costs.

Sometimes, vitamin supplements and fruit juices are enriched with freeze dried royal jelly. Royal jelly is widely used in beverages in Asia.

Royal jelly is also sold in a jelly made of honey, sugar, jam and pectin. Though simple enough to produce, there are no data available on the durability or residual efficacy of royal jelly presented in this way.

This category of products resembles medicines as far as their form of presentation is concerned, but in other respects these products are no different from the dietary supplements and foods described in the two preceding sections. However, they require more advanced technology for production and packaging and make higher demands on product stability as well as quality control. For the same reasons, many of these applications use freeze dried royal jelly. Unfortunately, the pricing of these products does not always reflect the quality of the product and many are grossly overpriced.

In medicine-like formulations royal jelly is generally included for its stimulatory effects. However, it is also used to solve specific health problems. A variety of formulations are available, often containing ingredients otherwise used to alleviate particular afflictions. As has been seen in an earlier section, there is no solid scientific base for any such uses. Advertising or other popular information should therefore be treated with great caution and royal jelly should never be used as a substitute for other treatments unless the treatment has been approved by a competent physician.

Whether royal jelly is the only active ingredient, or is mixed with others, the basic forms of presentation remain the same and are adapted to the desired applications or consumer preferences. Dosages may be presented in any of the following ways (see Figure 6.3):

- as a single dose package of dry royal jelly with separate solvent,
- as a single dose of mixed pulverized ingredients with or without solvent and in tablet or capsule form,
- as a single or multiple dose liquid solution for oral administration or injection

Single-dosage packages generally have to use a filler to bring the dose of the active ingredient (royal jelly or the ingredient mix) to a volume that can be easily handled by the consumer. An envelope containing only 250 mg of freeze-dried royal jelly would look very empty and the powder it contained might easily be lost. Sugar, salt, aromas, citric acid, glycine, a.o. may all serve as fillers (see Figure 6.4). As well as being mere fillers, they often render the product more pleasant to taste. Additional ingredients mixed with royal jelly are often other food supplements like plant extracts (ginseng), yeasts, pollen extracts and others.

Most packages provide the dry phase in a separate package, envelope or vial and a solvent in an appropriate container. Not only does this separation allow more effective treatment of the liquid phase (such as pasteurization or sterilization) but it also improves storage life and therefore facilitates shipping and marketing. Some refined packaging contains the dry phase in a special lid which upon opening releases the powder into the solvent.

In tablet form, the principal excipient is usually a powdered sugar plus a binding agent such as gum arabic (for simple recipes see 5.16.5 and 6.11.7). For larger production, tableting machines are necessary which can sometimes be purchased second-hand at reasonable prices. Hard and soft gelatine capsules can be used for similar formulations. The hard capsules can be filled by hand on a small scale or by machine on a more industrial level (see also Figures 3.10 and 3.11), but soft capsules and gelatine drops need expensive equipment and are usually manufactured only by larger enterprises or under contract by large enterprises for third parties.

Another form of presentation is in vials with a liquid solution of royal jelly. These are simple to prepare and can use fresh unprocessed royal jelly, but they present preservation problems both with regard to microbiological activity and the long-term stability of the royal jelly. The addition of a little alcohol or propolis extract increases protection against microbial growth. Such preparations are distributed widely and are now being imported mostly from Asia by Europe, the USA and some Latin American countries . One of the more common formulations contains honey, royal jelly and an alcohol extract of ginseng (see Figure 6.10). Since these products are not regulated as food or as medicines, they are not required to list all ingredients, particularly the different preservatives which are necessary in these liquid formulations.

The production of injectable royal jelly preparations must be left to qualified laboratories in order to avoid problems with contamination and toxicity. There are patents that protect the production of royal jelly extracts for human use (by injection), but up to now there is no actual production or use for these "medicines", at least in Western Europe.

The medicinal or pseudo-medicinal use of royal jelly is much more popular in Asia and Eastern Europe, where rules on medicinal formulations and applications are very different from those in Western Europe and North America. In Africa, very little use of royal jelly has been reported, either as a food supplement or as medicine.

Except in Asia, probably the largest use of royal jelly is in cosmetics. Royal jelly is included in many dermatological preparations, but mostly in those used for skin refreshing, and skin regeneration or rejuvenation. It is also used in creams or ointments for healing burns and other wounds. It is usually included in very small dosages (0.05 to 1 %) but it is likely that it deteriorates relatively quickly. No precise data on loss of effectiveness are available. The freeze-dried form of royal jelly is usually preferred because of ease of handling. A royal jelly/lactose paste mixed at 0⁰C is said to stabilize royal jelly (Rubinsstein, 1954). The paste can then be added to cosmetic preparations. More information and recipes can be found in Chapter 9.

The only other known uses for royal jelly are in animal nutrition. In particular, royal jelly has occasionally been used (fresh or freeze-dried) to stimulate race horses. For experimental purposes it is also used as a food for rearing mites and insects.

6.6 Royal jelly collection

Royal jelly is produced by stimulating colonies to produce queen bees outside the conditions in which they would naturally do so (swarming and queen replacement). It requires very little investment but is only possible with movable comb hives. Expert personnel are required, who are able to devote considerably more time than is commonly required for the production of other bee products. Without this prerequisite it is possible to only occasionally collect the contents from cells of natural swarms - and this amounts to no more than a gram or two per hive.

A well-managed hive during a season of 5-6 months can produce approximately SOOg of royal jelly. Since the product is perishable, producers must have immediate access to proper cold storage (e.g. a household refrigerator or freezer) in which the royal jelly is stored until it is sold or conveyed to a collection centre.

The most rational and economic methods for large scale production are variations of the Doolittle method of queen rearing. Usually, the starter colony is omitted and cell cups, with transferred larvae, are directly introduced into the finisher colonies. Strong queenright colonies are preferred, in which the queen chamber is separated from the cell rearing chamber by a queen excluder. The only required adaptation is to shorten the cycle in the finishing colonies (3 days versus 10) before cells are removed for harvesting (Figure 6.5). For occasional and small scale production any other queen rearing method can be used. However, there are many queen rearing methods which differ only in hive design and the use of starter and/or finisher colonies. For more details, it is recommended that the reader consult a regular beekeeping text or better, one specialized in queen rearing. Recommended English texts are Laidlaw, 1979; Laidlaw, 1992 and Ruttner, 1983.

a)

b) Figure 6.5 : a) Special frame with queen cells for queen rearing or royal jelly harvesting. These cells have already been sealed and are too old for collection of royal jelly. However, queens may be raised from these cells if they are introduced into queenless hives. b) Queen cells of the right age for royal jelly harvesting.

The basic requirements are movable comb hives, preferably some queen excluders, queen cups (made from wax or plastic), a transfer needle, a spoon or suction device to remove royal jelly, dark glass vials and a refrigerator. Special hive modifications may facilitate the work according to personal preferences, and centrifugal extractors for royal jelly may be used for large scale production. Feeding with sugar syrup (1:1 in sugar/water) increases cell acceptance, even when flowers are available.

Individual queen cells should not contain less than 200 mg of royal jelly. Low cell content means that there are too many cells for the finisher colony or that the colony is not in a condition to provide for queen rearing. There are racial differences in productivity and specially selected strains can be obtained. However, importing queens may not guarantee higher production in a different environment and carries a considerable risk of importing new or resistant diseases, thus reducing productivity and economic feasibility.

Mature queen cells, i.e. those with larvae four days old (3 days after grafting), must be brought quickly into the extraction room. The open, narrow part of the cells is cut to facilitate and speed up collection. Then the larvae are removed with a pair of soft forceps, taking care not to harm them and contaminate the jelly. The royal jelly is extracted by emptying each cell with a small spatula, by sucking it up with a special mouth operated device, with a pump operated device or by centrifugal extraction (see Figure 6.6). Following extraction, the cells are immediately ready for another rearing cycle.

The royal jelly must be filtered using a fine nylon net (nylon stockings are excellent) to eliminate fragments of wax and larvae. Metal filters should not be used. The jelly should be placed into dark glass vials or food-grade plastic containers, avoiding any excessive exposure to air. It should be refrigerated immediately. Any material or equipment contacting royal jelly - including hands - must be clean and disinfected using heat or pure alcohol. The laboratory must be kept impeccably clean and extraction should never be done outside or in sunlight.

The commercial production of royal jelly requires a methodical approach, good organization and precise timing. Constant attendance is essential as one day off can eliminate two days of production. In order to have a weekly day of rest (e.g. Sunday) no queen cells would be introduced on Thursday, which means that there will also be no collection on the following Wednesday.

These techniques are suitable for both small and quite large enterprises. Depending on the intended market, the approach can be either one of low cost or one in which all collecting, processing and distribution takes place in highly controlled environments. The latter will result in a product which is better suited for industrial use (see also section 6.11.1).

6.7 Storage

Royal jelly has a limited shelf-life. Early beliefs in the extreme instability of royal jelly activity, based on the alleged rapid loss of the "queen determination" factor (see 6.4.1) have not been confirmed. Since neither the mode of activity nor the actual effects of royal jelly are known, there are no data available on changes in its biological effectiveness on humans after long term storage.

Information is, however, available on changes in composition due to long term storage, such as a higher acid titre, a large unsoluble protein fraction, less free amino acids, less glucose oxidase and others (Takenaka et., 1986 and Karaali et al, 1988). Such changes make it appear likely that also biological activity is influenced by storage. Refrigeration and freezing delay and reduce the chemical changes. Although freeze-dried jelly is the most staable form of royal jelly, some changes still take place.

a)

b)

Figure 6.6: a) The steps for removing royal jelly from a queen cell and a diagram of a simple suction device for the collection of royal jelly from queen cups. b) A small vacuum pump can be adapted for the collection of larger quantities of royal jelly. Note that all the queen cells have been cut down in size to facilitate removal of the larvae and the royal jelly.

On the basis of the above, we can conclude that refrigeration of royal jelly at 0~ to 5 ⁰C is a minimum precaution. Still better is storage, whenever possible, at temperatures below -17⁰C, which is attainable in most household freezers. Since royal jelly is an emulsified product and not cellular tissue, freezing presents no particular problem and common household freezers can be used.

As there are no criteria for establishing "safety" limits for product activity, storage and shelf-life should be kept as brief as possible. For products sold in Europe, the average recommended storage time after production is 18 months under refrigeration. For products stored at - 170 C, storage can be extended to 24 months. After defrosting and packaging, the product should not be stored in a refrigerator for more than 12 months.

Freeze-dried royal jelly and royal jelly based products are generally stored at room temperature, sometimes for several years. Freeze-dried royal jelly is certainly more stable than the fresh product, but it was reported that only during the first two months of storage at room temperature no signs were observed of any deterioration (Okada et al., 1977). Therefore, also in this case cold storage is recommended to minimise changes and products should be kept on the shelf for as short a time as possible.

The storage recommendations for fresh and dried royal jelly are valid in the same way for all wet or dry products to which royal jelly has been added. Contrary to many recommendations on packages, these products should be stored in the same manner as the pure, fresh jelly.

In 1956, a French patent was granted for a method of stabilizing royal jelly by mixing it with an easily assimilable, adsorbent substance such as a carbohydrate or protein. A homogenised paste of 10 g fresh royal jelly with 100 g of lactose, mixed at 0⁰C was proposed by Jean (1956). However, no evaluation or verification of increased shelf-life is available. Such support substances, often sugars but also glycine are frequently used to increase the volume of single doses of freeze-dried royal jelly, to make handling easier for both packers (weighing of very small quantities is both difficult and imprecise) and customers.

Like all other bee products, royal jelly has its own microbiological protection and presents few microbiological storage problems when it is in its natural state. This protection however is not absolute and certain hygiene precautions must be observed during production (section 6.6) and storage. Hygienic working conditions and clean containers are a minimum requirement, and airtight containers should be used to provide additional protection not only against contamination but also against oxidation.

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